The Research On The Molecular Pathology Detection Method Of SLN Of Breast Cancer And Axillary Metastasis Marker

BackgroundThe SLN of breast cancer refers to the first lymph node that accepts the entire breast lymphatic drainage. SLNB for breast cancer can accurately predict the state of axillary NSLN, and has become the preferred method for axillary lymph node staging.When the SLNB was negative, patients do not need ALND. The intraoperative frozen sections have high false negative rate, and by detecting the expression of mammary epithelial specific gene molecular biology method to judge whether the metastasis of breast cancer cells in SLN, and it possess the merits of overall, objective, accurate and fast. We previously established molecular pathological method to detect SLN metastasis in breast cancer patients, which can quickly identify the SLN during the operation. It can provide guide for breast surgeon to perform operation. However, its sensitivity and specificity should be further verified.SLNB positive patients usually need ALND to control tumor cell metastasis. However 40~70% of patients have no metastasis of axillary NSLN, this part of patients do not need ALND, which can avoid a series of postoperative complications. Therefore, to identify what kind of SLN positive breast cancer without ALND is very necessary. Though the applying of molecular biomarkers in NSLN status predicting may make some SLN positive breast cancer patients avoid the suffering of ALND, the available choices remain limited and their practical values are still need additional verification.ObjectiveTo verify the reliability of our previously established RT-LAMP method for the detection of SLN metastasis in breast cancer patients. Through the comprehensive transcriptome analysis, explore and seek molecular markers for NSLN status predicting in breast cancer patients with metastatic SLN.MethodsAll the enrolled cases meet the following criteria:(1)Preoperative pathology confirmed breast cancer;(2)Clinical and radiographic examinations showed negative ALN;(3)No distant metastasis of tumor cells;(4)No preoperative chemotherapy,endocrine therapy and radiation therapy.134 cases of SLN samples were collected from seven hospitals in Beijing. SLN of breast cancer patient were analyzed by RT-LAMP and FDA-approved Gene Search method respectively, and the consistency of the two methods were assessed with a kappa concordance test.SLN positive patients with and without behind NSLN invasion were selected in the PLA 307 Hospital from November 2010 to April 2013, the differentially expressed and fused genes in SLN metastasis were screened by next-generation RNA sequencing on Illumina Hiseq 2000 Genome Analyzer platform.ResultsUsing the Gene Search assay as the gold standard, the sensitivity, specificity and consistent of RT-LAMP was 96.2%(25/26) 96.3%(104/108) and 96.3%(129/134),respectively. Statistical analysis showed that the two methods were consistent(Kappa=0.886, P<0.001).6 candidates(all ER/PR+, HER2-, Ki67 < 20%) with metastatic SLN were selected from 305 patients and equally divided into NSLN negative and positive group. Totally, 103 and 47 specifically expressed genes were identified in NSLN negative and positive group, respectively. Among them, FABP1(positive group) and CYP2A13(negative group) were the only 2 genes which encoding protein and with high expression levels. Meanwhile, with FDR < 0.05, 62 up-regulated genes(mainly enriched in KLK subfamily members) and 98 down-regulated genes(mainly enriched in B cell receptor signaling pathway) were discovered in NSLN positive group.Further, 10 different gene fusions were identified, and the most frequent gene in NSLN positive group was IGLL5, which may play significant roles in the process of lymph node metastasis.ConclusionOur RT-LAMP is highly consistent with Gene Search, possessing with the merits of rapid, convenient and low cost which demonstrating a good prospect of application in intra-operative detection of SLN metastasis for breast cancer patients.To our knowledge, this is the first research to explore molecular biomarkers for NSLN status predicating in SLN positive breast cancer patients by RNA sequencing.The specifically expressed genes and the fused genes screened by our study may have a hint for axillary NSLN metastasis.