Megumi Oda

FBXW7 and NOTCH1 mutations in childhood T cell acute lymphoblastic leukaemia and T cell non-Hodgkin lymphoma

Mutation analysis of FBXW7 and NOTCH1 genes was performed in 55 T cell acute lymphoblastic leukaemia (T‐ALL) and 14 T cell non‐Hodgkin lymphoma (T‐NHL) patients who were treated on the Japan Association of Childhood Leukaemia Study (JACLS) protocols ALL‐97 and NHL‐98. FBXW7 and/or NOTCH1 mutations were found in 22 (40·0%) of 55 T‐ALL and 7 (50·0%) of 14 T‐NHL patients. FBXW7 mutations were found in 8 (14·6%) of 55 T‐ALL and 3 (21·4%) of 14 T‐NHL patients, and NOTCH1 mutations in 17 (30·9%) of 55 T‐ALL and 6 (42·9%) of 14 T‐NHL patients. Three (5·4%) T‐ALL and two (1·4%) T‐NHL patients had mutations in both FBXW7 and NOTCH1. FBXW7 mutations included one insertion, one deletion, one deletion/insertion and nine missense mutations. NOTCH1 mutations were detected in the heterodimerization domain (HD) in 15 cases, in the PEST domain in seven cases, and in both the HD and PEST domains in one case. Five‐year event‐free survival and overall survival for patients with FBXW7 and/or NOTCH1 mutations were 95·5% (95% CI, 71·9–99·4%) and 100% respectively, suggesting that T‐ALL patients with FBXW7 and/or NOTCH1 mutation represent a good prognosis compared to those without FBXW7 and/or NOTCH1 mutations (63·6%, P = 0·007 and 78·8%, P = 0·023, respectively).

Outcome of risk-based therapy for infant acute lymphoblastic leukemia with or without an MLL gene rearrangement, with emphasis on late effects: a final report of two consecutive studies, MLL96 and MLL98, of the Japan Infant Leukemia Study Group

We evaluated the efficacy of a treatment strategy in which infants with acute lymphoblastic leukemia (ALL) were stratified by their MLL gene status and then assigned to different risk-based therapies. A total of 102 patients were registered on two consecutive multicenter trials, designated MLL96 and MLL98, between 1995 and 2001. Those with a rearranged MLL gene (MLL-R, n=80) were assigned to receive intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT), while those with germline MLL (MLL-G, n=22) were treated with chemotherapy alone. The 5-year event-free survival (EFS) rate for all 102 infants was 50.9% (95% confidence interval, 41.0–60.8%). The most prominent late effect was growth impairment, observed in 58.9% of all evaluable patients in the MLL-R group. This plan of risk-based therapy appears to have improved the overall prognosis for infants with ALL, compared with previously reported results. However, over half the events in patients with MLL rearrangement occurred before the instigation of HSCT, and that HSCT-related toxic events comprised 36.3% (8/22) of post-transplantation events, suggesting that further stratification within the MLL-R group and the development of more effective early-phase intensification chemotherapy will be needed before the full potential of this strategy is realized.

11p15 translocations involving the NUP98 gene in childhood therapy-related acute myeloid leukemia/myelodysplastic syndrome

In a survey of childhood therapy‐related acute myeloid leukemia/myelodysplastic syndrome (t‐AML/MDS) in Japan, we found 11p15 translocations in 5 (6%) of 81 children with t‐AML/MDS. t(11;17)(p15;q21), t(11;12)(p15;q13), t(7;11)(p15;p15), inv(11)(p15q22), and add(11)(p15) were each found in one patient. Southern blotting and/or RT‐PCR analyses revealed rearrangements of the NUP98 gene in tumor samples of all five patients. Rearrangements of DDX10 were detected in t‐AML/MDS cells with inv(11), and rearrangements of HOXA9 were detected in t‐AML cells with t(7;11). The 17q21 breakpoint of t(11;17) and the 12q13 breakpoint of t(11;12)(p15;q13) coincided with the loci of the HOXB and HOXC gene families, respectively. Therefore, it is reasonable to speculate that one of the HOXB genes and one of the HOXC genes were fused to NUP98 by t(11;17) and t(11;12), respectively, in t‐AML/MDS cells. We propose that NUP98 may be a target gene for t‐AML/MDS, and that t‐AML/MDS with a fusion of NUP98 and HOX or DDX10 genes may be more frequent in children than in patients of other age groups. Genes Chromosomes Cancer 26:215–220, 1999.

Suppressive effects of human herpesvirus 6 on in vitro colony formation of hematopoietic progenitor cells

Human herpesvirus 6 (HHV‐6) has been reported to be involved in bone marrow failure after bone marrow transplantation (BMT). To elucidate the role of HHV‐6 in the marrow failure, we examined the comparative effect of two variants of HHV‐6 (HHV‐6A and HHV‐6B) and human herpesvirus 7 (HHV‐7) on in vitro colony formation of hematopoietic progenitor cells in methylcellulose semi‐solid media. Progenitor cells prepared from cord blood mononuclear cells (CBMNCs) were infected with one of these viruses at various multiplicity of infection (MOI), and were subjected to methylcellulose colony assay. Formation of both granulocyte/macrophage (CFU‐GM) and erythroid (BFU‐E) colonies was MOI‐dependently suppressed after infection with the Z29 strain of HHV‐6B. Although HHV‐6A suppressed the formation of BFU‐E colonies as efficiently as HHV‐6B, the former did not exhibit significant suppressive effect on the formation of CFU‐GM colonies at an MOI 1. HHV‐7 had no effect on hematopoietic colony formation at all. Based on frequent positivity of viral DNA in single colonies obtained from HHV‐6‐infected progenitor cells by polymerase chain reaction and in situ hybridization, direct effects of HHV‐6 on the hematopoietic progenitor cells are suggested as the cause of the suppression rather than indirect effects via accessory cells of the bone marrow. J. Med. Virol. 52:406–412, 1997.

Risk-directed treatment of infant acute lymphoblastic leukaemia based on early assessment of MLL gene status: results of the Japan Infant Leukaemia Study (MLL96).

Summary.  We studied the effectiveness of risk‐directed therapy for infants younger than 13 months of age with acute lymphoblastic leukaemia (ALL). Fifty‐five infants were assigned to different treatment programs (from December 1995 to December 1998) on the basis of their MLL gene status at diagnosis. Forty‐two cases (76·3%) had a rearranged MLL gene (MLL+) and were treated with remission induction therapy followed by sequential intensive chemotherapy, including multiple genotoxic agents (MLL9601 protocol). Haematopoietic stem cell transplantation (HSCT) was attempted if suitable donors were available. Thirteen infants (23·7%) were classified as MLL– and treated for 2·5 years with intensive chemotherapy for high‐risk B‐ALL (MLL9602 protocol). Complete remission was induced in 38 of the 42 infants (90·5%) with MLL+ ALL and in all 13 patients (100%) with MLL– disease. In the MLL+ subgroup, the estimated event‐free survival (EFS) rate at 3 years post diagnosis was 34·0% ± 7·5%, compared with 92·3% ± 7·4% in the MLL– subgroup (overall comparison, P = 0·001 by log‐rank analysis). Both age less than 6 months (hazard ratio = 6·87, 95% CI = 0·91–52·3; P = 0·013) and central nervous system (CNS) involvement at diagnosis (hazard ratio = 2·92 95% CI = 1·29–6·63; P = 0·015) were significant independent predictors of an inferior outcome. These findings indicate a strategic advantage in classifying infant ALL as either MLL+ or MLL– early in the clinical course and selecting therapy accordingly. Standard chemotherapy for high‐risk B‐lineage ALL appeared adequate for MLL– cases. Novel therapeutic initiatives are warranted for infants with MLL+ disease, particularly those with initial CNS leukaemic involvement or age less than 6 months, or both.

Suppressive effects of human herpesvirus-6 on thrombopoietin-inducible megakaryocytic colony formation in vitro

Two clinical observations, the association of human herpesvirus-6 (HHV-6) with delayed engraftment after stem cell transplantation and thrombocytopenia concomitant with exanthema subitum, prompted us to evaluate the suppressive effects of HHV-6 on thrombopoiesis in vitro. Different culture conditions for thrombopoietin (TPO)-inducible colonies in semi-solid matrices were examined. Using cord blood mononuclear cells as the source of haematopoietic progenitors, two types of colonies, megakaryocyte colony-forming units (CFU-Meg) and non-CFU-Meg colonies, were established. The former colonies were identified by the presence of cells with translucent cytoplasm and highly refractile cell membrane, most of which were positive for the CD41 antigen. Although the plating efficiency of both types was much higher under serum-containing conditions than under serum-free conditions, the proportion of CFU-Meg to non-CFU-Meg colonies was consistently higher under serum-free conditions. The plating efficiency of CFU-Meg colonies was doubled by adding stem cell factor to the serum-free matrix. The effects of two variants of HHV-6 (HHV-6A and 6B) and human herpesvirus-7 (HHV-7) on TPO-inducible colonies were then compared. HHV-6B inhibited both CFU-Meg and non-CFU-Meg colony formation under serum-free and serum-containing conditions. HHV-6A had similar inhibitory effects. In contrast, HHV-7 had no effect on TPO-inducible colony formation. Heat-inactivation and ultra-filtration of the virus sample completely abolished the suppressive effect. After infection of CD34(+) cells with HHV-6, the viral genome was consistently detected by in situ hybridization. These data suggest that the direct effect of HHV-6 on haematopoietic progenitors is one of the major causes of the suppression of thrombopoiesis.

Management of severe neutropenia with cyclosporin during initial treatment of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis

Severe neutropenia (absolute neutrophil count <500/μ1) is probably due to the combined effects of dysregulated cytokine production and chemotherapeutic agents, and is one of the risk factors in the initial treatment of patients with Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH). We report here 9 cases of neutropenic HLH, of which 8 were treated with cyclosporin (CSA, 2-6 mg/kg/day; continuous infusion, or 6mg/kg/day; per os, for periods ranging from 9 days to 8 weeks) in the initial neutropenic phase during induction treatment using corticosteroids and etoposide. Five of the 6 cases, in which CSA treatment was started early (before the second week of induction), survived the critical period with recovery of neutrophil counts within a week. The remaining 3 cases, in which CSA was introduced later or not at all, died of infection. Based on these results, we recommend a prompt short-term CSA infusion during neutropenic episodes in the most common treatment regimen of etoposide and corticosteroids in patients with HLH. Improved neutrophil recovery as a result of CSA treatment makes it possible to continue immunochemotherapy safely and obtain improved patient outcomes.

Activation of Akt is Associated With Poor Prognosis and Chemotherapeutic Resistance in Pediatric B-Precursor Acute Lymphoblastic Leukemia

Activation of the phosphoinositide 3‐kinase (PI3K)/Akt pathway, a pro‐survival pathway, plays important roles in tumor cell growth. However, the role of Akt in the pathogenesis of pediatric B‐precursor acute lymphoblastic leukemia (B‐pre ALL) remains to be clarified. This study was undertaken to explore the clinical relevance and molecular mechanisms underlying the activation of Akt (i.e., phosphorylated Akt, P‐Akt) in pediatric B‐pre ALL.

IKZF1 deletion is associated with a poor outcome in pediatric B-cell precursor acute lymphoblastic leukemia in Japan

Genetic alterations of Ikaros family zinc finger protein 1 (IKZF1), point mutations in Janus kinase 2 (JAK2), and overexpression of cytokine receptor‐like factor 2 (CRLF2) were recently reported to be associated with poor outcomes in pediatric B‐cell precursor (BCP)‐ALL. Herein, we conducted genetic analyses of IKZF1 deletion, point mutation of JAK2 exon 16, 17, and 21, CRLF2 expression, the presence of P2RY8‐CRLF2 fusion and F232C mutation in CRLF2 in 202 pediatric BCP‐ALL patients newly diagnosed and registered in Japan Childhood Leukemia Study ALL02 protocol to find out if alterations in these genes are determinants of poor outcome. All patients showed good response to initial prednisolone (PSL) treatment. Ph+, infantile, and Down syndrome–associated ALL were excluded. Deletion of IKZF1 occurred in 19/202 patients (9.4%) and CRLF2 overexpression occurred in 16/107 (15.0%), which are similar to previous reports. Patients with IKZF1 deletion had reduced event‐free survival (EFS) and overall survival (OS) compared to those in patients without IKZF1 deletion (5‐year EFS, 62.7% vs. 88.8%, 5‐year OS, 71.8% vs. 90.2%). Our data also showed significantly inferior 5‐year EFS (48.6% vs. 84.7%, log rank P = 0.0003) and 5‐year OS (62.3% vs. 85.4%, log rank P = 0.009) in NCI‐HR patients (n = 97). JAK2 mutations and P2RY8‐CRLF2 fusion were rarely detected. IKZF1 deletion was identified as adverse prognostic factor even in pediatric BCP‐ALL in NCI‐HR showing good response to PSL.

Prognostic factors of Epstein–Barr virus-associated hemophagocytic lymphohistiocytosis in children: Report of the Japan Histiocytosis Study Group

Despite several advances in the treatment of Epstein–Barr virus (EBV) in recent years, patients with Epstein–Barr virus‐associated hemophagocytic lymphohistiocytosis (EBV‐HLH) do not always show satisfactory outcomes. We here conducted a nationwide survey in Japan to identify prognostic factors of EBV‐HLH in children with this disease in an effort to improve the management and the outcomes of these patients.