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3517 Background: The WW strategy for patients with rectal cancer who achieved a clinical complete response (cCR) after neoadjuvant therapy (nT) allows to avoid major resection and the associated morbidity and mortality. Standardized criteria to select and monitor WW patients, including biomarkers predicting recurrence after nT, are lacking. The prognostic impact of the immune infiltrate in colorectal cancers is now demonstrated and has been implemented into clinics through the Immunoscore, the first standardized digital-pathology-based assay, recommended by academic institutions. We evidenced that an Immunoscore adapted to biopsies (ISB) performed at diagnosis, predicts the response to nT and the risk of recurrence after nT. Its clinical utility was suggested in a test cohort of WW patients (El Sissy et al., Clin Cancer Res 2020). The aim of this study was to confirm the ability of the ISB to predict clinical outcomes, improve patients’ eligibility for the WW strategy, and optimize a follow-up schedule. Methods: A total of 304 WW patients from 10 centers across 7 countries were included. Tumor biopsies before treatment were immunostained for CD3+ and CD8+ T-cells and converted to ISB using the pre-defined cut-off. The primary endpoint was time-to-recurrence (TTR). Secondary endpoint was disease-free-survival (DFS). As immune response originates in draining lymph nodes, signs of immune activation were carried out in lymph nodes of additional patients managed by radical surgery with complete pathological response (pCR; n = 12) or non-pCR (n = 12) by 3' RNA-Seq and immunofluorescence technologies. Results: High-ISB patients presented with the lowest risk of recurrence after WW. 5-year recurrence-free rates were 97% (92%-100%), 61% (49%-76%), and 56% (44%-73%) with ISB High, Intermediate, and Low, respectively (HR [Low-vs-High] = 14.3, 95% CI 1.8-100). In patients with cCR after nT (n = 209), High-ISB showed a significant association with prolonged TTR and DFS (Logrank P = 0.005 and P = 0.006, respectively). When ISB was evaluated as a continuous variable, the risk of recurrence was increasing along with decreasing ISB (Wald tests, all P < 0.005). In multivariate analyses, ISB was independent of age, sex, location, and cTNM stage and was the single parameter correlated with TTR (HR [ISB High-vs-Low] = 0.08, 95% CI 0.01-0.6; P = 0.015) and DFS (P = 0.013). Unlike for patients with cCR, no difference according to ISB was observed for those with incomplete response (n = 41) or treated with brachytherapy (n = 34). Finally, intranodal signs of T-cell and B-cell activation were only evidenced in patients with pCR. Conclusions: ISB provides a reliable biomarker to predict clinical outcomes, improve eligibility, and optimize patients’ follow-up. Intranodal T-cell and B-cell activation further supports the immune benefit of both organ and lymph node preservation.

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.

3599 Background: Pre-operative CRT followed by total mesorectal excision (TME) is nowadays the standard of care for patient with LARC (cT3-T4N0 or cTxN+). Currently, pathologic complete response occurs in +/- 15% after CRT. Colorectal cancer T-cell infiltration is a strong prognostic factor for survival after primary tumor resection. Our aim was to determine whether T-cell infiltration in PTB could be predictive of tumor response and relapse after CRT + TME. Methods: Between 1999 and 2012, patients with LARC who underwent CRT + TME and with available clinical follow-up and PTB (with sufficient tumor cells density) were identified at the Cliniques universitaires St-Luc. The density of CD3 (T cells) and CD8 (cytotoxic) was quantified on immunostained PTB slides and analyzed with a dedicated image analysis software on whole-slide imaging. Comparisons were made using the Wilcoxon-Mann-Whitney test. Cumulative disease-free survival (DFS) was performed using the Kaplan-Meier estimator and compared by log-rank tests. Cox regression we used for uni- and multi-variate analysis. P value of less than 0.05 was considered statistically significant. Results: 154 patients (sex ratio M/F 1.8; mean age 65 years-old; upper (20%), mid (29%) and low rectum (51%), synchronous metastases (11%)) were analyzed. High CD3 and CD8 PTB densities were significantly associated with a higher pathological response (Dworak 3-4) and lower ypTNM stage after CRT +TME (p < 0,05). Higher CD3 and CD8 PTB densities were associated with higher patient DFS (CD3: HR = 2,30 (CI95%:1,15-4,59) p = 0,02; CD8: HR = 1,95 (CI95%: 1,01-3,75) p = 0,04). These results were confirmed in uni and multivariate analysis. CD3 and CD8 PTB densities added to pathological response (ypTNM/Dworak) but also clinical response (ycTNM) after CRT + TME increases significantly the accuracy prediction of tumor relapse. Conclusions: Pretherapeutic T-cell infiltration of LARC is predictive of tumor response and relapse after CRT +TME. This biomarker could be helpful for patient treatment decision. It must be validated in larger patient cohorts.

471 Background: To date the anatomic extent of tumor (TNM classifications) has been by far the most important factors to predict the prognosis of cancer patients. However, the impact of immune responses and tumor escape on patient prognosis in human cancer is poorly understood. Methods: We analyzed large retrospective cohorts of colorectal cancer patients. Results: We showed that tumors from human colorectal cancer with a high density of infiltrating memory and effector memory T-cells (T-EM) are less likely to disseminate to lymphovascular and perineural structures and to regional lymph-nodes (New Engl J Med, 2005). We showed that the combination of immune parameters associating the nature, the density, the functional orientation and the location of immune cells within the tumor was essential to accurately define the impact of the local host immune reaction on patients prognosis (Science, 2006). We proposed to define these immune criteria as “immune contexture.” Analysis of patients with early-stage colorectal cancer confirmed the major role of cyotoxic effector T cells in predicting the prognosis of the patients (J Clin Oncol, 2009). Investigation of the primary tumor microenvironment allowed us to uncover the association of favorable outcomes with efficient coordination of the intratumoral immune response. We described four major immune coordination profiles within primary tumors depending on the balance between tumor escape and immune coordination. Recent advances analyzing mechanisms responsible for lymphocytic infiltration will be discussed. Conclusions: The density and the immune-cell location within the tumor have a prognostic value that is superior of those of the TNM classifications. Tumor invasion is statistically dependent on the host-immune reaction. No significant financial relationships to disclose.