Melanoma antigen-specific effector T cell cytokine secretion patterns in patients treated with ipilimumab

Additional file 1. Summary of Luminex cytokine measurements

Immune monitoring of the circulation and the tumor microenvironment in patients with regionally advanced melanoma receiving neoadjuvant ipilimumab

We evaluated neoadjuvant ipilimumab in patients with surgically operable regionally advanced melanoma in order to define markers of activity in the blood and tumor as assessed at baseline (before ipilimumab) and early on-treatment. Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks x2 doses) bracketing surgery. Tumor and blood biospecimens were obtained at baseline and at surgery. Flow cytometry and immunohistochemistry for select biomarkers were performed. Thirty five patients were enrolled; IIIB (3; N2b), IIIC (32; N2c, N3), IV (2). Worst toxicities included Grade 3 diarrhea/colitis (5; 14%), hepatitis (2; 6%), rash (1; 3%), elevated lipase (3; 9%). Median follow up was 18 months: among 33 evaluable patients, median progression free survival (PFS) was 11 months, 95% CI (6.2-19.2). There was a significant decrease in circulating myeloid derived suppressor cells (MDSC). Greater decrease in circulating monocyte gate MDSC Lin1-/HLA-DR-/CD33+/CD11b+ was associated with improved PFS (p = 0.03). There was a significant increase in circulating regulatory T cells (Treg; CD4+CD25hi+Foxp3+) that, unexpectedly, was associated with improved PFS (HR = 0.57; p = 0.034). Baseline evidence of fully activated type I CD4+ and CD8+ antigen-specific T cell immunity against cancer-testis (NY-ESO-1) and melanocytic lineage (MART-1, gp100) antigens was detected and was significantly potentiated after ipilimumab. In tumor, there was a significant increase in CD8+ T cells after ipilimumab (p = 0.02). Ipilimumab induced increased tumor infiltration by fully activated (CD69+) CD3+/CD4+ and CD3 +/CD8+ T cells with evidence of induction/potentiation of memory T cells (CD45RO+). The change in Treg observed within the tumor showed an inverse relationship with clinical benefit and greater decrease in tumor MDSC subset Lin1-/HLA-DR-/CD33+/CD11b+ was associated with improved PFS at one year. Neoadjuvant evaluation revealed a significant immunomodulating role for ipilimumab on Treg, MDSC and effector T cells in the circulation and tumor microenvironment that warrants further pursuit in the quest for optimizing melanoma immunotherapy. © 2014 Tarhini et al

Anomaly mediated SUSY breaking scenarios in the light of cosmology and in the dark (matter)

Anomaly mediation is a popular and well motivated supersymmetry breaking scenario. Different possible detailed realisations of this set-up are studied and actively searched for at colliders. Apart from limits coming from flavour, low energy physics and direct collider searches, these models are usually constrained by the requirement of reproducing the observations on dark matter density in the universe. We reanalyse these bounds and in particular we focus on the dark matter bounds both considering the standard cosmological model and alternative cosmological scenarios. These scenarios do not change the observable cosmology but relic dark matter density bounds strongly depend on them. We consider few benchmark points excluded by standard cosmology dark matter bounds and suggest that loosening the dark matter constraints is necessary in order to avoid a too strong (cosmological) model dependence in the limits that are obtained for these models. We also discuss briefly the implications for phenomenology and in particular at the Large Hadron Collider.Comment: 37 pages, 20 figures, 1 tabl

Annual hospital volume of high dose interleukin-2 and inpatient mortality in melanoma and renal cell carcinoma patients

Background Immunotherapy using high dose interleukin-2 (HD IL2) in patients with renal cell carcinoma (RCC) and melanoma is associated with severe toxicities. The association between annual hospital volume of HD IL2 and inpatient mortality is not well studied. In this study we aim to quantify the impact of annual hospital volume of HD IL2 on inpatient mortality using National Inpatient Sample (NIS) data. Methods We did a cross-sectional study using NIS, one of the largest inpatient datasets in United States, from 2003 to 2011. Patients with melanoma and RCC receiving HD IL2 were identified by ICD9 procedure code 00.15. The primary outcome was inpatient mortality. Using Joinpoint regression, which detects change in trend of inpatient mortality with change in annual volume, the hospitals were classified in three volume categories (low: 1-40, medium: 41-120, high: >120). Multivariate logistic regression was used to identify predictors of inpatient mortality controlling for confounders. Results From 2003 to 2011, 29,532 patients with RCC or melanoma who received HD IL2 were identified, and 124 died during the hospitalization (0.4%). The hospitals with low, medium and high annual volume had significant difference in inpatient mortality (0.83%, 0.29%and 0.13% respectively, p = 0.0003). On multivariate analysis, low volume hospitals were associated with significantly higher odds of inpatient mortality (OR 6.1, 95%CI 1.6-23.2, p = 0.003) as compared to high volume hospitals. Additionally, the hospitals with annual volume of 1-20 had even higher rates (1.31% vs. 0.13%, p<0.0001) and multivariate odds (OR 8.9, 95%CI 2.4-33.2, p = 0.0006) of inpatientmortality as compared to high volume hospitals.Conclusions Lower annual hospital volume of HD IL2 is associated with worse outcomes. Annual hospital volume of 1-40 and 1-20 treatments per year is associated with 6 and 9 times higher odds of inpatient mortality respectively as compared to high volume hospitals. Our findings provide preliminary evidence for a volume-outcome relationship for RCC and melanoma patients undergoing HD IL2 treatment. They support future volume-outcome analyses in relation to other anti-cancer therapies that require special training and expertise

The helicase HAGE prevents interferon-a-induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

The tumour suppressor PML (promyelocytic leukaemia protein) regulates several cellular pathways involving cell growth, apoptosis, differentiation and senescence. PML also has an important role in the regulation of stem cell proliferation and differentiation. Here, we show the involvement of the helicase HAGE in the transcriptional repression of PML expression in ABCB5 + malignant melanoma-initiating cells (ABCB5 + MMICs), a population of cancer stem cells which are responsible for melanoma growth, progression and resistance to drug-based therapy. HAGE prevents PML gene expression by inhibiting the activation of the JAK-STAT (janus kinase-signal transducers and activators of transcription) pathway in a mechanism which implicates the suppressor of cytokine signalling 1 (SOCS1). Knockdown of HAGE led to a significant decrease in SOCS1 protein expression, activation of the JAK-STAT signalling cascade and a consequent increase of PML expression. To confirm that the reduction in SOCS1 expression was dependent on the HAGE helicase activity, we showed that SOCS1, effectively silenced by small interfering RNA, could be rescued by re-introduction of HAGE into cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes SOCS1 mRNA unwinding and protein expression in vitro

Baseline circulating IL-17 predicts toxicity while TGF-β1 and IL-10 are prognostic of relapse in ipilimumab neoadjuvant therapy of melanoma

Background: We evaluated candidate circulating serum cytokines, chemokines and growth factors in patients with locally/regionally advanced melanoma receiving neoadjuvant ipilimumab with toxicity and clinical outcome. Methods: Patients were treated with ipilimumab (10mg/kg IV every 3weeks, 2 doses) before and after surgery. xMAP multiplex serum testing for 36 functionally selected cytokines and chemokines was performed at baseline and at six weeks (following ipilimumab). Based on our prior data, the association of IL-17 and immune related colitis was tested. Serum cytokines were divided into functional groups (Th1, Th2, Regulatory, Proinflammatory) and were assessed at baseline and week 6 using sparse-group Lasso modeling to assess the association of various cytokine groups with progression free survival (PFS). The linear combination of the cytokines/chemokines in this model was then used as a risk score and a Kaplan-Meier curve was generated to examine the association of the dichotomized score and PFS. Results: Thirty-five patients were enrolled whose staging was: IIIB (3; N2b), IIIC (30; N2c, N3), IV (2). Median follow-up was 18months. Among 33 evaluable patients, median PFS was 11months (95% CI=6.2-19.2). IL-17 was found to correlate significantly with the incidence of grade 3 diarrhea/colitis when measured at baseline (p=0.02) with a trend towards significance at 6weeks (p=0.06). In the modeling analysis, at baseline, the linear combination of 2 regulatory cytokines [TGF- β1 (p=0.19) and IL-10 (p=-0.34)] was significantly associated with PFS (HR 2.66; p=0.035). No significant correlations with clinical outcomes were found in examining the week 6 cytokines. Conclusions: Baseline IL-17 level was significantly associated with the later development of severe diarrhea/colitis while the combination of baseline TGF- β1 and IL-10 levels were associated with therapeutic clinical outcome after neoadjuvant ipilimumab. These findings warrant further investigation and validation. Trial registration: ClinicalTrials.gov Identifier NCT00972933

CD8+ T cell responses in metastatic melanoma patients receiving an adenovirally antigen engineered dendritic cell vaccine +/- IFN-α

Dendritic cells (DC), the primary antigen presenting cells and stimulators of naïve immune cells, are uniquely positioned to promote anti-tumor immunity. We developed a DC vaccine which expresses three full length melanoma antigens tyrosinase, MART-1, and MAGE-A6 engineered with an Ad type 5 adenovirus “AdVTMM2” which can activate CD8+ and CD4+ T cells as well as natural killer (NK) cells. A clinical trial testing this vaccine as well as the potential effects of IFN-α administration post-vaccination has enrolled 36 patients to date (NCT01366144). Peripheral blood banked at baseline, post-DC vaccination, and after either observation or one month of high dose IFN-α was tested for anti-tumor immunity. Here, we present initial immune response testing of the 12 HLA-A2+ patients who were able to be assessed for circulating CD8+ T cell frequencies by HLA-A2-peptide dextramers. Patient PBMCs were analyzed by MHC dextramer binding assay to determine 1) the frequency of CD8+ cells specific to vaccine encoded antigens in the subset of HLA-A2+ patients and 2) potential determinant spreading to antigens not in the vaccine, 3) frequency and co-expression of the checkpoint inhibitor molecules CTLA-4, PD-1, and TIM-3 on CD8+ T cells, and 4) to characterize three NK cell subpopulations. On the CD8+ T cells, PD-1 was the checkpoint molecule most commonly expressed, while CTLA-4 was minimally expressed. TIM-3 was the checkpoint molecule most commonly expressed on all three subpopulations of NK cells. We observed that most patients developed vaccine-encoded antigen-specific responses, and a subset demonstrated determinant spreading to non-vaccine encoded antigens gp100 and/or NY-ESO-1. Expression of checkpoint molecules changed on both T and NK cells through the treatment periods, and the function (by IFNγ ELISPOT) was also assessed. This study will aid in the design of more effective dendritic cell vaccines and adjuvants for metastatic melanoma patients

Pro-inflammatory cytokines predict relapse-free survival after one month of interferon-α but not observation in intermediate risk melanoma patients

Background: E1697 was a phase III trial of adjuvant interferon (IFN)-α2b for one month (Arm B) versus observation (Arm A) in patients with resected melanoma at intermediate risk. We evaluated the levels of candidate serum cytokines, the HLA genotype, polymorphisms of CTLA4 and FOXP3 genes and the development of autoantibodies for their association with relapse free survival (RFS) in Arm A and Arm B among 268 patients with banked biospecimens. Methods: ELISA was used to test 5 autoantibodies. Luminex/One Lambda LABTypeRSSO was used for HLA Genotyping. Selected CTLA4 and FOXP3 Single nucleotide polymorphisms (SNPs) and microsatellites were tested for by polymerase chain reaction (PCR). Sixteen serum cytokines were tested at baseline and one month by Luminex xMAP multiplex technology. Cox Proportional Hazards model was applied and the Wald test was used to test the marginal association of each individual marker and RFS. We used the Lasso approach to select the markers to be included in a multi-marker Cox Proportional Hazards model. The ability of the resulting models to predict one year RFS was evaluated by the time-dependent ROC curve. The leave-one-out method of cross validation (LOOCV) was used to avoid over-fitting of the data. Results: In the multi-marker modeling analysis conducted in Arm B, one month serum IL2Rα, IL- 12p40 and IFNα levels predicted one year RFS with LOOCV AUC = 82%. Among the three markers selected, IL2Rα and IFNα were the most stable (selected in all the cross validation cycles). The risk score (linear combination of the 3 markers) separated the RFS curves of low and high risk groups well (p = 0.05). This model did not hold for Arm A, indicating a differential marker profile in Arm B linked to the intervention (adjuvant therapy). Conclusions: Early on-treatment proinflammatory serum markers (IL2Rα, IL-12p40, IFNα) significantly predict RFS in our cohort of patients treated with adjuvant IFN-α2b and warrant further study