ANATOMIC PATHOLOGY Original Article
Human Papillomavirus DNA in Anal Carcinomas Comparison of In Situ and Dot Blot Hybridization MAIRE A. DUGGAN, F.R.C.P.C.,1 VALERIE F. BORAS, M.D.,1 MASAFUMI INOUE, M.Sc.,' AND S. ELIZABETH McGREGOR, M.Sc.2
blot hybridization, 69% had HPV 16/6 and 15% had HPV 6/ 11. An HPV DNA frequency range of 0-85% in the same group of tumors with the use of three hybridization techniques indicates the influential role of the method on HPV DNA prevalences. HPV DNA was identified regardless of patient gender or type of squamous cell carcinoma. The presence of HPV 16 in 82% of the positive cases is supportive evidence of the carcinogenic role of the HPV in anal squamous cell carcinoma. (Key words: Anal squamous cell carcinomas; Human papillomavirus DNA prevalence; In situ hybridization; Dot blot hybridization) Am J Clin Pathol 1991;96:318-325
(i.e., 16 and 18 in cervical carcinogenesis),5 the roles of these factors in anal carcinogenesis are being investigated increasingly. Published frequencies on the prevalence of HPV DNA in anal squamous cell carcinomas range from 0 to 61%. 6 " 10 It has been suggested that demographic variables such as patient gender and sexual proclivity1 and tumor variables such as histologic type9 may affect the HPV DNA positivity rate. However, because the sensitivities of some hybridization techniques differ considerably," and because the referenced studies6"10 used different techniques to identify HPV DNA, the role of the hybridization technique in accounting for the range of published HPV DNA prevalences must be clarified. The assessment of the HPV DNA content of archival, paraffin-embedded tissue blocks of anal squamous cell From the department of Pathology, Foothills Hospital, The Tom carcinomas is feasible using either tissue in situ hybridBaker Cancer Centre, and the University of Calgary; and the department ofEpidemiology and Preventive Oncology, Alberta Cancer Board,ization Calgary,or by extracting the cellular DNA from the blocks—dot blot hybridization. In our previous study,6 Alberta, Canada. in situ hybridization with streptavidin-biotin-horseradish Received June 21, 1990; received revised manuscript and accepted peroxidase complex did not detect biotinylated DNAfor publication December 27, 1990. DNA HPV hybrids in 13 cases of anal squamous cell carSupported by grant 954-672 from the Research and Development Committee, Foothills Hospital. cinoma. As we performed it, this hybridization technique Address reprint requests to Dr. Duggan: Department of Pathology, Foothills Hospital, 1403-29 Street Northwest, Calgary, Alberta T2N 2T9, has a sensitivity of 500 viral copies per cell. By substituting streptavidin-alkaline phosphatase, we achieved a sensiCanada.
Anal squamous cell carcinoma, which sometimes is reported as basaloid, cloacogenic, epidermoid, or transitional cell carcinoma, is an uncommon tumor that occurs predominantly in elderly women.1 Its incidence has increased recently, especially among homosexual men. 2 ' 3 Epidemiologic evidence suggests that the human papillomavirus (HPV)-induced genital wart is associated etiologically with anal carcinoma risk.4 Other risk factors include a history of chronic anal irritation and cigarette smoking.4 Because part of the anus and lower female genital tract are derived embryologically from the cloaca,1 and given the tropism of certain HPV types (e.g., 6, 11, 16, 18, and 33 for the female genital epithelium) and the large body of evidence implicating specific HPV types
318
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Because the sensitivities of individual hybridization techniques differ considerably, their role in accounting for the published frequencies of human papillomavirus (HPV) DNA in anal squamous cell carcinomas, ranging from 0 to 61%, must be investigated. With the use of biotinylated probes to HPV 6, 11, 16, 18, and 33, three hybridization techniques were performed on the same paraffin-embedded tissue blocks selected from 13 cases of anal squamous cell carcinoma. HPV DNA was detected in 0%, 62%, and 85% of cases with the use of in situ hybridization with horseradish peroxidase, in situ hybridization with alkaline phosphatase, and dot blot hybridization, respectively. By dot
DUGGAN ET AL. HPV DNA in Anal Carcinomas tivity of approximately 1 -2 copies per cell with the same in situ hybridization method. Dot blot hybridization using biotinylated probes is more sensitive, with a minimum detection limit of 0.1 copies per cell.12 The current study was designed to determine the relative frequencies of HPV DNA in 13 cases of anal squamous cell carcinoma with the use of the above three hybridization techniques, each performed on the same paraffin-embedded tissue block, and to correlate the histologic features of the carcinoma with the identified HPV DNA type. MATERIALS AND METHODS
Probe Preparation In situ and dot blot hybridization were performed with cloned HPV DNA labeled with biotin. Plasmid pBR322 containing either HPV 6, 11, 16, 18, or 33 was digested with either restriction endonuclease BamH I or EcoR I and purified with the use of agarose gel electrophoresis. The probes were made by nick-translation of the 7-8kilobase genomes with the use of Biotin-11-dUTP (Enzo Diagnostics, New York, NY) and reduced in size to 3001,000 bases to increase their tissue permeability.13 Unincorporated nucleotides were removed by a sodium chloride-ethanol DNA precipitation method. In Situ
Hybridization
The full details of the method have been published previously.2 The tissue sections were deparaffinized in two changes of xylene, followed by two changes of absolute ethanol, and placed for 30 minutes in methanol containing 0.5% (volume/volume [v/v]) H 2 0 2 to inactivate the endogenous peroxidase. Then they were placed in 0.2 mol/ L HC1 for 10 minutes so that the nuclear proteins would be digested. The slides were treated with a purified 5 ng/ mL proteinase K solution and incubated at 37 °C for 15 minutes. The slides were washed in a phosphate-buffered saline (PBS) solution containing 0.2% (w/v) glycine and then immersed in a PBS solution containing 4% (w/v) paraformaldehyde to postfix (stabilize) the DNA. The slides were dehydrated through a graded ethanol-water series; then they were air-dried.
Air-dried sections were overlaid with 20 /iL hybridization medium containing 50% (v/v) deionized formamide; 10% (w/v) dextran sulfate; 2X SSC buffer (0.3 mol/L NaCl, 0.03 mol/L sodium citrate), pH 7.0; 400 Mg/mL sonicated carrier DNA; and 10 MS/mL biotin-labeled probe DNA. The tissue sections were covered with a silicon-coated coverslip and transferred to a glass humid chamber. DNA was denatured by incubating the humid chamber at 8 5 90 °C for 10 minutes and then overnight at 37 °C. After hybridization, the sections were washed with 0.5X PBS containing 30% (v/v) deionized formamide (Tm - 1 0 °C) for 15 minutes at 37 °C and then washed in 0.5X PBS containing 10% (v/v) deionized formamide for another 15 minutes. After the posthybridization wash, the sections were washed for another 3 minutes at room temperature in PBS containing 0.5% (v/v) Triton-X (Fisher Scientific, NJ) and finally in PBS for 3 minutes. Signal Generation with Streptavidin-Biotin Horseradish Peroxidase Complex In a humid chamber at room temperature, the slides were covered for 30 minutes with 50-100 pL 1:200 diluted streptavidin-biotin-horseradish peroxidase complex in PBS containing 1% (w/v) bovine serum albumin and 5 mmol/L ethylenediaminotetraacetate (EDTA). The slides were washed twice for 5 minutes in 2X SSC and PBS containing 0.1% (v/v) Triton-X and were developed for 20 minutes in a freshly prepared mixture of 100 mmol/ L sodium acetate, 0.4 mg/mL aminoethylcarbazole (AEC), and 0.25% (v/v) H 2 0 2 . The slides were counterstained with Gill's hematoxylin and blued in lithium carbonate. Signal Generation with Phosphatase Complex
Streptavidin-Alkaline
In a humid chamber at room temperature, the slides were covered with 20 yiL streptavidin-alkaline phosphatase complex (1.0 jig/mL) for 10 minutes in a buffer solution composed of 0.1 mol/L TRIS-HC1, pH 7.5; 0.15 mol/L NaCl; and 0.05% (v/v) Triton X-100. The slides were washed twice for 5 minutes in buffer solution. Two additional buffer washings were done, each for 15 minutes. There were two final rinses in a buffer solution containing 0.1 mol/L TRIS-HC1, pH 9.5; 0.1 mol/L NaCl; and 50 mmol/L MgCl2. The slides were developed in a solution containing 0.33 mg/mL nitroblue tetrazolium (NBT) and 0.17 mg/mL 5-bromo-4-chloro-3-indolylphosphate (BCIP) for 90 minutes. The reaction was stopped with a buffer composed of 20 mmol/L TRIS-HC1, pH 7.5; and 5 mmol/L EDTA. The slides were counterstained with nuclear fast red.
Vol. 96 • No. 3
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
A search of the 1966-1987 pathology files of the Foothills Hospital uncovered 13 cases of anal squamous cell carcinoma. All tissues were fixed in 10% (weight/volume [w/v]) phosphate-buffered formalin and processed routinely. The hematoxylin and eosin-stained slides were reviewed, and one representative paraffin block was selected from each case for the study. Serial sections were cut— first for in situ hybridization with horseradish peroxidase, and then for in situ hybridization with alkaline phosphatase. Tumor tissue was cut only from the residual block, and cellular DNA was extracted for dot blot hybridization.
319
320
ANATOMIC PATHOLOGY Article 0.1 mg/mL carrier DNA. Hybridization was performed at 42 °C for 16 hours with the use of a biotinylated probe concentration of 0.5 Mg/mL in a reaction mixture containing 50% (v/v) formamide, 4X SSPE, 5X Denhardt's solution, 5% (w/v) dextran sulfate, 0.1% (w/v) SDS, and 0.1 mg/mL carrier DNA (Tm —15 °C). The membranes were washed once for 15 minutes with 2X SSC/0.1% (w/ v) SDS at room temperature, twice with 0.5X SCC/0.1% (w/v) SDS for 30 minutes (Tm - 5 °C), and then once with 2X SSC for 10 minutes. Thereafter, the membranes were incubated for 60 minutes at 63 °C with 3% (w/v) bovine serum albumin and then with streptavidin-alkaline phosphatase conjugate, followed by the chromogenic substrate NBT/BCIP. Positive controls, composed of purified HPV DNA 6, 11, 16, 18, and 33 (100-pg target DNA and 200-ng carrier DNA), and a negative control, composed of 200-ng carrier DNA, were run simultaneously with the test samples. This technique has a sensitivity of 0.1 copies per cell, or 1 pg DNA.12 Samples were read as "positive" for HPV DNA when clear, purple dots could be distinguished from the background white color of the membrane. White dots and dot colors other than purple (e.g., yellow or pale brown) were read as "negative."
DNA Extraction from Paraffin Blocks The paraffin-embedded tissue blocks were cut with a microtome and rehydrated in TE (10 mmol/L TRIS-HC1, 1 mmol/L EDTA, pH 8.0). The rehydrated sections were ground to a powder with the use of liquid nitrogen and transferred to a digestion solution containing 50 mmol/ L TRIS-HC1, pH 8.0; 20 mmol/L EDTA; 1% (w/v) sodium dodecyl sulfate (SDS); and 100 Mg/mL proteinase K, and incubated overnight at 37 °C. The supernatant was discarded and the pellet further digested for 24-48 hours at 55 °C. After a phenol and chloroform extraction, DNA was precipitated by the addition of isopropanolNaCl. The DNA was incubated, centrifuged, washed in 70% ethanol, vacuum dried, and resuspended in TE. 13 The amount of DNA present was estimated with the use of agarose gel electrophoresis and ethidium bromide dye.12 Dot Blot
Hybridization
Full details of the method are available elsewhere.12 The sample volume of cellular DNA was reduced to 20fiL aliquots and spotted directly to the nitrocellulose membrane. The membranes were denatured and then air dried and baked for 60 minutes in an 80 °C vacuum oven. Membranes were incubated at 40 °C for at least 60 minutes in a prehybridization solution containing 50% (v/v) formamide, 4X SSPE (IX SSPE = 0.15 mmol/L NaCl; 10 mmol/L sodium phosphate, pH 7.0; and 1 mmol/L EDTA), 5X Denhardt's solution, 0.1% (w/v) SDS, and A.J.C.P. •
Statistical
Analysis
Exact binomial two-sided 95% confidence intervals (CIs) were calculated for the estimates of HPV DNA positivity obtained with the use of the three assay methods.15 Chi-square tests were used to test for an association between HPV DNA type and grade of keratinizing squamous cell carcinoma. A 5% level of significance was accepted. RESULTS Clinical Findings The patients ranged in age from 31 to 81 years (mean, 62 years), and consisted of nine women and four men. All of the patients had either rectal bleeding and/or an anal mass. Only one patient had a history of prior treatment for anal condylomata acuminata. Information with regard to the practice of anal-receptive intercourse was not available. Histologically, there were five basaloid carcinomas, diagnosed according to the criteria of Grinvalsky and Helwig16; seven keratinizing squamous cell carcinomas (four well differentiated and three moderately differentiated); and one verrucous carcinoma. A case each of basaloid and keratinizing squamous cell carcinoma had adjacent condylomata acuminata, whereas four cases of keratinizing squamous cell carcinoma (two well differentiated and two moderately differentiated) had koilo-
ember 1991
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Regardless of the bridging enzyme, all slides were mounted with aqueous mountant and reviewed for the presence or absence of a nuclear staining reaction, which was pink with AEC and purple with NBT/BCIP. Additional tissue sections fixed in a similar manner, and known to harbor HPV 6, 11, 16, 18, and 33, were run simultaneously as positive controls. Negative controls also were prepared by omitting the probe and replacing it with carrier DNA. The sensitivities of both in situ hybridization methods were tested with the use of identically processed HPVcontaining cell lines of known copy number (i.e., Siha [ 1 -2 copies of HPV 16], Hela [ 10-50 copies of HPV 18], and Caski [500 copies of HPV 16]).M In situ hybridization with horseradish peroxidase evoked a positive reaction in the Caski cell line only, indicating a sensitivity of 500 copies per cell for this method. In situ hybridization with alkaline phosphatase produced a positive signal in all three cell lines, indicating a sensitivity of 1-2 copies per cell for this method. Specificity tests also were performed for both methods by hybridizing the three cell lines with HPV types other than that present in the cell line. No cross-reaction occurred.
DUGGAN ET AL.
321
HPV DNA in Anal Carcinomas
HPV DNA Prevalence
ISH-HRP
ISH-AP
DBH
Method ISH-HRP = In situ hybridization with horseradish-peroxidase ISH-AP = In situ hybridization with alkaline phosphatase DBH = Dot blot hybridization cytotic features in the neoplasm.5 All patients were treated surgically. In Situ
Hybridization
With the use of horseradish peroxidase as the detecting enzyme, HPV DNA was not detected in a single case of anal carcinoma (95% CI, 0-25%); however, one adjacent condyloma contained HPV 6 (Table 1). With alkaline phosphatase as the detecting enzyme, a purple precipitate was seen in the malignant nuclei of eight cases (62%) (95% CI, 32-86%). Seven cases stained with HPV 16 (Fig. I A), and one stained with HPV 6. The quantity of nuclear staining was variable from case to case. Both adjacent condylomata acuminata contained HPV 6. By tumor type, three (43%) keratinizing squamous cell carcinomas, four (80%) basaloid carcinomas including the one with an adjacent condyloma, and the one (100%) verrucous carcinoma contained HPV DNA (Table 2). HPV 6 was identified in the one well-differentiated keratinizing squamous cell carcinoma with an adjacent con-
dyloma acuminatum, and HPV 16 was present in a case each of well- and moderately differentiated keratinizing squamous cell carcinoma. HPV DNA was not seen in any case of keratinizing squamous cell carcinoma with koilocytotic features. Dot Blot
Hybridization
The nitrocellulose membranes were read without the examiner's knowledge of the in situ hybridization results. Eleven cases (85%) (95% CI, 55-98%) contained HPV DNA (Table 1). Eight cases contained HPV 16, and there was a single case with HPV 6. There were two cases of mixed infection: a case each of HPV 16 + 6 and HPV 6 + 11. By tumor type, six (85%) keratinizing squamous cell carcinomas, four (80%) basaloid carcinomas, and the one (100%) verrucous carcinoma had HPV DNA (Table 2). HPV 6 + 1 1 was identified in the one case of well-differentiated keratinizing squamous cell carcinoma with an adjacent condyloma acuminatum, and HPV 16 (Fig. \B)
Vol. 96 • No. 3
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
• No HPV ^ HPV 16 • HPV 16+6 M HPV 6 • HPV 6+11
322
ANATOMIC PATHOLOGY Original Article
&4 ,
'
Human Papillomavirus DNA in Anal Carcinomas Comparison of In Situ and Dot Blot Hybridization MAIRE A. DUGGAN, F.R.C.P.C.,1 VALERIE F. BORAS, M.D.,1 MASAFUMI INOUE, M.Sc.,' AND S. ELIZABETH McGREGOR, M.Sc.2
blot hybridization, 69% had HPV 16/6 and 15% had HPV 6/ 11. An HPV DNA frequency range of 0-85% in the same group of tumors with the use of three hybridization techniques indicates the influential role of the method on HPV DNA prevalences. HPV DNA was identified regardless of patient gender or type of squamous cell carcinoma. The presence of HPV 16 in 82% of the positive cases is supportive evidence of the carcinogenic role of the HPV in anal squamous cell carcinoma. (Key words: Anal squamous cell carcinomas; Human papillomavirus DNA prevalence; In situ hybridization; Dot blot hybridization) Am J Clin Pathol 1991;96:318-325
(i.e., 16 and 18 in cervical carcinogenesis),5 the roles of these factors in anal carcinogenesis are being investigated increasingly. Published frequencies on the prevalence of HPV DNA in anal squamous cell carcinomas range from 0 to 61%. 6 " 10 It has been suggested that demographic variables such as patient gender and sexual proclivity1 and tumor variables such as histologic type9 may affect the HPV DNA positivity rate. However, because the sensitivities of some hybridization techniques differ considerably," and because the referenced studies6"10 used different techniques to identify HPV DNA, the role of the hybridization technique in accounting for the range of published HPV DNA prevalences must be clarified. The assessment of the HPV DNA content of archival, paraffin-embedded tissue blocks of anal squamous cell From the department of Pathology, Foothills Hospital, The Tom carcinomas is feasible using either tissue in situ hybridBaker Cancer Centre, and the University of Calgary; and the department ofEpidemiology and Preventive Oncology, Alberta Cancer Board,ization Calgary,or by extracting the cellular DNA from the blocks—dot blot hybridization. In our previous study,6 Alberta, Canada. in situ hybridization with streptavidin-biotin-horseradish Received June 21, 1990; received revised manuscript and accepted peroxidase complex did not detect biotinylated DNAfor publication December 27, 1990. DNA HPV hybrids in 13 cases of anal squamous cell carSupported by grant 954-672 from the Research and Development Committee, Foothills Hospital. cinoma. As we performed it, this hybridization technique Address reprint requests to Dr. Duggan: Department of Pathology, Foothills Hospital, 1403-29 Street Northwest, Calgary, Alberta T2N 2T9, has a sensitivity of 500 viral copies per cell. By substituting streptavidin-alkaline phosphatase, we achieved a sensiCanada.
Anal squamous cell carcinoma, which sometimes is reported as basaloid, cloacogenic, epidermoid, or transitional cell carcinoma, is an uncommon tumor that occurs predominantly in elderly women.1 Its incidence has increased recently, especially among homosexual men. 2 ' 3 Epidemiologic evidence suggests that the human papillomavirus (HPV)-induced genital wart is associated etiologically with anal carcinoma risk.4 Other risk factors include a history of chronic anal irritation and cigarette smoking.4 Because part of the anus and lower female genital tract are derived embryologically from the cloaca,1 and given the tropism of certain HPV types (e.g., 6, 11, 16, 18, and 33 for the female genital epithelium) and the large body of evidence implicating specific HPV types
318
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Because the sensitivities of individual hybridization techniques differ considerably, their role in accounting for the published frequencies of human papillomavirus (HPV) DNA in anal squamous cell carcinomas, ranging from 0 to 61%, must be investigated. With the use of biotinylated probes to HPV 6, 11, 16, 18, and 33, three hybridization techniques were performed on the same paraffin-embedded tissue blocks selected from 13 cases of anal squamous cell carcinoma. HPV DNA was detected in 0%, 62%, and 85% of cases with the use of in situ hybridization with horseradish peroxidase, in situ hybridization with alkaline phosphatase, and dot blot hybridization, respectively. By dot
DUGGAN ET AL. HPV DNA in Anal Carcinomas tivity of approximately 1 -2 copies per cell with the same in situ hybridization method. Dot blot hybridization using biotinylated probes is more sensitive, with a minimum detection limit of 0.1 copies per cell.12 The current study was designed to determine the relative frequencies of HPV DNA in 13 cases of anal squamous cell carcinoma with the use of the above three hybridization techniques, each performed on the same paraffin-embedded tissue block, and to correlate the histologic features of the carcinoma with the identified HPV DNA type. MATERIALS AND METHODS
Probe Preparation In situ and dot blot hybridization were performed with cloned HPV DNA labeled with biotin. Plasmid pBR322 containing either HPV 6, 11, 16, 18, or 33 was digested with either restriction endonuclease BamH I or EcoR I and purified with the use of agarose gel electrophoresis. The probes were made by nick-translation of the 7-8kilobase genomes with the use of Biotin-11-dUTP (Enzo Diagnostics, New York, NY) and reduced in size to 3001,000 bases to increase their tissue permeability.13 Unincorporated nucleotides were removed by a sodium chloride-ethanol DNA precipitation method. In Situ
Hybridization
The full details of the method have been published previously.2 The tissue sections were deparaffinized in two changes of xylene, followed by two changes of absolute ethanol, and placed for 30 minutes in methanol containing 0.5% (volume/volume [v/v]) H 2 0 2 to inactivate the endogenous peroxidase. Then they were placed in 0.2 mol/ L HC1 for 10 minutes so that the nuclear proteins would be digested. The slides were treated with a purified 5 ng/ mL proteinase K solution and incubated at 37 °C for 15 minutes. The slides were washed in a phosphate-buffered saline (PBS) solution containing 0.2% (w/v) glycine and then immersed in a PBS solution containing 4% (w/v) paraformaldehyde to postfix (stabilize) the DNA. The slides were dehydrated through a graded ethanol-water series; then they were air-dried.
Air-dried sections were overlaid with 20 /iL hybridization medium containing 50% (v/v) deionized formamide; 10% (w/v) dextran sulfate; 2X SSC buffer (0.3 mol/L NaCl, 0.03 mol/L sodium citrate), pH 7.0; 400 Mg/mL sonicated carrier DNA; and 10 MS/mL biotin-labeled probe DNA. The tissue sections were covered with a silicon-coated coverslip and transferred to a glass humid chamber. DNA was denatured by incubating the humid chamber at 8 5 90 °C for 10 minutes and then overnight at 37 °C. After hybridization, the sections were washed with 0.5X PBS containing 30% (v/v) deionized formamide (Tm - 1 0 °C) for 15 minutes at 37 °C and then washed in 0.5X PBS containing 10% (v/v) deionized formamide for another 15 minutes. After the posthybridization wash, the sections were washed for another 3 minutes at room temperature in PBS containing 0.5% (v/v) Triton-X (Fisher Scientific, NJ) and finally in PBS for 3 minutes. Signal Generation with Streptavidin-Biotin Horseradish Peroxidase Complex In a humid chamber at room temperature, the slides were covered for 30 minutes with 50-100 pL 1:200 diluted streptavidin-biotin-horseradish peroxidase complex in PBS containing 1% (w/v) bovine serum albumin and 5 mmol/L ethylenediaminotetraacetate (EDTA). The slides were washed twice for 5 minutes in 2X SSC and PBS containing 0.1% (v/v) Triton-X and were developed for 20 minutes in a freshly prepared mixture of 100 mmol/ L sodium acetate, 0.4 mg/mL aminoethylcarbazole (AEC), and 0.25% (v/v) H 2 0 2 . The slides were counterstained with Gill's hematoxylin and blued in lithium carbonate. Signal Generation with Phosphatase Complex
Streptavidin-Alkaline
In a humid chamber at room temperature, the slides were covered with 20 yiL streptavidin-alkaline phosphatase complex (1.0 jig/mL) for 10 minutes in a buffer solution composed of 0.1 mol/L TRIS-HC1, pH 7.5; 0.15 mol/L NaCl; and 0.05% (v/v) Triton X-100. The slides were washed twice for 5 minutes in buffer solution. Two additional buffer washings were done, each for 15 minutes. There were two final rinses in a buffer solution containing 0.1 mol/L TRIS-HC1, pH 9.5; 0.1 mol/L NaCl; and 50 mmol/L MgCl2. The slides were developed in a solution containing 0.33 mg/mL nitroblue tetrazolium (NBT) and 0.17 mg/mL 5-bromo-4-chloro-3-indolylphosphate (BCIP) for 90 minutes. The reaction was stopped with a buffer composed of 20 mmol/L TRIS-HC1, pH 7.5; and 5 mmol/L EDTA. The slides were counterstained with nuclear fast red.
Vol. 96 • No. 3
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
A search of the 1966-1987 pathology files of the Foothills Hospital uncovered 13 cases of anal squamous cell carcinoma. All tissues were fixed in 10% (weight/volume [w/v]) phosphate-buffered formalin and processed routinely. The hematoxylin and eosin-stained slides were reviewed, and one representative paraffin block was selected from each case for the study. Serial sections were cut— first for in situ hybridization with horseradish peroxidase, and then for in situ hybridization with alkaline phosphatase. Tumor tissue was cut only from the residual block, and cellular DNA was extracted for dot blot hybridization.
319
320
ANATOMIC PATHOLOGY Article 0.1 mg/mL carrier DNA. Hybridization was performed at 42 °C for 16 hours with the use of a biotinylated probe concentration of 0.5 Mg/mL in a reaction mixture containing 50% (v/v) formamide, 4X SSPE, 5X Denhardt's solution, 5% (w/v) dextran sulfate, 0.1% (w/v) SDS, and 0.1 mg/mL carrier DNA (Tm —15 °C). The membranes were washed once for 15 minutes with 2X SSC/0.1% (w/ v) SDS at room temperature, twice with 0.5X SCC/0.1% (w/v) SDS for 30 minutes (Tm - 5 °C), and then once with 2X SSC for 10 minutes. Thereafter, the membranes were incubated for 60 minutes at 63 °C with 3% (w/v) bovine serum albumin and then with streptavidin-alkaline phosphatase conjugate, followed by the chromogenic substrate NBT/BCIP. Positive controls, composed of purified HPV DNA 6, 11, 16, 18, and 33 (100-pg target DNA and 200-ng carrier DNA), and a negative control, composed of 200-ng carrier DNA, were run simultaneously with the test samples. This technique has a sensitivity of 0.1 copies per cell, or 1 pg DNA.12 Samples were read as "positive" for HPV DNA when clear, purple dots could be distinguished from the background white color of the membrane. White dots and dot colors other than purple (e.g., yellow or pale brown) were read as "negative."
DNA Extraction from Paraffin Blocks The paraffin-embedded tissue blocks were cut with a microtome and rehydrated in TE (10 mmol/L TRIS-HC1, 1 mmol/L EDTA, pH 8.0). The rehydrated sections were ground to a powder with the use of liquid nitrogen and transferred to a digestion solution containing 50 mmol/ L TRIS-HC1, pH 8.0; 20 mmol/L EDTA; 1% (w/v) sodium dodecyl sulfate (SDS); and 100 Mg/mL proteinase K, and incubated overnight at 37 °C. The supernatant was discarded and the pellet further digested for 24-48 hours at 55 °C. After a phenol and chloroform extraction, DNA was precipitated by the addition of isopropanolNaCl. The DNA was incubated, centrifuged, washed in 70% ethanol, vacuum dried, and resuspended in TE. 13 The amount of DNA present was estimated with the use of agarose gel electrophoresis and ethidium bromide dye.12 Dot Blot
Hybridization
Full details of the method are available elsewhere.12 The sample volume of cellular DNA was reduced to 20fiL aliquots and spotted directly to the nitrocellulose membrane. The membranes were denatured and then air dried and baked for 60 minutes in an 80 °C vacuum oven. Membranes were incubated at 40 °C for at least 60 minutes in a prehybridization solution containing 50% (v/v) formamide, 4X SSPE (IX SSPE = 0.15 mmol/L NaCl; 10 mmol/L sodium phosphate, pH 7.0; and 1 mmol/L EDTA), 5X Denhardt's solution, 0.1% (w/v) SDS, and A.J.C.P. •
Statistical
Analysis
Exact binomial two-sided 95% confidence intervals (CIs) were calculated for the estimates of HPV DNA positivity obtained with the use of the three assay methods.15 Chi-square tests were used to test for an association between HPV DNA type and grade of keratinizing squamous cell carcinoma. A 5% level of significance was accepted. RESULTS Clinical Findings The patients ranged in age from 31 to 81 years (mean, 62 years), and consisted of nine women and four men. All of the patients had either rectal bleeding and/or an anal mass. Only one patient had a history of prior treatment for anal condylomata acuminata. Information with regard to the practice of anal-receptive intercourse was not available. Histologically, there were five basaloid carcinomas, diagnosed according to the criteria of Grinvalsky and Helwig16; seven keratinizing squamous cell carcinomas (four well differentiated and three moderately differentiated); and one verrucous carcinoma. A case each of basaloid and keratinizing squamous cell carcinoma had adjacent condylomata acuminata, whereas four cases of keratinizing squamous cell carcinoma (two well differentiated and two moderately differentiated) had koilo-
ember 1991
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Regardless of the bridging enzyme, all slides were mounted with aqueous mountant and reviewed for the presence or absence of a nuclear staining reaction, which was pink with AEC and purple with NBT/BCIP. Additional tissue sections fixed in a similar manner, and known to harbor HPV 6, 11, 16, 18, and 33, were run simultaneously as positive controls. Negative controls also were prepared by omitting the probe and replacing it with carrier DNA. The sensitivities of both in situ hybridization methods were tested with the use of identically processed HPVcontaining cell lines of known copy number (i.e., Siha [ 1 -2 copies of HPV 16], Hela [ 10-50 copies of HPV 18], and Caski [500 copies of HPV 16]).M In situ hybridization with horseradish peroxidase evoked a positive reaction in the Caski cell line only, indicating a sensitivity of 500 copies per cell for this method. In situ hybridization with alkaline phosphatase produced a positive signal in all three cell lines, indicating a sensitivity of 1-2 copies per cell for this method. Specificity tests also were performed for both methods by hybridizing the three cell lines with HPV types other than that present in the cell line. No cross-reaction occurred.
DUGGAN ET AL.
321
HPV DNA in Anal Carcinomas
HPV DNA Prevalence
ISH-HRP
ISH-AP
DBH
Method ISH-HRP = In situ hybridization with horseradish-peroxidase ISH-AP = In situ hybridization with alkaline phosphatase DBH = Dot blot hybridization cytotic features in the neoplasm.5 All patients were treated surgically. In Situ
Hybridization
With the use of horseradish peroxidase as the detecting enzyme, HPV DNA was not detected in a single case of anal carcinoma (95% CI, 0-25%); however, one adjacent condyloma contained HPV 6 (Table 1). With alkaline phosphatase as the detecting enzyme, a purple precipitate was seen in the malignant nuclei of eight cases (62%) (95% CI, 32-86%). Seven cases stained with HPV 16 (Fig. I A), and one stained with HPV 6. The quantity of nuclear staining was variable from case to case. Both adjacent condylomata acuminata contained HPV 6. By tumor type, three (43%) keratinizing squamous cell carcinomas, four (80%) basaloid carcinomas including the one with an adjacent condyloma, and the one (100%) verrucous carcinoma contained HPV DNA (Table 2). HPV 6 was identified in the one well-differentiated keratinizing squamous cell carcinoma with an adjacent con-
dyloma acuminatum, and HPV 16 was present in a case each of well- and moderately differentiated keratinizing squamous cell carcinoma. HPV DNA was not seen in any case of keratinizing squamous cell carcinoma with koilocytotic features. Dot Blot
Hybridization
The nitrocellulose membranes were read without the examiner's knowledge of the in situ hybridization results. Eleven cases (85%) (95% CI, 55-98%) contained HPV DNA (Table 1). Eight cases contained HPV 16, and there was a single case with HPV 6. There were two cases of mixed infection: a case each of HPV 16 + 6 and HPV 6 + 11. By tumor type, six (85%) keratinizing squamous cell carcinomas, four (80%) basaloid carcinomas, and the one (100%) verrucous carcinoma had HPV DNA (Table 2). HPV 6 + 1 1 was identified in the one case of well-differentiated keratinizing squamous cell carcinoma with an adjacent condyloma acuminatum, and HPV 16 (Fig. \B)
Vol. 96 • No. 3
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
• No HPV ^ HPV 16 • HPV 16+6 M HPV 6 • HPV 6+11
322
ANATOMIC PATHOLOGY Original Article
&4 ,
'
