11q23 Rearrangements Research Articles

Post-Remission Treatment with Chemotherapy or Allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT) in Adult Patients with High-Risk (HR) Philadelphia Chromosome-Negative (Ph-neg) Acute Lymphoblastic Leukemia (ALL) According to Their Minimal Residual Disease (MRD). Final Results of the Pethema ALL-HR-11 Trial

Introduction: Recent studies have shown that young to middle-aged adults who receive a pediatric-inspired chemotherapy regimen for treatment of Ph-neg ALL do not appear to require an alloHSCT if they achieve good response on MRD testing after induction and/or consolidation therapy. Patients (pts) who are not good MRD responders achieve better outcomes with alloHSCT than their counterparts who do not receive alloHSCT. However, it is not clear if this approach can specifically apply to adult ALL pts with HR features at baseline. The aim of the prospective ALL-HR-11 trial (NCT01540812) from the Spanish PETHEMA Group was to evaluate response of HR Ph-neg adult ALL patients to a different post-induction therapy (chemotherapy or alloHSCT) according to MRD levels (centrally assessed by 8-color flow cytometry [FCM]) at the end of induction (week 5) and consolidation therapy (week 17).. Patients and methods: HR ALL included one or more of the following parameters at baseline: age 30-60y, WBC count >30x109/L for B-cell precursor ALL or >100x109/L for thymic T-ALL, pro-B, early or mature T-ALL, 11q23 or KMT2A rearrangements or complex karyotype. Induction therapy included vincristine, prednisone, daunorubicin and asparaginase (E coli native or PEG according to center availability) for 4 weeks (Induction-1). FLAG-Ida was administered as intensified induction (Induction-2) in pts not achieving CR or in those in CR with MRD≥0.1% at the end of induction. For pts in CR and MRD<0.1% early consolidation therapy included 3 cycles with rotating cytotoxic drugs with high-dose methotrexate, high-dose ARA-C and high-dose asparaginase (E coli native or PEG). These pts continued with delayed consolidation (identical to that of early consolidation) followed by maintenance therapy up to 2y in CR if MRD levels after consolidation were <0.01%; otherwise they were assigned to alloHSCT. Pts in CR after Induction-2 received one consolidation cycle and were assigned to alloHSCT. Main outcome measures were: complete response (CR), overall survival (OS) and cumulative incidence of relapse (CIR), assessed by competing risk analysis. Results: On April 2019, 307 HR ALL pts were evaluable. Median (range) age was 40 (15-60) y, 192 were males, 211 precursor B-ALL and 96 T-ALL, with a median WBC count of 12.9 (0.2-564) x109/L. Results of Induction-1 (n=304, 3 on induction): therapy-related death: 12(4%), resistance: 39(13%), CR: 253(83%). MRD<0.1% at the end of induction was observed in 77% of CR patients. Induction-2 was administered to 88 patients (due to no CR: 37, or to CR and MRD≥0.1%: 51). Overall response rate: 277 (91%). The 5y CIR and OS probabilities for the whole series were of 44%±8% and 48%±7% (median follow-up: 2.06y [range: 0-7.55y]). By intention-to treat after Induction-1, 94 pts were assigned to alloHSCT and 190 to delayed consolidation and maintenance. The 5y CIR and OS probabilities were of 37%±13% and 38%±11%, respectively, for pts assigned to alloHSCT, and of 48%±10% and 55%±10%, respectively, for those assigned to chemotherapy (P<0.001 for OS [Figure 1], and P=0.243 for CIR). Patients with MRD<0.1% at the end of induction and MRD<0.01% at the end of consolidation (n=137) showed a 5y CIR and OS of 42%±11% and 66%±11%, respectively (P<0.001 for both, Figure 2). Patients with MRD levels <0.01% on day14 of induction-1, end-induction-1 and end-consolidation (n=17) showed 5y CIR and OS probabilities of 17%±19% and 90%±19%, respectively. Conclusions: This trial, in which post-induction therapy was only based on MRD results assessed by FCM, suggests that avoiding alloHSCT does not hamper the outcome of HR Ph-neg adult ALL pts with adequate MRD response after induction and after consolidation. Better post-remission alternative therapies are specially needed for patients with poor MRD clearance. Supported by grants PI14/01971 FIS, Instituto Carlos III, and SGR225 (GRE), Spain. Disclosures Montesinos: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Teva: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: Research support; Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau. Esteve:Novartis: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy; Pfizer: Consultancy; Astellas: Consultancy, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy.

The Translational Switch of MYC Protein Aliases in Myeloma Tumor Cells

Our systematic investigation on MYC (C-Myc) - a crucial pro-regeneration transcription factor - has determined that genetic aberrations (chromosomal rearrangements) and overexpression of the gene are highly prevalent in multiple myeloma (MM). At diagnosis, the rearrangements of MYC locus (8q24) were detected in 51% of MM patients using karyotyping and interphase FISH with DNA probe sets to MYC and the flank genomic sequences. The abnormalities of 8q24 in MM include monosomy (5%), amplification (≥3 copies; 13%), break-apart (14%), jumping (11%), and others (8%). Such aberrations were present in 49% of low-risk MM (183/376) and in 62% of high-risk MM (51/82) defined by the gene expression profiling cancer-risk model (GEP-70). The GEP also indicated that 74% of MM patients had excessively high levels of MYC overexpression. Nonetheless, there was no significant correlation between the MYC cytogenetic abnormalities and the levels of MYC expression in myeloma tumor cells - unlike the primary IGH translocations that drive transcriptional spikes of its partner genes. MYC expression by GEP was unable to precisely predict the presence of MYC chromosomal rearrangements in MM. In fact, the clinical outcomes of MM patients with MYC aberrations vs. the patients without detectable 8q24 events showed that neither the numerical variations nor the structural rearrangements of 8q24 had significant impact on the overall survival of patients who were treated on Total Therapy protocols. Further, we have shown that chromosomal rearrangements and the levels of MYC overexpression were not intrinsically relevant to the translation of MYC protein in myeloma. Importantly, the fate of myeloma cells was associated with the presence of two endogenous polypeptide-isoforms of MYC - the canonical MYC protein alias (P01106; 48.8kD) and an alternative MYC protein alias (alt-MYC; > 50kD). In newly diagnosed MM, P01106 was absent in most of the patients; whereas, it was a constitutive alias well maintained across the myeloma tumor cells from the patients with relapsed and progressive diseases and in all of the self-renewable myeloma cell lines. In contrast, alt-MYC alias was present as a stand-alone alias or co-existing with P01106 alias in newly diagnosed myeloma. Most importantly, both MYC aliases were absent in some cell lines and primary tumor cells suggesting cancer cell growth may not be dependent on any of MYC aliases despite of high levels of MYC transcription. Since the MYC gene originated from a viral integration, MYC mRNA has dual-translation features due to an internal ribosome entry segment (IRES) at 5'UTR. Previous studies have determined that the translations can be alternatively initiated from an in-frame CUG (Leucine) codon to generate NP_002458 alias or at a downstream canonical cap-dependent AUG (Methionine) codon to generate P01106 alias. Although these two aliases share identical C-termini, the translational switch can extend the N-terminal in NP_002458 alias. In vitro, the switch of P01106 to alt-MYC has resulted in cell growth arrest. Such alternation was induced by a transient gene transfection of P53 tumor suppressor (TP53); seemingly, the alternative MYC alias acts as an antagonist to P01106 in cell proliferation. Overall, it was generally unpredictable which of the MYC aliases were the dominant driver; therefore, MYC cytogenetic rearrangements and overexpression may have unknown indirect impacts on the outcomes. We are currently examining the precise amino acid sequences on both N-terminal and C-terminal ends of MYC aliases to study the regulatory mechanisms of the translational switching. This may indicate a novel anti-cancer strategy to cease cancer proliferation by deliberately inducing alternative translation of the antagonistic alias of an oncogene, and eventually overcome cooperation of the chromosomal rearrangements and dysregulated oncogene transcriptions in progressive cancer cells. Disclosures van Rhee: Adicet Bio: Consultancy; Karyopharm Therapeutics: Consultancy; EUSA: Consultancy; Castleman Disease Collaborative Network: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy; Kite Pharma: Consultancy.

1731 Leukemic Infiltration Leading to Esophagitis: A Rare Initial Presentation of AML

INTRODUCTION: Acute Myeloid Leukemia (AML) is a group of blood cancers that arise from clonal proliferation of malignant hematopoietic precursor cells in bone marrow. We report a case of AML that presented as esophagitis secondary to leukemic infiltration of the esophagus. CASE DESCRIPTION/METHODS: A 60 year male who presented with 1 month history of worsening retrosternal pain, worsened with eating & drinking, leading to a 9 kg weight loss. Patient was taking increasing doses of NSAIDS to treat this pain unsuccessfully. Patient’s past medical history included hypertension, hyperlipidemia & Bell's palsy. Patient’s EKG & 3 troponins were normal. He had no melena, hematochezia but stool guaiac tested positive. Initial CBC showed a WBC 20.5 k/mm3, Hgb 7.7 g/dL, & Platelets 28 k/mm3 with peripheral smear revealing 86% blasts with rare Auer rods. Patient was diagnosed with AML with NSAID associated gastritis & was started on IV Proton pump inhibitor therapy. Patient underwent a bone marrow biopsy that showed 95% blasts and 5% erythroid. On flow cytometry 95% were myeloblasts. Fluorescence in-situ hybridizationtesting showed MLL mutation with 11q23 rearrangement. While awaiting induction 7 + 3 chemotherapy, patient underwent EGD due to continued odynophagia. It showed multiple discreet large non-bleeding ulcers in the proximal & middle third of the esophagus. CMV esophagitis was suspected & the patient received empiric ganciclovir for 3 days. The final biopsy showed no CMV and colonisation without invasion by Actinomycetes, but it also showed atypical mononucleated cells consistent with leukemic infiltrates in the ulcer bed. Patient received induction chemo 7 + 3 but had partial response based on day 14 bone marrow & was successfully reinducted. He is currently awaiting a bone marrow transplant on consolidation chemotherapy. He is symptom free of esophagitis. DISCUSSION: In AML, large numbers of immature myeloid cells are seen in the bone marrow & in peripheral blood resulting in a crowding out effect of other cell lines. Presentation is usually symptoms of anemia, bleeding from thrombocytopenia or immune deficiency. In previous autopsy series, gastrointestinal involvement of leukemia have been noted, with involvement of the esophagus being less common. There have been reports of leukemic infiltration of the esophagus developing during treatment for AML, but our case is a rare initial presentation with esophagitis from leukemic infiltration which led to the diagnosis and treatment of AML.

OP-10 - Role of [68Ga]-pentixafor-PET/CT as a novel imaging of cxcr4 expression in multiple myeloma; comparison to [18F]-FDG-PET/CT

We described the clinical features and outcomes for 63 adult patients with acute myeloid leukemia (AML) with a translocation involving the 11q23 locus (MLL) who were treated at Memorial Sloan Kettering Cancer Center (MSK). The population included 40 female (63 %) and 23 male (37 %) patients, with a median age of 51 years old (range 18–82 years). Of the 31 patients who had had an antecedent malignancy, 14 (45 %) had had breast cancer or DCIS and 22 (71 %) had received anthracycline-based systemic chemotherapy. The translocation partner for the 11q23 rearrangement was identified in 60 of the 63 patients (95 %) studied. The distribution of translocation partners differed for those who had previously received cytotoxic chemotherapy. Most patients with therapy-related disease had a 9p22 or 19p13 partner, as compared to those with de novo disease (95 % vs. 68 %, p = 0.023). Of the 30 patients who received all therapy under observation, 15 (50 %) patients had de novo disease and 15 (50 %) had received antecedent chemotherapy. No significant difference in survival was observed between groups (p = 0.44). Twenty-two patients received induction as up-front therapy, of whom 11 (50 %) achieved CR / CRi. The achievement of CR / CRi with one course of induction was associated with improved OS, with a 6-month OS of 73 % as compared to 23 % for those who did not (p = 0.018). The achievement of CR / CRi with a single course of induction may be a marker of favorable survival in this subtype of high-risk AML.Response to a single induction was associated with favorable survival in this population.

OP-31 - SH2-containing tyrosine phosphatase-1 (SHP-1) MRNA expression and its promoter methylation in imatinib response in Egyptian chronic myeloid leukemia patients

We described the clinical features and outcomes for 63 adult patients with acute myeloid leukemia (AML) with a translocation involving the 11q23 locus (MLL) who were treated at Memorial Sloan Kettering Cancer Center (MSK). The population included 40 female (63 %) and 23 male (37 %) patients, with a median age of 51 years old (range 18–82 years). Of the 31 patients who had had an antecedent malignancy, 14 (45 %) had had breast cancer or DCIS and 22 (71 %) had received anthracycline-based systemic chemotherapy. The translocation partner for the 11q23 rearrangement was identified in 60 of the 63 patients (95 %) studied. The distribution of translocation partners differed for those who had previously received cytotoxic chemotherapy. Most patients with therapy-related disease had a 9p22 or 19p13 partner, as compared to those with de novo disease (95 % vs. 68 %, p = 0.023). Of the 30 patients who received all therapy under observation, 15 (50 %) patients had de novo disease and 15 (50 %) had received antecedent chemotherapy. No significant difference in survival was observed between groups (p = 0.44). Twenty-two patients received induction as up-front therapy, of whom 11 (50 %) achieved CR / CRi. The achievement of CR / CRi with one course of induction was associated with improved OS, with a 6-month OS of 73 % as compared to 23 % for those who did not (p = 0.018). The achievement of CR / CRi with a single course of induction may be a marker of favorable survival in this subtype of high-risk AML.Response to a single induction was associated with favorable survival in this population.

OP-47 - Production of immortal squamous cell line from larynx carcinoma

We described the clinical features and outcomes for 63 adult patients with acute myeloid leukemia (AML) with a translocation involving the 11q23 locus (MLL) who were treated at Memorial Sloan Kettering Cancer Center (MSK). The population included 40 female (63 %) and 23 male (37 %) patients, with a median age of 51 years old (range 18–82 years). Of the 31 patients who had had an antecedent malignancy, 14 (45 %) had had breast cancer or DCIS and 22 (71 %) had received anthracycline-based systemic chemotherapy. The translocation partner for the 11q23 rearrangement was identified in 60 of the 63 patients (95 %) studied. The distribution of translocation partners differed for those who had previously received cytotoxic chemotherapy. Most patients with therapy-related disease had a 9p22 or 19p13 partner, as compared to those with de novo disease (95 % vs. 68 %, p = 0.023). Of the 30 patients who received all therapy under observation, 15 (50 %) patients had de novo disease and 15 (50 %) had received antecedent chemotherapy. No significant difference in survival was observed between groups (p = 0.44). Twenty-two patients received induction as up-front therapy, of whom 11 (50 %) achieved CR / CRi. The achievement of CR / CRi with one course of induction was associated with improved OS, with a 6-month OS of 73 % as compared to 23 % for those who did not (p = 0.018). The achievement of CR / CRi with a single course of induction may be a marker of favorable survival in this subtype of high-risk AML.Response to a single induction was associated with favorable survival in this population.

SP-34 - Transplant activities, experience

We described the clinical features and outcomes for 63 adult patients with acute myeloid leukemia (AML) with a translocation involving the 11q23 locus (MLL) who were treated at Memorial Sloan Kettering Cancer Center (MSK). The population included 40 female (63 %) and 23 male (37 %) patients, with a median age of 51 years old (range 18–82 years). Of the 31 patients who had had an antecedent malignancy, 14 (45 %) had had breast cancer or DCIS and 22 (71 %) had received anthracycline-based systemic chemotherapy. The translocation partner for the 11q23 rearrangement was identified in 60 of the 63 patients (95 %) studied. The distribution of translocation partners differed for those who had previously received cytotoxic chemotherapy. Most patients with therapy-related disease had a 9p22 or 19p13 partner, as compared to those with de novo disease (95 % vs. 68 %, p = 0.023). Of the 30 patients who received all therapy under observation, 15 (50 %) patients had de novo disease and 15 (50 %) had received antecedent chemotherapy. No significant difference in survival was observed between groups (p = 0.44). Twenty-two patients received induction as up-front therapy, of whom 11 (50 %) achieved CR / CRi. The achievement of CR / CRi with one course of induction was associated with improved OS, with a 6-month OS of 73 % as compared to 23 % for those who did not (p = 0.018). The achievement of CR / CRi with a single course of induction may be a marker of favorable survival in this subtype of high-risk AML.Response to a single induction was associated with favorable survival in this population.

Cytogenetic profile of a representative cohort of young adults with de novo acute myéloblastic leukaemia in Morocco

BackgroundWe analyzed the cytogenetic characteristics of a representative population of young adults with de novo acute myéloblastic leukemia (AML) treated in a single center institution. MethodsPatients with de novo AML included were aged between 20 and 60 years. Cytogenetic analysis was done at diagnosis. Twenty cells were analyzed, although examination of lower numbers of metaphases was also acceptable if an abnormal clone was detected. FindingsFrom 1/1/04 to 31/12/2014, among 1315 adult patients, 1055 (80%) patients were aged between 20 and 60 years. Karyotype was done in 927 (88%) patients and cytogenetic analysis failed in 32 cases (3·4%). Clonal abnormalities were observed in 520 (58%) patients. 175 (19·5%) were classified in the favorable group, 609 were in the intermediate group and 111 (12·5%) were in the adverse group. The most frequent chromosomal abnormality observed was t(8;21) in 112 (12·5%). Thirty three (3·7%) patients had t(15;17) and 30 (3·3%) had inv16, trisomy 8 was found in 47 (5·2%), 11q23 rearrangements in 32 (3·6%), 67 (7·4%) had a complex karyotype, −5/del(5q) and −7/del(7q) were seen in, respectively, 11(1%) and 27 (3%). Monosomy occurred in 115 (13%) patients, 70 (8%) responded to the definition of monosomal karyotypes. InterpretationThis is the largest study done in Morocco, Africa and Middle East. Epidemiological studies involving different ethnic populations and geographic regions of the world should help unfold the true nature of environmental and genetic interplay in the development of AML. Our cytogenetic profile reveals some particularities when compared to other populations; it does not seem to be similar neither to eastern or western countries.

CSPG4-Specific CAR T Cells for High-Risk Childhood B Cell Precursor Leukemia.

The advent of CD19-specific chimeric antigen receptor (CAR) T cells has proven to be a powerful asset in the arsenal of cancer immunotherapy of acute lymphoblastic leukemia and certain B cell lymphomas. However, a sizable portion of patients treated with CD19-CAR T cells relapse with CD19-negative cancer cells, necessitating the quest for back-up antigens. Chondroitin sulfate proteoglycan 4 (CSPG4) expression has been reported on leukemic blasts bearing the ill-fated MLL 11q23 rearrangement. We aimed at exploring the use of CSPG4-specific CAR T cells against mixed-lineage leukemia (MLL)-rearranged leukemic blasts, using the precursor B cell leukemia cell line KOPN8 (MLL–MLLT1 translocation) as a model. First, we confirmed CSPG4 expression on KOPN8 cells. Bulk T cells electroporated with mRNA encoding a CSPG4-specific CAR upregulated activation markers and secreted the Th1 cytokines TNF and IFNγ in an antigen-specific manner upon co-culture with KOPN8 cells. More importantly, CSPG4-specific CAR T cells evinced specific degranulation towards KOPN8 cells and specifically lysed KOPN8 target cells in chromium lysis experiments. CSPG4 is a well-established CAR target in cutaneous melanoma. Here, we provide proof-of-principle data for the use of CSPG4-specific CAR T cells against MLL-translocated leukemias.

JUN and ATF3 Regulate the Transcriptional Output of the Unfolded Protein Response to Support Acute Myeloid Leukemia

The genomic alterations that drive acute myeloid leukemia (AML) are well established, however, the molecular pathways that are deregulated as a consequence of these mutations and their impact on AML development and progression remain poorly understood. We and others have previously shown that the transcription factor (TF) JUN, though not mutated, is highly expressed in multiple AML sub-types (e.g. normal karyotype (NK-AML), complex karyotype and 11q23 rearrangements) relative to healthy hematopoietic stem and progenitor cells (HSPCs). We also recently reported that JUN supports cell survival in a panel of genetically diverse AML cell lines and patient-derived AML samples ex vivo. Moreover, we showed that JUN inhibition significantly delays disease onset and improves survival in a genetically engineered mouse model (GEMM) of AML driven by MLL-AF9. Of critical importance, we have now observed that deletion of JUN does not negatively impact steady-state hematopoiesis or the hematopoietic reconstituting potential of HSPCs. While these results position JUN as an attractive therapeutic target in AML, previous efforts to develop JUN inhibitors have been unsuccessful. Therefore, our objective is to elucidate the mechanisms by which JUN supports AML in order to identify putative therapeutic targets in this pathway.JUN regulates many transcriptional programs by forming hetero-dimers with one of many other TFs and our goal was to identify which dimeric partner cooperates with JUN to regulate transcriptional programs that support AML. We previously reported that JUN supports AML, in part, by regulating the transcriptional output of the unfolded protein response (UPR), which is a signal transduction network that helps cells negotiate endoplasmic reticulum (ER) stress. To identify dimerization partners that cooperate with JUN to drive transcriptional output of the UPR, we utilized the UCSC genome browser, which contains the chromatin immunoprecipitation-sequencing (ChIP-seq) data for JUN as well as 15 known dimerization partners of JUN. Using this database, we found that JUN localizes to the promoters of 35 UPR genes. Notably, we found that the stress-response factor ATF3 co-localizes with JUN at 31 of these 35 promoters (88.6%), exceeding the co-localization of the other 14 factors. Consistent with these observations, we found that shRNA-mediated inhibition of either JUN or ATF3 leads to decreased mRNA expression of the UPR targets genes (XBP1s, ATF4, DDIT3, HSPA5, GRP94, ATF6, EIF2AK3and ERN1) in human AML cell lines (e.g. THP-1, OCI-AML3, U937, MOLM14) as well as leukemia cells derived from the MLL-AF9-driven GEMM of AML. We also observed that inhibition of either JUN or ATF3 blocked the induction of UPR signaling and rendered human and mouse AML cells more susceptible to ER stress-induced death further suggesting that JUN and ATF3 regulate the transcriptional output of the UPR.To determine if ATF3 expression, like JUN, is deregulated in human AML, we performed a retrospective analysis of AML patient gene expression profiles and found that elevated expression of ATF3 is significantly associated with worse outcomes in two distinct datasets from NK-AML patients (p=0.0248, p=0.0049). Furthermore, we found that ATF3 expression is higher in AMLs with FLT3-ITD or 11q23 rearrangements, compared to healthy HSPCs. Based on these data, we investigated the functional role of ATF3 in AML. First, we found that shRNA-mediated inhibition of ATF3 significantly reduced the colony forming capacity of mouse AML cells expressing MLL-AF9 but, importantly, not healthy HSPCs. Furthermore, we found that ATF3 inhibition impeded the growth and survival of AML cells from a second GEMM of AML driven by the deletion of Dnmt3a and Tet2 in combination with expression of FLT3-ITD. We also observed that, similar to JUN inhibition, shRNA-mediated inhibition of ATF3 significantly reduced disease burden in MLL-AF9-driven AML in vivo (p=0.0028). At the cellular level, we found that AML cells depleted of ATF3 displayed increased CD11b expression and annexin V staining suggesting that ATF3 may support the differentiation blockade in AML.Collectively, our data suggest that JUN and ATF3 cooperate to support a variety of AML subtypes by regulating transcriptional programs, such as the UPR and our current efforts are focused on identifying additional targetable JUN/ATF3-regulated transcriptional programs that support AML. DisclosuresNo relevant conflicts of interest to declare.

Favorable Outcomes of Acute Leukemia of Ambiguous Lineage Treated with Hypercvad: A Multi-Center Retrospective Study

Background: Acute leukemias of ambiguous lineage (ALAL), including acute undifferentiated leukemia and mixed phenotype acute leukemia (MPAL), are rare hematologic malignancies, accounting for only 2-5% of cases of acute leukemia. Outcomes are generally poor, and retrospective studies have suggested that patients have better outcomes when treated with regimens used for acute lymphoblastic leukemia (ALL) rather than acute myeloid leukemia (AML). Due to heterogeneous definitions and rarity of these leukemias, no single ALL regimen has been studied extensively. Hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyperCVAD) is one of the most widely used adult ALL regimens in the United States. We retrospectively examined the effectiveness of this regimen as initial therapy in patients with ALAL.Methods: We retrospectively reviewed records from adult patients treated initially with hyperCVAD-based chemotherapy for ALAL as defined by the World Health Organization at five academic institutions. Only patients whose diagnosis was verified at a participating institution were examined. Philadelphia (Ph) chromosome-positive ALAL patients treated concurrently with tyrosine kinase inhibitors were included. The primary objective of the study was to determine the overall response rate (ORR) and median overall survival (OS). Descriptive statistics were used for baseline characteristics and responses, and Kaplan-Meier estimates were used to calculate all time-to-event outcomes.Results: Twenty-two patients were identified, and pre-treatment characteristics are shown in the Table. Median age was 50 years (range, 22-69), with an even distribution of male and female patients (50% each). The majority (77%) were Caucasian. Two patients (9%) had acute undifferentiated leukemia, 11 (50%) T/myeloid MPAL, 8 (36%) B/myeloid MPAL, and 1 (5%) B/T MPAL. Seven (32%) had a complex karyotype (at least 3 abnormalities), 6 (27%) had a normal karyotype, 3 (14%) were Ph-positive and were treated concurrently with dasatinib, and 1 (5%) had an 11q23 rearrangement. FLT3-ITD was detected in 5 of 8 patients (63%) with an available mutation panel. Three (15%) had central nervous system (CNS) involvement at presentation, and one additional patient developed CNS disease during the course of therapy.There were no deaths within the first 30 days, and the median number of cycles given was 4 (range, 1-8). The CR rate was 73% (n=16) and an additional 14% (n=3) had CR with incomplete count recovery (CRi), for an ORR rate of 86%. One patient had a partial response, and 2 patients had no response. Of the 19 patients achieving CR or CRi, median number of cycles to CR was 1.5, and 12 (63%) proceeded to allogeneic hematopoietic stem cell transplantation (alloHSCT). Of the 7 responders who did not receive alloHSCT, 3 relapsed shortly after initial CR and 4 are alive and in remission 77, 39, 37 and 10 months from treatment initiation. All 3 patients with Ph-positive disease achieved CR. Only 1 proceeded with alloHSCT and all 3 patients are alive and in remission at 51, 39, and 10 months. With a median follow up of 37 months, 14 (64%) patients were alive and in remission, including 11 who had undergone alloHSCT. Four of the 6 patients who died had a complex karyotype. Median overall survival for the entire cohort was not reached (Figure).Conclusions: HyperCVAD results in high remission rates in patients with ALAL, with low incidence of early mortality and high ability to proceed to allogeneic transplantation following response. HyperCVAD can be considered an effective front-line therapy for patients with ALAL. Additionally, hyperCVAD may be a backbone for incorporation of novel immunotherapies and targeted therapies, which may improve results in patients with appropriate targets. [Display omitted] DisclosuresSweet:Astellas: Consultancy; Agios: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Jazz: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Agios: Consultancy; Celgene: Honoraria, Speakers Bureau; BMS: Honoraria; Phizer: Consultancy; Jazz: Speakers Bureau; Astellas: Consultancy; Phizer: Consultancy; BMS: Honoraria. Wang:Novartis: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Consultancy. Al-Kali:Novartis: Research Funding.

Complex 3q26/EVI1 Rearrangements Genocopy Inv(3)/t(3;3) Acute Myeloid Leukemias By Enhancer Hijacking, EVI1 Overexpression, Absent MDS1-EVI1 and Low GATA2 Expression

IntroductionAcute myeloid leukemia (AML) with inv(3)(q21q26) or t(3;3)(q21;q26) overexpress EVI1 and have a very poor prognosis. EVI1 is part of the MECOM (MDS1-EVI1-Combination) locus from which MDS1-EVI1 and EVI1 can be transcribed from two different promoters. Although EVI1 is expressed at high levels, MDS1-EVI1 is absent or expressed at very low levels in inv(3)/t(3;3)-AMLs. Aberrant EVI1 expression in these leukemias is driven by the long-distant GATA2 enhancer, translocated from 3q21 to EVI1 at 3q26 (Gröschel et al, 2014). As a result of this GATA2 enhancer hijack by EVI1, GATA2 is switched off on the rearranged allele, resulting in mono-allelic and low GATA2 expression. We hypothesize that leukemic transformation of inv(3)/t(3;3)-AMLs is driven by EVI1 overexpression and by low GATA2 and that these leukemias are marked by the absence of MDS1-EVI1 expression. We previously reported about a group of AML patients that presented with complex rearrangements of 3q26 (refer to as variant-3q26-AML) with frequent MECOM involvement and very poor survival (Lugthart et al, 2010). Here we address the questions if these variant-3q26-AMLs 1) overexpress EVI1 by enhancer hijacking, 2) are marked by absent MDS1-EVI1 and 3) express low levels of GATA2. Accordingly, the variant-3q26-AMLs should be classified as inv(3)/t(3;3)-AMLs.ResultsWe identified 37 variant-3q26-AMLs with MECOM rearrangement as determined by Fluorescent in-situ hybridization (FISH). RNA-seq of these AMLs revealed EVI1 overexpression but also demonstrated the absence of MDS1-EVI1 in 90% of patient samples. Applying 3q-capture DNA-seq, we found that in 2 cases the patient cells harboured a “hidden” inv(3)(q21q26) with involvement of the GATA2 enhancer. In 7 cases recurrent 3q26/EVI1 translocations were identified, e.g. t(2;3)(p21;q26), t(3;8)(q26;q24), t(3;7)(q26;q11), involving the THADA, MYC or CDK6 loci respectively as previously described. Interestingly, we identified new translocations to the EVI1 locus in 13 AMLs, including a t(3;6)(q26;q21) and a t(3;4)(q26;p15), involving the CD164, and PROM1 loci respectively. In these samples we find clearly skewed expression of these genes to one allele, suggestively caused by the rearrangement and enhancer hijacking. CD164 plays a key role in adhesion, proliferation and migration of CD34+ hematopoietic progenitor cells (Watt et al, 2000). PROM1 (CD133) is expressed in human hematopoietic stem and progenitor cells and is thought to be involved in maintaining stem cell properties by suppressing differentiation (Bauer et al, 2008). We argue that EVI1 overexpression in these variant-3q26-AMLs is driven by hijacking enhancers of genes that are normally active in myeloid progenitors.In most of the patients the translocation breakpoints are in between the promoters of MDS1 and EVI1, explaining absence of MDS1-EVI1 expression. In addition, analysis of SNP-array data of these patients (N=33) showed Copy Number Loss (CNL) of the MDS1 exon(s) and not the EVI1 exons in at least 5 cases. Together these data suggest the importance of MDS1-EVI1 loss in 3q26-AMLs.Furthermore we wondered whether low GATA2 expression is an important event in variant-3q26-AMLs. Similar to inv(3)/t(3;3)-AMLs (Gröschel et al, 2014), RNA-seq revealed that the GATA2 expression was on average a two-fold lower in the variant-3q26-AMLs (N=37), compared to non-3q26 rearranged AMLs (N=114). Surprisingly, SNP-array analysis in 26 variant-3q26-AMLs revealed CNLs of GATA2 and/or its enhancer in 7 patients. Detailed SNP analysis in GATA2 exons by combined 3q-capture DNA-seq and RNA-seq uncovered another 7 cases with mono-allelic GATA2 expression or skewing to expression of one allele (allele_freq<0.4, P<0.05). Hence, in 53% of these patient samples one of the GATA2 alleles appeared to be affected. These results are unexpected, as the GATA2 locus was not involved in any of these rearrangements.ConclusionGiven their complex karyotype, variant-3q26-AMLs are often not recognised as 3q26/EVI1 AMLs. Although the exact mechanism remains elusive, the overall effect seems to be alike. EVI1 overexpression, potentially driven by enhancer hijacking of genes that are active in early myeloid progenitors, combined with absent MDS1-EVI1 and mono-allelic/low GATA2 expression results in AML with very poor survival. Given these data we believe variant-3q26-AMLs genocopy inv(3)/t(3;3)-AMLs and should be classified as such. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Chronic Lymphocytic Leukemia Patients with IGH Rearrangements Are Characterized By a Distinct Genetic Landscape with Prognostic Implications

Background: Chromosome 14q32 rearrangements involving the immunoglobulin heavy chain gene (IGH) affect less than 5% of chronic lymphocytic leukemia (CLL) patients. Their clinical course is aggressive and the outcome, worse than other CLL subtypes (Cavazzini et al, 2008; Gerrie et al, 2012). However, the biology of CLL showing IGH rearrangements (CLL-IGHR) is not completely defined. The identification of novel recurrent mutations in CLL by next generation-sequencing (NGS) has offered a more comprehensive view into the genomic landscape of the disease and improved the prognostication of CLL. Thus, mutational analysis might be especially useful in those patients with uncertain prognosis, such as those carrying IGH rearrangements.Aim: To analyze the mutational profile of CLL-IGHR patients by targeted NGS in order to improve our understanding of the genetic underpinnings of this subgroup.Methods: The study was based on 899 CLL patients, well characterized at cytogenetic, biological and clinical level, forty-two of them (4.7%) showing IGH rearrangements. Targeted NGS was performed in 231 CLL samples: 117 with 13q deletion, 27 with 11q deletion, 26 trisomy 12, 42 showing IGH rearrangements and the remaining 19 without any cytogenetic alteration. CD19+ B cells were isolated and DNA extracted. SureSelectQXT targeted enrichment technology and a custom-designed panel (MiSeq, Illumina), including 54 CLL-related and recurrent mutated genes, was carried out. The panel yielded 100x or greater coverage on 97% of the genomic regions of interest and the mean coverage obtained was 600x. Mutations were detected down to 3% allele frequency.Results: The mutational analysis of CLL-IGHR patients identified a total of 72 mutations in 32 genes. Seventy-one percent of patients (30/42) harbored at least one mutation. The most frequently mutated genes in this cohort were NOTCH1 (28.6%), POT1 (14.3%), TP53 (9.5%), SF3B1 (7%), BRAF (7%), EGR2 (7%), IGLL5 (7%) and MGA (7%), followed by BCL2, HIST1H1E and FBXW7 (4.8%), uncommonly mutated genes in CLL at these frequencies (Table 1). In fact, mutations in NOTCH1, BRAF, EGR2, BCL2, HIST1H1E and FBXW7 were significantly associated with CLL-IGHR patients (p=0.013, p=0.003, p=0.021, p=0.038, p=0.038 and p=0.021 respectively).In terms of time to the first therapy (TFT), CLL-IGHR had an intermediate-negative impact (median TFT=24 months) compared to the presence of cytogenetic alterations associated with good prognosis such as 13q deletions (median TFT>120 months; p<0.0001) (Figure 1A). Furthermore, the presence of mutations in the most frequently mutated genes (NOTCH1, POT1, TP53, SF3B1 or BRAF) within patients with IGH rearrangements had a negative clinical impact in the TFT and allowed us to refine the prognosis of this subgroup. Thus, the median TFT of patients with mutations was 1 month while the median TFT of patients without mutations was 14 months (p=0.014) (Figure 1B).A total of 17 out of 42 CLL-IGHR patients (40.5%) carried the t(14;18). Interestingly, patients with t(14;18) were characterized by: 1) A lower mutation frequency (average of mutations/patient=1.05) than the rest of rearrangements with unknown partners (average=2.16; p=0.039), and 2) The presence of mutations in BCL2 (11%) and HIST1H1E (11%). By contrast, CLL-IGHR without BCL2 rearrangement showed mutations in POT1 (20%), TP53 (16%), SF3B1 (12%) and BRAF (12%). Moreover, t(14;18) was significantly associated with good prognosis markers such as the mutated status of the variable region of the immunoglobulin genes (IGHV-M) (p=0.002). However, there was no significant difference in terms of TFT between patients with t(14;18) and patients with other IGH rearrangements (p=0.27).Conclusions: CLL patients with IGH rearrangements showed: i. A high gene mutation frequency; ii. A distinct mutational profile, with recurrent mutations in POT1, EGR2, BRAF, IGLL5 and MGA genes; iii. An adverse clinical outcome refined by the negative effect of genetic mutations. iv. Patients with t(14;18) presented a lower mutation frequency than the rest of rearrangements, carrying mutations in BCL2 and HIST1H1E, and associated with good-prognosis markers such as IGHV-M.Funding:PI15/01471; CIBERONC CB16/12/00233; FEHH-Janssen(MHS); JCyL(MQÁ) [Display omitted] DisclosuresMateos:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees.

EVI1 overexpression reprograms hematopoiesis via upregulation of Spi1 transcription

Inv(3q26) and t(3:3)(q21;q26) are specific to poor-prognosis myeloid malignancies, and result in marked overexpression of EVI1, a zinc-finger transcription factor and myeloid-specific oncoprotein. Despite extensive study, the mechanism by which EVI1 contributes to myeloid malignancy remains unclear. Here we describe a new mouse model that mimics the transcriptional effects of 3q26 rearrangement. We show that EVI1 overexpression causes global distortion of hematopoiesis, with suppression of erythropoiesis and lymphopoiesis, and marked premalignant expansion of myelopoiesis that eventually results in leukemic transformation. We show that myeloid skewing is dependent on DNA binding by EVI1, which upregulates Spi1, encoding master myeloid regulator PU.1. We show that EVI1 binds to the −14 kb upstream regulatory element (−14kbURE) at Spi1; knockdown of Spi1 dampens the myeloid skewing. Furthermore, deletion of the −14kbURE at Spi1 abrogates the effects of EVI1 on hematopoietic stem cells. These findings support a novel mechanism of leukemogenesis through EVI1 overexpression.

Identifying Prognostic Factors That Influence Outcome of Childhood Acute Myeloid Leukemia in First Relapse in Saudi Arabia: Results of the Multicenter SAPHOS Study

BackgroundThe outcome of childhood acute myeloid leukemia (AML) in first relapse (rAML) remains poor. Reported overall survival (OS) rates vary between high-income developed countries and those with fewer resources. The OS of rAML in high-income developing countries (HIDCs) has not been reported. Patients and MaterialsA multicenter study was performed in an HIDC. The outcome of patients with relapsed non-M3/non-Down syndrome AML was evaluated. Three-year OS was computed using the Kaplan-Meier method, and predictors of OS were analyzed using a Cox proportional hazards model. ResultsA total of 88 patients with non-M3/non-Down syndrome AML diagnosed between January 2005 and December 2012 with a first relapse were identified. Their 3-year OS was 22.6% ± 5.4%. Patients with inv(16) and t(8;21) had an OS of 75.0% ± 21.7% and 36.0% ± 16.1%, respectively. Worse outcomes were associated with “other intermediate” and 11q23 rearrangement AML (OS of 9.4% ± 8.7% and 10.7% ± 9.6%, respectively). Patients experiencing time to relapse (TTR) less than 1 year had shorter OS than those with a longer TTR (14.6% ± 5.4% vs. 41.1% ± 11.5%; P = .006). The outcome of patients after stem cell transplantation (SCT) in second complete remission (CR2) was superior compared with no SCT (50.9% ± 11.2% vs. 7.7% ± 4.6%; P = .001). TTR, risk group, CR2, and SCT in CR2 were the most significant predictors for survival. ConclusionsrAML remains a clinical challenge. Genetic variability in outcomes was observed. A majority of patients with inv(16) were successfully salvaged post-relapse, whereas patients with 11q23 rearrangement had a poor prognosis. Only one-third of those with t(8;21) rAML survived. Better access to SCT in HIDCs is needed.

A new adult AML case with an extremely complex karyotype, remission and relapse combined with high hyperdiploidy of a normal chromosome set in secondary AML

BackgroundChromosomal abnormalities are diagnostic and prognostic key factors in acute myeloid leukemia (AML) patients, as they play a central role for risk stratification algorithms. High hyperdiploidy (HH), a rare cytogenetic abnormality seen commonly in elder male AML patients, is normally categorized under AML with complex karyotype (CK). Accordingly, patients with HH generally are associated with low remission rates and a short overall survival.Case presentationHere we report a case of 21-year-old female, diagnosed with a de novo AML-M1 according to WHO classification and a CK at diagnosis. Cytogenetic, molecular cytogenetic approaches (standard fluorescence in situ hybridization (FISH), array-proven multicolor banding (aMCB)) and high resolution array comparative genomic hybridization (aCGH) analyses revealed a unique complex but still near diploid karyotype involving eleven chromosomes was identified. It included pentasomy 4, three yet unreported chromosomal aberrations t(1;2)(p35;p22), t(1;3)(p36.2;p26.2), and t(10;12)(p15.2;q24.11), and a combination of two cytogenetic events, yet unreported to appear in together, i.e. a reciprocal translocation t(1;3)(p36.2;p26.2) leading to EVI1/PRDM16 gene fusion, and monoallelic loss of tumor suppressor gene TP53. After successful chemotherapeutic treatment the patient experienced a relapse to AML-M1, and she developed secondary AML-M6 with tetraploidy and HH. Unfortunately, the young woman died 8.5 months after initial diagnosis.ConclusionsTo the best of our knowledge, a comparable adult AML associated with such a CK, coexistence of 3q rearrangements with loss of TP53 at diagnosis, and HH in secondary AML were not previously reported. Thus, the combination of the here seen chromosomal aberrations in adult primary AML seems to indicate for an adverse prognosis.

Recurrent 8q24 rearrangement in blastic plasmacytoid dendritic cell neoplasm: association with immunoblastoid cytomorphology, MYC expression, and drug response.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare skin-tropic hematological malignancy of uncertain pathogenesis and poor prognosis. We examined 118 BPDCN cases for cytomorphology, MYC locus rearrangement, and MYC expression. Sixty-two (53%) and 41 (35%) cases showed the classic and immunoblastoid cytomorphology, respectively. Forty-one (38%) MYC+BPDCN (positive for rearrangement and expression) and 59 (54%) MYC-BPDCN (both negative) cases were identified. Immunoblastoid cytomorphology was significantly associated with MYC+BPDCN. All examined MYC+BPDCNs were negative for MYB/MYBL1 rearrangement (0/36). Clinically, MYC+BPDCN showed older onset, poorer outcome, and localized skin tumors more commonly than MYC-BPDCN. MYC was demonstrated by expression profiling as one of the clearest discriminators between CAL-1 (MYC+BPDCN) and PMDC05 (MYC-BPDCN) cell lines, and its shRNA knockdown suppressed CAL-1 viability. Inhibitors for bromodomain and extra-terminal protein (BETis), and aurora kinases (AKis) inhibited CAL-1 growth more effectively than PMDC05. We further showed that a BCL2 inhibitor was effective in both CAL-1 and PMDC05, indicating that this inhibitor can be used to treat MYC-BPDCN, to which BETis and AKis are probably less effective. Our data will provide a rationale for the development of new treatment strategies for patients with BPDCN, in accordance with precision medicine.

Recurrent Cytogenetic Abnormalities in Intravascular Large B-Cell Lymphoma.

Data characterizing the cytogenetic landscape of intravascular large B-cell lymphoma (ILBCL) are limited. Here, we developed a comprehensive karyotypic data set to identify recurrent cytogenetic abnormalities in ILBCL. Cases of ILBCL with complete cytogenetic analysis were identified from an institutional database and the literature. The combined data were systematically reviewed for the presence of recurrent abnormalities. Four new cases were identified and combined with 25 karyotypes previously published in the literature. Karyotypes were uniformly complex with a median of 10 aberrations. In total, 72.4% had abnormalities involving chromosome 1, with 31.0% involving rearrangements of 1p13 or 1q21; 58.6% had abnormalities involving chromosome 6, which in almost all cases involved 6q; 34.5% had abnormalities involving chromosome 14, with 27.6% involving rearrangements of 14q32; and 55.2% had abnormalities of chromosome 18, with 37.9% harboring trisomy 18. Recurrent cytogenetic abnormalities involving chromosomes 1, 6q, and 18 are present in greater than 50% of ILBCL.

Impact of Fluorescent In Situ Hybridization Aberrations and CLLU1 Expression on the Prognosis of Chronic Lymphocytic Leukemia: Presentation of 156 Patients from Turkey.

Objective:This study evaluates the impact of CLLU1 expression and fluorescent in situ hybridization (FISH) analysis of a group of Turkish chronic lymphocytic leukemia (CLL) patients.Materials and Methods:A total of 156 CLL patients were analyzed by FISH method; 47 of them were also evaluated for CLLU1 expression. Results were correlated with clinical parameters.Results:FISH aberrations were found in 62% of patients. These aberrations were del13q14 (67%), trisomy 12 (27%), del11q22 (19%), del17p (8%), and 14q32 rearrangements (20%). Overall del11q22 and del17p were associated with the highest mortality rates, shortest overall survival (OS), and highest need for medication. Homozygous del13q14, 14q32 rearrangements, and higher CLLU1 expression correlated with shorter OS.Conclusion:Cytogenetics/FISH analysis is still indicated for routine evaluation of CLL. Special consideration is needed for the poor prognostic implications of del11q22, del17p, 14q32 rearrangements, and homozygous del13q14. The impact of CLLU1 expression is not yet clear and it requires more data before becoming routine in genetic testing in CLL patients.

H2AFY is a novel fusion partner of MECOM in acute myeloid leukemia

The MECOM gene encoding a zinc finger protein that functions as a transcription factor, was located on chromosome 3q26, and rearrangements of MECOM often cause its overexpression in acute myeloid leukemia (AML). We identified H2AFY as a novel fusion gene partner of MECOM in an elderly male AML patient with cryptic 3q26 rearrangement using the whole transcriptome sequencing, who carried out abnormal karyotype of 46,XY,t(3;5)(q27;q31),add(14)(p11). We validated the existence of the unreported H2AFY-MECOM fusion gene by RT-PCR and Sanger DNA sequencing, and detected mutations of NRAS and BCOR in this patient. In addition, we found abnormally elevated expression of MECOM in this patient by quantitative-polymerase chain reaction (RQ-PCR). Further research is needed to investigate functional characterizations of this novel fusion in the development of AML.