Leontovich, Alexey A.

AbstractWomen diagnosed with cutaneous melanoma have a survival advantage compared to men, which has been hypothesized to be due to difference in behavior and/or biology (sex hormones). It remains controversial whether this advantage is dependent on age or stage of disease. We sought to compare melanoma‐specific survival between females in pre, peri, and postmenopausal age groups to males in the same age group, adjusting for stage of disease. This is a retrospective population‐based cohort study using the Surveillance, Epidemiology, and End Results (SEER) database. Patients diagnosed from 1 January 1992 through 31 January 2011 with primary invasive cutaneous melanoma were included in our cohort. Melanoma‐specific survival was the main outcome studied. Of the 106,511 subjects that were included, 45% were female. Females in all age groups (18–45, 46–54, and ≥55) with localized and regional disease, were less likely to die from melanoma compared to males in the same age group. Among patients with localized and regional disease, the relative risk of death due to melanoma increased with advancing age at diagnosis; this increase was more pronounced among females than males. In contrast, we observed no female survival advantage among patients with distant disease and no effect of age on relative risk of death from melanoma. Females with localized and regional melanoma have a decreased risk of death compared to males within all age groups. Our data show no differences in survival between men and women with metastatic melanoma, indicating that the influence of sex on survival is limited to early stage disease but not confined to pre or perimenopausal age groups.

Abstract Background: NOTCH oncogenic signaling induces epithelial-to-mesenchymal transition (EMT) and tumor stemness that play a critical role in driving tumor progression. Following interaction of NOTCH receptors (NOTCH 1, 2, 3, and 4) with their ligands (DELTA-LIKE 1, 3, 4 and JAGGED 1, 2), γ- secretase complex performs an intra-membrane cleavage releasing the NOTCH intracellular domain (NNICD). NNICD translocates to the nucleus and induces the expression of HEY1 and HES1 transcription factors that in turn orchestrate NOTCH-mediated EMT and stemness reprogramming. Materials and Methods: Parental MCF-7 and unique metastatic MCF-7RAF-1 xenografts with constitutively active MAPK signaling (D’Assoro et al., Oncogene 2014: 33:599-610) were used to define the extent to which increased expression of NOTCH signaling was linked to distant metastases. SKY analysis was performed to assess the grade of chromosomal instability (CIN) in tumor xenografts. Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to generate mammospheres. Expression of CD24 luminal marker was characterized by FACS analysis and immunofluorescence. Global transcriptome analysis was performed using Affymetrix® microarray. Highly invasive MDA-MB 231 breast cancer cells were used to perform a tail vein cancer cell-seeding assay. The LY411575 γ-secretase inhibitor (Pan-NOTCH inhibitor) was used to block NOTCH signaling in MDA-MB 231 cells. Results: Metastasis Initiating Cells (MICs) isolated from the lungs of nude mice carrying MCF-7RAF-1 xenografts exhibited a higher degree of genomic stability than primary tumor xenografts that was characterized by the absence of centrosome amplification and non-clonal chromosomal abnormalities. MICs also showed higher mammosphere forming capacity that was linked to lower expression of luminal marker CD24. Significantly, global transcriptome analysis showed that expression of NOTCH3 and HES1 stemness genes was increased in MICs. To corroborate the role of NOTCH signaling in inducing metastatic seeding, MDA-MB 231 breast cancer cells were pre-treated with 1 µM of LY411575 After 72 hours viable cells were injected into the tail vein of immune-compromised mice to develop lung metastases. While animals injected with untreated MDA-MB 231 cells developed lung metastases, those injected with the LY411575-treated MDA-MB 231 cells failed to develop lung metastatic lesions. Conclusions: These preliminary findings demonstrate the role of NOTCH stemness signaling in promoting metastatic seeding of breast cancer cells. Since NOTCH signaling is a “druggable target”, these results also provide the rationale to develop novel NOTCH-targeted therapies to inhibit tumor stemness and early onset of distant metastases. Citation Format: Alexey Leontovich, Jeffrey Salisbury, Candace L. Haddox, Lisa Mills, Tufia Haddad, James McCubrey, Matthew Goetz, James Ingle, Evanthia Galanis, Antonino B. D'Assoro. NOTCH signaling promotes metastatic seeding and growth of human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4851. doi:10.1158/1538-7445.AM2017-4851

Abstract Background: Understanding the spatial interactions between tumor and immune cells in the heterogeneous tumor microenvironment is pivotal to improve clinical outcomes of immunotherapy. Various multiplexed imaging platforms have been recently developed to visualize the different immune cell subtypes with various distribution patterns that can impact the antitumor immunity. However, the lack of tools that allow for the spatial pattern modeling remains a major barrier for systematic analysis of cellular interactions in the TME. Methods: We used multiplexed immunohistochemistry (mIHC) to understand the cellular interplays in metastatic melanoma TME using FFPE section. This platform allows in situ quantitative single cell analysis with high-level of multiplexing while preserving the tumor heterogeneity. Single cell spatial data (position and location) in the context of TME can be obtained during mIHC, enabling further spatial modeling to characterize the cellular interactions between tumor immune cells. Novel spatial analysis algorithms are developed taking advantages of these spatial data. Results: Using R programming language, we developed two novel analytical approaches to reveal the spatial distributions of various immune cell subtypes relative to tumor cells. The cell neighborhood analysis algorithm uses the “applynbd()” function to traverse through every cellular point on a two-dimensional plane and creates neighborhoods of 24um in diameter centered by a tumor cell. This algorithm captures the counts of a specific type of immune cells that are in direct contact with a tumor cell, allowing the visualization and statistical analysis of cellular attraction/repulsion patterns. The cell aggregate analysis algorithm uses a sliding-window based approach to capture cell aggregates with certain densities and the areas within the TME covered by specific cell aggregates. Combined with the Ripley K-function, the cell aggregate algorithm captures the aggregation vs. segregation pattern between different cell types, enabling the clustering and distance analysis of immune cells relative to tumor cells. Our algorithms demonstrate that tumor cells either attract CD3+ tumor-infiltrating T cells or favor the aggregation of CD20+ B cell in peri-tumor areas depends on the levels of tumor cell human leukocyte antigen-1 (HLA-1) expression. Conclusion: Our novel spatial analysis algorithms enable the modeling of different interaction patterns between tumor and immune cells (direct contact vs. aggregation) at both single cell and TME level. It demonstrates that tumor cells with heterogeneous properties can impact the immune cells’ distributions in the TME with various biological outcomes. It also establishes tools that are necessary for systematic analysis of the TME, allowing the elucidation of the “homogeneous patterns” within the heterogeneous TME. Citation Format: Yiyi Yan, Alberto Santamaria-Pang, Michael Gerdes, Fiona Ginty, Anup Sood, Svetomir N. Markovic, Alexey Leontovich. Novel algorithms for spatial modeling of cellular interactions in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 547. doi:10.1158/1538-7445.AM2017-547

Abstract The variable clinical success with immune checkpoint inhibitors in the treatment of solid tumors challenges us to further understand the regulatory mechanisms of immune cells infiltration – a critical step of tumor elimination – in a highly heterogenous tumor microenvironment (TME). Systemic analyses of the tumor-immune cell interface and interactions are of particular significance with direct clinical impact. We employed a multiplexed immunofluorescence (MxIF) method, a novel technology that allows in situ quantitative single cell characterization with high order protein multiplexing on whole tumor sections, to elucidate the key components in TME governing the immune cells' composition and function in metastatic melanoma lymph node metastases. Here, we show that the expression of tumor cell Human Leukocyte Antigen 1 (HLA-1) is highly heterogeneous both within the TME and between patients. The level of tumor HLA-1 expression directly correlates with the magnitude of cytotoxic T lymphocytes (CTLs) infiltration in the TME. In addition, decreased tumor HLA-1 expression is associated with accumulation of CD20+ cells at the edge of the tumor as well as an increased population of CD4+FOXP3+ cells. The association between tumor HLA-1 expression and immune cell distribution is highly consistent yet heterogenous across the entire tumor mass. Moreover, using quantitative single cell data, we developed algorithms for computational and spatial modeling of tumor-immune cell interfaces, allowing statistical comparison of cellular interactions in the context of tumor heterogeneity. Spatial analysis demonstrates that tumor HLA-1 expression favors CTL invasion, while lack of tumor HLA-1 expression results in CTL evasion. Furthermore, in a cohort of 166 Stage III melanoma patients, we confirm that CTLs infiltration is only present in regions with high tumor HLA-1 expressions, and that patients with tumors high in both tumor HLA-1 and CTLs have significantly improved progression free survival (PFS) compared with those with tumors low in HLA-1 and/or CTLs. Our study demonstrates that the heterogeneous tumor HLA-1 expression results in various immune cell distribution patterns in the TME, directly contributing to the varied antitumor immunities and ultimately influencing tumor outcomes. As part of the antigen presentation machinery, HLA-1 expression on tumors is necessary for CTLs infiltration and is indispensable for T cell mediated tumor elimination with direct clinical benefits. We also establish a novel platform for visualization and spatial representation of the cellular heterogeneity within the TME, providing data processing and modeling algorithms necessary for understanding the cellular interplay mediating tumor surveillance, which will ultimately improve therapeutic efficacy and clinical outcomes. Citation Format: Yiyi Yan, Alexey Leontovich, Thomas Flotte, Michael Gerdes, Fiona Ginty, Alberto Santamaria-Pang, Anup Sood, Keyur Desai, Chrystal Chadwick, Rong Zhang, Svetomir N. Markovic. Tumor HLA Class I expression influences immune cells infiltration in metastatic melanoma tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A71.

Skin cancer affects more individuals in the USA than any other malignancy and malignant melanoma is particularly deadly because of its metastatic potential. Melanoma has been recognized as one of the most immunogenic malignancies; therefore, understanding the mechanisms of tumor–immune interaction is key for developing more efficient treatments. As the tumor microenvironment shows an immunosuppressive action, immunotherapeutic agents promoting endogenous immune response to cancer have been tested (interleukin-2, anticytotoxic-T-lymphocyte-associated antigen 4, and antiprogrammed cell death protein 1 monoclonal antibodies) as well as combinations of cytotoxic chemotherapy agents and inhibitors of angiogenesis (taxol/carboplatin/avastin). However, clinical outcomes are variable, with only a minority of patients achieving durable complete responses. The variability of immune homeostasis, which may be more active or more tolerant at any given time, in cancer patients and the interaction of the immune system with the tumor could explain the inconsistency in clinical outcomes among these patients. Recently, the role of the lymphocyte-to-monocyte-ratio (LMR) in the peripheral blood has been investigated and has been proven to be an independent predictor of survival in different hematological malignancies and in solid tumors. In melanoma, our group has validated the significance of LMR as a predictor of relapse after resection of advanced melanoma. In this study, we examined the dynamics in the immune system of patients with advanced melanoma by performing serial multiday concentration measurements of cytokines and immune cell subsets in the peripheral blood. The analysis of outcomes of chemotherapy administration as related to LMR on the day of treatment initiation showed that progression-free survival was improved in the patients who received chemotherapy on the day when LMR was elevated.

Galectin-9, a β-galactoside-binding protein, is defined as a negative regulator of T helper 1 (Th1) immune responses, favoring Th2 bias. Systemic immunity in patients with metastatic melanoma is predominantly Th2 biased. We hypothesized that galectin-9 can modulate systemic immunity toward Th2 polarization in patients with advanced melanoma. The presence or concentration of galectin-9 was assessed in tumors and plasma, in patients with metastatic melanoma. The immunomodulatory function of galectin-9 was determined by exposing human peripheral blood mononuclear cells to galectin-9 in vitro. Galectin-9 was expressed in 57% of tumors and was significantly (3.6-fold) increased in the plasma of patients with advanced melanoma compared with healthy controls (P<0.001). High plasma galectin-9 concentration was associated with systemic Th2 polarization and reduced 2-year survival compared with low/no galectin-9 expression. In-vitro, galectin-9 reduced proliferation of healthy peripheral blood mononuclear cells, and promoted Th1 cell apoptosis, Th2-biased cell phenotypes, and cytokine secretion. Galectin-9 also stimulated monocyte differentiation toward an M2 macrophage phenotype, as assessed by chemokine/cytokine secretion and CD206 expression, observed both in vitro as well as in patients with metastatic melanoma. Elevated galectin-9 in patient plasma correlated with Th2 systemic bias and less favorable clinical outcomes for patients with metastatic melanoma. This Th2 bias appears to be not only a feature of the known mechanisms of Th1 apoptosis by T-cell immunoglobulin and mucin-domain containing-3 binding, but also mediated by myeloid cell differentiation toward an M2 phenotype, that favors tumor progression. These data support galectin-9 as a novel therapeutic target for patients with metastatic melanoma.

Metabolic memory (MM) is defined as the persistence of diabetic (DM) complications even after glycemic control is pharmacologically achieved. Using a zebrafish diabetic model that induces a MM state, we previously reported that, in this model, tissue dysfunction was of a heritable nature based on cell proliferation studies in limb tissue and this correlated with epigenetic DNA methylation changes that paralleled alterations in gene expression. In the current study, control, DM, and MM excised fin tissues were further analyzed by MeDIP sequencing and microarray techniques. Bioinformatics analysis of the data found that genes of theDNA replication/DNA metabolism processgroup (with upregulation of theapex1, mcm2, mcm4, orc3,lig1, anddnmt1 genes) were altered in the DM state and these molecular changes continued into MM. Interestingly, DNA methylation changes could be found as far as 6–13 kb upstream of the transcription start site for these genes suggesting potential higher levels of epigenetic control. In conclusion, DNA methylation changes in members of theDNA replication/repair processgroup best explain the heritable nature of cell proliferation impairment found in the zebrafish DM/MM model. These results are consistent with human diabetic epigenetic studies and provide one explanation for the persistence of long term tissue complications as seen in diabetes.

AbstractAs previously reported by our laboratory, streptozocin‐induced diabetes mellitus (DM) in adult zebrafish results in an impairment of tissue regeneration as monitored by caudal fin regeneration. Following streptozocin withdrawal, a recovery phase occurs to reestablish euglycemia, via pancreatic beta‐cell regeneration. However, DM‐associated impaired fin regeneration continues indefinitely in the metabolic memory (MM) state, allowing for subsequent molecular analysis of the underlying mechanisms of MM. This study focuses on elucidating the molecular basis that explains the DM‐associated impaired fin regeneration and why it persists into the MM state with the aim of better understanding MM. Using a combination of microarray analysis and bioinformatics approaches, our study found that of the 14,900 transcripts analyzed, aberrant expression of 71 genes relating to tissue developmental and regeneration processes were identified in DM fish and the altered expression of these 71 genes persisted in MM fish. Key regulatory genes of major development and signal transduction pathways were identified among this group of 71. The aberrant expression of key regulatory genes in the DM state that persist into the MM state provides a plausible explanation on how hyperglycemia induced impaired fin regeneration in the adult zebrafish DM/MM model.