Significance of four MRD markers in MRD-based treatment strategy for childhood acute lymphoblastic leukemia.

Newly diagnosed children with ALL (n=32) were treated on a protocol incorporating minimal residual disease (MRD)-based treatment decisions. MRD was monitored at 4 time points by semi-quantitative PCR detection of antigen receptor gene rearrangement, flow cytometry, quantitative RT-PCR detection of chimeric gene transcripts and overexpressed WT1 mRNA. Four patients positive for MRD at week 5 were treated with an intensified regimen. Median follow-up was 5.0 years (range 3.8-6.6 years) with a 4-year event-free survival rate of 93.8+/-4.3%. This MRD-based treatment strategy seems to be highly successful and may improve the outcomes of children with ALL. A large study is warranted.

[Comparison of micafungin and fosfluconazole as prophylaxis for invasive fungal infection during neutropenia in children undergoing chemotherapy and hematopoietic stem cell transplantation].

Invasive fungal infection (IFI) is a serious complication of chemotherapy for hematological malignancies and autologous/allogeneic hematopoietic stem cell transplantation in children and shows a high mortality rate. We performed a randomized trial comparing micafungin (MCFG), a new anti-fungal agent, with fosfluconazole, a prodrug of fluconazole (FF) conventionally used as a prophylactic agent, for prophylaxis against IFI. Cefpirome was administered as prophylaxis against bacterial infection, and meropenem+minocycline as an empiric window therapy for febrile neutropenia. MCFG 2 mg/kg/day (max 100 mg/day) and FF 10 mg/kg/day (max 400 mg/day) were both safe and effective (event free ratio of IFI, MCFG 94.4% vs FF 94.3%) without significant difference. Thus, MCFG is safe and can be used for prophylaxis against IFI in children.

[Encouraging results of stem cell transplantation following a melphalan-preceding intensified preparative regimen for refractory acute leukemia in children].

The results of allogeneic stem cell transplantation for patients with chemotherapy-resistant non-remission acute leukemia have been very poor. We have used a melphalan-preceding intensified preparative regimen in which a six-day interval is set between melphalan 70 mg/m2 and the main part of the preparative regimen to avoid toxicity in 15 consecutive pediatric patients with refractory acute leukemia. Only one patient died of transplant-related toxicity within 100 days of transplant. One patient had refractory anemia originating from donor cells at three months after transplant. Eight patients relapsed at a median of six months after transplant; therefore, five of 15 patients have been in complete remission (CR) for a median of 61 months. Four of six patients who did not have blasts in their peripheral blood before melphalan are in CR This method seems to be safe and effective for refractory acute leukemia.

[The role of cord blood stem cell transplantation in children; a retrospective analysis of 39 cases in a single institute].

We report the results of 39 children who underwent cord blood stem cell transplantation (CBSCT) at our institute during the period from February 1996 to July 2005. The patients consisted of 9 with non-malignant disease, 26 with malignant disease and 4 with Epstein-Barr virus (EBV) associated disease. The median age of the patients was 4 years and 8 months (range, 6 months to 16 years 2 months). The median infused cell dose was 4.9 (range, 1.7-11.4) x 10(7)/kg. Thirty-four transplants were from HLA-mismatched donors, and 33 patients underwent a tacrolimus-containing regimen for GVHD prophylaxis. As for CBSCT as the first transplant, 3 out of 4 children with non-malignant disease achieved engraftment after CBSCT with the use of a reduced-intensity conditioning regimen. For acute leukemia, 3 patients out of 5 in their first remission and 2 out of 9 in advanced stage at CBSCT continue in remission at the time of writing. Fourteen patients received CBSCT as a second or a third transplant. None of 4 patients who underwent CBSCT as rescue therapy after rejection/graft failure achieved engraftment. It should be emphasized that EBV-associated disease seems to be a suitable disease for CBSCT, because all of the 4 patients who underwent CBSCT are still in CR.

NK-cell repertoire is feasible for diagnosing Epstein-Barr virus-infected NK-cell lymphoproliferative disease and evaluating the treatment effect.

Epstein-Barr virus (EBV) occasionally infects T and NK cells and causes EBV-infected T/NK-cell lymphoproliferative disease (LPD), which comprises chronic active EBV infection, EBV-associated hemophagocytic syndrome, mosquito allergy, hydroa vacciniforme, aggressive NK-cell leukemia, and NK/T-cell lymphoma. The diagnosis is proven by the monoclonal proliferation of EBV-infected T or NK cells, which is a time-consuming and complicated method. T-cell monoclonality is helpful for the screening of EBV-infected T-cell LPD in patients with EBV-genome burden and is easily shown with T-cell-receptor rearrangement or the T-cell repertoire, whereas NK-cell monoclonality is difficult to prove due to its lacking such rearranged receptors. We investigated a set of killer immunoglobulin-like receptors (KIRs) and also CD94-NKG2 heterodimers on NK cells, namely the NK-cell repertoire. Skewed repertoires were seen in all patients with EBV-infected NK-cell LPD, but not in any patients with EBV-infected T-cell LPD and were restored only after successful treatment. The normal KIR repertoire is variable for each individual and it seems difficult to detect minimal residual EBV-infected lymphocytes. However, the NK-cell repertoire is feasible for identifying EBV-infected NK-cell LPD and evaluating the treatment effect.

Activated proliferation of B-cell lymphomas/leukemias with the SHP1 gene silencing by aberrant CpG methylation.

Previously we showed reduced protein and mRNA expression of the SHP1 gene in lymphoma/leukemia cell lines and patient specimens by Northern blot, RT-PCR, Western blot, and immunohistochemical analyses. In this study, aberrant methylation in the SHP1 gene promoter was detected in many B-cell leukemia/lymphoma cell lines as well as in patient specimens, including diffuse large B-cell lymphoma (methylation frequency 93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%) by methylation-specific PCR, bisulfite sequencing, and restriction enzyme-mediated PCR analyses. The methylation frequency was significantly higher in high-grade MALT lymphoma cases (100%) than in low-grade MALT lymphoma cases (70%), which correlated well with the frequency of no expression of SHP1 protein in high-grade (80%) and low-grade MALT lymphoma (54%). It suggests that the SHP1 gene silencing with aberrant CpG methylation relates to the lymphoma progression. SHP1 protein expression was recovered in B-cell lines after the treatment of the demethylating reagent: 5-aza-2'-deoxycytidine. Transfection of the intact SHP1 gene to the hematopoietic cultured cells, which show no expression of the SHP1 gene, induced growth inhibition, indicating that gene silencing of the SHP1 gene by aberrant methylation plays an important role to get the growth advantage of the malignant lymphoma/leukemia cells. The extraordinarily high frequency (75 to 100%) of CpG methylation of the SHP1 gene in B-cell lymphoma/leukemia patient specimens indicates that the SHP1 gene silencing is one of the critical events to the onset of malignant lymphomas/leukemias as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. These data support the possibility that the SHP1 gene is one of the tumor suppressor genes.

Gene silencing of the tyrosine phosphatase SHP1 gene by aberrant methylation in leukemias/lymphomas.

High-frequent silencing of hematopoietic cell-specific protein-tyrosine phosphatase SHP1 gene by promoter methylation was detected in various kinds of leukemias and lymphomas, as well as in many hematopoietic cell lines, which is supported by our previous observation of strong decrease of SHP1 mRNA and protein. The promoter methylation of the SHP1 gene was clearly correlated with the clinical stage. Loss of heterozygosity with microsatellite markers near the SHP1 gene was shown in 79% of informative acute lymphoblastic leukemia cases. These results suggest that functional loss of SHP1 is associated with the pathogenesis of leukemias/lymphomas.