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Background: Oncogenic alterations (gene amplification, mutation, and fusion/rearrangements) of fibroblast growth factor receptor 2 (FGFR2) are rare and occur at varying frequencies across solid tumor types and have become critical therapeutic targets in drug development. First generation FGFR pan-kinase inhibitors non-selectively inhibit FGFR1-4 and are associated with dose limiting toxicities and narrow therapeutic windows. Off-isoform toxicity (FGFR1-hyperphosphatemia; FGFR4-diarrhea) and on-target acquired resistance have led to limited efficacy, which is primarily seen in FGFR2-fusion+ intrahepatic cholangiocarcinoma. Therapies that selectively target FGFR2 remain an unmet need in advanced breast cancer as well as other solid tumor types. RLY-4008 is a novel, oral FGFR2 inhibitor designed to overcome the limitations of pan-FGFR inhibitors (FGFRi) by potently and selectively targeting primary oncogenic FGFR2 alterations and acquired resistance mutations. RLY-4008 is > 200-fold selective over FGFR1, > 80- and > 5000-fold selective over FGFR3 and FGFR4 respectively. Here we describe a phase 1/2 study to investigate the safety and antitumor activity in advanced FGFR2 altered cancers, including breast cancer. Methods: ReFocus is a phase 1/2 open label global study evaluating the safety and efficacy of RLY-4008 (NCT04526106) in adult patients with advanced unresectable and/or metastatic cancers harboring an FGFR2 alteration. Key eligibility criteria include: documented FGFR2 alteration in blood or tissue per local assessment, ECOG performance status of 0-1, disease that is refractory or not adequately responding to standard therapy, has no available standard therapy, or patient is intolerant of, or declined standard therapy (including pan-FGFRi), and measurable or evaluable disease per RECIST 1.1. FGFR2 alteration will be confirmed retrospectively by central laboratory assessment. Part 1 dose escalation employed the Bayesian Optimal Interval (BOIN) design to determine the MTD/RP2D of RLY-4008. Part 2 dose expansion is presently enrolling patients at the RP2D of RLY-4008 and includes 5 cohorts comprised of patients with: 1. FGFR2 fusion+ cholangiocarcinoma previously treated with an FGFRi; 2. FGFR2 fusion+ cholangiocarcinoma not previously treated with an FGFRi; 3. FGFR2 fusion+ solid tumors; 4. FGFR2 mutation+ solid tumors and 5. FGFR2 amplified solid tumors. Solid tumors in cohorts 3, 4 and 5 will have a focus in breast cancer. The primary endpoint is objective response rate (ORR); key secondary endpoints include: duration of response, safety and tolerability, correlation of FGFR2 genotype by central tissue assessment with antitumor response, characterization of PK profile, and quality of life. US enrollment began September 2020 and has expanded into Europe and Asia. Clinical trial information: NCT04526106. Citation Format: Suneel Kamath, David Tai, Irene Moreno, Hani Babiker, Zhaohui Jin, Changhoon Yoo, Fabien Ricard, Kai Yu Jen, Jim Coward, Jia Liu, Frans Opdam, Michael Millward, Mariano Ponz-Sarvise, Jeffrey Yachnin, Richard Kim, Joon Oh Park, Vivek Subbiah, Alison M. Schram. ReFocus: A Phase 1/2 Study of the Highly Selective FGFR2 Inhibitor, RLY-4008, in Patients with Advanced Solid Tumors Including Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-24-01.

Background: Targeting constitutively active mutant kinases with selective small molecule inhibitors is a key therapeutic pillar of precision oncology. Phosphatidylinositol-4,5bisphosphate-3 kinase, catalytic subunit alpha (PIK3CA) mutations leading to oncogenic activation of PI3Kα represent the largest opportunity for this approach in solid tumors. However, there is no selective inhibitor that targets mutant PI3Kα in the clinic. Toxicity related to non-selective inhibition of WT PI3Kα (hyperglycemia) and other PI3K isoforms limits the tolerability, dosing and efficacy of the orthosteric inhibitor, alpelisib, the only approved solid tumor PI3K inhibitor. RLY-2608, a novel oral allosteric PI3Kα inhibitor, is uniquely designed to overcome these limitations via mutant- and isoform-selective PI3Kα inhibition for greater target coverage, improved tolerability and antitumor activity. We initiated a first-in-human (FIH), study to evaluate the clinical activity of RLY-2608 as a single agent in advanced solid tumor patients (pts) with PI3KCA mutations and in combination with fulvestrant in pts with PIK3CA mutant, HR+, HER2- metastatic breast cancer (MBC). Methods: This is a global, multi-center, dose escalation/expansion study (NCT05216432) of RLY2608 as a single agent in adults who have advanced solid tumors and are refractory, intolerant, or declined standard therapy and RLY-2608 in combination with fulvestrant in previously treated pts with HR+/HER2- MBC. Eligibility criteria include presence of PI3KCA mutation (blood or tumor) per local assessment, ECOG performance status 0-1, measurable or evaluable disease per RECIST 1.1 and no prior PI3K inhibitor (except combination group 2). RLY-2608 is administered on a continuous schedule with 4-week cycles. Adverse events (AEs) per CTCAE v5, PK, biomarkers (mutant ctDNAs and insulin pathway markers) and anti-tumor activity are assessed serially. Dose escalation employs a Bayesian Optimal Interval design to identify MTD and RP2D. Following dose escalation, pts will be treated with RLY-2608 at the MTD/RP2D in a monotherapy dose expansion with 5 groups (N=75, 15 each): 1. Clear cell ovarian carcinoma 2. Head and neck squamous cell carcinoma 3. Cervical cancer 4. Other solid tumors 5. PI3KCA double mutations. In addition, two expansion cohorts will enroll patients with HR+/HER2- MBC treated with RLY-2608 and fulvestrant combination (N = 30, 15 each): 1. No prior PI3K therapy 2. Intolerant to PI3K inhibitors. The primary endpoints are MTD/RP2D and AE profile for single agent and combination; key secondary endpoints are PI3KCA genotype in blood and tumor, PK, biomarkers, and overall response rate. US enrollment began December 2021 and ex-USA startup is under way. Citation Format: Andreas Varkaris, Erika Hamilton, Jason Henry, Alexander I. Spira, Alison M. Schram, Julia E. McGuinness, Gege Tan, Xiaoyan Li, Tamieka Hunter, Ramin Samadani, Alison Timm, Djuro Karanovic, Vivek Subbiah, Cesar A. Perez. First-in-human global multi-center study of RLY-2608, a pan mutant and isoform selective PI3Kα inhibitor, as a single agent in advanced solid tumor patients and in combination with fulvestrant in patients with advanced breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-22-01.

Supplementary Figure from Zenocutuzumab, a HER2xHER3 Bispecific Antibody, Is Effective Therapy for Tumors Driven by NRG1 Gene Rearrangements

NRG1 rearrangements are recurrent oncogenic drivers in solid tumors. NRG1 binds to HER3, leading to heterodimerization with other HER/ERBB kinases, increased downstream signaling, and tumorigenesis. Targeting ERBBs, therefore, represents a therapeutic strategy for these cancers. We investigated zenocutuzumab (Zeno; MCLA-128), an antibody-dependent cellular cytotoxicity–enhanced anti-HER2xHER3 bispecific antibody, in NRG1 fusion–positive isogenic and patient-derived cell lines and xenograft models. Zeno inhibited HER3 and AKT phosphorylation, induced expression of apoptosis markers, and inhibited growth. Three patients with chemotherapy-resistant NRG1 fusion–positive metastatic cancer were treated with Zeno. Two patients with ATP1B1–NRG1–positive pancreatic cancer achieved rapid symptomatic, biomarker, and radiographic responses and remained on treatment for over 12 months. A patient with CD74–NRG1-positive non–small cell lung cancer who had progressed on six prior lines of systemic therapy, including afatinib, responded rapidly to treatment with a partial response. Targeting HER2 and HER3 simultaneously with Zeno is a novel therapeutic paradigm for patients with NRG1 fusion–positive cancers.Significance:NRG1 rearrangements encode chimeric ligands that activate the ERBB receptor tyrosine kinase family. Here we show that targeting HER2 and HER3 simultaneously with the bispecific antibody Zeno leads to durable clinical responses in patients with NRG1 fusion–positive cancers and is thus an effective therapeutic strategy.This article is highlighted in the In This Issue feature, p. 1171

Supplementary Figures 1-7

Supplementary Methods Supplementary References Supplementary Figure 1. Effect of acquired resistance mutations on TRK inhibitor activity. Supplementary Figure 2. Further characterization of LOXO-195 inhibitory activity against acquired resistance mutations. Supplementary Figure 3. LOXO-195 is selectively cytotoxic to TRK-fusion cell lines. Supplementary Figure 4. Identification of a clonal NTRK1 (TRKA)-G595R mutation after larotrectinib treatment. Supplementary Table 1. LOXO-195 ADME properties. Supplementary Table 2. Selectivity of LOXO-195 for 228 kinases. Supplementary Table 3. Summary of LOXO-195 PK parameters for each patient during intra-patient dose escalation.

Supplementary Figure from CD74-NRG1 Fusions Are Oncogenic In Vivo and Induce Therapeutically Tractable ERBB2:ERBB3 Heterodimerization

Overall sequencing CONSORT diagram. Flow diagram of study patients and analyzed biospecimens processed through and selected for central sequencing using MSK-IMPACT and Guardant360. cfDNA, cell-free DNA; FFPE, formalin-fixed paraffin-embedded; MSK-IMPACT, Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets; QC, quality control.

On-target resistance to next-generation TRK inhibitors in TRK fusion–positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKAG667 and TRKCG696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations.Significance:In TRK fusion–positive cancers, TRK xDFG substitutions represent a shared liability for type I TRK inhibitors. In contrast, they represent a potential biomarker of type II TRK inhibitor activity. As all currently available type II agents are multikinase inhibitors, rational drug design should focus on selective type II inhibitor creation.This article is highlighted in the In This Issue feature, p. 1

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Preexistent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively, these data define HER2 mutations as a therapeutic target in breast cancer and suggest that coexistence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib.Significance:HER2 mutations define a targetable breast cancer subset, although sensitivity to irreversible HER kinase inhibition appears to be modified by the presence of concurrent activating genomic events in the pathway. These findings have implications for potential future combinatorial approaches and broader therapeutic development for this genomically defined subset of breast cancer.This article is highlighted in the In This Issue feature, p. 161

Comprehensive OncoPrint of nine patients with paired pre- and post-treatment tissue sequencing showing acquired alterations in the post-treatment tumor.

Supplementary Figures

Supplementary Tables 1-4

Supplementary data

NRG1 fusions are recurrent somatic genome alterations occurring across several tumor types, including invasive mucinous lung adenocarcinomas and pancreatic ductal adenocarcinomas and are potentially actionable genetic alterations in these cancers. We initially discovered CD74-NRG1 as the first NRG1 fusion in lung adenocarcinomas, and many additional fusion partners have since been identified. Here, we present the first CD74-NRG1 transgenic mouse model and provide evidence that ubiquitous expression of the CD74-NRG1 fusion protein in vivo leads to tumor development at high frequency. Furthermore, we show that ERBB2:ERBB3 heterodimerization is a mechanistic event in transformation by CD74-NRG1 binding physically to ERBB3 and that CD74-NRG1–expressing cells proliferate independent of supplemented NRG1 ligand. Thus, NRG1 gene fusions are recurrent driver oncogenes that cause oncogene dependency. Consistent with these findings, patients with NRG1 fusion-positive cancers respond to therapy targeting the ERBB2:ERBB3 receptors.

PFS of (A) neratinib plus fulvestrant combination cohort and (B) neratinib monotherapy cohort. CI, confidence interval; PFS, progression-free survival.

Supplementary Tables

Supplemental Methods 2

Supplemental Table 4. Patient level detail of use of immunotherapy in this cohort.

Supplemental Figure 1. Consort diagram summarizing the molecular workflow of cases.

Supplemental Table 2. Most commonly mutated genes in the TRK cohort.

Supplemental Figure 3. Comparison of overall and recurrence-free survival in TP53-wild type and TP53-mutant IMAs

Supplemental Methods 1

Supplemental Figure 3. Comparison of Tumor Types in Larotrectinib Study Population and MSK Cohort.

Purpose: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.Experimental Design: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).Results: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).Conclusions: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.

Supplemental Figure 6. Distribution of Surgery, Radiotherapy, Systemic Therapy, and Chemotherapy.

Supplemental Figure 5. Timeline for Patients with Emergent or Lost TRK Fusions.

Supplemental Table 3. Detail of patients who received chemotherapy by cancer type.

Supplemental Table 1. NTRK gene fusion partners.

Supplementary Tables 1-6

Supplemental Tables 1-11

Supplementary Figure1. ROS1 Intron 31 coverage by DNASeq. Supplementary Figure 2. Access to adequate material for RNA extraction.

Purpose:TRK inhibitors achieve marked tumor-agnostic efficacy in TRK fusion–positive cancers and consequently are now an established standard of care. Little is known, however, about the demographics, outcomes, response to alternative standard therapies, or genomic characteristics of TRK fusion–positive cancers.Experimental Design:Utilizing a center-wide screening program involving more than 26,000 prospectively sequenced patients, genomic and clinical data from all cases with TRK fusions were extracted. An integrated analysis was performed of genomic, therapeutic, and phenomic outcomes.Results:We identified 76 cases with confirmed TRK fusions (0.28% overall prevalence) involving 48 unique rearrangements and 17 cancer types. The presence of a TRK fusion was associated with depletion of concurrent oncogenic drivers (P < 0.001) and lower tumor mutation burden (P < 0.001), with the exception of colorectal cancer where TRK fusions cooccur with microsatellite instability (MSI-H). Longitudinal profiling in a subset of patients indicated that TRK fusions were present in all sampled timepoints in 82% (14/17) of cases. Progression-free survival on first-line therapy, excluding TRK inhibitors, administered for advanced disease was 9.6 months [95% confidence interval (CI), 4.8–13.2]. The best overall response rate achieved with chemotherapy containing–regimens across all lines of therapy was 63% (95% CI, 41–81). Among 12 patients treated with checkpoint inhibitors, a patient with MSI-H colorectal cancer had the only observed response.Conclusions:TRK fusion–positive cancers can respond to alternative standards of care, although efficacy of immunotherapy in the absence of other predictive biomarkers (MSI-H) appears limited. TRK fusions are present in tumors with simple genomes lacking in concurrent drivers that may partially explain the tumor-agnostic efficacy of TRK inhibitors.

Supplemental Figure 1. Venn Diagram of Patients in the Study.

Purpose:Targeted next-generation sequencing of DNA has become more widely used in the management of patients with lung adenocarcinoma; however, no clear mitogenic driver alteration is found in some cases. We evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions and MET exon 14 (METex14) alterations in DNA sequencing (DNAseq) driver–negative lung cancers.Experimental Design:Lung cancers driver negative by MSK-IMPACT underwent further analysis using a custom RNAseq panel (MSK-Fusion). Tumor mutation burden (TMB) was assessed as a potential prioritization criterion for targeted RNAseq.Results:As part of prospective clinical genomic testing, we profiled 2,522 lung adenocarcinomas using MSK-IMPACT, which identified 195 (7.7%) fusions and 119 (4.7%) METex14 alterations. Among 275 driver-negative cases with available tissue, 254 (92%) had sufficient material for RNAseq. A previously undetected alteration was identified in 14% (36/254) of cases, 33 of which were actionable (27 in-frame fusions, 6 METex14). Of these 33 patients, 10 then received matched targeted therapy, which achieved clinical benefit in 8 (80%). In the 32% (81/254) of DNAseq driver–negative cases with low TMB [0–5 mutations/Megabase (mut/Mb)], 25 (31%) were positive for previously undetected gene fusions on RNAseq, whereas, in 151 cases with TMB >5 mut/Mb, only 7% were positive for fusions (P < 0.0001).Conclusions:Targeted RNAseq assays should be used in all cases that appear driver negative by DNAseq assays to ensure comprehensive detection of actionable gene rearrangements. Furthermore, we observed a significant enrichment for fusions in DNAseq driver–negative samples with low TMB, supporting the prioritization of such cases for additional RNAseq.See related commentary by Davies and Aisner, p. 4586

Supplemental Figure 4. Comparison of overall and recurrence-free survival in CDKN2A-wild type and CDKN2A-mutant IMAs.

Supplemental Figure 4. Pathologic and preclinical characterization of melanoma with concurrent GON4L-NTRK1 and NRAS Q61R mutations.

Supplemental Figure 2. Detailed graphical visualizations of the F11R-NRG2 fusion.

NRG1 rearrangements are recurrent oncogenic drivers in solid tumors. NRG1 binds to HER3, leading to heterodimerization with other HER/ERBB kinases, increased downstream signaling, and tumorigenesis. Targeting ERBBs, therefore, represents a therapeutic strategy for these cancers. We investigated zenocutuzumab (Zeno; MCLA-128), an antibody-dependent cellular cytotoxicity–enhanced anti-HER2xHER3 bispecific antibody, in NRG1 fusion–positive isogenic and patient-derived cell lines and xenograft models. Zeno inhibited HER3 and AKT phosphorylation, induced expression of apoptosis markers, and inhibited growth. Three patients with chemotherapy-resistant NRG1 fusion–positive metastatic cancer were treated with Zeno. Two patients with ATP1B1–NRG1–positive pancreatic cancer achieved rapid symptomatic, biomarker, and radiographic responses and remained on treatment for over 12 months. A patient with CD74–NRG1-positive non–small cell lung cancer who had progressed on six prior lines of systemic therapy, including afatinib, responded rapidly to treatment with a partial response. Targeting HER2 and HER3 simultaneously with Zeno is a novel therapeutic paradigm for patients with NRG1 fusion–positive cancers. Significance: NRG1 rearrangements encode chimeric ligands that activate the ERBB receptor tyrosine kinase family. Here we show that targeting HER2 and HER3 simultaneously with the bispecific antibody Zeno leads to durable clinical responses in patients with NRG1 fusion–positive cancers and is thus an effective therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1171

Purpose To investigate clinical and morphologic characteristics of serous retinal disturbances in patients taking extracellular signal-regulated kinase (ERK) inhibitors. Design Single-center retrospective study of prospectively collected data. Participants Of 61 patients receiving ERK inhibitors for treatment of metastatic cancer, this study included 40 eyes of 20 patients with evidence of retinopathy confirmed by OCT. Methods Clinical examination, fundus photography, and OCT were used to evaluate ERK inhibitor retinopathy. The morphologic features, distribution, and location of fluid foci were evaluated serially. Visual acuity (VA) and choroidal thickness measurements were compared at baseline, fluid accumulation, and resolution. Main Outcome Measures Characteristics of treatment-emergent choroid and retinal OCT abnormalities as compared with baseline OCT findings and the impact of toxicity on VA. Results Of 20 patients with retinopathy, most showed fluid foci that were bilateral (100%), multifocal in each eye (75%), and with at least 1 focus involving the fovea (95%). All subretinal fluid foci occurred between the interdigitation zone and an intact retinal pigment epithelium. No statistical difference was found in choroidal thickness at fluid accumulation and resolution compared with baseline. Forty-five percent of eyes showed evidence of concomitant intraretinal edema localized to the outer nuclear layer. At the time of fluid accumulation, 57.5% eyes showed a decline in VA (mainly by 1–2 lines from baseline). For all eyes with follow-up, the subretinal fluid and intraretinal edema were reversible and resolved without medical intervention, and best-corrected VA at fluid resolution was not statistically different from baseline. Concomitant intraretinal fluid was not associated with worsening of VA. No patient discontinued or decreased drug dose because of retinopathy. Conclusions This study showed that ERK inhibitors may cause subretinal fluid foci with unique clinical and morphologic characteristics. The observed foci were similar to mitogen-activated protein kinase kinase (MEK) inhibitor–associated retinopathy and distinct from central serous chorioretinopathy. However, unlike with MEK inhibitors, an increased occurrence of concomitant intraretinal fluid without significant additive visual impact seems to occur with ERK inhibitors. In this series, ERK inhibitors did not cause irreversible loss of vision or serious eye damage; retinopathy was self-limited and did not require medical intervention.

PURPOSE Low-grade serous carcinoma (LGSC) is a rare type of ovarian cancer, which commonly arises from serous borderline tumor (SBT) and is characterized by frequent activating mutations in the mitogen-activated protein kinase pathway, including BRAF. The BRAFV600E mutation is associated with improved prognosis in SBT and LGSC, and responses to BRAF inhibitor therapy have been reported. We sought to characterize the clinicopathologic and molecular features of BRAF-driven tubo-ovarian and primary peritoneal serous tumors. METHODS Retrospective analysis of our institutional cohort of SBTs (n = 22), LGSCs (n = 119) and high-grade serous carcinomas (HGSCs, n = 1,290) subjected to targeted massively parallel sequencing was performed to identify cases with BRAF genetic alterations. Putative BRAF rearrangements were confirmed using targeted RNA sequencing and/or fluorescence in situ hybridization (FISH). BRAFV600E oncoprotein expression was assessed by immunohistochemistry on selected cases. RESULTS BRAF somatic genetic alterations were identified in 29 of 1,431 (2%) serous tumors and included mutations (n = 24), gene rearrangements (n = 3), and amplification (n = 2). BRAF mutations were more frequent in SBTs (7 of 22; 32%) compared with LGSCs (11 of 119; 9%, P = .009) and HGSCs (6 of 1,290; 0.5%; P < .0001, SBT/LGSC v HGSC). The BRAFV600E hotspot mutation was most common (n = 16); however, other BRAF driver mutations were also detected (n = 8). BRAF mutations were often clonal or truncal in SBTs and LGSCs, but subclonal in most HGSCs. Pathogenic BRAF gene fusions were identified in LGSCs (n = 2) and HGSC (n = 1) and involved distinct fusion partners ( AGK, MKRN1, and AGAP3). Three patients with BRAF-mutant LGSC were treated with targeted mitogen-activated protein kinase inhibitors, one of whom was maintained on therapy for over 3 years with clinical benefit. CONCLUSION Recognition of BRAF alterations beyond V600E mutation in LGSC may have clinical implications for appropriate targeted therapy selection.

Background Activating fusions of the NTRK1, NTRK2 and NTRK3 genes are drivers of carcinogenesis and proliferation across a broad range of tumour types in both adult and paediatric patients. Recently, the FDA granted tumour-agnostic approvals of TRK inhibitors, larotrectinib and entrectinib, based on significant and durable responses in multiple primary tumour types. Unfortunately, testing rates in clinical practice remain quite low. Adding plasma next-generation sequencing of circulating tumour DNA (ctDNA) to tissue-based testing increases the detection rate of oncogenic drivers and demonstrates high concordance with tissue genotyping. However, the clinical potential of ctDNA analysis to identify NTRK fusion-positive tumours has been largely unexplored. Methods We retrospectively reviewed a ctDNA database in advanced stage solid tumours for NTRK1 fusions. Results NTRK1 fusion events, with nine unique fusion partners, were identified in 37 patients. Of the cases for which clinical data were available, 44% had tissue testing for NTRK1 fusions; the NTRK1 fusion detected by ctDNA was confirmed in tissue in 88% of cases. Here, we report for the first time that minimally-invasive plasma NGS can detect NTRK fusions with a high positive predictive value. Conclusion Plasma ctDNA represents a rapid, non-invasive screening method for this rare genomic target that may improve identification of patients who can benefit from TRK-targeted therapy and potentially identify subsequent on- and off-target resistance mechanisms.

PURPOSEAlthough NRG1 fusions are oncogenic drivers across multiple tumor types including lung cancers, these are difficult to study because of their rarity. The global eNRGy1 registry was thus established to characterize NRG1 fusion–positive lung cancers in the largest and most diverse series to date.METHODSFrom June 2018 to February 2020, a consortium of 22 centers from nine countries in Europe, Asia, and the United States contributed data from patients with pathologically confirmed NRG1 fusion–positive lung cancers. Profiling included DNA-based and/or RNA-based next-generation sequencing and fluorescence in situ hybridization. Anonymized clinical, pathologic, molecular, and response (RECIST v1.1) data were centrally curated and analyzed.RESULTSAlthough the typified never smoking (57%), mucinous adenocarcinoma (57%), and nonmetastatic (71%) phenotype predominated in 110 patients with NRG1 fusion–positive lung cancer, further diversity, including in smoking history (43%) and histology (43% nonmucinous and 6% nonadenocarcinoma), was elucidated. RNA-based testing identified most fusions (74%). Molecularly, six (of 18) novel 5′ partners, 20 unique epidermal growth factor domain–inclusive chimeric events, and heterogeneous 5′/3′ breakpoints were found. Platinum-doublet and taxane-based (post–platinum-doublet) chemotherapy achieved low objective response rates (ORRs 13% and 14%, respectively) and modest progression-free survival medians (PFS 5.8 and 4.0 months, respectively). Consistent with a low programmed death ligand-1 expressing (28%) and low tumor mutational burden (median: 0.9 mutations/megabase) immunophenotype, the activity of chemoimmunotherapy and single-agent immunotherapy was poor (ORR 0%/PFS 3.3 months and ORR 20%/PFS 3.6 months, respectively). Afatinib achieved an ORR of 25%, not contingent on fusion type, and a 2.8-month median PFS.CONCLUSIONNRG1 fusion–positive lung cancers were molecularly, pathologically, and clinically more heterogeneous than previously recognized. The activity of cytotoxic, immune, and targeted therapies was disappointing. Further research examining NRG1-rearranged tumor biology is needed to develop new therapeutic strategies.

Five to ten percent of ER+ metastatic breast cancer (MBC) tumors harbor somatic PTEN mutations. Loss of function of this tumor-suppressor gene defines a highly aggressive, treatment-refractory disease for which new therapies are urgently needed. This Phase I multipart expansion study assessed oral capivasertib with fulvestrant in patients with PTEN-mutant ER+ MBC. Safety and tolerability were assessed by standard methods. Plasma and tumor were collected for NGS and immunohistochemistry analyses of PTEN protein expression. In 31 eligible patients (12 fulvestrant naive; 19 fulvestrant pretreated), the 24-week clinical benefit rate was 17% in fulvestrant-naive and 42% in fulvestrant-pretreated patients, with objective response rate of 8% and 21%, respectively. Non-functional PTEN was centrally confirmed in all cases by NGS or immunohistochemistry. Co­mutations occurred in PIK3CA (32%), with less ESR1 (10% vs 72%) and more TP53 (40% vs 28%) alterations in fulvestrant-naive versus fulvestrant-pretreated patients, respectively. PTEN was clonally dominant in most patients. Treatment-related grade ≥3 adverse events occurred in 32% of patients, most frequently diarrhea and maculopapular rash (both n = 2). In this clinical study, which selectively targeted the aggressive PTEN-mutant ER+ MBC, capivasertib plus fulvestrant was tolerable and clinically active. Phenotypic and genomic differences were apparent between fulvestrant-naive and -pretreated patients.Trial registration number for the study is NCT01226316.

Emerging efficacy data have led to the emergency use authorization or approval of COVID-19 vaccines in several countries worldwide. Most trials of COVID-19 vaccines excluded patients with active malignancies, and thus data on the safety, tolerability and efficacy of the vaccines in patients with cancer are currently limited. Given the risk posed by the COVID-19 pandemic, decisions regarding the use of vaccines against COVID-19 in patients participating in trials of investigational anticancer therapies need to be addressed promptly. Patients should not have to choose between enrolling on oncology clinical trials and receiving a COVID-19 vaccine. Clinical trial sponsors, investigators and treating physicians need operational guidance on COVID-19 vaccination for patients with cancer who are currently enrolled or might seek to enrol in clinical trials. Considering the high morbidity and mortality from COVID-19 in patients with cancer, the benefits of vaccination are likely to far outweigh the risks of vaccine-related adverse events. Herein, we provide operational COVID-19 vaccine guidance for patients participating in oncology clinical trials. In our perspective, continued quality oncological care requires that patients with cancer, including those involved in trials, be prioritized for COVID-19 vaccination, which should not affect trial eligibility., Patients with cancer have a high risk of morbidity and mortality from COVID-19. The rapid development of COVID-19 vaccines has provided new hope of mitigating the disease. Herein, the COVID19 and Cancer Clinical Trials Working Group calls for prioritization of patients with cancer, importantly including those participating in oncology clinical trials, for COVID-19 vaccination. The authors also provide operational COVID-19 vaccine guidance for patients participating in oncology clinical trials.

Background: BGB-3245 is a RAF dimer inhibitor with preclinical activity in MAPK-altered tumor models harboring BRAF V600 mutations, atypical BRAF mutations/fusions, and RAS mutations. This study is investigating the safety, pharmacokinetics, and preliminary antitumor activity of BGB-3245 in patients (pts) with advanced or refractory MAPK-altered solid tumors. Methods: Eligible pts were ≥18 yrs old with ECOG 0-1 and had solid tumors harboring MAPK pathway alterations. Dose-escalation and cohort-size decisions were made using the Modified Toxicity Probability Interval Design. The starting dose was 5 mg QD. Treatment emergent adverse events (TEAEs) were graded per NCI CTCAE v5.0. Tumor responses were assessed by investigators using RECIST v1.1. Results: As of 1 Sep 2022, 42 pts were treated across 6 cohorts (5-60 mg QD). The median age was 60 yrs; pts had received a median of 3 prior lines of treatment. All pts had TEAEs; 79% had treatment-related (TR) AEs. The most common TRAEs (≥ 10%) were rash acneiform (33%), rash maculopapular (24%), fever (17%), ALT elevations and nausea (both 12%). Gr ≥3 TRAEs were reported in 29% of pts, events in ≥ 2 pts included decreased platelet count and rash maculopapular (3 each), ALT and AST elevations, and fever (2 each). Dose reductions occurred in 5 pts: rhabdomyolysis (1), LVEF decreased (1), hand-foot syndrome (1), rash maculopapular (1), and liver function abnormalities (1). Dose interruptions due to AEs occurred in 60% of pts. Dose discontinuations occurred in 79% of pts, 57% due to disease progression or death, 21% due to AE. Dose limiting toxicities were observed at 10 mg, 40 mg, and 60 mg. 40 mg QD was determined as the MTD. PK results showed generally dose-proportional increases in exposure. Tmax was at ~2 h; BGB-3245 had a long terminal half-life and 7.4-fold average accumulation in exposure at steady-state. 79% of pts were efficacy evaluable. The disease control rate was 48% with 1 CR, 5 cPR, 2 uPR and 8 SD≥24 wks. Objective responders included BRAF V600E melanoma pts post-BRAF/MEK and checkpoint inhibitors (2; 1 CR, 1 cPR), 1 NRAS G12S melanoma and 1 NRAS Q61K melanoma (post checkpoint inhibitors), 1 BRAF V600E LGSOC (progressed on BRAF inhibitor), 1 BRAF V600E cholangiocarcinoma (progressed on BRAF/MEK inhibitors), 1 BRAF K601E/PIK3CA endometrial cancer, and 1 KRAS G12D appendiceal cancer. Preliminary analysis of circulating tumor DNA showed correspondence to clinical response. Conclusions: BGB-3245 has a manageable safety profile and a generally dose-proportional PK. Antitumor activity was observed in pts with no approved targeted therapy options. The safety and early efficacy profile of BGB-3245 supports further investigation in selected MAPK-altered tumors. Citation Format: Alison M. Schram, Vivek Subbiah, Ryan Sullivan, Rasha Cosman, Jia Liu, Eric I. Sbar, Thuy Hoang, Jiarong Chen, Mark Johnson, Vincent Amoruccio, Todd Shearer, Adeela Kamal, Jocelyn Lewis, Wenlin Shao, Badreddin Edris, Lusong Luo, Jayesh Desai. A first-in-human, phase 1a/1b, open-label, dose-escalation and expansion study to investigate the safety, pharmacokinetics, and antitumor activity of the RAF dimer inhibitor BGB-3245 in patients with advanced or refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT031.

Background: NRG1 fusions represent a rare and potentially actionable oncogenic alteration found in ~0.2% of solid tumors. Seribantumab is a fully human anti-HER3 IgG2 monoclonal antibody that blocks aberrant NRG1 fusion protein binding to HER3 thereby preventing ligand-dependent activation and dimerization resulting in complete inhibition of the PI3K/AKT and MAPK downstream signaling pathways. Seribantumab has shown significant anti-tumor activity in PDX models and in patients (pts) with solid tumors harboring NRG1 fusions. Methods: CRESTONE (NCT04383210) is a phase 2 study of seribantumab in adult pts with solid tumors harboring NRG1 fusions who received at least one prior therapy and were naïve to ERBB-targeted therapy (Cohort 1); pts previously treated with ERBB-targeted therapies (exploratory Cohort 2) and pts with tumors harboring additional molecular alterations (exploratory Cohort 3). Here, we present updated efficacy results for pts in Cohort 1 dosed at 3 g IV QW. Safety data are evaluated for pts enrolled in Cohort 1 and exploratory Cohorts 2 and 3. Results: As of Dec 2, 2022, 51 pts were enrolled across all cohorts with 30 pts in Cohort 1 dosed at 3 g IV QW. Median age was 62 years (range 19-84); median prior lines of therapy was 2 (range 1-7); tumor types included non-small cell lung cancer (NSCLC, n=30), pancreatic adenocarcinoma (PDAC, n=6), biliary tract/cholangiocarcinoma (CCA, n=6), breast (n=4), and others (n=5); 15 different NRG1 fusion partners were identified with CD74 (22%) and SLC3A2 (16%) as the most frequently reported. In the overall safety population, 41 pts (80%) reported at least one treatment-related adverse event (TRAE). The most common TRAEs (occurring in ≥20% of pts) were diarrhea (41%), fatigue (29%), rash (24%), and nausea (22%). Four pts (8%) experienced Gr 3/4 TRAEs; no Gr 5 TRAEs. The safety profile for Cohort 1 was similar to the overall safety population. Among the 30 pts in Cohort 1, 22 were evaluable for investigator assessed (INV) response per RECIST v1.1; 2 pts (9%) had confirmed complete response (CR), 6 pts (27%) had confirmed partial response (PR), and 13 pts (59%) had stable disease (SD) as their best overall response (BOR). The INV objective response rate (ORR, confirmed PR + CR) was 36% and disease control rate (DCR, confirmed PR+CR+SD) was 95%. The overall duration of response ranged from 1.4 to 17.2 months. In pts with NSCLC, the INV-ORR was 39% and DCR was 94%. Conclusions: Seribantumab has an acceptable safety and tolerability profile as a single agent in pts with solid tumors harboring NRG1 fusions. Updated efficacy data indicate seribantumab has robust and durable clinical activity, including CRs, across different tumor types harboring an NRG1 fusion and is a promising treatment option. Citation Format: Tejas Patil, Daniel R. Carrizosa, Mark E. Burkard, Karen L. Reckamp, Jayesh Desai, Young Kwang Chae, Stephen V. Liu, Kartik Konduri, Shirish M. Gadgeel, Jessica J. Lin, Parneet K. Cheema, David R. Spigel, Alison M. Schram, Valerie M. Jansen, Yasir Y. Elamin. CRESTONE: A Phase 2 study of seribantumab in adult patients with neuregulin-1 (NRG1) fusion positive locally advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT229.

Purpose: TRK inhibitors achieve marked tumor-agnostic efficacy in TRK fusion–positive cancers and consequently are now an established standard of care. Little is known, however, about the demographics, outcomes, response to alternative standard therapies, or genomic characteristics of TRK fusion–positive cancers. Experimental Design: Utilizing a center-wide screening program involving more than 26,000 prospectively sequenced patients, genomic and clinical data from all cases with TRK fusions were extracted. An integrated analysis was performed of genomic, therapeutic, and phenomic outcomes. Results: We identified 76 cases with confirmed TRK fusions (0.28% overall prevalence) involving 48 unique rearrangements and 17 cancer types. The presence of a TRK fusion was associated with depletion of concurrent oncogenic drivers (P < 0.001) and lower tumor mutation burden (P < 0.001), with the exception of colorectal cancer where TRK fusions cooccur with microsatellite instability (MSI-H). Longitudinal profiling in a subset of patients indicated that TRK fusions were present in all sampled timepoints in 82% (14/17) of cases. Progression-free survival on first-line therapy, excluding TRK inhibitors, administered for advanced disease was 9.6 months [95% confidence interval (CI), 4.8–13.2]. The best overall response rate achieved with chemotherapy containing–regimens across all lines of therapy was 63% (95% CI, 41–81). Among 12 patients treated with checkpoint inhibitors, a patient with MSI-H colorectal cancer had the only observed response. Conclusions: TRK fusion–positive cancers can respond to alternative standards of care, although efficacy of immunotherapy in the absence of other predictive biomarkers (MSI-H) appears limited. TRK fusions are present in tumors with simple genomes lacking in concurrent drivers that may partially explain the tumor-agnostic efficacy of TRK inhibitors.

Background: Loss of function in the tumor suppressor gene, PTEN, activates PI3K/AKT signaling, driving tumor growth. Somatic mutations in PTEN occur in 5-10% of estrogen-receptor-positive (ER+) breast cancer (BC), and PTEN loss/inactivation is associated with an aggressive BC phenotype and poor outcome. Capivasertib, a pan-AKT kinase inhibitor, has shown antitumor activity in solid tumors. In ER+ BC, suppression of PI3K/AKT signaling results in a compensatory increase in ER-dependent transcription, potentially limiting the efficacy of AKT inhibitors when given as monotherapy. We therefore investigated concurrent inhibition of AKT and ER with combination therapy of capivasertib and fulvestrant in PTEN-mutant ER+ metastatic BC (MBC). Methods: In an expansion cohort (part F) of a Phase I study (NCT01226316), oral capivasertib 400 mg twice daily, 4 days on 3 days off, and fulvestrant at labeled dose, was administered to ER+ MBC patients (pts) with tumors harboring a deleterious PTEN alteration (identified in tissue/plasma by local next-generation sequencing [NGS], with central NGS and immunohistochemistry [IHC] performed retrospectively). Pts were enrolled in fulvestrant-naïve (FN) or fulvestrant-resistant (FR) cohorts (max 24 pts/cohort). Key objectives included safety and efficacy based on 24-week clinical benefit rate (CBR). Results: At data cut-off, 31 pts (12 FN; 19 FR) received treatment. Median number of prior metastatic regimens was 7. FN pts had higher rates of visceral disease (100%) and prior chemotherapy receipt (median 4 [range 0-8]) than FR pts (84%; median 2 [1-7]), respectively]. CBR and median progression-free survival (PFS) were 17% and 2.6 months in FN pts, and 37% and 4.1 months in FR pts, respectively (Table). Twenty-four patients (77%) had PTEN mutations and 7 (23%) had PTEN gene deletions determined by local NGS. Central plasma NGS confirmed 79% (19/24) of the PTEN mutations, and IHC confirmed complete loss of the PTENprotein in 85% (22/26) of cases. Treatment-related grade ≥3 adverse events (AEs) occurred in 32%, most frequently diarrhea and maculopapular rash (both n=2 pts). Treatment-related AEs resulted in dose reduction in 2 pts. Table. Clinical efficacyFN, n=12FR, n=19ORR, % (95% CI)8 (0.2, 39)21 (6, 46)CBR, % (95% CI)17 (2, 48)37 (16, 62)Confirmed response, n (%)1 (8)4 (21)Stable disease ≥24 weeks, n (%)2 (17)3 (16)Median PFS, months (95% CI)2.6 (1.2–4.2)4.1 (1.5–6.7)Median duration of response, months (95% CI)5.5 (NC–NC)6.9 (1.4–NC)Data cut-off was 21 March 2019. Median time from last fulvestrant administration to study entry in FR pts (n=19) was 13.8 months (range 0.7–28.2). CBR was defined as confirmed responders and those with stable disease ≥24 weeks. NC, not calculable (because of limited pt numbers); ORR, objective response rate Conclusions: Capivasertib plus fulvestrant is clinically active in heavily pretreated PTEN-mutated ER+ MBC, including in pts with prior resistance to fulvestrant. Efficacy appeared marginally better in FR than FN pts, possibly due to enrichment of pts with more aggressive disease in the FN cohort. Further analyses of the relationship between genomic features, such as concurrent mutations with drug activity, will be reported. Citation Format: Lillian M Smyth, Gerald Batist, Funda Meric-Bernstam, Peter Kabos, Iben Spanggaard, Ana Lluch, Alison Schram, Andrea Varga, Andrea Wong, Helen Ambrose, Alan Barnicle, T. Hedley Carr, Elza C de Bruin, Carolina Salinas-Souza, Andrew Foxley, Joana Hauser, Justin PO Lindemann, Rhiannon Maudsley, Robert McEwen, Michele Moschetta, Martine Roudier, Gaia Schiavon, Pedram Razavi, Udai Banerji, Sarat Chandarlapaty, José Baselga, David M Hyman. Capivasertib (AZD5363) in combination with fulvestrant in PTEN-mutant ER+ metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-05.

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Preexistent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively, these data define HER2 mutations as a therapeutic target in breast cancer and suggest that coexistence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib. Significance: HER2 mutations define a targetable breast cancer subset, although sensitivity to irreversible HER kinase inhibition appears to be modified by the presence of concurrent activating genomic events in the pathway. These findings have implications for potential future combinatorial approaches and broader therapeutic development for this genomically defined subset of breast cancer. This article is highlighted in the In This Issue feature, p. 161