Methods

Isolation and enumeration of CTC.

CTCs were isolated and enumeratedusing the CellSearch System (Veridex LLC,Raritan, NJ). Blood samples of the patients were drawninto 10 mL CellSave tubes (Veridex LLC,Raritan, NJ) containing a cellular preservative agent. The samples were maintained at room temperature and processedwithin 72 h after collection. All CTC evaluations were doneat Veridex’s La Jolla facility. The CellSearch Systemconsists of an automated sample preparation system. TheCellSearch Epithelial Cell kit was used to immunomagnetically enrichcells expressing the epithelial cell adhesion molecule.The isolated cells were then fluorescently labeled with the nucleicacid dye 4',6-diamidino-2-phenylindole and labeled monoclonalantibodies specific for leukocytes (CD45-allophycocyanin) andepithelial cells (cytokeratin 8,18,19-phycoerythrin) (1). Identificationand enumeration of CTCs was done using the CellSpotter Analyzer(Veridex LLC,Raritan, NJ), a semiautomated fluorescence microscopy systemthat permits computer-generated reconstruction of cellular images. CTCs were defined as nucleated cells lacking CD45 and expressingcytokeratin.

Nucleic acid isolation from CTC

Blood samples were collected in 10 ml EDTA Vacutainer tube (Becton Dickinson, FranklinLakes, NJ) and processed within 36 hours collection using the CellSearch system with the CellSearch Profile kit (Veridex LLC,Raritan, NJ). The isolated CTCs were dispensed into the original AutoPrep tube (Veridex LLC,Raritan, NJ). Then, the AutoPrep tube with the sample from Celltracks AutoPrep System was removed and placed into MagCellect Magnet for a 10-minute incubation. A brownish line appeared during incubation. This line was the ferrofluid containing the bound cells. With conical tube still in MagCellect Magnet liquid was aspirated off with Pasteur pipette carefully, not to disrupt the ferrofluid bound cells. The tube was removed from the magnet. Using 1 ml pipetmen, 350l of Qiagen AllPrep DNA/RNA Micro Kit Lysis Buffer (RLT Plus) was added to the ferrofluid bound cells and the mixture was vortexed for 30 seconds to lyse the cells. If clumping of ferrofluid was evident after 30-second vortex, continue to vortex in 10 second intervals until ferrofluid was in solution. The sample was centrifuged at 800 x g for 10 seconds to pellet ferrofluid and insoluble debris. The supernatant was used for RNA isolation using Qiagen RNAeasy micro kit according to manufactory’s protocol. The nucleic acid concentration was measured using Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). The mean yield was 94.3 ng, ranging from 3.8 ng to 269.8 ng.

Exon amplification, SURVEYOR digestion, and dsDNA sizing.

Exon 3 through 8 of AR was amplified with specific primers designed according to the exon sequences (2). The sequences of these primers including nested primers are shown in Table 1 of the supplemental data file. Each amplicon covered 2 exons: amplicon 1 covered exons 3 and 4, amplicon 2 covered exons 5 and 6, and amplicon 3 covered exons 7 and 8. The cDNA (21 L) was synthesized using the Oligo-dT protocol of the SuperScript First-Strand RT-PCR kit (Invitrogen, Carlsbad, CA). The PCR amplification (50 L) of these exons with 30 cycles of pre-amplification and another 30 cycles ofnested amplification was performed according to a Transgenomic standard protocol using Taq-Pro CompletePolymerase (Denville Scientific, Metuchen, NJ). The PCR products were analyzed by gel electrophoresis using 2% agarose. The SURVEYOR Nuclease digestion reaction was performed to scan for heterozygous mutations and consisted of 10 l of heteroduplexed PCR product, 1 l of 0.15 mM MgCl2, 1 l SURVEYOR Nuclease Cofactor, 1 l SURVEYOR Nuclease Enhancer W,and 1 µL SURVEYOR Nuclease W (Transgenomic, Omaha, NE). The reaction was incubated at 42°Cfor 60 min and the reaction was terminated by adding 1.0 µL of Stop Solution.The analyses of DNA fragments were carried out by high-performanceliquid chromatography under non-denaturing conditions using the Transgenomic High Sensitivity WAVE Nucleic Acid Fragment Analysis System (Transgenomic,Omaha, NE) (3). Eight l of each digested sample was loaded onto a DNASep cartridge (Transgenomic) at45°C and eluted with an acetonitrile gradient in a0.1 M triethylammonium acetate buffer (pH 7) at a constantflow rate of 0.9 mL/min. The gradient for each amplicon was predicted using Navigator Software. Eluted DNA fragments were detectedby both UV and intercalation-based fluorescence, which improves the sensitivity of the SURVEYOR Nuclease-based mutation detection methodology. An intercalator dye, which is present in Transgenomic WAVE Optimized HS StainingSolution I, was mixedwith the separated fragments post-UV detection, and the fluorescence intensity measured by the fluorescence detector with excitation at 492nm and emission at 526 nm, according to the instructions of themanufacturer.

Size Fragment Detection and sequencing.

Sample surveyor traces were compared to a Wild Type reference to determine the presence heterozygous mutations down to 5% heterozygosity. Fragment sizes were determined using the Transgenomic Sizing Standard. All amplicons were sequenced bi-directionally to identify homozygous mutations as well as confirm the heterozygous mutations as determined by SURVEYOR Nuclease digestion (4); all amplicons were bi-directionally sequenced using the ABI3730 instrument (Applied Biosystems, Foster City, CA). Sequencingused nested primers and BigDye v3.1 (Applied Biosystems, Foster City, CA). The size of each amplicon and the estimated sequence reads from these amplicons are shown in Table 2 of this supplemental data file.

Table 1: Primer sequences

Exon / Forward / Reverse
3-4 / AAGACCTGCCTGATCTGTGG / CCTGGAGTTGACATTGGTGA
5-6 / GCCATTGAGCCAGGTGTAGT / GCAATGATACGATCGAGTTCC
7-8 / CGGATGTACAGCCAGTGTGT / GGCACTGCAGAGGAGTAGTG
3-4 nest / GCTTCTGGGTGTCACTATGGA / TGAATGACAGCCATCTGGTC
5-6 nest / AGCCCGACTCCTTTGCAG / CCTTGATGTAGTTCATTCGAAGTT
7-8 nest / TTGGATGGCTCCAAATCAC / ATCTGAAAGGGGGCATGAG

Table 2: Amplicon size and estimated sequence reads from each strand

References:

  1. Allard WJ, Matera J, Miller MC, Repollet M, Connelly MC, Rao C, et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res 2004;10:6897-904.
  2. Gottlieb B, Trifiro M, Lumbroso R, Vasiliou DM, Pinsky L.The androgen receptor gene mutations database. Nucleic Acids Res 1996;24:151-4.
  3. Underhill PA, Jin L, Lin AA, Mehdi SQ, Jenkins T, Vollrath D, et al. Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography. Genome Res 1997;7:996-1005.
  4. Jänne PA, Borras AM, Kuang Y, Rogers AM, Joshi VA, Liyanage H, et al.A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. Clin Cancer Res 2006;12:751-8.

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