Clinicopathological And Molecular Pathologic Features And Molecular Pathogenesis Of Encapsulated Papillary Carcinoma Of The Breast

BackgroundEncapsulted papillary carcinoma(EPC)is a kind of rare tumor subtypes of breast cancer,accounts for 0.5%to 0.2 of breast eancer.EPCs are prone to oecur in postmenopausal women.There have been a lot of clinical researches about the clinicopathological feature of this tumor,and most EPC cases are low-intermediate grade(LTIG)which have a good prognosis.Low?intermediated grade EPC has a low metastasis potential The reports about distant metastasis and recurrence were rare.The researches about irmunohistochemical staining of myoepithelial cell showed that EPC was similar to invasive breast cancer.In present,low-intermediate grade EPC constitutes a spectrum of intraductal and invasive carcinoma.In order to compare the molecular characteristics of EPC with those of DCIS and invasive carcinoma,the researchers used CNV analysis and genomic variation analysis to get inconsistent results.Recently,encapsulated tumors with fine fibrovascular cores lined by high-grade neoplastic epithelial cells,namely,high-grade(HG)EPC,have been documented.According to present study,pure HG EPC may present distant metastasis and cancer related death,and its clinicopathological features are more aggressive than low-intermediate grade subtype with prominent malignancy and poor prognosis.However,the specific differences in the clinicopathological characteristics between HG EPC and LTIG EPC are still unclear,and the report about EPC in Chinese population was rare.In the first part of this study,we compared the differences of clinical and pathological characteristics between HG and LTIG EPC in the 30 EPC we collected and identify some potentially distinguishing features and provide further infornation to facilitate the diagnosis and clinical management of HG EPC.We also review related literatures.In addition,to explore the molecular pathological characteristics and molecular pathogenesis of EPC and define the special clinicopathological characteristics of EPC,we used whole exome sequencing and cell experiments and revealed the mutation characteristics and important driver pathways of EPC for the first time.Part ? Comparison of clinicopathological features between high-grade EPC and low-intermediate grade EPC of the breastStudy AimsComparison of clinicopathological features between high-grade EPC and low-intermediate grade EPC of the breastMethod and MaterialWe collected EPC paraffin tissue samples staining by HE,FISH and immunohistochemitry and re-evaluate EPC grades,and follow-up.We compared the differences between high-grade and low,intermediate grade cases in clinical,histological,immunohistochemical and prognostie characteristies by statistieal analysis.Result(1)Among the 30 EPC patients,25 were diagnosed as LTIG and five as HG(median age:60 and 36 years,respectively).There Was a signifieant differenee in the age between the two subtypes.HG patients usually occurred in young women.(2)Histologically,880%of the HG EPCs exhibited predominantly solid architecture with prominent lymphoplasmacytic infiltrates,more crowded and thicker papillae,and greater stratification and irregular arrangement of malignant epithelial cells with significant atypia.Coexisting invasive components were observed in 32%and 80%of LTIG and HG cases,respectively.(3)Immunohistochemically,all the HG EPCs were triple negative immunophenotypes,while the LTIG EPCs were positive for ER and PR staining.48%of LTIG EPC cases were moderately positive for Her-2,among them,one case showed Her-2 gene amplification by FISH.(4)The basal-like markers cytokeratin 5/6 and epidermal growth factor receptor were detected in two and five HG cases,respectively.HG EPC was also characterized by a significantly high Ki-67 index(P<0.001).(5)HG EPC might be indicative of higher proliferative activity and potential aggressiveness.Long follow-up and rational treatment are required to characterize HG EPC.Future molecular pathology studies can reveal the characteristics of this subtype more comprehensively in a larger series.ConclusionCompared with low-intermediate grade EPC,high-grade EPC usually exhibits olid architecture,basal-like immunophenotype and invasive cancer components in young women.High-grade EPC should be distinguished from solide papillary carcinoma.HG EPC might be indicative of higher proliferative activity and potential aggressiveness.Long follow-up and further molecular studies are required to characterize HG EPC fully in a larger series.Part In The molecular pathological characteristics and pathogenesis of low-intermediate grade EPCStudy AimsTo explore the molecular pathological characteristics and pathogenesis of low-intermediate grade EPC.Method and MaterialOn basis of part 1,we expanded samples series to 27 pairs EPC for whole exome sequencing.According to the results of WES,we validated some important spots by Sanger sequencing.We also conducted tumor mutation load,mutation spectrum,mutation signature,GO enrichment analysis,mutation clone-subclone analysis.In terms of final results,we compared the driver genes in EPC with TCGA database.We expanded EPC series to 41 pairs based on WES.We designed targeted region sequencing panel,mapped significant mutation gene,enriched mutation gene pathway.And we valided some important mutations by Sanger sequencing and compared EPC significant mutation genes with several breast cancer dataset.We found ZFPM1 mutation was characteristic with a high frequency in EPC.We compared that with other diseases.In order to study the function of ZFPM1,we detected protein and RNA expression level by ZFPM1 immnunohistochemical staining,RT-PCR and qPCR.We designed mutant plasmid for cell transfection and analyzed the effect of mutant on cell function including immunofluorescence,luciferase report assay,cell migration assay and cell proliferation assay.Results(1)By WES,the main mutation spectrum characteristics of EPC are C>T/G>A,T>C/A>G and C>A/G>T according to the analysis of 27 EPC by WES.According to the TCGA dateset,we found that the mutation spectrum of EPC is similar to compared Luminal A invasive breast cance while it was different with that of DCIS.According to the analysis of mutation siginature,we defined that three signatures in EPC and two of them are similar to known Signature 5 and Signaturel3.(2)By WES,the average TMB of 27 pairs of samples was 1.07/Mb(Range 0.1-8.16/Mb).In TCGA dateset,the average TMB of these 245 Luminal A invasive breast cancer was 1,02/Mb(Range 0.07--6.67/Mb).There was no significant difference in TMB between the two subtypes of breast cancer.(3)By TRS,of all 41 EPC pairs,9 cases had ZFPM1 somatic mutation producing truncated or extended proteins without hotspots.ZFPM1 is a characteristic high frequency mutation in EPC.Compared with the database,the frequency of ZFPM1 mutation in EPC is high,but rare in other types of breast cancer(4)By TRS,PI3K-AKT-mTOR pathway mutations exist in 51%of LTIG EPC indicating that this pathway plays an important role in EPC tumorigenesis.The significant mutation genes in EPC included PIK3CA(51%),ZFPM1(21.9%)5GATA3(17%),AKTK 14.6%),CTCFC 14.6%),KMT2C(14.6%),CBFB(4.8%)and ULK1(4.8%)?In addition,several gene mutations related to transcription factors occur in EPC,such as GATA3,ZFPM1,CTCF and KMT2C.Previous studies have shown that these genes are involved in ER-mediated transcriptional regulation and play a role in ER dependent cell proliferation.(5)We found that ZFPM1 mutation coexisted with PI3K-AKT-mTOR pathway mutation,which was statistically significant.By clone analysis,PI3K-AKT-mTOR pathway and ZFPM1 might play as a driver in promoting the occurrence of the subtype of LTIG EPC.(6)Compared with Luminal A invasive carcinoma,PIK3CA is the only important driver gene found in EPC,while other high frequency driver genes do not appear in EPC.Only 10 DNA damage responsive mutations genes were detected in EPC.Mutations in the TP53 pathway gene were not detected in all the 41 EPCs,suggesting that most EPCs retained the activity of tumor suppressor genes TP53 and RBI,which may be one of the reasons for better prognosis of EPC than Luminal A invasive breast cancer.(7)We found that all or part of the subsequent loss of zinc fingers could induce GATA3 transcription effect if the N-terminal structure of ZFPM1 was retained by the dual-luciferase reporting experimentConclusion(1)ZFPM1 is a characteristic high-frequency mutation gene in EPC.ZFPM1 and PI3K-AKT-mTOR pathway mutation might constitute likely drivers of a subtype of EPC.(2)The mutation of ZFPM1 may induce the increase of GATA3 transcriptional activity in EPC.In addition to the PI3K-AKT-mTOR pathway mutations,EPC also involves a variety of transcriptional regulation gene mutations affecting ER transcriptional regulation process.(3)The molecular variation of EPC is similar with Luminal A subtype invasive carcinoma.The good prognosis for EPC attributes to owning with fewer key driving genes,fewer CGC-tumor first-line gene mutations and fewer DDR pathway gene mutations than Luninal A invasive cancer.