Mutations in the breakpoint cluster region-Abelson murine leukemia 1 gene in Brazilian patients with chronic myeloid leukemia

ABSTRACT Introduction: Mutations in the breakpoint cluster region-Abelson murine leukemia 1 gene are the leading cause of resistance to treatment with tyrosine kinase inhibitors in chronic myeloid leukemia patients. Mutations have been detected throughout the extension of the kinase domain of this gene and it is important to investigate their positions because there may be a difference in clinical relevance. Objective: To evaluate mutations in the transcripts of the BCR-ABL1 gene in Brazilian patients with chronic myeloid leukemia under tyrosine kinase inhibitor treatment in the Hospital de Clínicas of the Universidade Federal do Paraná. Methods: This retrospective observational cross-sectional study analyzed mutation data of BCR-ABL1 gene transcripts. Three hundred and thirty peripheral blood samples from 193 patients were evaluated with the search for mutations being achieved by Sanger sequencing. Results: Sixteen mutation types were identified in 48/193 (24.87%) patients with T315I (20.83%) being the most common. Furthermore, four polymorphisms (T240T, K247R, E275E and Y275Y) were identified. The highest incidence of mutations (19/53: 35.85%) occurred in the P-loop of the tyrosine kinase domain, whereas no mutation was found in the A-loop. In 43/48 (89.58%) patients only one mutation was found and more than one mutation was found in 5/48 (10.42%). The simultaneous presence of two mutations (E189G/V299L and E255K/T315I) was observed in 2/5 patients while the different mutations were seen in sequential samples of the other three patients (Y253Y/T315I, T315I/E255K and E255K/T315I). Conclusions: This molecular characterization contributed to the identification of the resistance profile to tyrosine kinase inhibitors in Brazilian patients, thus enabling the use of adequate therapeutic strategies in a timely manner.

Myeloproliferative neoplasms with t(8;22)(p11.2;q11.2)/BCR-FGFR1: a meta-analysis of 20 cases shows cytogenetic progression with B-lymphoid blast phase.

Rearrangements of FGFR1 result in the 8p11 myeloproliferative syndrome, a group of rare diseases that features a myeloproliferative neoplasm (MPN) that commonly progresses to lymphoblastic leukemia/lymphoma or acute myeloid leukemia. The most common partner of FGFR1 is ZMYM2, and patients with the ZMYM2-FGFR1 fusion often present with MPN and T-lymphoblastic lymphoma. There are 14 other partners that can fuse with FGFR1, and of interest is the BCR-FGFR1 fusion that results from t(8;22)(p11.2;q11.2). Patients with t(8;22) often show leukocytosis and present with an MPN resembling chronic myeloid leukemia or very rarely, with B-lymphoblastic leukemia (B-ALL). In this study, we analyzed the clinicopathological, cytogenetic, and molecular features of 2 new patients with the t(8;22)(p11.2;q11.2)/BCR-FGFR1 who presented with B-ALL. An underlying MPN became apparent when a morphologic remission of B-ALL was achieved after chemotherapy. We subsequently reviewed the literature and identified 18 additional cases reported with B-ALL in a background MPN or with the MPN as a chronic phase. Our data suggest that the t(8;22)(p11.2;q11.2)/BCR-FGFR1 may arise from a myeloid/B progenitor cell. It is important to recognize that neoplasms carrying the t(8;22)/BCR-FGFR1, although rare, can commonly with B lymphoblastic leukemia at the initial diagnosis, which could distract one from recognizing a possible underlying 8p11 myeloproliferative syndrome.

AAcute WT1-positive promyelocytic leukemia with hypogranular variant morphology, bcr-3 isoform of PML-RARα and Flt3-ITD mutation: a rare case report / Leucemia promielocítica aguda WT1-positivo com morfologia variante hypogranular, isoforma bcr-3 da PML-RARα e mutação Flt3-ITD: relato de caso raro

ABSTRACT CONTEXT: Acute promyelocytic leukemia (APL) accounts for 8% to 10% of cases of acute myeloid leukemia (AML). Remission in cases of high-risk APL is still difficult to achieve, and relapses occur readily. CASE REPORT: Here, we describe a case of APL with high white blood cell counts in blood tests and hypogranular variant morphology in bone marrow, together with fms-like tyrosine kinase-3 with internal tandem duplication mutations (FLT3-ITD), and bcr-3 isoform of PML-RARα. Most importantly, we detected high level of Wilms’ tumor gene (WT1) in marrow blasts, through the reverse transcription polymerase chain reaction (RT-PCR). To date, no clear conclusions about an association between WT1 expression levels and APL have been reached. This patient successively received a combined treatment regimen consisting of hydroxycarbamide, arsenic trioxide and idarubicin plus cytarabine, which ultimately enabled complete remission. Unfortunately, he subsequently died of sudden massive hemoptysis because of pulmonary infection. CONCLUSION: Based on our findings and a review of the literature, abnormal functioning of WT1 may be a high-risk factor in cases of APL. Further studies aimed towards evaluating the impact of WT1 expression on the prognosis for APL patients are of interest.

RESUMO CONTEXTO: Leucemia promielocítica aguda (LPA) compreende 8% a 10% dos casos de leucemia mieloide aguda (LMA). A remissão em casos de LPA de alto risco ainda é dificilmente conseguida, e recorrência é comum. RELATO DE CASO: Descrevemos aqui um caso de LPA com glóbulos brancos elevados no exame de sangue e a morfologia variante hipogranular na medula óssea, juntamente com fms-like tirosina-quinase-3 com mutações de duplicação em tandem interna (FLT3-ITD) e a isoforma bcr-3 de PML- RARα. Mais importante, detectamos alto nível de gene do tumor de Wilms (WT1) em blastos medulares por RT-PCR (reverse transcription polimerase chain reaction). Até agora, não há conclusões claras sobre a associação entre os níveis de expressão WT1 e APL. Este paciente recebeu sucessivamente regime de tratamento combinado, de hidroxicarbamida, trióxido de arsênico e idarrubicina e citarabina, alcançando finalmente a remissão completa. Infelizmente, em seguida, ele morreu de repente de hemoptise maciça devido a uma infecção pulmonar. CONCLUSÃO: Com base em nossos resultados e numa revisão da literatura, a função anormal de WT1 pode ser um fator de alto risco em casos de APL. Novos estudos, com o objetivo de avaliar o impacto da expressão de WT1 no prognóstico dos doentes com APL, são de interesse.

Acute WT1-positive promyelocytic leukemia with hypogranular variant morphology, bcr-3 isoform of PML-RARα and Flt3-ITD mutation: a rare case report.

CONTEXT:: Acute promyelocytic leukemia (APL) accounts for 8% to 10% of cases of acute myeloid leukemia (AML). Remission in cases of high-risk APL is still difficult to achieve, and relapses occur readily. CASE REPORT:: Here, we describe a case of APL with high white blood cell counts in blood tests and hypogranular variant morphology in bone marrow, together with fms-like tyrosine kinase-3 with internal tandem duplication mutations (FLT3-ITD), and bcr-3 isoform of PML-RARα. Most importantly, we detected high level of Wilms' tumor gene (WT1) in marrow blasts, through the reverse transcription polymerase chain reaction (RT-PCR). To date, no clear conclusions about an association between WT1 expression levels and APL have been reached. This patient successively received a combined treatment regimen consisting of hydroxycarbamide, arsenic trioxide and idarubicin plus cytarabine, which ultimately enabled complete remission. Unfortunately, he subsequently died of sudden massive hemoptysis because of pulmonary infection. CONCLUSION:: Based on our findings and a review of the literature, abnormal functioning of WT1 may be a high-risk factor in cases of APL. Further studies aimed towards evaluating the impact of WT1 expression on the prognosis for APL patients are of interest.

Caracterização do perfil de resistência da linhagem de leucemia mieloide crônica K-IM, resistente ao imatinibe / [Characterization of the resistance profile of the K-IM chronic myeloid leukemia strain, resistant to imatinib]

O advento da terapia alvo-específica com o imatinibe (IM) transformou o panorama da resposta ao tratamento, progressão e sobrevida dos pacientes de leucemia mieloide crônica (LMC). Apesar do sucesso e do desenvolvimento de inibidores de tirosina-quinase (TKIs) mais potentes, persiste a problemática da resistência ao tratamento. A relevância do estudo dos mecanismos de resistência aos TKIs atualmente reside na frequência das falhas terapêuticas não relacionadas à mutação em BCR-ABL, o principal alvo da terapia. Tendo por finalidade estudar os diversos mecanismos que cooperam para a aquisição de resistência, foi desenvolvida em nosso laboratório uma linhagem de LMC resistente ao IM. A linhagem resistente, denominada K-IM, foi selecionada pelo cultivo dalinhagem K562 em concentrações crescentes de IM, alcançando 1,0 μM do fármaco. Sem apresentar mutação no domínio quinase de BCR-ABL, ela se constitui um bom modelo para oestudo dos outros mecanismos de resistência ao IM. A linhagem apresentou aumento nos níveis deRNA mensageiro (RNAm) de BCR-ABL que não se traduziu em aumento da atividade de Bcr-Abl ou no impedimento da sua inibição pelo tratamento com IM. A linhagem K-IM se mostrou significativamente mais resistente do que a parental. Embora o tratamento com 1,0 μM de IM tenha promovido um acúmulo de células nas fases G0/G1 do ciclo celular, não foi acompanhado deindução à morte celular nem impediu o aumento do número de células em cultura. Foram avaliadas as proteínas transportadoras de efluxo por serem determinantes para a resistência à múltiplas drogas, porém a sua atividade não foi observada na linhagem K-IM...

The advent of target-specific therapy with imatinib (IM) has transformed the scenario of response to treatment, progression and survival of patients with chronic myeloid leukemia (CML). Despite the success and development of more potent tyrosine kinase inhibitors (TKIs), the problem of resistance to treatment remains. The relevance of the study of mechanisms of resistance to TKIs currently lies in the frequency of therapeutic failures unrelated to mutation in BCR-ABL, the main target of therapy. Aiming to study the various mechanisms that cooperate for the acquisition of resistance, a CML cell line resistant to IM was developed in our laboratory. The resistant cell line, called K-IM, was selected by culturing the K562 cell line at increasing concentrations of IM, reaching 1.0 μM of the drug. Without presenting mutation in the BCRABL kinase domain, it constitutes a good model for the study of the other mechanisms ofresistance to IM. The cell line showed an increase in BCR-ABL messenger RNA (mRNA) levels which did not result in increased Bcr-Abl activity or in the impairment of its inhibition by IMtreatment. The K-IM cell line was significantly more resistant to IM than the parental cell line. Although treatment with 1.0 μM of IM promoted cell accumulation in the G0/G1 phases of the cell cycle, it was not accompanied by induction of cell death nor prevented the increase in the number of cells in culture. The efflux transporter proteins were evaluated because they aredeterminant for multidrug resistance, but their activity was not observed in the K-IM cell line. Since the antiapoptotic proteins XIAP and survivin are studied as chemoresistance factors, their mRNA and protein levels were evaluated. K-IM showed similar levels to K562 of XIAP mRNA,but showed higher survivin levels in both instances, suggesting that this protein plays a role in its resistance...

Atypical chronic myeloid leukemia with t(9; 22)(p24,11. 2), a BCR-JAK2 fusion gene

We report here on a rare case of BCR-ABL1-negative atypical chronic myeloid leukemia with a t(9;22)(p24;q11.2)translocation and a BCR-JAK2 fusion gene, with resistance to the tyrosine kinase inhibitors imatinib and dasatinib.At two years of follow-up, the patient showed no hematologic response and was submitted to an allogeneic bonemarrow transplantation. Fifty-three days after the procedure, he died due to acute graft-versus-host disease. This BCR-JAK2 fusion gene has so far been found in only five patients in the whole world, with three clinical presentations: myeloproliferative neoplasm, acute lymphoblastic leukemia and acute myeloid leukemia.

Dissimilar expression of multidrug resistance mdr1 and bcrp by the replication of hepatitis C virus: role of the nonstructural 5A protein.

Multidrug resistance associated with the overexpression of ATP-dependent binding cassette (ABC) proteins is widely accepted as an important cause of treatment failure in patients with neoplastic or infectious diseases. Some of them play also a pivotal role in detoxification processes. Herein, we investigated the effect of hepatitis C virus (HCV) replication and nonstructural 5A (NS5A) protein on the expression and functional activity of two ABC transport proteins: MDR1 and BCRP. RT-quantitative real-time polymerase chain reaction (qPCR) was carried out for mdr1 and bcrp mRNAs in both Huh7 cells expressing NS5A and Huh7.5 cells containing either full-length- or subgenomic-HCV replicon systems. The functional activity of these pumps was studied by performing a dye efflux assay with DiOC2 and Rhodamine 123. A dose-dependent down-regulation of mdr1 expression was documented in Huh7 cells expressing the NS5A protein, as well as in both replicon systems. In contrast, a significant increase of bcrp expression in both systems was recorded, which were in full agreement with the dye efflux assay results. These results warrant further in vivo studies in HCV patients with cholestasis and/or patients that are refractive to the pharmacotherapy due to the activity of these pumps.

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.

PML-RARalpha fusion gene transcripts and biological features in acute promyelocytic leukemia patients.

Acute promyelocytic leukemia (APL) is characterized by the presence of rearrangements involving the retinoic acid receptor alpha (RARalpha) gene and a variable incidence in different populations. The hybrid gene PML-RARalpha, present in 98% of cases, encodes a fusion protein essential to the pathogenesis of the disease. Depending of the PML's gene breakpoint in chromosome 15, the transcript subtypes bcr1, bcr2 and bcr3 may be formed. The correlation between these transcript subtypes and clinical parameters is still controversial. The objective of this study was to determine the frequencies of the PML-RARalpha transcripts and subtypes in a series of 32 APL patients from Northeast Brazil and to evaluate the association of these subtypes to different parameters. The method used was RT-PCR. The frequency of our APL cases is approximately 28% of the acute leukemias. The results showed the presence of PML-RARalpha isoform in all patients and a higher frequency of the bcr1/2 subtype. No significant statistical association was found between molecular subtypes and age, sex, French-American-British (FAB) classification, leukocyte and platelet count, hemoglobin level or coagulation tests. In conclusion, these data suggest similar molecular and biological features for our APL patients at diagnosis in comparison with those reported in current scientific literature.

Frequencies of the breakpoint cluster region types of the BCR/ABL fusion gene in Mexican Mestizo patients with chronic myelogenous leukemia.

There is little information about the breakpoint cluster regions of the BCR/ABL fusion gene in Mexican Mestizos with Philadelphia chromosome positive chronic myelogenous leukemia; in a small study a different distribution of these as compared with Caucasians was recently described. We have now prospectively analyzed the breakpoint cluster regions of the BCR/ABL fusion gene in a group of 238 Mexican Mestizos patients with Philadelphia chromosome positive chronic myelogenous leukemia and found a prevalence of 54.2% for the b3/a2 subtype, 43.2% for the b2a2 and 2.5% for the b3a2/b2a2. These data are not different from those previously described in other populations and are consonant with the prevalence of chronic myelogenous leukemia in Mexico, which is not different from that described in other populations.

Renal and extrarenal congenital rhabdoid tumor: diagnosis by fine-needle aspiration biopsy and FISH.

We report on 2 patients with congenital malignant rhabdoid tumor, one located to the kidney and the other to the soft parts of the cheek. Initial diagnosis was performed through percutaneous fine-needle aspiration biopsies, which yielded cytologic smears exhibiting highly characteristic rhabdoid cells, i.e., cells with a large, vesicular nucleus with a prominent nucleolus and cytoplasm exhibiting a large, dense, paranuclear inclusion. Interphase FISH demonstrated only one signal (heterozygous deletion) for the BCR gene in both cases, supporting the diagnosis. Surgical pathology and immunohistochemistry of both cases confirmed the diagnosis. Both patients died within the following 6 mo to 1 yr.

The relationship of bone marrow histology with the molecular pattern in chronic myeloid leukemia.

The chromosomal abnormality in chronic myeloid leukemia (CML) results from a reciprocal translocation between chromosomes 9 and 22 transferring the c-abl proto-oncogene from chromosome 9 to the restricted breakpoint region on chromosome 22 M (bcr). In this study the breakpoint was determined within the M-bcr in 35 CML patients in the chronic phase, by Southern blotting analysis, and it was then correlated with bone marrow Granulocytic-Megakaryocytic (GRAN-MEG) and Granulocytic (GRAN) histological subgroups, as well as with the clinical findings and laboratory parameters. In the 35 patients analyzed, 46% were grouped as 5' and 54% as 3'. There was an increase in bone marrow basophils in 5' breakpoint patients compared to 3' breakpoint (p = 0.042) but the M-bcr breakpoint site did not differ significantly in the subgroup GRAN or GRAN-MEG (p = 0. 12). In conclusion, the patient population had a higher frequency of M-bcr breakpoint in zone 4 and 3' position; there was no correlation between 5' and 3' positions and clinical or haematological features, except a significant increase in bone marrow basophil cells in 5' breakpoint patients compared to 3' breakpoint. Although a higher frequency of the 3' breakpoint was found in patients with a low number of megakaryocytes compared to the cases with a granulocytic-megakaryocytic proliferation, this difference was not statistically significant.

Further evidence for the lack of correlation between the breakpoint site within M-BCR and CML prognosis and for the occasional involvement of p53 in transformation.

The actual significance of the type of BCR-ABL rearrangement in chronic myeloid leukemia (CML) prognosis remains controversial. Also, the molecular events that lead to CML progression are largely unknown. We analyzed the M-BCR breakpoint position in 64 CML patients by Southern blot and correlated the molecular findings with the cytogenetic, hematologic, and clinical data. No statistically significant differences were found with respect to the clinical and hematologic data presented at diagnosis or in the median duration of chronic phase (CP) and survival between the groups of patients with 5' and 3' breakpoints. We also studied by PCR-SSCP and direct sequencing the p53 gene in patients with specimens available in both chronic phase and blast crisis. We identified p53 mutations in 17% of the blast crisis samples analyzed, whereas no abnormalities were found in CP. This finding suggests that only in a minor fraction of cases are lesions in the p53 gene involved in transformation. Given the present findings, along with previous reports, we believe that a novel mechanism to explain the heterogeneity of CML should be postulated and actively pursued, as should the identification of secondary molecular events more consistently involved in progression.

[Atypical chronic myeloid leukemia with polymorphism of the BCR gene]. / Leucemia mieloide crónica atípica con polimorfismo del gen BCR.

The region of greatest rupture of the gene BCR (M-bcr) was analysed with the restriction enzyme Bgl II plus a molecular probe containing the 5' end of M-bcr in a woman with atypical, Ph'-negative chronic myelogenous leukaemia. An abnormal DNA fragment of 5.0 kb and an extra band were found, suggesting rearrangement. Nevertheless, the use of several restriction enzymes and the addition of a different probe showed the existence of polymorphism with the enzyme Bgl II, which is an unusual finding. These findings stress the usefulness of using different restriction enzymes and molecular probes in the molecular study of chronic myelogenous leukaemia.

Molecular differentiation of two different translocations producing two apparent Ph chromosomes in a patient with CML.

A case of chronic myelogenous leukemia (CML) is described whose leukemic cells appeared to contain two Philadelphia (Ph) chromosomes originating from different translocations involving the two chromosomes 22. The karyotype of the affected cells, established on two different occasions, was: 46,XY,t(9;22)(q34;q11),t(15;22)(p11;q11) with no normal chromosomes 22 and only one 9q+ in each of 115 marrow cells examined. The same findings were present in 50 peripheral blood cells cultured without phytohemagglutinin (PHA) stimulation. When stimulated with PHA, a normal male karyotype was present in the 11 cells examined. There were no additional chromosomal abnormalities and no indication of a blastic crisis after nearly 1 year following the original study. Analysis of the breakpoint cluster region (bcr) on chromosome 22 in the DNA of the affected cells (marrow) revealed evidence for one rearranged chromosome 22 and one normal chromosome 22, indicating that the t(15;22) was not due to the usual Ph translocation seen in CML. The results point to the crucial usefulness of molecular analysis in confirming cytogenetic results related to Ph translocations in CML.