Original Research

Multiple human papillomavirus infection in ASC-H and HSIL lesions in women with cervical biopsies and comparison between Cobas 4800 HPV and Hybrid capture High-risk 2 Test

¹Xavier Catteau (M.D), ²Michel Pétein (Ph.D), ³Michel Vanhaeverbeek (Ph.D), ¹Jean- Christophe Noël (Ph.D)

• ¹Unit of Gynaecopathology, Pathology Department, Erasme’s University Hospital- Université Libre de Bruxelles, Brussels, Belgium.
• ²Institute of Pathology and Genetics, Gosselies, Belgium.
• ³Laboratory of Experimental Medicine, Centre Hospitalo-Universitaire de Charleroi, André Vésale Hospital-Université Libre de Bruxelles, Montigny-Le-Tilleul, Belgium.

• Submitted: September 9, 2012
• Accepted:September 30, 2012
• Published: October 16, 2012

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objective

The primary aim of this study was to document the distribution of different HPV genotypes in women with cytological lesions of ASC-H and HSIL in whom a biopsy was performed. The secondary aim was to evaluate the performance of the hc2 test by comparing the results with those obtained with the Cobas 4800 test.

Methods

A total of 84 liquid-based cytology, 34 ASC-H and 50 HSIL, were collected for this study. Biopsy specimens of lesions were obtained in 68 cases. Cytlogical samples were tested with Hybrid Capture 2 and Cobas 4800.

Results

Eleven had no dysplasia, seven had CIN 1, twelve had CIN 2, eight had CIN2/3, twenty-six had CIN3, and four had SCC on biopsy. There was no statistically significant difference between age groups or histological lesions regarding the presence of co-infection. The detection rate of HR-HPV DNA was slightly higher using hc2 compared with Cobas 4800. Indeed, one specimen (lesion of ASC-H with CIN3 on biopsy) was hc2 positive, while Cobas 4800 negative. However, the results suggest that the clinical performances of both methods can be compatible.

Conclusions

There was no statistically significant difference in the presence of co-infection depending on the grade of dysplasia, nor according to age. The advantage of the Cobas 4800 is the ability to subtype HPV but at present it is not essential for the care of patients. In the case of our laboratory where hc2 is used, at present time there appears to be no particular reason to change the tests for HPV detection.

Keywords

Cobas; Hybrid Capture 2; HPV; Multiple; Vaccination; Dysplasia.

Introduction

Today, it is well known that a persistent infection, with a high-risk of human papillomaviruse (HR-HPV) increases significantly the risk of developing cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) [1-3]. Many studies have already been done to look into the value of adding an HR-HPV DNA test to the classical Pap smear in order to improve the efficacy of cervical cancer screening programs and also to improve the efficacy for the triage of women with ambiguous or borderline cervical smears [4-7].

Recently, interest in multiple HPV infection has increased due to the availability of vaccines against HPV 6, 11, 16, and 18. To date, 20-40% of HPV-positive women are reported to be infected with multiple HPV types [8]. Therefore, it would be of great interest to correctly identify which HPV infection type is present, as vaccination may modify the established equilibrium in the distribution of HPV types within immunized populations in the long term.

However, only a limited number of assays that detect DNA of HR-HPV types have proven to be of clinical value in longitudinal studies involving large cohorts of women. One of these involves the commercially available, hybrid capture 2 (hc2) test. The hc2 assay is based on signal amplification and was the first HPV DNA test approved by the US Food and Drug Administration (FDA). The Cobas 4800 HPV(Roche Molecular) test has recently been approved by the FDA [9]. This test is a qualitative single tube multiplex assay that simultaneously detects 14 high-risk genotypes, it identifies HPV type 16 and 18 and ß-globin is used as an internal control.

The primary aim of this study was to document the distribution of different HPV genotypes in women with cytological lesions of ASC-H and HSIL in whom a biopsy was performed. The secondary aim of this study was to evaluate the performance of the hc2 test by comparing the results with those obtained with the Cobas 4800 test.

Methods

The study protocol received approbation from the institutional ethical and research review boards of Erasme Hospital. A total of 84 liquid-based cytology, 34 ASC-H and 50 HSIL, were collected for this study. Cervical cells were first collected with a cytobrush for a conventional cytology. The cytobrush was washed into PreservCyt collection medium (Cytic Corporation, Boxborough, MA, USA). Cytomorphological analysis was performed according to the Bethesda 2001 classification by two pathologists (JCN, XC) [10]. Samples were kept at room temperature and processed within 1 week for HPV DNA testing. Colposcopy-guided biopsy specimens of lesions were obtained in 68 cases, and histological diagnosis was established without knowledge of HPV results. Biopsy samples with CIN 2/3 were confirmed by a second pathologist (JCN).

HPV DNA testing

Technicians performing each HPV DNA test were blinded as to the other test results, as well as to the cytology, colposcopy, and histology results. Amounts of 4 ml of samples in PreservCyt were processed in the sample conversion kit (Qiagen, Inc., Mississauga, Canada) and tested with hc2 according to the manufacturer’s recommendations, using the specific HPV RNA probe cocktail for carcinogenic HR-HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Presence or absence of HPV DNA in the specimen was defined according to the strength in relative light units (RLU) compared to 1 pg/ml HPV16 DNA-positive control (CO). The sample was considered positive when the ratio of RLU/CO was ≥ 1.

The Cobas 4800 system features fully automated sample preparation combined with real-time PCR technology plus software that integrates the two components. The test was designed to extract, amplify and detect a broad spectrum of HR-HPV genotypes, as well as to co-amplify the human cellular globin gene. In this study, PCR amplification and detection occurred in a single tube, where probes with four different reporter dyes tracked the different targets in the multiplex reaction. Reporter dye 1 tracked the HR- HPV pool with 12 HR targets (HPV-31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66 and -68); dyes 2 and 3 tracked HPV-16 and HPV-18, respectively; and dye 4 targeted ß-globin to provide a control for cell adequacy, extraction and amplification. The test was performed following the manufacturer’s instructions for the Cobas X 480 for DNA extraction and Cobas Z 480 for real-time PCR.

Statistical methods

Differences in the rates of detection of HR-HPV types were tested for statistical significance using a McNemar’s test. The agreement was determined using the kappa value. In general, a kappa value above 0.75 indicates excellent agreement, between 0.40 and 0.75 indicates fair to good agreement, and below 0.40 represents poor agreement beyond chance. Chi-square test was used to assess the association between the presence of co-infection and age and grade of dysplasia, respectively. All P values of < 0.05 were considered statistically significant. Statistical analyses were performed with STATISTICA version 7 software (StatSoft, Tulsa, UK).

Results

A total of 84 cervical scrape specimens were analyzed by two HR-HPV detection methods. Colposcopy-guided biopsy specimens of lesions were obtained in 68 cases.

Cytological and histological findings

Thirty-four ASC-H and fifty HSIL were collected for this study. The mean age of participants was 38 (range 20 to 84). Eleven had no dysplasia (metaplasia, atrophy, inflammatory changes or no contributory), seven had CIN 1, twelve had CIN 2, eight had CIN2/3, twenty-six had CIN3, and four had invasive squamous cell carcinoma on biopsy.

Table 1: Comparison of HPV results between hc2 and Cobas 4800

	HPV positive
	hc2	Cobas 4800
ASC-H	34	33
HSIL	50	50

HPV prevalence found by hc2 and Cobas 4800

The HPV detection results obtained with hc2 and Cobas 4800 are shown in (Table 1). The percentage of agreement between both tests was 98.8 % (83/84 cases). Considering the results of all participants, the detection rate of HR-HPV DNA was slightly higher using hc2 compared with Cobas 4800. Indeed, one specimen (lesion of ASC-H with CIN3 on biopsy) was hc2 positive, while Cobas 4800 negative. However, the results suggest that the clinical performances of both methods can be compatible (Mc Nemar’s test: p=1; kappa =0,98). In the case of single infections, 73.8% of the samples were positive for either HPV16 or HPV18. Multiple HPV types were detected in 26.2% of the samples (22/84): seven patients were under 30 years old, seven were between 30 and 50 years old and eight were over 50 years old. There was no statistically significant difference between age groups regarding the presence of co-infection (p=0.86). Co-infection was detected in 28.6% of CIN 1, 15.9% of CIN 2 > (CIN2+CIN2/3+CIN3) and 75% of invasive carcinoma. There was no statistically significant difference between histological lesions regarding the presence of co-infection (p=0.62). The distribution of HPV according to cytological and histological lesions is shown in (Table 2).

Table 2: Distribution of HPV by Cobas 4800 according to cytological and histological lesions

		HPV
Cytology	Histology	Total	16	18	Others HPV	Multiple infection
ASC-H	ND or NC*	10	4	3	-	3
	Negative**	5	2	1	-	2
	CIN 1	6	3	1	-	2
	CIN 2 >***	12	6	4	-	2
	Invasive	1	0	1	-	-
HSIL	ND or NC	10	4	2	-	4
	Negative	3	1	1	-	1
	CIN 1	1	1	-	-	-
	CIN 2 >	33	15	12	1	5
	Invasive	3	-	-	-	3
Total		84	36	25	1	22

Discussion

The prevalence of HPV genotypes in CIN2, CIN3 and cervical cancer has been reported from several countries and suggests that vaccination against HPV 16 and 18 could prevent up to 70-80% of cervical cancers worldwide [11]. We have found the same percentages in our series (76.5% of single infections with HPV 16 or 18). This obviously represents an important advance in the fight against cancer of the cervix. However, it also means that 20 to 30% of women, even those vaccinated may still develop invasive cancer. This rate is not negligible. Moreover, we have good knowledge about the types of HPV found in invasive cancers but the HPV type distribution for each grade of dysplasia is less well known. For this reason we were particularly interested in this study for genotyping HPV in the lesions of dysplasia. The underlying issues being: 1) What is the percentage of women with lesions of high-grade dysplasia, CIN2 and CIN3, which are infected with an oncogenic HPV other than 16 and 18? As this subset of women would not be covered by vaccination and would therefore remain at risk of developing invasive cancer. 2) Is there a higher risk of developing high-grade lesions if there is an infection with multiple HPV with the presence or absence of HPV 16 or 18? It may be that multiple types act synergistically to induce lesions, though this has not been clearly demonstrated. It is still unclear whether multiple type infections are the result of a risk factor shared with CIN or cancer or whether they are actually involved in the causation of CIN or cancer [12-15].

In several studies, the highest prevalence of multiple HPV infection were detected in high grade cervical intraepithelial neoplasia (CIN3: 43.3%) followed by decreasing prevalence in cases with decreasing grade of dysplasia (CIN2: 27.5%; CIN1: 18.9%) and this occurred in women between 25 and 34 years of age (35.5%), followed by the age group between 35–44 and <24 years, respectively [16-18]. In our study, there was no statistically significant difference in the presence of co-infection depending on the grade of dysplasia, nor according to age. This may be due to the fact that our series was relatively small.

New tools for detection of HPV DNA are emerging including the Cobas 4800. This new real-time PCR-based assay has several important advantages over other HPV detection and/or genotyping assays. First of all, the test not only reports the presence of HR-HPV genotypes, but it also simultaneously offers partial HPV genotyping for HPV-16 and HPV-18 separately. Secondly, the assay is very easy to use because it is adapted for primary specimens. Finally, results can be obtained approximately 4 h after receiving the specimen [19,20]. However, the Cobas 4800 also has some inconveniences, such as the need to simultaneously perform at least 22 tests to optimise the reactants. Given that the Cobas 4800 HPV test detects non-HPV-16/18 HR genotypes as a pool, it is not possible to assess the exact number of carcinogenic HPV types present. Moreover, there is also the risk of false negatives caused by viral integration of the region L1 which occurs mostly in high-grade lesions [21]. This risk does not exist with the hc2 test. In our study, the direct correlation between the Cobas 4800 HPV test and hc2 test were excellent (98.8%), and only 1 specimen (lesion of ASC-H with CIN3 on biopsy) was hc2 positive while, Cobas 4800 negative. Several groups compared hc2 and Cobas 4800 in different clinical settings and have reported good levels of agreement for HR- HPV DNA detection, ranging from 78% to 89%, and with kappa values ranging from 0.61 to 0.78 [22-28]. To further explain the occurrence of a hc2-positive while having a cobas 4800-negative case, we addressed the influence of viral integration events disrupting the Cobas primer binding region in L1. The usefulness of a full HPV genotyping is still uncertain. Indeed, there was no significantly increased risk for multiple HPV infections found in younger women (<30 years). Similarly, the prevalence of infection with multiple HPV types also varies significantly across studies from between 1% and 20% [1], particularly in relation to the HPV detection method used. Particularly in an era where HPV vaccination have started to improve the understanding of the natural history and dynamics of HPV infection and the pathogenic effect of multiple types it could be interest to monitor the impact of vaccination on the risk of acquiring an individual HPV type. But if HPV genotyping became indispensable, it might be of greater interest to do the hc2 test, due to it being easy and robust and therefore an ideal tool for application in large screening programs and then to use the Cobas 4800 as a reflex test (i.e. testing only of hc2-positive samples) for the genotyping of hc2-positive women.

Conclusion

In our study, there was no statistically significant difference in the presence of co-infection depending on the grade of dysplasia, nor according to age. This may be due to the fact that our series was relatively small. Further, larger studies are needed to confirm the impact of multiple infections in cervical carcinogenesis.

The hc2 and Cobas 4800 tests show similar results with a small advantage to using the hc2 if we refer to broader studies. The advantage of the Cobas 4800 is the ability to subtype HPV but at present it is not essential for the care of patients. In the case of our laboratory where hc2 is used, at present time there appears to be no particular reason to change the tests for HPV detection.

Authors’ Contribution

XC: Preparation of the manuscript
MO: Preparation of the manuscript
MV: Preparation of the manuscript
JCN: Preparation of the manuscript

Conflict of Interests

The authors declare that there are no conflicts of interests.

Acknowledgment

None

Funding

None

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