A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma

Aurélie Catteau, Hélène Girardi, Florence Monville, Cécile Poggionovo, Sabrina Carpentier, Véronique Frayssinet, Jesse Voss, Robert Brian Jenkins, Blandine Boisselier, Karima Mokhtari, Marc Sanson, Hélène Peyro-Saint-Paul, Caterina Giannini

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Abstract

INTRODUCTION: Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing.

RESULTS: The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results.

CONCLUSIONS: The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.

Original languageEnglish (US)
Pages (from-to)58
Number of pages1
JournalActa neuropathologica communications
Volume2
DOIs
StatePublished - 2014

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Glioma
Polymerase Chain Reaction
Mutation
Immunohistochemistry
Paraffin
Formaldehyde
Isocitrate Dehydrogenase
Exercise Test
Constriction
Patient Selection
Real-Time Polymerase Chain Reaction

ASJC Scopus subject areas

  • Medicine(all)

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A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma. / Catteau, Aurélie; Girardi, Hélène; Monville, Florence; Poggionovo, Cécile; Carpentier, Sabrina; Frayssinet, Véronique; Voss, Jesse; Jenkins, Robert Brian; Boisselier, Blandine; Mokhtari, Karima; Sanson, Marc; Peyro-Saint-Paul, Hélène; Giannini, Caterina.

In: Acta neuropathologica communications, Vol. 2, 2014, p. 58.

Research output: Contribution to journalArticle

Catteau, A, Girardi, H, Monville, F, Poggionovo, C, Carpentier, S, Frayssinet, V, Voss, J, Jenkins, RB, Boisselier, B, Mokhtari, K, Sanson, M, Peyro-Saint-Paul, H & Giannini, C 2014, 'A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma', Acta neuropathologica communications, vol. 2, pp. 58. https://doi.org/10.1186/2051-5960-2-58
Catteau, Aurélie ; Girardi, Hélène ; Monville, Florence ; Poggionovo, Cécile ; Carpentier, Sabrina ; Frayssinet, Véronique ; Voss, Jesse ; Jenkins, Robert Brian ; Boisselier, Blandine ; Mokhtari, Karima ; Sanson, Marc ; Peyro-Saint-Paul, Hélène ; Giannini, Caterina. / A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma. In: Acta neuropathologica communications. 2014 ; Vol. 2. pp. 58.
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title = "A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma",
abstract = "INTRODUCTION: Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing.RESULTS: The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5{\%} for 11/12 mutations (mean: 3.3{\%}), and sensitivity for mutation identification was very high (0.8{\%} for IDH1 R132H; 1.2{\%} for IDH1 R132C; 0.6{\%} for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91{\%} of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96{\%}). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100{\%} correct results.CONCLUSIONS: The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98{\%} with IHC for IDH1 R132H detection and 100{\%} with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.",
author = "Aur{\'e}lie Catteau and H{\'e}l{\`e}ne Girardi and Florence Monville and C{\'e}cile Poggionovo and Sabrina Carpentier and V{\'e}ronique Frayssinet and Jesse Voss and Jenkins, {Robert Brian} and Blandine Boisselier and Karima Mokhtari and Marc Sanson and H{\'e}l{\`e}ne Peyro-Saint-Paul and Caterina Giannini",
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T1 - A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma

AU - Catteau, Aurélie

AU - Girardi, Hélène

AU - Monville, Florence

AU - Poggionovo, Cécile

AU - Carpentier, Sabrina

AU - Frayssinet, Véronique

AU - Voss, Jesse

AU - Jenkins, Robert Brian

AU - Boisselier, Blandine

AU - Mokhtari, Karima

AU - Sanson, Marc

AU - Peyro-Saint-Paul, Hélène

AU - Giannini, Caterina

PY - 2014

Y1 - 2014

N2 - INTRODUCTION: Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing.RESULTS: The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results.CONCLUSIONS: The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.

AB - INTRODUCTION: Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing.RESULTS: The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results.CONCLUSIONS: The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.

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