BRIEF REPORT |
Correspondence to: Brigitte Samama, Institut d'Histologie, Faculté de Médecine, 4 rue Kirschleger, 67085 Strasbourg Cedex, France. E-mail: Brigitte.Samama@medecine.u-strasbg.fr
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Summary |
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Thin layer-based technology in cervical cancer screening now allows both Papanicolaou staining and HPV testing on the same sample. Here, we show that in situ hybridization with catalyzed reporter deposition is a powerful HPV detection method when applied on thin-layer cervical smears, allowing distinction between two staining patterns suggestive of two different physical states of HPV DNA, where diffuse signals are suggestive of episomes and punctate signals are suggestive of viral DNA integration
(J Histochem Cytochem 50:14171420, 2002)
Key Words: cervical smears, human papilloma virus, in situ hybridization, thin-layer cytology, tyramide amplification
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Introduction |
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Human papilloma viruses (HPVs) are implicated in the etiology of both benign and malignant cervical and vaginal lesions in human; they can be detected in virtually all cervical cancers (
ISH was applied on 221 of 2400 liquid-based thin-layer cervical smears addressed at our institute for screening. Pap-stained smears were examined by two independent readers and classified according to the Bethesda system. HPV testing was applied on all abnormal smears and on normal smears and smears with minor cellular changes either on request of the clinician or for diagnosis assessment. Human cervical cancer cell lines containing a known number of HPV 16 (SiHa and CaSki 12 and 400600 DNA copies, respectively) or HPV 18 (HeLa 1050 copies) copies served as intra- and interassay controls in our ISH technique. They were a kind gift from Dr. Wazilik (IGBMC; Strasbourg, France). Cultured cell lines were either paraffin-embedded after 24-hr buffered formalin fixation or prepared using the thin-layer protocol. Cells were suspended for 48 hr in 10 ml of the same preservation fluid used for collecting cervical samples and further processed as described for cervical smears. Thin layer slides of cervical smears were prepared using the CytoRich system (Microm; Francheville, France) according to the recommendations of the manufacturer. Remaining cell suspension was used for the Hybrid Capture II test (HC II) (Digene; Gaithersburg, MD). Thin-layer slides were postfixed in 10% buffered formalin for 15 min before ISH processing.
Protein digestion was carried out with proteinase K (Enzo Diagnostics; Farmingdale, NY) for 15 min at 37C (250 µg/ml for paraffin sections; 80 µg/ml for thin-layer slides). ISH was performed using commercial DNA probes (Enzo Diagnostics) diluted 1:10 with hybridization buffer for DNA probes (DAKO; Trappes, France). The available probes for HPV screening consisted of a mixture of HPV probes 6, 11, 16, 18, 31, 33, 51 (probe mix). For HPV typing, three probe cocktails were used: HPV 6/11 (low risk), HPV 16/18 (high risk), and HPV 31/33/51 (high risk). All slides were first processed with the probe mix. In cases of positivity and if enough cervical material remained, HPV typing was carried out. The probe was applied to the slides, overlaid with a coverglass, denatured for 12 min at 96C on a hot plate, and hybridized overnight at 37C in a moist chamber. After hybridization, the coverglass was removed and slides were rinsed with Signasure wash buffer (Enzo Diagnostics), followed by a stringency wash with In Situ Hybridization Washreagent (Enzo Diagnostics), for 10 min at 37C. The CSA system (DAKO) was used to enhance ISH staining, according to the recommendations of the manufacturer. All revelation and amplification steps were carried out at 25C on a calibrated hotplate. VIP (Vector Lab; Burlingame, CA) was used as substratechromogen reagent. Sections were counterstained with methyl green, rinsed, air-dried, and coverslipped with Eukitt.
Negative controls consisted of omission of the specific viral probe, use of probes showing no specificity for HPV or known human DNA sequences, and incubation of the specimens with VIP without any other previous treatment to evaluate the endogenous peroxidase activity. Under these conditions, no hybridization signals were observed.
Cervical smears with enough residual cells after ISH were processed with the HC II test according to the recommendations of the manufacturer. Seventy-three smears could be tested, including 18 smears with minor cellular changes, 33 ASCUS, 16 LSIL, five HSIL, and one carcinoma. The three cell lines were used as controls. In this test, the smears were analyzed for the presence of low-risk HPV types 6, 11, 42, 43, 44 and high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68.
For paraffin-embedded cell lines (Fig 1A1C) and thin-layer preparations of the cell lines (Fig 1D1F), we observed the same results. Punctate signals were present in cell nuclei with both mix probe and 16/18 probe, but not with 6/11 and 31/33/51 probes. The size of the signals increased with known copy number of HPV DNA: in SiHa cells one or two small dots, in HeLa cells usually two or three large dots, and in CaSki cells many confluent dots. This pattern of staining is comparable to that obtained with cells applied on slides by centrifugation (
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In our study group, 95% of the women screened had a normal smear or a smear with minor cellular changes, 2.75% showed an ASCUS, 1.7% LSIL, 0.6% HSIL and one smear was diagnosed as carcinoma (these percentages are in accordance with those of large cohorts). Comparison between HPV DNA testing and cytological findings showed that the rate of HPV detection increased with the severity of cytology, from 14% in minor cellular changes, 55% in ASCUS, 95% in LSIL, and up to 100% in HSIL and carcinoma. High-risk HPV types were mainly present, alone or associated with low-risk HPV types, whatever the cytological findings. Similar results have been observed in other published studies based on the HC II test, PCR, or indirect in situ PCR (
A good correlation between ISH and HC results was observed with the cervical smears. By using the HC test as the reference method, the sensitivity of our ISH protocol was 87.5% and the specificity 96%. For the discordant cases, it should be noted that the amount of cells used for HC was two- to eightfold higher than that used for ISH. The higher number of probes used in HC may also explain the discrepancy observed between the ISH and the HC results.
One advantage of in situ detection of HPV compared to liquid-based methods is to allow a count of positive nuclei to correlate with cytological data. Here we show that thin-layer smears are suitable for ISH, with no need for destaining before ISH as was necessary for classical Pap smears as previously reported (
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Acknowledgments |
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We thank Ms J. Meder and Mr J. Descamps for providing excellent technical assistance.
Received for publication January 30, 2002; accepted May 1, 2002.
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