Polymorphism of FGFR4 in cancer development and sensitivity to cisplatin and radiation in head and neck cancer

Tissue samples and DNA isolation 

DNA was isolated from 110 tumour biopsies from the head and neck region collected from patients at the University Hospital in Linköping between the years 2004–2006 (Table 1; approved by the Linköping University ethical committee). Of the patients, 67 (61%) were males and 43 (39%) were females with a mean age of 66 years (range 34–91). DNA was also isolated from 39 HNSCC cell lines established from patients at the University Hospital in Turku, Finland between the years 1990–2002. These cell lines were selected for this study to represent different parts of the spectrum of in vitro radiosensitivity and cisplatin sensitivity. Furthermore, 192 individuals from the South-East region of Sweden were used as controls, of which 97 were males and 95 were females with the mean age of 45 years (range 23–78). The controls were in Hardy–Weinberg equilibrium.

Table 1. Patient population characteristics

	Variable	Total number (110 cases)
	Age
	Range (years)	34–91
	Average (years)	66
	Sex
	Male	67 cases
	Female	43 cases
	Site
	Larynx	26 cases
	Oral cavity	62 cases
	Pharynx	22 cases
	Histologicalgrade
	Squamous cell carcinoma	109 cases
	Myoepithelial carcinoma	1 case
	TNMstage
	T1–T2	54 cases
	T3–T4	53 cases
	N0	59 cases
	N1	19 cases
	N2–N3	29 cases
	M0	102 cases
	M1	2 cases
	Treatment
	Radiotherapy	65 cases
	Surgery	5 cases
	Radiotherapy+surgery	37 cases
	No treatment	3 cases
	Follow-up
	Range (months)	0–53
	Average (months)	16

The DNA isolation of the tumour biopsies was performed by standard proteinase K treatment and phenol–chloroform extractions and the DNA isolation of the cell lines was performed by MaxwellTM 16 DNA Purification Kit (Promega, Madison, WI, USA).

Cells and culture conditions 

The HNSCC cell lines were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 2mM glutamine, 1% non-essential amino acids, 100IU/ml penicillin-G, 50μg/ml streptomycin, and 10% fetal bovine serum (all from GIBCO, Paisly, UK). The cells were incubated in humidified air with 5% CO2 at 37°C and subcultured once a week using 0.25% trypsin and 0.02% EDTA.

Normal oral human keratinocytes (NOK) were cultured as previously described.14 Biopsies were harvested during benign surgery in the oral cavity, mostly tonsillectomies and contained non-keratinized squamous cell epithelium (approved by the Linköping University ethical committee). Primary keratinocyte cultures were derived from trypsin-digested tissue using growth medium (Keratinocyte-SFM; GIBCO) supplemented with antibiotics solution (penicillin 100u/ml, streptomycin 100μg/ml) and culture flasks pre-coated with fibronectin and collagen. Medium was replaced at three day intervals, and the cultures were subcultured at about 75% confluence using 0.25% trypsin and 0.02% EDTA. Cultures in passage two and three were used for the analyses.

Assessment of radiation and cisplatin sensitivity 

Radiation sensitivity was determined with a clonogenic assay in 96 well plates and was given in terms of mean inactivation dose (AUC=area under curve) as earlier published by Grenman et al.15

The effect of cisplatin was determined in 35 of the cell lines by a colony-forming assay. Tumour cells were seeded into six well plates at concentrations of 200–400 cell/cm2 depending of the plating efficiency of each cell line. After 24h cells were exposed to cisplatin 1μg/ml for 1h and thereafter incubated for another nine days before fixation in 4% formalin, stained with 2% Giemsa, after which colonies containing 32 cells or more were counted. The cloning efficiency of untreated cells (control) was set at 100%, and the cloning efficiency of treated cells was expressed as a percentage of the control value. All cell lines were exposed in triplicate and the mean value was used for statistical analyses.

Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis 

The PCR was conducted on exon 9 for RFLP analysis and on the transmembrane and kinase domains (ranging from exons 9–18) for DNA sequencing. Primers were designed by our group, sequences are available upon request. Each PCR mixture had a total volume of 20μl and they consisted of 1.0μM of each primer (Invitrogen, Paisley, UK), 200μM of each dNTP, 2.0mM MgCl2, 10× Taq buffer (containing 200mM (NH4)2SO4, 750mM Tris (pH 9.0) and 0.1% Tween 20), 0.5U Taq DNA polymerase (Thermowhite, Saveen Werner AB, Limhamn, Sweden) and 50ng DNA. The amplifications were performed by annealing at 55–62°C for 35 cycles. The samples were loaded on a 1.5% agarose gel (Invitrogen) and stained with ethidium bromide for detection of PCR products on a UV-table. The 126-bp fragment from exon 9 for RFLP analysis was digested in 1h in 60°C with BstN1 (New England BioLabs, Beverly, MA, USA) according to the manufacturer’s instructions. Restriction fragments were resolved on a 3% Nusieve 1% agarose gel (Invitrogen) and stained with ethidium bromide for detection of the fragments. The FGFR4 Gly/Gly388 was characterized by two different fragments of 97 and 29 base pairs (bp), the FGFR4 Gly/Arg388 was characterized by three different fragments of 97, 68 and 29bp and the FGFR4 Arg/Arg388 by the two fragments of 68 and 29bp.

Western blot analysis 

Cells were washed in phosphate-buffered saline (PBS) and lysed in 63mM Tris–HCl buffer (pH 6.8) containing 10% glycerol, 2% SDS, 5% 2-mercaptoethanol and 0.05% bromphenol blue (all from Sigma, St Louis, MO, USA). The protein concentration was determined,16 and 100μg aliquots were separated by 4–20% SDS-PAGE (Bio-Rad Laboratories, Hercules, CA USA). Proteins were transferred onto a nitrocellulose membrane (pore size 0.45μm; Bio-Rad Laboratories), which was subsequently blocked for 90min at room temperature in Tris-buffered saline (TBS; 50mM Tris and 0.15M NaCl, pH 7.5) containing 5% skimmed milk and 0.1% Tween-20 (Sigma). After washing in TBS, the membranes were incubated with a rabbit anti-FGFR4 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibody was diluted in TBS containing 0.1% skimmed milk and 0.1% Tween-20 and incubated at 4°C overnight. The membrane was washed and incubated for 1h at room temperature with a peroxidase-conjugated anti-mouse (1:1500; Dakopatts, Älvsjö, Sweden) and the bands were visualized by Western Blotting Luminol Reagent (Santa Cruz Biotechnology). Equal loading was verified by reprobing the membrane with a goat anti-actin antibody (I-19; 1:1000; Santa Cruz Biotechnology), followed by a bovine anti-goat antibody (1:1000; Santa Cruz Biotechnology).

DNA sequencing with MegaBACE™ 

The PCR-products from the transmembrane and kinase domains were purified using the ExoSAPIT Purification Kit (Amersham Bioscience, Uppsala, Sweden). The purified PCR fragments were sequenced using the DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBACE™ DNA Analysis Systems (Amersham Bioscience) and the MegaBACE 1030 instrument (GE Healthcare, Uppsala, Sweden).

mRNA isolation and detection 

mRNA was extracted from cells using the PARIS™ Kit (Protein And RNA Isolation System; Ambion, Austin, Texas, USA) according to manufacturer’s instructions and RNA concentration was measured using a UV–vis spectrophotometer (NanoDrop ND-1000; Thermo Fisher Scientific, Wilmington, Delaware, USA). The cDNA synthesis was conducted with random primers and SuperScript II Reverse Transcriptase (Invitrogen). Primers (Invitrogen) were designed by our group, sequences available upon request. The PCR amplification and detection of cDNA was performed as described above but with an annealing temperature of 60°C and for 40 cycles.

Statistics 

χ2-test from Epi Info™, version 3.4.1., (Atlanta, Georgia, USA) was used to calculate Hardy–Weinberg equilibrium and also the statistical association between the frequency of the polymorphism and development of HNSCC. Overall survival was estimated with the Kaplan–Meier method and the significance of difference between survival rates for patients with different genotypes was assessed by log-rank test (SPSS 15.0 for Windows). Mann–Whitney U test was used to analyse the significance of the FGFR4 Gly/Arg polymorphism for radiation and cisplatin sensitivity (SPSS 15.0 for Windows).

FGFR4 genotyping and predisposition for developing HNSCC 

The FGFR4 Arg388 allele was found in 58% (111 out of 192) of the control individuals confirming the frequencies from previous studies,7, 8 whereas the frequencies of the Arg388 allele in the tumour biopsies from patients with HNSCC were 45% (49 out of 110). In the material from primary tumours we found that the Gly388 allele gave a statistically significant higher risk of developing cancer, OR 1.71 (p=0.026), suggesting the Gly388 allele to be a risk allele considering predisposition (see Table 2). Furthermore, males carrying the Gly388 allele had a 2-fold risk to develop HNSCC, OR 2.00 (p=0.031), while no significant increase of risk was seen for females. In addition, the Gly388 allele gave a statistically significant higher risk to develop cancer in the oral cavity, OR 2.49 (p=0.002) while no significance was seen for larynx.

Table 2. Predisposition and higher risk of developing cancer in correlation to the FGFR4 Arg388 allele

	SNP	Genotype	Control n (%)	HNSCCan (%)	ORb	95% CIb	p-value
	Total
	FGFR4 Gly388Arg	GA+AA	111 (58)	49 (45)	1.00
		GG	81 (42)	61 (55)	1.71	1.04–1.89	0.026
	Men
	FGFR4 Gly388Arg	GA+AA	60 (62)	30 (45)	1.00
		GG	37 (38)	37 (55)	2.00	1.02–2.17	0.031
	Women
	FGFR4 Gly388Arg	GA+AA	51 (54)	19 (44)	1.00
		GG	44 (46)	24 (56)	1.46	0.67–3.22	0.30
	Oral cavity
	FGFR4 Gly388Arg	GA+AA	111 (58)	22 (35)	1.00
		GG	81 (42)	40 (64)	2.49	1.32–4.71	0.002
	Larynx
	FGFR4 Gly388Arg	GA+AA	111 (58)	14 (54)	1.00
		GG	81 (42)	12 (46)	1.17	0.48–2.87	0.7

a HNSCC, head and neck squamous cell carcinoma. b χ2-test.

Since this tumour material is recently collected, we have only a one-year follow-up on the patients so far. Calculations for overall survival depending on FGFR4 Arg388 genotype show no significance overall or stratified for gender or age of onset with cut-off at 65 years.

The role of the FGFR4 Arg388 for radiation and cisplatin sensitivity 

According to Thussbas et al. the Arg388 allele is associated with resistance to adjuvant therapy in primary breast cancer.17 The most common treatments of HNSCC are radiotherapy, chemotherapy and surgery in various combinations. Therefore we investigated the role of the Arg388 allele for radiation sensitivity in 39 cell lines and for cisplatin sensitivity in 35 cell lines from HNSCC. Our calculations show no role of the Arg388 allele for the radiosensitivity (p=0.996; Fig. 1) but a tendency that the presence of Arg388 increases the sensitivity to cisplatin (p=0.141; Fig. 2).

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Figure 1. In 39 HNSCC cell lines the sensitivity to radiation was determined with a clonogenic assay and given in terms of mean inactivation dose (AUC=area under curve). The cell lines were divided into two groups: Gly388 and Arg388, the horizontal lines show the median value and with Mann–Whitney U test the p-value was 0.996.

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Figure 2. In 35 HNSCC cell lines the sensitivity to cisplatin was determined with a colony-forming assay. The cloning efficiency of untreated cells (control) was set at 100%, and the cloning efficiency of treated cells was express as a percentage of the control value. The cell lines were diluted into two groups: Gly388 and Arg388, the horizontal lines show the median value and with Mann–Whitney U test the p-value was 0.141.

Western blot analyses of the FGFR4 protein 

A significant reduction of overall survival in HNSCC patients was seen in a study correlating the Arg388 allele to a high FGFR4 expression.9 In the present study we investigate the hypothesis that the Arg388 allele in combination with high FGFR4 expression plays a role in treatment sensitivity. Therefore, we analysed the FGFR4 protein expression in eight cell lines with different radiation and cisplatin sensitivity (five cell lines with the Gly388 allele, three cell lines with the Arg388 allele; Table 3) and NOK. In two of the cell lines (UT-SCC-9 and UT-SCC-33) we found a low expression of FGFR4 while the cell lines UT-SCC-12 and 77 were the most similar to NOK. The remaining cell lines displayed 2–4 protein bands not represented in NOK (Fig. 3). However, these results did not show any correlation between protein expression and treatment sensitivity.

Table 3. Tumour characteristics

	Cell line	Sex	FGFR4	Primary tumourlocation	TNMa	Cisplatin Sensitivity (% survival)	AUCb
	UT-SCC-24A	M	Arg388	Mobile tongue	T2N0M0	100	2.6
	UT-SCC-77	M	Gly388	Mobile tongue	T1N0M0	31	2.5
	UT-SCC-33	F	Gly388	Gingiva of mandible	T2N0M0	91	2.3
	UT-SCC-34	M	Gly388	Supraglottic larynx	T4N0M0	68	2.1
	UT-SCC-12	F	Gly388	Skin of the nose	T2N0M0	0	2.1
	UT-SCC-2	M	Gly388	Floor of the mouth	T4N1M0	100	1.8
	UT-SCC-23	M	Arg388	Transglottic larynx	T3N0M0	26	1.6
	UT-SCC-9	M	Arg388	Glottic larynx	T2N1M0	85	1.4

a TNM classification according to the International Union against Cancer (UICC, 1977). b Radiation sensitivity given in terms of mean inactivation dose (AUC=area under curve). Previously published by Pekkola-Heino et al.24 and Erjala et al.25

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Figure 3. FGFR4 protein expression. Western blot analyses of FGFR4 in eight HNSCC cell lines with different radio- and cisplatin sensitivity from the two groups: Gly388 (five cell lines) and Arg388 (three cell lines) and normal oral keratinocytes (NOK). One representative blot out of two is shown.

DNA sequencing of the FGFR4 gene and RNA expression 

In order to explain the Western blot pattern we sequenced the transmembrane and kinase domains of the FGFR4 gene. A novel mutation was found in six (UT-SCC-2, -9, -12A, -33, -34 and -77) of the eight cell lines analysed with Western blot. A T>C in the consensus splice site, four nucleotides into intron 13 resulted in an amino acid exchange altering a stop codon to a glutamine. This leads to a six amino acid supplement to the protein before the next stop codon and could result in a truncated protein with a loss of exons 14–18.

The Western blot analysis showed only weak FGFR4 protein bands of cell lines UT-SCC-9 and -33 which both carried the novel mutation. Therefore, we analysed the mRNA expression in these cell lines and in NOK. However, mRNA was detected in both cell lines and NOK thus the solution to the weak bands in the Western blot analysis could not be explained on mRNA level.