Assessment of human papillomavirus mRNA detection over time in cervical specimens collected in liquid based cytology medium

Journal of Virological Methods 124 (2005) 211–215

Short communication

Assessment of human papillomavirus mRNA detection over time in cervical specimens collected in liquid based cytology medium Kate S. Cuschieria,∗ , Gerry Beattieb , Sameena Hassana , Kevin Robertsonc , Heather Cubiea a

Specialist Virology Centre, New Royal Infirmary of Edinburgh, 2nd Floor Microbiology, 51 Little France Crescent, Edinburgh EH16 4SA, UK b Department of Obstetrics and Gynaecology, St. Johns Hospital, Livingston, West Lothian EH64 6PP, UK c Scottish Centre for Genomic Technology and Informatics, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK Received 4 August 2004; received in revised form 1 November 2004; accepted 2 November 2004 Available online 16 December 2004

Abstract Little is known about the stability of human papillomavirus (HPV) RNA within cervical samples collected in liquid based cytology (LBC) preservation media. We addressed this by analysing patient LBC specimens for the presence of HPV RNA over a prospective time course. LBC samples in PreservCyt® were obtained from seven women referred to colposcopy due to a cytological diagnosis of moderate or severe dyskaryosis. Aliquots were removed and subject to RNA extraction at, 6 h (base-line), 4, 7 and 14 days, post-collection. HPV mRNA was detected using the PreTect® HPV Proofer, which detects HPV 16, 18, 31, 33 and 45 E6/E7 transcripts and human small ribonucleoprotein U1A mRNA as a sample control. HPV DNA genotyping was also performed at base-line to assess the range of types in our group. In addition to assessment of viral RNA, overall integrity of the cellular RNA extract was analysed by the RNA 6000 pico assay. Control human RNA was amplified successfully in all seven samples at each time point. Five of the seven women were HPV positive for E6/E7 viral transcripts at base-line and positivity was maintained in all five up to 14 days. Although the pattern of cellular RNA profiles generated from the samples was variable, results indicated that this extract could be amenable to gene expression profiling and that degradation did not increase as a result of storage time. It is concluded that HPV RNA in routinely collected LBC specimens in PreservCyt® can be detected for at least 14 days from sample collection. © 2004 Elsevier B.V. All rights reserved. Keywords: Human papillomavirus; RNA; Stability; Liquid based cytology

Replacement of the traditional Pap smear with liquid based cytology (LBC) for cervical screening is considered to have reduced the number of inadequate smears and improved the diagnostic accuracy of cytological reporting (McGoogan, 2004; Sass, 2004). Another benefit of the LBC approach is that in most cases, the majority of the suspension remains unused after cytology. This residual volume provides a specimen for ancillary testing for microbiological and other, cellular targets. Currently, the most relevant microbiological test is that for human papillomavirus (HPV), causally associated ∗

Corresponding author. Tel.: +44 131 242 6039; fax: +44 131 242 6008. E-mail address: [email protected] (K.S. Cuschieri).

0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2004.11.005

with the development of cervical intraepithelial neoplasia (CIN) and cervical cancer (Zur Hausen, 2002). In most large research studies where detection of HPV has been performed using LBC specimens (Clavel et al., 2001; ALTS Group, 2003; Cox, 2003), HPV DNA has been the target. DNA based HPV detection techniques include PCR methodologies, with common primer sets PGMY09/PGMY11 or GP5+/6+ (Coutlee et al., 2002; De Roda Husman et al., 1995), or the commercially available Digene Hybrid Capture® 2 (hc2) test. Consequently, most diagnostic HPV testing in different parts of the world is performed using DNA based tests. However, there has been growing interest in using detection of mRNA transcripts of

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known HPV oncogenes for diagnostic purposes. An advantage of such an approach could be an enhanced ability to differentiate between asymptomatic HPV infection and infection associated with disease (Wang-Johanning et al., 2002; Cuschieri et al., 2004). However, it is well recognised that RNA is a significantly less stable moiety than DNA and this could affect the suitability of RNA based testing in clinical samples. This is particularly relevant should HPV testing be performed after cytological assessment, as in countries with organised cytology screening programmes. In these cases, it is likely that the specimen would be at room temperature for several days, before being made available for virological testing. With this in mind, a longitudinal assessment of HPV RNA viability was undertaken in a collection medium used widely for liquid based cytology (PreservCyt® ). This was carried out by extracting aliquots of freshly collected LBC samples over a time course of 14 days. In addition to detection of RNA from the human small ribonucleoprotein control, detection of HPV E6/E7 mRNA was carried out to evaluate the consistency of HPV transcript detection throughout this time frame. Qualitative analysis of the cellular RNA fraction over the time course was undertaken to assess its candidacy for gene expression profiling, a technology which can be exploited to identify new putative markers of disease and disease susceptibility. A total of seven women were recruited from a local colposcopy clinic (St. Johns Hospital, Livingston, West Lothian, Scotland) after routine referral based on an earlier cytological report of moderate or severe dyskaryosis. An LBC sample in PreservCyt® was taken and consent for study participation obtained. After sample collection, a 4 ml aliquot of the LBC sample was removed within 6 h (base-line), 4, 7 and 14 days. Samples were spun for 15 min at 2800 × g. Subsequently all traces of PreservCyt® (Cytyc Corporation, Malborough, MA) medium were removed and pelleted material was extracted via the RNeasy system (QIAGEN LTD, Crawley, UK). RNA was eluted in 40–50 ␮l of RNAase free water and stored at −70 ◦ C pending analysis. A 14-day end-point was set to mimic the anticipated, maximum time that would be required before authorisation of a cyto-pathology result (i.e. before the sample could be released for additional testing). A 4 ml volume was chosen for RNA extraction to match with the volume required for the most frequently used, commercially available HPV detection assay—the Hybrid capture 2® (hc2). LBC samples were not refrigerated at any point during the process to reflect the normal situation of specimen transit and storage. Detection of HPV E6/E7 mRNA transcripts was performed on the RNA extracted at all time points using the PreTect® HPV Proofer Kit (NorChip AS Klokkarstua, Norway). The technology incorporates a nucleic acid sequence based amplification (NASBA) of RNA, prior to the detection of specific HPV E6/E7 mRNA by incorporation of fluorescent labelled molecular beacons corresponding to HPV 16, 18, 31, 33 and 45 viral E6/E7 sequences. Amplification of the human U1 small ribonucleo-

protein (U1A mRNA) was used as a kit-based control for RNA integrity/adequacy. The assay is described as “realtime” as RNA amplification and relative accumulation of target-specific fluorescent signal occurs simultaneously with fluorescence being detected on a fluorometer (NucliSens Easy Q analyser), which generate plots over time. Plots are ascribed positive/negative status via application of the supplied PreTect Analysis Software. A second 4 ml aliquot of the LBC specimens was taken for DNA extraction at base-line using the QIamp DNA Mini Kit. Subsequently, HPV DNA genotyping was performed by linear array (Roche Molecular Systems, CA, USA) in order to assess if the women were infected with HPV types that were outside the range of the five HPV probes included within the HPV Proofer Kit. Briefly, the process involved initial PCR amplification of an HPV-L1 fragment using PGMY primers (Coutlee et al., 2002; Gravitt et al., 1998). Amplicons were then hybridised to a nylon strip with immobilised probes for 22 high or intermediate risk HPV types (including all covered by the HPV Proofer Kit) and 15 low risk types. The assay also incorporates identification of a ␤-globin control to confirm an adequately cellular sample. A summary of HPV DNA and RNA positivity is presented in Table 1. At base-line a total of 6/7 samples were HPV DNA positive with four of the six testing positive for more than one HPV type and all testing positive for ␤-globin. In terms of HPV RNA detection, the U16 kit control was detected in all the samples at each time point. A total of 5/7 of the base-line samples were HPV E6/E7 mRNA positive, this positivity was consistent over the subsequent time points. On no occasion was >1 E6/E7 mRNA transcript detected within a sample. One sample was DNA positive yet RNA negative (sample 1) for a type within the HPV Proofer Kit range (HPV 45). However, this type appeared very weakly within a mixed infection of HPV 52, 45 and 42 and could not be detected on repeated DNA genotyping. In addition to assessing the longitudinal detection of the HPV mRNA trancripts, the integrity of total cellular RNA within all extracts (at each time point) was analysed using the Agilent RNA 6000 Pico Lab Chip® and bioanalyser system (Agilent Technologies, Deutschland GmbH) which is

Table 1 HPV genotypes detected in seven LBC specimens Sample ID

HPV DNA genotype(s) detecteda

HPV RNA genotype(s) detectedb

1 2 3 4 5 6 7

52, 45* , 42 16, 51, 59, 33* 45

16 45

16 16, 31 16, CP6108

16 16 16

(*) Denotes that the particular type was weakly positive. a DNA genotype detected in DNA extracted 6 h from sample collection. b RNA genotype detected at all time points (6 h, 4, 7 and 14 days).

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based on analysis of evident 5S, 18S and 28S ribosomal RNA. This was performed to assess the quality of the extract for down-stream gene expression profiling. A 1 ␮l volume of each extracted RNA sample was analysed according to the manufacturers’ instructions and electropherogram profiles were generated. These profiles were then compared to those generated from analyses of intact control total RNA extracted from mouse heart and degraded total RNA from breast tissue. When intact total RNA from a mouse heart was analysed using the bioanalyser system (Fig. 1, Control panel), peaks representing the 5S, 18S and 28S RNA species were clearly identifiable in the control sample. These features are usually interpreted as indicative of intact, un-degraded total RNA. In contrast, when negative control degraded total RNA from human breast tissue was analysed, the characteristic features of intact total RNA were absent. In this sample, a variety of peaks representing small lengths of RNA (the products of degradation) were observed. Variable profiles were obtained from the total cellular RNA obtained from the clinical samples collected in this study. In some, ribosomal RNA peaks were clearly identifiable, however, in others defined peaks were more difficult to differentiate from the rest of the profile. Bioanalyser profiles from two patients (samples 4 and 6) are depicted to represent this phenomenon in Fig. 1. It is notable, however, that the RNA profiles did not show evidence of degradation (an increase in small RNA fragments) as the period of storage increased from 6 h to 14 days. This study has shown that clinical cervical samples collected in PreservCyt are amenable to HPV mRNA testing up to 14 days after collection. Due to the limit of sample volume, we were not able to test beyond this (4th) time point, so it is possible that reliable HPV mRNA detection might still be achieved beyond 14 days. These results are consistent with the work of Tarkowski et al. (2001), who revealed that RNA was sufficiently intact for successful RT-PCR of the E6 gene after 1 year of storage in PreservCyt® . However, the latter longevity measurements were based on medium spiked with an HPV-16 containing cell line which, as noted by the authors, might not necessarily reflect the molecular environment in clinical samples as described in our study. No multiple HPV mRNA transcripts were detected in the samples, even in those which contained more than 1 HPV type that fell within the detection range of the kit (as evidenced by the DNA genotyping result). Presence of more than one highrisk HPV type is a common phenomenon associated with HPV infection and its clinical significance is undetermined (Peyton et al., 2001) although investigators have shown that within a population of infecting HPV types, only one type may be transforming (Soltar et al., 1998; Stoler and Baber, 2002). When we put this observation in the context of our study, the DNA based genotyping relies on amplification of a structural gene (L1) thereby denoting the presence or absence of virus(es) per se but not viral activity. Alternatively, the HPV RNA based test used is based on detection of tran-

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scribed oncogenes involved in transformation. These characteristics help to explain the differences observed in the number of HPV types detected within each sample by the separate tests. At the outset of this study, interpretation of the bioanalyser profiles obtained from the samples stored in PreservCyt® proved to be challenging due to a lack of technical data published in this field. Recently, however, data have been presented on the extraction of total RNA from other pathological, preserved samples, namely formalin-fixed paraffinembedded (FFPE) sections (Ding et al., 2004). This study demonstrated that total RNA obtained from this source does not often show the same features or characteristics as total RNA isolated from fresh tissue or cells. Encouragingly, however, it has been shown that RNA obtained from formalinfixed tissue is amenable to microarray analysis providing specialist tools such as the Affymetrix ‘X3P’ array are used. The X3P (Extreme 3 prime) array is designed to focus on interrogating sequences located closer to the 3 end of the transcripts (ARCTURUS, Mountain View, CA, USA). It is claimed that this approach allows the analysis of partially fragmented or chemically modified RNA. In this study, the profiles obtained from the cells stored in PreservCyt® were notably different from those obtained from the control RNA but were very similar to those obtained from FFPE sections. The data generated from FFPE sections provide hope that the RNA isolated from cells collected and stored in PreservCyt® will also be useful for array analyses in the future. We are aware that there are other types of collection media that we did not analyse within the context of this study. However, PreservCyt® is currently the most frequently used collection media in the UK for LBC. In the recent study by Habis et al. (2004), the authors assessed the cellular RNA quality, but not the viral RNA integrity of cervical cells in five different types of collection fluid. Two were found to be optimal for RNA preservation, namely PAXgene (PreAnalytiX, Franklin Lakes, NJ) and PreservCyt® . Considering the feasibility of RNA based testing in the future, a “one sample for all” approach would be practical and there are obvious benefits in using a medium that is already routinely used for cytology. Cells collected in medium specifically for RNA tests would require an additional sample alongside that for cytology and add to overall expense and inconvenience. RNAlater was found previously to be suitable for preservation of biopsy material (Florell et al., 2001). Interestingly however, Habis et al. (2004) found that RNAlater was not suitable for cellular RNA preservation. Preliminary experiments that were carried out at our site support this finding (unpublished data). In conclusion, we have shown that RNA from routinely collected cervical cells in PreservCyt® is a suitable target for HPV RNA transcript detection up to 14 days after sample collection and storage at room temperature. While four time points were the maximum that could be tested from a single specimen, longer time courses might show that adequate preservation could exceed our 14-day period.

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Fig. 1. Qualitative assessment of RNA extracted from cells stored in PreservCyt® . Depicted are representative profiles from Agilent Bioanalyser analyses of RNA samples from two patients (samples 4 and 6). Four mL aliquots of LBC sample were removed 6 h, 4, 7 and 14 days after sample collection. The control panel shows an electropherogram obtained by analysing 5 ng of total RNA extracted from snap frozen murine heart tissue. The degraded panel shows a typical electropherogram obtained by analysing 5 ng of degraded total RNA.

Acknowledgements The authors would like to thank the women who participated in this study, NorChip AS for providing the HPV RNA tests and Roche Molecular Systems Inc. for the HPV linear array tests.

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