Actionable Gene Fusions Research Articles

78P Real-world analysis of actionable gene fusions identified by NGS and correlation with IHC in 422 patients from the community

78P Real-world analysis of actionable gene fusions identified by NGS and correlation with IHC in 422 patients from the community

Evaluation of molecular residual disease in operable non-small cell lung cancer with gene fusions, MET exon skipping or de novo MET amplification.

Gene fusions and MET alterations are rare and difficult to detect in plasma samples. The clinical detection efficacy of molecular residual disease (MRD) based on circulating tumor DNA (ctDNA) in patients with non-small cell lung cancer (NSCLC) with these mutations remains unknown. This prospective, non-intervention study recruited 49 patients with operable NSCLC with actionable gene fusions (ALK, ROS1, RET, and FGFR1), MET exon 14 skipping or de novo MET amplification. We analyzed 43 tumor tissues and 111 serial perioperative plasma samples using 1021- and 338-gene panels, respectively. Detectable MRD correlated with a significantly higher recurrence rate (P < 0.001), yielding positive predictive values of 100% and 90.9%, and negative predictive values of 82.4% and 86.4% at landmark and longitudinal time points, respectively. Patients with detectable MRD showed reduced disease-free survival (DFS) compared to those with undetectable MRD (P < 0.001). Patients who harbored tissue-derived fusion/MET alterations in their MRD had reduced DFS compared to those who did not (P = 0.05). To our knowledge, this is the first comprehensive study on ctDNA-MRD clinical detection efficacy in operable NSCLC patients with gene fusions and MET alterations. Patients with detectable tissue-derived fusion/MET alterations in postoperative MRD had worse clinical outcomes.

Abstract 325: Validation of Aventa FusionPlus: An LDT NGS assay for comprehensive cancer genomic profiling

Abstract Next-generation sequencing (NGS) assays play a pivotal role in illuminating the genomic landscape of cancer by providing insights into gene regulation and biomarker detection for the purpose of guiding therapeutic decisions to advance personalized medicine. We validated the Aventa FusionPlus Test, a Laboratory-Developed Test (LDT) that integrates HiC technology to enhance detection of actionable gene fusions and rearrangements in solid tumors, including those that are missed by current standard of care tests. This is a DNA-based test that is robust to DNA degradation in FFPE specimens, in contrast with RNA-seq and long-read technologies. The test utilizes the Element Biosciences AVITI sequencing platform to achieve high quality sequencing data at low cost per sample. The Aventa FusionPlus test enables gene-level detection of structural variants near or within 361 medically actionable or diagnostically relevant cancer genes. The 3D structure and long-range linkages captured by the test amplify the signal counts for each rearrangement. The test integrates the Arima-HiC+ FFPE sample prep and the Arima Library Prep Kit workflow. After dewaxing and rehydrating the FFPE tissue, the chromatinized DNA is fragmented and the fragmented ends are filled in with a biotinylated nucleotide. Next, spatially proximal fragmented ends of DNA are ligated, capturing the 3D structure of the genome. The proximally-ligated DNA is then purified and further fragmented, and the biotinylated ligation fragments are enriched. DNA libraries are prepared from these enriched fragments and sequenced in “paired-end” mode. Genes involved in genomic rearrangements were detected using the Arima-SV pipeline along with manual curation. The test achieved robust detection of structural variants, with accuracy of 98.8% relative to reference methods. FFPE tumor specimens from 10 different tissue types and a wide range of tumor content (20%-100%) were included. No correlation between tumor cellularity and sensitivity was observed, suggesting that samples with as low as 20% tumor cellularity can be tested. When comparing to RNA-based and other targeted reference methods, we found additional fusions nearby oncogenes missed by these reference methods. As an example, in a breast cancer case, a fusion of genomic regions neighboring BRAF and MYC oncogenes on chromosomes 7 and 8 respectively, was missed by the reference method but was successfully detected by the Aventa FusionPlus test. The Aventa FusionPlus test is a highly sensitive, specific, and accurate method for comprehensive cancer genomic profiling. The enhanced understanding of gene fusions and rearrangements positions the test as a valuable tool for clinical decision-making in oncology. Citation Format: Shadi Melnyk, Jon Belton, Sofia Nomikou, Katelyn Powell, Daniel Heiney, Roy Khalife, Damien Jones, Anthony Schmitt, Catherine Wilson, Melissa Won, Anthony Magliocco, Chris Roberts. Validation of Aventa FusionPlus: An LDT NGS assay for comprehensive cancer genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 325.

Incidence and genomic characteristics of gene fusions in a large Chinese colorectal cancer cohort.

192 Background: Gene fusion is rare in colorectal cancer (CRC). With the advent of fusion-targeted therapeutics, such as entrectinib for NTRK fusion and selpercatinib for RET fusion, the identification of gene fusions holds clinical significance. Reports providing a comprehensive description of fusions in CRC are limited, and understanding the molecular characteristics of CRC with fusions will contribute to efficient fusion screening. Here, we investigate the incidence and genomic features of gene fusions in a large Chinese CRC population. Methods: We systematically analyzed next-generation sequencing results of tumor tissue from 5534 CRC patients between January 2020 and August 2023. The sequencing panel covers 769 cancer-related genes and can detect fusions, single nucleotide variants and indels, and microsatellite instability (MSI). Statistical significance was determined using chi-square test. Results: Overall, the median age of patients was 62 years (inter-quartile range [IQR]: 53-70), and 40% were female. Gene fusions were detected in 4.4% (242/5534) of all the patients. The incidence is 0.98% (54/5534) if only reported clinically actionable fusions are considered. The median age of the 242 fusion-positive patients was 64 years (IQR: 54-70), and 43% were female. The most highly detected potentially actionable gene fusions were NTRK (0.89%) and FGFR (0.81%), accounting for 39% of the fusion-positive patients. The other fusion genes were ERBB2 (0.52%), RET (0.38%), BRAF (0.31%), EGFR (0.27%), ALK (0.22%), MET (0.09%) and ROS1 (0.07%). Consistent with previous reports, patients with MSI-High were more likely to have fusions compared with those with microsatellite stability (MSS) or MSI-Low (9.9% vs 4.0%, p &lt; 0.001). Besides, RAS or BRAF mutated patients accounted for 59% of the cohort, of whom 2.5% (83/3273) had fusions detected, while 7.0% (159/2261) of the RAS and BRAF wildtype patients had positive fusions ( p &lt; 0.001). Moreover, 404 (7.3%) patients had RNF43 splice or truncating mutations, and gene fusions were detected in 12.9% of these patients. Combining these markers increased the detection rate of fusions, with 28% (33/118) of the RAS/ BRAF wildtype and MSI-H patients having fusions detected. Furthermore, in patients with wildtype RAS/BRAF and MSI-H and RNF43 splice or truncating mutations, the fusion detection rate was as high as 49% (32/65), including 24 patients with actionable fusions of definite clinical significance. Conclusions: Gene fusions were detected in 4.4% of the Chinese CRC population and tended to occur in patients with wildtype RAS/BRAF, MSI-H, or RNF43 mutations. Combining the mutational status of RAS, BRAF, and RNF43 along with MSI status can improve the fusion detection rate and help select candidates for fusion testing and targeted therapy.

Abstract A070: Genomic profiling of pancreatic cancer by KRAS status

Abstract Introduction: Ineffective treatments and late detection combine to give pancreatic cancer (PC) patients especially poor prognosis. However, as more targeted therapies are approved and are being investigated, there is hope for such therapies to play a pivotal role in improving PC patient outcomes. KRAS is an important oncogenic driver in most pancreatic cancers, making the RAS signaling pathway an obvious therapy target. In this study we examine the genomic profile of pancreatic cancers that do or do not harbor a RAS alteration to identify other oncogenic drivers that may represent therapy targets. Methods: Pancreatic tumor samples were profiled using the OncoExTraTM tumor-normal, whole exome, whole transcriptome sequencing assay. This assay detects somatic single base substitutions, indels, copy number alterations, gene fusions and alternative transcripts. Actionable altered genes, defined as those with associated FDA-approved targeted therapies in any cancer type, those in guidelines or with published evidence for matched therapies, or those required for eligibility for an active clinical trial, were identified. Results: A total of 384 pancreatic patients were profiled, 305 (79.4%) of which harbored 310 KRAS alterations. Most KRAS alterations were missense mutations (307 of 310; 99.0%). Most (283) missenses caused a substitution at amino acid position 12, with G12D (n=115), G12V (n=104) and G12R (n=54) being the most common. There were also 3 missense substitutions at position 13, and 21 at position 61. In addition, three patients harbored both a KRAS missense mutation and an amplification. In addition to KRAS, several other altered biomarkers were relatively prevalent, including TP53 (63.3%), CDKN2A (25.3%), ARID1A (9.1%), CDKN2B (7.0%), BRCA2 (4.4%), MEN1 (3.9%) and MTAP (3.6%). TP53 alterations were significantly overrepresented in KRAS-altered cancers. Alterations at MEN1, DAXX, ATRX, SETD2 and CREBBP, and high tumor mutational burden (TMB-high) were all overrepresented in cancers lacking KRAS alterations. In addition, all 19 (4.9%) actionable gene fusions involving oncogenic drivers (6 BRAF, 6 FGFR1/2, 3 NRG1, 2 RET, 1 RAF1, 1 ROS1) were found only in cancers lacking a KRAS alteration. Conclusions: Our study shows differences in the genomic profile based on KRAS status. We identified potentially targetable gene fusions and other alterations enriched in pancreatic tumors lacking KRAS alterations, suggesting several therapies may be reasonable options in such patients. Additionally, this analysis underscores the benefit of whole transcriptome profiling that identified actionable fusions in 4.9% of patient samples, all of which were unaltered at KRAS. Citation Format: Gargi D. Basu, Snehal G. Thakkar, David W. Hall, Cynthia A. Flannery, Satish Seerapu, Maen Abdelrahim. Genomic profiling of pancreatic cancer by KRAS status [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A070.

Analysis of actionable gene fusions in a large cohort of Chinese patients with colorectal cancer.

The prevalence of gene fusion is extremely low in unselected patients with colorectal cancer (CRC). Published data on gene fusions are limited by relatively small sample sizes, with a primary focus on Western populations. This study aimed to analyse actionable gene fusions in a large consecutive Chinese CRC population. This study included 5,534 consecutive CRC patients from the Genecast database. Genomic profiling was performed using a panel of 769 cancer-related genes. Data for 34 CRC patients with actionable gene fusions were also collected from cBioPortal and ChimerSeq. Among 5,534 CRC patients, 54 (0.98%) had actionable gene fusions, with NTRK1/2/3 being the most common fusion (0.38%), accounting for 38.9% (21/54) of those with fusions. Actionable gene fusion enrichment was higher in patients with microsatellite instability-high (MSI-H) (6.7% vs. 0.5%, P < 0.001), RAS/BRAF wildtype (2.0% vs. 0.2%, P < 0.001) and RNF43 mutation (7.7% vs. 0.4%, P < 0.001) than in patients with microsatellite stability/MSI-low, RAS/BRAF mutation and RNF43 wildtype, respectively. When these markers were combined, the fusion detection rate increased. Among patients with RAS/BRAF wildtype and MSI-H, fusions were detected in 20.3% of patients. The fusion detection rate further increased to 37.5% when RNF43 mutation was added. The fusion detection rate was also higher in colon cancer than in rectal cancer. No significant differences in clinical or molecular features were found in patients with actionable gene fusions between the Genecast, cBioPortal, and ChimerSeq databases. Approximately 1% of the unselected Chinese CRC population carries actionable gene fusions, mostly involving NTRK. Actionable gene fusions are more prevalent in MSI-H, RAS/BRAF wildtype, or RNF43-mutated CRC, as well as in colon cancer. Mapping of these molecular markers can markedly increase the fusion detection rate, which can help clinicians select candidates for fusion testing and targeted therapy.

Circulating Tumor DNA Enables Sensitive Detection of Actionable Gene Fusions and Rearrangements Across Cancer Types.

Genomic rearrangements can generate potent oncogenic drivers or disrupt tumor suppressor genes. This study examines the landscape of fusions and rearrangements detected by liquid biopsy (LBx) of circulating tumor DNA (ctDNA) across different cancer types. LBx from 53,842 patients with 66 solid tumor types were profiled using FoundationOneLiquid CDx, a hybrid-capture sequencing platform that queries 324 cancer-related genes. Tissue biopsies (TBx) profiled using FoundationOneCDx were used as a comparator. Among all LBx, 7,377 (14%) had ≥1 pathogenic rearrangement detected. A total of 3,648 (6.8%) LBx had ≥1 gain-of-function (GOF) oncogene rearrangement, and 4,428 (8.2%) LBx had ≥1 loss-of-function rearrangement detected. Cancer types with higher prevalence of GOF rearrangements included those with canonical fusion drivers: prostate cancer (19%), cholangiocarcinoma (6.4%), bladder (5.5%), and non-small cell lung cancer (4.4%). Although the prevalence of driver rearrangements was lower in LBx than TBx overall, the frequency of detection was comparable in LBx with a tumor fraction (TF) ≥1%. Rearrangements in FGFR2, BRAF, RET, and ALK, were detected across cancer types, but tended to be clonal variants in some cancer types and potential acquired resistance variants in others. In contrast to some prior literature, this study reports detection of a wide variety of rearrangements in ctDNA. The prevalence of driver rearrangements in tissue and LBx was comparable when TF ≥1%. LBx presents a viable alternative when TBx is not available, and there may be less value in confirmatory testing when TF is sufficient.

Clinical relevance of gene mutations and rearrangements in advanced differentiated thyroid cancer

Tumor genotyping is becoming crucial to optimize the clinical management of patients with advanced differentiated thyroid cancer (DTC); however, its implementation in clinical practice remains undefined. We herein report our single-center experience on molecular advanced DTC testing by next-generation sequencing approach, to better define how and when tumor genotyping can assist clinical decision making. We retrospectively collected data on all adult patients with advanced DTC who received molecular profiling at the IRCSS Sant'Orsola-Malpighi Hospital from 2008 to 2022. The genetic alterations were correlated with radioactive iodide refractory (RAI-R), RAI uptake/disease status, and time to RAI resistance (TTRR) development. A significant correlation was found between RAI-R development and genetic alterations (P= 0.0001). About 48.7% of RAI-R cases were positive for TERT/TP53 mutations (as both a single event and comutations with other driver gene alterations, such as BRAF mutations, RAS mutations, or gene fusions), while the great majority of RAI-sensitive cases carried gene fusions (41.9%) or were wild type (WT; 41.9%). RAI uptake/disease status and time to TTRR were significantly associated with genetic alterations (P= 0.0001). In particular, DTC with TERT/TP53 mutations as a single event or as comutations displayed a shorter median TTRR of 35.4 months (range 15.0-55.8 months), in comparison to the other molecular subgroups. TERT/TP53 mutations as a single event or as comutations remained independently associated with RAI-R after Cox multivariate analysis (hazard ratio 4.14, 95% CI 1.51-11.32; P= 0.006). Routine testing for genetic alterations should be included as part of the clinical workup, for identifying both the subset of more aggressive tumors and the subset of tumors harboring actionable gene fusions, thus ensuring the appropriate management for all patients with advanced DTC.

Detection of clinically actionable gene fusions by next-generation sequencing-based RNA sequencing of non-small cell lung cancer cytology specimens: A single-center experience with comparison to fluorescence insitu hybridization.

Genomic profiling is needed to identify actionable alterations in non-small cell lung cancer (NSCLC). Panel-based testing such as next-generation sequencing (NGS) is often preferred to interrogate multiple alterations simultaneously. In this study, we evaluate the utility of an RNA-based NGS assay to detect genomic alterations in NSCLC cytology specimens and compare these results to fluorescence in situ hybridization (FISH) testing. A retrospective review was performed of 264 NSCLC cytology specimens that were concurrently tested for gene fusions by RNA-based NGS and ALK, RET, and/or ROS1 by FISH. Genomic alterations were detected in 29 cases by NGS, including ALK, RET, ROS1, NTRK, NUTM1, and FGFR3 fusions and MET exon 14 skipping alterations. Of the 20 cases with ALK, RET, and ROS1 fusions detected by NGS, 16 (80%) were concordant with the corresponding FISH results. Three cases showed discordance, where EML4::ALK (n=2) and SLC34A2::ROS1 (n=1) fusions were not detected by the corresponding FISH assay; one case with EZR::ROS1 was inadequate for FISH. No gene fusions were detected in 181 cases by NGS and 54 cases failed testing. The concordance rates for detecting ALK, RET, and ROS1 fusions using NGS and FISH were 97%, 100%, and 99.5%, respectively. RNA-based NGS can be used to detect gene fusions in NSCLC cytology cases with high concordance with FISH results. However, RNA-based NGS may have high failure rates and therefore a low threshold for reflexing inadequate cases to an orthogonal testing method is essential for comprehensive genomic profiling.

Uterine Sarcoma With EML4::NTRK3 Fusion: A Spectrum of Mesenchymal Neoplasms Harboring Actionable Gene Fusions.

NTRK gene fusions are part of a paradigm shift in oncology, arising as one of the main genomic alterations with actionability in the so-called "agnostic setting." In gynecologic pathology, the recent description of uterine sarcoma resembling fibrosarcoma and with NTRK rearrangements (NTRK-rearranged uterine sarcoma) highlights the importance of recognizing clinicopathological cues that can lead to genomic profiling. Herein, we report the case of a 43-year-old woman presenting with vaginal bleeding and pelvic mass. Histopathology of the tumor showed moderately atypical spindle cells arranged in long fascicles reminiscent of fibrosarcoma, along with immunohistochemical positivity for S100, CD34, and pan-tropomyosin receptor kinase. This prompted RNA-sequencing and the finding of a rare EML4::NTRK3 fusion. Clinical, histologic, and molecular findings are described, in addition to discussions regarding differential diagnoses and possible implications of the findings in clinical practice.

3D genomics identifies previously unreported yet clinically actionable gene fusions and complex rearrangements in a cohort of driver-negative colorectal carcinoma tumors.

e15608 Background: Molecular characterization by next-generation sequencing (NGS) has been unable to detect pathogenic driver mutations in a significant number of tumors. For example, 40% of colorectal carcinomas (CRC) are RAS and BRAF wild-type and do not have therapeutic targets after comprehensive molecular characterization using FISH, IHC and NGS. Analysis of 3D genomics data produced by DNA-based chromosome conformation capture (Hi-C) assay has been shown to detect gene fusions and other rearrangements in tumor biopsies. We applied Hi-C in a cohort of CRC tumors previously characterized using FISH, IHC, whole exome sequencing (WES) and whole transcriptome sequencing (WTS) by a large commercial reference laboratory, where no targets had been reported, to evaluate the potential for Hi-C to identify novel therapeutic targets. Methods: FFPE tissue from 14 CRC tumors were subjected to Hi-C using the Arima HiC+ for FFPE kit. Hi-C libraries were sequencing to ~100M raw read-pairs on an Illumina NovaSeq, and fusions were called using the Arima-SV pipeline. To attribute clinical significance, we characterized fusions based on whether the fusion event involved a Tier 1 therapeutic level biomarker or a Tier 3 diagnostic or prognostic biomarker according to NCCN and OncoKb, or a Tier 2 biomarker if the biomarker was the target of a therapy in an ongoing clinical trial. Results: Hi-C detected fusions implicating a Tier 1 biomarker in 50% (7/14) of tumors, a Tier 2 level biomarker in 14% (2/14), and a Tier 3 level biomarker in 14% (2/14). Homologous recombination repair (HRR) gene rearrangements were detected in 29% (n = 4/14) of CRC tumors, disrupting HRR genes ( BRIP1, BRCA1, BRCA2, RAD51D). NRG1 gene fusions were detected in 14% (2/14), and amplified complex rearrangement carrying KRAS was detected in one CRC case. A complex rearrangement involving extrachromosomal DNA (ecDNA) amplification carrying EGFR was detected in two cases that were both previously found to have EGFR amplifications, but the ecDNA nature was not previously known and was confirmed by FISH. Lastly, Hi-C detected gene rearrangements involving POLE or APC in two CRC cases, alterations in which are known to increase risk for colonic polyposis. Conclusions: Fusion detection by Hi-C in CRC may uncover numerous therapeutic biomarkers not currently detected by molecular profiling. Our data suggests that homologous recombination deficiency (HRD) may frequently result from HRR gene rearrangements readily detectable by Hi-C, and this alternate mechanism for HRD may serve as a therapeutic biomarker for HRD directed therapies. Our data also suggests that therapeutically targetable gene fusions and complex rearrangements involving therapeutic level biomarkers are also readily detectable by Hi-C, detection of which may increase the number of CRC patients treatable with targeted therapies.

Abstract 4355: Prediction of cancer treatment response from histopathology images for a broad set of treatments and indications

Abstract Background: Advances in artificial intelligence have paved the way for predicting cancer patients’ survival and response to treatment from hematoxylin and eosin (H&amp;E)-stained tumor slides. Extant approaches do so either via prediction of actionable mutations and gene fusions, or directly from the H&amp;E images by training a task-specific model using large treatment outcome data. Methods: Here we present the first approach for predicting patient response to multiple targeted treatments and immunotherapies directly from H&amp;E slides. It is founded on two conceptual steps: (1) First, we developed DeepPT, a deep-learning framework that trains on formalin-fixed, paraffin-embedded (FFPE) TCGA whole slide images and their corresponding gene expression profiles to predict wide-scale tumor gene expression from the slides. (2) Second, we apply ENLIGHT, a published approach that predicts individual responses to a wide range of targeted and immunotherapies based on the tumor biopsy measured transcriptomics. Here we apply ENLIGHT to predict patient treatment response from the DeepPT predicted expression values instead of those measured directly from the tumor, notably, without any further training or adaptation of ENLIGHT. Results: First, we find that DeepPT generalizes well to predicting gene expression in all 13 TCGA cohorts tested in cross-validation and importantly, in two independent unseen breast and brain cancer datasets. DeepPT outperforms HE2RNA, a state-of-the-art algorithm for the same task. Second, we demonstrate that the combined DeepPT/ENLIGHT pipeline (termed ENLIGHT-DeepPT) successfully predicts true responders using the original ENLIGHT decision threshold with odds ratios of 1.5 - 4.5, increasing the baseline response rates by 15-85% among predicted responders in five independent unseen cohorts of diverse cancer types and treatments. Remarkably, in one dataset where matched data was available, ENLIGHT-DeepPT has similar performance to that obtained by a supervised learning algorithm that was recently published in Nature, trained on the same cohort. Conclusions: We present for the first time a general framework for predicting patient response to a broad array of targeted and checkpoint therapies from histopathological images, without reliance on treatment outcome data, which are yet scarce and challenging to obtain. Importantly, ENLIGHT-DeepPT offers clinicians real-time treatment recommendations when one cannot wait for sequencing results. Also, and due to its very low cost, we very much hope that it will facilitate the advent of precision oncology in developing countries. Citation Format: Danh-Tai Hoang, Gal Dinstag, Leandro C. Hermida, Doreen S. Ben-Zvi, Efrat Elis, Katherine Caley, Sanju Sinha, Neelam Sinha, Christopher H. Dampier, Tuvik Beker, Kenneth Aldape, Ranit Aharonov, Eric A. Stone, Eytan Ruppin. Prediction of cancer treatment response from histopathology images for a broad set of treatments and indications. [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 4355.

Abstract 1254: Actionable fusions detected by RNA-seq co-occur with PD-L1 expression and driver mutations in solid tumors patients

Abstract Background: Incorporating gene fusions into a comprehensive profile is critical not only because very effective therapies targeting oncogenic fusion proteins exist but they may also negate the response to therapy of an actionable SNV and InDel mutations. We examined the co-occurrence of actionable gene fusions detected by RNA-seq with actionable SNVs/InDels and with the immunotherapy response biomarker, PD-L1. Methods: In 2021, 5341 FFPE samples were analyzed by our clinical laboratory using a novel hybridization-based RNA sequencing assay. DNA (SNV/Indels) mutations were detected with a clinical grade NGS assay and PD-L1 protein expression was determined by IHC using an appropriate LDT or FDA approved assay. De-identified data were analyzed following an approved IRB protocol. Results: Of the 5341 patients tested for gene fusions only 0.7% were profiled with a comprehensive fusion detection panel for 250 clinically relevant fusion genes. Conversely, 67% of all patients were tested only for NTRK gene fusions. The prevalence of the following most relevant fusions was: ALK=3.16% (in particular EML4-ALK), NTRKs=0.85%, FGFR2=5.60%, RET=1.34% and ROS1=1.05%. Among NTRK fusions, NTRK3 was detected in 52.9% of the positive cases; in particular, dominant fusions are ETV6-NTRK3 (26%) and EML4-NTRK3 (20%). In the 163 fusion positive cases, 37 cases had available DNA mutation testing and PD-L1 expression testing. These patients presented 24 different actionable fusions, including ALK (2), FGFR1/2 (5), NTRK 1/2/3 (10), NRG1 (3), RET (3), and ROS1 (3) fusions. Interestingly, while 73% (27/37) of those tumors were PD-L1 positive, similar to the 75% found on the fusion negative samples, PD-L1 was positive in 88% (15/17) of the lung samples with pathogenic mutations from this subset. This was strikingly higher than the 45% (192/424) found in the fusion negative cohort. Finally, excluding TP53 mutations, NTRK fusions frequently co-occurred with RNF43, FBXW7, TERT promoter, and ARID1A pathogenic mutations. FGFR fusions co-occurred with BAP1, PBRM1, or KRAS mutations, which correlates with the fact that both FGFR2 fusions and swi/snf alterations are enriched in intrahepatic cholangiocarcinoma, where the FGFR fusions were detected. RET fusions co-occurred with ARID1A and TERT. ROS1 fusions co-occurred with SMARCA4 and KRAS pathogenic mutations simultaneously. NRG1 fusions co-occurred with ARID1A and FBXW7 mutations. EML4-ALK fusions co-occurred with FGFR2 and KMT2D variants of unknown significance. Conclusion: Actionable cancer-driving gene fusions were detected by RNA-sequencing and co-occurred with other biomarkers that can guide the selection of therapies, such as immune checkpoint inhibitors (ICI) and olaparib. The data suggest that gene fusion testing is an important addition to the genomic profiling for therapy selection in solid tumors. Citation Format: Miyoung Shin, Paris Petersen, Steven Lau-Rivera, Segun Jung, Sally Agersborg, Jacyln Hechtman, Fernando Lopez-Diaz, Vincent Funari. Actionable fusions detected by RNA-seq co-occur with PD-L1 expression and driver mutations in solid tumors patients [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 1254.

Abstract 84: Sensitive and unbiased detection of clinically actionable gene fusions from FFPE tumor biopsies using the Arima-HiC platform

Abstract Accurate and sensitive identification of gene fusions is critical to understanding disease mechanisms and the development and selection of optimal treatment regimens for cancer patients. Typically, these fusions are detected using low-resolution karyotyping, low throughput and biased FISH assays, or RNA sequencing approaches; however, the accuracy and sensitivity of gene fusion detection can be limited by factors such as low transcript abundance, transcript length, RNA degradation from formalin fixed paraffin embedded (FFPE) tissues, or the limited availability of fresh biopsy samples for RNA extraction. To address these limitations, we developed a novel approach to identifying gene fusions from FFPE samples using the Arima-HiC platform and short-read sequencing. We performed pan-cancer analysis on 12 FFPE adult tumor biopsies, each with gene fusions known to be clinically actionable. With this newly developed Hi-C workflow we identified all the known gene fusions with 100% sensitivity in the samples including those involving ALK, NTRK3, ROS1, FGFR2, and SS18 genes. This approach also revealed that the NTRK3 gene fusion was the result of a more complex rearrangement within chr12, within chr15, and between chr12 and chr15. Additionally, we detected the presence of numerous structural variants per sample in addition to the known gene fusion in each sample. For example in a bile duct tumor, Arima-HiC detected an FGFR2-EEA1 gene fusion, as well as 25 other structural variants genome-wide including in a CPD-LASP1 gene fusion This gene fusion has not been reported to our knowledge, however, LASP1 is a reported 3’ fusion partner with MLL in leukemia. While these findings are not clinically actionable today, the unbiased, accurate, and sensitive detection of structural variants from frozen and FFPE solid tumor biopsies may facilitate further research into their relationship between disease mechanisms and clinical outcomes. Taken together, these findings demonstrate the analytical utility of Arima Hi-C sequencing technology to provide both chromosome-scale and gene-level resolution for the detection of structural variants in tumor biopsies samples. This workflow can provide improved access to critical genomic information from FFPE blocks for the identification of pathognomonic and druggable gene fusion events and other structural variants across tumor types. Citation Format: David Jacob Hermel, Kristin Sikkink, Derek Reid, Anthony Schmitt, Darren S. Sigal. Sensitive and unbiased detection of clinically actionable gene fusions from FFPE tumor biopsies using the Arima-HiC platform [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 84.

A cell-free RNA-based next-generation sequencing (NGS) assay for the detection of actionable gene fusions in patients with non–small cell lung cancer (NSCLC).

3040 Background: Gene fusions and alternate RNA splice forms represent clinically actionable driver and resistance-conferring alterations in NSCLC and other cancers. Where tissue samples are inadequate for molecular testing, liquid biopsies could address the gap in detection of clinically relevant gene fusions. Targeted cell-free (cf) DNA-based NGS methods are typically used for non-invasive blood-based testing of gene fusions, but have limited sensitivity if fusion breakpoints involve long intronic regions or repetitive sequences (e.g. NTRKs and NRG1). RNA-based approaches are not affected by introns, can identify expressed fusion genes and can discriminate splicing. We developed an NGS assay optimized for cfRNA analyte for the detection of actionable gene fusions and exon deletion/skipping events in liquid biopsies. Methods: A highly multiplexed molecular-barcoded primer panel was designed for cfRNA-based detection of actionable fusion genes in NSCLC ( ALK, BRAF, FGFR2, FGFR3, MET (including exon 14 skipping), NRG1, NTRK1/2/3, RET and ROS1) covering &gt; 90% of reported driver and partner gene exons in COSMIC database. The panel also targets housekeeping genes as endogenous sample controls. We developed a custom bioinformatics pipeline to call fusions and exon skipping based on split and spanning reads. Results: In initial analytical validation using fragmented RNA from pre-characterized reference material (representing 32 unique fusions at known copy numbers), the assay could detect as low as 10 fusion copies with a sensitivity of 97.6% and a specificity of 100%. For clinical testing, cfRNA co-eluted with cfDNA in nucleic acid extracts from plasma was used. In plasma samples (n = 103) from advanced NSCLC, 76% (15/21) of fusions (5 ALK, 3 RET, 2 ROS1, 5 MET ex14 skipping, 1 FGFR3-TACC3) detected in a clinically validated cfDNA assay (LiquidHALLMARK) were also detected in corresponding cfRNA. In a subset of samples that were driver-negative untreated or tyrosine kinase-inhibitor treated, the cfRNA assay yielded 9 gene fusions or breakpoints (1 CD74-NRG1, 4 BRAF, 2 MET, 2 FGFR3-TACC3 fusions) that could not be detected by the cfDNA assay. This represents an increase of 8.7% (9/103) of actionable alterations (driver or resistance) identified using cfRNA. Together cfRNA and cfDNA resulted in 30 fusion events to be detected compared to 21 by the cfDNA assay alone, representing a 42.8% (9/21) increase in fusion-specific detection of the combined assay. Conclusions: This novel cfRNA assay can detect actionable gene fusions and exon skipping events in liquid biopsies with high sensitivity. Combining cfRNA with more routine cfDNA testing can increase the total actionable diagnostic information from non-invasive testing in NSCLC patients where tissue samples are lacking, especially for gene fusions not amenable to detection in cfDNA.

Systematic analysis of actionable gene fusions by NGS-based in pancancer for precision oncology in China.

e15072 Background: Detection of gene fusions with NGS is widely used for clinical diagnosis and drug development. To deeply understand clinically actionable gene fusions for implementation of precision oncology, we systematically analyzed the distribution of tumor types of each fused driver gene, and other mutation types in the same sample which were accompanied. Methods: Samples were performed with NGS based 450 tumor genes panel assay. Genomic alterations, including single nucleotide variations (SNV), short and long insertions/deletions (InDels), copy number variations and gene fusions were analyzed using a published algorithm. Results: We collected 1740 patients with 1478 known fusions and 338 novel fusions. Of all the patients, 48.5% were male, 6.5% were TMB-H (≥ 10 muts/Mb) and 0.6% were MSI-H. We found that breast carcinoma (BRCA) accounts for 28.6% of the ERBB2 fusions, intrahepatic cholangiocarcinoma (ICC) accounts for 60.4% of FGFR2 fusions, non-small cell lung cancer (NSCLC) accounts for 45.5% of FGFR1, 41.5% of FGFR3, and 87.3% of ALK fusions, respectively, renal cell carcinoma (RCC) accounts for 65.9% of TFE3 fusions, the soft tissue sarcoma (STS) accounts for 30.3% of NTRK1 fusions, 70.6% of PDGFB fusions, and 75.6% of SS18 fusions, respectively. We identified EGFR fusions in specific tumor types such as colorectal carcinoma (CRC) and Glioma (GBM), MET fusions in gallbladder carcinoma (GBC), PDGFRA fusions in NSCLC, BRAF fusions in ICC and FGFR3 fusions in urothelial carcinoma (UC). About novel fusions, many of them were detected in multiple tumor types, for example, PHF20-NTRK1 was detected in CRC, NSCLC and ovarian carcinoma (OC), FGFR2-INA was found in ICC and NSCLC, FGFR2-PAWR was detected in BRCA and ICC. Some partner genes were shared by multiple driver genes. For example, TTC28 presented as partner of FGFR2 in ICC, of NRG1 in ICC, of NTRK3 in hepatocellular carcinoma (HCC). Some novel fusions were found more than twice in one tumor type, for example, CMAHP-ALK was detected twice in NSCLC and EHBP1-MET was detected three times in ICC. These novel fusions are helpful to drug development and molecular diagnosis. Some co-occurring variations with fusions often existed in same patient. For example, patients with ALK fusion were also detected SNV of ALK such as L1196, F1174, and G1202. In addition, amplification of ERBB2 and EGFR were both 1.7% in patients with fusions, and CDKN2A/B deletion occupied 5.1% often with EML4-ALK and FGFR3-TACC3. Conclusions: In our study, many new novel fusions and known fusion in new tumor type were revealed. The regular pattern of co-occurring variations of fusion helps to improve the fusion detection rate and clinical application. This simultaneous detection of multiple meaningful variants is the advantage of NGS methods over other traditional methods. All of the above will helpful for improving the clinical benefit rate and accelerating target drug development.

Dissection of the expressed actionable fusions’ repertoire in solid tumors tested serially in the clinical setting across 14 cancer types.

e18804 Background: More frequently, select gene fusions have been tested in clinical practice as more approvals of very effective therapies targeting oncogenic chimeric fusion proteins. Large studies based on DNA-based NGS testing alongside clinical trials have established the prevalence of clinically actionable gene fusions such as NTRK1/2/3. However, much less is known about the broader repertoire and prevalence of bonafide expressed gene fusions. Methods: 6009 tumors across 14 cancer types were tested in our clinical laboratory between January and October 2021. Enrichment was performed using a custom hybridization-based capture after RNA to cDNA conversion with custom designed probes targeting &gt; 1104 different genes and enriched for 2230 specific RNA fusions to maximize sensitivity and breadth before sequencing ̃50 million 150x150 reads/sample on a NovaSeq 6000. Data was deidentified and aggregated according an approved IRB. Statistical significance was calculated by Fisher’s exact test. Results: We found that 37% of tumors tested carry &gt; 1 expressed chimeric transcript and that 7.2% of tumors (n = 431) express a gene fusion involving one of 19 actionable gene families/pathways targeted by an approved therapy. The most frequent involved FGFR1/2/3/4 (15%), ALK (14%), NTRK1/2/3 (12%), BRAF, ERBB2, RET and MET (6% each), RAS/RAF/MAPK and CDK4/6 pathway (5%). These fusions imply intra- and inter-chromosomal translocations on all chromosomes, especially on chromosomes 1, 3, 6, 7, 11, 12 and 17. NTRK1/2/3 fusions prevalence (0.9%) is significantly higher (p = 1.1e-10) than the 0.3% reported on a recent study from a large dataset (296K samples) based on DNA-sequencing testing (1). Moreover, we found that 32% of the NTRK partners were unique vs. 10% (p = 6.5e-08) from the Westphalen et al. study (1). 14 of 25 unique NTRK partners have not been described on large datasets (1) and only 3 have been reported on the literature as NTRK partners. Among them, we identified the hypoxia-induced neovascularization gene HIF1A, as a novel in frame 3’ fusion partner of NTRK3 [t(15;14)(15q25.3; 14q23.1). Finally, NTRK fusions were detected across 14 cancer types with higher frequency on brain tumors (17%), followed by lung cancers, colorectal and head and neck cancer (13% each) and sarcomas (12%). Conclusions: Gene fusions involving common actionable genes showed a high prevalence in the clinical setting. This study confirms that the number and breadth of gene fusions can present with common actionable information in &gt; 7% of clinical cases. We report here 14 new unique NTRK partners. Despite a 50 times smaller dataset, 3-fold more actionable expressed NTRK fusions involving novel partners were detected with a targeted RNA based hybridization approach in lieu of DNA based testing supporting the benefit of this strategy. Overexpression and translation can be confirmed only by this approach.

Targeted RNA sequencing for upfront analysis of actionable driver alterations in non-small cell lung cancer

ObjectivesTargeted RNA-based Next-Generation Sequencing (tRNA-seq) is increasingly being used in molecular diagnostics for gene fusion detection in non-small cell lung cancer (NSCLC). However, few data support its clinical application for the detection of single nucleotide variants (SNVs) and small insertions/deletions. In this study, we evaluated the performance of tRNA-seq using Archer FusionPlex for simultaneous detection of actionable gene fusions, splice variants, SNVs and indels in formalin-fixed, paraffin-embedded NSCLC tissue. Materials and methodsA total of 126 NSCLC samples, including 20 validation samples and 106 diagnostic cases, were analyzed by targeted DNA-based Next-Generation Sequencing (tDNA-seq) followed by tRNA-seq. ResultsAll 28 SNVs and indels in the validation set, and 34 out of 35 mutations in the diagnostic set were identified by tRNA-seq. The only mutation undetected by tRNA-seq, ERBB2 p.(Ser310Tyr), was not included in the current Archer panel design. tRNA-seq revealed one additional BRAF p.(Val600Glu) mutation not found by tDNA-seq. SNVs and indels were correctly called by the vendor supplied software, except for ERBB2 duplication p.(Tyr772_A775dup) which was only detected by an additional in-house developed bio-informatics pipeline. Variant allelic frequency (VAF) values were generally higher at the expression level compared to the genomic level (range 6–96% for tRNA-seq versus 6–61% for tDNA-seq) and low VAF mutations in DNA (6–8% VAF) were all confirmed by tRNA-seq. Finally, tRNA-seq additionally identified a driver fusion or splice variant in 10 diagnostic NSCLC samples including one MET exon 14 skipping variant not detected by tDNA-seq. ConclusionOur results demonstrate that tRNA-seq can be implemented in a diagnostic setting as an efficient strategy for simultaneous detection of actionable gene fusions, splice variants, SNVs and indels in NSCLC provided that adequate RNA-seq analysis tools are available, especially for the detection of indels. This approach allows upfront identification of currently recommended targetable molecular alterations in NSCLC samples.

A functional genomic approach to actionable gene fusions for precision oncology.

Fusion genes represent a class of attractive therapeutic targets. Thousands of fusion genes have been identified in patients with cancer, but the functional consequences and therapeutic implications of most of these remain largely unknown. Here, we develop a functional genomic approach that consists of efficient fusion reconstruction and sensitive cell viability and drug response assays. Applying this approach, we characterize ~100 fusion genes detected in patient samples of The Cancer Genome Atlas, revealing a notable fraction of low-frequency fusions with activating effects on tumor growth. Focusing on those in the RTK-RAS pathway, we identify a number of activating fusions that can markedly affect sensitivity to relevant drugs. Last, we propose an integrated, level-of-evidence classification system to prioritize gene fusions systematically. Our study reiterates the urgent clinical need to incorporate similar functional genomic approaches to characterize gene fusions, thereby maximizing the utility of gene fusions for precision oncology.

Fusion-positive non-small cell lung carcinoma: Biological principles, clinical practice, and diagnostic implications.

Based on superior efficacy and tolerability, targeted therapy is currently preferred over chemotherapy and/or immunotherapy for actionable gene fusions that occur in late-stage non-small cell lung carcinoma (NSCLC). Consequently, current clinical practice guidelines mandate testing for ALK, ROS1, NTRK, and RET gene fusions in all patients with newly diagnosed advanced non-squamous NSCLC (NS-NSCLC). Gene fusions can be detected using different approaches, but today RNA next-generation sequencing (NGS) or combined DNA/RNA NGS is the method of choice. The discovery of other gene fusions (involving, eg, NRG1, NUT, FGFR1, FGFR2, MET, BRAF, EGFR, SMARC fusions) and their partners has increased progressively in recent years, leading to the development of new and promising therapies and mandating the development and implementation of comprehensive detection methods. The purpose of this review is to focus on recent data concerning the main gene fusions identified in NSCLC, followed by the discussion of major challenges in this domain.