Relationship of human papillomavirus to ploidy in squamous cell carcinomas of the head and neck.

To establish the relationship between the presence of human papillomavirus (HPV) gene sequences and the development of genetic abnormalities, 31 squamous cell carcinomas of the head and neck were studied for the presence of HPV types 6b and 16 and the DNA content by flow cytometry. Eighteen (58%) cases were aneuploid. HPV DNA was present in seven (22.5%) tumors. Five of them were positive for the HPV type 6b and two for the HPV type 16. Aneuploidy was correlated with poorly differentiated tumors. No correlation was found between the presence of HPV, DNA content, or tumor differentiation. Consequently, the presence of HPV gene sequences does not seem to be related to a higher incidence of genetic abnormalities in squamous cell carcinomas of the head and neck.     

DNA ploidy in papillary tumours of the breast

The DNA content of benign and malignant papillary tumours of the breast as well as a group of papillary tumours where this distinction could not be made with certainty was measured by flow cytometry. All the benign papillomas were diploid; 5 of 19 carcinomas were aneuploid; and 1 of 15 tumours of undecided type was also aneuploid. Comparison of the DNA indices, proliferation indices and coefficients of variation were of no value in distinguishing between benign and malignant specimens.     

Human papillomavirus infection and anogenital condyloma in bone marrow transplant recipients

BACKGROUND: Secondary malignant diseases are late complications after allogeneic bone marrow transplantation (BMT). Anogenital lesions associated with human papillomavirus (HPV) infection have been described in renal transplant recipients but not after BMT. HPV types 16 and 18 are strongly linked to the malignant transformation. METHODS: In a series of 238 patients with allogeneic BMT, three had anogenital lesions. We looked for HPV in DNA extracted from embedded tissue to study HPV genotypes, p53 expression, and ploidy. RESULTS: In two patients, HPV sequences were detected. One of them, with giant condyloma, had HPV type 18 and two aneuploid clones, but p53 expression was not found. CONCLUSION: As in solid organ transplant recipients, anogenital condyloma may develop after BMT. Because the oncoprotein of HPV is able to bind and to degrade p53, it may lead to genetic instability, and subsequently to malignant transformation.     

Presence of human papillomavirus DNA and abnormal p53 protein accumulation in lung carcinoma.

BACKGROUND: In some carcinomas inactivation of the tumour suppressor gene product p53, either by point mutation or indirectly by the human papillomavirus (HPV), has been suggested as two alternative routes to malignant transformation. To test this hypothesis in lung tumours, 43 lung carcinomas were analysed by in situ hybridisation and polymerase chain reaction (PCR) for the presence of HPV DNA, and the results were compared with p53 protein immunohistochemical analysis. METHODS: The presence of HPV DNA in lung carcinoma was detected by nucleic acid in situ hybridisation for HPV types 6, 11, 16, 18, 31, and 33 using nonradioactively labelled DNA probes. Polymerase chain reaction (PCR) analysis was performed on all cases showing positive HPV DNA labelling by in situ hybridisation and in an additional 13 negative cases. Abnormal nuclear accumulation of the p53 protein was revealed by immunohistochemistry using the avidin-biotin-peroxidase complex method and a CM-1 polyclonal anti-human p53 antibody and a monoclonal mutation-specific Pab 240 p53 antibody. RESULTS: HPV DNA was found by in situ hybridisation in 13 lung carcinomas (30%). In all these cases subtype-specific HPV DNA could also be detected by PCR. Abnormal p53 protein accumulation was seen in 21 of the 43 carcinomas (49%), of which 18 were HPV negative. Twelve (57%) of the CM-1 positive cases were also positive for the mutation-specific antibody Pab 240. There was an obvious inverse relationship between the presence of papilloma viral DNA and abnormal p53 protein accumulation. CONCLUSIONS: p53 plays an important part in the development of lung carcinomas and, in some cases, HPV may contribute to it by binding and inactivating the p53 protein.     

Expression of p53 protein related to human papillomavirus and DNA ploidy in superficial esophageal carcinoma

We examined the p53 protein and human papilloma virus (HPV) by immunohistochemistry and DNA ploidy by cytofluorometry in paraffin-embedded esophageal carcinoma tissue specimens. Sixty-one patients with superficial esophageal carcinoma were operated on between 1983 and 1991 without any prior treatment. Immunostaining of the anti-p53 protein antibody (CM1) was positive in 32 carcinomas (52%). Patients with p53-positive tumors had a poorer outcome than those with p53-negative tumors (P<0.05). In addition, patients with p53-positive tumors did not have any characteristic site of relapse. Only 5 of the 61 patients (8.2%) had HPV-positive tumors. One of these 5 carcinomas expressed both p53 protein and HPV. Three patients with HPV-positive tumors which had invaded the submucosal layer died of relapse. A determination of DNA ploidy revealed 30 patients with aneuploid tumors, 13 with polyploid tumors and 18 with diploid tumors. The outcome of the patients with aneuploid tumors was worse than that of the patients with diploid tumor (P<0.05). p53 protein expression was not associated with DNA ploidy; however, the 16 patients who had both p53-positive and aneuploid tumors had a worse prognosis than patients with p53-negative and aneuploid tumors (P<0.01). These findings suggest that p53 protein expression in conjunction with DNA ploidy may be a useful indicator in evaluating the prognosis of patients with superficial esophageal carcinoma.     

Detection of human papillomavirus in small cell carcinomas of the anus and rectum

Small cell carcinomas represent <1% of colorectal/anal carcinomas and have a poor prognosis. Anorectal squamous cell carcinomas are often associated with high-risk human papillomavirus (HPV) infection, similar to squamous and small cell carcinomas of the uterine cervix. In HPV infection, the oncoprotein E7 inactivates the tumor suppressor Rb, leading to p16 upregulation; however, in small cell carcinomas, the Rb pathway is often blocked by other mechanisms; thus, increased p16 may not indicate HPV infection. P16 immunohistochemistry (IHC) may have a limited role in evaluating small cell carcinomas for HPV infection. Formalin-fixed, paraffin-embedded tissue sections of previously diagnosed small cell carcinomas of the anus (n=5) and rectum (n=11) were subjected to IHC for p16, CDX2, and p63, followed by in situ hybridization for high-risk HPV. All (100%) cases of anal and rectal small cell carcinomas were positive for p16, and 100% of anal and 82% of rectal small cell carcinomas were positive for high-risk HPV. The majority of cases showed low or very low HPV copy numbers. In 6 cases, HPV was detected only by using the HPV-16 genotype-specific assay detecting very low copy numbers (1 to 2 viral copies). The majority of tumors expressed p63, which was more pronounced in the anal tumors. CDX2 expression was observed predominantly in rectal tumors. High-risk HPV can be detected using in situ hybridization in the majority of anorectal small cell carcinomas, which are uniformly p16 positive by IHC. HPV-targeted therapy could result in better control of these aggressive neoplasms.     

Detection of human papillomavirus in formalin-fixed, invasive squamous carcinomas using the polymerase chain reaction

We analyzed 88 formalin-fixed, paraffin-embedded invasive squamous carcinomas for human papillomavirus-related DNA sequences (HPV types 16 and 18) following in vitro gene amplification using the polymerase chain reaction. HPV DNA sequences were found in 35 of 50 (70%) carcinomas of the anogenital region, including four of four (100%) anal, six of eight (75%) vulvar, nine of 14 (64%) vaginal, two of five (40%) penile, and 14 of 19 (74%) cervical tumors. Nine of 25 (36%) oropharyngeal squamous carcinomas contained HPV DNA sequences, including four of 10 (40%) laryngeal, three of eight (38%) buccal, and two of seven (29%) glossal tumors. HPV DNA sequences were not found in 13 esophageal carcinomas. Of the 44 cases that contained viral DNA, HPV-16 was detected in 41 cases (93%) and HPV-18 in five cases (11%), while both types were found in two cases (one anal and one vulvar). HPV DNA sequences were found in 43 of 83 (52%) nonverrucous and in one of five (20%) verrucous carcinomas, but this difference was not significant. These findings demonstrate that HPV DNA sequences are more frequently associated with anogenital than oropharyngeal squamous carcinomas and can be readily detected in routinely processed tissues using the polymerase chain reaction.     

Detection of human papillomavirus DNA and expression of p16, Rb, and p53 proteins in small cell carcinomas of the uterine cervix

Human papillomavirus (HPV) has been implicated as an etiologic agent for the development of primary small cell carcinoma of the uterine cervix, a rare but highly aggressive malignancy. It has been shown that the HPV E6 and E7 oncoproteins are able to inactivate the tumor suppressor functions of p53 and Rb. In squamous cell carcinoma and adenocarcinoma of the cervix, HPV infection is also associated with overexpression of p16, a cyclin-dependent kinase inhibitor. In this study, 22 cases of primary small cell carcinoma of the uterine cervix were subjected to broad-spectrum HPV DNA amplification and typing, and immunohistochemically examined for the expression of p16, Rb, and p53 proteins. The results show that HPV DNA was detected in every case (100%), with 18 cases (82%) harboring type 18. The tumor cells exhibited strong nuclear staining for p16 in 20 cases (91%). This was associated with a complete loss of Rb nuclear staining in tumor cells in 16 cases (73%). The p53 protein was essentially undetectable in all cases. In contrast, HPV DNA was not detected in 9 colorectal and 8 urinary bladder small cell carcinomas included in this study for comparison. While similar p16 and Rb expression patterns were observed in these HPV-negative tumors, a different expression pattern for p53 was noted where strong nuclear staining was seen in 8 cases (47%; P = 0.0004 compared with cervical tumors). These observations indicate that different mechanisms are involved in the pathogenesis of small cell carcinomas of the uterine cervix and support the notion that nuclear p16 overexpression serves as an indication of Rb defunctioning in tumor cells, which may or may not result from high-risk HPV infection.     

Detection of human papillomavirus DNA in cancer of the urinary bladder by in situ hybridisation.

The association of the human papillomavirus (HPV) with cancer of the urinary bladder was assessed by in situ hybridisation using probes selective for HPV types 6/11 and 16/18 DNA. No hybridisation signal was detected with the type 6/11 probe on 100 formalin-fixed, paraffin-embedded bladder tumours sampled. However, when the same samples were hybridised with the HPV type 16/18 DNA probe, 11 of 66 (16.6%) papillary and 1 of 10 (10%) solid transitional cell carcinomas gave positive signals. These results suggest the involvement of HPV in cancer of the bladder, although the frequency of multiple HPV types in these tumours is uncertain.     

Human papillomavirus (HPV)-related oropharyngeal nonkeratinizing squamous cell carcinoma: characterization of a distinct phenotype.

We have recently shown that HPV-positive tonsillar carcinoma in young patients exhibits nonkeratinizing basaloid morphology and a characteristic immunophenotype. The purpose of this study was to review a large number of cases of oropharyngeal carcinomas, in all age groups, and to identify tumors with nonkeratinizing morphology. Using polymerase chain reaction (PCR) the prevalence and type of HPV DNA was determined in representative cases and in a control group of conventional keratinizing squamous cell carcinomas. The tumors were further characterized with a panel of immunohistochemical stains. A total of 235 carcinomas were reviewed; 141 of the tonsils and 94 in the base of tongue. Ninety (36%) of the tonsillar and 30 (32%) of the base of tongue carcinomas were nonkeratinizing (NKCa) with basal cell features; the rest were classical keratinizing squamous cell carcinomas (KSCC). HPV DNA, particularly type 16, was identified in 10 (100%) of 10 of NKCA and in only 2 (20%) of 10 of KSCC (P = .0014). NKCas were strongly reactive to p16 antibodies while KSCC showed weak and focal reactivity. Higher Ki67 and lower p53 staining scores were observed in NKCa as compared to KSCC. It is concluded that NKCa of the tonsils and base of tongue is a distinct subtype of squamous cell carcinoma of the head and neck with high prevalence of HPV DNA and a characteristic immunophenotype.     

Nuclear DNA content in breast carcinoma with special reference to the parameters of malignant potentiality

Nuclear DNA ploidy pattern was examined in 22 breast carcinomas with a tumor mass of up to 2.2 cm and compared with the clinical and histological parameters of the malignant potentiality of breast carcinomas. The 22 carcinomas were divided into 11 aneuploid and 11 near-diploid carcinomas. It was found that the histological type, histological grade, nuclear grade, mitotic index and lymphatic invasion were either only slightly, or not correlated at all with the DNA ploidy pattern. Nevertheless, lymph node metastasis and negative estrogen receptor status was more frequently seen in the patients with aneuploid carcinoma than in those with near-diploid carcinoma.     

Nuclear DNA content in breast carcinoma with special reference to the parameters of malignant potentiality

Nuclear DNA ploidy pattern was examined in 22 breast carcinomas with a tumor mass of up to 2.2 cm and compared with the clinical and histological parameters of the malignant potentiality of breast carcinomas. The 22 carcinomas were divided into 11 aneuploid and 11 near-diploid carcinomas. It was found that the histological type, histological grade, nuclear grade, mitotic index and lymphatic invasion were either only slightly, or not correlated at all with the DNA ploidy pattern. Nevertheless, lymph node metastasis and negative estrogen receptor status was more frequently seen in the patients with aneuploid carcinoma than in those with near-diploid carcinoma.     

Proliferative activity assessed on the basis of DNA ploidy patterns in primary lung cancer

Flow cytometric cellular DNA-RNA content analysis by acridine orange staining was conducted on surgical fresh specimens of primary lung carcinomas from 66 patients (31 squamous cell carcinomas, 34 adenocarcinomas and 1 large cell carcinoma). The frequency of aneuploid tumors was 84.6% among the tumors. RNA content (RNA Index) in the DNA aneuploid tumor much more significantly (p less than 0.05) increased than the DNA diploid tumor. Tumor doubling time in the DNA aneuploid tumor was significantly (p less than 0.05) shorter than in the DNA diploid tumor. In the patients with lung cancers that recurred within 1 years, recurrence of the DNA aneuploid tumor was higher than the DNA diploid tumor. It is evident from the above results that proliferative activity in the DNA aneuploid tumor increases much more than in the DNA diploid tumor. This in turn may induce early recurrence in patients with lung cancer.     

Anal cloacogenic and squamous carcinomas. Comparative histologic analysis using in situ hybridization for human papillomavirus DNA

In order to evaluate the morphologic and possible etiologic distinctions between anal cloacogenic and squamous carcinomas, we performed histologic examination and in situ hybridization for human papillomavirus (HPV) DNA on anal canal and anal verge carcinomas from 37 patients. Twenty-one neoplasms were invasive or in situ squamous carcinomas, 14 were invasive cloacogenic carcinomas, and two were unclassified. In situ hybridization was positive for HPV types 16/18 in 12 cases and for types 6/11 in two cases of anal squamous carcinoma (67% HPV positivity overall). All 14 cases classified as anal cloacogenic carcinoma were negative for HPV DNA by this technique. One of the two unclassified carcinomas was positive for type 16/18 DNA. We conclude that anal cloacogenic and squamous carcinomas are histologically similar but distinct neoplasms. Differential expression of HPV DNA in these lesions may be a manifestation of separate mechanisms of pathogenesis, or it may be due to varying degrees of tumor cell differentiation.     

Human papillomavirus DNA sequences in cell lines derived from head and neck squamous cell carcinomas.

There is increasing evidence that human papillomaviruses (HPV) have a casual role in some neoplasms in human beings. As examples, DNA of HPV types 16, 18, and 31 are frequently present in genital cancers in humans. Recently, oncogenic HPV types have also been identified in neoplasms of the head and neck, including verrucous carcinoma of the larynx, squamous cell carcinomas of the oral cavity and larynx, and inverted papillomas of the nose. These findings and our resource of an extensive panel of head and neck squamous cell carcinoma (HNSCC) cell lines led us to begin to investigate how frequently HPV DNA was present in these tumor cell lines. For initial analysis, twenty-two HNSCC cell lines derived from 20 patients' tumors were selected as representative of our tumor cell line panel with respect to diversity of primary site, tumor stage, patient age, sex, and clinical course. For Southern analysis, cell line DNA was tested for hybridization with DNA probes for HPV types 6, 11, 16, 18, and 31. Polymerase chain reaction (PCR) analysis was also performed on five tumor cell lines using types 6, 11, 16, 18, and 52 as probes. Southern blot analysis revealed HPV-specific signals in two of the 22 HNSCC cell lines tested. One of these, UM-SCC-23, was HPV 31 positive, which to our knowledge is the first identification of HPV 31 in HNSCC. UM-SCC-63, the other HPV-positive tumor identified by Southern analysis, hybridized with both type 18 and 31. Of the five tumor cell lines tested with PCR, two were HPV positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA content in radiation-associated thyroid cancer

DNA content has been reported to be of prognostic significance in differentiated thyroid carcinoma. Since malignant tumors with irradiation as an initiator often contain DNA aberrations, the DNA content of well-differentiated thyroid carcinoma in patients with a prior history of low-dose head and neck irradiation was determined and compared with similar nonradiation-associated lesions. The DNA content of thyroid cancers from 53 patients was determined with use of flow cytometry. Sixteen radiation-associated thyroid carcinomas (11 papillary, 3 follicular, and 2 medullary) all were diploid. In a group of 37 nonradiation-associated tumors, 10 were aneuploid (10 of 29 papillary carcinomas and 0 of 2 follicular or 6 medullary carcinomas). This difference in DNA content is significant (p less than 0.02, Fisher's exact test). These findings were unexpected and suggest that if the initiating irradiation causes a DNA aberration, this aberration is not reflected in DNA content as measured by means of flow cytometry.     

Studies of human papillomavirus (HPV) in urological tumors

Urological tumors were examined for the presence of human papillomavirus (HPV) DNA by using Southern blot hybridization. In 20 male patients with condyloma acuminatum, HPV type 6 was found at 85% (17/20), HPV type 11 at 95% (19/20), HPV type 16 at 5% (1/20) and HPV type 18 at 0% (0/20). In 2 female patients with condyloma acuminatum, HPV types 6, 11, 16 and 18 were found at 100% (2/2), 100% (2/2), 50% (1/2) and 0% (0/2), respectively. All 6 of the patients who were positive for HPV type 6, were also positive for HPV type 11. Two patients were positive for HPV types 6, 11 and 16, the last of which was frequently found in penile cancer and uterine cervical cancer. In 6 patients with penile cancer, two patients were positive for HPV type 16 and negative for HPV types 6, 11 and 18. The remaining 4 patients were negative for all these HPV types. One patient who was positive for HPV type 16 had penile cancer after three previous episodes of penile condyloma acuminatum. From this information, a malignant change in the condyloma acuminatum was assumed to indicate the possible association of HPV type 16 with the process of malignant degeneration. HPV types, 6, 11, 16 and 18 were not detected in a female patient with vulvar cancer. Although HPV was thought to participate in the development of urological tumors except for external genital tumors, all patients examined, consisting of 2 with benign prostatic hypertrophy, 5 with prostatic cancer and 24 with bladder cancer, were negative for HPV types 6, 11, 16 and 18. Eight patients with bladder cancer were negative for HPV type 33.     

Heterogeneity of salivary gland tumors studied by flow cytometry

Intratumor DNA heterogeneity was investigated by flow cytometric analysis of multiple samples taken from different sites of 8 benign and 16 malignant primarily resected salivary gland tumors. All benign tumors had diploid DNA content. The overall incidence of DNA diploidy in 16 malignant cases examined was 50%. Intratumor differences in DNA ploidy were observed in four malignant tumors (25%); 2 of these 4 heterogenous tumors contained both aneuploid and diploid cell clones. The remaining 12 tumors showed a homogeneous DNA content in the different specimens; 8 were diploid, 3 aneuploid, and 1 was polypoid. The DNA nondiploid tumors were clinically more advanced than the DNA diploid ones (p < 0.01). The tumor proliferation rate (fraction of cells in S-phase) was higher in DNA nondiploid samples than in diploid ones (p < 0.01). The DNA nondiploid tumors seemed to recur more often than DNA diploid ones did. The data emphasize the usefulness of DNA measurements for the characterization of malignant salivary gland tumors but also the importance of adequate sampling in assessing their DNA ploidy.     

High prevalence of human papillomavirus in prostate tissues.

Specific human papillomavirus (HPV) types are associated with benign and malignant lesions of the anogenital region including the prostate gland. Using polymerase chain reaction (PCR) amplification of type-specific HPV sequences, we have assessed the prevalence of HPV DNA in prostate tissue from 88 individuals. Amplified sequences specific for HPV 16 were found in 34 of 56 benign prostatic hyperplasias and in 14 of 27 prostatic carcinomas. In contrast, HPV 18 was identified in only three benign hyperplasias and one carcinoma, all of which also contained HPV 16 DNA. Four of five normal prostates obtained at autopsy had no detectable HPV infection; one contained HPV 16 sequences. No significant difference in the prevalence of HPV DNA is observed between patients with benign disease and those with evidence of malignancy when fragments of surgical material are analyzed. Surgical method (transurethral resection or suprapubic prostatectomy) had no effect on the frequency of HPV detection. The prevalence of HPV DNA in the small number of normal prostates analyzed was not significantly different from that in the surgical samples. The presence of HPV in prostate tissues suggests a possible reservoir for sexual transmission of types with oncogenic potential. A role for the virus in the etiology of prostatic neoplasia remains to be demonstrated.     

Low frequency of human papillomavirus infection in initial papillary bladder tumors

The involvement of human papillomavirus (HPV) in bladder cancer remains controversial. We previously reported detection of L1-HPV DNA in 39% of bladder cancers of mixed grade and stage. To clarify the possible etiologic role of HPV we studied, using the same technique, a more homogeneous group of initial low-stage tumors. We investigated a total of 187 newly diagnosed superficial papillary bladder tumors for the presence of L1-HPV DNA by the polymerase chain reaction method and hybridization with specific probes for HPV 6, 11, 16, 18, 33. HPV DNA was detected in 16 (8.5%) of the 187 specimens tested, although in a low copy number compared with SiHa cervical cancer cells used as control. HPV type 16 was observed in eight tumors while HPV type 6 and type 11 were each observed in three tumors. Two tumor specimens contained two types of HPV: one tumor hybridized with type 6 and 16 and the other with type 11 and 18. This low rate of HPV detection (8.5%) in initial tumors does not favor a prominent role for HPV in bladder carcinogenesis. Received: 7 September 1998 / Accepted: 7 December 1998