Introduction: Myelodysplastic syndrome (MDS) is characterized by differentiation blockade, cytopenias with commontransfusion dependency and immune defects. Upon progression the myeloblasts accumulate ...and the patients become vulnerable to severe infection complications. Based on the Prague Charles University General Hospital registry (N=164, median age 73), the AZA therapy in higher-risk MDS patients results in median OS 13.8 Mo with ORR 48.5%. We also noted from our retrospective data that AZA-treated patients with higher G-CSF consumption had significantly reduced occurrence of Grade 4 neutropenias and longer OS (19 vs 16 Mo, p value 0.039).
Rationale: To improve poor clinical outcomes we initiated a randomized open labeled academic trial that compares standard AZA therapy (A) with novel AZA-based therapy combination involving use of G-CSF added prior AZA (GA). Both AZA and also decitabine were preclinically shown to induce myeloid differentiation upon G-CSF preincubation. G-CSF binds its receptor in granulocytic precursors and neutrophils to stimulate their survival, proliferation, and differentiation via myeloid master regulator transcription factor and leukemia-suppressor PU.1. We also have previously shown that AZA increases PU.1. expression.
Study design & Methods: GA/MDS-2013 (EudraCT No 2013-001639-38). Expected for enrollment are 134 patients, currently enrolled 53 patients (GA arm N=29, A arm N=24) with median age 74 years, M:F ratio 32:21 (GA 16:16, A 13:8),median IPSS-R 6, median follow up 11.2 Mo, median cycles of therapy 6. Diagnosis included:MDS (EB1, EB 2) with IPSS int-2/high (75%), MDS/AML<30% MB (22.5%), and CMML II (2.5%). Transplant candidates were excluded. Randomization is 2:1 for GA vs A arm. Primary endpoints: OS, PFS, time to AML transformation, ORR, infections & QoL. Secondary endpoints: biomarkers. Therapy schedule: 75mg/m2 of AZA 5-2-2, in GA: G-CSF s.c. injected 48 hrs before dose 1 and dose 6. G-CSF is measured in patient sera (prior therapy), myeloid surface markers are determined by flow cytometry (day -2, day 1, and day 9 of cycle 1). Genomic libraries from whole bone marrow are prepared by NEBNext Direct Kit involving 33 gene panel, sequencing runs are performed on Illumina platform. Statistics involved longitudinal multivariate data analysis including the joint models for the OS and response.
Results: The presented data include 2.5 years since the beginning of the trial. Median survival for GA arm was 11 vs 6 Mo in the A arm. ORR (CR, CRm, PR, HI) was 56% in GA arm vs 33% in the A arm. Transformation to AML for both arms was comparable. The stratified longitudinal Cox proportional hazards model containing time-varying covariates together with the ordinal multilevel logistic mixed model were utilized. From this joint fitted model, a negative coefficient for the G-AZA treatment (significant p-value 0.0442) can be noticed in the case of the Cox Proportional Hazard part of the model. This means that G-AZA treatment improves patient survival. The estimated odds for the GA arm that responded to the therapy with remission rather than progression is 12.4x higher than for the A arm, controlling for the remaining patients' characteristics (p-value 0.0016).Both the GA and A arms are comparably tolerated. Data on serious infections and neutropenia gr4 were not yet available. The levels of G-CSF in sera prior the study in both arms (GA vs A) were comparable. Flow cytometry revealed G-CSF mediated upregulation of FCgRI (CD64) in the GA but not in the A arm. Multivariate analysis indicates the following: mutated genes: DNMT3A (p-value 0.0157), EZH2 (p-value 0.0091), TP53 (borderline p-value 0.0510), & CSF3R (p-value 0.0057) shorten the overall survival. The significant negative effects on response was noted for mutated EZH2 (p-value 0.0208) and CSF3R (p-value 0.0424) genes.
Conclusions: The current results supported by different methods and statistics indicates a beneficial effect of G-CSF pre-treatment to standard AZA therapy in higher risk MDS patients. G-CSF pre-treatment to AZA increases OS and ORR. In addition, we identified biomarkers that are negatively associated with patient survival and response including EZH2, DNMT3A, TP53, & CSF3R.
Grant Support: Ministry of Health, #16-27790A. Institutional resources: Progres Q49 & Q26, UNCE/MED/016, LQ1604, SVV 260374/2017, RVO-64165.
No relevant conflicts of interest to declare.
Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene ...homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.
Purpose
Here, we studied whether amplicon next-generation deep sequencing (NGS) could improve the detection of emerging
BCR
-
ABL1
kinase domain mutations in chronic phase chronic myeloid leukemia ...(CML) patients under tyrosine kinase inhibitor (TKI) treatment and discussed the clinical relevance of such sensitive mutational detection.
Methods
For NGS data evaluation including extraction of biologically relevant low-level variants from background error noise, we established and applied a robust and versatile bioinformatics approach.
Results
Results from a retrospective longitudinal analysis of 135 samples of 15 CML patients showed that NGS could have revealed emerging resistant mutants 2–11 months earlier than conventional sequencing. Interestingly, in cases who later failed first-line imatinib treatment, NGS revealed that TKI-resistant mutations were already detectable at the time of major or deeper molecular response. Identification of emerging mutations by NGS was mirrored by
BCR
-
ABL1
transcript level expressed either fluctuations around 0.1 %
IS
or by slight transcript level increase. NGS also allowed tracing mutations that emerged during second-line TKI therapy back to the time of switchover. Compound mutants could be detected in three cases, but were not found to outcompete single mutants.
Conclusions
This work points out, that next-generation deep sequencing, coupled with a robust bioinformatics approach for mutation calling, may be just in place to ensure reliable detection of emerging
BCR
-
ABL1
mutations, allowing early therapy switch and selection of the most appropriate therapy. Further, prospective assessment of how to best integrate NGS in the molecular monitoring and clinical decision algorithms is warranted.
In this paper there are presented two nonlinear models within the area of linearized elasticity and some applications for them. First model aims to describe the degradation of linearized elastic ...solid by considering the Hooke's law with material moduli which depends on a concentration of a diffusing fluid. This model is solved numerically on a square sample with a elliptic hole and the fluid diffuses through the hole. Second model introduces constitutive relation where strain is nonlinear function of the stress into the framework of linearized elasticity. This model can be used to model materials for which maximal strain is a priori bounded. It has been recently shown that such models can be justified by means of implicit constitutive theory. Using this model is studied square sample with a V-notch subject to anti-plane stress. Described problems are solved using finite element method.
Introduction: Myelodysplastic syndromes (MDS) are clonal disorders of myeloid hematopoietic stem cells. Recent studies has shown that nearly 90% of patients with MDS carry somatic mutations in bone ...marrow (BM). These findings triggered a number of studies to identify potential uses of these mutations for diagnostics and prognostics purposes. We focused on a group of 38 patients with advanced stages of the disease that were selected for Azacitidine (AZA) therapy. We then utilized a set of 98 BM samples from the patient cohort that were collected in different stages before, during, and after the period of 4-12 cycles of the therapy. Each patient provided 3 samples on average. This study excludes patients that died early on AZA. Median OS on AZA therapy was 31 Mo. Most prevalent MDS subtypes were RAEB-2 (55%), RAEB-1 (24%), and MDS/AML (13%). 20% of patients had complex karyotype or poor cytogenetics (MedOS=22Mo) and the rest had mostly normal karyotype or intermediate cytogenetics (MedOS=40Mo) prior to AZA. Progression to AML was observed in 55% of patients (PFS= 16 Mo). After 4 cycles, PR was achieved in 59% of patients, CR in 12%, while SD was maintained in 21%, and 9% of patients progressed (PD) to AML.
Methods: We detected relevant mutations in MDS samples using the following approach. We collected genomic DNA from separated BM samples: either a CD3-negative population containing the myeloid compartment, or CD3-positive T cells representing an internal control. We prepared amplicon libraries from these samples using the Illumina TruSight Myeloid Panel that covers 54 key genes involved in myeloid malignancies (notably MDS and AML). We sequenced these libraries using the Illumina NGS platform. To achieve greater sensitivity in detecting SNVs and InDels we utilized two different variant calling pipelines (using samtools mpileup or FreeBayes). Since the (PCR) validation efficacy of each mutation from the single NGS run was below 60%, we improved specificity by using two independently prepared sequencing libraries. The intersection of the variant detections from both libraries was considered accurate and only these data were reported as variants.
Results: When we excluded all germinal variants, 43 somatic variants in ~18 genes were identified per patient on average. The majority (31/43) of these variants had an intermediate impact (on amino acid sequence), while 12/43 had high impact on the protein structure. Importantly, the majority of them had ~1% VAF (variant allele frequency) representing putative clones with low proliferative potential. In contrast, only 8 genes (~14 variants) were mutated with VAF>2%. The following genes were mutated most frequently: TET2, STAG2, ASXL1 in approximately 60-80% of patients. Data from repeatedly analyzed patient samples on AZA therapy led to an unexpected observation that the variants with WAF>2% often exhibit dynamically changing mutation pattern while the variants of non-proliferating clones (with VAF ~1%) remain very stable. We observed prominent development of some variants (ASXL1, STAG2, CUX1, BCOR) as well as an increase in VAF of others (TP53, RUNX1, CUX1) on AZA therapy. Most of these genes when mutated were reported previously as altering prognosis of MDS (Bejar R et al, 2014). Surprisingly, in some samples we found a mutation in the RUNX1 gene before AZA therapy that was not present after the treatment however, after the treatment another not previously observed mutation of RUNX1 emerged. Furthermore, the presence of any of the mutations before AZA including SF3B1 or TP53 did not have any prognostically significant association with OS or PFS. This contention is supported by the fact that many mutations actually disappeared on AZA.
Conclusions: Using an internal sample control combined with a duplicate NGS library preparation we achieved a very high accuracy of detecting somatic variants in MDS-BM sub-separated samples. We observed that variants above 2% VAF change dynamically over the course of AZA therapy while the variants with ~1% VAF remain stable. Our data suggest that development of somatic mutations in AZA-treated MDS patients is a dynamic process, which involves previously identified high risk genes including TP53, RUNX1, CUX1, ASXL1 and BCOR.
Grant support: GAČR 16-05649S & P305/12/1033, AZV: 16-27790A, CZ.1.05/1.1.00/02.0109, UNCE 204021, LH15170, PRVOUK P24, LQ1604 and RVO-VFN64165.
No relevant conflicts of interest to declare.
In this paper we study the deformation of a body with a notch subject to an anti-plane state of stress within the context of a new class of elastic models. These models stem as approximations of ...constitutive response functions for an elastic body that is defined within the context of an implicit constitutive relation between the stress and the deformation gradient. Gum metal and many metallic alloys are described well by such constitutive relations. We consider the state of anti-plane stress of a body with a smoothened V-notch within the context of constitutive relations for the linearized strain in terms of a power-law for the stretch. The problem is solved numerically and the convergence and the stability of the solution is studied.
Azacitidine (AZA) for higher risk MDS patients is a standard therapy with limited durability. To monitor mutation dynamics during AZA therapy we utilized massive parallel sequencing of 54 genes ...previously associated with MDS/AML pathogenesis. Serial sampling before and during AZA therapy of 38 patients (reaching median overall survival 24 months (Mo) with 60% clinical responses) identified 116 somatic pathogenic variants with allele frequency (VAF) exceeding 5%. High accuracy of data was achieved via duplicate libraries from myeloid cells and T-cell controls. We observed that nearly half of the variants were stable while other variants were highly dynamic. Patients with marked decrease of allelic burden upon AZA therapy achieved clinical responses. In contrast, early-progressing patients on AZA displayed minimal changes of the mutation pattern. We modeled the VAF dynamics on AZA and utilized a joint model for the overall survival and response duration. While the presence of certain variants associated with clinical outcomes, such as the mutations of
were adverse predictors while
mutations yield lower risk of dying, the data also indicate that allelic burden volatility represents additional important prognostic variable. In addition, preceding 5q- syndrome represents strong positive predictor of longer overall survival and response duration in high risk MDS patients treated with AZA. In conclusion, variants dynamics detected via serial sampling represents another parameter to consider when evaluating AZA efficacy and predicting outcome.
Introduction and hypothesis: Somatic gene mutations develop in ~78% Myelodysplastic syndrome (MDS) patients. MDS progresses into an unstable phase characterized by an accumulation of myeloblasts that ...is indicated for the DNA demethylation therapy with Azacitidine (AZA). To understand whether AZA is capable to eliminate tumor cells and whether a mutation pattern responds to AZA we herein tracked mutations in the bone marrow (BM) during AZA treatment. Two scenarios were postulated: 1) AZA treatment will/ will not eliminate clones characterized by specific mutations and this will relate to the clinical outcome, 2) during clinical progression on the AZA treatment either the new mutations will develop or the original mutations will be detected.
Patients: 40 int-2/high-risk MDS patients (176 samples, median age 70, 22F/18M) indicated for AZA (75mg/m2, 5+2+2) were sequenced. The MDS subtypes included RAEB1, RAEB2, and MDS/AML. Half of the patients progressed from the 5q-syndrome. Libraries were prepared using TruSight DNA amplicon kit. Set of 54 myeloid genes (associated with MDS or AML) were sequenced by Illumina platform HiSeq 2500 with depth >100 per mutation, mutation should be heterozygous, non-synonymous, exonic with frequency >10%. As controls: 4 normal BM samples and 2 cord bloods (also applied for data filtering) and MOLM-13 cell line carrying FLT3-ITD mutation were used. Additional controls included CD3+ T cells isolated from the patient BM samples.
Results: 70% of patients were informative for at least one somatic mutation with high impact on the amino acid sequence. Mutations in TET2 (in 13%) were overall the most frequent before AZA was started (followed by mutations in BCOR, RUNX1, STAG2, and NOTCH genes). In 36% of informative patients, the mutation pattern developed on AZA. Interestingly, the most frequent mutations after AZA were in ASXL1 in 10% (followed by mutations in TET2, BCOR, and CUX1 genes). While mutations in ASXL1 and NOTCH genes developped only in the non-5q derived patients, the mutations in RUNX1 developped only in the 5q-derived higher-risk MDS patients. Average number of mutations before AZA and on AZA was 2 per patient. Complete elimination of the mutation pattern was noted in 57% of informative patients during the first 8 months of AZA treatment and this was associated with the therapy responsiveness (PR or CR). In contrast in 7% of informative patients the mutation pattern remained the same and this was associated with the stable disease (leading to progression). Upon progression on the AZA treatment we have observed appearance of the new mutations in 73% of informative patients. 20% of patients progressing after 14 cycles of AZA were non-informative which suggests that these patients may carry mutations in genes not included within the tested set. We have assessed overall survival (OS) according to the mutation status in the following subgroups: non-informative patients, informative patients with mutations that were eliminated by AZA, and those retaining or gaining the mutations upon AZA. The group that developed or retained mutations during first 4-8 cycles of AZA displayed significantly lower OS (p=0.004, median OS:11 months) compared to either non-informative patients (26 months) or those where AZA completely eliminated the mutation pattern (28 months).
Conclusions: Tracking the mutations in MDS patients during AZA therapy provides opportunity to detect clonal development in 2/3 of the MDS patients and relate these data to the clinical outcome. Moreover, progression on AZA therapy is usually associated with the development of a new mutation pattern and this coincides with the significantly lower OS.
Grants: GACR P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/1.
Stopka:Celgene: Research Funding.
Mantle cell lymphoma (MCL) is a relatively distinct B-cell non-Hodgkin lymphoma subtype with aggressive and often recurrent clinical course. At diagnosis, MCL often manifests with leukemization, a ...feature more common to chronic lymphocytic leukemia (CLL). Common features and differences between MCL and CLL were not yet explored by comprehensive global approaches, despite such understanding potentially being very neat for deciphering pathogenesis and tailoring therapies of these clinically distinct diseases.
In our study, we have compared MCL(n=10), CLL(n=10) and normal control(n=8) B-cell samples using the Affymetrix Human Genome HG-U133 Plus 2.0 Array. We studied different mRNA levels of ~47.000 transcripts represented on the array. The comparative analyses identified a set of 892 differentially expressed genes between MCL and NBC; and 774 differentially expressed genes between CLL and NBC. In order to find MCL/CLL-specific biomarkers we focused on the intersection of differently expressed genes in both groups (CLL vs NBC and MCL vs NBC). There were 222 mRNAs in the intersection, 216 of them were deregulated in the same direction in both groups while 6 mRNAs were deregulated in the opposite direction. This set of 6 disease-specific mRNAs contained previously reported biomarkers (CD200, LEF1), and also the Myristoylated alanine-rich C-kinase substrate (MARCKS) that has not yet been studied in MCL. Thus we utilized the validation patient groups (NMCL=6, NCLL=8) and confirmed differential expression of MARCKS on protein levels by flow cytometry and immunofluorescence. As MARCKS was previously shown to either bound to the cell membrane, to reside in the cytosol, or alternatively become transmitted to nuclei, we investigated subcellular localization of MARCKS using immunofluorescence (IF). The cytoplasmic MARCKS signal in MCL was significantly higher than in CLL while the opposite was observed for the nuclear IF signal. The ratio between cytoplasmic and nuclear signal was 2.5 for MCL and 0.8 for CLL (p < 0,0001). The active forms of MARCKS were shown to become phosphorylated on serineresidues and this prompted us to study the phosphorylation forms of MARCKS in MCL. Indeed, one of the residues, Ser159/163, was hyperphosphorylated in the MCL cytoplasm and its level and distribution markedly differed from CLL or NBC. We next searched for regulatory mechanisms upstream of the MARCKS expression in MCL vs CLL. MARCKS is a predicted target of several microRNAs (according to DIANA-TarBase v7.0), among them also of miR-155 (that is differentially expressed between MCL and CLL). To further investigate the regulatory relationship between mir-155 and MARCKS we utilized a CLL cell line MEC-1 and using the CRISPR/Cas9 technology we prepared individual cell clones that were mutated within the mature miR-155 sequence that recognizes MARCKS mRNA. As expected, the miR-155-MEC-1 mutants expressed markedly higher level of MARCKS compared to the control MEC-1 cells.
In conclusion, our work identified a set of six differentially expressed mRNAs when comparing MCL and CLL, among them, MARCKS. We further showed that MARCKS is differentially expressed, localized, and phosphorylated between MCL and CLL, and that MARCKS is partly controlled by oncogenic microRNA miR-155. MARCKS may play an important role in MCL pathogenesis and can serve as useful MCL biomarker.
Grant support: GAČR 16-05649S & P305/12/1033, AZV: 16-27790A and 16-31586A. Institutional support: CZ.1.05/1.1.00/02.0109, UNCE 204021, LH15170, PRVOUK P24, LQ1604.
No relevant conflicts of interest to declare.
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