Loss of X chromosome in childhood acute lymphoblastic leukemia

We present six cases of childhood acute lymphoblastic leukemia (ALL) in which an acquired loss of the X chromosome was detected. The cases derive from a consecutive series of 178 childhood ALL, consisting of 80 girls and 98 boys. In five cases the presence of the TEL-AML1, t(12;21), fusion product was detected by FISH. The single negative case had an unusual t(1;19)(p13;q13). In addition, this was the only case that did not have a cytogenetically visible rearrangement involving one of the chromosome regions 6q, 9p, or 12p. The six cases showed the typical presentation features of an ALL of FAB type L1, a common ALL immunophenotype with myeloid marker co-expression, and a median presenting age of 7 years. We, therefore, conclude that loss of chromosome X may be a secondary event in older girls with TEL-AML1-positive ALL.     

Establishment of a human T-acute lymphoblastic leukemia cell line with a (16;20) chromosome translocation

A new T-cell line, Loucy, was established from the peripheral blood of a patient with T-cell acute lymphoblastic leukemia (T-ALL). The surface marker analysis of the cell line is OKT3+, OKT4+, THB4+, J5 +/-, OKT6-, TdT-, and HLA-DR-, indicating stage IV in T-cell lineage. Karyotype analysis revealed 45,X,5q-,t(16;20)(p12;q13). The translocation between chromosomes 16 and 20 has not been previously detected in ALL. This cell line may be of value in evaluating the role of t(16;20) in the etiology of T-ALL.     

Anomalies of chromosome 1 as a possible prognostic index in childhood acute lymphoblastic leukemia

Complete remission rates, remission duration, and survival were established in children with acute lymphoblastic leukemia (ALL) with chromosome #1 anomalies, and the results were compared with those in patients with normal karyotypes or other chromosomal changes. Even though the complete remission rate did not differ significantly among the three groups, remission duration and survival were significantly longer in children ALL and chromosome #1 anomalies as compared with those in the other two groups.     

High incidence and intraclonal heterogeneity of chromosome 11 aberrations in patients with newly diagnosed multiple myeloma detected by multiprobe interphase FISH

In multiple myeloma, additional copies of chromosome 11 material, reported to confer an unfavorable prognosis, have been found in 20-45% of patients. To assess the incidence and extent of chromosome 11 aberrations, we performed interphase fluorescence in situ hybridization on CD138+ bone marrow plasma cells of 50 newly diagnosed myeloma patients, using seven locus-specific probes for chromosome 11, one for 13q14.3, and a probe set for translocation t(11;14). In 33 of 50 patients, chromosome 11 aberrations were found. Results indicated a marked intraclonal heterogeneity: in 13 patients, trisomy 11; in 10 patients, subclones with trisomy 11 and partial trisomies 11q coexisted; in 6 patients, only a partial trisomy 11q; and in 6 patients, a tetrasomy or partial tetrasomy 11. The coexistence of subclones with varying extent and copy numbers of chromosome 11 material indicates ongoing structural changes and clonal evolution. Hybridization results delineated 11q23 and 11q25 as the most frequently gained regions, which supports a relevant pathogenetic role of genes on 11q23 and 11q25. To confirm the high incidence of 11q23 gains, a further 50 patients (total n=100) were analyzed for 11q23 and 13q14.3. Myeloma with gains of 11q23 showed a low frequency of deletion 13q14.3 and may prove to be a distinct subgroup of this disease.     

Lack of FLT3-ITD in Tunisian childhood acute lymphoblastic leukemia

BackgroundThe fms-like tyrosine kinase 3 (FLT3) gene belong to the class III receptor tyrosine kinases witch is predominantly expressed on hematopoietic progenitor cells, and plays an important role in haematopoiesis. Targeting the FMS-like tyrosine kinase receptor-3 (FLT3) in acute leukemia is mainly important. Therefore, activating mutations in FLT3, primarily the FLT3-internal tandem duplication (FLT3-ITD), was used as a prognostic marker especially in myeloid leukemia; however, in ALL, the prognostic relevance of FLT3 mutations is less clear.ObjectivesThis study was conducted to evaluate the frequency of FLT3-ITD mutation in Tunisian childhood acute lymphoblastic leukemia, and to correlate this mutation with prognostic parameters.MethodsGenomic DNA was extracted from EDTA-anticoagulant blood samples from a total of 25 children suffering from acute lymphoblastic leukemia (ALL). After DNA extraction, the polymerase chain reaction using specific primers was conducted to screen the FLT3-ITD.ResultsIn acute lymphoblastic leukemia (ALL), 9 cases with LAL-B were detected and the median age is 13 years. Chromosome abnormalities were detected in 5 with ALL and are correlated with worse prognosis (very high risk and relapse). At molecular lever, never FLT3-ITD was detected.ConclusionsOur findings suggest that FLT3 mutations are not common in Tunisian childhood ALL and thus do not affect clinical outcome.     

New chromosome rearrangement in acute lymphoblastic leukemia

Cytogenetic findings in leukemia can be used in the diagnosis, prognosis, and in the definition of different subgroups. The most common chromosome abnormalities associated with mature B-cell acute lymphoblastic leukemia (ALL) are t(8;14), t(8;22), t(2;8), and partial duplication of 1q. Various abnormalities involving chromosome 1 have also been reported in ALL. We present a 16-year-old male with mature B-cell ALL whose cytogenetic analysis of bone marrow showed the karyotype of 46,XY,t(8;14)(q24;q32), -15,der(1;15)(p10;q10). The case presented here carries one of the most common abnormalities, t(8;14) (q24;q32), and a new rearrangement, der(1;15)(p10;q10), which has not been described to date in mature B-cell ALL.     

Late-appearing Philadelphia chromosome in acute lymphoblastic leukemia

We describe the cytogenetic analysis of a patient affected by a common acute lymphoblastic leukemia, L2 type. At diagnosis and first relapse, the karyotype was normal, whereas at the second relapse more than 60% of the examined cells showed a Philadelphia chromosome, without any change in the morphological and immunophenotypical picture. This case confirms the observation that leukemic cells are susceptible to developing a Ph, considered a primary chromosomal abnormality, during the course of the disease.     

Cytogenetic findings in childhood acute lymphoblastic leukemia

Chromosome studies were performed on the bone marrow cells of 42 children with newly diagnosed acute lymphoblastic leukemia (ALL). All the children were subsequently treated with the same protocol. Chromosomal abnormalities were found in 25 patients, i.e., in 59.5% of the cases. Hyperdiploidy was observed in 21.4% hypodiploidy in 14.3%, and pseudodiploidy in 23.8% of the children. The most frequent structural aberrations were translocations, which were found in half of the patients with abnormal karyotypes. Chromosomes #5, #6, #7, #9, #14, #17, and #21 were involved in different types of changes most frequently. Because these findings correspond with observations published by others, they can be regarded as evidence of nonrandom involvement of these chromosomes in rearrangements in ALL. Special attention should be also paid to the deletion of 6q, which seems to be relatively common in ALL. In 12 cases, clonal evolution of karyotypic changes was observed.     

Comparative genomic hybridization in childhood acute lymphoblastic leukemia: correlation with interphase cytogenetics and loss of heterozygosity analysis

We used comparative genomic hybridization (CGH) to study DNA copy number changes in 71 children with acute lymphoblastic leukemia (ALL) including 50 B-lineage and 21 T-ALLs. Forty-two patients (59%) showed genomic imbalances whereby gains were more frequently observed than losses (127 vs. 29). Gains most commonly affected the entire chromosomes 21 and 10 (19.7% each), 6, 14, 18, X (15.5% each), 17 (14.1%) and 4 (11.3%). Highly hyperdiploid karyotypes (chromosome number >50) occurred more frequently in B-lineage than in T-lineage ALL (24% vs. 4.8%). In both cell lineages deletions were mainly detected on 9p (14.1%) and 12p (8.4%), and on 6q in T-lineage ALL (4.2%). These findings were compared with loss of heterozygosity (LOH) of 6q, 9p, 11q, and 12p previously performed in 56 of the 71 patients. Among 54 sites of LOH, CGH revealed losses of the respective chromosome arms in 17 LOH-positive regions (31.5%). G-banding analysis and interphase cytogenetics with subregional probes for 14 loci confirmed the presence of genomic imbalances as detected by CGH. We, therefore, conclude that, in the absence of cytogenetic data, CGH represents a suitable method for identifying hyperdiploid karyotypes as well as prognostically relevant deletions in ALL patients.     

High-throughput sequencing detects minimal residual disease in acute T lymphoblastic leukemia

High-throughput sequencing (HTS) of lymphoid receptor genes is an emerging technology that can comprehensively assess the diversity of the immune system. Here, we applied HTS to the diagnosis of T-lineage acute lymphoblastic leukemia/lymphoma. Using 43 paired patient samples, we then assessed minimal residual disease (MRD) at day 29 after treatment. The variable regions of TCRB and TCRG were sequenced using an Illumina HiSeq platform after performance of multiplexed polymerase chain reaction, which targeted all potential V-J rearrangement combinations. Pretreatment samples were used to define clonal T cell receptor (TCR) complementarity-determining region 3 (CDR3) sequences, and paired posttreatment samples were evaluated for MRD. Abnormal T lymphoblast identification by multiparametric flow cytometry was concurrently performed for comparison. We found that TCRB and TCRG HTS not only identified clonality at diagnosis in most cases (31 of 43 for TCRB and 27 of 43 for TCRG) but also detected subsequent MRD. As expected, HTS of TCRB and TCRG identified MRD that was not detected by flow cytometry in a subset of cases (25 of 35 HTS compared with 13 of 35, respectively), which highlights the potential of this technology to define lower detection thresholds for MRD that could affect clinical treatment decisions. Thus, next-generation sequencing of lymphoid receptor gene repertoire may improve clinical diagnosis and subsequent MRD monitoring of lymphoproliferative disorders.     

间期双色双融合荧光原位杂交技术检测成人B细胞急性淋巴细胞白血病费城染色体

目的 探讨成人B细胞急性淋巴细胞白血病(B-lineage acute lymphoblastic leukemia.BALL)中费城染色体(Philadelphia chromosome,Ph染色体)的发生率.方法 应用针对BCR-ABL的双色双融合探针和间期荧光原位杂交(dual-color dual-fusion interphase fluorescence in situ hybridization,DDFISH)技术前瞻性检测112例初诊成人B-ALL患者染色体标本中Ph染色体.并与常规细胞遗传学(conventional cytogenetics,CC)结果 进行比较.结果 成功进行CC检测的89例患者中有16例(17.98%)检出Ph染色体,而DD-FISH检测112例发现35例(31.25%)Ph染色体,阳性细胞率为18.50%～99.00%(平均66.23%).35例DD-FISH检测出Ph+的成人B-ALL患者中,10例未能成功进行CC检测,5例核型正常,16例检出Ph染色体,20例核型异常患者中13例为复杂异常.结论 Ph染色体在成人B-ALL中以DD-FISH检测发生率为31.25%;用BCR-ABL探针进行DD-FISH为检测Ph染色体提供了有效的方法 .是细胞遗传学检查的重要补充;对成人B-ALL患者应常规进行DD-FISH检测.     

Detection of chromosome aberrations in interphase tumor nuclei by nonradioactive in situ hybridization

In a blind study, chromosome aberrations in tumor cells were analyzed by conventional cytogenetic techniques (G banding) and nonradioactive in situ hybridization with chromosome-specific probes. The material was obtained directly from patients with hematologic diseases and from colon tumor derived cell lines. The cytogenetic data obtained with G banding were in accord with those obtained by in situ hybridization to metaphase chromosomes. Most importantly, in situ hybridization to interphase nuclei gave reliable results and even allowed detection of cell subpopulations that were not detected by analyzing metaphase chromosomes. Furthermore, in retrospect, even structural aberrations could be detected in interphase nuclei; abnormal cells with either an i(1q) or a translocation der(1)t(1;7) could be identified. Our results show that the application of in situ hybridization in combination with routine cytogenetic techniques offers significant advantages for cytogenetic analysis of solid tumors and hematologic malignancies.     

Comprehensive characterization of genomic aberrations in gangliogliomas by CGH, array-based CGH and interphase FISH

Gangliogliomas are generally benign neuroepithelial tumors composed of dysplastic neuronal and neoplastic glial elements. We screened 61 gangliogliomas [World Health Organization (WHO) grade I] for genomic alterations by chromosomal and array-based comparative genomic hybridization (CGH). Aberrations were detected in 66% of gangliogliomas (mean ± SEM = 2.5 ± 0.5 alterations/tumor). Frequent gains were on chromosomes 7 (21%), 5 (16%), 8 (13%), 12 (12%); frequent losses on 22q (16%), 9 (10%), 10 (8%). Recurrent partial imbalances comprised the minimal overlapping regions dim(10)(q25) and enh(12)(q13.3–q14.1). Unsupervised cluster analysis of genomic profiles detected two major subgroups (group I: complete gain of 7 and additional gains of 5, 8 or 12; group II: no major recurring imbalances, mainly losses). A comparison with low-grade gliomas (astrocytomas WHO grade II) showed chromosome 5 gain to be significantly more frequent in gangliogliomas. Interphase fluorescence in situ hybridization (FISH) identified the aberrations to be contained in a subpopulation of glial but not in neuronal cells. Two gangliogliomas and their anaplastic recurrences (WHO grade III) were analyzed. Losses of CDKN2A/B and DMBT1 or a gain/amplification of CDK4 found in the anaplastic tumors were already present in the respective gangliogliomas by array CGH and interphase FISH. In summary, genomic profiling in a large series of gangliogliomas could distinguish genetic subgroups even in this low-grade tumor.     

Monosomy 20 in childhood acute lymphoblastic leukemia.

We report two cases of acute lymphoblastic leukemia (ALL) with loss of chromosome 20 as the only karyotypic abnormality detected in the blast cells. The first patient is a 12-year-old boy studied at diagnosis. He represents the only case of monosomy 20 in our series of 90 pediatric ALL successfully karyotyped at diagnosis. In the second patient, monosomy 20 was detected at the second hematologic relapse, 12 years after the initial diagnosis; cytogenetic studies were not performed at disease onset.     

Second neoplasms after acute lymphoblastic leukemia in childhood

BACKGROUND. Effective forms of treatment for acute lymphoblastic leukemia (ALL) in childhood now result in survival rates above 70 percent at five years, but the treatments are potentially carcinogenic. To determine the magnitude of this risk and identify possible risk factors for the development of second neoplasms, we studied a large cohort of children treated for ALL. METHODS AND RESULTS. We undertook a retrospective cohort study of 9720 children who had been given a diagnosis of ALL between June 1972 and August 1988 and had been treated according to the therapeutic protocols of the Children's Cancer Study Group. The median follow-up was 4.7 years (range, 2 months to 16 years). We found that 43 second neoplasms occurred among the children in the cohort, including 24 neoplasms of the central nervous system, 10 new leukemias and lymphomas, and 9 other neoplasms. This represented a 7-fold excess of all cancers and a 22-fold excess of neoplasms of the central nervous system. The estimated cumulative proportion of children in whom a second neoplasm developed was 2.53 percent 15 years after diagnosis (95 percent confidence limits, 1.74 percent and 3.38 percent). An even higher risk, particularly of central nervous system tumors, was evident in children five years of age or less at the time of the diagnosis of ALL (P = 0.012). All central nervous system neoplasms developed in children who had previously undergone irradiation. There was no association with exposure to cyclophosphamide or anthracyclines. CONCLUSIONS. There is a substantial excess of second neoplasms, especially of the central nervous system, among children treated for ALL. Children five years old or younger and those receiving radiation are at higher risk, especially for second tumors arising in the central nervous system.     

Genomic aberrations in plasma cell leukemia shown by interphase fluorescence in situ hybridization

A combination of cytoplasmic immunofluorescence to detect the immunoglobulin light chain and an interphase fluorescence in situ hybridization technique was used to study the recurrent genetic abnormalities in 14 patients with plasma cell leukemia (PCL). Of the 14 patients studied, 5 had primary and 9 secondary PCL. Chromosomal abnormalities were detected in all 14 patients (100%). Deletions of 13q14 were detected in 11 (78%) cases and deletions of 17p13.1(TP53) in 6 (43%) cases. Translocations (11;14), (4;14), and (14;16) were found in 5 (35%), 2 (14%), and 1(7%) cases, respectively. Except for an association between t(4;14) and 13q14 deletions, no association was identified among the genetic abnormalities. Our study revealed that recurrent genetic changes are more frequent in PCL than in multiple myeloma. The frequent TP53 deletions may represent a marker of genetic instability giving rise to an increased propensity for myeloma cells to emigrate from the bone marrow environment and enter leukemic phase.     

Translocation (12;21) followed by insertion of chromosome 3 material in the derivative chromosome 12 in a case of childhood acute lymphoblastic leukemia

We report a 2-year-old boy with B-cell acute lymphoblastic leukemia. Cytogenetic studies at diagnosis with R-banding showed a 46,XY,ins(12;3)(p13;q?21q?22)/46,XY karyotype. Fluorescence in situ hybridization with TEL/AML1 probes and chromosome paints revealed complex rearrangements. The TEL/AML1 fusion gene was located on the derivative chromosome 21 but a segment of the long arm of a chromosome 3 was inserted between the proximal part of the short arm of the derivative chromosome 12 and the reciprocal part of the AML1 gene. This is consistent with an insertion of chromosome 3 into chromosome 12 after the t(12;21) took place, therefore indicating a probable secondary event due to clonal evolution.     

Detection of chromosome aberrations in interphase nuclei using fluorescence in situ hybridization technique.

We report here several experiences of interphase cytogenetics, using fluorescence in situ hybridization (FISH) technique, for the detection of chromosome aberrations. FISH, using alpha satellite specific probes of 18, X, Y chromosomes, was done in interphase nuclei from peripheral blood of patients with Edwards'' syndrome, Klinefelter''s syndrome and Turner''s syndrome with healthy male and female controls, respectively. The distributions of fluorescent signals in 100 interphase nuclei were well correlated with metaphase findings. Nowadays FISH plays an increasingly important role in a variety of research areas, including cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping.