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K-rasmutations in sinonasal cancers in relation to wood dust exposure
© Bornholdt et al; licensee BioMed Central Ltd. 2008
Received: 28 August 2007
Accepted: 20 February 2008
Published: 20 February 2008
Cancer in the sinonasal tract is rare, but persons who have been occupationally exposed to wood dust have a substantially increased risk. It has been estimated that approximately 3.6 million workers are exposed to inhalable wood dust in EU. In previous small studies of this cancer, ras mutations were suggested to be related to wood dust exposure, but these studies were too limited to detect statistically significant associations.
We examined 174 cases of sinonasal cancer diagnosed in Denmark in the period from 1991 to 2001. To ensure uniformity, all histological diagnoses were carefully reviewed pathologically before inclusion. Paraffin embedded tumour samples from 58 adenocarcinomas, 109 squamous cell carcinomas and 7 other carcinomas were analysed for K-ras codon 12, 13 and 61 point mutations by restriction fragment length polymorphisms and direct sequencing. Information on occupational exposure to wood dust and to potential confounders was obtained from telephone interviews and from registry data.
Among the patients in this study, exposure to wood dust was associated with a 21-fold increased risk of having an adenocarcinoma than a squamous cell carcinoma compared to unexposed [OR = 21.0, CI = 8.0–55.0]. K-ras was mutated in 13% of the adenocarcinomas (seven patients) and in 1% of squamous cell carcinomas (one patient). Of these eight mutations, five mutations were located in the codon 12. The exact sequence change of remaining three could not be identified unambiguously. Among the five identified mutations, the G→A transition was the most common, and it was present in tumour tissue from two wood dust exposed adenocarcinoma patients and one patient with unknown exposure. Previously published studies of sinonasal cancer also identify the GGT → GAT transition as the most common and often related to wood dust exposure.
Patients exposed to wood dust seemed more likely to develop adenocarcinoma compared to squamous cell carcinomas. K-ras mutations were detected in 13% of adenocarcinomas. In this study and previously published studies of sinonasal cancer the found K-ras mutations, were almost exclusively G → A transitions. In conclusion, our study, based on a large representative collection of human SNC tumours, indicates that K-ras mutations are relatively infrequent, and most commonly occur in adenocarcinomas. Wood dust exposure alone was not found to be explanatory for the G→A mutations, but combination of exposure to tobacco, wood dust, and possibly other occupational agents may be a more likely explanation. Overall, the study suggests a limited role for K-ras mutations in development of sinonasal cancer.
Cancer of the sinonasal cavities is rare with an annual standardized incidence rate of 0.46 per 100 000 person-years for men and 0.62 for women in Denmark year 2001 . In 1967, Acheson et al. reported of a cluster of patients with sinonasal adenocarcinomas in the furniture industry in Buckinghamshire, Great Britain . Since then, several studies have found an association between exposure to wood dust and sinonasal cancer [3–5]. In particular, adenocarcinoma has been strongly associated with exposure to hardwood dust . In a recent reanalysis of 12 pooled studies, an odds ratio of 45.5 [CI = 28.3–72.9] for adenocarcinoma was found among male workers occupied in jobs with high levels of wood dust exposure . The International Agency for Research on Cancer (IARC) classified "furniture and cabinet making" as carcinogenic to humans in 1987 and so was wood dust in 1995 .
Mutagenic carcinogens can leave fingerprints such as specific mutation patterns, which may help identify them as environmental risk factors . Because tumour suppressor genes and oncogenes are involved in carcinogenesis, they are good candidates for such investigations. One of the most well established links between a pattern of mutation and environmental exposure is ultraviolet light induced mutation in the p53 gene. UV-light damages DNA by the formation of dipyrimidine adducts between two adjacent pyrimidines. This adduct is converted to a unique UV induced transition, the CC→TT tandem mutation, which is detected at high frequencies in human skin cancers [10–12]. It is thus a fingerprint of UVB induced DNA damage.
Mutations in the activating codons of ras (12, 13 and 61) are detected in 20–30% of all human cancers, varying in frequency between the different organs. E.g. < 90% of pancreas cancers harbour ras mutations . Codon 12 is a preferential site for DNA adduct formation for several carcinogens . Smoking may be contributing to sinonasal cancer. Lung cancers from smokers frequently harbours mutations in the ras gene codon 12 , the most frequent type of mutations is a G•C→T•A transversion in K-ras codon 12 . The benzo(a)pyrene diol-epoxide-guanine adduct, that is most frequently produced when benzo(a)pyrene metabolites reacts with DNA, has been identified in the lungs of smokers. This adduct is most frequently transformed into G•C to T•A transversions . The nitrosamine NNK, a tobacco-specific lung carcinogen, is associated with G•C → A•T transitions in rodent lung tumours . Among non-smokers with lung cancer the G•C → A•T transition is the most frequent point mutation .
Genetic changes in sinonasal adenocarcinomas have been reported occasionally. Studies including a limited number of patients indicate, that the ras genes are mutated in some tumours [19–22]. In the studies of Saber et al  and Yom et al , K-ras was mutated in approximately 14% of the tumours. In the study of Spanish intestinal-type adenocarcinomas, 50% of the tumours harboured mutations . The G•C → A•T transition was among the most common mutations found, but interpretation of the results has been limited due to the small sample sizes. These previous studies indicated that wood dust exposure might be correlated to a specific mutation pattern.
Study subjects and tumour material
This study was reviewed and approved by the Danish National Committee on Biomedical Research Ethics (journal number: KF 01-048/02). Records on potential study subjects with sinonasal cancer were retrieved from the nationwide Danish Cancer Registry. This computerized register, which is regarded as almost complete, uses the unique Central Person Registry Number (CPR) as the key identifier of each patient. This CPR-number is applied to all citizens in Denmark, and is used all over the society for administrative identification of persons. It includes information on name and address, vital status, dates of eventual decease or emigration, and to some extent on relatives. From the start of the Danish Cancer registry in 1942 an extended version of the 7th revised of the International Classification of Disease for classification has been used for classification of cancers. Since 1978 tumours have also been classified according to the International Classification of Disease for Oncology, including detailed information of topography and morphology . By use of a combination of the two classification schemes we selected records of the in total 466 patients born after 1903 and registered with adenocarcinomas or squamous cell carcinomas between 1991 and 2001, with ICD7 code 160.
A detailed list of histological diagnoses included in the molecular epidemiology study, with reference to the WHO classification of tumours (WHO, 1991). SNOMED, Systemic Nomenclature of Medicine and the distribution of the histology of the samples.
Distribution in the study
SNUC, refer to IHC
Not othervice specified
Squamous cell carcinoma
keratinising and nonkeratinising
associated with inverted papilloma
Cylindrical cell carcinoma
Verrucous squamous cell carcinoma
Spindle cell carcinoma
low grade – acinic like
Small cell carcinoma
Probably metastasis from primary lung tumour
PET blocks were sectioned serially to prepare material for pathological review and molecular analysis: For the histopathological review, one section was stained with hematoxylin and eosin and one with alcien blue-PAS. Two times five 10-μm sections were collected for DNA extraction. A final section was cut for hematoxylin and eosin staining to confirm that there was tumour tissue in the intervening sections. On review the ratio of tumour cells to non-malignant cells in the tissue sections was estimated. In a few cases additional sections were cut for automated immunohistochemical staining.
Review of the histological diagnoses
To ensure uniform tumour classification according to modern criteria, the slides were coded and histological diagnoses were evaluated independently by two of us (M.D. and T.S) without knowledge of the original diagnosis. Both reviewers assigned a concordant diagnosis in almost all cases. For a few in which there was disagreement, both reviewers re-examined the slides in concert to reach consensus. In seven cases, immunohistochemical staining was performed to determine the final diagnosis.
Exposure to wood dust and potential confounders
Three different sources were used to obtain information on exposure to wood dust and other potential risk factors. First, all study subjects were retrieved based on the CPR-number in the computerized National Pension Fund, for which membership has been compulsory for all wage earners in Denmark since the start of this scheme in 1964. This register keeps historical information on all companies in Denmark. If a person has been an employee, a working history stating dates of start and end of each employment can be retrieved. Records have been kept even when a person has retired or deceased. Further, each company has by Statistics Denmark been classified according to an extended version of International Standard Industrial Classification of all economic activities, which includes detailed information on trades where exposure to wood dust is normal, e.g. wood furniture industry or carpentry shops, etc. Based on this information we reconstructed the employment- and potential wood dust exposure history from 1964 and until diagnosis. We also extracted a job title from the Central Person Registry and classified this according to potential wood dust exposure. Finally, we obtained information on occupational exposure to risk factors for sinonasal carcinomas including wood dust, tobacco smoking, formaldehyde, chromium(VI), nickel, textile and leather dust by a structured telephone interview either with the patient or if he or she was deceased with a next-of-kin, preferable the last spouse or a child. Information on vital status, relatives and contact addresses was obtained from the CPR-registry. The CPR-registry, which we used to identify contact addresses on patients, their spouses and children, was established in 1968 and included all persons alive from the first of April 1968 until today. Family relations were, at the onset of the CPR register presumed to be the persons living at the same address. Therefore, for the link between parents and their children, the CPR register is only regarded complete for persons born in 1930 and onwards. However, information before 1930 still exists in the registry, but is of more sporadic character. Since, the majority of our cases with sinonasal cancer (n = 103) was born before 1930, and had already died at the onset of this study, cases for whom we could identify relatives to contact, were primarily limited to those born after 1930. Among the 103 patients born before 1930, a contact person could only be identified for 31 patients (25 of these completed an interview). For the 71 patients born after 1930 a contact person could be identified for 67 persons (60 of these completed an interview). In total 85 interviews were successfully completed.
Genomic DNA was prepared from five 10-μm sections. The sections were extracted twice with 1 ml xylene (Merck) and twice with 1 ml 96% ethanol, each for 10 min at 55°C with centrifugation in between. After the last wash with ethanol, the tissue was dried and rehydrated with 500 μl lysis buffer (50 mM Tris-HCl pH 8.5, 1.0 mM EDTA, 0.5% Tween-20) for 2 hours (55°C) under rotation. The tissue was agitated with 50–100 μl 20 mg/ml proteinase K (Finnzymes, Finland) depending on sample size, overnight or until the tissue was totally degraded. The samples were extracted twice with 500 μl phenol/chloroform/water mixture (Applied Biosystems, cat. no 400765) and once with 500 μl chloroform (AMRESCO, Ohio, US). The DNA was precipitated for 2 hours at -20°C with 27 μl 3 M sodium acetate in ice-cold ethanol. The DNA was washed once with 70% ethanol, and dried at 50°C before it was dissolved in RNase free water. The DNA concentration was determined spectrophotometrically at 260 and 280 nm, and where possible, the concentration was adjusted to 100 μg/ml.
Primers and restriction enzymes used in the RFLP assays.
Sequence 5' to 3'
Restriction enzymes/recognition site
PCR product/cut fragments
ACT GAA TAT AAA CTT GTG GTA GTT GGA CCT
BstN I :
TCA AAG AAT GGT CCT GGA CC
ATA TAA ACT TGT GGT AGT TCC AGC TGG
Van 91 I :
TCA AAG AAT GGT CCT GGA CC
CTT GGA TAT TCT CGA CAC AGC TGA T
AAC TAT AAT TAC TCC TTA ATG TCA GCT TA
Reaction conditions for all the PCR reactions.
Primers forward + reverse
The PCR reactions were carried out on a PTC-100™ Programmable Thermal Controller (MJ Research, Inc). The cycling conditions for the RFLP12 assay and the RFLP13 assay were; denaturation at 94°C for 1 min, taken though 35 cycles at 94°C for 30 sec, 55°C for 45 sec, 72°C for 45 sec and a final incubation at 72°C for 10 min. For the RFLP61 assay the cycling conditions were: DNA was denatured at 94°C for 2 min, the reactions were taken through 12 cycles of 94°C for 30 sec and 67°C for 1 min with a decrement of -1°C for each cycle (67°C to 56°C), then taken through 28 cycles of 94°C for 40 sec, 56°C for 1 min and finally incubated at 72°C for 10 minutes. For all PCR reactions a no template control was included. All PCR products were visualised on a 1.5% NuSieve® 3:1 agarose gel, and only samples where a distinct band of the correct size was observed were used for restriction enzyme digestion.
Before restriction enzyme digestion, the PCR products for the RFLP13 assay were purified by GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences, UK). The restriction enzyme digestion of the RFLP12 assay was performed with 10 μl PCR product, 1× NEBuffer 2, 100 μg/ml BSA and 10 units of restriction endonuclease BstN1 I (Biolabs, New England), incubated overnight at 60°C. The digestion of the RFLP13 assay was performed in a total volume of 15 μl with 1× Surecut buffer B and 2 units of restriction endonuclease Van 91 I (Boeringer Manheim) with 6 hr of incubation at 37°C. The RFLP61 assay was digested in a total volume of 11.6 μl with 1× NEBuffer 2 and 6 units of restriction endonuclease Bcl I (Biolabs, New England) and 6 hr incubation at 50°C. The digested PCR fragments were visualised on a 3% NuSieve® 3:1 agarose gel, run in 1× TBE buffer.
To ensure that we were able to distinguish between a mutant sequence and wild type sequence, we included a negative and a positive control in all digestions. As a negative control, DNA from a healthy person was used. For positive controls in the codon 12 assay the A549 cell line, which contains a homozygous GGT→ AGT mutation was used . For codon 13 the MDA-MB231 cell line with a heterozygous GGC→GAT mutation  was used and for codon 61 DNA from a cancer patient with a heterozygous CAA→ CTA mutation was used. To determine the sensitivity of the assays, mutated DNA was diluted with wild type DNA. In the assay RFLP12, a distinct band of mutated PCR products was detected in a mixture containing 6.25 % homozygote DNA which corresponds to 12.5 % tumour tissue assuming a dominant mutation. In the other two assays, the RFLP13 and RFLP61, bands of mutated PCR product were detected with 12.5% mutated heterozygous DNA. A clear cut detection of mutations was therefore possible in samples with a tumour content of 10 % and above in the RFLP assays. Using direct sequencing, a mutation could be detected in samples containing 12.5 % mutated DNA which corresponds to 25 % tumour tissue heterozygous for mutation in a sample (data not shown).
In order to identify the mutations by direct sequencing, two assays were developed, the SEQ1213, SEQ61. In order to generate PCR products for sequencing the following primers were developed; forward1213 5' AAC CTT ATG TGT GAC ATG TTC'3, reverse1213 5' ATG GTC CTG CAC CAG TAA T'3, forward61 5' AAA GGT GCA CTG TAA TAA TCC'3 and reverse61 5' TGA TTT AGT ATT ATT TAT GGC AAA'3. The PCR conditions are listed in Table 3. The PCR reactions were performed in 50 μl reactions, on a PTC-100™ Programmable Thermal Controller (MJ Research, Inc). For the SEQ1213 assay the DNA was denatured at 94°C for 1 min, and amplified in 12 cycles of 94°C for 30 sec and 57°C for 1 min with decrement of -1°C for each cycle (57°C to 45°C). It was amplified through 28 cycles of 94°C for 40 sec, 45°C for 1 min and incubated for 72°C for 10 minutes. For the SEQ61 assay the amplification was done at: 95°C for 2 min then 40 cycles of 94°C for 1 min, 48°C for 30 sec and 72°C for 1 min. For all PCR reactions a control without template was included. The PCR products (3.0 μl) were visualised by electrophoresis, on a 1.5% NuSieve® 3:1 agarose gel, run in 1×TBE buffer. Only PCR reactions where a distinct band in the right size appeared were purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences, UK) according to the manufacturer. To generate the sequencing templates for the SEQ1213 and 61 the following primers were used; sequence1213 5' ATA TAG TCA CAT TTT CAT TAT T'3 and sequence61 5'TGT TTC TCC CTT CTC AGG ATT C'3 with the BigDye® Terminator Ready Reaction Kit (Applied Biosystems, part no 4303152) according to the manufacturer. The annealing temperatures were SEQ1213, 45°C and SEQ61, 50°C. The sequencing was performed on an ABI 310 Genetic Analyser, according to standard protocols. In order to confirm a mutation, both strands were sequenced a second time from an independent PCR reaction.
In this case-case study we compared historical occupational exposure to wood dust in cases with different histological types of sinonasal cancers, using squamous cell carcinoma cases as a reference group. We also compared this exposure to wood dust of the cases with and without K-ras mutations. To estimate the magnitude of the associations, multivariate adjusted odds ratios and their corresponding 95% confidence intervals were calculated by unconditional logistic regression with the statistical package Stata, version 9. Age and sex were included in all models as potential confounders. In the subgroup of 85 cases for which we have individual information on tobacco smoking and occupational exposure to formaldehyde, these were further included in the models. None of the cases with tumours with K-ras mutations were exposed to other potential confounders, i.e. leather and textile dust, nickel and chromium(VI).
Histology and wood dust exposure
Distribution of exposure to wood dust in relation to histological diagnosis of sinonasal cancers.
Wood dust exposure crude
Squamous cell carcinoma
Squamous cell carcinoma
No exposure (interview)
No exposure according to registers
Wood dust exposure confirmed by interview
Wood dust exposure according to registers
Exposure to wood dust by cases with squamous cell carcinomas and adenocarcinomas
Wood dust Yes/no
Squamous cell carcinomas
Lists the histology, exposure data and mutations from sinonasal patients harbouring K-ras mutations.
Most recent job title according to the Central person registry
Other potential confunders
Wood dust exposure according to interview
Ambiguous (codon 12 3rd or codon 13, nt 1 or 2)
Long distance driver
Squamos cell carcinoma
Ambiguous (codon 12, nt 1 or 2)
We identified 8 mutations in total. Three of these were ambiguous since a clear-cut sequencing result could not be obtained due to high background. Of the eight mutations, seven occurred in adenocarcinomas, only one occurred in a squamous cell carcinoma. The identified mutations were all located in codon 12. Three were G•C → A•T transitions causing an amino acid change from glycine to aspartic acid. In one patient a G•C → C•G transversion was identified leading to an amino acid change from glycine to alanine and finally, in one patient a G•C → T•A transversion had occurred changing glycine to valine.
Exposure to wood dust, tobacco smoking and formaldehyde in sinonasal cancer cases with and without K-ras mutations.
Wood dust exposure
We determined the sensitivity of the assays by diluting mutated DNA with wild type DNA. In the diluted positive controls we were able to detect mutations, corresponding to samples containing 12.5% tumour material harbouring a heterozygous K-ras mutation in the RFLP analysis. There was somewhat less sensitivity than with direct sequencing, but mutations could be detected in samples corresponding to 25% tumour tissue. The average amount of cancer tissue in the blocks was 51% with a standard deviation of 24% and a minimum content of 10%.
Cancers of the sinonasal cavities are strongly correlated to exposure to wood dust, in particular adenocarcinoma has been associated with exposure to hardwood dust in epidemiological studies [3–5, 7]. In the present study, we found that 23% of the patients had been exposed to wood dust. It has recently been estimated that 3.3% of the employed population in Denmark are exposed to wood dust . In our study exposure to wood dust adjusted for age was associated with a 21.0-fold [CI = 8.0–55.0] increase in the risk of having an adenocarcinoma than a squamous cell carcinoma. The frequency of wood dust exposure among the patients with adenocarcinomas was 8.9 times more common than among the patients with squamous cell carcinomas. Our findings are some what in accordance with a previously published pooled analysis on eight case/control studies by Mannetje et al . In this analysis the frequency of wood dust exposed adenocarcinomas (74.2%) were five times more common than wood dust exposed squamous cell carcinomas (14.7%).
In the present study, mutations in the K-ras gene were detected in 13% of the adenocarcinomas and in 1% of the squamous cell carcinomas. The found frequency of K-ras mutations was lower than expected. Due to the low frequency of K-ras mutations, even in this large study, there was not sufficient statistical power to detect statistically significant associations, if they exist. However, the study was conducted to include all cases of relevant sinonasal cancers in Denmark with in a reasonable time frame for conducting interview of the cases. The odds ratios in this study were influenced by some uncertainty, because some calculations were made on groups with less than 10 individuals.
We found an odds ratio for a non significant association between wood dust and K-ras mutation on 3.6 [CI = 0.9–15.2] Taking into consideration formaldehyde and smoking reduced the odds ratio (Table 7). However, the mutation frequency in adenocarcinomas were similar to those reported in the studies of Saber et al  and Yom et al , but it is lower than in a study by Frattini et al , who reported of a frequency of 50 % in 17 intestinal type adenocarcinomas from Italian patients. This was not explained by differences in the detection methods used. The sensitivity of the assays in the combination used should be sufficient to identify mutations in the sinonasal tumours. The differences in the prevalence of mutations might be related to differences in the type of exposures. For example hardwoods are used more frequently in Italy (55% of the wood consumed) compared to Northern European countries (8–21% of the wood consumed) .
The most frequently found mutation in this study was the GGTGLY → GATASP transitions, which were found in 3 out of 5 mutations. The GGT→GAT transition has been associated with a bad prognosis in studies of lung adenocarcinomas [30, 31]. It is also the most common among the previously published K-ras codon 12 and 13 mutations in sinonasal cancers. In the study by Saber et al , two out of four mutations were GAT transitions, and both were in wood dust exposed patients. In the study by Frattini et al , nine mutations were identified. Eight of these were GAT transitions, three of which were from wood dust exposed patients. The remaining one, a GGTGLY → AGTSER, was from a wood dust exposed patient as well. In the study by Perez et al  one mutation in the K-ras gene, a GAT transition from a wood dust exposed patient was identified.
Summarising mutation data from the present study and previously published ones, it appears that cancers harbouring K-ras mutations represent a small fraction of the sinonasal adenocarcinomas. The mutations were primarily located (16/20) at codon 12, and 85% of them were G→A transitions (17/20). All though the number is small, the high frequency of G→A transitions may suggest an environmental component involved in this subgroup of sinonasal cancers. In acute myeloid leukaemia increased frequency of activated ras genes identified a subgroup associated with occupational or chemical exposure .
The G→A transitions are typically produced by alkylating agents in experimental systems, consistent with O6-methyl-guanine-misparing [33, 34]. In humans this transition has been identified in human liver tumours after exposure to vinyl chloride [35, 36]. The G→A base transitions in K-ras are also very frequently identified in rodent lung neoplasms after long-term exposures to N-nitroso compounds . In tobacco-related lung adenocarcinomas of humans G→T transversions are the most frequent mutations, but G→A transitions are second most frequent [38, 39] and most frequent among non smokers . After chronic inhalation to mainstream cigarette smoke in rats the two identified mutations were G→A transitions and G→T transversions .
The combination of a G→A transition and exposure to wood dust was present among 60% (9/15) of the G→A mutations where the patient's occupational exposure was known. The frequency of G→A transitions did not differ between wood dust exposed patients and non-exposed (Fisher exact, P = 0.25) However, genotoxic effects have been detected in several studies of healthy wood workers [41–43] and wood dusts were tested positive in the comet assay in the human lung epithelial cell line A549 before an the inflammatory reaction was evoked [44, 43].
In addition, in the present study, the two patients with G→A transitions were both smokers. In the study by Saber et al , two of the three patients with G→A transitions were smokers. Since exposure to tobacco smoke carcinogens is also associated with G→A transitions, smoking, including involuntary smoking, may also play a role in the carcinogenesis of this subpopulation of sinonasal cancers. To determine the mechanisms behind the carcinogenesis induced by a potential synergistic effects of smoking and wood dust in this subpopulation of sinonasal cancers more studies are needed. Because of the difficulty in achieving statistical power in molecular epidemiology studies of this type cancer, due to the rarity of both sinonasal cancer and K-ras mutations, it might be more effective to study the mutagenesis of wood dust in experimental studies.
We sought to investigate whether exposure to wood dust was associated with a specific mutational pattern in the K-ras gene in a large, representative study population. We therefore examined all incident cases of sinonasal cancer in Denmark 1991–2001. Wood dust exposure occurred in 23% of our patients and was associated with a 21-fold [CI = 8.0–55.0] increase in the risk for having an adenocarcinoma than a squamous cell carcinoma compared to not being exposed. The frequency of K-ras mutations in adenocarcinoma (13%) was in the range reported in earlier studies, whereas the frequency was very low in the squamous cell carcinoma (1%). Collectively, taking into account the mutations we detected our and the ones in sinonasal cancers that have been reported before, the GGTGLY → GATASP transition is the most common mutation after exposure to wood dust. In conclusion, mutational activation of K-ras in SNC is restricted to a subgroup of the adenocarcinoma histology. However, since most of the mutation positive cases were smokers, active or passive exposure to cigarette smoke, may also be involved. The observed mutation types are in keeping with this. Overall, the study suggests a limited role for K-ras mutations in development of sinonasal cancer.
The authors gratefully acknowledge the help and collaboration of the departments of pathology in Denmark, without which this project could not have been completed. Technical assistance from Lourdes Pedersen is also gratefully acknowledged. This study was supported by EU 5th Framework Programme, Key Action 4, Environment and Health, Quality of Life and Management of Living Resources, project no. QLRT-2000-00573.
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