Cancer Pharmacogenomics

Cancer Pharmacogenomics
Gregory J. Tsongalis, Ph.D.
Director, Molecular Pathology
Dartmouth Medical School
Dartmouth Hitchcock Medical Center
Norris Cotton Cancer Center
Lebanon, NH
Laboratory Analysis of Human Cancer
(Management Based on Diagnosis)
H&E for
Morphology
Further Diagnostic Work-up
• Imaging (CT, MRI, PET)
• Immuno (IHC), molecular,
• Proteomic
1.4M New
Cancers
(US)
Gross Description
Therapeutic
management
Microscopic Description
Molecular Description
Carcinogenesis is a Multistage Process
Genetic
Change
Normal
Cell
Clonal
Expansion
Initiated
Cell
Genetic
Change
Preneoplastic
Lesion
1. Activation of Protooncogenes
2. Inactivation of Tumor Suppressor Genes
Malignant
Tumor
Molecular Oncology
G0
•Diagnostic
•Prognostic
•Predictive
•Therapeutic
G1
X
CELL
CYCLE
M
X
X
S
X
G2
Qualitative
Quantitative
Therapeutic
Genotyping
Therapies in Human Cancer
•Challenges:
–Limited efficacy
–Emergence of resistance
–Unexpected toxicities
What is Personalized Medicine?
(Pharmacogenomics)
Current Practice
Personalized Medicine
One
One size
size fits
fits all
all
Trial and Error
• Trial and error
• The right treatment
for the right person
at the right time
HIV Life Cycle
HIV
Nucleoside Analogues (NRTIs)
Reverse Transcriptase
RNA
DNA
Nucleus
Host Cell
Non-Nucleosides (NNRTIs)
Protease Inhibitors (PIs)
HIV-1 Protease Mutations
L
10
IDV
1
IRV
L
K
20
V
24
32
I
MR
36
I
I
M
M
46
I
AG
54
I
73 77
71
V
V I
V
VT SA
82
L
84
90
99
M
I AFTS V
L
RTV
10
32
20
33 36
46
F
54
IL
71
77
82
84
90
71
73 77
82
84
90
VL
G
SQV
10
48
54
V
S
A
D
NFV
10
30
FI
N
36
N
46
71
10
32
46
FIRV
LPV/RTV
10
24
46
84 88
90
D
I
47
I V
20
82
AFTS
IL
I
APV
77
50
V
84
54
VM
F
53
L
54
L
63
71
P
82
84
90
PGx: Personalized for the Individual Patient
Optimal drug response
Reduced adverse effects
Systemic response
PGx for the Cancer Patient
1)
2)
3)
4)
5)
6)
Not always systemic target
Tumors may be heterogenous
Metabolic vs target polymorphisms
Sporadic vs germline variants
Tissue based detection vs whole
blood
Same tumor, different site
(metastasis)
Technologies for Therapeutic Genotyping
ImmunoHistoChemistry
(IHC)
Fluorescence in situ
Hybridization (FISH)
Abbott Molecular
Dako
•
•
•
•
Inexpensive
• Fluorescent labeled DNA probes
hybridize to target
Subjective scoring
Semi-quantitative
• Hybridized DNA probes fluoresce,
giving off brightly lit signals that are
Protein target may be affected
easily viewed and analyzed
by tissue processing
Technologies for Therapeutic Genotyping
Real Time PCR - Allelic Discrimination
SNP Genotyping Assay
Vic (fluorescence)
5
4
3
2
1
0
0
1
2
3
Fam (fluorescence)
Vic and Fam represent two commonly used fluorescent
dyes to label real time detection probes.
Technologies for Therapeutic Genotyping
DNA Sequencing
Technologies for Therapeutic Genotyping
Luminex xMAP Technology
– Color-coded 5.6 micron microspheres
• 100 different colors, proprietary dyeing process
– Luminex 100 System
• Advanced optics, lasers, fluidics, DSPs, software
A Specific Example of PGx (Metabolism) for
Colorectal Cancer
•Topoisomerase inhibitor
•Irinotecan, CPT-11, 7-ethyl-10[4-(1(piperidion)-1piperidino]carbonylcamptothecin,
Camptosar®
•5-FU, leucovorin
• Uridine diphosphate glucuronosyl- transferase 1A1
• Reduced expression in Crigler-Najjar Type I and Gilbert’s
Syndrome (hyperbilirubinemia)
TA Repeats
Genotype
Frequency
TA6/6
*1/*1
46%
TA6/7
*1/*28
39%
TA6/8
*1/*37
3%
TA7/7
*28/*28
9%
TA7/8
*28/*37
1.5%
Irinotecan Metabolism
N
CH3
CH3
N
HO
O
O
N
N
N
N
O
HO
H3C
UGT1A1
O
CES
O
O
Irinotecan
CH3
O
HO
O
HO
H3C
O
N
HO
O
O
OH
N
OH
O
HO
O
SN-38
H3C
SN-38G
O
Personalized Medicine Is Also
Predicting Response to
Targeted Therapies
Pathways for Targeted Therapy
•
•
•
•
•
•
•
•
•
Angiogenesis
Apoptosis
Cell cycle control
Cell motility,
metastases
Gene expression
Growth factor
receptors
Immune responses
Mitochondrial
Signal transduction
Targeted Therapy
Hormonal Therapy Represents the Earliest Targeted Therapy
HER-2: The Poster Child for Targeted Therapy
HER-2 receptor
protein
HER-2
mRNA
Cytoplasm
HER-2 DNA
Nucleus
Cytoplasmic
membrane
Detecting HER-2
Protein expression by IHC
HER-2
Genes
HER-2
Protein
Herceptin
Gene amplification by FISH
CML: The New Poster Child for Targeted Therapy
• Diagnostic
• Therapeutic monitoring
• Resistance
The BCR-ABL Chimeric Fusion Protein
9
9+
22
Ph
bcr
abl
bcr-abl
FUSION PROTEIN
WITH TYROSINE
KINASE ACTIVITY
Detection of BCR-ABL
Positive for BCR-ABL
Flourescence
600
500
400
300
Internal Control
BCR-ABL
200
100
0
0
10
20
30
Cycles
Adapted from Cepheid
40
50
Therapeutic Options for CML
•ChemotherapyàHydroxyurea,
Busulfan
•Interferon-alpha
•Allogeneic Stem Cell
Transplantation
•Gleevec® (Imatinib Mesylate)
Gleevec®-Tyrosine Kinase Inhibitor
tra
bs
Gleevec
Su
tra
Su
P P P
bs
ATP
te
BCR-ABL
te
BCR-ABL
P
ATP in its specific binding site in the kinase domain of the protein is able
to phosphorylate tyrosine residues on selected substrates. The
phosphorylated substrate then binds with other molecules and activates
downstream pathways in leukaemogenesis. Gleevec occupies the ATP
pocket in the BCR-ABL kinase domain and substrates cannot be
phosphorylated.
Therapeutic Goals in CML
• Hematologic response: normal PB values and
spleen size.
• Cytogenetic response: reduction of Ph+ cells in
the blood or bone marrow.
Complete = 0% Ph+ cells
Partial CR = 1-35% Ph+ cells
Minor CR = 36-95% Ph+ cells
• Molecular response: reduction or elimination of
bcr-abl mRNA in marrow or PB.
Imatinib Resistance
>90% Imatinib resistance is due to reactivation of the bcr-abl
tyrosine kinase activity.
Persistance of bcr-abl kinase activity in the presence of imatinib
is mostly due to selected point mutations.
Mutations That Induce Resistance to Imatinib
48 MUTATIONS
Adapted from Kantarjian et al, Ann Intern Med 2006, 145:913-923
Another example of PGx-targeted
therapy in human cancer:
KRAS & EGFR
Prognostic vs Predictive Markers
•Prognostic marker- indicator of survival
independent of therapy; indicator of tumor
aggressiveness
•Predictive marker- indicator of response to
therapy, such as PFS or OS; (or in some
cases prediction of severe toxicity)
EGFR and KRAS
• EGFR- epidermal growth factor receptor, ErbB1
– Cell-surface receptor tyrosine kinase
– Activating mutations confer susceptibility to small
molecule TKI’
s
– Resistance mutations also occur
• KRAS- Kirsten rat sarcoma virus (human homolog)
– GTP binding protein; signal transduction downstream of
cell surface receptor
– Activating mutations (reduced GTPase activity) negate
the requirement for upstream receptor activation
EGFR and Targeted Therapies
mAb –Cetuximab or Panitumumab
Y
Ligand
EGFR
Tyrosine Kinase
Domain
Signaling
pathways
TKI – Gefitinib, Erlotinib
Ras is downstream of EGFR, activating mutations in
KRAS override EGFR status
mAb –Cetuximab or Panitumumab
Y
Ligand
EGFR
Tyrosine Kinase
Domain
Signaling
pathways
TKI – Gefitinib, Erlotinib
SOS
Ras
Raf
Evaluation of EGFR Status
Normal
Amplified
EGFR Mutation Analysis –Exon 21
Normal
Pre-digest
Positive
EGFR Mutation Analysis –Exon 21 Sequencing
Mutant
EGFR Mutation Analysis –Exon 19 del
KRAS Activating Mutations
• Found in 30-50% of CRCa’
s
• Associated with smoking in NSCLC
• Occur most often in codons 12 and 13
• Missense mutations (change of amino acid)
• 7 common mutations, account for at least 95%
of all identified
• A few mutations in other locations have been
reported ex: codon 61
KRAS mutation testing
•DNA extracted from archival FFPE tumor
sample (typically primary tumor)
•Detection limit: 5% mutation in wild-type
background
•Detects 7 mutations in codons 12 and 13
–Gly12Asp
–Gly12Ala
–Gly12Val
–Gly12Ser
Gly12Cys
Gly12Arg
Gly13Asp
Allelic Discrimination for KRAS Mutations
GLY12ASP, 35G>A
GLY12ARG, 34G>C
GLY12CYS, 34G>T
GLY12ALA, 35G>C
KRAS Mutation Detection by Sequencing
Codon
12
Codon
13
Gly
Gly
G>A mutation
(Gly12Asp)
Cancer PGx: Case report
The patient is an 85-year old woman who came to see her
primary care provider complaining of some fatigue and
inability to walk her usual distances. At that time she was
found to be anemic and an iron supplement was started
with recommendation for a colonoscopy. On colonoscopy,
a dense lesion was found at approximately 20 cm. A ringtype mass was confirmed by CT scan in the distal sigmoid
colon and diagnosed as a well differentiated
adenocarcinoma. The tumor was surgically excised.
Resected Specimen
Therapy Given
•5-FU, leucovorin, irinotecan
•Eloxatin, 5-FU, leucovorin (FOLFOX)
•Panitumumab
Clinical course
•Severe diarrhea
•Distal sensorineural symptoms worsen
and begin to ascend with secondary
unsteadiness of gait
•Unresponsive tumor
Clinical course???
• Severe diarrhea (homozygous *28 UGT1A1)
• Distal sensorineural symptoms worsen and begin
to ascend with secondary unsteadiness of gait
(homozygous GSTP1 105Ile)
• Unresponsive tumor (KRAS Gly12Asp)
Eloxatin (oxaliplatin; Sanofi-Aventis)
•Third generation platinum analog
•Less ototoxicity/nephrotoxicity than
Cisplatin or Carboplatin
•Main adverse effect: cumulative peripheral
neuropathy
Glutathione S-Transferase (GST)
• A family of cytosolic, mitochondrial and microsomal
enzymes
• Catalyze conjugation of xenobiotics to reduced
glutathione
• At least four classes, including pi (GSTP1), located at
11q13
– Polymorphisms are associated with enzyme activity variations
– SNP (exon 5; A313G/Ile105Val) predicts oxaliplatin neurotoxicity
(n=90)
• Homozygosity (wild-type, 105Ile) decreased activity in vitro
– Increased risk (OR 5.75, p=0.02) of Grade 3 peripheral neuropathy
• Heterozygosity/homozygosity (105Val) increased activity in vitro
– Decreased risk of specific neurotoxicity
Conclusions
Qualitative
Quantitative
Genotyping
1.
Diagnostics will become even more crucial to driving
therapeutics.
2.
Targeted therapies will play a critical role in the
management of the cancer patient.
3.
The use of these therapies will require target
identification by the clinical laboratory (genomic,
proteomic).
4.
Targeted therapies present new concepts and challenges
for personalized medicine (acquired vs inherited).
DHMC Molecular Pathology and Translational Research
Laboratories
Darcy Arendt, DMS IV
Claudine Bartels, Ph.D.
Heather Bentley
Samantha Calderon
Betty Dokus
Susan Gallagher
Carol Hart
Arnold Hawk
Joel Lefferts, Ph.D.
Angela Marchetti
Rebecca O’
Meara
Mary Schwab