Mutations In Fibroblast Growth Factor Receptor Research Articles

Updated analysis with longer follow up of a phase 2a study evaluating erdafitinib in Asian patients (pts) with advanced cholangiocarcinoma (CCA) and fibroblast growth factor receptor (FGFR) alterations.

430 Background: Pts with CCA progressing after first-line therapy have limited treatment options. We report an updated analysis of the ongoing LUC2001 open-label, multicenter, phase 2a study (NCT02699606) investigating the efficacy and safety of erdafitinib in Asian pts with advanced CCA and FGFR alterations who progressed after ≥1 prior systemic treatment. Methods: Eligible adults (aged ≥18 years) received erdafitinib 8 mg once daily (QD) with pharmacodynamically guided up-titration to 9 mg QD. The primary endpoint was objective response rate (ORR; RECIST 1.1); secondary endpoints included best overall response (BOR), disease control rate (DCR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Survival estimates were determined by the Kaplan–Meier method. Results: Of 232 patients with CCA who underwent molecular screening, 39 (16.8%) had FGFR alterations (21 [9.1%] fusions and 19 [8.2%] mutations). Overall, 22 (9.5%) eligible pts (median age, 52 [range, 29–69] years) were enrolled and received treatment. Median follow-up was 22.4 (range, 2.3–47.0) months; median treatment duration was 6.2 (range, 1.5–35.6) months. All 22 pts received ≥1 line of prior systemic therapy and 12 (55.0%) pts had ≥2 prior lines of therapy. The ORR was 40.9% (95% CI, 20.7%–63.6%) and median time to response was 1.8 (range, 1.5–5.6) months. Median DOR was 7.3 (95% CI, 3.7–17.5) months, median PFS was 5.6 (95% CI, 3.6–12.7) months, and median OS was 40.2 (95% CI: 9.9–not estimable) months (Table). Responses were observed in 8/14 pts with FGFR fusions and 1/8 pts with FGFR mutations and in pts who received 1 or ≥2 prior lines of therapy. All 22 pts had ≥1 treatment-emergent adverse event (TEAE), the most common being dry mouth (15/22 [68.2%]) and stomatitis (14/22 [63.6%]). Grade ≥3 TEAEs occurred in 15/22 (68.2%) pts (11/22 [50.0%] were treatment related), of which the most common were stomatitis (3/22 [13.6%]) and alanine aminotransferase (ALT) increased (3/22 [13.6%]); 11/22 (50.0%) pts had ≥1 serious TEAE (1/22 [4.5%] pts had a serious treatment-related TEAE). A TEAE leading to death occurred in 1 patient (sepsis; not treatment-related). Conclusions: Asian pts with advanced CCA and FGFR alterations treated with erdafitinib had durable efficacy and a manageable safety profile, supporting the earlier findings of erdafitinib benefit in this population. Clinical trial information: NCT02699606. [Table: see text]

Targeted therapies: Expanding the role of FGFR3 inhibition in urothelial carcinoma.

The management of urothelial carcinoma (UC) has rapidly advanced in recent years with new approvals for immune checkpoint inhibitors and antibody-drug conjugates. However, while many UC tumors contain potentially actionable mutations, the role for targeted small molecule inhibitors has been limited. One such target is the fibroblast growth factor receptor (FGFR) family of proteins. Activating mutations and amplifications of FGFR3 are common in UC with higher incidences seen in upper tract as compared to lower tract disease. Consequently, multiple FGFR-directed targeted therapies have been developed and trialed in both UC and other solid tumors harboring FGFR mutations. At current, erdafitinib, an inhibitor of FGFR1-4, is the only approved targeted therapy in metastatic UC following the BLC2001 study, which demonstrated a 49% overall response rate in patients with UC harboring an FGFR3 mutation. Additional FGFR-directed agents also continue to be investigated across multiple disease stages in FGFR-mutated UC including infigratinib, rogaratinib, and AZD4547, among others. Ongoing trials are combining these agents with immune checkpoint inhibitors and chemotherapy regimens. The precision medicine revolution has begun in UC, and FGFR3 inhibitors are leading the charge toward a more personalized, biomarker-driven treatment paradigm.

Frequent FGFR1 hotspot alterations in driver-unknown low-grade glioma and mixed neuronal-glial tumors

PurposeLow-grade gliomas (LGG) and mixed neuronal-glial tumors (MNGT) show frequent MAPK pathway alterations. Oncogenic fibroblast growth factor receptor 1 (FGFR1) tyrosinase kinase domain has been reported in brain tumors of various histologies. We sought to determine the frequency of FGFR1 hotspot mutations N546 and K656 in driver-unknown LGG/MNGT and examined FGFR1 immunohistochemistry as a potential tool to detect those alterations.MethodsWe analyzed 476 LGG/MNGT tumors for KIAA-1549-BRAF fusion, IDH1/2, TERT promotor, NF1, H3F3A and the remaining cases for FGFR1 mutation frequency and correlated FGFR1 immunohistochemistry in 106 cases.Results368 of 476 LGG/MNGT tumors contained non-FGFR1 alterations. We identified 9 FGFR1 p.N546K and 4 FGFR1 p.K656E mutations among the 108 remaining driver-unknown samples. Five tumors were classified as dysembryoplastic neuroepithelial tumor (DNT), 4 as pilocytic astrocytoma (PA) and 3 as rosette-forming glioneuronal tumor (RGNT). FGFR1 mutations were associated with oligodendroglia-like cells, but not with age or tumor location. FGFR1 immunohistochemical expression was observed in 92 cases. FGFR1 immunoreactivity score was higher in PA and DNT compared to diffuse astrocytoma, but no correlation between FGFR1 mutation in tumors and FGFR1 expression level was observed.ConclusionFGFR1 hotspot mutations are the fifth most prevailing alteration in LGG/MNGT. Performing FGFR1 sequencing analysis in driver-unknown low-grade brain tumors could yield up to 12% FGFR1 N546/K656 mutant cases.

Novel potential oncogenic and druggable mutations of FGFRs recur in the kinase domain across cancer types

Fibroblast growth factor receptors (FGFRs) are recurrently altered by single nucleotide variants (SNVs) in many human cancers. The prevalence of SNVs in FGFRs depends on the cancer type. In some tumors, such as the urothelial carcinoma, mutations of FGFRs occur at very high frequency (up to 60%). Many characterized mutations occur in the extracellular or transmembrane domains, while fewer known mutations are found in the kinase domain. In this study, we performed a bioinformatics analysis to identify novel putative cancer driver or therapeutically actionable mutations of the kinase domain of FGFRs. To pinpoint those mutations that may be clinically relevant, we exploited the recurrence of alterations on analogous amino acid residues within the kinase domain (PK_Tyr_Ser-Thr) of different kinases as a predictor of functional impact. By exploiting MutationAligner and LowMACA bioinformatics resources, we highlighted novel uncharacterized mutations of FGFRs which recur in other protein kinases. By revealing unanticipated correspondence with known variants, we were able to infer their functional effects, as alterations clustering on similar residues in analogous proteins have a high probability to elicit similar effects. As FGFRs represent an important class of oncogenes and drug targets, our study opens the way for further studies to validate their driver and/or actionable nature and, in the long term, for a more efficacious application of precision oncology.

DDRE-12. DETERMINATION OF TOTAL AND UNBOUND LEVELS OF A POTENT INHIBITOR OF FIBROBLAST GROWTH FACTOR RECEPTOR, INFIGRATINIB, IN HUMAN PLASMA, CEREBROSPINAL FLUID AND BRAIN TUMOR BY A VALIDATED LC-MS/MS ASSAY

Abstract BACKGROUND Infigratinib is an orally available potent inhibitor of fibroblast growth factor receptor (FGFR) under investigation in a Phase 0 clinical trial for recurrent high-grade gliomas harboring oncogenic FGFR mutation or translocation (NCT04424966). Here, we report on our development and validation of an LC-MS/MS method for determination of total and unbound levels of infigratinib in human plasma, cerebrospinal fluid (CSF) and glioblastoma tissue. METHODS Infigratinib was extracted from human plasma, CSF and glioblastoma homogenate samples by protein precipitation with methanol. Unbound fractions of infigratinib in plasma and brain tissues were determined by equilibrium dialysis. Validation was performed using Sciex ExionLC UHPLC system coupled with Sciex QTRAP 6500+ MS/MS detector using positive electrospray ionization mode. The method was validated according to FDA guidelines and CAP/CLIA regulations. RESULTS The method was validated for 0.2-1000 nmol/L concentration range using plasma as matrix for all biospecimens. The analytical separation was optimized on Phenomenex Kinetex™ F5 column with total run time of 3.5 min using isocratic mode. Maximum coefficient of variation for intra- and inter-day precision was 12.7% and the accuracy was within 88.5-115.0% in all matrixes. The analyte is stable in plasma for at least 19 hours at room temperature (RT). Plasma stability at -20°C was demonstrated for at least 119 days. Stability of stock and working solutions was demonstrated for at least 15 hours (RT) and 60 days (2-8°C). The unbound fraction of infigratinib in pooled human plasma and brain were determined to be 0.01. CONCLUSIONS A sensitive and rapid bioanalytical method is successfully developed and validated to quantify total and unbound infigratinib levels in human plasma, CSF and glioblastoma tissue. The method is presently employed to evaluate plasma pharmacokinetics and CNS penetration of infigratinib in recurrent glioblastoma patients in an ongoing Phase 0 clinical trial.

High-Resolution Melting Analysis for Rapid Detection of Mutations in Patients with FGFR3-Related Skeletal Dysplasias.

Background: Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are related to skeletal dysplasias (SDs): acondroplasia (ACH), hypochodroplasia (HCH) and type I (TDI) and II (TDII) tanatophoric dysplasias. This study was designed to standardize and implement a high-resolution melting (HRM) technique to identify mutations in patients with these phenotypes. Methods: Initially, FGFR3 gene segments from 84 patients were PCR amplified and subjected to Sanger sequencing. Samples from 29 patients positive for mutations were analyzed by HRM. Results: Twelve of the patients FGFR3 mutations had ACH (six g.16081 G > A, three g.16081 G > C and three g.16081 G > A + g.16002 C > T); thirteen of patients with HCH had FGFR3 mutations (eight g.17333 C > A, five g.17333 C > G and five were negative); and four patients with DTI had FGFR3 mutations (three g.13526 C > T and one g.16051G > T and two patients with DTII (presented mutation g.17852 A > G). When analyzing the four SDs altogether, an overlap of the dissociation curves was observed, making genotyping difficult. When analyzed separately, however, the HRM analysis method proved to be efficient for discriminating among the mutations for each SD type, except for those patients carrying additional polymorphism concomitant to the recurrent mutation. Conclusion: We conclude that for recurrent mutations in the FGFR3 gene, that the HRM technique can be used as a faster, reliable and less expensive genotyping routine for the diagnosis of these pathologies than Sanger sequencing.

The Effect of Yes-Associated Protein on the Interaction Between the MEK/Extracellular Signal-Regulated Kinase and Hippo Pathways in Osteoblasts Co-Cultured With Fibroblast Growth Factor Receptor 2-Mutated Dura Cells.

C342Y (Cys342Tyr) point mutation of FGFR2 (fibroblast growth factor receptor 2) is closely associated with the pathogenesis of Crouzon syndrome. The dura mater plays an important role in mediating the closure of cranial sutures. However, the underlying mechanisms of these pathological processes have been rarely investigated. in this study, the authors analyzed the effects of dura cells with FGFR2 mutations on the biological function of osteoblasts. Dura cells and cranial osteoblasts from C57BL/6 mice were extracted and cultured. C342Y-FGFR2 mutant constructs were established via lentivirus and applied to infect dura cells. A co-cultured trans-well system with dura cells and osteoblasts was established. Three experimental groups were set up: oste group, Oste + Dura-vector group, and Oste + Dura-C342Y group. The expression levels of key factors in MEK (Mitogen-activated protein kinase kinase, MAPKK)/extracellular signal-regulated kinase (ERK) and Hippo pathway were detected by western blot and RT-qPCR (Real Time Quantitative PCR). Finally, a rescue experiment was carried out with small interference RUA. The proliferation level of osteoblasts in Oste + Dura- C342Y group was significantly up-regulated. Our studies indicated that the activation of MEK/ERK pathway in Oste + Dura-C342Y group could inhibit the Hippo pathway, lead to down-regulation of large tumor suppressor 1 and promote the activation and nuclear localization of yes-associated protein, and the results of rescue experiments showed a reverse expression trend, further confirming the effects of C342Y-FGFR2 mutation in dura cells on osteoblasts and its potential mechanism. This study suggested that the C342Y-FGFR2 mutation in dura cells could promote osteoblastic proliferation, and shown the crosstalk between MEK/ERK and Hippo pathways. As the regulatory machinery center, yes-associated protein might play a bridging role in these pathways, and might influence the pathogenesis of craniosynostosis by activating downstream transcriptional factors.

712P Analysis of fibroblast growth factor receptor 3 aberrations in bladder cancer, for enabling personalized and effective therapy based on FGFR inhibitor

Bladder cancer is posing major clinical challenge one of the most common cancers. It is estimated that every year almost 550 thousand new cases is diagnosed worldwide. Fibroblast Growth Factor Receptor 3 (FGFR3) is considered as one of the most promising targets because of its essential role in the cancer cell proliferation, progression and angiogenesis. The study aimed was to analyze the frequency of the FGFR3 mutations in non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC) and the comparison of various diagnostic methods enabling the precise selection of patients for targeted therapies.

Molecular mechanisms of the anti-cancer drug, LY2874455, in overcoming the FGFR4 mutation-based resistance

In recent years, many strategies have been used to overcome the fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors (TKIs) resistance caused by different mutations. LY2874455 (or 6LF) is a pan-FGFR inhibitor which is identified as the most efficient TKI for all resistant mutations in FGFRs. Here, we perform a comparative dynamics study of wild type (WT) and the FGFR4 V550L mutant for better understanding of the 6LF inhibition mechanism. Our results confirm that the pan-FGFR inhibitor 6LF can bind efficiently to both WT and V550L FGFR4. Moreover, the communication network analysis indicates that in apo-WT FGFR4, αD–αE loop behaves like a switch between open and close states of the substrate-binding pocket in searching of its ligand. In contrast, V550L mutation induces the active conformation of the FGFR4 substrate-binding pocket through disruption of αD–αE loop and αG helix anti-correlation. Interestingly, 6LF binding causes the rigidity of hinge and αD helix regions, which results in overcoming V550L induced resistance. Collectively, the results of this study would be informative for designing more efficient TKIs for more effective targeting of the FGFR signaling pathway.

Molecular characterization of fast-growing melanomas

The rate of growth of primary melanoma is a robust predictor of aggressiveness, but the mutational profile of fast-growing melanomas (FGMM) and the potential to stratify patients at high risk of death has not been comprehensively studied. To investigate the epidemiologic, clinical, and mutational profile of primary cutaneous melanomas with a thickness≥1mm, stratified by rate of growth. Observational prospective study. Deep-targeted sequencing of 40 melanoma driver genes on formalin fixed, paraffin-embedded primary melanoma samples. Comparison of FGMM (rate of growth>0.5mm/month) and nonFGMM (rate of growth≤0.5mm/month). Two hundred patients were enrolled, among wom 70 had FGMM. The relapse-free survival was lower in the FGMM group (P=.014). FGMM had a higher number of predicted deleterious mutations within the 40 genes than nonFGMM (P=.033). Ulceration (P=.032), thickness (P=.006), lower sun exposure (P=.049), and fibroblast growth factor receptor 2 (FGFR2) mutations (P=.037) were significantly associated with fast growth. Single-center study, cohort size, potential memory bias, number of investigated genes. Fast growth is linked to specific tumor biology and environmental factors. Ulceration, thickness, and FGFR2 mutations are associated with fast growth. Screening for FGFR2 mutations might provide an additional tool to better identify FGMM, which are probably good candidates for adjuvant therapies.

Comprehensive functional evaluation of variants of fibroblast growth factor receptor genes in cancer

Various genetic alterations of the fibroblast growth factor receptor (FGFR) family have been detected across a wide range of cancers. However, inhibition of FGFR signaling by kinase inhibitors demonstrated limited clinical effectiveness. Herein, we evaluated the transforming activity and sensitivity of 160 nonsynonymous FGFR mutations and ten fusion genes to seven FGFR tyrosine kinase inhibitors (TKI) using the mixed-all-nominated-in-one (MANO) method, a high-throughput functional assay. The oncogenicity of 71 mutants was newly discovered in this study. The FGFR TKIs showed anti-proliferative activities against the wild-type FGFRs and their fusions, while several hotspot mutants were relatively resistant to those TKIs. The drug sensitivities assessed with the MANO method were well concordant with those evaluated using in vitro and in vivo assays. Comprehensive analysis of published FGFR structures revealed a possible mechanism through which oncogenic FGFR mutations reduce sensitivity to TKIs. It was further revealed that recurrent compound mutations within FGFRs affect the transforming potential and TKI-sensitivity of corresponding kinases. In conclusion, our study suggests the importance of selecting suitable inhibitors against individual FGFR variants. Moreover, it reveals the necessity to develop next-generation FGFR inhibitors, which are effective against all oncogenic FGFR variants.

Targeted genomic profiling revealed a unique clinical phenotype in intrahepatic cholangiocarcinoma with fibroblast growth factor receptor rearrangement

Genomic aberrations (GAs) in fibroblast growth factor receptors (FGFRs) are involved in the pathogenesis of intrahepatic cholangiocarcinoma (ICC), and clinical trials have shown efficacy of FGFR inhibitors in treating ICC patients with FGFR GAs such as FGFR2 rearrangement. To clarify the FGFRs GA profile and corresponding clinicopathological features in Chinese patients with ICC, a total of 257 cases were identified. Fourteen cases (5.45%) were positive for FGFR2 rearrangement. Further analysis on the 110 FGFR2 rearrangement negative cases showed that 13 patients present additional FGFRs GAs, including FGFR3 rearrangement (2.73%), and FGFRs mutations. When compared with patients without FGFRs GAs, those with FGFR2 or FGFR3 rearrangement presented more under the age of 58 years, female sex, HBsAb positivity, CD10 expression, and PD-L1 expression. The clinical characteristics between patients with FGFRs mutation and those without FGFRs GAs were similar, with the exception that cases with FGFRs mutation have more hepatolithiasis. We concluded that FGFR rearrangement is associated with unique clinical phenotypes in ICC.

Abstract 2476: The oncogenic role of p63 in altered-FGFR3 bladder tumors

Abstract Recently, a new classification of muscle-invasive bladder cancer (MIBCs) has been proposed (Kamoun, 2020). There are 6 classes of MIBC: the over-expression or activating mutations in Fibroblast Growth Factor Receptor 3 (FGFR3) are associated with luminal subtypes. As typically expected, basal tumors exhibit increased TP63 activity, but interestingly, this is also observed in luminal-papillary (LumP) tumors. LumP are enriched in both, FGFR3 mutations and p63 activation. The regulatory gene pathways and p63 functional role in these tumors remains poorly characterized. Therefore, this is our main objective, together with the study of the link with FGFR3 mutations. We hypothesize that p63 drives a protumoral program in bladder cancer (BC) commanded by the regulation of altered FGFR3. We used human BC cell lines bearing FGFR3 mutations with inducible silencing for p63 in order to identify the functional role of p63 in this context. Loss of p63 led to a decreased 2D and 3D growth (p<0.001). Gelatin degradation and migration ability were significantly lower in p63 depleted cells compared to CRL (p<0,01). In vivo assays showed that tumor volume was significantly lower in p63 depleted tumors compared to CRL (p<0.01), accompanied by a reduction of proliferating cell nuclear antigen (PCNA) expression, indicating that p63 is an essential protein through which the cell mediates its proliferative and migratory mechanism. In order to study the link between both, FGFR3 alterations and p63 activity, we used FGFR3 Knocked down cells and pharmacological inhibitors. FGFR3 inhibition decreased p63 expression associated with a reduction in TP63 activity. It reduced proliferation and cell migration in similar level to those observed by directly silencing p63 in FGFR3 altered cells. Conversely, FGFR3 wt cells were not affected by the inhibitor. Overall, these results show that FGFR3 inhibition blunted the carcinogenic program carried out by p63, suggesting there is a regulation of FGFR3 over p63. Supporting the regulation of p63 by FGFR3, analysis of mRNA levels in human bladder tumors showed that p63 expression was significantly higher in FGFR3 mutated tumors in two different sets of samples. In summary, we experimentally demonstrated the regulation of FGFR3 over the protumoral transcription factor p63 that induces proliferation, migration and tumor growth in bladder cancer. Citation Format: Macarena Zambrano, Aura I. Moreno-Vega, Florent Dufour, Marianela Sciacca, Yanina V. Langle, François Radvanyi, Ana M. Eiján, Isabelle Bernard-Pierrot, Catalina Lodillinsky. The oncogenic role of p63 in altered-FGFR3 bladder tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2476.

Using deep learning to identify bladder cancers with FGFR-activating mutations from histology images.

BackgroundIn recent years, the fibroblast growth factor receptor (FGFR) pathway has been proven to be an important therapeutic target in bladder cancer. FGFR‐targeted therapies are effective for patients with FGFR mutation, which can be discovered through genetic sequencing. However, genetic sequencing is not commonly performed at diagnosis, whereas a histologic assessment of the tumor is. We aim to computationally extract imaging biomarkers from existing tumor diagnostic slides in order to predict FGFR alterations in bladder cancer.MethodsThis study analyzed genomic profiles and H&E‐stained tumor diagnostic slides of bladder cancer cases from The Cancer Genome Atlas (n = 418 cases). A convolutional neural network (CNN) identified tumor‐infiltrating lymphocytes (TIL). The percentage of the tissue containing TIL (“TIL percentage”) was then used to predict FGFR activation status with a logistic regression model.ResultsThis predictive model could proficiently identify patients with any type of FGFR gene aberration using the CNN‐based TIL percentage (sensitivity = 0.89, specificity = 0.42, AUROC = 0.76). A similar model which focused on predicting patients with only FGFR2/FGFR3 mutation was also found to be highly sensitive, but also specific (sensitivity = 0.82, specificity = 0.85, AUROC = 0.86).ConclusionTIL percentage is a computationally derived image biomarker from routine tumor histology that can predict whether a tumor has FGFR mutations. CNNs and other digital pathology methods may complement genome sequencing and provide earlier screening options for candidates of targeted therapies.

Mutations in FGFR1 were associated with growth traits in sheep (Ovis aries)

For its role in the mediation of myoblast proliferation, fibroblast growth factor receptor 1 (FGFR1) was considered a functional candidate gene for growth performance in Tibetan sheep. Via the polymerase chain reaction-restriction fragment length polymorphism (PCR-PFLP) approach, four single nucleotide polymorphisms (SNPs) including g.14752C > T (intron 1), g.45361A > G (intron 7), g.49400A > G (3’UTR region) and g.49587A > T (3’UTR region), were identified in 422 ewes. The association analysis demonstrated that individuals carrying the AA genotype of g.49400A > G had significantly greater withers height, length than those with GG genotype (p < 0.05). Individuals with genotype AA of g.49587A > T had significantly greater weight and chest circumference than those with genotype TT (p < 0.01). Additionally, the individuals with Hap1/1 diplotypes (CAAA-CAAA) were highly significantly associated with weight and chest circumference than Hap1/2 diplotypes (CAAA-CAAT) (p < 0.05). The quantitative real-time polymerase chain reaction (qPCR) analysis revealed that the FGFR1 was detectable expressed in muscle tissues within three different age stage. Remarkably higher mRNA expression was detected at fetal lamb stage as compared with adult ewes (p < 0.01). The outcome of this research confirmed that both g.49400A > G and g.49587A > T of FGFR1 were involved in growth-related traits, which may be considered to be genetic markers for improving the growth traits of Tibetan sheep.

Novel Mutations in FGFR1 Causing Idiopathic Hypogonadotropic Hypogonadism: A Cohort Study in Eastern Indian Population

Introduction: IHH is a clinically and genetically heterogeneous condition with more than 80 different mutations described in literature, some of the important being KAL-1 (anosmin), fibroblast growth factor receptor-1 (FGFR1) and fibroblast growth factor-8 (FGF8). Objective: To characterize the clinical findings and molecular analysis of FGFR1 (KAL2) genes in 10 unrelated patients of idiopathic hypogonadotropic hypogonadism with and without hyposmia/anosmia. Methods: After confirming the diagnosis of IHH from detailed history, clinical and biochemical examination the patients were subjected to MRI of pituitary hypothalamic area and olfactory bulb and tract. For genetic analysis PCR of 18 exons was performed on the genomic DNA. The PCR products were purified, sequenced and analysed using BLAST search from NCBI GenBank. Results: We diagnosed ten unrelated sporadic cases of IHH (9 male and 1 female). Out of 10 cases 6 (60%) had anosmia (KS) 4 had normosmia. A variable degree of pubertal development was observed, including various clinical abnormalities, such as micropenis, dental agenesis, skeletal deformity, seizure disorder and bimanual synkinesis. All the patients who had anosmia (KS) had either hypoplasia or aplasia of olfactory bulb and tract on MRI. Two KS patients had novel sporadic FGFR1 mutation, one had a mutation n.2370 C>A detected at exon 10 of FGFR1 gene and another had a mutation n.1875 GA>CA at exon 8C of FGFR1 gene. Discussion: Loss-of-function sequence variants in FGFR1, encoding the fibroblast growth factor receptor-1, account for one autosomal dominant form of KS. Pathogenic changes in KAL1 or FGFR1 have been detected in approximately 20% of the KS patients, which indicates that other responsible genes are still to be discovered. FGFR1 comprises an extracellular region consisting of three immunoglobulin-like domains (D1-D3), a single transmembrane helix, and an intracellular region containing the tyrosine kinase domain. D2, D3 and the short linker between them house all the determinants of ligand (FGF) binding and specificity. In addition, alternative splicing of exon 8, encoding the D3 C-terminal half, leads to two FGFR1 isoforms, FGFR1b (exon 8A) and FGFR1c (exon 8B), with different specificities towards FGF ligands. All the pathogenic changes in FGFR1 that have been reported in KS so far disrupt both isoforms of FGFR1 i.e. FGF1b and FGF1c, therefore leaving open the question of which isoform(s) is (are) required in the olfactory bulb morphogenetic process. We identified a new mutation in exon 8C (patient no 2). Conclusions: IHH and KS patients present with broad spectrum of clinical manifestations. We identified two novel mutations in FGFR1 gene in two unrelated sporadic cases of KS in the Indian population.

A Pancancer Analysis of the Expression Landscape and Clinical Relevance of Fibroblast Growth Factor Receptor 2 in Human Cancers.

Background: Fibroblast growth factor receptor 2 (FGFR2) is frequently altered in tumors and one of the top therapeutic targets in cholangiocarcinoma (CHOL) with FGFR2 fusions. Although there have been several studies on individual tumors, a comprehensive analysis of FGFR2 genetic aberrations and their simultaneous clinical implications across different tumors have not been reported.Methods: In this study, we used the large comprehensive datasets available, covering over 10,000 tumor samples across more than 30 cancer types, to analyze FGFR2 abnormal expression, methylation, alteration (mutations/fusions and amplification/deletion), and their clinical associations.Results: Alteration frequency, mutation location distribution, oncogenic effects, and therapeutic implications varied among different cancers. The overall mutation rate of FGFR2 is low in pancancer. CHOL had the highest mutation frequency, and fusion accounted for the major proportion. All these fusion aberrations in CHOL were targetable, and an FDA-approved drug was approved recently. Uterine corpus endometrial carcinoma (UCEC) had the highest number of FGFR2 mutations, and the most frequently mutated positions were S252W and N549K, where the functional impact was oncogenic, but targeted therapy was less effective. Additionally, DNA methylation was associated with FGFR2 expression in several cancers. Moreover, FGFG2 expression and genetic aberrations showed clinical associations with patient survival in several cancers, indicating their potential for application as new tumor markers and therapeutic targets.Conclusions: This study showed the full FGFR2 alteration spectrum and provided a broad molecular perspective of FGFR2 in a comprehensive manner, suggesting some new directions for clinical targeted therapy of cancers.

MET-Pyk2 Axis Mediates Acquired Resistance to FGFR Inhibition in Cancer Cells.

Deregulation of fibroblast growth factor receptors (FGFRs) signaling, as a result of FGFR amplification, chromosomal translocation, or mutations, is involved in both initiation and progression of a wide range of human cancers. Clinical data demonstrating the dependence of cancer cells on FGFRs signaling clearly indicate these receptors as the molecular targets of anti-cancer therapies. Despite the increasing number of tyrosine kinase inhibitors (TKIs) being investigated in clinical trials, acquired resistance to these drugs poses a serious therapeutic problem. In this study, we focused on a novel pan-FGFR inhibitor—CPL304110, currently being investigated in phase I clinical trials in adults with advanced solid malignancies. We analyzed the sensitivity of 17 cell lines derived from cancers with aberrant FGFR signaling, i.e. non-small cell lung cancer, gastric and bladder cancer to CPL304110. In order to explore the mechanism of acquired resistance to this FGFR inhibitor, we developed from sensitive cell lines their variants resistant to CPL304110. Herein, for the first time we revealed that the process of acquired resistance to the novel FGFR inhibitor was associated with increased expression of MET in lung, gastric, and bladder cancer cells. Overexpression of MET in NCI-H1703, SNU-16, RT-112 cells as well as treatment with HGF resulted in the impaired response to inhibition of FGFR activity. Moreover, we demonstrated that cells with acquired resistance to FGFR inhibitor as well as cells overexpressing MET displayed enhanced migratory abilities what was accompanied with increased levels of Pyk2 expression. Importantly, inhibition of both MET and Pyk2 activity restored sensitivity to FGFR inhibition in these cells. Our results demonstrate that the HGF/MET-Pyk2 signaling axis confers resistance to the novel FGFR inhibitor, and this mechanism is common for lung, gastric, and bladder cancer cells. Our study suggests that targeting of MET/Pyk2 could be an approach to overcome resistance to FGFR inhibition.

Effects of PI3K and FGFR inhibitors alone and in combination, and with/without cytostatics in childhood neuroblastoma cell lines.

Neuroblastoma (NB) is a heterogenous disease with treatment varying from observation for low-risk tumors, to extensive therapy with chemotherapy, surgery, radiotherapy, and autologous bone-marrow-transplantation and immunotherapy. However, a high frequency of primary-chemo-refractory disease and recurrences urgently require novel treatment strategies. The present study therefore investigated the anti-NB efficacy of the recently FDA-approved phosphoinositide 3-kinase (PI3K) and fibroblast growth factor receptor (FGFR) inhibitors, alpelisib (BYL719) and erdafitinib (JNJ-42756493), alone and in combination with or without cisplatin, vincristine, or doxorubicin on 5 NB cell lines. For this purpose, the NB cell lines, SK-N-AS, SK-N-BE(2)-C, SK-N-DZ, SK-N-FI and SK-N-SH (where SK-N-DZ had a deletion of PIK3C2G and none had FGFR mutations according to the Cancer Program's Dependency Map, although some were chemoresistant), were tested for their sensitivity to FDA-approved inhibitors alone or in combination, or together with cytostatic drugs by viability, cytotoxicity, apoptosis and proliferation assays. The results revealed that monotherapy with alpelisib or erdafitinib resulted in a dose-dependent inhibition of cell viability and proliferation. Notably, the combined use of PI3K and FGFR inhibitors resulted in an enhanced efficacy, while their combined use with the canonical cytotoxic agents, cisplatin, vincristine and doxorubicin, resulted in variable synergistic, additive and antagonistic effects. Collectively, the present study provides pre-clinical evidence that PI3K and FGFR inhibitors exhibit promising anti-NB activity. The data presented herein also indicate that the incorporation of these inhibitors into chemotherapeutic regimens requires careful consideration and further research in order to obtain a beneficial efficacy. Nevertheless, the addition of PI3K and FGFR inhibitors to the treatment arsenal might reduce the occurrence of refractory and relapsing disease in NB without FGFR and PI3K mutations.

Clinical Characteristics and Spermatogenesis in Patients with Congenital Hypogonadotropic Hypogonadism Caused by FGFR1 Mutations.

Objective The aim of this study was to investigate the clinical characteristics of patients diagnosed with congenital hypogonadotropic hypogonadism (CHH) caused by FGFR1 (fibroblast growth factor receptor 1) gene mutations and to evaluate the effect of gonadotropin or pulsatile gonadotropin-releasing hormone (GnRH) therapy on spermatogenesis. Methods A retrospective study was conducted on CHH patients admitted to Peking Union Medical College Hospital from January 2012 to March 2020. Clinical features and laboratory results were recorded. Testicular volume and sperm count responding to gonadotropin and pulsatile GnRH therapy were compared between the FGFR1 mutation group and the mutation-negative group. Results (1) FGFR1 mutation group included 14 patients who received sperm-induction therapy, and the mutation-negative group enrolled 25 CHH patients. (2) The incidence of cryptorchidism was 50.0% (7/14) and 12.0% (3/25) in the FGFR1 group and the mutation-negative group, respectively (p=0.019). The baseline testicular volume of the FGFR1 mutation group was smaller than that of the mutation-negative group, 1.6 (0.5–2.0) mL vs. 2 (1.75–4) mL (p=0.033). The baseline luteinizing hormone (LH), Follicle-stimulating hormone (FSH), and testosterone levels were similar between the two groups. (3) Using the Kaplan–Meier and log-rank tests for the analysis of spermatogenesis, it was found that there was no significant difference in the first sperm appearance between the FGFR1 mutation group and the mutation-negative group (χ2 = 1.974, p=0.160). The median time of spermatogenesis in the FGFR1 mutation group was longer than that in the mutation-negative group, 16 months vs. 10 months, respectively. The cumulative spermatogenesis success rate at 12 months in the FGFR1 mutation group (35.71%) was lower than that in the mutation-negative group (68.75%) (p=0.047). The sperm concentration in the mutation-negative group was more easily achieved for different thresholds compared with that in the FGFR1 mutation group, but no significant difference was observed (p > 0.05) between the two groups. The last follow-up examination showed that the testicular volume was 7.00 (4.75–12.00) mL and 10.56 ± 4.82 mL (p=0.098), the ejaculate volume of sperm was 2.20 (1.40–2.26) mL and 3.06 ± 1.42 mL (p=0.175), and the sperm concentration was 7.19 (1.00–9.91) million/mL and 18.80 (4.58–53.62) million/mL (p=0.038) in the FGFR1 mutation and mutation-negative groups, respectively, while the sperm motility (A%, A + B%, and A + B + C%) was similar for the two groups (p=0.839, 0.909, and 0.759, respectively). The testosterone level during treatment was 366.02 ± 167.03 ng/dL and 362.27 ± 212.86 ng/dL in the FGFR1 mutation and mutation-negative groups, respectively (p=0.956). Conclusion Patients with FGFR1 mutations have a higher prevalence of cryptorchidism and smaller testicular volume. Although patients with FGFR1 mutations have a similar rate of success for spermatogenesis compared to that of the mutation-negative patients, a longer treatment period was required and a lower sperm concentration was achieved.