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The clinical and pathological responses to multimodal neoadjuvant therapy in locally advanced rectal cancers (LARCs) remain unpredictable, and robust biomarkers are still lacking. Recent studies have shown that tumors present somatic molecular alterations related to better treatment response, and it is also clear that tumor-associated bacteria are modulators of chemotherapy and immunotherapy efficacy, therefore having implications for long-term survivorship and a good potential as the biomarkers of outcome. Here, we performed whole exome sequencing and 16S ribosomal RNA (rRNA) amplicon sequencing from 44 pre-treatment LARC biopsies from Argentinian and Brazilian patients, treated with neoadjuvant chemoradiotherapy or total neoadjuvant treatment, searching for predictive biomarkers of response (responders, n = 17; non-responders, n = 27). In general, the somatic landscape of LARC was not capable to predict a response; however, a significant enrichment in mutational signature SBS5 was observed in non-responders (p = 0.0021), as well as the co-occurrence of APC and FAT4 mutations (p < 0.05). Microbiota studies revealed a similar alpha and beta diversity of bacteria between response groups. Yet, the linear discriminant analysis (LDA) of effect size indicated an enrichment of Hungatella, Flavonifractor, and Methanosphaera (LDA score ≥3) in the pre-treatment biopsies of responders, while non-responders had a higher abundance of Enhydrobacter, Paraprevotella (LDA score ≥3) and Finegoldia (LDA score ≥4). Altogether, the evaluation of these biomarkers in pre-treatment biopsies could eventually predict a neoadjuvant treatment response, while in post-treatment samples, it could help in guiding non-operative treatment strategies.

Anal squamous cell carcinoma (ASCC) is a rare malignancy with a rising incidence associated with human papillomavirus (HPV) infection. The locally advanced disease is associated with a 30% rate of treatment failure after standard chemoradiotherapy (CRT). We aimed to elucidate the prognostic factors for ASCC after curative CRT. A retrospective multicenter study of 176 consecutive patients with ASCC having completed CRT treated between 2010 and 2017 at two centers was performed. Complete response (CR), disease-free survival (DFS), and overall survival (OS) were analyzed by Kaplan–Meier estimates with log-rank tests. The hierarchical clustering on principal components (HCPC) method was employed in an unsupervised and multivariate approach. The CR rate was 70% and was predictive of DFS (p < 0.0001) and OS (p < 0.0001), where non-CR cases were associated with shorter DFS (HR = 16.5, 95% CI 8.19–33.21) and OS (HR = 8.42, 95% CI 3.77–18.81) in a univariate analysis. The median follow-up was 38 months, with a 3-year DFS of 71%. The prognostic factors for DFS were cT1-T2 (p = 0.0002), N0 (p = 0.035), HIV-positive (p = 0.047), HIV-HPV coinfection (p = 0.018), and well-differentiated tumors (p = 0.037). The three-year OS was 81.6%. Female sex (p = 0.05), cT1-T2 (p = 0.02) and well-differentiated tumors (p = 0.003) were associated with better OS. The unsupervised analysis demonstrated a clear segregation of patients in three clusters, identifying that poor prognosis clusters associated with shorter DFS (HR = 1.74 95% CI = 1.25–2.42, p = 0.0008) were enriched with the locally advanced disease, anal canal location, HIV-HPV coinfection, and non-CR. In conclusion, our results reinforce the prognostic value of T stage, N stage, sex, differentiation status, tumor location, and HIV-HPV coinfection in ASCC after CRT.

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 KRAS and TP53 mutated cases compared with the best prognosis cluster 1 (p

Simple Summary The most prevalent type of hereditary colorectal cancer is called Lynch syndrome and it is characterized by a tumor phenotype called microsatellite instability (MSI). This disease is a consequence of germline (inheritable) variants in any of the four mismatch repair (MMR) DNA genes, being their identification essential to ensure their appropriate diagnosis and implementation of preventive measurements. Nevertheless, only 50% of patients with MSI and suspected Lynch syndrome actually carry a germline pathogenic variant in an MMR gene that explains the clinical entity. The remaining 50% are termed Lynch-like syndrome, and their causes remain unknown. In this work, we tried to elucidate the molecular mechanisms that underlie this rare entity in a group of early-onset Lynch-like syndrome colorectal cancer, through whole-exome sequencing of germline and tumor samples. We observed that one-third of these patients have somatic alterations in genes associated with the MMR system and that these could be the mechanism causing their unexplained MSI. Furthermore, we found that patients who showed biallelic somatic alterations also carried germline variants in new candidate genes associated with DNA repair functions and that this could be, partly, the cause of the early onset in this cohort. Abstract Lynch-like syndrome (LLS) is an increasingly common clinical challenge with an underlying molecular basis mostly unknown. To shed light onto it, we focused on a very young LLS early-onset colorectal cancer (CRC) cohort (diagnosis ≤ 40 y.o.), performing germline and tumor whole-exome sequencing (WES) of 15 patients, and additionally analyzing their corresponding tumor mutational burden (TMB) and mutational signatures. We identified four cases (27%) with double somatic putative variants in mismatch repair (MMR) core genes, as well as three additional cases (20%) with double MSH3 somatic alterations in tumors with unexplained MSH2/MSH6 loss of expression, and two cases (13%) with POLD1 potential biallelic alterations. Average TMB was significantly higher for LLS cases with double somatic alterations. Lastly, nine predicted deleterious variants in genes involved in the DNA repair functions and/or previously associated with CRC were found in nine probands, four of which also showed MMR biallelic somatic inactivation. In conclusion, we contribute new insights into LLS CRC, postulating MSH3 and POLD1 double somatic alterations as an underlying cause of a microsatellite instability (MSI) phenotype, proposing intrinsic biological differences between LLS with and without somatic alterations, and suggesting new predisposing candidate genes in this scenario.

Lynch syndrome is the most common cause of hereditary colorectal cancer (CRC), and it is characterized by DNA mismatch repair (MMR) deficiency. The term Lynch-like syndrome (LLS) is used for patients with MMR-deficient tumors and neither germline mutation in MLH1, MSH2, MSH6, PMS2, or EPCAM nor MLH1 somatic methylation. Biallelic somatic inactivation or cryptic germline MMR variants undetected during genetic testing have been proposed to be involved. Sixteen patients with early-onset LLS CRC were selected for germline and tumor whole-exome sequencing. Two potentially pathogenic germline MCM8 variants were detected in a male patient with LLS with fertility problems. A knockout cellular model for MCM8 was generated by CRISPR/Cas9 and detected genetic variants were produced by mutagenesis. DNA damage, microsatellite instability, and mutational signatures were monitored. DNA damage was evident for MCM8KO cells and the analyzed genetic variants. Microsatellite instability and mutational signatures in MCM8KO cells were compatible with the involvement of MCM8 in MMR. Replication in an independent familial cancer cohort detected additional carriers. Unexplained MMR-deficient CRC cases, even showing somatic biallelic MMR inactivation, may be caused by underlying germline defects in genes different than MMR genes. We suggest MCM8 as a gene involved in CRC germline predisposition with a recessive pattern of inheritance., MCM8 may be involved in germline predisposition to colorectal cancer in Lynch-like syndrome cases.

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., Facultad de Ciencias Médicas, Centro de Investigaciones Inmunológicas Básicas y Aplicadas

Purpose: The most frequent cause of hereditary colorectal cancer (CRC) is Lynch syndrome, accounting for 3% of all CRC cases. The underlying germline predisposition is a mutation in any of the four mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2), while microsatellite instability (MSI) is a hallmark of these tumors. However, CRC cases with MSI, no MLH1 somatic hypermethylation and absence of MMR germline mutation account for up to 60% of cases of MMR-defective tumors. They have been termed Lynch-like syndrome (LLS) and treatment management decisions are complicated due to unconfirmed suspicions of hereditary cancer. The aim of the present study was to investigate whether LLS cases in patients under the age of 40 carry potentially pathogenic germline variants in new genes for CRC predisposition causing a MMR-defective tumor phenotype. Methods: We performed whole-exome sequencing (WES) in the germline and tumoral DNA of 16 early-onset LLS patients. We filtered for recessive germline variants in genes involved in DNA repair. An exhaustive functional evaluation for 2 MCM8 genetic variants detected in one patient was performed. The CRISPR/Cas9 system was used to generate MCM8ko clones in a cell model (DLD-1). Site-directed mutagenesis produced the genetic variants for ectopic expression in the MCM8ko model. Functional characterization included the comet assay, which detected DNA damage induced by oxaliplatin in the transfected cells. MCM8ko cells were evaluated for MSI after 30, 60 and 90 days of culture, and mutational signatures were assessed with SigProfiler. Results: The MCM8 variants c.351_354delAAAG (p.Lys118GlufsTer5, p.K118fs) and c.414A>G (p.Ile138Met, p.I138M) were identified in one early-onset LLS patient with biallelic somatic inactivation of MLH1. Manual evaluation of WES data showed that germline MCM8 variants were in trans. In the comet assay, both MCM8 variants revealed higher sensitivity to oxaliplatin and more DNA damage compared to the wild-type control. One of the MCM8KO clones showed MSI after 30 days of sub-culturing. After 120 days, MCM8KO clones had acquired a significant contribution of the SBS20 mutational signature, which is associated with defective MMR DNA. Conclusions: We postulate that MCM8 is a new germline CRC predisposing gene in early-onset LLS, which may be involved in both MMR and double-strand break repair. We further provide evidence that in some LLS CRC cases, especially in young patients, the biallelic somatic MMR inactivation may be caused by new CRC germline predisposing genes. Additional mutational screening is warranted for such cases. Citation Format: Mariano Golubicki, Laia Bonjoch, José G. Acuña-Ochoa, Marcos Diaz-Gay, Jennifer Muñoz, Miriam Cuatrecasas, Teresa Ocaña, Juan Robbio, Enrique L. Roca, Antoni Castells, Fracesc Balaguer, Marina Antelo, Sergi Castellvi-Bel. Germline biallelic mutations in MCM8 are associated with early-onset Lynch-like syndrome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2366.

4054 Background: NMASCC is a rising incidence disease with up to 30% of treatment failure to achieve complete response (CR) after standard chemoradiotherapy (CRT) leading to severe morbidity and death. Stage III-TNM, p53 mutations, HPV negativity, HIV infection are linked to treatment failure. We investigated the predictive/prognostic role of TNM, CR, HPV, PDL1 positivity and CD3/CD8 densities in NM-ASCC from a single institution. Methods: All 79 eligible consecutive NMASCC pts (available FFPE pre-treatment samples) seen from October-2009 to April-2019 having completed definitive CRT (50.4 Gy Pelvic Radiotherapy with Mitomycin-C 12mg/m2/IV/d1-5 / FU 1000mg/m2/d1-4 d29-32 (28%), Mitomycin-C/Capecitabine 825 mg/m2/bid (38%), Cisplatin 60 mg/m2/IV d1-29 and 5FU (34%) were analyzed. Mean age: 59 (range 26-87), 72% female, Stage III: 59%, HPV positive: 86% (HPV-16: 80%);14% HIV positive. IHC assessed by two pathologist for PD-L1 expression (ClonSP263) and CD3-CD8+ TILS densities (Clone 2GV6, Clone SP57). HPV-DNA assessed by PCR (BSGP5+/6+ multiplexed with beta-globin). Kaplan-Meier survival, CR, DFS, OS and Univariate analyses were performed using Cox proportional hazard model. Results: CR achieved within 6 months of treatment completion was 68%( 53pts). Median follow-up after treatment completion: 35 months (range 6 –149). As of February 2020, 82% (65 pts) are alive, no evidence of disease:(57%) 46 pts, recurrence rate: 26%(22 pts), cancer death: 18% (14 pts). PDL1+ tumors ( > 1% positivity-CPS score): 56%, expression levels: 1-5% (57%,26p), > 10%-100% (43%,19p). PDL1+ had a strong association with CR (p = 0.021); higher PDL1+ levels had 8-fold of CR-likelihood than PDL1 negative.(OR 8.50 vs. 1.12). Significative Spearman correlation between PDL1 tumors with CR and CD3-CD8 TILS density was observed (R = 0.43,p = 0.0017 and R = 0.36,p = 0.00094 respectively), albeit CD3-CD8 failed to reach significance as prognostic factors for either CR, DFS or OS. Only CR and PDL1 positive were strongly significantly associated to DFS (HR 0.10 [IC 95% 0.04-0.28] p < 0.001 and HR 0.28 [IC 95% 0.11-0.73] p = 0.006) and OS (HR 0.12 [IC 95% 0.03-0.45] p < 0.001 and HR 0.15 [IC 95% 0.03-0.68] p < 0.004). Low prevalence of HPV negative, early tumors, HIV positive cases in our series probably impacted in statistical power for prognosis correlation. Conclusions: PDL1 positivity was the strongest predictive/prognostic factor in NM-ASCC. Alternative therapeutics options to standard CRT should be explored on poor-risk patients as HPV-negative, P53-mutated and PDL1 negative patients.

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 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 < 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.