Shuya Kusumoto

Fusion of TEL/ETV6 to a Novel ACS2 in Myelodysplastic Syndrome and Acute Myelogenous Leukemia With t(5;12)(q31;p13)

We identified a novel human long fatty acyl CoA synthetase 2 gene, ACS2, as a new ETV6 fusion partner gene in a recurrent t(5;12)(q31;p13) translocation in a patient with refractory anemia with excess blasts (RAEB) with basophilia, a patient with acute myelogenous leukemia (AML) with eosinophilia, and a patient with acute eosinophilic leukemia (AEL). ACS2 is expressed in the brain and bone marrow and is highly conserved in man and rats. The resulting ETV6/ACS2 fusion transcripts showed an out‐frame fusion of exon 1 of ETV6 to exon 1 of ACS2 in the AEL case, an out‐frame fusion of exon 1 of ETV6 to exon 11 of ACS2 in the AML case, and a short in‐frame fusion of ETV6 exon 1 to the 3′ untranslated region of ACS2 in the RAEB case. Reciprocal ACS2/ETV6 transcripts were identified in two of the cases. Fluorescence in situ hybridization (FISH) analysis with ETV6 cosmids on 12p13, and BACs and P1s on 5q31, demonstrated that the 5q31 breakpoints of the AML and AEL cases involved the 5′ portion of the ACS2 gene, and that the 5q31, breakpoint of the RAEB case involved the 3′ portion of the ACS2 gene. None of the resulting chimeric transcripts except for the ACS2/ETV6 transcript in the RAEB case led to a fusion protein. Disruption of the second ETV6 allele by t(12;19) was detected in the AML case by FISH analysis. These observations suggest that the disruption of ETV6 and/or ACS2 may lead to the pathogenesis of hematologic malignancies with t(5;12)(q31;p13). Genes Chromosomes Cancer 26:192–202, 1999.

Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype.

Point mutations of the transcription factor AML1 are associated with leukemogenesis in acute myeloblastic leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and mutations in the second tyrosine kinase domain of the Fms-like tyrosine kinase 3 (FLT3) gene represent the most frequent genetic alterations in AML. However, such mutations per se appear to be insufficient for leukemic transformation. To evaluate whether both AML1 and FLT3 mutations contribute to leukemogenesis, we analyzed mutations of these genes in AML M0 subtype in whom AML1 mutations were predominantly observed. Of 51 patients, eight showed a mutation in the Runt domain of the AML1 gene: one heterozygous missense mutation with normal function, five heterozygous frameshift mutations and two biallelic nonsense or frameshift mutations, resulting in haploinsufficiency or complete loss of the AML1 activities. On the other hand, a total of 10 of 49 patients examined had the FLT3 mutation. We detected the FLT3 mutation in five of eight (63%) patients with AML1 mutation, whereas five of 41 (12%) without AML1 mutation showed the FLT3 mutation (P=0.0055). These observations suggest that reduced AML1 activities predispose cells to the acquisition of the activating FLT3 mutation as a secondary event leading to full transformation in AML M0.

Prognostic significance of the null genotype of glutathione S-transferase-T1 in patients with acute myeloid leukemia: increased early death after chemotherapy

We investigated the prognostic significance of genetic polymorphism in glutathione-S transferase mu 1 (GSTM1), glutathione-S transferase theta 1 (GSTT1), NAD(P)H:quinone oxidoreductase (NQO1) and myeloperoxidase (MPO), the products of which are associated with drug metabolism as well as with detoxication, in 193 patients with de novo acute myeloid leukemia (AML) other than M3. Of the patients, 64.2% were either homozygous or heterozygous for GSTT1 (GSTT1+), while 35.8% showed homozygous deletions of GSTT1 (GSTT1−). The GSTT1− group had a worse prognosis than the GSTT1+ group (P = 0.04), whereas other genotypes did not affect the outcome. Multivariate analysis revealed that GSTT1− was an independent prognostic factor for overall survival (relative risk: 1.53; P = 0.026) but not for disease-free survival of 140 patients who achieved complete remission (CR). The rate of early death after the initiation of chemotherapy was higher in the GSTT1− group than the GSTT1+ group (within 45 days after initial chemotherapy, P = 0.073; within 120 days, P = 0.028), whereas CR rates and relapse frequencies were similar. The null genotype of GSTT1 might be associated with increased toxicity after chemotherapy.

Prognostic value of p53 gene mutations and the product expression in de novo acute myeloid leukemia

Abstract: In acute myeloid leukemia (AML), p53 mutations are reportedly infrequent but associated with a poor prognosis. The majority of mutations are missense mutations, which generally lead to accumulation of nuclear p53 protein. However, the prognostic significance of the accumulation remains unknown in AML. In this study, we compared the prognostic value of p53 mutations versus accumulation of the product. p53 mutations were found in 9 (4.5%) of 200 patients with de novo AML. The p53 mutation detectable (mutation+) group had a worse prognosis (p=0.0009) than the mutation not detectable (mutation−) group. Multivariate analysis showed that the p53 mutation was an independent factor (p=0.005) for short overall survival as well as 60 yr or older (p=0.001) and unfavorable karyotypes (p=0.001). In 79 of the 200 patients, the expression of p53 was studied by immunocytochemistry (ICC) using anti‐p53 monoclonal antibody (DO‐7). All samples carrying missense mutations (N=6) were positive for ICC in over 15% of nuclei of each sample, chosen as the optimized cutoff value of p53 accumulation. Accumulation was thus found in 14 of the 79 patients. However, there was no prognostic difference according to the accumulation, because the mutation−/accumulation+ group (N=8) tended to have a good prognosis. These findings indicate that molecular detection of p53 mutations yields better prognostic information than ICC. In a subset of AML, p53 protein might be accumulated without mutation presumably due to upstream signals of p53.

Magnetic resonance imaging patterns in patients with multiple myeloma

Sixty‐one consecutive patients with multiple myeloma were studied with magnetic resonance (MR) imaging of the spine. Sagittal T1‐weighted and short inversion time (TI) inversion recovery (STIR) images were obtained. The MR patterns of the bone marrow were classified as diffuse (D) (n = 26), nodular (N) (n = 11), D + N (n = 13) or normal (n) (n = 11). Abnormal patterns were seen in 50 (82%) of the 61 patients. Correlations were found between the MR imaging patterns and some laboratory findings (WBC, haematocrit, platelet count, serum albumin, and percentage of marrow plasmacytosis). The survival of the patients with abnormal MRI patterns was significantly poorer than that of the patients with normal patterns. However, the survival of patients with a nodular pattern did not differ from those with a normal pattern. The MR imaging pattern of the bone marrow in patients with multiple myeloma is a useful factor in the assessment of prognosis.

Refractory anemia with severe dysplasia: clinical significance of morphological features in refractory anemia

Refractory anemia (RA) in myelodysplastic syndromes (MDS) are very heterogeneous diseases regarding their morphology, clinical features and survival. We proposed the new designations ‘RA with severe dysplasia (RASD)’ and ‘RA with minimal dysplasia (RAminiD)’. In our criteria, RASD is considered present if a bone marrow (BM) examination shows Pseudo-Pelger–Huet anomalies of mature neutrophils ⩾3% and/or micromegakaryocytes (mMgk) of megakaryocytes ⩾10% in RA patients. RAminiD is defined as RA cases other than RASD. After the reclassification of 58 primary RA patients, the group was composed of 45 RAminiD and 13 RASD patients. The blast percentage in the BM and the frequency of cytogenetic abnormalities observed in the RASD patients were intermediate between those in the RAminiD and RAEB patients. The analysis of survival curves revealed differences among the three groups; the RASD patients had lower survival probabilities than those of the RAminiD group, and significantly higher probabilities than those of the RAEB group. (RAminiD vs RASD, P = 0.06; RASD vs RAEB, P = 0.004.) Our data indicate that in RA patients, RASD is a distinct subset of RA with an unfavorable clinical outcome.

New system for assessing the prognosis of refractory anemia patients

Refractory anemia (RA) is a very heterogeneous disease regarding biological and clinical features. The International Prognostic Scoring System (IPSS) was useful for assessing the prognosis in the whole group of 219 myelodysplastic syndrome (MDS) patients. However, the IPSS was not sufficient in 132 RA patients. To predict survival and freedom from acute myeloid leukemia (AML) evolution, we investigated individual prognostic factors based on the clinical parameters (age, gender, morphologic features, cytopenias and cytogenetics) of 132 RA patients using univariate and multivariate analyses. Based on the results, we devised a new system for assessing the prognosis of RA patients. In our system, RA patients with pseudo-Pelger-Huët anomalies ⩾3% were classified as high risk (12 patients); of patients without pseudo-Pelger–Huët anomalies ⩾3%, those with intermediate/poor karyotype according to IPSS, Hb ⩽6 g/dl or mMgk ⩾10% were classified as intermediate risk (57 patients); and those without high or intermediate risk were classified as low risk (67 patients). In our system, the analyses of both survival times and leukemia-free survival times revealed significant differences among the three groups (P < 0.0001).

Bone marrow patterns in patients with aplastic anaemia and myelodysplastic syndrome: observations with magnetic resonance imaging

Abstract: Bone marrow magnetic resonance imaging (MRI) was obtained in 48 patients with myelodysplastic syndrome (MDS) (35 cases) or aplastic anaemia (AA) (13 cases). The lower thoracic and lumbar spine were evaluated on sagittal plane using a 1.5 Tesla superconducting MR unit with a surface coil. Pulse sequence of STIRs (TR 2000 msec, TI 160 msec, TE 20 msec) were applied. Four distinct patterns of signal intensity (SI) on the STIR images were classified as follows: pattern 1, homogeneously low SI; 2, marginally high SI; 3, heterogeneously high SI; 4, homogeneously high SI. In all 13 patients with AA, STIR images initially revealed pattern 1. In 25 of 35 cases with MDS patients, the STIR images were initially classified as pattern 3. The STIR images of 6 AA and 5 MDS patients with a clinical response to treatment showed pattern 2 similar to that of normal marrow distribution. The STIR images of MDS patients showed an abnormal distribution of SI. Significant signal changes in the STIR images can be observed in successive examinations of the patients, thus facilitating follow‐up of the disease and treatment. MRI of the bone marrow provides a noninvasive means of grossly examining a large fraction and is a useful technique in patients with aplastic anaemia or myelodysplastic syndrome.

Trisomy 8 may not be related to the pathogenesis of myelodysplastic syndromes: disappearance of trisomy 8 in a patient with refractory anaemia without haematological improvement

To the Editor: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders characterized by ineffective haemopoiesis. The most common single chromosomal aberrations are chromosome 5 ,7 and 8 abnormalities in MDS patients. Recently, several authors reported the close relationship between abnormality of genes in the long arm of chromosomes 7 and 5 and the genesis of myeloid malignancies. The majority of the patients with del(7) have been shown to have the smallest overlapping deleted segment within 7q22 (1). Although the segment of DNA on chromosome 7q22.1 was reported to contain specific gene(s) associated with MDS (2), the translocation breakpoint in a patient with acute myeloid leukaemia was mapped to 7q21.3 (3). These findings suggest that there are multiple genes in bands 7q21-7q22 which are related to the pathogenesis of myeloid malignancy. Critical regions on the long arm of chromosome 5 have been identified either as a common deleted segment at 5q31 (4) or as microdeletion at 5q33 (5) . This evidence suggests that these segments on 5q contain tumour suppressor genes that could play an important role in the pathogenesis of MDS. On the other hand, little is known about the MDSrelated genes on chromosome 8. We describe a patient with MDS whose chromosomal abnormality, trisomy 8, disappeared following treatment with oral la-hydroxyvitamin D, (vit D,) but without haematological improvement. We discuss the relationship between trisomy 8 and the pathogenesis of MDS. A 52-yr-old man was initially found to have thrombocytopenia in October 1991 at another hospital. The peripheral blood cell count revealed haemoglobin of 13.4 g/dl, platelet count of 89x109/1, and leucocyte count of 4.7x109/1. Although he had no symptoms, he consulted Saitama Medical School Hospital in April 1995 because his peripheral blood cell counts gradually decreased. Physical examination disclosed no enlargement of either the liver or the spleen. Laboratory tests revealed haemoglobin of 11.6 g/dl, platelet count of 77x 109/1, and leucocyte count of 3.9Ox1O9/1 with 52% neutrophils, 36% lymphocytes, 12% monocytes and 1 % eosinophils. The vitamin B,, and folic acid levels were within the normal range. Coombs tests were negative. The bone marrow (BM) showed normocellularity with 0.8% blasts. The dysplastic features of BM cells were minimal in the trilineages. The BM cells showed 47, XU, +8 karyotype in 9 metaphases on 20 analysed. The patient was diagnosed with MDS, refractory anaemia (RA). In May 1995, he started treatment with oral la-hydroxyvitamin D, (vit D3) (2 pg/d). The percentages of trisomy 8 cells were 40-60% on conventional cytogenetic analyses of the subsequent BM aspirations. In November 1996, 18 months after the initiation of treatment, conventional cytogenetic analysis of the BM cells showed a normal karyotype, 46XY [20/20]. Trisomy 8 was not detected by fluorescence in situ hybridization (FISH) analysis of the BM interface cells using a biotin-labelled chromosome 8 alpha satellite probe (D8Z2, Oncor, Gaithersburg, USA). Despite the disappearance of trisomy 8, his peripheral blood cell counts and BM findings were not improved. Trisomy 8 is one of the most frequent chromosomal aberrations observed in MDS patients. Patients with MDS with BM karyotypes that are normal, del(5q), del(20q) and -Y have relatively good prognoses, whereas relatively poor prognoses are present in those with complex abnormalities or chromosome 7 abnormalities (-7/7q-). Patients with trisomy 8 single aberration have intermediate prognoses (6). In RA patients with monosomy 7, the haematological improvement was closely associated

The pathogenetic mechanism of myeloid malignancies associated with deletions of the long arm of chromosome 20 can not be explained by a "one hit" model. An acute myeloid leukemia patient who developed with 20q- clone during complete remission for 9 years.

To the Editor: Acquired deletions of the long arm of chromosome 20 (20qx) are found in myeloid malignancies (1±3). Previous studies demonstrated a cytogenetic common deleted region spanning 20q11.2±q13.1 (4). In molecular studies, the critical region in myeloproliferative disorders (MPD) covered the 18 cM interval between SRC and D20S17, whereas the critical region in myelodysplastic syndromes (MDS) extends between D20S174 and D20S17 (4). There is the possibility that these segments on 20q may contain tumour suppressor gene(s), and the gene(s) may play an important role in the pathogenesis of myeloid malignancies. We report here the clinical and cytogenetic ®ndings in an acute myeloid leukaemia (AML) patient who developed with the 20qx clone during complete remission (CR) for 116 months and discuss the pathogenetic mechanism of myeloid malignancy associated with 20qx. A 52-yr-old man was initially found to have leukocytosis in February 1990 at another hospital. He had suffered from chronic hepatitis of hepatitis C virus (HCV) for 6 yr. Physical examination disclosed mild hepatomegaly. Laboratory tests revealed a haemoglobin level of 8.1 g/dl, a platelet count of 85r10/l, and a leukocyte count of 12.8r10/l with 95% blasts, 3% lymphocytes, 1% monocytes and 1% eosinophils. Serum GOT and GPT levels were slightly elevated at 47 IU/l and 46 IU/l, respectively. Bone marrow (BM) showed hypercellularity with 87.8% blasts. The blasts were positive for peroxidase in a cytochemical study, and CD13,34,7 and HLA-DR in ̄ow cytometry analysis. Initial cytogenetic analysis of BM cells revealed a normal karyotype. The patient was diagnosed as having AML (M1). He received intensive induction chemotherapy with Ara-C (120 mg) for 10 d, daunorubicin (DNR) (60 mg) for 6 d, 6-mercaptopurine (6-MP) (100 mg) for 10 d, and prednisolone (PSL) (60 mg) for 4 d, according to the Japan Adult Leukemia Study Group (JALSG) 89 protocol (5). After the ®rst induction therapy the blasts disappeared from the peripheral blood and the blood cell count was normalized. The blasts in the BM decreased to <5%. He achieved CR, and thereafter three courses of consolidation and six courses of maintenance therapy, according to the JALSG 89 protocol, were performed. Following the postremission chemotherapy, the laboratory tests revealed a haemoglobin level of 13.4 g/dl, a platelet count of 227r10/l, and a leukocyte count of 7.50r10/l with 62% neutrophils, 23% lymphocytes, 14% monocytes and 1% eosinophils. Then, 57 months after the start of chemotherapy, the BM cells showed a sole karyotypic abnormality, 46,XY,del(20)(q11), in nine of 20 metaphases analyzed. Despite the presence of 20qx, his peripheral blood ®ndings did not show cytopenia and the BM cells did not show dysplastic features. Thereafter, the percentage of the 20qx karyotype in BM cells was gradually reduced (Table 1). Ninety-nine months after the start of chemotherapy, the BM cells showed the 46,XY,del(20)(q11) karyotype in only two of 25 metaphases analyzed. Interphase ̄uorescence in situ hybridization (FISH) analysis using a D20S108 locus-speci®c probe (VYSIS, Tokyo, Japan), whose signal was con®rmed to be deleted in 20qx by metaphase FISH analysis, was performed. Interphase FISH analysis gave one signal in 2.4% (25 of 1032 BM monoEur J Haematol 2000: 65: 210±211 Printed in UK. All rights reserved Copyright # Munksgaard 2000