Increased detection of NRG1 fusions in non-squamous non-small cell lung cancer using combined DNA and RNA sequencing in a real-world cohort.

Background: We investigated the clinical implications of NRG1 fusion, a newly identified oncogenic rearrangement, in surgically resected invasive mucinous adenocarcinoma (IMA) of the lung. Patients and Methods: We performed RNA sequencing on 7 lung adenocarcinomas that had no known driver oncogenes and identified a novel fusion, SLC3A2-NRG1 in a case of IMA. The newly detected NRG1 fusion was verified in an IMA cohort of 59 cases using reverse transcription polymerase chain reaction (RT-PCR), direct sequencing and fluorescence in situ hybridization (FISH) analysis. Targeted caner panel sequencing and RT-PCR were performed to identify the possible coexistence of NRG1 fusions and other genetic alterations. Results: After screening 59 IMA, we found a total of 16 NRG1 fusions (13 SLC3A2-NRG1 and 3 CD74-NRG1). Among the 16 cases with NRG1 fusion, concurrent KRAS codon 12 mutations were found in 10 cases. We also found concurrent NRAS Q61L mutation and EML4-ALK fusion in additional two cases with NRG1 fusion. When comparing survival according to the presence of an NRG1 fusion, patients harboring NRG1 fusion showed inferior overall survival (OS) compared to those without NRG1 fusion (median 51.9 months [mo] vs. not reached [NR], P = 0.019). They also showed a trend toward shorter disease-free survival (DFS) (median 21.9 vs. 79.4 mo, P = 0.113). In the stage I IMA, patients with NRG1 fusion showed significantly inferior OS (median 48.1 mo vs. NR, P = 0.009) and DFS (median 18.9 vs. 91.1 mo, P = 0.013) compared to those without NRG1 fusion. Conclusion: NRG1 fusion is an important prognostic factor in IMA and should be considered a novel therapeutic target for IMA. Citation Format: Ji-Youn Han, Yeon-Su Lee, Dong Hoon Lee, Dong Wan Hong, Seung Hyun Hong, Jung-Ah Hwang, Byung Il Lee, Hye Jin You, Dong Hoon Shin, Geon Kook Lee. Clinical implications of NRG1 fusion in invasive mucinous adenocarcinoma of the lung. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 614. doi:10.1158/1538-7445.AM2015-614

Genomic Landscape of Resected Invasive Mucinous Adenocarcinoma of the Lung.

To identify druggable oncogenic fusions in invasive mucinous adenocarcinoma (IMA) of the lung, a malignant type of lung adenocarcinoma in which KRAS mutations frequently occur. From an IMA cohort of 90 cases, consisting of 56 cases (62%) with KRAS mutations and 34 cases without (38%), we conducted whole-transcriptome sequencing of 32 IMAs, including 27 cases without KRAS mutations. We used the sequencing data to identify gene fusions, and then performed functional analyses of the fusion gene products. We identified oncogenic fusions that occurred mutually exclusively with KRAS mutations: CD74-NRG1, SLC3A2-NRG1, EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET. NRG1 fusions were present in 17.6% (6/34) of KRAS-negative IMAs. The CD74-NRG1 fusion activated HER2:HER3 signaling, whereas the EZR-ERBB4 and TRIM24-BRAF fusions constitutively activated the ERBB4 and BRAF kinases, respectively. Signaling pathway activation and fusion-induced anchorage-independent growth/tumorigenicity of NIH3T3 cells expressing these fusions were suppressed by tyrosine kinase inhibitors approved for clinical use. Oncogenic fusions act as driver mutations in IMAs without KRAS mutations, and thus represent promising therapeutic targets for the treatment of such IMAs.

Invasive mucinous adenocarcinoma (IMA) is a unique subtype of lung adenocarcinoma, characterized genomically by frequent KRAS mutations or specific gene fusions, most commonly involving NRG1. Comprehensive analysis of a large series of IMAs using broad DNA- and RNA-sequencing methods is still lacking, and it remains unclear whether molecular subtypes of IMA differ clinicopathologically. A total of 200 IMAs were analyzed by 410-gene DNA next-generation sequencing (MSK-IMPACT; n = 136) or hotspot 8-oncogene genotyping (n = 64). Driver-negative cases were further analyzed by 62-gene RNA sequencing (MSK-Fusion) and those lacking fusions were further tested by whole-exome sequencing and whole-transcriptome sequencing (WTS). Combined MSK-IMPACT and MSK-Fusion testing identified mutually exclusive driver alterations in 96% of IMAs, including KRAS mutations (76%), NRG1 fusions (7%), ERBB2 alterations (6%), and other less common events. In addition, WTS identified a novel NRG2 fusion (F11R-NRG2). Overall, targetable gene fusions were identified in 51% of KRAS wild-type IMAs, leading to durable responses to targeted therapy in some patients. Compared with KRAS-mutant IMAs, NRG1-rearranged tumors exhibited several more aggressive characteristics, including worse recurrence-free survival (P < 0.0001). This is the largest molecular study of IMAs to date, where we demonstrate the presence of a major oncogenic driver in nearly all cases. This study is the first to document more aggressive characteristics of NRG1-rearranged IMAs, ERBB2 as the third most common alteration, and a novel NRG2 fusion in these tumors. Comprehensive molecular testing of KRAS wild-type IMAs that includes fusion testing is essential, given the high prevalence of alterations with established and investigational targeted therapies in this subset.

Introduction: Invasive mucinous adenocarcinoma (IMA) accounts for approximately 4-20% of non-small-cell lung carcinoma (NSCLC). Genetic alterations and RNA sequencing analysis of IMAs reveal a unique mucinous signature similar to gastrointestinal mucinous tumors. Due to its rarity, focused clinical trials for IMA patients are often not feasible. These patients respond poorly to platinum-based conventional chemotherapy, or immunotherapy. Lenvatinib is a multitargeted-TKI with demonstrated activity in patients with RET fusion-positive lung adenocarcinoma, thyroid, hepatocellular and gastric carcinoma. Methods: Here, we present two RET fusion-negative mucinous lung adenocarcinoma cases that received a multitargeted TKI lenvatinib with a clinical and survival benefit. Results: Case #1: An 81-year-old female initially diagnosed with cT4N0M0 IMA (TTF1 and CDX2 focally positive) was started on neoadjuvant treatment for a 12cm tumor. She underwent right thoracotomy/right lower lobectomy/mediastinal lymph node dissection, and pathology confirmed ypT4N1M0 mucinous adenocarcinoma. Three months post-operatively, the patient had disease progression in the lungs. She had no targetable mutations. She had four subsequent lines of treatment including nivolumab/nab-paclitaxel as the fourth-line treatment. After six months of nivolumab/nab-paclitaxel, lenvatinib was added to the combination due to clear disease progression evident on imaging. After twelve cycles of nivolumab/nab-paclitaxel/lenvatinib combination treatment, patient achieved a partial response per RECIST 1.1 (-35.7% change from baseline to current). The patient reported less dyspnea and increased activity tolerance. Approximately nine months after the start of the treatment, patient showed progressive lung disease. The patient died after one more line treatment with 41 months of total overall survival. Case#2: A 56-year-old male initially diagnosed with stage IVA IMA (TTF1 negative). After four lines of treatments, patient was started on lenvatinib alone. The patient reported marked improvement in dyspnea and pain and increased activity tolerance with lenvatinib. Imaging confirmed treatment response after three months of treatment. The patient was subsequently enrolled in the double lung transplant (DLT) registry aimed for lung-limited malignancies (D.R.E.A.M.) registry study (NCT05671887), and he is alive with 25 months follow-up since DLT. Conclusion: We report two cases of metastatic RET fusion-negative mucinous lung adenocarcinoma, in which treatment with the multitargeted TKI Lenvatinib led to significant clinical improvement. Biological rationale to explain the effect of lenvatinib in invasive mucinous lung adenocarcinoma needs to be explored. Citation Format: Tarik Demir, Carolyn Moloney, Liam Il-Young Chung, Young Kwang Chae. Lenvatinib as a potential treatment option for invasive mucinous lung adenocarcinoma [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 6495.

Lung Cancer ManagementVol. 6, No. 4 EditorialFree AccessNRG1: a cinderella fusion in lung cancer?Lucia Anna Muscarella & Antonio RossiLucia Anna Muscarella Laboratory of Oncology, Scientific Institute for Research & Health Care (IRCCS) 'Casa Sollievo della Sofferenza', San Giovanni Rotondo (FG), Italy & Antonio Rossi*Author for correspondence: Tel.: +39 0882 410 716; Fax: +39 0882 204 095; E-mail Address: arossi_it@yahoo.it Division of Medical Oncology, Scientific Institute for Research & Health Care (IRCCS), Casa Sollievo della Sofferenza Hospital, Viale Cappuccini 1, 71013, San Giovanni Rotondo (FG), ItalyPublished Online:5 Jan 2018https://doi.org/10.2217/lmt-2017-0018AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: ErbBNRG1 fusionNSCLCThe amazing bridge between gene fusions and lung cancer has been greatly consolidated during the last decades. The identification of ALK and ROS1 rearrangements gives the great opportunity to rewrite the standard-of-care for a portion of advanced non-small-cell lung cancer (NSCLC) patients. Despite their very low incidence, these well-known genomic rearrangements share common features strictly associated to specific phenotypes of lung adenocarcinoma. This makes them ideal for diagnostic procedures, patients' stratification and therapies [1,2]. In this scenario, the finding of further intriguing targetable gene fusions, despite with a low incidence (1–2%) in NSCLC, is now increasing and it is watched with attention by the clinical community [3].Recently, the NRG1 gene has been described as a new molecular feature of NSCLC [4]. The NRG1 gene is located at the long arm of chromosome 10 (10q23.1 region) and encodes for the neuregulin 1, a growth factor belonging to the complex family of proteins also called heregulins. These proteins are structurally related to the stimulation of ERBB receptors tyrosine kinase activity and EGF signals. Specifically, the NRG1-receptor binding induces the phosphorylation of the intrinsic kinase domains of ERBB3 and stimulates its dimerization with ERBB2 receptor and the activation of the downstream PI3K-AKT and MAPK pathways [5].The neuronal isoform NRG1 III-β3 is generally not expressed in normal lung tissue, but it was found to be ectopically activated in lung tumor cells by NRG1 genomic rearrangements involving mainly CD74 and SLC3A2 genes [6]. It is the first fusion associated with the mucinous subtype of lung adenocarcinoma with an occurrence ranging from 8 to 27%. By contrast, NRG1-gene fusions occur in 1–2% of NSCLC and occasionally reported in other solid tumors [7]. It has been more extensively investigated in Asian than Caucasian lung cancer patients in whom it has been not yet fully elucidated and remains only partially understood [6].ERBB signaling is one of the most deregulated lung cancer cascades. ERBB3 is not frequently affected by mutations or amplifications in lung cancer. Furthermore, MET or HER2 amplifications, which represent additional mechanisms of ERBB3 activation in lung tumors, rarely occur. Thus, the overproduction of NRG1 ligands could represent one of the leading mechanisms by which lung cancer cells aberrantly activate ERBB3-related receptor tyrosine kinase signaling.The first suggestion of a real clinical utility of NRG1 fusion comes by analyzing a large sample of mucinous lung adenocarcinoma in Asians for the NRG1 breaks. In fact, patients with stage I disease harboring tumors with NRG1 fusions showed an inferior overall survival and a trend toward a shorter disease-free survival compared with those without NRG1 fusions [8]. The powerfulness of this fusion has been highlighted in vitro. In fact, the expression of CD74–NRG1 fusion gene is able to promote cancer stem cell properties and it is involved in stem cell function of several types of cancers, including lung cancer. These data imply the existence of a mechanism by which the activated ERBB receptors contribute to the acquisition of cancer stem cell-like characteristics together with the ability of cancer cells to develop a resistance to chemotherapy [9]. Moreover, in the absence of RAS pathway mutations, NRG1 overexpression can play a major role in the primary cetuximab resistance in colon cancer cells and in primary resistance to trastuzumab in HER2 overexpressing breast cancer cells [10,11].Of interest, NRG1 fusions have been proved to be coexistent with ALK fusion or RAS mutation in NSCLC patients, both in primary and in metastatic sites of lung tumors [12–14]. Moreover, it has been recently considered as a potential mechanism of resistance after treatment with tyrosine kinase inhibitors in ALK-rearranged NSCLC cell lines. In fact, by using primary cultures of cancer cells from pleural effusion of an ALK-positive lung cancer patient, the increase of the NRG1 ligand levels and the consequent activation of ERBB3 pathway has been directly related to resistance to crizotinib treatment [15]. This assumption has been further confirmed showing that, under treatment with second-generation ALK inhibitors, NSCLC cells activated the EGFR family pathways directly through the NRG1–ERBB3–EGFR activation axis [16]. In support to these in vitro reports, the onset of the SLC3A2–NRG1 gene fusion during the natural history of two invasive mucinous lung adenocarcinoma in Asiatic patients has been described. These heavily pretreated patients received the combination of lumretuzumab, a monoclonal anti-ERBB3 antibody, plus erlotinib, an anti-EGFR small molecule, showing tumor shrinkage [17]. Furthermore, the use of afatinib, a pan-ERBB-family kinase inhibitor, in NRG1-positive samples resulted in a surprisingly durable response in patients with lung adenocarcinoma and cholangiocarcinoma [7,18].ERBB3 overexpression actually represents one of the targets of greater interest for the current pharmacological studies. In fact, its activation through phosphorylation has been detected in various cancers including metastatic lung carcinoma in presence of acquired resistance to other ERBB family inhibitors [19]. Moreover, due to its inactive tyrosine kinase domain, ERBB3 has been reported to have a critical role in the dimerization process in the context of acquired de novo resistance to ERBB3 targeted therapies. However, despite the well-tested efficacy of EGFR and HER2 inhibitors, ERBB3 specific upregulation has not been yet targeted with clear clinical efficacy. Of all the anti-ERBB3 agents, patritumab is in advanced clinical development, being currently investigated in a Phase III trial for treatment of NSCLC (JapicCTI-101262) [20]. Additional trials are investigating neratinib, alone and in combination with temsirolimus (NCT01827267) and trastuzumab emtansine (T-DM1; NCT02289833), in HER2 molecular profiled advanced NSCLC. Of great interest is MM-121. An ongoing open-label trial is investigating MM-121, a fully human monoclonal antibody targeting specifically ERBB3, in combination with docetaxel or pemetrexed compared with docetaxel or pemetrexed alone, in patients with heregulin-positive, advanced NSCLC with primary end point overall survival (NCT02387216).Overall, NRG1 fusions could represent new potential molecular alterations able to predict the therapy activity in a specific lung adenocarcinoma subtype. Since NRG1 fusions act through the activation of the ERBB receptor, blocking the activity of the NRG1–ERBB–PI3K–AKT pathway might be the best strategy for the treatment of NRG1-fused tumors.Financial & competing interests disclosureThis work was supported by the Italian Ministry of Health (Ricerca Corrente, RC1703LO41 to LA Muscarella and RC1703ON39 to A Rossi), by the '5x1000' voluntary contributions and by the AIRC/MFAG grant 12983 (to LA Muscarella) and FBNC Grant 2015–2016 to LA Muscarella. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.References1 Kwak El, Bang YJ, Camidge DR et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N. Engl. J. Med. 363(18), 1693–1703 (2010).Crossref, Medline, CAS, Google Scholar2 Davies KD, Le AT, Theodoro MF et al. Identifying and targeting ROS1 gene fusions in non-small-cell lung cancer. Clin. Cancer Res. 18(17), 4570–4579 (2012).Crossref, Medline, CAS, Google Scholar3 Schram AM, Chang MT, Jonhsson P, Drilon A. Fusions in solid tumours: diagnostic strategies, targeted therapy and acquired resistance. Nat. Rev. Clin. Oncol. doi: 10.1038/nrclinonc.2017.127 (2017) (Epub ahead of print).Crossref, Medline, Google Scholar4 Fernandez-Cuesta L, Plenker D, Osada H et al. CD74–NRG1 fusions in lung adenocarcinoma. Cancer Discov. 4(4), 415–422 (2014).Crossref, Medline, CAS, Google Scholar5 Yarden Y, Pines G. The ERBB network: at last, cancer therapy meets systems biology. Nat. Rev. Cancer 12(8), 553–563 (2012).Crossref, Medline, CAS, Google Scholar6 Trombetta D, Rossi A, Fabrizio FP et al. NRG1–ErbB lost in translation: a new paradigm for lung cancer? Curr. Med. Chem. doi: 10.2174/0929867324666170911170554 (2017) (Epub ahead of print).Crossref, Medline, Google Scholar7 Jones MR, Lim H, Shen Y et al. Successful targeting of the NRG1 pathway indicates novel treatment strategy for metastatic cancer. Ann. Oncol. doi: 10.1093/annonc/mdx523 (2017) (Epub ahead of print).Crossref, Google Scholar8 Shin DH, Lee D, Hong DW et al. Oncogenic function and clinical implications of SLC3A2–NRG1 fusion in invasive mucinous adenocarcinoma of the lung. Oncotarget 7(43), 69450–69465 (2016).Crossref, Medline, Google Scholar9 Murayama T, Nakaoku T, Enari M et al. Oncogenic fusion gene CD74–NRG1 confers cancer stem cell-like properties in lung cancer through a IGF2 autocrine/paracrine circuit. Cancer Res. 76(4), 974–983 (2016).Crossref, Medline, CAS, Google Scholar10 Luraghi P, Bigatto V, Cipriano E et al. A molecularly annotated model of patient-derived colon cancer stem-like cells to assess genetic and non-genetic mechanisms of resistance to anti-EGFR therapy. Clin. Cancer. Res. doi: 10.1158/1078-0432.CCR-17-2151 (2017) (Epub ahead of print).Crossref, Medline, Google Scholar11 Yang L, Li Y, Shen E et al. NRG1-dependent activation of HER3 induces primary resistance to trastuzumab in HER2-overexpressing breast cancer cells. Int. J. Oncol. 51(5), 1553–1562 (2017).Crossref, Medline, CAS, Google Scholar12 Xia D, Le LP, Iafrate AJ, Lennerz J. KIF13B–NRG1 gene fusion and KRAS amplification in a case of natural progression of lung cancer. Int. J. Surg. Pathol. 25(3), 238–240 (2017).Crossref, Medline, CAS, Google Scholar13 Muscarella LA, Trombetta D, Fabrizio FP et al. ALK and NRG1 fusions coexist in a patient with signet ring cell lung adenocarcinoma. J. Thorac. Oncol. 12(10), E161–E163 (2017).Crossref, Medline, Google Scholar14 Dhanasekaran SM, Balbin OA, Chen G et al. Transcriptome meta-analysis of lung cancer reveals recurrent aberrations in NRG1 and Hippo pathway genes. Nat. Commun. 5, 5893 (2014).Crossref, Medline, CAS, Google Scholar15 Dong X, Fernandez-Salas E, Li E, Wang S. Elucidation of resistance mechanisms to second-generation ALK inhibitors alectinib and ceritinib in non-small-cell lung cancer cells. Neoplasia 18(3), 162–171 (2016).Crossref, Medline, CAS, Google Scholar16 Kimura M, Endo H, Inoue T et al. Analysis of ERBB ligand-induced resistance mechanism to crizotinib by primary culture of lung adenocarcinoma with EML4–ALK fusion gene. J. Thorac. Oncol. 10(3), 527–530 (2015).Crossref, Medline, CAS, Google Scholar17 Ji-Youn H, Kun Young L, Jin Young K et al. EGFR and HER3 inhibition- A novel therapy for invasive mucinous non-small-cell lung cancer harbouring an NRG1 fusion gene. J. Thorac. Oncol. 12(S1), S669, Abstract P3.02C-006 (2016).Google Scholar18 Gay ND, Wang Y, Beadling C et al. Durable response to afatinib in lung adenocarcinoma harboring NRG1 gene fusions. J. Thorac. Oncol. 12(8), E107–E110 (2017).Crossref, Medline, Google Scholar19 Sun M, Behrens C, Feng L et al. HER family receptor abnormalities in lung cancer brain metastases and corresponding primary tumors. Clin. Cancer Res. 15(15), 4829–4837 (2009).Crossref, Medline, CAS, Google Scholar20 Malm M, Frejd FY, Ståhl S, Löfblom J. Targeting HER3 using mono- and bispecific antibodies or alternative scaffolds. MAbs 8(7), 1195–1209 (2016).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByCancer prognosis and immune systemThe impact of fusion genes on cancer stem cells and drug resistance7 June 2021 | Molecular and Cellular Biochemistry, Vol. 476, No. 10Neuregulin 1 Gene (NRG1). A Potentially New Targetable Alteration for the Treatment of Lung Cancer9 October 2021 | Cancers, Vol. 13, No. 20Clinicopathologic Features and Response to Therapy of NRG1 Fusion–Driven Lung Cancers: The eNRGy1 Global Multicenter RegistryJournal of Clinical Oncology, Vol. 39, No. 25Methods for actionable gene fusion detection in lung cancer: now and in the futurePasquale Pisapia, Francesco Pepe, Roberta Sgariglia, Mariantonia Nacchio, Gianluca Russo, Gianluca Gragnano, Floriana Conticelli, Maria Salatiello, Caterina De Luca, Ilaria Girolami, Albino Eccher, Antonino Iaccarino, Claudio Bellevicine, Elena Vigliar, Umberto Malapelle & Giancarlo Troncone16 September 2021 | Pharmacogenomics, Vol. 22, No. 13An improved assay for detection of theranostic gene translocations and MET exon 14 skipping in thoracic oncologyLaboratory Investigation, Vol. 101, No. 5Oncogenic driver mutations in non-small cell lung cancer: Past, present and futureWorld Journal of Clinical Oncology, Vol. 12, No. 4Tarloxotinib Is a Hypoxia-Activated Pan-HER Kinase Inhibitor Active Against a Broad Range of HER-Family Oncogenes1 March 2021 | Clinical Cancer Research, Vol. 27, No. 5Predictive biomarkers for molecular pathology in lung cancerPasquale Pisapia, Francesco Pepe, Giancarlo Troncone & Umberto Malapelle3 March 2020 | Biomarkers in Medicine, Vol. 14, No. 4Welcome to Volume 8 of Lung Cancer ManagementJennifer Straiton20 December 2018 | Lung Cancer Management, Vol. 8, No. 1 Vol. 6, No. 4 Follow us on social media for the latest updates Metrics History Received 11 November 2017 Accepted 21 November 2017 Published online 5 January 2018 Published in print December 2017 Information© 2018 Future Medicine LtdKeywordsErbB NRG1 fusionNSCLCFinancial & competing interests disclosureThis work was supported by the Italian Ministry of Health (Ricerca Corrente, RC1703LO41 to LA Muscarella and RC1703ON39 to A Rossi), by the '5x1000' voluntary contributions and by the AIRC/MFAG grant 12983 (to LA Muscarella) and FBNC Grant 2015–2016 to LA Muscarella. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

Background: NRG1 fusions are oncogenic drivers in NSCLC, with therapeutic implications underscored by FDA's designation of Zenocutuzumab for NRG1 fusion-positive cases. Despite this, the molecular characteristics and clinical impacts of distinct NRG1 fusion types remain poorly understood. Methods: We retrospectively enrolled 435 NSCLC patients, comprising 78 with NRG1 fusions and 357 wild-type (WT), from June 2016 to December 2023. We assessed mutational landscapes, tumor mutation burden (TMB), chromosomal instability scores (CIS), and gene expression using broad-panel targeted DNA and RNA sequencing analyses. Survival analyses were conducted in patients from our cohort (N=47) and online TCGA database (N=526). Results: Among the NRG1 fusion-positive patients, 65.8% harbored recurrent fusion partners like CD74-NRG1 and SLC3A2-NRG1, with CD74-NRG1 appearing most frequently (48.1%). These recurrent fusions were associated with significantly fewer EGFR and KRAS mutations and lower TMB and CIS compared to WT (P &amp;lt; 0.01). Additionally, 26 patients had unique singleton fusion partners, with 23 novel fusions identified, including CEBPD-NRG1 and BMP1-NRG1, although validation through tools such as RNA sequencing is warranted in the future. Interestingly, this group showed significant enrichment of mutations in genes like KRAS, MSH2, MSH6, FANCI, and MDM2, impacting key oncogenic and DNA-repair pathways including Fanconi anemia, mismatch repair, PI3K-AKT, and MAPK pathways (P &amp;lt; 0.01). Additionally, the RNA profiling showed upregulation of DNAJB1 (P &amp;lt; 0.01) and LMNA (P = 0.02) in the singleton group versus WT, linked to poorer overall survival (OS) in the TCGA cohort (HR: 1.52 for both). No significant differences in progression-free survival (PFS) were observed between uncommon and WT groups following the first-line anti-EGFR tyrosine kinase inhibitor (TKI) therapy. Conclusion: This study revealed the diversity of NRG1 fusions in NSCLC, identifying novel fusion partners and distinct molecular and clinical features across novel NRG1+ groups, especially the enriched DNA repair/oncogenic signaling pathways and uniquely upregulated transcription profile. Our study highlights the need of subtyping different NRG+ tumors to better understand their tumorigenesis and facilitates more personalized and targeted treatment of NRG1+ NSCLC patients. Citation Format: Minyi Zhu, Yang Xu, Haimeng Tang, Xue Wu, Qiuxiang Ou. Characterizing the molecular and clinical implications ofNRG1fusions in NSCLC through integrated RNA and DNA sequencing analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5369.

NRG1 fusion-driven tumors: biology, detection, and the therapeutic role of afatinib and other ErbB-targeting agents.

Neuregulin-1 (encoded by NRG1) serves as a ligand for ERBB3 and can lead to dysregulated cellular proliferation in cases of NRG1 fusions. The reported incidence of NRG1 fusions in solid tumors is ~0.1-0.3% with enrichment in invasive mucinous adenocarcinoma of the lung and KRAS wildtype pancreatic adenocarcinoma. However, the real-world incidence of NRG1 fusions across diverse tumor types continues to evolve with the clinical implementation of comprehensive next-generation sequencing (NGS) methods including RNA based NGS. The true diversity and prognostic significance of specific NRG1 fusion partners has yet to be elucidated. We report on NRG1 fusions from a large clinico-genomic database of patients (pts) in community-based oncology settings where NGS is ordered as standard clinical practice. We conducted a retrospective, records-based analysis of pts across Sarah Cannon’s network of over 299 partner community-based oncology clinics to identify pts with NRG1 fusions detected by commercial NGS testing ordered as a part of standard of care from 1/1/2017 - 7/7/2022. NRG1 fusions were reported in 0.05% (19 of 40,857) of pts with commercial NGS test results across a range of tumor types including lung (59%; n=11/19), pancreas (11%; n=2), breast (5%; n=1), colorectal (5%), esophageal (5%), endometrial (5%), soft tissue liposarcoma (5%), and carcinoma of unknown primary (5%). NGS identifying an NRG1 fusion was performed in 5 (26%) pts with early stage disease while 11 (58%) pts had NGS testing after initial diagnosis of or progression to advanced/metastatic disease. A majority of pts harboring NRG1 fusions were female (68%; n=13). NRG1 fusions were detected by DNA (58%; n=11) and RNA based (42%; n=8) NGS from liquid (5%; n=1) and tissue biopsies (95%; n=18). A total of 11 different fusion partners were reported. CD74 was the most prevalent fusion partner and was primarily detected by DNA NGS including in one pt from liquid biopsy. Five novel, as yet to be described, fusion partners (CDK13, IL1RL2, FUT10, PPP2R2A, PIM3) were detected - 3 by RNA sequencing and 2 by DNA sequencing of NRG1 (versus sequencing of the fusion partner). TP53 (42%; n=8), CDKN2A (32%; n=6), and MTAP (16%; n=3) were the most frequently co-altered genes. Other notable co-altered genes included mutations in PIK3CA (11%; n=2), BRAF (5%; n=1), EGFR (5%), ALK (5%), and NRAS (5%). Immune biomarkers were largely negative, with 16% (n=3) PD-L1 positive, 0% MSI-high, and 5% (n=1) TMB-high cases. These data highlight the importance of comprehensive molecular profiling for pts with solid tumors, as NRG1 fusions occur across tumor types and stage of disease. Appropriate test selection is paramount as novel NRG1 fusions are more robustly detected by RNA over DNA NGS and are more routinely detected in tissue over liquid biopsies. Clinical trials investigating therapies to target NRG1 fusions are ongoing [CRESTONE (NCT04383210); eNRGy (NCT02912949)]. Citation Format: Emma G. Sturgill, Jaya Srivastava, Jessica Correia, Cooper Schumacher, Daniel Luckett, Cesar A. Perez, Judy S. Wang, Stephen G. Divers, Babar Bashir, Jennifer Johnson, Valerie M. Jansen, Andrew J. McKenzie, David R. Spigel. Identification of NRG1 fusions in patients with solid tumors: analysis from a real-world community oncology network [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 921.

Pemetrexed maintenance significantly improved progression-free survival (PFS) and overall survival (OS) in advanced nonsquamous non-small-cell lung cancer (NSCLC) patients not progressing after induction chemotherapy. This study is aimed at examine the association of various clinical factor and survival in a real-world cohort analysis. One hundred ninety-four patients were included and classified as "PM" cohort ("Pemetrexed Maintenance", including patients given with pemetrexed maintenance after induction chemotherapy, n=112), and "noPM" cohort ("no Pemetrexed Maintenance" including those discontinuing pemetrexed, n=82). The median PFS was 8.8 and 5.4 months in the PM and noPM cohorts, respectively (p=0.001). The median OS was 19.6 months in the "PM" cohort and 13.2 months in the "noPM" cohort (p<0.02). In the multivariate analysis, ECOG Performance Status (PS) 0 and maintenance therapy were independently associated with improved PFS and OS. A longer median PFS was reported in patients given ≥5 cycles of pemetrexed maintenance (p<0.01). These results further confirm the survival benefit of pemetrexed maintenance in a real-word population. All eligible advanced NSCLC patients should be strongly considered for at least 5 of pemetrexed maintenance.

Invasive mucinous adenocarcinoma (IMA) is a unique histologic subtype of lung adenocarcinoma. Recent studies document distinctive genetic alterations (e.g., NRG1 fusions) and a "mucinous gene signature" in IMAs, as well as differences in clinical responses to traditional chemotherapies in IMAs versus non-mucinous adenocarcinomas. Our understanding of the genetic and clinical characteristics of IMAs has expanded, confirming the uniqueness of IMAs. Accordingly, IMAs require different therapeutic approaches than do lung adenocarcinomas in general. Here, we review recent updates on the genetic and clinical profiles of IMA of the lung.

Background: NRG1 fusions and rearrangements are oncogenic drivers that have been observed in a variety of tumor types and enriched in invasive mucinous adenocarcinomas (IMA) of the lung. The oncoprotein binds HER3-HER2 heterodimers and activates downstream signaling, supporting a therapeutic paradigm of ERBB3/ERBB2 inhibition. While patient responses have been observed with Afatinib, durable responses have largely been absent. Tarloxotinib, a clinical-stage prodrug that releases a potent, irreversible EGFR/HER2 inhibitor (Tarloxotinib-E) selectively in severely hypoxic regions of tumours has been shown to overcome the intrinsic resistance of EGFR exon 20 insertion mutations and HER2 activating mutations to existing TKIs in vitro and in vivo. Results: NRG1 altered cell lines (MDA-MB-175 with DOC4-NRG fusion, HCC95 with NRG gene amplification and H1793 with increased NRG1 mRNA) showed high sensitivity to Tarloxotinib-E whereas Tarloxotinib prodrug was &amp;gt;45 fold less potent under normoxic conditions, consistent with lower sensitivity of the prodrug and the requirement of hypoxia for activation. Tarloxotinib-E inhibited HER2, HER3, Akt and ERK phosphorylation between 10-100 nM whereas afatinib was 10-fold less potent. In vivo activity of Tarloxotinib was evaluated using the CLU-NRG1 patient-derived xenograft model. Nude mice bearing OV-10-0050 tumors implanted subcutaneously were treated with vehicle, the human equivalent dose (HED) of afatinib (6 mg/kg, qd, p.o.) and two doses of tarloxotinib (26 and 48 mg/kg, qw, i.p.) corresponding to Tarloxotinib HED of 75 and 150 mg/m2. Afatinib showed initial activity that reduced over time and was ineffective. In contrast, Tarloxotinib elicited a profound, durable and dose dependent anti-tumor response. Tarloxotinib dosed at 48 mg/kg showed dramatic tumor regression in all the mice while Tarloxotinib dosed at 26 mg/kg showed significant dose dependent tumor regression. Efforts are underway to define the PK/PD correlation by measuring Tarloxotinib and Tarloxotinib-E exposure in plasma and tumor along with the evaluation of hypoxia and the expression of STEAP4 reductase. Tarloxotinib mediated on-target and signaling effects in OV-10-0050 tumors will be presented at the meeting. Conclusions: Tarloxotinib, a prodrug of a potent irreversible inhibitor of EGFR/HER2 in clinical development demonstrated significant activity in CLU-NRG1 patient-derived xenograft model. NRG1 fusions and rearrangements represent an emerging actionable driver alteration in a variety of cancers. Clinical development of Tarloxotinib in NRG1 altered cancers presents an attractive opportunity. Citation Format: Vijaya G. Tirunagaru, Adriana Estrada-Bernal, Hui Yu, Chris Rivard, Fred Hirsch, Matthew Bull, Maria Abbatista, Jeff Smaill, Adam V. Patterson, Robert C. Doebele, Avanish Vellanki. Tarloxotinib exhibits potent activity in NRG1 fusion and rearranged cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2202.

Invasive mucinous lung adenocarcinoma (IMA) is a rare subtype of lung adenocarcinoma with no effective treatment option in advanced disease. KRAS mutations occur in 28–87% of the cases. NRG1 fusions were recently discovered in KRAS‐negative IMA cases and otherwise negative for known driver oncogenes and could represent an attractive therapeutic target. Published data suggest that NRG1 fusions occur essentially in nonsmoking Asian women. From an IMA cohort of 25 French patients of known ethnicity, driver oncogenes EGFR, KRAS, BRAF, ERBB2 mutations, and ALK and ROS1 rearrangements presence were analyzed. In the IMA samples remaining negative for these driver oncogenes, an NRG1 rearrangement detection was performed by FISH. A driver oncogene was identified in 14/25 IMA, namely 12 KRAS mutations (48%), one ROS1 rearrangement (4%), and one ALK rearrangement (4%). The detection of NRG1 rearrangement by FISH was conducted in the 11 pan‐negative IMA. One sample was NRG1FISH‐positive and 100% of the tumor nuclei analyzed were positive. This NRG1‐positive patient was a 61‐year‐old nonsmoking woman of Vietnamese ethnicity and was the sole patient of Asian ethnicity of the cohort. She died 6 months after the diagnosis with a pulmonary multifocal disease. NRG1FISH detection should be considered in patients with IMA pan‐negative for known driver oncogenes. These results might suggest that NRG1 fusion is more frequent in IMA from Asian patient. Larger studies are needed.

Does the histologic subtype of uterine carcinosarcoma correlate with outcomes?

&lt;div&gt;AbstractPurpose:&lt;p&gt;Invasive mucinous adenocarcinoma (IMA) is a unique subtype of lung adenocarcinoma, characterized genomically by frequent &lt;i&gt;KRAS&lt;/i&gt; mutations or specific gene fusions, most commonly involving &lt;i&gt;NRG1&lt;/i&gt;. Comprehensive analysis of a large series of IMAs using broad DNA- and RNA-sequencing methods is still lacking, and it remains unclear whether molecular subtypes of IMA differ clinicopathologically.&lt;/p&gt;Experimental Design:&lt;p&gt;A total of 200 IMAs were analyzed by 410-gene DNA next-generation sequencing (MSK-IMPACT; &lt;i&gt;n&lt;/i&gt; = 136) or hotspot 8-oncogene genotyping (&lt;i&gt;n&lt;/i&gt; = 64). Driver-negative cases were further analyzed by 62-gene RNA sequencing (MSK-Fusion) and those lacking fusions were further tested by whole-exome sequencing and whole-transcriptome sequencing (WTS).&lt;/p&gt;Results:&lt;p&gt;Combined MSK-IMPACT and MSK-Fusion testing identified mutually exclusive driver alterations in 96% of IMAs, including &lt;i&gt;KRAS&lt;/i&gt; mutations (76%), &lt;i&gt;NRG1&lt;/i&gt; fusions (7%), &lt;i&gt;ERBB2&lt;/i&gt; alterations (6%), and other less common events. In addition, WTS identified a novel &lt;i&gt;NRG2&lt;/i&gt; fusion (&lt;i&gt;F11R&lt;/i&gt;–&lt;i&gt;NRG2&lt;/i&gt;). Overall, targetable gene fusions were identified in 51% of &lt;i&gt;KRAS&lt;/i&gt; wild-type IMAs, leading to durable responses to targeted therapy in some patients. Compared with &lt;i&gt;KRAS&lt;/i&gt;-mutant IMAs, &lt;i&gt;NRG1&lt;/i&gt;-rearranged tumors exhibited several more aggressive characteristics, including worse recurrence-free survival (&lt;i&gt;P&lt;/i&gt; &lt; 0.0001).&lt;/p&gt;Conclusions:&lt;p&gt;This is the largest molecular study of IMAs to date, where we demonstrate the presence of a major oncogenic driver in nearly all cases. This study is the first to document more aggressive characteristics of &lt;i&gt;NRG1&lt;/i&gt;-rearranged IMAs, &lt;i&gt;ERBB2&lt;/i&gt; as the third most common alteration, and a novel &lt;i&gt;NRG2&lt;/i&gt; fusion in these tumors. Comprehensive molecular testing of &lt;i&gt;KRAS&lt;/i&gt; wild-type IMAs that includes fusion testing is essential, given the high prevalence of alterations with established and investigational targeted therapies in this subset.&lt;/p&gt;&lt;/div&gt;