Fgfr2 gene amplification and clinicopathological features in gastric cancer

ABSTRACT BACKGROUND: Frequency of _FGFR2_ amplification, its clinicopathological features, and the results of high-throughput screening assays in a large cohort of gastric clinical samples

remain largely unclear. METHODS: Drug sensitivity to a fibroblast growth factor receptor (FGFR) inhibitor was evaluated _in vitro_. The gene amplification of the _FGFRs_ in formalin-fixed,

paraffin-embedded (FFPE) gastric cancer tissues was determined by a real-time PCR-based copy number assay and fluorescence _in situ_ hybridisation (FISH). RESULTS: _FGFR2_ amplification

confers hypersensitivity to FGFR inhibitor in gastric cancer cell lines. The copy number assay revealed that 4.1% (11 out of 267) of the gastric cancers harboured _FGFR2_ amplification. No

amplification of the three other family members (_FGFR1_, _3_ and _4_) was detected. A FISH analysis was performed on 7 cases among 11 _FGFR2_-amplified cases and showed that 6 of these 7

cases were highly amplified, while the remaining 1 had a relatively low grade of amplification. Although the difference was not significant, patients with _FGFR2_ amplification tended to

exhibit a shorter overall survival period. CONCLUSION: _FGFR2_ amplification was observed in 4.1% of gastric cancers and our established PCR-based copy number assay could be a powerful tool

for detecting _FGFR2_ amplification using FFPE samples. Our results strongly encourage the development of FGFR-targeted therapy for gastric cancers with _FGFR2_ amplification. SIMILAR

CONTENT BEING VIEWED BY OTHERS ASSESSMENT OF COPY NUMBER IN PROTOONCOGENES ARE PREDICTIVE OF POOR SURVIVAL IN ADVANCED GASTRIC CANCER Article Open access 09 June 2021 DETECTING GENE COPY

NUMBER ALTERATIONS BY ONCOMINE COMPREHENSIVE GENOMIC PROFILING IN A COMPARATIVE STUDY ON FFPE TUMOR SAMPLES Article Open access 05 February 2025 _FGFR2_-FUSIONS DEFINE A CLINICALLY

ACTIONABLE MOLECULAR SUBSET OF PANCREATIC CANCER Article Open access 17 September 2024 MAIN Intensive investigations of anticancer treatments for gastric cancer have been done over the past

three decades; however, the prognosis for patients with unresectable advanced or recurrent gastric cancer remains poor (Bittoni et al, 2010; Fujii et al, 2010), and new therapeutic

modalities are needed. Fibroblast growth factors (FGFs) and their receptors are considered to be associated with multiple biological activities, including fundamental developmental pathways,

cellular proliferation, differentiation, motility and transforming activities (Itoh et al, 1994; Moffa et al, 2004; Grose and Dickson, 2005). Fibroblast growth factor signalling is also

involved in many physiological roles in the adult organism, such as the regulation of angiogenesis and wound repair, and FGF receptors (FGFRs) are expressed on many different cell types and

regulate key cell behaviours of cancer cells (Turner and Grose, 2010). Emerging evidence has demonstrated that the deregulation of FGF signalling is frequently observed in various solid

cancers and haematological malignancies (Beenken and Mohammadi, 2009). The most well-known association with _FGFR_ mutations is the _FGFR3_ mutation observed in bladder cancer, in which

somatic mutations in coding regions are observed in about 50% of all specimens (Cappellen et al, 1999; Turner and Grose, 2010). Other genetic alterations in _FGFR3_ include gene

amplification in bladder cancer and translocation in myeloma (Turner and Grose, 2010). Similarly, the deregulation of FGF signalling has been reported in various malignancies. Glioblastoma

exhibits FGFR1 kinase domain gain-of-function mutations, and FGFR1 is abnormally activated in malignant prostate cells. In 8p11 myeloproliferative syndrome, translocations fuse different

proteins in frame with the FGFR1 kinase domain, causing the constitutive dimerisation of the kinase (Giri et al, 1999; Rand et al, 2005; Beenken and Mohammadi, 2009). The _FGFR1_

amplification has been reported in approximately 10% of breast cancers (Courjal et al, 1997) and oral squamous carcinomas, and has been also found at a low incidence in ovarian cancer,

bladder cancer and rhabdomyosarcoma (Turner and Grose, 2010). _FGFR2_ mutations are observed in 12% of endometrial cancers but are reportedly rare in gastric cancers (Jang et al, 2001; Dutt

et al, 2008). The _K-sam_ gene was first identified and characterised as an amplified gene in the human gastric cancer cell line KATO-III (Hattori et al, 1990; Ueda et al, 1999), and its

product was later found to be identical to the bacteria-expressed kinase, or keratinocyte growth factor receptor, and FGF receptor 2 (FGFR2). _FGFR2_ amplification has been found in

diffuse-type gastric cancer-derived cell lines and the amplification was preferentially detected in diffuse-type gastric cancer. FGFR2 protein overexpression was detected using

immunohistochemical staining in 20 of 38 advanced cases of diffuse-type gastric cancer (Hattori et al, 1996). FGFR2 protein expression was observed in 31% of the gastric carcinomas and was

positively correlated with scirrhous cancer, a diffuse type, the invasion depth, the infiltration type and a poor prognosis (Toyokawa et al, 2009). On the other hand, along with another

group, we previously reported that _FGFR2_ amplification confers hypersensitivity to FGFR inhibitor in gastric cancer cell lines both _in vitro_ and _in vivo_ (Nakamura et al, 2006; Takeda

et al, 2007), strongly suggesting that _FGFR2_ amplification may be a promising molecular target for the treatment of _FGFR2_-amplified gastric cancer. However, very limited information on

_FGFR2_ amplification is available regarding the frequency, the degree of the increase in the copy number, the histology and a high-throughput screening method in gastric cancer. In this

report, we retrospectively studied these issues using formalin-fixed, paraffin-embedded (FFPE) samples in patients with gastric cancer who underwent surgery in an attempt to advance

FGFR2-targeted therapy for gastric cancer. MATERIALS AND METHODS CELL CULTURE All of the gastric cancer cell lines used in this study were maintained in RPMI-1640 medium (Sigma, St Louis,

MO, USA), except for IM95 (DMEM; Nissui Pharmaceutical, Tokyo, Japan), supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL, Grand Island, NY, USA), penicillin and

streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. IM95 and OCUM1 were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan) and the others were provided

from National Cancer Center Research Institute (Tokyo, Japan). PATIENTS A total of 267 patients with histologically confirmed gastric cancer who had undergone surgery at the National Cancer

Center Hospital between 1996 and 2006 were included in this study. All the patients in this series had an Eastern Cooperative Oncology Group performance status of 0 to 2 and had undergone

surgery. Of these patients, one subject was excluded because an insufficient quantity of DNA was extracted from the patient's specimen. Thus, samples from the remaining 267 patients

were analysed. This study was approved by the institutional review board of the National Cancer Center Hospital. ISOLATION OF GENOMIC DNA Genomic DNA samples were extracted from surgical

specimens preserved as FFPE tissue using a QIAamp DNA Micro kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Macro-dissection of the FFPE samples was

performed to select a cancer region, which was marked by a pathologist after deparaffinisation. The DNA concentration was determined using the NanoDrop2000 (Thermo Scientific, Waltham, MA,

USA). REAL-TIME REVERSE-TRANSCRIPTION PCR (RT–PCR) cDNA was prepared from the total RNA of each cultured cell line using a GeneAmp RNA-PCR kit (Applied Biosystems, Foster City, CA, USA).

Real-time RT–PCR amplification was carried out using a Thermal Cycler Dice (Takara, Otsu, Japan) in accordance with the manufacturer's instructions under the following conditions: 95 °C

for 5 min, and 50 cycles of 95 °C for 10 s and 60 °C for 30 s. The primers used for the real-time RT–PCR were as follows: _FGFR2_, forward 5′-GATAAATACTTCCAATGCAGAAGTGCT-3′ and reverse

5′-TGCCCTATATAATTGGAGACCTTACA-3′; _GAPDH_, forward 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse 5′-ATGGTGGTGAAGACGCCAGT-3′. _GAPDH_ was used to normalise the expression levels in the subsequent

quantitative analyses. IMMUNOBLOTTING A western blot analysis was performed as described previously (Matsumoto et al, 2009). The following antibodies were used: monoclonal FGFR2 antibody

(Santa Cruz Biotechnology, Santa Cruz, CA, USA), _β_-actin antibody and HRP-conjugated secondary antibody (Cell Signaling Technology, Beverly, MA, USA). CELL GROWTH INHIBITORY ASSAY To

evaluate growth inhibition in the presence of various concentrations of PD173074 (Sigma), we used an MTT assay and a previously described method (Kaneda et al, 2010). Briefly, the cells were

seeded at a density of 2 × 103 cells per well in 96-well plates. After 24 h, PD173074 was added and the incubation was further continued for 72 h at 37 °C. The assay was conducted in

triplicate. COPY NUMBER ASSAY FOR FOUR FGFR FAMILY GENES The copy numbers for _FGFR 1–4_ were determined using commercially available and pre-designed TaqMan Copy Number Assays according to

the manufacturer's instructions (Applied Biosystems). The primer IDs used for _FGFRs_ were as follows: _FGFR1,_ Hs02862256_cn; _FGFR2,_ HS05182482_cn (intron 14) and Hs05114211_cn

(intron 12); _FGFR3,_ Hs03518314_cn; and _FGFR4_, Hs01949336_cn. The _TERT_ locus was used for the internal reference copy number. Human Genomic DNA (Takara) was used as a normal control.

Real-time genomic PCR was performed in a total volume of 20 _μ_l in each well, containing 10 _μ_l of TaqMan genotyping master mix, 20 ng of genomic DNA and each primer. The PCR conditions

were 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min; the resulting products were detected using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems).

Data were analysed using SDS 2.2 software and CopyCaller software (Applied Biosystems). FLUORESCENCE _IN SITU_ HYBRIDISATION ANALYSIS The fluorescence _in situ_ hybridisation (FISH) method

was previously descried (Motoi et al, 2010). Probes designed to detect the _FGFR2_ gene and the _CEN10p_ on chromosome 10 were labelled with fluorescein isothiocyanate or Texas red and were

designed to hybridise to the adjacent genomic sequence spanning approximately 0.33 and 0.64 Mb, respectively. The probes were generated from appropriate clones from a library of human

genomic clones (GSP Laboratory, Kawasaki, Japan). Deparaffinised tissue sections were air dried and pre-treated with the GSP paraffin pre-treatment kit (GSP Laboratory). In all, 10 _μ_l of

fluorescent FISH probe was heated for 5 min at 73–75 °C in a waterbath for denaturation. The tissue sections were then placed in a denaturant solution (70% formamide/2 × saline sodium

citrate (SSC) pH 7-8) in a 73–75 °C waterbath, denatured for 5 min, dehydrated in 70 and 100% ethanol for 1 min each at room temperature, and air-dried. Denatured probes were applied, and

the specimens were covered with a coverglass and placed on a heated block at 45–50 °C. Then, the slides were sealed with rubber cement and placed in a pre-warmed humidified box overnight at

37 °C. Stringent washing was performed using 2 × SSC/0.3% NP-40 at room temperature and at 72 °C for 5 min and then with 2 × SSC at room temperature. The signals were observed using

fluorescence microscopy, and the FISH signals were evaluated by independent observers (TM and AK). After screening all the complete sections, images of the tumour cells were captured and

recorded and the signals for 20 random nuclei were counted for an area where individual cells were recognised on at least 10 representative images. The positive result of copy number gain is

determined as follows (FGFR2/CEN10p⩾2.0). STATISTICAL ANALYSIS The statistical analyses of the clinicopathological features were performed using the Student _t_-test and the _χ_2 test using

PAWS Statistics 18 (SPSS Japan Inc., Tokyo, Japan). The overall survival (OS) curves were estimated using the Kaplan–Meier method. RESULTS _FGFR2_ AMPLIFICATION CONFERS HYPERSENSITIVITY TO

FGFR INHIBITOR IN GASTRIC CANCER CELL LINES We examined the growth inhibitory effect of PD173074 (0.004–80 _μ_ M) on four _FGFR2_-amplified (HSC-43, TU-KATPIII, SNU-16 and HSC-39) and four

non-amplified (44As3, 58As1, IM95 and OCUM1) gastric cancer cell lines. The _FGFR2_ amplification status of each cell line had already been examined using a CGH analysis (unpublished data).

The mRNA and protein expressions of FGFR2 were overexpressed in the _FGFR2_-amplified cell lines (Figures 1A and B). A growth inhibitory assay showed that the IC50 values of the FGFR

inhibitor PD173074 in _FGFR2_-amplified cells were 0.01–0.07 _μ_ M, whereas those in non-amplified cells were 2.6–13.2 _μ_ M, indicating that _FGFR2_ amplification conferred an approximately

100-fold hypersensitivity to FGFR inhibitor in gastric cancer cell lines (Figure 1C). _FGFR2_ AMPLIFICATION IN CLINICAL GASTRIC CANCER CELL LINES AND SURGICAL SPECIMENS To develop a

high-throughput method for detecting _FGFR2_ gene amplification in a clinical setting, we verified a real-time PCR-based detection method, the TaqMan Copy Number Assay. The _FGFR2_ copy

number was 1.4–2.7 copies in the four non-amplified cell lines; however, the numbers in the four _FGFR2_-amplified cell lines were 28.2, 231.7, 88.2 and 36.3 copies, respectively (Figure

1D). In addition, another primer in intron 12 of _FGFR2_ produced a very similar result (_R_=0.99, Figure 1D). Collectively, these results suggested that a DNA copy number assay for _FGFR2_

was a sensitive and reproducible method. We also examined the copy numbers of _FGFR1_, _FGFR3_ and _FGFR4_, but no obvious gene amplification was observed in all of the eight cell lines

(Figure 1D). Next, _FGFR2_ amplification was evaluated using the copy number assay in 267 FFPE samples of primary gastric cancer specimens. _FGFR2_ amplification of more than 5 copies was

observed in 11 cases (92.0, 63.0, 41.4, 19.9, 18.4, 13.7, 8.3, 6.2, 6.2, 5.7 and 5.6 copies), with a frequency of 4.1% (Figure 2A). The mean copy number in the non-amplified cases was

2.4±0.6 copies. Meanwhile, no obvious gene amplification of _FGFR1_, _FGFR3_ or _FGFR4_ was observed (data not shown). FISH ANALYSIS FOR _FGFR2_ AMPLIFICATION We used a FISH analysis to

examine _FGFR2_ amplification in the same samples to verify the results of the above PCR-based DNA copy number assay. Highly amplified TU-KATOIII cells showed numerous and large clustered

signals, whereas non-amplified OCUM1 cells contained two normally paired signals (Figure 2B). A FISH analysis was performed on seven cases among 11 _FGFR2_-amplified cases and two

non-amplified cases. The FISH analysis revealed that _FGFR2_ was highly amplified in six of the seven _FGFR2_-amplified clinical samples (four showed multiple scattered signals and two

showed large clustered signals), while the remaining sample exhibited a relatively low grade of amplification (FGFR2/CEN10p=2.2, Figure 2B). The _FGFR2_ signals in the G3 and G10 samples,

which were determined not to be amplified based on the results of the DNA copy number assay, were not increased. These results clearly demonstrated the presence of _FGFR2_-amplified gastric

cancers among clinical samples. CLINICOPATHOLOGICAL FEATURES OF _FGFR2_-AMPLIFIED GASTRIC CANCER We evaluated the clinicopathological features including age, sex, histology and pathological

stage according to the _FGFR2_ amplification status. Patients age with _FGFR2_ amplification were significantly higher than the others, but sex and pathological stage were not associated

with _FGFR2_ amplification in this study (Table 1). Among the patients with _FGFR2_ amplification, the histologies of two cases were intestinal-type gastric cancer and one was unclassified

type, while the others were diffuse-type (Table 2). The tumours were located in either the upper or lower stomach. These results are summarised in Table 2. Finally, we examined the

prognostic impact of _FGFR2_ amplification on OS after surgery. _FGFR2_ amplification tended to be associated with a poorer outcome, compared with non-amplified cases, but no significant

difference was observed in the current study (log-rank test, _P_=0.075; Figure 2C). DISCUSSION To date, several studies have reported on the protein expression of FGFR2 and

clinicopathological analyses using immunohistochemistry, with 20 of 49 (41%) and 42 of 134 (31%) gastric cancers expressing FGFR2 protein when evaluated using positive or negative staining

(Hattori et al, 1996; Toyokawa et al, 2009). Regarding genomic alteration, the frequency of _FGFR2_ amplification has been reported to be 3 out of 19 (16%, among diffuse-type gastric

cancers) detected using comparative genomic hybridisation (CGH), 3 out of 57 (5%) detected using Southern blot analysis, and 2 out of 30 (7%) detected using CGH (Tsujimoto et al, 1997; Peng

et al, 2003; Kim et al, 2010). These results suggest that the frequency of _FGFR2_ amplification is around 5%, which is lower than the positive staining results obtained using

immunohistochemistry. However, the frequency of amplification has not been determined in a large cohort. Our results indicated that the frequency of _FGFR2_ amplification was 4.1% (11 out of

267), consistent with these previous reports on genomic alterations. To select a sub-population of gastric cancers sensitive to FGFR inhibitors in the future, gene amplification may be a

more suitable biomarker than positive staining using immunohistochemistry based on the results of preclinical studies (Figure 1, Takeda et al, 2007). In six cases, the copy number of _FGFR2_

was larger than 10 copies and numerous signals were observed by the FISH analysis (Figure 2B), indicating that these gastric cancer cells harboured high levels of amplification, similar to

the results obtained using gastric cancer cell lines. Preclinical studies suggest that these cases may be likely to respond to FGFR inhibitors. In the remaining case, _FGFR2_ amplification

was relatively low (4∼8 copies, G44). Such cases with low levels of _FGFR2_ amplification may require further investigation regarding their sensitivity to FGFR inhibitors in the future.

Meanwhile, we used a copy number assay to detect gene amplification in FFPE samples. Although DNA extracted from FFPE samples was considered to be of low quality with a DNA degradation in

general, a copy number assay was capable of detecting and screening amplification in the FFPE samples, which had been stored for as long as 10 years. The results were consistent with the

results of FISH studies in several cell lines, with seven positive cases and two negative cases. Our findings suggest that a copy number assay is a powerful tool for detecting and screening

gene amplification using FFPE samples. Recently, trastuzumab in combination with chemotherapy has been regarded as a new standard option for patients with HER2-positive advanced gastric or

gastro-oesophageal junction cancer (Bang et al, 2010). Therefore, the evaluation of both the HER2 and FGFR2 status before anti-cancer treatment may be needed in gastric cancer patients in

the near future. Many small molecules of VEGFR2 tyrosine kinase inhibitors, categorised as anti-angiogenic agents, are now under clinical evaluation, and some of them, including sorafenib

for hepatocellular carcinoma and sunitinib for renal cell carcinoma, are being clinically used as standard treatment options (Ellis and Hicklin, 2008). These compounds are also known to have

a potential kinase inhibitory effect on FGFRs (Takeda et al, 2007; Turner et al, 2010), indicating that the development of these multi-kinase inhibitors may be a promising approach to the

treatment of _FGFR2_-amplified gastric cancer. In addition to small molecular FGFR tyrosine kinase inhibitors, anti-FGFR antibodies, such as IMC-A1, PRO-001a and R3Mab, also offer promise as

molecular-based drugs (Turner and Grose, 2010). We plan to conduct a prospective study in a cohort of Japanese patients with _FGFR2_-amplified gastric cancers. In conclusion, we found that

_FGFR2_ amplification was observed in gastric cancer at a frequency of about 4.1%, and a copy number assay was a powerful tool for screening for _FGFR2_ amplifications using FFPE samples.

Our results warrant strong consideration of the development of FGFR inhibitors for the treatment of gastric cancers with _FGFR2_ amplification. CHANGE HISTORY * _ 29 MARCH 2012 This paper

was modified 12 months after initial publication to switch to Creative Commons licence terms, as noted at publication _ REFERENCES * Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L,

Sawaki A, Lordick F, Ohtsu A, Omuro Y, Satoh T, Aprile G, Kulikov E, Hill J, Lehle M, Rüschoff J, Kang YK (2010) Trastuzumab in combination with chemotherapy _vs_ chemotherapy alone for

treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. _Lancet_ 376 (9742): 687–697 Article  CAS 

Google Scholar  * Beenken A, Mohammadi M (2009) The FGF family: biology, pathophysiology and therapy. _Nat Rev Drug Discov_ 8 (3): 235–253 Article  CAS  Google Scholar  * Bittoni A,

Maccaroni E, Scartozzi M, Berardi R, Cascinu S (2010) Chemotherapy for locally advanced and metastatic gastric cancer: state of the art and future perspectives. _Eur Rev Med Pharmacol Sci_

14 (4): 309–314 CAS  PubMed  Google Scholar  * Cappellen D, De Oliveira C, Ricol D, de Medina S, Bourdin J, Sastre-Garau X, Chopin D, Thiery JP, Radvanyi F (1999) Frequent activating

mutations of FGFR3 in human bladder and cervix carcinomas. _Nature Genet_ 23: 18–20 Article  CAS  Google Scholar  * Courjal F, Cuny M, Simony-Lafontaine J, Louason G, Speiser P, Zeillinger

R, Rodriguez C, Theillet C (1997) Mapping of DNA amplifications at 15 chromosomal localizations in 1875 breast tumors: definition of phenotypic groups. _Cancer Res_ 57: 4360–4367 CAS  PubMed

  Google Scholar  * Dutt A, Salvesen HB, Chen TH, Ramos AH, Onofrio RC, Hatton C, Nicoletti R, Winckler W, Grewal R, Hanna M, Wyhs N, Ziaugra L, Richter DJ, Trovik J, Engelsen IB, Stefansson

IM, Fennell T, Cibulskis K, Zody MC, Akslen LA, Gabriel S, Wong KK, Sellers WR, Meyerson M, Greulich H (2008) Drug-sensitive FGFR2 mutations in endometrial carcinoma. _Proc Natl Acad Sci

USA_ 105: 8713–8717 Article  CAS  Google Scholar  * Ellis LM, Hicklin DJ (2008) VEGF-targeted therapy: mechanisms of anti-tumour activity. _Nat Rev Cancer_ 8 (8): 579–591 Article  CAS 

Google Scholar  * Fujii M, Kochi M, Takayama T (2010) Recent advances in chemotherapy for advanced gastric cancer in Japan. _Surg Today_ 40 (4): 295–300 Article  CAS  Google Scholar  * Giri

D, Ropiquet F, Ittmann M (1999) Alterations in expression of basic fibroblast growth factor (FGF) 2 and its receptor FGFR-1 in human prostate cancer. _Clin Cancer Res_ 5: 1063–1071 CAS 

PubMed  Google Scholar  * Grose R, Dickson C (2005) Fibroblast growth factor signaling in tumorigenesis. _Cytokine Growth Factor Rev_ 16: 179–186 Article  CAS  Google Scholar  * Hattori Y,

Itoh H, Uchino S, Hosokawa K, Ochiai A, Ino Y, Ishii H, Sakamoto H, Yamaguchi N, Yanagihara K, Hirohashi S, Sugimura T, Terada M (1996) Immunohistochemical detection of K-sam protein in

stomach cancer. _Clin Cancer Res_ 2: 1373–1381 CAS  PubMed  Google Scholar  * Hattori Y, Odagiri H, Nakatani H, Miyagawa K, Naito K, Sakamoto H, Katoh O, Yoshida T, Sugimura T, Terada M

(1990) K-sam, an amplified gene in stomach cancer, is a member of the heparin-binding growth factor receptor genes. _Proc Natl Acad Sci USA_ 87: 5983–5987 Article  CAS  Google Scholar  *

Itoh H, Hattori Y, Sakamoto H, Ishii H, Kishi T, Sasaki H, Yoshida T, Koono M, Sugimura T, Terada M (1994) Preferential alternative splicing in cancer generates a K-sam messenger RNA with

higher transforming activity. _Cancer Res_ 54: 3237–3241 CAS  PubMed  Google Scholar  * Jang JH, Shin KH, Park JG (2001) Mutations in fibroblast growth factor receptor 2 and fibroblast

growth factor receptor 3 genes associated with human gastric and colorectal cancers. _Cancer Res_ 61: 3541–3543 CAS  PubMed  Google Scholar  * Kaneda H, Arao T, Tanaka K, Tamura D, Aomatsu

K, Kudo K, Sakai K, De Velasco MA, Matsumoto K, Fujita Y, Yamada Y, Tsurutani J, Okamoto I, Nakagawa K, Nishio K (2010) FOXQ1 is overexpressed in colorectal cancer and enhances

tumorigenicity and tumor growth. _Cancer Res_ 70 (5): 2053–2063 Article  CAS  Google Scholar  * Kim HK, Choi IJ, Kim CG, Kim HS, Oshima A, Yamada Y, Arao T, Nishio K, Michalowski A, Green JE

(2010) Three-gene predictor of clinical outcome for gastric cancer patients treated with chemotherapy. _Pharmacogenomics J_; e-pub ahead of print 21 December 2010 * Matsumoto K, Arao T,

Tanaka K, Kaneda H, Kudo K, Fujita Y, Tamura D, Aomatsu K, Tamura T, Yamada Y, Saijo N, Nishio K (2009) mTOR signal and hypoxia-inducible factor-1 alpha regulate CD133 expression in cancer

cells. _Cancer Res_ 69 (18): 7160–7164 Article  CAS  Google Scholar  * Moffa AB, Tannheimer SL, Ethier SP (2004) Transforming potential of alternatively spliced variants of fibroblast growth

factor receptor 2 in human mammary epithelial cells. _Mol Cancer Res_ 2: 643–652 CAS  PubMed  Google Scholar  * Motoi T, Kumagai A, Tsuji K, Imamura T, Fukusato T (2010) Diagnostic utility

of dual-color break-apart chromogenic _in situ_ hybridization for the detection of rearranged SS18 in formalin-fixed, paraffin-embedded synovial sarcoma. _Hum Pathol_ 41 (10): 1397–1404

Article  CAS  Google Scholar  * Nakamura K, Yashiro M, Matsuoka T, Tendo M, Shimizu T, Miwa A, Hirakawa K (2006) A novel molecular targeting compound as K-samII/FGF-R2 phosphorylation

inhibitor, Ki23057, for Scirrhous gastric cancer. _Gastroenterology_ 131 (5): 1530–1541 Article  CAS  Google Scholar  * Peng DF, Sugihara H, Mukaisho K, Tsubosa Y, Hattori T (2003)

Alterations of chromosomal copy number during progression of diffuse-type gastric carcinomas: metaphase- and array-based comparative genomic hybridization analyses of multiple samples from

individual tumours. _J Pathol_ 201 (3): 439–450 Article  CAS  Google Scholar  * Rand V, Huang J, Stockwell T, Ferriera S, Buzko O, Levy S, Busam D, Li K, Edwards JB, Eberhart C, Murphy KM,

Tsiamouri A, Beeson K, Simpson AJ, Venter JC, Riggins GJ, Strausberg RL (2005) Sequence survey of receptor tyrosine kinases reveals mutations in glioblastomas. _Proc Natl Acad Sci USA_ 102:

14344–14349 Article  CAS  Google Scholar  * Takeda M, Arao T, Yokote H, Komatsu T, Yanagihara K, Sasaki H, Yamada Y, Tamura T, Fukuoka K, Kimura H, Saijo N, Nishio K (2007) AZD2171 shows

potent antitumor activity against gastric cancer over-expressing fibroblast growth factor receptor 2/keratinocyte growth factor receptor. _Clin Cancer Res_ 13 (10): 3051–3057 Article  CAS 

Google Scholar  * Toyokawa T, Yashiro M, Hirakawa K (2009) Co-expression of keratinocyte growth factor and K-sam is an independent prognostic factor in gastric carcinoma. _Oncol Rep_ 21 (4):

875–880 PubMed  Google Scholar  * Tsujimoto H, Sugihara H, Hagiwara A, Hattori T (1997) Amplification of growth factor receptor genes and DNA ploidy pattern in the progression of gastric

cancer. _Virchows Arch_ 431 (6): 383–389 Article  CAS  Google Scholar  * Turner N, Grose R (2010) Fibroblast growth factor signalling: from development to cancer. _Nat Rev Cancer_ 10 (2):

116–129 Article  CAS  Google Scholar  * Turner N, Pearson A, Sharpe R, Lambros M, Geyer F, Lopez-Garcia MA, Natrajan R, Marchio C, Iorns E, Mackay A, Gillett C, Grigoriadis A, Tutt A,

Reis-Filho JS, Ashworth A (2010) FGFR1 amplification drives endocrine therapy resistance and is a therapeutic target in breast cancer. _Cancer Res_ 70 (5): 2085–2094 Article  CAS  Google

Scholar  * Ueda T, Sasaki H, Kuwahara Y, Nezu M, Shibuya T, Sakamoto H, Ishii H, Yanagihara K, Mafune K, Makuuchi M, Terada M (1999) Deletion of the carboxyl-terminal exons of K-sam/FGFR2 by

short homology-mediated recombination, generating preferential expression of specific messenger RNAs. _Cancer Res_ 59 (24): 6080–6086 CAS  PubMed  Google Scholar  Download references

ACKNOWLEDGEMENTS We thank Miss Tomoko Kitayama and Miss Hideko Morita for their technical assistance. This study was supported by the Third-Term Comprehensive 10-Year Strategy for Cancer

Control and a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Genome Biology, Kinki University

Faculty of Medicine, Osaka, 589-8511, Japan K Matsumoto, T Arao & K Nishio * Gastrointestinal Medical Oncology, National Cancer Center Hospital, Tokyo, 104-0045, Japan T Hamaguchi, Y

Shimada, K Kato & Y Yamada * Endoscopic Division, National Cancer Center Hospital, Tokyo, 104-0045, Japan I Oda * Pathology Division, National Cancer Center Hospital, Tokyo, 104-0045,

Japan H Taniguchi * Shien Lab, National Cancer Center Hospital, Tokyo, 104-0045, Japan F Koizumi * Department of Life Sciences, Yasuda Women's University Faculty of Pharmacy, 6-13-1,

Ando, Asaminami, 731-0153, Hiroshima, Japan K Yanagihara * Division of Genetics, National Cancer Center Research Institute, Tokyo, 104-0045, Japan H Sasaki Authors * K Matsumoto View author

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ARTICLE CITE THIS ARTICLE Matsumoto, K., Arao, T., Hamaguchi, T. _et al._ _FGFR2_ gene amplification and clinicopathological features in gastric cancer. _Br J Cancer_ 106, 727–732 (2012).

https://doi.org/10.1038/bjc.2011.603 Download citation * Received: 14 October 2011 * Revised: 16 December 2011 * Accepted: 19 December 2011 * Published: 12 January 2012 * Issue Date: 14

February 2012 * DOI: https://doi.org/10.1038/bjc.2011.603 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable

link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * _FGFR2_ * gastric cancer * gene

amplification