Differential Gene Expression Of Polygonal Cells And Cuboidal Cells In So-called Pulmonary Sclerosing Hemangioma

ObjectivePulmonary sclerosing hemangioma(PSH) is an uncommon tumor of the lung and it s histogenesis and origin are uncertain to date.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 whethere the surface lining cuboidal cells of PSH are true tumor cells or the results of responsive proliferation in the tumor genesis and whethere this two major tumor cells have the same origin.A general consensus appears to have been reached that PSH is a benign neoplasm,but a few PSHs are found to invade and metastasize.The phenomenon which lesion tissues invaded bronchus and peripheral mesenchyma can be seen reported.In order to further veritfy the origin and the possible character of PSH,we captured two major cells of PSH by laser capture micro-dissection(LCM).We selectedthe P53 gene which was related to pathogenesis of this disease as the subject of study and used immunohistochemical 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.The expression of p53 protein and mutation of p53 gene are significant parameters which can reflect biological behavior of tumor cells.The aim of this study is to investigate the p53 protein expression and p53 gene mutation.Disclosing the different gene expression of the two cell types will provide the basis for the study of histogenesis of PSH tissue and helpful for differential diagnosis.Materials and Methods1,PatientsParaffin-embedded PSH samples and normal lung tissue adjacent to PSH were obtained from 40 patients who were diagnosed with PSH at the First Clinical College of China Medical University between 1995 and 2004.Among those cases,19 cases were celected which had whole clinic data to do gene expression analysis.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.2,ImmunohistochemistryLow molecular weight cytokeratin(CK-L) and epithelial membrane antigen (EMA) sign epithelia antigen,surfactant protein B(SP-B) and thyroid transcription factor- 1(TTF-1) sign respiratory multipotential primitive epithelium or pneumocytes typeⅡ,Vimentin(VT) signs mesenchymal antigen,CD34 signs endothelial cells of vessels,CD68 signs macrophage,neuron specific endolase(NSE) and chromograninA(ChA) sign endocrine cell.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 antibody(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.3,Laser capture microdissection of target cells,and extraction of RNAThe 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 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.According PicoPure^TM RNA Isolation Kit(Arcturus Bioscience,Mountain View,CA,USA),we extracted total RNA and stored in -80℃.4,RT-PCRRT-PCR was performed using a RiboAmps HS RNA Amplification kit(Arcturus Bioscience) according to the manufacturer's instructions.The PCR products were separated by electrophoresis on a 1.5%agarose gel and stained with ethidium bromide. The target bands were analyzed densitometrically using a Gel Imaging System (BioImaging System,UVP,CA,USA).The b-actin gene was amplified as an internal control.5.Laser capture microdissection of target cells,and extraction of DNAThe paraffin-embedded samples were sectioned successively at a thickness of 6 um.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.A 0.5-ml centrifuge tube containing 50 ml DNA lysis buffer(10 mmol/1 Tris HC1,pH8.0;1 mmol/1 EDTA,pH 8.0;1%Tween 20; 200 mg/ml proteinase K) was incubated at 48℃for 14 h,proteinase K was deactivated at 95℃for 10 min,and the tube was stored 220℃until further analysis.6,PCR-SSCP and SequencingPCR 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 106PCR 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).Results1,Gross and histological featureThe 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.2,ImmunohistochemistryEpithelial markers were all positive in cuboidal cells of all patterns.In the solid regions, some cell clusters showed positive immunoreactivity with CK-L.The results of SP-B staining were identical to the results seen with epithelial markers in the cuboidal cells. The multinuclear giant cells derived from cuboidal cells were also positive to SP-B staining but negative to CD68.Some cuboidal cells remaining in the solid region of PSH also expressed the surfactant proteins.However,both epithelial markers and SP-B staining were negative in polygonal cells,which lined the stroma.Both cuboidal cells and polygonal cells in the nucleus were strongly positive for TTF-1.The results of electron microscopy revealed a few short microvilli on the cuboidal cell surface.The cuboidal cells also had abundant rough endoplasmic reticulum,mitochondria,and typical lamellar bodies for pneumocytes typeⅡ.However,no neuroendocrine granule was seen in the cuboidal cells.Some polygonal cells contained sparse neuroendocrine granules and abundant microfilaments besides abundant rough endoplasmic reticulum and mitochondria.On the contrary,no lamellar body was seen in these cells.In addition, 2 neuroendocrine markers,such as NSE,were positive or dispersed positive in the polygonal cells of every pattern.Other neuroendocrine marker was faintly positive in polygonal cells,including synaptophysin and chromogranin A.Vimentin staining was positive in all cases.CD34 expressed positive results only in the endothelial cells of small vessels and was negative in cuboidal cells and polygonal cells.Some positive cells for MCT were seen sparsely in papillary,solid,and sclerosing regionsp53 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.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.3,RT-PCRGene expressions of polygonal and cuboidal cells were significantly different. Surface cuboidal cell strongly expressed cytokeratin,epithelial membrane antigen, surfactant protein B,and thyroid transcription factor-1 mRNA,whereas interstitial polygonal cell strongly expressed vimentin,synaptophysin,and thyroid transcription factor-1 mRNA and weakly expressed epithelial membrane antigen.Neither cell type expressed chromogranin-A.4,SSCP and sequencing analysesThe 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%.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.Conclusions1,The multinuclear giant cells in PSH are combined by the surface cuboidal cells. Multinuclear giant cells and sclerosing papillary are one of the reason of pathological misdiagnosis.2,The differences in morphological phenotype might result from differences in the state of differentiation,cuboidal cells may differentiate into typeⅡpneumocytes, while polygonal in stroma possess the multipotency differentiation.3,The expression of p53 protein may not be indicative of p53 gene mutation in PSH.The alteration of p53 gene and the expression of p53 protein are identified in both polygonal and cuboidal cells.The high mutation rate of p53 gene may indicate that PSH has potentially malignant biological behavior.