Targeting FGFR in non-small cell lung cancer: implications from the landscape of clinically actionable aberrations of FGFR kinases

Objective: Dysfunction in fibroblast growth factor receptor (FGFR) signaling has been reported in diverse cancer types, including non-small cell lung cancer (NSCLC). The frequency of FGFR aberrations in Chinese NSCLC patients is therefore of great clinical significance. Methods: A total of 10,966 NSCLC patients whose tumor specimen and/or circulating cell-free DNA (cfDNA) underwent hybridization capture-based next-generation sequencing were reviewed. Patients’ clinical characteristics and treatment histories were also evaluated. Results: FGFR aberrations, including mutations, fusions, and gene amplifications, were detected in 1.9% (210/10,966) of the population. FGFR abnormalities were more frequently observed in lung squamous cell carcinomas (6.8%, 65/954) than lung adenocarcinomas (1.3%, 128/9,596). FGFR oncogenic mutations were identified in 19 patients (∼0.17%), of which, 68% were male lung squamous cell carcinoma patients. Eleven out of the 19 patients (58%) had concurrent altered PI3K signaling, thus highlighting a potential combination therapeutic strategy of dual-targeting FGFR and PI3K signaling in such patients. Furthermore, FGFR fusions retaining the intact kinase domain were identified in 12 patients (0.11%), including 9 FGFR3-TACC3, 1 FGFR2-INA, 1 novel FGFR4-RAPGEFL1, and 1 novel fusion between the FGFR1 and SLC20A2 5′-untranslated regions, which may have caused FGFR1 overexpressions. Concomitant EGFR mutations or amplifications were observed in 6 patients, and 4 patients received anti-EGFR inhibitors, in whom FGFR fusions may have mediated resistance to anti-EGFR therapies. FGFR amplification was detected in 24 patients, with the majority being FGFR1 amplifications. Importantly, FGFR oncogenic mutations, fusions, and gene amplifications were almost always mutually exclusive events. Conclusions: We report the prevalence of FGFR anomalies in a large NSCLC population, including mutations, gene amplifications, and novel FGFR fusions.


Introduction
The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling pathway plays important roles in a variety of biological processes, including development, differentiation, cell proliferation, migration, angiogenesis, and carcinogenesis via several intracellular pathways, including the Ras/ Raf/MEK and the phosphatidylinositol 3-kinase (PI3K)-AKT pathways 1 . The FGF family contains 22 members, which are usually divided into 7 subfamilies according to their sequence similarities, biochemical functions, and evolutionary relationships 2 . All 4 FGFRs, including FGFR1, FGFR2, FGFR3, and FGFR4 are structurally homologous to vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptor (PDGFR), and other tyrosine kinase receptors 3 , and represent therapeutic targets of great potential.
Previous studies have shown that FGFR2/3 gene alterations, including FGFR3 activating mutations that affect either the extracellular (R248C and S249C) or transmembrane (G370C, S371C, Y373C, and G380R) domains of the protein, and gene fusions such as FGFR3-TACC3, are common in patients with urothelial carcinoma and cause constitutively activated FGF signaling, resulting in carcinogenesis 4 . Multiple FGFR inhibitors 5 , including erdafitinib 6,7 have shown antitumor activities in preclinical models and in early phase clinical trials involving patients with FGFR alterations. A recent study by Loriot et al. 8 reported that the use of erdafitinib was associated with an objective tumor response in 40% of previously treated patients who had locally advanced and unresectable or metastatic FGFR alteration-positive urothelial carcinomas. Such findings were superior to prior observations of an objective response rate of approximately 10% using second-line, single agent chemotherapy in an advanced urothelial carcinoma population [9][10][11] . Activation of FGF signaling has also been described in lung cancer, including non-small cell lung cancer (NSCLC). As previously described, the incidence of FGFR alterations, particularly FGFR1 amplification, was higher in squamous cell carcinoma (SCC) of the lung than in adenocarcinoma 12 . Moreover, FGFR2 mutations were also reported in NSCLC patients, including the extracellular domain mutations, W290C and S320C, and the kinase domain mutation, K660E/N 13 . In this study, we investigated the landscape of FGFR aberrations in a large Chinese NSCLC population by comprehensive genomic profiling using next-generation sequencing (NGS), to identify potential therapeutic options for FGFR-mutated NSCLC patients.

Materials and methods Patients
A total of 15,150 consecutive clinical lung cancer patients were analyzed using comprehensive genomic profiling targeting 400+ cancer-relevant genes, including all the exons of FGFR genes (FGFR1-4), as well as flanking intronic regions, and other introns selected by a Clinical Laboratory Improvement Amendments-certified, and College of American Pathologistsaccredited laboratory (Nanjing Geneseeq Technology, Jiangsu, China), as previously described 14 . We identified patients with FGFR alterations using a natural language search tool in the laboratory information management system database. Relevant demographic and clinical data were extracted from the database, including age, gender, date of diagnosis, histology, pathological stage, and evaluation of treatment response based on reports by clinical investigators.
For tumor tissue samples, the pathological diagnosis and tumor content of each case was confirmed by pathologists. Peripheral blood (8-10 mL) was collected in EDTA-coated tubes (BD Biosciences, San Jose, CA, USA) and centrifuged at 1,800 × g for 10 min within 2 h of collection to isolate the plasma for circulating tumor DNA (ctDNA) extraction, and white blood cells for genomic DNA extraction as the germline control.

DNA extraction and targeted enrichment
The ctDNA from plasma was purified using a Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Genomic DNA from white blood cells was extracted using the DNeasy Blood and Tissue Kit (Qiagen), while genomic DNA from formalin-fixed paraffin-embedded (FFPE) samples was purified using the QIAamp DNA FFPE Tissue Kit (Qiagen). All DNA was quantified using the dsDNA HS Assay Kit using a Qubit Fluorometer (Life Technologies, Carlsbad, CA, USA). Sequencing libraries were prepared using the KAPA Hyper Prep Kit (Roche, Basel, Switzerland), as described previously 14 . Indexed DNA libraries were pooled for probe-based hybridization capture of the targeted gene regions covering over 400 cancer-related genes for all solid tumors; all of which contained all exons of FGFR genes and selected introns for the detection of FGFR fusions.

Sequencing data processing
Sequencing was performed using the Illumina HiSeq4000 platform (Illumina, San Diego, CA, USA), followed by data analysis as previously described 15 . In brief, sequencing data were analyzed by Trimmomatic 16 to remove low quality (quality < 15) or n bases, and were then mapped to the human reference genome, hg19, using the Burrows-Wheeler Aligner (https://github.com/lh3/bwa/tree/master/bwakit). PCR duplicates were removed by Picard (https://broadinstitute.github. io/picard/). The Genome Analysis Toolkit (GATK) (https:// software.broadinstitute.org/gatk/) was used to perform local realignments around indels and for base quality reassurance. Single nucleotide polymorphisms (SNPs) and indels were analyzed by VarScan2 17 and HaplotypeCaller/UnifiedGenotyper in GATK, with the mutant allele frequency cutoff at 0.5% for tissue samples, 0.1% for cfDNA samples, and a minimum of three unique mutant reads. Common SNPs were excluded if they were present in > 1% population frequency in the 1,000 Genomes Project or the Exome Aggregation Consortium (ExAC) 65,000 exome database. The resulting mutation list was further filtered using an in-house list of recurrent artifacts based on a normal pool of whole blood samples. Gene fusions were identified by FACTERA 18 .

Ethical approval
The study was approved by the Ethics Committee of Guangdong General Hospital, China (Approval No. GDREC2016262H). Shanghai Chest Hospital served as one of the hospitals participating in the research project. The study was conducted in accordance with the tenets of the Declaration of Helsinki, and written informed consent was collected from each patient prior to sample collection.

The incidence of FGFR aberrations in NSCLC patients
From December 2016 to February 2019, a total of 15,150 individual clinical lung cancers were successfully evaluated by comprehensive genomic profiling using hybrid capture-based NGS. This work was based on the validated dataset for a total of 10,966 patients in our database system. Lung cancer tumor samples and liquid biopsies, if applicable, were compared to matched normal whole blood controls. A total of 87% of NSCLC samples examined were lung adenocarcinomas [lung adenocarcinoma (LUAC), n = 9,596], 9% were lung squamous cell carcinoma (LUSC, n = 954), and the remainder (4%) were of either mixed adenocarcinomas and squamous cell carcinomas or were missing sub-histological information in the database. Approximately 40% of the entire study population had only liquid biopsy specimens for genetic testing. A total of 210 patients (1.9%, 210/10,966) were identified with somatic aberrations of FGFRs (FGFR1-4), including mutations, gene rearrangements, and gene amplifications ( Figure 1A). Fifty-one patients (roughly 24%) had liquid biopsy samples including only plasma and pleural effusion samples. The median age of the cohort was 62 years of age (range: 34-84 years of age). Approximately 72% (152/210) of the patients were male. Approximately 61% of FGFR-positive patients were LUAC (n = 128), 31% were LUSC (n = 65), and the remaining 7 cases were of either mixed or unknown histology. Thus, FGFR alterations were more frequent in LUSC patients (6.8%, 65/954) than in LUAC patients (1.3%, 128/9,596). The majority of the FGFR aberrations were gene mutations (75%) with gene amplification and gene rearrangements being observed in similar frequencies (10% and 15%, respectively) ( Figure 1A). FGFR1 alterations were slightly more abundant than alterations in FGFR2-4 ( Figure 1B). Notably, we observed more amplification events in FGFR1s than in other FGFRs, and over 90% of FGFR4 alterations were mutations ( Figure 1C).

The identification of novel FGFR fusions in NSCLC patients
FGFR fusions retaining the intact kinase domain were identified in 0.11% (12/10,966) of NSCLC patients examined ( Figure 1D and Mutation Gene amplification Gene rearrangment   9/12) were positive for FGFR3-transforming acidic coiled-coil containing protein 3 gene (TACC3) fusions (FGFR3-TACC3), which were mostly reported in solid tumors 20 . Four of the 9 (45%) patients with FGFR3-TACC3 fusions had 5′ breakpoints in FGFR3 exon 17 and the remaining 55% were in exon 18, while TACC3 exons 10 and 11 were the most common 3′ breakpoint locations (Figure 2A). We observed 1 case of FGFR3 exon 17 fused to TACC3 exon 14 that may have resulted in a fusion protein with compromised dimerization capacity due to a truncated coiled-coil domain (Figure 2A). We also observed 1 gene rearrangement event involving  Figure 2B). The FGFR2-INA fusion was previously reported in low grade gliomas that drove oncogenesis via MAPK and PI3K/mTOR pathway activation 21 . Our observations represented the first case of a FGFR2-INA fusion in NSCLC, in particular, lung adenocarcinoma. Furthermore, 1 gene fusion event involving fibroblast growth factor receptor 4 (FGFR4) and the Rap guanine nucleotide exchange factor like 1 gene (RAPGEFL1) (FGFR4 F17: RAPGEFL1 R4) was detected in a lung adenocarcinoma patient (P26) (Figure 2C), which has not been previously documented, and therefore further validation of its function is necessary in future research. Notably, a concurrent activating EGFR ex19del mutation was also detected at an allele frequency of 21.71% in this patient. In addition, we observed 1 patient with a 5′-untranslated region of the Solute Carrier Family 20 Member 2 gene (SLC20A2) fused to FGFR1 exon 17 ( Figure 2D).
Of note, concomitant EGFR mutations or EGFR amplifications were observed in 6 of the 12 FGFR fusion patients  ( Table 2), 4 of which were previously treated with EGFR TKIs, but the disease had progressed prior to NGS tests. Although half the patients (n = 2) did not have pretreatment samples, the remaining 2 patients (P16 and P17) likely acquired FGFR fusions as alternative mechanisms to combat the anti-tumor activity of EGFR TKIs ( Table 2). Furthermore, a concurrent PIK3CA H1047R mutation was observed in 1 patient (P22) and may also have acted as a mechanism of acquired resistance to prior therapies including TKIs ( Table 2). No other known dominant driver mutations were detected in the remaining 6 patients ( Table 2).

Amplification of the FGF19 and FGFR genes in NSCLC patients
As previously mentioned, we observed more amplification events in FGFR1 than other FGFRs (Figure 1B). FGFR amplification was detected in a total of 24 patients, a majority of which (87.5%, 21/24) were FGFR1 amplifications ( Figure 1D). Similarly, the majority of FGFR-amplified patients (67%) were LUSC and 92% were male (Table 3). Notably, 25 patients (12%, 25/210) had multiple alterations in FGFR genes, but oncogenic FGFR mutations, fusions, or gene amplifications were almost mutually exclusive events, with the exception that 4 FGFR3-mutant patients had concurrent FGFR1 amplifications ( Figure 1D). Two patients had concurrent EGFR activating mutations and received prior EGFR-TKI treatments. However, no pretreatment samples were available for mutation profiling for these patients. The remaining patients (92%, 22/24) had no other dominant driver mutations and were either chemotherapy-refractory or treatment naïve ( Table 3).
We also identified 9 patients (0.08%, 9/10,966) who had amplifications of FGF19 (Figure 1D), which encodes a unique, high affinity ligand that specifically binds to FGFR4 in a heparin-dependent manner. Our observations were consistent with previous studies reporting on the role of the FGF19-FGFR4 signaling axis in human cancers, including hepatocellular carcinoma 22 and lung squamous cell carcinoma 23 . Two patients had concomitant aberrations of the PI3K signaling pathway, including PIK3CA amplification and the PIK3R2 G373R missense mutation ( Table 3). All patients were either chemotherapy-refractory or treatment naïve.

Discussion
This study represented the first comprehensive survey of FGFR aberrations in a large population of Chinese patients with NSCLC. Approximately 1.9% of the population had FGFR aberrations, including point mutations, gene rearrangements, and amplifications, with the most common abnormality being FGFR point mutations. The prevalence of FGFR alterations in this Chinese NSCLC population was relatively lower than that of a prior study (5.7%), as reported by Helsten et al. 24 in which the study population was unlikely to be only Chinese. Currently, there are a number of FGFR inhibitors approved by the Federal Drug Administration (FDA), including ponatinib, regorafenib, pazopanib, lenvatinib, and nintedanib, which were included in a trial specifically targeting NSCLC patients 25 . All these FGFR inhibitors are multi-kinase inhibitors that also exhibit nonspecific anti-tumor activities against other tyrosine kinases, including VEGFR, PDGFR, ROS1, and/ or RET. However, there are also specific FGFR inhibitors in clinical development. Notably, erdafitinib, a functionally selective pan-FGFR inhibitor, has been approved by the FDA to treat advanced metastatic urothelial cancers 6,8 . Different FGFR abnormalities responded differently to erdafitinib, with the highest response rate seen for patients with FGFR point mutations 8 . Another selective FGFR inhibitor, pemigatinib, was also recently granted accelerated approval for treatment of late stage FGFR2+ cholangiocarcinoma patients 26 . It is definitely of great clinical interest to study these FGFR inhibitors in NSCLC patients, so future trials may be warranted.
Unlike lung adenocarcinomas, no targeted molecular therapies have been developed for squamous cell lung cancers because targetable oncogenic aberrations are scarce in this tumor type. Here, we report that FGFR aberrations were present in approximately 6.8% of the LUSC cohort of this study, which was higher than the frequency (1.3%) in LUAC patients. Notably, over 75% of FGFR1 amplification events were observed in LUSC patients, which is consistent with previous findings 24,27 . More than half of the patients who carried FGFR activating/transforming mutations had concurrent dominant mutations in PI3K pathway genes, including PIK3CA and PIK3R2, consistent with previous reports [28][29][30] . Furthermore, we reported the overlapping of activated FGFR genes and genetic alterations of the PI3K pathway in NSCLC, including both LUAC and LUSC. A prior study by Packer et al. 31 revealed that PI3K inhibitors enhanced the anti-tumor efficacies of anti-FGFR inhibitors in vitro in endometrial cancers in which the activation of the PI3K pathway was observed in > 90% of FGFR2-mutated cases. The activation of the PI3K pathway was also reported to be enriched in breast cancer patients with activated FGFR/FGF signaling 32 . Together, our findings  highlighted an intriguing molecular feature and potential therapeutic target for combination therapies targeting the FGFR and PI3K pathways in FGFR-positive NSCLC patients exhibiting activated PI3K and MAPK pathways. Furthermore, we identified a total of 12 FGFR gene rearrangements in the NSCLC population that maintained intact FGFR kinase domains. FGFR fusions did not segregate well by histology or sex, as was previously reported by Wang et al. 33 which was likely due to the restricted cohort size. The majority of these patients were FGFR3-TACC3 positive, but we also observed 1 case of a FGFR2-INA fusion that was originally described in gliomas, and 2 novel FGFR fusions, including SLC20A2-FGFR1 and FGFR4-GAPGEFL1. A prior study by Wu et al. 34 reported a case of prostate cancer with the SLC45A3 non-coding exon 1 fused to the intact coding region of FGFR2, in which the SLC45A3-FGFR2 fusion was predicted to drive the overexpression of wildtype FGFR2. Thus, the SLC20A2-FGFR1 fusion observed in the current study may also have been able to drive the overexpression of wildtype FGFR1, although additional studies are needed to test this possibility. It is worth noting that half (n = 6) of the FGFR fusion patients carried EGFR aberrations, including EGFR ex19del, T790M, C797S, and EGFR amplifications. Two-thirds of those patients received prior EGFR TKI therapies. Reminiscent of a prior report by Ou et al. 35 , this observation suggested that FGFR fusions may act as a mechanism of acquired resistance to EGFR inhibitors in patients (P16, P17, P21, and P22) who were previously treated with EGFR TKIs.
Aside from point mutations and gene rearrangements, approximately 15% of all FGFR aberrations were amplifications, with FGFR1 amplifications being the most common anomalies. FGFR amplifications predominated in LUSC patients at a prevalence of 1.6%, in contrast to that of < 0.1% in the LUAC population. These frequencies were relatively lower than those reported by Helsten et al. 24 (9% and 4%, respectively), which could be attributed to a number of reasons including the ethnic differences underlying these two study populations, the restricted NSCLC cohort size of Helsten et al., as well as the inclusion of cases who had only liquid biopsy ctDNA samples in this work.
Previous studies have shown that FGFR1 amplification was common in breast cancer patients with early relapses and poor clinical outcomes 36 . Therefore, antibodies targeting FGFR represent a valid therapeutic strategy to treat breast cancer or other cancer histologies, including NSCLC. In addition, we also observed a low frequency of FGF19 amplifications in our NSCLC population. FGF19 encodes the ligand for FGFR4, and it was previously shown that FGF19 amplifications corresponded with constitutive activation of FGF receptor 4 (FGFR4)-dependent ERK/AKT-p70S6K-S6 signaling activation in head and neck squamous carcinoma cells 37 ; thus, raising the question as to whether the FGF19/FGFR4 axis also acts as an oncogenic driver in these NSCLC patients and represents a therapeutic target.

Conclusions
This study reported the frequency of FGFR aberrations, including activating mutations, gene rearrangements, and gene amplifications in a large population of Chinese NSCLC patients, and revealed the potential clinical utility of targeting FGFR aberrations with FGFR inhibitors in NSCLC patients. We also reported novel FGFR fusion events in NSCLC patients, including SLC20A2-FGFR1, FGFR2-INA, and FGFR4-GAPGEFL1; thus, highlighting potential therapeutic targets for the management of such patients.