PKCε promotes oncogenic functions of ATF2 in the nucleus while blocking its apoptotic function at mitochondria.

The transcription factor ATF2 elicits oncogenic activities in melanoma and tumor suppressor activities in nonmalignant skin cancer. Here, we identify that ATF2 tumor suppressor function is determined by its ability to localize at the mitochondria, where it alters membrane permeability following genotoxic stress. The ability of ATF2 to reach the mitochondria is determined by PKCε, which directs ATF2 nuclear localization. Genotoxic stress attenuates PKCε effect on ATF2; enables ATF2 nuclear export and localization at the mitochondria, where it perturbs the HK1-VDAC1 complex; increases mitochondrial permeability; and promotes apoptosis. Significantly, high levels of PKCε, as seen in melanoma cells, block ATF2 nuclear export and function at the mitochondria, thereby attenuating apoptosis following exposure to genotoxic stress. In melanoma tumor samples, high PKCε levels associate with poor prognosis. Overall, our findings provide the framework for understanding how subcellular localization enables ATF2 oncogenic or tumor suppressor functions.

KDM5B promotes immune evasion by recruiting SETDB1 to silence retroelements.

Tumours use various strategies to evade immune surveillance1,2. Immunotherapies targeting tumour immune evasion such as immune checkpoint blockade have shown considerable efficacy on multiple cancers3,4 but are ineffective for most patients due to primary or acquired resistance5-7. Recent studies showed that some epigenetic regulators suppress anti-tumour immunity2,8-12, suggesting that epigenetic therapies could boost anti-tumour immune responses and overcome resistance to current immunotherapies. Here we show that, in mouse melanoma models, depletion of KDM5B-an H3K4 demethylase that is critical for melanoma maintenance and drug resistance13-15-induces robust adaptive immune responses and enhances responses to immune checkpoint blockade. Mechanistically, KDM5B recruits the H3K9 methyltransferase SETDB1 to repress endogenous retroelements such as MMVL30 in a demethylase-independent manner. Derepression of these retroelements activates cytosolic RNA-sensing and DNA-sensing pathways and the subsequent type-I interferon response, leading to tumour rejection and induction of immune memory. Our results demonstrate that KDM5B suppresses anti-tumour immunity by epigenetic silencing of retroelements. We therefore reveal roles of KDM5B in heterochromatin regulation and immune evasion in melanoma, opening new paths for the development of KDM5B-targeting and SETDB1-targeting therapies to enhance tumour immunogenicity and overcome immunotherapy resistance.

IL-7R licenses a population of epigenetically poised memory CD8+ T cells with superior antitumor efficacy that are critical for melanoma memory.

Recurrence of advanced melanoma after therapy is a major risk factor for reduced survival, and treatment options are limited. Antitumor immune memory plays a critical role in preventing melanoma recurrence and memory T cells could be a potent cell-based therapy, but the identity, and functional properties of the required immune cells are incompletely understood. Here, we show that an IL-7Rhi tumor-specific CD8+ population is critical for antitumor memory and can be epigenetically augmented to drive powerful antitumor immune responses. Using a model of functional antimelanoma memory, we found that high IL-7R expression selectively marks a CD8+ population in lymphoid organs that plays critical roles in maintaining tumor remission after immunotherapy or surgical resection. This population has intrinsic cytotoxic activity, lacks markers of exhaustion and has superior antitumor efficacy. IL-7Rhi cells have a functionally poised epigenetic landscape regulated by DNA methylation, which can be augmented by hypomethylating agents to confer improved survival and complete melanoma clearance in naive mice. Importantly, greater than 95% of tumor-specific T cells in draining lymph nodes after therapy express high levels of IL-7R. This overlap between IL-7Rhi and antigen-specific T cells allows for enrichment of a potent functional CD8+ population without determining antigen-specificity, which we demonstrate in a melanoma model without a known antigen. We identify that IL-7R expression in human melanoma is an independent prognostic factor of improved survival. These findings advance our basic understanding of antitumor memory and suggest a cell-based therapy using high IL-7R expression to enrich for a lymph node population with superior antitumor activity that can be augmented by hypomethylating agents.

TCR-sequencing in cancer and autoimmunity: barcodes and beyond.

The T cell receptor (TCR) endows T cells with antigen specificity and is central to nearly all aspects of T cell function. Each naïve T cell has a unique TCR sequence that is stably maintained during cell division. In this way, the TCR serves as a molecular barcode that tracks processes such as migration, differentiation, and proliferation of T cells. Recent technological advances have enabled sequencing of the TCR from single cells alongside deep molecular phenotypes on an unprecedented scale. In this review, we discuss strengths and limitations of TCR sequences as molecular barcodes and their application to study immune responses following Programmed Death-1 (PD-1) blockade in cancer. Additionally, we consider applications of TCR data beyond use as a barcode.

Vascular mimicry as a facilitator of melanoma brain metastasis.

Melanoma has the highest propensity among solid tumors to metastasize to the brain. Melanoma brain metastases (MBM) are a leading cause of death in melanoma and affect 40-60% of patients with late-stage disease. Therefore, uncovering the molecular mechanisms behind MBM is necessary to enhance therapeutic interventions. Vascular mimicry (VM) is a form of neovascularization linked to invasion, increased risk of metastasis, and poor prognosis in many tumor types, but its significance in MBM remains poorly understood. We found that VM density is elevated in MBM compared to paired extracranial specimens and is associated with tumor volume and CNS edema. In addition, our studies indicate a relevant role of YAP and TAZ, two transcriptional co-factors scarcely studied in melanoma, in tumor cell-vasculogenesis and in brain metastasis. We recently demonstrated activation of the Hippo tumor suppressor pathway and increased degradation of its downstream targets YAP and TAZ in a metastasis impaired cell line model. In the current study we establish the utility of anti-YAP/TAZ therapy in mouse models of metastatic melanoma whereby treatment effectively inhibits VM and prolongs survival of mice with MBM. The data presented herein suggest that VM may be an important and targetable mechanism in melanoma and that VM inhibition might be useful for treating MBM, an area of high unmet clinical need, thus having important implications for future treatment regimens for these patients.

Clinical Predictors of Survival in Patients With BRAFV600-Mutated Metastatic Melanoma Treated With Combined BRAF and MEK Inhibitors After Immune Checkpoint Inhibitors.

Prospective and between trial comparisons indicate that first-line treatment with immune checkpoint inhibitors improves survival outcomes compared to first-line therapy with combined BRAF and MEK inhibitors in metastatic melanoma containing BRAFV600E/K mutations. Long-term outcomes for BRAF/MEK inhibition after progression on immunotherapy have not been reported. Moreover, clinical variables associated with outcome from treatment with combined BRAF/MEK inhibition were previously identified in the first-line setting but have not been investigated when targeted therapies are administered after progression on immune therapy. We performed a retrospective single institution analysis of 40 metastatic melanoma patients receiving combined BRAF/MEK inhibitors after progression on an anti-PD-1 or ipilimumab plus nivolumab to assess response rate by RECIST 1.1, progression-free and overall survival (PFS and OS). Pretreatment clinical variables were analyzed for association with OS. Ipilimumab/nivolumab was the first-line immunotherapy regimen in 39 patients (97.5%), and BRAFV600E/K mutations were present in 33 (83%) and 7 (17%) patients, respectively. The median OS from start of BRAF/MEK inhibitors was 20.3 months (1.73-106.4+, 95% CI of median 13.3-30.7). Clinical characteristics associated with worse survival prior to starting BRAF/MEK inhibitors included age > 60 years (median OS 14 vs. 28 months; HR 2.5; 95% CI 0.91-6.87, P = .023), ECOG-PS > 2 (median OS 7 vs. 33 months; HR 2.89; 95% CI 0.78-10.76, P = .018), and presence of bone metastases (median OS 9 vs. 52 months; HR 3.17; 95% CI 1.33-7.54, P = .002). These associations with shorter survival maintained their significance on multivariate analysis. If confirmed in larger cohorts, the identified prognostic variables can be used for stratification of patients in future randomized trials.

Educational Review: Clinical Application of Immune Checkpoint Blockade for the Treatment of Melanoma.

In the last decade, immunotherapy has become the cornerstone in the management of patients with melanoma, the foremost cause of skin-cancer-related death in the USA. The emergence of immune checkpoint blockade as a crucial element in current immunotherapy and combination strategies has significantly transformed the treatments of resectable and advanced (unresectable or metastatic) melanoma. This paper reviews the landmark clinical trials that formed the basis of management of melanoma in the perioperative and metastatic setting. Furthermore, we discuss the rationale for the applications of PD-1 blockade and its combination with anti-CTLA-4 and anti-LAG-3. The review also explores new experimental combinations of PD-1 blockade with other immunomodulatory agents, including targeted therapies, anti-TIGIT antibodies, TLR-9 agonists, antiangiogenic agents, and mRNA vaccines.

Patterns of brain metastases response to immunotherapy with pembrolizumab.

PURPOSE: Central nervous system (CNS) metastases from lung cancers and melanoma, significantly contribute to morbidity and mortality. Despite advances in local therapies, there is a need for effective systemic treatments. Pembrolizumab, a PD-1 inhibitor, has shown promise for some patients with untreated brain metastases from melanoma and non-small cell lung cancer (NSCLC). This study aims to analyze the response of brain metastasis to pembrolizumab and associate characteristics like size and location with treatment outcome. METHODS: This retrospective study used imaging data from a phase II trial of pembrolizumab in melanoma or NSCLC patients with untreated brain metastases. MRI evaluations were conducted at 2 month intervals, with each brain metastasis treated as a distinct tumor for response assessment, based on modified RECIST criteria (maximum 5 lesions, 5 mm target lesions). RESULTS: Of 130 individual target metastases (> 5 mm), in 65 patients with NSCLC (90 metastases) and Melanoma (40 metastases), 32 (24.6%) demonstrated complete resolution, 24 (18.5%) had partial resolution, 32 (24.6%) were SD and 42 (32.3%) demonstrated PD. Those smaller than 10 mm were more likely to show complete resolution (p = 0.0218), while those ≥ 10 mm were more likely to have PR. There was no significant association between size, number or location (supratentorial vs. infratentorial) and lesion progression. The median time to metastatic lesion progression in the brain was 5.7-7 weeks. CONCLUSION: Pembrolizumab is effective in brain metastases from NSCLC and melanoma, showing response (CR + PR) in 43% and progression (PD) in 32% of metastases. With the median time to CNS progression of 5.7-7 weeks, careful radiographic monitoring is essential to guide timely local treatment decisions.

A bedside to bench study of anti-PD-1, anti-CD40, and anti-CSF1R indicates that more is not necessarily better.

BACKGROUND: Stimulating inflammatory tumor associated macrophages can overcome resistance to PD-(L)1 blockade. We previously conducted a phase I trial of cabiralizumab (anti-CSF1R), sotigalimab (CD40-agonist) and nivolumab. Our current purpose was to study the activity and cellular effects of this three-drug regimen in anti-PD-1-resistant melanoma. METHODS: We employed a Simon's two-stage design and analyzed circulating immune cells from patients treated with this regimen for treatment-related changes. We assessed various dose levels of anti-CSF1R in murine melanoma models and studied the cellular and molecular effects. RESULTS: Thirteen patients were enrolled in the first stage. We observed one (7.7%) confirmed and one (7.7%) unconfirmed partial response, 5 patients had stable disease (38.5%) and 6 disease progression (42.6%). We elected not to proceed to the second stage. CyTOF analysis revealed a reduction in non-classical monocytes. Patients with prolonged stable disease or partial response who remained on study for longer had increased markers of antigen presentation after treatment compared to patients whose disease progressed rapidly. In a murine model, higher anti-CSF1R doses resulted in increased tumor growth and worse survival. Using single-cell RNA-sequencing, we identified a suppressive monocyte/macrophage population in murine tumors exposed to higher doses. CONCLUSIONS: Higher anti-CSF1R doses are inferior to lower doses in a preclinical model, inducing a suppressive macrophage population, and potentially explaining the disappointing results observed in patients. While it is impossible to directly infer human doses from murine studies, careful intra-species evaluation can provide important insight. Cabiralizumab dose optimization is necessary for this patient population with limited treatment options. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03502330.

Determinants of overall survival in patients with metastatic uveal melanoma.

BACKGROUND: Despite improvements in the treatment of primary uveal melanoma (UM), patients with metastatic disease continue to exhibit poor survival. METHODS: A retrospective review of metastatic UM patients at Yale (initial cohort) and Memorial Sloan Kettering (validation cohort) was conducted. Cox proportional hazards regression was used to determine baseline factors that are associated with overall survival, including sex, Eastern Cooperative Oncology Group (ECOG) Performance Status Scale, laboratory measurements, metastasis location, and use of anti-CTLA-4 and anti-PD-1 therapies. Differences in overall survival were analyzed using Kaplan-Meier analysis. RESULTS: A total of 89 patients with metastatic UM were identified; 71 and 18, in the initial and validation cohorts, respectively. In the initial cohort, median follow-up was 19.8 months (range, 2-127 months) and median overall survival was 21.8 months (95% CI, 16.6-31.3). Female sex, anti-CTLA-4, and anti-PD-1 therapy were associated with better survival outcomes with adjusted death hazard ratios (HRs) of 0.40 (95% CI, 0.20-0.78), 0.44 (0.20-0.97), and 0.42 (0.22-0.84), respectively, whereas development of hepatic metastases and ECOG score ≥1 (per 1 U/L) were associated with worse survival outcomes with HRs of 2.86 (1.28-7.13) and 2.84 (1.29-6.09), respectively. In both the initial and validation cohorts, use of immune checkpoint inhibitors was associated with improved overall survival after adjusting for sex and ECOG score, with death HRs of 0.22 (0.08-0.56) and 0.04 (0.002-0.26), respectively. CONCLUSIONS: Development of extrahepatic-only metastases, ECOG of 0, immune checkpoint therapy, and female sex were each associated with more than 2-fold reductions in risk of death. PLAIN LANGUAGE SUMMARY: Metastatic uveal melanoma patients face limited treatment options and poor survival rates. Results from this retrospective analysis indicate that immune checkpoint inhibitors, such as anti-CTLA-4 and anti-PD-1 therapies, were associated with improved survival outcomes. Factors such as extrahepatic-only metastases, better baseline performance status, and female sex contributed to a more than 2-fold reduction in death risk. These findings highlight the potential of immunotherapy in treating metastatic uveal melanoma.

Validation of the Prognostic Usefulness of the Gene Expression Profiling Test in Patients with Uveal Melanoma.

PURPOSE: To validate the prognostic usefulness of gene expression profile (GEP) testing in patients with uveal melanoma. To determine whether combining tumor size with the GEP classification provides additional prognostic value. DESIGN: Retrospective analysis. PARTICIPANTS: Patients with a diagnosis of choroidal melanoma examined at Yale New Haven Hospital; University of California, San Diego; and Memorial Sloan Kettering Cancer Center. METHODS: Patients' demographic and clinical data and tumor characteristics were collected. Univariate and multivariate Cox hazard regression analysis were used to assess the association between tumor characteristics and GEP classification with metastasis as an outcome. MAIN OUTCOME MEASURES: Metastasis-free survival (MFS). RESULTS: Of the 337 individuals included in the study, 87 demonstrated metastases. The mean follow-up time was 37.2 (standard deviation [SD], 40.2) months for patients with metastases and 55.0 (SD, 49.3) months for those without metastases. Tumors of larger thickness and GEP class 2 (vs. class 1) were associated significantly with increased risk of metastasis. Tumor thickness showed better prognostic usefulness than GEP classification (Wald statistic, 40.7 and 24.2, respectively). Class 2 tumors with a thickness of 7.0 mm or more were associated with increased risk of metastasis than tumors with a thickness of < 7.0 mm (hazard ratio [HR], 3.23; 95% confidence interval [CI], 1.61-6.51), whereas class 1 tumors with a thickness of 9.0 mm or more were associated with increased risk of metastasis than tumors with a thickness of < 9.0 mm (HR, 2.07; 95% CI, 0.86-4.99). No difference in MFS was found between patients with class 1A tumors compared with those with class 1B tumors (P = 0.8). Patients with class 2 tumors showed an observed 5-year MFS of 47.5% (95% CI, 36.0%-62.8%). CONCLUSIONS: Tumor size was the most significant predictor of metastasis and provided additional prognostic value independent of GEP classification. In addition, rates of metastasis for class 2 tumors were lower than estimates reported by Castle Bioscience, and no difference in rates of metastasis were found between class 1A and 1B tumors. This indicates that tumor size should be accounted for when relying on GEP for prognostication and that patients with GEP class 1A or 1B tumors may benefit from the same metastatic surveillance protocols. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Coupled fibromodulin and SOX2 signaling as a critical regulator of metastatic outgrowth in melanoma.

We aimed to study mechanisms controlling metastatic outgrowth of melanoma into clinically relevant lesions, a critical process responsible for the majority of melanoma deaths. To this end, we developed novel in vivo models and identified molecular events that can be ascribed to their distinct phenotypes, indolent or highly metastatic. Induction of a proliferative state at distant sites was associated with high levels of the stem-like/progenitor marker, SOX2, and required the upregulation of FMOD, an extracellular matrix component, which modulates tumor-stroma interactions. Functional studies revealed a possible link between FMOD and SOX2; dual FMOD and SOX2 silencing nearly abolished brain metastasis and had a similar effect on distant metastasis to other sites. Our in vitro data suggests that FMOD and SOX2 cooperation plays an important role in tumor vasculogenic mimicry. Furthermore, we found that FMOD and SOX2 functional roles might converge at the activation of transcriptional co-factors YAP and TAZ, possibly via crosstalk with the tumor suppressor Hippo pathway. Finally, high expression of both genes in patient specimens predicted early development of brain metastasis. Thus, our study identifies FMOD and SOX2 cooperation as a novel regulatory mechanism that might be linked functionally to melanoma metastatic competence.

Longitudinal single-cell analysis of a patient receiving adoptive cell therapy reveals potential mechanisms of treatment failure.

Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) is being studied in multiple tumor types. However, little is known about clonal cell expansion in vitro and persistence of the ACT product in vivo. We performed single-cell RNA and T-Cell Receptor (TCR) sequencing on serial blood and tumor samples from a patient undergoing ACT, who did not respond. We found that clonal expansion varied during preparation of the ACT product, and only one expanded clone was preserved in the ACT product. The TCR of the preserved clone which persisted and remained activated for five months was previously reported as specific for cytomegalovirus and had upregulation of granzyme family genes and genes associated with effector functions (HLA-DQB1, LAT, HLA-DQA1, and KLRD1). Clones that contracted during TIL preparation had features of exhaustion and apoptosis. At disease progression, all previously detected clonotypes were detected. New clonotypes appearing in blood or tumor at disease progression were enriched for genes associated with cytotoxicity or stemness (FGFBP2, GNLY, GZMH, GZMK, IL7R, SELL and KLF2), and these might be harnessed for alternative cellular therapy or cytokine therapy. In-depth single-cell analyses of serial samples from additional ACT-treated patients is warranted, and viral- versus tumor-specificity should be carefully analyzed.

Merkel Cell Carcinoma: Changing Practice Patterns and Impact on Recurrence-Free and Overall Survival at a Single Institution and Nationally.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin. Our report describes the evolution of management and characteristics associated with recurrence, disease-specific survival (DSS) and overall survival (OS) in the treatment of MCC. METHODS: A single institution retrospective review of MCC and SEER data to determine factors associated with RFS, DSS, and OS using a multivariable Cox regression on inverse-probability weighted cohorts. RESULTS: One hundred fifty-nine patients were identified with a median age of 75. Of these, 96% were Caucasian and 60% male. Fifty-eight out of 159 (36%) of all patients were deceased with 21/58 (36%) dead from MCC with a median follow-up of 3.1 years. Institutionally, trends over time demonstrated an increased use of immunotherapy with a concomitant decrease in chemotherapy and decreased use of radiotherapy alone. Institutionally and nationally, there has been increased surgical nodal staging. Institutionally, factors associated with shorter DSS included advanced age, active cigarette smoker (p = 0.002), cT2 disease (p = 0.007), and MCC with unknown primary (p < 0.001). Institutionally, factors associated with shorter OS included ages ≥ 75 years (p < 0.001), an immunocompromised state (p < 0.001), truncal primary site (p = 0.002), and cT2 disease (HR 9.59, p < 0.001). CONCLUSION: Changing practice patterns in MCC management have been driven by the adoption of immunotherapy. Our study highlights that competing risks of mortality in MCC patients likely prevents OS from being an accurate surrogate outcome measure to understand factors associated with DSS.

Emerging Studies of Melanoma Brain Metastasis.

PURPOSE OF REVIEW: Among solid tumors, melanoma has the highest propensity for brain dissemination. Although newer treatment approaches have resulted in excellent control or elimination of brain metastasis in many patients, they remain the cause of significant morbidity and mortality. Here, we review recent preclinical and clinical studies to detail current understanding of the incidence, prognosis, biological characteristics, and treatments for melanoma brain metastases. RECENT FINDINGS: Clinical trials tailored to this patient population have demonstrated prolonged disease control with immune checkpoint inhibitors. Emerging clinical challenges include radiation necrosis and perilesional edema, phenomena that are rarely seen in other organs. Recent preclinical studies have resulted in improved understanding of the tumor microenvironment in the brain, providing insights into additional treatment approaches. The biological basis of brain tumor homing and survival within the central nervous system remain understudied. Additional preclinical and clinical studies will enhance our ability to prevent and treat brain metastases.

Melanoma brain metastases have lower T-cell content and microvessel density compared to matched extracranial metastases.

BACKGROUND: Although melanoma brain metastases (MBM) tend to respond to systemic therapy concordantly with extracranial metastases, little is known about differences in immune cell and vascular content between the brain and other metastatic sites. Here we studied infiltrating immune cell subsets and microvessel density (MVD) in paired intracerebral and extracerebral melanoma metastases. METHODS: Paired intracerebral and extracerebral tumor tissue was obtained from 37 patients with metastatic melanoma who underwent craniotomy between 1997 and 2014. A tissue microarray was constructed to quantify subsets of tumor-infiltrating T-cell, B-cell, and macrophage content, PD-L1 expression, and MVD using quantitative immunofluorescence. RESULTS: MBM had lower CD3+ (p = 0.01) and CD4+ (p = 0.003) T-cell content, lower MVD (p = 0.006), and a trend for lower CD8+ (p = 0.17) T-cell content compared to matched extracerebral metastases. There were no significant differences in CD20+ B-cell or CD68+ macrophage content, or tumor or stroma PD-L1 expression. Low MVD (p = 0.008) and high CD68+ macrophage density (p = 0.04) in intracerebral metastases were associated with improved 1-year survival from time of first MBM diagnosis. CONCLUSIONS: Although responses to immune-modulating drugs in the body and the brain tend to be concordant, differences were found in MVD and T-cell content between these sites. Studies of these markers should be incorporated into prospective therapeutic clinical trials to determine their prognostic and predictive value.

Incidence and characteristics of metastatic intracranial lesions in stage III and IV melanoma: a single institute retrospective analysis.

INTRODUCTION: The study aimed to describe the brain metastases (BM) incidence, at diagnosis and follow-up, in patients initially presenting with stage III or IV melanoma and characterize their metastatic brain lesions. We also sought to describe the association of common genetic mutations and immunotherapy with BM development in advanced melanoma. METHODS: Using our institution's tumor registry, we identified patients with initial diagnoses of stage III and stage IV melanoma. In this cohort, we obtained BM incidence at diagnosis and follow-up, characterized the metastatic brain lesions and primary tumor's genetic profile. RESULTS: During the follow-up period, 22.9% of patients with an initial diagnosis of stage III developed BM. In this cohort, the median time for BM occurrence was 20 months; [95% CI (14-29)]. Likewise, 37.7% of patients with Stage IV melanoma presented with BM at the time of diagnosis, and 22.7% of remaining patients developed BM at follow-up over a median duration of 6 months [95% CI (4-11)]. Therefore, suggesting an overall incidence of 51.9% in stage IV melanoma. Next, we observed that the incidence of BM development during the follow-up period significantly decreased from 2012 to 2017 (p < 0.001). Lastly, we found a significantly higher frequency of mutational BRAF in the primary tumor of patients with BM (68.7% vs. 31.2%; p = 0.02). CONCLUSIONS: While the overall incidence of BM remains high, the decreasing incidence of BM over the follow-up period is promising. Similar BM incidence in patients with an initial diagnosis of stage III or stage IV warrants appropriate imaging surveillance regimen for stage III patients.

Pembrolizumab for management of patients with NSCLC and brain metastases: long-term results and biomarker analysis from a non-randomised, open-label, phase 2 trial.

BACKGROUND: We did a phase 2 trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) or melanoma with untreated brain metastases to determine the activity of PD-1 blockade in the CNS. Interim results were previously published, and we now report an updated analysis of the full NSCLC cohort. METHODS: This was an open-label, phase 2 study of patients from the Yale Cancer Center (CT, USA). Eligible patients were at least 18 years of age with stage IV NSCLC with at least one brain metastasis 5-20 mm in size, not previously treated or progressing after previous radiotherapy, no neurological symptoms or corticosteroid requirement, and Eastern Cooperative Oncology Group performance status less than two. Modified Response Evaluation Criteria in Solid Tumors (mRECIST) criteria was used to evaluate CNS disease; systemic disease was not required for participation. Patients were treated with pembrolizumab 10 mg/kg intravenously every 2 weeks. Patients were in two cohorts: cohort 1 was for those with PD-L1 expression of at least 1% and cohort 2 was patients with PD-L1 less than 1% or unevaluable. The primary endpoint was the proportion of patients achieving a brain metastasis response (partial response or complete response, according to mRECIST). All treated patients were analysed for response and safety endpoints. This study is closed to accrual and is registered with ClinicalTrials.gov, NCT02085070. FINDINGS: Between March 31, 2014, and May 21, 2018, 42 patients were treated. Median follow-up was 8·3 months (IQR 4·5-26·2). 11 (29·7% [95% CI 15·9-47·0]) of 37 patients in cohort 1 had a brain metastasis response. There were no responses in cohort 2. Grade 3-4 adverse events related to treatment included two patients with pneumonitis, and one each with constitutional symptoms, colitis, adrenal insufficiency, hyperglycaemia, and hypokalaemia. Treatment-related serious adverse events occurred in six (14%) of 42 patients and were pneumonitis (n=2), acute kidney injury, colitis, hypokalaemia, and adrenal insufficiency (n=1 each). There were no treatment-related deaths. INTERPRETATION: Pembrolizumab has activity in brain metastases from NSCLC with PD-L1 expression at least 1% and is safe in selected patients with untreated brain metastases. Further investigation of immunotherapy in patients with CNS disease from NSCLC is warranted. FUNDING: Merck and the Yale Cancer Center.

[11C]Methionine and [11C]PBR28 as PET Imaging Tracers to Differentiate Metastatic Tumor Recurrence or Radiation Necrosis.

PURPOSE: As stereotactic radiosurgery (SRS) and immunotherapy are increasingly used to treat brain metastases, incidence of radiation necrosis (RN) is consequently rising. Differentiating tumor regrowth (TR) from RN is vital in management but difficult to assess using MRI. We hypothesized that tumor methionine levels would be elevated given increased metabolism and high amino acid uptake, whereas RN would increase inflammation marked by upregulated translocator protein (PBR-TSPO), which can be quantified with specific PET tracers. PROCEDURES: We performed a feasibility study to prospectively evaluate [11C]methionine and [11C]PBR28 using PET in 5 patients with 7 previously SRS-treated brain metastases demonstrating regrowth to differentiate TR from RN. RESULTS: Sequential imaging with dual tracers was well-tolerated. [11C]methionine was accurate for detecting pathologically confirmed TR in 7/7 lesions, whereas [11C]PBR28 was only accurate in 3/7 lesions. Tumor PBR-TSPO expression was elevated in both melanoma and lung cancer cells, contributing to lack of specificity of [11C]PBR28-PET. CONCLUSION: Sequential use of PET tracers is safe and effective. [11C]Methionine was a reliable TR marker, but [11C]PBR28 was not a reliable marker of RN. Studies are needed to determine the causes of post-radiation inflammation and identify specific markers of RN to improve diagnostic imaging.

PLEKHA5 regulates tumor growth in metastatic melanoma.

BACKGROUND: PLEKHA5 has previously been identified as a novel molecule implicated in melanoma brain metastasis, a disease that continues to portend a poor prognosis. The aim of this study was to further investigate the functional role of PLEKHA5 in disseminated melanoma. METHODS: The impact of PLEKHA5 on proliferation and tumor growth was examined in vitro and in melanoma xenograft models, including brain-tropic melanomas (melanomas tending to disseminate to the brain). In vitro loss- and gain-of-function studies were used to explore the underlying mechanisms of PLEKHA5-mediated tumor growth and the crosstalk between PLEKHA5 and PI3K/AKT/mTOR or MAPK/ERK signaling. The clinical relevance of PLEKHA5 dysregulation was further investigated in a cohort of matched cranial and extracranial melanoma metastases. RESULTS: PLEKHA5 stable knockdown negatively regulated cell proliferation by inhibiting the G1 -to-S cell cycle transition, which coincided with upregulation of the cell cycle regulator PDCD4. Conversely, ectopic PLEKHA5 expression exhibited the inverse effect. PLEKHA5 knockdown significantly inhibited tumor growth, whereas its overexpression upregulated the growth of tumors, which was induced by cranial and subcutaneous inoculation of cells in nude mice. PLEKHA5 modulation affected PDCD4 protein stability and was coupled with changes in PI3K/AKT/mTOR pathway signaling. High PDCD4 expression in cerebral specimens was associated with better overall survival. CONCLUSIONS: This study further supports the role of PLEKHA5 as a regulator of melanoma growth at distant sites, including the brain. Furthermore, the results highlight the significance of PDCD4 dysregulation in disseminated melanoma and implicate PDCD4 as a possible causal link between PLEKHA5 and cell proliferation and growth.