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Journal for ImmunoTherapy of Cancer, Volume 7, pp 1-13; https://doi.org/10.1186/s40425-019-0797-4
Clinical response to MAPK inhibitors in metastatic melanoma patients is heterogeneous for reasons still needing to be elucidated. As the patient immune activity contributes to treatment clinical benefit, the pre-existing level of immunity at tumor site may provide biomarkers of disease outcome to therapy. Here we investigated whether assessing the density and spatial tissue distribution of key immune cells in the tumor microenvironment could identify patients predisposed to respond to MAPK inhibitors. Pretreatment tumor biopsies from a total of 213 patients (158 for the training set and 55 for the validation set) treated with BRAF or BRAF/MEK inhibitors within the Italian Melanoma Intergroup were stained with selected immune markers (CD8, CD163, β-catenin, PD-L1, PD-L2). Results, obtained by blinded immunohistochemical scoring and digital image analysis, were correlated with clinical response and outcome by multivariate logistic models on response to treatment and clinical outcome, adjusted for American Joint Committee on Cancer stage, performance status, lactate dehydrogenase and treatment received. Patients with high intratumoral, but not peritumoral, CD8+ T cells and concomitantly low CD163+ myeloid cells displayed higher probability of response (OR 9.91, 95% CI 2.23–44.0, p = 0.003) and longer overall survival (HR 0.34, 95% CI 0.16–0.72, p = 0.005) compared to those with intratumoral low CD8+ T cells and high CD163+ myeloid cells. The latter phenotype was instead associated with a shorter progression free survival (p = 0.010). In contrast, PD-L1 and PD-L2 did not correlate with clinical outcome while tumor β-catenin overexpression showed association with lower probability of response (OR 0.48, 95% CI 0.21–1.06, p = 0.068). Analysis of the spatially constrained distribution of CD8+ and CD163+ cells, representative of the opposite circuits of antitumor vs protumor immunity, respectively, may assist in identifying melanoma patients with improved response and better outcome upon treatment with MAPK inhibitors. These data underline the role of endogenous immune microenvironment in predisposing metastatic melanoma patients to benefit from therapies targeting driver-oncogenic pathways.
Journal for ImmunoTherapy of Cancer, Volume 7; https://doi.org/10.1186/s40425-019-0755-1
The interplay between the immune system and tumor progression is well recognized. However, current human breast cancer immunophenotyping studies are mostly focused on primary tumors with metastatic breast cancer lesions remaining largely understudied. To address this gap, we examined exome-capture RNA sequencing data from 50 primary breast tumors (PBTs) and their patient-matched metastatic tumors (METs) in brain, ovary, bone and gastrointestinal tract. We used gene expression signatures as surrogates for tumor infiltrating lymphocytes (TILs) and compared TIL patterns in PBTs and METs. Enrichment analysis and deconvolution methods both revealed that METs had a significantly lower abundance of total immune cells, including CD8+ T cells, regulatory T cells and dendritic cells. An exception was M2-like macrophages, which were significantly higher in METs across the organ sites examined. Multiplex immunohistochemistry results were consistent with data from the in-silico analysis and showed increased macrophages in METs. We confirmed the finding of a significant reduction in immune cells in brain METs (BRMs) by pathologic assessment of TILs in a set of 49 patient-matched pairs of PBT/BRMs. These findings indicate that METs have an overall lower infiltration of immune cells relative to their matched PBTs, possibly due to immune escape. RNAseq analysis suggests that the relative levels of M2-like macrophages are increased in METs, and their potential role in promoting breast cancer metastasis warrants further study.
Journal for ImmunoTherapy of Cancer, Volume 7; https://doi.org/10.1186/s40425-019-0579-z
Background The Italian Renal Cell Cancer Early Access Program was an expanded access program that allowed access to nivolumab, for patients (pts) with metastatic renal cell carcinoma (mRCC) prior to regulatory approval. Methods Pts with previously treated advanced or mRCC were eligible to receive nivolumab 3 mg/kg every 2 weeks. Pts included in the analysis had received ≥1 dose of nivolumab and were monitored for drug-related adverse events (drAEs) using CTCAE v.4.0. Immune-related (ir) AEs were defined as AEs displaying a certain, likely or possible correlation with immunotherapy (cutaneous, endocrine, hepatic, gastro-intestinal and pulmonary). The association between overall survival (OS) and irAEs was assessed, and associations between variables were evaluated with a logistic regression model. Results A total of 389 pts were enrolled between July 2015 and April 2016. Overall, the objective response rate was 23.1%. At a median follow-up of 12 months, the median progression-free survival was 4.5 months (95% CI 3.7–6.2) and the 12-month overall survival rate was 63%. Any grade and grade 3–4 drAEs were reported in 124 (32%) and 27 (7%) of pts, respectively, and there were no treatment-related deaths. Any grade irAEs occurred in 76 (20%) of patients, 8% cutaneous, 4% endocrine, 2% hepatic, 5% gastro-intestinal and 1% pulmonary. Of the 22 drAEs inducing treatment discontinuation, 10 (45%) were irAEs. Pts with drAEs had a significantly longer survival than those without drAEs (median OS 22.5 versus 16.4 months, p = 0.01). Pts with irAEs versus without irAEs had a more significant survival benefit (median OS not reached versus 16.8 months, p = 0.002), confirmed at the landmark analysis at 6 weeks. The occurrence of irAEs displayed a strong association with OS in univariable (HR 0.48, p = 0.003) and multivariable (HR 0.57, p = 0.02) analysis. Conclusions The appearance of irAEs strongly correlates with survival benefit in a real-life population of mRCC pts treated with nivolumab.
Journal for ImmunoTherapy of Cancer, Volume 7; https://doi.org/10.1186/s40425-019-0556-6
Background Tumor progression is accompanied by dramatic remodeling of the surrounding extracellular matrix leading to the formation of a tumor-specific ECM, which is often more collagen-rich and of increased stiffness. The altered ECM of the tumor supports cancer growth and metastasis, but it is unknown if this effect involves modulation of T cell activity. To investigate if a high-density tumor-specific ECM could influence the ability of T cells to kill cancer cells, we here studied how T cells respond to 3D culture in different collagen densities. Methods T cells cultured in 3D conditions surrounded by a high or low collagen density were imaged using confocal fluorescent microscopy. The effects of the different collagen densities on T cell proliferation, survival, and differentiation were examined using flow cytometry. Cancer cell proliferation in similar 3D conditions was also measured. Triple-negative breast cancer specimens were analyzed for the number of infiltrating CD8+ T cells and for the collagen density. Whole-transcriptome analyses were applied to investigate in detail the effects of collagen density on T cells. Computational analyses were used to identify transcription factors involved in the collagen density-induced gene regulation. Observed changes were confirmed by qRT-PCR analysis. Results T cell proliferation was significantly reduced in a high-density matrix compared to a low-density matrix and prolonged culture in a high-density matrix led to a higher ratio of CD4+ to CD8+ T cells. The proliferation of cancer cells was unaffected by the surrounding collagen-density. Consistently, we observed a reduction in the number of infiltrating CD8+ T-cells in mammary tumors with high collagen-density indicating that collagen-density has a role in regulating T cell abundance in human breast cancer. Whole-transcriptome analysis of 3D-cultured T cells revealed that a high-density matrix induces downregulation of cytotoxic activity markers and upregulation of regulatory T cell markers. These transcriptional changes were predicted to involve autocrine TGF-β signaling and they were accompanied by an impaired ability of tumor-infiltrating T cells to kill autologous cancer cells. Conclusions Our study identifies a new immune modulatory mechanism, which could be essential for suppression of T cell activity in the tumor microenvironment.
Journal for ImmunoTherapy of Cancer, Volume 5, pp 98-98; https://doi.org/10.1186/s40425-017-0305-7
Background We have previously shown that radiotherapy (RT) augments natural killer (NK) functions in pre-clinical models of human and mouse cancers, including sarcomas. Since dogs are an excellent outbred model for immunotherapy studies, we sought to assess RT plus local autologous NK transfer in canine sarcomas. Methods Dog NK cells (CD5dim, NKp46+) were isolated from PBMCs and expanded with irradiated K562-C9-mIL21 feeder cells and 100 IU/mL recombinant human IL-2. NK homing and cytotoxicity ± RT were evaluated using canine osteosarcoma tumor lines and dog patient-derived xenografts (PDX). In a first-in-dog clinical trial for spontaneous osteosarcoma, we evaluated RT and intra-tumoral autologous NK transfer. Results After 14 days, mean NK expansion and yield were 19.0-fold (±8.6) and 258.9(±76.1) ×106 cells, respectively. Post-RT, NK cytotoxicity increased in a dose-dependent fashion in vitro reaching ~ 80% at effector:target ratios of ≥10:1 (P< 0.001). In dog PDX models, allogeneic NK cells were cytotoxic in ex vivo killing assays and produced significant PDX tumor growth delay (P< 0.01) in vivo. After focal RT and intravenous NK transfer, we also observed significantly increased NK homing to tumors in vivo. Of 10 dogs with spontaneous osteosarcoma treated with focal RT and autologous NK transfer, 5 remain metastasis-free at the 6-month primary endpoint with resolution of suspicious pulmonary nodules in one patient. We also observed increased activation of circulating NK cells after treatment and persistence of labelled NK cells in vivo. Conclusions NK cell homing and cytotoxicity are increased following RT in canine models of sarcoma. Results from a first-in-dog clinical trial are promising, including possible abscopal effects.
Journal for ImmunoTherapy of Cancer, Volume 5, pp 95-95; https://doi.org/10.1186/s40425-017-0300-z
Cancer immunotherapy has transformed the treatment of cancer. However, increasing use of immune-based therapies, including the widely used class of agents known as immune checkpoint inhibitors, has exposed a discrete group of immune-related adverse events (irAEs). Many of these are driven by the same immunologic mechanisms responsible for the drugs’ therapeutic effects, namely blockade of inhibitory mechanisms that suppress the immune system and protect body tissues from an unconstrained acute or chronic immune response. Skin, gut, endocrine, lung and musculoskeletal irAEs are relatively common, whereas cardiovascular, hematologic, renal, neurologic and ophthalmologic irAEs occur much less frequently. The majority of irAEs are mild to moderate in severity; however, serious and occasionally life-threatening irAEs are reported in the literature, and treatment-related deaths occur in up to 2% of patients, varying by ICI. Immunotherapy-related irAEs typically have a delayed onset and prolonged duration compared to adverse events from chemotherapy, and effective management depends on early recognition and prompt intervention with immune suppression and/or immunomodulatory strategies. There is an urgent need for multidisciplinary guidance reflecting broad-based perspectives on how to recognize, report and manage organ-specific toxicities until evidence-based data are available to inform clinical decision-making. The Society for Immunotherapy of Cancer (SITC) established a multidisciplinary Toxicity Management Working Group, which met for a full-day workshop to develop recommendations to standardize management of irAEs. Here we present their consensus recommendations on managing toxicities associated with immune checkpoint inhibitor therapy.
Journal for ImmunoTherapy of Cancer, Volume 5; https://doi.org/10.1186/s40425-017-0215-8
Background Assays of the abundance of immune cell populations in the tumor microenvironment promise to inform immune oncology research and the choice of immunotherapy for individual patients. We propose to measure the intratumoral abundance of various immune cell populations with gene expression. In contrast to IHC and flow cytometry, gene expression assays yield high information content from a clinically practical workflow. Previous studies of gene expression in purified immune cells have reported hundreds of genes showing enrichment in a single cell type, but the utility of these genes in tumor samples is unknown. We use co-expression patterns in large tumor gene expression datasets to evaluate previously reported candidate cell type marker genes lists, eliminate numerous false positives and identify a subset of high confidence marker genes. Methods Using a novel statistical tool, we use co-expression patterns in 9986 samples from The Cancer Genome Atlas (TCGA) to evaluate previously reported cell type marker genes. We compare immune cell scores derived from these genes to measurements from flow cytometry and immunohistochemistry. We characterize the reproducibility of our cell scores in replicate runs of RNA extracted from FFPE tumor tissue. Results We identify a list of 60 marker genes whose expression levels measure 14 immune cell populations. Cell type scores calculated from these genes are concordant with flow cytometry and IHC readings, show high reproducibility in replicate RNA samples from FFPE tissue and enable detailed analyses of the anti-tumor immune response in TCGA. In an immunotherapy dataset, they separate responders and non-responders early on therapy and provide an intricate picture of the effects of checkpoint inhibition. Most genes previously reported to be enriched in a single cell type have co-expression patterns inconsistent with cell type specificity. Conclusions Due to their concise gene set, computational simplicity and utility in tumor samples, these cell type gene signatures may be useful in future discovery research and clinical trials to understand how tumors and therapeutic intervention shape the immune response.
Journal for ImmunoTherapy of Cancer, Volume 4; https://doi.org/10.1186/s40425-016-0118-0
The efficacy of PD-1/PD-L1 targeted therapies in addition to anti-CTLA-4 solidifies immunotherapy as a modality to add to the anticancer arsenal. Despite raising the bar of clinical efficacy, immunologically targeted agents raise new challenges to conventional drug development paradigms by highlighting the limited relevance of assessing standard pharmacokinetics (PK) and pharmacodynamics (PD). Specifically, systemic and intratumoral immune effects have not consistently correlated with standard relationships between systemic dose, toxicity, and efficacy for cytotoxic therapies. Hence, PK and PD paradigms remain inadequate to guide the selection of doses and schedules, both starting and recommended Phase 2 for immunotherapies. The promise of harnessing the immune response against cancer must also be considered in light of unique and potentially serious toxicities. Refining immune endpoints to better inform clinical trial design represents a high priority challenge. The Cancer Immunotherapy Trials Network investigators review the immunodynamic effects of specific classes of immunotherapeutic agents to focus immune assessment modalities and sites, both systemic and importantly intratumoral, which are critical to the success of the rapidly growing field of immuno-oncology.
Journal for ImmunoTherapy of Cancer, Volume 2, pp 10-10; https://doi.org/10.1186/2051-1426-2-10
Immunotherapeutic approaches, such as dendritic cell (DC) vaccination, have emerged as promising strategies in the treatment of glioblastoma. Despite their promise, however, the absence of objective biomarkers and/or immunological monitoring techniques to assess the clinical efficacy of immunotherapy still remains a primary limitation. To address this, we sought to identify a functional biomarker for anti-tumor immune responsiveness associated with extended survival in glioblastoma patients undergoing DC vaccination. 28 patients were enrolled and treated in two different Phase 1 DC vaccination clinical trials at UCLA. To assess the anti-tumor immune response elicited by therapy, we studied the functional responsiveness of pre- and post-vaccination peripheral blood lymphocytes (PBLs) to the immunostimulatory cytokines interferon-gamma (IFN-γ) and interleukin-2 (IL-2) in 21 of these patients for whom we had adequate material. Immune responsiveness was quantified by measuring downstream phosphorylation events of the transcription factors, STAT-1 and STAT-5, via phospho-specific flow cytometry. DC vaccination induced a significant decrease in the half-maximal concentration (EC-50) of IL-2 required to upregulate pSTAT-5 specifically in CD3(+)CD8(+) T lymphocytes (p < 0.045). Extended survival was also associated with an increased per cell phosphorylation of STAT-5 in cytotoxic T-cells following IL-2 stimulation when the median post/pre pSTAT-5 ratio was used to dichotomize the patients (p = 0.0015, log-rank survival; hazard ratio = 0.1834, p = 0.018). Patients whose survival was longer than two years had a significantly greater pSTAT-5 ratio (p = 0.015), but, contrary to our expectations, a significantly lower pSTAT-1 ratio (p = 0.038). Our results suggest that monitoring the pSTAT signaling changes in PBL may provide a functional immune monitoring measure predictive of clinical efficacy in DC-vaccinated patients.
Journal for ImmunoTherapy of Cancer, Volume 1, pp 19-19; https://doi.org/10.1186/2051-1426-1-19
Human Adenoviral vectors (HAdV) are immunogenic vectors which have been tested in many vaccination and gene therapy settings. Dendritic cells (DC) transduced by genetically engineered HAdV-5 (HAdV-5/DC), are investigational cancer vaccines being tested clinically. We have previously examined immune responses to HAdV-5 -encoded melanoma tumor antigens. Here, we determined whether the HAdV-5/DC also present immunogenic HAdV-5 vector-derived antigens, and characterized the cellular immune response to the viral as well as encoded melanoma tumor antigens. Both CD4(+) and CD8(+) HAdV-5-specific T cell responses were examined in vitro, with cells from both 8 healthy donors (HD) and 2 melanoma patients. PBMC were stimulated weekly with HAdV-5/DC and responses were examined after each stimulation. We also tested HAdV-5 neutralizing antibody levels and natural killer (NK) cell and regulatory T cell (Treg) activation and expansion in vitro. HAdV-5/DC rapidly induced a high frequency of type 1 cytokine producing HAdV-5-specific CD8(+) and CD4(+) T cells. IFNγ and TNFα-producing T cells predominate. Those with pre-existing cellular memory to HAdV-5 had more robust responses to the HAdV-5 as well as tumor-associated antigens. NK cells are activated while Treg are only minimally and transiently expanded. This study demonstrates that HAdV-5/DC promote strong type I cellular immunity to viral vector-derived antigens as well as to the encoded tumor antigens. The cytokine and chemokine milieu produced by HAdV-5/DC and the activated HAdV-5-specific T cells may enhance responses to encoded tumor antigens as well. These properties make HAdV-5/DC a cancer vaccine capable of activating type 1 virus and tumor antigen-specific immunity in a cooperative way.