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O22: A new recurrent 9q34 duplication in pediatric T-cell acute lymphoblastic leukemia
J.C. Strefford et al. / European Journal of Medical Genetics 48 (2005) 477–536
489
Bryan D. Young d, Vaskar Saha b, Christine J. Harrison a a Leukaemia Research Cytogenetics Group b Cancer Research UK Children’s Cancer Group, Queen Mary, University of London, London, UK c NimbleGen Systems, Inc., Madison, WI 53711, USA d Cancer Research UK Medical Oncology Unit, Queen Mary, University of London, London, UK Duplication of chromosome 21, involving 21q22 and amplification of the RUNX1 gene, dup(21), was discovered by chance whilst screening by FISH for the ETV6-RUNX1 fusion in childhood acute lymphoblastic leukaemia (ALL). Patients with this abnormality have a poor prognosis. Thus, their accurate detection is vital to ensure that the most appropriate treatment is given. It is imperative to fully characterise the genomic alterations in these patients, in order to develop the most appropriate method of detection. We describe the analysis of DNA from 15 patients with dup(21) using a combination of BAC (BAC aCGH) and oligonucleotide (Oligo aCGH)-based aCGH, confirmed with FISH and quantitative genomic PCR. The BAC aCGH platform contains 2621 genomic clones (Spectral Genomics) resulting in 1 Mb resolution whole-genome coverage. Oligo aCGH was performed with arrays designed to tile through chromosome 21 at a minimum spacing of 60 bp, resulting in approximately 45,000 features along the length of the chromosome (NimbleGen). In 30 patients with no available DNA, FISH analysis using clones defined by aCGH, was carried out on fixed cell suspensions. BAC aCGH revealed variable regions of amplification along chromosome 21, with a common region of amplification (CRA) (3–8.6 Mb) between genomic positions 31.5 and 40.1 Mb. A common region of deletion (CRD) (~4 Mb) was also identified telomeric to the CRA at genomic position 43.7–47 Mb. Copy number gains and losses involved all chromosome 21 clones in at least one patient, suggesting chromosomal instability. FISH, using a range of specific probes corresponding to the aCGH clones, confirmed the aCGH results, while indicating the precise copy number changes for each clone. The CRA resulted from 2.5- to 4-fold duplication with three to eight additional copies identified by FISH. Oligo aCGH refined the CRA and CRD to 6.527–6.604 and 3.541 Mb, respectively, allowing several breakpoint regions to be located to within 100 bps. In conjunction with genetic information from NCBI Build 35.1, gene lists were constructed for the CRA and CRD, highlighting 47 and 30 genes, respectively. Expression profiling and confirmatory RQ-RTPCR analysis of eight patients showed several genes in the region were differentially over-expressed when compared to other ALL samples. Parallel analysis has allowed an accurate correlation between expression and genomic changes, demonstrating that copy number gains and loss usually result in increased and decreased expression, respectively. Using state-of-the-art aCGH technology, supported by additional molecular methods, this study has defined the extent of the chromosome 21 amplified region surrounding RUNX1, demonstrated significant genomic instability in the region and allowed the development of an accurate FISH-based detection method. In addition, this study has identified candidate genes within these patients that are differentially over-expressed and allowed comparison between genomic and expression profiles. From a clinical perspective, it is likely that this work will result in improved methods for the detection of this high-risk chromosomal abnormality.
490
E.P. Nacheva et al. / European Journal of Medical Genetics 48 (2005) 477–536
O22: A new recurrent 9q34 duplication in pediatric T-cell acute lymphoblastic leukemia Pieter Van Vlierberghe, Jules P. Meijerink, Charles Lee, Adolfo A. Ferrando, A. Thomas Look, Elisabeth R. van Wering, H. Berna Beverloo, Jon C. Aster, Rob Pieters Over the last decade, genetic characterization of pediatric T cell acute lymphoblastic leukemia (T-ALL) has led to the identification of a variety of chromosomal abnormalities, including translocations, deletions and amplifications. In this study, we used array-comparative genome hybridization (array-CGH) to identify 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 slightly among patients, but the critical region involved VAV2, TRAF2 and NOTCH1. FISH analysis revealed that this 9q34 amplification was the result of a 9q34 duplication on one chromosome and could be identified in 17–39% of the leukemic cells at diagnosis. Although the presence of this leukemic subclone did not predict for poor clinical outcome in our patient cohort, leukemic cells carrying this duplication were still present at times of relapse, indicating that these cells effectively survived intensive 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 pediatric patient cohort. We showed that both genetic abnormalities are independent from this newly identified 9q34 duplication.
O23: Genomic profile of CML by aCGH Elisabeth P. Nacheva, Diana Brazma, Letizia Foroni, Colin Grace Royal Free and University College of London Med School, London, UK Chronic myelogenous leukaemia (CML), a clonal myeloproliferative disorder of the haematopoietic stem cell, typically evolves in three distinct clinical stages. The expression of the chimeric BCR/ABL fusion gene resulting from the disease specific t(9;22) is necessary for malignant transformation but not sufficient to maintain the disease progression. The appearance of various chromosomal and molecular alterations in the accelerated and terminal phase of CML is well documented without any causal relationship. The presence of the cryptic deletion at the translocation site in the der(9) chromosome detectable by FISH was shown to correlate with a short chronic phase and hence short survival. The consequences of the der(9) deletions and mechanism of formation remains unclear, as does our understanding of the molecular events behind the disease evolution. Here we present a genome wide screening for genetic imbalances by array CGH of 75 samples, including CML patients in chronic, accelerated or blast phase 10 CML cell lines and controls. We used commercially available BAC clones 1 Mbp array chip (Spectral Genomic 2600 chip). Twelve patients’ samples and all cell lines have a comprehensive molecular cytogenetics profile, including classical CGH and colour karyotyping (Gribble et al., 1999 and GCC, 2003). Fluorescence data were collected using a laser scanner (GenPix) and analysis performed with the aid of dedicated software ‘Formatter’. Our findings can be summarised as follows: ● Recurrent genomic imbalances of regions known to be aberrant in CML, which include (i) a common breakpoint at 8p12/p11.2 delineating extended (segmental) loss and gain of the region at 8q24.12, (ii) deletions at 9p21/p24 and (iii) a common break-
489
Bryan D. Young d, Vaskar Saha b, Christine J. Harrison a a Leukaemia Research Cytogenetics Group b Cancer Research UK Children’s Cancer Group, Queen Mary, University of London, London, UK c NimbleGen Systems, Inc., Madison, WI 53711, USA d Cancer Research UK Medical Oncology Unit, Queen Mary, University of London, London, UK Duplication of chromosome 21, involving 21q22 and amplification of the RUNX1 gene, dup(21), was discovered by chance whilst screening by FISH for the ETV6-RUNX1 fusion in childhood acute lymphoblastic leukaemia (ALL). Patients with this abnormality have a poor prognosis. Thus, their accurate detection is vital to ensure that the most appropriate treatment is given. It is imperative to fully characterise the genomic alterations in these patients, in order to develop the most appropriate method of detection. We describe the analysis of DNA from 15 patients with dup(21) using a combination of BAC (BAC aCGH) and oligonucleotide (Oligo aCGH)-based aCGH, confirmed with FISH and quantitative genomic PCR. The BAC aCGH platform contains 2621 genomic clones (Spectral Genomics) resulting in 1 Mb resolution whole-genome coverage. Oligo aCGH was performed with arrays designed to tile through chromosome 21 at a minimum spacing of 60 bp, resulting in approximately 45,000 features along the length of the chromosome (NimbleGen). In 30 patients with no available DNA, FISH analysis using clones defined by aCGH, was carried out on fixed cell suspensions. BAC aCGH revealed variable regions of amplification along chromosome 21, with a common region of amplification (CRA) (3–8.6 Mb) between genomic positions 31.5 and 40.1 Mb. A common region of deletion (CRD) (~4 Mb) was also identified telomeric to the CRA at genomic position 43.7–47 Mb. Copy number gains and losses involved all chromosome 21 clones in at least one patient, suggesting chromosomal instability. FISH, using a range of specific probes corresponding to the aCGH clones, confirmed the aCGH results, while indicating the precise copy number changes for each clone. The CRA resulted from 2.5- to 4-fold duplication with three to eight additional copies identified by FISH. Oligo aCGH refined the CRA and CRD to 6.527–6.604 and 3.541 Mb, respectively, allowing several breakpoint regions to be located to within 100 bps. In conjunction with genetic information from NCBI Build 35.1, gene lists were constructed for the CRA and CRD, highlighting 47 and 30 genes, respectively. Expression profiling and confirmatory RQ-RTPCR analysis of eight patients showed several genes in the region were differentially over-expressed when compared to other ALL samples. Parallel analysis has allowed an accurate correlation between expression and genomic changes, demonstrating that copy number gains and loss usually result in increased and decreased expression, respectively. Using state-of-the-art aCGH technology, supported by additional molecular methods, this study has defined the extent of the chromosome 21 amplified region surrounding RUNX1, demonstrated significant genomic instability in the region and allowed the development of an accurate FISH-based detection method. In addition, this study has identified candidate genes within these patients that are differentially over-expressed and allowed comparison between genomic and expression profiles. From a clinical perspective, it is likely that this work will result in improved methods for the detection of this high-risk chromosomal abnormality.
490
E.P. Nacheva et al. / European Journal of Medical Genetics 48 (2005) 477–536
O22: A new recurrent 9q34 duplication in pediatric T-cell acute lymphoblastic leukemia Pieter Van Vlierberghe, Jules P. Meijerink, Charles Lee, Adolfo A. Ferrando, A. Thomas Look, Elisabeth R. van Wering, H. Berna Beverloo, Jon C. Aster, Rob Pieters Over the last decade, genetic characterization of pediatric T cell acute lymphoblastic leukemia (T-ALL) has led to the identification of a variety of chromosomal abnormalities, including translocations, deletions and amplifications. In this study, we used array-comparative genome hybridization (array-CGH) to identify 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 slightly among patients, but the critical region involved VAV2, TRAF2 and NOTCH1. FISH analysis revealed that this 9q34 amplification was the result of a 9q34 duplication on one chromosome and could be identified in 17–39% of the leukemic cells at diagnosis. Although the presence of this leukemic subclone did not predict for poor clinical outcome in our patient cohort, leukemic cells carrying this duplication were still present at times of relapse, indicating that these cells effectively survived intensive 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 pediatric patient cohort. We showed that both genetic abnormalities are independent from this newly identified 9q34 duplication.
O23: Genomic profile of CML by aCGH Elisabeth P. Nacheva, Diana Brazma, Letizia Foroni, Colin Grace Royal Free and University College of London Med School, London, UK Chronic myelogenous leukaemia (CML), a clonal myeloproliferative disorder of the haematopoietic stem cell, typically evolves in three distinct clinical stages. The expression of the chimeric BCR/ABL fusion gene resulting from the disease specific t(9;22) is necessary for malignant transformation but not sufficient to maintain the disease progression. The appearance of various chromosomal and molecular alterations in the accelerated and terminal phase of CML is well documented without any causal relationship. The presence of the cryptic deletion at the translocation site in the der(9) chromosome detectable by FISH was shown to correlate with a short chronic phase and hence short survival. The consequences of the der(9) deletions and mechanism of formation remains unclear, as does our understanding of the molecular events behind the disease evolution. Here we present a genome wide screening for genetic imbalances by array CGH of 75 samples, including CML patients in chronic, accelerated or blast phase 10 CML cell lines and controls. We used commercially available BAC clones 1 Mbp array chip (Spectral Genomic 2600 chip). Twelve patients’ samples and all cell lines have a comprehensive molecular cytogenetics profile, including classical CGH and colour karyotyping (Gribble et al., 1999 and GCC, 2003). Fluorescence data were collected using a laser scanner (GenPix) and analysis performed with the aid of dedicated software ‘Formatter’. Our findings can be summarised as follows: ● Recurrent genomic imbalances of regions known to be aberrant in CML, which include (i) a common breakpoint at 8p12/p11.2 delineating extended (segmental) loss and gain of the region at 8q24.12, (ii) deletions at 9p21/p24 and (iii) a common break-