Gabriela Bindea

ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks

Summary: We have developed ClueGO, an easy to use Cytoscape plug-in that strongly improves biological interpretation of large lists of genes. ClueGO integrates Gene Ontology (GO) terms as well as KEGG/BioCarta pathways and creates a functionally organized GO/pathway term network. It can analyze one or compare two lists of genes and comprehensively visualizes functionally grouped terms. A one-click update option allows ClueGO to automatically download the most recent GO/KEGG release at any time. ClueGO provides an intuitive representation of the analysis results and can be optionally used in conjunction with the GOlorize plug-in. Availability: http://www.ici.upmc.fr/cluego/cluegoDownload.shtml Contact: jerome.galon@crc.jussieu.fr Supplementary information: Supplementary data are available at Bioinformatics online.

Clinical Impact of Different Classes of Infiltrating T Cytotoxic and Helper Cells (Th1, Th2, Treg, Th17) in Patients with Colorectal Cancer

The tumor microenvironment includes a complex network of immune T-cell subpopulations. In this study, we systematically analyzed the balance between cytotoxic T cells and different subsets of helper T cells in human colorectal cancers and we correlated their impact on disease-free survival. A panel of immune related genes were analyzed in 125 frozen colorectal tumor specimens. Infiltrating cytotoxic T cells, Treg, Th1, and Th17 cells were also quantified in the center and the invasive margin of the tumors. By hierarchical clustering of a correlation matrix we identified functional clusters of genes associated with Th17 (RORC, IL17A), Th2 (IL4, IL5, IL13), Th1 (Tbet, IRF1, IL12Rb2, STAT4), and cytotoxicity (GNLY, GZMB, PRF1). Patients with high expression of the Th17 cluster had a poor prognosis, whereas patients with high expression of the Th1 cluster had prolonged disease-free survival. In contrast, none of the Th2 clusters were predictive of prognosis. Combined analysis of cytotoxic/Th1 and Th17 clusters improved the ability to discriminate relapse. In situ analysis of the density of IL17+ cells and CD8+ cells in tumor tissues confirmed the results. Our findings argue that functional Th1 and Th17 clusters yield opposite effects on patient survival in colorectal cancer, and they provide complementary information that may improve prognosis.

Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression‐based stratification. These parameters rely on tumour‐cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named ‘Immunoscore’ has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM‐I (TNM‐Immune).

In Situ Cytotoxic and Memory T Cells Predict Outcome in Patients With Early-Stage Colorectal Cancer

PURPOSE Many patients who present with early-stage colorectal cancer (International Union Against Cancer TNM stages I and II) are nevertheless at high risk of relapse. We hypothesized that intratumoral immune reaction could influence their prognosis. PATIENTS AND METHODS The intratumoral immune reaction was investigated in 29 tumors by large-scale real-time polymerase chain reaction. Cytotoxic (CD8) and memory (CD45RO) T cells were quantified by immunohistochemical analyses of tissue microarrays from the center (CT) and the invasive margin (IM) of the 602 tumors from two independent cohorts. The results were correlated with tumor recurrence and patient survival. RESULTS Patients with a strong infiltration of CD45RO(+) cells in the tumor exhibited an increased expression of T-helper 1 and cytotoxicity-related genes. Densities of CD45RO(+) and CD8(+) cells in tumor regions (CT/IM) classified the patients into four distinct prognostic groups based on the presence of high density of each marker in each tumor region. The four groups were associated with dramatic differences in disease-free, disease-specific, and overall survival (all P < .0001). Five years after diagnosis, only 4.8% (95% CI, 0.6% to 8.8%) of patients with high densities of CD8(+) plus CD45RO(+) cells had tumor recurrence, and 86.2% (CI, 79.4% to 93.6%) survived. In contrast, the tumor recurred in 75% (95% CI, 17% to 92.5%) of patients with low densities of these cells, and only 27.5% (95% CI, 10.5% to 72%) survived (all P < .0001). Multivariate analyses showed that the immune criteria had independent effects on the rates of complete remission and survival. CONCLUSION The combined analysis of CD8(+) plus CD45RO(+) cells in specific tumor regions could provide a useful criterion for the prediction of tumor recurrence and survival in patients with early-stage colorectal cancer.

Histopathologic-Based Prognostic Factors of Colorectal Cancers Are Associated With the State of the Local Immune Reaction

PURPOSE The prognosis of patients with colorectal cancer has sometimes proved uncertain; thus, the prognostic significance of immune criteria was compared with that of the tumor extension criteria using the American Joint Committee on Cancer/International Union Against Cancer-TNM (AJCC/UICC-TNM) staging system. PATIENTS AND METHODS We studied the intratumoral immune infiltrates in the center of the tumor and in the invasive margin of 599 specimens of stage I to IV colorectal cancers from two independent cohorts. We analyzed these findings in relation to the degree of tumor extension and to the frequency of recurrence. RESULTS Growth of the primary tumor and metastatic spread were associated with decreased intratumoral immune T-cell densities. Sixty percent of patients with high densities of CD8(+) cytotoxic T-lymphocyte infiltrate presented with stage Tis/T1 tumor, whereas no patients with low densities presented with such early-stage tumor. In patients who did not relapse, the density of CD8 infiltrates was inversely correlated with T stage. In contrast, in patients whose tumor recurred, the number of CD8 cells was low regardless of the T stage of the tumor. Univariate analysis showed that the immune score was significantly associated with differences in disease-free, disease-specific, and overall survival (hazard ratio [HR], 0.64, 0.60, and 0.70, respectively; P < .005). Time-dependent receiver operating characteristic curve analysis illustrated the predictive accuracy of the immune parameters (c-index = 65.3%, time-dependent c-index [Cτ] = 66.5%). A final stepwise model for Cox multivariate analysis supports the advantage of the immune score (HR, 0.64; P < .001; Cτ = 67.9%) compared with histopathologic features in predicting recurrence as well as survival. CONCLUSION Assessment of CD8(+) cytotoxic T lymphocytes in combined tumor regions provides an indicator of tumor recurrence beyond that predicted by AJCC/UICC-TNM staging.

CluePedia Cytoscape plugin

Summary: The CluePedia Cytoscape plugin is a search tool for new markers potentially associated to pathways. CluePedia calculates linear and non-linear statistical dependencies from experimental data. Genes, proteins and miRNAs can be connected based on in silico and/or experimental information and integrated into a ClueGO network of terms/pathways. Interrelations within each pathway can be investigated, and new potential associations may be revealed through gene/protein/miRNA enrichments. A pathway-like visualization can be created using the Cerebral plugin layout. Combining all these features is essential for data interpretation and the generation of new hypotheses. The CluePedia Cytoscape plugin is user-friendly and has an expressive and intuitive visualization. Availability: http://www.ici.upmc.fr/cluepedia/ and via the Cytoscape plugin manager. The user manual is available at the CluePedia website. Contact: bernhard.mlecnik@crc.jussieu.fr or jerome.galon@crc.jussieu.fr Supplementary information: Supplementary data are available at Bioinformatics online.

Biomolecular Network Reconstruction Identifies T-Cell Homing Factors Associated With Survival in Colorectal Cancer

BACKGROUND & AIMS Colorectal cancer is a complex disease involving immune defense mechanisms within the tumor. Herein, we used data integration and biomolecular network reconstruction to generate hypotheses about the mechanisms underlying immune responses in colorectal cancer that are relevant to tumor recurrence. METHODS Mechanistic hypotheses were formulated on the basis of data from 108 patients and tested using different assays (gene expression, phenome mapping, tissue-microarrays, T-cell receptor [TCR] repertoire). RESULTS This integrative approach revealed that chemoattraction and adhesion play important roles in determining the density of intratumoral immune cells. The presence of specific chemokines (CX3CL1, CXCL10, CXCL9) and adhesion molecules (ICAM1, VCAM1, MADCAM1) correlated with different subsets of immune cells and with high densities of T-cell subpopulations within specific tumor regions. High expression of these molecules correlated with prolonged disease-free survival. Moreover, the expression of certain chemokines associated with particular TCR repertoire and specific TCR use predicted patient survival. CONCLUSIONS Data integration and biomolecular network reconstruction is a powerful approach to uncover molecular mechanisms. This study shows the utility of this approach for the investigation of malignant tumors and other diseases. In colorectal cancer, the expression of specific chemokines and adhesion molecules were found as being critical for high densities of T-cell subsets within the tumor and associated with particular TCR repertoire. Intratumoral-specific TCR use correlated with the prognosis of the patients.

Coordination of Intratumoral Immune Reaction and Human Colorectal Cancer Recurrence

A role for the immune system in controlling the progression of solid tumors has been established in several mouse models. However, the effect of immune responses and tumor escape on patient prognosis in the context of human cancer is poorly understood. Here, we investigate the cellular and molecular parameters that could describe in situ immune responses in human colorectal cancer according to clinical parameters of metastatic lymph node or distant organ invasion (META- or META+ patients). Primary tumor samples of colorectal carcinoma were analyzed by integrating large-scale phenotypic (flow cytometry, 39 patients) and gene expression (real time reverse transcription-PCR, 103 patients) data sets related to immune and protumoral processes. In META- colorectal cancer primary tumors with high densities of T cells, we observed significant positive correlations between markers of innate immune cells [tumor-associated macrophages, dendritic cells, natural killer (NK) cells, and NKT cells] and markers of early-activated T cells. Significant correlations were also observed between markers of cytotoxic and effector memory T-cell subpopulations. These correlation profiles were absent in tumors with low T-cell infiltrates and were altered in META+ tumors with high T-cell infiltrates. We show that the coexpression of genes mediating cytotoxicity (GNLY) and Th1 adaptive immune responses (IRF1) accurately predicted patient survival independently of the metastatic status. High intratumoral mRNA expression of the proangiogenic mediator vascular endothelial growth factor was associated with significantly reduced survival rates in patients expressing high mRNA levels of GNLY. Investigation of the colorectal cancer primary tumor microenvironment allowed us to uncover the association of favorable outcomes with efficient coordination of the intratumoral immune response.

Characterization of the immunophenotypes and antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy

BackgroundWhile large-scale cancer genomic projects are comprehensively characterizing the mutational spectrum of various cancers, so far little attention has been devoted to either define the antigenicity of these mutations or to characterize the immune responses they elicit. Here we present a strategy to characterize the immunophenotypes and the antigen-ome of human colorectal cancer.ResultsWe apply our strategy to a large colorectal cancer cohort (n = 598) and show that subpopulations of tumor-infiltrating lymphocytes are associated with distinct molecular phenotypes. The characterization of the antigenome shows that a large number of cancer-germline antigens are expressed in all patients. In contrast, neo-antigens are rarely shared between patients, indicating that cancer vaccination requires individualized strategy. Analysis of the genetic basis of the tumors reveals distinct tumor escape mechanisms for the patient subgroups. Hypermutated tumors are depleted of immunosuppressive cells and show upregulation of immunoinhibitory molecules. Non-hypermutated tumors are enriched with immunosuppressive cells, and the expression of immunoinhibitors and MHC molecules is downregulated. Reconstruction of the interaction network of tumor-infiltrating lymphocytes and immunomodulatory molecules followed by a validation with 11 independent cohorts (n = 1,945) identifies BCMA as a novel druggable target. Finally, linear regression modeling identifies major determinants of tumor immunogenicity, which include well-characterized modulators as well as a novel candidate, CCR8, which is then tested in an orthologous immunodeficient mouse model.ConclusionsThe immunophenotypes of the tumors and the cancer antigenome remain widely unexplored, and our findings represent a step toward the development of personalized cancer immunotherapies.

The tumor microenvironment and Immunoscore are critical determinants of dissemination to distant metastasis

Local lymphatic vessel density and immune cytotoxicity prevent metastatic invasion. Managing metastasis Because of the poor prognosis of metastatic cancer, it is critical to determine exactly how different factors contribute to cancer spread. Mlecnik et al. examined the impact of tumor-intrinsic, microenvironmental, and immunological factors on tumor metastasis in colorectal cancer patients. They found that decreased presence of lymphatic vessels and reduced immune cytotoxicity were more strongly associated with the metastatic process than tumor-intrinsic factors such as chromosomal instability or cancer-associated mutations. These data support testing the Immunoscore as a biomarker to predict metastasis and guide therapy. Although distant metastases account for most of the deaths in cancer patients, fundamental questions regarding mechanisms that promote or inhibit metastasis remain unanswered. We show the impact of mutations, genomic instability, lymphatic and blood vascularization, and the immune contexture of the tumor microenvironment on synchronous metastases in large cohorts of colorectal cancer patients. We observed large genetic heterogeneity among primary tumors, but no major differences in chromosomal instability or key cancer-associated mutations. Similar patterns of cancer-related gene expression levels were observed between patients. No cancer-associated genes or pathways were associated with M stage. Instead, mutations of FBXW7 were associated with the absence of metastasis and correlated with increased expression of T cell proliferation and antigen presentation functions. Analyzing the tumor microenvironment, we observed two hallmarks of the metastatic process: decreased presence of lymphatic vessels and reduced immune cytotoxicity. These events could be the initiating factors driving both synchronous and metachronous metastases. Our data demonstrate the protective impact of the Immunoscore, a cytotoxic immune signature, and increased marginal lymphatic vessels, against the generation of distant metastases, regardless of genomic instability.