Joseph et al , 2013

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Molecular etiology of second primary tumors in contralateral
tonsils of human papillomavirus-associatedindextonsillar
carcinomas
Andrew W. Josepha,†, Takenori Ogawaa,b,†, Justin A. Bishopc, Sofia Lyford-Pikea, Xiaofei
Changa, Timothy H. Phelpsd, William H. Westraa,c,e, and Sara I. Paia,e,*
aDepartment of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions,
Baltimore, MD, USA
bDepartment of Otolaryngology-Head and Neck Surgery, Tohoku University School of Medicine,
Sendai, Japan
cDepartment of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA
dDepartment of Art as Applied to Medicine, Johns Hopkins Medical Institutions, Baltimore, MD,
USA
eDepartment of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD, USA
Abstract
Objectives—For patients with tobacco-related head and neck squamous cell carcinoma
(HNSCC), the occurrence of a second primary tumor (SPT) is an ominous development that is
attributed to a field cancerization effect and portends a poor clinical outcome. The goal of this
study was to determine whether patients with human papillomavirus (HPV)-related index tonsillar
carcinomas can also develop SPTs in the contralateral tonsil, and to discern the molecular etiology
of HPV-related tumor multifocality.
Materials and Methods—The surgical pathology archives of The Johns Hopkins Hospital were
searched for all patients with primary HPV-related tonsillar squamous cell carcinoma who
developed a synchronous or metachronous carcinoma in the contralateral tonsil. The HPV-16 E6
exon was sequenced from each independent cancer site to determine whether the tumor pairs
harbored the same or a different HPV-16 variant.
Results—Four patients with bilateral HPV-related tonsillar carcinomas were identified. In every
case, the HPV DNA sequences derived from the index tumor and corresponding SPT were 100%
concordant, indicating that the index and SPTs were caused by the same HPV-16 variant.
Conclusion—For the small subset of patients with tonsillar carcinomas who develop SPTs in the
contralateral tonsil, the index case and the SPT consistently harbored the same HPV variant. This
finding suggests that HPV-related tumor multi-focality can be attributed either to independent
© 2012 Elsevier Ltd. All rights reserved.
*Correspondence: Sara I. Pai, MD, PhD, Departments of Otolaryngology-Head and Neck Surgery and Oncology, Johns Hopkins
Medical Institutions, JHOC 6th Floor, 601 North Caroline Street, Baltimore, MD, 21287. spai@jhmi.edu . Ph: +001-410-502-9825 .
†These authors contributed equally to this work.
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Disclosure: The authors have nothing to disclose.
NIH Public Access
Author Manuscript
Oral Oncol. Author manuscript; available in PMC 2014 March 01.
Published in final edited form as:
Oral Oncol. 2013 March ; 49(3): 244–248. doi:10.1016/j.oraloncology.2012.09.009.
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inoculation events by the same virus, or by migration of HPV-infected cells from a single
inoculation site to other regions of Waldeyer’s ring.
Keywords
Human papillomavirus; head and neck cancer; oropharyngeal cancer; squamous cell carcinoma;
second primary tumor
Introduction
The 20-year cumulative incidence of second primary tumors (SPTs) in head and neck
squamous cell carcinoma (HNSCC) is approximately 36%.1 Risk factors for the
development of SPTs are often the same risk factors responsible for the initial primary
process, which traditionally have included tobacco and alcohol use. The odds of being
diagnosed with a SPT are 2.9 (95% CI 1.8-4.1) times higher in patients who smoke, and 5.2
(95% CI 3.3-7.9) times higher in patients who drink, compared with those who do not use
these substances.2 Human papillomavirus (HPV)-associated HNSCCs (HPV-HNSCC) are
less often associated with excessive tobacco and alcohol use and, thus, patients with HPV-
HNSCCs are less likely to develop smoking-related SPTs.3-6 These same patients, however,
may be at an elevated risk of developing other HPV-associated carcinomas. Men with HPV-
associated oropharyngeal carcinoma are at increased risk for developing HPV-associated
anogenital carcinoma, and several recent reports have described patients diagnosed with
HPV-HNSCCs who subsequently developed second primary cancers of the head and neck:
McGovern et al. described a 46 year-old patient with three synchronous HPV-associated
carcinomas of Waldeyer’s ring,7 Roeser et al. described a case of synchronous HPV-
associated tonsillar carcinomas,8 and Singhi et al observed 3 patients with HPV-associated
oropharyngeal carcinomas in their series of patients with HPV-associated nasopharyngeal
carcinomas.9
The purpose of this study was to identify patients with human papillomavirus (HPV)-
associated index tonsillar carcinomas and a SPT in the contralateral tonsil in order to
determine whether these synchronous or metachronous HPV-HNSCC are infected with the
same or different variant of HPV. A better understanding of this relationship may provide
insight into the mechanisms underlying SPTs in patients with HPV-HNSCC. As the burden
of HPV-HNSCC continues to rise, it will become increasingly important to understand the
risk of SPT development in this patient population and adopt personalized treatment
strategies to manage these virus-related cancers.
Patients and Methods
Subjects
This study was approved by the Institutional Review Board of the Johns Hopkins Medical
Institutions. Briefly, we searched the Johns Hopkins Hospital pathology database for all
patients diagnosed with a primary squamous cell carcinoma of the tonsil between 1999 and
2006. This search yielded 135 patients with a confirmed tissue diagnosis of tonsillar
squamous cell carcinoma. We then reviewed all of the pathology reports for these patients to
identify those who had synchronous bilateral tonsillar carcinomas or who subsequently
developed metachronous tonsillar carcinomas. We were able to identifiy a total of six cases
(or 4% of tonsillar carcinomas). One case was then excluded from the analysis because the
tumor blocks were not available for HPV sequencing. Another case was excluded because
the tonsillar cancers were not HPV associated as determined by in-situ hybridization. The
medical records for each of the subjects included in this study were thoroughly reviewed to
ensure that each case involved a second primary carcinoma, rather than recurrence. In order
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to diagnose second primary carcinomas, we utilized a modified version of the criteria that
were originally described by Warren and Gates.10,11 In order to qualify as a SPT, the
following criteria were required: 1) each neoplasm must have been anatomically separate; 2)
the possibility that a second carcinoma represented a recurrence was excluded (each
malignancy must have been separated by time [at least three years] and/or anatomical space
[at least 2 cm of normal mucosa between each lesion]). To further exclude the possibility of
direct tumor extension into adjacent compartments of Waldeyer’s ring, we restricted our
cases to tonsillar carcinomas separated by a zone (i.e. base of tongue) of clinically
uninvolved tissue. Synchronous carcinomas were defined as two geographically separate
carcinomas that were diagnosed within a six-month period. Metachronous carcinomas were
defined as geographicallyseparate carcinomas which were diagnosed greater than six
months after an index carcinoma was diagnosed.
Comparison of HPV-16 Sequences and Identification of Variants
In order to investigate the molecular etiology of SPT development in HPV-HNSCC, we first
confirmed the presence of HPV 16 DNA in the index tumor and its paired SPT by a type 16
specific assay which was performed using the in situ hybridization catalyzed signal
amplification method for biotinylated probes (DAKO GenPoint, Carpinteria, CA).
Subsequently, the viral DNA derived from the index tumor and SPT were sequenced.
Paraffin embedded specimens were obtained and processed for genomic DNA extraction.
Rather than sequence the entire HPV-16 viral genome in order to identify viral variants,
previous research in cervical cancer has demonstrated that specific E6 nucleotide changes
are able to correctly identify viral variants with low potential for misclassification.12,13
Therefore, full-length E6 was amplified using the polymerase chain reaction (PCR), gel
purified, and subsequently sequenced. Sequencing data for the primary and secondary
tumors were then aligned to compare sequence similarity and to identify HPV variants.
Numerous HPV-16 variants have been described in the literature and have been grouped into
phylogenic lineages.14 The HPV-16 variants that were detected in the tumor samples were
assigned to a phylogenic class and subclass by detection of characteristic signature gene
sequences in the Open Reading Frame (ORF) of E6, as described previously.12 The
prototype sequence of the European lineage was used as the reference for aligning the
nucleotide position and comparison of sequences.15
Results
Patients
We found that 4% (6 of 135) of patients who were diagnosed with a primary tonsillar
carcinoma developed either a synchronous or metachronous SPT in the contralateral tonsil.
A total of four subjects met criteria for inclusion and had available tissue for analysis in this
study. All patients were male and had a mean age of 59 (range: 44 – 76 years old). Three of
the four identified cases presented with synchronous tonsillar carcinomas, while the
remaining case presented with a metachronous tonsillar carcinoma. The first case reported
that he had greater than 12 lifetime sexual partners; however, the sexual histories were
unavailable for the remaining cases. Three cases presented with a T1 tumor stage for the
index carcinoma, while one patient presented with a T3 tumor. The second primary tumors
for all cases were staged as early T1 disease or carcinoma in-situ. It should be noted that for
every case, the SPT occurred at a contralateral site that was distinct from the index tumor;
therefore, these carcinomas were classified as a contralateral primary cancer rather than
multifocal or multicentric carcinomas. Furthermore, no other secondary HPV-associated
cancer in the head and neck or at other sites (anal, penile, etc.) were diagnosed in these
patients. All of these head and neck cancer patients are alive and disease free 6 to 13 years
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after treatment. Complete demographic and clinicopathological characteristics are shown in
Table 1.
HPV-16 DNA Sequence Analysis
For each case, HPV-16 DNA sequencing from the index and SPTs were found to be 100%
concordant in the E6 ORF. For case 1, the isolates obtained from both tumors belonged to
the European class of HPV-16. A comparison of the isolates to the reference sequence
demonstrated that guanine substitutions occurred at positions 131 and 350, which defines the
G131/350G subclass. The prevalence of the G131G variant is approximately 10% in North
American cervical specimens.14 The exact sequence match between the isolates obtained
from each independent tumor site suggested that a common viral variant caused the index
tumor and the SPT. Similarly, comparison of the HPV-DNA isolates from the other cases
again demonstrated consensus genetic sequences between the primary and secondary tumors
suggesting infection with the same viral variant. The HPV isolate in case 2 was the
European-prototype variant P350G which is prevalent in approximately 30% of North
American cervical specimens and the HPV isolates in cases 3 and 4 were Asian-American
variants which are prevalent indeveloped simultaneous oropharyngeal SCC caused by identical HPV variants.18
Alternatively, the initial site of HPV inoculation and cancer development could itself serve
as an internal reservoir of HPV particles for subsequent infections of other susceptible
oropharyngeal locations (e.g. contralateral tonsil or other sites within Waldeyer’s ring). In
model 3, the initial site of HPV inoculation and cancer development could serve as the
epicenter for clonal expansion and migration of HPV-infected cells to other sites. Indeed,
clonal spread within the sheath of lymphoepithelium in Waldeyer’s ring has been favored by
some as the mechanism by which paired tonsillar and nasopharyngeal HPV-associated
carcinomas may develop.9 Although the development of SPTs in the contralateral tonsil
would require cellular migration across extended tracts of Waldeyer’s ring, this same
mechanism is now recognized as an important mechanism of tumor multifocality for
smoking-related HNSCCs 19, 20. To differentiate between models 1 and 3, further studies
that evaluate the integration site of the HPV DNA in the genome of the index and the SPT
will be necessary. Demonstration of a different integration site of the HPV DNA in the
primary and SPT genome would support model 1; whereas, identification of the same
integration site in the primary and SPT would support model 3.
Our study highlights how the concept of field cancerization may need to be interpreted
differently in the setting of virus-related cancers. In virus-related cancers, exposure to an
environmental carcinogen, such as HPV, can result in cellular infection and, subsequent,
intracellular changes in distinct sites within the oropharynx. We demonstrate that the same
viral variant is the causative agent of the index and SPT which suggests that these
independent tumors may be more genetically similar than in tobacco-related index and SPTs
that develop in the upper aerodigestive tract. Therefore, the diagnosis of SPT in the setting
of HPV-HNSCC may not necessarily portend a poor prognosis as in the case of tobacco-
related head and neck cancers given the similar genetic alterations present within the
primary and secondary cancers.
In this study, the second primary tumors were detected incidentally in three of the four
patients due to the routine practice of performing bilateral tonsillectomies during the work-
up of suspected tonsil cancers or of an unknown primary at the Johns Hopkins Hospital.19
The one patient who presented with a metachronous SPT underwent a diagnostic ipsilateral
tonsillectomy followed by radiation to the ipsilateral neck with Intensity Modulated
Radiation Therapy (IMRT) at an outside hospital and, subsequently, the SPT in his
contralateral tonsil was diagnosed 32 months after treatment of his primary cancer.
Therefore, the performance of bilateral tonsillectomies during the time of initial diagnostic
work-up may help to identify multiple occult sites of HPV infection and associated cancers
which may not be readily appreciated on physical examination. In addition, given the
existing practice by some radiation oncologists for treating unilateral necks with radiation,
our data further supports a potential benefit of performing bilateral tonsillectomies for
patients in whom a restricted radiation field might be planned.
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Conclusion
We report a series of HPV-associated second primary squamous cell carcinomas of the
palatine tonsils. For each case, molecular analysis of the index carcinoma and second
primary tumor indicated that the same viral variant was the etiologic cause of both cancers at
each independent site. We propose several models for the development of SPTs in HPV-
HNSCC. Although the development of SPTs does not appear to be common for patients
with HPV-related tonsillar carcinomas, removal of the contralateral tonsil may disclose
coincident cancers.
Acknowledgments
Grant Support: This study was supported by the National Institutes of Health/National Institute of Dental and
Craniofacial Research (NIH/NIDCR) Head and Neck SPORE grant P50 DE019032.
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Figure 1.
HPV-16 nucleotide sequence variations in the E6 open reading frame (ORF) from the index
tumor and second primary tumor (SPT). The DNA sequences from the index and SPT were
identical in each case. The prototype sequence of the European lineage (E-P-350T) was used
as the reference for alignment of the nucleotide position (Reference) and comparison of
sequences.11 Common positions where nucleotide polymorphisms occur in the E6 ORF are
listed at the top of the figure. Dashes indicate no variation was found between HPV-16 DNA
in the tumor specimen and the reference sequence. A, adenine; T, thymine; G, guanine; C,
cytosine. *Major phylogenic classes include European (E), European prototype-like (E-P),
and Asian American. Variants within each class (i.e. subclasses) are specified according to
the following convention: identity of substituted nucleotide -- position of the substituted
nucleotide -- identity of the nucleotide at position 350. For example, E-G131G denotes a
variant of the European class, G131G subclass (guanine substitutions at positions 131 and
350).
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Figure 2.
Three proposed models for the development of HPV-associated second primary tumors
(SPTs). Model 1: a single HPV infection creates a field effect by transforming multiple cells
at distinct anatomical locations. Model 2: in the setting of increased HPV infection
susceptibility, multiple independent infections from several HPV types infect different areas
of squamous epithelium and result in cancer formation at different sites. Model 3: SPTs are
clonally related to an index tumor and the result of a single infection. Aberrant oncogene
expression in an index tumor results in transformation of a clone which migrates to a second
location and develops into a SPT.
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Joseph et al. Page 10
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l
T
1N
2b
M
0
N
/A
R
 to
ns
il
T
is
N
0M
0
N
/A
0
R
he
um
at
oi
d
ar
th
ri
tis
Oral Oncol. Author manuscript; available in PMC 2014 March 01.