Immune checkpoint inhibitors (ICI), such as anti-PD-1 agents, have become part of the standard of care treatment of advanced non-small cell lung cancer (NSCLC). Predictive biomarkers are needed to identify patients that benefit from anti-PD-1 treatments. Tumor infiltrating lymphocytes (TILs) and PD-L1 are major players in the ICI mechanism of action. In this study, we assess the impact of real-world clinicopathological variables, including TILs and PD-L1, on anti-PD-1 efficacy. We performed a monocenter retrospective study in advanced NSCLC treated with nivolumab or pembrolizumab between January 2015 and February 2019. The impact of baseline clinical and pathological variables was assessed by univariate and multivariate models. TILs, defined as CD8+T-cells, and PD-L1 were scored in tumor and stroma, and correlated with progression free survival (PFS) and overall survival (OS). We included 366 patients of whom 141 were assessed for tumor and stromal TILs. The median follow-up time was 487 days. In the whole cohort, PFS was associated with high tumor PD-L1, high albumin and good performance. OS was associated with low LDH, high albumin, good performance and 'first-line treatment'. In the TILs subcohort, stromal TILs had the strongest impact on PFS and OS. Stromal TILs were a stronger marker for PFS and OS than tumoral TILs, tumoral PD-L1 or stromal PD-L1. Remaining factors for PFS and OS were albumin and albumin with LDH, respectively. This real-world study on clinicopathological features shows that stromal CD8 + TILs were the strongest predictor for PFS and OS in patients with advanced NSCLC on anti-PD-1 therapy. Other predictors for PFS and OS included albumin and albumin together with LDH, respectively. This study highlights the pivotal role of the stromal compartment in the mechanisms of action of ICI, and the need for further studies aiming to overcome this stromal firewall.

The EORTC-Lung Cancer Group initiated a Delphi consensus process to establish a consensual definition of resectable stage III non-small cell lung cancer (NSCLC) for the use in clinical trials, including a systematic review, survey, and review of clinical cases. Here, the survey results are presented, aimed to identify areas of controversy. A survey was distributed among the members of six international organizations related to lung cancer. Respondents were interrogated on the resectability (not limited to the technical resectability) of all stage III NSCLC TNM-subsets (8th edition). Additionally, four N2-subdivisions were used. The threshold for agreement was 75%. Answers with "yes" were considered upfront resectable. "Yes" and "maybe" were grouped together and considered potentially resectable. Answers with "no" were considered unresectable. 558 responses were collected from thoracic surgeons (38%), radiation oncologists (27%), medical oncologists (17%), pulmonologists (14%), and others (4%). Most worked in a specialized center (80%), had >5 years of experience (80%), were European (76%), male (73%), and treated >20 patients with stage III NSCLC annually (77%). Agreement was found in 26 (70%) out of 37 TNM-subsets: 9 (24%) were considered (potentially) resectable, and 17 (46%) unresectable. There was no agreement for 11 (30%) TNM-subsets: smaller tumors with N2-multistation, larger tumors with N2-single station, and invasive T4-tumors with maximum N2-single station involvement. This international and multidisciplinary survey showed agreement on the resectability for the majority of stage III NSCLC TNM-subsets, but also identified several TNM-subsets for which no agreement was found.

Cell‐free DNA (cfDNA) can be isolated and sequenced from blood and/or urine of cancer patients. Conventional short‐read sequencing lacks deployability and speed and can be biased for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies (ONT) sequencing we can achieve delivery of genomic and fragmentomic data from liquid biopsies. Copy number aberrations and cfDNA fragmentation patterns can be determined in less than 24 h from sample collection. The tumor‐derived cfDNA fraction calculated from plasma of lung cancer patients and urine of bladder cancer patients was highly correlated ( R = 0.98) with the tumor fraction calculated from short‐read sequencing of the same samples. cfDNA size profile, fragmentation patterns, fragment‐end composition, and nucleosome profiling near transcription start sites in plasma and urine exhibited the typical cfDNA features. Additionally, a high proportion of long tumor‐derived cfDNA fragments (> 300 bp) are recovered in plasma and urine using ONT sequencing. ONT sequencing is a cost‐effective, fast, and deployable approach for obtaining genomic and fragmentomic results from liquid biopsies, allowing the analysis of previously understudied cfDNA populations.

In advanced-stage NSCLC, tumor proportion score (TPS) is typically used to predict the efficacy of immune checkpoint inhibitors (ICIs). Nevertheless, in other cancer types, the combined positive score (CPS), which covers programmed death-ligand 1 (PD-L1) expression on both tumor and surrounding immune cells, is used. We investigated the predictive value of CPS in comparison to TPS in advanced NSCLC. A monocenter, retrospective study was performed in patients with advanced NSCLC treated with ICI monotherapy between 2015 and 2021. Hematoxylin and eosin and PD-L1 were stained on baseline tumor biopsy samples to score PD-L1 by both TPS and CPS. Positivity for TPS and CPS was defined as a score of 1% or above. Progression-free survival and overall survival (OS) were assessed for TPS and CPS scores. Among the 187 included patients, PD-L1 positivity was found in 112 patients (59.9%) by TPS and 135 patients (72.2%) by CPS. There was no significant difference in OS between TPS⁻ and TPS⁺ patients (p = 0.20). Nevertheless, CPS⁺ patients did have a longer OS than CPS⁻ patients (p = 0.006). OS was superior in both TPS⁻/CPS⁺ and TPS⁺/CPS⁺ as compared with TPS⁻/CPS⁻ cases (p = 0.018 and p = 0.015, respectively), whereas no considerable differences in OS were found between TPS⁻/CPS⁺ and TPS⁺/CPS⁺ cases. This retrospective real-world population study revealed that CPS differentiated OS better than TPS in patients with advanced NSCLC with ICI monotherapy. Remarkably, this was driven by the performance of the TPS⁻/CPS⁺ subgroup, indicating that CPS may be a better predictive biomarker for ICI efficacy.

Tumor-educated Platelets (TEPs) have emerged as rich biosources of cancer-related RNA profiles in liquid biopsies applicable for cancer detection. Although human blood platelets have been found to be enriched in circular RNA (circRNA), no studies have investigated the potential of circRNA as platelet-derived biomarkers for cancer. In this proof-of-concept study, we examine whether the circRNA signature of blood platelets can be used as a liquid biopsy biomarker for the detection of non-small cell lung cancer (NSCLC). We analyzed the total RNA, extracted from the platelet samples collected from NSCLC patients and asymptomatic individuals, using RNA sequencing (RNA-Seq). Identification and quantification of known and novel circRNAs were performed using the accurate CircRNA finder suite (ACFS), followed by the differential transcript expression analysis using a modified version of our thromboSeq software. Out of 4732 detected circRNAs, we identified 411 circRNAs that are significantly (p-value < 0.05) differentially expressed between asymptomatic individuals and NSCLC patients. Using the false discovery rate (FDR) of 0.05 as cutoff, we selected the nuclear receptor-interacting protein 1 (NRIP1) circRNA (circNRIP1) as a potential biomarker candidate for further validation by reverse transcription–quantitative PCR (RT-qPCR). This analysis was performed on an independent cohort of platelet samples. The RT-qPCR results confirmed the RNA-Seq data analysis, with significant downregulation of circNRIP1 in platelets derived from NSCLC patients. Our findings suggest that circRNAs found in blood platelets may hold diagnostic biomarkers potential for the detection of NSCLC using liquid biopsies.

Background: Assays that account for the biological properties and fragmentation of cell-free DNA (cfDNA) can improve the performance of liquid biopsy. However, preanalytic and physiological differences between individuals on fragmentomic analysis are poorly defined.Methods: We analyzed the impact of collection tube, plasma processing time, and physiology on the size distribution of cfDNA, their genome-wide representation, and sequence diversity at the cfDNA fragment ends using shallow whole-genome sequencing.Results: Neither different stabilizing collection tubes nor processing times affected the cfDNA fragment sizes, but could impact the genome-wide fragmentation patterns and fragment-end sequences of cfDNA. In addition, beyond differences depending on the gender, the physiological conditions tested between 63 individuals (age, body mass index, use of medication, and chronic conditions) minimally influenced the outcome of fragmentomic methods.Conclusions: Fragmentomic approaches have potential for implementation in the clinic, pending clear traceability of analytical and physiological factors.