Locally advanced rectal cancer (LARC) remains a medical challenge. Reliable biomarkers to predict which patients will significantly respond to neoadjuvant chemoradiotherapy (nCRT) have not been identified. We evaluated baseline genomic and transcriptomic features to detect differences that may help predict response to nCRT. Eligible LARC patients received nCRT (3D-LCRT 50.4 Gy plus capecitabine 825 mg/m2/bid), preceded by three cycles of CAPOX in high systemic-relapse risk tumors, and subsequent surgery. Frozen tumor biopsies at diagnosis were sequenced using a colorectal cancer panel. Transcriptomic data was used for pathway and cell deconvolution inferential algorithms, coupled with immunohistochemical validation. Clinical and molecular data were analyzed according to nCRT outcome. Pathways related to DNA repair and proliferation (p < 0.005), and co-occurrence of RAS and TP53 mutations (p = 0.001) were associated with poor response. Enrichment of expression signatures related to enhanced immune response, particularly B cells and interferon signaling (p < 0.005), was detected in good responders. Immunohistochemical analysis of CD20+ cells validated the association of good response with B cell infiltration (p = 0.047). Findings indicate that the presence of B cells is associated with successful tumor regression following nCRT in LARC. The prevalence of simultaneous RAS and TP53 mutations along with a proficient DNA repair system that may counteract chemoradio-induced DNA damage was associated with poor response.

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Rectal Cancer (RC) is a complex disease that involves highly variable treatment responses. Currently, there is a lack of reliable markers beyond TNM to deliver a personalized treatment in a cancer setting where the goal is a curative treatment. Here, we performed an integrated characterization of the predictive and prognostic role of clinical features, mismatch-repair deficiency markers, HER2, CDX2, PD-L1 expression, and CD3−CD8+ tumor-infiltrating lymphocytes (TILs) coupled with targeted DNA sequencing of 76 non-metastatic RC patients assigned to total mesorectal excision upfront (TME; n = 15) or neoadjuvant chemo-radiotherapy treatment (nCRT; n = 61) followed by TME. Eighty-two percent of RC cases displayed mutations affecting cancer driver genes such as TP53, APC, KRAS, ATM, and PIK3CA. Good response to nCRT treatment was observed in approximately 40% of the RC cases, and poor pathological tumor regression was significantly associated with worse disease-free survival (DFS, HR = 3.45; 95%CI = 1.14–10.4; p = 0.028). High neutrophils-platelets score (NPS) (OR = 10.52; 95%CI=1.34–82.6; p = 0.025) and KRAS mutated cases (OR = 5.49; 95%CI = 1.06–28.4; p = 0.042) were identified as independent predictive factors of poor response to nCRT treatment in a multivariate analysis. Furthermore, a Cox proportional-hazard model showed that the KRAS mutational status was an independent prognostic factor associated with higher risk of local recurrence (HR = 9.68; 95%CI = 1.01–93.2; p &lt;0.05) and shorter DFS (HR = 2.55; 95%CI = 1.05–6.21; p &lt;0.05), while high CEA serum levels were associated with poor DFS (HR = 2.63; 95%CI = 1.01–6.85; p &lt;0.05). Integrated clinical and molecular-based unsupervised analysis allowed us to identify two RC prognostic groups (cluster 1 and cluster 2) associated with disease-specific OS (HR = 20.64; 95%CI = 2.63–162.2; p &lt;0.0001), metastasis-free survival (HR = 3.67; 95%CI = 1.22–11; p = 0.012), local recurrence-free survival (HR = 3.34; 95%CI = 0.96–11.6; p = 0.043) and worse DFS (HR = 2.68; 95%CI = 1.18–6.06; p = 0.012). The worst prognosis cluster 2 was enriched by stage III high-risk clinical tumors, poor responders to nCRT, with low TILs density and high frequency of KRAS and TP53 mutated cases compared with the best prognosis cluster 1 (p &lt;0.05). Overall, this study provides a comprehensive and integrated characterization of non-metastatic RC cases as a new insight to deliver a personalized therapeutic approach.

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Purpose: Colorectal cancer is the second most frequent cancer and the second and third cause of cancer-related mortality in Argentina for men and women respectively. For locally advanced rectal cancer (LARC), preoperative chemoradiotherapy (CRT) ± induction chemotherapy (iCT) comprises the standard treatment. However, only 15-27% of patients achieve a pathological complete response, while 40-60% respond variably. This scenario poses a strong need for biomarkers able to predict response to CRT and provide guidance towards optimal treatment for each patient. Here we explore LARC gene expression data to identify baseline immune populations and pathways that might be associated with response to CRT. Methods: Using Agilent Human Genome Microarrays, we analyzed gene expression in pre-treatment tumor biopsies of 26 LARC patients as part of an ongoing prospective translational study in Argentina. After treatment with CRT (capecitabine + radiotherapy) ± iCT (capecitabine + oxaliplatin) and surgery, pathological response was assessed according to College of American Pathologists (CAP) guidelines. We grouped CAP scores 0-1 as responders and 2-3 as nonresponders. We estimated cellular heterogeneity of the microenvironment from transcriptomic data by applying two independent computational methods: GSVA and xCell, and inferred patient-specific pathway deregulation with PARADIGM. To elucidate differential features between both patient groups, we performed Two-sample t-Tests for GSVA and xCell, and Rank Product test for PARADIGM. All tests were adjusted for FDR&lt;0.01. Results: Of 26 analyzed patients, we focused on 16 with currently available response data. T-test yielded 98 differentially expressed genes. From the cell type estimators, among the responder group we observed upmodulation of genes related to B-cells (p-GSVA: 0.0001; p-xCell: 0.004), mast cells (p-GSVA: 0.038; p-xCell: 0.029), activated dendritic cells (p-GSVA: 0.035) and effector memory CD8+cells (p-xCell: 0.034). PARADIGM revealed upmodulated activities in responders such as ‘DNA damage', miR34A (regulation of tumor suppression), and TP73 (apoptotic response to DNA damage). Downmodulated pathways included E2F1/3 (transcriptional/cell cycle activation), PARP1 (DNA repair) and IL8 (tumorigenic/proangiogenic chemokine). For all cases, FDR-adjusted p &lt; 0.0001. Conclusions: Considering the caveats regarding our current sample size, we could detect higher involvement of adaptive immunity cell populations, upmodulation of proapoptotic features, and downmodulation of DNA repair and proliferation activities in baseline biopsies of responders when compared to nonresponders. Overall, a cell cycle-decelerated tumor with poor DNA repair abilities immersed in a proapoptotic environment seems to be the one in which CRT would be more effective. With necessary validation of these analyses underway, we envision this effort as the basis on which we will evaluate genomic biomarkers for response prediction in our LARC population. Citation Format: Juan M. Sendoya, Soledad Iseas, Mariano Golubicki, Juan Robbio, Mariana F. Coraglio, Ubaldo Gualdrini, Ana Cabanne, Mirta Kujaruk, Vanesa Mikolaitis, Mariana Rizzolo, Ruben Salanova, Cecilia Rotondaro, Martin Abba, Osvaldo Podhajcer, Enrique Roca, Andrea Llera. Transcriptome-based inference of immune cell subsets and pathway deregulation in rectal cancer reveals biological processes related to therapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-374.