One-instrument, objective microsatellite instability analysis using high-resolution melt

Kamilla Kolding Bendixen; Sofie Forsberg-Pho; Giulia Dazio; Emeli Elisabeth Hansen; Sarah Kronborg Eriksen; Samantha Epistolio; Elisabetta Merlo; Renzo Boldorini; Tiziana Venesio; Alessandra Movilia; Cecilia Caprera; Eva Christensen Arnspang; Michael Børgesen; Ulf Bech Christensen; Milo Frattini; Rasmus Koefoed Petersen

doi:10.1371/journal.pone.0302274

One-instrument, objective microsatellite instability analysis using high-resolution melt

Kamilla Kolding Bendixen, Sofie Forsberg-Pho, Giulia Dazio, Emeli Elisabeth Hansen, Sarah Kronborg Eriksen, Samantha Epistolio, Elisabetta Merlo, Renzo Boldorini, Tiziana Venesio, Alessandra Movilia, Cecilia Caprera, Eva Christensen Arnspang, Michael Børgesen, Ulf Bech Christensen, Milo Frattini, Rasmus Koefoed Petersen^*

^*Corresponding author for this work

Research output: Contribution to journal › Journal article › Research › peer-review

Abstract

In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers.The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type.The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status.The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance.Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors.Novel technologies have been applied and concepts such as tumor mutation burden have been introduced.However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator.In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue.The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles.The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study.The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.

Original language	English
Article number	e0302274
Journal	PLOS ONE
Volume	19
Issue number	4
ISSN	1932-6203
DOIs	https://doi.org/10.1371/journal.pone.0302274
Publication status	Published - Apr 2024

Bibliographical note

Publisher Copyright:
© 2024 Bendixen et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Keywords

DNA Mismatch Repair/genetics
Female
Humans
Male
Microsatellite Instability
Microsatellite Repeats/genetics
Real-Time Polymerase Chain Reaction/methods

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10.1371/journal.pone.0302274Licence: CC BY

Cite this

Bendixen, K. K., Forsberg-Pho, S., Dazio, G., Hansen, E. E., Eriksen, S. K., Epistolio, S., Merlo, E., Boldorini, R., Venesio, T., Movilia, A., Caprera, C., Arnspang, E. C., Børgesen, M., Christensen, U. B., Frattini, M., & Petersen, R. K. (2024). One-instrument, objective microsatellite instability analysis using high-resolution melt. PLOS ONE, 19(4), Article e0302274. https://doi.org/10.1371/journal.pone.0302274

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title = "One-instrument, objective microsatellite instability analysis using high-resolution melt",

abstract = "In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers.The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type.The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status.The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance.Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors.Novel technologies have been applied and concepts such as tumor mutation burden have been introduced.However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator.In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight{\textregistered} MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue.The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles.The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study.The MicroSight{\textregistered} MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.",

keywords = "DNA Mismatch Repair/genetics, Female, Humans, Male, Microsatellite Instability, Microsatellite Repeats/genetics, Real-Time Polymerase Chain Reaction/methods",

author = "Bendixen, {Kamilla Kolding} and Sofie Forsberg-Pho and Giulia Dazio and Hansen, {Emeli Elisabeth} and Eriksen, {Sarah Kronborg} and Samantha Epistolio and Elisabetta Merlo and Renzo Boldorini and Tiziana Venesio and Alessandra Movilia and Cecilia Caprera and Arnspang, {Eva Christensen} and Michael B{\o}rgesen and Christensen, {Ulf Bech} and Milo Frattini and Petersen, {Rasmus Koefoed}",

note = "Publisher Copyright: {\textcopyright} 2024 Bendixen et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",

year = "2024",

month = apr,

doi = "10.1371/journal.pone.0302274",

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Bendixen, KK, Forsberg-Pho, S, Dazio, G, Hansen, EE, Eriksen, SK, Epistolio, S, Merlo, E, Boldorini, R, Venesio, T, Movilia, A, Caprera, C, Arnspang, EC, Børgesen, M, Christensen, UB, Frattini, M & Petersen, RK 2024, 'One-instrument, objective microsatellite instability analysis using high-resolution melt', PLOS ONE, vol. 19, no. 4, e0302274. https://doi.org/10.1371/journal.pone.0302274

TY - JOUR

T1 - One-instrument, objective microsatellite instability analysis using high-resolution melt

AU - Bendixen, Kamilla Kolding

AU - Forsberg-Pho, Sofie

AU - Dazio, Giulia

AU - Hansen, Emeli Elisabeth

AU - Eriksen, Sarah Kronborg

AU - Epistolio, Samantha

AU - Merlo, Elisabetta

AU - Boldorini, Renzo

AU - Venesio, Tiziana

AU - Movilia, Alessandra

AU - Caprera, Cecilia

AU - Arnspang, Eva Christensen

AU - Børgesen, Michael

AU - Christensen, Ulf Bech

AU - Frattini, Milo

AU - Petersen, Rasmus Koefoed

N1 - Publisher Copyright: © 2024 Bendixen et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PY - 2024/4

Y1 - 2024/4

N2 - In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers.The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type.The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status.The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance.Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors.Novel technologies have been applied and concepts such as tumor mutation burden have been introduced.However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator.In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue.The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles.The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study.The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.

AB - In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers.The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type.The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status.The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance.Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors.Novel technologies have been applied and concepts such as tumor mutation burden have been introduced.However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator.In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue.The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles.The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study.The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.

KW - DNA Mismatch Repair/genetics

KW - Female

KW - Humans

KW - Male

KW - Microsatellite Instability

KW - Microsatellite Repeats/genetics

KW - Real-Time Polymerase Chain Reaction/methods

U2 - 10.1371/journal.pone.0302274

DO - 10.1371/journal.pone.0302274

M3 - Journal article

C2 - 38662796

AN - SCOPUS:85191491605

SN - 1932-6203

VL - 19

JO - PLOS ONE

JF - PLOS ONE

IS - 4

M1 - e0302274

ER -

One-instrument, objective microsatellite instability analysis using high-resolution melt

Abstract

Bibliographical note

Keywords

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