Volume 38, Issue 1, Pages 7-13 (January 2007)

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ABSTRACT

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Detection of human papillomavirus type 16 integration in pre-neoplastic cervical lesions and confirmation by DIPS-PCR and sequencing

Received 23 May 2006; received in revised form 12 September 2006; accepted 14 September 2006. published online 30 October 2006.

Abstract

Background

Persistent infections with high-risk types of human papillomavirus (HR-HPV) favour integration of viral DNA into the host cells and are associated with cervical carcinoma. HPV16 is the prevalent HR-type worldwide associated to cervical cancer. Integration of viral DNA promotes a selective cell growth advantage, resulting a risk factor for cancer development.

Objectives

To test physical status of HPV16 infection in pre-neoplastic cervical lesions using a quantitative real time-PCR (QRT-PCR) based method. To investigate reliability of this method in identification of HPV16 integrated sequences, by detection of integrated papillomavirus sequences (DIPS-PCR) assay and sequencing.

Study design

One hundred and seventy HR-HPV positive archival cervical specimens were tested for presence of HPV16 DNA. In HPV16 positive samples, viral load and physical status were evaluated.

Results

HPV16 DNA was detected in 74/170 (43%) HR-HPV positive specimens. In 52/74 a QRT-PCR was performed, and 3 integrated, 13 mixed and 36 episomal forms were detected. Presence of integrated forms was confirmed by DIPS-PCR and sequencing.

Conclusions

Presence of HPV integrated forms was detected and confirmed in pre-neoplastic cervical lesions. The QRT-PCR method we used is sensitive and specific for identification of HPV integration in cervical samples, and may be suitable for large scale investigations with prognostic and clinical implications in management of cervical cancer.

Keywords: Papillomavirus (HPV), Integration, Cervical cancer QRT-PCR, DIPS, Sequencing

Article Outline

• Abstract

• 1. Introduction

• 2. Materials and methods

• 2.1. Study population

• 2.2. Cell lines

• 2.3. DNA isolation

• 2.4. PCR assays

• 2.4.1. Qualitative PCR

• 2.4.2. Quantitative real time-PCR (QRT-PCR)

• 2.5. DIPS-PCR (detection of integrated papillomavirus sequences)

• 2.6. Direct sequencing analysis

• 3. Results

• 3.1. HPV16 detection

• 3.2. HPV viral load and physical status

• 3.3. DIPS-PCR and direct sequencing

• 4. Discussion

• Acknowledgment

• References

• Copyright

1. Introduction

Persistent infections with high-risk types of human papillomavirus (HR-HPV) are associated with dysplastic lesions of the uterine cervix, which may progress to invasive cervical cancer. Specifically, HPV type 16 is regarded as the major risk factor for development of cervical lesions and cancer (Munoz et al., 2003). Recent studies, focused on HPV life cycle and natural history of cervical cancer, allowed identification of at least three risk markers suitable for selection of subjects more prone to high grade cervical lesions and cancer: (i) persistence of HPV infection (Munoz et al., 2003, Nobbenhuis et al., 1999, Dalstein et al., 2003); (ii) high viral load (Dalstein et al., 2003, Lorincz et al., 2002, Snijders et al., 2003, Moberg et al., 2004); (iii) integration of HPV into the host genome (Kalantari et al., 1998, Klaes et al., 1999, Badaracco et al., 2002, Hudelist et al., 2004). These three events are tightly related each other, the last two likely being a consequence of the first. The main function of HPV integration seems to be the stabilization of transcription of viral oncoproteins which interact with many cellular factors involved in cell cycle regulation. This event favours the selective expansion of cell clones with optimised oncogene expression. Many studies showed evidence of HPV integration in advanced stages of cervical neoplasia (Hudelist et al., 2004, Cullen et al., 1991, Van Ranst et al., 1992, Jeon et al., 1995, Pirami et al., 1997, Park et al., 1997), although some authors detected only episomal forms in most of their investigated cervical malignancies (Matsukura et al., 1989). In any case, a selective growth advantage is described, and well documented by in vitro studies (Jeon et al., 1995), in cell populations harbouring integrated HPV genomes compared to those harbouring only episomal forms. Therefore, the early detection of HPV integration can represent a promising tool in prevention of cervical cancer. Between methods available for detection of HPV integrated sequences, a simple one suitable for large scale studies was evaluated in the present study. This method, previously described by Peitsaro et al. (2002), is focused on the crucial event which involves the HPV E2 open reading frame (ORF) sequence upon integration and which results in its disruption (Baker et al., 1987, Choo et al., 1987). E2 is a regulatory gene that controls the expression of the viral E6 and E7 oncoproteins, required to initiate and maintain the cell neoplastic growth (Zur Hausen, 2000). E2 deletion dramatically increases the amount of oncoproteins in the cell and, consequently, the cell proliferation rate (Zur Hausen, 2000, Von Knebel Doeberitz et al., 1998). The selected method points to the quantitative amplification of E2 sequences, which is inconsistent in case of viral integration. Some authors showed that this method allowed detection of integrated sequences even in pre-neoplastic diseases (Peitsaro et al., 2002, Gallo et al., 2003, Andersson et al., 2005). The present study aims to confirm integration as detected in pre-neoplastic lesions by this method, with the detection of integrated papillomavirus sequences-PCR (DIPS-PCR) (Luft et al., 2001), a technique which allows the identification of the fusion region between viral and cellular DNA, followed by DNA sequencing. For the first time, to our knowledge, we detected and confirmed the presence of HPV integrated forms, even mixed with episomal forms, in pre-neoplastic cervical lesions.

2. Materials and methods

2.1. Study population

Cervical specimens collected under informed consensus, from women involved in screening programs for cervical cancer organized in Turin between 1996 and 2002, were obtained with a Cervex brush and placed in Cytorich® medium (AutoCyte, Inc., Elon College, NC, USA). Analysis for HPV DNA and typing for high (HR) and low (LR) risk-HPVs were previously performed (Voglino et al., 2000, Van den Brule et al., 2002, Ronco et al., 2005). Residual material was stored at −80°C, to be used for other molecular analysis. In the present study 170 HR-HPV positive archival specimens with known cytology were selected. Cytologic and biopsy analysis were performed in the Pathology Departments of Saint Anna Hospital in Turin, Italy. Follow-up was not at present available for many patients.

2.2. Cell lines

SiHa cervical carcinoma cells (ATCC, Manassas, VA, USA) were used as control of HPV-DNA integration into the cellular genomic DNA. SiHa cells harbour 1–2 copies of integrated HPV16 genome into chromosome 13 (Durst et al., 1987), in which E2 gene is damaged but E6 is retained. CaSki cervical carcinoma cells (ATCC) were used as control of amplification of both E2 and E6 genes. CaSki cells may harbour from 400 to 600 copies of HPV16 integrated in head-to-tail tandem repeats (Wagatsuma et al., 1990). In this type of integration, only the viral copy flanking the cellular DNA is interrupted in E2 region, while internal copies have intact E2 ORF (Wagatsuma et al., 1990).

2.3. DNA isolation

Genomic DNA from both cell lines and cell cervical specimens was purified by silica-gel columns (Qiagen, Mi, Italy) according to the manufacturer's procedure. Final elution of DNA was performed in 100μl of distilled water.

2.4. PCR assays

2.4.1. Qualitative PCR

DNA samples were screened for DNA quality by amplification of a 268basepair (bp) fragment of the β-globin gene (Dong et al., 2002) and for detection of a HPV16 E6 region of 208bp according to Yoshinouchi et al. (1999). All amplifications were performed in a Gene® Amp PCR System 9700 Thermal Cycler (Applera, Foster City, CA, USA). Amplicons were analyzed on ethidium bromide stained 2% agarose gel.

2.4.2. Quantitative real time-PCR (QRT-PCR)

A quantitative real time-PCR (QRT-PCR) method recently developed by Peitsaro et al. (2002), based on the measurement of absolute copy number of HPV16 E2 and E6 ORFs, was used to evaluate the HPV16 physical status. To establish the status of infection (episomal, mixed or integrated) we considered E2/E6 ratio, as relationship between the amount of integrated and episomal forms. A ratio E2/E6 equal to 1, <1 or 0 indicates the presence of episomal, mixed or integrated forms respectively (Peitsaro et al., 2002).

Integrated viral load was calculated by subtracting the copy numbers of E2 (episomal) from the copy numbers of E6 (integrated and episomal).

Primers and TaqMan probes previously described (Peitsaro et al., 2002, Andersson et al., 2005) targeting the HPV16 E2 and E6 ORFs were used. E2 primers and probe recognize the E2 hinge region, deleted upon HPV16 integration. Amplification conditions were: 10min at 95°C; 15s at 95°C, 1min at 57°C for 50 cycles. QRT-PCR assays were performed with iCycler iQ™ Real Time PCR Detection System (Bio-Rad). Every sample was tested in triplicate. Each run included a HPV DNA negative sample and a sample with no DNA as negative controls; DNA from CaSki and SiHa cell lines as positive controls for intact E2 and E6 genes and for deleted E2 gene, respectively.

The threshold cycle (Ct) was calculated with a software for data analysis with an automatic setting of baseline (BioRad). An external calibration curve was generated automatically by plotting the Ct values against the logarithm of the copy numbers of serial 10-fold dilutions (106, 105, 104, 103 and 102 viral copies) of a plasmid carrying the complete HPV16 genome (Clonit, Mi, Italy). For E2 and E6 ORFs quantification, the absolute copy number of unknown samples was calculated by plotting the Ct values against the logarithm of the standard curve. The sensitivity of the method was 102 viral copies.

For DNA quantitation, a standard calibration curve using known amounts (200.0, 20.0, 2.0 and 0.2ng) of human genomic placentar DNA (Sigma–Aldrich, St. Louis, MO, USA) was generated (Wang-Johanning et al., 2002), and β-globin was amplified as previously described (Van Duin et al., 2002). DNA quantity was calculated by plotting the Ct values against the logarithm of the standard curve for each standard point.

Results were reported as viral copy numbers in 50ng of cellular DNA.

2.5. DIPS-PCR (detection of integrated papillomavirus sequences)

DIPS-PCR assay was performed on specimens, CaSki and SiHa DNA as previously described (Luft et al., 2001). In case of viral integration, DIPS-PCR amplicons included the site of viral disruption as well as the cell integration site. In case of HPV episomal status, DIPS-PCR amplified only a viral sequence of 791bp. PCR products were analyzed on ethidium bromide stained 2% agarose gel.

2.6. Direct sequencing analysis

DIPS-PCR products were excised from gel and purified with the “PCR Clean-up Gel Extraction Kit” (Macherey-Nagel, Dueren, Germany). DNA sequencing was carried out using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applera), and DNA analysis was performed with the ABI 310 automated capillary system (Applera), following the manufacturer's instructions. Sequences were analyzed with the BLAST Software (http://www.ncbi.nlm.nih.gov/BLAST).

3. Results

3.1. HPV16 detection

In the present study 170 HR-HPV positive archival cervical samples were tested for the detection of HPV type 16 DNA by PCR analysis targeting the E6 gene sequence (Yoshinouchi et al., 1999). Before HPV type specific PCR, samples were checked for DNA adequacy by PCR amplification of the β-globin gene and they all tested positive. Seventy-four samples out of 170 (43%) resulted HPV16 positive. Cytology was negative for intraepithelial lesion (NIL) in 31 cases, 7 of which showed benign cellular changes (BCC). Cytologic epithelial abnormalities were present in 43 cases [2 ASCUS (Atypical Squamous Cells of Uncertain Significance); 18 Low Grade Squamous Intraepithelial Lesions (LSIL), 22 High Grade Squamous Intraepithelial Lesions (HSIL), and 1 invasive cancer (CC)]. Biopsy follow-up information was available in 40 out of the 74 cytology cases and is presented in Table 1.

Table 1.

Viral load and physical status of HPV infection in cervical specimens


Sample no.	Cytology	Histology	E2/E6	E6 copies/50ng	E6−E2 copies/50ng	Physical status	Sequence homology
1	BCC		1	9.17×104		Episomal
2	BCC		1	1.81×105		Episomal
3	BCC		1	2.28×103		Episomal
4	BCC		0.19	2.95×106	2.38×106	Mixed	HPV16, HuGen DNA
5	BCC		1	6.76×105		Episomal
6	BCC		1	3.17×105		Episomal
7	ASCUS		0.37	1.07×107	7.80×106	Mixed	HPV16, HuGen DNA [Chr. 13]
8	ASCUS		0.23	6.30×105	4.75×105	Mixed	HPV16, HuGen DNA
9	H-SIL	CIN1	1	2.15×107		Episomal
10	L-SIL	CIN1	0.30	7.63×106	5.16×106	Mixed	HPV16, HuGen DNA [Chr. 4]
11	L-SIL	CIN1	0.45	3.43×107	1.87×107	Mixed
12	L-SIL	CIN1	1	1.88×104		Episomal
13	L-SIL	CIN1	1	8.81×105		Episomal
14	L-SIL	CIN1	0	3.56×104	3.56×104	Integrated	HPV16, HuGen DNA [Chr. 13]
15	H-SIL	CIN1	1	2.47×104		Episomal
16	L-SIL	CIN1	1	4.71×106		Episomal
17	H-SIL	CIN1	1	2.59×107		Episomal
18	L-SIL	CIN1	1	1.27×105		Episomal
19	L-SIL	CIN1	1	7.97×103		Episomal
20	H-SIL	CIN2	1	3.88×107		Episomal
21	H-SIL	CIN2	1	3.85×104		Episomal
22	L-SIL	CIN2	1	1.73×104		Episomal
23	H-SIL	CIN2	0.36	7.00×106	4.45×106	Mixed
24	H-SIL	CIN2	0.57	1.55×108	6.66×107	Mixed
25	H-SIL	CIN2	1	4.63×105		Episomal
26	L-SIL	CIN2	1	1.66×106		Episomal
27	H-SIL	CIN2	0.67	1.09×105	3.56×104	Mixed
28	L-SIL	CIN2	1	2.65×105		Episomal
29	L-SIL	CIN2	1	1.36×106		Episomal
30	H-SIL	CIN2	1	2.21×106		Episomal
31	L-SIL	CIN2	1	2.23×106		Episomal
32	H-SIL	CIN2	0.25	1.78×106	1.32×105	Mixed	HPV16, HuGen DNA
33	H-SIL	CIN3	1	8.98×104		Episomal
34	H-SIL	CIN3	1	3.60×106		Episomal
35	H-SIL	CIN3	1	7.55×104		Episomal
36	H-SIL	CIN3	0	1.71×105	1.71×105	Integrated	HPV16, HuGen DNA [Chr. 1]
37	H-SIL	CIN3	1	7.55×106		Episomal
38	L-SIL	CIN3	0.29	3.86×106	2.73×106	Mixed	HPV16, HuGen DNA
39	H-SIL	CIN3	0.14	8.43×106	7.19×106	Mixed
40	L-SIL	CIN3	1	4.62×106		Episomal
41	H-SIL	CIN3	1	4.21×105		Episomal
42	H-SIL	CIN3	0.60	5.63×106	2.42×106	Mixed
43	H-SIL	CIN3	0	2.88×105	2.88×105	Integrated	HPV16, HuGen DNA
44	H-SIL	CIN3	1	1.61×107		Episomal
45	H-SIL	CIN3	1	3.78×106		Episomal
46	CC	CC	0.12	3.48×107	3.03×107	Mixed	HPV16, HuGen DNA [Chr. 1]
47	NEG		1	4.61×107		Episomal
48	L-SIL	NEG	1	7.94×102		Episomal
49	L-SIL	NEG	1	2.25×107		Episomal
50	NEG		1	6.02×104		Episomal
51	NEG		1	3.21×103		Episomal
52	NEG		1	1.18×102		Episomal
CaSki			1	4.24×107
SiHa			0	3.06×105	3.06×105		HPV16, HuGen DNA [Chr. 13]

3.2. HPV viral load and physical status

HPV viral load and physical status were evaluated in 4 out of the 24 samples showing normal cytology and in all abnormal samples with exception of 2 (1 BCC and 1 CIN1) in which the material was not sufficient for further analysis, for a total of 52 out of the 74 HPV16 positive samples. HPV viral load and physical status were evaluated by QRT-PCR targeting E2 and E6 genes, as shown in Table 1. The relative amount of viral copy numbers, normalized with the β-globin standard curve and calculated per 50ng of input DNA for each sample, varied from 1.18×102 to 1.55×108. The relative amount of integrated copy numbers varied from 3.56×104 to 6.66×107. E2/E6 ratio revealed 3 integrated, 13 mixed and 36 episomal forms (Table 1). Table 2 shows the HPV status according to cervical lesions.

Table 2.

Distribution of HPV infection forms in cervical lesions


Cervical lesions	Episomal forms	Mixed forms	Integrated forms
No lesion	11/12 (92%)	1/12 (8%)	0
ASCUS	0	2/2 (100%)	0
CIN1	8/11 (73%)	2/11 (18%)	1/11 (9%)
CIN2	9/13 (69%)	4/13 (30%)	0
CIN3	8/13 (61%)	3/13 (23%)	2/13 (15%)
CC	0	1/1 (100%)	0

3.3. DIPS-PCR and direct sequencing

DIPS-PCR was performed in all the studied specimens with sufficient material for the analysis, that is 20/36 samples with episomal forms, 7/13 with mixed and 3/3 with integrated forms. SiHa cells served as a control of viral integration. In the episomal forms, DIPS-PCR amplified only the HPV16 DNA sequence of 791bp. In mixed and integrated forms (n=10), amplicons of different sizes were visualized. Direct sequencing was performed in mixed and integrated forms and showed the presence of a fusion region with homology for both HPV16 and human genomic (HuGen) DNA sequence (Table 1). In five samples (sample no. 7, 10, 14, 36 and 46, Table 1) and in SiHa cell line, homology with a specific human chromosome was detected (e.g. chromosome 1, 4 and 13). In the other five samples (sample no. 4, 8, 32, 38 and 43, Table 1), the limited number of nucleotides flanking the HPV sequence was not sufficient to specifically identify a unique human chromosome. Fig. 1 shows the DNA sequencing results of two samples (sample no. 36 and 10, Table 1) harbouring integrated (A) and mixed (B) viral forms respectively: in both the cases, the viral genome was flanking a specific human DNA sequence, that is chromosome 1 and 4, respectively.

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Fig. 1. Electropherograms showing direct sequencing of DIPS-PCR products. (A) Sample no. 36: CIN3 with HPV16 DNA fully integrated into human chromosome 1. (B) Sample no. 10: CIN1 with mixed viral forms.

4. Discussion

Our results show that HPV16 integration occurs in a subset of low grade cervical lesions, and that the QRT-PCR method we used is a sensitive, reliable and specific technique, suitable for the identification of HPV16 integration. The evaluation of E2/E6 copy number ratio allows to avoid technical problems due to the co-existence of infected and not infected cells, without misinterpretation. Moreover, as viral gene damaging upon integration may sometimes also involve HPV E1 gene (Durst et al., 1987, Kalantari et al., 1998), this analysis can be in future extended to the E1 ORF amplification.

Although the underlying mechanisms involved in cervical cancer development are still unclear, high HPV viral load and HPV integration seem to play an important role in its progression. With respect to viral load, many studies showed values of viral load irrespective of the amount of DNA extracted from specimens. We adjusted HPV viral load to known amount of a housekeeping gene (β-globin) (Van Duin et al., 2002), to cope with occurring DNA variability in samples. In our specimens, high variability was found in the amount of HPV viral load, in agreement with data reported by other authors (Swan et al., 1999, Peitsaro et al., 2002, Van Duin et al., 2002, Hudelist et al., 2004). High viral load was detected in the majority of specimens, regardless of the type of the lesions. However, a linear trend from lower to higher grade lesions as described by Swan et al. (1999) was not identifiable. Among specimens with abnormal cytology (n=43), including those with low grade lesions, 15 presented integrated sequences. One cytology specimen that was negative for intraepithelial lesion (NIL/BCC) also presented an integrated HPV sequence. This result supports the hypothesis that the integration event may occur early in pre-neoplastic lesions, when the viral genome still persists at an episomal status (Wentzensen et al., 2004). Although evidence of integration in low grade lesions is not always sustained (Hudelist et al., 2004), some authors detected the presence of integrated HPV sequences even in CIN1 lesions (Peitsaro et al., 2002, Gallo et al., 2003, Andersson et al., 2005). Our results are in line with these studies, as we found integration in three cases of biopsy-confirmed CIN1. We also found integration in two cases of cytologic ASCUS and one case of cytologic NIL/BCC. However, since follow-up biopsy information was not available for the latter three cases, it is difficult to determine the true disease state of the associated cervical epithelium. A part from one CIN1 lesion, in which the specimen was not sufficient for analysis, DIPS-PCR allowed to identify also in the above mentioned low grade lesions, the cellular-viral site of junction, confirming the QRT-PCR results, even when integrated forms were mixed with episomal forms. The cell genomic region involved in the integration process resulted different among our specimens, according to published data showing that the integration sites, mostly unique in each clinical specimen, are widely distributed over the whole human genome (Luft et al., 2001, Ziegert et al., 2003, Wentzensen et al., 2004). Indeed, while from the viral perspective a preferential region of integration (i.e. E2 ORF) has been identified, a preferred human genomic site for HPV integration has not been demonstrated so far. A predilection for human genomic fragile sites was documented (Thorland et al., 2003, Wentzensen et al., 2004), but no evidence of functional alterations in critical cellular genes has been described.

In three samples of our study (one CIN1 and two CIN3 lesions), the integrated form of HPV was detected. In the low grade CIN1 lesion, the integration site was within the human chromosome 13 as identified by DIPS-PCR and DNA sequencing. The early detection of integration in a low grade lesion, confirmed for the first time to our knowledge by a ‘gold standard’ method (i.e. DNA sequencing), may represent a relevant prognostic marker. As the association of viral integration with recurrent disease (Unger et al., 1995) and shortened disease-free survival (Unger et al., 1995, Vernon et al., 1997, Lindel et al., 2005) has been documented, the early detection of HPV integrated sequences may have consistent implications in the management of cervical cancer. This strategy could represent a helpful complementary tool in primary cervical screening to identify and monitor subjects at increased risk of neoplastic progression. Follow-up of a large population sample within screening programs would also allow the evaluation of its prognostic value.

Acknowledgements

This study was partly supported by the Special Project ‘Oncology’, Compagnia di San Paolo/FIRMS and by ‘Ricerca Sanitaria Finalizzata’, CIPE.

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a Unit of Cancer Epidemiology, C.E.R.M.S., University of Turin, via Santena 7, 10126 Turin, Italy

b Unit of Molecular Oncology, C.E.R.M.S., University of Turin, via Santena 7, 10126 Turin, Italy

c Unit of Microbiology, Ospedale S.Giovanni Battista, corso Bramante 80, 10126 Turin, Italy

d Center for Oncologic Prevention, via S.Francesco da Paola 31, 10126 Turin, Italy

Corresponding author. Tel.: +39 011 633 6863; fax: +39 011 633 4664.

PII: S1386-6532(06)00344-1

doi:10.1016/j.jcv.2006.09.008

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