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    A new approach to characterize the proteome of pathological samples stored in paraffin

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    Team Béatrice Romagnolo

    A new approach to characterize the proteome of pathologic samples stored in paraffin for the pathogenesis of colon cancer

    The “Intestinal self-renewal and tumorigenesis” team, in a study published in the Journal of Pathology, shows that a new proteomic analysis for samples preserved in paraffin (FFPE) recently developed by Université de Paris proteomic platform, 3P5, made it possible to better understand a new pathway of colonic carcinogenesis from rare pre-cancerous lesions preserved essentially in paraffin. This study was carried out in collaboration with the Pathology department of Cochin Hospital headed by Pr Benoit Terris and the proteomics platform 3P5.

    Besides the conventional sequence (adenoma-carcinoma) of tumor progression of colon cancer (CRC), where the activation of the Wnt/β-catenin pathway is the initiating event, there is an alternative pathway for CRC development, called "serrated" pathway. The histological characteristics of the pre-neoplastic lesions of the serrated route differ from those of the conventional route (CAD for “conventional adenoma”). They are subdivided into two classes, sessile serrated adenomas (SSA), at the origin of most CRCs called microsatellite unstable (MSI) with BRAF mutations, and traditional serrated adenomas (TSA). TSA are rare precancerous colonic lesions, the risk of progression to CRC with poor prognosis is increased compared to other CAD and SSA lesions. These lesions identified by endoscopy are mostly accessible only in the form of paraffin-fixed samples.

    In order to better understand the pathogenesis of TSA, we used a new approach developed by the proteomic platform of Université de Paris, 3P5, which allows a quantitative proteomic analysis of samples preserved in paraffin (FFPE: Formalin-Fixed Paraffin-Embedded). This approach includes dewaxing, reversion of chemical bridges induced by fixation, protein digestion, peptide fractionation and their analysis by mass spectrometry. In collaboration with the 3P5 proteomic platform, we carried out a global quantitative analysis using the label-free approach (LFQ) in order to establish the proteomic profile of 44 samples of colorectal adenomas (12 TSA, 15 CAD, 17 SSA) as well than 17 samples of normal colonic mucosa.

    This study allowed us to confirm that CAD and SSA constitute two very distinct, but homogeneous, entities of colorectal adenomas which follow two distinct pathways of colon carcinogenesis, in agreement with the data of the literature. We have shown that, unlike CAD and SSA, TSA constitutes a much more heterogeneous group of colorectal adenomas, which can be subdivided into two subgroups, one linked to SSA and enriched in BRAF mutations, and the other enriched in KRAS mutations which would be closer to CAD (Cf Figure A). We have also identified a marker specific for TSA, which is LEFTY1, which can be detected in immunohistochemistry (see figure B). The identification of LEFTY1 as a marker for TSA brings a new hypothesis regarding the pathogenesis of TSA which is poorly understood. LEFTY1 is a Nodal pathway inhibitor which belongs to the TGFβ/BMP pathway, known to play a role in the patterning of the intestinal epithelium by controlling the location of the crypts. LEFTY1 is plays an important role in embryonic development, especially in establishing the right-left symmetry, but its role in adult tissue is largely unknown. Combined with other work, our results suggest that LEFTY1 plays an important role in the control of colonic homeostasis, the mechanism of which remains to be understood, but also in the potential development of TSA.

    A. Unsupervised analysis of hierarchical clustering of proteomic data of colorectal adenomas (CAD, SSA and TSA). 

    B. Our results were chosen to cover J Pathol. This photo shows immunohistochemistry validation of various markers identified by proteomic analysis of FFPE samples of the adenomatous colonic lesions.

     

    This work should pave the way for further studies using such fixed material, which is abundantly present in the collections of anatomical pathology departments. Many cancerous, pre-cancerous, inflammatory, metabolic or other pathologies could thus be analyzed with this methodology and make it possible to characterize and validate in particular prognostic markers and help to identify therapeutic targets.

     

    Reference

    Pierre Sohier, Sanson R, Leduc M, Audebourg A, Broussard C, Salnot V, Just PA, Pasmant E, Mayeux P, Guillonneau F, Romagnolo B, Perret C, Terris B.  Proteome analysis of formalin-fixed paraffin-embedded colorectal adenomas reveals the heterogeneous nature of traditional serrated adenomas compared to other colorectal adenomas. J Pathol. 2020 Mar;250(3):251-261. doi: 10.1002/path.5366. Epub 2019 Dec 15.

    PMID: 31729028

     

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