PD-L1 blockade in combination with inhibition of MAPK oncogenic signaling in patients with advanced melanoma

Combining PD-L1 blockade with inhibition of oncogenic mitogen-activated protein kinase (MAPK) signaling may result in long-lasting responses in patients with advanced melanoma. This phase 1, open-label, dose-escalation and -expansion study (NCT02027961) investigated safety, tolerability and preliminary efficacy of durvalumab (anti–PD-L1) combined with dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) for patients with BRAF-mutated melanoma (cohort A, n = 26), or durvalumab and trametinib given concomitantly (cohort B, n = 20) or sequentially (cohort C, n = 22) for patients with BRAF-wild type melanoma. Adverse events and treatment discontinuation rates were more common than previously reported for these agents given as monotherapy. Objective responses were observed in 69.2% (cohort A), 20.0% (cohort B) and 31.8% (cohort C) of patients, with evidence of improved tumor immune infiltration and durable responses in a subset of patients with available biopsy samples. In conclusion, combined MAPK inhibition and anti–PD-L1 therapy may provide treatment options for patients with advanced melanoma.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability

Antoni Ribas
Oct 15, 2020 For the RNA sequencing (RNAseq), total RNA from each sample was prepared for sequencing using the Takara Bio SMART-Seq: SMART-Seq® v4 Ultra® Low Input RNA Kit. Between 0.8-1.3ng of RNA was used to prepare the RNA libraries. Eleven cycles of PCR were performed during cDNA amplification. Samples were then processed with the Nextera XT DNA sample preparation kits for Illumina. The cDNA was normalized to the modified recommended input amount of 100-150pg and ten cycles during Nextera library prep. The purified amplified libraries were then validated by Agilent High Sensitivity DNA chip on Agilent 2100 Bioanalyzer and quantitated via qPCR using KAPA Library Quantification Kit (KAPA Biosystems) according to manufacturer's instructions. The libraries were sequenced on Illumina HiSeq 2500 with the following run parameters: Paired-End/Dual-Indexed 2×75bp reads.
RNAseq data was aligned to the human reference genome (GRCh38) by Hisat2 (version 2.0.4). Gene expression was annotated using Ensembl (release 94) and summarized by HTSeq-counts (version 0.6.1). Gene expression values were normalized and compared across groups using the DESeq2 (version 1.28.1) R package (version 3.0.0). Gene expression was displayed as the z-score of the normalized gene expression using the ggplot2 (version 3.3.2) R package (version 3.0.0).
The clinical dataset analyzed here is available and may be obtained in accordance with AstraZeneca's data sharing policy, which is described at https:// astrazenecagrouptrials. A total of up to 69 patients were required for both the dose-escalation phase and the dose-expansion phase of the study. For the doseescalation phase, up to 24 evaluable patients were required, with 2 dose levels in Cohort A and 1 dose level each in Cohorts B and C. For the dose-expansion phase, a total of 42 subjects were needed in 3 expansion cohorts consisting of approximately 14 subjects in each of Cohorts A, B, and C. The goal was to have~20 subjects in each cohort treated at the MTD or 10 mg/kg dose level selected for each cohort. The sample size was primarily chosen to obtain preliminary assessment of antitumor activity (ORR).
No data were excluded from these analyses.
This article reports on a clinical trial and correlative studies in patient-derived biopsies. The clinical trial and the planning for sample analyses were conducted after prospective planning included in the clinical trial protocol.
Study participation began once written informed consent was obtained, and a subject identification (SID) number was assigned by a central system (Parexel). Once study eligibility (inclusion/exclusion) was assessed, the SID number was used to identify the subject during the screening process and throughout study participation. Patients were randomized to received either durvalumab in combination with dabrafenib and trametinib or with trametinib alone.
This was a Phase I, open-label study, therefore the study was not blinded. Each subject who met the eligibility criteria was assigned to a treatment arm. All immunoassays included in the study had been previously validated.