Validation of high-throughput sequencing-based test system for detection of microsatellite instability in colorectal cancer samples

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Abstract

Introduction. Currently in routine clinical practice, standard methods based on polymerase chain reaction (PCR) and immunohistochemical examination are used to determine microsatellite instability (MSI) and deficient mismatch repair system (dMMR). MSI identification using high-throughput sequencing (next generation sequencing, NGS) is of special interest because it allows to analyze a large number of microsatellites and simultaneously study alterations in therapeutically significant genes.
Aim. To validate of amplicon-based NGS panel for analysis of MSI and alterations in clinically significant genes in colorectal cancer.
Materials and methods. High-throughput sequencing for MSI analysis was performed on formalin-fixed paraffinembedded (FFPE) samples from patients with colorectal cancer of any stage using amplicon-based panel “Solo-test Driver” (Russia) which covers 38 genes and 39 short tandem repeats (mononucleotides). As a reference method, 5-loci (BAT25, BAT26, NR21, NR24 and NR27) PCR was used. In NGS, MSI was evaluated based on κmer distribution. Statistical analysis was performed using Cohen’s kappa (κ), Mann-Whitney u test, and Fisher’s exact test.
Results. An amplicon-based NGS panel for analysis of MSI in 39 loci and alterations in 39 genes was developed and validated on 160 archival FFPE samples of colorectal cancer. Per PCR, 42 (26.25 %) samples were MSI-positive, 118 (73.75 %) were MSI-negative. The results of PCR and NGS were concordant in 98.75 % (158/160) cases.
Conclusion. The κ coefficient was 0.97 which demonstrates high concordance of MSI analyses using PCR and the developed NGS-based assay system.

About the authors

A. A. Lebedeva

LLC OncoAtlas;
Sechenov University, Ministry of Health of Russia

Author for correspondence.
Email: lebedeva_a_a_1@staff.sechenov.ru
ORCID iD: 0000-0003-1920-5076

Alexandra Artemovna Lebedeva 

Bld. 4, 1A Leninsky Prospekt, Moscow 119049, Russia;

Bld. 2, 8 Trubetskaya St., Moscow 119991, Russia 

Russian Federation

A. N. Taraskina

LLC OncoAtlas

Email: fake@neicon.ru
ORCID iD: 0000-0003-1952-9783

 Bld. 4, 1A Leninsky Prospekt, Moscow 119049, Russia 

Russian Federation

A. I. Kavun

LLC OncoAtlas

Email: fake@neicon.ru
ORCID iD: 0000-0003-3861-5281

 Bld. 4, 1A Leninsky Prospekt, Moscow 119049, Russia 

Russian Federation

E. V. Belova

LLC OncoAtlas;
Sechenov University, Ministry of Health of Russia

Email: fake@neicon.ru
ORCID iD: 0000-0002-7701-8765

Bld. 4, 1A Leninsky Prospekt, Moscow 119049, Russia;

Bld. 2, 8 Trubetskaya St., Moscow 119991, Russia 

Russian Federation

T. V. Grigor'eva

LLC OncoAtlas;
Sechenov University, Ministry of Health of Russia

Email: fake@neicon.ru
ORCID iD: 0000-0001-9054-1481

Bld. 4, 1A Leninsky Prospekt, Moscow 119049, Russia;

Bld. 2, 8 Trubetskaya St., Moscow 119991, Russia 

Russian Federation

O. A. Kuznetsova

LLC OncoAtlas;
N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia

Email: fake@neicon.ru
ORCID iD: 0000-0001-7753-3081

Bld. 4, 1A Leninsky Prospekt, Moscow 119049, Russia;

24 Kashirskoe Shosse, Moscow 115522, Russia 

Russian Federation

D. A. Kravchuk

Moscow Multidisciplinary Clinical Center “Kommunarka”, Moscow Healthcare Department

Email: fake@neicon.ru
ORCID iD: 0000-0002-8780-6159

Bld. 3, 8 Sosenskiy Stan, Kommunarka Sosenskoye, Moscow 108814, Russia 

Russian Federation

L. D. Belyaeva

Sechenov University, Ministry of Health of Russia

Email: fake@neicon.ru
ORCID iD: 0009-0007-7177-2875

Bld. 2, 8 Trubetskaya St., Moscow 119991, Russia 

Russian Federation

V. E. Nikulin

N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia

Email: fake@neicon.ru
ORCID iD: 0000-0002-8958-9805

24 Kashirskoe Shosse, Moscow 115522, Russia 

Russian Federation

E. D. Khomenko

LLC OncoAtlas

Email: fake@neicon.ru
ORCID iD: 0009-0008-7852-7466

Bld. 4, 1A Leninsky Prospekt, Moscow 119049, Russia 

Russian Federation

V. A. Mileyko

LLC OncoAtlas;
Sechenov University, Ministry of Health of Russia

Email: fake@neicon.ru
ORCID iD: 0000-0002-0272-1747

Bld. 4, 1A Leninsky Prospekt, Moscow 119049, Russia

Bld. 2, 8 Trubetskaya St., Moscow 119991, Russia 

Russian Federation

A. A. Tryakin

N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia

Email: fake@neicon.ru
ORCID iD: 0000-0003-2245-214X

24 Kashirskoe Shosse, Moscow 115522, Russia

Russian Federation

M. Yu. Fedyanin

N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia;
Moscow Multidisciplinary Clinical Center “Kommunarka”, Moscow Healthcare Department;
N.I. Pirogov National Medical and Surgical Center, Ministry of Health of Russia

Email: fake@neicon.ru
ORCID iD: 0000-0001-5615-7806

24 Kashirskoe Shosse, Moscow 115522, Russia;

Bld. 3, 8 Sosenskiy Stan, Kommunarka Sosenskoye, Moscow 108814, Russia;

70 Nizhnyaya Pervomayskaya St., Moscow 105203, Russia 

Russian Federation

M. V. Ivanov

LLC OncoAtlas;
Sechenov University, Ministry of Health of Russia

Email: fake@neicon.ru
ORCID iD: 0000-0002-9961-0129

Bld. 4, 1A Leninsky Prospekt, Moscow 119049, Russia

Bld. 2, 8 Trubetskaya St., Moscow 119991, Russia 

Russian Federation

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