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The polymerase chain reaction (PCR) clonality assay based on the principle of random X chromosome methylation in females provides a potentially important tool in both cancer research and diagnostics. This assay, however, has not been compared to the standard Southern blot assay and is limited by the rate of heterozygosity of the X-linked phosphoglycerate kinase (PGK) and androgen receptor genes, the only two genes yet described with which this technique may be used. Using 46 marrow and blood specimens from females with and without hematological malignancies, the PCR and Southern blot methods of clonality were compared. In addition, a new technique based on the highly polymorphic fragile X (FMR1) locus was examined. The rate of heterozygosity was 25% for the PGK gene and 45% for the FMR1 gene. In the PCR assay, 7 of 8 and 11 of 14 normal control specimens showed a polyclonal methylation pattern in the PGK and FMR1 genes, respectively. Of the malignant specimens, 17 of 17 and 17 of 18 showed a monoclonal methylation pattern in the PGK and FMR1 genes, respectively. The Southern blot and PCR assay gave similar results with regards to the PGK gene. It is concluded that the PCR and Southern blot clonality assays are comparable with regards to the PGK gene and that both the PGK and FMR1 genes may be reliably used in the determination of clonality. The methods, however, are limited by the skewed methylation patterns seen in hematological specimens in a significant number of normal females.

Infection with human papillomavirus type 11 (HPV 11) is associated with benign epithelial proliferations and rarely with malignant and metastasizing tumors. Because of the biological diversity displayed in tissues infected with HPV 11, we have examined the capacity of various isolates of HPV 11 to transform cultured cells and compared their molecular differences by DNA sequence analysis. Five isolates of HPV 11 were examined for their ability to transform primary neonatal rat kidney epithelial cells and NIH 3T3 mouse fibroblasts in DNA transfection experiments using calcium phosphate precipitation. Included in these studies are the prototype isolate from a laryngeal papilloma (HPV 11P); HPV 11VC from a verrucous carcinoma of the penis; HPV 11Epi from the viral episomes of a primary squamous cell carcinoma; and two integrated genomes (HPV 11Int 1 and HPV 11Int 2) of the metastases. Only HPV 11VC cotransfected with the oncogene Ha-ras transformed neonatal rat kidney epithelial cells with an efficiency comparable to that of HPV 16 DNA. HPV 11VC DNA alone transformed NIH 3T3 cells. Analysis of the DNA sequence of HPV 11P and 11VC revealed 16 single nucleotide changes in the upstream regulatory region and open reading frames E1, E2, E4, and E5, five resulting in amino acid substitutions. This is the first demonstration of cellular transformation by a natural isolate HPV 11 DNA in vitro and illustrates that minimal changes in the DNA sequence of certain viruses confer oncogenicity to what are normally nontransforming viruses.

A primary perianal squamous cell carcinoma and two metastatic tumors from a renal transplant recipient with a previous history of condyloma acuminatum were analyzed by filter hybridization for the presence of human papillomavirus (HPV) DNA. Each of the DNA extracts from these three tissues was found to contain HPV DNA. Stringent hybridization and restriction endonuclease analysis identified this viral DNA as HPV 11 related, which largely comigrated with cellular DNA, suggesting the presence of integrated viral DNA. Each DNA extract was analyzed by two-dimensional gel electrophoresis, which separates circular and linear forms of DNA and can demonstrate linear viral DNA, which comigrated with high molecular weight linear cellular DNA, thus implying viral integration. In all three cases the vast majority of viral DNA was found to comigrate with linear DNA; in addition, a significant portion comigrated with high molecular weight cellular DNA, suggesting the presence of integrated viral DNA in these tumors. Restriction endonuclease analysis of high molecular weight cellular DNA from each of these tumors revealed identical banding patterns, indicating that the integration site in each tissue is identical and, therefore, that all three tumors most likely originated from a single clonal event. These molecular results are presented in light of the clinical history of this patient with a histologically "low grade," but biologically aggressive, squamous cell carcinoma and suggest that HPV 11 may be associated with the initiation of malignant epithelial neoplasms.