Spectral karyotyping and interphase FISH reveal abnormalities not detected by conventional G-banding. Implications for treatment stratification of childhood acute lymphoblastic leukaemia: detailed analysis of 70 cases

Seventy uniformly treated children with acute lymphoblastic leukemia were analysed for chromosomal abnormalities with conventional G-banding, spectral karyotyping (SKY) and interphase fluorescent in situ hybridisation (FISH) using probes to detect MLL, BCR/ABL, TEL/AML1 rearrangements and INK4 locus deletions. Numerical and/or structural changes could be identified in 80% of the patients by the use of molecular cytogenetic techniques, whereas abnormalities could be detected in 60% of the patients using G-banding alone. Altogether, 106 structural aberrations were defined by FISH compared to 34 using G-banding. Seventy-four percent of the patients had numerical aberrations, 54% structural aberrations and 20% had no identified aberrations. Twelve cases had prognostically unfavourable chromosomal aberrations that had not been detected in the G-banded analysis. We identified three novel TEL partner breakpoints on 1q41, 8q24 and 21p12, and a recurrent translocation t(1;12)(p32;p13) was found. In addition, two cases displayed amplification (7-15 copies) of AML1. Our results demonstrate the usefulness of SKY and interphase FISH for the identification of novel chromosome aberrations and cytogenetic abnormalities that provide prognostically important information in childhood ALL.     

High reliability and sensitivity of the BCR/ABL1 D-FISH test for the detection of BCR/ABL rearrangements

The BCR/ABL1 fusion gene is mainly caused by the t(9; 22)(q34; q11.2) translocation, which results in the Philadelphia (Ph) chromosome. The Ph chromosome is the typical hallmark in chronic myeloid leukemia (CML), but can also be present in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The BCR/ABL1 rearrangement is an important tumor classification marker and a useful prognostic factor allowing an adequate therapy management. Ph chromosome detection by conventional cytogenetics (CC) can be hampered by low quantity and quality of metaphases from tumor cells. Furthermore, BCR/ABL1 rearrangements may be hidden due to cryptic rearrangements or complex aberrations. Therefore, molecular cytogenetic methods turned out to be useful tools for the detection of BCR/ABL1 rearrangements. We performed fluorescent in situ hybridization (FISH) with the recently developed BCR/ABL1 D-FISH probe (QBIOgene, Illkirch, F) on cultured bone marrow and peripheral blood cells of 71 patients with CML, ALL, AML, and myeloproliferative disorder (MPD). FISH results and the results of banding methods were directly compared. Based on the analyses of >200 nuclei per patient, D-FISH correlated closely with CC and allowed an accurate quantification of BCR/ABL1 rearrangements even in a low percentage of aberrant cells. No false-positive or false-negative results were obtained. Furthermore, the D-FISH probe detected three cryptic and one complex BCR/ABL1 rearrangement, which were not visible by CC. We conclude that D-FISH reliably detects standard Ph chromosomes as well as its variant translocations and accurately quantifies BCR/ABL1 rearrangements prior and during cancer treatment as well as in the phase of remission, in daily routine tumor cytogenetic diagnostics.     

Acute lymphoblastic leukaemia.

In acute lymphoblastic leukaemia (ALL) the karyotype provides important prognostic information which is beginning to have an impact on treatment. The most significant structural chromosomal changes include: the poor-risk abnormalities; t(9;22)(q34;q11), giving rise to the BCR/ABL fusion and rearrangements of the MLL gene; abnormalities previously designated as poor-risk; t(1;19)(q23;p13), producing the E2A/PBX1 and rearrangements of MYC with the immunoglobulin genes; and the probable good risk translocation t(12;21)(p13;q22), which results in the ETV6/AML1 fusion. These abnormalities occur most frequently in B-lineage leukaemias, while rearrangements of the T cell receptor genes are associated with T-lineage ALL. Abnormalities of the short arm of chromosome 9, in particular homozygous deletions involving the tumour suppressor gene (TSG) p16(INK4A), are associated with a poor outcome. Numerical chromosomal abnormalities are of particular importance in relation to prognosis. High hyperdiploidy (51-65 chromosomes) is associated with a good risk, whereas the outlook for patients with near haploidy (23-29 chromosomes) is extremely poor. In view of the introduction of risk-adjusted therapy into the UK childhood ALL treatment trials, an interphase FISH screening programme has been developed to reveal chromosomal abnormalities with prognostic significance in childhood ALL. Novel techniques in molecular cytogenetics are identifying new, cryptic abnormalities in small groups of patients which may lead to further improvements in future treatment protocols.     

The detection and significance of chromosomal abnormalities in childhood acute lymphoblastic leukaemia

In childhood acute lymphoblastic leukaemia (ALL), cytogenetics plays an essential role in diagnosis and prediction of outcome. Conventional cytogenetic analysis, complemented by fluorescence in situ hybridization (FISH), is highly effective in the accurate detection of chromosomal abnormalities. For the precise identification of specific genetic changes, molecular techniques may be applied. Chromosomal changes in ALL may be of structural or numerical type. A large number of established structural chromosomal rearrangements have now been described for which the genetic alterations and effect on prognosis are well known. These include t(9;22)(q34;q11) and BCR/ABL, rearrangements of 11q23 involving MLL, t(12;21)(p13;q22) with the ETV6/AML1 fusion, t(1;19)(q23;p13) with E2A/PBX1, t(8;14)(q24;q32) and the immunoglobulin genes. Genetic changes associated with T ALL are also known, although their effect on outcome is less pronounced. Rare chromosomal abnormalities are continually being discovered in small patient subgroups leading to the identification of new ALL associated genetic changes. Alterations in chromosome number have a strong impact on outcome in childhood ALL. The association of a high hyperdiploid karyotype (51-65 chromosomes) with a good prognosis has been known for more than 20 years. Conversely, the loss of chromosomes in the near-haploid group (23-28 chromosomes) indicates a poor outcome. New methods of cancer classification involving gene expression profiling may eventually supercede cytogenetic analysis in the diagnosis and prediction of outcome in leukaemia. It is more likely that they will be used in a complementary approach alongside cytogenetic, FISH and molecular analysis to guide patient management in childhood ALL.     

BCR-ABL rearrangement in a child with acute myelogeneous leukaemia without a Philadelphia chromosome

Summary. We describe a BCR/ABL rearrangement positive but Philadelphia chromosome negative status in a 9-year-old boy suffering from an acute myelogeneous leukaemia (AML). This case was detected in a prospective PCR screening procedure including 21 children with newly diagnosed AML and 150 children with acute lymphoblastic leukaemia (ALL). We found a 5·4% incidence of BCR/ABL rearrangement positive cases in pre-B and c-ALL in childhood.     

The genetics of childhood acute lymphoblastic leukaemia.

In childhood acute lymphoblastic leukaemia (ALL) a number of genetic changes have been identified which provide diagnostic and prognostic information with a direct impact on patient management. The most significant abnormalities include the translocation, t(12;21)(p13;q22), giving rise to the ETV6/AML1 gene fusion; BCR/ABL arising from t(9;22)(q34;q11); re-arrangements of the MLL gene; the E2A/PBX1 from the t(1;19)(q23;p13); re-arrangements of MYC with the immunoglobulin genes and re-arrangements of the T cell receptor genes. Chromosomal deletions, particularly those of the short arms of chromosomes 9 and 12 and the long arm of chromosome 6, have been postulated to be the sites of tumour suppressor genes (TSG). Numerical chromosomal abnormalities are of particular importance in relation to prognosis. High hyperdiploidy (50-65 chromosomes) is associated with a good risk, whereas the outlook for patients with near haploidy (23-29 chromosomes) is extremely poor. In view of the introduction of risk-adjusted therapy into the UK childhood ALL treatment trials, an interphase FISH screening programme has been developed to reveal chromosomal abnormalities with prognostic significance in childhood ALL.     

Interphase molecular cytogenetic screening for chromosomal abnormalities of prognostic significance in childhood acute lymphoblastic leukaemia: a UK Cancer Cytogenetics Group Study

Summary Interphase fluorescence in situ hybridization (iFISH) was used independently to reveal chromosomal abnormalities of prognostic importance in a large, consecutive series of children (n = 2367) with acute lymphoblastic leukaemia (ALL). The fusions, TEL/AML1 and BCR/ABL, and rearrangements of the MLL gene occurred at frequencies of 22% (n = 447/2027) (25% in B-lineage ALL), 2% (n = 43/2027) and 2% (n = 47/2016) respectively. There was considerable variation in iFISH signal patterns both between and within patient samples. The TEL/AML1 probe showed the highest incidence of variation (59%, n = 524/884), which included 38 (2%) patients with clustered, multiple copies of AML1. We were thus able to define amplification of AML1 as a new recurrent abnormality in ALL, associated with a poor prognosis. Amplification involving the ABL gene, a rare recurrent abnormality confined to T ALL patients, was identified for the first time. The use of centromeric probes revealed significant hidden high hyperdiploidy of 33% and 59%, respectively, in patients with normal (n = 21/64) or failed (n = 32/54) cytogenetic results. The iFISH contributed significantly to the high success rate of 91% (n = 2114/2323) and the remarkable abnormality detection rate of 89% (n = 1879/2114). This study highlights the importance of iFISH as a complementary tool to cytogenetics in routine screening for significant chromosomal abnormalities in ALL.     

Spectral karyotyping and fluorescence in situ hybridization detect novel chromosomal aberrations, a recurring involvement of chromosome 21 and amplification of the MYC oncogene in acute myeloid leukaemia M2

Recurring chromosomal aberrations are of aetiological, diagnostic, prognostic and therapeutic importance in acute myeloid leukaemia (AML). However, aberrations are detected in only two thirds of AML cases at diagnosis and recurrent balanced translocations in only 50%. Spectral karyotyping (SKY) enables simultaneous visualization of all human chromosomes in different colours, facilitating the comprehensive evaluation of chromosomal abnormalities. Therefore, SKY was used to characterize 37 cases of newly diagnosed AML-M2, previously analysed using G-banding. In 15/23 patients it was possible to obtain metaphases from viably frozen cells; in 22 additional cases, fixed-cell suspensions were used. Of the 70 chromosomal aberrations identified by SKY, 30 aberrations were detected for the first time, 18 aberrations were redefined and 22 were confirmed. SKY detected two reciprocal translocations, t(X;3) and t(11;19). In five cases, eight structural aberrations resulted in partial gains of chromosome 21, six of which were undetected by G-banding. In 4/5 cases, these resulted in copy number increases for AML1. Amplification of MYC was detected in three cases. Using SKY and FISH, clonal aberrations were identified in 5/18 cases with a presumed normal karyotype; 3/5 aberrations were of known unfavourable prognostic significance. Karyotypes were entered into a custom-designed SKY database, which will be integrated with other cytogenetic and genomic databases.     

Complex chromosome rearrangements may locate the bcr/abl fusion gene sites other than 22q11

BACKGROUND AND OBJECTIVE: From 5-8% of Philadelphia (Ph) positive patients with chronic myeloid leukemia (CML) show variant translocations in which at least a third chromosome in addition to 9q34 and 22q11 is involved. The formation mechanisms and clinical significance of variant Ph translocations are still unclear. The BCR/ABL chimeric gene encoding for chimeric proteins is always present and maps on the 22q- regardless of the type of translocation. We studied two apparently Ph negative CML patients with unusual karyotypes both showing a typical b3a2 rearrangement. DESIGN AND METHODS: Dual-color fluorescence in situ hybridization (FISH) can visualize BCR and ABL genes and localize the BCR/ABL fusion gene. We used FISH to study the formation mechanisms of variant Ph translocations in two patients. RESULTS: The chimeric BCR/ABL gene was located on a locus other than the expected 22q11 in both patients. In the first case the fusion signal was present on the 9q34 band whereas in the second patient it was detected on chromosome 8, involved in masked Ph formation. INTERPRETATION AND CONCLUSIONS: The location of the hybrid BCR/ABL gene on chromosomes other than 22q- is a rare event which can only be observed using the FISH technique. When these unusual translocations occur the hypothesis most often put forward is that several consecutive cytogenetic events have taken place. The factors which regulate the formation of these breakpoints have yet to be clarified. The FISH technique allows the identification of chromosome rearrangements that could not otherwise be detected by conventional banding procedures. The location of the hybrid BCR/ABL gene on sites other than 22q11 represents a rare type of variant Ph translocation.The real frequency and clinical significance of such rearrangements need to be investigated.     

Prognostic cytogenetic markers in childhood acute lymphoblastic leukemia: cases from Mansoura Egypt

The objective of the work was to evaluate children with acute lymphoblastic leukemia (ALL) showing resistance to immediate induction chemotherapy in relation to conventional and advanced cytogenetic analysis. The study was conducted on 63 ALL children (40 males and 23 females) with age range 4.5 months-16 years (mean = 7.76 years). They included 37 cases who attained a true remission and 26 complicated by failure of remission, early relapse or death. They were subjected to history, clinical examination and investigations including CBC, BM examination, karyotyping, FISH for translocations and flowcytometry for immunophenotyping and minimal residual disease diagnosis. Cases aged < 5 years; male sex with organomegaly had better remission although statistically insignificant. Initially low HB < 8 gm/dl, high WBCs and platelet counts >50.000/mm(3) also showed better but non-significant remission rates. Most of our cases were L(2) with better remission compared to other immunophenotypes. About 40 informative karyotypes were subdivided into 15 hypodiploid, 10 pseudodiploid, 8 normal diploid and 7 hyperdiploid cases; the best remission rates were noticed among the most frequent ploidy patterns. Chromosomes 9, 11 and 22 were the most frequently involved by structural aberrations followed by chromosomes 5, 12 and 17. Resistance was noted with aberrations not encountered among remission group; deletions involving chromosomes 2p, 3q, 10p and 12q; translocations involving chromosome 5; trisomies of chromosomes 16 and 21; monosomies of 5 and X and inversions of 5 and 11. Our conclusions were that cytogenetic and molecular characterizations of childhood ALL could add prognostic criteria for proper therapy allocation.     

Clonal eosinophils are a morphologic hallmark of ETV6/ABL1 positive acute myeloid leukemia

BACKGROUND AND OBJECTIVES: The ETV6 gene undergoes rearrangements with tyrosine kinases in hematologic malignancies and solid tumors. ETV6/ABL1 chimeric proteins have been detected both in lymphoid and myeloid disorders. Our objective was to study two new cases of ETV6/ABL1-positive acute myeloid leukemia (AML) and to focus on bone marrow morphology and on molecular cytogenetics of eosinophilic cells. DESIGN AND METHODS: Fluorescence in situ hybridization (FISH) was performed in two AML cases with different translocations, i.e. t(8;12)(p21;p13) and t(9;12) (q34; p13). We used probes for the short arm of chromosome 12, for ABL1 and BCR, for centromeric regions, and for whole chromosome arms. Polymerase chain reaction (PCR) was carried out by applying primers selected for the ETV6 gene. RESULTS: In both cases, bone marrow morphology was characterized by trilineage dysplasia and increased abnormal eosinophils. FISH showed the 5'ETV6 translocated to chromosome 8 in patient #1, and to chromosome 9 in patient #2. A 3' PCR identified chimeric products resulting from fusion between ETV6 exon 4 or exon 5, and ABL1 exon 2. Accordingly, an ETV6/ABL1 fusion signal was detected on der(8) in patient #1, and on der(9) in patient #2. Using interphase FISH abnormal bone marrow eosinophils were proved to belong to the neoplastic clone, carrying the ETV6 rearrangement. INTERPRETATION AND CONCLUSIONS: Our findings provide new information on the heterogeneity of conventional cytogenetics in ETV6/ABL1 positive leukemias, and indicate the putative target cell in this AML is an immature precursor capable of terminally differentiating towards eosinophils.     

CD66c expression in B‐cell acute lymphoblastic leukemia: strength and weakness

Introduction: In B‐cell acute lymphoblastic leukemia (B‐ALL), testing at diagnosis for BCR/ABL1 gene rearrangements is mandatory for prognostic stratification and treatment decisions. Several diagnostic methods have been proposed using flow cytometry to identify BCR/ABL1⁺ B‐ALL. Methods: We evaluated expression of the myeloid antigen CD66c by flow cytometry in B‐ALL. We studied 94 patients with B‐ALL. The t(9;22)(q34;q11) or BCR/ABL1 rearrangement was detected by cytogenetic analysis or RT/PCR. Myeloid antigens CD66c, CD13, CD33, CD117, Myeloperoxidase, CD15 and CD65 were determined by flow cytometry. Results: Of these 94 cases, 17 (18%) cases displayed BCR/ABL1 gene rearrangements and 38 (40%) cases were CD66c positive. CD66c was the most common myeloid antigen expressed on malignant lymphoblasts. Its expression was correlated with BCR/ABL1 rearrangements (P = 0.0001): sensitivity 82%, specificity 69%, positive predictive value 37% and negative predictive value 95%. Co‐expression of CD66c⁺ CD13⁺ was more frequent in BCR/ABL1⁺ B‐ALL (29%) than BCR/ABL1^? cases (4%) (P = 0.0044). Some BCR/ABL1^? B‐ALL cases (including hyperdiploid or cases with normal karyotype) were CD66c positive (31%). Conclusion: CD66c expression is correlated, but not specifically, with BCR/ABL1 rearrangement. It would seem better to interpret the absence of CD66c expression with a lack of BCR/ABL1 rearrangement. This myeloid antigen could be interesting in the detection of minimal residual disease.     

Cytogenetic and FISH studies of a single center consecutive series of 152 childhood acute lymphoblastic leukemias

Between 1977 and 1996, cytogenetic investigations were performed on 182 childhood (< or = 16 yr) acute lymphoblastic leukemias (ALL), constituting 94% (182 of 194) of all ALL patients diagnosed and treated at the Departments of Pediatrics, Lund and Malmo University Hospitals, Sweden, during these two decades. The cytogenetic analyses were successful in 152 cases (84%). The failure rate was higher for the ALL investigated before 1987 (30% vs. 4%, p < 0.0001), and also the incidence of cytogenetically normal cases was higher during 1977-86 (43% vs. 25%, p < 0.05). Clonal chromosomal abnormalities were found in 103 (68%) ALL. Structural rearrangements were detected, by chromosome banding alone, in 76 cases (50%). Fluorescence in situ hybridization (FISH) was used to identify cases with t(12;21), 11q23 rearrangements, and 9p deletions, using probes for ETV6/CBFA2, MLL, and CDKN2A/B, in 72 cases from which cells in fixative and/or unstained metaphase preparations were available. In total, the most common structural rearrangements were del(9p) (17%), t(12;21) (15%), del(6q) (8%), and MLL rearrangements (4%). Six (32%) of nineteen cytogenetically normal ALL analyzed by FISH harbored cryptic abnormalities; three displayed t(12;21) and four had del(9p), one of which also carried a t(12;21). Five (45%) of the t(12;21)-positive ALL showed +der(21)t(12;21) or ider(21)(q10)t(12;21), resulting in the formation of double fusion genes. Among the more rare aberrations, eight structural rearrangements were identified as novel recurrent ALL-associated abnormalities, and nine cases harbored rearrangements previously not reported. Sixteen cases displayed karyotypically unrelated clones at different investigations. Seven ALL (5%) showed simple chromosomal changes, unrelated to the aberrations detected at diagnosis, during morphologic and clinical remission, and in all but one instance the patients remained in remission, with the abnormal clone disappearing in subsequent investigations. This indicates that the emergence of novel clonal chromosomal aberrations during remission in childhood ALL is rather common and does not by necessity predict a forthcoming relapse.     

The fusion of TEL and ABL in human acute lymphoblastic leukaemia is a rare event

We have recently identified a common ALL patient which harboured a chromosomal fusion between the TEL gene on chromosome 12 and the ABL gene on chromosome 9. We designed an RT-PCR assay to screen 186 adult ALL and 30 childhood ALL patients for this novel translocation. We were unable to identify any additional cases with a TEL/ABL fusion product.