Marcel Tauscher

Copy number alterations in childhood acute lymphoblastic leukemia and their association with minimal residual disease

In vivo response to initial therapy, as assessed by determination of minimal residual disease (MRD) after 5 and 12 weeks of treatment, has evolved as a strong prognostic factor in children with acute lymphoblastic leukemia (ALL) treated according to the BFM regime. Individual treatment response may be influenced by copy number alterations (CNA) leading to altered gene expression. We aimed to evaluate CNA using high‐resolution array‐comparative genomic hybridization (array‐CGH) in different treatment‐response groups. Leukemic genomic profiles of 25 standard risk (MRD‐SR) and 25 high risk (MRD‐HR) patients were compared. CNAs were found in 46/50 patients (92%). The most significant difference was a gain of 1q23‐qter because of an unbalanced t(1;19), found in 10/25 MRD‐SR patients, but in none of the MRD‐HR patients (P < 0.001). The most frequent CNAs in the MRD‐HR group were deletions of genomic regions harboring the immunoglobulin genes (Ig), e.g., 2p11.2 in 60% of MRD‐HR compared to 28% of MRD‐SR (P = 0.045). Combining all Ig loci, significantly more MRD‐HR than MRD‐SR patients displayed deletions (17:8 patients, P = 0.02). Frequency of other CNAs, such as loss of 9p21 or gains of 21q, did not differ strongly between the two patient groups. This is the first study evaluating the clinical significance of CNA as detected by array‐CGH in childhood ALL and the first to suggest that such analyses may provide clinically important data. This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

Constitutional trisomy 8p11.21-q11.21 mosaicism: a germline alteration predisposing to myeloid leukaemia

Juvenile myelomonocytic leukaemia (JMML) is a unique myeloproliferative disorder of early childhood. Frequently, mutations in NRAS, KRAS, PTPN11, NF1 or CBL are found in these patients. Monosomy 7 is the most common cytogenetic aberration. To identify submicroscopic genomic copy number alterations, 20 JMML samples were analysed by comparative genomic hybridization. Ten out of 20 samples displayed additional submicroscopic alterations. In two patients, an almost identical gain of chromosome 8 was identified. In both patients, fluorescence in situ hybridization confirmed a constitutional partial trisomy 8 mosaic (cT8M). A survey on 27 cT8M patients with neoplasms showed that 21 had myeloid malignancies, and five of these had a JMML. Notably, the region gained in our cases is the smallest gain of chromosome 8 reported in cT8M cases with malignancies so far. Our results dramatically reduce the critical region to 8p11.21q11.21 harbouring 31 protein coding genes and two non‐coding RNAs, e.g. MYST3, IKBKB, UBE2V2, GOLGA7, FNTA and MIR486– a finding with potential implications for the role of somatic trisomy 8 in myeloid malignancies. Further investigations are required to more comprehensively determine how constitutional partial trisomy 8 mosaicisms may contribute to leukaemogenesis in different mutational subtypes of JMML and other myeloid malignancies.

Clonal heterogeneity in childhood myelodysplastic syndromes--challenge for the detection of chromosomal imbalances by array-CGH.

To evaluate whether copy number alterations (CNAs) are present that may contribute to disease development and/or progression of childhood myelodysplastic syndromes (MDS), 36 pediatric MDS patients were analyzed using array‐based comparative genome hybridization (aCGH). In addition to monosomy 7, the most frequent chromosome aberration in childhood MDS, novel recurrent CNAs were detected. They included a loss of 3p14.3–p12.3, which contains the putative tumor suppressor gene FHIT, a loss of 7p21.3–p15.3, a loss of 9q33.3–q34.3 (D184) and microdeletions in 17p11.2, 6q23 containing MYB, and 17p13 containing TP53. In this small patient cohort, patients without CNA, patients with monosomy 7 only and patients with one CNA in addition to monosomy 7 did not differ in their survival. As expected, all patients with complex karyotypes, including two patients with deletions of TP53, died. A challenge inherent to aCGH analysis of MDS is the low percentage of tumor cells. We evaluated several approaches to overcome this limitation. Genomic profiles from isolated granulocytes were of higher quality than those from bone marrow mononuclear cells. Decreased breakpoint calling stringency increased recognition of CNAs present in small clonal populations. However, further analysis using a custom‐designed array showed that these CNAs often did not confirm the findings from 244k arrays. In contrast, constitutional CNVs were reliably detected on both arrays. Moreover, aCGH on amplified DNA from distinct myeloid clusters is a new approach to determine CNAs in small subpopulations. Our results clearly emphasize the need to verify array‐CGH results by independent methods like FISH or quantitative PCR.

Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase

Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puroΔtk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

Analysis of array-CGH data using the R and Bioconductor software suite.

Background. Array-based comparative genomic hybridization (array-CGH) is an emerging high-resolution and high-throughput molecular genetic technique that allows genome-wide screening for chromosome alterations. DNA copy number alterations (CNAs) are a hallmark of somatic mutations in tumor genomes and congenital abnormalities that lead to diseases such as mental retardation. However, accurate identification of amplified or deleted regions requires a sequence of different computational analysis steps of the microarray data. Results. We have developed a user-friendly and versatile tool for the normalization, visualization, breakpoint detection, and comparative analysis of array-CGH data which allows the accurate and sensitive detection of CNAs. Conclusion. The implemented option for the determination of minimal altered regions (MARs) from a series of tumor samples is a step forward in the identification of new tumor suppressor genes or oncogenes.

Induced G1 phase arrest of fast-dividing cells improves the quality of genomic profiles generated by array-CGH

Genome-wide profiling of copy number alterations by array-based high resolution comparative genomic hybridization (array-CGH) is an important method to ensure the genomic integrity of cells in diverse conditions. We observed that the analysis of genomic profiles, in particular of fast-dividing murine leukemia cell lines, is challenging due to characteristic patterns oscillating around the array-CGH baseline. Here we show array-CGH data can be drastically improved by reducing proliferation rates of cultured cells using deprivation protocols or cell cycle inhibitors. Arresting cell cycle in the G1 phase leads to smoother genomic profiles, and hence to a more reliable detection of copy number alterations.

Managing individuals with propensity to myeloid malignancies due to germline RUNX1 deficiency

With great interest we read the recent review on familial myelodysplastic syndromes (MDS) published in this journal by Liew and Owen.[1][1] Besides telomere disorders and familial monosomy 7, the review focused on familial platelet disorder with propensity to myeloid malignancies (FPDMM) and also

Mutations in the let-7 binding site - a mechanism of RAS activation in juvenile myelomonocytic leukemia?

Juvenile myelomonocytic leukemia (JMML) is a rare hematologic malignancy in childhood and accounts for less than 3% of all childhood hematologic malignancies. 1 The role of hyperactive RAS in JMML is underlined by the fact that approximately 80% of JMML develop due to gain-of-function mutations in NRAS, KRAS, PTPN11 and SOS1 or homozygous loss-of-function mutations in NF1 or c-CBL. 2-4 These genes are all components of the RAS/ERK signaling network, implicating deregulation of this signaling pathway in JMML pathogenesis. Therefore, it may be speculated that JMML lacking known mutations of genes playing a role in RAS signaling may carry other mutations, which result in activation of this pathway. Recently, germline mi-RNA gene variations were pro posed to affect the expression levels of tumor suppressor or oncogenes and, thereby, familial/hereditary cancer risk. 5 The let-7 mi-RNA family targets many important genes including cell cycle regulators such as CDC25A and CDK6, a number of early embryonic genes including HMGA2, Mlin-41 and IMP-1 and promoters of growth including RAS and C-MYC. In Caenorhabditis elegans let7 mutant seam cells fail to exit the cell cycle and to terminally differentiate, thus demonstrating continuous prolif eration, a hallmark of cancer. 6 Human RAS expression was also shown to be regulated by let-7. 7 Evidence of a role of let-7 in cancer came from the observation that lung tumor tissues display significantly reduced let-7 levels and significantly increased RAS protein levels relative to nor mal lung tissue. 8

Array-CGH in childhood MDS.

To study genomic imbalances potentially involved in disease development and/or progression of childhood MDS, array-based comparative genomic hybridization (aCGH) is a helpful tool. Copy number alterations (CNA) of subtle chromosomal regions containing potential candidate genes, e.g., TP53 or RUNX1 can be detected. However, characterizing small and/or heterogeneous tumor subpopulations by high-resolution aCGH within a majority of normal cells is a challenge in MDS and requires validation by independent methods like FISH or quantitative PCR. For the identification of tumor-relevant CNA, the analysis of DNA isolated from purified granulocytes or myeloid populations instead of DNA from whole bone marrow (BM) cells is helpful to overcome some of these limitations.