Citations

Abstract Background: The metastatic advancement of melanoma is largely dependent on its ability to evade immunity and gain access to the lymphatics. Characterization of tumor-draining, sentinel lymph nodes in patients has identified the nodes to express an immunosuppressed, Th2-dominant immune profile. Importantly, this profile is observed in sentinel lymph nodes even prior to evidence of metastasis supporting a model in which melanoma-derived factors prime tumor-draining, sentinel lymph nodes for tumor progression. Recent work in our lab has characterized the cellular interactions and the contribution of soluble factors in facilitating this immunosuppressive immune profile in fresh sentinel lymph nodes of patients; however, an appreciation for the role of subcellular counterparts remains to be explored. In this study, we considered a novel role for melanoma-derived, membrane-bound nanoparticles as drivers in polarizing immunity towards an immunosuppressive, tumor-promoting state. Methods and Results: Utilizing recent technology advances in Nanoparticle Tracking Analysis (NTA), characterization of melanoma derived vesicles from human melanoma cell lines showed a size distribution of particles ranging from 30nm to 1um, representing both exosomes and microvesicles. Following hypoxic culture, vesicle production was amplified, suggesting the increasingly hypoxic tumor microenvironment may trigger vesicle secretion. To evaluate the immune regulating potential of melanoma-derived vesicles, proteomic analysis of vesicle cargo by mass spectrometry identified over 100 proteins including known immune regulators Galectin 3, MHC I, ICAM1 and MIF. Functional assessment revealed that dendritic cells matured in vitro in the presence of melanoma-derived vesicles have significantly reduced surface marker expression of co-stimulatory markers CD80 and CD86 compared to controls. In addition to an activation defect, microarray analysis of dendritic cells matured in the presence of melanoma vesicles displayed a unique RNA expression profile. Pathway analysis of the expression arrays identified numerous overlapping signaling pathways involving proteins present in the melanoma-derived vesicles by mass spectrometry suggesting tumor-derived vesicle cargo has the ability to modulate dendritic cell function. Conclusion: Taken together, this study demonstrates a critical function for melanoma-derived vesicles in driving immunosuppression by characterizing their generation, protein cargo and immune regulating properties. The enrichment of melanoma-derived vesicles for immune mediators and ability of the vesicles to suppress dendritic cell function suggests a mechanism by which regional immunosuppression precedes nodal involvement and may foster an environment for metastasis to occur. Moving forward, we are currently translating these findings into fresh sentinel lymph nodes obtained from patients in order to elucidate the role of melanoma-derived vesicles in metastasis. Understanding this mechanism may yield valuable insight as we seek to develop efficacious immunotherapies targeting nodal metastasis. Citation Format: Rachel Goldenstein, Wendy Nevala, Svetomir N. Markovic. Melanoma-derived nanovesicles as drivers of immunosuppression. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A70.

Abstract Introduction: Galectin-9 (Gal-9) is a carbohydrate-binding protein that interacts with T-cell immunoglobulin mucin 3 (TIM3) to modulate immune homeostasis. The Gal-9/TIM3 interaction down regulates activated T cells which promotes tumor growth, much like program death 1 (PD-1) and its ligand (PD-L1). Still, the effect Gal-9 has on the immune environment of melanoma patients is weakly understood. This study focuses on the role of Gal-9 in immunoregulation of healthy persons and metastatic melanoma patients. Methods: Soluble Gal-9 levels were determined from plasma collected from patients and healthy persons using standard ELISA methods. Proliferation and apoptosis of cells in the presence and absence of Gal-9 was determined by mixed lymphocyte reactions and Tunnel staining. Supernatants from these reactions were collected and run using a cytokine bead assay to determine changes in immune parameters with and without Gal-9. Results: Soluble Gal-9 was significantly higher in metastatic melanoma patients compared to healthy controls (3929 vs. 877 pg/mL, p=0.0003 respectively). In the presence of Gal-9, the proliferative effect of an allostimulus could be overridden until the level of responder cells to stimulator cells was 4:1. Using an apoptosis assay, Gal-9 was found to increase the level of cellular apoptosis; however, apoptosis was decreased as the allostimulus increased. Healthy cells incubated with Gal-9 were found to increase secretion of growth factors (EGF and FGF-2), chemokines (CXCL1, CXCL8, CCL2 and CCL3), T helper 1 cytokines (IFN-g and IL-7) which have shown tumor promoting affects and T helper 2 cytokines (IL-13 and IL-4). Conclusions: Gal-9 may potentially play a role in suppressing anti-tumor immunity. This suggests the use of a Gal-9 neutralizing antibody for therapeutic benefit in melanoma. Citation Format: Elizabeth Ann L. Enninga, Wendy K. Nevala, Svetomir N. Markovic. Galectin-9 has tumor promoting properties that may be targetable in metastatic melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A41.

Abstract The success of clinical trials for patients with malignant melanoma (MM) designed from the “promising” results using pre-clinical models is minimal. With its increase in incidence, there is a dire need to develop both pre-clinical models comparable to clinical practice that have the capability to translate from bench-to-bedside and novel therapeutic agents for this life-threatening form of skin cancer. To address this concern, we have developed a panel of pre-clinical MM models including patient matched cell lines, patient cell-line derived xenograft models and patient tumor tissue-derived xenograft models. Patient derived xenograft (PDX) models are purported to most closely replicate a patient's response to therapy. Since the development of patient-derived cell lines and tumors grown in mice were more efficacious, their genetic, phenotypic, and therapeutic responses were compared to those of their corresponding patient-matched PDX model. Each model was molecularly characterized to confirm a match to that of the originating patient tumor tissue via protein immunohistochemistry (IHC), mRNA quantitative polymerase chain reaction (qPCR), DNA short tandem repeat (STR) analysis, and Sanger sequencing. Each pre-clinical model provided a genetic match to their respective patient tumor sample. All pre-clinical models were positive for the melanoma-specific markers (S-100 beta, HMB-45, and Melan-A) confirming a melanoma phenotype. Soft agar studies and tumor growth of our PDX and patient cell-line derived xenograft models confirmed tumorigenicity within our models providing the foundation to perform therapeutic studies. Mutation status data (i.e. BRAF, RAS), patient therapeutic clinical history and oncogene overexpression analysis (pERK, pAKT, PI3K, pmTOR, etc.) provide the platform and rationale behind the use of specific therapeutic agents per model. BRAF, NRAS and KRAS mutations were identified along with elevated pERK, pAKT and pmTOR in the models. Targeted therapies (trametinib/MEK inhibitor; vemurafenib/BRAF inhibitor; MK-2206/pAKT inhibitor; and everolimus/mTOR inhibitor) demonstrated dose dependent inhibition of cell proliferation in each of the models based upon oncogenic expression or mutations. In vivo experiments are in progress, which will allow comparison of the three models. The next steps include combination therapy to identify antitumor synergy targeting oncogenic signaling pathways. Citation Format: Antoneicka L. Harris, Laura Marlow, Adam Mathias, Louis Dawson, William Durham, Kenneth Meshaw, Robert Mullin, Daniel Small, Aidan Synnott, Kevin Wu, Dragana Milosevic, Brian Netzel, Stefan Grebe, Svetomir Markovic, John Copland. Characterization of novel patient derived melanoma xenografts and cell lines in response to targeted therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2417. doi:10.1158/1538-7445.AM2015-2417

Abstract Targeted therapies and immunotherapies have led to significant improvements in the treatment of advanced cancers, including metastatic melanoma. However, new strategies are desperately needed to overcome therapeutic resistance to these agents, as well as to identify effective treatment approaches for cancer patients that fall outside major targetable mutational subtypes (e.g., non–V600 BRAF melanoma). One such strategy is to extend the paradigm of individually tailored, molecularly targeted therapy into a broader spectrum of melanoma patients, particularly those bearing tumors without commonly recognized therapeutic targets, as well as having failed or were ineligible for immunotherapy. In this nontreatment pilot study, next-generation sequencing (NGS) technologies were utilized, including whole genome and whole transcriptome sequencing, to identify molecular aberrations in patients with non–V600 BRAF metastatic melanoma. This information was then rationally matched to an appropriate clinical treatment from a defined pharmacopeia. Five patients with advanced non–V600 BRAF metastatic melanoma were enrolled. We demonstrated successful performance of the following during a clinically relevant time period: patient tumor biopsy, quality DNA/RNA extraction, DNA/RNA-based sequencing for gene expression analysis, analysis utilizing a series of data integration methodologies, report generation, and tumor board review with formulated treatment plan. Streamlining measures were conducted based on the experiences of enrolling, collecting specimens, and analyzing the molecular signatures of patients. We demonstrated the feasibility of using NGS to identify molecular aberrations and generate an individualized treatment plan in this patient population. A randomized treatment study utilizing lessons learned from the conduct of this pilot study is currently underway. Mol Cancer Ther; 14(8); 1962–71. ©2015 AACR.

Key Points The mTOR pathway is constitutively activated in the TCL cells and is responsible for TCL proliferation. This is first trial to demonstrate that mTORC1 inhibitors (everolimus) have substantial antitumor activity (44% overall response rate) in patients with relapsed TCL.

Several recent studies have shown differences in the maternal immune milieu at different phases of pregnancy, but most studies have been cross-sectional or of relatively few time points. Levels of 42 cytokines were determined using a multiplex bead-based assay on archived serum from a cohort of pregnant womenN=16at median of 18 time points tested, from the first trimester through to parturition, per woman. Unconditional growth modeling was then used to determine time-dependent changes in levels of these cytokines. Macrophage-derived chemokine (MDC, aka CCL22) decreases as pregnancy progresses. IL-1β, IL-6, IL-8, IL-12p70, IL-13, IL-15, IP-10, and FLT3-ligand increase as a function of gestational weeks, and IFNα2, IL-1ra, IL-3, IL-9, IL-12p40, and soluble CD40 ligand increase as a function of trimester. As pregnancy normally progresses, a maternal shift away from a type 2-biased immune response and toward an inflammatory/counterregulatory response is observed.

Perineural spread is a well-known mechanism of dissemination of head and neck malignancies. There are few reports of melanoma involving the brachial plexus in the literature. To their knowledge, the authors report the first known case of perineural spread of malignant melanoma to the brachial plexus. Clinicoradiological and anatomopathological correlation is presented, highlighting the importance of peripheral nerve communications in perineural spread.

The serum immunoglobulin free light chain (FLC) assay quantitates free kappa (κ) and lambda (λ) light chains. FLC elevations in patients with diffuse large B‐cell lymphoma (DLBCL), Hodgkin lymphoma (HL), and chronic lymphocytic leukemia (CLL) are associated with an inferior survival. These increases in FLC can be monoclonal (as in myeloma) or polyclonal. The goal was to estimate the frequency of these elevations within distinct types of B‐cell and T‐cell non‐Hodgkin lymphoma (NHL) and whether the FLC measurements are associated with event‐free survival (EFS). We studied serum for FLC abnormalities using normal laboratory reference ranges to define an elevated κ or λ FLC. Elevations were further classified as polyclonal or monoclonal. Four hundred ninety‐two patients were studied: 453 B‐cell and 34 T‐cell NHL patients. Twenty‐nine % (142/453) of patients had an elevated FLC of which 10% were monoclonal elevations. Within B‐cell NHL, FLC abnormalities were most common in lymphoplasmacytic (79%), mantle cell (68%), and lymphomas of mucosa associated lymphoid tissue (31%); they were least common in follicular (15%). The hazard ratio (HR) for EFS in all patients was 1.41 (95% CI; 1.11–1.81); in all B‐cell NHL the HR was 1.44 (95% CI 1.11–1.96); in all T‐cell NHL the HR was 1.17 (95% CI 0.55–2.49). FLC abnormalities predicted an inferior OS (HR = 2.75, 95% CI: 1.93–3.90, P < 0.0001). The serum FLC assay is useful for prognosis in both B‐cell and T‐cell types of NHL. In B‐cell NHL further discrimination between a monoclonal and polyclonal elevation may be helpful and should be analyzed in prospective clinical trials. Am. J. Hematol. 89:1116–1120, 2014. © 2014 Wiley Periodicals, Inc.

Abstract Background: The prognosis for stage III melanoma is variable, with the 5-year survival ranging from 23-87%. Previous studies have demonstrated both immune cell activation and dysfunction within the sentinel lymph node (SLN) of melanoma patients. We hypothesize that differences within the host response, specifically the immune cell profile of the SLN, may predict the heterogeneous behavior of stage III melanoma. Methods: A retrospective review of clinically node negative patients undergoing a SLN biopsy at our institution was undertaken. Thirty four SLN positive cases in which the patients also underwent a complete lymph node dissection were identified and the archived FFPE SLN blocks were obtained. A single tumor positive SLN was selected and immunohistochemistry was performed for dendritic cell markers (CD123, CD86, CD11c and HLA-DR), cytotoxic T cells (CD8), anergic T cells (PD-1) and regulatory T cells (Foxp3). Slides were digitized and analyzed for percent of the SLN staining positive for each respective marker using IHCscore. These findings were correlated with risk of further non-SLN disease in the same nodal basin as well as effect on disease-free and overall survival. Results: The SLN exhibited expression of both immune activation (CD8, CD86, and CD11c) and immune suppressive (CD123, PD-1 and Foxp3) markers. HLA-DR expression was significantly higher in females (52.07% vs. 42.32%, p=0.02). No other immune marker was associated with any baseline demographic or clinical variable. Median disease-free survival and overall survival was 6 months and not yet reached and 16 months and not yet reached for patients with and without non-SLN involvement, respectively; however, no difference was noted in any of the SLN immune markers between patients with and without non-SLN involvement. Increased CD8 expression in the SLN was associated with an improvement in disease-free survival (HR 0.94 p = 0.048) but not overall survival (HR 0.93 p = 0.07). In two variable multivariate analyses, CD8 expression remained independent for disease-free survival when adjusted for Breslow depth but not non-SLN involvement. The median disease-free survival for patients with low CD8 expression within the SLN was 19.5 months and median disease-free survival has not been reached in those with high CD8 expression in the SLN after a median of 59 months follow up (log-rank p = 0.05). Conclusion: Previous reports suggest T cell infiltration of melanoma tumors is an important prognostic factor. In this study we found that CD8 T cell accumulation within the SLN was associated with a disease-free survival advantage. Cytotoxic T cell accumulation within the SLN declines early in melanoma and the degree of CD8 depletion corresponds to the risk of disease recurrence. These findings support a key role for effector T cells in the control of melanoma progression. Citation Format: Travis E. Grotz, Aaron S. Mansfield, James W. Jakub, Svetomir N. Markovic, Tina J. Hieken. Immune cell profile of the SLN has prognostic value in stage III melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1266. doi:10.1158/1538-7445.AM2013-1266