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High expression of the leukemia‐associated gene meningioma‐1 (MN1) is frequently found at diagnosis of acute myeloid leukemia (AML) and ...associates with adverse outcomes. The presence of measurable residual disease (MRD) in complete remission (CR) indicates high risk of relapse and worse outcome in AML patients. However, the prognostic impact of MN1 expression levels as MRD marker has not been evaluated. Digital droplet polymerase chain reaction (ddPCR) is a novel technique allowing sensitive and specific absolute gene expression quantification. We retrospectively analyzed 124 AML patients who received allogeneic hematopoietic stem cell transplantation (HSCT) in CR or CR with incomplete peripheral recovery. Absolute MN1 copy numbers in peripheral blood were assessed prior to HSCT (median 7; range 0–29 days) using ddPCR. High pre‐HSCT MN1/Abelson murine leukemia viral oncogene homolog 1 gene (ABL1) copy numbers associated with a higher cumulative incidence of relapse after HSCT and—in relapsing patients—shorter time to relapse. In multivariable analysis, high pre‐HSCT MN1/ABL1 copy numbers remained an independent prognosticator for relapse after HSCT. Patients with the highest pre‐HSCT MN1/ABL1 copy numbers also had the highest risk of relapse. MN1 copy number assessment also added prognostic information to nucleophosmin 1 gene (NPM1) mutation‐ and brain and acute leukemia, cytoplasmic (BAALC) and Wilm's tumor gene 1 (WT1) expression‐based MRD evaluation. Our study demonstrates the feasibility of the novel ddPCR technique for MN1/ABL1 copy number assessment as a marker for MRD. Evaluation of MN1/ABL1 copy numbers allows the identification of patients at high risk of relapse, independently of other diagnostic risk factors and MRD markers.
Allogeneic hematopoietic stem cell transplantation is an established consolidation therapy for patients with acute myeloid leukemia. However, relapse after transplantation remains a major clinical ...problem resulting in poor prognosis. Thus, detection of measurable (“minimal”) residual disease to identify patients at high risk of relapse is essential. A feasible method to determine measurable residual disease may be digital droplet PCR (ddPCR) that allows absolute quantification with high sensitivity and specificity without the necessity of standard curves. Using ddPCR, we analyzed pre-transplant peripheral blood and bone marrow of 51
NPM1
-mutated acute myeloid leukemia patients transplanted in complete remission or complete remission with incomplete recovery. Mutated
NPM1
measurable residual disease-positive patients had higher cumulative incidence of relapse (
P
< 0.001) and shorter overall survival (
P
= 0.014). Restricting the analyses to patients receiving non-myeloablative conditioning, mutated
NPM1
measurable residual disease positivity is associated with higher cumulative incidence of relapse (
P
< 0.001) and shorter overall survival (
P
= 0.006). Positive mutated
NPM1
measurable residual disease status determined by ddPCR before allogeneic stem cell transplantation is associated with worse prognosis independent of other known prognostic markers—also for those receiving non-myeloablative conditioning. In the future, mutated
NPM1
measurable residual disease status determined by ddPCR might guide treatment and improve patients’ outcomes.
Age-related somatic mutations linked to clonal hematopoiesis have been found in apparently healthy individuals and increase the risk of developing hematologic malignancies. In acute myeloid leukemia ...(AML) the clinical relevance of clonal hematopoiesis remains controversial and data on patients with detectable clonal hematopoiesis, consolidated with hematopoietic stem cell transplantation are limited. We analyzed samples from 113 AML patients in complete remission prior to hematopoietic stem cell transplantation for the presence of clonal hematopoiesis-associated mutations. The results were correlated with clinical and biological data. In complete remission we found 75 mutations previously linked to clonal hematopoiesis in 47 patients (41.6%). Twenty patients had ≥2 mutations linked to clonal hematopoiesis. DNMT3A, TET2, and ASXL1 were most frequently mutated. When compared to pre-treatment samples we found variable patterns of mutation persistence depending on the gene mutated. In AML patients after allogeneic hematopoietic stem cell transplantation the presence of clonal hematopoiesis-associated mutations in complete remission did not associate with inferior clinical outcome. This study demonstrates that clonal hematopoiesis is a frequent phenomenon in AML patients. Presence of clonal hematopoiesis has no negative prognostic impact in the context of an allogeneic hematopoietic stem cell transplantation and might be beneficial if certain genes are affected.
High
expression levels at acute myeloid leukemia diagnosis have been linked to adverse outcomes. Recent data indicate that high
expression levels may also be used as marker for residual disease ...following acute myeloid leukemia treatment. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a curative treatment for acute myeloid leukemia patients. However, disease recurrence remains a major clinical challenge and identification of high-risk patients prior to HSCT is crucial to improve outcomes. We performed absolute quantification of
copy numbers in peripheral blood prior (median 7 days) to HSCT in complete remission (CR) or CR with incomplete peripheral recovery in 82 acute myeloid leukemia patients using digital droplet PCR (ddPCR) technology. An optimal cut-off of 0.14
/
copy numbers was determined and applied to define patients with high or low
/
copy numbers. High pre-HSCT
/
copy numbers significantly associated with higher cumulative incidence of relapse and shorter overall survival in univariable and multivariable models. Patients with high pre-HSCT
/
copy numbers were more likely to experience relapse within 100 days after HSCT. Evaluation of pre-HSCT
/
copy numbers in peripheral blood by ddPCR represents a feasible and rapid way to identify acute myeloid leukemia patients at high risk of early relapse after HSCT. The prognostic impact was also observed independently of other known clinical, genetic, and molecular prognosticators. In the future, prospective studies should evaluate whether acute myeloid leukemia patients with high pre-HSCT
/
copy numbers benefit from additional treatment before or early intervention after HSCT.
Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) is defined as presence of hematologic malignancy-associated mutations, e.g. in the genes DNMT3A, TET2, ASXL1, but absence of ...hematologic neoplasms & is a frequent phenomenon in healthy individuals with increasing age. Some individuals with CHIP eventually develop acute myeloid leukemia (AML) & persistent clonal hematopoiesis-associated mutations (CH-mutations) in complete remission (CR) may give rise to relapse. To date in AML little is known about the biological & clinical implications of the presence of CH-mutations in CR, especially in the context of allogeneic hematopoietic stem cell transplantation (HSCT).
Patients & Methods: We analyzed peripheral blood samples of 113 AML patients (median age 63.6 years, range 31.9-75.8 years) in CR (CR1 61.9%, CR2 14.2%) or CR with incomplete peripheral recovery (CRi; 23.9%) for the presence of CH-mutations by targeted amplicon sequencing (mean amplicon coverage per sample 7205x) prior to HSCT. Genes evaluated for somatic CH-mutations based on database entries (dbSNP & COSMIC) & with a variant allele frequency (VAF) of ≥3% were DNMT3A, TET2, ASXL1, SRSF2, SF3B1, IDH1, IDH2, JAK2, PPM1D, &IKZF1 . Codon 646 ASXL1 mutations were validated using proof-reading polymerase Sanger sequencing. For 76 patients (67.3%) diagnostic bone marrow samples were available for applying a targeted amplicon panel comprising 54 recurrently mutated genes in myeloid malignancies (mean amplicon coverage per sample 8624x). At diagnosis, mutations in the genes NPM1, CEBPA, presence of FLT3 -ITD, & cytogenetics were determined using standard techniques. All patients received HSCT after non-myeloablative conditioning at our institution between 2001 & 2015. Samples were collected within 30 days before HSCT. Median follow-up for patients alive was 4.4 years after HSCT.
Results: We identified 70 CH-mutations present in CR/CRi in 48 AML patients (42.5%) with a mean VAF of 19.1% (58.6% of mutations with VAF>10%). The genes DNMT3A (31.4%), TET2 (28.6%) &ASXL1 (14.3%) were found mutated most frequently. We observed no associations between present CH-mutations in CR/CRi & clinical characteristics, except that patients with ≥1 CH-mutation were more often transplanted in CR2 compared to patients without CH-mutation by trend (20.8% vs 9.2%, P=0.10). The presence of ≥1 CH-mutation in CR/CRi did not impact leukemia-free survival (LFS; P=0.95, Figure 1A) or OS (P=0.37, Figure 1B), whereas patients with ≥2 CH-mutations had longer LFS (P=0.02, Figure 1C) & OS (P=0.007, Figure 1D) compared to patients with no or 1 CH-mutation. In CR/CRi DNMT3A mutations did not impact LFS (P=0.73) or OS (P=0.71), the presence of TET2 mutations did not influence LFS (P=0.25) but associated with longer OS (P=0.06) &ASXL1 mutations associated with longer LFS (P=0.11) & OS (P=0.13) by trend. At diagnosis, we identified 83 CH-mutations in 56/76 AML patients (73.7%) of which 29 (34.9%) persisted in 19/76 patients (25.0%) in CR/CRi. Mutations in the genes JAK2 (80.0%), SRSF2 (57.1%) &DNMT3A (50.0%) were most likely to persist. We identified 9 CH-mutations (3 DNMT3A, 3 TET2, 2 ASXL1, 1 SF3B1 mutation) in CR/CRi in 8 patients (10.5%), which were not detectable at diagnosis, possibly following the expansion of subclones under chemotherapy selection pressure. Whether patients had persistent, newly detected or lost CH-mutation in CR/CRi did not impact LFS (P=0.22) or OS (P=0.83).
Conclusion: In AML patients in CR/CRi CH-mutations are frequently present (42.5%) & show high VAFs (mean 19.1%) indicating presence of clonal hematopoiesis. Compared to diagnosis 34.9% of CH-mutations persisted in CR/CRi. 10.5% of patients had CH-mutations only detectable in CR/CRi prior to HSCT. While we did not observe a negative prognostic impact of presence of ≥1 CH-mutation in CR/CRi, the presence of distinct mutations (TET2, ASXL1) may positively influence outcome. Patients with ≥2 CH-mutations had longer LFS & OS. This finding may be explained by an increased immunogenic potential with higher number of CH-mutations leading to potent graft vs leukemia effects in the HSCT context.
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Lange:Novartis: Honoraria, Research Funding. Franke:Takeda: Consultancy; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Schwind:Novartis: Consultancy.
Introduction: Prognosis remains poor for most acute myeloid leukemia (AML) patients (pts). Enhanced risk-stratification, novel & individualized therapies to improve outcomes are needed. The RAS ...-pathway (NRAS, KRAS & HRAS& among others the regulator SHP-2 encoded by PTPN11) controls cell proliferation, cell differentiation & apoptosis & mutations in this pathway are common in many human malignancies, including AML. Previous studies of the prognostic impact of RAS mutations (mut) in AML demonstrated variable & context specific results. The prognostic impact in pts receiving allogeneic stem cell transplantation (HSCT) remains to be elucidated.
Patients & Methods: We analyzed the biologic & prognostic impact of RAS pathway mut(i.e. NRAS, KRAS, HRAS &PTPN11) in 113 AML pts (median age at HSCT 64 years y; range 32-75y) who received a non-myeloablative HSCT (Fludarabin 3x30mg/m^2 & 2Gy total body irradiation) at our institution. At diagnosis, cytogenetics & the mut status of CEBPA, NPM1 & presence of FLT3- ITD were assessed. We analyzed RAS pathway mut & the mut status of other AML-associated genes such as TET2, IDH1 &IDH2 on diagnostic bone marrow (BM) samples by targeted resequencing on a panel comprising 54 recurrently mutated (mut) genes in myeloid malignancies. For 104 pts receiving HSCT in complete remission (CR) or CR with incomplete recovery (CRi) outcome analysis was performed. Median follow up after HSCT was 6.0y.
Results: In the whole cohort we found 23 AML pts (20%) with RAS pathway mut. Nineteen pts had NRAS, 6 pts PTPN11, 3 pts KRAS mut. None of the pts had a HRAS mut. Sixteen of these 23 AML pts had one RAS pathway mut (NRAS mut n=13 &PTPN11 mut n=3), 6 had two RAS pathway mut (NRAS double mut n=2, NRAS &KRAS mut n=1, NRAS &PTPN11 mut n=2, KRAS &PTPN11 mut n=1) & one pts had three RAS pathway mut (KRAS & double NRAS mut). 59% of NRAS mut (13/22) & 50% of PTPN11 mut (3/6) were sole RAS pathway mut. All 3 KRAS mutwere subclonal and accompanied other RAS pathway mut. RAS pathway mutpts were more likely to have de novo AML (p=.05) & were more frequently CEBPA mutated (p=.03). RAS pathway mut were mutually exclusive with TET2 mut (n=23, 27% in RAS pathway wild type pts vs . n=0, 0% in RAS pathway mut pts, p=.01). In the whole cohort no significant differences in leukemia-free (LFS) or overall survival (OS) for pts with or without RAS pathway mut (Figure 1A & B) was observed. However, when we restricted our analysis to IDH1 (n=15)&IDH2 (n=18) mut AML pts we observed a significant shorter LFS (p=.01) & OS (p=.007 , Figure 1C & 1D, respectively) for pts with RAS pathway mut, which may indicate an interaction of IDH1 / IDH2 &RAS pathway mut. RAS pathway mut were equally distributed between IDH mut &IDH wildtype pts (p=.44). Fifteen IDH1 mut pts harbored 5 RAS pathway mut (NRAS mut n=1, NRAS double mut n=1, NRAS &KRAS mutn=1) & 18 IDH2 mut patients harbored 5 RAS pathway mut (i.e. NRAS mut n=4 &PTPN11 mut n=1). All RAS pathway mut were subclonal to the concomitant IDH mut. These findings suggest that RAS pathway mut may succeed the IDH mut, rendering the AML phenotype more aggressive by a yet to be identified mechanistical cooperation.
Conclusion: Our data revealed novel biological & clinical aspects of RAS pathway mut in AML. In our cohort, RAS pathway mut were mutually exclusive with TET2 mutations. In AML pts receiving HSCT no prognostic impact of RAS pathway mut was observed in the whole cohort. However, in pts with IDH1 or IDH2 mut subclonal RAS pathway mut associated with worse outcome in HSCT treated AML pts indicating a biological cooperation between theses genetic alterations. Especially, pts harboring these mut combinations may benefit from treatment with IDH1 & IDH2 and /or RAS pathway inhibitors.
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Franke:Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Takeda: Consultancy. Schwind:Novartis: Consultancy.
Introduction: MicroRNA miR-1301 is embedded in intron 1 in the DNMT3A gene, which encodes the DNA (cytosine-5)-methyltransferase 3A, is frequently mutated in older acute myeloid leukemia (AML) ...patients (pts) & has been associated with clonal hematopoiesis. In glioma cells miR-1301 suppresses tumor cell invasion by regulating the activity & function of the p53 pathway & inhibits cell proliferation by targeting NRAS . The role of miR-1301 in AML remains to be elucidated.
Here, we investigated the clinical & prognostic impact of pri- miR-1301 in a homogenously treated set of mainly older AML pts who received allogeneic stem cell transplantation (HSCT) as consolidation.
Patients & Methods: We analyzed 126 AML pts (median age at HSCT 64 years y; range 38-75 y) with available pretreatment bone marrow (BM). All received HSCT after non-myeloablative (NMA) conditioning (Fludarabine 30mg/m2 at day -4 to -2 & 2Gy total body irradiation at day 0) at our institution between January 2000 & June 2012 in complete remission (CR, 83%, n=104) or CR with incomplete peripheral recovery (Cri, 17%, n=22). Cytogenetics were determined at diagnosis using standard techniques for banding & fluorescence in-situ hybridization. Using flow cytometry, BM mononuclear cells were assessed for presence of CD2, CD7, CD11b, CD13, CD14, CD15, CD33, CD34, CD38, CD45, CD56, CD61, CD64, CD65, CD117 & Glycophorin A. For pts with material available, the presence of FLT3 -ITD, FLT3 -TKD & expression status of EVI1, as well as the mutation status of the CEBPA, DNMT3A, IDH1, IDH2 &NPM1 genes were determined. Pri- miR-1301 expression was measured using a SYBR-Green assay based quantitative reverse transcription polymerase chain reaction & normalized to 18S expression as internal control. The third quartile of normalized gene expression was used to define high & low pri- miR-1301 expressers. Median follow-up after HSCT was 6.0 y for pts alive.
Results: At diagnosis, high pri- miR-1301 expressers had lower white blood cell counts (P=.02). High pri- miR-1301 expression associated with higher percentage of CD7 (P=.04) & by trend of CD2 (P=.10) positive cells (both T-cell associated antigens) & lower percentage of CD15 (P=.01), CD33 (P=.002) & CD64 (P=.05) positive cells (myeloid antigens). Furthermore, high pri- miR-1301 expressers were less likely to be NPM1 mutated (P=.06) by trend. Since miR-1301 is embedded in the DNMT3A gene, we tested whether pri- miR-1301 expression associated with the presence of DNMT3A mutations, but did not observe any association (pts with DNMT3A mutations: high pri- miR-1301 expression: 18% vs . low pri- miR-1301 expression: 14%, P=.73). Pts with high pri- miR-1301 expression had longer leukemia-free survival (LFS, P=.07; Figure 1A) by trend & significantly longer overall survival (OS, P=.03; Figure 1B). In multivariate analysis, high pri- miR-1301 expression retained its prognostic impact on LFS (Hazard Ratio HR 0.51, Confidence Interval CI 0.28-0.93, P=.03) after adjustment for age at HSCT, hemoglobin level at diagnosis &EVI1 expression status at diagnosis & was the only significant factor for OS (HR 0.52, CI 0.29-0.95, P=.03).
Conclusion: High pri- miR-1301 expression associated with distinct clinical & prognostic features, including higher expression of the T-cell associated antigens CD2 & CD7, lower expression of myeloid antigens CD15, CD33 & CD64 & a lower incidence of NPM1 mutations by trend. Whereas DNMT3A embedded miR-1301, no association of pri- miR-1301 expression & occurrence of DNMT3A mutation was found. Importantly, high pri- miR-1301 expressers had by trend a longer LFS & significantly longer OS independent of other prognostic markers. Further studies are warranted to elucidate the AML biology associated with high expression of the DNMT3A embedded miR-1301 in AML.
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Franke:Novartis: Honoraria, Research Funding; Takeda: Consultancy; Pfizer: Honoraria. Schwind:Novartis: Consultancy.
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Background: Surface antigen expression evaluation is part of the standard work-up at acute myeloid leukemia (AML) diagnosis. The biological & prognostic implications of surface ...antigen expression patterns in normal karyotype (NK) AML patients (pts) remain unknown. Methods: The diagnostic antigen expression patterns of mononuclear cells in bone marrow (BM) of 111 NK-AML pts were assessed using a standard flow cytometric panel. At diagnosis common AML gene mutations (mut) & expression levels were analyzed. Pts received stem cell transplantation (SCT, 98% allogeneic, 2% autologous; median age 63 years y, range 26-74y) after induction therapy at our institution. Median follow up was 3.3y. With R’s gplot package unsupervised hierarchical clustering of surface antigens was performed & revealed 4 distinct clusters. Results: Pts in cluster 1 (n = 36) had higher expression of immature, in cluster 2 (n = 31) of thrombocytic/T-cell/erythroid, in cluster 3 (n = 24) of monocytic & in cluster 4 (n = 20) of myeloid surface antigens. All 4 clusters associated with distinct clinical & molecular features. At diagnosis, compared to all others, pts in cluster 1 had a higher CD34+/CD38- cell burden ( P< .001), higher blood blasts ( P< .03) & BM blasts ( P< .06) by trend. They had less NPM1 mut ( P< .001) & DNMT3A mut ( P= .02), were more likely to be EVI1 positive ( P= .03) & had higher EZH2 ( P= .02), RUNX1 ( P= .009), BAALC ( P< .001), ERG ( P= .02) & MN1 ( P< .001) expression. Compared to all others, pts in cluster 1 had a higher cumulative incidence of relapse (CIR, P= .002, at 1y 41% vs 15%) & shorter event-free survival (EFS, P= .02, at 1y 50% vs 69%). In multivariate analysis, cluster 1 pts had a significantly higher CIR (Hazard Ratio HR 5.4, P= .01) after adjustment for FLT3-ITD & shorter EFS (HR 2.1, P= .02) after adjustment for FLT3-ITD, age & disease status at SCT. Conclusions: Pts in cluster 1 had high expression of immature surface antigens (eg CD34, CD117, CD13), genes involved in stem cell renewal & worse outcome. Our data indicate a relationship between easily accessible surface antigen expression patterns at diagnosis, molecular disease features & aggressiveness of the NK-AML phenotype.
In recent years expression levels of several genes & microRNAs (miR) were identified as strong prognostic markers, capable to refine AML risk stratification. So far technical difficulties, including ...the limitations of established methods for comparable, absolute quantification & the lack of defined cut points prevented translation of these findings into clinical practice. Innovative digital droplet PCR (ddPCR) is a novel technique with high sensitivity, specificity that allows absolute quantification - without the need for standard curves - & promises better inter-laboratory comparability.
In AML pts, high miR-155 expression levels associate with the presence of FLT3-ITD & independently predict inferior outcome. Here, for the first time we applied ddPCR for absolute quantification of pre-miR-155 to define an absolute cut point to discriminate between high & lowexpressers, which was then validated in a second set of AML pts.
We analyzed a homogeneous test set of 71 AML pts treated between January 2000 & June 2012 in our institution. All pts received cytarabinebased induction therapies & were consolidated with allogeneic hematopoietic stem cell transplantation (HCT) after non-myeloablativeconditioning (NMA; consisting of fludarabine30mg / m² at days -4 to -2 & 2Gy total body irradiation day 0). At NMA-HCT ptswere in first (n=43; 60.6%) or second complete remission (CR; n=16; 22.5%) or CR with incomplete recovery (n=12; 16.9%). At diagnosis, cytogenetics & mutation status of the NPM1, CEBPA, IDH1, IDH2 &DNMT3A gene & presence of FLT3-ITD or FLT3-TKD mutation were assessed. The expression of the pre-miR-155 stem loop was measured using an EvaGreen-based ddPCR assay & normalized to the absolute copy numbers of ABL1.
The R Package OptimalCutpointswas used to determine a cut point of 1.104 copies pre-miR-155 per 100 ABL1 copies to discriminate between high (n =29; 40.8%) & low (n =42; 59.2 %) miR-155 expressers. High miR-155 expressers, more often had a FLT3-ITD (p=.039) & less frequently a mutation in the FLT3-TKD (p=.010). No significant association was found for other clinical or biological markers at diagnosis.
In the test set, pts with more than 1.104 copies pre-miR-155 per 100 ABL1 copies at diagnosis had a significant shorter event-free survival (EFS; p=.03, figure 1A) & overall survival (OS; p=.009, figure 1B).
To validate these findings, we used a second set of 71 pts (median age 63.4y range 37.1 to 74.7) with a median follow-up of 3.7y for pts alive that all received cytarabinebased induction therapies & NMA-HCT as consolidation. The ptsin the validation set also did not differ significantly in the analyzed clinical or molecular characteristics (i.e. white blood count, hemoglobin, platelets, blasts in bone marrow or peripheral blood at diagnosis, remission status at HCT CR1 vs. CR2 vs. CRi, ELN genetic group, mutational status of FLT3-TKD, NPM1, CEBPA, DNMT3A, IDH1 or IDH2 & presence of FLT3-ITD).
Using the determined cut point of 1.104 copies pre-miR-155 / 100 ABL1 copies in the test set, patients in the validation set were divided in 39 patients (54.9%) with a high miR-155 expression & 32 (45.1%) with a low miR-155 expression. Pts with high miR-155 expression in the validation set had shorter EFS (p=.11, figure 2A) by trend & a significant shorter OS (p=.05, figure 2B).
In conclusion, ddPCRis a novel, feasible method that allows absolute quantification of miRexpression. We defined an absolute cut point of 1.104 copies pre-miR-155 per 100 ABL1 copies for the prognosticator miR-155 in AML without the need for standard curves. Pts with pre-miR-155 expression above the cut point had a significant shorter EFS & OS. Remarkably, using a second clinically comparable set, we were able to validate our test set findings.
Future studies are planned to confirm the clinical impact of pre-miR-155 expression levels at diagnosis, as well as the identified absolute pre-miR-155 / ABL1 copy number cut point to distinguish high from low miR-155 expressers.
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Poenisch:Mundipharma: Research Funding.
Introduction: The transcription factor ZBTB7A regulates early differentiation of hematopoietic progenitors & has been associated with oncogenic as well as oncosuppressive functions. While it was ...shown that ectopic overexpression of Zbtb7a in immature lymphocytes leads to the development of an aggressive T-cell lymphoblastic leukemia, high ZBTB7A expression in cytogenetically normal acute myeloid leukemia (CN-AML) is associated with improved outcomes. Furthermore, recently a leukemogenic cooperation between RUNX1/RUNX1T1 &ZBTB7A mutations in t(8;21)-associated AML was suggested.
Here, we further evaluated the complex role of ZBTB7A expression in hematopoietic malignancies by assessing its potential prognostic impact in AML pts undergoing hematopoietic stem cell transplantation (HSCT) after non-myeloablative conditioning (NMA).
Methods: We analyzed bone marrow (BM) at diagnosis of 140 pts (median age 63 years y, range 37-75y) treated at our institution between 2000 & 2015. All pts received NMA conditioning (3x30mg/m2 Fludarabine on days -4 to -1 & 2Gy total body irradiation) followed by HSCT in complete remission with (CR; n=111; 79.3%) or without peripheral hematological recovery (CRi; n=29; 20.7%). Median follow-up for pts alive was 3.5y.
Our cohort included pts with CN-AML (n=62, 44.3%), complex karyotype (n=17; P=12.1%) & other cytogenetic abnormalities (n=61; 43.6%). At diagnosis mutations in the genes CEBPA, DNMT3A, IDH1, IDH2, NPM1 & the presence of FLT3-ITD were determined. In diagnostic BM cytogenetics were analyzed using standard techniques for banding & fluorescence in-situ hybridization & the expression of common surface markers was analyzed using flow cytometry. The expression of ZBTB7A was assessed using quantitative RT-PCR & normalized to ABL1 as internal control. As a cut-off the third quartile of normalized gene expression was identified to group high & low ZBTB7A expressers.
Results: At diagnosis pts with a high ZBTB7A expression more often had a complex karyotype (P=.02) & were less likely to have core-binding factor AML by trend (P=.18). Additionally, high ZBTB7A levels associated with significantly fewer blasts in peripheral blood (P=.008) & BM (P=.02). The BM mononuclear cells in high ZBTB7A expressers were to a smaller extent positive for myeloid markers (CD38 P=.03; CD33 P=.11; CD13 P=.13) & exhibited a higher percentage of erythroid (Glycophorin A P=.03) as well as monocytic (CD11b P=.04; CD14 P=.01) surface markers. We did not find any statistical associations between ZBTB7A levels & the mutation status of NPM1, CEBPA, IDH1, DNMT3A or the presence of FLT3-ITD. Yet, there was a trend for more IDH2 mutations in the group of high ZBTB7A expressers (P=.18).
At diagnosis a high expression of ZBTB7A associated with a significantly higher cumulative incidence of relapse (CIR; P=.002, Figure 1A). This finding also translated into a significantly shorter overall survival (OS; P=.01; Figure 1B) for AML pts with high ZBTB7A levels at diagnosis. When we restricted our analyses to CN-AML, high ZBTB7A expression remained a negative prognostic factor by trend (CIR P=.16; OS P=.11).
Conclusion: Expression of ZBTB7A associated with distinct biological features & surface marker pattern in AML. This underlines the results of recent studies which identified ZBTB7A as a novel player in leukemogenesis. However, our findings are in contrast with the previously shown favorable prognostic impact of high ZBTB7A levels in a CN-AML cohort mainly treated with chemotherapy. In contract all pts included in our studies were consolidated with NMA-HSCT. Since this treatment regimen is mainly based on the graft versus leukemia effect a high ZBTB7A expression could potentially interfere with the immunological recognition of the AML blasts resulting in a reduced response to NMA-HSCT.
Consequently, future functional studies & clinical trials should aim at further characterize the complex role of ZBTB7A in AML.
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Poenisch:Mundipharma: Research Funding.
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