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  • Author: Anca Coliţă x
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A new assay to identify recurrent mutations in acute myeloid leukemia using next-generation sequencing

Abstract

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by a late onset (it is rare in children), aggressive phenotype and dismal prognosis especially in patients in the older group (>65 years). For risk stratification of patients standard cytogenetic is used along with molecular techniques for point mutation identification. Here we describe a new method using next generation sequencing for identification of mutation in 5 AML recurrently mutated genes - RUNX1, FLT3, DNMT3A, IDH1 and IDH2. Materials and methods: Samples from 40 patients with normal karyotype AML referred to Fundeni Clinical Institute were sequenced. Primer design was performed using LaserGene Genomics suit. Next generation sequencing was performed on MiSeq (Illumina) and results were analyzed using LaserGene Genomics suit. Results of next generation sequencing were compared to Sanger sequencing. Results: No additional mutations were identified in samples from nine patients presenting FLT3-ITD and/or NPM1 mutations. In 25 out of 31 patients with normal karyotype and no FLT3-ITD and NPM1 mutations, we identified mutations in one of the 5 aforementioned genes. All these mutations identified by next generation sequencing were confirmed using the classical Sanger sequencing. Conclusions: We validated a very useful method for mutation identification in AML patients using next generation sequencing. There are many advantages exhibited by this method: it is more cost efficient and it has a higher sensitivity of mutation detection than Sanger sequencing, it has been described as being quantitative and in our case it allowed risk stratification for most of the normal karyotype AML samples which were FLT3-ITD and NPM1 negative.

Open access
The use of biomarkers in detecting subclinical cardiotoxicity in doxorubicin-based treatment for paediatric patients with acute lymphoblastic leukaemia

Abstract

The international standard protocol for acute lymphoblastic leukaemia (ALL), the most common haemato-oncological pathology at paediatric age, uses anthracyclines as antitumor agents, potentially associated with early or late onset cardiac damage. Currently, echocardiography is the gold standard in the diagnosis of cardiotoxicity, but several biomarkers are evaluated as a possible replacement, pending more extensive clinical studies. We started a prospective study in order to determine the role of two biomarkers, troponin and heart-type fatty acid binding protein, in the evaluation of cardiotoxicity in children over one year of age, diagnosed with ALL. Between February 2015 and April 2016, 20 patients were enrolled and monitored at diagnosis, during chemotherapy and four months after the end of reinduction, through cardiac evaluation and dosing of those two markers in five different points of the treatment protocol. During the first year of follow-up, the patients did not develop clinical signs of cardiac damage, but the study showed a slight increase in troponin levels during chemotherapy, with the return to baseline value after treatment cessation, and also a correlation with the total dose of anthracyclines given to the patient. On the other hand, the second biomarker, heart-type fatty acid binding protein, did not seem to be useful in detecting subclinical cardiac damage in these patients.

Open access
Acquired von Willebrand disease: from theory to practice. A single center experience - three case reports

Abstract

Acquired von Willebrand disease (AvWD) represents a rare, potentially severe and most likely underdiagnosed category of hemorrhagic syndromes determined by quantitative, qualitative or functional, nonhereditary, alterations of von Willebrand factor (vWF) that occur in the context of various underlying diseases. It is diagnosed mainly in adults, without any personal or familial history of bleeding. The etiopathogeny of AvWD is complex, marked by the intervention of multiple mechanisms, occuring in the evolution of neoplasia, autoimmune disorders, cardiovascular diseases and other conditions. The clinical and laboratory manifestations are similar to the congenital form with mucocutaneous hemorrhage in patients without bleeding history and demonstration of quantitative and/or functional anomalies of vWF. Treatment has two major objectives: control of bleeding and therapy of the underlying condition. As a practical illustration of the theoretical aspects we present 3 clinical cases of AvWD diagnosed in the Colţea Hospital Department of Hematology during the last 10 years.

Open access
First Successful Haploidentical Stem Cell Transplantation in Romania

Abstract

Hematopoietic stem cell transplantation is an established treatment for many malignant and non-malignant haematological disorders. In the current case report, we describe the first haploidentical stem cell transplantation, used for the first time in Romania, the case of a 33 year-old young woman diagnosed with Hodgkin’s lymphoma that has underwent a haploSCT after she relapsed from several chemotherapy regimens, as well as after an autologous stem cell transplantation. This success represents a prèmiere in Romanian clinical hematology, being the first case of a haploSCT in Romania, as well as in South-Eastern Europe.

Open access
Therapy-related myelodysplastic syndrome after successful treatment of acute promyelocytic leukemia: case report and literature review

Abstract

In the 2016 revision of the World Health Organization classification the term therapy-related myeloid neoplasia (t-MN) defines a subgroup of acute myeloid leukemia (AML) comprising patients who develop myelodysplastic syndrome (MDS-t) or acute myeloid leukemia (AML-t) after treatment with cytotoxic and/or radiation therapy for various malignancies or autoimmune disorders. We report the case of a 36 year old patient with t-MN (t-MDS) after achieving complete remission (CR) of a PML-RARA positive acute promyelocytic leukemia (APL) at 32 months after diagnosis. Initially classified as low risk APL and treated according to the AIDA protocol - induction and 3 consolidation cycles - the patient achieved a complete molecular response in September 2013 and started maintenance therapy. On follow-up PML-RARA transcript remained negative. In January 2016 leukopenia and thrombocytopenia developed and a peripheral blood smear revealed hypogranular and agranular neutrophils. Immunophenotyping in the bone marrow aspirate identified undifferentiated blast cells that did not express cytoplasmic myeloperoxidase. The cytogenetic study showed normal karyotype. The molecular biology tests not identified PMLRARA transcript. A diagnosis of t-MDS (AREB-2 - WHO 2008) was established. Treatment of AML was started with 2 “3+7” regimens and 1 MEC cycle. Two months from diagnosis, while in CR, an allogeneic HSCT from an unrelated HLA compatible donor was performed after myeloablative regimen. An unfavorable clinical evolution was followed by death on day 9 after transplantation. The occurrence of t-MNs during CR of APL represents a particular problem in terms of follow-up and differential diagnosis of relapse and constitutes a dramatic complication for a disease with a favorable prognosis.

This work was supported by the grants PN 41-087 /PN2-099 from the Romanian Ministry of Research and Technology

Open access
The importance of the new prognostic scoring system for evaluating patients with lower-risk myelodysplastic syndrome at diagnosis

Abstract

Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal stem cell disorders characterized by ineffective hematopoiesis with dysplastic changes in one or more myeloid cell lines and increased risk of progression to acute leukemia. The current diagnosis criteria include the morphology of peripheral blood (PB) and bone marrow (BM), bone marrow biopsy and cytogenetic exam. Material and method. For this study, we have analyzed 33 patients diagnosed with lower-risk MDS (IPSS 0 and intermediate-1) according to the World Health Organization (WHO) classification (2001) between 2008 and 2012. The diagnosis was confirmed by blood cell counts, bone marrow (aspirate and biopsy) exam and cytogenetic exam. Other causes of cytopenia or dysplastic changes were excluded. Results. The types of MDS according to the WHO classification were: nine patients with refractory anemia (RA) (27.27%), sixteen patients with refractory anemia with ringed sideroblasts (RARS) (48.48%), and eight patients with refractory cytopenia with multilineage dysplasia (RCMD) (24.24%) out of which two with refractory cytopenia with multilineage dysplasia with ringed sideroblasts (RCMD-RS). Cytogenetic exam was performed in all patients, but analyzable metaphases for cytogenetic exam were obtained only from twenty five patients. The patients who did not have analyzable metaphases on cytogenetic exam were considered low risk if: they had only one cytopenia and the percent of bone marrow blasts was less than 5%. For all patients who had analyzable metaphases at cytogenetic exam, the International Prognostic Scoring System (IPSS) and Revised International Prognostic Scoring System (R-IPSS) scores were determined, and their survival and the death leading events were observed. According to IPPS (1997), the cytogenetic exam was good in 17 cases, intermediate in 1 case and poor in 7 cases. The IPSS score was low in 13 cases and intermediate-1 in 12 cases. According to R-IPSS, cytogenetic exams had been very good and good in 17 cases, intermediate in 1 case, poor in 6 cases and very poor in 1 case. R-IPSS was very low and low in 17 cases and intermediate and high in 8 cases. Conclusions. This new R-IPSS score at diagnosis allows a more accurate classification of patients into risk groups and thus enables risk adapted therapy

Open access
How Epstein-Barr Virus “Manipulates” The Tumoral Microenvironment in Hodgkin Lymphoma?

Abstract

The Epstein-Barr virus (EBV) is a gamma-herpesvirus that colonizes the B-cell system of its human host, allowing it to persist asymptomatically in the majority of the world’s adult population. In most people primary infection goes unnoticed, whereas in a minority of individuals, primary infection results in infectious mononucleosis (IM), a benign condition that almost always resolves after several weeks or months. However, EBV is also causally linked with a number of malignancies, including B-cell lymphomas, such as classical Hodgkin lymphoma (cHL).

A proportion of patients with cHL harbor EBV within their tumor cells. Emerging evidence suggests that while EBV is able to subvert cellular processes to promote the growth and survival of HRS cells or their progenitors, mutations in key cell signalization pathways are probably required to do this when EBV is absent. The challenge is to unravel exactly how EBV and its latent genes contribute to the pathogenesis of cHL particularly with respect to how the virus co-operates with cellular genetic and epigenetic changes to drive transformation. It is hoped that the development of better in vitro and in vivo models of disease will reveal more fundamental aspects of EBV’s role in Hodgkin lymphoma pathogenesis and pave the way for targeted therapies for patients with EBV-positive cHL.

Open access
Present and Future in Treatment of Hodgkin Lymphoma

Abstract

Hodgkin lymphoma, formerly known as Hodgkin disease, has gone from an incurable disease to one with a cure rate of almost 75%. The disease is defined in terms of its microscopic appearance (histology) and the expression of cell surface markers (immunophenotype), but its biologic behavior and clinical characteristics are also important.

Treatment of Hodgkin lymphoma is tailored to disease type, disease stage, and an assessment of the risk of resistant disease. General treatment modalities include radiation therapy, induction chemotherapy, salvage chemotherapy, and hematopoietic stem cell transplantation.

Open access