ALBERT

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: 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: 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: 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: 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 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.