Anthony J. Faras

Human papillomavirus DNA in adenocarcinoma in situ, microinvasive adenocarcinoma of the uterine cervix, and coexisting cervical squamous intraepithelial neoplasia.

Previously, human papillomavirus (HPV) DNA, mainly HPV-18 DNA, was detected in more than 40% (17/40 cases) of invasive adenocarcinoma of the uterine cervix in our laboratory. In order to identify HPV DNA in the precursor lesions of adenocarcinoma of the cervix, 11 cases of adenocarcinoma in situ containing microinvasive adenocarcinoma and 10 cases of adenocarcinoma in situ were studied for the presence of HPV DNA by in situ hybridization using highly sensitive 3H-labeled HPV-16 and HPV-18 DNA probes. HPV types present in cervical squamous intraepithelial neoplasia (CIN) coexisting with adenocarcinoma in situ and microinvasive adenocarcinoma were also studied. Apart from the coexisting CIN II-III with glandular neoplasms, 48 cases of CIN III (severe dysplasia and squamous carcinoma in situ) removed by conization or hysterectomy and known to be free of adenocarcinoma were used for comparison. HPV DNA was detected in 64% of microinvasive adenocarcinoma, 70% of adenocarcinoma in situ, and 63% of the control CIN III. HPV-18 DNA was the preponderant type of HPV DNA found in adenocarcinoma in situ and microinvasive adenocarcinoma. All cases of HPV DNA-positive microinvasive adenocarcinoma contained the same type of HPV DNA as the lesions of coexisting adenocarcinoma in situ. CIN coexisting with microinvasive adenocarcinoma or adenocarcinoma in situ contained the same type of HPV as identified in the glandular lesions, whereas all of the HPV DNA-positive control CIN III cases contained HPV-16 DNA. These results suggest that adenocarcinoma in situ is a precursor lesion of adenocarcinoma of the cervix that contains HPV DNA, and that CIN coexisting with adenocarcinoma may be a result of a metaplastic process of adenocarcinoma or of bidirectional differentiation of the affected reserve cells.

Identification of human papillomavirus DNA in cervical and vaginal intraepithelial neoplasia with molecularly cloned virus-specific DNA probes

Summary:The presence of human papillomavirus (HPV) DNA was identified in the tissues of cervical and vaginal intraepithelial neoplasia by Southern blot DNA hybridization under conditions of low stringency. The specific types of HPV present in the tissues were identified by using molecularly cloned types 1 through 6 (HPV-1 through HPV-6) HPV DNA probes under high-stringency conditions. All tissues of cervical and vaginal intraepithelial neoplasia analyzed contained HPV genomes. Fifteen of 19 samples (79%) contained HPV-6 DNA, and 10 of 19 samples (53%) HPV-3 DNA. Hybridization with HPV-1, HPV-2, HPV-4, and HPV-5 DNAs was also observed in several of the samples. Four of the samples did not hybridize with any of the probes tested (HPV-1 through HPV-6); yet, all showed hybridization with an HPV-EV DNA (a type 3-related DNA) probe under low-stringency conditions, indicating the presence of HPV types other than those belonging to HPV-1 through HPV-6.

Sensitivity of koilocytosis, immunocytochemistry, and electron microscopy as compared to DNA hybridization in detecting human papillomavirus in cervical and vaginal condyloma and intraepithelial neoplasia.

The sensitivity in detecting human papillomavirus (HPV) by histological observation of koilocytosis, immunocytochemistry, and electron microscopy with reference to the results of Southern blot deoxyribonucleic acid (DNA) hybridization were reviewed in 41 lesions (37 patients) of cervical and vaginal condylomata acuminata and intraepithelial neoplasia. Human papillomavirus DNA was demonstrated in fresh tissues by Southern blot DNA hybridization in all but one lesion of moderate dysplasia (98%). The rate of koilocytosis observed in tissue sections was 80% in condyloma, and ranged from 89-20% in cervical intraepithelial neoplasia (CIN), with steady reduction as the grade of CIN or vaginal intraepithelial neoplasia (VaIN) was higher. The immunocytochemistry for HPV capsid antigens was positive in 80% of condylomata and ranged from 61-0% in CIN or VaIN. The rate declined in inverse proportion to the grade of CIN or VaIN. Electron microscopy of preselected areas containing intranuclear inclusions in paraffin sections of 10 lesions demonstrated HPV-like particles in 90% of the lesions. Although immunocytochemistry and observation of koilocytosis may be useful in detecting HPV in condylomata acuminata and mild dysplasia, their sensitivity was poor in CIN or VaIN of higher grades. Electron microscopy on preselected areas in paraffin blocks showed better sensitivity, presumably due to its ability to detect immature virions.

A clinical, histopathologic, and molecular biologic investigation of vulvar intraepithelial neoplasia

SummaryThe spectrum of vulvar intraepithelial neoplasia (VIN) was investigated with human papillomavirus (HPV) DNA probes by Southern blot hybridization technique. The results of vulvar tissue examinations from 25 patients were compared to prior genital and systemic diseases, clinical presentation, and histopathologic manifestations. The presence of HPV DNA in these lesions was largely associated with multifocality of the lesions, the presence of synchronous and metachronous multicentric genital neoplasia, and depressed immunocompetence. These findings indicate that VIN is associated with HPV and may have an infectious etiology.

The analysis of carcinomas of the vagina for human papillomavirus DNA.

SummaryInvasive cancers of the vagina are relatively rare and often resistant to effective treatment. While studies on the more abundant premalignant lesions of the vagina and premalignant and malignant tumors of the vulva and cervix have shown a frequent association with human papillomavirus (HPV) DNA infection, lack of fresh tissue samples has precluded similar studies on malignant tumors of the vagina. Using mostly in situ hybridization, we have retroactively examined 14 formalin-fixed, paraffin-embedded biopsies of invasive squamous cell carcinomas of the vagina. We have found 21% of the samples to have HPV DNA. These findings confirm a role for HPV in malignancies of the entire female lower genital tract.

Studies on the Replication of Reticuloendotheliosis Virus: Detection of Viral-Specific DNA Sequences in Infected Chick Cells

Infection of stationary chick embryo fibroblasts by reticuloendotheliosis virus (REV) is sensitive to cytosine arabinoside, an inhibitor of DNA synthesis. Furthermore, the majority of the nucleic acid sequences contained in the REV genome can be detected in infected cells in the form of DNA by RNA-DNA hybridization techniques. A small portion (ca. 5%) of the REV-specific sequences can be detected in uninfected chick embryo fibroblasts suggesting that these cells contain at least part of the REV genome as endogenous DNA sequences. These observations are consistent with an involvement of REV-specific proviral DNA as an intermediate in the replication of REV in chick embryo fibroblasts.

Serological and molecular evidence of rhesus papillomavirus type 1 infections in tissues from geographically distinct institutions.

We have previously demonstrated the presence of rhesus monkey papillomavirus type 1 (RhPV-1), from molecular and pathological evidence, in a mating group within a single institution. We have now also obtained a number of fresh or archival tissues of rhesus monkeys from other geographically distinct institutions. Using PCR amplification, we observed two animals from one of these institutions and five animals from another which demonstrated RhPV-1 DNA sequences. In addition we molecularly cloned the E7, E2, E4, L2 and L1 genes of RhPV-1 into bacterial expression vectors. The fusion gene products were used to test for serological response to RhPV-1 antigens by Western blot analysis. Responses were observed in up to 52% of the animals tested. While some serologically positive animals were also RhPV-1 DNA-positive, most were not.

DNA polymerase of reticuloendotheliosis virus: Inability to detect endogenous RNA-directed DNA synthesis

Abstract Purified preparations of reticuloendotheliosis virus (REV) appear to lack the endogenous RNase-sensitive DNA polymerase activity present in most, if not all, avian RNA tumor viruses. Although no endogenous RNA-directed DNA polymerase could be detected in REV, we have been able to demonstrate the presence of a virion-associated DNA polymerase by employing exogenous synthetic homopolymers as template · primer. A comparison of the REV-associated DNA polymerase activity with the RNA-directed DNA polymerase of Rous sarcoma virus (RSV) reveals similarities in the preference of the enzymes to utilize certain synthetic template · primer complexes containing ribopolymers as template. For example, both enzymes prefer poly(rA) · oligo(dT) 10 to poly(dA) · oligo(dT) 10 as template · primer. In addition, poly(rC) · oligo(dG) 10 appears to be efficiently utilized by the REV DNA polymerase under conditions whereby a DNA-directed DNA polymerase does not utilize this synthetic homopolymer as template · primer. Although the REV 70 S RNA genome is not transcribed by the DNA polymerase contained within virions of REV, it is as good a template · primer for the purified avian oncornavirus RNA-directed DNA polymerase as RSV 70 S RNA. Furthermore, the virion-associated REV DNA polymerase can transcribe REV 70 S RNA when oligo(dT) 12–18 is present as a source of primer. Therefore, the inability of the REV DNA polymerase to transcribe effectively the REV genome in vitro appears to reflect either some unique property of the REV enzyme or some structural feature of REV 70 S RNA, such as the lack of certain primer molecules required by the REV DNA polymerase for the initiation of DNA synthesis.

Human Papillomavirus DNA in Glandular Dysplasia and Microglandular Hyperplasia: Presumed Precursors of Adenocarcinoma of the Uterine Cervix

&NA; Presumed precursors of adenocarcinoma of the uterine cervix were investigated with specific techniques to identify human papillomavirus (HPV) DNA. The presence of HPV DNA in 36 lesions of glandular dysplasia and 16 lesions of microglandular hyperplasia of the uterine cervix was studied by in situ hybridization using 3H‐labeled HPV 16 and HPV 18 DNA probes. Only two of 36 lesions (6%) of glandular dysplasia contained HPV 18 DNA, although 64% of coexisting adenocarcinoma in situ, microinvasive adenocarcinoma, and cervical squamous intraepithelial neoplasia III lesions contained HPV 18 and/or HPV 16 DNA. Two lesions of HPV 18 DNA‐positive glandular dysplasia coexisted with adenocarcinoma in situ that contained the same type of HPV DNA. None of the microglandular hyperplasia lesions contained HPV 16 DNA or HPV 18 DNA. These results suggest that, if HPV infection is an initial step toward carcinogenesis, it is unlikely that glandular dysplasia and microglandular hyperplasia are precursor lesions of adenocarcinoma of the uterine cervix. A large proportion of glandular dysplasia may represent reactive lesions of endocervical columnar epithelium. Two lesions of HPV 18 DNA‐positive glandular dysplasia may represent well‐differentiated components of adenocarcinoma in situ of the uterine cervix. (Obstet Gynecol 73:1005, 1989)

The methionyl transfer RNAs of Rous sarcoma virus

Abstract Virions of Rous sarcoma virus contain the two major classes of methionyl tRNA present in uninfected chick embryo fibroblasts. The formylated species, N -formyl methionyl tRNA, appears to be selectively associated with the viral 70 S RNA genome.