Comprehensive clinical, pathological and molecular characterization of a cohort of locally advanced rectal cancer patients: Towards an integrative classification for rectal cancer management

Abstract

Introduction: The response of Locally Advanced Rectal Cancer (LARC) to preoperative CRT is heterogeneous. Prognosis of patients depends on several known factors: cT, nodal stage, EMVI, CRM, tumor regression grade and surgical margins. But these factors don't have a large impact on individualizing the therapy. Some patients may require an intensified regimen to increase tumor response, whereas standard 5-FU-based chemoradiotherapy may be sufficient for others. Molecular markers to predict treatment response in advance, could contribute to a better understanding of the underlying mechanisms of rectal cancer and its response to preoperative treatment. Gene expression is linked to cellular phenotype and tumour behaviour. The four consensus molecular subtype (CMS) groups represent the current best description of CRC heterogeneity at the gene-expression level. Our aim is to integrate CMS predictions with clinical and response assessment available data from LARC. Methods: We analyzed gene expression data from the first 12/42 consecutive patients with rectal adenocarcinoma from an ongoing recruiting prospective translational research trial in rectal cancer patients treated in a single institution since Nov 2015 in Argentina. All patients were radiologically staged using high-resolution abdominal-pelvic MRI, chest CT scans and CEA levels. LARC patients were stratified to arm A: preoperative chemoradiotherapy (CRT: 50Gy+ capecitabine 825mg/m2/bid) or arm B: induction chemotherapy (ICT: CAPOX 3 cycles) followed by CRT then TME surgery. MRI asessment was performed after ICT and 6-8w postCRT considering as Good response:mrTRG1-2, moderate response:mrTRG3, poor response: mrTRG4-5. Mutations in RAS exons 2, 3 and 4/BRAF V600E were measured by PCR Entrogen mutation panel. IHQ Protein Expression Deficit (MLH1,MSH2, MSH6,PMS2) was measured by Cell Marque monoclonal primary antibodies. High quality RNA extracted from baseline primary tumor biopsies was labeled and hybridized into Agilent Agi4x44K Whole Human Genome microarrays. We then analyzed the normalized datasets with the R package 'CMSclassifier' -developed by the Colorectal Cancer Subtyping Consortium-, using the similarity-to-centroid approach. Results: We analyzed 12 patients with rectal adenocarcinoma histologically confirmed, with a median age of 48 yo, male 67%, PS 0-1, and a median distance to anal verge: 80mm. 10/12 patients were LARC who completed preoperative treatment. Metastatic disease at diagnoses was observed in 2/12p. CMS 2-Canonical subtype: 10p (82%). Stage II/III:8p (T3-T4N0: 3p, N1/2: 5p, EMVI+: 4p, CRM+:7p). Stage IV:2p . MSH2 Defficit protein expresión: 1/10, K-RAS mut: 1/10. Arm A: 3p(CRT): Good response: 1p, Moderate response:1p and poor response 1p.) ARM B:5p (ICT+CRT): 5p (Good response: 2p, moderate response:1p, poor response 2p).CMS1- MSI immune subtype: 1/12p (8%), Stage III (mrT3N2,EMVI-,CRM+), Lynch's syndrome confirmed by MSH2 germinal mutation. Disease progression was observed after induction chemotherapy. CMS3-Metabolic subtype: 1/12p (8%). Stage III: mrT3N1, EMVI+,CRM+. MSS, KRAS mut. Poor response after ICT+CRT was observed. Conclusion: Our results confirmed CMS2 is the more prevalent group in rectal Cancer. CMS4 was not identified in our cohort probably due to the small sample. Within CMS2 patients, a heterogeneous grade of response was seen. We expect that an increase of analyzed cases can help us establishing molecular features to correlate with neoadjuvant response.