T-large granular lymphocyte leukemia (T-LGLL) is a clonal lymphoproliferative disorder of cytotoxic T-cells (CTL) that is associated with cytopenias, predominantly neutropenia and reticulocytopenic ...anemia. From a scientific point of view, T-LGLL provides a natural model to study the dynamics of CTL responses; the heterogeneity of the disorder allows for examining the diversity of CTL responses in both autoimmune disorders and putatively chronic reactive conditions. A proportion of patients may have an extreme reactive process that mimics an indolent neoplastic lymphoproliferation. NGS and deep T-cell repertoire (TCR) sequencing provide insight into the clonal dynamics at work in T-LGLL patients.
A large proportion of T-LGLL patients present with a bona-fide low-grade leukemia; this notion is supported by the discovery of recurrent somatic STAT3 mutations in some patients. STAT3 clonal burden represents an excellent marker that can be serially monitored along with clinical milestones to ultimately gain a more comprehensive understanding of disease etiology and natural history.
We collected a cohort of 183 LGLL patients and screened them via deep NGS for mutation status of STAT3. In 36% of patients, 4 distinct somatic mutations (Y640F, N647I, D661V, D661Y) were identified in the SH2 domain of STAT3. In patients with wildtype STAT3, no somatic mutation was implicated in clonal expansion except for a small minority with STAT5 mutations present.
We performed a longitudinal analysis of 20 representative STAT3-mutated T-LGLL patients with up to 10-year follow-up and an average of 7 analyzed blood samples per case. All serial samples were deep-sequenced to detect and determine the VAF of the known STAT3 mutations. Overall, STAT3 mutation VAF had a significant, inverse relationship to both hemoglobin and absolute neutrophil count (ANC) (both p<=0.001). In 7/11 cases harboring the Y640F mutation, chemotherapy led to remission accompanied by a decrease in VAF; 3 were asymptomatic and received no treatment. In patients with D661V or D661Y, 6/9 achieved remission with treatment. Only 1/3 cases with N647I entered remission.
This longitudinal cohort can be sub-categorized into distinct patterns of clonal dynamics: 1) emerging STAT3 mutation in 20% of patients with a decrease in ANC as VAF of STAT3 clones expand; 2) an opposite trend in 40% of patients where VAF decreased due to therapeutic manipulations; 3) stable VAF in 20% of patients with little change over time; 4) codominant or dominant/secondary STAT3 mutations with distinct subclonal burden in 20% of patients.
We performed deep TCR NGS on a representative subset of 9 patients to explore how STAT3 mutations correlated with T-cell clonal expansions. The data were processed by an extensive bioanalytic pipeline to quantify the relative abundance of each CDR3 rearrangement within a patient's TCR. Our cohort had an average of over 53,000 CDR3 templates per sample and was compared with 587 healthy controls.
Our results demonstrate multiple patterns of clonal dynamics over the course of T-LGLL. Within each case, the immunodominant clones in serial samples were identified and correlated with STAT3 VAF burden over time. When patients were in remission, both STAT3 VAF and clonality were typically low. Interestingly, functional remission occurred in 2 cases despite increases in both clonality and STAT3 VAF. In 5/9 cases, the T-LGLL process involved 1 STAT3 mutation and 1 corresponding pathogenic clonotype displaying similar dynamics over time. In patients with 2 mutations, multiple high-frequency clonotypes were observed. Most significantly, comparison of STAT3 VAF and the dominant clonotype(s) revealed that STAT3 mutation can arise within a pre-existing clonal expansion that may harbor 2 branching mutations in extreme cases.
Identification of CDR3 rearrangement sequences allowed for analysis of the distribution of clonotypes among patients and controls. The pathogenic clonotypes found in T-LGLL patients were detected in a high proportion of controls but at extremely low frequencies. This suggests that these potentially autoimmune clones exist in normal individuals but are effectively suppressed. No pathogenic clonotypes were shared among disease patients. In sum, analysis of clonal dynamics suggests that STAT3 mutations can occur in the context of pre-existing oligoclonal responses and involve otherwise low-frequency clonal specificities.
Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Mustjoki:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding.
A broad array of antibodies directed against the variable (V) region of the T-cell receptor (TCR) b (V sub(b)) chain has become available in a directly conjugated multicolor format that permits ...assessment of 19 of 25 V sub(b) families, covering 70% of the normal circulating T-cell repertoire. These antibodies were used to detect expanded T-cell populations in 43 peripheral blood samples submitted for suspected T-cell malignancy. Of 43 samples, 27 were diagnosed as follows: T-cell large granular lymphocyte leukemia, 14 samples; Sezary syndrome, 4 samples; T-cell prolymphocytic leukemia, 5 samples; or T-cell non-Hodgkin lymphoma or T-cell lymphoproliferative disorder not otherwise specified, 4 samples. The remaining 16 samples were determined to be nonneoplastic. All samples were diagnosed before assessment with anti-V sub(b) flow cytometry. By using a cutoff of 1.6 times the upper limit of normal range (ULN) to define malignant restriction of V sub(b) use, pathologic restriction of V sub(b) use was found directly or indirectly in all 27 samples carrying a diagnosis of malignancy and directly in 2 of 16 samples without a diagnosis of malignancy. TCR gene rearrangement studies were used to confirm V sub(b) flow cytometry results. By using a cutoff of 1.6 times the ULN for the detection of malignancy, the antibody panel had a diagnostic sensitivity of 89% for direct detection of pathologic V sub(b) restriction and a specificity of 88%, making it useful for rapid diagnosis of T-cell leukemia. chain use permits direct quantitation and immunophenotypic characterization of the V sub(b) family of the malignant clone. super(5) After characterization of the malignant clone, V sub(b) flow cyto-metric analysis also can be used to follow up the treatment and clinical course in a particular patient. super(8) In the present study, we demonstrated the usefulness of a multicolor panel of directly conjugated V sub(b) antibodies for rapid detection of T-cell malignancy in peripheral blood samples from patients with T-cell lymphoproliferative disorders.
Background:MDS pathogenesis is a multi-step molecular process resulting from somatic mutations acquired over time. Smoking is a known risk factor for MDS which may accelerate this process. To further ...investigate the contribution of tobacco use in MDS development, we studied the association between smoking and specific genomic alterations in MDS patients (pts).
Methods: MDS and CMML pts diagnosed between 1996 and 2016 with both smoking histories and sequencing data were included. The Center for Disease Control definition defined current, ex- and never smokers, the latter defined as pts with zero smoking duration/packs per day (PPD). Next generation targeted sequencing was performed for a panel of 60 genes known to be commonly mutated in myeloid malignancies. Fisher's exact test and Wilcoxon's test compared proportions and medians, respectively. Poisson regression modeled total numbers of mutations per pt. The probability of a mutation in a particular gene was modeled using conditional logistic regression. Adjustment was done for both sex and age, fits were stratified using age cut points of 0, 40, 50, 60, 65, 70, 75, 80, 85 and 90 years (y). Odds ratios (OR) were found as exponentials of gene status coefficients of such fits.
Results: In 672 pts, the median age at diagnosis was 68y (range, 20-103), for pt characteristics, see Table 1. Median OS 34 mo (95% CI 30-39) and median follow-up time was 21.5 mo (IQR, 10-45). Median tobacco exposures were 30 pack-years (py) in current smokers (range, 6-80) and 25 in ex-smokers (range, 0.3-175), P=.2. More men than women ever smoked (74% v 45%; P <.0001). Longer duration of smoking associated with higher IPSS-R categories (P=.012) and cytogenetic risk scores, P=.037. Most pts (502, 75%) had at least one mutation; 23% had a single gene mutation, 19% had 2, 15% had 3, and 18% had >3. The most frequently mutated genes were TET2 17%, ASXL1 16%, SRSF2 14%, SF3B1 12%, DNMT3A 10%, RUNX1 9%, STAG2 9%, and TP53 7%.
Poisson regression analysis was used to assess the impact of smoking duration (y) or PPD on mutation acquisition. A positive correlation of greater smoking intensity (> 2 PPD v <0.5, relative increase (RI) =1.46, P=.011) and longer duration of smoking (>40y v <10, RI = 1.31, P=.011) was seen with increased number of mutations Figure 1.
Impact of duration of smoking was evaluated using logistic regression. Duration of smoking >40y v <1 was significantly associated with the following molecular abnormalities: FLT3 (P= .036), EZH2/ del7 (P=.038) and NRAS (P=.046). Comparing 20-40y v <1 yielded SETBP1 (P=.005), DNMT3A (P=.010) and EZH2 /del7 (P=.036). Comparing 10-20y v <1 yielded STAG2 (P=.039) and NOTCH1 (P=.046). Finally, comparing durations of 1-10y v <1 yielded PHF6 (P=.016). OR per mutation, see Table 2.
Focusing on intensity of smoking, >2 PPD v <0.5 was significantly associated with higher rates of mutations in FLT3 (P=.013), EZH2 /del7 (P=.024), IDH1 (P=.041), and NPM1 (P=.044). Even low intensity smoking, comparing 0.5-1 PPD v <0.5, was significantly associated with mutations in SETBP1 (P=.002), DDX54 (P=.042) and DNMT3A (P=.046). For the combination of smoking duration and intensity, >40py v <10 was associated with IDH1 mutation (P=.046), while 20-30 py v <10 significantly associated with RAD21 (P=. 016), DDX54 (P=.018), SRSF2 (P=.041), del7/ EZH2 (P=.042), and NF1 (P=.045) mutations. Comparing 1-10py v <1, significant associations were found for IDH1 (P=.004), PRPF8 (P=.011), SETBP1 (P=.022), ETV6 (P= .040) and TET2 (P=. 047)mutations Table 2. Analyses were repeated after excluding CMML and MDS/MPN pts to control for the common genomic phenotype seen in these disorders, with identical results.
Conclusions: Tobacco smoking predisposes to either acquisition or selection of distinct somatic mutational patterns from those seen in “spontaneous/non-tobacco exposed” MDS. EZH2/ del7 abnormalitiesassociated with longer durations and heavier smoking patterns, while SETBP1 mutation associated with lighter smoking for prolonged durations. Lesions associated with light smoking for years may be related to chronic inflammatory changes, whereas those associated with higher intensity may reflect direct DNA damage. Validation of these findings warrants earlier sequencing in pts with pre-MDS conditions such as clonal hematopoiesis of indeterminate potential to better identify the onset of somatic mutations and their role in MDS pathogenesis.
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Advani:Takeda/ Millenium: Research Funding; Pfizer: Consultancy. Gerds:CTI BioPharma: Consultancy; Incyte: Consultancy. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.
La maladie de Behçet (MB) est une affection inflammatoire, systémique chronique évoluant par poussées. Elle est caractérisée par l’atteinte de plusieurs organes dont les muqueuses (aphtes) et la peau ...(pseudo-folliculites). La physiopathologie de la MB est en grande partie inconnue. Le rôle de l’immunité innée semble prépondérant avec notamment une activation des polynucléaires neutrophiles. La voie NFKB, engagée dès le contact d’un ligand sur le récepteur 1 du tumor necrosis factor (TNFR1), joue un rôle critique dans l’engagement du processus inflammatoire car elle produit des cytokines majeures comme l’IL-1, l’interleukine 6 et le TNF ; L’haploinsuffisance de A20 partage avec la maladie de Behçet l’aphtose bipolaire et l’uvéite, mais elle s’en distingue par sa transmission dominante, un début des symptômes plus précoce, et une atteinte digestive sévère au premier plan. Les polynucléaires neutrophiles sont retrouvés en abondances dans les lésions histologiques de patients, et plusieurs études ont suggéré leur état d’activation dans la MB, ils pourraient ainsi participer à la physiopathologie de la MB. La phosphodiestérase 4 (PDE4) une enzyme du système immunitaire qui (en dégradant l’AMPc) augmentent la sécrétion de médiateurs de l’inflammation. Sachant que l’AMPc induit l’inhibition de la voie NF-κb, sa dégradation entraîne une diminution de cette importante voie de l’inflammation. L’apremilast est une molécule administrée oralement qui inhibe la PDE4. Bien que ses mécanismes d’action précis ne soient pas connus dans la MB, l’apremilast s’est révélé efficace dans le traitement des manifestations cutanéo-articluraires de la MB.
L’objectif de ce travail était d’étudier l’activation des PNN et l’effet de l’inhibition de PDE4 dans la MB.
Vingt patients atteints de maladie de Behçet et 15 témoins sains ont été inclus.
Les PNN isolés de patients montraient des signes d’activations en comparaison aux témoins. Les marqueurs de surface d’activation CD11b, CD64, CD66b etCD11c étaient augmenté (2,7 %±3,7 vs 0,4 %±0,3, p=0,004, 1,5 %±1,2 vs 0,4 %±0,3, p=0,002, 6,7 %±4,3 vs 1,7 %±0,8, p=0,005, 6,4±4,5 vs 1,4 %±1, p=0,009, respectivement). La production de reactive oxygen species (ROS) et la NETose étaient augmentés dans les PNN de patients (11,1 MFI±5,7 vs 3,7 MFI±1,1, p=0,003 et 32 %±10,9 vs 5 %±3,9, p=0,002, respectivement). L’analyse transcriptomique des neutrophiles isolés de patients comparé aux témoins retrouvait une augmentation de la transcription des gènes relatif à l’immunité innée (TLR5, LY96, TLR1, TLR4), au signalement intracellulaire (IRAK4, JAK2, MAPK1, MAP3K1, STAT3) et au chemotaxisme (CCR3, CXCR2, CXCR1). L’analyse par immunofluorescence de lésions histologiques de patients a permis de confirmer l’infiltrat de PNN et de montrer qu’ils exprimaient la PDE4.
L’inhibition de PDE4 par le roflumilast in vitro permettait de diminuer la production de ROS et de NETs par les PNN de patients (11,1 MFI±5,7 vs 5,5 MFI±3,2, p=0,05).
Enfin, nous avons confirmé in vivo chez des patients traités par apremilast que ces marqueurs d’activations (marqueurs de surface, production de ROS, NETose) et la transcriptions des gènes relatif à l’immunité innée, au signalement intracellulaire et au chemotaxisme étaient grandement diminués à 12 semaines de traitement.
Nous avons démontré l’implication des PNN et le rôle de l’inhibition de PDE4 dans le contrôle de l’activation des PNN suggérant ainsi un rationnel physiopathologique de l’effet clinique de l’Apremilast dans la MB. Nous avons également pour la première fois montré au niveau transciptomique l’activation des PNN dans la MB.
We present Space Telescope Imaging Spectrograph emission-line spectra of the central regions of the spiral galaxies NGC 1300 and 2748. From the derived kinematics of the nuclear gas we have found ...evidence for central supermassive black holes in both galaxies. The estimated masses of the black holes in NGC 1300 and 2748 are (6.6+6.3−3.2) × 107 and (4.4+3.5−3.6) × 107 M⊙, respectively (both at the 95 per cent confidence level). These two black hole mass estimates contribute to the poorly sampled low-mass end of the nuclear black hole mass spectrum.
Background: The diagnosis of AML requires an integrated approach of clinical, morphologic, cytogenetic, and molecular assessments. The CONNECT MDS/AML Disease Registry (NCT01688011) is a prospective, ...longitudinal, multicenter, observational cohort study of patients (pts) in the United States with newly diagnosed AML, MDS, or idiopathic cytopenia of undetermined significance, designed to evaluate diagnostic and treatment (Tx) patterns, clinical and pt-reported outcomes, and correlative data, such as genetic mutations. This investigation was performed to assess how pts with AML are diagnosed at community and academic sites and compare how these diagnostic practices align with WHO 2008 recommendations for AML.
Methods: Pt enrollment began in December, 2013 and will continue until ~1500 pts from ~150 US sites are enrolled. Target enrollment for the AML cohort is 400 pts. To best capture the typical distribution of clinical practice settings in which AML pts are treated, ~80% of the sites will be community-based and ~20% will be academic institutions (ie, affiliated with a medical school). Pts aged ≥55 years (yrs) with newly diagnosed AML (excluding acute promyelocytic leukemia) are eligible. All decisions regarding pt care (Tx, testing) are determined by the study clinician, as the disease registry is non-interventional. AML diagnosis is confirmed by independent central review of all available diagnostic test reports, including bone marrow (BM) aspirates and biopsies, cytogenetics, molecular testing, and laboratory results. Diagnostic procedures are recorded in an electronic data capture system. Pt data will be entered quarterly for up to 8 years of follow-up.
Results: Between December 12, 2013 and May 19, 2016, 180 AML pts have enrolled in the registry from 73 sites; 125 pts (69%) enrolled at a community site (including 1 governmental site) and 55 pts (31%) enrolled at an academic center. Median age of all pts was 71 yrs (range 55-91), with 72% of pts aged ≥65 yrs (Table 1). Blast percentages were reported in 97% of cases, mainly in BM, although peripheral blood blasts were also assessed for 71% of pts (Table 2). Manual differential blast count was reported for 63% of pts, and flow cytometry and immunohistochemistry were used to determine blast counts for 10% of pts each. Immunophenotyping by flow cytometry was reported for 97% of pts. Erythroid, megakaryocytic, or neutrophilic dysplasia, and presence or absence of Auer rods, ring sideroblasts, or fibrosis were reported for <50% of all pts (Table 2). Conventional karyotyping was performed for 90% of pts and FISH testing was reported for 80% of pts (Table 3). Mutation testing was reported for 85 pts (47%).
Conclusions: The ongoing prospective CONNECT MDS/AML registry is uniquely positioned to assess current clinical practice patterns in AML. Flow cytometry and conventional karyotyping were done in most cases; however, manual blast count was missing for 37% of AML pt samples at diagnosis. Instead, ~10% of blasts counts were evaluated by flow cytometry or immunohistochemistry, and ~18% by 'other' means (ie, blast count estimations). Flow cytometry for blast enumeration is discouraged in WHO 2008 recommendations, as it may both under- and overestimate counts. Despite a 90% rate of conventional karyotyping, FISH testing was reported for 80% of pts, suggesting FISH may be used more than is necessary. Mutation testing was reported for approximately one-half of pts, which may reflect a lack of testing, a lag in mutational test reporting, or a combination of both. Diagnostic practices for AML pts enrolled thus far appear to be guided by WHO 2008 recommendations; awareness of updated recommendations may change diagnostic patterns as new AML pts enter the CONNECT MDS/AML registry.
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George:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Allakos: Research Funding; Allakos: Research Funding; Blueprint Medicines: Consultancy; Novartis: Consultancy; Seattle Genetics: Consultancy, Research Funding; Incyte: Consultancy; GLG: Consultancy; Wiley Blackwell: Consultancy; American Registry of Pathology: Patents & Royalties; Wolters Kluwer: Patents & Royalties; UpToDate: Patents & Royalties. Erba:Sunesis: Consultancy; Novartis: Consultancy, Speakers Bureau; Celator: Research Funding; Ariad: Consultancy; Jannsen: Consultancy, Research Funding; Juno: Research Funding; Astellas: Research Funding; Incyte: Consultancy, DSMB, Speakers Bureau; Agios: Research Funding; Pfizer: Consultancy; Gylcomimetics: Other: DSMB; Millennium Pharmaceuticals, Inc.: Research Funding; Celgene: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Daiichi Sankyo: Consultancy. Steensma:Amgen: Consultancy; Ariad: Equity Ownership; Millenium/Takeda: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Genoptix: Consultancy. Pollyea:Alexion: Other: advisory board; Ariad: Other: advisory board; Celgene: Other: advisory board, Research Funding; Glycomimetics: Other: DSMB member; Pfizer: Other: advisory board, Research Funding. Abedi:Celgene: Consultancy. Bejar:Genoptix: Consultancy. Grinblatt:Celgene Corporation: Consultancy, Speakers Bureau. Komrokji:Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Revicki:Celgene: Consultancy. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy. Savona:TG Therapeutics: Research Funding; Takeda: Research Funding; Amgen Inc.: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Incyte: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity’s Board of Directors or advisory committees; Sunesis: Research Funding; Ariad: Membership on an entity’s Board of Directors or advisory committees. Scott:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Sekeres:Millenium/Takeda: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees. Thompson:VIA Oncology: Membership on an entity’s Board of Directors or advisory committees; Takeda: Membership on an entity’s Board of Directors or advisory committees, Other: Multiple Myeloma International Registry; Celgene: Membership on an entity’s Board of Directors or advisory committees, Other: MDS/AML Registry; Doximity: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; AIM Specialty Health: Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity’s Board of Directors or advisory committees. Fliss:Celgene Corporation: Employment, Equity Ownership. Swern:Celgene: Employment, Equity Ownership. Nifenecker:Celgene: Employment, Equity Ownership. Kiselev:Celgene: Employment, Equity Ownership. Sugrue:Celgene Corporation: Employment, Equity Ownership. Foucar:Celgene: Membership on an entity’s Board of Directors or advisory committees; ASCP press: Other: Book royalties; Elsevier: Other: Book royalties; Lippincott WW: Other: Book royalties.
Retigabine represents an antiepileptic drug possessing a completely different mechanism of action when compared to the existing classical and newer antiepileptic drugs. In the therapeutic range, ...retigabine enhances potassium currents, very likely via destabilization of a closed conformation or stabilization of the open conformation of the potassium Kv7.2-7.3 channels. There are also data indicating that this drug may be a GABA enhancer. Kainate-induced status epilepticus in rats resulted in massive apoptosis in the pyriform cortex and hippocampal area - retigabine inhibited neurodegeneration only in the former brain structure. The metabolism of retigabine has nothing to do with cytochrome P450 enzymes and the drug undergoes glucuronidation and acetylation. Randomized, placebo-controlled multicenter studies have shown that retigabine produced a considerable improvement as an add-on drug in patients with partial drug-resistant epilepsy. The most prominent adverse effects due to retigabine combined with the existing antiepileptic treatment were dizziness, somnolence and fatigue. The preclinical data indicate that this antiepileptic drug may possibly be applied in patients with neuropathic pain and affective disorders. Initial clinical data suggest that retigabine may be also effective in Alzheimer's disease or stroke.
Ferroquine (FQ) is a new antimalarial agent with a high blood schizotoncidal activity. Previous studies on this compound were done with racemate mixtures. As FQ possesses planar chirality, pure ...enantiomers were obtained by enzymatic resolution in order to compare their antimalarial activities and cytotoxicities. (+)-FQ and (-)-FQ were equally active in vitro, at nanomolar concentrations. Both enantiomers were slightly less active than the racemate in vivo; cytotoxicities were similar. Actually, the racemate represents the optimal formulation. To the best of our knowledge, this is the first investigation of biological activities of compounds with metallocenic chirality.
CMML heterogeneous with a spectrum of dysplastic or proliferative clinical features. Except for rare balanced translocations (involving PDGFbR or PDGFaR), cytogenetic defects are overlapping with MDS ...and MPN. Recently, due to the introduction of NGS, many somatic mutations have been discovered in myeloid neoplasms. Analysis of mutational spectrum in CMML may facilitate understanding of the pathogenesis of this disease. However, with the recognition of the complexity of clonal architecture and intra-tumor heterogeneity, not only a share presence of individual mutations/their combinations, but also the clonal hierarchy may be of diagnostic importance. Ancestral events may correspond to clinically distinguished CMML subtypes or allow for a new sub-categorization reflective of common pathogenetic features. Here we studied somatic mutational spectra of 306 patients, including 150 CMML cases (97 CMML-1, 23 CMML-2, and 30 post CMML sAML) with 55% dysplastic (MD-CMML) and 44% proliferative form (MP-CMML), abnormal cytogenetics were found in 45% cases. In 10 cases serial analysis was performed. Comparisons were also performed with JMML (N=92) and M4/M5 AML (N=64). We performed analyses of WES in 108 paired cases with deep targeted DNA NGS in the remaining patients.
Within CMML cohort, the most frequently mutated somatic genes were TET2 (43%), SRSF2 (31%), ASXL1 (23%), RUNX1 (17%), CBL (13.3%), U2AF1 (12%) followed by EZH2, SETBP1, DNMT3A, and K/NRAS. Cross-sectional analysis demonstrated distinct variations in the distribution of individual mutations between subtypes. Similarly, global analysis of mutational frequencies showed only a few differences between clinical subtypes, e.g., SRSF2 mutations were found in 47% of MP-CMML vs. 24% in MD-CMML. JMML was showed a clearly distinct mutational pattern from CMML with PTPN11, NF1, K/NRAS and JAK3 mutations more commonly encountered in JMML. While ASXL1, EZH2 and TET2 (common in CMML) were not detected in JMML (p<0.001). CBL and SETBP1 were equally distributed in both diseases. When sAML from CMML was compared to Non-CBF AML M4/5, TET2 and ASXL1 mutations were more commonly found in sAML than in M4/M5 (40% vs. 6%; 30% vs. 0%, P<.0001). NPM1FLT3 and DNMT3A mutations were significantly more common in M4/M5.
Study of clonal architecture can allow for identification of ancestral vs. secondary events by serial or cross-sectional analyzes of variant allelic frequencies. Totally, 81% of TET2 mutations and 90% of SRSF2 mutations were ancestral, while 75% of U2AF1 and 78% of K/NRAS were secondary. Overall, 110 ancestral mutations were identified in 25 driver genes. CMML with founder clones included TET2 in 25%, SRSF2/ZRSR2 19%, ASXL1 12%, EZH2 5%, BCOR/BCORL1 8%, RUNX1 9%, SETBP1 5%, DNMT3A 5%, CBL/RAS 10% while all the others were present in 10% of CMML cases (of note is that 13% of them were co-dominant founder mutation). To examine whether this sub-classification corresponds to clinical phenotypes, they were correlated with clinical variables. For instance, ancestral mutations of TET2, ASXL1 and RUNX1 were commonly identified in both MD-CMML and MP-CMML while ancestral SETBP1, JAK2, SRSF2 and NRAS mutations were present only in MP-CMML, suggesting that initial genetic events of these 4 genes determine proliferative characteristics. In contrast, ancestral mutational events of SF3B1, DNMT3A, STAG2 and CBL were observed exclusively in MD-CMML. Secondary events further contribute to the clinical heterogeneity. For example, CBL secondary mutations were found in MP-CMML cases, while NRAS secondary mutations were observed in MD-CMML mainly in combination with ancestral STAG2 mutations. When we analyzed the impact of ancestral events on survival, EZH2 and U2AF1 mutant CMML conveyed worse prognosis (HR 3.7 and HR 2.2), but secondary EZH2 and U2AF1 mutations did not affect the outcome.
In sum, deep NGS allows for identification of specific ancestral events, which may determine the subsequent secondary mutational events in CMML. Classification of CMML based on ancestral events rather than on the global mutational spectrum correlates with clinical features and prognosis and may contribute to further clinical resolution of CMML based on the presence of specific founder mutations.
No relevant conflicts of interest to declare.
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