Prognostic implications of Fibroblast growth factor receptor 1 (FGFR1) gene amplification and protein overexpression in hypopharyngeal and laryngeal squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) arises from the squamous mucosa of the upper aerodigestive tract, which comprises the nasal cavity, paranasal sinus, oral cavity, nasopharynx, oropharynx, hypopharynx, and larynx. These distinct anatomic subsites contribute to the morphological, biological, and etiological heterogeneities of HNSCC. Cigarette smoking and alcohol consumption are the most common risk factors, especially for hypopharyngeal and laryngeal SCC [1, 2], while infection with high-risk human papilloma virus (HPV) has also been recognized as a risk factor associated with oropharyngeal SCC [3]. Recent studies have shown that HPV-positive SCC, mainly oropharyngeal SCC, exhibits significantly favorable survival outcomes [4‐6]. However, SCCs of the hypopharynx and larynx, the second most common respiratory tract cancer after lung cancer, that represent HPV-negative HNSCCs are related to little improvement in patient outcomes despite multidisciplinary treatments because of frequent locoregional recurrences, distant metastases, and second primary tumors [7, 8].

The Cancer Genome Atlas (TCGA) has recently identified potentially targetable somatic genomic alterations based on HPV infection status, smoking, and primary tumor sites in HNSCC [9]. HPV-negative tumors are characterized with recurrent focal amplifications in receptor tyrosine kinases and fibroblast growth factor receptor 1 (FGFR1; 10% frequency) is the second most commonly observed gene after EGFR (15% frequency). The gene encoding FGFR1 is located on chromosome 8p11.23 and encodes tyrosine kinase family, which plays crucial roles in cancer development. This gene is dysregulated by amplification, point mutation, translocation, and overexpression in various cancers [10]. These aberrant FGFR1 alterations, in general, lead to gain-of-function characteristics and constitutively activate the downstream RAS/mitogen-activated protein kinase (MAPK), PI3K/protein kinase B (AKT), and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways [11]. In previous studies of HNSCC, FGFR1 amplification has been reported in 3 to 17% of cases, and FGFR1 protein overexpression has been identified in about 11–82% of cases [12‐18]. However, they presented conflicting results for FGFR1 as a prognostic biomarker. In addition, most of the studies have been conducted on the whole HNSCCs showing biological heterogeneity, and site-specific studies have been rarely performed on SCC, especially in SCCs of hypopharynx and larynx, which represent the prevalent subsites of HPV-negative SCC [13‐19]. Therefore, more evidence is needed for the prognostic or predictive role of FGFR1 in HNSCC of hypopharynx and larynx.

As a predictive marker for drug response, FGFR1 has been identified in preclinical or clinical studies of lung SCC or breast cancer [19‐21]. Recently, several nonselective or selective tyrosine kinase inhibitors suppressing FGFR1 expression, such as lucitanib (E3810), dovitinib (TKI258), ponatinib (AP24534), AZD4547, BGJ398, and TAS-120, have shown promising data or are currently being investigated in preclinical models and clinical trials on solid tumors, including HNSCC (NCT02706691, NCT02795156) [22, 23]. However, effective targeted therapies for advanced HNSCC are still limited to the anti-EGFR monoclonal antibody, cetuximab, in HNSCC [24].

In this study, we evaluated FGFR1 gene amplification and protein overexpression and investigated its clinicopathologic and prognostic implications in hypopharyngeal and laryngeal SCC.

In the present study, we investigated the clinicopathologic and prognostic implications of FGFR1 gene amplification and protein overexpression in hypopharyngeal and laryngeal SCC. We analyzed a large number of cases that underwent standard management of curative surgery and appropriate adjuvant therapy in two different institutes.

In our series, hypopharyngeal and laryngeal SCCs were generally detected in late adulthood, and frequently related to smoking and alcohol consumption and p16 IHC negativity. Furthermore, advanced tumor stages, especially advanced pathologic lymph node stages, were not uncommon, and the 5-year DFS and OS rates were 74.1 and 67.6%, respectively, indicating that a proportion of patients with this disease exhibited unfavorable outcomes. These demographic features were generally similar to those previously reported and our series may be used as a surrogate of the population-based data [30, 31]. Hypopharyngeal SCCs showed more lymph node metastasis compared to laryngeal SCC in our study and they were expected to have a worse prognosis than laryngeal SCC. However, in Kaplan-Meier analysis and multivariate Cox regression model, the difference between OS and DFS according to larynx and hypopharynx was not statistically significant. Therefore, we think combining these two groups would not cause serious selection bias, but the concept of non-HPV related SCC can be approached.

FGFR1 gene amplification was observed in about 12% tested hypopharyngeal and laryngeal SCC cases that was slightly more frequent in hypopharyngeal SCC (14.3%) than in laryngeal SCC (8.3%). FGFR1 amplification has been suggested as an oncogenic driver mutation in tobacco-associated cancers of the aerodigestive tract [16, 32‐34]. Previous studies on FGFR1 in HNSCC have shown that FGFR1 amplification is more common in the SCC of hypopharynx and larynx than in that of oropharynx or oral cavity [14, 16]. In addition, FGFR1 amplification has no relationship with HPV infection [13]. All these findings are suggestive of the biological role of FGFR1 amplification in the tumorigenesis of these tobacco or alcohol-related cancers, hypopharyngeal and laryngeal SCCs, and may predict the role of targeted therapy for these tumors [30, 31].

Considering the clinical implications of FGFR1 amplification, we observed significant association with poor prognostic factors, specifically, lymphovascular invasion and advanced stages of lymph node metastasis. This observation may be related to our findings that cases with FGFR1 amplification were closely associated with advanced TNM tumor stages and poorer DFS. In particular, FGFR1 amplification was determined as an independent factor for disease progression and thus, may be involved in the invasion, metastasis, and drug resistance of tumor cells during the development of treatment-resistant, aggressive, advanced hypopharyngeal and laryngeal SCCs. Several studies have also shown that FGFR1 amplification plays a role in the invasion, metastasis, and drug resistance of various tumors [32, 35]. A recent study performed genomic profiling of HNSCC using targeted next-generation sequencing and identified FGFR1 amplification as an independent prognostic factor for OS [14], while it has been failed to impact on prognosis in other studies [15, 16, 36]. In our study, we failed to detect any association between FGFR1 amplification and OS. Cases positive for FGFR1 amplification were relatively fewer in number in previous and present studies; therefore, its prognostic role warrants validation in future studies, including meta-analysis.

In our series, high FGFR1 expression was observed in about 11% of tested cases with hypopharynx and larynx SCCs. Similar to FGFR1 amplification, FGFR1 protein overexpression was associated with lymph node metastasis and advanced TNM tumor stages. Considering the marginal association between high FGFR1 expression and FGFR1 amplification, gene amplification and the subsequent protein overexpression may be one of the related mechanisms underlying the invasion and metastasis of hypopharyngeal and laryngeal SCCs. However, FGFR1 protein overexpression showed no effect on survival outcomes. Previous studies have shown a disagreement with prognostic values of FGFR1 overexpression in HNSCC, probably owing to the use of different FGFR1 overexpression criteria, anti-FGFR1 antibodies, and cohorts with different anatomical locations [13, 17, 37].

FGFR1 demonstrated strong and diffused expression in poorly differentiated SCC, while normal, dysplastic squamous epithelium, or well differentiated SCC exhibited weak to moderate expression patterns. In a recent study on oral tongue SCC, FGFR1 expression was stronger in high-grade dysplasia than in low-grade dysplasia as well as in the nucleus of poorly differentiated SCC cells [37]. However, we failed to report any increase in nuclear staining in poorly differentiated SCC. These results suggest that FGFR1 may be one of the important factors in the carcinogenesis and progression of HPV-negative, smoking- and alcohol-related SCC represented by hypopharyngeal and laryngeal SCC.

Regarding the biologic implications of FGFR1 gene amplification or protein overexpression in hypopharyngeal and laryngeal SCC, we investigated a correlation of FGFR1 alteration with twist and snail, the well-known EMT markers, on the basis of previous studies showing that EMT is induced in tumors by abnormal activation of the FGFR signaling pathway in several types of cancers including HNSCC [38‐41]. However, we did not observe that amplification or high protein expression of FGFR1 is related to overexpression of these two EMT-related proteins. Therefore, further studies are needed to determine whether changes in FGFR1 affect EMT acquisition in hypopharyngeal and laryngeal SCC. In addition, further in-depth studies should be followed for the underlying mechanisms of aberrant FGFR1 alterations in the tumorigenesis of hypopharyngeal and laryngeal SCC in the aspect of the known downstream signals of RAS/MAPK, PI3K/AKT, and JAK/STAT signaling pathways [11].

FGFR1 gene amplification and protein or mRNA expression have demonstrated correlation in some previous studies [23, 28]. However, we could not find any strong correlation between FGFR1 gene amplification and protein overexpression. This discrepancy may be associated with the differences in the cutoff level of amplification and protein or mRNA expression among various studies. Furthermore, FGFR1 amplification may not always cause protein overexpression alone, and may be affected by other closely related receptor tyrosine kinases (RTK). In lung SCC, some researchers suggested the mechanism of discrepancy between protein expression and gene amplification of FGFR1 due to crosstalk between FGFR1 and co-activated RTKs in FGFR1-amplified lung cancers with low FGFR1 protein expression [42, 43].