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Purpose: Ocular adnexal (OA) sebaceous carcinoma is an aggressive malignancy of the eyelid and ocular adnexa that frequently recurs and metastasizes, and effective therapies beyond surgical excision are lacking. There remains a critical need to define the molecular-genetic drivers of the disease to understand carcinomagenesis and progression and to devise novel treatment strategies. Experimental Design: We present next-generation sequencing of a targeted panel of cancer-associated genes in 42 and whole transcriptome RNA sequencing from eight OA sebaceous carcinomas from 29 patients. Results: We delineate two potentially distinct molecular-genetic subtypes of OA sebaceous carcinoma. The first is defined by somatic mutations impacting TP53 and/or RB1 [20/29 (70%) patients, including 10 patients whose primary tumors contained coexisting TP53 and RB1 mutations] with frequent concomitant mutations affecting NOTCH genes. These tumors arise in older patients and show frequent local recurrence. The second subtype [9/29 (31%) patients] lacks mutations affecting TP53, RB1, or NOTCH family members, but in 44% (4/9) of these tumors, RNA sequencing and in situ hybridization studies confirm transcriptionally active high-risk human papillomavirus. These tumors arise in younger patients and have not shown local recurrence. Conclusions: Together, our findings establish a potential molecular-genetic framework by which to understand the development and progression of OA sebaceous carcinoma and provide key molecular-genetic insights to direct the design of novel therapeutic interventions.

Purpose Conjunctival squamous cell carcinoma (SCC), a type of ocular surface neoplasia, is primarily treated by surgical resection and topical immuno- or chemotherapy. Metastatic disease may be treated with systemic chemo- or immunotherapy, albeit with variable response. The purpose of this study was to determine whether immune checkpoint blockade might be considered in the management of conjunctival SCC. Methods In this retrospective study, we evaluated tumor programmed death-ligand 1 (PD-L1) expression, high-risk human papillomavirus (HPV) status, and immunohistochemical expression of cluster of differentiation 3 (CD3), cluster of differentiation 8 (CD8), and programmed death 1 (PD1) in tumor-associated immune infiltrate in a series of 31 conjunctival SCCs. Results PD-L1 expression in ≥1% of tumor cells was noted in 14 conjunctival SCCs (47%) and was more prevalent in invasive than in situ SCC and among tumors with higher American Joint Committee on Cancer (AJCC) T category (≥T3 versus ≤T2). The density of CD3-positive T cells was higher in primary than recurrent tumors and higher in invasive than in situ tumors. Density of CD3-positive and CD8-positive T cells was higher in higher AJCC stage tumors. Density of CD8-positive T cells was higher in HPV-positive than HPV-negative tumors. PD-L1 expression correlated with a higher density of CD3-, CD8-, and PD1-positive cells in the tumor-associated immune infiltrate but not with HPV status. Conclusions Our findings demonstrate that PD-L1 is expressed in almost half of conjunctival SCCs. The density of tumor-associated immune cells correlated with invasive SCC, stage, and HPV status in conjunctival SCC. Our findings support further studies to establish the potential application of immune checkpoint blockade in the management of conjunctival SCC.

Immunotherapies such as anti-CTLA-4 immune checkpoint blockade (ICB) have revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by off-target tissue damage or immune-related adverse events (irAEs). At present, there is limited understanding of irAE mechanisms, hampering development of approaches to mitigate their damage. We addressed this problem by generating animal models of intestinal irAE. Our results show that disruption of homeostatic immunity by genetic predisposition to intestinal inflammation or acute gastrointestinal infection sensitizes mice to anti-CTLA-4-mediated intestinal toxicity. Inflammation-prone mice treated with anti-CTLA-4 showed neutrophil accumulation, systemic interleukin-6 (IL-6) release, and dysbiosis. Significantly, IL-6 blockade combined with antibiotic treatment improved anti-CTLA-4 therapeutic efficacy and reduced intestinal irAEs. Immune signatures were validated in biopsies from patients who developed colitis during ICB, supporting the utility of our models. This study provides new pre-clinical models, mechanistic insight into irAEs, and potential approaches to enhance ICB efficacy while mitigating irAEs. Citation Format: Yifan Zhou, Yusra B. Medik, Bhakti Patel, Daniel B. Zamler, Sijie Chen, Thomas Chapman, Sarah Schneider, Rachel L. Babcock, Taylor T. Chrisikos, Laura M. Kahn, Allison M. Dyevoich, Elizabeth M. Park, Alexandria P. Cogdill, Daniel H. Johnson, Sarah B. Johnson, Khalida M. Wani, Debora A. Ledesma, Courtney W. Hudgens, Jingjing Wang, Md Abdul Wadud Khan, Aron Y. Joon, Weiyi Peng, Haiyan S. Li, Reetakshi Arora, Ximing Tang, Maria Gabriela Raso, Xuegong Zhang, Wai Chin Foo, Michael T. Tetzlaff, Gretchen E. Diehl, Karen Clise-Dwyer, Elizabeth M. Whitley, Matthew M. Gubin, James P. Allison, Patrick Hwu, Nadim J. Ajami, Adi Diab, Jennifer A. Wargo, Stephanie S. Watowich. Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5545.

BackgroundRecently, we showed that melanoma brain metastases (MBMs) are characterized by increased utilization of the oxidative phosphorylation (OXPHOS) metabolic pathway compared to melanoma extracranial metastases (ECMs). MBM growth was inhibited by a potent direct OXPHOS inhibitor, but observed toxicities support the need to identify alternative therapeutic strategies. Thus, we explored the features associated with OXPHOS to improve our understanding of the pathogenesis and potential therapeutic vulnerabilities of MBMs.MethodsWe applied an OXPHOS gene signature to our cohort of surgically resected MBMs that had undergone RNA-sequencing (RNA-seq) (n = 88). Clustering by curated gene sets identified MBMs with significant enrichment (High-OXPHOS; n = 21) and depletion (Low-OXPHOS; n = 25) of OXPHOS genes. Clinical data, RNA-seq analysis, and immunohistochemistry were utilized to identify significant clinical, molecular, metabolic, and immune associations with OXPHOS in MBMs. Preclinical models were used to further compare melanomas with High- and Low-OXPHOS and for functional validation.ResultsHigh-OXPHOS MBMs were associated with shorter survival from craniotomy compared to Low-OXPHOS MBMs. High-OXPHOS MBMs exhibited an increase in glutamine metabolism, and treatment with the glutaminase inhibitor CB839 improved survival in mice with MAPKi-resistant, High-OXPHOS intracranial xenografts. High-OXPHOS MBMs also exhibited a transcriptional signature of deficient immune activation, which was reversed in B16-F10 intracranial tumors with metformin treatment, an OXPHOS inhibitor.ConclusionsOXPHOS is associated with distinct clinical, molecular, metabolic, and immune phenotypes in MBMs. These associations suggest rational therapeutic strategies for further testing to improve outcomes in MBM patients.

Targeted and immunotherapy regimens have revolutionized the treatment of advanced melanoma patients. Despite this, only a subset of patients respond durably. Recently, combination strategies of BRAF/MEK inhibitors with immune checkpoint inhibitor monotherapy (��-CTLA-4 or ��-PD-1) have increased the rate of durable responses. Based on evidence from our group and others, these therapies appear synergistic, but at the cost of significant toxicity. We know from other treatment paradigms (e.g. hematologic malignancies) that combination strategies with multi-drug regimens (>4 drugs) are associated with more durable disease control. To better understand the mechanism of these improved outcomes, and to identify and prioritize new strategies for testing, we studied several multi-drug regimens combining BRAF/MEK targeted therapy and immunotherapy combinations in a Braf-mutant murine melanoma model (BrafV600E/Pten���/���). Short-term treatment with ��-PD-1 and ��-CTLA-4 monotherapies were relatively ineffective, while treatment with ��-OX40 demonstrated some efficacy [17% of mice with no evidence of disease, (NED), at 60-days]. Outcomes were improved in the combined ��-OX40/��-PD-1 group (42% NED). Short-term treatment with quadruplet therapy of immunotherapy doublets in combination with targeted therapy [dabrafenib and trametinib (DT)] was associated with excellent tumor control, with 100% of mice having NED after combined DT/��-CTLA-4/��-PD-1 or DT/��-OX40/��-PD-1. Notably, tumors from mice in these groups demonstrated a high proportion of effector memory T cells, and immunologic memory was maintained with tumor re-challenge. Together, these data provide important evidence regarding the potential utility of multi-drug therapy in treating advanced melanoma and suggest these models can be used to guide and prioritize combinatorial treatment strategies.

Immune-checkpoint inhibitors and adoptive tumor-infiltrating lymphocyte (TIL) therapies have profoundly improved the survival of patients with melanoma. However, a majority of patients do not respond to these agents, and many responders experience disease relapse. Although numerous innovative treatments are being explored to offset the limitations of these agents, novel therapeutic combinations with immunotherapies have the potential to improve patient responses. In this study, we evaluated the antimelanoma activity of immunotherapy combinations with Telaglenastat (CB-839), a potent glutaminase inhibitor (GLSi) that has favorable systemic tolerance. In in vitro TIL:tumor coculture studies, CB-839 treatment improved the cytotoxic activity of autologous TILs on patient-derived melanoma cells. CB-839 treatment decreased the conversion of glutamine to alpha-ketoglutarate (αKGA) more potently in tumor cells versus TILs in these cocultures. These results suggest that CB-839 may improve immune function in a tumor microenvironment by differentially altering tumor and immune cell metabolism. In vivo CB-839 treatment activated melanoma antigen–specific T cells and improved their tumor killing activity in an immune-competent mouse model of adoptive T-cell therapy. Additionally, the combination of CB-839 with anti-PD1 or anti-CTLA4 antibodies increased tumor infiltration by effector T cells and improved the antitumor activity of these checkpoint inhibitors in a high mutation burden mouse melanoma model. Responsiveness to these treatments was also accompanied by an increase of interferon gamma (IFNγ)–associated gene expression in the tumors. Together, these results provide a strong rationale for combining CB-839 with immune therapies to improve efficacy of these treatments against melanoma.

Uveal melanoma is a rare and aggressive malignancy and up to half of all patients will develop metastatic disease despite the effective treatment of the primary tumor. Insulin-like growth factors I/II play a fundamental role in the cell migration, proliferation, and apoptosis. IMC-A12, a mAb specifically targets insulin-like growth factor type I receptor, has shown promise in preclinical studies. We performed a multicenter phase II study for patients with metastatic uveal melanoma administered IMC-A12 10 mg/kg IV every two weeks until disease progression or unacceptable toxicity. The primary endpoint was objective response (proportion of patients with complete or partial response), and secondary endpoints were disease control rate, progression-free survival, and overall survival. A total of 18 patients enrolled in this study (10 males and eight females) with a median age. Ten patients (55%) had stable disease, seven patients (38%) had progression as best overall response. No partial response or complete response was observed; however, the disease control rate, defined as complete response + partial response + stable disease ≥3 months, was 50%. Median progression-free survival was 3.1 months, and median overall survival was 13.8 months. Adverse events of any grade occurred in 13 patients (72.2%). Treatment-related grade 3 adverse events were rare, and there were no grade 4 or 5 related adverse events. IMC-A12 was very well tolerated, however, showed limited clinical activity in uveal melanoma as a single agent. Due to its low toxicity profile it could be studied in combination with other pathway-specific agents.

Treatment with molecularly-targeted therapy has revolutionized cancer care, including BRAF/MEK-targeted melanoma therapy. However responses are heterogenous and frequently not long-lasting. Novel strategies to target resistance are needed. We studied a cohort of patients with resectable metastatic melanoma treated with neoadjuvant BRAF/MEK-targeted therapy (n=52) and noted a strong sexual dimorphism in response to treatment, with female patients demonstrating significantly higher rates of a major pathologic response (MPR) (p=0.0001). RNA sequencing of tumors demonstrated enrichment of androgen-related genes in those failing to achieve MPR. Pre-clinical studies validated these findings, with significantly increased tumor growth in male vs female mice treated with BRAF/MEK inhibitors (BRAF/MEKi) (p=0.0005). Androgen receptor (AR) expression was upregulated in tumors of BRAF/MEKi-treated mice, and modulation of AR signaling via AR-blockade or castration was associated with significantly slower tumor growth (p=0.0001 and p=0.00004, respectively). Together, these results have important implications in the context of treatment with BRAF/MEKi-targeted therapy.

Complex tumor microenvironmental (TME) features influence the outcome of cancer immunotherapy (IO). Here we perform immunogenomic analyses on 67 intratumor sub-regions of a PD-1 inhibitor-resistant melanoma tumor and 2 additional metastases arising over 8 years, to characterize TME interactions. We identify spatially distinct evolution of copy number alterations influencing local immune composition. Sub-regions with chromosome 7 gain display a relative lack of leukocyte infiltrate but evidence of neutrophil activation, recapitulated in The Cancer Genome Atlas (TCGA) samples, and associated with lack of response to IO across three clinical cohorts. Whether neutrophil activation represents cause or consequence of local tumor necrosis requires further study. Analyses of T-cell clonotypes reveal the presence of recurrent priming events manifesting in a dominant T-cell clonotype over many years. Our findings highlight the links between marked levels of genomic and immune heterogeneity within the physical space of a tumor, with implications for biomarker evaluation and immunotherapy response., Immunotherapies now dominate the treatment landscape for melanoma, but why they only work in a subset of patients remains unclear. Here, the authors perform an immunogenomic analysis on 67 intratumor sub-regions of a PD-1 inhibitor resistant melanoma, and 2 additional metastases from a single patient, mapping the spatial relationships between genomic and immune heterogeneity at high resolution.

Multiomic profiling of several cohorts of patients treated with immune checkpoint blockade highlights the presence and potential role of B cells and tertiary lymphoid structures in promoting therapy response.Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers(1-10) and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity(11-15), although these have been less well-studied in ICB treatment(16). A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling(17) that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter(18)) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

The integrity of the immune system represents a pivotal risk factor and prognostic biomarker for Merkel cell carcinoma. A higher density of tumor-associated T cells correlates with improved Merkel cell carcinoma–specific survival, but the prognostic importance of the T-cell infiltrate reactivity is unknown. We evaluated the T-cell receptor repertoire associated with 72 primary Merkel cell carcinomas and correlated metrics of the T-cell receptor repertoire with clinicopathologic characteristics and patient outcomes. We showed that a high Simpson's Dominance index (SDom) was significantly associated with fewer metastases (P = 0.01), lower stage at presentation (P = 0.02), lower final stage at last follow-up (P = 0.05), and longer time to first lymph node metastasis (P = 0.04). These correlations were mostly preserved in the Merkel cell polyomavirus–negative subgroup. Combining SDom with CD3+ or CD8+ T-cell density revealed three distinct prognostic groups with respect to disease-specific survival. Patients with both high SDom and high CD3+ or CD8+ T-cell density had markedly improved disease-specific survival compared with patients with low SDom and low CD3+ or CD8+ T-cell density (P = 0.002 and P = 0.03, respectively). Patients with either high SDom or high CD3+ or CD8+ had intermediate disease-specific survival. Our findings demonstrate that the quality of the tumor-associated T-cell infiltrate informs patient prognosis in primary Merkel cell carcinoma beyond the T-cell density.

Preclinical studies suggest that treatment with neoadjuvant immune checkpoint blockade is associated with enhanced survival and antigen-specific T cell responses over adjuvant treatment1; however, optimal regimens have not been defined. Herein, we report results from a randomized phase II study of neoadjuvant nivolumab versus combined ipilimumab with nivolumab in 23 patients with high-risk resectable melanoma (NCT02519322). RECIST overall response rates (ORR), pathologic complete response rates (pCR), treatment-related adverse events (trAEs), and immune correlates of response were assessed. Treatment with combined ipilimumab and nivolumab yielded high response rates (RECIST ORR 73%, pCR 45%) but substantial toxicity (73% grade 3 trAEs), whereas treatment with nivolumab monotherapy yielded modest responses (ORR 25%, pCR 25%) and low toxicity (8% grade 3 trAEs). Immune correlates of response were identified, demonstrating higher lymphoid infiltrates in responders to both therapies and a more clonal and diverse T cell infiltrate in responders to nivolumab monotherapy. These results are the first to describe the feasibility of neoadjuvant immune checkpoint blockade in melanoma and emphasize the need for additional studies to optimize treatment regimens and to validate putative biomarkers.

Summary Background Dual BRAF and MEK inhibition produces a response in a large number of patients with stage IV BRAF-mutant melanoma. The existing standard of care for patients with clinical stage III melanoma is upfront surgery and consideration for adjuvant therapy, which is insufficient to cure most patients. Neoadjuvant targeted therapy with BRAF and MEK inhibitors (such as dabrafenib and trametinib) might provide clinical benefit in this high-risk p opulation. Methods We undertook this single-centre, open-label, randomised phase 2 trial at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Eligible participants were adult patients (aged ≥18 years) with histologically or cytologically confirmed surgically resectable clinical stage III or oligometastatic stage IV BRAFV600E or BRAFV600K (ie, Val600Glu or Val600Lys)-mutated melanoma. Eligible patients had to have an Eastern Cooperative Oncology Group performance status of 0 or 1, a life expectancy of more than 3 years, and no previous exposure to BRAF or MEK inhibitors. Exclusion criteria included metastases to bone, brain, or other sites where complete surgical excision was in doubt. We randomly assigned patients (1:2) to either upfront surgery and consideration for adjuvant therapy (standard of care group) or neoadjuvant plus adjuvant dabrafenib and trametinib (8 weeks of neoadjuvant oral dabrafenib 150 mg twice per day and oral trametinib 2 mg per day followed by surgery, then up to 44 weeks of adjuvant dabrafenib plus trametinib starting 1 week after surgery for a total of 52 weeks of treatment). Randomisation was not masked and was implemented by the clinical trial conduct website maintained by the trial centre. Patients were stratified by disease stage. The primary endpoint was investigator-assessed event-free survival (ie, patients who were alive without disease progression) at 12 months in the intent-to-treat population. This trial is registered at ClinicalTrials.gov, number NCT02231775. Findings Between Oct 23, 2014, and April 13, 2016, we randomly assigned seven patients to standard of care, and 14 to neoadjuvant plus adjuvant dabrafenib and trametinib. The trial was stopped early after a prespecified interim safety analysis that occurred after a quarter of the participants had been accrued revealed significantly longer event-free survival with neoadjuvant plus adjuvant dabrafenib and trametinib than with standard of care. After a median follow-up of 18·6 months (IQR 14·6–23·1), significantly more patients receiving neoadjuvant plus adjuvant dabrafenib and trametinib were alive without disease progression than those receiving standard of care (ten [71%] of 14 patients vs none of seven in the standard of care group; median event-free survival was 19·7 months [16·2–not estimable] vs 2·9 months [95% CI 1·7–not estimable]; hazard ratio 0·016, 95% CI 0·00012–0·14, p Interpretation Neoadjuvant plus adjuvant dabrafenib and trametinib significantly improved event-free survival versus standard of care in patients with high-risk, surgically resectable, clinical stage III–IV melanoma. Although the trial finished early, limiting generalisability of the results, the findings provide proof-of-concept and support the rationale for further investigation of neoadjuvant approaches in this disease. This trial is currently continuing accrual as a single-arm study of neoadjuvant plus adjuvant dabrafenib and trametinib. Funding Novartis Pharmaceuticals Corporation.

We report here on a patient with metastatic melanoma who had extensive brain metastases. After being treated with the sequential combination of whole brain radiation therapy followed by the PD-1–inhibitory antibody, pembrolizumab, the patient had a durable complete response. Retrospective laboratory studies of T cells revealed that, after treatment with anti-PD-1 commenced, effector CD8+ T cells in the blood expanded and the ratio of CD8+:Treg T cells increased. A CD8+ T-cell clone present in the initial brain metastases was expanded in the blood after anti-PD-1 treatment, which suggested an antitumor role for this clone. Immunohistochemical analysis confirmed the presence of CD8+ T cells and low PD-L1 expression in the brain metastases before immunotherapy initiation. This sequence of therapy may provide an option for melanoma patients with unresponsive brain metastases. Cancer Immunol Res; 5(2); 100–5. ©2017 AACR.

Extramammary Paget disease (EMPD) is a locally aggressive cutaneous malignancy that usually arises in anogenital or axillary skin. Immune checkpoint inhibitors targeting programmed cell death receptor (PD-1) and/or its ligand (PD-L1) are approved for the treatment of several types of cancer, and response to these generally correlates with increased PD-L1 expression by tumor cells. The expression of PD-L1 and composition and density of the tumor-associated immune infiltrate in EMPD have been little studied. To determine whether EMPD might be amenable to immune checkpoint blockade, we analyzed the expression of PD-1 and PD-L1 and the composition and density of the tumor-associated immune infiltrate in EMPD and evaluated associations between biomarker expression and clinicopathologic parameters. Twenty-one EMPD tumors were evaluated for tumor cell PD-L1 expression and for relative expression and distribution of CD3, CD8, PD-1, and PD-L1 in the tumor-associated immune infiltrate by using a combination of visual and image analysis (Aperio ImageScope). In addition, PD-L1 expression was assessed in 10 cases of mammary Paget disease (MPD). In EMPD cases, PD-L1 was expressed by tumor cells (3/21, 14%) and the tumor-associated immune infiltrate (15/21, 71%), and PD-1 was expressed by the tumor-associated immune infiltrate in all cases analyzed (18/18). However, PD-L1 expression by EMPD tumor cells did not correlate with the density of CD3-, CD8-, or PD-1-positive cells in the tumor-associated immune infiltrate or other clinicopathologic parameters. Furthermore, the density of CD3, CD8, PD-1, and PD-L1 in the tumor-associated immune infiltrate did not correlate with any clinicopathologic parameters evaluated with the exception that CD3 positive values were significantly higher in patients who were still alive (median, 1310 cells/mm2, range, 543&ndash, 2115, ) than in those who died (median, 611 cells/mm2, range, 481&ndash, 908, p = 0.049). In all MPD cases, PD-L1 was absent in tumor cells but present in the tumor-associated immune infiltrate, and PD-L1 expression in lymphocytes was lower in patients with HER2/neu-positive than in those with HER2/neu-negative disease (p = 0.07). Our findings raise the possibility of therapeutic targeting of the PD-1/PD-L1 axis in EMPD.

Purpose: The purpose of this study is to determine if inhibition of mitochondrial oxidative phosphorylation (OxPhos) is an effective strategy against MAPK pathway inhibitor (MAPKi)–resistant BRAF-mutant melanomas. Experimental Design: The antimelanoma activity of IACS-010759 (OPi), a novel OxPhos complex I inhibitor, was evaluated in vitro and in vivo. Mechanistic studies and predictors of response were evaluated using molecularly and metabolically stratified melanoma cell lines. 13C-labeling and targeted metabolomics were used to evaluate the effect of OPi on cellular energy utilization. OxPhos inhibition in vivo was evaluated noninvasively by [18F]-fluoroazomycin arabinoside (FAZA) PET imaging. Results: OPi potently inhibited OxPhos and the in vivo growth of multiple MAPKi-resistant BRAF-mutant melanoma models with high OxPhos at well-tolerated doses. In vivo tumor regression with single-agent OPi treatment correlated with inhibition of both MAPK and mTOR complex I activity. Unexpectedly, antitumor activity was not improved by combined treatment with MAPKi in vitro or in vivo. Signaling and growth-inhibitory effects were mediated by LKB1–AMPK axis, and proportional to AMPK activation. OPi increased glucose incorporation into glycolysis, inhibited glucose and glutamine incorporation into the mitochondrial tricarboxylic acid cycle, and decreased cellular nucleotide and amino acid pools. Early changes in [18F]-FAZA PET uptake in vivo, and the degree of mTORC1 pathway inhibition in vitro, correlated with efficacy. Conclusions: Targeting OxPhos with OPi has significant antitumor activity in MAPKi-resistant, BRAF-mutant melanomas, and merits further clinical investigation as a potential new strategy to overcome intrinsic and acquired resistance to MAPKi in patients.

NKTR-214 (bempegaldesleukin) is a novel IL2 pathway agonist, designed to provide sustained signaling through heterodimeric IL2 receptor βγ to drive increased proliferation and activation of CD8+ T and natural killer cells without unwanted expansion of T regulatory cells (Treg) in the tumor microenvironment. In this first-in-human multicenter phase I study, NKTR-214 administered as an outpatient regimen was well tolerated and showed clinical activity including tumor shrinkage and durable disease stabilization in heavily pretreated patients. Immune activation and increased numbers of immune cells were observed in the periphery across all doses and cycles with no loss of NKTR-214 activity with repeated administration. On-treatment tumor biopsies demonstrated that NKTR-214 promoted immune cell increase with limited increase of Tregs. Transcriptional analysis of tumor biopsies showed that NKTR-214 engaged the IL2 receptor pathway and significantly increased genes associated with an effector phenotype. Based on safety and pharmacodynamic markers, the recommended phase II dose was determined to be 0.006 mg/kg every three weeks. Significance: We believe that IL2- and IL2 pathway–targeted agents such as NKTR-214 are key components to an optimal immunotherapy treatment algorithm. Based on its biological activity and tolerability, NKTR-214 is being studied with approved immuno-oncology agents including checkpoint inhibitors. See related commentary by Sullivan, p. 694. This article is highlighted in the In This Issue feature, p. 681

There is a critical need to improve our understanding of the pathogenesis of melanoma brain metastases (MBM). Thus, we performed RNA sequencing on 88 resected MBMs and 42 patient-matched extracranial metastases; tumors with sufficient tissue also underwent whole-exome sequencing, T-cell receptor sequencing, and IHC. MBMs demonstrated heterogeneity of immune infiltrates that correlated with prior radiation and post-craniotomy survival. Comparison with patient-matched extracranial metastases identified significant immunosuppression and enrichment of oxidative phosphorylation (OXPHOS) in MBMs. Gene-expression analysis of intracranial and subcutaneous xenografts, and a spontaneous MBM model, confirmed increased OXPHOS gene expression in MBMs, which was also detected by direct metabolite profiling and [U-13C]-glucose tracing in vivo. IACS-010759, an OXPHOS inhibitor currently in early-phase clinical trials, improved survival of mice bearing MAPK inhibitor–resistant intracranial melanoma xenografts and inhibited MBM formation in the spontaneous MBM model. The results provide new insights into the pathogenesis and therapeutic resistance of MBMs. Significance: Improving our understanding of the pathogenesis of MBMs will facilitate the rational development and prioritization of new therapeutic strategies. This study reports the most comprehensive molecular profiling of patient-matched MBMs and extracranial metastases to date. The data provide new insights into MBM biology and therapeutic resistance. See related commentary by Egelston and Margolin, p. 581. This article is highlighted in the In This Issue feature, p. 565

IMPACT: Melanoma leptomeningeal disease (LMD) is a devastating subtype of central nervous system (CNS) metastatic disease that is associated with limited treatment options and an extremely poor prognosis, thus requiring the development of preclinical models of LMD for therapeutic development. OBJECTIVES/GOALS: 1.Develop an immunocompetent murine model of melanoma LMD with tumors bearing genetic mutations commonly found in patients, specifically BRAF(V600E)/PTEN-/-2.Assess the safety of intrathecal (IT) immunotherapy, specifically anti-PD1 antibody (aPD1)3.Evaluate the therapeutic efficacy of IT aPD1 checkpoint blockade in murine melanoma LMD METHODS/STUDY POPULATION: To develop BRAF(V600E)/PTEN-/- LMD models, we acquired BP, D4M, and D4M-UV2 (irradiated) murine melanoma cell lines and luciferase-tagged them. 1.5x10^4 cells were suspended in 10 uL serum-free media and injected into the cisterna magna of female C57BL/6 mice. Brain and spinal cord were harvested for histologic assessment once mice were moribund. To assess safety of IT aPD1, we injected IT control IgG or IT aPD1 (13 ug, 26 ug, 39 ug) and monitored weights or harvested at days 7 or 14 for IHC staining of inflammation markers. To evaluate therapeutic efficacy of IT aPD1, BP cells were directly injected as above. After 3 days, mice underwent imaging to confirm tumor uptake and randomization to receive 13 ug IT control IgG or aPD1 once + 200 ug systemic (Sys) control IgG or aPD1 (days 0, 3, and 5), and then monitored for survival. RESULTS/ANTICIPATED RESULTS: For LMD development, all mice survived cisternal injection of BP, D4M, and D4M-UV2 cells and median survival was 17, 19, and 30 days, respectively. Presence of leptomeningeal deposits was confirmed for all tumor-bearing mice by IHC for MART1. For safety of IT aPD1, all mice survived the procedure and no mice displayed morbidity or >10% weight loss over 14 days of observation. IHC assessment of brain and spinal cord samples from mice treated with 13 ug aPD1 revealed focal ischemia related to injection site and no other signs of neurological damage or inflammation. IT aPD1 treatment of mice with BP leptomeningeal tumors demonstrated no significant survival advantage, although both IT aPD1 +/- Sys aPD1 had mice live up to days 29 and 26, respectively, compared to both IT control IgG +/- Sys aPD1, for which all mice died by day 22. DISCUSSION/SIGNIFICANCE OF FINDINGS: We demonstrate that cisternal injection of murine BRAF(V600E)/PTEN-/- melanoma cell lines yield LMD with reproducible survival and that treatment with IT aPD1 in this model is feasible and safe. Together these findings establish a new model to facilitate the development of more effective immunotherapy strategies for melanoma patients with LMD.

Sebaceous carcinoma (SC) is a rare but aggressive malignancy with frequent recurrence and metastases. Surgery is the mainstay of therapy, but effective systemic therapies are lacking because the molecular alterations driving SC remain poorly understood. To identify these, we performed whole-exome next-generation sequencing of 409 cancer-associated genes on 27 SCs (18 primary/locally recurrent ocular, 5 paired metastatic ocular, and 4 primary extraocular) from 20 patients. In ocular SC, we identified 139 non-synonymous somatic mutations (median/lesion 3; range 0-23). Twenty-five of 139 mutations (18%) occurred in potentially clinically actionable genes in 6 of 16 patients. The most common mutations were mutations in TP53 (n = 9), RB1 (n = 6), PIK3CA (n = 2), PTEN (n = 2), ERBB2 (n = 2), and NF1 (n = 2). TP53 and RB1 mutations were restricted to ocular SC and correlated with aberrant TP53 and RB protein expression. Systematic pathway analyses demonstrated convergence of these mutations to activation of the PI3K signalling cascade, and PI3K pathway activation was confirmed in tumours with PTEN and/or PIK3CA mutations. Considerable inter-tumoural heterogeneity was observed between paired primary and metastatic ocular SCs. In primary extraocular SC, we identified 77 non-synonymous somatic mutations (median/lesion 22.5; range 3-29). This overall higher mutational load was attributed to a microsatellite instability phenotype in three of four patients and somatically acquired mutations in mismatch repair genes in two of four patients. Eighteen of 77 mutations (23%) were in potentially clinically actionable genes in three of four patients, including BTK, FGFR2, PDGFRB, HRAS, and NF1 mutations. Identification of potentially clinically actionable mutations in 9 of 20 SC patients (45%) underscores the importance of next-generation sequencing to expand the spectrum of genotype-matched targeted therapies. Frequent activation of PI3K signalling pathways provides a strong rationale for application of mTOR inhibitors in the management of this disease. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Neoplasms and reactivation of latent viruses have been observed in individuals taking fingolimod. Merkel cell carcinoma (MCC), a rare neuroendocrine skin cancer, is associated with immunosuppression and can be triggered by the oncogenic Merkel cell polyoma virus (MCPyV). We report a case of a 61-year-old man with multiple sclerosis who developed MCPyV-positive MCC 4 years after starting fingolimod. This is the second report of MCC associated with MCPyV in an individual on fingolimod.

Background: OXPHOS mediates resistance to BRAF and MEK inhibitors in melanoma. Recently, we showed that MBMs have higher expression of OXPHOS genes than patient-matched extracranial metastases (ECMs). The induction of, and dependence upon, OXPHOS in MBMs was confirmed in mouse xenograft models. Notably, our studies demonstrated that OXPHOS varies among MBMs. In order to improve our understanding of the High-OXPHOS phenotype, we report here the molecular, immune, metabolic, and radiomic features that correlate with OXPHOS levels in MBMs. Methods: We applied an OXPHOS gene signature to RNA-sequencing (RNA-seq) data from surgically resected MBMs (n=88). Clustering by these genes identified MBMs with significant enrichment (High-OXPHOS; n=21) and depletion (Low-OXPHOS; n=25) of OXPHOS genes. The EdgeR/limma/voom pipeline was used to perform differential gene expression (DGE) analysis between High- and Low-OXPHOS MBMs. Pathway analyses were performed via Ensemble of Gene Set Enrichment Analyses (EGSEA). The ESTIMATE and MCP-Counter R packages were used to assess immune infiltrates from voom-transformed counts. Quantitative analysis of P-S6 and P-PRAS40 was performed by immunohistochemistry (IHC). Tumor phenotypes were segmented on conventional magnetic resonance (MR) images, and radiomic features were extracted using an in-house radiome-sequencing pipeline. Radiomic features were utilized to predict High- vs. Low-OXPHOS status. To evaluate the robustness of the estimates, Leave One Out cross-validation (LOOCV) was conducted on the patient set. Direct metabolite analysis was performed via liquid chromatography mass spectrometry (LC-MS) of Low-OXPHOS A375 and High-OXPHOS, MEKi-resistant A375-R1 brain xenografts. Results: RNA-seq analysis identified that High-OXPHOS MBMs are characterized by significant enrichment of PGC1α (FDR q-val=0.0002) and mTOR (FDR q-val=0.001) signaling and significant depletion of ImmuneScores, T cells, cytotoxic lymphocytes, B lineage cells, and NK cells (p Conclusions: Significant differences in mTOR (enriched) and immune (depleted) signaling were identified in High-OXPHOS clinical MBMs, and enrichment of glutamine signaling was observed in High-OXPHOS MBM xenografts. Further, our results demonstrated that changes in MR radiomic features can noninvasively discriminate between High- and Low-OXPHOS MBMs. Additional studies are ongoing to evaluate the clinical, prognostic, and predictive values of OXPHOS status in melanoma. Citation Format: Grant M. Fischer, Aikaterini Kotrotsou, Sara Ahmed, Pilar O’Neal, Iman Sahnoune, Y.N. Vashisht Gopal, Courtney Hudgens, Barbara Knighton, Rivka Colen, Michael T. Tetzlaff, Michael A. Davies. Molecular, immunologic, metabolic, and radiomic associations of oxidative phosphorylation (OXPHOS) in melanoma brain metastases (MBMs) [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A05.

Our understanding is limited concerning the tumor immune microenvironment of inflammatory breast cancer (IBC), an aggressive form of primary cancer with low rates of pathologic complete response to current neoadjuvant chemotherapy (NAC) regimens. We retrospectively identified pretreatment (N = 86) and matched posttreatment tissue (N = 27) from patients with stage III or de novo stage IV IBC who received NAC followed by a mastectomy. Immune profiling was performed including quantification of lymphoid and myeloid infiltrates by IHC and T-cell repertoire analysis. Thirty-four of 86 cases in this cohort (39.5%) achieved a pathologic complete response. Characterization of the tumor microenvironment revealed that having a lower pretreatment mast cell density was significantly associated with achieving a pathologic complete response to NAC (P = 0.004), with responders also having more stromal tumor-infiltrating lymphocytes (P = 0.035), CD8+ T cells (P = 0.047), and CD20+ B cells (P = 0.054). Spatial analysis showed close proximity of mast cells to CD8+ T cells, CD163+ monocytes/macrophages, and tumor cells when pathologic complete response was not achieved. PD-L1 positivity on tumor cells was found in fewer than 2% of cases and on immune cells in 27% of cases, but with no correlation to response. Our results highlight the strong association of mast cell infiltration with poor response to NAC, suggesting a mechanism of treatment resistance and a potential therapeutic target in IBC. Proximity of mast cells to immune and tumor cells may suggest immunosuppressive or tumor-promoting interactions of these mast cells.

Ocular adnexal sebaceous carcinoma (OASC) is an aggressive malignancy that frequently recurs locally and metastasizes. Surgical extirpation may produce significant aesthetic morbidity, and effective systemic therapies for locally advanced or metastatic disease are largely ineffective. Immune checkpoint inhibitors have shown efficacy in the management of several solid tumors where tumor cell PD-L1 expression correlates with improved response. To determine whether OASC might be amenable to immune checkpoint blockade, we performed comprehensive immune profiling for CD3, CD8, PD-1, FOXP3, and PD-L1 in 24 patients with primary OASC. The composition, distribution and density of the tumor associated immune infiltrate were quantified by automated image analysis and correlated with measures of clinical outcome. Tumor cells in 12 OASCs (50%) expressed PD-L1. Higher densities of CD3+ (p = 0.01), CD8+ (p = 0.006), and PD-1+ (p = 0.024) tumor-associated T cells were associated with higher T category (≥T3a per the 7th edition of the American Joint Committee on Cancer staging manual). Higher tumor cell expression of PD-L1 correlated with higher density of PD-1+ tumor-associated T cells (p = 0.021). Since a CD3+ CD8+ PD-1 + T-cell infiltrate represents a "suppressed T-cell phenotype" apparently permissive toward OASC progression, our findings provide a mechanistic rationale for the effective application of immune checkpoint blockade in OASC to abrogate PD-1/PD-L1 interaction and effectively unleash the immune infiltrate to treat higher-stage tumors.

Good bacteria help fight cancer Resident gut bacteria can affect patient responses to cancer immunotherapy (see the Perspective by Jobin). Routy et al. show that antibiotic consumption is associated with poor response to immunotherapeutic PD-1 blockade. They profiled samples from patients with lung and kidney cancers and found that nonresponding patients had low levels of the bacterium Akkermansia muciniphila . Oral supplementation of the bacteria to antibiotic-treated mice restored the response to immunotherapy. Matson et al. and Gopalakrishnan et al. studied melanoma patients receiving PD-1 blockade and found a greater abundance of “good” bacteria in the guts of responding patients. Nonresponders had an imbalance in gut flora composition, which correlated with impaired immune cell activity. Thus, maintaining healthy gut flora could help patients combat cancer. Science , this issue p. 91 , p. 104 , p. 97 ; see also p. 32

In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications., Resistance to BRAFV600E inhibitors often occurs in melanoma patients. Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.

Appreciation for genomic and immune heterogeneity in cancer has grown though the relationship of these factors to treatment response has not been thoroughly elucidated. To better understand this, we studied a large cohort of melanoma patients treated with targeted therapy or immune checkpoint blockade (n = 60). Heterogeneity in therapeutic responses via radiologic assessment was observed in the majority of patients. Synchronous melanoma metastases were analyzed via deep genomic and immune profiling, and revealed substantial genomic and immune heterogeneity in all patients studied, with considerable diversity in T cell frequency, and few shared T cell clones (, Melanoma: Tumor differences within a patient may explain heterogeneous responses Patients with metastatic melanoma display molecular and immune differences across tumor sites associated with differential drug responses. A team led by Jennifer Wargo from the University of Texas MD Anderson Cancer Center, Houston, USA, studied the radiological responses of 60 patients with metastatic melanoma, half of whom received targeted drug therapy and half of whom received an immune checkpoint inhibitor. The majority (83%) showed differences in responses across metastases. The group then profiled tumors in a subset, and found molecular and immune heterogeneity in different tumors within the same patient. Heterogeneity in mutational and immune profiles within tumors from individual patients could explain differences in treatment response. Knowing this, the authors emphasize the importance of acquiring biopsies from more than one tumor site in order to best tailor therapies to the features of metastatic cancer.

Background Immune related adverse events (irAEs) are common side effects of checkpoint inhibitory (CPI) therapies targeting CTLA-4 and PD-1/PD-L1. Grover’s disease is an uncommon dermatologic condition with unclear pathogenesis previously reported as an irAE with ipilimumab. Case Presentation We report an additional case of ipilimumab-induced Grover’s disease. Interestingly, this dermatologic side effect did not appear with use of anti-PD-1 therapy in our patient. Immune analysis was performed and suggests a possible role of Th2 cells in its patholgenesis. Conclusion This case suggests that Grover's disease is an irAE induced by Ipilimumab. Our immune analysis suggests that Th2 cells may be pathogenic mediators which warrants further study.

Normal pancreatic epithelium progresses through various stages of pancreatic intraepithelial neoplasms (PanINs) in the development of pancreatic ductal adenocarcinoma (PDAC). Transcriptional regulation of this progression is poorly understood. In mouse, the hepatic nuclear factor 6 (Hnf6) transcription factor is expressed in ductal cells and at lower levels in acinar cells of the adult pancreas, but not in mature endocrine cells. Hnf6 is critical for terminal differentiation of the ductal epithelium during embryonic development and for pancreatic endocrine cell specification. We previously showed that, in mice, loss of Hnf6 from the pancreatic epithelium during organogenesis results in increased duct proliferation and altered duct architecture, increased periductal fibrosis and acinar-to-ductal metaplasia. Here we show that decreased expression of HNF6 is strongly correlated with increased severity of PanIN lesions in samples of human pancreata and is absent from >90% of PDAC. Mouse models in which cancer progression can be analyzed from the earliest stages that are seldom accessible in humans support a role for Hnf6 loss in progression from early- to late-stage PanIN and PDAC. In addition, gene expression analyses of human pancreatic cancer reveal decreased expression of HNF6 and its direct and indirect target genes compared with normal tissue and upregulation of genes that act in opposition to HNF6 and its targets. The negative correlation between HNF6 expression and pancreatic cancer progression suggests that HNF6 maintains pancreatic epithelial homeostasis in humans, and that its loss contributes to the progression from PanIN to ductal adenocarcinoma. Insight on the role of HNF6 in pancreatic cancer development could lead to its use as a biomarker for early detection and prognosis.

Background: The gut microbiome is increasingly being recognized as a strong modulator of anti-PD1 based cancer immunotherapy. Compelling evidence demonstrates differential bacterial enrichment and diversity in responders (R) versus non-responders (NR), mediated by profound influences on systemic and anti-tumor immune infiltrates. However, this has not been studied in the setting of treatment with combined immune checkpoint blockade (CICB), which is associated with superior response rates, but higher rates of potentially debilitating toxicities. Methods: We assembled a cohort of patients with metastatic melanoma receiving CICB (n=54). All patients were classified as R (n=31, CR + PR) or NR (n=23, SD + PD) based on RECIST v1.1, and as having grade 3 or higher (T; n=29), or less than grade 3 (NT; n=25) immune related adverse event(s) by NCI CTCAE 4.0 criteria. Baseline stool samples were characterized by 16S rRNA sequencing. Correlative analyses of peripheral immune cell populations by flow cytometry (n=12) and circulating T cell repertoire by TCR-sequencing (n=12) were done on matched pre-treatment blood samples. Results: The overall gut microbial landscape in these patients was varied with high abundance of Bacteroidales and Clostridiales. Ordination of beta-diversity distances revealed a lack of clustering by subtype of primary tumor (uveal, mucosal, cutaneous) consistent with no significant effect of the tumor histology. While no apparent response or toxicity associations were evident based on diversity, notable compositional differences were appreciated. Comparison of relative abundances by LEfSe (LDA>2, p Conclusion: These findings build on our prior work and support the notion of a close link between the gut microbiome and therapeutic outcomes to checkpoint blockade therapy. Extensive studies are underway in both matched human biospecimens and in pre-clinical models to further understand mechanisms of interactions with immune markers, and to establish causality. Taken together, these data support a critical role for the gut microbiome as both a predictive tool and therapeutic target. Citation Format: Vancheswaran Gopalakrishnan, Miles Andrews, Wei-Shen Chen, Christine Spencer, Luis Vence, Alexandre Reuben, Zachary A. Cooper, Peter A. Prieto, Michael T. Tetzlaff, MA Abdul Wadud Khan, Alexander Lazar, Courtney W. Hudgens, Lauren E. Haydu, Hussein A. Tawbi, Patrick Hwu, Wen-Jen Hwu, Rodabe N. Amaria, Elizabeth M. Burton, Scottt E. Woodman, Adi Diab, Sapna P. Patel, Isabella C. Glitza, Jianhua Zhang, Joseph Petrosino, Robert R. Jenq, Michael A. Davies, Jeffrey E. Gershenwald, Padmanee Sharma, James P. Allison, Andrew Futreal, Jennifer A. Wargo. Therapeutic efficacy and tolerability of combined immune checkpoint blockade in metastatic melanoma patients is influenced by the gut microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1493.

Background: While treatment with immune checkpoint blockade (ICB) has markedly improved outcomes in advanced melanoma patients and other malignancies, predicting response remains a challenge. Biomarkers including tumor mutational burden (TMB), T-cell infiltration, and PD-L1 expression, have been identifiedbut remain inadequate. Other components of innate and adaptive immunity, including B-cells and tertiary lymphoid structures (TLS), have been implicated in the response to other cancer therapies, andpreclinical data suggests B cells may contribute in the response to immunotherapy. Here, we use targeted protein expression profiling-via NanoString digital spatial profiling (DSP) technology (research use only)-to demonstrate a role for B cells in the response to ICB in patients with high-risk resectable melanoma; furthermore, we characterize the B cell subsets that enable this function. Methods: We conducted a phase 2 clinical trial of neoadjuvant ICB therapy in patients with high-risk resectable melanoma (PD-1 blockade monotherapy or combined CTLA-4/PD-1 blockade) (NCT02519322). Longitudinal tumor samples were taken during therapy. Formalin-fixed paraffin embedded tissue sections from tumor samples (n=10 responders [R], n=10 non-responders [NR]) were analyzed by NanoString DSP technology and stained with a cocktail of S100B, CD45, CD19 and a 40-protein cocktail of antibodies conjugated to UV-photocleavable DNA barcodes. Regions of interest (ROI) were delineated using immunofluorescence followed by UV excitation of the defined ROIs, releasing the DNA barcodes for downstream quantitation on the NanoString nCounter®platform. Utilizing masking strategies, we define the unique expression pattern within discrete subsets of immune cells. These same tumors have concurrently been analyzed by RNAseq, immunohistochemistry, CYTOF, and single-cell RNAseq. Results: Biomarker counts are highly concordant across samples from the same patient’s tumor. High concordance between DSP and quantitative fluorescence is seen as a validation for the DSP method. We identify B cells as components of TLS; the B cells are closely integrated with CD4 and CD8 T cells and follicular dendritic cells. There are significantly more TLS identified in R compared to NR patients. Utilizing CYTOF, we concurrently identify specific subsets of B cells present within the TLS associated with response. We characterize the spatial relationship of these same B cell subsets with other components of the TLS and define the protein expression patterns of these cells. Conclusion: NanoString DSP data complement our deep molecular and immune profiling of tumors from melanoma patients treated with ICB; together, they provide a novel predictive role for B-cells and TLS in the response to ICB and, importantly, provide mechanistic insight into their potential contribution in the response to cancer therapy. Citation Format: Beth A. Helmink, Sangeetha Reddy, Jorge Blando, Yan Liang, Sarah Warren, Vancheswaran Gopalakrishnan, Hussein A. Tawbi, Rodabe N. Amaria, Michael Davies, Jeffrey E. Gershenwald, Elizabeth Burton, Rafet Basar, Alexander J. Lazar, Courtney W. Hudgens, Katy Rezvani, James P. Allison, Padmanee Sharma, Joseph M. Beechem, Jennifer A. Wargo, Michael T. Tetzlaff. NanoString®GeoMx®digital spatial profiling further defines the role of B cells in the response to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 499.

Sebaceous carcinoma (SC) is a rare but aggressive malignancy with frequent recurrence and metastases. Surgery is the mainstay of therapy, but effective systemic therapies are lacking because the molecular alterations driving SC remain poorly understood. To identify these, we performed whole-exome next-generation sequencing of 409 cancer-associated genes on 27 SCs (18 primary/locally recurrent ocular, 5 paired metastatic ocular, and 4 primary extraocular) from 20 patients. In ocular SC, we identified 139 non-synonymous somatic mutations (median/lesion 3; range 0-23). Twenty-five of 139 mutations (18%) occurred in potentially clinically actionable genes in 6 of 16 patients. The most common mutations were mutations in TP53 (n = 9), RB1 (n = 6), PIK3CA (n = 2), PTEN (n = 2), ERBB2 (n = 2), and NF1 (n = 2). TP53 and RB1 mutations were restricted to ocular SC and correlated with aberrant TP53 and RB protein expression. Systematic pathway analyses demonstrated convergence of these mutations to activation of the PI3K signalling cascade, and PI3K pathway activation was confirmed in tumours with PTEN and/or PIK3CA mutations. Considerable inter-tumoural heterogeneity was observed between paired primary and metastatic ocular SCs. In primary extraocular SC, we identified 77 non-synonymous somatic mutations (median/lesion 22.5; range 3-29). This overall higher mutational load was attributed to a microsatellite instability phenotype in three of four patients and somatically acquired mutations in mismatch repair genes in two of four patients. Eighteen of 77 mutations (23%) were in potentially clinically actionable genes in three of four patients, including BTK, FGFR2, PDGFRB, HRAS, and NF1 mutations. Identification of potentially clinically actionable mutations in 9 of 20 SC patients (45%) underscores the importance of next-generation sequencing to expand the spectrum of genotype-matched targeted therapies. Frequent activation of PI3K signalling pathways provides a strong rationale for application of mTOR inhibitors in the management of this disease. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

Diabetes results from an inadequate functional β cell mass, either due to autoimmune destruction (Type 1 diabetes) or insulin resistance combined with β cell failure (Type 2 diabetes). Strategies to enhance β cell regeneration or increase cell proliferation could improve outcomes for patients with diabetes. Research conducted over the past several years has revealed that factors regulating embryonic β cell mass expansion differ from those regulating replication of β cells post-weaning. This article aims to compare and contrast factors known to control embryonic and postnatal β cell replication. In addition, we explore the possibility that connective tissue growth factor (CTGF) could increase adult β cell replication. We have already shown that CTGF is required for embryonic β cell proliferation and is sufficient to induce replication of embryonic β cells. Here we examine whether adult β cell replication and expansion of β cell mass can be enhanced by increased CTGF expression in mature β cells.

In the version of this article originally published, there was an error in Fig. 1. In the neoadjuvant phase column, the n values for arms A and B were both reported to be 20. The n values for arms A and B were actually 12 and 11, respectively. Also, the URL underlying the accession code in the data availability section was incorrect. The URL was originally https://www.ebi.ac.uk/ega/studies/EGAS00001002698. It should have been https://www.ebi.ac.uk/ega/studies/EGAS00001003178. The errors have been corrected in the print, HTML and PDF versions of this article.

Introduction: Blockade of immune checkpoints has improved clinical outcomes for patients with metastatic non-small cell lung cancer (NSCLC), but its role in the perioperative setting for early-stage disease is unclear. We generated preclinical models of resectable NSCLC expressing an antigen that permits quantitative assessment of the immune response and compared survival, tumor recurrence, and immune response after neoadjuvant or adjuvant immunotherapy. Experimental Procedures: We transfected murine 344SQ NSCLC cells with an ovalbumin-expression plasmid to generate OVA+ cells that can be identified with an antibody against the peptide SIINFEKL bound to H-2Kb of MHC-I. We implanted 344SQ-OVA+ cells in the flank of syngeneic mice and then randomized mice with established tumors to either 3 doses of neoadjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4, or to observation. Primary tumors were resected in all mice 2 days after mice in the neoadjuvant group had received their last dose of therapy. Two days post-surgery, mice in the observation group were treated with 3 doses of adjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4. Mice were euthanized 4 weeks after injection or when moribund and their survival recorded and lung metastases counted. We determined the composition of CD3+CD8+ tumor-infiltrating lymphocytes (TILs) with flow cytometry using tetramers against SIINFEKL-specific T cell receptors and immunohistochemical staining of tumors. Results: Single agent and combined immunotherapy significantly prolonged survival compared to controls in both neoadjuvant and adjuvant groups (p Conclusions: Neoadjuvant combined immune checkpoint blockade is superior to adjuvant immunotherapy at prolonging survival, reducing distal recurrence and inducing anti-tumor immunity in preclinical models of resectable NSCLC. Citation Format: Tina Cascone, Haifa Hamdi, Fahao Zhang, Alissa Poteete, Lerong Li, Courtney W. Hudgens, Leila J. Williams, Qiuyu Wu, Jayanthi Gudikote, Weiyi Peng, Patrick Hwu, Jing Wang, Michael Tetzlaff, William N. William, John V. Heymach. Superior efficacy of neoadjuvant compared to adjuvant immune checkpoint blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1719.

BACKGROUND: MBMs are a common and lethal complication of advanced melanoma. An improved understanding of the molecular features of MBMs could facilitate the development of more effective treatments for patients (pts). Further, identification of differences between MBMs and ECMs would indicate the need for organ-specific treatment strategies. METHODS: Transcriptomic capture and Illumina RNA-sequencing (RNA-seq) were performed on (i) surgically resected formalin-fixed, paraffin-embedded (FFPE) MBMs (98 tumors from 84 pts) and (ii) surgically-resected ECMs from the same pts (54 from 38 pts). The EdgeR/limma/voom pipeline was used to perform differential gene expression (DGE) analyses. Pathway analyses were performed via Ensemble Gene Set Enrichment Analysis (EGSEA). The ESTIMATE and MCPCounter R packages were used to assess immune infiltrates from voom-transformed counts. Illumina whole exome sequencing (WES) was performed on 27 matched pairs of MBMs and ECMs from 17 pts. MuTect algorithm was utilized to assess non-synonymous, stop-gain, and stop-loss mutations. DNA methylation profiling was performed on 16 pt-matched pairs of MBMs and ECMs by the Illumina MethylationEPIC BeadChip platform; data was analyzed via the ChAMP pipeline. Quantitative analysis of CD3 and CD8 immune markers was performed by immunohistochemistry (IHC) on 31 MBMs and 49 pt-matched ECMs. RESULTS: Overall patterns of mRNA expression, mutational burden, and gene methylation were largely similar between pt-matched pairs of MBMs and ECMs, as unsupervised hierarchical clustering was driven primarily by patient identification. However, EGSEA of RNA-seq data identified significant (FDR q-value < 0.10) differences in immune networks (decreased in MBMs) and neuronal differentiation factors (increased in MBMs) in the pt-matched tumors. MCPCounter analysis revealed significant (p < 0.05) depletion of all classes of immune cells except neutrophils in MBMs compared to ECMs. IHC confirmed decreased TCD3 (p = 0.005) and TCD8 (p = 0.013) infiltrating cells in MBMs. ESTIMATE ImmuneScores among MBMs inversely correlated with expression of VSIG4, a relative of the B7 family believed to inhibit T-cell proliferation, and were significantly (p = 0.0088) increased in MBMs previously treated with radiation (XRT). Increased ImmuneScores also associated with significantly (p = 0.00002) improved overall survival (OS) from surgery for MBM. Significant correlations in mean promoter region methylation changes and RNA-seq log fold-changes were identified in 5/10 (50%) of the neuronal factors overexpressed in MBMs, suggesting an epigenetic mechanism for their differential expression. CONCLUSIONS: Significant differences in immune and neuronal gene networks were detected in MBMs compared to patient-matched ECMs, and expression of immune genes in MBMs positively correlated with previous XRT and OS. Citation Format: Grant M. Fischer, Ali Jalali, Aron Joon, Michael T. Tetzlaff, Alexander J. Lazar, Fernando Carapeto, Mariana P. Macedo, Courtney W. Hudgens, Jennifer L. McQuade, Khalida Wani, Brandy Conner, Bhavana Singh, Michael A. Davies. Comprehensive molecular profiling of melanoma brain metastases (MBMs) and patient (pt)-matched extracranial metastases (ECMs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3392.

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin tumor. MCC is immunogenic and highly responsive to immune checkpoint inhibitors. On rare occasions, MCC undergoes spontaneous regression of both local and metastatic disease in the absence of treatment. With at least 45 cases reported in the literature, this phenomenon has been described in 1.7-3% of MCCs but its mechanism is poorly understood. There has been speculation that biopsy-induced antigen shedding and subsequent recruitment of a host immune response drives spontaneous tumor regression. Here, we demonstrate activation of an adaptive immune response in a case of spontaneous MCC regression. A 65 year-old man presented with a rapidly-growing 3.5 cm skin tumor confirmed by histopathologic and immunohistochemical analyses to be Merkel cell polyomavirus-positive MCC. Eight days following the diagnostic shave biopsy, the tumor had clinically regressed. Histological analysis of a wide local excision specimen obtained 45 days after the initial biopsy revealed small, residual tumor nests and a surrounding lymphohistiocytic inflammatory infiltrate consistent with a regressing tumor. To evaluate the immune response at baseline and in the regressing tumor, we performed T cell receptor (TCR) sequencing as a metric of antigen-specific lymphocyte activation, and quantitative immunohistochemical analyses to phenotype the immune response. Both the baseline and regressing tumor contained intratumoral inflammatory infiltrates which were sparse relative to peritumoral infiltrates. There were notable reductions in CD4+ T cells and CD68+ macrophages in the regressing tumor relative to the initial biopsy. TCR clonality was increased in the regressing tumor relative to the baseline lesion, including both increased representation of the most dominant T cell clone from the baseline biopsy as well as emergence of new high-frequency clones. Together, these results suggest that spontaneous MCC regression is driven by expansion of novel and pre-existing adaptive cellular immune responses. Consistent with this, we observed analogous results with immunohistochemical staining and TCR sequencing performed in an MCC tumor regressing during treatment with the PD-L1 inhibitor, avelumab. Although further studies are needed to determine how biopsy unmasks immune responses and to elucidate target antigens of the T cell clones driving spontaneous MCC regression, our observations demonstrate that comparable immune activation underlies both spontaneous tumor regression and MCC response to immunotherapy. Citation Format: Mairead Baker, John W. Roman, Alexandre Reuben, Natasha Hill, Courtney W. Hudgens, Michael Tetzlaff, Nicholas F. Logemann, Isaac Brownell. Spontaneous regression of Merkel cell carcinoma is driven by adaptive immune activation and clonal T cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4676.

Prognosis is favorable in patients with primary localized melanoma but poor in patients with metastatic disease. With more than 76,000 cases expected to be diagnosed in 2016, more precise prognostic technologies and new therapies are needed. Although targeted treatment regimens have been approved in recent years, resistance has emerged in large part due to adaptive response mechanisms and intratumoral heterogeneity. Analysis of tumor samples across multiple molecular platforms will help elucidate the complexities within and across tumors which may underlie response to therapy as well as assist in identifying predictive biomarkers; however, these approaches require significant amounts of sample, time, and resources. In order to integrate the strengths of analyzing different molecular analytes, we have modularized Nanostring Technologies' molecular barcoding technology to permit simultaneous digital measurement of cancer-associated DNA mutation variants, mRNA expression, and protein expression in one assay from the same sample (3D Biology). Novel nucleotide variant probes enable sensitive and specific identification of DNA mutant allele sequences down to a level of detection of ≤ 5% from 5 ng of FFPE-extracted genomic DNA. Gene expression is measured via unique digital barcoding technology to measure mRNA transcripts, and protein expression and activity (via phosphorylation) is measured by DNA-labeled antibodies. The multi-omic workflow requires only two 5-10 micron sections of FFPE tissue, whereby DNA and RNA are extracted from one section and multiplex digital protein profiling is conducted on the second. As proof of concept demonstrating the utility of this 3D Biology platform, we have simultaneously analyzed DNA variants, RNA expression, and protein expression using NanoString’s nCounter Vantage 3D™ Solid Tumor Panel on 12 FFPE melanoma tumor samples and one normal tissue from six patients. This sample set included two metastatic tumors from each patient, and in one instance multiple regions from each metastatic site (7 and 2 regions) in order to assess intratumoral heterogeneity. Importantly, this sample set has associated mRNA expression measured using the nCounter® PanCancer Pathways for Human panel as well as whole exome sequencing (WES) data. Somatic variants seen in this latter dataset were compared with the results from the DNA SNV Solid Tumor Panel. Samples with variants that were detected in the nCounter assay but not WES were subjected to deep sequencing for validation. Overall, we show that this multiplex and multi-omic platform has the potential for rapid and sensitive assessment of patient samples that will impact clinical care. Citation Format: Jinho Lee, Christopher P. Vellano, Gavin Meredith, Jill Mckay-Fleisch, P. Martin Ross, Michael Tetzlaff, Alexandre Reuben, Courtney Hudgens, Jennifer Wargo, Jessica Garber, Andrew White, Joseph Pan, Mike Krouse, Mekala Pansalawatta, Lucas Dennis, Anisha Kharkia, Erin Piazza, Afshin Mashadi-Hossein, Rich Boykin, Nathan Elliott, Brian Filanoski, Gokhan Demirkan, Sarah Warren, Gary Geiss, Dae Kim, Joseph Beechem, Gordon B. Mills. 3D Biology™ view of cancer: Simultaneous detection of somatic DNA mutations and expression profiling of genes and signaling proteins from melanoma tumor FFPE samples [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 5563. doi:10.1158/1538-7445.AM2017-5563

Background: Melanoma therapy has benefitted greatly from immune checkpoint blockade, although responses are variable and not always durable. There is a growing appreciation of the role of the microbiome in cancer-related outcomes and recent evidence in murine models suggests that modulation of the gut microbiome may enhance responses to immune checkpoint blockade in melanoma. However this has not been investigated in patients. Here, we demonstrate that differential bacterial “signatures” exist in the gut microbiome of responders (R) and non-responders (NR) to anti-PD-1 therapy, and that insights gained could be used to derive actionable strategies to enhance responses. Methods: We collected buccal (n=105) and stool (n=53) samples from a cohort of anti-PD-1 treated metastatic melanoma patients (n=110). Patients were classified as either R or NR based on RECIST criteria, and 16S rDNA, and whole-genome shotgun sequencing was performed to characterize the diversity, composition and functional capabilities of the microbiomes. Immune profiling (via 7-marker IHC panel of CD3, CD8, PD-1, PD-L1, Granzyme B, RORγT and FoxP3) and cytokine analyses were also performed on available tumors and serum samples at baseline. Results: In these studies, we observed significant differences in the diversity and composition of the gut microbiome in R versus NR to PD-1 blockade at baseline, but no clear differences in buccal microbiomes. Specifically, R had a significantly higher alpha diversity compared to NR (p=0.017), and the Ruminococcaceae family of the Clostridiales order was enriched in R whereas Prevotellaceae family of the Bacteroidales order was enriched in NR. Immune profiling demonstrated significantly increased immune infiltrates in baseline tumor samples of R, with a positive correlation between CD8, CD3, PD-1 and FoxP3 T-cell density and abundance of specific bacteria enriched in R (e.g. Faecalibacterium). Low diversity was also associated with elevated levels of chronic inflammation markers in the serum at baseline. Lastly, we saw differentially abundant metabolic pathways in the gut microbiomes of R (pyrimidine nucleotide biosynthesis, fatty acid biosynthesis, shikimate pathway) vs NR (Tricarboxylic acid cycle, assimilatory sulphate and nitrate reduction, tryptophan biosynthesis). Conclusion: Differences exist in the diversity and composition of the gut microbiome in R vs NR to anti-PD-1 therapy and these microbiota could bridge the gap between host metabolism and anti-tumor immunity. These results have far-reaching implications and suggest that modifications to the gut microbiome could potentially enhance therapeutic responses to immune checkpoint blockade. Citation Format: Vancheswaran Gopalakrishnan, Christine Spencer, Alexandre Reuben, Peter Prieto, Diego Vicente, Tatiana V. Karpinets, Courtney W. Hudgens, Diane S. Hutchinson, Michael Tetzlaff, Alexander Lazar, Michael A. Davies, Jeffrey E. Gershenwald, Robert Jenq, Patrick Hwu, Padmanee Sharma, James Allison, Andrew Futreal, Nadim Ajami, Joseph Petrosino, Carrie Daniel-MacDougall, Jennifer A. Wargo. Response to anti-PD-1 based therapy in metastatic melanoma patients is associated with the diversity and composition of the gut microbiome [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 2672. doi:10.1158/1538-7445.AM2017-2672

9575 Background: Although both targeted and immune therapies have significantly improved outcomes for mm pts, only a minority of pts experience durable responses with many pts with multiple SMM demonstrating differential responses to therapy. We performed multidimensional spatial characterization of immune markers in SMM from mm patients treated with targeted and immune therapies to improve our understanding of correlations and determinants of response. Methods: NanoString's Digital Spatial Profiling research platform was used on 6 SMM from 3 pts (treatment-naïve; BRAF + MEK targeted therapy treated; anti-PD-1 immunotherapy treated) for 30 immune and signaling proteins. For analysis, we selected and compared immune-rich (CD45+) and tumor-rich (S100B+) regions across SMM. Results were compared to lesion-specific clinical responses. Results: Striking differences in patterns of expression across SMM from individual pts were detected, including in Ki67, CD68 myeloid cells, and the potent immunosuppressor B7-H3. SMM progressing after targeted therapy demonstrated higher pAKT and PD-L1 expression, consistent with described resistance mechanisms. Large differences in expression of PD-L1 were noted following anti-PD-1 therapy, which could contribute to heterogeneous responses. Differential expression patterns in the TME associated with response were also detected, including in increases in CD4 and CD14 cells in progressing lesions. Conclusions: Striking differences in responding and non-responding SMM were observed, providing potential explanations for the heterogeneous clinical responses frequently observed in mm pts. Studies are ongoing to further characterize interactions and spatial distribution of cell types, as well as integrate these findings with previous molecular and immune profiling data (whole exome sequencing, gene expression profiling, flow cytometry, IHC, TCR sequencing) in these and additional SMM to identify actionable strategies to homogenize responses across metastases in mm pts.

Importance Combined treatment with dabrafenib and trametinib (CombiDT) achieves clinical responses in only about 15% of patients with BRAF inhibitor (BRAFi)-refractory metastatic melanoma in contrast to the higher response rate observed in BRAFi-naive patients. Identifying correlates of response and mechanisms of resistance in this population will facilitate clinical management and rational therapeutic development. Objective To determine correlates of benefit from CombiDT therapy in patients with BRAFi-refractory metastatic melanoma. Design, Setting, and Participants Single-center, single-arm, open-label phase 2 trial of CombiDT treatment in patients with BRAF V600 metastatic melanoma resistant to BRAFi monotherapy conducted between September 2012 and October 2014 at the University of Texas MD Anderson Cancer Center. Key eligibility criteria for participants included BRAF V600 metastatic melanoma, prior BRAFi monotherapy, measurable disease (RECIST 1.1), and tumor accessible for biopsy. Interventions Patients were treated with dabrafenib (150 mg, twice daily) and trametinib (2 mg/d) continuously until disease progression or intolerance. All participants underwent a mandatory baseline biopsy, and optional biopsy specimens were obtained on treatment and at disease progression. Whole-exome sequencing, reverse transcription polymerase chain reaction analysis for BRAF splicing, RNA sequencing, and immunohistochemical analysis were performed on tumor samples, and blood was analyzed for levels of circulating BRAF V600. Main Outcomes and Measures The primary end point was overall response rate (ORR). Progression-free survival (PFS) and overall survival (OS) were secondary clinical end points. Results A total of 28 patients were screened, and 23 enrolled. Among evaluable patients, the confirmed ORR was 10%; disease control rate (DCR) was 45%, and median PFS was 13 weeks. Clinical benefit was associated with duration of prior BRAFi therapy greater than 6 months (DCR, 73% vs 11% for ≤6 months; P = .02) and decrease in circulating BRAF V600 at day 8 of cycle 1 (DCR, 75% vs 18% for no decrease; P = .02) but not with pretreatment mitogen-activated protein kinase (MAPK) pathway mutations or activation. Biopsy specimens obtained during treatment demonstrated that CombiDT therapy failed to achieve significant MAPK pathway inhibition or immune infiltration in most patients. Conclusions and Relevance The baseline presence of MAPK pathway alterations was not associated with benefit from CombiDT in patients with BRAFi-refractory metastatic melanoma. Failure to inhibit the MAPK pathway provides a likely explanation for the limited clinical benefit of CombiDT in this setting. Circulating BRAF V600 is a promising early biomarker of clinical response. Trial Registration clinicaltrials.gov Identifier:NCT01619774.

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.

Nature 532, 250–254 (2016); doi:10.1038/nature17392 In Fig. 5a of this Letter, the labels PBS and PLX4720 were inadvertently reversed. The corrected Fig. 5a is shown in Fig. 1 of this Corrigendum.

Background Immune related adverse events (irAEs) are common side effects of checkpoint inhibitory (CPI) therapies targeting CTLA-4 and PD-1/PD-L1. Grover’s disease is an uncommon dermatologic condition with unclear pathogenesis previously reported as an irAE with ipilimumab.