Molecular Screening in Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma: Anaplastic Lymphoma Kinase Analysis, Next-Generation Sequencing Fusion Gene Detection, and T-Cell Receptor Immunoprofiling.

Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) originates from the T-lineage and is marked by rearrangements of the ALK gene. More than 10 fusion partners with the ALK gene are known, with the most common being the t(2;5)(p23;q35) translocation resulting in the NPM1::ALK fusion. In 10% to 20% of the ALK+ ALCL cases, the ALK gene fuses with various other partners. Modern molecular techniques, especially next-generation sequencing (NGS), have eased the identification of ALK gene fusion partners and have allowed in-depth characterization of the T-cell receptor (TCR) repertoire. We devised a real-time quantitative reverse-transcription polymerase chain reaction to measure the expression of the translocated portion of the ALK gene. Fusion partners for the ALK gene were analyzed using rapid amplification of 5'cDNA ends (RACE) method or NGS. TCR immunoprofiling was performed by amplicon NGS. We studied 96 ALK+ ALCL patients. NPM1::ALK fusion gene was observed in 71 patients, ATIC::ALK in 9, and TPM3::ALK in 3. CLTC::ALK, MYH9::ALK, and RNF213::ALK fusions were identified in 2 patients each. We also discovered the TPM4::ALK and SATB1::ALK fusion genes, plus the following 2 previously unidentified ALK+ ALCL fusions: SQSTM1::ALK and CAPRIN1::ALK. High expression of the translocated ALK gene segment was observed in all 93 analyzed samples. TCR testing was conducted on 23 patients with available DNA. In 18 (78%) patients, we discerned at least one (ranging from 1 to 4) clonal TCR rearrangement. In 59% of the patients, clonal TCR beta junctions corresponded with sequences previously observed in both healthy donors and under various pathological conditions. Reverse-transcriptase quantitative detection of ALK expression is a fast and reliable method for both diagnosing and monitoring treatment response in ALK+ ALCL patients, irrespective of the ALK gene translocation. NGS reveals new ALK translocation partners. Both malignant and reactive TCR repertoires in ALK+ ALCL patients are unique and do not consistently occur among different patients.

Elevated erythroferrone distinguishes erythrocytosis with inherited defects in oxygen-sensing pathway from primary familial and congenital polycythaemia.

Congenital erythrocytoses represent a heterogenous group of rare defects of erythropoiesis characterized by elevated erythrocyte mass. We performed molecular-genetic analysis of 21 Czech patients with congenital erythrocytosis and assessed the mutual link between chronic erythrocyte overproduction and iron homoeostasis. Causative mutations in erythropoietin receptor (EPOR), hypoxia-inducible factor 2 alpha (HIF2A) or Von Hippel-Lindau (VHL) genes were detected in nine patients, including a novel p.A421Cfs*4 EPOR and a homozygous intronic c.340+770T>C VHL mutation. The association and possible cooperation of five identified missense germline EPOR or Janus kinase 2 (JAK2) variants with other genetic/non-genetic factors in erythrocytosis manifestation may involve variants of Piezo-type mechanosensitive ion channel component 1 (PIEZO1) or Ten-eleven translocation 2 (TET2), but this requires further research. In two families, hepcidin levels appeared to prevent or promote phenotypic expression of the disease. No major contribution of heterozygous haemochromatosis gene (HFE) mutations to the erythrocytic phenotype or hepcidin levels was observed in our cohort. VHL- and HIF2A-mutant erythrocytosis showed increased erythroferrone and suppressed hepcidin, whereas no overproduction of erythroferrone was detected in other patients regardless of molecular defect, age or therapy. Understanding the interplay between iron metabolism and erythropoiesis in different subgroups of congenital erythrocytosis may improve current treatment options.

Complex karyotype as a predictor of high-risk chronic lymphocytic leukemia: A single center experience over 12 years.

OBJECTIVES: A complex karyotype (CK) is considered a poor prognostic marker in chronic lymphocytic leukemia (CLL). METHODS: The study analyzed 644 untreated CLL patients (pts) using conventional/molecular cytogenetics to reveal the presence of a CK and its composition and to assess its predictive value. The mutational status ofTP53 was detected by next generation sequencing. RESULTS: A CK was detected in 79 pts (12.3%). Patients with a CK showed shorter overall survival (OS) compared to those without a CK (77 months vs. 115 months, p < 0.0001). Chromosomes most frequently included in a CK were 13, 11, 17, 8, 2, and 6. The most common aberrations in a CK were translocations, numerical changes and dicentric chromosomes (with no effect on OS). Patients with aberrations ofTP53 and ATM were shown to have adverse prognosis comparable to patients with a CK without these abnormalities. A stronger impact of a CK on OS of female and older CLL patients was observed. CONCLUSIONS: The determining of the presence of a CK is essential in modern clinical CLL practice. According to recent studies, the presence of a CK affects clinical and treatment decision-making.

Hemoglobinopathies.

This article summarize molecular-genetic basis of hemoglobinopathies, their classification and phenotypic manifestations. The description of individual subgroups is supplemented with a case reports of patients diagnosed in the Czech population. This paper provides an overview of 14 types of α-thalassemic mutations, 34 ß-thalassemic alleles, 4 δß-thalassemic alleles and 22 hemoglobin variants identified in the Czech population in 876 persons from 579 families. In more detail are described hemoglobinopathies, that have been diagnosed and described as novel: ß-thalassemic mutation CD 38/39 (-C); Hb Olomouc; Hb Hana; Hb Hradec Kralove and 18.3 kb deletion downstream of α-globin cluster leading to a new mechanism of α-thalassemia-2. The fact that until the end of 2017 hemoglobinopathies were diagnosed in nearly 900 patients shows that they are not rare in the Czech Republic. This brings increased demands for their diagnostics, including prenatal diagnosis. Key words: hemoglobinopathies - hemoglobinopathy with high affinity to oxygen - sickle cell anemia - thalassemia - thalassemic hemoglobinopathy - unstable hemoglobins.

Interferon-α Revisited: Individualized Treatment Management Eased the Selective Pressure of Tyrosine Kinase Inhibitors on BCR-ABL1 Mutations Resulting in a Molecular Response in High-Risk CML Patients.

Bone marrow transplantation or ponatinib treatment are currently recommended strategies for management of patients with chronic myeloid leukemia (CML) harboring the T315I mutation and compound or polyclonal mutations. However, in some individual cases, these treatment scenarios cannot be applied. We used an alternative treatment strategy with interferon-α (IFN-α) given solo, sequentially or together with TKI in a group of 6 cases of high risk CML patients, assuming that the TKI-independent mechanism of action may lead to mutant clone repression. IFN-α based individualized therapy decreases of T315I or compound mutations to undetectable levels as assessed by next-generation deep sequencing, which was associated with a molecular response in 4/6 patients. Based on the observed results from immune profiling, we assumed that the principal mechanism leading to the success of the treatment was the immune activation induced with dasatinib pre-treatment followed by restoration of immunological surveillance after application of IFN-α therapy. Moreover, we showed that sensitive measurement of mutated BCR-ABL1 transcript levels augments the safety of this individualized treatment strategy.

Molecular Characterization of ß-Thalassemia in the Czech and Slovak Populations: Mediterranean, Asian and Unique Mutations.

ß-Thalassemia (ß-thal) is considered rare in Central Europe. As in other malaria-free regions, the presence of ß-thal in Central Europe reflects historical and recent immigration, and demographic changes that have influenced the genetic variability of the current populations living in this area. This study assesses the frequency and spectrum of mutations on the ß-globin gene in Czech and Slovak subjects with clinical symptoms of thalassemia. The results of the initial part of this research were published more than two decades ago; the aim of this study was to update these original reports. During the period from 2002 to 2015, 400 cases from Czech and Slovak hematological centers were analyzed. Twenty-nine ß-thal mutations, identified in 356 heterozygotes from 218 unrelated families, involve five unique mutations including a recently described insertion of a transposable L1 element into the ß-globin gene. One mutation described here is reported for the first time. Most of the mutations were of Mediterranean origin and accounted for 82.0% of cases. All but one case studied were heterozygous carriers, manifesting ß-thal minor, with rare exceptions represented by the rare (ß(0)) codons 46/47 (+G) (HBB: c.142_142dupG) mutation associated with an α-globin gene quadruplication and by dominantly inherited ß-thal with a more severe phenotype. One double heterozygous ß-thal patient was a recent immigrant from Moldavia. The list of δß-thal alleles (26 carriers, 16 families) contains Hb Lepore and two types of δß(0)-thal deletions. In the past, genetic drift and migration as well as recent immigrations were responsible for the introduction of Mediterranean alleles, while several mutations described in single families were of local origin.

Characterization of iron metabolism and erythropoiesis in erythrocyte membrane defects and thalassemia traits.

BACKGROUND AND AIMS: Erythropoiesis is closely related to iron metabolism in a balanced homeostasis. Analyses of diverse erythroid and iron metabolism disorders have shown that disrupted erythropoiesis negatively affects iron homeostasis and vice versa. The aim of this study was to characterize the relationship between erythropoietic activity and iron homeostasis in pediatric patients with erythrocyte membrane defects and thalassemia traits. METHODS: Selected markers of erythropoietic activity (erythropoietin, soluble transferrin receptor - sTfR and growth differentiation factor 15) and iron status parameters (serum iron, ferritin and hepcidin) were evaluated in pediatric patients with erythrocyte membrane defects and thalassemia traits. RESULTS: The patients with erythrocyte membrane defects and thalassemia traits had altered iron homeostasis due to disturbed erythropoiesis. In comparison with healthy controls, they had a normal to low hepcidin/ferritin ratio and concomitantly elevated sTfR. CONCLUSION: The findings suggest that pediatric patients with erythrocyte membrane defects and thalassemia traits are more susceptible to iron overload than the general population and that the (hepcidin/ferritin)/sTfR ratio can be used to monitor any worsening of the disease.

A lower dosage of imatinib is sufficient to maintain undetectable disease in patients with chronic myeloid leukemia with long-term low-grade toxicity of the treatment.

The information about chronic myeloid leukemia (CML) patients with a deep molecular response of ≥ 4.5 log reduction (MR4.5) in whom the dose of imatinib (IM) had to be reduced to relieve toxicity is insufficient. In 205 CML patients the dose of IM was reduced in 19 (31.2%) out of 61 patients with MR4.5. The patients (12 pretreated with interferon-alpha) achieved MR4.5 after an average of 27.7 months. The duration of MR4.5 before the reduction of the dose was 16-123 (mean = 56.7) months. After the IM reduction (200 mg daily to 400 mg twice weekly for 15-90 (mean = 48) months) MR4.5 or major molecular response (MMR) was maintained in 14 (73.7%) and 2 (10.5%) patients, respectively. Three patients who lost MMR (15.8%) after the discontinuation of IM regained MR4.5 after the reintroduction of a lower dose. A lower dosage of IM should be tested for the management of side effects in patients with MR4.5 in prospective studies.

Comparison of reduced conditionings combining fludarabine with melphalan or 3-day busulfan in patients allografted for myeloid neoplasms.

In the present study we compared outcomes of patients with myeloid neoplasms undergoing allogeneic hematopoietic stem cell transplantation after fludarabine-based regimens with melphalan (FM140) or 3-day busulfan (FB3). The FM140 and FB3 combinations were administered to 21 and 27 patients, respectively. Efforts for early reduction (from day +30 to 60) and discontinuation (until day +100 to 130) of prophylactic immunosuppression were a component of the post-transplant approach. Following FB3 patients suffered from more severe stomatitis (P = 0.013). In contrast, other manifestations of regimen-related toxicity were more frequent in the FM140 group (P = 0.048). There were no statistically significant differences in the development of graft-versus-host disease, non-relapse mortality, post-transplant remission rate, or relapse incidence. Two-year disease-free survival rates were comparable in the two cohorts (66 vs. 55 %; P = 0.751), and so were the overall survival rates (64 vs. 62 %; P = 0.715). The outcomes of allografted patients with myeloid neoplasms were comparable after the FM140 and FB3 regimens. Relatively high therapeutic response in both groups may have been influenced by early reduction and discontinuation of prophylactic immunosuppression followed by effective immunological control of the malignant clone.

Array-based karyotyping in chronic lymphocytic leukemia (CLL) detects new unbalanced abnormalities that escape conventional cytogenetics and CLL FISH panel.

AIMS: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia with a very heterogeneous course. Progress in molecular genetic characterization of CLL has confirmed the prognostic role of unbalanced chromosomal abnormalities currently defined by molecular cytogenetic methods: conventional karyotyping and FISH. However, a significant percentage of genomic abnormalities escapes routine investigation due to the limitations of these methods. It is presently clear that some of these aberrations have impact on prognosis and disease progression. METHODS: We examined copy number changes in the tumor genomes of 50 CLL patients using bacterial artificial chromosome (BAC) and/or oligonucleotide array platforms. We compared the results of arrayCGH with those obtained by FISH and conventional cytogenetics and evaluated their clinical importance. RESULTS: A total of 111 copy number changes were detected in 43 patients (86%) with clonal abnormalities present in at least 23% of the cells. Moreover, 14 patients (28%) were found to have 39 genomic changes that had not been detected by standard cytogenetic and/or FISH analyses. These included possibly prognostically important recurrent 2p and 8q24 gains. The most frequent unbalanced changes involved chromosomes 18, 7, 3, 9 and 17. We also determined the minimal deleted region on chromosome 6q in 7 cases by chromosome 6/7 specific array. CONCLUSIONS: The results showed that a subset of potentially significant genomic aberrations in CLL is being missed by the current routine techniques. Further, we clearly demonstrated the robustness, high sensitivity and specificity of the arrayCGH analysis as well as its potential for use in routine screening of CLL.

Expression of miR-15a and miR-16-1 in patients with chronic lymphocytic leukemia.

INTRODUCTION: MicroRNAs (miRNAs) are small non-coding single-stranded RNA molecules that regulate gene expression at the post-transcriptional level. In the pathogenesis of chronic lymphocytic leukemia (CLL), miR-15a and miR-16-1 play an important role. These miRNAs are located on chromosome 13 in the 13q14.3 region, which is deleted in more than 55% of CLL patients. This aberration affects expression of miRNAs. OBJECTIVES: The study aimed at performing a molecular genetic analysis of miR-15a and miR-16-1 expression in a group of 39 patients diagnosed with CLL and determining the association between the expression of the two miRNAs and types of deletions in the 13q14 region. METHODS: We used fluorescence in situ hybridiziation (FISH) for determination of mono- or biallelic deletion 13q and quantitative polymerase chain reaction (Q-RT-PCR) to revealed expression miR-15a and miR-16-1 in 39 patients suffering from CLL. RESULTS: The analysis comprised 19 patients with monoallelic 13q14 deletion, 3 patients with biallelic deletion, 9 patients with both monoallelic and biallelic deletions, and 8 patients without 13q14 deletion serving as controls. The results showed different levels of miRNA expression in individual patients. Significantly higher normalized levels of miR-15a expression were found in the control group and patients with monoallelic 13q14 expression compared with patients with biallelic deletion. There was a significantly decreased expression of both miRNAs in patients with biallelic deletion of the 13q14 region but only when deletions were present in 77% or more of cells, as detected by fluorescent in situ hybridization (FISH).

ß-Thalassemia due to intronic LINE-1 insertion in the ß-globin gene (HBB): molecular mechanisms underlying reduced transcript levels of the ß-globin(L1) allele.

We describe the molecular etiology of ß(+)-thalassemia that is caused by the insertion of the full-length transposable element LINE-1 (L1) into the intron-2 of the ß-globin gene (HBB). The transcript level of the affected ß-globin gene was severely reduced. The remaining transcripts consisted of full-length, correctly processed ß-globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half-life due to activation of the nonsense-mediated decay pathway. The lower steady-state amount of mRNA produced by the ß-globin(L1) allele also resulted from a reduced rate of transcription and decreased production of full-length ß-globin primary transcripts. The promoter and enhancer sequences of the ß-globin(L1) allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the ß-globin(L1) transcription despite permanent ß-globin(L1) promoter CpG methylation. This result indicates that the decreased rate of transcription from the ß-globin(L1) allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the ß-globin gene represents a novel etiology of ß-thalassemia.

A significant proportion of patients with chronic myeloid leukemia and suboptimal response according to European Leukemia Net criteria have excellent prognosis without treatment change.

BACKGROUND: The Recommendations of the European Leukemia Net (ELN) have become an essential tool in the management and prognosis of patients with chronic myeloid leukemia (CML) treated with tyrosine kinase inhibitors (TKIs). However, the definition of suboptimal response remains under discussion. METHODS: We used conventional cytogenetics for the detection of clonal changes in Ph-positive and negative clones. RT-PCR and sequencing were carried out on peripheral blood leukocytes to detect the type of BCR-ABL1 transcript. The BCR-ABL1 mutational status was assessed using sequencing of RT-PCR products. High performance capillary electrophoresis for determination of imatinib (IMA) plasma concentration was used. RESULTS: A retrospective study of 110 patients diagnosed with chronic-phase (CP) CML treated with IMA or 2(nd) generation TKIs in the years 2000-2009 focused on analysis of patients with suboptimal response according to ELN criteria. 40 patients were administered IMA as first-line therapy and 70 had been pretreated with interferon-alpha (IFN-α) with or without Ara-C and/or hydroxyurea (HU) for a median 12 months (range, 1-92 months). After adjusting for the ELN criteria, major molecular response (MMR) was achieved after median 34 and 39 months in 66.7% and 41.7% of patients after the first and second-line IMA therapy with suboptimal response defined as lack of achievement of MMR at the 18(th) month of treatment, respectively. In comparison to patients with optimal response, patients with suboptimal response did not show significant differences in overall survival (OS) or progression-free survival (PFS). Cytogenetic assays demonstrated additional chromosome abnormalities (ACAs): chromosome 8 trisomy in a Ph-negative clone during the IMA treatment (in 1 case) and der(9q) in Ph-positive clone (in 2 cases); in patients receiving first-line IMA only chromosome 8 trisomy was observed which was associated with myelodysplastic syndrome - this was the only case where hematopoietic stem cell transplantation (HSCT) was performed. During the treatment with IMA in both subgroups no regulatory mutations in the ABL kinase domain were confirmed. CONCLUSION: We believe that the category of suboptimal response should be redefined or withdrawn from the ELN 2009 recommendations for management of CML patients treated with TKIs. Patients with suboptimal response who have no additional risks (additional cytogenetic abnormalities or BCR-ABL1 regulatory mutations) may remain on IMA treatment while patients with these risks should be switched to the 2(nd) generation TKIs.

Interferon-alpha in chronic myeloid leukemia revisited: a long-term retrospective study in Central and Northern Moravia.

AIMS: We assessed the long-term outcome of consecutive patients in the chronic phase of chronic myeloid leukemia (CML) treated with interferon-alpha (INF-α) in Central and Northern Moravia between 1989 and 2006. METHODS: A retrospective study focused on the response, prognostic factors and side-effects of INF-α. RESULTS: 118 patients (67 males and 51 females, median age 50 years; range 18-71) were analyzed. The median follow-up was 82.6 months (12.4-212.6). Thirty-six patients (30.5%) achieved major cytogenetic response (CyR) in median of 18.3 months (3.7-47.3) and maintained it for a median of 64.0 months (7.0-176.0). Sixty-one patients treated with INF-α for more than 12 months had an overall survival (OS) of 137.0 months (95% CI 117.6-156.4). Eighteen (29.5%) achieved complete CyR (CCyR). 109 patients discontinued the treatment with INF-α because of hematologic or cytogenetic resistance in 53 (48.7%), progression of CML in 31 (28.4%) and intolerance to INF-α in 17 (15.6%) patients. The percentage of peripheral blasts, leukocyte count (>50x10(9)/L), splenomegaly, anemia (Hgb≤110 g/L) and Sokal score had statistical impact on the OS in univariate assessment but only the Sokal score remained significant in multivariate analysis. Additional cytogenetic abnormalities at diagnosis were associated with poor prognosis. CONCLUSIONS: In most patients, treatment with INF-α had to be stopped because of a failure to induce response, progression of CML or side-effects but nearly one third of patients treated at least for one year had a long-term benefit from INF-α. The best prognosis was associated with achievement of CCyR and negativity of BCR-ABL in nested RT-PCR.

Imatinib trough plasma levels do not correlate with the response to therapy in patients with chronic myeloid leukemia in routine clinical setting.

Association of trough imatinib plasma levels (IPL) with cytogenetic or molecular response to treatment in patients with chronic myeloid leukemia (CML) was repeatedly reported. We analyzed their value in the routine clinical setting in 131 patients with chronic phase CML in whom imatinib was applied as first- or second-line treatment. A total of 1,118 measurements were obtained by ultra-performance liquid chromatography-tandem mass spectrometry assay in patients treated with daily dose of imatinib ranging from 100 to 800 mg. Samples were obtained from 1 to 96 h after drug ingestion. High inter (36%) and intraindividual variability (9-33%) of IPL was observed. For analysis of correlation of IPL with treatment response, two sets of samples were selected according to the European LeukemiaNet (ELN) criteria. The first set consisted of 241 samples taken 24 ± 2 h after dosing in 54 patients, and the second one consisted of 329 samples taken 24 ± 4 h after imatinib ingestion in 84 patients. In both sets, only patients treated with 400 mg imatinib once daily for at least 18 months were included. From multiple measurements in individual patients, mean IPL were used. In both sets, we were not able to demonstrate a statistically significant correlation between IPL and response to treatment according to the ELN. We believe that this was due to the differences in patients' compliance, leukemia biology, and other variables that are difficult to eliminate in the routine clinical practice. The use of IPL for prognostic estimation in CML treatment outside the clinical trials is probably limited.

Operational cures after interferon-alpha in patients with chronic myeloid leukemia in Central and Northern Moravia.

We assessed long-term outcome of 118 consecutive patients in chronic phase of chronic myeloid leukemia (CML) treated with interferon-alpha (IFN-α) in the Central and Northern Moravia region between 1989 and 2006 with focus on operational cure. The median follow-up was 82.6 months (range 12.4-212.6). Eighteen (15.3%) patients achieved complete cytogenetic response (CCyR) after median 16.7 (3.7-40.8) months. Nine of these patients (7.6%) achieved BCR-ABL negativity in nested reverse transcriptase-polymerase chain reaction ["complete" molecular response (CMR)] and 6 of them have been operationally cured without any treatment for median 6 (4-10) years, while 2 continue with IFN-α and 1 died from CML-unrelated cause. Operationally cured patients had a significantly lower percentage of initial peripheral promyelocytes, blasts, and erythroblasts than the rest of patients treated for more than 12 months (P=0.01-0.03). Unlike patients with sole CCyR, the majority of whom lost CCyR despite continuing IFN-α therapy and required imatinib, patients who achieved CMR had excellent long-term outcome.

Identification of e6a2 BCR-ABL fusion in a Philadelphia-positive CML with marked basophilia: implications for treatment strategy.

AIM: This is a case report of a 51 year old male with marked splenomegaly, basophilia, severe thrombocytopenia, anemia and high SFKL phosphorylation downstream of Bcr-Abl, investigated for association of the e6a2 BCR-ABL fusion gene and marked basophilia. The treatment strategy implications in patients with Philadelphia positive CML are described. METHODS: RT-PCR and sequencing were carried out on the peripheral blood leukocytes to detect the type of BCR-ABL transcript. The BCR-ABL mutational status was assessed using sequencing of the RT-PCR products. The in vitro test of sensitivity to TKIs was based on detecting inhibited phosphorylation of the Crkl and Phospho-Src family kinases (SFK, Tyr416) using immunodetection. RESULTS: The cytogenetics revealed 90% of Ph+ (Philadelphia) cells in the bone marrow aspirate with no additional clonal chromosomal abnormalities at diagnosis. This correlated with an accelerated phase of the CML. Sequencing analysis of reverse transcribed and PCR amplified BCR-ABL transcript revealed a rare e6a2 fusion, with no evidence for Bcr-Abl kinase domain mutation. Western blot analysis showed high phosphorylation (activation) of Crkl and the Src family of kinases (P-SFK). In vitro test of sensitivity of the patients' leukemic cells to imatinib demonstrated sensitivity of Bcr-Abl tyrosine kinase to imatinib, as assessed by a decrease in phosphorylated Crkl and the disappearance of P-SFK, suggesting that P-Src reflects only the Bcr-Abl-dependent Src activity. The initial treatment strategy was reduced imatinib and search for an unrelated hematopoietic stem cell donor (according to the ELN recommendations). The patient was allografted with peripheral stem cells from an HLA- identical male donor but on day +70 graft failure occurred. He was allografted again with the peripheral stem cells from an HLA-identical female donor, engrafted on day +15 and showed 100% donor chimerism with no evidence of the e6a2 BCR-ABL fusion transcript on day +30. CONCLUSION: The clinical disease course in patients with the rare e6a2 BCR-ABL transcript variant is aggressive. This may be the result of increased kinase activity due to partial loss of the guanine exchange factor/dbl-like domain which mediates the interaction with several Ras-like G-proteins involved in cell proliferation, signal transduction, and cytoskeletal organization. For the above reasons, these patients should receive stem cell transplant immediately after a short course of treatment with imatinib/ dual Src/Abl kinase inhibitor or they should be registered in clinical trials with experimental agents.

Imatinib dose escalation in two patients with chronic myeloid leukemia, with low trough imatinib plasma levels measured at various intervals from the beginning of therapy and with suboptimal treatment response, leads to the achievement of higher plasma levels and major molecular response.

Despite the prognostic value of trough imatinib plasma levels (IPL) identified in some studies, no recommendations for the use of IPL results in routine management of CML patients have been issued. We report two patients in whom daily imatinib dose was increased from 400 to 600 or 800 mg because of low IPL found at various intervals from the beginning of treatment (7 measurements; mean IPL values = 616.33 and 764.5 ng/mL, respectively). Both patients achieved suboptimal response according to the European LeukemiaNet criteria (complete cytogenetic response was not achieved after 1 year of treatment in patient 1 and major molecular response after 47 months of standard-dose imatinib therapy in patient 2). In addition, we have demonstrated low hOCT-1 expression at diagnosis in both patients, retrospectively. Escalation of imatinib daily dose resulted in a significant increase of IPL (6 measurements; mean = 1790 and 1416.66 ng/mL, respectively) and in the achievement of complete cytogenetic response in patient 1 after 3 months and major molecular response within 15 and 6 months in both patients. Our cases demonstrate that low IPL identified at various non-predefined intervals from the beginning of therapy may be used for deciding on dose escalation in selected CML patients in the routine clinical setting, especially in cases with suboptimal treatment response.

Cryptic MLL-AF10 fusion caused by insertion of duplicated 5' part of MLL into 10p12 in acute leukemia: a case report.

Chromosomal translocations involving the mixed lineage leukemia gene (MLL) located at 11q23 belong to common chromosomal abnormalities in both acute lymphoblastic (ALL) and acute myeloid leukemias (AML). It has been suggested that the mechanism of MLL leukemogenesis might be a result of a gain-of-function effect of the MLL fusion gene and simultaneous loss of function of one of the MLL alleles (haploinsufficiency). One of the recurrent translocations in AML-M5 involves chromosomal locus 10p12 and results in the MLL-AF10 fusion gene. Several mechanisms leading to MLL-AF10 fusion have been reported, and they have involved rearrangement of the 11q23 region. We present a detailed structural analysis of an AML case with an extra copy of the 5' part of MLL region and its insertion into the short arm of chromosome 10, resulting in an MLL-AF10 fusion without rearrangement of the MLL alleles on both chromosomes 11. Our observation supports a role for a simple MLL gain-of-function in leukemogenesis.