Validation of an LC-MS/MS method for the determination of sotorasib, a KRASG12C inhibitor, in human plasma.

Background: Sotorasib (AMG 510) is a first-in-class KRASG12C inhibitor that received accelerated US FDA approval in 2021 for the treatment of patients with KRASG12C-mutated locally advanced or metastatic non-small-cell lung cancer. Method: An LC-MS/MS method was developed and validated for the determination of sotorasib in human plasma to support clinical development studies. Samples were prepared using protein precipitation and analyzed by LC-MS/MS using gradient elution with a calibration standard curve range of 10.0-10,000 ng/ml. Stable isotope labeled [13C, D3]-sotorasib was used as an internal standard. Results & conclusion: The method fully met FDA guidelines for all validation parameters, including precision, accuracy, selectivity, matrix effect, recovery and stability and has been extensively used to support multiple clinical studies.

Bioanalytical Challenges due to Prior Checkpoint Inhibitor Exposure: Interference and Mitigation in Drug Concentration and Immunogenicity Assays.

Monoclonal antibodies (mAbs) are a leading class of biotherapeutics. In oncology, patients often fail on early lines of biologic therapy to a specific target. Some patients may then enroll in a new clinical trial with a mAb specific for the same target. Therefore, immunoassays designed to quantify the current mAb therapy or assess immunogenicity to the drug may be susceptible to cross-reactivity or interference with residual prior biologics. The impact of two approved anti-PD-1 mAbs, pembrolizumab and nivolumab, was tested in several immunoassays for cemiplimab, another approved anti-PD-1 mAb. The methods included a target-capture drug concentration assay, a bridging anti-drug antibody (ADA) assay and a competitive ligand-binding neutralizing antibody (NAb) assay. We also tested bioanalytical strategies to mitigate cross-reactivity or interference in these assays from other anti-PD-1 biologics. Both pembrolizumab and nivolumab cross-reacted in the cemiplimab drug concentration assay. This was mitigated by addition of antibodies specific to pembrolizumab or nivolumab. ADA specific for pembrolizumab and nivolumab did not interfere in the cemiplimab ADA assay. However, pembrolizumab and nivolumab generated a false-positive response in a target-capture NAb assay. Our results demonstrate that similar exogenous pre-existing anti-PD-1 mAbs (biotherapeutics) such as pembrolizumab and nivolumab are detected and accurately quantified in the cemiplimab drug concentration assay. However, once steady state is achieved for the new therapy, prior biologics would likely not be detected. Cross-reactivity and interference in immunoassays from previous treatment with class-specific biotherapeutic(s) pose significant bioanalytical challenges, especially in immuno-oncology.

Neutrophils in the era of immune checkpoint blockade.

The immune checkpoint blockade-based immunotherapies are revolutionizing cancer management. Tumor-associated neutrophils (TANs) were recently highlighted to have a pivotal role in modulating the tumor microenvironment and the antitumor immune response. However, these cells were largely ignored during the development of therapies based on programmed cell death receptor or ligand-1 and cytotoxic T lymphocyte antigen-4 immune checkpoint inhibitors (ICIs). Latest evidences of neutrophil functional diversity in tumor raised many questions and suggest that targeting these cells can offer new treatment opportunities in the context of ICI development. Here, we summarized key information on TAN origin, function, and plasticity that should be considered when developing ICIs and provide a detailed review of the ongoing clinical trials that combine ICIs and a second compound that might affect or be affected by TANs. This review article synthetizes important notions from the literature demonstrating that: (1) Cancer development associates with a profound alteration of neutrophil biogenesis and function that can predict and interfere with the response to ICIs, (2) Neutrophil infiltration in tumor is associated with key features of resistance to ICIs, and (3) TANs play an important role in resistance to antiangiogenic drugs reducing their clinical benefit when used in combination with ICIs. Finally, exploring the clinical/translational aspects of neutrophil impact on the response to ICIs offers the opportunity to propose new translational research avenues to better understand TAN biology and treat patients.

Overall survival in metastatic melanoma correlates with pembrolizumab exposure and T cell exhaustion markers.

Trial data support an absence of an exposure-survival relationship for pembrolizumab. As these relationships remain unexamined in a real-world setting, we determined them in metastatic melanoma prospectively in an observational study. Translational objectives included identifying biomarkers of progressive disease (PD). Checkpoint blockade naïve patients receiving 2 mg/kg Q3W pembrolizumab had pharmacokinetic and clinical outcome data collected. Trough, a valid surrogate for drug exposure, was assessed using ELISA. T-cell exhaustion and chemokine markers were determined using flow cytometry. Geometric means of exposures and biomarkers were tested against objective response groups using one-way ANOVA. The cohort was split by the median into high versus low pembrolizumab exposure groups. Kaplan-Meier progression-free survival (PFS) and overall survival (OS) curves were estimated for high versus low exposure, compared using the log rank test. The high pembrolizumab exposure group (n = 14) experienced substantially longer median OS (not reached vs. 48 months, p = .014), than the low exposure group (n = 14). A similar positive exposure PFS relationship was found (median not reached vs. 48 months, p = .045). The frequency of TIM-3 expression on CD4+ T cells was significantly higher in PD (mean 27.8%) than complete response (CR) (13.38%, p = .01) and partial response (12.4%, p = .05). There was a near doubling of CXCR6 and TIM-3 co-expression on CD4+ T cells in PD (mean 23.3%) versus CR (mean 11.4, p = .003) and partial response (9.8%, p = .0001). We describe positive exposure-PFS and exposure-OS relationships for pembrolizumab in metastatic melanoma. TIM-3, alongside co-expression of CXCR6 and TIM-3 on circulating CD4+ T cells are potential bio markers of treatment failure.

[Blood-based Biomarkers in the Immune Checkpoint Inhibitor Treatment in 
Non-small Cell Lung Cancer].

Immune checkpoint inhibitors (ICI) have transformed the treatment landscape of advanced non-small cell lung cancer (NSCLC). Biomarkers are essential for guiding precision immunotherapy. Tissue-based programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB) are currently widely used biomarkers for selecting patients for immunotherapy. However, tissue specimens are often difficult to reach and couldn't overcome spatial and temporal heterogeneity. Blood biomarkers offer an alternative non-invasive solution that could provide a complete insight on patient's immune status and tumor as well, and show their potential in predicting the outcome as well as in monitoring response to immunotherapy. In this article, we summarize current knowledge on blood biomarkers in NSCLC patients treated with ICI, and we hope to provide more references for development of novel biomarkers.
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Association Between First-Line Immune Checkpoint Inhibition and Survival for Medicare-Insured Patients With Advanced Non-Small Cell Lung Cancer.

Importance: Immunotherapy is now a cornerstone of treatment for advanced non-small cell lung cancer (NSCLC), but its uptake and effectiveness among older patients outside clinical trials remain poorly understood. Objective: To understand treatment patterns and evaluate the overall survival associated with checkpoint inhibitor immunotherapy, cytotoxic chemotherapy, and combined chemoimmunotherapy for older patients who have advanced NSCLC and Medicare coverage. Design, Setting, and Participants: This retrospective cohort study included Medicare-insured patients in the US aged 66 to 89 years who initiated first palliative-intent systemic therapy for lung cancer between January 1, 2016, and December 31, 2018. Survival follow-up continued through March 31, 2020. A total of 19 529 patients who had advanced lung cancer and were insured by a Medicare fee-for-service plan were included in the analysis. Exposures: Regimens included pembrolizumab monotherapy (n = 3079), combined platinum-based drug (ie, cisplatin or carboplatin [hereinafter, platinum]) and pemetrexed disodium (n = 5159), combined platinum and a taxane (ie, paclitaxel, nab-paclitaxel, or docetaxel) (n = 9866), and combined platinum, pemetrexed, and pembrolizumab (n = 1425), as ascertained using Medicare claims from the Centers for Medicare & Medicaid Services. Main Outcomes and Measures: The primary outcome was overall survival, which was measured using the restricted mean survival time (RMST) with propensity score adjustment for clinical and sociodemographic characteristics. Median survival was also reported for comparison with outcomes from registrational trials. Results: A total of 19 529 patients (54% male, 46% female; median age, 73.8 [interquartile range, 69.9-78.4] years) were identified for analysis. The uptake of pembrolizumab-containing regimens in the Medicare population was rapid, increasing from 0.7% of first-line treatments in the second quarter of 2016 to 42.4% in the third quarter of 2018. Patients who were older (≥70 years, 2484 [81%]), were female (1577 [51%]), and/or had higher Risk Stratification Index scores (highest quintile, 922 [30%]) were more likely to receive single-agent pembrolizumab than chemotherapy. After propensity score adjustment, pembrolizumab was associated with survival similar to platinum/pemetrexed (RMST difference, -0.2 [95% CI, -0.5 to 0.2] months) or platinum/taxane (RMST difference, -0.7 [95% CI, -1.0 to -0.4] months). Patients receiving platinum/pemetrexed/pembrolizumab chemoimmunotherapy also had adjusted survival similar to those receiving platinum/pemetrexed chemotherapy (RMST difference, 0.5 [95% CI, 0.1-0.9] months). The unadjusted median survival was 11.4 (95% CI, 10.5-12.3) months among patients receiving single-agent pembrolizumab, approximately 15 months shorter than observed among pembrolizumab-treated participants in the KEYNOTE-024 trial. The unadjusted median survival was 12.9 (95% CI, 11.8-14.0) months among patients receiving platinum/pemetrexed/pembrolizumab chemoimmunotherapy, approximately 10 months shorter than observed among platinum/pemetrexed/pembrolizumab-treated participants in the KEYNOTE-189 trial. Conclusions and Relevance: Immunotherapy has been incorporated rapidly into treatment for patients with advanced NSCLC. However, survival estimates in the Medicare population are much shorter than those reported in registrational trials. These results provide contemporary estimates of survival for older patients with advanced NSCLC treated in routine practice, facilitating patient-centered decision-making.

Biomarkers of Immune Checkpoint Inhibitors in Non-Small Cell Lung Cancer: Beyond PD-L1.

Immunotherapy has markedly improved the survival rate of patients with non-small cell lung cancer (NSCLC) and has introduced a new era in lung cancer treatment. Although some patients achieve durable responses to checkpoint blockade, not all experience such benefits, and some suffer from significant immunotoxicities. Thus, it is crucial to identify potential biomarkers suitable for screening the population that may benefit from immunotherapy. Based on the current clinical trials, the aim of the present study was to review the biomarkers for immune checkpoint inhibition that may have the potential to predict the response to immunotherapy in patients with lung cancer. A non-systematic literature review was done. We searched for eligible randomized controlled trials (RCTs) from PubMed, Embase, and the Cochrane Central Register of Controlled Trials from January 2015 to January 2021. The keywords included biomarkers, immunotherapy, immune checkpoint inhibition, programmed death ligand 1 (PD-L1), and non-small cell lung cancer. Additional biomarkers beyond PD-L1 that have been shown to have predictive capacity include tumor mutational burden, microsatellite instability, lung immune prognostic index, gut microbiome, and certain alterations in genes (eg, STK11 deletion, LKB1 kinase mutation, MDM2/4 amplification) that confer immunoresistance. The biomarkers reviewed in this article could help us better select the appropriate immunotherapy treatment for patients with NSCLC.

Potential of circulating immune cells as biomarkers of nivolumab treatment efficacy for advanced hepatocellular carcinoma.

BACKGROUND: Remarkable progress has been made in immunotherapy, specifically antibodies for programmed death 1 (PD-1) or programmed death-ligand 1 (PD-L1), for treating advanced cancers. In this study, we explored whether circulating immune cells can be used as biomarkers of the efficacy of such therapy. METHODS: We enrolled patients who received nivolumab, an anti-PD-1 antibody, for advanced hepatocellular carcinoma (HCC) in clinical trials and who consented to the collection of their peripheral blood. Using flow cytometry, we analyzed lymphocyte subclasses and the PD-1 or PD-L1 positivity of immune cells. These results were compared between patients with disease control (complete response, partial response, or stable disease) and those with disease progression. RESULTS: This study included 16 patients. The objective response rate was 19%, and the disease control rate was 75%. The hemogram results and the percentage of total αß T cells or CD4 T cells did not significantly change after nivolumab treatment; moreover, they were not associated with treatment outcomes. The number of CD8 T cells significantly increased after 4 weeks (p = 0.016); however, this change was not associated with treatment outcomes. Patients with disease control exhibited peripheral B cells with significantly lower pretreatment PD-1 positivity than did patients with disease progression (p = 0.042). Patients with disease progression were more likely to exhibit monocytes with increased PD-L1 positivity after 28 (p = 0.020) or 42 (p = 0.008) days of treatment. CONCLUSION: The low pretreatment PD-1 positivity of peripheral B cells and the constant posttreatment PD-L1 positivity of monocytes were associated with disease control after nivolumab treatment for advanced HCC.

A Canadian cancer trials group phase IB study of durvalumab (anti-PD-L1) plus tremelimumab (anti-CTLA-4) given concurrently or sequentially in patients with advanced, incurable solid malignancies.

Background The IND.226 study was a phase Ib study to determine the recommended phase II dose of durvalumab + tremelimumab in combination with standard platinum-doublet chemotherapy. Sequential administration of multiple agents increases total chair time adding costs overall and inconvenience for patients. This cohort of the IND.226 study evaluated the safety and tolerability of durvalumab + tremelimumab given either sequentially (SEQ) or concurrently (CON). Methods Patients with advanced solid tumours were enrolled and randomised to either SEQ tremelimumab 75 mg IV over 1 h followed by durvalumab 1500 mg IV over 1 h q4wks on the same day, or CON administration over 1 h. The serum pharmacokinetic profile of SEQ versus CON of durvalumab and tremelimumab administration was also evaluated. Results 14 patients either received SEQ (n = 7pts) or CON (n = 7 pts). There were no infusion related reactions. Drug related adverse events (AEs) were mainly low grade and manageable, and comparable in frequency between SEQ/CON- fatigue (43%/57%), rash (43%/43%), pruritus (43%/29%) and nausea (14%/29%). One patient in each cohort discontinued treatment due to toxicity. The PK profiles of durvalumab and tremelimumab were similar between CON and SEQ, and to historical reference data. Conclusions Concurrent administration of durvalumab and tremelimumab over 1 h is safe with a comparable PK profile to sequential administration.