Vertical Magnetic Separation of Circulating Tumor Cells and Somatic Genomic-Alteration Analysis in Lung Cancer Patients

Transcription

1 Vertical Magnetic Separation of Circulating Cells and Somatic Genomic-Alteration Analysis in Lung Cancer Patients Chang Eun Yoo 1,2#, Jong-Myeon Park 3#, Hui-Sung Moon 1,2, Je-Gun Joung 2, Dae-Soon Son 1,2, Hyo-Jeong Jeon 2, Yeon Jeong Kim 1,2, Kyung-Yeon Han 1,2, Jong- Mu Sun 4, Keunchil Park 4, Donghyun Park 1,2 *, Woong-Yang Park 2,5 * 1 Samsung Biomedical Research Institute (SBRI), Samsung Advanced Institute of Technology (SAIT), Samsung Electronics Co. Ltd., Seoul 06351, Korea 2 Samsung Genome Institute (SGI), Samsung Medical Center (SMC), Seoul 06351, Korea 3 Samsung Electronics Co., Ltd., Suwon, 16677, Korea 4 Department of Medicine, Sungkyunkwan University School of Medicine, Suwon 16416, Korea 5 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Korea # These authors contributed equally to this work * To whom correspondence should be addressed. Donghyun Park dh37.park@samsung.com Tel: Fax:

2 Woong-Yang Park Tel: Fax:

3 SUPPLEMENTARY INFORMATION Supplementary Figures S1 2 Supplementary Tables S1-4 3

4 (a) (b) (c) (d) (e) (f) Supplementary Figure S1. Photographs, dimensions, and schematic view of the modified well-plate. (a) Plate, (b) Well, (c) Insert, (d) Plate dimensions, (e) Well dimensions, (f) Schematic view of well fitted with an insert (green) 4

5 5

6 Supplementary Figure 2 Ratio profiles of copy numbers and percentage of CNAs in s and tumor samples for patient 20 (A), patient 23 (B), and patient 32 (C) 6

7 Supplementary Table S1 Histological information of patient samples and comparison of the number of s recovered by vertical magnetic separation and the CellSearch method 21. Number of s per 7.5 ml Patient # Histology Vertical Magnetic Separation CellSearch 1 ADC SQ ADC SCLC ADC ADC ADC SQ Pleomorphic ADC ADC ADC SCLC SCLC SCLC ADC 2 0 7

8 Supplementary Table S2 Histological information, the number of s collected, and sequencing performance of collected s for each patient sample Patient # Histology Specimen Candidates Collected Reads Mean Read Length (bp) Mapped Reads On Target Mean Depth Uniformity 16 NSCLC Frozen Tissue 8 8 Library Failed Base Proportion (>100 ) 17 NSCLC FFPE , , % 1, % NSCLC Frozen Tissue , , % 2, % SCLC Frozen Tissue , , % 1, % NSCLC Frozen Tissue , , % 1, % NSCLC Frozen Tissue 2 2 Library Failed 22 NSCLC Frozen Tissue , , % % NSCLC Frozen Tissue , , % % NSCLC Frozen Tissue , , % 1, % NSCLC Frozen Tissue , , % 2, % NSCLC No tumor , , % 1, % NSCLC Frozen Tissue , , % 1, % NSCLC Not Enough Tissue , , % 1, % NSCLC FFPE , , % 1, % NSCLC Frozen Tissue , , % 1, % NSCLC Frozen Tissue , , % 1, % SCLC Frozen Tissue , , % 1, % NSCLC Not Enough Tissue , , % 2, % NSCLC FFPE DNA , , % 1, % 92 8

9 35 NSCLC Frozen Tissue , , % 2, % SCLC Frozen Tissue , , % 3, % NSCLC Frozen Tissue , , % 1, % NSCLC Frozen Tissue , , % 2, % SCLC 40 NSCLC Not Enough Tissue Not Enough Tissue , , % 1, % , , % 1, % NSCLC Frozen Tissue , , % 1, % NSCLC FFPE 1 1 Library Failed 43 NSCLC Frozen Tissue , , % 2, % NSCLC No tumor NSCLC Frozen Tissue 0 0 No s 9

10 Supplementary Table S3 List of somatic, non-synonymous mutations identified by targeted sequencing in tumor samples and s, and digital PCR in s. VAF 1 and VAF 2 refer to the variant allele frequency measured by targeted sequencing and digital PCR, respectively. Patient # Sample Gene Genomic Position Amino Acid Change VAF 1(%) VAF 2 (%) Mutation Type Present in COSMIC Chr7_ L858R 17.9 COSM6224 Chr17_ T96S 12.2 Stopgain COSM10770 Chr17_ E349X 11.3 COSM Chr7_ G873R ATM Chr11_ Q3000* 4.3 N.A. b Stopgain Chr17_ V179M COSM43779 SMAD4 Chr18_ Q256L Chr7_ Chr7_ G724S M793I COSM13979 COSM NRAS CSF1R Chr1_ Chr5_ S17N Q311R COSM Chr7_ T790M 16.5 COSM6240 Chr7_ L858R 44.7 COSM SMAD4 Chr18_ G386D 22.5 COSM MET Chr7_ R988C COSM FGFR2 Chr10_ S372F FGFR2 Chr17_ Chr10_ M246V G298D COSM PIK3CA Chr3_ E545K 12.9 COSM763 10

11 FBXW7 Chr4_ R479G 22.5 COSM22967 KIT Chr4_ N665K 34.0 COSM FGFR3 Chr17_ Chr4_ R249S R399C c COSM10817 COSM Chr17_ P82L COSM ATM KRAS Chr11_ Chr12_ R3008H G12A COSM21626 COSM PIK3CA Chr3_ Chr17_ H1047R R249G COSM775 COSM ALK Chr17_ Chr2_ W91X P1191S Stopgain COSM STK11 Chr19_ Q170R c 41 CTNNB1 Chr3_ D32V 17.2 COSM5691 CDKN2A Chr9_ D74A PTEN Chr10_ F56V 34.3 COSM5257 Chr17_ E204X 49.0 COSM10804 a. Variants with the same amino acid position, but an alternative sequence in the COSMIC database b. Not available because a TaqMan probe for this mutation was not prepared c. Mutation also detected in matched WBCs 11

12 Supplementary Table S4 Sequence of PCR primers and TaqMan probes used for digital PCR Gene Genomic Position Ref/Alt Forward Primer Reverse Primer Wild Probe (VIC) Mutant Probe (FAM) Chr7_ G/A GGTGCGGAAGAGAAAGAATACCAT CTGACCTAAAGCCACCTT ACTTTGCCTTCTGC CTTTGCTTTCTGC ATM * Chr11_ C/T Chr17_ C/T CATCCAAATACACACGCAAATT GATTCACTGATTGTTAGGT CATCTTATCCGAGTGGAAG ATCTTATCCGAATGGAAG SMAD4 Chr18_ A/T AGGACAGCAGCAGAATGGATTT GGCTGCCTACTTTTTTAACTATTTAAA AAGTAGCTGGCTGACCAG TAGCTGGCAGACCAG Chr7_ G/A CACCGTGCAGATCAC TCTTTGTGTTCCCGGACATAGTC CAGATGCCCTTCG CAGATACCCTTCG NRAS Chr1_ C/T TGGTTCTGGATTAGCTGGATTGTC GTTGGAGCAGGTGGTGTTG AGTGCGCTTTTCC AGTGCGTTTTTCC CSF1R Chr5_ T/C TCCACCATGACTTTGAGGTTGAG CTTGAACTTGAGTGAGCAGAA CATCCAGGAGGTG ATCCGGGAGGTG MET Chr7_ C/T TGTTTTAAGATCTGGGCAGTGA GCCTATCCAAATGAGGAGTGTGTAC CATCGTAGCGAACTAA CATCGTAGCAAACTAA FGFR2 Chr10_ G/A TGCAGTAAATGGCTATCAGGTA CAATCTAGCGCCTGGAAGAGAAA AGGAGATTACAGCTTCCCCAGA AGGAGATTACAGCTTTCCCAGA FGFR2 Chr10_ C/T GTCACCTTGAGAACCTTGAG CCCAGCCCCACATCCA AAGAACGGCAGTAAAT AAAAGAACGACAGTAAAT FGFR3 Chr4_ C/T GTGGTGGCGGCTGTGA GGAGCCCAGGCCTTTCTTG CGCAGGCGGCAGAG CGCAGGCAGCAGAG Chr17_ G/A TGGGAAGGGACAGAAGATGACA GCCCCTGCACCAGCA CGCCGGTGTAGGAG CCGCCAGTGTAGGAG ALK Chr2_ G/A CCATGAGCAGCAGGAT GCAAATTCAACCACCAGAACATTGT ATCCCTGCCCCGGTTC AATCCCTGTCCCGGTTC STK11 Chr19_ A/G TGACGGCCTGGAGTACCT CCGGCTTGATGTCCTTGTG CAATGCCCTGGCTATG ATGCCCCGGCTATG * TaqMan probe for this mutation was not prepared 12