Sensitive detection of circulating breast cancer cells by reverse-transcriptase polymerase chain reaction of maspin gene.

BACKGROUND: Maspin, a recently identified protein related to the family of serpins, is believed to play a role in human breast cancer. In an effort to improve the present methods of detection, we have developed a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for maspin transcript to identify small numbers of mammary carcinoma cells in the peripheral blood and bone marrow of patients with breast cancer. PATIENTS AND METHODS: Five non-neoplastic mammary tissue samples, 13 breast cancer specimens as well as 17 peripheral blood and 4 bone marrow samples from normal subjects were screened for the presence of maspin mRNA by RT-PCR. The same assay was applied to peripheral blood or bone marrow samples obtained from 29 patients with stages I to IV breast cancer. RESULTS: By RT-PCR it was possible to amplify maspin mRNA in all of the primary and metastatic breast cancer specimens, but in none of the normal hemopoietic samples from healthy donors. Thus, detection of maspin transcript in the peripheral blood or marrow of a patient known to have breast cancer is indicative of the presence of mammary carcinoma cells. In reconstitution experiments, maspin RT-PCR reliably detected 10 mammary carcinoma cells in 1 million normal peripheral-blood mononuclear cells (PBMCs). None of the 9 patients with stages I, II, or III breast cancer had maspin transcript in peripheral blood. Of note, 3 of 9 patients with stage IV breast cancer receiving systemic therapy at the time of sample collection, but only 1 of 11 patients with stage IV not receiving therapy, had detectable maspin transcript in peripheral blood. Moreover, 3 marrow specimens from stage IV patients tested positive by this assay. CONCLUSIONS: This pilot study suggests that maspin RT-PCR assay is a sensitive, specific and sufficiently rapid method for detection of small numbers of circulating cells and marrow micrometastases in breast cancer patients. The possibility of applying this assay in the detection of tumor cell contamination of both marrow and stem-cell apheresis harvests of breast cancer patients merits further investigation.

Lymphotropic herpes virus (EBV, HHV-6, HHV-8) DNA sequences in HIV negative Castleman's disease.

Aim-To evaluate the possible involvement of lymphotropic herpes viruses in Castleman's disease.Methods-Archival formalin fixed, paraffin wax embedded biopsy specimens from 16 HIV negative patients (11 with localised and five of multicentric disease) were studied. Epstein-Barr virus (EBV), human herpes virus-6 (HHV-6) and human herpes virus-8 (HHV-8) DNA was detected using PCR. PCR was also used to characterise the EBV genomes and the clonal status of the lesions.Results-EBV sequences were identified in nine (56%) cases. The main EBV genotype detected was type 1. Two (12%) cases were positive for both HHV-6 and EBV sequences. HHV-8 sequences were detected in one case of localised Castleman's disease, the sequence of which differed from that of the HHV-8 prototype. No clonal immunoglobulin gene rearrangements were found.Conclusions-EBV DNA was detected in a substantial proportion of cases, suggesting that it may have a role in the pathogenesis of Castleman's disease, unlike HHV-6 which was detected rarely. This is the first report of HHV-8 specific sequences in the localised from of the disease.

Human herpesvirus-8 DNA sequences in human immunodeficiency virus-negative angioimmunoblastic lymphadenopathy and benign lymphadenopathy with giant germinal center hyperplasia and increased vascularity.

Human herpesvirus-8 (HHV-8) DNA sequences have been reported to be strictly associated not only with various forms of Kaposi's sarcoma but also with an unusual subgroup of acquired immunodeficiency syndrome (AIDS)-related B-cell lymphomas. A possible relation of this putative virus also with multicentric Castleman's disease (MCD) has been recently suggested. We used polymerase chain reaction to look for the presence of HHV-8 sequences in a well characterized series of benign, atypical, and malignant lymphoid tissues from 45 Hodgkin's disease and 43 non-Hodgkin's lymphoma (NHL) cases, as well as from 5 MCD, 15 angioimmunoblastic lymphadenopathy (AILD), and 23 benign lymphadenopathy cases. Among the 38 AIDS-related lymphoid lesions, only 1 NHL and 1 persistent generalized lymphadenopathy (PGL) case were positive. Furthermore, among the 92 non-AIDS-related lymphoproliferative disorders, HHV-8 sequences were detected in 3 classic AILD cases and in 4 reactive lymphadenopathies. Six of 9 HHV-8 positive lymphoid lesions (1 NHL, 1 PGL, 1 AILD, and 3 reactive lymphadenopathy cases) were also positive for Epstein-Barr viral sequences. The four human immunodeficiency virus (HIV) negative lymphadenopathies positive for HHV-8 sequences showed an almost identical histology, characterized by a predominantly follicular lesion, with giant germinal center hyperplasia, and increased vascularity, resembling HIV-related lymphadenopathy and MCD. Our results, while providing the first evidence of the presence of HHV-8 sequences in AILD cases, suggest a possible association of these herpes viral sequences with a distinct histologic type of non-neoplastic lymphadenopathy, not associated with other common herpes infections.

p53 gene point mutations in relation to p53 nuclear protein accumulation in colorectal cancers.

It is known that structural alterations of the p53 tumour suppressor gene cause malignant transformation and tumour progression in colorectal mucosa. In this study, 38 colorectal cancers were analysed for mutations detected in the p53 gene by single-strand conformational polymorphism and DNA sequence analysis, and the results were compared with p53 protein expression detected by immunohistochemistry. A very strict association (P < 0.0001) was found between genetic alterations and protein accumulation, as detected by the PAb 1801 monoclonal antibody. p53 expression and gene mutations were more frequent in rectal than in colonic cancers. No relation was observed with Dukes' stage, even though most of the mutations were at exon 7 in Dukes' A-B cancers and almost all mutations at exon 8 were observed in Dukes' C-D cancers. DNA ploidy was not generally associated with p53 protein expression or gene mutations. However, 83 per cent of cases with exon 5 and 6 mutations were diploid or near-diploid and 71 per cent of cases with mutations at exons 7 and 8 were aneuploid. Tumours with p53 gene mutations at exon 5 had a higher median [3H]thymidine labelling index (17 per cent) than those with mutations at exons 6, 7, and 8 (11.8 per cent).