Interphase fluorescence in situ hybridization and spectral karyotyping reveals hidden genetic aberrations in children with acute lymphoblastic leukaemia and a normal banded karyotype

Twenty-two cases of childhood acute lymphoblastic leukaemia (ALL) with normal G- or Q-banded karyotypes were studied by interphase fluorescence in situ hybridization (FISH) and spectral karyotyping. Probes detecting MLL, BCR/ABL and TEL/AML1 rearrangements were used for the interphase studies, along with centromere-specific probes from chromosomes 17 and X. In 10 patients (45%), previously undetected aberrations were demonstrable. Specific gene rearrangements and structural changes were found in six cases and numerical changes in five. Five of these aberrations have previously been reported to have an impact on prognosis. Three cases were massively hyperdiploid and, in one, the prognostically important BCR/ABL fusion was detected. In addition, a near-haploid karyotype with 27 chromosomes was found in one patient and TEL/AML1 rearrangements were detected in two cases. This study indicates that about half of childhood ALL cases with apparently normal karyotypes harbour genetic aberrations that may be detected using interphase FISH and spectral karyotyping.     

Location of the BCR/ABL Fusion Genes on Both Chromosomes 9 in Ph Negative Young CML Patients: An Indian Experience

The BCR/ABL gene rearrangement is cytogenetically visualized in most chronic myeloid leukemia (CML) cases. About 5–10 % of CML patients lack its cytogenetic evidence, however, shows BCR/ABL fusion by molecular methods. We describe two CML patients with Philadelphia (Ph) negative (−ve) and BCR/ABL positive by fluorescence in situ hybridization (FISH). Both the cases were in chronic phase at diagnosis. Conventional cytogenetics and different FISH assays were adopted using BCR/ABL probes. Home-brew FISH assay using bacterial artificial clone (BAC) for BAC-CTA/bk 299D3 for chromosomal region 22q13.31-q13.32 was performed in case 1. Both the patients were Ph-ve. In first case, dual color dual fusion (DCDF)-FISH studies revealed 1 Red (R) 2 Green (G) 1 Fusion (F) signal pattern in 80 % of cells indicating BCR/ABL fusion signals on chromosomes 9 instead of Ph and 2G2F signal pattern in 20 % of cells indicating two BCR/ABL fusions on both chromosomes 9q34 on presentation. In second case, FISH studies revealed the 1R1G1F signal pattern indicating BCR/ABL fusion signals on chromosomes 9 instead of Ph in 100 % of cells at presentation. During follow-up, both the patients exhibited 2G2F signal pattern indicating two BCR/ABL fusions on both chromosomes 9q34, which indicated a clonal evolution in 100 % cells. Both the patients did not achieve therapeutic response. Relocation of BCR/ABL fusion sequence on sites other than 22q11 represents a rare type of variant Ph, the present study highlights the hot spots involved in CML pathogenesis and signifies their implications in Ph−ve BCR/ABL positive CML. This study demonstrated the genetic heterogeneity of this subgroup of CML and strongly emphasized the role of metaphase FISH, especially in Ph−ve CML cases, as it detects variations of the classical t(9;22).     

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 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.     

Acute lymphoblastic leukaemia: diagnosis and classification

Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease with distinct biological and prognostic groupings. Diagnosis relies on traditional cytomorphological and immunohistochemical evaluation of the leukaemic blasts. Subsequently, cytogenetic analysis identifies clonal numeric and/or structural chromosomal abnormalities that may be present, thus confirming the subtype classification and providing important prognostic information for treatment planning. The major chromosomal abnormalities in ALL are t(9;22)(q34;q11), t(12;21)(p13;q22), t(4;11)(q21;q23), t(1;19)(q23;p13), 8q24 translocations and hyperdiploidy. Generally, hyperdiploidy, occurring most frequently in paediatric cases, is associated with a good prognosis, while hypodiploidy confers a poor prognosis. Among structural chromosomal abnormalities, the t(9;22)(q34;q11) resulting in the BCR/ABL fusion protein, and rearrangements of the MLL gene, confer a poor prognosis in both children and adults, while t(12;21)(p13;q22), resulting in the TEL/AML1 fusion protein, and del (12p) confer a good prognosis. More recently, additional diagnostic and prognostic information has been gained from fluorescence in situ hybridization (FISH) and DNA microarray techniques.     

Multiple clonal MLL fusions in a patient receiving CHOP-based chemotherapy

MLL rearrangements were analysed in the blood of a patient receiving chemotherapy for diffuse large B‐cell lymphoma using inverse polymerase chain reaction targeting exon 12, parallel sequencing and a custom algorithm design. Of thirteen MLL rearrangements detected, five were capable of generating MLL fusion genes, including MLL‐MLLT3, the most common fusion in acute myeloid leukaemia (AML). Other fusions, all previously clinically unobserved, included MLL‐NKD1, a fusion to the negative regulator of Wnt/β‐catenin signaling, a pathway linked to leukaemic cell proliferation. The majority of the fusions exhibited clonal persistence from before treatment until 6 months post‐chemotherapy, suggesting the fusions may confer a survival advantage to the mutant clone. MLL breakpoints were partly clustered at a specific location, indicating commonality in the process of their formation. Further, the same MLL breakpoint location exhibited a 50–100‐fold increase in C to T transitions, consistent with attack by activation‐induced cytidine deaminase (AICDA). As is also observed in AML and acute lymphoblastic leukaemia, in this single patient setting, MLL is capable of interacting with multiple fusion partners. This finding defines a discrete site of MLL susceptibility to fragmentation, linked to possible deregulation of AICDA function.     

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.     

Cytogenetic classification in Korean multiple myeloma patients: prognostic significance of hyperdiploidy with 47–50 chromosomes and the number of structural abnormalities

Chromosomal abnormalities are important prognostic factors for patients diagnosed with multiple myeloma (MM). We retrospectively reviewed the clinical and laboratory data of 525 MM patients to assess the abnormalities frequently found by conventional cytogenetic analysis and to determine their relationship to prognosis and clinical parameters. Samples from 222 (42.3%) patients had abnormal karyotypes. Hyperdiploidy‐1 (>50 chromosomes), hyperdiploidy‐2 (47–50 chromosomes), pseudodiploidy (46 with abnormalities), and hypodiploidy (<46 chromosomes) were found in 55, 44, 42, and 81 patients, respectively. The median overall survival (OS) was significantly shorter in patients with hyperdiploidy‐2 (20.9 months), pseudodiploidy (19.9 months), and hypodiploidy (18.3 months) compared with patients with normal karyotype (66 months) and hyperdiploidy‐1 (55.4 months) (P < 0.001). Among patients with chromosomal abnormalities, those with 1q amplification had a shorter median OS (17 vs. 25.1 months, P = 0.018). Patients with a chromosome 13 deletion in the pseudodiploidy group also had a shorter OS. A karyotype with more than six structural abnormalities was found to have the most significant independent prognostic value by multivariate analysis. These data show that hyperdiploidy with 47–50 chromosomes should be recategorized as an unfavorable risk group, and the number of structural abnormalities needs to be considered as an important factor for prognosis. In conclusion, our findings imply that subclassification of chromosomal abnormalities by conventional cytogenetics could be applied to the prognostic assessment of MM.     

Azurophilic inclusions in lymphocytes and plasma cells: a case of Sanfilippo disease

Correct action upon re‐emergence of minimal residual disease in acute myeloid leukaemia (AML) patients has not yet been established. The applicability of demethylating agents and use of allogeneic stem cell transplantation will be dependent on pre‐relapse AML growth rates. We here delineate molecular growth kinetics of AML harbouring MLL partial tandem duplication (MLL‐PTD; 37 cases) compared to those harbouring MLL translocations (43 cases). The kinetics of MLL‐PTD relapses was both significantly slower than those of MLL translocation positive ones (median doubling time: MLL‐PTD: 24 d, MLL‐translocations: 12 d, P = 0·015, Wilcoxon rank sum test), and displayed greater variation depending on additional mutations. Thus, MLL‐PTD+ cases with additional RUNX1 mutations or FLT3‐internal tandem duplication relapsed significantly faster than cases without one of those two mutations (Wilcoxon rank sum test, P = 0·042). As rapid relapses occurred in all MLL subgroups, frequent sampling are necessary to obtain acceptable relapse detection rates and times from molecular relapse to haematological relapse (blood sampling every second month: MLL‐PTD: 75%/50 d; MLL translocations: 85%/25 d). In conclusion, in this cohort relapse kinetics is heavily dependent on AML subtype as well as additional genetic aberrations, with possibly great consequences for the rational choice of pre‐emptive therapies.     

Del(5q) and MLL amplification in homogeneously staining region in acute myeloblastic leukemia: a recurrent cytogenetic association

We report here a 71 year-old female presenting with acute myeloblastic leukemia (FAB-M1) after treatment of essential thrombocythemia with Vercyte. Conventional cytogenetic techniques showed a complex karyotype, 44,XX,−5,−7,−11,add(11)(q23),−14,+mar,+r. The use of several fluorescent in situ hybridizations (FISH) lead to the identification of these complex rearrangements. The marker was found to be tricentric, with pericentromeric material of chromosome 7 inserted in the short arm of chromosome 5, resulting in monosomy 5q and 7q. The derivative chromosome 11 was dicentric and had subtelomeric sequences of 11p on both ends; several copies of the MLL gene were located in two different regions separated by a centromere of chromosome 11. Twenty-one cases, including ours, of myelodysplastic syndromes and acute myelogenous leukemia with MLL amplification present in hsr or dmin were found in the literature. Most of these patients shared some characteristics: they were old, they had de novo acute myeloid leukemia (AML) with a complex karyotype and a short survival, 90% of them having also a del(5q). Therefore, the simultaneous presence of MLL amplification and del(5q) appears to be a nonrandom association that could be the signature of AML in elderly patients with a poor prognosis.     

An In Vivo Topoisomerase II Cleavage Site and a DNase I Hypersensitive Site Colocalize Near Exon 9in the MLL Breakpoint Cluster Region

The human myeloid-lymphoid leukemia gene, MLL (also calledALL-1, Htrx, or HRX ), maps to chromosomal band11q23. MLL is involved in translocations that result in de novoacute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),mixed lineage leukemia, and also in therapy AML (t-AML) and therapy ALL(t-ALL) resulting from treatment with DNA topoisomerase II (topo II)targeting drugs. MLL can recombine with more than 30 otherchromosomal bands, of which 16 of the partner genes have been cloned.Breaks in MLL occur in an 8.3-kb breakpoint cluster region(BCR) encompassing exons 5 through 11. We recently demonstrated that75% of de novo patient breakpoints in MLL mapped in thecentromeric half of the BCR between two scaffold-associatedregions (SAR), whereas 75% of the t-AML patient breakpoints mapped tothe telomeric half of the BCR within a strong SAR. We have mappedadditional structural elements in the BCR. An in vivo DNA topo IIcleavage site (induced with several different drugs that target topoII) mapped near exon 9 in three leukemia cell lines. A strong DNase Ihypersensitive site (HS) also mapped near exon 9 in four leukemia celllines, including two in which MLL was rearranged [a t(6;11)and a t(9;11)], and in two lymphoblastoid cell lines with normalMLL. Two of the leukemia cell lines also showed an in vivo topoII cleavage site. Our results suggest that the chromatin structure ofthe MLL BCR may influence the location of DNA breaks in both denovo and therapy-related leukemias. We propose that topo II is enrichedin the MLL telomeric SAR and that it cleaves the DNase I HSsite after treatment with topo II inhibitors. These events may beinvolved in recombination associated with t-AML/t-ALL breakpointsmapping in the MLL SAR.     

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.     

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.     

The distribution of MLL breakpoints correlates with outcome in infant acute leukaemia

Acute leukaemia in early childhood ‐ and mainly infant leukaemia (IL) – is characterized by acquired genetic alterations, most commonly by the presence of distinct MLL rearrangements (MLL‐r). The aim of this study was to investigate possible correlations between clinical features and molecular analyses of a series of 545 childhood leukaemia (≤24 months of age) cases: 385 acute lymphoblastic leukaemia (ALL) and 160 acute myeloid leukaemia (AML). The location of the genomic breakpoints was determined in a subset of 30 MLL‐r cases. The overall survival of the investigated cohort was 60·5%, as determined by the Kaplan‐Meier method. Worse outcomes were associated with age at diagnosis ≤6 months (P < 0·001), high white blood cell count (P = 0·001), and MLL‐r (P = 0·002) in ALL, while children with AML displayed a poorer outcome (P = 0·009) regardless of their age strata. Moreover, we present first evidence that MLL‐r patients with poor outcome preferentially displayed chromosomal breakpoints within MLL intron 11. Based on the literature, most MLL‐r IL display a breakpoint localization towards intron 11, which in turn may explain their worse clinical course. In summary, the MLL breakpoint localization is of clinical importance and should be considered as a novel outcome predictor for MLL‐r patients.