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.
.
Measurable residual disease (MRD; previously termed minimal residual disease) is an independent, postdiagnosis, prognostic indicator in acute myeloid leukemia (AML) that is important for risk ...stratification and treatment planning, in conjunction with other well-established clinical, cytogenetic, and molecular data assessed at diagnosis. MRD can be evaluated using a variety of multiparameter flow cytometry and molecular protocols, but, to date, these approaches have not been qualitatively or quantitatively standardized, making their use in clinical practice challenging. The objective of this work was to identify key clinical and scientific issues in the measurement and application of MRD in AML, to achieve consensus on these issues, and to provide guidelines for the current and future use of MRD in clinical practice. The work was accomplished over 2 years, during 4 meetings by a specially designated MRD Working Party of the European LeukemiaNet. The group included 24 faculty with expertise in AML hematopathology, molecular diagnostics, clinical trials, and clinical medicine, from 19 institutions in Europe and the United States.
Risk stratification in acute myeloid leukemia (AML) is currently based on pretreatment characteristics. It remains to be established whether relapse risk can be better predicted through assessment of ...minimal residual disease (MRD). One proposed marker is the Wilms tumor gene WT1, which is overexpressed in most patients with AML, thus providing a putative target for immunotherapy, although in the absence of a standardized assay, its utility for MRD monitoring remains controversial.
Nine published and in-house real-time quantitative polymerase chain reaction WT1 assays were systematically evaluated within the European LeukemiaNet; the best-performing assay was applied to diagnostic AML samples (n = 620), follow-up samples from 129 patients treated with intensive combination chemotherapy, and 204 normal peripheral blood (PB) and bone marrow (BM) controls.
Considering relative levels of expression detected in normal PB and BM, WT1 was sufficiently overexpressed to discriminate > or = 2-log reduction in transcripts in 46% and 13% of AML patients, according to the respective follow-up sample source. In this informative group, greater WT1 transcript reduction after induction predicted reduced relapse risk (hazard ratio, 0.54 per log reduction; 95% CI, 0.36 to 0.83; P = .004) that remained significant when adjusted for age, WBC count, and cytogenetics. Failure to reduce WT1 transcripts below the threshold limits defined in normal controls by the end of consolidation also predicted increased relapse risk (P = .004).
Application of a standardized WT1 assay provides independent prognostic information in AML, lending support to incorporation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification, and to identify patients who may benefit from allogeneic transplantation.
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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Activation of the NF‐κB pathway requires the formation of Met1‐linked ‘linear’ ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of ...HOIP, HOIL‐1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING‐IBR‐RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two‐step mechanism involving both RING and HECT E3‐type activities. RING1‐IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT‐like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N‐terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C‐terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain’ (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2‐LDD region was found to be important for NF‐κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD‐dependent specificity for producing linear ubiquitin chains.
Like Parkin, the linear ubiquitin chain assembly complex LUBAC functions as a RING/HECT‐hybrid ubiquitin ligase, but includes a unique extension that dictates linear ubiquitin linkage specificity.
Abstract
Objectives
Acquired missense mutations in the
BCR::ABL1
kinase domain (KD) may cause tyrosine kinase inhibitor (TKI) treatment failure. Based on mutation‐specific in vitro derived ...IC50‐values, alternative TKI may be selected. We assessed clinical practice of
BCR::ABL1
KD mutation testing, clinical response in relation to IC50‐values, and clinical outcome of tested patients.
Methods
Patients from six Dutch CML reference centers and a national registry were included once a mutational analysis was performed. Reasons for testing were categorized as suboptimal TKI response, and primary or secondary TKI resistance.
Results
Four hundred twenty analyses were performed in 275 patients. Sixty‐nine patients harbored at least one mutation. Most analyses were performed because of suboptimal TKI response but with low mutation incidence (4%), while most mutations were found in primary and secondary resistant patients (21% and 51%, respectively). Harboring a
BCR::ABL1
mutation was associated with inferior overall survival (HR 3.2 95% CI, 1.7–6.1;
p
< .001). Clinically observed responses to TKI usually corresponded with the predicted TKI sensitivity based on the IC50‐values, but a high IC50‐value did not preclude a good clinical response per se.
Conclusions
We recommend
BCR::ABL1
KD mutation testing in particular in the context of primary or secondary resistance. IC50‐values can direct the TKI choice for CML patients, but clinical efficacy can be seen despite adverse in vitro resistance.
In myelodysplastic syndromes (MDS), deletions of chromosome 7 or 7q are common and correlate with a poor prognosis. The relevant genes on chromosome 7 are unknown. We report here that EZH2, located ...at 7q36.1, is frequently targeted in MDS. Analysis of EZH2 deletions, missense and frameshift mutations strongly suggests that EZH2 is a tumor suppressor. As EZH2 functions as a histone methyltransferase, abnormal histone modification may contribute to epigenetic deregulation in MDS.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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.
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.
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