FGFR3 Mutations Research Articles

Abstract 5058: Genomic alterations predictive of outcome in early staged cervical cancer: A translational investigation from the SENTICOL III trial

Abstract Background: The risk of relapse in patients with early-stage cervical cancer (ESCC) ranges from 4-14% (LACC study). Risk-factors for recurrence are mainly derived from histopathological factors. Senticol III is a large multicenter international randomized prospective study aims to validate sentinel lymph node biopsy in the surgical management of ESCC. Our objective is to characterize prognostic and actionable biomarkers in ESCC. Methods: FFPE-fixed tumoral slides of the first 150 randomized patients were analyzed based on SEDLIS criteria. We characterized TIL’s infiltration, PD-L1 expression and HPV status. Sequencing of tumor DNA was carried out using an in-house targeted next-generation sequencing panel (571 genes). Relapse-free survival (RFS) was defined as the time between randomization and relapse of any type. We performed Fisher’s exact test and Student’s t-test to compare distributions of categorical variables and Log Rank test. Results: The sequencing quality control criteria were met for 135 samples. PIK3CA was the most commonly altered gene (17%). Loss-of-function variants in KMT2-family genes (KTM2C 9%, KMT2D 6%, KMT2A 1%) were found in 16% of samples. The loss-of-function variant of ARID1A was mostly non-sense mutation (6%). Activations of ERBB2 (5%) were 3 focal amplifications and 4 hotspot missense mutations. Two samples were found to have microsatellite instability due to an alteration of MLH1. The Pik3/AKT/mTOR (26%) and the chromatin remodeling (23%) pathways were the most altered pathways. The median follow-up period was 20 months. There were 6 relapses, of which 80% were considered as low risk based on SEDLIS criteria. Recurrences were local and occurred 9-23 months following surgery. Multiparity was the only clinical feature associated with RFS (p < 0,001). Tumor size, histological subtypes, PD-L1 status, number of TIL’s, presence or absence of lymphovascular emboli and deep stromal invasion were not associated with RFS in our cohort.KMT2C (p=0,018) and ARID1A (p < 0,001) pathogen variants were significantly associated with RFS. The pathway chromatin remodeling (p=0,063) showed a trend toward association with RFS. We found 32% of genomic alterations actionable for a target therapy including mutations of PIK3CA, ERBB2, ARID1A, PTEN, BRCA2, KRAS, BRAF, CDK12, FGFR2, NF1, AKT1, BRCA1, TP53 and MSI-high. Conclusion: Our prospective, randomized and multicentric study suggests a genomic profile from a homogeneous cohort of 150 patients. We identified genes involved in chromatin remodeling (ARID1A and KMT2C) as prognostic biomarkers of clinical interest and suggesting the relevance of epidrugs in ESCC. Theranostic annotations of variants identify a matched to target therapy for 32% of alterations. Those targets represent multiple tracks for personalized therapy in ESCC. Citation Format: Maryame El Gani, Sabrina Ibadioune, Zakhia El Beaino, Abderraouf Hamza, Sophie Vacher, Emmanuelle Jeannot, Julien Masliah-Planchon, Anne Salomon, Alexandre Degnieau, Anne-Sophie Bats, Martin Koskas, Virginie Fourchotte, Estelle Wafo, Nicolas Bourdel, Raffaele Fauvet, Marie Plante, Patrice Mathevet, Maud Kamal, Fabrice Lecuru, Ivan Bièche. Genomic alterations predictive of outcome in early staged cervical cancer: A translational investigation from the SENTICOL III trial [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 5058.

Abstract 155: Clinico-radiological and histomolecular analyses identify of a new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered

Abstract Diffuse midline gliomas (DMG) H3 K27-altered are incurable grade 4 gliomas and represent a major challenge in neuro-oncology. This tumor type is now classified in four subtypes by the 2021 edition of the WHO Classification of the Central Nervous System (CNS) tumors. However, the H3.3-K27M subgroup still appears clinically and molecularly heterogenous. Recent publications reported that rare patients presenting a co-occurrence of H3.3K27M with BRAF or FGFR1 alterations tended to have a better prognosis. To better study the role of these co-driver alterations, we assembled a large pediatric and adult cohort of 29 tumors H3K27-altered with co-occurring activating mutation in BRAF or FGFR1 as well as 31 previous cases from the literature. We performed a comprehensive histological, radiological, genomic, transcriptomic and DNA methylation analysis. Interestingly, unsupervised t-distributed Stochastic Neighbor Embedding (tSNE) analysis of DNA methylation profiles regrouped BRAFV600E and all but one FGFR1MUT DMG in a unique methylation cluster, distinct from the other DMG subgroups and also from ganglioglioma (GG) or high-grade astrocytoma with piloid features (HGAP). This new DMG subtype harbors atypical radiological and histopathological profiles with calcification and/or a solid tumor component both for BRAFV600E and FGFR1MUT cases. Contrary to other DMG, these tumors occur more frequently in the thalamus (70% for BRAFV600E and 58% for FGFR1MUT) and patients have a longer overall survival with a median above three years. The analyses of a H3.3-K27M BRAFV600E tumor at diagnosis and corresponding in vitro cellular model showed that mutation in H3-3A was the first event in the oncogenesis, indicating a distinct oncogenesis from classical DMG despite an identical founder H3K27 alteration, and possible subclonal evolution.In conclusion, DMG, H3 K27 and BRAF/FGFR1 co-altered represent a new subtype of DMG with distinct genotype-phenotype characteristics, which deserve further attention with respect to trial interpretation and patient management. Our work also lays the foundations for therapeutic development highly-needed against this deadly disease. Citation Format: Lucie Auffret, Yassine Ajlil, Arnault Tauziede-Espariat, Thomas Kergrohen, Chloé Puiseux, Laurent Riffaud, Pascale Blouin, Anne-Isabelle Bertozzi, Pierre Leblond, Klas Blomgren, Sebastien Froelich, Alberto Picca, Mehdi Touat, Marc Sanson, Kevin Beccaria, Thomas Blauwblomme, Volodia Dangouloff-Ros, Nathalie Boddaert, Pascale Varlet, Marie-Anne Debily, Jacques Grill, David Castel. Clinico-radiological and histomolecular analyses identify of a new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered [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 155.

Abstract 5582: Discovery and preclinical characterization of ISM8001, a covalent and selective FGFR2/FGFR3 dual inhibitor with strong monotherapy anti-tumor activity against advanced solid tumors

Abstract Oncogenic alterations in fibroblast growth factor receptors 2 and 3 (FGFR2 and FGFR3) are key drivers in intrahepatic cholangiocarcinoma, urothelial carcinoma and multiple other solid tumors. Currently approved pan-FGFR inhibitor therapy is limited by off-isoform toxicity (FGFR1-mediated hyperphosphatemia and FGFR4-mediated diarrhea) and acquired FGFR2 or FGFR3 kinase domain resistance mutations. The second generation of FGFR inhibitors currently undergoing clinical evaluation such as RLY-4008 and LOXO-435 are primarily focused on achieving selectivity against either the FGFR2 or FGFR3 isoform, which may narrow their clinical potential. To overcome this limitation and to allow a favorable toxicity profile, we designed and evaluated the anti-cancer effects of a novel and selective FGFR2/FGFR3 dual inhibitor, ISM8001. Biochemically, ISM8001 showed superior inhibition against FGFR2 and FGFR3 with >90-fold and >800-fold selectivity of FGFR2 over FGFR1 and FGFR4, respectively. As an irreversible inhibitor, ISM8001 displayed strong affinity and high efficiency of covalent bond formation with FGFR2/3. Additionally, ISM8001 showed broad inhibition against multiple FGFR2/3 mutant isoforms, especially gatekeeper mutation (FGFR2-V564F and FGFR3-V555M). ISM8001 inhibited cell proliferation of multiple human cancer cell lines with FGFR2/3 aberration with IC50 <10 nM while IC50 >1 µM was observed in cell lines without FGFR2/3 alterations. Furthermore, ISM8001 demonstrated robust in vivo anti-tumor efficacy in multiple cell line-derived xenograft mouse models including the SNU-16 gastric cancer model (FGFR2-amplification), the AN3CA endometrial cancer model (FGFR2 N549K mutation) and the RT4 bladder cancer model (FGFR3-fusion), with tumor growth inhibition varying from 80%~120% while no significant serum phosphate increase or body weight loss. ISM8001 also displayed strong anti-tumor effects in xenograft models of Ba/F3-FGFR2/3 engineered cell lines with different gatekeeper mutations. ISM8001 is classified as a BCS II compound with favorable DMPK properties and druggability, high permeability, low efflux ratio and low DDI risk. In vivo PK profiles showed moderate to low clearance and high oral bioavailability across preclinical species. In addition, ISM8001 showed a promising in vitro safety profile with favorable selectivity in 468-kinase and Cerep-44 panel studies, and no hERG concerns. Results of 28-day non-clinical toxicology studies in rat and dog showed a safety window of approximately 2-5 fold based on efficacious exposure in different models. Taken together, these data support the clinical development of ISM8001 as a novel, selective FGFR2/FGFR3 dual inhibitor for the potential tissue-agnostic therapy of advanced solid tumors with FGFR2/3 aberrations. Citation Format: Yihong Zhang, Yazhou Wang, Xin Cai, Jinxin Liu, Hui Cui, Junwen Qiao, Xiaoxia Lin, Xiao Ding, Supriya Bavadekar, Sujata Rao, Man Zhang, Feng Ren, Alex Zhavoronkov. Discovery and preclinical characterization of ISM8001, a covalent and selective FGFR2/FGFR3 dual inhibitor with strong monotherapy anti-tumor activity against advanced solid tumors [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 5582.

Abstract 1965: 3HP-2827-high selective inhibitor of FGFR2, a best-in-class therapy for FGFR2-driven solid tumors

Abstract FDA-approved pan-FGFR inhibitors bring encouraging results in solid tumors. However, resistance mechanisms and side effect profiles may limit their clinical utility. We designed 3HP-2827, a highly selective and potent small molecule inhibitor of FGFR2, to treat solid tumors harboring FGFR2 alterations, including amplifications, mutations, and fusions to overcome these limitations. 3HP-2827 inhibits FGFR2 enzyme activity with more than 1623-fold selectivity over FGFR1 and FGFR3. 3HP-2827 inhibits cell proliferation with IC50 <10 nM and 30 nM in FGFR2 amplified and FGFR2-mutant cancer cell lines, respectively. 3HP-2827 demonstrates high kinome selectivity for FGFR2 against a panel of 485 human kinases, resulting in 90.1% inhibition of FGFR2, and no other kinases showed greater than 75% inhibition. 3HP-2827 leads only modest activity at 10 µM against PDE4D2 and LCK in 44 distinct common adverse drug reaction targets, supporting its high degree of safety. 3HP-2827 also demonstrates a dose-dependent inhibition of phosphorylation of FGFR2 and FGFR2 signaling pathway nodes, including ERK and AKT, in the FGFR2-dependent cancer cell line. In vivo, 3HP-2827 demonstrates potent antitumor activity in cell- and patient-derived xenograft mouse models, including FGFR2-amplified gastric cancer, FGFR2-mutant (N549K) endometrial cancer, FGFR2 fusion ICC and gastric cancers. Strikingly, 3HP-2827 demonstrates superior activity in patient-derived ICC and gastric cancer, cell-derived endometrial cancer (CDX) in tumor and pFGFR2 inhibitions. In the ICC PDX model, 3HP-2827 also induces the regression of pemigatinib-resistant tumors. 3HP-2827 is well tolerated in the toxicology studies, and no hyperphosphatemia or tissue mineralization is observed at 150 mg/kg/kg/day in rats and 120 mg/kg/day in dogs. The data support that 3HP-2827, a potential to be a best-in-class selective FGFR2 inhibitor, will be studying clinically at the beginning of 2024. Citation Format: Susan S. Liu-Chen, Jufang Lin, Jingyu Hu, Yanchao Liu, Weibo Zhang, Zhixiong Zhang, Yan Ding, Jinqiu Liu, Kevin Chen, Shaojing Hu. 3HP-2827-high selective inhibitor of FGFR2, a best-in-class therapy for FGFR2-driven solid tumors [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 1965.

Abstract 2098: A novel GCN2 kinase activator demonstrates therapeutic efficacy in preclinical PDX models of human cholangiocarcinoma

Abstract Introduction Cholangiocarcinoma (CCA) is a highly lethal, heterogenous biliary malignancy. Despite the development of targeted therapies, treatment success has been hindered by primary or acquired resistance. New therapeutic strategies are necessary to improve outcomes in these patients. NXP800 is a GCN2 small-molecule activator that phosphorylates eIF2a inducing selective ATF4 transcription which stimulates stress-induced genes leading to apoptosis. NXP800 has been previously validated in ovarian cancers with ARID1A mutations, well-known players in cholangiocarcinoma. We sought to determine the treatment efficacy of novel NXP800 in CCA utilizing patient derived xenograft (PDX) preclinical models. Methods In our preliminary studies, four human and two murine CCA cell lines were tested for cell viability using CellTiter-Glo to determine the maximal inhibitory concentration (IC50). Immunoblot analysis of HuCCT-1 cells incubated with 1 mM of NXP800 for 6 hours vs. vehicle was performed. We also evaluated response to GCN2 activation in multiple RNA sequenced PDX models. Xenografts were expanded into the flank of NOD/SCID mice and treatment begun when tumors averaged 120mm3. Tumor bearing mice were treated with NXP800 (35 mg/kg) or vehicle via oral gavage five days on, two days off, for 28 days. Tumor volume and animal weight were collected twice weekly. Predicted sensitive signatures were determined using multi-omics. Results IC50 dose response curves showed nanomolar activity in all six cell lines, ranging from 5-170 nM. Immunoblotting demonstrated an upregulation of phosphorylated eIF2a and in turn upregulation of ATF4 with treatment of NXP800 compared to vehicle. Five PDX tumors from patients with intrahepatic or distal CCA was established and validated by histologic analysis. Molecular characterization of the tumor identified tumor mutation and a total tumor mutational burden. RNA sequencing was completed and identified 21-gene signatures related to YAP activity, which were cross-referenced against our existing PDX model tumor bank. Treatment with NXP800 was associated with a statistically significant decrease in tumor size in three of five PDX models tested, which included an ARID1A, FGFR1, and ATM mutants. Preliminary multi-omics on the PDX models revealed that sensitivity mechanisms were centered around EGFR signaling. Conclusion The novel GCN2 kinase activator, NXP800, has nanomolar efficacy in both human and murine CCA cell lines in vitro. Additionally, NXP800 demonstrated therapeutic activity in multiple cholangiocarcinoma PDX models in vivo. We are opening a Phase 1b clinical trial investigating the effects of NXP800 use in patients with advanced CCA. Future studies determining the cellular effect of GCN2 activation utilizing NXP800 are being conducted. Citation Format: Danielle Marie Carlson, Hendrien Kuipers, Amro Abdelrahman, Erik Jessen, Joshua Roldan Kalil, Jack Sample, Jennifer Tomlinson, Mark Truty, Rory Smoot. A novel GCN2 kinase activator demonstrates therapeutic efficacy in preclinical PDX models of human cholangiocarcinoma [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 2098.

Abstract 3308: A selective FGFR1/2 degrader overcomes endocrine resistance in ER+/FGFR1-amplified andFGFR1/2-mutant breast cancer

Abstract FGFR1 amplification and FGFR1/2 activating mutations are associated with resistance to antiestrogens in the estrogen receptor-positive (ER+) breast cancer. In this study, we investigated selective degradation of FGFR1/2 using the proteolysis-targeted chimera (PROTAC) DGY-09-192 as a novel therapeutic strategy to overcome resistance to antiestrogens in ER+ breast cancers harboring FGFR1-amplification or FGFR1/2 activating mutations. Treatment of ER+/FGFR1-amplified CAMA1 and MDA-MB-134 breast cancer cells with DGY-09-192 degraded FGFR1 and suppressed phosphorylation of FGFR1 and its downstream targets FRS2, AKT, and ERK1/2. DGY-09-192-mediated FGFR1 degradation were rescued by a proteasome inhibitor, MG132, suggesting that the antitumor activity of DGY-09-192 depends on proteasome-mediated degradation. Growth of CAMA1 and MDA-MB-134 cells were modestly inhibited by single-agent DGY-09-192 or the ER degrader fulvestrant, but the combination completely arrested cell proliferation. Treatment of female NSG mice bearing established ER+/FGFR1-amplified patient-derived xenografts (PDXs) with single-agent DGY-09-192 (40 mg/kg/day, i.p.) or fulvestrant (5 mg/week, s.c.) delayed tumor growth, respectively. However, treatment with DGY-09-192 + fulvestrant induced tumor regression in all mice (n=7), suggesting that a FGFR1 degrader plus an antiestrogen can serve as an effective therapeutic combination for ER+/FGFR1-amplified breast cancer. Next, we examined whether ER+ breast cancer cells harboring FGFR1/2 hotspot mutations are sensitive to DGY-09-192. Ectopic expression of FGFR1N546K, the most common FGFR1 mutation in breast cancer, activated FGFR1 signaling and induced resistance to fulvestrant compared to MCF7 cells transduced with wild-type FGFR1. EFM-19 cells harboring FGFR2K659E exhibited resistance to fulvestrant in vitro. Treatment of both cell lines with DGY-09-192 degraded the mutant FGFR1N546K and FGFR2K659E, respectively, blocked phosphorylation of the FGFR downstream targets, and markedly inhibited cell proliferation. These results suggest that PROTAC-mediated degradation of FGFR1/2 effectively blocks the growth of ER+, FGFR1/2-mutant breast cancers resistant to antiestrogens. Conclusions: DGY-09-192 induced FGFR1/2 degradation and blocks FGFR downstream signaling in ER+/FGFR1-amplified and FGFR1/2-mutant breast cancer cells. The combination of DGY-09-192 and fulvestrant exhibited superior antitumor effects relative to each monotherapy alone in vitro and/or in vivo. Therefore, PROTAC-mediated FGFR1/2 degradation represents a promising therapeutic strategy for ER+ breast cancers harboring FGFR1-amplification or FGFR1/2 activating mutations. Citation Format: Yasuaki Uemoto, Chang-Ching Lin, Bingnan Wang, Dan Ye, Emmanuel Bikorimana, Alberto Servetto, Luigi Formisano, Fabiana Napolitano, Rosario Chica Parrado, Saurabh Mendiratta, Chuo Chen, Ariella B. Hanker, Carlos L. Arteaga. A selective FGFR1/2 degrader overcomes endocrine resistance in ER+/FGFR1-amplified andFGFR1/2-mutant breast cancer [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 3308.

Dysregulated FGFR3 signaling alters the immune landscape in bladder cancer and presents therapeutic possibilities in an agent-based model.

Bladder cancer is an increasingly prevalent global disease that continues to cause morbidity and mortality despite recent advances in treatment. Immune checkpoint inhibitors (ICI) and fibroblast growth factor receptor (FGFR)-targeted therapeutics have had modest success in bladder cancer when used as monotherapy. Emerging data suggests that the combination of these two therapies could lead to improved clinical outcomes, but the optimal strategy for combining these agents remains uncertain. Mathematical models, specifically agent-based models (ABMs), have shown recent successes in uncovering the multiscale dynamics that shape the trajectory of cancer. They have enabled the optimization of treatment methods and the identification of novel therapeutic strategies. To assess the combined effects of anti-PD-1 and anti-FGFR3 small molecule inhibitors (SMI) on tumor growth and the immune response, we built an ABM that captures key facets of tumor heterogeneity and CD8+ T cell phenotypes, their spatial interactions, and their response to therapeutic pressures. Our model quantifies how tumor antigenicity and FGFR3 activating mutations impact disease trajectory and response to anti-PD-1 antibodies and anti-FGFR3 SMI. We find that even a small population of weakly antigenic tumor cells bearing an FGFR3 mutation can render the tumor resistant to combination therapy. However, highly antigenic tumors can overcome therapeutic resistance mediated by FGFR3 mutation. The optimal therapy depends on the strength of the FGFR3 signaling pathway. Under certain conditions, ICI alone is optimal; in others, ICI followed by anti-FGFR3 therapy is best. These results indicate the need to quantify FGFR3 signaling and the fitness advantage conferred on bladder cancer cells harboring this mutation. This ABM approach may enable rationally designed treatment plans to improve clinical outcomes.

Abstract B102: Mutational and copy number-based ctDNA profiles mirror high-grade serous cancer tumors and enable detection of genetic changes appearing at recurrence

Abstract Circulating tumor DNA (ctDNA) is the cancer-derived fraction of extracellular DNA fragments present in the bloodstream as cell-free DNA (cfDNA). Consequently, ctDNA harbors the distinct genomic information specific to the cancer from which it derives. Therefore, ctDNA from liquid biopsies provides a non-invasive, universally available alternative to traditional tumor biopsies. ctDNA amount may provide insight into tumor burden, as its concentration is typically highest during diagnosis and relapse. The non-invasive nature of this method facilitates frequent monitoring, enabling longitudinal tracking of the disease's trajectory and response to treatments. By focusing on the key genomic changes, either short mutations or copy number aberrations (CNA), liquid biopsy allows the investigation of cancer progression and evolution over time, providing invaluable insight into the behavior and adaptability of the cancer. We collected 152 longitudinal plasma samples and 92 fresh tissue samples from 29 patients enrolled in the DECIDER cohort (NCT04846933), after a diagnosis of high-grade serous carcinoma (HGSC), the most common and deadliest histological subtype of ovarian cancer. With ultra-deep targeted sequencing of a gene panel of more than 700 cancer-related genes we were able to comprehensively evaluate the cancer-related genomic changes. The concordance rate between mutations in plasma compared to tumor tissue was 83 % at diagnosis and 90 % at relapse, confirming the reliability of exploiting ctDNA in monitoring genomic alterations. Furthermore, by comparing plasma- and tissue-derived mutations, we demonstrated that comparable levels of ctDNA are released from adnexa, intra-abdominal lesions, and ascites, proposing that a liquid biopsy represents mutations from various locations. Analysis of CNA showed that our approach successfully detected concordant profiles in most plasma samples, even with tumor content as low as 3%. Additionally, highly amplified regions were identified in samples with approximately 1% tumor content. Longitudinal analysis of both mutations and CNA revealed genomic changes in plasma samples at relapse. Twenty-four new exonic mutations were identified in eight patients at relapse; two of these alterations were related to HRD status, supporting PARP-inhibitors as a valid treatment option. Furthermore, mutations in FGFR3, JAK2, FLT3 and CDKN2A could be investigated as target indications for extended use of currently available therapies. Eleven patients had high tumor content in their plasma samples at the time of relapse. We detected altered CNA profiles in seven of these eleven patients, so that five patients harbored only a few novel focal amplifications or losses, while two patients showed significant changes in their copy-number profile during the therapy. In conclusion, our study identified recurrence-specific genomic aberrations, proposing that ctDNA more accurately represents the metastatic tumor and provides possibilities for personalized therapy options. Citation Format: Giovanni Marchi, Mai T.N. Nguyen, Anna Rajavuori, Taru Muranen, Kaisa Huhtinen, Sinikka Oksa, Sakari Hietanen, Johanna Hynninen, Jaana Oikkonen, Sampsa Hautaniemi. Mutational and copy number-based ctDNA profiles mirror high-grade serous cancer tumors and enable detection of genetic changes appearing at recurrence [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B102.

Abstract B035: A compendium of syngeneic, transplantable pediatric high-grade glioma models reveals subtype-specific therapeutic vulnerabilities

Abstract Pediatric high-grade gliomas (pHGGs) are lethal, incurable brain tumors frequently driven by clonal mutations in histone genes. They often harbor a range of additional genetic alterations that correlate with different ages, anatomical locations, and tumor subtypes. We developed models representing 16 pHGG subtypes driven by different combinations of alterations targeted to specific brain regions. Tumors developed with varying latencies and cell lines derived from these models engrafted in syngeneic, immunocompetent mice with high penetrance. Targeted drug screening revealed unexpected selective vulnerabilities. Moreover, H3.3K27M tumors with PIK3CA, NF1 and FGFR1 mutations were more invasive and harbored distinct additional phenotypes, such as exophytic spread, cranial nerve invasion and spinal dissemination. Collectively, these models reveal that different partner alterations produce distinct effects on pHGG cellular composition, latency, invasiveness, and treatment sensitivity. Citation Format: Michael McNicholas, Antonella De Cola, Zahedeh Bashardanesh, Amelia Foss, Cameron Lloyd, Steven Hebert, Damien Faury, Augusto Faria Andrade, Nada Jabado, Claudia Kleinman, Manav Pathania. A compendium of syngeneic, transplantable pediatric high-grade glioma models reveals subtype-specific therapeutic vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B035.

Clinicopathological analysis of rosette-forming glioneuronal tumors

BackgroundThis study aimed to investigate the clinicopathological characteristics, diagnostic indicators, and critical factors for the differential diagnosis of rosette-forming glioneuronal tumor (RGNT).Patients and methodsThis retrospective study included six surgically treated RGNT cases. We analyzed and summarized their clinical manifestations, radiological features, histological morphology, immunophenotype, and molecular genetic changes, supplemented with a literature review.ResultsThe patients comprised four males and two females with a mean age of 35 years. The tumors were located in the cerebellum (two cases); the fourth ventricle, quadrigeminal cistern, and third ventricle (one case each); and the fourth ventricle and brainstem (one case). Clinical manifestations included headaches in four cases, left eyelid ptosis in one case, and one asymptomatic case only identified during physical examination. Microscopically, the tumor cells were uniform in size and were marked by rosette-like or pseudorosette-like structures around the neuropil and blood vessels. Immunohistochemistry revealed biphasic patterns. The central neuropil components of the rosette-like structures around the neuropil and the pseudorosette structures of the perivascular regions expressed Syn, while the cells surrounding the rosettes expressed Olig2 and not GFAP. GFAP and S-100 were expressed in the glial components but not in the rosette or pseudorosette regions. The Ki-67 proliferation index was typically low. Molecular genetic analysis showed that the main molecular changes involved FGFR1 mutation accompanied by PIK3R1 mutation. None of the patients received chemoradiotherapy postoperatively. Follow-up durations varied between 4 and 23 months with no recorded recurrence or metastasis.ConclusionRGNT is a comparatively rare mixed glioneuronal tumor that occurs in the midline structures. Its morphology shows certain overlaps with other low-grade neuroepithelial tumors. Identifying the rosettes around the neuropil is critical for morphological diagnosis, and the molecular identification of FGFR1 mutations accompanied by PIK3R1 mutations can facilitate diagnosis.

Genetic landscape of breast cancer subtypes following radiation therapy: insights from comprehensive profiling.

In breast cancer, in the era of precision cancer therapy, different patterns of genetic mutations dictate different treatments options. However, it is not clear whether the genetic profiling of breast cancer patients undergoing breast-conserving surgery is related to the adverse reactions caused by radiotherapy. We collected formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 54 breast cancer patients treated with radiation after breast-conserving surgery and identified comprehensive molecular information in hundreds of cancer-associated genes by FoundationOne CDx (F1CDx), a next-generation sequencing (NGS)-based assay. Among our cohort of 54 breast cancer patients, we found high-frequency mutations in cancer-related genes such as TP53 (56%), RAD21 (39%), PIK3CA (35%), ERBB2 (24%), and MYC (22%). Strikingly, we detected that the WNT pathway appears to be a signaling pathway with specific high-frequency mutations in the HER2 subtype. We also compared the mutation frequencies of the two groups of patients with and without cutaneous radiation injury (CRI) after radiotherapy and found that the mutation frequencies of two genes, FGFR1 and KLHL6, were significantly higher in patients with CRI : No subgroup than in those with CRI : Yes. Different breast cancer subtypes have their own type-specific mutation patterns. FGFR1 and KLHL6 mutations are protective factors for radiation-induced skin toxicity in breast cancer patients.

Exploring FGFR3 Mutations in the Male Germline: Implications for Clonal Germline Expansions and Paternal Age-Related Dysplasias.

Delayed fatherhood results in a higher risk of inheriting a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early- or late-onset effects in the offspring. Here, we used digital polymerase chain reaction to assess the frequency and spatial distribution of 10 different FGFR3 missense substitutions in the sexually mature male germline. Our functional assessment of the receptor signaling of the variants with biophysical methods showed that 9 of these variants resulted in a higher activation of the receptor´s downstream signaling, resulting in 2 different expansion behaviors. Variants that form larger subclonal expansions in a dissected postmortem testis also showed a positive correlation of the substitution frequency with the sperm donor's age, and a high and ligand-independent FGFR3 activation. In contrast, variants that measured high FGFR3 signaling and elevated substitution frequencies independent of the donor's age did not result in measurable subclonal expansions in the testis. This suggests that promiscuous signal activation might also result in an accumulation of mutations before the sexual maturation of the male gonad with clones staying relatively constant in size throughout time. Collectively, these results provide novel insights into our understanding of the mutagenesis of driver mutations and their resulting mosaicism in the male germline with important consequences for the transmission and recurrence of associated disorders.

Comparison of tissue based FGFR mutation detection by Therascreen FGFR with UroTyper FGFR and ADC test and relevance for potential co-targeting with TROP2 and NECTIN4: Preview of Bladder BRIDGister.

679 Background: In view of the efficacy of FGFR targeting in early and advanced bladder cancer, as has been shown for erdafitinib in the THOR and NORSE trial, molecular testing of FGFR mutations and fusions will soon become clinical routine worldwide. Therascreen FGFR testing has been approved as companion diagnostic to select patients for erdafitinib. However, technical problems and shortages as well as a limited number of detectable mutations indicate the need of FGFR test systems that can be performed easily & fast worldwide. The objective was to evaluate concordance of UroTyper FGFR with Therascreen FGFR mutation testing and unravel molecular characteristics of FGFR mutated tumors to assess potential co-targeting options for erdafitinib to justify subsequent adopted clinical trials with the Bladder BRIDGister framework. Methods: FFPE tumor tissues from 100 TURB samples were prospectively collected as part of the Bladder BRIDGister. RNA from FFPE tissues were extracted by commercial kits to assess Therascreen FGFR & UroTyper FGFR MODAPLEX (BIOTYPE GmbH, Dresden) in conjunction with relative gene expression of subtyping markers (i.a.. PPARG), ADC targets (TROP2, NECTIN4) and CPI targets by RT-qPCR systems. Hierarchical clustering, Spearman correlation tests were done by JMP 9.0.0 (SAS software). Results: After exclusion of FGFR fusion samples, a total of 94 samples was available for comparative mutation analysis. Both PCR based FGFR tests concordantly identified S249C (n=15), R248C (n=2) and G370C (n=1) mutations, while the results for Y373C were in part discrepant with Therascreen FGFR detecting 9 mutations, while UroTyper FGFR did detect 6 mutations. Further examination of the 3 discrepant Y373C cases by SNaPshot and Uromonitor FGFR test revealed that the Therascreen FGFR test resulted in 3 false positive results. Moreover, one sample was invalid from Therascreen FGFR test. Overall, the concordance was very good and reached a kappa value of 0.92. Interestingly, FGFR3 mutations by UroTyper FGFR test were positively associated not only with FGFR3 mRNA overexpression (r=0.6699, p<0.0001), but also with TROP2 and NECTIN4 (r=0.5026 and r=0.4406, p<0.0001) indicating, that combinations of FGFR targeting with ADC targeting of TROP2 or NECTIN4 could have strong synergistic effects. Hierarchical clustering revealed a homogenous FGFR3 mutated group of tumors simultaneously co-expressing the target genes TROP2, NECTIN4 and PPARG. Conclusions: PCR-based FGFR assessment by Therascreen and UroTyper is highly concordant and enables fast, local FGFR assessment within few hours. FGFR3 mutations are associated with increased TROP2 & NECTIN4 expression indicating potential synergistic options which warrants further exploration as part of molecularly stratified clinical trials.

Genomic profiling of bladder cancer in waterpipe and cigarette smokers: Implications for carcinogenicity and genetic landscape.

657 Background: While cigarette smoking is a well-established risk factor for bladder cancer (BC), limited research has explored the contribution of waterpipe smoking (WPS) to BC. WPS is gaining popularity among Middle Eastern youth, often perceived as a safer alternative to cigarettes. Alarmingly, Lebanese males had an age-standardized incidence rate of 54.0 per 100,000 in 2019, the highest in the Arab World and five times the global average, prompting a critical investigation into potential risk factors. Mounting evidence suggests that WPS exposes users to toxicants and carcinogens that can induce chromosomal damage contributing to cancer. This study aims to assess the genomic and mutational profiles of BC in waterpipe smokers compared to cigarette smokers. Methods: This cross-sectional study enrolled adults with urothelial carcinoma (UC) who exclusively smoked waterpipe at the American University of Beirut Medical Center (AUBMC) between April 2017 and September 2019. Next-generation sequencing by MSK-IMPACT characterized mutational profiles, tumor mutation burden, and microsatellite instability (MSI) status in tumor-normal pairs. For comparison, a cohort from Memorial Sloan Kettering Cancer Center (MSKCC), comprising previously profiled BC tumors from cigarette smokers, was used. Results: A total of 14 exclusively WPS-BC patients were enrolled, with 12 males (85.7%) and a median age at diagnosis of 62.9 years (range: 30.7 to 78.9). Among these patients, 9 (64.3%) had non-muscle invasive BC, with 8 (57.2%) classified as high-grade UC. MSK-IMPACT sequencing successfully analyzed 13 WPS samples, revealing genomic patterns similar to the cigarette smoking cohort. Common genetic alterations in the WPS cohort included mutations in TERT promoter (62%), TP53 and FGFR3 (46% each), and PIK3CA (38%). Mutations in chromatin modifiers were less prevalent, with the highest frequencies observed in KDM6A (31%) and KMT2C (15%). The tumor mutation burden was higher in the WPS group (22 per MB) compared to the cigarette smoking cohort (9 per MB), with no definite evidence of MSI. PD-L1 expression was generally low, with only 2 samples exhibiting combined positivity scores (CPS) of 15 and 10, primarily associated with immune cells PD-L1 expression. Only one tumor demonstrated 3% PD-L1 expression on tumor cells, while 7 tumors displayed no expression on either tumor or immune cells (CPS of 0). Conclusions: This study underscores the genomic similarities between BC in waterpipe and cigarette smokers. The preliminary findings, limited by sample size, highlight the need for further investigations to elucidate the potential role of WPS in BC development. Future efforts will focus on cohort expansion to validate initial observations, raising awareness of health risks associated with WPS, and emphasizing the importance of continued research to inform preventive strategies.

Sunitinib in Patients With Breast Cancer With FGFR1 or FGFR2 Amplifications or Mutations: Results From the Targeted Agent and Profiling Utilization Registry Study.

The Targeted Agent and Profiling Utilization Registry Study is a phase II basket trial evaluating the antitumor activity of commercially available targeted agents in patients with advanced cancer and genomic alterations known to be drug targets. Results from cohorts of patients with metastatic breast cancer (BC) with FGFR1 and FGFR2 alterations treated with sunitinib are reported. Eligible patients had measurable disease, Eastern Cooperative Oncology Group performance status 0-2, adequate organ function, and no standard treatment options. Simon's two-stage design was used with a primary end point of disease control (DC), defined as objective response (OR) or stable disease of at least 16 weeks duration (SD16+) according to RECIST v1.1. Secondary end points included OR, progression-free survival, overall survival, duration of response, duration of stable disease, and safety. Forty patients with BC with FGFR1 (N = 30; amplification only n = 26, mutation only n = 1, both n = 3) or FGFR2 (N = 10; amplification only n = 2, mutation only n = 6, both n = 2) alterations were enrolled. Three patients in the FGFR1 cohort were not evaluable for efficacy; all patients in the FGFR2 cohort were evaluable. For the FGFR1 cohort, two patients with partial response and four with SD16+ were observed for DC and OR rates of 27% (90% CI, 13 to 100) and 7% (95% CI, 1 to 24), respectively. The null hypothesis of 15% DC rate was not rejected (P = .169). No patients achieved DC in the FGFR2 cohort (P = 1.00). Thirteen of the 40 total patients across both cohorts had at least one grade 3-4 adverse event or serious adverse event at least possibly related to sunitinib. Sunitinib did not meet prespecified criteria to declare a signal of antitumor activity in patients with BC with either FGFR1 or FGFR2 alterations. Other treatments and clinical trials should be considered for these patient populations.

Real world experience of FGFR gene alterations and clinical outcomes in advanced/metastatic urothelial cancer in Japan: MONSTAR-SCREEN database study.

647 Background: Gene alterations (GA) in fibroblast growth factor receptors ( FGFR) may be oncogenic drivers in urothelial cancer (UC). However, the association between FGFR GA status and the prognosis with platinum-based chemotherapy has not been well investigated, especially in Asian patients. This study aims to elucidate the proportion and prognosis of FGFR2 or 3 (2/3) GA-positive advanced or metastatic UC (a/m UC). Methods: This study included patients registered during 2019 and 2022 in the “MONSTAR-SCREEN” database, where FoundationOneLiquid (F1L) was used for detecting 324 cancer-related genes, including FGFR. Analyzed FGFR2/3 GA variants included FGFR3 mutations (R248C, S249C, G370C, and Y373C) and FGFR2/3 fusions (FGFR2-BICC1, FGFR2-CASP7, FGFR3-TACC3, and FGFR3-BAIAP2L1). The positive ratio of FGFR2/3 GA and other gene alterations except FGFR was obtained at registration in eligible patients; besides, FGFR2/3 GA status was also evaluated after systemic treatment. Progression-free survival (PFS) in each treatment line was estimated, summarized by FGFR2/3 GA positive/negative. Results: Among the registered 2,224 cancer patients in this database, 138 eligible a/m UC patients were included (median age [SD] 72.0 [9.1] years for all; 95 males [68.8%]). The primary tumor site was the bladder in 70 patients (50.7%), renal pelvis in 50 patients (36.2%), and ureter in 18 patients (13.0%). FGFR2/3 GA was positive in 16 patients (11.6% [95% confidence interval: 6.8, 18.1]); the proportion of other typical gene alterations was 92.8%, 41.3%, 23.2%, and 23.2% in TP53, TERT, ARID1A and BRCA2, respectively. F1L was performed both before and after systemic treatment twice or more in 55 patients (39.9%). The FGFR concordance was analyzed in 55 patients available for both before and after treatment F1L results. Among them, 3 of the 6 FGFR2/3 GA-positive patients (50.0%) remained positive, and 1 of the 49 FGFR2/3 GA-negative patients (2.0%) before systemic treatment turned positive after treatment. The median PFS after first-line treatment was 6.8 months in FGFR GA-positive and 6.4 months in the negative patients. The estimated survival rate after first-line treatment was also similar between FGFR GA-positive and -negative patients at 12 months (68.8% vs. 81.0%). Conclusions: The results showed a similar trend to previous studies, where FGFR GA was reported in approximately 20% of metastatic UC patients. No difference was found in the PFS and the estimated survival rate by FGFR2/3 GA-positive or -negative patients. Our data showed that treatment pressure did not alter the FGFR status commonly.

Identification of Piperazinyl-Difluoro-indene Derivatives Containing Pyridyl Groups as Potent FGFR Inhibitors against FGFR Mutant Tumor: Design, Synthesis, and Biological Evaluation.

The fibroblast growth factor receptor (FGFR) signaling pathway plays important roles in cellular processes such as proliferation, differentiation, and migration. In this study, we highlighted the potential of FGFR inhibitors bearing the (S)-3,3-difluoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-indene scaffold containing a crucial 3-pyridyl group for the treatment of FGFR mutant cancers. The representative compound (S)-23, which was identified through comprehensive evaluation, exhibited potent antiproliferative activity with GI50 in the range of 6.4-10.4 nM against FGFR1 fusion protein-carrying, FGFR2-amplified, and FGFR2 mutant cancer cell lines and good antiproliferative activity against FGFR3 translocation and mutant FGFR4 cancer cell lines, as well as potency assessment against FGFR1-4 kinases. Moreover, compound (S)-23 exhibited favorable pharmacokinetic properties, low potential for drug-drug interactions, and very potent antitumor activity in MFE-296 xenograft mouse models with a TGI of 99.1% at the dose of 10 mg/kg. These findings demonstrate that compound (S)-23 is a potential therapeutic agent for FGFR mutant tumors.

Exploring the Micro-Mosaic Landscape of FGFR3 Mutations in the Ageing Male Germline and Their Potential Implications in Meiotic Differentiation.

Advanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants-c.1138G>A (p.G380R) and c.1948A>G (p.K650E)-which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor's age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline.

A phase II telemedicine study of pemigatinib in adult patients with unresectable or metastatic pancreas cancer with FGFR2 gene fusions or other FGFR genetic alterations.

TPS717 Background: Advanced pancreas cancer has a poor prognosis with few effective targeted therapies. Given this current state, novel therapies for pancreas cancer are an unmet need. Genomic alterations in FGFR, including fusions and point mutations are known driver mutations in cancer. FGFR kinase inhibitors have been FDA approved in cholangiocarcinoma and urothelial carcinoma, however, have not been well studied in other cancers. Our team previously identified and treated four patients with advanced pancreas cancer and FGFR2 fusions with FGFR kinase inhibitors resulting in durable responses and prolonged survival. Additionally, through a review of Foundation Medicine’s clinical genomic database, consisting of 30,229 pancreas cancer tumors, we determined that FGFR1-3 fusions have a prevalence of approximately 1% in pancreas cancer, most commonly in FGFR2 (0.8%). Our observations provide preliminary evidence that FGFR targeted therapy results in excellent clinical outcomes. We hypothesize that FGFR altered pancreas cancer treated with a FGFR kinase inhibitor will result in durable responses. We developed a phase 2, telemedicine trial of pemigatinib, an FDA approved FGFR kinase inhibitor, in FGFR altered pancreas cancer. Since the COVID-19 pandemic, telemedicine is used in routine oncology care and clinical trials and is associated with cost savings for patients. The growing adoption of this technology provides an opportunity to enhance the study of ultra-rare cancers. Methods: The trial consists of unresectable or metastatic pancreas cancer of any histology in two cohorts: 1) Primary cohort of FGFR2 fusions (n=30) and 2) exploratory cohort of other known activating mutations in FGFR (n=10). Patients are identified from across the United States through Foundation Medicine, Caris Life Sciences, and the Pancreatic Cancer Action Network. Eligible patients will be treated with oral pemigatinib through telemedicine by investigators at The Ohio State University in collaboration with local oncologists for supportive care. This trial uses single-stage phase 2 design with sample size calculations based on an expected overall response rate of ≥ 33% and <10% as unfavorable response, at one‐sided Type I error of 0.05, 90% power and up to 13% attrition rate. Results: The primary objective is overall response rate. Secondary objectives are overall survival, progression free survival, and duration of response. Exploratory objectives are analysis of correlation of circulating tumor DNA with response to treatment and development of resistance. Conclusions: FGFR kinase inhibitorshave the potential to be an effective targeted therapy in pancreas cancer. Additionally, telemedicine is a novel tool that can aid in the study of ultra-rare cancers and reduce barriers to patient participation in clinical trials.

Clinical outcomes and molecular characteristics of patients with pancreatic acinar cell carcinoma.

688 Background: PACC is a rare malignancy originating from the exocrine pancreas, accounting for less than 1% of all pancreatic cancers. Due to the rarity of the disease entity, there is a paucity of data to guide treatment decisions. This retrospective study aims to provide additional clinical characteristics and outcomes to aid in future treatment decisions. Methods: Pathology records from Mayo Clinic (AZ, FL, MN) patients (pts) denoting acinar or mixed-acinar cell carcinoma were searched between 2002 to 2023 and selected for retrospective review. Pt demographics, treatment courses, and next generation sequencing (NGS) were collected. Results: A total of 62 pts (74% male, n = 46) were identified with median age 64 years old. Histologic subtypes were 67% pure acinar cell carcinoma (42/62), 24% mixed acinar-neuroendocrine (15/62), and 8% mixed acinar-adenocarcinoma (5/62). AJCC stages at diagnosis were 34% stage I/II (21/62), 16% stage III (10/62), and 50% stage IV (31/62). Early-stage disease was treated with upfront surgical resection (81%, 17/21). Locally advanced disease received neoadjuvant therapy (NAT) followed by surgical resection (80%, 8/10). FOLFIRINOX was most often chosen as NAT (65%, 11/17), while others received gemcitabine-based NAT. The R0 resection rate was 86%. The 3 and 5-year survival estimates for stage III were 90.9% (95% CI: 75.4 – 100%) and 67.3% (95% CI: 42.3 – 100%), respectively. The 3 and 5-year survival estimates for stage IV were 36.6% (95% CI: 18.6 – 71.9%) and 29.3% (95% CI: 13.1 – 65.5%), respectively. Median survival time for stage IV was 3 years. Somatic NGS testing was done in 24 pts with alterations found in: homologous recombination (HR) in 42% (10/24); RAF alterations in 25% (6/24); mismatch repair (MMR) in 13% (3/24; none of them with MMR germline mutations), 1 ETV6-NTRK3 fusion, and 1 FGFR mutation. Germline NGS testing was done in 21 pts and alterations in HR were seen in 33% (7/21) of pts. This led to additional therapeutic options in 46% (11/24) of pts. Conclusions: Unlike PDAC, PACC is a genomically diverse disease, with potentially targetable alterations in RAF, HRD, and MMR genes, offering additional therapeutic options for significant number of pts. In addition, more than one-third of the pts harbored germline mutations in HR genes. Therefore, somatic and germline testing should be considered for any pt diagnosed with PACC.