HIGH SENSITIVITY MLPA ASSAY FOR THE DETECTION OF THE RECURRENT CALR, JAK2, KIT AND MPL MUTATIONS WITH ? 1% ALLELE BURDEN IN MYELOPROLIFERATIVE NEOPLASMS
(Abstract release date: 05/19/16)
EHA Library. Steenkamer M. 06/09/16; 134913; PB2013
Ms. Maryvonne Steenkamer
Contributions
Contributions
Abstract
Abstract: PB2013
Type: Publication Only
Background
Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell malignancies, characterized by clonal proliferation of blood cells. Recurrent mutations in CALR, JAK2, KIT and MPL genes are important molecular markers for classification and prognostication of MPNs. Multiplex Ligation-dependent Probe Amplification (MLPA) is a widely used technique for gene copy number detection, also allowing simultaneous identification of known point mutations. However, the sensitivity of standard MLPA is limited to a mutant allele burden of ≥ 10%. To overcome this limitation, we have introduced a high sensitivity MLPA assay enabling detection of an allele burden as low as 1% for the most frequent mutations in MPN.
Aims
To demonstrate the feasibility of a modified MLPA assay to detect ≥ 1% mutant allele burden using artificial positive DNA and cell line samples. Validation of this assay is done with MPN patient samples.
Methods
Salsa MLPA P520 MPN probemix was designed and optimized to detect ≥ 1% mutant allele burden of the eight most frequent mutations in MPNs: CALR, 52-bp deletion and 5-bp insertion in exon 9 (L367fs*46 and K385fs*47), JAK2, deletions in exon 12 (N542_E543del and E543-D544del), JAK2, substitution in exon 14 (V617F), KIT, substitution in exon 17 (D816V) and MPL, substitutions in exon 10 (W515L and W515K). The performance of the newly developed MLPA assay was optimized and tested on artificial positive DNA samples with 1% mutation burden. The performance of the assay was further validated using commercial reference DNA samples with 1% allelic burden of JAK2 V617F and KIT D816V mutations, as well as by using dilution series of a JAK2 V617F positive, UKE-1 cell line, where the allelic burden was confirmed by the Ipsogen MutaQuant assay. Validation on diagnostic MPN patient samples (n=167) was performed in a single-blind setting.
Results
Results obtained by high sensitivity MLPA assay were concordant with an allele-specific PCR results in MPN patient samples. Moreover, 2 novel cases with 1.4 – 5% JAK2 V617F burden, which were not detected by allele-specific PCR, were identified with our novel assay and confirmed by the Ipsogen MutaQuant assay. Furthermore, no false positive calls for mutations were obtained when testing on healthy human DNA samples (n=143).
Conclusion
Our results demonstrate that the P520 MPN MLPA assay is a reliable method for simultaneous detection of eight frequent mutations in MPNs, even when the patient DNA sample has a low (1-5%) mutant allele burden. These results merit further consideration of MLPA as a possible alternative for mutation testing for newly diagnosed MPN patients.
Session topic: E-poster
Keyword(s): Molecular markers, Mutation analysis, Myeloproliferative disorder, PCR
Type: Publication Only
Background
Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell malignancies, characterized by clonal proliferation of blood cells. Recurrent mutations in CALR, JAK2, KIT and MPL genes are important molecular markers for classification and prognostication of MPNs. Multiplex Ligation-dependent Probe Amplification (MLPA) is a widely used technique for gene copy number detection, also allowing simultaneous identification of known point mutations. However, the sensitivity of standard MLPA is limited to a mutant allele burden of ≥ 10%. To overcome this limitation, we have introduced a high sensitivity MLPA assay enabling detection of an allele burden as low as 1% for the most frequent mutations in MPN.
Aims
To demonstrate the feasibility of a modified MLPA assay to detect ≥ 1% mutant allele burden using artificial positive DNA and cell line samples. Validation of this assay is done with MPN patient samples.
Methods
Salsa MLPA P520 MPN probemix was designed and optimized to detect ≥ 1% mutant allele burden of the eight most frequent mutations in MPNs: CALR, 52-bp deletion and 5-bp insertion in exon 9 (L367fs*46 and K385fs*47), JAK2, deletions in exon 12 (N542_E543del and E543-D544del), JAK2, substitution in exon 14 (V617F), KIT, substitution in exon 17 (D816V) and MPL, substitutions in exon 10 (W515L and W515K). The performance of the newly developed MLPA assay was optimized and tested on artificial positive DNA samples with 1% mutation burden. The performance of the assay was further validated using commercial reference DNA samples with 1% allelic burden of JAK2 V617F and KIT D816V mutations, as well as by using dilution series of a JAK2 V617F positive, UKE-1 cell line, where the allelic burden was confirmed by the Ipsogen MutaQuant assay. Validation on diagnostic MPN patient samples (n=167) was performed in a single-blind setting.
Results
Results obtained by high sensitivity MLPA assay were concordant with an allele-specific PCR results in MPN patient samples. Moreover, 2 novel cases with 1.4 – 5% JAK2 V617F burden, which were not detected by allele-specific PCR, were identified with our novel assay and confirmed by the Ipsogen MutaQuant assay. Furthermore, no false positive calls for mutations were obtained when testing on healthy human DNA samples (n=143).
Conclusion
Our results demonstrate that the P520 MPN MLPA assay is a reliable method for simultaneous detection of eight frequent mutations in MPNs, even when the patient DNA sample has a low (1-5%) mutant allele burden. These results merit further consideration of MLPA as a possible alternative for mutation testing for newly diagnosed MPN patients.
Session topic: E-poster
Keyword(s): Molecular markers, Mutation analysis, Myeloproliferative disorder, PCR
Abstract: PB2013
Type: Publication Only
Background
Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell malignancies, characterized by clonal proliferation of blood cells. Recurrent mutations in CALR, JAK2, KIT and MPL genes are important molecular markers for classification and prognostication of MPNs. Multiplex Ligation-dependent Probe Amplification (MLPA) is a widely used technique for gene copy number detection, also allowing simultaneous identification of known point mutations. However, the sensitivity of standard MLPA is limited to a mutant allele burden of ≥ 10%. To overcome this limitation, we have introduced a high sensitivity MLPA assay enabling detection of an allele burden as low as 1% for the most frequent mutations in MPN.
Aims
To demonstrate the feasibility of a modified MLPA assay to detect ≥ 1% mutant allele burden using artificial positive DNA and cell line samples. Validation of this assay is done with MPN patient samples.
Methods
Salsa MLPA P520 MPN probemix was designed and optimized to detect ≥ 1% mutant allele burden of the eight most frequent mutations in MPNs: CALR, 52-bp deletion and 5-bp insertion in exon 9 (L367fs*46 and K385fs*47), JAK2, deletions in exon 12 (N542_E543del and E543-D544del), JAK2, substitution in exon 14 (V617F), KIT, substitution in exon 17 (D816V) and MPL, substitutions in exon 10 (W515L and W515K). The performance of the newly developed MLPA assay was optimized and tested on artificial positive DNA samples with 1% mutation burden. The performance of the assay was further validated using commercial reference DNA samples with 1% allelic burden of JAK2 V617F and KIT D816V mutations, as well as by using dilution series of a JAK2 V617F positive, UKE-1 cell line, where the allelic burden was confirmed by the Ipsogen MutaQuant assay. Validation on diagnostic MPN patient samples (n=167) was performed in a single-blind setting.
Results
Results obtained by high sensitivity MLPA assay were concordant with an allele-specific PCR results in MPN patient samples. Moreover, 2 novel cases with 1.4 – 5% JAK2 V617F burden, which were not detected by allele-specific PCR, were identified with our novel assay and confirmed by the Ipsogen MutaQuant assay. Furthermore, no false positive calls for mutations were obtained when testing on healthy human DNA samples (n=143).
Conclusion
Our results demonstrate that the P520 MPN MLPA assay is a reliable method for simultaneous detection of eight frequent mutations in MPNs, even when the patient DNA sample has a low (1-5%) mutant allele burden. These results merit further consideration of MLPA as a possible alternative for mutation testing for newly diagnosed MPN patients.
Session topic: E-poster
Keyword(s): Molecular markers, Mutation analysis, Myeloproliferative disorder, PCR
Type: Publication Only
Background
Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell malignancies, characterized by clonal proliferation of blood cells. Recurrent mutations in CALR, JAK2, KIT and MPL genes are important molecular markers for classification and prognostication of MPNs. Multiplex Ligation-dependent Probe Amplification (MLPA) is a widely used technique for gene copy number detection, also allowing simultaneous identification of known point mutations. However, the sensitivity of standard MLPA is limited to a mutant allele burden of ≥ 10%. To overcome this limitation, we have introduced a high sensitivity MLPA assay enabling detection of an allele burden as low as 1% for the most frequent mutations in MPN.
Aims
To demonstrate the feasibility of a modified MLPA assay to detect ≥ 1% mutant allele burden using artificial positive DNA and cell line samples. Validation of this assay is done with MPN patient samples.
Methods
Salsa MLPA P520 MPN probemix was designed and optimized to detect ≥ 1% mutant allele burden of the eight most frequent mutations in MPNs: CALR, 52-bp deletion and 5-bp insertion in exon 9 (L367fs*46 and K385fs*47), JAK2, deletions in exon 12 (N542_E543del and E543-D544del), JAK2, substitution in exon 14 (V617F), KIT, substitution in exon 17 (D816V) and MPL, substitutions in exon 10 (W515L and W515K). The performance of the newly developed MLPA assay was optimized and tested on artificial positive DNA samples with 1% mutation burden. The performance of the assay was further validated using commercial reference DNA samples with 1% allelic burden of JAK2 V617F and KIT D816V mutations, as well as by using dilution series of a JAK2 V617F positive, UKE-1 cell line, where the allelic burden was confirmed by the Ipsogen MutaQuant assay. Validation on diagnostic MPN patient samples (n=167) was performed in a single-blind setting.
Results
Results obtained by high sensitivity MLPA assay were concordant with an allele-specific PCR results in MPN patient samples. Moreover, 2 novel cases with 1.4 – 5% JAK2 V617F burden, which were not detected by allele-specific PCR, were identified with our novel assay and confirmed by the Ipsogen MutaQuant assay. Furthermore, no false positive calls for mutations were obtained when testing on healthy human DNA samples (n=143).
Conclusion
Our results demonstrate that the P520 MPN MLPA assay is a reliable method for simultaneous detection of eight frequent mutations in MPNs, even when the patient DNA sample has a low (1-5%) mutant allele burden. These results merit further consideration of MLPA as a possible alternative for mutation testing for newly diagnosed MPN patients.
Session topic: E-poster
Keyword(s): Molecular markers, Mutation analysis, Myeloproliferative disorder, PCR
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