Assay Location Primer TaqMan probe Reference

Transcription

1 Table S1. Primers and TaqMan probes for picoliter-ddpcr Assay Location Primer TaqMan probe Reference Exon 19 deletion assay a Exon 19 GCACCATCTCACAATTGCCAG VIC-CAGAAGGTGAGAAAGTT-MGB Reference probe Original CACAGCAAAGCAGAAACTCACA FAM-ATGTTGCTTCTCTTAATTCC-MGB L858R mutation assay Exon 21 ACTTTGCCTCCTTCTGCATGG VIC-AGTTTGGCCAGCCCAA-MGB Mutant probe 17 CTACTTGGAGGACCGTCGC FAM-AGTTTGGCCCGCCCAA-MGB T790M mutation assay Exon 21 CGCCTGCTGGGCATCTG VIC-ATGAGCTGCGTGATGAG-MGB Mutant probe 17 GTCTTTGTGTTCCCGGACATAGT FAM-ATGAGCTGCATGATGAG-MGB a This assay was designed to detect 34 representative exon 19 deletions covering >95% of deletion mutations in LADC: ΔK745_E749, K745_T751>K, ΔE746_A750, E746_R748>E, E746_A750>IP, E746_T751>IP, E746_T751>I, E746_T751>V, E746_T751>A, E746_T751>VA, E746_S752>I, E746_751T>E, E746_S752>A, E746_S752>V, E746_P753>VS, E746_A755>E, ΔE746_T751, ΔE746_S752, E746_S752>D, ΔE746_S753, ΔL747_E749, ΔL747_A750, L747_A750>P, ΔL747_T751, L747_T751>Q, L747_T751>P, L747_T751>S, ΔL747_S752, L747_S752>Q, ΔL747_P753, L747_P753>V, L747_P753>S, L747_P753>Q, and S752-I 759del.

2 Table S2. EGFR-TKI sensitive and T790M-mutant DNAs detected by picoliter-ddpcr TKI-sensitive mutation assay T790M mutation assay Patient no. Pre or post TKI therapy Number of times All EGFR-WT No. of drops 19DEL or L858R Judgment TKI-sensitive mutation fraction (%) No. of drops All EGFR-WT T790M Judgment T790M mutation fraction (%) Patients resistant to EGFR-TKI therapy 1 pre #1 5,499, Positive ,772,087 1,322 2 Negative - post #1 5,801, Positive ,060, Positive #2 5,206, Positive ,419, Positive #3 5,529, Positive ,915, Positive post #1 5,426, Positive ,119, Positive 9.21 #2 5,079, Positive ,213, Positive 9.92 #3 5,090, Positive ,335, Positive post #1 6,630,426 1, Positive ,477,646 2, Positive 0.89 #2 6,066,868 1, Positive ,764,432 2, Positive 0.87 #3 5,153, Positive ,933,533 2, Positive post #1 5,277, Positive ,933, Positive post #1 6,054, Positive ,698,203 1, Positive post #1 6,198, Negative - 5,300, Negative - 7 post #1 5,824, Positive 3.4 6,692, Negative - 8 pre #1 5,861, Negative - 6,050, Negative - post #1 6,195, Positive ,515, Negative - 9 post #1 5,091, Positive ,941, Negative - 10 pre #1 5,716, Negative - 2,784, Negative - post #1 5,651, Positive 9.3 6,126, Negative - 11 pre #1 5,129, Positive ,238, Negative - post #1 6,897, Positive ,698, Negative - 12 pre #1 5,376, Positive ,184, Negative - post #1 3,714, Positive ,346, Negative - 13 post #1 5,617,971 1, Positive ,035,421 1,405 6 Negative - #2 6,240,017 1, Positive ,674,924 1,120 5 Negative - #3 4,979, Negative - 2,628, Negative - 14 post #1 4,222, Positive 5.8 4,236,300 1, Positive post #1 5,472, Positive ,448,473 1, Positive post #1 5,324, Positive ,823, Negative - Patients not resistant to EGFR-TKI therapy 17 pre #1 5,311, Negative - 5,626, Negative - #2 4,973, Negative - 4,736, Negative - #3 4,680, Negative - 4,765, Negative - 18 pre #1 5,969, Negative - 5,254, Negative - 19 pre #1 5,599, Negative - 4,946, Negative - 20 pre #1 5,942, Negative - 5,400, Negative - 21 pre #1 5,463, Negative - 5,151, Negative - 22 pre #1 3,004, Positive ,557, Negative - 23 pre #1 4,945, Positive ,458, Negative - 24 pre #1 4,524, Negative - 5,166, Negative - 25 pre #1 5,262, Negative - 5,166, Negative - 26 pre #1 6,006, Positive ,727, Negative - 27 pre #1 5,135, Negative - 5,491, Negative - 28 pre #1 4,808, Negative - 5,258, Negative - 29 pre #1 5,655, Negative - 5,291, Negative - 30 pre #1 5,092, Negative - 2,554, Negative -

3 31 pre #1 4,867, Negative - 1,244, Negative - 32 pre #2 3,858, Positive ,825, Negative - 33 pre #3 4,789, Negative - 4,789, Negative - 34 pre #4 6,058, Positive ,287, Negative - 35 pre #1 5,665, Positive ,143, Negative -

4 Table S3. Modes of disease progression and cfdna mutations Patient no. Modes of disease progression following EGFR-TKI therapy cfdna mutations Progression of tumors Appearance of new disease sites Sensitive mut (%) T790M mut (%) T790M fraction 1 Primary disease Malignant pericarditis Primary disease Brain metastasis Primary diseases and liver metastasis Malignant pleuritis, lymph node Brain metastasis Primary diseases and liver metastasis Liver metastasis Primary disease - Negative Negative - 7 Primary diseases Liver metastasis 3.4 Negative - 8 Malignant pleuritis Negative - 9 Primary diseases Malignant pleuritis 10.3 Negative - 10 Primary diseases and malignant pleuritis Negative - 11 Primary diseases Negative - 12 Primary diseases Brain metastases 69.3 Negative - 13 Malignant pleuritis Negative - 14 Liver and spinal metastases Primary diseases Bone metastases Primary diseases and spinal metastasis Negative -

5 Table S4. Pretreatment disease sites and cfdna mutations Patient no. cfdna mutations Pretreatment disease sites Sensitive mut (%) T790M mut (%) T790M fraction 1 Primary disease, pulmonary dissemination, and brain metastases 5.8 Negative - 8 Primary disease, lung metastasis, and malignant pleuritis Negative Negative - 10 Primary disease, malignant pleuritis and bone metastasis Negative Negative - 11 Primary disease, pulmonary dissemination, bone and brain metastases 3.2 Negative - 12 Primary disease, bone and liver metastases 60.6 Negative - 17 Malignant pleuritis Negative Negative - 18 Primary disease and malignant pleuritis Negative Negative - 19 Mediastinal lymph node metastases Negative Negative - 20 Mediastinal lymph node metastases Negative Negative - 21 Pulmonary dissemination Negative Negative - 22 Primary disease, malignant pleuritis and right breast metastasis 3.2 Negative - 23 Lung, liver, bone and lymph node metastases 21.4 Negative - 24 Brain metastasis Negative Negative - 25 Primary disease Negative Negative - 26 Mediastinal lymph node metastases 14.6 Negative - 27 Pulmonary dissemination and mediastinal lymph node metastases Negative Negative - 28 Brain, bone and lymph node metastases Negative Negative - 29 Mediastinal lymph node metastases Negative Negative - 30 Pulmonary dissemination and bone metastases Negative Negative - 31 Primary disease, bone and liver metastases Negative Negative - 32 Primary disease, malignant pleuritis, liver and brain metastases 17.8 Negative - 33 Mediastinal lymph node and adrenal metastases 1.3 Negative - 34 Primary disease, liver, bone and distant lymph node metastases 68.1 Negative - 35 Primary disease and brain metastases 0.8 Negative - -: not calculated due to lack of sensitive/resistant mutations.