ITRAQ proteomics of sentinel lymph nodes for the identification of extra cellular matrix protein signatures that can flag metastasis in early breast cancer

Abstract

BackgroundBreast cancer patients who present in the early stage of disease are affected by metastasis to the axillary group of lymph nodes. The first among this group that is affected is called as sentinel lymph node, and metastasis to this lymph node is crucial for the staging of cancer and the quality of surgical intervention. Sentinel Lymph Node Biopsy (SLNB) that is currently used to assess lymph node metastasis is neither sensitive, nor specific, is time‐consuming, thereby necessitating the identification of novel biomarkers that can flag sentinel lymph node metastasis.MethodsThis study was conducted after approval was obtained from the Institute Ethics Committee (Ref. No. IECPG‐ 27/23.01.2019, RT‐03/28.02.2019). Breast cancer patients were screened, and those with early stage were recruited in the study. Surgical resection of the breast was followed by identification of sentinel lymph nodes by methylene fluorescent technique. Histo‐pathology of fresh frozen section biopsy was used as the gold standard to assign the clinical phenotypes of metastatic (SLNM+) and non‐metastatic sentinel lymph nodes (SLNM‐). Discovery phase of the experiment included isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique comprising of six comparative experiments coupled with mass spectrometric analysis to identify differentially expressed proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD027668. Validation was done by ELISA and protein concentrations were used to estimate the ROC for computing diagnostic parameters. Pathway analysis and gene ontology analysis was done to understand the relevance of the differentially expressed proteins in this study.ResultsBased on MS/MS spectra there were 7510 differentially expressed proteins comparing SLNB + and SLNB ‐. Nineteen proteins up‐regulated, and eight proteins that were down regulated in SLNB+ as compared to SLNB‐. ELISA confirmed the up‐regulation of caveolin 1, collagen α‐1, desmin, fibrillin‐1, and microfibrillar associated glycoprotein 4 in metastatic, as compared to non‐metastatic lymph nodes. These proteins are known to be integral in tumorogenesis, cell proliferation, invasion, cell survival and anti‐apoptosis. These proteins have 80%‐100%, of sensitivity and specificity to differentiate the two clinical phenotypes.ConclusionIdentified protein biomarkers have requisite diagnostic parameters to be developed as a translational tool to assess the status of sentinel lymph nodes during mastectomy procedure to guide surgical therapy of axillary lymph nodes in early breast oncology.