Brakenhoff, R.H.

Joenje, H.

Snijders, P.J.F.

Zeeburg, H.J.T. van

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Re: Human Papillomavirus
DNA and p53 Polymorphisms
in Squamous Cell Carcinomas
From Fanconi Anemia Patients
Kutler et al. (1) recently reported that
human papillomavirus (HPV) DNA is
frequently detected in squamous cell
carcinomas (SCCs) of Fanconi anemia
patients, who are predisposed to develop
cancer at an early age, particularly acute
myeloid leukemia and SCCs of the head
and neck (HNSCCs) and the anogenital
region. Kutler et al. speculated that HPV
infection might be critically involved in
the pathogenesis of SCC in Fanconi ane-
mia patients, which raises hope that vac-
cination against HPV might prevent
such tumors. HPV is known to be etio-
logically involved in SCCs that arise in
the anogenital region (cervix) and in a
minority of HNSCCs, particularly those
in the oropharynx (2,3). Inhibitory on-
coproteins expressed by HPV cause in-
activation of the p53 and pRb pathways.
HNSCCs that contain transcriptionally
active HPV DNA typically lack muta-
tions in TP53, the gene encoding p53,
whereas 50%–60% of HNSCCs that do
not contain HPV DNA have mutations
in TP53 (2).
Kutler et al. (1) report that 15 of the
18 HNSCCs (all in the oral cavity) from
the Fanconi anemia patients in their
study had detectable HPV DNA and that
none of the 18 HNSCCs contained a
mutation in TP53. HPV DNA was de-
tected by polymerase chain reaction
(PCR) in laser-microdissected tissue, and
quantitative PCR was used to determine
the HPV DNA copy number per tumor
cell. A cutoff value set at more than one
copy per 10 cells was used to designate a
sample positive for HPV DNA.
We have collected similar informa-
tion on HNSCCs from Fanconi anemia
patients and have obtained different re-
sults. Of the five HNSCCs studied, none
were HPV DNA-positive and four had a
mutation in TP53. Although we studied
fewer tumors than Kutler et al., the fre-
quencies of HPV DNA positivity and
TP53 mutations are statistically signifi-
cantly different when comparing their
results with ours (P  .002 for presence
of HPV DNA, and P.001 for muta-
tions in TP53). The basis for these dif-
ferences is unclear. They may reflect
differences in ethnic backgrounds or
life-style factors (i.e., tobacco and/or al-
cohol use, disease history, or sexual be-
havior) between the patients we studied
and those studied by Kutler et al. In
addition to patient-related differences,
slight differences in HPV DNA detec-
tion methodologies might account for
part of the discrepancy between our re-
sults and those of Kutler et al. We used
a semiquantitative PCR-based method
with consensus primers and an oligo-
probe cocktail to detect the DNA of 14
high-risk HPV types in total tumor
DNA; this method can detect as few as
one copy of HPV16 DNA per 5000 cells
(4). However, none of the HNSCC spec-
imens from the Fanconi anemia patients
we tested were positive for HPV DNA,
which made further quantitative analysis
redundant. Results of previous studies
suggest that the likely involvement of
HPV in the etiology of a tumor is re-
flected by the presence of at least one
copy of HPV DNA in every cancer cell
(2,5). To obtain independent confirma-
tion of the HPV DNA status of HNSCCs
from Fanconi anemia patients, we sug-
gest that loss-of-heterozygosity patterns
in the tumor DNA could be evaluated,
because we have found that HNSCCs with
transcriptionally active HPV DNA ap-
pear to exhibit a loss-of-heterozygosity
pattern that is strikingly different from
that observed among HNSCCs without
detectable HPV DNA (6).
Until these issues have been exam-
ined in more detail, we wish to caution
against raising too much hope that HPV
vaccination (7) will be an option to pre-
vent HNSCC in Fanconi anemia pa-
tients. The current policy of frequent
surveillance and examination of biopsy
samples of suspected tissues in the oral
cavity and oropharynx should remain
part of the standard practice in the clin-
ical management of Fanconi anemia pa-
tients, including those who are enrolled
in preventive vaccination trials.
HESTER J. T. VAN ZEEBURG
PETER J. F. SNIJDERS
HANS JOENJE
RUUD H. BRAKENHOFF
REFERENCES
(1) Kutler DI, Wreesmann VB, Goberdhan A,
Ben-Porat L, Satagopan J, Ngai I, et al.
Human papillomavirus DNA and p53 poly-
morphisms in squamous cell carcinomas from
Fanconi anemia patients. J Natl Cancer Inst
2003;95:1718–21.
(2) van Houten VM, Snijders PJ, van den Brekel
MW, Kummer JA, Meijer CJ, van Leeuwen B,
et al. Biological evidence that human papillo-
maviruses are etiologically involved in a sub-
group of head and neck squamous cell carci-
nomas. Int J Cancer 2001;93:232–5.
(3) Gillison ML, Koch WM, Capone RB, Spaf-
ford M, Westra WH, Wu L, et al. Evidence for
a causal association between human papillo-
mavirus and a subset of head and neck can-
cers. J Natl Cancer Inst 2000;92:709–20.
(4) van den Brule AJ, Pol R, Fransen-Daalmeijer
N, Schouls LM, Meijer CJ, Snijders PJ.
GP5/6 PCR followed by reverse line blot
analysis enables rapid and high-throughput
identification of human papillomavirus geno-
types. J Clin Microbiol 2002;40:779–87.
(5) Ha PK, Pai SI, Westra WH, Gillison ML,
Tong BC, Sidransky D, et al. Real-time quan-
titative PCR demonstrates low prevalence of
human papillomavirus type 16 in premalig-
nant and malignant lesions of the oral cavity.
Clin Cancer Res 2002;8:1203–9.
(6) Braakhuis BJ, Snijders PJ, Keune WH, Meijer
CJ, Ruijter-Schippers HJ, Leemans CR, et al.
Genetic patterns in head and neck cancers that
contain or lack transcriptionally active human
papillomavirus. J Natl Cancer Inst. In press
2004.
(7) Koutsky LA, Ault KA, Wheeler CM, Brown
DR, Barr E, Alvarez FB, et al. A controlled
trial of a human papillomavirus type 16 vac-
cine. N Engl J Med 2002;347:1645–51.
NOTES
Affiliations of authors: Department of
Otolaryngology/Head-Neck Surgery (HJTVZ,
RHB), Department of Pathology (PJFS), and De-
partment of Clinical Genetics and Human Genet-
ics (HJ), Vrije Universiteit Medical Center, Am-
sterdam, the Netherlands.
Correspondence to: R. H. Brakenhoff, PhD,
Tumor Biology Section, Department of Otolaryn-
gology/Head-Neck Surgery, Vrije Universiteit
Medical Center, P.O. Box 7057, 1007 MB, Am-
sterdam, The Netherlands (e-mail: rh.brakenhoff@
vumc.nl).
DOI: 10.1093/jnci/djh178
RESPONSE
We reported a high prevalence of hu-
man papillomavirus (HPV) DNA and
the absence of p53 mutations among 24
squamous cell carcinomas (SCCs) from
25 Fanconi anemia patients (1). By con-
968 CORRESPONDENCE Journal of the National Cancer Institute, Vol. 96, No. 12, June 16, 2004
 by guest on April 3, 2011
jnci.oxfordjournals.org
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nloaded from
 
trast, van Zeeburg et al. describe an un-
published series of five cases of head
and neck squamous cell carcinoma
(HNSCC) from Fanconi anemia patients
in which they identified mutations in
p53 in four cases and the absence of
detectable HPV DNA in all cases.
Several factors may explain the dif-
ferent results obtained in these two stud-
ies, the least persuasive of which are the
small sample size in the van Zeeberg et
al. study and methodologic differences
between the studies, including the
source of DNA (we used laser capture
microdissected specimens), the oligonu-
cleotide primers used for HPV DNA
detection (we used highly efficient CPI
and CPIIG consensus primers and vali-
dated all findings with real-time poly-
merase chain reaction), and stringency
of analyses performed (our experiments
were repeated at least three times) (2,3).
One of the more important reasons
for the differing results may be case
selection. We included all cases of SCC
reported in the International Fanconi
Anemia Registry (IFAR) without re-
strictions. Conversely, the cases van
Zeeburg et al. report on were presum-
ably selected on the basis of the authors’
ability to establish long-term tumor cell
cultures (at least for four of the five
reported cases). This is an important dis-
tinction, because SCCs from Fanconi
anemia patients are known to be difficult
to put into culture, and therefore the
cases reported by van Zeeburg et al. may
not be representative of the HNSCCs
found in Fanconi anemia patients.
A major clinical difference between
SCCs in Fanconi anemia patients and
those in the general population is the
lower prevalence of tobacco and/or al-
cohol use among Fanconi anemia pa-
tients, which is known to have a sub-
stantial impact on the pattern of
genomic aberrations in SCCs (4). Al-
though van Zeeburg et al. agree that this
is an important issue, they do not pro-
vide the carcinogen exposure histories
for their case patients. Interestingly, one
of the authors has previously published
data on the absence of detectable HPV
DNA in two of the five cases included in
the current series (5). In that study, both
cases of HNSCC occurred in patients
who had a clinically significant history
of tobacco or alcohol exposure, which
sharply contrasts with the tobacco and/or
alcohol exposures of most Fanconi ane-
mia patients who develop SCC (5).
Our unpublished observations sug-
gest that the incidence, pathogenesis,
and genomic composition of Fanconi
anemia-associated HNSCC that develop
in patients who suffer graft-versus-host
disease after having a bone marrow
transplant may be different from those
occurring in the absence of this disease
(Kutler DI, Wreesmann VB, Goberdhan
A, Ben-Porat L, Satagopan J, Ngai I, et
al.: unpublished observations). In this
respect, it is interesting to note that the
rate of graft-versus-host disease is his-
torically higher in Europe than in the
United States due to differences in trans-
plantation protocols. This clinical infor-
mation should also be provided by van
Zeeburg et al. to allow a meaningful
assessment of their results.
Finally, on the basis of their unpub-
lished findings, van Zeeburg et al. sug-
gest that a unique pattern of loss-of-
heterozygosity may help identify HPV
DNA-positive HNSCCs. However,
there are several problems with this ap-
proach. Fewer than 10% of patients with
HNSCC in the general population report
having no history of tobacco or alcohol
use. Accordingly, the substantial amount
of tobacco and/or alcohol consumption
has complicated delineation of the con-
tributions of HPV to the pathogenesis of
HNSCC in the general population, not to
mention the identification of reproduc-
ible differences in the genetic composi-
tion in HPV-related and HPV-unrelated
cases of HNSCC. We performed a
genomic screening study comparing all
cases of HNSCC in Fanconi anemia pa-
tients (N  18) with a group of ran-
domly selected cases of HNSCC from
the general population (n  42; Kutler
DI, Wreesmann VB, Goberdhan A, Ben-
Porat L, Satagopan J, Ngai I, et al.:
unpublished data). We found statisti-
cally significant differences in the pat-
tern and frequency of chromosomal
aberrations detected by comparative
genomic hybridization between HNSCCs
from the two populations. This result
differs considerably from that in a pre-
vious report on two of the five cases in
the authors’ series, in which no differ-
ences in chromosomal aberrations were
found between the HNSCCs in two
Fanconi anemia patients and those in
the general population, reflecting the
selection bias for the cases in their se-
ries (5).
Overall, the high prevalence of
HNSCC in Fanconi anemia patients re-
quires diligent surveillance of this pa-
tient population. On the basis of our
findings, the role of HPV in the patho-
genesis of HNSCC in Fanconi anemia
patients merits further investigation, as
does HPV vaccination as a mode of can-
cer prevention.
DAVID I. KUTLER
VOLKERT B. WREESMANN
BHUVANESH SINGH
REFERENCES
(1) Kutler DI, Wreesmann VB, Goberdhan A,
Ben-Porat L, Satagopan J, Ngai I, et al. Hu-
man papillomavirus DBA and p53 polymor-
phisms in squamous cell carcinomas from
Fanconi anemia patients. J Natl Cancer Inst
2003;95:1718–21.
(2) van den Brule AJ, Pol R, Fransen-Daalmeijer
N, Schouls LM, Meijer CJ, Snijders PJ.
GP5/6 PCR followed by reverse line blot
analysis enables rapid and high-throughput
identification of human papillomavirus geno-
types. J Clin Microbiol 2002;40:779–87.
(3) Ha PK, Pai SI, Westra WH, Gillison ML,
Tong BC, Sidransky D, et al. Real-time quan-
titative PCR demonstrates low prevalence of
human papillomavirus type 16 in premalig-
nant and malignant lesions of the oral cavity.
Clin Cancer Res 2002;8:1203–9.
(4) Brennan JA, Boyle JO, Koch WM, Goodman
SN, Hruban RH, Eby YJ, 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.
(5) Hermsen MA, Xie Y, Rooimans MA, Meijer
GA, Baak JP, Plukker JT, et al. Cytogenetic
characteristics of oral squamous cell carcino-
mas in Fanconi anemia. Fam Cancer
2001;1:39–43.
NOTES
Affiliation of authors: Laboratory of Epithelial
Cancer Biology, Memorial Sloan-Kettering Can-
cer Center, New York, NY.
Correspondence to: Bhuvanesh Singh, MD,
Laboratory of Epithelial Cancer Biology, Memo-
rial Sloan-Kettering Cancer Center, 1275 York
Ave., New York, NY 10021 (e-mail: singhb@
mskcc.org).
DOI: 10.1093/jnci/djh179
Journal of the National Cancer Institute, Vol. 96, No. 12, June 16, 2004 CORRESPONDENCE 969
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Re: Human papillomavirus DNA and p53 polymorphisms in squamous cell carcinomas from Fanconi anemia patients

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Re: Human papillomavirus DNA and p53 polymorphisms in squamous cell carcinomas from Fanconi anemia patients

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