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p53 protein expression and genetic mutation in
two primary cell types in pulmonary sclerosing
haemangioma
Y Wang, S-D Dai, F-J Qi, H-T Xu and E-H Wang
J. Clin. Pathol. 2008;61;192-196; originally published online 17 Aug 2007;
doi:10.1136/jcp.2007.050401
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Original article
p53 protein expression and genetic mutation in two
primary cell types in pulmonary sclerosing
haemangioma
Y Wang, S-D Dai, F-J Qi, H-T Xu, E-H Wang
Department of Pathology,
College of Basic Medical
Sciences, China Medical
University and Department of
Pathology, First Affiliated
Hospital of China Medical
University, Shenyang 110001,
China
Correspondence to:
Dr En-Hua Wang, Department of
Pathology, College of Basic
Medical Sciences, China
Medical University and
Department of Pathology, First
Affiliated Hospital of China
Medical University, Shenyang
110001, China; wangeh@
hotmail.com
Accepted 2 August 2007
Published Online First
1 October 2007
ABSTRACT
Aims: To investigate the significance of p53 protein
expression and genetic mutations in two primary cell
types in pulmonary sclerosing haemangioma (PSH).
Methods: p53 protein expression in polygonal cells and
cuboidal cells in 19 patients with PSH was detected using
immunohistochemistry. The two major cell types were
captured using laser capture microdissection technology.
Mutations in the p53 gene (exons 5–8) were examined
using single-stranded conformation polymorphism and
DNA sequencing analysis.
Results: p53 protein expression and gene mutations
were observed in 15.8% (3/19) of cases. In these cases,
p53 protein was expressed in the nucleus of both cell
types, with higher expression levels and mutation rates in
polygonal cells than in surface cuboidal cells. Two cases
showed mutation only in the polygonal cells, while one
case showed double (separate) mutations in both the
polygonal and cuboidal cells.
Conclusions: p53 mutation was exhibited in PSH. The
mutation rate in polygonal cells was higher than that in
surface cuboidal cells.
Pulmonary sclerosing haemangioma (PSH) is a rare
pulmonary tumour of unknown origin, although
the histological characterisation and biological
behaviour of PSH have long been a focus of
research.1–5 PSH is widely believed to be a benign
tumour because of its clinical course; however,
Miyagawa-Hayashino et al6 reported metastases in
approximately 2–4% of PSH cases, necessitating
further exploration of the development and progression of this tumour.
Mutations in the p53 gene, and associated p53
protein accumulation, are the most common
events associated with the development and
clinical progression of malignant tumours.7–12
Several authors have detected expression of p53
mRNA and protein in PSH, although their findings
differed significantly.5 13 14 In the present study, we
evaluated expression of the p53 protein in two
types of cells affected by PSH via streptavidin
peroxidase immunohistochemistry. Mutations in
the p53 gene were detected by single-stranded
conformation polymorphism (SSCP) and sequencing analyses, providing data for investigating the
biological behaviour of PSH.
METHODS
Subjects
Paraffin-embedded PSH samples and normal lung
tissue adjacent to PSH were obtained from 19
patients (two men and 17 women) who were
192
diagnosed with PSH at the First Clinical College of
China Medical University between 1995 and 2004.
This study was conducted according to the
regulations of the institutional review boards
(China Medical University). The age range was
24–46 years, and the mean age was 34.5 years. Of
the 19 patients, 12 had no symptoms and their
PSH was found upon routine examination; the
other seven patients presented with symptoms
including chest pain, cough and bloody phlegm.
x Rays or CT scans revealed unitary masses,
frequently in the periphery of the lungs. These
masses were round or oval and of high density,
with a smooth boundary, and with no burs or
lobules. The masses were located in the right lung
in 11 cases and the left lung in eight cases. The
follow-up time (after surgery, to December 2005)
ranged from 13–129 months; all patients survived,
without recurrence or metastasis.
Immunohistochemistry
Formalin-fixed paraffin-embedded tissue blocks
were cut into 4 mm sections, de-waxed and
hydrated. Immunostaining was performed with
the streptavidin peroxidase system (Ultrasensitive;
MaiXin, Fuzhou, China) according to manufacturer’s instructions. The sections were incubated
with a primary anti-p53 antibody (DO-7, dilution
1:50; Santa Cruz Biotechnology, Santa Cruz,
California, USA). Biotinylated goat anti-mouse
serum IgG was used as a secondary antibody.
After washing three times in phosphate-buffered
saline (PBS), the sections were incubated with
streptavidin-biotin conjugated with horseradish
peroxidase, and visualised by demonstration of
conjugated peroxidase with diaminobenzidine as
the substrate. The sections were counterstained
with haematoxylin. For the negative control,
primary antibodies were replaced with PBS; for
the positive control, known positive tissue was
used. A slide was considered negative or positive
according to the absence or presence of positive
staining: no staining or fewer than 5% of total cells
positive for p53 was considered negative; greater
than 5% of total cells positive for p53 was
considered positive staining.
Laser capture microdissection of target cells, and
extraction of DNA
The paraffin-embedded samples were sectioned
successively at a thickness of 6 mm. The sections
were subjected to Mayer’s H&E staining, dehydration with gradient alcohol, and lucidification with
xylene (for 5 min). After drying, the sections were
J Clin Pathol 2008;61:192–196. doi:10.1136/jcp.2007.050401
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Original article
placed onto the object stage of an inverted microscope
connected to a laser capture microdissection system (model
LM200; Olympus, Tokyo, Japan). The target cells were
identified with an orientating beam and then captured by a
laser beam. A total of 5000–10000 surface cuboidal cells and
polygonal cells were captured (fig 1). A 0.5-ml centrifuge tube
containing 50 ml DNA lysis buffer (10 mmol/l Tris HCl, pH8.0;
1 mmol/l EDTA, pH 8.0; 1% Tween 20; 200 mg/ml proteinase
K) was incubated at 48uC for 14 h, proteinase K was deactivated
at 95uC for 10 min, and the tube was stored 220uC until further
analysis.
PCR-SSCP and sequencing
PCR reaction mixture (50 ml) containing 10 ml DNA isolated
from polygonal or surface cuboidal cells served as a template,
and was mixed with 1 ml primers (30 pmol/ml; 5–8 exons), 0.4 ml
Taq polymerase, 4 ml dNTP, and 5 ml 106 PCR buffer. The PCR
conditions included initial denaturing at 94uC for 2 min, then
40 cycles at 94uC for 40 s, after which samples were subjected
to annealing (see table 1 for temperatures and times), and a final
extension at 72uC for 1 min. The PCR products (4 ml) were
loaded onto a 2% agarose gel to confirm successful amplification
and non-specific bands, followed by SSCP analysis. The PCR
product (6 ml) was mixed with an equal volume of loading dye
and denatured at 100uC for 10 min, and placed on ice for 5 min.
The samples were then separated on 10% non-denaturing
polyacrylamide gel (49:1). For each condition, we used adjacent
normal lung tissue as a control. After electrophoresis, the gel
was fixed, silver-stained, developed, photographed and analysed. The sample was considered normal if the band position
was the same as that of the normal tissue. The same PCR
products were used for sequencing (Shanghai United Gene
Biotechnology, Shanghai, China).
RESULTS
Gross and histological study
The nodules were 1.4–4.9 cm in diameter and were well
circumscribed with or without capsule. They were medium
soft and often had a pale-brown region caused by haemorrhage.
The tumour showed expansive growth pattern without multiple masses, infiltration and metastasis in any case.
Histologically, all cases showed varying degrees of solid,
papillary, haemorrhagic and sclerotic patterns. The tumours
were composed entirely of polygonal and cuboidal cells.
Polygonal cells were located in the solid and papillary areas,
which had faintly stained or eosinophilic cytoplasm, round or
oval nuclei, and small nucleoli, but rare or no karyomitosis.
Surface cuboidal cells covered the papilla or were located in the
Table 1 p53 primer sequences, length of amplified fragments and PCR
annealing temperatures
Exon
Primer sequence
5
59-GCTGCCGTGTTCCAGTTGCT-39
59-CCAGCCCTGTCGTCTCTCCA-39
59-TTGCTCTTAGGTCTGGCCCC-39
59-CAGACCTCAGGCGGCTCATA-39
59-TAGGTTGGCTCTGACTGTACC-39
59-TGACCTGGAGTCTTCCAGTGT-39
59-TATCCTGAGTAGTGGTAATC-39
59-AAGTGAATCTGAGGCATAAC-39
6
7
8
Product
size
PCR annealing
temperature and time
294 bp
61uC, 40 s
127 bp
58.8uC, 40 s
116 bp
58.8uC, 40 s
213 bp
51uC, 40 s
Figure 1 Laser captured pulmonary sclerosing haemangioma (PSH) polygonal cells and surface cuboidal cells. (A) PSH tissue, showing cuboidal cells
on the papillary surface and polygonal cells in the stroma, (H&E, not mounted). (B) PSH tissue after laser capture of papillary surface cuboidal cells. (C)
Papillary surface cuboidal cells captured by laser from PSH tissue. (D) PSH tissue, showing polygonal cells in the solid areas (H&E, not mounted). (E)
PSH tissue after laser capture of polygonal cells. (F) Polygonal cells captured by laser from PSH tissue.
J Clin Pathol 2008;61:192–196. doi:10.1136/jcp.2007.050401
193
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Original article
interspaces of the haemorrhagic areas and in the lacuna spaces
of the solid areas. These cells were mostly cuboidal, and some
were thin and flat, or cylindrical. These cells had merged into
multinucleated giant cells,3 but did not present as heteromorphic. Infiltration of inflammatory cells such as lymphocytes, infiltrated haemosiderin deposition, calcification, and
ossification could be observed in the interstitium (fig 2A–F).
Immunohistochemistry
p53 protein expression was observed in both cell types in three
out of the 19 PSH tissue samples (15.8%), with more
immunoreactive polygonal cells than immunoreactive surface
cuboidal cells (fig 3). Of the other 15 samples showing no p53
protein expression, one case exhibited immunoreactivity, but in
fewer than 5% of cells. There was no p53 protein expression in
any samples of normal lung tissue.
SSCP and sequencing analyses
As shown in table 2, the SSCP analysis and DNA sequencing
revealed that abnormal mobility bands and mutations were
observed in three p53-immunoreactive cases, with a mutation
rate of 15.8% (fig 4A,B). A mutation in the p53 gene occurred in
exon 6 in one case, and exon 7 in two cases. No mutations were
found in exons 5 and 8. Two cases showed mutation only in the
polygonal cells, while one case shows double (separate)
mutations in both the polygonal and cuboidal cells. Four
missense mutations were identified and their amino acid
sequences were predicted (table 2).
DISCUSSION
It is generally believed that PSHs are benign tumours because of
a clear demarcation of the haemangioma from surrounding
tissues, low cellular heteromorphism, rare or no karyomitosis,
and good clinical prognosis. However, some authors classify
PSH as a potential malignant tumour because the tumour tissue
has been found to infiltrate the surrounding interstitium or
bronchi, and because local lymph node metastasis of the tumour
has been reported.1 6 15–21
Although several markers, including cytokeratin, Vimentin,
carcinoembryonic antigen and surfactant protein A, are
differentially expressed in polygonal cells and surface cuboidal
cells in PSH, and there are characteristic lamellar bodies in
surface cuboidal cells, both types of cell can express epithelial
membrane antigen and thyroid transcription factor 1. Because
thyroid transcription factor 1 can be specifically expressed in the
fetal lung epithelial cell nuclei, it is thought that both types of
cells derive from primitive respiratory epithelium, and that the
Figure 2 Histomorphological changes in
pulmonary sclerosing haemangioma
(PSH). PSH is composed of sclerotic (A,
upper left), hemorrhagic (A, lower right),
solid (B), and papillary (C) areas. Papillary
surface cuboidal cells (C) were not
immunoreactive to Vimentin but
immunoreactive to surfactant protein B;
these cells occasionally fused into
multinucleated giant cells (D). There are
many mast cells (F) and widespread
calcification (E).
194
J Clin Pathol 2008;61:192–196. doi:10.1136/jcp.2007.050401
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Original article
Figure 3 p53 protein expression in
pulmonary sclerosing haemangioma
(PSH). (A) Significantly more polygonal
cells were p53 immunoreactive than
cuboidal cells in PSH (streptavidin
peroxidase immunostaining, 6400). (B)
The PSH sample from case 2 was not p53
immunoreactive (cells with
immunoreactive nuclei ,5%)
(streptavidin peroxidase immunostaining,
6400).
differences in their morphological phenotype are due to
disparate differentiation states.1 2 22 However, it remains
unknown whether the two cell types behave similarly in vivo.
To further investigate the role of these two cell types in PSH,
we studied the cell-specific expression of the p53 gene, a gene
that is heavily implicated in human tumourigenesis. Previous
reports suggest that approximately 50% of human tumours are
associated with mutations in, or overexpression or loss of
heterozygosity of, the p53 gene.7 8 Greenblatt et al18 studied the
p53 gene sequence related to human tumours and found that
approximately 87% of p53 gene mutations occur in exons 5–8.
Based on these findings, we investigated potential mutations in
exons 5–8 of the p53 gene in PSH tissue. The present results
demonstrate that the p53 protein is expressed in 15.8% of the
PSH samples, with a higher expression in polygonal cells than in
surface cuboidal cells. The SSCP and sequencing analyses
demonstrated that p53 gene mutations occurred in both cell
types, with a higher gene mutation rate in polygonal cells than
in surface cuboidal cells. We have also found with the same
tissue samples that E-cadherin, b-catenin, and p120ctn are
abundantly expressed on surface cuboidal cell membrane, but
are poorly or not expressed in polygonal cell cytoplasm,23
suggesting a marked formation of the cad–cat complex in the
former and a lack of formation of the cad–cat complex in the
latter. These findings support the hypothesis that the two cell
types are at different differentiation states, and may help
explain why polygonal cells, rather than surface cuboidal cells,
have been observed in PSH metastases.2 6 15
p53 gene sequencing revealed an mutation rate of 15.8% (3/
19) in PSH, a figure that confirms the positive rates for the SSCP
and immunohistochemical analyses. The results suggest that
PSH exhibits potential malignant biological behaviour and may
help explain the clinical findings of occasional infiltration and
metastasis of PSH.
In summary, the finding of p53 mutations in a small number
of PSH is supportive of PSH being a neoplastic process. p53 gene
Figure 4 Mutations in exon 7 in the
polygonal cells of the pulmonary
sclerosing haemangioma sample from
case no. 14, as demonstrated by the
single-stranded conformation
polymorphism (SSCP) and sequencing
analyses. (A) SSCP analysis of exon 7 of
the p53 gene in normal lung tissue (N)
and samples. Polygonal cells (P) show
abnormal mobility bands. M, marker; C,
surface cuboidal cells. (B) p53 exon 7
sequencing. No mutations were found in
surface cuboidal cells (C) while a
mutation was identified in polygonal cells
(P) of case no. 14, GCCRGGC (ie,
glycineRalanine).
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Original article
Table 2 p53 Mutation and expression analysis of pulmonary sclerosing haemangioma
p53 gene sequence
Case no.
Cell type
SSCP
Exon, mutation
Predicted
effect
8
Polygonal
Cuboidal
Polygonal
Cuboidal
Polygonal
Cuboidal
Shift band
None
Shift band
Shift band
Shift band
None
6, GTGRGCG
5–8, none
7, ATCRTTC
7, AACRACC
7, GGCRGCC
5–8, none
V197A
None
I232L
N247C
G244A
None
12
14
No. of
cited db
p53
protein
expression
6
6
137
43
22
22
+
–
+
+
+
–
No. of cited db, number of times the mutation has been found in human cancers in the p53 database.24
6.
Take-home messages
7.
c
c
We investigated p53 protein expression and genetic mutations
in two primary cell types in pulmonary sclerosing
haemangioma using laser capture microdissection,
immunohistochemistry, single-stranded conformation
polymorphism and DNA sequencing.
The key findings of our work were that we discovered the p53
mutation exhibited in pulmonary sclerosing haemangioma, and
that the mutation rate in polygonal cells was higher than in
surface cuboidal cells.
8.
9.
10.
11.
12.
mutations were found to occur in PSH, and does this indicate
potential malignant biological behaviour of this type of PSH?
The question requires further support from future investigations with larger sample sizes and case follow-up, particularly
cases with lymph node metastases and recurrent PSH.
13.
14.
15.
Competing interests: None.
Ethics approval: Ethics committee approval was obtained.
REFERENCES
1.
2.
3.
4.
5.
196
Devouassoux-Shisheboran M, Hayashi T, Linnoila RI, et al. A clinicopathologic
study of 100 cases of pulmonary sclerosing hemangioma with immunohistochemical
studies: TTF-1 is expressed in both round and surface cells, suggesting an origin from
primitive respiratory epithelium. Am J Surg Pathol 2000;24:906–16.
Chan AC, Chan JK. Pulmonary sclerosing hemangioma consistently expresses
thyroid transcription facter-1: a new clue to its histogenesis. Arch Pathol Lab Med
2001;125:1531–6.
Wang E, Lin D, Wang Y, et al. Immunohistochemical and ultrastructural markers
suggest different origins for cuboidal and polygonal cells in pulmonary sclerosing
hemangioma. Hum Pathol 2004;35:503–8.
Niho S, Suzuki K, Yokose T, et al. Monoclonality of both pale cells and cuboidal cells
of sclerosing hemangioma of the lung. Am J Pathol 1998;152:1065–9.
Dacic S, Sasatomi E, Swalsky PA, et al. Loss of heterozygosity patterns of sclerosing
hemangioma of the lung and bronchioloalveolar carcinoma indicate a similar
molecular pathogenesis. Arch Pathol Lab Med 2004;128:880–4.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Miyagawa-Hayashino A, Tazelaar HD, Langel DJ, et al. Pulmonary sclerosing
hemangioma with lymph node metastases: report of 4 cases. Arch Pathol Lab Med
2003;127:321–5.
Shabnam MS, Srinivasan R, Wali A, et al. Expression of p53 protein and the
apoptotic regulatory molecules Bcl-2, Bcl-XL, and Bax in locally advanced squamous
cell carcinoma of the lung. Lung Cancer 2004;45:181–8.
Miller LD, Smeds J, George J, et al. An expression signature for p53 status in
human breast cancer predicts mutation status, transcriptional effects, and patient
survival. Proc Natl Acad Sci U S A 2005;102:3550–5.
Brambilla E, Negoescu A, Gazzeri S, et al. Apoptosis-related factors p53, Bcl2, and
Bax in neuroendocrine lung tumors. Am J Pathol 1996;149:1941–52.
Jackel MC, Sellmann L, Youssef S, et al. Prognostic significance of expression of
p53, bcl-2 and bax in squamous epithelial carcinoma of the larynx—a multivariate
analysis. HNO 2001;49:204–11.
Sirvent JJ, Aguilar MC, Olona M, et al. Prognostic value of apoptosis in breast
cancer (pT1-pT2). A TUNEL, p53, bcl-2, bag-1 and Bax immunohistochemical study.
Histol Histopathol 2004;19:759–70.
Bandoh N, Hayashi T, Kishibe K, et al. Prognostic value of p53 mutations, bax, and
spontaneous apoptosis in maxillary sinus squamous cell carcinoma. Cancer
2002;94:1968–80.
Chiba W, Sawai S, Yasuda Y, et al. Positive expression of c-myc and p53 products in
two cases of pulmonary sclerosing hemangioma. Nihon Kyobu Shikkan Gakkai Zasshi
1995;33:1348–54.
Iyoda A, Hiroshima K, Shiba M, et al. Clinicopathological analysis of pulmonary
sclerosing hemangioma. Ann Thorac Surg 2004;78:1928–31.
Yano M, Yamakawa Y, Kiriyama M, et al. Sclerosing hemangioma with metastases
to multiple nodal stations. Ann Thorac Surg 2002;73:981–3.
Jungraithmayr W, Eggeling S, Ludwig C, et al. Sclerosing hemangioma of the lung:
a benign tumour with potential for malignancy? Ann Thorac Cardiovasc Surg
2006;12:352–4.
Chan NG, Melega DE, Inculet RI, et al. Pulmonary sclerosing hemangioma with
lymph node metastases. Can Respir J 2003;10:391–2.
Katakura H, Sato M, Tanaka F, et al. Pulmonary sclerosing hemangioma with
metastasis to the mediastinal lymph node. Ann Thorac Surg 2005;80:2351–3.
Komatsu T, Fukuse T, Wada H, et al. Pulmonary sclerosing hemangioma with
pulmonary metastasis. Thorac Cardiovasc Surg 2006;54:348–9.
Kim KH, Sul HJ, Kang DY. Sclerosing hemangioma with lymph node metastasis.
Yonsei Med J 2003;44:150–4.
Tanaka I, Inoue M, Matsui Y, et al. A case of pneumocytoma (so-called sclerosing
hemangioma) with lymph node metastasis. Jpn J Clin Oncol 1986;16:77–86.
Nagata N, Dairaku M, Sueishi K, et al. Sclerosing hemangioma of the lung. An
epithelial tumor composed of immunohistochemically heterogenous cells. Am J Clin
Pathol 1987;8:552–9.
Dai Shun-Dong, Zhang Xiu-Wei, Qi Feng-Jie, et al. Expression of E-cadherin, bcatenin and p120ctn in the pulmonary sclerosing hemangioma. Lung Cancer
2007;57:54–9.
The p53 Web Site. http://p53.free.fr (accessed 10 December 2007).
J Clin Pathol 2008;61:192–196. doi:10.1136/jcp.2007.050401