RU2018138508A - Способы выявления рака легкого - Google Patents
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- 208000020816 lung neoplasm Diseases 0.000 title claims 23
- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims 21
- 201000005202 lung cancer Diseases 0.000 title claims 21
- 239000002773 nucleotide Substances 0.000 claims 72
- 125000003729 nucleotide group Chemical group 0.000 claims 69
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- 108020004707 nucleic acids Proteins 0.000 claims 23
- 102000039446 nucleic acids Human genes 0.000 claims 23
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- 230000003321 amplification Effects 0.000 claims 16
- 201000005243 lung squamous cell carcinoma Diseases 0.000 claims 16
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- 230000015572 biosynthetic process Effects 0.000 claims 3
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- JJWKPURADFRFRB-UHFFFAOYSA-N carbonyl sulfide Chemical compound O=C=S JJWKPURADFRFRB-UHFFFAOYSA-N 0.000 claims 2
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- 239000012634 fragment Substances 0.000 claims 2
- 208000037841 lung tumor Diseases 0.000 claims 2
- 238000002844 melting Methods 0.000 claims 2
- 230000008018 melting Effects 0.000 claims 2
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- 239000007787 solid Substances 0.000 claims 2
- 238000011477 surgical intervention Methods 0.000 claims 2
- 238000001712 DNA sequencing Methods 0.000 claims 1
- -1 nucleotide triphosphates Chemical class 0.000 claims 1
- 231100000915 pathological change Toxicity 0.000 claims 1
- 230000036285 pathological change Effects 0.000 claims 1
- 239000001226 triphosphate Substances 0.000 claims 1
- 235000011178 triphosphate Nutrition 0.000 claims 1
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Claims (70)
1. Способ определения однонуклеотидных вариантов, присутствующих в плоскоклеточной карциноме легкого, предусматривающий
получение набора ампликонов путем осуществления мультиплексной реакции амплификации в отношении нуклеиновых кислот, выделенных из образца крови или ее фракции от индивидуума с подозрением на плоскоклеточную карциному легкого, где каждый ампликон из набора ампликонов перекрывает по меньшей мере один локус однонуклеотидного варианта из набора локусов однонуклеотидных вариантов, которые, как известно, ассоциированы с раком легкого; и
определение последовательности по меньшей мере сегмента каждого ампликона из набора ампликонов, где сегмент содержит локус однонуклеотидного варианта, за счет чего осуществляется определение однонуклеотидных вариантов, присутствующих в плоскоклеточной карциноме.
2. Способ по п. 1, где плоскоклеточная карцинома представляет собой плоскоклеточную карциному стадии 1a, 1b или 2a.
3. Способ по п. 1, где плоскоклеточная карцинома представляет собой плоскоклеточную карциному стадии 1a или 1b.
4. Способ по п. 1, где индивидуум не подвергается хирургическому вмешательству.
5. Способ по п. 1, где индивидуум не подвергается биопсии.
6. Способ по п. 1, дополнительно предусматривающий введение индивидууму соединения, при этом известно, что соединение является особенно эффективным в лечении плоскоклеточной карциномы легкого c одним или более определенными однонуклеотидными вариантами.
7. Способ по п. 1, где способ дополнительно предусматривает определение частоты вариантного аллеля для каждого из однонуклеотидных вариантов на основании определения последовательности.
8. Способ по п. 7, где план лечения рака легкого идентифицируют, исходя из определений частоты вариантного аллеля.
9. Способ по п. 7, дополнительно предусматривающий введение индивидууму соединения, при этом известно, что соединение является особенно эффективным в лечении плоскоклеточной карциномы легкого, характеризующейся одним из однонуклеотидных вариантов с частотой вариабельного аллеля, превышающей частоту по меньшей мере половины других однонуклеотидных вариантов, которые были определены.
10. Способ по п. 1, где нуклеиновые кислоты выделены из опухоли индивидуума, а однонуклеотидные варианты идентифицированы в опухоли из набора локусов однонуклеотидных вариантов перед определением последовательности по меньшей мере сегмента каждого ампликона из набора ампликонов для образца крови или ее фракции.
11. Способ подтверждения диагноза рак легкого у индивидуума с подозрением на рак легкого на основании образца крови или ее фракции от индивидуума, предусматривающий
получение набора ампликонов путем осуществления мультиплексной реакции амплификации в отношении нуклеиновых кислот, выделенных из образца, где каждый ампликон из набора ампликонов перекрывает по меньшей мере один локус однонуклеотидного варианта из набора локусов однонуклеотидных вариантов, которые, как известно, ассоциированы с раком легкого; и
определение последовательности по меньшей мере сегмента каждого ампликона из набора ампликонов, где сегмент содержит локус однонуклеотидного варианта, за счет чего осуществляется определение того, присутствует ли один или более однонуклеотидных вариантов в нескольких локусах однонуклеотидных вариантов, где
a) отсутствие однонуклеотидного варианта подтверждает диагноз аденокарциномы стадии 1a, 2a или 2b,
b) присутствие однонуклеотидного варианта подтверждает диагноз плоскоклеточной карциномы или аденокарциномы стадии 2b или 3a, или
c) присутствие десяти или более однонуклеотидных вариантов подтверждает диагноз плоскоклеточной карциномы или аденокарциномы стадии 2b или 3.
12. Способ по п. 11, где способ дополнительно предусматривает определение стадии патологического изменения при раке легкого с помощью неинвазивного способа.
13. Способ выявления плоскоклеточной карциномы легкого в образце крови или ее фракции от индивидуума с подозрением на рак легкого, предусматривающий
получение набора ампликонов путем осуществления мультиплексной реакции амплификации в отношении нуклеиновых кислот, выделенных из образца, где каждый ампликон из набора ампликонов перекрывает по меньшей мере один локус однонуклеотидного варианта из набора локусов однонуклеотидных вариантов, которые, как известно, ассоциированы с раком легкого; и
определение последовательности по меньшей мере сегмента каждого ампликона из набора ампликонов, где сегмент содержит локус однонуклеотидного варианта, за счет чего осуществляется определение того, присутствует ли один или более однонуклеотидных вариантов из нескольких локусов однонуклеотидных вариантов, где присутствие однонуклеотидного варианта в образце для любого из нескольких однонуклеотидных локусов является показателем присутствия плоскоклеточной карциномы.
14. Способ выявления плоскоклеточной карциномы легкого у индивидуума, предусматривающий определение того, присутствует ли однонуклеотидный вариант в нескольких локусах однонуклеотидных вариантов, которые, как известно, ассоциированы с плоскоклеточной карциномой легкого, путем осуществления высокопроизводительного секвенирования ДНК в отношении нескольких локусов однонуклеотидной изменчивости, на основании образца крови или ее фракции от индивидуума, где присутствие однонуклеотидного варианта в образце для любого из нескольких однонуклеотидных локусов является показателем присутствия плоскоклеточной карциномы легкого.
15. Способ выявления клонального однонуклеотидного варианта в опухоли легкого у индивидуума, предусматривающий
получение набора ампликонов путем осуществления мультиплексной реакции амплификации в отношении нуклеиновых кислот, выделенных из образца, где каждый ампликон из набора ампликонов перекрывает по меньшей мере один локус однонуклеотидного варианта из набора локусов однонуклеотидных вариантов, которые, как известно, ассоциированы с раком легкого;
определение последовательности по меньшей мере сегмента каждого ампликона из набора ампликонов, где сегмент содержит локус однонуклеотидного варианта; и
определение частоты вариантного аллеля для каждого из локусов SNV, исходя из последовательности нескольких копий из серии ампликонов, где более высокая относительная частота аллеля по сравнению с другими однонуклеотидными вариантами из нескольких локусов однонуклеотидных вариантов является показателем клонального однонуклеотидного варианта в опухоли.
16. Способ по п. 15, дополнительно предусматривающий введение индивидууму соединения, которое нацеливается на один или более клональных однонуклеотидных вариантов, но не на другие однонуклеотидные варианты.
17. Способ по п. 15, где частота вариантного аллеля, превышающая 1,0%, является показателем клонального однонуклеотидного варианта.
18. Способ определения того, нужно ли выделять нуклеиновые кислоты из циркулирующих свободных нуклеиновых кислот от индивидуума с раком легкого, предусматривающий
определение того, является ли рак легкого аденокарциномой или плоскоклеточной карциномой, где, если рак легкого представляет собой плоскоклеточную карциному, у индивидуума выделяют циркулирующие свободные нуклеиновые кислоты.
19. Способ по п. 18, дополнительно предусматривающий определение стадии рака легкого, где, если рак легкого представляет собой плоскоклеточную карциному или аденокарциному стадии 3a, у индивидуума выделяют циркулирующие свободные нуклеиновые кислоты.
20. Способ по п. 19, где, если рак легкого представляет собой плоскоклеточную карциному или аденокарциному стадии 3a, у индивидуума выделяют циркулирующие свободные нуклеиновые кислоты.
21. Способ по п. 20, где, если рак легкого представляет собой стадию плоскоклеточной карциномы или аденокарциному стадии 3a, нуклеиновые кислоты не выделяют из опухоли легкого у индивидуума.
22. Способ определения однонуклеотидных вариантов, присутствующих в плоскоклеточной карциноме легкого, в образце крови или ее фракции от индивидуума с подозрением на плоскоклеточную карциному, при этом способ предусматривает
образование реакционной смеси для амплификации путем объединения полимеразы, нуклеотидтрифосфатов, фрагментов нуклеиновой кислоты из библиотеки нуклеиновых кислот, полученной на основании образца, и набора праймеров, каждый из которых связывается в пределах 150 пар оснований от локуса однонуклеотидного варианта, или набора пар праймеров, каждый из которых перекрывает участок из 160 пар оснований или меньше, содержащий локус однонуклеотидного варианта, где локусы однонуклеотидного варианта, как известно, ассоциированы с плоскоклеточной карциномой;
воздействие на реакционную смесь для амплификации с помощью условий амплификации с получением набора ампликонов, содержащих по меньшей мере один локус однонуклеотидного варианта из набора локусов однонуклеотидных вариантов, как известно, ассоциированных с раком легкого; и
определение последовательности по меньшей мере сегмента каждого ампликона из набора ампликонов, где сегмент содержит локус однонуклеотидного варианта, за счет чего осуществляется определение однонуклеотидных вариантов, присутствующих в плоскоклеточной карциноме.
23. Способ по п. 22, где плоскоклеточная карцинома представляет собой плоскоклеточную карциному стадии 1a, 1b или 2a.
24. Способ по п. 22, где плоскоклеточная карцинома представляет собой плоскоклеточную карциному стадии 1a или 1b.
25. Способ по п. 22, где индивидуум не подвергается хирургическому вмешательству.
26. Способ по п. 22, где индивидуум не подвергается биопсии.
27. Способ по п. 22, дополнительно предусматривающий введение индивидууму соединения, при этом известно, что соединение является особенно эффективным в лечении плоскоклеточной карциномы легкого c одним или более определенными однонуклеотидными вариантами.
28. Способ по п. 22, где способ дополнительно предусматривает определение частоты вариантного аллеля для каждого из однонуклеотидных вариантов на основании определения последовательности.
29. Способ по п. 22, дополнительно предусматривающий разработку плана лечения рака легкого, исходя из определений частоты вариантного аллеля.
30. Способ по п. 22, дополнительно предусматривающий введение индивидууму соединения, при этом известно, что соединение является особенно эффективным в лечении плоскоклеточной карциномы легкого, характеризующейся одним из однонуклеотидных вариантов с частотой вариабельного аллеля, превышающей частоту по меньшей мере половины других однонуклеотидных вариантов, которые были определены.
31. Способ по п. 22, где нуклеиновые кислоты выделены из опухоли индивидуума, а однонуклеотидные варианты идентифицированы в опухоли из набора локусов однонуклеотидных вариантов перед определением последовательности по меньшей мере сегмента каждого ампликона из набора ампликонов для образца крови или ее фракции.
32. Композиция, содержащая фрагменты циркулирующих опухолевых нуклеиновых кислот, содержащие универсальный адаптер, где циркулирующие опухолевые нуклеиновые кислоты происходят из опухоли, представляющей собой плоскоклеточную карциному легкого.
33. Композиция, содержащая фрагменты циркулирующих опухолевых нуклеиновых кислот, содержащих универсальный адаптер, где циркулирующие опухолевые нуклеиновые кислоты происходят из образца крови или ее фракции от индивидуума с плоскоклеточной карциномой легкого.
34. Твердая подложка, содержащая несколько клональных популяций нуклеиновых кислот, где клональные популяции содержат ампликоны, полученные из образца циркулирующих свободных нуклеиновых кислот, где циркулирующие опухолевые нуклеиновые кислоты происходят из опухоли, представляющей собой плоскоклеточную карциному легкого.
35. Твердая подложка, содержащая несколько клональных популяций нуклеиновых кислот, где клональные популяции содержат фрагменты нуклеиновой кислоты, полученные из образца циркулирующих свободных нуклеиновых кислот из образца крови или ее фракции от индивидуума с плоскоклеточной карциномой легкого.
36. Способ по п. 35, где фрагменты нуклеиновой кислоты в различных клональных популяциях содержат один и тот же универсальный адаптер.
37. Способ по п. 36, где клональные популяции нуклеиновых кислот получены из фрагментов нуклеиновой кислоты на основании набора образцов от двух или более индивидуумов.
38. Способ по п. 37, где фрагменты нуклеиновой кислоты содержат один из серии молекулярных штрихкодов, соответствующих образцу в наборе образцов.
39. Способ по любому из предыдущих пунктов, где выявляются по меньшей мере 5 SNV, и где присутствие по меньшей мере 5 SNV является показателем плоскоклеточной карциномы.
40. Способ по любому из пп. 1-31, где определение того, присутствует ли в образце однонуклеотидный вариант, предусматривает идентификацию значения достоверности для каждого определения аллеля в каждом из наборов локусов однонуклеотидной изменчивости, исходя по меньшей мере частично из глубины прочтения для локуса.
41. Способ по любому из пп. 1-31, где распознавание однонуклеотидного варианта осуществляют, если значение достоверности в отношении присутствия однонуклеотидного варианта превышает 90%.
42. Способ по любому из пп. 1-31, где распознавание однонуклеотидного варианта осуществляют, если значение достоверности в отношении присутствия однонуклеотидного варианта превышает 95%.
43. Способ по любому из пп. 1-31, где набор локусов однонуклеотидной изменчивости содержит все локусы однонуклеотидной изменчивости, идентифицированные в наборах данных TCGA и COSMIC для рака легкого.
44. Способ по любому из пп. 1-31, где набор сайтов однонуклеотидной изменчивости содержит все сайты однонуклеотидной изменчивости, идентифицированные в наборах данных TCGA и COSMIC для плоскоклеточной карциномы легкого.
45. Способ по любому из пп. 1-31, где способ осуществляют при глубине прочтения для набора локусов однонуклеотидной изменчивости, составляющей по меньшей мере 1000.
46. Способ по любому из пп. 1-31, где набор локусов однонуклеотидных вариантов содержит 25-1000 локусов однонуклеотидной изменчивости, которые, как известно, ассоциированы с раком легкого.
47. Способ по любому из пп. 1-31, где эффективность и частоту ошибок на цикл определяют для каждой реакции амплификации из мультиплексной реакции амплификации локусов однонуклеотидной изменчивости, и эффективность и частоту ошибок применяют для определения того, присутствует ли в образце однонуклеотидный вариант из набора локусов однонуклеотидных вариантов.
48. Способ по любому из пп. 1-31, где реакция амплификации представляет собой ПЦР-реакцию, а температура отжига на 1-15°C превышает температуру плавления по меньшей мере 50% праймеров из набора праймеров.
49. Способ по любому из пп. 1-31, где реакция амплификации представляет собой ПЦР-реакцию, и продолжительность стадии отжига в ПЦР-реакции составляет 15-120 минут.
50. Способ по любому из пп. 1-31, где реакция амплификации представляет собой ПЦР-реакцию, и продолжительность стадии отжига в ПЦР-реакции составляет 15-120 минут.
51. Способ по любому из пп. 1-31, где концентрация праймеров в реакции амплификации составляет 1-10 нМ.
52. Способ по любому из пп. 1-31, где праймеры в наборе праймеров разработаны для сведения к минимуму образования димеров из праймеров.
53. Способ по любому из пп. 1-31, где реакция амплификации представляет собой ПЦР-реакцию, причем температура отжига на 1-15°C превышает температуру плавления по меньшей мере 50% праймеров из набора праймеров, продолжительность стадии отжига в ПЦР-реакции составляет 15-120 минут, концентрация праймеров в реакции амплификации составляет 1-10 нМ, и праймеры в наборе праймеров разработаны для сведения к минимуму образования димеров из праймеров.
54. Способ по любому из пп. 1-31, где мультиплексную реакцию амплификации осуществляют в условиях ограничения праймеров.
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| EP3443119B1 (en) | 2022-02-23 |
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