ALBERT

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Description: We have recently demonstrated the presence of Kaposi's sarcoma–associated herpesvirus (KSHV) in cultured bone marrow (BM) stromal dendritic cells from all patients with myeloma studied. To show that these findings were not an artifact of tissue culture, we performed in situ hybridization (ISH) and polymerase chain reaction (PCR) to detect KSHV in BM core biopsies. Using ISH to open reading frame-72 (ORF 72), we localized KSHV to BM dendritic cells in 17 of 20 patients with myeloma, 2 patients with plasmacytosis associated with the acquired immunodeficiency syndrome, and 1 case of aplastic anemia. In contrast, BM from normal subjects (n = 4) and patients with lymphoma and leukemia (n = 21) did not contain KSHV. PCR amplification with KSHV primers demonstrated product in fresh BM biopsy samples from 6 of 7 myeloma patients, whereas three normal marrows contained no amplified product. These findings suggest that KSHV, possibly through alterations in the BM microenvironment and production of viral interleukin-6 (vIL-6), may stimulate and maintain abnormal plasma cell proliferation in myeloma and related disorders.

Description: p53 mutations are found in a variety of neoplasia. B-immunoblastic lymphoma (BIBL) is a rapidly progressive, aggressive lymphoma. As patients with acquired immunodeficiency syndrome (AIDS) live longer, BIBL is becoming an increasing problem. We asked three questions in our study. What is the frequency of p53 mutations in BIBL? Is it more frequent in patients with AIDS? Can immunohistochemical staining of lymph nodes for expression of p53 substitute for mutational analysis of p53 to detect lymphomas with mutated p53? Exons 5, 6, 7, 8 of the p53 gene (hot-spots for mutations) were amplified and examined for mutations by single-strand conformation polymorphism (SSCP) analysis. Altered migration was observed in 7 of 52 BIBL samples. Of these, 4 of 25 were from individuals infected with human immunodeficiency virus (HIV) and 3 of 27 were not infected with HIV. Direct sequencing of amplified material confirmed the presence of mutations in exons 5, 7, 8 of p53. A total of 26 BIBL as well as other lymphoma/leukemia samples, stained strongly by immunohistochemistry with three antibodies directed against human p53. Five of 6 BIBL samples with p53 mutations stained strongly for p53, but 20 lymphoma samples with no detectable p53 mutations also stained strongly for p53. Of note, however, 10 hyperplastic, nonmalignant lymph nodes from individuals either infected or not infected with HIV had negligible staining for p53 protein. In conclusion, p53 mutations occur in about 14% BIBL samples; the frequency of p53 mutations in BIBL in individuals with and without AIDS was similar. Positive p53 immunohistochemistry did not correlate with detectable p53 mutations in the same tissue, but positive immunohistochemical staining for p53 was only found in neoplastic lymph nodes. This latter finding provides a strong warning that p53 immunochemistry with available reagents cannot be used to determine which tumors have mutations of p53.

Description: We have recently demonstrated the presence of Kaposi's sarcoma–associated herpesvirus (KSHV) in cultured bone marrow (BM) stromal dendritic cells from all patients with myeloma studied. To show that these findings were not an artifact of tissue culture, we performed in situ hybridization (ISH) and polymerase chain reaction (PCR) to detect KSHV in BM core biopsies. Using ISH to open reading frame-72 (ORF 72), we localized KSHV to BM dendritic cells in 17 of 20 patients with myeloma, 2 patients with plasmacytosis associated with the acquired immunodeficiency syndrome, and 1 case of aplastic anemia. In contrast, BM from normal subjects (n = 4) and patients with lymphoma and leukemia (n = 21) did not contain KSHV. PCR amplification with KSHV primers demonstrated product in fresh BM biopsy samples from 6 of 7 myeloma patients, whereas three normal marrows contained no amplified product. These findings suggest that KSHV, possibly through alterations in the BM microenvironment and production of viral interleukin-6 (vIL-6), may stimulate and maintain abnormal plasma cell proliferation in myeloma and related disorders.

Description: p53 mutations are found in a variety of neoplasia. B-immunoblastic lymphoma (BIBL) is a rapidly progressive, aggressive lymphoma. As patients with acquired immunodeficiency syndrome (AIDS) live longer, BIBL is becoming an increasing problem. We asked three questions in our study. What is the frequency of p53 mutations in BIBL? Is it more frequent in patients with AIDS? Can immunohistochemical staining of lymph nodes for expression of p53 substitute for mutational analysis of p53 to detect lymphomas with mutated p53? Exons 5, 6, 7, 8 of the p53 gene (hot-spots for mutations) were amplified and examined for mutations by single-strand conformation polymorphism (SSCP) analysis. Altered migration was observed in 7 of 52 BIBL samples. Of these, 4 of 25 were from individuals infected with human immunodeficiency virus (HIV) and 3 of 27 were not infected with HIV. Direct sequencing of amplified material confirmed the presence of mutations in exons 5, 7, 8 of p53. A total of 26 BIBL as well as other lymphoma/leukemia samples, stained strongly by immunohistochemistry with three antibodies directed against human p53. Five of 6 BIBL samples with p53 mutations stained strongly for p53, but 20 lymphoma samples with no detectable p53 mutations also stained strongly for p53. Of note, however, 10 hyperplastic, nonmalignant lymph nodes from individuals either infected or not infected with HIV had negligible staining for p53 protein. In conclusion, p53 mutations occur in about 14% BIBL samples; the frequency of p53 mutations in BIBL in individuals with and without AIDS was similar. Positive p53 immunohistochemistry did not correlate with detectable p53 mutations in the same tissue, but positive immunohistochemical staining for p53 was only found in neoplastic lymph nodes. This latter finding provides a strong warning that p53 immunochemistry with available reagents cannot be used to determine which tumors have mutations of p53.