Markovic, Svetomir N.

The peripheral blood lymphocyte-to-monocyte ratio (LMR) has been associated with prognosis in many malignancies including metastatic melanoma. However, it has not been studied in patients treated with immune checkpoint inhibitors. In this study, we analyzed the baseline LMR with progression-free survival (PFS) and overall survival (OS) in metastatic melanoma patients treated with pembrolizumab. A total of 133 patients with metastatic melanoma treated with pembrolizumab were included in this retrospective study. LMR was calculated from pretherapy peripheral blood counts and the optimal cutoff value was determined by a receiver operator characteristic curve. PFS and OS were evaluated using the Kaplan–Meier method and multivariate Cox proportional hazard modeling. Patients with an LMR of at least 1.7 showed improved PFS (hazard ratio=0.55; 95% confidence interval: 0.34–0.92; P=0.024) and OS (hazard ratio=0.29; 95% confidence interval: 0.15–0.59; P=0.0007). The baseline LMR is associated with PFS and OS in metastatic melanoma patients treated with pembrolizumab, and could represent a convenient and cost-effective prognostic biomarker. Validation of these findings in an independent cohort is needed.

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 Metastatic melanoma (MM) is the most aggressive type of skin cancer contributing to approximately 80% of all skin cancer related deaths. In 2016, it was estimated that more 76,000 cases would be diagnosed in the United States. The high metastatic potential and aggressive clinical behavior of this disease makes it a major health problem. As a consequence there has been notable development in for novel targeted (i.e. BRAF inhibitors) and immune therapies (i.e. anti-PD1 and CTLA4 inhibitors). However, despite improvements in patient outcomes, most patients develop resistance within 6-11 months on dual BRAF/MEK inhibition, and 5 months on immune therapy, highlighting the need to identify new therapies that improve disease management and patient survival. Increased expression of CyclinD1 has been reported to occur as a mechanism of resistance to BRAF inhibition, resulting in reactivation of the canonical activating pathway in melanoma, MAPK. Thus, we hypothesize that combined inhibition of CDK4/6 and MAPK signaling will provide a novel anti-tumor synergy drug combination superior to current standard of care in BRAF mutant patient-derived preclinical models. Using MM human tissue from patients we developed 3 BRAF mutant preclinical models investigating a novel triple therapy drug combination BRAFi/MEKi/CDK4/6i. Protein analysis and drug proliferation assays were used to design the novel combination. In vivo, we found statistically significant differences in tumor inhibition between the novel combination and the current standard of care. The triple therapy also showed promising results, in vivo, when assessed as a salvage therapy following tumor escape from BRAFi/MEKi combination treatment. Citation Format: Antoneicka L. Harris, Michael Thompson, Svetomir Markovic, William Durham, Louis Dawson, Daniel Small, John A. Copland. Tumor regression in novel triple therapy BRAF mutant metastatic melanoma patient-derived preclinical models [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 LB-130. doi:10.1158/1538-7445.AM2017-LB-130

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

Objective: To report the clinical features, treatment, and outcomes of patients with immune checkpoint inhibitor-induced hepatotoxicity. Patients and Methods: In this retrospective observational study, we identified patients with metastatic malignant melanoma seen in consultation and/or treated between March 2011 and March 2016. Hepatotoxicity was assessed using the Common Terminology Criteria for Adverse Events, v4.0. Results: Seventeen patients were identified as having any degree of hepatotoxicity by history (grade 1 to 4). Twelve of 17 were diagnosed after ipilimumab, 3 of 17 were diagnosed after pembrolizumab, and 2 of 17 after ipilimumab combined with nivolumab. Median time from first dose of immune therapy to hepatotoxicity was 52 days. Clinical symptoms were variable: asymptomatic, fatigue, myalgias, headache, abdominal pain, nausea, vomiting, confusion, and/or jaundice. Eight patients had concurrent adverse events including colitis, hypophysitis, pneumonitis, and/or rash. Immune therapy was discontinued in all patients except 3. The patients were most commonly treated with systemic corticosteroids such as prednisone. Immunosuppression was discontinued by taper over a median of 42 days; in 3 patients steroids had to be reinitiated based on clinical or laboratory worsening of liver tests. Normalization of liver tests was seen within a median of 31 days of immunosuppression initiation. One patient with grade 4 hepatotoxicity had normalization with the addition of cyclosporine. Conclusions: Melanoma patients treated with immune checkpoint inhibitors should be monitored regularly for hepatotoxicity. Treatment with discontinuation of therapy and initiation of corticosteroids is indicated with grade 3 or 4 hepatotoxicity. Cyclosporine may be beneficial in steroid-refractory hepatotoxicity.

Abstract Lessons Learned Percutaneous thermal ablation combined with in situ granulocyte-macrophage colony-stimulating factor cytokine therapy was technically feasible and well tolerated. No significant clinical or immunologic responses were seen. Background Melanoma tumor-derived heat-shock proteins (HSPs) and HSP-peptide complexes can elicit protective antitumor responses. The granulocyte-macrophage colony-stimulating factor (GM-CSF) chemokine can also promote uptake and processing by professional antigen presenting cells (APCs). On this basis, we designed a pilot study of percutaneous thermal ablation as a means to induce heat-shock protein vaccination plus GM-CSF to determine safety and preliminary antitumor activity of this combination. Materials and Methods This study was designed to assess overall safety of percutaneous ablation combined with GM-CSF for unresectable, metastatic melanoma including uveal and mucosal types. All patients received heat-shock therapy (42°C for 30 minutes), then received one of three treatments: (a) intralesional GM-CSF (500 mcg standard dose); (b) radiofrequency ablation (RFA) + GM-CSF; or (c) cryoablation plus GM-CSF. The primary endpoint of the study was the induction of endogenous HSP70 and melanoma-specific cytotoxic T lymphocytes (CTL). Results Nine patients (three per study arm) were enrolled. No dose-limiting toxicity was observed as specified per protocol. All patients developed progressive disease and went on to receive alternative therapy. Median overall survival (OS) was 8.2 months (95% confidence interval [CI] 2–17.2). The study was not powered to detect a difference in clinical outcome among treatment groups. Conclusion Percutaneous thermal ablation plus GM-CSF was well tolerated, technically feasible, and demonstrated an acceptable adverse event profile comparable to conventional RFA and cryoablation. While HSP70 was induced following therapy, the degree of HSP70 elevation was not associated with clinical outcome or induced CTL responses. While percutaneous thermal ablation plus GM-CSF combinations including checkpoint inhibitors could be considered in future studies, the use of GM-CSF remains experimental and for use in the context of clinical trials.

SummaryHistological transformation (HT) of follicular lymphoma (FL) to an aggressive lymphoma after chemotherapy remains a key issue. The incidence of HT after radioimmunotherapy (RIT) is unknown. This single institution study analysed the risk of HT in FL after treatment with yttrium‐90 ibritumomab tiuxetan in 115 consecutive patients treated during 1987–2012. RIT was administered for progressive FL in 111 (97%) patients and as first‐line therapy in the remaining 4. 28% (n = 32) had HT, occurring at a median of 60 months from diagnosis and 20 months after RIT. 48% (12/25) of patients who received fludarabine developed HT. The estimated 10‐year risk of HT in the fludarabine and non‐fludarabine groups was 67% and 26% respectively (P = 0·015). Only prior fludarabine was significantly associated with predicting the risk of HT after RIT. 8% (9/115) of patients developed therapy‐related myelodysplastic syndrome/acute myeloid leukaemia (tMDS/AML) at a median of 41·4 months (range, 5–89). The estimated 10‐year risk of tMDS/AML in non‐fludarabine treated patients (n = 90) versus fludarabine treated (n = 25) was 13% and 29%, respectively. The estimated overall risk of FL undergoing HT at 10 years without fludarabine exposure appears similar to patients reported in the literature that have not received RIT. Patients with prior purine‐analogue therapy are at significantly higher risk of HT.

AbstractHigher ratios of the pre‐treatment peripheral blood absolute lymphocyte (ALC) to absolute monocyte counts (AMC) are associated with improved outcomes in lymphoma. Conversely, elevated pre‐treatment serum cytokines are associated with inferior outcomes. The relationship between pre‐treatment serum cytokines and ALC/AMC ratios remains unknown. We studied twelve serum cytokines and the ALC/AMC ratios in 390 patients with untreated diffuse large B‐cell, follicular, mantle cell, T‐cell, and Hodgkin lymphoma. Different pre‐treatment serum cytokine concentrations correlated with ALC, AMC, and ALC/AMC ratios depending on the lymphoma type. In the entire cohort (n = 390) lower ALC/AMC ratios modestly correlated with higher IL‐2R (r = −0.36), IL‐12 (r = −0.17), IP‐10 (r = −0.23), and MIG (r = −0.32) concentrations (p < 0.001). Elevated IL‐2R was independently associated with suppressed ALC (OR 2.69, 95% CI 1.77–4.07,p < 0.001), elevated AMC (OR 2.05, 95% CI 1.34–3.14,p < 0.001), and suppressed ALC/AMC ratios (OR 3.51, 95% CI 2.31–5.34,p < 0.001). Both elevated IL‐2R (HR 2.27, 95% CI 1.48–3.49,p < 0.001) and suppressed ALC/AMC ratios (HR 1.53, 95% CI 1.03–2.28,p = 0.037) were independently associated with inferior overall survival. These data support the notion that elevated serum cytokines are immunosuppressive and provide further rationale to target the tumor microenvironment for therapeutic benefit.