Unbalanced translocation der(1;7)(q10;p10) defines a unique clinicopathological subgroup of myeloid neoplasms.

The unbalanced translocation, der(1;7)(q10;p10), is one of the characteristic cytogenetic abnormalities found in myelodysplastic syndromes (MDS) and other myeloid neoplasms. Although described frequently with very poor clinical outcome and possible relationship with monosomy 7 or 7q- (-7/7q-), this recurrent cytogenetic abnormality has not been explored fully. Here we analyzed retrospectively 77 cases with der(1;7)(q10;p10) in terms of their clinical and cytogenetic features, comparing with other 46 adult -7/7q- cases without der(1;7)(q10;p10). In contrast with other -7/7q- cases, where the abnormality tends to be found in one or more partial karyotypes, der(1;7)(q10;p10) represents the abnormality common to all the abnormal clones and usually appears as a sole chromosomal abnormality during the entire clinical courses, or if not, is accompanied only by a limited number and variety of additional abnormalities, mostly trisomy 8 and/or loss of 20q. der(1;7)(q10;p10)-positive MDS cases showed lower blast counts (P<0.0001) and higher hemoglobin concentrations (P<0.0075) at diagnosis and slower progression to acute myeloid leukemia (P=0.0043) than other -7/7q- cases. der(1;7)(q10;p10) cases showed significantly better clinical outcome than other -7/7q cases (P<0.0001). In conclusion, der(1;7)(q10;p10) defines a discrete entity among myeloid neoplasms, showing unique clinical and cytogenetic characteristics.

Mutations of the Smad4 gene in acute myelogeneous leukemia and their functional implications in leukemogenesis.

The Smad family proteins are critical components of the transforming growth factor (TGF)-beta signaling pathway. TGF-beta is a multipotent cytokine that elicits many biological functions. In particular, TGF-beta exhibits effects on the cell cycle manifested by G1-phase arrest, differentiation, or apoptosis of several target cells, suggesting that disruption of TGF-beta signaling pathway could be involved in cancer formation. Here we show one missense mutation of the Smad4 gene in the MH1 domain (P102L) and one frame shift mutation resulting in termination in the MH2 domain (Delta(483 - 552)) in acute myelogeneous leukemia. Both of the mutated Smad4 proteins lack transcriptional activities. Concomitant expression of the P102L mutant with wild-type Smad4 inactivates wild-type Smad4 through inhibiting its DNA-binding ability. The Delta(483 - 552) mutant blocks nuclear translocation of wild-type Smad4 and thus disrupts TGF-beta signaling. This is the first report showing that mutations in the Smad4 gene are associated with the pathogenesis of acute myelogeneous leukemia and the obtained results should provide useful insights into the mechanism whereby disruption of TGF-beta signaling pathway could lead to acute myelogeneous leukemia. Oncogene (2001) 20, 88 - 96.

Structurally altered Evi-1 protein generated in the 3q21q26 syndrome.

Overexpression of the Evi-1 gene appears to be a consistent feature of the 3q21q26 syndrome, an association of myeloid leukemias/myelodysplastic syndrome with a specific chromosomal aberration involving both 3q21 and 3q26, such as t(3;3)(q21;q26) or inv(3)(q21q26). The rearrangement in 3q26 has been reported to occur near the Evi-1 locus, implicating that it is the critical gene deregulated in the 3q21q26 syndrome. Here we present a structural abnormality of Evi-1 protein in a case with the 3q21q26 syndrome. In this case carrying typical inv(3)(q21q26), the 3q26 breakpoint is located within an intron of the Evi-1 gene, and resulted in overexpression of normally unexpressed, an aberrant form of Evi-1 protein, in which the C-terminal 44 amino acids of wild-type Evi-1 protein were truncated and replaced by five amino acids. The truncated Evi-1 protein is shown to increase AP1 activity when expressed in NIH3T3 cells as its wild-type counterpart. We also show that the origin of this peculiar type of rearrangement of the Evi-1 gene is not an artifact during establishment of the cell line, but is the event that occurred in the primary leukemic cells. Our results strongly support that the primary target for the 3q21q26 syndrome is the Evi-1 gene, and provide the first evidence that the structurally altered Evi-1 gene may be involved in the 3q21q26 syndrome.

High-grade cytomegalovirus antigenemia after hematopoietic stem cell transplantation.

Clinical impact of high-grade (HG) cytomegalovirus (CMV) antigenemia after hematopoietic stem cell transplantation has not been clarified. Therefore, in order to investigate the risk factors and outcome for HG-CMV antigenemia, we retrospectively analyzed the records of 154 Japanese adult patients who underwent allogeneic hematopoietic stem cell transplantation for the first time from 1995 to 2002 at the University of Tokyo Hospital. Among 107 patients who developed positive CMV antigenemia at any level, 74 received risk-adapted preemptive therapy with ganciclovir (GCV), and 17 of these developed HG-antigenemia defined as > or = 50 positive cells per two slides. The use of systemic corticosteroids at > or = 0.5 mg/kg/day at the initiation of GCV was identified as an independent significant risk factor for HG-antigenemia. Seven of the 17 HG-antigenemia patients developed CMV disease, with a cumulative incidence of 49.5%, which was significantly higher than that in the low-grade antigenemia patients (4%, P<0.001). However, overall survival was almost equivalent in the two groups. In conclusion, the development of HG-antigenemia appeared to depend on the profound immune suppression of the recipient. Although CMV disease frequently developed in HG-antigenemia patients, antiviral therapy could prevent a fatal outcome.

No concomitant occurrence of the N-ras and p53 gene mutations in myelodysplastic syndromes.

Mutations of the N-ras oncogene and p53 tumor suppressor gene were simultaneously investigated in bone marrow cells from 44 patients with myelodysplastic syndrome (MDS) or MDS-derived leukemia by single-strand conformation polymorphism (SSCP) analysis followed by direct sequencing. The mutations of the N-ras gene were detected only in two cases with MDS-derived leukemia. Three patients with MDS-derived leukemia and one with refractory anemia with excess of blasts exhibited five mutations of the p53 gene. No concomitant mutations of both genes were observed in our study, suggesting that alterations of both genes could play an important role in the progression of MDS in a non-cooperative manner.

Recent progress in molecular mechanisms of leukemogenesis: the cyclin-dependent kinase 4-inhibitor gene in human leukemias.

In order to clarify the significance of p16 gene (CDKN2) inactivation and its disease specificity among hematopoietic tumors, configurations of the p16 gene as well as those of the adjacent p15 and interferon alpha (IFN alpha) genes were examined in primary hematopoietic tumors. Loss of the p16 gene is frequent in and highly specific to lymphoid tumors among hematopoietic tumors. Gene deletions but not minute mutations should be the predominant mechanism of p16 gene inactivation in these types of tumors. The p16 gene is most frequently deleted among the p16, p15 and IFN alpha genes and thus should be the target of deletions in this locus. Deletions of the p16 gene were frequently observed in tumors carrying chromosome 9p abnormalities while a significant number of cases showed loss of the p16 gene without chromosome 9p abnormalities. So far inactivation of p53 and Rb tumor suppressors have also been found in lymphoid tumors. In our study, we detected homozygous deletions of p16 gene in 20%, loss of Rb protein in 28%, and p53 gene alterations in 8% of lymphoid tumors. Notably, 44% of lymphoid tumors showed inactivation of at least one of the three tumor suppressors, suggesting these tumor suppressors are important for lymphoid tumorigenesis. Inactivations of these tumor suppressors should independently occur in development of lymphoid tumors.

Increased Evi-1 expression is frequently observed in blastic crisis of chronic myelocytic leukemia.

Evi-1 is a transforming gene originally identified in a common integration site of murine leukemia retrovirus and mapped in human chromosome 3q26. It is not normally expressed in either human or murine hematopoietic cells, but is overexpressed in retrovirus-induced murine myeloid leukemias as well as human myeloid leukemias with 3q26 abnormalities, and thus thought to be responsible for both human and murine leukemogenesis. In this study, possible involvement of the Evi-1 gene in human leukemias was evaluated by Northern blot analysis in a total of 73 patients with various types of leukemias. We found that increased expression of the Evi-1 gene was most frequently observed in patients with CML in blastic crisis. It was found in 10 of 14 (71.0%) samples from CML in blastic crisis, three of 15 (20.0%) from acute myelocytic leukemia, three of 11 (27.3%) from MDS-derived leukemia, and one of 11 (9.1%) from acute lymphoblastic leukemia. Among 18 patients showing increased Evi-1 expression, none of 17 informative patients showed cytogenetic abnormalities involving 3q26. In addition, Southern blot analysis revealed neither amplification nor rearrangements of the Evi-1 gene in 11 Evi-1-positive patients whose DNA samples were available. Our results suggest that increased expression of the Evi-1 gene may play an important role in development of human leukemias, especially in progression from chronic phase to blastic crisis of CML even without 3q26 abnormalities.

Identification of a novel fusion gene, TTL, fused to ETV6 in acute lymphoblastic leukemia with t(12;13)(p13;q14), and its implication in leukemogenesis.

ETS variant gene 6 (ETV6)/translocation, ETS, leukemia (TEL)-involving chromosomal translocations are frequently observed in various hematologic neoplasms. We describe here a novel ETV6-involving translocation, t(12;13)(p13;q14), found in the case of acute lymphoblastic leukemia, in which ETV6 fused with a previously unknown gene, named Twelve-thirteen Translocation Leukemia gene (TTL), at 13q14. TTL was weakly but ubiquitously expressed in normal human tissues as detected by reverse transcribed-PCR. Three TTL splicing forms were identified, TTL-T from a human testis cDNA library, with an open-reading frame of 402 bp encoding 133 amino acids (aa), and TTL-B1 and -B2 from a human brain cDNA library. These proteins have no homology to known proteins. In leukemic cells from the patient, both reciprocal fusion transcripts, ETV6/TTL and TTL/ETV6, were expressed. The predominant fusion transcript, TTL/ETV6-1, encodes a predicted 530 aa fusion protein containing 89 aa of the N-terminal TTL fusing to the helix-loop-helix domain and ETS-binding domain of ETV6. Although the function of TTL is yet to be elucidated, our findings will provide another insight into the molecular pathogenesis of leukemia having ETV6-involving translocations.

Predictors for severe cardiac complications after hematopoietic stem cell transplantation.

The value of pre-transplant factors for predicting the development of cardiac complications after transplantation has been inconsistent among studies. We analyzed the impact of pre-transplant factors on the incidence of severe cardiac complications in 164 hematopoietic stem cell transplant recipients. We identified eight patients (4.8%) who experienced grade III or IV cardiac complications according to the Bearman criteria. Seven died of cardiac causes a median of 3 days after the onset of cardiac complications. On univariate analysis, both the cumulative dose of anthracyclines and the use of anthracyclines within 60 days before transplantation affected the incidence of severe cardiac complications (P=0.0091 and 0.011). The dissociation of heart rate and body temperature, which reflects "relative tachycardia", was also associated with a higher incidence of cardiac complications (P=0.024). None of the variables obtained by electrocardiography or echocardiography were useful for predicting cardiac complications after transplantation, although the statistical power might not be sufficient to detect the usefulness of ejection fraction. On a multivariate analysis, the cumulative dose of anthracyclines was the only independent significant risk factor for severe cardiac complications. We conclude that the cumulative dose of anthracyclines is the most potent predictor of cardiac complications and the administration of anthracyclines should be avoided within two months before transplantation.

Increased incidence of acute graft-versus-host disease with the continuous infusion of cyclosporine A compared to twice-daily infusion.

We retrospectively compared the incidence of acute graft-versus-host disease (GVHD) before and after September 1999, when we changed the mode of cyclosporine A (CsA) administration from twice-daily infusions (TD) (n=58) to continuous infusion (CIF) (n=71). The incidence of grade II-IV acute GVHD in the CIF group (56%) was significantly higher than that in the TD group (27%, P=0.00022). Multivariate analysis identified only two independent significant risk factors for the development of grade II-IV acute GVHD; CIF of CsA (relative risk 2.59, 95% CI 1.46-4.60, P=0.0011) and the presence of HLA mismatch (2.01, 95% CI 1.15-3.53, P=0.014). The incidence of relapse was significantly lower in the CIF group when adjusted for disease status before transplantation (0.41, 95% CI 0.18-0.95, P=0.038), which resulted in better disease-free survival in high-risk patients (43 vs 16% at 2 years, P=0.039), but not in standard-risk patients (72 vs 80%, P=0.45). CIF of CsA with a target level of 250-400 ng/ml may not be appropriate for GVHD prophylaxis in standard-risk patients.

Male predominance among Japanese adult patients with late-onset hemorrhagic cystitis after hematopoietic stem cell transplantation.

Late-onset hemorrhagic cystitis (LHC) after hematopoietic stem cell transplantation (HSCT) is mainly caused by viral infections. We retrospectively analyzed the records of 141 Japanese adult patients who underwent a first allogeneic HSCT from 1995 to 2002. In all, 19 patients developed LHC a median of 51 days after HSCT. Adenovirus (AdV) was detected in the urine of 10 LHC patients, of whom eight had AdV type 11. Five of the six available serum samples from these patients were also positive for AdV type 11, but the detection of AdV in serum was not associated with a worse outcome. Male sex and the development of grade II-IV acute graft-versus-host disease were identified as independent significant risk factors for LHC. Male predominance was detected in LHC after HSCT, as has been previously shown in children with AdV-induced acute HC. The detection of AdV DNA in serum did not predict a poor outcome.

Efficacy and safety of modified rituximab-ESHAP therapy for relapsed/refractory B-cell lymphoma.

Combined therapy of rituximab, etoposide, methylprednisolone, high-dose cytarabine, and cisplatin (R-ESHAP) has been one of the most frequently used salvage regimens for relapsed/refractory non-Hodgkin's lymphoma. In 2002, we introduced the modified R-ESHAP regimen in which cisplatin was switched to carboplatin. we evaluated the safety and effectiveness of this modified regimen by reviewing the records of 35 patients who had been administered R-ESHAP. Our cohort included 21 patients with diffuse large b cell lymphoma (DLBCL) and 14 patients with follicular lymphoma (FL). The overall response rate (ORR) was 48% for DLBCL and 93% for FL. The overall survival (OS) for patients with DLBCL was 51% with a median follow-up of 11 months, and 91% for patients with Fl with a median follow-up of 36 months. Our study indicates that modified R-ESHAP is an effective and safe salvage regimen for relapsed/refractory FL, however, its efficacy for relapsed DLBCL is limited.

Outcome and treatment of late-onset noninfectious pulmonary complications after allogeneic haematopoietic SCT.

Late-onset noninfectious pulmonary complications (LONIPCs) are life threatening for allogeneic hematopoietic SCT (allo-HSCT) recipients. However, the impact of LONIPCs on survival has not been properly evaluated and little is known about treatment efficacy. We retrospectively investigated 290 allo-HSCT recipients in our institute and reviewed the clinical aspects of 44 patients who had been diagnosed with LONIPCs. LONIPCs were significantly associated with higher rates of chronic GVHD (P<0001) and nonrelapse mortality (P=0.013), and lower rates of relapse (P=0.009). As a result of these effects, OS was significantly worse in those with LONIPCs (P=0.003). This result differs from a previous report. We then assessed short-term treatment response and final outcome. These results were defined by radiological findings, subjective symptoms, oxygen requirement and survival. Use of inhaled and systemic steroids did not affect either short-term response or final outcomes. However, administration of systemic corticosteroids earlier than at 21 days (median interval of time from onset of symptoms to systemic corticosteroids administration) was associated with a better outcome (P=0.054 for short-term response, and 0.016 for final outcome). Our study indicates that LONIPCs reduce OS, and early intervention with systemic corticosteroids may be effective.

Mutations and loss of expression of a mismatch repair gene, hMLH1, in leukemia and lymphoma cell lines.

Defects in genes involved in DNA mismatch repair have been detected in both hereditary and sporadic tumors of colon, endometrium, and ovary and suggested to be associated with tumorigenesis. To investigate disruptions of the mismatch repair system in hematological malignancies, we examined alterations of the human mutL homologue 1 (hMLH1) gene, a member of the mismatch repair gene family, in a total of 43 human leukemia and lymphoma cell lines, by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing analyses. Mutations of the hMLH1 gene were detected in three cell lines established from lymphoid leukemias. Moreover, Northern and Western blot analyses showed that expression of hMLH1 transcript or protein was abrogated in these three leukemia cell lines. Further studies for microsatellite loci showed that these cell lines without hMLH1 expression showed microsatellite instability. This is the first report that describes mutations and inactivation of the hMLH1 gene in human leukemia cells, suggesting that disruption of DNA mismatch repair system may play an important role in the development of human lymphoid leukemias.

Frameshift mutations of the hMSH6 gene in human leukemia cell lines.

Defects in DNA mismatch repair mechanisms, including frameshift mutations of the hMSH6 and hMSH3 genes at their (C)8 and (A)8 tracks, respectively, have been shown to be associated with human malignancies. To clarify the possible involvement of these mutations in hematopoietic malignancies, we screened a total of forty-four human leukemia and lymphoma cell lines for mutations in the hMSH6 and hMSH3 genes, as well as in other genes required for DNA replication or repair, by polymerase chain reaction single-strand conformation polymorphism analysis and sequencing analysis. Frameshift mutations at the (C)8 track of the hMSH6 gene were detected in two cell lines established from lymphoid leukemias. These two cell lines had no wild-type alleles, and both of them showed microsatellite instability. This is the first report that describes mutations and inactivation of the hMSH6 gene in hematological malignancies, suggesting that defects of the hMSH6 gene may be associated with development of hematological malignancies.

Homozygous loss of the cyclin-dependent kinase 4-inhibitor (p16) gene in human leukemias.

Recently, it has been shown that the homozygous deletion of the cyclin-dependent kinase-4 inhibitor (CDK4I;p16) gene, which is mapped to chromosome 9p21, is frequently observed in a wide spectrum of human cancers, including leukemias. Therefore, the CDK4I gene is thought to be a putative tumor-suppressor gene. We report here that both alleles of the CDK4I gene were completely or partially deleted in human leukemia cells derived from both patients and established cell lines. Thirty-seven hematopoietic cell lines and samples from 72 patients with leukemias were examined for homozygous loss of the CDK4I gene locus by Southern blot analysis. We found that a part or the whole of the CDK4I gene was homozygously deleted in 14 of the 37 (38%) cell lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute lymphocytic leukemia (ALL), and 13 with chronic myelocytic leukemia in blastic crisis. In the cell lines, the homozygous deletion of the CDK4I gene was detected in a variety of cell lineages, whereas all 4 cases showing the homozygous deletion were confined to ALL. It should be noted that 2 of them had no cytogenetic abnormalities of chromosome 9. Our results suggest that loss of the CDK4I function may contribute to immortalization of human leukemia cells and play a causative role at least in development of human lymphocytic leukemias.