Clonal Origins And PIK3CA Mutations Of Benign And Malignant Proliferative Lesions Of The Breast

IntroductionIntraductal proliferative lesions are a group of cytologically and architectureally diverse proliferations,typically originating from the terminal duct-lobular unit and confined to the mammary duct lobular system.They can be categorized generally as usual ductal hyperplasia(UDH),ductal intraepithelial neoplasia(DIN),including grade 1A,grade 1B,grade 1C,grade 2 and grade 3.A step-wise increase in cancer risk,as suggested by epidemiological data and morphological findings,led many investigators favor the view that these lesions represent a continuum of histological events which may lead to invasive carcinoma.But,what type of intraductal proliferative lesions is already neoplasia?It is well known that most neoplasm is monoclonal in origin while common benign lesions are polyclonal.Furthermore,clonality even has been suggested as a potentially useful marker to distinguish early premalignancy from benign hyperplasia. X-inactivation HUMARA assay is the most common and best method of clonal analysis.Generally,tumor progression is the result of accumulation of gene alterations and new genetic injure is the turning point of tumor advancement.The biological consequences of PI3K/AKT activation are broad and can be subdivided into regulation of cell proliferation,survival and motility.Genetic alterations in PI3K/AKT pathway members have been identified,including PTEN gene deletion or mutations,amplification of AKT or PIK3CA and so on.Particularly exciting was the more recent discovery that PIK3CA was somatically mutated in a significant fraction of diverse tumor types,such as colorectal and breast cancer.Most of the reported mutations in PIK3CA cluster in the conserved regions coding for the helical and kinase domains of p110 a.These mutations constitutively activate its kinase activity.PIK3CA mutations,because they are found in more than 20%of breast cancers,are considered to be one of the most commonly observed genetic changes besides p53 mutations and HER2 amplification.What stage did PIK3CA mutations occur and what roles did they play in the oncogenesis of breast? Nothing is known about them. The clinic pathologic characteristics,such as ER,PR,HER2 and lymph node status,is related with therapy and prognosis of breast cancer,but only a few reports have been available thus far on their relationship with PIK3CA mutations.What's more,their conclusion is controversial.Therefore,in the present study,we used HUMARA gene analysis to examine the clonality of intraductal proliferative lesions and to further analyze the PIK3CA mutations in intraductal proliferative lesions and infiltrating carcinoma of the breast as well as their relationship with the various clinic pathologic characteristicsPartⅠClonal Analysis of Intraduetal Proliferative Lesions of the BreastObjective to investigate clonal origins of intraductal proliferative lesionsMethods 101 breast specimens were paraffin-embedded blocks of formalin-fixed tissues.According to the criteria of WHO Classification of Tumors,the lesions were diagnosed and classified on H&E-stained sections by two experienced pathologists. Lesional and normal breast gland cells were microdissected using a Laser Capture Microdissection(LCM) system.Genomic DNA was extracted.PCR primers of human androgen receptor(HUMARA) exon 1 was designed and labeled at the 5'end with 6-carboxyflurorescein,whose product contained a methylation site and a high polymorphism(CAG)n short tandem repeat.After digestion by restriction enzyme HpaⅡ,HUMARA exon 1 was amplified by a fluorescent nested-PCR procedure and the PCR products were separated on DNA sequencer and analyzed the fluorescent intensity of the two HUMARA alleles.Results DNA from 88 of 101(87.13%) patients was able to be amplified at the HUMARA exon 1 locus and 68 of them(77.27%) were heterozygous and informative. PCR amplification of digested and undigested DNA from 9/12 UDH and 5/18 DIN 1A,yielded 2 bands of almost identical intensity(0.33＜CR＜3.00),suggesting that these tissues were polyclonal and that X-inactivation was not skewed.Digested with HpaⅡblocked to amplification of one of the two HUMARA alleles(CR＜0.33,或＞3.00) in 3/12 UDH,13/18 DIN 1A,28/28 DIN 1B,10/10 carcinoma in situ, suggesting that these lesions are of monoclonal origin.DIN 1B and in situ carcinoma had a significantly higher frequency of monoclonal origin than DIN 1A(P=0.006, Fish exact test).Similarly,the frequency of monoclonality was significantly higher in DIN 1A than in UDH(P=0.003,Fish exact test).Conclusion Most of DIN 1A,1B and carcinoma in situ,are monoclonal and real tumors.PartⅡPIK3CA Mutations in Intraductal Proliferative Lesions and Invasive Carcinoma of the BreastObjective to screen the PIK3CA mutations in intraductal proliferative lesions and invasive carcinoma of the breastMethods 127 intraductal proliferative lesions and 114 invasive cancer tissues of the breast were analyzed for PIK3CA mutations.Target cells were microdissected using a laser capture microdissection(LCM) system.Surrounding nontumorous tissue or tissue derived from a tumor-free block of each case served as the corresponding normal control.Genomic DNA was extracted using QIAmp DNA Micro Kit.PCR amplification was done for exons 9 and 20 of PIK3CA,where 90%of mutations cluster.The purred PCR products were directly sequenced.Results 50 missense mutations were identified in total,of which,17 and 33 mutations clustered in exon 9 and exon 20,respectively.The most common mutations were E542K(7 cases) and E545K(9 cases) in exon 9 and H1047R(30 cases) in exon 20.In addition,two novel mutations,G3292T and I543T were found.Mutations were found less frequently in grade lower DIN1B(3 of 68;4.41%) than carcinoma in situ (15 of 59;25.42%,P=0.001,Chi-square test) or infiltrating carcinoma(32 of 114; 28.07%,P=0.000,Chi-square test).However,there is no difference in the frequency of PIK3CA mutations between carcinoma in situ and invasive carcinoma(P=0.711, Chi-square test).Conclusion PIK3CA mutations mostly began to occur in the stage of carcinoma in situ,which was a late event of oncogenesis of breast.PartⅢThe Relationship of PIK3CA Mutations with Clinic Pathologic Characteristics of Breast Cancer Objective to investigate the relationship of PIK3CA mutations with clinic pathologic characteristics of breast cancerMethods EnVision two-step immunohistochemical staining showed the protein level of ER,PR,HER2,p53,Ki67 and PTEN.Relationship of PIK3CA mutations with various clinic pathologic charactricters(tumor size,histological grade,tumor stage, lymph node status,ER,PR,HER2,p53,Ki67 and PTEN) was further explored.Results The frequency of PIK3CA mutations in ER~+(44/141,31.21%) and PR~+(37/104,35.58%)tumors tended to be higher than those in ER~-(3/32,9.38%) and PR~-(10/69,14.49%) tumors(P=0.012 and P=0.002,respectively,Chi-square test). PIK3CA mutations and PTEN loss occur in a mutually exclusive fashion(P=0.006, Chi-square test).PIK3CA mutation status was not significantly associated with tumor size(P=0.486,Wilcoxon-Mann-Whitney test) and lymph node status(P=0.733, Chi-square test).Frenquency of PIK3CA mutations tended to be same among stageⅠ(16/45,35.55%),stageⅡA(7/42,16.67%) and stageⅡB(9/27,33.33%)(P=0.115, Chi-square test).There is no difference in PIK3CA mutations among various grade tumor(P=0.097,Chi-square test):grade 1 13/62(20.97%),grade 2 13/41(31.71%), grade 3 6/11(54.55%).HER2 and p53 expression were not significantly associated with PIK3CA mutations(P=0.336,P=0.320,respectively,Chi-square test).There is no difference in the Ki67 proliferation index between PIK3CA wild-type and mutatant tumor(17.30%±13.62%vs 12.30%±13.22%,P=0.866,Wilcoxon-Mann-Whitney test).Conclusions The frequency of PIK3CA mutations in ER~+ tumors and PR~+ tumor was significantly higher than those in ER~- tumors and PR~- tumor.PIK3CA mutations and PTEN loss occur in a mutually exclusive fashion.PIK3CA mutation status was not significantly associated with tumor size,histological grade,tumor stage,lymph node status,p53,and HER2 and Ki67 proliferation index.