ALBERT

Description: Introduction: Myelodysplastic syndromes (MDS) are considered as a "stem cell disorders", in which hematopoietic stem cells and lineage-committed progenitor cells acquire genetic and epigenetic alterations and provide aberrant, clonal hematopoiesis, sometimes resulted in the progression to acute myeloid leukemia (Elias HK et al, Oncogene 2014). We previously reported a rare case of which the patient developed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) 2.5 years after being diagnosed with MDS (Kohno T et al, Br J Haematol 1996). p190 BCR-ABL1 mRNA was detected in the Ph+ALL cells. Metaphase cytogenetics showed the karyotypes: 46, XY, 20q- in MDS phase and 46, XY, t(9;22)(q34;q11), 20q- in ALL phase, indicating that MDS and Ph+ALL in this patient were of the same clonal origin. To uncover the detail of the clonal evolution, we analyzed bone marrow samples of MDS and Ph+ALL in this patient by targeted massively parallel sequencing with a panel of 154 genes including known driver genes of hematologic malignancies. Methods: Genomic DNAs (gDNAs) were extracted from the bone marrow mononuclear cells of MDS and Ph+ALL in this patient. Targeted sequencing was performed on the Illumina HiSeq2500 platform. Single nucleotide variants (SNVs) and small insertions and deletions (INDELs) were called using HaplotypeCaller of Genome Analysis Toolkit (GATK) version 3.4-46. We also attempted to detect the breakpoint of BCR-ABL1 translocation from the targeted sequencing data using the computational method, BreaKmer (Abo RP et al, Nucleic Acids Research 2015). The candidates of the mutations and structural variations were validated by amplicon-based deep sequencing and Sanger sequencing. Copy number variations were analyzed using Affymetrix CytoScan HD Array. Results: The mutations in ASXL1 and U2AF1 genes were identified in the MDS sample with variant allele frequencies (VAFs) of about 45%. At the progression of Ph+ALL, the mutations in SETBP1, SMC1A, and SLC5A8 genes were newly acquired while the ASXL1 and U2AF1 mutations were also identified with the same level of VAFs (about 50%) as the other mutations. VAFs of all of the mutations were decreased to about 20% after the chemotherapy for Ph+ALL, and then increased to about 40% at the recurrence of the disease. Furthermore, we identified the breakpoint of BCR-ABL1 translocation at intron 1 of ABL1 genes and intron 1 of BCR genes, that is the well-known cluster region, m-bcr, only among the samples of Ph+ALL. Copy number analysis confirmed that both MDS and Ph+ALL samples harbored the deletion of chromosome 20q. And the deletion of IKZF1 gene, which is frequently identified in Ph+ALL cases (Mullighan CG et al, Nature 2008), was not identified during the progression from MDS to Ph+ALL. These results demonstrated that the MDS clone harboring 20q- and ASXL1 and U2AF1 mutations acquired the mutations in SETBP1, SMC1A, and SLC5A8 genes and the p190 BCR-ABL1, resulted in the development of Ph+ALL in this patient. Conclusion: The alterations of SETBP1, SMC1A, and SLC5A8 genes are usually reported in myeloid malignancies (Makishima H et al, Nat Genet 2013, Kon A et al, Nat Genet 2013, Whitman SP et al, Blood 2008). Previous study in transgenic mouse demonstrated the distinct role of p190 BCR-ABL1 in the development of an ALL (Voncken JW et al, Blood 1995). Recapitulating this scenario, p190 BCR-ABL1 may play a critical role in the development of Ph+ALL from the MDS stem cells in this patient. This study may provide a new insight into the stem cell origin of MDS and the role of p190 BCR-ABL1 in the development of Ph+ALL. Disclosures No relevant conflicts of interest to declare.

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: Interleukin-15 receptor (IL-15R) and IL-2R have the same β and γ chains, but IL-15R has a specific α chain distinct from that of IL-2Rα, which is indispensable for the high affinity binding of IL-15. In the present study, we examined four IL-2-dependent adult T-cell leukemia (ATL) cell lines for their IL-15R expression. All cell lines bound IL-15, which was not inhibited by a 100-fold excess amount of IL-2, proliferated in response to IL-15 to the same degree as to the stimulation with IL-2, and were maintained without IL-2. The responses to 1L-15 were inhibited by the antibodies against IL-2R β or γ chains but was not by the IL-2R α chain antibody. [125I]–IL-15 exhibited a single high-affinity binding with an apparent kd of 0.17 nmol/L. Reverse transcription–coupled polymerase chain reaction (RT-PCR) showed that the cell lines had the mRNA of IL-15R α. The cell lines also had IL-15 mRNA. Despite the presence of IL-15 mRNA, the cell lines did not secrete IL-15, and the culture supernatants of fresh ATL cells and plasma from the patients did not contain a detectable amount of IL-15 with a few exceptional cases, although fresh ATL cells also responded to IL-15. These results suggest that ATL cells have the complete form of IL-15R and respond to IL-15. Such an IL-15–dependent cell proliferation mechanism might be used in the development of ATL and for the invasion and proliferation of ATL cells in the visceral organs.

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 Nilotinib has shown superiority over imatinib for treating pts with newly diagnosed CML-CP. Additionally, pts who are resistant or intolerant to imatinib can often achieve improved responses by switching to nilotinib. However, the value of switching pts to nilotinib due to molecular SoR on imatinib is not well characterized. The Study to Evaluate Nilotinib in CML pts with SubOptimal Response (SENSOR) was therefore conducted to evaluate the efficacy and safety of nilotinib 400 mg BID in pts with CML-CP with SoR to frontline imatinib. Here, we present results of a planned 12-mo interim analysis. Methods SENSOR (NCT01043874) is an open-label, multicenter, phase IV study. Adult pts with CML-CP in complete cytogenetic response but not in major molecular response (MMR; defined as BCR-ABL ≤ 0.1% on the International Scale [IS]) after ≥ 18 mo on frontline imatinib (SoR as defined by 2009 European LeukemiaNet criteria) were eligible to enroll and switch to nilotinib 400 mg BID. Molecular responses were assessed prior to enrollment, at 1, 2, and 3 mo and every 3 mo thereafter. Mutational analyses were performed at baseline (BL) and at end of study in all pts, every 3 mo in pts with BL mutations, and with any ≥ 5-fold increase in BCR-ABL from the lowest level in pts not in MMR. All of these assays were done at a single central laboratory. The primary endpoint was the rate of MMR at 12 mo. Planned enrollment was 45 pts in order to provide a ≥ 25% lower limit of the 95% CI with an expected MMR rate of 40%. Planned follow-up was 24 mo. Results Planned enrollment of 45 pts was completed between Dec 2009 and Feb 2012. Median age of pts was 47 years (range, 19-80). Pts had received imatinib for a median of 23.0 mo (range, 17.0-103.3) at a median daily dose of 400 mg (range, 297-628). Median BL BCR-ABL/ABL ratio was 0.24% (range, 0.11-3.49). At the cutoff date, 19 pts (42%) had completed the study (24 mo follow-up), and 21 (47%) had nilotinib treatment ongoing. Median nilotinib duration was 19.1 mo (range, 0.1-22.7) and median dose intensity was 733 mg/day (range, 183-800). Five (11%) pts discontinued from study treatment for the following reasons: adverse events (AEs; n = 2), disease progression (n = 1), withdrawal of consent (n = 1), and administrative problems (n = 1). At 12 mo, the MMR rate was 51.1% and the median BCR-ABL/ABL ratio was 0.10% (range, ≤ 0.0032-1.62). The cumulative rate of MMR by 12 mo was 66.7%. Among 34 pts with MMR at any time, median time to first MMR was 2.28 mo. No pt who achieved MMR had confirmed loss of MMR (BCR-ABLIS 〉 0.1% with a ≥ 5-fold increase in BCR-ABL transcripts from the lowest level achieved on study in 2 consecutive samples) by the cutoff date (Table). Two pts (4.4%) achieved BCR-ABLIS ≤ 0.0032% at 12 mo. BCR-ABL mutations were detected at BL in 5 pts (E255K, E459K + exon 8/9 35 base pair insertion, Q252R, M244V, and exon 7 deletion), all of whom achieved MMR on nilotinib. Mutations were identified during treatment in 13 pts, 2 of whom had the same mutation at BL; 10 of these pts achieved MMR. One pt developed a T315I mutation, progressed at 5.4 mo, and died at 9.4 mo. These were the only reported events for progression-free survival and overall survival (1 each). The safety profile of nilotinib was consistent with previous studies. Drug-related AEs (any grade) occurring in ≥ 20% of pts included hyperbilirubinemia (53.3%), increased ALT (28.9%), headache (28.9%), hypophosphatemia (26.7%), rash (26.7%), and increased lipase (24.4%). Grade 3/4 newly occurring/worsening abnormal hematology values were hemoglobin (6.7%), neutrophils (4.4%), platelet count (2.2%), and decreased total white blood cells (2.2%). Six pts had serious AEs (2 with suspected relation to study drug [anemia and erythema multiforme]). Conclusions After switching to nilotinib, MMR was rapidly achieved in the majority of pts. These results support the clinical benefit of switching to nilotinib in pts with molecular SoR to imatinib. Disclosures: Miyamura: Novartis: Speakers Bureau; JRC Nagoya 1st Hospital: Employment. Miyamoto:Kyushu University Hospital: Employment. Kurokawa:Celgene: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Consultancy, Research Funding. Yamamoto:Novartis: Honoraria, Research Funding. Taniwaki:Novartis: Honoraria. Kimura:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Ohyashiki:Novartis: Honoraria, Research Funding. Kawaguchi:Novartis: Honoraria. Matsumura:Bristol Myers Squibb: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Tsurumi:Novartis: Honoraria. Hino:Novartis: Research Funding. Tadokoro:Novartis: Guest speaker in a scientific meeting sponsored by Novartis Other. Hyodo:Hiroshima University: Employment. Kubo:Aomori Prefectural Central Hospital: Employment; Novartis: Honoraria. Kondo:Novartis: Employment. Amagasaki:Novartis: Employment. Kawahara:Novartis: Employment. Yanada:Novartis: Consultancy.

Description: Myelodysplastic syndrome (MDS) is a heterogeneous group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis and a high propensity to transform into acute myeloid leukemia (AML). Although hematopoietic stem cell transplantation can cure some MDS patients, most patients are not eligible to undergo the treatment because of advanced age or comorbidities. In order to choose the optimal treatments, predicting prognosis is crucial. Chromosomal karyotype, bone marrow (BM) blast percentage, and cytopenia are well-known prognostic factors for MDS patients. These factors are incorporated into the international prognostic scoring system and its revised version (IPSS-R). However, the impact of morphological abnormalities has not been fully determined. Thus, to elucidate this, we examined the influence of the number of dysplastic lineages and the presence of increased ring sideroblasts (RS) on the survival of MDS patients whose BM blast percentages were