Pre-Analytical Modification of Serum miRNAs: Diagnostic Reliability of Serum miRNAs in Hemolytic Diseases.

Circulating microRNAs (miRNAs) are useful biomarkers of hemolysis. Since blood cells are the main origins of circulating miRNAs, we evaluated blood cell-related pre-analytical modification of the miRNA signatures during blood drawing and serum processing. The levels of miRNA before and after ex vivo blood drawing were analyzed with the reverse transcriptase-based polymerase chain reaction method. Furthermore, the changes of miRNA signatures caused by different time-lag between blood drawing and serum preparation by 24 h were evaluated. Finally, we compared the miRNA levels between leftover samples and samples of hemolytic diseases. Blood drawing procedure induced increments of red blood cell (RBC)-related miRNAs (miR-451a, miR-486) about 2-fold. One hour standing of blood samples before serum separation induced almost the same increases in RBC-related miRNAs. To test the clinical usefulness of miR-451a as a biomarker of hemolytic diseases, we analyzed miRNAs of samples from 10 normal subjects, 30 leftover samples in the clinical laboratory, and 20 samples from patients with hemolytic diseases. Serum miR-451a significantly increased in patients with hemolytic anemia more than the levels of pre-analytical modification. In conclusion, the pre-analytical modification of serum miRNAs did not disturb the usefulness of RBC-derived miRNAs as biomarkers of hemolytic diseases.

Aberrant methylation and decreased expression of the RIZ1 gene are frequent in adult acute lymphoblastic leukemia of T-cell phenotype.

Retinoblastoma protein-interacting zinc finger, RIZ1, is a tumor suppressor gene that is inactivated in various solid tumors. However, the role of the RIZ1 gene has not been well examined in adult acute lymphoblastic leukemia (ALL). We analyzed the expression and promoter methylation status of the RIZ1 gene in patients with newly diagnosed ALL by quantitative real-time reverse transcription polymerase chain reaction (PCR) and methylation-specific PCR, respectively. RIZ1 expression in 67 cases of ALL (mean 1.043) was decreased compared with that in normal bone marrow (mean 1.471) (p = 0.030). Methylation was detected in 11 of 71 patients (15.5%) but not in healthy controls. Methylation was associated with decreased RIZ1 expression in many ALL cases examined, but this was not statistically significant. In T-ALL, RIZ1 methylation was more frequent (63.6%) than in B-ALL (6.7%) (p < 0.0001) and the decrease of RIZ1 expression was more significant than in B-ALL (p = 0.045). 5-Aza-2'-deoxycytidine treatment of MOLT-4 cells with RIZ1 methylation induced demethylation of RIZ1 and restoration of expression. Forced RIZ1 expression in T-ALL cell lines suppressed cell growth accompanied by G2/M arrest and apoptosis. No mutations were found by PCR-single strand conformation polymorphism analysis in hotspots of the gene. These results suggest that RIZ1 is inactivated in adult ALL, and this inactivation is associated with methylation in T-ALL.

[Long-term remission after nonmyeloablative allogeneic bone marrow transplantation in a relapsed Hodgkin lymphoma patient with CNS involvement].

Intracranial involvement of relapsed Hodgkin lymphoma (HL) is quite rare and its prognosis is very poor. We report a patient with relapsed HL with central nervous system (CNS) involvement after autologous stem cell transplantation successfully treated with allogeneic bone marrow transplantation with reduced intensity conditioning regimen. A standard therapy for relapsed CNS HL has not yet established. To the best of our knowledge, this is the first case report describing allogeneic stem cell transplantation for relapsed CNS HL in an elderly patient. Our results in this case suggest that allogeneic stem cell transplantation could be a useful therapeutic option in relapsed CNS HL patients, if their CNS lesions are controlled before stem cell transplantation.

Aberrant methylation of the RIZ1 gene in myelodysplastic syndrome and acute myeloid leukemia.

We performed methylation specific PCR analysis on the RIZ1 promoter in MDS and AML. Methylation was detected in 17 of 34 MDS (50%) and 22 of 72 AML (31%) (p=0.053). Methylation was detected in eleven of 17 secondary AML from MDS (65%), and eleven of 55 de novo AML (20%) (p=0.0005). Bisulfite sequence revealed methylation at many CpG sites in the promoter. Decreased RIZ1 expression was accompanied by methylation in six of nine samples examined, while it was also observed in seven of 13 without methylation. Treatment of AML cells, that have RIZ1 methylation, with 5-Aza-dC, induced growth suppression with RIZ1 restoration. Our results suggest that the RIZ1 gene is inactivated in MDS and AML in part by methylation, whereas another mechanism should be involved in others.