The effect of dexamethasone on defective nephrin transport caused by ER stress: a potential mechanism for the therapeutic action of glucocorticoids in the acquired glomerular diseases.

The mechanism by which glucocorticoids govern antiproteinuric effect in nephrotic syndrome remains unknown. Present study examined the protective role of dexamethasone (DEX) in the intracellular trafficking of nephrin under endoplasmic reticulum (ER) stress. Human embryonic kidney-293 cell line expressing a full-length human nephrin was cultured in mediums containing 5.5 or 25 mM glucose with or without DEX. The result revealed that glucose starvation evoked a rapid ER stress leading to formation of underglycosylated nephrin that was remained in the ER as a complex with calreticulin/calnexin. DEX rescued this interfered trafficking through binding to its receptor and stimulating the mitochondrial transcripts and adenosine 5' triphosphate (ATP) production, leading to synthesis of fully glycosylated nephrin. These results suggest that ER-stress in podocytes may cause alteration of nephrin N-glycosylation, which may be an underlying factor in the pathomechanism of the proteinuria in nephrotic syndrome. DEX may restore this imbalance by stimulating expression of mitochondrial genes, resulted in the production of ATP that is essential factor for proper folding machinery aided by the ER chaperones.

Codon 201(Gly) polymorphic type of the DCC gene is related to disseminated neuroblastoma.

The deleted in colorectal carcinoma (DCC) gene is a potential tumor-suppressor gene on chromosome 18q21.3. The relatively high frequency of loss of heterozygosity (LOH) and loss of expression of this gene in neuroblastoma, especially in the advanced stages, imply the possibility of involvement of the DCC gene in progression of neuroblastoma. However, only few typical mutations have been identified in this gene, indicating that other possible mechanisms for the inactivation of this gene may exist. A polymorphic change (Arg to Gly) at DCC codon 201 is related to advanced colorectal carcinoma and increases in the tumors with absent DCC protein expression. In order to understand whether this change is associated with the development or progression of neuroblastoma, we investigated codon 201 polymorphism of the DCC gene in 102 primary neuroblastomas by polymerase chain reaction single-strand conformation polymorphism. We found no missense or nonsense mutations, but a polymorphic change from CGA (Arg) to GGA (Gly) at codon 201 resulting in three types of polymorphism: codon 201(Gly) type, codon 201(Arg/Gly) type, and codon 201(Arg) type. The codon 201(Gly) type occurred more frequently in disseminated (stages IV and IVs) neuroblastomas (72%) than in localized (stages I, II, and III) tumors (48%) (P=.035), and normal controls (38%) (P=.024). In addition, the codon 201(Gly) type was significantly more common in tumors found clinically (65%) than in those found by mass screening (35%) (P=.002). The results suggested that the codon 201(Gly) type of the DCC gene might be associated with a higher risk of disseminating neuroblastoma.

Deficient activity of von Willebrand factor-cleaving protease in patients with Upshaw-Schulman syndrome.

We identified unusually large von Willebrand factor (vWF) multimers caused by deficient activity of vWF-cleaving protease in 2 patients with Upshaw-Schulman syndrome. The autoantibodies that inhibited the protease activity were not detected in the plasma of either patient. Periodic fresh-frozen plasma transfusion was effective for management of the hemolysis and thrombocytopenia. We detected enriched enzyme activity in a particular plasma fraction, although molecular cloning of this specific protease is needed to determine a more detailed pathogenesis and to develop new therapeutic approaches.

Delay of the diagnostic lumbar puncture and intrathecal chemotherapy in children with acute lymphoblastic leukemia who undergo routine corticosteroid testing: Tokyo Children's Cancer Study Group study L89-12.

PURPOSE: To determine the effects of eliminating initial lumbar punctures in 418 consecutively treated children with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Patients were enrolled onto a trial conducted in central Japan between 1989 and 1992. Treatment consisted of standard four-drug induction therapy followed by a risk-based intensification phase, reinduction therapy, late intensification, and remission maintenance therapy (total of 104 weeks). The initial lumbar puncture, with an intrathecal injection of chemotherapy, was performed after 1 week of prednisolone sensitivity testing (day 8). End points included response to prednisolone, CNS status at the time of the day 8 lumbar puncture, subsequent adverse events in CNS and bone marrow, and event-free survival (EFS). RESULTS: The remission induction rate was 93.1% with a 6-year EFS rate (+/- SE) of 68.7% +/- 2.4%, which is similar to historical results for patients who received their diagnostic lumbar puncture and first instillation of intrathecal chemotherapy on day 0. Overall, 84.5% of the patients had good responses to prednisolone, whereas 15.5% had poor responses. Clinical outcome was strikingly better for the good responders (6-year EFS, 74.1% +/- 2.5% compared with 40.1% +/- 6.4% for patients with poor responses), suggesting that omission of intrathecal chemotherapy did not alter the predictive value of drug sensitivity testing. Eighteen patients experienced CNS relapse as their first adverse event (cumulative risk, 5.1%; 95% confidence interval, 2.7% to 7.4%), coincident with reports from groups using conventional strategies of CNS clinical management. Bleeding into the CSF at the time of the day 8 lumbar puncture was apparent in 29 cases (8.1%), but leukemic blasts were identified in only two. CONCLUSION: Delay of the initial lumbar puncture and intrathecal injection of chemotherapy seems to be feasible in children with ALL. Further controlled evaluations are needed to establish the validity of this conclusion.

Coduplication of the MLL and FLT3 genes in patients with acute myeloid leukemia.

Tandem duplication (TD) of the MLL or FLT3 gene in acute myeloid leukemia (AML) has been reported. We examined whether TD of these two genes occurs simultaneously. We analyzed 13 AML and 2 myelodysplastic syndrome patients, including 6 adult patients with trisomy 11 and 9 pediatric patients with TD of the FLT3 gene, using RT-PCR followed by sequencing. Among these, TD of the MLL and FLT3 genes was found in 5 and 10 patients, respectively. Notably, TD of both the MLL and FLT3 genes (coduplication) was detected in two AML patients, who died 6 and 14 months after diagnosis. TD of these two genes in AML is rare; thus, coduplication of these genes in the same patient is predicted to be very rare. Although the mechanisms of TD of both genes are different, development of TD of both genes may be related to an unknown similar etiology in leukemia because the frequency of coduplication of these genes in a single patient is considered to be very low. Further studies of the coduplication of these genes in AML patients may lead to the clarification of its mechanism and clinical implications.

The CDCREL1 gene fused to MLL in de novo acute myeloid leukemia with t(11;22)(q23;q11.2) and its frequent expression in myeloid leukemia cell lines.

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.

Absent or reduced expression of the caspase 8 gene occurs frequently in neuroblastoma, but not commonly in Ewing sarcoma or rhabdomyosarcoma.

PROCEDURE: To clarify whether the caspase 8 gene is involved in the pathogenesis of neuroblastoma (NB), we examined alterations of the caspase 8 gene in 15 NB, seven Ewing sarcoma (ES), and eight rhabdomyosarcoma (RMS) cell lines, using reverse transcription-polymerase chain reaction (RT-PCR) and RT-PCR single-strand conformation polymorphism (SSCP) analyses. RESULTS: The caspase 8 gene was not expressed in 11 (73%) of 15 NB cell lines, it was absent in only one of seven ES cell lines, but was present in all eight RMS cell lines examined. No mutations were detected in any cell lines examined. CONCLUSIONS: Inactivation of the caspase 8 gene is considered to be involved in the pathogenesis of NB, but not ES or RMS.

Down's syndrome with myelodysplastic syndrome showing t(7;11)(p13;p14).

We report a boy with Down's syndrome (DS) who developed myelodysplastic syndrome (MDS) after spontaneous remission of transient myeloproliferative disorder (TMD) at birth. Chromosomal analysis of the blasts in the MDS phase demonstrated t(7;11)(p13;p14) which had not been detected in the TMD phase. NUP98-HOXA9 chimera mRNA, which is known to be involved in t(7;11)(p15;p15) translocation in acute myeloid leukemia (AML), was not detected by reverse transcriptase-polymerase chain reaction, and NUP98 rearrangement was not detected by Southern blot analysis of the blasts in the MDS phase. Reciprocal translocation is very rare in AML/MDS in DS, and the t(7;11)(p13;p14) found in our patient was different from the recurrent translocation t(7;11)(p15;p15) previously reported.

[Fanconi anemia].

Fanconi anemia is a rare autosomal recessive disorder which is characterized with chromosomal fragility and hypersensitivity to DNA cross-link agents. There are large variability in phenotype even among patients of single family. Therefore, diagnosis depends solely on hypersensitivity to DNA cross-link agents. At present at least 8 complement groups are known and 3 genes, FANCA, FANCC and FANCG, have been cloned. There is controversies concerning intracellular localization and interaction between these genes. Although the mechanism of the final effect of products of these genes leading to Fanconi anemia phenotype, chromosomal fragility and ultimately development of cancers is not fully understand, increasing evidence shows that gene products seems to work to prevent oxygen injury by reactive oxygen species rather than repair of DNA injury.

Adult survivors of children's cancer and their offspring.

BACKGROUND: Although it is anticipated in Japan that the number of long-term survivors of children's cancer will rapidly increase and that they will have children, reports of studies concerning the offspring of such survivors have come mainly from western countries. For this reason, it seems that the results of this study will be important. METHODS: Ninety-seven survivors of children's cancer, who were diagnosed between 1962 and 1989 and are now older than 20 years old, and their offspring were studied. RESULTS: Of 97 survivors, 30 have married and 23 survivors or their spouses had been pregnant (33 total pregnancies). Twenty-five babies were born and seven pregnancies ended in spontaneous abortion. The abortion rate was not different from that of the Japanese general population. The birthweights of babies born to survivors tended to be lower than those of control subjects. The proportion of babies with birthweights under 2500 g was significantly greater for babies born to survivors than to the control subjects. None of the 25 babies born had congenital anomalies. Subsequent development of these children has been uneventful with no malignancies after a median follow-up period of 4 years 10 months (range: 10 months to 16 years, 3 months). CONCLUSIONS: The reproductive activity of children's cancer survivors and the health status of their offspring seem to be satisfactory. However, the sample size of the present study is too small to draw any definite conclusions. Because it is anticipated that the number of long-term survivors of children's cancer will rapidly increase in Japan, it is important to establish an effective system of following up these survivors and their offspring in order to provide them with appropriate suggestions for a better quality of life.

Six months of maintenance chemotherapy after intensified treatment for acute lymphoblastic leukemia of childhood.

PURPOSE: We postulated that intensification of chemotherapy immediately after remission induction might reduce the leukemic cell burden sufficiently to allow an abbreviated period of antimetabolite therapy. PATIENTS AND METHODS: Three hundred forty-seven children (ages 1 to 15 years) with previously untreated acute lymphoblastic leukemia (ALL) were enrolled onto the Tokyo L92-13 study, which excluded patients with mature B-cell ALL and patients less than 1 year old. One hundred twenty-four patients were classified as standard risk, 122 as high risk, and 101 as extremely high risk, according to age, peripheral-blood leukocyte count, selected genetic abnormalities, and immunophenotype. All subjects received four drugs for remission induction, followed by a risk-directed multidrug intensification phase and therapy for presymptomatic leukemia in the CNS. Maintenance chemotherapy with oral mercaptopurine and methotrexate was administered for 6 months, with all treatment stopped by 1 year after diagnosis. RESULTS: The mean (+/- SD) event-free survival (EFS) and overall survival rates for all patients were 59.5% +/- 3.4% and 81.5% +/- 2.2%, respectively, at 5. 5 years after diagnosis. EFS rates by risk category were similar (60. 2% +/- 6.0% for standard risk, 57.7% +/- 5.6% for high risk, and 62. 5% +/- 5.7% for extremely high risk), whereas overall survival rates differed significantly (91.2% +/- 2.7%, 80.0% +/- 4.1%, and 72.1% +/- 4.5%, respectively, P <.0001 by the log-rank test). There were 107 relapses. Eighty-five (79.4%) of these 107 patients achieved second complete remissions, with subsequent EFS rates of 61.5% +/- 7. 9% (standard risk), 42.6% +/- 8.1% (high risk), and 9.6% +/- 6.4% (extremely high risk). Of the five risk factors analyzed, only the response to prednisolone monotherapy among extremely high-risk patients proved important. CONCLUSION: Early treatment intensification did not compensate for a truncated phase of maintenance chemotherapy in children with standard- or high-risk ALL. However, 6 months of antimetabolite treatment seemed adequate for extremely high-risk patients who were good responders to prednisolone and received intensified chemotherapy that included high-dose cytarabine early in the clinical course.

SN-1, a novel leukemic cell line with t(11;16)(q23;p13): myeloid characteristics and resistance to retinoids and vitamin D3.

The MLL gene is fused with the cAMP-responsive element binding protein-binding protein (CBP) gene in t(11;16)(q23;p13), which has been reported to be associated with therapy-related acute leukemia. We established a novel myeloid cell line, SN-1, from a patient with T-cell acute lymphoblastic leukemia with t(11;16)(q23;p13) having in-frame MLL-CBP fusion transcripts. The majority of the SN-1 cells were positive for myeloperoxidase when examined using an electron microscope and expressed CD13, CD33, CD56, and HLA-DR antigens, but not CD7, CD10, CD19, CD34, or CD41 antigens, suggesting that these cells are of myeloid origin. SN-1 cells underwent functional and morphological differentiation when treated with actinomycin D or sodium butyrate, but not with all-trans-retinoic acid (ATRA) or 1alpha,25-dihydroxyvitamin D3 (VD3). Exposure of SN-1 cells to ATRA hardly affected cell growth and differentiation, whereas the growth of HL-60 and NB4 cells treated with ATRA was effectively inhibited, and differentiation into mature granulocytes was induced. SN-1 cells were relatively insensitive to VD3 with respect to inhibiting the cell growth and inducing the ability to reduce nitroblue tetrazolium, lysozyme activity, and morphological differentiation, although the expression of CD11b was slightly induced by VD3. These results suggest that the cell line was impaired in the signal transduction systems of ATRA and VD3. This cell line should be useful for the study of the role of CBP as a transcriptional regulator in leukemia differentiation and for the functional analysis of the MLL-CBP fusion gene, which will provide new insights into leukemogenesis caused by 11q23 translocations.

The p73 gene is less involved in the development but involved in the progression of neuroblastoma.

We performed expression, mutation, loss of heterozygosity (LOH) and fluorescence in situ hybridization (FISH) analyses of the p73 gene in neuroblastomas (NBs). Reverse transcription-polymerase chain reaction (RT-PCR) using primers which can detect both the p73alpha and p73beta transcripts was performed on 30 fresh NBs and 22 NB cell lines. Aberrant expression of the p73 gene was found in 4 (25%) of 16 primary tumors found by mass screening and in 10 (71.4%) of 14 primary tumors found clinically. The rates of expression in these two types of tumors were significantly different (p=0.026, Fisher's exact test). The incidence of aberrant expression of the p73 gene was significantly higher in stage IV patients than in stages I, II, III plus IVS patients (p=0.0236, Fisher's exact test). No homozygous deletions or rearrangements of the p73 gene were found in any samples examined. In addition to the polymorphism in exon 2, a silent mutation (codon 336 GCC/GCT) was found in one primary tumor. LOH of the p73 gene was detected in 5 (15%) of 33 primary NBs using PCR-LOH analysis. FISH analysis showed that all 17 NB cell lines used in this study revealed allelic loss of the p73 gene, while most of them expressed the p73 gene. These results suggest that the p73 gene is not monoallelically expressed in NB. We conclude that the p73 gene is less involved in the development but involved in the progression of neuroblastoma.

Hypermethylation of p16 and p15 genes and RB protein expression in acute leukemia.

Both p16 and p15, encoded by genes located on chromosome 9p21, are inhibitors of cyclin-dependent kinases 4/6 (CDK4/6) and upstream regulators of RB function, and set up the RB/p16 tumor suppressive pathway, which is abrogated frequently in human neoplasms, either through inactivation of the RB or p16 tumor-suppressor protein, or alteration of the cyclin D1 or CDK4 oncoproteins. In hematological malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid malignancies, and more particularly to T-cell acute lymphoblastic leukemia (T-ALL). However, in the other subsets of ALL, deletions of p16 and p15 are relatively rare events. To investigate whether these genes are inactivated by methylation of the 5' CpG islands, we examined 35 leukemia cell lines and 29 childhood acute myeloid leukemia (AML) patients by Southern blot, polymerase chain reaction (PCR) and Western blot analyses. We found methylation of p16 in 12 (50%) of 24 ALL cell lines, 5 (50%) of 10 AML cell lines without homozygous deletion of p16, and 11 (38%) of 29 AML patients. Those leukemia cell lines subjected to p16 methylation were found to have lost p16 protein expression. The p15 gene was methylated in 10 (34%) of 29 ALL cell lines, 6 (60%) of 10 AML cell lines without homozygous deletion of p15, and 15 (52%) of 29 AML patients. These results revealed the frequent methylation of p16 and p15 genes in B-ALL and AML despite a low frequency of p16 and p15 deletions and mutations in these leukemias. In the study for expression of RB protein, we found no expression of RB in 4 of 16 leukemia cell lines. Inactivation of the p16 gene was found in all the cell lines with expression of RB. Neither amplification nor rearrangement of cyclin D1 gene was found in any cell lines. These results suggest that inactivation of p16 and p15 genes is one of the most common genetic events in acute leukemia, and plays an important role for the RB/p16 pathway in the pathogenesis of acute leukemia.

A prospective, randomized trial of conventional, dose-accelerated corticosteroids and intravenous immunoglobulin in children with newly diagnosed idiopathic thrombocytopenic purpura.

To determine the minimal essential treatment for childhood acute idiopathic thrombocytopenic purpura (ITP), a prospective, randomized trial was conducted focusing on hemorrhagic manifestation as well as platelet count. Subjects with a platelet count of <10 x 10(3)/microL or 10 to 29 x 10(3)/microL and mucosal bleeding (group 1) were randomly assigned to receive intravenous immunoglobulin (IVIg) at 1 to 2 g/kg, conventional oral prednisolone (o-PSL) (2 mg/kg for 2 weeks). parenteral methylprednisolone (mPSL) (5 mg/kg for 5 days), or pulsed parenteral methylprednisolone (PmPSL) (30 mg/kg for 3 days). Subjects with a platelet count of 10 to 29 x 10(3)/microL without mucosal bleeding (group 2) were randomized to receive either o-PSL or no treatment. In subjects with a platelet count of 30 x 10(3)/microL or higher (group 3), patients undergoing no specific treatment were monitored. In group 1, IVIg offered faster platelet enhancement compared with o-PSL and mPSL, although neither mPSL no PmPSL showed any advantage, even over o-PSL. Platelet response was uniformly excellent when pretreatment platelet coun was > or = 10 x 10(3)/microL. Furthermore, the presence or absence of mucosal bleeding in subjects with a platelet count <10 x 10(3)/microL had no effect on the response to treatment. In group 2, platelet increase was indifferently attained with or without o-PSL. These data suggest that childhood acute ITP with a platelet count > or = 10 x 10(3)/microL may be left untreated or may be treated with o-PSL when mucosal bleeding is evident, whereas for those with a platelet count <10 x 10(3)/microL, IVIg is the most predictable platelet enhancer. Thus, a platelet count of 10 x 10(3)/microL seems to be informative enough to decide whether to treat childhood acute ITP.

Long-term follow-up of childhood acute lymphoblastic leukemia in Tokyo Children's Cancer Study Group 1981-1995.

The objectives were as follows: Firstly, to estimate the overall probability of event-free survival (EFS) and isolated CNS relapse in the studies for children with acute lymphoblastic leukemia (ALL) during the 1980s and 1990s. Secondly, to report the EFS according to presenting features and lineage. Thirdly, to evaluate the treatment results re-classified by the risks of NCI criteria. Four consecutive protocol studies were performed in the Tokyo Children's Cancer Study Group: L81-10 protocol (1981-1984, 189 patients), L84-11 (1984-1989, 484 patents), L89-12 (1989-1992, 418 patients) and L92-13 (1992-1995, 347 patients). Overall EFS at 5 years in each protocol was 56.5 +/- 3.8(1 s.e.)%, 71.0 +/- 2.1%, 67.8 +/- 2.3%, and 63.4 +/- 2.7%, respectively. The cumulative isolated CNS relapse rate at 5 years was 8.1 +/- 2.1%, 3.5 +/- 0.9%, 3.6 +/- 1.0%, 1.0 +/- 0.6. The EFS in SR/HR (standard risk/high risk) according to the NCI criteria in B-precursor ALL at 5 years was 61.9 +/- 4.3%/41.4 +/- 7.4% (lineage was not confirmed.), 72.5 +/- 2.6%/63.4 +/- 5.0%, 77.4 +/- 2.7%/56.3 +/- 4.7%, and 67.8 +/- 3.4%/56.7 +/- 5.4% in each protocol. Also EFSs according to NCI SR/HR at 5 years of T-ALL in protocols L84-11, L89-12 and L92-13 were 55.6 +/- 16.6%/60.9 +/- 10.1%, 72.7 +/- 13.4%/51.6 +/- 9.1%, and 77.1 +/- 14.4%/53.6/10.1%, respectively. The truncation of maintenance therapy to 6 months resulted in a decreased EFS in L92-13, particularly due to an increase of bone marrow relapse after cessation of therapy in SR and HR. The NCI risk criteria work properly even in the patients treated by different intensities, so that it makes the comparison possible among the patients in various groups. The overall EFSs in childhood ALL improved in 1980s, but it seemed stable or decreased in 1990s. The short maintenance therapy resulted in poor outcome in SR on the L92-13 protocol. Many of these late relapsers were effectively rescued and overall survival remained at a high level. The proportion of patients who received cranial irradiation reduced without any increase of the CNS events.

Intracranial hemorrhage in children with immune thrombocytopenic purpura

We sent questionnaires to hospitals in Japan in order to study the incidence and conditions of intracranial hemorrhage (ICH) in children with immune thrombocytopenic purpura (ITP). From 1980 to 1995, 11 cases of ICH were reported in eight patients with ITP at 35 institutions. One patient had ICH four times, but only one patient died of the condition. From 1990 through 1995, ICH occurred in four (0.52%) of 772 patients with ITP. None of the patients died. The platelet count when ICH occurred was 5.2 +/- 3.7 x 10(9)/l (mean +/- SD) (n = 11). Four of the eight patients (1980-1995) had received active treatment [e.g. intravenous immunoglobulin G (i.v. IgG)] immediately before ICH occurred. In seven cases (1980-1995), possible causes of ICH, including menstruation (n = 2) and viral infections (n = 3), were identified. Systemic lupus erythematosus (SLE) later developed in three patients. Although the incidence of ICH in children with ITP has not decreased compared with the rates in earlier studies, the mortality rate has decreased markedly. Our results suggest that menstruation, infection, and risk factors for progression to SLE may help to predict ICH in children with ITP. Large-scale prospective trials are needed to identify risk factors for ICH.

Consistent detection of CALM-AF10 chimaeric transcripts in haematological malignancies with t(10;11)(p13;q14) and identification of novel transcripts.

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.