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MUC1 IN THE DIAGNOSIS AND
PATHOGENESIS OF MALIGNANT
MESOTHELIOMA
Alina Miranda BSc (Hons)
This thesis
is presented for the degree of
Doctor of Philosophy
At
The University of Western Australia
School
Of Medicine and Pharmacology
2011
SUMMARY
Malignant mesothelioma is a highly aggressive tumour arising from the serosal surfaces
of the pleural, peritoneal and occasionally pericardial cavities. Despite the universal
ban of asbestos use in mining and in many industries, the incidence of mesothelioma is
expected to increase in Australia until 2020, mainly because of the long latency period
between initial asbestos exposure to development of disease, making it an extremely
important health issue for our society. The new wave of asbestos related disease is now
seen from individuals in the construction industries and the everyday home handy man.
The diagnosis of mesothelioma is an extremely difficult process and is usually made in
conjunction with clinical, radiological and pathological findings.
It is therefore
understandable that the interest to identify a technique to aid in the early diagnosis of
mesothelioma is extensive.
Several biomarkers and combinations of biomarkers have been evaluated to aid in the
non-invasive diagnosis of malignant mesothelioma. Pleural effusion is one of the
earliest signs and symptoms that patients present with, however, despite recurrent
pleural effusions and serial chest x-rays including computed tomography (CT) scans, it
is not unusual that a definite diagnosis is not possible until tumour burden increases
where it is more visible on radiology while the patient’s symptoms become more
debilitating and the overall prognosis becomes worse. One of the main diagnostic
difficulties is the ability to differentiate between benign reactive mesothelial
proliferation and mesothelioma.
Currently, the ‘gold standard’ for a diagnosis of
mesothelioma requires a histological biopsy specimen with invasion being the only
reliable marker between benign reactive mesothelial proliferation and malignant
Page i
mesothelioma. Unlike epithelial adenocarcinomas, there are no sensitive and specific
tumour markers available to detect mesothelioma. Although pleural effusion is readily
drained and easily collected for cytological investigation, the cytological diagnosis of
mesothelioma is highly controversial and unacceptable at an international level.
However, some centres have been successfully diagnosing mesothelioma on cytological
effusion samples for more than 30 yrs and one of the main reasons for this success is the
use of the anti-MUC1 antibody epithelial membrane antigen (EMA).
The anti-MUC1 EMA antibody has been used extensively as a differential marker
between reactive mesothelial cells and mesothelioma; however, internationally there has
been less confidence in its ability to equivocally differentiate between these two
processes, mainly due to inconsistent results. This is the first study designed on a
prospective patient cohort with reviewers blinded to clinical, surgical, radiological and
previous pathology results to examine the performance of EMA as a diagnostic marker
in the cytological diagnosis of mesothelioma.
The results demonstrate the sensitivity
and specificity of five anti-MUC1 EMA antibodies highlighting that the EMA E29
clone has the highest sensitivity and specificity for a final diagnosis of mesothelioma.
Several previous studies have implicated that MUC1 plays a vital role in the
pathogenesis of epithelial malignancies including breast, lung, prostate and pancreatic
tumours however this is the first time that MUC1 has shown a functional role in cell
migration, invasion and tumorigenesis of mesothelioma.
Understanding these
molecular differences seen in the malignant phenotype enables us to understand the
pathway to tumour progression and ultimately it enables us to design specific
immunotherapies targeted towards treating aggressive tumours such as mesothelioma.
Page ii
ACKNOWLEDGEMENTS
Whatever possessed me to do this at my age I will never know, but it was certainly one of the
most challenging experiences that I have had to face to date.
I would sincerely like to thank my two principal supervisors Professor Jenette Creaney and
Professor Anna Nowak for their continuous support, encouragement and understanding. I know
it must be hard to teach an ‘old’ dog new tricks and I thank you both for your patience,
encouragement and persistence especially towards the end.
To the PathWest research committee, thank you for providing me with the opportunity to reach
this goal.
I sincerely thank all the staff within the Histology and Cytology departments,
especially Mike Platten, Chris, Felicia, Fran, Tara, John, Hendrica and Matt, for all your help in
preparing and collecting the material. A special thank you goes to Dr Sterrett and Dr Segal for
the valuable hours that they have contributed and to Dr DeBoer, Dr Amanuel, Dr Frost, Dr
Kumarasinghe and Dr Williams for all their continuous encouragement and support.
A special thank you goes to Deborah Yeoman who guided me through the early days, thanks for
keeping me driven and focused. Thanks to all the staff from the Biomarker and Discovery area,
Ian, Hanne, Justine, Sarah, David and Crystal. Thanks to all other TIG staff who kept me
entertained on a daily basis in the labs, Bruce, Richard, Cleo, Amanda, Ali, Dozi, Amy, Joanne,
Sam, Ann, Karen and Kelly. Sincere thanks go to Scott for your molecular assistance and to
Mel for her continued support and encouragement while trying to put this thesis together.
Thanks to the LIWA staff for their assistance, AiLing, Baharah, Sally, Hui Ling, Cecilia and
Steve. I would like to thank Lisa and Marie for their help and support while I was working with
them at SJOG research.
To my family and friends, in particular, Cath who initially encouraged me to take this challenge,
David, Alice, Shane and Sandy, thanks guys for being there continually for my kids and for me.
To all the mums and teachers from Kindy to Year 3 at JTC primary thanks for keeping me
informed with all the kids activities, excursions, school drop offs and school pickups. Sincere
thanks goes to Linda for being there for me during my most difficult moments.
Finally, to my beloved family and especially to my dedicated husband Sunil, thank you for your
unconditional love, continued support and encouragement. To my three beautiful children,
Aimee, Shaila and Kanan, I know it has been a rough ride for all of us but I hope that somehow
I can repay those lost days? I love and thank you all for helping me to survive this journey and
for your continued love, hugs and kisses that kept me sane through this extremely difficult
process.
Page iii
DECLARATION
All experiments presented in this thesis were performed by the PhD candidate, except as stated
below. This thesis has not been previously accepted for any other degree at this, or any other
institution, and contains fewer than 100, 000 words.
Mrs Hanne Dare, performed the CA15-3 assays required for Chapter 4, results.
Ms Sarah Wong performed most of the FACS experiments of cell lines described in Chapter 5.
Signature:
………………………………………….
Date:
………………………………….............
Page iv
TABLE OF CONTENTS
Summary…………………………………………………………………………………i
Acknowledgments……………………………………………………………………...iii
Declaration……………………………………………………………………………...iv
Table of Contents………………………………………………………………………..v
List of Figures………………………………………………………………………......xi
List of Tables..................................................................................................................xiv
List of Abbreviations…………………………………………………………………..xvi
CHAPTER 1:
1
LITERATURE REVIEW
1
1.1
1.1.1
MALIGNANT MESOTHELIOMA
2
Asbestos
3
1.1.1.1 History of Asbestos use in Australia
1.1.2
Other aetiological agents
5
5
1.1.2.1 Simian Virus 40 (SV-40)
6
1.1.2.2 Radiation
7
1.1.2.3 Erionite
8
1.1.3
Genetic Predisposition
9
1.1.4
Clinical presentation of mesothelioma
9
1.1.5
Establishing a diagnosis of malignant mesothelioma
10
1.1.5.1 Histological assessment of malignant mesothelioma
10
1.1.5.2 Cytological diagnosis of malignant mesothelioma
12
1.1.6
1.1.5.2.1
Histochemistry analysis of malignant mesothelioma
14
1.1.5.2.2
Immunohistochemistry of malignant mesothelioma
15
1.1.5.2.3
Ultrastructural analysis of malignant mesothelioma
18
Therapeutic options for mesothelioma
19
1.1.6.1 Surgery
20
1.1.6.2 Chemotherapy
21
Page v
1.1.6.3 Radiotherapy
22
1.1.6.4 Multimodality approach to treatment
23
1.2
1.2.1
MUC1
23
Structure of MUC1
24
1.2.1.1. Gene structure of MUC1
24
1.2.1.2 Protein structure of MUC1
25
1.2.1.3 Alternatively spliced variants of MUC1
26
1.2.1.4 Soluble MUC1
28
1.2.2
Function of MUC1
29
1.2.2.1 MUC1 in protection of cells and as an adhesion molecule
29
1.2.2.2 MUC1 in reproduction
30
1.2.2.3 MUC1 mediated immunoregulatory mechanisms
31
1.2.2.4 The role of MUC1 in signal transduction
32
1.2.3
1.3
1.3.1
MUC1 antibodies
33
MUC1 EXPRESSION IN MALIGNANCY
35
MUC1 protein expression in malignancy
35
1.3.1.1. CA15-3 levels in malignancy
38
1.3.2.
MUC1 mRNA expression in malignancy
39
1.3.3.
MUC1 cytoplasmic tail in malignancy
40
1.3.4.
MUC1 as an adhesion molecule in malignancy
41
1.3.5.
MUC1 resistance to apoptosis
42
1.3.6.
Why is MUC1 disregulated in cancer?
43
THESIS OVERVIEW
44
1.4
CHAPTER 2
47
MATERIALS AND METHODS
47
2.1
TISSUE CULTURE
48
Page vi
2.1.1
Maintenance of cell lines
48
2.1.2
Splitting cells
49
2.1.3
Viable cell counting
49
2.1.4
Freezing cells
50
2.1.5
Cell blocks from cell lines
50
PATIENT SAMPLES
50
Cell block microarray (CBMA)
52
2.2
2.2.1
2.2.1.1 Immunohistochemistry staining of CBMA
52
2.2.1.2 Manual immunohistochemistry staining of CBMA
52
2.2.1.3 Automated immunohistochemistry staining of CBMA
53
2.3.
ANALYSIS OF CBMA
54
2.3.1
Immunohistochemical scoring of CBMA
56
2.3.2.
Statistical Analysis of CBMA Immunohistochemical Staining
56
2.4
CA15-3 ASSAY
57
2.5
MESOTHELIN ASSAY
58
2.6
HOMOGENISATION OF CELL LINES IN ULTRASPEC RNA ISOLATION
REAGENT
58
2.7
RNA EXTRACTION AND REVERSE TRANSCRIPTION
59
2.8
QUANTITATIVE POLYMERASE CHAIN REACTION (PCR)
59
2.9
IMMUNOHISTOCHEMISTRY STAINING OF CELL LINES
60
2.10
SOFT AGAR ASSAY
61
2.11
CyQUANT CELL PROLIFERATION ASSAY
61
2.12
WST-1 CELL PROLIFERATION ASSAY
62
2.13
TRANSWELL MIGRATION ASSAY
63
Page vii
2.14
WOUND HEALING ASSAY
64
2.15
FLOW CYTOMETRY
64
2.16
SH-RNA MUC1 STABLE CELL LINE TRANSFECTION
65
2.16.1
Transformation of plasmids in competent cells
66
2.16.2
Quick plasmid mini preparation
66
2.16.3
Restriction digest of plasmid
67
2.16.4
Amplification and maxipreparation of purified plasmid
67
2.16.5
Selection for antibiotic resistance
67
2.16.6
Plasmid DNA transfection
68
GENERAL STATISTICAL ANALYSIS
69
2.17
70
CHAPTER 3
SENSITIVITY AND SPECIFICITY OF MUC1 IN THE CYTOLOGICAL
DIAGNOSIS OF EPITHELIAL MALIGNANACY AND MALIGNANT
MESOTHELIOMA
70
3.1
INTRODUCTION
71
3.2
RESULTS
73
3.2.1
Description of patient cohort
74
3.2.2
Description of sample scoring
75
3.2.2.1 Dichotomisation of samples based upon epithelial cellularity
75
3.2.2.2 Categorisation of samples based on cellular morphology
78
3.2.2.3 Scoring of immunoperoxidase staining
81
3.2.3
Immunoreactivity of anti-MUC1 monoclonal antibodies
82
3.2.3.1 Anti-MUC1 antibody EMA (E29) on interim diagnosis
82
3.2.3.2 Anti-MUC1 antibody (Mc5) on interim diagnosis
89
3.2.3.3 Anti-MUC1 antibody (VU4H5) on interim diagnosis
89
Page viii
3.2.3.4 Anti-MUC1 antibody (SM3) on interim diagnosis
90
3.2.3.5 Anti-MUC1 antibody (BC2) on interim diagnosis
90
3.2.3.6 Summary of Anti-MUC1 antibody staining related to interim diagnosis
94
3.2.4
Summary of Anti-MUC1 antibody staining related to Final Diagnosis
97
3.2.5
Sensitivity and Specificity of anti-MUC1 monoclonal antibodies determined on a subset
of 35 mesothelioma patients
101
3.2.6
Comparison of the sensitivity of MUC1 for mesothelioma diagnosis
102
3.2.7
Diagnosing mesothelioma without the anti-MUC1 antibody EMA (E29)
103
3.3
DISCUSSION
112
3.4
CHAPTER SUMMARY
120
122
CHAPTER 4
SENSITIVITY AND SPECIFICITY OF SOLUBLE MUC1 CONCENTRATIONS IN
SUPERNATANT SAMPLES OF PLEURAL MALIGNANCIES
122
4.1
INTRODUCTION
123
4.2
RESULTS
127
4.2.1
Patient characteristics
127
4.2.2
Sensitivity and Specificity of CA15-3 in effusion supernatant samples
128
4.2.2.1 Concordance of MUC1 cell surface expression and effusion CA15-3 levels
132
4.2.3
Sensitivity and specificity of mesothelin in effusion supernatant samples
132
4.2.4
Correlation of CA15-3 and mesothelin levels in effusions
138
4.2.5
Comparing and combining the performance of CA15-3 and mesothelin
138
4.2.6
Relationship between the CA15-3 levels in effusions with survival in patients with
mesothelioma
142
4.3
DISCUSSION
144
4.4
CHAPTER SUMMARY
147
Page ix
149
CHAPTER 5
EFFECT OF MUC1 ON MALIGNANT MESOTHELIOMA CELL FUNCTION
AND CHARACTERISTICS
149
5.1
INTRODUCTION
150
5.2
RESULTS
153
Characterising MUC1 in mesothelioma cell lines
153
5.2.1
5.2.1.1 mRNA levels of MUC1 in mesothelioma cell lines
153
5.2.1.2 Protein expression of MUC1 on mesothelioma cell lines using immunohistochemistry154
5.2.1.3 Protein expression of MUC1 on normal mesothelial cell lines using
immunohistochemistry
5.2.2
ShRNA MUC1 knockdown in mesothelioma cell lines
158
160
5.2.2.1 MUC1 mRNA expression in mesothelioma cell lines following shRNA MUC1
knockdown
160
5.2.2.2 Cell surface expression of MUC1 in mesothelioma cell lines following shRNA MUC1
knockdown using flow cytometry
162
5.2.2.3 Protein expression of MUC1 in mesothelioma cell lines following shRNA MUC1
knockdown using chamber slide immunohistochemistry
165
5.2.3
Effect of reduced MUC1 expression on in vitro mesothelioma cell proliferation
167
5.2.4
Effect of reduced MUC1 expression level on in vitro mesothelioma cell migration
168
5.2.4.1 Invitro scratch assays on mesothelioma and shRNA MUC1 knockdown cell lines
171
5.2.4.2 Cell migration assays on mesothelioma and shRNA MUC1 knockdown cell lines
174
5.2.5
Effect of reduced MUC1 expression on in vitro mesothelioma tumourigenic potential 176
5.2.5.1 Cell migration assays on parental and shRNA MUC1 knockdown cell lines
179
5.3
DISCUSSION
182
5.4
CHAPTER SUMMARY
187
CHAPTER 6
189
Page x
FINAL DISCUSSION
189
6.1
GENERAL DISCUSSION
190
6.2
CONCLUSIONS
196
BIBLIOGRAPHY
198
APPENDIX
220
Page xi
LIST OF FIGURES
Figure
Page
1.1
Histological sections of mesothelioma stained with H and E.........................……......13
1.2
Cytological smears of mesothelioma stained with Papanicolaou stain.........................13
1.3
Schematic representation of MUC1………...…………………………........................26
3.1
Tissue morphology divided into low and high cellularity……..………………...........77
3.2A
H and E section of a case diagnosed as malignant………………………….................79
3.2B
H and E section of a case diagnosed as malignant possibly
adenocarcinoma.............................................................................................................79
3.2C
H and E section of a case diagnosed as benign.................................………................80
3.2D
H and E section of a case diagnosed as atypical/equivocal...........................................80
3.3
Optimisation of cell block microarray..........................................................................85
3.4
Comparison between anti-MUC1 antibodies……………………………….................88
3.5
Cell block microarray stained with the manual E29 antibody….....……..….…...........91
3.6
Cell block microarray stained with the Mc5 antibody....……………………...............92
3.7
Cell block microarray stained with the VU4H5 antibody…………………….............93
3.8
Cell block microarray stained with the SM3 antibody....………………………..........94
3.9
Cell block microarray stained with the BC2 antibody.....…………………….............95
3.10
Characteristics of patient cohort determined from interim pathology diagnosis..…....98
3.11
Hypothetical analysis of the patient cohort in the absence of the EMA (E29)
antibody.......................................................................................................................104
3.12
Hypothetical analysis of the patient cohort in the presence of the EMA (E29)
antibody.......................................................................................................................105
Page xii
Figure
Page
3.12A Hypothetical analysis of the patient cohort in the presence of the EMA (E29) antibody
distinguishing between mesothelioma and other malignancy......................................106
3.12B Hypothetical analysis of the patient cohort in the presence of the EMA (E29) antibody
distinguishing equivocal cell morphology....................................................................107
4.1
CA15-3 concentrations determined in effusions from individual patients................. 130
4.2
ROC analysis to determine the ability of CA15-3 to distinguish between mesothelioma,
benign and malignant cases........…….........................................................................133
4.3
Mesothelin concentrations determined in effusions from individual patients..............135
4.4
ROC analysis to determine the ability of Mesothelin to distinguish between
mesothelioma, benign and all malignant cases........................………….....................137
Spearman correlation of CA15-3 and mesothelin values in effusion samples from
4.5
patients with mesothelioma….………….....................................................................139
4.6
ROC analysis for all epithelial malignancies........…....……………………...............140
4.7
ROC analysis for differentiating mesothelioma from all other samples.…….............141
4.8
Relationship between CA15-3 levels in effusions and survival of patients with
mesothelioma.........................................……………………………….......................143
5.1
Innate levels of MUC1 expression on cell lines...........................................................157
5.2
Normal mesothelial cells stained with anti-MUC1 antibodies....................................159
5.3
mRNA confirmation of shRNA MUC1 knockdown in stably transfected
cell line.........................................................................................................................161
FACS cell surface expression of anti-MUC1 E29 antibody on the VGE62 shRNA
5.4
cell line........................................................................................................................163
5.5
Comparison of FACS cell surface expression on five mesothelioma cell lines..........164
5.6
Innate levels of MUC1 expression of shRNA MUC1 cell line VGE62......................166
ƒ‰‡š‹‹‹
Figure
Page
5.7
Cell proliferation of shRNA MUC1 in VGE62 cell line..........…………...................169
5.8
Cell proliferation of shRNA MUC1 cell lines and parental control............................170
5.9
Wound scratch assay of VGE62 shRNA MUC1 transfectants and parental
control..........................................................................................................................172
5.10
Wound scratch assay of all shRNA MUC1 transfectants and parental
control..........................................................................................................................173
5.11
Transwell migration of all shRNA MUC1 transfectants and parental control cell
lines............................................................................................................................. 175
5.12
Anchorage independent growth of STY51 and normal mesothelial cells..….............177
5.13
Anchorage independent growth shRNA MUC1 transfectant and parental cell
lines...….......................................................................................................................178
5.14
Matrigel cell invasion of shRNA MUC1 cell lines and parental cell
lines...................….......................................................................................................180
Diagnostic algorithm for the proposed utility of MUC1 immunocytochemistry and
6.1
CA15-3 in the differential diagnosis of reactive mesothelial cells, malignant
mesothelioma and epithelial malignancy....................................................................193
ƒ‰‡š‹˜
LIST OF TABLES
Table
1.1
Page
Summary of markers used in the differential diagnosis of mesothelioma and
adenocarcinoma.........................................................................................................16
1.2
Summary of the major differences between the MUC1
variants……………………………………………………………………………...28
2.1
Panel of antibodies used for diagnosis of cell block microarray...............................55
3.1
Patient characteristics grouped by Interim pathology diagnosis................................75
3.2
Characteristics of samples categorized as benign….…………………………….....76
3.3
Summary of immunohistochemical staining of 266 patients...…..............................83
3.3A
A summary of the anti-MUC1 antibody staining on control tissue sections..............84
3.4
Correlation of immunoperoxidase scoring of all cases using EMA clone (E29).......86
3.5
Correlation of immunoperoxidase scoring of all mesothelioma cases using EMA clone
(E29)...........................................................................................................................87
3.6
Correlation of immunoperoxidase scoring of epithelial malignancies using EMA clone
(E29)..........................................................................................................................89
3.7
Summary of sensitivity and specificity of anti-MUC1 antibodies for an interim diagnosis
of epithelioid
malignancy.................................................................................................................96
3.8
Summary of sensitivity and specificity of anti-MUC1 antibodies for an interim diagnosis
of mesothelioma.........................................................................................................97
3.9
Summary of the sensitivity and specificity of anti-MUC1 antibodies for a final diagnosis
of epithelioid
malignancy..................................………………………………………………......101
3.10
Summary of the sensitivity and specificity of anti-MUC1 antibodies for a final diagnosis
of mesothelioma.........................................………………………………………...101
Page xv
Table
3.11
Page
Compilation of data relevant to mesothelioma diagnosis from tables 3.8 and
3.10..............................................................................................................................103
3.12
Comparison of study characteristics for anti-MUC1 antibody for a diagnosis of
mesothelioma...............................................................................................................118
4.1
Patient demographics of effusion supernatant samples collected....…………...........128
4.2
CA15-3 concentrations from the interim diagnosis category...………………...........131
4.3
CA15-3 concentrations in effusions from the final diagnosis category..........……....131
4.4
Mesothelin levels from interim diagnosis category........………………………........134
4.5
Mesothelin levels from the final diagnosis category........…………...………...........136
4.6
Combined performance of CA15-3 and mesothelin...................................................142
5.1
Characteristics of all cell lines used in this study and relative to MUC1 mRNA.....155
5.2
Immunohistochemistry scores of tested anti-MUC1 antibodies in cell lines.............156
5.3
Percentage of cells expressing MUC1 on cells surface relative to isotype control....162
Page xvi
LIST OF ABBREVIATIONS
A
Ampicillan
ATCC
American Type Culture Collection
AUC
Area under the curve
Bcl-XL
B cell lymphoma extra large transmembrane molecule
BFS
Buffered formal saline 10%
bp
Base pairs
BSA
Bovine serum Albumin
C
Celsius
CBMA
Cell block microarray
cDNA
Complementary DNA
CEA
Carcinoembryonic antigen
CK
Cytokeratin
c-Src
Cellular proto oncogene tyrosine kinase
Ct
Threshold cycle
CT
Computed tomography
dATP
Deoxyadenosine-5’-triphosphate
DC
Dendritic cells
dCTP
Deoxycytidine-5’triphosphate
dGTP
Deoxyguanosine-5’triphosphate
dNTP
Deoxynucleotide Triphosphate
dTTP
Deoxythymidine-5’-triphosphate
ddH2O
Double deionised water
DDT
Dithiothreitol
DMSO
Dimethylsulphoxide
Page xvii
DNA
Deoxyribonucleic acid
DPX
Distyrene, Plasticizer and Xylene mounting media
ECL™
Enhanced Chemiluminescence
EDTA
Ethylenediamine tetra-acetic acid
EGFR
Epidermal growth factor receptor
EMA
Epithelial membrane antigen
EPP
Extra pleural pneumonectomy
ErbB
Erythrocyte leukaemia viral oncogene
EtOH
Ethanol
FCS
Fetal calf serum
FNA
Fine needle aspiration
G
Gleason grade
GAPDH
Glyceraldehyde 3-phosphate dehydrogenase
H2O2
Hydrogen peroxide
H and E
Haematoxylin and Eosin
HBBS
Hanks Buffered salt solution
HCl
Hydrochloric acid
HEPES
4-(2-Hydroxyl)-1-piperazineethanesulfonic acid
HMFG
Human milk fat globulin
HREC
Human Research Ethics Committee
HRP
Horse radish peroxidase
IASLC
International association for the study of lung cancer
ISOBM
International Society for Oncodevelopmental Biology
and Medicine
kbp
Kilo base pairs
Page xviii
kDa
Kilo Dalton
Lck
Lymphocyte specific tyrosine kinase
LDR
Length to diameter ratio
M
Molar
MAP2K1
Mitogen activated protein kinase 1
MM
Malignant mesothelioma
mAB
Monoclonal antibody
Mc5
anti-MUC1 antibody
MeOH
Methanol
μg
Microgram
μL
Microlitre
mg
Milligram
min
Minute
mL
Millilitre
mRNA
Messenger RNA
MUC
Mucin
NaN3
Sodium azide
Na2CO3
Sodium bicarbonate
NaCl
Sodium chloride
NCARD
National Centre for Asbestos Related Disease
NaOH
Sodium hydroxide
NPV
Negative predictive value
NSCLC
Non small cell lung cancer
OD
Optical density
PAS
Periodic acid Schiff
Page xix
PBL
Peripheral blood lymphocyte
PBS
Phosphate buffered saline
PC
Pericardial cells
PCAM
Intercellular cell adhesion molecule
P/D
Pleurectomy and decortication
PDGF-A
Platelet derived growth factor
PEFL
Peritoneal fluid
PEM
Polymorphic epithelial mucin
PHA
Phytohaemagglutinin
PLFL
Pleural fluid
PMSF
Phenylmethylsulfonyl fluoride
PO
Periodate oxidation
PPV
Positive predictive value
PUM
Peanut reactive urinary mucin
PVDF
Polyvinylidene difluoride
RB
Retinoblastoma
RNA
Ribonucleic acid
RPMI
Roswell Park Memorial Institute
ROC
Receiver operating characteristics
RT-PCR
Reverse transcription polymerase chain reaction
SEA
Sea urchin sperm protein enterokinase and agrin
sec
Second
SCGH
Sir Charles Gairdner Hospital, Nedlands
SJOG
St John of God Hospital, Subiaco
SDS
Sodium dodecyl sulphate
Page xx
SNOP
Systematized Nomenclature of Pathology
STAT
Signal transducer and activator of transcription
SV-40
Simian virus 40
TAA
Tumour associated antigen
TACE
Tumour necrosis α converting enzyme
TE
Trypsin-EDTA
Tm
Melting temperature
TMA
Tissue microarray
TN
Total negative
TP
Total positive
TTF-1
Thyroid transcription factor 1
VATS
Video assisted thoracoscopic surgery
VNTR
Variable number of tandem repeats
Page xxi
CHAPTER 1:
LITERATURE REVIEW
1
1.1
MALIGNANT MESOTHELIOMA
This chapter will discuss the incidence and aetiology of malignant mesothelioma
(hereafter ‘mesothelioma’), including the diagnostic algorithms and clinical
management currently available, before addressing in more detail the topic of this
thesis.
Malignant mesothelioma is a highly aggressive malignancy involving the pleural,
peritoneal and occasionally the pericardial cavity. Mesothelioma is a fatal disease with
a median survival from initial diagnosis between 9 and 12 months, even with optimal
evidence-based therapy (Vogelzang, NJ, Rusthoven, JJ et al. 2003; Nowak, AK and
Bydder, S 2007). Australia has one of the highest incidence of mesothelioma in the
world, with approximately 600 cases per annum (SafeWorkAustralia 2010).
Approximately 90% of cases occur in the pleura with the remaining cases involving the
peritoneum and isolated cases occurring in the pericardium or testis.
The major causative agent of mesothelioma is exposure to asbestos. Mesothelioma has
an extremely long latency period and usually takes 20 to 40 years to become clinically
apparent after asbestos exposure. Incidence patterns of this disease therefore reflect the
production and use of asbestos in any given region, and currently in Australia are
reflecting a period just before regulatory mechanisms were put in place to minimise
asbestos exposure. In Australia the incidence of mesothelioma has been estimated to
peak in the next 10 years (Clements, M, Berry, G et al. 2007). More males than females
develop mesothelioma as a result of the greater use of asbestos in male dominated
industries.
Incidence rates for Australian males and females aged >20yrs were
53.3/million and 10.2/million respectively (Leigh, J and Driscoll, T 2003). In Great
2
Britain, the occurrence of mesothelioma is predicted to peak in 2016 with an
approximate number of 2040 mesothelioma related deaths per annum (Tan, E, Warren,
N et al. 2010). The peak incidence of mesothelioma in the United States of America
may have already occurred, currently there are between 2500 and 3000 cases diagnosed
annually (Yang, H, Testa, JR et al. 2008).
Significant restrictions have been put in place on the mining and use of asbestos in
many industrialised countries over the last 30 years (Kazan-Allen, L 2005; Bianchi, C
and Bianchi, T 2007; Yang, H, Testa, JR et al. 2008). However, asbestos manufacturers
have found alternative markets for the use of asbestos in the Asia-Pacific region,
targeting underdeveloped countries with minimal regulations limiting asbestos use.
This has resulted in an increased number of mesothelioma cases diagnosed in countries
such as; Brazil, China, Japan, Korea, Malaysia, Thailand, Vietnam, India, Indonesia and
The Philippines. Several of these countries have already consumed more than 60 000
tonnes of asbestos since 2000 (Kazan-Allen, L 2005).
1.1.1
Asbestos
Asbestos has been used for thousands of years for household items from the wicks of oil
lamps to pottery.
Its widespread use began in Canada in 1880, when serpentine
asbestos was used for the construction of heat resistant fabrics. Asbestos has many
properties that make it a good industrial product including extreme flexibility, with high
tensile strength and heat resistance (Rom, WN and Palmer, PE 1974) .
Asbestos has been known to cause disease since 1906, when Montague Murray first
described a case of asbestosis in England (Lemen, R, Dement, J et al. 1980). This
3
report was followed in 1927 by a more detailed account of asbestosis by Cooke and
McDonald who described ‘curious bodies’ originating from asbestos fibres that had
reached the lungs (McDonald, S 1927). In 1930, the first case of ‘asbestos bodies’
found in sputum samples of asbestos workers in the United States was described
(Lynch, KM 1955).
These early studies formed the basis of the hypotheses that
individuals exposed to the asbestos dust develop asbestosis, especially if the exposure
was high or over an extended period of time. However, subsequent authors found that
asbestosis was also occurring in individuals working in industries such as pipe
insulation and ship building.
Cases of extensive pleural calcification were also
observed in workers from sand asphalt and vinyl tile flooring industries (Rom, WN and
Palmer, PE 1974). Further studies into the mechanical trades highlighted that 25% of
individuals exposed to asbestos had x-ray abnormalities consistent with asbestosis and
restricted pulmonary function (Rom, WN and Palmer, PE 1974; Wagner, JC, Berry, G
et al. 1974).
There are two major forms of asbestos, serpentine or chrysotile (white asbestos) and
amphibole which include both crocidolite (blue asbestos) and amosite (brown asbestos).
Crocidolite is an extremely fine, thin, straight fibre which is generally less than 0.1μm
in diameter. In animal models inhaled crocidolite fibres align towards the centre of
airflow and have been shown to extend to the distal alveolar spaces. In comparison,
chrysotile is spiral in structure and adheres to the respiratory mucous membranes via the
ciliary tufts of the bronchial epithelial cells. Chrysotile can be eliminated from the body
more readily than crocidolite or amosite (Bernstein, DM, Rogers, RA et al. 2010).
4
A meta-analysis of over 29 epidemiological studies concluded that amphibole types of
asbestos (such as crocidolite and amosite) have much greater oncogenic potential than
chrysotile for the development of asbestos related diseases (Berman, DW and Crump,
KS 2008). The authors hypothesised that fibres longer than 10μm are more potent than
shorter fibres for inducing mesothelioma (Berman, DW and Crump, KS 2008).
1.1.1.1
History of Asbestos use in Australia
In Australia, there were two main asbestos mining operations. Chrysotile was mined in
Baryulgil in northern New South Wales from 1939 to 1983. Crocidolite asbestos was
mined in Wittenoom, in northwest Western Australia from 1937 until 1966. Asbestos
fibre was also imported from Canada (chrysotile) and South Africa (crocidolite and
amosite) and asbestos containing products were imported from the United Kingdom,
United States of America, Germany and Japan (Bianchi, C and Bianchi, T 2007).
In Australia, the consumption of crocidolite began to be phased out in 1967, although
the use of amosite continued until the mid 1980’s.
Regulations prohibiting the
importation, use or sale of chrysotile came into place on December 31st 2003 (Jamrozik,
E, de Klerk, N et al. 2011). However, asbestos is still present in many Australian homes
and can be found in a range of products including brake pads, clutch linings, insulation,
asbestos cement sheeting, gaskets and adhesives.
1.1.2
Other aetiological agents
Aetiological agents in addition to asbestos have been linked to the development of
mesothelioma, including simian virus 40 (SV-40), radiation and erionite and these will
be discussed below in more detail.
5
1.1.2.1
Simian Virus 40 (SV-40)
The evidence that suggests SV-40 plays a role in the development of mesothelioma
comes from two findings. Firstly that SV-40 infected hamsters develop mesothelioma
(Carbone, M, Rizzo, P et al. 1997).
Secondly that 60% of human mesothelioma
tumours express SV-40-like DNA sequences (Jasani, B, Jones, CJ et al. 2001; Klein, G,
Powers, A et al. 2002; Garcea, RL and Imperiale, MJ 2003).
As SV-40 virus
contaminated monkey kidney cells were used to manufacture early batches of polio
vaccines it has been postulated that this was a mechanism by which SV40 could have
infected vaccinated humans. Carbone et al, hypothesized that SV-40 may not play a
direct carcinogenic role on its own, but that the activity of the SV-40 large T antigen
mediated through the inactivation of tumour protein p53 (p53) and retinoblastoma
tumour suppressor may give rise to mesothelial cells that were more susceptible to the
transforming effects of asbestos (Carbone, M, Rizzo, P et al. 1997). Other studies have
indicated that asbestos fibres and SV-40 may act synergistically as co-carcinogens in
the development of mesothelioma (Robinson, C, van Bruggen, I et al. 2006).
The role of SV-40 as an aetiological agent for mesothelioma is however controversial,
with equivocal findings being reported from a multi-laboratory analysis of reference
tumours designed to explain the conflicting findings in the field (Strickler, HD 2001).
A meta-analysis which reviewed molecular, pathological and clinical data from 1793
cancer patients, concluded that there was no significant role for SV-40 infection in
human brain and bone cancers, mesothelioma and non-Hodgkins lymphoma (Vilchez,
RA, Kozinetz, CA et al. 2003) .
6
In Denmark the polio virus vaccine was administered to most children between 1955
and 1961 and there was no increase in the incidence of mesothelioma (Engels, EA,
Katki, HA et al. 2003). A further study by Manfredi et al (2005) demonstrated that SV40 T antigen DNA and protein were not detectable in tumour tissue of 69 mesothelioma
patients including 16 samples obtained from the United States (Manfredi, JJ, Dong, J et
al. 2005). So, despite the strong mechanistic support for the role of SV40 infection in
mesothelioma pathogenesis, the findings are conflicting and controversial.
1.1.2.2
Radiation
Radiation has also been shown to play a role in the development of mesothelioma. This
is of importance because of the increased use of multimodality approaches including
surgery, chemotherapy and radiation therapy in the treatment of malignancies. The
development of mesothelioma has been reported following radiation therapy for breast
cancer (Hill, JK, Heitmiller, RF, 2nd et al. 1997), cervical cancer (Babcock, TL, Powell,
DH et al. 1976), Hodgkin’s disease (Lerman, Y, Learman, Y et al. 1991), Wilm’s
tumour (Anderson, KA, Hurley, WC et al. 1985) and seminoma (Gilks, B, Hegedus, C
et al. 1988). The most conclusive evidence comes from a study which excluded patients
with previous asbestos exposure as a risk factor in cases of post radiation mesothelioma
(Weissmann, LB, Corson, JM et al. 1996). In this study, levels of asbestos bodies were
measured from a patient’s lung that developed mesothelioma following irradiation
therapy for Hodgkin’s lymphoma. A count of less than 250 asbestos bodies per gram of
lung tissue suggested no significant prior asbestos exposure consistent with normal
exposure for a population. The lack of known asbestos exposure, the younger age of
mesothelioma at presentation and the presence of disease within the field of radiation
7
treatment all suggested that radiation was an aetiological factor for the development of
disease (Weissmann, LB, Corson, JM et al. 1996).
1.1.2.3
Erionite
Erionite is a naturally occurring fibrous mineral which is found in several countries
including Antarctica, Austria, Canada, France, Germany, Italy, Russia, Scotland and
Turkey (Dogan, AU and Dogan, M 2008). Erionite forms brittle fibrous mass-like
structures within rock hollows which appear white to transparent, almost glass-like in
appearance. Erionite was first listed as a carcinogen in 1987 when inhalation studies in
rats confirmed that erionite caused mesothelioma and in animal models eronite has been
shown to be more carcinogenic than asbestos. This is supported by animal experiments
which showed that mesothelioma developed in 27 out of 28 rats exposed to erionite
compared with 11 of 648 rats exposed to asbestos alone (Dogan, AU and Dogan, M
2008). A study performed by Wagner et al 1985, confirmed that 48% of animals
injected with asbestos developed mesothelioma compared to almost 100%
mesothelioma incidence in animals injected with erionite (Wagner, JC, Skidmore, JW et
al. 1985). The morphology of erionite bundles into ‘fibres’ and ‘fibrils’ which increase
surface area to volume ratio may explain the carcinogenetic properties of the mineral.
An extremely high incidence of mesothelioma has been observed in three small villages
in Turkey with approximately 50% of all resident deaths due to mesothelioma. These
villages are highly contaminated with erionite and this is believed to be the causative
agent of mesothelioma. Statistically erionite is considered to be more carcinogenic than
amphibole asbestos (Carbone, M, Emri, S et al. 2007; Constantopoulos, SH 2007).
8
1.1.3
Genetic Predisposition
Genetic factors may also have a role in an individual’s susceptibility to develop
mesothelioma. Evidence comes from two cohort studies. In studies of the Turkish
villages with high incidences of mesothelioma six families were identified which
showed an obvious familial clustering of mesothelioma suggesting an autosomal
dominant pattern of inheritance with incomplete penetrance. Children diagnosed with
mesothelioma had affected parents and 50% of each generation was affected (RoushdyHammady, I, Siegel, J et al. 2001). In a second study the incidence of mesothelioma
observed in immigrants from Turkish villages who showed similarities to the earlier
study, suggesting that the development of mesothelioma was not directly related to the
duration of erionite exposure (Carbone, M, Emri, S et al. 2007).
1.1.4
Clinical presentation of mesothelioma
Characteristically, patients with mesothelioma present with symptoms including chest
pain and shortness of breath. Patients presenting with malaise, fever, severe weight
loss, or persistent cough are less common however these symptoms occur during the
course of the disease. Patients with early disease may present with a pleural effusion
alone, either incidentally identified in the course of another investigation, or following
investigation for dyspnoea. The pleural cavity is the most common site of occurrence
for mesothelioma, however it is also known to occur in the peritoneal, pericardial and
tunica vaginalis.
Establishing a diagnosis of mesothelioma can be extremely difficult especially in the
early stage of the disease (Addis, B and Roche, H 2009). Whilst many patients first
present with advanced disease, in others, the diagnosis is made some months or even
9
years after an initial presentation with pleural effusion, despite multiple investigations
attempting to reach a diagnosis.
1.1.5
Establishing a diagnosis of malignant mesothelioma
Mesothelioma often forms diffuse tumours characterised by serosal thickening with an
associated pleural effusion. Pleural mesothelioma classically forms a thickened growth
along the parietal or visceral surface which encapsulates and constricts the underlying
lung. Direct local invasion of the lung may not be present, but tumour may also involve
the pleural fissures and the chest wall. Destruction of the pleural cavity and invasion
into the chest wall may also be seen at biopsy (Henderson, D, Shilken, K et al. 1992).
The most common first investigative tool for patients with a suspected diagnosis of
mesothelioma is a plain chest x-ray which will often confirm the presence of a pleural
effusion with or without pleural based masses. Computed tomography (CT) scans may
then confirm pleural effusions, pleural thickening, interlobular fissures or possible chest
wall invasion (Nowak, AK and Bydder, S 2007). A CT scan cannot differentiate
between mesothelioma and primary adenocarcinoma of the lung, or metastatic
malignancies to the pleural cavity and hence additional tests are required to differentiate
these types of malignancies.
1.1.5.1
Histological assessment of malignant mesothelioma
The macroscopic tumour tissue may vary in consistency and gross appearance; some
tumours may feel rubbery and firm while others are soft and gelatinous to touch, this
especially applies to epithelioid mesotheliomas which may produce abundant
hyaluronic acid. The tumour colour may also vary from pale yellow to grey-white in
10
appearance and some areas of haemorrhage and necrosis may be evident. In advanced
peritoneal mesothelioma, a large mass may be present in the omentum which may
eventually encase the abdominal viscera including the liver and spleen.
Tissue samples from pleural tumours can be obtained by thoracotomy, blind biopsy or
with the assistance of video-assisted thoracoscopic surgery (VATS). Each method is
associated with its own risks and benefits. VATS is the most common method used, as
it has relatively reduced risk of surgical complications compared to thoracotomy and an
increased chance of identifying tumours compared with blind biopsies (Powell, TI,
Jangra, D et al. 2004).
There are three main histological subtypes of mesothelioma that are recognized;
epithelioid, mixed (biphasic) and sarcomatoid variants. Approximately 70% of cases
are epithelioid mesothelioma in type. These tumours are usually well differentiated
epithelial neoplasms which characteristically have a tubular or tubulopapillary structure
in keeping with papillary architecture (Corrin, B and Nicholson, A 2006) (Figure 1.1A).
Microscopically epithelioid mesothelioma appears quite cytologically bland. The nuclei
are usually round in appearance with a granular chromatin pattern, small prominent
nucleoli and isolated mitoses (Henderson, D, Shilken, K et al. 1992; Corrin, B and
Nicholson, A 2006) and may be difficult to differentiate from benign mesothelial
hyperplasia.
Sarcomatoid mesothelioma constitutes 10-20% of cases and often consists of elongated
spindle cells accompanied by collagen deposition, varying numbers of mitotic figures
and neoplastic cells showing variable nuclear pleomorphism (Figure 1.1C).
This
11
subtype of mesothelioma may be indistinguishable from malignant fibrous
histiocytoma, leiomyosarcoma, and areas within chondrosarcoma and osteosarcoma
(Henderson, D, Shilken, K et al. 1992; Corrin, B and Nicholson, A 2006).
Histologically, 20-30% of mesothelioma’s are mixed (biphasic) in type with components that
are unequivocally epithelioid and sarcomatoid in appearance (Figure 1.1B). The proportion of
the two components is variable and depends on the amount of tumour sampled (Corrin, B and
Nicholson, A 2006).
1.1.5.2
Cytological diagnosis of malignant mesothelioma
Approximately 80% of mesothelioma patients present with serous effusions which are
often drained for cytological diagnosis and for symptom palliation.
Cytological
assessment of the effusion usually demonstrates a highly cellular sample with abundant
large cohesive aggregates of epithelioid cells (Figure 1.2A) displaying mesothelial
characteristics, such as abundant dense basophilic cytoplasm, cell engulfment, and
intercellular windows as a result of microvilli borders, small peripheral glycogen
vacuoles and occasional large hard edged hyaluronic acid vacuoles (Figure 1.2B).
Fragments of collagen or basement membrane material can sometimes be seen in the
background or centrally within papillary groups. Often large or giant mesothelial cells
may be present scattered throughout the sample even though the nuclear to cytoplasmic
ratio may appear within normal limits. Occasionally small keratinised pyknotic cells
are noted singly in the background, representing degenerate mesothelial cells.
12
Figure 1.1: Histological sections of mesothelioma stained with H & E. A: Epithelioid
mesothelioma, demonstrating the characteristic tubular appearance of tumour cells. B:
Mixed biphasic mesothelioma, where there are features of epithelioid and sarcomatoid
appearance and C: Sarcomatoid mesothelioma, a typically spindled celled presentation with
areas of collagen deposition.
Epithelioid Features
A
B
C
Spindled cells, sarcomatoid features
Figure 1.2: Cytological preparations of mesothelioma stained with Papanicolaou Stain. A:
Demonstrates a low power field of view containing several large cohesive aggregates of
malignant cells. B: High power magnification highlights large cells with dense basophilic
cytoplasm, large hard edge vacuoles, peripheral nuclear glycogen lakes and intercellular
windows. The nuclei are enlarged with prominent macronucleoli.
Glycogen lakes
A
B
Macronucleoli
Intercellular windows
Macrophages
13
Nuclear abnormality such as nuclear enlargement, irregularity of nuclear contours and
anisocytosis can occur in mesothelioma. Marked nuclear hyperchromasia is usually
absent and macronucleoli are considered a poor prognostic feature (Hasteh, F, Lin, GY
et al. 2010) (Figure 1.2B).
The difficulty in differentiating a mesothelioma from benign reactive mesothelial
proliferation is well recognized (Wolanski, KD, Whitaker, D et al. 1998; Cury, PM,
Butcher, DN et al. 1999; Attanoos, RL, Griffin, A et al. 2003; Westfall, DE, Fan, X et
al. 2009; Hasteh, F, Lin, GY et al. 2010; Su, XY, Li, GD et al. 2010). Diagnosis is
usually a two steps procedure: firstly establishing the presence of a malignant
population and secondly determining the phenotype of the malignant cells.
The
differential diagnosis of epithelioid mesothelioma is usually metastatic adenocarcinoma.
Ancillary tests (histochemistry, immunohistochemistry and occasionally electron
microscopy) are required to establish a definitive diagnosis.
1.1.5.2.1
Histochemistry analysis of malignant mesothelioma
In pleural tumours the presence of periodic acid Schiff (PAS) diastase positive neutral
epithelial, mucin found within glandular lumina is a helpful diagnostic feature used to
differentiate mesothelioma from metastatic adenocarcinoma.
Some mesothelioma
tumours may contain glycogen or hyaluronic acid and histochemical stains for these
may also be used to differentiate mesothelioma from metastatic adenocarcinomas
(Adams, SA, Sherwood, AJ et al. 2002).
14
1.1.5.2.2
Immunohistochemistry of malignant mesothelioma
As yet there is no sensitive or specific markers to identify mesothelioma although some
specific markers are expressed preferentially on mesothelial cells such as calretinin,
mesothelin
and
cytokeratin
(CK)
5/6
whilst
epithelial
markers
such
as
carcinoembryonic antigen (CEA), B72.3, thyroid transcription factor (TTF-1), BerEP4
(also known as EpCAM) and CD15 (also known as LeuM1) are infrequently expressed
on this tumour (Ordonez, NG 2007).
The use of immunohistochemical staining to diagnose mesothelioma has been
extensively investigated (Silverman, JF, Nance, K et al. 1987; Brown, RW, Clark, GM
et al. 1993; Ordonez, NG 2003a; Porcel, JM, Vives, M et al. 2004; Ordonez, NG 2007).
Most studies have focused on the differential diagnosis of mesothelioma and metastatic
adenocarcinoma which is important to establish treatment regimes, clinical trial
eligibility and eligibility for financial compensation for development of disease due to
asbestos exposure. Current recommendations are that a panel of at least two mesothelial
cell markers and two carcinoma markers be used for the diagnosis of epithelial
mesothelioma. With the recommendation that each laboratory should establish a panel
of markers for the diagnosis of mesothelioma (Husain, AN, Colby, TV et al.
2009)(Table 1.1).
The best combination of two immunohistochemical markers is CEA and B72.3, both are
shown to stain positive in adenocarcinomas with 97% sensitivity and specificity and
both negative in mesothelioma with a 99% sensitivity and a 97% specificity (Brown,
RW, Clark, GM et al. 1993). The range of staining reported in literature is conflicting
with results for B72.3 ranging from 35%-100% staining intensity for adenocarcinoma
15
and 0-48% for mesothelioma (Ordonez, NG 2003a). CD15 has been shown to react
with 75% of adenocarcinomas and 32% of mesotheliomas (Ordonez, NG 2003a),
BerEP4 reacts with 99% of adenocarcinomas and 7% of mesotheliomas (Ordonez, NG
2007).
Calretinin has been shown to be a sensitive marker in epithelioid mesotheliomas with
100% sensitivity and specificity however the incidence of calretinin positive
adenocarcinomas ranges from 10-48% showing a small focal diffusing staining reaction
(Leers, MP, Aarts, MM et al. 1998; Ordonez, NG 2003a).
Table 1.1: Summary of markers commonly used in the differential diagnosis of
mesothelioma and adenocarcinoma in cytological and histological specimens.
The
percentage of cases reportedly reactive with each marker is shown.
Specific Marker
Mesothelioma Adenocarcinoma
PAS +/- D
<1%
60-75%a
EMA (E29)
73 – 95%bf
95 – 100%c
100%d
0 – 48%cd
64 – 100%c
22 – 35%c
CEA
<1%e
40 – 90%e
B72.3
0 – 48%c
35 – 100%c
CD15
32%c
75%c
93 – 100%cf
39%cg
<1%h
42 – 97%g
Calretinin
CK5/6
Mesothelin
TTF-1
a (Hammar, SP, Bockus, DE et al. 1996)
b (Wolanski, KD, Whitaker, D et al. 1998; Kushitani, K, Takeshima, Y et al. 2007; Creaney, J, Segal, A et al. 2008)
c (Ordonez, NG 2003a) d (Leers, MP, Aarts, MM et al. 1998)
e (Davidson, B 2011) f (Su, XY, Li, GD et al. 2010) g (Ordonez, NG 2007)
h (Khoor, A, Byrd-Gloster, AL et al. 2011)
16
CK5/6 has emerged as one of the most useful diagnostic markers for a diagnosis of
mesothelioma,
however
CK5/6
shows
positive
staining
in
19%
of
lung
adenocarcinomas and in 22% - 35% of serous adenocarcinomas (Chu, PG and Weiss,
LM 2002; Ordonez, NG 2007).
Squamous cell carcinomas also express CK 5/6
demonstrating strong diffuse cytoplasmic positivity and although metastatic squamous
cell carcinomas are rare to the pleural cavity it is difficult to differentiate this tumour
from mesothelioma (Ordonez, NG 2007).
Mesothelin is a 40kDa glycosylphosphatidylinositol linked glycoprotein expressed in
both normal and malignant mesothelial cells (Chang, K, Pai, LH et al. 1992).
Mesothelin however is not specific for the mesothelial cell linage, as ovarian serous and
pancreatic cancers and up to 39% of lung adenocarcinomas express the molecule
(Ordonez, NG 2003b).
In contrast to the amount of research into antibodies to differentially diagnose
mesothelioma from metastatic adenocarcinoma little research has been done to identify
markers to differentiate malignant from benign reactive mesothelial cells. Epithelial
membrane antigen (EMA), the anti-MUC1 antibody was shown to have strong
membranous positivity in mesothelioma cells while in reactive proliferating mesothelial
cells the EMA staining is usually weak or diffusely cytoplasmic (Wolanski, KD,
Whitaker, D et al. 1998; Saad, RS, Cho, P et al. 2005). The X-linked inhibitor of
apoptosis protein (XIAP) and an isoform of glucose transporter (GLUT1) have also
been shown to be overexpressed in mesothelioma relative to benign reactive mesothelial
cells although with lower sensitivity and diagnostic accuracy of 80% (Shen, J, Pinkus,
GS et al. 2009).
17
Whilst the diagnosis of mesothelioma is possible on cytology effusion samples, the
sensitivity of such diagnosis varies between approximately 30% (DiBonito, L,
Falconieri, G et al. 1993; Renshaw, AA, Dean, BR et al. 1997; Teirstein, AS 1998;
Ordonez, NG 2003a) and 73% (Wolanski, KD, Whitaker, D et al. 1998). One of the
reasons for the differences in the reported sensitivities for a cytological diagnosis of
mesothelioma is the difficulty in differentiating between malignant and benign reactive
mesothelial cells.
The value of the anti-MUC1 antibody in the diagnosis of
mesothelioma has not been fully investigated and may prove to play a vital role in the
cytological diagnosis of mesothelioma and forms the topic of discussion in Chapter 3 of
this thesis.
1.1.5.2.3
Ultrastructural analysis of malignant mesothelioma
When immunohistochemical and histochemical results are inconclusive, ultrastructural
analysis by electron microscopy may facilitate the diagnosis of mesothelioma compared
to other malignancies. In epithelioid mesothelioma abundant cytoplasmic intermediate
filaments with formation of tonofibrils, elongated and complex desmosomes, an
absence of mucin droplets and the presence of long branching plasmalammel microvilli
demonstrating a length to diameter ratio of 10:1 or more are present.
Other
ultrastructural features specific for mesothelioma include cytoplasmic glycogen
deposits, dilated intercellular spaces and intracellular lumina, which are often lined by
microvilli. Basal lamina can be present around mesothelioma cells with the microvilli
often coated with an amorphous granular material (Wick, MR, Loy, T et al. 1990;
Butnor, KJ 2006).
18
In contrast metastatic adenocarcinomas exhibit short truncated microvilli and absence of
tonofibrils. These cells often demonstrate intracytoplasmic mucin granules a feature
absent in mesothelioma (Wick, MR, Loy, T et al. 1990). Intracytoplasmic lumina,
subplasmalemmel intermediate filaments and cytoplasmic surfactant bodies are features
usually absent in metastatic adenocarcinomas (Wick, MR, Loy, T et al. 1990).
1.1.6
Therapeutic options for mesothelioma
Therapeutic options for mesothelioma are determined by tumour stage and patient
health. Staging mesothelioma is extremely difficult compared to other organ specific
malignancies for a variety of reasons. Firstly, the staging system evolved as a ‘surgical’
stage, and is often difficult to apply in patients not undergoing surgical exploration.
Secondly, the nodal staging system for mesothelioma has followed drainage patterns
established for non-small cell lung cancer, whereas it is likely that nodal involvement in
mesothelioma may occur in the mediastinal and subpleural nodes before involving the
hilar nodes. The International Association for the Study of Lung Cancer (IASLC) is
currently reviewing the staging of mesothelioma and establishing a comprehensive
prospective staging and outcomes database from around the world (Rice, D, Rusch, V et
al. 2011). Early diagnosis may be as important as staging in determining therapeutic
options in mesothelioma.
In small subsets of patients undergoing multimodality
treatment regimes which include extra pleural pneumonectomy (EPP), cures have been
observed (van Ruth, S, Baas, P et al. 2003; Flores, RM, Krug, LM et al. 2006),
however, this is controversial and with the caveat that these are a highly selected group
of individuals (Sugarbaker, DJ, Garcia, JP et al. 1996; van Ruth, S, Baas, P et al. 2003;
Flores, RM, Krug, LM et al. 2006; Treasure, T, Lang-Lazdunski, L et al. 2011).
19
Patients with advanced unresectable disease undergo chemotherapy and palliative
interventions. These are the best resources available to relieve pain and dyspnoea,
recurrent pleural effusions, enlarging chest wall masses and physical discomfort often
experienced by these patients (Nowak, AK and Bydder, S 2007).
1.1.6.1
Surgery
The role of surgery as a treatment option for mesothelioma is highly controversial
(Treasure, T, Lang-Lazdunski, L et al. 2011). Three common surgical procedures are
available and include surgical pleurodesis via VATS, pleurectomy/decortication (P/D),
and EPP.
EPP has been successful in the treatment of tuberculosis empyema and other pleural
diseases including mesothelioma. EPP is a radical surgical procedure for mesothelioma
which involves the removal of the parietal, pleural, lung, pericardium and diaphragm.
This technique is successful in some mesothelioma patients resulting in an increase in
patient survival with significant tumour reduction (van Ruth, S, Baas, P et al. 2003).
Unfortunately, not all patients are eligible for this surgical procedure.
Also high
morbidity rates of 60% (Sugarbaker, DJ, Jaklitsch, MT et al. 2004) and mortality rates
of 4-9% have been reported (Sugarbaker, DJ, Garcia, JP et al. 1996; Sugarbaker, DJ,
Jaklitsch, MT et al. 2004). The addition of high dose radiotherapy to EPP has been
suggested to reduce local recurrence and prolong survival in early stage disease,
although this was a non-randomised cohort study only and cannot be supported by the
single arm study data (Rusch, VW, Rosenzweig, K et al. 2001).
20
P/D enables radical resection of the early stage tumour while preserving the lung
parenchyma. P/D involves the removal of the visceral, parietal and pericardial pleural
as well as debulking the tumour and therefore is technically less difficult compared to
EPP and is associated with reduced mortality rates of <5% (Rusch, VW 1997).
However, in recent years a more aggressive approach to P/D has been suggested, and a
large case series of both procedures has suggested that survival results may be
comparable or better than EPP, although these data are not randomised and there is
likely to be an effect of biased patient selection on these results (Flores, RM, Pass, HI et
al. 2008).
1.1.6.2
Chemotherapy
The use of chemotherapeutic agents in mesothelioma aims to improve quality of life,
provide symptomatic relief and improve patient survival. The study which current
standard practice is based was performed by Vogelzang et al 2003, who conducted a
phase III trial comparing pemetrexed and cisplatin with cisplatin alone. In the trial 226
patients were randomised to receive the combined pemetrexed and cisplatin treatment,
and 222 to receive cisplatin. Patients on the combination treatment arm had a 12.1
month median survival time compared to 9.3 months for individuals treated with
cisplatin alone (Vogelzang, NJ, Rusthoven, JJ et al. 2003).
The pemetrexed and
cisplatin regime was well tolerated, improving the patient’s quality of life within the
first three months of therapy, with no apparent effect on efficacy. This regime is now
the benchmark standard of care and treatment for unresectable mesothelioma.
Over 50 phase II studies have been performed in mesothelioma which have tested a
wide range of drugs including anthracyclines, alkylating agents, platinum compounds,
21
taxanes, vinka alkaloids and antifolates, response rates have been in general poor and
the trials have tended not to be reproducible in different patient sets (Vorobiof, DA and
Mafafo, K 2009).
Other antifolates have been investigated but are less commonly used than pemetrexed.
A phase III trial of raltitrexed, a thymidine synthase inhibitor, in conjunction with
cisplatin in a first line setting improved the survival rate of mesothelioma patient’s
compared to cisplatin alone (van Meerbeeck, JP, Gaafar, R et al. 2005). The study
demonstrated that raltitrexed combined with cisplatin improved the overall response rate
compared to cisplatin alone; however there was no reported difference in the overall
health status and quality of life in patient’s (van Meerbeeck, JP, Gaafar, R et al. 2005).
The median overall survival rate for patients receiving raltitrexed and cisplatin was
significantly increased to 11.4 months, with a 1 year survival rate of 46% in the
combination arm compared with 39% with the cisplatin alone (van Meerbeeck, JP,
Gaafar, R et al. 2005).
1.1.6.3
Radiotherapy
The role of radiation therapy in the treatment of unresectable mesothelioma is still
undefined. Literature suggests that since mesothelioma typically involves large areas of
the pleura at diagnosis, curative radiotherapy is difficult to deliver because of the
proximity of lung, heart, liver and spinal cord that would receive toxic doses of
radiation (Senan, S 2003). However, radiotherapy may play an important role in the
palliation of pain at localised sites and when used as a component in a multimodality
treatment regime. The use of radiotherapy to port sites and sites of operative
intervention remains controversial (Boutin, C, Rey, F et al. 1995; Bydder, S, Phillips, M
et al. 2004; O'Rourke, N, Garcia, JC et al. 2007; Kara, P, Ugur, I et al. 2010).
22
1.1.6.4
Multimodality approach to treatment
Flores et al prospectively studied patients on cisplatin and gemcitabine followed by EPP
and subsequent hemithoracic radiation therapy. In this study a median survival of 19
months for all patients was reported. Those patients who completed chemotherapy and
underwent EPP showed a median survival of 33.5 months suggesting that multimodality
treatment with cisplatin and gemcitabine, followed by EPP and radiotherapy for locally
advanced mesothelioma is feasible. The improved overall survival compared to EPP
and radiotherapy alone was encouraging (Flores, RM, Krug, LM et al. 2006).
Recent treatment pathways include the use of neoadjuvant chemotherapy followed by
EPP and radiotherapy for mesothelioma.
In a prospective trial of neoadjuvant
chemotherapy (cisplatin and gemcitabine) followed by EPP, a response rate of 32%
with a median survival of 23 months was reported (Weder, W, Stahel, RA et al. 2007).
1.2
MUC1
MUC1 is a member of the mucin family of proteins and is produced by all epithelial
cells including cells of the breast, gastrointestinal tract, pancreas and ovary, prostate and
to a lesser degree by some haemopoietic cells.
The mucin family is characterised by proteins which contain the mucin domain, also
known as the variable number of tandem repeats (VNTR) domain. Mucins can be
heavily glycosylated with up to 50-90% of the molecular mass of MUC1 being
contributed to by the carbohydrate side chains (Lan, MS, Batra, SK et al. 1990).
Mucins can be subdivided into two subfamilies based upon their location with reference
23
to the cell surface; secreted mucins which are entirely extracellular, and cell
transmembrane mucins such as MUC1 which contain both extracellular and intracellular
regions.
MUC1 has been extensively studied for many years and has been known by several
names, including polymorphic epithelial mucin gene (PEM), epithelial membrane
antigen (EMA), peanut reactive urinary mucin (PUM), CD227, human milk fat globulin
(HMFG) and H23 antigen. One of the main reasons for this variation in nomenclature
is that investigators were studying the target of a wide range of monoclonal antibodies,
which later were shown to all react to MUC1.
1.2.1 Structure of MUC1
1.2.1.1.
Gene structure of MUC1
The MUC1 gene is located on chromosome 1q21and encodes 9 exons spanning 2312bp.
The MUC1 gene promoter comprises of 2872bp located at the 5’terminal sequence and
contains elements that bind a selection of transcriptional regulators such as specificity
protein 1, activation protein 1, nuclear factor 1, E-box (lies upstream from a promoter
region), GC box, oestrogen and progesterone receptor site (Zaretsky, JZ, Sarid, R et al.
1999). Although MUC1 gene expression is observed mostly in epithelial cells, its
transcription at low levels has been detected in other types of cells including normal and
neoplastic plasma cells, follicular dendritic cells, myofibroblasts, perineural cells,
Hodgkins and Non-Hodgkins lymphoma (Zaretsky, JZ, Sarid, R et al. 1999).
24
1.2.1.2
Protein structure of MUC1
The archetypal MUC1 gene product is a large transmembrane protein with molecular
weight ranging from 500kDa to more than 1000kDa that may extend 200 to 500nm
beyond the cell membrane. Variation in the size of the protein is due to differences in
the number of VNTR domains and the degree of glycosylation of the protein. MUC1
contains a large extracellular region, a small transmembrane region and a cytoplasmic
tail (Figure 1.3). The larger subunit of MUC1 is extracellular and contains the Nterminal sequence and the heavily O-glycosylated VNTR domain, which consists of 25125 repeats of the amino acid sequence ‘GSTAPAHGVTSAPDTRPAP’. At the cell
surface MUC1 does not exist as a linear molecule. Following translation, the MUC1
precursor polypeptide is autocatalytically cleaved within the N-terminal extracellular
region at a G↓SVVV motif found in the SEA (sea urchin sperm protein, enterokinase,
and agrin) domain (Rose, MC and Voynow, JA 2006).
The extracellular subunit
remains associated with the transmembrane and cytoplasmic domain throughout
intracellular processing and the fully glycosylated mature mucin protein is packaged
and stored within secretory granules until it is transported to the cell membrane as a
stable heterodimeric complex.
The N-terminal signal peptide of MUC1 directs
localisation of the mature protein to the apical membrane in epithelial cells.
The smaller fragment contains the short transmembrane domain and the intracellular
cytoplasmic tail. The two fragments are held together by noncovalent sodium dodecyl
sulphate labile bonds. The small subunit is referred to as MUC1-Cytoplasmic tail
(MUC1-CT) and contains a small 58 amino acid extracellular region together with a 21
amino acid transmembrane domain and a 72 amino acid cytoplasmic tail. The MUC1CT
peptide
core
is
approximately
14kDa
following
glycosylation
and/or
25
phosphorylation the size of the CT domain may increase to approximately 25-30kDa
(Gendler, SJ 2001).
Figure 1.3: Schematic representation of MUC1 adapted from Hartrup and Gendler,
2008 (SEA- sperm protein enterokinase and agrin).
Extracellular Domain
Transmembrane Domain
Cyto
Cytoplasmic tail
1.2.1.3
Alternatively spliced variants of MUC1
One of the major hallmarks of cell membrane mucins is the presence of alternative
splice forms.
Several molecular studies have identified variable MUC1 isoforms
including MUC1 A, B, C, D, X, Y, Z, REP and SEC (Table 1.2). MUC1 isoforms
demonstrate diversity of cellular function and properties, which may explain the crucial
role MUC1 plays in many physiological and pathological conditions.
MUC1/REP is the full length archetypal MUC1 protein, consisting of an extracellular
domain containing the VNTR region and a cytoplasmic transmembrane region
(Obermair, A, Schmid, BC et al. 2002). In this thesis MUC1 refers to this isoform
unless otherwise specified.
26
The isoforms MUC1/X, MUC1/Y and MUC1/Z all lack the VNTR domain and do not
exhibit mucin-like characteristics (Baruch, A, Hartmann, M et al. 1999) they differ from
each other in the length of the translated product of the second exon. MUC1/Y is the
most extensively studied of these three isoforms and signal transduction initiation by the
cytoplasmic domain of the MUC1/Y protein has been shown to enhance tumour
progression in vivo and may therefore play a significant role in oncogenesis (Baruch, A,
Hartmann, M et al. 1999).
MUC1/Y may bind to MUC1/SEC, which induces
phosphorylation of the MUC1/Y protein, profoundly affecting cell morphology
(Baruch, A, Hartmann, M et al. 1999).
MUC1/A, MUC1/B, MUC1/C and MUC1/D differ from MUC1/REP in the size of the
translated product of the second exon. MUC1/A has been suggested to be a cancer
specific splice variant found in thyroid carcinoma (Weiss, M, Baruch, A et al. 1996).
Splice variants MUC1/C and MUC1/D were initially described in malignant ovarian
tumours (Obermair, A, Schmid, BC et al. 2002). Expression of these splice forms has
recently been reported in other human tissues (Ng, W, Loh, AX et al. 2008;
Strawbridge, RJ, Nister, M et al. 2008).
MUC1/SEC lacks the hydrophobic transmembrane and cytoplasmic domains required
for anchorage to the cells membrane and encodes a protein, which is secreted from the
cell and can be detected in the serum of cancer patients. As previously described
MUC1/SEC has been suggested to interact with MUC1/Y cytoplasmic domain. The
secreted form MUC1/SEC may act as a cognate ligand for the MUC1/Y isoform, which
has been shown to play a role in oncogenesis (Obermair, A, Schmid, BC et al. 2001).
27
Table 1.2: Summary of the major differences between the MUC1 variants.
MUC1
ISOFORM
REP1
CHARACTERISTICS
Classical isoform
DOMAINS PRESENT
Extracellulara
VNTRb
SEAc
TMd
CTe
+
+
+
+
-
-
+
+
+
+
+
+
+
+
+
+
4
no data
4
oncogenic;
binds MUC1/SEC
oncogenic
+
Cancer specific
(thyroid)
Expressed in normal
tissue
Cancer specific
(ovarian)
Cancer specific
(ovarian)
Secreted form
+#
+
+
+
+
+#
+
+
+
+
+#
+
+
+
+
+#
+
+
+
+
+
+
+
-
-
X
Y
Z4
2
A
B2,3
C2,1
D2,1
SEC4
+
1. Obermair, Schmid et al 2001
2. Weiss, Baruch et al.1996
3. Strawbridge, Nister et al. 2008
4. Baruch, Hartman et al. 1999
a – Extracellular domain
b – Variable number tandem repeat domain
c – Sea urchin sperm protein, enterokinase and agrin domain
d – transmembrane domain
e – cytoplasmic tail domain
# - The extracellular domain of isoforms A, B, C and D are truncated relative to MUC1/REP
1.2.1.4
Soluble MUC1
Although MUC1 is a transmembrane glycoprotein a soluble form is also present in
tissue culture supernatants and body fluids. There are at least two possible mechanisms
to account for the presence of soluble MUC1. Firstly, the extracellular domain of the
MUC1 heterodimeric complex can be cleaved into the surrounding lumen by the action
of enzymes such as tumour necrosis factor α converting enzyme (TACE/ADAM17), a
disintegrin and metalloprotease family member. MUC1 cleavage by either TACE or
28
ADAM has been inhibited following alteration or mutation of the G↓SVVV cleavage
site within the SEA domain (Thathiah, A and Carson, DD 2004).
Secondly, the alternative splice product MUC1/SEC which lacks the transmembrane
domain and cytosolic components of MUC1 archetypal protein may be present in
circulation (Hattrup, CL and Gendler, SJ 2008). Levels of soluble MUC1 in serum and
effusions can be detected by several methods including the CA15-3 assay, an ELISA
incorporating two antibodies directed against a different epitopes within the VNTR
domain.
Circulating tumour marker antigens can be used clinically and the
measurement of circulating CA15-3 levels is widely used as an aid in monitoring
tumour progression and response to therapy in patients with metastatic breast cancer
(discussed below).
1.2.2
Function of MUC1
MUC1, like all mucins plays a functional role in hydration and lubrication of cell
surfaces, and protects cells from proteases and pathogenic organisms (Gendler, SJ
2001). MUC1 has 88% homology in the cytoplasmic tail and transmembrane regions
between species which suggests an important functional role for the molecule (Spicer,
AP, Duhig, T et al. 1995).
1.2.2.1
MUC1 in protection of cells and as an adhesion molecule
MUC1 has been suggested to display anti adhesive properties due to its large
extracellular structure which may extend 100nm from the cell surface. In addition the
MUC1 protein core is covered in dense sugar chains. These physical features of MUC1
protect the underlying epithelium from proteases and pathogens and may inhibit cell to
29
cell interactions (Hattrup, CL and Gendler, SJ 2008). In normal colon epithelium,
MUC1 is found to be more heavily glycosylated than in other tissue locations which
may be necessary for the resistance to proteases in the gut (Cao, Y, Blohm, D et al.
1997).
In addition to the physical barrier, MUC1 may play a role in cell adhesion through its
interaction with β-catenin. Phosphorylation of MUC1 CT, at the ‘TDRSPYEKV’ motif,
by the tyrosine kinases, cellular proto oncogene tyrosine kinase (c-Src) and lymphocyte
specific tyrosine kinase (Lck) increases MUC1 affinity for β-catenin. This reduces the
amount of β-catenin available for binding to E-cadherin, and thus reducing E-cadherin
mediated cell adhesion (Li, Y, Kuwahara, H et al. 2001). This is a highly regulated
process as phosphorylation of the serine residue in this motif by glycogen synthase 3-β
inhibits the binding of MUC1 to β-catenin (Ren, J, Li, Y et al. 2002).
1.2.2.2
MUC1 in reproduction
During the normal, nonpregnant state, MUC1 plays a significant role in protecting the
endometrium from pathogen attack. During the reproductive process MUC1 plays a
complex, and not fully understood role, with MUC1 expression in mammary tissue and
in the female reproductive tract, being tightly regulated during pregnancy and lactation
by steroid hormones (Brayman, M, Thathiah, A et al. 2004). In humans endometrial
MUC1 expression is reduced in response to high progesterone levels which facilitates
embryo implantation (Brayman, M, Thathiah, A et al. 2004). Regulation of human
MUC1 by oestrogen may be indirect and there is evidence that MUC1 CT binds to the
oestrogen receptor α (ERα) and protects the receptor from proteosomal degradation.
Different splice forms of MUC1 are expressed in response to oestrogen (Zaretsky, JZ,
30
Barnea, I et al. 2006).
Defects in MUC1 expression in endometrium may affect
implantation resulting in recurrent abortions; there may a synergistic relationship
involving MUC1 between maternal and embryonic cells (Meseguer, M, Aplin, JD et al.
2001).
MUC1 downregulation was demonstrated in vitro at the site of blastocyst attachment in
humans. Acute loss of MUC1 may be mediated by locally activated sheddases such as
TNFα
converting
enzyme
TACE/ADAM17
a
membrane
type
1
matrix
metalloproteinase (Dharmaraj, N, Wang, P et al. 2010). MUC1 expression can also be
downregulated by a protein inhibitor of activated signal transducer and activator of
transcription (STAT) family members. Endometrial MUC1 expression is regulated by
ovarian steroid hormones oestrogen and progesterone.
In humans progesterone
stimulates MUC1 expression and the regulation of the female reproduction system is
mediated by oestrogen receptor (ER) and progesterone receptor (PR). Although the ER
does not directly regulate the human MUC1 gene, some isoforms of MUC1 mRNA may
be regulated by ERα in breast cancer cells (Zaretsky, JZ, Barnea, I et al. 2006). The PR
isoforms differentially regulate MUC1 gene expression in uterine epithelial cells
(Brayman, MJ, Julian, J et al. 2006).
1.2.2.3
MUC1 mediated immunoregulatory mechanisms
Reports have suggested that MUC1 may play a pivotal role in normal immune
regulation (Agrawal, B, Krantz, MJ et al. 1998). MUC1 is synthesised and expressed
on the cell surface of a majority of T cells following activation (Agrawal, B, Krantz, MJ
et al. 1998). The MUC1 expressed on activated T cells is a hypoglycosylated form
similar to that seen in the cancer state (Agrawal, B and Longenecker, BM 2005), which
31
suggested that MUC1 may function to down regulate ongoing T cell response under
appropriate conditions.
MUC1 expression on stimulated T cells is upregulated by IL-12, whereas IFN-γ, IL-2,
IL-4, IL-5, IL-10, and TNF-α have no effect on MUC1 expression (Agrawal, B and
Longenecker, BM 2005).
1.2.2.4
The role of MUC1 in signal transduction
The extracellular domain and cytoplasmic tail of MUC1 play a significant role in cell
signalling. The structure of MUC1 enables cells to respond to external stimuli via
internal signal transduction pathways. The CT contains seven tyrosine residues, along
with eight serine and five threonine residues all of which may act as potential
phosphorylation sites for a variety of kinases including epithelial growth factor receptor
(EGFR), erythroblastic leukemia viral oncogene (ErbB), glycogen synthase 3β and
protein kinase Cδ (Li, X, Wang, L et al. 2005). The EGFR and ErbB family of
transmembrane receptor tyrosine kinases are involved in the development and
progression of a range of human malignancies. Hetero and homodimerization of these
receptors may activate a selection of different signalling cascades, including those
mediated by mitogen activated protein kinases (MAP), extracellular signal regulated
kinases (ERK1/2 pathways), p38, and Jun N terminal kinases (JNKs) (Li, X, Wang, L et
al. 2005).
The c-Src tyrosine kinase, phosphorylates MUC1 CT at the ‘YEKV’ motif, and is
involved in GSK3β and β-catenin binding. Studies in vivo and in vitro suggest that c-
32
Src mediated phosphorylation of MUC1 increases MUC1 affinity to β-catenin, and thus
influence cell adhesion as described above (Li, Y, Kuwahara, H et al. 2001).
The interaction of the MUC1 extracellular domain with viruses, bacteria, growth factors
and markers on other cells such as selectins and intercellular cell adhesion molecule
(ICAM) may trigger intracellular signalling events mediated by the CT of the MUC1
heterodimer, however the large range of intercellular signalling events have been more
fully explored in the tumour context (Carson, DD 2008).
1.2.3
MUC1 antibodies
There are over 50 monoclonal antibodies directed to MUC1. Towards the end of 1997 a
review of 56 anti-MUC1 monoclonal antibodies was carried out by the International
Society for Oncodevelopmental Biology and Medicine (ISOBM). These monoclonal
antibodies were divided into 4 major groups based on the staining reaction in paraffin
sections, from normal human small intestine, colon and breast, with and without
periodate oxidation (PO) treatment a method which removes the sugar molecules and
unmasks the epitope in the native protein (Cao, Y, Karsten, U et al. 1998).
In the first group, 14 antibodies were found to detect MUC1 independent of
glycosylation and were classified as pan-MUC1 monoclonal antibodies.
These
antibodies detected MUC1 in normal epithelium and in malignant tissue and were
capable of immunoaffinity purification and quantification of MUC1. Some of the
antibodies in this category include; BC3, BC2, B27.29, E29 and Mc5 (Cao, Y, Karsten,
U et al. 1998). The main patterns of reactivity were dependent upon the antibody
33
staining behaviour before and after PO treatment. The first group of antibodies stained
independent of PO treatment.
The second group of 24 monoclonal antibodies were not reactive with one or more
types of the tissues examined. However following treatment with PO, these tissues
became reactive to some of these antibodies. These antibodies may differentiate MUC1
glycosylation during physiological or pathological processes, especially during
carcinogenesis. Antibodies in this category include; DF3, HMFG-1, VU4H5 and SM3
(Cao, Y, Karsten, U et al. 1998).
In the third group, six monoclonal antibodies detected periodate sensitive carbohydrate
epitopes.
The antibodies in this group may be applied to the analysis of MUC1
glycosylation in immunohistochemistry as a double staining method in conjunction with
MUC1 peptide specific antibodies (Cao, Y, Karsten, U et al. 1998).
The final group consisted of five antibodies showing reactivity different from the
patterns of the other three groups, but it was unclear what their target epitopes were
(Cao, Y, Karsten, U et al. 1998). There were six monoclonal antibodies that were found
to be nonreactive with any of the tissues examined highlighting that the specificity of
MUC1 is dependent upon tissue type and glycosylation status.
The study concluded that MUC1 is glycosylated differently in different tissues and that
these glycosylation patterns influence its immunohistochemical detection with
monoclonal antibodies.
PO treatment is an appropriate step to include in MUC1
34
immunohistochemistry to detect epitopes masked by glycosylation and to improve and
enhance the staining reaction (Cao, Y, Karsten, U et al. 1998).
1.3
MUC1 EXPRESSION IN MALIGNANCY
Various aspects of MUC1 expression and biology have been shown to be altered in a
wide range of glandular malignancies including breast, lung, gastrointestinal (including
colon), pancreas, prostate, ovarian and uterine (Obermair, A, Schmid, BC et al. 2002;
Tsutsumida, H, Swanson, BJ et al. 2006; van der Vegt, B, de Roos, MA et al. 2007).
Recently, a small preliminary study identified that MUC1 expression was also altered in
mesothelioma (Creaney, J, Segal, A et al. 2008). Altered MUC1 expression has been
observed in haematological malignancies including multiple myeloma, B cell nonHodgkins lymphoma and leukaemia (Brugger, W, Buhring, HJ et al. 1999). At a
cellular level MUC1 expression is generally increased in malignancy, the protein may
be expressed in altered cell compartments, locations and glycosylation may be reduced.
Changes in MUC1 have been associated with tumorigenesis and pathogenesis of cancer,
particularly in tumour cell proliferation, apoptosis and invasion and in some instances
this has a direct correlation with clinical outcomes for patients and this will be discussed
below.
1.3.1
MUC1 protein expression in malignancy
Increased MUC1 expression appears to correlate with more advanced stages of
malignancy. This has been quantified in a study of prostate cancer, whereby MUC1
was not overexpressed in samples of normal, benign prostate, or in prostate
intraepithelial neoplasia, but was overexpressed in 10% of samples of low Gleason
score (G) <7, 47% of moderate (G=7) and 78% of high score tumour (G>7).
35
Furthermore, 90% of cases with lymph node metastases overexpressed MUC1 (Cozzi,
PJ, Wang, J et al. 2005). In pancreatic cancer, some gastrointestinal, gall bladder, breast
and other cancers, MUC1 expression has been linked to the more invasive phenotype of
the disease (Yonezawa, S, Nakamura, A et al. 2002; Kawamoto, T, Shoda, J et al. 2004;
van der Vegt, B, de Roos, MA et al. 2007). Not surprisingly overexpression of MUC1
has been correlated with poor patient outcomes in several types of malignancies. Lung
adenocarcinoma patients with upregulated protein expression of MUC1, determined by
immunohistochemistry had a poorer survival than those with low expression.
Multivariate analysis in this study demonstrated that MUC1 expression was an
independent prognostic factor for early stage non small cell lung cancer (NSCLC) (Situ,
D, Wang, J et al. 2010).
However, in some cases the depolarisation of MUC1 expression that occurs in glandular
epithelial tumours may complicate quantification.
For example, in normal breast
epithelium MUC1 is expressed in 12% of the apical surfaces (Lacunza, E, Baudis, M et
al. 2010) compared to 32% of 73 cases of ductal carcinoma in situ (van der Vegt, B, de
Roos, MA et al. 2007). Breast tumours showing diffuse cytoplasmic MUC1 expression
were of higher grade and patients had a poorer prognosis than those with tumours
showing localised apical MUC1 expression (van der Vegt, B, de Roos, MA et al. 2007).
One confounding observation has been that very undifferentiated high grade tumours
may have no MUC1 expression and be associated with poor prognosis (Luna-More, S,
Rius, F et al. 2001; Rahn, JJ, Dabbagh, L et al. 2001; van der Vegt, B, de Roos, MA et
al. 2007).
36
Some MUC1 immunohistochemical studies have been difficult to compare because of
the choice of antibodies in a given study.
As discussed in Section 1.2.3, many
antibodies to MUC1 have been generated. A study of different anti-MUC1 antibodies
in prostate cancer found when using an antibody that recognised hyperglycosylated
MUC1 (Clone NCL-MUC1) 14% of benign prostate and 13.5% of prostate cancer were
positive, but using an antibody that only reacted with the hypoglycosylated form (Clone
SM3), 37.5% of normal and 70% of prostate cancers were positive (Burke, PA, Gregg,
JP et al. 2006). However, the targets of many of the antibodies are not as easily
delineated with the Clone E29 being described as binding both hyper and
hypoglycosylated forms and recognising 33% of normal and 51% of prostate cancer
(Burke, PA, Gregg, JP et al. 2006). In general the observation has been that in more
advanced malignancy, MUC1 is hypoglycosylated.
The mechanism by which an
alteration in the level of glycosylation occurs is still not completely known, however,
one suggestion is that defects may occur in the glycosltransferase activities in colorectal
carcinoma which may explain the loss of MUC1 glycosylation in malignancy (Cao, Y,
Blohm, D et al. 1997).
Tumour derived unglycosylated VNTR epitopes act as a potent chemo-attractant for
immature human myeloid dendritic cells (DCs) (Carlos, CA, Dong, HF et al. 2005). It
has been observed that MUC1 may inhibit monocyte maturation into immature DCs
leading to the generation of dysfunctional DC’s which are not capable of priming the T
cell population to attack the tumour. This incorrect processing of the secreted MUC1
fragments by DCs prevents cleavage and inhibits presentation, therefore activation of
the T cells fails to occur. Additionally, immature DCs are drawn to MUC1 positive
tumours through the production of cytokines such as TNF-α and IL-6, enhancing local
37
immunosuppression and increasing tumour invasion (Carlos, CA, Dong, HF et al.
2005).
1.3.1.1.
CA15-3 levels in malignancy
Not only is cell localised MUC1 protein overexpressed in some forms of epithelial
malignancy, increased levels of the soluble form of MUC1 can be detected in patients’
blood and effusions. Most studies have utilised the CA15-3 assay developed by Hayes
et al, (Hayes, DF, Zurawski, VR, Jr. et al. 1986). The normal range of CA15-3 in
apparently healthy individuals is below 36kU/L and this value is used as a threshold for
determining assay positivity. Approximately 70% of patients’ with metastatic breast
cancer are CA15-3 positive whereas CA15-3 was elevated in no patients with stage 1
disease, 8% of stage 2 and 37% of stage 3 cases (Velaiutham, S, Taib, NA et al. 2008).
CA15-3 levels in breast cancer correlate with tumour size and lymph node metastatic
status (Park, BW, Oh, JW et al. 2008). Preoperative CA15-3 levels in serum were
shown to significantly predict patient survival independent of patient age, tumour grade
and hormone receptor status (Park, BW, Oh, JW et al. 2008).
However, current
international guidelines do not recommend its use for the routine diagnosis of breast
cancers because of its moderate sensitivity and absence of clinical impact (Harris, L,
Fritsche, H et al. 2007). Current guidelines do however recommend the clinical use of
CA15-3 for monitoring disease progression in women with metastatic disease because
of its correlation with tumour burden (Bensouda, Y, Andre, F et al. 2009).
In addition, CA15-3 has been shown to aid in the differential diagnosis of benign and
malignant pleural effusions (Ghayumi, SM, Mehrabi, S et al. 2005). CA15-3 levels are
elevated in 80% of malignant effusions at 93% specificity (Miedouge, M, Rouzaud, P et
38
al. 1999; Alatas, F, Alatas, O et al. 2001). However, only a small number of studies
have looked at the diagnostic value of the CA15-3 antigen as a diagnostic tool to
differentiate mesothelioma from carcinoma and from benign effusions (Shimokata, K,
Totani, Y et al. 1988; Miedouge, M, Rouzaud, P et al. 1999; Alatas, F, Alatas, O et al.
2001; Ghayumi, SM, Mehrabi, S et al. 2005). These studies found that by combining
the CA15-3 assay with other tumour markers increased the sensitivity and specificity
for a diagnosis of malignancy.
1.3.2.
MUC1 mRNA expression in malignancy
MUC1 mRNA is overexpressed in thyroid carcinoma (Morari, EC, Silva, JR et al. 2010)
endometrial and cervical tumours (Hebbar, V, Damera, G et al. 2005). Whether there is
a common molecular pathway for MUC1 overexpression is not known. Overexpression
of MUC1 in human breast carcinoma may be a result of the abnormal chromosomal
rearrangement or gene duplication (Zaretsky, JZ, Sarid, R et al. 1999; Khodarev, NN,
Pitroda, SP et al. 2009). Whilst the presence of a large number of transcriptional cisregulatory elements including TATA, CCAATT, E and GC boxes, binding sites for
activator protein 1,2,3 and 4, CAAT box transcription factor /nuclear factor 1
(CTF/NFI), specificity protein 1, STAT 1, 3, 5, and transcriptional repressor protein
YY1, together with binding sites for the oestrogen and progesterone receptors suggest
the potential for multiple mechanisms of regulating MUC1 expression (Zaretsky, JZ,
Sarid, R et al. 1999), there are elements that regulate transcription in mammary
epithelial cells, elements that are specific for transcription in haematopoetic cells,
elements that drive transcription in immunospecific cells, elements that are specific for
transcription in hepatocytes, elements that control transcription in muscle and elements
specific for viral promotors (Zaretsky, JZ, Sarid, R et al. 1999).
39
The expression of MUC1 splice variants A, D, X, Y, Z and REP is strongly associated
with malignancy (Smorodinsky, N, Weiss, M et al. 1996; Weiss, M, Baruch, A et al.
1996; Baruch, A, Hartmann, M et al. 1997; Oosterkamp, HM, Scheiner, L et al. 1997;
Baruch, A, Hartmann, M et al. 1999).
The splicing variations seen in the 5’ exon 2 region of MUC1 are also reported to be
cancer associated.
The variant A transcript was reported to be more abundant in
thyroid, cervical and breast cancer tissues (Weiss, M, Baruch, A et al. 1996; Obermair,
A, Schmid, BC et al. 2001; Obermair, A, Schmid, BC et al. 2002; Schmid, BC,
Buluwela, L et al. 2002) with splice variants A, B, C and D shown to be determined by
a single nucleotide polymorphism in exon 2 of the MUC1 gene in these tumours.
Variants B and C have been associated with increased in vitro invasiveness of breast
tumours (Schmid, BC, Buluwela, L et al. 2002). While variants C and D have been
described in malignant ovarian tumours (Obermair, A, Schmid, BC et al. 2001).
Overexpression of MUC1/Y in mammary mouse epithelial cells increases their
metastatic potential in vitro and is related to the uncontrolled growth and proliferation
of breast cancer cells (Baruch, A, Hartmann, M et al. 1997; Schmid, BC, Buluwela, L et
al. 2002). Therefore the MUC1 gene has the potential to be transcribed in a wide
selection of cell types.
1.3.3.
MUC1 cytoplasmic tail in malignancy
Intracellular translocation from the cytoplasm to the nucleus of the MUC1 CT is
observed in various cancers (Behrens, ME, Grandgenett, PM et al. 2010). Several
oncogenic effects of MUC1 occur through the interaction of the CT with a selection of
40
signalling molecules including Src, GSK3β and epidermal growth factor suggesting that
MUC1 is linked to the regulation of cell proliferation, apoptosis, invasion and
transcription (Li, Y, Ren, J et al. 2001; Schroeder, JA, Adriance, MC et al. 2003;
Pochampalli, MR, Bitler, BG et al. 2007).
The CT has been found in the nucleus of various cells in association with potent
transcription factors including β-catenin (Huang, L, Ren, J et al. 2003; Wen, Y, Caffrey,
TC et al. 2003), p53 (Wei, X, Xu, H et al. 2005), oestrogen receptor α (Wei, X, Xu, H et
al. 2006) and affects nuclear localisation, and activation of NF-κB and Wnts
(Thompson, EJ, Shanmugam, K et al. 2006; Ahmad, R, Raina, D et al. 2007). The Wnts
are secreted glycoproteins that bind the frizzled receptor resulting in an extracellular
signalling cascade that inactives β-catenin degradation and results in nuclear
transformation possibly leading to oncogenic transformations in MUC1 expression (Li,
Y, Ren, J et al. 2001).
1.3.4.
MUC1 as an adhesion molecule in malignancy
Alterations in the location, the total amount and the glycosylation of MUC1 may affect
the adhesive properties of cancer cells. Firstly the increase in MUC1 expression on the
whole cell membrane, rather than on the apical border may result in destabilisation of
the cell to cell adhesion encouraging tumour cells to migrate and metastasise
(Kawamoto, T, Shoda, J et al. 2004).
Specifically it has been demonstrated in
pancreatic and colon cancer that the expression of sialyl Lewis
x
and sialyl Lewis
a
carbohydrate epitopes may act as ligands for selectin-like molecules on endothelial
cells, which may facilitate metastatic dissemination (Baldus, SE, Monig, SP et al.
2002). Soluble MUC1 can inhibit adhesive interactions with ICAM-1 suggesting that
41
MUC1 may play a role in haematogenous spread by epithelial bound MUC1 adhering to
ICAM-1 on endothelial venules or bone marrow stromal cells, further suggesting that
MUC1 may play a pivotal role in promoting tumour cell survival and metastases
(Regimbald, LH, Pilarski, LM et al. 1996).
1.3.5.
MUC1 resistance to apoptosis
Recently, overexpression of MUC1 has been associated with the apoptotic response to
genotoxic stress by localising within the mitochondrial outer membrane whereby
disrupting the intrinsic mitochrondrial apoptopic pathway in dysplastic and malignant
gastric carcinoma (Benjamin, JB, Jayanthi, V et al. 2010). In cell line models MUC1
expression correlates with increased sensitivity to genotoxic anticancer agents,
including UV irradiation, cisplatin and doxetaxal (Ren, J, Agata, N et al. 2004). One of
the possible mechanisms for this increased sensitivity is a reduction in B cell lymphoma
extra large transmembrane molecule (Bcl-XL), cyclin D, Myc transcription factors, and
vascular endothelial growth factor (VEGF), all critical downstream genes important for
the oncogenic effect of signal transducer and activator of transcription 3.
The MUC1 knockdown cell line MDA-MB-468 showed an increased expression of
MMP2 a metalloprotease which degrades type IV collagen. In breast cancers, MMP2
increases with tumour progression suggesting that it may also facilitate angiogenesis
and metastasis. The increased MMP2 observed in the reduced MUC1 cell line MDAMB-468 may have resulted from two mechanisms; firstly that MMP2 expression may
be confined to the stroma, suggesting that the increased transcription of MMP2 after the
loss of epithelial MUC1 may reflect a mesenchymal phenotype.
Secondly, the
increased expression of MMP2 result from the overexpression of erbB2, implying that
42
MMP2 may be part of a transcriptional profile linked to low levels of MUC1 resulting
in decreased cellular invasion (Hattrup, CL and Gendler, SJ 2006).
There may be multiple mechanisms by which MUC1 may function as an oncogene as
different studies have shown links of MUC1 over or aberrant expression to processes of
apoptosis, cell proliferation and transcription in cancer however these factors may
depend on cell type and signalling pathways.
Although previous results have
strengthened the links between MUC1 and transcriptional regulation, the role of MUC1
in cancer may be more complex than a direct correlation between MUC1 expression
levels and oncogenic function.
1.3.6.
Why is MUC1 disregulated in cancer?
In vitro studies have suggested a mechanism through which MUC1 could be involved in
various aspects of tumourigenic processes, including cell proliferation, apoptosis and
invasion. In breast cancer cell lines in which MUC1 expression was reduced via short
interfering RNA (siRNA) technology a reduction in transcription of several genes
including VEGF, platelet derived growth factor (PDGF-A), PDGF-B and mitogen
activated protein kinase 1 (MAP2K1) has been demonstrated. As these molecules play
important roles in angiogenesis and tumour growth and metastasis, such studies have
demonstrated a role for MUC1 in the cancerous state (Hattrup, CL and Gendler, SJ
2008). Various studies have shown links of MUC1 over or aberrant expression to
processes of apoptosis, cell proliferation and transcription in cancer (dependant on cell
type). However the role of MUC1 in cancer may be more complex than a direct
correlation between MUC1 expression levels and tumour functionality. MUC1 has also
been shown to play a role in the initiating events of tumourigenesis. Firstly, MUC1
43
transformation of normal rat fibroblasts caused tumourigenesis and induced anchorage
independent growth (Li, Y, Liu, D et al. 2003). Further studies demonstrated that the
CT domain was sufficient for this transformation (Huang, L, Ren, J et al. 2003), In
another model, cigarette smoke, a known aetiological agent for lung cancer induced a
loss of expression of MUC1 on the apical membrane of pre-neoplastic human bronchial
epithelial cells and the intracellular translocation of the MUC1 CT to the nucleus, and
effected EGFR expression as early steps in the tumourigenic process, (Chen, YT,
Gallup, M et al. 2010).
Finally the signature of genes expressed in MUC1 over-expressing cells are
preferentially involved in proliferation and cell cycle regulation (MacDermed, DM,
Khodarev, NN et al. 2010). Patients with lung adenocarcinoma that expressed this gene
signature had poorer survival compared to those who did not express the signature
(MacDermed, DM, Khodarev, NN et al. 2010).
1.4
THESIS OVERVIEW
The aim of this thesis is to examine the characteristics of MUC1 in mesothelioma with
two main purposes: firstly to define the performance of MUC1 in the cytological
diagnosis of this disease, and secondly to further understand the role of MUC1 in the
biology of mesothelioma. The cytological diagnosis of mesothelioma is extremely
controversial and although it has been performed successfully for the past 30 years at
PathWest, this success has not been replicated at a national or international level. At a
recent International mesothelioma interest group (IMIG) conference in 2008, a French
pathologist performed a 10 year review and reported that the sensitivity and specificity
of diagnosing mesothelioma in cytology was only 39%. The lack of acceptance of
44
cytological diagnosis of mesothelioma may in part be due to the reported low
sensitivity, often quoted at around 30% which is deemed too low to be diagnostically
useful (Renshaw, AA, Dean, BR et al. 1997; Churg, A, Colby, TV et al. 2000; Husain,
AN, Colby, TV et al. 2009). While a recent retrospective audit performed between
1988 and 2007 demonstrated that the sensitivity of diagnosing mesothelioma in
cytology was 78% with a specificity of 99% (Personal communication, Professor J
Creaney). This thesis will aim to address the sensitivity and specificity of diagnosing
mesothelioma in cytology samples based upon a prospective cohort of patients. If an
earlier diagnosis of mesothelioma can be established on cytological samples then this
may decrease the time required to make a definitive diagnosis, reduce the need for
additional invasive investigations, and allow the instigation of earlier treatment regimes
which may prolong the survival rate of patients.
Several studies have implicated MUC1 in the pathogenesis of breast, prostate, pancreas,
gastrointestinal and ovarian tumours however the role of MUC1 in mesothelioma is still
unknown.
Understanding the molecular differences associated with the malignant
phenotype may help us understand the molecular pathway to tumour progression. The
second purpose of this study is to evaluate the role MUC1 plays in mesothelioma and to
investigate whether MUC1 contributes to the malignant phenotype. If this is the case,
there is potential to explore future MUC1 directed therapies in mesothelioma.
This thesis tested the following hypothesis:
1. MUC1/EMA can be used as a diagnostic marker for mesothelioma in cytological
samples.
45
2. CA15-3 concentrations in effusions will differentiate between mesothelioma and
effusions of benign causes. Increased supernatant CA15-3 is useful in the diagnosis of
mesothelioma in cytological samples.
3. CA15-3 concentrations in effusions / or expression on tumour will correlate with
patient prognosis.
4. MUC1 expression correlates with cell proliferation, invasion, migration and
tumorigenicity of mesothelioma in vitro.
46
CHAPTER 2
MATERIALS AND METHODS
47
2.1
TISSUE CULTURE
2.1.1
Maintenance of cell lines
Adherent, MCF7 breast cancer, HeLA cervical cancer, U87MG glioma and A549 lung
cancer cell lines originally purchased from American Type Culture Collection (ATCC)
were maintained in RPMI-1640 (Invitrogen, Auckland, NZ ) supplemented with
between 5 to 15% foetal calf serum (FCS, Invitrogen), 105U/L benzylpenicillin (CSL,
Parkville, Victoria, Australia), 50μg/L gentamycin (Pfizer, Bentley, WA, Australia) and
20mM HEPES (Sigma, St Louis, MO, USA). Cells were maintained under sterile
conditions in 75cm2 tissue culture grade flasks (Falcon, Becton Dickinson, North Ryde,
Australia) at 37ºC, in water saturated atmosphere of 5% carbon dioxide (CO2). All cell
lines were tested for the presence of mycoplasma species at three monthly intervals and
found to be free of contamination. These conditions are subsequently referred to as
‘standard conditions’.
Human mesothelioma cell lines were established from patient’s pleural fluid in the
National Centre for Asbestos Related Disease (NCARD) laboratory as per Manning et
al (Manning, LS, Whitaker, D et al. 1991). Cell lines were confirmed to be mesothelial
in origin by ultrastructural analysis using electron microscopy. Cell lines were cultured
under standard conditions.
Short term cultures of normal mesothelial cells were generated from pericardial fluid
collected from patients undergoing coronary artery bypass graft surgery with no
documented history of previous malignancy.
Pericardial fluid (5–15mL) was
centrifuged at 400 x g for 10 min, supernatant removed and cells resuspended in 5mL of
48
15% FCS/RPMI. The cells were cultured under standard conditions and cells from
passage two to five were used for experimental work. The mesothelial nature of
cultured cells was confirmed by immunohistochemistry using an antibody directed
against calretinin (Zymed, California, USA). This protocol generated cultures in which
>90% of cells stained positive with calretinin.
Patients were recruited from those attending the Respiratory and Thoracic clinics at Sir
Charles Gairdner Hospital (SCGH), Nedlands WA. Patients were excluded from the
study if they were under 18 years of age. All patient samples were obtained following
written informed consent. This study was approved by SCGH Human Research Ethics
Committee (HREC).
2.1.2
Splitting cells
Cells were maintained on a weekly basis by removing media and washing cells with
sterile phosphate buffered saline (PBS, Sigma) to remove serum. Cells were removed
from the flask with the addition of 0.25% trypsin (Sigma) and incubation at 37ºC for 5
min. Dislodged cells were resuspended in appropriate prewarmed culture medium and
transferred into 15mL centrifuge tubes. The resuspended cells were centrifuged at 400
x g for 5 min, supernatant was removed and pelleted cells were resuspended in fresh
prewarmed culture media.
Cells were diluted for continued culture or used for
experimental purposes.
2.1.3
Viable cell counting
Cell viability was determined by trypan blue exclusion. A 10μL aliquot of a cell
suspension was diluted 1 in 2 in 0.4% trypan blue solution (Sigma) and resuspended
gently before transfer to a Neubauer haemocytometer chamber and viable (unstained)
49
cells in 4 major grids counted. Total viable cell numbers were calculated using the
following formula:
Viable cells / mL = unstained cells / 2 x dilution factor x 104
2.1.4
Freezing cells
Cells were frozen for long term storage when no more than 80% confluent. Cells were
harvested from 75cm2 flasks as previously described (section 2.1.2) and centrifuged at
400 x g for 5 min, supernatant was discarded and cells were resuspended in 1mL of cold
freezing media. Freeze media contained standard culture media for the specified cell
line supplemented with 10% dimethyl suloxide (DMSO, Sigma). Cells were transferred
to cryovial tubes (NUNC, Denmark) labelled appropriately and stored at -80ºC until
required.
2.1.5
Cell blocks from cell lines
Cells were harvested from 75cm2 flasks as previously described (section 2.1.2) and
resuspended in 0.5mL of human plasma to which 0.5mL of thrombin (DADE, Behring)
was subsequently added. The resultant clot was separated gently from the edge of the
tube with a sterile Pasteur pipette and then resuspended in 10mL of 10% buffered
formal saline (BFS, Amber scientific, Midvale, WA, Australia). Cell blocks were sent
to PathWest, QEII Medical Centre, Nedlands, Perth WA, for paraffin embedding.
2.2
PATIENT SAMPLES
Effusion samples received at PathWest from in-patients at SCGH and from regional
laboratories were collected by laboratory staff unaware of patient history. Samples were
collected from July 2007 to July 2010. Only those samples in excess of diagnostic
50
requirement with volumes greater than 200mL were included in this study. This study
was approved by SCGH HREC.
The study effusion sample was divided into two aliquots and centrifuged at 400 x g for
15 min. One resultant cell pellet was processed to make a cell block as described in
section 2.1.5.
The remaining pellet was resuspended in Ultraspec RNA isolation
reagent (Fisher Biotec, Houston, Texas) and stored at -80oC until subsequent RNA
extraction (section 2.4). The supernatant was pooled and stored at -80ºC until assayed
for CA15-3 (section 2.3).
Each sample was allocated a unique consecutive study number, and patient
demographic data including sex, date of birth and sample type and sample date were
recorded. Sample details remained coded until the study censor date (1/10/2010) when
pathological and clinical data were reviewed. The pathology report for the parallel
original clinical sample was reviewed and the Systematized Nomenclature of Pathology
(SNOP) (College of American Pathologists Committee on Nomenclature and
Classification of Disease 1965) diagnostic coding was recorded. Clinical data for the
individual cases were reviewed and where available the cause of the effusion was noted
as was date of death or date last seen alive.
Criteria for ‘final diagnosis’ was
determined six months after the first effusion sample and on the ‘final’ disease status of
all individuals in the study through clinical follow up, which included clinical history,
additional pathology and radiological evidence until the time of patient death or end of
study was reached. A small subset analysis was performed on 35 mesothelioma patients
that had confirmation of mesothelioma by biopsy or autopsy was performed.
51
2.2.1
Cell block microarray (CBMA)
A haematoxylin and eosin stain (H and E) of a 5μm section of each patient sample cell
block was prepared by PathWest histopathology staff. Each H and E section was
reviewed under light microscopy and an area of representative cells was identified and
marked with a permanent marker for inclusion in the CBMA.
The Beecher tissue microarray (Wisconsin, USA) was used to construct the CBMA for
this study. A 4cm x 2cm solid paraffin block was constructed and this was designated
the recipient block. A 0.4mm core was punched from the recipient paraffin block and
the marked H and E section was manually aligned onto the surface of the corresponding
donor cell block. A 0.4mm core was punched from the area of interest of the donor
block and this core was inserted into the 0.4mm space of the recipient block. Duplicate
samples from each patient’s cell block were included. Control samples were included
on each cell block microarray. These were a selection of cell lines, normal tonsil, colon,
lung, breast and appendix together with reactive appendix, malignant colon, lung and
breast tissue.
2.2.1.1
Immunohistochemistry staining of CBMA
5μm sections were cut from the CBMA and stained with a panel of antibodies (Table
2.1) either using the automated ‘Benchmark ultra system’ (Ventana Medical Systems,
Arizona) or manually.
2.2.1.2
Manual immunohistochemistry staining of CBMA
For manual immunohistochemical staining, sections were deparaffinised in xylene and
rehydrated in a series of decreasing alcohol concentrations. Antigen retrieval was
52
performed for 40 mins in a 95ºC water bath using DakoCytomation target retrieval
solution (Dako, Glostrup, Denmark).
Concentrations of primary antibody were
determined empirically in preliminary experiments to ensure optimal discrimination
between malignant cells and normal mesothelial cells. Experimental dilutions are listed
in Table 2.1 Primary antibody was diluted to a final volume of 1mL (sufficient to cover
the entire CBMA section) and slides were incubated at room temperature for 1 hour. For
negative controls, the primary antibody was omitted. Following incubation sections
were washed with 1x PBS and 500μL of secondary antibody ‘Superpicture Polymerconjugated with horse radish peroxidase (HRP)’ (Zymed) was added to each section and
incubated for 10 min.
Following incubation, sections were washed with 1x PBS and immunodetection was
performed using the 3,3-diaminobenzidine (DAB) reagent (Sigma). This produced a
brown staining reaction where primary and secondary antibodies were attached.
The DAB solution was incubated on each section for 30 min at room temperature.
Sections were washed with 1x PBS, counterstained in Mayer’s haematoxylin (Sigma),
and Scott’s tap water substitute (Amber Scientific, Midvale, WA) followed by
dehydration through increasing alcohol concentrations. Slides were cleared in two
changes of xylene and coverslips mounted with Pertex DPX mounting media (Medite,
Germany).
2.2.1.3
Automated immunohistochemistry staining of CBMA
The automated immunohistochemical staining of several of the antibodies identified in
Table 2.1 was performed using the Ventana Benchmark™ Ultra system (Ventana
53
Medical Systems, Arizona). The automated method for the anti-MUC1 monoclonal
antibody is described as follows. The machine was initially warmed to 75°C and slides
were incubated for 4 minutes. Sections were deparaffinised in six wash steps with a
reaction buffer supplied by Ventana Medical Systems. Slides were warmed to 76°C and
incubated for a further 4 minutes followed by an antigen retrieval method using Ventana
Ultra cell conditioning solution (95°C for 60mins). Sections were washed with an
endogenous peroxidase and then incubated with anti-MUC1 antibody for 60mins at a
1:800 dilution.
Following incubation sections were washed with reaction buffer
(Ventana Medical Systems, Arizona), amplified for 8 mins, washed, and incubated for a
further 8 mins with UV HRP universal secondary antibody. Sections were washed with
reaction buffer followed by visualisation with Ventana Ultraview Universal DAB
incubation for a further 8 mins and then washed with reaction buffer. Sections were
enhanced with Ventana universal copper solution and incubated for a further 4 mins,
washed with reaction buffer and counterstained with Ventana Haematoxylin II (Ventana
Medical Systems, Arizona). Sections are washed in reaction buffer, blued in Ventana
bluing reagent as a post counterstain for 4 mins, washed in reaction buffer and
automatically coverslipped.
2.3.
ANALYSIS OF CBMA
Morphological analysis was performed on the H and E section of each patient sample
cell block. Microscopic assessment was based upon the cytological identification of
cells Samples were classified as malignant when cells with increased nuclear to
cytoplasmic ratio or hyperchromatic nuclei with irregular nuclear membranes were
present, particularly when cohesive aggregates of such cells were observed.
54
Table 2.1: Panel of antibodies used for diagnosis of the cell block microarray.
ANTIBODY
NAME
CLONE
DILUTION
Carcinoembryonic
Antigen
Calretinin
Polyclonal
CEA
1:2000
Z11-E3
1:200
IgG2b
Cytokeratin 5/6
D5/16B4
1:50
IgG1
Mesothelin
5B2
1:100
IgG1
Thyroid
transcription
factor 1
Tumour
associated
glycoprotein 72
B72.3
8G7G3/1
1:100
IgG1/K
DAKO,
Denmark
Automatic
B72.3
Neat
IgG1/K
Cell Marque,
Ventana
Automatic
CD15
MMA
1:50
IgM/K
Automatic
MUC1
1:800
1:100
1:400
IgG2a/K
MUC1
E29
E29
VU4H5
BD
Biosciences
DAKO,
Denmark
Santa Cruz,
CA, USA
MUC1
Mc5
1:100
IgG1
MUC1
SM3
1:50
IgG1
MUC1
BC2
1:400
IgG1
ISOTYPE
IgG1
SOURCE
AUTOMATIC
OR MANUAL
DAKO,
Automatic
Denmark
Zymed
laboratories,
Invitrogen
Zymed
laboratories,
Invitrogen
Leica,
Automatic
Automatic
Automatic
Automatic
Manual
Manual
Neomarkers
Fremont, CA,
USA
Santa Cruz,
CA, USA
Manual
Santa Cruz,
CA, USA
Manual
Manual
55
Cases were classified as benign when no malignant cells were observed and included
cases which contained polymorphonuclear leukocytes, macrophages, lymphocytes and
mesothelial cells.
Cases were called equivocal when marked reactive mesothelial
proliferation was observed and/or small numbers of cells showed a degree of nuclear
atypia (Shidham, V and Falzon, M 2010).
2.3.1
Immunohistochemical scoring of CBMA
Stained sections of the CBMA were independently reviewed by three individuals
experienced in diagnostic cytopathology, (the candidate, Dr A Segal (PathWest) and Dr
G Sterrett, (PathWest). Reviewers were blinded to patient details. In discrepant cases a
consensus opinion was reached. Only those cases which had at least one full core
available for review of the duplicates were included in the study. A representative core
contained sufficient material for a cytological diagnosis. Since MUC1 is an epithelial
marker it was important to identify the number of epithelial cells present in each section
and each pair of core samples from an individual case was initially assessed as having
high or low epithelial cellularity. Each antibody stain was scored based on Remmele’s
method; a sliding scale of zero, 1+ to 3+. (no reactivity = 0% of cells stained; “1+ or
equivocal” = 10%-20%; “2+” = 21%-50% and “3+” = 51%-100%) (Remmele, W,
Hildebrand, U et al. 1986). For some antibodies, staining was further evaluated as
nuclear, cytoplasmic or membranous.
2.3.2.
Statistical Analysis of CBMA Immunohistochemical Staining
Sensitivity and specificity are statistical measures defining the performance of a binary
classification test and can be calculated using a 2 x 2 table. Sensitivity is defined as the
proportion of individuals with disease who have a positive result. Specificity is defined
56
as the proportion of individuals without disease who have a negative test result. While
the positive and negative predictive values describe the patient’s probability of having
disease once the results of the tests are known. The positive predictive value (PPV) is a
score defined as the proportion of individuals with a positive test result who actually
have the disease. Negative predictive value (NPV) is the proportion of individuals with
a negative test result who do not have disease.
2.4
CA15-3 ASSAY
CA15-3 concentration was determined in pleural fluid supernatants and cell line
supernatants using the CanAg CA15-3 enzyme immune assay kit (Fujirebio, Sweden)
following the manufacturer’s instructions as described for serum. The assay is based on
a sandwich ELISA utilising two mouse monoclonal antibodies, MA695 as a capture
antibody, which recognises a sialylated carbohydrate epitope expressed on the MUC1
antigen and a MA552 detector antibody which targets the PDTRPAPG region of the
protein core of MUC1. Briefly calibrators, controls or samples were incubated with a
solution of both biotinylated anti-CA15-3 (MA695) monoclonal antibody (mAb) and
HRP-conjugated anti-CA15-3 (MA552) mAb in streptavidin coated microstrips at room
temperature with shaking.
After two hours wells were washed and 100 L of
chromogen reagent (hydrogen peroxide and 3, 3’, 5, 5’tetra-methylbenzidine) added.
Following a 30 min incubation, stop solution was added. The plate was read on a
microplate spectrophotometer (Spectromax, Molecular Devices) at 405nm. Sample
concentrations were determined from standard curves run concurrently. Values greater
than 36kU/L were considered to be outside the normal range of healthy individuals.
Assays were performed in duplicate on all samples.
57
2.5
MESOTHELIN ASSAY
Soluble mesothelin concentration was determined in pleural fluid supernatants using the
MESOMARK ™ assay kit (Fujirebio, Malvern, PA) following the manufacturer’s
instructions as described for serum. The assay is based on a sandwich ELISA utilising
two mouse monoclonal antibodies, OV569 and 4H3 (Scholler, N, Fu, N et al. 1999).
Calibrators, controls or samples in 100 L aliquots were incubated for one hour at room
temperature with shaking on pre-coated plates.
Following washing, 100 L of
secondary antibody conjugated to HRP was added to each well. After incubation for one
hour as above, wells were washed and 100 L of chromogen substrate was added to each
well. Stop solution was added after 30 min incubation and the absorbance values at
450nm were determined using a microplate spectrophotometer (Spectromax).
Mesothelin concentrations were determined from a standard curve performed on each
plate and expressed as nM. Dilution of samples was carried out if necessary using the
diluent supplied by the manufacturer. Values greater than 20nM were considered to be
positive as previously reported (Creaney, J, van Bruggen, I et al. 2007). Assays were
performed in duplicate on all samples.
2.6
HOMOGENISATION OF CELL LINES IN ULTRASPEC RNA
ISOLATION REAGENT
Cultured cells at least 70% confluent were harvested from flasks (section 2.1.1) and
resuspended in prewarmed culture medium then transferred into a 15mL centrifuge
tube. Cells were centrifuged at 400 g x 5mins. Supernatant was removed and the cell
pellet was resuspended in 1mL of Ultraspec solution (Fisherbiotec).
Cells were
transferred to a 1.5mL microfuge tube, allowed to equilibrate on ice for 30 min and
stored at -80ºC until RNA extraction.
58
2.7
RNA EXTRACTION AND REVERSE TRANSCRIPTION
Total RNA was extracted from cell lines and patient samples stored at -80 in Ultraspec
using an RNeasy kit (QIAGEN, Victoria, Australia) as per manufacturer’s instructions
and
included
DNase
digestion.
Total
RNA
quantity
was
determined
spectrophotometrically (Pharmacia LKB-Ultrospec III) by analysing a 1/50 dilution of
purified RNA solution. Reverse transcription of total RNA was performed using Oligo
dT primers (Promega) and Omnioscript RT kit (QIAGEN, Victoria, Australia) as per
manufacturer’s instructions. Purified cDNA was stored at -20ºC.
2.8
QUANTITATIVE POLYMERASE CHAIN REACTION (PCR)
Quantitative PCR reactions (25μL volume) were performed on the iCycler iQ Real time
detection system (BioRad, New South Wales, Australia) using a specific primer set
(MUC1
forward
5’-AGACGTCAGCGTGAGTGATG
3’
and
reverse
5’-
GACAGCCAAGGCAATGAGAT-3’) (Ohuchida, K, Mizumoto, K et al. 2006) using
1μL cDNA, 0.2μM primers, 1/1000 fluorescein (BioRad, Hercules, California, USA)
and 12.5μL of 2 x SYBR Green PCR mix (QIAGEN, Victoria, Australia). PCR was
initiated at 95°C for 5mins followed by a 45 cycle amplification (95°C for 30 sec, 58°C
for 30 sec, 72°C for 30 sec) and 1 cycle for melt analysis (72°C for 10 sec and 10°C
hold). The primer set corresponds to the nucleotide sequence 4757 – 5017 of the human
polymorphic epithelial mucin gene (Genbank accession number M61170) and amplifies
the full length MUC1 product designated MUC1-TM. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) or βActin were the internal control genes used in PCR
experiments. The relative expression of all quantitative PCR reactions was calculated
using the standard comparative delta delta Ct formula (Livak, KJ and Schmittgen, TD
59
2001) relative to a cDNA standard reverse transcribed from Stratagene QPCR Human
Reference Total RNA.
Fold change = 2-ΔΔCt
Where ΔΔCt = (CT gene of interest - CT internal control) test sample
- (CT gene of interest - CT internal control) reference sample
2.9
IMMUNOHISTOCHEMISTRY STAINING OF CELL LINES
Cultured cells were harvested (section 2.1.2) and 1 x 105 cells were plated into each
well of an 8 chamber polystyrene vessel tissue culture treated glass slide (Becton
Dickinson Biosciences) with appropriate culture medium. Cells were incubated at
37ºC, in water saturated atmosphere of 5% CO2 until each well displayed cells close to
90% confluence. Medium was removed and 500μL of 10% buffered formal saline
(BFS) was added to each well for 30 mins at 4ºC to fix cells. Following incubation BFS
was removed and cells washed three times with 1% FCS/PBS solution.
Primary
antibodies (Table 2.1) were added in a 1/100 concentration. For negative control, the
primary antibody was omitted. Slides were incubated at room temperature for 1 hour or
overnight in a humidifying chamber box. The primary antibody was removed by
washing three times with 1% FCS/PBS solution and 100μL of the secondary antibody
‘Superpicture Polymer detection kit enhanced with HRP polymer’ (Zymed) was added
and incubated for a further 10 mins at room temperature. Cells were washed three times
with 1 % FCS/PBS and 100μL of DAB was added to each well for a further incubation
period of 30 mins.
Following a final wash with 1%FCS/PBS, the 8 chamber
polystyrene tissue culture vessels are removed from the glass slide using the
manufacturer’s device. The nuclei are stained with Mayer’s haematoxylin (Sigma) and
60
Scott’s tap water substitute followed by dehydration of the slide through increasing
alcohol concentrations, clearing the slide in two changes of xylene and mounting the
slide on cover slips using DPX mounting media.
Each slide was scored using
Remmele’s method (section 2.3.1.)
2.10
SOFT AGAR ASSAY
The soft agar assay was used as an in vitro based growth assay to determine the ability
of a single cell to proliferate into a colony in anchorage independent conditions
(Franken, NA, Rodermond, HM et al. 2006). Six well flat bottomed tissue culture
plates (Falcon) were pre-coated with 2mL per well of 0.7% agarose (Promega,
Wisconsin, USA) in 15% FCS/RPMI and allowed to set at room temperature for at least
1 hour in sterile conditions. Cultured cells (1 x 104 cells/mL) were resuspended in
duplicate, pre-heated (at 40ºC) 0.35% low melting agarose (Promega) in 15%
FCS/RPMI then added drop wise into duplicate wells in a final volume of 1mL before
being allowed to set at room temperature for 15 min. 500μL of 15%FCS/RPMI was
added to each well and replaced every 3 days. After 21 days incubation the number of
colonies present in each well was counted manually using a light microscope under 10X
magnification. The assay is a semiquantitative method and colonies greater than 30
cells were counted and recorded from two separate wells of each cell line. Eight random
fields of views were counted from each well and recorded. Primary mesothelial cells
were used as a negative control.
2.11
CyQUANT CELL PROLIFERATION ASSAY
Cell proliferation assays were performed using the CyQuant NF cell proliferation kit
(Invitrogen). The assay is based on the principle that cellular DNA content is highly
61
regulated and is proportional to cell number. The assay incorporates a cell DNA
binding dye in combination with a membrane permeabilization reagent. Cells were
plated in triplicate wells (2 x 103 cells per well) of a 96 well plate with increasing FCS
concentration ranging from 0 – 15% FCS on four separate plates. The plates were
incubated at 37ºC in a water saturated atmosphere of 5% CO2. At selected time points
media was removed from the wells and replaced with 100μL of 1x Hanks balanced salt
solution containing 1x CyQuant NF dye reagent. The plate was then covered in foil and
incubated for an hour under standard conditions. Sample fluorescence was measured
using the Wallac 1420 VictorTM (Perkin Elmer, Australia) microplate reader with an
excitation at 485nm and emission detection at 530nm. Wells were assessed minus the
blank well on each plate and the doubling time of each cell line was calculated using a
nonlinear regression of the exponential growth curve function in the graphical package
Prism 4 (GraphPad Software, Inc. La Jolla, CA).
2.12
WST-1 CELL PROLIFERATION ASSAY
Cell proliferation assays were also performed using the WST-1 cell proliferation reagent
(Roche Applied Sciences, Australia). The assay measures the metabolic activity of
viable cells. The method was similar to the Cyquant cell proliferation assay and cells
were plated in triplicate (2 x 103 cells per well) of a 96 well plate with increasing FCS
concentration ranging from 0 – 15% FCS on four separate plates. The plates were
incubated at 37ºC in a water saturated atmosphere of 5% CO2. At selected time points
10uL of the WST-1 reagent was added to each well and plates were incubated for 2
hours at 37°C in a water saturated atmosphere of 5% CO2. Absorbance was measured
at 450nm against blank controls on the Wallac 1420 VictorTM (Perkin Elmer, Australia)
multiplate reader. Wells were assessed minus the blank well on each plate and the
62
doubling time of each cell line was calculated using a nonlinear regression of the
exponential growth curve function in the graphical package Prism 4 (GraphPad
Software, Inc. La Jolla, CA).
2.13
TRANSWELL MIGRATION ASSAY
Transwell migration assays were performed on cell lines to investigate the migration of
cells from one area to another area in response to a chemoattractant. The MCF7 human
breast adenocarcinoma cell line was used as a non-invasive control (Hinton, A,
Sennoune, SR et al. 2009).
Cells were incubated under standard tissue culture
conditions in 75cm2 flasks until 80% confluency was reached. Cells were then serum
starved overnight in 0.4% FCS/RPMI and transwell inserts (Corning, Life Sciences)
were rehydrated overnight in 0.4%FCS/RPMI. Media was removed from each well and
cells were harvested off the flasks the following day and seeded (1 x 105cells/well) in
duplicate with 0.4%FCS/RPMI. The bottom of each well contained 0% and 15%
FCS/RPMI for each cell line. One control insert contained no cells.
Plates were
incubated for 24 hours under standard tissue culture conditions. Following incubation,
non-invading cells were removed from the upper surface of the insert membrane by
gently scrubbing the surface with a moistened cotton bud in RPMI. Cells on the under
surface of the membrane were stained with the Romanowski stain or the commercially
available “Diff Quik” stain (Australian Biostain, Victoria) and inserts were allowed to
dry. The membrane was then carefully removed from the insert using a sharp scalpel
blade and forceps to detach the membrane completely. A glass slide was dipped in
xylene and the membrane gently placed onto the centre of a slide where it is mounted
with DPX and a glass coverslip. Migrating cells were counted at 40X magnification at
63
3 random fields of views. Results were calculated semiquantitatively as the percentage
of invading cells over the number of cells plated.
2.14
WOUND HEALING ASSAY
Wound healing assays were performed to study directional cell migration in vitro
Cells were seeded at high density (5 x 105
(Liang, CC, Park, AY et al. 2007).
cells/well) into flat bottom 6 well plates in duplicate.
Cells were cultured under
standard tissue culture conditions until 90% confluence was reached.
Cells were
washed in PBS and serum starved in 0.4%FCS/RPMI overnight. On the following day,
media was removed from wells and cells were washed with PBS. A straight line was
drawn vertically on the bottom of each well with a marker pen. Scratch marks (3/well)
were made in each well perpendicular to the line drawn using a sterile 200μL pipette tip
at a 30º angle. Media was replaced and an image of each well was taken using an
inverted microscope and a Nikon digital camera at 0, 6, 12, 24 and 48 hour intervals or
until wound closure was evident.
Images were analysed using Image J analysis
(Rashband 2004). The area of each scratch was determined at the 0 time point and the
same area was superimposed on each image to determine the area/number of particles
(μm) within that area for each given time. Each cell line was plotted as a percentage of
the wound closure that had been covered by migrating cells and determined at the 6, 12,
24 and 48 hour time point.
2.15
FLOW CYTOMETRY
Flow cytometry was performed on cell lines to examine the expression of MUC1 using
the EMA antibody (clone E29). Cells were harvested by cell scraping or trypsining
flasks and 5 x 105cells/mL were resuspended in cold FACS buffer (1X Hanks Buffered
64
Salt solution (HBBS) containing 0.1% EDTA and 200μL aliquots were added to four
wells in a 96 well plate. The plate was centrifuged at400g for 5 min and supernatant
removed. Primary antibody (EMA, E29 clone) was added to appropriate wells and the
plate was incubated at room temperature for 30 min. Following incubation, 200μL of
cold FACS buffer was added to each well and the plate was centrifuged at 400g for 5
min. The supernatant was removed and 50μL of secondary antibody (Goat anti-mouse
IgG FITC, Australian Biosearch, Karrinyup, WA) added to appropriate wells, the plate
was covered in foil and incubated on ice for a further 30 min. The plate was centrifuged
at 1200rpm for 5 min, supernatant removed and the plate washed twice with cold FACS
buffer and centrifuged following each wash.
Propidium iodide (1μL of 500nM
solution) (Sigma) was added to each well to assess the percentage of cell death. For
each sample there were two negative controls included, firstly the primary antibody was
omitted, and secondly the secondary antibody was omitted. Cells were analysed on the
Quava EasyCyte Plus, (Millipore, USA) and results were assessed on FlowJo 887
software (Treestar Inc, Ashland, OR, USA). Forward and side scatter dot plots were
used to gate live cells and this gate was then used to assess for the percentage of cells
stained with the EMA (E29) monoclonal antibody.
2.16
SH-RNA MUC1 STABLE CELL LINE TRANSFECTION
Mesothelioma cell lines were transfected with the commercially
available
SureSilencing™ Hygromycin vector, shRNA MUC1 plasmid purchased from SA
Biosciences (Qiagen, Victoria, Australia) to produce stable shRNA MUC1 knockdown
cell lines. The constructs specifically knock down the expression of specific genes by
RNA interference. Each vector expresses a short hairpin RNA under the control of a U1
promoter and the Hygromycin resistance gene enabling the selection of a stable cell line
65
transfectant. The vector sequence is designed from the MUC1 reference sequence
NM_001018016, which correlates to transcript variant MUC1/Y.
The scrambled
negative vector sequence does not match any human, mouse or rat gene.
2.16.1
Transformation of plasmids in competent cells
To amplify shRNA plasmids E. Coli DH5α competent cells (Invitrogen) were incubated
with 2μL of each plasmid on ice for 30 min. The E. coli cells were transformed by heat
shocking at 42ºC for 1 min following which 900μL of super optimal broth media (SOC,
Invitrogen) was added at room temperature. The tubes were placed on a shaker at 37ºC
at 225rpm for 1 hour. Following incubation 50μL and 100μL aliquots of each plasmid
preparation was evenly distributed onto Luria Bertani (LB) ampicillin plates and
incubated at 37ºC overnight. Following overnight incubation, a single colony was
removed off each 50μL plate and streaked onto a grid plate containing 20 separate
colonies from each plasmid preparation. These grid plates were incubated for at least 5
hours at 37ºC and a third of the bacterial growth was removed from four separate clones
using a sterile loop which was then inoculated into 5mLs of LB media containing
100μg/μL of ampicillin. These tubes were then incubated overnight on a small shaker
in preparation for a rapid isolation of plasmid.
2.16.2
Quick plasmid mini preparation
Following overnight incubation 4mLs of each bacterial stock sample was transferred
into a 15mL tube which was centrifuged to pellet the cells. A quick plasmid miniprep
(Invitrogen) was used to isolate the plasmid following manufacturer’s instructions and a
Pst 1 restriction digest (see below) was performed to confirm purification of the
hygromycin shRNA vector.
66
2.16.3
Restriction digest of plasmid
A Pst 1 restriction enzyme digest (Roche) was performed from a 1μL aliquot of each
plasmid with 1 unit of Pst 1 incubated at 37°C for 1 hour. The product was separated
on a 1% agarose gel with undigested samples in parallel to confirm purified plasmid
containing the shRNA insert. The SureSilencing™ Hygromycin vector generates Pst 1
fragments of 3892 bp and 1147 bp when separated on a 1% agarose gel.
2.16.4
Amplification and maxipreparation of purified plasmid
From the original grid plate (section 2.16.1), the remainder of the bacterial growth was
removed from the clones confirmed to contain the shRNA plasmid and these were
amplified into a 250mL volume of LB media containing ampicillin (100mg/mL). The
bacterial culture was placed on a shaker overnight to amplify bacterial stock. The
overnight LB culture was pelleted at 600 x g for 15 min at 4ºC. An Endofree plasmid
purification maxi kit (Qiagen) was used to purify each plasmid, according to
manufacturer’s instructions. Briefly, bacteria were harvested, lysed, clearing of the
bacterial lysate and precipitation following manufacturer’s instructions. The plasmid
DNA was redissolved in 500μL of TE buffer and incubated in a water bath at 37ºC for 5
min to help dissolve the pellet. Following incubation the pellet was centrifuged briefly
and samples were then placed on ice while 1μL of each plasmid underwent a Pst 1
restriction digest (Section 2.16.2) which was then visualised on a 1% agarose gel to
confirm isolated plasmid contained the products of interest.
2.16.5
Selection for antibiotic resistance
The minimum antibiotic concentration necessary to kill tissue culture cell lines was
determined by generating a dose response curve for each line using Hygromycin
67
(Sigma). Cells were plated (1 x 104 cells/well) in a 24 well plate with increasing
concentrations of hygromycin from 0, 20, 40, 60, 80, 100μg/mL. Cells were allowed to
grow until the well with no added drug reached confluence. Media was replaced every
2 days during the selection process and the minimal concentration of antibiotic that
killed all the cells was determined by a cell count as the effective concentration used for
selection of each transfected cell line.
2.16.6
Plasmid DNA transfection
Lipofectamine 2000 (Invitrogen) was used to transfect the purified shRNA MUC1 and
control plasmid into required cell lines. One day before transfection, cells were plated
(1 x 105 cells/well) in a 24 well plate so that cells were 80% confluent at the time of
transfection. Plasmid DNA (0.8μg) was diluted in 50μL of OptiMem 1 (Invitrogen) and
2μL of Lipofectamine 2000 was diluted in 50μL of OptiMem 1 and incubated for 5 min
at room temperature. Following incubation diluted plasmid DNA was combined with
the diluted Lipofectamine and incubated for a further 20 min at room temperature. Each
100μL complex was added to each well containing cells and medium. The plates were
mixed by gentle rocking and incubated under standard tissue culture conditions.
Following 24 hour incubation, cells were harvested off the plate and transferred into a
12 well plate with fresh 15%FCS/RPMI and incubated under standard tissue culture
conditions for a further 24 hours. Media was removed and replaced the following day
with effective concentration of hygromycin in 15%FCS/RPMI. Cells were incubated
under standard tissue culture conditions with the minimal concentration of hygromycin
required in 15%FCS/RPMI until stable cell line tranfectants were established and
confirmed by RT quantitative-PCR using primers specific for MUC1.
68
2.17
GENERAL STATISTICAL ANALYSIS
All variables were assessed for normality. For data that was non-normally distributed
log transformation was performed before parametric testing. Statistical analysis was
performed using GraphPad Prism software version 4.0 (San Diego, CA, USA) and
SPSS software version 18 (Armonk, NY, USA). A p value of 0.05 or less was regarded
as significant and represented as an asterisk in figures.
The Cramer’s V analysis was performed using SPSS and is a measure of association
based upon the Chi square test where a value of ‘0’ means no association and ‘1’
represents perfect association.
A two proportion z test was used to determine
frequencies between groups.
Biomarker levels were compared using non parametric Mann-Whitney U test. The
biomarker correlation between CA15-3 and mesothelin was determined using Spearman
rank analysis (rs).
Diagnostic performance was assessed with receiver operating
characteristics (ROC) curve analysis with the area under the curve (AUC) calculated.
The combined tumour marker was determined by standardizing the logarithmically
transformed biomarker levels and comparing the diagnostic power of the individual
markers and the combined marker for both the mesothelioma and epithelial malignancy
cohorts. This was done by using logistic regression models to derive coefficients for
mesothelin and CA15-3, adjusted for gender, for the prediction of either mesothelioma
or all epithelial cancer. The combined marker was calculated as a probability from
these coefficients for mesothelioma or all epithelial cancer. ROC analysis was then the
used to calculate AUC in order to compare the predictive power of mesothelin, CA15-3,
and the combined marker, for differentiating mesothelioma and all epithelial cancer.
Survival plots were generated by the Kaplan Meier method.
69
CHAPTER 3
SENSITIVITY
AND
SPECIFICITY
OF
MUC1
IN
THE
CYTOLOGICAL DIAGNOSIS OF EPITHELIAL MALIGNANACY
AND MALIGNANT MESOTHELIOMA
70
3.1
INTRODUCTION
The diagnosis of mesothelioma is difficult and is often made based on the combination
of clinical, radiological and pathological findings.
Recent Guidelines from the
International Mesothelioma Interest Group (Husain, AN, Colby, TV et al. 2009) and the
European Respiratory Society and European Society of Thoracic Surgeons (Scherpereel,
A, Astoul, P et al. 2010) state that a histopathological biopsy is the ‘Gold Standard’ for
a diagnosis of mesothelioma with invasion being considered the most important
diagnostic feature (Churg, A, Colby, TV et al. 2000; Hasteh, F, Lin, GY et al. 2010).
Pathological diagnosis based on the cytological examination of pleural effusion or fine
needle aspirations is controversial as invasion cannot be ascertained and therefore
considered by many experts insufficient because of the high risk of diagnostic error
(Scherpereel, A, Astoul, P et al. 2010).
Currently there is no sensitive or specific immunohistochemical marker available to
confirm a diagnosis of mesothelioma. Several studies have investigated the ability of a
wide range of immunohistochemical markers to distinguish between reactive
mesothelial hyperplasia, mesothelioma and metastatic disease (Ordonez, NG 2003a;
Ordonez, NG 2007; Westfall, DE, Fan, X et al. 2009) and the current consensus is that a
panel of antibodies is required (Husain, AN, Colby, TV et al. 2009). Whilst most of
these antibody evaluation studies have been performed in tissue biopsy specimens, a
small number have been performed in cytologic effusion samples (Wolanski, KD,
Whitaker, D et al. 1998; Saad, RS, Cho, P et al. 2005; Hasteh, F, Lin, GY et al. 2010).
Some authors suggest that methods and antibodies applied to histological specimens
cannot be directly applied to cytological material due to differences in methodology,
71
interpretation and possible changes to the cells when they are shed into effusion
(Dejmek, A and Hjerpe, A 2000).
One of the primary concerns regarding cytological diagnosis is the establishment of the
malignant phenotype as cellular invasion can not be conclusively determined in
cytological specimens. Morphologic characteristics of cells such as large cellular
aggregates, papillary-like fragments, nuclear pleomorphism, macronucleoli, large
cellular aggregates, and cell-in-cell engulfment can be indicative of malignancy but
such features may also occur in reactive hyperplastic mesothelial cells (Hasteh, F, Lin,
GY et al. 2010). The anti-MUC1 EMA (E29) antibody has been shown to be a useful
marker for distinguishing between benign reactive mesothelial cells and mesothelioma
in a cytological setting (Henderson, D, Shilken, K et al. 1992; Wolanski, KD, Whitaker,
D et al. 1998; Whitaker, D 2000; Saad, RS, Cho, P et al. 2005; Creaney, J, Segal, A et
al. 2008; Hasteh, F, Lin, GY et al. 2010). These studies have generally been performed
on retrospective collections of samples with known diagnosis. The aim of the current
study is to evaluate the sensitivity of antibodies against MUC1/EMA for mesothelioma
diagnosis in cytological effusion samples in a prospective series of samples.
72
3.2
RESULTS
The performance of anti-MUC1 antibodies in the cytological diagnosis of mesothelioma
was assessed in terms of quantitative diagnostic accuracy. As this study used patient
samples where a histologically proven biopsy, autopsy or electron microscopy result of
mesothelioma was not uniformly available, we determined diagnostic sensitivity and
specificity of MUC1 in the context of four potential determinants of pathological
diagnosis.
1:
In the first method, anti-MUC1 antibody EMA (E29), Mc5, VU4H5, SM3 and
BC2 sensitivities was analysed relative to the official PathWest Pathology
reported diagnosis of the sample; this was referred to as the “interim pathology
diagnosis”. In some cases this interim diagnosis was based on evidence which
included the reactivity of the anti-MUC1 antibody, EMA (E29).
2:
The second approach was to determine the “final” disease status of all
individuals in the study through clinical follow up, which included clinical
history, additional pathology and radiological test results until the time of patient
death or the end of the study.
3:
The third method was to perform a subset analysis of only those mesothelioma
patients that had a confirmation of mesothelioma by biopsy or autopsy.
4:
The final method was to perform a “study” based diagnosis in which the results
from cytological morphology evaluation determined on the H and E sections and
from only the antibody panel used in the present study, excluding any of the
results from MUC1/EMA antibody staining.
73
3.2.1
Description of patient cohort
Patient samples were prospectively collected from effusions in excess of diagnostic
requirement from PathWest Cytology laboratory, Nedlands. Of the 280 patient samples
collected, there were 224 pleural, 46 peritoneal and 10 pericardial effusions. Following
blinded study review of sample morphology and all antibody staining results, case
details were decoded identifying that 14 samples were multiples collected from patients
already represented in the cohort. In each of these cases, the multiple specimens were
re-evaluated and given that the diagnostic composition of each multiple case was similar
the first sample collected was chosen for statistical purposes reducing the patient cohort
to 266.
For interim analysis, pathology reports were reviewed and categorised into five groups
based upon the reported Systematized Nomenclature of Pathology (SNOP) code for that
sample (Table 3.1). From the 266 patient samples evaluated in this study, 45% were
reported as benign effusions, 4% atypical or equivocal effusions, 3% non-epithelial
malignancy, 30% epithelioid malignancy and 17% mesothelioma.
There was no
statistical difference in the male/female ratio or the age between the groups (Table 3.1).
Of the 121 samples reported as being benign 51% were classified based on SNOP
coding as normal, 27% as containing inflammatory cells and 22% were reported as
containing benign reactive mesothelial cells. There was not enough biochemical data
available on the samples to accurately determine the percentage of effusions that were
exudative in nature, although 13 samples (10%) were classified as transudates and
therefore unlikely to be due to malignancy (Light, RW, Macgregor, MI et al. 1972).
74
Approximately 40% of samples were from patients with a known history of malignancy
or with symptoms suggestive of malignancy (Table 3.2).
Table 3.1 Patient characteristics grouped by Interim Pathology Diagnosis
Interim
Diagnosis
SNOP
codes
Number
(% of total)
Number
Females
Median
Age (range)
Benign
0001
4000
7300
6903
6971
9593
9803
8006
8143
9053
-
121 (45.5%)
42
66.6 (23 – 88)
11(4.1%)
3
71 (69 – 73)
7 (2.6%)
4
77.25 (75 – 81)
81 (30.5%)
45
69.5 (32 – 96)
46 (17.3%)
266
8
102
70.6 (55 – 90)
71.2
Atypical
suspicious
Non-epithelial
Malignancy
Epithelial
Malignancy
Mesothelioma
TOTAL
3.2.2
Description of sample scoring
Each section was assessed with the reviewer blinded to clinical, surgical, radiological
and previous pathological data. To assess the ability of MUC1 to differentiate epithelial
from non-epithelial cells each case was initially dichotomised as low or high epithelial
cellularity (Figure 3.1). Each case was further categorised based on established criteria
as being of benign, malignant or equivocal morphology (Shidham, V and Falzon, M
2010). Finally, the immunoperoxidase staining of each section was scored.
3.2.2.1
Dichotomisation of samples based upon epithelial cellularity
A range of cellularity was observed in samples. In total 64% cases were of high
epithelial cellularity while 36% of cases demonstrated low epithelial cellularity.
Representative samples are shown in Figure 3.1.
75
Table 3.2 Characteristics of samples categorized as benign.
Characteristics
SNOPa code
Biochemistryb
Clinical historyc
a
Categories
No. Cases
0001/0002
4000
7300
7718
Transudate
Exudate
Unknown
History of malignancy
Symptoms suspicious of malignancy
Low level of suspicion of malignancy
Unknown
56
33
26
6
13
21
87
11
36
30
44
– Effusions were classified based upon the Systematized Nomenclature of Pathology
(SNOP) as being normal (i.e SNOP codes 0001/0002), containing inflammatory cells
(SNOP 4000), reactive mesothelial cells (SNOP 7300) and lymphocytosis (SNOP 7718)
b
– Effusions were classified as transudates or exudates on the basis of Light’s criteria
(Light, RW, Macgregor, MI et al. 1972)
c
– Effusions were separated into four groups on the basis of the documented clinical
history.
A case with high epithelial cellularity showed a mixed population of cells including
small loosely cohesive clusters of regular (normal) mesothelial cells, scattered small
round lymphocytes and macrophages (Figure 3.1A).
A case with low epithelial
cellularity did not include as many cells in total but still had a representative mixed
population of mesothelial cells, lymphocytes and macrophages (Figure 3.1B).
76
Figure 3.1: Tissue morphology divided into high and low cellularity.
A
MacroMesothelial
Lymphocytes
Image A: Represents Patient 92 stained with EMA (E29) on the automated staining
system. The section is cellular and shows a mixed population of cells including
mesothelial cells, lymphocytes and macrophages, staining negative with EMA E29.
B
LymphoMacro-
Mesothelial
Image B: Represents Patient 89 stained with EMA (E29) on the automated staining
system. This section is not as cellular as Image A, however a mixed population of
mesothelial cells, lymphocytes and macrophages are identified. The section is
negative for EMA (E29) staining.
77
3.2.2.2
Categorisation of samples based on cellular morphology
Cellular morphology was determined from each H and E section from all cell blocks
within the study. This was not ideal as cytological features usually demonstrated on the
Papanicolaou and Diff Quik stain were not available.
Figure 3.2 A and B demonstrates H and E sections of two cases classified on
morphological criteria as malignant. Figure 3.2 A shows a homogenous population of
enlarged epithelial cells, showing a mild degree of nuclear pleomorphism, with
prominent nucleoli. The cytoplasm is dense with predominant glycogen lakes noted
within the cytoplasm.
The H and E section in Figure 3.2 B shows several cohesive aggregates of cells with
predominant vacuolated cytoplasm and a mild degree of nuclear irregularity. A mixed
lymphocytic population is visible within the background.
Figure 3.2 C shows a sample classified based on morphological criteria as benign. The
sample contains a mixed population of mesothelial cells (with normal nuclei),
macrophages, lymphocytes and polymorphonuclear leukocytes. Figure 3.2 D represents
a case categorised as atypical/equivocal.
population
of
mesothelial
cells,
The H and E sections shows a mixed
macrophages,
isolated
lymphocytes
and
polymorphonuclear leukocytes, however, a number of mesothelial cells showed mild
nuclear irregularity though not sufficient to classify the case as malignant, and therefore
the case was placed into the equivocal category, with insufficient cytological features
demonstrated for a diagnosis of malignancy.
78
Figure 3.2 A: H and E section demonstrating the cytological morphology from a case
diagnosed as malignant. The section a monotonous population of enlarged
mesothelial-like cells showing a degree of cellular pleomorphism. Nuclei are variable
with prominent nucleoli evident. Predominant glycogen lakes are identified within the
cytoplasm.
Patient 13
Figure 3.2 B: H and E section from patient 29 demonstrates cohesive aggregates of
malignant cells showing a mild degree of nuclear pleomorphism and hyperchromasia.
Predominent mucin-like vacuoles are noted displacing a few nuclei, with a mixed
lymphocytic population seen in the background together with isolated mesothelial
cells.
Patient 29
79
Figure 3.2 C: H & E section demonstrating the cytological morphology from a case
diagnosed as benign. The section shows a mixed population of mesothelial cells,
macrophages, lymphocytes and polymorphonuclear leukocytes.
Patient 100
Figure 3.2 D: H & E section demonstrating the cytological morphology from a case
categorized as atypical/equivocal. The section shows a mixed population of
mesothelial cells, showing mild degree of nuclear abnormality, macrophages,
lymphocytes and polymorphonuclear leukocytes.
Patient 28
80
There was a total of 118 cases categorised as benign, 32 cases categorised as
atypical/equivocal and 116 cases categorised as malignant on the basis of morphological
criteria. More cases were classified as atypical/equivocal than were classed as benign or
malignant on the interim report compared to the final report (Table 3.1).
3.2.2.3
Scoring of immunoperoxidase staining
Before the immunoperoxidase staining of the entire sample cohort was scored, scoring
parameters were defined and standardised for each antibody in a selection of 20 effusion
sections. Scoring was performed in collaboration with Dr Greg Sterrett and Dr Amanda
Segal, two experienced Consultant Pathologists at PathWest Laboratories.
Examples of scored samples are shown in Figure 3.3. For the anti-MUC1 antibody
(clone E29) sections were recorded as negative when no immunoreactivity was seen
(Figure 3.3A). Equivocal or 1+ positive staining was noted when there were <20% of
cells showing membranous staining pattern or cells were stained only as a cytoplasmic
blush (Figure 3.3B). A positive 2+ staining was reported for cases in which >21% but
<50% of cells were immunoreactive (Figure 3.3C) and sections with >50% of cells
stained intensity were scored as 3+ positive (Figure 3.3D).
The entire sample cohort was contained on four cell block microarrays (CBMA) and
immunoperoxidase staining was performed by the three readers independently. For
some stains, less than 266 cases were evaluated due to loss of sections during the
preparation of the individual CBMA slides. Discrepant cases were reviewed at a double
headed microscope and a consensus score was achieved. Results for the five antiMUC1 antibodies (EMA-E29, Mc5, VU4H5, BC2 and SM3), three mesothelial
81
antibodies (calretinin, cytokeratin 5/6 and mesothelin), four glandular antibodies (CEA,
TTF-1, B72.3 and CD15) and for the PAS-D stain are presented in Table 3.3.
3.2.3
Immunoreactivity of anti-MUC1 monoclonal antibodies
Five different anti-MUC1 antibody clones were evaluated in this study.
Three
antibodies (EMA-E29, Mc5 and BC2) belong to Group 1, the pan-MUC1 antibodies
which were found to react with MUC1 independent of the glycosylation state. The
remaining two antibodies (VU4H5 and SM3) belong to Group 2, which recognise only
specific glycosylated forms of MUC1 (Cao, Y, Karsten, U et al. 1998).
3.2.3.1
Anti-MUC1 antibody EMA (E29) on interim diagnosis
During the period of this research project the Pathology laboratory was transitioning
from traditional manual immunohistochemical staining methods to the use of a high
throughput automated platform using the Benchmark Ultra Automatic Staining system.
Both techniques were evaluated in this study.
Figure 3.4 gives an overview of the different staining patterns observed using the EMA
(E29) antibody by the manual staining method. The majority of mesothelioma cases
42/46 (91.3%) stained with MUC1/EMA (E29) exhibited a strong membrane
accentuated stain (Figure 3.4), in some mesothelioma cases, cells had strong
cytoplasmic as well as membranous staining patterns and in two cases no staining of
mesothelioma cells was observed. EMA (E29) stain was strongly positive in 87%
(71/82) of non-mesothelioma epithelial malignancies with cells primarily staining
strongly cytoplasmic with or without membranous accentuation.
82
Table 3.3 Summary of immunohistochemical staining for 266 cases relative to the
interim pathology diagnosis.
Staining
Method
Score
EMA (E29)
Manual
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
Negative
Equivocal 1+
Positive 2/3
Missing
EMA
Auto
(E29)
Mc5
VU4H5
BC2
SM3
Calretinin
CK5/6
Mesothelin
CEA
TTF-1
B72.3
CD15
PAS+D
Mesothelioma
Epithelial
malignancy
Non-epithelial
malignancy
Atypical/
suspicious
Benign
(n=46)
2
2
42
(n=81)
10
1
69
1
10
8
62
1
11
7
61
2
13
8
57
3
22
15
43
1
48
12
20
1
66
10
1
4
62
14
4
1
49
15
14
3
9
13
57
2
59
8
10
4
35
15
28
3
29
11
37
4
50
10
16
5
(n=7)
5
1
1
(n=11)
5
2
4
(n=121)
113
8
6
1
5
5
1
118
3
5
4
2
5
9
10
27
2
6
37
1
13
11
20
2
19
12
13
2
40
4
1
1
6
5
34
1
5
3
38
7
8
30
1
19
12
13
2
45
1
44
2
36
9
1
45
1
7
9
2
65
27
23
6
105
9
2
5
117
2
7
11
2
118
6
6
4
1
1
1
7
1
6
1
7
3
1
6
3
2
7
5
1
5
6
7
4
3
86
32
3
83
31
4
3
96
21
1
3
113
3
1
7
11
5
116
2
7
11
3
115
1
5
2
8
2
7
1
10
1
5
114
2
1
4
109
1
11
83
Table 3.3A: Summary of the anti-MUC1 antibody staining on control tissue sections on
CBMA.
Tissue
Colon
Tonsil
Appendix
Lung
Breast
Tonsil reactive
Colon Ca
Appendix reactive
Breast Ca
Lung Ca
E29 manual
1
0
0
2
1
0
3
0
3
3
E29 auto
1
1
0
1
1
0
2
0
3
2
Mc5
0
0
0
2
2
0
2
0
3
0
VU4H5
1
0
0
1
1
0
2
0
3
2
SM3
0
0
0
0
0
0
0
0
2
0
BC2
0
0
0
1
0
0
0
0
2
2
Two of the seven non-epithelial malignancies showed strong cytoplasmic staining; both
cases were diagnosed on follow up as acute myeloid leukaemia. Seven of the 26 cases
reported to contain benign reactive mesothelial cells contained cells with focal,
dispersed cytoplasmic EMA (E29) staining (Figure 3.4E and F).
In total 44% (116/265) of samples stained positive, 56% (148/265) were negative, and
5% (14/265) were equivocal when the EMA (E29) antibody was used (Table 3.3).
A similar pattern of cellular localisation was observed when the anti-MUC1 (EMA
clone E29) antibody was used on the automated platform. Of note however, none of the
seven cases with dispersed cytoplasmic staining of the benign reactive mesothelial cells
when the manual staining method was used were stained by the automated method.
Using the EMA (E29) antibody on the automated staining system, 34% (90/266) cases
were positive, 56% (148/266) were negative and 10% (27/266) were equivocal (Table
3.3).
84
Figure 3.3: Optimisation of cell block microarray analysis. Below are four representative
images of sections stained with the E29 antibody on the automated staining platform. Image
A represents no staining (0), image B represents an equivocal case where 10-20% of cells
are staining positive (1+), image C demonstrates a metastatic adenocarcinoma with 21-50%
of cells showing cytoplasmic positivity (2+) image D represents the strong membranous
stain seen in mesothelioma where >50% of cells stain positive (3+). All images were
photographed using Aperio imagescope analysis at 19.2X magnification.
A
B
C
D
85
Anecdotally concerns were raised about discrepancies in results obtained when sections
were stained with the anti-MUC1 (clone E29) antibody using the automatic staining
system versus the traditional manual staining method. This issue was addressed in the
current study as serial sections from the 266 cases were stained with the EMA (E29)
antibody by both the manual method and on the automated platform. Overall there was
a strong correlation between the two staining methods when the results from all samples
were analysed (Cramer’s V test = 0.544; p < 0.0001) (Table 3.4).
With approximately 50% (131/265) of cases scored as negative, and nearly 20%
(50/265) scored as positive by both methods.
However, when only the results from the mesothelioma cases were analysed a
discrepancy between the two methods was revealed (Table 3.5).
Table 3.4: Correlation in immunoperoxidase scoring of all cases (n=265*) using the
No. cases / category
Staining
Automated E29
EMA (clone E29) antibody and either the Manual or Automated staining methods.
0
0
131
Manual E29 Staining
No. cases / category
1+
2+
8
6
1+
4
4
6
11
25
2+
0
0
10
25
35
3+
0
0
7
50
57
135
12
29
89
265*
TOTAL
3+
3
TOTAL
148
* 1 section missing on cell block microarray during preparation.
86
Table 3.5: Correlation in immunoperoxidase scoring of mesothelioma cases (n=46)
using the EMA (clone E29) antibody and either the Manual or Automated staining
methods.
No.cases/category
Staining
Automated E29
0
Manual E29 Staining
No. cases / category
1+
2+
3+
TOTAL
0
2
1
8
2
13
1+
0
1
3
4
8
2+
0
0
0
9
9
3+
0
0
3
13
16
2
2
14
28
46
TOTAL
Of the 28 cases which were scored as 3+ following manual staining, 2 were negative, 4
were equivocal and 9 scored 2+ by the automated method. Sensitivity of each staining
method for mesothelioma was calculated by grouping cases scored 2+ and 3+ as
positive, and 0 and 1+ as negative. The sensitivity for mesothelioma using the manual
staining method was 91% (42/46) and for the automated method was 54% (25/46); the
proportion of mesothelioma cases stained using the manual EMA (E29) staining method
was significantly higher than those stained with the automated staining system (z =
2.981, p < 0.01) (Figure 3.4A and B).
To determine if the difference in EMA (E29) staining was related to mesothelioma or
epithelial malignancy in general, the sensitivity of each of the staining methods was
reviewed relative to the non-mesothelioma epithelial malignancies in this study (Table
3.6). The sensitivity of the manual staining method was 86% (69/80) and the automated
method was 78% (62/80). The proportion of epithelial malignancies stained using the
87
Figure 3.4: Comparison between the anti-MUC1 antibodies. The sections below
demonstrate the staining variation seen between the manual EMA(E29) stain (A) and
the automated EMA (E29) stain (B). Section (C) stained with the anti-MUC1 (Mc5),
section (D) stained with the anti-MUC1 (VU4H5), section (E) stained with anti-MUC1
(SM3) and section (F) stained with the anti-MUC1 (BC2) monoclonal antibodies. All
sections are from the same patient diagnosed with mesothelioma.
B
A
C
D
E
F
88
manual EMA (E29) staining was not significantly different to those stained with the
automated staining machine (z = 1.456 p > 0.01)(Figure 3.5).
Table 3.6: Correlation in immunoperoxidase scoring of epithelial malignancy (n=80*)
using EMA (clone E29) antibody and either the Manual or Automated staining methods.
No.cases/category
E29 Staining
Automated
0
Manual E29 Staining
No. cases / category
1+
2+
3+
TOTAL
0
9
0
0
1
10
1+
1
1
0
6
8
2+
0
0
6
15
21
3+
0
0
4
37
41
10
1
10
59
80*
TOTAL
* 1 missing section from cell block microarray during preparation.
3.2.3.2
Anti-MUC1 antibody (Mc5) on interim diagnosis
The anti-MUC1 (Mc5) monoclonal antibody demonstrated strong accentuated
membrane staining with diffuse cytoplasmic staining in mesothelioma and nonmesothelioma epithelial malignant cells (Figure 3.6). In addition a high number of
benign samples 24% (23/94) that contained non-malignant mesothelial cells stained
positive with the Mc5 clone (Figure 3.6E and F). From the 256 patients with samples
49% (127/256) were Mc5 positive and 50% (129/256) were Mc5 negative (Table 3.3).
3.2.3.3
Anti-MUC1 antibody (VU4H5) on interim diagnosis
The staining pattern of the anti-MUC1 antibody (VU4H5) was quite variable from
strong membranous accentuation to focal diffuse cytoplasmic staining in mesothelioma
(Figure 3.7A and B).
There were 2% (2/116) of benign cases showing positive
89
immunoreactivity and in some mesothelioma cells nuclear staining was also noted
(Figure 3.4D). From the 256 patients with samples 31% (80/256) patients were positive
for VU4H5 staining while 68% (176/256) showed negative VU4H5 staining (Table
3.3).
3.2.3.4
Anti-MUC1 antibody (SM3) on interim diagnosis
The majority of mesothelioma and non-mesothelioma samples did not stain with the
anti-MUC1 antibody (SM3), in total only 8% (21/261) showed positive SM3 staining
with 92% (240/261) showing no SM3 staining at all (Table 3.3).
In the five
mesothelioma cases that stained with this antibody, only one had strong membrane
staining in a few mesothelioma cells (Figure 3.8B).
In non-mesothelioma epithelial malignant cells the staining pattern demonstrated strong
positive cytoplasmic and membranous accentuation (Figure 3.8C and D).
3.2.3.5
Anti-MUC1 antibody (BC2) on interim diagnosis
The anti-MUC1 (BC2) antibody showed strong cytoplasmic staining pattern in nonmesothelioma epithelial malignancies with a small number of mesothelioma cells
showing mild membranous accentuation (Figure 3.9A and B). From the 261 patients
with evaluable samples, 56/261 (21%) showed positive staining for BC2 with 205/261
(78%) showing no staining pattern (Table 3.3).
90
Figure 3.5: Cell block microarray stained with a manual E29 antibody. Sections A
and B represent mesothelioma cases staining strongly positive. Sections C and D
represent epithelial malignancies while sections E and F represent benign mesothelial
cells showing focal membranous staining pattern.
A
B
C
D
E
F
91
Figure 3.6: Cell block microarray stained with Mc5 antibody. Sections A and B
represent mesothelioma cases staining strongly positive. Sections C and D represent
metastatic adenocarcinomas while sections E and F represent benign mesothelial cells
showing prominent membranous staining patterns.
A
B
C
D
E
F
92
Figure 3.7: Cell block microarray stained with VU4H5 antibody. Sections A and B
represent mesothelioma. Sections C and D represent metastatic adenocarcinomas
while sections E and F represent benign mesothelial cells showing prominent
membranous staining patterns.
A
B
C
D
E
F
93
Figure 3.8: Cell block microarray stained with SM3 antibody. Sections A and B represent
mesothelioma. Sections C and D represent metastatic adenocarcinomas while sections E
and F represent benign mesothelial cells staining negative with the SM3 antibody.
A
B
C
D
E
F
94
Figure 3.9: Cell block microarray stained with BC2 antibody. Sections A and B represent
mesothelioma cases. Sections C and D represent metastatic adenocarcinomas while sections
E and F represent benign mesothelial cells.
A
B
C
D
E
F
95
3.2.3.6
Summary of Anti-MUC1 antibody staining related to interim diagnosis
The immunoreactivity of a panel of anti-MUC1 antibodies was examined in samples
from 266 individuals with effusion. To compare the sensitivity and specificity of the
antibodies for firstly epithelial malignancy and secondly mesothelioma, data were
initially analysed relative to the Interim Pathological Diagnosis of each sample. Using
this criteria there were firstly a total of 81 cases of epithelial malignancy and secondly
46 cases of mesothelioma. (Data for the calculation of the sensitivity and specificity of
each antibody are included in Appendix, Chapter 8).
The sensitivities of the five anti-MUC1 clones for epithelial malignancy ranged from
17% to 84% with the E29 clone having the highest sensitivity. Specificities ranged
from 77% to 100% with the SM3 and BC2 clones being the most specific, but clearly at
the cost of sensitivity (17% and 43% respectively) (Table 3.7).
Table 3.7: Summary of the sensitivity and specificity of anti-MUC1 antibodies for an
interim diagnosis of epithelioid malignancy including mesothelioma (n=127) relative to
all other cases studied (n=139). (Note: calculation of individual sensitivities and
specificities are presented in Appendix A).
Antibodies
Sensitivity (%)
Specificity (%)
Accuracy (%)
E29 manual
84
97
90
E29 auto
67
99
83
Mc5
76
77
77
VU4H5
60
98
78
SM3
17
100
58
BC2
43
100
71
96
Table 3.8: Summary of the sensitivity and specificity of anti-MUC1 antibodies for an
interim diagnosis of mesothelioma (n=46) relative to all other cases studied including
non-mesothelioma epithelial malignancies (n=220) (Appendix B).
Antibodies
Sensitivity (%)
Specificity (%)
Accuracy (%)
E29 manual
91
65
77
E29 auto
58
63
62
Mc5
82
57
62
VU4H5
45
71
67
SM3
2
91
75
BC2
30
80
72
When examining only the mesothelioma cases of epithelial malignancy sensitivities for
the E29 and Mc5 antibodies improved to 91% and 82% respectively but the specificity
of all antibodies was reduced (Table 3.8). In terms of diagnostic accuracy the E29 clone
is the best of the five antibodies examined.
3.2.4
Summary of Anti-MUC1 antibody staining related to Final Diagnosis
Of the 266 individuals in this study, follow-up was achieved for 90% of the population
(Figure 3.10). At the close of study 30 of the 121 cases which were benign on the
interim pathology were malignant on follow-up including five cases which had a
diagnosis of mesothelioma. Also follow-up of the 11 atypical/equivocal cases revealed
90% (10/11) to be malignant on follow-up, with seven cases of mesothelioma identified.
Because of the temporal nature of disease, two major situations arose in which case
diagnosis changed between the interim and the final diagnosis. In the first situation
samples were collected before a final diagnosis and these are referred to as
97
Figure 3.10: Characteristics of patient cohort as determined from the interim pathology
diagnosis. The red boxes represent the distribution of diagnoses determined from at least
six months follow up.
Multiple samples on the same
patient were excluded (n = 14)
Pathology diagnosis
from effusion cytology
Follow-up
6 months
following
diagnosis.
Report from interim Pathology diagnosis
Confirmed on follow-up
98
prediagnostic samples. In the second situation, post-diagnostic samples were collected
from patients with an established diagnosis of malignancy however, these effusions
contained no malignant cells and the effusion may have been related to other disease
processes.
The latter situation was observed in 50% (15/30) of benign cases which were classed as
benign on the interim pathology report but following clinical data review were found to
be from patients with an established diagnosis of malignancy.
Therefore after follow-up there were 107 benign cases, 12 non-epithelial malignancies,
89 non-mesothelioma epithelial malignancies and 58 cases of mesothelioma. Of the 12
pre-mesothelioma diagnosis samples, five (42%) were scored strongly positive by the
blinded scoring of the MUC1 (E29) manual staining method.
The sensitivities of the five anti-MUC1 clones for a final diagnosis of epithelial
malignancy (including mesothelioma) ranged from 14% for the SM3 clone to 75% for
the E29 clone. The specificity of the different antibody clones ranged from 81% for the
Mc5 to 100% for the SM3 and E29 clones. With the Mc5 clone reacting strongly in
23/115 non epithelioid malignancy effusion samples, this resulted in a false positive rate
of approximately 20% (Table 3.9.).
The E29 clone demonstrated a sensitivity of 75% with a specificity of 100% for a
diagnosis of epithelioid malignancy including mesothelioma. For a final diagnosis of
mesothelioma the sensitivity of the different antibody clones ranged from 1.8% for the
SM3 clone to 83% for the E29 clone. The specificity ranged from 58% for the Mc5
99
clone to 90% for the SM3 clone (Table 3.10). For a final diagnosis of mesothelioma the
sensitivity and specificity of the E29 clone was 83% and 66% respectively, relative to
all other cases studied. However, the specificity of the E29 clone increases to 100% for
a final diagnosis of mesothelioma in the absence of epithelial malignancy (Appendix E).
Therefore after follow-up there were 107 benign cases, 12 non-epithelial malignancies,
89 non-mesothelioma epithelial malignancies and 58 cases of mesothelioma. Of the 12
pre-mesothelioma diagnosis samples, five (42%) were scored strongly positive by the
blinded scoring of the MUC1 (E29) manual staining method.
The sensitivities of the five anti-MUC1 clones for a final diagnosis of epithelial
malignancy (including mesothelioma) ranged from 14% for the SM3 clone to 75% for
the E29 clone. The specificity of the different antibody clones ranged from 81% for the
Mc5 to 100% for the SM3 and E29 clones. With the Mc5 clone reacting strongly in
23/115 non epithelioid malignancy effusion samples, this resulted in a false positive rate
of approximately 20% (Table 3.9.).
The E29 clone demonstrated a sensitivity of 75% with a specificity of 100% for a
diagnosis of epithelioid malignancy including mesothelioma. For a final diagnosis of
mesothelioma the sensitivity of the different antibody clones ranged from 1.8% for the
SM3 clone to 83% for the E29 clone. The specificity ranged from 58% for the Mc5
clone to 90% for the SM3 clone (Table 3.10). For a final diagnosis of mesothelioma the
sensitivity and specificity of the E29 clone was 83% and 66% respectively, relative to
all other cases studied. However, the specificity of the E29 clone increases to 100% for
a final diagnosis of mesothelioma in the absence of epithelial malignancy (Appendix E).
100
Table 3.9: Summary of the sensitivity and specificity of anti-MUC1 antibodies for a
final diagnosis of epithelioid malignancy including mesothelioma (n = 144) relative to
all other cases in the study (n = 122) (Appendix C).
Antibodies
Sensitivity (%)
Specificity (%)
Accuracy (%)
E29 manual
75
100
86
E29 auto
60
100
76
Mc5
73
81
77
VU4H5
55
97
74
SM3
14
100
52
BC2
38
99
65
Table 3.10: Summary of the sensitivity and specificity of anti-MUC1 antibodies for a
final diagnosis of mesothelioma (n = 58) relative to all other cases studied including
non-mesothelioma epithelial malignancies (n=208) (Appendix D).
Antibodies
Sensitivity (%)
Specificity (%)
Accuracy (%)
E29 manual
83
66
70
E29 auto
52
70
65
Mc5
79
58
62
VU4H5
37
70
63
SM3
1.8
90
70
BC2
23
78
67
3.2.5
Sensitivity and Specificity of anti-MUC1 monoclonal antibodies
determined on a subset of 35 mesothelioma patients
As previously discussed, the validity of a diagnosis of mesothelioma based upon
cytology samples has been questioned. Therefore a sub-set analysis was performed on
those cases in the cohort on which a diagnosis of mesothelioma was made on tissue
101
obtained from biopsy or autopsy or where there was EM confirmation of the cytology
results. Thirty five patients had a diagnosis of mesothelioma confirmed either on
biopsy, electron microscopy or at autopsy. There were 24 mesothelioma cases with
biopsy, nine from EM and two on autopsy. Of the 35 patients with a confirmed
diagnosis, 26 cases showed a strong MUC1 (E29) positivity on the manual staining
method while only 14 stained MUC1 (E29) strongly positive on the automated staining
system.
The sensitivity for diagnosing mesothelioma from a cytological effusion
sample in these patients with a histologically confirmed biopsy, autopsy or EM
diagnosis was 74% with a specificity of 61% and a percentage accuracy of 63% when
the samples were stained using the manual MUC1 (E29) method (Appendix F). The
automated staining method showed a sensitivity of 40% with a specificity of 65% for a
final histological diagnosis of mesothelioma, with a percentage accuracy of 63%
(Appendix F).
3.2.6
Comparison of the sensitivity of MUC1 for mesothelioma diagnosis
Because of potential issues relating to the definition of mesothelioma cases in this study
three separate analysis were performed. In any of the analyses, when the sensitivities of
the different MUC1 antibodies under study were compared the E29 clone, using the
manual staining method, had the highest diagnostic accuracy for mesothelioma (Table
3.8 and 3.10).
Comparing the sensitivity of the E29 anti-MUC1 antibody for
mesothelioma as determined by the three separate study methods, that of using the (i)
interim diagnosis, (ii) the final diagnosis and (iii) the diagnosis confirmed by other
pathology methods demonstrated that there was no statistically significant difference in
the results for the groups (Table 3.11).
102
Table 3.11: Compliation of data relevant to mesothelioma diagnosis from tables 3.8
and 3.10.
Study Method for
Mesothelioma Diagnosis
Interim diagnosis
1
No
Mesothelioma
cases
46
Sensitivity E29
p-value1
91%
ref
p-value1
-
Final diagnosis
58
83%
p=0.75
ref
Pathology confirmation
35
74%
p=0.08
p=0.2
Summary of results of statistical comparison between the reference group (ref/interim
diagnosis) and the other study group (final diagnosis) by the Chi-square test.
3.2.7
Diagnosing mesothelioma without the anti-MUC1 antibody EMA (E29)
As a final method to circumvent the potential confounding effect of the inclusion of the
MUC1 EMA (E29) staining in the clinical diagnostic panel, all 266 cases in the study
were re-categorised as being benign, atypical/equivocal, mesothelioma or as an
epithelial malignancy based on a defined decision-tree with and without the results from
the anti-MUC1 antibody (Figure 3.11 and 3.12).
In the first analysis (Figure 3.11) cases were categorised based on morphological
characteristics and 118 cases were classified as benign, 32 equivocal and 116 malignant.
For the malignant and equivocal cases (n = 148), the first branch of the tree was based
on Periodic Acid Schiff (PAS) with Diastase (PAS+D) reactivity. This cytochemical
stain is positive for neutral mucins in epithelial malignancies and generally negative in
mesotheliomas which tend to be glycogen-rich (Whitaker, D 2000; Adams, SA,
Sherwood, AJ et al. 2002). Sixteen cases were PAS+D positive and classified as
epithelial malignancy.
103
Figure 3.11: Hypothetical analysis of the patient cohort in the absence of the EMA
(E29) antibody.
(*) Denotes mesothelioma antibodies including Calretinin, CK 5/6 and Mesothelin.
(+) Denotes epithelial markers including CEA, B72.3, TTF-1 and CD15
Dx without the EMA Ab
266
cases
Cytological
Morphology
116
Malignant
32
Equivocal
118
Benign
PAS +D
16 +ve
Epithelial
Malig
132 -ve
Mesothelioma Markers *
38 +ve
MM
94
Others
Glandular Markers +
43 +ve Epithelial
Malig
51
Others
29 Malignant
7 Non Epith
Malignancy
22 Malignant
NOS
22 Others
22 Equivocal
104
Figure 3.12: Hypothetical analysis of the patient cohort in the presence of the EMA
(E29) antibody.
(*) Denotes mesothelioma markers Calretinin, CK 5/6 and Mesothelin.
(+) Denotes epithelial markers CEA, B72.3, TTF-1 and CD15.
105
Figure 3.12A: Hypothetical analysis of the patient cohort in the presence of the EMA
(E29) antibody distinguishing between mesothelioma and other malignancy.
(*) Denotes mesothelioma markers Calretinin, CK 5/6 and Mesothelin.
(+) Denotes epithelial markers CEA, B72.3, TTF-1 and CD15.
106
Figure 3.12B: Hypothetical analysis of the patient cohort in the presence of the EMA
(E29) antibody distinguishing equivocal cell morphology.
107
The remaining 132 cases negative for PAS+D were evaluated using three mesothelioma
markers, calretinin, cytokeratin (CK) 5/6 and mesothelin. Calretinin is a 29kD calcium
binding protein expressed on both benign and malignant mesothelial cells (Chhieng,
DC, Yee, H et al. 2000; Westfall, DE, Fan, X et al. 2009). The CK 5/6 markers are
intermediate sized basic keratins which are mainly expressed in keratinising (epidermis)
and non-keratinising (mucosa) squamous epithelium and in myoepithelial cell layers of
the prostate, breast and salivary glands (Chu, PG and Weiss, LM 2002). CK 5/6 has
emerged as one of the most diagnostic markers for mesothelioma (Chu, PG and Weiss,
LM 2002). Mesothelin is a 40kDa glycosylphosphatidylinositol linked glycoprotein
expressed in both normal and malignant mesothelial cells (Ordonez, NG 2003b). Two
or more of the mesothelial cell markers, stained in 38 of the 132 cases and these cases
were classified as mesothelioma for the purposes of this study.
The staining of the remaining 94 cases was evaluated using four glandular cell markers.
The glandular cell markers were carcinoembryonic antigen (CEA), B72.3, TTF-1 and
CD15. CEA is one of the most widely used tumour markers expressed in tumours
which are epithelial in origin including lung adenocarcinoma, colorectal carcinoma and
mucinous ovarian carcinoma (Hammarstrom, S 1999). B72.3 is a monoclonal antibody
that reacts with a TAG 72 protein, a tumour associated glycoprotein complex expressed
in epithelial carcinomas (Ordonez, NG 2007). TTF-1 is expressed in the thyroid and in
Type II pneumocytes of the bronchioalveolar and non-ciliated bronchiolar epithelial
(Clara) cells of the lung and within tumours derived from these organs (Khoor, A, ByrdGloster, AL et al. 2011). Several studies have shown that TTF-1 can be used to
differentiate
lung
adenocarcinoma
from
non-pulmonary
adenocarcinoma
or
mesothelioma (Khoor, A, Whitsett, JA et al. 1999; Moldvay, J, Jackel, M et al. 2004).
108
CD15 (also known as Leu-M1) is a monocyte granulocyte marker expressed by tumour
cells in Hodgkins disease as well as many types of pulmonary adenocarcinomas and is
therefore
considered
to
help
differentiate
metastatic
mesothelioma (Ordonez, NG 2007; Davidson, B 2011).
adenocarcinomas
from
Forty three cases stained
positive for CEA alone or with at least two or more of the glandular cell markers, and
were classified as epithelial malignancy for the purpose of this study.
There were 51 cases that could not be differentiated using this analysis tree. Of the 51
cases remaining, 29 cases morphologically looked malignant on cytology, 7 of these
cases were suspicious for a non-epithelial malignancy while 22 cases showed
cytological features suggestive of an epithelial malignancy however differentiation of
these cases was considered inconclusive based upon this analysis. The final 22 cases
showed cytological features that were difficult to differentiate as benign or malignant.
From this hypothetical review, 44 cases would require further investigation including
electron microscopy, additional immunohistochemistry or a biopsy follow up.
Therefore using this decision tree which did not include the MUC1 antibody, from the
original set of 266 cases, 118 were classed as benign, 59 as epithelial malignancy and
38 as mesothelioma. It was not possible to classify 51 cases on the basis of criteria used
in the decision tree.
In comparison, a decision algorithm was generated that incorporated the results obtained
from the anti-MUC1 EMA (E29) antibody (Figure 3.12) to classify cases. As above,
based on morphological characteristics 118 cases were classified as benign, 32
equivocal and 116 malignant. Of the 148 malignant and equivocal cases stained with
109
the MUC1 antibody, 118 showed strong positivity (>2) suggesting malignancy while 30
cases demonstrated minimal or no MUC1 positivity. These 30 MUC1 negative cases
were excluded from the rest of the tree. The 118 malignant cases were then stained with
PAS+D with 16 cases demonstrating positivity for neutral mucins suggesting an
epithelial malignancy. The remaining 102 cases were separated using the staining
profile with mesothelioma markers, calretinin, CK 5/6 and mesothelin. Forty two of
these cases were positive for at least two of these markers suggesting a diagnosis of
mesothelioma. The remaining 60 cases were differentiated using the glandular marker
panel of CEA, B72.3, TTF-1 and CD15. A strong positive reaction in CEA alone or in
at least two glandular markers was observed in 39 cases, favouring an epithelial
malignancy. Of the remaining 21 cases, 1 cytologically demonstrated a malignant
pattern showing cellular features suggestive of a metastatic lymphoma. Compared to
the previous decision tree (Figure 3.11), 20 cases remained showing definite malignant
morphology requiring further investigation including electron microscopy, further
immunoperoxidase staining or biopsy follow-up to help differentiate tumour type.
The decision tree was a hypothetical scenario aimed to highlight the significance of the
MUC1 (E29) stain for a differential diagnosis of mesothelioma compared to epithelial
malignancies. The first tree was based upon the diagnosis of mesothelioma in the
absence of the MUC1 (E29) antibody. By incorporating a panel of mesothelioma and
epithelial markers for the first decision tree, the final results came down to 22 cases
which were malignant but not otherwise specified with 22 cases which still remained
equivocal for a diagnosis. The second decision tree incorporates the MUC1 (E29)
antibody before the PAS+D stain. After analysis with the panel of mesothelioma and
epithelial markers the final results on the second decision tree came down to 13 cases
110
which were malignant but not otherwise specified with 20 cases which still remained
equivocal for a diagnosis. From the second decision tree there were four extra cases of
mesothelioma which stained strongly MUC1 (E29) positive, which were not identified
from the first decision tree.
From both hypothetical scenarios the non-epithelial
malignancies were mainly identified on cytological morphology and excluded as an
epithelial malignancy from the mesothelioma and epithelial markers. This hypothetical
scenario highlights the potential of the MUC1 (E29) marker to be incorporated within
the panel of immunohistochemical stains to help differentiate mesothelioma from
epithelial malignancies and benign reactive mesothelial cells.
111
3.3
DISCUSSION
Establishing a diagnosis of mesothelioma can be extremely difficult especially in the
early development of disease and several markers have been extensively investigated in
serum and effusions to discriminate mesothelioma and non mesothelioma abnormalities.
The diagnosis of mesothelioma is possible on cytological effusion samples and this
study demonstrated that the inclusion of MUC1 immunoperoxidase staining in an
antibody panel may be of fundamental importance.
The anti-MUC1 antibodies examined in this study all reacted with the tandem repeat
region of the MUC1 extracellular domain. In mesothelioma cases which stained with
each of these anti-MUC1 antibodies, reactivity was mainly accentuated on the
membrane. In samples that contained benign reactive mesothelial cells that stained with
MUC1, expression was mainly confined within the cytoplasm. These results were
similar to those observed by Hasteh et al, who demonstrated that 23% of benign reactive
mesothelial proliferations showed weakly or focal positive MUC1 staining (Hasteh, F,
Lin, GY et al. 2010).
In non-mesothelioma epithelial malignancies staining with
MUC1, expression was in both the membrane and cytoplasmic compartments, notably
however membrane staining was not as accentuated as seen in mesothelioma. As
mesothelioma cells have a well defined microvillous border compared with
adenocarcinomas (Wick, MR, Loy, T et al. 1990) and MUC1 is expressed on the
microvilli (van der Kwast, TH, Versnel, MA et al. 1988) this may account for the
distinctive membrane accentuated staining observed.
Although MUC1 is predominantly expressed on the cell membrane, during the
malignant process MUC1 cell localisation has been frequently observed to be
112
disregulated (Luna-More, S, Rius, F et al. 2001; Cozzi, PJ, Wang, J et al. 2005; van der
Vegt, B, de Roos, MA et al. 2007). Several reports have also demonstrated that the CT
of MUC1 translocates to the nucleus (Leng, Y, Cao, C et al. 2007; Bitler, BG,
Goverdhan, A et al. 2010) and/or the mitochondria (Ren, J, Bharti, A et al. 2006)
through interactions with heat shock proteins. The anti-MUC1 antibodies used in the
current study do not specifically react with the CT domain but detected cytoplasmic
MUC1 expression in some benign and some epithelial malignant cells, and the VU4H5
antibody showed nuclear staining in some mesothelioma cells, whether this is a
precursor or differentially expressed full length MUC1 protein will require further
investigation.
The SM3 and the BC2 anti-MUC1 antibodies both recognise a hypoglycosylated form
of MUC1 (Cao, Y, Karsten, U et al. 1998). The lowest sensitivity for mesothelioma
was detected with the SM3 antibody (1/45 mesothelioma positive), suggesting that the
majority of mesothelioma cases express a hyperglycosylated form of MUC1 in
comparison to 25% of cases of non-mesothelioma epithelial malignancy.
MUC1 is a well established marker for epithelial malignancy (Silverman, JF, Nance, K
et al. 1987; Cao, Y, Blohm, D et al. 1997; Kirschenbaum, A, Itzkowitz, SH et al. 1999;
Luna-More, S, Rius, F et al. 2001; Obermair, A, Schmid, BC et al. 2002; Kawamoto, T,
Shoda, J et al. 2004; Hebbar, V, Damera, G et al. 2005; de Roos, MA, van der Vegt, B
et al. 2007; Duncan, TJ, Watson, NF et al. 2007). In this study five different antibodies
demonstrated a very high specificity for epithelial malignancy (97 to 100%). However
there was a wide range of sensitivities (17 to 84%) for the epithelial malignancies in this
study possibly reflecting differences in the glycol-forms of MUC1 being detected by the
113
different antibodies and the variety of different primary phenotypes of tumours
represented. The E29 anti-MUC1 clone had the highest diagnostic accuracy, classifying
90% of cases correctly distinguishing effusions resulting from epithelial malignancy
from other causes. Similarly a wide range of sensitivities (2 to 91%) was observed for
the five MUC1 antibodies for mesothelioma, with the E29 clone having the highest
diagnostic accuracy.
When looking exclusively at the diagnostic accuracy of the MUC1 antibodies for
mesothelioma, there is a perceived problem with the establishment of the reference
group. In order to cover the various requirements for firstly, a world-wide acceptable
diagnosis of mesothelioma and secondly a diagnosis of mesothelioma that does not
incorporate the results from the anti-MUC1, E29 antibody, several different strategies
were employed to define the mesothelioma cohort. In the most strictly defined cohort
there were 35 cases of mesothelioma confirmed by histopathology or electron
microscopy.
Using the manual staining method the E29 anti-MUC1 clone had a
sensitivity of 74% in these 35 cases. In the less stringently defined situations where the
results from the MUC1 antibody (46 cases), or clinical and radiological follow-up (58
cases) was used to identify cases of mesothelioma, sensitivity was not statistically
different.
The attempt to develop a decision-tree based algorithm to identify mesothelioma cases
similarly did not reveal any statistical difference in sensitivity of cytological diagnosis
without or with the inclusion of MUC1 (E29), although approximately 10% (ie 38 to 42
cases) more cases where identified in the latter scenario. This result may have reached
significance if more cases could have been recruited. Therefore with sensitivity greater
114
than 74% the results suggest that the anti-MUC1 (E29) antibody should be included
when examining cytological specimens, especially when there is a high suspicion for
mesothelioma. One of the fundamental points that need to be noted is that cytological
diagnosis is a multistep process, and this study was evaluating one step of that process,
the value of the anti-MUC1 antibodies to differentiate benign from malignant cells.
This is the first prospective study to demonstrate the diagnostic utility of MUC1 for
mesothelioma in a cytological setting.
Clearly, as already established epithelial malignancies are generally MUC1 positive and
this is reflected in the current study when the specificity results are presented for the
ability of MUC1 to diagnose mesothelioma for the entire study. Specificities ranged
from 57% to 90% for a diagnosis of mesothelioma. Previously reported ranges for the
sensitivity of the E29 antibody ranged from 45% to 100% (Wolanski, KD, Whitaker, D
et al. 1998; Saad, RS, Cho, P et al. 2005; Aerts, JG, Delahaye, M et al. 2006; Ordonez,
NG 2007; Creaney, J, Segal, A et al. 2008; Grefte, JM, de Wilde, PC et al. 2008; Shen,
J, Pinkus, GS et al. 2009), these results are similar to those observed by previous
authors in retrospective and/or case control studies.
The more diagnostically relevant question is the specificity of MUC1 to distinguish
mesothelioma from benign reactive mesothelial cells. The specificities for a diagnosis
of mesothelioma ranged from 80% for the Mc5 clone to 100% for the E29 clone when
cases of epithelial malignancies were excluded from analysis (data not shown). There
were no false positives cases identified on the E29 stain, compared to 23% of false
positives identified with the Mc5 clone. Highlighting the specificity for a diagnosis of
malignancy compared to benign reactive proliferations, which is required in the
115
diagnostic setting. The reason why these antibodies are different is unknown given that
they fall into the same staining category defined by Cao et al (Cao, Y, Karsten, U et al.
1998). It is this ability to distinguish benign reactive from malignant mesothelioma
which is of vital importance for mesothelioma diagnosis in cytology specimens. From
this study, it is not clear what the epitope is that is recognised by the E29 clone that
facilitates this differentiation and maybe the reactivity of the E29 antibody is dependent
on fixation or processing. The results from the parallel staining using the manual or
automatic staining method suggesting that increased temperatures during the staining
protocol increases the sensitivity for a positive reaction with the E29 clone in
mesothelioma cases.
This is the first observation of a difference in the staining results using the MUC1 (E29)
antibody in a manual or automated staining platform and its affect on a diagnosis of
mesothelioma. Whilst a strong correlation existed between the two staining methods, a
significant difference was found in the sensitivity of identifying mesothelioma cases
using the manual E29 staining method (sensitivity 91%) compared to the automated
platform (sensitivity 58%).
As there was no significant difference in diagnosing
epithelial malignancies from the E29 manual or automated platform there may be a
difference in the MUC1 expression on mesothelioma and other epithelial cancers. This
may be a result of the glycosylation pattern on the MUC1 epitope compared to that
previously seen in epithelial carcinomas (Cao, Y, Blohm, D et al. 1997; Cao, Y,
Karsten, U et al. 1998; Burke, PA, Gregg, JP et al. 2006; de Roos, MA, van der Vegt, B
et al. 2007; Duncan, TJ, Watson, NF et al. 2007). In this study, we have suggested that
the majority of mesothelioma cases express a hyperglycosylated form of MUC1. So
perhaps, the MUC1 epitope on mesothelioma cells is larger than those seen in epithelial
116
malignancies however further study would need to be performed to investigate this
hypothesis.
The difference in results obtained with the two staining methods may reflect differences
in the antigen retrieval step. In the manual method, antigen retrieval is performed at
180°C using a pressure cooker system.
This is a higher temperature than that achieved with the automated system which is not
capable of reaching temperatures greater than 100°C. Considering a high percentage of
MUC1’s mass is composed of carbohydrate sugars then perhaps the higher temperatures
enable the removal of these sugars from the immunodominant region of the EMA
(MUC1) epitope resulting in a positive MUC1 (E29) staining reaction.
This is a potential explanation for the reason that the sensitivity of cytology has
previously been reported to be approximately 30% (To, A, Dearnaley, DP et al. 1982).
Perhaps a study should be performed to evaluate cases in a blinded fashion from
laboratories around the world which would finally answer this question and perhaps
then the diagnosis of mesothelioma based on cytology effusion samples may be
accepted at an international level.
Another explanation is that previous studies investigating the diagnostic utility of the
anti-MUC1 monoclonal antibody to differentiate mesothelioma from adenocarcinoma
have not previously used antigen retrieval methods for immunohistochemical staining.
117
Table 3.12: Comparison of study characteristic for anti-MUC1 antibody for a diagnosis
of mesothelioma.
Author – year
EMA
clone
E29
E29
E29
E29
Dilution
E29
E29
E29
1:2
1:40
1:100
Su, 2010h
Van
der
Kwast, 1988i
Wolanski,
1998j
E29
E29
1:100
1:100
E29
1:100
Attanoos, 2003k
Roberts, 2001l
Cury, 1999m
E29
E29
E29
1:100
1:50
1:20
Grefte, 2007a
Shen , 2009b
Saad , 2005c
Motherby,
1999d
Hasteh, 2010e
Cibas, 1987f
Creaney, 2008g
1:100
1:300
1:30
1:1600
Antigen
Retrieval
No
No
No
No
SENS/SPEC
%
100/91
100/54
70/40
100/70
Sample
Size (MM)
33(11)
73(35)
40(20)
154(14)
Source
100/91
75/50
84/93
116(52)
141(20)
36(20)
Dako
Dako
Dako
94/83
92/97
113(18)
97(25)
Dako
Dako
No
73/30
214(141)
Dako
No
No
No
80/56
67/92
96/65
100(60)
188(76)
65(31)
Dako
Dako
Dako
No
No
HIEP*/
citrate buffer
HIEP*
No
Dako
Dako
Dako
Dako
*Heat induced epitope antigen retrieval
a(Grefte, JM, de Wilde, PC et al. 2008)
b(Shen, J, Pinkus, GS et al. 2009)
c(Saad, RS, Cho, P et al. 2005)
d(Motherby, H, Kube, M et al. 1999)
e(Hasteh, F, Lin, GY et al. 2010)
f(Cibas, ES, Corson, JM et al. 1987)
g(Creaney, J, Segal, A et al. 2008)
h(Su, XY, Li, GD et al. 2010)
i(van der Kwast, TH, Versnel, MA et al. 1988)
j(Wolanski, KD, Whitaker, D et al. 1998)
k(Attanoos, RL, Griffin, A et al. 2003)
l(Roberts, F, Harper, CM et al. 2001)
m(Cury, PM, Butcher, DN et al. 1999)
Table 3.12, demonstrates 13 studies investigating the validity of the anti-MUC1
monoclonal antibody as a diagnostic tool for mesothelioma showing that only 15% of
these studies used some kind of antigen retrieval method including heat induced epitope
antigen retrieval methods during the immunohistochemical staining of this anti-MUC1
antibody. However, if the antigen retrieval method was paramount in the staining
regime then one would expect the two cases that used an antigen retrieval method to
118
show a higher sensitivity and specificity for this anti-MUC1 antibody, however this
clearly was not the case in comparison to the other studies. In addition only three
studies had sample sizes greater than 50 cases diagnosed with mesothelioma. The
comparison between studies also demonstrates the variation in the working dilution of
the EMA (E29) antibody used and perhaps this may also contribute to the differences in
results observed with the EMA (E29) antibody in a diagnosis of mesothelioma.
Multiple publications have tried to demonstrate the best possible marker available to aid
in the differential diagnosis between mesothelioma and adenocarcinoma in serous
effusions. As yet there are not single predictive tumour markers routinely included in
the histological work up of mesothelioma. Both the calretinin and CK 5/6 have been
widely accepted markers for the diagnosis of mesothelioma in effusion samples
(Chhieng, DC, Yee, H et al. 2000; Ordonez, NG 2007). In this current study, 11/77
(14%) of epithelioid mesotheliomas stained positive for calretinin and this is similar to
that seen in previous studies (Ordonez, NG 2007). Cytokeratin 5 is expressed in normal
mesothelial cells, transitional epithelium and myoepithelial cells. It is often used as a
diagnostic marker for mesothelioma and squamous cell carcinoma, however cytokeratin
5 is also expressed in a significant proportion of breast and lung carcinomas (Ordonez,
NG 2007). As previously observed serous adenocarcinomas express CK 5/6, therefore
a differential diagnosis only on the basis of this antibody is difficult. Mesothelin was
positive in 84% of mesotheliomas and 37% of epithelial malignancies as previously
observed (Ordonez, NG 2003b).
The prevalence of mesothelioma in Western Australia (WA) is in part due to the history
of Wittenoom mining between the years of 1937 to 1966 when the mine finally closed.
119
The Australian mesothelioma surveillance program commenced in 1980, which
incorporates a voluntary notification of cases from a network of respiratory physicians,
pathologists, general and thoracic surgeons, medical records administration, State and
Territory departments of occupational health and cancer registries (Leigh, J and
Driscoll, T 2003). Therefore, because the prevalence of mesothelioma is high in WA,
most patients are referred to a tertiary hospital and for this reason there is a high number
of mesothelioma cases in this prospective study and this may not be reflective of all
centres where a smaller percentage of mesothelioma cases may be seen.
There was a potential bias in the samples collected in the study. Whilst the aim was to
collect a consecutive cohort of effusion samples, only samples in excess of diagnostic
requirements could ethically be collected. Therefore to ensure adequate samples for
diagnostic requirements, including the preparation of four smears and a diagnostic cell
block for immunohistochemical stains (Thomas, SC, Davidson, LR et al. 2010), only
samples greater than 200ml were collected for the purpose of this study. It is possible
therefore that the study was biased towards cases such as malignancies and exudates
where larger effusion samples are more frequently collected compared to transudate
samples (Porcel, JM 2011).
3.4
CHAPTER SUMMARY
A diagnosis of mesothelioma can be extremely difficult to establish even after recurrent
pleural effusions, repeated cytological examinations, fine needle aspirates and serial
chest x-rays or computed tomography. A definite diagnosis may not be achieved until
the tumour burden increases or until further invasive investigation is undertaken.
120
Effusion collection is easily obtained and widely accepted by patients and is often the
only identifiable pathology in the early stage of disease, however at present neoplastic
invasion is the only consistently reliable marker for the discrimination between benign
versus malignant mesothelial proliferation which is difficult to assess in a cytological
sample.
Immunohistochemistry is often used in conjunction with morphology in
effusion samples to help distinguish benign, reactive and malignant mesothelial cell
proliferations and the correct diagnostic antibody panel is valuable in achieving the
correct possible diagnosis.
However, currently, the cytological diagnosis of
mesothelioma determined from an effusion sample can only act as a suspicion for
disease and further invasive investigation is warranted (Husain, AN, Colby, TV et al.
2009; Scherpereel, A, Astoul, P et al. 2010). Conversely, the British Thoracic Society,
2007 provided a statement supporting the cytological diagnosis obtained from an
effusion sample or by percutaneous fine needle aspiration (BTS 2007). If the effusion
cytology and the associated immunohistochemistry equivocally favours a diagnosis of
mesothelioma and given that clinical and radiological appearance correlates with the
pathological findings, then perhaps these results may be sufficient for a diagnosis of
mesothelioma.
121
CHAPTER 4
SENSITIVITY AND SPECIFICITY OF SOLUBLE MUC1
CONCENTRATIONS IN SUPERNATANT SAMPLES OF PLEURAL
MALIGNANCIES
122
4.1
INTRODUCTION
Differential diagnosis of malignant and non-malignant effusions can be difficult, with
cytological diagnosis only correctly identifying 60% of malignant effusions (Wolanski,
KD, Whitaker, D et al. 1998; Saad, RS, Cho, P et al. 2005; Hasteh, F, Lin, GY et al.
2010). Therefore, a number of tumour biomarkers have been evaluated as
complementary tools to aid this differential diagnosis, including carcinoembryonic
antigen (CEA), carbohydrate antigen 15-3 (CA15-3), CA19-9, lactate dehydrogenase
(LDH), adenosine deaminase (ADA), neuron-specific enolase (NSE), cytokeratin
fragment 19 (CYFRA 21-1), squamous cell carcinoma antigen (SCC) and total sialic
acid (TSA) (Alatas, F, Alatas, O et al. 2001; Korczynski, P, Krenke, R et al. 2009;
Hackbarth, JS, Murata, K et al. 2010). However, the sensitivity of these biomarkers has
varied between studies ranging from 28% to 98% CA15-3, the soluble form of MUC1
was reported to be one of the most sensitive of the markers studied (Miedouge, M,
Rouzaud, P et al. 1999; Alatas, F, Alatas, O et al. 2001; Ghayumi, SM, Mehrabi, S et al.
2005) with sensitivities above 70% (at specificities >83%) in different patient groups.
Therefore the clinical utility of these different markers is unclear.
Only a small number of studies have looked at the diagnostic value of the CA15-3 as a
tool to differentiate between mesothelioma and carcinoma in effusion samples
(Shimokata, K, Totani, Y et al. 1988; Miedouge, M, Rouzaud, P et al. 1999; Alatas, F,
Alatas, O et al. 2001; Ghayumi, SM, Mehrabi, S et al. 2005). In the study from Alatas
et al (2001) the differentiation of mesothelioma from bronchial malignancies was
questioned in serum and pleural fluid. From a total of 74 effusions, 30/74 (40.5%) were
benign, 20/74 (27%) were mesothelioma and 24/74 (32.4%) were bronchial
malignancies. The sensitivities for CA15-3 alone were improved by combining the
123
markers CA15-3 and CYFRA 21-1 in both serum and pleural effusion (Alatas, F,
Alatas, O et al. 2001). Miedouge et al showed that the best combination of tumour
markers demonstrating a sensitivity of 88.4% and a specificity of 95% for a diagnosis of
malignancy was the panel of markers including CEA, CA15-3, CYFRA and NSE,
however, CA15-3 showed a sensitivity of 45.5% in only a small number of
mesothelioma cases (Miedouge, M, Rouzaud, P et al. 1999). The only study to examine
the specificity of CA15-3 for differential diagnosis of mesothelioma reported that 38%
of mesothelioma cases were CA15-3 positive and there was no significant difference in
CA15-3 levels in effusions related to mesothelioma or other malignancies metastatic to
the pleura (Creaney, J, Segal, A et al. 2008). However, this was a retrospective study
with a known number of mesothelioma, benign and reactive effusions. The current
study uses a blinded prospective consecutive patient cohort to study the utility of CA153 in malignant and non-malignant effusions.
Soluble mesothelin has recently been intensely investigated as a mesothelioma-specific
biomarker
(Creaney,
J
and
Robinson,
BW
2009).
Mesothelin
is
a
glycophosphatidylinositol-linked cell surface glycoprotein which can be released from
the cell surface (Hellstrom, I, Raycraft, J et al. 2006).
Serum levels of soluble
mesothelin are higher in patients with mesothelioma compared with other diseases of
the lung and pleura (Robinson, BW, Creaney, J et al. 2003). Similarly mesothelin
concentrations are elevated in effusions from patients with mesothelioma compared to
effusions of benign and other malignant causes (Scherpereel, A, Grigoriu, B et al. 2006;
Creaney, J, Yeoman, D et al. 2007; Davies, HE, Sadler, RS et al. 2009). Mesothelin
concentrations are approximately 10-fold higher in effusions compared to sera with less
than 2% of patients with benign effusions having mesothelin concentrations greater than
124
20nM (Creaney, Yeoman et al. 2007).
At this cut-off, over 2/3 of mesothelioma
patients and approximately 15% of patients with other malignancies have elevated
levels of mesothelin in their effusions (Creaney, J, Yeoman, D et al. 2007; Davies, HE,
Sadler, RS et al. 2009). It has been suggested that soluble mesothelin may provide an
adjunct tool to assist with mesothelioma diagnosis in effusions, however it must be
noted that at this sensitivity almost a third of mesothelioma cases are not detected.
Given that over 80% of mesothelioma tumours express MUC1, and that elevated levels
of soluble MUC1 have previously been reported in the effusions of mesothelioma
patients the current study was conducted to firstly determine the ability of CA15-3
levels in serous effusions to differentiate between malignant and non-malignant
effusions, and secondly to differentiate between mesothelioma and other metastatic
malignancies from the consecutive prospective cohort.
In other cancers improved sensitivity has been achieved through combining biomarkers
(Schorge, JO, Drake, RD et al. 2004; Zhang, Z, Yu, Y et al. 2007; Yurkovetsky, Z,
Skates, S et al. 2010).
Therefore the current study also determined whether the
diagnostic accuracy of the mesothelin assay for mesothelioma could be improved by
combining the results with those from the CA15-3 assay.
Previous studies have suggested a relationship between patient prognosis and serum
levels of both the CA15-3 (Bielsa, S, Esquerda, A et al. 2009) and mesothelin
biomarker (Cheng, WF, Huang, CY et al. 2009) in pleural effusions from metastatic
ovarian adenocarcinoma.
Therefore, as a secondary aim the prognostic value of
effusion CA15-3 was examined in patients with mesothelioma. Additionally, given that
125
soluble mesothelin is a promising biomarker for mesothelioma, this study compared the
sensitivity and specificity of the combination of the two assays.
The hypothesis is that increased supernatant CA15-3 levels are useful in the diagnosis
of mesothelioma and correlate with poor patient prognosis. An additional hypothesis
was that combining CA15-3 and mesothelin further increased the sensitivity for a
diagnosis of mesothelioma.
126
4.2
RESULTS
4.2.1
Patient characteristics
Patient effusion samples in excess of diagnostic requirement were prospectively
collected from PathWest Cytology Laboratory, Nedlands as described in Chapter 2.2
and Chapter 3.2.1. Of the 266 patients reported in Chapter 3.2.1, effusion supernatants
were collected from 252 and included in this current study; 202 pleural fluids, 40
peritoneal fluids and 10 pericardial fluids.
For primary analysis, samples were
classified according to the reported pathological diagnosis of the given sample and
patient characteristics are presented in Table 4.1. The pathological diagnosis of each
specimen as reported by PathWest was recorded as the Systematized Nomenclature of
Pathology code (SNOP). For this study the mesothelioma category included all samples
with a SNOP code of 9053. Epithelial malignancy was defined by the SNOP codes
8003, 8006, 8143 and included metastatic adenocarcinomas from, breast, lung, thyroid
and ovarian primaries and metastatic squamous and small cell anaplastic carcinoma.
Non-epithelial malignancies included acute myeloid leukaemia, lymphoma, B cell
lymphoma, melanoma and Non-Hodgkins lymphoma (SNOP 9593, 9733, 9803).
Benign effusions included those cases with non-malignant cytological features
including lymphocytosis, eosinophilia, inflammation and reactive mesothelial
proliferation. Cases defined as atypical/equivocal included those cases containing small
numbers of highly atypical cells of uncertain origin with features insufficient for a
definite diagnosis of malignancy. From the 252 effusion supernatant samples, 153 were
received from men and 99 received from women, with a mean age of 68.6 years (range
19 - 94).
127
Table: 4.1: Patient demographics for effusion supernatant samples collected.
Interim
Pathology
Diagnosis
Mesothelioma
Age Median
(Range)
Sex
Pleural
Fluid
Peritoneal Pericardial
Fluid
Fluid
70.6(55-90)
F
8
-
-
69.6(50-94)
M
34
1
2
Epithelial
70.8(38-96)
F
26
14
3
Malignancy
74(48-93)
M
31
4
1
Non-Epithelial
78(75-81)
F
2
1
-
Malignancy
56.6(19-72)
M
2
-
1
Atyp/Equivocal
71(69-73)
F
3
1
-
76(61-86)
M
5
65.9(23-88)
F
33
8
-
66.2(26-93)
M
58
11
3
113
202
40
10
252
Benign
Total
4.2.2
-
Total
45
79
6
9
Sensitivity and Specificity of CA15-3 in effusion supernatant samples
CA15-3 concentrations were determined in the effusion supernatants from 252 cases
using the CANAg CA15-3 enzyme immunoassay kit (Chapter 2.4) (Figure 4.1)
Analysis of peritoneal and pericardial effusions did not show any significant difference
in interpretation of the CA15-3 levels compared to pleural effusions as a group and
therefore it was decided to analyse the entire effusion set as a whole.
The
manufacturer’s cut-off values for elevated levels were used for this study without
modification.
128
CA15-3 concentrations ranged from below the limit of detection to 2413kU/L. The
effusions from patients with mesothelioma had significantly higher concentrations of
CA15-3 than those patients with benign/reactive effusions (p = < 0.0001), non-epithelial
malignancies (p = 0.02) and atypical/equivocal cases (p = 0.03).
Effusions from
patients with metastatic epithelial malignancies had significantly higher levels of CA153 than patients with benign effusions (p = <0.0001) or mesothelioma (p=0.0009). At
the manufacturer’s defined cut-off of 36kU/L, 58 of the 79 (73%) epithelial malignancy
effusions showed CA15-3 levels above reference range
and 25 of the 45 (56%)
mesothelioma effusions were CA15-3 positive (Table 4.2).
Follow-up was performed on all patients (average 6-18 months) as described Chapter
3.2.4, and 16 cases were identified from whom the study sample was either reported as
atypical or benign but was identified as being either pre-diagnostic or from people with
an established malignancy. Of the three cases with a sample reported as atypical or
equivocal none had elevated CA15-3. Three of the 13 cases with a benign sample were
CA15-3 positive.
After follow up there were 52 mesothelioma cases, 85 epithelial malignancies, and nine
non-epithelial malignancies, 6 atypical/equivocal and 100 benign cases (Table 4.3).
CA15-3 had a sensitivity of 50% and a specificity of 96% for a diagnosis of
mesothelioma relative to all benign cases.
129
Figure 4.1: CA15-3 concentrations determined in effusions from individual patients.
Effusions were grouped into mesothelioma patients, patients with metastatic epithelial
malignancies, non-epithelial malignancies, those cases reported as atypical/equivocal and
benign effusions as reported in the Pathology report for each sample. Horizontal dashed line
indicate the manufacturer’s defined upper limit of normal in serum (36 kU/L). The black
points represent 16 cases that were diagnosed as a non malignant effusion but were
subsequently reported as malignant on follow up. (* p<0.05; ** p<0.01; *** p<0.001).
130
Table 4.2: CA15-3 concentrations in effusions from the Interim Diagnosis Category
Interim Diagnosis
Category
n
Mean
±SD
Median
(IQR 25-75)
Positive
CA15-3
a
(percentage)
25 (55.5%)
Significance
P valuesb
Mesothelioma
45
98.72
±208.3
46.19
(12.4 – 96.6)
Epithelial
Malignancy
79
348.2
±900.3
107.1
(29.7– 250.7)
58 (73.4%)
P < 0.0009
Non-epithelial
Malignancy
6
10.9
± 5.8
11.5
(4.7 – 16.5)
0 (0%)
P < 0.0186
Atypical/
Equivocal
9
38.2
±79.2
11.5
(7.6 – 19.2)
1(11.1%)
P < 0.0309
Benign
113
9.3
±14.5
5.4
(2.8 – 10.2)
3(2.65%)
P < 0.0001
REF
a
Positive CA15-3 defined as greater than 36 kU/L.b
b
Differences between groups of patients were assessed by Student’s t test after
transforming CA15-3 values to the log scale.
Table 4.3: CA15-3 concentrations in effusions from the Final diagnosis category.
Final Diagnosis
Category
n
Mesothelioma
52
Mean
±SD
Median
(IQR 25-75)
98.98
38.1
±195.4
(11.7-82.5)
Epithelial
85 324.5
86.18
Malignancy
±871.9
(21.7-243.9)
Non-epithelial
9
9
7.4
Malignancy
± 5.59
(4.0-12.3)
Atypical/
6
50.6
11.7
Equivocal
±97.3
(7.6-132.5)
Benign
100 7.67
5.4
±7.8
(2.6-9.8)
a
Positive CA15-3 defined as greater than 36 kU/L.b
b
Positive
CA15-3
a
(percentage)
26 (50%)
Significance
P valuesb
58 (68.2%)
P < 0.0014
0 (0%)
P < 0.0019
1c(16%)
P < 0.2157
3d(3%)
P < 0.0001
REF
Differences between groups of patients were assessed by Student’s t test after
transforming CA15-3 values to the log scale.
c
Patient did not develop a malignancy at the close of the study.
d
2/3 patients developed mesothelioma, 1 patient had a prediagnosed epithelial
malignancy with a negative effusion and an increased CA15-3 level.
131
Analysis of ROC curves generated from this data set showed that CA15-3 levels in
effusions could differentiate mesothelioma cases from cases of benign effusion (AUC =
0.89 (95% CI 0.84 to 0.94) p = 0.0001).
differentiate between
Similarly, CA15-3 levels were able to
cases of non-mesothelioma epithelial malignancy and
mesothelioma (AUC = 0.64 (95% CI 0.54 to 0.74) p = 0.006) (Figure 4.2).
4.2.2.1
Concordance of MUC1 cell surface expression and effusion CA15-3
levels
The level of cell surface expression of MUC1 was correlated to the effusion CA15-3
levels from 252 patients. From a final diagnosis of 52 cases reported as mesothelioma
50% (26/52) of these demonstrated a positive CA15-3 level while 85% (44/52)
demonstrated cell surface expression of MUC1 with the E29 clone. Of the 85 epithelial
malignancies expressing CA15-3, excluding mesothelioma 81% (69/85) of these
expressed cell surface MUC1 and 68.2% (58/85) had elevated CA15-3. While only
11% (1/9) of the non-epithelial malignancies showed cell surface expression of MUC1
with no CA15-3 levels detected.
4.2.3
Sensitivity and specificity of mesothelin in effusion supernatant
samples
Mesothelin concentrations were determined in the effusion supernatants from 252
samples, and results presented relative to the pathology diagnosis of a given sample.
Mesothelin levels ranged from below the limit of detection to 2366 nmol/L.
Significantly higher mesothelin levels were observed in epithelial malignancy than
benign fluids (p = < 0.0001).
132
Figure 4.2: Receiver operating characteristic (ROC) analysis performed to determine the
ability of CA15-3 concentrations to distinguish between a) mesothelioma and benign cases,
and b) mesothelioma and all non-mesothelioma malignant cases based on the interim
diagnosis. Area under the ROC curve were a) 0.89 (95% CI 0.84 to 0.94) and b) 0.64 (95% CI
0.55 to 0.74).
ROC for CA15-3 levels
Sensitivity
1.00
MM vs All non-MM
malignant
MM vs All Benign
0.75
0.50
0.25
0.00
0.00
0.25
0.50
0.75
1 - Specificity
1.00
1.25
133
Effusion supernatants from patients with mesothelioma also had significantly higher
concentrations of mesothelin than patients with benign effusions (p = <0.0001),
epithelial malignancy (p = <0.0001), non-epithelial malignancy (p = <0.0001) and
atypical/equivocal effusions (p = <0.02) (Figure 4.3) (Table 4.4).
Table 4.4: Mesothelin levels from interim diagnosis category.
Interim Pathology n
Diagnosis
Mesothelioma
Mean±SD
Median
(IQR 25-75)
164.1
± 41.3
445.1
(18.7 – 90.2)
Epithelial
79 20.3 ± 64.4
5.4
Malignancy
(3.1 – 16.0)
Non-epithelial
6
3.3 ± 2.1
2.8
Malignancy
(1.4 – 5.6)
Atypical/Equivocal 9
87.5 ± 221.8 12.8
(5.0– 26.6)
Benign
113 6.1 ± 8.1
3.6
(1.9 – 6.8)
α
Positive mesothelin levels >20nmol/L.
b
45
Positive
Significance
α
mesothelin P valuesb
34 (75.5%)
REF
17 (21.5%)
P < 0.0001
0 (0%)
P < 0.0002
4 (44.4%)
P < 0.02
7 (6.2%)
P < 0.0001
Differences between groups of patients were assessed by Student’s t test after
transforming mesothelin values to the log scale.
Sensitivity and specificity data were determined in the context of the final diagnosis
based upon patient follow up (Table 4.5). Samples from 18 of 85 (21%) patients with
epithelial malignancy and 34 of 52 (65%) patients with mesothelioma had mesothelin
levels above the reference range.
No samples from patients with non-epithelial
malignancies were positive for mesothelin (Table 4.4). At a threshold of 20nmol/L
(Creaney, J, Yeoman, D et al. 2007) mesothelin levels in this study demonstrated a
sensitivity of 69% and a specificity of 92%, for a final diagnosis of mesothelioma,
relative to all other benign cases (ie benign and atypical/equivocal) (Table 4.6).
134
Figure 4.3: Mesothelin concentrations determined in effusions from individual patients.
Effusions were grouped into MM patients, patients with metastatic epithelial malignancies,
non-epithelial malignancies, those cases reported as atypical/equivocal and benign
effusions, as reported in the Pathology report for each sample. Horizontal dashed line
indicate the defined upper limit of normal in supernatant (20nmol/L) (Creaney et al. 2007).
The black points represent 16 cases that were diagnosed as a non malignant effusion but
were subsequently reported as malignant on follow up. (* p<0.05; ** p<0.01; *** p<0.001).
135
Analysis of ROC curves generated from this data set showed that mesothelin levels in
effusions could differentiate mesothelioma cases from cases of benign effusion (AUC
0.933 (95% CI 0.89 to 0.97) p = < 0.0001) and also differentiate between cases of non
mesothelioma epithelial malignancies and mesothelioma (AUC 0.8465 (95% CI 0.788
to 0.911) p = < 0.0001) (Figure 4.4).
Table 4.5: Mesothelin levels from the final diagnosis category
Final Diagnosis
n
Mean±SD
Median
(IQR 25-75)
Positive
Significance
α
mesothelin P valuesb
Mesothelioma
52
144.3 ± 416.6
36 (69.2%)
REF
Epithelial
Malignancy
Non-epithelial
Malignancy
Atypical/Equivocal
85
19.6 ± 62.1
18 (21%)
P < 0.0001
9
3.7 ± 2.0
0 (0%)
P < 0.0001
6
125 ± 271.4
3c (50%)
P < 0.188
Benign
100 5.3 ± 6.6
40.2
(14.87-82.7)
5.7
(3.1 – 15.7)
3.4
(1.9 – 5.5)
17.3
(2.7-354.9)
3.2
(1.8 – 6.4)
5d (5%)
P < 0.0001
α
Positive mesothelin levels >20nmol/L.
b
Differences between groups of patients were assessed by Student’s t test after
transforming mesothelin values to the log scale.
c
2/3 patients developed mesothelioma 1 with sarcomatoid variant. 1 patient did not
develop malignancy.
d
2/5 patients had a diagnosis of epithelioid malignancy with a negative effusion, while
the remaining 3 patients did not develop any malignancy.
Mesothelin concentrations were determined on the effusion supernatant in a subset of
252 patients. Sensitivity and specificity data was determined on the context of the final
diagnosis based on patient follow up excluding all non mesothelioma malignancies;
44/158 cases were mesothelin positive.
136
Figure 4.4: Receiver operating characteristic (ROC) analysis performed to determine the
ability of mesothelin concentrations to distinguish between a) mesothelioma and benign
effusions, and b) mesothelioma and all non-mesothelioma malignant cases. Area under
the curve were a) 0.93(95% CI 0.89 to 0.97); and b) 0.8465 (95% CI 0.788 to 0.911).
Sensitivity
ROC curve for Mesothelin levels
1.00
MM vs All Non-MM
Malignant (b)
0.75
MM vs All benign (a)
0.50
0.25
0.00
0.00
0.25
0.50
0.75
1 - Specificity
1.00
137
The overall sensitivity was 69% with specificity of 92% for a final diagnosis of
mesothelioma relative to all benign cases (ie, benign and atypical/equivocal).
4.2.4
Correlation of CA15-3 and mesothelin levels in effusions
To examine the relationship between CA15-3 and soluble mesothelin concentrations in
mesothelioma samples a bivariant scatter plot was generated (Figure 4.5). There was a
significant correlation between CA15-3 and mesothelin concentrations in effusion
samples from the 52 patients with a final diagnosis of mesothelioma (rSpearman = 0.56; p
= 0.0001, 95% CI 0.33 to 0.72) (Figure 4.5). Out of the 52 mesothelioma patients, 22
showed high levels for both mesothelin and CA15-3, 14 cases showed a high level for
mesothelin alone, 4 showed a high level for CA15-3 alone and 12 cases showed a low
level for both mesothelin and CA15-3.
4.2.5
Comparing and combining the performance of CA15-3 and mesothelin
The CA15-3 and mesothelin results were combined in a logistic regression model,
adjusting for gender, following standardizing the logarithmically transformed biomarker
levels (Methods Section 2.16). The relative diagnostic power of the individual markers
and the combined marker for differentiating (a) benign cases from all malignancies and
(b) mesothelioma from all other cases were compared. Results from ROC analysis are
presented in Figure 4.6 and 4.7.
To distinguish all malignancies including
mesothelioma from benign cases, the AUC (±SE) for CA15-3 was 0.88±0.02 and for
mesothelin was 0.73±0.032 which were significantly different from each other
(p<0.001) indicating the superior diagnostic value of CA15-3 for differentiating cancer
from benign cases (Figure 4.6A).
138
E ffusio n M e so the lin (nm o l/L )
Figure 4.5: Spearman correlation of CA15-3 and mesothelin values in effusion
samples from patients with mesothelioma. Dashed lines represent upper limit of
normal for each marker.
10000
1000
100
10
Spearmans r = 0.56
(p = < 0.0001)
1
0.1
1
10
100
1000
Effusion CA15-3 (kU/L)
10000
139
Figure 4.6: A: Receiver operating characteristic curves for all epithelial
malignancies. The AUC is 0.88 ± 0.02 for CA15-3 and 0.73 ± 0.32 for mesothelin.
B: The AUC for the combine marker was not significantly different from CA15-3
alone.
A
B
Combined marker
140
Figure 4.7: A: Receiver operating characteristic curves for differentiating
mesothelioma from all other samples. The AUC for mesothelin is 0.88 ± 0.02 for
mesothelioma and 0.613 ± 0.03 for CA15-3. B: The AUC for the combined
marker was 0.91 ± 0.023 which is not significantly different from the AUC for
mesothelin alone.
A
B
Combined marker
141
For differentiation of mesothelioma from all other samples in this study the AUC for
CA15-3 was 0.61 ± 0.37 and for mesothelin was 0.88 ± 0.26 (Figure 4.7A). The AUC
for the combined marker was 0.91±0.023, which was not significantly better than
mesothelin alone (Figure 4.7B and Table 4.6).
Table 4.6: Combined performance of CA15-3 and mesothelin.
Assays
MM vs Benign
All Malignant MM vs other
vs Benign
malignant
CA15-3
Mesothelin
CA15-3 + Mesothelin
0.89
0.93
0.94
0.88
0.73
0.89
4.2.6
0.61
0.88
0.91
Relationship between the CA15-3 levels in effusions with survival in
patients with mesothelioma
To determine the prognostic value of effusion CA15-3 concentrations, patients with
mesothelioma were categorised according to effusion CA15-3 levels above or below the
median for the group (median = 47.8kU/L). Survival was calculated from the time of
sample collection to death (n = 39) or until censoring at last follow-up for patients who
were still alive (n = 13). Median survival for 21 mesothelioma patients with high
CA15-3 was 10 months, not significantly different from median survival of 9 months
for the low CA15-3 group (p = 0.734) using the log-rank test (Figure 4.8).
No
significant difference in the survival of patients with mesothelioma relative to CA15-3
concentrations was demonstrated when data were analysed in tertiles or relative to 36
kU/L threshold (data not shown).
142
Percent survival
Figure 4.8: Relationship between CA15-3 levels in effusions and survival of patients
with mesothelioma. Patients were dichotomized above and below the median CA15-3
level for the group (i.e. 48 kU/L). Time in months from date of sample until death or
censor.
110
100
90
80
70
60
50
40
30
20
10
0
Below median
Above median
p = 0.7354
0
10
20
30
40
Months
50
60
143
4.3
DISCUSSION
An early diagnosis is one of the key factors to achieving appropriate management of
effusion, prognostication, legal compensation and expert opinion in mesothelioma. If a
tumour marker could be identified with a high sensitivity and specificity for
mesothelioma, this could improve the speed and accuracy of the diagnosis. As yet there
are no sensitive tumour biomarkers available other than for soluble mesothelin,
currently the only test considered as the reference serum biomarker for mesothelioma.
The likelihood of serum mesothelin being utilized as a stand-alone diagnostic test is
minimal, but the assay could play a vital role in combination with other diagnostic tools.
Although previous studies have described the use of biomarkers for the differential
diagnosis of pleural effusion malignancies, to date, none have entered routine clinical
diagnostic use.
The purpose of this study was to firstly confirm the ability of CA15-3 levels in effusions
to differentiate between malignant and non-malignant samples. Results from this study
demonstrate that from 252 effusion samples, 87 were CA15-3 positive and 84 of these
cases actually turned out to be malignant, resulting in a specificity of 97% with a
sensitivity of 57% (including all cancers, mesothelioma, epithelial and non-epithelial).
These results are similar to those observed from other studies (Miedouge, M, Rouzaud,
P et al. 1999; Alatas, F, Alatas, O et al. 2001; Ghayumi, SM, Mehrabi, S et al. 2005).
This sensitivity is similar to reported sensitivities for differential diagnosis of malignant
from benign effusions by cytological examination (Ghayumi, SM, Mehrabi, S et al.
2005). Therefore, possibly as is currently the practise for effusions to be biochemically
analysed to determine whether they are exudative or transudative in nature, the
144
inclusion of CA15-3 during the diagnostic work-up could add weight to the cytological
diagnosis in this context.
However, as previously reported, CA15-3 concentrations do not differentiate
mesothelioma from other epithelial malignancies. Indeed, for the sample set under
study CA15-3 levels were significantly lower for the group of mesothelioma patients
relative to the patients with other epithelial malignancies. The reason why there is a
difference in the levels of soluble MUC1 between the two groups is not clear. A
correlation of the individual patients MUC1 tissue expression and CA15-3 levels clearly
shows that whilst the majority of epithelial tumours that express surface MUC1 have
elevated CA15-3 in the effusion, in approximately 40% of the mesothelioma cases
CA15-3 was not elevated despite strong cell surface MUC1 staining of the
mesothelioma tumour cells. This suggests that perhaps different mechanisms are in
operation between mesothelioma and epithelial tumours, possibly as a result of a
different mechanism of MUC1 cleavage, or due to a variation in MUC1 splice forms.
Pleural effusions from patients with mesothelioma have significantly higher
concentrations of soluble mesothelin compared to patients with non-mesothelioma
malignancies (Creaney, J, Yeoman, D et al. 2007). In this current study, with a cutoff
of 20nmol/L, a sensitivity of 65.3% and specificity of 95% was observed for a diagnosis
of mesothelioma similar to previously observed (Creaney, J, Yeoman, D et al. 2007;
Grigoriu, BD, Chahine, B et al. 2009) suggesting that this tumour biomarker may be
valuable in differentiating mesothelioma from benign effusions.
145
This is the first study to demonstrate a correlation between mesothelin and CA15-3 in
samples from mesothelioma patients. The reason for the common expression profile of
these biomarkers in mesothelioma patients is not known, but it is possible that as both
are cell surface adhesion molecules, capable of being shed from the cell surface that the
same enzyme(s) are responsible for cleavage of both molecules.
Alternatively,
expression of these markers could be a reflection of the degree of differentiation of the
mesothelioma tumours and therefore the most epithelial-like tumours express both
molecules. Of note there were only two cases confirmed on biopsy follow-up as
sarcomatoid variant mesothelioma and both demonstrated CA15-3 levels below the
threshold.
By combining the tumour markers our aim was to improve diagnostic accuracy and
although we did see this with both markers used individually, unfortunately combining
the two tumour markers did not improve the diagnostic accuracy above mesothelin
alone. This observation is similar to previous studies in which others have tried to
combine other markers with mesothelin in serum as a clinical adjunct to detecting
recurrent ovarian carcinoma and mesothelioma (Hellstrom, I, Raycraft, J et al. 2006;
Creaney, J, van Bruggen, I et al. 2007; Creaney, J, Yeoman, D et al. 2007; Grigoriu,
BD, Scherpereel, A et al. 2007; Creaney, J, Yeoman, D et al. 2008). This study
however does suggest that both CA15-3 and mesothelin may be valuable to aid in the
differential diagnosis of mesothelioma, especially in those cases where the cytological
morphology between mesothelioma and marked reactive mesothelial proliferation is
difficult to establish and when the immunohistochemical stains are inconclusive.
146
High serum levels of CA15-3 from women with breast cancer are a potential prognostic
indicator, correlating with preoperative tumour size and the presence of lymph node
metastases of breast cancer (Martin, A, Corte, MD et al. 2006; Park, BW, Oh, JW et al.
2008). In the present study an increased concentration of CA15-3 in the effusion was
not associated with survival in mesothelioma patients. This difference likely reflects the
different tumour settings early breast cancer is curable when no micrometastases are
present, or when small volume micrometastases can be cured by adjuvant therapy. A
high CA15-3 in this setting may purely reflect the inability of surgery and adjuvant
therapy to cure a greater volume of systemic disease. In contrast, in mesothelioma,
therapy is palliative rather than potentially curative, and the concentration of CA15-3 in
effusions may not reflect the systemic burden of disease and may not reflect tumour size
which is often quite bulky in mesothelioma patients.
4.4
CHAPTER SUMMARY
An early non-invasive method of diagnosis for mesothelioma is still elusive, and
although several potential biomarkers have been described, mesothelin is currently the
only tumour biomarker utilized in a clinical diagnostic setting. In this study we have
demonstrated the high specificity of CA15-3 as a differentiating tumour marker between
benign effusions and epithelioid malignancies. The diagnostic differentiation between
reactive mesothelial cells and mesothelioma is difficult and perhaps in those cases
where immunohistochemistry is inconclusive, determining the CA15-3 level may assist
clinicians in deciding when to pursue a more invasive approach to definitive diagnosis,
and when close observation may be a reasonable strategy.
147
Additionally, this is the first study to demonstrate a strong correlation between
mesothelin and CA15-3 levels in effusion supernatant samples from patients with
mesothelioma. When used as a combination marker, a high level of mesothelin in the
fluid strongly suggests a diagnosis of malignancy, in particular mesothelioma.
In
comparison, a high level of CA15-3 in effusion samples correlated with a higher
probability of an epithelial malignancy. Given that effusion samples are often drained
for palliation and diagnostic purposes it may also be valuable to perform this simple
assay on inconclusive cases to aid in the diagnosis of malignancy, leading to earlier
appropriate management of the patient.
148
CHAPTER 5
EFFECT OF MUC1 ON MALIGNANT MESOTHELIOMA CELL
FUNCTION AND CHARACTERISTICS
149
5.1
INTRODUCTION
The exact function of MUC1 in tumour progression has only been partially elucidated in
other malignancies including breast, prostate, lung and gastrointestinal cancer. An
understanding of the structure of MUC1 may help generate hypotheses about its
function in mesothelioma.
MUC1 has been shown to undergo post translational
proteolytic cleavage at the SEA domain generating two subunits that form a stable
heterodimer on the cells surface (Ligtenberg, MJ, Kruijshaar, L et al. 1992; Macao, B,
Johansson, DG et al. 2006). The larger subunit contains the extracellular domain,
including a variable number of tandem repeats modified by O-glycans, which have been
shown to play an important role in cell adhesion (Wesseling, J, van der Valk, SW et al.
1996). The smaller subunit contains a short extra cellular domain, a transmembrane
domain and a cytoplasmic tail. MUC1 can bind to the epidermal growth factor receptor
(EGFR) (Li, Y, Ren, J et al. 2001), c-Src (Li, Y, Bharti, A et al. 1998) and β-catenin
(Schroeder, JA, Adriance, MC et al. 2003; Huang, L, Chen, D et al. 2005) through the
cytoplasmic domain, suggesting a potential role for MUC1 in cell signalling and cell
growth related pathways.
Several studies have investigated the effects of silencing MUC1 expression in a
selection of malignant cell lines using RNA interference (siRNA) or small hairpin RNA
(shRNA) to alter the regulation of malignant phenotypes. This technology has been a
powerful tool to suppress the expression of other specific gene products that have been
shown to play important functional roles in tumour progression.
MUC1 silencing has resulted in decreased cell proliferation in a range of malignant cell
lines in vitro, including epithelial tumours such as oral epidermoid (Li, Y, Liu, D et al.
150
2003), and in pancreatic cancers (Tsutsumida, H, Swanson, BJ et al. 2006; Yuan, Z,
Liu, X et al. 2009; Xu, H, Inagaki, Y et al. 2011a; Xu, H, Inagaki, Y et al. 2011b) and
from haematological malignancies (Kawano, T, Ahmad, R et al. 2008; Hasegawa, H,
Komoda, M et al. 2011) Decreased growth of tumours from cell lines with reduced
MUC1 expression was also observed in murine model systems (Tsutsumida, H,
Swanson, BJ et al. 2006; Yuan, Z, Liu, X et al. 2009). However, there is also some
conflicting data regarding the effect of reduced MUC1 on proliferation, as in one study
where MUC1 expression was reduced in two independent breast cancer cell lines, one
line (MDA-MB-468) had reduced proliferation whilst the other (BT-20) showed an
increased rate of proliferation (Hattrup, CL and Gendler, SJ 2006).
Furthermore,
reduced proliferation was only observed in the T cell line, Jurkat, in response to CD3
activation and not under normal conditions following reduced MUC1 expression,
(Mukherjee, P, Tinder, TL et al. 2005).
In model systems reduced MUC1 has correlated with a decrease in cell invasion in
pancreatic (Tsutsumida, H, Swanson, BJ et al. 2006; Xu, H, Inagaki, Y et al. 2011a; Xu,
H, Inagaki, Y et al. 2011b) and breast cancer cell lines (Hattrup, CL and Gendler, SJ
2006). In an orthotopic pancreatic cancer model not only was the invasiveness of the
tumours with decreased MUC1 expression reduced but a reduction in the metastatic
capacity was also observed (Tsutsumida, H, Swanson, BJ et al. 2006). However, in two
other model systems an effect of reduced MUC1 expression on cell invasiveness was
only apparent after cell stimulation. Firstly, MUC1 knockdown significantly reduced
renal cell cancer invasion and migration but only under hypoxic conditions (Aubert, S,
Fauquette, V et al. 2009), and secondly, virus induced cell invasiveness was blocked
following MUC1 knockdown (Kondo, S, Yoshizaki, T et al. 2007). The effect of
151
reduced MUC1 expression on cell invasiveness may in part be mediated by increased Ecadherin expression, and E-cadherin/B-catenin complex expression (Xu, H, Inagaki, Y
et al. 2011b).
These contradictory findings highlight the possibility that MUC1 may not always play a
specific role in the pathogenesis of the disease process.
Although several molecular tools have been established to investigate the function of
MUC1 in a range of tumour types, there are no previous studies investigating the effects
of MUC1 on mesothelioma cell function and characteristics.
The aim of this chapter is to investigate the functional role of MUC1 in human
mesothelioma cell lines. Initially, levels of MUC1 expression in a series of human
mesothelioma cells lines were examined in order to understand the range and variability
of MUC1 expression in this tumour type, and to select appropriate cell lines with high,
medium and low expression levels of MUC1 expression for additional study.
Subsequent experiments used shRNA to suppress MUC1 in mesothelioma cell lines
with varied levels of MUC1 expression. Cell lines were stably transfected with four
shMUC1 specific shRNA plasmids (SA Biosciences) and a control shRNA scrambled
sequence (See Chapter 2.14). The stably transfected cell lines were examined for levels
of MUC1 expression and the effects of shRNA MUC1 knockdown on cell proliferation,
cell migration and tumorigenicity in vitro.
152
5.2
RESULTS
5.2.1
Characterising MUC1 in mesothelioma cell lines
To further examine the hypothesis that MUC1 plays an important functional role in the
malignant behaviour of mesothelioma, the innate levels of MUC1 expression were
examined in a panel of mesothelioma cell lines established from patient pleural
effusions. Samples were all from males, with a median age of 60 (range 23 – 94 years)
and were predominantly of an epithelial histology (Table 5.1).
Mesothelioma cells were compared to normal mesothelial cells in culture from
pericardial fluid and also to a small panel of epithelial cancer cell line controls. HELA,
A549 and MCF7 cell lines have been utilised in several publications when investigating
the role MUC1 plays in cervical, lung and breast carcinomas respectively (Obermair, A,
Schmid, BC et al. 2001; Koga, T, Kuwahara, I et al. 2007; Kuwahara, I, Lillehoj, EP et
al. 2007; Engelmann, K, Shen, H et al. 2008). The glioblastoma cell line U87MG was
used as a negative control as it has previously been shown not to express MUC1 (Kalra,
AV and Campbell, RB 2007). MCF7 cells were used as a negative control for cell
migration experiments (Chernov, AV, Baranovskaya, S et al. 2010).
5.2.1.1
mRNA levels of MUC1 in mesothelioma cell lines
Total RNA was extracted from 16 established mesothelioma cell lines, a lung
adenocarcinoma (A549) and a cervical cancer (HELA) cell line along with two nonmalignant mesothelial samples (PC163 and PC159), and the relative expression of full
length archetypical MUC1 was determined by quantitative real time PCR as described
(Chapter 2.7).
153
Full length MUC1 mRNA was detected in all cell lines examined including normal,
non-malignant mesothelial cells. There was a 5-fold range in the expression of MUC1
in mesothelioma cell lines. Two of the cell lines expressed significantly less MUC1
than normal mesothelial cells. Six of the lines had a similar level of MUC1 to normal
mesothelial cells. Eight lines expressed MUC1 at higher levels than the normal cells,
comparable to the relative amount expressed by the epithelial cervical and lung cancer
cell lines (Table 5.1).
5.2.1.2
Protein expression of MUC1 on mesothelioma cell lines using
immunohistochemistry
Protein expression of MUC1 was examined in 14 mesothelioma cell lines, two benign
mesothelial cell cultures and three control cell lines, using immunohistochemistry on
cells grown on chamber slides. Five anti-MUC1 antibodies (E29, SM3, VU4H5, BC2
and Mc5) were selected for this study. These were directed against peptides within the
tandem repeat region of MUC1. In addition, two antibodies (N19 and C20) directed
against the N terminus and C terminus of the MUC1 protein core respectively were used
in this study.
Staining of MUC1 positive cells grown in tissue culture chamber slides were diffusely
cytoplasmic (Figure 5.1), which is different from the predominantly membranous
staining observed in mesothelioma cells from pleural effusions prepared in thrombin
clot for paraffin embedding and when examined in sections (Chapter 3, Figure 3.2.4).
154
Table 5.1: Characteristics of cell lines used in this study and relative MUC1 mRNA
levels.
MM CELL
LINES
Sex
Age
MM
Histology
Survival
(months)
MUC1
mRNA
ST45
M
52
Epithelioid
16
10.6 ±0.1
VGE62
M
74
Epithelioid
14
9.7 ± 0.7
HEW215
M
63
Not specified
22
9.3 ± 0.5
OLD1612
M
58
Biphasic
2
9.1 ± 0.3
LO68
M
57
Not specified
7
8.6 ± 0.1
DI24A
M
68
Not specified
10
8.4 ±0.3
HO275
M
94
Epithelioid
13
8.1 ± 0.6
ONE58
M
58
Not specified
6
8.1 ±0.00
JU77
M
62
Not specified
18
7.9 ± 0.1
GAY2911
M
70
Epithelioid
15
7.9 ± 0.7
NO36
M
36
Not specified
2
7.8 ± 0.6
JO38
M
48
Biphasic
2
6.5 ±0.2
PA45
M
69
Not specified
10
6.4 ±0.5
DI24B
M
68
Not specified
10
5.9 ± 1.3
STY51
M
23
Not specified
23
3.8 ± 0.1
VAN148
M
53
Epithelioid
21
1.6 ±2.3
Normal
Mesothelial
PC163
PC159
F
M
77
63
na
na
na
na
PC166
PC168
GIB406
Control
Cell lines
A549
M
M
M
74
50
52
na
na
na
na
na
na
M
58
na
HELA
F
31
U87MG
F
44
Adenocarcinoma
Lung
Adenocarcinoma
Cervical
Glioblastoma
MCF7
F
69
Adenocarcinoma
Breast
na
na
na
Comment
MUC1
mRNA
6 ± 0.28
6.45
±0.57
nd
nd
nd
MUC1
mRNA
9.85
±0.14
8.75
±0.71
No MUC 1
expression
No
migratory
phenotype
155
Representative examples of MUC1 staining and scoring with three different antibodies is
shown in Figure 5.1. Staining intensity was assessed semi-quantitatively using structured
criteria (Section 2.3.1) and results presented in Table 5.2.
Table 5.2:
Immunohistochemistry scores of tested anti-MUC1 antibodies in 14
mesothelioma, lung carcinoma (A549*), cervical cancer (HELA*), glioblastoma
(U87MG* negative control) and benign mesothelial cells. Staining was performed on
cells grown in 8 well chamber slides and staining intensity was scored based on a
sliding scale of 0 (no stain); 10—20% (1+), 21—50% (2+) and 51—100% (3+) of cells
staining.
Cell Line
EPITOPE
E29
PDTRP
MC5
DTRPAP
ONE
3+
3+
1+
-
-
-
-
JU77
3+
2+
2+
1+
2+
-
-
GAY
3+
3+
2+
2+
1+
-
-
OLD
3+
3+
3+
2+
-
1+
-
DI24B
3+
1+
1+
-
-
-
-
ST45
3+
3+
2+
1+
2+
-
-
LO68
3+
2+
2+
-
-
-
-
VGE62
3+
3+
3+
1+
2+
-
-
NO36
3+
3+
2+
1+
1+
1+
-
STY51
3+
3+
2+
1+
1+
-
-
JO38
3+
3+
1+
1+
-
-
-
HEW215
3+
2+
1+
1+
1+
1+
-
PA45
3+
2+
1+
1+
1+
1+
-
VAN
3+
2+
2+
-
-
-
-
PC166
2+
3+
1+
1+
-
-
-
PC168
-
2+
2+
-
1+
-
-
HELA*
3+
3+
2+
-
1+
-
-
A549*
3+
2+
1+
1+
-
2+
-
-
1+
-
-
-
-
-
U87MG*
VU4H5
SM3
BC2
APDTRPAP APDTRP APDTR
N19
N-TERM
C20
C-TERM
156
Figure 5.1: Innate levels of MUC1 expression was performed on all cell lines in 8
well chamber slides. This image represents the JU77 MM cell line showing the
glycosylation profiles of 4 different monoclonal antibodies; E29, Mc5, SM3 and a
negative control incubated with no primary antibody.
157
In summary, all mesothelioma cells examined stained strongly with the E29 mAb. All
mesothelioma cell lines stained with the Mc5 and VU4H5 mAbs but intensities of staining
ranged from weak to strong with 8 lines strongly staining with Mc5 and only 2 with
VU4H5. None of the mesothelioma cell lines stained strongly with SM3 or BC2, and
indeed 4 and 6 cell lines respectively did not stain with these antibodies. Only weak
staining was observed with the N19 antibody and no staining was observed with the C20
antibody even in control lines.
5.2.1.3
Protein expression of MUC1 on normal mesothelial cell lines using
immunohistochemistry
Normal mesothelial cells, propagated from pericardial effusions and grown in chamber
slides did not stain as strongly as mesothelioma cell lines with the E29 mAb, but did
show diffuse cytoplasmic staining in a similar pattern to that observed with
mesothelioma cell lines when stained with other anti-MUC1 antibodies directed to the
tandem repeat region (Figure 5.2 A and B). This result was in contrast to what was
observed in Chapter 3, where few benign reactive mesothelial cells were observed to
stain with the panel of anti-MUC1 antibodies (Figure 3.5E). To address whether this
difference in staining was due to the different methods in which cells were processed a
normal mesothelial cell GIB406 sample was divided in two and half was processed as
per Chapter 2.1.5 such that a thrombin clot was made and embedded in paraffin, and
sectioned onto glass slides, while the other half was grown in chamber wells for 24
hours before being directly fixed in situ (Chapter 2.9). Both samples were processed in
parallel and results are shown in (Figure 5.2 C and D), suggesting that the differences
observed may be attributable to processing methods.
158
Figure 5.2: Normal mesothelial cells stained with anti-MUC1 monoclonal antibodies in
chamber well slides and by thrombin clot preparation. Image A and B represent normal
mesothelial cells PC 168. Image A demonstrates the strong positive staining reaction seen
when stained with the anti-MUC1 antibody VU4H5 while image B represents the negative
staining effect when the same cell line was stained with the anti-MUC1 antibody E29.
Images C and D represent normal mesothelial cells named ‘GIB406’ obtained from
pericardial fluid and stained with EMA (E29) clone. Cells stained within the chamber slides
(image C) show weak cytoplasmic positivity for EMA (E29) however when cells are
paraffin blocked no positivity was demonstrated.
A
B
C
D
159
5.2.2
ShRNA MUC1 knockdown in mesothelioma cell lines
No previous studies have examined the effect of reduced MUC1 expression in
mesothelioma cells. A selection of two MUC1 mRNA high (LO68 and VGE62), two
medium (NO36 and GAY2911) and one low (STY51) expressing cell lines were selected
for further study. Cell lines were transfected with four commercially available shRNA
plasmids targeted to different MUC1 sequences and with a control shRNA scrambled
plasmid as per Chapter 2.14. Stably transfected cells were identified by hygromycin
resistance and MUC1 expression was quantitated at the mRNA and protein levels.
5.2.2.1
MUC1 mRNA expression in mesothelioma cell lines following shRNA
MUC1 knockdown
Total RNA was extracted from 1 x107 cells as described (Chapter 2.7). Quantitative
PCR was performed using the primer set designed to amplify the full length MUC1
product (Ohuchida, K, Mizumoto, K et al. 2006).
The five cell lines transfected with the clone #1 MUC1 shRNA showed a significant
reduction in the relative expression of MUC1mRNA compared to the scrambled (NC)
cell lines (Figure 5.3). No consistent statistically significant reduction in MUC1 mRNA
levels was observed with the other three shRNA plasmids examined (data not shown).
Therefore clone #1 was chosen for further experimental study of the effects of MUC1
knockdown in mesothelioma cells.
160
Figure 5.3: A: mRNA confirmation of shRNA MUC1 knockdown in all stably
transfected cell lines. The graph also demonstrates the shRNA MUC1 knockdown in
Clone #1 cell lines. All samples have been normalized against shRNA MUC1
negative clone (NC). Each RT PCR reaction was performed in duplicate and repeated
at least twice (mean +/- std dev). B: A Stratagene cDNA standard curve was used to
determine the efficiency of each PCR reaction and as an independent
calibrator.
A
shRNA of MM cell lines
Relative MUC1
Expression
2.0
1.5
**
*
**
*
*
Parent
Neg Control
#1 Clone
1.0
0.5
0.0
P NC #1
P NC #1
P NC #1
P NC #1
P NC #1
VGE62
LO68
NO36
GAY2911
STY58
B
OHUC Primer
ȕActin Primer
161
5.2.2.2
Cell surface expression of MUC1 in mesothelioma cell lines following
shRNA MUC1 knockdown using flow cytometry
Exponentially growing cells were harvested from culture and stained with E29 antiMUC1 antibody as described in Chapter 2.15 and cell surface expression of MUC1 was
determined using FACS. To determine the percentage of cells stained each cell line was
compared with the respective cells stained with an isotype control antibody (Figure 5.4).
The percentage of cells that exhibited cell surface expression of MUC1 was reduced in
the shRNA MUC1 clone #1 lines compared with the respective parental cell lines,
however only 3/5 mesothelioma cell lines showed a significant reduction in cell surface
MUC1 expression compared to the control scrambled NC lines (Table 5.3)(Figure 5.5).
Table 5.3: Percentage of cells expressing MUC1 on cells surface relative to isotype
control cells.
Clone #1
(%)a
Parental
(%)a
p valueb
Scrambled
NC (%)a
p valuec
LO68
1.8±0.4
7.8±1.1
<0.01
10.1±1.2
<0.01
VGE62
21.1±2.4
78.4±1.4
<0.01
50.2±14.3
ns
NO36
35.5±18.3
80.4±4.6
<0.05
30.5±7.4
ns
GAY2911
21.3±6.7
35.8±6.1
<0.02
20.5±7.2
ns
1.7±1.
5.9±0.4
<0.01
4.2±2.1
ns
Cell Line
STY51
a
–mean percentage of cells (±SD) staining positive compared to isotype control.
b
– p value for the difference between the means of MUC1 staining in the parental versus clone
#1 cell lines using the Student’s t-test (ns – not significant).
c
- p value for the difference between the means of MUC1 staining in the scrambled control
versus clone #1 cell lines using the Student’s t-test.
162
Figure 5.4: FACS cell surface expression of anti-MUC1 (E29) antibody was
performed in duplicate on all shRNA MUC1 cell lines compared to the scrambled and
parental controls. Below is a representative FACs plot demonstrating the VGE parental cell line, scrambled control cell line and Clone #1 cell line together with control
parameters required to demonstrate MUC1 (E29) cell surface expression.
163
Figure 5.5: Comparison of FACs cell surface expression on 5 mesothelioma cell
lines performed in duplicate. The parental cell line demonstrates that the cell surface expression of MUC1 varies from 78.4% for the VGE parental cell line down
to 5.92% for the STY51 cell line.
164
5.2.2.3
Protein expression of MUC1 in mesothelioma cell lines following shRNA
MUC1 knockdown using chamber slide immunohistochemistry
Chamber slide immunohistochemistry was used to determine the MUC1 protein
expression in cells. Briefly, 1 x 105 cells were plated into each well of an 8 chamber slide
and incubated until cells were close to 90% confluence. Cells were then stained with a
panel of anti-MUC1 monoclonal antibodies as described in (Table 2.1) and each slide
was microscopically assessed and scored.
As previously observed (Figure 5.6) parental cell lines had strong cytoplasmically diffuse
staining with the anti-MUC1 E29 antibody. The scrambled NC cell line showed cellular
morphology similar to the parental clone however the intensity of anti-MUC1 E29
staining was less than that observed for the parental clone however still cytoplasmically
diffuse. The shRNA MUC1 clone #1 showed slight variability in cell morphology.
The shRNA MUC1 clone #1 cells appeared round with a central round nucleus and
displayed only minimal anti-MUC1 E29 staining mainly localised around the nucleus
(Figure 5.6).
Consistent reduction was observed in MUC1 mRNA levels following MUC1 knockdown
however less consistent results were obtained for protein expression, with the transfection
of mesothelioma cells with the scrambled NC plasmid variably affecting MUC1 cell
surface expression and expression of MUC1 in in vitro growing cells.
165
Figure 5.6: Innate levels of MUC1 expression of shRNA MUC1 knockdown cell
lines and parental control was performed in 8 well chamber slides then stained with 5
anti-MUC1 monoclonal antibodies SM3, E29, VU4H5, Mc5 and BC2. Images
represent the cells stained with anti-MUC1 E29 antibody.
VGE62 Parent E29
VGE62 #1 Clone E29
VGE62 #2 Clone E29
VGE62 #3 Clone E29
VGE62 #4 Clone E29
VGE62 NC Clone E29
166
As antibody-mediated determination of MUC1 protein expression is very dependent on
the glycosylation state it was decided to proceed with these clones and examine the
effects of MUC1 knockdown on mesothelioma cell proliferation, migration and
tumourigenic potential.
5.2.3
Effect of reduced MUC1 expression on in vitro mesothelioma cell
proliferation
The effect of decreased MUC1 mRNA expression on in vitro cell proliferation of
mesothelioma cell lines was evaluated by CyQUANT and WST-1 proliferation assays
as described Sections 2.10 and 2.11 respectively. The CyQUANT cell proliferation
assay is based on the principle that cellular DNA content is highly regulated and
proportional to cell number and sample fluorescence measured with an excitation of
485nm and emission detection at 530nm. The WST-1 cell proliferation assay is based
upon the addition of a substrate that measures the metabolic activity of viable cells, with
absorbance measured at 450nm. Parental, clone #1 and the scrambled vector of each
cell line were assessed using both assays in triplicate in four 96 well plates at different
serum concentrations. Plates were incubated for 72 hours and sample fluorescence
and/or absorbance were measured at 24, 48 and 72 hour time points. The doubling time
of each cell line was calculated using a nonlinear regression of the exponential growth
curve function in the graphical package Prism 4 (GraphPad Software, Inc. La Jolla,
CA).
Proliferation results are presented for the VGE62 parental and transfected cell lines as
determined by the CyQUANT assay (Figure 5.7). At the 24, 48 and 72 hour time
points, there is no difference in cell proliferation between the VGE62 parent and
167
transfected cell lines when cells were grown in 15% FCS. However, at lower FCS
concentrations the VGE62 #1 and scrambled clones show significantly reduced
proliferation rates compared with the parental cell line. At some serum concentrations
doubling time could not be calculated for the transfected lines using the CyQuant assay
data. There was no difference in doubling time of the VGE62 cell lines as measured by
the CyQuant assay at 5% FCS concentration (Figure 5.7). No significant difference in
doubling time, as measured by the WST-1 assay, was observed for the VGE62 cell lines
at the different serum concentrations examined (Figure 5.7). This finding was similar in
the LO68 and GAY2911 cell lines however the NO36 and STY51 cell lines showed no
difference in cell proliferation between the parental and shRNA MUC1 knockout in
both methods of cell proliferation (Figure 5.8). However under standard tissue culture
conditions the NO36 and STY51 cell lines are usually quite difficult to maintain in
lower FCS concentrations.
5.2.4
Effect of reduced MUC1 expression level on in vitro mesothelioma cell
migration
A small proportion of specialized cells are capable of actively migrating within an
organism; these include stem cells, leukocytes and fibroblasts (Entschladen, F, Drell,
TLt et al. 2005). In addition to the normal physiological migration of cells, some
tumour cells are also capable of cell migration, a process that facilitates tumour
progression, invasion and metastasis. In pancreatic and breast carcinoma decreased
MUC1 expression is found to decrease cell migration (Yuan, Z, Wong, S et al. 2007).
There are different assays available to assess tumour cell migration. Two dimensional
assays provide an insight into the migratory activity of cells in response to a natural or
pharmacological mediator.
168
Figure 5.7: Cell proliferation assays for VGE62 cell lines; data presented for the parental
line (P), the control line transfected with scrambled (NC) shRNA and the MUC1 knockdown
line (#1). (A) Cell proliferation determined using the CyQuant assay. Data is presented as
mean ±SD of triplicate assays at each time point and the dashed line represents the nonlinear fit of an exponential growth curve. (B) Doubling time (mean ± 95%CI) for each cell
line calculated from the data presented in (A). Note doubling time could not be calculated
for some lines. (C) Doubling time (mean ± 95%CI) for each cell line calculated from the
data generated by WST-1 assays. .
A
VGE62
0% FCS
0.4% FCS
VGE#1 Clone
VGE Neg Control
VGE Parent
1.0×10 7
1.0×10 6
1.0×10 8
Fluorescence
Fluorescence
1.0×10 8
1.0×10 5
VGE#1 Clone
VGE Neg Control
VGE Parent
1.0×10 7
1.0×10 6
1.0×10 5
0
10
20
30
40
50
60
70
0
80
10
20
30
1% FCS
VGE#1 Clone
VGE Neg Control
VGE Parent
1.0×10 7
1.0×10 6
Fluorescence
Fluorescence
60
70
80
1.0×10 8
1.0×10 5
VGE#1 Clone
VGE Neg Control
VGE Parent
1.0×10 7
1.0×10 6
1.0×10 5
0
10
20
30
40
50
60
70
80
0
10
20
30
HOURS
40
50
60
70
80
HOURS
5% FCS
15% FCS
VGE#1 Clone
VGE Neg Control
VGE Parent
7
1.0×10 6
1.0×10 8
Fluorescence
1.0×10 8
Fluorescence
50
2% FCS
1.0×10 8
1.0×10
40
HOURS
HOURS
1.0×10 5
1.0×10
VGE#1 Clone
VGE Neg Control
VGE Parent
7
1.0×10 6
1.0×10 5
0
10
20
30
40
50
60
70
80
0
10
20
HOURS
30
40
50
60
70
80
HOURS
B
C
VGE WST-1
VGE62 CYQUANT
1024
256
VGE Neg Control
0%
0.4%
1%
not calculatable
8
not calculatable
16
not calculatable
32
not calculatable
64
not calculatable
128
not calculatable
Doubling Time
VGE #1 Clone
2%
5%
Serum concentration
15%
Doubling Time
VGE Parent
512
1024
512
256
128
64
32
16
8
4
2
1
VGE Parent
VGE #1 Clone
VGE Neg Control
0%
0.4%
1%
2%
5%
15%
Serum Concentration
169
Figure 5.8: Cell proliferation of shRNA MUC1 cell lines and parental control. The graphs
below represent the Cyquant and WST-1 assays demonstrating the cell fluorescence and
absorbance of the parental, negative control (NC) and shRNA MUC1 Clone #1 knockdown
at 5% FCS concentration across the five representative cell lines.
CYQUANT
Fluorescence
5.0×10 7
4.0×10 7
3.0×10 7
2.0×10 7
1.0×10 7
V
VGGE
E P
VG N
C
LOE #
LO 6 1
6 8
LO 8 N P
6 C
N 8#
O
N 3 1
O 6
N 36 NP
O C
36
G #1
G AY
A P
Y
G N
A C
Y
ST #1
ST Y
Y P
ST N
Y C
#1
0
WST-1
2.5
1.5
1.0
0.5
0.0
V
VGGE
E P
VG N
C
LOE #
LO 6 1
68 8 P
LO N
6 C
N 8#
N O3 1
O 6
N 36 N P
O C
36
G #1
G AY
A P
G YN
A C
Y
ST #1
ST Y
Y P
ST N
Y C
#1
Absorbance
2.0
170
To investigate the role of MUC1 in cell migration in mesothelioma cells, 2D wound
scratch assays and transwell migration assays were performed. Since reducing MUC1
expression has been shown to decrease cell migration in pancreatic carcinoma (Yuan, Z,
Wong, S et al. 2007), and mesothelial cells exhibit different migratory behaviour to
epithelial cells (Mutsaers, SE 2002), we hypothesise that reduced MUC1 expression in
mesothelioma cells would decrease cell migration.
5.2.4.1
Invitro scratch assays on mesothelioma and shRNA MUC1 knockdown
cell lines
In vitro scratch assay were performed as described Chapter 2.12 with a scratch being
made horizontally through a monolayer of cells when at 90% confluency.
The
percentage area of the scratch that had been covered by migrating cells was determined
at three time points (6, 24 and 48 hours) and representative micrographs are shown
(Figure 5.9).
All five parental cell lines and 4/5 control scrambled cell lines showed complete wound
closure by 48 hours (Figure 5.10). Of the five cell lines, the GAY2911 cell line showed
complete wound closure at 24 hours while VGE62, LO68, NO36 and STY51 did not
achieve this until 48 hours (Figure 5.10). The LO68 parental cell line showed complete
wound closure at 48 hours however at this time point the wound was only 48% ± 4.1
closed for the NC cell line (p=<0.0001).
171
Figure 5.9: Wound scratch assay of shRNA MUC1 transfectants and parental
Control of the VGE62 cell line using image J analysis.
VGE62 Parent at 6, 24 and 48 Hr time
VGE62 #1 Clone at 6, 24 and 48 Hr
VGE62 NC Clone at 6, 24 and 48 Hr
172
Figure 5.10: Wound scratch assay of shRNA MUC1 transfectants and parental
control. Statistical significance is denoted with an * where *** P < 0.0001, ** P < 0.005,
* P < 0.05.
LO68
VGE62
***
*
***
LO68 Parent
LO68 Neg Control
LO68 #1 Clone
**
100
80
% of wound closure
120
150
100
60
40
20
50
0
6h
12 h
24 h
48 h
6h
12h
24h
48h
6h
12h
24h
48h
LO68 P
6h
12 h
24 h
48 h
6h
12 h
24 h
48 h
0
LO68 NC
LO68 #1
VGE62 P
VGE62 NC VGE62 #1
GAY2911
NO36
***
*
*
*
140
150
100
% o f w o u n d c lo s u r e
NO36 Parent
NO36 Neg Control
NO36 #1 Clone
120
80
60
40
20
GAY Parent
GAY Neg Control
GAY #1 Clone
100
50
0
6h
12h
24h
48h
6h
12h
24h
48h
6h
12h
24h
48h
6h
12h
24h
6h
12h
24h
6h
12h
24h
0
NO36 P
NO36 NC
NO36 #1
GAY P
GAY NC
GAY #1
STY51
**
*
% of wound closure
125
STYParent
STYNegControl
STY#1Clone
100
75
50
25
6h
12h
24h
48h
STYP
6h
12h
24h
48h
0
6h
12h
24h
48h
% of wound closure
VGE Parent
VGE Neg Control
VGE #1 Clone
6h
12h
24h
48h
% of w ou n d clos u re
*
200
STYNC
STY#1
173
The ability of mesothelioma cells with reduced MUC1 to close the wound was
significantly reduced in all five of the cell lines examined compared with the respective
scrambled control cell line, ranging at the 48 hour time point from 10 ± 0.8% for the
LO68 to 44 ± 11% for the VEG62 MUC1 knockdown cell lines (Figure 5.10).
5.2.4.2
Cell migration assays on mesothelioma and shRNA MUC1 knockdown
cell lines
Cell migration was also assayed by determining the number of cells migrating through a
non-coated 8μm polycarbonate filter in a modified Boyden Chamber (Chapter 2.13).
The total number of cells that migrated through the filter ranged from 75 ± 10 cells for
the VGE62 to 180 ± 10 cells for the LO68 parental cell lines (Figure 5.11).
Approximately 10 cells were found to migrate through the filter for the control, nonmigrating cell line MCF7. Compared to the parental cell line, the number of cells
migrating through the filter was significantly reduced in the control scrambled NC cell
lines for the VGE62 and NO36 cells. The reason for this is unclear. Nevertheless, for
each of the five cell lines, the number of cells migrating through the membrane was
significantly reduced in the clones with reduced MUC1 expression compared to the
parental cell line (p<0.01). For all but the VGE62 cell line, migration of the MUC1
knockdown clone #1 was significantly less than the respective negative control (NC)
cell line (p<0.05). Of note, in particular, the number of cells migrating for the VGE62,
NO36, GAY2911 and STY51 lines with reduced MUC1 expression was comparable to
that of the MCF7 control line.
174
Figure 5.11: Transwell migration of all cell lines show a significant inhibition of the
shRNA MUC1 #1 clones compared to the parental cell line and shRNAMUC1 negative
control. The photomicrographs under the graph represents each cell line after MGG
staining. Statistical significance is denoted with an * where *** P<0.0001, ** P<0.005,
* P < 0.05.
LO68
VGE
150
VGE Parent
VGE Neg Control
VGE #1 Clone
**
100
ns
50
VGE62 P
VGE NC
150
100
50
0
VGE #1
LO68 P
VGE62
VGE62 #1
LO68 NC
LO68 P
LO68 #1
LO68 NC
LO68 #1
GAY 2911
NO36
***
200
NO36 Parent
NO36 Neg Control
NO36 #1 Clone
***
150
No. of m igrating cells
200
100
50
**
GAY P
GAY NC
GAY #1
*
150
100
50
0
0
NO36 P
NO36 P
NO36 NC
NO36 #1
GAY P
NO36 NC
NO36 #1
GAY2911
GAY NC
GAY #1
GAY2911 NC
GAY2911 #1
STY51
***
***
200
N o. of m igrating cells
N o. of m igr ating cells
LO68 Parent
LO68 Neg Control
LO68 #1 Clone
*
200
0
VGE P
**
250
N o . o f m ig r atin g c e lls
N o . o f m ig r a tin g c e lls
200
STY Parent
STY Neg Control
STY #1 Clone
150
100
50
0
STY P
STY51 P
STY NC
STY #1
STY51
STY51 #1
175
In conclusion, using a scratch and modified Boyden Chamber assay reduced MUC1
expression resulted in reduction in migration of mesothelioma cells.
5.2.5
Effect of reduced MUC1 expression on in vitro mesothelioma
tumourigenic potential
To assess the effect of MUC1 expression on tumourigenic potential, the ability of cells
to form colonies under anchorage independent conditions and their ability to invade
through a polycarbonate membrane coated with Matrigel basement membrane matrix
was analysed.
Growing cells under anchorage independent growth conditions is
indicative of the cells ability to form tumours in vivo (Franken, NA, Rodermond, HM et
al. 2006).
Cells were seeded in duplicate wells and after 21 days in culture, colony formation was
assessed and quantitated. Colonies were defined as consisting of greater than 30 cells
per aggregate (Chapter 2.9). Anchorage independent growth was evident in all parental
cell lines except for the STY51 and representative micrographs are shown in (Figure
5.12.A). No colonies were formed by control or non-malignant mesothelial cell cultures
(Figure 5.12.A). The number of colonies formed by the four mesothelioma parental cell
lines ranged from 15±5 for LO68 (Figure 5.13) to 4±1 for VGE62 (Figure 5.13). Whilst
the numbers of colonies formed by the scrambled clones were not statistically different
from the respective parental line, for the LO68 cell line the colonies appeared to be
smaller and have a flatter 2D growth pattern (data not shown).
176
Figure 5.12: (A) STY51 and normal mesothelial cell lines demonstrating the lack of
colony formation capabilities. Arrows demonstrate single cell presentation.
(B) Representative photomicrographs of soft agar assays are shown for GAY2911 and
NO36 cell lines at 4x magnification.
A
B
177
Figure 5.13: MUC1 confers anchorage independent growth in soft agar assays of
mesothelioma cell lines. 1 x 104 cells/well were suspended in soft agar and incubated for
21 days. Colony formation of >30 cells were counted for each of the indicated cell lines.
The results are expressed as the number of colonies (mean +/- s.d.) obtained from two
independent experiments. After several attempts the STY51 P and transfected cell lines
did not form colonies in soft agar assays. They resembled the benign mesothelial cells
and showed single cell formation. Statistical significance is demonstrated with * where
*** P<0.0001, ** P < 0.005, * P < 0.05
LO68
VGE
***
**
VGE Parent
VGE Neg Control
VGE #1 Clone
*
8
6
4
2
**
20
C olony form ation
>30cells/aggregate
C o lo n y fo r m atio n
> 30 ce lls/ag g r eg a te
10
0
VGE P
VGE NC
LO68 Parent
LO68 Neg Control
LO68 #1 Clone
15
10
5
0
VGE #1
LO68 P
NO36
LO68 #1
GAY2911
**
*
NO36 Parent
NO36 Neg Control
NO36 #1 Clone
*
15
10
5
C o lo n y fo r m a tio n
> 3 0 c e lls /a g g r e g a te
20
20
C o lo ny form atio n
> 30 cells/ag gr egate
LO68 NC
GAY Parent
GAY Neg Control
GAY #1 Clone
15
10
5
0
0
NO36 P
NO36 NC
NO36 #1
GAY P
GAY NC
GAY #1
178
In the four mesothelioma cell lines that demonstrated anchorage independent growth,
the number of colonies formed from lines with reduced MUC1 expression was
significantly less than for the respective parental lines (p<0.05) and for three of the four
lines the respective scrambled NC control (p<0.05) (Figure 5.13).
No anchorage
independent colonies were formed by non-malignant mesothelial cells.
5.2.5.1
Cell migration assays on parental and shRNA MUC1 knockdown cell
lines
To further investigate the tumourigenic potential of mesothelioma in vitro, Matrigel
transwell membrane assays were used to assess the potential for mesothelioma cells to
invade a basement membrane-like barrier. Three random fields of views were counted
at 400x magnification (Figure 5.14).
MCF7 cells were included in the assay as a non-invasive control.
Under the
experimental conditions examined there was no statistically significant difference
between the number of MCF7 cells (15.3±3.8 cells) and the number of VGE62
(26.7±6.7 cells) and NO36 (21.7±2.5 cells) parental cells invading through the Matrigel
coated membrane. Therefore the VGE62 and NO36 mesothelioma cell lines could be
described as non-invasive. The remaining three mesothelioma cell lines examined were
found to have an invasive phenotype, having approximately five-fold (GAY2911) and
ten-fold (LO68 and STY51) more cells invading through the Matrigel than the control
MCF7 cell line. For each of these three parental mesothelioma lines approximately
75% of the cells capable of cell migration (ie transversing a nylon membrane) displayed
an invasive phenotype (ie transversing the matrigel coated membrane) (Figure 5.14).
179
Figure 5.14: Matrigel cell invasion of parental, shRNA negative control and shRNA
MUC1 Clone #1 from LO68, GAY2911 and STY51 cell lines. MCF7 cells were
included in the assay as a non-invasive control. Statistical significance is demonstrated
with * where *** P < 0.0001, ** P < 0.005, * P < 0.05.
LO68
% of migrating cells
that are invasive
100
Parent
Neg Control
#1 Clone
75
50
25
0
P
NC
#1
GAY2911
% of m igrating cells
that are invasive
175
Parent
Neg Control
#1 Clone
150
125
100
75
50
25
0
P
NC
#1
STY
***
% of migrating cells
that are invasive
100
***
Parent
Neg Control
#1 Clone
75
50
25
0
P
NC
#1
180
Only in one of the three cell lines (STY51) was a statistically significant difference
demonstrated in the percentage of migrating cells capable of invading through the
matrigel matrix following reduced MUC1 expression (Figure 5.14).
181
5.3
DISCUSSION
Changes in MUC1 expression have been associated with tumourigenesis and other
characteristics of cancers, particularly cell proliferation, apoptosis, and invasion in a
selection of glandular malignancies including breast, prostate, pancreas and uterine
cancer (Obermair, A, Schmid, BC et al. 2002; Tsutsumida, H, Swanson, BJ et al. 2006;
van der Vegt, B, de Roos, MA et al. 2007). The innate mRNA levels and protein
MUC1 expression were initially examined in a selection of normal mesothelial and
mesothelioma cell lines.
The exact function of MUC1 in mesothelioma tumour
progression has never been investigated and the aim of this chapter was to investigate
the functional role of MUC1 on benign and mesothelioma cell lines.
The level of mRNA MUC1 expression ranged from 10.6 ± 0.07 for the ST45
mesothelioma cell line to 1.65 ± 2.26 for the VAN mesothelioma cell line. Normal
mesothelial cells expressed a moderate amount of MUC1 with eight mesothelioma cell
lines expressing mRNA MUC1 at higher levels to the normal mesothelial cells,
comparable to the relative amount expressed by epithelial colon or lung cell lines.
MUC1 has been shown to be distributed apically in normal glandular epithelial cells and
its glycosylation patterns have been observed to be tissue specific (Gendler, SJ 2001),
however the glycosylation pattern of MUC1 has not previously been demonstrated in
mesothelioma.
This study demonstrates that the protein distribution of MUC1 in
mesothelioma cell lines is variable and dependent on the variation of MUC1 epitopes
present on mesothelioma cell lines. The E29, Mc5 and VU4H5 monoclonal antibody
which recognize the tandem repeat domain of the MUC1 protein were the most
sensitive monoclonal antibodies detecting MUC1 expression within the cytoplasm of all
182
mesothelioma cells and in normal mesothelial cells to a weaker intensity. The E29
antibody detects all forms of MUC1 irrespective of the degree of glycosylation. Since
VU4H5 staining is only detected when the threonine residue of the PDTRPAP epitope
of the MUC1 tandem region is non-glycosylated then perhaps this accounts for the
positive staining effect seen in the PC168 normal mesothelial cell line compared to the
E29 clone (ten Berge, RL, Snijdewint, FG et al. 2001). The SM3 and BC2 antibodies,
which are directed against a hypoglycosylated epitope of MUC1, showed weak to
moderate staining intensity in the mesothelioma cell lines and were not as sensitive as
the anti-MUC1 E29 monoclonal antibody in mesothelioma.
Given that a positive
reaction was demonstrated with the E29, Mc5 and VU4H5 antibodies this suggests that
MUC1 on mesothelioma cell lines is hyperglycosylated as both the hypoglycosylated
antibodies, SM3 and BC2 only showed weak staining intensities.
Establishing the innate levels of mRNA and protein expression of MUC1 in a selection
of normal and mesothelioma cell lines, enabled a selection of high, medium and low
expressing MUC1 cell lines to be transfected with commercially available shRNA
MUC1 and control plasmid. From the five cell lines transfected with shRNA MUC1 the
VGE62, LO68 and NO36 showed mRNA expression of MUC1 which was greater than
or equal to the scrambled negative control cell line, however, the GAY2911 and the low
MUC1 expressing STY51 cell line showed that MUC1 expression from the parental cell
lines was less than the scrambled negative control. Nevertheless in all of these cell lines
the clone #1 was consistently reduced compared to the scrambled negative control.
The level of cell surface expression of MUC1 from flow cytometry correlated to the
quantitative PCR enabling us to confirm that shRNA MUC1 knockdown cell line was
183
clone #1. Therefore, the shRNA MUC1 knockdown clone #1 from each mesothelioma
cell line was selected to investigate further biological relationships between
mesothelioma cells and MUC1 expression.
Other studies have demonstrated that MUC1 inhibition by peptides, vectors and siRNA
in breast, kidney and pancreatic carcinomas all resulted in reduced cell proliferation of
those tumours in vitro (Schroeder, JA, Masri, AA et al. 2004; Mahanta, S, Fessler, SP et
al. 2008; Bitler, BG, Menzl, I et al. 2009; Yuan, Z, Liu, X et al. 2009). Although cell
proliferation was reduced at lower FCS concentrations in clone #1cell line compared to
the parental cell lines in both LO68 and GAY2911 cell lines, the NO36 and STY51 cell
lines showed no difference in cell proliferation between the parental and shRNA MUC1
knockout. These results suggest that MUC1 may not play a substantial functional role
in determining the cell proliferative ability of mesothelioma. This finding was similar
to that seen by Mukherjee et al, in a Jurkat lymphoma cell line, in which siRNA knock
down of MUC1 did not completely inhibit cell proliferation (Mukherjee, P, Tinder, TL
et al. 2005). There are likely to be multiple determinants of proliferative ability. It
seems probable that MUC1 is not a critical determinant of the cell proliferation cascade
in mesothelioma but may be more important in epithelial carcinomas.
Further
investigations into potential cell proliferation signalling cascades in these shRNA
MUC1 mesothelioma cell lines may help us understand the biology of cell proliferation
in mesothelioma
Cancer cell migration and invasion is the fundamental property of tumour metastasis
and to address the hypothesis that MUC1 expression was a determinant of these
properties, a 2D scratch assay and a 2D modified Boyden chamber assay was used with
184
the parental and shRNA MUC1 transfected mesothelioma cell lines. Results from the
2D scratch assay demonstrated that all five parental cell lines showed complete wound
closure within a 48 hour period. The scrambled control cell line showed complete
wound closure at 48 hours for the VGE62, NO36 and STY51 cell lines. The LO68
scrambled control cell line was only 44%±10 closed at the 48 hour time point. The
lower mRNA expressing MUC1 GAY2911 cell line showed complete wound closure of
the parental and scrambled control cell lines within a 24 hour period, while clone #1 cell
lines did not achieve complete wound closure for any of the mesothelioma transfected
cell lines suggesting that reduced MUC1 expression inhibits cell migration in
mesothelioma.
These results were concordant with results from the 2D modified
Boyden chamber assay. For each of the five mesothelioma cell lines, the number of
cells migrating through the membrane was significantly reduced in the clone #1 cell line
compared to the parental cell lines. For all but the VGE62 cell line the migration of
clone #1 was significantly less than the scrambled negative clone, suggesting that
MUC1 expression in mesothelioma cells plays a functional role in cell migration and
tumour progression.
Tumour cells acquire the ability to invade extra cellular matrix barriers by expressing a
range of proteases such as MMP. Proteases, protease inhibitors and ECM proteins
represent important components that control tumour microenvironment, tumour
vascularisation and tumour progression. Tumour cell invasion is a tightly regulated
process involving the reorganisation of the actin cytoskeleton including stress fibre and
focal adhesion reassembly. The membrane bound mucins form a physical barrier that
protects the apical borders of epithelial cells from damage induced by toxins,
microorganisms and other forms of stress that may occur at the interface to the external
185
environment. Since MUC1 is a large transmembrane type 1 protein that extends beyond
the cell membrane, it is likely that the structure of MUC1 is a critical determinant of its
vital role in its surrounding environment.
One plausible mechanism by which
downregulation of MUC1 may inhibit cell migration is through the β-catenin pathway
(Bitler, BG, Menzl, I et al. 2009).
Previous studies have suggested that overexpression of the MUC1 heterodimer confers
anchorage independent growth and tumorigenicity in part through stabilization of βcatenin (Li, Y, Liu, D et al. 2003; Raina, D, Kharbanda, S et al. 2004; Ren, J, Agata, N
et al. 2004).
The effects of MUC1 on anchorage independent cell growth and
tumorigenicity might be attributable to disruption of cell adhesion by the heavily
glycosylated ectodomain. Anchorage independent growth was observed in all parental
cell lines except the STY51 cell line which failed to form colonies compared to the
shRNA MUC1 clone #1 cell line. The ability for a population of cells to form colonies
in anchorage independent environment is a feature that has been associated with the
tumorigenicity of cells. Tumour cells are similar to stem cells and are defined by their
ability to be pluripotent, high clongenicity with self replicating potential (Engelmann,
K, Shen, H et al. 2008).
From the hypothesis that MUC1 expression increases
tumorigenicity of mesothelioma cells, and given that the STY51 cell line did not form
colonies in an anchorage independent environment, then the question remains ‘is the
STY51’a tumour cell line’? However, tumour cells can form a single cell population
similar to that seen in Lobular carcinoma of the breast.
By altering MUC1 expression from the selection of mesothelioma cell lines we have
observed a reduction in anchorage independent growth, cell migration and invasion of
186
the LO68, VGE62, NO36, GAY2911 and STY51 cell lines.
Matrigel transwell
membrane assays were also used to assess the invasive potential of mesothelioma cells
to invade a basement membrane barrier. These results divided the mesothelioma cell
lines into those with non-invasive and invasive potential. Both the VGE62 and NO36
cell lines showed no significant differences compared to the non-invading cell line,
MCF7 cells and therefore could be described as not having invasive potential. The
LO68, GAY2911 and STY51 cell lines were all found to demonstrate an invasive
phenotype. Typically mesotheliomas are usually diffuse tumours which form a
thickened growth along the parietal or visceral surface, encapsulating and constricting
the underlying lung (Henderson, D, Shilken, K et al. 1992). From these results we can
suggest that mesothelioma cells have the capacity to migrate and spread within its
microenvironment. However, metastatic mesothelioma is rare and usually correlates
with advanced disease (Ishikawa, T, Wanifuchi, H et al. 2010). Apart from STY51, the
remaining four mesothelioma cell lines do not appear to display invasive potential and
are unlikely to represent an aggressive tumour type in vivo. The STY51 cell line is
however interesting, as it does not display an anchorage independent growth pattern, but
is capable of invading through a matrigel matrix, displaying an invasive phenotype
capable of metastatic disease. The results in this thesis suggest that reduced expression
of MUC1 results in a loss of cell-cell adhesions and maintenance of the colony
formation in vitro but it does not highlight the signalling pathway involved in the
inhibition of MUC1 in mesothelioma cell lines in vitro.
5.4
CHAPTER SUMMARY
The function of MUC1 in tumour progression has been extensively investigated in a
wide range of epithelial malignancies including breast, prostate, lung and
187
gastrointestinal cancer, however this is the first time that the function and role of MUC1
in mesothelioma has been examined. This study has demonstrated that MUC1 mRNA
expression and cell surface protein expression of MUC1 are variable in a range of
normal mesothelial and mesothelioma cell lines. When mesothelioma cell lines were
transfected with shRNA MUC1 plasmid, reduced MUC1 expression was observed in
the mRNA expression of MUC1 and on the cell surface expression of MUC1. Cell
proliferation assays with the transfected shRNA MUC1 cell lines suggested that perhaps
MUC1 may not play a key functional role in the proliferation of mesothelioma cells and
perhaps alternative signalling mechanisms are responsible for promoting tumour
proliferation in this tumour type. This study has, however, suggested that MUC1 may
play a functional role in cell migration and tumorigenicity of mesothelioma cell lines.
This evidence for the role of MUC1 in cell migration and invasion in mesothelioma
suggests that MUC1is a potential target for immunotherapy treatment of mesothelioma.
MUC1 targeting agents such as the recombinant viral vector vaccine TG4010 and the
Liposomal vaccine L-BLP25 are currently in advanced clinical testing in NSCLC and
other cancers (Mellstedt, H, Vansteenkiste, J et al. 2011; Sharma, S, Srivastava, MK et
al. 2011). Our results lend some support testing this approach in mesothelioma.
188
CHAPTER 6
FINAL DISCUSSION
189
6.1
GENERAL DISCUSSION
Malignant mesothelioma is a highly aggressive tumour of the serosal cavities with an
extremely poor prognosis and no curative treatment. The diagnosis of mesothelioma is
extremely difficult and is usually made in conjunction with clinical, radiological and
pathological findings.
The symptoms are nonspecific and accumulation of serous
effusion is usually one of the first signs of disease.
The diagnostic dilemma for
cytopathologists is the differentiation of metastatic adenocarcinomas, malignant
epithelial mesotheliomas and benign reactive mesothelial cells in serous effusions.
Numerous studies have investigated an extensive range of biomarkers to facilitate an
early non-invasive diagnosis of mesothelioma.
However, currently, there are no
specific or sensitive biomarkers available to equivocally diagnose mesothelioma and
only in experienced centres should a diagnosis be established on serous effusion
cytology (van der Bij, S, Schaake, E et al. 2011).
The cytological diagnosis of
mesothelioma is controversial and not widely accepted at an international level. This
thesis examined the diagnostic utility of the anti-MUC1 EMA antibody to differentiate
between malignant epithelial mesothelioma, reactive mesothelial proliferation and
metastatic adenocarcinoma. Although several previous studies have tried to address this
question this current study differs by being a prospective collection of serous effusions
and addresses potential issues that could explain the staining variation seen with the
anti-MUC1 antibody, in particular the E29 antibody. In addition for the first time, the
function and biology of the MUC1 antigen was evaluated in mesothelioma and the
results suggest that it may play a functional role in cell migration and invasion in this
tumour.
Five anti-MUC1 antibodies selected for this study were directed against peptides within
the tandem repeat region of the MUC1 protein. In general terms the SM3 and BC2
190
antibodies both recognise a hypoglycosylated form of MUC1 and these antibodies
demonstrated a lower sensitivity in diagnosing mesothelioma, compared to the E29,
Mc5 and VU4H5 antibodies which recognise the hyperglycosylated form of MUC1
(Cao, Y, Karsten, U et al. 1998). Since a majority of mesothelioma cases in this study
demonstrated staining with the E29, Mc5 and VU4H5 antibodies this suggests that
MUC1 on mesothelioma cells is hyperglycosylated compared to the typical MUC1
expressed on epithelial malignancies (Burke, PA, Gregg, JP et al. 2006). However there
must be subtle differences in the epitopes recognized by these three antibodies to
account for the superior diagnostic accuracy for mesothelioma of the E29 clone over the
Mc5 and VU4H5 clones. It appears that the differences in staining of benign reactive
and malignant mesothelial cells accounts for the greater specificity of the E29 clone
over the Mc5 clone. As benign mesothelial cells express MUC1 mRNA at similar or
higher levels than some malignant mesothelioma cells, and MUC1 protein both within
the cytoplasm and on the cell membrane may be a specific mesothelioma-specific
epitope recognized by the E29 antibody. Results from the comparison of the automatic
and manual staining protocols suggest that this epitope is more heat-sensitive in
mesothelioma than in other epithelial cancers and further studies are warranted to try
and identify the epitope.
There is no doubt that MUC1 is overexpressed in epithelial malignancies.
By
performing a hypothetical analysis based on the staining results in the presence or
absence of MUC1 its value in the cytological evaluation of effusions was quantitated.
In this study, 44 cases required further investigation when the diagnostic panel excluded
the MUC1 antibody, in comparison, less than half this number required clinical followup (ie 20 cases) when the MUC1 antibody was incorporated within the diagnostic panel.
These results mostly reflect the expression of MUC1 on epithelial cancers in general.
191
However by including MUC1 in the diagnostic panel approximately 10% more
mesothelioma cases were identified.
From a diagnostic and clinical stand point one of the main results from this study was
the variable staining results observed with the two different staining platforms, manual
and automatic, for the diagnosis of mesothelioma with the MUC1 (E29) antibody.
Clearly differences in sample processing can influence immunohistochemistry results.
Previously, following a systematic review of published reports that distinguished
between benign and malignant pleural disease it was highlighted that no methodological
homogeneity existed between laboratories in the method of staining with anti-MUC1
antibodies and it was suggested that this may account for the inconsistencies in the
published literature regarding the value of MUC1 for differentiating benign reactive
pleural disease and mesothelioma (King, J, Thatcher, N et al. 2006).
Although
traditionally individual laboratories have optimized staining methodologies internally,
there is a precedent for the development of uniform / universal staining methods for
specific antibodies notably the HER2 antibody staining regime for breast cancer
(Farshid, G, Armes, JE et al. 2010). Therefore it is possible that a staining regime for
MUC1 in mesothelioma could be standardized and applied at an international level. A
potential algorithm for a differential diagnosis of reactive mesothelial proliferation,
malignant mesothelioma and epithelial malignancy can be seen in Figure 6.1.
This study also examined the diagnostic value of soluble MUC1 (CA15-3)
concentrations:
As previously reported effusion CA15-3 concentrations were a
sensitive and specific means of differentiating between benign and malignant effusions.
In this study a sensitivity of 57% at a specificity of 97% was demonstrated in this
setting. Given that biochemical analysis of serous effusions are routinely performed in
192
Figure 6.1: A diagnostic algorithm outlining the proposed utility of MUC1 immunocytochemistry and CA15-3 levels in pleural effusion samples to differentiate between reactive
mesothelial cells, malignant mesothelioma and epithelial malignancy.
193
laboratories then incorporation of the CA15-3 assay represents an easy, effective and
inexpensive mechanism to aid in the differential diagnosis of benign reactive
mesothelial cells compared to epithelial malignant diseases. This result would assist to
aid in the diagnosis of serous effusion cytology evaluation.
However, as previously reported effusion CA15-3 concentrations could not differentiate
between mesothelioma and epithelial cancers.
Previously effusion mesothelin
concentrations have been shown to differentiate cases of mesothelioma from other
cancers. This study was the first to demonstrate a correlation between CA15-3 and
mesothelin in mesothelioma patients. By combining these two tumour markers no
improvement in overall diagnostic accuracy above mesothelin alone was demonstrated,
however, the potential role for the combination of these two tumour markers in the
differential diagnosis of mesothelioma, especially in those cases where cytological
morphology between mesothelioma and benign reactive mesothelial proliferation is
difficult to evaluate was demonstrated.
In patients with epithelial tumours MUC1 over-expression has been associated with
poor prognosis. One common hypothesis to explain this relationship is that MUC1
positive tumours have a higher rate of proliferation than MUC1 negative tumours.
However, this relationship between MUC1 expression and survival was not observed in
the mesothelioma patients in the current study. Considering that the in vitro MUC1
knockdown experiments did not show any relationship between MUC1 expression and
proliferation this may be a true reflection of differences between mesothelioma and
other epithelial tumours.
194
A correlation was observed between surface MUC1 expression and soluble levels of
MUC1 in effusions of patients with epithelial tumours, which was not seen in the
mesothelioma patients. This disconnect may have resulted from either differences in
MUC1 isoforms expressed by the different tumour types or differences in enzymes
required to cleave surface MUC1 dependent upon the degree of glycosylation in
mesothelioma relative to other epithelial tumours. In this study, no soluble MUC1
(CA15-3) was detected in the supernatants of mesothelioma cell lines (data not shown).
Whilst further studies are required we hypothesize that in mesothelioma MUC1
expression may reflect the degree of tumour differentiation, the more differentiated the
tumour a larger amount of MUC1 is expressed on the surface and in general poorly
differentiated mesothelial tumours have a poor prognosis.
Tumour associated antigens (TAA) have become a major area of investigation as
immunotherapeutic targets for the treatment of cancer. Peptide based epitopes have
previously been used to investigate cytotoxic immune responses in the clinical setting
however more specific and efficient approaches could be applied using TAA’s as
potential targets for specific immune modulation and therapy. The antigenic structures
recognized by T cells are highly diverse and includes glycopeptides, lipids and
glycolipids (Rudd, PM, Elliott, T et al. 2001). The aberrant glycosylation of MUC1
observed in cancer suggests that glycosylated peptides may be formed and presented on
major histocompatibility complex (MHC) class 1 and II molecules resulting in
glycopeptide specific immune responses. The human epidermal growth factor receptor
2 (HER2) overexpressed in 25-30% of breast cancers is another example of an
extracellular receptor which is currently used in the management of metastatic breast
cancer (Tsang, RY and Finn, RS 2012). The HER2 gene is a member of tyrosine kinase
195
receptor which leads to increased homo and heterodimerisation and subsequent
activation of downstream signalling pathways associated with cell differentiation, cell
proliferation, survival and angiogenesis. Trastuzumab, was the first available HER2
directed therapy, a monoclonal antibody that targets the HER2 extracellular domain and
approved by the US Food and Drug Administration in 1988 for the management of
HER2 positive metastatic breast cancers. Like HER2, in vitro experiments suggest that
MUC1 expression is related to the adhesive properties of the mesothelioma cells and
this was evident by decreased migration and invasion of the mesothelioma cell lines
with reduced MUC1 expression. In mesothelioma there is debate regarding the role of
metastatic disease and patient prognosis therefore, targeting MUC1 may represent the
ideal scenario in mesothelioma whereby decreasing tumour growth and metastatic
potential.
6.2
CONCLUSIONS
The results in this thesis have for the first time demonstrated the importance of the
tumour marker anti-MUC1 (E29) antibody as a diagnostic marker to aid in the
differential diagnosis of benign reactive mesothelial cells, mesothelioma and metastatic
epithelial malignancy. Furthermore, this study has highlighted a possible reason for
variations observed with staining intensities of the EMA antibody suggesting that an
algorithm should be established to improve the quality assurance and to maintain
reproducibility of results. Although the CA15-3 assay did not show any significant
difference between diagnosing mesothelioma from metastatic epithelial malignancy,
this was the first study to demonstrate a correlation between CA15-3 and mesothelin in
mesothelioma patients. By combining these two tumour markers we did not improve
diagnostic accuracy above Mesothelin alone, however, we did observe that the CA15-3
assay played a significant role in differentiating benign reactive mesothelial
196
proliferation from mesothelioma, one of the main diagnostic challenges in cytology.
Finally, to investigate the functional biology of mesothelioma, this is the first study to
investigate the role MUC1 plays in mesothelioma. We have demonstrated that by
reducing MUC1 in a selection of mesothelioma cell lines, MUC1 may not play a
functional role in the cell proliferation of mesothelioma however; we have observed that
MUC1 is important in cell migration, invasion and tumorigenicity of mesothelioma cell
lines. This further suggests that MUC1 may be a promising target for mesothelioma
treatment.
197
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APPENDIX
220
APPENDIX FOR CHAPTER 3
A: EPITHELIAL MALIGNANCY FROM INTERIM PATHOLOGY REPORT
ANTIBODIES TOTAL TP
TN
FP FN SENSITIVITY % SPECIFICITY % PPV % NPV %
E29 MANUAL 264
112 127
4
21
84.2
97
96
86
E29 AUTO
265
89
131
1
44
67
99
76
88.5
Mc5
256
99
98
28 31
76.1
77
78
76
VU4H5
256
77
124
3
52
60
97.6
96.2
70.4
SM3
261
21
129
0 111
17
100
100
53.7
BC2
261
56
130
0
75
42.7
100
100
63.4
ACCURACY %
90
83
77
78.5
57.4
71.2
B: MESOTHELIOMA FROM INTERIM PATHOLOGY REPORT
ANTIBODIES TOTAL TP
E29 MANUAL 264
42
E29 AUTO
265
27
Mc5
256
37
VU4H5
256
20
SM3
261
1
BC2
261
13
TN
FP FN SENSITIVITY % SPECIFICITY % PPV % NPV %
144
74
4
91.3
66
35
97.2
139
80 19
58
63
23
94
121
90
8
82
57.3
29.1
94
152
60 24
45.4
71.6
25
86.3
196
20 44
2
90.7
5
82
174
43 31
30
80
23.2
85
ACCURACY %
70
62
62
67
75.4
72
C: EPITHELIAL MALIGNANCY ON FINAL FU
ANTIBODIES TOTAL TP
TN
FP FN SENSITIVITY % SPECIFICITY % PPV % NPV %
E29 MANUAL 265
117 110
0
38
75
100
100
74
E29 AUTO
265
93
110
0
62
60
100
100
64
Mc5
256
105
92
21 38
73
81
83
71
VU4H5
256
78
112
2
64
55
97
96
64
SM3
261
21
116
0 124
14
100
100
48
BC2
261
55
115
1
90
38
99
98
56
ACCURACY %
86
76
77
74
52
65
D: MESOTHELIOMA ON FINAL FU
ANTIBODIES TOTAL TP
E29 MANUAL 265
48
E29 AUTO
265
30
Mc5
256
43
VU4H5
256
21
SM3
261
1
BC2
261
13
TN
FP FN SENSITIVITY % SPECIFICITY % PPV % NPV %
137
70 10
83
66
41
95
144
63 28
52
70
32
85
117
83 13
79
58
34
92
141
59 35
37
70
26
82
184
20 56
1.8
90
5
78
162
43 43
23
79
23
80
ACCURACY %
70
65
62
63
70
67
E: MESOTHELIOMA ON FINAL FU IN ABSENCE OF EPITHELIAL MALIGNANCY
ANTIBODIES TOTAL TP
E29 MANUAL 179
48
E29 AUTO
179
30
Mc5
169
43
VU4H5
168
21
SM3
174
1
BC2
173
13
TN
FP FN SENSITIVITY % SPECIFICITY % PPV % NPV %
121
0
10
83
100
100
95
121
0
28
51
100
100
83
90
22 14
75
80
66
89
112
2
35
37
98
91
78
117
0
56
2
100
100
69
116
1
43
23
99
93
75
ACCURACY %
94
84
78
79
67
74
F: MESOTHELIOMA ON A SUBSET OF 35 INDIVIDUALS WITH CONFIRMED FU
ANTIBODIES TOTAL TP
E29 MANUAL 266
26
E29 AUTO
266
13
TN
FP FN SENSITIVITY % SPECIFICITY % PPV % NPV %
141
90
9
74
61
155
76 21
40
65
ACCURACY %
63
63
TP: Total positive
TN: Total negative
FP: False positive
FN: False negative
221