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Adoptive T cell therapy (ACT) produces durable responses in some cancer patients; however, most tumors are refractory to ACT and the molecular mechanisms underlying resistance are unclear. Using two independent approaches, we identified tumor glycolysis as a pathway associated with immune resistance in melanoma. Glycolysis-related genes were upregulated in melanoma and lung cancer patient samples poorly infiltrated by T cells. Overexpression of glycolysis-related molecules impaired T cell killing of tumor cells, whereas inhibition of glycolysis enhanced T cell-mediated antitumor immunity in vitro and in vivo. Moreover, glycolysis-related gene expression was higher in melanoma tissues from ACT-refractory patients, and tumor cells derived from these patients exhibited higher glycolytic activity. We identified reduced levels of IRF1 and CXCL10 immunostimulatory molecules in highly glycolytic melanoma cells. Our findings demonstrate that tumor glycolysis is associated with the efficacy of ACT and identify the glycolysis pathway as a candidate target for combinatorial therapeutic intervention.

199 Background: KIT-mutated MM is a rare melanoma entity. Response rates of KIT-mutated MM to anti CTLA-4 and anti PD-1 have not previously been reported. Methods: A single-institution retrospective review identified patients (pts) with KIT-mutated MM treated with at least two doses of anti CTLA-4 or anti PD-1 between 2008-2017 with response determined by immune-related response criteria (irRC). Overall survival (OS) was from ICI start to death or last follow-up date, and progression-free-survival (PFS) was from ICI start to date of progression or death if pts deceased without disease progression. Results: Thirty-five pts treated with ipilimumab were identified. Median follow-up was 63.7 months. Median age at MM diagnosis was 65.4 years (range 26-81) and 54.3% were male. Subtypes were 51.4% mucosal, 22.9% acral-lentiginous, 22.9% cutaneous, and 2.9% unknown primary. KIT mutations were 57.1% exon 11, 25.7% exon 17, 11.4% exon 13, and 5.7% exon 2. Anatomical M stage at treatment initiation was 11.4% M1a, 34.3% M1b, and 54.3% M1c. Median OS was 11.8 months (8.4-35.6) and PFS was 3.0 months (2.8-5.8). Responses to ipilimumab were 8.6% complete response (CR), 11.4% partial response (PR), 28.6% stable disease (SD), and 51.4% progressive disease (PD) for a 48.6% clinical response rate (CRR). Twenty pts treated with anti PD-1 were identified. Median follow-up was 7.9 months. Median age at MM diagnosis was 66.7 years (range 42.6-86.8) and 70% were male. Subtypes were 40% mucosal, 30% cutaneous, 20% acral-lentiginous, and 10% unknown primary. KIT mutations were 45% exon 11, 35% exon 17, 10% exon 2, 5% exon 13 and and 5% exon 10. Anatomical M stage at treatment initiation was 10% M1a, 15% M1b, and 75% M1c. Median OS was 22.5 months (7.4-NA ) and PFS was 3.2 months (2.7-NA). Responses to anti PD-1 were 10.0% CR, 25.0% PR, 20.0% SD, and 45.0% PD for a 55.0% CRR. For both anti CTLA-4 and anti PD-1 treated cohorts, univariate analysis demonstrated no statistical significance between tumor response to ICI and clinical pt characteristics or KIT mutation exon. Conclusions: KIT-mutated MM demonstrated CRR of 48.6-55.0% to ICI. There was no correlation between pt characteristics or KIT exon mutation and response to therapy; however, analysis was limited by sample size.

Point mutations in the KIT receptor tyrosine kinase gene have recently been identified in mucosal, acral lentiginous, and chronically sun-damaged melanomas. We have identified the first human melanoma cell line with an endogenous L576P mutation, the most common KIT mutation in melanoma (∼30-40%). In vitro testing showed that the cell viability of the L576P mutant cell line was not reduced by imatinib, nilotinib, or sorafenib small molecule KIT inhibitors effective in nonmelanoma cells with other KIT mutations. However, the viability of the mutant cells was reduced by dasatinib at concentrations as low as 10 nM (P = 0.004). Molecular modeling studies found that the L576P mutation induces structural changes in KIT that reduce the affinity for imatinib (ΔΔGbind = −2.52 kcal/mol) but not for dasatinib (ΔΔGbind = +0.32 kcal/mol). Two metastatic melanoma patients with the L576P KIT mutation were treated with dasatinib, including one patient previously treated with imatinib. Both patients had marked reduction (>50%) and elimination of tumor F18-fluorodeoxyglucose (FDG)-avidity by positron emission tomography (PET) imaging after dasatinib treatment. These data support the selective inhibitory effect of dasatinib against cells harboring the most common KIT mutation in melanoma, and thus has therapeutic implications for acrallentiginous, chronic sun-damaged, and mucosal melanomas. [Mol Cancer Ther 2009;8(8):2079–85]

Anal mucosal melanoma is a disorder with limited treatment options and is associated with a poor prognosis. The authors describe the case of a 79-year-old man who was diagnosed with stage IV anal mucosal melanoma expressing the KIT Val560Asp mutation, and was treated with surgery, radiotherapy, and a combination of sorafenib and temozolomide therapy. The authors discuss the potential therapeutic use of sorafenib in anal mucosal melanoma patients with specific mutations such as the KIT Val560Asp mutation. Background A 79-year-old man presented to his primary care physician with a 2-month history of pruritus ani and a pigmented nodular lesion was discovered in the posterior rectum. The patient had no other symptoms, or any family history of malignancy. Investigations Physical examination; excisional biopsy; CT scan of the chest, abdomen and pelvis; lung biopsy; blood tests; tumor immunohistochemistry for KIT, vascular endothelial growth factor platelet-derived growth factor receptor α and β, and mismatch-repair proteins MLH1, MSH2, and MSH6; and KIT and BRAF tumor genotyping. Diagnosis Stage IV M1b metastatic anal mucosal melanoma. Management Wide local excision with mucosal advancement of the rectal wall, external-beam radiation, and sorafenib–temozolomide therapy.

Purpose: Inflammatory marker expression in stage III melanoma tumors was evaluated for association with outcome, using two independent cohorts of stage III melanoma patients' tumor tissues. Experimental Design: Fifteen markers of interest were selected for analysis, and their expression in melanoma tissues was determined by immunohistochemistry. Proteins associating with either overall survival (OS) or recurrence-free survival (RFS) in the retrospective discovery tissue microarray (TMA; n = 158) were subsequently evaluated in an independent validation TMA (n = 114). Cox proportional hazards regression models were used to assess the association between survival parameters and covariates, the Kaplan–Meier method to estimate the distribution of survival, and the log-rank test to compare distributions. Results: Expression of CD74 on melanoma cells was unique, and in the discovery TMA, it associated with favorable patient outcome (OS: HR, 0.53; P = 0.01 and RFS: HR, 0.56; P = 0.01). The validation data set confirmed the CD74 prognostic significance and revealed that the absence of macrophage migration inhibitory factor (MIF) and inducible nitric oxide synthase (iNOS) was also associated with poor survival parameters. Consistent with the protein observation, tumor CD74 mRNA expression also correlated positively (P = 0.003) with OS in the melanoma TCGA data set. Conclusions: Our data validate CD74 as a useful prognostic tumor cell protein marker associated with favorable RFS and OS in stage III melanoma. Low or negative expression of MIF in both TMAs and of iNOS in the validation set also provided useful prognostic data. A disease-specific investigation of CD74's functional significance is warranted, and other markers appear intriguing to pursue. Clin Cancer Res; 22(12); 3016–24. ©2016 AACR.

Melanomas of the choroid, ciliary body, and iris of the eye are collectively known as uveal melanomas. These cancers represent 5% of all melanoma diagnoses in the United States, and their age-adjusted risk is 5 per 1 million population. These less frequent melanomas are dissimilar to their more common cutaneous melanoma relative, with differing risk factors, primary treatment, anatomic spread, molecular changes, and responses to systemic therapy. Once uveal melanoma becomes metastatic, therapy options are limited and are often extrapolated from cutaneous melanoma therapies despite the routine exclusion of patients with uveal melanoma from clinical trials. Clinical trials directed at uveal melanoma have been completed or are in progress, and data from these well designed investigations will help guide future directions in this orphan disease. Cancer 2016;122:2299-2312. © 2016 American Cancer Society.

Summary We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF , mutant RAS , mutant NF1 , and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multi-dimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

Purpose: Copy number alterations have been shown to be involved in melanoma pathogenesis. The randomized phase III clinical trial E2603: carboplatin, paclitaxel, ± sorafenib (CP vs. CPS) offers a large collection of tumor samples to evaluate association of somatic mutations, genomic alterations, and clinical outcomes, prior to current FDA-approved therapies. Experimental Design: Copy number and mutational analysis on 119 pretreatment samples was performed. Results: CPS therapy was associated with improved progression-free survival (PFS) compared with CP in patients with tumors with RAF1 (cRAF) gene copy gains (HR, 0.372; P = 0.025) or CCND1 gene copy gains (HR, 0.45; P = 0.035). CPS therapy was associated with improved overall survival (OS) compared with CP in patients with tumors with KRAS gene copy gains (HR, 0.25; P = 0.035). BRAF gene copy gain and MET amplification were more common in samples with V600K versus V600E mutations (P < 0.001), which was validated in The Cancer Genome Atlas (TCGA) dataset. Conclusions: We observed improved treatment response with CPS in patients with melanoma whose tumors have RAF1 (cRAF), KRAS, or CCND1 amplification, all of which can be attributed to sorafenib targeting CRAF. These genomic alterations should be incorporated in future studies for evaluation as biomarkers. Clin Cancer Res; 22(2); 374–82. ©2015 AACR.

Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as β-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and β-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.

Driving cancer cells into a more differentiated state is a rational therapeutic approach. Primary uveal melanoma cells with a propensity to metastasize have less-differentiated features than their less aggressive counterparts. Treatment of uveal melanoma cells with histone deacetylase inhibitors induces a more differentiated phenotype with resultant lower growth capacity. Clin Cancer Res; 18(2); 323–5. ©2011 AACR.

Background: While checkpoint inhibitor (CPI) therapy has transformed metastatic melanoma (MM) treatment, many patients remain refractory. We reasoned that combining CPI with an agent that activates antigen presenting cells and improves T-cell priming may result in improved response. Our approach is to modulate the tumor microenvironment through image-guided intratumoral (i.t.) injection of the TLR9 agonist, IMO-2125, in combination with either ipilimumab (ipi) or pembrolizumab (pembro). We hypothesize that this will result in dendritic cell (DC) activation and induction of tumor-specific CD8+T-cells which will synergize with ipilimumab or pembrolizumab to overcome immune-escape. Based on this rationale we initiated a phase I/II clinical trial. Study Design/Methods: Adults with refractory MM that have had prior PD-1 blockade therapy (with or without a BRAF inhibitor) are eligible. IMO-2125, in doses escalating from 4mg to 32mg, is given i.t. weeks 1, 2, 3, 5, 8, and 11 along with standard doses of ipilimumab or pembrolizumab. Primary endpoints are safety, tumor response, and PK. Multiple biopsies are obtained in both the injected and distant tumor pre- and on-treatment. Immune analyses include DC subsets and their activation status as well as T cell activation, function and proliferation. T-cell repertoire diversity is evaluated by high-throughput CDR3 sequencing and changes in gene expression signatures are assessed by nanoString. Changes in circulating cytokines are also being assessed during therapy. Results: Enrollment is proceeding on both the IMO-ipilimumab and IMO-pembrolizumab dose-escalation arms of the trial. Safety is acceptable with no DLT recorded to date. Durable clinical responses (including 1 CR) have been observed with IMO-ipilimumab in patients who were refractory to PD-1 inhibitor. Fresh tumor biopsies show maturation (upregulation of HLA-DR) of the myeloid DC1 subset (CD1c+CD303-), upregulation of PD-L1 by malignant cells, as well as an IFNα response gene signature in the IMO-2125 injected tumor lesion 24 hrs post-treatment compared to pre-treatment biopsy. On-treatment biopsy results show a higher expression of CD56+ and Ki67+ effector CD8+ T-cells in responding patients. Initial CDR3 sequencing demonstrates increased diversity in the injected lesions of all patients assessed and expansion of top clones present in the distant lesion in a responding patient. Plasma analysis showed an increase in IFNγ levels in the plasma of responding patients. Conclusions: These results demonstrate that IMO-2125 with a checkpoint inhibitor is a viable strategy to revive the immune response in tumors that are refractory to PD-1 inhibitors. Further clinical evaluation of both the IMO-ipilimumab and IMO-pembrolizumab combination is planned in a Phase 2 expansion of the current trial. Citation Format: Cara Haymaker, Marc Uemura, Ravi Murthy, Marihella James, Daqing Wang, Julie Brevard, Suzanne Swann, James Geib, Mark Cornfeld, Srinivas Chunduru, Sudhir Agrawal, Cassian Yee, Jennifer Wargo, Rodabe Amaria, Sapna Patel, Hussein Tawbi, Isabella Glitza, Scott Woodman, Wen-Jen Hwu, Michael A. Davies, Patrick Hwu, Willem Overwjik, Chantale Bernatchez, Adi Diab. Translational evidence of reactivated innate and adaptive immunity with intratumoral IMO-2125 in combination with systemic checkpoint inhibitors from a Phase I/II study in patients with anti-PD-1 refractory metastatic melanoma [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 5652. doi:10.1158/1538-7445.AM2017-5652

Background: Major advances have been made in the treatment of metastatic melanoma through the use of molecularly targeted therapy and immunotherapy, and trials incorporating these agents are now being extended to patients with earlier-stage disease. The current standard of care (SOC) therapy for high-risk resectable melanoma (stage IIIB/IIIC) is upfront surgery and SOC adjuvant therapy; however, relapse rates are high (~70%). We hypothesized that treatment with neoadjuvant + adjuvant targeted therapy (dabrafenib + trametinib) in this patient population would result in lower relapse rates and prolonged survival over SOC therapy. Methods: To test this hypothesis, we designed a randomized clinical trial (NCT02231775) in patients with resectable Stage IIIB/C or oligometastatic stage IV BRAF-mutant melanoma. Patients were randomized 1:2 to SOC (Arm A) versus neoadjuvant + adjuvant D+T (Arm B, 8 wks neoadjuvant + 44 wks adjuvant). The primary endpoint was relapse-free survival (RFS) with additional secondary endpoints. Importantly longitudinal sampling of tumor tissue was obtained (at baseline, week 3, and surgery) and molecular profiling was performed to gain insights into mechanisms of therapeutic response. Results: 21 of 84 patients were enrolled (arm A=7, arm B=14). Arms were well matched, and toxicity to targeted therapy was limited. RECIST response rate to 8 wks D+T was 77%, with a pathologic complete response rate (pCR) of 58%. Interim analysis revealed a significantly higher RFS in the D+T arm over SOC (p Conclusion: Neoadjuvant + adjuvant D+T is associated with a high pCR rate and improved RFS over SOC in patients with high-risk resectable metastatic melanoma. Pathological and molecular correlative analysis revealed both pre- and on-treatment tumor features to be highly associated with the high pCR rate. Citation Format: Scott E. Woodman, Peter Prieto, Miles C. Andrews, Rodabe N. Amaria, Michael Tetzlaff, Adi Diab, Sapna P. Patel, Hwu Wen-Jen, Isabella Glitza, Hussein Tawbi, Patrick Hwu, Janice Cormier, Anthony Lucci, Richard Royal, Jeffrey Lee, Roland Bassett, Lauren Simpson, Elizabeth Burton, Li Zhao, Elizabeth Grimm, Alexandre Reuben, Christine Spencer, Junna Oba, Merrick Ross, Jeffrey Gershenwald, Michael Davies, Jennifer A. Wargo. Novel neoadjuvant targeted therapy trial yields insight into molecular mechanisms of response [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 CT156. doi:10.1158/1538-7445.AM2017-CT156

11567 Background: Deleterious mutations in DDR genes are frequently associated with response to poly(ADP-ribose) polymerase (PARP) inhibitors and platinum chemotherapy. However, much remains unknown about their association with specific molecular signaling pathways. We report the prevalence of pathogenic variants in DDR genes and their co-alteration with other somatic variants. Methods: Targeted exome sequencing of 201 genes was performed in 1,189 patients (pts) with advanced solid tumors enrolled in a molecular testing protocol (NCT01772771), using matched normal and tumor DNA. We assessed germline and somatic alterations in 15 cancer-related DDR genes, their co-occuring genomic alterations and the tumor mutation burden (TMB), defined as number of somatic non-synonymous mutations. Results: A total of 124 pathogenic or likely pathogenic variants in DDR genes were identified in 111/1189 (9%) pts with 57% of these alterations being somatic. These variants were found in the following genes: ATM 17 pts (1.4%); BAP1 5 pts (0.4%); BRCA1 18 pts (1.5%); BRCA2 17pts (1.4%); CHEK1 8 pts (0.7%); CHEK2 16 pts (1.3%); ERCC3 4 pts (0.3%); ERCC4 2 pts (0.2%); ERCC5 3 pts (0.2%); MLH1 4pts (0.3%); MSH2 8 pts (0.7%); MSH6 6 pts (0.5%); PALB2 3 pts (0.2%) and RAD51 1pt (0.1%). DDR alterations were found more frequently in the following tumor types tested: breast 14%, colorectal 12%, melanoma 8%, glioblastoma 6% and ovarian 6%. The most relevant somatic co-alterations with DDR mutations were activation of the PI3K/AKT/mTOR pathway through mutations or copy-number variations in AKT1, MTOR, NF1, PIK3CA, PIK3R1, PTEN, TSC1 and TSC2 (p = 0.008). Patients with deleterious variants in mismatch excision repair genes (MLH1, MSH2 or MSH6) had a significantly higher TMB than all other patients enrolled (median TMB = 62 vs 5, p = 0.002). Patients with somatic pathogenic DDR variants had a significantly higher TMB (median = 13) compared to patients with germline DDR variants (median = 5) (p = 0.004). Conclusions: The association of DNA repair mutations with alterations in signaling pathways provide rationale for novel therapeutic combinations. Variations in TMB based on distinct types of DDR gene alterations may have implications for immunotherapy.

9587 Background: Targeted and immune therapies have dramatically improved outcomes in stage IV metastatic melanoma pts. These agents are now being tested in earlier-stage disease. SOC surgery for high-risk resectable melanoma (AJCC stage IIIB/IIIC), with or without adjuvant therapy, is associated with a high risk of relapse (~70%). We hypothesized that neoadjuvant (neo) + adjuvant treatment with D+T improves RFS in these pts. Longitudinally collected biospecimens from pts receiving this treatment were analyzed to identify candidate strategies to further improve outcomes. Methods: A prospective single-institution randomized clinical trial (NCT02231775) was conducted in BRAF-mutant pts with resectable Stage IIIB/C or oligometastatic stage IV melanoma. Pts were randomized 1:2 to SOC (Arm A) versus neo + adjuvant D+T (Arm B; 8 wks neo + 44 wks adjuvant). The primary endpoint was RFS. Tumor biopsies were collected at baseline, week 3, and at surgery for molecular and immune profiling (whole exome sequencing, gene expression profiling, IHC, flow cytometry). Results: 21 of a planned 84 patients were enrolled (Arm A = 7, Arm B = 14). Arms were well balanced for standard prognostic factors, and toxicity was manageable. RECIST response rate with neo D+T was 77%, and the pathologic complete response rate (pCR) was 58%. First interim analysis revealed significantly improved RFS in the D+T arm over SOC (HR 62.5, p < 0.0001), leading to early closure to enrollment. Pts with a pCR at surgery had significantly improved RFS versus pts without pCR (p = 0.04) on neo D+T. Tumor profiling revealed incomplete MAPK pathway blockade and higher levels of CD8+ T cells expressing immunomodulators Tim-3 and Lag-3 in pts who did not achieve a pCR. Conclusions: Neo + adjuvant D+T is associated with a high pCR rate and markedly improved RFS over SOC in pts with high-risk resectable BRAF-mutant metastatic melanoma. pCR at surgery is associated with improved RFS. Tumor analyses reveal candidate targets for testing in future trials to enhance responses to neo D+T. Clinical trial information: NCT02231775.

TPS9594 Background: Immune checkpoint inhibitors have impressive response rates and improved survival for many pts with MM, offering durable responses in up to 35% of pts while others have short-lived or no response ( > 40%). A potentially targetable mechanism for immune escape is for cancers to subvert the cellular epigenetic machinery, affecting multiple aspects of the immune response from suppression of tumor antigen expression [such as Cancer Testis Antigens (CTA)] to antigen processing and presentation, thus allowing tumor proliferation to continue undetected. In preclinical models, decitabine (DAC), a DNA hypomethylating agent (HMA), has been able to revert heterogeneous CTA expression profiles, increasing cancer cell immunogenicity. HMAs also increased TH1-chemokine expression, T cell tumor infiltration, T cell-mediated tumor killing, and have been shown to be synergistic with checkpoint inhibitors in preclinical models. This suggests that epigenetic therapy with checkpoint inhibition is a rational combination to target MM. Clinical advantages of the HMA CC-486 over DAC include oral bioavailability and potential versatility in dosing and schedule. We hypothesize that CC-486 + PEMBRO will be tolerated at biologically relevant doses; enhance response to PEMBRO in pts with mm who are PD-1 naïve; and reverse resistance to immunotherapy in pts refractory/resistant to PD-1 directed therapy. Methods: This study will evaluate the safety and efficacy of CC-486 + PEMBRO defined by Objective Response Rate (ORR) by RECIST 1.1 in pts with MM. Pts who are PD-1 naïve (Arm A, n = 36) and pts who have progressed on prior PD-1 directed therapy (Arm B, n = 35) will be enrolled. Unlimited prior systemic therapies will be allowed. Pts will receive 300mg PO of CC-486 on days 1-14 and 200mg IV of PEMBRO every 3 weeks. Continuous monitoring for toxicity and futility will be performed and assumes an ORR of > 35% and > 15% for Arms A and B, respectively (95% power). Tumor biopsies at baseline and post treatment are required. Effects of CC-486 + PEMBRO on CTAs, MDSCs and Tregs, and correlation between mutation burden and response will be studied. This study is open for enrollment. Clinical trial information: NCT02816021.

9579 Background: Combined treatment with ipilimumab and nivolumab (Ipi/Nivo) achieves clinical responses in > 50% of mm pts. However, responses are not universal and toxicity may be limiting, thus biomarkers of response and toxicity are needed to optimize and personalize this therapy. Methods: Tumor biopsies were collected before (n = 29) and on treatment (n = 7) from mm pts (n = 40) treated with Ipi/Nivo. Whole exome sequencing (WES), gene expression profiling, TCR sequencing, and immunohistochemistry (IHC) were performed to define molecular and immune features of the tumors. Radiographic responses in patients were assessed via RECIST 1.1criteria, and patients were classified as responders (R) deriving clinical benefit (with SD, PR, CR) and non-responders (NR) not deriving clinical benefit (PD). Toxicity was also scored, with patients dichotomized into low toxicity ( < grade 2) versus high toxicity ( > grade 3) re: immune-related (IR) toxicities. Results: In this cohort, the response rate was 80%, with 53% of patients experiencing > grade 3 toxicity. There was no significant difference in baseline mutational load in responders (R) vs non-responders (NR) to Ipi/Nivo, but NR had a higher burden of copy number alterations (CNA; p = 0.013), with frequent alterations detected in PTEN, JAK2, and B2M. There were no significant differences in baseline CD8+ T cell density, expression of immune-related genes, or T cell clonality for R vs NR pts. Ipi/Nivo treatment increased intratumoral T cell clonality, but this did not correlate with response. A more diverse peripheral T cell repertoire at baseline was detected in pts who developed IR toxicity (p < 0.05). Conclusions: This data suggests that responses to Ipi/Nivo in mm may occur in the absence of high mutational load or brisk immune infiltrate at baseline. Putative mechanisms of resistance to Ipi/Nivo include high burden of CNA and alterations in PTEN, JAK2, and B2M. Together these studies identify candidate biomarkers of resistance and toxicity for Ipi/Nivo, though they need to be tested in larger cohorts and across cancer types.

9582 Background: Toll like receptor (TLR) agonists may enhance vaccination or direct immune activation at the tumor microenvironment. This trial evaluates the biologic and clinical effects of Resiquimod, a TLR 7/8 agonist that can activate both myeloid (mDC, TLR 8) and plasmacytoid (pDC, TLR 7) dendritic cells, in patients with advanced stage melanoma. Methods: Class I HLA-A0201+ subjects with in-transit melanoma metastases or high risk for recurrence were vaccinated weekly with peptide vaccination (class I restricted peptide GP100209-2m and, if HLA-DP4+, also with class II restricted peptide MAGE-3243-258). Subjects were randomized 1:1 to receive Resiquimod as an adjuvant applied to the GP100 vaccination site. Subjects with in-transit disease were thereafter treated with resiquimod topically on half of the target lesions. Results: All patients (n = 47) underwent GP100209-2m vaccination, a majority (39) also received the MAGE-3243-258 peptide. The type I interferon-inducible genes (Mx A and IRF7), IFNg, and IP-10 RNA expression were up-regulated only in vaccination sites treated with Resiquimod (each p < 0.01) , demonstrating pDC activation (Type I interferon) and possibly T and NK cell activation (IFNg and IP-10). Nineteen subjects had in-transit disease at entry into the trial. In response to peptide vaccination alone, tumor regression was more likely in patients who received Resiquimod at the vaccination site (group A) compared to those who did not (group B). (4/9 vs 0/10, p = 0.033). In group A, 5 patients continued treatment with Resiquimod topically on the tumors, and all had tumor response (4PR, 1CR). In group B, 5 continued to tumoral resiquimod and 3 had regression (3 PR). Conclusions: Resiquimod increases Type I interferon and IFNg at the peptide vaccination site by activation of pDC/mDC and increases the antitumor response sufficiently to mediate regression of in-transit melanoma metastasis. Resiquimod on in-transit melanoma, in vaccinated hosts, drives regression of metastases, regardless of previous exposure at vaccination. Clinical trial information: NCT00960752.

136 Background: Checkpoint inhibitors (CPI) have transformed melanoma treatment but many patients remain refractory. CPI plus i.t. IMO may improve response by activating innate immune function. Based on this we initiated a trial of IMO in combination with ipi or pembrolizumab (pem) in RM. Methods: Adults with RM that progressed during or after ≥ 12 weeks of PD-1 therapy are eligible. IMO, 4 – 32 mg, is given i.t. for 6 doses, along with either ipi or pem. Endpoints are safety, response, biomarkers, and PK. Injected and distant tumors are biopsied pre-treatment and again at 24 hrs (injected tumor), weeks 8 and 13 for immune analyses. Results: As of October 7, 2016, 10 pts have been treated; median age 55 (range: 39-76), 8 with visceral and 1 with brain metastases. Two pts have mucosal histology. 60% have BRAF mutations. Prior duration of anti-PD-(L)1 therapy ranges from 8 to 63 weeks and median time from last PD-1 therapy to onset of study treatment is 6 (4,57) weeks. IMO has been administered at 4, 8, and 16 mg. No DLTs have been observed and there have been no treatment-related discontinuations or deaths. Ipi was discontinued after the second dose in one subject with previous ipi-related hepatitis for recurrent transaminase elevations (grade 4). Grade 3 hypophysitis is the only other immune-related AE (2 pts). Most frequent TEAE (N > 2) are nausea, vomiting, anemia, diarrhea, increases in ALT/AST/GGT/triglycerides, chills, fatigue, pyrexia, and leukopenia; the majority are low-grade. 6 patients are evaluable for response - CR (1), PR (2), SD (2), PD (1) by RECIST1.1. Tumor biopsies show consistent maturation of the myeloid DC1 subset in IMO injected tumors at 24 hrs. Week 8 results are consistent with a higher rate of proliferative (Ki67) effector CD4+ and CD8+ T-cells in responders. Circulating IFNγ shows 2-3 fold increase in responders. Conclusions: The combination of IMO and ipi is tolerable and has activity in PD-1 refractory melanoma. Dose escalation is ongoing and a Phase 2 expansion with both combinations is planned. Updated safety, antitumor activity, PK, and biomarker data will be presented at the meeting. Clinical trial information: NCT02644967.

138 Background: Adoptive cell therapy (ACT) of autologous tumor-infiltrating lymphocytes (TIL) can produce long lasting treatment responses in patients (pts) with metastatic melanoma. In our initial report of 31 treated pts, we demonstrated overall response rate (ORR) of 48%. Due to the evolution of treatment options for metastatic melanoma with heavy reliance on immunomodulatory therapies, we sought to re-evaluate responses in the era of checkpoint blockade. Methods: Pts receive treatment consisting of 7 days of lymphodepleting chemotherapy consisting of cyclophosphamide and fludarabine. High dose interleukin-2 (720,000 IU/kg IV q 8 hrs up to 15 doses) was infused after TIL infusion. Responses were assessed by imaging per irRC. Results: A total of 74 pts have been treated in our initial TIL study with an updated ORR assessment of 43%. Stratification of pts according to their checkpoint blockade immune-modulator therapy prior to TIL ACT, demonstrated that prior Ipilimumab (Ipi) decreased ORR to 33% compared to 51% in checkpoint naïve pts and decreased overall survival (OS) post-TIL therapy (median 7.7 vs 24.6 months respectively). There were too few pts to assess the impact of anti-PD1 as a single agent. A multiparametric analysis revealed that LDH levels at the time of therapy mainly influences OS and ORR to TIL but still could not invalidate the impact of Ipi prior to TIL ACT. The durability of the response was also found to be different between the 2 groups (30% for Ipi prior and 50% No Ipi prior). This new reality also impacted previously reported parameters that correlated with ORR to TIL ACT such as the expression of B-and-T lymphocyte attenuator (BTLA) on CD8+ TIL. Interestingly, assessment of mutation load (ML) of the tumors prior to TIL ACT showed that the check point naïve group display a high ML ( > 100) within their tumor whereas some patients who had Ipi prior to TIL have a low ML ( < 100). Conclusions: Our preliminary analysis shows that pre-treatment with Ipi decreases ORR to TIL ACT. Understanding how prior ipi modifies TIL, the tumor and microenvironment will help to define the full extent of the impact and how to best treat Ipi refractory pts with TIL. Clinical trial information: NCT00338377.

Metastatic solid tumors are almost invariably fatal. Patients with disseminated small-cell cancers have a particularly unfavorable prognosis, with most succumbing to their disease within two years. Here, we report on the genetic and functional analysis of an outlier curative response of a patient with metastatic small-cell cancer to combined checkpoint kinase 1 (CHK1) inhibition and DNA-damaging chemotherapy. Whole-genome sequencing revealed a clonal hemizygous mutation in the Mre11 complex gene RAD50 that attenuated ATM signaling which in the context of CHK1 inhibition contributed, via synthetic lethality, to extreme sensitivity to irinotecan. As Mre11 mutations occur in a diversity of human tumors, the results suggest a tumor-specific combination therapy strategy in which checkpoint inhibition in combination with DNA-damaging chemotherapy is synthetically lethal in tumor cells but not normal cells with somatic mutations that impair Mre11 complex function.Strategies to effect deep and lasting responses to cancer therapy in patients with metastatic disease have remained difficult to attain, especially in early-phase clinical trials. Here, we present an in-depth genomic and functional genetic analysis identifying RAD50 hypomorphism as a contributing factor to a curative response to systemic combination therapy in a patient with recurrent, metastatic small-cell cancer.

The management of melanoma has evolved owing to improved understanding of its molecular drivers. To augment the current understanding of the prevalence, patterns, and associations of mutations in this disease, the results of clinical testing of 699 advanced melanoma patients using a pan-cancer next-generation sequencing (NGS) panel of hotspot regions in 46 genes were reviewed. Mutations were identified in 43 of the 46 genes on the panel. The most common mutations were BRAFV600 (36%), NRAS (21%), TP53 (16%), BRAFNon-V600 (6%), and KIT (4%). Approximately one-third of melanomas had >1 mutation detected, and the number of mutations per tumor was associated with melanoma subtype. Concurrent TP53 mutations were the most frequent events in tumors with BRAFV600and NRAS mutations. Melanomas with BRAFNon-V600mutations frequently harbored concurrent NRAS mutations (18%), which were rare in tumors with BRAFV600 mutations (1.6%). The prevalence of BRAFV600 and KIT mutations were significantly associated with melanoma subtypes, and BRAFV600 and TP53 mutations were significantly associated with cutaneous primary tumor location. Multiple potential therapeutic targets were identified in metastatic unknown primary and cutaneous melanomas that lacked BRAFV600and NRAS mutations. These results enrich our understanding of the patterns and clinical associations of oncogenic mutations in melanoma.

The primary objective of this study was to determine the safety, toxicity, and maximum tolerated dose of nanoparticle albumin-bound (nab)-paclitaxel as part of biochemotherapy for metastatic melanoma and to determine whether substituting nab-paclitaxel for less potent agents could increase response rates and duration. Treatment consisted of intravenous cisplatin (20 mg/m) on days 1-4, oral temozolomide (250 mg/m) on days 1-3, subcutaneous interferon-α (5×10 IU/m) on days 1-5, and continuous intravenous interleukin-2 (9×10 IU/m) for 96 h on days 1-4. A standard 3+3 dose escalation method was used; the nab-paclitaxel starting dose was 100 mg/m on day 1 and 70 mg/m on day 5. The treatment cycle was repeated every 3 weeks and toxicity was assessed weekly. Ten patients were enrolled. Dose-limiting toxicities included diarrhea, transaminasemia, and neutropenia. The maximum tolerated dose was not identified because the nab-paclitaxel dose on day 1 at the lowest planned dose (80 mg/m) caused dose-limiting toxicity in two of five patients. Of the nine patients who were evaluable for response, five had a partial response. The median time to disease progression was 5.30 months and the median overall survival was 8.73 months. Six patients developed central nervous system metastasis at a median of 5.33 months after treatment initiation. Biochemotherapy including nab-paclitaxel according to the doses and schedule regimen used in the present study has significant toxicity. Substituting dacarbazine with temozolomide did not prevent central nervous system metastasis in patients with metastatic melanoma.

Purpose Nearly all primary uveal melanoma (UM) that metastasize involve the liver. Hepatocyte growth factor (HGF) is proposed to be an important microenvironmental element in attracting/supporting UM metastasis through activation of MET. The majority (>85%) of UM express mutations in the G-alpha proteins, that drive the MEK-ERK1/2 pathway. Thus, we proposed that the combination of MET and MEK inhibition would inhibit the growth and migration of G-alpha protein mutant versus non-mutant UM cells. Methods Western-blots demonstrated the relative protein levels of ERK1/2 and MET in UM cells. Cells were treated with the small molecule inhibitors AZD6244 (MEKi) and/or MK-8033 (METi) and downstream markers evaluated. Further studies determined the effect of combination MEKi and METi treatment on cell growth, apoptosis and migration. Results All G-alpha protein mutant UM cell lines express MET mRNA and protein. The level of mRNA expression correlates with protein expression. MEKi, but not METi treatment results in markedly reduced ERK1/2 phosphorylation. Either MEKi or METi treatment alone results in reduced cell proliferation, but only modest induction of apoptosis. The combination MEKi+METi results in significant reduction of proliferation in G-alpha protein mutant cells. UM cell migration was blocked by METi, but not MEKi treatment. Conclusions MET protein expression showed no correlation with G-alpha protein mutation status. Combining MEKi with METi treatment has added benefit to either treatment alone in reducing G-alpha protein mutant UM cell growth. Combining METi with MEKi treatment adds the effect of limiting uveal melanoma cell migration.

Antibody blockade of the inhibitory CTLA-4 pathway has led to clinical benefit in a subset of patients with metastatic melanoma. Anti-CTLA-4 enhances T cell responses, including production of IFN-γ, which is a critical cytokine for host immune responses. However, the role of IFN-γ signaling in tumor cells in the setting of anti-CTLA-4 therapy remains unknown. Here, we demonstrate that patients identified as non-responders to anti-CTLA-4 (ipilimumab) have tumors with genomic defects in IFN-γ pathway genes. Furthermore, mice bearing melanoma tumors with knockdown of IFN-γ receptor 1 (IFNGR1) have impaired tumor rejection upon anti-CTLA-4 therapy. These data highlight that loss of the IFN-γ signaling pathway is associated with primary resistance to anti-CTLA-4 therapy. Our findings demonstrate the importance of tumor genomic data, especially IFN-γ related genes, as prognostic information for patients selected to receive treatment with immune checkpoint therapy.

Purpose: Conjunctival melanoma is a rare but potentially deadly tumor of the eye. Despite effective local therapies, recurrence and metastasis remain frequent. Once the tumor has metastasized, treatment options are limited and the prognosis is poor. To date, little is known of the genetic alterations in conjunctival melanomas. Experimental Design: We conducted genetic analysis of 78 conjunctival melanomas, to our knowledge the largest cohort reported to date. An oncogene hotspot array was run on 38 samples, screening for a panel of known cancer-relevant mutations. Thirty tumors were analyzed for genome-wide copy number alterations (CNA) using array-based comparative genomic hybridization. Sanger sequencing of selected target mutations was conducted in all samples. Results: BRAF mutations were identified in 23 of 78 (29%) tumors. NRAS mutations, previously not recognized as relevant in conjunctival melanoma, were detected in 14 of 78 (18%) tumors. We found CNAs affecting various chromosomes distributed across the genome in a pattern reminiscent of cutaneous and mucosal melanoma but differing markedly from uveal melanoma. Conclusions: The presence of NRAS or BRAF mutations in a mutually exclusive pattern in roughly half (47%) of conjunctival melanomas and the pattern of CNAs argue for conjunctival melanoma being closely related to cutaneous and mucosal melanoma but entirely distinct from uveal melanoma. Patients with metastatic conjunctival melanoma should be considered for therapeutic modalities available for metastatic cutaneous and mucosal melanoma including clinical trials of novel agents. Clin Cancer Res; 19(12); 3143–52. ©2013 AACR.

There have been significant advances in the treatment of metastatic melanoma through targeted and immunotherapy, however a significant proportion of patients still progress on these regimens with many experiencing mixed responses. Intense research efforts to better understand resistance are underway, and multiple molecular resistance mechanisms to targeted therapy have been identified. The appreciation of genetic heterogeneity as a contributor to resistance to therapy has grown, though immune heterogeneity has been poorly characterized. The goal of the present study is to better understand the molecular and immune heterogeneity in synchronous melanoma metastases at the time of disease progression. In this study, we prospectively evaluated 32 tumors from 15 patients who were treatment-naïve (n = 4), or had received prior targeted (n = 4) or immunotherapy (n = 7). Whole exome sequencing demonstrated between 4-41% of non-synonymous exonic mutations (NSEM) were restricted to individual tumors within a patient. Deep profiling of infiltrating immune cell subsets by flow cytometry and immunohistochemistry analyses confirmed the immune infiltrate between synchronous metastases to be highly heterogeneous, specifically in regards to CD4 and CD8 T lymphocytes. In aggregate, 92% of these T cell clones were unique to distinct tumors within the same patient, with limited overlap with clones detected in the blood. NetMHC3.4 neoantigen prediction demonstrated a large fraction of predicted neoantigens were restricted to individual tumors, with over 10% of these presenting extremely high predicted affinity. Importantly, analysis of RECIST measurements of individual lesions within the same patient suggested this molecular and immune heterogeneity could contribute to differential tumor growth and response to therapy within the same patient. This has important clinical implications, and suggests a single tumor biopsy may not be sufficiently representative of the molecular and immune landscape of multiple tumors within the same individual. Citation Format: Alexandre Reuben, Christine N. Spencer, Peter A. Prieto, John P. Miller, Xizeng Mao, Wei-Shen Chen, Hannah Cheung, Hong Jiang, Cara Haymaker, Mariana Petaccia De Macedo, Haven R. Garber, Pei-Ling Chen, Vancheswaran Gopalakrishnan, Jacob Austin-Breneman, Courtney W. Hudgens, Jason Roszik, Patrick Hwu, Scott E. Woodman, Lynda Chin, Michael A. Davies, Rodabe N. Amaria, Sapna P. Patel, Alexander J. Lazar, Michael T. Tetzlaff, Karen C. Dwyer, Ignacio I. Wistuba, Padmanee Sharma, James P. Allison, Jianhua Zhang, Andrew Futreal, Zachary A. Cooper, Jennifer A. Wargo. Genomic and immune heterogeneity in synchronous melanoma metastases is associated with differential tumor growth and response to therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2392.

Somatic copy number alterations (SCNAs) affecting oncogenic drivers have a firmly established role in promoting cancer. However, no agreed-upon standard exists for calling locus-specific amplifications and deletions in each patient sample. Here, we report the correlative analysis of copy number amplitude and length with gene expression across 6,109 samples from The Cancer Genome Atlas (TCGA) dataset across 16 cancer types. Using specificity, sensitivity, and precision-based scores, we analyzed over 30 recurrently focally altered loci that had sufficient pan-cancer penetrance for robust statistical assessment. We were able to assign optimized amplitude and length cutoffs for nine recurrent SCNAs affecting known oncogenic drivers, using mRNA expression as a functional readout. These cutoffs captured the majority of SCNA-driven, highly-expression-altered samples. The majority of oncogenes required only amplitude cutoffs, as high amplitude samples were almost invariably focal; however, CDKN2A and PTEN uniquely required both amplitude and length cutoffs as primary predictors. For PTEN, these extended to downstream AKT activation. In contrast, the remaining 20+ assessed SCNA genes, particularly ones located peri-telomerically or in fragile sites, showed poor expression-copy number correlations. Finally, we describe several novel associations between copy number and gene expression that have implications for how SCNAs are assessed as harboring candidate cancer driver genes. Overall, our analyses identify optimized amplitude and length cutoffs as efficient predictors of gene expression changes for specific oncogenic SCNAs, yet warn against one-size-fits-all interpretations across all loci. Our results have implications for cancer data analyses and the clinic, where copy number and mutation data are increasingly used to personalize cancer therapy. Citation Format: Jason Roszik, Ching Jiun Wu, Alan E. Siroy, Alexander J. Lazar, Michael A. Davies, Scott Woodman, Lawrence N. Kwong. Somatic copy number alterations at oncogenic loci show diverse correlations with gene expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-330.

T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. Here, we interrogated the role of loss of expression of the tumor suppressor, PTEN, in immune resistance. In preclinical studies, we found that silencing PTEN in tumor cells inhibited T cell-mediated tumor killing and decreased T cell trafficking into tumors. In clinical studies, we observed that tumors with loss of PTEN had significantly less CD8+ T cell infiltration than PTEN-present tumors. In addition, 26% of melanomas that did not yield successful TIL growth demonstrated PTEN loss, which was more frequent than was observed in tumors that yielded successful TIL growth (11%). We further validated the association between reduced number and impaired function of TIL with PTEN loss using another independent cohort, TCGA dataset for SKCM. More importantly, we analyzed clinical outcomes of metastatic melanoma patients treated with the FDA-approved anti-PD-1 antibodies. Our analysis demonstrates that a greater reduction in tumor burden was achieved by PD-1 blockade in PTEN present patients, when compared with PTEN absent patients. To decipher the factors mediating the immunosuppressive effects of PTEN loss, we determined the expression profiles of tumor cells with or without PTEN expression. Our results indicated that PTEN loss increased the production of immunosuppressive factors, including CCL2 and VEGF. Anti-VEGF blocking antibody improved anti-tumor activity of transferred tumor-reactive T cells and enhanced tumor infiltration of transferred T cells in PTEN-silenced tumors. These results suggest that loss of PTEN can facilitate the resistance of T cell-mediated immune responses by increasing the expression of immunosuppressive factors. Given that PTEN loss results in activation of the PI3K pathway, we evaluated the efficacy of immunotherapy in combination with a selective PI3Kβinhibitor to treat spontaneously developed BRAF mutant, PTEN null melanomas in genetically engineered mouse models. Our result showed that the combination of PI3Kβ inhibitor and anti-PD-1 significantly delayed tumor growth in tumor-bearing mice. Mice treated with this combination had a median survival time of 28 days, which is longer than the survival time of mice treated with either therapy. Increased numbers of T cells at tumor sites were found in mice receiving the combination therapy compared with mice receiving either agent alone. Taken together, our results demonstrate that PTEN loss contributes to the generation of immunosuppressive tumor microenvironment. Notably, this study provides the first direct clinical evidence to support the association between PTEN loss and poor clinical outcome in immunotherapy treated patients. In addition, our study indicates that inhibition of the PI3K-AKT pathway can improve the efficacy of immunotherapy in cancer. Citation Format: Weiyi Peng, Jie Qing Chen, Chengwen Liu, Shruti Malu, Caitlin Creasy, Michael Tetzlaff, Chunyu Xu, Jodi McKenzie, Chunlei Zhang, Xiaoxuan Liang, Leila Williams, Wanleng Deng, Guo Chen, Rina Mbofung, Alexander Lazar, Carlos Torres-Cabala, Zachary Cooper, Pei-Ling Chen, Trang Tieu, Stefani Spranger, Xiaoxing Yu, Chantale Bernatchez, Marie-Andree Forget, Cara Haymaker, Rodabe Amaria, Jennifer McQuade, Isabella Glitza, Tina Cascone, Haiyan Li, Lawrence Kwong, Timothy Heffernan, Jianhua Hu, Roland Bassett, Marcus Bosenberg, Scott Woodman, Willem Overwijk, Gregory Lizée, Jason Roszik, Thomas Gajewski, Jennifer Wargo, Jeffrey Gershenwald, Laszlo Radvanyi, Michael Davies, Patrick Hwu. Loss of PTEN promotes resistance to T cell-mediated immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4363.

TPS9594Background: Improving T cell trafficking to tumors may help augment anti-tumor responses in ACT using tumor infiltrating lymphocytes (TILs). mm produces the chemokines CXCL1 and CXCL8, which...

9535Background: Our previous analysis of advanced CM pts and the TCGA demonstrated that BRAFV600 (V600), NRAS, TP53 and BRAFNon-V600 (NonV600) muts are common in CMs. As V600 and NRAS muts are both...

We have made major advances in the treatment of melanoma through the use of targeted therapy and immune checkpoint blockade; however, clinicians are posed with therapeutic dilemmas regarding timing and sequence of therapy. There is a growing appreciation of the impact of antitumor immune responses to these therapies, and we performed studies to test the hypothesis that clinical patterns and immune infiltrates differ at progression on these treatments. We observed rapid clinical progression kinetics in patients on targeted therapy compared to immune checkpoint blockade. To gain insight into possible immune mechanisms behind these differences, we performed deep immune profiling in tumors of patients on therapy. We demonstrated low CD8+ T-cell infiltrate on targeted therapy and high CD8+ T-cell infiltrate on immune checkpoint blockade at clinical progression. These data have important implications, and suggest that antitumor immune responses should be assessed when considering therapeutic options for patients with melanoma.

Spindle cell melanoma represents a rare but distinct subset of melanoma, and its genomic spectrum has not been fully defined.We searched our institutional database for patients with a diagnosis of pure spindle cell-type melanoma whose tumors had been analyzed for BRAF, NRAS, and KIT mutations using pyrosequencing technique.We identified 24 patients with spindle cell melanoma, including 10 patients with desmoplastic melanoma, whose tumors had been analyzed for at least one of the three genes. The median Breslow thickness was 2.6 mm, and the most common site of the primary melanoma was the trunk, followed by the head and neck region. BRAF, NRAS and KIT genomic sequencing was performed successfully in 20, 18 and 14 patients, respectively. Among the 20 melanomas with completed BRAF-sequencing analysis, 6 (30%) harbored a mutation, of which 5 (83%) had a V600E mutation and 1 (17%) had a V600R mutation. None of the melanomas harbored NRAS or KIT mutations.As has been reported in other common types of melanoma, V600 BRAF mutation is the most common mutation of those tested in spindle cell melanoma. NRAS or KIT mutation appears to be rare, if not completely absent.

Uveal melanoma is the most common primary intraocular cancer in adults. Nearly half of primary uveal melanoma tumors metastasize, but there are currently no effective therapies for metastatic uveal melanoma. The recent discovery of mutations that underlie uveal melanoma metastasis, growth, and survival provide a key to the molecular understanding of this disease. Much work is now underway to leverage this knowledge to develop effective therapies. This review summarizes recently discovered molecular features of uveal melanoma and therapies being explored to capitalize on this knowledge.

BACKGROUND: Melanoma patients with LMD have a median survival of 1.8 months; there is significant medical need for innovative therapeutic strategies. METHODS: Stage IV melanoma patients were diagnosed with LMD by positive CSF cytology and/or leptomeningeal enhancement on post-contrast MRI. Patients received treatment with 1-5 doses/week of intrathecal IL-2 (IT IL2) for 4 weeks, followed by weekly injections for 4 weeks, then every other week depending on tolerance and response. The patients were ultimately given maintenance dosing of a single injection every 1-3 months under FDA approved compassionate-use investigational new drug application. Log-rank tests and Cox proportional hazards regression were used to assess statistical significance. RESULTS: The median age among 42 pts (32 male, 10 female) was 46.7 years (range 18.8-71.0). Symptoms due to increased intracranial pressure developed initially in all pts, and were controlled with supportive medications and/or CSF removal. The median overall survival (OS) was 9.1 months (range 0.7 - 86.2); 16% of pts had OS > 24 mos. Pts with LMD only without extracranial disease (ECD) (n = 4) had a median OS of 38.4 months. Pts with LMD with controlled ECD (n = 19, median OS 11.0 months) survived longer than pts with progressive ECD (n = 11, 6.5 mos; p = 0.30). Previous or concurrent brain metastases had no impact on OS. Shorter OS was associated with the presence of neurological symptoms at baseline (HR 2.1, p = 0.03). OS was not significantly associated with age, gender, LDH, or BRAF mutation status. CONCLUSIONS: The results support that IT IL2 can potentially improve the OS in a subset of pts, particularly those with no or controlled ECD, negative cytology, and no neurological symptoms. Furthermore, these results provide a strong rationale to evaluate IT immunotherapy in prospective clinical trials.

Meeting abstracts Cryo is an effective modality for pain palliation and local control of soft tissue and bone metastases. Cryo induces necrotic cell death, and combination cryo with anti-CTLA-4 generates potent systemic anti-tumor immune responses in preclinical models and small clinical studies.

Purpose: Activation of the phosphoinositide 3-kinase (PI3K)-AKT pathway has been implicated in melanoma based primarily on the prevalence of mutations in PTEN and NRAS. To improve our understanding of the regulation and clinical significance of the PI3K-AKT pathway in melanoma, we quantitatively measured the levels of phosphorylated AKT, its substrate GSK3α/β, and its negative regulator PTEN in clinical metastases. Results were compared with mutational status, clinical outcomes, and sites of metastasis. Experimental Design: DNA and protein were isolated from dissected frozen melanoma metastases (n = 96). Activating mutations of BRAF, NRAS, AKT, PIK3CA, and KIT were detected by mass spectroscopy genotyping. Phosphorylated AKT (Ser473 and Thr308), P-GSK3α/β, and PTEN protein expression were measured by reverse-phase protein array. A panel of human melanoma cells lines (n = 58) was analyzed for comparison. Results: BRAF-mutant tumors had higher levels of P-AKT-Ser473 (P = 0.01), P-AKT-Thr308 (P = 0.002), and P-GSK3α/β (P = 0.08) than NRAS-mutant tumors. Analysis of individual tumors showed that almost all tumors with elevated P-AKT had low PTEN levels; NRAS-mutant tumors had normal PTEN and lower P-AKT. Similar results were observed in melanoma cell lines. Stage III melanoma patients did not differ in overall survival based on activation status of the PI3K-AKT pathway. Brain metastases had significantly higher P-AKT and lower PTEN than lung or liver metastases. Conclusions: Quantitative interrogation of the PI3K-AKT pathway in melanoma reveals unexpected significant differences in AKT activation by NRAS mutation and PTEN loss, and hyperactivation of AKT in brain metastases. These findings have implications for the rational development of targeted therapy for this disease. (Clin Cancer Res 2009;15(24):7538–46)

The role of immunohistochemistry in the assessment of KIT status in melanomas, especially acral lentiginous/mucosal, is not well established. Although the reported prevalence of KIT mutations in acral lentiginous/ mucosal melanomas is relatively low, detection of mutations in KIT can have profound therapeutic implications. We evaluated the efficacy of immunohistochemistry to predict mutations in KIT. One hundred seventy-three tumors, comprising primary and metastatic melanomas (141 acral lentiginous/mucosal, 5 nodular, 4 lentigo maligna, 3 superficial spreading, 2 uveal, 1 melanoma of soft parts, 8 metastases from unclassified primaries, and 9 metastases from unknown primaries) were studied. Immunohistochemical expression of KIT using an anti-CD117 antibody and KIT mutational analysis by gene sequencing of exons 11, 13, and 17 were performed. Eighty-one percent of acral lentiginous/mucosal melanomas, primary and metastatic, showed KIT expression by at least 5% of the tumor cells. The overall frequency of activating KIT gene mutations in acral lentiginous/ mucosal melanomas was 15% (14 out of 91 cases), being the L576P mutation in exon 11 the most frequently detected (4 of 14 cases). Cases showing less than 10% positive tumor cells were negative for KIT mutations. Eighty-two percent (12 of 14) of cases positive for KIT mutation showed KIT expression in more than 50% of the cells. An association between immunohistochemical expression of KIT and mutation status was found (P= 0.007). Immunohistochemical expression of KIT in less than 10% of the cells of the invasive component of acral lentiginous/mucosal melanomas appears to be a strong negative predictor of KIT mutation and therefore can potentially be used to triage cases for additional KIT genotyping.

9039 Background: Activating mutations in KIT are present in 15-20% of acral lentiginous (AL), mucosal (MM) and chronic sun-damaged melanomas. Functional studies in KIT mutant melanoma have been ham...

9057 Background: Cutaneous melanomas (CM) have a high rate of somatic mutations. We analyzed the prevalence and correlations of mutations in 201 cancer-related genes to identify clinically signific...

9064 Background: The success of personalized cancer therapies targeting patients with specific actionable mutations underscores the need to expand the search for relevant mutations with functional ...

9071 Background: Next-generation sequencing (NGS) panels of cancer-related genes are increasingly utilized in oncology. We developed an algorithm to estimate total tumor mutation burden in melanoma using genes present in local and commercial NGS panels. The algorithm was validated using independent datasets and analyzed for clinical significance in two cohorts of advanced melanoma patients (pts). Methods: Overlapping genes (n = 170) from two NGS panels were used for algorithm development. Publicly available whole exome sequencing (WES) data (n = 345) from the cutaneous melanoma TCGA was employed to develop a mutation score for each gene in the panel. The summation of the mutation scores yields the Predicted Total Mutation Load (PTML) of the tumor. The algorithm was applied to 3 independent melanoma WES datasets to test the correlation of predicted with actual mutation burden. The PTML was then determined for cohorts of melanoma pts treated with immune-therapy regimens (e.g., adoptive cell transfer (ACT)),...

e20014 Background: Chemotherapy induced tumor cell death triggers immune response which can be potentiated by the use of immune stimulating checkpoint blockade. We have conducted a first in human, ...

Previous co-immunoprecipitation studies have shown that endogenous PFK-M (phosphofructokinase, muscle-specific isoform) associates with caveolin (Cav)-3 under certain metabolic conditions. However, it remains unknown whether Cav-3 expression is required for the plasma membrane recruitment and caveolar targeting of PFK-M. Here, we demonstrate that recombinant expression of Cav-3 dramatically affects the subcellular localization of PFK-M, by targeting PFK-M to the plasma membrane, and by trans-locating PFK-M to caveolae-enriched membrane domains. In addition, we show that the membrane recruitment and caveolar targeting of PFK-M appears to be strictly dependent on the concentration of extracellular glucose. Interestingly, recombinant expression of PFK-M with three Cav-3 mutants [DeltaTFT (63 to 65), P104L, and R26Q], which harbor the same mutations as seen in the human patients with Cav-3-related muscle diseases, causes a substantial reduction in PFK-M expression levels, and impedes the membrane recruitment of PFK-M. Analysis of skeletal muscle tissue samples from Cav-3(-/-) mice directly demonstrates that Cav-3 expression regulates the phenotypic behavior of PFK-M. More specifically, in Cav-3-null mice, PFK-M is no longer targeted to the plasma membrane, and is excluded from caveolar membrane domains. As such, our current results may be important in understanding the pathogenesis of Cav-3-related muscle diseases, such as limb-girdle muscular dystrophy-1C, distal myopathy, and rippling muscle disease, that are caused by mutations within the human Cav-3 gene.

Microvascular permeability is mediated by (i) the caveolar transcytosis of molecules across endothelial cells and (ii) the paracellular movement of ions and nutrients. Recently, we derived Cav-1 (-/-) knock-out mice using standard homologous recombination techniques. These mice are viable but show a loss of endothelial cell caveolae and striking defects in caveolae-mediated endocytosis. Thus, a compensatory mechanism must be operating in these mice. One possible compensatory response would be an increase in the paracellular pathway, resulting in increased microvascular permeability. To test this hypothesis directly, we studied the microvascular permeability of Cav-1 null mice using a variety of complementary in vivo approaches. Radio-iodinated bovine serum albumin was injected into Cav-1-deficient mice, and its rate of clearance from the circulatory system was compared with that of wild type control mice. Our results indicate that iodinated bovine serum albumin is removed from the circulatory system of Cav-1-deficient mice at a substantially faster rate. To determine whether this defect is restricted to the paracellular movement of albumin, lungs from Cav-1-deficient mice were next perfused with the electron dense dye Ruthenium Red. Micrographs of lung endothelial cells from Cav-1-deficient mice demonstrate that the paracellular movement of Ruthenium Red is dramatically increased. In addition, electron micrographs of Cav-1-deficient lung capillaries reveal defects in tight junction morphology and abnormalities in capillary endothelial cell adhesion to the basement membrane. This defect in cell-substrate attachment is consistent with the postulated role of caveolin-1 in positively regulating integrin signaling. Because loss of caveolin-1 expression results in constitutive activation of eNOS activity, we also examined whether these increases in microvascular permeability are NO-dependent. Interestingly, treatment with l-NAME (a well established nitric-oxide synthase inhibitor) successfully reversed the microvascular hyperpermeability phenotype of Cav-1 knock-out mice. Thus, caveolin-1 plays a dual regulatory role in controlling microvascular permeability: (i) as a structural protein that is required for caveolae formation and caveolar transcytosis and (ii) as a tonic inhibitor of eNOS activity to negatively regulate the paracellular pathway.

9079 Background: In this report we describe the association of specific patient attributes, such as mutation status, prior treatment with checkpoint blockade, and location of primary tumor with tre...

9100 Background: V600E and V600K mutations are the most common BRAF mutations in melanoma. We previously showed that non-V600 BRAF mutations account for 12% of all BRAF mutations in melanoma. Limited clinical data are available for non-V600 BRAF mutant melanomas. To better understand non-V600 BRAF mutations in melanoma, characteristics of tumor and clinical outcomes were investigated. Methods: Clinical and pathologic data were collected retrospectively in patients (pts) with melanoma who had multi-gene mutation analysis (next-gene sequencing) at MD Anderson Cancer Center. Fisher's exact test was used for all comparisons. Results: We found 52 pts with non-V600 BRAF mutations, among whom 8 had NRAS/non-V600 co-mutations. Common non-V600 mutations were K601E (17.3%), G469E (15.4%), G469R (7.7%), D594G (5.8%), L597S (5.8%), and S467L (5.8%). Compared with V600E mutations as a control (n=82) , the presence of a non-V600 mutations was significantly associated with older age (median, 58 years vs 50; P=0.001), le...

A fundamental aim of cancer research is to identify the molecular changes that cause normal cells to evolve into malignant tumors. Malignant transformation has been proposed to occur as a consequence of the accumulation of genomic aberrations that successively overcome the cellular barriers to malignancy. Although a concerted effort has been made to discover the complement of molecular aberrations required for malignant transformation, the spectrum of events that must occur in a single cell, including which oncogene activation and tumor suppressor gene inactivation events can cooperate to induce malignant transformation, has remained elusive. A number of benign lesions without malignant potential accumulate genomic aberrations and have the potential to inform on the changes that are tolerated in benign tumors; however, they are insufficient to induce malignant transformation. In PNAS, Hafner et al. (1) comprehensively investigate the mutation status, mechanistic barriers to malignancy, and clonal relationship of seborrheic keratoses (SK), a tumor lineage that is without malignant potential.

e20036 Background: BRAF mutations are found in ~50% of melanoma. V600E/K substitutions are the most common and well-characterized. We analyzed the clinical characteristics of patients with non-V600E/K BRAF mutations. Methods: We identified 38 melanoma patients whose tumor contained a non-V600E/K BRAF mutation and reviewed the clinical characteristics. The sequencing analysis was performed with either pyrosequencing or next generation sequencing assay. Results: 38 patients were identified with non-V600E/K BRAF mutations. Mutations detected in more than 1 patient included V600R (n=14, 37%), K601E (5, 13%), G469E (3, 8%); L597S (3, 7%), D594G (2, 5%); 11 other mutations were identified in single patients. Median age was 57 yrs; 82% were men; 95% were white. The common primary subtypes were nodular (26%), superficial spreading (24%), unknown primary (21%); no one were acral, mucosal or uveal melanomas. Ten (26%) of 27 with known ulceration status had ulceration, and 3 (7%) of 22 with known mitosis status had < 1 mitosis /mm2. The sites of primary melanoma were located mostly in the head/neck and the trunk (63%), extremities (16%) and unknown primary (21%). The stage at diagnosis was I /II (29%), III (40%), IV (18%) and unknown (13%). Among 33 (87%) patients who ultimately developed distant metastases, 23(67%) had metastasis in the soft tissue/nodes, 21 (63%) in the lung, 9 (24%) in the brain, 7 (21%) in the liver, 6 (16%) in the bone, and 5 (15%) in the adrenal gland. Among patients (n=25) with initial stage I-III melanoma who later developed distant metastasis, the median duration between the time of initial diagnosis and distant metastases was 36 months. Among the 32 (84%) of the patients who developed stage IV melanoma, the median survival from the time of stage IV diagnosis was 18 months. Five patients received vemurafenib treatment, and 2 patients (K601E; T599_V600ins2) had stable disease and 2 patients (L597Q; G466E) had disease progression and 1 lost to follow up. Conclusions: Unexpectedly higher proportion of the patients with non-V600E/K mutant-melanoma had unknown primary melanoma and higher mitotic rate. In a small number of patients who received vemurafenib, the clinical response appears to be low.