BACKGROUND:KRAS is the most heterogeneous and frequent oncogenic driver in non-small cell lung cancer (NSCLC). KRAS alleles and co-mutations shape the tumour microenvironment, potentially influencing benefit from immune checkpoint inhibitors (ICI). The aim of our study was to evaluate the patterns of KRAS mutations, their correlation with clinical and pathological features and their association with ICI outcomes. METHODS:We conducted a multicentre retrospective analysis of patients with advanced KRAS-mutant NSCLC treated with ICI. Clinical, pathological and molecular data were collected, including KRAS mutation type (G12C vs non-G12C), key co-mutations and PD-L1 expression. These variables were correlated with clinical outcomes. Survival outcomes were analysed using Kaplan-Meier estimates and multivariate Cox regression models. RESULTS:A total of 198 patients with KRAS mutant NSCLC were evaluated, with 49.5% carrying a G12C mutation. Overall, 81% of patients received first-line ICI, either as monotherapy or combined with chemotherapy. After a median follow-up of 48 months, G12C cases showed significantly longer median overall survival (OS) (15 vs 9 months; HR 0.71; p = 0.031). Multivariate analysis indicated that G12C mutations correlated with improved OS, as well as good performance status and absence of central nervous system (CNS) metastases. CONCLUSIONS:In this retrospective real-world cohort, KRAS mutation subtype was associated with differences in ICI outcomes, particularly OS. These findings should be considered hypothesis-generating and warrant prospective validation in molecularly characterized cohorts.

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.

Introduction Immunotherapy is firmly established as a treatment regimen in various solid tumors, driven by its exceptional benefits in a selected group of patients. Despite widespread adoption of immune checkpoint blockade (ICB) across diverse solid tumors, the quest for a clinically informative biomarker for long-term benefit remains unmet.Methods A total of 49 patients with metastatic NSCLC treated with ICB were included. Long-term (LTR) and short-term responders (STR) were defined as those with a response to ICB lasting more than 24 months or less than 6 months, respectively. Longitudinal blood specimens were collected before ICB treatment initiation and early-on treatment. Plasma ctDNA next-generation sequencing panel (NGS) and serum proteomics were performed. GeoMx DSP on baseline tumor tissue was performed in a subset of patients.Results Our analysis revealed specific characteristics of LTR compared with STR, namely higher PD-L1 in tumor cells (p = 0.005) and higher incidence of irAEs (p = 0.001). Genomic features associated with lack of benefit from ICB included co-occurring mutations in KRAS/STK11 and TP53/KMT2D (p < 0.05). At a baseline, LTR patients exhibited higher serum levels of proteins related with apoptosis (CASP8, PRKRA), chemotaxis, immune proteasome, processing of MHC class I (S100A4, PSMD9, RNF41) and immune homeostasis (HAVCR1, ARG1) (p < 0.05). Protein spatial profiling of tumor samples showed higher levels of proteins linked with the presence of immune cells (CD45), T cells (CD8), antigen presentation (HLA-DR) and immune regulation proteins (PD-L1, IDO1) within the tumor and tumor stroma component (p < 0.05) in LTR patients. Serum longitudinal analysis identified a set of proteins that presented distinct dynamics in LTR compared to STR, making them interesting candidates to evaluate as early predictors of treatment efficacy.Conclusions Our multimodal analysis of patients with metastatic NSCLC treated with ICB identified clinicopathological and immunological features associated with long-term benefits. The presence of preexisting antitumor immunity emerged as a strong predictor of long-term benefits, providing insights for potential biomarkers and therapeutic strategies for enhancing ICB outcomes in metastatic NSCLC.

Assessment of molecular response (mR) to targeted and immunotherapies (IO) in solid cancer is an emerging clinical liquid biopsy application. Current approaches have demonstrated promising results but have predominately relied on tumor-informed strategies and small to intermediate-sized next generation sequencing (NGS) gene panels. Lack of tissue availability and emergence of comprehensive genomic profiling (CGP), however, limit the practicality of implementing these approaches in a clinical setting. Furthermore, biological signals from non-tumor derived alterations arising from clonal hematopoiesis (CH) may confound detection of tumor-specific alterations for therapy selection and longitudinal monitoring. To address these limitations, we evaluated the 521-gene PGDx elio™ plasma complete assay with an integrated matched white blood cell (WBC) analysis algorithm to longitudinally assess cell-free tumor DNA (ctDNA) through a baseline plasma-informed approach and correlate mR to radiographic response and outcomes. Plasma from 57 patients with mNSCLC who received IO from 2017-2019 at Hospital del Mar had detectable pre-treatment ctDNA. Blood was collected prior to the start of therapy, early on-treatment (median 4 weeks) and longitudinally throughout clinical follow-up and disease progression. Germline and CH variants were identified and removed through matched WBC NGS analyses. mR was calculated as the percent change in mean variant allele frequency of the tumor-specific alterations assessed at the early on-treatment timepoint compared to the pre-treatment timepoint and then compared to radiographic response (RR) and overall survival (OS). Thirty percent (17/57) of patients had a complete or partial radiographic response, with median follow up of >12 months. A tumor-specific genomic landscape analysis at baseline demonstrated associations between mR, recurrently mutated genes, and clinical outcomes. Removal of non-tumor derived cell-free DNA (cfDNA) variants improved characterization of mR and associations with radiographic and survival-based endpoints. A mR rate of 33% (19/57) was observed with a median OS of 53 months compared to 8.4 months for patients experiencing molecular progression at the early on-treatment timepoint (HR 4.8, 95% CI 2.2-10.2, p<0.001). Of all patients that survived ≤12 months after initiation of treatment, 85.7% (24/28) were categorized as experiencing molecular progression through early on-treatment cfDNA analyses. CGP of cfDNA with integrated matched WBC analyses improves accuracy of mR and associations with RR and outcomes. This tumor-agnostic approach for assessment of genomic alterations at baseline and mR early on-treatment improves our ability to associate liquid biopsy-based biomarkers with patient and treatment-level outcomes. Jennifer B. Jackson, Pedro Rocha, James White, Andrew Georgiadis, Ellen Verner, Amy Greer, Rafael Bach Mora, Laura Masfarré, Nil Navarro, Beatriz Bellosillo, Sergi Clavé, Mark Sausen, Edurne Arriola. Comprehensive genomic profiling of cell-free DNA and matched white blood cells improves accuracy of ctDNA molecular response in immunotherapy treated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 726.

Abstract Background: Immunotherapy is firmly established as a treatment regimen in various solid tumors due to its exceptional benefits observed in a select group of patients. Despite widespread use of immune checkpoint blockade (ICB) across diverse solid tumors, including non-small cell lung cancer (NSCLC), the quest for a clinically informative biomarker for long-term benefit remains unmet. Here we investigate the potential utility of clinicopathological and spatial proteomic profiling of tumor specimens from NSCLC patients to identify predictive biomarkers of long-term benefit to ICB. Methods: Forty-nine patients diagnosed with advanced NSCLC who received ICB at Hospital del Mar, Barcelona between 2017-2021 were included. Long-term responders (LTR, n=21) were defined as patients achieving a maintained radiologic response for more than 2 years, and short-term responders (STR, n=28) as patients presenting disease progression within the first six months of ICB initiation. Clinicopathological information, incidence of immune related adverse (irAES) and PD-L1 tumor proportion score assessed by IHC was available for all of them. DSP GeoMx was performed in subset of patients (LTR n=14 and STR n=14), to assess 49 immune biomarkers. Pancytokeratin (panCK; epithelial), CD3, CD20 and SYTO 13 (nuclear) were utilized as morphology biomarkers. Regions of interest were placed in tissue areas containing tumor and segmented in Tumor (PanCK+) and tumor microenvironment (TME) (PanCK-) that was used on downstream analysis. Digital counts were normalized using the protein expression of housekeeper proteins. Statistical analysis was performed using linear mixed models. A P value less than 0.05 was considered significant. Results: Our analysis revealed specific characteristics of LTR patients compared with STR, namely LTR population was enriched for PD-L1 positive in tumors (p=0.005) and a higher incidence of immune-related adverse events (irAEs) (p=0.001). Within the Tumor compartment, LTR patients displayed significantly higher levels of IDO1, CD8, PD-L1, CD45, HLA-DR and STING, while STR patients exhibit higher levels of B7-H3, CD56 and OX40L (p<0.05 for all proteins). Comparison analysis of the TME compartment between LTR and STR revealed augmented levels of IDO1, CD8, CD45, CD11c, CD27 and CD3 in LTR patients, and lower levels of B7H3 and OX40L compared with STR patients (p<0.05 for all proteins). Conclusions: Our comprehensive analysis of metastatic NSCLC patients treated with ICB has unveiled distinct clinicopathological and immunological features associated with long-term benefit of ICB, highlighting the presence of pre-existing antitumor immunity as a stronger predictor of long-term benefit. These findings offer insights into potential biomarkers and therapeutic strategies for enhancing ICB outcomes in metastatic NSCLC. Citation Format: Sharia Hernandez, Rafael Bach, Mario Giner, Wei Lu, Larisa Kostousov, Sean Barnes, Khaja Khan, Laura Masfarré, Xavier Villanueva, Ignacio Sanchéz, Nil Navarro, Álvaro Taus, Miguel Galindo, Max Hardy, Raúl Del Rey-Vergara, Albert Iñañez, Beatriz Sanchez-Espiridion, Laura Moliner, Sergi Clavé, Beatriz Bellosillo, Ana Rovira, Júlia Perera-Bel, Ignacio Wistuba, Edurne Arriola, Luisa M. Solis, Pedro Rocha. Spatial proteomic profiling unveils pre-existing anti-tumor immunity as a hallmark of exceptional benefit from immunotherapy in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6391.

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.

Abstract Introduction: Immune checkpoint inhibitors (ICIs) have greatly improved clinical outcomes for patients with non-small cell lung cancer (NSCLC), however, programmed death-ligand 1 (PD-L1) and tumor mutation burden (TMB) have failed to consistently predict therapeutic response and outcomes in this population. There remains a critical unmet need to identify patients who are likely to experience clinical benefit from treatment, particularly as additional treatment regimens become available. Longitudinal cell-free tumor DNA (ctDNA) assessments have recently been applied as a strategy to identify molecular response (mR) and better predict long term clinical response. Through this real-world analysis of NSCLC, we explore the technical and biological considerations associated with determination of ctDNA mR through matched cell-free and white blood cell (WBC) DNA analyses and apply this optimized approach to assess therapeutic response to ICIs. Methods: Plasma from 50 patients with advanced or metastatic NSCLC who received ICIs from 2017-2019 at Hospital del Mar had detectable pre-treatment ctDNA and underwent analysis using the 521 gene PGDx elio plasma complete liquid biopsy assay. Patient blood was collected prior to the start of ICI therapy, early on-treatment (median 4 weeks; range 2.4-9 weeks) and longitudinally throughout clinical follow-up and disease progression. Matched WBCs were also analyzed for each patient to identify alterations arising from germline polymorphisms and clonal hematopoiesis (CH). mR was calculated as the percent change in mean variant allele frequency of the tumor-specific alterations assessed at the early on-treatment timepoint compared to the pre-treatment timepoint, and compared to radiographic response, progression-free, and overall survival (OS). Results: Best overall response for patients within the cohort was 4% complete response, 26% partial response, 34% stable disease, and 36% progressive disease. The removal of germline or CH variants allowed for improved characterization of tumor-specific somatic alterations and associations with radiographic and survival-based endpoints. With the integrated ctDNA and matched WBC approach, we observed a mR rate of 34% (17/50) with a median OS of 36.8 months compared to 9.2 months for patients experiencing molecular progression at the early on-treatment timepoint (HR 5.6, 95% CI 2.4-12.7, p<0.001, log-rank test). Specifically, of all patients that survived ≤12 months after initiation of treatment, 84% (21/25) were categorized as experiencing molecular progression through early on-treatment ctDNA analyses. Conclusions: This real-world study of ICI treated, advanced NSCLC highlights the opportunity afforded through integrated analyses of ctDNA and matched WBCs to accurately determine mR and the associations with radiographic response and OS. Citation Format: Jennifer B. Jackson, Pedro Rocha, James R. White, Andrew P. Georgiadis, Ellen L. Verner, Amy Greer, Rafael Bach Mora, Laura Masfarré Pintó, Nil Navarro, Álvaro Taus, Beatriz Bellosillo Paricio, Sergi Clavé, Mark T. Sausen, Edurne Arriola. Enhanced detection of ctDNA molecular response for immunotherapy treated non-small cell lung cancer through analyses of cell-free and matched white blood cell DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6443.

Non-small cell lung cancer (NSCLC) can harbor different KRAS mutations. Although targeted therapy is available for KRAS G12C-mutant (mt) NSCLC, immune checkpoint inhibitors (ICIs) are still the first line treatment (tx) for these patients (pts). Here we aimed to assess the outcomes on ICIs for KRAS G12C compared to KRAS non-G12C-mt pts. This is an updated observational, retrospective, multicenter study of pts with KRAS-mt NSCLC treated with ICIs between January 2017 and October 2023. 14 pts received anti-KRAS G12C tx. Targeted sequencing was performed in 59% cases and polymerase chain reaction in the rest. Clinicopathological and molecular data were collected. We evaluated the characteristics, tx response and survival outcomes on ICIs of pts with KRAS G12C vs non-G12C-mt tumors. 189 pts were included with a median follow-up of 34.3 months (m). STK11 and TP53 were the most frequent co-mutated genes present in 4.3%/18.5% G12C/non-G12C and 21.3%/44.7% G12C/non-G12C, respectively. In all KRAS mt tumors, harboring a TP53 co-mutation was associated to a positive PD-L1 and a better ECOG (p < 0.001 and p=0.006, respectively). In addition, a trend to a better overall survival (OS) was seen in TP53 vs STK11 tumors (14.7 vs 6.1m, respectively, p = 0.195). No differences were seen in the median duration of response or progression free survival between G12C/non-G12C (10.7 vs 9.5m p=0.202 and 8 vs 5m p= 0.554, respectively). KRAS G12C tumors were associated with a better median OS compared with non-G12C tumors (16.2 vs 9.2m p=0.024). In the multivariate analysis for OS, PD-L1 negative tumors and ECOG ≥1 were independently associated with worse OS (p=0.004 and p<0.001, respectively).Table: 55PN (%)G12C (n=92)Non-G12C (n=97)p-valueMedian age (range)62.9 (61.8-64.9)65 (63-66.3)Sex0.237Male36 (39.1)67 (69.1)Female56 (60.9)30 (30.9)Tobacco0.866Former47 (51.1)49 (50.5)Current44 (47.8)46 (47.4)Stage0.530I-III18 (19.6)23 (23.7)IV74 (80.4)74 (76.3)M1 CNS17 (18.5)26 (26.8)0.172M1 liver16 (17.4)19 (19.6)0.698ICIs treatment line0.466172 (78.3)80 (82.5)≥220 (21.7)17 (17.5)PD-L10.234Negative (0%)22 (23.9)32 (33)Positive (≥1%)64 (69.6)63 (65)ECOG0.241034 (37)44 (45.4)≥158 (63)53 (54.6) Open table in a new tab Our work shows that pts with KRAS G12C tumors are associated with better OS on ICIs tx when compared with pts with KRAS non-G12C tumors. Harboring a TP53 co-mutation associated to a KRAS mutation might determine a different tumor microenvironment and therefore a better response to ICI tx.

MET gene amplification is a potential therapeutic target in lung adenocarcinoma (LUAD) currently identified by fluorescence in situ hybridization (FISH). Next-generation sequencing (NGS) has become an essential diagnostic tool for routine biomarker profiling of cancer patients, including the detection of copy number alterations (CNAs). The aim of this study was to evaluate the concordance between these two methods and identify potential limitations of NGS implementation. Thirty cases with MET amplification detected by FISH were retrospectively selected from a cohort of 567 LUAD diagnosed between 2018 and 2022 (prevalence: 5.3%). Cases were classified according to the current Camidge-2021 FISH criteria as 5 low-amplified (MET/CEP7 ratio ≥1.8 to ≤2.2), 11 medium-amplified (MET/CEP7 ratio >2.2 to <4) and 14 high-amplified (MET/CEP7 ratio ≥4). Tumor area and tumor cell percentage were annotated to later evaluate complete molecular alterations by NGS using the Oncomine Precision Assay. Detection of MET amplification was concordant in only 16 out of 30 cases (5 cases were excluded due to insufficient DNA), and no correlation was found between MET copy number determined by FISH and NGS (R2=0.346). Distribution of amplification categories was random: 2 cases showed low-amplification, 7 medium-amplification, and 7 high-amplification. Slight differences were observed between concordant and discordant cases in: MET enumeration (12.4 vs. 9.3 gene copies), MET/CEP7 ratio (4.8 vs. 3.2), tumor area (15 vs. 11mm2), and DNA concentration (12 vs. 7 ng/μl), but none of them were statistically significant. FISH results of the 9 discordant cases were reviewed and high copy number heterogeneity was observed in 7 of them and focal amplifications were observed in 2. In 7 out of 9 discordant cases, co-alterations were detected: 2 KRAS (p.G12C and p.Q61H), 2 BRAF non-V600, 2 MET exon 14 skipping and one EGFR p.L858R. The detection of MET amplification by NGS did not show satisfactory concordance with the results obtained by FISH, likely due to the high heterogeneity in MET gene copy number enumeration. As a result, complementary determination by FISH is necessary, given the lack of standardized criteria for establishing CNAs positivity by NGS.

Small cell lung cancer (SCLC) subtypes are driven by dominant transcriptional programs. Preclinical work suggests that transcriptomic subtyping (ASCL1, NEUROD1, POU2F3 and Inflamed) could inform potential tumor vulnerabilities and support therapeutic decisions. Here we explore the association of SCLC subtypes with clinicopathological and EMT characteristics.

RET rearrangements have emerged as a new actionable alteration in papillary thyroid cancers (PTC). These fusions may involve many partner genes, are thought to be mutually exclusive with other oncogenic drivers and their screening is recommended based on fluorescence in situ hybridization (FISH), RT-PCR or next-generation sequencing (NGS). The aim of this study is to evaluate the use of NGS as a standard molecular technique to characterize RET gene fusions. Sixty PTC cases without the BRAF p.V600E and/or TERT promoter mutations were selected retrospectively in two institutions. Molecular alterations were evaluated using the Oncomine Focus Assay, and all samples were analyzed by RT-PCR using the Idylla GeneFusion Assay. Fusion positive samples were confirmed with FISH with break-apart probes. All DNA samples were acceptable for analysis, whereas 4 RNA samples failed quality control NGS parameters. RET fusions were detected in 11 cases (18%): CCDC6::RET (C1:R12) in 8 samples, NCOA4::RET (N6:R12) in 2 samples, and 1 RET expression imbalance positive sample without gene partner characterization. All 11 cases were validated by both FISH and RT-PCR. Besides, 4 more cases presented NGS RET expression imbalance but none of them were confirmed neither by RT-PCR nor by FISH and considered false positive results. There were no false negative NGS cases identified in parallel by RT-PCR. Overall, a positive predictive value of 89% and a negative predictive value of 90% were estimated, reaching a concordance of 89% compared to NGS. Regarding DNA analysis, NRAS p.Q61R/K was the most prevalent alteration in 13 cases (22%) followed by 2 cases with KRAS mutations (p.G12S and p.Q61R). Other potentially actionable fusions found in our series were 2 ETV6::NTRK3 (E4:N14), 2 STRN::ALK (S3:A20) and 1 EML4::ALK (E6:A20). Molecular screening in non-BRAF PTC patients is useful to identify patients harboring RET fusions who may benefit from targeted therapies. As other potentially actionable gene fusions are also found in these patients, routine implementation of NGS analysis warrants a comprehensive biomarker study.

Introduction: Neoadjuvant and adjuvant immune checkpoint blockade (ICB) have recently become standard of care in resectable non-small cell lung cancer (NSCLC). Yet, biomarkers that inform patients who benefit from this approach remain largely unknown. Here, we interrogated the tumor immune microenvironment (TIME) in earlystage NSCLC patients that underwent up-front surgery. Methods: A total of 185 treatment-naive patients with early-stage NSCLC, that underwent up-front surgical treatment between 2006 and 2018 at Hospital del Mar were included. 124 lung adenocarcinomas (LUADs), and 61 squamous cell carcinoma (LUSCs) were included in a tissue microarray. Immunohistochemistry for CD3, CD4, CD8, CD68, CD80, CD103, FOXP3, PD-1, PD-L1, PD-L2 and HLA class II were evaluated by digital image analysis (QuPath software). TIME was categorized into four groups using PD-L1 expression in tumor cells (<1 % or & GE;1 %) and tumor resident memory (CD103+) immune cells (using the median as cut-off). We explored the association between different TIME dimensions and patient's clinicopathological features and outcomes.Results: We found increased levels of T cell markers (CD3+, CD4+, CD8+ cells), functional immune markers (FOXP3+ cells) as well as, higher HLA-II tumor membrane expression in LUADs compared to LUSCs (p < 0.05 for all). In contrast, LUSCs displayed higher percentage of intratumor macrophages (CD68+ cells) as well as, higher PD-L1 and PD-L2 tumor membrane expression (p < 0.05 for all). Unsupervised analysis revealed three different tumor subsets characterized by membrane tumor expression of PD-L1, PD-L2 and HLA-class II. Enrichment of T cells (CD3+, CD8+ cells), regulatory T cells (FOXP3+ cells) and macrophages (CD68+ cells) was observed in the CD103+/PD-L1+ group (p < 0.05 for all). Multivariate analysis showed that infiltration by CD103+ immune cells was associated with improved OS (p = 0.009).Conclusions: TIME analysis in resected NSCLC highlighted differences by histology, PD-L1 expression and molecular subgroups. Biomarker studies using IHC might aid to individually tailor adjuvant treatment in early-stage NSCLC.

Distinct KRAS mutations subtypes can be found in NSCLC (Non-small cell lung cancer). Targeted therapy has become a treatment choice for patients with KRAS G12C NSCLC although immunotherapy (ICI) is widely used in these patients with good outcomes in a subset of patients. Response to immunotherapy of non-KRAS G12C NSCL is less characterized. Here we aimed to assess the outcomes on immunotherapy for patients with tumors harboring KRAS G12C mutation compared to other KRAS mutations.

IntroductionNext-generation sequencing (NGS) is currently widely used for biomarker studies and molecular profiling to identify concurrent alterations that can lead to the better characterization of a tumor’s molecular landscape. However, further evaluation of technical aspects related to the detection of gene rearrangements and copy number alterations is warranted.MethodsThere were 12 ALK rearrangement-positive tumor specimens from patients with non-small cell lung cancer (NSCLC) previously detected via fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and an RNA-based NGS assay, and 26 MET high gene copy number (GCN) cases detected by FISH, selected for this retrospective study. All 38 pre-characterized cases were reassessed utilizing the PGDx™ elio™ tissue complete assay, a 505 gene targeted NGS panel, to evaluate concordance with these conventional diagnostic techniques.ResultsThe detection of ALK rearrangements using the DNA-based NGS assay demonstrated excellent sensitivity with the added benefit of characterizing gene fusion partners and genomic breakpoints. MET copy number alterations were also detected; however, some discordances were observed likely attributed to differences in algorithm, reporting thresholds and gene copy number state. TMB was also assessed by the assay and correlated to the presence of NSCLC driver alterations and was found to be significantly lower in cases with NGS-confirmed canonical driver mutations compared with those without (p=0.0019).DiscussionOverall, this study validates NGS as an accurate approach for detecting structural variants while also highlighting the need for further optimization to enable harmonization across methodologies for amplifications.

Supplementary Figures and Material and Methods

Immune checkpoint blockade (ICB) has significantly impacted the treatment landscape in non-small cell lung cancer (NSCLC). However, this benefit is restricted to a subset of patients. While these results are encouraging, it is crucial to identify those patients who benefit the most from ICB and those who are refractory. Here we investigate the potential utility of longitudinal serum proteomic profiling to identify predictive biomarkers. We included 40 metastatic NSCLC patients who received ICB treatment at Hospital del Mar, Barcelona between 2017-2021. Patients were categorized as long-term responders (LTR, n=20) if they had maintained radiologic response for more than 2 years or non-responders (NR, n=20) if they had disease progression at first radiological evaluation. Serum protein levels were assessed longitudinally (before treatment, previous cycle 2) using the Olink® Explore 384 Oncology panel. LTR had a greater proportion of high PD-L1 expression (p=0.01). Analysis of baseline serum samples showed an increase in LTR of proteins levels related to apoptosis and autophagy (CASP8, ATG4A), TCR signaling pathway (STAT5B, CRACR2A) and myeloid differentiation (ZBTB16, ARG1) (p<0.05 for all proteins). Higher levels of proteins related to immunosuppression and tumorigenesis (PDCD1, TGFBR2, KLK4, KLK6) prior to the second cycle of ICB were associated with poorer response to ICB, with non-responders exhibiting significantly higher levels (p<0.05 for all). Gene set enrichment analysis showed that LTR had an increase in myeloid cell differentiation process, while higher levels of immunoregulatory interactions between lymphoid and non-lymphoid cells were observed in NR (p<0.001 for all). Our findings suggest that patients with high levels of proteins related to apoptosis, autophagy, TCR signaling, and myeloid differentiation at baseline may identify LTR to ICB. Our study underscores the potential use of baseline and longitudinal assessment of serum proteomics as valuable tools for patient selection who are suitable candidates for ICB. Further studies are needed to validate these findings and establish their potential clinical utility.

Non-small cell lung cancer (NSCLC) can harbor different KRAS mutations. Although targeted therapy is available for KRAS G12C-mutant NSCLC, immune checkpoint inhibitors (ICIs) are still the first line treatment (tx) for these patients (pts). Here we aimed to assess the outcomes on ICIs for KRAS G12C compared to KRAS non-G12C-mutant pts. This is an observational, retrospective, multicenter study in pts with KRAS-mutant NSCLC treated with ICIs (monotherapy or combination) between January 2017 and January 2023. Targeted sequencing was performed in most cases. Clinicopathological and molecular data were collected. We evaluated pts characteristics, tx response and survival outcomes from the beginning of ICIs in KRAS G12C vs non-G12C tumors. A total of 169 pts were included. NGS was performed in 105 pts, STK11 and TP53 were the most frequent co-mutated genes present in 4.4%/15% G12C/non-G12C (p=0.110) and 20%/35% G12C/non-G12C (p=0.143), respectively. There were no differences in median progression free survival (PFS) between G12C/non-G12C (8.8 vs 7.1 months(m) respectively p=0.727). In the multivariate analysis, PD-L1 negative tumors and ECOG ≥1 remained independently associated with worse PFS (p=0.010 and p= 0.002 respectively). Regarding overall survival (OS), harboring a G12C mutation was associated with a better OS compared with non-G12C tumors (mOS 16.2m vs 10.4m p=0.033), conversely PD-L1 negative tumors and basal ECOG of ≥1 was associated with worse OS (p= 0.002 and p<0.001 respectively). Table: 1408PPatient’s characteristicsN (%)G12C (n=75)Non-G12c (n=94)p-valueMedian age62.963.80.471Sex0.220Male45 (60)66 (70.2)Female30 (40)28 (29.7)Tobacco0.203Former34 (45.4)53 (56,4)Current41 (54.6)41 (43.6)Stage0.630I-III17 (21.7)22 (23,4)IV58 (78.3)72 (76.6)M1 CNS14 (18.6)24 (25.5)0.381M1 liver14 (18.6)20 (21.3)0.820ICIs treatment line0.604157 (76)75 (80)≥218 (24)19 (20)PD-L10.397Negative (0%)17 (22.7)30 (32)Positive (≥0%)58 (77.3)64 (68)ECOG0.887031 (41.4)41 (43.6)≥144 (58.6)53 (56.4) Open table in a new tab . Our work shows that patients with KRAS non-G12C tumors are more frequently PD-L1 negative and harbor a higher proportion of STK11 co-mutations conditioning a worse OS on immunotherapy treatment. Innovative therapeutic strategies need to be investigated for these patients.