Stina Syrjänen

Detection of human papillomavirus (HPV) DNA in oral squamous cell carcinomas by in situ hybridization and polymerase chain reaction.

SummaryA series of 156 formalin-fixed, paraffin-embedded biopsies from 40 patients with surgically-treated oral squamous cell carcinomas was analysed for the presence of human papillomavirus (HPV) infection by histopathological evaluation, in situ DNA hybridization and polymerase chain reaction (PCR). Epithelial changes suggesting a HPV lesion within, or adjacent to, the carcinoma lesions were found in 16 out of 40 patients (40%). Morphological signs of a flat HPV lesion were found in four cases (10%), those of inverted type in three cases (7.5%), and those of papillary type in nine cases (22.5%). HPV DNA was demonstrated in one of the lesions by in situ hybridization with biotin-labelled DNA cocktail probe containing HPV types 6, 11, 16 and 18. With the PCR technique, samples from 11 (27.5%) of the 40 patients proved to contain HPV DNA. Of these, HPV 6 was demonstrated in one case, HPV 16 in ten cases and HPV 18 in one case. HPV DNA was exclusively detected in the biopsies showing carcinoma tissue or its adjacent precancer lesions. No viral DNA was found in the biopsies derived from the tumour-free resection margins. These results provide further evidence to support the concept of HPV involvement in the aetiology of oral squamous cell carcinomas, most probably acting synergistically with other carcinogens.

Factors associated with progression of cervical human papillomavirus (HPV) infections into carcinoma in situ during a long‐term prospective follow‐up

Summary. In the course of a prospective study of 508 women with papillomavirus (HPV) lesions of the uterine cervix, 66 lesions that progressed into carcinoma in situ (CIS) were identified and treated by conization during a mean follow‐up period of 35 months. The lesions were investigated with light microscopy and with in‐situ DNA hybridization using 35S‐labelled probes for HPV 6,11,16,18, 31 and 33. After radical cone treatment, 11 of the 66 women (16‐7%) have presented with a recurrent HPV infection. The recurrence rate increased with the duration of the follow‐up period from <10% at the mean follow‐up of 25 months to 16.7% at the most recent follow‐up at 35 months. Most of these 66 HPV lesions (89%) presented with concomitant CIN in the first punch biopsy, but it is noteworthy that the other 11 % presented without concomitant CIN. HPV DNA of at least one of the six types examined was found in 73% of the first biopsies and it is noteworthy that the so‐called ‘low‐risk’ types, HPV 6 and 11, were found as frequently as the ‘high‐risk’ types, HPV 16 and 18 (18% and 17%, respectively). This would suggest a similarity in the biological behaviour of these two HPV groups. Although the concept of the ‘high‐risk’ and ‘low‐risk’ HPV types may remain at least partially valid, it is imperative to realize that infection by HPV 6 and 11 by no means excludes the possibility for clinical progession into CIS and eventually to an invasive carcinoma.

Human Papilloma Virus (HPV) Antigens in Lesions of Laryngeal Squamous Cell Carcinomas

Paraffin sections from 36 laryngeal squamous cell carcinomas with histological features of coexistent condylomatous changes (the flat, inverted and papillomatous lesions caused by human papilloma virus; HPV) were stained for HPV antigens with an indirect immunoperoxidase technique. The HPV antiserum used was prepared in guinea pigs immunized against the purified HPV virions from human common wart tissue. Of the tumors studied, 36% showed the presence of HPV antigens. In that, one-third of the flat and inverted lesions and 42% of the papillomatous lesions contained cells with intranuclear HPV-staining reaction. The role of HPV as the etiologic agent of laryngeal squamous cell papillomas is discussed, as is the potential malignant transformation of them. Based on the present results, the previously expressed view on the probable role of HPV in the development of laryngeal squamous cell carcinoma seems to gain further substantiation.

Detection of human papillomavirus DNA in anogenital condylomata in men using in situ DNA hybridisation applied to paraffin sections.

An in situ DNA hybridisation method was used to detect human papillomavirus (HPV) DNA (HPV types 6, 11, 16, and 18), and an immunoperoxidase (IP-PAP) method to detect HPV structural protein expression in paraffin sections of biopsy specimens from 133 men treated for penile (in 114 cases) and anal (in 19 cases) warts. The anatomical distribution on the penis of classic condyloma acuminatum and of papular and flat condylomata was practically identical. The gross appearance of the warts did not correlate with their morphology on light microscopy. The detection rate of dysplasia was very different in the three types of lesions (25% in flat, 50% in acuminatum, and 75% in papular warts). Of 133 lesions, 59 (44.4%) contained HPV antigens, their expression being inversely related to the grade of dysplasia; only 17% of HPV 16 lesions had detectable HPV antigen compared with 50% to 67% in lesions of the other three HPV types. HPV 16 and HPV 18 DNA were most commonly (11%) detectable in Bowenoid lesions; however, most of the HPV 16 and 18 positive cases were found among the flat and acuminatum type of lesions. Though the overall detection rate of HPV DNA (76%) did not correlate with the grade of dysplasia, a clear cut association of HPV 16 and HPV 18 with dysplastic lesions was found, none of the HPV 16 and 25% of the HPV 18 positive cases being devoid of concomitant dysplasia. The corresponding figures for HPV 6 and HPV 11 were 59.2% and 68.8%, respectively. The implications of these findings are discussed in terms of epidemiologically established connections between penile and cervical cancer, with special emphasis of the high risk HPV types 16 and 18. The applicability of in situ DNA hybridisation as a powerful tool in the analysis of specific HPV DNA sequences in routinely processed biopsy specimens from these lesions is emphasised.

Cationic liposomes mediated delivery of antisense oligonucleotides targeted to HPV 16 E7 mRNA in CaSki cells.

The high risk types 16 and 18 of human papillomavirus (HPV) are involved in the etiology of genital squamous cell carcinoma. The early genes 6 and 7 (E6-E7) of these viruses code for the major transforming proteins, capable of inducing cell transformation alone or acting synergistically with other oncogenes. Antisense oligonucleotides, recently applied to inhibit the functions of a number of cellular and viral proteins, might provide the basis for a new therapeutic strategy against HPV-induced malignancies. We studied the proliferation of CaSki cells by the MTT assay after their exposure to HPV 16 E7 mRNA antisense oligonucleotides with and without cationic liposomes (containing dimethyldioctadecylammonium bromide DDAB, and dioleylphosphatidylethanolamine, DOPE). Unmodified oligonucleotides (either 12- or 23-mers) did not have any effect on either CaSki cell proliferation or morphology when compared with the untreated cells. The cellular uptake of oligonucleotides was significantly enhanced by the cationic liposomes as assessed by confocal laser scanning microscopy (CLSM). The cationic liposomes were toxic to the cells as demonstrated by the reduced cell number and altered cell morphology. Only a slight reduction of the cell proliferation was seen when antisense 12-mer was protected from its 3- and 5-ends with thiolate and FITC, respectively. Both the 12- and the 23-mers with the cationic liposomes inhibited cell proliferation, the inhibitory effect being longer with the 23-mer. Overall, the MTT assay was less sensitive than light microscopy to reveal the toxic effects on CaSki cells. The results suggest that antisense oligonucleotides targeted to HPV 16 E7 mRNA can be introduced into CaSki cells with cationic liposomes.

The acetic acid test in evaluation of subclinical genital papillomavirus infection: a comparative study on penoscopy, histopathology, virology and scanning electron microscopy findings.

OBJECTIVES--To evaluate colposcopic criteria in acetowhite lesions of the penis (penoscopy) for the diagnosis of subclinical genitoanal papillomavirus infection (GPVI) compared with histopathological criteria of HPV involvement and to various hybridisation assays for HPV DNA detection, and to depict typical lesions by scanning electron microscopy. DESIGN--The study included 101 randomly selected male partners of females with known GPVI, or with penile symptoms such as itching, burning and dyspareunia who did not exhibit overt genital warts but appeared to be afflicted with acetowhite penile lesions after topical application of 5% acqueous acetic acid. Lesions were judged by penoscopy as either typical, conspicuous or nontypical for underlying HPV infection. Biopsy specimens from 91 men were examined by light microscopy and by either Southern blot (SB), polymerase chain reaction (PCR) and/or in situ hybridisation (ISH) assays for the presence of HPV DNA of the HPV types 6, 11, 16, 18, 31, 33 and 42 (Group A). From another ten men lesions clinically typical for GPVI were also examined topographically by scanning electronic microscopy (Group B). SETTING--The STD out-patient clinic of the Department of Dermatovenereology of Karolinska Hospital, Stockholm, Sweden. RESULTS--Group A Seventy eight (86%) of the biopsied lesions met the penoscopy criteria of being either typical of or conspicuous for GVPI. The agreement between penoscopy and histopathology was fairly good, as HPV diagnosis was made by both methods in 56 (62%) of the cases. The reliability of applying strict colposcopic hallmarks was further substantiated by the finding that 55 (60%) of the biopsy specimens taken from penoscopically typical/conspicuous lesions contained HPV DNA. However, there are diagnostic pitfalls for the acetic acid test. Coexistence of an eczematoid reaction with changes indicative of HPV influence was detected in six (7%) of the cases, while an inflammatory response only occurred in 17 (19%) of the specimens. Additional histopathological diagnoses (normal epithelium, lichen sclerosus et atrophicus, balanitis circinata parakeratotica, verruca plana) were established in another eight (9%) of the cases. Among the HPV DNA positive cases, all of the HPV types tested for were detected with the exception of HPV 18. A severe penile intraepithelial neoplasia (PIN III) was revealed in five (5%) of biopsies; HPV 16 was present in two and HPV 42 in one of these biopsy specimens. GROUP B--Scanning electron microscopy depiction harmonised with the penoscopy findings showing that subclinical GPVI characteristically exhibits a well demarcated, slightly elevated border and that the central area of lesions often displays a groove in which the epithelium appears to be thin with protrusions from beneath that probably represent capillaries. CONCLUSION--Use of the acetic acid test for evaluation of GPVI should be combined with a colposcopic evaluation based on strict topographic hallmarks, followed by a directed biopsy for light microscopic evaluation. We found that the positive predictive value of colposcopy was as high when correlated with histopathological findings (72%) as when virological methods were used, whether HPV DNA hybridisation testing was performed with the well established SB and ISH assays (45%), or by applying the newly introduced and highly sensitive PCR assay as well (71%). False positivity from the acetic acid test occurs and is mainly due to inflammatory conditions but also to the presence of other conditions. Epithelial fissures are evidently associated with some subclinical GPVI lesions and may potentially represent loci minores for infectious stimuli and perhaps facilitate the transmission of some blood-borne STDs. We prose that the term papillomavirus balanoposthitis should be used for penile HPV infection associated with inflammatory responses. Our study indicates that PIN III frequently occurs in a subclinical form and may be associated with not only previously identified high-risk HPV types such as type 16, but also with the HPV type 42 that has not previously been considered as oncogenic.

Anal condylomas in men. 1. Histopathological and virological assessment.

A series of 128 biopsy specimens from anal condylomas in 73 homosexual or bisexual and 38 heterosexual men (mean (SD) age 31.8 (9.6) years) were subjected to histological assessment and human papillomavirus (HPV) typing by in situ DNA hybridisation with 35S-labelled HPV 6, 11, 16, 18, 31, and 33 probes. Most patients were also tested serologically for antibodies to human immunodeficiency virus (HIV). As evaluated on light microscopy, most (74%, 95/128) of the lesions were exophytic (papillary) acuminate warts, 15% (19) were flat, and 11% (14) were pigmented papulous lesions. No signs of anal intraepithelial neoplasia (AIN) were seen in 70% (90) of the 128 biopsy specimens (NAIN), 27% (35) were classified as showing AIN I, and another 2% (three) as AIN II. AIN was significantly (p less than 0.05) more often associated with papulous lesions, only 43% (6/14) of which showed NAIN compared with 72% (68/98) of acuminate condylomas. The duration of disease was directly related to the presence and severity of AIN in the lesions; thus in 47 lesions that had been present for more than 12 months, NAIN was found in 31 (66%), AIN I in 14 (30%), and AIN II in two (4%). HPV DNA of at least one of the six types tested for was detected in 109/125 (87%) lesions. HPV 6 and HPV 11 were the two most common types, comprising 57% (62) and 37% (40), respectively, of the 109 HPV DNA positive cases. Only seven (6%) biopsy specimens were associated with any of HPV types 16, 18, 31, or 33, which carry a high risk of potential malignant transformation. No association was found between sexual preferences of patients and the incidence of any of the various HPV types. Neither did the distribution of the various HPV types differ between men with antibody to HIV and those without antibody. All the men with antibody to HIV were homosexual or bisexual. On microscopy, 93% (38) of 41 lesions containing HPV 11 and 75% (48/64) of HPV 6 lesions were of the acuminate wart type; in comparison, the remaining 16 HPV 6 lesions were equally either flat or papulous (eight, 13% each). Of the 64 HPV 6 and 41 HPV 11 associated lesions, 73% (47) and 63% (26), respectively, were classified as NAIN. Only two lesions were associated with HPV 16, and both showed mild dysplasia. On the other hand, two HPV 6 induced lesions were associated with AIN II. No differences were found between HPV 6 and HPV 11 in duration of disease; (39%, and 27% respectively, had been present for more than 12 months). The results showed that overt anal wart disease was associated with HPV types 6 and 11 in most cases. Although HPV types considered as being of higher oncogenic potential were detected relatively rarely, the associated AIN in a relatively high proportion (31% 32/105) of HPV 6 or 11 induced lesions indicated that a malignant potential, even for HPV 11 associated anal warts, cannot be excluded.

Genital human papillomavirus lesions of the male sexual partners: the diagnostic accuracy of peniscopy.

OBJECTIVES--To evaluate the accuracy of peniscopy for identifying human papillomavirus (HPV) lesions in male sexual partners of women with HPV infection. The predictive value of the medical history for HPV infection was also evaluated. DESIGN--Examination of voluntary male partners of the women with HPV infection using colposcopy (peniscopy after acetic acid), cytology and surgical biopsy, the latter being analysed by light microscopy, in situ hybridisation (ISH) and polymerase chain reaction (PCR) for HPV DNA. A detailed medical history was to be taken, too. SETTING--Department of Gynaecology and Obstetrics, Kuopio University Central Hospital, Finland. SUBJECTS--A series of 101 voluntary male partners of 101 women invited for examination, treatment and follow-up for their genital HPV infections on the basis of abnormal Papanicolaou (PAP) smears. RESULTS--On peniscopy 64 (63.4%) of the men presented with lesions either typical of (34.7%) or suspicious for (28.7%) HPV infection. Of the latter, 89% were flat lesions mostly undetectable by the naked eye. The cytologic smear was positive in only nine men. On light microscope, 85.7% of the peniscopically typical lesions were found to be consistent with (68.6%) or suspicious for (17.1%) HPV infection. HPV DNA was found in 33 (34.5%) of the 96 typed biopsies, and never in biopsies from peniscopically healthy areas. In logistic regression analysis of the historical data recorded, only the contact time with the current sexual partner was of predictive value for histologically proven HPV infection. CONCLUSIONS--Peniscopy is an applicable means for the identification of penile lesions due to HPV infection, but it is not a conclusive diagnostic tool capable of differentiating HPV from non-HPV findings.

Diagnosis of genital human papillomavirus (HPV) lesions in the male: correlation of peniscopy, histology and in situ hybridisation.

OBJECTIVE--To assess the diagnostic criteria of genital HPV lesions in male sexual partners of HPV infected women. METHODS--Peniscopically directed biopsy specimens (from 693 lesions in 300 men) were examined on light microscopy and in situ hybridisation (ISH) for HPV types 6,11,16,18,31,33 and 42. The predictive value of different histological criteria for ISH positivity was also evaluated using stepwise logistic regression analysis. RESULTS--Flat HPV lesions were most accurately predicted by the punctuation pattern on peniscopy, giving the concordance between peniscopy and histology between peniscopy and histology of 79.5% (66/83) and that between peniscopy and ISH of 56.6% (47/83). Diffuse acetowhite pattern disclosed a typical HPV lesion in only 17.8% (13/73), and HPV DNA was found in 11.0% (8/73) of cases. Of the 114 biopsy specimens from peniscopically healthy areas adjacent (0.5-1 cm) to the lesions, 93.0% (106/114) were normal on light microscopy, and HPV DNA was found in only 2.6%. Penile intraepithelial neoplasia (PIN) lesions were most frequently ISH positive, 81.1% (30/37), 50% showing HPV 16 and/or 18 DNA. Lesions classified as HPV-suspicious or nonspecific on light microscopy were HPV DNA-positive in 16.9% (11/65) and 8.1% (13/160), the frequency of high-risk HPV types being 3.1% and 1.3%, respectively. In logistic regression analysis, koilocytosis was the most powerful predictor of ISH-positivity in the flat lesions (without PIN), the risk ratio being 3.7. CONCLUSION--No conclusive peniscopic criteria for male HPV infections could be established, making histological evaluation mandatory. Care should be exercised in interpreting as HPV lesions the cases devoid of koilocytosis, HPV typing being essential in confirming the diagnosis in doubtful cases.

Prevalence of genital human papillomavirus infections in a mass-screened Finnish female population aged 20-65 years.

The results of the nationwide, population-based cervical cancer screening programme (organized by the Finnish Cancer Society since early 1960s) were analysed to establish the prevalence figures (and their changes) for genital human papillomavirus (HPV) infections in an unselected Finnish female population (aged between 20 and 65 years) screened in Kuopio Province between 1981 and 1989. During the study period 82,393 women were invited on a regular basis for the mass-screening, and also 4131 women in a risk group. Of these, a total of 63,115 and 3249 women attended, resulting in the attendance rates of 76.6% and 78.6%, respectively. As a result of the screening, a total of 509 (0.80%) of the 63,115 smears were diagnosed as having the cytological changes consistent with HPV infection in the mass screening. The corresponding figures in the risk group screening were 58/3249 (1.78%). There was a sharply increasing trend in the prevalence of genital HPV infections from 1981 through 1987, from 0.04% to 1.76% (ie a 44-fold increase in 7 years) which, surprisingly, then declined to 1.43% in 1988 and 1.04% in 1989. Based on a random sample of 2084 routine (non-mass-screening) Pap smears (out of 28,861) collected from the files of our laboratory, the prevalence of HPV infections was stratified by age groups. The highest prevalence (6.1%) was observed in women aged between 20 and 29 years, followed by 2.2% in those aged 30–39 years. Using the figures of the relative risk (RR) of HPV infections by age, an estimation was made to assess the prevalence of clinical HPV infections in the Finnish female population in general. With the annual number of female births (approximately 30,000), it was estimated that some 18,000 women aged between 20 and 29 years will harbour a clinically manifest genital HPV infection. It is noteworthy that the above established figures do not include any data of the subclinical and latent HPV infections, not detectable by the Pap smear. Beyond any doubt, genital HPV infections are the most frequent STD in Finland.