Prevalence of JC polyomavirus genomic sequences from the large T-antigen and non-coding control regions among Bulgarian patients with primary brain tumors.

A total of 111 fresh brain biopsies from patients with primary brain tumors were examined for JC polyomavirus sequences from the Large T antigen encoding region (LT) and the viral non-coding control region (NCCR). SYBR Green and TaqMan real-time polymerase chain reaction assays were used. In the glioblastoma group of 39 patients 48.7% were positive for LT sequences. Among the astrocytoma group (19 patients) and the oligodendroglioma group (12 patients) 31.6% and 33.3% were also positive. The prevalence of LT genomic sequences among the other groups was as follows: in 2 out of 3 oligoastrocytomas; in 3 out 5 gangliogliomas; in 2 out of 5 meduloblastomas; in 1 out 3 pineocytomas; and in none of the tested 5 ependimomas. All positive samples had a late threshold cycle that varied from 36 to 49, indicative of very low starting viral number. Only 21 of all the 111 samples were positive for NCCR. Low copy number in range of 10-1,000 was present. Notably, only 8 of all NCCR positive specimens were also LT positive. It might be suggested that the disproportion between the results for LT and NCCR is either due to clonally integrated LT fragments, with loss of genetic material, or changes in the NCCR. The latter would alter the productive course of the infection and may establish a premise for continuous interaction of viral regulatory proteins with cell molecules that are responsible for the control of the cell cycle. This may lead subsequently to malignant transformation.

Detection of JC virus DNA sequences in brain tumors in pediatric patients.

OBJECT: The JC virus is a human neurotropic polyomavirus that causes progressive multifocal leukoencephalopathy and is closely related to simian virus 40. Several recent reports have indicated a possible association between the JC virus and the development of various human brain tumors. The authors examined the presence of JC virus DNA sequences in primary brain tumors in pediatric patients to evaluate the hypothesis that particular brain tumors can arise in the pediatric population as a consequence of infection with the JC virus. METHODS: Genomic DNA sequences were isolated from 62 brain tumors (32 medulloblastomas, 18 ependymomas, five choroid plexus papillomas, and seven pilocytic astrocytomas) and analyzed for the presence of JC virus DNA by Southern blot hybridization and direct sequencing. The JC virus DNA sequence was detected in five ependymomas and one choroid plexus papilloma. Immunohistochemical studies revealed nuclear expression of the large T-antigen in a choroid plexus papilloma. None of the medulloblastomas or pilocytic astrocytomas contained JC virus DNA. CONCLUSIONS: The results of this study provide molecular evidence of the association between JC virus and the development of certain ependymomas and choroid plexus papillomas.

Low frequency of SV40, JC and BK polyomavirus sequences in human medulloblastomas, meningiomas and ependymomas.

Several reports have suggested a role for polyomaviruses in the pathogenesis of human brain tumors. This potential involvement is not conclusively resolved. For the present study, a highly sensitive PCR-assay with fluorescence-labelled primers was developed to search for polyomavirus sequences in human brain tumor and control DNA samples. The assay was shown to detect approximately one viral large T-antigen (TAg) gene per 250 cells. We identified simian virus 40 (SV40)-like sequences in 2/116 medulloblastomas, in 1/131 meningiomas, in 1/25 ependymomas and in 1/2 subependymomas. A single case of ependymoma contained SV40 VP-1 late gene sequences. Moreover, one of the meningioma samples showed JC virus sequences. In contrast, 60 hepatoblastoma samples and 31 brain samples from schizophrenic patients were consistently negative. BK virus sequences were not detectable in any of our samples. Immunohistochemical analysis of two SV40 positive tumor biopsies failed to detect large TAg in the tumor cells. In the JC positive meningioma, immunoreactivity for the viral late gene product (VP-1) was not observed. Our data do not entirely rule out SV40 and JC virus as an initiative agent with a hit-and-run mechanism. However the low frequency of virus sequences and the absence of TAg protein expression argue against a major role of these viruses in the pathogenesis of human medulloblastomas, meningiomas and ependymomas.

Simian virus 40 is present in most United States human mesotheliomas, but it is rarely present in non-Hodgkin's lymphoma.

Simian virus 40 (SV40) causes mesotheliomas, osteosarcomas, ependymomas, choroid plexus tumors, and lymphomas in hamsters. In humans, SV40 has been detected in tumors of the first four types. Using the polymerase chain reaction (PCR), we tested 29 non-Hodgkin's lymphomas (intermediate and high-grade), 25 posttransplant lymphoproliferative disorders, and 5 AIDS lymphomas for SV40 DNA. PCR analysis revealed that 3 of 29 lymphomas, 6 of 25 posttransplant lymphoproliferative disorders, and 2 of 5 AIDS lymphomas contained SV40 sequences corresponding to the retinoblastoma (RB)-pocket binding domain of SV40 tumor antigen (Tag). However, among positive samples, only one posttransplant lymphoproliferative disorder and one AIDS lymphoma contained the SV40 regulatory region, which suggest a higher viral load in these patients. In parallel experiments, 8 of 12 mesotheliomas tested positive for SV40 for both the RB-pocket binding domain of Tag and the SV40 regulatory region. These data confirm the presence of SV40 in most United States mesotheliomas and indicate that in human non-Hodgkin's lymphomas, the prevalence of SV40 is low.

Detection of simian virus 40 DNA sequence in human primary glioblastomas multiforme.

OBJECT: Deoxyribonucleic acid oncoviruses can induce neoplastic transformation of cells because their viral proteins interfere with antiproliferative cellular proteins. Simian virus 40 (SV40) is a DNA virus that induces the emergence of ependymomas, choroid plexus tumors, mesotheliomas, osteosarcomas, sarcomas, and various tumors when injected into newborn hamsters. Recently, approximately 60% of human ependymomas, choroid plexus tumors, and mesotheliomas were reported to contain and express SV40 DNA sequences. In this study the presence of SV40 DNA sequences was investigated in human brain tumors. METHODS: Three of 32 glioblastomas mutiforme (GBMs), but none of two ependymomas and five medulloblastomas, were found to possess SV40 DNA sequences when examined using polymerase chain reaction (PCR). The DNA sequence analysis of PCR-amplified fragments disclosed that the samples were identical to the regulatory region of SV40. All three GBMs, which arose in elderly patients with wild-type p53, were considered to be primary (de novo) tumors. Although each of the three tumors was immunohistochemically negative for SV40 T antigen, in situ hybridization successfully demonstrated the messenger RNA for SV40 T antigen. CONCLUSIONS: The results of this study indicate that latent infection of SV40 in elderly people may be implicated in the tumorigenesis of certain primary GBMs.

BK virus-induced osteosarcoma (Os515) as a model of human osteosarcoma.

BK virus was isolated by Gardner et al in 1971 from the urine of an immunosuppressed patient with a kidney allograft. Antibodies to this virus are ubiquitous in the general populations worldwide, but the oncogenic capacity of BKV in humans had not been reported. The virus transformed in vitro permissive human and non-permissive animal cells, and the transformed cells had the T antigen. Intracerebral and intravenous inoculation of BKV in newborn hamsters induced malignant tumours (mainly ependymomas, malignant insulinomas, and osteosarcomas). Subcutaneous and intraperitoneal routes were also effective. The virus was only rescued from a few tumours by fusion with human embryonic cells or Vero cells. Brain tumours appeared earlier and osteosarcomas developed in animals which survived for more than 6 months. Many of the osteosarcomas were bony and grew slowly with frequent lung metastases, and a few osteosarcomas were soft and grew rapidly without lung metastases. Experimental targeting chemotherapy with doxorubicin (DX)-containing immunoliposomes was performed against Os515 osteosarcoma. In in vitro experiments, DX-Lip-MoAb29 showed a more significant inhibitory effect on cultured Os515 cells than free Dx and DX-Lip. DX-Lip DNP had less effect. In in vivo experiments, DX-Lip-MoAb29 suppressed the growth of Os515 tumour isografts in hamsters and prolonged the survival of recipients more significantly than free DX.

Simian virus 40 DNA sequences in human brain and bone tumours.

This report reviews recent observations regarding the association of simian virus 40 (SV40) DNA sequences with brain and bone tumours of childhood [1-3]. Our initial investigation was suggested by the tumorigenicity of SV40 in animals, and the transgenic mouse expression of SV40 large T-antigen in which all animals developed choroid plexus (CP) tumours. Polymerase chain reaction (PCR) analysis and DNA sequencing demonstrated SV40-like DNA sequences, amplified from the "Rb-pocket" binding domain of the viral large T-antigen, in 10/20 CP and 10/11 ependymoma tumours of children. The PCR analysis was subsequently extended to three additional regions of the viral genome: the carboxy-terminal region of large T-antigen, the viral enhancer/origin, and the VP1 gene. All amplified products were related to SV40 sequences. Furthermore, because one individual in the original brain tumour study was a member of a Li-Fraumeni kindred, 151 DNA samples from such families were analysed. Only 18 were positive for viral sequences and 11 of these were isolated from individuals with osteosarcomas. This observation led to a further analysis of DNA from bone tumours, in which 54/160 samples contained SV40-like sequences. These studies associate SV40-like sequences with human CP, ependymoma, and bone tumours. A causal relationship to human oncogenesis remains a subject for further study.

Low incidence of SV40-like sequences in ependymal tumours.

Between 1955 and 1963, millions of children and adults were exposed to SV40-contaminated poliovirus vaccines. The oncogenic potential of this polyomavirus was revealed when intracerebral inoculation of SV40 into newborn hamsters resulted in the development of ependymomas and choroid plexus papillomas. Subsequently, SV40-like sequences were repeatedly detected in human ependymomas with broadly ranging incidence rates of 7-90%. Most epidemiological studies, however, have not described an increased occurrence of ependymomas. To gain more data on this controversial issue, this study examined 62 archived ependymal tumours from 31 children and 31 adults who underwent surgery between 1990 and 1999. Only three (5%) of the tumours--including 24 classical, 20 anaplastic, and 12 myxopapillary ependymomas; one subependymoma; and five ependymoblastomas--revealed subgenomic SV40 sequences. None of the ependymomas in patients born between 1920 and 1960 demonstrated SV40-like sequences. The positive tumours represent 7% of grade II and III ependymomas (two paediatric and one adult tumour). DNA sequencing of the PCR product revealed identical sequences of SV40 in the positive ependymal tumours. Compared with the results from other countries, this incidence rate is relatively low. Therefore, it seems likely that significant differences between individual countries exist regarding the prevalence of SV40-positive ependymomas. These differences may reflect different degrees of exposure to SV40-contaminated polio vaccine.

Natural simian virus 40 strains are present in human choroid plexus and ependymoma tumors.

Simian virus 40 (SV40) sequences for large tumor antigen (T-ag) were recently detected in a significant fraction of certain human brain tumors of early childhood (Bergsagel et al., N. Engl. J. Med. 326, 988-993, 1992). In the current study, we sought to determine whether authentic SV40 was present in the choroid plexus and ependymoma tumors previously examined. Polymerase chain reaction and DNA sequence analysis revealed authentic SV40 regulatory region and major capsid (VP1) sequences in 14 of 17 tumors tested. Only one 72-basepair element was detected in the SV40 enhancer region of positive tumor samples, an arrangement designated as "archetypal." The C terminus of the T-ag gene was detected in the same 14 tumors and was sequenced from 5 tumors; some nucleotide changes were found that would result in amino acid changes in T-ag. Infectious SV40 was isolated from one sample after lipofection of tumor DNA into monkey kidney cells. Sequence analysis of the rescued virus SVCPC revealed (i) an archetypal regulatory region, (ii) nucleotide changes in the C terminus of the T-ag gene that distinguished it from SV40 laboratory strains 776 and SV40-B2 and from human isolate SVPML-1, and (iii) identity with previous human brain tumor isolate SVMEN in the three genomic regions sequenced. No human-isolate-specific distinguishing features were detected among the viral sequences analyzed. Thus, authentic SV40 is present in humans and associated with two tumor types known to be induced experimentally by the virus.

Absence of simian virus 40 in human brain tumors from northern India.

Simian virus 40 (SV40), a monkey polyomavirus, was a contaminant of early poliovirus vaccines administered to millions of individuals in the 1950s and early 1960s. SV40 causes brain tumors in laboratory animals, and SV40 DNA sequences have been variably identified in human choroid plexus tumors and ependymomas. We studied the possible association between SV40 and human brain tumors in northern India, where humans have frequent contact with SV40-infected rhesus macaques. DNA from pathologic specimens from 33 ependymomas, 14 choroid plexus tumors and 18 control brain tissues (contused brain, brain metastases) was extracted and analyzed under masked conditions. We used real-time PCR to detect and quantify SV40 (T antigen) and human (GAPDH) DNA sequences. The SV40 PCR assay detected as few as 10 copies of SV40 DNA and had a linear range from 1 x 10(2) to 1 x 10(6) copies. SV40 DNA was detected in 1 specimen (an ependymoma). However, few SV40 DNA copies were detected in this sample (<10 copies, equivalent to <1 copy/350 cells, based on simultaneous GAPDH quantification), and SV40 was not detected when this sample was retested. Our findings do not support a role for SV40 in choroid plexus tumors or ependymomas from northern India.

Detection of authentic SV40 DNA sequences in human brain and bone tumours.

This report summarizes our follow-up studies of SV40 DNA sequences in human brain tumors of early childhood and our confirmation of the presence of SV40 DNA in human osteosarcomas. We examined brain tumors and osteosarcoma samples by the polymerase chain reaction (PCR) using primers from four separated regions of the SV40 genome. Sequence analysis confirmed that authentic SV40 DNA was present. The regulatory region of each tumor-associated viral DNA was of archetypal length (non-duplicated enhancer); sequence variation was noted at the extreme C-terminus of the large T-antigen (T-ag) genes. Infectious SV40 was recovered from one brain tumor. We sequenced the entire early genomic region from three human isolates of SV40 and two laboratory strains originally recovered from monkeys. The predicted amino acid sequence of the large T-ags showed remarkable sequence conservation among isolates, except for a small variable region identified at the C-terminus of the protein. There were no human-isolate-specific changes detected that could serve to distinguish a human variant of SV40 nor were any tumor-type-specific viral markers observed. Based on these data, we conclude that authentic SV40 is associated with some human brain and bone tumors and that multiple SV40 strains can infect humans.

Potential exposure to SV40 in polio vaccines used in Sweden during 1957: no impact on cancer incidence rates 1960 to 1993.

U.S. polio vaccines produced during the 1950s were potentially contaminated by simian virus 40 (SV40). Recently DNA from SV40 has been detected in brain ependymoma, pleural mesothelioma and osteosarcoma. In 1957, when national polio vaccination was started in Sweden, vaccine potentially contaminated with SV40 was given to approximately 700,000 individuals, mainly pre-school and school children born between 1946 and 1953. From 1958, a Swedish inactivated polio vaccine was exclusively used, which has been claimed to be free of SV40. We explored cancer incidence rates in the cohorts exposed to the potentially contaminated polio vaccines in Sweden. The Swedish Cancer Registry provided annual cancer incidence rates in five-year age groups for the years 1960-93. Cancer incidence in cohorts maximally exposed was followed during this period, and the incidence when these cohorts reached a specific age was compared to the incidence when unexposed cohorts reached the same age. For osteosarcoma and brain ependymoma overall age-standardised incidence rates were essentially unchanged between 1960 and 1993, and age specific rates were similar in the exposed and unexposed male and female cohorts. During the same period, overall age standardised incidence rates in males of brain cancers increased from 9.0 to 13.1 and of pleural mesotheliomas from 0.2 to 2.1 per 100,000. None of these increased rates was associated with the exposed cohorts. The use of potentially SV40 contaminated inactivated polio vaccines in Sweden has not been shown to be associated with increased cancer incidence. However, the exposed cohorts have not yet reached the age of increased risk of brain cancer or mesothelioma.

Contamination of poliovirus vaccines with simian virus 40 (1955-1963) and subsequent cancer rates.

CONTEXT: Poliovirus vaccine contaminated with live simian virus 40 (SV40), a macaque polyomavirus that is tumorigenic in rodents, was used extensively in the United States between 1955 and 1963. Simian virus 40 DNA has recently been detected in several rare human tumors, including ependymomas, osteosarcomas, and mesotheliomas. OBJECTIVE: To determine the risk of ependymoma, osteosarcoma, and mesothelioma among Americans who as children received SV40-contaminated poliovirus vaccine. DESIGN: Retrospective cohort study using data from the Surveillance, Epidemiology, and End Results program (1973-1993) and the Connecticut Tumor Registry (1950-1969), as well as national mortality statistics (1947-1973). SETTING: United States. PARTICIPANTS: Birth cohorts that were likely to have received SV40-contaminated poliovirus vaccine as infants, born 1956 through 1962 (60 811730 person-years of observation); as children, born 1947 through 1952 (46430953 person-years); or that were unexposed, born 1964 through 1969 (44959979 person-years). MAIN OUTCOME MEASURES: Relative risk (RR) of each cancer among exposed compared with unexposed birth cohorts. RESULTS: Age-specific cancer rates were generally low and were not significantly elevated in birth cohorts exposed to SV40-contaminated vaccine. Specifically, compared with the unexposed, the relative risk of ependymoma was not increased in the cohorts exposed as infants (RR, 1.06; 95% confidence interval [CI], 0.69-1.63), or as children (RR, 0.98; 95% CI, 0.57-1.69) nor did the exposed have an increased risk of all brain cancers. Osteosarcoma incidence also showed no relation to exposure as infants (RR, 0.87; 95% CI, 0.71-1.06) or children (RR, 0.85; 95% CI, 0.59-1.22). Last, mesotheliomas were not significantly associated with exposure, although the cohorts studied have not yet reached the age at which these tumors tend to occur. CONCLUSIONS: After more than 30 years of follow-up, exposure to SV40-contaminated poliovirus vaccine was not associated with significantly increased rates of ependymomas and other brain cancers, osteosarcomas, or mesotheliomas in the United States.