ALBERT

Description: A human herpesvirus-8 (HHV-8) enzyme-linked immunosorbent assay (ELISA) with a whole virus lysate as antigen was developed and used to measure the seroprevalence rate and levels of IgG antibodies to HHV-8 in sera/plasma of various patient groups and blood donors. The virus antigen was prepared from the KS-1 cell line, which produces lytic virus, and therefore contains a broad array of viral proteins. Seroprevalence studies using this ELISA showed the following: 10 of 91 blood donors (11%) had an average HHV-8 antibody titer of 118; 67 of 72 (93%) classic Kaposi's sarcoma (KS) patients were positive with an average titer of 14,111; and 57 of 62 (92%) KS/human immunodeficiency virus (HIV) patients were positive with an average titer of 4,000. A study on a very limited number of serial serum samples from patients before and after diagnosis with KS showed highly elevated antibody titers to HHV-8 virus after KS lesions developed. Preliminary data show that 50% of the sera from HIV-1+ homosexual patients contain IgG antibodies to HHV-8 suggesting that this population is at high risk for developing KS. Antibody results correlated well with the confirmatory immunofluorescent assays (IFA) using KS-1 cells as the substrate. This HHV-8 IgG antibody detection ELISA is sensitive and specific and does not cross-react with Epstein-Barr virus (EBV) or other human herpesviruses. The results of this HHV-8 antibody survey suggest that this rapid ELISA assay can be used to screen large numbers of sera to find those at risk for developing KS.

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: The tumor suppressor genes p16INK4A and p15INK4B map to the 9p21 chromosomal locus and are either homozygously deleted or mutated in a wide range of human cancer cell lines and tumors. Although chromosome 9 abnormalities have been described in non-Hodgkin's lymphomas (NHLs), to date, the mutational status of these genes has not been determined for these malignancies. A total of five cell lines and 75 NHLs were examined for homozygous deletions or point mutations in the coding regions of both the p15 and p16 genes using Southern blot and/or polymerase chain reaction-single-strand conformation polymorphism analyses. Homozygous deletions of either the p16 gene or both the p15 and p16 genes were observed in one diffuse large B-cell lymphoma cell line and two uncultured lymphomas consisting of one large B-cell and one mixed T-cell lymphoma. In contrast, point mutations were not detected in either the cell lines or lymphomas. These results indicate that the rate of alterations in the p15 and p16 genes is low for lymphomas, but loss of p16 and/or p15 may be involved in the development of some lymphomas.

Description: Sequence analysis of the retinoic acid receptor-alpha (RAR alpha) gene from a subline of HL-60 cells (RA-res) stably resistant to all-trans retinoic acid (RA) disclosed a single-base change in codon number 411, the same C to T transition previously reported in an independently selected HL-60 RA resistant clone by Robertson et al (Blood 80:1885, 1992). This mutation eliminates a FokI restriction endonuclease site. Using primers framing this mutation in exon 9 of the RAR alpha gene, we showed that polymerase chain reaction products amplified from either mRNA or genomic DNA templates from the RA-res subline were completely resistant to FokI digestion whereas those from wild-type (wt) HL-60 cells could be digested to completion. The lack of a normal allele in the RA-res cells was confirmed by mixing experiments and hybridization analyses. Southern blot analysis of DNA from the RA-res and wt cells versus control placental DNA indicated that the RAR alpha gene is not haploid. The independent isolation of the same RAR alpha mutation in different laboratories suggests either that the mutation exits in a small subpopulation in the wt line or that this is a mutational “hot spot.” Furthermore, the results indicate that if a dominant negative mode of resistance is involved in the RA-res subline, this must involve interference with the function of heterologous receptor proteins such as the retinoid X receptors. The lack of any normal RAR alpha in this subline may facilitate studies of the mode of action of retinoids.

Description: Somatic hypermutation (SHM) targets primarily the immunoglobulin variable region (IgV) genes in germinal center (GC) B cells, thereby allowing antibody affinity maturation. A malfunction of SHM, termed aberrant somatic hypermutation (ASHM), was found in about 50% of diffuse large B-cell lymphomas (DLBCLs), leading to mutations in the 5′ sequences of multiple genes, including oncogenes. Although the SHM mechanism is largely unknown, it was shown to require the activation-induced cytidine deaminase (AID) gene. AID mRNA is expressed in GC B cells and GC-derived lymphomas, but the pattern of expression of the AID protein is not known. Using 2 specific antibodies, here we show that the AID protein can be detected in GC centroblasts and their transformed counterpart (Burkitt lymphoma) but not in pre-GC B cells and post-GC neoplasms, including B-cell chronic lymphocytic leukemia and multiple myeloma. DLBCLs displayed variable levels of AID expression, which did not correlate with IgV ongoing hypermutation, ASHM, or disease subtype. Finally, both in normal and malignant B cells the AID protein appeared predominantly localized in the cytoplasm. These results indicate that the AID protein is specifically expressed in normal and transformed GC B cells; nonetheless, its predominantly cytoplasmic localization suggests that additional mechanisms may regulate its function and may be altered during lymphomagenesis. (Blood. 2004;104:3318-3325)

Description: Previously, we have found that the loss of heterozygosity (LOH) was frequently observed on chromosome 6q in acute/lymphoma-type adult T-cell leukemia (ATL), suggesting a putative tumor-suppressor gene for ATL may be present on chromosome 6q. To further define a region containing this gene, we performed fine-scale deletional mapping of chromosome 6q in 22 acute/lymphomatous ATL samples using 24 highly informative microsatellite markers. LOH was found in 9 samples (40.9%) at 1 or more of the loci examined. Of the 9 samples, 8 shared the same smallest commonly deleted region flanked by D6S1652 and D6S1644 (6q15-21). The genetic distance between these two loci is approximately 4 cM. These results suggest that a putative tumor-suppressor gene on chromosome 6q15-21 probably plays a very important role in the evolution of acute/lymphomatous ATL. Our map provides key information toward cloning the gene.

Description: The 3′ end of the VWF gene was screened in the affected members of 3 different families with type 2A (phenotype IID) von Willebrand disease (vWD). Exons 49 to 52 of the VWF gene were amplified and screened for mutations by chemical cleavage mismatch detection. Mismatched bands were detected in exon 52 of 2 patients and in exon 51 of a third patient. Using direct DNA sequencing, a heterozygous G8562A transition leading to a Cys2008Tyr substitution was found in all the patients in family 1, and a T8561A transversion leading to a Cys2008Ser substitution was found in both patients from family 2. In a patient from a third family, an 8-base deletion from nucleotide 8437 to 8444 was identified in exon 51. The 2 mutations in exon 52 were reproduced by in vitro site-directed mutagenesis of full-length von Willebrand factor (vWF) cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWFs for these 2 mutations exhibited the typical aberrant vWF:Ag multimer pattern seen in the plasma of the patients. These 3 mutations demonstrate the importance of other carboxy-terminal cysteines in addition to the reported Cys2010 residue, in the normal dimerization of vWF, and their essential role in the assembly of normal multimeric vWF.

Description: R115777 is a potent farnesyltransferase (FTase) inhibitor with substantial antitumor activity in preclinical models. We conducted a phase 1 study (3 + 3 design) of R115777 in patients with myelodysplastic syndrome (MDS). R115777 was administered twice daily (3-weeks-on/1-week-off schedule for 8 weeks) (starting dosage, 300 mg by mouth twice daily; total, 600 mg). Maintenance therapy at the dose/schedule tolerated during induction could be continued until toxicity or lack of benefit. Twenty-one patients with MDS were treated (median age, 66 years). Four (19%) patients had ras mutations (n-ras,3; k-ras, 1). Objective responses (hematologic improvement, 3; partial remission, 2; or complete remission, 1) were seen in 6 of 20 (30%) evaluable patients, only 2 of whom had ras mutations. Response sequences were unusual in some patients who had increases in platelet counts without intervening aplasia. Other responders demonstrated an initial, albeit modest, myelosuppressive effect. The maximum tolerated dose was 400 mg by mouth twice a day. The most frequent side effect was myelosuppression. Dose-limiting toxicities (fatigue and confusion) occurred at 900 mg by mouth total daily dose. R115777 inhibited HDJ-2 prenylation and suppressed the activity of FTase, but not of the related geranylgeranyltransferase I enzyme, in peripheral blood mononuclear cells. Modulation of Akt, Erk, and signal transducer and activator of transcription 3 (STAT3) phosphorylation was variable, and responses occurred even without their down-regulation. Reductions in serum tumor necrosis factor-α (TNF-α) levels by day 7 showed a trend toward correlation with response (P = .09). We conclude that, at doses that are well tolerated, R115777 markedly inhibits the FTase target and has antitumor activity in MDS. (Blood. 2003;102:4527-4534)

Description: The mechanism involved in the association between antiphospholipid antibodies and thrombosis or fetal loss remains unclear. We assessed the biosynthesis of thromboxane A2 and prostacyclin in 31 samples from 25 patients with lupus anticoagulant and in 32 controls. The urinary excretion of the major thromboxane metabolite of platelet origin (11- dehydrothromboxane B2) was very significantly increased (P less than .0003) in the patients. In contrast, the urinary metabolite reflecting the vascular production of prostacyclin (2,3-dinor-6-keto-prostaglandin F1 alpha) was much less increased (P less than .02). We found no correlation between the levels of anticardiolipin antibodies and the urinary excretion of 11-dehydro-thromboxane B2. Six patients with elevated urinary 11-dehydrothromboxane B2 were treated with low-dose aspirin (20 mg/d during 7 days). In these patients, there was a close relationship between the extent of inhibition of the thromboxane urinary metabolite (72%) and serum thromboxane B2 (79%). In contrast, the urinary excretion of 2,3-dinor-6-ketoprostaglandin F1 alpha was nearly unchanged (13% reduction). In addition, the F(ab')2 fragments isolated from six patients presenting increased urinary 11-dehydro- thromboxane B2 enhanced the generation of thromboxane B2 (P = .04) and the release of 14C serotonin (P = .009) by normal washed platelets, as compared with F(ab')2 from controls. In summary, our study shows that in patients with lupus anticoagulant, platelet activation may occur without a compensatory increment in the vascular biosynthesis of prostacyclin. This observation may be crucial to cause or reflect an increased risk for thrombosis. In addition, our results may suggest a rationale for antiplatelet agents for the prophylaxis of thrombosis in many patients with the antiphospholipid syndrome.

Description: Allelotype analysis of adult T-cell leukemia (ATL) was undertaken for the first time to identify chromosomal loci relevant to the development of acute/lymphomatous ATL. Loss of heterozygosity (LOH) was screened using 94 highly polymorphic microsatellite markers, distributed among all nonacrocentric, autosomal chromosomes. In each of the 22 cases, DNA obtained from their leukemic cells in acute/lymphomatous phase was compared with their constitutional DNA from mononuclear cells in chronic or remission phase. Allelic losses of at least on one chromosome arm occurred in 91% of the cases (20 individuals). Among 39 chromosome arms, allelic losses were observed on 31 arms at least for one sample. A high frequency of allelic loss (〉30%) was seen on chromosome arms 6q (41%) and 17p (48%). The mean fractional allelic loss (FAL) was 0.109. These findings suggest that a novel tumor suppressor gene on chromosome arm 6q, as well as the p53 gene on chromosome arm 17p, probably have an important role in the development of acute/lymphomatous ATL. © 1998 by The American Society of Hematology.