Download Epigenetic Inactivation of RASSF1A in Head and Neck Cancer1

Vol. 9, 3635–3640, September 1, 2003
Clinical Cancer Research 3635
Epigenetic Inactivation of RASSF1A in Head and Neck Cancer1
Seung Myung Dong,2 Dong-Il Sun,2
Nicole E. Benoit, Igor Kuzmin,
Michael I. Lerman, and David Sidransky3
moter methylation and HPV infection abrogate the same
pathway in tumorigenesis.
Department of Otolaryngology-Head and Neck Surgery, Head and
Neck Cancer Research Division, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205-2196 [S. M. D., D-I. S.,
N. E. B., D. S.]; Laboratory of Immunobiology, National Cancer
Institute, Frederick, Maryland 21702 [I. K., M. I. L.]; and Department
of Otolaryngology-Head and Neck Surgery, The Catholic University
of Korea, College of Medicine, Seoul, Korea [D-I. S.]
INTRODUCTION
ABSTRACT
Purpose: RASSF1A, a recently identified candidate tumor suppressor gene, was found to be inactivated in lung
cancer and other tumor types. We sought to understand the
role of RASSF1A in head and neck cancer.
Experimental Design: We analyzed the status of
RASSF1A and presence of high-risk human papilloma virus
(HPV) in head and neck cancer squamous cell carcinoma
(HNSCC) cell lines and primary tumors. We used methylation-specific PCR to detect promoter hypermethylation and
direct sequence analysis to detect point mutations in primary tumors and cell lines. 5-aza-2-deoxycytidine was used
to demethylate the RASSF1A promoter in cell lines.
Results: Promoter methylation of RASSF1A was detected in 42.9% (3 of 7) cell lines and 15% (7 of 46) primary
tumors but not in the normal control DNA. Direct sequence
analysis revealed a point mutation in a cell line and another
in a primary HNSCC. After treatment with 5-aza-2-deoxycytidine, re-expression and demethylation of RASSF1A gene
were detected in cell lines with promoter hypermethylation.
HPV DNA was detected in 34.7% (16 of 46) primary
HNSCC. We found a significant inverse correlation between
RASSF1A promoter methylation and HPV infection (P ⴝ
0.038).
Conclusions: Our results suggest that RASSF1A is inactivated in a subset of HNSCC primary tumors. Moreover,
an inverse correlation between RASSF1A and HPV supports a biological mechanism in which both RASSF1A pro-
Received 10/15/02; revised 3/20/03; accepted 4/14/03.
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.
1
Supported by NIH Grants RO1-DEO12588 entitled, “Molecular Progression Model for HNSCC” and NO1-CO-12400.
2
S. M. D. and D-I. S. contributed equally to this study.
3
To whom requests for reprints should be addressed, at Department of
Otolaryngology–Head and Neck Surgery, Division of Head and Neck
Cancer Research, The Johns Hopkins University School of Medicine,
818 Ross Research Building, 720 Rutland Avenue, Baltimore, MD
21205-2196. Phone: (410) 502-5153; Fax: (410) 614-1411; E-mail:
[email protected].
HNSCC4 occurs in ⬎50,000 Americans each year, and the
incidence of this type of tumor is expected to rise as a result of
the increasing number of female and adolescent smokers. Despite the great emphasis on early diagnosis and the efforts to
improve surgical and radiation treatment, ⬃50% of HNSCC
patients do not survive for ⬎5 years after diagnosis (1). The
growing epidemiological problem and lack of progress in head
and neck oncology emphasizes the need for basic studies on the
molecular biology of HNSCC. Over the past decade, molecular
and cytogenetic studies have contributed significantly to the
identification of genetic and epigenetic changes associated with
this cancer.
Tobacco and alcohol consumption are well-established risk
factors of HNSCC (2). Loss of chromosomal areas and mutations in oncogenes are crucial events in tumor formation (3–7).
In addition, inactivation of TSGs by promoter methylation is
also common. However, a small proportion (15–20%) of
HNSCC occurs in nonsmokers and nondrinkers, suggesting the
presence of other risk factors. Recent epidemiological and molecular data suggest that HPV infection of the upper airway may
promote head and neck tumorigenesis (8 –10). HPV-positive
oropharyngeal cancers display distinct pathological molecular
features as well as a different clinical course from HPV-negative
oropharyngeal cancers, whose etiology is linked to smoking and
drinking (9).
Allelic loss on chromosomal arm 3p is common in head
and neck cancers (11–15) and other cancers, including lung,
kidney, breast, and bladder (14 –16). Dammann et al. (20)
described the cloning and characterization of a human RAS
effector homologue, RASSF1, located in the 120-kb region of
minimal homozygous deletion at 3p21.3 in lung cancer (17).
The RASSF1A isoform was frequently inactivated in several
types of tumors (18 –20). Huang et al. (21) also provided evidence that promoter hypermethylation and allelic loss are the
major mechanisms for inactivation of RASSF1A in nasopharyngeal tumorigenesis.
In this study, we investigated whether RASSF1A alterations
might play a role in the tumorigenesis of head and neck cancer.
We also tested for HPV status based on previous evidence that
HPV and RASSF1A alterations rarely occur together in cancer
cell lines.
4
The abbreviations used are: HNSCC, head and neck cancer squamous
cell carcinoma; MSP, methylation-specific PCR; RT-PCR, reverse transcription-PCR; HPV, human papilloma virus; LOH, loss of heterozygosity; TSG, tumor suppressor gene; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; Rb, retinoblastoma gene.
Downloaded from clincancerres.aacrjournals.org on June 12, 2017. © 2003 American Association for Cancer
Research.
3636 RASSF1A Inactivation in Head and Neck Cancer
MATERIALS AND METHODS
Samples. Forty-six head and neck primary tumors from
the Johns Hopkins Hospital were selected to include a high
proportion of oropharyngeal tumors with a high frequency of
HPV positivity. Seven head and neck cancer squamous cell lines
(06, 011, 013, 019, 023, 028, and 029) were also included. DNA
was purified by phenol-chloroform extraction and ethanol precipitation and dissolved in distilled water as described previously (22).
DNA Sequence Analysis. Samples from 26 primary tumors and seven cell lines were subjected to DNA sequencing for
screen of RASSF1A gene mutation. All six exons of RASSF1A
gene were examined using the 10 pairs of primers, as described
previously (21). After detection of a PCR product, direct PCR
sequencing reactions were performed using the Amplicycle Sequencing Kit (Perkin-Elmer, Branchburg, NJ).
Bisulfite Treatment. DNA from primary tumor and cell
lines was subjected to bisulfite treatment, as described previously (23). Briefly, 1 ␮g of DNA was denatured by sodium
hydroxide and modified by sodium bisulfite. DNA samples were
then purified using the Wizard purification resin (Promega
Corp.), again treated with sodium hydroxide, precipitated with
ethanol, and resuspended in water.
MSP. Genomic DNAs, modified by bisulfite treatment
and sodium hydroxide, were used as a template for MSP. The
RASSF1A gene primer pairs specific for methylated and unmethylated DNA were listed as described previously (21).
Each PCR product was directly loaded on 4% agarose gels,
stained with ethidium bromide, and visualized under UV illumination.
Microsatellite Analysis of DNA. A panel of four dinucleotide microsatellite repeats PCR primers (D3S1588,
D3S1621, D3S1568, and D3S3582; Research Genetics, Huntsville, AL) was used to identify losses at the 3p21.3 region. PCR
conditions and criteria for LOH and homozygous deletion were
described previously (22).
Expression Analysis of RASSF1 Isoforms and RNA
Analysis. Isoform-specific RT-PCR assays were used for
analysis of RASSF1A and RASSF1C expression. Primers for
RASSF1A and RASSF1C included R182 and either NF or
NOX182 as described previously (19). Total RNA was isolated
from the head and neck cancer cell lines by TRIzol extraction
(Life Technologies, Inc., Rockville, MD). Five ␮g of total RNA
were reverse transcribed by use of Super Script First Strand
cDNA kit (Invitrogen Co.). To induce expression of RASSF1A
after exposure to 5-aza-2-deoxycytidine, a drug that inhibits
DNA methylation, subconfluent cultures of either RASSF1Aexpressing or nonexpressing cell lines, were exposed to 1 ␮M
5-aza-2deoxycytidine for 7 days. After isolation of total RNA
using RNeasy extraction kit (Qiagen), multiplex RT-PCR was
performed for GAPDH and either RASSF1A or RASSF1C.
RT-PCR of GAPDH transcripts was performed with PCR primers described previously (19). RT-PCR products were separated
by 4% agarose gel electrophoresis and visualized after staining
with ethidium bromide.
Analysis of p53. Using the Affymetrix GeneChip p53
reagent kit, genomic DNA from tumor or cell line samples was
PCR amplified for p53 exons 2–11. Next, the amplified DNA
Fig. 1 Expression of RASSF1A after treatment of head and neck
cancer cells with 5-aza-2-deoxycytidine. 013, 019, 028 head and neck
squamous carcinoma cell lines that express RASSF1C (data not shown)
but not RASSF1A were grown in the presence (⫹ lanes) and absence (⫺
lanes) of 1 ␮M 5-aza-2-deoxycytidine for 7 days. Total RNA was
isolated, cDNA was prepared, and isoform-specific RT-PCR was performed for RASSF1A and GAPDH as a control.
was fragmented with calf intestine alkaline phosphatase
(Roche). The fragmented DNA was fluorescently end-labeled
using Enzo’s BioArray Terminal Labeling Kit. The samples
were hybridized to the GeneChip p53 array and washed then
visualized using a GeneArray scanner. A reference DNA sample
(provided in the Affymetrix kit) was also run to use as a baseline
sequence for analysis. Samples read as mutated with the GeneChip p53 array were manually sequenced for confirmation.
Analysis of HPV. Purified genomic DNA was amplified
by PCR for the HPV-16 and HPV-18 E7 genes as well as for an
internal reference gene, ␤-globin. Oligonucleotide primers and
PCR conditions were described previously (24).
Statistical Analysis. Statistical analysis was performed
by use of ␹2 and Fisher’s exact tests to test for differences
between groups. All analyses were performed using SPSS Window version 9.0 (SPSS, Inc., Chicago, IL).
RESULTS
To investigate epigenetic silencing of RASSF1A in
HNSCC, we first tested for promoter methylation in seven head
and neck HNSCC cell lines (06, 011, 013, 019, 023, 028, and
029). Using MSP, 42.9% (3 of 7) of cell lines demonstrated
RASSF1A methylation. To determine the precise pattern of CpG
methylation within the RASSF1A CpG islands, we directly sequenced all three MSP products from the methylated cell lines.
All three samples demonstrated methylation of all 16 CpG sites
within the amplified fragment. RT-PCR analysis of the three
HNSCC cell lines with RASSF1A methylation demonstrated
complete transcriptional silencing, whereas RASSF1A expression was detected in the other four cell lines without RASSF1A
methylation. To confirm that promoter hypermethylation contributes to the lack of expression of RASSF1A in the HNSCC
cell lines, we assessed the effect of 5-aza-2-deoxycytidine, a
drug that inhibits DNA methylation. We exposed the
RASSF1A-nonexpressing HNSCC cell lines (013, 019, and
028) to 5-aza-2-deoxycytidine for 3 days and found re-expression of RASSF1A by these cell lines but little or no change in the
expression of the housekeeping gene GAPDH (Fig. 1) or in the
expression of the other isoform RASSF1C (data not shown).
We then tested 46 HNSCC primary tumors for allelic loss
using microsatellite markers, which map close to the RASSF1A
gene. We found allelic loss in 43.5% (20 of 46) of the HNSCC
primary tumors in at least one closely mapped marker. We then
investigated the frequency of RASSF1A promoter methylation in
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Clinical Cancer Research 3637
Fig. 2 MSP for the detection of methylated RASSF1A 5⬘ CpG sequences in primary resected HNSCCs. Representative samples are
shown. M, DNA size marker (50, 100, and 150 bp); U, results with
primers specific for unmethylated sequences; M, results with primers
specific for methylated sequences. MC, in vitro methylated positive
control; UC, in vitro unmethylated positive control; H2O, negative
control with water blanks. Each lane shows the PCR results for the
methylated (#3– 42) and unmethylated (#43) sequences from HNSCCs.
all 46 HNSCC primary tumors by MSP (Fig. 2). Aberrant
promoter methylation of RASSF1A was found in 15% (7 of 46)
primary tumors but not in the paired normal DNA (Table 1).
Only three cases with RASSF1A promoter methylation demonstrated an allelic imbalance in the 3p21.3 region by microsatellite analysis.
To determine whether the RASSF1A gene was altered by
mutation in a subset of 26 primary tumors (including all four
with RASSF1A promoter methylation and absence of LOH) and
seven cell lines, we performed extensive mutational analysis for
all six exons (including intron/exon boundaries) of the
RASSF1A gene using direct PCR sequencing. We found two
missense mutations, one mutation in a cell line and another in a
primary HNSCC without evidence of promoter hypermethylation of the RASSF1A gene. The mutation was at codon 133
(GCT to TCT) that predicts Ala to Ser in the 011-cell line and
at codon 281 (TTT to TCT) that predicts Phe to Ser in HNSCC
primary tumor. The latter mutation was somatic, i.e., not present
in the matched lymphocyte DNA. The identical mutation at
codon 133 was detected previously in a breast tumor by Burbee
et al. (19).
We then screened for the presence of HPV DNA in these
tumors by PCR for both E6 and E7 genes, 34.7% (16 of 46) of
the primary tumors demonstrated the presence of HPV-16
genes, and HPV was not present in any of the cell lines. There
was an inverse correlation between RASSF1A promoter methylation and the presence of HPV in the 46 primary HNSCCs (P ⫽
0.038 by ␹2).
We further investigated the correlation between p53 gene
mutation and the presence of HPV from our data bank. Among
the 46 head and neck cancers, a result of p53 sequence information analysis was available from Affymetrix p53 chip array
followed by direct sequence analysis in all cases. A portion
(34.8%; 16 of 46) of HNSCCs had p53 gene mutation. Among
nine tumors with p53 gene mutation, only one tumor sample
also harbored HPV. Although p53 mutations were found in
one-third of all tumors, there was a marked difference in p53
mutation frequency between HPV-positive and -negative tumors
in head and neck (P ⫽ 0.026).
DISCUSSION
Genetic aberrations have been investigated as markers of
disease progression and/or outcome in HNSCC, including gain
or loss of chromosomal regions (7, 25–27). More recently,
several studies involving promoter hypermethylation have iden-
tified genes such as p16, MGMT, GST-␲, and DAP-kinase,
whose expression is down-regulated in HNSCC samples compared with normal tissue (28 –29). Mapping studies of chromosome 3 deletions provide evidence for the presence of three
discrete 3p deleted regions (3p14, 3p21, and 3p24-p25) in lung
and head and neck cancers (13, 15). It has been suggested that
the short arm of chromosome 3 harbors several TSGs, which
may be of diagnostic and therapeutic importance (12, 30).
Extensive LOH studies in carcinoma of the lung, breast, cervix,
kidney, and head and neck suggest that either a single TSG or a
group of different ones reside on 3p and contribute to the
pathophysiology of these cancers (31, 32). More recently, homozygous deletions of 3p21.3 have been reported in several
breast cancer and lung cancer cell lines, and deletion may be a
major mechanism of inactivation for RASSF1A in these cancers
(12, 15).
Ectopic expression of RASSF1A decreases in vitro colony
formation, suppresses anchorage-dependent growth, and dramatically reduces tumorigenecity in vivo (17, 19). With these
tumor suppression effects, the presence of a RAS association
domain suggests that RASSF1A may function as an effecter
molecule in the RAS-activated growth inhibition signaling pathways. Recent studies suggest that RASSF1A inhibits accumulation of native cyclin D1, and RASSF1A-induced cell cycle
arrest can be relieved by ectopic expression of cyclin D1 or of
other downstream activators of the G1-S phase transition (33).
There is strong evidence that RASSF1A functions as a bonafide
TSG that undergoes epigenetic inactivation in cancer by methylation of the CpG islands in the promoter region (16 –21, 34).
We have confirmed the presence of RASSF1A methylation in
15% of primary HNSCCs and higher frequency in cell lines.
After treatment of 5-aza-2-deoxycytidine in HNSCC cell
lines with RASSF1A gene promoter hypermethylation, reexpression and demethylation of the promoter region of
RASSF1A gene were demonstrated. Our results suggest that
aberrant hypermethylation of the RASSF1A promoter region is
directly responsible for transcriptional inactivation of its expression in HNSCC cell line as shown in other tumor types (18, 19).
Half (50%) of primary HNSCCs showed LOH at the 3p21.3
region. These results are consistent with reports that methylation
and LOH are the major loss function pathways for RASSF1A
inactivation because we detected only one primary tumor with a
somatic mutation. Other studies have also reported RASSF1A
methylation in head and neck and other tumors and the observation that point mutations of this gene are rare (18 –21, 35–38).
Inactivation in one case (primary tumor #3) by point mutation of
one allele and promoter hypermethylation in the other provides
additional support for a complete abrogation of RASSF1A function in tumor cells.
HPV has also been implicated in the etiology of HNSCC.
Recent observations suggest that patients with HPV-associated
HNSCC may display a clinical course different from those of
patients with HNSCC whose etiology in linked to smoking and
drinking (9). HPV was detected in primary HNSCC tumors
without RASSF1A promoter methylation status. This result
points to a strong inverse correlation between HPV infection
and RASSF1A silencing/promoter hypermethylation. This correlation could also reflect a functional role of the cellular
RASSF1A and viral proteins in the G1 cell cycle check point.
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3638 RASSF1A Inactivation in Head and Neck Cancer
Table 1
LOH at RASSF1A
3p21.3 methylation
Cell line
06
011
013
019
022
028
029
Primary tumor
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
a
b
HNSCC primary tumora
RASSF1A mutation
HPV infection
p53 mutation
Site
nd
nd
nd
nd
nd
nd
nd
⫺
⫺
⫹
⫹
⫺
⫹
⫺
⫺
133, GCT(Ala) ⫺ TCT(Ser)
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
36, CCG(Pro) ⫺ CCA(Pro)b
248, CGG(Arg) ⫺ CTG(Leu)
138, ⫺1 bp
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫹
⫺
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫹
⫹
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫹
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
281, TTT(Phe) ⫺ TCT(Ser)
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
⫺
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
⫺
⫺
⫺
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫹
⫹
⫺
⫺
⫹
⫹
⫹
⫺
Tongue
281, GAC(Asp) ⫺ GAG(Gln)
Tongue
278, CCT(Pro) ⫺ GCT(Ala)
Tongue
⫺
Tongue
⫺
Tongue
247, AAC(Asn) ⫺ ATC(Ile)
Tongue
⫺
Tongue
342, CGA(Arg) ⫺ TGA(stop)
Tongue
193, CAT(His) ⫺ CTT(Leu)
Floor of mouth
⫺
Floor of mouth
⫺
Floor of mouth
220, TAT(Tyr) ⫺ TGT(Cys)
Floor of mouth
272, GTG(Val) ⫺ ATG(Met)
Alveolus
⫺
Alveolus
⫺
Supraglottis
146, TGG(Trp) ⫺ TGA(stop)
RMT
248, CGG(Arg) ⫺ TGG(Trp)
RMT/Alveolus
⫺
Hypophar, wall
243–244, ⫺3 bp
Pyriform sinus
⫺
Glottic larynx
⫺
Tonsil
⫺
Tonsil
⫺
Tonsil
285, GAG(Glu) ⫺ TAG(stop)
Tonsil
342, ⫺1 bp
Tonsil
⫺
Tonsil
⫺
Tonsil
⫺
Tonsil
⫺
Tonsil
⫺
Tonsil
⫺
Tonsil
⫺
Tonsil
⫺
Base of tongue
⫺
Base of tongue
⫺
Base of tongue
⫺
Base of tongue
192–193, CAGCAT ⫺ CATTAT (2 aa) Base of tongue
237, ATG(Met) ⫺ ATT(Ile)
Base of tongue
36, CCG(Pro) ⫺ CCA(Pro)b
Base of tongue
⫺
Base of tongue
⫺
Base of tongue
⫺
Base of tongue
196, CGA(Arg) ⫺ TGA(stop)
Base of tongue
⫺
Base of tongue
⫺
Base of tongue
⫺
Base of tongue
Tongue
Larynx
Unknown
Base of tongue
Larynx
Unknown
Base of tongue
⫹, positive; ⫺, negative; nd, not detected; RMT, retromolar trigone; ALV, alveolar ridge.
Possible splice site mutation, attributable to proximity to intron:exon boundary.
P16, cyclin D1 (and rarely, Rb) are critical regulators of the G1
checkpoint and play important roles in the tumorigenesis of
head and neck cancer. The E7 papillomavirus protein can bypass
Rb family-dependent cell cycle regulation by directly inhibiting
the interaction of Rb protein with E2F transcription factors and
other pocket-binding proteins. H1299 and HeLa CCL2 cell lines
are known to be insensitive to RASSF1A-induced cell cycle
arrest. This is likely attributable to the expression of the papillomavirus E7 protein that can bypass the Rb family cell cycle
checkpoint control (9).
Recently, Agathanggelou et al. (20) also described the
absence of RASSF1A promoter methylation in primary cervical
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Research.
Clinical Cancer Research 3639
cancers. Although the report did not demonstrate the presence of
HPV in cervical cancers, we can postulate a low frequency of
RASSF1A promoter methylation because of a high rate HPV
infection in these tumors. Thus, an inverse correlation between
RASSF1A and HPV in cancers may be common.
There was no association between RASSF1A inactivation
and p53 status. The inverse association between p53 mutations
and HPV presence in head and neck cancer continues to support
two parallel pathways of HNSCC development (9, 39, 40). Our
results further suggest that there may be important overlapping
pathways in the development of head and neck cancers between
HPV and RASSF1A. Although the E6 protein inactivates p53,
E7 may block Rb family-mediated cell cycle arrest in oropharyngeal tumors. Thus, RASSF1A inactivation now represents
another important genetic feature of HPV-negative tumors.
Our study provides evidence that promoter hypermethylation is the major mechanism for inactivation of RASSF1A in
HNSCC and indicates this gene may be a key tumor suppressor
target at 3p21.3. Some sites were underrepresented in our study
(e.g., larynx) and could harbor a higher frequency of RASSF1A
methylation. The inverse correlation between HPV infection and
RASSF1A silencing in the development of head and neck cancers is based on a limited number of tumors and remains to be
fully explored.
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Epigenetic Inactivation of RASSF1A in Head and Neck
Cancer
Seung Myung Dong, Dong-Il Sun, Nicole E. Benoit, et al.
Clin Cancer Res 2003;9:3635-3640.
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