Shifting from Immunohistochemistry to Screen for ALK Rearrangements: Real-World Experience in a Large Single-Center Cohort of Patients with Non-Small-Cell Lung Cancer.

The identification of ALK fusions in advanced non-small-cell lung carcinoma (aNSCLC) is mandatory for targeted therapy. The current diagnostic approach employs an algorithm using ALK immunohistochemistry (IHC) screening, followed by confirmation through ALK FISH and/or next-generation sequencing (NGS). Challenges arise due to the infrequency of ALK fusions (3-7% of aNSCLC), the suboptimal specificity of ALK IHC and ALK FISH, and the growing molecular demands placed on small tissue samples, leading to interpretative, tissue availability, and time-related issues. This study investigates the effectiveness of RNA NGS as a reflex test for identifying ALK fusions in NSCLC, with the goal of replacing ALK IHC in the systematic screening process. The evaluation included 1246 NSCLC cases using paired techniques: ALK IHC, ALK FISH, and ALK NGS. ALK IHC identified 51 positive cases (4%), while RNA NGS detected ALK alterations in 59 cases (4.8%). Of the 59 ALK-positive cases identified via NGS, 53 (89.8%) were confirmed to be positive. This included 51 cases detected via both FISH and IHC, and 2 cases detected only via FISH, as they were completely negative according to IHC. The combined reporting time for ALK IHC and ALK FISH averaged 13 days, whereas ALK IHC and RNA NGS reports were obtained in an average of 4 days. These results emphasize the advantage of replacing systematic ALK IHC screening with RNA NGS reflex testing for a more comprehensive and accurate assessment of ALK status.

Navigating Change in Tumor Naming: Exploring the Complexities and Considerations of Shifting Toward Molecular Classifications.

Navigating change in tumor naming. Balance organ-based and molecular classifications for optimal treatment.

The evolution of lung adenocarcinoma precursors is associated with chromosomal instability and transition from innate to adaptive immune response/evasion.

Studying lung adenocarcinoma (LUAD) early carcinogenesis is challenging, primarily due to the lack of LUAD precursors specimens. We amassed multi-omics data from 213 LUAD and LUAD precursors to identify molecular features underlying LUAD precancer evolution. We observed progressively increasing mutations, chromosomal aberrations, whole genome doubling and genomic instability from precancer to invasive LUAD, indicating aggravating chromosomal instability (CIN). Telomere shortening, a crucial genomic alteration linked to CIN, emerged at precancer stage. Moreover, later-stage lesions demonstrated increasing cancer stemness and decreasing alveolar identity, suggesting epithelial de-differentiation during early LUAD carcinogenesis. The innate immune cells progressively diminished from precancer to invasive LUAD, concomitant with a gradual recruitment of adaptive immune cells (except CD8+ and gamma-delta T cells that decreased in later stages) and upregulation of numerous immune checkpoints, suggesting LUAD precancer evolution is associated with a shift from innate to adaptive immune response and immune evasion mediated by various mechanisms.

The Glucose Transporter 5 Enhances CAR-T Cell Metabolic Function and Anti-tumour Durability.

Activated T cells undergo a metabolic shift to aerobic glycolysis to support the energetic demands of proliferation, differentiation, and cytolytic function. Transmembrane glucose flux is facilitated by glucose transporters (GLUT) that play a vital role in T cell metabolic reprogramming and anti-tumour function. GLUT isoforms are regulated at the level of expression and subcellular distribution. GLUTs also display preferential selectivity for carbohydrate macronutrients including glucose, galactose, and fructose. GLUT5, which selectively transports fructose over glucose, has never been explored as a genetic engineering strategy to enhance CAR-T cells in fructose-rich tumour environments. Fructose levels are significantly elevated in the bone marrow and the plasma of acute myeloid leukaemia (AML) patients. Here, we demonstrate that the expression of wild-type GLUT5 restores T cell metabolic fitness in glucose-free, high fructose conditions. We find that fructose supports maximal glycolytic capacity and ATP replenishment rates in GLUT5-expressing T cells. Using steady state tracer technology, we show that 13C6 fructose supports glycolytic reprogramming and TCA anaplerosis in CAR-T cells undergoing log phase expansion. In cytotoxicity assays, GLUT5 rescues T cell cytolytic function in glucose-free medium. The fructose/GLUT5 metabolic axis also supports maximal migratory velocity, which provides mechanistic insight into why GLUT5-expressing CAR-Ts have superior effector function as they undergo "hit-and-run" serial killing. These findings translate to superior anti-tumour function in a xenograft model of AML. In fact, we found that GLUT5 enhances CAR-T cell anti-tumour function in vivo without any need for fructose intervention. Accordingly, we hypothesize that GLUT5 is sufficient to enhance CAR-T resilience by increasing the cells' competitiveness for glucose at physiologic metabolite levels. Our findings have immediate translational relevance by providing the first evidence that GLUT5 confers a competitive edge in a fructose-enriched milieu, and is a novel approach to overcome glucose depletion in hostile tumour microenvironments (TMEs).

Enhancing NSCLC recurrence prediction with PET/CT habitat imaging, ctDNA, and integrative radiogenomics-blood insights.

While we recognize the prognostic importance of clinicopathological measures and circulating tumor DNA (ctDNA), the independent contribution of quantitative image markers to prognosis in non-small cell lung cancer (NSCLC) remains underexplored. In our multi-institutional study of 394 NSCLC patients, we utilize pre-treatment computed tomography (CT) and 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) to establish a habitat imaging framework for assessing regional heterogeneity within individual tumors. This framework identifies three PET/CT subtypes, which maintain prognostic value after adjusting for clinicopathologic risk factors including tumor volume. Additionally, these subtypes complement ctDNA in predicting disease recurrence. Radiogenomics analysis unveil the molecular underpinnings of these imaging subtypes, highlighting downregulation in interferon alpha and gamma pathways in the high-risk subtype. In summary, our study demonstrates that these habitat imaging subtypes effectively stratify NSCLC patients based on their risk levels for disease recurrence after initial curative surgery or radiotherapy, providing valuable insights for personalized treatment approaches.

Comprehensive analysis of transcription factor-based molecular subtypes and their correlation to clinical outcomes in small-cell lung cancer.

BACKGROUND: Recent studies have reported the predictive and prognostic value of novel transcriptional factor-based molecular subtypes in small-cell lung cancer (SCLC). We conducted an in-depth analysis pairing multi-omics data with immunohistochemistry (IHC) to elucidate the underlying characteristics associated with differences in clinical outcomes between subtypes. METHODS: IHC (n = 252), target exome sequencing (n = 422), and whole transcriptome sequencing (WTS, n = 189) data generated from 427 patients (86.4% males, 13.6% females) with SCLC were comprehensively analysed. The differences in the mutation profile, gene expression profile, and inflammed signatures were analysed according to the IHC-based molecular subtype. FINDINGS: IHC-based molecular subtyping, comprised of 90 limited-disease (35.7%) and 162 extensive-disease (64.3%), revealed a high incidence of ASCL1 subtype (IHC-A, 56.3%) followed by ASCL1/NEUROD1 co-expressed (IHC-AN, 17.9%), NEUROD1 (IHC-N, 12.3%), POU2F3 (IHC-P, 9.1%), triple-negative (IHC-TN, 4.4%) subtypes. IHC-based subtype showing high concordance with WTS-based subtyping and non-negative matrix factorization (NMF) clusterization method. IHC-AN subtype resembled IHC-A (rather than IHC-N) in terms of both gene expression profiles and clinical outcomes. Favourable median overall survival was observed in IHC-A (15.2 months) compared to IHC-N (8.0 months, adjusted HR 2.3, 95% CI 1.4-3.9, p = 0.002) and IHC-P (8.3 months, adjusted HR 1.7, 95% CI 0.9-3.2, p = 0.076). Inflamed tumours made up 25% of cases (including 53% of IHC-P, 26% of IHC-A, 17% of IHC-AN, but only 11% of IHC-N). Consistent with recent findings, inflamed tumours were more likely to benefit from first-line immunotherapy treatment than non-inflamed phenotype (p = 0.002). INTERPRETATION: This study provides fundamental data, including the incidence and basic demographics of molecular subtypes of SCLC using both IHC and WTS from a comparably large, real-world Asian/non-Western patient cohort, showing high concordance with the previous NMF-based SCLC model. In addition, we revealed underlying biological pathway activities, immunogenicity, and treatment outcomes based on molecular subtype, possibly related to the difference in clinical outcomes, including immunotherapy response. FUNDING: This work was supported by AstraZeneca, Future Medicine 2030 Project of the Samsung Medical Center [grant number SMX1240011], the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) [grant number 2020R1C1C1010626] and the 7th AstraZeneca-KHIDI (Korea Health Industry Development Institute) oncology research program.

Small cells - big issues: biological implications and preclinical advancements in small cell lung cancer.

Current treatment guidelines refer to small cell lung cancer (SCLC), one of the deadliest human malignancies, as a homogeneous disease. Accordingly, SCLC therapy comprises chemoradiation with or without immunotherapy. Meanwhile, recent studies have made significant advances in subclassifying SCLC based on the elevated expression of the transcription factors ASCL1, NEUROD1, and POU2F3, as well as on certain inflammatory characteristics. The role of the transcription regulator YAP1 in defining a unique SCLC subset remains to be established. Although preclinical analyses have described numerous subtype-specific characteristics and vulnerabilities, the so far non-existing clinical subtype distinction may be a contributor to negative clinical trial outcomes. This comprehensive review aims to provide a framework for the development of novel personalized therapeutic approaches by compiling the most recent discoveries achieved by preclinical SCLC research. We highlight the challenges faced due to limited access to patient material as well as the advances accomplished by implementing state-of-the-art models and methodologies.

Brief Report: Comprehensive Clinicogenomic Profiling of Small Cell Transformation From EGFR-Mutant NSCLC Informs Potential Therapeutic Targets.

Introduction: NSCLC transformation to SCLC has been best characterized with EGFR-mutant NSCLC, with emerging case reports seen in ALK, RET, and KRAS-altered NSCLC. Previous reports revealed transformed SCLC from EGFR-mutant NSCLC portends very poor prognosis and lack effective treatment. Genomic analyses revealed TP53 and RB1 loss of function increase the risk of SCLC transformation. Little has been reported on the detailed clinicogenomic characteristics and potential therapeutic targets for this patient population. Methods: In this study, we conducted a single-center retrospective analysis of clinical and genomic characteristics of patients with EGFR-mutant NSCLC transformed to SCLC. Demographic data, treatment course, and clinical molecular testing reports were extracted from electronic medical records. Kaplan-Meier analyses were used to estimate survival outcomes. Next generation sequencing-based assays was used to identify EGFR and co-occurring genetic alterations in tissue or plasma before and after SCLC transformation. Single-cell RNA sequencing (scRNA-seq) was performed on a patient-derived-xenograft model generated from a patient with EGFR-NSCLC transformed SCLC tumor. Results: A total of 34 patients were identified in our study. Median age at initial diagnosis was 58, and median time to SCLC transformation was 24.2 months. 68% were female and 82% were never smokers. 79% of patients were diagnosed as stage IV disease, and over half had brain metastases at baseline. Median overall survival of the entire cohort was 38.3 months from initial diagnoses and 12.4 months from time of SCLC transformation. Most patients harbored EGFR exon19 deletions as opposed to exon21 L858R alteration. Continuing EGFR tyrosine kinase inhibitor post-transformation did not improve overall survival compared with those patients where tyrosine kinase inhibitor was stopped in our cohort. In the 20 paired pretransformed and post-transformed patient samples, statistically significant enrichment was seen with PIK3CA alterations (p = 0.04) post-transformation. Profiling of longitudinal liquid biopsy samples suggest emergence of SCLC genetic alterations before biopsy-proven SCLC, as shown by increasing variant allele frequency of TP53, RB1, PIK3CA alterations. ScRNA-seq revealed potential therapeutic targets including DLL3, CD276 (B7-H3) and PTK7 were widely expressed in transformed SCLC. Conclusions: SCLC transformation is a potential treatment resistance mechanism in driver-mutant NSCLC. In our cohort of 34 EGFR-mutant NSCLC, poor prognosis was observed after SCLC transformation. Clinicogenomic analyses of paired and longitudinal samples identified genomic alterations emerging post-transformation and scRNA-seq reveal potential therapeutic targets in this population. Further studies are needed to rigorously validate biomarkers and therapeutic targets for this patient population.

Tumor- and circulating-free DNA methylation identifies clinically relevant small cell lung cancer subtypes.

Small cell lung cancer (SCLC) is an aggressive malignancy composed of distinct transcriptional subtypes, but implementing subtyping in the clinic has remained challenging, particularly due to limited tissue availability. Given the known epigenetic regulation of critical SCLC transcriptional programs, we hypothesized that subtype-specific patterns of DNA methylation could be detected in tumor or blood from SCLC patients. Using genomic-wide reduced-representation bisulfite sequencing (RRBS) in two cohorts totaling 179 SCLC patients and using machine learning approaches, we report a highly accurate DNA methylation-based classifier (SCLC-DMC) that can distinguish SCLC subtypes. We further adjust the classifier for circulating-free DNA (cfDNA) to subtype SCLC from plasma. Using the cfDNA classifier (cfDMC), we demonstrate that SCLC phenotypes can evolve during disease progression, highlighting the need for longitudinal tracking of SCLC during clinical treatment. These data establish that tumor and cfDNA methylation can be used to identify SCLC subtypes and might guide precision SCLC therapy.

HER4 and EGFR Activate Cell Signaling in NRG1 Fusion-Driven Cancers: Implications for HER2-HER3-specific Versus Pan-HER Targeting Strategies.

INTRODUCTION: NRG1 gene fusions are clinically actionable alterations identified in NSCLC and other tumors. Previous studies have reported that NRG1 fusions signal through HER2 and HER3 but, thus far, strategies targeting HER3 specifically or HER2-HER3 signaling have exhibited modest activity in patients with NSCLC bearing NRG1 fusions. Although NRG1 fusion proteins can bind HER4 in addition to HER3, the contribution of HER4 and other HER family members in NRG1 fusion-positive cancers is not fully understood. METHODS: We investigated the role of HER4 and EGFR-HER3 signaling in NRG1 fusion-positive cancers using Ba/F3 models engineered to express various HER family members in combination with NRG1 fusions and in vitro and in vivo models of NRG1 fusion-positive cancer. RESULTS: We determined that NRG1 fusions can stimulate downstream signaling and tumor cell growth through HER4, independent of other HER family members. Moreover, EGFR-HER3 signaling is also activated in cells expressing NRG1 fusions, and inhibition of these receptors is also necessary to effectively inhibit tumor cell growth. We observed that cetuximab, an anti-EGFR antibody, in combination with anti-HER2 antibodies, trastuzumab and pertuzumab, yielded a synergistic effect. Furthermore, pan-HER tyrosine kinase inhibitors were more effective than tyrosine kinase inhibitors with greater specificity for EGFR, EGFR-HER2, or HER2-HER4, although the relative degree of dependence on EGFR or HER4 signaling varied between different NRG1 fusion-positive cancers. CONCLUSIONS: Our findings indicate that pan-HER inhibition including HER4 and EGFR blockade is more effective than selectively targeting HER3 or HER2-HER3 in NRG1 fusion-positive cancers.

c-Met immunohistochemistry as reflex test at diagnosis for non-small cell lung cancer: a real-world experience from a monocentric case series.

AIMS: Recent clinical trials have shown promising results with drugs targeting the hepatocyte growth factor receptor (c-Met) for advanced non-small cell lung cancers overexpressing c-Met. We assessed reflex testing of c-Met immunohistochemistry (IHC) at diagnosis for NSCLC in the real-world. METHODS: We retrospectively collected clinical, pathological and molecular data of cases diagnosed with NSCLC in our institution from January 2021 to June 2023. We performed c-Met IHC (SP44 clone) and scored the expression using a H-score and a three-tier classification. RESULTS: 391 cases with interpretable c-Met IHC staining were included. The median age at diagnosis was 70 years (range 25-89 years) including 234 males (male/female ratio 1:5). 58% of the samples came from surgical resections, 35% from biopsies and 8% from cytological procedures. 52% of cases were classified as c-Met-positive (H-score≥150) and 19% were classified as c-Methigh (≥50%, 3+). 43% of the c-Metneg presented with lymph node and/or visceral metastases at diagnosis vs 55% for c-Methigh (p=0.042). 23% of the adenocarcinomas showed c-Methigh expression vs 3% for squamous cell carcinomas (p=0.004). 27% of the c-Metneg cases had a high PD-L1 expression vs 58% of c-Methigh cases (p<0.001). MET ex14 skipping was present in 8% of the c-Methigh cases. CONCLUSIONS: Systematic c-Met testing in daily routine for NSCLC patients is feasible, highlighting a potential correlation with clinicopathological and molecular features.

Unfolding the secrets of small cell lung cancer progression: Novel approaches and insights through rapid autopsies.

The understanding of small cell lung cancer (SCLC) biology has increased dramatically in recent years, but the processes that allow SCLC to progress rapidly remain poorly understood. Here, we advocate the integration of rapid autopsies and preclinical models into SCLC research as a comprehensive strategy with the potential to revolutionize current treatment paradigms.

Clinical insights into small cell lung cancer: Tumor heterogeneity, diagnosis, therapy, and future directions.

Small cell lung cancer (SCLC) is characterized by rapid growth and high metastatic capacity. It has strong epidemiologic and biologic links to tobacco carcinogens. Although the majority of SCLCs exhibit neuroendocrine features, an important subset of tumors lacks these properties. Genomic profiling of SCLC reveals genetic instability, almost universal inactivation of the tumor suppressor genes TP53 and RB1, and a high mutation burden. Because of early metastasis, only a small fraction of patients are amenable to curative-intent lung resection, and these individuals require adjuvant platinum-etoposide chemotherapy. Therefore, the vast majority of patients are currently being treated with chemoradiation with or without immunotherapy. In patients with disease confined to the chest, standard therapy includes thoracic radiotherapy and concurrent platinum-etoposide chemotherapy. Patients with metastatic (extensive-stage) disease are treated with a combination of platinum-etoposide chemotherapy plus immunotherapy with an anti-programmed death-ligand 1 monoclonal antibody. Although SCLC is initially very responsive to platinum-based chemotherapy, these responses are transient because of the development of drug resistance. In recent years, the authors have witnessed an accelerating pace of biologic insights into the disease, leading to the redefinition of the SCLC classification scheme. This emerging knowledge of SCLC molecular subtypes has the potential to define unique therapeutic vulnerabilities. Synthesizing these new discoveries with the current knowledge of SCLC biology and clinical management may lead to unprecedented advances in SCLC patient care. Here, the authors present an overview of multimodal clinical approaches in SCLC, with a special focus on illuminating how recent advancements in SCLC research could accelerate clinical development.

Lack of correlation between MET and PD-L1 expression in non-small cell lung cancer revealed by comparative study of matched biopsies and surgical resection samples.

INTRODUCTION: Both MET expression and the PD-L1 tumor proportion score (TPS) are companion diagnostics for treatment of advanced non-small cell lung carcinoma (aNSCLC) patients. We evaluated the rate of correlation between MET expression and the PD-L1 TPS in matched biopsies and surgically resected specimens from NSCLC patients. PATIENTS AND METHODS: This retrospective analysis assessed the prevalence and correlation between MET expression (SP44 clone) and the PD-L1 TPS (22C3 clone) by immunohistochemistry together with molecular alterations determined by targeted next-generation sequencing in matched lung biopsy and surgically lung resected specimens from 70 patients with NSCLC. RESULTS: The study found a significant correlation between the MET H-score in surgical samples and matched biopsies (P-value < 0.0001), as well as between the PD-L1 TPS in paired biopsies and surgical samples (P-value < 0.0001). However, there was no significant correlation between the MET H-score or expression subgroups and the PD-L1 TPS in both types of paired samples (P-value = 0.47, and P-value = 0.90). The MET H-score was significantly higher in adenocarcinoma compared to squamous cell carcinoma (P-value < 0.0001). A mutational analysis showed that the MET H-score was significantly higher in NSCLC cases with targetable molecular alterations (P-value = 0.0095), while no significant correlation was found for the PD-L1 TPS. CONCLUSIONS: Our study found no significant correlation between PD-L1 and MET expression in samples from NSCLC patients, highlighting the importance of personalized treatment strategies based on individual expression profiles. These findings provide valuable insight into the development of effective immunotherapy and targeted therapy for NSCLC patients.

CD70 is a therapeutic target upregulated in EMT-associated EGFR tyrosine kinase inhibitor resistance.

Effective therapeutic strategies are needed for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations that acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) mediated by epithelial-to-mesenchymal transition (EMT). We investigate cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches and identify CD70 as being highly upregulated in EMT-associated resistance. Moreover, CD70 upregulation is an early event in the evolution of resistance and occurs in drug-tolerant persister cells (DTPCs). CD70 promotes cell survival and invasiveness, and stimulation of CD70 triggers signal transduction pathways known to be re-activated with acquired TKI resistance. Anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting chimeric antigen receptor (CAR) T cell and CAR NK cells show potent activity against EGFR TKI-resistant cells and DTPCs. These results identify CD70 as a therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits clinical investigation.

Ultrafast Gene Fusion Assessment for Nonsquamous NSCLC.

Introduction: Gene fusion testing of ALK, ROS1, RET, NTRK, and MET exon 14 skipping mutations is guideline recommended in nonsquamous NSCLC (NS-NSCLC). Nevertheless, assessment is often hindered by the limited availability of tissue and prolonged next-generation sequencing (NGS) testing, which can protract the initiation of a targeted therapy. Therefore, the development of faster gene fusion assessment is critical for optimal clinical decision-making. Here, we compared two ultrafast gene fusion assays (UFGFAs) using NGS (Genexus, Oncomine Precision Assay, Thermo Fisher Scientific) and a multiplex reverse-transcriptase polymerase chain reaction (Idylla, GeneFusion Assay, Biocartis) approach at diagnosis in a retrospective series of 195 NS-NSCLC cases and five extrapulmonary tumors with a known NTRK fusion. Methods: A total of 195 NS-NSCLC cases (113 known gene fusions and 82 wild-type tumors) were included retrospectively. To validate the detection of a NTRK fusion, we added five NTRK-positive extrathoracic tumors. The diagnostic performance of the two UFGFAs and standard procedures was compared. Results: The accuracy was 92.3% and 93.1% for Idylla and Genexus, respectively. Both systems improved the sensitivity for detection by including a 5'-3' imbalance analysis. Although detection of ROS1, MET exon 14 skipping, and RET was excellent with both systems, ALK fusion detection was reduced with sensitivities of 87% and 88%, respectively. Idylla had a limited sensitivity of 67% for NTRK fusions, in which only an imbalance assessment was used. Conclusions: UFGFA using NGS and reverse-transcriptase polymerase chain reaction approaches had an equal level of detection of gene fusion but with some technique-specific limitations. Nevertheless, UFGFA detection in routine clinical care is feasible with both systems allowing faster initiation of therapy and a broad degree of screening.

Deciphering the Impact of HER2 Alterations on Non-Small-Cell Lung Cancer: From Biological Mechanisms to Therapeutic Approaches.

Despite the recent increase in the number of types of treatments, non-small-cell lung cancer (NSCLC) remains the major cause of death from cancer worldwide. So, there is an urgent need to develop new therapeutic strategies. The HER2 gene codes for tyrosine kinase receptor whose alterations are known to drive carcinogenesis. HER2 alterations, including amplification, mutations, and overexpression, have been mainly described in breast and gastric cancers, but up to 4% of NSCLC harbor actionable HER2 mutations. HER2-targeted therapy for NSCLC with trastuzumab, pertuzumab, and trastuzumab emtansine has failed to demonstrate an improvement in survival. Nevertheless, recent data from phase II trials have shed light on promising specific therapies for HER2-mutant NSCLC such as trastuzumab deruxtecan. Herein, we aimed to provide an updated review on the biology, epidemiology, molecular testing, and therapeutic strategies for NSCLC with HER2 molecular alterations.

Molecular Profiling in Non-Squamous Non-Small Cell Lung Carcinoma: Towards a Switch to Next-Generation Sequencing Reflex Testing.

Molecular diagnosis of lung cancer is a constantly evolving field thanks to major advances in precision oncology. The wide range of actionable molecular alterations in non-squamous non-small cell lung carcinoma (NS-NSCLC) and the multiplicity of mechanisms of resistance to treatment resulted in the need for repeated testing to establish an accurate molecular diagnosis, as well as to track disease evolution over time. While assessing the increasing complexity of the molecular composition of tumors at baseline, as well as over time, has become increasingly challenging, the emergence and implementation of next-generation sequencing (NGS) testing has extensively facilitated molecular profiling in NS-NSCLC. In this review, we discuss recent developments in the molecular profiling of NS-NSCLC and how NGS addresses current needs, as well as how it can be implemented to address future challenges in the management of NS-NSCLC.

Limited Benefit from the Addition of Immunotherapy to Chemotherapy in TKI-Refractory EGFR-Mutant Lung Adenocarcinoma.

BACKGROUND: The benefit of chemotherapy combined with immunotherapy in EGFR-mutant lung adenocarcinoma (LUAD) patients whose tumor developed resistance to EGFR tyrosine kinase inhibitors (TKIs) is not thoroughly investigated. The goal of this retrospective cohort study is to assess the clinical efficiency of immunotherapy alone or in combination with chemotherapy in a real-world setting. METHODS: This retrospective cohort study enrolled LUAD patients with EGFR sensitive mutations whose tumor had acquired resistance to EGFR TKIs and received systemic treatment with chemotherapy (chemo; n = 84), chemotherapy combined with immunotherapy (chemoIO; n = 30), chemotherapy plus bevacizumab with or without IO (withBev; n = 42), and IO monotherapy (IO-mono; n = 22). Clinical progression-free survival (PFS) and overall survival (OS) were evaluated. Associations of clinical characteristics with outcomes were assessed using univariable and multi-covariate Cox Proportional Hazards regression models. RESULTS: A total of 178 patients (median age = 63.3; 57.9% females) with a median follow-up time of 42.0 (Interquartile range: 22.9-67.8) months were enrolled. There was no significant difference in PFS between chemoIO vs. chemo groups (5.3 vs. 4.8 months, p = 0.8). Compared to the chemo group, patients who received withBev therapy trended towards better PFS (6.1 months vs. 4.8; p = 0.3; HR 0.79; 95% CI: 0.52-1.20), while patients treated with IO-mono had inferior PFS (2.2 months; p = 0.001; HR 2.22; 95% CI: 1.37-3.59). Furthermore, PD-L1 level was not associated with PFS benefit in the chemoIO group. Patients with EGFR-mutant LUAD with high PD-L1 (≥50%) had shorter PFS (5.8 months) than non-EGFR/ALK LUAD patients who received chemoIO (12.8 months, p = 0.002; HR 0.22; 95% CI: 0.08-0.56) as first-line treatment. Chemotherapy-based therapy rendered similar benefit to patients with either EGFR exon19 deletion vs. L858R in the LUAD. CONCLUSIONS: This retrospective analysis revealed that immunotherapy provided limited additional benefit to chemotherapy in TKI-refractory EGFR-mutant LUAD. Chemotherapy alone or combined with bevacizumab remain good choices for patients with actionable EGFR mutations.