Ilana Zalcberg

Granulocytic sarcoma of the small intestine with CBFβ/MYH11 fusion gene: report of an aleukaemic case and review of the literature

Granulocytic sarcomas (GS) are rare extramedullary tumours composed of immature myeloid cells. Inversion of chromosome 16 [inv(16)] is a cytogenetic marker for M4Eo subtype of acute myeloid leukaemia (AML). The possibility of an association between the development of granulocytic sarcoma of the small intestine (GSSI) and the M4Eo subtype of AML was suggested in nine previous case reports. Here we report an aleukaemic case of GSSI with inv(16) and its molecular equivalent, the CBFbeta/MYH11 fusion gene, detected by reverse transcriptase-polymerase chain reaction (RT-PCR), that after treatment with conventional AML chemotherapy followed by autologous bone marrow transplantation, achieved complete haematological and molecular remission on bone marrow examination. After chemotherapy, a thickened ileum wall positive for CBFbeta/MYH11 on tumour mass samples was still observed on computed tomography (CT) studies, raising the question of residual GS representing a reservoir of malignant cells. This case demonstrates the critical need of multidisciplinary diagnosis and follow-up of this entity combining immunopathologic, cytogenetic and molecular studies, reinforcing the potentiality of risk-adapted therapy strategies, as it is increasingly claimed for patients with overt AML.

miRNA-451: A putative predictor marker of Imatinib therapy response in chronic myeloid leukemia

In a recent issue of Leukemia Research, Lopotova et al. [1] sug-gest theexistenceofareciprocalregulatoryloopbetweenBCR-ABLand microRNA-451 (miR-451) as a maintenance mechanism of theleukemic stateofCMLcells.Theauthorsalsoreportthatdownreg-ulation of miR-451 might be inversely related to BCR-ABL kinaseactivity in chronic myeloid leukemia (CML) cells. MiRNAs are non-coding RNAs of 21–25 nucleotides that have been implicated ina number of biological processes, regulating gene expression bypromoting mRNA degradation or repressing its translation. Aber-rant miRNA expression has been described for a variety of solidtumors andhematologicalmalignancies,includingCML.SomemiR-NAs, as for example miR-150, miR-151, miR-10a and miR-96 wereseen aberrantly expressed in purified CD34+ population and totalleukocytes from peripheral blood of CML patients [2]. The role ofmiRNAs in CML resistance has not been thus far fully evaluated. Byreal-time PCR Lopotova et al. [1] investigated miR-451 expressionin samples of CML patients treated with Imatinib (IM) at the timeof diagnosis (n=14), in major molecular response (MMR; n=14),in hematological relapse (n=17) and in suboptimal response to IM(n =7) and found miR-451 down regulated in most of the diagno-sis and hematological relapse samples in contrast with normal orslightly increasedlevelsobservedinMMRandsuboptimalresponsesamples.

Clinical relevance of FLT3 gene abnormalities in Brazilian patients with infant leukemia

Infant leukemia (IL) is characterised by the presence of MLL rearrangements and a poor outcome. FLT3 gene is consistently highly expressed in MLL+ patients. To correlate the clinical aspects of IL with FLT3 sequence alterations, we have analysed 159 children included in the Brazilian Collaborative Study Group of Infant Acute Leukemia. FLT3-D835 mutations and FLT3-ITD were detected by PCR-RFLP assay and standard PCR amplification, respectively. Mean age at diagnosis was 11.3 months. Overall, 7.5% (ITDs n = 6 and D835 n = 6) of patients contained FLT3 mutations. FLT3 mutated cases exhibited significantly higher white blood cells (WBC) than wild-type patients (p = 0.013). Median overall survival time was 9.2 months (SE 3.3, 95% CI 2.8–15.6). Variables with significant poorer outcomes were age <6 months (p = 0.0043), MLL+ (p = 0.0292), AML subtype (p = 0.0008), high WBC (p = 0.0179) and FLT3-D835 mutation (p = 0.042). The concomitant presence of FLT3 and MLL abnormalities displayed the worst survival (p = 0.0032). Cox regression analysis, with survival as endpoint, showed that leukemia subtype and WBC were independent prognostic factors. Although FLT3 mutations were not a frequent genetic abnormality in this cohort, they might be prognostically important in IL, but this will need to be confirmed in the analyses of larger patient cohorts.

Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCR&ggr; PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.

Clinical and demographic characteristics of Epstein-Barr virus-associated childhood Burkitt’s lymphoma in Southeastern Brazil: epidemiological insights from an intermediate risk region

This report describes clinical and demographic characteristics of Epstein-Barr virus-associated childhood Burkitt’s lymphoma in Southeastern Brazil. We studied a group of 54 children with Burkitt’s lymphoma from Southeastern Brazil, where epidemiological status of Burkitt’s lymphoma is poorly understood. Epstein-Barr virus association showed an intermediate frequency (~60%) between endemic and sporadic subtypes. Median age was five years. Epstein-Barr virus infection was significantly associated to low age (Epstein-Barr virus+ four years vs. Epstein-Barr virus− eight years). Sex ratio (M:F) was 2:1, with a significantly higher number of males in old age classes. Young age at diagnosis and excess of males at older ages, as well as a causal relationship between low age, epstein-barr virus and Burkitt’s lymphoma risk, may characterize Burkitt’s lymphoma in Brazil.

Detection of BCR-ABL transcripts by multiplex and nested PCR in different haematological disorders.

Single-step Multiplex RT-PCR was used as a rapid and highly sensitive method for screening patients with myeloproliferative conditions and ALL for the presence of underlying BCR-ABL gene fusions. Positive and negative results obtained with the multiplex assay were subsequently confirmed by nested PCR. We studied 21 patients for detecting the presence of b3a2, b2a2 and ela2 BCR-ABL transcripts at diagnosis and following treatment with different therapeutical procedures. These studies allowed the molecular characterisation of patients with different haematological disorders and for demonstrating BCR-ABL transcripts in Ph− CML. In a Ph+ CML patient, a switch of isoforms was detected after bone marrow transplantation and infusion with donor lymphocytes, implying substitution of ela2 for b3a2 coexisting with a myeloid/lymphoid biphenotypic profile. In ALL, one Ph+ patient showed coexpression of ela2 and b2a2 at diagnosis followed by persistence of e1a2 after bone marrow transplantation. Our results were compared to previous findings in the literature on molecular diagnosis of leukaemias.

Hepatosplenic γδ T-cell lymphoma following seven malaria infections

Hepatosplenic γδ T‐cell lymphoma (HSTL) is a clinicopathological entity associated with an immunocompromised status in approximately 25% of patients. Herein is described a case of HSTL in a 53‐year‐old Brazilian man with seven previous malaria infections, initially misdiagnosed as a hyperreactive splenomegaly due to chronic malaria. A characteristic lymphoid infiltrate was observed in spleen, liver and bone marrow sinusoids/sinuses. Neoplastic cells had a CD45RO+, CD2+, CD7+, CD3+, CD5–, CD8+, CD56+, perforin+, FasL‐negative, T‐cell receptor (TCR)αβ‐negative, TCRγδ+ profile. Analyses of γ and δ TCR rearrangements confirmed diagnosis of γδ T‐cell lymphoma by detecting VγI/Vδ1‐Jδ1 clonal rearrangements. Sensitive polymerase chain reaction (PCR) for Plasmodium falciparum, Epstein–Barr virus and herpesvirus‐8 failed to demonstrate infection. The disease progressed to a fatal outcome following cutaneous infiltration and leukemic proliferation. The authors also comment on the association of lymphoma and infection, focusing on PCR diagnosis of TCRγ and δ clonal rearrangements and the presumed pathogenic events leading to HSTL in the context of chronic malaria infection. Initial lymphomagenic stages might not be direct consequences of antigenic stimulation of Vδ1 T‐cells, but might depend on interactions between γδ T and B cells during cooperative or regulatory responses to Plasmodium sp.

TET2 expression level and 5-hydroxymethylcytosine are decreased in refractory cytopenia of childhood.

Myelodysplastic syndromes (MDS) are myeloid malignancies characterized by ineffective hematopoiesis, dysplasia, peripheral cytopenia and increased risk of progression to acute myeloid leukemia. Refractory cytopenia of childhood (RCC) is the most common subtype of pediatric MDS and has overlapping clinical features with viral infections and autoimmune disorders. Mutations in TET2 gene are found in about 20-25% of adult MDS and are associated with a decrease in 5-hydroxymethylcytosine (5-hmC) content. TET2 deregulation and its malignant potential were reported in adult but not in pediatric MDS. We evaluated the gene expression and the presence of mutations in TET2 gene in 19 patients with RCC. TET2 expression level was correlated with 5-hmC amount in DNA and possible regulatory epigenetic mechanisms. One out of 19 pediatric patients with RCC was a carrier of a TET2 mutation. TET2 expression and 5-hmC levels were decreased in patients when compared to a disease-free group. Lower expression was not associated to the presence of mutation or with the status of promoter methylation, but a significant correlation with microRNA-22 expression was found. These findings suggested that TET2 downregulation and low levels of 5-hmC are inversely related to miR-22 expression. The existence of a regulatory loop between microRNA-22 and TET2 may play a role in MDS pathogenesis.

Imatinib resistance: a review of alternative inhibitors in chronic myeloid leukemia

The development of point mutations in the BCR-ABL kinase domain is the main reason for imatinib resistance in chronic myeloid leukemia. Different detection methods are used in chronic myeloid leukemia monitoring, such as direct sequencing, denaturing high performance liquid chromatography and allele specific polymerase chain reaction. Mutation analysis has become mandatory during patient workup of chronic myeloid leukemia in order for the physician to choose the most suitable tyrosine kinase inhibitor. This article, a review of possible therapies used to overcome imatinib resistance, investigates the current position by searching the PubMed electronic database using the following keywords: imatinib, dasatinib, nilotinib, aurora kinase, SRC kinase, mutation, treatment, drugs and resistance. New tyrosine kinase inhibitors include BCR-ABL kinase selective inhibitors, dual ABL/SRC kinase inhibitors and aurora kinase inhibitors. Awareness of the spectrum of new drugs against mutations, in particular the T315I mutation, makes it possible to properly select the best therapy for each patient.

Relationship of Epstein-Barr Virus and Interleukin 10 Promoter Polymorphisms with the Risk and Clinical Outcome of Childhood Burkitt Lymphoma

Epstein-Barr virus (EBV) is an important environmental factor associated to the development of Burkitt lymphoma (BL) in endemic and intermediate risk regions. However, little is known about the contribution of genetic constitution to the development and clinical response of the disease. The aim of this work was to investigate the role of EBV and Interleukin 10 (IL10) single nucleotide polymorphisms (−1082A/G, −819C/T, −592C/A) and microsatellites (IL10.R and IL10.G) in susceptibility and clinical outcome in pediatric BL patients, in a region with intermediate EBV association frequency. The frequencies of IL10 promoter Single nucleotide polymorphisms −1082A/G, −819C/T, −592C/A, and IL10.R and IL10.G microsatellites were compared in 62 pediatric patients and 216 healthy donors. IL10 −1082GG and GCC/GCC genotypes were more frequent in patients than in controls, and associated to a higher risk of BL development (GG genotype OR 2.62, 95% CI, 1.25–5.51; P = 0.008; Pc = 0.024). EBV was detected in tumor samples by EBER-ISH in 54.1% of cases. EBV+ patients exhibited a better event free survival (EFS) (P = 0.019) than EBV− patients. Carriers of IL10 R3-GCC had worse EFS (P = 0.028). Our results suggest a risk effect and an independent prognostic value of IL10 polymorphisms and EBV in childhood BL patients.