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Key Points Dasatinib-based 2-step induction resulted in a 100% CR rate with minimal toxicities and 53% MRD negativity.This protocol treatment increased the number of HSCTs in CR1, thereby improving 3-year EFS., Visual Abstract, The standard treatment for adults with Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) in Japan is imatinib-based chemotherapy followed by allogeneic hematopoietic stem cell transplantation (HSCT). However, ∼40% of patients cannot undergo HSCT in their first complete remission (CR1) because of chemotherapy-related toxicities or relapse before HSCT or older age. In this study, we evaluated dasatinib-based 2-step induction with the primary end point of 3-year event-free survival (EFS). The first induction (IND1) was dasatinib plus prednisolone to achieve CR, and IND2 was dasatinib plus intensive chemotherapy to achieve minimal residual disease (MRD) negativity. For patients who achieved CR and had an appropriate donor, HSCT during a consolidation phase later than the first consolidation, which included high-dose methotrexate, was recommended. Patients with pretransplantation MRD positivity were assigned to receive prophylactic dasatinib after HSCT. All 78 eligible patients achieved CR or incomplete CR after IND1, and 52.6% achieved MRD negativity after IND2. Nonrelapse mortality (NRM) was not reported. T315I mutation was detected in all 4 hematological relapses before HSCT. Fifty-eight patients (74.4%) underwent HSCT in CR1, and 44 (75.9%) had negative pretransplantation MRD. At a median follow-up of 4.0 years, 3-year EFS and overall survival were 66.2% (95% confidence interval [CI], 54.4-75.5) and 80.5% (95% CI, 69.7-87.7), respectively. The cumulative incidence of relapse and NRM at 3 years from enrollment were 26.1% and 7.8%, respectively. Dasatinib-based 2-step induction was demonstrated to improve 3-year EFS in Ph+ ALL. This study was registered in the UMIN Clinical Trial Registry as #UMIN000012173.

Background The efficacy of conventional chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been controversial as post-remission therapies for adult Philadelphia chromosome-negative acute lymphoblastic leukemia patients. Methods We retrospectively analyzed 96 adolescent and adult cases of Philadelphia chromosome-negative acute lymphoblastic leukemia to evaluate whether allo-HSCT should be performed after first complete remission (1CR). Results In total, 34 patients received chemotherapy followed by allo-HSCT (HSCT group) and 62 received chemotherapy alone (chemotherapy group). No significant differences in the event-free survival (EFS) or overall survival were observed between the two groups. In the chemotherapy group, use of pediatric regimens was significantly associated with favorable EFS, while high white blood cell (WBC) count and CD20 positivity were associated with poor outcome. In patients who received pediatric regimens, subsequent allo-HSCT did not influence EFS. In patients who received conventional chemotherapy (adult regimen), subsequent allo-HSCT did not improve EFS. High WBC count and CD20 positivity were also significantly associated with poor EFS in patients who received adult regimens. Patients with low WBC count and absence of CD20 who received adult regimens did not benefit from allo-HSCT. Conclusions Allo-HSCT may not be required in the pediatric regimen-eligible patients; however, pediatric regimen-ineligible patients with either CD20 positivity or high WBC count should receive allo-HSCT after achieving 1CR. This study was registered at http://www.umin.ac.jp/ctr/ as #C000016287.

Background: The Japan Adult Leukemia Study Group (JALSG) conducted the Ph+ALL202 and Ph+ALL208 studies for newly diagnosed (ND) Philadelphia-chromosome (Ph)-positive acute lymphoblastic leukemia (ALL), and successfully introduced imatinib into intensive chemotherapy followed by allogeneic hematopoietic cell transplantation (alloHCT). However, 30-40% of patients could not undergo alloHCT at CR1 because of older age, early relapse, or therapy-related death. We report the outcome of the Ph+ALL213 study, which introduced dasatinib (DA) to improve the efficacy and two-step combination to minimize the toxicity of treatments prior to alloHCT. Methods: The Ph+ALL213 study was a single-arm, multicenter phase II study for ND Ph+ALL. The protocol was reviewed and approved by the institutional review board of each hospital and registered in the UMIN Clinical Trials Registry (#UMIN000012173) and the Japan Registry of Clinical Trials (#jRCTs041180136). Patients with ND BCR/ABL1-positive ALL aged 16-64 years with PS 0-3 and sufficient organ function were included. All patients provided written informed consent before enrollment. The first step of treatment was induction (IND) targeting hematological complete remission (HCR) by 28 days of 140-mg DA and 14 days of 60-mg/m2 prednisone (PSL). IND was preceded by 7 days of PSL pre-phase treatment, during which BCR/ABL1 was confirmed by multiplex RQ-PCR. The second step was intensive consolidation (IC) targeting molecular CR (MCR), which was defined as no BCR/ABL1 signal by RQ-PCR, by 28 days of 100-mg DA in combination with CALGB BFM-like intensive chemotherapy. Consolidation comprised 4 cycles alternating between two regimens: high-dose methotrexate/cytarabine followed by 21 days of 100-mg DA (C1) and a CHOP-like regimen using vincristine (VCR)/cyclophosphamide/daunorubicin followed by 21 days of 100-mg DA (C2). The maintenance regimen was 12 cycles of 24 days of 100-mg DA with VCR /PSL. Patients who achieved HCR and had an appropriate donor proceeded to alloHCT after the first C1 consolidation (C1-1). DA was started after alloHCT only when the BCR/ABL1 signal was positive by RQ-PCR just before transplantation. The primary endpoint was event-free survival (EFS) from the induction at 3 years. Major secondary endpoints were hematological and molecular responses, adverse events (AEs), hematological relapse, non-relapse mortality (NRM), and overall survival (OS) from the induction at 3 years. Results: Of the 81 patients enrolled between Feb 2014 and April 2016 from 46 hospitals, 78 were evaluable, and the median age was 45 years (range, 16-64 years). Hematological response after IND were 73 (93.6%) CR, 4 (5.1%) CRi, and 1 (1.3%) PD, and 40 (56%) patients achieved MCR after IC. Grade 4 neutropenia/ thrombocytopenia in IND, IC, and C1-1 was reported in 51.3%/ 48.7%, 93.5%/ 5.2%, and 97.2%/ 70.8%, respectively, whereas grade 4 non-hematological AEs were noted in 2.6%, 9.1%, and 8.5%, respectively. No patients died during the course of IND, IC, or C1-1. All of 4 patients who relapsed before alloHCT had T315I mutations. One pt relapsed only in the CNS. Six patients completed the planned chemotherapy, and 8 patients discontinued the planned treatment because of AEs (4), physician's decision (2), and transfer (2) to non-registered hospitals. AlloHCT at CR1 was performed for 58 patients (74.4%). Forty-one patients (70.7%) had CMR just before transplantation. MAC and RIC conditioning was intensified for 69.0% and 31.0%, respectively, and the donor type was related, unrelated, and cord blood for 29.3%, 48.3%, and 20.7%, respectively. Eleven of 13 patients who discontinued the treatment underwent alloHCT afterwards. Three patients had NRM, but 7 patients survived with CR. At the median follow-up of 48.1 months, the 3-year EFS and OS were 67.2% (95%CI, 55.4-76.5%) and 82.8% (95%CI, 72.3-89.7%), respectively (figure 1). Of the patients who transplanted at CR1, the 3-year EFS and OS were 74.1% (95%CI, 60.8-83.5%) and 86.1% (95%CI, 74.2-92.8%), respectively. Among these patients, hematological relapse, relapse mortality, and NRM were noted in 10 (17.2%), 5 (8.6%), and 6 (10.3%), respectively. Conclusions: In this study, we reduced the relapse and toxicity before alloHCT, and improved the EFS and OS at 3 years. JALSG is starting a Ph+ALL219 phase II study to introduce ponatinib for patients who did not achieve CMR after INC following the schedule in the Ph+ALL213 study. Disclosures Kako: Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Honoraria. Yokoyama:Astellas: Other: Travel expenses. Onishi:Pfizer Japan Inc.: Honoraria; MSD: Honoraria, Research Funding; Novartis Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; Astellas Pharma Inc.: Honoraria; Celgene: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinyaku: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Kyowa-Hakko Kirin: Honoraria. Shiro:Bristol-Myers Squibb: Honoraria. Atsuta:CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria.

Dexamethasone (Dex) is an effective therapeutic agent against multiple myeloma (MM); however, resistance to it often becomes a clinical issue. CD44 is an adhesion molecule that serves as a cell surface receptor for extracellular matrix components, including hyaluronan (HA). HA is an extracellular matrix component that is involved in survival and progression in MM. In the present report, we describe isolation of a CD44-expressing population from a Dex-sensitive MM cell line, RPMI8226, in which the CD44-high population had a significantly higher potential to resist Dex than did the CD44-low population. Furthermore, we demonstrate that CD44 engagement by an anti-CD44 monoclonal antibody (mAb) or HA protects MM cells from Dex-induced growth inhibition. The activity of HA was partially inhibited by blocking its binding to CD44, indicating that CD44 mediates HA activity promoting MM cell survival. CD44 engagement by an anti-CD44 mAb led to phosphorylation and degradation of IkappaB-alpha, thus preventing its Dex-induced up-regulation. Our data suggest that CD44 is not only an important mediator for the survival activity of HA, but it may also contribute to MM cell resistance to Dex.

Clonal expansion of non-leukemic cells expressing two novel MLL – ELL variants differing in transforming activity

Late-onset noninfectious pulmonary complications (LONIPCs) occurring beyond 3 months after allogeneic stem cell transplantation (allo-SCT) have become recognized as life-threatening complications, and they reduce the recipient's quality of life. However, the pathogenesis and optimal treatment for LONIPCs are still unclear. In this study, we retrospectively analyzed the incidence and outcome of LONIPCs among allo-SCT recipients. Between October 1993 and September 2001, 96 patients underwent allo-SCT and 76 patients who survived and were free of disease for more than 3 months after SCT were enrolled. Among the 76 patients, 18 patients (23.7%) developed LONIPCs at a median interval of 227 days after allo-SCT (range, 91-1105 days). The patients with LONIPCs were subclassified into those with bronchiolitis obliterans (BO) (6 patients), with interstitial pneumonia (IP) (11 patients), or with both BO and IP (1 patient). The presence of extensive chronic graft-versus-host disease (GVHD) was significantly associated with the development of LONIPCs (P = .0008). Liver or skin involvement in chronic GVHD was not associated, but sicca syndrome was significantly associated with the development of LONIPCs (P < .0001). Most of the IP patients (58.3%) responded well to immunosuppressive treatment, while BO patients did not respond to the therapy. Eight of the 18 patients with LONIPCs died. The major cause of death was respiratory failure (62.5%). The relapse rate of primary malignant disease in the LONIPC patients was significantly lower than that of non-LONIPC patients (1 of 17 [5.9%] versus 16 of 52 [30.8%]; P = .0387). These results indicate that the development of LONIPCs was strongly associated with chronic GVHD and especially with sicca syndrome and the graft-versus-leukemia (GVL) effect. (Blood. 2003;102:4236-4242)

As a source of hematopoietic stem cells for transplantation, the use of peripheral blood stem cells (PBSCs) has become routine and comparable to that of the use of bone marrow. Recently, elderly patients with hematological malignancies also have been allowed to receive minitransplantations with nonmyeloablative conditioning regimens under sufficient PBSC infusion. As a result of these minitransplantations, elderly donors have been chosen increasingly from the siblings of elderly patients. We analyzed factors influencing the condition of CD34+ cells in the first days of collection in 49 healthy donors from July 1995 to January 2001. The median dose of recombinant human granulocyte colony-stimulating factor was 8 microg/kg/day (range 8 - 10) over 3 days. The target number of CD34+ cells used in this study was > or = 3 x 10(6) cells/kg of recipient body weight. The median apheresis volume was 12 L. Except for one 60 year old man, we obtained an adequate number of stem cells. In the regression analysis, a negative correlation was seen between donor age and the number of CD34+ cells/kg of recipient body weight per 12 L volume (Y = aX + b; a = -0.07507; b = 6.629996; r = -0.50985; p = 0.000252). Significantly higher apheresis results were obtained in donors younger than 45 years compared with donors 45 years old and older (p < 0.0227). There were no correlations among the number of CD34+ cells, donor body weight, and the number of leukocytes in peripheral blood on the first day of apheresis.

Polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes syndrome is a rare multisystem disorder. Overproduction of vascular endothelial growth factor (VEGF) by plasmocytoma could be responsible for the symptoms. The authors treated four patients with high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Within 6 months, symptoms associated with rapid normalization of serum VEGF levels improved.

Background. To the authors' knowledge, there have been no reports describing a case of malignant paraganglioma that produces both adrenocorticotropic hormone (ACTH) and interleukin-6 (IL-6). Methods. The clinical course and pathophysiology of a patient with Cushing's syndrome induced by ectopic ACTH syndrome caused by a cervical malignant paraganglioma with elevated plasma levels of IL-6 was investigated. Results. Ultrasonography, computed tomography, and magnetic resonance imaging of the neck revealed the presence of a tumor around the area from which the blood sample that showed the highest levels of ACTH and IL-6 was obtained by selective catheterization. Immunohistochemical staining of the removed tissue demonstrated the presence of ACTH and IL-6 proteins in the tumor cells. Reverse transcriptase-polymerase chain reaction also revealed the existence of IL-6 mRNA in those cells. Conclusion. The malignant paraganglioma of ectopic ACTH syndrome may produce IL-6. The present investigation provides new observations concerning ectopic ACTH syndrome.

POEMS syndrome is a rare plasma cell disorder characterized by peripheral neuropathy, monoclonal gammopathy, and high levels of serum vascular endothelial growth factor, the pathogenesis of which remains unclear. A unique feature of this syndrome is that the proliferating monoclonal plasma cells are essentially λ-restricted. Here we determined complete nucleotide sequences of monoclonal immunoglobulin λ light chain (IGL) variable regions in 11 patients with POEMS syndrome. The V-region of the Igλ gene of all 11 patients was restricted to the Vλ1 subfamily. Searching for homologies with IGL germlines revealed that 2 germlines, IGLV1-44*01 (9/11) and IGLV1-40*01 (2/10), were identified, with an average homology of 91.1%. The IGLJ3*02 gene was used in 11 of 11 re-arrangements with an average homology of 92.2%. These data suggest that the highly restricted use of IGL Vλ1 germlines plays an important role in the pathogenesis of POEMS syndrome.

New strategies are needed to overcome the resistance of multiple myeloma (MM) to dexamethasone (Dex). Several recent in vitro studies demonstrated the antitumor effect of nitrogen-containing amino-bisphosphonates (N-BPs) in various tumor cell lines. Inhibition of the prenylation of small G proteins is assumed to be one of the principal mechanisms by which N-BPs exert their effects. There have been few reports on N-BP treatment of MM cells that are resistant to Dex. Additionally, it is not known how small G proteins are altered in N-BP-treated MM cells. In this study, we evaluated the effect of the most potent N-BP, zoledronate (ZOL), on a Dex-resistant human MM cell subline (Dex-R) that we established from the well-documented RPMI8226 cell line. ZOL reduced the viability and induced apoptosis of Dex-R cells. Some of the ZOL-treated RPMI8226 cells and ZOL-treated Dex-R cells were elongated; however, elongated cells were not seen among the Dex-treated RPMI8226 cells. Furthermore, we found that portions of the small G proteins, Rho and Rap1A, were unprenylated in the ZOL-treated MM cells. Geranylgeraniol reduced the above-mentioned ZOL-induced effects. These findings suggest that ZOL may be beneficial for the treatment of Dex-resistant MM by suppressing the processing of RhoA and Rap1A.

Molecular remission of multiple myeloma with cytoreductive autografting followed by a dose-reduced allograft

Chronic graft-versus-host disease (GVHD) remains the major cause of late morbidity and mortality after allogeneic stem cell transplantation. We retrospectively analysed 2937 patients who underwent bone marrow transplantation from an unrelated donor (UR-BMT) facilitated by the Japan Marrow Donor Program (JMDP) and survived beyond day 100 after transplantation. The cumulative incidence of chronic GVHD (limited + extensive) or extensive chronic GVHD at 5 years post-transplant was 45.8% and 28.2%, respectively. On multivariate analysis, seven variables predicting chronic GVHD were identified: recipient age over 20 years, donor age over 30 years, primary diagnosis of chronic myeloid leukaemia, human leucocyte antigen (HLA)-A or -B mismatch, total body irradiation-containing regimen, platelet count not having reached 50 x 10(9)/l by day 100, and prior acute GVHD. Among 2609 patients with haematological malignancy, overall survival was significantly higher in patients with limited chronic GVHD but lower in patients with extensive chronic GVHD compared with those without chronic GVHD. The cumulative incidence of relapse among patients with limited or extensive chronic GVHD was significantly lower than that among patients without chronic GVHD. Our results suggest that limited chronic GVHD provides a survival benefit to patients with haematological malignancies by reducing the risk of relapse without increasing the risk of death from chronic GVHD.

Successful matched unrelated BMT for secondary AML which developed simultaneously with relapsed Hodgkin's lymphoma

Successful combination treatment with bevacizumab, thalidomide and autologous PBSC for severe POEMS syndrome

Successful engraftment by second cord blood transplantation with reduced-intensity conditioning after graft rejection due to hemophagocytic syndrome following initial CBT

Successful cord blood transplantation in a minor BCR-ABL + CML patient who had been in lymphoid blast crisis at presentation

Second cord blood transplantation (CBT) with reduced-intensity conditioning for graft failure after the first CBT for AML

Background: POEMS syndrome is a rare plasma cell disorder characterized by polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes associated with multiorgan involvement and monoclonal plasma cell dyscrasia. The pathogenesis of POEMS syndrome is not well understood, but overproduction of vascular endothelial growth factor (VEGF) probably secreted by plasma cells is considered to be responsible. High-dose melphalan (Mel) with autologous stem cell transplantation (ASCT) has been recently considered to be an effective strategy, but its treatment related mortality is considered to be high. Here, we report the results of ASCT for POEMS syndrome. Methods: Between January 2004 and June 2008, twelve patients with POEMS syndrome underwent ASCT at Chiba University Hospital and were retrospectively analyzed in the study. Conditioning treatment for ASCT was Mel 200 mg/m2, while Mel dose was reduced to 140 mg/m2 if a patient had poor performance status (PS). Results: Ten out of twelve patients were male and their median age was 48 years (range 34–61). Median time from first symptom and diagnosis to ASCT was 21 and 10 months, respectively. Seven patients received induction therapy before preparation of ASCT, thalidomide-dexamethasone in four, intravenous Mel in two, and bevacizumab followed by thalidomide in one patient. Peripheral blood stem cells were mobilized by cyclophosphamide/G-CSF in nine cases and G-CSF alone in three. Median serum VEGF level before ASCT was 2835 pg/ml (range; 334–5300). Median number of infused CD34+ cells was 2.3 × 106/kg (range; 2.1–4.3 × 106/kg). Eleven patients were conditioned with Mel 200 mg/m2, while one patient with poor PS (Karnofsky score 30%) received Mel 140 mg/m2. Median time to neutrophil recovery (>500/mm3) was 13 days post transplant (range 9–17 days). Only two patients developed respiratory distress during neutrophil engraftment period. Both cases were successfully treated with corticosteroid therapy, none of them required intubation. During a median follow up period of 16.5 months (range 2–54), there was no transplant related death. Neurologic improvement was significantly observed in all patients and median post transplantation serum VEGF level was decreased to 330 pg/ml (range 87–3250). Only one patient experienced relapse of polyneuropathy at 43 months post transplant following reincrease of serum VEGF level. Conclusions: ASCT is an effective therapy for POEMS syndrome. Despite previous published reports arousing potential risk of life-threatening engraftment syndrome, treatment related mortality and periengraftment pulmonary complications were significantly low in our patient series. We speculate that stabilization of serum VEGF level by induction therapy before ASCT could play a potential role for reducing the risk of peritransplant complications.

Background: POEMS syndrome (also known as Crow-Fukase or Takatsuki syndrome) is a rare plasma cell disorder characterized by peripheral neuropathy, a monoclonal plasma cell disorder, and other paraneoplastic features including organomegaly, endocrinopathy, skin changes, edema and effusions. High levels of vascular endothelial growth factor (VEGF) in the serum may contribute to pathology seen in this disease but exact pathogenetic mechanisms are still unclear. A unique feature of this syndrome is that the proliferating monoclonal plasma cells are virtually always λ-restricted. Here, we determined complete nucleotide sequences of monoclonal immunoglobulin λ light chain (IGL) variable regions in 10 patients with POEMS syndrome and found that Vλ germline usage is highly restricted to the Vλ1 family and oligoclonal germlines. Materials and methods: Total RNA was extracted from bone marrow mononuclear cells of patients with POEMS syndrome and the V-J region of the IGL gene was amplified by RT-PCR using 5′ degenerative primers for the Vλ consensus leader lesion (5′-ATGGCCKGSWYYSYTCTCCTC-3′) and 3′ primers matching the consensus upstream part of the cl exon (5′-CTCCCGGGTAGAGAAGTCACT-3′). Monoclonal bands were detected by heteroduplex analysis or TA cloning and subjected to nucleotide sequencing. Sequence data were analyzed using the International ImMunoGeneTics information system (IMGT, http://imgt.cines.fr) and mutations were identified by comparison with the germline sequence databases. Results: Of 13 POEMS syndrome patients with λ-type M protein, nucleotide sequencing of the IGL gene was successful in 10. The V-region of the IGL gene of all 10 patients was restricted to the Vλ1 family. Searching for homologies with IGL germlines revealed that only two were used, with an average homology of 90.1%. IGLV1-40*01 was used in 8 patients and IGLV1-44*01 in the remaining two. In the IGL-J region, the IGLJ3 gene was found in 9 of 10 patients and IGLJ2 in one (10%). All IGLV-J rearrangements were mutated with homologies to the germline sequence ranging from 77.66% to 96.10% (average homology: 90.1%). All CDR3 were composed of 11 amino acids, with identical acidic isoelectric point (pI) values (13.0) and similar molecular weights ranging from 1394.7 to 1552.8 (median, 1506.2). Conclusions: IGL-M protein in POEMS syndrome belongs to the Vλ1 family and is markedly restricted to specific oligoclonal germlines. Our results suggest that the restricted use of IGL plays an important role in the pathogenesis of POEMS syndrome. These data provide an important insight for elucidating the pathogenesis of POEMS syndrome. To the best of our knowledge, this the first report of restricted oligo-clonal usage of monoclonal immunoglobulin germlines in plasma cell disorders.

Introduction The MLL gene located on 11q13 is a frequent target of chromosomal translocation and induces acute leukemia with a poor prognosis. The ELL gene, one of partner genes with MLL translocation, is located on 19p13.1 and involved in about 5% of spontaneous acute leukemias and about 30% of secondary acute leukemias with MLL gene rearrangement. Here we present a case who had clonal expansion of MLL-ELL- positive cells in bone marrow (BM) with normal morphology after achieving complete remission (CR) and successfully underwent unrelated bone marrow transplantation (UR-BMT). We also analyzed the breakpoint of MLL-ELL by RT-PCR and sequencing and identified a novel type of breakpoint in MLL gene. Patient and methods A 32-years-old Japanese woman, who had no previous medical history, suffered from dyspnea and severe pancytopenia and referred to a hospital in Dec. 2001. BM aspiration showed hypercellular marrow with 89% of blasts which were positive for myeloperoxidase staining and expressed myeloid immunophenotypes (i.e. CD7, CD13, CD33, CD34 and HLA-DR), leading to the diagnosis of AML. BM cytogenetics revealed t(11;19)(q23;p13.1), along with trisomy 8. After one course of induction therapy, she achieved CR. BM cytogenetics showed normal chromosome (46, XX) and FISH analysis of MLL gene showed negative for MLL rearranged cells. Consolidation chemotherapies were carried out and BM aspirations showed CR throughout the entire course. However, repeated FISH analyses revealed the number of MLL rearrangement positive cells were gradually increasing without expanding of leukemic blasts. Since MLL-ELL positive leukemia shows a very poor prognosis, UR-BMT was planned through Japan Marrow Donor Program. She was re-admitted on April 2003. Before transplantation, her complete blood counts were normal and BM aspirate showed hypercellular marrow with 0.4% blasts with myeloid/erythroid ratio 4.5. There were no morphological abnormalities, however, cytogenetic study showed 100 % of cells were positive for t(11;19)(q23;p13.1) without trisomy 8. FISH analysis showed 90.1% of cells were positive for MLL rearrangement and negative for trisomy 8. She underwent UR-BMT successfully and is still alive in good health. We analyzed MLL-ELL gene rearrangement by RT-PCR and determined DNA sequence of MLL-ELL fusions. Results We obtained unexpected longer PCR products than that of previously reported and sequencing analysis revealed exon 12 of MLL gene fused to ELL gene, although it has been reported that exons 10 or 11 of MLL is fused in frame with ELL in most cases of MLL-ELL translocations. Discussion We identified new type of MLL gene breakpoint which contains exon 12 of MLL gene in MLL-ELL positive AML. This type of MLL fusion, which contains an intact PHD domain, may cause clonal expansion of MLL-ELL rearrangement positive cells with no obvious differentiation block. Now, we are analyzing biological differences between common type MLL-ELL and exon12 type MLL-ELL by using retroviral gene transfer system.

Background: Chronic GVHD (cGVHD) remains the major cause of late morbidity and mortality after allogeneic stem cell transplantation. However, there are limited data available on cGVHD after unrelated BMT (UR-BMT). We retrospectively analyzed the data of 5,660 patients who underwent UR-BMT through the Japan Marrow Donor Program (JMDP) between January 1993 and June 2004. Methods: Data were collected by the JMDP using a standard report form. Follow-up reports were submitted at 100 days, 1 year, and then annually after transplantation. Overall survival (OS) was estimated by the Kaplan-Meier method and patients surviving beyond day 100 after transplant were analyzed for the development of cGVHD. The log-rank test was used for univariate analysis and time-dependent Cox proportional hazards modeling was used for multivariate analysis. The cumulative incidence of cGVHD and of relapse was calculated using the Gray method considering death without cGVHD and death without relapse as respective competing risks. Results: The median age of all patients was 28 years and the median follow-up was 433.5 days after transplant. Estimated 5-year OS of all patients and those with hematological malignancies was 47.4% and 45.5%, respectively. A total of 3,974 patients survived beyond day 100 after transplant and their cumulative incidence of cGVHD was 43.2% at day 500 and 44.9% at day 2,000 post-transplant. The cumulative incidence of extensive cGVHD was 28.8% at day 2,000 post-transplant. In multivariate analysis, variables predicting cGVHD were recipient age (p=0.000), donor age (p=0.002), diagnosis of hematological malignancy (HR=1.99, p=0.000), HLA class I mismatch by either serology or DNA typing (HR=1.24, p=0.020), acute GVHD (I: HR=1.50, p=0.000; II: HR=2.07, p=0.000; III and IV: HR=2.25, p=0.000) and no platelet recovery over 50,000/mm3 before day 100 (HR=1.36, P=0.002). There was a significant difference between patients 100 days post-transplant was 62.4% without cGVHD, 68.0% with limited cGVHD, and 55.4% with extensive cGVHD (p=0.000). In the patients with hematological malignancies, OS at 5 years was 58.8%, 67.3% and 55.8%, respectively (p=0.000). Cumulative incidence of relapse of hematological malignancies at day 2,000 in patients surviving >100 days post-transplant was 17.6% with limited cGVHD, 18.4% with extensive cGVHD and 27.1% without cGVHD (P=0.000). Conclusions: This study provides strong evidence of risk factors for developing cGVHD after UR-BMT and suggests that limited cGVHD provides a survival benefit to patients with hematological malignancies by reducing the risk of relapse without increasing the risk of death from cGVHD. There was a significant difference in occurrence of cGVHD between patients

Multiple myeloma (MM) is almost invariably fatal despite all available chemotherapeutic and supportive treatment. Initial treatment of MM with dexamethasone (Dex), a key drug for MM chemotherapy, effectively induces myeloma cell death. However, prolonged drug exposure results in the development of Dex-resistance and clinical failures. Therefore, one of the most important issues in myeloma therapy is to overcome resistance to Dex at the stage of clinical refractoriness. Several recent in-vitro studies have demonstrated an antitumor effect of nitrogen-containing amino-bisphosphonates (N-BPs) in some tumor cell lines, including myeloma cell lines. However, few data are available concerning the effects of N-BPs on Dex resistant myeloma cell lines. In this study, we have established a Dex-resistant human myeloma cell line and investigated the antitumor effect of the third generation bisphosphonate Zoledronate (ZOL) on the Dex-resistant subline. 1) Development of de novo resistance through continuous exposure to Dex. A Dex-resistant human myeloma cell line (Dex-R) was selected from the Dex-sensitive myeloma cell line RPMI8226 by continuously exposing cells to gradually increasing doses of Dex. Resistance was validated by flow cytometry. 2) Apoptosis assay. Dual-color FACS with propidium iodide (PI) and Annexin V was used to detect Dex-induced apoptosis in RPMI8226 and Dex-R. 3) Surface markers. Phenotypes of sensitive and resistant cells were compared by FACS analysis. 4) Cell Proliferation and DNA synthesis assay. RPMI8226 and Dex-R cells were cultured in 96 wells plates for 72 hours in the absence or presence of Dex (1 μM) or increasing concentrations of ZOL (2.5 to 50 μM). Cell growth was assessed by WST-8 assay kit. DNA synthesis was measured according to thymidine uptake. 5) Actin staining. RPMI8226 and Dex-R cells were cultured for 48 hours in the absence or presence of Dex (2 μM) or ZOL (40 μM). The distribution of f-actin stained by fluorescent phalloidin was investigated by confocal fluorescence microscopy. The developed subline Dex-R showed reduced apoptotic and antiproliferative responses to Dex treatment. Flow cytometry on 24 hours Dex exposure revealed significantly low percentage of apoptotic (Annexin V positive and PI negative) population in Dex-R cells compared with RPMI8226 cells (6.7% vs. 29.9%). Our data demonstrate that Dex-R cells showed increased CD38, CD44, CD49d (VLA-4) expression, and decreased CD45, CD95 (Fas), CD138 (Syndecan-I) expression as compared to native RPMI8226 cells. ZOL induced apoptosis and inhibited DNA synthesis and cell proliferation in both RPMI8226 and Dex-R cell lines in a dose-dependant manner. Interestingly, some modifications of the cellular morphology were observed in ZOL-treated cells. Rhodamine-phalloidin staining of the f-actin cytoskeleton showed disruption of the cytoskeleton in these deformed cells. This fibroblast-like cellular morphology was common to Dex-R and RPMI8226 cells treated with ZOL, but was not detected under Dex treatment. This result might reflect different mechanism of antitumor effect between Dex and ZOL.. Our results suggest that ZOL can induce myeloma cell death in vitro in a different mechanistic way than Dex. ZOL might be effective as an antitumor drug for Dex resistant myeloma.