Somatic copy number variations (CNV; i.e., amplifications and deletions) have been implicated in the origin and development of multiple cancers and some of these aberrations are designated targets for therapies. Although FISH is still considered the gold standard for CNV detection, the increasing number of potentially druggable amplifications to be assessed makes a gene-by-gene approach time- and tissue-consuming. Here we investigated the potential of next generation sequencing (NGS) custom panels to simultaneously determine CNVs across FFPE solid tumor samples. DNA was purified from cell lines and FFPE samples and analyzed by NGS sequencing using a 20-gene custom panel in the GeneReader Platform®. CNVs were identified using an in-house algorithm based on the UMI read coverage. Retrospective validation of in-house algorithm to identify CNVs showed 97.1% concordance rate with the NGS custom panel. The prospective analysis was performed in a cohort of 243 FFPE samples from patients arriving at our hospital, which included 74 NSCLC tumors, 148 CRC tumors, and 21 other tumors. Of them, 33% presented CNVs by NGS and in 14 cases (5.9%) the CNV was the only alteration detected. We have identified CNV alterations in about one-third of our cohort, including FGFR1, CDK6, CDK4, EGFR, MET, ERBB2, BRAF, or KRAS. Our work highlights the need to include CNV testing as a part of routine NGS analysis in order to uncover clinically relevant gene amplifications that can guide the selection of therapies.

No abstract

Background: Fusions involving the tyrosine kinase receptor genes ALK, ROS1, RET, NTRK or MET exon 14 skipping variant (METex14) are present in a significant percentage of advanced solid tumors and their accurate identification is critical to guide targeted therapies. While FISH has traditionally been considered the gold standard for fusion analysis, it is costly and shows biases. GeneReader Next Generation Sequencing (NGS) (Qiagen) and nCounter (Nanostring) are two technologies allowing simultaneous detection of fusion transcripts and splicing variants. In this work we compared the performance of both platforms for fusion and splicing variant detection in advanced solid tumor patients. Methods: RNAs from 40 selected solid tumors were purified using High Pure FFPET RNA Isolation Kit (Hoffman-La Roche) and prospectively analyzed by GeneReader and nCounter. The custom nCounter codeset used targets fusions involving ALK, ROS1, RET, NTRK1-3, NRG1 and MET exon 14 skipping variant based on a dual strategy: detection of specific fusion transcripts and imbalances between the 3' and 5' mRNA regions, enabling the recognition of even those fusions not identified with the specific primers. Reporter counts from nCounter were collected with the nSolver Analysis software (Nanostring) and analyzed using an algorithm developed in the laboratory. The design of QIAact Lung Fusion Custom GeneReader panel contains specific junction probes for the detection of fusions in ALK, ROS1, RET, FGFR1- 3, NRG1, NTRK1- 3, EGFR, BRAF, and MET exon 14 skipping variant. GeneReader analysis and interpretation were performed with the QCI-Analyze and QCI-Interpret software's (Qiagen). Results: Valid results were obtained for 40/40 (100%) of samples tested. Paired analysis showed a 97.5% concordance (39/40 cases) between the results obtained by nCounter and GeneReader NGS, corresponding to a Cohen's kappa of 0.935 [CI=0.809-1.0]. Overall, 8 samples tested positive for fusion transcripts, namely EML4-ALK (n=4), CCDC6-RET (n=1), KIF5B-RET (n=1), EZR-ROS1 (n=1) and ETV6-NTRK3 (n=1). In addition, MET exon 14 skipping variant was detected in two samples. The discordant case between nCounter and NGS corresponded to a rare RET fusion only detected by 3'-5' imbalance using nCounter, while the remaining 29 patients were pan-negative. In one of them, an uncommon HLA-DRB1-MET fusion not included in the nCounter codeset, was found by the GeneReader custom panel. Conclusions: RNA-based NGS and nCounter show excellent concordance for detection of gene fusions and MET splicing variant in advanced solid tumors. Citation Format: Mónica Garzón Ibañez, Ana Gimenez-Capitán, Marta Vives Usano, Ruth Román Lladó, Sonia Rodriguez, Erika Aldeguer, Beatriz García Pelaez, Nuria Jordana Ariza, Cristina Aguado, Santiago Viteri, Andrés Aguilar, Irene Moya, Carlos Cabrera, Maria Jose Catalán, Maria Gonzalez cao, Silvia Garcia-Roman, Jordi Bertran Alamillo, Florencia Garcia Casabal, Rafael Rosell, Miguel Angel Molina-Vila, Clara De La Caridad Mayo De Las Casas. Comparison of clinically relevant fusions detection using two multiplexing RNA based platforms: nCounter and GeneReader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 290.

Introduction: Although immune checkpoint inhibitors have become a standard of care for multiple cancer types; currently used biomarkers, such as PD-L1 expression, MSI or TMB, fail to identify all responders. In addition, little is known about markers of response to immunotherapy in some infrequent histological subtypes, such as mucinous adenocarcinoma of the lung. Gene expression signatures incorporating not only PD-L1 but also other immune modulators can have a better predictive power, particularly in these histological subtypes. Methods: A cohort of 165 baseline samples from with stage I-IV non-small cell lung cancer (NSCLC) patients was retrospectively analyzed, 55 stage I-IIIA mucinous adenocarcinomas (MA), 31 stage I-IIIA nonmucinous adenocarcinomas (NMA) and 79 stage IIIB-IV NMAs. RNA was extracted from FFPE tumor samples and the expression levels of a 7-gene immune signature (CD4, CD8A, FOXP3, GZMM, INFG, CD274 and PDCD1) were determined using a nCounter panel. Krustal-Wallis test was used for statistical comparisons. Results: No significant differences were observed in the expression levels of FOXP3 and PDCD1 (codifying forkhead box P3 and PD-1) between the three groups analyzed. In contrast, GZMM, IFNG and CD4 expression (codifying granzyme M, interferon gamma and CD4) was significantly higher in early stage MA compared to stage I-IIIA or stage IIIB-IV NMA. CD8A expression was significantly higher in early stage tumors, independently of the presence of mucinous component. In addition, the levels of CD274 mRNA (codifying PD-L1) were significantly higher in early stage MA compared to advanced stage NMA. Finally, among the stage I-IIIA MA, 27/55 (49%) were KRAS-mutant, but they did not show significant differences with KRAS-wt MAs in the expression of any of the genes tested. Conclusions: Mucinous adenocarcinoma of the lung shows a different pattern of expression of immune-related genes, which may influence response to immunotherapy. Citation Format: Ruth Román Lladó, Cristina Aguado Esteban, Ana Giménez-Capitán, Ueda Daisuke, Masaoki Ito, Yasuhiro Tsutani, Yoshihiro Miyata, Cristina Teixidó, Noemí Reguart, Morihito Okada, Sonia Rodríguez, Ariadna Balada, Erika Aldeguer, Santiago Viteri Ramirez, Maria Gonzalez Cao, Andrés Aguilar, Rafael Rosell, Miguel Angel Molina Vila. Inmune gene expression by nCounter in mucinous adenocarcinoma lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5594.

Background Non-small cell lung cancer (NSCLC) tumors with mutations in the EGF receptor (EGFR) relapse to therapy with EGFR tyrosine kinase inhibitors (EGFR TKIs) due to a variety of mechanisms, such as emergence resistance mutations, dysregulation of AXL, MET, HER2 or FGFR1 receptors or histological transformation. The CL1-49076-003 trial of the MET/AXL/FGFR inhibitor S49076 in combination with gefitinib enrolled T790M-negative patients in progression to first-line EGF TKIs and showing dysregulation of MET and/or AXL. Molecular screening was performed in 47 patients, 23 of them met the molecular eligibility criteria and 14 were enrolled in the trial. Baseline biopsies of the 14 patients were submitted to molecular profiling. Methods Ten patients had enough material available for Next Generation Sequencing (NGS), that was performed using the GeneRead QIAact Lung UMI Panel (Qiagen, Hilden, FRG). The panel included mutation detection in 16 genes, copy number variations in 5 and the MET exon 14 splicing variant. Amplifications detected by NGS were confirmed by FISH. Of the 4 patients with insufficient material for NGS, 3 were submitted to FISH for HER2 and MET and quantitative PCR for BRAF and PIK3CA hotspot mutations. Finally, of the remaining patient only FISH for MET could be performed. Results Copy number gains were the most prevalent alterations in patients progressing to EGFR TKIs. Four of 14 evaluable patients (29%) showed MET amplification, 4/13 (31%) HER2 amplification and 3/10 (30%) EGFR copy number gains. FGFR1 amplifications were absent in the patient cohort. The baseline sensitizing mutation was confirmed in all cases. Two patients showed additional mutations that could be related to resistance, namely a p.G724S mutation in EGFR and a p.N784fs*2 mutation in MET. Remarkably, these two patients did not present gene amplifications. No other mutations were detected in the rest of genes analyzed, including BRAF, PIK3CA, KRAS, NRAS or ERBB2. Among the 4 patients with more than 6 months of progression free survival, 2 had MET amplifications. The patient with the p.G724S experienced rapid progression of target lesions. Conclusion Next Generation Sequencing can be used to determine mechanisms of resistance to EGFR TKIs at progression, and can give useful clinical information in order to select therapies for second line treatment Citation Format: Monica Garzon Ibanez, Nuria Jordana Ariza, María González Cao, Ruth Román Lladó, Alejandro Martínez Bueno, Lidia Alonso Landeira, María de los Llanos Gil, Miguel Ángel Molina Vila, Niki Karachaliou, Veronika Smutna, Valerie Cattan, Rafael Rosell, Santiago Viteri. Molecular profiling of T790M-negative NSCLC patients progressing on EGFR-TKI enrolled in the CL1-49076-003 trial with a MET/AXL/FGFR inhibitor in combination with gefitinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2521.