JOURNAL OF CLINICAL MICROBIOLOGY, May 2006, p. 1755–1762
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 44, No. 5
Human Papillomavirus Type 16 Integration in Cervical Carcinoma
In Situ and in Invasive Cervical Cancer
Hugo Arias-Pulido,1Cheri L. Peyton,1† Nancy E. Joste,2Hernan Vargas,3,4‡ and Cosette M. Wheeler1*
Departments of Molecular Genetics and Microbiology1and Department of Pathology,2University of New Mexico, Health Sciences Center,
School of Medicine, Albuquerque, New Mexico; Grupo Farmacogene ´tica del Ca ´ncer, Dep. Farmacia,
Universidad Nacional de Colombia, Bogota ´, Colombia3; and Grupo de Biologı ´a Molecular Tumoral,
Instituto Nacional de Cancerologı ´a, Bogota ´, Colombia4
Received 24 October 2006/Returned for modification 5 January 2006/Accepted 20 February 2006
Integration of human papillomavirus type 16 (HPV-16) into the host DNA has been proposed as a potential
marker of cervical neoplastic progression. In this study, a quantitative real-time PCR (qRT-PCR) was used to
examine the physical status of HPV-16 in 126 cervical carcinoma in situ and 92 invasive cervical cancers. Based
on criteria applied to results from this qRT-PCR assay, HPV-16 was characterized in carcinoma in situ cases
as episomal (61.9%), mixed (i.e., episomal and integrated; 29.4%), and integrated (8.7%) forms. In invasive
cervical cancer samples, HPV-16 was similarly characterized as episomal (39.1%), mixed (45.7%), and inte-
grated (15.2%) forms. The difference in the frequency of integrated or episomal status estimated for carcinoma
in situ and invasive cervical cancer cases was statistically significant (P ? 0.003). Extensive mapping analysis
of HPV-16 E1 and E2 genes in 37 selected tumors demonstrated deletions in both E1 and E2 genes with the
maximum number of losses (78.4%) observed within the HPV-16 E2 hinge region. Specifically, deletions within
the E2 hinge region were detected most often between nucleotides (nt) 3243 and 3539. The capacity to detect
low-frequency HPV-16 integration events was highly limited due to the common presence and abundance of
HPV episomal forms. HPV-16 E2 expressed from intact episomes may act in trans to regulate integrated
genome expression of E6 and E7.
With about 400,000 new cases and nearly 250,000 deaths
each year, cervical cancer contributes significantly to world-
wide cancer-related morbidity and mortality (37). In the
United States, approximately 11,000 new cases of invasive cer-
vical cancer (CC) were expected in 2004, and approximately
4,000 women would die of the disease (12). Epidemiologic
studies have shown that low-grade squamous intraepithelial
lesions (LSIL) and high-grade squamous intraepithelial lesions
(HSIL) present different genetic as well as viral characteristics
that, under certain circumstances, give cells an advantage to
progress to more severe lesions and CC (21, 30). Further,
epidemiologic and molecular evidence is sufficient to conclude
that high-risk human papillomaviruses (HPVs), specifically
HPV types 16 and 18 (HPV-16 and -18), are etiologic agents
for CC (39). In the majority of cases, however, most HPV-
associated lesions regress spontaneously (21), indicating that
additional genomic alterations may also be necessary for pro-
gression to cancer.
HPV-16 integration into the host genome has been sug-
gested as a step associated with neoplastic progression. In vitro
studies have demonstrated that cell populations with inte-
grated HPV-16 posses a selective growth advantage compared
to cells that maintain HPV-16 viral genomes as episomes (13,
14, 27). This growth advantage is thought to result from dis-
ruption of the HPV-16 E2 open reading frame, which may lead
to increased cell immortalization capacity by augmenting the
steady-state levels of mRNAs encoding the viral oncogenes E6
and E7 (14, 27). HPV-16 integration has been reported with
different frequencies in all spectrums of cervical neoplasias
from LSIL (6, 7, 24, 32) to HSIL and CC (2, 4, 8, 10, 11, 15–18,
23, 24, 29, 31, 34–36, 40, 41). In general, the frequency of
HPV-16 viral integration increased in parallel with the severity
of cervical lesions. These reports have suggested that viral
integration could represent a risk for tumor progression (7, 8,
10, 11, 17, 24, 32, 34). In addition, HPV-16 integration has
been associated with poor clinical outcome (16, 35, 36).
The purpose of this study was to examine the physical status
of HPV-16 in a set of carcinomas in situ (CIS) and CC tumors
and map the specific viral integration sites when possible. To-
wards this end, a previously reported quantitative real-time
PCR (qRT-PCR) was applied based on the assumption that
the quantitative ratio of HPV-16 E2 to E6 gene targets would
allow discrimination of the physical status of HPV-16 (24).
MATERIALS AND METHODS
Clinical specimens and cell lines. A total of 166 HPV-16-positive tumor
biopsies representing formalin-fixed, paraffin-embedded specimens from 126 CIS
and 40 CC cases comprised part of the material. The tissues were obtained from
a New Mexico population-based study, and the characterization of a portion of
the population and HPV status has been published elsewhere (25). In addition,
52 HPV-16-positive tumor biopsies derived from patients diagnosed with inva-
sive cervical cancer at the National Cancer Institute in Bogota ´, Colombia, were
also analyzed (26). All samples were provided without patient identifiers. The
studies were approved by local internal review boards.
Paraffin-embedded tissue microdissection and processing. Tumor cells from
paraffin-embedded tissue specimens were microdissected as described by Arias-
* Corresponding author. Mailing address: Department of Molecular
Genetics and Microbiology, School of Medicine, Health Sciences Cen-
ter, University of New Mexico, Albuquerque, NM 87111. Phone: (505)
272-5785. Fax: (505) 277-0265. E-mail: firstname.lastname@example.org.
† Present address: Department of Zoology, Oregon State University,
‡ Presentaddress:GrupoFarmacogene ´ticadelCa ´ncer,Dep.Farmacia,
Universidad Nacional de Colombia, Bogota ´, Colombia.
Pulido et al. (1). Briefly, cells were isolated by manual microdissection after
toluidine blue staining (0.05% [wt/vol]), and normal and tumor DNA from each
individual specimen was crudely extracted by digestion with proteinase K (10
mM Tris, 1 mM EDTA [pH 8.5], proteinase K, 0.2 mg/ml, 0.1% Laureth-12) for
24 h at 55°C, followed by proteinase K inactivation at 95°C for 10 min. The cell
lysate was diluted 10-fold with water, and 2 ?l was used for each amplification
Fresh tissue and cell line processing. Fresh tumor biopsies (Colombian sam-
ples) were snap-frozen in liquid nitrogen and stored at ?80°C immediately
following a clinical examination. All frozen specimens used in the study were
determined to contain at least 80% tumor cells by hematoxylin and eosin stain-
ing. Tumor DNA was obtained as previously described (26). Briefly, DNA from
tumor samples and cell lines was isolated using standard procedures of protein-
ase K digestion, phenol-chloroform extraction, and ethanol precipitation. Tumor
and cell line DNAs were quantitated using Pico Green (Molecular Probes,
SiHa (HPV-16-positive) and C-33A (HPV-negative) cell lines were obtained
from the American Tissue Culture Collection. The SiHa cell line contains one to
two copies per cell of HPV-16 and presents a disruption of the E2 open reading
frame at the 3134–3384 region as a result of viral integration into the host
genome (19). The W12 cell line was kindly provided by M. A. Stanley (Depart-
ment of Pathology, University of Cambridge, United Kingdom). At low passage
levels, W12 cells stably maintain HPV-16 replicons at a copy number of approx-
imately 1,000 copies per cell (13). In our laboratory we measured HPV-16
episomes in W12 cells by qRT-PCR and detected on average of between 1 ? 103
and 5 ? 103copies of HPV-16 per cell.
Detection of HPV-16 physical status.(i) qRT-PCR. Real-time PCR for
HPV-16 E2 and E6 was performed as described elsewhere (24) except that the
E6 and E2 probes were labeled with 6-carboxyfluorescein at the 5? end and
6-carboxytetramethylrhodamine at the 3? end (Sigma-Genosys, The Woodlands,
TX). The sequences of these primers are provided in Table 1. The qRT-PCR was
performed with the GeneAmp 5700 sequence detection system (Applied Biosys-
tems, Foster City, CA). Briefly, a standard PCR was carried out in a 50-?l
reaction volume containing 1? PCR GeneAmp PCR gold buffer supplemented
with 3.0 mM MgCl2(Applied Biosystems, Foster City, CA), 0.2 mM deoxynucle-
otide triphosphates, 2 ?l of the cell lysate (1 to 5 ng of DNA in the case of
purified tumor DNA), and 1 unit of AmpliTaq DNA polymerase (Applied
Biosystems, Foster City, CA). The amplification conditions were 2 min at 50°C,
10 min at 95°C, and a two-step cycle of 95°C for 15 s and 60°C for 60 s for a total
of 40 cycles. The final primer and probe concentrations, in a total volume of 50
?l, were 0.3 and 0.1 mM, respectively. The DNA quality of each specimen was
assessed by a separate qRT-PCR for beta-globin using the forward (PCO3) and
reverse (PCO4) primers shown in Table 1. The PCR conditions were the same as
for the HPV-16 E6 and E2 genes, except the concentration of Mg was 4 mM and
a total of 50 cycles were used.
For each set of samples analyzed, a standard curve was obtained by amplifi-
cation of HPV-16 plasmid using a 10-fold dilution series (1 ? 104to 1 ? 100
copies). The standard curve was used to extrapolate the copy numbers of selected
TABLE 1. Primers used for mapping HPV-16 E1 and E2 genes and detection of beta-globin target regions
Primer5? to 3? sequenceLocationa
aNucleotide positions based on the revised, or HPV-16R, sequence (20, 22) and GenBank accession no. U01317 for beta-globin. Primers used to detect HPV-16 E6
and E2 by qPCR were described elsewhere (24).
1756ARIAS-PULIDO ET AL.J. CLIN. MICROBIOL.
gene targets present in each clinical specimen. Dilutions were prepared in a
background of a crude C-33A cell lysate (1 ? 104cells per reaction). All samples
were analyzed in triplicate. In addition, 10-fold dilutions of SiHa (1 ? 104to 1 ?
101HPV-16 genome equivalents per reaction) and W12 cell lysate (1 ? 103to
1 ? 101HPV-16 genome equivalents per reaction) and a no-template control
reaction mixture (water) were included in each microplate analysis. Assignment
of integrated, mixed or episomal physical status was calculated for each clinical
specimen as proposed elsewhere (24). This calculation assumed that E2 and E6
gene segments are present in equivalent proportions within each episomal HPV
genome and that integrated HPV genome forms would have the E2 target
deleted or absent. Thus, integration was determined by subtracting the copy
numbers of E2 (episomal) from the total copy numbers of E6 (episomal and
integrated). The ratio of episomal E2 gene target to the integrated E6 gene
target represents the amount of the HPV episomal form in relation to the
integrated form. Integration was defined by absence of the E2 signal or ratios of
0.001 to 0.003. This ratio cutoff was obtained in reconstitutions experiments when
SiHa (integrated form) was in a 10-fold excess of W12 (episomal form) (Fig. 1;
also see details in the Results section). Ratios of less than 1 (range, 0.004 to 0.99)
indicated the presence of both integrated and episomal forms, and ratios of
greater than 1 (range, 1.00 to 7.29) indicated a predominance of episomal forms.
(ii) Deletion mapping of HPV-16 E1/E2 genes by PCR. Fifty-two fresh frozen
invasive cervical cancer tumors were used initially to examine the general integ-
rity of HPV-16 E1 and E2 genes by PCR targeting of three larger gene frag-
ments. HPV-16 E1 gene targets were detected by PCR amplification of two
segments using overlapping primers E1F1 and E1R5 (1,114 bp) and E1F5 and
E2R9 (1,089 bp) (Table 1). A single HPV-16 E2 gene fragment was amplified
with primers E1F9 and E2R14 (1,228 bp; Table 1). These PCR fragments were
obtained using the Takara LA Taq amplification kit (Takara, Madison, WI).
Reactions were performed in 50 ?l of buffer that contained 2.5 mM MgCl2, 0.2
mM deoxynucleotide triphosphates, 0.5 U LA Taq polymerase, and 0.3 ?M of
each primer. Five cycles of 93°C, 30 s; 55°C, 60 s; and 72°C, 60 s were followed
by 37 cycles with the following thermal cycling: 93°C, 30 s; 50°C, 60 s; 72°C, 90 s.
All amplicons were visualized on 1.5% agarose gels following staining with
Overlapping primer pairs used to more finely map HPV-16 E1 and E2 regions
are shown in Table 1. The forward (F) and reverse (R) primers are noted for 14
primer pairs employed. HPV-16 primers were selected in an attempt to achieve
HPV type specificity by sequence alignments of all HPV genomes using the
MultAli program (3). Subsequently, 10-ng samples of HeLa, SW756 (both
HPV-18 positive), ME180 (positive for HPV 68), and C-33A were examined in
the PCR. While signals from SiHa and W12 were obtained, no signals were
observed in C-33A, HeLa, SW756, or ME180 samples. Optimization of primer
conditions was performed with a 10-fold dilution series of SiHa (1 ? 103to 1 ?
101HPV-16 genome equivalents per reaction) and W12 cells (1 ? 102to 1 ? 101
HPV-16 genome equivalents per reaction). The PCR conditions allowed detec-
tion of approximately 10 HPV-16 genome equivalents from both SiHa and W12
cells with the various primer pairs. Intensities varied slightly when PCR products
were analyzed in agarose gels.
Standard PCRs consisted of 1? GeneAmp PCR buffer supplemented with 2.5
mM MgCl2and 0.001% (wt/vol) gelatin (Applied Biosystems, Foster City, CA),
0.2 mM deoxynucleotide triphosphates, 2 ?l of the cell lysate, 30 pmol of each
forward and reverse primer, and 0.5 U of AmpliTaq DNA polymerase (Applied
Biosystems, Foster City, CA) in a total volume of 50 ?l. The amplification
conditions were 3 min at 93°C, followed by 45 cycles of 93°C for 1 min, 50°C for
1 min, and 72°C for 2 min. A final extension of 10 min at 72°C was added to
complete the PCR. Ten-fold dilutions of SiHa (1 ? 102to 1 ? 101HPV-16
genome equivalents per reaction) and W-12 (1 ? 101to 1 ? 100HPV-16 genome
equivalents per reaction) cell lysate reaction mixtures were included in each run
as positive controls along with no template (water) and C-33A (1 ? 104cells per
reaction) as negative controls. To assess DNA sample quality for this analysis,
PCR was carried out for the beta-globin gene target under the same conditions,
except that a total of 40 cycles were performed. A fragment of 473 bp beta-globin
was obtained with the primers GlobF and GlobR shown in Table 1. PCR prod-
ucts were visualized in agarose gels following staining with ethidium bromide.
(iii) HPV-16 nucleotide position numbering. HPV-16 nucleotide positions and
comparisons are based on HPV-16R (22), the sequence revised to include cor-
rections previously reported (20).
Statistical analyses. The Pearson chi-square test was used to test for indepen-
dence between the physical status of HPV-16 in CIS and CC. P values were
considered significant if less than 0.05.
Integration status of HPV-16. Reconstitution experiments
were performed using W12 and SiHa cell lines to represent
different amounts and thus ratios of episomal and integrated
forms of HPV-16. The assumption was made that in clinical
specimens, integration could be a single event potentially oc-
curring in a background of high levels of episomal HPV-16
DNA. For SiHa, given an estimated one to two HPV-16 inte-
gration events per cell, HPV-16 genome equivalents directly
approximate the number of SiHa cells assayed. For W12 cells,
approximately 1,000 to 5,000 HPV-16 genome equivalents per
cell were measured in our studies, which is in relative agree-
ment with previous information (13).
As shown in Fig. 1, ratio values close to zero (median, 0.005;
range, 0.001 to 0.006) were obtained only when SiHa (inte-
grated DNA; 1 ? 104HPV-16 genome equivalents) was in
100-fold excess of W12 (episomal DNA; 1 ? 102HPV-16
genome equivalents). A ratio value of 0.038 (range, 0.02 to
0.04) was obtained in mixtures that contained at least 10-fold
excess of SiHa. A one-to-one mixture of integrated and episo-
mal HPV-16 forms gave a ratio value of 0.25 (range, 0.15 to
0.41). A ratio value of 0.56 (range, 0.4 to 0.8) was obtained
when a 10-fold excess of episomal DNA (10 genome equiva-
lents of HPV-16 integrated forms combined with 100 genome
equivalents of HPV-16 episomal forms) was present in the
mixture. Experiments were performed in triplicate for each
qRT-PCR analysis and repeated at least three times. Similar
results were obtained when 1 ? 105to 1 ? 100HPV-16 ge-
nome equivalents per reaction of SiHa were combined with
1 ? 106to 1 ? 100HPV-16 genome equivalents per reaction of
W12. All possible combinations were examined.
PCR efficiencies for E2 and E6 targets were determined for
FIG. 1. Detection threshold of the HPV-16 qRT-PCR assay. Val-
ues in fluorescence units for the E6 and E2 gene targets are shown on
the y axis. The ratio of integrated HPV (SiHa) to episomal HPV-16
(W12) DNA in each reaction is shown on the x axis. Below the x axis
scale in parentheses is the mean ratio derived from E6:E2 qRT-PCR
values at each defined combination of integrated and episomal forms.
SiHa (1 ? 104to 1 ? 101HPV-16 genome equivalents per reaction)
was combined with 102genome equivalents of W12. Bars indicate
standard errors; open and filled circles indicate individual quantitative
PCR values obtained for E6 and E2, respectively (see the text for
VOL. 44, 2006 VIRAL INTEGRATION IN CERVICAL CANCER1757
each individual microplate assayed by the following calculation
(5): E ? 10?1/S?1, where E is run efficiency and S is the slope
of the generated standard curve. A total of 10 individual assay
runs were used to calculate the mean and median efficiency
values for each target. The E6 mean efficiency was 100.6, the
median was 93.1, and the range was 89 to 115. The E2 mean
efficiency was 99.1, the median was 93.1, and the range was 83
to 115. In our study, E6 and E2 were quantitated in triplicate
and the coefficient of variation for the assay was less than 29%,
which is in agreement with other studies of HPV qRT-PCR
assays (6, 9). In this work, we defined integration events only
for results that gave ratios of 0.001 to 0.003 or no signals for
HPV-16 E2 targets but that were positive for HPV-16 E6 and
beta-globin gene targets.
Analysis of HPV-16 physical status by the described qRT-
PCR in 126 paraffin-embedded CIS samples showed the pres-
ence of episomal forms in 78 (61.9%), mixed (episomal and
integrated) forms in 37 (29.4%), and integrated forms in 11
(8.7%) CIS samples. The analysis of the physical status in 40
paraffin-embedded CC showed that 14 (35%), 19 (47.5%), and
7 (17.5%) samples presented the virus in episomal, mixed, and
integrated forms, respectively. In 52 fresh-frozen tumors, 22
(42.3%), 23 (44.2%), and 7 (13.5%) were characterized as
episomal, mixed, and integrated HPV forms, respectively.
Combining results from archival paraffin-embedded and fresh
frozen invasive cervical tumors, the HPV-16 episomal, mixed,
and integrated forms were found in 36 (39.1%), 42 (45.7%),
and 14 (15.2%) tumors, respectively. The difference between
HPV-16 genomic forms characterized in CIS and CC was sta-
tistically significant (P ? 0.003).
The microdissected paraffin-embedded tissues were gener-
ally positive only for HPV-16 (126 CIS and 40 CC). Coinfec-
tions with other HPV types were detected in six samples.
HPV-33 (n ? 2), -35, -45, and -59 were detected as single
additional HPV infections. One sample presented coinfection
with both HPV-31 and HPV-53. This is consistent with a gen-
eral clonal origin of cancer and its true precursors. Three, two,
and one of the six samples were determined to have HPV-16 in
episomal, mixed, and integrated forms, respectively. Four sam-
ples in the fresh-frozen tumor series were positive for HPV-31
(mixed), -35 (mixed), -39 (episomal), and -61 (mixed) in addi-
tion to HPV-16. One additional sample contained both
HPV-39 and HPV-62 (mixed). Given the limited number of
HPV coinfections we observed, there was no association of
mixed infections with HPV integration.
Extensive mapping analysis of HPV-16 E1 and E2 genes was
performed with samples that demonstrated HPV-16 integrated
as well as mixed and episomal forms with the qRT-PCR assay.
In the representative samples shown in Fig. 2, 35 samples
demonstrated deletions that were sometimes accompanied by
rearrangement. Two additional samples shown (C29 and C57)
had no deletions but contained rearrangements. The pattern of
E1 and E2 losses found in 18 paraffin-embedded and 19 fresh-
frozen tumors is shown in Fig. 2. This analysis indicated that
loss of E2 was more common than losses at E1 in both CIS and
CC samples. The 3243–3539 fragment, a region that contains
the HPV-16 E2 hinge region, was not detected and was pre-
sumed deleted in 29 (78.4%) of 37 samples (Fig. 2). The
second most commonly deleted region was located in the N
terminus (2783 to 3063) of E2. This deletion was observed in
26 (70.3%) of 37 samples. Nineteen of 37 (51.4%) samples
showed losses in the E1 gene region.
No detectable deletions in the full-length HPV-16 E1 and
E2 gene fragments were found in 46 (88.5%) and 35 (67.3%)
of the 52 fresh-frozen tumors, respectively. Thus, no deletions
or potential integration sites were identified in a significant
proportion of tumors.
Analysis of rearranged sequences. Possible rearrangements
were considered when multiple bands were observed upon gel
analysis of PCR amplicons. A few samples demonstrated a
complex pattern of integration: losses of single E1/E2 frag-
ments were interspersed with PCR positivity in adjacent E1-E2
regions (samples C22, C49, C96, 111, 124, i256, and 266) (Fig.
2). Sequence analyses were conducted on four samples (C28,
C29, C34, and C57) with presumed rearrangements located
within the primer pair E1F9-E2R14. A fragment of 583 bp was
isolated with this primer pair from C28, C29, and C57 samples.
A fragment of 520 bp was obtained with this primer pair for
C34. Alignment of the HPV-16 reference clone sequence dem-
onstrated no HPV-16 similarities for the 520-bp fragment gen-
erated from specimen C34. However, a fragment that consisted
of two HPV-16 segments that were head-to-tail linked was
reconstructed for C28, C29, and C57 (Fig. 3).
Several studies reported in this journal (23, 24, 40) have
suggested that detection of HPV-16 integration status through
evaluating the HPV-16 E6/E2 ratio is a sensitive method that
might be useful in assessing cervical cancer risk. Given the
inter- and intra-assay variations in qRT-PCR (6, 9) and the fact
that HSIL are often surrounded by LSIL containing a high
copy number of HPV episomal forms (28), we believed that
use of this technique may not be readily amenable to routine
detection of HPV-16 integration. We conducted reconstitution
experiments to assess the sensitivity of the assay. In our hands,
this method allowed distinction of integrated versus episomal
HPV-16 DNA only when integrated forms were in 100-fold
excess of episomal DNA forms. A 10-fold excess of integrated
viral DNA above the episomal form gave values close to 0
(range, 0.02 to 0.04), and a slight decrease in the copy number
of integrated viral DNA shifted the values, which by this assay
would indicate the presence of mixed forms (i.e., both inte-
grated and episomal forms) of HPV-16 (Fig. 1). Similar results
have been reported that suggested a lack of sensitivity of the
assay examined here (23).
In spite of these observations, we attempted to apply this
technique to assess the physical status of HPV-16 in cervical
tumors. Review of the literature indicates the presence of both
integrated and episomal HPV genomic forms in cervical can-
cers, but the extent of this phenomenon is unclear. Analysis of
the viral status using the qRT-PCR assay showed that, in gen-
eral, exclusively integrated HPV-16 genomes were detectable
as an infrequent event in both CIS (8.7%) and CC (15.2%)
microdissected tumors. Instead, episomal and mixed forms
were commonly detected. Analyses using microdissected par-
affin-embedded samples versus DNA from fresh tissues
showed that the frequencies of episomal (35.0% versus 42.3%),
mixed (47.5% versus 44.2%), and integrated (17.5% versus
13.5%) forms of HPV-16 were similar by this assay. As ex-
1758ARIAS-PULIDO ET AL.J. CLIN. MICROBIOL.
pected, our results suggest that the E2/E6 qRT-PCR assay was
not able to commonly detect HPV-16 integration events even
in microdissected, tumor-enriched populations of cells. This is
presumably due to the common presence of HPV-16 episomal
forms in the majority of cervical cancers. In addition, the ex-
istence of deletions outside of the proposed E2 target and the
presence of concatemers that, though integrated, still conserve
intact copies of the E2 gene could mask the detection of inte-
gration in some samples.
Mapping analyses of E1 and E2 amplicons demonstrated
FIG. 2. Deletion mapping of HPV-16 E1 and E2 genes in CIS and CC samples. Selected samples demonstrating E1 and E2 losses (n ? 35) and
two tumors containing rearrangements with no losses (C29 and C57) are included. ID column indicates individual specimen identifiers; fresh frozen
cervical tumors are designated by C followed by numeric assignments; paraffin-embedded specimens are denoted by numeric assignments with no
alpha modifier for CIS and the addition of an i preceding the number for invasive CC tumors; qPCR column designates integrated, episomal, and
mixed forms based on E2:E6 ratio determinations. Various columns indicated by F represent the upstream primer for each pair specified in Table
1. R, rearrangements detected by gel analysis; ND, not determined.
VOL. 44, 2006VIRAL INTEGRATION IN CERVICAL CANCER1759
the extent of deletions outside of the E2 region targeted by
the qRT-PCR assay. In addition, a few cases demonstrated
a complex pattern of deletions, suggesting the presence of
rearranged HPV-16 sequences (Fig. 2). Rearrangements of
HPV-16 sequences have been previously described, but no
attempt to characterize the involved regions was made (15, 33,
41). Because short identical nucleotides (up to 10 bases) at the
viral and cellular sites seem to facilitate viral integration, it has
been suggested that nonhomologous end-joining recombina-
tion may occur in vivo (41). We isolated and identified in three
samples a 17-bp fragment that could potentially allow recom-
bination of HPV fragments by this mechanism (Fig. 3). It is
interesting that this short segment is found in the two regions
that showed the highest integration frequency in all samples
analyzed: the first location is at nucleotides 2854 to 2870, a
region covered by primers E2F10 and E2R10, and the second
location is at nucleotides 3452 to 3468, a region targeted by
primers E2F12 and E2R12 (Fig. 3). Whether the 17-nucleotide
sequence is involved in sequence-specific viral recombination
events, such as those consistent with molecular “baiting” (38),
remains unknown. Further systematic studies of both E1 and
E2 and cellular integration junctions are warranted.
Loss of E1 and E2 open reading frame upon HPV-16 inte-
gration in cervical lesions and cancer has been reported in
several studies (2, 4, 8, 10, 11, 15–18, 23, 24, 29, 31, 34–36, 40,
41). We found in our study that integration occurred more
frequently in the E2 gene. Specifically, the E2 hinge region,
targeted by primers E2F12 and E2R12 (nt 3243 to 3539), was
deleted in 78.4% of 37 selected samples, followed by deletions
in 67.6% of specimens in the region covered by primers E2F10
and E2R10 (nt 2783 to 3063). Although HPV-16 integration
has been found in both the E1 and E2 genes, a “hot” spot has
not been described. Reports have varied considerably on the
frequencies and sites of viral integration identified, which may
be explained by differences in technical approaches employed
or by differences in the populations studied.
A close review of the few published systematic analyses of
HPV-16 integration demonstrated that the E2 region 3132 to
3599, which includes the regions examined in our study, was
deleted in ?60% tumors (16, 29, 36), and it was also deleted in
a high percentage of cervical intraepithelial neoplasia I to
cervical intraepithelial neoplasia III lesions (29). A second
region, 2738 to 3189, which includes our most commonly de-
leted region targeted by E2F10 and E2R10, was also found to
be deleted in ?71% of tumors (16, 36). These studies were
performed in tumors derived from populations with different
ethnic backgrounds. Our study includes samples from two dif-
ferent populations, from the United States and Colombia. We
found that the E2F10-E2R10 (2783 to 3063) and the E2F12-
E2R12 (3243 to 3539) regions are deleted in 25 (67.6%) tu-
mors and 29 (78.4%) of 37 tumors, respectively. Our data
therefore support the existence of a hot spot for HPV-16 viral
integration that may be present in a majority of cervical tumors
regardless of the ethnic background of the population studied.
A systematic study of E1 deletions has not been previously
reported. One group reported that a common region of dele-
tion was found close to the segment encompassed by primers
E1F6 and E1R6 (nt 1952 to 2186), followed by deletions in
regions flanked by primers E1F4 to E1R7 (nt 1503 to 2485)
(18, 41) (Fig. 2). These integration sites were also found in a
few of our samples (Fig. 2).
The suggestion that methodologies employed to detect
HPV-16 integration may be responsible for the reported dis-
crepancies is consistent with the higher detection of integration
events in cervical cancer using more sensitive techniques (15,
17). For example, Kalantari et al., using a system aimed at
specifically detecting integrated forms of HPV-16, reported
integration in 14 (70%) of 20 samples that previously were
reported as containing episomal forms (15). Higher frequen-
cies (88%) of HPV-16 integration in CC have been described
with a method based on amplification of transcripts (17).
The role of HPV-16 integration in cervical neoplastic
progression has been proposed from studies that demon-
strated that disruption of HPV-16 E2 releases the repressor
activities of this gene product on HPV-16 E6 and E7 genes.
This in turn could lead to an increase in the expression
levels of HPV-16 E6 and E7 oncogenes, conferring a growth
advantage on the affected cells (13, 14, 27). Our results,
however, showed that HPV-16 integration events exist in the
presence of hundreds to thousands of viral episomes and
that strict viral integration occurs in a low percentage of CIS
and CC tumors. This observation suggests that HPV-16 E2
may often be available in trans to modulate or regulate
HPV-16 E6 and E7 expression. After performing experi-
FIG. 3. Rearrangements in HPV-16 E2 sequences. A. Nucleotide positions per HPV-16R sequence are noted at the top of the sequence
information and correspond to nucleotide positions 2690 to 2870 and 3469 to 3871. The 17 nucleotides that serve as the recombination site are
shown in italics. B. Illustration of the proposed end-joining recombination event shown here as an example in tumor C28 that leads to
reconstruction of the 583-bp fragment obtained.
1760 ARIAS-PULIDO ET AL.J. CLIN. MICROBIOL.
ments using a W12 culture model of cervical carcinogenesis,
Pett et al. recently reported that loss of inhibitory high-risk
HPV episomes is required for the spontaneous selection of
cells containing integrated HPV-16 (24a). Extant data and
current dogma must be given serious reconsideration.
We thank V. V. V. S. Murty for providing the HeLa, SW756, and
ME180 DNA, E.-M. de Villiers for kindly providing the HPV-16 ref-
erence plasmid, Curtis Hunt for assistance with statistical analyses,
Michelle Ozbun for propagating and preparing the W12 cells used as
controls, and the gynecologists from the Department of Gynecology at
the INC (Colombia).
This work was supported by a RAC grant, C-2239-T, to H.A.-P., and
an NIH grant, AI32917, to C.M.W. Support from Colciencias (Colom-
bia) (grant 2101-04-11896) is also acknowledged.
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