Genomic Landscape of Acute Myeloid Leukemia (AML) on the Basis of 2017 ELN Classification and Other Mutations in Adult AML - Single Healthcare System Data

Introduction Recently, genomic mutation profiling of leukemic cells has been actively studied and some results have been integrated into the 2017 ELN classification with cytogenetic analysis for risk assessment of AML populations.1 However, only a few mutations are well identified and included in the 2017 ELN classification. In addition, except interaction between NPM1 and FLT-ITD, correlation and co-occurrence among various mutations have not been well studied. Here we describe our single center genomic landscapes of 2017 ELN guideline components with other NGS mutations in adult AML. Methodology We performed hematopoietic tumor profiling assay by next generation sequencing (NGS) testing and cytogenetic chromosome analyses in 193 AML patients diagnosed from 2018-08-01 to 2020-03-10. Treatment-related AML patients were excluded. All NGS and cytogenetic analyses were performed before starting chemotherapy. On the basis on 2017 ELN cytogenetic and mutations components, we analyzed significantly other co-occurred and exclusively occurred NGS mutations by using Fischer's exact test. Based on 2017 ELN classification, either one of t(6;9), t(v;11q23.3), t(9;22), inv(3) or t(3;3),-5, del(5q), -7, -17, or complex/monsoonal karyotype meeting the ELN criteria positive was grouped as Adverse Karyotype. 43 AML related genes, including 6 ELN components of NPM1, FLT-ITD low, CEBPA biallelic, TP53, RUNX1, ASXL1 and other 37 mutations including DNMT3A, NRAS and KRAS were analyzed as a single component. In addition, we grouped some mutations into larger sets or pathways and analyzed them in the same way. DNMT3A, TET2, IDH1, IDH2, and SETBP1 were grouped as DNA methylation. SFSB1, SRSF2, U2AF1, and ZRSR2 were grouped as spliceosome. BCOR, CBORL1, EXH2, and KDM6A were grouped as chromatin modifier. ASXL1, which is also chromatin modifier, was analyzed as a separated component because it is a part of the 2017 ELN. NRAS and KRAS were analyzed separately and also as one group. One-tailed statistical significance is at level of 5% for statistical analysis. Results Our cohort was male predominant (57%, 110/193) with median age of 64 YO (range 18 - 93). 29%, 25% and 46% of patients were 2017 ELN favorable, intermediate, and adverse group respectively. 27.5% and 40% were Adverse and Normal Karyotype respectively. 4%, 4% and 1% of patients were RUNX1-RUNX1T1, CBFB-MYH11 and MLLT3-KMT2A positive respectively. Figure1 describes the occurrence rate of significantly occurred mutations. Regarding 2017 ELN components, 37 patients (19.2%) were positive to NPM1, 34 patients (17.6%) had FLT-ITD low, 3 (1.6%) had CEBPA biallelic, 40 (20.7%) had TP53, 27 (14.0%) had ASXL, and 24 (12.4%) had RUNX. There was no FLT-ITD high mutation in our cohort. In total 63 patients (32.6%) had at least one DNA methylation mutation. 44 patients (22.8%) had at least one spliceosome mutation and 9 patients (4.6%) had at least one chromatin modifier mutation other than ASXL1. In activated signaling, NRAS, KRAS, FLT-TKD were significantly occurred. Table 1 describes significantly co-occurred and exclusively occurred mutations and Figure 2 visualizes significantly co-occurred and exclusively occurred mutations. TP53 exclusively occurred with normal karyotype and many different mutations including NPM1, FLT-ITD low, DNA methylation group, and KRAS/NRAS, and significantly co-occurred with Adverse Karyotype. NPM1 significantly co-occurred with Normal Karyotype and FLT-ITD mutations. Interestingly, DNA methylation group and especially IDH2 and DNMT3A significantly co-occurred with NPM1. Also, RUNX1, ASXL1 and Spliceosome group co-occurred with each other. KRAS/NRAS co-occurred with CBFB-MYH1. KIT co-occurred with CBFB-MYH1 and RUNX-RUNX1T1, although the sample sizes were relatively small. Discussion Based on strong accumulated evidence, ELN established new guidelines in 2017. Therefore, it would be a sophisticated way to find other significant mutations and their interactions on the basis of already well established 2017 ELN components. Our findings suggest DNA methylation regulatory genes, especially DNMT3A and IDH2, significantly co-occurred with NPM1, which is a component of the favorable group. Also, spliceosome mutations significantly co-occurred with RUNX1 and ASXL1, which are components of the adverse group. Further research is needed to determine whether those cooccurrences affect each group's prognosis. Figure Disclosures No relevant conflicts of interest to declare.

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Profile Of Microparticles In Patients With Acute Leukemia At Diagnosis and Upon Remission Induction

Blood ◽

10.1182/blood.v122.21.4741.4741 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4741-4741

Author(s):

Inna Tsoran-Rosenthal ◽

Benjamin Brenner ◽

Anat Aharon

Keyword(s):

Tissue Factor ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Annexin V ◽

Leukemic Cells ◽

Hypercoagulable State ◽

Platelet Glycoprotein ◽

Remission Induction ◽

Induction Treatment ◽

Hla Dr

Introduction Microparticles (MPs) shedding from the cell surface are assumed to express antigens reflecting their cell origin. MPs bearing tissue factor (TF) can play a pivotal role in the pathogenesis of the prothtombotic condition in patients with malignancies. Patients with acute myeloid leukemia (AML) can develop venous thromboembolism. This study was aimed to evaluate whether circulating MPs could serve as a biomarker, indicating changes in blood cells and predicting development of a thrombogenic state in AML patients at diagnosis and remission. Methods Blood samples were obtained from healthy controls and patients with AML at diagnosis, 2 weeks after the start of therapy and at remission achievement. The MP cell origin was assessed using fluorescent antibodies and was analyzed by fluorescence-activated cell sorting (FACS). The following fluorescent antibodies were used: CD41 (platelet glycoprotein complex), CD144 (the endothelial marker), CD11 (leukocyte marker) and Annexin V (for MPs expressing negative phospholipids on their surface). To identify MPs originating from leukemic cells, MPs were also labeled with CD34, CD117, CD33, and HLA-DR. The procoagulant and anticoagulant activity of MPs was evaluated in the study groups using the FACS analysis: each MP sample was labeled with florescent antibodies against TF and tissue factor pathway inhibitor (TFPI) and the TF/TFPI ratio, potentially indicating a hypercoagulable state, was calculated. Results Forty patients with AML were enrolled in the study: 25 patients achieved remission with induction treatment. In AML patients, the average MP count at diagnosis was higher than in controls and than that observed at nadir and remission, although the difference did not reach statistical significance. Notably, the Annexin V expression was significantly higher in controls compared to patients at diagnosis (33.24% vs. 8.27%; p<0.01), or those at nadir (33.24% vs. 8%; p<0.01). CD144 levels were found to be higher in patients’ MPs at diagnosis compared to controls (28.4% vs. 7.5%; p<0.05). The levels of CD34 and CD33 were significantly greater in patients at diagnosis compared to remission (4.4% vs. 1.7%; p<0.05 and 34.9% vs. 15.3%; p<0.05, respectively) and controls (4.4% vs. 1.4%; p<0.001 and 34.9% vs. 15.7%; p<0.01, respectively). A similar trend was observed with CD117 and HLA-DR. The TF expression was found to be higher in patients at diagnosis compared to nadir (6.4% vs. 4.1%; p<0.05), remission (6.4% vs. 2.7%; p<0.001). The TFPI level appeared to be greater in controls’ MPs compared to that of patients (9% vs. 3.5%; p<0.05), while the TF/TFPI ratio was higher at diagnosis compared to remission (8.5 vs. 0.46; p<0.01) and controls (8.5 vs. 0.35; p<0.05). Conclusion MPs of AML patients at diagnosis express markers of blast cells and may potentially serve as a biomarker. The increased level of endothelial MPs in AML and high MP thrombogenicity at diagnosis may be suggestive of a hypercoagulable state. Disclosures: No relevant conflicts of interest to declare.

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Predicting outcome based on minimal residual disease (MRD) using multiparameter flow cytometry (FC) in acute myeloid leukemia (AML) patients (Pts).

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.6565 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 6565-6565

Author(s):

Sagar D Sardesai ◽

Wei Tan ◽

Laurie Ann Ford ◽

George Deeb ◽

Anne Marie W. Block ◽

...

Keyword(s):

Residual Disease ◽

Statistical Significance ◽

Expression Patterns ◽

Intensive Therapy ◽

Normal Karyotype ◽

Population Based ◽

Leukemic Cells ◽

Multiparameter Flow Cytometry ◽

Predicting Outcome

6565 Background: Most AML pts will experience relapse caused by persistent leukemic cells during complete remission (CR). The aim of our study was to predict outcome in AML pts in CR by assessing their MRD using standard 4-colour FC panels available at our institute. Methods: We queried our AML database between 1/2004 to 10/2010 for newly diagnosed untreated AML pts >18 years of age who achieved CR after one induction regimen. Treatment included 7+3 or similar intensive approaches. The gating strategy used a series of Boolean regions that best defined the diagnostic abnormal population based on its expression patterns using three 4-colour FC panels (F7-CD38,CD10,CD19,CD34; F9-C11b,CD33,CD13,CD34; F38-CD15,CD56,CD7,CD34). The same regions were applied to post-induction samples, and the number of residual events was determined. Results: 140 AML patients with a median age of 64 (range 24-93) years, including 74 females (52.8%) were analyzed; 67 (47.8%) patients relapsed. Eighty-eight (62.8%) pts were CD34-positive (CD34+) at diagnosis. Normal karyotype was detected in 27 (30.7%): FLT-3 ITD was detected in 4/23 (17.4%) and NPM-1 mutation was detected in 1/15 (0.07%) samples. Median follow-up for CD34+ pts was 17.9 months. Forty-two (30%) pts underwent stem cell transplantation (SCT) in first CR: 31 (22.1 %) were allogeneic and 11 (7.9%) autologous. In multivariate analysis, a detectable MRD in any of the 3 panels at or beyond week 16 among CD34+ AML pts in CR was significantly associated with inferior relapse free survival (F7: P=0.035 , F9: P=0.027, F38: P=0.042). In addition, MRD ≥ week 16 using panel F9 was also significantly associated with inferior overall survival (P=0.0224). In pair-wise comparison, those who were MRD-negative ≥ week 16 did not benefit from SCT compared to the non-SCT group. Similar analyses among CD34– AML pts did not achieve statistical significance. Conclusions: MRD+ by FC in CD34+ AML is an independent prognostic factor and can identify pts who may benefit from SCT/more intensive therapy to maintain remission. However, the value of studying MRD in CD34– AML requires further consideration. More effort is needed to identify stem cells in CD34– AML.

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DNA Methylation Changes In Aging Human CD34+ Cells Coincide With Hotspots Of Disordered Methylation In AML At Imprinted and Allelically Methylated Regions

Blood ◽

10.1182/blood.v122.21.1193.1193 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1193-1193

Author(s):

Tim Triche ◽

Stephen Capone ◽

Akil Merchant ◽

Preet Chaudhary ◽

Giridharan Ramsingh

Keyword(s):

Dna Methylation ◽

Conflicts Of Interest ◽

Wilms Tumor ◽

Statistical Significance ◽

Differentially Methylated Regions ◽

Hematopoietic Stem ◽

Loss Of Imprinting ◽

Age Related ◽

Cd34 Cells ◽

Epigenetic Machinery

Abstract Aging of the hematopoietic system in humans is associated with increased incidence of myeloid malignancies. Epigenetic machinery such as DNA methylation is known to change with age, and is disproportionately impacted by recurrent genetic mutations in acute myeloid leukemia (AML). We hypothesized that epigenetic changes in CD34+ hematopoietic stem and progenitor cells (HSPCs) may precede recurrent genetic changes in AML, and might be detected in normal aging HSPCs with increasing frequency. We also hypothesized that areas with increased variability in methylation may be hot-spots for the emergence of epigenetically distinct HSPC clones. In order to characterize these changes, we performed methylome-wide profiles of human HSPCs from different age groups (20-25 years (10), 40-45 years (10) and >60 years (9)).Adult HSPCs were obtained from Leukocyte Reduction System cones from healthy platelet donors. CD34+ cells were then isolated by Fluorescence Assisted Cell Sorting. 200 ng of DNA extracted from the CD34+ cells was processed using the Infinium Methylation 450k Beadchip (Illumina). Differentially methylated regions (DMRs) were identified using the bump hunting procedure of Jaffe (2011) to pool information across CpG loci into regions of consistent change and to quantify statistical significance. 893 differentially methylated regions (DMRs) varied linearly with age in HSPCs; a set of 31 such regions yielded an accurate predictor of age in lineage-sorted cells (N=48, Reinius et al., 2012) and whole blood (N=656, Hannum et al., 2011), with a root-mean-squared error of 5.3 years. While age-related lymphopenia has previously been reported, DNA methylation marks for lineage commitment (Houseman et al., 2012) were nearly uniform within our subjects’ CD34+ cells, and exhibited no relationship with age. However, regional summaries of methylation provided more accurate age predictions than specific CpG loci. We reasoned that differential variability at individual loci might be the cause. We thus investigated regions where methylation variability increased with age. Known imprinted clusters and allelically methylated regions (AMRs) identified by Fang (2012, PNAS) were disproportionately represented among these; 27% of known imprinting regions and 33.3% of allelically methylated regions in the genome coincided with at least one such region, while comprising only 0.3% of the genome and 0.7% of loci assayed. Among these, the H19 imprinting control region has been shown to crucially regulate long-term HSPC homeostasis in mice via IGF2, and the allelically methylated WT1/WT1-AS region on chromosome 11p is a highly recurrent hotspot for disordered methylation in AML, as well as sequential epigenetic defects in Wilms’ tumor. The allelically methylated vault RNA VTRNA2-1 (recently shown to predict survival in AML) on chromosome 5q, and the monoallelically expressed TP73 and DIRAS3 genes on chromosome 1p, were also sites of greater methylation variability with age in normal HSPCs. Wu et al. (1997) showed that loss of imprinting at H19/IGF2 is common in AML, seemingly conferring a selective metabolic advantage, and global loss of imprinting in mice leads to widespread tumorigenesis (Holm et al., 2005). Recurrent methylation aberrations in induced pluripotent stem cells favor imprinted clusters (Nazor, 2012), and epigenetic polymorphisms arise in these regions over time in cultured cells (Tanay et al, 2012). However, to our knowledge, ours is the first report of this type of heterogeneity with age in normal human adult HSPCs. Clonal hematopoiesis has previously been documented in healthy elderly adults (Levine 2012), and the majority of patients in the Cancer Genome Atlas (TCGA) AML study exhibited mutations in one or more genes regulating epigenetic machinery. We propose that increased epigenetic heterogeneity in aging HSPCs, particularly at regions with allele-specific methylation (such as H19/IGF2), may precede malignant evolution in some leukemias, and warrants further investigation. Disclosures: No relevant conflicts of interest to declare.

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Intra Bone Route of Donor Lymphocyte Infusions May Result with an Anti-Leukemic Effect Associated with an Accumulation of CD279+ Lymphocytes in the Marrow

Blood ◽

10.1182/blood.v128.22.5763.5763 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5763-5763

Author(s):

Andrzej Lange ◽

Monika Mordak-Domagala ◽

Mariola Sedzimirska ◽

Michalina Krydel ◽

Helena Pakos ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Normal Karyotype ◽

Wilcoxon Test ◽

Leukemic Cells ◽

Sorafenib Treatment ◽

Marrow Cavity ◽

Normal Individuals ◽

Bone Marrow Cavity ◽

Anti Cd20

Abstract Immunologic surveillance of leukemia is employed for the prevention and treatment of relapse post alloHSCT. The rate of success depends on the characteristics of leukemia which is more beneficial if the cells are recognized in the way of alloreactivity. Intra venous DLI is associated with a high risk of aGvHD and we believe that this route of administration may not make the direct contact between infused cells and blasts the optimal one. The previous reports documented a lower risk of aGvHD if the transplant material was given to the bone marrow cavity which at relapse is usually infiltrated by the blasts. To address these issues, we started delivering donor lymphocytes directly to the bone marrow cavity (IB-DLI) in patients post alloHSCT at relapse. This technique was employed in 4 patients, 3 with AML and one with CLL, all relapsed post alloHSCT: 3 alloSIB: 50-year-old female AML patient with normal karyotype (relapsed 2 years post HSCT) and 22-year-old AML male 7q31 del (relapsed at 3 years post HSCT) and 64-year-old CLL male, TP53 del, recurrent EBV reactivation with vasculitis (progressed 7 years), and 25-year-old male AML FLT3 ITD+ received MUD HSCT (relapsed 9 months). Two patients with a proportion of 26% and 12% blasts in the marrow respectively received IB-DLI up-front and two others due to the excess (52.50%, 57.70% (CD5+CD19+)) of leukemic cells received either FLAG (AML case) or anti-CD20 MoAB (CLL case) followed by IB-DLI. The patients received from 3 to 5 IB-DLIs according to the escalating dose regimen starting from 10E6 and ending with a dose of 10E8 of CD3+ cells/kg b. w. (blood MNC were harvested with use of Spectra Optia (Terumo BTC) for SIB transplantation, in MUD HSCT they were taken from the PBPC material (J Hasskarl et al. 2012). The intervals between IB DLI varied from 1 to 2 months. One patient received azacitidine between the DL infusions (Schroeder T et al. 2013) which was associated with longer intervals. The cells were injected directly to the bone marrow cavity under local anaesthesia with a low molecular Heparin prophylaxis. The blood and marrow specimens were taken prior each IB-DLI for: cytology, cytometry (including CD8, CD279, CD26, CD28 MoAb in addition to the routine staining used for blast cells), genetic work (chimerism, mutations associated with the disease). Clinical outcome:No side effects were noticed (including GvHD).The patients are alive.Anti-leukemic effect: 3.1 Responding AML ITD+ case was free of blasts 6 months after the initiation of the treatment. However, after the third IB-DLI blasts appeared in the marrow but the patient responded favorably to the Sorafenib treatment and the following course of IB-DLI. 3.2 Partial remission (stable proportion of blasts and sustained hematopoiesis); the patient (22-year-old AML male) was transplanted a second time but blasts at the range of 38% were still observed in the marrow without progression after 9 months. 3.3 One case which failed to respond to IB-DLI (female AML patient) was transplanted a second time but this approach also failed and this patient now has full blown leukemia. The laboratory work up:Proportions of lymphocytes in the marrows tended to increase after completion of each IB-DLI (32.0 ± 4.6% vs 37.1 ± 3.7%, Wilcoxon test for pairs, p=0.078).Collectively CD279+ cells contributed to the lymphocyte pool in the marrow to a greater extent than it was seen in the blood at the same time (16.6 ± 2.9% vs 33.1 ± 4.0%, p<0.001); this was also valid for CD8+CD279+ cells (12.4 ± 3.2% vs 27.1 ± 4.5%, p<0.001).Microarray analysis of the transcriptome in the marrows of patients who received three IB-DLI courses revealed that in the patients responded favorably (CR or PR) their transcriptome profiles formed a cluster together with the transcriptomes of normal individuals in contrast to the patients who failed to respond, whose transcriptome profiles clustered separately. IB-DLI was safe and not associated with GvHD. In all cases the infusion was associated with an increase in the marrow of lymphocytes being CD8+CD279+. The response may result in CR or PR and the patients were in a good physical shape during the treatment, which makes it possible to consolidate the treatment with a second alloHSCT if required. A lack of any response after the first courses of IB-DLI was associated with a failure to respond, even if the second transplant is performed. Supported by The National Centre for Research and Development grant (INNOMED/I/1/NCBR/2014) Disclosures No relevant conflicts of interest to declare.

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Residual HSPC in the Leukemia Microenvironment Are Reprogrammed Via Extracellular Vesicle Trafficking

Blood ◽

10.1182/blood.v128.22.888.888 ◽

2016 ◽

Vol 128 (22) ◽

pp. 888-888 ◽

Cited By ~ 1

Author(s):

Sherif Abdelhamed ◽

Noah I Hornick ◽

Peter Kurre

Keyword(s):

Cell Cycle ◽

Dna Methylation ◽

Dna Damage ◽

High Throughput Sequencing ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Leukemic Cells ◽

Alternative Mechanism ◽

Proteomic Profiling ◽

Hematopoietic Stem

Several groups have shown that leukemic cells create a self-reinforcing bone marrow (BM) niche that functionally impairs normal hematopoietic stem and progenitor cells (HSPC) indirectly through stroma-secreted factors. We recently demonstrated an alternative mechanism whereby extracellular vesicles (EVs) from acute myeloid leukemia (AML) patients and cell lines, but not BM CD34 controls, suppress their clonogenicity through EV trafficking of microRNA that directly downregulate critical transcription factors (c-Myb and HoxA9). Here, we aimed to clarify the fate of residual HSPC in in vivo AML xenografts, as well as ex vivo intrafemural (IF) injection and in vitro exposure of EVs experiments. Among KSL cells we observed a significant increase in the frequency of the long-term hematopoietic stem cell (SLAM, CD150+CD48−) subpopulation, but not the multipotent progenitors even at low levels of AML infiltration or direct IF injection of EVs. The HSPC pool redistribution was accompanied by cell cycle alterations in residual HSPC that showed AML EVs consistently induced quiescence (G0) in KSL (cKit+Sca1+Lin−) HSPC populations. When we assessed their DNA damage, residual HSPC showed a distinct increase in the gH2AX foci relative to control non-engrafted mice as well as the transcriptional upregulation of Rad51 and P21 genes along with gains in phosphorylation of the tumor suppressor p53. Yet, the reprogrammed KSL showed no evidence of apoptosis indicated by the lack of upregulation of the p53 target, Puma, and Annexin V staining, nor evidence of senescence (P16 and Sparc transcripts). To gain additional insight, we performed a tandem mass tag (TMT) proteomic profiling of AML-EV exposed HSPC with or without exposure to EVs derived from AML cells. The results showed significant enrichment of DNA methylation regulatory pathway such as DNMT1, HELLS and UHRF1 as well as inflammatory pathways including IL1b, NOS, CEBPB and NFkB pathway-targets, confirmed by transcriptional profiling of KSL from xeno-transplanted mice. Based on our recent report that miR-1246 is one of the most highly enriched miRNA in AML derived EVs and proceeded to determine its target transcripts using an attenuated RISC complex (RISC-Trap), followed by high-throughput sequencing. Bioinformatics analysis identified a set of 27 miR-1246-specific targets relative to control microRNAs. Strikingly, the target set was selectively enriched for a panel of negative cell-cycle regulator genes (CDK1, CDK7, CDK11, CCNF, HDAC2 and GATA3) as well as the DNA methylation regulators (DNMT1 and HELLS).Collectively, our results demonstrated that residual HSPC in the AML BM are phenotypically reprogrammed and suppressed in their proliferation along with DNA damage accumulation via paracrine EV microRNA trafficking. Our study provides insight into HSPC fates in the AML niche and echoes observations of cell competition, as a mode of non-cell autonomous regulation where p53 activation in the reprogrammed cells leads to a progressive decline in proliferation and fitness. We propose that AML EV trafficking of miR-1246 specifically may contribute to the altered fate of residual HSPC via transcriptional regulation of proliferation-related genes. Disclosures No relevant conflicts of interest to declare.

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Relapse Mechanism of FLT3-ITD Positive AML and Cell Differentiation Blockage

Blood ◽

10.1182/blood.v126.23.4961.4961 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4961-4961

Author(s):

Michal Hayun ◽

Maria Zaatra ◽

Chen Itzkovitch ◽

Dvora Sahar ◽

Eldad J. Dann ◽

...

Keyword(s):

Cell Lines ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Leukemia Cell ◽

Normal Karyotype ◽

Leukemic Cells ◽

Genetic Profile ◽

Relapse Risk ◽

Induction Failure ◽

Cd34 Expression

Abstract Introduction: Activating mutation of FLT3 by internal tandem duplications (ITD) is the most common molecular aberration found in AML. FLT3-ITD mutation induces a proliferation signal and is associated with leukocytosis and poor prognosis. The co-existence of FLT3-ITD with translocation t(5:11) that yields fusion transcript of Nucleoporin98 and the nuclear receptor binding SET-domain protein 1 (NUP98/NSD1) was recently reported to be associated with chemo-resistance. Relapse risk of the majority of FLT3-ITD positive AML patients who are negative to NUP98/NSD1 is increased compared to other AML with normal karyotype, despite similar remission rates. The mechanism of FLT3-ITD contribution to relapse development in patients who achieved remission is unknown. Aims: To explore potential mechanisms that allow relapse in FLT3-ITD positive AML in the presence or absence of NUP98/NSD1 fusion gene. In addition, time sequence of the emergence of both mutations is suggested from a case with relapse. Methods: Leukemic blasts derived from newly diagnosed or relapsed FLT3-ITD positive AML patients were enriched for FLT3-ITD mutated sub-clones by sorting according to CD34 expression. Genomic DNA was extracted from each sorted fraction and FLT3-ITD mutation allele load was quantitatively determined by GeneScane. Total RNA was extracted from FLT3-ITD positive patients and RT-PCR was performed to detect NUP98/NSD1 fusion mRNA. All patients received induction with intensive chemotherapy combination of Daunorubicin and Cytarabine and their outcome was recorded. Chemo-sensitivity assays using Ara-C were conducted on different leukemia cell lines sub-populations divided by their CD34 expression. Results: NUP98/NSD1 was identified in 4 of 19 (21%) FLT3- ITD positive adult AML patients with normal karyotype who had a compatible donor and were considered transplant eligible. Patients harboring both NUP98/NSD1 and FLT3-ITD (75%) had higher rate of induction failure than FLT3-ITD patients without NUP98/NSD1 (40%). To explore the correlation between FLT3-ITD and differentiation capacity, primary AML FLT3-ITD positive blasts were sorted into two distinct sub-populations according to CD34 expression (example is shown in fig. 1). In 14/19 patients DNA from CD34+ and CD34- leukemic blasts was successfully extracted and FLT3- ITD allele load was tested and recorded as the ratio between FLT3 normal and mutated alleles. Of these 14 patients, 3 experienced induction failure and 8 (58%) achieved complete remission but unfortunately eventually relapsed. FLT3-ITD allelic ratio (AR) was equally measured in both CD34+ and CD34- sub-populations in 7 patients (50%) while in 5 patients (35.7%) the mutated allele was restricted to the CD34 positive cells and 4/5 (80%) patients experienced relapse. In two patients (14%) who also expressed NUP98/NSD1, the FLT3-ITD mutated allele was restricted to CD34 negative sub-population. In one patient, NUP98/NSD1 was detected in relapse but not in diagnostic specimen. Cytotoxic assay confirmed that differentiation stage as determined by CD34 expression is fundamental for chemo-sensitivity of leukemic cells regardless of their genetic profile. CD34 positive cell lines as well as CD34+ sub-population of Kasumi-1 cell line were resistant to the Ara-C compare to CD34 negative cell lines or the matched Kasumi-1 CD34- sub-population (fig. 2). Conclusions: FLT3-IDT mutation is a late event during leukemogenesis. We observed NUP98/NSD1 that emerged as a new mutation on relapse in a FLT3-IDT positive patient, assuming this may also be a later event. Our strategy to sort blasts according to their CD34 expression enable us to describe the accumulation of FLT3-ITD sub-clones at the primitive stage that express CD34, therefore, these leukemic sub-clones may be enriched with early leukemic precursors. Such differentiating blockage results a higher portion of cells which survive chemotherapy among FLT3-ITD and hence increases relapse risk as an indirect effect. Inducing differentiation in FLT3-ITD positive AML should be further studied as a therapeutic strategy. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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Low Expression of p14ARF in De Novo AML with Normal Karyotype Is Associated with Short Survival but Increased in Vitro Sensitivity to p53 Modulating Drugs

Blood ◽

10.1182/blood.v112.11.2937.2937 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2937-2937

Author(s):

Esbjörn Paul ◽

Bertil Uggla ◽

Kals G Wiman ◽

Christer Paul ◽

Soren Lehmann ◽

...

Keyword(s):

De Novo ◽

P53 Protein ◽

Statistical Significance ◽

Normal Karyotype ◽

Leukemic Cell ◽

Leukemic Cells ◽

Prognostic Impact ◽

Future Possibility ◽

De Novo Aml

Abstract Background: The p53 protein is strongly regulated by the E3 ligase HDM-2, which specifically binds to p53 and causes proteosomal degradation. The p14ARF protein activates p53 by binding to and inhibiting HDM-2. Design and methods: To further study the prognostic impact of p14ARF in AML, leukemic cells from 57 adult patients with normal karyotype de novo AML were analysed for p14ARF mRNA expression level using real-time PCR. We also measured the cytotoxicity against conventional cytostatics and PRIMA-1, a novel small molecule shown to activate mutated p53 (Nature Medicine2002; 8: 282–288) using an ATP-assay. Intracellular p53 protein was measured by FACS after incubation with PRIMA-1 in combination with the HDM-2 blocking agent RITA (Nature Medicine2004; 10: 1321–1328) Results: Patients whose cells expressed more p14ARF mRNA than the 75th percentile (0.26) showed significantly better survival compared with those with lower levels, 61% vs. 30% 3-year survival (p=0.033). The difference remained significant also when NPM1/FLT3 status was considered. The mean effects of all tested conventional antileukemic drugs were greater in leukemic cell samples expressing p14ARF mRNA ≥0.26, but the differences did not reach statistical significance. In contrast, PRIMA-1 had a significantly greater effect on leukemic cell samples with low levels of p14ARF mRNA and PRIMA-1 and RITA significantly increased intracellular p53 levels. Conclusions: Low level of p14ARF mRNA in leukemic cells from patients with normal karyotype AML is a strong marker for poor prognosis and decreased sensitivity to conventional cytostatics. Treatment with drugs targeting p53 can be a future possibility to improve the outcome for these patients.

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Clinical, Biological Profile and Outcome of Biphenotypic Acute Leukemia: a Case Series.

Blood ◽

10.1182/blood.v116.21.1683.1683 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1683-1683

Author(s):

Yanming Zhang ◽

Wu Depei ◽

Aining Sun ◽

Huiying Qiu ◽

Guangsheng He ◽

...

Keyword(s):

Acute Leukemia ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Case Series ◽

Disease Free Survival ◽

Normal Karyotype ◽

Structural Rearrangement ◽

Complex Karyotype ◽

B Lymphoid ◽

Biphenotypic Acute Leukemia

Abstract Abstract 1683 Biphenotypic acute leukemia (BAL) is a very rare type of acute leukemia, which presents a high heterogeneity and poor prognosis. We identified 51 cases (3.0%) BAL from 1693 newly diagnosed acute leukemia patients according to the EGIL scoring system between January 2003 and July 2009. The biological features, treatment and outcome of 39 evaluable BAL patients were analyzed retrospectively. There were 23 (59.0%) cases of myeloid and B-lymphoid (M/B) phenotype, 14 (35.9%) cases of myeloid and T-lymphoid (M/T) phenotype, one case (2.6%) of trilineage phenotype or B-lymphoid and T-lymphoid phenotype respectively. The high expressions of CD34 (84.6%) and HLA-DR (54.5%) on the blast cells of BAL support the notion that BAL probably arises from hemopoietic stem/progenitor cells. It seemed that CD7-positive patients had poorer median survivals comparing with those CD7-negative patients, however, there was no statistical difference (P=0.076). Abnormal karyotypes were detected in 75.7% of 37 BAL patients with valid analysis and displayed a high heterogeneity, which were associated with structural rearrangement and numerical abnormalities including t(8;21) (16.2%), t(9;22) (13.5%), structural rearrangement of 11 chromosome (16.2%), 11q23 (5.4%), complex karyotype (21.6%) and other abnormal karyotypes (10.8%). Combined regimens for both AML and ALL, ALL-type regimens appeared a better complete remission (CR) rate than AML-type regimens (71.4% vs. 63.6% vs. 33.3%). The median survival for overall survival (OS) and disease-free survival (DFS) in our series was 14 and 12 months, the probability of OS and DFS at 2 years was 26.0% and 18.5%, respectively. No statistical differences were observed in CR rate, OS and DFS between M/B and M/T cases. It showed that BAL patients with complex karyotype or rearrangement of 11 chromosome had a significantly worse survival in contrast to normal karyotype, t(8;21) and t(9;22) group (P=0.001). Although BAL with t(8;21) seemed to be appeared a better survival than normal karyotype and t(9;22) group, there were no statistical significance (P=0.436). Our data indicate that combined-type regimens or ALL-based protocols are more effective and complex karyotype, rearrangement of 11 chromosome have the unfavorable prognosis for BAL. Disclosures: No relevant conflicts of interest to declare.

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Use of SAMe-TT₂R₂ Score to Predict the Quality of Anticoagulation Control in Patients with Atrial Fibrillation Receiving Warfarin in Thailand

Journal of the Medical Association of Thailand ◽

10.35755/jmedassocthai.2020.06.10827 ◽

2020 ◽

Vol 103 (6) ◽

pp. 548-552

Keyword(s):

Atrial Fibrillation ◽

Therapeutic Range ◽

Statistical Significance ◽

Discrimination Performance ◽

Poor Quality ◽

Chi Square ◽

Exact Test ◽

Time In Therapeutic Range ◽

Anticoagulation Control

Objective: To predict the quality of anticoagulation control in patients with atrial fibrillation (AF) receiving warfarin in Thailand. Materials and Methods: The present study retrospectively recruited Thai AF patients receiving warfarin for three months or longer between June 2012 and December 2017 in Central Chest Institute of Thailand. The patients were classified into those with SAMe-TT₂R₂ of 2 or less, and 3 or more. The Chi-square test or Fisher’s exact test was used to compare the proportion of the patients with poor time in therapeutic range (TTR) between the two groups of SAMe-TT₂R₂ score. The discrimination performance of SAMe-TT₂R₂ score was demonstrated with c-statistics. Results: Ninety AF patients were enrolled. An average age was 69.89±10.04 years. Most patients were persistent AF. An average CHA₂DS₂-VASc, SAMe-TT₂R₂, and HAS-BLED score were 3.68±1.51, 3.26±0.88, and 1.98±0.85, respectively. The present study showed the increased proportion of AF patients with poor TTR with higher SAMe-TT₂R₂ score. The AF patients with SAMe-TT₂R₂ score of 3 or more had a larger proportion of patients with poor TTR than those with SAMe-TT₂R₂ score of 2 or less with statistical significance when TTR was below 70% (p=0.03) and 65% (p=0.04), respectively. The discrimination performance of SAMe-TT₂R₂ score was demonstrated with c-statistics of 0.60, 0.59, and 0.55 when TTR was below 70%, 65% and 60%, respectively. Conclusion: Thai AF patients receiving warfarin had a larger proportion of patients with poor TTR when the SAMe-TT₂R₂ score was higher. The score of 3 or more could predict poor quality of anticoagulation control in those patients. Keywords: Time in therapeutic range, Poor quality of anticoagulation control, Warfarin, SAMe-TT₂R₂, Labile INR

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PITX2 DNA-Methylation: Predictive versus Prognostic Value for Anthracycline-Based Chemotherapy in Triple-Negative Breast Cancer Patients

Breast Care ◽

10.1159/000510468 ◽

2020 ◽

pp. 1-9

Author(s):

Rudolf Napieralski ◽

Gabriele Schricker ◽

Gert Auer ◽

Michaela Aubele ◽

Jonathan Perkins ◽

...

Keyword(s):

Breast Cancer ◽

Dna Methylation ◽

Cancer Patients ◽

Triple Negative Breast Cancer ◽

Predictive Value ◽

Untreated Patient ◽

Triple Negative ◽

Statistical Significance ◽

Breast Cancer Patients ◽

The Impact

Background: PITX2 DNA methylation has been shown to predict outcomes in high-risk breast cancer patients after anthracycline-based chemotherapy. To determine its prognostic versus predictive value, the impact of PITX2 DNA methylation on outcomes was studied in an untreated cohort vs. an anthracycline-treated triple-negative breast cancer (TNBC) cohort. Material and Methods: The percent DNA methylation ratio (PMR) of paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time PCR test. Patient samples of routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue and clinical data from 144 TNBC patients of 2 independent cohorts (i.e., 66 untreated patients and 78 patients treated with anthracycline-based chemotherapy) were analyzed. Results: The risk of 5- and 10-year overall survival (OS) increased continuously with rising PITX2 DNA methylation in the anthracycline-treated population, but it increased only slightly during 10-year follow-up time in the untreated patient population. PITX2 DNA methylation with a PMR cutoff of 2 did not show significance for poor vs. good outcomes (OS) in the untreated patient cohort (HR = 1.55; p = 0.259). In contrast, the PITX2 PMR cutoff of 2 identified patients with poor (PMR >2) vs. good (PMR ≤2) outcomes (OS) with statistical significance in the anthracycline-treated cohort (HR = 3.96; p = 0.011). The results in the subgroup of patients who did receive anthracyclines only (no taxanes) confirmed this finding (HR = 5.71; p = 0.014). Conclusion: In this hypothesis-generating study PITX2 DNA methylation demonstrated predominantly predictive value in anthracycline treatment in TNBC patients. The risk of poor outcome (OS) correlates with increasing PITX2 DNA methylation.

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