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Purpose: To determine whether the tumor immune infiltrate, as recently evaluated with the Immunoscore methodology, could be a useful prognostic marker in patients with rectal cancers.Experimental design: The influence of the immune infiltrate on patient's outcome was investigated in patients with or without preoperative chemoradiation therapy (pCRT). The density of total (CD3+) and cytotoxic (CD8+) T lymphocytes was evaluated by immunohistochemistry and quantified by a dedicated image analysis software in surgical specimens of patients with rectal cancer (n = 111) who did not receive pCRT and in tumor biopsies performed before pCRT from additional 55 patients. The results were correlated with tumor recurrence, patient's survival, and response to pCRT.Results: The densities of CD3+ and CD8+ lymphocytes and the associated Immunoscore (from I0 to I4) were significantly correlated with differences in disease-free and overall survival (HR, 1.81 and 1.72, respectively; all P < 0.005). Cox multivariate analysis supports the advantage of the Immunoscore compared with the tumor–node–metastasis (TNM) staging in predicting recurrence and survival (all P < 0.001). Lymph node ratio added information in a prognostic model (all P < 0.05). In addition, high infiltration of CD3+ and CD8+ lymphocytes in tumor biopsies was associated with downstaging of the tumor after pCRT (CD3+ cells; Fisher exact test P = 0.01).Conclusions: The Immunoscore could be a useful prognostic marker in patients with rectal cancer treated by primary surgery. The determination of the immune infiltrate in biopsies before treatment could be a valuable information for the prediction of response to pCRT. Clin Cancer Res; 20(7); 1891–9. ©2014 AACR.

PDF file - 83KB, Kaplan Meier curves for the duration of OS according A, to T cell (CD3+) density evaluated in combined tumor regions (CT and IM). B, to T cell (CD8+) density evaluated in combined tumor regions (CT and IM).

Supplementary Table 1: characteristics of the HEGP cohort of rectal cancer patients Supplementary Table 2: Characteristics of the St Spiridon Hospital cohort of patients Supplementary Table 3: Immune infiltration of CD3+ and CD8+ cells in tumor regions and clinical outcome. Cohort of 111 patients eligible to primary surgery. Supplementary Table 4: Patients at risk at each interval in the Kaplan Meier survival curves for the duration of DFS and OS according to the Immunoscore (CD3-CD8). Supplementary Table 5: Repartition of the "surgery cohort" patient's according to Stage and Immunoscore

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.

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Surgical resection of colorectal liver metastases combined with systemic treatment aims to maximize patient survival. However, recurrence rates are very high postsurgery. In order to assess patient prognosis after metastasis resection, we evaluated the main patho‐molecular and immune parameters of all surgical specimens. Two hundred twenty‐one patients who underwent, after different preoperative treatment, curative resection of 582 metastases were analyzed. Clinicopathological parameters, RAS tumor mutation, and the consensus Immunoscore (I) were assessed for all patients. Overall survival (OS) and time to relapse (TTR) were estimated using the Kaplan–Meier method and compared by log‐rank tests. Cox proportional hazard models were used for uni‐ and multivariate analysis. Immunoscore and clinicopathological parameters (number of metastases, surgical margin, histopathological growth pattern, and steatohepatitis) were associated with relapse in multivariate analysis. Overall, pathological score (PS) that combines relevant clinicopathological factors for relapse, and I, were prognostic for TTR (2‐year TTR rate PS 0–1: 49.8.% (95% CI: 42.2–58.8) versus PS 2–4: 20.9% (95% CI: 13.4–32.8), hazard ratio (HR) = 2.54 (95% CI: 1.82–3.53), p

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.

The role of the immune response at the tumor site is now recognized as crucial in the clinical course of patients with cancer. The importance of the immune cell type, their functional orientation, their density and location within the tumor's regions (tumor/invasion margin) has recently been shown and were grouped together under the term "immune contexture". A strong infiltration by cytotoxic and memory T cells in a Th1-polarized tumor microenvironment appears to have a major prognosis impact. A test called Immunoscore taking into account these various parameters has been suggested to measure in a simple, reproducible and robust manner the intra- and peritumoral immune response. The prognostic value of Immunoscore has recently been validated in colon cancers by a large international retrospective study under the aegis of the Society for Immunotherapy of Cancer (SITC). The Immunoscore could have several potential clinical applications such as prognostic as well as theranostic.

Contains fulltext : 193579.pdf (Publisher’s version ) (Closed access) BACKGROUND: The estimation of risk of recurrence for patients with colon carcinoma must be improved. A robust immune score quantification is needed to introduce immune parameters into cancer classification. The aim of the study was to assess the prognostic value of total tumour-infiltrating T-cell counts and cytotoxic tumour-infiltrating T-cells counts with the consensus Immunoscore assay in patients with stage I-III colon cancer. METHODS: An international consortium of 14 centres in 13 countries, led by the Society for Immunotherapy of Cancer, assessed the Immunoscore assay in patients with TNM stage I-III colon cancer. Patients were randomly assigned to a training set, an internal validation set, or an external validation set. Paraffin sections of the colon tumour and invasive margin from each patient were processed by immunohistochemistry, and the densities of CD3+ and cytotoxic CD8+ T cells in the tumour and in the invasive margin were quantified by digital pathology. An Immunoscore for each patient was derived from the mean of four density percentiles. The primary endpoint was to evaluate the prognostic value of the Immunoscore for time to recurrence, defined as time from surgery to disease recurrence. Stratified multivariable Cox models were used to assess the associations between Immunoscore and outcomes, adjusting for potential confounders. Harrell's C-statistics was used to assess model performance. FINDINGS: Tissue samples from 3539 patients were processed, and samples from 2681 patients were included in the analyses after quality controls (700 patients in the training set, 636 patients in the internal validation set, and 1345 patients in the external validation set). The Immunoscore assay showed a high level of reproducibility between observers and centres (r=0.97 for colon tumour; r=0.97 for invasive margin; p

In the fine balance between tumor invasion and our defensive systems, the role played by the adaptive immune response at the tumor site is critical. Beyond the fact that all the immune components of the innate and adaptive response can be observed to varying degrees in the tumor microenvironment, it appears that a high density of T cytotoxic and memory lymphocytes, in a context of Th1 immune orientation in the tumor and its invasion front, provides a prognostic marker of paramount importance for colorectal cancer and more generally all solid tumors. The understanding of the role of immunity in cancer, tailored during one century of intensive research, has led to a complete paradigm shift.based on a sharp dissection In order to show the major impact of this conceptual revolution, we herein retrace through the example of colorectal cancer, how an effective immune test, namely the "Immunoscore", has been developed. We also provide up to date data demonstrating the capacity of the Immunoscore to prognosticate with a better accuracy than the TME classification clinical outcomes and to guide therapeutic strategies.

Treatment of metastatic colorectal cancer is based upon the assumption that metastases are homogeneous within a patient. We quantified immune cell types of 603 whole-slide metastases and primary colorectal tumors from 222 patients. Primary lesions, and synchronous and metachronous metastases, had a heterogeneous immune infiltrate and mutational diversity. Small metastases had frequently a low Immunoscore and T and B cell score, while a high Immunoscore was associated with a lower number of metastases. Anti-epidermal growth factor receptor treatment modified immune gene expression and significantly increased T cell densities in the metastasis core. The predictive accuracy of the Immunoscore from a single biopsy was superior to the one of programmed cell death ligand 1 (PD-L1). The immune phenotype of the least-infiltrated metastasis had a stronger association with patient outcome than other metastases. ispartof: CANCER CELL vol:34 issue:6 pages:1012-+ ispartof: location:United States status: published

Resume Dans des modeles precliniques et dans certains cancers les lymphocytes T specifiques d’antigenes de tumeurs jouent un role important dans le controle de la proliferation des tumeurs. Neanmoins, dans de nombreuses situations tumorales, leur presence ne semble pas influer sur la progression de la tumeur. Une meilleure analyse de l’heterogeneite phenotypique et fonctionnelle de ces lymphocytes T au contact des tumeurs et des mecanismes de resistance a l’action cytotoxique de ces cellules developpes par la tumeur permettraient d’optimiser les traitements d’immunotherapie anti-tumorale.

Interleukin 17 (IL-17) is a proinflammatory cytokine produced by activated CD4+ memory T cells. We previously showed that IL-17 increased the growth rate of human cervical tumors transplanted into athymic nude mice. To address the possible role of T cells in the biologic activity of IL-17 for tumor control, we grafted 2 murine hematopoietic immunogenic tumors (P815 and J558L) transfected with a complementary DNA encoding murine IL-17 into syngeneic immunocompetent mice. We found that growth of the 2 IL-17–producing tumors was significantly inhibited compared with that of mock-transfected tumors. In contrast to the antitumor activity of IL-17 observed in immunocompetent mice, we observed no difference in the in vivo growth of IL-17–transfected or mock-transfected P815 cells (P815–IL-17 and P815-Neo, respectively) transplanted into nude mice. We then showed that IL-17 increased generation of specific cytolytic T lymphocytes (CTLs) directed against the immunodominant antigens from P815 called A, B, C, D, and E, since all mice injected with P815–IL-17 developed a P815-specific CTL response, whereas only 6 of 16 mice immunized with P815-Neo had a specific CTL response against the antigens. The induction of CTLs was associated with establishment of a tumor-protective immunity. These experiments suggest that T lymphocytes are involved in the antitumor activity of IL-17. Therefore, IL-17, like other cytokines, appears to be a pleiotropic cytokine with possible protumor or antitumor effects on tumor development, which often depends on the immunogenicity of tumor models.

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.

Purpose: To determine whether the tumor immune infiltrate, as recently evaluated with the Immunoscore methodology, could be a useful prognostic marker in patients with rectal cancers. Experimental design: The influence of the immune infiltrate on patient's outcome was investigated in patients with or without preoperative chemoradiation therapy (pCRT). The density of total (CD3+) and cytotoxic (CD8+) T lymphocytes was evaluated by immunohistochemistry and quantified by a dedicated image analysis software in surgical specimens of patients with rectal cancer (n = 111) who did not receive pCRT and in tumor biopsies performed before pCRT from additional 55 patients. The results were correlated with tumor recurrence, patient's survival, and response to pCRT. Results: The densities of CD3+ and CD8+ lymphocytes and the associated Immunoscore (from I0 to I4) were significantly correlated with differences in disease-free and overall survival (HR, 1.81 and 1.72, respectively; all P < 0.005). Cox multivariate analysis supports the advantage of the Immunoscore compared with the tumor–node–metastasis (TNM) staging in predicting recurrence and survival (all P < 0.001). Lymph node ratio added information in a prognostic model (all P < 0.05). In addition, high infiltration of CD3+ and CD8+ lymphocytes in tumor biopsies was associated with downstaging of the tumor after pCRT (CD3+ cells; Fisher exact test P = 0.01). Conclusions: The Immunoscore could be a useful prognostic marker in patients with rectal cancer treated by primary surgery. The determination of the immune infiltrate in biopsies before treatment could be a valuable information for the prediction of response to pCRT. Clin Cancer Res; 20(7); 1891–9. ©2014 AACR.

Recombinant virus encoding tumor antigens are the most used vectors in human clinical trials of cancer vaccines because of their ability to target exogenous antigen in the endogenous MHC class I pathway and to elicit CTL. However, their use requires different constraining procedures to avoid their spreading. The immunosuppression of cancer patients may also increase their intrinsic toxicity. Therefore, the development of non-live vectors may avoid these drawbacks. Different groups now clearly demonstrated that particulate antigens when they are phagocytosed could be targeted in the MHC class I pathway. They also induce CTL in mice which when immunized with these particulate antigens were protected against a challenge with tumors expressing this antigen. Other strategies using toxins or antigens fused or incorporated into various oil or lipid based chemical adjuvants have also suceeded in the induction of CTL response and in some cases have been shown to be efficient as cancer vaccine.

Sunitinib, an antiangiogenic molecule, is one of the first-line standard of care in the treatment of patients with metastatic renal cell carcinoma. However, it only benefits to a subgroup of patients and no predictive markers of sunitinib efficacy have been identified. Twenty-eight metastatic renal cell carcinomas were treated with sunitinib-based therapy and another subgroup of 7 primary renal cell cancer patients were also treated by sunitinib in a neoadjuvant trial. Measurements of CD3 + CD4 + CD25 hi Foxp3 + regulatory T cells, an immunosuppressive cell population, were performed before and after each cycle of treatment in blood and tumor in a prospective study. We observed a decrease in the number of peripheral blood Foxp3 + regulatory T cells after each cycle of sunitinib-based therapy. The overall survival was significantly longer in patients showing a decrease in the number of Foxp3 + regulatory T cells after 2 or 3 cycles of treatment (P

The primary goal of cancer vaccines is to induce CD8+ T cells specific for tumor-associated antigens (TAA) but the characterization of these cells has been difficult because of the low sensitivity of ex vivo assays. Here, we focused on TAA-specific CD8+ T-cell responses in melanoma patients after vaccination with autologous dendritic cells loaded with lysates derived from allogeneic tumor-cell lines (Lysate-DC). Out of 40 patients treated, 16 patients developed immune response to tumor-cell lysate and/or CD8+ T cells specific for differentiation and cancer-testis antigens. TAA-specific CD8+ T-cell responses were detected by interferon (IFN)-gamma enzyme-linked immunospot after in vitro sensitization and were, either transient during the treatment period or delayed, that is, observed after completion of all vaccinations. We could not correlate these immune responses to clinical data as none of the patients achieved an overall objective response according to Response Evaluation Criteria in Solid Tumors criteria. Three patients were reported as stable disease and 10 patients presented evidence of antitumor activity. We found that TAA-specific T cells characterized in 4 patients produced perforin ex vivo, but no IFN-gamma in enzyme-linked immunospot. Differential expression of IFN-gamma and perforin was also observed for viral-specific T cells. Altogether, our results show that Lysate-DC therapy elicited tumor-specific CD8+ T cells nonlimited to human leukocyte antigen-A2+ patients, with some T cells secreting perforin ex vivo and IFN-gamma only after restimulation. The differential expression of perforin and IFN-gamma by antitumor and antiviral CD8+ T cells supports that the sole use of IFN-gamma production to monitor T cells overlooks functional T-cell subpopulations triggered by vaccines.

The homopentameric B-subunit of Shiga toxin (STxB) is used as a tool to deliver antigenic peptides and proteins to the cytosolic compartment of dendritic cells (DCs). In this study, a series of interface mutants of STxB has been constructed. All mutants retained their overall conformation, while a loss in thermal stability was observed. This effect was even more pronounced in trifluoroethanol solutions that mimic the membrane environment. Despite this, all mutants were equally efficient at delivering a model antigenic protein into the MHC class I-restricted antigen presentation pathway of mouse DCs, suggesting that the structural stability of STxB is not a key factor in the membrane translocation process.

The nontoxic B subunit of Shiga toxin (STxB) targets in vivo Ag to dendritic cells that preferentially express the glycolipid Gb3 receptor. After administration of STxB chemically coupled to OVA (STxB-OVA) or E7, a polypeptide derived from HPV, in mice, we showed that the addition of α-galactosylceramide (α-GalCer) resulted in a dramatic improvement of the STxB Ag delivery system, as reflected by the more powerful and longer lasting CD8+ T cell response observed even at very low dose of immunogen (50 ng). This synergy was not found with other adjuvants (CpG, poly(I:C), IFN-α) also known to promote dendritic cell maturation. With respect to the possible mechanism explaining this synergy, mice immunized with α-GalCer presented in vivo the OVA257–264/Kb complex more significantly and for longer period than mice vaccinated with STxB alone or mixed with other adjuvants. To test whether this vaccine could break tolerance against self Ag, OVA transgenic mice were immunized with STxB-OVA alone or mixed with α-GalCer. Although no CTL induction was observed after immunization of OVA transgenic mice with STxB-OVA, tetramer assay clearly detected specific anti-OVA CD8+ T cells in 8 of 11 mice immunized with STxB-OVA combined with α-GalCer. In addition, vaccination with STxB-OVA and α-GalCer conferred strong protection against a challenge with vaccinia virus encoding OVA with virus titers in the ovaries reduced by 5 log compared with nonimmunized mice. STxB combined with α-GalCer therefore appears as a promising vaccine strategy to more successfully establish protective CD8+ T cell memory against intracellular pathogens and tumors.

e14643 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. ...

3610 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 fac...

A number of studies in animal models and humans have shown that both humoral andcell-mediated immune responses play an important role in the control of viral infection and tumor development. In most cases, vaccination with non-vectorized peptides or proteins induces low antibody responses and fails to elicit specific cytotoxic T lymphocytes (CTL). In order to make vaccination more efficient, we chemically coupled the non-toxic B subunit of Shiga toxin (STxB) to a full-size antigenic model protein, ovalbumin (OVA), yielding STxB-OVA. We found that STxB-OVA delivers OVA-derived peptides into both the MHC class I- and II-restricted presentation pathways in mouse dendritic cells. Accordingly, the study of STxB trafficking in these cells revealed that, after internalization, a fraction of STxB followed the retrograde transport pathway to the endoplasmic reticulum, while another fraction was targeted to late endosomes/lysosomes. Vaccination of mice with STxB-OVA primed a specific anti-OVA CTL response without the use of adjuvants. Splenocytes and, particularly, CD4(+) T cells from mice immunized with STxB-OVA also produced higher amounts of the T(h)1 cytokine IFN-gamma and IgG2a-type antibodies than mice immunized with non-vectorized ovalbumin. In conclusion, this study identifies a unique non-live vaccine delivery system for polyepitopic antigens that elicits antigen-specific CTL, a humoral immune response and a T(h)1-type polarization without the use of adjuvant.

602 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

Different arguments suggest that cytotoxic CD8 T lymphocytes (CTL) play a key role in the protection against tumors and in the establishment of anti-tumor immunity. Unfortunately, administration of soluble proteins alone generally does not induce CD8+ T cells presumably because antigen derived peptides are not introduced into the major histocompatibility complex (MHC) class I antigen presentation pathway. Attenuated recombinant live vectors such as viruses or bacteria which have the ability to deliver antigen into the cytosol of cells have been shown to induce cytotoxic T cell response. However, there are safety concerns associated with these approaches especially in immunodeficient patients. Synthetic vectors such as heat shock proteins, virus like particles (VLP) and liposomes could deliver exogenous protein into the cytosol of cells associated with the induction of CTL and tumor immunity. We and other groups have successfully exploited the original intracellular traffic of toxins to use them as vectors for tumor antigens.

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). LCM/patient IS Survival HR Logrank (IS0-2 vs 3-4; 95%IC) p-value Mean of all CD8-CD20 DFS 0.44 (0.22-0.91) 0.5 OS 0.29 (0.09-1.00) 0.03 Less of all CD8-CD20 DFS 0.31 (0.18-0.53) 0.0005 OS 0.25 (0.12-0.51) 0.00003 Most of all CD8-CD20 DFS 0.92 (0.53-1.56) 0.7 OS 0.95 (0.42-2.12) 0.9 Conclusions: B cells (CD20+) associated with cytotoxic T cells (CD8+) in both tumor regions (CT/IM) of the least infiltrated LCM/pt are highly prognostic after curative resection. These results confirm the important role of tumor regions and of B-cells (CD20+) for the tumor immune response. Disclosure: All authors have declared no conflicts of interest.

Immunization with peptide or recombinant proteins generally fails to elicit CTL, which are thought to play a key role in the control of virus-infected cells and tumor growth. In this study we show that the nontoxic B subunit of Shiga toxin fused to a tumor peptide derived from the mouse mastocytoma P815 can induce specific CTL in mice without the use of adjuvant. The Shiga B subunit acts as a vector rather than as an adjuvant, because coinjection of the tumor peptide and the B subunit as separate entities does not lead to CTL induction. We also demonstrated that in vitro the B subunit mediates the delivery of various exogenous CD8 T cell epitopes into the conventional MHC class I-restricted pathway, as this process is inhibited by brefeldin A and lactacystin and requires a functional TAP system. In contrast to other nonviral methods for transport of exogenous Ags into the endogenous MHC class I pathway that involve macropinocytosis or phagocytosis, the Shiga B subunit targets this pathway in a receptor-dependent manner, namely via binding to the glycolipid Gb3. Because this receptor is highly expressed on various dendritic cells, it should allow preferential targeting of the Shiga B subunit to these professional APCs. Therefore, the Shiga B subunit appears to represent an attractive vector for vaccine development due to its ability to target dendritic cells and to induce specific CTL without the need for adjuvant.