Carmen Ballesteros-Merino

International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study

BACKGROUNDnThe 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.nnnMETHODSnAn 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. Harrells C-statistics was used to assess model performance.nnnFINDINGSnTissue 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<0·0001). In the training set, patients with a high Immunoscore had the lowest risk of recurrence at 5 years (14 [8%] patients with a high Immunoscore vs 65 (19%) patients with an intermediate Immunoscore vs 51 (32%) patients with a low Immunoscore; hazard ratio [HR] for high vs low Immunoscore 0·20, 95% CI 0·10-0·38; p<0·0001). The findings were confirmed in the two validation sets (n=1981). In the stratified Cox multivariable analysis, the Immunoscore association with time to recurrence was independent of patient age, sex, T stage, N stage, microsatellite instability, and existing prognostic factors (p<0·0001). Of 1434 patients with stage II cancer, the difference in risk of recurrence at 5 years was significant (HR for high vs low Immunoscore 0·33, 95% CI 0·21-0·52; p<0·0001), including in Cox multivariable analysis (p<0·0001). Immunoscore had the highest relative contribution to the risk of all clinical parameters, including the American Joint Committee on Cancer and Union for International Cancer Control TNM classification system.nnnINTERPRETATIONnThe Immunoscore provides a reliable estimate of the risk of recurrence in patients with colon cancer. These results support the implementation of the consensus Immunoscore as a new component of a TNM-Immune classification of cancer.nnnFUNDINGnFrench National Institute of Health and Medical Research, the LabEx Immuno-oncology, the Transcan ERAnet Immunoscore European project, Association pour la Recherche contre le Cancer, CARPEM, AP-HP, Institut National du Cancer, Italian Association for Cancer Research, national grants and the Society for Immunotherapy of Cancer.

Multiparametric immune profiling in HPV– oral squamous cell cancer

Evaluation of T lymphocyte frequency provides prognostic information for patients with oral squamous cell cancer (OSCC). However, the effect of simultaneously evaluating T cell frequency and assessing suppressive elements and defects in antigen-processing machinery (APM) has not been clarified. Simultaneous characterization of CD3+, CD8+, FoxP3+, CD163+, and PD-L1+ cells using multispectral imaging was performed on sections from 119 patients with HPV- OSCC. Expression of β2-microglobulin, MHC class I heavy chain, and large multifunctional peptidase 10 was quantified, and all data were correlated with patient outcome. We found that, consistent with previous reports, high numbers of CD8+ T cells at the invasive margin correlated significantly with prolonged overall survival (OS), while the number of FoxP3+ or PD-L1+ cells did not. Compiling the number of FoxP3+ or PD-L1+ cells within 30 μm of CD8+ T cells identified a significant association with a high number of suppressive elements close to CD8+ T cells and reduced OS. Integrating this information into a cumulative suppression index (CSI) increased correlation with OS. Incorporating tumor expression levels of APM components with CSI further improved prognostic power. This multiparametric immune profiling may be useful for stratifying patients with OSCC for clinical trials.

Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination

BackgroundOne of today’s greatest hurdles for cancer immunotherapy is the absence of information regarding which tumor antigens are already recognized by patients receiving immunotherapies, and whether those therapies then boost or generate an immune response against tumor proteins. For CD8+ T cells in particular, patient-specific immune recognition and responses at the level of individual tumor antigens are rarely characterized. Because of this, some immunologists have turned to serum antibodies as an alternative measure of antigen-specific anti-tumor immunity. In this work, we sought to simultaneously interrogate serum IgG and CD8+ T cell recognition of individual tumor antigens to determine whether antigen-specific serum IgG antibodies provide a window into the behavior of antigen-specific CD8+ T cell responses. Using antibody-based assays to evaluate immune response repertoires and focus T cell antigen exploration could afford substantial advantages for discovering and monitoring the anti-cancer immune responses of patients enrolled on clinical trials.MethodsWe vaccinated female BALB/c mice with a novel combination of an autophagosome-enriched vaccine derived from 4T1 mammary carcinoma along with poly-I:C adjuvant, then screened serum for IgG binding to arrays of 15mer peptides containing known mutation sites in 4T1. Simultaneously, we primed CD8+ T cell cultures from these same animals with 8-11mer peptides derived from these antigens. These primed T cells were then stimulated to measure recognition of the peptides or live 4T1 cells by IFNγ release.ResultsVaccinated animals demonstrate increases in antigen-specific CD8+ T cell recognition of 4T1 tumor cells and peptides. For proteins confirmed in 4T1 cells and vaccine by mass spectrometry, there is a correlation between this increased CD8+ T cell IFNγ release and serum IgG binding to individual peptide antigens.ConclusionsThese results suggest it is possible to observe some features of a patient’s antigen-specific T cell repertoire via an antibody surrogate, which has implications for tumor antigen discovery and clinical monitoring of antigen-specific anti-tumor immunity.

Novel frontiers in detecting cancer metastasis

Cancer microenvironment is the critical battle ground between the cancer cells and host response. Thus, more emphasis is directed to study the relationship between cancer cells and the stromal cells. Multiplex microscopy is an emerging technique in which multiple cell populations within the cancer microenvironment may be stained so that spatial relationship between cancer cells and, in particular, the immune cells may be studied during different stages of cancer development. Recent discovery of mutational burden and neoantigens in cancer has opened new landscapes in the interaction of host immune cells and cancer neoantigens. The emerging role of miRNAs may become an added dimension to study cancer beyond traditional pathway of DNA directed RNA being associated with the malignant behavior of cancer. Circulating tumor cells, cancer markers and ctDNA can be used as markers for circulating cancer cells in the blood. Further studies are needed to validate if liquid biopsy of cancer may become a routine clinical tool to screen cancer or follow patients for recurrence or responses to treatment.

Association of Immunologic Markers With Survival in Upfront Resectable Pancreatic Cancer

in agricultural areas because of atrazine exposure,4,5 one of the most common herbicides used in the United States, this herbicide is rarely used in forested areas and is more commonly used for corn in the Midwest. A previous study in the San Joaquin Valley in California from 1997 to 2006 also did not find evidence of this association.6 The primary limitation of this study is that it was retrospective and observational, using an administrative database, and thus relied on accurate coding. The rising risk of gastroschisis in rural counties in California is not clearly owing to agricultural exposures. Future studies should consider other risk factors that may be more common in these areas.

The Epigenomic Landscape of Pituitary Adenomas Reveals Specific Alterations and Differentiates Among Acromegaly, Cushing's Disease and Endocrine-Inactive Subtypes

Purpose: Pituitary adenomas are one of the most common benign neoplasms of the central nervous system. Although emerging evidence suggests roles for both genetic and epigenetic factors in tumorigenesis, the degree to which these factors contribute to disease remains poorly understood. Experimental Design: A multiplatform analysis was performed to identify the genomic and epigenomic underpinnings of disease among the three major subtypes of surgically resected pituitary adenomas in 48 patients: growth hormone (GH)–secreting (n = 17), adrenocorticotropic hormone (ACTH)–secreting (n = 13, including 3 silent-ACTH adenomas), and endocrine-inactive (n = 18). Whole-exome sequencing was used to profile the somatic mutational landscape, whole-transcriptome sequencing was used to identify disease-specific patterns of gene expression, and array-based DNA methylation profiling was used to examine genome-wide patterns of DNA methylation. Results: Recurrent single-nucleotide and small indel somatic mutations were infrequent among the three adenoma subtypes. However, somatic copy-number alterations (SCNA) were identified in all three pituitary adenoma subtypes. Methylation analysis revealed adenoma subtype-specific DNA methylation profiles, with GH-secreting adenomas being dominated by hypomethylated sites. Likewise, gene-expression patterns revealed adenoma subtype-specific profiles. Integrating DNA methylation and gene-expression data revealed that hypomethylation of promoter regions are related with increased expression of GH1 and SSTR5 genes in GH-secreting adenomas and POMC gene in ACTH-secreting adenomas. Finally, multispectral IHC staining of immune-related proteins showed abundant expression of PD-L1 among all three adenoma subtypes. Conclusions: Taken together, these data stress the contribution of epigenomic alterations to disease-specific etiology among adenoma subtypes and highlight potential targets for future immunotherapy-based treatments. This article reveals novel insights into the epigenomics underlying pituitary adenomas and highlights how differences in epigenomic states are related to important transcriptome alterations that define adenoma subtypes. Clin Cancer Res; 24(17); 4126–36. ©2018 AACR.

PD-L1 expression with immune-infiltrate evaluation and outcome prediction in melanoma patients treated with ipilimumab

ABSTRACT Background: Tumor microenvironment may have a key role in providing immunological markers that can help predict clinical response to treatment with checkpoint inhibitors. We investigated whether the baseline expression of PD-L1 in advanced melanoma patients treated with ipilimumab may correlate with clinical outcome. Methods: PD-L1 expression was assessed in 114 patients with advanced melanoma treated with ipilimumab and, in a cohort of 77 patients, a comprehensive assessment using multispectral imaging to assess the presence and distribution of CD3+, CD8+, CD163+, FOXP3+ and PD-L1+ cells inside and at periphery of the tumor was performed. Results: PD-L1 status alone was not a predictive biomarker for response or survival. There was an association between clinical benefit from ipilimumab therapy with the coexistence of low densities of CD8+ and high densities of CD163+ PD-L1+ cells at the periphery of the tumor. Conclusions: To explain the association of this peculiar microenvironment with clinical benefit from ipilimumab, we proposed a model where baseline CD8 cells levels are low due to inhibitory effect of Tregs and to pro-tumor activity of TAM M2 (CD163+ PD-L1+ cells). Ipilimumab treatment causes a decrease of Treg cells, mediated by ADCC from macrophages, with a concomitant change in TAM polarization that switches from M2 to M1 with a subsequent attraction of CD8 cells and the increase of antitumor response.

Abstract 37: Anti-OX40 (MEDI6469) prior to definitive surgical resection in patients with head and neck squamous cell carcinoma

Background: Head and neck squamous cell carcinomas (HNSCC) produce suppressive factors that impair the immune system, thus limiting effective antitumor immunity. OX40 is a member of the tumor necrosis factor (TNF) receptor family and its biologic activity leads to potent co-stimulation, which can enhance T-cell memory, proliferation and antitumor activity in patients with metastatic cancer. However, its effect on wound healing and the optimal timing of administration in relation to surgery to induce immune changes within the tumor microenvironment (TME) is not known. Objectives: To determine the safety and peak immunologic activity of neoadjuvant anti-OX40 treatment administered prior to definitive surgical resection in patients with locoregionally advanced HNSCC. Methods: Between January 2016 and July 2016, 10 patients with locoregionally advanced HNSCC were enrolled into this phase Ib neoadjuvant time course trial testing a murine antibody to OX40 (MEDI6469) administered 2 days, 1 week and 2 weeks prior to definitive surgical resection. In order to assess changes in the tumor microenvironment (TME), a tissue biopsy and peripheral blood samples were obtained prior to MEDI6469 infusion and tissue was also harvested at the time of surgical resection from the primary tumor site, metastatic and draining lymph nodes along with peripheral blood. Assessments of tumor infiltrating lymphocyte (TIL) populations were performed based on flow cytometry and fluorescent multiplex immunohistochemistry (mIHC); other circulating immunologic parameters that correlate with changes induced by MEDI6469 administration were also measured. These immune changes were assessed and compiled in a “cumulative suppression index,” which incorporates immunosuppressive elements within the tumor, such as FoxP3+ and PD-L1+ cells, to be correlated with clinical variables and outcome. Surgical complications were described using the Clavien-Dindo grading scale. Clinical trial information: NCT02274155. Results: MEDI6469 administration was well tolerated and there were no grade 3 or 4 adverse events (AEs) attributable to anti-OX40 treatment. The toxicity profile was mild, most commonly consisting of low-grade fever prior to surgery, which was performed in all patients without delay. Postoperative grade 3 and 4 complications per Clavien-Dindo scale were observed in two patients. Immunologic changes were observed at all time courses with significant activation and proliferation of CD4+ and CD8+ central and effector memory T-cell populations in both the TME and circulation occurring between 12 and 19 days following MEDI6469 infusion. Ki67 was specifically induced in the TME and on peripheral blood PBMCs after MEDI6469 administration, returning to baseline at Day 55. Up-regulation of PD-L1 was also seen in the tumor post treatment in the majority of specimens. In the tumor, expression of CD39, ICOS and PD-1 is increased on CD4+ T cells in almost all patients and a recently identified tumor-reactive T-cell subset of CD39+CD103+CD8+ T cells, with resident memory phenotype, was increased in some patients. Conclusion: Preoperative MEDI6469 administration prior to surgery is feasible and safe in patients with HNSCC and results in activation and proliferation of T cell populations and up-regulation of PD-L1 in tumor cells occurring between 12 and 19 days following infusion. Citation Format: R. Bryan Bell, Rom S. Leidner, Rebekka A. Duhen, Carmen Ballesteros-Merino, Zipei Feng, Yoshinobu Koguchi, Carlo B. Bifulco, Brendan D. Curti, Walter J. Urba, Bernard A. Fox, Andrew D. Weinberg. Anti-OX40 (MEDI6469) prior to definitive surgical resection in patients with head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 37.

New method for immunoprofiling of the tumor microenvironment of cancer patients by opal multiplex quantitative immunofluorescence (IF) assay

Meeting abstractsnnBavituximab is a novel chimeric IgG1 monoclonal antibody targeting the membrane phospholipid, phosphatidylserine (PS), externalized on the luminal surface of endothelium in tumors, tumor cells, and tumor exosomes under stressor conditions in the tumor microenvironment. PS exposure

Multispectral imaging and objective assessment of immune-tumor interactions in non-small cell lung cancer

Lung cancer is currently the most common cause of cancer-related death in the world and approximately 85% of all lung cancers are non-small cell lung cancer (NSCLC). Previous studies have shown that patients with NSCLC containing high densities of CD8+ memory T cells have a survival advantage. Others have identified an association between relatively high numbers of regulatory T cells and poor prognosis. Our group is interested in better characterizing the immune infiltrates and refining prognostic biomarkers that identify patients at risk of recurrence. Recent advances in multispectral imaging provide an opportunity to evaluate up to 7 markers in a single 4 micron section of formalin-fixed paraffin-embedded (FFPE) tissue. Using this technology, we reported that a high ratio of CD8:FoxP3 at the tumor correlated with the ability to isolate tumor-specific T cells from melanomas (Feng et al., submitted). This determination could be further improved by incorporating the ratio of CD8:PD-L1 present in the tumor into the evaluation. Now we are applying these same strategies to the study of NSCLC. Initial analyses have been performed on more than 450 tumor cores from 77 patients with Stage 3A/3B/4 NSCLC and included the following markers: CD3, CD8, FoxP3, CD163 and PD-L1. Additional markers are being evaluated. Ultimately, we expect that this approach could be used to stratify patients for clinical trials. We anticipate that some biomarkers will identify suppressive pathways and be associated with a short progression-free survival. While we expect there will be heterogeneity in escape mechanisms, identification of a specific mechanism could be used to tailor therapy with an agent or agents to overcome the specific immune suppressive pathway operational in that specific tumor. Patients who lack evidence for a specific suppressive pathway would be randomized to treatment with combination immunotherapy that includes a cancer vaccine.