Combining measures of immune infiltration shows additive effect on survival prediction in high-grade serous ovarian carcinoma.

BACKGROUND: In colorectal and breast cancer, the density and localisation of immune infiltrates provides strong prognostic information. We asked whether similar automated quantitation and combined analysis of immune infiltrates could refine prognostic information in high-grade serous ovarian carcinoma (HGSOC) and tested associations between patterns of immune response and genomic driver alterations. METHODS: Epithelium and stroma were semi-automatically segmented and the infiltration of CD45RO+, CD8+ and CD68+ cells was automatically quantified from images of 332 HGSOC patient tissue microarray cores. RESULTS: Epithelial CD8 [p = 0.027, hazard ratio (HR) = 0.83], stromal CD68 (p = 3 × 10-4, HR = 0.44) and stromal CD45RO (p = 7 × 10-4, HR = 0.76) were positively associated with survival and remained so when averaged across the tumour and stromal compartments. Using principal component analysis, we identified optimised multiparameter survival models combining information from all immune markers (p = 0.016, HR = 0.88). There was no significant association between PTEN expression, type of TP53 mutation or presence of BRCA1/BRCA2 mutations and immune infiltrate densities or principal components. CONCLUSIONS: Combining measures of immune infiltration provided improved survival modelling and evidence for the multiple effects of different immune factors on survival. The presence of stromal CD68+ and CD45RO+ populations was associated with survival, underscoring the benefits evaluating stromal immune populations may bring for prognostic immunoscores in HGSOC.

B regulatory cells in cancer.

B regulatory cells are a newly described subpopulation of B cells that appear to play important roles in autoimmunity and more recently, in cancer. In this review we summarize our current knowledge of B regulatory cells, as well as the body of evidence pointing towards a role for B cells in general, and B regulatory cells in particular, in promoting tumor growth.

FAN stimulates TNF(alpha)-induced gene expression, leukocyte recruitment, and humoral response.

Factor associated with neutral sphingomyelinase activation (FAN) is an adaptor protein that constitutively binds to TNF-R1. Microarray analysis was performed in fibroblasts derived from wild-type or FAN knockout mouse embryos to evaluate the role of FAN in TNF-induced gene expression. Approximately 70% of TNF-induced genes exhibited lower expression levels in FAN-deficient than in wild-type fibroblasts. Of particular interest, TNF-induced expression of cytokines/chemokines, such as IL-6 and CXCL-2, was impaired in FAN-deficient cells. This was confirmed by real time RT-PCR and ELISA. Upon i.p. TNF or thioglycollate injection, neutrophil recruitment into the peritoneal cavity was reduced by more than 50% in FAN-deficient mice. Nevertheless, FAN-deficient animals did not exhibit an increased susceptibility to different microorganisms including bacteria and parasites, indicating that FAN is not essential for pathogen clearance. Specific Ab response to BSA was substantially impaired in FAN-deficient mice and this was associated with a reduced content of leukocytes in the spleen of BSA-challenged FAN-deficient mice as compared with their wild-type counterparts. Altogether, our results indicate the involvement of FAN in TNF-induced gene expression and leukocyte recruitment, contributing to the establishment of the specific immune response.

Combining Nivolumab and Ipilimumab with Infliximab or Certolizumab in Patients with Advanced Melanoma: First Results of a Phase Ib Clinical Trial.

PURPOSE: TNF blockers can be used to manage gastrointestinal inflammatory side effects following nivolumab and/or ipilimumab treatment in patients with advanced melanoma. Our preclinical data showed that anti-TNF could promote the efficacy of immune checkpoint inhibitors. PATIENTS AND METHODS: TICIMEL (NTC03293784) is an open-label, two-arm phase Ib clinical trial. Fourteen patients with advanced and/or metastatic melanoma (stage IIIc/IV) were enrolled. Patients were treated with nivolumab (1 mg/kg) and ipilimumab (3 mg/kg) combined to infliximab (5 mg/kg, N = 6) or certolizumab (400/200 mg, N = 8). The primary endpoint was safety and the secondary endpoint was antitumor activity. Adverse events (AEs) were graded according to the NCI Common Terminology Criteria for Adverse Events and response was assessed following RECIST 1.1. RESULTS: Only one dose-limiting toxicity was observed in the infliximab cohort. The two different combinations were found to be safe. We observed lower treatment-related AEs with infliximab as compared with certolizumab. In the certolizumab cohort, one patient was not evaluable for response. In this cohort, four of eight patients exhibited hepatobiliary disorders and seven of seven evaluable patients achieved objective response including four complete responses (CRs) and three partial responses (PRs). In the infliximab cohort, we observed one CR, two PRs, and three progressive diseases. Signs of activation and maturation of systemic T-cell responses were seen in patients from both cohorts. CONCLUSIONS: Our results show that both combinations are safe in human and provide clinical and biological activities. The high response rate in the certolizumab-treated patient cohort deserves further investigations.

Neutral Sphingomyelinase 2 Heightens Anti-Melanoma Immune Responses and Anti-PD-1 Therapy Efficacy.

Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3, catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro, small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow-derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti-PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8+ T-cell-dependent immune responses and overcome resistance to anti-PD-1.

New Insights into the Role of Sphingolipid Metabolism in Melanoma.

Cutaneous melanoma is a deadly skin cancer whose aggressiveness is directly linked to its metastatic potency. Despite remarkable breakthroughs in term of treatments with the emergence of targeted therapy and immunotherapy, the prognosis for metastatic patients remains uncertain mainly because of resistances. Better understanding the mechanisms responsible for melanoma progression is therefore essential to uncover new therapeutic targets. Interestingly, the sphingolipid metabolism is dysregulated in melanoma and is associated with melanoma progression and resistance to treatment. This review summarises the impact of the sphingolipid metabolism on melanoma from the initiation to metastatic dissemination with emphasis on melanoma plasticity, immune responses and resistance to treatments.

Resistance of melanoma to immune checkpoint inhibitors is overcome by targeting the sphingosine kinase-1.

Immune checkpoint inhibitors (ICIs) have dramatically modified the prognosis of several advanced cancers, however many patients still do not respond to treatment. Optimal results might be obtained by targeting cancer cell metabolism to modulate the immunosuppressive tumor microenvironment. Here, we identify sphingosine kinase-1 (SK1) as a key regulator of anti-tumor immunity. Increased expression of SK1 in tumor cells is significantly associated with shorter survival in metastatic melanoma patients treated with anti-PD-1. Targeting SK1 markedly enhances the responses to ICI in murine models of melanoma, breast and colon cancer. Mechanistically, SK1 silencing decreases the expression of various immunosuppressive factors in the tumor microenvironment to limit regulatory T cell (Treg) infiltration. Accordingly, a SK1-dependent immunosuppressive signature is also observed in human melanoma biopsies. Altogether, this study identifies SK1 as a checkpoint lipid kinase that could be targeted to enhance immunotherapy.

Corrigendum: The TNF Paradox in Cancer Progression and Immunotherapy.

[This corrects the article DOI: 10.3389/fimmu.2019.01818.].

Anti-TNF, a magic bullet in cancer immunotherapy?

Immune checkpoint blockers (ICB) have revolutionized cancer therapy. However, complete response is observed in a minority of patients and most patients develop immune-related adverse events (irAEs). These include colitis, which can be treated with anti-tumor necrosis factor (TNF) antibodies such as Infliximab. In a recent issue of the Journal for ImmunoTherapy of Cancer, Badran et al. reported that co-administering Infliximab together with ICB to five cancer patients prevents colitis recurrence, with four of them exhibiting overall disease stability. The basis for this treatment strategy stemmed from our pre-clinical demonstration that TNF contributes to resistance to anti-PD-1 therapy. In agreement with this concept, we have shown that TNF blockers improve the anti-tumor therapeutic activity of ICB in mice and based on these findings we are currently evaluating the combination in melanoma patients enrolled in the TICIMEL clinical trial. Herein, (i) we discuss the scientific rationale for combining anti-TNF and ICB in cancer patients, (ii) comment on the paper published by Badran et al. and (iii) provide the TICIMEL clinical trial design.

Sphingomyelin Synthase 1 (SMS1) Downregulation Is Associated With Sphingolipid Reprogramming and a Worse Prognosis in Melanoma.

Sphingolipid (SL) metabolism alterations have been frequently reported in cancer including in melanoma, a bad-prognosis skin cancer. In normal cells, de novo synthesized ceramide is mainly converted to sphingomyelin (SM), the most abundant SL, by sphingomyelin synthase 1 (SMS1) and, albeit to a lesser extent, SMS2, encoded by the SGMS1 and SGMS2 genes, respectively. Alternatively, ceramide can be converted to glucosylceramide (GlcCer) by the GlcCer synthase (GCS), encoded by the UGCG gene. Herein, we provide evidence for the first time that SMS1 is frequently downregulated in various solid cancers, more particularly in melanoma. Accordingly, various human melanoma cells displayed a SL metabolism signature associated with (i) a robust and a low expression of UGCG and SGMS1/2, respectively, (ii) higher in situ enzyme activity of GCS than SMS, and (iii) higher intracellular levels of GlcCer than SM. SMS1 was expressed at low levels in most of the human melanoma biopsies. In addition, several mutations and increased CpG island methylation in the SGMS1 gene were identified that likely affect SMS1 expression. Finally, low SMS1 expression was associated with a worse prognosis in metastatic melanoma patients. Collectively, our study indicates that SMS1 downregulation in melanoma enhances GlcCer synthesis, triggering an imbalance in the SM/GlcCer homeostasis, which likely contributes to melanoma progression. Evaluating SMS1 expression level in tumor samples might serve as a biomarker to predict clinical outcome in advanced melanoma patients.

Deconstruction of a Metastatic Tumor Microenvironment Reveals a Common Matrix Response in Human Cancers.

We have profiled, for the first time, an evolving human metastatic microenvironment by measuring gene expression, matrisome proteomics, cytokine and chemokine levels, cellularity, extracellular matrix organization, and biomechanical properties, all on the same sample. Using biopsies of high-grade serous ovarian cancer metastases that ranged from minimal to extensive disease, we show how nonmalignant cell densities and cytokine networks evolve with disease progression. Multivariate integration of the different components allowed us to define, for the first time, gene and protein profiles that predict extent of disease and tissue stiffness, while also revealing the complexity and dynamic nature of matrisome remodeling during development of metastases. Although we studied a single metastatic site from one human malignancy, a pattern of expression of 22 matrisome genes distinguished patients with a shorter overall survival in ovarian and 12 other primary solid cancers, suggesting that there may be a common matrix response to human cancer.Significance: Conducting multilevel analysis with data integration on biopsies with a range of disease involvement identifies important features of the evolving tumor microenvironment. The data suggest that despite the large spectrum of genomic alterations, some human malignancies may have a common and potentially targetable matrix response that influences the course of disease. Cancer Discov; 8(3); 304-19. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 253.

TNFα blockade overcomes resistance to anti-PD-1 in experimental melanoma.

Antibodies against programmed cell death-1 (PD-1) have considerably changed the treatment for melanoma. However, many patients do not display therapeutic response or eventually relapse. Moreover, patients treated with anti-PD-1 develop immune-related adverse events that can be cured with anti-tumor necrosis factor α (TNF) antibodies. Whether anti-TNF antibodies affect the anti-cancer immune response remains unknown. Our recent work has highlighted that TNFR1-dependent TNF signalling impairs the accumulation of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in mouse melanoma. Herein, our results indicate that TNF or TNFR1 blockade synergizes with anti-PD-1 on anti-cancer immune responses towards solid cancers. Mechanistically, TNF blockade prevents anti-PD-1-induced TIL cell death as well as PD-L1 and TIM-3 expression. TNF expression positively correlates with expression of PD-L1 and TIM-3 in human melanoma specimens. This study provides a strong rationale to develop a combination therapy based on the use of anti-PD-1 and anti-TNF in cancer patients.

A Strong B-cell Response Is Part of the Immune Landscape in Human High-Grade Serous Ovarian Metastases.

PURPOSE: In high-grade serous ovarian cancer (HGSOC), higher densities of both B cells and the CD8+ T-cell infiltrate were associated with a better prognosis. However, the precise role of B cells in the antitumor response remains unknown. As peritoneal metastases are often responsible for relapse, our aim was to characterize the role of B cells in the antitumor immune response in HGSOC metastases. EXPERIMENTAL DESIGN: Unmatched pre and post-chemotherapy HGSOC metastases were studied. B-cell localization was assessed by immunostaining. Their cytokines and chemokines were measured by a multiplex assay, and their phenotype was assessed by flow cytometry. Further in vitro and in vivo assays highlighted the role of B cells and plasma cell IgGs in the development of cytotoxic responses and dendritic cell activation. RESULTS: B cells mainly infiltrated lymphoid structures in the stroma of HGSOC metastases. There was a strong B-cell memory response directed at a restricted repertoire of antigens and production of tumor-specific IgGs by plasma cells. These responses were enhanced by chemotherapy. Interestingly, transcript levels of CD20 correlated with markers of immune cytolytic responses and immune complexes with tumor-derived IgGs stimulated the expression of the costimulatory molecule CD86 on antigen-presenting cells. A positive role for B cells in the antitumor response was also supported by B-cell depletion in a syngeneic mouse model of peritoneal metastasis. CONCLUSIONS: Our data showed that B cells infiltrating HGSOC omental metastases support the development of an antitumor response. Clin Cancer Res; 23(1); 250-62. ©2016 AACR.

A CCR4 antagonist reverses the tumor-promoting microenvironment of renal cancer.

Elevated expression of the chemokine receptor CCR4 in tumors is associated with poor prognosis in several cancers. Here, we have determined that CCR4 was highly expressed in human renal cell carcinoma (RCC) biopsies and observed abnormal levels of CCR4 ligands in RCC patient plasma. An antagonistic anti-CCR4 antibody had antitumor activity in the RENCA mouse model of RCC. CCR4 inhibition did not reduce the proportion of infiltrating leukocytes in the tumor microenvironment but altered the phenotype of myeloid cells, increased NK cell and Th1 cytokine levels, and reduced immature myeloid cell infiltrate and blood chemokine levels. In spite of prominent changes in the myeloid compartment, the anti-CCR4 antibody did not affect RENCA tumors in T cell-deficient mice, and treatment with an anti-class II MHC antibody abrogated its antitumor activity. We concluded that the effects of the anti-CCR4 antibody required the adaptive immune system and CD4+ T cells. Moreover, CCL17-induced IFN-γ production was reduced when Th1-polarized normal CD4+ T cells were exposed to the CCR4 ligand, evidencing the involvement of CCR4 in Th1/Th2 regulation. The anti-CCR4 antibody, alone or in combination with other immune modulators, is a potential treatment approach to human solid cancers with high levels of CCR4-expressing tumor-infiltrating leukocytes and abnormal plasma CCR4 ligand levels.

Targeting TNF alpha as a novel strategy to enhance CD8+ T cell-dependent immune response in melanoma?

Tumor Necrosis Factor α (TNF) is a pleiotropic cytokine exhibiting a dual activity in oncoimmunology, either acting as a cytotoxic effector produced by leukocytes or behaving as an immunosuppressive molecule. We have just discovered that TNF signaling impairs the accumulation of tumor-infiltrating CD8+ T lymphocytes in experimental melanoma.

Neoadjuvant Chemotherapy Modulates the Immune Microenvironment in Metastases of Tubo-Ovarian High-Grade Serous Carcinoma.

PURPOSE: The purpose of this study was to assess the effect of neoadjuvant chemotherapy (NACT) on immune activation in stage IIIC/IV tubo-ovarian high-grade serous carcinoma (HGSC), and its relationship to treatment response. EXPERIMENTAL DESIGN: We obtained pre- and posttreatment omental biopsies and blood samples from a total of 54 patients undergoing platinum-based NACT and 6 patients undergoing primary debulking surgery. We measured T-cell density and phenotype, immune activation, and markers of cancer-related inflammation using IHC, flow cytometry, electrochemiluminescence assays, and RNA sequencing and related our findings to the histopathologic treatment response. RESULTS: There was evidence of T-cell activation in omental biopsies after NACT: CD4(+) T cells showed enhanced IFNγ production and antitumor Th1 gene signatures were increased. T-cell activation was more pronounced with good response to NACT. The CD8(+) T-cell and CD45RO(+) memory cell density in the tumor microenvironment was unchanged after NACT but biopsies showing a good therapeutic response had significantly fewer FoxP3(+) T regulatory (Treg) cells. This finding was supported by a reduction in a Treg cell gene signature in post- versus pre-NACT samples that was more pronounced in good responders. Plasma levels of proinflammatory cytokines decreased in all patients after NACT. However, a high proportion of T cells in biopsies expressed immune checkpoint molecules PD-1 and CTLA4, and PD-L1 levels were significantly increased after NACT. CONCLUSIONS: NACT may enhance host immune response but this effect is tempered by high/increased levels of PD-1, CTLA4, and PD-L1. Sequential chemoimmunotherapy may improve disease control in advanced HGSC. Clin Cancer Res; 22(12); 3025-36. ©2016 AACR.

Blocking Tumor Necrosis Factor α Enhances CD8 T-cell-Dependent Immunity in Experimental Melanoma.

TNF plays a dual, still enigmatic role in melanoma, either acting as a cytotoxic cytokine or favoring a tumorigenic inflammatory microenvironment. Herein, the tumor growth of melanoma cell lines expressing major histocompatibility complex class I molecules at high levels (MHC-I(high)) was dramatically impaired in TNF-deficient mice, and this was associated with enhanced tumor-infiltrating CD8(+) T lymphocytes. Immunodepletion of CD8 T cells fully restored melanoma growth in TNF(-/-) mice. Systemic administration of Etanercept inhibited MHC-I(high) melanoma growth in immunocompetent but not in immunodeficient (IFNγ(-/-), nude, or CD8(-/-)) mice. MHC-I(high) melanoma growth was also reduced in mice lacking TNF-R1, but not TNF-R2. TNF(-/-) and TNF-R1(-/-) mice as well as Etanercept-treated WT mice displayed enhanced intratumor content of high endothelial venules surrounded by high CD8(+) T-cell density. Adoptive transfer of activated TNF-R1-deficient or -proficient CD8(+) T cells in CD8-deficient mice bearing B16K1 tumors demonstrated that TNF-R1 deficiency facilitates the accumulation of live CD8(+) T cells into the tumors. Moreover, in vitro experiments indicated that TNF triggered activated CD8(+) T cell death in a TNF-R1-dependent manner, likely limiting the accumulation of tumor-infiltrating CD8(+) T cells in TNF/TNF-R1-proficient animals. Collectively, our observations indicate that TNF-R1-dependent TNF signaling impairs tumor-infiltrating CD8(+) T-cell accumulation and may serve as a putative target to favor CD8(+) T-cell-dependent immune response in melanoma.