Search

Epidermal growth factor receptor (EGFR) mutations typically occur in exons 18–21 and are established driver mutations in non-small cell lung cancer (NSCLC)1–3. Targeted therapies are approved for patients with ‘classical’ mutations and a small number of other mutations4–6. However, effective therapies have not been identified for additional EGFR mutations. Furthermore, the frequency and effects of atypical EGFR mutations on drug sensitivity are unknown1,3,7–10. Here we characterize the mutational landscape in 16,715 patients with EGFR-mutant NSCLC, and establish the structure–function relationship of EGFR mutations on drug sensitivity. We found that EGFR mutations can be separated into four distinct subgroups on the basis of sensitivity and structural changes that retrospectively predict patient outcomes following treatment with EGFR inhibitors better than traditional exon-based groups. Together, these data delineate a structure-based approach for defining functional groups of EGFR mutations that can effectively guide treatment and clinical trial choices for patients with EGFR-mutant NSCLC and suggest that a structure–function-based approach may improve the prediction of drug sensitivity to targeted therapies in oncogenes with diverse mutations.

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.

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

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.

Background: ERBB2 (HER2) insertions and point mutations are oncogenic drivers in non-small cell lung cancer (NSCLC) and now has approved targeted therapy for this patient population. Here, we present the landscape of ERBB2 genetic alterations (alts) and co-mutations, and the comparison between ERBB2 and EGFR exon 20 insertions in NSCLC. Methods: Two large retrospective cohorts were analyzed. In the Geneplus (China) cohort, 1,281 newly diagnosed NSCLC patients (pts) harboring ERBB2 alt were analyzed based on tissue and/or ctDNA. In the Guardant360 (Guardant Health, Redwood City, USA) cohort, 1719 ctDNA profiles of newly diagnosed NSCLC samples harboring ERBB2 alts were evaluated, with clonal alterations defined as those with ≥50% ratio of the maximum somatic variant allele fraction. Results: In the Geneplus cohort of 1,281 pts with ERBB2 alts, 55% were female with a median age of 58 years. ERBB2 mutations or insertions were identified in 930 pts, ERBB2 amplification(amp)-only in 351 pts, with mutation and co-amp at 7.5%. Most common alterations were in the tyrosine kinase domain exon 20 (85%), with Y772_A775dupYVMA to be the most frequent (57.4%), followed by G776delinsVC/LC/VV/IC (10.6%), and S310X (10.4%). EBRR2-mutation NSCLC were frequently having PDL1 Conclusion: In two large independent cohorts, Chinese and Western, ERBB2 mutation and co-mutation patterns were similar. ERBB2 exon 20 insertions/mutations were dominant at over 80% with Y772_A775dupYVMA being the most common driver mutation; TP53 and EGFR were the most frequently co-occurred genes. ERBB2 mutation lung cancers had low TMB and PDL1, as expected in female dominant lung adenocarcinomas, similar to EGFR exon 20 NSCLC. Clonal VUSs may represent a novel subset of mutations to functionally characterize. Citation Format: Lingzhi Hong, Leylah Drusbosky, Yinyi Wang, Yuanyuan Xiong, Rongrong Chen, Simon Heeke, Monique B. Nilsson, Jianjun Zhang, John V. Heymach, Xiuning Le. Mutation and co-mutation landscape ofERBB2 alterations in advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6087.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the US. Targeted agents, such as EGFR tyrosine kinase inhibitors (TKIs) and KRAS G12C inhibitors, have provided clinical benefits for patients and can yield significant tumor shrinkage. Inevitably, however, there remain residual tumors that harbor drug-tolerant persister cells (DTPCs) not killed by initial treatment, leading to emerging of drug-resistant cells (DRCs) and tumor recurrence. The identification of effective treatment strategies to target DTPCs is a critical and unmet need. We hypothesized that DTPCs can be targeted using chimeric antigen receptor (CAR)-based cellular therapy approaches directed at cell surface proteins expressed on DTPCs and that the combination of such approaches with targeted agents will more effectively delay the emergence of fully drug-resistant cells than targeted agents alone. To this end, we first performed luciferase-based killing assays and observed that EGFR-targeting CAR-T and CAR-NK cells displayed potent cytotoxicity against DTPCs of osimertinib-treated EGFR mutant NSCLC cells. Moreover, osimertinib DTPCs showed increased sensitivity to EGFR CAR-T and CAR-NK cells compared to their parental cells, likely due to the upregulation of EGFR on the cell surface following osimertinib treatment. In a patient-derived xenograft model of HER2YVMA mutant lung cancer, we observed that HER2 CAR-NK cells showed enhanced antitumor activity in mice treated with the HER2 inhibitor, poziotinib, for two weeks, compared to CAR-NK cells only, poziotinib only, or mice treated upfront with HER2 CAR-NK cells plus poziotinib. These in vitro and in vivo data support that CAR-based cellular therapy can effectively eliminate DTPCs. To identify additional candidate cell surface proteins expressed on DTPCs that could be targeted by CAR-based cell therapy approaches, we interrogated RNA expression from our panel of DTPCs as well as public datasets containing DTPCs of targeted agents. Likewise, we investigated the expression of cell surface proteins using datasets of parental and DRCs to identify the common candidate targets that could be exploited on both DTPCs and DRCs. Our analysis identified CD70 as being consistently upregulated in both EGFR TKI-resistant cells and DTPCs of osimertinib-treated EGFR mutant NSCLC. Moreover, we observed CD70 upregulation in DTPCs and DRCs of other targeted agents, including KRAS, ALK, and RET inhibitors, suggesting that CD70 may be a common target in DTPCs across oncogenic drivers. Furthermore, we determined that CD70-targeting CAR-T and CAR-NK cells showed promising in vitro activity against the DTPCs of osimertinib-treated EGFR mutant NSCLC. These results demonstrate CAR-based cellular therapy as an effective approach to target DTPCs and identify CD70 as a novel therapeutic target for combatting DTPCs in NSCLC. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Xiaoxing Yu, Alissa Poteete, Qian Huang, Simon Heeke, John Heymach. CD70-targeting CAR-T and CAR-NK cells demonstrate potent activity against NSCLC drug-tolerant persister cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 891.

Purpose: RET fusions drive oncogenesis in 1-2% of non-small cell lung cancer (NSCLC) and are sensitive to the selective RET inhibitors selpercatinib and pralsetinib. They have also emerged as mechanisms of acquired resistance to other targeted therapies. The genomic landscape of RET fusions and the impact of co-occurring genomic alterations on the efficacy of selective RET inhibitors is yet to be fully described. Methods: A total of 678 RET fusion-positive samples were analyzed from three cohorts: Guardant360® circulating tumor DNA (ctDNA) (n=467), tissue and/or plasma from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (AACR Project GENIE) non-squamous NSCLC cohort (n=161), and the MD Anderson Cancer Center (MDACC) cohort of patients treated with selpercatinib or pralsetinib (n=50). Molecular characteristics were compared across all three cohorts and to the entire non-RET non-squamous NSCLC AACR Project GENIE cohort (n=17770). Fisher’s exact test was used to evaluate the difference in response rate between patient groups, and Cox proportional hazard models were used to calculate hazard ratios for time-to-event endpoints for patients in the MDACC clinical cohort (n=51, 50 patients with RET fusion, 1 patient with RET point mutation). Results: The most common fusion partner in all cohorts was KIF5B (ctDNA: 73%; GENIE: 69%; MDACC: 71%), followed by CCDC6 (19%; 17%; 18%) and NCOA4 (5%; 1%; 7%). Across all cohorts, co-mutations in TP53 were most common (60%; 34%; 40%) and EGFR, CDKN2A/B, MET, and ATM consistently co-occurred across all cohorts. EGFR co-occurred more frequently with non-KIF5B fusion partners (chi-square p < 0.006). MYC and CCND1 amplifications were enriched in all three cohorts compared to the non-RET cohort. In the MDACC clinical cohort (n=51), the majority were female, never-smokers, with adenocarcinoma histology. The median follow-up time was 28.3 months (95% CI: 15.2 ~ 35 months). Overall response rate (ORR) was 74.4% (95% CI: 58.8 ~ 86.5%), where patients with TP53 mutation had numerically lower ORR compared to patients without TP53 mutation (58.5% vs. 87.5%; p = 0.0632). Median progression-free survival (PFS) time was 16.5 months (95% CI: 13.5 ~ 27.4 months) and KIF5B fusions trended towards worse PFS outcome compared to non-KIF5B fusions (HR: 2.24; 95% CI: 0.88 ~ 5.67; p = 0.0896). TP53 alterations were associated with a significantly worse overall survival (OS) (HR: 2.93; 95% CI: 1.08 ~ 7.95; p = 0.0346). Conclusion: In the largest RET fusion-positive NSCLC cohort to date, RET fusions frequently co-occurred with other genomic alterations, most commonly in TP53. TP53 alterations are associated with a significant reduction in overall survival in patients treated with the selective RET inhibitors. Additional analysis is underway. Citation Format: Tuqa Al Khalaf, Simon Heeke, Lei Feng, Leylah M. Drusbosky, Jeff Lewis, Waree Rinsurongkawong, Vadeerat Rinsurongkawong, Jack Lee, Jianjun Zhang, Don Gibbons, Ara Vaporciyan, Vincent Lam, Vivek Subbiah, John Heymach, Yasir Elamin. The genomic landscape of RET fusions in non-small cell lung cancer and the impact of co-occurring genomic alterations on the efficacy of selective RET inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4264.

The number of genomic alterations required for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has increased and become more complex these last few years. These molecular abnormalities lead to treatment that provides improvement in overall survival for certain patients. However, these treated tumors inexorably develop mechanisms of resistance, some of which can be targeted with new therapies. The characterization of the genomic alterations needs to be performed in a short turnaround time (TAT), as indicated by the international guidelines. The origin of the tissue biopsies used for the analyses is diverse, but their size is progressively decreasing due to the development of less invasive methods. In this respect, the pathologists are facing a number of different challenges requiring them to set up efficient molecular technologies while maintaining a strategy that allows rapid diagnosis. We report here our experience concerning the development of an optimal workflow for genomic alteration assessment as reflex testing in routine clinical practice at diagnosis for NS-NSCLC patients by using an ultra-fast-next generation sequencing approach (Ion Torrent Genexus Sequencer, Thermo Fisher Scientific). We show that the molecular targets currently available to personalized medicine in thoracic oncology can be identified using this system in an appropriate TAT, notably when only a small amount of nucleic acids is available. We discuss the new challenges and the perspectives of using such an ultra-fast NGS in daily practice.

Introduction International guidelines recommend BRAF mutational status assessment in treatment-naive advanced non-squamous non-small cell lung carcinoma (NSCLC) patients since the presence of a BRAFV600 mutation enables specific BRAF inhibitor treatment. For this purpose, the mutational status needs to be obtained in 10 working days. Herein, we prospectively evaluated the feasibility of systematic assessment of the BRAF status using immunohistochemistry (IHC) in a single institution (LPCE, Nice) at baseline for NSCLC diagnosed. Methods 1317 NSCLC were evaluated using BRAF IHC from 2011 to 2019. Initially the BRAF status was prospectively assessed using NGS and/or pyrosequencing in 618 consecutively diagnosed NSCLC patients from 2012 to 2016; BRAFV600E and BRAF nonV600E mutated tumors detected in this cohort were retrospectively evaluated using BRAF IHC. Secondarily, 699 biopsies of NSCLC were prospectively analyzed between 2017 and 2019 using BRAF IHC. BRAF IHC positive tumors were tested using a rapid BRAF specific PCR based assay. Results Initially, 21/618 (3%) of tumors (15 early and 6 late stage tumors) were BRAFV600E mutated according to the results of NGS and/or pyrosequencing. BRAF IHC was positive in 21/21 of these cases and negative in 51/51 (100 %) BRAF non V600E mutated cases. In the prospective BRAF IHC tested cohort of patients, 24/699 (3%) tumors (13 early and 11 late stage tumors) were positive with VE1 IHC. The BRAF PCR assay was positive in 20/24 (83 %) of these cases. Conclusion BRAFV600E IHC screening of treatment-naive NSCLC patients is a rapid, specific and very sensitive method which can lead in advanced stage positive NSCLC tumors to a BRAF inhibitor treatment. This test can be routinely integrated into mandatory predictive biomarker ‘testing of NSCLC. According to the organization of patient care and the physician’s request, this practice can be proposed as an alternative to NGS-based tissue biopsy made at baseline.

Rationale: The characterization of new theranostic biomarkers is crucial to improving the clinical outcome of patients with advanced lung cancer. Here, we aimed at characterizing the P2RX7 receptor, a positive modulator of the anti-tumor immune response, in patients with lung adenocarcinoma. Methods: The expression of P2RX7 and its splice variants was analyzed by RT-qPCR using areas of tumor and non-tumor lung adenocarcinoma (LUAD) tissues on both immune and non-immune cells. The biological activity of P2RX7 was studied by flow cytometry using fluorescent dyes. Bi-molecular fluorescence complementation and confocal microscopy were used to assess the oligomerization of P2RX7. Tumor immune infiltrates were characterized by immunohistochemistry. Results: Fifty-three patients with LUAD were evaluated. P2RX7A, and 3 alternative splice variants were expressed in LUAD tissues and expression was down regulated in tumor versus adjacent non-tumor tissues. The protein retained biological activity only in immune cells. The P2RX7B splice variant was differentially upregulated in immune cells (P < 0.001) of the tumor and strong evidence of oligomerization of P2RX7A and B was observed in the HEK expression model, which correlated with a default in the activity of P2RX7. Finally, LUAD patients with a high level of P2RX7B had non-inflamed tumors (P = 0.001). Conclusion: Our findings identified P2RX7B as a new theranostic tool to restore functional P2RX7 activity and open alternative therapeutic opportunities to improve LUAD patient outcome.

International audience; The histological distinction of lung neuroendocrine carcinoma, including small cell lung carcinoma (SCLC), large cell neuroendocrine carcinoma (LCNEC) and atypical carcinoid (AC), can be challenging in some cases, while bearing prognostic and therapeutic significance. To assist pathologists with the differentiation of histologic subtyping, we applied a deep learning classifier equipped with a convolutional neural network (CNN) to recognize lung neuroendocrine neoplasms. Slides of primary lung SCLC, LCNEC and AC were obtained from the Laboratory of Clinical and Experimental Pathology (University Hospital Nice, France). Three thoracic pathologists blindly established gold standard diagnoses. The HALO-AI module (Indica Labs, UK) trained with 18,752 image tiles extracted from 60 slides (SCLC = 20, LCNEC = 20, AC = 20 cases) was then tested on 90 slides (SCLC = 26, LCNEC = 22, AC = 13 and combined SCLC with LCNEC = 4 cases; NSCLC = 25 cases) by F1-score and accuracy. A HALO-AI correct area distribution (AD) cutoff of 50% or more was required to credit the CNN with the correct diagnosis. The tumor maps were false colored and displayed side by side to original hematoxylin and eosin slides with superimposed pathologist annotations. The trained HALO-AI yielded a mean F1-score of 0.99 (95% CI, 0.939–0.999) on the testing set. Our CNN model, providing further larger validation, has the potential to work side by side with the pathologist to accurately differentiate between the different lung neuroendocrine carcinoma in challenging cases.

Due to increased demand for testing, as well as restricted supply chain resources, testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to face many hurdles. Pooling several samples has been proposed as an alternative approach to address these issues. We investigated the feasibility of pooling nasopharyngeal swab (NPS) or saliva samples for SARS-CoV-2 testing with a commercial assay (Idylla SARS-CoV-2 test; Biocartis). We evaluated the 10-pool and 20-pool approaches for 149 subjects, with 30 positive samples and 119 negative samples. The 10-pool approach had sensitivity of 78.95% (95% confidence interval [CI], 54.43% to 93.95%) and specificity of 100% (95% CI, 71.51% to 100%), whereas the 20-pool approach had sensitivity of 55.56% (95% CI, 21.20% to 86.30%) and specificity of 100% (95% CI, 25% to 100%). No significant difference was observed between the results obtained with pooled NPS and saliva samples. Given the rapidity, full automation, and practical advantages of the Idylla SARS-CoV-2 assay, pooling of 10 samples has the potential to significantly increase testing capacity for both NPS and saliva samples, with good sensitivity. IMPORTANCE To control outbreaks of coronavirus disease 2019 (COVID-19) and to avoid reagent shortages, testing strategies must be adapted and maintained for the foreseeable future. We analyzed the feasibility of pooling NPS and saliva samples for SARS-CoV-2 testing with the Idylla SARS-CoV-2 test, and we found that sensitivity was dependent on the pool size. The SARS-CoV-2 testing capacity with both NPS and saliva samples could be significantly expanded by pooling 10 samples; however, pooling 20 samples resulted in lower sensitivity.

Background: SARS-CoV-2 infection is the cause of the respiratory illness COVID-19, which presents most frequently with respiratory symptoms SARS-CoV-2 cell entry requires interactions with ACE2 and TMPRSS2 on the surface of the host cell Cancer patients and, specifically, those with thoracic malignancies seem to experience poorer clinical outcomes Methods: We utilized bulk and single-cell transcriptional data from a combination of normal and malignant tissues and cells from aerodigestive and respiratory tracts to explore mechanisms governing the expression of ACE2 and TMPRSS2 Additionally, we determined the effect of EMT induction, ZEB1 modulation, and SARS-CoV-2 infection on ACE2 expression Results: Our bulk data suggests that aerodigestive and lung cancer models express a broad range of ACE2 and TMRPSS2, particularly in epithelial cells, and would serve as good models for studying SARS-CoV-2 infection We assessed the relationship between ACE2 and epithelial differentiation in numerous datasets, and found consistent positive correlations with transcriptional and microRNA signifiers of epithelial differentiation The miR-200 family – zinc finger E-box-binding homeobox 1 (ZEB1) pathway, which is an established regulator of EMT, also directly regulates ACE2 expression, likely via putative ZEB1 repressor sites located in the ACE2 promoter Furthermore, SARS-CoV-2 infection reduces ACE2 expression and shifts cells to a more mesenchymal phenotype with loss of EPCAM and upregulation of ZEB1 and other EMT-associated genes Conclusions: ACE2-positive cells are almost exclusively epithelial and unexpectedly rare, considering the devastating impact of this infection Following viral entry, SARS-CoV-2 infection induces molecular changes within the cells that are reminiscent of EMT, including increased ZEB1 ZEB1, in turn, appears to directly repress the expression of ACE2 This SARS-CoV-2-induced ACE2 deficiency, compounded by the downregulation of genes, including claudins, which play a critical role in restricting epithelial and endothelial permeability, exposes respiratory cells to increased risk of edema and acute respiratory distress syndrome (ARDS) Legal entity responsible for the study: The authors Funding: NIH/NCI R01-CA207295 (L A B ), NIH/NCI U01-CA213273 (L A B , J V H ), CCSG P30-CA01667 (L A B ), University of Texas SPORE in Lung Cancer P5-CA070907 (L A B , D L G , J V H , C M G ), the Department of Defense (LC170171;L A B ), Khalifa Bin Zayed Al Nahyan Foundation (C M G ), RP170067 (EMP), through generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shot Program and Andrew Sabin Family Fellowship, and The Rexanna Foundation for Fighting Lung Cancer Disclosure: C Gay: Research grant/Funding (self): Astra Zeneca J V Heymach: Advisory/Consultancy: AstraZeneca;Advisory/Consultancy: Boehringer Ingelheim;Advisory/Consultancy: Exelixis;Advisory/Consultancy: Genentech;Advisory/Consultancy: GlaxoSmithKline;Advisory/Consultancy: Guardant Health;Advisory/Consultancy: Hengrui;Advisory/Consultancy: Spectrum L A Byers: Advisory/Consultancy, Research grant/Funding (self): AstraZeneca;Advisory/Consultancy, Research grant/Funding (self): AbbVie;Advisory/Consultancy, Research grant/Funding (self): GenMab;Advisory/Consultancy: BergenBio;Advisory/Consultancy: Pharma Mar SA;Advisory/Consultancy, Research grant/Funding (self): Sierra Oncology;Advisory/Consultancy: Merck;Advisory/Consultancy: Bristol Myers Squibb;Advisory/Consultancy: Genentech;Advisory/Consultancy: Pfizer;Research grant/Funding (self): Tolero Pharmaceuticals All other authors have declared no conflicts of interest

Introduction: Aside from the reverse transcription-PCR tests for the diagnosis of the COVID-19 in routine clinical care and population-scale screening, there is an urgent need to increase the number and the efficiency for full viral genome sequencing to detect the variants of SARS-CoV-2. SARS-CoV-2 variants assessment should be easily, rapidly, and routinely available in any academic hospital.Materials and Methods: SARS-CoV-2 full genome sequencing was performed retrospectively in a single laboratory (LPCE, Louis Pasteur Hospital, Nice, France) in 103 SARS-CoV-2 positive individuals. An automated workflow used the Ion Ampliseq SARS-CoV-2 panel on the Genexus Sequencer. The analyses were made from nasopharyngeal swab (NSP) (n = 64) and/or saliva (n = 39) samples. All samples were collected in the metropolitan area of the Nice city (France) from September 2020 to March 2021.Results: The mean turnaround time between RNA extraction and result reports was 30 h for each run of 15 samples. A strong correlation was noted for the results obtained between NSP and saliva paired samples, regardless of low viral load and high (>28) Ct values. After repeated sequencing runs, complete failure of obtaining a valid sequencing result was observed in 4% of samples. Besides the European strain (B.1.160), various variants were identified, including one variant of concern (B.1.1.7), and different variants under monitoring.Discussion: Our data highlight the current feasibility of developing the SARS-CoV-2 next-generation sequencing approach in a single hospital center. Moreover, these data showed that using the Ion Ampliseq SARS-CoV-2 Assay, the SARS-CoV-2 genome sequencing is rapid and efficient not only in NSP but also in saliva samples with a low viral load. The advantages and limitations of this setup are discussed.

Small cell lung cancer (SCLC) is a highly aggressive cancer with limited treatment options and generally poor prognosis. Treatment of SCLC has not considerably changed over the last decades with therapeutic options focusing on unselected populations. Although in the past SCLC was thought to be a relatively homogenous malignancy, recent reports from our group and others identified four major distinct subgroups of SCLC, each with different therapeutic vulnerabilities. Three of the subtypes are defined by the expression of a specific transcription factor, ASCL1 (SCLC-A), NEUROD1 (SCLC-N) and POU2F3 (SCLC-P) while the fourth subtype is defined by an inflamed phenotype (SCLC-I). While our initial subtyping of SCLC is based on a gene expression signature comprised of ~1300 genes, which makes routine implementation challenging, we hypothesized that DNA methylation as a proxy to gene expression might be a more suitable approach for biomarker development in SCLC. We assembled a cohort of 105 SCLC formalin-fixed paraffin embedded (FFPE) samples (82/105 Stage > IIIb) and performed matched RNA-Sequencing (RNAseq) and methylation profiling using reduced-representation bisulfite sequencing (RRBS). To validate our findings and expand our analysis across different sample types, we profiled a panel of 59 fully characterized SCLC cell lines as well as 68 patient-derived xenograft models. We found that methylation levels differ markedly between the four subtypes, with the SCLC-N presenting with a hypermethylated phenotype and the SCLC-P with a hypomethylated phenotype across the genome, highlighting the profound differences in the underlying epigenetic regulation among the SCLC subtypes and supporting DNA methylation analysis as a potential readout for identifying SCLC subtypes. Furthermore, in order to subtype the clinical SCLC samples, we developed a predictive model using an extreme gradient boost model using RNA expression and DNA methylation, respectively, to allow the classification with 94.5% accuracy in the tissue testing cohort. Using a cohort of matched plasma samples, we further demonstrated that the DNA methylation differences were indeed preserved in cell-free DNA (cfDNA) allowing subtype classification with an accuracy of 87.5%. These data indicate that DNA methylation can be used for reliable subtyping of SCLC in tissue and in liquid biopsy samples. In summary, using a large cohort of predominantly extensive stage SCLC clinical samples, we were able to identify profound differences in DNA methylation that can be exploited as a novel biomarker for the classification of SCLC into four distinct subtypes with both tissue biopsy and non-invasive using plasma. Considering the previously shown therapeutic vulnerabilities of the four subtypes, these findings will enable the rapid initiation of personalized clinical trials in SCLC. Citation Format: Simon Heeke, Carl M. Gay, Marcos R. Estecio, Allison Stewart, Hai Tran, Bingnan Zhang, Ximing Tang, Gabriela Raso, Kyle Concannon, Luana Guimaraes De Sousa, Whitney E. Lewis, Monique Nilsson, Yuanxin Xi, Lixia Diao, Qi Wang, Jianjun Zhang, Jing Wang, Ignacio I. Wistuba, Lauren A. Byers, John V. Heymach. Use of DNA methylation from tumor and plasma to identify four major small cell lung cancer subtypes with distinct biology and therapeutic vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3473.

Approximately 15% of all patients with non-small cell lung cancer (NSCLC) and nearly 35% of Asian patients with NSCLC harbor activating mutations within the epidermal growth factor receptor (EGFR). Although these patients are initially highly sensitive to first or second generation EGFR tyrosine kinase inhibitors (TKIs) including erlotinib or third-generation inhibitors including osimertinib, EGFR TKI-refractory disease inevitably emerges. While therapeutic strategies to target resistant disease that emerges though secondary EGFR mutations or MET amplification have been developed, there remains a void of therapeutic options for patients where resistance occurs through EGFR-independent mechanism such as epithelial to mesenchymal transition (EMT) or transformation to small cell lung cancer (SCLC). To identify cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches we interrogated RNAseq data from EGFR mutant NSCLC cell lines (HCC827 and HCC4006) and their associated EGFR TKI resistant variants previously shown to have developed resistance through EMT and filtered gene expression data to include only genes which transcribed proteins localized to the cell surface. We identified CD70 as a being highly upregulated in EGFR TKI resistant cells (p = 7.2e-42). Given that CD70 expression is highly restricted and only transiently expressed on immune cells, CD70 was selected as a top candidate cell surface protein for targeting studies. Western blotting and flow cytometry analysis confirmed CD70 protein levels to be highly upregulated in EGFR TKI resistant cells that had undergone EMT but not in cells harboring secondary EGFR mutations or MET amplifications. We also observed CD70 upregulation in osimertinib-treated drug tolerant persister cells, indicating that CD70 upregulation is an early event in the evolution of TKI resistance. Moreover, patient-derived models of acquired EGFR TKI resistance also exhibited CD70 positivity. Our data also indicated that in EGFR mutant NSCLC cells, CD70 could be upregulated through decreased CD70 promoter methylation as well as by the EMT regulators, transforming growth factor-β (TGF-β) and ZEB1, both of which were upregulated in TKI resistant cells. In EGFR TKI resistant cells, CD70 knockdown impaired cell viability and invasiveness, and stimulation of CD70 using the exogenous binding partner CD27 resulted in activation of AKT and MAPK, pathways known to be re-activated with acquired TKI resistance. CD70-targeting approaches including anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting CAR T cell and CAR NK cells showed promising in vitro and in vivo activity against CD70 positive tumor cells and in osimertinib drug-tolerant persister cells. These results identify CD70 as a novel therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits further investigation in the clinic. Citation Format: Monique B. Nilsson, Yan Yang, Sonia Patel, Simon Heeke, Xiuning Le, Thiru Aruguman, Jacqulyne Robichaux, Xiaoxing Yu, Alissa Poteete, Xiaoyang Ren, Lixia Diao, Li Shen, Qi Wang, Fahao Zhang, Leticia Campos Clemente, Luisa Solis Soto, Chunhua Shi, Hai Tran, Jason Bock, Jing Wang, Ignacio I. Wistuba, John D. Minna, John V. Heymach. CD70 is a novel therapeutic target for EGFR mutant NSCLC with acquired, EMT-associated EGFR TKI resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1827.

Introduction After the recent advent of Immune Checkpoint Inhibitors (ICIs), one of current challenge of clinical cancer trials is certainly to develop the optimal combinations of ICIs with DNA-Damaging Agents (chemotherapy and/or radiotherapy, named DDAs hereafter). Elucidating resistance mechanisms to these different treatments is pivotal to propose new predictive biomarkers and to develop therapeutic strategies to improve ICI efficiency. We hypothesized that resistance to DDA and ICIs is mediated in part by intrinsic tumor mechanisms, some of which may be shared. Methods To address this knowledge gap, we compared RNA expression signatures, antigen presentation, and PD-L1 expression from cohorts of cancer patients treated with radiotherapy, chemotherapy, and immunotherapy to identify shared molecular pathways that may mediate cross-resistance. Using a panel of lung cancer cell lines, we then confirmed that the tumor-cell-intrinsic expression of SQSTM1 is positively correlated with antigen presentation, and inversely with DNA damage repair and DDA/ICIs resistance gene signatures. This SQSTM1/HERV pathway was then supported in vitro using engineered silenced cell-lines (SQSTM1, MAVS, and STING ShRNA) treated with FDA-approved DDA (cisplatin, oxaliplatin, doxorubicin, and ionizing radiations) or synthetic double-stranded RNA (5’ppp dsRNAs and poly IC). Results Through three complementary approaches (in silico, ex vivo on patient cohorts, and in vitro), we identify the p62/SQSTM1 scaffold protein as a key molecular mediator able of predicting and controlling sensitivity for DDA and ICIs. Mechanistically, in response to DNA damage, we found that SQSTM1 is essential for the inhibition of DNA repair and the reactivation of human endogenous retroviruses (hERV). Analogous to a “viral alarm”, the hERV are sensed and activate IFN responses that drive the expression of MHC-I and PD-L1, leading to tumor immune evasion. Targeting the hERV pathways in SQSTM1-depleted tumor cells by poly(I:C)/Lyovec, or docetaxel treatment can rescue the hERV, IFN, and MHC pathway, providing a promising therapeutic avenue turning a “cold” into a “hot” tumor in non-responders cancer patients. Conclusion Depending on its levels, we thus propose SQSTM1 as a predictive biomarker for guiding treatment decisions from i) ICI alone, ii) ICI combined with cisplatin, or; iii) ICI combined with poly(I:C)/Lyovec or docetaxel, with the aim to increase immunotherapy efficacy. Citation Format: Iris Grosjean, Grégoire D’Andréa, Nathalie Yazbeck, Amine Belaid, Barnabé Roméo, Marie Angela Domdom, Nicolas Guillot, Eric Gilson, Emmanuel Chamorey, Gérard Milano, Véronique Hofman, Marius Ilié, Isabelle Benard-Thiery, Simon Heeke, Patrick Brest, François Ghiringhelli, Paul Hofman, Baharia Mograbi. The tumor scaffold protein SQSTM1 at the crossroads of DNA damage and immunotherapy responses in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1284.

Background: Resistance to targeted tyrosine kinase inhibitors (TKI) inevitably develops in metastatic EGFR-mutant non-small cell lung cancer (NSCLC). Resistance mechanisms are diverse, and mechanisms beyond receptor tyrosine kinase (RTK) pathway mutations are poorly understood. We hypothesized that the use of osimertinib as first-line therapy is increasing the prevalence of RTK-independent resistance mechanisms, and that RTK-independent resistant tumors undergo enhanced tumor cell lineage plasticity as an escape mechanism to EGFR TKI therapy. Methods: We identified patients who developed osimertinib resistance (OR) after first line (1L, n=54) and second line (2L, n=42) treatments and determined the resistance mechanisms based on clinical sequencing and histopathology. We also performed single-cell RNA-seq of 24 samples from 13 patients with EGFRm NSCLC at TKI treatment-naïve (TN, n=2), residual disease (RD, n=4), and progression disease (PD, n=7) stages. Results: Compared to 2L OR tumors, 1L OR tumors had increased RTK-independent mechanisms of resistance (76% vs. 46%, p=0.002), including 8% with small cell transformation (n=4), 2% with squamous transformation (n=1) and 66% with unknown mechanisms (n=34). To understand inter- and intra-tumor heterogeneity, we analyzed transcriptomic profiles of 76,266 single cells. Lung developmental lineages were assigned to 10,250 EpCAM+ cells, including 4,735 cells classified as malignant cells by inferCNV and RTK signaling analysis. In the two EGFRm TN tumors, the malignant cells demonstrated bronchoalveolar lineage and moderate EGFR expression. In the TKI resistant cases (PD, n=7), both RTK-dependent and RTK-independent resistance were observed. The RTK-dependent tumors (EGFR T790M n=1; ERBB2 amplification n=1) demonstrated preserved bronchoalveolar lineage identity. In the RTK-independent resistant tumors (n=5), one had complete lineage switch from epithelial to small cell neuroendocrine and very low expression level of EGFR. The remaining 4 PD tumors displayed varying expression of epithelial-to-mesenchymal transformation (EMT) features. One tumor had sarcomatoid histology and a high proportion of cells having positive VIM expression (84%) and 92% of cells having complete loss of NAPSA expression; 3 tumors had partial EMT demonstrated by heterogeneous proportion of cells having VIM expression (18-56%) and loss of NAPSA (26-67%). Interestingly, some of the cells with EMT and partial-EMT had moderate levels of EGFR expression, similar to the levels in the TN tumors. Conclusion: With osimertinib use at 1L, the incidence of RTK-independent resistance has increased to become the dominant mechanism, whereas RTK-dependent resistance has decreased. Increased lineage plasticity (small cell neuroendocrine, squamous and EMT) potentially serves as an RTK-independent TKI-resistance mechanism in EGFRm NSCLC. Citation Format: Xiuning Le, Ruiping Wang, Natalie Vokes, Yasir Elamin, Neda Kalhor, Daniel McGrail, Yuanxin Xi, Santiago Treviño III, Lingzhi Hong, Robyn Du, George Blumenschein, Carl Gay, Marcelo Negrao, Mehmet Altan, Hai Tran, Limei Hu, Jing Wang, Simon Heeke, Monique Nilsson, Jacqulyne Robichaux, Minghao Dang, Guangchun Han, Lauren Byers, Anne Tsao, Boris Sepesi, Chantale Bernatchez, Jianjun Zhang, Linghua Wang, John Heymach. Enhanced lineage plasticity in RTK-independent TKI-resistant EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3260.

In non-small cell lung cancer (NSCLC), response to immune checkpoint blockade (ICB) is associated with programmed cell death ligand 1 expression that is induced by interferon-γ-produced by tumor-infiltrating CD8+ T cells. However, not all tumors with a PD-L1 expression and/or CD8+ T cell infiltrate respond to ICB, and some tumors without any PD-L1 expression respond to ICB. Moreover, little is known about all the mechanisms governing ICB resistance in NSCLC. The objective of the study was to investigate a circulating immunological signature (cytokines, chemokines and immune checkpoints) which could be predictive of resistance to ICB in patients with advanced NSCLC. We performed a multiplexed analysis on 23 TruCulture® (in vitro T cells activation system) and 41 plasma samples using the Luminex® platform (Bio-Techne, MN USA). We investigated the relationship between the levels at baseline of 30 circulating analytes and the response to ICB of advanced NSCLC patients. Through the TruCulture® samples analysis, we identified two types of responders depending on T cell functionality. The responders with a functional T cell activation had lower levels of neutrophil associated analytes (CXCL5/6; p-value Citation Format: Wassila Khatir, Olivier Humbert, Jaap Neels, Léa Berland, Jonathan Benzaquen, Fabian Andrés Gallardo Rivera, Maryline Allegra, Myriam Salah, Virginie Tanga, Olivier Bordone, Julien Fayada, Virginie Lespinet-Fabre, Elodie Long-Mira, Sandra Lassalle, Patrick Brest, Valérie Vouret, Charlotte Maniel, Jacques Boutros, Simon Heeke, Véronique Hofman, Charles-Hugo Marquette, Paul Hofman, Marius Ilie. Identification of a predictive circulating immunological signature of response to immune checkpoint inhibitors in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1265.

The leading cause of cancer-related deaths in the US is non-small cell lung cancer (NSCLC), and about 10-15% of NSCLC cases harbor EGFR activating mutations. While these patients are initially responsive to EGFR tyrosine kinase inhibitors (TKIs), they eventually experience progression and develop acquired TKI resistance. Currently, there are no approved therapies for NSCLC patients with acquired resistance to the third-generation EGFR TKI osimertinib (OSI). Moreover, EGFR-TKI resistant NSCLCs are refractory to immune checkpoint inhibitors. Therefore, novel treatment strategies are urgently needed. Chimeric antigen receptors (CARs) have been used to enhance the anti-tumor activity of immune effector cells. Here, we developed CAR-based therapies targeting EGFR as a novel strategy to overcome immune suppression and target EGFR TKI-resistant NSCLC. We designed multi-generational EGFR CAR constructs and transduced them into T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. We then evaluated their activity against NSCLC cell lines in vitro using firefly luciferase assays. EGFR CAR-T and CAR-NK cells showed potent cytotoxicity against NSCLC cells expressing EGFR including EGFR wild-type and mutant NSCLCs, as well as NSCLC cells with acquired resistance to OSI. However, EGFR CAR-T cells showed decreased killing activity against OSI-resistant NSCLC cells compared to their parental cells. In contrast, EGFR CAR-NK cells showed stronger cytotoxicity against NSCLC with acquired OSI resistance as compared to parental cells. Transcriptomic analysis revealed that NSCLC cells with acquired resistance to TKI had undergone epithelial-mesenchymal transition and had downregulated expression of genes related to antigen presentation and upregulated expression of B7-H6 (NCR3LG1), which have been reported to be associated with increased responsiveness to NK cells. Moreover, treatment of TKI resistant cells with OSI resulted in upregulation of cell surface EGFR and potentiated CAR-NK cells-mediated killing. TGF-β is a critical immune-suppressive cytokine. We found that EGFR-TKI resistant NSCLC cells elaborated expression of TGF-β as compared to parental cells. Blockade of the TGF-β pathway using galunisertib significantly enhanced the activity of EGFR CAR-NK cells against OSI-resistant NSCLC cells. Next, we engineered the dominant-negative TGF-β receptor II (DNTGFBRII) into our EGFR CAR constructs. The expression of DNTGFBRII inhibited the phosphorylation of SMAD2 and the downregulation of granzyme B and perforin induced by TGF-β. DNTGFBRII-CAR-NK cells showed higher killing activity against OSI-resistant NSCLC cells and were resistant to TGF-β-mediated immunosuppression. In conclusion, EGFR CAR-NK cells show significant anti-tumor activity to EGFR TKI-resistant NSCLC cells, and the cytotoxicity is enhanced in combination with OSI treatment and blockade of the TGF-β pathway. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Jacqulyne Robichaux, Alissa Poteete, Xiaoxing Yu, Fahao Zhang, Simon Heeke, Yu Qian, Junqin He, John Heymach. EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 548.

Epidermal growth factor receptor (EGFR) mutations typically occur in exons 18-21 and are established driver mutations in non-small cell lung cancer (NSCLC)

Despite the recent implementation of immunotherapy as a single treatment or in combination with chemotherapy for first-line treatment of advanced non-small cell lung cancer (NSCLC), many patients do not benefit from this regimen due to primary treatment resistance or toxicity. Consequently, there is an urgent need to develop efficient biomarkers that can select patients who will benefit from immunotherapy thereby providing the appropriate treatment and avoiding toxicity. One of the biomarkers recently described for the stratification of NSCLC patients undergoing immunotherapy are mutations in STK11/LKB1, which are often associated with a lack of response to immunotherapy in some patients. Therefore, the purpose of this review is to describe the different cellular mechanisms associated with STK11/LKB1 mutations, which may explain the lack of response to immunotherapy. Moreover the review addresses the co-occurrence of additional mutations that may influence the response to immunotherapy and the current clinical studies that have further explored STK11/LKB1 as a predictive biomarker. Additionally this work includes the opportunities and limitations to look for the STK11/LKB1 status in the therapeutic strategy for NSCLC patients.

Despite the recent implementation of immunotherapy as a single treatment or in combination with chemotherapy for first-line treatment of advanced non-small cell lung cancer (NSCLC), many patients do not benefit from this regimen due to primary treatment resistance or toxicity. Consequently, there is an urgent need to develop efficient biomarkers that can select patients who will benefit from immunotherapy thereby providing the appropriate treatment and avoiding toxicity. One of the biomarkers recently described for the stratification of NSCLC patients undergoing immunotherapy are mutations in STK11/LKB1, which are often associated with a lack of response to immunotherapy in some patients. Therefore, the purpose of this review is to describe the different cellular mechanisms associated with STK11/LKB1 mutations, which may explain the lack of response to immunotherapy. Moreover the review addresses the co-occurrence of additional mutations that may influence the response to immunotherapy and the current clinical studies that have further explored STK11/LKB1 as a predictive biomarker. Additionally this work includes the opportunities and limitations to look for the STK11/LKB1 status in the therapeutic strategy for NSCLC patients.

Iron is an essential nutrient for mammals as well as for pathogens. Inflammation-driven changes in systemic and cellular iron homeostasis are central for host-mediated antimicrobial strategies. Here, we studied the role of the iron storage protein ferritin H (FTH) for the control of infections with the intracellular pathogen Salmonella enterica serovar Typhimurium by macrophages. Mice lacking FTH in the myeloid lineage (LysM-Cre+/+Fthfl/fl mice) displayed impaired iron storage capacities in the tissue leukocyte compartment, increased levels of labile iron in macrophages, and an accelerated macrophage-mediated iron turnover. While under steady-state conditions, LysM-Cre+/+Fth+/+ and LysM-Cre+/+Fthfl/fl animals showed comparable susceptibility to Salmonella infection, i.v. iron supplementation drastically shortened survival of LysM-Cre+/+Fthfl/fl mice. Mechanistically, these animals displayed increased bacterial burden, which contributed to uncontrolled triggering of NF-κB and inflammasome signaling and development of cytokine storm and death. Importantly, pharmacologic inhibition of the inflammasome and IL-1β pathways reduced cytokine levels and mortality and partly restored infection control in iron-treated ferritin-deficient mice. These findings uncover incompletely characterized roles of ferritin and cellular iron turnover in myeloid cells in controlling bacterial spread and for modulating NF-κB and inflammasome-mediated cytokine activation, which may be of vital importance in iron-overloaded individuals suffering from severe infections and sepsis.

International audience; Background: Detection of genomic rearrangements, like anaplastic lymphoma kinase (ALK) fusions, is a pivotal requirement in non-small cell lung cancer (NSCLC) for the initiation of a targeted treatment. While tissue testing remains the gold standard, detection of these alterations using liquid biopsies is an unmet need. To enable the detection of ALK rearrangements from circulating-free RNA (cfRNA) from NSCLC patients, we have evaluated a novel reverse transcription PCR (RT-PCR) based assay.Methods: Sixty-six patients with advanced stage NSCLC were included in the study. ALK status was determined by immunohistochemistry (IHC) and/or FISH on tissue sections. For the detection of ALK rearrangements from 2ml plasma collected in EDTA or Streck BCT DNA tubes, cfRNA was extracted using a prototype cfRNA sample preparation method and tested by a novel multiplex ALK/RET RT-PCR assay (Roche).Results: Of the forty-two patients with an ALK rearrangement, 30 (71%) were included at baseline. In 10 of the baseline patients, an ALK rearrangement was detected by RT-PCR [baseline sensitivity 33.33% (95% CI: 17.29–52.81%)]. All 24 negative ALK IHC/FISH-negative patients were negative using the RT-PCR based assay (specificity =100%).Conclusions: The prototype Roche ALK/RET RT-PCR assay was able to detect ALK fusion transcripts in the plasma of NSCLC patients at baseline as well as at disease progression with limited sensitivity but high specificity. Consequently, this assay could potentially be considered to select patients for an ALK-targeting therapy when tissue samples are lacking.

Despite molecular and clinical heterogeneity, small cell lung cancer (SCLC) is treated as a single entity with predictably poor results. Using tumor expression data and non-negative matrix factorization, we identify four SCLC subtypes defined largely by differential expression of transcription factors ASCL1, NEUROD1, and POU2F3 or low expression of all three transcription factor signatures accompanied by an Inflamed gene signature (SCLC-A, N, P, and I, respectively). SCLC-I experiences the greatest benefit from the addition of immunotherapy to chemotherapy, while the other subtypes each have distinct vulnerabilities, including to inhibitors of PARP, Aurora kinases, or BCL-2. Cisplatin treatment of SCLC-A patient-derived xenografts induces intratumoral shifts toward SCLC-I, supporting subtype switching as a mechanism of acquired platinum resistance. We propose that matching baseline tumor subtype to therapy, as well as manipulating subtype switching on therapy, may enhance depth and duration of response for SCLC patients. Gay et al. provide a classification for four subtypes of small cell lung cancer, each with unique molecular features and therapeutic vulnerabilities. An inflamed, mesenchymal subtype predicts benefit with the addition of immunotherapy to chemotherapy. Intratumoral switching between chemosensitive and chemoresistant subtypes accompanies therapeutic resistance.

Introduction Tumor mutational burden (TMB) has been proposed as a novel predictive biomarker for the stratification of patients with NSCLC undergoing immune checkpoint inhibitor (ICI) treatment. The assessment of TMB has recently been established using large targeted sequencing panels, and numerous studies are ongoing to harmonize TMB assessment. "Correlation" or the coefficient of determination has generally been used to evaluate the association between different panels. We hypothesized that these metrics might overestimate the comparability, especially for lower TMB values. Methods A total of 30 samples from patients with NSCLC undergoing ICI treatment were consecutively sequenced using the following three large, targeted sequencing panels: FoundationOne, Oncomine TML, and QiaSeq TMB. The TMB values were compared in the whole patient population and in a subset of patients in which the TMB assessed by FoundationOne was between 5 and 25 mutations/Mb. Prediction of durable clinical benefit (>6 mo with no progression) was assessed using receiver operator characteristics, and optimal cutoff values were calculated using the Youden J statistic. Results Correlation between the three targeted sequencing panels was strong in the whole patient population (R2 > 0.79) but was dramatically reduced in the subset of patients with TMB of 5 to 25 mutations/Mb. The agreement assessed using the Bland-Altman method was also very low. All panels were able to predict durable clinical benefit in the TMB-high population. Conclusions Assessment of TMB using the three targeted sequencing panels was possible and predictive of response to ICI treatment, but correlation was an inappropriate measurement to assess the association between the respective panels.

Background NGS from plasma samples in non-squamous cell lung carcinoma (NSCC) can aid in the detection of actionable genomic alterations. However, the absolute clinical value of NGS in liquid biopsy (LB) made at baseline is currently uncertain. We assessed the impact of plasma-based NGS using an in-house test and an outsourced test in comparison to a routine molecular pathology workflow. Methods Twenty-four advanced/metastatic treatment-naïve NSCC patients were prospectively included. NGS analyses were conducted both in-house using the Oncomine cfTNA Panel and in an external testing center using the Foundation Liquid assay. NGS analysis and/or specific molecular based assays were conducted in parallel on tissue or cytological samples. Results Both LB tests were well correlated. Tissue NGS results were obtained in 67% of patients and demonstrated good correlation with LB assays. Activating EGFR mutations were detected using LB tests in three patients. PD-L1 expression assessed in tissue sections enabled the initiation of pembrolizumab treatment in five patients. Conclusion NGS from LB is feasible in routine clinical practice using an in-house or an outsourced test at baseline. However, the impact on therapy selection was limited in this small series of patients and LB was not able to replace tissue-based testing in our hands.

Background: Assessment of actionable EGFR mutations is mandatory for treatment-naïve advanced or metastatic non-squamous lung carcinoma (NSLC), but the results need to be obtained in less than 10 working days. For rapid EGFR testing, an EGFR-specific polymerase chain reaction (PCR) assay is an alternative and simple approach compared to next generation sequencing (NGS). Here, we describe how a rapid EGFR-specific PCR assay can be implemented in a single laboratory center (LPCE, Nice, France) as reflex testing in treatment-naïve NSLC. Methods: A total of 901 biopsies from NSLC with more than 10% of tumor cells were prospectively and consecutively evaluated for EGFR mutation status between November 2017 and December 2019 using the Idylla system (Biocartis NV, Mechelen, Belgium). NGS was performed for nonsmokers with NSLC wild type for EGFR, ALK, ROS1, and BRAF and with less than 50% PD-L1 positive cells using the Hotspot panel (Thermo Fisher Scientific, Waltham, MA, USA). Results: Results were obtained from 889/901 (97%) biopsies with detection of EGFR mutations in 114/889 (13%) cases using the Idylla system. Among the 562 EGFR wild type tumors identified with Idylla, NGS detected one actionable and one nonactionable EGFR mutation. Conclusions: Rapid and targeted assessment of EGFR mutations in treatment-naïve NSLC can be implemented in routine clinical practice. However, it is mandatory to integrate this approach into a molecular algorithm that allows evaluation of potentially actionable genomic alterations other than EGFR mutations.

International audience; Background - The introduction of liquid biopsy using PCR-based assays into routine practice has had a strong impact on the treatment of EGFR-mutated lung adenocarcinoma and is now commonly used for routine testing of EGFR mutations in certain clinical settings. To assess whether the claimed benefits of PCR-based assays hold true in daily practice at a multicenter clinical institution, we assessed how treatment decisions are affected by PCR-based assays for the analysis of EGFR mutations from plasma samples in a centralized laboratory (LPCE, Nice, France). Patients and methods - A total of 345 samples were analyzed using the US Food and Drug Administration-approved Cobas EGFR Mutation Test v2 and 103 using the Therascreen EGFR Plasma RGQ PCR Kit over 3 years (395 samples from 324 patients). Eleven plasma samples were validated independently using Cobas at 3 institutions, and 130 samples were analyzed using Stilla digital PCR. Clinical data were collected for 175 (54%) of 324 patients. Results - Cobas was superior to the Therascreen assay and demonstrated 100% reproducibility. Digital PCR showed only 48%, 83%, and 58% concordance with Cobas for exon 19 deletions, L858R mutations, and T790M mutations, respectively. Liquid biopsies helped inform and change treatment when resistance occurred and enabled the detection of EGFR mutations in patients when biopsy tissue results were unavailable. Conclusion - PCR-based assays are a fast and convenient test, allowing the detection of primary and secondary EGFR mutations from plasma. Cobas proved to be a reliable test, whereas digital PCR produced too many inconclusive results to be currently recommended as a principal testing device.

Background: Circulating tumor cells (CTCs) hold potential for noninvasive diagnosis, prognosis and prediction testing in non-small cell lung cancer (NSCLC) patients. Minimizing degradation or loss of CTCs is pivotal for detection and profiling of the low abundance and fragile CTCs, particularly in clinical trials. We prospectively investigated (NCT02372448) whether a new blood collection device performed better compared to commonly used K3EDTA tubes, when subjected to long-term sample storage. Methods: Blood samples were drawn into K3EDTA and blood collection tubes (BCT) (Streck), and filtered by the Isolation by SizE of Tumor/Trophoblastic Cells (ISET® system), for CTC detection in two study populations of NSCLC patients; the training set of 14 patients with stage II/IV NSCLC, and the validation set of 36 patients with stage IV NSCLC). MET expression was evaluated by immunocytochemistry (ICC) and anaplastic lymphoma kinase (ALK) gene rearrangement by break-apart fluorescence in situ hybridization (FISH) on ISET-enriched CTCs. Results: Blood processed after 24 h and 48 h in BCT tubes showed stable CTCs counts and integrity, whereas CTCs in K3EDTA tubes showed an altered morphology in all patients. CTCs recovered in BCT or K3EDTA tubes at 24 and 48 h were evaluable by ICC for MET expression and by FISH for ALK rearrangement. Conclusions: The BCT tubes gave a high yield and preserved the integrity of CTCs after 24 and 48 h of storage at room temperature, which facilitate their molecular characterization in NSCLC patients entering clinical trials.