Human Papillomavirus Infection and - Europe PMC

American Journal of Pathology, Vol. 140, No. 6, June 1992

Copyright © American Association ofPathologists

Human Papillomavirus Infection and Anal Carcinoma Retrospective Analysis by In Situ Hybridization and the Polymerase Chain Reaction

Sherif R. Zaki,* Randy Judd,t Lisa M. Coffield,* Patricia Greer,* Florence Rolston,* and Bruce L. Evattt From the Divisions of Viral and Rickettsial Diseases,* and HIV/AIDS,* National Center for Infectious Diseases, Centers for Disease Control, and the Department of Pathology and Laboratory Medicine, t Emory University School of Medicine,

Atlanta, Georgia

To examine the association of human papillomavirus (HPV) infection with anal squamous cell carcinoma the authors applied the highly sensitive polymerase chain reaction (PCR) and in situ hybridization (ISH) techniques to detectHPVDNA informalinfixed, paraffin-embedded tissues from 18 patients. The presence of HPV types 16/18 in 3 (16 7%) of 18 patients with anal carcinoma was found, using a colorimetric ISH technique for HPV types 6, 11, 16, 18, 31, 35, and 51. Results from one of these three patients were also positive for HPV 31, 35, 51 by ISH techniques. When the same series was analyzed using the PCR and consensus primers to the LI open reading frame of the HPV genomes, the frequency ofpositive patients rose to 14 (77.8%) of 18. PCR analysis of the 14 lesions containing HPV DNA, using typespecific primers andprobesforHPV 6, 11, 16, 18, and 33, showed that 1 contained HPV 6, 1 contained HPV 11, 4 contained HPV 16, 1 contained HPV 18, 1 contained HPV 33, 5 contained HPV of unclassified type(s), and 1 contained a mixture of three HPV types. There was concordance between typing ofcases that were positive by ISH and PCR methods. These data agree with the concept that HPV, in particular type 16, is implicated in the pathogenesis of anal cancer. (Am J Pathol 1992, 140:1345-1355)

The human papillomaviruses (HPV) are a group of epitheliotrophic DNA viruses, containing a double-stranded DNA genome of approximately 8000 bp, associated with

a variety of benign and

mucous

malignant tumors of the skin and

membranes.15 Genotyping of HPVs is accom-

plished by DNA hybridization under controlled conditions of stringency. HPVs differing by more than 50% DNA homology when tested under stringent conditions are considered to be of different types.6'7 These methods, have been used to identify more than 60 types of HPV.8 There is a growing body of evidence linking specific types of HPV with particular squamous cell neoplasms of the anogenital region.6 HPV types 16 and 18, and to a lesser extent types 31, 33, and 35, are usually found in high-grade intraepithelial neoplasias and invasive squamous cell carcinomas of the cervix, vulva, vagina, and penis, whereas HPV 6 and 11 are more commonly detected in genital condylomas and lower grades of intraepithelial neoplasia.9-15 Anal squamous cell carcinoma is an uncommon malignancy of unknown cause. Associated factors include chronic irritation, homosexual contact, inflammatory bowel disease, smoking, anal condylomata, and immunosuppression.1619 Epidemiologic studies suggest that a sexually transmitted agent, such as HPV, may play an important role in the development of anal carcinoma.20'21 Moreover, recent studies have demonstrated the presence of HPV antigens and nucleic acids in anal carcinomas. However, large variations in HPV prevalence have been reported in anal squamous cell carcinomas studied. 9'22 34 Factors such as selection of study group or use of different hybridization techniques for HPV detection that vary in their sensitivity and virus type specificity may explain this variation. The polymerase chain reaction (PCR)35 is the most sensitive method for HPV nucleic acid detection currently available. Novel PCR methods have been described for Presented in part at the United States and Canadian Academy of Pathology 80th Annual Meeting, Chicago, IL, March 17, 1991. Accepted for publication December 31, 1991. Address reprint requests to Dr. Sherif Zaki, Molecular Pathology and Ultrastructural Activity, Centers for Disease Control, G-32, 1600 Clifton Rd., Atlanta, GA 30333.

1345

1346

Zaki et al

AJP June 1992, Vol. 140, No. 6

the detection of a broad spectrum of HPV genotypes by using general or consensus primers. 3 Furthermore, these methods can be applied to a single section of routinely processed, formalin-fixed, paraffin-embedded tissue. This study describes the use of the PCR with generic and type-specific primers, coupled by the in situ hybridization (ISH) technique, to examine the prevalence and possible role of HPV in the development of anal squamous cell carcinoma.

and parakeratosis, and could be assessed only in lesions demonstrating a surface or in situ component. All lesions diagnosed as CIS contained malignant cells extending the entire epithelial thickness. Histopathologic examination, PCR, and ISH were all done in a double-blind manner, without prior knowledge of results using any of the other assays.

In Situ Hybridization In situ hybridization was completed using biotinylated probes specific for HPV types 6/11, 16/18, and 31/35/51 (Enzo Diagnostics Inc., New York, NY). Briefly, the procedure involves predigestion of deparaffinized sections mounted on silanized slides with proteinase K followed by simultaneous denaturation of the cellular DNA and probe for 10 minutes at 950C, and then by hybridization for 2 hours at 370C. Proteinase K was used at a concentration of 0.25 mg/ml, and the length of the predigestion step was varied empirically to achieve optimal signal with preservation of morphologic features. The reaction was visualized after incubation with avidin-biotin-horseradish peroxidase complex, followed by addition of hydrogen peroxide and aminoethylcarbazole (AEC). Biotinylated pBR322 and cytomegalovirus (CMV) DNA were used as negative control probes, whereas biotin-labeled human placental DNA was used as an endogenous positive control probe. Control tissue included known HPV-positive genital lesions and normal skin sections. The following additional control slides of HPVinfected cell lines were used: HeLa cells containing 10 to 50 copies per cell of HPV type 18, and CaSki cells containing approximately 500 copies per cell of HPV type 16.41,42 The ISH technique was considered adequate

Materials and Methods Patients Eighteen anal carcinomas accessioned between 1982 and 1989 at Grady Memorial Hospital, Atlanta, were identified. This series included 11 invasive squamous cell carcinomas (ISCC) and 7 carcinomas in situ (CIS). Control paraffin blocks from 18 hemorrhoid specimens accessioned during 1988 and 1989 were also available for ISH and PCR analysis. All specimens received were fixed in buffered formalin, processed routinely, and embedded in paraffin. For each case, all available hematoxylin and eosin stained sections were reviewed and a representative block was selected for further studies. Blocks from lymph-node metastases or from coexisting vulvar CIS were available in four cases. Histologic sections were evaluated for various pathologic features (Table 1), following accepted criteria as applied to the uterine cervix.40 Nuclear atypia reflected nuclear enlargement, hyperchromasia, pleomorphism, and nucleolar size. Koilocytotic features included nuclear enlargement, irregularity, multinucleation, and perinuclear clearing. Proliferation referred to the degree of acanthosis, papillomatosis,

Table 1. Clinical and Pathologic Features of Patients with Anal Carcinomas

Case 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18

Age/Sex

Year

Diagnosis

46/F 81 /M 63/F 58/F 40/F 64/F 51/M 40/F 74/M 47/F 48/F 34/F 42/F 52/F 20/F 58/F 31/M 38/M

1982 1982 1983 1983 1984 1984 1983 1984 1984 1984 1985 1985 1985 1987 1987 1987 1988 1989

ISSC ISSC CIS

Atypia

ISSC ISSC ISSC

Proliferation

Keratinization

+

+++

ISSC

ISSC

Koilocytosis

++

+

+++ + +

++

NP

++

NP NP

++ ++ + ++ + ++ + ++

CIS CIS

+

ISSC CIS

CIS ISSC CIS

++

++

NP

++

++ ++

++

NP

++

+ +

+

CIS

++ +

++

+

+ +

ISSC

++ + +

ISSC

++ ++

++

Year = year specimen accessioned; atypia = nuclear enlargement/pleomorphism; proliferation = acanthosis/papillomatosis of surface component; ISSC = invasive squamous cell carcinoma; CIS = carcinoma in situ; -, absent; +, mild; + +, moderate; + + +, marked; NP = no surface component present.

HPV and Anal Carcinoma 1347 AJP June 1992, Vol. 140, No. 6

Figure 1. he detection ofbuman DNAsequences in tissuesections using a biotin-labelled human placentalpositive controlprobe. A: Adequatepositive control response. B: Inadequatepositive control response (AEC substrate witb light-green counterstain). Figure 2. Detection of HPV 16/18 DNA by in situ hybridization (case no. 12). A: Prominent staining of cells in superficial part of biopsy. B: Higherpower magnification of tissue shown in (A) demonstrating nuclear pattern of staining (AEC substrate with ligbt bematoxylin counterstain).

1348 Zaki et al AJPJune 1992, Vol. 140, No. 6

Table 2. Human Papillomavirus in Anal Carcinoma by In Situ Hybridization and the Polymerase Chain Reaction Analysis Patient Sex HPV PCR ISH Other Age Diagnosis 1 46 F ISSC 16 - ve 2 81 M ISSC 16 -ve LN HPV 16+ve by PCR 3 53 F CIS 18 IT 4 58 F ISSC ns IT 5 40 F ISSC -ve LN HPV -ve -ve 6 64 F ISSC - ve - ve 7 M 51 ISSC ns - ve 8 40 F ISSC - ve IT 9 74 M CIS -ve -ve 10 47 CIS F ns IT 11 F 48 ISSC 6 - ve 12 34 F CIS 16 16/18 13 42 F CIS 16 16/18 Vulvar CIS HPV 6 + ve by ISH and PCR 14 52 F 11 ISSC - ve 15 20 F CIS ns - ve HIV+ 16 CIS 58 F 33 -ve VulvarCIS HPV + ve 17 31 M ISSC 16,18 16/18 HIV+ ns 31/35/51 M 18 38 ISSC ns - ve HIV+ PCR, polymerase chain reaction; ISH, in situ hybridization; F, female; M, male; ISSC, invasive cell squamous cell carcinoma; CIS, carcinoma in situ; LN, lymph node; HIV, human immunodeficiency virus; HPV, human papilloma virus; +ve, HPV positive; -ve, HPV negative; ns, not subtyped; IT, inadequate tissue.

when the placental DNA probe gave a uniform signal over most nuclei, the plasmid control gave no signal, and the control tissues stained appropriately.

placed directly in sterile 1.5-mi microcentrifuge tubes were deparaffinized, followed by digestion for 2 hours at 550C in 200 ,ul of TET buffer (TET = 50 mmol/l Tris-HCI [pH 8.5y1 mmol/l ethylenediamine tetracetic acid [EDTA] 0.5% Tween 20) containing proteinase K at a concentration of 200 ,ug/ml. After the proteinase K was inactivated for 10 minutes at 950C, the supernatant was used for PCR analysis. Because of differences in tissue section size, and hence DNA content, various volumes of supernatant

PCR Analysis DNA for PCR analysis was extracted from paraffin sections as described previously.43 Briefly, 8-,um sections

I

2A 2

Anal Carcinomas 3 4 5B 6

ail*R 7

a

Anal Carcinomas 10 I1 12 13A 1 514 16 1A16B 17 18

a

A -450 bp

g 7

1

bp 1353 1078 872

603 310

281 ~~~~~~~~~~~~~271,

-

a -450 bp

+'

Hemorrhoid Controls 1

I

C,

'4850 bpA

2

3

4

5

6

7

8

9

4 z

40" /

CD0

Hemorrhoid Controls -

10 11

12 13I

-'~ ~~~t

Is 1inll

18

b

1353 872 003 310

271. 281

D -o45o bo. Figure 3. Detection ofHPVDNA in anal squamous cell carcinomas (A,B), and hemorrhoidal tissue (C,D), using the PCR and L1 consensus primers. PCR products are shown after agarose gel electrophoresis, ethidium-bromide staining, and ultraviolet illumination (A,C) as well as after subsequent blotting and hybridization with a 32P labeled HPV oligonucleotide generic probe mixture (B,D).

HPV and Anal Carcinoma 1349 AJPJune 1992, Vol. 140, No. 6

Table 3. Polymerase Chain Reaction Results of Humzan Papillomavirus DNA

Total Diagnosis CIS 7 11 ISCC * ns, not subtyped.

Positive 6 8

Negative 1 3

2A2B3 4

281

SAB7

1

8

/

18 1 0

16 2 2

33 1 0

ns

2 3

mal cycler (Perkin-Elmer Cetus, Norwalk, CT). The procedures for PCR amplification of the control myeloperoxidase gene, as well as amplification and typing of DNA extracts using HPV type-specific primers and probes, have been described previously.4 Amplification and typing of DNA extracts, using generic primers and probes, were performed as described elsewhere.45 Necessary precautions to avoid crosscontamination were taken at all stages of extraction and amplification.46 Some of these precautions included the use of disposable microtome blades, and physical isolation of sample extraction, preparation for PCR, amplification, and analysis.

(1-5 ,ul) were initially used to optimize PCR conditions with control (myeloperoxidase) primers. Optimal volumes of sample, yielding maximal signal on ethidium-stained gels, were subsequently used for HPV PCR analysis. The oligonucleotides used as primers and probes were synthesized on an Applied Biosystems 381 A synthesizer (Foster City, CA) by the phosphoramidite method.44 Consensus oligonucleotide primers and probes, described by Tang and Manos45 and capable of amplifying and detecting regions within the Li open reading frame from more than 25 of the genital HPVs, including HPV types 6, 11, 16, 18, and 33, were synthesized. Specific probes and primers for HPV types 6, 11, 16, 18, and 33 were synthesized as previously described. Myeloperoxidase-specific primers capable of amplifying a 359-bp genomic DNA fragment were used as an internal control of DNA structural integrity.43 Amplification of the extracted DNA was performed using a thermostable Thermus aquaticus (Taq) DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT) in a DNA ther-

Anal Carchiomas

16 18 ns* 0 1

11 0 1

6 0 1

Typing by Histologic Type HPV type

Results Clinical and Pathologic Features The pertinent clinical and pathologic features from the patients studied are summarized in Table 1. All patients

,Y

Anal Carcinomas

6a,

.........13.

In

11

I9t.AIBRIAI

17

i6 l17

/1/

bp-I*

361 bp

313 bp

216 bp

45ibw Figure 4. Detection ofHPV type-specific DNA in anal carcinoma by PCR analysis. Autoradiograms ofPCR products obtained using HPV type specific primers and detected with type-specific probes are shown.

1350

Zaki et al

AJP June 1992, Vol. 140, No. 6

3il

HPV and Anal Carcinoma

1351

AJPJune 1992, Vol 140, No. 6

(13 women and 5 men) presented with rectal bleeding and/or an anal mass. In one patient, the carcinoma was discovered as an incidental finding in a hemorrhoid specimen. We were unable to determine the sexual behaviors of the patients from the available hospital records. Three of five patients from the 1987-1989 period (2 men and 1 woman) had positive test results for antibodies to the human immunodeficiency virus (HIV); no information on HIV status of the other patients was available. Of the 18 patients, 2 had lymph-node metastases, and another two had coexisting vulvar CIS. Pathologic examination revealed 11 cases of ISSC and 7 cases of CIS. Histopathologic evidence of HPV infection (koilocytosis) was present in 5 of 18 cases. We were unable to correlate any histopathologic features with the subsequent detection of HPV by either ISH or PCR analyses.

In Situ Hybridization Several control probes were critical to the development and interpretation of the ISH assay. Sections from tissue blocks, predigested for various intervals, were hybridized with a biotinylated endogenous positive control probe (human placental DNA). This allowed for optimizal digestion conditions for differences in tissue fixation, and enabled us to achieve maximal signal intensity and more than 90% nuclear staining in tissue sections (Figure 1A). Under optimized conditions the negative control probes (pBR 322 and CMV) did not generate any signal. Four of our study cases were considered unsuitable for ISH analysis, on the basis of a negative or inadequate positive control response (Figure 1 B, Table 2). HPV DNA was detected in 3 of 18 (16.7%) patients by ISH analysis (Table 2). All three patients were positive for HPV 16/18. Moreover, one of the three patients was also positive for HPV 31/35/51. The signal was confined to the epithelium and varied from a strong, coarsely granular, patchy staining of 5 to 15% of the nuclei in cases 13 and 17, to only rare positive cells in case 12. The coexisting vulvar CIS in case 13 was positive for HPV 6/11 by ISH analysis. None of the anal cancer tissues examined were positive for HPV 6/11 by ISH analysis. Staining was strongest in the superficial nuclei and areas exhibiting histologic changes suggestive of HPV infection (Figure 2). Only rare nuclei

stained in the deep and invasive elements of the tumors. Lymph-node metastases in cases 2 and 5 were both negative using the ISH technique. The epithelium of the hemorrhoid control tissue was negative for HPV DNA by ISH techniques.

PCR Analysis PCR analysis was completed and results were analyzed without bias concerning either histopathologic findings or ISH results. In all cases, amplification of a part of the myeloperoxidase gene was successful, indicating the presence of intact target DNA (data not shown). Several negative controls, such as placental and pancreatic DNA, as well as distilled water instead of DNA, were consistently negative throughout the procedures. When PCR was used the number of cases positive for HPV DNA rose to 14 of 18 (77.8%) (Table 2). Figure 3 shows the results of experiments in which DNA extracted from anal cancer specimens and control hemorrhoid tissue were amplified with HPV consensus primers and analyzed by Southern blotting, using a generic probe mix. Figure 4 represents a Southern blot analysis of products of amplification generated using HPV type-specific primers and hybridized with oligonucleotides specific for HPV types 6, 11, 16, 18, and 33. There was concordance between typing of positive cases by ISH and the corresponding PCR HPV type. No case was positive by ISH analysis and negative by PCR technique. With respect to histology, HPV was detected in 6 of 7 cases of CIS and in 8 of 11 cases of ISCC (Table 3). HPV 16 was the most commonly detected HPV type found alone or in combination with other types in five cases. HPV 18 was detected in two cases, whereas HPV 6, 11, and 33 were seen in one case each. Nonsubtyped genomes were detected in five cases. All three specimens from known HIV-positive patients in this study contained untyped HPV DNA by PCR analysis. Case 17 also contained HPV 16 and 18 DNA, in addition to the untyped HPV genome. ISH results suggest that the untyped HPV in this particular case may be HPV 31, 35, or 51. However, the probes used for ISH analysis contain the highly homologous late regions of the virus. Therefore, the possibility of crossreaction with an unidentified HPV on ISH cannot be excluded. PCR analysis of this case with HPV 31 type-specific primers and probes was neg-

Figure 5. Examples ofsome histologicfeatures present in anal squamous cell cancers and hemorrhoid controls studied. A: Case no. 12. Area of carcinoma in situ showing epithelial thickening, byperkeratosis, parakeratosis, and acanthosis. HPV 16 was identified by PCR (H&E X 100). B: Case no. 12. Koilocytotic cells (arrows) demonstrate nuclear enlargement, irregularity, and hyperchromasia, with perinuclear clearing (H&E x400). C: Case no. 1. Area of thickened surface epithelium overlying invasive squamous cell carcinoma. HPVwas not detected by PCR analysis (H&E X 100). D: Case no. 1. Large multinucleated koilocytotic cells (arrows) from HPV-negative patient (H&E X 400). E: Case no.

18. Koilocytotic cells (arrows)from HPV-negativepatient (H&E X 400). F: Case no. 1 7. Invasive squamous cell carcinoma lacking koilocytotic cells, from HIV-positivepatient. Untyped HPVas well as HPV 16 and HPV 18 were detected by PCR analysis (H&E X400). G: Case no. 3. Area of carcinoma in situ with nuclei showing homogeneous, ground-glass chromatin (arrows). HPV 18 was detected by PCR analysis (H&E x 400). H: Focus of moderate dysplasia in hemorrhoid specimen that was positive for HPV by PCR analysis (H&E X200). I: Focus ofsevere dysplasia in hemorrhoid specimen tha was positive for HPV by PCR analysis (H&E X200).

1352

Zaki et al

AJP June 1992, Vol. 140, No. 6

ative (data not shown). When HPV 33 primers were used, an intense band, slightly larger in size than expected, was seen on the ethidium-stained gel. Southern hybridization with a type 33-specific probe resulted in a weak hybridization signal, suggesting that the HPV may be HPV type 33-related (Figure 4). None of the histologic features evaluated in this study (Table 1) correlated with the presence of HPV by PCR (Table 2). Although some cases that tested positive for HPV did demonstrate epithelial proliferation (Figure 5A) and focal koilcytosis (Figure 5B), features associated with HPV infection of the vulva and cervix, other cases with equally prominent epithelial proliferation (Figure 5C) and koilocytosis (Figure 5D) yielded negative results for HPV infection. No unique histologic features were noted in the specimens from HIV-positive patients that were infected with untyped HPV. Cases associated with HIV infection also did not demonstrate any correlation between the presence (Figure 5E) or absence (Figure 5F) of koilocytosis and HPV infection. One of the HPV-positive cases contained foci where the nuclear chromatin had homogeneous or ground-glass appearance (Figure 5G). However, this finding lacked sufficient sensitivity or specificity to be of any diagnostic value. When control hemorrhoid tissue was examined by PCR and generic HPV primers, 3 of 18 were positive (Figure 3, Table 4). Review of the histologic sections from these patients revealed a microscopic focus of moderate dyplasia in one (Figure 5H), and extensive moderate with focal severe dysplasia in another (Figure 51); neither dysplasia had been detected during the initial pathologic evaluation.

Discussion Much information about the role of HPV in neoplasia has come from studies using molecular hybridization techniques such as ISH and Southern blotting. ISH offers the unique advantage of detecting specific nucleic acid sequences in intact tissue sections, allowing correlations with histopathologic changes. Furthermore, compared with conventional Southern analysis, which requires 1020 million cells to generate an adequate signal, ISH can detect unique DNA and RNA sequences at the level of the single cell in tissue sections. However, ISH is insensitive, since 50 to 100 genomic copies or more are usuTable 4. Prevalence of HPV infected Squamous Cell Carcinoma Compared with Nonmalignant Hemorrboidal Tissue Diagnosis Negative Positive Total Hemorrhoids 15 3 18 4 14 SCC 18

ally needed in a single cell, whereas Southern hybridization is far more sensitive since it can detect as little as one copy per cell, when multiple cells are infected. The technique of in vitro DNA amplification with the use of the PCR, like ISH, can be performed on a single section of routinely processed, formalin-fixed, paraffin-embedded tissue. Furthermore, its sensitivity supersedes that of Southern blotting since a single copy of target DNA present in a tissue sample can be detected after multiplying it approximately one million-fold by repeated cycles of amplification. One important drawback of PCR, however, is the possibility of false-positive reactions due to the exquisite sensitivity of the technique. Therefore, careful precautions must be taken to avoid this problem when using PCR analysis. The prevalence of HPV in the female genital tract and the strong association of specific HPV types with benign and malignant lesions in this anatomical area are well documented. There is mounting evidence suggesting that anal cancer is also associated with HPV infection. Recently, several groups have demonstrated the presence of HPV antigens, DNA, and RNA in anal carcinomas. The large variation in reported HPV prevalence rates and the occasional low HPV rates observed in some studies may reflect the variable sensitivity of detection techniques and the spectrum and specificity of HPV probes used (Table 5). Furthermore, the degree to which the frequency of HPV infection in anal cancer tissue is increased above nonmalignant anal tissue is not evident from these studies. In the present study of 18 patients with anal squamous cell carcinoma, we detected HPV DNA in 14 (77.8%) patients. Our frequency of HPV infection is slightly higher, but in general agreement with currently available data on the prevalence of HPV in anal cancer (Table 5). Although we used both generic and typespecific PCR primers and probes, capable of detecting more than 25 HPV types, the actual prevalence of HPV in anal cancer is probably even higher. This underestimation of HPV prevalence cannot be attributed to the sensitivity of the PCR technique or inadequate tissue fixation since we were able to amplify a single copy (myeloperoxidase) control gene from all patient specimens. Rather, given the large number of known HPV types and the fact that new types continue to be discovered on a regular basis, other less common or presently unknown subtypes may have escaped detection with the primers and probes used. Further studies using additional typespecific primers and probes, or generic HPV primers and probes outside of the Li region, will be needed to clarify this point. The detection of HPV DNA in 14 of 18 cases by PCR analysis and in 3 of 18 patients by ISH technique is consistent with the greater potential sensitivity of the PCR

HPV and Anal Carcinoma 1353 AJP June 1992, Vol. 140, No. 6

Table 5. Studies of HPV in Anal Squamous Cell Carcinoma

Investigations/Year Scheurlen et al/1986 Hill and Coghill/1986 Palmer et al/1986 Gal et al/i 987 Wells et al/1987 Yun et al/1988

Technique/Subtype studied SBH/HPV 16 DNA hybridization/HPV 6, 11, 16, 18 SBH/HPV 16

IHC/polyclonal antibody IHC/ISH/polyclonal antibody and HPV 6, 11,

ISH/HPV 6, 11, 16, 18

Prevalence no. (%) 1/2 (50) 1/1 (100) 6/10 (60) 5/8 (62.5) 4/9 (44.4)

0/1 (0)

16, 18, 31, 33

Duggan et al/1989 Beckman et al/1989 Gal et al/1989 Taxy et al/1989

ISH/6, 11, 16, 18 ISH/6, 11, 16, 18, 31 ISH/6, 11, 16, 18, 31 IHC,ISH/polyclonal antibody and HPV 6, 1 1,

Palefsky et al/1990 Wolber et al/1990 Palefsky et al/1991

DBH/6, 11, 16, 18, 31, 33, 35 ISH/6, 11, 16, 18 ISH,PCR/HPV type specific primers for types 6, 11, 16, 18, 31,33 ISH,PCR/HPV type specific primers for HPV

16, 18, 31

Koulos et al/1991 Present study 1991

16, 18, 31, 35 ISH,PCR/HPV type specific and generic primers; roughly 25 HPV types, including 6, 11, 16, 18, 33

See legend to Table 2 for abbreviations. Additional abbreviations: IHC = immunochemistry; SBH blot hybridization; HPV ns = non-subtyped HPV.

technique. The DNA extracted from all 18 patients was considered adequate for PCR analysis, as determined by amplification of a single copy gene in each specimen. However, tissue sections from only 14 cases were judged adequate for ISH analysis after hybridization with a human genomic probe. This could have resulted in some false-negatives by ISH in this study. Optimal ISH is achieved only when tissue specimens are properly fixed and predigested under carefully controlled conditions. Although we could not control the fixation process, attempts at optimizing digestion conditions were unsuccessful in generating an adequate signal with human placental DNA in four of our study cases. Another possible explanation for the low rate of HPV DNA detection by our DNA-ISH technique is its inherent lower sensitivity as compared with RNA-ISH methods.30 Our results indicate that infection with high-risk HPV types, mainly HPV 16, is closely associated with anal squamous cell carcinoma. This conclusion is in agreement with previous studies that have demonstrated an association between various HPV types and anogential neoplasms. The fact that only high-risk HPV types were detected by ISH analysis, and that the distinction between the prevalence of high-risk and low-risk HPV types in anal cancer became less obvious after PCR analysis, is interesting. This finding reflects the sensitivity of PCR and may indicate that high copy numbers of high-risk HPV are necessary for oncogenesis. Unclassified HPV types were detected in five patients (28%), three of which had HIV-positive test results. Recent studies indicate that concurrent immune dysfunction resulting from HIV infection may be a factor promoting the

4/19 (21) 23/70 (32.8)

12/18(67) 4/16 (25) 10/15 (67) 14/21 (67) 11/13 (84.6) 2/2 (100) 14/18 (77.8)

=

Most common type detected

HPV 16 HPV 16 HPV 16 HPV 16 HPV 6/16 HPV 16 HPV 16 HPV 16 HPV 16 HPV 16/18 HPV 16 HPV 16

HPV 16 HPV ns

Southern blot hybridization; DBH

=

dot

effect of HPV infection in the pathogenesis of anal cancer.19 Although the number of cases is small, an unidentified, sexually transmitted, oncogenic, or opportunistic HPV type may be operative in such patient populations. DNA sequencing and restriction analysis of these unclassified types as well as carefully controlled studies will be necessary to clarify this point. Little is known about the prevalence of HPV in normal anal tissues. Although hemorrhoids do not represent normal tissues, the study of such a control group gives some indication about the prevalence of papillomaviruses in nonmalignant anal tissue. The finding of HPV DNA by PCR analysis in 16.7% of control tissues is interesting. Histologic review of HPV-positive patients revealed foci of moderate and severe dysplasia in two cases and unremarkable histology in the third. No significant histologic changes were seen in the remaining cases. These findings suggest that infection of anorectal mucosal cells with papillomavirus may predispose to dysplasia and subsequent carcinoma. They also demonstrate that careful histopathologic examination of all hemorrhoid specimens for evidence of dysplasia is certainly warranted. HPV RNA was detected in seven of nine neoplasms examined by ISH analysis in a recent study of 19 cases of intraepithelial neoplasia arising in hemorrhoidal tissue.47 In summary, our study strongly links HPV infection with anal squamous cell carcinoma. Recent studies suggest that interaction of the E6 and E7 HPV products with cellular tumor suppressor gene products, such as the pRB and p53, may be one mechanism by which papillomavirus induces malignancy.48i50 However, factors other than the simple presence of a putatively oncogenic

1354

Zaki et al

AJPJune 1992, Vol. 140, No. 6

virus probably affect anal carcinogenesis. Epidemiologic evidence strongly suggests that other carcinogenic cofactors, such as smoking and alcohol, play an important role in the development of anal cancer. Several different approaches will clearly be necessary to elucidate these associations and the role of HPV in anogenital disease.

15.

Acknowledgments

16.

The authors thank Carol Young and Ann Alvarez for manuscript preparation, and Billie Swisher for the cutting of the histological sections used in this study.

17.

18.

References 1. Pfister H: Biology and biochemistry of papillomaviruses. Rev Physiol Biochem Pharmacol 1984, 99:111-181 2. Zur Hausen H: The role of viruses in human tumors. Adv Cancer Res 1980, 33:77-107 3. Zur Hausen H: Human papillomaviruses and their possible role in squamous cell carcinomas. Curr Top Microbiol 1977, 78:1-30 4. Pfister H: Human papillomaviruses and genital cancer. Adv Cancer Res 1987, 48:113-147 5. Bonfiglio TA, Stoler MH: Human papillomavirus and cancer of the uterine cervix. Hum Pathol 1988, 19:621-622 6. Howley PM: On human papillomaviruses (editorial). N Engl J Med 1986, 315:1089-1090 7. Lutzner MA: Papillomaviruses and neoplasia in man. New Concepts in Neoplasias as Applied to Diagnostic Pathology. Edited by Fenoglio-Preiser CM, Weinstein RS, Kaufman N. Baltimore, Williams and Wilkins, 1986, pp 126-170 8. DeVilliers EM: Heterogeneity of the human papillomavirus group. J Virol 1989, 63:4898-4903 9. Zur Hausen H, Schneider A: The role of papillomaviruses in human anogenital cancer. The Papillomaviruses. Edited by Howley PM, Salzman NP. New York, Plenum Press, 1987, pp 245-263 10. Grissmann L, Wolnick L, Ikenberg H, Koldovosky U, Schnurch HG, Zur Hausen H: Human papillomavirus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancers. Proc Natl Acad Sci USA 1983, 80:560-563 11. Wilezynski SP, Walker J, Liao S-Y, Bergen S, Berman M: Adenocarcinoma of the cervix associated with human papillomavirus. Cancer 1988, 62:1331-1336 12. Lorincz AT, Lancaster WD, Temple GF: Cloning and characterization of the DNA of a new human papillomavirus from a woman with dysplasia of the cervix. J Virol 1986, 58:225229 13. Durst M, Gissmann L, lkenberg H, zur Hausen H: A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc Natl Acad Sci USA 1983, 80:3812-3815 14. Beaudenon S, Kreinsdorf D, Croissant 0, Jablonska S,

19.

20.

21.

22.

23. 24.

25.

26.

27.

28.

29.

Wain-Hobson S, Orth G: A novel type of human papillomavirus associated with genital neoplasias. Nature 1986, 321:246-249 Reid R, Greenberg M, Jenson AB, Husain M, Willett J, Daoud Y, Temple G, Stanhope CR, Sherman Al, Phibbs GD, Lorincz AT. Sexually transmitted papillomavirus infections. I. The anatomic distribution and pathologic grade of neoplastic lesions associated with different viral types. Am J Obstet Gynecol 1987,156:212-222 Peters RK, Mack TM: Pattems of anal carcinoma by gender and marital status in Los Angeles County. Br J Cancer 1983, 48:629-636 Peters RK, Mack TM, Bernstein L: Parallels in the epidemiology of selected anogenital carcinomas. J Natl Cancer Inst 1984, 72:609-615 Holly EA, Whittemore AS, Aston DA, Ahn DK, Nickoloff BJ, Kristiansen JJ: Anal cancer incidence: genital warts, anal fissure or fistule, hemorrhoids, and smoking. J Natl Cancer Inst 1989,1:1726-1731 Palefsky JM, Gonzales J, Greenblatt RM, Ahn DK, Hollander H: Anal intraepithelial neoplasia and anal papillomavirus infection among homosexual males with group IV HIV disease. JAMA 1990, 21:2911-2916 Daling JR, Weiss NS, Hislop TG, Maden CM, Coates RJ, Sherman KJ, Ashley RL, Beagrie M, Ryan JA, Corey L: Sexual practices, sexually transmitted diseases, and the incidence of anal cancer. N EngI J Med 1987, 317:973-977 Austin, DF: Etiological clues from descriptive epidemiology: squamous carcinoma of the rectum or anus. Natl Cancer Inst Monogr 1982, 62:89-90 Scheurlen W, Stremlau A, Gissman L, HoIn D, Zenner HP, zur Hausen H: Rearranged HPV 16 molecules in an anal and in a laryngeal carcinoma. Int J Cancer 1986, 38:671676 Hill SA, Coghill SB: Human papillomavirus in squamous cell carcinoma of the anus. Lancet 1986, 2:1333 Palmer JG, Shepherd NA, Jass JR, Crawford LV, Northover JMA: Human papillomavirus type 16 DNA in anal squamous cell carcinoma. Lancet 1987, 2:42 Gal AA, Meyer PR, Taylor CR: Papillomavirus antigens in anorectal condyloma and carcinoma in homosexual men. JAMA 1987, 257:337-340 Wells M, Griffiths S, Lewis F, Bird CC: Demonstration of human papillomavirus types in paraffin processed tissue from human anogenital lessions by in situ DNA hybridization. J Pathol 1987, 152:77-82 Yun K, Molenaar AJ, Wilkins RJ: Detection of human papillomavirus DNA in anogenital exophytic lesions by in situ hybridization to paraffin sections. Acta Pathol Jpn 1988, 38:1131-1139 Duggan MA, Boras VF, Inoue M, McGregor SE, Robertson Dl: Human papillomavirus DNA determination of anal condylomata, dysplasias, and squamous carcinomas with in situ hybridization. Am J Clin Pathol 1989, 92:16-21 Beckmann AM, Daling JR, Sherman KJ, Maden C, Miller BA, Coates RJ, Kiviat NB, Myerson D, Weiss NS, Hislop TG, Beagrie M, and McDougall JK: Human papillomavirus infection and anal cancer. Int J Cancer 1989, 43:1042-1049

HPV and Anal Carcinoma

1355

AJPJune 1992, Vol. 140, No. 6

30. Gal AA, Saul SH, Stoler MH: In situ hybridization analysis of human papillomavirus in anal squamous cell carcinoma. Mod Pathol 1989, 2:439-443 31. Taxy JB, Gupta PK, Gupta JW, Shah KV: Anal cancer: Microscopic condyloma and tissue demonstration of human papillomavirus capsid antigen and viral DNA. Arch Pathol Lab Med 1989,113:1127-1131 32. Wolber R, Dupuis B, Thiyagaratnam P, Owen D: Anal cloacogenic and squamous carcinomas: Comparative histologic analysis using in situ hybridization for human papillomavirus DNA. Am J Surg Pathol 1990,14:176-182 33. Koulos J, Symmans F, Chumas J, Nuovo G: Human papillomavirus detection in adenocarcinoma of the anus. Mod Pathol 1991, 4:58-61 34. Palefsky JM, Holly EA, Gonzales J, Berlinc J, Ahn DK, Greenspan JS: Detection of human papillomavirus in anal intraepithelial neoplasia and anal cancer. Cancer Res 1991, 51:1014-1019 35. Saiki RK, Geflund DH, Stoffel S, Scharl SJ, Higuchi R, Horn GT, Mullis KB, Ehrlich HA: Primer directed amplification of DNA with a thermostable DNA polymerase. Science 1988, 239:487-491 36. Manos MM, Ting Y, Wright DK, Lewis AJ, Broker TR, Wolinsky SM: Use of the polymerase chain reaction amplification for the detection of genital human papillomaviruses. Cancer Cells 1989, 7:209-214 37. Gregoire L, Arella M, Campione-Piccardo J, Lancaster WD: Amplification of human papillomavirus DNA sequence by using consensus primers. J Clin Microbiol 1989, 27:26602665 38. Snijders PJF, Van Der Brule AJC, Shrijnemakers HFJ, Snow G, Meijer CJLM, Walboomers JMM: The use of general primers in the polymerase chain reaction permits the detection of a broad spectrum of human papillomavirus genotypes. J Gen Virol 1990, 71:173-181 39. Van Den Brule AJC, Snijders PJF, Gordijn RLJ, Bleker OP, Meijer CJLM, Walboomers JMM: General primer-mediated polymerase chain reaction permits the detection of sequenced and still unsequenced human papillomavirus genotypes in cervical scrapes and carcinomas. Int J Cancer 1990, 45:644-649

40. Ferenczy A, Winkler B: Cervical intraepithelial neoplasia and condyloma. Blaustein's Pathology of the Female Genital Tract. Edited by Kurman RJ. New York, Springer-Verlag, 1987, pp 177-217 41. Schwarz E, Freese UK, Gissmann L, Mayer W, Rooggenbuck G, Stremlau A, zur Hausen H: Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 1985, 314:111-114 42. Yee C, Krishnan-Hewlett I, Baker CC, Schiegel R, Howley PM: Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines. Am J Pathol 1985, 119:361-366 43. Judd R, Zaki SR, Coffield LM, Evatt BL: Sinonasal papillomas and human papillomavirus: HPV 11 detected in fungiform schneiderian papillomas by in situ hybridization and the polymerase chain reaction. Hum Pathol 1991, 22:550556 44. Caruthers MH, Baron AD, Beaucage SL, Dodds DR, Fisher EF, McBride W, Matteucci M, Stabinsky Z, Tany JY: Chemical synthesis of deoxyoligonucleotides by the phosphoraramidite method. Methods Enzymol 1987, 154:287-313 45. Ting Y, Manos MM: Detection and typing of genital human papillomaviruses. PCR Protocols, a Guide to Methods and Applications. Edited by Innis MA, Gelfand DH, Sninsky JJ, White TJ. San Diego, Academic Press, Inc., 1990, pp 356367 46. Kwok S, Higuchi R: Avoiding false positives with PCR. Nature 1989, 339:237-238 47. Foust RL, Dean PJ, Stoler MH, Moinuddin SM: Intraepithelial neoplasia of the anal canal in hemorrhoidal tissue. Hum Pathol 1991, 22:528-534 48. Dyson N, Howley PM, Munger K, Harlow E: The human papillomavirus - 16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 1989, 243:934-937 49. Munger K, Werness BA, Dyson N, Phelps WC, Harlow E, Howley PM: Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J 1989, 8:4099-4105 50. Werness BA, Levine AJ, Howley PM: Association of human papillomaviorus type 16 and 18 E6 proteins with p53. Science 1990, 248:76-79