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Due to advances in diagnosis and treatment, cancer–related mortality has decreased, and by the year 2030, there will be 22 million cancer survivors in the United States. This success comes with an increased incidence of serious adverse effects, mainly in the cardiovascular system. While new treatment modalities are emerging for Triple-negative breast cancer (TNBC), most current strategies include anthracycline-based regimens to manage disease. Therefore, novel strategies are needed to overcome anthracycline-induced cardiotoxicities in this patient population. Activation of the TSP1/CD47 signaling axis is implicated in the progression of heart failure, with reported increases in TSP1 levels following myocardial infarction. Therefore, we examined the potential of CD47 blockade as a strategy to prevent cardiac injury as a consequence of cancer chemotherapy. Our data in a syngeneic orthotopic breast cancer model shows that blockade of CD47 using an in vivo anti-sense phosphodiesterase morpholino (PMO) preserved ejection fraction, fractional shortening, and cardiac output when compared to DOX treatment while preserving oncologic efficacy of chemotherapy. To determine a potential mechanism of cardioprotection, hearts of control and CD47 PMO-treated mice were subjected to RNA sequencing. Gene set enrichment analysis (GSEA) showed significant positive enrichment for metabolic pathways including pyruvate metabolism (NES= 2.3 , p< 0.002), and oxidative phosphorylation (NES=2.0, p< 0.01). During cardiac insult, metabolic flexibility of cardiomyocytes results in metabolic reprogramming from fatty acid oxidation to a glycolytic mechanism to overcome injury. Thus, DOX-associated cardiotoxicity may be mediated by an increase in TSP1 and a decrease in glycolysis, leading to the inability to overcome acute cellular stress. In vitro cellular bioenergetics, analysis revealed that TSP1 caused a dose-dependent reduction in glycolytic flux and glycolytic capacity in cardiac myoblast. This, coupled with preserved cardiac viability of cardiac cells treated with CD47 PMO in the presence of DOX, suggests that TSP1 may act through CD47 to prevent cardiac cell metabolic reprogramming needed to overcome injury. Furthermore, anti-sense experiments with siRNAs to Glut-4 and Hexokinase-II showed that the protection conferred by CD47 is mediated by activating these proteins. Therefore our studies suggest that the TSP1/CD47 axis may be central to the interplay of metabolism to preserve cardiac tissue integrity; thus, targeting this pathway may prevent the onset of chronic cardiac disease due to chemotherapy in cancer patients. Citation Format: Steven M. Bronson, Jessica D. Mackert, Mitra Kooshki, Adam S. Wilson, Nildris Cruz-Diaz, Pierre L Triozzi, Alexandra Thomas, Katherine L. Cook, David R. Soto-Pantoja. Blockade of CD47/Thrombospondin-1 signaling increases glycolytic metabolism as a protective mechanism against chemotherapy-associated cardiac injury in a model of Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-08-14.

Purpose:Immunotherapy with checkpoint inhibitors is improving the outcomes of several cancers. However, only a subset of patients respond. Therefore, predictive biomarkers are critically needed to guide treatment decisions and develop approaches to the treatment of therapeutic resistance.Experimental Design:We compared bioenergetics of circulating immune cells and metabolomic profiles of plasma obtained at baseline from patients with melanoma treated with anti–PD-1 therapy. We also performed single-cell RNA sequencing (scRNAseq) to correlate transcriptional changes associated with metabolic changes observed in peripheral blood mononuclear cells (PBMC) and patient plasma.Results:Pretreatment PBMC from responders had a higher reserve respiratory capacity and higher basal glycolytic activity compared with nonresponders. Metabolomic analysis revealed that responder and nonresponder patient samples cluster differently, suggesting differences in metabolic signatures at baseline. Differential levels of specific lipid, amino acid, and glycolytic pathway metabolites were observed by response. Further, scRNAseq analysis revealed upregulation of T-cell genes regulating glycolysis. Our analysis showed that SLC2A14 (Glut-14; a glucose transporter) was the most significant gene upregulated in responder patients' T-cell population. Flow cytometry analysis confirmed significantly elevated cell surface expression of the Glut-14 in CD3+, CD8+, and CD4+ circulating populations in responder patients. Moreover, LDHC was also upregulated in the responder population.Conclusions:Our results suggest a glycolytic signature characterizes checkpoint inhibitor responders; consistently, both ECAR and lactate-to-pyruvate ratio were significantly associated with overall survival. Together, these findings support the use of blood bioenergetics and metabolomics as predictive biomarkers of patient response to immune checkpoint inhibitor therapy.

Supplementary Figure from Circulating Immune Bioenergetic, Metabolic, and Genetic Signatures Predict Melanoma Patients' Response to Anti–PD-1 Immune Checkpoint Blockade

Purpose: Immunotherapy with checkpoint inhibitors is improving the outcomes of several cancers. However, only a subset of patients respond. Therefore, predictive biomarkers are critically needed to guide treatment decisions and develop approaches to the treatment of therapeutic resistance. Experimental Design: We compared bioenergetics of circulating immune cells and metabolomic profiles of plasma obtained at baseline from patients with melanoma treated with anti–PD-1 therapy. We also performed single-cell RNA sequencing (scRNAseq) to correlate transcriptional changes associated with metabolic changes observed in peripheral blood mononuclear cells (PBMC) and patient plasma. Results: Pretreatment PBMC from responders had a higher reserve respiratory capacity and higher basal glycolytic activity compared with nonresponders. Metabolomic analysis revealed that responder and nonresponder patient samples cluster differently, suggesting differences in metabolic signatures at baseline. Differential levels of specific lipid, amino acid, and glycolytic pathway metabolites were observed by response. Further, scRNAseq analysis revealed upregulation of T-cell genes regulating glycolysis. Our analysis showed that SLC2A14 (Glut-14; a glucose transporter) was the most significant gene upregulated in responder patients' T-cell population. Flow cytometry analysis confirmed significantly elevated cell surface expression of the Glut-14 in CD3+, CD8+, and CD4+ circulating populations in responder patients. Moreover, LDHC was also upregulated in the responder population. Conclusions: Our results suggest a glycolytic signature characterizes checkpoint inhibitor responders; consistently, both ECAR and lactate-to-pyruvate ratio were significantly associated with overall survival. Together, these findings support the use of blood bioenergetics and metabolomics as predictive biomarkers of patient response to immune checkpoint inhibitor therapy.

BackgroundTargeting programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase (IDO1) pathways is an appealing option for cancer treatment.MethodsThe open-label, phase 1/2 ECHO-203 study evaluated the safety, tolerability, and efficacy of the IDO1 inhibitor epacadostat in combination with durvalumab, a human anti-PD-L1 monoclonal antibody in adult patients with advanced solid tumors.ResultsThe most common treatment-related adverse events were fatigue (30.7%), nausea (21.0%), decreased appetite (13.1%), pruritus (12.5%), maculopapular rash (10.8%), and diarrhea (10.2%). Objective response rate (ORR) in the overall phase 2 population was 12.0%. Higher ORR was observed in immune checkpoint inhibitor (CPI)-naïve patients (16.1%) compared with patients who had received previous CPI (4.1%). Epacadostat pharmacodynamics were evaluated by comparing baseline kynurenine levels with those on therapy at various time points. Only the 300-mg epacadostat dose showed evidence of kynurenine modulation, albeit unsustained.ConclusionsEpacadostat plus durvalumab was generally well tolerated in patients with advanced solid tumors. ORR was low, and evaluation of kynurenine concentration from baseline to cycle 2,day 1, and cycle 5,day 1, suggested >300mg epacadostat twice daily is needed to ensure sufficient drug effect.Clinical trial informationA study of epacadostat (INCB024360) in combination with durvalumab (MEDI4736) in subjects with selected advanced solid tumors (ECHO-203) (NCT02318277).

Introduction: Tumor infiltrating lymphocytes (TILs), hold promise in advancing adoptive cell therapy for patients with otherwise limited treatment options. However, many tumors do not attract TILs, or the TILs themselves are exhausted which limits treatment availability and efficacy. Consequently, there is a need for alternative sources of tumor reactive T cells. Here, we address this need through the ex vivo 3D cell culture biofabrication of immune competent patient tumor organoids (iPTOs). These constructs, composed of patient-matched cancer cells, antigen presenting cells (APCs), and stromal cells encapsulated in extracellular matrix (ECM)-like hydrogel, closely mimic the in vivo tumor microenvironment. We demonstrate that constant circulation of peripheral blood mononuclear cells (PBMCs) through a microfluidic device housing iPTOs can emulate lymph node activation of immune cells to the tumor, yielding organoid interacting lymphocytes (OILs) that possess increased markers of activation, cytotoxicity, proliferation, homing markers, and inflammatory cytokine production relative to uncirculated PBMCs. Methods: Tumor tissue, APCs (from lymph nodes or spleen), and peripheral blood were collected from 8 patients with mesothelioma (3), melanoma (2), and appendiceal cancer (3). Chips were built from a glass slide and laser cut Polymethyl methacrylate. IPTOs were made such that a concentration of 3 APCs for every 1 tumor cell were added to a collagen and hyaluronic acid mixture that was then photocrosslinked in the chip chamber. PBMCs were circulated through chips housing the photocrosslinked iPTOs for 7 days and then expanded. Following expansion, a subpopulation of these cells were cocultured with tumor only organoids for a 7 day period. Single and secondary tumor exposure populations of effector, memory, and reactive T cells were analyzed via flow cytometry, immunohistochemistry, and cell culture medium proteomic analysis, and compared with both uncirculated PBMCs and patient TILs. Results: Data collected from patients with appendiceal, melanoma, and mesothelioma tumors shows generation of OILS with increase in cytotoxic T lymphocyte, effector memory, and central memory phenotypes when compared to uncirculated PBMCs, greater than or comparable to TILs and with similar effector cytokine and enzyme expression. OILs were also found to express less immunosuppressive signals (i.e., Tim3, PD1, and IL 10). Critically, chip-based T cell activation resulted in 10x more viable OILs than were typically observed with TILs following parallel growth period of 14 days. Conclusions: This data suggests that microfluidic incubation of lymphocytes with lymph node and tumor cells can generate large amounts of viable tumor-specific T cells on-demand with characteristics similar to TILs but with increased viability, cell expansion propensity, and cytotoxic responsiveness. Citation Format: Damian C. Hutchins, Cecilia R. Schaaf, Nicholas P. Edenhoffer, Mitra Kooshki, Robyn Greissenger, Steven D. Forsythe, Adam R. Hall, Lance D. Miller, Shay Soker, Pierre L. Triozzi, Konstantinos Votanopoulos. Microfluidic incubation of patient derived tumor and immune cells boosts lymphocyte cytotoxic phenotype. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4051.

We describe a first suspected case of fibrosing mediastinitis following anti-programmed death (PD)-1 therapy, pembrolizumab. Multimodality imaging, including cardiac magnetic resonance imaging, and a multidisciplinary team approach were integral to the diagnosis. If further substantiated, systematic surveillance after anti-PD-1 therapy for fibrosing mediastinitis may be warranted. (

Cellular and intrinsic markers of sarcoma immunogenicity are poorly understood. To gain insight into whether tumor-immune interactions correlate with clinical aggressiveness, the authors examined the prognostic significance of immune gene signatures in combination with tumor mutational burden (TMB) and cancer-testis antigen (CTA) expression.RNA sequencing and clinical data of 259 soft tissue sarcomas from The Cancer Genome Atlas project were used to investigate associations between published immune gene signatures and patient overall survival (OS) in the contexts of TMB, as computed from whole-exome sequencing data, and CTA gene expression. Multivariate Cox proportional hazards regression models and log-rank tests were used to assess survival associations.Immune signature scores that reflected in part the intratumoral abundance of cytotoxic T cells showed significant positive associations with OS. However, the prognostic power of the T-cell signatures was highly dependent on TMB-high status, consistent with protective effects of tumor-infiltrating T cells in tumors with elevated antigenicity. In TMB-low tumors, a signature of infiltrating plasma B cells was significantly and positively associated with OS, independent of T-cell signature status. Although tumor subtypes based on differential expression patterns of CTA genes showed different survival associations within leiomyosarcoma and myxofibrosarcoma histologies, neither CTA nor histologic subtype interacted with the T-cell-survival association.Signatures of T-cell and plasma B-cell infiltrates were associated with a survival benefit in soft tissue sarcomas. TMB, but not CTA expression, influenced the prognostic power of T-cell-associated, but not plasma B-cell-associated, survival.Clinical data and RNA analysis of 259 soft tissue sarcomas from The Cancer Genome Atlas project were used to investigate associations between five published gene immune cell expression signatures and survival in the context of tumor mutations. Activated T cells had a significant positive association with patient survival. Although high tumor mutation burden was associated with good survival, the prognostic power of T-cell signatures was highly dependent on tumor mutational status, consistent with protective effects of tumor-infiltrating T cells in tumors with high levels of antigens. In low tumor mutation-bearing tumors, plasma B cells were positively associated with survival.

PURPOSE Immune checkpoint inhibitor (ICI) therapy has recently been found to improve survival in patients with a number of cancers, including those with metastatic disease. There is an association of adverse radiation effect (ARE) in patients with brain metastases who have been treated with stereotactic radiosurgery (SRS) and ICIs. METHODS AND MATERIALS Single-institution retrospective review identified 1118 brain metastases treated with SRS between 2013 and 2018 that had received ICI therapy and 886 metastases that did not receive ICI. Toxicity grading was done via the Common Terminology Criteria for Adverse Events v4.0 grading criteria. Cumulative incidence of ARE was estimated using competing risks methodology; univariate and multivariable regression models were generated to estimate the subdistribution hazard (sHR) of ARE. RESULTS Two-year cumulative incidence of ARE was 4.5% and 2.1% in patients treated with and without ICI, respectively (Gray's P = .004). Of the 52 metastases exhibiting ARE during the follow-up period, ARE severity by Common Terminology Criteria for Adverse Events v4 was grade 1 in 14 patients, grade 2 in 15, grade 3 in 9, and grade 4 in 14. There were no grade 5 events. Factors associated with an increased sHR of ARE on univariate analysis included ICI, metastasis volume, SRS dose, prescription isodose line, cavity-directed SRS, and V12. Multivariable analysis revealed prescription isodose line (sHR 0.95, P < .01) and ICI (sHR 2.58, P < .01) as significant predictors of ARE. Increasing V12 was associated with a rapidly increasing risk of adverse radiation effect in patients who received ICI. CONCLUSIONS Our findings suggest that patients receiving ICI have an increased risk of ARE after radiosurgery for brain metastases, with large metastases being at particularly high risk.

There is a lack of robust generalizable predictive biomarkers of response to immune checkpoint blockade in multiple types of cancer. We develop hDirect-MAP, an algorithm that maps T cells into a shared high-dimensional (HD) expression space of diverse T cell functional signatures in which cells group by the common T cell phenotypes rather than dimensional reduced features or a distorted view of these features. Using projection-free single-cell modeling, hDirect-MAP first removed a large group of cells that did not contribute to response and then clearly distinguished T cells into response-specific subpopulations that were defined by critical T cell functional markers of strong differential expression patterns. We found that these grouped cells cannot be distinguished by dimensional-reduction algorithms but are blended by diluted expression patterns. Moreover, these identified response-specific T cell subpopulations enabled a generalizable prediction by their HD metrics. Tested using five single-cell RNA-seq or mass cytometry datasets from basal cell carcinoma, squamous cell carcinoma and melanoma, hDirect-MAP demonstrated common response-specific T cell phenotypes that defined a generalizable and accurate predictive biomarker.

BackgroundTriple-negative breast cancer(TNBC) lacks druggable targets and has high metastatic incidence. Immune checkpoint blockades (ICB) are FDA approved for TNBC treatment, but therapeutic response and biomarkers are limited. CD47 is an integral membrane protein overexpressed on cancer cells that alters anti-tumor immunosurveillance, resulting in tumor progression. CD47 is involved in metabolic reprogramming but whether CD47 is a marker of progression and its role in ICB response for TNBC remains unknown.MethodsHuman TNBC biopsies were subjected to immunohistochemical analysis to determine CD47 role in TNBC progression. To determine CD47 impact on tumor burden, a carcinogen-induced TNBC model was performed in female wild type(WT) and cd47 null(cd47-/-) C57Bl/6 mice. To evaluate immune infiltrate signaling, tumors underwent spatial tissue proteomics by multiplexing photo-cleavable antibodies in Formalin-Fixed Paraffin-Embedded samples. An orthotopic EMT-6 murine TNBC model was performed to investigate tumor burden for CD47 monotherapy or in combination with anti-PD-L1 therapy.ResultsHuman matched primary, and metastatic TNBC biopsies increased immunoreactivity to CD47, signifying a potential therapeutic target(n=24). CD47 deficiency in the carcinogen-induced DMBA model decreased tumor incidence, weight, and area compared to WT(n=8/group,*pConclusionsOur data indicates that CD47 may serve as a marker of anti-PD-L1 response, and targeting CD47 enhances immunogenicity and decreases immunosuppressive molecules, sensitizing TNBC tumors to anti-PD-L1 therapy to reduce tumor burden.AcknowledgementsDSP is supported by the NCI R21 (CA249349) and the American Cancer Society Research Scholar Grant (133727-RSG-19-150-01-LIB). ERS is supported by the NIAID Immunology and Pathogenesis T32 Training Grant (T32AI007401).Ethics ApprovalAnimal studies were approved by the Institutional Care and Use Committee, Wake Forest Health Sciences.

BackgroundCD47 is an integral membrane protein that alters adaptive immunosurveillance when bound to the matricellular glycoprotein thrombospondin-1 (TSP1). We examined the impact of the CD47/TSP1 signaling axis on melanoma patient response to anti-PD-1 therapy due to alterations in T cell activation, proliferation, effector function, and bioenergetics.MethodsA syngeneic B16 mouse melanoma model was performed to determine if targeting CD47 as monotherapy or in combination with anti-PD-1 impacted tumor burden. Cytotoxic (CD8+) T cells from Pmel-1 transgenic mice were used for T cell activation, cytotoxic T lymphocyte, and cellular bioenergetic assays. Single-cell RNA-sequencing, ELISA, and flow cytometry was performed on peripheral blood mononuclear cells and plasma of melanoma patients receiving anti-PD-1 therapy to examine CD47/TSP1 expression.ResultsHuman malignant melanoma tissue had increased CD47 and TSP1 expression within the tumor microenvironment compared with benign tissue. Due to the negative implications CD47/TSP1 can have on antitumor immune responses, we targeted CD47 in a melanoma model and observed a decrease in tumor burden due to increased tumor oxygen saturation and granzyme B secreting CD8+ T cells compared with wild-type tumors. Additionally, Pmel-1 CD8+ T cells exposed to TSP1 had reduced activation, proliferation, and effector function against B16 melanoma cells. Targeting CD47 allowed CD8+ T cells to overcome this TSP1 interaction to sustain these functions. TSP1 exposed CD8+ T cells have a decreased rate of glycolysis; however, targeting CD47 restored glycolysis when CD8+ T cells were exposed to TSP1, suggesting CD47 mediated metabolic reprogramming of T cells. Additionally, non-responding patients to anti-PD-1 therapy had increased T cells expressing CD47 and circulating levels of TSP1 compared with responding patients. Since CD47/TSP1 signaling axis negatively impacts CD8+ T cells and non-responding patients to anti-PD-1 therapy have increased CD47/TSP1 expression, we targeted CD47 in combination with anti-PD-1 in a melanoma model. Targeting CD47 in combination with anti-PD-1 treatment further decreased tumor burden compared with monotherapy and control.ConclusionCD47/TSP1 expression could serve as a marker to predict patient response to immune checkpoint blockade treatment, and targeting this pathway may preserve T cell activation, proliferation, effector function, and bioenergetics to reduce tumor burden as a monotherapy or in combination with anti-PD-1.

Background/aimsSurvival after diagnosis of metastasis from uveal melanoma is poor. Identifying individuals at high risk for metastasis and developing adjuvant therapy to prevent clinically apparent metastasis could improve survival. We conducted an adjuvant trial of sequential, low-dose dacarbazine (DTIC) and interferon-alpha-2b (IFN-α−2b) in patients with cytogenetic high-risk uveal melanoma.MethodsPatients diagnosed with iris, ciliary body or choroidal melanoma with high-risk tumour cytogenetics (monosomy 3) were offered adjuvant treatment with low-dose DTIC and IFN-α−2b following primary therapy. Eligible but not enrolled patients were observed for comparison. DTIC was administered at 850 mg/m2 intravenously on days 1 and 28. IFN-α−2b was administered at 3 million units three times a week subcutaneously for 24 weeks beginning at week 9. Hepatic imaging was performed prior to adjuvant therapy and then at least every 6 months. Survival data were collected for 5 years after enrolment.Results33 patients (22%) were enrolled (treatment group), 29 (19%) were eligible but did not enrol (observation group) and 88 (59%) were not eligible. The 5-year metastasis-free survival (MFS) was 64%±9% for treated and 33%±10% for observed patients (p=0.05). The 5-year overall survival (OS) rate was 66%±9% for treated and 37%±10% for observed patients (p=0.02).ConclusionsWhen adjusted for differences in age, tumour size and initial treatment, survival between treated and observed patients was no longer significant (p=0.56 MFS and p=0.92 OS). Differences in baseline tumour characteristics between treated and observed patients can influence interpretation of results.Trial registration numberNCT01100528.

Background Immune checkpoint blockade therapy targeting PD-L1 has recently been approved for metastatic triple negative breast cancer (TNBC) patients. However, a 7% response rate calls for better models and strategies to stimulate TN-tumor immunogenicity to increase patient response. Overexpression of the receptor CD47 impairs innate and adaptive tumor immunosurveillance when engaged to its counter receptor SIRPα or ligand thrombospondin-1. Co-expression of CD47 and PD-L1 is implicated with disease progression in TNBC patients. We examined through murine models and tumor organoid platforms whether targeting CD47 sensitizes TNBC tumors to PD-L1 therapy, focusing on the modulation of cellular bioenergetics as a potential mechanism and potentially predict response. Methods The effects of targeting CD47 and PD-L1 were examined through orthotopic syngenic 4T1 and EMT-6 TNBC murine models. Due to predicting patient therapeutic response challenges, tumor organoid platforms investigated mechanisms of tumor sensitization to anti-PD-L1 by targeting CD47. Organoids were constructed by embedding murine TNBC tumor tissue and AH1 CD8+ T cells in a specialized ECM mimicking hydrogel. Immunohistochemistry was performed on organoid, human and murine TNBC tumor tissue. Cellular bioenergetics was analyzed through Seahorse® bioanalyzer. Results Staining of human TNBC biopsies found elevated CD47 expression, signifying a potential therapeutic target. Targeting CD47 or in combination with anti-PD-L1 resulted in decreased tumor volume and weight in a TNBC murine model. The decrease in tumor burden was correlated with increased intratumoral granzyme B secreting CD8+ T cells. Additionally, targeting CD47 within organoids increased IFNγ and granzyme B released, indicating enhanced CD8+ T cell cytolytic capacity. Differential cellular bioenergetics was observed between cancer and T cells suggesting a shift in metabolism in the tumor microenvironment. CD47 targeted T cells had an increased glycolytic rate compared to WT T cells. Conversely CD47 targeted TNBC cells had a decreased glycolytic rate, which may be correlated with decreased PD-L1 expression. Conclusions Targeting CD47 enhanced granzyme B and IFNγ expression suggesting potential mechanisms to increase tumor immunogenicity. CD47 targeted monotherapy or combination with anti-PD-L1 preserves T cell bioenergetics and antitumor function, resulting in decreased TNBC tumor burden. Alternatively, CD47 targeted TNBC had a decreased glycolytic rate and decreased PD-L1 expression, which is reported to regulate glycolysis through Akt/mTOR signaling. Targeting CD47 on T cells enhances their bioenergetics and antitumor function while decreasing TNBC cell bioenergetics, making them more susceptible to immune cell killing. Our data indicates that CD47 targeted monotherapy or combination with anti-PD-L1 may enhance TNBC patient response and improve overall survival. Acknowledgements DSP and SS are supported by the Wake Forest Comprehensive Cancer Center Breast Cancer Center of Excellence Pilot Award. DSP is also supported by the ASTRO-BCRF Career Development Award (637969) while ERS is supported by NIGMS T32 (GM127261). Ethics Approval Animal studies were approved by the Institutional Care and Use Committee, Wake Forest Health Sciences.

Expression of immune checkpoint proteins restrict immunosurveillance in the tumor microenvironment; thus, FDA-approved checkpoint inhibitor drugs, specifically PD-1/PD-L1 and CTLA-4 inhibitors, promote a cytotoxic antitumor immune response. Aside from inflammatory signaling, immune checkpoint proteins invoke metabolic reprogramming that affects immune cell function, autonomous cancer cell bioenergetics, and patient response. Therefore, this review will focus on the metabolic alterations in immune and cancer cells regulated by currently approved immune checkpoint target proteins and the effect of costimulatory receptor signaling on immunometabolism. Additionally, we explore how diet and the microbiome impact immune checkpoint blockade therapy response. The metabolic reprogramming caused by targeting these proteins is essential in understanding immune-related adverse events and therapeutic resistance. This can provide valuable information for potential biomarkers or combination therapy strategies targeting metabolic pathways with immune checkpoint blockade to enhance patient response.

BackgroundTriple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by a lack of specific targets and a 35% incidence of brain metastasis. There is no targeted treatment for managing brain metastasis associated with TNBC; therefore, new strategies are urgently needed to overcome disease mortality. The presence of cell surface protein CD47 allows cancer cells to evade innate and adaptive immune surveillance resulting in metastatic spread. CD47 binds to and activates SIRPα on the surface of myeloid cells, inhibiting their phagocytic activity. On the other hand, CD47 binds the matricellular protein Thrombospondin-1 limiting T cell activation. Thus, blocking the CD47 is a potential therapeutic strategy for the prevention of brain metastasis.MethodsBreast cancer patient biopsies were stained with antibodies against CD47. An anti-CD47 antibody was used in a syngeneic model of orthotopic triple-negative breast cancer. CD47 null mice were used in a breast cancer brain metastasis model by intracardiac injection of the E0071-Br-Luc cell line. Brain metastatic burden was measured by the in vivo imaging system (IVIS) and quantified by luciferase luminescence. Immunohistochemical analysis was performed to quantify tumor-infiltrating macrophages. Gene expression analysis of tumors was carried out by RNA-sequencing.ResultsImmunohistochemical staining of patient biopsies revealed an 89% increase in CD47 expression in metastatic brain tumors compared to primary lesions (p ≤ 0.05). Anti-CD47 treatment in mice bearing brain metastatic 4T1br3 orthotopic tumors reduced tumor volume and tumor weight by over 50% compared to control mice (p ≤ 0.05) and increased F4/80 macrophage marker 5-fold tumors compared to control (p ≤ 0.05). CD47 null mice had a 60% increase in survival (p ≤ 0.05) and an 89% decrease in metastatic brain lesions (p ≤ 0.05) compared to control mice in a brain metastasis model. Additionally, RNA sequencing revealed 318 uniquely expressed genes and a significant reduction of genes related to extracellular matrix organization in tumors treated with an anti-CD47 antibody.ConclusionsCD47 was increased in metastatic brain tumors compared to primary lesions in breast cancer patients. CD47 blockade reduced orthotopic brain-metastatic tumor burden associated with increased macrophage infiltration and reduced extracellular matrix-associated gene expression. Additionally, CD47 null mice had improved overall survival and reduced brain metastatic lesions. Thus, CD47 blockade may be an effective therapeutic for triple-negative breast cancer brain metastasis.AcknowledgementsThis work is supported by a NCI R21 CA249349 and the American Cancer Society Research Scholar Grant (133727-RSG-19-150-01-LIB).Ethics ApprovalAnimal studies were approved by the Wake Forest School of Medicine Animal Care and UseCommittee (ACAUC). Human subject studies were approved by the institutional review board (IRB).

BackgroundImmune checkpoint blockade (ICB) is now a mainstay of cancer therapy with success in extending the survival time in several cancers, including melanoma. Still, these modalities result in immune-related adverse events (irAEs), with approximately 80% of melanoma patients experiencing toxicity. IrAEs can lead to treatment discontinuation, contributing to mortality. In addition, one uncommon irAE with a high mortality rate is ICB-related myocarditis. In addition to myocarditis, ICB can also cause arrhythmias and heart failure. Currently, early markers to predict cardiotoxicity are unknown. During cardiac insult, cardiomyocyte metabolic flexibility results in metabolic reprogramming from fatty acid oxidation to glycolysis to overcome injury; however prolonged metabolic remodeling precedes most pathological alterations in the heart, suggesting that changes in metabolism may contribute to immunotherapy-related cardiac damage.MethodsMale C57BL/6 mice were injected with B16 melanoma cells. Once tumors reached 100 mm3, the animals were treated with three doses of anti-PD-1 antibody (200 µg IP). Echocardiograms were performed prior to necropsy using Vevo LAZR ultrasound to assess cardiac function. Bulk RNA sequencing (RNA-seq) and immunohistochemistry were performed on cardiac tissue. Single-cell RNA sequencing (scRNA-seq) was performed on human peripheral blood mononuclear cells (PMBCs) in patients treated with anti-PD-1 therapy.ResultsEchocardiogram showed that anti-PD1 treatment attenuated stroke volume and increase heart rate compared to WT mice, suggesting that anti-PD1 treatment may be associated with changes in cardiac function. Histological examination showed no evidence of cardiac inflammation. RNA-seq was performed to further examine mechanisms of anti-PD1 therapy on the heart. Our data shows that over 230 genes were uniquely expressed in cardiac tissue of mice treated with anti-PD-1 therapy compared to isotype control. While no overt changes in immune infiltrate were seen, gene set enrichment analysis (GSEA) showed significant positive enrichment in chemokine receptor interactions with anti-PD-1 treatment potentially playing a role in the differentiation of cardiac, immune cell populations.Furthermore, gene enrichment was also observed among metabolic pathways. Interestingly, upregulation of PDK4 was observed in the hearts of animals treated with anti-PD1 antibody. PDK4 activation restricts cardiomyocyte metabolic flexibility during stress. scRNA-seq data from patient PBMCs also indicates an increase in PDK4 levels in the monocytic population after anti-PD1 treatment, suggesting that this protein may be regulated due to checkpoint blockade.ConclusionsCardiac inflammation due to checkpoint blockade is implicated in ICB-myocarditis However, RNA-seq data suggest that PD-1 therapy alters chemokine and metabolic pathways that may contribute to cardiac damage, suggesting potential early markers to identify cardiac irAEsAcknowledgementsT32 from the Office of The Director, National Institutes of Health (T32-OD010957, SMB) (R21CA249349, DSP), an American Cancer Society Research Scholar Grant (133727-RSG-19-150-01-LIB, DSP)

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by the lack of druggable targets and an incidence of brain metastasis from the primary site of approximately 35%. There is no standard treatment for managing brain metastasis associated with TNBC; therefore, new strategies are urgently needed to overcome disease mortality. The CD47/SIRPα signaling pathway is implicated in tumor progression due to bypassing innate and adaptive immune surveillance. Most strategies targeting this pathway focus on targeting the receptor CD47; however, targeting SIRPα as a potential strategy to mitigate tumor burden remains understudied. Analysis of gene expression database shows that SIRPα expression is significantly elevated in invasive breast cancer when compared to primary. Furthermore, single-cell data indicates that SIRPα is expressed in basal epithelial cells in TNBC tumors aside from the myeloid compartment. Our immune staining against SIRPα in patient biopsies shows a five-fold increase in SIRPα expression in metastatic brain tumors compared to the primary lesions. Therefore, targeting SIRPα may be a new immunotherapeutic strategy to treat breast cancer brain metastases. Anti-SIRPα treatment of mice bearing brain metastatic 4T1br3 orthotopic tumors showed reduced tumor volume and tumor weight by over 50% compared to isotype control-treated mice. Furthermore, in a model of intracardial brain metastasis, treatment with SIRPα antibody was associated with a 60% increase in survival compared to isotype control-treated mice. RNA sequencing of tumors indicated that SIRPα blockade is associated with a reduction in genes linked to mitochondrial respiratory chain and increases in negative regulation of the cell cycle. Furthermore, in vitro SIRPα targeting enhanced the cell-mediated cytotoxicity of microglia against 4T1Br3 breast cancer cells. This suggests that SIRPα blockade may influence both tumor and innate immune cells to limit brain metastatic breast cancer growth and enhance survival.

// David R. Soto-Pantoja 1, 3 , Adam S. Wilson 1 , Kenysha YJ. Clear 1 , Brian Westwood 1 , Pierre L. Triozzi 2, 3 and Katherine L. Cook 1, 3 1 Department of Surgery, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 2 Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 3 Comprehensive Cancer Center of Wake Forest Baptist Medical Center, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA Correspondence to: Katherine L. Cook, email: klcook@wakehealth.edu Keywords: breast cancer, melanoma, immune therapy, ipilimumab, CTLA4 Received: March 22, 2017 Accepted: July 23, 2017 Published: August 03, 2017 ABSTRACT The unfolded protein response (UPR) is a stress pathway controlled by GRP78 to mediate IRE1, PERK, and ATF6 signaling. We show that targeting GRP78, IRE1, and PERK differentially regulates macrophage polarization. Specifically, PERK targeting enhanced macrophage proliferation and macrophage-mediated killing but not GRP78 or IRE1. Targeting UPR in cancer cells also differentially affected macrophage cytolytic capacity. Tumoral IRE1 or GRP78 inhibition enhanced macrophage-mediated cancer cell clearance. Conditioned media from GRP78-silenced cancer cells caused reciprocal regulation of CD80 and CD206, suggesting control of plasticity by secreted factors. GRP78 targeting in mice resulted in a cytokine shift and increased tumoral CD80+/CD68+ cells, suggesting an M1-like profile. Targeting UPR in both macrophage and cancer cells indicates that PERK or GRP78 reduction enhances macrophage clearance of cancer cells. Recent evidence suggests that macrophage polarization influences immune checkpoint therapy resistance. To determine whether UPR effects immunotherapy resistance, analysis of matched melanoma patient PBMC before/after developing ipilimumab resistance demonstrated increased UPR signaling and an M2-like macrophage population, supporting a novel role of UPR signaling and innate immune regulation in anti-CTLA-4 therapy resistance. These data suggest that targeting GRP78 or PERK promotes an anti-tumor immune response by either directly promoting macrophage cytolytic activity or indirectly by shifting tumoral cytokine secretion.

We sought to identify candidate biomarkers for early brain metastasis (BM) recurrence in patients who underwent craniotomy followed by adjuvant stereotactic radiosurgery. RNA sequencing was performed on eight resected brain metastasis tissue samples and revealed B-cell related genes to be highly expressed in patients who did not experience a distant brain failure and had prolonged overall survival. To translate the findings from RNA sequencing data, we performed immunohistochemistry to stain for B and T cell markers from formalin-fixed parffin-embedded tissue blocks on 13 patients. CD138 expressing plasma cells were identified and quantitatively assessed for each tumor sample. Patients’ tumor tissues that expressed high levels of CD138 plasma cells (N = 4) had a statistically significant improvement in OS compared to low levels of CD138 (N = 9) (p = 0.01). Although these findings are preliminary, the significance of CD138 expressing plasma cells within BM specimens should be investigated in a larger cohort. Immunologic markers based on resection cavity analysis could be predictive for determining patient outcomes following cavity-directed SRS.

Background The effect of immunotherapy on brain metastasis patients remains incompletely understood. Our goal was to evaluate its effect on survival, neurologic death, and patterns of failure after stereotactic radiosurgery (SRS) without prior whole-brain radiation therapy (WBRT) in patients with lung and melanoma primaries metastatic to the brain. Methods We performed a retrospective analysis of 271 consecutive lung or melanoma patients treated with upfront SRS for brain metastases between 2013 and 2018. Of these patients, 101 (37%) received immunotherapy and 170 (63%) did not. Forty-three percent were treated with nivolumab. Thirty-seven percent were treated with pembrolizumab. Fifteen percent were treated with ipilimumab. One percent were treated with a combination of nivolumab and ipilimumab. One percent were treated with atezolizumab. Three percent were treated with another immunotherapy regimen. Survival was estimated by the Kaplan–Meier method and cumulative incidences of neurologic death, and local and distant brain failure were estimated using death as a competing risk. Results The median overall survival (OS) of patients treated with immunotherapy vs without was 15.9 (95% CI: 13.3 to 24.8) vs 6.1 (95% CI: 5.1 to 8.8) months (P < .01). The 1-year cumulative incidence of neurologic death was 9% in patients treated with immunotherapy vs 23% in those treated without (P = .01), while nonneurologic death was not significantly different (29% vs 41%, P = .51). Median brain metastasis velocity (BMV) did not differ between groups, and rates of salvage SRS and WBRT were similar. Conclusions The use of immunotherapy in patients with lung cancer or melanoma metastatic to the brain treated with SRS is associated with improved OS and decreased incidence of neurologic death.

New effective therapies are greatly needed for metastatic uveal melanoma, which has a very poor prognosis with a median survival of less than 1 y. The melanocortin 1 receptor (MC1R) is expressed in 94% of uveal melanoma metastases, and a MC1R-specific ligand (MC1RL) with high affinity and selectivity for MC1R was previously developed. Methods: The (225)Ac-DOTA-MC1RL conjugate was synthesized in high radiochemical yield and purity and was tested in vitro for biostability and for MC1R-specific cytotoxicity in uveal melanoma cells, and the lanthanum-DOTA-MC1RL analog was tested for binding affinity. Non–tumor-bearing BALB/c mice were tested for maximum tolerated dose and biodistribution. Severe combined immunodeficient mice bearing uveal melanoma tumors or engineered MC1R-positive and -negative tumors were studied for biodistribution and efficacy. Radiation dosimetry was calculated using mouse biodistribution data and blood clearance kinetics from Sprague–Dawley rat data. Results: High biostability, MC1R-specific cytotoxicity, and high binding affinity were observed. Limiting toxicities were not observed at even the highest administered activities. Pharmacokinetics and biodistribution studies revealed rapid blood clearance (

INTRODUCTION. We hypothesized that engineering a combined lymph node/melanoma organoid from the same patient would allow tumor, stroma, and immune system to remain viable for personalized immunotherapy screening. METHODS. Surgically obtained matched melanoma and lymph node biospecimens from the same patient were transferred to the laboratory and washed with saline, antibiotic, and red blood cell lysis buffer. Biospecimens were dissociated, incorporated into an extracellular matrix (ECM)-based hydrogel system, and biofabricated into three dimensional (3D) mixed melanoma/node organoids. Cells were not sorted, so as to preserve tumor heterogeneity, including stroma and immune cell components, resulting in immune-enhanced patient tumor organoids (iPTOs). Organoid sets were screened in parallel with nivolumab, pembrolizumab, ipilimumab, and dabrafenib/trametinib for 72 h. LIVE/DEAD staining and quantitative metabolism assays recorded relative drug efficacy. Histology and immunohistochemistry were used to compare tumor melanoma cells with organoid melanoma cells. Lastly, node-enhanced iPTOs were employed to activate patient-matched peripheral blood T cells for killing of tumor cells in naïve PTOs. RESULTS. Ten biospecimen sets obtained from eight stage III and IV melanoma patients were reconstructed as symbiotic immune/tumor organoids between September 2017 and June 2018. Successful establishment of viable organoid sets was 90% (9/10), although organoid yield varied with biospecimen size. Average time from organoid development to initiation of immunotherapy testing was 7 days. In three patients for whom a node was not available, it was substituted with peripheral blood mononuclear cells. iPTO response to immunotherapy was similar to specimen clinical response in 85% (6/7) patients. In an additional pilot study, peripheral T cells were circulated through iPTOs, and subsequently transferred to naïve PTOs from the same patient, resulting in tumor killing, suggesting a possible role of iPTOs in generating adaptive immunity. CONCLUSION. Development of 3D mixed immune-enhanced tumor/node organoids is a feasible platform, allowing individual patient immune system and tumor cells to remain viable for studying of personalized immunotherapy response.

433 Background: Single agent checkpoint inhibition is effective in a small proportion of platinum-refractory UC patients but improvements are needed. UC is highly inflammatory, and low-dose chemotherapy may enhance the response to immunotherapy. We evaluated whether combination therapy with low-dose paclitaxel and pembrolizumab is more efficacious than single-agent pembrolizumab which had an objective response rate (ORR) of 21% in a similar patient population in the KEYNOTE-045 study. We also incorporated multiple novel biomarker studies to explore immune regulatory mechanisms in UC. Methods: This is a prospective, single-arm phase II trial (NCT02581982) of pembrolizumab combined with low-dose paclitaxel in patients with platinum-refractory metastatic UC. Key inclusion criteria included measurable progression of disease within 12 months of platinum therapy and ECOG ≤1. Patients received pembrolizumab 200mg day 1 and paclitaxel 80 mg/m2 days 1 and 8 of a 21 day cycle for up to 8 cycles unless clinical or radiographic disease progression or unacceptable adverse events (AEs) were observed. Responding patients could remain on pembrolizumab maintenance for up to 2 years. The primary endpoint was ORR; key secondary endpoints included overall survival (OS), 6-month progression free survival (PFS), and safety. Results: Twenty-seven patients were treated between 4/2016 - 6/2020, with a median follow up of 9.9 months. At baseline, the median age was 68 years (range 49-80), with 81% men and 78% non-Hispanic white. The majority (59%) were ECOG 1. Twenty-one of 27 (78%) received prior definitive therapy: chemoradiation in 24% and surgery in 76%. The majority (78%) of patients received prior cisplatin. 70% progressed on a cisplatin-based regimen while 30% progressed on carboplatin-based regimen within 12 months of study entry. The ORR by intention to treat was 9 of 27 patients (33%) and in patients evaluable for response by imaging was 9 of 25 (36%), including 3 with complete response. Disease control rate in evaluable patients was 72%. Six-month PFS was 46.8% (95% CI: 27.2%, 64.2%) and median OS was 11.7 months (95% CI: 8.7 mo, NR). Common ≥ grade 2 AEs were anemia (44%), lymphopenia (37%), hyperglycemia (33%), and fatigue (33%). Possible treatment-related at least grade 3 or 4 AEs occurred in 56% of subjects, including 2 immune-mediated AEs (pneumonitis and nephritis) resulting in therapy cessation but a durable partial response. There were no grade 5 events. Conclusions: This study illustrates that the addition of low-dose paclitaxel to pembrolizumab improves outcomes in patients with platinum-refractory UC, relative to single-agent pembrolizumab. No unanticipated safety signals emerged. Exploratory analyses including PDL1 status, tumor mutational burden, and change in circulating microRNAs and in immune cell populations are ongoing. Clinical trial information: NCT02581982.

Though treatment with immune checkpoint blockage (ICB) has greatly improved clinical outcomes, not all patients respond to this treatment. Being able to predict which patients are likely to respond would be a significant clinical advance. Thus there remains a need for predictive biomarkers to determine in advance those with the most potential to benefit from immune checkpoint blockade. To date, most biomarkers of response are identified in tumor tissue, whereas biomarkers that can be assessed from peripheral blood are more desirable, due to the ease of access and reproducibility of sampling. To identify biomarkers associated with ICB response from peripheral blood, we apply single cell RNA sequencing to 24 peripheral blood mononuclear cell (PBMC) samples, collected from 12 stage III and IV melanoma patients before and after treatment with anti-PD1 monotherapy. Among these 12 patients, 6 are responders and 6 are non-responders. After quality control, a total of 62,273 single cells remains for further analysis. To define the cell type landscape in an unbiased manner, unsupervised clustering is used. We identify 20 robust cell clusters, including two B cell clusters (B-cell_1; B-cell_2), three myeloid clusters (monocytes, myeloid-derived suppressor cells, and M2 macrophages), twelve clusters enriched for T/NK cells, two clusters of platelets, and Hematopoietic Stem and Progenitor Cells (HSPCs). Across all patients, cell composition differs between pre- and post-treatment samples. Strikingly, responders have significant greater proportions of B-cell_1 cells in their pre-treatment samples than non-responders. At gene level, our analysis identifies an individual overexpressed marker in B-cell_1, i.e. IGLC3, which is highly enriched in responders than non-responders before treatment. In summary, our analysis identifies specific cell type and gene within patients’ PBMC samples that can serve as predictive biomarkers for patient’s response to ICB. Citation Format: Qianqian Song, Elizabeth Forbes, Lance D. Miller, Pierre L. Triozzi, Liang Liu, Wei Zhang, David R. Soto-Pantoja. Single-cell liquid biopsy reveals circulating heterogeneity and converging subpopulations in relation to immunotherapy response in melanoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-127.

The combination of standard-dose chemotherapy and immunotherapy has been shown to be beneficial for patients with non-small cell lung cancer (NSCLC) with good performance status (PS). However, treatment options for patients with poor PS are limited. In the present study, the feasibility and immunological effects of low-dose chemotherapy with carboplatin and paclitaxel combined with immunotherapy with pembrolizumab were examined in patients with metastatic NSCLC and a poor PS. Patients with advanced NSCLC and a PS of 2 were randomized to single-agent pembrolizumab at 200 mg every 3 weeks or pembrolizumab combined with weekly carboplatin area under the curve 1 and paclitaxel 25 mg/m2. Blood for circulating immune cell phenotyping, soluble program death ligand 1 (sPD-L1) and immune-modulatory microRNAs (miRNAs) was collected prior to treatment and at weeks 4 and 7. Ten patients were randomized to the combination arm and 10 to the single-agent arm. Therapy was well tolerated. Four patients discontinued carboplatin due to hypersensitivity reactions but continued pembrolizumab and paclitaxel treatments. Increases in activated CD4+ T cells and in immune-regulatory miRNA, and decreases in myeloid derived suppressor cells were observed in the blood of patients in the combination arm and not in the single-agent arm. Changes in circulating regulatory T cells and sPD-L1 were not observed. Seven patients in the combination arm manifested a partial response compared with only two in the single-agent arm. Weekly low-dose chemotherapy carboplatin and paclitaxel was well tolerated and immunologically active when combined with pembrolizumab in patients with advanced NSCLC and a PS of 2. This combination merits further study in this patient population.

Sarcoma is comprised of a heterogeneous group of tumors originating from the mesenchyme. Sarcoma is also the first tumor that responded to immunotherapeutic agents often termed as “Coley’s toxins.” However, immunotherapy is yet to establish its presence in sarcomas. Complex interactions between tumor and immune cells in the tumor microenvironment play a crucial role in response to immunotherapy. There is a dynamic equilibrium created by the immune cells infiltrating the tumor, and this forms the basis of tumor evasion. Manipulating the intratumoral microenvironment will help overcome tumor evasion.

Mounting evidence supports a role for the immune system in breast cancer outcomes. The ability to distinguish highly immunogenic tumors susceptible to anti-tumor immunity from weakly immunogenic or inherently immune-resistant tumors would guide development of therapeutic strategies in breast cancer. Genomic, transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohorts were used to examine statistical associations between tumor mutational burden (TMB) and the survival of patients whose tumors were assigned to previously-described prognostic immune subclasses reflecting favorable, weak or poor immune-infiltrate dispositions (FID, WID or PID, respectively). Tumor immune subclasses were associated with survival in patients with high TMB (TMB-Hi, P

Cerenkov luminescence imaging (CLI) is a relatively new imaging modality that utilizes conventional optical imaging instrumentation to detect Cerenkov radiation derived from standard and often clinically approved radiotracers. Its research versatility, low cost, and ease of use have increased its popularity within the molecular imaging community and at institutions that are interested in conducting radiotracer-based molecular imaging research, but that lack the necessary resources and infrastructure. Here, we provide a description of the materials and procedures necessary to conduct a Cerenkov luminescence imaging experiment using a variety of imaging instrumentation, radionuclides, and animal models.

Introduction The roles of early whole brain radiotherapy (WBRT) and upfront stereotactic radiosurgery (SRS) alone in the treatment of melanoma patients with brain metastasis remain uncertain. We investigated the volumetric kinetics of brain metastasis development and associations with clinical outcomes for melanoma patients who received upfront SRS alone. Methods Volumetric brain metastasis velocity (vBMV) was defined as the volume of new intracranial disease at the time of distant brain failure (DBF) for the first DBF (DBF1) and second DBF (DBF2) averaged over the time since initial or most recent SRS. Non-volumetric brain metastasis velocity (BMV) was calculated for comparison. Results Median overall survival (OS) for all patients was 7.7 months. Increasing vBMVDBF1 was associated with worsened OS (hazard ratio (HR): 1.10, confidence interval (CI): 1.02 - 1.18, p = .01). Non-volumetric BMVDBF1 was not predictive of OS after DBF1 (HR: 1.00, CI: 0.97 - 1.02, p = .77). Cumulative incidence of DBF2 at three months after DBF1 was 50.0% for vBMVDBF1 > 4 cc/yr versus (vs) 15.1% for vBMVDBF1 ≤ 4 cc/yr, (Gray’s p-value = .02). Cumulative incidence of salvage WBRT at three months after DBF1 was 50.0% for vBMVDBF1 > 4 cc/yr vs 2.3% for vBMVDBF1 ≤ 4 cc/yr (Gray’s p-value < .001). Conclusion In melanoma patients with brain metastasis, volumetric BMV was predictive of survival, shorter time to second DBF, and the need for salvage WBRT. Non-volumetric BMV, however, did not predict for these outcomes, suggesting that vBMV is a stronger predictor in melanoma.

Purpose To report outcomes and complications of fine-needle aspiration biopsy (FNAB) of uveal melanoma performed for diagnostic and prognostic purposes. Methods Prospective interventional case series of 150 consecutive patients with a clinical diagnosis of uveal melanoma. The FNAB approach (transcorneal (TCO), transscleral (TSC) and transvitreal (TVT) was primarily determined by the location of the tumour. The FNAB was performed using a 25-gauge needle using a previously published technique. Prognostication was done using fluorescent in situ hybridisation detection of monosomy of chromosome 3. Results FNAB was obtained via TCO (8), TSC (71) and TVT (64) approach and impression smear in seven cases. Diagnostic yield was 92%. False-negative results were observed in 8%. Diagnostic yield was significantly correlated to biopsy approach (TCO 100%, TSC 96%, TSV 86%; p=0.029) and tumour size (basal diameter >5.0 mm; height >2.5 mm). Persistent haemorrhage (subretinal haemorrhage or vitreous) requiring surgical intervention (1%) and rhegmatogenous retinal detachment (1%) were rare. Endophthalmitis, hypotony, tumour recurrence, episcleral seeding were not observed over the average follow-up of 37 months. Prognostication could be performed in 85% of cases. Overall, only 47% of eligible patients enrolled into the adjuvant therapy trial. Conclusions FNAB for uveal melanoma with 25-gauge needle is a safe procedure that can yield diagnostic and prognostic information in vast majority of cases (92% and 85%, respectively). Even so, only about half of the eligible cases eventually enrolled into the adjuvant therapy trial. Possibility of negative FNAB yield should be considered when counselling patients with small tumours. Alternative means of diagnostic biopsy and methods of prognostication need to be assessed for small tumours.

Talimogene laherparepvec is a genetically modified herpes simplex virus-1-based oncolytic immunotherapy for the local treatment of unresectable cutaneous, subcutaneous, and nodal tumors in patients with melanoma recurrence following surgery. We aim to describe the safety of talimogene laherparepvec. Intralesional talimogene laherparepvec was administered at less than or equal to 4 ml×10 PFU/ml at protocol day 1, then less than or equal to 4 ml×10 PFU/ml 21 days later, and then every 14 days. Treatment continued until complete response, absence of injectable tumors, progressive disease, intolerance, or US Food and Drug Administration approval. Adverse events were graded during and 30 days after the end of treatment. Lesions suspected to have herpetic origin were tested for talimogene laherparepvec DNA by quantitative PCR (qPCR). Between September 2014 and October 2015, 41 patients were enrolled with stage IIIB (22%), IIIC (37%), IVM1a (34%), IVM1b (5%), and IVM1c (2%) melanoma. The median age was 72 (range: 32-96) years and 54% of the patients were men. Patients had an ECOG performance status of 0 (68%) or 1 (32%). The median treatment duration was 13.1 (3.0-41.1) weeks. Treatment-related adverse events of greater than or equal to grade 3 were reported in three (7.3%) patients and included vomiting, upper abdominal pain, chills, hyperhidrosis, nausea, pyrexia, and wound infection. Suspected herpetic lesions were swabbed in five (12%) patients. One of the five tested positive for talimogene laherparepvec DNA by qPCR, but this lesion had been injected previously with talimogene laherparepvec. During the study, five patients completed treatment because of complete response per investigators. In the clinical practice setting, talimogene laherparepvec has a safety profile comparable to that observed in previous clinical trials. Talimogene laherparepvec (IMLYGIC) is now approved in the US, European Union, and Australia.

The survival of patients with uveal melanoma remains poor because of the development of metastatic disease. Adjuvant therapy after treatment of the primary tumor has been tested but has not been shown to prevent the development of metastasis. Several new approaches are being developed. Cytotoxic and immunotherapeutic regimens are being more rationally applied using tumor genetic criteria to better identify patients at risk. Trials in the adjuvant setting of novel immunotherapeutic and targeted agents active in the metastatic setting are being developed, as are approaches to promote cellular differentiation and dormancy. The rarity and biology of uveal melanoma present challenges. Participation in well-designed, scientifically sound clinical trials is critical.

Circulating tumor cells have emerged as prognostic biomarkers in the treatment of metastatic cancers of epithelial origins viz., breast, colorectal and prostate. These tumors express Epithelial Cell Adhesion Molecule (EpCAM) on their cell surface which is used as an antigen for immunoaffinity capture. However, EpCAM capture technologies are of limited utility for non-epithelial cancers such as melanoma. We report a method to enrich Circulating Melanoma Cells (CMCs) that does not presuppose malignant cell characteristics. CMCs were enriched by centrifugation of blood samples from healthy (N = 10) and patient (N = 11) donors, followed by RBC lysis and immunomagnetic depletion of CD45-positive leukocytes in a specialized magnetic separator. CMCs were identified by immunocytochemistry using Melan-A or S100B as melanoma markers and enumerated using automated microscopy image analyses. Separation was optimized for maximum sensitivity and recovery of CMCs. Our results indicate large number of CMCs in Stage IV melanoma patients. Analysis of survival suggested a trend toward decreased survival with increased number of CMCs. Moreover, melanoma-associated miRs were found to be higher in CMC-enriched fractions in two patients when compared with the unseparated samples, validating this method as applicable for molecular analyses. Negative selection is a promising approach for isolation of CMCs and other EpCAM -negative CTCs, and is amenable to molecular analysis of CMCs. Further studies are required to validate its efficacy at capturing specific circulating cells for genomic analysis, and xenograft studies.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer. The Merkel cell polyomavirus (MCPyV) is implicated in its pathogenesis. Immune mechanisms are also implicated. Patients who are immunosuppressed have an increased risk. There is evidence that high intratumoral T-cell counts and immune transcripts are associated with favorable survival. Spontaneous regressions implicate immune effector mechanisms. Immunogenicity is also supported by observation of autoimmune paraneoplastic syndromes. Case reports suggest that immune modulation, including reduction of immune suppression, can result in tumor regression. The relationships between MCPyV infection, the immune response, and clinical outcome, however, remain poorly understood. Circulating antibodies against MCPyV antigens are present in most individuals. MCPyV-reactive T cells have been detected in both MCC patients and control subjects. High intratumoral T-cell counts are also associated with favorable survival in MCPyV-negative MCC. That the immune system plays a central role in preventing and controlling MCC is supported by several observations. MCCs often develop, however, despite the presence of humoral and cellular immune responses. A better understanding on how MCPyV and MCC evade the immune response will be necessary to develop effective immunotherapies.

Natural killer (NK) cells are implicated in the control of metastasis in uveal melanoma, a process that has been ascribed to its cancer stem cell subpopulation. NK cell activation is regulated by specific microRNA (miR). The NK cell sensitivity and regulatory miR production of uveal melanoma cancer stem cells was examined. Cancer stem cells enriched from aggressively metastatic MUM2B uveal melanoma cells by selecting CD271(+) cells or propagating as non-adherent spheres in stem-cell supportive were more resistant to NK cell cytolysis than cancer stem cells enriched from less aggressively metastatic OCM1 uveal melanoma cells. Both MUM2B and OCM1 cells expressed and secreted NK cell regulatory miRs, including miR 146a, 181a, 20a, and 223. MUM2B cells expressed and secreted miR-155; OCM1 cells did not. Transfecting MUM2B cells with anti-miR-155 increased NK cell sensitivity. CD271(+) cells were identified in the blood of patients with metastatic uveal melanoma and were characterized by low expression of melanocyte differentiation determinants and by the ability to form non-adherent spheres in stem-cell supportive media. These cells also expressed NK cell regulatory miRs, including miR-155. These results indicate that uveal melanoma cancer stem cells can vary in their sensitivity to NK cell lysis and their expression of NK cell regulatory miRs. Circulating CD271(+) cells from patients with metastatic uveal melanoma manifest cancer stem cell features and express miRs associated with NK cell suppression, including miR-155, that may contribute to metastatic progression.

Recombinant human (rh) granulocyte/macrophage colony stimulating factor (GM-CSF) has demonstrated antitumor immunologic activity in prostate and other cancers. Dosing has been empiric, and biomarkers of effect have not been established. Eight patients with biochemical relapse of prostate cancer were randomized into group A, in which they received rhGM-CSF at 250 µg/m days 1-14 of a 28-day cycle, and 8 were randomized into group B, in which they received 250 µg 3 times a week continuously. Blood dendritic cell (DC), immune suppressor cell, cytokine, and microRNA (miR) levels were examined using flow cytometric, enzyme-linked immunosorbent, and/or polymerase chain reaction-based assays. Group A had greater increases in myeloid DC and in granulocyte and monocytes. In croup B, plasmacytoid DC decreased. In group A, DC production of interleukin-12 relative to interleukin-10 decreased; this ratio increased in group B. Increases in myeloid-derived suppressor cells were observed in both the groups with increases greater in group A. Increases in regulatory T cells and in serum tumor necrosis and vascular endothelial growth factors were only observed in group A. Serum miR-155 decreased in group A. An increase in serum miR-223 and a decrease in miR-125b and miR-146a were observed in group B. The dosing of rhGM-CSF influences immune and miR effects. More DC activation and fewer myeloid-derived suppressor and regulatory T cells are observed when administered at lower doses intermittently and continuously compared with when administered at higher doses daily and cyclically. Serum levels of miRs are potentially useful biomarkers of these effects.

Uveal melanoma disseminates hematogenously, and blood biomarkers may be useful for prognosis and for monitoring disease progression. Melanoma-associated, metastatic and immune factors have been measured in the blood of patients with uveal melanoma, as have circulating melanoma cells. Most of the biomarkers were derived from studies in cutaneous melanoma. For various biological and/or technical reasons, these assessments have not demonstrated the accuracy required for effective prognostic or monitoring assays. Advances in uveal melanoma genomics and proteomics have generated many candidate biomarkers that are potentially measurable in blood. Measuring circulating nucleic acids may also be possible. Improvements in molecular profiling techniques that accurately predict metastatic risk in uveal melanoma patients should facilitate biomarker discovery and aid implementation in clinical testing. The stage is set to translate the advances made in understanding the molecular characteristics of uveal melanoma in order to identify and test clinically useful blood biomarkers of tumor dissemination and/or progression.

Antibody, small molecule and protein inhibitors of angiogenesis are used in the management of several cancers. These do not specifically target tumor vascularity, and resistance can be problematic. VB-111 is a vascular-targeting agent consisting of a non-replicating adenovirus vector with a pre-proendothelin-1 promoter that encodes an apoptotic receptor.The rationale and design of VB-111, its mechanism of action, and preclinical studies examining antitumor activity, toxicology and pharmacodynamics are reviewed. Phase I and Phase II clinical trials are also reviewed.VB-111 is a vascular-targeting gene therapeutic that is both tissue- and condition-specific, with effects limited to endothelial cells undergoing angiogenesis. Systemic administration produces selective destruction of tumor vascularity. Synergistic antitumor activity can be observed when combined with chemotherapy. VB-111 has been found to be safe and well tolerated in a Phase I clinical trial in patients with advanced solid tumors. Phase II clinical trials are in progress. VB-111 is novel agent for cancer that may have application as monotherapy and in combination with other therapies.

Apoptosis genes, such as TP53 and p16/CDKN2A, that mediate responses to cytotoxic chemotherapy, are frequently nonfunctional in melanoma. Differentiation may be an alternative to apoptosis for inducing melanoma cell cycle exit. Epigenetic mechanisms regulate differentiation, and DNA methylation alterations are associated with the abnormal differentiation of melanoma cells. The effects of the deoxycytidine analogue decitabine (5-aza-2'-deoxycytidine), which depletes DNA methyl transferase 1 (DNMT1), on melanoma differentiation were examined. Treatment of human and murine melanoma cells in vitro with concentrations of decitabine that did not cause apoptosis inhibited proliferation accompanied by cellular differentiation. A decrease in promoter methylation, and increase in expression of the melanocyte late-differentiation driver SOX9, was followed by increases in cyclin-dependent kinase inhibitors (CDKN) p27/CDKN1B and p21/CDKN1A that mediate cell cycle exit with differentiation. Effects were independent of the TP53, p16/CDKN2A and also the BRAF status of the melanoma cells. Resistance, when observed, was pharmacologic, characterized by diminished ability of decitabine to deplete DNMT1. Treatment of murine melanoma models in vivo with intermittent, low-dose decitabine, administered sub-cutaneously to limit high peak drug levels that cause cytotoxicity and increase exposure time for DNMT1 depletion, and with tetrahydrouridine to decrease decitabine metabolism and further increase exposure time, inhibited tumor growth and increased molecular and tumor stromal factors implicated in melanocyte differentiation. Modification of decitabine dose, schedule and formulation for differentiation rather than cytotoxic objectives inhibits the growth of melanoma cells in vitro and in vivo. Copyright © 2011 UICC. Fil: Alcazar, Oscar. Cleveland Clinic Foundation; Estados Unidos Fil: Achberger, Susan. Cleveland Clinic Foundation; Estados Unidos Fil: Aldrich, Wayne. Cleveland Clinic Foundation; Estados Unidos Fil: Hu, Zhenbo. Cleveland Clinic Foundation; Estados Unidos Fil: Negrotto, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Cleveland Clinic Foundation; Estados Unidos Fil: Saunthararajah, Yogen. Cleveland Clinic Foundation; Estados Unidos Fil: Triozzi, Pierre. Cleveland Clinic Foundation; Estados Unidos

Carcinoembryonic antigen (CEA) is a cancer vaccines' target. Several features of recombinant adeno-associated virus (rAAV) are attractive for vaccine applications. Combining other viral vector vaccines with Toll-like receptor (TLR) agonists enhances antitumor immunity. Wild-type and CEA transgenic (Tg) mice were immunized with rAAV-expressing CEA, the TLR9 agonist, oligodinucleotide (ODN)1826 and the TLR7 agonist, imiquimod. Mice were challenged with MC38 colon tumor cells and MC38 cells expressing CEA. rAAV-CEA immunization combined with ODN1826 or imiquimod enhanced CEA-specific T-helper 1 immunity and protected against tumor challenge in wild-type but not in CEA-Tg mice. In contrast, immunization with rAAV-CEA in CEA-Tg mice could abrogate the antitumor effects of ODN1826 and promote tumor growth. Compared to wild-type, CEA-Tg mice were characterized by a greater myeloid suppressor cell and T-helper 2 response to TLR agonists and to syngeneic tumors. Depleting PDCA1(+) plasmacytoid dendritic cells and Gr1(+) myeloid cells increased anti-CEA immune responses in CEA-Tg mice to rAAV-CEA-ODN1826 immunization, whereas depleting CD25(+) T cells did not. There are differences in the response of wild-type and CEA-Tg mice to rAAV-CEA, TLR agonists and syngeneic tumor. In CEA-Tg mice, tumor growth can be promoted with rAAV-CEA and TLR agonists. Dendritic and myeloid cells play a regulatory role.

Interferon (IFN)-β in preclinical studies, compared to IFN-α2, bound with higher affinity to its receptor, induced to higher levels of IFN-stimulated gene products, induced more apoptosis in melanoma cells, and had antitumor effects against melanoma. A maximally tolerated dose of 12 × 10(6) international units/m(2) after 2 weeks subcutaneously daily with dose escalation to 18 × 10(6) international units/m(2) was thus used in a phase II trial of IFN-β1a in cutaneous metastatic melanoma (n = 17) and uveal melanoma (n = 4). It resulted in expected but reversible drug-related severe (grade 3) adverse events in 13/21 patients; anorexia and fatigue were mostly of mild or moderate severity and infrequently needed dose reduction. Although a single patient had a sustained regression, overall IFN-β1a did not have clinical benefit (response rate10%; median progression-free survival 1.8 months). Effective and potent induction in peripheral blood cells and into serum of products of IFN-stimulated genes such as the pro-apoptotic cytokine, TRAIL, and the immunomodulatory and anti-angiogenic chemokines, CXCL10 and CCL8, confirmed gene regulatory actions. To probe further anti-angiogenic mechanisms, both VEGF-A and CXCL-5 were assessed; compared to before treatment, both proteins decreased. Continued improvements in understanding of antitumor mechanisms will enhance usefulness of IFNs for nodal or distant metastases from melanoma.

Immunotherapeutics have been applied intratumorally to manage accessible lesions and to induce systemic immunity in malignant melanoma. Intratumoral bacillus Calmette-Guérin (BCG) has been used for 40 years, and intratumoral BCG, IL-2, IFN-α and imiquimod are recommended as treatment options for patients with in-transit melanoma metastases. Regression of cutaneous metastases can be achieved. Subcutaneous metastases are more refractory, and regression of uninjected, visceral metastases is infrequent. Other microbial products, cytokines, chemicals, immune cells, antibody and viral and plasmid vectors expressing immunologically active molecules have been tested. Antitumor activity has not been demonstrated to be superior to that of intratumoral BCG. There are few controlled trials, and whether survival is impacted with any approach has not yet been established. The immunotherapeutics applied and the intratumoral administration procedure itself can activate responses that are immune inhibitory. More rigorous clinical testing and improved understanding and modulation of regulatory immune responses are necessary.

Cycloxygenase-2 (COX-2) is an enzyme involved in prostaglandin E2 (PGE(2)) synthesis associated with higher renal cell carcinoma stage. COX-2 inhibition enhances interferon (IFN-α) anti-tumor immune effects in pre-clinical models. A phase II trial of celecoxib and IFN-α in a targeted population of metastatic renal cell carcinoma patients with maximal COX-2 expression was conducted.Cytokine-naive metastatic renal cell carcinoma patients with tumors expressing ≥10% maximal COX-2 staining by immunohistochemistry received IFN-α 5 million units daily and celecoxib 400 mg orally twice daily in an open-label, single-arm phase II trial.There were 3 partial responses among 17 patients (objective response rate 18%; 95% confidence interval, 4-43%). Time to progression was 5.6 months. Increased tumor staining 3+ for COX-2 was associated with increased baseline peripheral blood PGE(2) levels, and these patients demonstrated less PGE(2) decrease with therapy. Patients with more 3+ COX-2 staining had significantly more CD3(+) (p = 0.004) and CD4(+) (p = 0.002) IFN-γ T cells at baseline and a significantly greater decrease in these cells with therapy.Celecoxib plus IFN-α in renal cell carcinoma (RCC) patients with maximally staining COX-2 tumors does not significantly enhance overall response rates over IFN monotherapy.COX-2-expressing RCC demonstrates inherent immunosuppression. COX-2 inhibition with IFN results in minimal immunomodulation and no augmented clinical activity in RCC.

Despite advances in the diagnosis and local tumor control, the overall mortality rate for uveal melanoma remains high because of the development of metastatic disease. The clinical and histopathological systems currently being used to classify patients are not sufficiently accurate to predict metastasis. Tumor genotyping has demonstrated significant promise but obtaining tumor tissue can be problematic. Furthermore, assessment of tumor tissue does not indicate whether tumor cells have actually been shed and cannot indicate whether treatment is reducing metastasis. The detection of circulating tumor cells in blood has been shown to be a prognostic biomarker that can be used to monitor the effectiveness of therapy in patients with metastatic carcinoma. Uveal melanoma disseminates hematogenously, and the detection of circulating melanoma cells may potentially be useful for diagnosis, risk stratification, and the monitoring of disease progression and treatment efficacy. PCR-based and immunomagnetic cell isolation techniques, derived from studies in patients with cutaneous melanoma, have been tested. For various biological and technical reasons, they have not demonstrated the accuracy and reproducibility required for an effective prognostic assay in patients with uveal melanoma. Assessments have been confounded by false positives and negatives and thus, correlations between circulating melanoma cells and survival have not yet been established. Circulating melanoma cell detection is a valuable tool for investigating metastasis in uveal melanoma and also has the potential to become a standard part of uveal melanoma management. However, more research on the biology of uveal melanoma as well as improvements upon the current technologies are needed.