Fibroblast Growth Factor Receptor Research Articles

Efficacy of post-lenvatinib treatments in patients (pts) with advanced renal cell carcinoma (aRCC).

4538 Background: Lenvatinib is a tyrosine kinase inhibitor (TKI) that targets both vascular endothelial growth factor (VEGF) receptor and fibroblast growth factor receptor. It has demonstrated efficacy both in the upfront and refractory disease settings. However, there is a lack of data surrounding the efficacy of TKIs post-lenvatinib exposure. In this study, we investigate the activity of post-lenvatinib therapies in pts with aRCC. Methods: We conducted a retrospective analysis utilizing the International Metastatic Database Consortium (IMDC). Pts having received treatment post lenvatinib exposure were eligible and divided into two cohort: pts post-1st line lenvatinib (2nd line cohort) and pts post-2nd line lenvatinib (3rd line cohort). The primary objective was objective response rate (ORR) and time to treatment failure (TTF). ORR was summarized with 95% two-sided exact binomial confidence interval. TTF was defined as time from treatment initiation to drug cessation for any reason censored at the date of last follow-up. Results: Overall, 84 pts received 1st line lenvatinib of whom 43 (51%) remain on therapy, 20 (24%) received 2nd line treatment, and 21 (25%) received no subsequent treatment. All pts received 1st line pembrolizumab + lenvatinib (ORR 50%, median TTF 9.7 months). Reason for lenvatinib discontinuation was progression (50%), progression + toxicity (20%), toxicity (15%), or other (15%). For the 2nd line cohort, median age was 61 years, most pts were male (85%), had prior nephrectomy (75%), clear cell histology (85%), and were IMDC intermediate/poor risk (55%). 2nd line therapy regimens included TKI monotherapy (80%), TKI-IO (5%), and other (15%). The ORR to 2nd line treatment was 5% (95% CI 0.2-25) and median TTF was 5.8 months (95% CI 1.9-14.9). Of 2nd line lenvatinib-exposed pts (n=84), 24 (29%) remain on treatment, 34 (40%) received 3rd line treatment, and 26 (31%) did not receive additional therapy. Most pts received 2nd line everolimus + lenvatinib (97%) (ORR 39%, median TTF 5.9 months). Reason for lenvatinib discontinuation was progression (59%), progression + toxicity (9%), toxicity (12%), or other (21%). For the 3rd line cohort, median age was 67 years, most pts were male (68%), had prior nephrectomy (88%), clear cell histology (68%), and were IMDC intermediate/poor risk (77%). 3rd line treatments included TKI alone (50%), IO-TKI (38%), and other (12%). The ORR to 3rd line treatment was 12% (95% CI 3.3-27) and median TTF was 2.8 months (95% CI 1.9-7.4). Conclusions: In this analysis, we demonstrate modest activity of TKI-based therapy post-lenvatinib exposure. Our study highlights the need for improved treatment options for pts progressing on lenvatinib-based therapies.

SCR-A002, a novel FGFR2b-targeting antibody-drug-conjugate for solid tumors.

e15019 Background: FGFR2b (fibroblast growth factor receptor 2 IIIb), a transmembrane tyrosine kinase, is overexpressed in various cancers, including gastric, esophageal, breast, hepatocellular, pancreatic, ovary, uterine, cervical, endometrial, lung, colorectal cancers. Bemarituzumab (FPA144), an afucosylated humanized IgG1 monoclonal antibody targeting FGFR2b with enhanced ADCC activity, showed promising clinical efficacy in phase 2 study of FGFR2b-selected gastric or gastroesophageal junction adenocarcinoma. We have developed a potential first-in-class FGFR2b-targeting ADC (SCR-A002) at IND-enabling status, displaying significantly superior anti-tumor potency compared to Bemarituzumab. Methods: SCR-A002 is comprised of a humanized anti-FGFR2b IgG1 monoclonal antibody conjugated with proprietary TOPO1 inhibitor payload (DAR 8) via a cleavable linker. Anti-FGFR2b antibody was generated by immunization of FGFR2b protein in mice followed by hybridoma technique. Binding activity and cytotoxicity were assessed in FGFR2b+ tumor cell line. And anti-tumor efficacy was evaluated in several CDX and PDX models. Results: SCR-A002 could specifically bind to FGFR2b on cancer cells and be internalized, and the TOPO1 inhibitor could be cleaved intracellularly to kill the cancer cells potently in vitro. After single-dose administration of SCR-A002, tumor regression was achieved in different FGFR2b expression level CDX and PDX models, even with big-size tumor or Bemarituzumab resistance. Conclusions: SCR-A002 showed potent anti-tumor efficacy in preclinical data, which suggest that SCR-A002 is expected to provide a new treatment option as single agent or combo with SoC for patients with FGFR2b-overexpressing solid tumor.

Incidence and potential prognostic implications of fibroblast growth factor receptor gene alterations and coexisting mutations in colorectal cancer.

e15558 Background: The fibroblast growth factor receptor (FGFR) family comprises four tyrosine kinase receptors (FGFR1-4). FGFR1 amplification (6.3%) as well as overexpression of FGFR1 (10.1%), FGFR2 (5.5%) and FGFR3 (16.2%) have been demonstrated in primary colorectal cancer (CRC, incidence in brackets). The prognostic utility of testing FGFR alterations and association with response to FGFR-inhibitors are still emerging. Here, we report FGFR alteration data with clinicopathological correlations and survival outcomes from a Sydney based Local Health District in Australia. Methods: Clinicopathological data was analysed from 341 ethnically diverse patients with CRC treated at Southwestern Sydney Local Health District from 2014-2021. Next generation sequencing for point mutations, deletions, and amplifications was performed using the Qiagen 30-gene panel. Mismatch repair (MMR) status was determined using immunohistochemical staining. Demographic characteristics, histological features and survival outcomes were recorded from electronic medical records. Results: Of the 341 patients, 42 (12.3%) had FGFR alterations of which 52% were men and 48% women. 32 patients were Caucasian, 4 Middle Eastern, 5 Asian and 1 South American. Median age at diagnosis was 66 years. Of the 42 patients, FGFR1 mutations were present in 26.2%, FGFR1 amplifications in 4.7% and FGFR1 deletions in 4.7%. FGFR2 mutations were present in 16.7%, and 50% had FGFR3 mutations. One patient had concurrent FGFR1 and FGFR3 mutations. The vast majority were adenocarcinoma (81%), followed by mucinous adenocarcinoma (17%) and adenocarcinoma with neuroendocrine features (2%). 21.4% of patients with FGFR alterations had concurrent KRAS mutations, 28.6% had concurrent BRAF mutations (91.6% V600E) and MMR-deficiency was present in 19%. Median overall survival of patients with FGFR alterations was 44 months, for those who also had deficient MMR 48 months, concurrent BRAF V600E mutations 51 months and concurrent KRAS mutations 71 months. 43% (18 patients) were stage 4 at presentation. Of these 18 patients, 7 had FGFR1 mutations, 1 had a FGFR2 mutation and 10 had FGFR3 mutations. The median overall survival of patients with stage 4 disease was 11 months and those with concurrent BRAF V600E mutations 17 months. Two patients had concurrent KRAS mutations (both G12V), with median overall survival of 5.5 months and 1 patient had MMR deficiency, with an overall survival of 9 months. Conclusions: FGFR alterations were identified in 12.3% of patients with colorectal cancer in an ethnically diverse cohort in Australia. Concurrent KRAS and BRAF mutations appear to affect survival outcomes. Further multivariate analyses are underway.

Efficacy and safety of erdafitinib in adults with breast cancer and prespecified fibroblast growth factor receptor alterations in the phase 2 open-label, single-arm RAGNAR trial.

1088 Background: Erdafitinib is an oral selective pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor approved in the US for adult patients (pts) with locally advanced or metastatic urothelial carcinoma with susceptible FGFR3 genetic alterations, as determined by an FDA-approved companion diagnostic test, whose disease has progressed on or after ≥1 line of prior systemic therapy. The primary analysis results from the RAGNAR study demonstrated tumor agnostic efficacy in patients with advanced solid tumors with predefined FGFR alterations after failure of standard therapies (Pant 2023). Here, we report results on patients with breast cancer in the RAGNAR study. Methods: Patients with breast cancer and prespecified FGFR1-4 alterations (mutations or fusions) who had documented disease progression after exhausting standard therapies received oral erdafitinib 8 mg daily with optional up-titration until disease progression or intolerable toxicity. The primary end point was objective response rate by Independent Review Committee (IRC). Secondary endpoints include duration of response, disease control rate, progression-free survival, overall survival, and safety. Results: At data cutoff (median follow up, 30.3 months), 16 patients with breast cancer received erdafitinib. Median age was 54 y (range 37-74); all (100%) patients were female and had visceral metastases. Patients had a median of 5 prior lines of systemic therapies (range 2-12); one (6%) patient had responded to the last line of therapy. Ten (63%) patients had FGFR fusions, and 6 (38%) had FGFR mutations. Mutations of TP53 and PIK3CA were found in 6/16 patients. Objective response rate and disease control rate by IRC were 31% (95% CI 11-59) and 69% (95% CI 41-89); objective response rate and disease control rate by investigator were 38% (95% CI 15-65) and 69% (95% CI 41-89), respectively. Median time to onset of response was 1.4 months. Responses were observed in patients with both FGFR2 fusions and with FGFR2/3 mutations. Median duration of response, progression free survival, and overall survival were 6.9 months, 5.7 months, and 8.9 months, respectively. Common adverse events (AEs) included stomatitis (81%), diarrhea (69%), and hyperphosphatemia (69%), dry mouth (63%), and palmar-plantar erythrodysesthesia (44%). Six (38%) patients had serious AEs. No patients discontinued erdafitinib due to AEs. No treatment-related deaths were observed. Conclusions: Erdafitinib demonstrated clinically meaningful activity in heavily pretreated patients with breast cancer and prespecified FGFR alterations. Safety data were consistent with the erdafitinib safety profile. Clinical trial information: NCT04083976 .

304P Clinical response to futibatinib in intrahepatic cholangiocarcinoma with acquired resistance to fibroblast growth factor receptor 2 inhibitors

304P Clinical response to futibatinib in intrahepatic cholangiocarcinoma with acquired resistance to fibroblast growth factor receptor 2 inhibitors

Efficacy of erdafitinib in adults with advanced solid tumors and non-prespecified fibroblast growth factor receptor mutations in the phase 2 RAGNAR trial: Exploratory cohort.

Efficacy of erdafitinib in adults with advanced solid tumors and non-prespecified fibroblast growth factor receptor mutations in the phase 2 RAGNAR trial: Exploratory cohort.

Hypericin Alleviates Chronic Kidney Disease-induced Left Ventricular Hypertrophy by Regulation of FGF23-FGFR4 Signaling Pathway.

Chronic kidney disease (CKD) is a significant global health threat that imposes a substantial burden on both individuals and societies. CKD frequently correlates with cardiovascular events, particularly left ventricular hypertrophy (LVH), which contributes to the high mortality rate associated with CKD. Fibroblast growth factor 23 (FGF23), a hormone primarily involved in regulating calcium and phosphorus metabolism, has been identified as a major risk factor for LVH in CKD patients. Elevated serum FGF23 levels are known to induce LVH and myocardial fibrosis by activating the fibroblast growth factor receptor 4 (FGFR4) signal pathway. Therefore, targeting FGFR4 and its downstream signaling pathways holds potential as a treatment strategy for cardiac dysfunction in CKD. In our current study, we have discovered that Hypericin, a key component derived from Hypericum perforatum , has the ability to alleviate CKD-related LVH by targeting the FGFR4/phospholipase C gamma 1 (PLCγ1) signaling pathway. Through in vitro experiments using rat cardiac myocyte H9c2 cells, we observed that Hypericin effectively inhibits FGF23-induced hypertrophy and fibrosis by suppressing the FGFR4/PLCγ1/calcineurin/nuclear factor of activated T-cell (NFAT3) signaling pathway. In addition, our in vivo studies using mice on a high-phosphate diet and rat models of 5/6 nephrectomy demonstrated that Hypericin has therapeutic effects against CKD-induced LVH by modulating the FGFR4/PLCγ1/calcineurin/NFAT3 signaling pathway. In conclusion, our research highlights the potential of Hypericin as a candidate for the treatment of CKD-induced cardiomyopathy. By suppressing the FGFR4/PLCγ1 signaling pathway, Hypericin shows promise in attenuating LVH and myocardial fibrosis associated with CKD.

Efficacy and safety of erdafitinib in adults with NSCLC and prespecified fibroblast growth factor receptor alterations in the phase 2 open-label, single-arm RAGNAR trial.

8515 Background: Erdafitinib is an oral selective pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor approved in the US for treatment of locally advanced or metastatic urothelial carcinoma in adults with susceptible FGFR3 alterations, as determined by FDA-approved companion diagnostic test, who have progressed during or after ≥1 line of prior systemic therapy. Primary analysis results from the RAGNAR study demonstrated tumor agnostic efficacy in patients with advanced solid tumors with predefined FGFR alterations after failure of standard therapies (Pant 2023). Here we report results on patients with non-small-cell lung cancer (NSCLC) in the RAGNAR study. Methods: Patients with advanced or metastatic squamous or non-squamous NSCLC with prespecified FGFR1-4 alterations (mutations/fusions) and with documented disease progression after exhausting standard therapies were included. Patients with other targetable alterations (eg, EGFR, ALK, ROS1) were excluded. Patients received oral erdafitinib until disease progression or intolerable toxicity. The primary end point was objective response rate by independent review committee. Secondary endpoints include duration of response, disease control rate, progression-free survival, overall survival, and safety. Results: At data cutoff (median survival follow-up of 23.7 months), 23 patients with NSCLC received erdafitinib. Median age was 63 years (range 50-79); 22 (96%) had metastases. Patients had a median of 2 prior lines of systemic therapies (range 1-7; 48% had 3+ prior lines); only 2 (9%) patients had responded to their prior line of therapy. Fourteen patients (61%) had squamous and 9 (39%) non-squamous histology; 13 patients (57%) had FGFRfusions and 10 (43%) had FGFR mutations. Three patients had CDKN2Aand 3 patients had PIK3CA co-alterations. Objective response rate by independent review committee was 26% (95% CI 10-48); disease control rate was 74% (95% CI 52-90). Median time to onset of response was 1.5 months. Responses were observed in 21% (3/14) of patients with squamous; 33% (3/9) with non-squamous histology, 29% (2/7) of patients with FGFR2, 25% (4/16) with FGFR3alterations, and in 20% (2/10) of patients with FGFR mutations and 31% (4/13) with fusions. Median duration of response, progression-free survival and overall survival were 4.6 months, 4.1 months, and 10.5 months, respectively. Most common adverse events (AEs) were diarrhea (65%), stomatitis (61%), dry mouth (44%), hyperphosphatemia (65%), dry skin (30%), and fatigue (22%); 6 (26%) had serious AEs; 2 (9%) discontinued erdafitinib due to AEs. No treatment-related deaths were observed. Conclusions: Erdafitinib demonstrated clinically meaningful activity in pre-treated patients with NSCLC and pre-specified FGFR alterations. Safety data were consistent with erdafitinib safety profile. Clinical trial information: NCT04083976 .

Antitumor Activity of Tasurgratinib as an Orally Available FGFR1-3 Inhibitor in Cholangiocarcinoma Models With FGFR2-fusion.

Cholangiocarcinoma (CCA) is an aggressive tumor with limited treatment options especially in 2nd line or later treatments. Targeting fibroblast growth factor receptor (FGFR) 2 has recently emerged as a promising treatment option for patients with CCA harboring FGFR2-fusion. This study investigated the antitumor activities of tasurgratinib as an orally available FGFR1-3 inhibitor, in preclinical FGFR2-driven CCA models. Antitumor activities of tasurgratinib were examined in vitro and in vivo using NIH/3T3 cells expressing FGFR2-fusion as FGFR2-driven CCA models, and in vivo using a CCA patient-derived xenograft model. The molecular mechanism of action of tasurgratinib was elucidated through co-crystal structure analysis with FGFR1, manual complex model analysis with FGFR2, and binding kinetics analysis with FGFR2. Furthermore, the cell-based inhibitory activities against acquired resistant FGFR2 mutations in patients with CCA treated with FGFR inhibitors were evaluated. Tasurgratinib showed antitumor activity in preclinical FGFR2-driven CCA models by inhibiting the FGFR signaling pathway in vitro and in vivo. Furthermore, cell-based target engagement assays indicated that tasurgratinib had potent inhibitory activities against FGFR2 mutations, such as N549H/K, which are the major acquired mutations in CCA. We also confirmed that tasurgratinib exhibited fast association and slow dissociation kinetics with FGFR2, binding to the ATP-binding site and the neighboring region, and adopting an Asp-Phe-Gly (DFG)-"in" conformation. These data demonstrate the therapeutic potential of tasurgratinib in FGFR2-driven CCA and provide molecular mechanistic insights into its unique inhibitory profile against secondary FGFR2 resistance mutations in patients with CCA treated with FGFR inhibitors.

Spectrofluorometric determination of futibatinib in human plasma and pharmaceutical formulations

Futibatinib is a powerful inhibitor of fibroblast growth factor receptors that impedes its phosphorylation and subsequently leading to a reduction in in cell viability across various cell lines. Futibatinib was approved for initial use as an effective treatment for several diseases, including non-small cell lung cancer and breast cancer. Herein, a novel selective fluorescence probe was created for futibatinib quantification in various matrices, including pharmaceutical formulation and human plasma. The technique primarily depends on futibatinib’s chemical conversion into a fluorescent product through a reaction with trimethylamine and bromoacetyl bromide. The created fluorescent probe exhibits maximum emission peak at 338 nm upon excitation at 248 nm. The method provided a low detection limit of 0.120 ng/mL and maintained a linear concentration-dependent relationship across the range of 1–200 ng/mL. High sensitivity, accuracy and precision were demonstrated for futibatinib quantification in pharmaceutical formulation and spiked plasma matrix by the method, which was validated in accordance with ICH requirements.

The combination of gemcitabine plus an anti-FGFR inhibitor can have a synergistic antitumor effect on FGF-activating cholangiocarcinoma

Anti-FGFR treatment for cholangiocarcinoma (CCA) with fibroblast growth factor receptor (FGFR) alteration is a promising treatment option. Since the antitumor mechanisms of anti-FGFR inhibitors and conventional cytotoxic drugs differ, synergistic effects can be possible. This study aimed to evaluate the efficacy of the combined administration of gemcitabine (GEM) and pemigatinib in CCA cells with FGFR2 alterations. To simulate the treatment for patients with 3 kinds of CCA, chemonaïve CCA with activation of the FGF pathway, chemo-resistant CCA with activation of the FGF pathway, and CCA without FGF pathway activation (as controls), we evaluated 3 different CCA cell lines, CCLP-1 (with a FGFR2 fusion mutation), CCLP-GR (GEM-resistant cells established from CCLP-1), and HuCCT1 (without FGFR mutations). There was no significant difference between CCLP-1 and HuCCT1 in GEM suspensibility (IC50 = 19.3, 22.6 mg/dl, p = 0.1187), and the drug sensitivity to pemigatinib did not differ between CCLP-1 and CCLP-GR (IC50 = 7.18,7.60 nM, p = 0.3089). Interestingly, only CCLP-1 showed a synergistic effect with combination therapy consisting of GEM plus pemigatinib in vitro and in vivo. In a comparison of the reaction to GEM exposure, only CCLP-1 cells showed an increase in the activation of downstream proteins in the FGF pathway, especially FRS2 and ERK. In association with this reaction, cell cycle and mitosis were increased with GEM exposure in CCLP-1, but HuCCT1/CCLP-GR did not show this reaction. Our results suggested that combination therapy with GEM plus pemigatinib is a promising treatment for chemonaïve patients with CCA with activation of the FGF pathway.

Role of serum endotoxin, FGF19, TLR2, TNF-α, IL-12 and IL-10 in NAFLD-associated T2DM pathogenesis: Insights into Th1 bias and protective mechanisms.

Non-alcoholic fatty liver disease (NAFLD) in non-obese patients is pathophysiologically distinct, exhibiting common immunological link with type-2 diabetes mellitus (T2DM). This study aims to delineate the role of Toll-like receptor 2 (TLR2)-mediated immuno-modulation along with its association with fibroblast growth factor receptor 4 (FGFR4) and its ligand fibroblast growth factor 19 (FGF19) in the pathogenesis of NAFLD without or with T2DM. Blood samples were collected from patients with NAFLD (n = 90), NAFLD with T2DM (n = 90) and healthy cohorts (n = 90) with consent and clinical records. Real-time polymerase chain reaction (PCR), enzyme-linked immunoassay (ELIZA) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to analyze messenger ribonucleic acid (mRNA), protein expression and gene polymorphism. The molecular genetic analysis revealed the prevalence of variant allele(A) in FGFR4 gene in both cases compared to controls. The mRNA expression of FGF19 and TLR2 exhibited significant upregulation in NAFLD without T2DM compared to NAFLD with T2DM. Tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12) showed upregulation in both disease cohorts compared to control while IL-10 showed significant downregulation in NAFLD with T2DM compared to the other two cohorts. Correlation analysis between FGF19 and TLR2 revealed significant positive association in both NAFLD with and without T2DM. The Th1:Th2 ratio showed significant upregulation in NAFLD with T2DM compared to NAFLD without T2DM. In conclusion, elevated serum endotoxin levels appear to contribute to NAFLD and T2DM development. Upregulated FGF19 seems to be protective against developing T2DM in NAFLD patients. Higher TLR2, TNF-α and IL-12 expression in NAFLD without T2DM suggests a Th1 bias in its pathogenesis, while reduced IL-10 in NAFLD with T2DM implies a more skewed Th1 state in this condition.

Essential growth factor receptors for fibroblast homeostasis and activation: Fibroblast Growth Factor Receptor (FGFR), Platelet Derived Growth Factor Receptor (PDGFR), and Transforming Growth Factor β Receptor (TGFβR).

Fibroblasts are cells of mesenchymal origin that are found throughout the body. While these cells have several functions, their integral roles include maintaining tissue architecture through the production of key extracellular matrix components, and participation in wound healing after injury. Fibroblasts are also key mediators in disease progression during fibrosis, cancer, and other inflammatory diseases. Under these perturbed states, fibroblasts can activate into inflammatory fibroblasts or contractile myofibroblasts. Fibroblasts require various growth factors and mitogenic molecules for survival, proliferation, and differentiation. While the activity of mitogenic growth factors on fibroblasts in vitro was characterized as early as the 1970s, the proliferation and differentiation effects of growth factors on these cells in vivo are unclear. Recent work exploring the heterogeneity of fibroblasts raises questions as to whether all fibroblast cell states exhibit the same growth factor requirements. Here, we will examine and review existing studies on the influence of fibroblast growth factor receptors (FGFRs), platelet-derived growth factor receptors (PDGFRs), and transforming growth factor β receptor (TGFβR) on fibroblast cell states.

HSPA5 and FGFR1 genes in the mesenchymal subtype of glioblastoma can improve a treatment efficacy

ABSTRACT Tyrosine kinase inhibitors (TKIs) have emerged as a potential treatment strategy for glioblastoma multiforme (GBM). However, their efficacy is limited by various drug resistance mechanisms. To devise more effective treatments for GBM, genetic characteristics must be considered in addition to pre-existing treatments. We performed an integrative analysis with heterogeneous GBM datasets of genomic, transcriptomic, and proteomic data from DepMap, TCGA and CPTAC. We found that poor prognosis was induced by co-upregulation of heat shock protein family A member 5 (HSPA5) and fibroblast growth factor receptor 1 (FGFR1). Co-up regulation of these two genes could regulate the PI3K/AKT pathway. GBM cell lines with co-upregulation of these two genes showed higher drug sensitivity to PI3K inhibitors. In the mesenchymal subtype, the co-upregulation of FGFR1 and HSPA5 resulted in the most malignant subtype of GBM. Furthermore, we found this newly discovered subtype was correlated with homologous recombination deficiency (HRD) In conclusion, we discovered novel druggable candidates within the group exhibiting co-upregulation of these two genes in GBM, suggest potential strategies for combination therapy.

Abstract A004: Clinical test design affects tumor tissue and ctDNA FGFR gene status in metastatic urothelial cancer: a prospective study

Abstract Fibroblast growth factor receptor (FGFR) gene alterations are common in urothelial cancer (UC) and targetable with erdafitinib. The co-approved companion diagnostic is an amplicon-based tumor tissue test that detects a limited number of recurrent FGFR alterations. However, FGFR status may change during disease progression, and rare FGFR alterations not typically covered by existing tests may be of clinical importance. Therefore, in a prospective pan-Canadian study (NCT06129084), we compared standard archival tissue testing to broad targeted sequencing of cell-free circulating tumor DNA (ctDNA) to detect FGFR alterations in UC. Eligible patients had progressing metastatic UC and were undergoing tissue FGFR testing. Blood was drawn at study enrollment and plasma cell-free DNA and matched white blood cell DNA underwent deep-targeted sequencing with a hybridization capture panel including the introns and exons of FGFR1-4 and the common breakpoint region of TACC3. Archival tissue selection and testing was initiated by the treating physician and typically used the Oncomine Focus assay. 210 patients were enrolled across 12 sites, and 140 patients have tissue and ctDNA results available for concordance assessment. 43/140 (31%) of cell-free DNA samples had low tumor fraction (<0.5%) and were considered inconclusive. For the remaining 97 same-patient tissue and ctDNA pairs, FGFR status was identical in 88 (91%). In 7 of 9 discordant cases, the tissue was negative, but ctDNA was positive for FGFR alterations: these were enriched for FGFR3-TACC3 fusions (4/7). Two discordant fusions had canonical breakpoints that can be identified by current amplicon-based companion diagnostics, indicating potential spatiotemporal heterogeneity in somatic FGFR status. The other two discordant (ctDNA-only) fusions had non-canonical breakpoints that are not amenable to detection by current amplicon-based companion diagnostics. In two mutation discordant cases (S249C positive in ctDNA only), lack of signal in tumor tissue could be explained by the presence of additional FGFR3 variants within 25bp of S249 and in cis - disrupting primer binding sites. There were only two cases of tissue-only FGFR alterations among the 97 evaluable pairs (FGFR3-TACC3 fusion, S249C). ctDNA testing of metastatic UC with a broad capture-based approach covering all exons and introns of FGFR genes can identify additional alterations not detected with current amplicon-based companion diagnostics applied to archival tissue. Our results support ctDNA testing as a valuable adjunct to tissue testing, but not as a replacement due to the high frequency of inconclusive ctDNA results, secondary to tumor fraction below the threshold. Assay design, including target enrichment technique, is a significant source of variability in tissue and ctDNA FGFR status and should be carefully considered in future clinical biomarker testing strategies. Citation Format: David C. Müller, Gillian Vandekerkhove, Andrew J. Murtha, Jack V.W. Bacon, Carlos Vasquez-Rios, Maria Stephenson, Kimia Rostin, Elena Schönlau, Connor Wells, Sunil Parimi, Krista Noonan, Naveen S. Basappa, Jenny J. Ko, Daygen Finch, Nimira Alimohamed, Tarek A. Bismar, Gang Wang, Andreas Papadakis, Lucia Nappi, Matti Annala, Cecily Q. Bernales, Alan Spatz, Kim N. Chi, Alexander W. Wyatt, Bernhard J. Eigl. Clinical test design affects tumor tissue and ctDNA FGFR gene status in metastatic urothelial cancer: a prospective study [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A004.

Abstract A024: Genomic peroxisome proliferator-activated receptor gamma (PPARG) study: A sponsored testing program to identify patients harboring genetic alterations in advanced UC in support of the FX-909 Phase 1 study

Abstract The transcription factor PPARG is associated with the luminal lineage subtype representing ~65% of advanced urothelial cancer (UC) patients. Recurrent genetic alterations in PPARG, fibroblast growth factor receptor 3 (FGFR3), and the heterodimeric binding partner for PPARG, retinoid X receptor alpha (RXRA), are characteristic of this luminal subtype. FX-909, a covalent PPARG inverse agonist, represents a promising first-in-class therapy for patients with advanced UC. FX-909-CLINPRO-1 [NCT05929235] is an open-label Phase 1 study to evaluate the safety, tolerability, pharmacokinetics, biologic activity, and preliminary clinical activity of FX-909. This study includes a Phase 1b expansion cohort, which will enroll advanced UC patients with genetic alterations in PPARG, RXRA, and/or FGFR3. FlareTx is sponsoring a testing program called Genomic PPARG Study (GPS) to enable investigators to identify patients with these relevant genetic alterations. Tumors collected from patients with advanced UC were sequenced using the Tempus xT assay (DNA-seq of 648 genes at 500x coverage; RNA-seq). A clinical report is generated and returned to the physician to guide selection of approved therapies, and if appropriate to support eligibility for inclusion in the study. FlareTx is provided with de-identified genomic data, where assessment of the prevalence of genetic alternations such as copy number variation (CNV), fusions and mutations (single nucleotide variants) in PPARG, RXRA and FGFR3 are undertaken. Additionally, luminal molecular classification is performed using the Robertson method [1]. PPARG mean expression value are normalized by transcripts-per-million (TPM) and reported as Log2[TPM+1]. In the initial data set of 27 patients, 3 patients had PPARG amplification (CN >/= 3), 3 patients had RXRA hotspot mutation (S427F) [2], 6 patients had FGFR3 mutation(s), and 1 patient had both PPARG amplification and a FGFR3 mutation. Twenty of 27 patients tested had sufficient starting material of good quality to also perform whole transcriptome analysis. The mean mRNA expression level of PPARG was 6.77 Log2[TPM+1] (range 2.91 Log2[TPM+1] – 9.22 Log2[TPM+1]). Consistent with the Robertson subtype distribution, 65% of patients were luminal and 35% were non-luminal (basal-squamous and neuronal); 10 of 13 luminal patients had one or more genetic alteration in PPARG, RXRA and/or FGFR3. High PPARG expression was associated with luminal subtype compared to non-luminal subtype (7.90 Log2[TPM+1] vs 4.67 Log2[TPM+1]; p=0.00042). GPS is open at two active Phase 1 sites, with several more planned as the study advances towards the Phase 1B dose expansion stage. GPS runs concurrently with the FX-909 study and is a novel precision strategy to maximize identifying advanced UC patients with actionable genetic alterations to potentially enroll in the clinical study.

Abstract B014: FGFR inhibition upregulates Nectin-4 expression in FGFR3 altered urothelial carcinoma

Abstract Bladder urothelial carcinoma (UC) is a common malignancy and remains a significant cause of mortality; however, in recent years there have been substantial advances in the treatment of metastatic UC (mUC). This includes the approval of the antibody drug conjugate (ADC) enfortumab vedotin, which targets Nectin-4, and the fibroblast growth factor receptor (FGFR) inhibitor erdafitinib (in FGFR2/3 altered tumors). Despite the impressive efficacy of these agents, most patients will ultimately develop progressive disease, underlining the need for further therapeutic development. Previous work has demonstrated that NECTIN4 expression and FGFR3 alterations are enriched in luminal subtypes of UC and NECTIN4 expression is higher in FGFR altered tumors. We validate these findings in scRNAseq from primary tumors as well as bulk RNAseq from murine models. Given the positive correlation between NECTIN4 and FGFR3 alterations we hypothesized that FGFR3 inhibition would downregulate Nectin-4 expression. To our surprise we found in vitro treatment with erdafitinib in cell lines with FGFR3 fusion proteins (RT112, RT4, SW780) lead to an increase in Nectin-4 protein levels. Additionally, in vitro FGFR inhibition in the UMUC-14 cell line which harbors an FGFR3S249C hotspot mutation, upregulates Nectin-4 protein levels. To further investigate this phenomenon, we engineered the FGFR3 WT cell line, 5637 to overexpress FGFR3-TACC3 or FGFR3S249C. We saw that FGFR3-TACC3 and FGFR3S249C overexpression both down regulated Nectin-4 indicating that FGFR3 activity negatively regulates Nectin-4 expression. We have also investigated the association between FGFR inhibition and Nectin-4 expression in two in vivo models: (1) in a xenograft model using the RT112 line we found an overall increase in Nectin-4 expression in tumors treated with a 5-day course of erdafitinib; (2) in a UPFL1 (a cell line derived from a genetically engineered mouse with a S249C mutation) syngeneic model we also saw an increase in Nectin-4 expression after erdafitinib treatment. We have observed that the increase in Nectin-4 expression persists in vitro even after withdrawal of erdafitinib treatment, indicating that intermittent dosing of erdafitinib maybe be sufficient to prime a tumor for Nectin-4 targeted therapy. Lastly, preliminary in vitro investigation of the combination of erdafitinib and enfortumab vedotin in RT112 cells showed synergy, particularly with lower doses of both agents, providing further support of our hypothesis that FGFR3 inhibition may act to sensitize UC to Nectin-4 ADC therapy. A potential synergistic relationship is further supported by the recent results of the phase 1 trial investigating the combination of erdafitinib plus enfortumab vedotin in which the objective response rate was 100% (9/9 patients: 8 PRs & 1CR). Citation Format: Sean Clark-Garvey, Mi Zhou, Michael Sturdivant, Wolfgang Beckabir, Lucia Kim, E. Drew Toomer, Ian McCabe, Akihiro Hamada, Daniel Crona, Jeffrey S. Damrauer, William Y. Kim. FGFR inhibition upregulates Nectin-4 expression in FGFR3 altered urothelial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B014.

A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host–pathogen interactions

BackgroundSporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host–pathogen protein–protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host–pathogen protein–protein interactions involved are poorly understood.MethodsTo gain a better understanding of the protein–protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites.ResultsA total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified.Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes.ConclusionPf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.

The diverse dependence of galectin-1 and -8 on multivalency for the modulation of FGFR1 endocytosis

Fibroblast growth factor receptor 1 (FGFR1) is a N-glycosylated cell surface receptor tyrosine kinase, which upon recognition of specific extracellular ligands, fibroblast growth factors (FGFs), initiates an intracellular signaling. FGFR1 signaling ensures homeostasis of cells by fine-tuning essential cellular processes, like differentiation, division, motility and death. FGFR1 activity is coordinated at multiple steps and unbalanced FGFR1 signaling contributes to developmental diseases and cancers. One of the crucial control mechanisms over FGFR1 signaling is receptor endocytosis, which allows for rapid targeting of FGF-activated FGFR1 to lysosomes for degradation and the signal termination. We have recently demonstrated that N-glycans of FGFR1 are recognized by a precise set of extracellular galectins, secreted and intracellular multivalent lectins implicated in a plethora of cellular processes and altered in immune responses and cancers. Specific galectins trigger FGFR1 clustering, resulting in activation of the receptor and in initiation of intracellular signaling cascades that shape the cell physiology. Although some of galectin family members emerged recently as key players in the clathrin-independent endocytosis of specific cargoes, their impact on endocytosis of FGFR1 was largely unknown.Here we assessed the contribution of extracellular galectins to the cellular uptake of FGFR1. We demonstrate that only galectin-1 induces internalization of FGFR1, whereas the majority of galectins predominantly inhibit endocytosis of the receptor. We focused on three representative galectins: galectin-1, -7 and -8 and we demonstrate that although all these galectins directly activate FGFR1 by the receptor crosslinking mechanism, they exert different effects on FGFR1 endocytosis. Galectin-1-mediated internalization of FGFR1 doesn’t require galectin-1 multivalency and occurs via clathrin-mediated endocytosis, resembling in this way the uptake of FGF/FGFR1 complex. In contrast galectin-7 and -8 impede FGFR1 endocytosis, causing stabilization of the receptor on the cell surface and prolonged propagation of the signals. Furthermore, using protein engineering approaches we demonstrate that it is possible to modulate or even fully reverse the endocytic potential of galectins.

Erdafitinib (Balversa) for urothelial carcinoma.

Erdafitinib (Balversa – Janssen), an oral kinase inhibitor, has received full approval from the FDA for treatment of locally advanced or metastatic urothelial carcinoma in adults with susceptible FGFR3 (fibroblast growth factor receptor) genetic alterations who had disease progression on or after at least one prior line of systemic therapy.