Variants In Non-small Cell Lung Cancer Research Articles

Concomitant double-fusion of PLEKHA7-ALK and INPP5D-ALK reveals favorable alectinib sensitivity in lung adenocarcinoma: a case report and literature review

Anaplastic lymphoma kinase (ALK) gene fusion is a classic driver mutation in non-small cell lung cancer (NSCLC); however, ALK double-fusion variants in NSCLC have rarely been reported. In this study, we reported a case with extremely uncommon ALK double-fusion variants. A 32-year-old female diagnosed with lung adenocarcinoma, who had developed multiple intrapulmonary and brain metastases, experienced worsening of her condition despite undergoing prior chemotherapy. Subsequent testing using next-generation sequencing (NGS) detected the presence of PLEKHA7-ALK and INPP5D-ALK double-fusion. The prescription of alectinib revealed potent efficacy and resulted in an increase in the survival rate. This case presented two uncommon and concomitant ALK fusion partners in NSCLC; more importantly, the INPP5D-ALK subtype has not been reported, therefore this study broadens the spectrum of ALK double-fusion variants and provides insight into the use of ALK inhibitors for the treatment of NSCLC in patients with double ALK fusions.

Estudio de variantes genéticas en 169 pacientes de cáncer de pulmón no microcítico

IntroductionLung cancer is the leading cause of cancer death in our country. Non-small cell lung cancer (NSCLC) represents the paradigm of personalized medicine. The main objective of this study is analysing the distribution of the most frequently described clinically significant variants in NSCLC, in our environment. Material and methodsWe studied the immunohistochemical expression of TTF1, p40 and PD-L1 and the genetic variants frequency using Next-Generation Sequencing (NGS) with a panel of 52 genes, in 174 NSCLC paraffin-embedded samples in 169 patients (111 men and 52 women) from the province of Cádiz. ResultsThe immunohistochemical expression of TTF1, p40 and PD-L1 was positive in 87%, 0% and 46% in adenocarcinoma, and 0%, 100% and 41% in squamous cell carcinoma. In NGS, the most common single nucleotide variants (SNVs) were KRAS (36%), EGFR (14%), BRAF (10%), PIK3CA (8%), and MET (3%). The most frequent copy number variants (CNVs) were amplifications in NF1 (30%), EGFR (18%), CCND1 (9%), MYC (9%) and KRAS (7%). In women, SNV in EGFR are more frequent than in men (P<.0001). Adenocarcinoma is the most frequent histological type with SNV in KRAS (P=.007361) or in EGFR (P<.0001). Gene fusions were detected in 16 patients (9.47%), in 9 cases in the MET gene. ConclusionsWe detected associations, not described so far, between immunohistochemical expression and specific gene variants, which could have an impact on the treatment of NSCLC patients.

The impact of single gene testing (SGT) on subsequent comprehensive genomic profiling (CGP) success in community oncology practice for advanced non-small cell lung cancer (NSCLC): Results from a prospective observational reference laboratory testing program.

6506 Background: CGP concurrently tests tumor tissue for guideline-recommended predictive biomarkers for optimal therapy selection and identification of clinical trials in NSCLC patients. A common practice in the community oncology setting is to first order SGT, then consider CGP when SGT results are negative or after treatment failure. Given the limited tissue available and the scope of alterations not tested by SGT, we sought to characterize the effects of prior SGT on subsequent CGP testing success, and therapeutic opportunities identified by CGP beyond SGT for NSCLC patients in the community setting. Methods: Reference laboratory information systems were used to prospectively contact clinicians across 80 community practices who ordered at least 1 SGT for guideline-recommended genomic variants in NSCLC. CGP was offered for their patients prior to SGT or upon receipt of negative SGT results. SGT included individual assays for BRAF, EGFR, KRAS, MET exon 14 skipping mutations; ALK, RET, and ROS1 rearrangements; and PD-L1 IHC . CGP included DNA-seq for mutations, copy number variants in 523 genes including guideline-recommended genomic variants, MSI, and tumor mutational burden; RNA-seq for rearrangements/fusions/splice variants; and PD-L1 IHC. Results: Among a total of 580 NSCLC patients with CGP ordered (May 2021-December 2022), 168 (29%) had ≥1 SGT ordered prior to CGP (median=5). No patients had all SGT performed, with untested cases ranging from 10% for ALK to 90% for MET exon 14. The same FFPE tissue block was used for CGP in 150/168 (89%) of cases with prior negative SGT. Compared to CGP-only cases, CGP for cases with prior negative SGT was canceled twice as often at tissue review (16% vs 7%; p=.001), had higher DNA extraction failures (13% vs 8%; p=.09), and lower DNA sequencing success (70% vs 83%, p&lt;.001). CGP detected guideline-recommended variants in 51% of all cases. Among prior negative SGT cases, frequencies were higher in genes where SGT was performed less often and is less sensitive than CGP RNA-seq ( RET fusions , MET exon 14 skipping). CGP also identified guideline-recommended variants in genes with no SGT offered during the study period ( ERBB2 mutations , NTRK2/3 fusions), as well as variants with emerging evidence for FDA expedited program-designated clinical trial therapies in 28% of patients, including NRG1 fusions and BRAF non-V600E , KEAP1, KRAS non-G12C, NFE2L2, STK11, and TP53 Y220C mutations. Conclusions: Prior negative SGT doubled subsequent CGP test cancellations for NSCLC due to tissue insufficiency and increased CGP DNA extraction failures. SGT practice in the community oncology setting does not meet practice guideline recommendations and negatively impacts the potential benefit of subsequent CGP for NSCLC patients.

Clinical characteristics and targeted therapy of different gene fusions in non-small cell lung cancer: a narrative review.

Lung cancer is the most fatal malignant tumor in the world. Since the discovery of driver genes, targeted therapy has been demonstrated to be superior to traditional chemotherapy and has revolutionized the therapeutic landscape of non-small cell lung cancer (NSCLC). The remarkable success of tyrosine kinase inhibitors (TKIs) in patients with epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusions has shifted the treatment from platinum-based combination chemotherapy to targeted therapy. Although the incidence rate of gene fusion is low in NSCLC, it is of great significance in advanced refractory patients. However, the clinical characteristics and the latest treatment progress of patients with gene fusions in lung cancer have not been thoroughly explored. The objective of this narrative review was to summarize the latest research progress of targeted therapy for gene fusion variants in NSCLC to improve understanding for clinicians. We conducted a search of PubMed database and American Society of Clinical Oncology (ASCO), the European Society for Medical Oncology (ESMO), and World Conference on Lung Cancer (WCLC) abstracts meeting proceedings from 1 January 2005 to 31 August 2022 with the following keywords "non-small cell lung cancer", "fusion", "rearrangement", "targeted therapy" and "tyrosine kinase inhibitor". We comprehensively listed the targeted therapy of various gene fusions in NSCLC. Fusions of ALK, ROS proto-oncogene 1 (ROS1), and rearranged during transfection proto-oncogene (RET) are relatively more common than others (NTRK fusions, NRG1 fusions, FGFR fusions, etc.). Among ALK-rearranged NSCLC patients treated with crizotinib, alectinib, brigatinib, or ensartinib, the Asian population exhibited a slightly better effect than the non-Asian population in first-line therapy. It was revealed that ceritinib may have a slightly better effect in the non-Asian ALK-rearranged population as first-line therapy. The effect of crizotinib might be similar in Asians and non-Asians with ROS1-fusion-positive NSCLC in first-line therapy. The non-Asian population were shown to be more likely to be treated with selpercatinib and pralsetinib for RET-rearranged NSCLC than the Asian population. The present report summarizes the current state of fusion gene research and the associated therapeutic methods to improve understanding for clinicians, but how to better overcome drug resistance remains a problem that needs to be explored.

Abstract 4120: Acquired TPM3-NTRK1 fusion resistant to larotrectinib in a non-small cell lung cancer with EML4-ALK fusion progressed on lorlatinib

Abstract Background: As oncogenic driver genes have been detected, targeted therapy plays a crucial role in non-small cell lung cancer (NSCLC) with targeted mutation. Lorlatinib, a selective third-generation inhibitor of anaplastic lymphoma kinase (ALK), is a treatment option for ALK-positive NSCLC patients who previously failed on ALK tyrosine kinase inhibitors (TKIs). Larotrectinib, a highly selective tropomyosin receptor kinase (TRK) inhibitor, is a treatment option for NSCLC with neurotrophic tyrosine receptor kinase (NTRK) mutation. Herein, we present an acquired TPM3-NTRK1 fusion resistant to larotrectinib in lung adenocarcinoma with EML4-ALK fusion progressed on lorlatinib. Case Presentation: A 48-year-old male with stage IV adenocarcinoma of lung with metastatic disease of bone was initially treated with carboplatin, pemetrexed, and pembrolizumab. Follow up CT in 4 months showed numerous hepatic lesions and liver biopsy demonstrated large cell neuroendocrine carcinoma. Upon progression of disease (PD), systemic therapy regimen was switched to combination of carboplatin, paclitaxel, and bevacizumab. After two months since starting the new regimen, a second PD was observed. Follow-up MRI brain demonstrated multiple small metastatic lesions. Subsequently, he was started on alectinib 600 mg twice a day given EML 4-ALK fusion mutation [4.8% of variant allele frequency (VAF)] in circulating tumor DNA (ctDNA) NGS assay. Follow-up CT chest in 5 months showed 5 mm left lower lobe nodule which remained unchanged in size. He maintained SD for 9 months before repeat MRI brain demonstrated PD with new leptomeningeal carcinomatosis. Subsequently, he received whole brain radiotherapy and the treatment regimen was switched to lorlatinib 100 mg daily. In 7-week follow-up CT, the 5 mm nodule in the left lower lobe was stable. Follow-up MRI brain in 16 weeks showed decrease in size and number of numerous enhancing parenchymal and leptomeningeal lesions. Overall SD was maintained for 8 months before follow-up MRI brain showed new punctate lesions. Repeat ctDNA NGS assay revealed new TPM3-NTRK1 fusion (VAF 0.2%) and ALK L1196M (VAF 2.8%), and a decision was made to add larotrectinib 100 mg daily. The 6-week follow-up MRI brain showed new parenchymal brain metastases. Discussion: It has been reported that the development of ALK resistance mutations may be associated with specific EML4-ALK variants. However, the influence of the specific EML4-ALK variants in NSCLC was not fully investigated. We report the case of acquired TPM3-NTRK1 fusion resistant to larotrectinib and acquired ALK L1196M in lung adenocarcinoma with EML4-ALK fusion progressed on lorlatinib. Further investigations are warranted to explore the mechanism of resistance to ALK TKIs. Citation Format: Horyun Choi, Jinah Kim, Yeun Ho Lee, Leeseul Kim, Na Hyun Kim, Young Kwang Chae. Acquired TPM3-NTRK1 fusion resistant to larotrectinib in a non-small cell lung cancer with EML4-ALK fusion progressed on lorlatinib [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 4120.

Abstract 5795: Validation of FDA approved oncoReveal Dx lung and colon cancer assay (oRDx-LCCA)

Abstract Introduction: The oncoReveal™ Dx Lung and Colon Cancer Assay (oRDx-LCCA) is an amplicon-based targeted next generation sequencing (NGS), in vitro diagnostic test that detects DNA variants across 22 genes, including single nucleotide variants (SNVs) and insertions and deletions (INDELs) in KRAS and EGFR, from DNA isolated from formalin-fixed paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) tumor tissue specimens. It is approved by the FDA as a companion diagnostic (CDx) for identifying approved therapeutic treatments for KRAS G12/G13 wild-type variants in CRC, and EGFR L858R variants and exon 19 in-frame deletions for NSCLC. oRDx-LCCA can detect non-CDx variants such as EGFR T790M, G719X and exon 20 insertions in NSCLC and KRAS Q61X and A146T and BRAF V600E in CRC. Variants are detected using Pillar Biosciences’ proprietary PiVAT® (Pillar Variant Analysis Toolkit) software. Methods: DNA input range was evaluated at 6 concentrations between 5 - 160ng in duplicate using DNA extracted from 15 clinical FFPE samples containing SNVs and INDELs from representative targeted mutations. Accuracy was determined by comparing detected variants with an externally validated comparator (CompO) across 208 samples (84 CRC, 124 NSCLC). Limit of detection (LoD) was determined by serial dilution of KRAS, EGFR, or BRAF positive FFPE samples in normal DNA to detect variant allele frequency (VAF) between 1 - 10%. Reproducibility tests were performed across 3 sites, 9 runs, 6 operators, 3 reagent lots and 3 thermocyclers, resulting in a total of 360 libraries. The non-inferiority (NI) statistical test was used to compare the oRDx-LCCA with an FDA approved CDx assay for EGFR exon 19 deletions and L858R mutations (CompQ) and for KRAS G12/G13 mutations (CompC). Synthetic FASTQ files were created at VAFs above and below the variant detection threshold for CDx mutations and used to validate quality control parameters, mean coverage, and on-target rate. Results: Effective DNA input range was determined to be between 10 - 80ng. LoD’s for KRAS G12/G13, EGFR L858 and exon 19 deletion, and BRAF V600E were established at &amp;lt; 4%. Positive percent agreement (PPA) and negative percent agreement (NPA) between oRDx-LCCA and CompO were &amp;gt; 99% each. Across all variables tested for reproducibility, the average percent positive agreement (APA) and average percent negative agreement (ANA) were each &amp;gt; 95%. NI margin was calculated to be &amp;lt; 10% between oRDx-LCCA and CompQ across all NSCLC samples and between oRDx-LCCA and CompC across all CRC samples. A total of 61 synthetic FASTQ datasets were created to validate the functionality and accuracy of PiVAT including VAF thresholds for 126 SNV and INDEL CDx variants. Conclusion: oRDx-LCCA is a highly accurate FDA approved IVD assay and PiVAT is a powerful FDA approved software tool for the detection of clinically relevant KRAS variants in CRC and EGFR variants in NSCLC and determination of approved therapy. Citation Format: Gloria Chan Johnson, Jordan Aldersley, Nicholas Lodato, Jamie Lim, Ian Flockhart, Mark Urbin, Yue Ke, Sean Polvino, Martin Zillmann, Gang Song, Zhaohui Wang. Validation of FDA approved oncoReveal Dx lung and colon cancer assay (oRDx-LCCA) [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 5795.

Associations of genetic risk, BMI trajectories, and the risk of non-small cell lung cancer: a population-based cohort study

BackgroundBody mass index (BMI) has been found to be associated with a decreased risk of non-small cell lung cancer (NSCLC); however, the effect of BMI trajectories and potential interactions with genetic variants on NSCLC risk remain unknown.MethodsCox proportional hazards regression model was applied to assess the association between BMI trajectory and NSCLC risk in a cohort of 138,110 participants from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. One-sample Mendelian randomization (MR) analysis was further used to access the causality between BMI trajectories and NSCLC risk. Additionally, polygenic risk score (PRS) and genome-wide interaction analysis (GWIA) were used to evaluate the multiplicative interaction between BMI trajectories and genetic variants in NSCLC risk.ResultsCompared with individuals maintaining a stable normal BMI (n = 47,982, 34.74%), BMI trajectories from normal to overweight (n = 64,498, 46.70%), from normal to obese (n = 21,259, 15.39%), and from overweight to obese (n = 4,371, 3.16%) were associated with a decreased risk of NSCLC (hazard ratio [HR] for trend = 0.78, P < 2×10−16). An MR study using BMI trajectory associated with genetic variants revealed no significant association between BMI trajectories and NSCLC risk. Further analysis of PRS showed that a higher GWAS-identified PRS (PRSGWAS) was associated with an increased risk of NSCLC, while the interaction between BMI trajectories and PRSGWAS with the NSCLC risk was not significant (PsPRS= 0.863 and PwPRS= 0.704). In GWIA analysis, four independent susceptibility loci (P < 1×10−6) were found to be associated with BMI trajectories on NSCLC risk, including rs79297227 (12q14.1, located in SLC16A7, Pinteraction = 1.01×10−7), rs2336652 (3p22.3, near CLASP2, Pinteraction = 3.92×10−7), rs16018 (19p13.2, in CACNA1A, Pinteraction = 3.92×10−7), and rs4726760 (7q34, near BRAF, Pinteraction = 9.19×10−7). Functional annotation demonstrated that these loci may be involved in the development of NSCLC by regulating cell growth, differentiation, and inflammation.ConclusionsOur study has shown an association between BMI trajectories, genetic factors, and NSCLC risk. Interestingly, four novel genetic loci were identified to interact with BMI trajectories on NSCLC risk, providing more support for the aetiology research of NSCLC.Trial registrationhttp://www.clinicaltrials.gov, NCT01696968.

Intratumoral heterogeneity as a predictive biomarker in immunotherapy for advanced non-small cell lung cancer.

e21058 Background: Intratumoral heterogeneity (ITH) is associated with the poor prognosis in a variety of solid tumors. However, the use of ITH as a predictive biomarker for immunotherapy in patients with advanced non-small cell lung cancer (NSCLC) is still lacking. Methods: We used the Bioconductor R package Maftools to analyze somatic variants of NSCLC from an independent cohort (Hellmman CM012 study cohort) with data from 75 patients with advanced NSCLC. We then used a mutant-allele tumor heterogeneity (MATH) algorithm to measure ITH and analyzed the correlation with immunogenic marker, and the prognostic effect of ITH on immunotherapy. Results: The objective response rate (ORR) and durable clinical benefit (DCB) of immunotherapy was 36.5% and 53.8% for the patients with low ITH (ITH-L), and 21.7% and 39.1% for the high ITH (ITH-H) subgroup (P = 0.0427, P = 0.3180). The ITH-L patients had significantly improved median progression free survival (mPFS) than the ITH-H group (7.98 versus 2.60 months, hazard ratio (HR) = 1.88, 95%CI 1.06−3.34, P = 0.0280). The ORR and DCB of immunotherapy was 45.9% and 59.5% for the patients with high TMB (TMB-H), and 18.4% and 39.5% for the low TMB (TMB-L) subgroup (P = 0.0354, P = 0.1077). The TMB-H patients had significantly better mPFS than the TMB-L group (8.12 versus 3.78 months, HR = 0.49, 95%CI 0.28−0.87, P = 0.0130). A pooled analysis of ITH and TMB show that mPFS and ORR in the TMB-H+ITH-L group was significantly better than that in the TMB-H+ITH-H, TMB-L+ITH-H, TMB-L+ITH-L group (P = 0.0099, P = 0.0121). A subgroup analysis of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) found that ITH-L was associated with better mPFS (ITH-L versus ITH-H = 8.71 versus 2.17, HR = 3.42, 95%CI 0.96−12.23, P = 0.0460) in LUSC, and no significantly mPFS benefit was found in the ITH-L group in LUAD (ITH-L versus ITH-H = 7.98 versus 5.09, HR = 1.69, 95%CI 0.88−3.26, P = 0.1100). There was no significant difference in TMB levels between ITH-L and ITH-H groups (13.8 Muts/Mb versus 14.2 Muts/Mb, P = 0.2139). Conclusions: ITH may serve as a positive predictor of immunotherapy in patients with advanced NSCLC, especially in LUSC and their clinical value warrants further investigation.

A Polytherapy Strategy Using Vincristine and ALK Inhibitors to Sensitise EML4-ALK-Positive NSCLC.

Simple SummaryLung cancer is the third most common cancer worldwide with poor survival after initial diagnosis. A large proportion, approximately 85%, of patients with lung cancer are classified as non-small cell lung cancer (NSCLC). The most common genetic alterations in NSCLCs are KRAS, EGFR and ALK mutations. Patients carrying the EML4-ALK mutation are treated with potent ALK drugs. Up to date, there are 15 different EML4-ALK mutations found in NSCLC patients. Of note, patients carrying the EML4-ALK V3 mutation respond poorly to ALK chemotherapeutic regime and acquire resistance. The aim of the present study was to assess the potential of a combination chemotherapy using vincristine, a traditional chemotherapeutic drug, and potent ALK drugs. We demonstrated that vincristine sensitises cells carrying EML4-ALK V1 mutation but not V3. Cells carrying the EML4-ALK V3 mutation confer low response to drug combination due to high levels of tubulin acetylation and active proliferation pathways compared to V1 cells.The oncogenic fusion of EML4-ALK is present in about 4–6% of non-small cell lung cancer (NSCLC). A targeted approach with ALK tyrosine kinase inhibitors (TKIs) has been proven highly effective in ALK-positive NSCLC patients. However, despite the initial responses, the outcome of the treatment is variable. Previous studies have shown that the differential response depends in part on the type of EML4-ALK variant. Here, we examined the combination of ALK inhibitors and microtubule poison, vincristine, in cells expressing EML4-ALK V1 and V3, the two most common variants in NSCLC. We showed that combination therapy of ALK-TKIs with vincristine had anti-proliferative effects and blocked RAS/MAPK, PI3K/AKT and JAK/STAT3 signalling pathways in EML4-ALK V1 but not V3 cells. Our results demonstrate that high levels of tubulin acetylation are associated with poor response to vincristine in EML4-ALK V3 cells. Additionally, we demonstrated differences in microtubule stability between the two EML4-ALK fusions. EML4-ALK V3 cells exhibited dynamic microtubules that confer poor response to vincristine compared to V1 cells. Hence, we suggested that the portion of EML4 in the fusion has an important role for the outcome of the combination treatment.

Targeting rare and non-canonical driver variants in NSCLC – An uncharted clinical field

ObjectivesImplementation of tyrosine kinase inhibitors (TKI) and other targeted therapies was a main advance in thoracic oncology with survival gains ranging from several months to years for non-small-cell lung cancer (NSCLC) patients. High-throughput comprehensive molecular profiling is of key importance to identify patients that can potentially benefit from these novel treatments. Material and MethodsNext-generation sequencing (NGS) was performed on 4500 consecutive formalin-fixed, paraffin-embedded specimens of advanced NSCLC (n = 4172 patients) after automated extraction of DNA and RNA for parallel detection of mutations and gene fusions, respectively. Results and ConclusionBesides the 24.9 % (n = 1040) of cases eligible for approved targeted therapies based on the presence of canonical alterations in EGFR exons 18–21, BRAF, ROS1, ALK, NTRK, and RET, an additional n = 1260 patients (30.2 %) displayed rare or non-canonical mutations in EGFR (n = 748), BRAF (n = 135), ERBB2 (n = 30), KIT (n = 32), PIK3CA (n = 221), and CTNNB1 (n = 94), for which targeted therapies could also be potentially effective. A systematic literature search in conjunction with in silico evaluation identified n = 232 (5.5 %) patients, for which a trial of targeted treatment would be warranted according to available evidence (NCT level 1, i.e. published data showing efficacy in the same tumor entity).In conclusion, a sizeable fraction of NSCLC patients harbors rare or non-canonical alterations that may be associated with clinical benefit from currently available targeted drugs. Systematic identification and individualized management of these cases can expand applicability of precision oncology in NSCLC and extend clinical gain from established molecular targets. These results can also inform clinical trials.

Abstract B029: Comparison of a comprehensive NGS profiling assay and conventional molecular testing approaches for detection of clinically relevant alterations in NSCLC

Abstract Introduction: Genomic analysis using next-generation sequencing (NGS) enables simultaneous detection of targetable alterations and biomarkers with emerging clinical utility in non-small cell lung cancer (NSCLC) patients. Importantly, many of these alterations are mutually exclusive and tumor tissue for molecular testing is often limited. As such, we developed PGDx elioTM tissue complete (ETC) as a comprehensive NGS assay capable of detecting somatic single nucleotide variants (SNVs), insertions and deletions (indels), amplifications, and rearrangements, as well as microsatellite stability (MSI) and tumor mutational burden (TMB). Here, we present the performance of this assay in detecting key clinical variants in NSCLC. Methods: Studies comprising &amp;gt;300 NSCLC specimens (FFPE tissue and characterized cell lines) were analyzed using ETC, a 500+ gene assay (under development), across key clinically relevant variants in NSCLC, including tumor mutation burden (TMB). Accuracy of the results were compared to orthogonal methods (e.g. whole-exome sequencing (WES), IHC, FISH) and analyzed for the overall percent agreement (OPA). Additionally, archival FFPE samples from 46 NSCLC patients previously found to harbor ALK translocations, MET amplifications, MET exon 14 skipping mutations, EGFR mutations, and/or ROS1 translocations were also analyzed. The results were compared to orthogonal methods and the overall genomic landscape evaluated. Results: Clinical FFPE and characterized cell line specimens were evaluated for the following alterations: EGFR mutations (L858R, T790M, and Exon 19 deletions), BRAF V600E mutations, and ALK and ROS1 translocations. Compared to orthogonal methods, the NGS assay demonstrated &amp;gt;93% OPA across all variants. Comparison of TMB results to WES data demonstrated high accuracy and precision, across a range of DNA inputs (50-200 ng) and tumor purities (10-30%). In the 46 retrospective NSCLC cases, the NGS assay identified 15 ALK translocations, 6 MET amplifications, 1 MET exon 14 skipping mutation, and 5 EGFR mutations, with most being mutually exclusive. The majority of cases were confirmed by orthogonal assays, with the few apparent discordances likely due to tumor heterogeneity, assay distinctions, or analyte input. Higher TMB was found in cases without targetable alterations. Conclusions: ETC provides accurate and reproducible results for the detection of clinically relevant alterations in NSCLC. Further verification and validation studies of this gene panel are ongoing. Overall NGS showed excellent concordance with orthogonal variant detection methods. Importantly, ETC demonstrated added value in assessing all genomic alteration types in a single assay, as well as reporting composite genomic scores, suggesting that NGS may offer a comprehensive solution to molecular testing. Citation Format: Elizabeth Weingartner, Kelly M.R. Gerding, Gustavo Cerquiera, Christopher Gault, James Hernandez, Kenneth Valkenburg, Laurel Keefer, Eileen Sagini, Dorhyun Johng, Caitlin Gilley, Colby Ganey, Leila Ettehadieh, Diandra Denier, Christina Oliveras, Kartikeya Joshi, Eric Kong, Eniko Papp, Amy Greer, James R. White, Donna Nichol, John Simmons. Comparison of a comprehensive NGS profiling assay and conventional molecular testing approaches for detection of clinically relevant alterations in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B029. doi:10.1158/1535-7163.TARG-19-B029

Abstract 1764: Accurate and reproducible detection of fusions and exon skipping events in NSCLC-derived samples using a comprehensive, targeted RNA-Seq system across multiple laboratories

Abstract Introduction: Reliable assessment of cancer-associated RNA markers in lung cancer produced by gene-fusions or exon skipping events by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We evaluated a comprehensive system for targeted RNA-Seq that includes reagents for nucleic acid quantification, library prep, run controls, and companion bioinformatics software. The reproducibility of this system was evaluated in a multi-phase study design at 5 independent laboratories. Methods: Total nucleic acid (TNA) was isolated from formalin-fixed, paraffin-embedded (FFPE) residual non-small cell lung cancer (NSCLC) tumor biopsies and cell-lines (RT-112, H596, HCC78). These TNA isolates were used to prepare a set of 30 test samples, including a dilution series to assess assay sensitivity. A non-template control and kit positive control were also included. The sample set was evaluated using the QuantideX® NGS RNA Lung Cancer Kit RUO (Asuragen) and sequenced on the MiSeq® system (Illumina) at Asuragen and 4 independent laboratories. Analyses were conducted using QuantideX® NGS Reporter RUO (Asuragen), a software suite that includes a FASTQ processing pipeline and incorporates pre-analytical QC information into the fusion-caller algorithm and reporting tool. Results: Laboratories were trained on the assay workflow and companion bioinformatics software in less than two days followed by independent library preparation and sequencing workflows. A total of 266 sample libraries were evaluated from inputs down to &amp;lt;10 ng. A single library was excluded from sequencing due to a failed QC status. Specific targeted fusions (ALK, ROS1, FGFR3) and splice variants (MET exon 14 skipping) were detected in 132 libraries and were concordant at all sites. Designs to detect 3’/5’ expression imbalances reported the presence of gene fusions for ALK and ROS1 in a total of 96 libraries with one aberrant call and one missed call, which occurred in libraries flagged as “at risk” by the interpretive software. Conclusions: The accuracy of this novel targeted NGS assay for RNA fusions and splice variants in NSCLC was demonstrated in a multi-site laboratory evaluation using clinically-relevant specimens and low inputs of TNA. The ability of the panel to detect both common and rare gene fusion transcripts and exon skipping events within an integrated wet- and dry-bench workflow provides a foundation for the reliable detection of oncogenic RNA fusions and aberrant splicing events that can respond to current and emerging targeted therapies. This study highlighted the ease of implementation and consistent performance that can be achieved in different laboratories when the process from sample-to-report is highly integrated. Citation Format: Gary J. Latham, Richard Blidner, Brian C. Haynes, Shobha Gokul, Maria L. Aguirre, Stephen Hyter, Ziyan Y. Pessetto, Maria Curtis, Dan Su, Tom Halsey, Victor Weigman, Patrick Hurban, Andrew K. Godwin, Leon C. van Kempen. Accurate and reproducible detection of fusions and exon skipping events in NSCLC-derived samples using a comprehensive, targeted RNA-Seq system across multiple laboratories [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1764. doi:10.1158/1538-7445.AM2017-1764

7 - Optimization of Blood-Based Liquid Biopsy Assays for the Rapid Identification of 19 Actionable RNA Fusion Variants in NSCLC

Background: Non-small-cell lung cancer (NSCLC) remains a diagnostic challenge for physicians. Considering the lethality of this disease, rapid diagnostic turnaround times are a critical factor in patient care. While the highest risk factor for NSCLC is smoking history, nonsmokers who develop the disease more commonly have chromosomal rearrangements, including fusions in anaplastic lymphoma kinase (ALK), c-rosoncogene 1, receptor tyrosine kinase (ROS1), and rearranged during transfection (RET) proto-oncogene. The incidence of these variants in the total NSCLC population is estimated to be 5%, 2%, and 1%, respectively. Biodesix has developed commercially available blood-based tests for circulating tumor RNA that detects the presence of actionable RNA fusion variants in NSCLC for use in its CLIA certified laboratory. Methods: Starting with up to 4 mL of patient plasma, circulating RNA is isolated and converted into cDNA in a single reaction. The cDNA produced is tested with multiplexed ALK, ROS1, and RET ddPCR assays covering 78%, 87.5% and 99% of the prevalent fusion variants, respectively. Results are provided to physicians within 72 hours of sample receipt. Results: 19 fusion variants were detected with an analytic sensitivity of 0.2% or lower and 100% analytic specificity. With EML4-ALK variants, a clinical concordance of 92% was achieved. Conclusion: We have developed novel blood-based tests for the most common NSCLC RNA fusion variants that result from ALK, ROS1, and RET rearrangements. The rapid turn-around offered by these blood-based tests has the potential to significantly shorten time to treatment for patients.

Abstract 1535: Next generation sequencing of brain metastasis in non-small cell lung cancer

Abstract Introduction: Molecular analysis of non-small cell lung cancer (NSCLC) has provided a detailed classification of NSCLC. Activating mutations in the EGFR gene are both prognostic and predictive in that they are associated with improved survival irrespective of therapy and are associated with a significant response to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib or erlotinib. Patients with EGFR mutations show a significant improvement in progression free survival compared to standard chemotherapy. In addition, a recent study showed that ALK gene rearrangement was significantly associated with pericardial spread and pleural disease and those patients with either ALK gene rearrangements or EGFR mutations were predisposed to liver metastasis. These results support the hypothesis that the dominant molecular oncogenes in NSCLC are associated with different biological behaviors which manifest as distinct patterns of metastatic spread at the time of diagnosis. To date, no pattern of molecular variation has been linked to brain metastasis. Here we use next generation sequencing to identify a pattern of genomic variation associated with the development of brain metastases in NSCLC. Methods: Three patient groups were analyzed: samples from NSCLC patients who did not develop metastasis (n = 8), samples from NSCLC patients who developed metastasis not associated with the central nervous system (n = 7), and brain metastasis samples of NSCLC patients (n = 16). DNA was isolated, and libraries prepared using Qiagen's Comprehensive Cancer Panel which focuses on 160 cancer-related genes. Libraries were sequenced on the Illumina NextSeq 500. Alignment was done using SoftGenetic's NextGENe with subsequent biological interpretation with Ingenuity Variant Analysis. Results: At the variant level, a missense mutation in CDK12 (chr1:37686934) was found in 7 of the 16 cases with brain metastasis while only 1 of 8 cases with no metastasis and 0 of 7 cases with non-CNS metastasis had the variant. However, no other variants were found in ≥6 cases of brain metastasis and 1 or less of the control cases. After filtering variants that occurred in more than 1 control (no metastasis or non-CNS metastasis), the TP53 gene was altered in 11 of the 16 cases with brain metastasis. This included 14 different variants with none of these variants present in the controls. Additionally, BRCA1 and CDK12 had variants in 9 cases with brain metastasis, but the variant was also present in 1 of the controls. Taken together, all 16 cases contained alterations in at least 1 gene involved in PI3K/AKT signaling including variants in JAK1, MTOR, TP53, TSC1, and TSC2. Conclusion: This pilot study has delivered valuable results to better characterize the pattern of genomic variants in NSCLC. While no single variant was associated with brain metastasis, this study implicates PI3K/AKT signaling and, in particular, variants of TP53 as crucial for determining the potential development of NSCLC brain metastasis. Citation Format: Bryan Thibodeau, Paola Yumpo Cardenas, Samreen Ahmed, Marc Dunn, Matthew Johnson, George Wilson. Next generation sequencing of brain metastasis in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1535.

Abstract C148: Rapid blood-based test for ALK, RET and ROS1 mRNA fusions and somatic variants in NSCLC

Abstract Tissue based methods for the detection of ALK fusions include FISH and IHC. Patients with advanced non-small cell lung cancer (NSCLC) may not be good candidates for biopsies and in cases where tissue is obtained, it is not always sufficient for testing. Additionally, determination of the full range of tumor heterogeneity and any impact of molecular variants on therapeutic responses may not be fully captured by the current FISH and IHC tests being conducted for ALK. For these reasons, blood-based testing that uses PCR and NGS methods for detection of mRNA fusion transcripts and somatic variants in circulation are becoming more clinically relevant. Pre-analytic as well as primer design are critical parameters in the development of circulating RNA tests. We have approached the pre-analytic variables by assessing multiple potential sources of fusion RNAs in prospectively-collected plasma samples, including cell-free RNA (cfRNA) and exosomal RNA. Additionally, test development for ALK, RET and ROS1 (ARR) utilized fusion-partner agnostic primers, only requiring knowledge of the fusion kinase sequence. This test technology has previously been shown in FFPE-extracted mRNA specimens to have the unique capability to identify common fusions, including EML4-ALK variants 1 and 3a/b, as well as rare fusions and novel transcript variants. In this study we have extended the capability of this technology to plasma samples from donors previously diagnosed with NSCLC. Data generated during the development of this test involved extraction of RNA from cell lines and donor plasma, processing to cDNA, adaptor ligation and two-step anchored PCR with specificity for human ALK, RET and ROS1. Amplicons were sequenced on a MiSeq (v2 chemistry) and reads were mapped to the human genome and a fusion specific database in order to identify the specific fusion partner(s). Test turn-around time from RNA extraction to results averaged 72 hours. With this test system we evaluated known variant-positive cell lines for EML4-ALK, SLC34A2-ROS1 and CCDC6-RET, as well as remnant prospectively-collected plasma from NSCLC donors (females of less than 65 years of age, and wild-type for circulating EGFR sensitizing and KRAS somatic variants). All cell lines were positive for the respective fusions (lower limit of input RNA at 25ng) and negative in test cross-reactivity experiments (up to 250ng input RNA). The presence of the fusions in the cell lines were independently confirmed with variant-specific qPCR assays. We additionally tested cell-line spiked human donor samples and successfully demonstrated detection of cell-line derived ARR fusions and donor-derived wild-type ALK, RET &amp; ROS1. All human donor samples passed QC measures based on control gene expression. Ongoing experiments are being conducted to further develop this test and evaluate its relevance for routine blood-based clinical laboratory testing using plasma from patients diagnosed with NSCLC. Citation Format: Hestia Mellert, Kristina Koch, Shannon Campbell, Gary Pestano. Rapid blood-based test for ALK, RET and ROS1 mRNA fusions and somatic variants in NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C148.

Twenty-One Novel EGFR Kinase Domain variants in Patients with Nonsmall Cell Lung Cancer.

Somatic sequence variants in the epidermal growth factor receptor (EGFR) kinase domain are associated with sensitivity to tyrosine kinase inhibitors (TKIs) in patients with nonsmall cell lung cancer (NSCLC). Patients exhibiting sequence variants in this domain that produce kinase activity enhancement, are more likely to benefit from TKIs than patients with EGFR wild-type disease. Although most NSCLC EGFR-related alleles are concentrated in a few positions, established protocols recommend sequencing EGFR exons 18-21. In this study, 21 novel somatic variants belonging to such exons in adult Argentinean patients affected with NSCLC are reported. Of these, 18 were single amino acid substitutions (SASs), occurring alone or in combination with another genetic alteration (complex cases), one was a short deletion, one was a short deletion-short insertion combination, and one was a duplication. New variants and different combinations of previously reported variants were also found. Moreover, two of the reported SASs occurred in previously unreported positions of the EGFR kinase domain. In order to characterize the new sequence variants, physicochemical, sequence and conformational analyses were also performed. A better understanding of sequence variants in NSCLC may facilitate the most appropriate treatment choice for this complex disease.

Quantitative Real-Time Reverse Transcription–PCR Study of the Expression of Vascular Endothelial Growth Factor (VEGF) Splice Variants and VEGF Receptors (VEGFR-1 and VEGFR-2) in Non–Small Cell Lung Cancer

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and its expression is increased in non-small cell lung cancer (NSCLC). We aimed to determine the expression pattern of VEGF splice variants in NSCLC and its correlation with the clinicopathological characteristics of tumors. We used real-time reverse transcription PCR to quantify the mRNA expression of total VEGF, 4 VEGF splice variants (VEGF(121), VEGF(165), VEGF(183), and VEGF(189)), and 2 VEGF receptors (VEGFR-1 and VEGFR-2) in 27 pairs of cancerous and adjacent noncancerous tissues originating from patients with NSCLC. Total VEGF, VEGF(121), and VEGF(165) were expressed in all specimens, whereas VEGF(183) and VEGF(189) were present in small amounts in certain samples. Total VEGF, VEGF(121), and VEGF(165) mRNA was upregulated in cancerous compared with healthy tissues, whereas VEGF(183) and VEGF(189) expression tended to be higher in healthy tissues. The expression of VEGFRs was similar between matched specimens. No correlation was found between the expression of total VEGF or VEGF splice variants and the clinicopathological characteristics of tumors. The expression patterns of VEGF splice variants differed between tissue pairs. VEGF(121) was the major variant expressed in all samples; however, its relative expression was higher in cancerous tissues. The relative expression of VEGF(183) and VEGF(189) was upregulated in healthy lung tissues, whereas the ratio of VEGF(165) to total VEGF was similar between matched specimens. The expression pattern of certain VEGF splice variants is altered during tumorigenesis. Our data support the hypothesis that during malignant progression an angiogenic switch favoring the shorter diffusible isoforms occurs.

Real-Time RT-PCR Quantification of Human Telomerase Reverse Transcriptase Splice Variants in Tumor Cell Lines and Non–Small Cell Lung Cancer

We developed and validated a real-time reverse transcription (RT)-PCR for the quantification of 4 individual human telomerase reverse transcriptase (TERT) splice variants (alpha+beta+, alpha-beta+, alpha+beta-, alpha-beta-) in tumor cell lines and non-small cell lung cancer (NSCLC). We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript-specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients. In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The alpha+beta- splice variant showed the highest expression and alpha-beta+ and alpha-beta- the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the alpha+beta- variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by alpha+beta+, with the alpha-beta+ and alpha-beta- splice variants having the lowest expression. In the NSCLC tumors, the alpha+beta+ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival. Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT alpha+beta+ splice variant may be an independent negative prognostic factor for NSCLC patients.

A new era for bronchioloalveolar carcinoma: current state of the art and recent advances in biologically targeted therapy

Bronchioloalveolar carcinoma is a fascinating and unusual variant of nonsmall-cell lung cancer that has a tendency towards an indolent course and to metastasize to the lung rather than distant organs. Chemotherapy has shown activity in advanced bronchioloalveolar carcinoma but response rates remain low. Epidermal growth factor receptor tyrosine kinase inhibitors have shown impressive activity against bronchioloalveolar carcinoma in trials. New data suggest that epidermal growth factor receptor mutations and gene copy number may predict subsets of patients who could most benefit from these novel agents. These new findings may point the way to a new era in which we can predict which patients will respond to epidermal growth factor receptor tyrosine kinase inhibitors and thus allow us to tailor therapy to the individual patient.