Fibroblast Growth Factor Receptor 3 Protein Research Articles

Elucidating the Role of Wildtype and Variant FGFR2 Structural Dynamics in (Dys)Function and Disorder.

The fibroblast growth factor receptor 2 (FGFR2) gene is one of the most extensively studied genes with many known mutations implicated in several human disorders, including oncogenic ones. Most FGFR2 disease-associated gene mutations are missense mutations that result in constitutive activation of the FGFR2 protein and downstream molecular pathways. Many tertiary structures of the FGFR2 kinase domain are publicly available in the wildtype and mutated forms and in the inactive and activated state of the receptor. The current literature suggests a molecular brake inhibiting the ATP-binding A loop from adopting the activated state. Mutations relieve this brake, triggering allosteric changes between active and inactive states. However, the existing analysis relies on static structures and fails to account for the intrinsic structural dynamics. In this study, we utilize experimentally resolved structures of the FGFR2 tyrosine kinase domain and machine learning to capture the intrinsic structural dynamics, correlate it with functional regions and disease types, and enrich it with predicted structures of variants with currently no experimentally resolved structures. Our findings demonstrate the value of machine learning-enabled characterizations of structure dynamics in revealing the impact of mutations on (dys)function and disorder in FGFR2.

Discovery of N-(4-((6-(3,5- Dimethoxyphenyl)-9H-purine derivatives as irreversible covalent FGFR inhibitors

Fibroblast growth factor receptor (FGFR) is an attractive target for cancer therapy, but existing FGFR inhibitors appear to hardly meet the demand for clinical application. Herein, a number of irreversible covalent FGFR inhibitors were designed and synthesized by selecting several five- and six-membered azaheterocycles as parent scaffold with different substituents to take over the hydrophobic region in the active pocket of FGFR proteins. Among the resulting target compounds, III-30 showed the most potent effect on enzyme activity inhibition and anti-proliferative activity against the tested cancer cell lines. Significantly, III-30 could inhibit the enzyme activity by achieving irreversible covalent binding with FGFR1 and FGFR4 proteins. It could also regulate FGFR-mediated signaling pathway and mitochondrial apoptotic pathway to promote cancer cell apoptosis and inhibit cancer cell invasion and metastasis. Moreover, III-30 had a good metabolic stability and showed relatively potent anti-tumor activity in the MDA-MB-231 xenograft tumor mice model.

FGFR2 and miR-889-3p expression in oral cancer is associated with cervical lymph node metastasis.

Fibroblast growth factor receptor-2 (FGFR2) and miR-889-3p expression in oral squamous cell carcinoma (OSCC) tumours were compared to normal controls. We then examined the relationship between miR-889-3p, FGFR2 expression and patient clinicopathological features. The interaction of FGFR2 and miR-889-3p was investigated using bioinformatics. Then, OSCC tumour biopsies and normal gingiva were collected and processed for expression analysis of FGFR2-specific mRNA and miR-889-3p using real-time PCR. Immunohistochemistry evaluated the expression of the FGFR2 protein. The protein and mRNA expression levels of FGFR2 were significantly greater in tumours when contrasted with controls. Expression of miR-889-3p was significantly lower in OSCC compared to normal tissues. The FGFR2 and miR-889-3p expressions were inversely related (-0.86 and -0.73, respectively) in both cases and controls. Changes in miR-889-3p and FGFR2 expression in tumour tissues were associated with lymph node metastasis (LNM), with ~0.57 and ~3.0 folds of change in positive-LNM patients, respectively. Decreased expression of miR-889-3p in OSCC tumours suggests that miR-889-3p functions as a tumour suppressor gene. Overexpression of FGFR2 further proves the role of miR-889-3p in the regulation of the FGFR2 pathway. This was further confirmed by showing differences in miR-889-3p expression in positive and negative LNM cases.

Clinical Implication of Concurrent Amplification of MET and FGFR2 in Metastatic Gastric Cancer.

c-mesenchymal epithelial transition factor receptor (c-MET) and fibroblast growth factor receptor 2 (FGFR2) amplification have been identified as factors associated with advanced stage and poor prognosis in gastric cancer (GC). While they are typically considered mutually exclusive, concurrent amplifications have been reported in a small subset of GC patients. in this retrospective study, we analyzed the clinical outcomes of GC patients with MET and FGFR2 amplification using the next-generation sequencing (NGS) database cohort at Samsung Medical Center, which included a total of 2119 patients between October 2019 and April 2021. Of 2119 cancer patients surveyed, the number of GC patients was 614 (29.0%). Out of 614 GC patients, 39 (6.4%) had FGFR2 amplification alone, 22 (3.6%) had MET amplification, and 2 GC patients (0.3%) had concurrent FGFR2 and MET amplification. Two patients with concurrent FGFR2 and MET amplification did not respond to first-line chemotherapy. These two patients had significantly shorter overall survival (3.6 months) compared to patients with FGFR2 or MET amplification alone (13.6 months and 8.4 months, respectively) (p = 0.004). Lastly, we tested the existence of FGFR2 and MET in tumor specimens from different organ sites. Initially, the NGS was tested in a primary tumor specimen from stomach cancer, where the MET copy number was 14.1 and the FGFR2 copy number was 5.3. We confirmed that both MET and FGFR2 were highly amplified in the primary tumor using FISH (MET-CEP7 ratio = 5 and FGFR2-CEP7 ratio = 3). However, although the MET copy number was normal in peritoneal seeding using FISH, FGFR2 remained amplified using FISH (FGFR2-CEP7 ratio = 7) with high FGFR2 protein overexpression. Hence, there was intra-patient molecular heterogeneity. our findings suggest that concurrent amplification of FGFR2 and MET in GC patients is associated with clinical aggressiveness and may contribute to non-responsiveness to chemotherapy or targeted therapy.

Discovery of Novel Naphthoquinone-Chalcone Hybrids as Potent FGFR1 Tyrosine Kinase Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling.

This work presents a flexible synthesis of 10 novel naphthoquinone-chalcone derivatives (1-10) by nucleophilic substitution of readily accessible aminochalcones and 2,3-dichloro-1,4-naphthoquinone. All compounds displayed broad-spectrum cytotoxic activities against all the tested cancer cell lines (i.e., HuCCA-1, HepG2, A549, MOLT-3, T47D, and MDA-MB-231) with IC50 values in the range of 0.81-62.06 μM, especially the four most potent compounds 1, 3, 8, and 9. The in vitro investigation on the fibroblast growth factor receptor 1 (FGFR1) inhibitory effect indicated that eight derivatives (1-2, 4-5, and 7-10) were active FGFR1 inhibitors (IC50 = 0.33-3.13 nM) with more potency than that of the known FGFR1 inhibitor, AZD4547 (IC50 = 12.17 nM). Promisingly, compounds 5 (IC50 = 0.33 ± 0.01 nM), 9 (IC50 = 0.50 ± 0.04 nM), and 7 (IC50 = 0.85 ± 0.08 nM) were the three most potent FGFR1 inhibitors. Molecular docking, molecular dynamics simulations, and MM/GBSA-based free energy calculation revealed that the key amino acid residues involved in the binding of the compounds 5, 7, and 9 and the target FGFR1 protein were similar with those of the AZD4547 (i.e., Val492, Lys514, Ile545, Val561, Ala640, and Asp641). These findings revealed that the newly synthesized naphthoquinone-chalcone scaffold is a promising structural feature for an efficient inhibition of FGFR1.

Fibroblast growth factor receptors 1 and 4 combined with lymph node metastasis predicts poor prognosis in oral cancer.

The fibroblast growth factor receptor (FGFR) members including FGFR1-4 have been identified as promising novel therapeutic targets and prognostic markers in multiple solid tumors. However, the predictive role of the expression of FGFR proteins in oral squamous cell carcinoma (OSCC) requires further exploration. Immunohistochemical evaluation of FGFR1-4 was performed on 161 paired OSCC samples. The associations of FGFRs with clinicopathologic and prognostic parameters were analyzed. To further assess the contribution of FGFRs to OSCC proliferation, cell lines, and one PDX model was utilized to examine the anti-tumor effect of the pan-FGFR inhibitor AZD4547. All FGFR members were found to be overexpressed in OSCC tumors when compared to normal tissues, and their expression was significantly associated with poor overall survival and disease-free survival. Multivariate Cox regression analysis revealed high expression of FGFR1 (p = 0.014) and FGFR4 (p = 0.009) were independent prognostic factors and co-overexpression of FGFR1 and FGFR4 with lymph node metastasis increased HR for death (p = 0.02). The pan-FGFR inhibitor AZD4547 showed anti-tumor activity in cell lines and in a patient-derived xenograft of OSCC. This study highlights the co-overexpression of FGFR1 and FGFR4 as a significantly poor prognosis indicator in OSCC when combined with lymph node metastasis.

Abstract 2592: Helicobacter pylori-induced FGFR4 mediates nuclear accumulation of NRF2 in gastric tumorigenesis

Abstract Background: Fibroblast growth factor receptor 4 (FGFR4) is a member of the transmembrane tyrosine kinase receptor family and has been linked to a variety of malignancies. NRF2 (Nuclear factor-erythroid 2-related factor) is a cytoprotective factor and a critical regulator in the antioxidant response pathway. In this study, we sought to uncover novel functions of FGFR4 and its role in regulating the antioxidant response in gastric carcinogenesis. Methods and Results: Using western blot and immunofluorescence, H. pylori infection in gastric cancer cell lines demonstrated high levels of reactive oxygen species and induction of both FGFR4 and NRF2. Using Flow cytometry, FGFR4 silencing resulted in decrease of NRF2 with a significant spike in ROS levels and an increase in DNA damage and cell death. FGFR4 knockdown showed a significant decrease in NRF2 transcriptional activity as measured by NRF2 ARE luciferase reporter assay and resulted in reduced mRNA levels of HO-1 (Heme Oxygenase-1), which is a classical target of NRF2. These results were confirmed by immunofluorescence showing a significant increase of nuclear accumulation of NRF2 after H. pylori infection which was abolished after FGFR4 knockdown. Similar results were found using recombinant protein FGF19, a FGFR4 ligand. C57/B6 wild-type mice were infected with the pylori strain (PMSS1). An increase in FGFR4, NRF2, and HO-1 was seen by immunofluorescence, Western blot, and quantitative real-time PCR in H. pylori-infected mice vs control mice. We observed a reduction in NRF2 in our in vitro and in vivo models using H3B-6527, a specific FGFR4 inhibitor. We also discovered an association between an increase in FGFR4 and P62 protein expressions and NRF2 protein stability. We detected a significant increase in FGFR4, NRF2, and HO1 in dysplastic and neoplastic gastric lesions using the TFF1-KO mouse model which was further aggravated by H. pylori infection. In terms of the mechanism, utilizing proximity ligation and immunoprecipitation assays, we found that FGFR4 binds to P62 to inhibit the interaction between NRF2 and KEAP1, allowing NRF2 to avoid degradation facilitating its translocation and accumulation in the nucleus. Conclusion: These findings revealed that FGFR4 has a unique functional role in promoting gastric carcinogenesis by mediating accumulation and activation of the NRF2 antioxidant response. The use of FGFR4 inhibitors is a viable treatment option that warrants further research in patients with gastric cancer. Citation Format: Nadeem S. Bhat, Mohammed Soutto, Xing Zhang, Zheng Chen, Ahmed Gomaa, Marwah Al-Mathkour, Selma Maacha, Heng Lu, Dunfa Peng, Zekuan Xu, Wael El-Rifai. Helicobacter pylori-induced FGFR4 mediates nuclear accumulation of NRF2 in gastric tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2592.

Abstract 5071: Olverembatinib (HQP1351) enhances antitumor effects of immunotherapy in renal cell carcinoma (RCC)

Abstract In solid tumors, resistance to checkpoint inhibitors (CPIs) is frequently observed, partially due to upregulation of vascular endothelial growth factor A (VEGFA) and programmed death-ligand 1 (PD-L1). This culminates in an immunosuppressive tumor microenvironment and immune escape. Inhibitors against VEGF and the VEGF receptor (VEGFR) foster tumor vessel normalization and immunostimulatory reprogramming, in turn promoting treatment effects of immunotherapies. In recent years, TKIs, including axitinib, lenvatinib, and cabozantinib, plus immunotherapy have been approved to treat advanced RCC. Currently under clinical development for relapsed or refractory chronic myeloid leukemia and gastrointestinal tumor, olverembatinib (HQP1351) is a new-generation multikinase inhibitor with targets including VEGFR, fibroblast growth factor receptor (FGFR), SRC, BCR-ABL1, c-KIT, and platelet-derived growth factor receptor. The aim of this study was to assess whether olverembatinib combined with immunotherapy can promote inhibitory effects on RCC. In cell-free kinase assays, olverembatinib inhibited VEGFR1, -2, and -3 with IC50 values of 4.2, 6.1, and 4.1 nM, respectively. Compared to lenvatinib, olverembatinib had more potent antiproliferative effects on human umbilical vein endothelial cells. Olverembatinib also had antiproliferative activity in murine RCC lines RANCA and RAG, with IC50 values of 141 and 53 nM, respectively. When olverembatinib was coadministered with an anti-PD-1 antibody in a RANCA-derived syngeneic model, both agents exerted synergistic effects, with tumor growth inhibition rates reaching 60.6%. Mechanistically, olverembatinib influenced cancer cell proliferation directly by inhibiting phosphorylation of FGFR and downstream proteins. Increased cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 were observed, suggesting induction of apoptosis. Olverembatinib also influenced proliferation of vascular endothelial cells by inhibiting phosphorylation of VEGFR, SRC, and downstream proteins Akt and extracellular signal-regulated kinases. Olverembatinib also reduced expression of PD-L1 in RCC cells. In tumor-infiltrating lymphocyte assays, olverembatinib increased numbers of cytotoxic T cells (CTL, CD8+) and natural-killer cells (NK, CD3−/CD49B+) in RANCA tumor tissues. Combined with an anti-PD-1 antibody, olverembatinib increased CTLs, NK cells, dendritic cells (DCs, MHC-II+/CD11C+), and M1 macrophages (F4/80+/CD11B+/CD86+) in RANCA tumor tissues, indicating an immunoregulatory effect of olverembatinib. Taken together, these data suggest that combining olverembatinib with a CPI confers synergistic antitumor effects in an RCC cancer mouse model by targeting tumor growth, angiogenesis, and immune regulation. This novel combination may provide an alternative approach to enhance treatment effects with CPIs in renal cancers. Citation Format: Guangfeng Wang, Eric Liang, Ping Min, Huidan Yu, Bingxing Wu, Dajun Yang, Yifan Zhai. Olverembatinib (HQP1351) enhances antitumor effects of immunotherapy in renal cell carcinoma (RCC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5071.

High expression of FGFR3 predicts a better prognosis for patients with non-small cell lung cancer in a Chinese population.

This study sought to examine the expression and mutation status of fibroblast growth factor receptor 3 (FGFR3) in non-small cell lung cancer (NSCLC) tissues and explore the prognostic potential of FGFR3 in NSCLC. Immunohistochemistry (IHC) was used to evaluate the FGFR3 protein expression of 116 NSCLC tissues. Sanger sequencing was used to examine the mutation status of exons 7, 10, and 15 in FGFR3. A Kaplan‑Meier survival analysis was conducted to evaluate the association between the expression level of FGFR3 and the overall survival (OS) and disease-free survival (DFS) of NSCLC patients. Univariate and multivariate Cox analyses were conducted to examine the association between the risk score and clinical features. FGFR3 was immunoreactive in 26 of the 86 NSCLC cases. Further, FGFR3 was positively expressed in 84.6% of the lung adenocarcinoma (AC) cases and 15.4% of the lung squamous cell carcinoma (SCC) cases. FGFR3 mutations were detected in 2 NSCLC patients (2/72, 2.8%), who both harbored the T450M mutation, a novel mutation in exon 10 of FGFR3. In NSCLC, a high expression of FGFR3 was positively correlated with gender, smoking, histology type, T stage, and the epidermal growth factor receptor (EGFR) mutation (P<0.05). FGFR3 expression was also correlated with better OS and DFS. The multivariate analysis revealed that FGFR3 served as an independent prognostic factor (P=0.024) for the OS of NSCLC patients. This study showed that FGFR3 was highly expressed in NSCLC tissues, and the frequency rate for the FGFR3 mutation at T450 M in NSCLC tissues was low. The survival analysis suggested that FGFR3 may be a useful prognostic biomarker in NSCLC.

Severe iatrogenic hypoglycaemia modulates the fibroblast growth factor protein response.

There is evidence that fibroblast growth factor (FGF) levels may be implicated in hypoglycaemia, with FGF19 being a potential contributor to insulin-independent pathways driving postprandial hypoglycaemia following bariatric surgery and basic FGF (FGF2) being elevated following mild hypoglycaemia occurring after the glucose tolerance test. However, their response following severe iatrogenic hypoglycaemia is unknown and therefore this pilot exploratory study was undertaken. A case-control study of aged-matched type 2 diabetes (T2D; n=23) and control (n=23) subjects who underwent a hyperinsulinaemic clamp, initially to euglycaemia in T2D (5 mmol/L; 90 mg/dl), and then to hypoglycaemia (<2 mmol/L; <36 mg/dl) with subsequent follow-up time course to 24 h. FGF and FGF receptor proteins were determined by Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement. At baseline, FGF12 (p= .006) was higher and FGF20 (p= .004) was lower in T2D versus controls. At hypoglycaemia, FGF7 was lower in T2D. Post-hypoglycaemic levels of FGF18, FGF19, FGF20 and FGF23 were lower while FGF12 and FGF16 were higher in T2D versus control at different time points. No differences between T2D and controls were seen for FGF1, FGF2, FGF4, FGF6, FGF8, FGF9, FGF10, FGF21 or any of the FGF receptors. At 24 h post-hypoglycaemia, FGF20 (p= .01) differed between controls and T2D, while the levels for the other proteins measured returned to baseline. None of the FGF proteins altered from baseline to euglycaemia when clamped in T2D subjects. FGF23 negatively correlated with fasting blood glucose, but no FGFs correlated with body mass index in T2D. Severe transient hypoglycaemia modulated FGF7, 16, 19, 20 and 23 (known to be associated with diabetes), together with FGF18 and 12, not previously reported to be associated with diabetes but that may be important in the pathophysiology of hypoglycaemia; FGF20 remained low at 24 h. Taken together, these data suggest that recurrent hypoglycaemia may contribute to the development of complications through changes in FGF proteins.

FGFR2 overexpression and compromised survival in diffuse-type gastric cancer in a large central European cohort.

The significance of fibroblast growth factor receptor 2 (FGFR2) in gastric cancer (GC) has been studied predominantly in Asian patient cohorts. Data on White patients are scarce. Here, we aimed to independently validate the expression and putative tumor biological significance of FGFR2 in a large non-Asian GC cohort. Immunohistochemistry (IHC) was performed on large-area tissue sections from 493 patients with GC and evaluated using the HScore. GCs with moderate and strong FGFR2 expression were studied for Fgfr2 amplification using chromogenic in situ hybridization (CISH). Median overall survival was determined using the Kaplan-Meier method. The majority [240 (99.1%)] of FGFR2-positive GCs showed a variable combination of staining intensities with marked intratumoral heterogeneity, including weak [198 (40.2%) cases], moderate [145 (29.4%)], and strong [108 (21.9%)] staining in diverse combinations. 250 (50.9%) GCs expressed no FGFR2. Fgfr2 gene amplification was found in 40% of selected cases with high protein expression and was also heterogeneous at the cell level. FGFR2 protein expression did not correlate with patient survival in the entire cohort However, using different cutoff values, a negative correlation between FGFR2-expression and patient outcome was found for diffuse-type GC. FGFR2 expression was associated with a lower tumor grade and intestinal phenotype (p≤0.0001). FGFR2-positive diffuse-type GCs classify a small subset of patients with a poor tumor specific survival (5.29±1.3 vs. 14.67±1.9 months; p = 0.004).

Abstract 1414: Effect of E7090, an FGFR1-3 selective inhibitor, on resistance to a CDK4/6 inhibitor and endocrine therapy in ER(+)/HER2(-) breast cancer preclinical models

Abstract Introduction: The fibroblast growth factor receptor (FGFR) signaling has a crucial role in proliferation, survival, migration, and drug resistance of cancer cells, as well as tumor angiogenesis. Moreover, gene alterations of FGF/FGFR were reported after treatment of a CDK4/6 inhibitor and endocrine therapy (ET) in ER(+)/HER2(-) breast cancer (BC) patients, suggesting FGF/FGFR signaling mediates resistance to these therapies. E7090, an orally available FGFR1-3 selective inhibitor, is currently under evaluation in a Phase 2 study in patients with unresectable advanced or metastatic cholangiocarcinoma with FGFR2 gene fusion (NCT04238715), and a Phase 1 study as a monotherapy or in combination with fulvestrant (FUL) or exemestane for ER(+)/HER2(-) BC patients (NCT04572295). In this study, we present the roles of FGF/FGFR signaling in resistance to a CDK4/6 inhibitor and/or ET, and activities of E7090 on drug resistance in preclinical ER(+)/HER2(-) BC cell lines and PDx models. Results: Antitumor activities of E7090 (25 or 50mg/kg, Q1Dx14 or x21, p.o.) were evaluated using five ER(+)/HER2(-) BC PDx models, OD-BRE-0438, -0450, -0188, -0704, and IM-BRE-556 with or without prior treatment of FUL (5 mg/mouse, Q7Dx2, s.c.)+palbociclib (PAL, 100mg/kg, Q1Dx14, p.o.). Among them, OD-BRE-0438 and -0704 showed higher sensitivities to E7090 with prior FUL+PAL than without it, and E7090 showed tumor regression in the OD-BRE-0438 model only with prior FUL+PAL. In these two models, FUL+PAL treatment upregulated expressions of several FGF ligands mRNA (qPCR) and FGFRs protein (IHC) in tumors. To evaluate the activities of FGF/FGFR signaling on sensitivities to FUL or FUL+PAL, in vitro cell proliferation assay using ER(+)/HER2(-) BC cell lines, MCF-7, ZR-75-1, and HCC1428 were performed in the presence of FGF2 and FGF10, and decreased sensitivities to FUL or FUL+PAL were observed. Furthermore, co-treatment with E7090 restored the sensitivity to those drugs in cultured cell lines. In consistent with in vitro results, FUL in combination with E7090 showed a combination antitumor activity in the OD-BRE-0438 model, in which expressions of several FGF ligands mRNA and FGFR2 protein in tumors showed a tendency of upregulation by FUL. Conclusion: In ER(+)/HER2(-) BC PDx models, FGFs/FGFRs expression in tumors were induced by treatment of FUL and/or PAL, and antitumor activity of E7090 was enhanced with prior FUL+PAL. In vitro study using cultured ER(+)/HER2(-) BC cell lines indicated that sensitivity against FUL or FUL+PAL was decreased by FGFR activation. Furthermore, a combination antitumor activity of E7090 and FUL was observed in the OD-BRE-0438 model. These data suggest that activation of FGF/FGFR signaling confer the resistance to a CDK4/6 inhibitor and/or ET, and E7090 shows antitumor activity against ER(+)/HER2(-) BC with prior a CDK4/6 inhibitor and/or ET. Citation Format: Satoshi Kawano, Sayo Fukushima, Kyoko Nishibata, Ryu Gejima, Yuki Niwa, Yasuhiro Funahashi, Saori Watanabe Miyano. Effect of E7090, an FGFR1-3 selective inhibitor, on resistance to a CDK4/6 inhibitor and endocrine therapy in ER(+)/HER2(-) breast cancer preclinical models [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 1414.

Abstract 353: The translocations of FGFR2 and NTRK1 in intrahepatic cholangiocarcinoma and their discordance in status by FISH and IHC

Abstract Objectives Intrahepatic cholangiocarcinoma(ICC) is a subtype of primary liver cancer lacking effective therapeutic agents. Chromosomal translocations of the neurotrophic tyrosine receptor kinase (NTRK) and fibroblast growth factor receptor 2 (FGFR2) have become promising therapeutic targets in several kinds of malignancies, but their incidence and optimum testing standards have not been fully assessed in ICC. The current study investigated the status of FGFR2 and NTRK1 genes in ICCs by different methods, and their correlation with clinicopathological features. Methods We examined FGFR2 and NTRK protein expression by immunohistochemistry (IHC), FGFR2 and NTRK1 gene translocation by fluorescence in situ hybridization (FISH). Samples were collected from 160 patients who underwent surgical resection for ICC. Break-apart probes were employed and the ratios of FGFR2 or NTRK1/chromosome enumeration probe (CEP) 10 double-color probes were determined. Furthermore, we analyzed the clinicopathological significances of the patients. Results FGFR2-positive status was detected in 13.2% of ICCs. Twenty-one patients (13.2%) were detected FGFR2 translocation. Compared with the patients with normal FGFR2, FGFR2-positivity tended to correlate with low histological grade, less mucus(P=0.024), and lower preoperative serum CA199 level, showed(P&amp;lt;0.01)in certain tumors, and were accompanied by hepatitis B virus infection more frequently (P&amp;lt;0.01). Histologically, tumors with FGFR2 translocation had a tendency to manifest small-duct subtype. The Kaplan-Meier survival analysis showed that patients with FGFR2 translocation had better prognosis in 5-year survival than those with normal FGFR2 (P=0.007),( median overall survival, 57 months vs 33 months). With regard to the NTRK1 translocated ICCs, 5.6% (9/160) were detected to have NTRK1 translocation, 5 cases were female, 4 were with hepatitis B virus infection, and 6 showed histologically large-duct subtype. Interestingly, two patients demonstrated both FGFR2 and NTRK1 translocation by FISH. However, among the 9 cases with NTRK1 translocation, only 2 cases showed moderately positive on IHC, with the remaining 7 cases weak or negative. Similarly, only 14.3% (3/21) FGFR2 translocation FISH-positive cases showed moderate to strong positive by IHC. Concordance rates between IHC and FISH were 22.2% for NTRK1 and 14.3% for FGFR2. The positive rate by IHC is significantly lower than that by FISH on both NTRK1 (2.5% vs 5%) and FGFR2 (3.7% vs 13%) according to our cohort. Conclusions The positive rate by IHC is significantly lower than that by FISH on both genes. The findings of this study suggest that a large subgroup of FGFR2-positive and NTRK-positive cases can be considered for targeted therapy. Moreover, the FGFR2 and NTRK status in ICCs should be determined carefully using a sensitive approach toward larger cancer tissues. Citation Format: Yining Zou, Yuan Ji. The translocations of FGFR2 and NTRK1 in intrahepatic cholangiocarcinoma and their discordance in status by FISH and IHC [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 353.

FGFR1 overexpression in non-small cell lung cancer is mediated by genetic and epigenetic mechanisms and is a determinant of FGFR1 inhibitor response

Amplification of fibroblast growth factor receptor 1 (FGFR1) in non-small cell lung cancer (NSCLC) has been considered as an actionable drug target. However, pan-FGFR tyrosine kinase inhibitors did not demonstrate convincing clinical efficacy in FGFR1-amplified NSCLC patients. This study aimed to characterise the molecular context of FGFR1 expression and to define biomarkers predictive of FGFR1 inhibitor response.In this study, 635 NSCLC samples were characterised for FGFR1 protein expression by immunohistochemistry and copy number gain (CNG) by in situ hybridisation (n = 298) or DNA microarray (n = 189). FGFR1 gene expression (n = 369) and immune cell profiles (n = 309) were also examined. Furthermore, gene expression, methylation and microRNA data from The Cancer Genome Atlas (TCGA) were compared. A panel of FGFR1-amplified NSCLC patient-derived xenograft (PDX) models were tested for response to the selective FGFR1 antagonist M6123.A minority of patients demonstrated FGFR1 CNG (10.5%) or increased FGFR1 mRNA (8.7%) and protein expression (4.4%). FGFR1 CNG correlated weakly with FGFR1 gene and protein expression. Tumours overexpressing FGFR1 protein were typically devoid of driver alterations (e.g. EGFR, KRAS) and showed reduced infiltration of T-lymphocytes and lower PD-L1 expression. Promoter methylation and microRNA were identified as regulators of FGFR1 expression in NSCLC and other cancers. Finally, NSCLC PDX models demonstrating FGFR1 amplification and FGFR1 protein overexpression were sensitive to M6123.The unique molecular and immune features of tumours with high FGFR1 expression provide a rationale to stratify patients in future clinical trials of FGFR1 pathway-targeting agents.

Expression of fibroblast growth factor 9 and its receptors in the dentate gyrus of hippocampus in poststroke depression rats.

Studies have found that fibroblast growth factor 9 (FGF9) might have a negative effect in the psychiatric diseases, such as depression or anxiety, but its potential role in the pathophysiology of poststroke depression (PSD) remains uncertain. Here, we set out to investigate the expression changes of FGF9 and its receptors in PSD rats. Middle cerebral artery occlusion (MCAO) combined with chronic unpredictable mild stress was used to establish the PSD rat model. Then, the rats were randomly divided into four groups: control (sham-operation), MCAO, PSD and treated (fluoxetine injection by intraperitoneal). Weight measurement, sucrose preference test, open-field test and forced swim test were performed to evaluate the behavioral changes, and then Western blot and real-time quantitative PCR were used to detect the expression level of FGF9, fibroblast growth factor receptor 1 (FGFR1) and receptor 3 (FGFR3) in the dentate gyrus of rat hippocampus. We found that FGF9 protein and mRNA expression increased significantly in the MCAO and PSD groups; FGFR3 protein and mRNA expression decreased significantly in the MCAO and PSD groups; FGFR1 protein and mRNA expression decreased significantly in the PSD group, but increased in the treated group. Furthermore, the changes mentioned above were reversed obviously by fluoxetine. These results indicated that upregulated FGF9 expression and downregulated FGFR1 and FGFR3 expression may be involved in the pathogenesis of PSD, and the FGF9/FGFR signaling pathway may be considered as an attractive target for further study.

FGFR1 Is Associated With Tamoxifen Resistance and Poor Prognosis of ER-Positive Breast Cancers by Suppressing ER Protein Expression.

Fibroblast growth factor receptor 1 (FGFR1) is widely recognized as a key player in mammary carcinogenesis and associated with the prognosis and therapeutic response of breast cancers. With the aim of investigating the correlation between FGFR1 expression and estrogen receptor (ER) and exploring the effect of FGFR1 on endocrine therapy response and ER+ breast cancer prognosis, we examined the FGFR1 protein expression among 184 ER-positive breast cancers by the immunohistochemistry (IHC) method, analyzed the association between FGFR1 expression and disease characters using the Pearson’s chi-square test, and assessed the prognostic role of FGFR1 among breast cancers using Cox regression and Kaplan-Meier analyses. Moreover, in vitro assays were conducted to confirm the correlation between FGFR1 and ER expression and investigate the effect of FGFR1 on tamoxifen (TAM) sensitivity in ER+ breast cancer. The results showed that ER expression was negatively correlated with FGFR1 expression (P = 0.011, r = -0.221). Moreover, FGFR1 expression was one of the prognostic factors of ER-positive breast cancer (OR = 1.974, 95% CI = 1.043-3.633), and high FGFR1 expression was correlated with decreased breast cancer overall survival. In addition, knocking down FGFR1 inhibited cell proliferation and enhanced TAM sensitivity in TAM-resistant cells. In conclusion, we found that there was a significant negative correlation between FGFR1 and ER levels in ER+ breast cancers, high FGFR1 protein expression was associated with poor breast cancer prognosis, down-regulating FGFR1 could elevate ER expression and is associated with enhanced TAM sensitivity in ER+ breast cancers.

Fibroblast growth factor receptor 4 as a prognostic indicator in triple-negative breast cancer.

BackgroundTriple-negative breast cancer (TNBC) constitutes up to 15% of all breast cancers. It is one of the most aggressive breast cancers and is more prone to metastasize compared with other subtypes. Breast cancer patients with this subtype usually have a poor prognosis. Fibroblast growth factor receptor 4 (FGFR4) belongs to the receptor tyrosine kinase (RTK) family, and early analyses identified that FGFR4 was involved in breast cancer. However, the prognostic effect of FGFR4 on TNBC is unknown. In the present study, we investigated the association between FGFR4 and TNBC prognosis.MethodsA total of 282 TNBC patients were enrolled. FGFR4 protein expression was detected in these 282 TNBC patients using immunohistochemistry (IHC).ResultsIn the present study, FGFR4 was highly expressed in TNBC patients. Lymph node metastasis (LNM) (P=0.033) and p53 status (P=0.019) were associated with high FGFR4 expression. Univariate analysis identified high FGFR4 expression (P=0.016) as a prognostic predictor, and multivariate analysis found that high FGFR4 expression (P=0.016) was an independent prognostic factor. The Kaplan-Meier survival curve showed that high FGFR4 protein expression was correlated with poorer overall survival (OS).ConclusionsThe results of our present study show that FGFR4 protein expression is correlated with a worse prognosis in TNBC.

The Relationship between Leptin, the Leptin Receptor and FGFR1 in Primary Human Breast Tumors.

Obesity is associated with increased breast cancer risk and poorer cancer outcomes; however, the precise etiology of these observations has not been fully identified. Our previous research suggests that adipose tissue-derived fibroblast growth factor-2 (FGF2) promotes the malignant transformation of epithelial cells through the activation of fibroblast growth factor receptor-1 (FGFR1). FGF2 is increased in the context of obesity, and increased sera levels have been associated with endocrine-resistant breast cancer. Leptin is a marker of obesity and promotes breast carcinogenesis through several mechanisms. In this study, we leverage public gene expression datasets to evaluate the associations between FGFR1, leptin, and the leptin receptor (LepR) in breast cancer. We show a positive association between FGFR1 and leptin protein copy number in primary breast tumors. These observations coincided with a positive association between Janus kinase 2 (Jak2) mRNA with both leptin receptor (LepR) mRNA and FGFR1 mRNA. Moreover, two separate Jak2 inhibitors attenuated both leptin+FGF2-stimulated and mouse adipose tissue-stimulated MCF-10A transformation. These results demonstrate how elevated sera FGF2 and leptin in obese patients may promote cancer progression in tumors that express elevated FGFR1 and LepR through Jak2 signaling. Therefore, Jak2 is a potential therapeutic target for FGFR1 amplified breast cancer, especially in the context of obesity.

Abstract B23: FGFR3 mutations and their relation to FGFR3 expression and clinical outcome in a large radical cystectomy cohort: Implications for anti-FGFR3 bladder cancer treatment?

Abstract Objective: Fibroblast growth factor receptor 3 (FGFR3) is a potentially actionable target in bladder cancer (BC). FGFR3 mutations are associated with favorable prognosis in non-muscle invasive (NMI) BC and MIBC. Overexpression of FGFR3 was reported in up to 40% of FGFR3 wild-type MIBC. p53 alterations rarely coincide with FGFR3 mutations. We analyzed FGFR3 mutations and protein-expression of FGFR3 and p53 and assessed their prognostic value in a multicenter, multilaboratory setting. Methods: We included 1,000 cN0M0, chemotherapy-naive patients who underwent radical cystectomy (RC) with pelvic node dissection. Specimens were reviewed by eight uropathologists. At seven laboratories, FGFR3 mutation status was examined using PCR-SNaPshot. p53 and FGFR3 expression were determined by immunohistochemistry (IHC). FGFR3 mutation status, p53 and FGFR3 protein expression were correlated to each other, clinicopathologic parameters, and disease-specific survival (DSS). Results: FGFR3 mutations were found in 107/1,000 RCs (11%), of which 67 were S249C. Overexpression of FGFR3 was found in 279/1,000 (28%) of tumors. p53 overexpression (cut-off&amp;gt;10%) was found in 638/926 (69%) of available cases. Among FGFR3 mutant tumors, 73% had FGFR3 overexpression. Among FGFR3 wild-type tumors, 22% had FGFR3 overexpression. FGFR3 mutations were associated with lower pT-stage (P&amp;lt;0.001), lower grade (G2-WHO 1973) (P&amp;lt;0.001), absence of CIS (P=0.009), pN0 (P&amp;lt;0.001), normal p53 (P&amp;lt;0.001), and prolonged DSS (Plog-rank=0.001). FGFR3 overexpression was associated with lower pT-stage (P&amp;lt;0.001) and G2 (P&amp;lt;0.001) but not with absence of CIS (P=0.860), pN0 (P=0.230), normal p53, (P=0.330) nor prolonged DSS (Plog-rank=0.204). We found no significant difference in DSS for patients with FGFR3 mutant tumors comparing normal vs. overexpression of FGFR3 (Plog-rank=0.444). Furthermore, we also found no significant difference in DSS for patients with FGFR3 wild-type tumors comparing normal vs. overexpression of FGFR3 (Plog-rank=0.754). Conclusions: FGFR3 mutations identified patients with favorable BC with fewer p53 alterations at RC. FGFR3 overexpression was not associated with DSS in patients with FGFR3 wild-type tumors. Our results suggest that FGFR3 mutations (driver effect) have a distinct functional role than FGFR3 overexpression (passenger effect). Hence, patients with FGFR3 mutations may be more likely to benefit from anti-FGFR3 therapy than patients with only overexpression of FGFR3. Citation Format: Bas van Rhijn, Laura Mertens, Roman Mayr, Peter Bostrom, Mirari Marques, Geert van Leenders, Stefanie Gotz, Michiel van der Heijden, Michael Jewett, Francisco Real, Robert Stohr, Alexandre Zlotta, Markus Eckstein, Yanish Soorojebally, Max Burger, Wolfgang Otto, Francois Radvanyi, Damien Pouessel, Theo van der Kwast, Nuria Malats, Arndt Hartmann, Yves Allory, Deric van der Schoot, Ellen Zwarthoff, Tahlita Zuiverloon. FGFR3 mutations and their relation to FGFR3 expression and clinical outcome in a large radical cystectomy cohort: Implications for anti-FGFR3 bladder cancer treatment? [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B23.

FGFR3 Mutation Status and FGFR3 Expression in a Large Bladder Cancer Cohort Treated by Radical Cystectomy: Implications for Anti-FGFR3 Treatment?†

Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer (BC). FGFR3 mutations are common in noninvasive BC and associated with favorable BC prognosis. Overexpression was reported in up to 40% of FGFR3 wild-type muscle-invasive BC. We analyzed FGFR3 mutations, FGFR3, and p53 protein expression and assessed their prognostic value in a cohort of 1000 chemotherapy-naive radical cystectomy specimens. FGFR3 mutations were found in 11%, FGFR3 overexpression was found in 28%, and p53 overexpression was found in 69% of tumors. Among FGFR3 mutant tumors, 73% had FGFR3 overexpression versus 22% among FGFR3 wild-type tumors. FGFR3 mutations were significantly associated with lower pT stage, tumor grade, absence of carcinoma in situ, pN0, low-level p53, and longer disease-specific survival (DSS). FGFR3 overexpression was associated only with lower pT stage and tumor grade. Moreover, FGFR3 overexpression was not associated with DSS in patients with FGFR3 wild-type tumors. In conclusion, FGFR3 mutations identified patients with favorable BC at cystectomy. Our results suggest that FGFR3 mutations have a driver role and are functionally distinct from FGFR3 overexpression. Hence, patients with FGFR3 mutations would be more likely to benefit from anti-FGFR3 therapy. Ideally, further research is needed to test this hypothesis. Patient summaryOncogenic fibroblast growth factor receptor 3 (FGFR3) mutations are very common in bladder cancer. In this report, we found that these FGFR3 mutations were associated with favorable features and prognosis of bladder cancer compared with patients with FGFR3 overexpressed tumors only. As a consequence, patients with FGFR3 mutant tumors would be more likely to benefit from anti-FGFR3 therapy than patients with FGFR3 protein overexpression only.