FGFR1 Gene Amplification Research Articles

Use of 3′ Rapid Amplification of cDNA Ends (3′ RACE)-Based Targeted RNA Sequencing for Profiling of Druggable Genetic Alterations in Urothelial Carcinomas

Targeted treatment of advanced or metastatic urothelial carcinomas (UCs) requires the identification of druggable mutations. This study describes the development of a 3′ Rapid Amplification of cDNA Ends (3′ RACE)-based targeted RNA sequencing panel which accounts for the status of all genes relevant to UC treatment, namely, FGFR1-4, KRAS, NRAS, BRAF, ERBB2 (HER2), CD274 (PD-L1) and PIK3CA. FGFR2/3-activating point mutations or fusions were found in 54/233 (23.2%) tumors. FGFR3 rearrangements were identified in 11 patients, with eight of them being undetectable by commonly used PCR kits. In addition, one tumor contained a high-copy FGFR2 gene amplification accompanied by strong overexpression of the gene. Mutations in RAS/RAF genes were present in 30/233 (12.9%) UCs and were mutually exclusive with alterations affecting FGFR2/3 genes. On the contrary, activating events in the HER2 oncogene (point mutations and overexpression), as well as PIK3CA mutations, which were relatively common, occurred with similar frequencies in RAS/RAF- or FGFR2/3-positive vs. negative samples. High PD-L1 mRNA expression was associated with advanced disease stage and was not observed in tumors with increased HER2 mRNA expression or in UCs with evidence for FGFR2/3 activation. Three of the studied carcinomas had high-level microsatellite instability (MSI). Overall, more than half of the UCs had potentially druggable genetic alterations. The proposed NGS panel permits comprehensive and cost-efficient analysis of UC-specific molecular targets and may be considered in clinical routine.

Synergic activity of FGFR2 and MEK inhibitors in the treatment of FGFR2-amplified cancers of unknown primary

Patients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96), and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. The tissue-of-origin prediction was obtained from the tumor microRNA expression profile and confirmed by single-cell transcriptomics. Genomic testing and FISH analysis identified FGFR2 gene amplification in both models, in the form of homogenously staining region (HSR) in CUP#55 and double minutes in CUP#96. FGFR2 was recognized as the main oncogenic driver and therapeutic target. FGFR2-targeting drug BGJ-398 (infigratinib) in combination with the MEK inhibitor trametinib proved to be synergic and exceptionally active, both in vitro and in vivo. The effects of the combined treatment by single-cell gene expression analysis revealed a remarkable plasticity of tumor cells and the greater sensitivity of cells with epithelial phenotype. This study brings personalized therapy closer to CUP patients and provides the rationale for FGFR2 and MEK targeting in metastatic tumors with FGFR2 pathway activation.

Abstract PO4-06-07: Efficacy and safety of futibatinib in patients with locally advanced/metastatic triple-negative breast cancer harboring FGFR2 gene amplification: final results from the phase 2, open-label FOENIX-MBC2 study

Abstract Background: Triple-negative breast cancer (TNBC) accounts for ~15% of breast cancers and represents an aggressive subtype with limited targeted therapeutic options. Fibroblast growth factor receptor (FGFR) signaling plays an important role in breast tumorigenesis and metastasis. The phase 2 FOENIX-MBC2 study (NCT04024436) was designed to evaluate responses to futibatinib, a highly selective and potent irreversible covalent inhibitor of FGFR1–4 (FDA-approved for intrahepatic cholangiocarcinoma), in patients with metastatic breast cancer. Here, we report final efficacy and safety data for futibatinib in the cohort of patients with advanced/metastatic TNBC harboring FGFR2 gene amplification. Methods: Eligible patients had locally advanced/metastatic TNBC with measurable disease per RECIST v1.1, an ECOG performance status of 0/1, and progressive disease after ≥1 prior chemotherapy-containing regimen for advanced disease. Patients were positive for FGFR2 gene amplification determined by analysis of tumor tissue or circulating tumor DNA. Patients received oral futibatinib 20 mg once daily until disease progression, unacceptable toxicity, or other discontinuation criteria were met. The primary endpoint was objective response rate (ORR) by RECIST v1.1 in patients with centrally confirmed FGFR2 amplification. Responses (complete response and partial response) required confirmation after at least 4 weeks. Secondary endpoints included the 6-month progression-free survival (PFS) rate, PFS, duration of response (DOR), and overall safety. Results: Overall, 21 female patients with TNBC were enrolled (median age: 53 years) in this cohort. Patients had received a median 5 lines of prior systemic therapy. Overall, 2 patients had a confirmed partial response (ORR: 9.5%; 95% confidence interval [CI]: 1.2, 30.4), with a median DOR of 6.1 months (range: 3.1−9.2 months). Five (23.8%) patients had stable disease. PFS at 6 months was observed in 4 patients (19.0%; 95% CI: 5.4, 41.9) and the median PFS was 1.9 months (95% CI: 1.6, 4.0). Twenty patients (95.2%) had ≥1 treatment-related adverse event (TRAE), including 7 (33.3%) with a Grade 3 TRAE (no Grade 4 or 5). Common TRAEs (any grade) included hyperphosphatemia (85.7% of patients), constipation, diarrhea, fatigue, and dry skin (each in 23.8% of patients). Anemia was the only Grade 3 TRAE observed in more than 1 patient (n=2, 9.5%). Overall, 28.6% of patients had a serious adverse event (SAE; any cause), and 1 patient had an SAE leading to death (ischemic stroke). None of the SAEs or deaths were considered treatment-related. Conclusions: In heavily pretreated patients with metastatic TNBC harboring FGFR2 gene amplification, futibatinib monotherapy demonstrated modest anticancer activity. Futibatinib was tolerable and had an acceptable safety profile, consistent with previous data. Further biomarker work to identify patients who might benefit most from futibatinib is ongoing. Clinical trial information: NCT04024436 Citation Format: Antonio Giordano, Nisha Unni, Senthil Damodaran, Hope Rugo, Timothy Crook, Thomas Bachelot, Federico Piacentini, Hector Soto Parra, Ian Krop, Masashi Shimura, Gareth Tomlinson, Maciej Gil, Nicholas Turner, Fabrice André. Efficacy and safety of futibatinib in patients with locally advanced/metastatic triple-negative breast cancer harboring FGFR2 gene amplification: final results from the phase 2, open-label FOENIX-MBC2 study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-07.

Abstract 6957: Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary

Abstract The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cancer cells release EVs in the extracellular space, where they can interact with recipient cells inducing regulation of gene expression, activation of specific signaling pathways and tumor microenvironment remodeling. We investigated the EVs released by two cell lines of cancer of unknown primary site (CUP) that we established from patient’s tumor cells, namely CUP#55 and CUP#96, as described in https://www.medrxiv.org/content/10.1101/2023.03.12.23287041v1). CUP comprises 3-5% of new cancer cases and presents with metastases of unknown or uncertain origin and no apparent primary tumor. Our CUP cell lines are both characterized by FGFR2 gene amplification. It has been recognized that tumors use circular extrachromosomal DNA (ecDNA) as a way to increase oncogenic amplification. We hypothesized that FGFR2 amplification in CUP cell lines could be associated with ecDNA generation and that this ecDNA could be loaded as cargo inside EVs. FGFR2 copy number was quantified using a probe-based droplet digital PCR assay then we identified the different nature of FGFR2 amplification in the two models using FGFR2 FISH assay: CUP#55 presented a chromosomal homogeneously staining region while CUP#96 displayed double minute chromosomes. FGFR2 DNA was detectable in the cytoplasm fraction, therefore we isolated extracellular vesicles from culture medium using an ultracentrifugation-based protocol and confirmed the presence of the entire length of FGFR2 gene inside small and large vesicles. Since ecDNA contributes to cancer genome remodeling also forming chimeric circles, we confirmed the circular nature of a fraction of FGFR2 ecDNA using the Plasmid-Safe ATP-dependent DNAse test. Finally, we tested the oncogene delivery capability of CUP EVs by administering CUP#96 and CUP#55 small and large vesicles to recipient cell lines. We observed an increase of FGFR2 DNA at 24 hours upon EV treatments, and its functional transcription in FGFR2 mRNA after 48 hours. In conclusion, we identified a model of oncogene amplification and delivery in two cell lines of cancer of unknown primary, which could explain the high metastatic potential of this cancer type. (The research leading to these results has received funding from AIRC under IG 2021 - ID. 25789 project - P.I. Ferracin Manuela) Citation Format: Irene Salamon, Gianluca Storci, Beatrice Fontana, Salvatore Serravalle, Giulia Gallerani, Roberta Roncarati, Spartaco Santi, Massimiliano Bonafè, Manuela Ferracin. Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6957.

Exploration of molecular markers related to chemotherapy efficacy of hepatoid adenocarcinoma of the stomach.

Preoperative neoadjuvant chemotherapy may not improve the prognosis of patients with hepatoid adenocarcinoma of the stomach (HAS), a rare pathological type of gastric cancer. Thus, the study aimed at the genomic and transcriptomic impacts of preoperative chemotherapy on HAS. Patients with HAS who underwent surgical resection at Peking University Cancer Hospital were retrospectively included in this study. Whole exome sequencing and transcriptome sequencing were performed on pre-chemotherapy, non-chemotherapy and post-chemotherapy samples. We then compared the alterations in molecular markers between the post-chemotherapy and non-chemotherapy groups, and between the chemotherapy-effective and chemotherapy-ineffective groups, respectively. A total of 79 tumor samples from 72 patients were collected. Compared to the non-chemotherapy group, the mutation frequencies of several genes were changed after chemotherapy, including TP53. In addition, there was a significant increase in the frequency of frameshift mutations and cytosine transversion to adenine (C > A), appearance of COSMIC signature 6 and 14, and a reduced gene copy number amplification. Interestingly, the same phenomenon was observed in chemotherapy-ineffective patients. In addition, many HAS patients had ERBB2, FGFR2, MET and HGF gene amplification. Moreover, the expression of immune-related genes, especially those related to lymphocyte activation, was down-regulated after chemotherapy. Chemotherapy is closely associated with changes in the molecular characteristics of HAS. After chemotherapy, at genomic and transcriptome level, many features were altered. These changes may be molecular markers of poor chemotherapeutic efficacy and play an important role in chemoresistance in HAS. In addition, ERBB2, FGFR2, MET and HGF gene amplification may be potential therapeutic targets for HAS.

1527P Efficacy and safety of infigratinib in locally advanced or metastatic gastric cancer or gastroesophageal junction adenocarcinoma patients with FGFR2 gene amplification

1527P Efficacy and safety of infigratinib in locally advanced or metastatic gastric cancer or gastroesophageal junction adenocarcinoma patients with FGFR2 gene amplification

Decoding the Conformational Selective Mechanism of FGFR Isoforms: A Comparative Molecular Dynamics Simulation

Fibroblast growth factor receptors (FGFRs) play critical roles in the regulation of cell growth, differentiation, and proliferation. Specifically, FGFR2 gene amplification has been implicated in gastric and breast cancer. Pan-FGFR inhibitors often cause large toxic side effects, and the highly conserved ATP-binding pocket in the FGFR1/2/3 isoforms poses an immense challenge in designing selective FGFR2 inhibitors. Recently, an indazole-based inhibitor has been discovered that can selectively target FGFR2. However, the detailed mechanism involved in selective inhibition remains to be clarified. To this end, we performed extensive molecular dynamics simulations of the apo and inhibitor-bound systems along with multiple analyses, including Markov state models, principal component analysis, a cross-correlation matrix, binding free energy calculation, and community network analysis. Our results indicated that inhibitor binding induced the phosphate-binding loop (P-loop) of FGFR2 to switch from the open to the closed conformation. This effect enhanced extensive hydrophobic FGFR2-inhibitor contacts, contributing to inhibitor selectivity. Moreover, the key conformational intermediate states, dynamics, and driving forces of this transformation were uncovered. Overall, these findings not only provided a structural basis for understanding the closed P-loop conformation for therapeutic potential but also shed light on the design of selective inhibitors for treating specific types of cancer.

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).

FGFR1 Amplification and Response to Neoadjuvant Anti-HER2 Treatment in Early HER2-Positive Breast Cancer

HER2-positive breast cancer (BC) is an aggressive subtype that affects 20–25% of BC patients. For these patients, neoadjuvant therapy is a good option that targets a pathological complete response (pCR) and more breast-conserving surgery. In effect, the outcomes of patients with HER2-positive BC have dramatically improved since the introduction of anti-HER2 antibodies such as trastuzumab (TZ) and/or pertuzumab (PZ) added to chemotherapy. This study sought to examine whether correlation exists between copy number variations (CNVs) in several genes related to the PI3K/AKT pathway (HER2, FGFR1, PIK3CA, AKT3 and MDM2) and the efficacy of anti-HER2 neoadjuvant treatment in patients with early HER2-positive BC. Forty-nine patients received TZ or PZ/TZ and chemotherapy as neoadjuvant treatment. Gene CNVs were determined by quantitative polymerase chain reaction on paraffin-embedded biopsy specimens. The response to 6 months of therapy was assessed by Miller–Payne grading of the tumor on surgical resection; grades 4 and 5, indicating >90% tumor reduction, were defined as a good response. A good response was shown by 64.5% and a pCR by 31.2% of patients. When stratified by anti-HER2 antibody received and gene CNV, it was found that patients with FGFR1 gene amplification or those with FGFR1 amplification treated with TZ alone showed a poor response (p = 0.024 and p = 0.037, respectively). In the subset of patients treated with TZ/PZ combined, the pCR rate was significantly lower among those showing FGFR1 amplification (p = 0.021). Although based on a small sample size, our findings suggest that patients with FGFR1 amplification might benefit less from anti-HER2 antibody therapy.

Predictive role of CCND1, FGFR1 gene amplifications and PIK3CA mutations for endocrine therapy of primary metastatic breast cancer

Background. More than half of breast carcinomas express estrogen and progesterone receptors (ER and PR) and remain estrogen-dependent. Endocrine therapy with inhibitors of the estrogen cascade allows long-term and effective control of the disease in a significant number of patients, however, some patients demonstrate primary, and the majority - secondary resistance to such treatment.
 The aim of the study was to evaluate the predictive significance of the CCND1 and FGFR1 gene amplifications and PIK3CA mutations for endocrine therapy.
 Materials and methods. The study included 138 women with ER-positive primary metastatic breast cancer (BC) who received hormone therapy with aromatase inhibitors (AIs) (n = 69), tamoxifen (n = 65), goserelin (n = 2), or a combination of goserelin and tamoxifen (n = 2) as the first line treatment. CCND1 and FGFR1 gene amplifications were tested by digital droplet PCR, while mutations in exons 7, 9, and 20 of the PIK3CA gene were determined using high-resolution melting analysis and allele-specific PCR. We analyzed the associations between the presence of the mentioned genetic lesions, progression-free-survival (PFS) and response to treatment.
 Results. Amplifications of CCND1 and FGFR1 genes were identified in 24 (17.9%) and 28 (20.9%) of 134 successfully tested cases, respectively; 9 tumors were positive for both alterations. Amplifications were more prevalent in less differentiated tumors (p = 0.018). CCND1 amplification was associated with shorter PFS in patients receiving aromatase inhibitors (16.0 months vs. 32.4 months, HR = 3.16 [95% CI: 1.26-7.93], p = 0.014). FGFR1 status did not significantly affect PFS of AI-treated women, however, partial regress as a result of AI implementation was less frequent in FGFR1-amplified BC as compared to cases with normal FGFR1 copy number (p = 0.031).
 The frequency of PIK3CA mutations was 40.2% (49/122). They were more often observed in smaller tumors (p = 0.034), in PR-positive carcinomas (p = 0.012), and in cases with more extensive metastatic involvement (p = 0.029). The presence of PIK3CA mutations did not affect the results of treatment with AI or tamoxifen.
 Conclusion The presence of CCND1 and/or FGFR1 amplification is associated with worse results of AI therapy of metastatic breast cancer.

Cancer-related FGFR2 overexpression and gene amplification in Japanese patients with gastric cancer

ObjectiveFibroblast growth factor receptor 2 (FGFR2) has been proposed as a novel druggable target in unresectable gastric cancer. FGFR2 alteration has been reported as associated with poor prognosis even in patients with gastric cancer who received systemic chemotherapy. This study aimed to evaluate the frequency of FGFR2 overexpression and gene amplification in clinical specimens from Japanese patients with recurrent or unresectable gastric cancer.MethodsThis observational study enrolled patients who were histologically or cytologically confirmed with unresectable HER2-negative or unknown gastric or gastroesophageal junctional adenocarcinoma treated with at least one previous chemotherapy. FGFR2 overexpression and gene amplification in the specimens were evaluated by immunohistochemical staining and fluorescence in situ hybridization methods, respectively.ResultsIn a total of 173 eligible cases, FGFR2 immunohistochemistry score was evaluated as 0, 1, 2, 3 and 4 for 20, 80, 35, 28 and 10 cases, respectively. In 151 evaluable cases with FGFR2 immunohistochemistry scores of 1–4, FGFR2 copy number expressed as fluorescence in situ hybridization signals were detected as <4, ≥4 < 10 and ≥10 copies for 123, 16 and 12 cases, respectively. FGFR2 copy number showed an increasing tendency along with higher FGFR2 immunohistochemistry scores in the corresponding specimen. The response rate and time to treatment failure for first line chemotherapy did not have any obvious relationship to FGFR2 immunohistochemistry score and FGFR2 copy number.ConclusionsAlthough FGFR2 overexpression and gene amplification were shown in Japanese patients with unresectable gastric cancer, these alterations did not impact the effects of cytotoxic agents as first line chemotherapy.

Abstract 1043: Enhancing cytotoxic chemotherapy effects by nintedanib in gastric cancer preclinical models

Abstract Background: Gastric adenocarcinoma (GAC) remains the 3rd most common cause of cancer mortality in the world. Systemic chemotherapy is a preferred treatment option for advanced and recurrent GAC, but response rates and survival extension remain limited. Tumor angiogenesis plays a critical role in GAC growth, invasion and metastasis. In this study, we determined the antitumor efficacy of nintedanib, a potent triple angiokinase inhibitor for VEGFR-1/2/3, PDGFR-α/β and FGFR-1/2/3, alone or in combination with chemotherapy, in preclinical models of GAC. Methods: Animal survival and tumor growth studies were performed in 4-6 week-old female NOD/SCID mice with human GAC cell xenografts (MKN-45, KATO-III, SNU-5). The mechanistic evaluation involved Immunohistochemistry and Immunoblot analyses of subcutaneous tumors. In vitro cell viability assays were performed using colorimetric WST-1 reagent. Results: In MKN-45 cell-derived peritoneal dissemination xenografts, animal survival was improved by nintedanib (33%), docetaxel (100%) or irinotecan (181%), while oxaliplatin, 5-FU and epirubicin had no effect. The addition of nintedanib to docetaxel (157%) or irinotecan (214%) led to a further extension in animal survival. In KATO-III cell-derived xenografts carrying FGFR2 gene amplification, nintedanib monotherapy extended survival much more (209%). Docetaxel and irinotecan effects were again further enhanced by nintedanib. In MKN-45 subcutaneous xenografts, nintedanib, epirubicin, docetaxel or irinotecan reduced tumor growth (range: 68-87%), while 5-FU and oxaliplatin had a smaller effect (40%). The addition of nintedanib to all chemotherapy groups demonstrated a further reduction in tumor growth. Subcutaneous tumor analysis revealed that nintedanib attenuated tumor cell proliferation, reduced tumor vasculature and increased tumor cell death. Nintedanib (10 μM) inhibited in vitro cell proliferation of mutationally different GAC cells by 25% (MKN-45), 75% (KATO-III) or 82% (SNU-5), and confirmed additive inhibitory effects in combinations with chemotherapy agents. Conclusion: Nintedanib showed notable antitumor efficacy and significantly improved taxane or irinotecan chemotherapy responses. Combination regimens with nintedanib have the potential for improving clinical GAC therapy. Citation Format: Niranjan Awasthi, Margaret A. Schwarz, Frank Hilberg, Roderich E. Schwarz. Enhancing cytotoxic chemotherapy effects by nintedanib in gastric 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 1043.

Genetic Characterization of Cancer of Unknown Primary Using Liquid Biopsy Approaches.

Cancers of unknown primary (CUPs) comprise a heterogeneous group of rare metastatic tumors whose primary site cannot be identified after extensive clinical–pathological investigations. CUP patients are generally treated with empirical chemotherapy and have dismal prognosis. As recently reported, CUP genome presents potentially druggable alterations for which targeted therapies could be proposed. The paucity of tumor tissue, as well as the difficult DNA testing and the lack of dedicated panels for target gene sequencing are further relevant limitations. Here, we propose that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) could be used to identify actionable mutations in CUP patients. Blood was longitudinally collected from two CUP patients. CTCs were isolated with CELLSEARCH® and DEPArrayTM NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with Ampli1TM kits. Circulating cell-free DNA (ccfDNA), purified from plasma at different time points, was tested for tumor mutations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology. In parallel, FFPE tumor tissue was analyzed with three different assays: FoundationOne CDx assay, DEPArray LibPrep and OncoSeek Panel, and the SureSelect custom panel. These approaches identified the same mutations, when the gene was covered by the panel, with the exception of an insertion in APC gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. FGFR2 and CCNE1 gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in ARID1A gene (p.R1276∗) was detected in the tumor tissue and ccfDNAs. The alterations were validated by Droplet Digital PCR in all ccfDNA samples collected during tumor evolution. CTCs from a second patient presented a pattern of recurrent amplifications in ASPM and SEPT9 genes and loss of FANCC. The 92-gene custom panel identified 16 non-synonymous somatic alterations in ccfDNA, including a deletion (I1485Rfs∗19) and a somatic mutation (p. A1487V) in ARID1A gene and a point mutation in FGFR2 gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy testing in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes.

Evidence for frequent concurrent DCUN1D1, FGFR1, BCL9 gene copy number amplification in squamous cell lung cancer

Non-small cell lung cancer (NSCLC) targeted therapies are mostly based on activating mutations and rearrangements which are rare events in Lung Squamous Cell Carcinomas (LUSC). Recently advances in immunotherapy have improved the therapeutic repository for LUSC, but there is still an urgent need for novel targets and biomarkers. We examined 73 cases of LUSC for relative copy number amplification of DCUN1D1, BCL9, FGFR1 and ERBB2 genes and searched for correlations with molecular alterations and clinicopathological characteristics. In our cohort BCL9 gene was amplified in 57.5 % of the cases, followed by DCUN1D1 in 37 %, FGFR1 in 19 % whereas none of the cases were amplified in ERBB2 gene. The majority of the samples exhibited amplification in at least one gene while half of them displayed concurrent amplification of two/three genes. Interestingly, 93 % of the FGFR1 amplified cases were also found co amplified with DCUN1D1 and/or BCL9 genes. Linear correlations were found between BCL9 and DCUN1D1 as well as BCL9 and FGFR1 gene amplification. BCL9 and DCUN1D1 genes’ amplification was correlated with poorly differentiated tumors (p = 0.035 and p = 0.056 respectively), implying their possible role in tumor aggressiveness. This is the first study, to the best of our knowledge that examines the correlation of DCUN1D1 and BCL9 genes relative copy number amplification with molecular alterations and clinicopathologic characteristics of squamous cell lung cancer tissue samples. Our findings show concurrent amplification of genes in different chromosomes, with possible involvement in tumor aggressiveness. These results support the complexity of LUSC tumorigenesis and imply the necessity of multiple biomarkers / targets for a more effective therapeutic result in LUSC.

CCND1 and FGFR1 gene amplifications are associated with reduced benefit from aromatase inhibitors in metastatic breast cancer.

Endocrine therapy is a mainstay for the treatment of hormone receptor-positive breast cancer (BC); however, only a fraction of patients experience a pronounced response to antagonists of estrogen signaling. There is a need to identify predictors for efficacy of this treatment. This study included 138 patients with newly diagnosed metastatic BC, who received upfront endocrine therapy. Archival biopsy specimens were tested for CCND1 and FGFR1 gene amplification and mRNA expression by PCR-based methods. CCND1 and FGFR1 amplification was detected in 24 (17.9%) and 28 (20.9%) of 134 evaluable cases, respectively; 9 carcinomas had concurrent alterations of these two genes. Presence of amplification in at least one locus was more common in tumors of higher grade (p = 0.018) and was associated with higher Ki-67 proliferation index (p = 0.036). CCND1 gene amplification was associated with shorter progression-free survival (PFS) in patients receiving aromatase inhibitors (AI) [16.0months vs. 32.4months, HR = 3.16 (95% CI 1.26-7.93), p = 0.014]. FGFR1 status did not significantly affect PFS of AI-treated women; however, objective response to AI was observed less frequently in FGFR1-amplified BC as compared to cases with normal FGFR1 copy number [2/15 (13.3%) vs. 22/46 (47.8%), p = 0.031]. Meanwhile, CCND1/FGFR1 gene status did not influence the outcome of tamoxifen-treated patients. Presence of CCND1 and/or FGFR1 amplification is associated with worse outcomes of AI therapy in patients with metastatic BC.

Clinical Significance of FGFR1 Gene Abnormalities in Blood Tumors

To study the potential significance and clinical application of FGFR1 gene abnormality in the diagnosis, clinical features, pathological mechanism and treatment in hematological tumors. Clinical data of total of 29 patient with chromosome of 8 short arm (8P) abnormality who had more comprehensive medical history from 2013 to 2018 were collected. The karyotype analysis of bone marrow chromosomes in patients was carried out by using chromosome R band banding technique. FGFR1 gene was detected by using fluorescence in situ hybridization (FISH). Seven cases of FGFR1 gene abnormalities were decteted, including 3 cases of FGFR1 gene amplification, 2 cases of translocation, and 2 cases of deletion. Five patients with FGFR1 gene amplification or deletion not accompaned with eosinophilia, moreover the chromosome was a complex karyotype with poor prognosis; Two cases of FGFR1 gene translocation were non-complex chromosomal translocation and one of which survived for 6 years after bone marrow transplantation, the other chromosome karyotype showed no rearrangement of 8 short arm. However, FGFR1 gene rearrangement was confirmed by FISH analysis, which was a rare insertional translocation. FGFR1 gene amplification or deletion often occur in cases with complex karyotype, which not accompany eosinophilia, moreover have poor prognosis. The patients with FGFR1 gene translocation accompany eosinophilia which is consistent with the clinical characteristics of myeloid / lymphoid neoplasms with FGFR1 abnormality. Karyotype analysis combined with FISH method can improve the detection of abnormal clones.

Genetic alterations in epidermal growth factor receptor-tyrosine kinase inhibitor-naïve non-small cell lung carcinoma.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are an approved first-line therapy against unresectable or advanced non-small cell lung cancer (NSCLC) harboring EGFR gene activating mutations. However, the majority of tumors develop acquired resistance against EGFR-TKIs and some tumors exhibit natural resistance. A number of resistance mechanisms against the latest third-generation EGFR-TKIs have been reported, including tertiary EGFR C797S mutation and several gene alterations activating EGFR or other signaling pathways. The current study aimed to identify the frequency of natural EGFR-TKI resistance in pretreatment NSCLC and to predict the therapeutic effect of EGFR-TKIs. A total of 246 EGFR-TKI-naïve NSCLC patients harboring known EGFR gene mutations were identified. The presence of EGFR C797S and T790M mutations were determined using the peptide nucleic acid-locked nucleic acid PCR clamp method. ERBB2, MET, EGFR, ALK, BRAF, FGFR1, MYC, RET, CCND1, CCND2, CDK4, CDK6, MDM2 and MDM4 gene amplification, which can lead to resistance against any generation EGFR-TKIs, was determined using the multiplex ligation-dependent probe amplification assay. No concurrent C797S mutation with known EGFR mutations were identified. T790M mutation was identified in 12 patients (4.9%). ERBB2 or MET gene amplification was found in some patients (0.0-0.4%). MDM2 gene amplification was associated with tumor recurrence and shorter progression-free survival (PFS) for first- or second-generation EGFR-TKIs. De novo EGFR C797S mutation was not identified. Other resistance mechanisms against EGFR-TKIs were indicated in some patients with EGFR-TKI-naïve NSCLC. MDM2 gene amplification, which can lead to altered cell cycle, was associated with tumor recurrence and shorter PFS in EGFR-TKI therapy.

Abstract OT2-07-01: A phase 2 study of futibatinib (TAS-120) in metastatic breast cancers harboring fibroblast growth factor receptor (FGFR) amplifications (FOENIX-MBC2)

Abstract Background: FGFR1 gene amplifications occur in ~15% of invasive breast cancers (BCs) andhave been shown to be associated with endocrine therapy resistance, whereas FGFR2 amplifications occur in ~2% of invasive BCs (4% of triple-negative BCs [TNBCs]). Futibatinib is an oral, highly selective, irreversible FGFR1-4 inhibitor that has been shown to inhibit both mutant and wild-type FGFR isoforms. In a phase 1 study, futibatinib showed promising clinical activity and tolerability across tumor types, including BC. This phase 2 trial (FOENIX-MBC2) is designed to evaluate futibatinib alone or in combination with fulvestrant (a selective estrogen receptor down-regulator administered intramuscularly) in patients with metastatic BC. Trial design: FOENIX-MBC2 is a multicenter, phase 2, open-label, non-randomized study planned to be conducted in patients with locally advanced/metastatic BC harboring FGFR1/2 amplifications who have experienced disease progression after prior therapy for advanced/metastatic disease. Eligibility criteria include an Eastern Cooperative Oncology Group performance status of 0 or 1 and no prior FGFR inhibitor treatment. Approximately 168 patients are planned to be enrolled in one of four cohorts based on a recurrent BC diagnosis and FGFR amplification status (table) as determined by local testing. Patients will receive single-agent futibatinib (cohorts 1-3) or futibatinib plus fulvestrant (cohort 4) until disease progression, unacceptable toxicity, or other discontinuation criteria are met. Primary and secondary endpoints are detailed in the table. Sample sizes for cohorts 1, 2, and 3 are based on a Simon’s optimal 2-stage design; that for cohort 4 is based on a proof-of-concept phase 2 design. The anticipated start date is August 30, 2019. Patients, treatment, and endpoints in cohorts 1-4 of FOENIX-MBC2 CohortApprox. target enrollment (n)TreatmentKey patient inclusion criteriaEndpointsa1≤55FutibatinibHR+ HER2– BC, measurable disease per RECIST v1.1, FGFR2 amplification, and 1–3 prior endocrine therapies and ≤2 prior chemotherapy regimens for advanced/metastatic diseasePrimary: ORR Secondary: CBR, DOR, OS, PFS, 6-month PFS rate, safety2≤55FutibatinibTNBC, measurable disease per RECIST v1.1, FGFR2 amplification, and ≥1 prior chemotherapy or chemotherapy/immunotherapy regimen for advanced/metastatic diseasePrimary: ORR Secondary: CBR, DOR, OS, PFS, 6-month PFS rate, safety3≤24FutibatinibHR+ HER2– BC or TNBC; non-measurable, evaluable disease; FGFR2 amplification; and prior therapy as per cohort 1 (HR+ HER2– BC) or cohort 2 (TNBC)Primary: CBR Secondary: CR rate, DOR, OS, PFS, 6-month PFS rate, safety4≤34Futibatinib + fulvestrantHR+ HER2– BC, measurable disease per RECIST v1.1, high levels of FGFR1 amplification (FGFR1:CEN8 ratio ≥5.0 or FGFR1 copy number ≥10), and 1–2 prior endocrine-containing regimens and ≤1 prior chemotherapy regimen for advanced/metastatic disease, but no prior fulvestrantPrimary: 6-month PFS rate Secondary: ORR, CBR, DOR, OS, PFS, safetyCBR, clinical benefit rate; CR, complete response; DOR, duration of response; HER2-, human epidermal growth factor-negative; HR+, hormone-receptor-positive; ORR, objective response rate; OS, overall survival; PFS, progression-free survival; RECIST v1.1, Response Evaluation Criteria in Solid Tumors version 1.1aORR is defined as the proportion of all treated patients with a best overall response of CR or PR; CBR is defined as the proportion of all treated patients with a best overall response of CR, PR, or SD ≥24 weeks. ORR and CBR will be summarized descriptively. The 6-month PFS rate is the percentage of patients who remain alive and progression-free at 6 months estimated using the Kaplan-Meier method. This study is funded by Taiho Oncology, Inc. and Taiho Pharmaceutical Co., Ltd. Citation Format: Nick C. Turner, Ian E. Krop, Aditya Bardia, Senthil Damodaran, Miguel Martin, Karim A. Benhadji, Yaohua He, Mieke Ptaszynski, Carlos L. Arteaga. A phase 2 study of futibatinib (TAS-120) in metastatic breast cancers harboring fibroblast growth factor receptor (FGFR) amplifications (FOENIX-MBC2) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT2-07-01.

Expansion part of phase I study of E7090 in patients with cholangiocarcinoma harboring FGFR2 gene fusion and with gastric cancer harboring FGFR2 gene amplification or FGFR2 protein high expression.

538 Background: E7090 is a selective tyrosine kinase inhibitor against FGFR1-3. This first-in-human phase I study has been conducted in Japan and consists of 2 parts. Based on the toxic, pharmacokinetic, and pharmacodynamic profiles in Part 1, dose escalation study, the recommended dose was determined to be 140 mg QD for Part 2, an expansion study part restricted to patients with tumors harboring FGFR gene alterations. Here we provide the interim analysis results of Part 2. Methods: In Part 2, patients with cholangiocarcinoma harboring FGFR2 gene fusion (CCA cohort) and gastric cancer harboring FGFR2 gene amplification or FGFR2 protein high expression (GC cohort) were enrolled. Patients received oral dosing of E7090 until disease progression or unacceptable toxicity. Safety was assessed using CTCAE version 4.03. Tumor response was evaluated by site investigators using RECIST 1.1. Results: As of 31 March 2019, 16 patients including 6 patients in CCA cohort and 10 patients in GC cohort received E7090 in Part2. Median age was 63 years, 25% were female, ECOG PS of 0 and 1 were 50% respectively. 5 patients (83%) in CCA cohort and 1 patient (10%) in GC cohort achieved partial response as the best overall response. The disease control rates were 100% in CCA cohort and 30% in GC cohort, respectively. Median progression-free survivals were 8.3 months in CCA cohort and 2.8 months in GC cohort at the cut-off date. 2 patients with CCA remain on treatment. The most common treatment-related TEAEs occurring in 30% or more of patients in Part 2 were hyperphosphatemia (100%), palmar–plantar erythrodysesthesia syndrome (56%), paronychia (50%), dysgeusia (38%), stomatitis (31%), diarrhea (31%), increased AST (31%) and blood creatinine increased (31%). Grade≥3 treatment-related TEAEs were reported in 2 out of 16 patients (13%); they were increased AST, lipase increased and retinopathy. Conclusions: This study indicated that E7090 has a manageable safety profile in Part 2 and the promising clinical activity in CCA patients with FGFR2 gene fusion.

Abstract A033: Evaluation of FGFR2b in context of relevant biomarkers supports potential combination of anti-FGFR2b with anti-PD-(L)1 therapy in gastric cancer

Abstract Background: Gastric cancer is the third leading cause of global cancer-related death, with 782,685 deaths in 2018 (Jemal, A. et al. 2018). Currently available treatments have limited benefits. Hence there’s a pressing need for more effective treatment strategies. FGFR2 gene amplification or protein overexpression in gastric cancer tumors is associated with poor prognosis (Su et al., 2014, Ahn et al., 2016). Five Prime has developed an FGFR2b-specific first-in-class humanized monoclonal IgG1 antibody, bemarituzumab (FPA144, or bema), that selectively blocks FGFR2b and triggers antibody-dependent cell-mediated cytotoxicity that showed monotherapy efficacy in a phase 1 trial (Lee et al. ASCO 2016, Catenacci et al. ASCO 2017). In this study, we evaluated the expression of FGFR2b in the context of relevant biomarkers for other approved and potential drug combinations. Methods: RNA and IHC analysis were performed using public datasets and procured tumor samples, respectively. 97 tissue samples from gastric cancer patients were assessed for expression of FGFR2b, PD-(L)1, and CD8 by IHC. In addition, TCGA-STAD database was evaluated for the status of FGFR2 overexpression and the expression of relevant targeted combination drug partners, e.g., ERBB2, PD-(L)1 and cytolytic score. For PD-(L)1 and CD8 staining, SP263 and SP57 antibodies were used, and results were reported as percent positive cells. For FGFR2b staining, the FPR2-D antibody was used (Kim, KM. et al. 2016). Staining with intensity greater than 2+ was considered FGFR2b-positive and anything less than or equal to 2 was considered negative for FGFR2b overexpression. We also investigated the literature and past and ongoing clinical trials to assess PD-(L)1 prevalence in gastric cancer and its correlation to efficacy. Results: Based on RNA expression and copy number data from samples in the TCGA-STAD dataset, combination of bemarituzumab and HER2 targeting agents would not be expected to have synergistic activity, as both populations appear mutually exclusive. In contrast, FGFR2 positive/overexpressing tumors showed good representation of high RNA expression levels of PD-(L)1, PD-1 and cytolytic score. Clinical studies using anti-PD-(L)1 therapies such as pembrolizumab indicate that most benefit (higher ORR) was observed in patients that express PD-(L)1 above a predefined threshold, measured either as a combined positive score of immune and tumor cell staining, or tumor cell staining alone (&amp;gt;1 as cutoff). In our dataset, we calculated % positive PD-(L)1 for both tumor cells and immune cells. Both FGFR2b+ and FGFR2b- tumors showed similar distribution of CD8 and PD-(L)1 positive cells. 60% of FGFR2b+ tumors were PD-(L)1 positive using a greater than 1% PD-(L)1 (immune cell + tumor cell) cut off. Conclusion: In our dataset, 60% of FGR2b+ population is also PD-(L)1 positive (&amp;gt;1%) suggesting that a combination of bemarituzumab with checkpoint inhibitors such as anti-PD-(L)1 could potentially benefit patients with FGFR2b+ tumors. Further mechanistic insight and support comes from preclinical studies (Pierce et al. AACR 2016), which showed an increase in efficacy with the combination treatment of bemarituzumab and anti-PD1 in an orthotopic mouse model. Future studies will be performed to evaluate and get a better understanding of the underlying mechanism of action of combination activity, and confirm co-expression in a larger, confirmatory dataset. Citation Format: Khushboo Sharma, Maike Schmidt, Kevin Hestir, Nerissa Mendoza, Janine Powers, James Hnatyszyn. Evaluation of FGFR2b in context of relevant biomarkers supports potential combination of anti-FGFR2b with anti-PD-(L)1 therapy in gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A033. doi:10.1158/1535-7163.TARG-19-A033