Journal of Histochemistry and Cytochemistry, Vol. 46, 535-540, April 1998, Copyright © 1998, The Histochemical Society, Inc.


TECHNICAL NOTE

Detection of Human Papillomavirus in Archival Tissues: Comparison of In Situ Hybridization and Polymerase Chain Reaction

Elizabeth R. Ungera, Suzanne D. Vernonb, Daisy R. Leea, Donna L. Millerb, and William C. Reevesb
a Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia
b Division of Viral and Rickettsial Disease, Center for Infectious Disease, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia

Correspondence to: Elizabeth R. Unger, Centers for Disease Control and Prevention, 1600 Clifton Road, MSG18, Atlanta, GA 30333, eru0{at}cdc.gov (E-mail).


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Formalin-fixed, paraffin-embedded tissues in pathology archives are an important resource for molecular epidemiology studies. Use of these tissues requires that assays be optimized to account for inevitable variations in tissue fixation and processing that occur in the performance of routine histology. We compared results of colorimetric in situ hybridization (ISH) to L1 consensus polymerase chain reaction (PCR) for detection and typing of human papillomavirus (HPV) in 180 blocks of archival tissues (up to 9 years in storage) from cervical cancer patients. Fifteen samples could not be amplified by PCR, but assays were concordant in 75.1% (124/165) of samples that could be analyzed by both methods. Similar numbers of ISH+/PCR- (23) and ISH-/PCR+ (18) cases were found. Eight of the 18 ISH-/PCR+ cases were attributable to PCR detection of HPV types not included in the ISH assay. This degree of concordance required individual optimization of assay conditions for each block. ISH and PCR assays for HPV yield complementary results, and both can be successfully applied to archival tissues. (J Histochem Cytochem 46:535–540, 1998)

Key Words: colorimetric in situ, hybridization, human papillomavirus, polymerase chain reaction, archival tissues, formalin fixation


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Patient tissues obtained for diagnosis are routinely stored in pathology archives. These samples are an increasingly important resource for the study of disease. The tissues are well-characterized by classical histopathology, and blocks can be selected to be representative of the disease. Retrospective analysis permits rapid correlation of assay results with clinical outcome. For diseases with low prevalence, such as invasive cervical cancer in the United States, the accrual of cases is often inadequate for prospective analysis.

Application of modern molecular biology techniques to this material is challenging. "Routine" tissue processing in clinical histology laboratories does not mean consistently reproducible processing. There are unavoidable variations in tissue handling before fixation as well as variable conditions of time and temperature during fixation. Reagents in automated tissue processors vary in potency, depending on the numbers of cases processed and the frequency of reagent replacement. The environmental conditions for storage of processed tissue are not controlled. These variables are all well-tolerated for histologic analysis but contribute to irreproducibililty in DNA and RNA preservation.

To be able to rely on the results of studies based on archival tissues, molecular assays must be optimized to compensate for variable fixation and processing. In situ hybridization (ISH) and polymerase chain reaction (PCR) assays have both been reported for DNA analysis in formalin-fixed archival tissues. Both assay formats require small amounts of tissue and thus conserve material essential for clinical management. In addition, both formats tolerate some degradation of target nucleic acids and can utilize nonradioactive detection methods.

Our interest in the molecular epidemiology of human papillomavirus (HPV) in cervical cancer required us to carefully evaluate methods for HPV detection in archival tissues. Even small errors in HPV detection have the potential to influence results of epidemiological associations (Franco 1992 ). In this study we compared the results of HPV testing in archival specimens of cervical cancer with carefully optimized ISH and PCR assays. Each assay has potential advantages. The PCR assay can be easily adapted to detect the majority of HPV types associated with anogenital tract disease by using multiple degenerate primer pairs in the amplification reaction. The ISH assay localizes HPV within a histological context and allows evaluation of integration status. Although some of the findings may be unique to HPV detection, the results will help to define the usefulness of each assay format for molecular testing in archival material.


  Materials and Methods
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Summary
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Materials and Methods
Results
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Study Material
Archival tissues from cervical cancer patients diagnosed and treated at Grady Memorial Hospital, Atlanta, Georgia, from January of 1986 through June of 1995 were analyzed. All samples had been fixed in formalin and processed into paraffin blocks according to routine surgical pathology practice. Blocks were selected to include the highest ratio of well-preserved diagnostic tumor to normal tissue. Consecutive serial sections were used for the assays, with histological confirmation of the lesion in initial and final sections. A new disposable microtome blade was used for each block to minimize the potential for cross-contamination of samples. The study was conducted with the approval of the human investigation committees of Emory University School of Medicine and the Centers for Disease Control and Prevention.

HPV ISH
Clones for HPV Types 16, 18, 31, 33, and 35, containing the complete viral genome in pBR322 or pT713, were obtained as a gift from Bethesda Research Laboratories (Gaithersburg, MD). The plasmid DNA from each of these clones was isolated and purified by standard techniques. Unmodified plasmid DNA (pBR322; Bethesda Research Laboratories) was the negative control probe. Human placental DNA (Sigma Chemical; St Louis, MO) was the endogenous positive control probe to allow optimization of digestion conditions. All probes were labeled as previously described (Unger et al. 1988 ) using nick-translation with Bio-11-dUTP (Enzo Biochemical; New York, NY), resulting in fragments of less than 300 bases.

Colorimetric ISH as adapted for automation was used as previously described in detail (Unger et al. 1988 , Unger et al. 1991 ). Briefly, the slides were treated with xylene to remove paraffin, dehydrated in absolute alcohol, and digested with pepsin (30 min, 37C) to make the target DNA available to the probe. (Pepsin varied between 0.5 mg/ml 0.01 N HCl and 6 mg/ml 0.1 N HCl, optimum selected by results with endogenous positive control probe described below.) The tissues were then neutralized with Tris buffer, dehydrated, and covered with a hybridization cocktail that contained 1.0 µg/ml of the biotin-labeled DNA probe in a 36% formamide cocktail. Tissue and probe were simultaneously denatured in the100–105C oven of the instrument for 20 min (first 10 min for temperature equilibration). The hybridization was performed for 2 hr at 37C, followed by a series of graded salt washes to 0.1 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M sodium citrate) at 42C.

Specifically hybridized probe was detected with an avidin–alkaline phosphatase conjugate (Dako; Santa Barbara, CA), followed by 1-hr color development with McGadey reagent (5-bromo-4-chloro-3 indoyl phosphate/nitroblue tetrazolium; BCIP/NBT) at 37C for 1 hr. Slides were counterstained with nuclear fast red, covered with CrystalMount (Biomeda; Foster City, CA), air-dried, and mounted with Permount (Fisher Scientific; Pittsburgh, PA). These conditions resulted in moderate stringency, minimizing the extent of cross-hybridization and maintaining an adequate signal. The signal was stable for over 1 year at room temperature when slides were coverslipped as described.

The in situ hybridization was considered satisfactory when (a) the endogenous positive control probe gave a dark, even signal over almost every nucleus, indicating adequate digestion and availability of the target DNA, (b) the negative control probe gave no signal, (c) control sections gave the appropriate reaction. Control sections included formalin-fixed paraffin blocks of CaSki cells (400–600 copies of HPV 16 per cell), HeLa (10–50 copies of HPV 18 per cell), HTB35 (1–2 copies of HPV 16 per cell), and HTB31 (no HPV). In practice, each block was initally tested with the human placental DNA probe to establish optimal digestion conditions. Once optimum was established, the samples were reassayed under established conditions with the full set of probes (endogenous positive, negative, and HPV).

Sections were evaluated by light microscopy for type and integration status of HPV. As described and illustrated previously (Cooper et al. 1991 ; Kristiansen et al. 1994 ; Unger et al. 1995 ), a dot-like signal evenly distributed over most tumor nuclei was interpreted as integrated HPV, and a patchy signal unevenly distributed over tumor nuclei was interpreted as episomal. Mixtures of the two signal patterns were interpreted as both integrated and episomal (mixed).

HPV PCR
PCR assays were performed on DNA extracted from tissue sections as described (Shibata 1994 ). Briefly, two 5-µm paraffin sections were dewaxed in xylene, washed in alcohol, and dried under vacuum. Sections were digested for 1 hr at 55C with 200 µl of proteinase K (Boehringer-Mannheim Biochemicals, Indianapolis, IN; 200 µg proteinase K/ml of 50 mM Tris-HCl, pH 8.5, 1 mM EDTA, 0.5% Tween 20), followed by boiling for 10 min to inactivate the enzyme. Digested samples were stored at -20C until analysis.

Amplification of the HPV L1 consensus region was performed using consensus primers MY11 and MY09 to generate a PCR product of approximately 450 BP (Ting and Manos 1990 ). Every PCR assay included a positive control of CaSki cellular DNA (HPV 16 DNA), a negative control of human placental DNA (HPV-negative DNA; Sigma), and a negative template control. DNA quantity and integrity were monitored by amplification of a 268-BP fragment of the ß-globin gene (Perkin–Elmer; Foster City, CA) in replicate tubes. The standard 50-µl PCR reaction included 5 µl of the DNA template with 1.25 U of Taq (Perkin–Elmer) and 0.1 µM of each primer. For cases that did not amplify ß-globin, increased template concentration was tried in repeat assays. This template titration was required in 5% of samples.

Products from the L1 consensus PCR were typed by using a high-stringency dot-blot hybridization assay on nylon membranes with individual HPV type-specific (16, 18, 31, 33, 35, 56) oligonucleotide probes (Hildesheim et al. 1994 ) synthesized with digoxigenin at the 3' end. Hybrids were detected with alkaline phosphatase-conjugated anti-digoxigenin and chemiluminescent detection with Lumiphos (Boehringer-Mannheim).

Sensitivity of HPV detection by this protocol was estimated by serial dilutions of DNA extracts prepared from sections of formalin-fixed, paraffin-embedded SiHa and CaSki cells. Human placental DNA was included in the diluent. Using cell counts and the known copy number of HPV 16 in the cell lines, the assay could detect 50 copies of HPV 16 in one 50-µl PCR reaction (5 µl of extract).

L1 consensus PCR products that did not hybridize to any of the type-specific probes were sequenced by using cycle sequencing with fluorescent-labeled dideoxynucleotides. Each PCR product (200 ng) was purified by centrifugation in a Centricon 30 microconcentrator (Amicon; Beverly, MA) and sequenced in both directions, using MY11 and MY09 primers and Taq DyeDeoxy Terminators Cycle Sequencing Kits (Applied Biosystems; Foster City, CA) according to the manufacturer's instructions. Sequence information was collected with Applied Biosystems 373A DNA Collection and Analysis Software and was analyzed using the University of Wisconsin Genetics Computer Group package.

Samples that failed to amplify ß-globin were not interpreted. Results of the L1 consensus PCR reflected the final assessment of HPV presence and type as confirmed by type-specific hybridization and, if required, sequencing of the PCR products. L1 consensus PCR products that did not hybridize and failed to yield an HPV sequence were considered negative.

Data Analysis
The ISH and PCR assays were performed and interpreted in different laboratories and the results entered into separate Paradox (Borland International; Scotts Valley, CA) databases so as not to bias interpretation of either assay. The database for each assay included block number (used to link results), date of biopsy, date of assay, and HPV results. Results for each sample were tabulated and compared for detection and typing of HPV.


  Results
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Correlation of ISH and L1 PCR Results for HPV
Results were matched by block number and concordance was defined as agreement between assays for the detection of HPV. If the type(s) of HPV detected did not agree, the match was considered concordant with type discrepancy. If results were discordant, the type of discordance was recorded, i.e., ISH+/PCR- or ISH-/PCR+.

Table 1 summarizes the results for the 180 blocks included in this study. Assay concordance was 75.1% (124/165; excluding the 15 blocks that could not be amplified by PCR). McNemar's test for correlated proportions indicates that the probability of a sample being positive by either test (ISH or PCR) is not statistically different (McNemar's exact p=0.53).


 
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Table 1. Comparison of ISH and PCR for detection of HPV

There were 23 ISH+/PCR- cases. In seven instances an additional tissue sample from the same patient did yield concordant ISH and PCR results, providing support for the ISH result. Review of the ISH slides from these discordant cases demonstrated unequivocal signal, often as intense as shown in Figure 1. Although the example illustrated is of integrated HPV, integration status did not differ significantly between ISH+/PCR+ and ISH+/PCR- cases (respectively, 53 integrated, 18 mixed, and 18 episomal compared to 13 integrated, seven mixed, and three episomal; Fisher's exact test, exact p=0.52). Slightly fewer cases (18) were ISH-/PCR+. In eight of these instances the type of HPV detected by the PCR assay was not included in the ISH assay (four HPV 58, two HPV 26, and one each HPV 51 and 56).



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Figure 1. In situ hybridization result of cervical cancer negative in L1 PCR assay. Nuclear fast red counterstain, BCIP/NBT substrate. (A) Human placental DNA (endogenous positive control probe). All nuclei show a dark, even signal, indicating adequate availability of tissue nucleic acid. (B) HPV 18 probe. No signal is present, identical to results with negative control probe. (C) HPV 16 probe. Dot-like signal is present in most tumor nuclei, indicating integrated HPV 16. Bar = 5 µm.

The HPV types detected by ISH and PCR for those cases with type discrepancies are shown in Table 2. In seven instances the PCR assay detected multiple types, only one of which was detected by ISH. There were six examples (6.7% of positive cases) in which the HPV types detected by the two assays did not agree.


 
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Table 2. Instances of discrepant HPV typing by ISH and PCR

Influence of Year of Block Preparation and Block Storage Time on Assay Concordance and Successful Amplification
Reagents and conditions of processing undoubtedly varied over the course of the 9 years that the blocks were prepared. Table 3 shows the assay results and ability to amplify DNA by the year of block preparation. The percentage of blocks that could not be amplified ranged from 18.7 to 0, and the assay concordance varied between 64.3 and 94.1%, with no discernible trend for either measurement (chi-square for trend, p=0.58 and 0.22, respectively).


 
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Table 3. Assay concordance by year of block preparation

Time of block storage, calculated as the difference between the date of block preparation and the date of the PCR assay, could also influence the stability and accessibility of target DNA. Time of block storage is related to year of block preparation, but because the assays on primary tumors were performed over the course of the 4-year study as patients presented for follow-up, a block prepared, for example, in 1990 could be stored between 2 and 4 years before being assayed. The time of block storage ranged from 0.01 to 8.96 years, distributed as follows: less than 1 year, 41; 1–3 years, 63; greater than 3 years, 76. There was no significant variation in samples failing amplification nor in the concordance of ISH and PCR for blocks stored within the three time frames (chi-square for trend, p=0.80 and 0.74, respectively).


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Our findings indicate that detection of HPV in formalin-fixed, paraffin-embedded blocks by colorimetric ISH and L1 consensus PCR yields complementary results. For the 165 (of 180) archival blocks that could be assayed by both methods, the assays were concordant in 75.1% (124). When cases positive for types not included in the ISH assay were excluded, the concordance between the assays was 79.0%. HPV types detected by both assays were concordant (or overlapping) in 95.2% of the positive cases. The explanation for the remaining type-discordant cases (6) has not been determined but could be due to cross-hybridization of closely related HPV types by the genomic probes used in the ISH assay.

Under the conditions described, the probability of a sample being positive by either method was not statistically different (McNemar's exact p=0.53). In addition, ISH provided results for 15 cases (8.3%) that failed to amplify by PCR. The apparent similarity between the sensitivity of the two assays may be a reflection of the ability of the ISH assay to detect very few positive cells in a largely negative background. The ISH assay's sensitivity is determined by the number of copies of HPV per cell, whereas the PCR assay's sensitivity is determined by the number of copies in the assay tube.

The L1 consensus assay was selected to allow a single assay to detect multiple HPV types. Deletions in the L1 region are not common, but any mutation or deletion could result in a negative PCR assay while yielding positive results with ISH. Another potential limitation of the L1 consensus assay is that the product is 450 BP, at the upper limits of size that can be amplified from formalin-fixed paraffin sections (Frank et al. 1996 ). The control for template integrity, ß-globin, yields a 268-BP product. In some instances a minor inhibition to amplification might not be reflected in the control but might be enough to negate the L1 consensus assay. It could be anticipated that a type-specific PCR assay would be more sensitive than the L1 consensus assay. Baay et al. 1996 directly compared L1 consensus PCR and two additional general primer systems for HPV with type-specific PCR. These researchers report increased sensitivity with the type-specific assays, with further improvement when the type-specific assays were designed to yield a smaller product (approximately 100 BP). For screening in a large epidemiological study, individual type-specific HPV PCR reactions are not practical.

The method of DNA extraction is another variable in the PCR assay. Although some investigators include phenol extraction (Karlsen et al. 1994 ), our initial experiments, in agreement with Frank et al. 1996 , determined that optimal results were obtained without phenol treatment. Increasing the time of digestion could also influence the final yield of DNA.

Storage time, up to 9 years in this study, did not significantly influence the ability to amplify material. This observation is in agreement with those of Iwamoto et al. 1996 , who used tissues 20 or more years old in a PCR assay for p53. Storage time also did not influence concordance between ISH and PCR. The year of block preparation also had no influence of assay concordance or ability to amplify DNA.

It must be emphasized that these results were not obtained by simply performing the same assay on each block. Considerable effort was spent in determining the optimal conditions for each tissue. For ISH, digestion conditions varied between 0.5 mg pepsin/ml 0.01 N HCl and 6 mg pepsin/ml 0.1 N HCl. The conditions were selected to yield the strongest signal with the endogenous positive control probe (human placental DNA). In addition, the ISH sensitivity was monitored by including a low positive control in each assay (SiHa cells) to ensure that probe and detection reagents performed at maximal efficiency. Similarly, we closely monitored the PCR assay by adjusting the template concentrations to yield positive results for the ß-globin amplification.

There have been few detailed comparisons of the results of ISH and PCR assays on archival material. A much smaller study of HPV in penile cancer (Varma et al. 1991 ) found a similar concordance between optimized colorimetric ISH and type-specific assay for HPV. Other workers looked at the comparison of colorimetric ISH and L1 consensus PCR for HPV detection in parallel cervical smears (Herrington et al. 1995 ). Sampling errors significantly contributed to the variation in results. When the analysis was restricted to those assays performed on aliquots of the same sample (n = 50), the concordance between the two assays was 74%. These authors noted type discrepancies between the two assays similar to our findings and attributed them to cross-hybridization of the complex probes used in ISH. Problems unique to archival materials could not be addressed by that study.

In summary, our study of 180 blocks of archival tissue demonstrates that this material can be used for molecular epidemiology studies if care is taken to optimize results. ISH and PCR provide complementary results, and the use of two independent assays increases the likelihood of accurate determination of HPV within the tissue. PCR allows detection of HPV types not included in the ISH assay, and ISH provides data on HPV integration. Refinements in each assay format—such as signal amplification for ISH (Kerstens et al. 1995 ) or adjustment of extraction conditions (Frank et al. 1996 ), and amplimer length (Baay et al. 1996 ) for PCR—have the potential to change the degree of concordance between the two assays. The results in this study reflect our experience with the assays described and emphasize that, regardless of the methods used, variability in tissue fixation and processing encountered in routinely processed tissue in pathology archives requires conditions to be optimized for each individual tissue. Assays for endogenous targets, such as genomic human DNA for ISH and ß-globin for PCR, are useful for optimization.


  Acknowledgments

Preliminary results were presented in abstract form at the joint meeting of The Histochemical Society and the Microscopy Society of America, Kansas City, MO, August, 1995.

Received for publication July 18, 1997; accepted November 13, 1997.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Baay MF, Quint WG, Koudstaal J, Hollema H, Duk JM, Burger MP, Stolz E, Herbrink P (1996) Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. J Clin Microbiol 34:745-747[Abstract]

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