Evaluating the feasibility of detecting HPV type 16, 18 and 52 DNA in the peripheral blood of patients with cervical cancer using real-time PCR and to determine its clinical significance:
A hospital-based study was performed to determine the prevalence of type 16, 18 and 52 HPV infections not only in cervical swabs but also in blood among women with pre-invasive and cervical cancer residing in Taipei, Taiwan. Blood and cervical swab specimens from 135 consecutive patients with 60 invasive cervical cancers, 10 microinvasions, 20 cervical intraepithelial neoplasias (CIN III, 10 CIN II, 10 CIN I) and 25 controls, were collected and examined for HPV type 16, 18 and 52 DNA using real-time PCR to investigate the prevalence and viral load of HPV DNA at the time of diagnosis and during follow-up in patients with positive blood samples.
This study was designed to provide new information regarding the occurrence of cervical cancer in patients with HPV subtype 52 detected in circulating blood, a relatively uncommon finding among cervical cancers in the Americas, Europe, Africa and Southeast Asia. The study protocol was reviewed and approved by the Institutional Review Board and Research Grant Committee of Cathay General Hospital. A total of 135 patients treated in the gynecologic cancer center of Cathay General Hospital (CGH) from January 2003 to December 2003 were recruited. Patients were recruited consecutively from those diagnosed with CIN lesions during examinations between January and June 2003. Among the 135 patients, 25 patients with benign tumors who received a simple hysterectomy performed by the same surgeon were recruited between January and March 2003 to serve as controls. The controls had no pathological findings of CIN or cancer of the cervix.
Justification of the sample size is that this study was designed to detect a clinically meaningful difference in the prevalence rates of positive test results for HPV DNA in blood samples between patients with invasive cervical cancer and patients with CIN or the controls. Assuming that the incidence rate of positive test results for HPV DNA in blood samples for patients with CIN or the controls is less than 2% (Dong et al, 2002), the selected sample size of 130 (65 per group) will give a 97% probability of correctly
detecting a 25% difference in the prevalence rate of positive test results for HPV DNA in blood samples at the 5% level of significance. Both blood specimens and cervical swabs were collected from all patients for the viral load detection of HPV types 16, 18 and 52 using real-time PCR. However, two of the 60 invasive cervical cancer specimens from cervical swabs could not be obtained due to lack of permission from patients.
All patients underwent complete physical and gynecologic examination, and the cervical cancer was staged according to the guidelines of the International Federation of Gynecology and Obstetrics (FIGO). By the end of December 31, 2004, all patients had follow-up after primary treatment at 3-month intervals for the first two years or until death. All surviving patients were followed up for at least one year. The follow-up investigations included physical examination, cervical cytology, chemistry profiling and analysis of tumor markers such as serum squamous cell carcinoma antigen (SCC-Ag) or CA-125. Further examinations, such as computed tomography (CT) of the pelvis, bone scan, chest radiography or biopsy of any suspected lesions, were performed when clinically indicated. Distant metastasis was defined as disease that occurred outside the pelvis. The association was evaluated between the HPV DNA level in the blood and clinical parameters such as tumor size, clinical staging, tumor marker, lymph node metastasis, lymphovascular space involvement (LVSI), histological type and adjuvant therapy. Among patients with a HPV-positive blood sample, the correlation between the amount of HPV DNA in blood and clinical parameters was evaluated before treatment and at three months after treatment.
DNA from cervical swab or blood samples was extracted using the QIAamp Blood Kit following the manufacturer’s instructions (Qiagen, Hilden, Germany). Quantitative real-time PCR fluorescent assays for each of the HPV genotypes and for the HLA-DQα gene were performed with the Qgene HPV 16, HPV 18, HPV 52 and housekeeping gene HLADQα detector kits (Qgene Biotechnology, Kaohsiung, Taiwan) along with SYBR Green dye using the ABI 5700 apparatus (Applied Biosystems, Foster City, CA). The cervical swab was taken with an Ayre spatula and agitated into 3 ml of Tris-HCl (pH 8.3), 0.2% Triton X-100, then stored at –20°C. Two ml of whole blood was obtained and stored in a tube containing citric acid. The real-time PCR assay had a dynamic range from 100 to more than 107 copies, allowing documentation of a wide range of HPV DNA copies found in the clinical specimens.
Samples were stratified by type of cervical disease, viral type and the range of viral DNA copies per microgram of cellular DNA, also the ability of these parameters to predict the progression of cervical carcinoma was analyzed. Blood and cervical swabs from women with CIN or cervical cancer were tested for the presence of HPV type 16, 18 and 52 DNA using real-time quantitative PCR and the presence and viral load of HPV NDA in blood and cervical swabs was correlated with CIN lesion status and the different stages of cervical cancer.
Preparation of DNA from blood and cervical swabs
DNA was extracted for PCR by adding a 400-μl aliquot of a swab sample to 500 μl of DNA extraction solution (Qiagen, Hilden, Germany) with proteinase K, and then incubated at 56°C for 1 h. The Proteinase K was then heat inactivated at 100°C for 30 minutes. After centrifugation at 10,000 xg for 10 minutes at 4°C, the supernatant was collected and transferred to a new microcentrifuge tube. Next, 400 μl of the solution from the cervical swab sample was processed using the QIAamp Blood Kit (Qiagen) according to the protocol recommended by the manufacturer. For blood samples, a 400-μl aliquot lysate was incubated with the Qiagen protease and buffer AL from the QIAamp Blood Kit (Qiagen) at 56°C for 10 minutes. The lysate was applied to a QIAamp spin column, and finally eluted with 100 μl nuclease free water (QIAamp Blood DNA mini kit protocol), and vortexed for one minute before PCR amplification.
Real-time quantitative PCR of HPV type 16, 18, 52 and HLA-DQα DNA
Real-time PCR was performed with the Qgene HPV 16, HPV 18, HPV 52 and housekeeping gene HLA-DQα detector kits (Qgene Biotechnology, Kaohsiung, Taiwan) in a 15 μl reaction mixture and was monitored after each elongation step by SYBR Green 1 dye binding to amplify product using the ABI 5700 apparatus (Applied Biosystems, Foster City, CA). Highly specific primers were selected and hot start PCR was used to reduce the interference of non-specific primer annealing.
Quantitation was done using an external standard curve. The HPV type 16 viral DNA fragment (415 bp) was amplified from genome of the CaSki cell line (Baker et al, 1987), and a 415-bp long HPV 18 viral DNA fragment was amplified from the genome of
a HeLa cell line (Schwarz et al, 1985). A 50-bp long HPV type 52 viral DNA was amplified from the positive clinical samples. The accurate molecular weight and copy numbers of each viral DNA fragment were determined based on UV absorption at a wavelength of 260 nm, and then used as the template DNA for establishment of the standard curve. A standard curve with each template at 106, 105, 104, 103, 102, 101, 100 copy numbers was generated in parallel with the clinical samples. Concentrations of HPV DNA were expressed as copies of HPV genome per 1 μg of cellular genome from the cervical swab or copies of HPV genome in each ml of blood.
The HPV 16, HPV 18, HPV 52 and HLA-DQα primer sequences from each kit are shown in Table 1. An aliquot of 5 μl of DNA sample was added to 10 μl of PCR reagent mixture consisting of HPV optimal buffer (15 mM KCl, 20 mM Tris-HCl (pH 8.3), 0.2%
Triton X 100, nuclease-free bovine serum albumin, and MgCl2), which was optimized to obtain a specific and efficient amplification - 0.3 pmol/μl of each primer, 1 mM each dATP, dCTP, dTTP, dGTP, 3.75 μl 1X SYBR Green and 0.07 U/μl AmpliTaq Gold (Roche Molecular Systems, Foster City, CA). Following the addition of the sample, the microamp tubes were capped and then centrifuged at 1,000 xg briefly. The reaction was started with a 10 minute incubation at 95°C to activate the AmpliTaq Gold, followed by 50 cycles at 95°C for 30 seconds, then 60oC for 30 seconds and finally 72°C for 45 seconds. The specificity was verified on the dissociation curve as well as by electrophoresis on 2% agarose gel. A linear plot of the log of copy numbers vs. numbers of threshold cycles was consistently obtained for HPV type 16, 18 and 52 genes, and the correlation coefficient for each target gene was between 0.995 and 1.00 in each run.
Validation of Real-time PCR quantitation
Both the HPV and HLA-DQα PCR reactions were performed in duplicate. Multiple negative water blanks were included in every analysis. Standard curves were run in parallel with each analysis using DNA extracted from HPV-positive cell lines (CaSki cells were derived from HPV 16 integrated human cervical carcinoma and HeLa cells from HPV 18 integrated human cervical adenocarcinoma; both obtained from the ATCC).
The PCR product of HPV DNA 52 was obtained as type 52 positive controls from a clinical sample confirmed by direct sequencing. CaSki DNA and HeLa DNA were used to further validate the accuracy of the real-time PCR. CaSki cells are known to contain about 600 copies of HPV 16 genome per cell (equivalent to 6.6 pg of DNA/genome)
(Baker et al, 1987). Because the weight of one genome per cell is approximately equal to 1.1 (6.6/600) pg, the total cellular CaSki DNA was estimated to contain about 10,000 copies of the HPV genome. The real-time PCR results revealed that at 1.1 ng of CaSki DNA input, the amplification plot overlapped with the standard at 10,000 copies of amplified DNA from genomes of the Caski cell line and also of the HeLa cells.
The normal and affected samples from the 135 patients were run in duplicate.
Primers and probes to a housekeeping gene (HLA-DQα) were run in parallel to standardize the input DNA. Concentrations of blood HPV DNA were expressed as copies of HPV genome/ml of blood and were calculated using the following equation (Lo et al, 1999) C = Q x Vdna/Vpcr x 1/Vext, where C represents the target concentration in blood, expressed as copies/ml; Q represents the copy number as determined by the sequence detector; Vdna represents the total volume of DNA obtained after DNA extraction (50 μl);
Vpcr represents the volume of DNA used for the PCR reaction (5 μl); and Vext represents the volume of blood used to extract the DNA.
Direct sequencing of products of the Real-time PCR
All products of the real-time PCR were purified using a pre-sequencing kit and then sequenced with the HPV type 16, 18 and 52 specific primers (Table 1) and a DNA sequencing kit. Finally, the sequencing products were purified using ethanol precipitation and were analyzed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster, CA). Sequence alignments were obtained using returned results from the GeneBank on-line Blast server (from URL: http://www.ncbi.nlm.nih.gov/BLAST/).
Differences in proportions were evaluated using Fisher’s Exact Test. A value p<0.05 was considered significant. Statistical analysis was performed using Statistical Package for the Social Sciences software (SPSS, Chicago, IL).
Analyzing whether integration or high viral loads of human papillomavirus (HPV) is essential for malignant transformation of HPV type 52 and 58 as well as type 16 and 18:
Sample collections
The study protocol was reviewed and approved by the Institutional Review Board
and Research Grant Committee of Cathay General Hospital (CGH). This prospective study was conducted to determine the prevalence, physical status and viral load of the HPV types 16, 18, 52 and 58 DNA in the cervical swabs of patients with CIN II-III and cervical cancer using genechips and real-time PCR analysis. Between January 2003 and March 2004, at the gynecologic cancer center of CGH, cervical swabs were collected at the time of diagnosis of cervical carcinoma from 81 consecutive Taiwanese patients and from 97 consecutive Taiwanese patients at the time of their diagnosis with CIN II-III.
Informed consent for participation was obtained from each patient. The cervical swab was collected with an Ayre spatula and agitated into 3 ml of Tris-HCl (pH 8.3), 0.2%
Triton X-100, then stored at -20oC.
DNA Extraction
DNA was extracted with the QIAamp DNA Blood Mini Kit (Qiagen Catalog No.51106) according to the manufacturer’s instructions. Extracted DNA was eluted with 100 μl AE buffer (10 mM Tris, pH8.5) and stored at -20oC until analysis.
Diagnosis of disease and follow-up of patients
All patients with CIN II-III or cervical cancer were examined by colposcopy and the diagnosis was confirmed by biopsy. All patients underwent complete physical and gynecologic examination, and the cervical cancer was staged according to the guidelines of the International Federation of Gynecology and Obstetrics (FIGO). All patients were followed up after primary treatment at 3 month intervals for the first 2 years or until death.
Follow ups, including physical examination, cervical cytology, blood chemistry profile, and tumor marker detection such as SCC or CA-125, and further work up such as computer tomography (CT) scans of the pelvis, bone scan, chest radiograph, or biopsy of any suspected lesions, were performed when clinically indicated. Associations were evaluated between HPV status and viral load in swabs and clinical parameters such as tumor size, clinical staging, tumor marker, lymph node metastasis, lymphovascular space involvement (LVSI), histological type, adjuvant therapy and clinical outcome.
HPV DNA genotyping
The frequency of HPV DNA and genotypes were determined by a polymerase chain reaction-based (PCR) genechip method with HPV L1 gene MY11/Gp6+ consensus primers as previously described (Huang et al, 2004). The MY11/GP6+ consensus primers
were used to amplify a fragment of 190 bp in the L1 open reading frame.
E2, E6 viral load absolute quantitation of HPV type 16, 18, 52 and 58 using real-time PCR
DNA amplifications were carried out in a 96-well plate in an ABI Prism 5700 Sequence Detection System (Applied Biosystems). Both E2 and E6 real-time polymerase chain reactions were carried out in triplicate for each sample. Amplification and quantification of the E2 and E6 genes were carried out simultaneously in separate reactions in a 96-well PCR plate. Additionally, one sibling control sample, which had been quantified previously, was included in each reaction to serve as quality assurance of the quantification system. Numbers of the threshold cycle (Ct) obtained from E2 PCR and those from E6 PCR were regressed to the standard curve to obtain the HPV copy number. For evaluation of triplicate data, the mean value and standard deviation (SD) were calculated. Data between the ranges of mean ±1.96x SD was considered acceptable.
Multiple negative water blanks were included in every analysis.
The reaction was performed in a 25 μl mixture containing 1x reaction buffer (HPTM HotStart Taq SYBR Green Kit Cat No.PTM767B, Protech) and 100 nM of primers for both E2 and E6 regions. Fifty nanograms of total DNA were added to the reaction mixture. The primer sequences of E2 and E6 for HPV type 16, 18, 52 and 58 were as shown in Table 3. The amplification conditions were as follows: 10 min at 95oC, a two-step cycle at 95oC for 10s and 60oC for one minute for a total of 45 cycles. The specificity was verified by the additional dissociation curve and followed by 2% agarose gel electrophoresis. Two standard curves were obtained by amplification of serial dilutions (ranging from 10 to 10,000,000 copies per μl) of cloned plasmid containing either partial HPV 16 (from base 28 to base 3890), HPV 18 (from base 45 to base 3993), HPV 52 (from base 95 to base 3895) or HPV 58 (from base 45 to base 3994) DNA, which included equivalent amounts of E2 and E6 genes in pGEM T-Easy vector (Promega). The number of threshold cycles for E2 PCR and E6 PCR were equivalent in each run. A linear plot of the log of the copy number vs. number of threshold cycles was consistent for both genes, and the correlation coefficient was between 0.995 and 1.00 in each run.
Assumptions of physical status using E2/E6 ratio
The real-time PCR methods used in this study were developed based on the
following assumptions: (1) preferential disruption of E2 will cause the absence of E2 gene sequences in the PCR product following integration, (2) copy numbers of both genes (E2 and E6) should be equal when viral DNA presents in episomal forms, and (3) E2 gene copy numbers will be smaller than that for E6 when viral DNA presents in concomitant form.
Measurement of E2/E6 ratio in relation to physical status
Concentrations of HPV DNA were expressed as copies of HPV genome in 50 ng of cellular DNA. Ratios of E2 to E6 less than one indicated the presence of both integrated and episomal forms. The amount of integrated E6 was calculated by subtracting the copy numbers of E2 (episomal). The ratio of E2 to integrated E6 represented the amount of the episomal form in relation to the integrated form.
Statistical analysis
Statistical analysis was mainly performed using SAS 9.1.3 software. The viral load was analyzed using a Wilcoxon rank sum test based on log transformed data (y = log(x+1)) to compare differences between groups. The frequency distribution of physical status was analyzed by Fisher’s exact test. A diagnostic test based on log transformation of E6 viral loads was used to perform receiver operating characteristic (ROC) curve analysis.
Analyzing whether integration or high viral loads of HPV predict prospectively the risk of progression of low-grade squamous intraepithelial lesions (LSILs) of the uterine cervix in women with human papillomavirus (HPV) infections:
To examine this issue, the 2-year cumulative risk were evaluated for HSIL attributable to HPV 16, 18, 52, and 58, the most common oncogenic types in pre-invasive cervical lesions including LSILs and HSILs in Asia, and questioned whether the integration of HPV oncogenes into a host genome contributed to the risk of LSILs progressing to HSILs. In addition, it was determined if E6 viral load and its change contributed to the risk of LSILs progressing to HSILs during the 6 month interval between baseline diagnosis of LSIL by Pap smear and the 6 month follow-up visit by repeat Pap smear.
Subjects and Methods
The Taiwan Cooperative Oncology Group (TCOG) (T1899), a multicenter study, was conducted under the supervision of the National Health Research Institute of Taiwan.
A total of 1246 women with abnormal Pap smears, including those diagnosed with ASCUS or AGUS (n = 431), LSIL (n = 437), and HSIL (n = 373), from August 1999 to March 2004, were enrolled. The details of the study design and population have been published (Chen et al, 2006; Sun et al, 2005). Of the 1246 participants, 936 underwent cervical biopsies for histologic examination.
Women with LSIL (n = 437) had Pap smears, HPV testing, and colposcopic examinations every 3 months during the follow-up period. Participants were excluded from the study if they had no follow-up data (n = 60), baseline cancer on pathological examination (n = 1), fewer than 4 follow-up visits (n = 82), or no baseline HPV data (n = 2). Women with histologically confirmed HSIL were defined as having disease progression and were treated and exited from the study. Women with LSIL with two consecutive normal Pap smears and who showed HPV clearance during the follow-up period were defined as in remission and also exited. Women with LSIL not in remission or progression were defined as having persistent disease. HPV persistence was defined as HPV positivity for a given type tested on two consecutive occasions versus clearance.
A total of 294 women with LSIL, having at least four follow-up visits every 3 months, and 460 women with HSIL were tested for HPV DNA using both HC2 and PCR-reverse line blotting and were included in the longitudinal follow-up study (unpublished data). Among the 294 patients with LSIL, 187 specimens from 65 women with HPV 16 (n = 14), 18 (n = 8), 52 (n = 30) and 58 (n = 13) were collected at baseline and followed up every 6 months until follow-up showed disease progression. The specimens were further tested for viral load, E2/E6 ratio and viral load change using real-time PCR. Four who lacked samples of follow-up HPV DNA were excluded from final analysis. In addition, 212 HSIL positive patients with HPV 16 (n = 92), 18 (n = 5), 52 (n = 57), or 58 (n = 58) infections were also obtained and tested for viral load, E2/E6 ratio, and physical status of HPV DNA using real-time PCR to compare with data from LSIL specimens. Specimens for viral load and viral load change by hybrid capture two
A total of 294 women with LSIL, having at least four follow-up visits every 3 months, and 460 women with HSIL were tested for HPV DNA using both HC2 and PCR-reverse line blotting and were included in the longitudinal follow-up study (unpublished data). Among the 294 patients with LSIL, 187 specimens from 65 women with HPV 16 (n = 14), 18 (n = 8), 52 (n = 30) and 58 (n = 13) were collected at baseline and followed up every 6 months until follow-up showed disease progression. The specimens were further tested for viral load, E2/E6 ratio and viral load change using real-time PCR. Four who lacked samples of follow-up HPV DNA were excluded from final analysis. In addition, 212 HSIL positive patients with HPV 16 (n = 92), 18 (n = 5), 52 (n = 57), or 58 (n = 58) infections were also obtained and tested for viral load, E2/E6 ratio, and physical status of HPV DNA using real-time PCR to compare with data from LSIL specimens. Specimens for viral load and viral load change by hybrid capture two