Chronic Myeloid Leukemia Blast Crisis Research Articles

European CML Blast Crisis Register

European CML Blast Crisis Register

Spectrum and Immunophenotypic Profile of Acute Leukemia: A Tertiary Center Flow Cytometry Experience.

BackgroundFor diagnosis, sub-categorization and follow up of Acute Leukemia (AL), phenotypic analysis using flow cytometry is mandatory.Material and methodsWe retrospectively analyzed immunophenotypic data along with cytogenetics/molecular genetics data (wherever available) from 631 consecutive cases of AL diagnosed at our flow cytometry laboratory from January 2014 to August 2017.ResultsOf the total 631 cases, 52.9% (n=334) were acute lymphoblastic leukemia (ALL), 43.9% (n=277) acute myeloid leukemia (AML), 2.2% (n=14) mixed phenotypic acute leukemia (MPAL), 0.5% (n=3) acute undifferentiated leukemia (AUL) and 0.5% (n=3) chronic myeloid leukemia in blast crisis (CML-BC). ALL cases comprised of 81.7% (n=273/334) B-cell ALLs (95.2%, n=260/273 common B-ALLs and 4.8%, n=13/273 Pro B-ALLs). CD13 was the commonest cross lineage antigen, expressed in B-ALL (25.6%, n=70/273), followed by CD33 (17.9%, n=49) and combined CD13/CD33 (11.3%, n=31/273) expression. T-ALLs constituted 18.3% (n=61/334) of total ALLs and included 27.9% (n=17/61) cortical T- ALLs. CD13 was commonest (32.7%, n=20/61) aberrantly expressed antigen in T-ALLs, followed by CD117 (19.1%, n=9/47). AML cases included 32.1% (n=89/277) AML with recurrent genetic abnormalities, 9.0% (n=25/277) with FLT3/NPM1c mutation and 58.9% (n=163/277) AML NOS including 14.7% (n=24/163) AML M4/M5, 1.8% (n=3/163) AML M6 and 3.7% (n=6/163) AML M7. In AMLs, CD19 aberrancy was the most common (20.2%, n=56/277) followed by CD56 (15.8%, n=42/265).ConclusionsIn this study, we document the spectrum, correlate the immunophenotype with genetic data of all leukemias, especially concerning T-ALL where the data from India is scarce.

PTBP2 exon 10 inclusion is associated with the progression of CML and it is BCR-ABL1 dependent.

Altered or aberrant expression of several splicing factors leads to the progression of different cancers. Though there are several ongoing studies underscoring the role of the splicing regulator polypyrimidine tract binding protein 2 (PTBP2) in neuronal cells, we unveil the role of PTBP2 in chronic myeloid leukemia (CML). Different RNA binding proteins (RBP's) earlier reported in chronic myeloid leukemia blast crisis (CML-BC) cases (n = 28) from Radich Oncomine leukemia dataset, were compared. We observed increased expression of MSI2 followed by PTBP2 in BC cases and increased PTBP2 expression in relapsed cases (n = 10) from the same dataset compared to other RBPs. We also observed increased PTBP2 exon 10 inclusion in KCL22, a granulocytic lineage CML cell line when compared to other CML cell lines of different lineages. As PTBP2 protein expression is associated with PTBP2 exon 10 inclusion, we observed in cell lines and in a set of progressed cases (n = 4) that increased BCR-ABL1 expression potentiates PTBP2 exon 10 inclusion and thus confers the existence of a functional protein. Inhibition of BCR-ABL1 with imatinib not only blocks the inclusion of exon 10 but also deregulates PTBP2 expression in CML cells. Knockdown of PTBP2 in KCL22 cells leads to reduced cell proliferation, increased G2/M cell cycle arrest and increased apoptosis. Taken together our study portrays PTBP2 as a new possible target for CML and progressive inclusion/exclusion of PTBP2 exon 10 might play an important role in CML progression.

Hyper-Editing of Cell-Cycle Regulatory and Tumor Suppressor RNA Promotes Malignant Progenitor Propagation

Adenosine deaminase associated with RNA1 (ADAR1) deregulation contributes to therapeutic resistance in many malignancies. Here we show that ADAR1-induced hyper-editing in normal human hematopoietic progenitors impairs miR-26a maturation, which represses CDKN1A expression indirectly via EZH2, thereby accelerating cell-cycle transit. However, in blast crisis chronic myeloid leukemia progenitors, loss of EZH2 expression and increased CDKN1A oppose cell-cycle transit. Moreover, A-to-I editing of both the MDM2 regulatory microRNA and its binding site within the 3' UTR region stabilizes MDM2 transcripts, thereby enhancing blast crisis progenitor propagation. These data reveal a dual mechanism governing malignant transformation of progenitors that is predicated on hyper-editing of cell-cycle-regulatory miRNAs and the3' UTR binding site of tumor suppressor miRNAs.

A study to determine the clinical, hematological, cytogenetic, and molecular profile in CML patient in and around Eastern UP, India.

Introduction:Myeloproliferative disorders are characterized by proliferation of one or more myeloid lineages cells. In order to assess the burden of these illness for public health planning, it is important to know their frequency.Objectives:A study to determine the clinical, hematological, cytogenetic, and molecular profile in chronic myeloid leukemia (CML) patient in and around Eastern UP, India.Materials and Methods:Newly diagnosed and follow-up adult and pediatric cases of myeloproliferative disorder were taken into study. Detailed history, physical, and systemic examination was done with informed consent. Investigations like complete blood count including hemoglobin level, platelet count, total and differential leucocyte count, general blood picture, and bone marrow aspiration/biopsy were done. Molecular and cytogenetic studies were also done whenever required.Results:In total, 90 patients were enrolled in the study. The median age of presentation of CML was 37 years and the mean age was 38.6 years. M: F ratio of 1.4:1.75 patients (83%) were in CML-chronic phase (CP), 11 patients (12%) in CML-accelerated phase (AP) phase, and 4 patients (5%) were found in CML-blast crisis (BC) phase. The common symptoms of the patients were fullness of the abdomen (66.6%). Among these 69 cases, Philadelphia chromosome was present in 65 (94.2%) cases. Revers transcriptase polymerase chain reaction (RT–PCR) was done in 40 out of 90 cases, breakpoint cluster region (BCR)-Abelson oncogene (ABL) gene came out to be positive in all the 40 cases.Conclusion:Most CML patients in eastern UP (India) are relatively young (31–40 years). In addition, males were more commonly affected.

Clinical Significance of Additional Chromosomal 8 Clonal Evolution in CML Progression

To explore the clinical significance of clonal evolution of additional chromosomal 8 in CML progression. An unusual case with the clonal evolution from trisomy 8 to tetrasomy 8 accompanied by 2 time of CML blast crisis (BC) was reported. This patient suffered from 2 time of CML blast crisis and the additional chromosome 8 aberrations were accompanied. Trisomy 8 and tetrasomy 8 were detected at first CML blast crisis and second CML blast crisis, respectively. After tetrasomy 8 was developed, the c-Myc was over-expressed and the central nervous system leukemia happened in this case. Only high dose Ara-C and MTX regimen could induce remission for a short period. These findings suggested that additional chromosome 8 aberrations are important marker for poor prognosis of CML patients and contribute to a poor prognosis.

Allogeneic Stem Cell Transplantation for Blast Crisis Chronic Myeloid Leukemia in the Era of Tyrosine Kinase Inhibitors — a Retrospective Study By the EBMT Chronic Malignancies Working Party

Introduction: The prognosis of patients diagnosed with blast crisis (BC) chronic myeloid leukemia (CML) is dismal. Allogeneic stem cell transplantation (alloSCT) represents the only curative treatment option. In the current tyrosine kinase inhibitor (TKI) era, however, data on transplant outcomes in patients with BC CML, particularly those with active BC at transplant, are scarce. We hereby report on a multicentre, EBMT-registry based retrospective study of adult patients allografted for BC CML focusing on patients with active disease at transplant and pre-transplant prognostic factors.Patients and methods: Patients with BC CML at transplant (i.e. prior to the start of the conditioning) who underwent alloSCT after the year 2004 within the EBMT database were identified. Next, transplant centers were asked to report the exact disease status at transplant (including blood count, blast count in peripheral blood and bone marrow, achievement and type of remission with corresponding assessment dates, and the reason to proceed with alloSCT in BC CML). A total of 170 patients allografted for BC CML between 2004 and 2016 had complete data for analysis. Overall survival (OS) and leukemia-free survival (LFS) were calculated from date of alloSCT to the appropriate endpoint. For multivariable analysis of predictors of OS and LFS, Cox proportional hazard regression models were performed. Confounding prognostic factors (full models) were: age, disease status prior to alloSCT, Karnofsky performance status (KPS) prior to transplant, interval from diagnosis to transplant, year of transplant, stem cell source, conditioning intensity, donor type, and donor/recipient sex match. All patients provided informed consent for data collection and analysis.Results: Median age at alloSCT was 45 years (range [r], 18-75). Median time from diagnosis to alloSCT was 13.9 months (r, 1.6-367.4). Median follow-up time was 54.7 months (r, 0.1-135.2). Stem cell source was peripheral blood, bone marrow and cord blood in 145 (85%), 18 (11%) and 7 (4%) patients, respectively. Donor types were: unrelated (UD), matched related, and mismatched related in 91 (54%), 64 (38%), and 15 (9%) patients, respectively. Conditioning was myeloablative in 108 (64%) of patients. KPS at alloSCT was ≤80% in 31% of patients. Information on BCR-ABL mutations was available for 41 patients; T315I was present in 28 patients. After thorough analysis of disease parameters, a total of 95 patients had any kind of remission of BC CML (including secondary chronic phase) prior to transplant (termed BC in remission); 75 patients had active BC CML prior to transplant (termed BC active). Main reason for proceeding with alloSCT despite active disease was resistance/refractoriness towards TKI in combination with polychemotherapy. Extramedullary disease was documented in 4 patients. In uni- and multivariable analyses of the entire cohort, besides low KPS, only disease status prior to transplant was significantly associated with shorter OS and LFS (for BC active: HR 2.00, 95%CI 1.35-2.96, p=0.001 and HR 1.80 95%CI 1.27-2.57, p=0.001, respectively). Accordingly, for patients allografted for active BC estimated 3-year OS and LFS was rather short (23.8% 95%CI 13.6-34.0 and 11.6% 95%CI 3.0-20.2, respectively) and significantly lower as compared to patients allografted for BC in remission (3-year OS and LFS: 51.1% 95%CI 40.5-61.7 and 33.8% 95% CI 23.6-44.0, respectively) (Figure 1A and B). Consequently, prognostic factors for survival were analyzed separately according to disease status at alloSCT (slim models, Table 1). For patients with BC in remission at transplant advanced age, lower KPS, shorter interval from diagnosis to transplant, myeloablative conditioning, and UD transplant were risk factors for inferior survival, whereas in patients allografted for active BC, only UD transplant was associated with prolonged LFS and with a trend towards improved OS (Table 1).Conclusion: Survival of BC CML patients after alloSCT in the TKI era remains poor unless disease remission could be achieved. In patients who achieve remission prior to alloSCT, conventional prognostic indicators remain the determinants of transplant outcomes. In patients with active BC CML, UD transplantation appears to be associated with a survival advantage in our study. [Display omitted] DisclosuresFinke:Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Medac: Consultancy, Honoraria, Other: travel grants, Research Funding; Riemser: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding. Tischer:Jazz Pharmaceuticals: Other: Jazz Advisory Board. Mayer:Eisai: Research Funding; Roche: Research Funding; Affimed: Research Funding; Novartis: Research Funding; Johnson & Johnson: Research Funding. Byrne:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Chalandon:Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel costs.

RNA Editing of Tumor Suppressor Mir-26a Promotes Malignant Progenitor Propagation in Chronic Myeloid Leukemia

Recent studies demonstrate the importance of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) in normal fetal and adult hematopoiesis. RNA-sequencing studies have shown that elevated levels of the ADAR1 editase has emerged as a dominant driver of cancer progression and therapeutic resistance. Specifically, the deregulation of ADAR1 promotes the transformation of chronic myeloid leukemia (CML) from chronic phase (CP) to a therapy resistant blast crisis (BC) phase. Through the regulation of mRNA and microRNA (miRNA) stability, ADAR1 plays a pivotal role in embryonic development and stem cell regulation. We have previously shown that inflammation-responsive ADAR1 heavily contributes to stem cell differentiation and self-renewal in CML disease progression. Here, we describe a novel role of ADAR1 in cell cycle regulation of BC leukemia cells through regulation of miRNA biogenesis.To investigate the role of ADAR1 in miRNA regulation, we performed miRNome miScript PCR array analysis of 1008 miRNAs in cord blood CD34+ expressing hematopoietic stem and progenitor cells (HSPCs) overexpressing ADAR1 wild type (WT) and A-to-I editing deficient ADAR1 mutant. Overall, a total of 112 miRNAs were significantly differentially expressed following ADAR1 expression with cell cycle identified as the top cellular pathway significantly targeted by miRNAs regulated by ADAR1. Notably, ADAR1 editase activity inhibits the expression of miR-26a-5p, a tumor suppressor miRNA that is frequently downregulated in BC CML. ADAR1 inhibits miR-26a-5p through direct editing of the DROSHA cleavage site of primary miR-26a-5p, preventing miR-26a-5p maturation and processing. In normal hematopoietic progenitors, ADAR1-mediated inhibition of miR-26a results in repression of cyclin-dependent kinase inhibitor 1A (CDKN1A) expression indirectly via suppression of the polycomb repressive complex, enhancer of zeste homolog 2 (EZH2), thereby accelerating cell cycle transit. However, in BC CML progenitors, decreased EZH2 and increased CDKN1A oppose the cell cycle accelerating effect of ADAR1. Moreover, we found that the miR-26a targets a different set of mRNA in BC CML compared to cord blood HSPC and has divergent roles in cell cycle regulation. Lentiviral miR-26a overexpression reduced BC leukemia stem cell (LSC) dormancy in the bone marrow and reverses the functional effects of ADAR1, including inhibition of BC cell proliferation in vivo and impaired LSC self-renewal capacity as measured by colony forming assays.Our finding reveals the effects of ADAR1 in LSC generation through impairing biogenesis of cell cycle regulatory miRNAs. The deregulation of ADAR1 contributes to the malignant reprogramming of progenitors into dormant LSCs that are resistant to therapeutic treatments. Future development of ADAR1 inhibitors may be effective in the elimination of dormant BC CML LSCs that evade tyrosine kinase inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Identification of Syndecan-1 As a Key Dependency of Myeloid Leukemia Growth and Dissemination

Intratumoral heterogeneity is a common feature of many myeloid leukemias and a significant reason for treatment failure and relapse. Thus identifying the cells responsible for residual disease and leukemia re-growth is critical to better understand how they are regulated. Here we show that a knock-in reporter mouse for the stem cell gene Musashi 2 (Msi2) allows identification of therapy resistant leukemia propagating cells in aggressive myeloid malignancies, and provides a new strategy for defining their core dependencies. Specifically, we carried out a high throughput screen using Msi2 reporter blast crisis chronic myeloid leukemia (bcCML) and identified syndecan-1 (Sdc1), a cell surface proteoglycan, as preferentially expressed in therapy resistant bcCML cells, and critical for bcCML function. Specifically, in Sdc1-/- mice, Sdc1 loss led to a defect in bcCML growth and propagation in vitro and in vivo, and markedly improved survival. Further, live imaging revealed that Sdc1 loss had a striking impact on the spatiotemporal dynamics of leukemia cells, impairing their localization, migration and systemic dissemination. Mechanistically, distinct elements of Sdc1 contributed to leukemia growth and dissemination, with the core protein alone being able to rescue the growth defect, but the heparin sulfate chains that mediate matrix attachment being needed for migration. These data present a new platform for delineating the biological underpinnings of leukemia stem cell function, and identify Sdc1 as a central regulator of leukemia stem cell growth and dissemination. DisclosuresNo relevant conflicts of interest to declare.

Blinatumumab Induces Responses in Extramedulary B-Cell Acute Lymphoid Leukemia (B-ALL) and Lymphoid Blast Crisis Chronic Myelogenous Leukemia (CML), and Rarely Results in CD19 Negative Relapse

Blinatumumab Induces Responses in Extramedulary B-Cell Acute Lymphoid Leukemia (B-ALL) and Lymphoid Blast Crisis Chronic Myelogenous Leukemia (CML), and Rarely Results in CD19 Negative Relapse

An In Vivo Genome-Wide CRISPR Screen Identifies Novel Dependencies for Blast Crisis Chronic Myelogenous Leukemia

Poorly differentiated aggressive myeloid diseases such as Acute Myelogenous Leukemia (AML) and blast crisis Chronic Myelogenous Leukemia (bcCML) are often resistant to standard therapy and associated with significantly poor survival in both children and adults. There is thus a significant need for a better understanding of the mechanisms that drive disease progression and for finding novel therapeutic targets. Thus, to determine the molecular effectors of myeloid leukemia growth in vivo, we carried out a genome-wide CRISPR/Cas9 dropout screen using the lentiviral Brie gRNA library. This library targets 19,674 genes, and has on average 3 gRNAs for each gene, and 1000 control non-targeting gRNAs. We carried out this whole-genome screen in a mouse model of Cas9+ blast crisis CML (bcCML) driven by BCR-ABL/ NUP98-HOXA9 since this represents a very aggressive phase of myeloid cancer where 90% of the leukemic blasts are undifferentiated and cancer stem cell-like. This in vivo screen led to the identification of 3636 genes essential for leukemic growth and propagation in the bone marrow of recipient mice, constituting pathways such as metabolism, protein translation and DNA replication. The genes that were significantly depleted included known drivers of myeloid cancer progression and regulators of myeloid cancer stem cells (for example, Brd4, Kdm1a, Pafah1b1/Lis1, Rptor), indicating that our screening strategy can successfully identify functional drivers of cancer growth.While intrinsic signals that drive myeloid cancer progression are well described, little is known about how interactions with the surrounding microenvironment can control leukemic growth and propagation. Our whole-genome screen identified ~130 cell surface genes that are significantly depleted in the bcCML stem cells transplanted in vivo. Since environmental factors commonly signal through receptors on the surface of leukemic cells, this subset is likely to include most, if not all, genetic effectors of niche driven signals required for in vivo growth and propagation of aggressive myeloid leukemia cells. Of these 130 genes, several have earlier been shown by us and others to be essential for myeloid cancer progression including Itgb1, Cxcr4 and Cd44. We are currently testing the functional contribution of novel candidate cell surface molecules, which can integrate signals from the environment, on the in vivo growth and progression of myeloid malignancies. We anticipate that these studies will provide a basis for testing antibody-mediated therapeutic inhibition of specific microenvironmental signals on myeloid leukemia growth and propagation. DisclosuresNo relevant conflicts of interest to declare.

GATA2 Zinc Finger Mutations Affect DNA-Binding and Promote Granulopoietic Differentiation

GATA2 Zinc-Finger (ZF) mutations are associated with specific subgroups of myeloid malignancies. Alterations of the N-terminal ZF1 were identified in AML patients with biallelic CEBPA mutations, whereas the C-terminal ZF2 is typically affected by germline mutations, predisposing to MDS and AML, or by somatic lesions in CML blast crisis. Nevertheless, the context-dependent mechanism underlying GATA2 ZF2 mutations remains mostly unclear. Here, we set out to study the functional consequences of GATA2 ZF mutations in myeloid malignancies.In particular, we performed DNA-pulldown experiments, using FLAG-tagged full length GATA2 ZF WT and mutant proteins after expression in HEK293T cells and incubation of the cell lysates with biotinylated oligonucleotides encoding the binding-motif GATC. These experiments showed disruption of DNA binding for all GATA2 ZF mutants tested in vitro - regardless of the mutant positions within the ZF domains (Figure 1 A).Moreover, we studied the impact of GATA2 ZF mutations on the protein-interaction with Friend of GATA Protein 1 (FOG1; HGNC symbol ZFPM1). The influence of FOG1 on GATA2-dependent transcriptional activation was evaluated using a GATA-specific luciferase reporter. All GATA2 mutants tested were able to activate this reporter, although to variable extent. While co-expression of FOG1 overall counteracted GATA2-dependent transcriptional activation, this effect was significantly reduced for the GATA2 mutants L321F (located in ZF1) and T354M (located in ZF2). This suggested that both ZF domains are involved in the FOG1-interaction.To gain insights into the influence of GATA2 ZF1 mutation on hematopoiesis we performed colony forming cell (CFC)-assays. Lin- primary murine bone marrow cells expressing GATA2 WT or mutants were plated in methylcellulose supplemented with cytokines. Consistent with previous reports, GATA2 WT expression led to a reduced colony number, while this effect was decreased for both mutants A318T (ZF1) and L359V (ZF2). In particular, a higher number of CFU-G colonies were observed for the GATA2 mutant-expressing cells indicating a lineage shift towards granulopoiesis (Figure 1 B).In summary, we have shown that GATA2 mutations influence DNA-binding, protein-interactions and myeloid differentiation. Our findings further suggest that GATA2 ZF1 mutations may contribute to myeloid leukemogenesis through increased proliferation of granulocytic progenitors. Understanding the oncogenic collaboration of GATA2 mutations with other driver genes in distinct patient subgroups is a challenge ahead. [Display omitted] DisclosuresHiddemann:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding.

EBV-Related Post-Transplant Lymphoproliferative Disease in Allogeneic Hematopoietic Cell Transplantation: Single-Center Experience and Considerations Regarding CNS Involvement

EBV-related post-transplant lymphoproliferative disease (EBV-PTLD) is a serious complication of allo-HCT, for which antithymocyte globulin (ATG) administration has been identified as the most important predisposing factor. Although the development of EBV-PTLD used to be detrimental to patients, use of rituximab has largely changed both the incidence and the outcome of this complication, either administered as preemptive treatment based on peripheral blood (PB) EBV titles, or for the management of the disease itself.We determined the incidence of EBV-PTLD through chart review of 797 consecutive allo-HCT recipients transplanted in our center (7/1990-7/2018) and evaluated factors potentially influencing EBV-PTLD occurrence and outcome.Among 797 allo-HCT recipients (n=465 sibling, n=277 MUD, n=47 haploidentical, n=2 twin and n=6 cord blood), 14 (1.7%) patients developed EBV-PTLD. The diagnosis was confirmed by biopsy in 9/12 cases (7 monomorphic, 1 polymorphic and 1 HL-like PTLD), 1 case developed sole EBV encephalitis, 1 case developed CNS lymphoma, and the remaining 3 cases were clinically diagnosed on the basis of generalized lymphadenopathy coupled with high PB EBV title. The patients suffered from ALL (n=6), AML (n=2), CML (n=2), severe aplastic anemia (n=2), MDS (n=1) and plasmacytic leukemia (n=1). Notably, 6/14 patients had high tumor burden at the time of transplantation [refractory AML (n=1), relapsed refractory ALL (n=2), CML blastic crisis (n=2), refractory MDS RAEB II (n=1)]. EBV-PTLD incidence was significantly higher in MUD versus sibling allo-HCT (3.4% versus 0.6%, p=0.006), in haploidentical versus sibling allo-HCT (6.4% versus 0.6%, p<0.001), and in haploidentical versus conventional allo-HCT (6.4% versus 1.5%, p=0.01). Among the haploidentical allo-HCTs complicated with EBV-PTLD, 2 were T-cell depleted with add-back infusions of transduced donor lymphocytes, and 1 was T-cell replete. These results possibly reflect the standard use of ATG as acute GVHD prophylaxis in MUD and haploidentical allo-HCT. Indeed, 12/14 (85.7%) patients who developed EBV-PTLD had received ATG as part of their conditioning regimen, at doses ranging from 5-10mg/kg (median 5mg/kg), and 1 additional patient had received ATG 20mg/kg as 2nd-line treatment for hyperacute GVHD. The conditioning intensity was not significantly associated with EBV-PTLD development [classic myeloablative (n=8), reduced-toxicity (n=3), reduced-intensity (n=3)]. EBV-PTLD occurred early in the post-transplant period (median: 73, range 41-603 days). In 7/14 cases, it was preceded by the onset of aGVHD (median interval from aGVHD diagnosis: 58 days), in one case it coincided with aGVHD diagnosis +45 days from allo-HCT, and in one case it developed soon (+55 days) after induction of GVHD through DLIs. At EBV-PTLD diagnosis, the median EBV title in PB was 78,400 copies/ml (range 84-2,860,000). Of note, one patient developed sole CNS EBV-PTLD with extremely low PB EBV viral load. This patient had received preemptive rituximab 500mg 7 months prior to CNS EBV-PTLD development, on the basis of elevated PB EBV title (70,900 copies/ml). Since then, PB EBV title was always tested below <500 copies/ml by conventional PCR, and was practically negative at the time of EBV-PTLD diagnosis (84 copies/ml). Ten patients were treated with intravenous rituximab: in six patients the disease resolved, but 3/4 cases with CNS involvement succumbed. The fourth patient with CNS involvement is currently under treatment with high-dose MTX and intravenous rituximab and has achieved partial remission. The remaining four patients were unsuccessfully treated with combinations of bleomycin/vindesine/IFNa/immunoglobulins/DLIs.In conclusion, EBV-PTLD is an early complication of alloHCT, associated mainly with defects in T cell immunity. Selective ATG administration to MUD and haploidentical allo-HCT may explain the higher incidence of EBV-PTLD as compared to sibling allo-HCT, yet we identified higher incidence among haploidentical alloHCTs that cannot be attributed to a particular lymphodepletion scheme. Prompt administration of intravenous rituximab is very effective, except for CNS involvement. Importantly, PB EBV monitoring may be misleading in CNS EBV-PTLD, particularly following preemptive intravenous rituximab administration. Taking into account the dismal outcome, intrathecal rituximab should be considered. DisclosuresVardi:Gilead: Research Funding; Janssen: Honoraria. Gavriilaki:European Hematology Association: Research Funding.

Successful Salvage with Inotuzumab Ozogamicin in Relapsed/Refractory Lymphoid Blast Crisis of Chronic Myeloid Leukemia after Failure of Multiple Lines of Therapy Including Blinatumomab

Background: Lymphoid blast crisis, one of the two major forms of chronic myeloid leukemia (CML) blast crisis (BC), is comprised of lymphoblasts and occurs in about 30% of BC patients. These individuals often respond to the same treatment strategies used in Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). Blinatumomab, a bispecific T engager, or BiTE, is an antibody with dual specificity against both CD3 and CD19, and is approved for use in relapsed/refractory ALL. We present the first known case of relapsed/refractory lymphoid blast phase CML, including treatment failure with blinatumomab, who achieved a complete response to inotuzumab ozogamicin.Case report: In 2004, the patient was initially diagnosed with CML and received treatment with hydroxyurea followed by imatinib. He was intermittently compliant, later changed to nilotinib, and was ultimately lost to follow-up. Approximately 11 years after diagnosis, he returned with pancytopenia. Bone marrow aspiration and biopsy showed a hypercellular bone marrow with 10-15% lymphoid blasts that expressed CD34, CD19, cCD79a, HLA-Dr, CD10 and nTdt. Testing for the BCR-ABL translocation (at breakpoint P210) was positive confirming lymphoid blast phase CML. He received hyperCVAD + dasatinib, responded, and was referred for allogeneic hematopoietic cell transplantation (HCT) but deferred for social reasons. He was ultimately lost to follow-up.The patient returned with relapsed disease in February 2017 and bone marrow aspiration and biopsy was consistent with CD19+ lymphoid blast phase CML with BCR-ABL major breakpoint p210 and new F317L mutation of ABL kinase domain. He received salvage therapy with vincristine/prednisone/bosutinib, however, repeat bone marrow testing confirmed persistent CD19+ lymphoid blasts (20%). He was treated with blinatumomab, entered a minimal residual disease (MRD)-positive morphologic remission with 1.9% abnormal B lymphoblasts by flow cytometry. A second cycle of blinatumomab was complicated by bone pain and repeat bone marrow testing showed progressive disease with 50-60% CD19+, CD22+ lymphoblasts. Salvage therapy with fludarabine, cytarabine, and idarubicin (FLAG-Ida) in September 2017 was unsuccessful and palliative treatment with vincristine, prednisone, and methotrexate was initiated. He developed new onset neurologic symptoms in March 2018 and lumbar puncture showed evidence of disease in his central nervous system (CNS) The patient received concurrent intrathecal methotrexate and inotuzumab ozogamicin which led to clearance of CNS disease and a morphologic remission in his marrow. He continues with MRD-positivity with 0.15% B lymphoblasts and is on ponatinib maintenance while allogeneic HCT evaluation is underway.Discussion: This is the first reported patient with lymphoid blast phase CML, refractory to BiTE therapy, that was successfully salvaged with inotuzumab ozogamycin. Recent data from acute lymphoblastic leukemia (ALL) reported that the majority of BiTE failures remained CD19+ and the mechanisms of CD19 immunotherapy failure are poorly understood (Jabbour E, et al, AJH 2017). These patients may be successfully salvaged by CD19-directed chimeric antigen receptor (CAR) T cells or other directed therapies such as inotuzumab ozogamycin.Conclusion: Patients with relapsed/refractory lymphoid blast phase CML should receive directed salvage therapies. This case report demonstrates that failure of one line of directed therapy does not predict for future failures. DisclosuresNo relevant conflicts of interest to declare.

Inactivation of Stage-Specific B-Cell Commitment Genes Generates Distinct Molecular Subtypes of BCR-ABL1 Lymphoblastic Leukemia

Background Philadelphia-chromosome positive acute lymphoblastic leukemia (Ph+ ALL), the most common form of ALL in adults, is a highly aggressive blood malignancy defined by the BCR-ABL1 fusion. Although inhibitors targeting the BCR-ABL1 oncoprotein, such as imatinib, have significantly improved clinical response rates for this disease, a subset of patients are refractory to therapy or respond initially but relapse soon after. ABL1 kinase domain mutations partly explain differential responses in patients; however, for the majority of cases, a molecular basis that can reconcile this clinical observation is lacking.Methods Flow-sorted blasts from 53 primary samples, 49 de novo Ph+ ALL and 4 lymphoid blast crisis CML, were subjected to RNA sequencing (RNA-seq) and whole genome sequencing (WGS). Response rates were tracked using BCR-ABL1 transcript levels from patient blood.Results Hierarchical clustering of transcriptome data produced two molecular subgroups of Ph+ ALL. One subgroup, which we termed ‘Core-B’, upregulated key regulators of B-lymphoid differentiation including IL7R and MS4A1 (CD20). By contrast, the second subgroup upregulated an expression program related to hematopoietic stem cell (HSC) and myeloid differentiation, with upregulation of KIT, CD34, MPO, CSF3R, and GATA3. We termed this subgroup ‘Aberrant-Stem-Myeloid’ (ASM). These subgroups displayed a striking disparity in response rates to intensive chemotherapy with imatinib. Whereas ‘Core-B’ patients showed highly durable responses often lasting many years, ‘ASM’ patients frequently relapsed (4% vs 43% relapse; p=0.007). We used WGS analysis to investigate the genetic basis of these molecular subtypes. ‘Core-B’ Ph+ ALLs were enriched for deletions in PAX5, a B-cell specification gene, and CDKN2A/B, tumor suppressors. The ‘ASM’ subtype lacked these genetic alterations; instead, these leukemias were enriched for deletions in EBF1, an early B-cell lineage factor that represses T-lymphoid and myeloid lineages and is expressed before PAX5 in B-cell lineage differentiation. Accordingly, blasts from ‘ASM’ leukemias with EBF1 deletions showed decreased CD19 antigen expression and upregulation of myeloid antigens by clinical flow cytometry. Rare cases with concurrent EBF1 and PAX5 deletions showed expression features of both ‘ASM’ and ‘Core-B’ leukemias. Mutations observed in myeloid leukemias (TET2, RUNX1) were only present in the ‘ASM’ subtype. Loss of IKZF1, found in 77% of cases, also displayed distinct patterns between the two subgroups; deletions leading to the dominant negative isoform (Ik6) were enriched in the ‘Core-B’ subgroup (45% vs. 14%; p=0.019) while monosomy 7 and large deletions encompassing IKZF1 were enriched in the ‘ASM’ leukemias (41% vs. 10%; p=0.017). In 1 of 4 diagnosis/relapse patients analyzed, a molecular switch from ‘Core-B’ at diagnosis to ‘ASM’ at relapse was observed. The diagnostic ‘Core-B’ clone from this patient harbored a PAX5 mutation that was lost at relapse, whereas the relapsed ‘ASM’ clone harbored trisomy 21 and a RUNX1 mutation. Altogether, our data suggest that the ‘ASM’ leukemias emerge through dysregulation of genes earlier in lympho-myeloid specification compared to ‘Core-B’ leukemias.These findings led us to investigate if the ‘ASM’ subtype originates from an HSC and the ‘Core-B’ subtype originates from a B-cell progenitor. We first looked at the distribution of the long (p210) and short (p190) isoforms of BCR-ABL1 in the two subtypes. The p210 isoform, also the hallmark of CML, is speculated to arise in an HSC, and the p190 is thought to arise in a B-cell progenitor. Neither the p190 or p210 BCR-ABL1 isoform was enriched in either subgroup. We resolved highly purified HSC and progenitor subsets from CD34+CD19- cells, functionally evaluated by methylcellulose assays, and subjected them to a sensitive nested-PCR strategy. Cases from both the ‘ASM’ and ‘Core-B’ subtypes showed HSC/myeloid progenitor involvement regardless of the BCR-ABL1 isoform. This data suggest that the cell-of-origin does not play a role in establishing the molecular subtype of leukemia blasts.Conclusion There are two distinct molecular subtypes of Ph+ ALL that demonstrate differential responses to treatment and emerge from independent mutational routes. Moreover, the key genetic determinants that form the molecular subtype are secondary driver alterations that lie downstream of BCR-ABL1. DisclosuresNo relevant conflicts of interest to declare.

CD8+ Effector T Cell Migration into the Bone Marrow of Transplanted Mice: Influence of Gvhd, Leukemia Cells and Variable Dependence on Chemokine Receptors

The graft-versus-leukemia (GVL) effect, mediated by alloreactive T cells, reduces the risk of leukemia relapse after allogeneic hematopoietic stem cell transplantation. While much work has focused on how T cells kill leukemia cells, less attention has been paid to the process whereby activated alloreactive T cells find leukemic targets. We initially focused on how chemokine receptor signaling impacts effector T cells egress from blood, entry into the marrow space and subsequent scanning of leukemia cells. We further tested how this process is influenced by transplantation and the consequences of alloreactive T cells. We tracked the migration of CD8+ RAG1-/- OT-1 TCR transgenic effectors (generated by in vivo vaccination of B6 mice seeded with naïve OT-1 cells) in recipients. OT-1 cells recognize the SIINFEKL peptide from ovalbumin and are not reactive in our models. OT-1 effectors were treated (or not) with pertussis toxin (PTX) to block chemokine receptor signaling, and labeled with fluorescent dyes so as they could be identified by flow cytometry and two photon intravital microscopy (2PIM). PTX+ and PTX- OT-1 cells were transferred together into the following recipients: unmanipulated B6 mice, irradiated (B6xBALB/c) CB6F1mice transplanted with B6 bone marrow (BM) cell, with or without B6 T cells. In some experiments on day 12 post-transplant, F1 mice also received WT or IFN-γR-/- B6 blast crisis CML cells (BC-CML) generated by transduction of BM with retrovirus encoding bcr-abl and the NUP98/HOXA9 fusion as we have described (J Clin Invest. 2017;127(7):2765-2776). OT-1 effectors were infused on day 14 and 3 hours later the calvarium was analyzed by 2PIM to enumerate PTX treated or untreated OT-1 effectors that were inside or outside the blood vessels. Blood vessels were identified using labeled dextran or Qdot 655.PTX treatment was highly effective as PTX-treated OT-1 were nearly completely excluded from lymph node (LN). PTX treatment reduced OT1 migration into the BM of unmanipulated B6 mice and similarly in F1 recipients without T cells. However, the presence of a GVH response reduced the recruitment of PTX- OT-1 cells into BM. The ratio of OT-1 outside/inside vessels was still higher in the PTX- group; however, this was mostly driven by an increase of PTX+ OT-1 in vessels in GVH mice. These data suggest that GVH may reduce effector T cell recruitment to the BM, though some of this effect could be due to effectors trafficking instead to other locations, perhaps GVHD target tissues.The presence of WT BC-CML leukemia cells in F1 GVH recipients increased recruitment of OT-1 PTX- effectors into the BM, both relative to PTX+ OT-1 and in total numbers compared to experiments wherein no BC-CML cells were infused. We considered the possibility that IFN-g-stimulation of BC-CML cells may have released chemokines that recruited PTX- OT-1. However, IFNγR-/- BC-CML also increased PTX- OT-1 recruitment in GVHD mice to a similar degree. Experiments are ongoing to better understand how BC-CML cells promote PTX-inhibitable effector T cell recruitment. DisclosuresShlomchik:NapaJen: Consultancy.

Blast Crisis of CML After TKI Discontinuation in a Patient With Previous Stable Deep Molecular Response: Is It Safe to Stop?

Blast Crisis of CML After TKI Discontinuation in a Patient With Previous Stable Deep Molecular Response: Is It Safe to Stop?

Adoptive Immunotherapy with Cord Blood for the Treatment of Refractory Acute Myelogenous Leukemia: Feasibility, Safety, and Preliminary Outcomes

Adoptive immunotherapy has shown efficacy in patients with relapsed/refractory acute myelogenous leukemia (AML). We conducted a prospective evaluation of cord blood (CB)-based adoptive cell therapy following salvage chemotherapy in patients with AML or myelodysplastic syndrome (MDS) and describe the safety and early outcomes of this approach. To enhance the antileukemic effect, we selected CB units (CBUs) with a shared inherited paternal antigen (IPA) and/or noninherited maternal antigen (NIMA) match with the recipients. Furthermore, the CBUs had total nucleated cell (TNC) dose <2.5 × 107/kg and were at least 4/6 HLA-matched with the patients; a higher allele-level match was preferred. Heavily pretreated adult patients with AML/MDS were enrolled. CBU searches were performed for 50 patients. CBUs with shared IPA targets were identified for all, and CBUs with NIMA matches were found for 80%. Twenty-one patients underwent treatment (AML, primary induction failure, n = 8; refractory relapse, n = 10, including 7 recipients of previous allogeneic HSCT; blast crisis chronic myelogenous leukemia, n = 1; MDS, n = 2). Most received combination chemotherapy; those not fit for intensive treatment received a hypomethylating agent. Response was defined as <10% residual blasts in hypocellular bone marrow at approximately 2 weeks after treatment. Ten of the 19 evaluable patients responded, including 5 of the 7 recipients of previous transplant. Response was seen in 4 of 4 patients with full CBU-derived chimerism, 2 of 2 of those with partial, low-level chimerism and 4 of 12 of the recipients with no detectable CBU chimerism. The most common adverse events were infections (bacterial, n = 5; viral, n = 2; fungal, n = 5). Grade IV acute graft-versus-host disease (GVHD) developed in 2 patients with full CBU chimerism; 2 other patients had grade 1 skin GVHD. A total of 11 patients died, 7 from disease recurrence and 4 from infections (1 early death; the other 3 in remission at the time of death). Overall, 12 patients proceeded to allogeneic HSCT; of those, 7 had responded to treatment, 3 had not (and had received additional therapy), and 2 had persistent minimal residual disease. In conclusion, the use of CB as adoptive immunotherapy in combination with salvage chemotherapy for patients with refractory AML/MDS is feasible, can induce disease control, can serve as a bridge to allogeneic HSCT, and has an acceptable incidence of adverse events. Alloreactivity was enhanced through the selection of CBUs targeting a shared IPA and/or NIMA match with the patients. CBUs with lower cell doses, already available in the CB bank and unlikely to be adequate grafts for adult transplants, can be used for cell therapy within a short time frame.

Transcriptional activation of the miR-17-92 cluster is involved in the growth-promoting effects of MYB in human Ph-positive leukemia cells.

MicroRNAs, non-coding regulators of gene expression, are likely to function as important downstream effectors of many transcription factors including MYB. Optimal levels of MYB are required for transformation/maintenance of BCR-ABL-expressing cells. We investigated whether MYB silencing modulates microRNA expression in Philadelphia-positive (Ph+) leukemia cells and if MYB-regulated microRNAs are important for the “MYB addiction” of these cells. Thirty-five microRNAs were modulated by MYB silencing in lymphoid and erythromyeloid chronic myeloid leukemia-blast crisis BV173 and K562 cells; 15 of these were concordantly modulated in both lines. We focused on the miR-17-92 cluster because of its oncogenic role in tumors and found that: i) it is a direct MYB target; ii) it partially rescued the impaired proliferation and enhanced apoptosis of MYB-silenced BV173 cells. Moreover, we identified FRZB, a Wnt/β-catenin pathway inhibitor, as a novel target of the miR-17-92 cluster. High expression of MYB in blast cells from 2 Ph+leukemia patients correlated positively with the miR-17-92 cluster and inversely with FRZB. This expression pattern was also observed in a microarray dataset of 122 Ph+acute lymphoblastic leukemias. In vivo experiments in NOD scid gamma mice injected with BV173 cells confirmed that FRZB functions as a Wnt/β-catenin inhibitor even as they failed to demonstrate that this pathway is important for BV173-dependent leukemogenesis. These studies illustrate the global effects of MYB expression on the microRNAs profile of Ph+cells and supports the concept that the “MYB addiction” of these cells is, in part, caused by modulation of microRNA-regulated pathways affecting cell proliferation and survival.

Crosstalk between BCR-ABL and protease-activated receptor 1 (PAR1) suggests a novel target in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome, which generates the oncogene BCR-ABL1. Protease-activated receptor 1 (PAR1) is involved in tumor progression and angiogenesis. We have previously reported that PAR1 expression is elevated in human leukemias that display a more aggressive clinical behavior, including the blast crisis of CML. In this study, we analyzed the crosstalk between the oncoprotein BCR-ABL and PAR1 in CML. Leukemic cell lines transfected with the BCR-ABL1 oncogene showed significantly higher expression levels of PAR1 compared with that of wild-type counterparts. This phenomenon was reversed by treatment with tyrosine kinase inhibitors (TKIs). Conversely, treatment with the PAR1 antagonist SCH79797 inhibited BCR-ABL expression. The PAR1 antagonist induced apoptosis in a dose- and time-dependent manner. Higher vascular endothelial growth factor (VEGF) levels were observed in cells transfected with BCR-ABL1 than in their wild-type counterparts. VEGF expression was strongly inhibited after treatment with either TKIs or the PAR1 antagonist. Finally, we evaluated PAR1 expression in CML patients who were either in the blast or chronic phases and had either received TKI treatment or no treatment. A significant decrease in PAR1 expression was observed in treatment-responsive patients, as opposed to a significant increase in PAR1 expression levels in treatment-resistant patients. Patients classified as high risk according to the Sokal index showed higher PAR1 expression levels. Our results demonstrate the crosstalk between BCR-ABL and PAR1. These data may offer important insight into the development of new therapeutic strategies for CML.