The Studies On FHIT Aberrance And Immune Markers Of Myoepithelial Cell In Breast Cancer

PrefaceBreast cancer is one of the most common malignancy and the main cause of cancer mortality among women . About 250000 women in the world and 13000 women die of breast cancer in China each year.It is important to investigate the mechnism of breast cancer development and progression, because of high incidence of breast cancer pathogenesis and mortality. Only find the mechnism of breast cancer development and progression, could we obtain more basis to the research work on prevention and therapy of breast cancer. That is the most useful pathway to decrease the incidence of breast cancer tumorigenes is ard mortality.The early and correct diagnosis is another useful way to decrease the mortality incidence of breast cancer. First,the the correct diagnosis is the basis to the treatment. Second, the early diagnosis and treatment cf breast microcancer and carcinoma in situ could prevent the breast cancer progression, it is an important pathyway to improve the prognosis of breast cancer. The corrct diagnosis of breast cancer is also very important like the investigation of breast cancer tumorigenesis and progression.It is well known the tumor development and progression are the consequence of multistep process activation of oncogenes and inactivation of tumor suppressor genge(TSG). Consequencely,the study on the inactivation of tumor suppressor gene could help us learn about the tumorgenesis. It is belived that TSG could be inactivated by two hits. The first hit is the loss of heterozygosity (LOH) ,which could cause the inactivation of the single allele, but could not completely suppress theTSG expression because the remaining allele can express the protein. The second hit is mutation, which can inactivate the other single allele . Only by two hits, the TSGs are inactivated fully and lost the downregulation on cell growth,thus make tumor develop and progress. DMA methylation has been associated with transcriptionl inactivation of genes in human cancer and can serve as an alternative to mutational inactivation. This mechnism of gene methylation is called epigenetics mechanism because the gene sequence is not changed. As the third mechnism of the TSG inactivation, the DNA methylation is corelated closely to the tumor development and progression,and is becoming one of the hot spots in the cancer research.The fragile histidine triad (FHIT)gene is a new putative tumor suppressor gene and the FHIT aberrance has been found in many types of cancer .Now there is much research on the genetics mechanism (mutation and LOH) to investigate the FHIT inactivation, however the research on the epigenetics mechanism(DNA methylation) is very rare. The research about the pathway (cell cycle,cell proliferation and apoptosis)by which the inactivated FHIT gene cause tumorigenesis is also rare. As a result , we will investigate the mechanism of FHIT gene inactivation and the pathway by which inactivated FHIT gene cause tumorigenesis in breast cancer.The precise identification of myoepithelial cells(MEC) is a valuable diagnostic clue in breast pathology to differentiate several benigh epithelial lesions from malignant epithelial lesions. MEC are present in breast benigh lesions with exception of microglandular adenosis while lost in malignancy with exception of adenoid cystic carcinoma and epithelial-myoepithelial carcinoma. The identification of MEC may be difficult in routinely HE stained sections because the MEC is similar to luminal epithelial cell(EC), stromal myofibroblasts, and vascular smooth muscle cells in morphology.It is important to choose the accurate immunohistochemical markers to highlight these cells.The MEC markers used frequently are S-100,CDIO,p63 and smooth muscle-specific proteins, such as smooth muscle actin(SMA), smooth muscle myosin heavy chain(SMMHC) and calponin. The previous researchers paied their attention to the single marker's sensitivity and specificity. There is no report about the comparison of sensitivity and specificity between one marker with the other. Consquencely, the purpose of this study is to make a series comparison of specificity and sensitivity in above MEC markers.Methods(1) 38 cases of breast cancer fresh samples and their normal paired breast tissue which was at least 5cm far from the cancer foci were collected. The genome DNA was extracted from each fresh samples. Analysis of PCR-based LOH was performed by using three microsdtellite markers flanking 3pl4. 2: D3S1295, D3S1313 and D3S4260.Abnormal methylation of 5-CpG islands of FHIT was analyzed by methylation-specific PCR. The FHIT expression in breast cancer was detected by immunohistochemistry.The correlation each other among LOH in FHIT gene, abnormal methylation, FHIT protein expression was investigated. The relationship between clinicopathologic parameters of breast cancer and LOH in FHIT gene, abnormal methylation,FHIT protein expression was investigated too.(2) 50 cases of breast disease paraffin embedded samples were collected.The FHIT, cyclinDl, Ki-67 and Bax expression were detected by means of immunohistochemistry.The correlation between each other of FHIT, cyclinDl,Ki-67 and Bax expression were investigated.The relationship between breast cancer clinicopathologic parameters and FHIT,cyclinDl,Ki-67, Bax expression were analysed.(3) 60 cases of paraffin embedded samples of breast lesions were collected. The sensitivity and specificity of S-100, SMA, calponin, CDIO and p63 in labeling myoepithelial cells were detected by single label immunohistochemistry and double label immunohistochemistry.Results(1) LOH frequency in FHIT gene in breast cancer12 of 36 informative cases were LOH positive at 3pl4.2 involving at least one markers and LOH frequency was 33%(12/36).The incidency of LOH in microsatellite D3S1295, D3S1313 and D3S4260 was 23%> 23% and 17% respectively.(2) FHIT aberrant methylation in breast cancerFHIT aberrant methylation was found in 16 of 38 (42%)cases of breast carcinoma. Of 48 cases of normal control breast tissue,5 cases were methylation positive, the methylation incidency was 13%. The FHIT methylation was positively correlated to histologic grade (P<0. 05). "he unmethylated form was found in 100% of breast cancer and normal control breast tissue.(3) FHIT expression in breast cancer13 of 38 cases of breast cancer were FHIT protein positive(34%) We compared the FHIT expression results with breast cancel' clinicopathologic parameters and found a negative correlation between the FHIT expression and histologic grade.(4) The correlation analysis between each other among FHIT LOH .methylation and protein expression.9 of 12 cases of breast cancer that were LOH positive were methylated for FHIT,whereas only 7 of 26 cases that were retention of heterozygosity(ROH) were methylated. The incidency of methylation in LOH cases was significantly higher than that in ROH cases (P<0.01) .As a result, there was a positive correlation between LOH and aberrant, methylation in FHIT.Only 2 of 12 cases of breast cancer that were allelic loss in FHIT were FHIT protein positive, however, 12 of 26 cases that were ROH in FHIT were FHIT protein positive. The allelic loss in FHIT was negat ively correlated to FHIT protein positive (P<0. 05) .(5) cyclinDl expression in breast diseasesThe cyclinDl incidency in breast benign lesions without dysplasia, breast benign lesions with dysplasia, intraductal carcinoma and invasive carcinoma was 0%(0/8),50%(l/2),17%(l/6) and 41%(14/34) respectively.The cyclinDl incidency in breast benign lesions is lower than that in breast malignancies and there was no significant difference in comparison (P>0.05) .However the incidency in breast benign lesions without dysplasia was signicicantly lower than that in breast benign lesions with dysplasia and breast malignancy ( P<0. 05 ) .We compared the cyclinDl expression and breast cancer clinicopathologic parameters and found there was no correlation.(6) Ki-67 expression in breast benign and malignant diseasesAlthough a few cells (almost of which were MECs and few of which were ECs) in 10 cases of breast binign lesions were Ki-67 positive, the 10 cases were detected negative for Ki-67 because the number of Ki-67 positive cells was less than 10% of breast epithelial cells. The incidency of Ki-67 expression in intraductal carcinoma and invasive carcinoma was 50% and65%. The Ki-67 expression frequency in malignancy was statistically higher than that in benign lesions (P<0. 01) . We compared the Ki-67 expression and breast cancer clinicopathologic parameters and found there was positive correlation between Ki-67 expression and histologic grade.(7) Bax expression in breast benign and malignant diseasesThe Bax expression frequency in breast benign ana malignant lesions were 30% and 67.5% respectively, there was significantly statistical difference in comparison of the two expression frequency (P<0.05) . The Bax expression was positively correlated to histologic grade and lymphaden metastasis.(8) The correlation analyses of Fhit, Ki-67, cyclinDl and Bax expressionBy statistical analysis, we found FHIT expression was negativelycorrelated to Ki-67 and cyclinDl expression in breast cancer (P<0.05)(P<0. 05) ; The Ki-67 expression was positively correlated to cyclinDl andBax expression (P<0. 05) (P<0. 05) ;There was no correlation between Fhitand Bax expression.(9) S-100 expression in benign and malignant breast diseasesIn benign breast diseases ,such as fibrocysticdisease,fibroadenoma, intraductal papilloma and benign phyllode tumor,the S-100 positive MECs were found around ECs .But the number of positive MECs were more or less,the S-100 frequency of MECs was 30—80%,mainly 50% , in 30 cases of benign lesions.The breast ECs also expressed S-100,and the positive frequency was 20~50% which was lower than MEC.In 8 cases of intraductal carcinoma,a few S-100 positive cells were found around the intraductal carcinoma cell nests. Of 50 intraductal carcinoma cell nests detected at random,there were only 6 nests around which few positive cells were found.S-100 positive cells were found in nests.In 2 2 cases of invasive carcinoma,no S-100 positive cell was found around carcinoma nests, but many tumor cells were S-100 positive.In 60 cases of breast benign and malignant lesions above mentioned, stromal myofibroblasts, and vascular smooth muscle cells were all S-100 negative,and neurofiber was found S-100 positive.(10) SMA and calponin expression in breast benign and malignant lesions In benign lesions, the MECs were SMA positive continuously surrounding the ductules, looking like garland, and all of the ECs were SMA negative. The larger the ductal epithelial cell nests became due to ductal epithelial hyperplasia,the flater the periphery cells of glands became, and the continuous positive cells changed from garland to line-like in some cases and same as positive cells in fibrocystic breast diseases. SMA showed positive cells in apocrine cysts outside and negative in ECs Papillaryly hyperplastic ECs were SMA negative , but there were continuous SMA positive cells between EC and fibrovascular stromal core in intraductal papilloma. There were many SMA positive cells in the fibrovascular core , however,the positive cells were not MECs but smooth muscle cells in intracystic papillocarcinoma. There were SMA positive cells which consisted of MECs, smooth muscle cells and myofibroblasts around the intraductal carcinoma nests. The MECs around the intraductal carcinoma nests were from continuous to discontinuous,or absolutelyabsent.In invasive carcinomas,around carcinoma nests there were lots of SMA positive cells which were smooth muscle cells and myofibroblasts but MECs. In all of cases, the stromal myofibrablasts and smooth muscle cells were SMA positive.As to calponin,the immunostaining was similar to that of SMA more or less. In some cases, the calponin immunos ta i n i ng of stromal myof ibrablasts and smooth muscle cells were weaker than that of SMA, and calponin positive cells in stroiral myof ibrablasts and smooth muscle cells were less than SMA positive cells.(11) CD10 expression in breast benign and malignant lesions.In benign lesions .almost all of MECs expressed CD10 and EC did rot express CD10. Of benign lesions a few ECs expressed CD10 in only 3 cases. There were CD10 positive cells in the circumference of apocrine gland and apocrine metaplastic cells were CD10 positive.In intraductal carcinoma, the positive cells surrounding the carcinoma nests were compressed and produced an appearance of line. The tumor cells did not expressed CD10 in invasive carcinomas except for 3 cases of them.In benign lesions,the myofibroblasts and smooth muscle cells did not express CD10. In 30 cases of malignant lesions, vascular smooth muscle cells did not express CD10 and myofibroblasts expressed CD10 in 3 cases of malignant lesions.(12) p63 expression in breast benign and malignant lesionsIn 30 cases of benign lesions, almost all of the MEC expressed p63 and EC did not express p63. The p63 expression was almost similar to that of CD10 in intraductal carcinoma. A few tumor cells expressed p63 in 2 cases of intraductal carcinoma.In invasive carcinomas,the carcinoma cells did not express p63 and there were no p63 positive cells around the carcinoma nests.In above 60 cases of benign and malignant lesions, all of the stromal myofibroblasts and vascular smooth muscle cells were p63 negative. (13) Double label immunoh i s t ochemi s try (p63/SMA)There were p63/SMA positive MECs ( black nucleus and dark red cytoplasma) like corella around the negative EC.The positive cells surrounded the neoplastic ductules like thread in intraductle carcinomas.In invasive carcinomas, there were no p63/SMA positive cells,but there were lots of myofibroblasts and vascular smooth muscle cells whose cytoplasma were dark red.ConclusionAs a new candidate TSG, the LOH, methylation and abnormal expression of FHIT were frequent events in breast cancer development and progression. The aberrant FHIT methylation was positively correlated to histologic grade and clinic stage of breast cancer, and FHIT expression was correlated to histologic grade. As a result, FHIT methylation and expression could be prognosis marker of breast cancer.There was positive relationship between LOH and methylation and they could be considered as the "two hits" which inactivated the FHIT, namely, LOH was the first hit, methylation was the second hit.We concluded that the FHIT inhibited the cell proliferation through downregulation the expression of cyclinDl from the findings that the FHIT expression was negatively correlated to Ki-67 and cyclinDl.As MEC markers, SMA, calponin and p63 showed higher sensitivity and S-100 showd lower sensitivity. p63 had higher specificity and SMA, calponin and S-100 had lower specificity. As to sensitivity and specificity, p63 was the better MEC marker.