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Introduction: Molecular profiling of NSCLC is essential for optimising treatment decisions, but often incomplete. We assessed the efficacy of protocolised molecular profiling in the current standard-of-care (SoC) in a prospective observational study in the Netherlands and measured the effect of providing standardised diagnostic procedures. We also explored the potential of plasma-based molecular profiling in the primary diagnostic setting. Methods: This multi-centre prospective study was designed to explore the performance of current clinical practice during the run-in phase using local SoC tissue profiling procedures. The subsequent phase was designed to investigate the extent to which comprehensive molecular profiling (CMP) can be maximized by protocolising tumour profiling. Successful molecular profiling was defined as completion of at least EGFR and ALK testing. Additionally, PD-L1 tumour proportions scores were explored. Lastly, the additional value of centralised plasma-based testing for EGFR and KRAS mutations using droplet digital PCR was evaluated. Results: Total accrual was 878 patients, 22.0% had squamous cell carcinoma and 78.0% had non-squamous NSCLC. Stage I-III was seen in 54.0%, stage IV in 46.0%. Profiling of EGFR and ALK was performed in 69.9% of 136 patients included in the run-in phase, significantly more than real-world data estimates of 55% (p<0.001). Protocolised molecular profiling increased the rate to 77.0% (p = 0.049). EGFR and ALK profiling rates increased from 77.9% to 82.1% in non-squamous NSCLC and from 43.8% to 57.5% in squamous NSCLC. Plasma-based testing was feasible in 98.4% and identified oncogenic driver mutations in 7.1% of patients for whom tissue profiling was unfeasible. Conclusion: This study shows a high success rate of tissue-based molecular profiling that was significantly improved by a protocolised approach. Tissue-based profiling remains unfeasible for a substantial proportion of patients. Combined analysis of tumour tissue and circulating tumour DNA is a promising approach to allow adequate molecular profiling of more patients. [ABSTRACT FROM AUTHOR]

Background: The efficacy of PD-1 blocking agents in advanced NSCLC has shown prolonged effectiveness, but only in a minority of patients. Multiple biomarkers have been explored to predict treatment benefit, yet their combined performance remains inadequately examined. In this study, we assessed the combined predictive performance of multiple biomarkers in NSCLC patients treated with nivolumab., Methods: Pretreatment samples from 135 patients receiving nivolumab were used to evaluate the predictive performance of CD8 tumor-infiltrating lymphocytes (TILs), intratumoral (IT) localization of CD8 TILs, PD-1 high expressing TILs (PD1T TILs), CD3 TILs, CD20 B-cells, tertiary lymphoid structures (TLS), PD-L1 tumor proportion score (TPS) and the Tumor Inflammation score (TIS). Patients were randomly assigned to a training (n = 55) and validation cohort (n = 80). The primary outcome measure was Disease Control at 6 months (DC 6m) and the secondary outcome measure was DC at 12 months (DC 12m)., Results: In the validation cohort, the two best performing composite biomarkers (i.e. CD8+IT-CD8 and CD3+IT-CD8) demonstrated similar or lower sensitivity (64% and 83%) and NPV (76% and 85%) compared to individual biomarkers PD-1T TILs and TIS (sensitivity: 72% and 83%, NPV: 86% and 84%) for DC 6m, respectively. Additionally, at 12 months, both selected composite biomarkers (CD8+IT-CD8 and CD8+TIS) demonstrated inferior predictive performance compared to PD-1T TILs and TIS alone. PD-1T TILs and TIS showed high sensitivity (86% and 100%) and NPV (95% and 100%) for DC 12m. PD-1T TILs could more accurately discriminate patients with no long-term benefit, as specificity was substantially higher compared to TIS (74% versus 39%)., Conclusion: Composite biomarkers did not show improved predictive performance compared to PD-1T TILs and TIS alone for both the 6- and 12-month endpoints. PD-1T TILs and TIS identified patients with DC 12m with high sensitivity. Patients with no long-term benefit to PD-1 blockade were most accurately identified by PD-1T TILs., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: E.F.S. reports financial compensation to the institution for attendance of advisory boards and speaker engagements from AstraZeneca, Bristol Myers Squibb, Bayer, DSI, Eli Lilly, MSD, Merck, Novartis, Pfizer, Takeda, Regeneron, Roche Genentech, Roche Diagnostics, Boehringer Ingelheim, Sanofi. Research support from Astra Zeneca, Bristol Myers Squibb, Merck, MSD. All are outside the submitted work. GA Meijer is co-founder and board member (CSO) of CRCbioscreen BV, CSO of Health-RI (Dutch National Health Data infrastructure for research & innovation), and member of the supervisory board of IKNL (Netherlands Comprehensive Cancer Organisation). He has a research collaboration with CZ Health Insurances (cash matching to ZonMw grant) and he has research collaborations with Exact Sciences, Sysmex, Sentinel Ch. SpA, Personal Genome Diagnostics (PGDX), DELFi and Hartwig Medical Foundation; these companies provide materials, equipment and/or sample/genomic analyses. All are outside the submitted work. K.M. reports grants from AstraZeneca, non-financial support from Roche, Takeda, Pfizer, PGDx and DELFi, speakers fee from MSD, Roche, AstraZeneca, Benecke and consultant fee from Pfizer, BMS, Roche, MSD, Abbvie, AstraZeneca, Diaceutics, Lilly, Bayer, Boehringer Ingelheim and Merck outside the submitted work. K.H., V.N, M.M, R.D.S., M.M.H, D.S.T. have nothing to disclose. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Hummelink et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: In non-small cell lung cancer (NSCLC), PD-L1 expression on either tumor cells (TC) or both TC and tumor-infiltrating immune cells (IC) is currently the most used biomarker in cancer immunotherapy. However, the mechanisms involved in PD-L1 regulation are not fully understood. To provide better insight in these mechanisms, a multiangular analysis approach was used to combine protein and mRNA expression with several clinicopathological characteristics., Patients and Methods: Archival tissues from 640 early stage, resected NSCLC patients were analyzed with immunohistochemistry for expression of PD-L1 and CD8 infiltration. In addition, mutational status and expression of a selection of immune genes involved in the PD-L1/PD-1 axis and T-cell response was determined., Results: Tumors with high PD-L1 expression on TC or on IC represent two subsets of NSCLC with minimal overlap. We observed that PD-L1 expression on IC irrespective of expression on TC is a good marker for inflammation within tumors. In the tumors with the highest IC expression and absent TC expression an association with reduced IFNγ downstream signaling in tumor cells was observed., Conclusions: These results show that PD-L1 expression on TC and IC are both independent hallmarks of the inflamed phenotype in NSCLC, and TC-negative/IC-high tumors can also be categorized as inflamed. The lack of correlation between PD-L1 TC and IC expression in this subgroup may be caused by impaired IFNγ signaling in tumor cells. These findings may bring a better understanding of the tumor-immune system interaction and the clinical relevance of PD-L1 expression on IC irrespective of PD-L1 expression on TC., Competing Interests: Willemijn SME Theelen: none. Thomas Kuilman: none. Katja Schulze: Genentech employee and holder of Roche stock. Wei Zou: Genentech employee and holder of Roche stock. Oscar Krijgsman: none. Dennis DGC Peters: none. Sten Cornelissen: none. Kim Monkhorst: personal fees from Roche, Pfizer, BMS, Abbvie, AstraZeneca, grants from AstraZeneca, non -financial support from Roche, non-financial support from Genentech outside the submitted work. Pranamee Sarma: Genentech employee and holder of Roche stock. Teiko Sumiyoshi: Genentech employee and holder of Roche stock Lukas C Amler: Genentech employee and holder of Roche stock. Stefan M Willems: medical advisor for and/or receives research grants from Roche, Pfizer, MDS, AstraZeneca, Cergentis, Merck Hans LG Blaauwgeers: none. Carel JM van Noesel: none. Daniel S Peeper: none. Michel M van den Heuvel: none Marcin Kowanetz: Genentech employee and holder of Roche stock. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: In female mammalian cells, random X chromosome inactivation (XCI) equalizes the dosage of X-encoded gene products to that in male cells. XCI is a stochastic process, in which each X chromosome has a probability to be inactivated. To obtain more insight in the factors setting up this probability, we studied the role of the X to autosome (X ratio A) ratio in initiation of XCI, and have used the experimental data in a computer simulation model to study the cellular population dynamics of XCI., Methodology/principal Findings: To obtain more insight in the role of the XratioA ratio in initiation of XCI, we generated triploid mouse ES cells by fusion of haploid round spermatids with diploid female and male ES cells. These fusion experiments resulted in only XXY triploid ES cells. XYY and XXX ES lines were absent, suggesting cell death related either to insufficient X-chromosomal gene dosage (XYY) or to inheritance of an epigenetically modified X chromosome (XXX). Analysis of active (Xa) and inactive (Xi) X chromosomes in the obtained triploid XXY lines indicated that the initiation frequency of XCI is low, resulting in a mixed population of XaXiY and XaXaY cells, in which the XaXiY cells have a small proliferative advantage. This result, and findings on XCI in diploid and tetraploid ES cell lines with different X ratio A ratios, provides evidence that the X ratio A ratio determines the probability for a given X chromosome to be inactivated. Furthermore, we found that the kinetics of the XCI process can be simulated using a probability for an X chromosome to be inactivated that is proportional to the X ratio A ratio. These simulation studies re-emphasize our hypothesis that the probability is a function of the concentration of an X-encoded activator of XCI, and of X chromosome specific allelic properties determining the threshold for this activator., Conclusions: The present findings reveal that the probability for an X chromosome to be inactivated is proportional to the X ratio A ratio. This finding supports the presence of an X-encoded activator of the XCI process.