Multiple mechanisms have been advanced to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated suppression of CD4+ effector T cells (Teffs) but none appear to completely explain suppression. ...Previous data indicates that Tregs may affect the microenvironment redox state. Given the inherent redox sensitivity of T cells, we tested the hypothesis that oxidants may mediate the direct suppression of Teffs by Tregs.
Tregs and Teffs were isolated from the spleens of wild type (WT) C57BL/6 mice or Ncf1(p47phox)-deficient C57BL/6 mice which lack NADPH oxidase function. Teffs were labeled with CFSE and co-cultured with unlabeled Tregs at varying Treg:Teff ratios in the presence of anti-CD3/CD28 coated beads for 3 days in suppression assays. Treg-mediated suppression was quantified by flow cytometric analysis of CFSE dilution in Teffs. The presence of the antioxidants n-acetylcysteine (NAC) or 2-mercaptoethanol or inhibitors of NADPH oxidase (diphenyleneiodonium and VAS-2870) resulted in reduced WT Treg-mediated suppression. The observed suppression was in part dependent upon TGFβ as it was partially blocked with neutralizing antibodies. The suppression of Teff proliferation induced by exogenous TGFβ treatment could be overcome with NAC. Ncf1-deficient Teff were slightly but significantly less sensitive than WT Teff to suppression by exogenous TGFβ. Ncf1-deficient Tregs suppressed Ncf1-deficient Teff very poorly compared to wild type controls. There was partial but incomplete reconstitution of suppression in assays with WT Tregs and Ncf1-deficient Teff.
We present evidence that NADPH oxidase derived ROS plays a role in the direct Treg mediated suppression of CD4+ effector T cells in a process that is blocked by thiol-containing antioxidants, NADPH oxidase inhibitors or a lack of Ncf1 expression in Tregs and Teffs. Oxidants may represent a potential new target for therapeutic modulation of Treg function.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph+) leukemia is effectively managed with several approved drugs, including ...ponatinib for BCR-ABL1T315I-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants. We demonstrate that combining asciminib with ATP site TKIs enhances target inhibition and suppression of resistant outgrowth in Ph+ clinical isolates and cell lines. Inclusion of asciminib restores ponatinib's effectiveness against currently untreatable compound mutants at clinically achievable concentrations. Our findings support combining asciminib with ponatinib as a treatment strategy for this molecularly defined group of patients.
•Most BCR-ABL1 point mutants but not compound mutants are sensitive to asciminib•Variants at BCR-ABL1 position F359 are linked with asciminib resistance in patients•Combination of asciminib with ponatinib restores efficacy against compound mutants
Most clinical BCR-ABL1 compound mutants are insensitive to current clinical tyrosine kinase inhibitors. Eide et al. show that adding asciminib, an allosteric inhibitor, to ponatinib, an ATP site inhibitor, effectively targets compound mutants and provides a potential mechanism for the collaborative effect.
BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point ...mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.
•For CML patients on TKI therapy, 70% of double mutations in the BCR-ABL1 kinase domain detected by direct sequencing are compound mutations.•Sequential, branching, and parallel routes to compound mutations were observed, suggesting complex patterns of emergence.
Background T-cell receptor (TCR) clonality assessment is a principal diagnostic test in the management of mycosis fungoides (MF). However, current polymerase chain reaction–based methods may produce ...ambiguous results, often because of low abundance of clonal T lymphocytes, resulting in weak clonal peaks that cannot be size-resolved by contemporary capillary electrophoresis (CE). Objective We sought to determine if next-generation sequencing (NGS)-based detection has increased sensitivity for T-cell clonality over CE-based detection in MF. Methods Clonality was determined by an NGS-based method in which the TCR-γ variable region was polymerase chain reaction amplified and the products sequenced to establish the identity of rearranged variable and joining regions. Results Of the 35 MF cases tested, 29 (85%) showed a clonal T-cell rearrangement by NGS, compared with 15 (44%) by standard CE detection. Three patients with MF had follow-up testing that showed identical, clonal TCR sequences in subsequent skin biopsy specimens. Limitations Clonal T-cell populations have been described in benign conditions; evidence of clonality alone, by any method, is not sufficient for diagnosis. Conclusion TCR clonality assessment by NGS has superior sensitivity compared with CE-based detection. Further, NGS enables tracking of specific clones across multiple time points for more accurate identification of recurrent MF.
Highlights • NPM1 mutations in acute myeloid leukemia were accompanied by additional mutations. • NPM1 had the highest variant allele fraction (VAF) in only one of 120 cases. • Nearly all NPM1 ...-mutated AML patients showed a pattern of concurrent gene mutations. • Concurrent gene mutations had higher median VAFs than the co-existing NPM1 mutation.
Leukemias are currently subclassified based on the presence of recurrent cytogenetic abnormalities and gene mutations. These molecular findings are the basis for risk-adapted therapy; however, such ...data are generally obtained by disparate methods in the clinical laboratory, and often rely on low-resolution techniques such as fluorescent in situ hybridization. Using targeted next generation sequencing, we demonstrate that the full spectrum of prognostically significant gene mutations including translocations, single nucleotide variants (SNVs), and insertions/deletions (indels) can be identified simultaneously in multiplexed sequence data. As proof of concept, we performed hybrid capture using a panel of 20 genes implicated in leukemia prognosis (covering a total of 1 Mbp) from five leukemia cell lines including K562, NB4, OCI-AML3, kasumi-1, and MV4–11. Captured DNA was then sequenced in multiplex on an Illumina HiSeq. Using an analysis pipeline based on freely available software we correctly identified DNA-level translocations in three of the three cell lines where translocations were covered by our capture probes. Furthermore, we found all published gene mutations in commonly tested genes including NPM1, FLT3, and KIT. The same methodology was applied to DNA extracted from the bone marrow of a patient with acute myeloid leukemia, and identified a t(9;11) translocation with single base accuracy as well other gene mutations. These results indicate that targeted next generation sequencing can be successfully applied in the clinical laboratory to identify a full spectrum of DNA mutations ranging from SNVs and indels to translocations. Such methods have the potential to both greatly streamline and improve the accuracy of DNA-based diagnostics.
A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene ( FLT3 ). This ...mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3 ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3 ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (>1000-fold) of FLT3 and 26 other genes from 22 FLT3 ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3 ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3 ITDs can be reliably detected in panel-based, next-generation sequencing assays.
Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based detection of abnormal fusion transcripts is an important strategy for the diagnosis and monitoring of patients ...with acute myeloid leukemia (AML) with t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1;q22); CBFB-MYH11 or t(15;17)(q22;q12); PML-RARA. In RT-qPCR assays, patient-derived cDNA is subjected to amplification using PCR primers directed against the fusion transcript of interest as well as a reference gene for normalization. Quantification is typically performed by constructing standard curves for each PCR run using a series of plasmid standards of known concentration that harbor the same fusion transcript or the same reference gene of interest. Fusion transcripts and reference gene copy numbers are then calculated in patient samples using these standard curves. The process of constructing standard curves is laborious and consumes additional reagents. In this chapter, we give the method details for a multiplex RT-qPCR strategy to detect and quantify the acute myeloid leukemia (AML)-associated fusion transcripts PML-RARA in patients with t(15;17) without the need for standard curves. This general method can also be applied to other AML-associated fusion transcripts such as CBFB-MYH11 and RUNX1-RUNX1T1.
The distinction between chronic eosinophilic leukemia, not otherwise specified and idiopathic hypereosinophilic syndrome largely relies on clonality assessment. Prior to the advent of next-generation ...sequencing, clonality was usually determined by cytogenetic analysis. We applied targeted next-generation sequencing panels designed for myeloid neoplasms to bone marrow specimens from a cohort of idiopathic hypereosinophilic syndrome patients (n=51), and assessed the significance of mutations in conjunction with clinicopathological features. The findings were further compared with those of 17 chronic eosinophilic leukemia, not otherwise specified patients defined by their abnormal cytogenetics and/or increased blasts. Mutations were detected in 14/51 idiopathic hypereosinophilic syndrome patients (idiopathic hypereosinophilic syndrome/next-generation sequencing-positive) (28%), involving single gene in 7 and ≥2 in 7 patients. The more frequently mutated genes included ASXL1 (43%), TET2 (36%), EZH2 (29%), SETBP1 (22%), CBL (14%), and NOTCH1 (14%). Idiopathic hypereosinophilic syndrome/next-generation sequencing-positive patients showed a number of clinical features and bone marrow findings resembling chronic eosinophilic leukemia, not otherwise specified. Chronic eosinophilic leukemia, not otherwise specified patients showed a disease-specific survival of 14.4 months, markedly inferior to idiopathic hypereosinophilic syndrome/next-generation sequencing-negative (P<0.001), but not significantly different from idiopathic hypereosinophilic syndrome/next-generation sequencing-positive (P=0.117). These data suggest that targeted next-generation sequencing helps to establish clonality in a subset of patients with hypereosinophilia that would otherwise be classified as idiopathic hypereosinophilic syndrome. In conjunction with other diagnostic features, mutation data can be used to establish a diagnosis of chronic eosinophilic leukemia, not otherwise specified in patients presenting with hypereosinophilia.
Objectives: The 2017 Workshop of the Society for Hematopathology/European Association for Haematopathology reviewed the role of genetic testing in the diagnosis of hematopoietic neoplasms, including ...non-acute leukemia myeloid malignancies. Methods: The workshop panel assigned 98 submitted cases to the category of non-acute leukemia myeloid neoplasms, of which 13 were selected for oral presentation. Results: Data from both conventional karyotyping and genetic sequencing had important impact on diagnosis, classification, and prognostication. However, some cases had genetic results that appeared discordant from the morphology and/or clinical features. Thus, the workshop underscored the need for careful management of genetic data by the pathologist and clinician, in the context of other findings. Conclusions: The workshop cases highlighted the significance of genetic aberrations in the diagnosis and treatment of non-acute leukemia myeloid neoplasms. Many genetic data have already been incorporated in the most recent World Health Organization classification, and undoubtedly they will factor increasingly in future classifications. Key Words: Myelodysplastic syndrome; Myeloproliferative neoplasm; Molecular diagnostics; Cytogenetics; Targeted therapy; WHO classification
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