Jun Imagawa

Discontinuation of dasatinib in patients with chronic myeloid leukaemia who have maintained deep molecular response for longer than 1 year (DADI trial): a multicentre phase 2 trial

BACKGROUND First-line imatinib treatment can be successfully discontinued in patients with chronic myeloid leukaemia after deep molecular response has been sustained for at least 2 years. We investigated the safety and efficacy of discontinuing second-line or subsequent dasatinib after at least 1 year of deep molecular response. METHODS The Dasatinib Discontinuation trial was a prospective multicentre trial done in Japan. Eligible patients taking dasatinib and with confirmed stable deep molecular response were enrolled between April 1, 2011, and March 31, 2012. All patients received dasatinib consolidation therapy for at least 1 year. In those with sustained deep molecular response, dasatinib was discontinued. Patients were followed up every month in year 1 (clinical cutoff), every 3 months in year 2, and every 6 months in year 3 for deep molecular response and immunological profiles. The primary endpoint was the proportion of patients with treatment-free remission at 6 months after discontinuation. Molecular relapse was defined as loss of deep molecular response at any assessment. This study is registered, number UMIN000005130. FINDINGS 88 patients were enrolled in the consolidation phase, 24 were excluded from the discontinuation phase due to fluctuations in BCR-ABL1 transcript levels. One patient was excluded because of positive expression of major and minor BCR-ABL1 transcripts in chronic myeloid leukaemia cells and the detection of minor BCR-ABL1 transcripts during consolidation. Thus, 63 patients discontinued dasatinib treatment. The 25 patients who were excluded from discontinuation continued to receive dasatinib and none showed disease progression. Median follow-up was 20.0 months (IQR 16.5-24.0). Of the 63 patients who discontinued and were not excluded, 30 patients maintained deep molecular response while 33 patients had molecular relapses, all within the first 7 months after discontinuation. The estimated overall treatment-free remission was 49% (95% CI 36-61) at 6 months. No severe treatment-related toxic effects were seen. Treatment was restarted in the 33 patients with relapse; rapid molecular responses were seen in all 33 patients, of whom 29 (88%) regained deep molecular response within 3 months, as did the remaining four by 6 months. INTERPRETATION Dasatinib discontinuation after sustained deep molecular response for more than 1 year is feasible. FUNDING Epidemiological and Clinical Research Information Network (ECRIN).

AML1/RUNX1 point mutation possibly promotes leukemic transformation in myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by proliferation of one or more myeloid cell lineages. Some patients exhibit leukemic transformation (LT) by unknown mechanisms, and chemotherapy may increase the risk of LT. To clarify the molecular mechanisms of LT, gene alterations involved in LT from patients in the chronic phase (CP) of MPNs were identified. Among 18 patients who progressed to leukemia, AML1/RUNX1 mutations were detected in 5 patients at the LT but in none at the CP. To investigate the leukemogenic effect of AML1/RUNX1 mutants, the AML1D171N mutant was transduced into CD34(+) cells from patients in the CP of MPNs. The D171N transduction resulted in proliferation of immature myeloid cells, enhanced self-renewal capacity, and proliferation of primitive progenitors. Taken together, these results indicate that AML1/RUNX1 point mutations may have a leukemogenic potential in MPN stem cells, and they may promote leukemic transformation in MPN.

Hyperactivation of the RAS signaling pathway in myelodysplastic syndrome with AML1/RUNX1 point mutations.

AML1/RUNX1 mutations have been reported frequently in myelodysplastic syndrome (MDS) patients, especially those diagnosed with refractory anemia with excess blast (RAEB), RAEB in transformation (RAEBt), or AML following MDS (these categories are defined as MDS/AML). Although AML1 mutations are suspected to play a pivotal role in the development of MDS/AML, acquisition of additional genetic alterations is also necessary. We analyzed gene alterations in MDS/AML patients with AML1 mutations, comparing them to alterations in those without an AML1 mutation. AML1 mutations were significantly associated with −7/7q-, whereas MDS/AML patients without AML1 mutations showed a high frequency of −5/5q- and a complex karyotype. Patients with AML1 mutations showed more mutations of their FLT3, N-RAS, PTPN11, and NF1 genes, resulting in a significantly higher mutation frequency for receptor tyrosine kinase (RTK)–RAS signaling pathways in AML1-mutated MDS/AML patients compared to AML1-wild-type MDS/AML patients (38% versus 6.3%, P<0.0001). Conversely, p53 mutations were detected only in patients without AML1 mutations. Furthermore, blast cells of the AML1-mutated patients expressing surface c-KIT, and SHP-2 mutants contributed to prolonged and enhanced extracellular signal-regulated kinase activation following stem cell factor stimulation. Our results suggest that MDS/AML arising from AML1/RUNX1 mutations has a significant association with −7/7q- alteration, and frequently involves RTK–RAS signaling pathway activation.

RUNX1/AML1 mutant collaborates with BMI1 overexpression in the development of human and murine myelodysplastic syndromes

RUNX1/AML1 mutations have been identified in myelodysplastic syndromes (MDSs). In a mouse bone marrow transplantation model, a RUNX1 mutant, D171N, was shown to collaborate with Evi1 in the development of MDSs; however, this is rare in humans. Using enforced expression in human CD34(+) cells, we showed that the D171N mutant, the most frequent target of mutation in the RUNX1 gene, had an increased self-renewal capacity, blocked differentiation, dysplasia in all 3 lineages, and tendency for immaturity, but no proliferation ability. BMI1 overexpression was observed in CD34(+) cells from the majority of MDS patients with RUNX1 mutations, but not in D171N-transduced human CD34(+) cells. Cotransduction of D171N and BMI1 demonstrated that BMI1 overexpression conferred proliferation ability to D171N-transduced cells in both human CD34(+) cells and a mouse bone marrow transplantation model. Stepwise transduction of D171N followed by BMI1 in human CD34(+) cells resulted in long-term proliferation with a retained CD34(+) cell fraction, which is quite similar to the phenotype in patients with higher-risk MDSs. Our results indicate that BMI1 overexpression is one of the second hit partner genes of RUNX1 mutations that contribute to the development of MDSs.

Clinical and genetic features of therapy-related myeloid neoplasms after chemotherapy for acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is a highly curable disease with excellent complete remission and long-term survival rates. However, the development of therapy-related myeloid neoplasms (t-MN) is being reported with increasing frequency in patients successfully treated for APL. We attempted to clarify the different clinical features and hematologic findings between t-MN and relapse cases, and to identify gene alterations involved in t-MN. We compared 10 relapse and 11 t-MN cases that developed in 108 patients during their first complete remission from APL. At APL diagnosis, t-MN patients had lower white blood cell counts than did relapse patients (P = .048). Overall survival starting from chemotherapy was significantly worse in t-MN patients than in relapse patients (P = .022). The t-MN cases were characterized as CD34(+)/HLA-DR(+) and PML-RARA(-), and 4 RUNX1/AML1 mutations were detected. T-MN is easily distinguished from APL relapse by evaluating these hematologic features, and it may originate from primitive myeloid cells by chemotherapy-induced RUNX1 mutations.

The Maturation of Myeloma Cells Correlates with Sensitivity to Chemotherapeutic Agents

We analyzed both morphologic and phenotypic findings of myeloma cells before and after chemotherapy in 21 patients with multiple myeloma. The morphologic analysis was based on the Greipp classification, and phenotypic analysis was performed by 3-color flow cytometry using the CD38 plasma gating method (Marrow plasma 38). Results with flow cytometry using a combination of MPC1, CD49e, and CD45 supported the morphologic findings for the myeloma cells. Treatment with 3 or 4 cycles of VAD (vincristine, doxorubicin, and dexamethasone) therapy was effective in reducing the total numbers of myeloma cells, but the proportion of immature myeloma cells increased after this treatment. However, the immature myeloma cells were reduced by high-dose melphalan (HD-Mel) therapy followed by autologous stem cell transplantation (ASCT). High-dose cyclophosphamide treatment for stem cell harvesting did not show an effect on the residual immature myeloma cells after VAD treatment. In addition, thalidomide was not effective in reducing the numbers of immature myeloma cells. These results suggest that VAD (3 or 4 cycles) therapy plus HD-Mel followed by ASCT is a reasonable treatment for multiple myeloma and that Marrow plasma 38 analysis is a useful method for monitoring the response of multiple myeloma to chemotherapy.

Progressive myeloma after thalidomide therapy in a patient with immature phenotype of myeloma (plasma) cells.

In our experience with thalidomide treatment for refractory multiple myeloma (MM), most patients with progressive disease (PD) did not show an increase in M-protein despite the tumor burden of myeloma cells. This finding led us to suspect that proliferation of immature myeloma cells showing MPC-1-/CD49e- phenotype may be a sign of PD. We report the results of consecutive analysis of the phenotype of myeloma (plasma) cells in an MM patient with PD during treatment with thalidomide. The myeloma cells decreased by thalidomide therapy were mature (MPC-1+/CD49e+) and intermediate (MPC-1+/CD49e-) types. When the patient was in the PD state, extramedullary plasmacytoma was recognized without proliferation of myeloma cells in the bone marrow (BM). The phenotype of myeloma (plasma) cells in both of these locations was that of immature myeloma cells (MPC-1-/CD49e-), and they showed decreased intensity of CD38 expression. The level of immunoglobulin G (IgG) in serum was decreased, and myeloma (plasma) cells in BM did not increase in PD. Although these clinical features may not be specific to MM patients in PD undergoing treatment with thalidomide, we suggest that immature myeloma cells may be resistant to thalidomide.

High early death rate in elderly patients with acute promyelocytic leukemia treated with all-trans retinoic acid combined chemotherapy

In a recent article in Cancer Science, Ono et al. [1] reported that elderly patients (60–70 years) with acute promyelocytic leukemia (APL) showed a higher induction death rate and non-relapse mortality during consolidation therapy, resulting in a significantly inferior overall survival rate. Many APL clinical trials have found that intensive chemotherapy combined with all-trans retinoic acid (ATRA) dramatically improved patient outcomes. Therefore, it has been generally thought that even elderly APL patients should be treated aggressively to increase the likelihood of curing the disease. However, most clinical trials have excluded elderly patients. Indeed, the German Acute Myeloid Leukemia Cooperative Group showed that 30 % of their newly diagnosed APL patients were age C60 years, and of these, 25 % were ineligible for the therapeutic study due to death before therapy, reduced performance status, comorbidity, or concomitant malignancy [2]. Moreover, elderly patients show lower tolerance to chemotherapy, resulting in a higher incidence of early death during chemotherapy [3]. To determine the responses to therapy in all relevant patient groups, we investigated all APL cases in our hospitals. Unselected patients with newly diagnosed APL were consecutively enrolled at Hiroshima University Hospital and its affiliated hospitals between 1996 and 2008. The patients received front-line intensive treatment consisting of ATRA and anthracycline/cytarabine-based chemotherapy with dose adjustment to WBC counts, as per the recommendations of the Japan Leukemia Study Group studies. Patients 70 years or older received a dose reduction of 30 % in order to prevent severe adverse effects. Patients were examined as approved by the Institutional Review Board at Hiroshima University. We identified a total of 124 patients and conducted a follow-up study [4]. The patient ages were: 10 patients \30 years, 22 patients 30–39 years, 21 patients 40–49 years, 25 patients 50–59 years, 27 patients 60–69 years, 16 patients 70–79 years and three patients 80 years or older. Thirty-two (26 %) patients 65 years or older were regarded as elderly; two of them received ATRA without chemotherapy. Differential clinical outcomes between younger and elderly patients with APL were compared. Complete remission (CR) for patients age C65 years was 78 %, which was lower than that for the whole population (95 %) and for patients age \65 years (100 %). The 5-year overall survival (OS) rate for the whole population was 76 %. The 5-year OS rate of J. Imagawa H. Harada (&) Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan e-mail: herf1@hiroshima-u.ac.jp; hharada@juntendo.ac.jp

Giant granulocytic sarcoma of the vagina concurrent with acute myeloid leukemia with t(8;21)(q22;q22) translocation

Granulocytic sarcoma (GS) is a localized extramedullary solid tumor consisting of immature myeloid cells [1]. Its occurrence in the female genital tract is rare and mainly in ovary or uterus, and the most frequent symptom of GS in the female genital tract is vaginal bleeding [2, 3]. Here we report an extremely rare case of vaginal GS, noticed by vaginal discharge, concurrent with acute myeloid leukemia (AML). A previously healthy and unmarried 25-year-old female presented with profuse stinking vaginal discharge. Magnetic resonance imaging (MRI) of the pelvis demonstrated a massive 7.9 cm 9 4.4 cm tumor between the posterior vaginal wall and the rectum, with propagation into the vaginal cavity (Fig. 1a). A pathological examination of the vaginal biopsy revealed normal squamous cells of the vagina and diffuse subepithelial infiltrates of medium-sized rounded cells in its stroma (Fig. 1d, e). Immunohistochemistry revealed CD45CD56CD3CD5CD15CD20CD30 CD79 (Fig. 1f). The histological and immunological findings were suggestive of NK-cell lymphoma. The patient’s blood cell count and laboratory data were normal. Fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) examination and bone marrow aspiration were performed for the clinical staging. The vaginal tumor demonstrated an increased FDG uptake with a standard uptake value (SUV) of 7.6 (Fig. 1b, c). No other region including bone marrow showed pathological FDG uptake. However, bone marrow aspiration showed hypercellular marrow comprised predominantly of myeloblasts with Auer rods (FAB classification, M2). Surface antigens of blast cells were CD34CD13CD33 CD117CD4CD56CD3CD19CD57. A cytogenetic study of the bone marrow cells revealed the chromosomal abnormality, 46,XX,del(7)(q32),t(8;21)(q22;q22), del(12)(p?). With concern for the vaginal tumor possibly being a GS, additional immunohistochemical studies were performed. The vaginal tumor cells were positive for lysozyme, CD68, and CD34 (Fig. 1g), resulting in the alteration of the diagnosis to GS following AML. Furthermore, an RTPCR analysis of the AML1-ETO fusion gene was positive in both the bone marrow cells and the vaginal tumor tissue (Fig. 1h). Immediately, the patient underwent standard induction chemotherapy with idarubicin (12 mg/m for 3 days) and cytarabine (100 mg/m for 7 days), and achieved complete hematological remission. The vaginal tumor also disappeared and the FDG uptake became negative. After five additional cycles of consolidation therapy including a cycle of high-dose cytarabine, she has been in complete cytogenetical remission for 3 years. We have shown here the first reported case of giant GS in the vagina presenting with t(8;21) AML. This case is exceptional because vaginal discharge was the first sign of an AML. Several retrospective studies have reported that GS has been found with a relatively higher incidence (9–38%) in patients with t(8;21)(q22;q22) translocation [4, 5]. The majority of these GS cases presenting with t(8;21) AML J. Imagawa T. Yoshida A. Sakai A. Kimura H. Harada (&) Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Miniami-ku, Hiroshima 734-8553, Japan e-mail: herf1@hiroshima-u.ac.jp

Final 3-year Results of the Dasatinib Discontinuation Trial in Patients With Chronic Myeloid Leukemia Who Received Dasatinib as a Second-line Treatment

Micro‐Abstract We describe the results of a prospective trial of the discontinuation of second‐line dasatinib treatment in chronic myeloid leukemia patients who maintained a deep molecular response for > 1 year. The treatment‐free remission rate at 36 months was 44.4%. High natural killer cell counts before discontinuation correlated significantly with successful therapy discontinuation. Introduction We previously reported an interim analysis of the DADI (dasatinib discontinuation) trial. The results showed that 48% of patients with chronic myeloid leukemia in the chronic phase who maintained a deep molecular response (DMR) for ≥ 1 year could discontinue second‐ or subsequent‐line dasatinib treatment safely at a median follow‐up of 20 months. However, the results from longer follow‐up periods would be much more useful from a clinical perspective. Patients and Methods The DADI trial was a prospective, multicenter trial conducted in Japan. After confirming a stable DMR for ≥ 1 year, dasatinib treatment subsequent to imatinib or nilotinib was discontinued. After discontinuation, the loss of DMR (even of 1 point) was defined as stringent molecular relapse, thereby triggering therapy resumption. The predictive factors of treatment‐free remission (TFR) were analyzed. Results The median follow‐up period was 44.0 months (interquartile range, 40.5‐48.0 months). The estimated overall TFR rate at 36 months was 44.4% (95% confidence interval, 32.0%‐56.2%). Only 2 patients developed a molecular relapse after the 1‐year cutoff point. The presence of imatinib resistance was a significant risk factor for molecular relapse. Moreover, high natural killer cell and low &ggr;&dgr;+ T‐cell and CD4+ regulatory T‐cell (CD25+CD127low) counts before discontinuation correlated significantly with successful therapy discontinuation. Conclusion These findings suggest that discontinuation of second‐ or subsequent‐line dasatinib after a sustained DMR of ≥ 1 year is feasible, especially for patients with no history of imatinib resistance. In addition, the natural killer cell count was associated with the TFR.