FGFR3 Mutations Research Articles

189-OR: FGF21-Mediated Signaling Defects and Their Effects on Insulin Resistance in Insulin-Mediated Pseudoacromegaly

Insulin-mediated pseudoacromegaly (IMPA) is a rare, severe insulin resistance syndrome linked to digenic mutations in FGFR1 and KLB. These proteins form the receptor complex for FGF21, a hepatokine that plays a critical role in adipose tissue insulin sensitivity. Patients with IMPA have normal growth hormone and IGF-1. However, they have markedly increased insulin levels, tall stature, and obesity. To assess the pathogenicity of these mutations, we isolated iPSC from the proband with IMPA. We used CRISPR gene editing to correct the FGFR1 and KLB mutations and differentiated the iPSC lines into adipocytes. Western immunoblotting demonstrated that the uncorrected (mutant) cells demonstrated decreased levels of FGFR1 and pERK1/2 in response to insulin and/or FGF21 compared to corrected (wild-type) cells. Additionally, mutant adipocytes demonstrated significantly larger lipid droplets when stained with BODIPY. To complement the iPSC work, a humanized transgenic knock-in mouse line was created using CRISPR gene editing. When exposed to a high fat diet (HFD), female mice harboring variants in FGFR1 and KLB (DM - Double Mutant) developed significant weight gain compared to wild-type (WT) littermates. The difference in body weight was attributed to a difference in body fat. Similar to the human phenotype, the DM mice experience intermittent hyperglycemia and significant hyperinsulinemia. Explants of gonadal white adipose tissue demonstrated decreased lipolysis and pAKT and pERK. Mac-2 staining of white adipose tissue demonstrated abundant adipose tissue macrophages forming crown-like structures, suggesting adipose tissue inflammation. Additionally, we measured gene expression in brown adipose tissue, and noting that Ucp1 expression in DM mice was 37% when compared to WT. These results suggest pathogenic mechanisms in which FGFR1 and KLB mutations lead to IMPA via decreased FGF21-mediated signaling in the adipose tissue. Disclosure N.Phan: None. D.M.Ornitz: None. S.I.Stone: None. Funding Eunice Kennedy Shriver National Institute of Child Health and Human Development (R21HD098872); National Institute of Diabetes and Digestive and Kidney Diseases (K08 DK124574, P30DK020579, P30DK056341); National Center for Advancing Translational Sciences

Frequency of PDGFRA, PDGFRB and FGFR1 Gene Rearrangements in Patients with Eosinophilia and Their Clinico-Haematologic Parameters

Objective: To determine the frequency and clinico-haematological features of PDGFRA, PDGFRB and FGFR1 gene rearrangements in patients with persistent Eosinophilia using Fluorescence in situ hybridization.
 Study Design: Cross-sectional study.
 Place and Duration of Study: Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Dec 2018 to Dec 2019.
 Methodology: All Patients who presented to AFIP having absolute eosinophil count >1.5x109/L persistent for over six months or with Myeloid or Lymphoid neoplasms with persistent Eosinophilia were studied. Patients having reactive Eosinophilia and those on treatment were excluded. Interphase FISH studies were performed. In addition, 2.5ml of sodium heparin blood was taken. After the denaturation of DNA, slides were set up according to standard protocol. FIP1L1/CHIC2/PDGFRA dual colour probe was applied for PDGFRA, 5q32 PDGFRA break apart probe for PDGFRB and XL FGFR1 break apart probe for FGFR1 gene rearrangement.
 Results: A total of 60 patients were included in the study. Of these, 50(83.3%) were males, and 10(16.7%) were females, with an average absolute Eosinophilia count of 5.92±7.10x109/L. The only rearrangement detected in patients with Eosinophilia was FIPILI-PDGFRA gene fusion, detected in 20% of the patients. No other rearrangement was found.
 Conclusion: PDGFRA, PDGFRB and FGFR1 mutations are rare yet most prominent in patients with clonal Eosinophilia. About 80% of eosinophilic patients were found to have idiopathic Eosinophilia, which requires further consideration to address the disease prevalence.

LGG-24. DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR MANAGEMENT AND OUTCOME: A SINGLE CENTER EXPERIENCE

Abstract Diffuse leptomeningeal glioneuronal tumor (DLGNT) is a rare diagnosis that was first incorporated into the WHO Classification in 2016. DLGNTs are usually low-grade tumors, with no current standard of care. A retrospective review of patients with a histologic diagnosis of DLGNT, from 2010 to 2022, was done with IRB approval. Eight patients were identified with a median age at diagnosis of 5.5 years (range 4 months - 21 years). Imaging findings included diffuse leptomeningeal enhancement and multifocal nodular lesions in the supratentorial and infratentorial locations. One patient had localized pineal disease and 2 patients had isolated spinal cord involvement. Histologically, 6/8 (75%) patients showed low-grade tumor (Ki-67 index = 2-15%). BRAF-KIAA1549 fusion was reported in 3, NTRK fusion in 1, PTPN11 mutation in 1, and FGFR1 mutation in 1 patient. Seven patients (88%) were treated with proton radiation, 3 at diagnosis and 4 upon progression. Chemotherapy was used at diagnosis on 4 patients (temozolomide, carboplatin and vincristine, and vinblastine). Targeted therapy (NTRK or BRAF-inhibitors) was utilized upon progression in four patients, and at diagnosis in one. All patients showed disease progression at a median time of 24 months from diagnosis. The median progression-free survival (PFS) in the group receiving upfront RT was 2.2 years (range = 0.4 – 6.6 years), versus chemotherapy of 1.4 years (range = 0.15 - 2.6 years) The 2 years PFS and OS are 57% and 100%, respectively. At a median follow-up of 4.5 years (range = 3 months to 9 years), 6 patients have stable disease (of whom 2 are off therapy), and 2 patients have progressive disease. DLGNTs are rare tumors with scarce data about radiological and molecular characteristics, and no established standard of care. Our case series emphasizes the need for further studies on molecular profiling and exploring targeted therapies.

LGG-20. LANDSCAPE OF FGFR ALTERATIONS IN PEDIATRIC AND AYA GLIOMAS

Abstract FGFR alterations including single nucleotide variants (SNV) and rearrangements represent common oncogenic alterations found in pediatric low-grade gliomas. From a population-based cohort of over 1800 pediatric and AYA (adolescent and young adult) gliomas, FGFR mutations were found in 6% of pediatric low-grade gliomas (LGG) and 16 % of IDH-WT AYA gliomas. Given the frequency of FGFR driver alterations, we assembled a large cohort of 370 FGFR mutated glioma across all ages (6 months to 87 years) 53% of which were LGG. In the pediatric patients, most (87%) of FGFR altered gliomas were low-grade; while in AYA patients only 67%. Fusions were more frequent in pediatric LGG (60%), while SNVs were more common in AYA LGG (57%). The rearranged genes also differed; pediatric LGG: 26% FGFR1 ITD, 20% FGFR1 fusions, 15% FGFR2 fusions. AYA LGG had equal frequency of FGFR1 and FGFR2 fusions (30%). In adults, high grade gliomas were more common (73%), and frequently had additional oncogenic drivers. In general, additional oncogenic drivers were more common with increasing age and grade. In contrast to pediatric and AYA LGG, FGFR3-TACC3 fusions represented 40% of adult cases. Interestingly, this fusion was also found in 10% of FGFR LGGs overall. Although analysis of clinical outcomes are still ongoing, in our preliminary data 18 patients with FGFR altered low grade gliomas were treated with targeted agents; 3 stopped due to toxicity and were not evaluated for response. From patients on MAPKi 1/6 had a minor response and 3/6 stable disease; while 6/9 on FGFRi had partial or minor responses with additional 3 patients recently initiated on the drug. FGFR alterations are frequently encountered in AYA and pediatric population, molecular characterization is important in guiding prognosis and therapy. Although encouraging, further studies are needed to assess the benefit of FGFR inhibition in these patients.

DIPG-47. DISCOVERING A MOLECULAR BASIS FOR VULNERABILITY OF H3K27M-DMG TO HISTONE DEACETYLASE INHIBITORS

Abstract Using two H3K27M-DMG treatment-naive preclinical models (PBT22 and PBT29), we detected a 20-fold differential response to the histone deacetylase inhibitor, Quisinostat. PBT-22 harbors mutations in H3F3A, TP53, and ASXL2, while PBT-29 has mutations in H3F3A, TP53, PIK3CA and FGFR1. Acetylation and deacetylation of wtH3 alter chromatin structure as part of normal regulation of gene expression. The methionine substitution for Lysine in H3K27M removes this histone from participating in acetylation/deacetylation regulated gene expression. Concomitantly, H3K27M induces a marked reduction in global acetylation of histone tails on wtH3. In the native state of H3K27M DMG with hypoacetylated H3, we posit that the specific pattern of nucleosome integration with genes responsible for cell survival/death underlies the differential vulnerability of DMGs to HDACi. Additionally, the nucleosome integration pattern with cell survival/death genes may be based on the relative abundance of wtH3wt versus H3K27M- histones. Following Quis treatment (48 hrs) in both preclinical models, total wtH3ac protein abundance increased 3-fold, suggesting HDACi stabilizes or impedes K27 acetylated H3 histone turnover. Comparison of differentially expressed genes (DEGs) from control and Quis-treated PBT22 and PBT29 will seed gene ontology analysis focusing on chromatin remodeling, cell death, and growth arrest pathways accounting for differential vulnerability. Findings will be validated by measuring transcripts and proteins (qRT-PCR and ELISA or mass spectrometry) from analytes from a larger panel of DMG cell lines. The data depicts DMG preclinical models with large differential sensitivity to Quis. The long-term goal is to discover a molecular profile of DMGs predictive of the vulnerability to HDACi. The discovery exercise is also likely to yield insight into the development of resistance to HDAC inhibitors.

Inhibition of FGFR3 upregulates MHC-I and PD-L1 via TLR3/NF-kB pathway in muscle-invasive bladder cancer.

Improving the potency of immune response is paramount among issues concerning immunotherapy of muscle-invasive bladder cancer (MIBC). On the basis of immune subtypes, we investigated possible molecular mechanisms involved in tumor immune escape in MIBC. According to the 312 immune-related genes, three MIBC immune subtypes were clustered. Cluster 2 subtype is characterized by FGFR3 mutations and has a better clinical prognosis. However, the expression levels of MHC-I and immune checkpoints genes were the lowest, indicating that this subtype is subject to immune escape and has a low response rate to immunotherapy. Bioinformatics analysis and immunofluorescence staining of clinical samples revealed that the FGFR3 is involved in the immune escape in MIBC. Besides, after FGFR3 knockout with siRNA in RT112 and UMUC14 cells, the TLR3/NF-kB pathway was significantly activated and was accompanied by upregulation of MHC-I and PD-L1 gene expression. Furthermore, the use of TLR3 agonists poly(I:C) can further improve the effect. Together, our results suggest that FGFR3 might involve in immunosuppression by inhibition of NF-kB pathway in BC. Considering that TLR3 agonists are currently approved for clinical treatment as immunoadjuvants, our study might provide more insights for improving the efficacy of immunotherapy in MIBC.

Elucidating the potential effects of point mutations on FGFR3 inhibitor resistance via combined molecular dynamics simulation and community network analysis.

FGFR3 kinase mutations are associated with a variety of malignancies, but FGFR3 mutant inhibitors have rarely been studied. Furthermore, the mechanism of pan-FGFR inhibitors resistance caused by kinase domain mutations is still unclear. In this study, we try to explain the mechanism of drug resistance to FGFR3 mutation through global analysis and local analysis based on molecular dynamics simulation, binding free energy analysis, umbrella sampling and community network analysis. The results showed that FGFR3 mutations caused a decrease in the affinity between drugs and FGFR3 kinase, which was consistent with the reported experimental results. Possible mechanisms are that mutations affect drug-protein affinity by altering the environment of residues near the hinge region where the protein binds to the drug, or by affecting the A-loop and interfering with the allosteric communication networks. In conclusion, we systematically elucidated the underlying mechanism of pan-FGFR inhibitor resistance caused by FGFR3 mutation based on molecular dynamics simulation strategy, which provided theoretical guidance for the development of FGFR3 mutant kinase inhibitors.

AI-based identification of FGFR3 mutation status from routine histology slides of muscle-invasive bladder cancer.

e16580 Background: Gain of function mutations of the FGFR3 gene have been reported to be important driver mutations in a subset of muscle-invasive bladder urothelial cancer (MIBC). Subsequently, gene specific kinase inhibitors (e.g., erdafitinib) have been developed to target FGFR3 mutant metastasized MIBC with promising clinical activity. FGFR3 mutational status testing is required for erdafitinib treatment, but FGFR3 mutations are rare in metastatic MIBC (10-15%). Thus, cheap and fast pre-screening tools such as artificial intelligence (AI) based image classifiers could help to reduce turnover times of FGFR3 mutational testing and save costs arising from universal mutational testing. Herein, we propose an AI-based approach to identify MIBC FGFR3 mutated patients on routine histological slides. Methods: Slides from 612 patients with MIBC from two different sources (CCC-EMN Erlangen cohort n = 239, mutant = 20; TCGA-BLCA n = 373, mutant = 50) were used. The CCC-EMN cohort was used as a discovery set while TCGA-BLCA was used as an independent validation set. A two-step AI model was used to 1) extract features from each slide and 2) predict FGFR3 status from these features. The AUC, sensitivity and specificity were used as metrics to assess the performance of our model. Most predictive regions of FGFR3 mutation were reviewed by an expert pathologist. Results: The model reached an AUC/Sensitivity/Specificity of 0.82 [0.76-0.87] / 0.90 [0.82-0.97] / 0.57 [0.53-0.62] on TCGA-BLCA, substantially improving upon results previously reported on the same cohort (Loeffler et al. 2021: AUC = 0.70). Most predictive regions of FGFR3 mutation revealed monomorphous conventional urothelial tumor morphology and low desmoplastic tumor stroma content, while least predictive regions contained pleomorphic tumor morphology and high amounts of desmoplastic tumor content. Conclusions: By ruling out 57% of MIBC FGFR3 wild type tumors with high sensitivity, our model is the first to reach valuable performance for clinical use. Consequently, this study paves the way for the development of FGFR3 pre-screening tools to ease the management of patients with MIBC.

An in-progress phase 2 window of opportunity (WOO) trial of targeted therapy with erdafitinib for patients with recurrent FGFR3-altered non–muscle-invasive bladder cancer (NMIBC).

TPS4614 Background: Alterations to FGFR3 are the most common somatic mutations in non-muscle invasive bladder cancer (NMIBC), present in 40-60% of high-grade Ta and > 80% of recurrent low grade Ta tumors. FGFR3 mutations are also known to be associated with a “cold” tumor microenvironment (TME) that may mediate BCG resistance. Erdafitinib, a targeted inhibitor of FGFR 1-4, is the first FDA approved targeted therapy for locally advanced/metastatic bladder cancer. As FGFR3 mutations are present 3-5x more commonly in NMIBC than metastatic disease, erdafitinib is a logical therapeutic candidate for FGFR3-altered NMIBC. Through this ongoing phase II trial, we seek to determine the safety and efficacy of erdafitinib for the treatment of intermediate and high risk FGFR3-altered NMIBC after prior intravesical therapy. Methods: To address the need for expanded targeted therapeutics for NMIBC,, we launched an investigator-initiated phase II window of opportunity (WOO) trial of erdafitinib (NCT04917809, PI Pietzak). We are currently enrolling patients with intermediate or high risk NMIBC with history of ≥1 or more induction courses of BCG and/or intravesical chemotherapy who present with a clinically staged Ta papillary recurrence on office cystoscopy. Patients are also required to have an oncogenic FGFR3 mutation or fusion, as confirmed by OncoKB on MSK-IMPACT, to be included in the trial. Trial patients are treated with 6 mg oral erdafitinib daily for 28 days during the “window of opportunity” time period between office cystoscopy and transurethral resection of the recurrent bladder tumor (TURBT). The primary outcome is the rate of objective response, defined as complete (no visible tumor) or partial ( > 50% reduction of measured diameter by Response Evaluation Criteria for Tumors of the Bladder [RECIT-Bladder]) as compared to cystoscopy at TURBT. We are employing an optimal Simon two-stage binomial design, assuming an objective response of > 15% would and < 2% would not be worthy of further study. We plan to enroll 16 patients in the first stage and 9 in the second (87% power). Secondary outcomes include safety (CTAE 5.0), patient reported outcomes, and recurrence-free survival. Correlative studies will be conducted with urine, blood, and tumor tissue samples. Urine and blood are collected before (day 1), during (day 14) and after treatment (day 28) and will undergo immune and molecular studies to understand shifts in the microenvironment in the setting of response and resistance. This trial is being conducted in parallel with a co-clinical trial of patient derived organoid/xenograft models (MSK 19-015, PI Pietzak) to further refine the understanding of FGFR3 mutational resistance to standard treatment and the effects of targeted immunotherapy on tumor clinicogenomics. Clinical trial information: NCT04917809 .

Erdafitinb in patients with FGFR-altered tumors: Results from the NCI-COG Pediatric MATCH trial arm B (APEC1621B).

10007 Background: The NCI-Children’s Oncology Group (COG) Pediatric Molecular Analysis for Therapy Choice (MATCH) trial assigns patients aged 1-21 years with relapsed or refractory solid tumors, CNS tumors, lymphomas, and histiocytic disorders to phase 2 treatment arms of molecularly-targeted therapies based on genetic alterations in their tumor. Arm B (APEC1621B) evaluated the orally bioavailable FGFR inhibitor erdafitinib in patients whose tumors harbored activating alterations in FGFR 1/2/3/4. Methods: Patients received oral erdafitinib 4.7 mg/m2/dose (Max dose: 8 mg) daily for 28-day cycles until disease progression or drug related dose limiting toxicity (Max 26 cycles). Disease assessments were performed every 2-3 cycles. The primary endpoint was objective response rate; secondary endpoints included progression free survival (PFS). Results: A total of 20 evaluable patients (median age: 15 yrs) were enrolled from June 2018 to July 2022 in this first study of erdafitinib in pediatric patients. The most common diagnosis (n = 17) was glioma or glioneuronal tumor (11 low grade and 6 high grade). Three patients had non-CNS solid tumors (1 each with neuroblastoma, osteosarcoma, and rhabdomyosarcoma). FGFR alterations detected consisted of activating hotspot mutations in FGFR1 (n = 16), FGFR2 (n = 1), FGFR4 (n = 1), and FGFR1 fusions (n = 2; FGFR1: TACC1, FGFR1: TACC3). Partial responses were confirmed by central review in 2/20 patients (10%, 90%CI: 3.4%, 26.2%), both with low grade gliomas or glioneuronal tumors (LGG, LGGNT) and FGFR1 hotspot mutations (K687E and N577K). A best response of stable disease (SD) was observed in 6 additional patients with gliomas with a median duration of SD of 6.5 cycles. Six-month PFS was 45% (95% CI: 23.1%, 64.7%) and six-month OS was 89.7% (95%CI: 64.8%, 97.3%). Treatment related adverse events included those previously reported with FGFR inhibitors including hyperphosphatemia and nail changes or infections. Grade 1 vision changes were also reported. There was one Grade 3 spinal cord compression and one Grade 4 intracranial hemorrhage. Conclusions: Erdafitinib was generally well tolerated in this heavily pre-treated cohort of children with relapsed tumors harboring FGFR alterations. Partial responses or stable disease were observed in the majority (6/11, 54%) of patients with FGFR1-mutant low grade gliomas or glioneuronal tumors, suggesting the possible benefit of FGFR inhibitor therapy for these patients. Further studies would be necessary to more thoroughly evaluate the activity of erdafitinib for patients with other histologies and FGFR alteration types or combination approaches that include other targeted and/or cytotoxic agents. Clinical trial information: NCT03210714 .

MYC amplification segment-size mutual exclusivity analysis with other oncogenic events in a pan-cancer sequencing cohort.

3073 Background: MYC gene alterations resulting in increased copy number are recurrent oncogenic alterations across human malignancies. Presently no clear data exists to aid in interpreting MYC amplification characteristics for identifying MYC-dependent tumors. Methods: Analysis was conducted on a center-wide cohort of 68,920 tumor-normal matched samples from pediatric and adult patients sequenced with an FDA-authorized targeted sequencing panel (MSK-IMPACT). To ascertain tumor content, ploidy and allelic specific gene copy number, tumor-normal samples were analyzed with FACETS. Samples with tumor purity <20% were excluded. MYC amplification was defined as total copy number (TCN) minus ploidy ≥ 4. All samples were queried for presence of concurrent oncogenic or likely-oncogenic alterations based on OncoKb annotation. Co-occurrence (Co) and mutual-exclusivity (Me) analysis between MYC amplification according to segment size and other oncogenic alterations was performed with DISCOVER (false discovery rate threshold of 0.01) for each tumor type. Results: After removing low-quality samples (n=19,010/68,920), 49,910 samples were included in the analysis. 3,219 (6.4%) samples harbored MYC amplification, with segment size ranging from less than 100Kb to more than 100Mb, with 38% samples displaying focal amplifications shorter than 10 Mb and 62% broad amplifications longer than 10 Mb. TCN was inversely correlated with segment size (R= -0.34, p=2.2e-16). Triple-negative breast cancer (13.3%), hepatocellular carcinoma (8.7%), neuroendocrine tumors of the rectum (8.3%), high-grade serous ovarian cancer (7.6%) and large cell neuroendocrine carcinomas of the lung (6.7%) had the highest prevalence of MYC amplification without concurrent potentially driver oncogene mutations, amplifications or fusions. CoMe analysis demonstrated mutual exclusivity of MYC gene amplification regardless of segment size with most major tumor-type specific oncogenic events. All MYC amplifications were mutually exclusive with MET splice variants and mutations (q< 0.001) and KRAS mutations (q< 0.001) in lung adenocarcinoma (LUAD), FGFR3 mutations (q< 0.001) and fusions (q< 0.001) in bladder cancer, PTEN mutations (q< 0.001) in hormone receptor-positive breast cancer, KRAS mutations (q< 0.001) in colorectal cancer and CDK4 amplifications (q< 0.001) in sarcomas. Broad MYC amplifications were mutually exclusive with RET, ROS1 and ALK fusions in LUAD, while focal ones co-occurred with RET (q=0.007) and ALK fusions (q< 0.001) or secondary mutations (q< 0.001). Conclusions: This dataset defines a subgroup of MYC amplified cancers without co-occurring drivers and further subdivided by segment size, providing information that may help better identify patients for trials of novel MYC-directed therapies.

Real world genomics in metastatic urothelial carcinoma (mUC).

e16583 Background: Next generation sequencing (NGS) has therapeutic value in mUC. Beyond FGFR3 alteration and tumor mutational burden, genetic testing can identify novel predictive and prognostic biomarkers. In clinical practice, variability is introduced between assays due to genomic coverage, sequencing depth, timing, and tissue used. Methods: A single-institution retrospective database of patients (pts) diagnosed with mUC between 2013 and 2022 was generated. Clinicopathologic features and NGS results, including mutations, fusions, and copy number alterations, were abstracted from pt records. Progression free survival was calculated from start of first-line (1L) treatment (PFSFLT) and of 1L/maintenance/2L immunotherapy (PFSIO) to date of disease progression or death, censored at last follow up for pts alive without progression. Overall survival (OS) was calculated from diagnosis date of mUC to the date of death or censored at last follow up. Association between two categorical factors was estimated by chi-square test. Time-to-event data were analyzed by Kaplan-Meier method with log-rank test and Cox model. Results: 251 records were reviewed, of which 79 unique mUC pts with somatic NGS testing were identified. Pts were predominantly male (70.9%), white (86.1%), past/present smokers (65.8%), with bladder primary site (78.5%) and urothelial histology (72.2%). NGS was performed on 37 (46.8%) primary and 42 (53.2%) metastatic specimens, including 57 solid tumor (72.2%), 12 liquid biopsy (15.2%), and 10 (12.7%) with both. NGS was performed prior to or during 1L treatment for 61 pts (77.2%) and later for 18 pts (22.8%). Median TMB, where measured (n = 43), was 9 mut/Mb (2-40). The most prevalent genes with pathogenic variants identified were TP53 (37, 46.8%), TERT (32, 40.5%), PIK3CA (22, 27.8%), FGFR3 (18, 22.8%), ARID1A (16, 20.3%), CDKN2A/B (13, 16.5%) and ERBB2 (13, 16.5%). Several TP53 (45) and ARID1A variants (19) were observed, and fewer TERT (3) and FGFR3 (6) variants were seen. Among prevalent mutant genes, TP53 and TERT were most commonly concordant (68.4%, p = 0.0068). TERT was significantly co-mutated with ARID1A (64.6% concordant, p = 0.00479) and exclusive with mutant PIK3CA (41.8% concordant, p = 0.012). ERBB2 and FGFR3 alterations were mutually exclusive. ERBB2 alterations significantly correlated with lack of metastasis to lung, liver, or bone (p = 0.00355), though no genes predicted metastasis to those sites. ERBB2 alterations also correlated with improved PFSIO (HR 0.3, 95% CI 0.09-0.99, p = 0.049), but not with PFSFLT. FGFR3 mutation is associated with inferior OS (HR 1.88., 95% CI: 1.03 – 3.43, p = 0.04), especially for male pts (HR 2.85, 95% CI: 1.26-6.51, p = 0.013). Conclusions: A diverse array of NGS testing was performed on mUC pts, collected at varying timepoints. Despite inherent heterogeneity, these results yielded prognostic and predictive information for ERBB2 and FGFR3 alterations to be further studied in larger cohorts.

Efficacy and safety of futibatinib for refractory advanced solid malignancies with FGFR alterations identified in circulating tumor DNA: TiFFANY, A GOZILA-affiliated Trial.

3102 Background: FGFR alterations are observed in approximately 7% of advanced solid malignancies and are associated with poor prognosis and resistance to traditional anti-cancer therapy. Despite this, optimal therapeutic strategies with FGFR inhibitors for most of FGFR-altered solid malignancies are yet to be defined. Circulating tumor DNA (ctDNA) analysis has the potential to accurately detect FGFR alterations by assessing spatial and temporal intratumoral heterogeneity. Methods: We conducted a multicenter, investigator-initiated, phase II basket-type trial, TiFFANY, to evaluate the efficacy and safety of futibatinib, a highly selective covalent pan-FGFR inhibitor, in patients (pts) with advanced solid malignancies with FGFR alterations identified by ctDNA analysis who were refractory or intolerant to standard-of-care treatment. ctDNA analysis was performed in the GOZILA study. Enrolled pts received futibatinib at a dose of 20 mg once daily in a 21 day-cycle. The primary endpoint was investigator-assessed objective response rate (ORR). We set the threshold ORR of 5% and expected one of 25%. Planned sample size was 26 with one-sided alpha of 2.5% and power of 80%. Blood and tissue samples collected before treatment (baseline), at week 3, and after disease progression were analyzed for biomarkers and resistance mechanisms. Results: Twenty-six pts with FGFR alterations (mutation, 9; amplification, 13; fusion, 4) in ctDNA were enrolled between August 2019 and March 2021. The primary endpoint was met with five (19.2%; 95% CI, 6.6–39.4%) achieving a confirmed partial response (PR) in various cancer types (biliary tract, gastric, urothelial, and urachal cancer). Median progression-free survival and overall survival were 2.6 and 8.9 months. The most common treatment-associated adverse events were hyperphosphatemia (100%), eye toxicity (46.2%) and diarrhea (42.3%), all of which were manageable. The median proportional change in ctDNA fraction from baseline to 3 weeks after treatment initiation in pts who achieved PR was significantly less than in pts with stable or progressive disease (0.11 vs. 1.0 vs. 1.6, respectively). Pts with no concurrent RTK/RAS/PI3K and cell cycle alterations in ctDNA were significantly more likely to respond to futibatinib than those with at least one of these alterations (ORR 50% vs. 0%; P=0.0038). Acquired gene alterations in ctDNA after progression tended to be more common in pts with FGFR amplification (83.3%) than in those with FGFR mutation or fusion (60% or 66.7%). Conclusions: Futibatinib demonstrated promising efficacy in refractory advanced solid malignancies with FGFR alterations in ctDNA with an acceptable toxicity profile. Oncogenic co-alterations detected by ctDNA genotyping may predict primary resistance. Clinical trial information: JapicCTI-194624 .

Validation of a 56 gene panel for the detection of somatic variants in metastatic breast cancer.

e13131 Background: The use of ctDNA and next generation sequencing has unprecedented potential to improve genomic profiling and predicting actionable drug treatment in patients with metastatic breast cancer (MBC). Liquid biopsy is uniquely suited for patients where tissue biopsy is not feasible. We specifically focus on actionable variants found in liquid biopsy for MBC including bTMB and MSI. Here we report on the validation of a 56 gene sub panel for the determination of somatic mutations in MBC. Methods: Commercially available, well-characterized, biological ctDNA reference samples with allele frequency ranging from 0.5 -2.5% and bTMB scores (7 – 20) were obtained to analytically evaluate sensitivity, specificity and precision of a range of clinically significant variants (SNV, indels, fusions and copy number variants) in the TSO-500 ctDNA NGS assay. Additionally, 48 Whole blood samples from well characterized metastatic breast cancer patients and 13 healthy donors were obtained from commercial sources. cfDNA was extracted from the plasma separated from whole blood and analyzed on both TS0-500 ctDNA assay and PGDx Elio Complete. TSO-500 ctDNA sequencing data was further analyzed with the DRAGEN pipeline for variant calling and all variants were annotated and curated with the Clinical Genomic Workspace from PierianDx. PGDx utilized a proprietary pipeline and reporting of all variants detected. Results: Analytical validation determined excellent sensitivity (PPA: 99.4 – 100%), specificity (NPA: 99.6 -99.8%) and reproducibility (95 – 98.6%) for SNV, indels, fusions and Copy number variants. bTMB, with a cutoff for bTMB high as >20 mut/Mb, exhibited a sensitivity of 75% and specificity of 93.3%. Limit of detection (LOD) was established at 0.5 % VAF for SNV, Indels and fusions at a minimum read depth of 1000x. LOD for Copy number amplifications was established at 1.26-fold change or 3 copies. Clinical samples also exhibited excellent concordance across both TS0-500 ctDNA and PGDx Elo Complete. SNV, indels and copy number variants exhibited 100% (PPA) sensitivity and 99-100% Specificity (NPA). bTMB exhibited 87.5 % sensitivity and 96.22% specificity. As expected, fusions and microsatellite instability were observed in the clinical population but were rare. No reportable mutations were found in the healthy donors. Actionable mutations were reported for PIK3CA, ESR1, BRCA1 and BRCA2, TP53 and ERBB2 variants. Rare mutations in ATM, CHEC2, PALB2, FGFR1 and CCND1 were also observed. Conclusions: Clinical utility of liquid biopsy assays requires excellent sensitivity, specificity and precision. The Epic 56 gene panel has established clinical validity and high sensitivity and specificity across all relevant variants including bTMB in MBC. This clinically available NGS panel for metastatic breast cancer allows for routine genomic profiling, specifically in patients where tissue biopsy cannot be obtained.

Identification of novel actionable SNVs via targeted comprehensive genome profiling in patients with gallbladder and cholangiocarcinoma cancer.

e16006 Background: Liquid biopsy approach has paved the way for selecting targeted therapies. When the tissue is absent, the advent of comprehensive Next Generation Sequencing (NGS) using CcfDNA has further allowed significant advances in precision oncology by pinpointing rare, novel, and actionable alterations in oncogenes, tumor suppressor genes, and cellular signaling pathways. Methods: Retrospective NGS-based genome profiling was performed on ccfDNA (circulating cell-free DNA) samples from 11 patients using OncoIndx proprietary gene panel that targets exonic regions of 600 cancer-related genes. Illumina-compatible libraries were prepared by the target hybridization method and sequenced on Nextseq 2000 platform in a pair-end fashion. Variant calling was performed using an in-house developed bioinformatics pipeline. The 11 cases comprised 5 gallbladder and 6 cholangiocarcinoma patients. Results: We uncovered several novel mutations such as MTORK1452N, ERBB2H878Y, JAK1A634T, SMAD3R287W, IDH1R132C, PRKNK161N and FGFR2Y105C. Although ERBB2H878Y and IDH1R132C are known in cholangiocarcinoma, this is the first report of gallbladder cancer. In addition to SMAD3 and PARKIN, alterations were also observed in tumor suppressor proteins APC, BARD1, BRCA1/2, and GATA2. High genomic instability as demonstrated by HRD and LOH correlated with FGFR2, PRKN, and IDH1 mutations. FAT domain mutations in mTOR such as MTORK1452N are known to be responsive to rapamycin. Similarly, ERBB2H878Y is known to respond to rapamycin as well as a TKI. The FGFR2Y105C activating mutation has previously been reported in one case of atypical Crouzon Syndrome but has never been reported in cancer. This could have potential implications for the use of FGFR2 targeting agents in cholangiocarcinoma. IDH1R132C mutation is known in ~11% of cholangiocarcinoma patients, however, this alteration has not been reported in gallbladder cancer. This observation has therapeutic implications for the use of IDH1 inhibitors in gallbladder cancer based on its promising data in cholangiocarcinoma. Conclusions: We report, for the first-time novel and actionable SNVs in key oncogenes and tumor suppressors related to critical signaling pathways in a population of gallbladder and cholangiocarcinoma patients. The data strongly underscores the importance of comprehensive NGS-based in-depth assessment in gaining clinically viable therapeutic insights for patients.

The impact of next generation sequencing on the management of urothelial cell carcinoma.

e16576 Background: The urothelial carcinoma is commonly seen in urethra, bladder, ureter and renal pelvis. Targetable mutations included PD-L1, FGFR, PIK3CA, BRCA2, FBXW7, and Tumor mutation burden (TMB) greater than 10 Muts/Mb. Next genome sequencing is widely used to guide treatment of oncology patients; however, the efficacy of this practice is still not clear. Methods: Patients used in this study are from the Northwell Health Zuckerberg Cancer Center. This is a retrospective cohort study of patients with advanced urothelial carcinoma, who underwent next genome sequencing and received treatment based on targetable mutations. The primary objective for this study is to measure the response rate for patients with actionable gene aberrations on targetable treatment. Results: The RECIST 1.1 guidelines were used when identifying measurable lesions. Measurable lesions were identified as lymph nodes with a short axis ≥ 15 mm, or soft tissue lesions with a long axis ≥ 10 mm. Our sample size included 48 patients with urothelial carcinoma, these include bladder, ureter and renal pelvis cancers. Of these 48 patients, 21 received targetable treatment (16 males, 5 females), from those 21 only 20 had imaging before and after treatment. Most common mutations were TMB, PD-L1 and FGFR. 4 patients out of 20 had an overall response rate (20%), 3 had partial response (PR) and 1 had complete response (CR) to their respective targeted treatment. 11 patients had stable disease (SD). Thus disease control rate (DCR) was 15 out of 20 patients (75%) (when including PR, CR and SD out of 20 patients) had no progression of disease. Finally, 5 of the 20 patients had an overall progression of disease (PD). Conclusions: The results of our study indicate that next genome sequencing led to good treatment outcomes for patients previously heavily treated with advanced urothelial malignancy.

Genomic characterization of FGFR-altered solid tumors using a clinically annotated pan-cancer repository.

3074 Background: Somatic mutations, gene fusions, and amplifications in FGFR1/2/3 are found across solid tumors. FGFR2/3 fusions and select mutations have matched FDA-approved therapies in intrahepatic cholangiocarcinoma (iCCA) and urothelial carcinoma (UC). Identification of FGFR-altered tumors may inform patient selection for future drug development. Methods: All solid tumors that underwent MSK-IMPACT, a DNA-based NGS panel with coverage of FGFR1-4, were queried for FGFR1-4 alterations. FGFR structural variants (SV) were manually reviewed and compared to RNA sequencing data (RNAseq, Archer FusionPlex) when available. Alteration (alt) frequencies of iCCA and UC tumors with target FGFR alt (level 1 OncoKB evidence), non-target FGFR alt (lacking level 1 evidence) and no FGFR alt were compared using chi-squared tests and odds ratios. Euclidean distance was calculated between cohorts based on prevalence of frequently altered genes (>5%). Consensus clustering was used to identify optimal number of clusters and compare genomic profiles between clusters. Results: At data lock of June 1, 2022, 5695 tumors among 48,416 patients harbored an FGFR alteration (11.8%). FGFR SV were identified in 347 tumors on MSK-IMPACT. Of these 111/140 (79.2%) with RNAseq were positive. 98% (203/207) of SV without RNAseq were considered likely activating (canonical SV, n=125; in-frame inclusive of kinase domain, n=78). The most frequent fusion was FGFR3-TACC3 (n=124), followed by fusions unique to a single patient (n=100). Fusions were discovered in >15 tumor types, most frequently in iCCA (n=93), UC (n=50) and high-grade glioma (n=44). Among 1176 iCCA tumors, 9.9% (n = 117) had a target FGFR2 fusion. Target FGFR tumors were enriched for concurrent BAP1 alt (18%) compared to tumors without FGFR alt (58/969, 5.9%) and with non-target FGFR alt (7/90, 7.8%). Both groups without target alt were enriched in TP53, ARID1A and IDH1 alts compared to target group. Four optimal genomic clusters were identified, with FGFR2 fusions enriching in cluster 4 (29% vs 3%, 7% and ≤1%); 21 non-target FGFR alt were also in cluster 4. Among 2720 UC tumors, 24.7% (n=672) had target FGFR alt (select FGFR3 mutations and FGFR2/3 fusions). Three genomic clusters were identified, with target FGFR enriched in cluster 3 (42% target FGFR vs cluster 1: 11%, cluster 2: 18%) and non-target FGFR alterations enriched in cluster 1 (13% vs. cluster 2: 9.4 %, cluster 3: 7.0%); 68 non-target FGFR alt were in cluster 3. Tumors with non-target FGFR alterations and no FGFR alterations had close genomic association (Euclidean distance: 0.18). Conclusions: Activating FGFR fusions, which occur across tumor types with marked diversity in fusion partners, present opportunities for future drug development. Non-target FGFR alterations in iCCA and UC, particularly those that cluster with target FGFR alterations, may be candidates for future FGFR-targeted strategies.

Genomic profiling of biliary tract carcinomas by their location.

e16175 Background: Historically, biliary tract cancers have had a poor prognosis. Genomic profiling allows identification of alterations in genes with FDA-approved matched therapies either in these cancers ( FGFR2/3 fusions, certain IDH1 missense mutations), in all solid tumors (e.g. BRAF V600E mutations), or in other cancers (e.g. BRCA1/2 loss-of-function mutations) that could inform treatment. Here we examine the frequency of matched therapies in biliary tract carcinomas and the association with tumor location. Methods: All biliary tract carcinoma patient samples receiving the OncoExTra assay between April 2018 and November 2022 were included. The assay uses tumor-normal, whole-exome, whole-transcriptome sequencing to identify genomic alterations. Alterations in genes with FDA-approved matched therapies in any cancer, with matched clinical trials, or with evidence in cancer guidelines or the literature for possible matched therapies were recorded. Results: A total of 155 patient samples were processed: 92 intrahepatic CCA, 34 gall bladder carcinomas, 9 extrahepatic CCA, and 20 ampulla of Vater carcinomas. Targetable alterations were found in 153 (98.7%) samples; 31 (20.3%) had FDA-approved matched therapies, including 5 (3.3%) with microsatellite unstable and/or tumor mutational burden-high (eligible for immunotherapy) and 7 (4.6%) homologous recombinational repair deficient. Alterations of several genes showed a significant association with tumor type (Table). Cancers in the ampulla of Vater were enriched for KRAS, APC and ARID2 alterations; TP53 alterations were less frequent in biliary tract/intrahepatic cancers. Among the 15 FGFR1/2/3 alterations, there were 3 amplifications (1 FGFR1, 1 FGFR2, 1 FGFR3), 3 missense FGFR2 mutations, and 9 FGFR2 fusions. Conclusions: Comprehensive genomic profiling of biliary tract cancers identifies numerous targetable alterations. The distribution of altered genes varies, suggesting that appropriate therapies may differ by location, reflecting different lineage types. [Table: see text]

Landscape of FGFR2/3 alterations in hepatobiliary malignancies.

e16152 Background: Genomic aberrations (GAs) in FGFRs are involved in the pathogenesis of Hepatobiliary Malignancies, and clinical trials have shown efficacy of FGFR inhibitors in treating hepatobiliary malignancies patients with FGFR GAs. However, the molecular profile of FGFR2/3 in hepatobiliary malignancies is still lacking. Here, we focused on mutational landscape of FGFR2/3 GAs in hepatobiliary malignancies. Methods: We prospectively sequenced 876 tumors and matched blood DNA from Chinese and TCGA patients with hepatobiliary malignancies using next-generation sequencing techniques. Comprehensive molecular characterization was analyzed. Results: 13.2% (116/876) hepatobiliary malignancies carried FGFR2/3 GAs, including somatic mutation (56, 6.4%), CNV (41, 4.7%), and fusion (23, 2.62%). In FGFR2/3 mutation cohorts, the five most frequently somatic mutated genes were TP53 (46%), NCOR2 (26%), LRP1B (18%), KMT2C (17%), and FAT1 (16%), except for FGFR2 and FGFR3 gene. For FGFR2/3 wildtype cohorts, the five most frequently mutated genes were TP53 (46%), NCOR2 (13%), CTNNB1 (12%), LRP1B (12%), and ARID1A (10%). 77 mutated genes were significant difference between FGFR2/3 mutation and FGFR2/3 wildtype cohorts, such as NCOR2, KMT2C, FAT1, ROS1 and KMT2B, excluding the FGFR2 and FGFR3 gene. In addition, patients with FGFR2/3 GAs harbored a significantly higher TMB than those in patients without FGFR2/3 GAs (12.6 vs 9.0, p=0.003). There was no significant difference in genomic instability between the FGFR3 mutant group and the wild group (p>0.05), including ploidy, wGII, and ITH. Conclusions: FGFR2/3 GAs are frequently altered in hepatobiliary malignancies. Comprehensive genomic profiling of FGFR2/3 GAs highlights the clinical and therapeutic implication. There were characterized the genomic differences and similarities, stratified by the FGFR2/3 status, which may reflect the hepatobiliary malignancies patients with FGFR2/3 GAs harbored specific molecular mechanism.

Genomics driven precision oncology in advanced biliary tract cancer improves survival

BackgroundBiliary tract cancers (BTCs) including intrahepatic, perihilar, and distal cholangiocarcinoma as well as gallbladder cancer, are rare but aggressive malignancies with few effective standard of care therapies. MethodsWe implemented integrative clinical sequencing of advanced BTC tumors from 124 consecutive patients who progressed on standard therapies (N=92 with MI-ONCOSEQ and N=32 with commercial gene panels) enrolled between 2011-2020. ResultsGenomic profiling of paired tumor and normal DNA and tumor transcriptome (RNA) sequencing identified actionable somatic and germline genomic alterations in 54 patients (43.5%), and potentially actionable alterations in 79 (63.7%) of the cohort. Of these, patients who received matched targeted therapy (22; 40.7%) had a median overall survival of 28.1 months compared to 13.3 months in those who did not receive matched targeted therapy (32; P < 0.01), or 13.9 months in those without actionable mutations (70; P < 0.01). Additionally, we discovered recurrent activating mutations in FGFR2, and a novel association between KRAS and BRAF mutant tumors with high expression of immune modulatory protein NT5E (CD73) that may represent novel therapeutic avenues. ConclusionsOverall, the identification of actionable/ potentially actionable aberrations in a large proportion of cases, and improvement in survival with precision oncology supports molecular analysis and clinical sequencing for all patients with advanced BTC.