Markovic, Svetomir

e22052 Background: Metastatic Uveal Melanoma (MUM) is a rare tumor with poor prognosis following development of liver metastasis. We hypothesized that patterns of metastasis in Uveal Melanoma correlate with clinical outcomes. Methods: We retrospectively reviewed patients with MUM at Mayo Clinic, Rochester from January 1999 to August 2019. Patients were stratified into two groups based on the pattern of hepatic and pulmonary metastasis at the time of diagnosis of metastatic disease: Group 1 (≤5 liver metastasis or lung metastasis) and Group 2 ( > 5 liver metastasis without lung metastasis). Baseline characteristics were compared between both groups. Survival analysis was performed using the Kaplan Meier method. Univariate and multivariate analysis were performed for Overall Survival (OS). Results: 147 patients were included in the study (n = 67 Group 1; n = 80 Group 2). In Group 1, 49/67 patients presented with ≤5 liver metastasis and 18/67 had lung metastasis without liver metastasis. Median OS for Group 1 was significantly longer than Group 2 (38 vs. 15 months; p < 0.0001) (Table). On univariate analysis, predictors for OS were: Pattern of Metastasis, ECOG PS > 0, Time to metastasis > 60 months, and Surgical metastatectomy. Pattern of Metastasis was an independent predictor for OS in a multivariate model that included these predictors (p = 0.0004). Group 1 patients were more likely to undergo surgical metastatectomy compared to group 2 (21.5% vs. 1.3%; p < 0.0001). Interestingly, the median time to metastasis from diagnosis of UM was significantly longer for Group 1 as compared to Group 2 (67 vs. 24.5 months; p < 0.0001). Conclusions: Limited (≤5) liver metastasis or lung metastasis (without liver metastasis) at diagnosis predict favorable clinical outcomes in MUM. The occurrence of such metastasis following a significantly longer time from primary diagnosis suggests the existence of a distinct sub-type of metastatic disease with relatively indolent behavior. [Table: see text]

3037 Background: Tumor immunotherapies directed towards enhancing natural or endogenous anti-tumor T-cell immunity in patients with advanced malignancies are currently being implemented in clinic with promising results. In order to optimize therapeutic protocols and monitor the effectiveness of such therapies, a reliable T-cell marker is needed. Methods: We utilized CD11a (LFA-1, lymphocyte functional-associated antigen 1), an integrin up-regulated on effector and memory CD8 T-cells, and PD-1 (programmed death-1), an immunoregulatory receptor expressed by activated T cells, as biomarkers to identify, quantitate and monitor endogenous tumor-reactive cytotoxic lymphocytes (CTLs) in two mouse tumor models and the peripheral blood (PB) of 12 patients with stage IV melanoma. Results: High expression of CD11a and PD-1 was identified among CD8 T-cells residing within primary and metastatic murine tumor sites, as well as in spontaneous murine breast cancer tissues. In the PB of melanoma patients, tumor antigen-specific CD8 T cells were associated with a population of CD11a high CD8 T-cells which co-expressed high levels of PD-1, as opposed to eleven healthy donors who had a much lower frequency of PD-1+ CD11a high CD8 T-cells (p <0.0001). Phenotypic analysis showed that CD11a high CD8 T-cells are proliferating (Ki67 positive) activated (CD62L low, CD69 high) T-cells. Increased CD11a high CD8 T-cells and delayed tumor growth were observed in PD-1 deficient mice, suggesting that the antitumor effector function of CD8 T cells is compromised by co-expression of elevated levels of PD-1. Conclusions: CD11a high CD8 T-cell population expresses high levels of PD-1 and is likely the cellular target of PD-1 blockade therapy. High expression of CD11a (LFA-1) and PD-1 (CD279) by CD8 T-cells may represent a novel biomarker to identify and monitor endogenous tumor-reactive CTLs. This may not only provide an immunological readout for evaluating the efficacy of therapy, but also contribute to the selection of patients with solid malignancies likely to benefit from anti-PD-1 therapy.

e14507 Background: Serious gastrointestinal injury (SGI) is a rare but documented side effect that can occur independently with agents that affect the vascular endothelial growth factor (VEGF) receptor or with stereotactic body radiotherapy (SBRT). The risk of SGI in patients treated with the combination of SBRT sequentially followed by a VEGF inhibitor (VEGF-i) is not yet quantified. We explored the incidence of SGI in patients treated with SBRT with or without VEGF-i therapy at a single institution. Methods: From May 2008 to August 2011, 76 patients with 84 primary or metastatic intraabdominal lesions underwent SBRT (median dose, 50 Gy in 5 fractions). Twenty of these patients (26%) received a VEGF-i within two years of completing SBRT (bevacizumab, n=14; sorafenib, n=4; pazopanib, n=1; sunitinib, n=1). The most common site treated with SBRT was the liver (n=43, 57%), and the most common histology was colorectal adenocarcinoma (n=18, 24%). Other common histologies included melanoma of the skin (n=14, 18%) and non-small cell lung cancer (n=10, 13%). The incidence of SGI (CTCAE v4.0 grade 3 to 5 ulceration or perforation) after SBRT was obtained, and the relationship between SGI and VEGF-i was examined. Other factors that could potentially contribute to SGI, such as radiotherapy dose to bowel, were also analyzed. Results: Seven patients (9%) experienced SGI at a median 4.6 months (range, 3-17 months) after SBRT. All seven had received a VEGF-i prior to SGI and within 13 months of completing SBRT, and five received the VEGF-i within three months of SBRT. The six-month estimate of SGI in patients receiving a VEGF-i within three months of SBRT was 38%. No SGI was noted in the 63 patients not receiving VEGF-i therapy after SBRT. The Log-Rank test showed a significant correlation between SGI and VEGF-i therapy within three months of SBRT (p=0.0006) but not between SGI and maximum radiotherapy bowel dose (p=0.20). Conclusions: The combination of SBRT and VEGF-i therapy results in a much higher risk of SGI than would be expected with either treatment given independently. Local therapies other than SBRT should be considered if a patient is likely to be treated with a VEGF-i in the future.

8599 Background: Chemotherapy resistance is a common, difficult problem for melanoma patients needing long term care and those suffering from recurrence. Neural growth factor receptor (NGFR), also known as CD271, is commonly expressed on mesenchymal stem cells and is important for development, survival and differentiation of cells. This has previously shown to be expressed on melanoma stem cells. We hypothesized that treatment with carboplatin enriches the population of tumor cells for cancer stem cells (CSC) as a mechanism of chemotherapy resistance. Methods: Core tumor samples from 118 patients with metastatic melanoma were obtained from formalin-fixed paraffin embedded specimens. Slides were stained using an anti-CD271 antibody for IHC. A375 melanoma cells were treated with increasing concentrations of carboplatin for approximately 1 year. WT, 6 mg/mL and 10 mg/mL resistant cells were tagged with CD271-PE antibody and run on a flow cytometer. These cells were then tested for gene expression by microarray using an Affymetrix human 133 plus 2.0 chip, and analyzed on Partek software. Pathway analysis of these carboplatin resistant cells was preformed using Ingenuity. Results: Virtually all melanoma tumor samples stained positively for CD271 by IHC, with a median of 50% cells within tumor samples expressing the cytoplasmic marker. Flow cytometry demonstrated an increase in the percentage of cells expressing CD271 as resistance to carboplatin increased. Wild type A375 cells contained 30% positive CD271, A375 6mg/mL resistant contained 42% positive and A375 10mg/mL resistant showed 66% positive. Microarray also exhibited a direct correlation between CD271 gene expression with increasing carboplatin resistance. Functional ontology enrichment revealed development and regulation of the EMT pathway as an important process impacted by our resistant cell line. Conclusions: The data suggests that as melanoma cells become resistant to certain chemotherapy drugs they essentially loose their differentiated phenotype and become more stem cell-like. Monitoring melanoma expression of CD271 could help individualize therapy and anticipate chemotherapeutic resistance in this high-risk group of patients.

362 Background: Most pts with mGEA do not respond to ICI or ramucirumab/paclitaxel (RAM/TAX). Emerging data suggest that ICI may potentiate subsequent therapy (Tx). In KN059 we unexpectedly observed durable responses in 2 pts on RAM/TAX after ICI (PMID 29674442). We examined if ICI impacts efficacy of subsequent RAM/TAX in a larger cohort and explored alterations in the tumor microenvironment. Methods: All pts with mGEA at Mayo Clinic (MN, AZ, FL) who received RAM/TAX (2014-19) were included. We compared best objective response rate (ORR: complete [CR] or partial response [PR]) per RECIST1.1 and overall survival (OS) in pts who received RAM/TAX with (Group A) vs without (Group B) immediately preceding PD-1 blockade. Chi square and multivariate logistic and Cox regression were used. We adjusted for total lines of Tx received and the line of Tx in which RAM/TAX was given, to control for potential differences in the biology of heavily treated pts or those receiving RAM/TAX as 2nd vs 3rd line Tx. We also adjusted for age and ECOG PS. Results: In total 87 pts met inclusion criteria. In the 19 pts (Group A) who received RAM/TAX (usually as 3rd line Tx) after progression on ICI, there was a 77% relative risk reduction of death after initiation of RAM/TAX compared to the 68 pts (Group B) who received RAM/TAX (usually as 2nd line) without preceding ICI (median OS 15.0 vs 7.6 mo; HR 0.23; P < .001). The ORR was also significantly higher (58% vs 18%; P < .001) including the CR rate (16% vs 1%; P = .006). Two CRs in Group A are ongoing (10 - 34 mo). Of note, 95% of Group A pts did not respond to ICI, and all had irRECIST progression on ICI. Immunofluorescent analysis from a responder showed 65-fold reduction (post vs pre-Tx) in intratumoral density of immunosuppressive FOXP3+ Tregs, with preserved density of antitumor CD8+ T cells. Conclusions: Higher than expected efficacy was observed on RAM/TAX when immediately preceded by ICI, suggesting ICI may enhance efficacy of subsequent anti-VEGFR/taxane therapy. This serial immunotherapy combination may be a novel option for pts with primary resistance to ICI and will be tested prospectively in a new randomized phase 2 trial (NCT04069273).

65 Background: The addition of chemotherapy to immune checkpoint inhibitors has been explored clinically in solid tumors in order to overcome one of the challenges of immunotherapy: clinical outcomes are variable, with only a subset of patients achieving durable responses. Further understanding of the immunoregulatory mechanisms of chemotherapy in the setting of immunotherapy is crucial for the development of optimal combinatorial chemo-immunotherapeutic strategies to improve clinical outcomes in patients with advanced cancer. Methods: Metastatic melanoma (MM) patients who failed anti-PD-1 single therapy were subsequently treated with paclitaxel and carboplatin in addition to PD-1 blockade. Using peripheral blood (PB) from MM patients and healthy donor, the phenotypic and functional changes in T cells induced by chemotherapy, in the setting of PD-1 blockade, were examined. Results: Paclitaxel and carboplatin, in combination with PD-1 blockade, were able to induce significant clinical responses in a subset of MM patients who were resistant to anti-PD-1 therapy alone. In patients who responded to this combination, a subset of tumor-reactive effector T cells (CD8+CD11a+PD1+GranzymeB+) survived the treatment, with increased frequency after the addition of chemotherapy. This T cell population preserved its effector phenotype, although ongoing PD-1 blockade was needed to improve its anti-tumor activity. In vitro experiments, using CD8+CD11a+PD1+GranzymeB+isolated from healthy donor PB, also demonstrated its ability to survive chemotherapy treatment. In addition, after cultured with chemotherapy, this T cell subset demonstrated higher cytotoxic T cell functions as well as increased chemotherapy efflux. Conclusions: Chemotherapy regulates a subset of tumor-reactive effector T cells (CD8+CD11a+PD1+GranzymeB+) and increases clinical efficacy of PD-1 blockade. This novel effector T cell population underlies the key cellular and molecular immunoregulatory mechanisms of chemotherapy. It serves as a meaningful marker to measure these collaborative effects and to develop the optimal chemo-immunotherapy strategy to improve clinical responses to current immune checkpoint blocking agents.

Abstract Background: The phosphatidylinositol 3-kinase/mammalian target of rapamycin (mTOR) signal transduction pathway integrates signals from multiple receptor tyrosine kinases to control cell proliferation and survival. Everolimus (RAD001, Novartis Pharmaceuticals) is an oral investigational antineoplastic agent that targets mTOR. Objectives: To learn the anti-tumor activity and toxicity of single-agent RAD001 in pts with relapsed/refractory aggressive NHL. Patients and Methods: Patients were eligible if they had measurable disease, a platelet count >75,000, an absolute neutrophil count >1,000, and a creatinine and bilirubin <2x laboratory upper limit of normal. Patients received RAD001 10 mg PO daily and were evaluated monthly. Dose reductions to 5 mg daily and 5 mg every other day were allowed. Response was assessed after 2 cycles and periodically thereafter. Patients could remain on drug until progression or toxicity. Results: Thirty-seven pts were treated - 20 (54%) with relapsed diffuse large cell (DLC), 14 (38%) with relapsed mantle cell (MCL), 2 high grade, and 1 follicular grade III. The median age was 72 years (range, 45–92). Patients had received a median of 4 prior therapies (range, 1–15). The overall response rate was 32% (12/37; 95% CI:20–49%) with 1 complete response and 11 partial responses. The ORR was 35% (7/20) in the DLC group and 29% (4/14) in the MCL patients. Patients have received a median of 2 cycles (range, 1–16+) of therapy. The median time to progression for all 37 patients is 3.1 months (95%CI; 2.0 – 4.7). The median duration of response for the 12 responders is 5.5 months (95% CI; 2.3 – 9.2) and 5 pts remained progression free at 6 months. Three patients are currently maintaining a response with a median time of 10.5 months (range, 2.9–15.6+months). RAD001 was well tolerated. The incidence of grade 3 anemia, neutropenia, and thrombocytopenia in this heavily pretreated pt population was 11%, 16%, and 30%. Two pts developed grade 4 neutropenia. Grade 2 hypercholesterolemia occurred in 11%; grade 2 hyperglycemia in 16%; grade 3 hyperglycemia in 11% and 1 pt had grade 4 hypertriglyceridemia. Summary: RAD001 has single-agent activity in relapsed aggressive NHL and provides proof-of-concept that targeting the mTOR pathway in NHL is clinically relevant.

138 Background: Clinical management of metastatic melanoma (MM) after PD-1 blockade failure remains challenging and lacks a standard of care. Chemo-immunotherapy (CIT) combinations have demonstrated favorable efficacy and safety profiles in lung cancer patients. In this study, we compared the clinical outcomes of CIT with immunotherapy or chemotherapy alone after PD-1 blockade failure. Methods: We reviewed MM patients seen at Mayo Clinic between Jan, 2012 and Jun, 2018 who failed anti-PD1 therapy and received subsequent CIT, or immune checkpoint inhibitors (ICI) or chemotherapy alone. A total of 60 patients were analyzed, the CIT cohort [n=33 (55%)] treatment consisted of carboplatin/paclitaxel (n=29), nab-paclitaxel (n=2), paclitaxel (n=1), and temozolomide (n=1). In the ICI (n=9) or chemotherapy alone cohort (n=18) [n=27 (45%)], treatment consisted of carboplatin/paclitaxel (n=11), temozolomide (n=4), nab-paclitaxel (n=3), ipilimumab/nivolumab (n=4), pembrolizumab (n=4), or nivolumab (n=1). Results: Patients in the CIT cohort had a median OS of 3.5 years (95% CI: 1.7-NR) compared to 1.8 years (95% CI: 0.9-2) in the ICI or chemotherapy alone cohort, p=0.02. The median EFS following CIT was 7.6 months (95% CI: 6-10) compared to 3.4 months (95% CI: 2.8-4.1) following either ICI or chemotherapy alone, p=0.0005. A trend towards longer median EFS with use of CIT was seen in patients with BRAF wild-type [median 9 months (95% CI: 6-12)] compared to those harboring a BRAF mutation [median 6.5 months (95% CI: 1.8-9.1), p=0.29]. Side effects were similar among both groups. Conclusions: In MM patients who have failed anti-PD-1 therapy, the CIT combination showed favorable clinical outcomes and acceptable safety profile. This regimen should be considered for MM pts in this setting who have limited treatment options. [Table: see text]

6592 Background: Sex, age and ECOG performance status (PS) may affect immune response and the efficacy of cancer immunotherapy with immune checkpoint inhibitors (ICI). We did a meta-analysis to assess the potential sex, age and ECOG PS differences of immunotherapy efficacy in advanced cancer. Methods: PubMed was searched up to January 15, 2019 for randomized controlled trials (RCT) comparing overall survival (OS) in patients with advanced cancer treated with ICI immunotherapy vs control therapy (without ICI). For sex difference analysis, pooled hazard ratio (HR) of death for men and women was calculated separately, and the heterogeneity between the two estimates was assessed using an interaction test by pooling study-specific interaction HR. Age ( < 65 vs ≥65) and ECOG PS (0 vs ≥1) difference was analyzed similarly. Subgroup analysis by cancer type and line of therapy (frontline vs subsequent) was explored. All analyses were done in Comprehensive Meta Analysis (v2) with random effects models. Results: Thirty phase 2/3 RCTs involving 17,728 patients were included. An OS benefit of immunotherapy was found for both men (HR 0.75, 95% confidence interval [CI] 0.69-0.81, P < 0.01) and women (HR 0.79, 95% CI 0.69-0.90, P < 0.01); for both younger ( < 65; HR 0.75, 95% CI 0.68-0.83, P < 0.01) and older (≥65; HR 0.76, 95% CI 0.69-0.83, P < 0.01) patients; and for both ECOG PS 0 (HR 0.78, 95% CI 0.69-0.89, P < 0.01) and PS ≥1 (HR 0.77, 95% CI 0.71-0.84, P < 0.01) patients. No significant difference of relative benefit from immunotherapy over control therapy was found in patients with different sex ( P = 0.283), age ( P = 0.906) or ECOG PS ( P = 0.783). In melanoma RCTs, compared with women (HR 0.77, 95% CI 0.64-0.94, P < 0.01), men (HR 0.56, 95% CI 0.42-0.76, P < 0.01) had more OS benefit from immunotherapy ( P = 0.037). No significant difference was found in other subgroup analyses by cancer types or line of therapy. Conclusions: Overall we found no evidence of association of sex, age, or ECOG PS with cancer immunotherapy efficacy. However, in melanoma, men might benefit more from immunotherapy than women.