A NOVEL REAL-TIME PCR ASSAY SET FOR DETECTION OF DIVERSE CALR MUTATIONS IN MYELOPROLIFERATIVE NEOPLASMS
(Abstract release date: 05/19/16)
EHA Library. Jager R. 06/09/16; 134911; PB2011
Dr. Roland Jager
Contributions
Contributions
Abstract
Abstract: PB2011
Type: Publication Only
Background
Mutations in the CALR gene have been found in a large proportion of patients suffering from myeloproliferative neoplasms (MPN). While 52bp deletions (Type 1) and 5bp insertions (Type 2) constitute >80% of CALR mutations, a variety of other insertion-deletion mutations have been reported, all within a 66bp mutational hotspot in exon 9 of CALR. Reliable and efficient detection of those somatic driver mutations is crucial for MPN diagnosis and patient management.
Aims
We previously characterized our MPN patient cohort for insertions and deletions in the CALR gene using a PCR-based fragment sizing assay in combination with Sanger sequencing. Here we compare the characteristics of this fragment analysis with a novel real-time PCR (qPCR) based assay set (ipsogen CALR RGQ PCR Kit, provided pre-launch by QIAGEN, Hilden, Germany).
Methods
Fragment analysis of CALR exon 9 was performed using high-resolution sizing of fluorescent dye labeled PCR products on a 3130xl Genetic Analyzer (ABI, Foster City, CA). The ipsogen CALR RGQ PCR Kit was applied according to the manufacturer’s instructions in 7 separate hydrolysis-probe-based qPCR reactions per sample within the same run. The kit identifies Type 1 and Type 2 mutations using ARMS PCR, in which a primer mismatch prevents elongation on wild-type (WT) DNA. Minor variants of CALR mutations are detected through a PCR clamping approach using 3'-phosphate blocked oligonucleotides complementary to WT sequence. For both fragment analysis and the ipsogen assay we determined dynamic range and detection limits through serial dilution of mutant genomic DNA (gDNA) in WT gDNA, generating CALR mutational burden standards down to 0.01%.
Results
Using the ipsogen CALR RGQ PCR Kit, we re-tested gDNA from 48 MPN patients (10 Type 1, 10 Type 2, 18 minor variants, 10 CALR mutation negative) previously evaluated by fragment analysis. Concordance between both assays was 100%. While the ipsogen assay formally requires a per reaction input of 50ng gDNA freshly isolated from peripheral blood, our samples were from a mixed cohort of various storage time and isolation protocols. Moreover, we applied the ipsogen assay on a large proportion of our samples using DNA inputs of 25ng and 10ng, respectively. CALR mutational status could be confirmed for all those cases. We next determined the limit of detection (LoD) of Type 1 and Type 2 mutations for both the ipsogen assay and fragment analysis. While limit-of-blank-based calculation of LoD revealed 0.39% and 0.08% for Type 1 and Type 2 ipsogen assays, respectively, fragment analysis could reliably detect 2.5% but not 1% CALR mutational burdens. Determination of the dynamic range revealed linearity for the fragment analysis down to the 2.5% detection limit. For the ipsogen assays, linearity on the log-scale could be observed down to 1% and 0.5% CALR mutant burden for Type 1 and Type 2 assays, respectively. Within these dynamic ranges, standard curves could be produced with a correlation coefficient of >0.98.
Conclusion
While fragment analysis represents a rapid, cost-effective assay for screening larger cohorts in a multi-well format, the specific identification of Type 1 and Type 2 mutations requires Sanger sequencing in addition. The ipsogen CALR RGQ PCR Kit offers, at once, reliable detection of CALR mutations and specific identification of Type 1 and Type 2 mutations, requiring minimal processing and equipment (Rotor-Gene Q MDx, QIAGEN). Moreover, the ipsogen assay showed superior sensitivity over fragment analysis and might therefore be most suitable for tracking the dynamics of the malignant MPN clone.
Session topic: E-poster
Keyword(s): Assay, Myeloproliferative disorder, Somatic mutation
Type: Publication Only
Background
Mutations in the CALR gene have been found in a large proportion of patients suffering from myeloproliferative neoplasms (MPN). While 52bp deletions (Type 1) and 5bp insertions (Type 2) constitute >80% of CALR mutations, a variety of other insertion-deletion mutations have been reported, all within a 66bp mutational hotspot in exon 9 of CALR. Reliable and efficient detection of those somatic driver mutations is crucial for MPN diagnosis and patient management.
Aims
We previously characterized our MPN patient cohort for insertions and deletions in the CALR gene using a PCR-based fragment sizing assay in combination with Sanger sequencing. Here we compare the characteristics of this fragment analysis with a novel real-time PCR (qPCR) based assay set (ipsogen CALR RGQ PCR Kit, provided pre-launch by QIAGEN, Hilden, Germany).
Methods
Fragment analysis of CALR exon 9 was performed using high-resolution sizing of fluorescent dye labeled PCR products on a 3130xl Genetic Analyzer (ABI, Foster City, CA). The ipsogen CALR RGQ PCR Kit was applied according to the manufacturer’s instructions in 7 separate hydrolysis-probe-based qPCR reactions per sample within the same run. The kit identifies Type 1 and Type 2 mutations using ARMS PCR, in which a primer mismatch prevents elongation on wild-type (WT) DNA. Minor variants of CALR mutations are detected through a PCR clamping approach using 3'-phosphate blocked oligonucleotides complementary to WT sequence. For both fragment analysis and the ipsogen assay we determined dynamic range and detection limits through serial dilution of mutant genomic DNA (gDNA) in WT gDNA, generating CALR mutational burden standards down to 0.01%.
Results
Using the ipsogen CALR RGQ PCR Kit, we re-tested gDNA from 48 MPN patients (10 Type 1, 10 Type 2, 18 minor variants, 10 CALR mutation negative) previously evaluated by fragment analysis. Concordance between both assays was 100%. While the ipsogen assay formally requires a per reaction input of 50ng gDNA freshly isolated from peripheral blood, our samples were from a mixed cohort of various storage time and isolation protocols. Moreover, we applied the ipsogen assay on a large proportion of our samples using DNA inputs of 25ng and 10ng, respectively. CALR mutational status could be confirmed for all those cases. We next determined the limit of detection (LoD) of Type 1 and Type 2 mutations for both the ipsogen assay and fragment analysis. While limit-of-blank-based calculation of LoD revealed 0.39% and 0.08% for Type 1 and Type 2 ipsogen assays, respectively, fragment analysis could reliably detect 2.5% but not 1% CALR mutational burdens. Determination of the dynamic range revealed linearity for the fragment analysis down to the 2.5% detection limit. For the ipsogen assays, linearity on the log-scale could be observed down to 1% and 0.5% CALR mutant burden for Type 1 and Type 2 assays, respectively. Within these dynamic ranges, standard curves could be produced with a correlation coefficient of >0.98.
Conclusion
While fragment analysis represents a rapid, cost-effective assay for screening larger cohorts in a multi-well format, the specific identification of Type 1 and Type 2 mutations requires Sanger sequencing in addition. The ipsogen CALR RGQ PCR Kit offers, at once, reliable detection of CALR mutations and specific identification of Type 1 and Type 2 mutations, requiring minimal processing and equipment (Rotor-Gene Q MDx, QIAGEN). Moreover, the ipsogen assay showed superior sensitivity over fragment analysis and might therefore be most suitable for tracking the dynamics of the malignant MPN clone.
Session topic: E-poster
Keyword(s): Assay, Myeloproliferative disorder, Somatic mutation
Abstract: PB2011
Type: Publication Only
Background
Mutations in the CALR gene have been found in a large proportion of patients suffering from myeloproliferative neoplasms (MPN). While 52bp deletions (Type 1) and 5bp insertions (Type 2) constitute >80% of CALR mutations, a variety of other insertion-deletion mutations have been reported, all within a 66bp mutational hotspot in exon 9 of CALR. Reliable and efficient detection of those somatic driver mutations is crucial for MPN diagnosis and patient management.
Aims
We previously characterized our MPN patient cohort for insertions and deletions in the CALR gene using a PCR-based fragment sizing assay in combination with Sanger sequencing. Here we compare the characteristics of this fragment analysis with a novel real-time PCR (qPCR) based assay set (ipsogen CALR RGQ PCR Kit, provided pre-launch by QIAGEN, Hilden, Germany).
Methods
Fragment analysis of CALR exon 9 was performed using high-resolution sizing of fluorescent dye labeled PCR products on a 3130xl Genetic Analyzer (ABI, Foster City, CA). The ipsogen CALR RGQ PCR Kit was applied according to the manufacturer’s instructions in 7 separate hydrolysis-probe-based qPCR reactions per sample within the same run. The kit identifies Type 1 and Type 2 mutations using ARMS PCR, in which a primer mismatch prevents elongation on wild-type (WT) DNA. Minor variants of CALR mutations are detected through a PCR clamping approach using 3'-phosphate blocked oligonucleotides complementary to WT sequence. For both fragment analysis and the ipsogen assay we determined dynamic range and detection limits through serial dilution of mutant genomic DNA (gDNA) in WT gDNA, generating CALR mutational burden standards down to 0.01%.
Results
Using the ipsogen CALR RGQ PCR Kit, we re-tested gDNA from 48 MPN patients (10 Type 1, 10 Type 2, 18 minor variants, 10 CALR mutation negative) previously evaluated by fragment analysis. Concordance between both assays was 100%. While the ipsogen assay formally requires a per reaction input of 50ng gDNA freshly isolated from peripheral blood, our samples were from a mixed cohort of various storage time and isolation protocols. Moreover, we applied the ipsogen assay on a large proportion of our samples using DNA inputs of 25ng and 10ng, respectively. CALR mutational status could be confirmed for all those cases. We next determined the limit of detection (LoD) of Type 1 and Type 2 mutations for both the ipsogen assay and fragment analysis. While limit-of-blank-based calculation of LoD revealed 0.39% and 0.08% for Type 1 and Type 2 ipsogen assays, respectively, fragment analysis could reliably detect 2.5% but not 1% CALR mutational burdens. Determination of the dynamic range revealed linearity for the fragment analysis down to the 2.5% detection limit. For the ipsogen assays, linearity on the log-scale could be observed down to 1% and 0.5% CALR mutant burden for Type 1 and Type 2 assays, respectively. Within these dynamic ranges, standard curves could be produced with a correlation coefficient of >0.98.
Conclusion
While fragment analysis represents a rapid, cost-effective assay for screening larger cohorts in a multi-well format, the specific identification of Type 1 and Type 2 mutations requires Sanger sequencing in addition. The ipsogen CALR RGQ PCR Kit offers, at once, reliable detection of CALR mutations and specific identification of Type 1 and Type 2 mutations, requiring minimal processing and equipment (Rotor-Gene Q MDx, QIAGEN). Moreover, the ipsogen assay showed superior sensitivity over fragment analysis and might therefore be most suitable for tracking the dynamics of the malignant MPN clone.
Session topic: E-poster
Keyword(s): Assay, Myeloproliferative disorder, Somatic mutation
Type: Publication Only
Background
Mutations in the CALR gene have been found in a large proportion of patients suffering from myeloproliferative neoplasms (MPN). While 52bp deletions (Type 1) and 5bp insertions (Type 2) constitute >80% of CALR mutations, a variety of other insertion-deletion mutations have been reported, all within a 66bp mutational hotspot in exon 9 of CALR. Reliable and efficient detection of those somatic driver mutations is crucial for MPN diagnosis and patient management.
Aims
We previously characterized our MPN patient cohort for insertions and deletions in the CALR gene using a PCR-based fragment sizing assay in combination with Sanger sequencing. Here we compare the characteristics of this fragment analysis with a novel real-time PCR (qPCR) based assay set (ipsogen CALR RGQ PCR Kit, provided pre-launch by QIAGEN, Hilden, Germany).
Methods
Fragment analysis of CALR exon 9 was performed using high-resolution sizing of fluorescent dye labeled PCR products on a 3130xl Genetic Analyzer (ABI, Foster City, CA). The ipsogen CALR RGQ PCR Kit was applied according to the manufacturer’s instructions in 7 separate hydrolysis-probe-based qPCR reactions per sample within the same run. The kit identifies Type 1 and Type 2 mutations using ARMS PCR, in which a primer mismatch prevents elongation on wild-type (WT) DNA. Minor variants of CALR mutations are detected through a PCR clamping approach using 3'-phosphate blocked oligonucleotides complementary to WT sequence. For both fragment analysis and the ipsogen assay we determined dynamic range and detection limits through serial dilution of mutant genomic DNA (gDNA) in WT gDNA, generating CALR mutational burden standards down to 0.01%.
Results
Using the ipsogen CALR RGQ PCR Kit, we re-tested gDNA from 48 MPN patients (10 Type 1, 10 Type 2, 18 minor variants, 10 CALR mutation negative) previously evaluated by fragment analysis. Concordance between both assays was 100%. While the ipsogen assay formally requires a per reaction input of 50ng gDNA freshly isolated from peripheral blood, our samples were from a mixed cohort of various storage time and isolation protocols. Moreover, we applied the ipsogen assay on a large proportion of our samples using DNA inputs of 25ng and 10ng, respectively. CALR mutational status could be confirmed for all those cases. We next determined the limit of detection (LoD) of Type 1 and Type 2 mutations for both the ipsogen assay and fragment analysis. While limit-of-blank-based calculation of LoD revealed 0.39% and 0.08% for Type 1 and Type 2 ipsogen assays, respectively, fragment analysis could reliably detect 2.5% but not 1% CALR mutational burdens. Determination of the dynamic range revealed linearity for the fragment analysis down to the 2.5% detection limit. For the ipsogen assays, linearity on the log-scale could be observed down to 1% and 0.5% CALR mutant burden for Type 1 and Type 2 assays, respectively. Within these dynamic ranges, standard curves could be produced with a correlation coefficient of >0.98.
Conclusion
While fragment analysis represents a rapid, cost-effective assay for screening larger cohorts in a multi-well format, the specific identification of Type 1 and Type 2 mutations requires Sanger sequencing in addition. The ipsogen CALR RGQ PCR Kit offers, at once, reliable detection of CALR mutations and specific identification of Type 1 and Type 2 mutations, requiring minimal processing and equipment (Rotor-Gene Q MDx, QIAGEN). Moreover, the ipsogen assay showed superior sensitivity over fragment analysis and might therefore be most suitable for tracking the dynamics of the malignant MPN clone.
Session topic: E-poster
Keyword(s): Assay, Myeloproliferative disorder, Somatic mutation
{{ help_message }}
{{filter}}