Extracellular vesicle miRNAs drive aberrant macrophage responses in NSAID-exacerbated respiratory disease.

BACKGROUND: Extracellular vesicles (EVs) have been implicated in the pathogenesis of asthma, however, how EVs contribute to immune dysfunction and type 2 airway inflammation remains incompletely understood. We aimed to elucidate roles of airway EVs and their miRNA cargo in the pathogenesis of NSAID-exacerbated respiratory disease (N-ERD), a severe type 2 inflammatory condition. METHODS: EVs were isolated from induced sputum or supernatants of cultured nasal polyp or turbinate tissues of N-ERD patients or healthy controls by size-exclusion chromatography and characterized by particle tracking, electron microscopy and miRNA sequencing. Functional effects of EV miRNAs on gene expression and mediator release by human macrophages or normal human bronchial epithelial cells (NHBEs) were studied by RNA sequencing, LC-MS/MS and multiplex cytokine assays. RESULTS: EVs were highly abundant in secretions from the upper and lower airways of N-ERD patients. N-ERD airway EVs displayed profoundly altered immunostimulatory capacities and miRNA profiles compared to airway EVs of healthy individuals. Airway EVs of N-ERD patients, but not of healthy individuals induced inflammatory cytokine (GM-CSF and IL-8) production by NHBEs. In macrophages, N-ERD airway EVs exhibited an impaired potential to induce cytokine and prostanoid production, while enhancing M2 macrophage activation. Let-7 family miRNAs were highly enriched in sputum EVs from N-ERD patients and mimicked suppressive effects of N-ERD EVs on macrophage activation. CONCLUSION: Aberrant airway EV miRNA profiles may contribute to immune dysfunction and chronic type 2 inflammation in N-ERD. Let-7 family miRNAs represent targets for correcting aberrant macrophage activation and mediator responses in N-ERD.

Inflammatory macrophage memory in nonsteroidal anti-inflammatory drug-exacerbated respiratory disease.

BACKGROUND: Nonsteroidal anti-inflammatory drug-exacerbated respiratory disease (N-ERD) is a chronic inflammatory condition, which is driven by an aberrant arachidonic acid metabolism. Macrophages are major producers of arachidonic acid metabolites and subject to metabolic reprogramming, but they have been neglected in N-ERD. OBJECTIVE: This study sought to elucidate a potential metabolic and epigenetic macrophage reprogramming in N-ERD. METHODS: Transcriptional, metabolic, and lipid mediator profiles in macrophages from patients with N-ERD and healthy controls were assessed by RNA sequencing, Seahorse assays, and LC-MS/MS. Metabolites in nasal lining fluid, sputum, and plasma from patients with N-ERD (n = 15) and healthy individuals (n = 10) were quantified by targeted metabolomics analyses. Genome-wide methylomics were deployed to define epigenetic mechanisms of macrophage reprogramming in N-ERD. RESULTS: This study shows that N-ERD monocytes/macrophages exhibit an overall reduction in DNA methylation, aberrant metabolic profiles, and an increased expression of chemokines, indicative of a persistent proinflammatory activation. Differentially methylated regions in N-ERD macrophages included genes involved in chemokine signaling and acylcarnitine metabolism. Acylcarnitines were increased in macrophages, sputum, nasal lining fluid, and plasma of patients with N-ERD. On inflammatory challenge, N-ERD macrophages produced increased levels of acylcarnitines, proinflammatory arachidonic acid metabolites, cytokines, and chemokines as compared to healthy macrophages. CONCLUSIONS: Together, these findings decipher a proinflammatory metabolic and epigenetic reprogramming of macrophages in N-ERD.

An anti-inflammatory eicosanoid switch mediates the suppression of type-2 inflammation by helminth larval products.

Eicosanoids are key mediators of type-2 inflammation, e.g., in allergy and asthma. Helminth products have been suggested as remedies against inflammatory diseases, but their effects on eicosanoids are unknown. Here, we show that larval products of the helminth Heligmosomoides polygyrus bakeri (HpbE), known to modulate type-2 responses, trigger a broad anti-inflammatory eicosanoid shift by suppressing the 5-lipoxygenase pathway, but inducing the cyclooxygenase (COX) pathway. In human macrophages and granulocytes, the HpbE-driven induction of the COX pathway resulted in the production of anti-inflammatory mediators [e.g., prostaglandin E2 (PGE2) and IL-10] and suppressed chemotaxis. HpbE also abrogated the chemotaxis of granulocytes from patients suffering from aspirin-exacerbated respiratory disease (AERD), a severe type-2 inflammatory condition. Intranasal treatment with HpbE extract attenuated allergic airway inflammation in mice, and intranasal transfer of HpbE-conditioned macrophages led to reduced airway eosinophilia in a COX/PGE2-dependent fashion. The induction of regulatory mediators in macrophages depended on p38 mitogen-activated protein kinase (MAPK), hypoxia-inducible factor-1α (HIF-1α), and Hpb glutamate dehydrogenase (GDH), which we identify as a major immunoregulatory protein in HpbE Hpb GDH activity was required for anti-inflammatory effects of HpbE in macrophages, and local administration of recombinant Hpb GDH to the airways abrogated allergic airway inflammation in mice. Thus, a metabolic enzyme present in helminth larvae can suppress type-2 inflammation by inducing an anti-inflammatory eicosanoid switch, which has important implications for the therapy of allergy and asthma.

Ultra-deep sequencing leads to earlier and more sensitive detection of the tyrosine kinase inhibitor resistance mutation T315I in chronic myeloid leukemia.

Chronic myeloid leukemia cells acquire resistance to tyrosine kinase inhibitors through mutations in the ABL1 kinase domain. The T315I mutation mediates resistance to imatinib, dasatinib, nilotinib and bosutinib, whereas sensitivity to ponatinib remains. Mutation detection by conventional Sanger sequencing requires 10%-20% expansion of the mutated subclone. We studied the T315I mutation development by ultra-deep sequencing on the 454 XL+ platform (Roche) in comparison to Sanger sequencing. By ultra-deep sequencing, mutations were detected at loads of 1%-2%. We selected 40 patients who had failed first-line to third-line treatment (imatinib, dasatinib, nilotinib) and had high loads of the T315I mutation detected by Sanger sequencing. We confirmed T315I mutations by ultra-deep sequencing and investigated the mutation dynamics by backtracking earlier samples. In 20 of 40 patients, we identified the T315I three months (median) before Sanger sequencing detection limits were reached. To exclude sporadic low percentage mutation development without subsequent mutation outgrowth, we selected 42 patients without resistance mutations detected by Sanger sequencing but loss of major molecular response. Here, no mutation was detected by ultradeep sequencing. Additional non-T315I resistance mutations were found in 20 of 40 patients. Only 15% had two mutations per cell; the other cases showed multiple independently mutated clones and the T315I clone demonstrated a rapid outgrowth. In conclusion, T315I mutations could be detected earlier by ultra-deep sequencing compared to Sanger sequencing in a selected group of cases. Earlier mutation detection by ultra-deep sequencing might allow treatment to be changed before clonal increase of cells with the T315I mutation.

Selective BCL-2 inhibition by ABT-199 causes on-target cell death in acute myeloid leukemia.

B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here, we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nmol/L, and cell death occurred within 2 hours. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on-target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML.

The molecular profile of adult T-cell acute lymphoblastic leukemia: mutations in RUNX1 and DNMT3A are associated with poor prognosis in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and heterogeneous disease. The diagnosis is predominantly based on immunophenotyping. In addition to known cytogenetic abnormalities molecular mutations were recently identified. Here, 90 adult T-ALL cases were investigated for mutations in NOTCH1, FBXW7, PHF6, CDKN2A, DNMT3A, FLT3, PTEN, and RUNX1 using 454 next-generation amplicon sequencing and melting curve analyses. These data were further complemented by FISH, chromosome banding, array CGH, and CDKN2B promoter methylation analyses. NOTCH1 was the most frequently mutated gene with a 71.1% frequency followed by FBXW7 (18.9%), PHF6 (39.5%), DNMT3A (17.8%), RUNX1 (15.5%), PTEN (10.0%), CDKN2A (4.4%), FLT3-ITD (2.2%), and FLT3-TKD (1.1%). In total, 84/90 (93.3%) cases harbored at least one mutation. Combining these data with CDKN2A/B deletions and CDKN2B methylation status, we detected minimum one aberration in 89/90 (98.9%) patients. Survival analyses revealed the subtype as defined by the immunophenotype as the strongest independent prognostic factor. When restricting the survival analysis to the early T-ALL subtype, a strong association of RUNX1 (P = 0.027) and DNMT3A (P = 0.005) mutations with shorter overall survival was observed. In conclusion, RUNX1 and DNMT3A are frequently mutated in T-ALL and are associated with poor prognosis in early T-ALL.

Three novel cytogenetically cryptic EVI1 rearrangements associated with increased EVI1 expression and poor prognosis identified in 27 acute myeloid leukemia cases.

In acute myeloid leukemia (AML), increased ecotropic virus integration site 1 protein homolog (EVI1) gene expression is prognostically unfavorable. Subsets of cases show 3q26 rearrangements, such as inv(3)(q21q26)/t(3;3)(q21;q26), frequently accompanied by chromosome 7 abnormalities. We investigated whether cytogenetically cryptic EVI1 rearrangements may cause EVI1 overexpression in myeloid malignancies without 3q26 abnormalities and investigated 983 patients with AML (n = 606) or myelodysplastic syndromes (MDS; n = 377) with normal karyotype (CN-AML/CN-MDS, n = 594) or chromosome 7 abnormalities (n = 389) for EVI1 rearrangements using interphase FISH. We identified cytogenetically cryptic EVI1 rearrangements in 27 patients (19 AML, 8 MDS): inv(3)(p24q26) [n = 10]; t(3;21)(q26;q11) [n = 9]; and der(7)t(3;7)(q26;q21) [n = 8]. Elevated EVI1 expression was detected in nearly all cases with cryptic EVI1 rearrangements: Median %EVI1/ABL1 was 92.8 (range: 29.8-146.1) in inv(3)(p24q26), 104.9 (41.4-176.3) in t(3;21)(q26;q11), and 101.8 (4.4-210.4) in der(7)t(3;7)(q26;q21). This was similar to median %EVI1/ABL1 of 73.9 (range: 7.3-585.6) in an independent cohort of inv(3)(q21q26)/t(3;3)(q21;q26) and 67.1 (2.3-410.7) in other 3q26/EVI1 rearrangements. Healthy controls showed median EVI1 expression of 0.5 (range: 0.0-5.8). Using SNP microarray and sequencing analyses, the breakpoints of der(7)t(3;7)(q26;q21) were assigned to CDK6 and centromeric of EVI1, and of t(3;21)(q26;q11) to be within EVI1 and NRIP1. Median overall survival in patients with cryptic EVI1 rearrangements was short, comparable to patients with inv(3)(q21q26)/t(3;3)(q21;q26) or other EVI1 rearrangements. Cryptic EVI1 rearrangements contribute to explain the clinical heterogeneity of CN-AML and are associated with elevated EVI1 expression and an unfavorable prognosis. Screening for cryptic EVI1 rearrangements by FISH may be particularly appropriate in CN-AML with elevated EVI1 expression or in AML/MDS patients with chromosome 7 abnormalities.

Gene expression of BAALC, CDKN1B, ERG, and MN1 adds independent prognostic information to cytogenetics and molecular mutations in adult acute myeloid leukemia.

Expression of BAALC, ERG, and MN1 is associated with outcome in normal karyotype acute myeloid leukemia (AML). In this study, the expression of these markers and of EVI1 and CDKN1B was determined using oligonucleotide microarrays in 286 AML comprising all cytogenetic groups. Higher expression of each gene was associated with an inferior outcome: CDKN1B, median overall survival (mOS): 14.9 months vs. not reached (nr), P = 0.005, median event-free survival (mEFS): 9.7 vs. 31.0 months, P = 0.013; BAALC, no impact on OS, mEFS: 6.2 vs. 13.0 months, P = 0.03; ERG: mOS: 12.5 months vs. nr, P = 0.002, mEFS: 8.1 vs. 15.7 months, P = 0.001; MN1: mOS: 12.3 months vs. nr, P = 0.004, mEFS: 8.1 vs. 16.7 months, P = 0.001. A multivariate analysis revealed an independent impact on OS for CDKN1B, ERG, and MN1 expression. A novel score based on BAALC, CDKN1B, ERG, and MN1 expression had an impact on OS and EFS independent of cytogenetics and age. A score taking into account gene expression and karyotype allowed the separation of four prognostic groups with significant differences in OS and EFS (OS at 2 years: 90.4%, 56.4%, 34.0%, 12.6%; mEFS: n.r., 18.1 months, 8.7 months, 2.5 months). The impact on outcome of this score was independent of NPM1mut/FLT3-ITD- status, MLL-PTD, and age.

TET2 deletions are a recurrent but rare phenomenon in myeloid malignancies and are frequently accompanied by TET2 mutations on the remaining allele.

TET2 mutations have been identified in 10-25% of patients with myeloid malignancies, but TET2 gene copy number alterations remain to be evaluated. We performed interphase and metaphase fluorescence in situ hybridization (FISH), using newly designed probes that span TET2, in 893 patients with acute myeloid leukaemia (AML) and chronic myeloid malignancies and validated the new assay by single nucleotide polymorphism arrays. Next-generation sequencing for molecular TET2 mutations was performed in 37/50 del(TET2) patients. We identified TET2 deletions in 50/893 patients (26 males, 24 females; 44-87 years) resulting in a 5.6% frequency [22/425 AML (5.2%), 15/217 chronic myelomonocytic leukaemia (CMML; 6.9%), 9/188 myelodysplastic syndromes (4.8%), 4/63 myeloproliferative neoplasms (6.3%)]. Cytogenetic alterations (mostly complex) were detected in 35/50 (70.0%) of del(TET2) cases. TET2 deletions were cytogenetically cryptic in 25/50 (50.0%). Sequencing detected TET2mut in 19/37 (51.4%) del(TET2) cases investigated, predominantly CMML (10/14; 71.4%). In de novo AML with intermediate cytogenetics, presence of any TET2 alteration was related to worse median overall survival (347 d vs. not reached; P = 0.052) and event-free survival (164 vs. 457 d; P = 0.024). JAK2(V617F) was found in 6/18 (33.3%) del(TET2) cases analysed. Thus, TET2 haploinsufficiency recurrently occurs in myeloid malignancies, frequently combined with TET2 mutations on the remaining allele. The prognostic impact of del(TET2) in myeloid malignancies should be further evaluated.

Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype.

Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.

Strategy for robust detection of insertions, deletions, and point mutations in CEBPA, a GC-rich content gene, using 454 next-generation deep-sequencing technology.

CEBPA mutations are of prognostic relevance in acute myeloid leukemia (AML) and are currently detected using a combination of denaturing high-performance liquid chromatography (DHPLC), gene scan/fragment length analysis, and direct Sanger sequencing. Next-generation deep pyrosequencing, principally, allows for the highly sensitive detection of molecular mutations. However, standard 454 chemistry laboratory procedures lack efficient amplification of guanine-cytosine (GC)-rich amplicons during the emulsion PCR (emPCR) steps allowing direct massively parallel clonal amplification of PCR products. To solve this problem, we investigated six distinct emPCR conditions. The coding sequence of CEBPA was subdivided into four overlapping amplicons: GC content for amplicon 1, 74%; amplicon 2, 76%; amplicon 3, 77%; and amplicon 4, 69%. A new emPCR condition, improving the standard titanium assay, presents a robust solution to sequence amplicons with a GC content of up to 77%. Moreover, this assay was subsequently tested on a larger independent cohort of 23 AML patients. For each patient, a median of 737 reads was generated (coverage range, 397-fold to 1194-fold) and therefore allowed a robust detection of insertions, deletions, and point mutations. In conclusion, next-generation amplicon sequencing enables the highly sensitive detection of molecular mutations and is a feasible assay for routine assessment of GC-rich content amplicons.

CDKN1B, encoding the cyclin-dependent kinase inhibitor 1B (p27), is located in the minimally deleted region of 12p abnormalities in myeloid malignancies and its low expression is a favorable prognostic marker in acute myeloid leukemia.

BACKGROUND: Alterations of the short arm of chromosome 12 (12p) occur in various hematologic malignancies and ETV6 and CDKN1B, which are located on 12p, have been implicated as leukemogenic genes of interest. DESIGN AND METHODS: We selected seven patients with myeloid malignancies and small 12p deletions detected by fluorescence in situ hybridization encompassing only the region centromeric of ETV6 and further evaluated them by single nucleotide polymorphism microarrays. RESULTS: The minimally deleted region contained only nine genes. These genes were subsequently analyzed by microarray expression profiling in an independent cohort of 781 patients, most, but not all, of whom had different hematologic malignancies CREBL2, MANSC1, and CDKN1B were expressed in more than 25% of cases, while the other six genes were expressed in only a minority of cases. As CDKN1B is a cell cycle regulator and functions as a tumor suppressor gene, this gene was selected for further expression studies in 286 patients with acute myeloid leukemia. When comparing patients with low CDKN1B expression (expression level<1,160; 1st quartile) with those with intermediate or high expression (2nd-4th quartiles), certain mutations were observed more frequently in the former: RUNX1-RUNX1T1 (11/83, 13.3% versus 5/203; 2.5%; P=0.001), PML-RARA rearrangements (11/83, 13.3% versus 4/203, 2.0%; P<0.001), 11q23/MLL rearrangements (6/83, 7.2% versus 4/203, 2.0%; P=0.038), and FLT3-TKD mutations (7/63, 11.1% versus 6/167, 3.6%; P=0.047). The median overall survival of patients with low CDKN1B expression was longer than that of patients with intermediate/high expression (not reached versus 14.9 months; P=0.005). Likewise, patients with low CDKN1B expression had a longer event-free survival than those with intermediate/high expression (31.0 versus 9.7 months; P=0.013). CONCLUSIONS: CDKN1B is an interesting candidate gene as a potential biomarker for prognostication in acute myeloid leukemia.

Next-generation sequencing technology reveals a characteristic pattern of molecular mutations in 72.8% of chronic myelomonocytic leukemia by detecting frequent alterations in TET2, CBL, RAS, and RUNX1.

PURPOSE: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. Thus far, data on a comprehensive cytogenetic or molecular genetic characterization are limited. PATIENTS AND METHODS: Here, we analyzed 81 thoroughly characterized patients with CMML (CMML type 1, n = 45; CMML type 2, n = 36) by applying next-generation sequencing (NGS) technology to investigate CBL, JAK2, MPL, NRAS, and KRAS at known mutational hotspot regions. In addition, complete coding regions were analyzed for RUNX1 (beta isoform) and TET2 aberrations. RESULTS: Cytogenetic aberrations were found in 18.2% of patients (14 of 77 patients). In contrast, at least one molecular mutation was observed in 72.8% of patients (59 of 81 patients). A mean of 1.6 mutations per patient was observed by this unprecedented screening. In total, 105 variances were detected by this comprehensive molecular screening. After excluding known polymorphisms or silent mutations, 82 distinct mutations remained (CBL, n = 15; JAK2V617F, n = 8; MPL, n = 0; NRAS, n = 10; KRAS, n = 12; RUNX1, n = 7; and TET2, n = 41). With respect to clinical data, a better outcome was seen for patients carrying TET2 mutations (P = .013). CONCLUSION: The number of molecular markers used to categorize myeloid neoplasms is constantly increasing. Here, NGS screening has been demonstrated to support a comprehensive characterization of the molecular background in CMML. A pattern of molecular mutations translates into different biologic and prognostic categories of CMML.