DNA recovery from Hybrid Capture II samples stored in specimen transport medium with denaturing reagent, for the detection of human papillomavirus by PCR

Journal of Virological Methods 126 (2005) 197–201

DNA recovery from Hybrid Capture II samples stored in specimen transport medium with denaturing reagent, for the detection of human papillomavirus by PCR Silvia H. Rabelo-Santos a,c , Jos´e Eduardo Levi b , Sophie F.M. Derchain a,∗ , Luis Ot´avio Z. Sarian a , Luiz Carlos Zeferino a , Samara Messias a , Denise Lima de Moraes a , Elisabete A. Campos a , Kari Juhani Syrj¨anen d a

Department of Obstetrics and Gynecology, Universidade Estadual de Campinas (UNICAMP), Caminas, SP, Brazil b Virology Laboratory, Institute of Tropical Medicine, University of S˜ ao Paulo (USP), S˜ao Paulo, Brazil c Federal University of Goi´ as (UFG), Goiˆania, GO, Brazil d Department of Oncology and Radiotherapy, Turku University Central Hospital Savitehtaankatu, Turku, Finland Received 24 November 2004; received in revised form 17 February 2005; accepted 22 February 2005 Available online 31 March 2005

Abstract The purpose of this study was to examine the quality of DNA recovered for human papillomavirus (HPV) detection using polymerase chain reaction (PCR) in samples that had been collected for Hybrid Capture II (HCII), testing and stored in specimen transport medium (STM) with denaturing reagent at −20 ◦ C for 18 months. Endocervical tissue was collected from 92 women for HCII assay using the Digene STM, and a Papanicolaou smear was carried out in all cases. Seven women had normal colposcopy results. The remaining 85 patients underwent colposcopy-directed biopsy or cervical conization for histological investigation. Of the 92 samples tested, 84 were HCII-positive and 8 were negative. Quality control for amplification was carried out with ␤-globin primers G73 and G74, and HPV was tested using PGMY09 and PGMY11. DNA was recovered from 83 of the 92 samples (90%). Among the 84 samples HCII-positive initially, HPV was detected by PCR in 56 (67%). PCR did not detect HPV DNA in the eight samples that were HCII-negative, although five of them were positive for ␤-globin. This paper describes a novel DNA extraction technique that may permit exact HPV typing in stored samples collected originally for HCII testing, making it possible to carry out retrospective investigations to retrieve information on specific HPV types in large HCII series. © 2005 Elsevier B.V. All rights reserved. Keywords: DNA extraction; HPV; Hybrid Capture II; Specimen transporting medium; Polymerase chain reaction

1. Introduction Currently, more than 100 human papillomavirus (HPV) genotypes have been described, and over 40 different types are known to infect the genital tract (Bosch et al., 1995; Walboomers et al., 1999; Syrj¨anen and Syrj¨anen, 2000). Genital HPV genotypes are grouped commonly into high-risk and ∗ Corresponding author. Present address: Rua Antˆ onio Hossri, 629 Cidade Universit´aria, 13083-370s Campinas, SP, Brazil. Tel.: +55 19 37889305; fax: +55 19 37889302. E-mail address: [email protected] (S.F.M. Derchain).

0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.02.009

low-risk categories on the basis of known epidemiological association with preneoplastic/neoplastic and benign lesions, respectively (Zur Hausen, 1996). Persistent infection with high-risk HPV genotypes appears to be a necessary prerequisite for the development of cervical carcinoma, which has an annual global incidence rate of over 400,000 cases (Bosch et al., 1995; Walboomers et al., 1999; Dillner, 2001). Several methods for the detection of HPV DNA have been described over the past two decades, allowing the identification of a wide spectrum of HPV genotypes (Solomon et al., 2001; Hubbard, 2003). Hybrid Capture II HPV test (HCII) is the only commercially available assay approved by the Food

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and Drug Administration of the USA for the routine detection of HPV infections. Mixtures of unlabeled, full-length genomic RNA probes may be included in the HCII assay: probe A cocktail which contains low-risk HPV genotypes 6, 11, 43 and 44, and probe B cocktail containing high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 (Perrons et al., 2002; Poljak et al., 2002; Hubbard, 2003). Despite the fact that the HCII test is unable to determine the specific HPV type, this test has become the HPV detection standard in many countries, and has been tested intensively in large clinical studies (Cuzick et al., 2000; Solomon et al., 2001). A target amplification technique, polymerase chain reaction (PCR), constitutes the most flexible and most sensitive of all DNA detection techniques, and is applicable for HPV detection, viral load determination, DNA sequencing and mutation analysis. When carried out in the multiplex mode, multiple target DNA sequences are analyzed simultaneously. The disadvantage of PCR is that the “in-house” PCR tests, by their very nature, suffer from deficient inter-laboratory standardization. PCR assays are also subject to environmental contamination when material amplified previously may contaminate negative specimens, leading to false positive results (Hubbard, 2003). Although the relative risk of most phylogenetically highrisk types of HPV is still unknown and since they seem to differ in their oncogenic potential, detection of individual HPV genotypes by PCR in cervical specimens is, therefore, valuable for assessing their risk. HCII has been used extensively since 1998; consequently, there are large series of stored samples in many laboratories. It should be possible to use PCR to identify the specific virus type in these denatured frozen samples that have been stored in specimen transport medium for HCII tests (A. L¨orincz, personal communication). Analysis of specific HPV types in HCII-tested samples from women with adequate follow-up would be helpful for retrospective evaluation of the natural history of these lesions. The objective of this study was, therefore, to determine the recovery of DNA extracted from samples taken from women with abnormal Pap smears, stored with a denaturing reagent in standard transport medium and analyzed by HCII, to test the quality of these samples for HPV detection by PCR.

2. Materials and methods 2.1. Samples for HCII testing A total of 92 cervical specimens were obtained between January and June 2001 from women referred for cervicitis (n = 9), atypical squamous cell of undetermined significance (n = 3), low-grade squamous intraepithelial lesion (n = 63) and high-grade squamous intraepithelial lesions (n = 17) based on the result of a Papanicolaou smear. All women enrolled to this study had endocervical material collected for HCII assay, using Digene Specimen Collection Medium. All

Table 1 HCII results according to cytological and histological diagnostic Morphological diagnosis

HCII-positive

HCII-negative

Total

Cytology result Negative ASC-US LSIL HSIL

8 (88) 3 (100) 57 (90) 16 (94)

1 (12) 0 6 (10) 1 (6)

9 (10) 3 (3) 63 (68) 17 (18)

Histological result No biopsy Cervicitis CIN 1 CIN 2 CIN 3

6 (86) 3 (75) 57 (92) 15 (94) 3 (100)

1 (14) 1 (25) 5 (8) 1 (6) 0

7 (8) 4 (4) 62 (68) 16 (17) 3 (3)

Total

84 (91)

8 (9)

92 (100)

The values in parenthesis are in percent.

except seven women, who had normal colposcopy results, underwent colposcopy-directed biopsy or cervical conization for histological examination, which revealed 4 cases of cervicitis, 62 cases of cervical intraepithelial neoplasia grade 1, 16 of grade 2 and 3 of grade 3. In the HCII assay, the samples with the relative light unit/positive control (RLU/PC) cut-off value of 1.0 or above were considered positive for the HPV genotypes included in probe cocktail B (HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) (Table 1). Of the 92 samples tested, 84 were HCII-positive and 8 were negative. In HCII-positive cases, viral loads ranged between 1.3 and 2575.0 RLU/PC. All HCII samples were stored in standard transport medium (Digene Corporation Gaithersburg, MD), which contains the denaturing reagent, diluted sodium hydroxide solution with dark purple indicator dye, supplied as a part of the HCII collection kit and used as the first step in the HCII assay. All samples were stored in a freezer at −20 ◦ C for 18 months before DNA extraction for PCR amplification. 2.2. DNA extraction Total DNA was extracted from the samples containing the denaturing reagent using the extraction technique provided by Dr. A.T. L¨orincz (personal communication). Firstly, an aliquot (450 ␮L) of the Hybrid Capture denatured specimen was removed and transferred to a 1.5 mL microtube, with 1.0 mL precipitation solution composed of 2 mL of NaAc 3 M, pH 5.2, 200 ␮g of glycogen (prepared with 10 ␮L of 20 mg/mL glycogen solution), and 100 mL of absolute ethanol (0.13 mM sodium acetate, 0.04 ng glycogen in absolute ethanol). The solution was first shaken, and then left to rest overnight in a freezer at −70 ◦ C. On the following day, the solution was centrifuged at 12,000 × g for 15 min at 4 ◦ C. After rinsing the pellet in 400 ␮L of 70% ethanol, it was centrifuged at 3000 × g for 5 min. The tube was inverted for 30 min to allow the ethanol to evaporate and the dry pellet was solubilized in 100–200 ␮L of TE solution (Tris 1 mM EDTA 100 uM, pH 8.2).

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2.3. HPV detection

Table 2 HPV detection by HCII and PCR

The quality of DNA was monitored by amplification of part of the ␤-globin gene in replicate tubes using G73 and G74 primers. Two microliters of DNA were added to a PCR mix containing 0.4 ␮M G73/G74 primers, 3 mM MgCl2 , 0.1 mM dNTPs and five units of Taq polymerase in a final volume of 50 ␮L. HPV DNA was amplified in replicate tubes by using L1 consensus primers PGMY09 and PGMY11 that amplify a 450 bp fragment. Two microliters of DNA were added to a PCR mix containing 0.08 mM PGMY09/PGMY11 primers, 4 mM MgCl2 , 0.2 mM dNTPs and eight units of Taq polymerase in a final volume of 25 ␮L. Positive controls consisted of cervical samples collected from patients not included in this study using the HCII sample collection kit and processed under conditions similar to those described in detail by Levi et al. (2002). ER20 contains HPV 6 and presents a positive HCII result for probe A (3.67). ER136 is an HPV 44- and 66-positive sample for HCII probe A (8.07) and probe B (204.49). The PCR negative/HCII-positive samples were tested in duplicate. In this demonstration study, specific HPV types were not determined in the HPV-positive PCR samples.

PCR**

2.4. Statistical analysis Kappa (␬) coefficient was calculated to assess the agreement between HPV detection using HCII and 18 months later, PCR. Output tables were obtained and statistical calculations were performed using the software package R Environment for Statistical Computing (R Development Core Team, 2004), with confidence intervals of 95% (95% CI).

3. Results Almost 90% of women with negative cytology tested positive for HPV DNA, although approximately the same proportions of positive HCII tests were found among women with atypical squamous cells of unknown significance (100%), low-grade squamous intraepithelial lesion (90%) and highgrade squamous intraepithelial lesion (94%). Fourteen percent of women with a normal cervix and 25% of those with cervicitis were HPV DNA-positive, whereas 92% of patients with cervical intraepithelial neoplasia grade 1, 94% of women with cervical intraepithelial neoplasia grade 2 and 100% of women with cervical intraepithelial neoplasia grade 3 had a positive HPV test (Table 1). Initial control amplification with ␤-globin primers G73 and G74 revealed that DNA was recovered from 83 of the 92 samples (90%). Among the 84 samples initially positive for HCII, HPV was detected by PCR in 56 (67%). No HPV DNA was detected by PCR in any of the eight HCIInegative samples although five were positive for ␤-globin (Table 2).

199

HCII-positive**

HCII-negative**

n

n

%

%

Positive Negative Not performed*

56 23 6

66 27 7

0 5 3

0 62 38

Total

84

100

8

100

∗ ∗∗

␤-Globin negative. ␬ = 0.22.

4. Discussion The present study was designed to assess whether sufficient intact DNA could be recovered from HCII samples, stored in STM (denatured) for 18 months in the freezer at −20 ◦ C, using a novel HPV DNA extraction method. This technique was capable of detecting HPV DNA using a conventional PCR amplification assay. If reproduced in other laboratories, this novel technique may have important implications such as allowing subsequent HPV typing of stored HCII samples. Importantly, following a careful extraction protocol, adequate amounts of well-preserved DNA were recovered from 83 out of the 92 samples (90%) stored for 18 months in STM at −20 ◦ C. The adequacy of the DNA sample was confirmed by the conventional amplification of the ␤-globin single-copy gene. These samples should be adequate for PCR amplification of HPV DNA. No PCR inhibitors were detected. There have been few previous studies in which DNA extraction for HPV testing from denatured samples has been attempted. Recently, Poljak et al. (2002) described a method of DNA extraction from samples containing denaturing reagent. Their technique was completely different from that described in this paper, in that a fully automated specimen preparation instrument was used (MagNa Pure LC, Roche and MagNa Pure LC DNA isolation kit) to extract DNA from a wide variety of human tissue. Using the KM29/RS42 primers, these authors succeeded in demonstrating the amplification of ␤globin in 183 out of 185 HCII-positive samples, which had been frozen at −70 ◦ C from completion of HCII testing until the beginning of their study. The quality of the sample was also different, in that these investigators had stored 140 samples prior to adding the denaturing reagent. It should be noted that at the sampling stage, prior to the addition of the denaturing reagent, storage is valid for only 14 days, according to the manufacturer’s instructions. Therefore, in practice, only those samples collected and stored in denaturing reagent are valid. In this report, a novel technique is described, designed to extract DNA from denatured HCII specimens. There have been no data published from studies using a similar extraction technique or with comparable sample quality and storage conditions. It is not certain which of these differences may account for the discrepancies found between our results and those reported by Poljak et al. (2002). Poor sampling is not

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a plausible explanation for the lower DNA detection rate in the present series compared to that reported by Poljak et al. (2002), because the HPV viral loads, as measured by HCII, ranged from 1.23 to 2575.6RLU (mean 454.56), which are good baseline HPV DNA amounts. Therefore, the extraction technique described in the current study deserves further investigation, and the results should be controlled according to the storage conditions of the samples, time of storage and initial HCII viral loads. The results of the present study have potential practical value for HPV diagnosis. The signal-amplified, hybridization microplate-based HCII assay is the only commercially available HPV DNA detection assay that has sufficient scientific data to support its performance in different clinical settings. Furthermore, the HCII assay is the only test approved by the Food and Drug Administration for the detection of HPV infections. However, although based on HPV probe cocktails, the test is not capable of detecting specific HPV types. As pointed out previously, assessment of specific HPV types has important implications for HPV diagnosis and research. Therefore, detection of the specific HPV types by PCR in specimens stored over prolonged periods of time would enable retrospective assessment of the lesions, permitting establishment of clinical management and prognosis. As such, this technology should provide valuable information concerning the natural history of CIN and specifically on the role played by the different HPV types (Davies et al., 2001; Gravitt et al., 2000). One of the major advantages of detecting adequate amounts of intact DNA for HPV DNA amplification by PCR in stored HCII-tested samples is the identification of the specific virus genotype. This in turn could provide valuable information regarding the persistence of HPV DNA, which would be helpful in making decisions on individual treatment, in epidemiological settings and in vaccine development (L¨orincz, 1996; Zehbe et al., 1998; Poljak et al., 2002). Another important advantage of PCR is to avoid crossreactivity of the two probe cocktails used in HCII (A and B) that could compromise the specificity and accuracy of the HCII assay (Peyton et al., 1998; Poljak et al., 2002) and others have found another 17 HPV genotypes that are potentially cross-reactive, in addition to the 5 HPV types already declared by the manufacturer to cross-react with the HCII probes cocktail. Cross-reactivity between HPV 6 and HPV 42 could lead to mistaken classification of a specimen as being positive for a high-risk type in situations in which a high concentration of the low-risk genotype is present (Hubbard, 2003). The number of samples used in this study was relatively small, but representative of both low- and high-grade squamous intraepithelial lesion samples that tested positive and negative according to the HCII assay. Not unexpectedly, the concordance between the HCII test and PCR was not particularly good. This is in alignment, however, with most of the large-scale studies (based on hundreds or thousands of samples) comparing the HCII assay with PCR, in which the

concordance between the two techniques has only been moderate (␬ values between 0.6 and 0.7) (Syrj¨anen and Syrj¨anen). A larger series needs to be tested before drawing any conclusions about the concordance between the HCII assay and the new extraction technique for PCR described in this article. In general, all studies, including the present one, report that HCII detects more HPV-positive samples than PCR. It may be that the DNA quality for hybridization is less strict than for PCR amplification. This was evident when a similar comparison was performed in the laboratory of one of the authors (J.E. Levi, personal communication). Initially, roughly 70% of the HCII-positive samples were also PCR-positive, but when a more sensitive PCR technique was used, 98% of the samples were found positive (Levi et al., 2002). This method consists of amplification of a small amplicon of approximately 70 bp, which is probably easier to amplify than the 450 bp spanned by PGMY09/11 that was used in the current study. In conclusion, a DNA extraction technique was evaluated, which was capable of recovering adequate amounts of DNA in 90% of the denatured samples stored over a prolonged time period in the standard transport medium for HCII assay. This technique offers the potential advantage of permitting an exact HPV type determination in the vast amounts of stored samples originally collected for HPV testing using the HCII assay. The application of the new technique can only be fully established after testing specific HPV types in a larger series of samples.

Acknowledgments This study was supported by Fundac¸a˜ o de Apoio a` Pesquisa do Estado de S˜ao Paulo (FAPESP grant number 99/11264-0 and 00/697-7). The skillful technical assistance of L´ucia Fagian is gratefully acknowledged.

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