A Rare Case of Transfusion Transmission of Hepatitis A Virus to Two Patients with Haematological Disease.

BACKGROUND: This paper describes the transmission of hepatitis A virus (HAV) to two blood recipients from a healthy donor that later presented to the blood bank with jaundice. METHODS: The RNA of HAV was detected by qualitative nested reverse transcription polymerase chain reaction (nested RT-PCR) and quantified by real-time RT-PCR. HAV RNA samples were genotyped by direct sequencing of PCR products. A sequence from a fragment of 168 bp from the VP1/2A HAV region was used to construct a phylogenetic tree. CASE REPORT: A 31-year-old male donor accepted for donation of a whole blood unit returned to the blood bank with clinical jaundice 20 days after donation. His serological and NAT tests were negative for HBV and HCV. Serological tests for HAV IgM and IgG were negative on donation sample but positive on follow-up sample, confirming donor's HAV acute infection. Both recipients of red blood cells (R1) and platelet concentrate (R2) from the same implicated donation were HAV IgM-negative and IgG-positive. Qualitative PCR was positive on samples from all three individuals and phylogenetic analysis of viruses proved HAV transmission to the two recipients of blood products. HAV viral load on donor follow-up sample and the platelet recipient was 1.3 and 1.5 × 10(3) IU/ml, respectively. The RBC recipient, also infected by HCV, was undergoing bone marrow transplantation and died from fulminant hepatitis, 26 days after the implicated HAV transfusion. CONCLUSION: The blood donor, a garbage collector, spontaneously returned to the blood bank when developing jaundice. This highlights the importance of donor education to immediately report to blood banks of any signs and symptoms related to infectious disease developed after blood donation. The fact that one immunocompromised patient with HCV infection died from fulminant hepatitis after receiving a HAV-contaminated platelet transfusion underpins the importance of a HAV vaccination program for these group of patients.

Rapid and sensitive allele-specific (AS)-RT-PCR assay for detection of T315I mutation in chronic myeloid leukemiapatients treated with tyrosine-kinase inhibitors

Abstract Point mutations in the kinase domain of BCRABLwere described in 40–90% of patients with chronic myeloid leukemia (CML) resistant to Imatinib. We hereindescribe the development of a rapid allele-specific (AS)-RT-PCR assay to identify the T315I mutation, which confers full resistance to all available tyrosine-kinaseinhibitors (TKI). The mutation status of 65 patients with resistant CML was evaluated, and the T315I was detectedin 3/65 (4.6%). Comparisons between sequencing and AS-RT-PCR results, as well as serial dilutions experimentsproved that the method is specific and reproducible, with maximum sensitivity of 1 9 10-3. The developed assay isa convenient and easy tool to be used in research of CML resistance for rapid mutation screening and, together withsequencing, may be included in efficient strategies for early detection of TKI resistance in patients with CML.(AU)

SUZ12 is a candidate target of the non-canonical WNT pathway in the progression of chronic myeloid leukemia.

Polycomb proteins form multiprotein complexes that repress target genes by chromatin remodeling. In this work, we report that the SUZ12 polycomb gene is over-expressed in bone marrow samples of patients at the blastic phase of chronic myeloid leukemia. We also found a direct interaction between polycomb group genes and the WNT signaling pathway in chronic myeloid leukemia transformation. Electrophoretic mobility shift assay (EMSA), Chromatin immunoprecipitation assay (ChIP), and mass spectrometry assays identified noncanonical WNT pathway members, such as WNT5A and WNT11, bound to the SUZ12 promoter. Immunohistochemistry and immunofluorescence with WNT5A and WNT11 antibodies confirmed nuclear localization. Knockdown of WNTs 1, 5A, and 11 with RNAi approaches showed that WNT members are capable of activating SUZ12 transcription with varying promoter affinities. Finally, we suggest that SUZ12 is blocking cellular differentiation, as SUZ12 knockdown release differentiation programs in chronic myeloid blastic phase (CML-BP) transformed cell line.

Burkitt lymphoma/leukaemia transformed from a precursor B cell: clinical and molecular aspects.

Burkitt lymphoma/leukaemia (BL/L) is a heterogeneous disease with respect to epidemiological patterns and cell origin. The occurrence of BL/L with an immature phenotype raises the question whether this phenotype might be a consequence of early B-cell transformation or, alternatively, a secondary feature of transformed, mature B cells. It also poses important clinical questions regarding diagnosis and therapeutic procedures. Here we describe the case of a 4-yr-old child with BL/L and FAB L3 morphology, with phenotypic and genotypic characteristics of a CD10+ precursor B-cell acute lymphoid leukaemia (ALL) associated with t(8;14)(q24;q32). Molecular analysis showed expression of RAG1 and RAG2 and an unmutated VDJCmu immunoglobulin rearrangement coinciding with a lack of AICDA expression, indicating an immature B-cell origin. His clinical response suggested that FAB L3 ALL with MYC rearrangement and an aberrant precursor B-cell phenotype is clinically similar to BL/L. Moreover, short, intensive chemotherapeutic protocols seemed to be beneficial. This case also allowed us to refine the description of cellular and molecular variants of BL/L regarding the cell origin and pathogenesis of this biologically heterogeneous disease.

Coexpression of p53 Protein and MDR FunctionalPhenotype in Leukemias: The Predominant Associationin Chronic Myeloid Leukemia

Background: One of the best characterized resistance mechanisms of leukemias is multidrug resistance(MDR) mediated by P-glycoprotein (Pgp) and multidrug-resistant related protein (MRP). In addition to Pgpand MRP, p53 mutation or inactivation might play a relevant role in therapeutic failure. Some studies havedemonstrated that Pgp and MRP may be activated in association with overexpression of mutant or inactivatedp53 protein. The aim of this study was to investigate the association between p53 expression and MDRfunctional phenotype analyzed by flow cytometry (FCM).Methods: Rhodamine-123 assay analyzed by FCM was used to detect the MDR phenotype that was positivein 18 out of 41 (43.9%) cases of chronic myeloid leukemia (CML), 16 out of 28 (57.1%) chronic lymphoidleukemia (CLL) cases, 11 out of 28 (39.3%) acute myeloid leukemia (AML) cases, and four out of 22(18.2%) acute lymphoid leukemia (ALL) cases.Results: Variable levels of p53 expression were observed in leukemic cells: 12 out of 41 (29.2%) in CML,nine out of 28 (32.1%) in CLL, 15 out of 28 (53.6%) in AML, and eight out of 22 (36.4%) in ALL samples.Conclusions: In our study, no significant association between p53 expression and MDR functionalphenotype was observed in ALL, CLL, and AML. On the other hand, a significant association (P 0.0003)of the coexpression was observed in CML. The p53 overexpression was more frequently seen in theaccelerated phase and the blastic phase of this disease. Our results suggest that an MDR functionalphenotype could be associated with p53 mutation in the advanced stage of leukemias.(AU)

Genetic mutation and early onset of T-cell leukemia in pediatric patients infected at birth with HTLV-I.

T-cell leukemia/lymphoma (T-c LL) associated with prior infection with HTLV-I is rarely described in children. We present herein, the clinical, morphological, and virologic features of T-c LL, which occurred in eight pediatric cases with similar features of ATLL described in adults. There were three girls and five boys with age ranging from 2 to 18 years. Lymphoadenopathy, hepatosplenomegaly and marked skin lesions were presented in all cases. Five patients had hypercalcemia. The diagnostic criteria of T-c LL were based on both morphological and immunophenotypical analyses characterized by T-cell markers positively. Seven cases were cCD3+, CD4/CD25+, whereas CD1a and TdT were negative in all cases tested. HTLV-I antibodies were detected in all cases. HTLV-I provirus integration of at least one provirus was seen in all cases tested by molecular analysis. Mother-to-child transmission of HTLV-I was demonstrated in six cases. Interestingly, a homozygous deletion in p16 gene locus was observed in all four cases studied, while exons 7 and 8 of p53 were deleted in one child. The deletion of the p16(INK4A)/p14(ARF) or mutation of p53, key regulatory protein of cell cycle checkpoint in G1/S progression, found in five of the eight pediatric patients suggests that in these cases genetic lesions associated with HTLV-I infection may predispose for an early onset of leukemia.

Coexpression of p53 protein and MDR functional phenotype in leukemias: the predominant association in chronic myeloid leukemia.

BACKGROUND: One of the best characterized resistance mechanisms of leukemias is multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) and multidrug-resistant related protein (MRP). In addition to Pgp and MRP, p53 mutation or inactivation might play a relevant role in therapeutic failure. Some studies have demonstrated that Pgp and MRP may be activated in association with overexpression of mutant or inactivated p53 protein. The aim of this study was to investigate the association between p53 expression and MDR functional phenotype analyzed by flow cytometry (FCM). METHODS: Rhodamine-123 assay analyzed by FCM was used to detect the MDR phenotype that was positive in 18 out of 41 (43.9%) cases of chronic myeloid leukemia (CML), 16 out of 28 (57.1%) chronic lymphoid leukemia (CLL) cases, 11 out of 28 (39.3%) acute myeloid leukemia (AML) cases, and four out of 22 (18.2%) acute lymphoid leukemia (ALL) cases. RESULTS: Variable levels of p53 expression were observed in leukemic cells: 12 out of 41 (29.2%) in CML, nine out of 28 (32.1%) in CLL, 15 out of 28 (53.6%) in AML, and eight out of 22 (36.4%) in ALL samples. CONCLUSIONS: In our study, no significant association between p53 expression and MDR functional phenotype was observed in ALL, CLL, and AML. On the other hand, a significant association (P = 0.0003) of the coexpression was observed in CML. The p53 overexpression was more frequently seen in the accelerated phase and the blastic phase of this disease. Our results suggest that an MDR functional phenotype could be associated with p53 mutation in the advanced stage of leukemias.

Decreased serum inhibin B/FSH ratio as a marker of Sertoli cell function in male survivors after chemotherapy in childhood and adolescence.

OBJECTIVE: Inhibin B produced by Sertoli cells may be an important marker of seminiferous tubule function in patients treated with chemotherapy (CT). The aim of this study was to evaluate the inhibin B/FSH ratio to detect male gonadal dysfunction in cancer survivors treated in childhood and adolescence. PATIENTS: Twenty-one male patients (group A) treated with 6-10 courses of CT for Hodgkin's disease during childhood and adolescence were examined 3-11 years after the conclusion of treatment. Twenty healthy young men (18-23 years old) were used as controls (group B). METHODS: Serum samples for the determination of inhibin B, follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), sex hormone-binding globulin (SHBG) and semen for analysis were collected. RESULTS: The median testicular volume of patients of group A was lower than those of group B (p = 0.001) and a positive correlation was found between testicular size and sperm count (r = -0.5, p = 0.01). Semen analysis revealed azoospermia in 11 patients, severe oligospermia in four and normal sperm count in three. No significant difference was found in the median of T, LH, SHBG, inhibin B concentrations and T/LH ratio between the groups. Serum inhibin B was correlated with the serum FSH levels (r = -0.5, p = 0.02). Median FSH was significantly higher (p = 0.0001), and median inhibin B/FSH ratio was significantly lower in group A than in controls (p = 0.0002), but the inhibin B/FSH ratio was higher in the patients with normal sperm count than in those with oligospermia (p = 0.00004). CONCLUSIONS: These results show that the cytotoxic effects of CT cause severe damage to the germinal epithelium with subtle effects on Sertoli cells. To assess Sertoli cell function in men with primary testicular damage after treatment with CT in childhood and adolescence, the inhibin B level needs to be interpreted in the context of the circulating FSH, especially when normal FSH levels are observed.

Leucemia-linfoma de c‚lulas T do adulto no Brasil: epidemiologia, tratamento e aspectos controversos / Adult T cell leukemia-lymphoma in Brasil: epidemiology, treatment and controverstial aspects

A leucemia-linfoma de c‚lulas T do adulto (LLcTA) ‚ uma neoplasia de linf¢citos T maduros, associada … infec‡Æo pelo v¡rus linfotr¢pico de c‚lulas T humanas do tipo I (HTLV-I). LLcTA ocorre mais freqentemente em regiäes onde a infec‡Æo pelo HTLV-I ‚ endˆmica, como no JapÆo e ilhas caribenhas, por‚m casos espor dicos j  foram descritos em regiäes nÆo-endˆmicas para infec‡Æo por HTLV-I. No Brasil, as formas cl¡nicas mais reconhecidas sÆo as formas agudas e linfomatosas. Um fato interessante da coorte brasileira ‚ a ocorrˆncia desta doen‡a em crian‡as e jovens (2-21 anos). Rio de Janeiro e Salvador sÆo as cidades com maior ocorrˆncia de casos diagnosticados, seguidos de Recife e SÆo Paulo. LLcTA nas formas mais agressivas nÆo responde aos tratamentos quimioter picos convencionais com insucesso em termos de sobrevida > 5 anos. No entanto, nos casos que receberam a consolida‡Æo do tratamento com a-interferon (aIFN) associado a zidovudine (AZT) como esquema de manuten‡Æo de remissÆo, demonstraram melhor sobrevida. Recentemente o tratamento com anti-CD25, apesar de estar em fase experimental, vem apresentado resultados promissores. Com o crescente reconhecimento da LLcTA dentre as doen‡as linfoproliferativas no Brasil, tornam-se necess rios estudos terapˆuticos diferenciados desta doen‡a. Procuramos neste artigo descrever as caracter¡sticas cl¡nico-epidemiol¢gicas dos casos brasileiros, e com a revisÆo da literatura sobre os resultados terapˆuticos no tratamento do LLcTA, pretendemos propor um estudo com fins terapˆuticos utilizando drogas moderadoras biol¢gicas e anti-retrovirais.(AU)

Fun‡„o gonadal de sobreviventes de doen‡a de hodgkin tratados na infƒncia e adolescˆncia com quimioterapia / Gonadal function of survivor of Hodgkin's disease treated in infancy and adolescence with chemotherapy

Avaliamos a fun‡„o gonadal de 21 pacientes do sexo masculino com doen‡a de Hodgkin (grupo A), que receberam quimioterapia durante a infƒncia e adolescˆncia, e comparamos com 20 indiv¡duos adultos jovens sadios (grupo B). A mediana da idade dos pacientes no momento do estudo foi de 18 anos (17-23 anos), e do in¡cio da quimioterapia 10 anos (6-19 anos). Na ‚poca do tratamento , 14 pacientes eram imp£beres e 7 j  estavam na puberdade. No momento da investiga‡„o todos se encontravam no est gio puberal V de Tanner e tinham completado quimioterapia entre 3 e 11 anos previamente. A mediana do volume testicular foi menor no grupo A do que no B, p = 0,001. N„o houve diferen‡as significativas da TT, SHBG, PRL e LH entre os grupos. A mediana dos n¡veis basais do FSH do grupo A foi maior do que no B, p = 0,001. Houve significativa diferen‡a entre as medianas do pico m ximo do FSH e do LH ap¢s est¡mulo com GnRH entre os grupos, p = 0,002 e o p = 0,0002 respectivamente. encontramos uma correla‡„o positiva entre a idade do paciente na ‚poca do tratamento e o valor m ximo do LH ao est¡mulo com GnRH (r + 0,4; p = 0,03) e uma correla‡„o negativa com o tempo decorrido entre o t‚rmino do tratamento e o estudo (r = -0,5; p = 0,008). Onze pacientes apresentavam azoospermia, 4 oligospermia e 3 pacientes apresentavam espermograma normal. Um paciente recuperou a fertilidade, com normaliza‡„o do espermograma, 11 anos ap¢s o t‚rmino do tratamento. Conclu¡mos que pacientes tratados na infƒncia e adolescˆncia com quimioterapia apresentam importante dano no epit‚lio germinativo, mantendo n¡veis normais de testosterona …s custas do aumento da secre‡„o de LH. A presen‡a de redu‡„o do volume testicular nestes pacientes ‚ sugestiva de dano no epit‚lio germinativo, sendo necess rio um longo per¡odo de acompanhamento para avaliar poss¡vel recupera‡„o da fun‡„o gonadal. (AU)

We studied the gonadal function in 21 male patients with Hodgkin's disease(group A), who had received chemotherapy during childhood andadolescent, and compared them to 20 healthy young men (group B).The median age at the time of the study was 18 years (17-23), and at thetime of chemotherapy, 10 years (6-19). At that time, 14 were prepubertaland 7 pubertal; by the time of the study all were Tanner V, and had completedchemotherapy 3 to 11 years previously. The median testicular volumewas significantly higher in patients than controls (p= 0.001). No significantdifferences were found in TT, SHBG, PRL and LH concentrationsbetween patients and controls. The median serum FSH concentrationwas significantly higher in patients than controls (p= 0.0001). We detectedan appreciable difference in peak FSH and LH levels after a GnRH test in group A and B (p= 0.002 and p= 0.0002, respectively).We observed a positive correlation betweenthe age of the patients at the time of treatment andpeak LH levels (r= 0.4; p= 0.03), and a negative correlationwith the period of time between the end oftreatment and the study (r= -0.5; p= 0.008). Elevenpatients had azoospermia, 4 oligospermia and 3patients had a normal semen analysis; one hadrecovered fertility, with normalization of sperm count11 years after the end of the treatment. We concludethat chemotherapy causes severe damage togerminal epithelium in children treated during prepubertaland adolescent age. Normal testosterone levelsmay be secondary to compensated LH secretion.Reduction of testicular size in these patients suggestsgerm cell damage. Long-term follow up is necessaryto establish whether the gonadal function will recover.

SUZ12 is a candidate target of the non-canonical WNT pathway in the progression of chronic myeloid leukemia / Traduzido

Polycomb proteins form multiprotein complexes that repress target genes by chromatin remodeling. In this work, we report that the SUZ12 polycomb gene is over-expressed in bone marrow samples of patients at the blastic phase of chronic myeloid leukemia. We also found a direct interaction between polycomb group genes and the WNT signaling pathway in chronic myeloid leukemia transformation. Electrophoretic mobility shift assay (EMSA), Chromatin immunoprecipitation assay (ChIP), and mass spectrometry assays identified noncanonical WNT pathway members, such as WNT5A and WNT11, bound to the SUZ12 promoter. Immunohistochemistry and immunofluorescence with WNT5A and WNT11 antibodies confirmed nuclear localization. Knockdown of WNTs 1, 5A, and 11 with RNAi approaches showed that WNT members are capable of activating SUZ12 transcription with varying promoter affinities. Finally, we suggest that SUZ12 is blocking cellular differentiation, as SUZ12 knockdown release differentiation programs in chronic myeloid blastic phase (CML-BP) transformed cell line(AU)

Rapid and sensitive allele-specific (AS)-RT-PCR assay for detection of T315I mutation in chronic myeloid leukemia patients treated with tyrosine-kinase inhibitors.

Point mutations in the kinase domain of BCR-ABL were described in 40-90% of patients with chronic myeloid leukemia (CML) resistant to Imatinib. We herein describe the development of a rapid allele-specific (AS)-RT-PCR assay to identify the T315I mutation, which confers full resistance to all available tyrosine-kinase inhibitors (TKI). The mutation status of 65 patients with resistant CML was evaluated, and the T315I was detected in 3/65 (4.6%). Comparisons between sequencing and AS-RT-PCR results, as well as serial dilutions experiments proved that the method is specific and reproducible, with maximum sensitivity of 1 × 10(-3). The developed assay is a convenient and easy tool to be used in research of CML resistance for rapid mutation screening and, together with sequencing, may be included in efficient strategies for early detection of TKI resistance in patients with CML.

Acute myeloid leukemia: update in diagnosis and treatment in Brazil.

OBJECTIVE: To identify how the Brazilian hematology centers treated and diagnosed cases of acute myeloid leukemia in 2009. METHODS: An epidemiological observational multicenter study of 11 listed Brazilian centers that treat acute myeloid leukemia and perform bone marrow transplantation. Data were collected from clinical charts of patients with acute myeloid leukemia treated at the said centers between 2005 and 2009. The availability for immunophenotyping and cytogenetic tests was assessed. RESULTS: During 2009, a total of 345 new cases of acute myeloid leukemia were diagnosed. Differences were noted in the tests performed between patients who initiated treatment at the center and those referred for treatment. Of the participating centers, 72% conducted some type of molecular study in acute myeloid leukemia upon diagnosis. CONCLUSION: Treatment for acute myeloid leukemia in Brazil shows significantly inferior results when compared to other centers worldwide.

Burkitt lymphoma/leukaemia transformed from a precursor B cell: clinical and molecular aspects

Burkitt lymphoma ⁄ leukaemia (BL ⁄ L) is a heterogeneous disease with respect to epidemiological patternsand cell origin. The occurrence of BL⁄ L with an immature phenotype raises the question whether thisphenotype might be a consequence of early B-cell transformation or, alternatively, a secondary feature oftransformed, mature B cells. It also poses important clinical questions regarding diagnosis and therapeuticprocedures. Here we describe the case of a 4-yr-old child with BL ⁄ L and FAB L3 morphology, with phenotypicand genotypic characteristics of a CD10+ precursor B-cell acute lymphoid leukaemia (ALL) associatedwith t(8;14)(q24;q32). Molecular analysis showed expression of RAG1 and RAG2 and an unmutated VDJClimmunoglobulin rearrangement coinciding with a lack of AICDA expression, indicating an immature B-cellorigin. His clinical response suggested that FAB L3 ALL with MYC rearrangement and an aberrant precursorB-cell phenotype is clinically similar to BL ⁄ L. Moreover, short, intensive chemotherapeutic protocolsseemed to be beneficial. This case also allowed us to refine the description of cellular and molecularvariants of BL⁄ L regarding the cell origin and pathogenesis of this biologically heterogeneous disease.(AU)

Epigenetic alterations of p15(INK4B) and p16(INK4A) genes in pediatric primary myelodysplastic syndrome.

We studied the methylation status of the p15(INK4B) and p16(INK4A) genes in 47 pediatric patients with primary MDS, its correlation with subtype, and the role of p15(INK4B) and p16(INK4A) in the evolution of MDS toward AML. Aberrant methylation of the p15(INK4B) gene was detected in 15 of 47 patients (32%), whereas only four patients demonstrated methylation of the p16(INK4A) gene (8%). The frequency of p15(INK4B) methylation was significantly higher in RAEB and RAEB-t subtypes (p<0.003). Aberrant methylation of the p16(INK4A) gene was also more frequent in the subtypes that characterize advanced stages of the disease (p<0.05). Evolution of disease was verified in 17 (36%) of the 47 patients. The association of p15(INK4B) and p16(INK4A) methylation status with evolution of disease was clearly significant (p<0.008 and p<0.05, respectively). These results suggest that methylation of the p15(INK4B) and p16(INK4A) genes is an epigenetic biomarker of pediatric disease evolution.