PURPOSE:Clinical outcomes with immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) range from durable disease control to rapid progression. Current biomarkers have limited ability to predict these divergent outcomes. EXPERIMENTAL DESIGN:Spatially-resolved immune subtyping in the TRACERx421 multiregion cohort was used to assess intratumoral immune heterogeneity (ITIH), defined as coexisting immune-enriched and immune-depleted regions. A computational model was used to infer ITIH from single samples, classifying tumors as homogenously immune-enriched (Hom-IE), heterogeneously immune-enriched (Het-IE), heterogeneously immune-depleted (Het-D), and homogenously immune-depleted (Hom-D). ITIH was validated by digital pathology and evaluated in three independent cohorts (n=1,085) of advanced NSCLC patients treated with ICIs. RESULTS:In TRACERx421, ITIH was associated with differences in immune programs among tumors otherwise classified within the same immune subtype. Single-sample ITIH inference recapitulated these patterns, as supported by gradients of tumor-infiltrating lymphocytes detected via digital pathology. Across cohorts, Hom-IE tumors demonstrated longer OS compared to other groups (34.6 vs. 12.1-16.6 months in cohort A; 21.9 vs. 8.4-13.3 months in cohort B; 22.6 vs. 11.3-13.4 months in cohort C). Hom-IE tumors were associated with longer OS regardless of PD-L1 status. Consistently, among PD-L1 low/negative tumors, Hom-IE tumors showed significantly longer OS compared to the non-Hom-IE counterpart. Conversely, the detrimental effects of KEAP1STK11 mutations persisted in the Hom-IE background. CONCLUSIONS:These findings demonstrate that ITIH can be assessed from individual tumor samples, identifies a microenvironment state associated with long-term benefit from immunotherapy, and provides an additional layer of resolution to established and emerging biomarkers.

INTRODUCTION:Adjuvant osimertinib was shown to improve survival outcomes in resected, EGFR mutated, stage IB-IIIA NSCLC. However, the prognostic implications of co-occurring mutations in resected, stage I tumours are unclear. Due to the poor prognosis of EGFR/TP53 co-mutations in patients with advanced NSCLC, we focused on this event in a cohort of patients with untreated, resected-stage I-LUAD. METHODS:Patients who underwent radical surgical treatment for stage I LUAD in two high volume centers with available postoperative molecular testing were collected. Survival curves were estimated using the Kaplan-Meier product-limit method and were compared using the log-rank test; multivariable Cox regression analyses were used to assess the association between clinical and biological variables and RFS, and results were reported in the form of hazard ratios and their 95 % confidence intervals. We then leveraged publicly available datasets (OncoSG, MSK-LUAD604) to further test the hypothesis in external cohorts. RESULTS:A total of 370 patients were included from two high volume centers of which 97 had an EGFR mutation. In the McGill/IRE cohort, patients with EGFR+TP53 co-mutation had a significantly worse RFS when compared to patients with EGFR-mutated LUAD (p = 0.001). In the multivariable analysis, EGFR+TP53 co-mutations (HR 5.32) were significantly associated with worse RFS. In the MSKCC cohort, EGFR+TP53 co-mutations were associated with shorter RFS and similar OS. CONCLUSION:Our results suggest that, among patients with EGFR-mutated, resected-stage I-LUAD, the presence of a TP53 co-mutation is associated with worse RFS. These findings corroborate the aggressive biology associated with these molecular findings in patients with metastatic, EGFR-mutated LUAD.

Supplementary Figure 7. Exploratory analysis of median overall survival (A-C) and progression-free survival (B-D) according to TMB status in NI (A-B) and N-CT (C-D) treatment arm. TMB low (yellow line; n = 30/40) vs TMB high (blue line; n = 43/23). OS: Overall survival; PFS: progression-free survival.

Abstract Purpose: The role of activating alterations in the MAPK pathway in predicting immunotherapy efficacy in patients with lung squamous cell carcinoma (LSCC) is largely unknown. The aims of the randomized, phase II SQUINT trial were to assess the efficacy of nivolumab plus ipilimumab (NI) versus platinum-based chemotherapy plus nivolumab (N-CT) and to identify clinically available biomarkers of response to immunotherapy in patients with advanced or metastatic LSCC. Patients and Methods: SQUINT was an open-label, randomized, parallel, noncomparative, phase II trial of NI versus N-CT in chemo-naïve adult patients with metastatic or recurrent LSCC. The study was conducted across 15 Italian centers from September 2017 to February 2022 (ClinicalTrials.gov ID: NCT03823625). Results: Forty-five patients were included in the NI arm and 46 patients in the N-CT arm. At 12 months, the overall survival (OS) rate was 62% in the NI arm and 50% in the N-CT arm. In total, 74 patients were included in the analyses for individual biomarkers. In patients with mutations or copy-number variations of genes involved in the MAPK pathway, we observed higher response to immunotherapy (43% vs. 15%), longer progression-free survival (P = 0.03), and OS (P < 0.001). A higher density of CD8+PD1+ T cells (P = 0.04) among MAPK-altered tumors versus that in wild-type tumors, together with an increased CD8+PD1+/FOXP3 ratio (P = 0.047), was observed. In the validation cohort of patients not exposed to immunotherapy, OS was similar between MAPK1/3-mutant and wild-type LSCC. Conclusions: We showed for the first time that MAPK pathway–activating alterations influence the outcome of LSCC treated with immunotherapy, highlighting the relevance of gene profiling.

In this study the relation between preoperative fast tumor growth, as estimated by tumor doubling time < 400 days, and Kirsten Rat Sarcoma Virus (KRAS) mutations is investigated in a cohort of clinical stage I lung adenocarcinoma presenting as solid dominant nodules (consolidation-to-tumor ratio > 0.5). Moreover, in the patients with pathologic stage I disease, a fast growth in the preoperative period identifies a subgroup of KRAS-mutated lung adenocarcinomas at higher risk of recurrence. Introduction: To analyze the impact of Kirsten-Rat-Sarcoma Virus (KRAS) mutations on tumor-growth as estimated by tumor-doubling-time (TDT) among solid-dominant clinical-stage I lung adenocarcinoma. Moreover, to evaluate the prognostic role of KRAS mutations, TDT and their combination in completely-resected pathologic-stage I adenocarcinomas. Methods: In this single-center retrospective analysis, completely resected clinical-stage I adenocarcinomas presenting as solid-dominant nodules (consolidation-to-tumor ratio > 0.5) in at least 2 preoperative computed tomography scans were enrolled. Nodules' growth was scored as fast (TDT < 400 days) or slow (TDT > 400 days). KRAS-mutated adenocarcinomas were identified with next-generation sequencing. Logistic- and Cox-regressions were used to identify predictors of fast-growth and disease-free survival (DFS), respectively. Results: Among 151 patients, 83 (55%) had fast-growing nodules and 64 (42.4%) were KRAS-mutated. Fast-growing nodules outnumbered in the KRAS-mutated group (n = 45; 70.3%), median TDT 95-days (interquartile range, IQR 43.5-151.5) compared to the KRAS wild-type group (38, 43.7%), median TDT 138-days (IQR 70.3-278.5). KRAS-mutations predicted faster-growth at multivariable analysis (P = .009). In a subgroup analysis including 108 pathologic-stage I adenocarcinomas, neither KRAS-mutations (P = .081) nor fast-growing pattern (P = .146) affected DFS. Nevertheless, the association of KRAS-mutations and fast-growing pattern identified a subgroup of patients with worse DFS (P = .02). The combination of fast-growing and KRAS-mutations (hazard-ratio 2.97 [95%CI 1.22-7.25]; P = .017) and average nodule diameter at diagnosis (hazard-ratio 1.08 [95%CI 1.03-1.14]; P = .004) were independent predictors of worse DFS. Conclusion: KRAS mutations are associated to faster growth, in clinical-stage I adenocarcinoma presenting at diagnosis as solid dominant nodules undergoing complete resection. Moreover, faster-growth identifies a subgroup of pathologic-stage I KRAS-mutated adenocarcinomas with higher recurrences.

The Telomere Repeat-Binding factor 2 (TRF2) contributes to cancer progression by both telomere-dependent and independent mechanisms, including immune escape and angiogenesis. Here, we found that TRF2, through its Basic domain, directly interacts with Emerin forming a complex, including Lamin A/C, Lamin B1, SUN1, and SUN2. Importantly, TRF2 association with the inner nuclear membrane is functional to the proper establishment of cell polarity, finally promoting productive 1D and 3D migration in triple negative breast cancer cells (TNBC). In line with this, a spontaneous model of TNBC metastasis, combined with intravital imaging, allowed us to demonstrate that TRF2 promotes cell migration at the primary tumor site and is required for the early steps of the metastatic cascade. In human breast cancers, aberrantly elevated TRF2 expression positively correlates with cancer progression, metastasis, and poor prognosis, identifying TRF2 as a potential target for novel therapeutic strategies against TNBC.

Supplementary Figure 4. Overall survival (A) and progression-free survival (B) according to PD-L1 status. PD-L1 negative (<1%; yellow line, n = 52) vs PD-L1 positive (≥1%; blue line, n = 17).

Next-generation sequencing (NGS) allows the detection of multiple genetic targets in different tumor types. This study aimed to confirm the benefits of implementing in-house NGS testing for non-small-cell lung cancer (NSCLC) samples in molecular pathology laboratories. A multi-institutional study was conducted to evaluate the analytical performance, turnaround time, and feasibility of in-house NGS testing of 50 genes from 283 NSCLC samples. The first phase was a retrospective study with inter-laboratory testing (21 samples), and the second phase was a prospective study with intralaboratory testing (262 samples). The retrospective study showed a 100% sequencing success rate for DNA and RNA, high interlaboratory concordance (95.2%), and a strong correlation (R2 Z 0.94) between observed and expected single-nucleotide variant/insertion and/or deletion variant allele fraction. The prospective study showed a sequencing success rate of 99.2% for DNA and 98% for RNA. NGS identified 285 relevant variants (81.1% single-nucleotide variants/insertion and/or deletion variants, 9.8% copy number variants, and 9.1% gene fusions). Co-mutations with potential clinical relevance were detected in 20.5% of samples positive for the main oncogenic drivers in NSCLC. Additionally, 11% of samples wild type for the main oncogenic drivers carried alterations in other relevant genes. The in-house NGS testing had a median turnaround time from sample processing to molecular report of 4 days. This study demonstrates the advantages of implementing in-house NGS testing in molecular pathology laboratories.

Objectives To analyze the impact of Kirsten-Rat-Sarcoma Virus (KRAS) mutations on tumor-growth as estimated by tumor-doubling-time (TDT) among solid-dominant clinical-stage I lung adenocarcinoma. Moreover, to evaluate the prognostic role of KRAS mutations, TDT and their combination in completely-resected pathologic-stage I adenocarcinomas. Methods In this single-center retrospective analysis, completely resected clinical-stage I adenocarcinomas presenting as solid-dominant nodules (consolidation-to-tumor ratio>0.5) in at least two preoperative computed-tomography scans were enrolled. Nodules’ growth was scored as fast (TDT<400 days) or slow (TDT>400 days). KRAS-mutated adenocarcinomas were identified with next-generation sequencing. Logistic- and Cox-regressions were used to identify predictors of fast-growth and disease-free survival (DFS), respectively. Results Among 151 patients, 83 (55%) had fast-growing nodules and 64 (42.4%) were KRAS-mutated.Fast-growing nodules outnumbered in the KRAS-mutated group (n=45; 70.3%), median TDT 95-days (interquartile range, IQR 43.5–151.5) compared to the KRAS wild-type group (38, 43.7%), median TDT 138-days (IQR 70.3–278.5). KRAS-mutations predicted faster-growth at multivariable analysis (p=0.009).In a subgroup analysis including 108 pathologic-stage I adenocarcinomas, neither KRAS-mutations (p=0.081) nor fast-growing pattern (p=0.146) affected DFS. Nevertheless, the association of KRAS-mutations and fast-growing pattern identified a subgroup of patients with worse DFS (p=0.02). The combination of fast-growing and KRAS-mutations (hazard-ratio 2.97 [95%CI 1.22–7.25]; p=0.017) and average nodule diameter at diagnosis (hazard-ratio 1.08 [95%CI 1.03-1.14]; p=0.004) were independent predictors of worse DFS. Conclusion KRAS mutations are associated to faster growth, in clinical-stage I adenocarcinoma presenting at diagnosis as solid-dominant nodules undergoing complete resection. Moreover, faster-growth identifies a subgroup of pathologic-stage I KRAS-mutated adenocarcinomas with higher recurrences. MICROABSTRACT In this study the relation between preoperative fast tumor growth, as estimated by tumor doubling time <400 days, and Kirsten Rat Sarcoma Virus (KRAS) mutations is investigated in a cohort of clinical stage I lung adenocarcinoma presenting as solid dominant nodules (consolidation-to-tumor ratio >0.5). Moreover, in the patients with pathologic stage I disease, a fast growth in the preoperative period identifies a subgroup of KRAS-mutated lung adenocarcinomas at higher risk of recurrence.

Alterations in the phosphoinositide 3-kinase (PI3K)/AKT/PTEN signaling pathway are a well-recognized mechanism of resistance in hormone receptor-positive, HER2-negative metastatic breast cancer (HR+/HER2- mBC). These alterations are present in approximately half of patients with HR+/HER2- mBC. The major alterations in the pathway are somatic mutations in the PIK3CA (40-45%) and AKT1 (5%) genes, and loss-of-function alterations in PTEN (5-10%). New targeted agents that act against these alterations have been developed. Therefore, it is important to determine the mutational status of genes in this pathway to potentially offer a therapeutic alternative for these patients. In this review, we discuss the clinical and biological significance of PI3K pathway alterations in HR+/HER2- mBC, focusing on tumors that progress following endocrine therapy and CDK4/6 inhibitor treatment. We then highlight how different diagnostic strategies, including sample type, testing methodology, and timing, can improve the identification of patients who are eligible for targeted therapies and promote the effective integration of molecular diagnostics into routine clinical care.

Supplementary Figure 9. Median overall survival (A-C) and progression-free survival (B-D) according to MAPK status in NI (A-B) and N-CT (C-D) treatment arm. MAPK1/3 WT (yellow line; n = 26/31) vs MAPK1–3 altered (blue line; n = 48/32). OS: Overall survival; PFS: progression-free survival; WT: wild-type.

Subsolid nodules emerged as frequent radiological variants of lung adenocarcinoma. Radiological features including solid-component prevalence and larger tumour dimensions prompt tumoral invasiveness guiding prognosis and management. Thus, we aimed to clarify the molecular grounds that dictate these radiological appearances and clinical behaviour in a real-life European-cohort. Additionally, following the growing interest toward targeted-therapies in early-stage diseases, we aimed to present real-life epidemiological data of actionable mutations in these patients. In this retrospective single-centre study, targeted next-generation sequencing was performed continuatively in all the resected subsolid lung adenocarcinomas in the period between May 2016 and December 2023. Clinico-radiological data were collected. The genetic landscape of our real-life European subsolid adenocarcinoma population is defined. Common and actionable mutations (frequency > 5

INTRODUCTION:The CAN.HEAL consortium, comprising 47 cancer centers and academic institutions across 17 EU countries, has developed a set of recommendations for Molecular Tumor Boards (MTBs) to address the lack of standardized guidelines in personalized cancer medicine. METHODS:Over the past 2 years, through extensive collaboration and seven dedicated online meetings, CAN.HEAL experts developed consensus-based recommendations across 10 critical domains. RESULTS:The consortium agreed that MTBs' primary role is to perform molecular and clinical assessments for patients requiring care beyond standard treatment. Core MTB composition should include medical oncologists, molecular biologists, pathologists, and bioinformaticians. Patient eligibility criteria should prioritize performance status, with flexibility for rare cases. Shared informed consent is crucial for sample collection, data use, and research. A two-tiered IT workflow, with minimal and maximal datasets, is recommended, along with a comprehensive decision support tool. These recommendations focus on genomic testing, acknowledging diversity of NGS assays and proposing general guidelines. MTB reports should be concise, with technical details provided in the molecular diagnostic report. Innovative approaches like the Drug Rediscovery Protocol support access to off-label therapies. Harmonized training for MTB members is essential to bridging knowledge gaps in this evolving field. Indicators are needed to assess MTB effectiveness over time. Expanding MTB benefits to underserved populations depends on creating a shared European MTB database. CONCLUSION:Standardizing MTB practices represents a key step toward equitable access to personalized medicine and improved cancer care across Europe. Sustainable implementation requires coordinated EU efforts, and dynamic MTBs that continuously refine genomic-driven decisions within real-world contexts.

Supplementary Figure 14. Representative cases of multiplexed immunofluorescence showing two lung squamous cell carcinoma cases with (A) and without (B) MAPK alterations.

Supplementary Figure 1. Median overall survival according to the presence of bone metastases in nivolumab plus ipilimumab (A) and nivolumab plus platinum-based chemotherapy (B) arms. Patients with bone metastases (green line) vs patients without (blue line). OS: overall survival.

Supplementary Table 6. Clinical characteristics of MAPK1/MAPK3-positive vs -negative patients in the Dana Farber Cancer Institute validation cohort.

Importance:Patients with borderline resectable or unresectable stage III non-small cell lung cancer (NSCLC) with T4 and/or N2-N3 involvement face limited treatment options and poor outcomes. Neoadjuvant chemoimmunotherapy has shown promise in improving resectability and pathological responses. Objective:To evaluate the efficacy of neoadjuvant programmed cell death 1 protein (PD-1) or programmed cell death 1 ligand 1 (PD-L1) blockade combined with chemotherapy in enhancing surgical outcomes and pathological responses in patients with T4 and/or N2-N3 stage III NSCLC. Design, Setting, and Participants:This multicenter cohort study analyzed data from patients treated between February 2018 and January 2024 with neoadjuvant PD-1/PD-L1 inhibitors plus chemotherapy at academic and tertiary care centers across the US and Italy. Pathological and survival outcomes were assessed. Patients with stage III NSCLC and T4 and/or N2-N3 involvement were included. Data were collected from February 2018 to January 2024. Exposures:Neoadjuvant PD-1/PD-L1 blockade combined with platinum-based chemotherapy. Main Outcomes and Measures:Pathological complete response (pCR), major pathological response, surgical resectability, and event-free survival (EFS). Results:Of 112 patients, 58 (51.8%) were female, and the median (range) age was 66 (41-84) years. A total of 84(75.0%) underwent surgical resection, achieving a pCR rate of 29.0% (24 of 83 with available final pathology) and a major pathological response rate of 42.2% (35 of 83). Patients with both PD-L1 expression of 50% or more and high tumor mutational burden achieved the highest pCR rate (4 of 9 [44.4%]; P = .03). Conversely, covariants in KRAS/STK11 or KRAS/KEAP1 were associated with lack of pCR. Patients with single-station or multistation N2/N3 disease exhibited comparable pathological outcomes. The median EFS for all resected patients was 52.6 months (95% CI, 27.8 to not reached), and this was significantly longer in patients with pCR (not reached vs 27.8 months [95% CI, 19.5 to not reached]; P < .001). Conclusions and Relevance:In this study, neoadjuvant PD-1/PD-L1 blockade combined with chemotherapy resulted in high pathological response rates and surgical resectability in patients with T4 and/or N2-N3 stage III NSCLC. This approach offers a viable treatment option for patients with borderline resectable or unresectable NSCLC but requires further validation through prospective studies.

Supplementary Figure 15. Median overall survival (A) and progression-free survival (B) according to TP53 mutation status. (A) TP53 (mutant; blue line; n = 41) vs TP53 (wild-type) (yellow line; n = 33). (B) TP53 (mutant; blue line; n = 41) vs TP53 (wild-type; yellow line; n = 33).

Immunotherapy has significantly transformed the landscape of non–oncogene-driven NSCLC. Multiple trials have reported that immunotherapy, either alone or in combination with chemotherapy, can induce pathologic complete responses—essentially tumor eradication—in non–oncogene-driven patients, at least in resectable stages. In contrast, tumors with specific molecular alterations, such as EGFR mutations or ALK rearrangements, are generally considered refractory to immune checkpoint inhibitors. Data on other lung cancer drivers, including HER2 mutations, are scarce and limited to retrospective series, raising the question of whether immunotherapy should be avoided in all oncogene-addicted lung cancers. Here, we present the case of a patient with advanced HER2 mutant NSCLC who achieved complete pathologic response to chemoimmunotherapy. Our observation suggests that not all driver mutations equally drive resistance to immunotherapy, and some molecular events, such as HER2 mutations, need additional investigations.