Study Of The Origin And Character In So-Called Pulmonary Sclerosing Hemangioma

ObjectivePSH is a rare benign neoplasm, but it has uncertain about its histogenesis and character up to now. PSH consists of two major tumor cells: polyangular cells and cuboidal cells. Polyangular cells are true tumor cells which derive from primitive respiratory epithelium, but it remains controversial over whether the surface lining cuboidal cells of PSH are true tumor cells or the results of responsive proliferation in the tumor genesis and whether this two major tumor cells have the same origin. Although most PSHs have well clinical process, yet the phenomenon which lesion tissues invaded bronchus and peripheral mesenchyma can be seen by microscopic observe and the cases with lymph metastasis have been reported. In order to further verify the origin and the possible character of PSH,we captured two major cells of PSH by laser capture micro - dissection (LCM) and adopted cional analysis of X -linked androgen receptor (AR) and phosphoglycerate kinase (PGK) gene to explore whether the cuboidal or polyangular cells have the same origin;We selected the p53 gene which was related to pathogenesis of this disease as the subject of study and used Immunohistochemi-cal method, laser capture micro - dissection ( LCM ) technology, Single - stranded Conformation Polymorphism (SSCP) and DNA sequencing analysis to examine the expression of p53 protein and mutation of p53 gene in polyangular cells and cuboidal cells in PSH and valued its significance, providing reference to histogenesis and biological conduct of PSH.Materials and Methods PatientsAll 19 diagnosed cases of PSH (from 1995 to 2005) were obtained from the Pathology Diagnostic Center of the First Affiliated Hospital of China Medical U-niversity. 2 of the patients were male, and the other 17 were female.ImmunohistochemistryAnti - p53 (DO - 7 ) , SP and DAB kit were the product of Fuzhou Maixin Biological Technique Company. The paraffin sections were sliced into 4jxm thick samples. HE and the avidin - biotin immunoperoxidase method (SP) were performed after deparaffinized, dehydrated. The positive controls were sections that were known to have a p53 protein accumulation. The negative control consisted of replacing the primary antibody with PBS. The presence of nuclear staining or nuclear and cytoplasmic staining in any number of cells seen at 100 magnification was considered a positive reaction. Semiquantitative evaluations of immunohistochemistry results were performed according to the percentages of positive cells;no positive cells or positive cells^5% ( - ) , positive cells >5% ( + ) 0LCM and DNA extraction6 - u-m sections were cut from the paraffin blocks, and deparaffinized, dehydrated , stained with Mayer's hematoxylin 3min and eosin 20s, and then lucid-itied by xylene 5 min. Slides are dried and immediately was fixed on the object stage of LCM (laser capture micro - dissection, LCM) 200 system. Then a thin and flat cap with ethylene vinyl acetate polymer ( EVA ) membrane was tightly covered onto the surface of section. After a beam of light located the intended cells, the laser beam was projected to capture the lining cuboidal cells and poly-angular cells. When 5000 intended cells were captured from each specimen, and DNA is isolated from captured cells by DNA lysate. Processed by 48*0 water - baths for 14 hours, then the proteinase K was inactivated by heating at 95° C for 10 min and stored in -20TI until use.Nested - Polymerase Chain Reaction for AR,PGK geneBriefly, approximately 5ui of extracted genomicDNA in a total reaction volume of 20 jxl was digested with ljxl Hhal or Hpall, forlO h at 37°C. For each specimen, a control sample containing only deionised H2O was run simultaneously. The restriction enzyme was then inactivated by heating at 70° C for 15 min. For the first PCR, 5ui of each digested products was added to 25 jxl ofPCR reaction mixture consisting of 0. 5 jxl of each primer ARIA, AR1B or PGK1A, PGK1B. luJ of this PCR mixture was added to the second PCR reaction mixture. The second PCR was performed under the same conditions as the first PCR for 35 cycles using primers AR2A, AR2B or PGK2A, PGK2B. 8|xl the second PCR products of AR and 8ui loading buflfer were used to denature polyacrylamide gel electrophoresis ( containing 6mol/L carbamide) for 3 hours at 80V. Gels were then silver - stained. 5ui the second PCR products of PGK were digested with BstXI for 14h at 45 C. The residual BstXI was then inactivated by heating at 65 C for 15 min. BstXI - digested products were electrophore-sed in a 2% agarose gel, and bands were detected with ethidium bromide staining for lOmin.As the criterion of Lee et al, the allele inactivation ratio was definited as the allele amplification ratio of the Hhal -digested sample (allelel/allele2) divided by the allele amplification ratio of the Hhal -nondigested sample (allelel/ allele2 ). A clonality ratio was determined by dividing the allele inactivation ratio of the lesion by that of the normal tissue. If 0.25 < the clonality ratio of the lesion ^ 1, the lesion was judged to polyclonal proliferation;If 0 ^ the clonality ratio of the lesion ^ 0. 25, it was considered evidence of monoclonal proliferation;If 0 ^ the allele inactivation ratio of the normal tissue ^ 0. 25, it was considered to be skewed patterns of X - chromosome inactivation.p53 polymerase chain reation,Single -stranded Conformation Polymorphism analysis and DNA sequencinglOui genomic DNA extracted from the cuboidal and polyangular cells respectively was used for amplification of p53 gene( exon 5 ~ 8) in 50jxl of reaction mixture. 6jxl PCR products of p53 were mixed with 6jxl loading and denaturing buffer were heated at 100t for 10 min, and immediately dipped into the mixture of ice - water for 5 min and then loaded onto 10% nondenaturing polyacrylamide gel electrophoresis (49:1). Electrophoresis was carried out at constant temperature. Gels were then silver - stained. Compared with the normal control bands, the sample which showed additional bands or abscent bands or shifts revealed the presence of p53 gene mutation. 40jxl of PCR products for p53 (exon 5 ~8)were used to direct DNA sequencing(Shang Hai Gene SequencingCompany).ResultsHistological featuresMost tumors displayed four histological patterns: solid, papillary, hemor-rhagic and sclerotic. The Four histological patterns transit among each other. There are two major tumor cells: polyangular cells and cuboidal cells. Polyangu-lar cells lie in solid and papillary regions with uniform in shape, clear or eosino-philic cytoplasm and a round or oval nucleus. Mitotic figures were rarely seen. Cuboidal cells are on the surfaces of papillary and monolayer and arrange tightly, with flat and column cells rarely seen. Cuboidal cells can merge into multi-nuclear giant cells, but have not heteromorphism. There are large luman spaces with a lot of erythrocytes in hemorrhagic areas. In the stroma was infiltrated with lymphocytes, hemosiderin sediment,calcification and ossification.Immunohlstochemical resultsOf 19 PSH, 4 cases (21. 1% ) was the positive expression of p53 protein (the number of positive cells is more than 5% ) and the intensity of p53 expression in polyangular cells was higher than that in surface cuboidal cells;15 cases (78.9% ) of 19 was the negative expression of p53 protein, and one of the im-munonegative PSHs had p53 positive cells, but the number of positive cells is less than 5%.Clonal analysis of AR, PGK gene1. Clonal analysis of AR geneIn all 17 cases of female PSH, 15 samples were successfully amplified for AR gene. 2 major cells of 8 samples showed two DNA bands between 200bp to 300bp. But there were 2 cases unsuitable for the clonality test for the highly skewed patterns of X - chromosome inactivation in normal lung tissues of adjacency lesion (the allele inactivation ratio was 0.12 and 0.14, respectively) , so only 6 cases of 8 were utilized in clonal analysis. Among the 6 samples suitable for the clonality test, PCR product bands showed the same loss of allele ( clonality ratio =0) or unbalanced methylation pattern (clonality ratio =0.19).2. Clonal analysis of PGK geneThere were 15 female samples and 1 male sample successfully amplified for PGK gene. Only 4 cases of PSHs have polymorphism of PGK gene (the polymorphic rate was 27% ). With Hpall digestion and treated with BstXI, the two types of cells showed the same loss of allele (clonality ratio =0).Single - stranded Conformation Polymorphism and DNA sequencing analysis for p53 gene(5 ~8)5 cases of 19 PSHs showed abnormal shift bands by SSCP analysis. The mutant ratio of p53 gene by DNA sequencing analysis was 42.1% (8/19). For the mutant cells, unitary p53 gene mutations were identified in 5 polyangular cells and 2 cuboidal cells, and 1 case mutation was detected in polyangular cells and cuboidal cells simultaneously by direct sequencing analysis. For the mutant types, 3 cases were missense mutations and 4 cases were frameshift mutations and 1 case was both missense and frameshift mutations simultaneously.The comparison between the mutation of p53 gene and the expression of p53 protein2 of 4 cases p53 immunopositive PSHs were missense mutations and 1 was both missense and frameshift mutations;4 of 15 cases (33. 3% ) immunonega-tive PSHs tissue samples which had almost no positive cells were frameshift mutations and 1 case which positive cells was less than 5% was missense mutations by sequencing analysis. The all samples showing abnormal shift bands by SSCP analysis were confirmed to be mutations by DNA sequencing analysis.Conclusions1. The polyangular cells and cuboidal cells of PSH exhibited a uniform pattern of monoclonality which indicated that they both might be the true tumor cells.2. The alteration of p53 gene and the overexpression of p53 protein are i-dentified in polyangular cells and cuboidal cells. The mutant ratio of p53 gene in polyangular cells was higher than that in surface cuboidal cells. The high mutant ratio of p53 gene indicated that PSH may be have potentially malignant biologi-cal behavior.3. The expression of p53 protein may not be indicative of p53 gene mutations in PSHs.