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Int. J. Cancer: 92, 9 –17 (2001) Publication of the International Union Against Cancer

© 2001 Wiley-Liss, Inc.

DETECTION OF INTEGRATED PAPILLOMAVIRUS SEQUENCES BY

LIGATION-MEDIATED PCR (DIPS-PCR) AND MOLECULAR
CHARACTERIZATION IN CERVICAL CANCER CELLS
Frank LUFT1, Ruediger KLAES1, Matthias NEES1, Matthias DÜRST2, Volker HEILMANN3, Peter MELSHEIMER4 and
Magnus VON KNEBEL DOEBERITZ1*
1
Division of Molecular Diagnostics and Therapy, Department of Surgery, University of Heidelberg, Germany
2
Department of Obstetrics and Gynecology, University of Jena, Germany
3
Division of Obstetrics and Gynecology, University of Ulm, Germany
4
Department of Obstetrics and Gynecology, University of Heidelberg, Germany

Human papillomavirus (HPV) genomes usually persist as
episomal molecules in HPV associated preneoplastic lesions
whereas they are frequently integrated into the host cell
genome in HPV-related cancers cells. This suggests that ma-
lignant conversion of HPV-infected epithelia is linked to re-
combination of cellular and viral sequences. Due to technical
limitations, precise sequence information on viral– cellular
junctions were obtained only for few cell lines and primary
lesions. In order to facilitate the molecular analysis of
genomic HPV integration, we established a ligation-medi-
ated PCR assay for the detection of integrated papillomavirus
sequences (DIPS-PCR). DIPS-PCR was initially used to amplify
genomic viral– cellular junctions from HPV-associated cervi-
cal cancer cell lines (C4-I, C4-II, SW756, and HeLa) and
HPV-immortalized keratinocyte lines (HPKIA, HPKII). In ad-
dition to junctions already reported in public data bases,
various new fusion fragments were identified. Subsequently,
22 different viral– cellular junctions were amplified from 17
cervical carcinomas and 1 vulval intraepithelial neoplasia
(VIN III). Sequence analysis of each junction revealed that
the viral E1 open reading frame (ORF) was fused to cellular
sequences in 20 of 22 (91%) cases. Chromosomal integration
loci mapped to chromosomes 1 (2n), 2 (3n), 7 (2n), 8 (3n), 10
(1n), 14 (5n), 16 (1n), 17 (2n), and mitochondrial DNA (1n),
suggesting random distribution of chromosomal integration
sites. Precise sequence information obtained by DIPS-PCR
was further used to monitor the monoclonal origin of 4
cervical cancers, 1 case of recurrent premalignant lesions and
1 lymph node metastasis. Therefore, DIPS-PCR might allow
efficient therapy control and prediction of relapse in patients
with HPV-associated anogenital cancers.
© 2001 Wiley-Liss, Inc.
Key words: cervical cancer; human papillomavirus; viral integra-
tion; ligation-mediated PCR; molecular marker
Persistent infections with high-risk human papillomavirus types
(HR HPV) induce dysplastic lesions (cervical intraepithelial neo-
plasia grades I–III, CIN I–III) of the cervix uteri that may progress
to invasive cancers.1,2 In productive HPV infections, which are
histologically defined as low-grade pre-malignant lesions (CIN I),
HPV genomes persist as episomal molecules. However, in a subset
of the high-grade lesions (CIN II–III) and most cervical cancers,
HPV genomes are integrated into the host cell genome.3–5 The
frequency of spontaneous regression declines from CIN I to CIN
III, which correlates with increasing frequency of HPV integra-
tion.6,7 This change might suggest that recombination of viral and
cellular sequences results in a selective growth advantage for the
emerging malignant cancer cells.8 HPV integration might repre-
sent a crucial event in cervical carcinogenesis and could therefore
correlate directly with the malignant potential.
Integration of HPV genomes in cervical cancers frequently
disrupts the E1 and/or E2 open reading frames (ORFs), but con-
sistently results in functional conservation and activation of tran-
scription of the viral oncogenes E6 and E7.3,9,10 The continuous
expression of these viral oncogenes is required to induce and
maintain the neoplastic phenotype of cervical cancer cells.11,12
Therefore, detection of integrated HPV genomes might represent a
suitable marker for the identification of cervical dysplastic lesions
at high risk for malignant progression.
Currently, all high-grade lesions are surgically removed by
conization, despite the high rate of spontaneously regressing le-
sions. Novel diagnostic tests are necessary that allow the precise
and reliable identification of patients with lesions at high risk of
neoplastic progression from those whose lesions will most likely
regress. These diagnostic tests would be highly beneficial and
could reduce the number and costs of unnecessary conizations,
provided the patients are followed up in reasonable intervals.
RNA-based assays for viral integration are generally feasible,5
but show significant technical and biological limitations because of
the limited stability of mRNA in clinical biopsies. In contrast,
DNA-based assays are less demanding applications in clinical
settings. Furthermore, because of technical limitations, only lim-
ited data on the precise molecular characteristics of genomic HPV
integration in clinical samples are available. This circumstance still
impedes the elucidation of the exact role of viral integration in
carcinogenesis of HPV-associated lesions.
In this report, we describe a single-side-specific ligation-medi-
ated polymerase chain reaction (PCR) protocol. This strategy
permits amplification of DNA sequences directly adjacent to any
known sequence.13 To improve specific target amplification,
a specifically designed double-stranded (ds) adapter is used
that comprises features of “vectorette-PCR”14 and “suppression-
PCR.”15 The PCR protocol presented here for the detection of
integrated papillomavirus sequences (DIPS-PCR) was established
for the most common cancer-associated HPV-types 16 and 18.
After initial studies on HPV-containing cervical cancer and kera-
tinocyte cell lines, we applied the DIPS-PCR protocol to primary
clinical samples. DNA sequencing of the viral– cellular junctions
obtained in DIPS-PCR was performed to permit the molecular
characterization and precise localization of HPV integration sites
in cell lines and primary lesions.
Abbreviations: CIN, cervical intraepithelial neoplasia; CxCa, cancer of
the cervix; DIPS, detection of integrated papillomavirus sequences; ds,
double stranded; HPV, human papillomavirus; HR HPV, high-risk human
papillomavirus; ORF, open reading frame; VIN III, vulval intraepithelial
neoplasia grade III
*Correspondence to: Division of Molecular Diagnostics and Therapy,
Department of Surgery, University of Heidelberg, Im Neuenheimer Feld
110, D-69120 Heidelberg, Germany. Fax: ⫹49-622-56-59-81.
E-mail: knebel@med.uni-heidelberg.de
Received 1 September 2000; Revised 27 November 2000; Accepted 4
December 2000
Published online 9 February 2001.
 10970215, 2001, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/1097-0215(200102)9999:9999<::AID-IJC1144>3.0.CO;2-L by Readcube (Labtiva Inc.), Wiley Online Library on [13/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
10 LUFT ET AL.

MATERIAL AND METHODS the second PCR was AP1 (adapter primer 1): 5⬘-ggccatcagtcag-
Cervical carcinoma cell lines and clinical samples cagtcgtag-3⬘.
Cell lines derived from HPV-18-positive cervical carcinomas PCR analysis of the adapter-ligated DNA fragments
(C4-I, C4-II, SW756 and HeLa) and the HPV-16-immortalized cell
lines HPKIA and HPKII16,17 were cultured in Dulbecco’s modified PCR amplification was performed in a total volume of 25 ␮l in
Eagle’s medium, supplemented with 10% fetal bovine serum. a thermal cycler (GeneAmp 2400, Perkin Elmer). First-round,
linear PCR (40 cycles) was carried out in 1⫻ PCR buffer (Gibco
Genomic DNA was extracted from cell lines and various passages
BRL, Gaithersburg, MD), containing 1.5 mM MgCl2, 200 ␮M
during the immortalization process of HPKIA (p5⫹3, p49⫹12, p92,
each dNTP, 0.2 ␮M primer (viral primer 1, Table I), 1 unit Taq
p269) and HPKII (p1, p3⫹3, p4, p48, p80, p156⫹2, p289⫹78). polymerase (Gibco BRL) and 2 ␮l of the diluted ligation product.
Clinical tumor samples (punch biopsies and cervical swabs) were PCR parameters were: initial denaturation at 96°C for 2 min,
collected at the Departments of Obstetrics and Gynecology at the followed by 40 cycles consisting of denaturation at 96°C for 0.5
Universities of Jena, Ulm and Heidelberg, Germany. min, primer annealing for 0.5 min, primer extension 72°C for 3
min and final extension 72°C for 7 min. For the second, exponen-
DNA isolation from cell lines and clinical samples tial PCR, 2 ␮l of the PCR product was subjected to amplification
Genomic DNA from cell lines and clinical samples was ex- as described above, except that 2 primers (the viral primer 2 in
tracted by proteinase K digestion and standard phenol extraction.18 Table I, together with the adapter-specific AP1 primer, each 0.4 ␮M)
HPV typing was performed by PCR and restriction fragment were used and 30 PCR cycles carried out. PCR products were ana-
length polymorphism (RFLP) analysis as described elsewhere.19 lyzed on 2% agarose gels stained with ethidium bromide and read by
transillumination. As control, amplification (⬇1.4 kb, accession num-
Digestion of genomic DNA and ligation of the adapter ber ap001068) of a genomic locus on chromosome 21 was performed
using primers DIPS1L (5⬘-ttctctatgtgcgttctctccctg-3⬘; Ta 64°C) in the
Digestion of genomic DNA (0.6 ␮g) with either TaqI or Sau3AI first linear PCR and DIPS2L (5⬘-caaactccaggtctccaaccag-3⬘; Ta 64°C)
(10 units) was performed in a volume of 20 ␮l in a thermal cycler together with AP1 in the second PCR.
(GeneAmp 2400, Perkin Elmer, Foster City, CA) overnight, followed
by heat inactivation of the enzyme, according to the manufacturer’s Sequencing analysis of the PCR products
instructions (New England Biolabs, Beverly, MA). Ligation of the PCR products of interest were excised from the gel and DNA
enzyme-specific adapters (50 pmol) to the restriction-digested extracted using the Qiagen Gel-Extraction-Kit (Qiagen, Hilden,
genomic DNA was performed by adding 5 U of T4-DNA ligase Germany). PCR fragments were analyzed further by direct se-
(Roche Mannheim, Germany), ATP and DTT (final concentration of quencing using the Big-Dye terminator DNA-sequencing Kit (Per-
2 mM each) in a total volume of 24 ␮l at 14°C overnight. The ligation kin Elmer) and analyzed on an Abi Prism 310 Genetic Analyzer
reaction was diluted to 40 ␮l with sterile water. Then 2 ␮l of the (Applied Biosystems, Foster City, CA). Sequencing results were
ligation products were subjected to the first round of linear PCR analyzed and compared with public data bases using the HUSAR
amplification. analysis program package (German Cancer Research Center, Hei-
delberg, Germany).
Sequences and formation of the ds adapter
Analysis of the clonal status of HPV integration
The special properties of the adapters used prevent efficient
amplification of the vast majority of genomic fragments with Tissue sections (5 ␮m) from fresh-frozen and archival paraffin-
adapter sequences at both ends, but lacking an internal viral embedded clinical samples were stained by hematoxylin and eosin
sequence (vectorette-feature and suppression effect). The method (H&E) using standard protocols. Microdissection of distinct neo-
is discussed in detail elsewhere.14,15. Briefly, the ds adapter is plastic and stromal regions was performed and the dissected cells
were digested in a buffer with proteinase K at 56°C for 3– 4 hr.
constituted of 1 short oligo (17mer or 19mer) that is extension-
Proteinase K was heat inactivated at 95°C for 10 min. Specific
deficient at its 3⬘ end by an amine group and a second longer oligo
(45mer). This structure contains both a ds and a single-stranded
part. The “vectorette-feature” of the adapter is generated by the TABLE I – HPV PRIMER SETS USED IN DIPS-PCR ANALYSIS
absence of an adapter primer (AP1) binding site. The AP1 primer
is complementary to the missing strand of the single-stranded Primer 1 Primer 2
HPV Primer set (1.PCR) (2.PCR) Sau3AI ORF Product
portion. Elongation of the shorter oligo creating an AP1 binding
5⬘ nt Length 5⬘ nt Length
site cannot occur because the 3⬘ end is blocked. This feature
reduces non-specific amplification with AP1 primer alone. How- 16 16for1 902 24 952 27 3478 E2/E4 2575
ever, on rare occasions, molecules are generated that contain 16 16for2 1308 22 1347 20 3478 E2/E4 2180
extended adapter sequences at both ends. These products are 16 16for3 1870 25 1942 24 3478 E2/E4 1585
potential targets for non-specific priming using only AP1 primer. 16 16for4 2725 20 2736 25 3478 E2/E4 791
16 16for5 3482 20 3496 20 4519 L2 1072
However, this artificial amplification is addressed by the “suppres-
sion effect,” which is achieved by the use of an adapter primer that 18 18for1 923 21 948 24 4732 L2 3833
is about 2 times shorter than the adapter itself. Intramolecular 18 18for2 1415 21 1425 20 4732 L2 3356
annealing of the adapter sequences at both ends of the same 18 18for3 1859 22 1929 27 4732 L2 2852
18 18for4 2804 23 2814 24 4732 L2 1967
fragment creating a panhandle-like structure is favored and more 18 18for5 3629 23 3634 24 4732 L2 1147
stable than intermolecular annealing of AP1 to the adapter. The ds
adapter (25␮M) was pre-annealed by mixing equimolar amounts 18 18rev11 6223 28 6217 28 5038 L2 1223
18 18rev21 5026 21 4982 26 4732 L2 294
of 2 oligonucleotides (AL1 and either AS or AS1) in 66 mM Tris 18 18rev31 4702 27 4629 24 919 E1 3754
HCl (pH 7.4) and gradually cooling the mix from 90°C (2 min) to 18 18rev41 4072 27 4016 22 919 E1 3141
4°C (overnight) in a water bath. Aliquots were stored at ⫺20°C.
DNA sequences for AL1 (adapter strand long 1): 5⬘-gggccatcagt- HPV, human papilloma virus; PCR, polymerase chain reaction;
cagcagtcgtagccggatccagacttacacgttg-3⬘; AS (adapter strand ORF, open reading frame.–1Antisense primer sets were used for the
detection of 5⬘ viral-cellular junctions in HPV 18-positive cell lines
short): 5⬘-PO4-cgcaacgtgtaagtctg-NH2-3⬘ (for TaqI-specific (Fig. 2, Fig. 3). The primers were used with an annealing temperature
DIPS-PCR analysis) and AS1 (adapter strand short 1): 5⬘-PO4- of 62°C in PCR. Nucleotide positions of primers, corresponding down-
gatccaacgtgtaagtctg-NH2-3⬘ (for Sau3AI-specific DIPS-PCR stream Sau3AI sites, and respective viral ORFs were based on Gen-
analysis). The primer specific to the adapter sequence used in bank accession numbers K02718 for HPV 16 and X05015 for HPV 18.
 10970215, 2001, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/1097-0215(200102)9999:9999<::AID-IJC1144>3.0.CO;2-L by Readcube (Labtiva Inc.), Wiley Online Library on [13/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
MOLECULAR CHARACTERIZATION OF PAPILLOMAVIRUS INTEGRATION 11

FIGURE 1 – Schematic representation of the DIPS-PCR assay. (a) Possible structures of integrated HPV 16 or HPV 18. Viral open reading
frames (ORFs) are noted below the respective boxes. Endonuclease restriction sites (vertical dotted lines), classes of possible restriction
fragments generated (A, B, C and D) and locations of polymerase chain reaction (PCR) primers (arrowheads) are indicated. (b) Before PCR,
restriction of genomic DNA results in 4 categories of possible DNA fragments (A, B, C and D). Episomal human papilloma virus (HPV) forms
are not shown (comparable to restriction fragments of class B). DNA fragments of particular interest contain both viral and cellular sequences
(classes A and C). (c) Ligation of endonuclease-specific double-stranded adapters (black arrows) to restriction fragments introduces primer
binding sites for PCR. (d) First step of DIPS-PCR: linear amplification of DNA fragments containing HPV sequences with HPV-specific primers
(classes A, B and C). Single-stranded PCR products are generated. (e) Second step of DIPS-PCR: exponential PCR amplification, using
combinations of nested HPV- and adapter-specific primers (adapter primer 1, AP1). Double-stranded PCR products are generated.

PCR amplification of integrated HPV sequences was performed RESULTS

using 1 primer targeting the HPV sequence, and a second primer
corresponding to the adjacent cellular part of the viral– cellular
junction determined by DIPS-PCR analysis. The PCR products
were analyzed on 2% agarose gels. Information on PCR specific
for HPV integration and primer sequences used may be obtained
from the authors on request.
Detection of integrated papillomavirus sequences by
ligation-mediated PCR (DIPS-PCR)
The DIPS-PCR assay (outlined in Fig. 1) is based on the
principle of ligation-mediated PCR technique13 and was specifi-
cally adapted for the detection of integrated papillomavirus se-
quences. Enzymatic restriction of cellular DNA with Sau3AI
 10970215, 2001, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/1097-0215(200102)9999:9999<::AID-IJC1144>3.0.CO;2-L by Readcube (Labtiva Inc.), Wiley Online Library on [13/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
12 LUFT ET AL.

(TaqI) was followed by ligation of specific ds DNA adapters that

are compatible to the sticky ends of each fragment. Therefore, each
DNA fragment generated has a defined sequence at both ends,
allowing exact priming and amplification in PCR. Significant
improvements could be achieved by the 2-step, single-side-specific
PCR strategy using only 1 viral primer in the first linear PCR
amplification and 2 primers (1 virus-specific, 1 adapter-specific) in
the second, exponential PCR (data not shown). This protocol
permits selective enrichment and detection of DNA fragments
containing HPV sequences. PCR products that contain exclusively
viral sequences were easily identified according to their expected
size. Such PCR products can result from both episomal and inte-
grated HPV molecules. Recombinant PCR molecules, which con-
tain both viral and cellular sequences, deviate from the expected
product length. The chimeric structure was confirmed by DNA
sequence analysis. As a control for PCR, approximately 1.4 kb of
a genomic locus on chromosome 21 were amplified.
Evaluation of the DIPS-PCR assay using HPV-containing
cell lines
To establish and optimize the DIPS assay and to obtain chimeric
viral– cellular junctions, we analyzed 4 HPV-18-positive carcinoma-
derived cell lines (C4-I, C4-II, SW756 and HeLa) and 2 HPV-16-
immortalized human cell lines (HPKIA and HPKII). To evaluate
extended applications of the DIPS-PCR analysis for integrated HPVs,
the HPV-18-positive cell lines were tested for both 3⬘ and 5⬘ viral–
cellular junctions. For the analysis of 3⬘ viral– cellular junctions in the
HPV-18-containing cell lines, 5 nested primer sets were used (18for1,
18for2, 18for3, 18for4 and 18for5, compare Table I). These primers
cover all of the potential 3⬘ part of integrated HPV 18. In addition, we
analyzed the potential 5⬘ viral– cellular junctions with 4 additional
nested primer sets (18rev1, 18rev2, 18rev3 and 18rev4, compare
Table I) that cover the potential 5⬘ part of integrated HPV 18. Figure
2 shows a representative DIPS-PCR analysis of the HPV-18-positive
cell lines C4-I, C4-II, SW756 and HeLa using the primer sets de-
scribed above.
The cell lines C4-I and C4-II were established independently
from the same cervical carcinoma biopsy.20 Not surprisingly,
DIPS-PCR analysis of C4-I and C4-II resulted in identical PCR
products, thus pointing out the common clonal origin of both cell
lines. For both C4-I and C4-II, PCR amplimers generated by the
primer sets 18for3 and 18for4 originated from the same viral–
cellular junction as confirmed by DNA sequence analysis and were
identical with respect to the cellular sequence. PCR with primer
sets 18for1 and 18for2 was not able to amplify integration-specific
DNA fragments because the resulting PCR products would exceed
the size of approximately 1.5 kb that can be readily amplified using
the PCR conditions for the DIPS assay. Similarly, analysis of the
5⬘ viral– cellular junctions in both C4-I and C4-II with primer set
18rev1 resulted in identical ⬇1.7-kb PCR products. The 3⬘ and 5⬘
viral– cellular junctions have not been published.
In SW756 cells, the PCR products generated using the primer sets
18for1, 18for2 and 18for3 all originated from 1 identical viral–
cellular junction. No products could be amplified using primer 18for4. FIGURE 2 – DIPS-PCR analysis of HPV 18 integration in cell lines
The ⬇1.1-kb PCR product obtained by using primer set 18for5 derived from cervical cancers. The human papilloma virus (HPV)
contains exclusively viral sequences that start at the 18for5 primer 18-positive cell lines C4-I, C4-II, SW756 and HeLa were analyzed by
DIPS-PCR for 3⬘ and 5⬘ viral– cellular junctions and polymerase chain
position and extend to the Sau3AI site at nucleotide position 4732. reaction (PCR) products separated on 2% agarose gels; 1-kb and
This amplification presumable took place at the 5⬘ part of integrated 100-bp ladders (Gibco) were used as DNA size markers. A control
HPV 18 in SW756 (Fig. 3). Analysis of the 5⬘ viral– cellular junction amplification (control) of a genomic locus (⬇1.4 kb) was performed to
in SW756 cells using primer set 18rev1 resulted in a ⬇1.5-kb PCR confirm DNA integrity. No template: negative control. Data are dis-
product that contained only viral sequences. This fragment extended cussed in detail in the Results section. A schematic representation of
up to the Sau3AI site at nucleotide position 4732. The expected the results is shown in Figure 3.
Sau3AI site at nucleotide position 5038 was mutated in SW756 cells,
which caused a read-through at this position. The 294-bp PCR prod- from the same viral– cellular junction as described recently.22,23
uct generated by using 18rev2 was confirmed as the expected am- Interestingly, the PCR product generated by 18for4 originated
plimer that extends to the conserved viral Sau3AI site (see Table I). from a second, independent viral– cellular junction. Figure 4 illus-
PCR fragments produced by 18rev3 and 18rev4 originated from the trates this viral-genomic junction that has not been previously
same viral– cellular junction previously described by Gallego et al.21 determined. The analysis of 5⬘ viral– cellular junctions using
DIPS analysis of HPV integrates in HeLa cells using primers primer 18rev1 produced a 1.4-kb PCR product that originated from
18for1, 18for2 and 18for3 resulted in PCR products that originated a recently described viral-cellular breakpoint.23 The results for the
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MOLECULAR CHARACTERIZATION OF PAPILLOMAVIRUS INTEGRATION 13

FIGURE 3 – Schematic representation (approximately to scale) of DIPS-PCR results for human papilloma virus (HPV) 18-positive cell lines.
The primer sets used and orientation of viral primers are indicated by arrowheads and dotted lines. Resulting polymerase chain reaction (PCR)
products are shown as bars. Black areas represent viral sequences and shaded areas represent cellular sequences. The viral open reading frames
are indicated, Sau3AI restriction sites are shown by lines. Numbers indicate the exact position of disruption of the viral genome upon integration.
For C4-I and C4-II, identical PCR products and integration sites were found. *For SW756, the PCR product generated using primer set 18for5
(Fig. 2) results from amplification at the 5⬘ viral sequences. The Sau3AI site, which is mutant in SW756 cells, is shown in parenthesis. For HeLa,
DIPS-PCR analysis revealed 2 independent 3⬘ viral– cellular junctions and 1 5⬘ chimeric junction.

FIGURE 4 – Sequence analysis of 2 representative viral– cellular junctions found in HeLa cells that resulted from human papilloma virus (HPV)
18 integration into human chromosome 8. HPV 18 sequences (top), cellular sequences (bottom) and sequence of viral– cellular junctions (middle)
were aligned. Identical sequences are highlighted. Numbering of sequences according to GenBank accession numbers X05015 for HPV 18 and
AC027531, a genomic clone that maps to chromosome 8.

HPV-18-containing cell lines are summarized in Table II and 16for4 and 16for5 in Table I) were used that cover the potential 3⬘
schematically presented in Figure 3. flank of the integrated HPV 16 genome. Using primer set 16for1,
For DIPS-PCR analysis of the HPV 16-positive cell lines 1 characteristic viral– cellular junction was identified for each cell
HPKIA and HPKII, 5 nested primer sets (16for1, 16for2, 16for3, line that was previously unknown (data not shown). The viral–
 10970215, 2001, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/1097-0215(200102)9999:9999<::AID-IJC1144>3.0.CO;2-L by Readcube (Labtiva Inc.), Wiley Online Library on [13/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
14 LUFT ET AL.

TABLE II – DIPS-PCR ANALYSIS OF CELL LINES

Primer Viral HPV Chromosomal

Cell line HPV set disruption ORF location

C4-I 18 18for4 2952 E2 8

C4-I1 18 18rev1 5442 L1/L2 8
SW756 18 18for3 2643 E1
SW7561 18 18rev4 3419 E2/E4
HeLa 18 18for3 2497 E1 8
HeLa 18 18for4 3100 E2 8
HeLa1 18 18rev1 5736 L1 8
HPKIA 16 16for1 1054 E1 17
HPKII 16 16for1 1296 E1 3
HPV, human papilloma virus; ORF, open reading frame.–1The HPV
18-positive cell lines were additionally tested for 5⬘ viral-cellular
junctions. For C4-II, the results were identical to C4-I and not shown.
Sequence analysis of the viral-cellular junctions defines the most 3⬘ or
5⬘ viral nucleotides of integrated HPV sequences and the respective
ORFs. Chromosomal locations of cellular sequences were determined
from database comparisons. For HPV numbering see accession num-
bers used in Table I.

TABLE III – DIPS-PCR ANALYSIS OF CLINICAL SAMPLES

Primer Viral HPV Chromosomal

Sample Number Histology HPV set disruption ORF location

T1 1 CxCa16 16for1 1248 E1 2q22

T2 2 CxCa16 16for1 978 E1 Mitoch.
T3 3 CxCa16 16for2 1470 E1 2
4 16for2 1641 E1 16
T4 5 CxCa 16 16for3 2156 E1 2
T5 6 CxCa 16 16for3 2387 E1 1
T6 7 CxCa 18 18for3 1972 E1 7q31
T7 8 CxCa 16 16for1 1162 E1 14 FIGURE 5 – Representative results from DIPS-PCR analysis of hu-
9 16for2 1547 E1 14 man papilloma virus (HPV) 16-positive cervical lesions. Four cervical
T8 10 CxCa 16 16for1 1508 E1 14 carcinomas and 1 vulval intraepithelial neoplasia (VIN III) lesion were
T9 11 CxCa 16 16for1 1353 E1 17q22 analyzed for 3⬘ viral– cellular junctions, using different polymerase
T10 12 CxCa 16 16for3 2387 E1 Unknown chain reaction (PCR) primer sets (16for1-16for4). PCR products were
T11 13 CxCa 18 18for3 2096 E1 14 separated on 2% agarose gels. Samples and primer sets used are
V1 14 VIN III 16 16for2 1830 E1 10 indicated; 1-kb and 100-bp ladders were used as DNA size markers.
T12 15 CxCa 18 18for3 2213 E1 8p11.2 Control amplification (control) of a genomic locus (⬇1.4 kb) was
16 18for4 2892 E2 8p11.2 performed to confirm DNA integrity. No template: negative control.
T13 17 CxCa 16 16for2 1857 E1 7q31 PCR products for T9, T17, V1 and T5 represent amplification of
T14 18 CxCa 16 16for4 2762 E1/E2 Unknown viral– cellular junctions. For tumor T14, DIPS-PCR analysis with the
T15 19 CxCa 16 16for1 1165 E1 14q24.3 16for4 primer set revealed 2 independent amplimers. The 791-bp
20 16for3 2000 E1 8 product (arrowhead) resulted from amplification of viral sequences
T16 21 CxCa 18 18for3 1988 E1 17 adjacent to the Sau3AI site (nucleotide position 3478) and had the
T17 22 CxCa 16 16for2 1783 E1 1q41 expected size. The second, smaller product (⬇200 bp) originates from
a viral– cellular junction.
HPV, human papilloma virus; ORF, open reading frame; CxCa,
cancer of the cervix; VIN III, vulval intraepithelial neoplasia grade III;
mitoch., mitochondrial sequence.–Sequence analysis of the viral-cel- between 24 and 652 bp. In 20 of 22 cases (91%), the HPV E1 ORF
lular junctions defines the most 3⬘ viral nucleotides that were disrupted was disrupted; in 1 case the E2 ORF was disrupted; and 1 disrup-
upon integration and the respective ORFs. Chromosomal locations of tion occurred in the region where the E1 and E2 ORFs overlap.
cellular sequences were determined from database comparisons. For
HPV numbering see accession numbers used in Table I. Wherever possible, chromosomal localizations of genomic se-
quences were determined. In 4 lesions, 2 viral– cellular junctions
were detected (T3, T7, T12 and T15 in Table III). In 2 of these
cellular junction in HPKIA was obtained using TaqI for genomic lesions, loci on different chromosomes were affected (T3, T15). In
DNA restriction. TaqI cuts only once in the HPV 16 genome the remaining 2 lesions (T7, T12), loci mapped to identical chro-
(nucleotide position 505) and was initially used in DIPS-PCR mosomal locations. In general, HPV integration shows random
analysis. Sequence analysis of the junctions revealed that in distribution and no apparent preferences for specific genomic loci.
HPKIA cells, HPV integration targets chromosome 17, which However, in 4 of 18 lesions from different individuals, loci on
confirms previous work that mapped viral integration to chromo- chromosome 14 were affected, and in 2 independent lesions,
some 17q25 by in situ hybridization.24 For the HPKII cells, se- chromosomal localizations at 7q31 were affected. It might be
quence analysis showed an integration locus on chromosome 3. interesting to investigate further if specific integration of the HPV
genomes into particular loci contributes to a selective growth
DIPS-PCR analysis of viral– cellular junctions advantage and specifically promotes the development of cervical
in clinical samples cancer (compare Table III).
In 18 HPV-16- or -18-positive primary lesions with integrated
viral genomes, a total of 22 viral– cellular junctions were identified Stability and clonality of HPV integration in HPV-transfected
(Table III and Fig. 5). All PCR products encompassing the viral– human keratinocytes during immortalization
cellular junctions were verified by direct DNA sequence analysis. The HPV-16-containing cell lines HPKIA and HPKII, derived
PCR products of viral– cellular junctions ranged from 135 bp to 1.1 from human primary foreskin keratinocytes, show a number of chro-
kb. The cellular portions of the determined sequences varied mosomal imbalances during propagation.16,25 DIPS-PCR analysis re-
 10970215, 2001, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/1097-0215(200102)9999:9999<::AID-IJC1144>3.0.CO;2-L by Readcube (Labtiva Inc.), Wiley Online Library on [13/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
MOLECULAR CHARACTERIZATION OF PAPILLOMAVIRUS INTEGRATION 15

sulted in the characterization of 1 viral– cellular junction (Table II) for

each cell line. Various passages available after transfection with
cloned HPV 16 DNA during the immortalization process were ana-
lyzed by integration-specific PCR. For HPKIA cells, the specific viral
integration pattern could be demonstrated in all of the analyzed
passages (p5⫹3, p49⫹12, p92, p269). Similarly, the presence of the
viral integration pattern specific for HPKII was observed in all pas-
sages obtained following p1 (p3⫹3, p4, p48, p80, p156⫹2,
p289⫹78). These results (data not shown) indicate the clonal origin of
both cell lines. The remarkable stability of HPV integration loci even
after prolonged in vitro culture and genome-wide chromosomal im-
balances might reflect the importance of HPV integration for the
maintenance of the immortal phenotype.
Clonality of HPV integration in primary lesions
and recurrent disease
To confirm the monoclonal nature of HPV integration in 4
different primary cervical carcinomas from 4 individuals, we iso-
lated tumor tissue from distinct tumor areas by microdissection. In
each case, 4 distinct microdissected tumor areas were analyzed.
These adjacent tumor regions were tested for the presence of
specific integration events determined by DIPS-PCR. This analysis
verified the presence of identical integration-specific amplimers in
all 4 regions analyzed in each individual case (Fig. 6). In addition,
the clonal origin of 1 case of regional lymph node metastasis was
confirmed using the same approach following DIPS-PCR analysis
of the primary tumor. As a pre-screening for this purpose, the
presence of tumor cells could even be demonstrated using genomic
DNA extracted from whole tissue sections that contain at least
80% non-tumorigenic cells (data not shown).
For 1 patient, the clonal origin of 2 independent VIN III lesions
could be demonstrated by retrospective PCR analysis of indepen-
dent biopsies (data not shown). From a VIN III lesion that devel-
oped in 1996, a specific viral integration locus was determined
using DIPS-PCR. The identical viral integration locus could be
amplified from archival paraffin-embedded tissue of the VIN III
lesion that was removed in 1995 by laser evaporation. These data
underline the clonal nature of primary and recurrent HPV-associ-
ated lesions.

DISCUSSION

Integration of HPV DNA into the host genome is an important

event that might be of critical significance for cervical carcinogen-
esis. The knowledge of exact genomic viral– cellular junctions
offers a variety of applications for detailed analysis of HPV
integration for diagnostic and research purposes as demonstrated
in this study.
A number of PCR-based techniques have been used to analyze
the physical status of HPV genomes in primary, HPV-associated
lesions. For example, Carmody et al.26 used viral primers in
combination with IRS-specific (interspersed repetitive sequences)
priming. This assay is specific for integrated HPV, but is restricted
by the average distribution of IRS elements, which is estimated to
be 3,000 – 6,000 bp for ALU repeats. Different PCR protocols have
been described in which the failure to amplify distinct targets
within the E1 or E2 ORFs potentially indicates viral integra-
tion.27–31 However, co-existence of integrated and episomal viral
molecules or the presence of concatemerized, tandemly repeated or
otherwise rearranged HPV genomes could complicate the precise
analysis of the physical HPV status.32 For instance, Cullen et al.4
reported rearranged episomes found in 20 of 22 (91%) carcinomas
with episomal HPV genomes, which may cause misinterpretation
concerning the HPV status.
In a recent study we used an RT-PCR based assay for the
amplification of papillomavirus-derived oncogene transcripts
(APOT assay) to detect mRNA transcripts derived from integrated
HPV 16 and HPV 18 genomes.5 Using the APOT-PCR approach
to amplify HPV transcripts from clinical lesions, we identified
integrate-derived transcripts in 88% of cervical cancers, but in only
FIGURE 6 – Clonality of human papilloma virus (HPV) 16 integra-
tion in a cervical tumor biopsy was demonstrated by polymerase chain
reaction (PCR) specific for the viral– cellular junction in 4 adjacent
microdissected tumor regions. One viral primer and 1 primer that
targets the neighboring cellular DNA were used. (a) Hematoxylin-and-
eosin-stained sections of tumor T9 with 4 tumor regions (A, B, C and
D) before (above) and after (below) microdissection (magnification
25⫻). (b) Schematic illustration of the viral– cellular junction in tumor
sample T9 (compare Table III) with locations and orientation of the
primers P1 and P2 indicated by arrowheads. The viral genome was
disrupted within the E1 open reading frame at nucleotide position
1353. The cellular sequence mapped to chromosome 17q22. (c) De-
tection of integration-specific DNA fragments in tumor regions (A, B,
C and D) by PCR (2% agarose gel). In normal tissue samples from the
same patient (N1 and N2), the integration-specific fusion fragment was
not detected. In HPV 16-positive cervical tumors from a different
patient (C1) and in an HPV-negative DNA sample (C2), the specific
amplimer was not generated. Amplification of genomic locus
D5S10726 served as control for DNA integrity and PCR performance;
1-kb and 100-bp ladders (Gibco) were used as DNA size markers. No
template: negative control.
16% of CIN III lesions and 5% of CIN II lesions. These data
clearly show that the detection of integrate-derived oncogene tran-
scripts is a sensitive and specific marker for advanced dysplasia or
invasive cervical cancer. Furthermore, HPV integration might cor-

16 LUFT ET AL.
relate with the potential of particular CIN lesions to progress to
invasive cervical cancer.
However, integrate-derived viral transcription may be sup-
pressed by E2 proteins that are expressed from episomal viral
genomes in lesions with concomitant episomal and integrated
HPV.32 Moreover, precise genomic integration loci cannot be
determined by RNA-based assays because integrate-derived fusion
transcripts are often spliced over large stretches that can be located
far downstream of the viral integration sites. Furthermore, HPV
integration into cellular DNA is a prerequisite for integrate-spe-
cific transcription and could therefore probably precede transcrip-
tional activity.
The DIPS-PCR assay, as described here, overcomes all techni-
cal limitations inherent to RNA instability in clinical biopsies.
Instead, DIPS relies entirely on genomic DNA, which is much
more robust and easily extracted from most clinical samples,
including punch biopsies and cervical swabs or samples that were
not properly stored or rapidly processed. Furthermore, DIPS-PCR
analysis is also applicable to 5⬘ viral– cellular junctions as shown
for the HPV-18-positive cancer cell lines (Figs. 2, 3).
The identification of viral– cellular junctions in each of the cell
lines clearly demonstrates the value of the DIPS-PCR assay. For
the C4-I, C4-II, HeLa, HPKIA and HPKII cell lines, viral break-
points and viral-cellular fusion fragments could be determined that
had not yet been reported. In addition, DIPS-PCR results in the
identification of a large number of viral– cellular junctions from
clinical samples that are unique for each tumor or pre-malignant
lesion. Sequence analysis of the chimeric junctions obtained by
PCR from primary lesions (Table III) permits the determination of
precise cellular integration loci. In many cases, integration sites
can be exactly mapped to subchromosomal localizations. Although
no striking evidence was obvious for certain recurrent chromo-
somal integration loci, some interesting observations still could be
made. Loci on chromosome 14 were affected in 4 (22%) of 18
independent lesions (T7, T8, T11 and T15 in Table III). Interest-
ingly, Koopman et al.33 recently mapped HPV 16 integration to a
derivative chromosome 14 near a (3p;14) translocation breakpoint
in a cell line established from an invasive cervical carcinoma. In
our study, 1 subchromosomal locus could be mapped to 14q24.3
(T15), a region that harbors the human c-fos proto-oncogene.34
HPV integration near cellular oncogenes and consequences for
their structure and expression have been reported previously.35,36
Another interesting locus might be on chromosomal band 7q31,
which was affected in 2 independent lesions. Recently, functional
evidence was reported for a tumor suppressor gene located on
7q31.1 neighboring the Fra7G fragile site.37 This finding might
indicate a genomic region that is generally vulnerable to recom-
bination and genetic rearrangements. Reuter et al.38 recently dem-
onstrated that the detailed analysis of HPV integration loci can
result in the identification of novel cellular genes that are involved
in HPV-associated pathogenesis. Similarly, detailed analysis of
genomic integration loci characterized in this study could further
address the issue of preferential HPV integration and could con-
tribute to identify genomic regions that are particularly prone to
genetic rearrangements.33,39
The loss of the E1 and/or E2 ORFs upon HPV integration in
cervical lesions has been reported in a number of studies. Chen et
al.28 mapped a general viral deletion region primarily to nucleotide
positions 1417–2881, which represents mostly the E1 ORF. In
contrast, Vernon et al.30 found disruption most frequently in the E2
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