Loss Of Heterozygosity On Chromosome 9 And PTCH1 Expression In Cutaneous Squamous Cell Carcinoma And Basal Cell Carcinoma

ObjectiveIt has been established that the progressions of squamous cell carcinoma and basal cell carcinoma are due to the combined effects of multipl factors,multisteps and multigenes,invovling activations of oncogenes and inactivations of tumor suppressor genes(TSGs).The loss of heterozygosity(LOH) of polymorphic markers on chromosomes is widely used of screening,locating the candidate TSGs and studying the mechanisms.SCCs and BCCs have different biological behaviors and pathological features,indicating the different genetic mechanisms.PTCH1 protein,expressed by tumor suppressor gene PTCH,plays a very important part in Hedgehog signaling pathway in many tumors.So we would detect PTCH1 protein expression in SCC and BCC and expected to explain the role of Hedgehog signaling pathway in SCC and BCC.Probablely we could offer an ideal biomarker for the diagonosis and therapy of SCC and BCC.Recent studies show that LOH on chromosome 9p21-22 and 9q22-23 were more prevalent suggests the presence of TSGs are associated with both tumors.To identify the relationship,laser capture microdissection(LCM) was used to obtain homogeneous tumor cells.Subsequently,microdissected cell DNA and peripheral venous blood cell DNA were extracted and amplified with PCR.Then the amplified DNA product was used for LOH analysis by combing 8%denaturing polyacrylamind gel containing 7M urea and silver stain.We detect 5 microsatellites markers,3 markers are located on 9p21-22 and the others two are located on 9q22.3,.Through the LOH analysis of 26 BCC and 10 SCC specimens,we hope to know the difference in their genetic pathway. And the PTCH1 expression difference between SCC and BCC was analyzed by immunohistostaining.Materials and methodsTumor specimens:26 BCC and 10 SCC fresh tissues,embeded with OCT,were stored at-80℃.A11 the diagnosis was histologically confirmed.DNA extraction of LCM-capture cells and peripheral venous blood cells:7μm thickness H-E stained sections were microdissected using LCM system.About 4000 tumor cells were selected and captured.We extrate DNA from microdissected cells according to QIAamp DNA Mini Kit instructions and peripheral blood cells according to TIANamp Blood DNA Kit instructions.Selection of microsatellite markers and PCR:The 5 microsatellite markers was selected from NCBI database and UCSC database.PCR was performed in a 25μl reaction volume containing 60ng of genomic DNA,1μM of each primer,10μl PCR Master Mix.The reactions were denatured for 5 min at 94℃,and subsequently amplified for 35 cycles of 30s at 94℃,30s at 55℃-59℃,30s at 72℃,and a final extension 7 min at 72℃.LOH analysis:The PCR product was analyzed on 8%denaturing polyacrylamide gel containing 7M urea and 0.2%silver stained.LOH was confirmed at a particular locus only if one allele showed a reduction in intensity of 50%or more in the tumor relative to normal.MSI was diagnosed in case of an addition or deletion of one or more repeat units resulting in novel alleles.Immunohistochemistry:8 BCCs,28 SCCs and 5 normal epidermis tissues were embeded with paraffin and 5μm thickness sections were used for immunohistochemistry with PTCH1 antibody(1:200).Immunohistochemistry was carried out as previous report.Positive and negative controls were set up.It was considered as positive if the positive cells counted for more than 25%.The data was analyzed using SPSS13.0 statistical software.Results1.In the informative cases of 26 BCCs,LOH was detected at marker D9S196(11.5%,3/26),D9S916(7.7%,2/26),D9S1814(15.4%,4/26),MSI was found at D9S974(15.4%,4/26).LOH and MSI was not found at marker D9S176.2.In 10 SCC specimens,one of 10(10%) displayed MSI for marker D9S974.The other four observed markers D9S176,D9S196,D9S1814 and D9S916 showed no AI.3.Immunohistochemistry:the positive rate of normal tissue is 20%(1/5),100%(8/8) in BCC,92.3%(24/26) in SCC.Conclusion1.LOH for D9S1814 and MSI for D9S974 had similar freqency implies that the markerd pl5 and pl6 gene both had a possible relationship with the development and progression of BCC.2.The two markers of D9S176 and D9S196,marking PTCH1 gene,had significant lower freqency of LOH than that had been reported.PTCH1 may have ethnic differences in the development of BCC and SCC.3.BCC and SCC have different LOH mechanism on Chromosome 9.4.The expression of PTCH1 protein in SCC and BCC is significantly stronger compared with normal tissues.(P＜0.05),but no significant difference between SCC and BCC(P＞0.05).Hedgehog signal transduction pathway plays an important role in SCC and BCC.5.The expression of PTCH1 in SCC had no association with the tumor stage (P＞0.05).