Genomic Alterations And Clonal Evolution Of Invasive Micropapillary Carcinoma Of The Breast Identified By Topographic Cell Cluster Sequencing

Objective Invasive micropapillary carcinoma(IMPC)is a special histopathology type,and has very high rates of lymphovascular invasion and lymph node metastasis and has been reported in several organs.Morphologically,IMPC tumor cells are adhered to cell clustered structures with polarity reversed and located within empty stromal spaces,and tumor cells in recurrence,invasion,and metastasis are also arranged into cell cluster structures.Clinically,IMPC exhibits higher rates of lymphovascular invasion,lymph node metastasis,recurrence,and distant metastasis,and a poorer prognosis.The unique clustered growth pattern and aggressive biological behaviors make IMPC a good model for studying the mechanism of breast cancer invasion and metastasis.We have discovered and forward the research hypothesis of “IMPC tumor cells clustered metastasis” in the previous series of studies on breast IMPC.But the genomic characteristics of clustered invasion and metastasis in IMPC are unclear.Therefore,this study uses the topographic cell cluster sequencing(TCCS)method to study and consolidate this hypothesis from the perspective of genomics.MethodsIn this study,whole-exome sequencing(WES)and whole-genome sequencing(WGS)were performed on freshly frozen IMPC tumors and matched normal breast tissues of17 IMPC patients to reveal the genomic mutation spectrum of IMPC.In addition,lasercapture microdissection(LCM)and WGS were performed on cell clusters in primary tumors and matched axillary lymph node metastases of 29 IMPC patients by TCCS,while retaining the spatial location information in tissue sections.According to the CNV information and spatial location information of the tumor cell clusters in the tissue section,the evolutionary relationship,and the invasion path of the tumor cell clusters in the primary tumor were analyzed by constructing the phylogenetic tree.And analyzed the evolutionary relationship and metastatic routes of the primary tumor cell clusters to lymph node metastases,and to find the key branch genes in the process of IMPC lymph node metastasis.According to the clinicopathological record data,86 IMPC samples were randomly selected,and the immunohistochemistry(IHC)method was performed to detect the expression level and expression location of IGSF9,PRDM16 and ALDH2 in IMPC.Analyze the correlation between IGSF9,PRDM16 and ALDH2 protein expression and lymph node metastasis in IMPC patients based on the clinical-pathological data of the patients,and the influence of protein expression on the prognosis of IMPC patients.Results(1)The high lymph node metastasis potential of IMPC is significantly positively correlated with the degree of genomic CNV.(2)There are significant genomic CNV differences between pure IMPC and mixed IMPC.There are significant differences in the genomic CNV between the IMPC component and the Invasive ductal carcinoma not otherwise specified(IDC)component in the mixed IMPC,and the pathological component of IMPC is evolved from the pathological component of IDC.(3)There is a circular evolution from the inside to the outside between different cell clusters in the IMPC primary foci.And there are monoclonal metastatic seeds in the primary tumor of IMPC,which can be transferred to the lymph node metastases.The monoclonal metastatic seeds have a common CNV characteristic of PRDM16 and IGSF9 gene copy-number losses and ALDH2 gene copy-number gain.(4)IHC results further demonstrate that PRDM16,IGSF9,and ALDH2 protein expression are closely associated with lymph node metastasis and poor prognosis of IMPC patients.ConclusionIn this study,the TCCS method revealed the presence of monoclonal metastatic seed in the primary lesions of IMPC.IMPC tumor cell clusters with IGSF9 and PRDM16 gene copy-number losses and ALDH2 gene copy-number gains are the seed of IMPC tumor cells.This study provides further genomics theoretical basis for the clustered invasion and metastasis of IMPC.