BACKGROUND:PD-L1 tumor proportion score (TPS) is used to guide immunotherapy in non-small cell lung cancer (NSCLC), yet its ability to predict pathological response in the neoadjuvant setting remains limited. METHODS:Thirty pathologists from 11 countries independently assessed PD-L1 TPS in pre-treatment biopsies and residual viable tumor (RVT) in matched resection specimens after neoadjuvant chemoimmunotherapy in 30 digitized cases from the ReGraDE (regression grading in Germany) study. Interobserver agreement was evaluated using intraclass correlation coefficients (ICC) and Fleiss' kappa. Associations between TPS and RVT were analyzed using Pearson correlation, and correlations derived from single-rater and averaged TPS were compared using the Williams test. RESULTS:Interobserver agreement was moderate for both TPS and RVT (ICC = 0.74, 95% CI 0.60-0.87, and ICC = 0.74, 95% CI 0.62-0.85, respectively) and became near-perfect when mean scores per case were calculated across observers (ICC = 0.99, for both). However, TPS remained poorly correlated with RVT for both single-rater assessment (r = -0.17, p = 0.38) and averaged TPS (r = -0.16, p = 0.39) with no significant difference between these approaches (p = 0.96). Differences in interpretation thresholds were observed particularly in borderline cases around the 1% TPS cut-off and in distinguishing 0% from minimal RVT, but did not account for the lack of association between TPS and RVT. Systematic differences were observed depending on individual professional experience, particularly in borderline cases. CONCLUSION:Interobserver variability does not explain the limited predictive value of PD-L1 TPS in the neoadjuvant setting, suggesting an intrinsic limitation of the biomarker.

Precision oncology (PO) has revolutionized cancer treatment by aligning therapy with a tumor's molecular profile. The integration of next-generation sequencing (NGS) has allowed for actionable genomic alterations to inform treatment decisions. However, challenges remain in streamlining NGS results and integrating them into clinical workflows, particularly in the context of Molecular Tumor Boards (MTBs). This study presents an integrated platform designed to support MTB decision-making by consolidating clinical and molecular data from heterogeneous sources using an ETL (Extract, Transform, Load) process. The platform automates data loading, significantly reducing clinicians' time on manual data entry and provides a data integration view of the patient status helping decision making. A total of 69 variables, encompassing clinical, molecular, and MTB recommendations, were integrated into the platform. From February 2024 to September 2025, the platform collected data from 1.119 patients and 1.221 NGS results, with 30% of cases receiving therapeutic recommendations. This approach enhances clinical care, promotes data interoperability, and supports PO in a real-world MTB workflow.

Artificial intelligence (AI) can predict genomic alterations from histology, yet its adoption is slowed by a lack of trust. We argue that deliberate morphology (i.e., a cognitive understanding of histological features supported by standardized annotations) creates a bidirectional feedback loop between clinical practice and model outputs.We translate these observations into an actionable hypothesis for clinical and computational teams: that by enhancing explainability, deliberate morphology could facilitate the responsible deployment of AI biomarkers in oncology.

INTRODUCTION:Tumor spread through air spaces (STAS) and histologic tumor grade have been reported as adverse prognostic factors in non-small cell lung cancer (NSCLC), yet their impact on the extent of surgical resection remains insufficiently defined. This study aimed to evaluate the prognostic impacts of STAS and tumor grade in stage IA patients (pT1a/bN0) from JCOG0802/WJOG4607L comparing segmentectomy and lobectomy. METHODS:STAS and tumor grading were assessed in 593 tumors by 32 expert pulmonary pathologists across the world. The prognostic impact of STAS in lobectomy (n=294) versus segmentectomy (299) in relation to relapse-free survival (RFS) and overall survival (OS) was evaluated, as well as the association of STAS with histologic grading. RESULTS:STAS was identified in 227 cases (38.3%) and was significantly associated with shorter relapse-free survival (RFS) and overall survival (OS) (RFS HR=2.204; OS HR=1.917; both p<0.001). Multivariable analysis confirmed STAS as an independent adverse factor for RFS (p<0.001) and OS (p=0.009). STAS was associated with higher local recurrence in both lobectomy and segmentectomy approaches (p=0.012 and p=0.002, respectively). STAS independently predicted shorter RFS in both lobectomy and segmentectomy subgroups, but only OS in the lobectomy subgroup. Among invasive non-mucinous adenocarcinomas, histologic grade 3 was strongly associated with STAS (p<0.001) and poor outcomes. Both STAS and grade 3 independently predicted shorter RFS, whereas only grade 3 predicted OS. CONCLUSIONS:These findings reinforce STAS and high-grade adenocarcinoma as clinically significant prognostic factors and support their incorporation into risk stratification for stage IA (pT1a/bN0) NSCLC patients.

Antibody-drug conjugates (ADC) as a new treatment modality have enabled novel, promising treatment options in lung cancer. However, biomarkers for the selection of appropriate treatments are still under development, posing novel challenges for tissue selection and development of companion diagnostics (CDx). In this review, we address the challenges and discuss best practice for fast, reliable and robust implementation of novel biomarkers in treatment selection of ADCs in lung cancer.

INTRODUCTION:Biomarker testing is an essential component of optimal therapeutic management in NSCLC, enabling the use of both Food and Drug Administration-approved and emerging targeted therapies. Despite well-established biomarker testing guidelines and the availability of many approved targeted therapies, a substantial proportion of patients with advanced NSCLC are not benefiting from precision oncology. In this study, we analyze the distribution of actionable genomic alterations across histologic subtypes and clinicodemographic subgroups of NSCLC using data in 82,328 samples profiled with a single comprehensive genomic profiling assay, aiming to support universal molecular testing across all NSCLC subtypes to ensure equitable access to available therapeutics. METHODS:This is an observational retrospective analysis on histologically confirmed NSCLC cases tested with comprehensive genomic profiling by next-generation sequencing between 2014 and 2022 using Foundation One/Foundation CDx. All cases were centrally reviewed by board-certified anatomic pathologist to determine histologic type and subtype. RESULTS:A total of 82,328 patients with NSCLC were included. An actionable genomic alteration (GA) was found in 35.1% of the cases. Lung adenocarcinoma (LUAD) and adenosquamous carcinoma were more frequently associated with actionable GA (45.8% and 40.9%, respectively) as compared with sarcomatoid (29.1%), not otherwise specified (27.6%), large cell (21.1%), and squamous cell (6.5%) histologies. Sarcomatoid histology had the highest METex14 skipping mutation (mut) frequency (9.95% versus 2.43% in LUAD). Tumor mutation burden more than or equal to 10 mut/Mb was associated with histology (50.91% in large cell, 40.79% in not otherwise specified, 39.08% in squamous cell, and 36.30% in sarcomatoid versus 31.22% in LUAD and 29.22% in adenosquamous carcinoma). Patients with actionable GA had usually a low tumor mutation burden (80.88%). A significant correlation (p < 0.005) between age and actionable GA was reported for BRAF/ERBB2 muts, ALK/RET/ROS1 rearrangements, and MET amplification. EGFR actionable muts and KRAS G12C were more frequently observed in females, whereas no significant correlation between sex and other GA was observed. Finally, genetic ancestry analyses revealed a strong correlation for EGFR actionable muts and South/East Asia and America, but not for other GA. CONCLUSIONS:This is the largest NSCLC data set analyzed for biomarker distribution across histologies, age, sex, and genetic ancestry. This data set confirms sufficient enough biomarker prevalence across many histologic subtypes of NSCLC, providing reassurance that all NSCLC cases should be considered for biomarker workup.

Non-small cell lung carcinoma (NSCLC) encompasses a diverse range of molecular subtypes that require precise identification to guide therapy selection. Among these molecular alterations, gene fusions involving ALK, ROS1, RET, and NTRK present significant challenges for reliable detection in routine clinical practice, particularly due to formalin fixation and the limited availability of nucleic acids, which often hinder molecular analyses. This article highlights key considerations spanning the pre-analytical, analytical, and post-analytical stages, emphasizing the critical need to standardize the entire workflow to ensure high-quality results in gene fusion detection through molecular biology techniques. Particular attention is given to next-generation sequencing (NGS) approaches, with RNA-based NGS strategies being highlighted for their superior sensitivity and accuracy in identifying gene fusions. In conclusion, this work consolidates practical recommendations for integrating optimized gene fusion detection into routine clinical workflows. These include guidance on assay design, quality control measures, and method validation, to ensure the delivery of reliable results to support personalized treatment strategies in NSCLC.

BackgroundLung adenocarcinoma (LUAD) develops through a multistep histologic continuum from normal lung epithelium to premalignant lesions and invasive carcinoma. While genomic alterations in invasive LUAD have been extensively characterized, early immune and microenvironmental reprogramming events occurring at premalignant stages remain incompletely defined. We aimed to characterize spatial transcriptomic dynamics across this continuum, with particular focus on atypical adenomatous hyperplasia (AAH).MethodsFormalin-fixed paraffin-embedded surgical specimens from 23 patients with resected LUAD containing matched areas of normal lung tissue, AAH, and invasive carcinoma were analyzed using GeoMx® Digital Spatial Profiling. Regions of interest (ROIs) were selected by expert thoracic pathologists. Differential gene expression, pathway enrichment, unsupervised clustering, and immune-related gene signature analyses were performed. A spatially derived 11-gene signature capturing progressive molecular changes was developed and externally explored in public LUAD datasets.ResultsAAH lesions exhibited a distinct transcriptomic profile positioned between normal lung and invasive carcinoma. Compared with normal tissue, AAH demonstrated early activation of oncogenic pathways, including MAPK and PI3K-AKT signaling, as well as extracellular matrix remodeling and interferon-related immune programs. Invasive tumors showed further amplification of immune regulatory and stromal activation signatures. Unsupervised clustering revealed molecular heterogeneity within AAH, identifying a subset of lesions with tumor-like transcriptional features. An 11-gene spatial signature captured progressive reprogramming across histologic stages and was associated with adverse biological characteristics in external LUAD cohorts.ConclusionsSpatial transcriptomic profiling reveals that molecular and immune reprogramming begins at premalignant stages of LUAD development. AAH represents a biologically active and heterogeneous intermediate state within lung carcinogenesis. These findings support the biological relevance of early microenvironmental reprogramming and may inform future risk stratification and interception strategies.

Introduction: Tissue , plasma-based next-generation sequencing (NGS) have complementary roles in patients with advanced NSCLC. Nevertheless, whether there is any added clinical value in combining both methods in the treatment of na & iuml;ve patients remains unclear. Methods: We retrospectively collected clinical and genomic data from 275 patients with treatment-na & iuml;ve advanced NSCLC who had undergone plasma-based NGS at diagnosis in our institution. We analyzed patient data in two separate cohorts, each assessed with a different plasma-based NGS method: cohort 1 (n = 127, Guardant360) , cohort 2 (n = 148, FoundationACT/FoundationOne Liquid CDx). Ninety-five patients (75%) in cohort 1 and 108 patients (73%) in cohort 2 underwent concurrent amplicon-based tissue NGS testing locally. Results: Forty-three patients in cohort 1 (34%) and 49 patients in cohort 2 (33%) harbored European Society for Medical Oncology Scale for Clinical Actionability of Molec- ular Targets (ESCAT) I or II targetable driver alterations. The addition of orthogonal biopsy (tissue to liquid, or liquid to tissue) offered no relevant clinical value in cases with ESCAT I or II targetable drivers already detected by one method. In contrast, adding orthogonal biopsy incremented the detection of ESCAT I or II targetable drivers not only in cases with uninformative testing (undetectable circulating tumor DNA, unavailable/inadequate tissue) but also in about 5% of the patients with seemingly informative but driver undetected molecular results. The prevalence of ESCAT I or II targetable drivers in plasma was significantly higher in patients with adenocarcinoma, 20 pack-year or less smoking history , abdominal metastases.Conclusions: Our study suggests that the addition of sequential orthogonal biopsy should be considered when- ever an ESCAT I or II targetable driver has not been detected by the initial method, including cases with seem- ingly informative molecular analysis. (c) 2024 The Authors. Published by Elsevier Inc. on behalf of the International Association for the Study of Lung Cancer. This is an open access article under the CC BY-NC-ND li- cense (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

BACKGROUND:Non-small cell lung carcinoma (NSCLC) is a leading example of precision oncology, with a growing number of actionable targets. However, long turnaround times (TAT) for biomarker results can delay optimal treatment decisions. We evaluated whether a streamlined workflow could deliver comprehensive molecular reports within 72 h. METHODS:In this prospective cohort study (UTOPIA protocol), 96 patients with early-stage or advanced NSCLC at Hospital Universitario 12 de Octubre underwent molecular tumor board (MTB)-centered triage, automated NGS processing, and integrated data review. TAT was defined (in working days) from MTB triage to electronic report validation. Communication was supported by daily operational huddles and intralaboratory pre-MTB meetings using a standardized checklist. RESULTS:All 96 NGS reports met the 72-hour TAT target (100%). The NGS failure rate was 1%. Potentially actionable genomic alterations were identified in 45.8% of patients, most frequently EGFR (24%) and KRAS G12C (8.3%). Other targetable alterations included six ALK fusions (6.3%), four MET exon 14 skipping mutations (4.2%), two BRAF V600E mutations (2.1%), and one RET fusion (1%). CONCLUSION:An ultrafast biomarker testing workflow for lung cancer, enabled by MTB-driven triage and structured team communication, can reliably generate comprehensive molecular reports within 72 h. This approach may reduce TAT-related treatment delays and support timely biomarker-guided therapy for patients with NSCLC.

Background/Objectives: The non-small-cell lung cancer (NSCLC) therapeutic landscape has undergone a profound transformation with the introduction of multiple personalized treatment options. Mutations in ERBB2 (HER2) have recently emerged as promising novel targets for the treatment of non-squamous NSCLC (nsNSCLC). Accurate, rapid, and efficient molecular profiling is crucial for identifying patients who may benefit from targeted therapies, including HER2-directed agents. Materials and Methods: Here, we aimed to retrospectively assess the performance of the Oncomine™ Precision Assay* (OPA) in combination with the Ion Torrent Genexus™ Integrated Sequencer* (Thermo Fisher Scientific. Waltham, MA, USA) for detecting ERBB2 mutations in nsNSCLC. A total of 108 archived nsNSCLC samples, consisting of biopsies, resections, and cytological specimens, were used to assess concordance with in-house-validated orthogonal tests. Results: The OPA showed high sensitivity and specificity with an overall accuracy of 100% for single-nucleotide variants (SNVs) and insertions and deletions (Indels). SNVs and Indels with allele frequencies as low as 5% were correctly identified across samples with a tumor cell content ranging from 5% to 95%. Additionally, the assay demonstrated high reproducibility across the six participating laboratories. The turnaround time of the OPA was notably shorter compared to traditional orthogonal methods, facilitating rapid molecular report generation. Conclusions: The OPA in combination with the Ion Torrent Genexus™ System allows for highly sensitive and specific detection of relevant ERBB2 mutations. The assay’s streamlined workflow, coupled with its automated data analysis pipeline, enables a fast turnaround time for testing across a range of sample types. This includes samples with reduced tumor cell content and limited available input. This study demonstrates the future potential of using this assay in a clinical setting.

The implementation of biomarker testing for targeted therapies and immune checkpoint inhibitors is a cornerstone in the management of metastatic and locally advanced non-small cell lung cancer (NSCLC), playing a pivotal role in guiding treatment decisions and patient care. The emergence of precision medicine in the realm of operable NSCLC has been marked by the recent approvals of osimertinib, atezolizumab, nivolumab, pembrolizumab and alectinib for early-stage disease, signifying a shift towards more tailored therapeutic strategies. Concurrently, the landscape of this disease is rapidly evolving, with several further pending approvals and numerous clinical trials in progress.To harness the benefits of these innovative neo-adjuvant and adjuvant therapies, the integration of predictive biomarker testing into standard clinical protocols is imperative for patients with operable NSCLC. A multidisciplinary international consortium has identified three primary obstacles impeding the effective testing of patients with operable NSCLC. These challenges encompass the limited number of test requests by physicians, the inadequacy of tissue samples for comprehensive testing, and the prevalence of cost-reduction measures leading to suboptimal testing practices.This review delineates the aforementioned challenges and proposed solutions, and strategic recommendations aimed at enhancing the testing process. By addressing these issues, we strive to optimize patient outcomes in operable NSCLC, ensuring that individuals receive the most appropriate and effective care based on their unique disease profile.

CONTEXT:Molecular tumor boards (MTBs) are multidisciplinary meetings of specialists dedicated to analyzing biomarker test results to provide personalized treatment recommendations. However, global disparities in the successful implementation of MTBs exist, driven by unequal access to molecular diagnostics and supportive multidimensional expertise. OBJECTIVE:To establish recommendations for MTB implementation, outline practical frameworks for their operation, and address disparities in expertise and resources between new and established MTBs. DESIGN:A modified Delphi method involved 37 international experts in three survey rounds and online meetings, with consensus defined as more than or equal to 75% agreement. RESULTS:The panel identified a molecular biologist or pathologist with expertise in molecular diagnostics and tumor-specific medical oncologists as indispensable MTB members. Case selection should reflect institutional expertise and volume, with newer MTBs reviewing less selected cases to gain experience. Regular meetings are advised to avoid delays beyond 14 days from result availability to discussion. Reporting should be standardized to include clinicopathologic data (tumor characteristics, treatment history), biomarker findings (testing results, sample details), and recommendations (treatment, retesting, genetic counseling). Treatment options should be ranked by the level of evidence for actionability and may include options not available locally. Performance evaluation should consider changes in patient management based on MTB input and matched therapy rates. CONCLUSIONS:These MTB consensus recommendations are applicable across tumor types, despite being developed by lung cancer and molecular specialists and initiated by the International Association for the Study of Lung Cancer. They provide a structured framework for MTB implementation, report standardization, case selection, and quality assessment, aiming to standardize practice and address gaps in expertise for personalized cancer care.

PD-L1 expression in ROS1-positive non-small cell lung carcinoma (NSCLC) patients remains unclear regarding its possible clinical-biological role. A retrospective, descriptive study of 24 ROS1-positive NSCLC patients (2013–2023) from five Spanish hospitals were evaluated. Manual (clone SP263) PD-L1 tumour proportion score (TPS) was also subjected to two artificial intelligence (AI) testing algorithms. Clinical and molecular data categorized according to PD-L1 levels by double independent review, were retrospectively collected and, potential correlations were analysed. The median age was 60 years (46–85), female (54.1

INTRODUCTION:Tumor grading informs therapy and patient management across many organs, yet no consensus exists for grading invasive squamous cell carcinoma of the lung (LUSC). This study aimed to develop a globally applicable grading system using international cohorts. METHODS:Histologic features, including tumor budding, smallest tumor nest size, nuclear size, and tumor spread through air spaces (STAS), were evaluated in two training sets comprising 262 and 427 LUSCs resected without neoadjuvant therapy from three institutions. Kaplan-Meier and Cox proportional hazards models were used to identify features associated with recurrence-free survival (RFS) and overall survival (OS). Features significant in both training sets were used to construct a grading system, which was then validated in a test set (n = 827, five institutions). Interobserver agreement was assessed among 10 pathologists on 25 cases. RESULTS:Tumor budding (two-tier: cutoff at 10 buds per 0.785 mm2) was the only histologic feature significantly associated with both RFS and OS in multivariable analyses across both training sets. The proposed two-tier grading system-low-grade (0-9 buds), high-grade (≥10 buds)-was validated in the test set, demonstrating median RFS of 4.8 versus 1.6 years for low- versus high-grade tumors in the entire cohort and 7.2 versus 3.4 years within stage I patients. Interobserver agreement was moderate (Fleiss' kappa = 0.524). CONCLUSIONS:The authors propose a simple, prognostically relevant grading system for resected invasive LUSC based on tumor budding. It is reproducible across international data sets and practical for routine pathology, offering a unified framework for clinical and research use.

Context.— The neurotrophic tropomyosin receptor kinase (NTRK) family gene rearrangements have been recently incorporated as predictive biomarkers in a “tumor-agnostic” manner. However, the identification of these patients is extremely challenging because the overall frequency of NTRK fusions is below 1%. Academic groups and professional organizations have released recommendations on the algorithms to detect NTRK fusions. The European Society for Medical Oncology proposal encourages the use of next-generation sequencing (NGS) if available, or alternatively immunohistochemistry (IHC) could be used for screening with NGS confirmation of all positive IHC results. Other academic groups have included histologic and genomic information in the testing algorithm. Objective.— To apply some of these triaging strategies for a more efficient identification of NTRK fusions within a single institution, so pathologists can gain practical insight on how to start looking for NTRK fusions. Design.— A multiparametric strategy combining histologic (secretory carcinomas of the breast and salivary gland; papillary thyroid carcinomas; infantile fibrosarcoma) and genomic (driver-negative non–small cell lung carcinomas, microsatellite instability–high colorectal adenocarcinomas, and wild-type gastrointestinal stromal tumors) triaging was put forward. Results.— Samples from 323 tumors were stained with the VENTANA pan-TRK EPR17341 Assay as a screening method. All positive IHC cases were simultaneously studied by 2 NGS tests, Oncomine Comprehensive Assay v3 and FoundationOne CDx. With this approach, the detection rate of NTRK fusions was 20 times higher (5.57%) by only screening 323 patients than the largest cohort in the literature (0.30%) comprising several hundred thousand patients. Conclusions.— Based on our findings, we propose a multiparametric strategy (ie, “supervised tumor-agnostic approach”) when pathologists start searching for NTRK fusions.

Introduction: RET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC. Methods: This situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion–positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay. Results: The most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively). Conclusions: In-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.