BackgroundAdjuvant immune checkpoint inhibitors (ICI) have improved survival in stage III cutaneous melanoma, yet many patients do not benefit. The tumor microenvironment is pivotal for durable responses; defining cellular composition can pinpoint immune components promoting anti-tumor activity and identify biomarkers associated with improved outcomes.MethodRegional lymph nodes (RLNs) were obtained at surgery from 29 patients with stage III melanoma. Eligible patients received adjuvant anti-PD-1 (αPD-1; pembrolizumab or nivolumab). Mass cytometry (CyTOF) was used to determine cellular composition of pre-treatment surgical specimens. NanoString bulk gene expression data from 125 patients receiving surgery without adjuvant therapy were used to evaluate trends observed in the CyTOF dataset.ResultsHigher proportions of CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cells were associated with improved prognosis in the CyTOF cohort. In the untreated cohort, a “B cell excluded” subgroup (< 2.5% tumor-infiltrating lymphocyte pathology score) had worse outcome, showing reduced B cell score and lower expression of activation genes including CD38, without change in CD8+ T cell score.ConclusionBaseline infiltration of CD8+ TRM and plasmablast-like CD38hiCD19dim cells in RLN is strongly associated with prolonged distant metastasis-free survival in patients receiving αPD-1, supporting their potential as prognostic biomarkers in stage III melanoma.

Adjuvant immunotherapy has significantly improved survival for patients with stage III cutaneous melanoma, yet a fraction of patients will not benefit from immune checkpoint inhibitors (ICI). The tumor microenvironment plays a pivotal role in generating durable responses to ICI. By analyzing the cellular composition of tumor-associated subsets, key immune components essential for promoting an anti-tumor environment can be pinpointed. This will allow for both patient stratification and identification of biomarkers associated with improved patient outcome. Regional lymph nodes were obtained from patients with stage III melanoma at surgery (n=29). Patients eligible for anti-PD-1 therapy (αPD-1; pembrolizumab or nivolumab) received adjuvant treatment for up to one year. CyTOF was used to determine cellular composition in pre-treatment surgical specimens. Bulk gene expression data generated by NanoString from patients receiving surgery without adjuvant therapy (n=125) was implemented for evaluating trends observed in the CyTOF dataset. Although no significant differences were observed across major hierarchical immune cell types between patients who developed distant metastasis after surgery and those that did not, an increased proportion of CD103 + PD-1 + CD8 + (T RM ) T cells and plasmablast-like CD38 hi CD19 dim cells were associated with improved prognosis in the CyTOF cohort. In the untreated cohort, a subset of patients defined as “Ultra-cold” (< 2.5 % tumor-infiltrating lymphocyte (TIL) scored by a pathologist) had significantly worse outcome than those with higher TIL infiltration. This Ultra-cold TIL group was associated with reduced B cell score, but not CD8 + T cell score, as well as reduced expression of activation genes like CD38 . In this study, CD103 + PD-1 + CD8 + (T RM ) T cells and plasmablast-like CD38 hi CD19 dim cell populations were found to be strongly associated with prolonged distant metastasis-free survival in regional lymph nodes from patients with stage III melanoma treated with αPD-1. This suggests an association between progression and infiltration of these cell types at baseline and highlights the potential of using immune cell subsets as prognostic biomarkers. What is already known on this topic – Patients being diagnosed with stage III melanoma will receive immune checkpoint inhibitors but will often be cured by surgery alone. Selection of which patients might benefit from treatment is still unresolved. Accurate biomarkers would aid in treatment stratification, to avoid overtreatment, and unnecessary toxicities. What this study adds – This study highlights how baseline CD103 + PD-1 + CD8 + (T RM ) T cells and plasmablast-like CD38 hi CD19 dim cell populations in the regional lymph node, is strongly associated with improved outcome in patients receiving anti-PD-1 therapy. How this study might affect research, practice or policy – The strong association between baseline plasmablast-like cell infiltration in RLN and prolonged distant metastasis free- survival, highlights this cell type as a potential treatment stratification criterion to identify patients with good prognosis.

Background Adjuvant immunotherapy has significantly improved survival for patients with stage III cutaneous melanoma, yet a fraction of patients will not benefit from immune checkpoint inhibitors (ICI). The tumor microenvironment plays a pivotal role in generating durable responses to ICI. By analyzing the cellular composition of tumor-associated subsets, key immune components essential for promoting an anti-tumor environment can be pinpointed. This will allow for both patient stratification and identification of biomarkers associated with improved patient outcome. Method Regional lymph nodes were obtained from patients with stage III melanoma at surgery (n=29). Patients eligible for anti-PD-1 therapy (αPD-1; pembrolizumab or nivolumab) received adjuvant treatment for up to one year. CyTOF was used to determine cellular composition in pre-treatment surgical specimens. Bulk gene expression data generated by NanoString from patients receiving surgery without adjuvant therapy (n=125) was implemented for evaluating trends observed in the CyTOF dataset. Results Although no significant differences were observed across major hierarchical immune cell types between patients who developed distant metastasis after surgery and those that did not, an increased proportion of CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cells were associated with improved prognosis in the CyTOF cohort. In the untreated cohort, a subset of patients defined as “Ultra-cold” (< 2.5 % tumor-infiltrating lymphocyte (TIL) scored by a pathologist) had significantly worse outcome than those with higher TIL infiltration. This Ultra-cold TIL group was associated with reduced B cell score, but not CD8+ T cell score, as well as reduced expression of activation genes like CD38 . Conclusion In this study, CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cell populations were found to be strongly associated with prolonged distant metastasis-free survival in regional lymph nodes from patients with stage III melanoma treated with αPD-1. This suggests an association between progression and infiltration of these cell types at baseline and highlights the potential of using immune cell subsets as prognostic biomarkers. What is already known on this topic – Patients being diagnosed with stage III melanoma will receive immune checkpoint inhibitors but will often be cured by surgery alone. Selection of which patients might benefit from treatment is still unresolved. Accurate biomarkers would aid in treatment stratification, to avoid overtreatment, and unnecessary toxicities. What this study adds – This study highlights how baseline CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cell populations in the regional lymph node, is strongly associated with improved outcome in patients receiving anti-PD-1 therapy. How this study might affect research, practice or policy – The strong association between baseline plasmablast-like cell infiltration in RLN and prolonged distant metastasis free- survival, highlights this cell type as a potential treatment stratification criterion to identify patients with good prognosis. ### Competing Interest Statement The authors have declared no competing interest. Southern and Eastern Norway Regional Health Authority, https://ror.org/02qx2s478, 2019048, 2016117, 2017100 Norwegian Ministry of Health and Care Services Fondsstiftelsen at Oslo University Hospital

Supplementary Figure 3 shows cell cycle profiles for Melmet 1 and WM1366 cells and YH2AX expression measured by flow cytometry for WM1366 cells. Further, the figure shows immunoblots of indicated proteins for cells treated with BGB324 and/or AZD7762 or BGB324 and/or VE-822.

Supplementary Figure 1 shows AXL expression in a melanoma cell line panel, dose response of BGB324 and how BGB324 treatment affects invasion and migration.

Supplementary Figure 2 shows animal weight throughout the treatment period.

Supplementary Table 1 from Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma

Supplementary Table 2 from Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma

More than half of metastatic melanoma patients receiving standard therapy fail to achieve a long-term survival due to primary and/or acquired resistance. Tumor cell ability to switch from epithelial to a more aggressive mesenchymal phenotype, attributed with AXLhigh molecular profile in melanoma, has been recently linked to such event, limiting treatment efficacy. In the current study, we investigated the therapeutic potential of the AXL inhibitor (AXLi) BGB324 alone or in combination with the clinically relevant BRAF inhibitor (BRAFi) vemurafenib. Firstly, AXL was shown to be expressed in majority of melanoma lymph node metastases. When treated ex vivo, the largest reduction in cell viability was observed when the two drugs were combined. In addition, a therapeutic benefit of adding AXLi to the BRAF-targeted therapy was observed in pre-clinical AXLhigh melanoma models in vitro and in vivo. When searching for mechanistic insights, AXLi was found to potentiate BRAFi-induced apoptosis, stimulate ferroptosis and inhibit autophagy. Altogether, our findings propose AXLi as a promising treatment in combination with standard therapy to improve therapeutic outcome in metastatic melanoma.

Abstract Receptor tyrosine kinase AXL is found upregulated in various types of cancer, including melanoma, and correlates with an aggressive cancer phenotype, inducing cell proliferation and epithelial-to-mesenchymal transition. In addition, AXL has recently been linked to chemotherapy resistance, and inhibition of AXL is found to increase DNA damage and reduce expression of DNA repair proteins. In light of this, we aimed to investigate whether targeting AXL together with DNA damage response proteins would be therapeutically beneficial. Using melanoma cell lines, we observed that combined reduction of AXL and CHK1/CHK2 signaling decreased proliferation, deregulated cell-cycle progression, increased apoptosis, and reduced expression of DNA damage response proteins. Enhanced therapeutic effect of combined treatment, as compared with mono-treatment, was further observed in a patient-derived xenograft model and, of particular interest, when applying a three-dimensional ex vivo spheroid drug sensitivity assay on tumor cells harvested directly from 27 patients with melanoma lymph node metastases. Together, these results indicate that targeting AXL together with the DNA damage response pathway could be a promising treatment strategy in melanoma, and that further investigations in patient groups lacking treatment alternatives should be pursued.

The objective of this study was to analyze the expression and clinical role of mitosis regulators α-thalassemia/mental retardation syndrome X-linked (ATRX) and death-domain-associated protein (DAXX) in metastatic high-grade serous carcinoma (HGSC). ATRX and DAXX protein expression by immunohistochemistry was analyzed in 400 HGSC effusions. DAXX expression was additionally studied in 15 cancer cell lines, including 4 ovarian carcinoma lines, and in 81 of the 400 HGSC effusions using Western blotting. ATRX and DAXX were expressed in HGSC cells in 386/400 (96%) and 348/400 (87%) effusions, respectively. Western blotting showed DAXX expression in all 15 cell lines and in 70/81 (86%) HGSC effusions. DAXX expression by immunohistochemistry was higher in pleural compared to peritoneal effusions (p = 0.006) and in post-chemotherapy compared to pre-chemotherapy effusions (p = 0.004), and its expression was significantly associated with poor overall survival in univariate of the entire cohort (p = 0.014), as well as analysis limited to chemo-naïve effusions tapped at diagnosis (p = 0.038). The former association retained its prognostic role in Cox multivariate survival analysis (p = 0.011). ATRX expression was unrelated to clinicopathologic parameters or survival. In conclusion, DAXX is associated with disease progression and could be a prognostic marker in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

Objective: The aim of this study was to analyze the expression and clinical role of the mitosis regulators athalassemia/mental retardation syndrome X-linked (ATRX) and death-domain-associated protein (DAXX) in metastatic high-grade serous carcinoma (HGSC).

T cell receptor (TCR)-engineered T cell therapy is a promising cancer treatment approach. Human telomerase reverse transcriptase (hTERT) is overexpressed in the majority of tumors and a potential target for adoptive cell therapy. We isolated a novel hTERT-specific TCR sequence, named Radium-4, from a clinically responding pancreatic cancer patient vaccinated with a long hTERT peptide. Radium-4 TCR-redirected primary CD4(+) and CD8(+) T cells demonstrated in vitro efficacy, producing inflammatory cytokines and killing hTERT(+) melanoma cells in both 2D and 3D settings, as well as malignant, patient-derived ascites cells. Importantly, T cells expressing Radium-4 TCR displayed no toxicity against bone marrow stem cells or mature hematopoietic cells. Notably, Radium-4 TCR+ T cells also significantly reduced tumor growth and improved survival in a xenograft mouse model. Since hTERT is a universal cancer antigen, and the very frequently expressed HLA class II molecules presenting the hTERT peptide to this TCR provide a very high (>75%) population coverage, this TCR represents an attractive candidate for immunotherapy of solid tumors.

Receptor tyrosine kinase AXL is a one-pass transmembrane protein upregulated in cancers and associated with lower survival and therapy resistance. AXL can be cleaved by the A Disintegrin and Metalloproteinases (ADAM)10 and ADAM17, yielding a soluble version of the protein. Elevated soluble AXL (sAXL) has been reported to be associated with disease progression in hepatocellular carcinoma, renal cancer, neurofibromatosis type 1 and inflammatory diseases. In the present work, we analyzed sAXL levels in blood from melanoma patients and showed that sAXL increases with disease progression. Additionally, increased sAXL levels were found correlated with shorter two-year survival in stage IV patients treated with ipilimumab. Furthermore, we showed that sAXL levels were related to the percentage of cells expressing AXL in resected melanoma lymph node metastases. This finding was verified in vitro, where sAXL levels in the cell media corresponded to AXL expression in the cells. AXL inhibition using the small-molecular inhibitor BGB324 reduced sAXL levels, while the cellular expression was elevated through increased protein stability. Our findings signify that quantification of sAXL blood levels is a simple and easily assessable method to determine cellular AXL levels and should be further evaluated for its use as a biomarker of disease progression and treatment response.

MX2 protein is a dynamin-like GTPase2 that has recently been identified as an interferon-induced restriction factor of HIV-1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome-wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context-dependent.

Although clinical management of melanoma has changed considerably in recent years, intrinsic treatment resistance remains a severe problem and strategies to design personal treatment regimens are highly warranted. We have applied a three-dimensional (3D) ex vivo drug efficacy assay, exposing disaggregated cells from 38 freshly harvested melanoma lymph node metastases and 21 patient derived xenografts (PDXs) to clinical relevant drugs for 7 days, and examined its potential to evaluate therapy response. A strong association between Vemurafenib response and BRAF mutation status was achieved (P < .0001), while enhanced viability was seen in some NRAS mutated tumors. BRAF and NRAS mutated tumors responded comparably to the MEK inhibitor Cobimetinib. Based on the ex vivo results, two tumors diagnosed as BRAF wild-type by routine pathology examinations had to be re-evaluated; one was subsequently found to have a complex V600E mutation, the other a double BRAF mutation (V600E/K601 N). No BRAF inhibitor resistance mechanisms were identified, but PIK3CA and NF1 mutations were identified in two highly responsive tumors. Concordance between ex vivo drug responses using tissue from PDXs and corresponding patient tumors demonstrate that PDX models represent an indefinite source of tumor material that may allow ex vivo evaluation of numerous drugs and combinations, as well as studies of underlying molecular mechanisms. In conclusion, we have established a rapid and low cost ex vivo drug efficacy assay applicable on tumor tissue from patient biopsies. The 3D/spheroid format, limiting the influence from normal adjacent cells and allowing assessment of drug sensitivity to numerous drugs in one week, confirms its potential as a supplement to guide clinical decision, in particular in identifying non-responding patients.

Objective. To analyze the expression and clinical role of CHK1 and CHK2 in metastatic high-grade serous carcinoma (HGSC). Methods. HGSC effusions (n = 335; 280 peritoneal, 55 pleural) were analyzed for protein expression of total CHK1 and its phosphorylated forms p-ser317 and p-ser296, as well as total CHK2 and its phosphorylated form pthr68 using immunohistochemistry. Expression was analyzed for association with clinicopathologic parameters, including chemotherapy response, and survival. Results. Carcinoma cells stained positive, predominantly at the nuclei, in the majority of cases (range 83-100% for the five antibodies), while expression in reactive mesothelial cells and tumor-associated macrophages was more variable. Total CHK1 (p = 0.037), p-CHK1ser317 (p = 0.001), p-CHKlser296 (p = 0.002) and pCHK2thr68 (p < 0.001) expression was significantly higher in post-chemotherapy disease recurrence compared to pre-chemotherapy effusions obtained at diagnosis. CHK1, p-CHKlser296, p-CHK2thr68 and p-CHK1ser317 nuclear expression was positively related to expression of the checkpoint regulator WEE1, previously studied in this cohort (p = 0.003, p = 0.013, p = 0.001 and p = 0.01, respectively). Higher total CHK1 (p = 0.007), p-CHK1ser317 (p = 0.004), CHK2 (p = 0.01) and p-CHK2thr68 (p = 0.048) expression was significantly related to shorter overall survival in univariate analysis, and CHK1ser317 was an independent prognostic marker in multivariate analysis (p = 0.025). Higher p-CHK1ser317 (p = 0.03) and CHK2 (p = 0.034) expression was additionally associated with poor progression-free survival. Conclusions. CHK1 and CHK2 and their activated forms are frequently expressed in HGSC effusions, with higher expression following exposure to chemotherapy, and their expression is related to survival. (C) 2018 Elsevier Inc. All rights reserved.

Objective. The objective of this study was to analyze the expression level and clinical role of soluble AXL (sAXL) in cancers affecting the serosal surfaces, with focus on ovarian carcinoma. Methods. sAXL protein expression by ELISA was analyzed in 572 effusion supernatants, including 424 peritoneal, 147 pleural and 1 pericardial specimens. Results. sAXL was overexpressed in peritoneal effusions compared to pleural and pericardial specimens (p < 0.001). sAXL levels were additionally significantly higher in effusions from patients with ovarian carcinoma, malignant mesothelioma and breast carcinoma compared to specimens from patients with other cancers (predominantly carcinomas of lung, gastrointestinal or uterine corpus/cervix origin) or benign reactive effusions (p < 0.001). sAXL was further overexpressed in high-grade serous carcinoma (HGSC; n = 373) compared to low-grade serous carcinoma (LGSC; n = 32; p = 0.036). In HGSC, sAXL levels were significantly lower in post-chemotherapy effusions compared to primary diagnosis pre-chemotherapy specimens (p = 0.002). sAXL levels in HGSC were unrelated to chemoresponse at diagnosis, progression-free survival or overall survival. Levels were similarly unrelated to survival in LGSC and breast carcinoma. Conclusions. sAXL is widely expressed in malignant effusions, particularly in ovarian and breast carcinoma and in malignant mesothelioma. sAXL is overexpressed in HGSC compared to LGSC and its levels are lower following exposure to chemotherapy. However, sAXL levels are not informative of chemoresponse or survival. (C) 2018 Elsevier Inc. All rights reserved.