A randomized controlled phase II clinical trial on mRNA electroporated autologous monocyte-derived dendritic cells (TriMixDC-MEL) as adjuvant treatment for stage III/IV melanoma patients who are disease-free following the resection of macrometastases.

BACKGROUND: Autologous monocyte-derived mRNA co-electroporated dendritic cells with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively activated TLR4 (caTLR4) (referred to as TriMixDC-MEL) have anti-tumor activity in advanced melanoma patients. We investigated the safety and activity of adjuvant TriMixDC-MEL in stage III/IV melanoma patients. MATERIALS AND METHODS: Forty-one patients were randomly assigned to treatment with TriMixDC-MEL (n = 21) and standard follow-up (n = 20). "Cross-over" was allowed at the time of non-salvageable recurrence. The primary endpoint was the percentage of patients alive and disease-free at 1-year. For a subset of patients, (formalin-fixed paraffin-embedded), tumor tissue samples were available for mRNA expression profiling and PD-L1 immunohistochemical staining. RESULTS: Baseline characteristics were well balanced. One-year after randomization, 71% of patients in the study arm were alive and free of disease compared to 35% in the control arm. After a median follow-up of 53 months (range 3-67), 23 patients experienced a non-salvageable melanoma recurrence (TriMixDC-Mel arm n = 9 and control arm n = 14).The median time to non-salvageable recurrence was superior in the TriMixDC-MEL arm (median 8 months (range 1-6) vs. not reached; log-rank p 0.044). TriMixDC-MEL-related adverse events (AE) consisted of transient local skin reactions, flu-like symptoms and post-infusion chills. No grade ≥ 3 AE's occurred. The mRNA expression profiling revealed four genes (STAT2, TPSAB1, CD9 and CSF2) as potential predictive biomarkers. CONCLUSION: TriMixDC-MEL id/iv as adjuvant therapy is tolerable and may improve the 1-year disease-free survival rate. Combination of optimized autologous monocyte-derived DC-formulations warrants further investigation in combination with currently approved adjuvant therapy options.

Validated programmed cell death ligand 1 immunohistochemistry assays (E1L3N and SP142) reveal similar immune cell staining patterns in melanoma when using the same sensitive detection system.

AIMS: Tumour cell and/or immune cell programmed cell death ligand 1 (PD-L1) expression is considered as a potential biomarker for anti-PD1 and anti-PD-L1 immunotherapy. Currently, different PD-L1 assays are used. This study aims to compare the staining patterns of two PD-L1 antibody clones in melanoma metastases and correlate them with PD-L1 mRNA expression. METHODS AND RESULTS: The immunohistochemistry assays were optimized and validated independently on a Ventana Benchmark Ultra (Ventana Medical Systems Inc., Tucson, AZ, USA) (E1L3N) and XT (SP142), using the same detection system. In total, 46 melanoma metastases were stained with both validated immunohistochemistry assays. Stained slides were digitized for qualitative and semi-quantitative evaluation; done by pathologist and semi-automated software analysis. A subset of 21 melanoma metastases was selected for quantification of the PD-L1 mRNA expression. Accuracy and precision criteria were met for both assays. PD-L1 protein and mRNA expression showed remarkably good Spearman's coefficients of 0.90 (E1L3N) and 0.87 (SP142). Despite the remarkable correlation between both PD-L1 assays in expression patterns and quantification values (ρ > 0.90), E1L3N showed significantly more tumour cell staining than SP142. CONCLUSIONS: E1L3N and SP142 IHC assays were optimized and validated successfully and independently for sensitive and accurate PD-L1 detection. Concordance was best for immune cell scoring, while E1L3N tended to detect more tumour cells. Determination of the clinically relevant cut-off values for immune cell versus tumour cell detection requires further research.

Molecular and epigenetic features of melanomas and tumor immune microenvironment linked to durable remission to ipilimumab-based immunotherapy in metastatic patients.

BACKGROUND: Ipilimumab (Ipi) improves the survival of advanced melanoma patients with an incremental long-term benefit in 10-15 % of patients. A tumor signature that correlates with this survival benefit could help optimizing individualized treatment strategies. METHODS: Freshly frozen melanoma metastases were collected from patients treated with either Ipi alone (n: 7) or Ipi combined with a dendritic cell vaccine (TriMixDC-MEL) (n: 11). Samples were profiled by immunohistochemistry (IHC), whole transcriptome (RNA-seq) and methyl-DNA sequencing (MBD-seq). RESULTS: Patients were divided in two groups according to clinical evolution: durable benefit (DB; 5 patients) and no clinical benefit (NB; 13 patients). 20 metastases were profiled by IHC and 12 were profiled by RNA- and MBD-seq. 325 genes were identified as differentially expressed between DB and NB. Many of these genes reflected a humoral and cellular immune response. MBD-seq revealed differences between DB and NB patients in the methylation of genes linked to nervous system development and neuron differentiation. DB tumors were more infiltrated by CD8(+) and PD-L1(+) cells than NB tumors. B cells (CD20(+)) and macrophages (CD163(+)) co-localized with T cells. Focal loss of HLA class I and TAP-1 expression was observed in several NB samples. CONCLUSION: Combined analyses of melanoma metastases with IHC, gene expression and methylation profiling can potentially identify durable responders to Ipi-based immunotherapy.

Identifying MAGE-A4-positive tumors for TCR T cell therapies in HLA-A∗02-eligible patients.

T cell receptor (TCR) T cell therapies target tumor antigens in a human leukocyte antigen (HLA)-restricted manner. Biomarker-defined therapies require validation of assays suitable for determination of patient eligibility. For clinical trials evaluating TCR T cell therapies targeting melanoma-associated antigen A4 (MAGE-A4), screening in studies NCT02636855 and NCT04044768 assesses patient eligibility based on: (1) high-resolution HLA typing and (2) tumor MAGE-A4 testing via an immunohistochemical assay in HLA-eligible patients. The HLA/MAGE-A4 assays validation, biomarker data, and their relationship to covariates (demographics, cancer type, histopathology, tissue location) are reported here. HLA-A∗02 eligibility was 44.8% (2,959/6,606) in patients from 43 sites across North America and Europe. While HLA-A∗02:01 was the most frequent HLA-A∗02 allele, others (A∗02:02, A∗02:03, A∗02:06) considerably increased HLA eligibility in Hispanic, Black, and Asian populations. Overall, MAGE-A4 prevalence based on clinical trial enrollment was 26% (447/1,750) across 10 solid tumor types, and was highest in synovial sarcoma (70%) and lowest in gastric cancer (9%). The covariates were generally not associated with MAGE-A4 expression, except for patient age in ovarian cancer and histology in non-small cell lung cancer. This report shows the eligibility rate from biomarker screening for TCR T cell therapies and provides epidemiological data for future clinical development of MAGE-A4-targeted therapies.

A Comprehensive Analysis of Baseline Clinical Characteristics and Biomarkers Associated with Outcome in Advanced Melanoma Patients Treated with Pembrolizumab.

BACKGROUND: Pembrolizumab improves the survival of patients with advanced melanoma. A comprehensive analysis of baseline variables that predict the benefit of pembrolizumab monotherapy has not been conducted. METHODS: Survival data of patients with advanced melanoma who were treated with pembrolizumab in a single university hospital were collected. A multivariate Cox regression analysis was performed to correlate baseline clinical, laboratory, and radiologic characteristics and NanoString IO360 gene expression profiling (GEP) with survival. RESULTS: 183 patients were included (stage IV 85.2%, WHO performance status ≥1 31.1%; pembrolizumab first-line 25.7%), of whom 112 underwent baseline 18F-FDG-PET/CT imaging, 58 had circulating tumor DNA (ctDNA) assessments, and GEP was available in 27 patients. Active brain metastases, a higher number of metastatic sites, lower albumin and absolute lymphocyte count (ALC), higher C-reactive protein (CRP) and neutrophil-to-lymphocyte ratio, higher total metabolic tumor volume (TMTV), and higher ctDNA levels were associated with worse survival. Elevated lactate dehydrogenase (LDH) ≥ 2ULN (upper limit of normal), CRP ≥ 10ULN, or ALC < 750/mm3 delineate a subpopulation where treatment with pembrolizumab is futile. A TMTV ≥ 80 mL encompassed 17/21 patients with LDH ≥ 2ULN, CRP ≥ 10ULN, or ALC < 750/mm3. No significant associations were observed between baseline GEP scores and survival. CONCLUSION: Multiple baseline variables correlate with survival on pembrolizumab. TMTV is a more comprehensive baseline biomarker than CRP, LDH, or ALC in predicting the futility of pembrolizumab.

Phase I clinical trial of decitabine (5-aza-2'-deoxycytidine) administered by hepatic arterial infusion in patients with unresectable liver-predominant metastases.

DNA demethylating agents may increase the immunogenicity of malignant tumours and increase the efficacy of subsequent treatment with immune check point inhibitors. We investigated the safety of administrating the demethylating agent decitabine by hepatic arterial infusionin patients with unresectable liver meta stases from solid tumours in a dose escalation phase I clinical trial. A total of nine eligible patients were enrolled and initiated study treatment at three different dose levels (two patients at 10, four at 15 and six at a dose level of 20mg decitabine/m2/day) (per protocol there was no intent to escalate the dose above the median tolerated intravenous dose level). Decitabine was administered as a 1-hour hepatic arterial infusion on five consecutive days every 4 weeks. Intrapatient dose escalation was applied in five patients. Grades 1 and 2 haematological toxicity was the most frequent treatment-related adverse event. None of the patients experienced treatment-limiting adverse events. Expression analysis of 30 cancer test is antigens (CTA) in pretreatment and post-treatment biopsies from patients indicated an increased expression of 21 CTAs after treatment. There were no objective tumour responses on study treatment or during post study exposure to immune checkpoint therapy in four patients with uveal melanoma liver metastases. We conclude that the investigate d hepatic arterial administration regimen for decitabine can be safely applied, and a dose level of 20 mg/m2/day on five consecutive days every 4 weeks can be considered for further investigation in combinatorial immunotherapy regimens. TRIAL REGISTRATION NUMBER: NCT02316028.

Optimal Evaluation of Programmed Death Ligand-1 on Tumor Cells Versus Immune Cells Requires Different Detection Methods.

CONTEXT: - The benefit of programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) as a method to select patients who may benefit from programmed death receptor-1 (PD-1)/PD-L1 immunotherapies remains uncertain in many tumor indications. OBJECTIVES: - To compare the commercially available, approved PD-L1 IHC assays (22C3, 28-8, SP142, SP263), specifically identifying the changes in staining output created by altering the detection method. DESIGN: - This pilot study investigates the respective PD-L1 kit assay staining patterns and related scoring of tumor cells and immune cells on lung carcinoma and melanoma. Furthermore, the influence of the detection method (platform and related reagents) on PD-L1 antibody performance is studied. RESULTS: - The SP142 kit reveals more immune cell staining but less tumor cell staining than the other PD-L1 kits. Alternatively, the 22C3 and 28-8 kits show good tumor cell sensitivity, but less pronounced immune cell staining, even in tonsil. Tumor cell staining by the SP263 kit is comparable to that of 22C3 and 28-8 kits, while immune cell staining is better. Strikingly, the selection of the detection method has a major impact on the sensitivity of the assay for PD-L1 detection per cell type. Switching the detection method of the kits could largely circumvent the observed staining differences. CONCLUSIONS: - The diverse sensitivities caused by the choice of the detection method should be taken into consideration when selecting PD-L1 kits or developing PD-L1 IHC laboratory-developed tests. When using alternative kits or laboratory-developed tests, it is strongly recommended to reestablish their clinical utility per therapeutic agent or compare them with the original kit.