Toshihiko Sugao

FLT3 internal tandem duplication is associated with a high relapse rate and central nervous system involvement in acute promyelocytic leukemia cases: single institutional analysis

To the Editor: Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is observed in 20–30% of the newly diagnosed acute myelogenous leukemia (AML) and indicates a significantly poorer prognosis in AML with normal karyotype (1). It has also been reported that a high prevalence of FLT3-ITD was observed in patients with AML who presented with central nervous system (CNS) involvement (2). In the all-trans retinoic acid era, acute promyelocytic leukemia (APL) has become the most curative subtype of AML (2); however, relapses still occur in 10–30% of patients after achieving the first complete remission (CR) (3). To improve prognosis further, risk-adapted therapy has also been utilized in APL (4). For APL, it remains under discussion whether FLT3ITD is a significant prognostic factor. Recently, it was reported that FLT3-ITD is associated with a high white blood cell (WBC) count at the onset and with poor overall and disease-free survival (5). However, there have not been any reports indicating whether FLT3-ITD is associated with CNS involvement among patients with APL. To clarify this issue, we retrospectively observed FLT3ITD and clinical outcomes of patients with APL. The institutional ethical committee approved this clinical observation, and we analyzed patients with APL who were hospitalized in our institute between November 1998 and December 2009. Patient characteristics are summarized in Table 1. Bone marrow cells were collected from informed patients, and the presence of FLT3-ITD was confirmed by the RT-PCR method (6). There were 26 patients who were screened for FLT3-ITD. Fifteen patients were men and 11 were women. FLT3-ITD was demonstrated on presentation in 6 (23%) (positive group). All patients were treated according to the Japan Adult Leukemia Study Group APL97 protocol (7). For remission induction therapy, patients received 45 mg ⁄m ⁄d of ATRA orally with or without idarubicin and cytarabine or idarubicin alone. After achieving CR, patients received three courses of consolidation chemotherapy. The first consolidation consisted of mitoxantrone on days 1–3 and Ara-C on days 1–5. The second consolidation contained Ara-C for 5 days, etoposide for 5 days, and daunorubicin on days 1 through 3. The third consolidation consisted of Ara-C for 5 days and idarubicin for 3 days. Methotrexate (15 mg), cytarabine (40 mg), and steroid were administered intrathecally for prophylaxis of CNS involvement to all patients after 1st or 2nd consolidation chemotherapy. CNS involvement was not revealed in any patients at their 1st lumabar puncture. All cases in both groups achieved CR. The relapse rates of FLT3-ITD-positive and FLT3-ITD-negative patients were 100% and 5%, respectively. Among positive patients, median duration from diagnosis to 1st relapse was 469 d (range 240–737). The rates of CNS involvement were 50% in the positive and 5% in the negative group. Among negative patients, only one patient had developed relapse. She presented with CNS relapse despite maintaining CR in the bone marrow. Thereafter, systemic relapse was demonstrated. Interest-

Vascular endothelial growth factor acted as autocrine growth factor in an acute promyelocytic leukemia case.

Vascular endothelial growth factor (VEGF) plays an important role in normal and neoplastic hematopoiesis [1,2]. In embryonic hematopoietic tissues, VEGF receptor type-1 (VEGFR-1) is expressed as a negative regulator, while VEGFR-2 plays an important role in embryonic and adult stable hematopoiesis; in the recovery phase following the suppression of hematopoiesis, VEGFR-1 acts to regulate hematopoiesis [2,3]. It is now known that, in some hematological malignancies, VEGF is expressed [2,4], and the autocrine VEGF stimulation of immature blasts has been reported [5]. In acute promyelocytic leukemia (M3), prominent production of VEGF has been reported, and angiogenesis is observed in bone marrow specimens [6]; however, VEGFRs are not expressed in M3 cells, and the VEGF-mediated activation of stromal cells induces leukemia cell proliferation in a paracrine fashion [7,8]. We encountered one case of M3 who relapsed soon after achieving complete remission (CR). We analyzed the molecular characteristics of M3 cells in this case, focusing on the VEGF system. A 43-year-old Japanese woman was admitted to our hospital in 2008 because of a bleeding tendency (petechiae in the skin, and gastrointestinal bleeding). The white blood cell count was 71,400/mL, in which myeloblasts comprised 41% and promyelocytes 9%. The blood hemoglobin level was 9.7 g/dL, and the platelet count was 75,000/mL. The bone marrow was hyperplastic (nuclear cell count of 480 000/mL), and promyelocytes had expanded to 97% with faggot cells, which were positive on CD13 and CD33 staining [Figure 1(C)]. Chromosomal analysis revealed 49,X,add(X)(q22),der(15)t(15;17)(q22;q12), –17,þmar16 3,þmar2, and fluorescence in situ hybridization analysis showed a 99% fusion signal for promyelocytic leukemia and retinoic acid receptor a. Disseminated intravascular coagulation and hyperfibrinolysis were demonstrated (prothrombin time international normalized ratio 2.35, activated partial thromboplastin time 33.1 s, fibrinogen 0.80 g/L, soluble fibrin monomers 156.8 mg/mL, plasma fibrinogen/fibrin degradation products 145.2 mg/mL, D dimer 61.5 mg/mL, thrombin–antithrombin complex 68.1 mg/L, plasmin–a2-plasmin inhibitor complex 23.9 mg/mL). We diagnosed the patient with M3, and all-trans retinoic acid was administered in combination with chemotherapeutic agents because of hyperleukocytosis. The patient achieved cytogenetic CR; however, during consolidation chemotherapy, M3 relapsed, and chemotherapy including arsenic trioxide was started. Bone marrow cells were collected after obtaining informed consent, mononuclear cells were prepared, and RNA was extracted, and this was used for reverse transcription-polymerase chain reaction (RT-PCR). Figure 1(A) shows the results, in which VEGF-A and VEGFR-1 and -2 were expressed in M3 cells in this patient, but not

Chronic Myelogenous Leukemia Cells Contribute to the Stromal Myofibroblasts in Leukemic NOD/SCID Mouse In Vivo

We recently reported that chronic myelogenous leukemia (CML) cells converted into myofibroblasts to create a microenvironment for proliferation of CML cells in vitro. To analyze a biological contribution of CML-derived myofibroblasts in vivo, we observed the characters of leukemic nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mouse. Bone marrow nonadherent mononuclear cells as well as human CD45-positive cells obtained from CML patients were injected to the irradiated NOD/SCID mice. When the chimeric BCR-ABL transcript was demonstrated in blood, human CML cells were detected in NOD/SCID murine bone marrow. And CML-derived myofibroblasts composed with the bone marrow-stroma, which produced significant amounts of human vascular endothelial growth factor A. When the parental CML cells were cultured with myofibroblasts separated from CML cell-engrafted NOD/SCID murine bone marrow, CML cells proliferated significantly. These observations indicate that CML cells make an adequate microenvironment for their own proliferation in vivo.

Vascular endothelial growth factor-C and its receptor type-3 expressed in acute lymphocytic leukemia cases with t(1;19)

The vascular endothelial growth factor (VEGF)-C system was analyzed in two cases of acute lymphocytic leukemia (ALL) with TCF3/PBX1 fusion to determine whether the VEGF-C system influences the growth of these ALL blasts. Bone marrow non-adherent mononuclear cells were prepared from the patients, and expressions of VEGFs and VEGF receptors (VEGFRs) were analyzed based on RNA and protein levels. Cell proliferation was also assayed with or without neutralizing antibodies to VEGFs. The patients’ leukemic blasts expressed a significant amount of VEGF-C and VEGFR type-3. When anti-VEGF-C antibody was added to the blast cell cultures, cell proliferation was suppressed. These observations indicate that, in our ALL cases with TCF3/PBX1 fusion, VEGF-C autocrine stimulation plays an important role in the proliferation of ALL.