Cancer prevalence in 129 breast-ovarian cancer families tested for BRCA1 and BRCA2 mutations.

BACKGROUND: Women who carry germline mutations in the breast-ovarian cancer susceptibility genes, BRCA1 and BRCA2, are at very high risk of developing breast and/or ovarian cancer. Both genes are tumour suppressor genes that protect all cells from deregulation, and there are reports of their involvement in other cancers that vary and seem to depend on the population investigated. It is therefore important to investigate the other associated cancers in different populations to assist with risk assessments. OBJECTIVES: To assess the cancer risk profile in BRCA-mutation-positive and negative South African breast-ovarian cancer families, mainly of Caucasian origin. DESIGN: Descriptive study in which the prevalence of all cancers in the pedigrees of BRCA1- and BRCA2-mutation-positive groups and a group of families without mutations in either gene were compared with the general population. RESULTS: As expected, female breast and ovarian cancer was significantly increased in all three groups. Furthermore, male breast cancer was significantly elevated in the BRCA2-positive and BRCA-negative groups. Stomach cancer prevalence was significantly elevated in the BRCA2-positive families compared with the general population. CONCLUSIONS: These results can be applied in estimation of cancer risks and may contribute to more comprehensive counselling of mutation-positive Caucasian breast and/or ovarian cancer families.

PTEN mutations in uterine sarcomas.

OBJECTIVE: Uterinesarcomas comprise three main types: carcinosarcomas, leiomyosarcomas, and endometrial stromal sarcomas. Carcinosarcomas are highly aggressive neoplasms with a biphasic histology of carcinomatous and sarcomatous elements. It is now generally accepted that carcinosarcomas are biphasic tumors that have to be regarded as endometrial carcinomas where metaplasia occurs. Mutations of the PTEN tumor suppressor gene, located on 10q23, play a significant role in the pathogenesis of the endometrioid type of endometrial carcinoma. Loss of heterozygosity of chromosome 10q has been reported in uterine leiomyosarcoma. Since little is known about the molecular pathobiology, our goal was to investigate the potential role of the PTEN gene in the carcinogenesis of uterine sarcomas. METHODS: We examined 21 carcinosarcomas, 21 leiomyosarcomas, and 5 endometrial stromal sarcomas using exon-by-exon polymerase chain reaction-single-strand conformation polymorphism analysis. RESULTS: Overall 8.5% (4/47) of uterine sarcomas were found to harbor somatic PTEN mutations. Of these, approximately 17% (3/18) were carcinosarcomas with endometrioid-type carcinoma components and approximately 5% (1/21) were leiomyosarcomas. No mutations were detected in carcinosarcomas with nonendometrioid carcinoma components (0/3) and in endometrial stromal sarcomas (0/5). CONCLUSIONS: These data suggest that intragenic PTEN mutations are involved in the genesis of uterine carcinosarcomas with endometrioid-type carcinoma components but rarely contribute to the pathobiology of uterine leiomyosarcomas.

FHIT RNA and protein expression in oral squamous cell carcinomas.

BACKGROUND: To investigate the possible role of FHIT, a possible tumour suppressor gene, in oral carcinogenesis, we examined 17 oral squamous cell carcinomas (OSCCs) for genetic alterations. MATERIALS AND METHODS: Fresh tissue was obtained during surgery, snap-frozen in liquid nitrogen and stored at -70 degrees C. Nested PCR amplification to examine the integrity of FHIT mRNA was performed on the reverse transcribed complementary DNA obtained from the frozen normal and tumour tissue. Immunohistochemistry was done on formal in-fixed paraffin-embedded tissue protein from the same cases using a polyclonal antiserum against the full length Fhit. RESULTS: Twelve out 17 (71%) OSCCs showed reduced or absent Fhit protein and half of the cases with reduced Fhit protein exhibited aberrant RT-PCR products. CONCLUSION: Immunohistochemical detection of Fhit protein expression in OSCCs is the more sensitive method to determine the status of Fhit in these tumours, in agreement with previous studies of other tumour types.

Microsatellite instability in uterine sarcomas.

Studies have shown a 15-30% frequency of microsatellite instability in endometrial cancer. In addition, we found a 21% frequency of microsatellite instability in endometrial cancer. Our aim was to investigate the presence of microsatellite instability and loss of heterozygosity in uterine sarcomas. The records of 69 women referred to Kalafong Academic and Pretoria Academic Hospital with a primary diagnosis of uterine sarcoma were reviewed. At histological review of 43 cases with a primary diagnosis of leiomyosarcoma, diagnosis of mitotically active leiomyoma was made in 21. Diagnosis of carcinosarcoma was made in 21 cases and endometrial stromal sarcoma in five. In all cases, genomic DNA was extracted from normal myometrium and tumor and analyzed for microsatellite instability and loss of heterozygosity. High-frequency microsatellite instability was absent in leiomyosarcoma, endometrial stromal sarcoma, and mitotically active leiomyomas and was observed in 1 (5%) carcinosarcoma. Loss of heterozygosity for chromosome 11 was present in 8/48 (17%) of uterine sarcomas, equally distributed between leiomyosarcomas (4/22 = 18%) and carcinosarcomas (4/21 = 19%). There was no loss of alleles in endometrial stromal sarcoma nor mitotically active leiomyomas. In conclusion, it is suggested that tumor suppressor genes may play a role in the tumorigenesis of uterine mesenchymal cells, whereas mismatch repair genes contribute to the carcinogenesis of endometrial cancer.

The prevalence of antibodies to simian T-cell leukaemia/lymphotropic virus (STLV-I) in non-human primate colonies in Kenya.

Retroviruses closely related to the human T-cell leukaemia/lymphotrophic virus type I (HTLV-I) have been detected in several, non-human, primate species. These retroviruses are called simian T-lymphotrophic virus type I (STLV-I). Infection with STLV-I has been associated with lymphoma and leukaemia in macaques, baboons, African green monkeys and gorillas. However, no STLV-I infection has been detected in New World primates, although STLV-II has been detected in spider monkeys. When sera from 10 species of non-human primates maintained at the Institute of Primate Research were screened for STLV-I infection, anti-STLV-I antibodies were detected in 12%, 12%, 23% and 38% of the olive baboons, yellow baboons, African green monkeys and lowland Sykes' monkeys, respectively. Western-blot studies confirmed these results. To date, no clinical disease has been linked with STLV-I infection in these colonies. The relatively high prevalence of anti-STLV-I antibodies in these non-human primates offers an opportunity for studies on the transmission, phylogenetic relationships and natural history of STLV-I in primate colonies.

Immunohistochemical evaluation of Fhit protein expression in oral squamous cell carcinomas.

The expression of Fhit (fragile histidine triad) protein in oral squamous cell carcinoma (OSCC) and adjacent oral epithelium was evaluated by immunohistochemistry on formalin-fixed paraffin-embedded blocks of 32 cases of OSCC. Rabbit polyclonal anti-GST-Fhit antiserum at 1:600 was used, after antigen enhancement in a microwave pressure cooker, in a saturated lead thiocyanate solution. This antiserum has been shown specifically to detect human Fhit by immunohistochemistry at dilutions up to 1:10,000. The Fhit protein expression was evaluated using both the intensity and extent of staining. Normal stratified squamous epithelium showed strong positivity, especially in the stratum spinosum and areas of keratinisation. Basal and parabasal cells were negative or expressed low levels of Fhit relative to the squamous epithelium. Mild and moderate epithelial dysplasia showed Fhit expression in the superficial layers, while Fhit expression was absent from severely dysplastic lesions. A reduction or loss of Fhit expression was found in 21 (66%) of the OSCC. The alterations in Fhit protein expression in OSCC, and not in normal tissues, are consistent with the proposal that Fhit inactivation plays a role in oral carcinogenesis.

Warthin's tumour is not an Epstein-Barr virus related disease.

BACKGROUND: The histogenesis of Warthin's tumour (WT) is controversial. A possible role for Epstein-Barr virus (EBV) has been suggested. MATERIALS AND METHODS: Twenty formaldehyde-fixed, paraffin-embedded blocks of WT from the parotid gland were examined for the presence of EBV. In situ hybridisation was performed using EBV encoded small nuclear RNAs (EBER1/2) probes labelled with fluorescein isothiocyanate. An EBV-positive P3HR-1 cell line processed to paraffin wax was used as a positive control and a brain section as negative control. RESULTS: EBER1/2 could not be found in the neoplastic epithelial cells in any of the tumours nor in the adjacent normal parotid tissues. Individual positive lymphocytes were present in 7 tumours. CONCLUSIONS: These results indicated that EBV is not involved in the pathogenesis of WT.

Detection and subtyping of human herpesvirus-8 in renal transplant patients before and after remission of Kaposi's sarcoma.

BACKGROUND: Kaposi's sarcoma (KS) is a complication of renal transplantation. If the human herpesvirus-8 (HHV-8) causes KS, the virus should be present in all KS lesions and be drastically reduced or cleared from involved tissue on remission of the KS. METHODS: Fourteen renal transplant patients with cutaneous KS, including autopsy material from two cases, were investigated for the presence of HHV-8. A second skin biopsy was taken from 11 survivors, after remission of KS, from normal skin in the same anatomical region as the first biopsy. Remission was induced by reduction or cessation of immunosuppression. A peripheral blood sample was collected simultaneously with the repeat biopsy. A nested polymerase chain reaction assay was used to detect HHV-8 DNA in the biopsy tissue and peripheral blood mononuclear cells followed by direct sequencing of polymerase chain reaction product to detect any nucleotide changes. RESULTS: HHV-8 DNA was detected in all the cutaneous KS and all the visceral KS samples, as well as a number of KS-free organs including the thyroid, salivary gland, and myocardium that have not been described before. Mutations in the viral DNA could be demonstrated in all patients. The mutations found were related more to that seen in AIDS-KS cases than that found in African endemic KS cases. HHV-8 sequences could be detected in follow-up frozen skin biopsies of five patients but were negative in the equivalent formalin-fixed specimens. Viral DNA was also detected in 2 of 11 peripheral blood mononuclear cell samples collected at the time of the follow-up skin biopsies. CONCLUSION: Reduction or withdrawal of immunosuppression allows the immune system to recover sufficiently to reduce viral replication with subsequent viral persistence and low grade viral replication that coincides with clinical remission of the KS lesions. This provides further evidence for the important etiological role played by HHV-8 in the pathogenesis of posttransplant KS.

Simian immunodeficiency viruses (SIVs) from eastern and southern Africa: detection of a SIVagm variant from a chacma baboon.

Simian immunodeficiency viruses (SIVs) have been shown to infect many Old World African primate species. Thus far, no work has been published on southern African primates. In this study we investigated the genetic diversity between SIV strains from Kenyan and South African vervets (Cercopithecus aethiops pygerythrus). We amplified and sequenced a 1113 bp region of the env gene. Phylogenetic analysis of these sequences showed that all strains clustered with members of the vervet subgroup of SIVagm. The SIVs from South African vervets differed by 7% from each other and by 8-14% from the Kenyan SIV strains, while the Kenyan SIV strains differed by 10-21% from SIVagm of other east African vervets. We also isolated and sequenced, for the first time, a SIV strain from a healthy chacma baboon (Papio ursinus), caught in South Africa. Phylogenetic analysis of the env region showed the virus to be closely related to the South African vervet SIV strains, while analysis of its pol region confirmed the virus to be a SIVagm variant.

Correlation between p53 gene mutation, p53 protein labeling and PCNA expression in oral squamous cell carcinomas.

BACKGROUND: The prevalence of oral squamous cell carcinoma (OSCC) among the Black community in South Africa is unacceptably high. The association between p53 protein, and PCNA overexpression and the presence of p53 gene mutations was evaluated. MATERIALS AND METHODS: One hundred and ten formalin-fixed, paraffin-embedded blocks of OSCC were selected for immunohistochemical studies for p53 protein and PCNA expression using the DO-7 and PC10 monoclonal antibodies, respectively. DNA was extracted from fifty-five blocks and exons 5 to 9 of the p53 gene were amplified with nested primers, thereafter sequencing was performed to confirm the presence of mutations detected by single stranded conformational polymorphism. RESULTS: Fifty-six cases (51%) showed p53 expression, while fourteen mutations (25%) were detected. A significant difference was found between the PCNA index in p53 positive and p53 negative tumors while the mean PCNA index for the tumors with p53 mutations was not significantly different from the tumors without mutations. CONCLUSIONS: No association between p53 protein overexpression and p53 gene mutations could be demonstrated.

Detection of p53 gene mutations in oral squamous cell carcinomas of a black African population sample.

Mutations in the p53 gene have been reported in head and neck carcinomas. We determined the p53 mutation profile in 55 oral squamous cell carcinomas (OSCCs) from a black African population sample. DNA from all the patients were investigated using PCR amplification of the p53 gene (exons 5-9), followed by heteroduplex single-stranded conformational polymorphism (HEX-SSCP) analysis on the PCR products. Direct sequencing was performed on cases where mutations were identified. The results showed mutations in 13 of 55 (23.6%) tumours. Eleven of 13 (85%) were single base pair substitutions (9 transitions and 2 transversions), and 2 were deletions. Two novel mutations were identified: a large 63-base pair deletion, and a single base pair substitution. The mutations in our study occurred outside the head and neck tumour hot spot region (codons 238-248).

Detection of human herpes virus 8 DNA and sequence polymorphism in classical, epidemic, and iatrogenic Kaposi's sarcoma in South Africa.

The aetiology and detection of human herpes virus type 8 (HHV-8) DNA sequences in Kaposi's sarcoma (KS) is a matter of intense investigation. We report on the detection of HHV-8 DNA and sequence polymorphism in different clinicopathological subtypes of cutaneous KS samples from South Africa. The diagnosis was confirmed by histological examination in all cases. Six patients had classic KS (CKS), 3 epidemic KS (EKS), and 3 iatrogenic KS (IKS). A nested polymerase chain reaction (PCR) assay was used to detect HHV-8 DNA in cell lysates, prepared from formalin fixed, paraffin embedded sections. We investigated polymorphism in the HHV-8 DNA using single-stranded conformational polymorphism (SSCP) analysis on the PCR products, followed by direct sequencing. HHV-8 DNA was detected in all the patients with KS, irrespective of the clinicopathological subtype. Direct sequencing was performed on 5 selected cases and showed single base pair substitutions in all. The spectrum of mutations was similar to those described previously. No correlation was found between the different types of KS and sequence variation. The results support the hypothesis that HHV-8 is strongly associated with different clinicopathological subtypes of KS and confirm the occurrence of HHV-8 in patients with CKS, EKS, and IKS in South Africa.

The riminophenazines, clofazimine and B669, inhibit potassium transport in gram-positive bacteria by a lysophospholipid-dependent mechanism.

The effects of the riminophenazine antimicrobial agents clofazimine and B669 as well as those of lysophosphatidylcholine (LPC), on microbial K(+)-transporting systems were investigated in a range of Gram-positive and Gram-negative bacteria using 42K and 86Rubidium (86Rb) as tracers. Exposing the Gram-positive bacteria to 0.1-10 mg/L of the drugs resulted in a dose-related inhibition of uptake of both radiolabelled cations due primarily to the inhibition of their influx which was prevented by pretreating the microorganisms with 25 mg/L alpha-tocopherol (vitamin E) which forms a complex with lysophospholipids. In contrast, Gram-negative bacteria were resistant to riminophenazine-mediated inhibition of K(+)-transport, with only one of four well-characterised K(+)-transport system mutants of Escherichia coli, namely Kup, being affected by the antimicrobial agents. The selective antimicrobial activity of riminophenazines against Gram-positive bacteria is probably achieved by lysophospholipid-mediated inactivation of K(+)-transport, while Gram-negative microorganisms possess several K(+)-transport systems which are either inaccessible and/or insensitive to lysophospholipids. Thus, K(+)-transport systems may represent novel targets for antimicrobial agents.

Sequence variation and subtyping of human and simian T-cell lymphotropic virus type I strains from South Africa.

We report on the subtyping of South African primate T-cell lymphotropic virus type I (PTLV-I) strains by investigating the LTR region using sequence analysis and restriction fragment length polymorphism (RFLP) techniques. DNA from either uncultured peripheral blood mononuclear cells (PBMCs); cultured PBMC or cell lines of eight human T-cell lymphotropic virus type I (HTLV-I); and two simian T-cell lymphotropic virus type I (STLV-I) strains (Cercopithecus aethiops pygerythrus) were amplified by polymerase chain reaction (PCR), cloned, and sequenced. The samples originated from different geographical regions in South Africa. Phylogenetic relationships were estimated using the neighbor-joining method. The South African HTLV-I strains were of Cosmopolitan origin and similar to each other. RFLP analysis confirmed this subtyping. A divergence of 0.3 to 1.6% between the Cosmopolitan strains was observed, while the divergence between the HTLV-I and STLV-I strains ranged from 6.3 to 7%. The STLV-I strains were closely related to that of a chimpanzee, providing evidence of interspecies transmission.

Human papillomavirus DNA in oral squamous cell carcinomas from an African population sample.

BACKGROUND: The incidence of oral squamous cell carcinoma (OSCC) is on the increase in developing countries. MATERIALS AND METHODS: Formalin fixed paraffin embedded blocks of OSCCs from a Black South African population sample of peri-urban and rural origin were selected as follows: Group 1 - 57 OSCCs with a mean age of 59 years; Group 2 - 43 OSCCs all cases younger than 40 years; Group 3 - 46 OSCCs with blocks containing only tumour tissue without any normal epithelium and Group 4, a control group of 38 non-neoplastic epithelial lesions. Type specific primers were used in a standard PCR to amplify a segment of the E6 region of HPV 6, 11, 16 and 18. RESULTS: HPV 11 and 16 DNA were found in one sample each from groups 1 and 2 respectively. CONCLUSION: HPV is not an etiologic factor in the development of OSCC in the population studied.

Germline mutations of the BRCA1 gene in breast and ovarian cancer families provide evidence for a genotype-phenotype correlation.

Mutations in the BRCA1 gene, discovered in 1994, are associated with an 80-90% lifetime risk of breast cancer. We have analysed 60 families with a history of breast and/or ovarian cancer for germline mutations in BRCA1. Twenty-two different mutations were detected in 32 families (53%), of which 14 are previously unreported. We observed a significant correlation between the location of the mutation in the gene and the ratio of breast to ovarian cancer incidence within each family. Our data suggest a transition in risk such that mutations in the 3' third of the gene are associated with a lower proportion of ovarian cancer. Haplotype analysis supports previous data which suggest some BRCA1 mutation carriers have common ancestors; however, we have found at least two examples where recurrent mutations appear to have arisen independently.