PD-L1 is the main ligand for the immune inhibitory receptor PD-1. This ligand is frequently expressed by melanoma cells. In this study, we investigated whether PD-L1 expression is controlled by ...melanoma driver mutations and modified by oncogenic signaling inhibition.
Expression of PD-L1 was investigated in a panel of 51 melanoma cell lines containing different oncogenic mutations, including cell lines with innate and acquired resistance to BRAF inhibitors (BRAFi). The effects of targeted therapy drugs on expression of PD-L1 by melanoma cells were investigated.
No association was found between the level of PD-L1 expression and mutations in BRAF, NRAS, PTEN, or amplification of AKT. Resistance to vemurafenib due to the activation of alternative signaling pathways was accompanied with the induction of PD-L1 expression, whereas the resistance due to the reactivation of the MAPK pathway had no effect on PD-L1 expression. In melanoma cell lines, the effects of BRAF, MEK, and PI3K inhibitors on expression of PD-L1 were variable from reduction to induction, particularly in the presence of INFγ. In PD-L1-exposed lymphocytes, vemurafenib paradoxically restored activity of the MAPK pathway and increased the secretion of cytokines.
In melanoma cell lines, including BRAFi-resistant cells, PD-L1 expression is variably regulated by oncogenic signaling pathways. PD-L1-exposed lymphocytes decrease MAPK signaling, which is corrected by exposure to vemurafenib, providing potential benefits of combining this drug with immunotherapies.
Clinical deterioration (ICU transfer and cardiac arrest) occurs during approximately 5-10% of hospital admissions. Existing prediction models have a high false positive rate, leading to multiple ...false alarms and alarm fatigue. We used routine vital signs and laboratory values obtained from the electronic medical record (EMR) along with a machine learning algorithm called a neural network to develop a prediction model that would increase the predictive accuracy and decrease false alarm rates.
Retrospective cohort study.
The hematologic malignancy unit in an academic medical center in the United States.
Adult patients admitted to the hematologic malignancy unit from 2009 to 2010.
None.
Vital signs and laboratory values were obtained from the electronic medical record system and then used as predictors (features). A neural network was used to build a model to predict clinical deterioration events (ICU transfer and cardiac arrest). The performance of the neural network model was compared to the VitalPac Early Warning Score (ViEWS). Five hundred sixty five consecutive total admissions were available with 43 admissions resulting in clinical deterioration. Using simulation, the neural network outperformed the ViEWS model with a positive predictive value of 82% compared to 24%, respectively.
We developed and tested a neural network-based prediction model for clinical deterioration in patients hospitalized in the hematologic malignancy unit. Our neural network model outperformed an existing model, substantially increasing the positive predictive value, allowing the clinician to be confident in the alarm raised. This system can be readily implemented in a real-time fashion in existing EMR systems.
Summary Background Avelumab, a human Ig-G1 monoclonal antibody targeting PD-L1 and approved in the USA for the treatment of metastatic Merkel cell carcinoma, has shown antitumour activity and an ...acceptable safety profile in patients with advanced solid tumours in a dose-escalation phase 1a trial. In this dose-expansion cohort of that trial, we assess avelumab treatment in a cohort of patients with advanced, platinum-treated non-small-cell lung cancer (NSCLC). Methods In this dose-expansion cohort of a multicentre, open-label, phase 1 study, patients with progressive or platinum-resistant metastatic or recurrent NSCLC were enrolled at 58 cancer treatment centres and academic hospitals in the USA. Eligible patients had confirmed stage IIIB or IV NSCLC with squamous or non-squamous histology, measurable disease by Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST v1.1), tumour biopsy or archival sample for biomarker assessment, and Eastern Cooperative Oncology Group performance status 0 or 1, among other criteria. Patient selection was not based on PD-L1 expression or expression of other biomarkers, including EGFR or KRAS mutation or ALK translocation status. Patients received infusional avelumab monotherapy 10 mg/kg every 2 weeks until disease progression or toxicity. The primary objective was to assess safety and tolerability. This trial is registered with ClinicalTrials.gov , number NCT01772004 ; enrolment in this cohort is closed and the trial is ongoing. Findings Between Sept 10, 2013, and June 24, 2014, 184 patients were enrolled and initiated treatment with avelumab. Median follow-up duration was 8·8 months (IQR 7·2–11·9). The most common treatment-related adverse events of any grade were fatigue (46 25% of 184 patients), infusion-related reaction (38 21%), and nausea (23 13%). Grade 3 or worse treatment-related adverse events occurred in 23 (13%) of 184 patients; the most common (occurring in more than two patients) were infusion-related reaction (four 2% patients) and increased lipase level (three 2%). 16 (9%) of 184 patients had a serious adverse event related to treatment with avelumab, with infusion-related reaction (in four 2% patients) and dyspnoea (in two 1%) occurring in more than one patient. Serious adverse events irrespective of cause occurred in 80 (44%) of 184 patients. Those occurring in more than five patients (≥3%) were dyspnoea (ten patients 5%), pneumonia (nine 5%), and chronic obstructive pulmonary disease (six 3%). Immune-related treatment-related events occurred in 22 patients (12%). Of 184 patients, 22 (12% 95% CI 8–18) achieved a confirmed objective response, including one complete response and 21 partial responses. 70 (38%) had stable disease. Overall, 92 (50%) of 184 patients achieved disease control (they had a confirmed response or stable disease as their best overall response). One patient was initially thought to have died from grade 5 radiation pneumonitis during the study; however, this adverse event was subsequently regraded to grade 3 and the death was attributed to disease progression. Interpretation Avelumab showed an acceptable safety profile and antitumour activity in patients with progressive or treatment-resistant NSCLC, providing a rationale for further studies of avelumab in this disease setting. Funding Merck KGaA and Pfizer.
Malignant transformation can result in melanoma cells that resemble different stages of their embryonic development. Our gene expression analysis of human melanoma cell lines and patient tumors ...revealed that melanoma follows a two-dimensional differentiation trajectory that can be subclassified into four progressive subtypes. This differentiation model is associated with subtype-specific sensitivity to iron-dependent oxidative stress and cell death known as ferroptosis. Receptor tyrosine kinase-mediated resistance to mitogen-activated protein kinase targeted therapies and activation of the inflammatory signaling associated with immune therapy involves transitions along this differentiation trajectory, which lead to increased sensitivity to ferroptosis. Therefore, ferroptosis-inducing drugs present an orthogonal therapeutic approach to target the differentiation plasticity of melanoma cells to increase the efficacy of targeted and immune therapies.
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•Melanoma differentiation involves four progressive stepwise states•Differentiation is two-dimensional with an intermediate neural crest signature•Melanoma dedifferentiation increases sensitivity to ferroptosis•Co-targeting ferroptosis can block the dedifferentiation resistance escape route
Tsoi et al. show that melanoma can be categorized into four subtypes following a differentiation trajectory with subtype-specific sensitivity to ferroptosis induction, which presents a therapeutic approach to target the differentiation plasticity to increase the efficacy of targeted and immune therapies.
Pembrolizumab (PD-1 inhibitor) and cetuximab (EGFR inhibitor) are active as single agents and in combination with cytotoxic chemotherapy for recurrent or metastatic head and neck squamous cell ...carcinoma (HNSCC). Given each drug's single agent activity and unique mechanism of action, we aimed to evaluate the anti-tumour activity of PD-1 blockade with EGFR inhibition in recurrent or metastatic HNSCC.
This study is an open-label, non-randomised, multi-arm, phase 2 trial done at four academic centres in the USA. Participants were required to have platinum-resistant or platinum-ineligible, recurrent or metastatic HNSCC, be at least 18 years old, have an Eastern Cooperative Oncology Group performance status 0–1, have measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, and to have received no previous immunotherapy or EGFR inhibition. All participants received pembrolizumab 200 mg intravenously every 3 weeks, combined with an initial loading dose of cetuximab 400 mg/m2 intravenously followed by 250 mg/m2 intravenously weekly (21 day cycle). The primary endpoint was overall response rate defined as the proportion of participants with a partial or complete responses (per RECIST version 1.1) by 6 months in the intention-to-treat population. The safety population included all participants who received at least one dose of pembrolizumab. Herein, the final analysis of cohort 1 (no previous PD-1, PD-L1, or EGFR inhibition for recurrent or metastatic HNSCC) is reported. Three additional cohorts (two for participants with a previous response to immunotherapy followed by relapse or progression, with or without previous cetuximab exposure, and one for cutaneous HNSCC) will be reported separately once fully accrued. This study is registered with ClinicalTrials.gov, NCT03082534, and remains open as the three additional cohorts are actively accruing participants.
Between March 22, 2017, and July 16, 2019, 33 participants were enrolled to cohort 1. All 33 participants received at least one dose of pembrolizumab. Median follow-up duration was 7·3 months (IQR 3·9–10·9). By 6 months, the overall response rate was 45% (95% CI 28–62), with 15 of 33 participants achieving a partial response. The most common grade 3–4 treatment-related adverse event was oral mucositis (three 9% of 33 participants), and serious treatment-related adverse events occurred in five (15%) participants. No treatment-related deaths occurred.
Pembrolizumab combined with cetuximab shows promising clinical activity for recurrent or metastatic HNSCC, and merits further investigation.
Merck Sharp & Dohme.
It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes ...and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy.
A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation.
A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells.
Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses.
Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA ...methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing.
In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16-20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×).
Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.
Background In melanoma, dysregulation of the MAPK pathway, usually via BRAF.sup.V600 or NRAS.sup.Q61 somatic mutations, leads to constitutive ERK signaling. While BRAF inhibitors are initially ...effective for BRAF-mutant melanoma, no FDA-approved targeted therapies exist for BRAF-inhibitor-resistant BRAF.sup.V600, NRAS mutant, or wild-type melanoma. Methods The 50% inhibitory concentration (IC50) of SCH772984, a novel inhibitor of ERK1/2, was determined in a panel of 50 melanoma cell lines. Effects on MAPK and AKT signaling by western blotting and cell cycle by flow cytometry were determined. Results Sensitivity fell into three groups: sensitive, 50% inhibitory concentration (IC.sub.50) < 1 muM; intermediately sensitive, IC.sub.50 1-2 muM; and resistant, >2 muM. Fifteen of 21 (71%) BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH772984. All three (100%) BRAF/NRAS double mutants, 11 of 14 (78%) NRAS mutants and 5 of 7 (71%) wild-type melanomas were sensitive. Among BRAF.sup.V600 mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH772984. SCH772984 caused G1 arrest and induced apoptosis. Conclusions Combining vemurafenib and SCH722984 in BRAF mutant melanoma was synergistic in a majority of cell lines and significantly delayed the onset of acquired resistance in long term in vitro assays. Therefore, SCH772984 may be clinically applicable as a treatment for non-BRAF mutant melanoma or in BRAF-mutant melanoma with innate or acquired resistance, alone or in combination with BRAF inhibitors. Keywords: ERK inhibitor, BRAF inhibitor, Melanoma, Targeted therapy, Acquired resistance
The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance. In this study we investigated whether co-targeting the MAPK and the PI3K-AKT ...pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro.
Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures.
More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 μM). The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line.
AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations.
Approximately 20% of melanomas contain a mutation in NRAS. However no direct inhibitor of NRAS is available. One of the main signaling pathways downstream of NRAS is the MAPK pathway. In this study ...we investigated the possibility of blocking oncogenic signaling of NRAS by inhibiting two signaling points in the MAPK pathway.
Fourteen NRAS mutated human melanoma cell lines were treated with a pan-RAF inhibitor (PRi, Amgen Compd A), a MEK inhibitor (MEKi, trametinib) or their combination and the effects on proliferation, cell cycle progression, apoptosis, transcription profile and signaling of the cells were investigated.
The majority of the cell lines showed a significant growth inhibition, with high levels of synergism of the PRi and MEKi combination. Sensitive cell lines showed induction of apoptosis by the combination treatment and there was a correlation between p-MEK levels and synergistic effect of the combination treatment. Proliferation of sensitive cell lines was blocked by the inhibition of the MAPK pathway, which also blocked expression of cyclin D1. However, in resistant cell lines, proliferation was blocked by combined inhibition of the MAPK pathway and cyclin D3, which is not regulated by the MAPK pathway. Resistant cell lines also showed higher levels of p-GSK3β and less perturbation of the apoptotic profile upon the treatment in comparison with the sensitive cell lines.
The combination of PRi + MEKi can be an effective regimen for blocking proliferation of NRAS mutant melanomas when there is higher activity of the MAPK pathway and dependence of proliferation and survival on this pathway.
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