The presence of additional cytogenetic aberrations in chronic myeloid leukemia cells at the time of diagnosis or their appearance on tyrosine kinase inhibitor therapy predicts the imatinib treatment failure.

Currently used treatment of CML dramatically improved the prognosis of disease. However, additional chromosome aberrations (ACA/Ph+) are still one of the adverse prognostic factors. OBJECTIVES: evaluation of the impact of ACA/Ph+ appearance during disease outcome on the response to treatment. THE STUDY GROUP: consisted of 203 patients. The median time of follow-up was 72 months. ACA/Ph+ was found in 53 patients. RESULTS: patients were divided into four groups: standard risk, intermediate, high and very high risk. When ACA/Ph+ presence was documented at diagnosis time the optimal response was observed in 41.2%, 25%, and 0% of pts with intermediate, high and very high risk, respectively. If ACA/Ph+ were detected during imatinib treatment the optimal response was in 4.8% of patients. The risk of blastic transformation for patients with standard risk, intermediate, high and very high risk was 2.7%, 18.4%, 20% and 50%, respectively. CONCLUSIONS: the presence of ACA/Ph+ at diagnosis time or their appearance on therapy seems to be clinically relevant not only in terms of the risk of blastic transformation but also in terms of the treatment failure. Gathering patients with various karyotypes and their responses to treatment would allow to set better guidelines and predictions.

Defining the mutational profile of lower-risk myelodysplastic neoplasm patients with respect to disease progression using next-generation sequencing and pyrosequencing.

Introduction: Lower-risk myelodysplastic neoplasms (LR-MDS) comprise the majority of MDS. Despite favourable prognoses, some patients remain at risk of rapid progression. We aimed to define the mutational profile of LR-MDS using next-generation sequencing (NGS), Sanger Sequencing (SSeq), and pyrosequencing. Material and methods: Samples from 5 primary LR-MDS (67 exons of SF3B1, U2AF1, SRSF2, ZRSR2, TET2, ASXL1, DNMT3A, TP53, and RUNX1 genes) were subjected to NGS. Next, a genomic study was performed to test for the presence of identified DNA sequence variants on a larger group of LR-MDS patients (25 bone marrow [BM], 3 saliva [SAL], and one peripheral blood [PB] sample/s). Both SSeq (all selected DNA sequence variants) and pyrosequencing (9 selected DNA sequence variants) were performed. Results: Next-generation sequencing results identified 13 DNA sequence variants in 7 genes, comprising 8 mutations in 6 genes (ASXL1, DNMT3A, RUNX1, SF3B1, TET2, ZRSR2) in LR-MDS. The presence of 8 DNA variants was detected in the expanded LR-MDS group using SSeq and pyrosequencing. Mutation acquisition was observed during LR-MDS progression. Four LR-MDS and one acute myeloid leukaemia myelodysplasia-related patient exhibited the presence of at least one mutation. ASXL1 and SF3B1 alterations were most commonly observed (2 patients). Five DNA sequence variants detected in BM (patients: 9, 13) were also present in SAL. Conclusions: We suggest using NGS to determine the LR-MDS mutational profile at diagnosis and suspicion of disease progression. Moreover, PB and SAL molecular testing represent useful tools for monitoring LR-MDS at higher risk of progression. However, the results need to be confirmed in a larger group.

New genetic variants of TET2 and ASXL1 identified by next generation sequencing and pyrosequencing in a patient with MDS-RS-MLD and secondary acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by the presence of cytopenias, ineffective hematopoiesis and frequent transformation into secondary acute myeloid leukemia (secAML). Recent genomic studies provide unprecedented insight into the molecular landscape of clonal proliferation in MDS. Genetic diversity of both MDS and secAML subclones cannot be defined by a single somatic mutation. Mutations of the founding clone may survive over implemented chemotherapy and allogenic hematopoietic cell transplantation (alloHCT), but new subclonal mutations may also appear. Next generation sequencing (NGS) makes it possible to define the mutational profile of disease subclones during the treatment course and has a potential in pre- and post-alloHCT monitoring. Understanding the molecular pathophysiology of MDS may soon allow for monitoring the course of disease and personalized treatment depending on the mutational landscape. In the present paper we report, for the first time in MDS, ASXL1 c.1945G>T, TET2 c.4044+2dupT and c.4076G>T sequence variants. Moreover, we detected RUNX1 c.509-2A>C and SF3B1 c.1874G>T sequence variants. Furthermore, we verify the clinical utility of NGS and pyrosequencing in MDS and secAML.

Comparative proteomic profiling of sera from patients with refractory multiple myeloma reveals potential biomarkers predicting response to bortezomib-based therapy.

INTRODUCTION: In the era of implementing novel agents in multiple myeloma (MM) regimens, drug resistance has become a key factor undermining the results of treatment. Identifying biomarkers allows the prediction of therapy outcomes with specific agents and may lead to the avoidance of resistance. OBJECTIVES: This study aimed to identify biomarkers in the pretreatment sera of patients with refractory/ relapsed MM that differ from those in the sera of patients who achieved a better depth of response with bortezomib-containing therapy. PATIENTS AND METHODS: Pretreatment serum samples were obtained from 61 proteasome inhibitor-naive, transplant-eligible patients who were eligible for salvage PAD (bortezomib, doxorubicin, and dexamethasone) or VTD (bortezomib, thalidomide, and dexamethasone) chemotherapy. Based on their response to therapy, patients were classified into 3 groups: complete or very good partial response, partial response, and progressive or stable disease. A comparative proteomic analysis of the groups was performed. RESULTS: The analyzed groups significantly differed in terms of both overall survival and progression­free survival. In total, 632 proteins were identified. The proteomic signature revealed 54 proteins that differentiated each analyzed experimental group. Functional analysis revealed that the main identified pathways (17 proteins) involved the regulation of hydrolase activity and cellular response to stimuli. The identified proteins included apolipoprotein C1, complement components, and sulfhydryl oxidase 1. CONCLUSIONS: Our results demonstrated that the label-free proteomic analysis is a useful method for describing proteins differentially expressed in the sera of patients with MM. Further studies are needed to analyze the use of identified proteins as biomarkers.

Translocation t(8;14)(q24;q11) with concurrent PTEN alterations and deletions of STIL/TAL1 and CDKN2A/B in a pediatric case of acute T-lymphoblastic leukemia: A genetic profile associated with adverse prognosis.

We report a pediatric case of acute T-lymphoblastic leukemia (T-ALL) with NOTCH1wt , FBXW7wt , STIL/TAL1, and PTEN (exons 2, 3, 4, 5) monoallelic deletions, biallelic CDKN2A/B deletion, and a minor t(8;14)(q24;q11)-positive subclone. Undetectable by a flow cytometric minimal residual disease assay, the t(8;14)(q24;q11) subclone expanded as detected by fluorescence in situ hybridization from 5% at diagnosis to 26% before consolidation and 100% at relapse bearing a monoallelic deletion (exons 2, 3) with a new frameshift mutation of PTEN and the same set of remaining molecular alterations. This case documents an unfavorable prognostic potential of a co-occurrence of this set of molecular genetic events and addresses risk stratification in T-ALL.

Comparative proteomic profiling of refractory/relapsed multiple myeloma reveals biomarkers involved in resistance to bortezomib-based therapy.

Identifying biomarkers of the resistance in multiple myeloma (MM) is a key research challenge. We aimed to identify proteins that differentiate plasma cells in patients with refractory/relapsed MM (RRMM) who achieved at least very good partial response (VGPR) and in those with reduced response to PAD chemotherapy (bortezomib, doxorubicin and dexamethasone). Comparative proteomic analysis was conducted on pretreatment plasma cells from 77 proteasome inhibitor naïve patients treated subsequently with PAD due to RRMM. To increase data confidence we used two independent proteomic platforms: isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and label free (LF). Proteins were considered as differentially expressed when their accumulation between groups differed by at least 50% in iTRAQ and LF. The proteomic signature revealed 118 proteins (35 up-regulated and 83 down-regulated in ≥ VGPR group). Proteins were classified into four classes: (1) involved in proteasome function; (2) involved in the response to oxidative stress; (3) related to defense response; and (4) regulating the apoptotic process. We confirmed the differential expression of proteasome activator complex subunit 1 (PSME1) by enzyme-linked immunosorbent assay. Increased expression of proteasomes and proteins involved in protection from oxidative stress (eg., TXN, TXNDC5) plays a major role in bortezomib resistance.

[Point mutations of genes encoding proteins involvedin RNA splicing in patients with myelodysplastic syndromes]. / Mutacje punktowe genów kodujacych bialka biorace udzial w splicingu RNA u chorych z zespolami mielodysplastycznymi.

The myelodysplastic syndromes (MDS) constitute heterogeneous group of clonal disorders, characterized by ineffective hematopoiesis, peripheral cytopenia and increased risk of acute myeloid leukemia development. Molecular mechanisms behind MDS have not been fully explained, however recent studies based on new technologies confirmed that epigenetic abnormalities and somatic mutation in the spliceasome machinery are crucial in pathogenesis of these diseases. Abnormal mRNA splicing (excision of intronic sequences from mRNA) has been found in over half of all MDS patients and resulted in accumulation of cytogenetical and molecular changes. The biological impact of splicing factor genes mutations has been evaluated only in a limited extend and current studies concentrate on analysis of MDS transcriptome. Molecular characteristic of classical and alternative splicing is presented in the paper, according to current knowledge. We review the most prominent findings from recent years concerning mutation in the spliceasome machinery with respect to MDS phenotype and disease prognosis. Perspectives in applying of novel diagnostic and therapeutic possibilities for myelodysplasia, based on spliceosome mutations identification are also presented.