Trisomy 21 is a recurrent secondary aberration in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion.

TEL/AML1 gene fusion is the most frequent genetic lesion in pediatric acute lymphoblastic leukemia (ALL). It occurs as a consequence of the cryptic chromosomal translocation t(12;21)(p13;q22). In a cohort of 50 RT-PCR-positive TEL/AML1 patients, karyotype examination by GTG banding and fluorescence in situ hybridization (FISH) allowed us to identify chromosome anomalies in addition to the already existing t(12;21). Secondary aberrations were found in 29 out of 41 patients (71%) at initial diagnosis and in all 9 patients with relapse. Structural rearrangements affected chromosome arms 2p, 2q, 5q, 9p, 12p (n = 2), 6q, 11p (n = 3), and 21q (n = 4). An extra chromosome 21 was found to be the most frequent anomaly. It was detected in 6 out of 41 patients at initial diagnosis (15%) and in 7 out of the 9 patients at relapse. No karyotype with trisomy 21 exceeded 47 chromosomes. Gain of chromosome 21 was the sole anomaly in GTG-banding analysis in 2/41 patients at initial diagnosis and in 4/9 at relapse. Notably, chromosome painting analysis performed in 11 out of the 13 patients with an extra chromosome 21 revealed duplication of the normal chromosome 21 in 8, and duplication of der(21)t(12;21) in 3 patients. Furthermore, gain of der(21)t(12;21) chromosome was confined exclusively to the relapse patients.     

Prevalence of TEL/AML1 fusion gene in Brazilian pediatric patients with acute lymphoblastic leukemia

We studied 58 childhood B-lineage acute lymphoblastic leukemia (B-ALL) in Brazilian sample patients at the time of diagnosis to investigate the prevalence of the cryptic t(12;21)(p13;q22). All bone marrow specimens were G-band karyotyped, and commercial dual-color DNA probes were used to search for fusion signals in nuclei. The karyotype analysis showed hyperdiploidy as the most frequent abnormality. The frequency of patients with TEL/AML1 gene fusion was 19% (11 out of 58 cases). Six of the positive samples had normal karyotypes. Deletion of the wild-type TEL allele was observed in 27.3% of TEL/AML1 fusion-positive cases, but it was also identified in 4.2% of the negative cases. Three cases presented two fusion signals, indicating possible duplication of the der(21). The mean age of the patients with TEL/AML1 fusion was 4.8 years and the mean amount of peripheral leukocytes was 44,270 x 10(6)/L. The higher frequency of females with B-ALL (33/58 cases) observed in our sample was probably due to the selection mode of the study cases. The prevalence of TEL/AML1 fusion in Brazilian children in our study is similar to that found in other populations.     

The presence of TEL/AML1 rearrangement and cryptic deletion of the TEL gene in adult acute lymphoblastic leukemia (ALL)

TEL/AML1 (also known as ETV6/RUNX1) rearrangement is the most frequent genetic change in childhood B-acute lymphoblastic leukemia (ALL) and is associated with a favorable prognosis. Its presence in adult ALL, however, has been questionable, and any association between TEL/AML1 rearrangement and clinical prognosis is unknown. To reveal the presence and incidence of the TEL/AML1 rearrangement in adult ALL, we applied fluorescence in situ hybridization (FISH). We conducted extra-signal, dual-color fluorescence in situ hybridization (ES-FISH) for TEL/AML1 rearrangement on bone marrow cells from 74 adult ALL patients and analyzed the survival time. We demonstrated the TEL/AML1 rearrangement in 2 patients (2.7%) among 74 patients with ALL. Of 74 adult ALL patients, 3 (4.0%) showed deletion of the TEL gene without TEL/AML1 rearrangement. The mean survival time of patients with TEL/AML1+ALL and patients with cryptic TEL deletion was 33 and 5 months, respectively, whereas the mean survival time of 53 TEL/AML1-ALL patients was 16.7 months. TEL/AML1 rearrangement is not unique in childhood ALL, and cryptic TEL deletion without TEL/AML1 rearrangement was more frequent than the TEL/AML1 rearrangement in adult ALL. We recommend TEL/AML1 FISH in adult ALL patients because conventional cytogenetic studies so far have yielded uninformative results.     

TEL/AML-1 fusion gene. its frequency and prognostic significance in childhood acute lymphoblastic leukemia

TEL gene rearrangement due to the 12;21 chromosome translocation is believed to be the most common molecular genetic abnormality in childhood acute lymphoblastic leukemia (ALL). A study was conducted to investigate the frequency and prognostic significance of TEL/AML-1 fusion gene resulting from a cryptic t(12;21)(p13;q22). Bone marrow samples from 86 patients diagnosed over the past 5 years at Columbus Children's Hospital were analyzed by fluorescence in situ hybridization (FISH) technique for TEL/AML-1 fusion gene, using LSI((R)) DNA probes. The positive cases were analyzed for clinical outcome. Patients in this study received treatment according to Children's Cancer Group (CCG) protocols. Fifteen of the 86 cases (17%) were positive for the fusion gene. All were B-cell lineage and except for one, all were CD10 positive. TEL/AML-1 was not found in any T-cell ALL. The mean overall survival (OS) following diagnosis for the TEL/AML-1-positive group was significantly longer than for the TEL/AML-1-negative group by log-rank = 7.84, P = 0.005. Similarly, the event-free survival (EFS) after remission for the positive group (median 94.5 months) was longer than the negative group (median 57 months) by log-rank = 7.19, P = 0.007. This study confirms that the TEL/AML-1 fusion gene may be the most common genetic event in childhood ALL, occurring in 17% of the patients. It appears restricted to the B-cell lineage. In this study, the presence of a TEL/AML-1 fusion gene was statistically significant in predicting both OS and EFS, indicating a favorable clinical outcome for these patients. Screening for TEL/AML-1 should become routine at diagnosis and a useful biological variable for risk stratification in future clinical trials.     

Near haploid childhood acute lymphoblastic leukemia masked by hyperdiploid line: detection by fluorescence in situ hybridization.

Near-haploid (<30 chromosomes) acute lymphoblastic leukemia (ALL) is a rare and unique subgroup of childhood common ALL associated with a very poor outcome. It may be underdiagnosed when masked by a co-existing hyperdiploid line, which has to be distinguished from the common good-prognostic hyperdiploid (>50 chromosomes) ALL. We present three children in whom, by conventional cytogenetics, near-haploid ALL was detected on relapse. Using interphase FISH probes of chromosomes X, Y, 4, 12, and 21, we were able, in two cases, to trace the hidden near-haploid lines of approximately 5% and 20% of the cells, masked by hyperdiploid cells of approximately 80% and 70%, respectively; at relapse, the proportion was reversed, with predominant near-haploid lines of over 80% and residual hyperdiploidy of less than 10%. The near-haploid lines consisted of 24 and 27 chromosomes, and always retained the second copy of chromosome 21 or its derivative, as detected in one of our patients by SKY. The hyperdiploid clones were the exact duplicates of the near-haploid ones and contained four and two copies of the chromosomes represented in two and one copies in the near-haploid stem line, respectively. Unlike the common hyperdiploid ALL, no trisomies were observed. The patients were all aged >10 years, with WBC 0.7-30 x 10(9)/L, and a common ALL phenotype. They were treated with the ALL-BFM-95 protocol, medium risk group, and responded well to 8 days of steroid therapy, but relapsed early, within 11 months, and died a few months later. Interphase FISH technique is recommended for the detection of cryptic near-haploid clones in the diagnostic survey of ALL. To assess the prognostic value of near-haploidy in the context of the ALL-BFM protocols, a larger cohort of patients is required.     

New highly sensitive fluorescence in situ hybridization method to detect PML/RARA fusion in acute promyelocytic leukemia

We investigated a new fluorescence in situ hybridization (FISH) method to detect PML/RARA fusion and/or anomalies of the RARA gene (alias RARalpha) in interphase nuclei from patients with acute promyelocytic leukemia (APL). This method uses a commercially available product with two different colored fluorescent probes to detect both PML/RARA gene fusion products (double fusion signal or dual-color fluorescence in situ hybridization [D-FISH]). A total of 82 bone marrow specimens were studied, including 30 from normal bone marrow transplant donors, 33 from patients with untreated APL, 14 from patients with treated APL, and 5 from APL patients with known translocation variants or alternate translocations. The signal patterns and percentage of abnormal nuclei were determined in a blinded study on 500 interphase nuclei for each specimen. Based on 25 normal specimens, the normal cutoff was >0.6% and >1.6% for t(15;17) and t(17;var), respectively. The clinical sensitivity for this series of patients was 98% and the clinical specificity was 100%. The results suggest that the new D-FISH probe set can detect all t(15;17)(q22;q21) and all variant forms of this translocation associated with PML and RARA. In addition, this FISH method can detect all alternate translocations involving RARA and not PML. This FISH method can be used both for the accurate diagnosis of APL and to monitor low levels of disease in treated patients.     

Detection of t(12;21) in childhood acute lymphoblastic leukemia by fluorescence in situ hybridization

Metaphase preparations from 36 patients with acute lymphoblastic leukemia (ALL) have been retrospectively screened by fluorescence in situ hybridization (FISH) to determine the incidence of translocation (12;21) and the potential usefulness of FISH as an adjunct to conventional cytogenetic analysis. With the use of specific chromosome paints, 4 of 31 patients with B-lineage childhood ALL (13%) demonstrated rearrangements of chromosomes 12 and 21, and therefore, were considered to harbor the translocation, which had not previously been detected by conventional karyotyping. However, none of these positive cases revealed the standard reciprocal t(12;21)(p12;q22) as the sole abnormality involving chromosomes 12 and 21. The study confirms the feasibility and advantages of introducing FISH screening for t(12;21) in pediatric ALL cases and demonstrates the usefulness of FISH screening as a backup to concurrent cytogenetic analysis to resolve variant translocations and aberrant results. The presence of t(12;21) has also been correlated to clinical data to assess the prognostic significance of this translocation on its own or in association with other prognostic features.     

AML1 amplification in a case of childhood acute lymphoblastic leukemia

We report an 8-year-old girl with B-cell acute lymphoblastic leukemia (ALL). The blast cell karyotype at diagnosis included a marker chromosome revealed by fluorescence in situ hybridization to be a derivative of chromosome 21. A high level amplification of the AML1 gene was identified, but it disappeared upon complete remission. This rare but recurrent abnormality warrants research of B-cell ALL, especially when a marker chromosome is present in the blast cell karyotype.     

Automated detection of residual leukemic cells by consecutive immunolabeling for CD10 and fluorescence in situ hybridization for ETV6/RUNX1 rearrangement in childhood acute lymphoblastic leukemia

Among the various methods available for analyzing minimal residual disease, a new procedure for the cell-based approaches using consecutive phenotypic and genotypic analysis as revealed by immunofluorescent labeling and subsequent fluorescent in situ hybridization (FISH) has been developed. We are introducing a fluorescent microscopy-based technique by which not only cellular targets and immunological marker positivity, but also the FISH pattern was identified by automated scanning. For the latter one translocation-specific FISH pattern recognition was accomplished by using an automated scanning mode for the 3D determination of valid distances between FISH signals, to define the cutoff value for the shortest green-red spot distance differentiating positive cells from negative ones. The procedure was tested with CD10(+) acute lymphoblastic leukemia cell line harboring the t(12;21)(p13;q22) resulting in the ETV6/RUNX1 rearrangement (formerly TEL/AML1), as well as peripheral blood lymphocytes of healthy individuals. Using the combined, automated method, a sensitivity of 98.67% and a specificity of 99.97% were obtained. The mean false positivity + 2 standard deviations cutoff level (0.09%) allows detection of leukemic cells with high accuracy, even a bit below the tumor load dilution of 10(-3), a value reported to be critical in clinical decision making.     

应用RT/PCR技术检测急性粒细胞白血病M2型AML1/ETO融合基因转录本的研究

目的：搪塞检测ＡＭＬ１－ＥＴＯ融合转录本在ｔ（８;２１）急性粒细胞白血病Ｍ２型「Ｍ２／ｔ（８;２１」的临床诊断和预后判断中的意义。方法：用半筑巢式逆录聚合酶链反应（ＲＴ／ＰＣＲ）技术检测ＡＭＬ１／ＥＴＯ融合基因转录本,应用ＳｏｕｔｈｅｒｎＢｌｏｔ技术及限制性内切酶技术检测ＰＣＲ产物的特生。结果：在１９例ＡＭ加共检出１０例存在ＡＭＬ１／ＥＴＯ融合基因转录本,阳性率为５２．６％,２例ＭＤＳ中１例为阳性     

Characterization of two marker chromosomes in a patient with acute nonlymphocytic leukemia by two-color fluorescence in situ hybridization

A patient with acute nonlymphocytic leukemia (ANLL), M5b according to French-American-British (FAB) classification, showed monosomy 16, an extra 1p−, and a 21q+. These derivative chromosomes could not be defined by GTG-banding. For better characterization, we performed two-color fluorescence in situ hybridization (FISH) experiments applying DNA libraries from sorted human chromosomes, chromosome-specific repetitive probes, and a band-specific YAC-clone. With these FISH studies the karyotype could be characterized as 46,XY,+der(1)t(1;21)(p11;?),−16,der(21)t(16;21)(p11.1;q22).     

A study on the incidence of ABL gene deletion on derivative chromosome 9 in chronic myelogenous leukemia by interphase fluorescence in situ hybridization and its association with disease progression

Fluorescence in situ hybridization for the BCR/ABL rearrangement in 138 bone marrow specimens from 59 Philadelphia(+) (Ph(+)) chronic myelogenous leukemia (CML) patients, 35 Ph(+) acute lymphoblastic leukemia (ALL) patients, and 57 Ph(-) ALL patients was used. Sixteen (27.1%) of the 59 CML patients had deletions of the residual ABL gene on the derivative chromosome 9. During the study period, 32 of the 59 CML patients progressed to blast crisis or accelerated phase. Of these, nine patients had residual ABL gene deletions on the derivative chromosomes 9 and 23 patients had no deletions. The mean duration from first diagnosis to blast crisis or accelerated phase for the nine patients with ABL deletions was 32.8 months, and for the 23 patients without ABL deletions, it was 62.4 months (P = 0.017). The overall survival time for the 16 patients with deletions was 32.8 months, and for the 43 patients without deletions, it was 60.1 months (P = 0.164). ABL deletions were not detected among the 35 ALL patients (17 with major BCR/ABL, 18 with minor BCR/ABL), and it appears that this deletion occurs rarely or not at all in Ph(+) ALL patients, which is in contrast to the CML patients (27.1%). However, we detected two ALL cases with ABL deletion but without BCR/ABL rearrangement among 49 Ph(-) ALL and 66 Ph(-) AML patients. In conclusion, patients with ABL deletions progress to blast crisis or accelerated phase in a significantly shorter time than do those without such deletions. It is therefore suggested that the ABL deletion is an indicator of a poor prognosis in CML.     

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.     

Screening by fluorescence in situ hybridization for MLL status at diagnosis in 239 unselected patients with acute myeloblastic leukemia

A large number of abnormalities involving the MLL gene have been associated with hematological malignancies, including acute myeloblastic leukemias (AML). Given the overall unfavorable prognosis of AML with an MLL abnormality, its reliable and accurate detection is needed for informed treatment decision. We therefore investigated the occurrence of MLL abnormalities in 239 unselected consecutive AML patients, using conventional cytogenetic and fluorescent in situ hybridization (FISH) analyses. FISH analysis for MLL was performed using a commercial dual-color probe. Of the 239 patients, 30 (12.6%) showed MLL abnormalities under FISH analysis, 10 (4.2%) showed a split signal indicating the disruption of the MLL gene by translocation or insertion, and 20 (8.4%) showed overrepresentation of the MLL gene without evidence of rearrangement. MLL abnormalities were more frequently found in AML-M5 and M4, mainly as rearrangements, and in AML-M2, mainly as overrepresentation. Our results are in agreement with those reported in other studies. All M2, M4, and M5 AML patients without known recurrent translocations, such as t(8;21) and inv(16), should be investigated by FISH with an MLL probe because it allows the detection of MLL rearrangement that would go undetected by conventional cytogenetics and because it has the ability of detecting multiple copies of the MLL gene in, for example, marker chromosomes and double minutes.     

荧光原位杂交技术检测BCR/ABL融合基因的研究

目的建立荧光原位杂交技术平台,应用FISH技术检测在CML检测BCR/ABL融合基因,探讨FISH技术在在CML中应用的价值。方法应用FISH对BCR/ABL探针进行前期的验证,建立正常阈值,再应用该探针检测CML中BCR/ABL融合基因,进行临床检测评估。结果主要假阳性信号模式的正常阈值为1G1R1F 11%、1G1R2F 2%。75例样本FISH检测出48例阳性,15例经细胞遗传学检测,12例检测结果与FISH结果一致,3例CG为阴性,FISH检测为阳性。结论荧光原位杂交技术应用于临床检测之前应进行探针的前期验证,制定一套规范实验流程,且FISH技术在CML诊断、分型、临床治疗方案的制定、预后的判断以及微小残留病变检测上均有重要的价值。     

多探针荧光原位杂交检测急性髓系白血病常见细胞遗传学异常

目的:评价多探针荧光原位杂交(FISH)在检测急性髓系白血病(AML)常见细胞遗传学异常中的价值,探讨细胞遗传学异常与临床诊断、治疗、预后的关系。方法:采用针对AML/MDS的FISH多探针诊断系统,即以针对AML1/ETO融合基因、PML-RARα融合基因、CBFβ/MYH11融合基因、MLL基因、P53基因、Del(5q)、Del(7q)、Del(20q)8种DNA探针对40例患者进行多探针FISH检测,同时联合染色体核型、临床资料进行研究。结果:40例AML中,共22例多探针FISH检出了细胞遗传学改变,包括:AML1/ETO、PML-RARα、MLL基因断裂重排、Del(5q)、Del(7q)、P53基因缺失、8号染色体三体7种细胞遗传学异常。而常规染色体核型分析仅检出11例遗传学异常。多探针FISH与染色体核型分析的总阳性率分别为57.50%及27.50%。AML1/ETO、PML/RARα阳性者首次诱导化疗效果较理想;而Del(7q)、MLL基因断裂重排阳性、伴复杂细胞遗传学改变者可能预示不良预后。结论:FISH多探针诊断系统检测AML患者常见遗传学异常更省时、准确、高效,有利于完善白血病的分层诊断及指导临床个体化治疗。     

Assessment of 1p/19q deletions by fluorescence in situ hybridization in gliomas

This study assessed the efficacy of FISH for detecting 1p/19q deletion in gliomas on 77 paraffin-embedded tissue samples, of which 42 cases (55%) were positive for 1p/19q codeletion; 7 of the 42 had a previous history of glioma. In one case, analysis failed. The remaining 34 cases were negative; of these, three had a previous history of glioma and one had the reverse 1q/19p deletion. A majority of the 10 recurrent gliomas had progressed, and 70% had a 1p/19q deletion. The signal pattern in a majority of 1p/19q codeletion cases was a single red marker signal and two green reference signals (1R2G) for both probe sets. One case had a different signal pattern for chromosomes 1 and 19 (1R2G and 2R4G), as seen in polysomy cells. Three cases had two target and four control signals (2R4G), as seen in tetraploid cells. Eleven had complex signal patterns seen in diploid and polyploid cells (1R2G/相似文献