European recommendations and quality assurance for cytogenomic analysis of haematological neoplasms.

Cytogenomic investigations of haematological neoplasms, including chromosome banding analysis, fluorescence in situ hybridisation (FISH) and microarray analyses have become increasingly important in the clinical management of patients with haematological neoplasms. The widespread implementation of these techniques in genetic diagnostics has highlighted the need for guidance on the essential criteria to follow when providing cytogenomic testing, regardless of choice of methodology. These recommendations provide an updated, practical and easily available document that will assist laboratories in the choice of testing and methodology enabling them to operate within acceptable standards and maintain a quality service.

Improved survival for children and young adolescents with acute myeloid leukemia: a Dutch study on incidence, survival and mortality.

Variation in survival of pediatric acute myeloid leukemia (pAML) over time and between Western European countries exists. The aim of the current study is to assess the progress made for the Dutch pAML population (0-17 years) during 1990-2015, based on trends in incidence, survival and mortality. Data from the population-based Netherlands Cancer Registry were merged with leukemia-related characteristics and treatment specifics from the Dutch Childhood Leukemia Study Group (Dutch Childhood Oncology Group (DCOG) from 2002 onwards). Mortality data (1980-2016) were obtained from the cause of death registry of Statistics Netherlands. Trend analyses were performed over time and by treatment protocol. Between 1990 and 2015, a total of 635 children aged 0-17 years were diagnosed with AML for an average of 25 patients (range 18-36) per year. There was a slight increase in the incidence at age 1-4 years (average annual percentage change (AAPC) of +2.2% per year (95% CI 0.8-3.5, p < 0.01)). Overall, the 5-year survival significantly improved over the past 26 years and nearly doubled from 40% in the early 1990s to 74% in 2010-2015. Multivariable analysis showed a 49% reduction in risk of death for pAML patients treated according to the latest DB-AML 01 protocol (p = 0.03). The continuing decrease of mortality (AAPC -2.8% per year (95% CI -4.1 to -1.5)) supports the conclusion of true progress against pAML in the Netherlands.

Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange.

Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.

A new recurrent 9q34 duplication in pediatric T-cell acute lymphoblastic leukemia.

Over the last decade, genetic characterization of T-cell acute lymphoblastic leukemia (T-ALL) has led to the identification of a variety of chromosomal abnormalities. In this study, we used array-comparative genome hybridization (array-CGH) and identified a novel recurrent 9q34 amplification in 33% (12/36) of pediatric T-ALL samples, which is therefore one of the most frequent cytogenetic abnormalities observed in T-ALL thus far. The exact size of the amplified region differed among patients, but the critical region encloses approximately 4 Mb and includes NOTCH1. The 9q34 amplification may lead to elevated expression of various genes, and MRLP41, SSNA1 and PHPT1 were found significantly expressed at higher levels. Fluorescence in situ hybridization (FISH) analysis revealed that this 9q34 amplification was in fact a 9q34 duplication on one chromosome and could be identified in 17-39 percent of leukemic cells at diagnosis. Although this leukemic subclone did not predict for poor outcome, leukemic cells carrying this duplication were still present at relapse, indicating that these cells survived chemotherapeutic treatment. Episomal NUP214-ABL1 amplification and activating mutations in NOTCH1, two other recently identified 9q34 abnormalities in T-ALL, were also detected in our patient cohort. We showed that both of these genetic abnormalities occur independently from this newly identified 9q34 duplication.

Incidence of additional genetic changes in the TEL and AML1 genes in DCOG and COALL-treated t(12;21)-positive pediatric ALL, and their relation with drug sensitivity and clinical outcome.

Clinical heterogeneity within t(12;21) or TEL/AML1-positive ALL (25% of childhood common/preB ALL) indicates that additional genetic changes might contribute to outcome. We studied the relation between additional genetic changes in TEL(ETV6) and AML1(RUNX1) (FISH), drug sensitivity (MTT assay) and clinical outcome in 143 DCOG and COALL-treated t(12;21)-positive ALL patients. Additional genetic changes in TEL and AML1 were present in 83% of the patients, and consisted of (partial) deletion of the second TEL gene (70%), an extra AML1 gene (23%) or an extra der(21)t(12;21) (10%). More than one additional change was observed in 20%. Disease-free survival (pDFS) of DCOG patients without additional genetic changes (4 years pDFS +/- s.e. 53 +/- 17%) and of those with an extra der(21)t(12;21) (60 +/- 22%) is poorer than that of compared to patients with other additional genetic changes in TEL or AML1 (79 +/- 6%; P-trend = 0.02). This was mainly due to the occurrence of early relapses within 2.5 years after the first diagnosis. Similar observations were found in the COALL cohort, albeit not significant owing to limited follow-up. Multivariate analysis including age, WBC and genetic abnormalities in TEL and/or AML1 showed that especially, in vitro resistance to prednisolone (hazard ratio 5.78, 95% CI 1.45-23.0; P=0.01) is an independent prognostic factor in DCOG- and COALL-treated t(12;21)-positive ALL.

Breakpoint heterogeneity in t(10;11) translocation in AML-M4/M5 resulting in fusion of AF10 and MLL is resolved by fluorescent in situ hybridization analysis.

Ten AML-M4/M5 patients' samples containing a t(10;11) translocation, but with different cytogenetic breakpoints on chromosome 11q (11q13-23), were studied by G- and R-banding and fluorescent in situ hybridization. Southern blotting analysis, studied in five patients, revealed a rearranged MLL gene. Reverse transcription-PCR analysis carried out in six patients showed a 5' MLL-3' AF-10 fusion transcript. Fluorescent in situ hybridization studies suggested that in 8 of 10 patients, the rearrangement/fusion transcript resulted from an inversion of a part of 11q (q13q23) translocated to 10p12. In the other two patients, it is assumed that an inversion/translocation has occurred of a part of 10p to the der(11). The results suggest that the orientation of the AF-10 gene on 10p is 5' telomeric and 3' centromeric. This is the first example of opposite-oriented genes being involved in translocation to yield fusion transcripts.

Fusion of the homeobox gene HLXB9 and the ETV6 gene in infant acute myeloid leukemias with the t(7;12)(q36;p13).

Recently, we and others reported a recurrent t(7;12)(q36;p13) found in myeloid malignancies in children < or =18 months of age and associated with a poor prognosis. Fluorescence in situ hybridization studies mapped the 12p13 breakpoint to the first intron of ETV6 and narrowed down the region of 7q36 involved. By using the sequences made public recently by the Human Genome Project, two candidate genes in 7q36 were identified: the homeobox gene HLXB9 and c7orf3, a gene with unknown function. Reverse transcription-PCR of two cases with t(7;12), using primers for c7orf3 and ETV6, was negative. However, reverse transcription-PCR for HLXB9-ETV6 demonstrated alternative splicing; the two major bands corresponded to fusion of exon 1 of HLXB9 to exons 2 and 3, respectively, of ETV6. The reciprocal ETV6-HLXB9 transcript was not detected. It remains to be elucidated if the leukemic phenotype is attributable to the formation of the HLXB9-ETV6 fusion protein, which includes the helix-loop-helix and E26 transformation-specific DNA binding domains of ETV6 or to the disruption of the normal ETV6 protein.

Translocation (11;15)(q23;q14) in three patients with acute non-lymphoblastic leukemia (ANLL): clinical, cytogenetic and molecular studies.

We report on three patients with acute non-lymphoblastic leukemia (ANLL) displaying the same chromosomal translocation t(11;15)(q23;q14). The clinical course of the disease was aggressive, and survival was short. The FAB subtype was M-2 in two cases, and M-1 in the remaining patient. Immunologically two cases showed aberrant expression of a lymphoid antigen (CD19 and TdT, respectively). HTRX1/MLL gene was rearranged in one patient studied at the time of diagnosis. These results plus data scattered in the literature show that the t(11;15)(q23;q14) can be added to the list of recurrent rearrangements in ANLL involving 11q23.

Rapid and sensitive detection of all types of MLL gene translocations with a single FISH probe set.

The MLL gene on chromosome 11 band q23 is frequently involved in chromosome translocations in acute lymphoblastic leukemia and acute myeloid leukemia. The translocation results in the formation of a fusion gene on the derivative 11 chromosome consisting of the 5' part of the MLL gene and the 3' part of another gene; already more than 30 different partner chromosome regions have been described. MLL gene rearrangements are generally correlated with a poor prognosis. Therefore the presence of an 11q23 aberration has direct implications for treatment stratification, making early and rapid detection of utmost importance. In this study, we developed a FISH probe set for detection of MLL gene rearrangements according to strict design criteria. The cosmid probes are derived from the flanking regions of the MLL breakpoint region on chromosome 11 and when used in dual colored FISH experiments give rise to a split of the normally colocalizing (fused) signals in case of a translocation. This split signal was observed in seven out of 10 cases with an 11q23 translocation with various partner chromosomes. In the three other cases, a deletion of the 3' part of the MLL gene, downstream of the breakpoint region was also found. A low false positive value of only 1.7% was obtained for interphase cells in contrast to conventional dual colored FISH where the creation of a fusion signal has cut off values of at least 5-10%. A major advantage of our type of probe set is the application of a single FISH experiment to detect all types of MLL translocations. Moreover, since this cosmid probe set can be used for either interphase or metaphase studies, metaphases are no longer a prerequisite for detecting the presence of an 11q23 translocation. Nevertheless, metaphase FISH with the new probe set is helpful in determining the partner chromosome and therefore may lead to the identification of new partner genes.

Split-signal FISH for detection of chromosome aberrations in acute lymphoblastic leukemia.

Chromosome aberrations are frequently observed in precursor-B-acute lymphoblastic leukemias (ALL) and T-cell acute lymphoblastic leukemias (T-ALL). These translocations can form leukemia-specific chimeric fusion proteins or they can deregulate expression of an (onco)gene, resulting in aberrant expression or overexpression. Detection of chromosome aberrations is an important tool for risk classification. We developed rapid and sensitive split-signal fluorescent in situ hybridization (FISH) assays for six of the most frequent chromosome aberrations in precursor-B-ALL and T-ALL. The split-signal FISH approach uses two differentially labeled probes, located in one gene at opposite sites of the breakpoint region. Probe sets were developed for the genes TCF3 (E2A) at 19p13, MLL at 11q23, ETV6 at 12p13, BCR at 22q11, SIL-TAL1 at 1q32 and TLX3 (HOX11L2) at 5q35. In normal karyotypes, two colocalized green/red signals are visible, but a translocation results in a split of one of the colocalized signals. Split-signal FISH has three main advantages over the classical fusion-signal FISH approach, which uses two labeled probes located in two genes. First, the detection of a chromosome aberration is independent of the involved partner gene. Second, split-signal FISH allows the identification of the partner gene or chromosome region if metaphase spreads are present, and finally it reduces false-positivity.

A single split-signal FISH probe set allows detection of TAL1 translocations as well as SIL-TAL1 fusion genes in a single test.

About 30% of T cell acute lymphoblastic leukemias (T-ALL) carry TAL1 gene aberrations. In the majority of cases (approximately 25%), this concerns a submicroscopic deletion of approximately 90 kb in chromosome region 1p32, which deletes the coding regions of the SIL gene and the untranslated region of the TAL1 gene, thereby placing the TAL1 gene under control of the SIL promoter region. Translocation (1;14)(p32;q11) involving the TAL1 gene occurs at a much lower frequency (3%), whereas some other rare variant translocations have been described as well. In this study we developed a set of TAL1 FISH probes based on the split-signal FISH principle that enables detection of both types of TAL1 gene aberrations in single test. For this purpose, one probe was designed downstream of the TAL1 gene (TAL1-D) and the second probe in the region upstream of the TAL1 gene, partly covering the SIL gene (SIL-U). We show that this split-signal FISH probe set allows reliable detection of the unaffected SIL-TAL1 gene region with a fusion signal, SIL-TAL1 fusion genes with loss of the SIL-U signal, and TAL1 gene translocations with a split-signal, independent of the involved partner gene.

In vitro drug-resistance profile in infant acute lymphoblastic leukemia in relation to age, MLL rearrangements and immunophenotype.

Acute lymphoblastic leukemia (ALL) in infants under 1 year is strongly associated with translocations involving 11q23 (MLL gene), CD10-negative B-lineage (proB) immunophenotype, and poor outcome. The present study analyses the relationship between age, MLL rearrangements, proB-lineage, and in vitro drug resistance determined using the MTT assay. Compared to 425 children aged over 1 year with common/preB (c/preB) ALL, the 44 infants were highly resistant to steroids (for prednisolone (PRED) more than 580-fold, P=0.001) and L-asparaginase (L-ASP) (12-fold, P=0.001), but more sensitive to cytarabine (AraC) (1.9-fold, P=0.001) and 2-chlorodeoxyadenosine (2-CdA) (1.7-fold, P<0.001). No differences were found for vincristine, anthracyclines, thiopurines, epipodophyllotoxines, or 4-hydroperoxy (HOO)-ifosfamide. ProB ALL of all ages had a profile similar to infant ALL when compared with the group of c/preB ALL: relatively more resistant to L-ASP and PRED (and in addition thiopurines), and more sensitive to AraC and 2-CdA. Age was not related to cellular drug resistance within the proB ALL group (<1 year, n=32, vs >/=1 year, n=19), nor within the MLL-rearranged ALL (<1 year, n=34, vs >/=1 year, n=8). The translocation t(4;11)(q21;q23)-positive ALL cases were more resistant to PRED (>7.4-fold, P=0.033) and 4-HOO-ifosfamide (4.4-fold, P=0.006) than those with other 11q23 abnormalities. The expression of P-glycoprotein, multidrug-resistance protein, and lung-resistance protein (LRP) was not higher in infants compared to older c/preB ALL patients, but LRP was higher in proB ALL and MLL-rearranged ALL of all ages. In conclusion, infants with ALL appear to have a distinct in vitro resistance profile with the proB immunophenotype being of importance. The role of MLL cannot be excluded, with the t(4;11) being of special significance, while age appears to play a smaller role.

t(7;12)(q36;p13) and t(7;12)(q32;p13)--translocations involving ETV6 in children 18 months of age or younger with myeloid disorders.

Our retrospective karyotype review revealed two rare recurrent translocations affecting ETV6 (TEL): t(7;12)(q36;p13) and t(7;12)(q32;p13). Five patients with a t(7;12) were from a group of 125 successfully karyotyped pediatric patients enrolled in consecutive clinical AML trials of the Dutch Childhood Leukemia Study Group over a period of 7 years. During a search of available cytogenetic databases, we found 7q and 12p abnormalities in two additional Dutch patients and in three participants in Pediatric Oncology Group trials. A del(12p) had been initially identified in four of these patients and re-examination of the original karyograms revealed a t(7;12)(q36;p13) in two instances and a probable t(7;12) in the other two. FISH confirmed the presence of a t(7;12)(q36;p13) in the latter. Most (n = 7) also had trisomy 19. The t(7;12)(q36;p13) (n = 9) was more common than the t(7;12)(q32;p13) (n = 1). These subtle translocations were found only in children 18 months of age or younger. A literature search revealed that the t(7;12) with break-points at 7q31-q36 and 12p12-p13 had been reported in six children with myeloid disorders and in two with acute lymphoblastic leukemia; all were 12 months of age or younger. Only two of the 17 for whom survival data were available, were alive after at least 22 months of continuous complete remission. Our findings suggest that ETV6 rearrangements due to a t(7;12) may play an adverse role in myeloid disorders in children 18 months of age or younger. Therefore, children in this age group with myeloid disorders should be screened for both MLL and ETV6 rearrangements.

Comparative therapeutic value of post-remission approaches in patients with acute myeloid leukemia aged 40-60 years.

The preferred type of post-remission therapy (PRT) in patients with acute myeloid leukemia (AML) in first complete remission (CR1) is a subject of continued debate, especially in patients at higher risk of nonrelapse mortality (NRM), including patients >40 years of age. We report results of a time-dependent multivariable analysis of allogenic hematopoietic stem cell transplantation (alloHSCT) (n=337) versus chemotherapy (n=271) or autologous HSCT (autoHSCT) (n=152) in 760 patients aged 40-60 years with AML in CR1. Patients receiving alloHSCT showed improved overall survival (OS) as compared with chemotherapy (respectively, 57±3% vs 40±3% at 5 years, P<0.001). Comparable OS was observed following alloHSCT and autoHSCT in patients with intermediate-risk AML (60±4 vs 54±5%). However, alloHSCT was associated with less relapse (hazard ratio (HR) 0.51, P<0.001) and better relapse-free survival (RFS) (HR 0.74, P=0.029) as compared with autoHSCT in intermediate-risk AMLs. AlloHSCT was applied following myeloablative conditioning (n=157) or reduced intensity conditioning (n=180), resulting in less NRM, but comparable outcome with respect to OS, RFS and relapse. Collectively, these results show that alloHSCT is to be preferred over chemotherapy as PRT in patients with intermediate- and poor-risk AML aged 40-60 years, whereas autoHSCT remains a treatment option to be considered in patients with intermediate-risk AML.

A comparison of the detection sensitivity of lymphocyte membrane antigens using fluorescein and phosphor immunoconjugates.

In this study we compared the sensitivity of immunocytochemical procedures, using conventional and time-resolved fluorescent dyes, in a model system consisting of paraformaldehyde-fixed human lymphocytes. The lymphocytes were stained for the presence of the CD4 epitope by indirect immunofluorescence using FITC as label or by using time-resolved luminescent immunophosphors. These immunophosphors were primarily developed for use under time-resolved fluorescence conditions, but they are also very well suited for use in conventional fluorescence microscopes. The differently labeled cells were first examined visually with a conventional fluorescence microscope in a double-blind study. The fluorescence was also measured with a CCD camera mounted on a specially constructed time-resolved fluorescence microscope which allows the suppression of the fast decaying fluorescence, thereby permitting visualization of the specific, slowing decaying luminescence of the phosphor label. With this microscope FITC and immunophosphor labeled lymphocytes were compared under normal conditions (i.e., continuous excitation) and under conditions of time-resolved registration. Conventional fluorescence microscopy revealed a better sensitivity in favor of the phosphor conjugates. This difference became more prominent when the preparations were quantitatively assessed with the CCD-time-resolved microscope. Time-resolved microscopy permitted a suppression of fast decaying fluorescence better than 1 to 10(6).

T-cell receptor Vß skewing frequently occurs in refractory cytopenia of childhood and is associated with an expansion of effector cytotoxic T cells: a prospective study by EWOG-MDS.

Immunosuppressive therapy (IST), consisting of antithymocyte globulin and cyclosporine A, is effective in refractory cytopenia of childhood (RCC), suggesting that, similar to low-grade myelodysplastic syndromes in adult patients, T lymphocytes are involved in suppressing hematopoiesis in a subset of RCC patients. However, the potential role of a T-cell-mediated pathophysiology in RCC remains poorly explored. In a cohort of 92 RCC patients, we prospectively assessed the frequency of T-cell receptor (TCR) ß-chain variable (Vß) domain skewing in bone marrow and peripheral blood by heteroduplex PCR, and analyzed T-cell subsets in peripheral blood by flow cytometry. TCRVß skewing was present in 40% of RCC patients. TCRVß skewing did not correlate with bone marrow cellularity, karyotype, transfusion history, HLA-DR15 or the presence of a PNH clone. In 28 patients treated with IST, TCRVß skewing was not clearly related with treatment response. However, TCRVß skewing did correlate with a disturbed CD4(+)/CD8(+) T-cell ratio, a reduction in naive CD8(+) T cells, an expansion of effector CD8(+) T cells and an increase in activated CD8(+) T cells (defined as HLA-DR(+), CD57(+) or CD56(+)). These data suggest that T lymphocytes contribute to RCC pathogenesis in a proportion of patients, and provide a rationale for treatment with IST in selected patients with RCC.