Article Text
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.
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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 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 μm 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 μm. 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 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 μl DNA lysis buffer (10 mmol/l Tris HCl, pH8.0; 1 mmol/l EDTA, pH 8.0; 1% Tween 20; 200 μg/ml proteinase K) was incubated at 48°C for 14 h, proteinase K was deactivated at 95°C for 10 min, and the tube was stored −20°C until further analysis.
PCR-SSCP and sequencing
PCR reaction mixture (50 μl) containing 10 μl DNA isolated from polygonal or surface cuboidal cells served as a template, and was mixed with 1 μl primers (30 pmol/μl; 5–8 exons), 0.4 μl Taq polymerase, 4 μl dNTP, and 5 μl 10× PCR buffer. The PCR conditions included initial denaturing at 94°C for 2 min, then 40 cycles at 94°C for 40 s, after which samples were subjected to annealing (see table 1 for temperatures and times), and a final extension at 72°C for 1 min. The PCR products (4 μl) were loaded onto a 2% agarose gel to confirm successful amplification and non-specific bands, followed by SSCP analysis. The PCR product (6 μl) was mixed with an equal volume of loading dye and denatured at 100°C 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 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).
Take-home messages
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.
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 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, β-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 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.
REFERENCES
Footnotes
Competing interests: None.
Ethics approval: Ethics committee approval was obtained.