Advances in Molecular OncologyAdvances in Molecular OncologyУспехи молекулярной онкологии2313-805X2413-3787Publishing House ABV Press38910.17650/2313-805X-2021-8-4-53-60RESEARCH ARTICLESЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИLiquid biopsy of colorectal cancer: a new approach to evaluation of aberrant methylation of the SEPT9 geneЖидкостная биопсия колоректального рака: новый подход к оценке аберрантного метилирования гена SEPT9https://orcid.org/0000-0002-0297-4963BotezatuI. V.БотезатуИ. В.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

https://orcid.org/0000-0003-0614-8789KondratovaV. N.КондратоваВ. Н.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

https://orcid.org/0000-0002-7297-5240StroganovaA. M.СтрогановаА. М.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

https://orcid.org/0000-0003-3315-0817DrankoS. L.ДранкоС. Л.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

https://orcid.org/0000-0002-0190-5069LichtensteinA. V.ЛихтенштейнА. В.
Russian Federation

Anatoly Vladimirovitch Lichtenstein

24 Kashirskoe Shosse, Moscow 115478

Анатолий Владимирович Лихтенштейн

115478 Москва, Каширское шоссе, 24

alicht@mail.ruN.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of RussiaФГБУ «Национальный медицинский исследовательский центр онкологии им. Н.Н. Блохина» Минздрава России151220218453601812202118122021Copyright ©; 2021, Botezatu I.V., Kondratova V.N., Stroganova A.M., Dranko S.L., Lichtenstein A.V.Copyright ©; 2021, Ботезату И.В., Кондратова В.Н., Строганова А.М., Дранко С.Л., Лихтенштейн А.В.2021Botezatu I.V., Kondratova V.N., Stroganova A.M., Dranko S.L., Lichtenstein A.V.Ботезату И.В., Кондратова В.Н., Строганова А.М., Дранко С.Л., Лихтенштейн А.В.https://creativecommons.org/licenses/by/4.0https://umo.abvpress.ru/jour/article/view/389

Introduction. Hypermethylated CpG islands in the promoters of suppressor genes (in particular, SEPT9) are clinically significant markers of malignant growth that are widely used in liquid biopsy. Real-time polymerase chain reaction with methylation-specific primers is commonly used to quantify hypermethylated DNA. The method requires data normalization, depends on copy number variability of the calibrator genes, and is rather laborious.

The study subject is to develop an alternative qDMA method (quantitative DNA Melting Analysis).

Materials and methods. DNA samples isolated from blood plasma of healthy donors and colorectal cancer patients were analyzed by the method including: 1) asymmetric polymerase chain reaction with methylation-independent individually selected primers for the SEPT9 gene; 2) using the TaqMan probe hybridizing to two CpG dinucleotides in the amplicon; 3) post-amplification melting of probe/amplicon hybrids; 4) quantitative analysis of DNA melting.

Results. The method was tested on the SEPT9 gene in liquid biopsy of colorectal cancer. Differences in SEPT9 methylation in healthy donors (n = 41) and cancer patients (n = 39) were statistically significant (p <0.0001). Analytical sensitivity and diagnostic efficiency of qDMA were determined: AUC (area under curve) ROC– 0.812 (according to the result of 10-fold cross-validation AUC ROC – 0.801), sensitivity – 90%, specificity – 66%.

Conclusion. The proposed method for the quantitative assessment of aberrantly methylated DNA is simple, implemented in the closed-tube format, does not require normalization and usage of standard curves. The possibility of optimization through the use of a multiplex variant with simultaneous analysis of several markers is assumed.

Введение. Гиперметилированные CpG-островки в промоторах генов-супрессоров (в частности, SEPT9) – клинически значимые опухолевые маркеры, широко используемые в жидкостной биопсии. Для количественной оценки гиперметилированных ДНК обычно используют полимеразную цепную реакцию в реальном времени с метилспецифическими праймерами. Этот метод требует нормализации данных, зависит от степени вариабельности числа копий генов-калибраторов и весьма трудоемок.

Цель исследования – разработка альтернативного метода оценки аберрантного метилирования посредством количественного анализа плавления ДНК (qDMA, quantitative DNA melting analysis).

Материалы и методы. Образцы ДНК, выделенные из плазмы крови здоровых доноров и больных колоректальным раком, анализировали методом qDMA, включающим: 1) асимметричную полимеразную цепную реакцию с метилнезависимыми индивидуально подобранными праймерами к гену SEPT9; 2) использование зонда TaqMan, гибридизующегося с 2 CpG-динуклеотидами ампликона; 3) постамплификационное плавление гибридов зонд/ампликон; 4) количественный анализ плавления ДНК.

Результаты. Метод испытан на гене SEPT9 (применялся в жидкостной биопсии колоректального рака). Различия в степени метилирования SEPT9 между группами здоровых (n = 41) и больных (n = 39) людей статистически значимы (p <0,0001). Определена диагностическая эффективность qDMA: AUC ROC (area under curve – площадь под ROC-кривой) – 0,812 (после перекрестной валидизации – 0,801), чувствительность – 90 %, специфичность – 66 %.

Заключение. Предлагаемый метод количественной оценки аберрантно метилированных ДНК прост, реализуется в закрытом формате, не требует нормализации и построения стандартных кривых. Предполагается возможность его оптимизации посредством применения мультиплексного варианта с одновременным анализом нескольких маркеров.

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