B cells (CD20+) associated to tumor infiltrating cytotoxic T-cells observed on resected liver colorectal metastases (LCM) are prognostic

Aim: Colorectal cancer infiltrating cytotoxic T-cells (CD8+ cells) are a strong prognostic factor for survival after primary tumor and metastases resection. The impact of B cells for prognosis is less characterized. Methods: Metastatic colorectal patients (pts) engaged for curative liver surgery with available FFPE blocks for all resected LCM, were included. The density of CD8+ and CD20+ cells in the metastasis (CT) and the invasive margin (IM) for all LCM was determined by whole-slide immunohistochemistry and quantified with dedicated image analysis software. The mean value of the 3 most infiltrated areas (0.8 mm2) was calculated. The densities of CD8 and CD20 (CT and IM regions) were classified into Hi or Lo according to cut-off values (minimal p-value approach). The total number of Hi densities was calculated to determine the immunoscore (IS) 0-2 Hi (low IS) or 3-4 Hi (high IS). For pts with multiple LCM, all LCM were quantified. The mean value of all densities, the least and the most infiltrated LCM/pt were analyzed. Cumulative DFS/OS analyses were performed using the Kaplan-Meier estimator. The hazard ratio (HR) for OS/DFS comparing (IS0-2 vs 3-4) was determined using univariate Cox regression and the significance by log-rank tests. Results: 294 LCM from 88 patients (M/F 1.1, mean 3.3/pt, synchr/metachr 5.8) were included. For the least infiltrated metastasis: a high IS is prognostic for DFS and OS. Independently of CD8 cells, a high CD20 density associated concomitantly with both regions (CT/IM) is prognostic for OS (HR: 0.36; p = 0.00004) but not for DFS (HR 0.58, p = 0.1). [...

Prognostic association of FoxP3 regulatory T cells with tumor infiltrating CD8 cytotoxic T cells quantified on resected liver colorectal metastases (LCM)

Background: The adaptive Th1 immune response (CD3/CD8/CD45RO T-cells) is a strong prognostic factor for the survival of the patients after colorectal primary tumor and metastases resection. The prognostic impact of regulatory T-cells (FoxP3) in metastatic settings is less characterized. Methods: Metastatic colorectal patients undergoing curative liver surgery for all resected LCM were investigated. For this cohort, we previously reported the prognostic role of an immune score based (IS) for T (CD3/CD8/CD45RO) and B (CD20) cells infiltration . The FoxP3 and CD8 cells infiltrating the center (CT ) and the invasive margin (IM) of LCM were immunohistochemically stained and their densities quantified on whole slides with a dedicated image analysis software. The mean value of the 3 most infiltrated areas (0.64 mm²) was calculated. The CT and IM densities of FoxP3 and CD8 were classified into high (Hi) or low (Lo) according to the minimum p-value cut-off. The total number of Hi densities determined the IS : 0-2 Hi (low IS) or 3-4 Hi (high IS). Cumulative DFS/OS analyses were performed using the Kaplan-Meier estimator. The hazard ratio for OS/DFS comparing (IS 0-2 vs 3-4) was determined using univariate Cox regression and the significance by log-rank tests. Results: 294 LCM from 88 patients (Male/Female 1.1, mean 3.3/patient, synchronous/metachronous 5.8) were included. Inter-metastatic heterogeneity for FoxP3 and CD8 (CT/IM) is high and observed among all patients. For the least infiltrated LCM/patient: a high IS is prognostic for DFS (HR:0.31 95%IC 0.18-0.53; p = 0.0006) and OS (HR:0.29 95%IC 0.14-0.64; p = 0.001). Independently of CD8 cells, a high FoxP3 density in both regions (CT/IM) is not prognostic for DFS (HR:0.53 95%IC 0.28-0.97; p = 0.74) but for OS (HR:0.16 95%IC 0.02-1.17; p = 0.04). Conclusions: Regulatory T cells associated with cytotoxic T cells in both tumor regions (CT/IM) of the least infiltrated LCM/patient are highly prognostic after curative resection. These results confirm the important role of tumor regions and FoxP3 for modulating the tumor immune response

Characterization of the immune microenvironment of synchronous primary tumor and liver colorectal metastases

Background: The adaptive Th1 immune response (CD3, CD8, CD45RO T cells) observed in resected primary colorectal tumor (PCT) and liver colorectal metastases (LCM) is an important prognostic factor. Preoperative treatment modifies the LCM immune microenvironment with a significant association between pathological response and increase of T (CD3, CD8) and B cells (CD20) and downregulated T regulatory cells (FoxP3). We investigated the impact of different preoperative treatments on the quality and density of the immune infiltrates in synchronous resected PCT and LCM. Methods: Metastatic colorectal patients (n = 107) undergoing curative liver surgery for all resected LCM (n = 338) were investigated. 28 patients have been operated concomitantly for synchronous LCM (n = 102) and PCT (n = 23 available) after surgery alone (S), chemotherapy (Cht) alone, Cht + anti-VEGF and Cht + anti-EGFR. The density of CD3 (T cells), CD8 (cytotoxic), CD45RO (memory), CD20 (B cells) and FoxP3 (regulatory) in the core (CT) and invasive margin (IM) of all synchronous LCM and PCT was quantified on immunostained slides. The mean density value (CT, IM) was calculated for each marker with a dedicated image analysis software on whole-slide imaging. Comparisons were made using the Wilcoxon-Mann-Whitney test. Results: Global analysis of immune cell density in LCM and corresponding PCT showed no significant correlation even when further subcategorizing by treatment (preoperative or S), metastasic burden or subdividing by the least, the mean or the highest infiltrated LCM for patients with multiple LCM.Compared to PCT, LCM were more frequently associated with a high immune infiltrate for CD3 and CD45RO in the IM (p < 0.001) and CD8 in CT and IM (p < 0.001). Conversely, high CD20 and FoxP3 density were higher in the CT of PCT (p < 0.005). When comparing preoperative treatment to S, only Cht+anti-EGFR significantly increases CD3 and CD8 in CT of LCM but not PCT (p < 0.001). Conclusions: Cytotoxic/memory T cells and B/regulatory T cell densities were significantly higher in LCM and PCT, respectively. Only anti-EGFR treatment increases T cells densities in the CT of the LCM but not in the PCT, suggesting a specific treatment effect in this tumor region

Lymph node ratio and surgical quality are strong prognostic factors of rectal cancer: results from a single referral centre.

AIM: Nodal stage is a strong prognostic factor of oncological outcome of rectal cancer. To compensate for the variation in total number of harvested nodes, calculation of the lymph node ratio (LNR) has been advocated. The aim of the study was to compare the impact, on the long-term oncological outcome, of the LNR with other predictive factors, including the quality of total mesorectal excision (TME) and the state of the circumferential resection margin. METHOD: Consecutive patients having elective surgery for nonmetastatic rectal cancer were extracted from a prospectively maintained database. Retrospective uni- and multivariate analyses were performed based on patient-, surgical- and tumour-related factors. The prognostic value of the LNR on overall survival (OS) and on overall recurrence-free survival (ORFS) was assessed and a cut-off value was determined. RESULTS: From 1998 to 2013, out of 456 patients, 357 with nonmetastatic disease were operated on for rectal cancer. Neoadjuvant radiochemotherapy was administered to 66.7% of the patients. The mean number of lymph nodes retrieved was 12.8 ± 8.78 per surgical specimen. A lower lymph node yield was obtained in patients who received neoadjuvant chemoradiotherapy (11.8 vs 14.2; P = 0.014). The 5-year ORFS was 71.8% and the 5-year OS was 80.1%. Multivariate analysis confirmed LNR, the quality of TME and age to be independent prognostic factors of OS. LNR, age and perineural infiltration were independently associated with ORFS. Low- and high-risk patients could be discriminated using an LNR cut-off value of 0.2. CONCLUSION: LNR is an independent prognostic factor of OS and ORFS. In line with the principles of optimal surgical management, the quality of TME and lymph node yield are essential technical requirements

Preoperative treatment to modify the immune microenvironnement of liver colorectal metastases.

Background: We previously reported that an adaptive Th1 immune response (CD3/CD8/CD45RO T-cells) observed in resected primary colorectal tumor and liver colorectal metastases (LCM) is an important prognostic factor. B and FoxP3 regulatory lymphocytes participate to the modulation of this response. We aimed to investigate whether the preoperative treatments influenced the quality and the density of the immune infiltrates previously reported in the LCM. Methods: We used a cohort of metastatic colorectal patients (n=107) engaged for curative liver surgery with available FFPE blocks for all resected LCM to confirm the prognostic impact of the immune response. Among this cohort of 338 LCMs, 46 were completely resected after chemotherapy (CT) alone, 130 after CT + anti-VEGF, 118 after CT + anti-EGFR and 44 after surgery alone. LCMs were analyzed for histological response according the Tumor Regression Grade (TRG) and regrouped as Response (R, TRG1-3) or No Response (NR, TRG4-5). The density of CD3+ (T-cells), CD8+ (cytotoxic), CD45RO+ (memory), CD20+ (B-cells) and FoxP3+ (regulatory) in the core (CT) and invasive margin (IM) of all LCM was quantified on immunostained slides. The mean density value (CT/IM) was calculated for each marker with a dedicated image analysis software on whole-slide imaging. Comparisons were made using the Wilcoxon-Mann-Whitney test. Results: LCMs showing R (compared to NR and untreated LCM) were more frequently associated with a high immune infiltrate for CD3+ (CT: p<0.005; IM: p<0.05), CD8+ (CT: p<0.005; IM: p<0.005) and CD20+ (CT: p<0.05). Conversely, high FoxP3+ density in the CT and IM was related to NR and untreated LCMs (p<0.01). LCMs treated with an anti-EGFR therapy showed higher densities of CD3+ (CT: p<0.005; IM: p<0.01), CD8+ (CT: p<0.005), CD45RO+ (CT: p<0.005), CD20+ (CT: p<0.005) and FoxP3+ (CT: p<0.05; IM: p<0.005) compared to other treatments and untreated LCMs. Conclusions: Preoperative treatment modifies the LCM immune microenvironnement. LCMs with a histological response show a cytotoxic immune response (CD3+/CD8+) with associated B-cells (CD20+) and downregulated Tregs (FoxP3+). The use of an anti-EGFR therapy significantly increases immune infiltration in the CT

Comprehensive Intrametastatic Immune Quantification and Major Impact of Immunoscore on Survival.

This study assesses how the metastatic immune landscape is impacting the response to treatment and the outcome of colorectal cancer (CRC) patients. Complete curative resection of metastases (n = 441) was performed for two patient cohorts (n = 153). Immune densities were quantified in the center and invasive margin of all metastases. Immunoscore and T and B cell (TB) score were analyzed in relation to radiological and pathological responses and patient's disease-free (DFS) and overall survival (OS) using multivariable Cox proportional hazards models. All statistical tests were two-sided. The spatial distribution of immune cells within metastases was nonuniform. Patients, as well as metastases of the same patient, had variable immune infiltrates and response to therapy. A beneficial response was statistically significantly associated with increased immune densities. Among all metastases, Immunoscore (I) and TB score evaluated in the least immune-infiltrated metastases were the strongest predictors for DFS and OS (five-year follow-up, Immunoscore: I 3-4: DFS rate = 27.9%, 95% CI = 15.2 to 51.3; vs I 0-1-2: DFS rate = 12.3%, 95% CI = 4.9 to 30.6; HR = 0.45, 95% CI = 0.28 to 0.70, P = .02; I 3-4: OS rate = 64.6%, 95% CI = 46.6 to 89.6; vs I 0-1-2: OS rate = 32.5%, 95% CI = 17.2 to 61.4; HR = 0.32, 95% CI = 0.15 to 0.66, P = .001, C-index = 65.9%; five-year follow-up, TB score: TB 3-4: DFS rate = 25.7%, 95% CI = 14.2 to 46.6; vs TB 0-1-2: DFS rate = 5.0%, 95% CI = 0.8 to 32.4; HR = 0.36, 95% CI = 0.22 to 0.57, P < .001; TB 3-4: OS rate = 63.7%, 95% CI = 46.4 to 87.5; vs TB 0-1-2: OS rate: 21.4%, 95% CI = 9.2 to 49.8; HR = 0.25, 95% CI = 0.12 to 0.51, P < .001, C-index = 67.8%). High TB score and Immunoscore patients had a median survival of 70.5 months, while low patients survived only 25.1 to 38.3 months. Nonresponding patients with high-immune infiltrates had prolonged DFS (HR = 0.28, 95% CI = 0.15 to 0.52, P = .001) and OS (HR = 0.25, 95% CI = 0.1 to 0.62, P = .001). The immune parameters remained the only statistically significant prognostic factor associated with DFS and OS in multivariable analysis (P < .001), while response to treatment was not. Response to treatment and prolonged survival of metastatic CRC patients were statistically significantly associated with high-immune densities quantified into the least immune-infiltrated metastasis