Natural killer (NK) cells are a key component of an innate immune system. They are important not only in initiating, but also in augmenting adaptive immune responses. NK cell activation is mediated by a carefully orchestrated balance between the signals from inhibitory and activating NK cell receptors. NK cells are potent producers of proinflammatory cytokines and are also able to elicit strong antitumor responses through secretion of perforin and granzyme B. Tumors can develop many mechanisms to evade NK cell antitumor responses, such as upregulating ligands for inhibitory receptors, secreting anti-inflammatory cytokines and recruiting immunosuppressive cells. Enhancing NK cell responses will likely augment the effectiveness of immunotherapies, and strategies to accomplish this are currently being evaluated in clinical trials. A comprehensive understanding of NK cell biology will likely provide additional opportunities to further leverage the antitumor effects of NK cells. In this review, we therefore sought to highlight NK cell biology, tumor evasion of NK cells and clinical trials that target NK cells.

Immunotherapy targeting T-cell inhibitory receptors, namely programmed cell death-1 (PD-1) and/or cytotoxic T-lymphocyte associated protein-4 (CTLA-4), leads to durable responses in a proportion of patients with advanced metastatic melanoma. Combination immunotherapy results in higher rates of response compared to anti-PD-1 monotherapy, at the expense of higher toxicity. Currently, there are no robust molecular biomarkers for the selection of first-line immunotherapy. We used flow cytometry to profile pretreatment tumor biopsies from 36 melanoma patients treated with anti-PD-1 or combination (anti-PD-1 plus anti-CTLA-4) immunotherapy. A novel quantitative score was developed to determine the tumor cell expression of antigen-presenting MHC class I (MHC-I) molecules, and to correlate expression data with treatment response. Melanoma MHC-I expression was intact in all tumors derived from patients who demonstrated durable response to anti-PD-1 monotherapy. In contrast, melanoma MHC-I expression was low in 67% of tumors derived from patients with durable response to combination immunotherapy. Compared to MHC-I high tumors, MHC-I low tumors displayed reduced T-cell infiltration and a myeloid cell-enriched microenvironment. Our data emphasize the importance of robust MHC-I expression for anti-PD-1 monotherapy response and provide a rationale for the selection of combination immunotherapy as the first-line treatment in MHC-I low melanoma.

More evidence is needed to support using GEP testing to inform recommendations regarding SLNB, intensity of follow-up or imaging surveillance, and postoperative adjuvant therapy. The MPWG recommends further research to assess the validity and clinical applicability of existing and emerging GEP tests. Decisions on performing GEP testing and patient management based on these results should only be made in the context of discussion of testing limitations with the patient or within a multidisciplinary group.

This population-based validation confirmed the value of the EORTC nomogram in predicting recurrence-free survival in patients with SN-negative melanoma. The EORTC nomogram could be used in clinical practice for personalizing follow-up and selecting high-risk patients for trials of adjuvant systemic therapy.

In 2 large patient cohorts from 2 different continents, regression was a favorable prognostic factor for patients with stage 1 and 2 melanomas, especially in those with thin and intermediate thickness tumors and those with SSM subtype.

Abstract Melanoma patients (pts) with liver mets have a lower response rate (RR), and shorter progression-free (PFS) and overall survival (OS) compared to pts without liver mets when treated with anti-PD-1 (PD1) therapy. The liver microenvironment (ME) induces T-cell tolerance through the interaction of T cells with liver sinusoidal endothelial cells. To explore this further we compared clinicopathological features, circulating cytokines and tumor gene expression (GE) profiles of pts with and without liver mets treated with PD1 combined with ipilimumab (PD1+IPI), and treated with BRAF targeted therapy (TT). Demographics, disease characteristics and outcome data were collected from 140 pts treated with PD1+IPI and from 76 pts treated with BRAF+/-MEKi. Tumor-infiltrating lymphocytes (TILs) immunoreactivity score (TILs density x % of tumor with TILS), % of tumor content, necrosis and fibrosis were assessed by immunohistochemistry in liver and lung samples. Pre-treatment circulating cytokines and tumor GE data (RNA seq) were compared between pts with and without liver mets. In pts treated with IPI+PD1, liver mets had the lowest tumor regression (med -7%) compared to all other sites of disease (med -66%), while in TT-treated pts the response was similar across all sites of disease. Pts with liver mets (n=39) had lower RR (44% v 75%), and shorter median PFS and OS (p<0.05) when treated with IPI+PD1 than those without liver mets (n=101). In contrast, in pts treated with TT, RR, PFS and OS were similar between pts with (n=19) vs without (n=57) liver mets. Pts with liver mets had less response in adrenal and LN mets when treated with IPI+PD1 compared to pts without liver mets, but not with TT. In a multivariate analysis performed on the PD1+IPI cohort and validated in the TT group, presence of bone (OR 4.6 p=0.004) and spleen mets (OR 13.5 p=0.01) were associated with the presence of liver mets. Compared to lung mets (n=22), liver mets ME (n=22) had a lower TILs immunoreactivity score (med 30 vs 80, p=0.05), while there was no difference in the % of tumor content, fibrosis or necrosis. The expression of 65 cytokines was measured in plasma of treatment-naive pts; pts with liver mets (n=37) had higher levels of Eotaxin 2 (p=0.01), IP-10 (p=0.02) & IL-8 (p=0.03) compared to pts without liver mets (n=99). GE analysis of melanoma samples showed higher expression of MMP-8 and HIF1a in pts with (n=58) vs without (n=28) liver mets, validated in an independent cohort (n=58). Pts with liver mets display distinct clinicopathological features, distinct circulating cytokines and melanoma GE profiles, and are less responsive to PD1+IPI compared to pts without liver mets. The levels of Eotaxin-2, IP-10 and IL-8 are higher in melanoma pts with liver mets compared to pts without liver mets, similar to what is seen in pts with colon cancer liver mets. Liver mets’ ME may hold unique immunosuppressive mechanisms that are amenable to therapeutic targeting. Citation Format: Ines Silva, Annie Tasker, Camelia Quek, Robert Rawson, Su Yin Lim, Kevin Wang, Jordan Conway, Rebecca Velickovic, Tasnia Ahmed, Serigne Lo, Jean Yang, Helen Rizos, James S. Wilmott, Richard A. Scolyer, Alexander M. Menzies, Georgina V. Long. Liver metastases (mets) induce systemic immunosuppression and immunotherapy resistance in metastatic melanoma [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 975.

Similar rates of CNS metastasis were observed in 2 large, geographically distinct cohorts of patients with stage III melanoma. The results highlight the importance of primary tumor mitotic rate. Furthermore, they provide a framework for developing evidence-based surveillance strategies and evaluating the impact of contemporary adjuvant therapies on the risk of CNS metastasis development.

Abstract Background High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity amongst complex human cancers. scRNA-Seq studies using fresh human surgical tissue is logistically difficult, precludes histopathological triage of samples and limits the ability to perform batch processing. This hinderance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers, and on high throughput scRNA-Seq platforms. Results We show that the viable cryopreservation of human cancers provides high quality single-cell transcriptomes using the Chromium 10X platform. We sequenced a total of ∼120,000 cells from fresh and cryopreserved replicates across three breast cancers, two prostate cancers and a cutaneous melanoma. Importantly, tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We then show that cryopreservation had minimal impacts on results of downstream analyses such as biological pathway enrichment. Further, we demonstrate the advantage of cryopreserving whole-cells for immunophenotyping methods such as CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

ABSTRACT IMPORTANCE Brain metastases occur in 60% of patients with advanced melanoma and are a major cause of melanoma-related mortality and morbidity. Although our understanding of the molecular alterations associated with melanoma progression is improving, there are currently no validated biomarkers which might help identify those patients at highest risk of developing brain metastases. OBJECTIVE To examine the somatic mutational and copy-number landscape of brain metastases that develop as the isolated first visceral site of recurrence – “early brain-metastasis” compared to extracranial melanoma metastases. DESIGN, SETTING AND PARTICIPANTS Whole-exome sequencing of 50 tumors from patients undergoing surgical resection of one or more brain metastasis occurring as the first site of visceral relapse were identified from prospectively maintained databases in Sydney, Wellington, New York and Cambridge. Whole exome sequencing analyses allowed mutational profiles to be compared to cutaneous melanomas in The Cancer Genome Atlas (SKCM-TCGA; n=358) and the Memorial Sloan Kettering (SKCM-MSK-IMPACT; n=186) datasets. An external dataset comprising a further 18 patients with surgically resected early brain metastasis from two additional academic centers served as an independent validation cohort. MAIN OUTCOMES AND MEASURES To assess the frequency of driver mutations in early brain metastasis and their influence on survival. RESULTS In concordance with the landmark melanoma sequencing studies, we identified mutations in BRAF (21/50, 42%), NRAS (14/50, 28%) and NF1 (11/50, 22%) as the most frequently mutated melanoma driver genes. When compared to the mutational landscape of cutaneous melanomas in TCGA (SKCM-TCGA), KRAS was the most significantly enriched driver gene, with 5/50 (10%) of brain metastases harboring non-synonymous mutations, of which 4/5 (80%) were in the hotspot positions of codons 12 and 61. This was significantly higher than the corresponding frequency of KRAS -mutations within the entire SKCM-TCGA (2% (7/358), p=0.009, Fisher’s Exact Test) as well as the SKCM-MSK-IMPACT cohort (1.6% (3/186), p=0.016). Variants in KRAS were mutually exclusive from BRAF V600 , NRAS and HRAS mutations and were associated with a significantly reduced overall survival from resection of brain metastasis (relative to KRAS -wild type brain metastases) in multivariate Cox proportional hazard models (HR 1.80, 95% CI 1.46-24.89, p=0.013). Mutations in KRAS were also clonal and concordant with extracranial disease, which suggests these mutations are present within the primary tumor CONCLUSIONS AND RELEVANCE Our analysis, the largest to date, suggests that early metastases to the brain (presenting as the first site of visceral relapse) are characterized by significant enrichment of hotspot KRAS mutations, potentially implicating constitutive RAS-driven cellular programs in neurotropic metastatic behavior in these cases. Based on these data, we suggest that screening for KRAS mutations might help identify those patients with primary melanoma at higher risk of brain metastases or poor survival, and could help inform future surveillance strategies. Key Points Question What is the frequency of driver mutations in early melanoma brain metastases? Findings In this study of 50 patients with melanoma metastasizing first to the brain, KRAS mutations were the most significantly enriched driver gene (n=5, 10% of patients) when compared to landmark cutaneous melanoma studies. The high KRAS mutation frequency was also observed in an external validation cohort of 18 patients with early brain metastases. Mutations in KRAS were mutually exclusive from mutations in the key RAS signaling genes and conferred a worse overall survival from resection of brain metastasis. Meaning Hotspot KRAS mutations could help identify those patients with primary melanoma at higher risk of brain metastases that may benefit from more intensive, protracted surveillance as well as earlier use of adjuvant therapy.

The prognosis for patients with UM is poor, and recent clinical trials have failed to prolong overall survival (OS) of these patients. Over 95% of UM harbor activating driver mutations, and this allows for the investigation of ctDNA. In this study, we investigated the value of ctDNA for adaptive clinical trial design in metastatic UM. Longitudinal plasma samples were analyzed for ctDNA in 17 metastatic UM patients treated with PKCi-based therapy in a phase 1 clinical trial setting. Plasma ctDNA was assessed using digital droplet PCR (ddPCR) and a custom melanoma gene panel for targeted next generation sequencing (NGS). Baseline ctDNA strongly correlated with baseline lactate dehydrogenase (LDH) (<i>p</i> < 0.001) and baseline disease burden (<i>p</i> = 0.002). Early during treatment (EDT) ctDNA accurately predicted patients with clinical benefit to PKCi using receiver operator characteristic (ROC) curves (AUC 0.84, [95% confidence interval 0.65-1.0, <i>p</i> = 0.026]). Longitudinal ctDNA assessment was informative for establishing clinical benefit and detecting disease progression with 7/8 (88%) of patients showing a rise in ctDNA and targeted NGS of ctDNA revealed putative resistance mechanisms prior to radiological progression. The inclusion of longitudinal ctDNA monitoring in metastatic UM can advance adaptive clinical trial design.

<h3>Background</h3> Pembrolizumab is a standard of care for the treatment of unresectable or metastatic melanoma and an adjuvant treatment of melanoma with involvement of lymph node(s) following complete resection. However, new treatment options are needed to reduce the tumor burden before surgery and improve overall outcomes in patients with advanced melanoma. <h3>Methods</h3> MK-3475-U02 is a phase 1/2, rolling arm, multicenter, open-label, adaptive design study to evaluate the safety and efficacy of investigational agents with or without pembrolizumab or pembrolizumab alone for the treatment of melanoma. Patients will be enrolled in 1 of the 3 substudies. Substudy 02A will include patients with programmed death-1 (PD-1)–refractory melanoma (progressed after ≥2 doses of anti-PD-1/programmed death ligand-1 [PD-L1] therapy) randomized equally to treatment arms evaluating ≥1 investigational agent(s) with or without pembrolizumab. Enrollment is planned for up to ~100 patients per arm.Substudy 02B will include patients with unresectable stage III or stage IV melanoma not amenable to local therapy. Patients will be randomized 2:1 to combination (≥1 investigational agent(s) with or without pembrolizumab) or monotherapy (pembrolizumab alone) stratified by baseline lactate dehydrogenase status (normal/elevated) and prior adjuvant therapy with a PD-1 inhibitor (yes/no). Enrollment is planned for ~90 patients in the combination arm and ~45 in the control arm.Substudy 02C will include patients with stage IIIB/IIIC/IIID melanoma who are candidates for neoadjuvant therapy. Patients will be randomly assigned to combination (≥1 investigational agent(s) with or without pembrolizumab) or monotherapy (pembrolizumab alone). Surgical resection will be performed 6 weeks after the first dose of neoadjuvant study intervention. Enrollment is planned for ~25 patients in combination and ~15 in the pembrolizumab monotherapy arms.Treatment will continue for up to 2 years (up to 1 year neoadjuvant/adjuvant therapy for substudy 02C), until disease progression, unacceptable toxicity, or study discontinuation. The primary end points include safety (adverse events and study intervention discontinuations) for all 3 substudies; objective response rate by blinded independent central review per Response Evaluation Criteria in Solid Tumors 1.1 for substudies 02A and 02B, and pathological complete response (pCR) as assessed by central review of the pathology results for substudy 02C. Secondary end points include duration of response for substudies 02A and 02B, and recurrence-free survival, near pCR, and pathological partial response rates for substudy 02C. <h3>Results</h3> N/A <h3>Conclusions</h3> N/A <h3>Acknowledgements</h3> The authors thank the patients and their families for participating in these trials and all investigators and site personnel. Medical writing and/or editorial assistance was provided by Neha Tuli-Wildemore, MBBS, PhD, and Doyel Mitra, PhD, of the ApotheCom pembrolizumab team (Yardley, PA, USA). This assistance was funded by Merck Sharp &amp; Dohme Corp., a subsidiary of Merck &amp; Co., Inc., Kenilworth, NJ, USA. <h3>Trial Registration</h3> NCT04305041, NCT04305054, NCT04303169 <h3>Ethics Approval</h3> The study protocol and all amendments were approved by the relevant Institutional Review Board or ethics committee at each study site. All patients provided written informed consent to participate in the clinical trial. <h3>Consent</h3> N/A <h3>Reference</h3> N/A