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.

Carcinoid syndrome (CS) is a rare and serious condition that occurs in about 20-25% of patients with neuroendocrine tumors (NETs). Refractory carcinoid syndrome (RCS) refers to persistent symptoms associated with hormone release despite treatment with somatostatin analogues (SSA). Nowadays, treatment decisions for patients with RCS are a matter of debate. Liver Locoregional Therapies (LLRT), such as hepatic artery embolization, radiofrequency ablation, cryoablation, and surgical segment resection are increasingly being used to treat RCS, given their effectiveness in tumor debulking and symptom relief. A retrospective cohort comprising patients with CS of two institutions from Argentina was analyzed. This cohort was a patient subgroup analysis form a registry of patients with NET diagnosis and liver metastases between March 2016 to December 2022. Descriptive statistics was used to summarize main patient characteristics. Survival analysis was performed using the Kaplan-Meier method, and we evaluated the presence of prognostic factors using multivariate Cox regression models. 81 (26.9%) out of 301 patients included in our registry had CS diagnosis. Mean age was 67.4 (IQR 57-75), 47 (58%) were men, 68 (84%) had multicentric tumors, and small bowel was the primary localization for 58(71.6%) of the cases. NETs was G1 in 52 (64.2%) cases, extrahepatic involvement was evidenced in 50 (61.7%) patients, and carcinoid heart disease was documented in 11 (13.6%) patients. 72 (88.8%) patients were treated with SSA. Median follow up was 50 months. After the first-line treatment for CS, the overall survival at 54 months was 78% (CI95: 67.6-90.7%). Progression free survival (PFS) at 50 months was 56.3% (CI95: 43.8-72.3%). Histological grade and tumor localization were significantly associated with PFS in the multivariate model (p < 0.005). A total of 18 (22.2%) patients required subsequent treatment after RCS diagnosis, 12 received LLRT and 6 systemic therapies. At a median follow up of 64 months, PFS after the subsequent therapy was initiated was 52.1% (CI95 28.8-94.3) and 30% (CI95 6.3-100%) for LLRT and subsequent therapies respectively (p=0.26). In RCS, LLRT is an effective treatment approach, particularly for patients with G1 NETs and limited tumor burden. In this study, RCS patients that received LLRT, and other systemic therapies had comparable PFS. Treatment decisions should be made after a comprehensive patient evaluation, considering factors such as ECOG, liver involvement patterns and primary tumor localization.

Los tumores neuroendocrinos G2 (TNE) del tracto digestivo son un grupo heterogéneo de tumores con múltiples opciones de tratamiento, pero los ensayos prospectivos que evalúan el papel de los factores predictivos en estos pacientes son escasos. El objetivo fue analizar factores pronósticos y características clínicas en una población con G2-TNE y determinar el papel de Ki-67 en la estratificación de la población G2. La supervivencia se estimó mediante el método de Kaplan-Meier y los cuartiles Ki-67 se compararon mediante la prueba de log-rank. El valor de Ki-67 para discriminar la mortalidad se evaluó con un análisis de curva ROC. De 144 pacientes, la edad media fue 54.9 (DS 14.7), 46.7% hombres, 102 (70.8%) con enfermedad metastásica, hepática en 97 (67.4%). El 67.9% se sometió a cirugía. El 34% recibió quimioterapia y el 10.9% terapia dirigida. La mediana de Ki-67 fue 6 (IQR 4-10), curva ROC = 0.62 (IC 95% 0.53 a 0.72 p = 0.021); corte: 6.5 (sensibilidad 62.2%, especificidad 57.7%). La supervivencia mediana fue 97, 67, 51 y 27 meses según la estratificación por cuartil (p.001). Murieron 45 (31.7%). Nuestros resultados sugieren que entre los tumores G2 TNE-GEP existe un grupo heterogéneo de neoplasias con diferencias significativas en la supervivencia. Las recomendaciones actuales de la quimioterapia se utilizaron en nuestra serie, con mayor frecuencia en subgrupos de mayor índice proliferativo. Nuestro estudio no tiene el poder suficiente para detectar diferencias entre los cuartiles Ki-67, aunque se observa una clara tendencia según puntos de corte establecidos.

Los tumores neuroendocrinos son una patología relativamente poco frecuente. Se caracterizan por ser, en general, tumores con un comportamiento biológico indolente.1 Así comienzan los artículos sobre esta entidad, no somos originales, pero nos permite hacer algunas reflexiones en esta isla de los pequeños tumores.2

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 <0.05) and shorter DFS (HR = 2.55; 95%CI = 1.05–6.21; p <0.05), while high CEA serum levels were associated with poor DFS (HR = 2.63; 95%CI = 1.01–6.85; p <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 <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 <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.

Anal squamous cell carcinoma (ASCC) is a rare gastrointestinal malignancy associated with high-risk Human papillomavirus (HPV) infection. Despite improved outcomes in non-metastatic ASCC, definitive chemoradiotherapy constitutes the standard treatment for localized disease. Evidences for predictive and prognostic biomarkers are limited. Here, we performed a viral, immune, and mutational characterization of 79 non-metastatic ASCC patients with complete definitive chemoradiotherapy. HPV-16 was detected in 91% of positive cases in single infections (78%) or in coinfections with multiple genotypes (22%). Fifty-four percent of non-metastatic ASCC cases displayed mutations affecting cancer driver genes such as PIK3CA (21% of cases), TP53 (15%), FBXW7 (9%), and APC (6%). PD-L1 expression was detected in 57% of non-metastatic ASCC. Increased PD-L1 positive cases (67%) were detected in patients with complete response compared with non-complete response to treatment (37%) (p = 0.021). Furthermore, patients with PD-L1 positive tumors were significantly associated with better disease-free survival (DFS) and overall survival (OS) compared with patients with PD-L1 negative tumors (p = 0.006 and p = 0.002, respectively). PD-L1 expression strongly impacts CR rate and survival of non-metastatic ASCC patients after standard definitive chemoradiotherapy. PD-L1 expression could be used to stratify good versus poor responders avoiding the associated morbidity with abdominal perineal resection.

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.

Background Nonoperative management after neoadjuvant treatment in low rectal cancer enables organ preservation and avoids surgical morbidity. Our aim is to compare oncological outcomes in patients with clinical complete response in watch and wait strategy with those who received neoadjuvant therapy followed by surgery with a pathological complete response. Methods Patients with non-metastatic rectal cancer after neoadjuvant treatment with clinical complete response in watch and wait approach (group 1, n = 26) and complete pathological responders (ypT0N0) after chemoradiotherapy and surgery (group 2, n = 22), between January 2011 and October 2018, were included retrospectively, and all of them evaluated and followed in a multidisciplinary team. A comparative analysis of local and distant recurrence rates and disease-free and overall survival between both groups was carried out. Statistical analysis was performed using log-rank test, Cox proportional hazards regression model, and Kaplan-Meier curves. Results No differences were found between patient’s demographic characteristics in both groups. Group 1: distance from the anal verge mean 5 cm ( r = 1–12), 10 (38%) stage III, and 7 (27%) circumferential resection margin involved. The median follow-up of 47 months ( r = 6, a 108). Group 2: distance from the anal verge mean 7 cm ( r = 2–12), 16 (72%) stage III, and 13 (59%) circumferential resection margin involved. The median follow-up 49.5 months ( r = 3, a 112). Local recurrence: 2 patients in group 1 (8.3%) and 1 in group 2 (4.8%) ( p = 0.6235). Distant recurrence: 1 patient in group 1 (3.8%) and 3 in group 2 (19.2%) ( p = 0.2237). Disease-free survival: 87.9% in group 1, 80% in group 2 ( p = 0.7546). Overall survival: 86% in group 1 and 85% in group 2 ( p = 0.5367). Conclusion Oncological results in operated patients with pathological complete response were similar to those in patients under a watch and wait strategy mediating a systematic and personalized evaluation. Surgery can safely be deferred in clinical complete responders.

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.

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.

Abstract 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.

Abstract Purpose: No biomarker to personalize treatment in locally advanced rectal cancer (LARC) is currently available. We assessed in LARC whether a diagnostic biopsy-adapted immunoscore (ISB) could predict response to neoadjuvant treatment (nT) and better define patients eligible to an organ preservation strategy (“Watch-and-Wait”). Experimental Design: Biopsies from two independent cohorts (n1 = 131, n2 = 118) of patients with LARC treated with nT followed by radical surgery were immunostained for CD3+ and CD8+ T cells and quantified by digital pathology to determine ISB. The expression of immune-related genes post-nT was investigated (n = 64 patients). Results were correlated with response to nT and disease-free survival (DFS). The ISB prognostic performance was further assessed in a multicentric cohort (n = 73 patients) treated by Watch-and-Wait. Results: ISB positively correlated with the degree of histologic response (P < 0.001) and gene expression levels for Th1 orientation and cytotoxic immune response, post-nT (P = 0.006). ISB high identified patients at lower risk of relapse or death compared with ISB low [HR, 0.21; 95% confidence interval (CI), 0.06–0.78; P = 0.009]. Prognostic performance of ISB for DFS was confirmed in a validation cohort. ISB was an independent parameter, more informative than pre- (P < 0.001) and post-nT (P < 0.05) imaging to predict DFS. ISB combined with imaging post-nT discriminated very good responders that could benefit from organ preservation strategy. In the “Watch-and-Wait” cohort (n = 73), no relapse was observed in patients with ISB high (23.3%). Conclusions: ISB predicts response to nT and survival in patients with LARC treated by surgery. Its usefulness in the selection of patients eligible for a Watch-and-Wait strategy is strongly suggested.

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.

SummaryPurpose The vasopressin analog desmopressin (dDAVP) is known to increase plasma levels of hemostatic factors, and preclinical studies in colorectal cancer models have demonstrated that it hampers tumor vascularization and metastatic progression. We evaluated safety and preliminary efficacy of dDAVP in rectal cancer patients with bleeding, before receiving specific oncologic treatment with surgery, chemotherapy and/or radiotherapy. Methods Patients with rectal cancer having moderate or severe rectal bleeding were enrolled in an open-label, dose-finding trial. Intravenous infusions of dDAVP were administered during two consecutive days in doses from 0.25 to 2.0 µg/kg, using single or twice daily regimen. Bleeding was graded using a score based on the Chutkan scale and tumor perfusion was evaluated by dynamic contrast-enhanced magnetic resonance imaging. Results The trial accrued a total of 32 patients. Dose-limiting toxicity occurred in patients receiving 1 µg/kg or higher. The most prominent treatment-related severe adverse event was hyponatremia. Most patients receiving the maximum tolerated dose of 0.5 µg/kg showed at least a partial hemostatic response and 58% developed a complete response with absence of bleeding at day 4 and/or at the last follow-up at day 14. Tumor perfusion was decreased in two-thirds of patients after dDAVP treatment. Conclusions dDAVP appeared as a promising hemostatic agent in rectal cancer patients with bleeding. Randomized clinical trials to confirm its effectiveness are warranted.Clinical trial registrationwww.clinicaltrials.gov NCT01623206

Introduction: In the most current WHO classification for neuroendocrine tumors (NET) well-differentiated G3 has been distinguished from poorly differentiated neuroendocrine carcinoma (NEC). Neuroendocrine neoplasia G3 (NEN G3) group consisting of both NEC and NET G3 has recently been shown to be a quite heterogeneous patient group concerning prognosis and treatment benefit. Currently, there are little prospective data available for optimal diagnosis, treatment and biomarkers selection and the availability of checkpoint inhibitors lends promise to treat these patients (pts). Methods: From 2006 to 2018, 64 pts with Gastroenteropancreatic (GEP) NEN G3 with available tumor tissue samples were identified at our institution. Clinical data were retrospectively extracted from medical records. Samples were evaluated by three physicians with expertise in gastrointestinal pathology to determine inter-observer differences to identify NET G3 and NEC. We studied the immunohistochemistry expression of mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, MSH6), PDL1, Rb and p53 by optic microscopy. Results: Mean age was 54.9 years and 57.8% were male. Most had NEC (n = 49, 76.6%) and primary tumors were located in the oesophagus (23.9%), rectum (15.2%), pancreas (13.0%) and other sites (34.8%) with 13.0% having unknown primary location; 64.3% had metastasis at diagnosis with a median Ki 67 of 57% (range 22% to 92%). Among NET G3 patients (n = 15, 23.4%) primary tumors were located in the pancreas (54,5%) rectum (9.1%) and other sites (27,3%), whereas 9.1% had unknown primary location and 76.9% were advanced at diagnosis, with a median Ki 67 of 28% (range 21% to 62%) (NEC vs NET G3, P < .001). For the pathological diagnosis, the intraclass correlation coefficient for Ki 67 was 0.74 (95% CI, 0.60 – 0.84; P < .001) and the kappa index for inter-observer concordance in differentiation grade was 0.52 (P < .001). With a median follow‐up of 19.6 months (interquartile range 4.5 to 18.9 months), the median OS was 13.4 months (n = 41). Based on previous publications (1) three different prognostic categories were identified according to the degree of morphologic differentiation (well versus poorly‐differentiated) and Ki67 index (<55% versus ≥55%). On this basis, median OS was: 21.8 months in well‐differentiated neoplasms with Ki67 index 20‐55% (named Type A); 12.7 months in poorly‐differentiated neoplasms with Ki67 index 20‐55% (Type B); and 12.1 months (P) in poorly‐differentiated neoplasms with Ki67 index ≥55% (Type C) (P = .339). All NEC cases (n = 41) had expression alterations in Rb and/or P53, but this was also present in some NET G3 cases (n = 3). Deficient MMR expression was detected in only one patient with esophageal NEC (2.0%). PDL1-positive tumors (n = 5, 7.8%) included 1 (6.7%) NET and 4 (8.2%) NEC, none of them with more than 30% of staining. Conclusion: The diagnosis of GEP NEN G3 is challenging, with considerable discordance even for expert pathologists. The Rb and p53 expression could be considered to confirm NEC in selected cases. A small subsample in our cohort showed deficient MMR expression and PDL1 positive tumors. The heterogeneity in this group of pts is still far from producing validated specific therapies for specific subcategories but they represent a solid basis for designing prospective therapeutic clinical trials in homogenous clinical settings.

2628 Background: We investigated whether an adaptation to rectal biopsies of the recently validated consensus Immunoscore, could predict the response to neoadjuvant treatment and delineate clinical responders that could benefit from a “Watch and Wait” (W&W) strategy with acceptable outcomes. Methods: Initial biopsies from 273 patients with locally advanced rectal cancer (LARC) treated by neoadjuvant chemoradiotherapy (nCRT) followed by Total Mesorectal Excision (TME), were immunostained for CD3+ and cytotoxic CD8+ T cells and quantified by digital pathology to determine the Immunoscore within pre-treatment Biopsy (ISB). Expression level of 44 immune related genes post-neoadjuvant treatment was investigated by Nanostring technology (n = 64 patients). Results were correlated with response to neoadjuvant treatment, disease free survival (DFS) and time to recurrence (TTR). Prognostic performance of ISB was finally assessed in 73 LARC treated by W&W strategy. Results: ISB Low, Intermediate and High were respectively observed in 23.3, 50.4 and 26.3 % of the cohort. ISB was positively and significantly correlated with the response to nCRT, as evaluated by Dworak classification (P = .0034), ypTNM (P = .0003), down-staging (P = .0014), and neoadjuvant rectal (NAR) score, (P < .0001). ISB status was also positively associated with the degree of local immune activation post-neoadjuvant treatment. ISB High patients were at low risk of relapse, with 5-year DFS rates of 81.1 % (CI, 71.3-92.1 %) as compared to 57.8 % (CI, 45.9-72.9 %) in ISB low patients. In multivariate analysis, ISB was the only significant parameter at presentation associated with DFS (High vs Low: P = .001). Among W&W patients, significant difference was observed for TTR according to ISB status (High vs Low: P = .025). Conclusions: ISB could provide a reliable estimate of the response to nCRT and risk of recurrence in LARC patients' treated by TME or W&W strategy.

Early‐onset (<50 years‐old) nonpolyposis nonfamilial colorectal cancer (EO NP NF CRC) is a common clinical challenge. Although Lynch syndrome (LS) is associated with EO CRC, the frequency of this syndrome in the EO NF cases remains unknown. Besides, mismatch repair deficient (MMRd) CRCs with negative MMR gene testing have recently been described in up to 60% of cases and termed “Lynch‐like syndrome” (LLS). Management and counseling decisions of these patients are complicated because of unconfirmed suspicions of hereditary cancer. To define the prevalence of MMR deficient CRCs, LS and LLS in patients with EO NP NF CRC, we recruited 102 patients with a first diagnosis of NP NF CRC ≤ 50 years old during 2003–2009 who underwent genetic counseling at our institution in Argentina. Tumor immunohistochemical (IHC) MMR for protein expression and microsatellite instability (MSI) status were evaluated, and in those with loss of MLH1 expression by IHC, somatic BRAF V600E mutation and both somatic and germline MLH1 methylation levels were studied. Tumors characterized as MMRd without somatic BRAF mutation nor MLH1 methylation were sent for germline analysis. Twenty one (20.6%) tumors were MMRd. Fourteen of 16 putative LS cases underwent germline testing: 6 pathogenic mutations were identified and 8 cases had no identifiable pathogenic mutations. The prevalence of LS and LLS in this cohort was 5.8% (6/102) and 7.8% (8/102), respectively. As a conclusion we found that 20% of patients with EO NP NF CRC have MMRd tumors, and at least half of these are likely to have LLS.