ALBERT

Description: Adult T-cell leukemia/lymphoma (ATL) relapse is a serious therapeutic challenge after allogeneic hematopoietic stem cell transplantation (allo-SCT). In the present study, we retrospectively analyzed 35 patients who experienced progression of or relapsed persistent ATL after a first allo-SCT at 3 institutions in Nagasaki prefecture (Japan) between 1997 and 2010. Twenty-nine patients were treated by the withdrawal of immune suppressants as the initial intervention, which resulted in complete remission (CR) in 2 patients. As the second intervention, 9 patients went on to receive a combination of donor lymphocyte infusion and cytoreductive therapy and CR was achieved in 4 patients. Of 6 patients who had already had their immune suppressants discontinued before the relapse, 3 patients with local recurrence received local cytoreductive therapy as the initial treatment, which resulted in CR for more than 19 months. Donor lymphocyte infusion–induced remissions of ATL were durable, with 3 cases of long-term remission of more than 3 years and, interestingly, the emergence or progression of chronic GVHD was observed in all of these cases. For all 35 patients, overall survival after relapse was 19.3% at 3 years. The results of the present study suggest that induction of a graft-versus-ATL effect may be crucial to obtaining durable remission for ATL patients with relapse or progression after allo-SCT.

Description: Background Adult T-cell leukemia-lymphoma (ATL) is a chemo-resistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. The HSP90 inhibitor 17-AAG, derived from geldanamycin, has potent antitumor activity against ATL. However, geldanamycin derivatives have several limitations, including poor solubility, formulation difficulties, and severe hepatotoxicity in clinical settings, which have prompted development of second generation synthetic HSP90 inhibitors including NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor that inhibits the ATPase activity of HSP90. AUY922 has shown nanomolar efficacy against a wide range of human cancer cells in vitro and also inhibits progression of a variety of tumors in vivo. Phase I/II studies of AUY922 with advanced solid tumors and hematological malignancies are presently underway. Here, we studied the effects of AUY922 on ATL in vitro and in vivo. Results We initially analyzed the effects of AUY922 (Novartis Pharmaceuticals) on survival of ATL-derived cell lines (KK1, SO4, LM-Y1, KOB, ST1) and HTLV-I-infected T-cell lines (MT2, HuT102). Cells cultured with various concentrations of AUY922 for 72 hours showed survival suppression in a dose-dependent manner in MTS assay findings. The concentrations of AUY922 required to inhibit cell survival by 50% (IC50) varied from 12.5 to 25.0 nM. We also found that the inhibitory effect of AUY was superior to that of 17-AAG. We further assessed AUY922-induced cell survival inhibition with peripheral blood mononuclear cells (PBMCs) obtained from patients with ATL and healthy donors. AUY922 induced apparent cell survival suppression in primary ATL cells, but not in normal PBMCs, while FACS analysis revealed that AUY922 induced cell-cycle arrest and apoptosis in these cell lines. Interestingly, AUY922 induced down-regulation of PIM kinases, which was confirmed by DNA microarray, qRT-PCR, and WB analysis results. Furthermore, SGI-1776, a PIM kinase inhibitor, successfully induced cell survival suppression in ATL and HTLV-1 infected cell lines in both dose- and cell-dependent manners. To elucidate the molecular mechanisms of cytotoxicity, we also examined the expressions of several client proteins using WB analysis. AUY922 treatment led to strong up-regulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease of HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including p-Akt, Akt, IκBα, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. In a xenograft model created with C.B-17/Icr-SCID mice, intraperitoneal administration of the vehicle or AUY922 was given after injection of HuT102 cells. In the control mice, bulky tumors grew within 4 weeks, whereas daily administrations of AUY922 significantly impaired tumor growth. Conclusion Together, our findings suggest that AUY922 may be an effective therapeutic agent for ATL and PIM kinases are a novel therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Description: To examine whether donor-derived cells could exist in nonhematopoietic tissues of recipients after allogeneic hematopoietic stem-cell transplantation, we examined the patterns of the short tandem repeat (STR) of DNA extracted from fingernail clippings of recipients so that the contamination of blood cells was excluded. All 21 patients reached donor-derived hematopoiesis after transplantation and 20 of them were in remission of the primary diseases at the time of sampling. Compared with the STRs of donor cells, among 9 of 21 patients, DNA extracted from fingernail samples showed coexistence of the donor pattern of the STRs, sharing from 8.9% to 72.9% of total STR areas. Time from transplantation to sampling was from 305 to 2399 days among positive cases. These results demonstrate for the first time the existence of stable contribution of donor cells in fingernails among recipients of allogeneic hematopoietic stem cells.

Description: It is reported that fonder mutations affects the development these disorders closely. However, it is unclear whether hematopoiesis carrying these mutations could contribute to the development of the different diseases, directly. We here report a case of acute monoblastic leukemia (AMoL) followed by myeloproliferative neoplasm, unclassifiable (MPN-U), 2 years later, both derived from the same clonal hematopoiesis. [Case report and Methods] A 75-year-old male was admitted to our hospital duo to fever and fatigue. WBC count was 114.7 x 109/L with 9% blasts and 82% monocytes. Bone marrow aspiration revealed 93% of blasts and promonocytes, which resulted in the diagnosis of AMoL with normal karyotype. He received three courses of chemotherapy, which brought CR, but minor monocytosis (〉1 x 109/L) persisted without proliferation of immature blasts. After 2 years from the diagnosis of AMoL, platelet count started to increase. WBC count was between 10 and 20 x 109/L without blast, and platelet count reached 500 x 109/L. Bone marrow examination (2 years from the diagnosis of AMoL) revealed normal cellularity, and less than 5% of blast with normal karyotype. Considering the leukocytosis and thrombocytosis, we evaluated his status as similar to MPN, since dysplasia was not apparent on his hematopoietic cells. Although he had previously received chemotherapy for AMoL, it was diagnosed as MPN-U. Two years after the diagnosis of MPN-U, the disease transformed into AML, not AMoL. He died of AML, four years after AMoL developed. To study clonal status of AMoL and MPN-U in this case, bone marrow mononuclear cells of AMoL (at diagnosis) and MPN-U phases, and buccal swab collected during MPN-U phase were subjected to whole exome sequencing (WES). Validation of mutations was performed by amplicon-base deep sequencing. DNA obtained from bone marrow smear during CR was tested whether mutations existed or not by deep sequencing. Mean depth of WES was 89.15, 140.18, and 98.02 for AMoL, MPN-U, and buccal mucosa samples, respectively. Clonal evolution analysis was performed using clonality inference in tumors using phylogeny. [Results] We found total 22 mutations in samples of AMoL and MPN-U phase with different variant allele frequency (VAF) by WES. When AMoL was diagnosed, 16 genes were mutated with more than 10% of VAF, including genes such as ASXL1, CBL, GATA2, NPM1 (4 bp insertion), SRSF2, and TET2. On the other hand, when MPN-U was diagnosed, there were 16 genes mutated with more than 10% of VAF, and 10 mutations out of 16 were the same mutation as found in the sample of AMoL phase including mutations in SRSF2, GATA2, and TET2. Six mutated genes found only in the sample of MPN-U phase included GNAS and JAK2 (V617F mutation). Since 10 out of 22 genes were commonly mutated in both the AMoL and MPN-U samples, we next analyzed the mutational status of these 22 genes in the CR sample using deep sequencing. It was found that the commonly mutated 10 genes in both AMoL and MPN-U were also mutated in the CR sample with high VAF (〉 25%). Some gene mutations found only in AMoL, and some only in MPN-U were also present during CR phase but with low VAF (〈 10%). Clonal evolution pathway generated by using deep sequence data suggested that before AMoL, there was a founder clone with mutations of TET2, SRSF2, and GATA2, although we could not analyze the sample before AMoL. Mutations in NPM1, ASXL1, CBL, and ASH1L seemed to contribute to the development of AMoL from the founder clone, and those of JAK2 and GNAS for MPN-U. It is also suggested that small amount of hematopoietic stem / progenitor cells containing the JAK2 mutation already existed in CR sample, and these developed into MPN-U probably with gaining other gene mutations. Our data prospectively showed that clonal hematological disorder would arise from a pre-malignant, clonal hematopoiesis that produced normal levels of mature hematopoietic cells. Our analysis clearly demonstrated that two different clonal hematological disorders developed from the same clonal hematopoiesis that had TET2, SRSF2, and GATA2 mutation, which were previously reported as driver mutations. To our knowledge, this is the first report to analyze the progression of AML and MPN from the identical hematopoietic stem cells by WES and deep sequencing validation to model clonal evolution and a case to give a new suggestion about the development of the myeloid malignancies. Disclosures Miyazaki: Celgene Japan: Honoraria; Chugai: Honoraria, Research Funding; Sumitomo Dainippon: Honoraria; Shin-bio: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Moriuchi:Celgene: Other: Research Funding to my institution; travel, accommodations, expenses, Speakers Bureau.

Description: Background Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus which is known as the cause of adult T-cell leukemia/lymphoma (ATLL). Some reports indicated that HTLV-1 carriers were immunologically compromised host. Although there were several reports about the survival impact of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for ATLL, no large study concerning HSCT for the HTLV-1 carrier with the diseases other than ATLL has been reported. Japan is one of endemic countries of HTLV-1, and the prevalence of HTLV-1 infection was reported to be almost 1% of Japanese population. On behalf of the Japan Society for Hematopoietic Cell Transplantation (JSHCT) Complication Working Group, we here report the impact of HTLV-1 serological status on survival in those with HSCT for the diseases other than ATLL by using the Transplant Registry Unified Management Program (TRUMP) which is the nationwide survey database of the Japanese Data Center for Hematopoietic Cell Transplantation. Patients and Method There were 25840 patients (24400 adult and 1440 child), who had the information about the pretransplant serological status of HTLV-1, received their first HSCT between Jan 2007 and Dec 2015 in TRUMP database. We analyzed an overall survival (OS) and non-relapse mortality (NRM) after HSCT in relation to HTLV-1 serological status using Kaplan-Meir method, Gray's test. A Gray test for NRM calculated considering with disease related death as the competing risk. In multivariate analyses using Cox proportional hazard model for OS and Fine-Gray proportional hazards models for competing risk of NRM. And a p-value of less than 0.05 was considered as statistically significant. A statistical analysis was performed with 'EZR'. Results Median age of HTLV-1 carrier and non-carrier in adult patients were 57 years (17-76) and 53 years (16-88) and in pediatric patients were 11 years (0-15) and 8 years (0-15), respectively. The number of HTLV-1 carrier/non-carrier in adult patients in each disease were 80/7511 in AML, 133/6673 in malignant lymphoma, 34/3265 in plasma cell neoplasm, 30/2885 in ALL, 31/2178 in MDS, 8/446 in CML, 5/539 in aplastic anemia, and 11/570 in others, respectively. The number of HTLV-1 carrier/non-carrier in pediatric patients in each disease were 3/684 in ALL, 6/394 in AML, 2/170 in MDS, 2/76 in congenital metabolic disease, and 3/100 in others. The number of HTLV-1 carrier/non-carrier in adult patients who received allo-HSCT or auto-HSCT were 237/15777 and 95/8920, and in pediatric patients who received allo-HSCT was 16/1424, respectively. No HTLV-1 carrier child recipients who recieved auto-HSCT were identified in TRUMP database. There were no significant differences about stem cell sources, disease risk, and HCT-CI score before HSCT between HTLV-1 carriers and non-carriers. The OS rates at 3yr after allo-HSCT in adult and pediatric HTLV-1 carrier vs non-carrier were 40.7% vs. 50.2% (P=0.003) and 49.1% vs. 67.1% (P=0.318), and NRM rates at 1yr after allo-HSCT were 46.9% vs 23.6% (P=0.001) and 18.8% vs 11.7% (P=0.05), respectively. In multivariate analyses in terms of OS and NRM, HTLV-1 carrier was a significant prognostic factor in adult patients (HR 1.23, 95% CI 1.03-1.47, P‹0.001 for OS, and HR 1.27, 95% CI 1.05-1.61, P‹0.001 for NRM) and in pediatric patients (no statistical significance was seen on OS, and HR 2.58, 95% CI 1.17-5.70, P‹0.001 for NRM). The incidence of non-infectious NRM, such as pulmonary complications (IPS/DAH, ARDS), acute and chronic GVHD, MOF, and VOD/SOS was significantly higher in adult HTLV-1 carriers who received allo-HSCT when compared with that in HTLV-1 non-carriers. On the other hand, Infectious NRM was significantly higher incidence in child HTLV-1 carrier. With respect to disease related death, there were no differences between HTLV-1 carrier and non-carrier both in adult and pediatric patients. Among the adult patients who received auto-HSCT, there was no statistically significant difference in terms of OS, NRM, and disease related death between HTLV-1 carrier and non-carrier. Conclusion This is the first large study showing the survival impact of HTLV-1 serological status in patients with diseases other than ATLL who received HSCT. It demonstrated that HTLV-1 antibody-positivity was the poor prognostic factor in terms of OS and NRM after allo-HSCT in adult patients and NRM after allo-HSCT in pediatric patients. Disclosures Nakasone: Phizer: Honoraria; Novartis: Honoraria; Kyowa Hakko Kirin: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen: Honoraria; Takeda: Honoraria. Suzuki:Kyowa-Hakko Kirin: Honoraria; Chugai Pharmaceutical: Honoraria; Mochida Pharmaceutical: Honoraria; Novartis: Honoraria; Shionogi: Honoraria; Takeda Pharmaceuticals: Honoraria; Meiji Seika Pharma: Honoraria; MSD: Research Funding; Ohtsuka: Honoraria; Sawai Pharmaceutical: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Gilead Sciences: Consultancy; MundiPharma: Consultancy; Jazz Pharmaceuticals: Consultancy.

Description: [Background] The 67-kDa laminin receptor (67LR) is a non-integrin cell surface receptor that mediates high-affinity interactions with laminin. Although 67LR was reported to interact with GM-CSF receptor (GM-CSFR) modulating the signaling pathway in hematopoietic cells, little report regarding its expression and the characteristics of AML. [Objective] We addressed whether the surface expression of 67LR be related to the characteristics of AML, and its molecular mechanisms. [Methods] The surface expression of 67LR on CD34-positive AML cells was assessed since it is widely expressed on the mature hematopoietic cells. After purification of CD34 positive cells, flowcytometric analysis of 67LR in 27 AML (M0 [1], M1 [3], M2 [11], M4 [6], M5 [2], M6 [2], MDS/AML [2]) was performed. A GM-CSF dependent TF-1 leukemia cell line (established from M6) was used for the in vitro analysis. cDNA or short interfering RNA of LR was transfected into TF-1 (TF-1LR and TF-1si). Using these cell lines, cell growth, colony formation, cell cycle distribution, and protein phosphorylation were tested. [Results] AML cases were divided into two groups by the surface-expression of 67LR: 9cases in the high-expression group (positive in 〉25% of cells, LR-H) and 18 in the low-expression group (LR-L).The median WBC was significantly higher in LR-H than LH-L (36,700 and 3,250/μl, respectively, p=0.015). Nucleated cell count of bone marrow was also higher in LR-H (p=0.01). Overall survival of LR-H was significantly poor than LR-L (20% and 54%, respectively, p=0.01). Since the expression of 67LR was related to the increased AML cells in clinical samples, we hypothesized that the expression of 67LR influenced the growth of leukemia cells. Using TF-1, the surface-expression of 67LR was modulated by the over expression or interfering of mRNA of 67LR. The surface expression of 67LR on wild type TF-1, TF-1LR and TF-1si was 64%, 92% and 36%, respectively. In WST1 assay, the expression of 67LR was related to the proliferation of TF-1 cells: TF-1LR proliferated rapidly than control (absorbance 1.71±0.06 and 1.24±0.10, respectively. p=0.0023), whereas TF-1si cells grew slowly (absorbance of TF-1si, 0.790±0.004; control, 1.149±0.052, p=0.0018). The number of colony in semi-solid media was related to the expression of 67LR: TF-1LR formed 300±10, the control cells made 91±8colonies/5000cells (p

Description: Myeloperoxidase (MPO) is a specific enzyme whose expression is restricted to the late stage of myeloid differentiation such as promyelocytes and more matured cells. In a pathological situation, however, morphologically immature blasts of acute myeloid leukemia (AML) express MPO at variety of percentages that has been utilized for the diagnosis of AML. On the other hand, the expression of MPO has been shown to have prognostic value for AML cases. Several groups including ours reported that the high percentage of MPO positive blasts at diagnosis was related to the favorable prognosis. Recently, we also found that the expression of the MPO gene in CD133 positive cells also related to the better prognosis of AML cases, suggesting the MPO in the very immature fraction, that is thought to contain leukemia stem cells, is related to the chemosensitivity of AML cells. These results made us hypothesize that MPO directly changes chemosensitivity of leukemia cells through its enzymatic activity. To test this hypothesis, we transfected wild type and enzymaticaly defective MPO (dMPO) cDNA into MPO-negative leukemia cells line, K562, generating K562-MPO and K562-dMPO. Control cells were K562-vector that were transfected with empty vector. The expression of MPO protein was shown in K562-MPO cells, but only precursor of MPO protein, apo-pro-MPO was expressed in K562-dMPO cells as expected. The enzymatic activity was shown only in K562-MPO cells. When these cells were treated with cytosine arabinoside (AraC), K562-MPO cells showed decreased proliferation by WST1 assay compared with wild type K562, K562-vector and K562-dMPO. The expression of Annexin V was increased in AraC-treated K562-MPO suggesting these cells died through apoptosis. Using flowcytometer or confocal microscopy with aminophenyl fluorescein (APF), reactive oxygen species (ROS) were detected in wild type K562 and K562-MPO cells after treatment with AraC. The amount of ROS shown by the intensity of APF-fluorescence increased more in K562-MPO compared with wild type K562 or K562-vector. The difference in the APF-fluorescence observed AraC-treated K562-vector and K562-MPO cells was strongly enhanced by the addition of hydrogen peroxide as a donor of ROS. However, this difference completely disappeared with the addition of N-acetyl cystein (NAC), an inhibitor of ROS, suggesting the involvement of MPO to generate ROS in K562-MPO cells. Although flowcytometer with DAF-2DA failed to detect the significant differences in the amount of reactive nitrogen species (RNS) among AraC-treated wild type K562, K562-vector and K562-MPO cells, immunoblotting against the nitrated tyrosine residue demonstrated the increased nitration of protein in K562-MPO cells compared with wild type or K562-vector. It was suggested that RNS was generated in K562-MPO treated with AraC. These results supported our working hypothesis that MPO in leukemia cells enhanced the sensitivity against chemotherapeutic agents through the generation of ROS and RNS.