Increased tryptophan, but not increased glucose metabolism, predict resistance of pembrolizumab in stage III/IV melanoma.

Clinical trials of combined IDO/PD1 blockade in metastatic melanoma (MM) failed to show additional clinical benefit compared to PD1-alone inhibition. We reasoned that a tryptophan-metabolizing pathway other than the kynurenine one is essential. We immunohistochemically stained tissues along the nevus-to-MM progression pathway for tryptophan-metabolizing enzymes (TMEs; TPH1, TPH2, TDO2, IDO1) and the tryptophan transporter, LAT1. We assessed tryptophan and glucose metabolism by performing baseline C11-labeled α-methyl tryptophan (C11-AMT) and fluorodeoxyglucose (FDG) PET imaging of tumor lesions in a prospective clinical trial of pembrolizumab in MM (clinicaltrials.gov, NCT03089606). We found higher protein expression of all TMEs and LAT1 in melanoma cells than tumor-infiltrating lymphocytes (TILs) within MM tumors (n = 68). Melanoma cell-specific TPH1 and LAT1 expressions were significantly anti-correlated with TIL presence in MM. High melanoma cell-specific LAT1 and low IDO1 expression were associated with worse overall survival (OS) in MM. Exploratory optimal cutpoint survival analysis of pretreatment 'high' vs. 'low' C11-AMT SUVmax of the hottest tumor lesion per patient revealed that the 'low' C11-AMT SUVmax was associated with longer progression-free survival in our clinical trial (n = 26). We saw no such trends with pretreatment FDG PET SUVmax. Treatment of melanoma cell lines with telotristat, a TPH1 inhibitor, increased IDO expression and kynurenine production in addition to suppression of serotonin production. High melanoma tryptophan metabolism is a poor predictor of pembrolizumab response and an adverse prognostic factor. Serotoninergic but not kynurenine pathway activation may be significant. Melanoma cells outcompete adjacent TILs, eventually depriving the latter of an essential amino acid.

InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma.

INTRODUCTION: We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. METHODS: Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay). RESULTS: Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p = 0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p<0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001). CONCLUSION: Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.

Multi-organ landscape of therapy-resistant melanoma.

Metastasis and failure of present-day therapies represent the most common causes of mortality in patients with cutaneous melanoma. To identify the underlying genetic and transcriptomic landscapes, in this study we analyzed multi-organ metastases and tumor-adjacent tissues from 11 rapid autopsies after treatment with MAPK inhibitor (MAPKi) and/or immune checkpoint blockade (ICB) and death due to acquired resistance. Either treatment elicits shared genetic alterations that suggest immune-evasive, cross-therapy resistance mechanisms. Large, non-clustered deletions, inversions and inter-chromosomal translocations dominate rearrangements. Analyzing data from separate melanoma cohorts including 345 therapy-naive patients and 35 patients with patient-matched pre-treatment and post-acquired resistance tumor samples, we performed cross-cohort analyses to identify MAPKi and ICB as respective contributors to gene amplifications and deletions enriched in autopsy versus therapy-naive tumors. In the autopsy cohort, private/late mutations and structural variants display shifted mutational and rearrangement signatures, with MAPKi specifically selecting for signatures of defective homologous-recombination, mismatch and base-excision repair. Transcriptomic signatures and crosstalks with tumor-adjacent macroenvironments nominated organ-specific adaptive pathways. An immune-desert, CD8+-macrophage-biased archetype, T-cell exhaustion and type-2 immunity characterized the immune contexture. This multi-organ analysis of therapy-resistant melanoma presents preliminary insights with potential to improve therapeutic strategies.

Development of [18F]F-5-OMe-Tryptophans through Photoredox Radiofluorination: A New Method to Access Tryptophan-Based PET Agents.

Although various radiolabeled tryptophan analogs have been developed to monitor tryptophan metabolism using positron emission tomography (PET) for various human diseases including melanoma and other cancers, their application can be limited due to the complicated synthesis process. In this study, we demonstrated that photoredox radiofluorination represents a simple method to access novel tryptophan-based PET agents. In brief, 4-F-5-OMe-tryptophans (l/d-T13) and 6-F-5-OMe-tryptophans (l/d-T18) were easily synthesized. The 18F-labeled analogs were produced by photoredox radiofluorination with radiochemical yields ranging from 2.6 ± 0.5% to 32.4 ± 4.1% (3 ≤ n ≤ 5, enantiomeric excess ≥ 99.0%) and over 98.0% radiochemical purity. Small animal imaging showed that l-[18F]T13 achieved 9.58 ± 0.26%ID/g tumor uptake and good contrast in B16F10 tumor-bearing mice (n = 3). Clearly, l-[18F]T13 exhibited prominent tumor uptake, warranting future evaluations of its potential usage in precise immunotherapy monitoring.

Blocking Genomic Instability Prevents Acquired Resistance to MAPK Inhibitor Therapy in Melanoma.

Blocking cancer genomic instability may prevent tumor diversification and escape from therapies. We show that, after MAPK inhibitor (MAPKi) therapy in patients and mice bearing patient-derived xenografts (PDX), acquired resistant genomes of metastatic cutaneous melanoma specifically amplify resistance-driver, nonhomologous end-joining (NHEJ), and homologous recombination repair (HRR) genes via complex genomic rearrangements (CGR) and extrachromosomal DNAs (ecDNA). Almost all sensitive and acquired-resistant genomes harbor pervasive chromothriptic regions with disproportionately high mutational burdens and significant overlaps with ecDNA and CGR spans. Recurrently, somatic mutations within ecDNA and CGR amplicons enrich for HRR signatures, particularly within acquired resistant tumors. Regardless of sensitivity or resistance, breakpoint-junctional sequence analysis suggests NHEJ as critical to double-stranded DNA break repair underlying CGR and ecDNA formation. In human melanoma cell lines and PDXs, NHEJ targeting by a DNA-PKCS inhibitor prevents/delays acquired MAPKi resistance by reducing the size of ecDNAs and CGRs early on combination treatment. Thus, targeting the causes of genomic instability prevents acquired resistance. SIGNIFICANCE: Acquired resistance often results in heterogeneous, redundant survival mechanisms, which challenge strategies aimed at reversing resistance. Acquired-resistant melanomas recurrently evolve resistance-driving and resistance-specific amplicons via ecDNAs and CGRs, thereby nominating chromothripsis-ecDNA-CGR biogenesis as a resistance-preventive target. Specifically, targeting DNA-PKCS/NHEJ prevents resistance by suppressing ecDNA/CGR rearrangements in MAPKi-treated melanomas. This article is highlighted in the In This Issue feature, p. 799.

Targeting wild-type TP53 using AMG 232 in combination with MAPK inhibition in Metastatic Melanoma; a phase 1 study.

BACKGROUND: Targeting the MDM2-p53 interaction using AMG 232 is synergistic with MAPK inhibitors (MAPKi) in preclinical melanoma models. We postulated that AMG 232 plus MAPKi is safe and more effective than MAPKi alone in TP53-wild type, MAPKi-naïve metastatic melanoma. METHODS: Patients were treated with increasing (120 mg, 180 mg, 240 mg) oral doses of AMG 232 (seven-days-on, 15-days-off, 21-day cycle) plus dabrafenib (D) and trametinib (T) (Arm 1, BRAFV600-mutant) or T alone (Arm 2, BRAFV600-wild type). Patients were treated for seven days with AMG 232 alone before adding T±D. Safety and efficacy were assessed using CTCAE v4.0 and RECIST v1.1 criteria, respectively. Pharmacokinetic (PK) analysis was performed at baseline and steady-state levels for AMG 232. RESULTS: 31 patients were enrolled. Ten and 21 patients were enrolled in Arm 1 and Arm 2, respectively. The most common AMG 232-related adverse events (AEs) were nausea (87%), diarrhea (77%), and fatigue (74%). Seven patients (23%) were withdrawn from the study due to AMG 232-related AEs. Three dose-limiting AEs occurred (Arm 1, 180 mg, nausea; Arm 2, 240 mg, grade 3 pulmonary embolism; Arm 2, 180 mg, grade 4 thrombocytopenia). AMG 232 PK exposures were not altered when AMG 232 was combined with T±D. Objective responses were seen in 8/10 (Arm 1) and 3/20 (Arm 2) evaluable patients. The median progression-free survival for Arm 1 and Arm 2 was 19.0 months-not reached and 2.8 months, respectively. CONCLUSION: The maximum tolerated dose of AMG 232 for both arms was 120 mg. AMG 232 plus T±D exhibited a favorable PK profile. Although objective responses occurred in both arms, adding AMG 232 to T±D did not confer additional clinical benefit.

Melanoma Brain Metastases: An Update on the Use of Immune Checkpoint Inhibitors and Molecularly Targeted Agents.

Brain metastases from melanoma are no longer uniformly associated with dismal outcomes. Impressive tumor tissue-based (craniotomy) translational research has consistently shown that distinct patient subgroups may have a favorable prognosis. This review provides a historical overview of the standard-of-care treatments until the early 2010s. It subsequently summarizes more recent advances in understanding the biology of melanoma brain metastases (MBMs) and treating patients with MBMs, mainly focusing upon prospective clinical trials of BRAF/MEK and PD-1/CTLA-4 inhibitors in patients with previously untreated MBMs. These additional systemic treatments have provided effective complementary treatment approaches and/or alternatives to radiation and craniotomy. The current role of radiation therapy, especially in conjunction with systemic therapies, is also discussed through the lens of various retrospective studies. The combined efficacy of systemic treatments with radiation has improved overall survival over the last 10 years and has sparked considerable research interest regarding optimal dosing and sequencing of radiation treatments with systemic treatments. Finally, the review describes ongoing clinical trials in patients with MBMs.

War against NRAS-Mutant Melanoma Using Targeted Therapies Remains Challenging.

In the search for targeting MAPK plus other pathways in NRAS-mutant melanoma, a phase Ib/II trial tested binimetinib plus ribociclib in metastatic melanoma. The response rate in the phase II trial was 19.5%, and the median progression-free survival was 3.7 months. See related article by Schuler et al., p. 3002.

Theragnostic significance of tumor-infiltrating lymphocytes and Ki67 in BRAFV600-mutant metastatic melanoma (BRIM-3 trial).

We conducted a retrospective tumor tissue analysis as part of the BRIM3 trial to evaluate the theragnostic significance of tumor-infiltrating lymphocytes (TILs) and melanoma cell proliferation. Using manual semi-quantitative analyses, we assessed the density of TILs by pathology review of tissue sections stained with hematoxylin and eosin (H&E TIL score) and by immunohistochemistry (IHC) with an anti-CD8 antibody (CD8 TIL score); also, the melanoma cell proliferation by IHC with an anti-Ki67 antibody. Three hundred and fifty-three, 280, and 172 patients' tumor tissue samples were available for H&E, CD8, and Ki67 IHC analysis, respectively. There was no association between high (2+, 3+) peritumoral and intratumoral H&E and/or TIL CD8 score or high Ki67 proliferation index (>15%) with serum LDH level and stage IV melanoma. Neither high Ki67 proliferation, nor high peritumoral and/or intratumoral TIL score was significantly associated with objective antitumor response in any treatment arm. High intratumoral and high peritumoral CD8 TIL score was significantly associated with progression-free survival (PFS) only in DTIC-treated patients (P = 0.002 and 0.037, respectively); in vemurafenib-treated patients, high intratumoral and/or peritumoral CD8 TIL score was not significant (log-rank P = 0.053 and 0.062, respectively). Nevertheless, a high peritumoral CD8 TIL score was a significant predictor of PFS and overall survival after adjustment for age, sex, serum LDH, ECOG performance status, and treatment arm in a Cox regression model. Vemurafenib does not only benefit patients bearing brisk TILs; even vemurafenib-treated patients with absent and/or non-brisk TILs tend to have longer PFS compared to DTIC-treated patients with brisk TILs. High peritumoral CD8 TIL score is a favorable prognostic factor independent of well-established AJCC staging factors.

The clinical significance of adenomatous polyposis coli (APC) and catenin Beta 1 (CTNNB1) genetic aberrations in patients with melanoma.

BACKGROUND: Melanoma-intrinsic activated ß-catenin pathway, the product of the catenin beta 1 (CTNNB1) gene, has been associated with low/absent tumor-infiltrating lymphocytes, accelerated tumor growth, metastases development, and resistance to anti-PD-L1/anti-CTLA-4 agents in mouse melanoma models. Little is known about the association between the adenomatous polyposis coli (APC) and CTNNB1 gene mutations in stage IV melanoma with immunotherapy response and overall survival (OS). METHODS: We examined the prognostic significance of somatic APC/CTNNB1 mutations in the Cancer Genome Atlas Project for Skin Cutaneous Melanoma (TCGA-SKCM) database. We assessed APC/CTNNB1 mutations as predictors of response to immunotherapies in a clinicopathologically annotated metastatic patient cohort from three US melanoma centers. RESULTS: In the TCGA-SKCM patient cohort (n = 434) presence of a somatic APC/CTNNB1 mutation was associated with a worse outcome only in stage IV melanoma (n = 82, median OS of APC/CTNNB1 mutants vs. wild-type was 8.15 vs. 22.8 months; log-rank hazard ratio 4.20, p = 0.011). APC/CTNNB1 mutation did not significantly affect lymphocyte distribution and density. In the 3-melanoma institution cohort, tumor tissues underwent targeted panel sequencing using two standards of care assays. We identified 55 patients with stage IV melanoma and APC/CTNNB1 genetic aberrations (mut) and 169 patients without (wt). At a median follow-up of more than 25 months for both groups, mut compared with wt patients had slightly more frequent (44% vs. 39%) and earlier (66% vs. 45% within six months from original diagnosis of stage IV melanoma) development of brain metastases. Nevertheless, time-to-development of brain metastases was not significantly different between the two groups. Fortunately, mut patients had similar clinical benefits from PD-1 inhibitor-based treatments compared to wt patients (median OS 26.1 months vs. 29.9 months, respectively, log-rank p = 0.23). Less frequent mutations in the NF1, RAC1, and PTEN genes were seen in the mut compared with wt patients from the 3-melanoma institution cohort. Analysis of brain melanoma tumor tissues from a separate craniotomy patient cohort (n = 55) showed that melanoma-specific, activated ß-catenin (i.e., nuclear localization) was infrequent (n = 3, 6%) and not prognostic in established brain metastases. CONCLUSIONS: APC/CTNNB1 mutations are associated with a worse outcome in stage IV melanoma and early brain metastases independent of tumor-infiltrating lymphocyte density. However, PD1 inhibitor-based treatments provide comparable benefits to both mut and wt patients with stage IV melanoma.

Long-term outcomes of patients with active melanoma brain metastases treated with combination nivolumab plus ipilimumab (CheckMate 204): final results of an open-label, multicentre, phase 2 study.

BACKGROUND: Combination nivolumab plus ipilimumab was efficacious in patients with asymptomatic melanoma brain metastases (MBM) in CheckMate 204, but showed low efficacy in patients with symptomatic MBM. Here, we provide final 3-year follow-up data from the trial. METHODS: This open-label, multicentre, phase 2 study (CheckMate 204) included adults (aged ≥18 years) with measurable MBM (0·5-3·0 cm in diameter). Asymptomatic patients (cohort A) had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and no neurological symptoms or baseline corticosteroid use; symptomatic patients (cohort B) had an ECOG performance status of 0-2 with stable neurological symptoms and could be receiving low-dose dexamethasone. Nivolumab 1 mg/kg plus ipilimumab 3 mg/kg was given intravenously every 3 weeks for four doses, followed by nivolumab 3 mg/kg every 2 weeks for up to 2 years, until disease progression or unacceptable toxicity. The primary endpoint was intracranial clinical benefit rate (complete responses, partial responses, or stable disease lasting ≥6 months) assessed in all treated patients. Intracranial progression-free survival and overall survival were key secondary endpoints. This study is registered with ClinicalTrials.gov, NCT02320058. FINDINGS: Between Feb 19, 2015, and Nov 1, 2017, 119 (72%) of 165 screened patients were enrolled and treated: 101 patients were asymptomatic (cohort A; median follow-up 34·3 months [IQR 14·7-36·4]) and 18 were symptomatic (cohort B; median follow-up 7·5 months [1·2-35·2]). Investigator-assessed intracranial clinical benefit was observed in 58 (57·4% [95% CI 47·2-67·2]) of 101 patients in cohort A and three (16·7% [3·6-41·4]) of 18 patients in cohort B; investigator-assessed objective response was observed in 54 (53·5% [43·3-63·5]) patients in cohort A and three (16·7% [3·6-41·4]) patients in cohort B. 33 (33%) patients in cohort A and three (17%) patients in cohort B had an investigator-assessed intracranial complete response. For patients in cohort A, 36-month intracranial progression-free survival was 54·1% (95% CI 42·7-64·1) and overall survival was 71·9% (61·8-79·8). For patients in cohort B, 36-month intracranial progression-free survival was 18·9% (95% CI 4·6-40·5) and overall survival was 36·6% (14·0-59·8). The most common grade 3-4 treatment-related adverse events (TRAEs) were increased alanine aminotransferase and aspartate aminotransferase (15 [15%] of 101 patients each) in cohort A; no grade 3 TRAEs occurred in more than one patient each in cohort B, and no grade 4 events occurred. The most common serious TRAEs were colitis, diarrhoea, hypophysitis, and increased alanine aminotransferase (five [5%] of each among the 101 patients in cohort A); no serious TRAE occurred in more than one patient each in cohort B. There was one treatment-related death (myocarditis in cohort A). INTERPRETATION: The durable 3-year response, overall survival, and progression-free survival rates for asymptomatic patients support first-line use of nivolumab plus ipilimumab. Symptomatic disease in patients with MBM remains difficult to treat, but some patients achieve a long-term response with the combination. FUNDING: Bristol Myers Squibb.

Multiregional genetic evolution of metastatic uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults and leads to deadly metastases for which there is no approved treatment. Genetic events driving early tumor development are well-described, but those occurring later during metastatic progression remain poorly understood. We performed multiregional genomic sequencing on 22 tumors collected from two patients with widely metastatic UM who underwent rapid autopsy. We observed multiple seeding events from the primary tumors, metastasis-to-metastasis seeding, polyclonal seeding, and late driver variants in ATM, KRAS, and other genes previously unreported in UM. These findings reveal previously unrecognized temporal and anatomic complexity in the genetic evolution of metastatic uveal melanoma, and they highlight the distinction between early and late phases of UM genetic evolution with implications for novel therapeutic approaches.

Safety and efficacy of the combination of nivolumab plus ipilimumab in patients with melanoma and asymptomatic or symptomatic brain metastases (CheckMate 204).

BACKGROUND: In patients with melanoma and asymptomatic brain metastases (MBM), nivolumab plus ipilimumab provided an intracranial response rate of 55%. Here, we present the first report for patients who were symptomatic and/or required corticosteroids and updated data for asymptomatic patients. METHODS: Patients with measurable MBM, 0.5-3.0 cm, were enrolled into Cohort A (asymptomatic) or Cohort B (stable neurologic symptoms and/or receiving corticosteroids). Nivolumab, 1 mg/kg, and ipilimumab, 3 mg/kg, were given intravenously every 3 weeks ×4, followed by nivolumab, 3 mg/kg, every 2 weeks until progression, unacceptable toxicity, or 24 months. The primary endpoint was intracranial clinical benefit rate (CBR; complete response [CR], partial response [PR], or stable disease ≥6 months). RESULTS: Symptomatic patients (N = 18) received a median of one nivolumab and ipilimumab combination dose and had an intracranial CBR of 22.2%. Two of 12 patients on corticosteroids had CR; 2 responded among the 6 not on corticosteroids. Median intracranial progression-free survival (PFS) and overall survival (OS) were 1.2 and 8.7 months, respectively. In contrast, with 20.6 months of follow-up, we confirmed an intracranial CBR of 58.4% in asymptomatic patients (N = 101); median duration of response, PFS, and OS were not reached. No new safety signals were observed. CONCLUSIONS: Nivolumab plus ipilimumab provides durable clinical benefit for asymptomatic patients with MBM and should be considered for first-line therapy. This regimen has limited activity in MBM patients with neurologic symptoms and/or requiring corticosteroids, supporting the need for alternative approaches and methods to reduce the dependency on corticosteroids. Clinical trial registration. ClinicalTrials.gov, NCT02320058.

Melanoma-specific expression of the tumor suppressor proteins p16 and PTEN is a favorable prognostic factor in established melanoma brain metastases.

PTEN and p16 frequently undergo (epi)genetic aberrations in melanoma resulting in decreased, or absent, protein levels. We investigated the prognostic significance of these tumor suppressor genes in melanoma brain metastases (MBMs). Immunohistochemical analysis was performed on archived tissue sections from craniotomies. Expression of PTEN and p16 was semiquantitatively scored (0-3 scale) in melanoma cells, glia, TILs, and endothelial cells of tumor-associated vessels and was compared among the different brain tumor cell compartments. Overall survival (OS) analysis was performed according to PTEN and p16 protein expression in melanoma cells. 58 patients (median age 56, 37 male) underwent craniotomy for MBMs before February 2014. The OS of patients with decreased, or absent, protein expression (0, 1+) of PTEN and p16 in melanoma cells was significantly shorter compared to that of patients with high (2+, 3+) expression (median OS 2.40 vs. 10.75 months and 4.1 vs. 8.1 months, respectively; Gehan-Breslow-Wilcoxon test P = 0.026 and P = 0.037, respectively). PTEN and p16 protein expression were significantly lower in TILs compared to melanoma cells (Mann-Whitney test P = 0.023 and P < 0.0001, respectively). Low/absent protein expression of PTEN/p16 is an adverse prognostic factor in MBMs. Surprisingly, expression of both PTEN and p16 proteins was significantly lower in TILs compared to melanoma cells. Proliferating (p16 absent/low) TILs within the brain with or without an active PI3K-Akt pathway (PTEN absent/low) may represent a favorable host response in MBMs. Thus, treatment of patients with MBMs with CDK4/6 or PI3K pathway inhibitors may result in an unfavorable, bystander, off-target effect on host immune response.

Targeting the IL-2 inducible kinase in melanoma; a phase 2 study of ibrutinib in systemic treatment-refractory distant metastatic cutaneous melanoma: preclinical rationale, biology, and clinical activity (NCI9922).

BACKGROUND: IL-2 inducible kinase (ITK) is highly expressed in metastatic melanomas and its inhibition suppresses melanoma cell proliferation. We hypothesize that ibrutinib has a direct antitumor effect in melanoma cell lines and that treatment of metastatic melanomas with ibrutinib induces antitumor responses. METHODS: We assessed the ibrutinib effect on melanoma cell proliferation, apoptosis, and motility. Patients with metastatic melanoma refractory to PD-1 and MAPK inhibitors (if BRAFV600-mutant) were treated with ibrutinib, 840 mg PO QD, as part of a phase II clinical trial (clinicaltrials.gov NCT02581930). RESULTS: Melanoma cell lines frequently express ITK, YES1, and EGFR. Ibrutinib suppressed cell motility and proliferation in most cell lines. Eighteen patients (13 male; median age 63.5 years, range 37-82; 12 with ipilimumab resistance) were enrolled. The most frequent side effects were fatigue (61%), anorexia (50%), hyponatremia (28%), nausea, and vomiting (22% each). No antitumor responses were seen. At a median follow-up of 6 months (0.3-35.8 months), the median progression-free survival was 1.3 months (range 0.2-5.5 months). Fifteen patients were discontinued from the study due to progression, and 14 patients had died from metastatic melanoma. All archived tumors expressed ITK, 41% had no expression of p16 and PTEN, and 61% had absent tumor-infiltrating lymphocytes (TILs). Ibrutinib significantly suppressed proliferating (Ki67+) CD19+ peripheral blood mononuclear cells and had no significant effect on other lymphocyte subsets. CONCLUSION: Ibrutinib did not induce any meaningful clinical benefit. ITK expression may not be clinically relevant. Treatment-refractory metastatic melanomas have other fundamental defects (i.e. absent PTEN and p16 expression, absent TILs) that may contribute to an adverse prognosis.

Immune Checkpoint Markers in Superficial Angiosarcomas: PD-L1, PD-1, CD8, LAG-3, and Tumor-Infiltrating Lymphocytes.

ABSTRACT: Cutaneous angiosarcomas may express programmed death ligand-1 (PD-L1) and PD-L1 expression, and the presence of tumor-infiltrating lymphocytes (TILs) correlates with outcome. These observations provide a basis for PD-1/PD-L1 inhibitor therapy. Lymphocyte activation gene 3 (LAG-3) is an inhibitory receptor that interacts with the PD-L1 axis and is considered to be a marker of immune exhaustion. The presence of LAG-3-positive lymphocytes in cutaneous angiosarcoma has not been established. We reviewed 10 cases of treatment naive angiosarcoma of skin and superficial soft tissue and assessed for PD-L1 (ZR3) expression, presence of TILs, and expression of CD8, PD1, and LAG-3 by tumor-associated inflammatory cells by immunohistochemistry. All 10 angiosarcomas were positive for PD-L1: 7 with high expression and 3 with low expression. TILs were present in all tumors: brisk in 7 and nonbrisk in 3. CD8 lymphocytes were present in all tumors with a range of 212-1274 cells per square millimeter (mean 557 CD8 cells/mm2). LAG-3-positive lymphocytes were present in 9 of 10 angiosarcomas with a range of 0-728 cells/mm2 (mean 146 LAG-3 cells cells/mm2). The ratio of LAG-3 lymphocytes to CD8 lymphocytes was 0%-59% (mean 27%). The PD1 cell counts were intermediate between CD8 and LAG3 counts. Cutaneous angiosarcomas frequently express PD-L1, have prominent numbers of CD8 positive, and have smaller numbers of LAG-3-positive and PD-1-positive TILs. Our findings provide further evidence of PD-L1 expression in cutaneous angiosarcoma and the promise for immune checkpoint inhibitor therapy.

Brain Tumor Microenvironment and Angiogenesis in Melanoma Brain Metastases.

BACKGROUND: High tumor-infiltrating lymphocytes (TILs) and hemorrhage are important prognostic factors in patients who have undergone craniotomy for melanoma brain metastases (MBM) before 2011 at the University of Pittsburgh Medical Center (UPMC). We have investigated the prognostic or predictive role of these histopathologic factors in a more contemporary craniotomy cohort from the University of North Carolina at Chapel Hill (UNC-CH). We have also sought to understand better how various immune cell subsets, angiogenic factors, and blood vessels may be associated with clinical and radiographic features in MBM. METHODS: Brain tumors from the UPMC and UNC-CH patient cohorts were (re)analyzed by standard histopathology, tumor tissue imaging, and gene expression profiling. Variables were associated with overall survival (OS) and radiographic features. RESULTS: The patient subgroup with high TILs in craniotomy specimens and subsequent treatment with immune checkpoint inhibitors (ICIs, n=7) trended to have longer OS compared to the subgroup with high TILs and no treatment with ICIs (n=11, p=0.059). Bleeding was significantly associated with tumor volume before craniotomy, high melanoma-specific expression of basic fibroblast growth factor (bFGF), and high density of CD31+αSMA- blood vessels. Brain tumors with high versus low peritumoral edema before craniotomy had low (17%) versus high (41%) incidence of brisk TILs. Melanoma-specific expression of the vascular endothelial growth factor (VEGF) was comparable to VEGF expression by TILs and was not associated with any particular prognostic, radiographic, or histopathologic features. A gene signature associated with gamma delta (gd) T cells was significantly higher in intracranial than same-patient extracranial metastases and primary melanoma. However, gdT cell density in MBM was not prognostic. CONCLUSIONS: ICIs may provide greater clinical benefit in patients with brisk TILs in MBM. Intratumoral hemorrhage in brain metastases, a significant clinical problem, is not merely associated with tumor volume but also with underlying biology. bFGF may be an essential pathway to target. VEGF, a factor principally associated with peritumoral edema, is not only produced by melanoma cells but also by TILs. Therefore, suppressing low-grade peritumoral edema using corticosteroids may harm TIL function in 41% of cases. Ongoing clinical trials targeting VEGF in MBM may predict a lack of unfavorable impacts on TIL density and/or intratumoral hemorrhage.

Chemotherapy Following PD-1 Inhibitor Blockade in Patients with Unresectable Stage III/Stage IV Metastatic Melanoma: A Single Academic Institution Experience.

Retrospective case studies in various cancers have shown clinical benefit from chemotherapy following PD-1 inhibitor progression. We asked whether we see a similar clinical benefit with chemotherapy following PD-1 inhibitor progression in metastatic melanoma. We performed a retrospective study in patients with metastatic melanoma, who had received PD-1 inhibitor-based treatments, subsequently progressed, and eventually received chemotherapy. We identified 25 patients (median age 58 years; range 31-77 years; 13 females). Most patients had cutaneous melanoma (72%), were BRAFV600E-negative (75%), and received single-agent temozolomide (84%). At a median follow-up of 21.0 months (range: 4.1-154.2 months), 2 patients had durable response to chemotherapy (progression-free survival is 31.9+ and 21.6+ months, respectively), and 1 patient had a partial, short-term response. We conclude that in this poor prognosis group administration of chemotherapy has a 12% response rate that can be durable. Overall, the clinical benefit is not inferior to that of PD-1 inhibitor-based treatments.

Prognostic value of B cells in cutaneous melanoma.

BACKGROUND: Measures of the adaptive immune response have prognostic and predictive associations in melanoma and other cancer types. Specifically, intratumoral T cell density and function have considerable prognostic and predictive value in skin cutaneous melanoma (SKCM). Less is known about the significance of tumor-infiltrating B cells in SKCM. Our goal was to understand the prognostic and predictive value of B cell phenotypic subsets in SKCM using RNA sequencing. METHODS: We used our previously published algorithm, V'DJer, to assemble B cell receptor (BCR) repertoires and estimate diversity from short-read RNA sequencing (RNA-seq). We applied machine learning-based cellular phenotype classifiers to measure relative similarity of bulk tumor sample gene expression profiles and different B cell phenotypes. We assessed these aspects of B cell biology in 473 SKCM from the Cancer Genome Atlas Project (TCGA) as well as in RNA-seq data corresponding to tumor samples procured from patients who received CTLA-4 and PD-1 inhibitors for metastatic SKCM. RESULTS: We found that the BCR repertoire was associated with different clinical factors, such as tumor tissue site and sex. However, increased clonality of the BCR repertoire was favorably prognostic in SKCM and was prognostic even after first conditioning on various clinical factors. Mutation burden was not correlated with any BCR measurement, and no specific mutation had an altered BCR repertoire. Lack of an assembled BCR in pre-treatment tumor tissues was associated with a lack of anti-tumor response to a CTLA-4 inhibitor in metastatic SKCM. CONCLUSIONS: These findings suggest an important prognostic and predictive role for B cell characteristics in SKCM. This has implications for melanoma immunobiology and potential development of immunogenomics features to predict survival and response to immunotherapy.

Examination and prognostic implications of the unique microenvironment of breast cancer brain metastases.

PURPOSE: Brain metastases (BM) are a complication of advanced breast cancer (BC). Histology of melanoma BM offers prognostic value; however, understanding the microenvironment of breast cancer brain metastases (BCBM) is less characterized. This study reports on four histological biomarkers, gliosis, immune infiltrate, hemorrhage, necrosis, and their prognostic significance in BCBM. METHODS: A biobank of 203 human tissues from patients who underwent craniotomy for BCBM was created across four academic institutions. Degree of gliosis, immune infiltrate, hemorrhage, and necrosis were identified and scored via representative H&E stain (0-3+). Overall survival (OS) was estimated using the Kaplan-Meier method. Cox proportional hazards regression evaluated prognostic value of the biomarkers in the context of standard clinical characteristics. RESULTS: BCBM subtype (available for n = 158) was 36% Her2+, 26% hormone receptor (HR)+/Her2- 38% HR-/Her2- (triple negative, TN). Gliosis was observed in 82% (116/141) of BCBM, with immune infiltrate 44% (90/201), hemorrhage 82% (166/141), and necrosis 87% (176/201). Necrosis was significantly higher in TNBC (p < 0.01). Presence of gliosis, immune infiltrate, and hemorrhage correlated with improved OS (p = 0.03, p = 0.03, p = 0.1), while necrosis correlated with inferior OS (p = 0.01). Improved OS was associated with gliosis in TN (p = 0.02), and immune infiltrate (p = 0.001) and hemorrhage (p = 0.07) in HER2+. In a multivariable model for OS, incorporating these biomarkers with traditional clinical variables improved the model fit (p < 0.001). CONCLUSION: Gliosis confers superior prognosis in TNBC BM; immune infiltrate and hemorrhage correlate with superior prognosis in HER2+ BCBM. Understanding the metastatic microenvironment of BCBM refines prognostic considerations and may unveil novel therapeutic strategies.