Background: Lung cancer is a highly heterogeneous disease in which molecular characterization has become essential for guiding personalized therapies. The implementation of next-generation sequencing (NGS) allows for the simultaneous detection of multiple genomic alterations, improving tumor profiling and therapeutic decision-making. This study aimed to characterize the molecular landscape of lung cancer using NGS and to evaluate its association with histological subtypes and programmed death-ligand 1 (PD-L1) expression. Methods: A retrospective observational study was conducted on 96 patients diagnosed with lung cancer between 2023 and 2025. Molecular profiling was performed using the Action OncoKitDx panel. Associations between genetic alterations, histological subtypes, and PD-L1 expression were analyzed using Fisher's exact test, with p < 0.05 considered statistically significant. Results: Adenocarcinoma was the most common histological subtype (67.7%), followed by squamous cell carcinoma (26%). The most common mutations were KRAS (34.4%), TP53 (29.2%), and EGFR (11.5%). KRAS mutations were significantly associated with adenocarcinoma (p = 0.001), while squamous cell carcinoma showed a higher frequency of cases without molecular alterations detected by the NGS panel (p = 0.002). Co-mutations were identified in 22.9% of cases, with KRAS-TP53 being the most common combination. Tumors harboring EGFR mutations showed a significantly lower frequency of co-mutations (p = 0.012). No significant associations were found between PD-L1 expression and either histological subtypes or the analyzed genetic alterations. Conclusions: Lung cancer exhibits marked molecular heterogeneity, with a predominance of KRAS mutations in adenocarcinoma. The low frequency of co-mutations in EGFR-mutated tumors supports their role as dominant driver alterations. The lack of association between PD-L1 expression and genomic alterations highlights the complexity of its regulation and suggests the involvement of multiple biological factors. These findings reinforce the clinical value of NGS in comprehensive tumor profiling and in the development of precision medicine strategies.

Lung cancer is one of the most commonly diagnosed cancers worldwide. It is the leading cause of cancer-related deaths in both men and women. In 2020, there were an estimated 2.2 million new cases of lung cancer and 1.8 million deaths due to the disease. Historically, lung cancer has been more common in men, but the gap has been closing. Smoking tobacco is the leading cause of lung cancer. Survival rates for lung cancer vary greatly depending on the stage at diagnosis and other factors. Overall, the prognosis for lung cancer is often poor, with a relatively low five-year survival rate compared to some other cancers. In this work we aim to show new paths in the diagnosis of lung cancer, through the study of several mutations and proteins, mostly detected by Next-generation sequencing (NGS) which has significantly transformed our understanding of cancer, by providing high-throughput and cost-effective methods for analyzing genomic information. In the context of lung cancer, NGS has played a crucial role in advancing our knowledge of the disease, improving diagnosis and treatment, and guiding personalized medicine approaches. key points highlighting the importance of next-generation sequencing in lung cancer: Comprehensive Genomic Profiling Identification of Driver Mutations Stratification of Patients Predicting Treatment Response Monitoring Disease Progression Clinical Trials and Drug Development Early Detection and Prognosis A large meta-analysis has been done, as well as a detailed study of 86 patients diagnosed with lung cancer in the ANALIZA laboratory. In this sense the most frequently implicated mutations in this tumor have been analyzed, ALK, ROS1 and EGFR, the positions they occupy in the genes, in addition to the programmed death ligand 1 (PD-L1), an immune control protein, which is expressed in activated immune cells and in tumor cells, and how its identification allows us to direct treatment in a more optimal way. In summary, next-generation sequencing has revolutionized the field of lung cancer research and clinical practice. By providing detailed insights into the genomic landscape of tumors, NGS facilitates personalized treatment approaches, early detection, and ongoing monitoring, ultimately leading to improved patient outcomes.

Introduction: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. Methods: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). Results: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2thorn or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). Conclusions: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm. (C) 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

It has been proposed that a combination of assays may refine the prediction of response to checkpoint inhibitors, with tumour mutation burden (TMB) being lately the strongest biomarker associated with efficacy. Although some targeted next generation sequencing (NGS) assays provide a good estimate of whole exome sequencing TMB, they are only available throughout referral laboratories. We sought to investigate the possibility of profiling TMB in-house with a commercially available targeted NGS assay, and to correlate the results with survival, the status of TP53, PD-L1 overexpression and tumor-infiltrating lymphocytes (TILs). The study included samples from 40 patients diagnosed with early-stage lung squamous cell carcinoma. PD-L1 immunohistochemistry (IHC) was performed with two clones (SP263 and SP142, Ventana Medical Systems). CD8+ TILs were scored with a digital algorithm. The status of TP53 (exons 4–10) was investigated with direct sequencing. Ion TorrentTM OncomineTM Tumor Mutation Load Assay (ThermoFisher Scientific), a targeted NGS assay which covers 1.7Mb across 409 cancer driver genes, was used to assess TMB. NGS was performed on genomic DNA from FFPE tumor samples using the Ion S5TM system. The results were analyzed with the Ion ReporterTM software. According to the manufacturer’s instructions, TMB was calculated based on the number of non-synonymous somatic mutations, after removing polymorphisms and known or predicted driver mutations from all the variants. We investigated the correlation between TMB and PD-L1 expression, lymphocyte density, TP53 status and survival. TMB data was available for all 40 patients. The mean and median number of mutations was 13 and 11, respectively. CD8+/mm2 TILs within the peritumoral stromal compartment ranged from 312 to 4793 and within the intraepithelial compartment ranged from 17 to 2002. Sixty percent and 32% of cases were positive in tumor cells with SP263 or SP142, respectively (1% cut-off). Immune cells PD-L1 expression (1% cut-off) was observed in 92% of samples with both clones. No correlation was found between PD-L1 expression or lymphocyte density and TMB. Tumors with TP53 mutations showed non-significant higher numbers of mutations than those wild-type (p=0.075). Accordingly, there was a similar trend for overall survival using the median number of mutations as a cut-off (p=0.137, HR, 5.29). It is feasible to use targeted NGS to estimate TMB. In our pilot study, there was no obvious correlation with other immunooncology predictive biomarkers. Funding: AES 2017/Feder. ISCIII: PI17/01001

BACKGROUNDHER2-positive gastric cancer (GC) affects 7%-34% of patients with GC. Trastuzumab-based first-line treatment has become the standard of care for HER2-positive advanced gastric cancer (AGC). However, there are no clinically validated biomarkers for resistance to HER2-targeted therapies. Upregulation of PI3K pathway and tyrosine kinase receptor (TKR) alterations have been noted as molecular mechanisms of resistance in breast cancer. Our study aimed to perform a molecular characterization of HER2-positive AGC and investigate the role of PI3K/Akt/mTOR signaling pathway activation and TKR gene copy number (GCN) gains as predictive biomarkers in HER2-positive AGC treated with trastuzumab.PATIENTS AND METHODSForty-two HER2-positive GC samples from patients treated with trastuzumab-based first-line chemotherapy were selected. DNA samples were sequenced. PTEN and MET immunohistochemistry were also performed.RESULTSConcurrent genetic alterations were detected in 97.1% of HER2-positive AGC. We found activation of PI3K/Akt/mTOR pathway in 52.4% of patients and TKR GCN gains in 38.1%. TKR GCN gains did not correlate with overall survival (OS) or progression-free survival (PFS). Multivariate Cox models showed that PI3K/Akt/mTOR activation negatively affects the effectiveness of trastuzumab-based chemotherapy in terms of OS and PFS.CONCLUSIONOur results provide for the first time a detailed molecular profile of concurrent genetic alterations in HER2-positive AGC. PI3K pathway activation could be used as a predictive marker of worse outcome in this patient population. In addition, gains in copy number of other TKR genes in this subgroup may also influence the survival benefit obtained with trastuzumab.IMPLICATIONS FOR PRACTICEThis article reports, for the first time, a detailed molecular profile of genomic alterations in patients with HER2-positive advanced gastric cancer (AGC). PI3K/Akt/mTOR signaling pathway activation seems to have a differentially negative effect on overall survival and progression-free survival in AGC treated with trastuzumab-based chemotherapy. Combining different targeted agents could be a successful therapeutic strategy to improve the prognosis of HER2-positive AGC.

Although the use of NGS (next-generation sequencing) is indeed feasible for the study of druggable rearrangements, the sensitivity and specificity of targeted NGS has been challenged on several grounds: use of fixed tissue, poor quality/insufficient sample, RNA-contamination risk or long turn-around time. Our relatively high rate of failures (7.6%) with the RNA workflow of targeted NGS (data not shown), prompted us to investigate possible alternatives. The objective of this study is to demonstrate an efficient solution based on multiplexed fluorescence in situ hybridization (FISH) for the comprehensive characterization of fusions (ALK, ROS1, RET and NTRK1) in lung adenocarcinomas (ACs). We have implemented for the first time in the literature a novel four-color ALK/ROS1 probe and used an automated scanning system for scoring these four translocations. A total of 64 patients with early stage lung ACs who underwent surgery at HM Sanchinarro University Hospital were considered. All slides were carefully reviewed to identify the different histological patterns. Afterwards we performed FISH to study ALK, ROS1, RET and NTRK1 in each pattern. We used both commercially (RET and NTRK1) and non-commercially (ALK/ROS1 dual) available Vysis break-apart probes (Abbott Molecular, USA). Slides were captured and scored with the BioView (Rehovot, Israel) automated imaging and analysis system, using dedicated applications for each probe. Positive cases were all confirmed with targeted NGS (Oncomine Focus AssayTM, Life Technologies, USA). Seven out of 420 slides did not hybridize (1.6%). We found two ALK (2.8%) and two ROS1 (2.8%) positive tumors, while no translocations were identified in RET or NTRK1. The rearrangements were present in all the different histological patterns within a given tumor, with a similar proportion of positive cells. The two major patterns of positivity were represented. The mean percentage of positive cells was 65% (range 46 to 84%). The NGS results confirmed the FISH findings. Simultaneous FISH testing for ALK and ROS1 is feasible on a single slide. The strategy reported herein provided reduced overall scoring time and ensured sensitive counting. This model might be easier to reconcile (lower cost, shorter turn-around time, and less failure rate) with subsequent comprehensive genotyping. Therefore, it could be used prospectively in advanced lung ACs to align the use of several technologies.

AIMS:To study programmed death ligand 1 (PD-L1) expression, tumour-infiltrating T lymphocytes (TILs) and the molecular context in patients with early-stage squamous cell lung carcinomas (SCCs). METHODS AND RESULTS:The study included samples from 40 patients (discovery cohort) and 29 patients (validation cohort) diagnosed with early-stage SCC. PD-L1 immunohistochemistry (IHC) was performed with three commercially available clones (E1L3N, SP263 and SP142). CD8+ TILs were scored with a digital algorithm. All tumours were analysed with targeted next-generation sequencing (NGS). Additionally, TP53 mutations were investigated with direct sequencing. In both cohorts, we observed a significant association between CD8+ TILs density and high PD-L1 IHC expression in tumour cells (TCs). Furthermore, high SP142 PD-L1 expression in immune cells (ICs) was also associated significantly with CD8+ TILs density. Therefore, CD8+ TILs density discriminated between patients with high versus low PD-L1 IHC expression with excellent sensitivity and specificity. Interestingly, the highest percentages of PD-L1-positive TCs with the three antibodies were found in samples with cyclin-dependent kinase 6 (CDK6) amplification, with high amplification of proto-oncogene C-Myc (CMYC) or with cyclin D1-PI3 kinase subunit alpha (CCND1-PIK3CA) co-amplification. High SP142 PD-L1 IHC expression in ICs showed a non-significant correlation with TP53 mutations. Conversely, most cases with fibroblast growth factor receptor 1 (FGFR1) amplification were negative for all PD-L1 clones. CONCLUSIONS:Our preliminary results support the use of digital CD8+ TILs scoring and targeted NGS alongside PD-L1 expression. The approach presented herein could help define patients with SCCs candidates to immune checkpoints inhibitors.

e15567 Background: Use of Trastuzumab accrues significant clinical benefit in the HER2 positive metastatic Gastric Cancer (GC) population. However, many patients do not respond to therapy or develop refractory disease. The identification of potential mechanisms of Trastuzumab resistance may provide a rationale for the development of more effective targeted therapies. Upregulation of the PI3K pathway has been noted as a molecular mechanism of resistance in breast cancer. Our aim was to determine if PI3K activated pathway determined by PTEN loss and/or PI3KCA mutation could be used as a negative predictive factor in a HER2amplified metastatic GC cohort treated with Trastuzumab. Methods: Forty-two formalin-fixed paraffin embedded HER2 amplified GC samples from patients (pts) treated with Trastuzumab-based 1stline chemotherapy were selected. PTEN expression was analyzed with immunohistochemistry and DNA samples were sequenced with the Ion AmpliSeq Cancer Hotspot Panel v2, using the Ion Torrent Personal Genome Machine (PGM) sequencing platform (Life Technologies, Carlsbad, CA, USA). Results: We found PI3K pathway activation in 40.5% (n = 17) of pts: PTEN loss was found in 33.3% (n = 14) and PI3KCA mutation in 11.9% (n = 5). The Kaplan-Meier survival curves displayed a statistically significant difference in overall survival (OS) according to the PI3K pathway status: median OS was 19.5 months in pts without PI3K activation and 14.9 months in pts with PI3K activation (log-rank test, p = 0.042). Cox regression analysis estimated that the PI3K upregulated group had a higher likelihood of death (hazard ratio 2.06, CI 95% 1.01 to 4.20, p = 0.046). After adjustment for RAS and other tyrosine kinase receptors (TKR) alterations, the PI3K status remains as a negative predictive factor only in the non-activated RAS/TKR group (median OS 14.3 vs 19.5 months in patients without PI3K activation, log-rank test, p = 0.047). Conclusions: PI3K pathway activation could be used as a predictive marker for worse outcome in patients with HER2 amplified metastatic gastric cancer treated with trastuzumab. In addition, co-activation of RAS and other TKR in this subgroup may influence the survival benefit of Trastuzumab.

67 Background: HER2 amplified cases are the only subset of gastric carcinoma (GC) patients with an approved targeted therapy (≈20%). In GC it is still unknown if there is a mutually exclusive pattern of mutations in major driver oncogenes. We performed a systematic search for targetable oncogenes in a cohort of HER2 amplified GC patients. Methods: 53 Formalin-fixed paraffin embedded samples from HER2 amplified GC patients (43 tumor and 10 normal samples) were selected for next generation sequencing (NGS). Before DNA extraction a macrodissection procedure was performed to guarantee at least 30% tumor in all cases. DNA samples were sequenced using the Ion Torrent Personal Genome Machine (PGM) sequencing platform (Life Technologies, Carlsbad, CA, USA). The Ion AmpliSeq Cancer Hotspot Panel v2 was used. This panel encompasses more than 2800 mutational hotspots of 50 oncogenes and tumor suppressor genes. Data were processed using the Ion Torrent platform-specific pipeline software Torrent suite v4.2. Moreover, sequencing data were analyzed with Ion Reporter software 4.2 to detect any copy number alteration of the genes included in the panel. Results: We successfully sequenced all samples. We identified 89 mutations in 12 genes (range from 1 ~ 9). The most frequent significant mutations included TP53 mutations (30), PI3KCA (3), SMAD4 (3), CDKN2A (4), CTNNB1 (3) and MET (3). Other mutations were found in KRAS, NOTCH, APC, and VHL genes. We also detected potential amplifications in the KRAS (4), EGFR (9), PI3KCA (11), AKT (6), FGFR (6), CDKN2A (4) and CDH1(8) genes. Among 43 tumor specimens, 86% of specimens harbored at least one genetic alteration, most of them linked to actionable mutations or amplifications Conclusions: Within HER2 amplified GCs, there are additional subsets with a potentially targetable oncogene. Future testing for these targets will benefit from including HER2 amplified GC patients Supported by the Spanish Ministry of Health, Fondo de Investigaciones Sanitarias grant PI11/01005 and European FEDER (PN I+D+I 2008‐20011).

The cobas® (Roche) portfolio of companion diagnostics in oncology currently has three assays CE-marked for in vitro diagnostics. Two of these (EGFR and BRAF) are also US FDA-approved. These assays detect clinically relevant mutations that are correlated with response (BRAF, EGFR) or lack of response (KRAS) to targeted therapies such as selective mutant BRAF inhibitors in malignant melanoma, tyrosine kinases inhibitor in non-small cell lung cancer and anti-EGFR monoclonal antibodies in colorectal cancer, respectively. All these assays are run on a single platform using DNA extracted from a single 5 µm section of a formalin-fixed paraffin-embedded tissue block. The assays provide an 'end-to-end' solution from extraction of DNA to automated analysis and report on the cobas z 480. The cobas tests have shown robust and reproducible performance, with high sensitivity and specificity and low limit of detection, making them suitable as companion diagnostics for clinical use.

The cobas® (Roche) portfolio of companion diagnostics in oncology currently has three assays CE-marked for in vitro diagnostics. Two of these (EGFR and BRAF) are also US FDA-approved. These assays detect clinically relevant mutations that are correlated with response (BRAF, EGFR) or lack of response (KRAS) to targeted therapies such as selective mutant BRAF inhibitors in malignant melanoma, tyrosine kinases inhibitor in non-small cell lung cancer and anti-EGFR monoclonal antibodies in colorectal cancer, respectively. All these assays are run on a single platform using DNA extracted from a single 5 µm section of a formalin-fixed paraffin-embedded tissue block. The assays provide an ‘end-to-end’ solution from extraction of DNA to automated analysis and report on the cobas z 480. The cobas tests have shown robust and reproducible performance, with high sensitivity and specificity and low limit of detection, making them suitable as companion diagnostics for clinical use.

AIMS:To study the ALK translocation in patients with advanced non-small-cell lung carcinomas (NSCLCs) seen at a European cancer centre, and its association with EGFR mutations, KRAS mutations and MET amplification. METHODS AND RESULTS:The study included samples from 86 patients diagnosed with advanced NSCLC. ALK fluorescence in-situ hybridization (FISH) was performed using the ALK break-apart probe set (Vysis). ALK FISH-positive cases were defined as those with more than 15% break-apart signals or isolated red signals in 50 cells. EGFR and KRAS mutations were determined by direct sequencing. All ALK-positive cases were analysed retrospectively for MET amplification using a FISH assay, and for ALK mutations by sequencing. We found nine (10.5%) ALK-positive cases, all in adenocarcinomas and the majority in female patients (88.9%). Signet ring cells were observed in four (44.4%) of the nine patients. None of the ALK translocated cases showed MET amplifications or EGFR, KRAS and ALK mutations. CONCLUSIONS:The prevalence of ALK translocation in an unselected population of European patients with advanced NSCLCs was 10%. This alteration was mutually exclusive with EGFR and KRAS mutations, as well as with MET amplification. If multiplexing is considered at the preanalytical phase, lung biopsy specimens are sufficient for performing several predictive assays.

The arrival of targeted therapies has presented both a conceptual and a practical challenge in the treatment of patients with advanced non-small cell lung carcinomas (NSCLCs). The relationship of these treatments with specific histologies and predictive biomarkers has made the handling of biopsies the key factor for success. In this study, we highlight the balance between precise histological diagnosis and the practice of conducting multiple predictive assays simultaneously. This can only be achieved where there is a commitment to multidisciplinary working by the tumor board to ensure that a sensible protocol is applied. This proposal for prioritizing samples includes both recent technological advances and the some of the latest discoveries in the molecular classification of NSCLCs.

Aim To conduct a methods correlation study of three different assays for the detection of mutations at EGFR gene in human formalin-fixed paraffin-embedded tumour (FFPET) specimens of non-small cell lung carcinomas (NSCLC). Methods We conducted a 2-site method comparison study of two european conformity (CE) in vitro diagnostic (IVD)-marked assays, the cobas EGFR Mutation Test and the Therascreen EGFR29 Mutation Kit, and 2× bidirectional Sanger sequencing. We blind-tested 124 NSCLC FFPET specimens with all three methods; the cobas test was performed at both sites. Positive (PPA) and negative percent agreements (NPA) were determined for the cobas test versus each of the other two methods. Specimens yielding discordant test results between methods were further tested using quantitative massively parallel pyrosequencing (MPP). Results PPA between cobas and Sanger was 98.8%; NPA was 79.3%. Overall there were seven discordant results. MPP confirmed an exon 19 deletion in two cases and L858R mutation in four cases. PPA between cobas and Therascreen was 98.9% and NPA was 100%. There was one discordant result. Reproducibility of the cobas test between the two sites was 99.2%. Conclusions The invalid rates for the cobas test and Therascreen were lower than Sanger sequencing. The cobas and Therascreen assays showed a high degree of concordance, and both were more sensitive for the detection of exon 19 deletion and L858R mutations than Sanger. The cobas test was highly reproducible between the two testing sites, used the least amount of DNA input and was the only test with automated results reporting.

BACKGROUND:The cobas 4800 BRAF V600 Mutation Test is a CE-marked and FDA-approved in vitro diagnostic assay used to select patients with metastatic melanoma for treatment with the selective BRAF inhibitor vemurafenib. We describe the pre-approval validation of this test in two external laboratories.METHODS:Melanoma specimens were tested for BRAF V600 mutations at two laboratories with the: cobas BRAF Mutation Test; ABI BRAF test; and bidirectional direct sequencing. Positive (PPA) and negative (NPA) percent agreements were determined between the cobas test and the other assays. Specimens with discordant results were tested with massively parallel pyrosequencing (454). DNA blends with 5% mutant alleles were tested to assess detection rates.RESULTS:Invalid results were observed in 8/116 specimens (6·9%) with Sanger, 10/116 (8·6%) with ABI BRAF, and 0/232 (0%) with the cobas BRAF test. PPA was 97·7% for V600E mutation for the cobas BRAF test and Sanger, and NPA was 95·3%. For the cobas BRAF test and ABI BRAF, PPA was 71·9% and NPA 83·7%. For 16 cobas BRAF test-negative/ABI BRAF-positive specimens, 454 sequencing detected no codon 600 mutations in 12 and variant codon 600 mutations in four. For eight cobas BRAF test-positive/ABI BRAF-negative specimens, four were V600E and four V600K by 454 sequencing. Detection rates for 5% mutation blends were 100% for the cobas BRAF test, 33% for Sanger, and 21% for the ABI BRAF. Reproducibility of the cobas BRAF test was 111/116 (96%) between the two sites.CONCLUSIONS:It is feasible to evaluate potential companion diagnostic tests in external laboratories simultaneously to the pivotal clinical trial validation. The health authority approved assay had substantially better performance characteristics than the two other methods. The overall success of the cobas BRAF test is a proof of concept for future biomarker development.