Accuracy of four mononucleotide-repeat markers for the identification of DNA mismatch-repair deficiency in solid tumors

Yuko Takehara, Takeshi Nagasaka, Akihiro Nyuya, Tomoko Haruma, Junko Haraga, Yoshiko Mori, Keiichiro Nakamura, Toshiyoshi Fujiwara, C. Richard Boland, Ajay Goel

Research output: Contribution to journalArticle

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Abstract

Background: To screen tumors with microsatellite instability (MSI) arising due to DNA mismatch repair deficiency (dMMR), a panel of five quasi-monomorphic mononucleotide-repeat markers amplified in a multiplex PCR (Pentaplex) are commonly used. In spite of its several strengths, the pentaplex assay is not robust at detecting the loss of MSH6-deficiency (dMSH6). In order to overcome this challenge, we designed this study to develop and optimize a panel of four quasi-monomorphic mononucleotide-repeat markers (Tetraplex) for identifying solid tumors with dMMR, especially dMSH6. Methods: To improve the sensitivity for tumors with dMMR, we established a quasi-monomorphic variant range (QMVR) of 3-4bp for the four Tetraplex markers. Thereafter, to confirm the accuracy of this assay, we examined 317 colorectal cancer (CRC) specimens, comprising of 105 dMMR [45 MutL homolog (MLH)1-deficient, 45 MutS protein homolog (MSH)2-deficient, and 15 MSH6-deficient tumors] and 212 MMR-proficient (pMMR) tumors as a test set. In addition, we analyzed a cohort of 138 endometrial cancers (EC) by immunohistochemistry to determine MMR protein expression and validation of our new MSI assay. Results: Using the criteria of ≥1 unstable markers as MSI-positive tumor, our assay resulted in a sensitivity of 97.1% [95% confidence interval (CI)=91.9-99.0%] for dMMR, and a specificity of 95.3% (95% CI=91.5-97.4%) for pMMR CRC specimens. Among the 138 EC specimens, 41 were dMMR according to immunohistochemistry. Herein, our Tetraplex assay detected dMMR tumors with a sensitivity of 92.7% (95% CI=80.6-97.5%) and a specificity of 97.9% (95% CI=92.8-99.4%) for pMMR tumors. With respect to tumors with dMSH6, in the CRC-validation set, Tetraplex detected dMSH6 tumors with a sensitivity of 86.7% (13 of 15 dMSH6 CRCs), which was subsequently validated in the EC test set as well (sensitivity, 75.0%; 6 of 8 dMSH6 ECs). Conclusions: Our newly optimized Tetraplex system will help offer a robust and highly sensitive assay for the identification of dMMR in solid tumors.

Original languageEnglish
Article number5
JournalJournal of Translational Medicine
Volume16
Issue number1
DOIs
Publication statusPublished - Jan 12 2018

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DNA Repair-Deficiency Disorders
DNA Mismatch Repair
Tumors
Repair
DNA
Assays
Neoplasms
Microsatellite Instability
Endometrial Neoplasms
Microsatellite Repeats
Confidence Intervals
Colorectal Neoplasms
MutS Homolog 2 Protein
Turcot syndrome
Immunohistochemistry
Multiplex Polymerase Chain Reaction

Keywords

  • Colorectal cancer
  • DNA mismatch repair
  • Endometrial cancer
  • Hypermutated tumors
  • Microsatellite instability

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Accuracy of four mononucleotide-repeat markers for the identification of DNA mismatch-repair deficiency in solid tumors. / Takehara, Yuko; Nagasaka, Takeshi; Nyuya, Akihiro; Haruma, Tomoko; Haraga, Junko; Mori, Yoshiko; Nakamura, Keiichiro; Fujiwara, Toshiyoshi; Boland, C. Richard; Goel, Ajay.

In: Journal of Translational Medicine, Vol. 16, No. 1, 5, 12.01.2018.

Research output: Contribution to journalArticle

Takehara, Yuko ; Nagasaka, Takeshi ; Nyuya, Akihiro ; Haruma, Tomoko ; Haraga, Junko ; Mori, Yoshiko ; Nakamura, Keiichiro ; Fujiwara, Toshiyoshi ; Boland, C. Richard ; Goel, Ajay. / Accuracy of four mononucleotide-repeat markers for the identification of DNA mismatch-repair deficiency in solid tumors. In: Journal of Translational Medicine. 2018 ; Vol. 16, No. 1.
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abstract = "Background: To screen tumors with microsatellite instability (MSI) arising due to DNA mismatch repair deficiency (dMMR), a panel of five quasi-monomorphic mononucleotide-repeat markers amplified in a multiplex PCR (Pentaplex) are commonly used. In spite of its several strengths, the pentaplex assay is not robust at detecting the loss of MSH6-deficiency (dMSH6). In order to overcome this challenge, we designed this study to develop and optimize a panel of four quasi-monomorphic mononucleotide-repeat markers (Tetraplex) for identifying solid tumors with dMMR, especially dMSH6. Methods: To improve the sensitivity for tumors with dMMR, we established a quasi-monomorphic variant range (QMVR) of 3-4bp for the four Tetraplex markers. Thereafter, to confirm the accuracy of this assay, we examined 317 colorectal cancer (CRC) specimens, comprising of 105 dMMR [45 MutL homolog (MLH)1-deficient, 45 MutS protein homolog (MSH)2-deficient, and 15 MSH6-deficient tumors] and 212 MMR-proficient (pMMR) tumors as a test set. In addition, we analyzed a cohort of 138 endometrial cancers (EC) by immunohistochemistry to determine MMR protein expression and validation of our new MSI assay. Results: Using the criteria of ≥1 unstable markers as MSI-positive tumor, our assay resulted in a sensitivity of 97.1{\%} [95{\%} confidence interval (CI)=91.9-99.0{\%}] for dMMR, and a specificity of 95.3{\%} (95{\%} CI=91.5-97.4{\%}) for pMMR CRC specimens. Among the 138 EC specimens, 41 were dMMR according to immunohistochemistry. Herein, our Tetraplex assay detected dMMR tumors with a sensitivity of 92.7{\%} (95{\%} CI=80.6-97.5{\%}) and a specificity of 97.9{\%} (95{\%} CI=92.8-99.4{\%}) for pMMR tumors. With respect to tumors with dMSH6, in the CRC-validation set, Tetraplex detected dMSH6 tumors with a sensitivity of 86.7{\%} (13 of 15 dMSH6 CRCs), which was subsequently validated in the EC test set as well (sensitivity, 75.0{\%}; 6 of 8 dMSH6 ECs). Conclusions: Our newly optimized Tetraplex system will help offer a robust and highly sensitive assay for the identification of dMMR in solid tumors.",
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author = "Yuko Takehara and Takeshi Nagasaka and Akihiro Nyuya and Tomoko Haruma and Junko Haraga and Yoshiko Mori and Keiichiro Nakamura and Toshiyoshi Fujiwara and Boland, {C. Richard} and Ajay Goel",
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T1 - Accuracy of four mononucleotide-repeat markers for the identification of DNA mismatch-repair deficiency in solid tumors

AU - Takehara, Yuko

AU - Nagasaka, Takeshi

AU - Nyuya, Akihiro

AU - Haruma, Tomoko

AU - Haraga, Junko

AU - Mori, Yoshiko

AU - Nakamura, Keiichiro

AU - Fujiwara, Toshiyoshi

AU - Boland, C. Richard

AU - Goel, Ajay

PY - 2018/1/12

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N2 - Background: To screen tumors with microsatellite instability (MSI) arising due to DNA mismatch repair deficiency (dMMR), a panel of five quasi-monomorphic mononucleotide-repeat markers amplified in a multiplex PCR (Pentaplex) are commonly used. In spite of its several strengths, the pentaplex assay is not robust at detecting the loss of MSH6-deficiency (dMSH6). In order to overcome this challenge, we designed this study to develop and optimize a panel of four quasi-monomorphic mononucleotide-repeat markers (Tetraplex) for identifying solid tumors with dMMR, especially dMSH6. Methods: To improve the sensitivity for tumors with dMMR, we established a quasi-monomorphic variant range (QMVR) of 3-4bp for the four Tetraplex markers. Thereafter, to confirm the accuracy of this assay, we examined 317 colorectal cancer (CRC) specimens, comprising of 105 dMMR [45 MutL homolog (MLH)1-deficient, 45 MutS protein homolog (MSH)2-deficient, and 15 MSH6-deficient tumors] and 212 MMR-proficient (pMMR) tumors as a test set. In addition, we analyzed a cohort of 138 endometrial cancers (EC) by immunohistochemistry to determine MMR protein expression and validation of our new MSI assay. Results: Using the criteria of ≥1 unstable markers as MSI-positive tumor, our assay resulted in a sensitivity of 97.1% [95% confidence interval (CI)=91.9-99.0%] for dMMR, and a specificity of 95.3% (95% CI=91.5-97.4%) for pMMR CRC specimens. Among the 138 EC specimens, 41 were dMMR according to immunohistochemistry. Herein, our Tetraplex assay detected dMMR tumors with a sensitivity of 92.7% (95% CI=80.6-97.5%) and a specificity of 97.9% (95% CI=92.8-99.4%) for pMMR tumors. With respect to tumors with dMSH6, in the CRC-validation set, Tetraplex detected dMSH6 tumors with a sensitivity of 86.7% (13 of 15 dMSH6 CRCs), which was subsequently validated in the EC test set as well (sensitivity, 75.0%; 6 of 8 dMSH6 ECs). Conclusions: Our newly optimized Tetraplex system will help offer a robust and highly sensitive assay for the identification of dMMR in solid tumors.

AB - Background: To screen tumors with microsatellite instability (MSI) arising due to DNA mismatch repair deficiency (dMMR), a panel of five quasi-monomorphic mononucleotide-repeat markers amplified in a multiplex PCR (Pentaplex) are commonly used. In spite of its several strengths, the pentaplex assay is not robust at detecting the loss of MSH6-deficiency (dMSH6). In order to overcome this challenge, we designed this study to develop and optimize a panel of four quasi-monomorphic mononucleotide-repeat markers (Tetraplex) for identifying solid tumors with dMMR, especially dMSH6. Methods: To improve the sensitivity for tumors with dMMR, we established a quasi-monomorphic variant range (QMVR) of 3-4bp for the four Tetraplex markers. Thereafter, to confirm the accuracy of this assay, we examined 317 colorectal cancer (CRC) specimens, comprising of 105 dMMR [45 MutL homolog (MLH)1-deficient, 45 MutS protein homolog (MSH)2-deficient, and 15 MSH6-deficient tumors] and 212 MMR-proficient (pMMR) tumors as a test set. In addition, we analyzed a cohort of 138 endometrial cancers (EC) by immunohistochemistry to determine MMR protein expression and validation of our new MSI assay. Results: Using the criteria of ≥1 unstable markers as MSI-positive tumor, our assay resulted in a sensitivity of 97.1% [95% confidence interval (CI)=91.9-99.0%] for dMMR, and a specificity of 95.3% (95% CI=91.5-97.4%) for pMMR CRC specimens. Among the 138 EC specimens, 41 were dMMR according to immunohistochemistry. Herein, our Tetraplex assay detected dMMR tumors with a sensitivity of 92.7% (95% CI=80.6-97.5%) and a specificity of 97.9% (95% CI=92.8-99.4%) for pMMR tumors. With respect to tumors with dMSH6, in the CRC-validation set, Tetraplex detected dMSH6 tumors with a sensitivity of 86.7% (13 of 15 dMSH6 CRCs), which was subsequently validated in the EC test set as well (sensitivity, 75.0%; 6 of 8 dMSH6 ECs). Conclusions: Our newly optimized Tetraplex system will help offer a robust and highly sensitive assay for the identification of dMMR in solid tumors.

KW - Colorectal cancer

KW - DNA mismatch repair

KW - Endometrial cancer

KW - Hypermutated tumors

KW - Microsatellite instability

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