Escape of leukemia blasts from HLA-specific CTL pressure in a recipient of HLA one locus-mismatched bone marrow transplantation.

A case of leukemia escape from an HLA-specific cytotoxic T lymphocyte (CTL) response in a recipient of bone marrow transplantation is presented. Only the expression of HLA-B51, which was a mismatched HLA locus in the graft-versus-host direction, was down-regulated in post-transplant leukemia blasts compared with that in pre-transplant blasts. All CTL clones, that were isolated from the recipient's blood when acute graft-versus-host disease developed, recognized the mismatched B(∗)51:01 molecule in a peptide-dependent manner. The pre-transplant leukemia blasts were lysed by CTL clones, whereas the post-transplant leukemia blasts were not lysed by any CTL clones. The IFN-γ ELISPOT assay revealed that B(∗)51:01-reactive T lymphocytes accounted for the majority of the total alloreactive T lymphocytes in the blood just before leukemia relapse. These data suggest that immune escape of leukemia blasts from CTL pressure toward a certain HLA molecule can lead to clinical relapse after bone marrow transplantation.

BCR-ABL-independent and RAS / MAPK pathway-dependent form of imatinib resistance in Ph-positive acute lymphoblastic leukemia cell line with activation of EphB4.

OBJECTIVE: We investigated the mechanism responsible for imatinib (IM) resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) cell lines. METHODS: We established cell lines from a patient with Ph(+) ALL at the time of first diagnosis and relapsed phase and designated as NPhA1 and NPhA2, respectively. We also derived IM-resistant cells, NPhA2/STIR, from NPhA2 under gradually increasing IM concentrations. RESULTS: NPhA1 was sensitive to IM (IC(50) 0.05 microm) and NPhA2 showed mild IM resistance (IC(50) 0.3 microm). NPhA2/STIR could be maintained in the presence of 10 microm IM. Phosphorylation of MEK and ERK was slightly elevated in NPhA2 and significantly elevated in NPhA2/STIR compared to NPhA1 cells. After treatment with IM, phosphorylation of MEK and ERK was not suppressed but rather increased in NPhA2 and NPhA2/STIR. Active RAS was also increased markedly in NPhA2/STIR after IM treatment. The expression of BCL-2 was increased in NPhA2 compared to NPhA1, but no further increase in NPhA2/STIR. Proliferation of NPhA2/STIR was significantly inhibited by a combination of MEK inhibitor and IM. Analysis of tyrosine phosphorylation status with a protein tyrosine kinase array showed increased phosphorylation of EphB4 in NPhA2/STIR after IM treatment. Although transcription of EphB4 was suppressed in NPhA1 and NPhA2 after IM treatment, it was not suppressed and its ligand, ephrinB2, was increased in NPhA2/STIR. Suppression of EphB4 transcripts by introducing short hairpin RNA into NPhA2/STIR partially restored their sensitivity to IM. CONCLUSIONS: These results suggest a new mechanism of IM resistance mediated by the activation of RAS/MAPK pathway and EphB4.

Clinical significance of nuclear non-phosphorylated beta-catenin in acute myeloid leukaemia and myelodysplastic syndrome.

Wnt signaling activates the canonical pathway and induces the accumulation of non-phosphorylated beta-catenin (NPBC) in the nucleus. Although this pathway plays an important role in the maintenance of haematopoietic stem cells as well as in oncogenesis, the significance of nuclear NPBC remains unclear in malignant haematopoiesis. This study examined the expression of nuclear NPBC in bone marrow specimens from 54 and 44 patients with de novo acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS), respectively. On immunohistochemistry with an anti-NPBC antibody, the nuclei were positively stained in 22 and 18 of AML and MDS specimens, respectively. Staining of nuclear NPBC was associated with AML subtypes (M6 and M7), low complete remission (CR) rate, and poor prognosis. Nuclear NPBC was also associated with a high score when using the International Prognostic Scoring System (IPSS) for MDS and with -7/-7q and complex karyotypes. These findings suggest that in situ detection of nuclear NPBC by immunohistochemistry could provide new insights into the pathogenesis and prognosis of AML and MDS.

LRP16 is fused to RUNX1 in monocytic leukemia cell line with t(11;21)(q13;q22).

OBJECTIVE: The RUNX1 (also known as AML1) gene is observed frequently as the target of chromosomal rearrangements in human acute leukemia. We describe here a previously unreported rearrangement, t(11;21)(q13;q22), that disrupts the RUNX1 gene in a patient with acute leukemia and the molecular analysis of the fusion gene. METHODS: We have established a monocytic leukemia cell line, ELAM-1, from a patient with acute leukemia evolving from myelodysplastic syndrome (MDS). Translocation (11;21) (q13;q22) was observed in both patient leukemia cells and ELAM-1. RESULTS: The split signal of RUNX1 was detected by fluorescence in situ hybridization and indicated the involvement of RUNX1 in ELAM-1. Using 3'- Rapid amplification of cDNA ends and reverse transcription-Polymerase chain reaction analysis, we detected both RUNX1 (exon 5)-LRP16 and RUNX1 (exon 6)-LRP16 transcripts, suggesting that the RUNX1 breakpoint lies in intron 6 and that alternative fusion splice variants are generated. Reciprocal LRP16-RUNX1 fusion was also detected. CONCLUSIONS: We identified a novel RUNX1 fusion partner, LRP16 on 11q13 involving t(11;21)(q13;q22). Although it was reported that overexpression of LRP16 promotes human breast cancer cell proliferation, the function of LRP16 in leukemia remains to be studied. This fusion gene and cell line may provide a new research tool to investigate the mechanism of leukemogenesis generated by the RUNX1 fusion gene.

Establishment of a stroma-dependent human acute myelomonocytic leukemia cell line, NAMO-2, with FLT3 tandem duplication.

We have established a stroma-dependent myelomonocytic cell line, NAMO-2, with FLT3 internal tandem duplication (FLT3/ITD). Leukemia cells from a patient with acute myelomonocytic leukemia were administered to form subcutaneous tumors in nude mice, which were maintained successively, although we failed to establish continuously growing cells from the original leukemia cell culture. In the cultures of cells from subcutaneous tumors, there were stroma cells that had originated from the nude mice and showed continuous growth. The leukemia cells showed continuous growth dependent on this stroma, and this cell line was named NAMO-2. Detection of FLT3/ITD by the reverse transcriptase polymerase chain reaction (PCR) and genomic PCR showed that NAMO-2 was homozygous for FLT3/ITD. Constitutive activation of FLT3 was detected by Western blotting, and the phosphorylation of Akt, MEK, and STAT5 was also observed. FLT3 kinase inhibitor AG1296 specifically inhibited cell growth. NAMO-2 provides a useful tool to analyze adherence-dependent survival signaling of leukemia with FLT3/ITD and a model for the screening of FLT3 kinase inhibitors.

Disseminated intravascular coagulation in acute leukemia: clinical and laboratory features at presentation.

BACKGROUND: Although there are two major scoring systems for the clinical diagnosis of disseminated intravascular coagulation (DIC), the validity of these systems for leukemia-associated DIC remains to be confirmed. METHODS: By analyzing 125 newly diagnosed acute leukemia patients, we investigated clinical and laboratory features of leukemia-associated DIC, and determined the validity of the two established criteria. RESULTS: A total of 36 patients (29%) were diagnosed with DIC according to expert opinion, a method regarded as the de facto gold standard. Leukemia-associated DIC is characterized by rare manifestation of organ failure because of thrombosis and no relevance of the platelet count for the diagnosis. The results of receiver operating characteristics analysis favored fibrin degradation product (FDP) rather than D-dimer as the fibrin-related marker test. Although prothrombin time, plasma fibrinogen, and serum FDP levels were significantly different for patients with and without DIC, multivariate analysis identified FDP levels to be the only factor associated with DIC diagnosis. The cut-off level of 15 microg/mL for FDP was found to be the most effective to differentiate DIC from non-DIC, resulting in diagnostic sensitivity and specificity of 92% and 96%, respectively. The diagnostic results for our patients produced with this FDP-based system were at least comparable with or superior to those obtained with the two currently available scoring systems. CONCLUSIONS: Our findings suggest that an FDP-based criterion may be applicable for the diagnosis of leukemia-associated DIC. Although it appears to be simple and practicable enough for clinical use, prospective validation of this criterion is needed.

Micafungin, a novel antifungal agent, as empirical therapy in acute leukemia patients with febrile neutropenia.

OBJECTIVE: Invasive fungal infection is a major cause of morbidity and mortality in patients with febrile neutropenia unresponsive to antibacterial treatment. Empirical antifungal therapy with amphotericin B has been the standard of care for these patients; however, there remains a need for less toxic alternative drugs. PATIENTS AND METHODS: We conducted a prospective study to evaluate the efficacy and safety of micafungin (MCFG), a novel antifungal agent of the echinocandin class, in an empirical therapy setting for patients with febrile neutropenia. RESULTS: A total of 31 patients with acute leukemia who developed febrile neutropenia were enrolled in the study. Among them, 18 patients fulfilling the protocol-defined criteria, including 10 with persistent fever and 8 with recurrent fever, received MCFG empirically. Underlying diseases consisted of acute myeloid leukemia (n=15) and acute lymphoblastic leukemia (n=3). The median duration of neutropenia and drug administration was 22 and 9.5 days, respectively. Treatment success, defined as defervescence during the neutropenic period, absence of breakthrough fungal infections, and requiring no replacement of antifungal drugs, was achieved in 14 patients (78%). None of the patients required discontinuation or dose reduction due to adverse events except for one patient with severe hypokalemia. CONCLUSIONS: Although the studied patients were limited in number, our results indicate that MCFG is an encouraging agent for empirical antifungal therapy in patients with febrile neutropenia, and deserves further investigation in large-scale studies.

Long-term outcomes for unselected patients with acute myeloid leukemia categorized according to the World Health Organization classification: a single-center experience.

The actual utility of a new classification system of acute myeloid leukemia (AML) recently introduced by the World Health Organization (WHO) has not been thoroughly investigated yet. In this study, we evaluated long-term outcomes of unselected AML patients categorized according to the new WHO classification. Between 1990 and 2002, 109 adult AML cases were referred to our hospital. For the entire population, the median survival duration was 1.2 yr with a 5-yr survival rate of 31%. AML with recurrent genetic abnormalities accounted for 26%, AML with multilineage dysplasia for 29%, therapy-related AML for 13%, and AML not otherwise categorized for 32% of classifiable cases. Among the four groups, a significant difference was observed in terms of overall survival (P < 0.0001). Univariate analysis showed that six variables affected survival: cytogenetic risk, age, multilineage dysplasia, prior chemo/radiotherapy, type of treatment (intensive or palliative), and transplantation. However, in multivariate analysis no adverse prognostic impact of multilineage dysplasia and prior chemo/radiotherapy was detected (P = 0.4979 and 0.8702), whereas cytogenetic risk and patient age maintained their prognostic value (P = 0.0005 and 0.0100). These results indicate that outcomes for AML patients appear to be distinguished on the basis of the WHO classification, but the prognostic significance of multilineage dysplasia and prior therapy is lost after adjusting for cytogenetic risk and age. Our findings suggest that the WHO classification may be strengthened by greater emphasis on genetic/cytogenetic information.