EVALUATION OF MULTIPLEX LIGATION DEPENDENT PROBE AMPLIFICATION FOR IDENTIFICATION OF ACUTE LYMPHOBLASTIC LEUKEMIA WITH AN INTRACHROMOSOMAL AMPLIFICATION OF CHROMOSOME 21 IN A BRAZILIAN POPULATION
(Abstract release date: 05/21/15)
EHA Library. Fuka G. 06/12/15; 102818; PB1592
Disclosure(s): Instituto Nacional de Câncer (INCA)
Gerhard Fuka
Contributions
Contributions
Abstract
Abstract: PB1592
Type: Publication Only
Background
An intrachromosomal amplification of chromosome 21 (iAMP21) defines a unique subgroup of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The finding of three or more extra copies of the RUNX1 gene by fluorescence in situ hybridization (FISH) is internationally used to define of an iAMP21. Recently, genomic profiling of chromosome 21 has been suggested for assisting the identification of an iAMP21.
Aims
In this work, we aimed to test the multiplex ligation-dependent probe amplification (MLPA) SALSA P327-A1 probe set for evaluating copy number alterations (CNA) on chromosome 21.
Methods
A series of consecutive diagnostic samples from Brazilian childhood BCP-ALL patients collected between the years 2002 to 2012 were selected according to availability of material of good quality providing that bone marrow aspirates harbored at least 30% of blast cells. The MLPA SALSA P327-A1 probe set was employed to detect an iAMP21 (MRC Holland, Amsterdam, The Netherlands). Test fragments of the P327-A1 probe set were designed to detect CNA of 24 genes distributed from centromeric to telomeric regions (q11.2-q22.3, 14.668-46.356) of chromosome 21. Relative copy numbers were obtained after normalization of peaks against cord blood derived controls. Relative peak heights between 0.75 and 1.3 were considered normal, while those below 0.75 and above 1.3 indicated losses or gains of genomic material, respectively. FISH was performed using the “LPH012 TEL/AML1 Translocation, Dual Fusion Probe”, designed to detect the ETV6/RUNX1 fusion gene. Cases were considered positive for an iAMP21 when ≥5 RUNX1 FISH signals were observed in at least 7% of metaphase nuclei (standard deviation +/- 2%).
Results
In 74 out of 368 BCP-ALL patients, gain of genetic material on chromosome 21 was detected. MLPA peak profiles segregated patients into three groups by hierarchical clustering. Next, patient material was subjected to FISH. Cells with ≥5 RUNX1 FISH signals (n=9) were considered as “true iAMP21”. By contrast those with 3-4 RUNX1 signals (n=35) or a normal RUNX1 FISH pattern (n=6) probably acquired additional copies of an intact chromosome 21, even though not detectable by FISH in all cases. Peak ratios of RUNX1 and DYRK1A genes, located within the common region of amplification (CRA), were in “true iAMP21” patients higher, an opposite pattern was observed for telomeric SNF1LK and TFF1 genes. Since we observed variations in MLPA peak heights between different patients, the correlation between RUNX1, DYRK1A, ETS2 and ERG gene load and expression was tested. For illustration we stepwise arranged cases from the lowest to the highest MLPA peak ratios and depicted corresponding delta CT (threshold cycle) values. Positive correlation between the MLPA peak ratios and CT values were observed, since those patients with high MLPA peak ratios were the ones with low gene expression. This observation particularly held true for the genes RUNX1, DYRK1A, and ERG.
Summary
Altogether, we confirmed that aberrations on chromosome 21 represent a high level of complexity and individuality and provided evidence for the existence of gene regulatory mechanisms that might modulate the expression of amplified genes. In BCP ALL with an iAMP21 we consider MLPA tests highly desirable for data replication of array based comparative genomic hybridization analyzes. MLPA profiles of high peak ratios of CRA genes might be especially helpful in case identification whenever metaphase nuclei for FISH are not available. However, we want to stress that a precise separation of BCP-ALL with an iAMP21 from hyperdiploid ALL with additional copies of chromosome 21 is still under revision. Control fragments binding on chromosomes that are usually over represented should be included in next generation MLPA probe sets.
Keyword(s): Acute lymphoblastic leukemia
Session topic: Publication Only
Type: Publication Only
Background
An intrachromosomal amplification of chromosome 21 (iAMP21) defines a unique subgroup of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The finding of three or more extra copies of the RUNX1 gene by fluorescence in situ hybridization (FISH) is internationally used to define of an iAMP21. Recently, genomic profiling of chromosome 21 has been suggested for assisting the identification of an iAMP21.
Aims
In this work, we aimed to test the multiplex ligation-dependent probe amplification (MLPA) SALSA P327-A1 probe set for evaluating copy number alterations (CNA) on chromosome 21.
Methods
A series of consecutive diagnostic samples from Brazilian childhood BCP-ALL patients collected between the years 2002 to 2012 were selected according to availability of material of good quality providing that bone marrow aspirates harbored at least 30% of blast cells. The MLPA SALSA P327-A1 probe set was employed to detect an iAMP21 (MRC Holland, Amsterdam, The Netherlands). Test fragments of the P327-A1 probe set were designed to detect CNA of 24 genes distributed from centromeric to telomeric regions (q11.2-q22.3, 14.668-46.356) of chromosome 21. Relative copy numbers were obtained after normalization of peaks against cord blood derived controls. Relative peak heights between 0.75 and 1.3 were considered normal, while those below 0.75 and above 1.3 indicated losses or gains of genomic material, respectively. FISH was performed using the “LPH012 TEL/AML1 Translocation, Dual Fusion Probe”, designed to detect the ETV6/RUNX1 fusion gene. Cases were considered positive for an iAMP21 when ≥5 RUNX1 FISH signals were observed in at least 7% of metaphase nuclei (standard deviation +/- 2%).
Results
In 74 out of 368 BCP-ALL patients, gain of genetic material on chromosome 21 was detected. MLPA peak profiles segregated patients into three groups by hierarchical clustering. Next, patient material was subjected to FISH. Cells with ≥5 RUNX1 FISH signals (n=9) were considered as “true iAMP21”. By contrast those with 3-4 RUNX1 signals (n=35) or a normal RUNX1 FISH pattern (n=6) probably acquired additional copies of an intact chromosome 21, even though not detectable by FISH in all cases. Peak ratios of RUNX1 and DYRK1A genes, located within the common region of amplification (CRA), were in “true iAMP21” patients higher, an opposite pattern was observed for telomeric SNF1LK and TFF1 genes. Since we observed variations in MLPA peak heights between different patients, the correlation between RUNX1, DYRK1A, ETS2 and ERG gene load and expression was tested. For illustration we stepwise arranged cases from the lowest to the highest MLPA peak ratios and depicted corresponding delta CT (threshold cycle) values. Positive correlation between the MLPA peak ratios and CT values were observed, since those patients with high MLPA peak ratios were the ones with low gene expression. This observation particularly held true for the genes RUNX1, DYRK1A, and ERG.
Summary
Altogether, we confirmed that aberrations on chromosome 21 represent a high level of complexity and individuality and provided evidence for the existence of gene regulatory mechanisms that might modulate the expression of amplified genes. In BCP ALL with an iAMP21 we consider MLPA tests highly desirable for data replication of array based comparative genomic hybridization analyzes. MLPA profiles of high peak ratios of CRA genes might be especially helpful in case identification whenever metaphase nuclei for FISH are not available. However, we want to stress that a precise separation of BCP-ALL with an iAMP21 from hyperdiploid ALL with additional copies of chromosome 21 is still under revision. Control fragments binding on chromosomes that are usually over represented should be included in next generation MLPA probe sets.
Keyword(s): Acute lymphoblastic leukemia
Session topic: Publication Only
Abstract: PB1592
Type: Publication Only
Background
An intrachromosomal amplification of chromosome 21 (iAMP21) defines a unique subgroup of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The finding of three or more extra copies of the RUNX1 gene by fluorescence in situ hybridization (FISH) is internationally used to define of an iAMP21. Recently, genomic profiling of chromosome 21 has been suggested for assisting the identification of an iAMP21.
Aims
In this work, we aimed to test the multiplex ligation-dependent probe amplification (MLPA) SALSA P327-A1 probe set for evaluating copy number alterations (CNA) on chromosome 21.
Methods
A series of consecutive diagnostic samples from Brazilian childhood BCP-ALL patients collected between the years 2002 to 2012 were selected according to availability of material of good quality providing that bone marrow aspirates harbored at least 30% of blast cells. The MLPA SALSA P327-A1 probe set was employed to detect an iAMP21 (MRC Holland, Amsterdam, The Netherlands). Test fragments of the P327-A1 probe set were designed to detect CNA of 24 genes distributed from centromeric to telomeric regions (q11.2-q22.3, 14.668-46.356) of chromosome 21. Relative copy numbers were obtained after normalization of peaks against cord blood derived controls. Relative peak heights between 0.75 and 1.3 were considered normal, while those below 0.75 and above 1.3 indicated losses or gains of genomic material, respectively. FISH was performed using the “LPH012 TEL/AML1 Translocation, Dual Fusion Probe”, designed to detect the ETV6/RUNX1 fusion gene. Cases were considered positive for an iAMP21 when ≥5 RUNX1 FISH signals were observed in at least 7% of metaphase nuclei (standard deviation +/- 2%).
Results
In 74 out of 368 BCP-ALL patients, gain of genetic material on chromosome 21 was detected. MLPA peak profiles segregated patients into three groups by hierarchical clustering. Next, patient material was subjected to FISH. Cells with ≥5 RUNX1 FISH signals (n=9) were considered as “true iAMP21”. By contrast those with 3-4 RUNX1 signals (n=35) or a normal RUNX1 FISH pattern (n=6) probably acquired additional copies of an intact chromosome 21, even though not detectable by FISH in all cases. Peak ratios of RUNX1 and DYRK1A genes, located within the common region of amplification (CRA), were in “true iAMP21” patients higher, an opposite pattern was observed for telomeric SNF1LK and TFF1 genes. Since we observed variations in MLPA peak heights between different patients, the correlation between RUNX1, DYRK1A, ETS2 and ERG gene load and expression was tested. For illustration we stepwise arranged cases from the lowest to the highest MLPA peak ratios and depicted corresponding delta CT (threshold cycle) values. Positive correlation between the MLPA peak ratios and CT values were observed, since those patients with high MLPA peak ratios were the ones with low gene expression. This observation particularly held true for the genes RUNX1, DYRK1A, and ERG.
Summary
Altogether, we confirmed that aberrations on chromosome 21 represent a high level of complexity and individuality and provided evidence for the existence of gene regulatory mechanisms that might modulate the expression of amplified genes. In BCP ALL with an iAMP21 we consider MLPA tests highly desirable for data replication of array based comparative genomic hybridization analyzes. MLPA profiles of high peak ratios of CRA genes might be especially helpful in case identification whenever metaphase nuclei for FISH are not available. However, we want to stress that a precise separation of BCP-ALL with an iAMP21 from hyperdiploid ALL with additional copies of chromosome 21 is still under revision. Control fragments binding on chromosomes that are usually over represented should be included in next generation MLPA probe sets.
Keyword(s): Acute lymphoblastic leukemia
Session topic: Publication Only
Type: Publication Only
Background
An intrachromosomal amplification of chromosome 21 (iAMP21) defines a unique subgroup of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The finding of three or more extra copies of the RUNX1 gene by fluorescence in situ hybridization (FISH) is internationally used to define of an iAMP21. Recently, genomic profiling of chromosome 21 has been suggested for assisting the identification of an iAMP21.
Aims
In this work, we aimed to test the multiplex ligation-dependent probe amplification (MLPA) SALSA P327-A1 probe set for evaluating copy number alterations (CNA) on chromosome 21.
Methods
A series of consecutive diagnostic samples from Brazilian childhood BCP-ALL patients collected between the years 2002 to 2012 were selected according to availability of material of good quality providing that bone marrow aspirates harbored at least 30% of blast cells. The MLPA SALSA P327-A1 probe set was employed to detect an iAMP21 (MRC Holland, Amsterdam, The Netherlands). Test fragments of the P327-A1 probe set were designed to detect CNA of 24 genes distributed from centromeric to telomeric regions (q11.2-q22.3, 14.668-46.356) of chromosome 21. Relative copy numbers were obtained after normalization of peaks against cord blood derived controls. Relative peak heights between 0.75 and 1.3 were considered normal, while those below 0.75 and above 1.3 indicated losses or gains of genomic material, respectively. FISH was performed using the “LPH012 TEL/AML1 Translocation, Dual Fusion Probe”, designed to detect the ETV6/RUNX1 fusion gene. Cases were considered positive for an iAMP21 when ≥5 RUNX1 FISH signals were observed in at least 7% of metaphase nuclei (standard deviation +/- 2%).
Results
In 74 out of 368 BCP-ALL patients, gain of genetic material on chromosome 21 was detected. MLPA peak profiles segregated patients into three groups by hierarchical clustering. Next, patient material was subjected to FISH. Cells with ≥5 RUNX1 FISH signals (n=9) were considered as “true iAMP21”. By contrast those with 3-4 RUNX1 signals (n=35) or a normal RUNX1 FISH pattern (n=6) probably acquired additional copies of an intact chromosome 21, even though not detectable by FISH in all cases. Peak ratios of RUNX1 and DYRK1A genes, located within the common region of amplification (CRA), were in “true iAMP21” patients higher, an opposite pattern was observed for telomeric SNF1LK and TFF1 genes. Since we observed variations in MLPA peak heights between different patients, the correlation between RUNX1, DYRK1A, ETS2 and ERG gene load and expression was tested. For illustration we stepwise arranged cases from the lowest to the highest MLPA peak ratios and depicted corresponding delta CT (threshold cycle) values. Positive correlation between the MLPA peak ratios and CT values were observed, since those patients with high MLPA peak ratios were the ones with low gene expression. This observation particularly held true for the genes RUNX1, DYRK1A, and ERG.
Summary
Altogether, we confirmed that aberrations on chromosome 21 represent a high level of complexity and individuality and provided evidence for the existence of gene regulatory mechanisms that might modulate the expression of amplified genes. In BCP ALL with an iAMP21 we consider MLPA tests highly desirable for data replication of array based comparative genomic hybridization analyzes. MLPA profiles of high peak ratios of CRA genes might be especially helpful in case identification whenever metaphase nuclei for FISH are not available. However, we want to stress that a precise separation of BCP-ALL with an iAMP21 from hyperdiploid ALL with additional copies of chromosome 21 is still under revision. Control fragments binding on chromosomes that are usually over represented should be included in next generation MLPA probe sets.
Keyword(s): Acute lymphoblastic leukemia
Session topic: Publication Only
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