荧光原位杂交技术检测慢性粒细胞白血病微小残留病

目的 研究应用荧光原位杂交技术(FISH)检测慢性粒细胞白血病微小残留病，及用FISH技术对缓解期慢性粒细胞白血病(CML)患者外周血进行检测，评价其体内微小残留病的意义。方法 应用CG和I-FI舛对30例CML(13例给予化疗、17例移植后)患者初发和／或缓解期的骨髓及外周血标本进行分析，分别检测Ph染色体和BCR/ABL融合基因的存在。结果 对13例临床给予化疗的患者初发期的外周血和骨髓进行CG分析，Ph检出率分别为15％(2／13)、100％(13／13)。同时进行I-FISH分析，均可检出BCR/ABL融合基因。对初发期骨髓的CG、I-FISH分析结果进行统计学分析，两组无显著差异；对外周血的CG、I-FISH分析结果进行统计学分析，两组有显著差异。对其缓解期骨髓CG、I-FISH分析结果进行统计学分析，两组有显著差异；对缓解期外周血CG、I-FISH结果进行统计学分析，两组有显著差异；对缓解期外周血I-FISH和骨髓I-FISH结果进行统计学分析，两组呈显著相关。对17例移植后患者CG、I-FISH结果进行统计学分析，两组有显著差异。结论 FISH技术检测慢性粒细胞白血病微小残留病敏感性大大高于常规细胞遗传学分析；采集缓解期外周血进行荧光原位杂交分析，可作为一种方便易行的手段评价微小残留病。     

A BCR-JAK2 fusion gene as the result of a t(9;22)(p24;q11.2) translocation in a patient with a clinically typical chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by the presence of a t(9;22)(q34;q11.2), which leads to the well-known BCR-ABL1 fusion protein. We describe a patient who was diagnosed clinically with a typical CML but on cytogenetic analysis was found to have a t(9;22)(p24;q11.2). Chromosomal fluorescence in situ hybridization showed that the BCR gene locus spanned the breakpoint at band 22q11.2 but that the ABL1 gene was not rearranged. By means of a candidate gene approach, the JAK2 gene, at 9p24, was identified as the fusion partner of BCR in this case. The BCR-JAK2 fusion protein contains the coiled-coil dimerization domain of BCR and the protein tyrosine kinase domain (JH1) of JAK2. The patient's disease did not respond to Imatinib, and this unresponsiveness was most likely a result of the BCR-JAK2 fusion protein.     

Dasatinib

Dasatinib is approved for the treatment of chronic myeloid leukemia (CML) in patients with resistance or intolerance to imatinib. This article reviews pharmacokinetic, pharmacodynamic, and clinical data on dasatinib, and highlights some of the most important issues that need to be addressed. Imatinib and dasatinib both target the tyrosine kinase activity of the BCR/ABL oncogenic fusion protein. In terms of pharmacodynamics, the two agents differ in several ways: (i) dasatinib is >300-fold more potent than imatinib in inhibiting BCR/ABL activity; (ii) inhibition profiles on other tyrosine kinases differ between imatinib and dasatinib; and (iii) dasatinib has other peculiar effects on the leukemogenic signaling, including activation of p38 mitogen-activated protein kinase (MAPK) and reduction of the apoptotic-inactive form of the BCL2-associated agonist of cell death (BAD) protein. Recent pharmacodynamic data suggested combination therapy with dasatinib and signaling inhibitors (e.g. flavopiridol, farnesyl transferase inhibitors, or histone deacetylase inhibitors) may be beneficial. In contrast to other tyrosine kinase inhibitors (TKIs), dasatinib has a reduced half-life and no active metabolites. In a randomized, open-label, phase III trial, dasatinib 100 mg once daily demonstrated similar efficacy and a better tolerability profile than 70 mg twice daily. This unexpected result has been confirmed in recent studies, in which a dose of dasatinib 100 mg once daily was sufficient to trigger apoptosis in leukemic cells. Furthermore, cytogenetic responses correlate with BCR/ABL inhibition. Data suggest dasatinib 100 mg once daily achieves oncogenic shock and chronic inhibition of BCR/ABL activity, suggesting that in the future, pulse therapy with TKIs may be an option in some specific patients with CML.     

Analysis of gene expression profiles in an imatinib-resistant cell line,KCL22/SR

The BCR/ABL tyrosine kinase inhibitor, imatinib, has shown substantial effects in blast crises of chronic myelogenous leukemia. However, most patients relapse after an initial clinical response, indicating that drug resistance is a major problem for patients being treated with imatinib. In this study, we generated a new imatinib-resistant BCR/ABL-positive cell line, KCL22/SR. The 50% inhibitory concentration of imatinib was 11-fold higher in KCL22/SR than in the imatinib-sensitive parental cell line, KCL22. However, KCL22/SR showed no mutations in the BCR/ABL gene and no increase in the levels of BCR/ABL protein and P-glycoprotein. Furthermore, the level of phosphorylated BCR/ABL protein was suppressed by imatinib treatment, suggesting that mechanisms independent of BCR/ABL signaling are involved in the imatinib resistance in KCL22/SR cells. DNA microarray analyses demonstrated that the signal transduction-related molecules, RAS p21 protein activator and RhoA, which could affect Ras signaling, and a surface tumor antigen, L6, were upregulated, while c-Myb and activin A receptor were downregulated in KCL22/SR cells. Furthermore, imatinib treatment significantly suppressed the level of phosphorylated p44/42 in KCL22 cells but not in KCL22/SR cells, even when BCR/ABL was inhibited by imatinib. These results suggest that various mechanisms, including disturbance of Ras-mitogen-activated protein kinase signaling, are involved in imatinib resistance.     

Deletion of any part of the BCR or ABL gene on the derivative chromosome 9 is a poor prognostic marker in chronic myelogenous leukemia

To evaluate the prognostic significance of submicroscopic deletions of the ABL or BCR gene associated with t(9;22) in chronic myelogenous leukemia (CML), we investigated the incidence of an ABL or BCR deletion on derivative chromosome 9 using fluorescence in situ hybridization (FISH). FISH was performed using the LSI BCR/ABL dual-fusion translocation probe on bone marrow cells of 86 patients with CML. Of 86 patients, ABL deletion was detected in 13 (15.1%) patients and BCR deletion in 8 patients (9.3%). Patients with ABL deletion showed shorter event-free survival time (EFS) than those without ABL deletion (P = 0.020). Patients with BCR deletion showed significantly short overall survival time (OS; P = 0.039). Patients with ABL and/or BCR deletion (14/86 patients, 16.3%) showed significantly short OS and EFS (median OS, 43.0 months; median EFS, 40.0 months), compared to the patients without any BCR or ABL gene deletions (median OS, 94.0 months; median EFS, 90.0 months; P = 0.041 for OS, P = 0.008 for EFS). All the patients with BCR deletion, except for one, had a concomitant ABL deletion, suggesting that BCR deletion occurs in conjunction with ABL deletion. In patients with ABL deletion only, BCR/ABL rearrangement with b2a2 mRNA type tended to be more frequent than in patients without any deletion of the two genes (P = 0.073). Deletion of any of the BCR or ABL genes on derivative chromosome 9 was associated with both short OS and EFS. We conclude that deletion of not only the ABL gene, but also of the BCR gene, is a poor prognostic marker that indicates rapid disease progression in CML.     

Terapia przewlekłej białaczki szpikowej – teraźniejszość i wyzwania na przyszłość

Introduction of imatinib to the treatment of chronic myelogenous leukemia (CML) more than a decade ago may be considered as one of the milestones in the history of cancer treatment and oncology. Small molecule inhibitor, which specifically inhibits BCR/ABL oncogenic tyrosine kinase, responsible for malignant transformation of hematopoietic stem cell proved to be unexpectedly effective in the majority of patients and has revolutionized CML therapy. Unfortunately, a significant group of patients develops resistance or is intolerant to the drug which necessitate search for new better drugs. In the last 5 years 2^nd generation inhibitors have been approved (dasatinib, nilotinib and bosutinib), clinical trials are ongoing with ^3rd generation inhibitors (among them ponatinib, active against BCR/ABL with T315I mutation) and allosteric inhibitors. None of the available drugs eliminates leukemia stem cells, which are the roots of the disease, therefore therapy must be continued indefinitely. Since ultimate goal is to cure the disease there are number of trials to eradicate the disease with combination therapies. We may expect that such like imatinib opened new therapeutic horizons in oncology, complete eradication of CML will help to find cure other cancers.     

BCR/ABL fusion gene detected on 9q34 by fluorescence in situ hybridization in an acute leukemia with two BCR/ABL positive clones, one Ph-negative and one Ph-positive.

We report cytogenetic, fluorescence in situ hybridization (FISH), and molecular analyses in the first reported case of an acute leukemia with two BCR-positive clones: one cell Ph-positive and all others Ph-negative. A BCR/ABL fusion gene on 9q34 was detected only with a BCR/ABL dual color translocation probe. These FISH interphase signals must be confirmed on a metaphase to avoid an erroneous interpretation. This observation appears to indicate a 2-step mechanism for this aberrant fusion gene localization: first, a classical t(9;22), and then the transfer of the fusion gene formed on chromosome 22 to chromosome 9 by a second translocation between the long arms of the derivative chromosomes 9q+ and 22q-, masking the first chromosome exchange.     

Associations between imatinib resistance conferring mutations and Philadelphia positive clonal cytogenetic evolution in CML

In patients with chronic myeloid leukemia (CML), resistance against imatinib is associated with mutations in the kinase domain (KD) of the BCR‐ABL1 fusion gene and/or with additional chromosomal abnormalities (ACAs) secondary to the Philadelphia chromosome. To evaluate associations between these molecular and cytogenetic events, we correlated cytogenetic data with results of KD mutation analysis in 205 CML patients with acquired resistance to imatinib (100 females, 105 males, 17.9‐90.3 years). In 79/205 patients (38.5%), at least one KD mutation was detected; in 13/79 (16.5%) even two different mutations were observed. A second KD mutation was frequent in cases with G250E (4/9), E255V (1/3), T315I (5/18), F317L (2/7), F359C/V (4/7), or H396R (2/3), but was never detected in cases with V299L (n = 3) or Y253H (n = 4). With respect to cytogenetics, ACAs were identified in 76/205 patients (37.1%), in 29 (36.7%) together with KD mutations. ACAs were frequent in cases with E255V (2/3), T315I (8/18), F317L (4/7), F359C/V (4/7), or H396R (2/3), but rare in those with M351T (1/9). Therefore, some KD mutations (e.g., T315I) might be associated with higher genetic instability paving the way to additional cytogenetic and molecular genetic events. © 2010 Wiley‐Liss, Inc.     

Molecular targeted treatment--new treatment strategy for patients with chronic myeloid leukemia

Imatinib mesylate is a new drug that can inhibit the tyrosine kinase activity of Bcr-Abl, the receptors for platelet-derived growth factor receptor(PDGF) and stem cell factor, or c-kit. Chronic myeloid leukemia (CML) is distinguished by the presence of a reciprocal translocation between chromosomes 9 and 22 that results in a shortened chromosome 22, termed the Philadelphia(Ph) chromosome. As a result of the translocation, a fusion gene called the Bcr-Abl gene is created from two normal cellular genes, encoding a chimeric Bcr-Abl protein with a deregulated tyrosine kinase activity. The expression of Bcr-Abl tyrosine kinase has been shown to be necessary and sufficient for the transformed phenotype of CML cells. Imatinib can block the kinase activity of Bcr-Abl, thus inhibiting the proliferation of Ph-positive progenitors, and has shown activity against all phases of CML, though responses are most substantial and durable in patients in the chronic phase. An international phase III study which compared the efficacy of imatinib with that of interferon alpha combined with low-dose cytarabine in newly diagnosed chronic-phase CML showed the rate of major cytogenetic response at 24 months was 90%, including 82% of complete cytogenetic response. These results indicated that imatinib was superior to interferon-containing treatment as a first-line therapy. More than 10,000 patients worldwide, including those in Japan, have been treated with imatinib in clinical trials, and a lot of information has been accumulated on the use of this drug. The aim of this article is to review the use of this drug and the practical management of patients with chronic myeloid leukemia.     

A near‐haploid clone harboring a BCR/ABL1 gene fusion in an adult patient with newly diagnosed B‐lymphoblastic leukemia

The detection of recurrent genetic abnormalities in B‐lymphoblastic leukemia (B‐ALL) is critical for risk stratification and therapy‐related decisions. Near‐haploidy (24‐30 chromosomes), a subgroup of hypodiploidy (<46 chromosomes), and BCR/ABL1 gene fusions are both recurrent genetic abnormalities in B‐ALL and are considered adverse prognostic findings, although outcomes in BCR/ABL1‐positive patients have improved with tyrosine kinase inhibitor therapy. While near‐haploid clones are primarily observed in children and rarely harbor structural abnormalities, BCR/ABL1‐positive B‐ALL is primarily observed in adults. Importantly, recurrent genetic abnormalities are considered mutually exclusive and rarely exist within the same neoplastic clone. We report only the second case to our knowledge of a near‐haploid clone that harbors a BCR/ABL1 fusion in an adult with newly diagnosed B‐ALL. Conventional chromosome studies revealed a near‐haploid clone (27 chromosomes) along with a der(22)t(9;22)(q34.1;q11.2) in 17 of 20 metaphases analyzed. Our B‐ALL fluorescence in situ hybridization (FISH) panel confirmed the BCR/ABL1 fusion and monosomies consistent with chromosome studies in approximately 95% of interphase nuclei. Moreover, no evidence of a “doubled” near‐haploid clone was observed by chromosome or FISH studies. This highly unusual case illustrates that while rare, recurrent genetic abnormalities in B‐ALL can exist within the same neoplastic clone.     

Bosutinib: A Review of Its Use in Patients with Philadelphia Chromosome-Positive Chronic Myelogenous Leukemia

Bosutinib (Bosulif^®) is an orally administered small molecule tyrosine kinase inhibitor (TKI) of BCR–ABL and SRC family kinases. It is indicated for the treatment of adult patients with chronic-, accelerated-, or blast-phase Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) with resistance or intolerance to prior therapy (imatinib, dasatinib, or nilotinib) [USA] or for a small subpopulation of these patients for whom imatinib, nilotinib, and dasatinib are not considered appropriate treatment options (EU). In a multinational pivotal trial (n = 547), bosutinib treatment resulted in a major cytogenetic response (MCyR) at 24 weeks in one-third of all treated patients with imatinib-resistant chronic-phase CML who had no previous exposure to any TKIs other than imatinib (primary endpoint), with similar results observed in chronic-phase CML patients who were intolerant of imatinib and naïve to all other TKIs. MCyRs were also seen in more than one–quarter of evaluable patients with chronic-phase CML previously treated with multiple TKIs. Most of the patients with chronic-phase CML achieved a complete hematologic response with bosutinib and some patients with advanced phases of CML achieved an overall hematologic response. Responses were seen irrespective of the type of BCR-ABL mutation at baseline, except T315I. Bosutinib had a manageable tolerability profile in the pivotal trial, with ≤21 % of patients with chronic-phase CML discontinuing the treatment because of adverse events. Diarrhea was the most common adverse event but was generally manageable, with only few patients discontinuing the treatment because of diarrhea. Therefore, bosutinib is a useful TKI option for patients with Ph+ CML in second-line or greater settings.     

Molecular and cytogenetic characterization of a novel translocation t(4;22) involving the breakpoint cluster region and platelet-derived growth factor receptor-alpha genes in a patient with atypical chronic myeloid leukemia

We report a case of BCR-ABL-negative atypical chronic myeloid leukemia (CML) with translocation t(4;22) (q12;q11.2) juxtaposing the breakpoint cluster region (BCR) and platelet-derived growth factor receptor-alpha (PDGFRA) genes. The patient was a 57-year-old man with a history of stage IV diffuse large B-cell lymphoma, status post-6 cycles of combination chemotherapy in 1999, who presented in August 2002 with enlarged lymph nodes, anemia, and marked leukocytosis (50 x 10(9) g/dL) consistent with a myeloproliferative disorder (MPD). A bone marrow biopsy showed granulocytic hyperplasia, neutrophilia, and mild eosinophilia. Initial cytogenetic evaluation by interphase FISH for BCR-ABL, to rule out a translocation 9;22, showed a variant signal pattern consistent with rearrangement of BCR at 22q11.2, but not ABL at 9q34. Analysis of the patient's cDNA by polymerase chain reaction (PCR) for BCR-ABL was negative. Cytogenetic analysis showed an abnormal karyotype with rearrangement of chromosomes 4 and 22. PCR amplification and subsequent sequence analysis demonstrated an in-frame 5'-BCR/3'-PDGFRA fusion in the patient's cDNA. PDGFRA encodes a receptor tyrosine kinase and shares structural and organizational homology with the KIT and CSf1R receptor genes. However, although the incidence of MPD involving translocations of PDGFRB has been well established, to our knowledge there are only two previous reports describing a BCR-PDGFRA fusion gene, in 3 patients diagnosed with atypical CML. Here, we report the molecular and cytogenetic characterization of a patient with BCR-PDGFRA-positive MPD who had a complete hematologic response after treatment with imatinib mesylate.     

Integration of amplified BCR/ABL fusion genes into the short arm of chromosome 17 as a novel mechanism of disease progression in chronic myeloid leukemia

We describe the cases of two patients with Philadelphia chromosome-positive chronic myeloid leukemia (CML), in whom the extramedullary blastic phase developed during disease progression. The similar clinical presentations of these patients was accompanied by gain of identical secondary chromosome abnormalities, that is, monosomies 9, 14, and 22, and by a clustered amplification of the BCR/ABL fusion gene. The additional copies of the BCR/ABL fusion gene were integrated into the short arm of structurally abnormal chromosomes 17 in both patients. The conformity of these genetic features in two patients with a rare disease manifestation leads us to the assumption that either the clustered amplification of the BCR/ABL fusion gene or the integration of this cluster into the short arm of chromosome 17 or both are associated with extramedullar disease progression in CML. Furthermore, the insertion of amplified BCR/ABL fusion genes into structurally abnormal chromosomes provides a novel mechanism of disease progression in BCR/ABL-positive CML.     

Molecular cytogenetic analysis of gene rearrangements in childhood acute lymphoblastic leukemia

The aims of this study were to estimate the incidences of BCR/ABL, MLL, TEL/AML1 rearrangements, and p16 deletions in childhood acute lymphoblastic leukemia (ALL), to identify new abnormalities, and to demonstrate the usefulness of interphase fluorescence in situ hybridization (FISH). We performed G-banding analysis and FISH using probes for BCR/ABL, MLL, TEL/AML1 rearrangements, and p16 deletions on 65 childhood ALL patients diagnosed and uniformly treated at a single hospital. Gene rearrangements were identified in 73.8% of the patients using the combination of G-banding and FISH, while the chromosomal abnormalities were identified in 49.2% using G-banding alone. Gene rearrangements were disclosed by FISH in 24 (72.7%) of 33 patients with normal karyotype or no mitotic cell in G-banding. Among the gene rearrangements detected by FISH, the most common gene rearrangement was p16 deletion (20.3%) and the incidences of others were 14.1% for TEL/AML1, 11.3% for MLL, and 1.8% for BCR/ABL translocations. Infrequent or new aberrations such as AML1 amplification, MLL deletion, ABL deletion, and TEL/AML1 fusion with AML1 deletion were also observed. We established the rough incidences of gene rearrangements in childhood ALL, found new abnormalities and demonstrated the diagnostic capability of interphase FISH to identify cryptic chromosome aberrations.