Iwona Solarska

Cladribine added to daunorubicin-cytarabine induction prolongs survival of FLT3-ITD+ normal karyotype AML patients.

To the editor: Internal tandem duplication in the FLT3 gene ( FLT3- ITD) has been recognized as a marker conferring poor outcome in patients with normal karyotype acute myeloid leukemia (NK-AML).[1][1] Because of the inferior outcome of FLT3- ITD+ NK-AML patients when treated with standard

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia

Fms-like tyrosine kinase 3–internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene–partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl’s rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

Mild chronic graft-versus-host disease may alleviate poor prognosis associated with FLT3 internal tandem duplication for adult acute myeloid leukemia following allogeneic stem cell transplantation with myeloablative conditioning in first complete remission: a retrospective study

Internal tandem duplication (ITD) of the FLT3 gene (Fms‐like tyrosine kinase 3) is the most commonly found mutation in acute myeloid leukemia (AML). The significance of FLT3‐ITD at diagnosis was retrospectively estimated for allo‐HSCT (allogeneic hematopoietic stem cell transplantation) outcomes in 140 patients, median age of 38, undergoing allo‐HSCT after myeloablative conditioning in first complete remission of AML. FLT3‐ITD was detected at AML diagnosis in 42/140 (30%) of included into this study patients. At 3 years, relapse incidence (RI) following allo‐HSCT in AML patients with intermediate or normal karyotype was significantly higher in those FLT3‐ITD positive than FLT3‐ITD negative [52.9 vs. 20.4%, P = 0.002]. Additionally, patients with mild chronic graft‐versus‐host disease (cGvHD) had significantly lower RI compared to patients with moderate or severe grade cGvHD or those not experiencing cGvHD, respectively, 4.8 vs. 36.0 vs. 27.8%, P = 0.032. FLT3‐ITD was harboring a poor prognosis in AML with intermediate or normal karyotype and significantly increased risk of relapse following allo‐HSCT. It appears that allo‐HSCT does not cure patients with FLT3‐ITD, unless they develop symptoms of mild cGvHD and graft versus leukemia, which may decrease RI.

CEBPA copy number variations in normal karyotype acute myeloid leukemia: Possible role of breakpoint-associated microhomology and chromatin status in CEBPA mutagenesis

Copy number variations (CNV) in CEBPA locus represent heterogeneous group of mutations accompanying acute myeloid leukemia (AML). The aim of this study was to characterize different CEBPA mutation categories in regard to biological data like age, cytology, CD7, and molecular markers, and identify possible factors affecting their etiology. We report here the incidence of 12.6% of CEBPA mutants in the population of 262 normal karyotype AML (NK-AML) patients. We confirmed that double mutant AMLs presented uniform biological features when compared to single CEBPA mutations and accompanied mostly younger patients. We hypothesized that pathogenesis of distinct CEBPA mutation categories might be influenced by different factors. The detailed sequence analysis revealed frequent breakpoint-associated microhomologies of 2 to 12bp. The analysis of distribution of microhomology motifs along CEBPA gene showed that longer stretches of microhomology at the mutational junctions were relatively rare by chance which suggests their functional role in the CEBPA mutagenesis. Additionally, accurate quantification of CEBPA transcript levels showed that double CEBPA mutations correlated with high-level CEBPA expression, whereas single N-terminal CEBPA mutations were associated with low-level CEBPA expression. This might suggest that high-level CEBPA expression and/or accessibility of CEBPA locus contribute to B-ZIP in-frame duplications.

Differential expression of BIRC family genes in chronic myeloid leukaemia – BIRC3 and BIRC8 as potential new candidates to identify disease progression

Although the therapy of chronic myeloid leukaemia (CML) with tyrosine kinase inhibitors (TKI) is considered a major advance in oncology, a significant group of patients still develops drug resistance and does not benefit from targeted therapy (Marin et al, 2013). These patients are at high risk of progression from chronic phase (CML-CP) to almost inevitably fatal blastic phase (CML-BP) (or blast crisis). Therefore, there is a need to identify suboptimal responders to imatinib who should be switched to secondor third-generation TKI as early as possible (Marin et al, 2013). Although the Sokal and Hasford risk scores remain the most used prognostic indicators at diagnosis, reliable markers of suboptimal response and/or progression of the disease are needed. BCR-ABL1 transcript level monitoring is used to evaluate response to the therapy, and its predictive value at 3 and/or 6 months after starting TKI therapy has recently been shown (Neelakantan et al, 2013). Among other possible candidates, high levels of cancerous inhibitor of PP2A (CIP2A) may be an independent determinant of progression to blast crisis (Lucas et al, 2011). Searching for potential candidates of disease progression we have focused on the BIRC (baculoviral IAP repeat-containing; BIRC) gene family expression in various stages of CML. To date, there is no comprehensive analysis of the whole family of BIRC genes in CML. The BIRC family comprises eight functionallyand structurally-related members. Their common structural feature, a motif termed the baculovirus IAP repeat, is required for their cytoprotective function, hence the majority of BIRC serve as endogenous inhibitors of apoptosis (Deveraux & Reed, 1999). While the first human BIRC gene (originally termed BIRC1, now NAIP, encoding the NLR family, apoptosis inhibitory protein) was described in the neurodegenerative disorder, spinal muscular atrophy (Roy et al, 1995), most recent studies focus on the role of BIRC genes in various types of neoplasms. Overexpression of BIRC genes has been associated with cancer progression, multidrug resistance, poor prognosis and short survival in several cancers including haematological malignancies. One of the most extensively studied members of BIRC family – BIRC5 (BIRC5, also known as survivin) is upregulated in haematological malignancies and solid tumours, and was also shown to be overexpressed in CML-BP in comparison to CML-CP (Hernandez-Boluda et al, 2005). Higher expression of BIRC5 was also linked to higher Sokal score and was positively correlated with P-glycoprotein expression in late CML-CP (Reis et al, 2011). Recently, the disruption of BIRC3, leading to truncated protein expression has been implicated in resistance to fludarabine in chronic lymphocytic leukaemia (CLL) (Rossi et al, 2012). To elucidate the potential role of the BIRC family of genes in CML we investigated the relative expression of all eight known BIRC family members. We analysed sequential samples of cDNA from peripheral blood obtained from CML patients at various stages of the disease. Blood samples were taken after informed consent was obtained and reverse transcription (RT)-quantitative real time polymerase chain reaction (qPCR) experiments were performed according to MIQE (Minimum Information for Publication of qPCR Experiments) guidelines (Bustin et al, 2009). Initially we looked at samples from CML-CP patients at diagnosis and after development of TKI resistance (confirmed as a loss of cytogenetic response, n = 5). Four patients developed resistance to imatinib (one had V299L mutation); the fifth was resistant to imatinib, dasatinib and nilotinib (no mutation detected) (Fig. 1A). We then analysed sequential samples from patients with CML who progressed to either accelerated phase (CMLAccPh) (n = 2) or to CML-BP (n = 4). Contrary to the general view of the role of BIRC genes in tumour progression, we found a significant decrease in BIRC3 and BIRC8 expression in CML-CP after development of TKI resistance (Fig. 1A) and also in progression to CML-AccPh/BP (Fig. 1B). We also observed a marked increase in BIRC5 expression after progression to CML-AccPh/BP (as previously shown by Hernandez-Boluda et al, 2005) (Fig. 1B) but not in CML-CP samples obtained after development of TKI resistance (Fig. 1A). Expression of other BIRC genes, namely: NAIP (BIRC1), BIRC2, XIAP (BIRC4), BIRC6 and BIRC7 was comparable in all the studied stages of the disease (data not shown). To confirm these results analysis of a larger group of patients was performed. Samples were obtained from patients in either CML-CP at diagnosis prior to any treatment (n = 15) or in CML-BP (n = 11) (Fig. 2). To compare BIRC family genes expression in CML with that of normal haematopoietic cells, cDNA from healthy blood donors was also used (n = 10). In accordance with paired samples analysis, we observed downregulation of BIRC3 and BIRC8 expression in CML-BP, while BIRC5 was upregulated in CML-BP patients (as compared to CML-CP and healthy blood donors) (Fig. 2). There was no significant difference in the relative expression of other BIRC family members (data not shown). correspondence

Planowana ciąża u chorej na przewlekłą białaczkę szpikową leczoną imatynibem

Tyrosine kinase inhibitors (TKI) used in the treatment of chronic myelogenous leukemia (CML) can cause disturbances in fetal development in pregnant women. Women of childbearing potential are recommended to practice effective contraception. Women who want to become pregnant should stopped treatment with TKI before and during pregnancy. The optimal time to stop treatment is deep molecular response after minimum 2 years of treatment. We present a case study of a 24-year- -old female patient with CML, diagnosed in myeloblastic phase. After treatment with imatinib deep and permanent molecular remission was achieved. Treatment was interrupted after 5 years in order to become pregnant. During pregnancy increase in BCR-ABL1 transcript was observed and treatment with interferon a was introduced. The baby was healthy. The imatinib was reintroduced, after 3 months deep molecular response was achieved.

BCRP mRNA and FLT3-ITD are independent poor risk factors in adult patients with acute myeloid leukemia and intermediate or normal karyotype

FMS‐like tyrosine kinase 3‐internal tandem duplication (FLT3‐ITD) is aberration associated with poor prognosis in AML. We have analyzed the expression of MDR‐1, MRP‐1, and BCRP mRNA in relation to FLT3‐ITD in 100 AML adult patients with normal and intermediate karyotype.

Negative impact of FLT3-ITD mutation on expression of MDR-1 mRNA in adult acute myeloid leukemia.

Th e treatment outcome of patients with acute myeloid leukemia (AML) varies considerably, and refl ects the heterogeneity of the disease. Although the development of new therapeutic agents and treatment regimens has improved the cure rate of patients with AML, from 20 to 50% of patients are primarily resistant to induction chemotherapy and most of the remaining will relapse and die of their disease. Outcomes are poor in part due to resistance to chemotherapeutic drugs. While some mechanisms of drug resistance have been well characterized, this phenomenon still remains incompletely understood. One mechanism behind drug resistance is altered drug transport, which could be due to overexpression of the transport proteins: p-glycoprotein (Pgp), multidrug resistance protein-1 (MRP-1), breast cancer resistance protein (BCRP) and lung resistance-related protein (LRP). Th ree of them – Pgp, MRP-1 and BCRP – are members of the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, and function as a membrane pump responsible for the effl ux of a range of chemotherapeutic drugs. Pgp is the protein product of the multidrug resistance-1 ( MDR-1 ) gene, and is still considered by many researchers as the prognostic marker and major determinant for poor outcomes in AML [1 – 4]. It was shown that Pgp also has an anti-apoptotic function in AML that is independent of its role in drug effl ux. Pgp can inhibit ceramide-mediated apoptosis [5]. Compounds that inhibit the effl ux function of Pgp in AML cells may also be active against its anti-apoptotic function. Th ere has been much recent interest in identifying new molecular markers of prognostic value in AML. Of these, internal tandem duplication (ITD) in the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) has been found to have a strong correlation with drug resistance and a poor outcome in about 30% of cases of AML [6]. It has been reported in some research articles that FLT3 -ITD may negatively infl uence MDR - 1 /Pgp expression in AML blasts [3,7,8]. Th is is of especial interest as MDR-1 /Pgp and FLT3 -ITD mutation are well known targets for inhibition by several agents tested in preclinical and clinical trials. In this study, we estimated the expression of MDR-1 , MRP-1 , BCRP and LRP mRNA and detected the FLT3 -ITD mutation in patients with AML. We divided all patients into two groups according to FLT3 -ITD mutation (positive and negative), and compared the expression of the tested genes and other prognostic factors in AML: age (� 60 vs. � 60 years), cytogenetic aberrations (good, intermediate, poor according to Southwest Oncology Group [SWOG] classifi cation), de novo /secondary AML, white blood cell count (WBC), the percentage of blasts in the bone marrow (BM) and peripheral blood (PB), and the type of AML according to the French – American – British (FAB) classifi cation between groups. Moreover, the rates of complete remission (CR) and relapse (RR), and disease-free survival (DFS) and overall survival (OS), in both groups were estimated. A total of 126 consecutive adult patients with a median age of 53 years (range 21 – 87), comprising 59 (46.8%) women and 67 (53.2%) men with previously untreated AML, newly diagnosed between June 2007 and March 2010, and 40 healthy donors were included in this study. Diagnosis of AML was based on standard morphological and immunophenotypic criteria in accordance with the World Health Organization (WHO) classifi cation. Baseline characteristics of patients are given in Table I.