Therapy-related myeloid neoplasms (tMNs) are severe adverse events that can occur after treatment with autologous hematopoietic stem cell transplantation (ASCT). This study aimed to investigate the ...development of tMNs following ASCT at the molecular level by whole-exome sequencing (WES) and targeted deep sequencing (TDS) in sequential (pre-) tMN samples. WES identified a significantly higher number of mutations in tMNs as compared with de novo myelodysplastic syndrome (MDS) (median 27 vs 12 mutations; P = .001). The mutations found in tMNs did not carry a clear aging-signature, unlike the mutations found in de novo MDS, indicating a different mutational mechanism. In some patients, tMN mutations were identified in both myeloid and T cells, suggesting that tMNs may originate from early hematopoietic stem cells (HSCs). However, the mutational spectra of tMNs and the preceding malignancies did not overlap, excluding common ancestry for these malignancies. By use of TDS, tMN mutations were identified at low variant allele frequencies (VAFs) in transplant material in 70% of the patients with tMNs. Reconstruction of clonal patterns based on VAFs revealed that premalignant clones can be present more than 7 years preceding a tMN diagnosis, a finding that was confirmed by immunohistochemistry on bone marrow biopsies. Our results indicate that tMN development after ASCT originates in HSCs bearing (pre-)tMN mutations that are present years before disease onset and that post-ASCT treatment can influence the selection of these clones. Early detection of premalignant clones and monitoring of their evolutionary trajectory may help to predict the development of tMNs and guide early intervention in the future.
•tMNs after ASCT originate from HSCs bearing (pre-)tMN mutations that are present years before disease onset.•Post-ASCT treatment can influence selection and outgrowth of (pre)leukemic clones.
Patients with acute myeloid leukemia (AML) frequently harbor mutations in genes involved in the DNA (hydroxy)methylation pathway (DNMT3A, TET2, IDH1, and IDH2). In this study, we measured ...5-hydroxymethylcytosine (5hmC) levels in 206 clinically and molecularly well-characterized younger adult AML patients (≤60 years) included in the European Organization for Research and Treatment of Cancer/Gruppo Italiano Malattie Ematologiche dell'Adulto (EORTC/GIMEMA) AML-12 06991 clinical trial and correlated the 5hmC levels with mutational status and overall survival (OS). In healthy control cells, 5hmC levels were confined to a narrow range (1.5-fold difference), whereas in AML cells, a much wider range was detected (15-fold difference). We identified 3 5hmC subpopulations in our patient cohort (low, intermediate, and high). The low 5hmC group consisted almost entirely of patients with TET2 or IDH mutations. As expected, TET2 and IDH mutated patients had significantly lower levels of 5hmC compared with patients without mutated TET2 and IDH1/2 (both P < .001). Interestingly, high 5hmC levels correlated with inferior OS (high vs intermediate 5hmC: P = .047, hazard ratio HR = 1.81). Multivariate analysis revealed that high 5hmC is an independent poor prognostic indicator for OS (high vs intermediate 5hmC: P = .01, HR = 2.10). This trial was registered at www.clinicaltrials.gov as NCT00004128.
•5hmC levels vary considerably in patients with AML.•High levels of 5hmC independently correlate with inferior overall survival in AML.
Monocytosis may occur in numerous inflammatory conditions but is also the defining feature of chronic myelomonocytic leukemia (CMML). Clonal somatic mutations detectable in CMML may occur with ageing ...in otherwise healthy individuals, so-called “clonal hematopoiesis” (CH). We investigated whether the combination of CH and monocytosis would represent an early developmental stage of CMML. We studied community-dwelling individuals with monocytosis (≥ 1 x 109/L and ≥10% of leukocytes) in the population-based Lifelines cohort (n=144676 adults). The prevalence and spectrum of CH were evaluated for individuals ≥60 years with monocytosis (n=167, 0.8%) and controls 1:3 matched for age and sex (n=501). Diagnoses of hematological malignancies were retrieved by linkage to the Netherlands Cancer Registry. Monocyte counts and the prevalence of monocytosis increased with advancing age. Older individuals with monocytosis more frequently carried CH (50.9% versus 35.5%, P<0.001). Monocytosis associated with enrichment of multiple gene mutations (P=0.006) and spliceosome mutations (P=0.007) but not isolated mutated DNMT3A, TET2 or ASXL1. Persistent monocytosis over four years was observed in 30/102 evaluable individuals and associated with higher prevalence of CH (63%). Myeloid malignancies, including one case of CMML, developed in four individuals with monocytosis that all carried CH.In conclusion, monocytosis and CH both occur at older age and do not necessarily reflect clonal monocytic proliferation. In a fraction of older subjects with monocytosis, CH might constitute early clonal dominance in developing malignant myelomonocytic disease. Mutational spectra deviating from age-related CH require attention.
Ring sideroblasts (RS) emerge as result of aberrant erythroid differentiation leading to excessive mitochondrial iron accumulation, a characteristic feature for myelodysplastic syndromes (MDS) with ...mutations in the spliceosome gene SF3B1. However, RS can also be observed in patients diagnosed with acute myeloid leukemia (AML). The objective of this study was to characterize RS in patients with AML. Clinically, RS-AML is enriched for ELN adverse risk (55%). In line with this finding, 35% of all cases had complex cytogenetic aberrancies, and TP53 was most recurrently mutated in this cohort (37%), followed by DNMT3A (26%), RUNX1 (25%), TET2 (20%), and ASXL1 (19%). In contrast to RS-MDS, the incidence of SF3B1 mutations was low (8%). Whole-exome sequencing and SNP array analysis on a subset of patients did not uncover a single genetic defect underlying the RS phenotype. Shared genetic defects between erythroblasts and total mononuclear cell fraction indicate common ancestry for the erythroid lineage and the myeloid blast cells in patients with RS-AML. RNA sequencing analysis on CD34+ AML cells revealed differential gene expression between RS-AML and non RS-AML cases, including genes involved in megakaryocyte and erythroid differentiation. Furthermore, several heme metabolism-related genes were found to be upregulated in RS- CD34+ AML cells, as was observed in SF3B1mut MDS. These results demonstrate that although the genetic background of RS-AML differs from that of RS-MDS, they have certain downstream effector pathways in common.
•Ring sideroblasts in AML are associated with complex karyotypes and TP53 mutations.•Gene expression studies in CD34+ AML cells suggest an altered erythroid differentiation program in AML with ring sideroblasts.
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We assessed the prognostic impact of
TET2
mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and ...Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell’ Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of
TET2
mutations were investigated.
TET2
mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (
p
= 0.024).
TET2
and
IDH1
/
2
mutations strongly associated with aberrations in the DNA methyltransferase
DNMT3A
. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant
TET2
forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function
TET2
mutations predicted poor outcome, we questioned whether low
TET2
mRNA expression in cases of AML without
TET2
mutations would affect overall survival. Notably, also AML patients with low
TET2
mRNA expression levels showed inferior overall survival.
Patients with acute myeloid leukemia (AML) frequently harbor mutations in genes involved in the DNA (hydroxy)methylation pathway (DNMT3A, TET2, IDH1, and IDH2). In addition, changes in DNA ...methylation have been implicated in the pathogenesis of AML. Recently it was discovered that TET proteins are able to convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), which is an important intermediate in the demethylation pathway. In this study, we measured 5-hydroxymethylcytosine levels in AML patients, and correlated these with mutational status and overall survival (OS).
Samples from 206 clinically and molecularly well-characterized younger adult AML patients (≤60 years), included in the EORTC/GIMEMA AML-12 06991 clinical trial, were analyzed for mutations in DNMT3A, TET2, IDH1 and IDH2. 5-hydroxymethylcytosine levels were measured using HPLC-MS/MS.
In healthy control cells, 5hmC levels were confined to a narrow range (1.5 fold difference), whereas in AML cells, a much wider range was detected (15 fold difference). In remission, 5hmC values were normalized to levels comparable to healthy bone marrow and peripheral blood, indicating that the aberrant 5hmC levels at diagnosis are intrinsic to the leukemic cells. Patients with mutations in TET2 and patients with mutations in IDH1/2 had significantly lower levels of 5hmC compared to patients without mutated TET2 and IDH1/2 (both P<.001), whereas mutations in DNMT3A did not influence 5hmC levels. Patients with bi-allelic TET2 inactivation displayed lower 5hmC levels than patients with one affected allele (P=.003). Among the patients that did not harbor TET2 or IDH mutations, still a wide variation in 5hmC levels was observed, and importantly, both low and very high 5hmC levels correlated with inferior OS (P=.02, HR=1.80 and P=.04, HR=1.97, respectively). Multivariate analysis revealed that abnormal levels of 5hmC were independent prognostic indicators for OS. The difference in OS could not be explained by an initial inferior response to therapy, however, the relapse rate was considerably higher in patients with low and very high 5hmC levels compared to patients with intermediate 5hmC.
Both low and very high levels of 5hmC are markers of poor prognosis in AML, lending further support for testing therapies targeting the DNA hydroxymethylation pathway.
No relevant conflicts of interest to declare.
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