Prognostic and predictive value of the Immunoscore in stage III colon cancer patients treated with oxaliplatin in the prospective IDEA France PRODIGE-GERCOR cohort study.

BACKGROUND: The Immunoscore (IS), which prognostically classifies stage I-III colon cancer (CC) patients, was evaluated in the International Duration Evaluation of Adjuvant Therapy (IDEA) France cohort study investigating 3 versus 6 months of oxaliplatin-based adjuvant chemotherapy in stage III CC patients. PATIENTS AND METHODS: Densities of CD3+ and CD8+ T cells in the tumor and invasive margin were determined by immunohistochemistry, quantified by digital pathology, and converted to IS. Mismatch repair status was determined by immunohistochemistry or by pentaplex PCR. Prediction of disease-free survival (DFS) by IS was analyzed by a multivariable Cox regression model in each study arm. Harrell's C-statistics were used to investigate the IS performance. RESULTS: Samples of 1322 patients were available. IS Low, Intermediate (Int), and High were observed in 43.6%, 47.0%, and 9.4% of patients, respectively. IS Low identified patients at higher risk of relapse or death compared with Int + High [hazard ratio (HR) = 1.54; 95% confidence interval (CI) 1.24-1.93, P = 0.0001]. The 3-year DFS was 66.80% (95% CI 62.23-70.94) for IS Low and 77.14% (95% CI 73.50-80.35) for IS Int + High. In multivariable analysis, IS remained significantly independently associated with DFS (P = 0.003) when adjusted for sex, histological grade, T/N stage, and microsatellite instability. For mFOLFOX6-treated patients (91.6% of the cohort), a statistical significant interaction was observed for the predictive value of IS for treatment duration (3 versus 6 months) in terms of DFS (P = 0.057). IS Int + High significantly predicted benefit of 6 months of treatment (HR = 0.53; 95% CI 0.37-0.75; P = 0.0004), including clinically low- and high-risk stage III CC (all P < 0.001). Conversely, patients with IS Low (46.4%) did not significantly benefit from the 6-month mFOLFOX6 versus the 3-month mFOLFOX6. CONCLUSIONS: The prognostic value of IS for DFS was confirmed in patients with stage III CC treated with oxaliplatin-based chemotherapy. Its predictive value for DFS benefit of longer duration of mFOLFOX6 adjuvant treatment was found in IS Int + High. These results will be validated in an external independent cohort. CLINICALTRIALS. GOV REGISTRATION: NCT03422601; EudraCT Number: 2009-010384-16.

Immunoscore and Immunoprofiling in cancer: an update from the melanoma and immunotherapy bridge 2015.

The fifth "Melanoma Bridge Meeting" took place in Naples, December 1-5th, 2015. The main topics discussed at this meeting were: Molecular and Immuno advances, Immunotherapies and Combination Therapies, Tumor Microenvironment and Biomarkers and Immunoscore. The natural history of cancer involves interactions between the tumor and the immune system of the host. The immune infiltration at the tumor site may be indicative of host response. Significant correlations were shown between the levels of immune cell infiltration in tumors and patient's clinical outcome. Moreover, incredible progress comes from the discovery of mutation-encoded tumor neoantigens. In fact, as tumors grow, they acquire mutations that are able to influence the response of patients to immune checkpoint inhibitors. It has been demonstrated that sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. The road ahead is still very long, but the knowledge of the mechanisms of immune escape, the study of tumor neo-antigens as well as of tumor microenvironment and the development of new immunotherapy strategies, will make cancer a more and more treatable disease.

Immune infiltration in human tumors: a prognostic factor that should not be ignored.

The natural history of a tumor includes phases of 'in situ' growth, invasion, extravasation and metastasis. During these phases, tumor cells interact with their microenvironment and are influenced by signals coming from stromal, endothelial, inflammatory and immune cells. Indeed, tumors are often infiltrated by various numbers of lymphocytes, macrophages or mast cells. It is generally believed that the latter produce factors that maintain chronic inflammation and promote tumor growth, whereas lymphocytes may control cancer outcome, as evidenced in mouse models. In this study, we analyze data from large cohorts of human tumors, clearly establishing that infiltration of the primary tumor by memory T cells, particularly of the Th1 and cytotoxic types, is the strongest prognostic factor in terms of freedom from disease and overall survival at all stages of clinical disease. We review data suggesting that tertiary lymphoid structures adjacent to tumors and composed of mature dendritic cells (T and B cells organized as germinal centers) may be the site of an antitumor reaction. We propose an immune scoring based on the type, density and location of lymphocyte infiltrates as a novel prognostic factor for use in addition to tumor node metastasis staging to predict disease-free survival and to aid in decisions regarding adjuvant therapies in early stage human cancers.

[Not Available]. / Implications immunologiques potentielles du curage ganglionnaire : Exemple du cancer colorectal.

F. Pagès, A. Berger, F. Zinzindohoué, A. Kirilovsky, J. Galon, W.-H. Fridman Lymph node dissection is an integral part of the surgical resection of colon cancers; it completes the wide regional resection of tumor and it allows prognostic evaluation through accurate staging. Studies have demonstrated an immune reaction to the tumoral site which attests to an ongoing dialog between the tumor and systemic defenses. The regional lymph nodes constitute an important first line of immune defense where initial host response is initiated or, inversely, they may participate in a local state of immunosuppression. This article reviews current knowledge on intra-tumoral and nodal immune status in colorectal cancers and attempts to evaluate the potential immunologic implications of lymph node dissection.

[Not Available]. / Implications immunologiques potentielles du curage ganglionnaire : Exemple du cancer colorectal.

F. Pagès, A. Berger, F. Zinzindohoué, A. Kirilovsky, J. Galon, W.-H. Fridman Lymph node dissection is an integral part of the surgical resection of colon cancers; it completes the wide regional resection of tumor and it allows prognostic evaluation through accurate staging. Studies have demonstrated an immune reaction to the tumoral site which attests to an ongoing dialog between the tumor and systemic defenses. The regional lymph nodes constitute an important first line of immune defense where initial host response is initiated or, inversely, they may participate in a local state of immunosuppression. This article reviews current knowledge on intra-tumoral and nodal immune status in colorectal cancers and attempts to evaluate the potential immunologic implications of lymph node dissection.

[Potential immunologic consequences of lymph node dissection in colorectal cancer]. / Implications immunologiques potentielles du curage ganglionnaire: Exemple du cancer colorectal.

Lymph node dissection is an integral part of the surgical resection of colon cancers; it completes the wide regional resection of tumor and it allows prognostic evaluation through accurate staging. Studies have demonstrated an immune reaction to the tumoral site which attests to an ongoing dialog between the tumor and systemic defenses. The regional lymph nodes constitute an important first line of immune defense where initial host response is initiated or, inversely, they may participate in a local state of immunosuppression. This article reviews current knowledge on intra-tumoral and nodal immune status in colorectal cancers and attempts to evaluate the potential immunologic implications of lymph node dissection.

Differential gene expression profile of glucocorticoids, testosterone, and dehydroepiandrosterone in human cells.

Glucocorticoids are the major immunomodulating hormones in the human body. Recently, increasing interest in androgens as immunomodulators has emerged. In particular, Dehydroepiandrosterone (DHEA) has been suggested as beneficial in the treatment of some autoimmune disorders. However, the action and role of testicular and adrenal androgens on human immune cells remains unclear. This is the first study to provide large-scale gene expression data on the action of different steroids (DHEA, glucocorticoids, and testosterone) on human peripheral blood mononuclear cells using the recently developed genomic-scale technology of microarrays. Novel computational tools and techniques such as Principal Component Analysis (PCA) were used for analysis, clustering and visualization. We have demonstrated that each steroid has its distinct gene expression profile, although DHEA and testosterone co-regulated most genes in a similar direction while glucocorticoids frequently regulated the same genes in an opposite direction. Our data suggest an important and a complex regulatory role for androgens on human immune cells that should be considered in androgen replacement or treatment strategies.

The tumor-necrosis-factor receptor-associated periodic syndrome: new mutations in TNFRSF1A, ancestral origins, genotype-phenotype studies, and evidence for further genetic heterogeneity of periodic fevers.

Mutations in the extracellular domain of the 55-kD tumor-necrosis factor (TNF) receptor (TNFRSF1A), a key regulator of inflammation, define a periodic-fever syndrome, TRAPS (TNF receptor-associated periodic syndrome [MIM 142680]), which is characterized by attacks of fever, sterile peritonitis, arthralgia, myalgia, skin rash, and/or conjunctivitis; some patients also develop systemic amyloidosis. Elsewhere we have described six disease-associated TNFRSF1A mutations, five of which disrupt extracellular cysteines involved in disulfide bonds; four other mutations have subsequently been reported. Among 150 additional patients with unexplained periodic fevers, we have identified four novel TNFRSF1A mutations (H22Y, C33G, S86P, and c.193-14 G-->A), one mutation (C30S) described by another group, and two substitutions (P46L and R92Q) present in approximately 1% of control chromosomes. The increased frequency of P46L and R92Q among patients with periodic fever, as well as functional studies of TNFRSF1A, argue that these are low-penetrance mutations rather than benign polymorphisms. The c.193-14 G-->A mutation creates a splice-acceptor site upstream of exon 3, resulting in a transcript encoding four additional extracellular amino acids. T50M and c.193-14 G-->A occur at CpG hotspots, and haplotype analysis is consistent with recurrent mutations at these sites. In contrast, although R92Q also arises at a CpG motif, we identified a common founder chromosome in unrelated individuals with this substitution. Genotype-phenotype studies identified, as carriers of cysteine mutations, 13 of 14 patients with TRAPS and amyloidosis and indicated a lower penetrance of TRAPS symptoms in individuals with noncysteine mutations. In two families with dominantly inherited disease and in 90 sporadic cases that presented with a compatible clinical history, we have not identified any TNFRSF1A mutation, despite comprehensive genomic sequencing of all of the exons, therefore suggesting further genetic heterogeneity of the periodic-fever syndromes.

Soluble CD16 inhibits CR3 (CD11b/CD18)-mediated infection of monocytes/macrophages by opsonized primary R5 HIV-1.

We demonstrate that soluble CD16 (sCD16; soluble Fc gamma RIII), a natural ligand of CR3, inhibits the infection of monocytes by primary R5 HIV-1 strain opsonized with serum of seronegative individuals. Inhibition of monocyte infection by sCD16 was similar to that observed with anti-CR3 mAbs, indicating that opsonized HIV may use a CR3-dependent pathway for entry in monocytic cells. Cultured human monocytes express both CR3 (CD11b/CD18) and CCR5 receptors. RANTES, the natural ligand of CCR5, inhibited infection of monocytes with unopsonized HIV particles and partially that of monocytes infected with HIV particles opsonized with complement-derived fragments. Although HIV-infected monocytes from homozygous CCR5 Delta 32/Delta 32 (CCR5(-/-)) individuals produce low levels of p24, cells infected with opsonized particles produced higher levels of p24 than cells infected with unopsonized particles. Our results thus suggest that CR3 may represent an alternative coreceptor to CCR5 of opsonized primary R5 virus entry into monocytes/macrophages. We also observed that the concentration of sCD16 is greatly decreased in sera of HIV-infected patients with low lymphocyte CD4(+) counts. Taken together, our findings suggest that sCD16, present in plasma, may play an important role in controlling HIV-1 spread.

Adrenal cortical activation in murine colitis.

BACKGROUND & AIMS: Proper adrenal glucocorticoid secretion is crucial in the course of inflammatory diseases. However, the function and structure of the adrenal glands have not been examined in inflammatory bowel diseases. METHODS: After induction of trinitrobenzene sulfonic acid (TNBS) colitis in SJL/J mice, plasma hormone and cytokine levels were measured, adrenal structure was analyzed by immunohistochemistry and electron microscopy, and adrenal cytokine/cytokine receptor expression were studied by RNase protection. RESULTS: Adrenals of colitic animals were enlarged and hypervascularized. These animals had a marked increase in plasma corticosterone levels during the course of colitis (270 +/- 34 vs. 16 +/- 11 ng/mL; P < 0.0001) but only a modest elevation of their concurrent adrenocorticotropin levels (57 +/- 13 vs. 29 +/- 9 pmol/L; NS). On electron microscopy, adrenocortical cells showed ultrastructural signs of marked stimulation, and intra-adrenal lymphocytes were frequently found in direct contact with these cells. Concurrent plasma levels of interleukin (IL)-6, the major cytokine activating the hypothalamic-pituitary-adrenal axis, were markedly increased (495 +/- 131 vs. 20 +/- 1.5 pg/mL; P < 0.0001), and this cytokine directly stimulated corticosterone secretion by adrenocortical cells in vitro. Intra-adrenal expression of IL-6 in animals with colitis was increased 80-fold, and the IL-6 receptor subunits IL-6R alpha and gp130 were present in the adrenal cells. Treatment of animals with neutralizing anti-IL-6 antibody reduced the TNBS-induced growth and activation of the adrenal cortices. CONCLUSIONS: Colitis is associated with a profound stimulation of adrenocortical cell function and glucocorticoid release. Direct immune-adrenal interactions seem to contribute to this activation of the adrenal glands during colitis.

TNFRSF1A mutations and autoinflammatory syndromes.

The autoinflammatory syndromes are systemic disorders characterized by apparently unprovoked inflammation in the absence of high-titer autoantibodies or antigen-specific T lymphocytes. One such illness, TNF-receptor-associated periodic syndrome (TRAPS), presents with prolonged attacks of fever and severe localized inflammation. TRAPS is caused by dominantly inherited mutations in TNFRSF1A (formerly termed TNFR1), the gene encoding the 55 kDa TNF receptor. All known mutations affect the first two cysteine-rich extracellular subdomains of the receptor, and several mutations are substitutions directly disrupting conserved disulfide bonds. One likely mechanism of inflammation in TRAPS is the impaired cleavage of TNFRSF1A ectodomain upon cellular activation, with diminished shedding of the potentially antagonistic soluble receptor. Preliminary experience with recombinant p75 TNFR-Fc fusion protein in the treatment of TRAPS has been favorable.

The gene for familial Mediterranean fever, MEFV, is expressed in early leukocyte development and is regulated in response to inflammatory mediators.

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever and neutrophil-mediated serosal inflammation. We recently identified the gene causing FMF, designated MEFV, and found it to be expressed in mature neutrophils, suggesting that it functions as an inflammatory regulator. To facilitate our understanding of the normal function of MEFV, we extended our previous studies. MEFV messenger RNA was detected by reverse transcriptase-polymerase chain reaction in bone marrow leukocytes, with differential expression observed among cells by in situ hybridization. CD34 hematopoietic stem-cell cultures induced toward the granulocytic lineage expressed MEFV at the myelocyte stage, concurrently with lineage commitment. The prepromyelocytic cell line HL60 expressed MEFV only at granulocytic and monocytic differentiation. MEFV was also expressed in the monocytic cell lines U937 and THP-1. Among peripheral blood leukocytes, MEFV expression was detected in neutrophils, eosinophils, and to varying degrees, monocytes. Consistent with the tissue specificity of expression, complete sequencing and analysis of upstream regulatory regions of MEFV revealed homology to myeloid-specific promoters and to more broadly expressed inflammatory promoter elements. In vitro stimulation of monocytes with the proinflammatory agents interferon (IFN) gamma, tumor necrosis factor, and lipopolysaccharide induced MEFV expression, whereas the antiinflammatory cytokines interleukin (IL) 4, IL-10, and transforming growth factor beta inhibited such expression. Induction by IFN-gamma occurred rapidly and was resistant to cycloheximide. IFN-alpha also induced MEFV expression. In granulocytes, MEFV was up-regulated by IFN-gamma and the combination of IFN-alpha and colchicine. These results refine understanding of MEFV by placing the gene in the myelomonocytic-specific proinflammatory pathway and identifying it as an IFN-gamma immediate early gene.

Hierarchy of protein tyrosine kinases in interleukin-2 (IL-2) signaling: activation of syk depends on Jak3; however, neither Syk nor Lck is required for IL-2-mediated STAT activation.

Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.

Stat4 is expressed in activated peripheral blood monocytes, dendritic cells, and macrophages at sites of Th1-mediated inflammation.

Stat4 is a key transcription factor involved in promoting cell-mediated immunity, whose expression in mature cells has been reported to be restricted to T and NK cells. We demonstrate here, however, that Stat4 expression is not restricted to lymphoid cells. In their basal state, monocytes do not express Stat4. Upon activation, however, IFN-gamma- and LPS-treated monocytes and dendritic cells express high levels of Stat4. Monocyte-expressed Stat4 in humans is phosphorylated in response to IFN-alpha, but not IL-12. In contrast, the Th2 cytokines, IL-4 and IL-10, specifically down-regulate Stat4 expression in activated monocytes, while having little effect on Stat6 expression. Moreover, macrophages in synovial tissue obtained from patients with rheumatoid arthritis express Stat4 in vivo, suggesting a potential role in a prototypical Th1-mediated human disease. IFN-alpha-induced Stat4 activation in human monocytes represents a previously unrecognized signaling pathway at sites of Th1 inflammation.

Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes.

Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.

Regulation of production of soluble Fc gamma receptors type III in normal and pathological conditions.

CD16 (Fc gamma R type III), a low affinity IgG Fc receptor, is found in two forms, a transmembrane Fc gamma RIIIa expressed by NK cells and monocytes and a phosphatidylinositol-linked Fc gamma RIIIb present on neutrophils. Exposure of neutrophils to inflammatory signals induces a rapid loss of CD16 expression and release of a soluble form of CD16 (sCD16). Soluble CD16 circulates in plasma, levels being reduced in sera from patients with multiple myeloma. In the present manuscript the authors summarize work that aimed to better understand: (i) the role of proteinases in sCD16 production and CD16 membrane shedding; and (ii) the regulation of sCD16 levels in multiple myeloma patients and the possible biological consequences of its decrease in this disease. Soluble CD16 was purified from human serum. Its N-terminal sequencing demonstrated that it originates from neutrophil CD16 and its C-terminal sequencing showed that the cleavage site was between Val 196 and Ser 197, close to the membrane anchor. Analysis of the effect of protease inhibitors revealed that the cleavage leading to sCD16 production by PMA-activated neutrophils was metalloproteinase-dependent. In addition, membrane and sCD16 were sensitive to serine proteinases released by azurophil granules or added under purified form. The reduction of sCD16 levels that occurs in patients with multiple myeloma was associated with a slight decrease in circulating neutrophils, but not with a significant defect in sCD16 production by neutrophils, as detected in vitro. Moreover, addition of a recombinant sCD16 to plasmocytoma lines did not significantly modify their proliferation and Ig secretion.

IL-12 induces IFN regulating factor-1 (IRF-1) gene expression in human NK and T cells.

IL-12 is a critical immunoregulatory cytokine that promotes cell-mediated immune responses and the differentiation of naive CD4+ cells to Th1 cells; however, relatively few IL-12 target genes have been identified. To better clarify the molecular basis of IL-12 action, we set out to characterize genes up-regulated by IL-12, first by contrasting IL-12- and IFN-alpha-inducible genes. We identified several genes up-regulated by IL-12, namely, MIP-1alpha, MIP-1beta, IL-1RA, and IFN regulatory factor-1 (IRF-1). IRF-1 is a transcription factor regulated by IFNs that is also essential for Th1 responses. We demonstrated that IL-12 directly up-regulates IRF-1 to the same extent as IFN-alpha in normal human T cells and in NK cells. We showed that IL-12 had a direct effect on IRF-1, an effect not mediated indirectly by the induction of IFN-gamma production. Furthermore, IL-2 and IL-12 synergistically induced IRF-1, whereas IFN-alpha and IL-12 did not. The participation of STAT4 in the regulation of IRF-1 was demonstrated in two ways. First, STAT4 was required for the IL-12-dependent transactivation of an IRF-1 reporter construct, and second, STAT4 binding to the IRF-1 promoter was shown using EMSA. In contrast to IL-12, no up-regulation of IRF-1 was found in IL-4-stimulated cells, and IL-4 did not block IL-12-dependent up-regulation of IRF-1. Therefore, IRF-1 may be an important contributor to IL-12 signaling, and we speculate that the defective IL-12 responses seen in IRF-1-/- mice might be attributable, in part, to the absence of this transcription factor.