11q23 Rearrangements Research Articles

Outcome of 3q26.2/MECOM rearrangements in chronic myeloid leukemia.

To evaluate the outcomes of patients with 3q26.2/MECOM-rearranged chronic myeloid leukemia (CML). We reviewed consecutive adult patients with 3q26.2/MECOM-rearranged CML between January 1, 1998 and February 16, 2023. Rearrangements of 3q26.2/MECOM were confirmed by conventional cytogenetics, and fluorescence in situ hybridization starting in 2015. We identified 55 patients with MECOM-rearranged CML, including 23 in chronic phase (CP) or accelerated phase (AP) and 32 in blast phase (BP). Nine patients (16%) achieved a major cytogenetic response (MCyR) or deeper. At a median follow-up of 89months, median survival was 14months. The 5-year survival rate was 19% overall, 23% in CML-CP/AP, and 15% in CML-BP. In the 6-month landmark analysis, the 5-year survival rate was 41% for allogeneic stem cell transplantation (allo-SCT) recipients versus 17% for non-recipients (P = 0.050). Multivariate analysis showed that the percentage of marrow blasts and achievement of MCyR or deeper could predict survival. Outcomes of 3q26.2/MECOM-rearranged CML are poor despite the availability of multiple BCR::ABL1 tyrosine kinase inhibitors (TKIs). Third-generation TKIs in combination with novel agents and possible allo-SCT could be considered given the poor outcomes and resistance to second-generation TKIs.

Aberrant ecotropic viral integration site-1 (EVI-1) and myocyte enhancer factor 2 C gene (MEF2C) in adult acute myeloid leukemia are associated with adverse t (9:22) & 11q23 rearrangements

Acute myeloid leukemia (AML) shows multiple chromosomal translocations & point mutations which can be used to refine risk-adapted therapy in AML patients. Ecotropic viral integration site-1 (EVI-1) & myocyte enhancer factor 2 C gene (MEF2C) are key regulatory transcription factors in hematopoiesis and leukemogenesis & both drive immune escape.This prospective study involved 80 adult de novo AML patients recruited from Oncology Center, Mansoura University, between March 2019 and July 2021. The MEF2C and EVI1 expression were measured using a Taqman probe-based qPCR assay.The results revealed that EVI1 and MEF2C expression were significantly elevated in AML patients as compared to control subjects (p = 0.001. 0.007 respectively). Aberrant expressions of EVI1 and MEF2C showed a significant negative correlation with hemoglobin levels (p = 0.034, 0.025 respectively), & bone marrow blasts (p = 0.007, 0.002 respectively). 11q23 translocation was significantly associated with EVI1 and MEF2C (p = 0.004 and 0.02 respectively). Also, t (9;22) was significantly associated with EVI1 and MEF2C (p = 0.01 and 0.03 respectively), higher expression of EVI1 and MEF2C were significantly associated with inferior outcome after induction therapy (p = 0.001 and 0.018 respectively) and shorter overall survival (p = 0.001, 0.014 respectively).In conclusion, EVI1 & MEF2C were significantly expressed in AML cases. EVI1 & MEF2C overexpression were significantly associated with 11q23 rearrangements and t (9;22) and were indicators for poor outcome in adult AML patients; These results could be a step towards personalized therapy in those patients.

Allo-HSCT Improved Outcome of High Risk ALL in Pediatric-Inspired PDT-ALL-2016 Cohort

Background Pediatric-inspired regimen have remarkably improved the prognosis of adult ALL, particularly for adolescent and young adult (AYA) patients. The role of allo-HSCT was controversy in the era of pediatric-inspired regimen. Some study revealed that patients treated with pediatric-inspired regimen those who achieved MRD negative may not require allo-HSCT (Ref1. Ribera et.al, Blood 2021; Ref2. Dhedin et.al, Blood, 2015). To address this issue, we performed study on HR ALL population in pediatric-inspired PDT-ALL-2016 cohort, by comparing outcome of allo-HSCT and chemotherapy groups. Methods PDT-ALL-2016 cohort is a GRAALL-backbone pediatric-inspired regimen (NCT03564470), patients diagnosed with ALL in Nanfang hospital were enrolled in JAN-2016 to DEC-2021. High-risk features included: WBC higher than 30×10^9/L for B-ALL or 100×10^9/L for T-ALL; pro-B or ETP; presence of t(9;22), t(1;19), t(4;11) or 11q23 rearrangements, Ph-like, complex karyotype, hypodiploid or near triploid; IKZF1 deletion/muation. Patients diagnosed with HR-ALL and achieved CR1 after induction were enrolled in this study. The left-truncated Kaplan-Meier estimator was used to compare the overall survival (OS), event-free survival (EFS), cumulative incidence of relapse (CIR) and non-relapse mortality (NRM). Results A total of 265 HR-ALL patients were enrolled in this study, 168 patients in allo-HSCT and 97 patients in chemotherapy cohorts, respectively. The baseline characteristics between two cohorts were comparable. With a median follow-up time of 43.6 months (3.8-60.0 months), the 3-yr OS and EFS were better in allo-HSCT cohort compared with chemo-only cohort (3-yr OS, 76.8% VS 55.6%, P＜0.001; 3-yr EFS, 66.5% VS 38.8%, P＜0.001). Meanwhile, the allo-HSCT cohort had lower CIR and higher but acceptable NRM compared with chemo-only cohort (3-yr CIR, 15.5% VS 55.5%, P＜0.001; 3-yr NRM, 13.8% VS 3.2%, P＜0.001; Table 1). For MRD positive subgroup (allo-HSCT, n=64; chemo-only, n=37), patients who underwent allo-HSCT had longer EFS and OS, and lower CIR compared with chemo-only cohort (3-yr OS, 71.0% VS 35.3%, P＜0.001; 3-yr EFS, 59.3% VS 25.6%, P＜0.001; 3-yr CIR, 19.8% VS 69.8%, P＜0.001). Of note, for MRD negative subgroup (allo-HSCT, n=96; chemo-only, n=51), the allo-HSCT cohort had longer EFS and lower CIR compared with chemo-only cohort, the OS also improved by allo-HSCT (3-yr OS, 81.2% VS 71.0%, P=0.173; 3-yr EFS, 71.1% VS 50.0%, P=0.014; 3-yr CIR, 14.6% VS 48.8%, P＜0.001; Fig 1). In multivariable analyses, allo-HSCT was protective factors for OS (HR=0.37), EFS (HR=0.56) and CIR (HR=0.45), and negatively infected NRM (HR=4.66). Conclusion Here, we showed that allo-HSCT further improved the outcome of HR ALL regardless of the MRD status, in the era of pediatric-inspired regimen. The survival advantage of allo-HSCT came from lower CIR compared with chemotherapy only. Furthermore, compared with published data, the NRM was more acceptable in our cohort. It is the high NRM (20-40%) in other studies ( Ref1. Ribera et.al, Blood 2021; Ref2. Dhedin et.al, Blood, 2015; Ref3. Wieduwilt et.al, leukemia, 2021; Ref4. Seftel et.al, AJH, 2015) that counterbalanced the benefit of CIR by allo-HSCT, which highlight the importance of experienced management for total therapy in adult ALL. This research was supported by the National Natural Science Foundation of China(NFSC82170163, 81970147), Clinical Study of Nanfang Hospital(LC2016ZD009/2019CR012).

Impact of High-Risk Cytogenetics (HR-CTG) on the Outcome of Newly Diagnosed Adult Patients with Philadelphia Negative B-Cell Acute Lymphoblastic Leukemia (B-ALL) Treated with Frontline Blinatumomab (Blina) and/or Inotuzumab Ozogamicin (Ino) Containing Hypercvad (HCVAD) Therapy

Introduction: HR-CTG in B-ALL is considered an adverse prognostic factor along with age and measurable residual disease (MRD) status. The use of potent B-cell targeted drugs like InO and Blina as part of frontline therapy has improved outcomes in adult pts with B-ALL ( Jabbour et al, Lancet Haematology 2022 and 2023) but the impact of baseline HR-CTG needs to be further elucidated . Methods: From Oct 2014-Feb 2023 we identified 71 consecutive patients (pts) with Philadelphia (Ph)-negative B-ALL and adequate CTG data who were treated on frontline therapy that included InO and/or Blina along with HCVAD chemotherapy. The intensive phase of treatment protocol consisted of HCVAD 4 courses followed by 4 courses of blina which could be initiated post HCVAD course 2 for high-risk or MRD positive pts and has been published before (Jabbour et al, Lancet Haematology 2022). The protocol was later amended to include fractionated InO from course 2 of HCVAD at a cumulative dose of 2.4 mg/m 2(Short et al, ASCO 2023). Pts with confirmed Ph-like ALL were excluded. CTG was evaluated and graded as HR based on a combination of conventional karyotyping and fluorescent in situ hybridization data and included low-hypoploidy or near triploidy (Ho-Tr), complex karyotype (CK; ≥5 abnormalities), 14q32 rearrangement ( IGH) and KMT2A re-arrangement as described before (Issa et al, Cancer 2017). Results: 31 of 71 pts (44%) with Ph- B-ALL had HR-CTG; 14 pts (45%) with Ho-Tr, 8 (26%) pts with KMT2A rearrangement, 6 pts (19%) with CK and 3 pts (10%) with IGH rearrangement (Table 1). The median age of the whole cohort was 33 years (range, 18-60 years); median age was 29 yrs in HR-CTG group compared to 34 yrs in non-HR-CTG group. 12 pts (39%) in the HR group compared to 5 pts (12%) in the non-HR group had a TP53 mutation ( p=0.01). 5/12 (42%) pts in the HR group with TP53 mutation had low-hypoploidy. 7 pts (23%) and 13 (32%) pts in the HR-CTG and non-HR-CTG group respectively had RAS mutations, with 1 pt in each group having both TP53 and RAS mutation. 17 pts (55%) received Blina and 14 pts (45%) received both InO and Blina in the HR group compared to 20 pts (50%) each who received Blina and Blina+ InO in the non-HR group. A best response of complete remission (CR)/CR with incomplete count recovery (CRi) was obtained in 100% pts (CR=30, CRi=1) in the HR-CTG group, 17 (55%) of whom had concurrent MRD negativity ; in the non-HR-CTG group 39 pts (98%) attained CR and one pt a partial remission, with concurrent MRD negativity attained in 21/39 (54%) pts with CR. At a median follow-up of 31 months (mos) for the full cohort (43 mos for HR pts and 28 mos for the non-HR group) the median overall survival (OS) was not reached (NR) and 3-yr OS was 85% (95% CI, 78-92%); median OS and 3-yr OS were NR and 89% in the HR-CTG group and 74.7 mos and 82% in the non-HR-CTG group ( p=0.64). The median relapse free survival (RFS) was NR in the HR-CTG group and 74 mos in the non-HR group ( p=0.73) while the 3-yr RFS were 74% and 80% respectively. There was no intergroup difference in RFS and OS based on the MRD status at response assessment. 11 HR-CTG pts (35%) underwent allogeneic stem cell transplantation (SCT) (5 Ho-Tr, 4 KMT2Ar, 1 CK, 1 IGHr) in continued remission at a median of 4.8 mos from therapy initiation (IQR 4.5-8.2 mos), 9 of whom went directly to SCT without introduction of a new therapy between the frontline therapy and SCT. Two pts received additional lines of therapy for MRD relapse/progression without morphological relapse before SCT. The 3-year OS in the HR-CTG pts who underwent SCT was 87% and similar to HR pts who did not undergo SCT (89%, p=0.78). 5 pts (12%) underwent SCT in the non-HR-CTG group, 4 for persistent or recurrent MRD positivity and one with Down's syndrome. No pts in either groups developed hepatic sinusoidal obstruction syndrome or a secondary myeloid neoplasm. Conclusion: In our analysis, in a prospectively maintained dataset where >90% pts were treated on clinical trial, HR-CTG did not have any significant impact on B-ALL survival outcomes when treated with frontline therapy containing B-cell targeted agents. Further analysis is underway.

Identification of ZFP91 As the Master Transcription Factor for EVI1 Gene Expression

Ecotropic viral integration site 1 (EVI1) is essential for hematopoiesis, particularly for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is widely known to be an independent poor prognostic factor in acute myeloid leukemia (AML). Previous studies have shown that enhancer hijacking or gene fusion caused by chromosomal translocation or inversion involving 3q26 leads to EVI1 overexpression. Intriguingly, however, aberrant EVI1 expression is seen in many cases of AML without 3q26 rearrangement. The prognosis of these cases is similarly dismal, but the mechanism that causes aberrant EVI1 expression in the absence of 3q26 chromosomal abnormalities is largely unknown. To identify key regulatory factors essential for EVI1 expression in AML, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout screen. We knocked in the green fluorescent protein (GFP) coding sequence at the EVI1 locus in the human myeloid leukemia cell line K562, which expresses high levels of EVI1 without a 3q26 chromosomal aberration. After transduction of a genome-wide CRISPR/Cas9 knockout library, transduced cells were separated into GFP High and GFP Low subpopulations by flow cytometry. By sequencing the uniquely attached barcodes, we estimated the effect of gene knockout for each gene. Genes counted more frequently in the GFP Low fraction were listed as candidates for activators of EVI1. The top 13 genes, including three apparent positive controls such as EVI1 and two ribosome-related genes, had a false discovery rate (FDR) of less than 1%. We validated these candidate genes in K562 and two other EVI1-overexpressing AML cell lines by transducing individual single guide RNAs targeting these genes. These analyses identified zinc finger protein 91 (ZFP91) as a potential regulatory factor that significantly upregulates EVI1 expression common to the three independent myeloid leukemia cell lines. ZFP91 is an atypical E3 ubiquitin ligase, a DNA-binding protein that functions as a transcription factor. ZFP91 promotes cell growth and inhibits apoptosis in AML and has been shown in previous studies to activate multiple oncogenic pathways, including nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and hypoxia-inducible factor-1α (HIF-1α). We demonstrated that knockout of ZFP91 in K562 substantially reduced EVI1 protein expression to undetectable levels by Western blotting (Figure 1). EVI1 mRNA expression also decreased dramatically after ZFP91 knockout. These results suggest that ZFP91 may play an indispensable role in EVI1 transcription. We performed chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) targeting the EVI1 promoter in K562 and demonstrated that ZFP91 knockout clones showed approximately 10-fold weaker signals for active histone modifications (H3K4me3 and H3K27ac) at the EVI1 promoter (Figure 2). In addition, chromatin immunoprecipitation sequencing (ChIP-seq) for ZFP91 in K562 revealed the presence of a distinct peak as well as the ZFP91-specific motif sequence within the EVI1 promoter region. These data strongly suggest that ZFP91 functions as an essential transcription factor for EVI1 gene overexpression in some EVI1-associated myeloid leukemias. We confirmed that ZFP91 knockout in K562 inhibited cell proliferation, which is consistent with previous studies in other AML cell lines. Based on these results, we believe that ZFP91 has the potential to serve as a therapeutic target in EVI1-associated AML. To conclude, we have identified the transcription factor ZFP91 as a novel prominent activator of EVI1 expression in some EVI1-associated AML. Our study suggests that ZFP91 may be the key to unraveling the pathogenesis of EVI1-associated AML and an attractive therapeutic target in EVI1-associated AML.

A Genomic Approach for High-Grade B Cell Lymphoma with MYC and BCL2 and / or BCL6 Rearrangements: Two Clusters Stood out on Next Generation Sequencing Results

Introduction High-grade B cell lymphomas with double or triple hit rearrangement (HGBL DH/TH), which appeared on the 4th revised edition WHO classification of lymphoid neoplasms, are aggressive mature B-cell lymphomas with high-grade cytology (Burkitt-like, blastoid or DLBCL-like) that have a MYC (8q24) rearrangement in combination with a BCL2 (18q21) and / or a BCL6 (3q27) rearrangement. There are currently no treatment guidelines for this subtype, and the usual immunochemotherapy leads to few complete remissions. While the NGS technology has been used in diffuse large B cell lymphoma, there has been no specific study on additional genetic events which would predict the clinical outcome in HGBL DH/TH. Here, we report our preliminary results using a targeted DNA capture panel approach (NGS) to identify subgroups with different clinical outcomes among HGBL DH/TH. Methods We conducted a retrospective study on 44 patients diagnosed with DH or TH HGBL and treated at Centre Hospitalier Lyon Sud or Centre Léon Bérard starting in 2013. We collected the medical information regarding the treatment, time to response to treatment or relapse, progression free survival, and overall survival. They all benefited from a DNA-targeted NGS analysis covering 54 genes. Results The cohort included 20 females and 24 males, with a median age of 65 (31-87). Among them, 22 patients were MYC/ BCL2 DH (50%), 12 were MYC/ BCL6 DH (27,3%) and 10 were TH (22,7%). Eleven patients had B symptoms (25%). The Ann Arbor stage was high (III-IV) in 97.5%. The WHO Performance Status was low (0-1) for most of the patients (70%). The majority of them presented with an IPI above to 2 (82,5%) and an elevated LDH level (80%). The Ki67 index was above 70% (33/42) in 78,5% of the cases and above 90% (19/42) in nearly half of them. The major finding of the NGS analysis was the identification of 2 groups based on the TP53/ CDKN2A_p14ARF mutational status. The CDKN2A locus encodes for 2 alternative open reading frames leading to 2 unrelated proteins p14ARF and p16INK4A. P14ARF is involved in the regulation of the TP53 pathway whereas p16INK4A is not. We therefor defined 2 groups : the first group included patients with either TP53 or CDKN2A_p14ARF-only alterations (group A, n=10) while the second group had no alteration in these two coding sequences (group B, n=34) although 12 patients had a CDKN2A_p16INK4A loss or mutation affecting both CDKN2A transcripts. Most of TP53 mutations were missense mutations mostly in TP53 DNA-binding domain and 2 cases appeared to be single hit mutations with an allele frequency below 50%. The patient with a p14ARF only mutation had a start codon mutation p.(M1*) with an allele frequency of 96% without CDKN2A copy number loss. The median overall survival (OS) (Figure 1) was 6,2 months for group A, and 55,5 months for group B (p = 0,0076). The median follow-up time was 14 months (7,5 months in the group A, 18 in group B). Only 2 patients in group A survived and are still in complete remission, one was DH BCL6 and the other DH BCL2 Conclusion Our preliminary results indicate a strong negative prognostic impact of either CDKN2A_p14ARF or TP53 gene alterations on the OS of HGBL DH/TH patients. The biological basis for the difference between groups would be in favour of the alteration of the p14ARF-TP53 tumor suppressive pathway as major contributor of chemoresistance. A more detailed analysis is on-going to further identify mutation profiles of double hit and triple hit lymphomas and compare our finding with existing data. Our results confirm the more favourable outcome of DH BCL6 patients (data not shown), which is consistent with the 5 th revised OMS classification that removed that specific entity. Our results also indicate that routine NGS analysis of HGBL DH/TH patients offers new perspectives for patient stratification. Regarding their survival, patients with TP53 pathway alterations should probably not be treated with the standard RCHOP chemo-immunotherapy. Instead, a more intensive immunochemotherapy regimen or novel strategies such as first-line bispecific antibodies or CARTCells might be considered.

Genetic characteristics and survival analysis of 27 cases of juvenile myelomonocytic leukemia

Objective: To investigate the genetic and genomic profiling of juvenile myelomonocytic leukemia (JMML) and factors affecting its survival rate. Methods: Clinical characteristics, cytogenetics, molecular biology results and survival status of children with 27 JMML cases admitted to the Hematology Department of Children's Hospital, Capital Institute of Pediatrics from December 2012 to December 2021 were analyzed retrospectively, and the outcomes of the children were followed up. Kaplan-Meier method was used for survival analysis. Univariate analysis was used for analyzing factors affecting the overall survival (OS) rates of patients who received hematopoietic stem cell transplantation (HSCT). Log-Rank test was used for comparison of survival curves. Results: Among 27 JMML cases, there were 11 males and 16 females. The age of disease onset was 28 (11,52) months. There are 20 cases of normal karyotype, 4 cases of monosomy 7, 1 case of trisomy 8,1 case of 11q23 rearrangement and 1 case of complex karyotype. A total of 39 somatic mutations were detected.Those involved in RAS signal pathway were the highest (64%(25/39)), among which PTPN11 mutation was the most frequent (44% (11/25)). A total of 17 cases (63%) received HSCT, 8 cases (30%) did not receive HSCT, and 2 cases (7%) lost follow-up. For children receiving transplantation, the follow-up time after transplantation was 47 (11,57) months. The 1-year OS rate of high-risk transplantation group (17 cases) and high-risk non transplantation group (6 cases) was (88±8)% and (50±20)% respectively, with a statistically significant difference (χ2=5.01, P=0.025). The 5-year OS rate of the high-risk transplantation group was (75±11)%. The survival time of those who relapsed or progressed to acute myeloid leukemia after transplantation was significantly shorter than that of those who did not relapse (χ2=6.80, P=0.009). The OS rate of patients with or without PTPN11 mutation was (81±12) % and (67±19)% respectively (χ2=0.85, P=0.356). Conclusions: The main pathogenesis involved in JMML is gene mutation related to RAS signaling pathway, and the most common driver gene of mutation is PTPN11. Allogeneic HSCT can significantly improve the survival rate of high-risk JMML patients. The recurrence or progression after transplantation was related to poor prognosis.

Oncogene EVI1 Drives Acute Myeloid Leukemia Via a Targetable EVI1-CtBP1/2 Interaction and By Repressing Myeloid Regulator CEBPA

Overexpression of EVI1 (located at 3q26), as a result of 3q26 rearrangements, causes the development of one of the most aggressive, chemotherapy-resistant forms of AML. We hypothesize that insight into the function of EVI1 in AML will lead to the identification of targetable vulnerabilities for those patients. EVI1 encodes a DNA-binding zinc-finger protein that has a critical role in transcriptional regulation and maintenance of quiescent hematopoietic stem cells. Here, we aim to understand the mechanism by which EVI1 overexpression in bone marrow progenitors drives AML development. To identify its target genes, we made EVI1 rapidly degradable by endogenous fusion with an auxin-inducible degron (AID) tag in inv(3q21;3q26) rearranged MUTZ3 leukemia cells. Degradation of EVI1 strongly increased myeloid differentiation of MUTZ3 cells. Using metabolic labeling of newly produced RNA (SLAM-seq), we found 6 genes to be upregulated after 1.5 hrs of degradation. Out of those genes, CEBPA was also upregulated upon shRNA knockdown of EVI1 or after introducing inactivating mutations in the hijacked GATA2 enhancer that drives EVI1 expression. Those findings suggest persistent upregulation of CEBPA when EVI1 levels are reduced. In line with this, CEBPA expression is 1.83 fold (p=0.00024) lower in AML patients with 3q26 rearrangements (n=18) compared to non-3q26 rearranged cases (n=97). Interestingly, overexpression of CEBPA in MUTZ3 cells bypassed EVI1 driven repression and caused myeloid differentiation as well. We next addressed the question how EVI1 represses CEBPA transcription. The CEBPA locus contains 14 enhancers in a 100kb region around the gene body, among which the myeloid specific +42kb enhancer, essential for neutrophil development. EVI1 binds this +42kb enhancer of CEBPA as determined by chromatin immunoprecipitation sequencing (ChIP-seq) in 3q26-rearranged AML and MUTZ3 cells. After degradation of EVI1, H3K27Ac levels increased at this enhancer, indicating that EVI1 reduces activity of the +42kb CEBPA enhancer. Since EVI1 lacks any enzymatic domains that could modulate chromatin, we sought to further understand how EVI1 represses activity of the +42kb CEBPA enhancer. EVI1 protein immunoprecipitation followed by mass spectrometry revealed interaction with CtBP1/2, factors known to interact with repressors such as Polycomb proteins or HDACs. ChIP-seq showed a strong overlap between CtBP2 and EVI1 binding sites in MUTZ3 cells. Moreover, EVI1 elimination caused loss of CtBP2 binding to the chromatin including to the +42kb CEBPA enhancer. EVI1 harbors a PLDLS domain essential for binding to CtBP1/2. Mutation of this domain abolished CtBP2 binding and reduced transformation of mouse bone marrow progenitors by EVI1, as determined by in vitro replating assays. We reasoned we could exploit the high affinity of EVI1 for CtBP through this single domain by overexpressing a repeat of a small section of EVI1 containing only the CtBP-binding PLDLS-site. Overexpression of "4X-PLDLS competitor peptide" was able to sequester CtBP and thereby prevent it's binding to EVI1. The competitor peptide was able to abolish in vitro replating capacity and re-enabled differentiation of mouse bone marrow progenitors transformed with EVI1. In line with this, transduction of the competitor peptide into MUTZ3 cells led to upregulation of CEBPA as well as growth inhibition. Furthermore, in vivo leukemia outgrowth in a xenotransplant model of 3q26 rearranged AML cell lines MUTZ3 and SB1690CB was fully abolished by the competitor peptide, but not by a mutant control peptide. In conclusion, EVI1 is a repressor of active chromatin regions, including CEBPA via the +42kb myeloid specific enhancer. Repression by EVI1 is dependent on its interaction with CtBP1/2, proteins that recruit repressors of transcription via an essential . A PLDLS domain in EVI1 is essential for this interaction. The competitor peptide experiments point to exploiting this interaction with repressive molecules as a potential therapeutic target in AML with overexpression of EVI1.

A Genomic Approach in High-Grade B Cell Lymphoma with MYC and BCL2 and / or BCL6 Rearrangements: Two Clusters Stood out on Next Generation Sequencing Results

Introduction Double or triple hit high-Grade B cell Lymphoma (HGBL), which appeared on the 4th revised edition WHO classification of lymphoid neoplasms in 2016, is a group of agressive mature B-cell lymphomas which morphogically ressemble diffuse large B-cell lymphoma (DLBCL) or with features intermediate between DLBCL and Burkitt lymphoma, that have a MYC (8q24) rearrangement in combination with a BCL2 (18q21) and / or a BCL6 (3q27) rearrangement. There are currently no treatment guidelines for DH/TH HGBL, and the usual chemotherapy leads to few complete remissions. While the whole exome sequencing technology has been used in diffuse large B cell lymphoma, there has been few studies on additional DH/TH HGBL genetic events which would predict the clinical outcome. Here, we report our preliminary results on DH/TH HGBL patients using a targeted DNA capture panel approach (NGS) to identify subgroups with different clinical outcomes among DH/TH HGBL. Methods We conducted a retrospective study on 66 patients diagnosed with DH or TH HGBL and treated at Centre Hospitalier Lyon Sud or Centre Léon Bérard starting in 2013. We collected the medical information regarding the treatment, time to response to treatment or relapse, progression free survival, and overall survival. In addition, we performed a preliminary NGS analysis on the most recent samples using SophiaGenetics LYS panel (54 genes) in Centre Léon Bérard. Results The cohort included 30 females and 36 males, with a median age of 65 (IQR: 56.25-72.75). Among them, 32 patients were MYC/BCL2 DH (48.49%), 14 were MYC/BCL6 DH (21.21%) and 20 were TH (30.30%). Nine patients had B symptoms (9/62 14.52%). The Ann Arbor stage was high (III-IV) (59/66, 89.39%). The WHO Performance Status was low (0-1-2) for most of the patients (52/59, 88.14%). The majority of them presented with an IPI above to 2 (39/51, 76.47%) and an elevated LDH level (48/62, 77.42). The Ki67 positivity was above 70% (52/62, 83.87%) in most of the patients and above 90% (27/62, 43.55%) in nearly half of them. The median follow-up time was 11 months (IQR: 6-37.75) for the cohort. The major finding of the NGS analysis was the identification of 2 clusters based on the TP53/CDKN2A mutational status. The first group included patients with mutually exclusive TP53 or CDKN2A alterations (cluster A, n=7) while the second group had no alteration in these two genes (cluster B, n=7). TP53 alteration consisted exclusively in gene mutations while we found loss of heterozygoty or point mutation for CDKN2A. The median progression free survival (PFS) (Figure 1) and overall survival (OS) (Figure 2) were 5.39 months (IQR: 6.51-NA) and 6.18 month (IQR: 5.88-NA) for group A respectively, and were not achieved for cluster B. The median follow-up was 5.88 months (IQR: 4.24-7.69) for cluster A and 11.56 months (IQR: 9.3-31.6) for cluster B, the difference being due to the higher mortality rate in cluster A. Conclusion Our preliminary results indicate a strong negative prognostic impact of either CDKN2A or TP53 gene alterations on the PFS and OS in HGBL DH/TH patients. Although the difference was not statistically significant between clusters with only 14 patients having NGS results, we plan to include other samples to further consolidate the data. More importantly, the biological basis for the difference between clusters would be in favour of the alteration of the ARF-TP53 tumor suppressive pathway as major contributor to survival since one of our sample had only a point mutation in ARF start codon. Taken together, our results suggest that routine NGS analysis of HGBL DH/TH patients may offer new perspectives on patient stratification and possibly new classification and therapeutic opportunities such as MDM2 or BCL2 inhibitors, in combination with conventional chemotherapy and need to be confirmed in a larger population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PB1801: CHROMOSOMAL ABNORMALITIES DURING THE CLINICAL COURSE OF ADULT ACUTE MYELOID LEUKEMIA

Background: Using conventional cytogenetics, an abnormal karyotype can be detected in 60-70% of adult acute myeloid leukemia (AML) patients. Taking into consideration the identified cytogenetic abnormalities AML patients are currently classified into three risk groups: favorable, intermediate and adverse. These genetic abnormalities are the most important factors in determining response to chemotherapy as well as outcome in AML. Aims: Aim of this study was to detect the diagnostic and prognostic significance of chromosomal abnormalities in leukemic cells at different phases of AML. Methods: Cytogenetic investigations of bone marrow and/or peripheral blood cells from 140 adult patients were performed during clinical evolution of AML: in newly diagnosed patients, in remission and at relapse. The methods of conventional cytogenetics (GTG) and fluorescence in situ hybridization (FISH) were used. Cytogenetic methods were performed using standard techniques and karyotypes were described according to the International System for Human Cytogenetic Nomenclature. Results: Cytogenetic investigations of 140 adult AML patients were performed in newly diagnosed patients, in remission and at relapse. Structural (del(5q), del(7q), rearrangements of 3q, 12p, 17p and 11q23, t(8;21)(q22;q22), t(9;22)(q34;q11), t(15;17)(q22;q21), t(16;16)(p13;q22), inv(16)(p13q22), marker and ring chromosomes, acentric structures and others) and numerical (-5, -7, -Y, +8, monosomal karyotype and others) chromosomal abnormalities in leukemic cells were found. Some genetic abnormalities (BCR/ABL1, PML/RARa, AML1/ETO, CBFβ/MYH11 fusion genes) were detected by molecular genetic methods (FISH). Spectrum of cytogenetic abnormalities had an important diagnostic and prognostic significance. Diagnosis of AML was possible due to the presence of specific genetic markers that can confirm some types of AML (AML with t(8;21)(q22;q22), t(9;22)(q34;q11), t(15;17)(q22;q21), t(16;16)(p13;q22)/inv(16)(p13q22), 3q21q26 rearrangements, BCR/ABL1, PML/RARa, AML1/ETO, CBFβ/MYH11 fusion genes). Taking into consideration the identified cytogenetic abnormalities AML patients were classified by risk groups: the group of patients with adverse cytogenetic markers (3q21q26 rearrangements, -5, -7, t(9;22)(q34;q11), BCR/ABL1 fusion gene, multiple changes (≥3), monosomal karyotype), the intermediate risk group without significant prognostic markers and the group with favorable prognostic factors (t(8;21)(q22;q22), t(15;17)(q22;q21), t(16;16)(p13;q22)/inv(16)(p13q22), AML1/ETO, PML/RARa, CBFβ/MYH11 fusion genes). In remission cytogenetic investigations showed a normal karyotype without cytogenetically visible changes in all patients. Cytogenetic investigations at relapse were performed in 5 of 140 AML cases. Additional/secondary chromosomal aberrations in leukemic cells or new cells clones with multiple structural and numerical changes at relapse was not detected. Summary/Conclusion: Cytogenetic investigations had an important significance for diagnosis, prognosis and selection the optimal treatment strategy of AML. Besides the analysis of chromosome banding patterns it is necessary for patients with AML to apply molecular genetic studies, namely FISH and polymerase chain reaction (PCR). Taking into consideration the identified cytogenetic abnormalities AML patients were classified by risk groups. Presence of the additional/secondary chromosomal aberrations in leukemic cells or new cells clones with multiple structural and numerical changes at AML relapse is the one sign of clonal evolution and progression of disease.

Acute lymphoblastic leukemia (ALL) with KMT2A gene rearrangement in infants - characteristic clinical picture based on a case report

The most common malignancy affecting children is leukemia, which in infants refers to be diagnosed before 1 year of age and is relatively rare, but remains a problem for clinicians due to its aggressive clinical presentation, poor response to current treatments, and molecular biology. Infants with acute leukemia tend to present with aggressive features, including hyperleukocytosis, hepatosplenomegaly, central nervous system (CNS) involvement, and cutaneous infiltration. In infant leukemia, the KMT2A gene rearrangement at chromosome 11q23 is quite common. Infants diagnosed with acute leukemia harboring a 11q23 rearrangement have a particularly poor prognosis when compared to other children with acute leukemia.
 A 10-month-old girl was admitted in December 2017 to the Department of Pediatric Hematology, Oncology and Transplantology in Lublin with the suspicion of leukemia. One week before hospitalization minor bruises began to appear on the skin, unrelated to an injury. The child was admitted to the hospital, and there a complete blood count showed anemia, hiperleukocytosis and thorombocytopenia. On the admission to the Clinic: the severe general condition of the patient, with features of cardiopulmonary insufficiency were confirmed. Due to the patient's age and the presence of the KMT2A/MLLT3-t(9;11) (p22;q23) gene rearrangement, the infant was stratified as a high-risk group (HRG) and chemotherapy was started in accordance with the therapeutic program.
 Infant leukemia is one of the most difficult clinical situations encountered in pediatric oncology. Given the infant’s vulnerability and unique genetic rearrangements, there is a need to develop new protocols and therapies for infants.

The retinoic acid receptor co-factor NRIP1 is uniquely upregulated and represents a therapeutic target in acute myeloid leukemia with chromosome 3q rearrangements

Aberrant expression of Ecotropic Viral Integration Site 1 (EVI1) is a hallmark of acute myeloid leukemia (AML) with inv(3) or t(3;3), which is a disease subtype with especially poor outcome. In studying transcriptomes from AML patients with chromosome 3q rearrangements, we identified a significant upregulation of the Nuclear Receptor Interacting Protein 1 (NRIP1) as well as its adjacent non-coding RNA LOC101927745. Utilizing transcriptomic and epigenomic data from over 900 primary samples from patients as well as genetic and transcriptional engineering approaches, we have identified several mechanisms that can lead to upregulation of NRIP1 in AML. We hypothesize that the LOC101927745 transcription start site harbors a context-dependent enhancer that is bound by EVI1, causing upregulation of NRIP1 in AML with chromosome 3 abnormalities. Furthermore, we showed that NRIP1 knockdown negatively affects the proliferation and survival of 3qrearranged AML cells and increases their sensitivity to all-trans retinoic acid, suggesting that NRIP1 is relevant for the pathogenesis of inv(3)/t(3;3) AML and could serve as a novel therapeutic target in myeloid malignancies with 3q abnormalities.

The retinoic acid receptor co-factor NRIP1 is uniquely upregulated and represents a therapeutic target in acute myeloid leukemia with chromosome 3q rearrangements

Aberrant expression of Ecotropic Viral Integration Site 1 (EVI1) is a hallmark of acute myeloid leukemia (AML) with inv(3) or t(3;3), which is a disease subtype with especially poor outcome. In studying transcriptomes from AML patients with chromosome 3q rearrangements, we identified a significant upregulation of the Nuclear Receptor Interacting Protein 1 (NRIP1) as well as its adjacent non-coding RNA LOC101927745. Utilizing transcriptomic and epigenomic data from over 900 primary samples from patients as well as genetic and transcriptional engineering approaches, we have identified several mechanisms that can lead to upregulation of NRIP1 in AML. We hypothesize that the LOC101927745 transcription start site harbors a context-dependent enhancer that is bound by EVI1, causing upregulation of NRIP1 in AML with chromosome 3 abnormalities. Furthermore, we showed that NRIP1 knockdown negatively affects the proliferation and survival of 3q-rearranged AML cells and increases their sensitivity to all-trans retinoic acid, suggesting that NRIP1 is relevant for the pathogenesis of inv(3)/t(3;3) AML and could serve as a novel therapeutic target in myeloid malignancies with 3q abnormalities.

Myeloproliferative Neoplasms with Monocytosis.

Myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic stem cell neoplasms comprising of polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) that share driver mutations (JAK2/CALR/MPL) resulting in constitutive activation of JAK/STAT and other signaling pathways. Patients with MPN have shortened survival and an inherent risk for leukemic evolution. Prognostically relevant clinical and genetic parameters have been incorporated into mutation-enhanced scoring systems (MIPSS70-plus version 2.0, MIPSS-ET/PV). In the current review, we describe clinical and pathological features along with prognostic significance of MPN with monocytosis. Monocytosis, defined by an absolute monocyte count (AMC) ≥ 1 × 10 9/L, is a typical manifestation of chronic myelomonocytic leukemia (CMML) but is also associated with 21% and 17% of PV and PMF patients, respectively. Recent studies on the subject have reported that MPN patients with monocytosis are older and present with concomitant leukocytosis. In regard to PV, patients with monocytosis harbor unfavorable cytogenetic abnormalities including +8, 7/7q, i(17q), 5/5q-,12p-, inv(3), or 11q23 rearrangement and SRSF2 mutations, whereas PMF patients with monocytosis had significant thrombocytopenia, higher circulating blasts, higher symptom burden, and ASXL1 mutations. Moreover, presence of monocytosis predicted inferior survival in both PV and PMF. Monocytosis in MPN is associated with a distinct clinical and genetic profile and may serve as a marker of aggressive disease biology.

Impact of Gender on Molecular AML Subclasses - a Harmony Alliance Study

Impact of Gender on Molecular AML Subclasses - a Harmony Alliance Study

T-MDS Is a Distinct Clinical and Pathological Entity Characterized By Better Survival Compared to t-AML

Introduction: Therapy-related myeloid neoplasm (t-MN) includes acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) that occur as a complication of DNA-damaging therapies. The World Health Organization recommends considering t-MN as a single entity. Whether t-MDS and t-AML have distinct characteristics and outcomes is not known. The aim of this study was to compare clinicopathological characteristics and outcomes of t-MDS and t-AML.Methods: All patients diagnosed with t-MN based on the World Health Organization criteria were identified. Bone marrow biopsies, cytogenetic, and next-generation sequencing (NGS) were obtained at the treating physician's discretion. Pathogenic/likely pathogenic variants (PV) were called based on the standard criteria. Overall survival (OS) was calculated from the time of t-MN diagnosis until the last follow up using Kaplan-Meier analysis using Wilcoxon test. For survival analysis comparing chemotherapy to best supportive care (BSC) only, patients were censored at the time of allogeneic stem cell transplant (SCT). Multivariate analysis was performed using Cox proportional hazard method and corrected using false discovery rate (FDR). Statistical analysis was performed using JMP (v14.1, SAS Institute) and significance was defined as P<0.05.Results: We identified 554 patients, of which 180 (32.4%), 365 (65.8%), and 9 (1.6%) presented as t-AML, t-MDS, and t-MDS/MPN respectively. t-MDS/MPN patients were excluded from further analysis due to a small number. Clinical and laboratory characteristics of the t-AML and t-MDS cohorts is shown in Table 1. t-AML patients were significantly more anemic and thrombocytopenic at presentation. As expected, t-AML had a higher peripheral blood and BM blast count. There was no difference in proportion of patients with chromosomal abnormalities, though a statistically significantly higher proportion of t-MDS patients had chromosome 5 abnormality, 5q deletion, monosomy 5, chromosome 7 abnormality, monosomy 7 compared to t-AML patients. On other hand, 11q23 (mixed lineage leukemia, MLL) rearrangement was more common in t-AML compared to t-MDS (9.3% vs. 2.8%, P=0.005).A higher proportion of t-MDS patients had PV detected by NGS compared to t-AML (92.9% vs. 85.6%, P=0.038). A higher proportion of t-MDS patients had PV in TP53 (37.6% vs. 21.4%, P=0.004) and ASXL1 (23% vs. 11.7%, P=0.016) genes; whereas a higher proportion of t-AML patients had PV in RAS (18.9% vs. 9.1%, P=0.013) and WT1 (8.1% vs. 2.9%, P=0.05) genes.One hundred twenty-eight (35%) of 365 t-MDS patients progressed to t-AML. The presence of any chromosomal abnormality at t-MDS diagnosis predicted a higher risk of transformation to t-AML (χ 2 3.9, P=0.03).t-AML patients had a significantly shorter OS compared to t-MDS (9.2 vs. 19.7 months, P<0.0001, Figure A). This difference persisted when stratified by no disease modifying therapies (BSC only) 2 vs. 17 months (P<0.0001, Figure B), as well as among those who received at least one line of chemotherapy (14 vs. 24.6 months, P<0.001). Finally, a higher proportion of patients with t-AML underwent SCT and there was a trend towards improved survival for t-AML patients (vs. t-MDS 52.9 vs. 20.7 months, P=0.07) from the time of transplant.Multivariate analysis for OS performed to control for all the variables that were different at presentation (except for blast count), showed that t-MDS (as opposed to t-AML) phenotype at diagnosis, and undergoing SCT were independent predictors of improved survival (Table 2).Conclusion: t-MDS and t-AML have distinct clinical, cytogenetic, and genetic features at presentation. In the absence of disease modifying therapies, t-AML is a more aggressive phenotype, consistently associated with a shorter survival. Even after controlling adverse risk features, t-AML phenotype had a shorter survival compared to t-MDS. [Display omitted] DisclosuresLitzow: AbbVie: Research Funding; Astellas: Research Funding; Amgen: Research Funding; Actinium: Research Funding; Pluristem: Research Funding; Jazz: Other: Advisory Board; Omeros: Other: Advisory Board; Biosight: Other: Data monitoring committee. Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Results of the Full Pediatric, Minimal Residual Disease (MRD)-Oriented Trial LAL-RI/2008 for Adolescents and Young Adults (AYA) with Standard Risk (SR), Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia (ALL)

Background and objective. Full pediatric or pediatric-derived trials constitute the treatment of choice for AYA with SR-ALL. In the MRD era, patients (pts) with SR factors at baseline with poor MRD clearance should be considered as HR patients. The objectives of the full pediatric LAL-RI/2008 trial (NCT02036489) from the Spanish PETHEMA group were to assess the proportion of AYA with SR-ALL at baseline who have HR features based on poor early cytological response and/or poor MRD clearance, and to compare their prognosis with that from those pts who achieve good early cytological response and good MRD clearance.Patients and methods. SR ALL was defined in adolescents (age 15 to 18 years) and young adults (age 19 to 29 years) fulfilling all the following criteria: WBC count ≤30 × 109/L, and absence of t(9;22), t(1;19), t(4;11) or any other 11q23 rearrangements. Induction therapy included vincristine (VCR), prednisone (PDN), daunorubicin (DNR) and native E. coli asparaginase (ASP) for 4 weeks. Pts who achieved complete remission (CR) and MRD<0.1% proceeded to consolidation-1 (high-dose methotrexate [MTX], intermediate-dose ARA-C, etoposide and mercaptopurine [MP]), consolidation-2/reinduction (VCR, dexamethasone, DNR, cyclophosphamide and ASP), maintenance-1 (MP and MTX with monthly reinductions with VCR, PDN and ASP until the end of the 1st year) and maintenance-2 (MP and MTX without reinductions until the end of the 2nd year). CNS prophylaxis consisted of triple intrathecal therapy (MTX, ARA-C and hydrocortisone, 16 doses throughout the treatment). Pts with poor early cytological response (BM blast cells >10% on day 14 of induction), MRD level >0.1% (assessed by flow cytometry) at CR or MRD level >0.05% at any time after CR were considered as high risk (HR) pts and received the ALL-HR trial consisting of intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (alloHSCT) (Ribera JM et al. J Clin Oncol 2014; 32:1595-604).Results. Between 2008 and 2017, 79 eligible pts were treated according to the ALL-RI-08 protocol. Median age (range) was 20 years (15-29), 34 (43%) were adolescents and 45 (57%) YA. The mean (SD) WBC count was 10 (9) x109/L, and 73 patients (94%) had B-cell precursor ALL. Two patients are on treatment and 74 out of the remaining 77 (96%) achieved the CR. From the 77 pts, 15 had poor early cytological response, 3 had MRD >0.1% at the time of CR and 1 pt showed reappearance of MRD level >0.05% during maintenance. In all, 19 patients were considered as HR. Of the 58 patients who remained SR and followed the LAL-RI/2008 protocol, 14 relapsed (7 during treatment and 7 off therapy), whereas in the HR group, 3 were resistant, 4 relapsed and 2 died by toxicity (transplant-related in 1 out of 4 pts submitted to alloHSCT). Table 1 shows the DFS, OS and cumulative incidence of relapse (CIR) at 5-yrs for the whole series, the patients remaining SR and those who turned HR. Of note, OS was lower in the HR pts (Figure 1). On comparison with the results of the ALL-RI-96 trial (same inclusion criteria, same treatment but no follow-up by MRD, Ribera JM et al. J Clin Oncol 2008; 26:1843-9), no differences were observed in DFS, OS and CIR (Table 1).ConclusionsTwenty-five percent of AYA pts with SR ALL at baseline are in fact HR based on slow early cytological response to therapy and/or poor MRD clearance. High-risk pts showed lower OS despite being moved to a HR protocol.Supported in part by grants RD12/0036/0029 (RTICC, FEDER), PI14/01971 FIS, Instituto Carlos III, and SGR225 (GRE), Spain. [Display omitted] DisclosuresRibera:Celgene: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Roche: Honoraria; ARIAD: Research Funding, Speakers Bureau; Incyte: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Amgen Inc.: Research Funding, Speakers Bureau. Montesinos:Celgene Corporation: Honoraria, Research Funding.

Integrative Genomic Analysis of Adult Mixed Phenotype Acute Leukemia (MPAL)

IntroductionMixed phenotype acute leukemia (MPAL) is a rare subgroup of acute leukemia characterized by blasts presenting both myeloid and lymphoid lineages. Standard of care for MPAL is not defined and prognosis of MPAL is poor. Genetic basis for mixed immunophynotype and distinct molecular characteristics from other leukemia is not well understood.MethodsWe studied 31 bone marrow samples from previously untreated MPAL patients (median age 53) meeting WHO criteria. Genomic DNA/RNA from bone marrow samples were analyzed by comprehensive genomic analysis including targeted capture sequencing of 295 genes (median 393x coverage), RNA sequencing, and Infinium methylation EPIC array (Illumina). Copy number alterations (CNA) were analyzed from methylation array data using ChAMP algorithm. Mutational landscape of MPAL was compared with194 AML, 71 B-ALL, and 14 T-ALL cases sequenced with the same platform. CpG methylation pattern of MPAL was also compared with 194 AML (TCGA), 505 B-ALL and 101 T-ALL cases (data shared by Nordlund et al. Genome Biology. 2013).ResultsOf the 31 MPAL cases, 18 (58%) had myeloid-T and 13 (42%) had myeloid-B phenotype. Four (13%) cases had Philadelphia chromosome, one (3%) had 11q23 rearrangement, and 8 (26%) had complex karyotypes. Eighteen patients (62%) were treated with ALL regimen, 8 (28%) received AML regimen, and 3 (10%) received other therapy.Targeted sequencing revealed 86 driver mutations (55 SNVs and 31 indels) in 35 genes in 29 samples (94%). MPAL carried median 2 (range 0-6) mutations per case that was similar to AML (P = 0.79) and T-ALL (P = 0.92) but was significantly higher than B-ALL(P < 0.001). The most frequently mutated genes were DNMT3A , IDH2 , NOTCH1 , and RUNX1 in 7 (23%) cases each (Figure 1). Myeloid-T and myeloid-B showed distinct patterns of mutations, in which mutations in NOTCH pathway genes had strong trend of enrichment in myeloid-T (P = 0.06), whereas RUNX1 mutations were significantly enriched in myeloid-B (P = 0.02). When mutation landscape was compared among AML, B-ALL, T-ALL and MPAL, TP53 , NRAS , and DNMT3A mutations were shared across all leukemia. In contrast, NPM1 mutation was specific to AML and mutations in NOTCH1 , FBXW7 , and IL7R were specific to myeloid-T MPAL and T-ALL. CNA analysis showed that CDKN2B as a significantly deleted gene in MPAL.Unsupervised hierarchical clustering of promoter methylation revelaed two distinct clusters. This methylation cluster had significant correlation with immunophenotype (73% of cluster 1 was myeloid-B and 88% of cluster 2 was myeloid-T, P < 0.001). Overall, myeloid-T had more hypermethylated CpG loci than myeloid-B (mean beta value 0.28 vs. 0.52 in myeloid-B vs. myeloid-T, P = 0.002). This difference in methylation was independent from IDH mutation. Molecular pathway analysis of differentially methylated probes (DMP) showed that T-cell receptor pathway, T cell differentiation, and activation pathway genes (CD3D , CD247 , LCK , PRF1 , RHOH , and PRKCQ ) were differentially methylated and were expressed between myeloid-B and myeloid-T. Furthermore, motif enrichment analysis of promoter DMP revealed that IRF8 and IRF4 binding motifs were significantly hyper-methylated in myeloid-T compared to myeloid-B. Consistent with this, RAG1 , RAG2 , CXCR4 and PAX5 , downstream targets of IRF4/IRF8 andessential genes in B-cell development, were differentially expressed between two phenotypes. RNA sequencing revealed two known translocations (NSD1-NUP98 , KMT2A-MLLT4 ) in addition to the novel translocations such as FOXP1-DNAJC15 , TNKS - LYZ , and NOP14 - PLEC in MPAL. We did not detect ZN384 fusions that have been reported in pediatric MPAL (Alexander et al. ASH 2016). Unsupervised hierarchical clustering of methylation pattern among MPAL, AML, B-ALL and T-ALL revealed that 41% and 59% of MPAL had AML-like and ALL-like methylation pattern, respectively (Figure 2). Patients who received therapy that matched with their methylation pattern had significantly better response to treatment than patients who received unmatched therapy (CR rate: 77% vs. 31%, P = 0.024).ConclusionMyeloid-T and myeloid-B MPAL showed distinct patterns of mutation, methylation and gene expressions. Methylation data could identify AML-like and ALL-like MPAL which corresponded to better response to therapy when matched therapy was given. These data suggest that molecularly guided therapy may be beneficial in the treatment of MPAL. [Display omitted] DisclosuresTakahashi:Symbio Pharmaceuticals: Consultancy. DiNardo:Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy. Andreeff:Daiichi Sankyo: Consultancy. Kantarjian:Bristol-Meyers Squibb: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

Durable Remission after Salvage Chemotherapy with Combining Bortezomib and Vorinostat Followed By CD19 CAR-T Therapy in a Patient with MLL-Rearranged ALL after Early Relapse Post Allogeneic Hematopoietic Stem Cell Transplantation

Durable Remission after Salvage Chemotherapy with Combining Bortezomib and Vorinostat Followed By CD19 CAR-T Therapy in a Patient with MLL-Rearranged ALL after Early Relapse Post Allogeneic Hematopoietic Stem Cell Transplantation

High-Level EVI1 Perturbs Hematopoietic Function By Skewing Cells Towards Myeloid Phenotype Via Induction of PU.1/ Spi1 Â Transcription

Inv(3q26) and t(3:3)(q21;q26) are specific to poor prognosis acute myeloid leukemia (AML) and myelodysplastic syndrome, and result in marked overexpression of EVI1 isoforms. Despite extensive study, the mechanism by which this induces myeloid malignancy is unclear. We describe a mouse model that mimics the transcriptional effects of 3q26 rearrangement, and study of this has revealed a novel mechanism of leukemogenesis, specifically myeloid skewing with concomitant suppression of erythropoiesis and lymphopoiesis. In an effort to characterize the effects of high-level EVI1 expression on hematopoietic cells in vivo, we created a tetracycline (Tet)-inducible allele of Evi1 (termed Evi1TO) in the mouse by inserting seven repeats of the Tet operon within the first exon of Evi1. The advantage of this design is that it allows for the induction of all spliced isoforms of the locus, of which at least three exist, those encoding 135, 123, and 103 kDa. To assess whether Evi1 can be induced in vivo, we tested induction in mice harboring Evi1TO/TO / Rosa26rtTA/rtTA alleles: treatment of these mice with DOX results in dramatic up-regulation of the major Evi1 transcript in hematopoietic cells to levels commonly seen in leukemic cells. This accurately models the pattern of Mecom gene expression most commonly seen in human AML with rearrangements at 3q26, in which setting the EVI1 isoforms are upregulated, but typically not the longer, MDS1-EVI1/PRDM3 isoform; and this particular expression pattern is associated with the poorest prognosis. To determine the direct effect of EVI1 overexpression on hematopoietic stem/progenitor cells (HSPCs) and to minimize secondary and non-cell autonomous effects, we enumerated HSPCs in Evi1TO/TO / Rosa26rtTA/rtTA mice following three days of induction administered by DOX chow. This revealed a doubling of LSK cells, due primarily to increases in HSCs and MPP3 cells, the latter representing myeloid-poised progenitors. In long-term competitive transplantation experiments, EVI1 overexpression resulted in marked suppression of both erythropoiesis and lymphopoiesis, due to both increases in apoptosis and decreases in proliferation. In the same setting, there was a marked expansion of myeloid cells, such that EVI1-overexpressing cells constituted the majority of myeloid cells in the marrow, despite the presence of normal competitor bone marrow. Our data indicate that when Evi1 is induced in vivo in an admixture with normal hematopoietic cells, there is expansion of the Evi1 -expressing cells indicating they have a proliferative and/or survival advantage, with specific predilection for myeloid differentiation; we found that EVI1 overexpression led to significant increases in both bone marrow and circulating granulocytes and their progenitors, and marked decreases in erythrocytes and T and B lymphocytes; monocytes and platelets were largely unaffected. To understand the mechanism, we employed EML cells, which are immortalized progenitor cells. EVI1 overexpression caused marked upregulation of Sca-1, a marker of myeloid skewing, with concomitant inhibition of erythropoiesis; these effects are dependent on EVI1 binding to DNA. This myeloid skewing correlated with a two-fold increase in PU.1 expression. ChIP-seq experiments revealed several binding sites for EVI1 upstream of PU.1, and knock-out of one of these sites in vivo led to blunting of PU.1 induction by EVI1, and abrogated EVI1-mediated upregulation of Sca-1; in vitro knockdown studies showed similar results. These data are consistent with a mechanism whereby EVI1 overexpression upregulates PU.1, and together these factors suppress erythropoiesis and promote myelopoiesis. However, by itself EVI1 overexpression does not result in leukemia. To see if leukemia would result if additional mutations were induced, we treated competitively transplanted mice with the mutagen, ethylnitrosourea (ENU). This induced myeloid leukemias in 100% of the mice, and all were derived from EVI1-overexpressing cells. These data suggest that EVI1 overexpression requires additional cooperating genetic events for induction of leukemia. DisclosuresNo relevant conflicts of interest to declare.