Mariko Eguchi

An effective chemotherapeutic regimen for acute myeloid leukemia and myelodysplastic syndrome in children with Down's syndrome

In recent pediatric collaborative studies of acute myeloid leukemia (AML), patients with Downs syndrome (DS) have better outcome than other patients when they were treated according to their intensive AML protocols. This may be attributed to enhanced sensitivity of DS AML cells to selected chemotherapeutic agents. We evaluated a less intensive chemotherapeutic regimen which was specifically designed for children with AML-DS. Remission induction chemotherapy consisted of daunorubicin (25 mg/m2/day for 2 days), cytosine arabinoside (100 mg/m2/day for 7 days), and etoposide (150 mg/m2/day for 3 days). Patients received one to seven courses of consolidation therapy of the same regimen. Thirty-three patients were enrolled on the study and their clinical, hematologic and immunophenotypic features were analyzed. Of the 33 patients, all were younger than 4 years and diagnosed as having acute megakaryoblastic leukemia or myelodysplastic syndrome. All patients achieved a complete remission and estimated 8 year event-free survival rate was 80 ± 7%. Three patients relapsed and two died due to cardiac toxicity and one due to septic shock. The results of our study showed that patients with AML-DS constitute a unique biologic subgroup and should be treated according to a less intensive protocol designed for AML-DS.

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.

Tandem duplication of the FLT3 gene is infrequent in infant acute leukemia

Leukemia CBFb/PEBP2b and a myosin heavy chain in acute myeloid leukemia. Science 1993; 261: 1041–1044. 3 Langabeer SE, Walker H, Gale RE, Wheatley K, Burnett AK, Goldstone AH, Linch DC. Frequency of CBFb/MYH11 fusion transcripts in patients entered into the UK MRC AML trials. Br J Haematol 1997; 96: 736–739. 4 Poirel H, Radford-Weiss I, Rack K, Troussard X, Veil A, Valensi F, Picard F, Guesnu M, Leboeuf D, Melle J, Dreyfus F, Flandrin G, Macintyre E. Detection of the chromosome 16 CBFb-MYH11 fusion transcript in myelomonocytic leukemias. Blood 1995; 85: 1313–1322. 5 Ritter M, Thiede C, Schackel U, Schmidt M, Alpen B, Paschenberg U, Mohr B, Ehninger G, Neubauer A. Underestimation of inversion (16) in acute myeloid leukaemia using standard cytogenetics as compared with polymerase chain reaction: results of a prospective investigation. Br J Haematol 1997; 98: 969–972. 6 Costello R, Sainty D, Lecine P, Cusenier A, Mozziconacci M-J, Arnoulet C, Maraninchi D, Gastaut J-A, Imbert J, Lafage-Pochitaloff M, Gabert J. Detection of CBFb/MYH11 fusion transcripts in acute myeloid leukemia: heterogeneity of cytological and molecular characteristics. Leukemia 1997; 11: 644–650. 7 Wong KF, Kwong YL. Trisomy 22 in acute myeloid leukemia: a marker for myeloid leukemia with monocytic features and cytogenetically cryptic inversion 16. Cancer Genet Cytogenet 1999; 109: 131–133. 8 Langabeer SE, Walker H, Rogers JR, Burnett AK. Wheatley K, Swirsky D, Goldstone AH, Linch DC, Incidence of AMLI/ETO fusion transcripts in patients entered into the MRC AML trials. Br J Haematol 1997; 99: 925–928. 9 Hackwell SM, Robinson DO, Harvey JF, Ross FM. Identification of false-positive CBFb/MYH11 RT-PCR results. Leukemia 1999; 13: 1617–1619.

Application of fluorescence in situ hybridization to detect residual leukemic cells with 9;22 and 15;17 translocations

We performed fluorescence in situ hybridization (FISH) upon 9;22 and 15;17 translocation-positive bone marrow cells to monitor the clinical course of 46 patients with chronic myelocytic leukemia (CML) and nine with acute promyelocytic leukemia (AML M3) who received chemotherapy and/or bone marrow transplantation (BMT). M-BCR-ABL and PML-RARα probes were used to detect translocations of t(9;22) and t(15;17), respectively. Signals from CML patients treated with interferon (17 patients) or BMT (29 patients) were 0.5–15% positive for the 9;22 translocation. Among nine M3 patients who received extensive chemotherapy or BMT, 1–5% were positive for the 15;17 translocation. A highly sensitive FISH procedure using both translocation probes and a whole chromosome Y probe was established and applied to eight sex-mismatched BMT patients (seven CML and one AML M3), in which 0.1–0.6% of signals positive for the specific translocations were detected. These results suggested that interphase FISH is powerful enough to identify minor cell populations of 9;22 or 15;17 translocations after therapy, as well as to detect specific chromosome abnormalities at diagnosis.

Interphase Fluorescence In Situ Hybridization Overcomes Pitfalls of G-Banding Analysis with Special Reference to Underestimation of Chromosomal Aberration Rates

Fluorescence in situ hybridization (FISH) is suitable for detecting different types of chromosome aberrations on interphase nuclei even in specimens with no or few chromosome metaphases. However, it is not known why FISH is superior to conventional G-banding analysis. The sensitivity of interphase FISH was compared to that of G-banding analysis in 288 leukemia/lymphoma patients for 10 different types of chromosome aberrations: t(9;22) (M- and m-BCR), t(8;21), 11q23 abnormalities, t(15;17), del(5)/-5, del(13)/-13, +8, -7, and +12. The results revealed that t(15;17) positive cells could not proliferate well in culture, leading to underestimation of abnormality by G-banding. Monosomy 7 in acute myelocytic leukemia (AML) and myelodysplastic syndrome (MDS) as well as trisomy 12 and deletion chromosome 13 in chronic lymphocytic leukemias (CLL) were also severely underestimated by G-banding. On the other hand, no discrepancies were observed in t(8;21), t(9;22), translations involving 11q23, or in trisomy 8. These findings indicate the superiority of interphase FISH over conventional cytogenetics for detecting chromosome abnormalities in small clones, especially for monosomy 7 or (15;17) translocations.

Fluorescence in situ Hybridization Analysis of 12;21 Translocation in Japanese Childhood Acute Lymphoblastic Leukemia

Fluorescence in situ hybridization (FISH) analysis was applied to detect t(12;21) using two yeast artificial chromosome probes and cosmid probes covering the TEL(ETV6) and the AML1 gene to clarify the incidence of abnormality of t(12;21) in Japanese childhood acute lymphoblastic leukemia (ALL). We detected seven TEL/AML1 fusion positive patients (9.5%), all of whom were diagnosed as B‐lineage ALL, among 74 childhood ALL. On the other hand, no TEL/AML1 fusion positive patients were found among 37 adult ALL. The incidence among Japanese seemed to be lower than that among other nations. Of the seven patients with the TEL/AML1 fusion, five exhibited normal karyotype, one was t(8;12)(q11;p13), i(21q) and the remaining one exhibited a near‐triploid karyotype in conventional G‐banding. The FISH method clearly demonstrated that all patients with the TEL/AML1 fusion had subpopulations of leukemic cells with deletion of the normal TEL allele, which is significant for understanding the progression of leukemia with t(12;21).

Frequent allelic loss of the RB , D13S319 and D13S25 locus in myeloid malignancies with deletion/translocation at 13q14 of chromosome 13, but not in lymphoid malignancies

In order to identify a commonly deleted region of 13q14 on chromosome 13, we performed fluorescence in situ hybridization (FISH) on 17 patients with myeloid malignancies and 12 patients with lymphoid leukemia/lymphoma who exhibited either deletion or translocation at 13q14. Three cosmid probes (RB, D13S319 and D13S25) hybridizing to sequences on 13q14 were used. Fourteen of the 17 patients with myeloid malignancies (82.4%) exhibited allelic loss at the RB, D13S319 and D13S25 locus, whereas only three of the 12 patients with lymphoid malignancies (25.0%) exhibited loss within these loci. These three patients had chronic lymphocytic leukemia (CLL). Six, two and one of the remaining nine lymphoid leukemia/lymphoma patients had breakpoints centromeric to the RB gene, telomeric to D13S25 and within the D13S319 locus, respectively. A high frequency of allelic loss was found using these probes in patients with myeloid malignancies, compared to in patients with leukemia in the lymphoid origin, except CLL patients. These results indicate that loss of the RB gene itself or a region between RB and D13S319, which includes commonly deleted loci, may play an important role in myeloid leukemogenesis.

Establishment of a myeloid leukaemia cell line (Kasumi-4) with t(9;22;11)(q34;q11;q13), inv(3)(q21q26) and the EVI1 gene activation from a patient with chronic myelogenous leukaemia in blast crisis

A novel human leukaemia cell line (Kasumi‐4) was established from the peripheral blood of a 6‐year‐old girl suffering from chronic myelogenous leukaemia (CML) in blast crisis. The Kasumi‐4 cells had the following characteristic features: undifferentiated blasts which were positive for CD34, CD33 and CD13 surface markers, but negative for myeloperoxidase platelet peroxidase, CD36, CD41 and CD42; chromosome abnormalities of t(9;22;11) (q34;q11;q13), inv(3)(q21q26); and elevated expression of EVI1 gene which is located at chromosome band 3q26. Megakaryocytic maturation was not observed in the liquid culture following the addition of TPA, IL‐3, IL‐6 or GM‐CSF. b2‐a2 type of BCR‐ABL chimaeric messenger RNA was detected by RT‐PCR analysis. This is the first leukaemia cell line with a three‐way translocation containing the Ph chromosome and the second cell line with an inv(3)(q21q26). This cell line appears to be useful for studying the mechanisms of leukaemogenesis involving these chromosomal abnormalities and related oncogenes.

Establishment of a novel cell line (TS-2) of pre-B acute lymphoblastic leukemia with a t(1;19) not involving the E2A gene

The t(1;19)(q23;p13) translocation involving the E2A gene on chromosome 19p13.3 is a nonrandom translocation that is often seen in childhood pre-B-cell acute lymphoblastic leukemia (ALL). However, recent studies have demonstrated the presence of immunophenotypic and molecular heterogeneity among patients with the cytogenetically identical chromosome translocation. Here we report a novel pre-B ALL cell line, TS-2, with t(1;19) translocation not involving the E2A gene. The breakpoint of t(1;19) in TS-2 was demonstrated to be at 19p13.3, a region indistinguishable from the locus of the E2A gene, by cytogenetic study and fluorescence in situ hybridization. However, rearrangement of the E2A gene was not detected in TS-2 by Southern blot analysis. Moreover, the expressions of PBX1 or E2A/PBX1 fusion genes were not detected by an extensive study with Northern blot analysis and reverse transcription-polymerase chain reaction. These findings suggest that TS-2 may have a genetic abnormality involving uncharacterized gene(s) at 19p13.3 distinct from the E2A gene and, therefore, may be useful for investigating the heterogeneity of molecular pathogenesis in leukemias with t(1;19)(q23;p13) translocation.

Detection of Residual Host Cells in Sex–mismatched Bone Marrow Transplantation in Various Hematological Diseases by Fluorescence in situ Hybridization

Thirty–eight sex–mismatched bone marrow transplantation patients with various hematological diseases were followed–up using fluorescence in situ hybridization. Probes specific for various transloca–tions, the X chromosome (DXZ1) and the whole Y chromosome (WCPY), were used to assess successful engraftment and residual host cells. The combination of translocation and WCP Y probes enabled the identification of host and donor cells in addition to the identification of malignant vs. normal cells in the transplant recipient. Fifteen patients were sequentially followed up. The results obtained using the combination of translocation plus WCP Y probes were more reliable than those with DXZ1 plus WCP Y probes, or the translocation probe alone, especially when the percentage of residua] leukemic cells detected by the translocation probe alone was around the cut–off level.