Circulating L-arginine predicts the survival of cancer patients treated with immune checkpoint inhibitors.

BACKGROUND: The discovery of immune checkpoint inhibitors (ICIs) has revolutionized the systemic approach to cancer treatment. Most patients receiving ICIs, however, do not derive benefits. Therefore, it is crucial to identify reliable predictive biomarkers of response to ICIs. One important pathway in regulating immune cell reactivity is L-arginine (ARG) metabolism, essential to T-cell activation. We therefore aimed to evaluate the association between baseline plasma ARG levels and the clinical benefit of ICIs. PATIENTS AND METHODS: The correlation between ARG levels and clinical ICI activity was assessed by analyzing plasma samples obtained before treatment onset in two independent cohorts of patients with advanced cancer included in two institutional molecular profiling programs (BIP, NCT02534649, n = 77; PREMIS, NCT03984318, n = 296) and from patients in a phase 1 first-in-human study of budigalimab monotherapy (NCT03000257). Additionally, the correlation between ARG levels and ICI efficacy in preclinical settings was evaluated using a syngeneic mouse model of colorectal cancer responsive to ICIs. Using matched peripheral blood mononuclear cell (PBMC) plasma samples, we analyzed the correlation between ARG levels and PBMC features through multiplexed flow cytometry analysis. RESULTS: In both discovery and validation cohorts, low ARG levels at baseline (<42 µM) were significantly and independently associated with a worse clinical benefit rate, progression-free survival, and overall survival. Moreover, at the preclinical level, the tumor rejection rate was significantly higher in mice with high baseline ARG levels than in those with low ARG levels (85.7% versus 23.8%; P = 0.004). Finally, PBMC immunophenotyping showed that low ARG levels were significantly associated with increased programmed death-ligand 1 expression in several immune cell subsets from the myeloid lineage. CONCLUSIONS: We demonstrate that baseline ARG levels predict ICI response. Plasma ARG quantification may therefore represent an attractive biomarker to tailor novel therapeutic regimens targeting the ARG pathway in combination with ICIs.

Impact of acetaminophen on the efficacy of immunotherapy in cancer patients.

BACKGROUND: Acetaminophen (APAP) use has been associated with blunted vaccine immune responses. This study aimed to assess APAP impact on immunotherapy efficacy in patients with cancer. PATIENTS AND METHODS: Exposure to APAP was assessed by plasma analysis and was correlated with clinical outcome in three independent cohorts of patients with advanced cancer who were treated with immune checkpoint blockers (ICBs). The immunomodulatory effects of APAP were evaluated on a preclinical tumor model and on human peripheral blood mononuclear cells (PBMCs) from healthy donors. RESULTS: Detectable plasma APAP levels at treatment onset were associated with a significantly worse clinical outcome in ICB-treated cancer patients, independently of other prognostic factors. APAP significantly reduced ICB efficacy in the preclinical MC38 model, as well as the production of PD-1 blockade-related interferon-γ secretion by human PBMCs. Moreover, reduction of ICB efficacy in vivo was associated with significantly increased tumor infiltration by regulatory T cells (Tregs). Administration of APAP over 24 h induced a significant expansion of peripheral Tregs in healthy individuals. In addition, interleukin-10, a crucial mediator of Treg-induced immune suppression, was significantly up-regulated upon treatment with ICB in cancer patients taking APAP. CONCLUSIONS: This study provides strong preclinical and clinical evidence of the role of APAP as a potential suppressor of antitumor immunity. Hence, APAP should be used with caution in patients treated with ICB.

Plasma proteomics identifies leukemia inhibitory factor (LIF) as a novel predictive biomarker of immune-checkpoint blockade resistance.

BACKGROUND: Immune checkpoint blockers (ICBs) are now widely used in oncology. Most patients, however, do not derive benefit from these agents. Therefore, there is a crucial need to identify novel and reliable biomarkers of resistance to such treatments in order to prescribe potentially toxic and costly treatments only to patients with expected therapeutic benefits. In the wake of genomics, the study of proteins is now emerging as the new frontier for understanding real-time human biology. PATIENTS AND METHODS: We analyzed the proteome of plasma samples, collected before treatment onset, from two independent prospective cohorts of cancer patients treated with ICB (discovery cohort n = 95, validation cohort n = 292). We then investigated the correlation between protein plasma levels, clinical benefit rate, progression-free survival and overall survival by Cox proportional hazards models. RESULTS: By using an unbiased proteomics approach, we show that, in both discovery and validation cohorts, elevated baseline serum level of leukemia inhibitory factor (LIF) is associated with a poor clinical outcome in cancer patients treated with ICB, independently of other prognostic factors. We also demonstrated that the circulating level of LIF is inversely correlated with the presence of tertiary lymphoid structures in the tumor microenvironment. CONCLUSION: This novel clinical dataset brings strong evidence for the role of LIF as a potential suppressor of antitumor immunity and suggests that targeting LIF or its pathway may represent a promising approach to improve efficacy of cancer immunotherapy in combination with ICB.

Circulating innate immune markers and outcomes in treatment-naïve advanced non-small cell lung cancer patients.

INTRODUCTION: Innate immunity represents the first step of activation of the immune system and dictates the quality of adaptive immune responses. Studies have reported links between systemic inflammatory or innate immune markers and prognosis in patients with lung cancer. To our knowledge, the prospective and concomitant study of these systemic markers has never been performed. METHODS: Advanced treatment-naive non-small cell lung cancer (NSCLC) patients eligible for first-line platinum-based chemotherapy were prospectively included from December 2012 to July 2015 (N = 148). Blood samples of patients were collected before the first cycle for fresh NK cell phenotyping. Peripheral blood mononuclear cells were cryopreserved for natural cytotoxicity receptor (NCR) genotyping as well as sera for NCR's ligand quantification. Data on leukocytes, neutrophils and monocyte counts and lactate dehydrogenase (LDH) levels were extracted from electronic medical records. RESULTS: Among all studied markers, monocytosis, neutrophilia, leucocytosis, high LDH and sBAG6 levels and reduced levels of NCR3 transcripts were associated with poor overall survival (OS) in univariate analysis. The levels of NCR3 transcripts was linked to age, number of metastatic sites, monocyte counts, LDH and sBAG6 levels. Neutrophilia was associated to high sBAG6 levels. NCR3 was the unique innate immune parameter that remained as an independent factor associated with both OS (P = 0.003) and progression-free survival (P = 0.009) in the multivariate analysis. CONCLUSION: This study brought evidence that these biomarkers are entangled; parameters associated with an inflammatory process were related to reduced levels of NCR3 transcripts. Finally, the level of NCR3 transcripts was independently associated with outcomes in treatment-naive patients with advanced NSCLC.

Immunotherapy in advanced gastric cancer, is it the future?

The prognosis of advanced gastric cancer remains extremely poor despite the use of standard therapies such as chemotherapy and biological agents. Blocking immune checkpoint especially programmed cell death-1 (PD-1) and its ligand (PD-L1 or B7-H1), has proven efficacy in several solid cancers, and seems to become a potential option in gastric cancer treatment. This review will focus on data describing the immune microenvironment of gastric tumors on which blocking PD-1/PD-L1 axis may have an anti-tumor efficacy. Then, the encouraging results of clinical trials evaluating anti-PD-1/PD-L1-based therapeutic strategy in first or later-line settings will be discuss. Finally, clinical outcomes according to PD-L1 expression, mismatch repair phenotype and other potential predictive biomarkers of anti-tumor response will be described. Altogether, immunotherapy seems promising in advanced gastric cancer in monotherapy or in combining strategies probably for a specific subgroup of patients who need to be better identified.

Inflammatory gastrointestinal diseases associated with PD-1 blockade antibodies.

BACKGROUND: Immune check-point blockade agents have shown clinical activity in cancer patients but are associated with immune-related adverse events that could limit their development. The aim of this study was to describe the gastrointestinal immune-related adverse events (GI-irAE) in patients with cancer treated with anti-PD-1. METHODS: this is a retrospective study of consecutive adult patients who had a suspected GI-irAE due to anti-PD-1 antibodies between 2013 and 2016. Patients were recruited through a pharmacovigilance registry. Patients' data were reviewed by a multidisciplinary committee that included gastroenterologists, oncologists and a pathologist. Quantitative variables are described by median (range), qualitative variable by frequency (percentage). RESULTS: Forty-four patients were addressed to a Gastroenterology unit for a suspected GI-IrAE. Twenty patients had a confirmed GI-irAE related to anti-PD-1, which occurred 4.2 months (0.2; 22.1) after the initiation of anti-PD-1. GI-IrAE incidence rate under anti-PD-1 treatment was estimated to be 1.5%. Among patients with GI-IrAE, main symptoms were diarrhoea (n = 16, 80%), abdominal pain (n = 13, 65%), nausea and vomiting (n = 11, 55%), intestinal obstruction (n = 1, 5%), and haematochezia (n = 2, 10%). No patient had colectomy. Four distinct categories of GI-irAE were observed: acute colitis (n = 8, 40%), microscopic colitis (n = 7, 35%), upper gastrointestinal tract inflammation (n = 4, 20%) and pseudo-obstruction (n = 1, 5%). Response rates to corticosteroids were 87.5% (7/8) in acute colitis, 57% (4/7) in microscopic colitis and 75% (3/4) in upper gastrointestinal tract inflammation. Median time to resolution was 36 days (6-172) in acute colitis, and 98 days (42-226) in microscopic colitis. CONCLUSION: This study suggests that GI-irAE are different and less frequent with anti PD-1 than with anti CTLA-4.

Baseline gut microbiota predicts clinical response and colitis in metastatic melanoma patients treated with ipilimumab.

BACKGROUND: Ipilimumab, an immune checkpoint inhibitor targeting CTLA-4, prolongs survival in a subset of patients with metastatic melanoma (MM) but can induce immune-related adverse events, including enterocolitis. We hypothesized that baseline gut microbiota could predict ipilimumab anti-tumor response and/or intestinal toxicity. PATIENTS AND METHODS: Twenty-six patients with MM treated with ipilimumab were prospectively enrolled. Fecal microbiota composition was assessed using 16S rRNA gene sequencing at baseline and before each ipilimumab infusion. Patients were further clustered based on microbiota patterns. Peripheral blood lymphocytes immunophenotypes were studied in parallel. RESULTS: A distinct baseline gut microbiota composition was associated with both clinical response and colitis. Compared with patients whose baseline microbiota was driven by Bacteroides (cluster B, n = 10), patients whose baseline microbiota was enriched with Faecalibacterium genus and other Firmicutes (cluster A, n = 12) had longer progression-free survival (P = 0.0039) and overall survival (P = 0.051). Most of the baseline colitis-associated phylotypes were related to Firmicutes (e.g. relatives of Faecalibacterium prausnitzii and Gemmiger formicilis), whereas no colitis-related phylotypes were assigned to Bacteroidetes. A low proportion of peripheral blood regulatory T cells was associated with cluster A, long-term clinical benefit and colitis. Ipilimumab led to a higher inducible T-cell COStimulator induction on CD4+ T cells and to a higher increase in serum CD25 in patients who belonged to Faecalibacterium-driven cluster A. CONCLUSION: Baseline gut microbiota enriched with Faecalibacterium and other Firmicutes is associated with beneficial clinical response to ipilimumab and more frequent occurrence of ipilimumab-induced colitis.

Cancer Immunotherapy with Anti-CTLA-4 Monoclonal Antibodies Induces an Inflammatory Bowel Disease.

BACKGROUND: Therapeutic monoclonal anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibodies are associated with immune-mediated enterocolitis. The aim of this study was to provide a detailed description of this entity. METHODS: We included patients with endoscopic signs of inflammation after anti-CTLA-4 infusions for cancer treatment. Other causes of enterocolitis were excluded. Clinical, biological and endoscopic data were recorded. A single pathologist reviewed endoscopic biopsies and colectomy specimens from 27 patients. Patients with and without enterocolitis after ipilimumab-treated melanoma were compared, to identify clinical factors associated with enterocolitis. RESULTS: Thirty-nine patients with anti-CTLA-4 enterocolitis were included (ipilimumab n = 37; tremelimumab n = 2). The most frequent symptom was diarrhoea. Ten patients had extra-intestinal manifestations. Most colonoscopies showed ulcerations involving the rectum and sigmoid, 66% of patients had extensive colitis, 55% had patchy distribution and 20% had ileal inflammation. Endoscopic colonic biopsies showed acute colitis in most patients, while half of the patients had chronic duodenitis. Thirty-five patients received steroids that led to complete clinical remission in 13 patients (37%). Twelve patients required infliximab, of whom 10 (83%) responded. Six patients underwent colectomy (perforation n = 5; toxic megacolon n = 1); one of them died postoperatively. Four patients had a persistent enterocolitis at follow-up colonoscopy. Patients with enterocolitis were more frequently prescribed NSAIDs compared with patients without enterocolitis (31 vs 5%, p = 0.003). CONCLUSIONS: Ipilimumab and tremelimumab may induce a severe and extensive form of inflammatory bowel disease. Rapid escalation to infliximab should be advocated in patients who do not respond to steroids. Patients treated with anti-CTLA-4 should be advised to avoid NSAIDs.

Tumour-infiltrating CD68+ and CD57+ cells predict patient outcome in stage II-III colorectal cancer.

BACKGROUND: The aim of our study was to evaluate the prognostic role of immunological microenvironnement in stage II-III CRC patients. METHODS: We constructed a tissue microarray from 196 consecutive patients with stage II-III CRC and compared CD3, CD4, CD8, CD57, CD68, CXCL9/MIG, CXCL13, and PPARγ immunoreactivity in tumour samples and their matched non-tumour tissue. We assessed their association with relapse-free survival (RFS; primary endpoint) and overall survival (OS) in multivariate Cox models. RESULTS: Low densities of CD57+ and CD68+ tumour-infiltrating cells (TIC) independently predicted worse outcomes. A prognostic score combining CD57 (+, > vs -, ≤2 cells per spot) and CD68 (+, >0 vs -, =0 cells per spot) TIC density discriminated CRC patients at low (CD68+/CD57+), intermediate (CD68+/CD57-), or high (CD68-/CD57-) risk, with hazard ratios for the intermediate-risk and high-risk groups of 2.7 (95% confidence interval (CI): 1.3-5.8) and 9.0 (3.2-25.4) for RFS, and 2.5 (1.2-5.1) and 10.6 (3.8-29.2) for OS, respectively, as compared with the low-risk group. Corresponding 5-year survival rates (95% CI) in the low-, moderate- and high-risk groups were 84% (71-91), 65% (54-74), and 12% (2-47), respectively, for RFS, and 91% (80-96), 76% (66-84), and 25% (7-59), respectively, for OS. CONCLUSION: Tumour CD57+ and CD68+ TIC density assessment independently predicts survival in patients with stage II-III CRC. If validated, our score based on a quick, inexpensive, and well-established method such as point counting on diagnostic tissue sections could be used routinely as a prognostic tool in CRC patients.

Experience in daily practice with ipilimumab for the treatment of patients with metastatic melanoma: an early increase in lymphocyte and eosinophil counts is associated with improved survival.

BACKGROUND: Ipilimumab is a recently approved immunotherapy that has demonstrated an improvement in the overall survival (OS) of patients with metastatic melanoma. We report a single-institution experience in patients treated in a compassionate-use program. PATIENTS AND METHODS: In this prospective study, patients were treated between June 2010 and September 2011. Inclusion criteria were a diagnosis of unresectable stage III or IV melanoma, at least one previous line of chemotherapy, and survival 12 weeks after the first perfusion. Four courses of ipilimumab were administered at a dose of 3 mg/kg every 3 weeks. RESULTS: Seventy-three patients were included. Median OS was 9.1 months (95% CI 6.4-11.3) from the start of ipilimumab. Immune-related adverse events were observed in 45 patients (62%), including 19 grade 3-4 events (26%). No drug-related death occurred. A lymphocyte count >1000/mm(3) at the start of the second course and an increase in the eosinophil count >100/mm(3) between the first and second infusions were correlated with an improved OS. CONCLUSION: Ipilimumab toxic effect is manageable in real life. Biological data such as lymphocyte and eosinophil counts at the time of the second ipilimumab infusion appear to be early markers associated with better OS.

Calreticulin exposure on malignant blasts predicts a cellular anticancer immune response in patients with acute myeloid leukemia.

Experiments performed in mice revealed that anthracyclines stimulate immunogenic cell death that is characterized by the pre-apoptotic exposure of calreticulin (CRT) on the surface of dying tumor cells. Here, we determined whether CRT exposure at the cell surface (ecto-CRT) occurs in human cancer in response to anthracyclines in vivo, focusing on acute myeloid leukemia (AML), which is currently treated with a combination of aracytine and anthracyclines. Most of the patients benefit from the induction chemotherapy but relapse within 1-12 months. In this study, we investigated ecto-CRT expression on malignant blasts before and after induction chemotherapy. We observed that leukemic cells from some patients exhibited ecto-CRT regardless of chemotherapy and that this parameter was not modulated by in vivo chemotherapy. Ecto-CRT correlated with the presence of phosphorylated eIF2α within the blasts, in line with the possibility that CRT exposure results from an endoplasmic reticulum stress response. Importantly, high levels of ecto-CRT on malignant myeloblasts positively correlated with the ability of autologous T cells to secrete interferon-γ on stimulation with blast-derived dendritic cell. We conclude that the presence of ecto-CRT on leukemia cells facilitates cellular anticancer immune responses in AML patients.

Identification of CD8+CD25+Foxp3+ suppressive T cells in colorectal cancer tissue.

BACKGROUND: The antitumoral immune response is one determinant of colorectal cancer (CRC) outcome. Recent work suggests that Foxp3(+)CD25(+)CD4(+) regulatory T cells (T4reg) might hamper effective immunosurveillance of emerging cancer cells and impede effective immune responses to established tumours. In this descriptive study, we analysed blood and tissue regulatory T cell populations in patients with CRC. METHODS: Blood and tissue regulatory Foxp3(+) T cells from 40 patients with CRC were compared to regulatory Foxp3(+) T cells from normal colonic tissue and from blood of 26 healthy volunteers. Flow cytometry was used to quantify and phenotype all Foxp3(+) T cell populations. Correlations were sought with the tumour stage and with micro-invasive status. The suppressive capacity of regulatory Foxp3(+) T cells was assessed by their effect on CD4(+)CD25(-) T cell proliferation in vitro and by their capacity to inhibit cytokine production by conventional T cells. RESULTS: We found a significant increase of CD8(+)CD25(+)Foxp3(+) cells (T8reg) in blood and CRC tissue; their phenotype was close to that of T4reg. T8reg cells infiltrating CRC were activated, as suggested by increased cytoxic T lymphocyte-associated antigen-4, glucocorticoid-induced tumour necrosis factor-related protein, and transforming growth factor (TGF)beta1 expression compared to T8reg from normal autologous colonic tissue. Moreover, T8reg were able to suppress CD4(+)CD25(-) T cell proliferation and Th1 cytokine production ex vivo, demonstrating that tumour-infiltrating T8reg have strong suppressive capacities. T8reg numbers correlated with the tumour stage and with micro-invasive status. Finally, interleukin 6 and TGF beta 1 synergistically induced the generation of CD8(+)CD25(+)Foxp3(+) T cells ex vivo. CONCLUSIONS: We have identified a new regulatory T cell population (CD8(+)Foxp3(+)) in colorectal tumours. After isolation from cancer tissue these CD8(+)Foxp3(+) cells demonstrated strong immunosuppressive properties in vitro. These data suggest that these cells may contribute to tumoral immune escape and disease progression.

The Janus face of dendritic cells in cancer.

On the basis of experimental models and some human data, we can assume that tumor outgrowth results from the balance between immunosurveillance (the extrinsic tumor suppressor mechanisms) and immunosubversion dictated by transformed cells and/or the corrupted surrounding microenvironment. Cancer immunosurveillance relies mainly upon conventional lymphocytes exerting either lytic or secretory functions, whereas immunosubversion results from the activity of regulatory T or suppressor myeloid cells and soluble mediators. Although specific tools to target or ablate dendritic cells (DCs) became only recently available, accumulating evidence points to the critical role of the specialized DC system in dictating most of the conventional and regulatory functions of tumor-specific T lymphocytes. Although DC can be harnessed to silence tumor development, tumors in turn can exploit DC to evade immunity. Indeed, DCs harbor defects in their differentiation and stimulatory functions in cancer-bearing hosts and can actively promote T-cell tolerance to self-tumor antigens. In this review, we will focus on the dual role of DC during tumor progression and discuss pharmacoimmunological strategies to harness DC against cancer.

Killer dendritic cells and their potential role in immunotherapy.

Tumor immunosurveillance is mediated by innate and adaptive components of cellular immunity. A complex network of cellular interactions is needed to elicit protective antitumoral CD4+and CD8+T cell responses. Thereby dendritic cells (DCs) play a central role as professional antigen presenting cells (APCs) that take up antigens, process, and present them to prime naïve T cells. Recognition and lysis of tumor cells has been attributed to innate effectors such as natural killer (NK), NKT and gammadeltaT cells. Recently, novel subsets of cytotoxic DCs, called "killer DCs" (KDCs), have been reported in rodents and humans. Killer dendritic cells could directly link innate and adaptive immunity. This review aims at comparing the different KDC populations, their phenotypes, killer function, and their potential application for anticancer immunotherapy.

Exosomes for the treatment of human malignancies.

Exosomes are nanometer particles (50-100 nm) secreted by most living cells. The first description of exosomes was made in 1987 by Rose Johnstone, who described a vesicle formation during the maturation process of reticulocytes. At this time it has been suggested that exosome release could represent a major route for the externalization of obsolete membrane proteins. A renewed vision of exosome function was raised when Graça Raposo demonstrated in 1996 that exosomes derived from B cells could have immunogenic capacities. Since then, exosomes have been described in numerous cell types IN VITRO, including hematopoietic and nonhematopoietic cells. The physiological relevance of exosomes IN VIVO still remains unclear. Studies have demonstrated that exosomes can play a role in the physiology of originating cells (i.e., reticulocyte-derived exosomes). Furthermore, exosomes can act on intercellular communication by allowing exchange of proteins, lipids, and also mRNA between cells. Finally, exosomes have been shown to modulate the immune system (i.e., dendritic cells, B cells, and tumor cells). In the present review, we have focused on the potential therapeutic role of exosomes as a cell free vaccine in cancer.

Tumor-derived exosomes: a new source of tumor rejection antigens.

Exosomes are small vesicles released by a broad array of hematopoietic cells. Previous studies showed that exosomes released by antigen loaded dendritic cells induce immune-mediated anti-tumor response in mice. Here, we will describe the biochemical properties of tumor-derived exosomes and, their pre-clinical activity as cancer vaccines.

Dendritic cell maturation overrules H-2D-mediated natural killer T (NKT) cell inhibition: critical role for B7 in CD1d-dependent NKT cell interferon gamma production.

Given the broad expression of H-2 class Ib molecules on hematopoietic cells, antigen presentation pathways among CD1d expressing cells might tightly regulate CD1d-restricted natural killer T (NKT) cells. Bone marrow-derived dendritic cells (BM-DCs) and not adherent splenocytes become capable of triggering NK1.1(+)/T cell receptor (TCR)(int) hepatic NKT cell activation when (a) immature BM-DCs lack H-2D(b)-/- molecules or (b) BM-DCs undergo a stress signal of activation. In such conditions, BM-DCs promote T helper type 1 predominant CD1d-restricted NKT cell stimulation. H-2 class Ia-mediated inhibition involves more the direct H-2D(b) presentation than the indirect Qa-1(b) pathway. Such inhibition can be overruled by B7/CD28 interactions and marginally by CD40/CD40L or interleukin 12. These data point to a unique regulatory role of DCs in NKT cell innate immune responses and suggest that H-2 class Ia and Ib pathways differentially control NKT cell recognition of DC antigens.