ES2531994T3 - Cebadores de polinucleótidos para detectar mutaciones de PIK3CA - Google Patents
Cebadores de polinucleótidos para detectar mutaciones de PIK3CA Download PDFInfo
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- ES2531994T3 ES2531994T3 ES10158523.0T ES10158523T ES2531994T3 ES 2531994 T3 ES2531994 T3 ES 2531994T3 ES 10158523 T ES10158523 T ES 10158523T ES 2531994 T3 ES2531994 T3 ES 2531994T3
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- 230000035772 mutation Effects 0.000 title abstract description 28
- 101000605639 Homo sapiens Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Proteins 0.000 title abstract description 11
- 102100038332 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Human genes 0.000 title abstract description 11
- 102000040430 polynucleotide Human genes 0.000 title abstract 5
- 108091033319 polynucleotide Proteins 0.000 title abstract 5
- 239000002157 polynucleotide Substances 0.000 title abstract 5
- 239000000203 mixture Substances 0.000 abstract description 7
- 101150063858 Pik3ca gene Proteins 0.000 abstract description 3
- 239000012634 fragment Substances 0.000 abstract 2
- 238000009396 hybridization Methods 0.000 abstract 2
- 108020004707 nucleic acids Proteins 0.000 abstract 2
- 102000039446 nucleic acids Human genes 0.000 abstract 2
- 150000007523 nucleic acids Chemical class 0.000 abstract 2
- 230000000295 complement effect Effects 0.000 abstract 1
- 238000000034 method Methods 0.000 abstract 1
- 238000012163 sequencing technique Methods 0.000 description 10
- 241000239226 Scorpiones Species 0.000 description 9
- 238000003556 assay Methods 0.000 description 9
- 102200085789 rs121913279 Human genes 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 102200085788 rs121913279 Human genes 0.000 description 5
- 206010006187 Breast cancer Diseases 0.000 description 4
- 208000026310 Breast neoplasm Diseases 0.000 description 4
- 206010061535 Ovarian neoplasm Diseases 0.000 description 4
- 206010033128 Ovarian cancer Diseases 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 239000004114 Ammonium polyphosphate Substances 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 208000019694 serous adenocarcinoma Diseases 0.000 description 1
- 208000004548 serous cystadenocarcinoma Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
Un método de PCR para detectar la presencia o la ausencia de una mutación en el gen PIK3CA que comprende las etapas de: a) mezclar una muestra de ácido nucleico que comprende al menos un fragmento del gen PIK3CA con una secuencia de polinucleótido cebador que consiste en la SEQ ID NO. 21, donde el polinucleótido se usa como cebador y el segundo cebador corresponde a una región del fragmento de la secuencia de PIK3CA por debajo de la región a la cual el polinucleótido es complementario, y llevar a cabo una PCR con la mezcla; y b) detectar la hibridación del polinucleótido a la muestra de ácido nucleico en donde la hibridación indica la presencia de una mutación.
Description
E10158523
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Scorpions utilizable con las secuencias de cebador se muestra como las SEQ ID NO. 17 y 18. Las regiones de correspondencia entre el cebador Scorpions y las regiones diana se muestran subrayadas o destacadas de forma idéntica.
Región de exón 9
- Mutación
- Secuencia de cebador SEQ. ID. NO.
- E542K-0
- 5'-CTTTCTCCTGCTCAGTGATTTT-3' 3
- E542K-1
- 5'-CTTTCTCCTGCTCAGTGATTAT-3' 4
- E542K-2
- 5'-CTTTCTCCTGCTCAGTGATTCT-3' 5
- E542K-3
- 5'-CTTTCTCCTGCTCAGTGATTGT-3' 6
- E542K-4
- 5'-CmCTCCTGCTCAGTGATATT-3' 7
- E542K-5
- 5'-CTTTCTCCTGCTCAGTGATCTT-3' 8
- E542K-6
- 5'-CTTTCTCCTGCTCAGTGATGTT-3' 9
- E545K-0
- 5'-ACTCCATAGAAAATCTTTCTCCTGCTT-3' 10
- E545 K-1
- 5'-ACTCCATAGAAAATCTTTCTCCTGCAT-3' 11
- E545K-2
- 5'-ACTCCATAGAAAATCTTTCTCCTGCCT-3' 12
- E545K-3
- 5'-ACTCCATAGAAAATCTTTCTCCTGCGT-3' 13
- E545K-4
- 5'-ACTCCATAGAAAATCTTTCTCCTGATT-3' 14
- E545K-5
- 5'-ACTCCATAGAAAATCMCTCCTGGTT-3' 15
- E545K-6
- 5'-ACTCCATAGAAAATCTTTCTCCTGTTT-3' 16
- Scorpion de exón 9
-
imagen5 17 y 18
Región de exón 20
La región diana para las mutaciones H1047R y H1047L se muestra a continuación como la SEQ ID NO. 19 (las
10 bases mutantes se muestran entre corchetes con la variante normal en primer lugar). Los cebadores directos para las mutaciones también se muestran a continuación (SEQ ID NOS. 20 a 33). Para potenciar la especificidad de dichas reacciones, se usaron discordancias de cebador adicionales cercanas al extremo 3' (mostradas subrayadas en las secuencias de cebador). Se usaron los cebadores óptimos (H1047R-1 y H1047L-1) para los experimentos descritos. El cebador Scorpions utilizable con las secuencias de cebador se muestra como las SEQ ID NO. 34 y 35.
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Las regiones de correspondencia entre el cebador Scorpions y las regiones diana se muestran subrayadas o destacadas de forma idéntica.
- Mutación
- Secuencia de cebador SEQ. ID. NO.
- H1047R-0
- 5'-TGTTGTCCAGCCACCATGAC-3' 20
- H1047R-1
- 5'-TGTTGTCCAGCCACCATGCC-3' 21
- H1047R-2
- 5'-TGTTGTCCAGCCACCATGGC-3' 22
- H1047R-3
- 5'-TGTTGTCCAGCCACCATGTC-3' 23
- H1047R-4
- 5'-TGTTGTCCAGCCACCATAAC-3' 24
- H1047R-5
- 5'-TGTTGTCCAGCCACCATCAC-3' 25
- H1047R-6
- 5'-TGTTGTCCAGCCACCATTAC-3' 26
- H1047L-0
- 5'-TGTTGTCCAGCCACCATGAA-3' 27
- H1047L-1
- 5'-TGTTGTCCAGCCACCATGCA-3' 28
- H1047L-2
- 5'-TGTTGTCCAGCCACCATGGA-3' 29
- H1047L-3
- 5'-TGTTGTCCAGCCACCATGTA-3' 30
- H1047L-4
- 5'-TGTTGTCCAGCCACCATAAA-3' 31
- H1047L-5
- 5'-TGTTGTCCAGCCACCATCAA-3' 32
- H1047L-6
- 5'-TGTTGTCCAGCCACCATTAA-3' 33
- Scorpion de exón 20
-
imagen7 34 y 35
5
10
15
20
25
30
35
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9 fueron capaces de detectar 5 copias de ADN a una concentración del 1% con un ΔCt dentro de los valores de corte predefinidos (Tabla 1).
Tabla 1:
- ADN MUT /reacción (copias)
- ADN MUT (copias) /reacción % relativo de alelos MUT ΔCT
- H1047L
- H1047R E542K E545K ΔCt CORTE de
- imagen9
-
imagen10 imagen11 imagen12 imagen13 imagen14 imagen15 imagen16 imagen17
- 100
-
5
imagen18 5% 5,9 4,3 5,1 5,8imagen19 imagen20
- 250
-
5
imagen21 2% 7,2 6,7 7,2 6,4 H1047 12
- 500
-
5
imagen22 1% 8,3 7,6 8,4 7,0 H1047R 12
- 1000
-
5
imagen23 0,5% 9,3 9,0 10,2 8,1 E542K 9
- 5,000
-
5
imagen24 0,1% 11,5 10,5 12,1 10,1 E545K 8
Ejemplo 3
Las mezclas de líneas celulares que contiene la mutación H1047R (HCT-116) y la E545K (MCF-7) se usaron para comparar las sensibilidades relativas de los ensayos ARMS en comparación con el secuenciamiento. Ambas líneas celulares eran heterocigotas para la mutación. La secuenciación se realizó usando cebadores y condiciones de ciclación PCR como es descrito por Samuels et al[1], los ensayos ARMS y la secuenciación se realizaron a concentraciones del gen mutante de 100%, 50%, 30%, 10% y 1% de la mezcla total. Los resultados se muestran en la Figura 1 en la que los resultados bajo el título "Scorpions" muestran el aumento del número de copias de amplicón tras rondas sucesivas de PCR (los resultados obtenidos usando cebadores de control y cebadores mutantes se muestran como líneas separadas). Bajo el título de "Secuenciamiento de ADN" se muestran los resultados del secuenciamiento de la cadena inversa del gen de la mezcla. El secuenciamiento no fue capaz de detectar la presencia de mutante H1047R cuando estaba presente en menos del 50% de la mezcla total, y fue incapaz de detectar la presencia del mutante E545K cuando estaba presente en menos del 30% de la mezcla total. Los ensayos que usan los cebadores de la invención, por el contrario, fueron capaces de detectar la presencia de mutantes en una concentración del %.
Ejemplo 4
Este ensayo se aplicó a ADN extraído de tejido congelado fresco procedente de una variedad de tipos tumorales que fueron evaluados para determinar la presencia de mutaciones PIK3CA usando el ensayo ARMS/Scorpion. En total se disponía de ADN de 279 muestras de tumores. El ensayo evidenció mutaciones en 5 de 49 (10,2%) de las muestras de cáncer colorrectal, en 19 de 49 (38,7%) de las muestras de cáncer de mama, en 1 de 51 (1,9%) de las muestras de cáncer de pulmón, y en 1 de 34 (2,9%) de las muestras de melanoma. No se detectaron mutaciones en 50 muestras de cáncer de próstata ni en 46 muestras de cáncer de ovario. De las muestras colorrectales positivas para mutaciones de PIK3CA, 3 fueron H1047R, 1 fue H1047L y 1 fue E542K; de las muestras de cáncer de mama positivas para PIK3CA, 15 fueron H1047R, 1 fue H1047L y 3 fueron E545K; tanto las mutaciones en muestras de cáncer de pulmón como de melanoma positivas para mutaciones PIK3CA fueron H1047R. El secuenciamiento solo identificó 14 del total de 26 (53%) de mutaciones detectadas. El secuenciamiento detectó una mutación en un espécimen de cáncer de mama que el ensayo ARMS no estaba diseñado para detectar (c.1634 A>G; p E545G). Ésta no es una mutación nueva y ha sido descrita previamente en cánceres de mama y colorrectales [3-5].
La incidencia de mutaciones PIK3CA en las muestras analizadas fue consistente con los estudios previos, excepto para el cáncer de ovario [1, 3-9]. Las mutaciones PIK3CA han sido descritas previamente en cánceres de ovario pero se ha sugerido que puede haber una asociación con cánceres de células endometroides y claras [8, 10]. Todos los cánceres de ovario evaluados en este estudio fueron adenocarcinomas serosos, lo que puede explicar la ausencia de mutaciones PIK3CA.
El ensayo ARMS identificó significativamente más mutaciones en las muestras clínicas que las observadas mediante secuenciamiento directo. Las mezclas de línea celular confirman que este ensayo es más sensible que el secuenciamiento para detectar las mutaciones PI3KCA de interés. Es probable que la heterogeneidad de las
E10158523
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muestras clínicas que contienen tanto tejido normal como tumoral signifique que en algunos casos la incidencia de la mutación esté por debajo de la detectable por métodos de secuenciamiento, y como tal el ensayo ARMS es más adecuado para una aplicación clínica. La desventaja es que solo se detectan determinadas mutaciones específicas de ARMS. Sin embargo, en esta serie de 279 muestras solo se detectó una única mutación en el exón 9 ó 20 del gen PI3KCA para cuya detección el ensayo ARMS no estaba diseñado.
En resumen, los ejemplos demuestran que la presente invención proporciona un ensayo sensible de alta capacidad para la detección de las 4 mutaciones más comunes del gen PIK3CA. Dicho ensayo puede aplicarse a cantidades pequeñas de ADN y puede detectar niveles bajos de PIK3CA mutante en una muestra.
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- <223> Exón 9 Scorpion
- <210> 18
- 5
- <213> Exón 9 Scorpion2
- <210> 19
- 10
- <223> Regiones diana de H1047R y H1047L <210> 20
- <223> Cebador directo H1047R-0
- 15
- <210> 21
- <223> Cebador directo H1047R-1
- 20
- <210> 22 <223> Cebador directo H1047R-2
- <210> 23
- 25
- <223> Cebador directo H1047R-3
- <210> 24
- 30
- <223> Cebador directo H1047R-4 <290> 25
- <223> Cebador directo H1047R-5
- 35
- <210> 26
- <223> Cebador directo H1047R-6
- 40
- <210> 27 <223> Cebador directo de H1047L-0
- <210> 28
- 45
- <223> Cebador directo de H1047L-1
- <210> 29
- 50
- <223> Cebador directo de H1047L-3 <210> 30
- <223> Cebador directo de H1047-3
- 55
- <210> 31
- <223> Cebador directo de H1047L-4
- 60
- <210> 32 <223> Cebador directo de H1047L-5
- <210> 33
- 65
- <223> Cebador directo de H1047L-6
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Referencias:
1. 1. Samuels Y, Wang Z, Bardelli A, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004; 304(5670): 554.
2. 2. Higuchi R, Krummel B, Saiki RK. A general method of in vitro preparation and specific mutagenesis of DNA 5 fragments: study of protein and DNA interactions. Nucleic Acids Res 1988; 16(15): 7351-67.
- 3.
- 3. Wu G, Xing M, Mambo E, et al. Somatic mutation and gain of copy number of PIK3CA in human breast cancer. Breast Cancer Res 2005; 7(5): R609-16.
- 4.
- 4. Levine DA, Bogomolniy F, Yee CJ, et al. Frequent mutation of the PIK3CA gene in ovarian and breast cancers, Clin Cancer Res 2005;11(8):2875-8.
10 5. 5. Velho S, Oliveira C, Ferreira A, et al. The prevalence of PIK3CA mutations In gastric and colon cancer. Eur J Cancer 2005;41(11):1649-54.
6. 6. Lee JW, Soung YH, Kim SY, et al. PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas. Oncogene 2005; 24(8): 1477-80.
7. 7. Bachman KE, Argani P, Samuels Y, et al. The PIK3CA gene is mutated with high frequency in human breast 15 cancers. Cancer Biol Ther 2004; 3(8): 772-5.
- 8.
- 8. Campbell IG, Russell SE, Choong DY, et al. Mutation of the PIK3CA gene in ovarian and breast cancer. Cancer Res 2004; 64(21): 7678-81.
- 9.
- 9. Omholt K, Krockel D, Ringborg U, Hansson J. Mutations of PIK3CA are rare in cutaneous melanoma. Melanoma Res 2006; 11(2): 197-200.
20 10. 10.Wang Y, Helland A, Holm R, Kristensen GB, Borresen-Dale AL. PIK3CA mutations In advanced ovarian carcinomas. Hum Mutat 2005;25(3):322.
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-
imagen1
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Application Number | Priority Date | Filing Date | Title |
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GB0719034A GB2453173A (en) | 2007-09-28 | 2007-09-28 | Polynucleotide primers |
GB0719034 | 2007-09-28 |
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ES12169599.3T Active ES2620289T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores polinucleótidos para detectar mutaciones PIK3CA |
ES10158519.8T Active ES2532141T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
ES12169586.0T Active ES2622885T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores polinucleótidos para detectar mutaciones PIK3CA |
ES12169604T Active ES2531055T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
ES12169594.4T Active ES2532225T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
ES10158523.0T Active ES2531994T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
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ES12169599.3T Active ES2620289T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores polinucleótidos para detectar mutaciones PIK3CA |
ES10158519.8T Active ES2532141T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
ES12169586.0T Active ES2622885T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores polinucleótidos para detectar mutaciones PIK3CA |
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ES12169594.4T Active ES2532225T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
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US10190171B2 (en) | 2007-09-28 | 2019-01-29 | Qiagen Manchester Limited | Polynucleotide primers for detecting PIK3CA mutations |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US10190171B2 (en) | 2007-09-28 | 2019-01-29 | Qiagen Manchester Limited | Polynucleotide primers for detecting PIK3CA mutations |
US10196692B2 (en) | 2007-09-28 | 2019-02-05 | Qiagen Manchester Limited | Polynucleotide primers for detecting PIK3CA mutations |
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