Three way translocation in a new variant of t(8;21) acute myeloid leukemia involving Xp22

The t(8;21)(q22;q22) is one of the most frequent chromosomal abnormality associated with acute myeloid leukemia (AML) M2 sub type. The additional chromosomal abnormalities including structural and numerical are frequently reported with the translocation, t (8;21)(q22;q22). We report a case of AML-M2 with t(X;8;21)(p22;q22;q22) associated with loss of Y chromosome. Using a dual color fluorescence in situ hybridization (FISH) analysis with ETO and AML1 probes, we demonstrated an ETO/AML1 fusion signal on the derivative chromosome 8 and one ETO signal on derivative Chromosome Xp22. The patient did not respond to therapy and follow-up of cytogenetics revealed same chromosome abnormality. Hence, this three way translocation involving X chromosome might be associated with poor prognosis.     

T(1;21;8)(p34;q22;q22): a novel variant of t(8;21) in acute myeloblastic leukemia with maturation

The complex variants of t(8;21) involving chromosomes 8 and 21 as well as another chromosome account for approximately 3% of acute myeloid leukemia patients. We report here a 30-year-old male patient with AML-M2. Fluorescence in situ hybridization analysis using dual-color fluorescence ETO and AML1 probes located at 8q22 and 21q22 respectively showed an AML1/ETO fusion signal on the derivative chromosome 8. Whole chromosome painting probes were used for chromosome 1, 8 and 21 and revealed a three-way translocation (1;21;8)(p34 ~ p35;q22;q22). Involvement of chromosome region 1p34 has never been reported earlier, although region 1p35 as a variant in AML with t(8;21) has been reported with an AML1/ETO fusion signal on the 1p35 rather than der(8). In conclusion, combining conventional karyotype, FISH or RT-PCR analyses are a rational strategy for the identification of the complex variants of t(8;21) translocation which could be critical events responsible for leukemogenesis.     

急性髓系白血病伴新的t(8;21)变异易位——t(7;21)(p21;q22)易位

目的:探讨1例急性髓系白血病(acute myeloid leukemia,AML)伴新的t(8; 21)变异易位即t(7; 21)(p21;q22)易位患者的临床与分子生物学特点.方法:将AML患者的骨髓细胞经短期培养后按常规方法制备染色体,R显带进行核型分析;利用AML1/ETO双色双融合探针进行荧光原位杂交检测;实时荧光定量PCR法检测AML1/ETO融合基因的转录本拷贝数.结果:患者的常规细胞遗传学分析结果显示为t(7; 21)(p21;q22)易位.86％的骨髓细胞为AML1/ETO融合基因阳性,融合基因转录本为51 440个拷贝/10 000个内参Ab1基因拷贝.结论:t(7;21)(p21; q22)是一种新的t(8; 21)(q22; q22)变异易位,与其他类型的t(8; 21)变异易位相似,预示有良好预后.     

Molecular and clinical advances in core binding factor primary acute myeloid leukemia: a paradigm for translational research in malignant hematology

Clonal chromosomal abnormalities are the most important prognostic indicators in acute myeloid leukemia (AML). Recent advances in molecular biology have allowed structural and functional characterization of many of these genomic rearrangements and have provided evidence for their primary role in leukemogenesis. Two of the most prevalent cytogenetic subtypes of adult primary or de novo AML, t(8;21)(q22;q22) and inv(16)(p13q22), are characterized by disruption of the AML1(CBF alpha 2) gene at 21q22 and the CBF beta gene at 16q22, respectively. Both genes encode a subunit of core binding factor (CBF), a regulator of normal hematopoiesis. At the molecular level, t(8;21)(q22;q22) and inv(16)(p13q22) result in the creation of novel fusion genes, AML1/ETO and CBF beta/MYH11, whose structures and functions are being successfully characterized by in vitro studies and transgenic animal models. Detection of t(8;21)(q22;q22) or inv(16)(p13q22) in adult patients with primary AML is a favorable independent prognostic indicator for achievement of cure after intensive chemotherapy or bone marrow transplantation and may serve as a paradigm for risk-adapted treatment in AML. The purpose of this review is to summarize the recent advances in the molecular biology and clinical management of t(8;21)(q22;q22) and inv(16)(p13q22) primary AML, collectively referred to here as CBF AML.     

Incidence of MLL rearrangement in acute myeloid leukemia, and a CALM-AF10 fusion in M4 type acute myeloblastic leukemia

To determine the incidence of the mixed lineage leukemia (MLL) gene rearrangements in acute myeloid leukemia (AML) without cytogenetically-detected 11q23 abnormalities, we screened 64 cases of AML at diagnosis for MLL rearrangement by FISH. Three cases (4.7%) had a MLL rearrangement detected; one was shown to have a cryptic t(11;22)(q23;q11) and another to have a t(9;11)(p21-22;q23) which had been missed by the conventional cytogenetic study. No 11q23 structural abnormality was visible in the third case. Twenty-six of the 64 cases were further studied by Southern blotting and DNA hybridization, and four of these cases (15%) were found to have MLL rearrangement: in three of these, FISH had not detected any abnormality. FISH was also used to confirm MLL involvement in eight cases of AML that had a cytogenetic abnormality at 11q23; in one of these, Southern blot did not show a rearrangement. The survival of patients with MLL abnormalities identified by cytogenetics, FISH and/or DNA analysis was significantly worse than that of patients without MLL abnormalities (event-free survival p = 0.016) although two patients with a t(9;11)(p21-22;q23) were long-term survivors, consistent with this particular translocation having a better prognosis. One further case with a cytogenetic abnormality close to 11q23 was studied; it was found to have a t(10;11)(p13;q21), and the breakpoints were shown by FISH to involve the Clathrin Assembly Lymphoid Myeloid (CALM) gene at 11q21 and the AF10 gene at 10p13. Our data confirm the value of combining cytogenetic, FISH and molecular analyses to define the incidence and precise nature of MLL and 11q23 abnormalities in AML.     

Identification of novel chromosomal rearrangements in acute myelogenous leukemia involving loci on chromosome 2p23, 15q22 and 17q21.

Chromosomal translocations are frequently linked to multiple hematological malignancies. The study of the resulting abnormal gene products has led to fundamental advances in the understanding of cancer biology. This is the first report of t(2;15)(p23;q22) and t(2;17)(p23;q21) translocations in human malignancy. Patient 1, a 73-year-old male, was diagnosed with myeloblastic (FAB M1 sub-type) AML. Cytogenetic analysis showed a 47,XY,t(2;15)(p23;q22),+13 karyotype. Fluorescent in situ hybridization (FISH) showed that the PML gene was transferred intact to the short arm of chromosome 2 while the ALK gene on chromosome 2p23 was passively transferred to the long arm of chromosome 15. Patient 2 was a 60-year-old male diagnosed with monocytic (FAB M4-type) AML. Cytogenetic analysis showed 46,XY,t(2;17)(p23;q21) karyotype. FISH analysis showed that neither RARalpha nor ALK were disrupted by the translocation. None of the coding region of the three genes studied were translocated in these patients. This raises the possibilities that other neighboring genes could be involved or that noncoding regulatory sequences of the studied genes could be put in contact and deregulate expression of other genes. Alternatively, displacement of ALK, RARalpha and PML to novel positions could lead to loss of their normal regulation     

ETV6/AML1 fusion by FISH in adult acute lymphoblastic leukemia.

Dual-color interphase fluorescence in situ hybridization (FISH) with ETV6 and AML1 probes was used for the first time on a series of 159 adult patients with acute lymphoblastic leukemia (ALL), for detection of the t(12;21)(p13;q22) translocation. Seven patients (4.4%) were found, with 50-100% of positive cells, of whom one of two tested, proved negative for the fusion product by RT-PCR. Two of them, aged 43 and 50 years, are the oldest patients so far confirmed to have the translocation. Three who relapsed at 10, 11 and 24 months, suggest that adults may not enjoy the good short-term prognosis reported for t(12;21)-positive children. Thirty-one-negative cases had signal numbers differing from the two expected for each gene. In 15 cases these results were consistent with the karyotype. In nine cases with uninformative cytogenetics, the numbers were consistent with those for centromeres and indicated a hidden aneuploidy. Loss of ETV6 genes in two cases and AML1 amplification in three others were not suspected from the cytogenetics. In conclusion, FISH proved to be reliable in defining ETV6/AML1 positivity in this group of patients as well as providing valuable insights into negative cases.     

Identification of signature genes by microarray for acute myeloid leukemia without maturation and acute promyelocytic leukemia with t(15;17)(q22;q12)(PML/RARalpha)

Acute myeloid leukemia (AML) has distinct subgroups characterized by different maturation and specific chromosomal translocation. In order to gain insight into the gene expression activities in AML, we carried out a gene expression profiling study with 21 AML samples using cDNA microarrays, focusing on acute promyelocytic leukemia with specific translocation t(15;17)(q22;q12) [French-American-British or FAB-M3 with t(15;17)] and AML without maturation (FAB-M1) characterized by morphologically and phenotypically immature AML blasts and no recurrent chromosomal abnormalities. Using a multivariate sigma-classifier algorithm, we identified 33 strong feature genes that distinguish FAB-M3 with t(15;17) from other AML samples, and 24 strong feature genes that classify FAB-M1. A direct comparison between FAB-M3 with t(15;17) and FAB-M1 led to selection of 13 strong feature genes. Those genes include some known to be related to leukemogenesis and cell differentiation. RIN1, a gene in the ras pathway, was up-regulated in FAB-M3 with t(15;17). Growth factor-binding protein 2 gene was down-regulated in FAB-M1. Huntingtin gene was up-regulated in FAB-M1. Others include syndecan 4, interleukin-2 receptor beta, folate receptor beta, low affinity immunoglobulin gamma, Fc receptor IIC precursor, insulin-like growth factor binding protein 2, and myeloperoxidase, which are involved in cell differentiation. Overexpression of myeloperoxidase in FAB-M3 cells with t(15;17) compared to FAB-M1 cells is consistent with the conventional cytochemical staining pattern. Thus, the study revealed that a morphologically-defined FAB-M1 subtype has a distinct gene expression signature that contributes to its cell differentiation and proliferation as well as FAB-M3 with a recurrent cytogenetic abnormality t(15;17)(q22;q12).     

Cytogenetic Characteristics of de novo Acute Myeloid Leukemia in Southern Vietnam

Background: The cytogenetic characteristics are important factors for risk stratification at diagnosis of acute myeloid leukemia (AML); however, cytogenetic profile of Vietnamese patients with AML remains undetermined. In this study, we present the chromosomal data of de novo AML patients in Southern Vietnam. Methods: We performed cytogenetic testing for 336 AML patients using G banding. If the patients had suspected abnormalities, fluorescence in situ hybridization with probes of inv(3)(q21q26)/t(3;3)(q21;q26), 5q31, 7q31, t(8;21)(q21.3;q22), 11q23, t(15;17)(q24;q21), inv(16)(p13q22)/t(16;16)(p13;q22)were analyzed. Patients without above aberrations or with normal karyotype were tested by fluorescence in situ hybridization using probe 11q23. Results: We found that the median age was 39 years. According to French – American – British classification, AML-M2 is the most frequent type with 35.1%. Chromosomal abnormalities were detected in 208 cases, accounting for 61.9%. Among structural abnormalities, t(15;17) was the most common (19.6%), followed by t(8;21) and inv (16)/t(16;16) in 10.1% and 6.2%, respectively. In perspective of chromosomal numerical abnornmalities, loss of sex chromosomes are the most common (7.7%), followed by +8 in 6.8%, -7/del(7q) in 4.4%, +21 in 3.9% and -5/del (5q) in 2.1%. The prevalence of addditional cytogenetic aberrations accompanying with t(8;21) and inv(16)/t(16;16) were 82.4% and 52.4%, repectively. None of +8 cases was associated with t(8;21). Regarding cytogenetic risk assessment according to European Leukemia Net 2017, there were 121 (36%) patients in favorable-risk, 180 (53.6%) in intermediate-risk and 35 (10.4%) in adverse-risk group. Conclusion: In conclusion, this is the first comprehensive cytogenetic profile of Vietnamese patients diagnosed with de novo AML, which helps clinical doctors in prognostic classification for AML patients in Southern Vietnam.     

Variant Philadelphia translocations in chronic myeloid leukemia: correlation with cancer breakpoints, fragile sites and oncogenes

Four cases of variant Philadelphia (Ph1) translocations were found in 72 patients (5.5%) with Ph1-positive chronic myeloid leukemia (CML). One previously unreported case was a simple variant translocation, namely, 46,XY,t(11;17)(q13;p13),t(17;22)(q25;q22); 46,XY,t(1;21)(q32;q11),t(11;17)(q13;p13), t(17;22)(q25;q11). Complex variant translocations were observed in three cases, namely, 46,XY,t(5;9;22)(q31;q34;q11),46,XX,t(8;9;22) (q22;q34;q11) and 46,XX,t(9;15;22) (q34;q15;q11). The chromosomal breakpoints in the cases of variant Ph1 translocations were the following: 1q32, 5q31, 8q22, 11q13, 15q15, 17p13, 17q25 and 21q11. Eight of the eight (100%) breakpoints were located in Giemsa-negative bands. Furthermore, seven of the eight (87%) variant Ph1 breakpoints correspond to the breakpoints present in consistent cancer arrangements. Three of the eight (38%) correspond to fragile sites and four of the eight (50%) correspond to oncogenes.     

Submicroscopic deletions in an acute myeloid leukemia case with a four-way t(8;11;16;21)

The t(8;21)(q22;q22) rearrangement is observed in about 15% of acute myelocitic leukemia (AML) cases, while variant t(8;21) translocations are detected in 6-10% of AML patients positive for the 5'RUNX1/3'CBFA2T1 fusion gene. We report a detailed molecular cytogenetic analysis of a four-way variant t(8;11;16;21)(q22;q14;q12;q22) performed by fluorescence in situ hybridization with specific BAC and PAC clones. The study demonstrated the loss of several megabases belonging to chromosomes 11 and 16 whereas no deletion was detected on der(21). These findings suggest that a precise breakpoint characterization could identify submicroscopic genomic deletions whose meaning remains to be defined.     

实时定量荧光反转录-聚合酶链反应检测急性白血病AML1／ETO融合基因

  【摘要】　目的　建立实时定量荧光反转录-聚合酶链反应（RQ RT-PCR）的方法并用来检测AML-M2患者中AML1／ETO融合基因的拷贝数,观察患者体内融合基因阳性率、该融合基因转录水平的变化情况及AML1／ETO融合基因阳性患者对治疗的反应。方法　利用含AML1／ETO融合基因的Kasumi-1细胞株构建质粒标准品并制作标准曲线,检测25例AML-M2患者的骨髓及外周血标本45份,个别患者连续监测融合基因转录表达水平。25例患者均同时行流式细胞术免疫分型检测及骨髓细胞染色体检查,确诊后给予MA方案进行诱导缓解。结果　在28 %（7／25）的AML-M2初发确诊患者中检测到AML1／ETO阳性（AML1／ETO：ABL为0.01～19.2）,其中5例（20 %）有t(8;21)(q22;q22)。连续监测患者融合基因转录表达水平与临床缓解和复发的变化情况相吻合。7例AML1／ETO融合基因阳性患者均在MA治疗1个疗程后达完全缓解,AML1／ETO融合基因下降3个数量级。其余18例完全缓解11例。连续监测7例AML1／ETO融合基因阳性患者6个月均处于完全缓解状态。结论　实时定量荧光PCR技术成熟、操作简便,检测白血病融合基因结果准确稳定,对于临床明确诊断、具体分型、动态观测肿瘤负荷、选择治疗方案、评估治疗效果和预后都有较大价值。     

Combined Study of Cytogenetics and Fluorescence in Situ Hybridization (FISH) Analysis in Childhood AcuteLymphoblastic Leukemia (ALL) in a Tertiary Cancer Centre in South India

FISH is one of the most sensitive molecular methods to detect genetic abnormalities with DNA probes.When cytogenetic studies are normal or insufficient, FISH may detect cryptic rearrangements, rare or slowlyproliferative abnormal populations in non-mitotic cells. We cytogenetically evaluated 70 childhood ALL - 67.1%were found to have an abnormal karyotype. The 23 patients (32.9%) with a normal karyotype were analyzedby FISH applying two probes; TEL/AML1 and MYB which detect cryptic rearrangements of t(12;21)(p13;q22)and deletion of (6q) respectively, associated with a good prognosis. Out of 23 patients, one was positive fort(12;21)(p13;q22) (4.3%). None of our patients were positive for MYB del(6q). Two patients showed an extrasignal for MYB on chromosomes other than 6 (8.6 %) indicating amplification or duplication. Findings werecompared with the available literature. Our study clearly indicated the integrated FISH screening method toincrease the abnormality detection rate in a narrow range. FISH is less useful for diagnostic study of patientswith suspected del(6q) but it helps in detecting known cryptic rearrangements as well as identification of newabnormalities(translocation , duplication and amplification) at the gene level.     

High frequency of t(12;21)(p13;q22) in children with acute lymphoblastic leukemia and known clinical outcome in southern Brazil

The presence of the t(12;21)(p13;q22) distinguishes a subset of children with acute lymphoblastic leukemia (ALL) that present a favorable prognosis. This is a cryptic translocation difficult to detect through conventional cytogenetics. In this study, bone marrow samples from 30 children with ALL from southern Brazil were evaluated by fluorescence in situ hybridization (FISH) for the t(12;21), using locus specific probes to detect the TEL/AML1 rearrangement. The selection criteria included: age (0-12 years old); FAB classification (L1 or L2), absence of specific clonal chromosomal aberrations; and adequate cellular integrity to perform FISH analysis. A frequency of 40% of the t(12;21) was observed, in addition to extra copies of the AML1 gene in 7.5% of patients. These findings were analyzed in relation to the patient's clinical parameters and compared with other pediatric populations.