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Generation and Molecular Characterization of Head and Neck Squamous Cell Lines of Fanconi Anemia Patients

Departments of ¹ Otolaryngology/Head-Neck Surgery, ² Pathology, and ³ Clinical Genetics and Human Genetics, Vrije University Medical Center, Amsterdam, The Netherlands; ⁴ Oregon Cancer Institute, Oregon Health and Science University, Portland, Oregon; ⁵ Department of Hematology and Institut Universitaire d'Hematologie, Hôpital Saint-Louis, Paris, France; and ⁶ Department of Otorhinolaryngology Head and Neck Surgery, University Hospital Lund, Sweden

Requests for reprints: R.H. Brakenhoff, Department of Otolaryngology/Head-Neck Surgery, Vrije Universiteit Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. Phone 31-20-444-0953; Fax: 31-20-444-3688; E-mail: rh.brakenhoff{at}vumc.nl.

	Abstract

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Patients with Fanconi anemia (FA) are prone to develop malignancies at an early age. Besides hematologic malignancies, squamous cell carcinomas in the anogenital region and head and neck are also frequently found in these patients. The aim of this study was to generate a panel of head and neck squamous cell carcinoma (HNSCC) cell lines and xenografts of FA HNSCC, and to characterize these cell lines in comparison with a panel of seven cell lines from patients with sporadic HNSCC. Analyses have been done on sensitivity to DNA cross-linking agents, loss of heterozygosity profile, TP53 mutations, TP53 polymorphisms and the presence of human papillomavirus. Four FA HNSCC cell lines were established. Sensitivity to DNA cross-linking agents (cisplatin) in the FA HNSCC cell lines was on average 10 times higher as compared with the sporadic HNSCC cell lines. Human papillomavirus was not detected in any of the FA or sporadic cell lines. No differences were found in loss of heterozygosity pattern, TP53 mutation frequency and TP53 polymorphism between FA and sporadic HNSCC cell lines. This is the first report on the generation of squamous cell lines of FA patients. The FA HNSCC cell lines we have generated may be utilized for future studies and might aid in the development of new preventive therapies for FA patients. The genetic characteristics of these cell lines suggest that FA HNSCC are not very different from sporadic HNSCC, except for the sensitivity to cisplatin which is consistent with the known cellular FA phenotype.

Key Words: Head and neck cancer • Fanconi anemia • Human papillomavirus • allelic loss • cell lines

	Introduction

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Head and neck squamous cell carcinomas (HNSCC) comprise about 5% of all newly diagnosed cancer cases in the Netherlands and has a worldwide prevalence of 500,000 new cases each year. HNSCC arises from the squamous epithelial cells in the upper aerodigestive tract, which includes the oral cavity, nasopharynx, hypopharynx, and larynx. Risk factors for developing HNSCC are smoking and/or alcohol abuse. A second risk factor is infection with human papillomavirus (HPV), particularly for development of carcinomas in the oropharynx and oral cavity. Finally, a third risk factor for development of HNSCC is genetic predisposition. It has been noted that patients with Fanconi anemia (FA) are genetically predisposed to head and neck cancer (1, 2).

FA is an autosomal recessive disease and has a worldwide prevalence of one to five per million. Cells of FA patients have spontaneous chromosomal instability and a cellular hypersensitivity to DNA cross-linking agents, such as mitomycin C and cisplatin. This hypersensitivity has been exploited for complementation analysis to answer the question of whether one or more genes causes FA (3). Today, 11 complementation groups have been classified (A, B, C, D1, D2, E, F, G, I, J, and L) and genes have been identified from 8 of these complementation groups (4, 5). The most common complementation groups are A and C, accounting for, respectively, 65% and 15% of all FA cases. Mutations in one of these FA genes prohibit FA protein complex formation, resulting in excessive DNA damage. The disease is characterized by diverse congenital malformations, progressive bone marrow failure and predisposition to develop malignancies. The life expectancy of FA patients is reduced to an average of 20 years, mainly because, at an early age, FA patients develop bone marrow failure and malignancies, particularly acute myeloid leukemia. They also frequently develop squamous cell carcinomas (SCC), particularly in the head and neck, anogenital region, and esophagus (3).

Most FA HNSCC patients seem to develop carcinomas in the oral cavity (63%), in particular on the tongue (32%; ref. 6). The high incidence of HNSCC and anogenital SCC in FA patients in combination with the specific locations of the tumors led to the hypothesis that an environmental factor might be associated with HNSCC development in FA patients (6). Because HPV is involved in the carcinogenesis of some SCC of the anogenital (cervix) as well as head and neck (oropharynx) regions (1), the environmental factor that might play a role in FA HNSCC development could therefore be this virus (6).

A recent report showed that HPV is present in most FA HNSCCs (15/18) examined. In addition, none of these 18 FA HNSCCs were found to have a TP53 mutation (7), which is in concordance with previous results on HPV-mediated carcinogenesis (1). HPV expresses two viral oncoproteins, and one of these, E6, inactivates the p53 protein. The exact role of HPV in the pathogenesis of these tumors, however, needs further investigation because in other HNSCC of FA patients, no HPV could be detected, whereas the TP53 gene was mutated (8).

To facilitate these studies, and to evaluate therapeutic strategies for FA HNSCC tumors, we aimed at generating cell lines of these tumors. Here, we describe the establishment of four FA HNSCC cell lines. We compared the genetic profiles, HPV status, TP53 mutation frequency, and TP53 codon 72 polymorphism of these FA HNSCC cell lines with cell lines derived from sporadic HNSCC.

	Materials and Methods

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Patient Material. Tumor samples were obtained from various sources: OHSU-974, VU-1131-T2.8, and VU1365 cell lines were established according to methods described previously (9). UHLU-539 could only be obtained as a xenograft. Relevant clinical data of the tumors and patients are depicted in Table 1. Cell lines of sporadic tumors were established previously, indicated as 92VU040, 92VU041, 92VU059, 92VU078, 92VU080, 93VU094, and 93VU120 (9); clinical data for sporadic HNSCCs are summarized in Table 2. The study was approved by the Institutional Review Board.

Sulforhodamine B Assay. Inhibition of cell growth in response to cisplatin and methotrexate was determined with the semiautomated proliferation assay using sulforhodamine B (SRB; Sigma, Zwijndrecht, The Netherlands), as described previously (10). In short, 1,000 to 3,000 cells in 150 µL medium were plated in 96-well plates (Greiner, Mannheim, Germany) and incubated for 72 hours at 37°C and 5% CO₂. A total of 50 µL of medium containing cisplatin or methotrexate (VUmc Pharmacy) was added and cells were incubated for 72 hours at 37°C and 5% CO₂. Cells were exposed to different cisplatin and methotrexate concentrations (1, 10, and 100 nmol/L, and 1 and 10 µmol/L cisplatin; or 0.1, 1, 10, and 100 nmol/L, and 1 µmol/L methotrexate). Finally cells were fixed with 25% trichloroacetic acid for at least 1 hour at 4°C, washed and stained for 15 minutes with 0.4% SRB dissolved in 1% acetic acid. Plates were washed with 1% acetic acid and dried. The dye was solubilized with 10 mmol Tris-HCl (pH 9.0) by shaking for 1 hour, absorbances were measured at 540 nm in the microplate reader (Multiskan Bichromatic, Labsystem, Helsinki, Finland).

RNA and DNA Isolation. RNA was isolated using RNA-bee (Campro Scientific, Veenendaal, The Netherlands); the cell-pellet was resuspended into 1 mL RNA-bee and 200 µL chloroform was added, mixed, and placed on ice for 5 minutes. This was followed by centrifugation for 15 minutes at 17,000 x g at 4°C. The aqueous layer was transferred to a new vial and the interphase was used for DNA isolation. RNA (450 µL) was precipitated by addition of 450 µL isopropanol, mixed, placed on ice for 5 minutes and centrifuged for 15 minutes at 17,000 x g at 4°C in an Eppendorf (Eppendorf, Hamburg, Germany) tabletop centrifuge. The pellet was washed with 500 µL 70% ethanol, dried, and dissolved in 30 µL RNase-free water. For DNA reverse extraction, 800 µL DNA-STAT60 (Campro Scientific) and 200 µL chloroform was added to the interphase, mixed, and centrifuged for 15 minutes at 17,000 x g at 4°C. The aqueous layer was transferred into a new vial and DNA was precipitated as described for RNA. As reference, normal DNA was extracted from stored T lymphocytes of all patients (sporadic SCCs). DNA isolation was done by treatment with proteinase K as described previously (see also below; ref. 11).

Isolation of DNA from Microdissected Tumors. Snap-frozen tissue of VU-1131-T2.8, VU-1365, and UHLU-539 was serially sectioned (10-20 sections of 5 µm) on a cryostate microtome. Sections were directly used for HPV analysis or the DNA was isolated as follows. Sections were put in 90 µL proteinase K buffer [100 mmol/L Tris-HCl (pH 9.0), 10 mmol/L NaCl, 1% SDS, and 5 mmol/L EDTA] with 10 µL Proteinase K (1 mg/mL; Boehringer-Ingelheim, Alkmaar, The Netherlands) for 24 hours at 52°C with shaking. Thereafter, DNA was isolated by phenol/chloroform/isoamylalcohol (25:24:1) extraction, followed by ethanol precipitation. DNA was dissolved in LoTE-buffer [3 mmol/L Tris-HCl, 0.2 mmol/L EDTA (pH 7.5)]. One 5-µm section of paraffin-embedded tissue of the sporadic cell lines 92VU040, 92VU041, 92VU059, 92VU078, 92VU080, 93VU094, and 93VU120 was directly used for HPV analysis.

HPV analysis. Firstly, a ß-globin PCR generating a 209-bp product was done to determine the DNA integrity of the samples. All samples positive for ß-globin PCR were subjected to HPV PCR analysis. HPV detection was done by GP5+/6+ PCR enzyme immunoassay as previously described using an oligoprobe cocktail for 14 high-risk HPV types (i.e., HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) in the enzyme immunoassay (12). This method targets L1 sequences within the HPV genomes. GP5+/6+ PCR enzyme immunoassay–positive cases were subsequently typed by reverse line blot genotyping. Because failure of HPV detection by GP5+/6+ PCR may be due to integration of the viral genome and disruption of the GP5+/6+ primer region within L1, samples negative in the GP5+/6+ PCR enzyme immunoassay were additionally tested by HPV-16 E7 region type–specific PCR, as previously described (12).

Microsatellite Analysis. Microsatellite analysis to assess loss of heterozygosity (LOH) profiles were done as previously described (11). The following markers were used: D3S1284 (3p12), D3S1766 (3p14), D3S1293 (3p24), D3S1029 (3p21), D3S1217 (3p13), D3S1274 (3p12), D9S171 (9p21), D9S1748 (9p21), D9S157 (9p22), D9S162 (9p22), IFNA (9p21), D9S1751 (9p21), D17S1866 (17p13.3), CHRNB1 (17p11-12), TP53 (17p13.1), D13S170 (13q31), D13S168 (13q14.3), D13S158 (13q32), D13S294 (13q14.3), D18S57 (18q12), D18S34 (18q12), D8S1130 (8p23), and LPL-GZ (8p22). Statistical analysis of differences in LOH frequencies between FA HNSCC and sporadic HNSCC cell lines of all 23 markers was done using Fisher's exact test. Two-tailed analyses were done, and P values < 0.05 were considered statistically significant.

TP53 Mutation Analysis. TP53 mutation analysis was done on both RNA and DNA of all cell lines. Transcript sequencing was done as previously described (13) with a few adaptations. RNA primer III reversed was adapted to (5' TTA TGG CGG GAG GTA GAC TG 3'); cycling conditions were 3 minutes at 94°C, followed by 35 cycles (94°C for 30 seconds, 58°C or 60°C for 1 minute, and 72°C for 1 minute) and finally an extension for 5 minutes at 72°C, holding at 4°C with the MJ cycler (Biozym, Landgraaf, The Netherlands). PCR products were purified using the Qiagen PCR purification kit (Westburg, Leusden, The Netherlands). Sequencing was done using the Big Dye primer -21M13 or M13 kit (Amersham Biosciences, Buckinghamshire, United Kingdom) using 1 µL of purified PCR product. Sequencing products were mixed (GATC) and precipitated using 100 µL 70% ethanol for 20 minutes on ice followed by 30 minutes centrifugation at 17,000 x g at 4°C. The pellet was vacuum-dried and dissolved in 15 µL TSR (Applied Biosystems, Buckinghamshire, United Kingdom) and analyzed on the ABI310 (Amersham Biosciences). TP53 mutation analysis was also done on genomic DNA in exons 5 to 9 as described before (11). For analysis of codon 72 polymorphism, exon 4 of normal DNA from blood was sequenced.

	Results

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Cell Lines. Four FA HNSCC cell lines could be established from the patients listed in Table 1. Three of them (OHSU-974, VU1131-T2.8, and VU1365) could be cultured in standard medium under standard conditions. Xenograft take rates of these cell lines were 5/6 for OHSU-974, 6/6 for VU1131-T2.8, and 0/6 for VU1365. UHLU-539 could only be propagated as xenograft, and is not available as a cultured cell line.

HPV and TP53 Mutation. HPV DNA was not detected in any of the FA and sporadic HNSCC cell lines. Mutations in TP53 were detected in all FA HNSCC cell lines. VU1131-T2.8 and VU1365 were found to have TP53 mutations on well-known hotspots (R273 and R282, respectively). A 1-bp deletion in OHSU-974 at codon 41 (exon 4) results in a very short p53 protein by introduction of a stop codon downstream of this frameshift mutation. UHLU-539 was found to have a deletion of 1.8 kb encompassing exons 8 and 9. Mutations of VU1131-T2.8 and VU1365 were confirmed on the original tumor. The TP53 mutations of OHSU-974 and UHLU539 could not be confirmed as the original tumor material was not available. In the seven sporadic HNSCC cell lines, four were found to have a TP53 mutation. Cell line 92VU078 had mutation A161T (exon 5), 92VU080 had a deletion of 300 bp, including a part of exon 6, 93VU094 had a deletion of exon 8 and 93VU120 had mutation A159F (exon 5). The frequencies of p53 mutations in FA and sporadic HNSCC cell lines were not significantly different (P = 0.2, two-tailed Fisher's exact test).

TP53 Codon 72 Polymorphism. The TP53 exon 4 codon 72 polymorphism was analyzed for all FA and sporadic HNSCC. The results are summarized in Tables 1 and 2 for the FA and sporadic HNSCC cell lines, respectively.

Microsatellite Analysis of Fanconi Anemia HNSCC and Sporadic HNSCC Cell Lines. The LOH profiles of the four FA HNSCC and seven sporadic HNSCC cell lines are depicted in Fig. 1. Losses at all chromosome arms studied were detected in most FA HNSCC and sporadic HNSCC cell lines. Exceptions were the FA cell lines, VU1365 and OHLU539, and the sporadic cell lines, 92VU040 and 93VU120. VU1365 showed no losses at 17p and 18q and OHLU539 had no losses at 13q and 18q. Cell line 92VU040 only showed LOH at 9p and 93VU120 only at 3p and 18q. No statistical differences of LOH frequencies between FA HNSCC and sporadic HNSCC could be found for any of the 23 markers tested. Recently, results were published on the different LOH patterns of HPV-positive versus HPV-negative tumors (14), using E6 expression as an indicator of positivity or negativity. It was shown that tumors that do contain HPV DNA and which are E6-positive show much less LOH than tumors that do not contain HPV DNA. We compared the LOH frequencies of this previous study (14) with those of the FA HNSCC and the sporadic HNSCC of the present study. LOH at some regions was significantly more frequent in both FA and sporadic HNSCC than in the HPV DNA/E6-positive tumors reported previously (12/23 and 10/23 markers, respectively; P < 0.05). No statistical differences were detected between both FA HNSCC and sporadic HNSCC versus the HPV DNA/E6-negative tumors (14). This suggests that HPV was not involved in the genesis of the studied FA HNSCC and sporadic HNSCC.

View larger version (35K): [in this window] [in a new window]   Figure 1. Results of microsatellite analysis of 23 markers on six chromosome arms of four FA HNSCC and seven sporadic HNSCC cell lines. Left, chromosome arms. Right, the microsatellite marker used is depicted. Note that the FA HNSCCs show the LOH patterns as has been described for sporadic HNSCCs (14), which are not induced by HPV.

View larger version (23K): [in this window] [in a new window]   Figure 2. Sensitivity for cisplatin (A) and methotrexate (B) of two FA HNSCC cell lines (OHSU-974 and VU1131-T2.8) and three sporadic cell lines (92VU078, 92VU094, and 93VU120). IC50 values are given in nanomoles per liter. Results from two independent experiments (done in duplicate) are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our data show that the FA HNSCC cell lines are very similar to the majority of sporadic HNSCC cell lines. We detected no HPV DNA, although previously, a role for HPV in FA HNSCC was proposed (7). It could be argued that these cell lines were HPV-negative for various reasons including that the virus could be integrated, that the specific sequences for PCR detection are not present, or that the subtype is not recognized by the general primer PCR used. Not all of these reasons seem valid as the majority of HPV types in HNSCC is HPV16 (1) and one copy of HPV can be detected in 5,000 cells by the general primer PCR. Nevertheless, we decided to analyze several surrogate markers including the presence of TP53 mutations, TP53 codon 72 Arg/Pro polymorphism, and LOH patterns.

In 50% to 60% of all HNSCC cases, mutations in the TP53 gene are detected. We analyzed both FA and sporadic HNSCC for mutations of TP53 and found mutations of TP53 in all (4/4) FA HNSCC. In two cases, these were confirmed on the DNA from the primary tumor and in two cases, this was not available. Mutations of TP53 were also found in four (4/7) sporadic HNSCC. It is known that this type of mutation could be related to etiology. Particularly, tobacco use causes G to T transitions, which are frequently found in HNSCC. G to A transitions are also frequently found in HNSCC, but this transition might not be associated with tobacco use (15). In our study, one FA patient (VU-1131T2.8) had used tobacco (6 pack-years) and the TP53 mutation was indeed a G to T transition. The other FA patients had no history of tobacco use and consistent with this, the TP53 mutations were not tobacco-related [C to T transition (VU-1365) and two deletions (OHSU-974 and UHLU-539)]. Of the four sporadic HNSCC that showed a TP53 mutation, three had a history of tobacco and/or alcohol use. Cell line 92VU078 (3.5 pack-years) had a G to A transition, which might not be related with tobacco use, cell line 92VU080 (2.5 pack-years) had a deletion of 300 bp, which is not thought to be tobacco-related, whereas cell line 93VU120 (37 pack-years) had two transitions, G to T and C to T, with the G to T transition probably being tobacco-related.

Previously, it was shown that the known codon 72 polymorphism of TP53 (arginine or proline) might play a role in the development of HPV-induced HNSCC. The Arg⁷² p53 protein is considered to be a risk factor for HPV-induced HNSCC because it is more easily degraded by the E6 protein of HPV16 and HPV18 (16), and therefore we compared the allele frequencies of the two variants. In a control population (no HNSCC), allele frequencies were around 0.75 and 0.25 for arginine and proline, respectively (17), and we found no major differences in allele frequencies and genotypes (Table 3) in the FA HNSCC cell lines and sporadic HNSCC cell lines, although it should be noted that the number of samples for these comparisons is small, and these data should be interpreted with caution. The codon 72 polymorphism status of FA patients with and without SCC was compared previously, and it was shown that FA patients with Arg⁷² homozygosity had an increased risk (OR 5.6) to develop SCC (7). Although the Arg⁷² homozygosity in FA SCC patients is relatively high, it is important to note that this is mainly due to a remarkably low frequency of Arg/Pro heterozygotes (4/24) in the FA SCC patients as compared with the FA patients without SCC (7). We did not observe this low frequency of Arg/Pro heterozygotes (2/4) in the FA-HNSCC patients, but it should again be stressed that the number of samples is very small for these comparisons.

Based on the observations that none of the FA HNSCCs contained HPV DNA, all showed a TP53 mutation and all exhibited a pattern of allelic loss associated with sporadic cancers without an HPV etiology, we conclude that HPV is not likely to have played a role in the genesis of these FA HNSCCs. We do not have a good explanation why these data are in contrast with those reported previously (7), except for possible differences in the clinical history of the patients.

Besides a comparative genetic characterization of the cell lines, we did an SRB-assay for testing the sensitivity of the cell lines to DNA cross-linking drugs. FA cells are known to be sensitive to DNA cross-linking drugs, a trait that is used for diagnosis of FA. Two FA HNSCC cell lines were tested in the SRB assay. One of the FA cell lines (VU-1365) did not grow when diluted to low cell concentrations and the sensitivity for cytotoxic drugs could therefore not be established for this cell line. The SRB assay showed that the FA HNSCC cell lines were on average 10 times more sensitive to cisplatin compared with the sporadic HNSCC cell lines. This observation, although expected, raises hope for future treatment of sporadic HNSCC and other cancers. The Fanconi anemia genes might not only play a role in FA-HNSCC but also in sporadic HNSCC. Recently it was observed that Fanconi anemia gene FANCF can be methylated in sporadic head and neck carcinoma, lung carcinoma, and ovary carcinoma (19), and it was shown that this can result in abrogation of the FA pathway. Abrogation of the FA pathway will cause a FA-like phenotype of the tumor characterized by a high sensitivity to DNA cross-linkers as cisplatin. The observation that SCCs arising in FA patients indeed remain cisplatin-sensitive, despite all accumulating genetic changes during carcinogenesis, indicates that sporadic HNSCC with an inactivated FA pathway will also become sensitive to cross-linking drugs such as cisplatin and might thus be more effectively treated by chemoradiotherapy.

In conclusion, we established four FA HNSCC cell lines and these seem to be genetically similar to sporadic HNSCC cell lines, which was also previously reported by cytogenetic characterization on both FA and sporadic HNSCC (20). Whether the process of carcinogenesis, the progression through precursor stages and the ordered accumulation of genetic changes (21) is comparable with that of sporadic HNSCC is not known at present. If so, diagnosis and monitoring of preneoplastic fields might be a quantum step forward to early diagnosis and even prevention of HNSCC in FA patients. The FA HNSCC cell lines we have generated may be used further for development of new preventive therapies for FA patients.

	Acknowledgments

 
Grant support: Fanconi Anemia Research Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	Footnotes

 
Note: Present address of M.A.J.A. Hermsen: Department of Otolaryngology, IUOPA, Hospital Central de Asturias, Oviedo, Spain.

Received 10/12/04. Revised 11/23/04. Accepted 12/ 1/04.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Van Houten VMM, Snijders PJF, Van Den Brekel MWM, et al. Biological evidence that human papillomaviruses are etiologically involved in a subgroup of head and neck squamous cell carcinomas. Int J Cancer 2001;93:232–5.[CrossRef][Medline]
2. Gillison ML, Koch WM, Capone RB, et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000;92:709–20.[Abstract/Free Full Text]
3. Joenje H, Patel KJ. The emerging genetic and molecular basis of Fanconi anaemia. Nat Rev Genet, 2001;2:446–57.[CrossRef][Medline]
4. Meetei AR, De Winter JP, Medhurst AL, et al. A novel ubiquitin ligase is deficient in Fanconi anemia. Nat Genet 2003;35:165–70.[CrossRef][Medline]
5. Levitus M, Rooimans MA, Steltenpool J, et al. Heterogeneity in Fanconi anemia: evidence for 2 new genetic subtypes. Blood 2004;103:2498–503.[Abstract/Free Full Text]
6. Kutler DI, Auerbach AD, Satagopan J, et al. High incidence of head and neck squamous cell carcinoma in patients with Fanconi anemia. Arch Otolaryngol Head Neck Surg 2003;129:106–12.[Abstract/Free Full Text]
7. Kutler DI, Wreesmann VB, Goberdhan A, et al. Human papillomavirus DNA and p53 polymorphisms in squamous cell carcinomas from Fanconi anemia patients. J Natl Cancer Inst 2003;95:1718–21.[Abstract/Free Full Text]
8. van Zeeburg HJT, Snijders PJF, Joenje H, Brakenhoff RH. Re: Human papillomavirus DNA and p53 polymorphisms in squamous cell carcinomas from Fanconi anemia patients. J Natl Cancer Inst 2004;96:968.[Free Full Text]
9. Hermsen MAJA, Joenje H, Arwert F, et al. Centromeric breakage as a major cause of cytogenetic abnormalities in oral squamous cell carcinoma. Genes Chromosomes Cancer 1996;15:1–9.[CrossRef][Medline]
10. Braakhuis BJM, Jansen G, Noordhuis P, Kegel A, Peters GJ. Importance of pharmacodynamics in the in vitro antiproliferative activity of the antifolates methotrexate and 10-ethyl-10-deazaaminopterin against human head and neck squamous-cell carcinoma. Biochem Pharmacol 1993;46:2155–61.[CrossRef][Medline]
11. Tabor MP, Van Houten VMM, Kummer JA, et al. Discordance of genetic alterations between primary head and neck tumors and corresponding metastases associated with mutational status of the TP53 Gene. Genes Chromosomes Cancer 2002;33:168–77.[CrossRef][Medline]
12. Van Den Brule AJC, Pol R, Fransen-Daalmeijer N, Schouls LM, Meijer CJLM, Snijders PJF. GP5+/6+ PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes. J Clin Microbiol 2002;40:779–87.[Abstract/Free Full Text]
13. Rozemuller EH, Kropveld A, Kreyveld E, et al. Sensitive detection of P53 mutation: analysis by direct sequencing and multisequence analysis. Cancer Detect Prev 2001;25:109–16.[Medline]
14. Braakhuis BJM, Snijders PJF, Keune WH, et al. Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst 2004;96:998–1006.[Abstract/Free Full Text]
15. Brennan JA, Boyle JO, Koch WM, et al. Association between cigarette smoking and mutation of the p53 gene in squamous-cell carcinoma of the head and neck. N Engl J Med 1995;332:712–7.[Abstract/Free Full Text]
16. Storey A, Thomas M, Kalita A, et al. Role of a p53 polymorphism in the development of human papillomavirus-associated cancer. Nature 1998;393:229–34.[CrossRef][Medline]
17. Hamel N, Black MJ, Ghadirian P, Foulkes WD. No association between p53 codon 72 polymorphism and risk of squamous cell carcinoma of the head and neck. Br J Cancer 2000;82:757–9.[CrossRef][Medline]
18. Hermsen MAJA, Joenje H, Arwert F, et al. Assessment of chromosomal gains and losses in oral squamous cell carcinoma by comparative genomic hybridisation. Oral Oncol 1997;33:414–8.[Medline]
19. Taniguchi T, Tischkowitz M, Ameziane N, et al. Disruption of the Fanconi anemia-BRCA pathway in cisplatin-sensitive ovarian tumors. Nat Med 2003;9:568–74.[CrossRef][Medline]
20. Hermsen MAJA, Xie Y, Rooimans MA, et al. Cytogenetic characteristics of oral squamous cell carcinomas in Fanconi anemia. Fam Cancer 2001;1:39–43.[CrossRef][Medline]
21. Braakhuis BJM, Tabor MP, Kummer JA, Leemans CR, Brakenhoff RH. A genetic explanation of Slaughter's concept of field cancerization: evidence and clinical implications. Cancer Res 2003;63:1727–30.[Abstract/Free Full Text]

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