Anemia is a major and currently poorly understood clinical manifestation of hematopoietic aging. Upon aging, hematopoietic clones harboring acquired leukemia-associated mutations expand and become ...detectable, now referred to as clonal hematopoiesis (CH). To investigate the relationship between CH and anemia of the elderly, we explored the landscape and dynamics of CH in older individuals with anemia. From the prospective, population-based Lifelines cohort (n = 167 729), we selected all individuals at least 60 years old who have anemia according to World Health Organization criteria (n = 676) and 1:1 matched control participants. Peripheral blood of 1298 individuals was analyzed for acquired mutations at a variant allele frequency (VAF) of 1% or higher in 27 driver genes. To track clonal evolution over time, we included all available follow-up samples (n = 943). CH was more frequently detected in individuals with anemia (46.6%) compared with control individuals (39.1%; P = .007). Although no differences were observed regarding commonly detected DTA mutations (DNMT3A, TET2, ASXL1) in individuals with anemia compared with control individuals, other mutations were enriched in the anemia cohort, including TP53 and SF3B1. Unlike individuals with nutrient deficiency (P = .84), individuals with anemia of chronic inflammation and unexplained anemia revealed a higher prevalence of CH (P = .035 and P = .017, respectively) compared with their matched control individuals. Follow-up analyses revealed that clones may expand and decline, generally showing only a subtle increase in VAF (mean, 0.56%) over the course of 44 months, irrespective of the presence of anemia. Specific mutations were associated with different growth rates and propensities to acquire an additional hit. In contrast to smaller clones (<5% VAF), which did not affect overall survival, larger clones were associated with increased risk for death.
•Specific mutations, including TP53 and SF3B1, but not DNMT3A, TET2, and ASXL1, were enriched in older individuals with anemia.•In general, clones only confer a limited selective advantage over time, which is differentially affected by specific driver genes.
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Cancer development is a dynamic process during which the successive accumulation of mutations results in cells with increasingly malignant characteristics. Here, we show the clonal evolution pattern ...in myelodysplastic syndrome (MDS) patients receiving supportive care, with or without lenalidomide (follow-up 2.5-11 years). Whole-exome and targeted deep sequencing at multiple time points during the disease course reveals that both linear and branched evolutionary patterns occur with and without disease-modifying treatment. The application of disease-modifying therapy may create an evolutionary bottleneck after which more complex MDS, but also unrelated clones of haematopoietic cells, may emerge. In addition, subclones that acquired an additional mutation associated with treatment resistance (TP53) or disease progression (NRAS, KRAS) may be detected months before clinical changes become apparent. Monitoring the genetic landscape during the disease may help to guide treatment decisions.
Valid variant calling results are crucial for the use of next-generation sequencing in clinical routine. However, there are numerous variant calling tools that usually differ in algorithms, filtering ...strategies, recommendations and thus, also in the output. We evaluated eight open-source tools regarding their ability to call single nucleotide variants and short indels with allelic frequencies as low as 1% in non-matched next-generation sequencing data: GATK HaplotypeCaller, Platypus, VarScan, LoFreq, FreeBayes, SNVer, SAMtools and VarDict. We analysed two real datasets from patients with myelodysplastic syndrome, covering 54 Illumina HiSeq samples and 111 Illumina NextSeq samples. Mutations were validated by re-sequencing on the same platform, on a different platform and expert based review. In addition we considered two simulated datasets with varying coverage and error profiles, covering 50 samples each. In all cases an identical target region consisting of 19 genes (42,322 bp) was analysed. Altogether, no tool succeeded in calling all mutations. High sensitivity was always accompanied by low precision. Influence of varying coverages- and background noise on variant calling was generally low. Taking everything into account, VarDict performed best. However, our results indicate that there is a need to improve reproducibility of the results in the context of multithreading.
Aim
We aimed to analyse the association of clonal haematopoiesis of indeterminate potential (CHIP) with incident heart failure (HF) in a European population cohort.
Methods and results
From the ...prospective Prevention of Renal and Vascular End‐stage Disease (PREVEND) cohort, we included all 374 participants with incident HF and selected 1:1 age‐ and sex‐matched control subjects. Peripheral blood samples of 705 individuals were successfully analysed by error‐corrected next generation sequencing for acquired mutations at a variant allele frequency ≥2% in 27 CHIP driver genes. The median age of the study population was 65 years (interquartile range 58–70) and 35.6% were female. CHIP mutations positively correlated with age, smoking, hypertension and cardiovascular biomarkers including N‐terminal pro‐B‐type natriuretic peptide and mid‐regional pro‐A‐type natriuretic peptide, but the frequency of CHIP was comparable in individuals with incident HF and in control participants (18.4% vs. 17.3%; p = 0.69). In multivariable Cox regression models, CHIP was not significantly associated with incident HF (hazard ratio HR 1.24, 95% confidence interval CI 0.93–1.65; p = 0.144). This association, however, was modified by age (p for CHIP–age interaction = 0.002). Among people younger than 65 years, CHIP mutations were more frequently detected in the case cohort compared to the control cohort (14.2% vs. 5.8%; p = 0.009), and were significantly associated with new‐onset HF (HR 2.07, 95% CI 1.30–3.29; p = 0.002).
Conclusion
Clonal haematopoiesis of indeterminate potential correlates with HF risk factors and biomarkers, and is associated with incident HF in subjects <65 years of age.
Association of clonal haematopoiesis of indeterminate potential (CHIP) with incident heart failure (HF).
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.
Knowledge about evolution of clonal hematopoiesis, which may drive malignant progression, is crucial for clinical decision-making. We investigated the landscape of clonal evolution by error-corrected ...sequencing on 7,045 sequential samples from 3,359 individuals in the prospective population-based Lifelines cohort, with a special focus on cytosis and cytopenia. Spliceosome (SRSF2/U2AF1/SF3B1) and JAK2 mutated clones show highest growth rates over a median 3.6-year period, while clone sizes for DNMT3A and TP53 increase only marginally, independent of cytosis or cytopenia. Nevertheless, large differences are observed between individuals carrying the same mutation, indicative of modulation by non-mutation-related factors. Clonal expansion is not dependent on classical cancer risk factors (e.g., smoking). Risk for incident myeloid malignancy diagnosis is highest for JAK2, spliceosome, or TP53 mutations and absent for DNMT3A, and it is mostly preceded by cytosis or cytopenia. The results provide important insight into high-risk evolutionary patterns to guide monitoring of “CHIP” and “CCUS.”
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•Spliceosome (SF3B1/SRSF2/U2AF1) and JAK2 mutated clones show highest growth rates•DNMT3A and TP53 mutated clones increase only marginally over time•Clonal expansion is independent of general cancer risk factors or cytopenia/cytosis•Spliceosome, TP53, JAK2, and K/NRAS mutations confer highest risk for myeloid cancer
Zeventer et al. report on longitudinal analyses of clonal hematopoiesis in 3,359 community-based individuals. Patterns of clonal evolution reveal large heterogeneity, irrespective of blood count abnormalities. Clonal expansion, patterns of sequential mutation acquisition, and the risk to develop myeloid malignancy are dependent on the specific mutated gene. The results warrant a gene-specific approach in the management of “CHIP” and “CCUS.”
There are various next-generation sequencing techniques, all of them striving to replace Sanger sequencing as the gold standard. However, false positive calls of single nucleotide variants and ...especially indels are a widely known problem of basically all sequencing platforms.
We considered three common next-generation sequencers-Roche 454, Ion Torrent PGM and Illumina NextSeq-and applied standard as well as optimized variant calling pipelines. Optimization was achieved by combining information of 23 diverse parameters characterizing the reported variants and generating individually calibrated generalized linear models. Models were calibrated using amplicon-based targeted sequencing data (19 genes, 28,775 bp) from seven to 12 myelodysplastic syndrome patients. Evaluation of the optimized pipelines and platforms was performed using sequencing data from three additional myelodysplastic syndrome patients.
Using standard analysis methods, true mutations were missed and the obtained results contained many artifacts-no matter which platform was considered. Analysis of the parameters characterizing the true and false positive calls revealed significant platform- and variant specific differences. Application of optimized variant calling pipelines considerably improved results. 76% of all false positive single nucleotide variants and 97% of all false positive indels could be filtered out. Positive predictive values could be increased by factors of 1.07 to 1.27 in case of single nucleotide variant calling and by factors of 3.33 to 53.87 in case of indel calling. Application of the optimized variant calling pipelines leads to comparable results for all next-generation sequencing platforms analyzed. However, regarding clinical diagnostics it needs to be considered that even the optimized results still contained false positive as well as false negative calls.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Splicing factor 3B subunit 1 (SF3B1) mutations define a distinct myelodysplastic syndromes (MDS) patient group with a relatively favourable disease course and high response rates to ...luspatercept. Few data are available on bone marrow phenotype beyond ring sideroblasts in this subgroup of patients with MDS. In the present study, we identified immunophenotypic erythroid, myelomonocyte and progenitor features associated with SF3B1 mutations. In addition, we illustrate that SF3B1‐mutation type is associated with distinct immunophenotypic features, and show the impact of co‐occurrence of a SF3B1 mutation and a deletion of chromosome 5q on bone marrow immunophenotype. These genotype–phenotype associations and phenotypic subtypes within SF3B1‐MDS provide leads that may further refine prognostication and therapeutic strategies for this particular MDS subgroup.
Older acute myeloid leukemia (AML) patients have a poor prognosis; therefore, novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical ...results. Here, we report the first-in-human study exploiting a unique scalable NK-cell product generated
from CD34
hematopoietic stem and progenitor cells (HSPC) from partially HLA-matched umbilical cord blood units.
Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30 × 10
/kg body weight) after lymphodepleting chemotherapy without cytokine boosting.
HSPC-NK cell products contained a median of 75% highly activated NK cells, with <1 × 10
T cells/kg and <3 × 10
B cells/kg body weight. HSPC-NK cells were well tolerated, and neither graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting being given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, which was accompanied by augmented IL15 plasma levels. Moreover, donor chimerism up to 3.5% was found in bone marrow. Interestingly,
HSPC-NK cell maturation was observed, indicated by the rapid acquisition of CD16 and KIR expression, while expression of most activating receptors was sustained. Notably, 2 of 4 patients with minimal residual disease (MRD) in bone marrow before infusion became MRD negative (<0.1%), which lasted for 6 months.
These findings indicate that HSPC-NK cell adoptive transfer is a promising, potential "off-the-shelf" translational immunotherapy approach in AML.
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