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Cytogenetic/molecular heterogeneity is the hallmark of multiple myeloma (MM). However, we recently showed that the serine/threonine kinase PDPK1 and its substrate RPS6KA3 (also termed RSK2) are universally active in MM, and play pivotal roles in myeloma pathophysiology. In this study, we assessed involvement of aberrant miR-375 repression in PDPK1 overexpression in MM. An analysis of plasma cells from 30 pre-malignant monoclonal gammopathies of undetermined significance and 73 MM patients showed a significant decrease in miR-375 expression in patient-derived plasma cells regardless of the clinical stage, compared to normal plasma cells. Introduction of miR-375 reduced PDPK1 expression in human myeloma cell lines (HMCLs), indicating that miR-375 is the dominant regulator of PDPK1 expression. In addition, miR-375 introduction also downregulated IGF1R and JAK2 in HMCLs. CpG islands in the MIR375 promoter were pathologically hypermethylated in all 8 HMCLs examined and in most of 58 patient-derived myeloma cells. Treatment with SGI-110, a hypomethylating agent, and/or trichostatin A, a histone deacetylase inhibitor, increased miR-375 expression, but repressed PDPK1, IGF1R and JAK2 in HMCLs. Collectively, these results show the universal involvement of overlapping epigenetic dysregulation for abnormal miR-375 repression in MM, which is likely to contribute to myelomagenesis and to subsequent myeloma progression by activating oncogenic signalling pathways.

Pediatric acute megakaryoblastic leukemia in non-Down syndrome (AMKL) is a unique subtype of acute myeloid leukemia (AML). Novel CBFA2T3-GLIS2 and NUP98-KDM5A fusions recurrently found in AMKL were recently reported as poor prognostic factors. However, their detailed clinical and molecular characteristics in patients treated with recent improved therapies remain uncertain. We analyzed molecular features of 44 AMKL patients treated on two recent Japanese AML protocols, the AML99 and AML-05 trials. We identified CBFA2T3-GLIS2, NUP98-KDM5A, RBM15-MKL1, and KMT2A rearrangements in 12 (27%), 4 (9%), 2 (5%), and 3 (7%) patients, respectively. Among 459 other AML patients, NUP98-KDM5A was identified in 3 patients, whereas CBFA2T3-GLIS2 and RBM15-MKL1 were only present in AMKL. GATA1 mutations were found in 5 patients (11%). Four-year overall survival (OS) and event-free survival (EFS) rates of CBFA2T3-GLIS2-positive patients in AMKL were 41.7% and 16.7%, respectively. Three-year cumulative incidence of relapse in CBFA2T3-GLIS2-positive patients was significantly higher than that of CBFA2T3-GLIS2-negative patients (75.0% vs. 35.7%, P = 0.024). In multivariate analyses, CBFA2T3-GLIS2 was an independent poor prognostic factor for OS (HR, 4.34; 95% CI, 1.31-14.38) and EFS (HR, 2.95; 95% CI, 1.20-7.23). Furthermore, seven (54%) of 13 infant AMKL patients were CBFA2T3-GLIS2-positive. Notably, out of 7 CBFA2T3-GLIS2-positive infants, six (86%) relapsed and five (71%) died. Moreover, all of CBFA2T3-GLIS2-positive patients who experienced induction failure (n = 3) were infants, indicating worse prognosis of CBFA2T3-GLIS2-positive infants. These findings indicated the significance of CBFA2T3-GLIS2 as a poor prognostic factor in AMKL patients, particularly in infants.

Background Overexpression of CXC chemokine receptor 4 (CXCR4+) is a poor prognostic factor in adult acute myeloid leukemia (AML); however, its prognostic significance in pediatric AML is unclear. Procedure This retrospective study examined the prognostic significance of CXCR4+ in pediatric AML patients enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 study. Results In the total cohort (n = 248), no significant differences were observed between CXCR4+ patients (n = 81) and CXCR4− patients (n = 167) in terms of 3-year overall survival (OS) (69.4% vs. 75.2%, P = 0.44). However, there was a significant difference in 3-year OS between CXCR4+ and CXCR4− patients in the low-risk (LR) group (n = 93; 79.2% vs. 98.3%, P = 0.007). CXCR4+ patients in the t(8;21) AML without KIT mutation group had a significantly worse 3-year OS than CXCR4− patients (n = 44; 76.1% vs. 100.0%, P = 0.01). Multivariate Cox regression analysis identified CXCR4+ as a poor prognostic factor for OS in LR AML patients (hazard ratio, 11.47; P = 0.01). Consistent with the data for survival analysis, CXCR4+ patients in the t(8;21) AML group had a higher incidence of splenomegaly than CXCR4− patients (25.9% vs. 5.9%, P = 0.03). Conclusions These results suggest that CXCR4+ is a poor prognostic factor for LR patients, particularly t(8;21) patients without KIT mutation. The poor outcome was only applicable to OS, not relapse-free survival (RFS); thus, CXCR4+ may be associated with a poor prognosis after recurrence. Intensive therapy, including administration of CXCR4 antagonists, may be promising for pediatric AML patients with LR.

Background On the basis of results of previous Japanese trials for myeloid leukemia in Down syndrome (ML-DS), the efficacy of risk-oriented therapy was evaluated in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-D05 study. Procedure All patients received induction chemotherapy that consisted of pirarubicin, intermediate-dose cytarabine, and etoposide. Patients who achieved complete remission (CR) after initial induction therapy were stratified to the standard risk (SR) group and received four courses of reduced-dose intensification therapy. Patients who did not achieve CR were stratified to the high risk (HR) group and received intensified therapy that consisted of continuous or high-dose cytarabine. Results A total of 72 patients were eligible and evaluated. One patient died of sepsis during initial induction therapy. Sixty-nine patients were stratified to SR and two patients to HR. No therapy-related deaths were observed during intensification therapy. The 3-year event-free and overall survival rates were 83.3% ± 4.4% and 87.5% ± 3.9%, respectively. Age at diagnosis less than 2 years was a significant favorable prognostic factor for risk of relapse (P = 0.009). Conclusions The attempt of risk-oriented prospective study for ML-DS was unsuccessful, but despite the dose reduction of chemotherapeutic agents, the overall outcome was good, and further dose reduction might be possible for specific subgroups.

The deleted in colorectal carcinoma (DCC) gene at 18q21 encodes a netrin-1 receptor, a tumor suppressor that prevents cell growth. While allele loss or decreased expression of DCC has been associated with the progression of solid tumors and hematologic malignancies, including leukemias and malignant lymphomas, its involvement has not been evaluated in multiple myeloma (MM), a plasma cell malignancy characterized by complex and heterogenous molecular abnormalities. We here show that 10 of 11 human myeloma-derived cell lines (HMCLs) expressed non-translated aberrant DCC transcriptional variants, in which exon 2 fuses with intron 1 instead of exon 1 (mt.DCC). Among them, two co-expressed wild type transcripts (wt.DCC), while eight co-expressed the splicing variant (sv.DCC) lacking exon 1. The remaining HMCL expressed only sv.DCC. In addition, analyses revealed that there were two types of mt.DCC that differed in their fusion of intron 1 with exon 2. In patient-derived samples from 30 MM and 8 monoclonal gammopathy of undetermined significance (MGUS) patients, wt.DCC was expressed in 53% of MM, but not in MGUS, while 23% of MM and 75% of MGUS expressed only sv.DCC. Considering that 25% of MGUS, 57% of MM, and 91% HMCLs expressed mt.DCC, our results suggest that the acquisition of mt.DCC might be a secondary genetic change in plasma cell dyscrasia.

Myeloid malignancy with Down syndrome (ML-DS) is estimated to have a step-wise leukemogenesis including GATA1 mutation. Trisomy 21 is essential for ML-DS; however, we do not know exactly which gene or genes located on chromosome 21 are necessary for the ML-DS. We report a female infant with transient myeloproliferative disorder (TMD) and partial trisomy 21. SNP array analysis showed 10 Mb amplification of 21q22.12-21q22.3, which included DYRK1A, ERG, and ETS but not the RUNX1 gene. With two other reported TMD cases having partial trisomy 21, DYRK1A, ERG, and ETS were the most likely genes involved in collaboration with the GATA1 mutation.

We have identified a novel SPAG9-JAK2 fusion in a B-cell precursor acute lymphoblastic leukemia (ALL) with t(9;17)(p24;q21) and a poor outcome, using paired-end transcriptome sequencing. Homozygous and hemizygous deletions of CDKN2A/2B, and hemizygous deletions of PAX5, BTG1, CDK6, ADARB2, and IKZF1 were also identified by multiple ligation-dependent probe amplification and single nucleotide polymorphism array analyses. Having both a tyrosine kinase-activating rearrangement and genomic lesions affecting lymphoid transcription factors suggested that the leukemia was of the Philadelphia chromosome (Ph)/BCR-ABL1-like ALL subtype and that JAK2 inhibitors might be able to overcome this aggressive ALL with SPAG9-JAK2.

Acute lymphoblastic leukemia (ALL) occasionally develops before or after the onset of Langerhans cell histiocytosis (LCH). The mechanism of LCH developing after ALL remains unclear; thus the clonality of LCH developing during maintenance chemotherapy for T-cell ALL (T-ALL) was investigated. The T-ALL and LCH cells tested had the same T-cell receptor (TCR) gamma rearrangement. Mutation analysis of the NOTCH1 gene revealed 7213C>T (Q2405X) in exon 34 in T-ALL and LCH cells, but 5156T>C (I1719T) in exon 27 only in T-ALL. Polymerase chain reaction-restriction fragment length polymorphism analysis revealed three patterns of NOTCH1 mutations in T-ALL cells. The results suggest that the T-ALL and LCH cells were derived from a common precursor with TCR rearrangement and a single NOTCH1 mutation, rather than LCH cells developing from a minor clone of T-ALL with single NOTCH1 mutation.

The monocytic leukemia zinc finger protein KAT6A (formerly MOZ) gene is recurrently rearranged by chromosomal translocations in acute myeloid leukemia (AML). KAT6A is known to be fused to several genes, all of which have histone acetyltransferase (HAT) activity and interact with a number of transcription factors as a transcriptional coactivator. The present study shows that the leucine twenty homeobox (LEUTX) gene on 19q13 is fused to the KAT6A gene on 8p11 in a therapy-related AML with t(8;19)(p11;q13) using the cDNA bubble PCR method. The fusion transcripts contained 83 nucleotides upstream of the first ATG of LEUTX and are presumed to create in-frame fusion proteins. LEUTX is known to have a homeobox domain. Expression of the LEUTX gene was only detected in placenta RNA by RT-PCR, but not in any tissues by Northern blot analysis. The putative LEUTX protein does not contain any HAT domain, and this is the first study to report that KAT6A can fuse to the homeobox gene. The current study, with identification of a new partner gene to KAT6A in a therapy-related AML, does not elucidate the mechanisms of leukemogenesis in KAT6A-related AML but describes a new gene with a different putative function.

The development of oral squamous cell carcinoma (OSCC) is a multistep process that requires the accumulation of genetic alterations. To identify genes responsible for OSCC development, we performed high‐density single nucleotide polymorphism array analysis and genome‐wide gene expression profiling on OSCC tumors. These analyses indicated that the absent in melanoma 2 (AIM2) gene and the interferon‐inducible gene 16 (IFI16) mapped to the hematopoietic interferon‐inducible nuclear proteins. The 200‐amino‐acid repeat gene cluster in the amplified region of chromosome 1q23 is overexpressed in OSCC. Both AIM2 and IFI16 are cytoplasmic double‐stranded DNA sensors for innate immunity and act as tumor suppressors in several human cancers. Knockdown of AIM2 or IFI16 in OSCC cells results in the suppression of cell growth and apoptosis, accompanied by the downregulation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells activation. Because all OSCC cell lines have reduced p53 activity, wild‐type p53 was introduced in p53‐deficient OSCC cells. The expression of wild‐type p53 suppressed cell growth and induced apoptosis via suppression of nuclear factor kappa‐light‐chain‐enhancer of activated B cells activity. Finally, the co‐expression of AIM2 and IFI16 significantly enhanced cell growth in p53‐deficient cells; in contrast, the expression of AIM2 and/or IFI16 in cells bearing wild‐type p53 suppressed cell growth. Moreover, AIM2 and IFI16 synergistically enhanced nuclear factor kappa‐light‐chain‐enhancer of activated B cells signaling in p53‐deficient cells. Thus, expression of AIM2 and IFI16 may have oncogenic activities in the OSCC cells that have inactivated the p53 system. (Cancer Sci 2012; 103: 782–790)

Adult T-cell leukemia/lymphoma (ATLL) is a malignant tumor caused by latent human T-lymphotropic virus 1 (HTLV-1) infection. We previously identified a common breakpoint cluster region at 10p11.2 in acute-type ATLL by spectral karyotyping. Single nucleotide polymorphism array comparative genomic hybridization analysis of the breakpoint region in three ATLL-related cell lines and four patient samples revealed that the chromosomal breakpoints are localized within the enhancer of polycomb 1 (EPC1) gene locus in an ATLL-derived cell line (SO4) and in one patient with acute-type ATLL. EPC1 is a human homologue of the E(Pc) enhancer of polycomb gene of Drosophila. Inappropriate expression of the polycomb group gene family has been linked to the loss of normal gene silencing pathways, which can contribute to the loss of cell identity and malignant transformation in many kinds of cancers. In the case of the SO4 cell line, which carried a der(10)t(2;10)(p23;p11.2) translocation, EPC1 was fused with the additional sex combs-like 2 (ASXL2) gene at 2p23.3 (EPC1/ASXL2). In the case with an acute-type ATLL, who carried a der(10)del(10)(p11.2)del(10)(q22q24) translocation, a putative truncated EPC1 gene (EPC1tr) was identified. Overexpression of EPC1/ASXL2 enhanced cell growth in T-leukemia cells, and a GAL4-EPC1/ASXL2 fusion protein showed high transcriptional activity. Although a GAL4-EPC1tr fusion protein did not activate transcription, overexpression of EPC1tr accelerated cell growth in leukemia cells, suggesting that the EPC1 structural abnormalities in the SO4 cell line and in the patient with acute-type ATLL may contribute to leukemogenesis. © 2009 Wiley-Liss, Inc.

Background. Mixed-lineage leukemia (MLL)-partial tandem duplication (PTD) is associated with poor prognosis in adult acute myeloid leukemia (AML), but its relationship to pediatric AML is unknown. Procedure. One hundred fifty-eight newly diagnosed AML patients, including 13 FAB-M3 and 10 Down syndrome (DS) patients, who were treated on the Japanese Childhood AML Cooperative Treatment Protocol AML 99 were analyzed for MLLPTD, as well as internal tandem duplication (ITD) and the kinase domain mutation (D835Mt) in the FLT3 gene. Results. We found MLL-PTD in 21 (13.3%) of 158 AML patients, but not in FAB-M3 or DS patients. The differences between patients with and without MLLPTD were significant for 3-year overall survival (OS) (56.3% vs. 83.2%, P ¼0.018), disease-free survival (DFS) (41.7% vs. 69.6%, P ¼0.010), and relapse rate (RR) (54.3% vs. 27.6%, P ¼0.0085) of 135 AML patients excluding the FAB-M3 and DS patients. Furthermore, ITD and D835Mt in the FLT3 gene were found in 17 (12.6%) and 8 (5.9%) of these 135 patients, respectively. The differences between patients with FLT3-ITD and the wild-type allele were significant for 3-year OS (35.3% and 84.3%, P

We have identified a novel fusion partner of MLL, namely the mastermind like 2 (MAML2 gene), in secondary acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with inv(11)(q21q23). RT-PCR and sequencing revealed that exon 7 of MLL was fused to exon 2 of MAML2 in the AML and MDS cells. The inv(11)(q21q23) results in the creation of a chimeric RNA encoding a putative fusion protein containing 1,408 amino acids from the NH2-terminal part of MLL and 952 amino acids from the COOH-terminal part of MAML2. The NH2-terminal part of MAML2, a basic domain including a binding site of the intracellular domain of NOTCH, was deleted in MLL-MAML2. MLL-MAML2 in secondary AML/MDS and MECT1-MAML2 in mucoepithelioid carcinoma, benign Wartin's tumor, and clear cell hidradenoma consist of the same COOH-terminal part of MAML2. A luciferase assay revealed that MLL-MAML2 suppressed HES1 promoter activation by the NOTCH1 intracellular domain. MAML2 involving a chimeric gene might contribute to carcinogenesis in multiple neoplasms by the disruption of NOTCH signaling.

The recurring chromosome translocation t(11;16)(q23;p13) is detected in leukemia patients, virtually all of whom have received previous chemotherapy with topoisomerase (topo) II inhibitors. In the t(11;16), 3' CBP, on 16p13, is fused to 5' MLL, on 11q23, resulting in an MLL-CBP fusion gene that plays an important role in leukemogenesis. In this study, we cloned genomic breakpoints of the MLL and CBP genes in the t(11;16) in the SN-1 cell line and in five patients with therapy-related leukemia, all of whom had received topo II inhibitors for previous tumors. In all patients except one, both the genomic MLL-CBP and the reciprocal fusions were cloned. Genomic breakpoints in MLL occurred in the 8.3-kb breakpoint cluster region in all patients, whereas the breakpoints in CBP clustered in an 8.2-kb region of intron 3 in four patients. Genomic breakpoints in MLL occurred in intron 11 near the topo II cleavage site in the SN-1 cell line and in one patient, and they were close to LINE repetitive sequences in two other patients. In the remaining two patients, genomic breakpoints were in intron 9 in Alu repeats. Genomic breakpoints in CBP occurred in and around Alu repeats in one and two patients, respectively. In two patients, the breaks were near LINE repetitive sequences, suggesting that repetitive DNA sequences may play a role. No specific recombination motifs were identified at or near the breakpoint junctions. No topo II cleavage sites were detected in introns 2 and 3 of CBP. However, there were deletions and duplications at the breakpoints in both MLL and CBP and microhomologies or nontemplated nucleotides at most of the genomic fusion junctions, suggesting that a nonhomologous end-joining repair mechanism was involved in the t(11;16).

The nucleoporin gene NUP98 has been reported to be fused to 9 partner genes in hematologic malignancies with 11p15 translocations. The NUP98-HOXA9 fusion gene has been identified in acute myeloid leukemia (AML) and chronic myelogenous leukemia with t(7;11)(p15;p15). We report here a novel NUP98 partner gene, HOXA13, in a patient with de novo AML having t(7;11)(p15;p15). The HOXA13 gene is part of the HOXA cluster genes and contains 2 exons, encoding a protein of 338 amino acids with a homeodomain. The NUP98-HOXA13 fusion protein consists of the N-terminal phenylalanine-glycine repeat motif of NUP98 and the C-terminal homeodomain of HOXA13, similar to the NUP98-HOXA9 fusion protein. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis in various leukemic cell lines showed that the HOXA13 gene was expressed significantly more frequently in acute monocytic leukemic cell lines than in other leukemic cell lines (P = 0.039). HOXA13 and three HOXA cluster genes (A9, A10, A11) located at the 5′ end of the HOXA9 gene were frequently expressed in myeloid leukemic cell lines. Our results revealed that t(7;11)(p15;p15) was not a single chromosomal abnormality at the molecular level. The protein encoded by the NUP98-HOXA13 fusion gene is similar to that encoded by NUP98-HOXA9, and the expression pattern of the HOXA13 gene in leukemic cell lines is similar to that of the HOXA9 gene, suggesting that the NUP98-HOXA13 fusion protein may play a role in leukemogenesis through a mechanism similar to that of the NUP98-HOXA9 fusion protein. © 2002 Wiley-Liss, Inc.

We report on an adult patient with de novo acute myeloid leukemia (AML) with a t(11;22)(q23;q11.2) involving CDCREL1 and MLL genes. Reverse transcriptase (RT)-polymerase chain reaction (PCR) followed by direct sequencing analysis revealed the MLL-CDCREL1 fusion transcript in his leukemic cells. Analysis of the fusion transcript showed that exon 6 of MLL was fused to exon 4 of CDCREL1, which contains an AT-hook domain of MLL and a GTP binding domain of CDCREL1. To investigate the roles of CDCREL1 further, we examined the expression of the CDCREL1 gene in various cell lines. Expression of CDCREL1 was detected in 11 (85%) of 13 AML cell lines and 3 (21%) of 14 acute lymphoblastic leukemia (ALL) cell lines, but none of 11 EB virus transformed B-cell lines by RT-PCR. The expression rate of CDCREL1 was significantly higher in AML cell lines than in ALL cell lines (P = 0.0035). Platelet glycoprotein 1B beta (GP1B beta), which is located downstream of CDCREL1 and is cotranscribed with CDCREL1 due to a nonconsensus polyadenylation sequence, was expressed in all these cell lines. The higher expression rate of CDCREL1 in AML cell lines than in ALL cell lines suggests that this gene may play some role in myeloid leukemogenesis.

We describe a boy with Fanconi anemia (FA) who developed acute lymphoblastic leukemia (ALL) (FAB-LI) followed by acute myeloid leukemia (AML) (FAB-M5) at relapse. The patient was diagnosed with early pre-B-cell ALL without preceding aplastic anemia and was treated with ALL-oriented chemotherapy which included doxorubicin (a total dose of 140 mg/m2 administered), which is a topoisomerase II inhibitor. Complete remission was obtained, but after 38 weeks AML developed. The karyotype of ALL cells at diagnosis showed 46,XY, and that of AML cells at relapse was 46,XY, t(11;16)(q23;p13). An MLL gene rearrangement and MLL-CBP chimeric mRNA were found in AML, but not in ALL. A diagnosis of FA was confirmed by an increased number of chromosomal breaks and rearrangements in peripheral blood lymphocytes cultured with mitogen in the presence of mitomycin C. We conclude that this FA patient developed ALL followed by a therapy-related t(11;16)-AML resulting in an MLL-CBP fusion. Further examination of such patients would shed light on leukemogenesis in FA patients. Genes Chromosomes Cancer 27:264–269, 2000. © 2000 Wiley-Liss, Inc.

The t(7;11)(p15;p15) translocation has been reported as a rare and recurrent chromosomal abnormality in acute myeloid leukemia (AML) patients. The NUP98-HOXA9 fusion gene with t(7;11)(p15;p15) was identified and revealed to be essential for leukemogenesis and myeloproliferative disease. To date, t(7;11)(p15;p15) with NUP98-HOXA11 fusion has been reported only in one case of ph-negative chronic myeloid leukemia (CML). Here, we report a case of a 3-year-old girl with juvenile myelomonocytic leukemia (JMML) carrying t(7;11)(p15;p15) abnormality with NUP98-HOXA11 fusion. AML chemotherapy followed by bone marrow transplantation (BMT) was found to be effective in treating this disorder, and she remains in complete remission for 3 years after BMT. We suggest the possibility that AML chemotherapy might be effective for treating JMML with t(7;11)(p15;p15) abnormality and NUP98-HOXA11 fusion.

Chromosomal aberrations and inactivation of tumour suppressor genes are frequent in acute leukaemia. To determine whether the FHIT gene is involved in the development of leukaemia, we examined the FHIT transcript in 65 leukaemia cell lines, 5 fresh acute leukaemia patients at diagnosis and in complete remission, normal peripheral blood lymphocytes obtained from 14 healthy volunteers and Epstein-Barr (EB) virus transformed 5 B-cell lines (EB-lines), using nested reverse transcription-polymerase chain reaction and direct sequencing. The transcripts were classified into 4 patterns: pattern I revealed the normal transcripts only, pattern II the altered transcripts in addition to the normal transcripts, pattern III the altered transcripts without the normal transcripts and pattern IV an absence of normal and altered FHIT transcripts. Nineteen cell lines were classified as pattern I, 32 as pattern II, 2 as pattern III and 12 as pattern IV. The frequency of loss of FHIT expression (pattern III or IV) varied in each type of leukaemia cell line; the order ranked from the highest incidence was acute myeloid leukaemia (AML), T-cell acute lymphoblastic leukaemia (T-ALL), B-precursor ALL, B-ALL, and chronic myeloid leukaemia (CML). No genomic rearrangement was found in any samples examined. All of 5 patients showed same pattern II FHIT transcripts at 2 different stages of the disease. All normal peripheral blood lymphocytes and EB-lines were classified as pattern I or II. Our results suggested that patterns III and IV of FHIT transcripts might be associated with the development of a subset of leukaemia, while pattern II which has so far been reported as an aberrant transcript in varieties of malignant tumours might not be associated with leukaemogenesis.

The t(10;11)(p13-14;q14-21) is a rare but recurring translocation associated with acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML). Recently the CALM gene was cloned from the t(10;11) breakpoint of U937 and fused to AF10, a putative transcription factor, which had been identified as one of the fusion partners of the MLL gene. In order to define the involvement of these genes in primary leukaemias and cell lines with t(10;11), we analysed the expression of fusion transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) in five patient samples including ALL, AML and lymphoblastic lymphoma, and three monocytic cell lines (P31/Fujioka, KP-Mo-TS and U937). The CALM-AF10 fusion transcript was detected in all samples; however, the AF10-CALM fusion was not detected in two patient samples and one cell line. In RT-PCR analysis there were six isoforms of the CALM-AF10 fusion transcripts and five of AF10-CALM fusion transcripts. We also detected novel transcripts in U937. Sequence analysis revealed that all these isoforms had in-frame junctions and that some of them resulted from alternative splicing at different exons of CALM and others from different breakpoints at CALM and/or AF10. There were at least two different breakpoints of CALM and three of AF10 gene. Our results suggest that the CALM-AF10 fusion gene is a constant feature and is involved in the pathogenesis of haematological malignancies with t(10;11)(p13-14;q14-21), showing various and often multilineage phenotypes. Thus, t(10;11) needs to be investigated by RT-PCR for identification of the genes involved.

Recurrent translocations involving chromosome band 11q23 are often found in human acute leukemias. Recently, the MLL gene on 11q23 and 10 partner genes involved in these translocations have been cloned and characterized. We performed a reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the resultant der(11) chimeric mRNAs of the 3 types of 11q23 translocations including t(4;11), t(9;11), or t(11;19), in 14 leukemia patients with MLL gene rearrangements. At diagnosis or relapse, chimeric mRNA could be detected in all of the 4 patients with t(4;11), 2 of 3 with t(9;11), 2 of 3 with t(11;19), and 1 of 4 with unsuccessful karyotype. In 5 patients, we could monitor minimal residual disease (MRD) serially through the clinical course. One patient, in whom chi-meric mRNA was detected during complete remission (CR) just after the induction chemotheraphy, relapsed within 2 months and died, while 2 patients in which chimeric mRNA was not detected remained in CR from 10–23 months. These findings suggest that RT-PCR is a useful approach for detecting which partner gene is involved in the translocation and monitoring MRD in patients with MLL gene rearrangement. Nonetheless, the clinical relevance of MRD evaluation by RT-PCR monitoring remains controversial. Long-term and prospective investigation of a larger series of patients is needed to confirm the clinical significance of monitoring MRD by RT-PCR method. Med. Pediatr. Oncol. 28:325–332, 1997. © 1997 Wiley-Liss, Inc.

The cryptic t(5;11)(q35;p15.5) creates a fusion gene between the NUP98 and NSD1 genes. To ascertain the significance of this gene fusion, we explored its frequency, clinical impact, and gene expression pattern using DNA microarray in pediatric acute myeloid leukemia (AML) patients. NUP98-NSD1 fusion transcripts were detected in 6 (4.8%) of 124 pediatric AML patients. Supervised hierarchical clustering analyses using probe sets that were differentially expressed in these patients detected a characteristic gene expression pattern, including 18 NUP98-NSD1-negative patients (NUP98-NSD1-like patients). In total, a NUP98-NSD1-related gene expression signature (NUP98-NSD1 signature) was found in 19% (24/124) and in 58% (15/26) of cytogenetically normal cases. Their 4-year overall survival (OS) and event-free survival (EFS) were poor (33.3% in NUP98-NSD1-positive and 38.9% in NUP98-NSD1-like patients) compared with 100 NUP98-NSD1 signature-negative patients (4-year OS: 86.0%, 4-year EFS: 72.0%). Interestingly, t(7;11)(p15;p15)/NUP98-HOXA13, t(6;11)(q27;q23)/MLL-MLLT4 and t(6;9)(p22;q34)/DEK-NUP214, which are known as poor prognostic markers, were found in NUP98-NSD1-like patients. Furthermore, another type of NUP98-NSD1 fusion transcript was identified by additional RT-PCR analyses using other primers in a NUP98-NSD1-like patient, revealing the significance of this signature to detect NUP98-NSD1 gene fusions and to identify a new poor prognostic subgroup in AML.

Thde t( 11;22)(q24;q 12) and t(21;22)(q22;q 12) are specific chromosomal translocations found in the Ewinhd family of tumors including ES, PNET and Askin tumors. In these translocations, the amino-terminal portion of the EWS gene located in 22q 12 fuses to the carboxyl-terminal portion of the FLI-I gene located in 11 q24 or the ERG gene located in 21 q22, which belong to the ets oncogene super-family of transcription activators. We investigated the chimeric mRNAs of 15 ESs (7 cell lines and 8 tumor samples) and 7 PNETs (3 cell lines and 4 tumor samples) using the RT-PCR method and sequencing. We detected 2 types of EWS-ERG chimeric mRNA in 2 ES cell lines and I PNET tumor sample in addition to 4 types of EWS-FU-1 chimeric mRNA in 11 ESs (4 cell lines and 7 tumor samples) and 4 PNETs (2 cell lines and 2 tumor samples). There seemed to be no association between the type of chimeric mRNA and clinical features such as sex, age, primary site and histopathology of the patients. All of the chimeric mRNAs are generated from in-frame junctions and are thought to encode fusion proteins that may be the molecular mechanism involved in the Ewing family of tumors.