Entrectinib in ROS1-positive advanced non-small cell lung cancer: the phase 2/3 BFAST trial.

Although comprehensive biomarker testing is recommended for all patients with advanced/metastatic non-small cell lung cancer (NSCLC) before initiation of first-line treatment, tissue availability can limit testing. Genomic testing in liquid biopsies can be utilized to overcome the inherent limitations of tissue sampling and identify the most appropriate biomarker-informed treatment option for patients. The Blood First Assay Screening Trial is a global, open-label, multicohort trial that evaluates the efficacy and safety of multiple therapies in patients with advanced/metastatic NSCLC and targetable alterations identified by liquid biopsy. We present data from Cohort D (ROS1-positive). Patients ≥18 years of age with stage IIIB/IV, ROS1-positive NSCLC detected by liquid biopsies received entrectinib 600 mg daily. At data cutoff (November 2021), 55 patients were enrolled and 54 had measurable disease. Cohort D met its primary endpoint: the confirmed objective response rate (ORR) by investigator was 81.5%, which was consistent with the ORR from the integrated analysis of entrectinib (investigator-assessed ORR, 73.4%; data cutoff May 2019, ≥12 months of follow-up). The safety profile of entrectinib was consistent with previous reports. These results demonstrate consistency with those from the integrated analysis of entrectinib in patients with ROS1-positive NSCLC identified by tissue-based testing, and support the clinical value of liquid biopsies to inform clinical decision-making. The integration of liquid biopsies into clinical practice provides patients with a less invasive diagnostic method than tissue-based testing and has faster turnaround times that may expedite the reaching of clinical decisions in the advanced/metastatic NSCLC setting. ClinicalTrials.gov registration: NCT03178552 .

A targeted amplicon next-generation sequencing assay for tryptase genotyping to support personalized therapy in mast cell-related disorders.

Tryptase, the most abundant mast cell granule protein, is elevated in severe asthma patients independent of type 2 inflammation status. Higher active ß tryptase allele counts are associated with higher levels of peripheral tryptase and lower clinical benefit from anti-IgE therapies. Tryptase is a therapeutic target of interest in severe asthma and chronic spontaneous urticaria. Active and inactive allele counts may enable stratification to assess response to therapies in asthmatic patient subpopulations. Tryptase gene loci TPSAB1 and TPSB2 have high levels of sequence identity, which makes genotyping a challenging task. Here, we report a targeted next-generation sequencing (NGS) assay and downstream bioinformatics analysis for determining polymorphisms at tryptase TPSAB1 and TPSB2 loci. Machine learning modeling using multiple polymorphisms in the tryptase loci was used to improve the accuracy of genotyping calls. The assay was tested and qualified on DNA extracted from whole blood of healthy donors and asthma patients, achieving accuracy of 96%, 96% and 94% for estimation of inactive α and ßΙΙΙFS tryptase alleles and α duplication on TPSAB1, respectively. The reported NGS assay is a cost-effective method that is more efficient than Sanger sequencing and provides coverage to evaluate known as well as unreported tryptase polymorphisms.

Intratumoral CD103+ CD8+ T cells predict response to PD-L1 blockade.

BACKGROUND: CD8+ tissue-resident memory T (TRM) cells, marked by CD103 (ITGAE) expression, are thought to actively suppress cancer progression, leading to the hypothesis that their presence in tumors may predict response to immunotherapy. METHODS: Here, we test this by combining high-dimensional single-cell modalities with bulk tumor transcriptomics from 1868 patients enrolled in lung and bladder cancer clinical trials of atezolizumab (anti-programmed cell death ligand 1 (PD-L1)). RESULTS: ITGAE was identified as the most significantly upregulated gene in inflamed tumors. Tumor CD103+ CD8+ TRM cells exhibited a complex phenotype defined by the expression of checkpoint regulators, cytotoxic proteins, and increased clonal expansion. CONCLUSIONS: Our analyses indeed demonstrate that the presence of CD103+ CD8+ TRM cells, quantified by tracking intratumoral CD103 expression, can predict treatment outcome, suggesting that patients who respond to PD-1/PD-L1 blockade are those who exhibit an ongoing antitumor T-cell response.

Survey of CF mutations in the clinical laboratory.

BACKGROUND: Since it is impossible to sequence the complete CFTR gene routinely, clinical laboratories must rely on test systems that screen for a panel of the most frequent mutations causing disease in a high percentage of patients. Thus, in a cohort of 257 persons that were referred to our laboratory for analysis of CF gene mutations, reverse line probe assays for the most common CF mutations were performed. These techniques were evaluated as routine first-line analyses of the CFTR gene status. METHODS: DNA from whole blood specimens was extracted and subjected to PCR amplification of 9 exons and 6 introns of the CFTR gene. The resulting amplicons were hybridised to probes for CF mutations and polymorphisms, immobilised on membranes supplied by Roche Molecular Systems, Inc. and Innogenetics, Inc. Denaturing gradient gel electrophoresis and sequencing of suspicious fragments indicating mutations were done with CF exon and intron specific primers. RESULTS: Of the 257 persons tested over the last three years (referrals based on 1) clinical symptoms typical for/indicative of CF, 2) indication for in vitro fertilisation, and 3) gene status determination because of anticipated parenthood and partners or relatives affected by CF), the reverse line blots detected heterozygote or homozygote mutations in the CFTR gene in 68 persons (26%). Eighty-three percent of those affected were heterozygous (47 persons) or homozygous (10 persons) for the DeltaF508 allele. The only other CF-alleles that we found with these tests were the G542X allele (3 persons), the G551D allele (3 persons), the 3849+10kb C-T allele (2 persons) the R117H allele (2 persons) and the 621+1G-T allele (1 person).Of the fifteen IVS8-5T-polymorphisms detected in intron 8, seven (47%) were found in males referred to us from IVF clinics. These seven 5T-alleles were all coupled with a heterozygous DeltaF508 allele, they make up 35% of the males with fertility problems (20 men) referred to us. CONCLUSIONS: In summary, the frequency of CF chromosomes in the cohort examined with these tests was 26%, with the DeltaF508 allele affecting 83% of the CF chromosomes. It is a substantial improvement for routine CF diagnostics to have available a test system for 30 mutations plus the polypyrimidine length variants in intron 8. Our results show that this test system allows a routine first-line analyses of the CFTR gene status.