diagnosis: Breast cancer tumor stage: Metastatic gender: Female tissue: Peripheral Blood cell type: Circulating Tumor Cells
Treatment protocol
Array CGH experiments of Circulating Tumor, Primary Tumor, or cell line genomic DNA as test samples and normal human genomic DNA as reference sample.
Extracted molecule
genomic DNA
Extraction protocol
Magnetic beads coated with EpCAM mAb are used to enrich for tumor cells and then subjected to FACS analysis using differentially labeled mAbs to distinguish tumor cells (EpCAM+) from leukocytes (CD45+) during sorting. Using this protocol, we isolated small pools of highly purified CTCs from breast cancer patients. DNA from Tumor cells sorted in 10µL of TE was amplified using the GenomePlex® Single Cell Whole Genome Amplification Kit (Sigma-Aldrich) according to manufacturer’s protocol. The approach was evaluated using breast tumor cell model systems, BT474 and MCF7, and CTCs metastatic breast cancer (MBC) patients. Microdissection and DNA extraction of archival formalin-fixed paraffin-embedded (FFPE) primary tumor were done as previously described in Waldman FM, DeVries S, Chew KL, Moore DH, 2nd, Kerlikowske K, Ljung BM. Chromosomal alterations in ductal carcinomas in situ and their in situ recurrences. J Natl Cancer Inst 2000;92(4):313-20.
Label
Cy3
Label protocol
BioPrime Array CGH Labeling System (Invitrogen) was used to enzymatically label approximately 600ng of test or reference DNA with Cyanine 3-dCTP or Cyanine 5-dCTP (Amersham), respectively.Briefly, DNA suspended in 11µL of water and 10µL of random prime mix was heated for 10mins at 100°C and snap cooled on ice. 2.5uL of 10x-dNTP mix (8µL of 100mM dATP, 8µL of 100mM dGTP, 8µL of 100mM dTTP, 4µL of 100mM dCTP, 2µL 1M Tris, 0.4µL of 0.5M EDTA, and water), 1µL of Cy3 or Cy5 labeled dCTP( 1mM) and 0.6µL Exo-Klenow (40 U/µL) were added to the denatured DNA and incubated for 12 -20 hours at 37°C. Unincorporated nucleotides were removed using a Sephadex G-50 spin-columns.
Array CGH experiments of Circulating Tumor, Primary Tumor, or cell line genomic DNA as test samples and normal human genomic DNA as reference sample.
Extracted molecule
genomic DNA
Extraction protocol
Magnetic beads coated with EpCAM mAb are used to enrich for tumor cells and then subjected to FACS analysis using differentially labeled mAbs to distinguish tumor cells (EpCAM+) from leukocytes (CD45+) during sorting. Using this protocol, we isolated small pools of highly purified CTCs from breast cancer patients. DNA from Tumor cells sorted in 10µL of TE was amplified using the GenomePlex® Single Cell Whole Genome Amplification Kit (Sigma-Aldrich) according to manufacturer’s protocol. The approach was evaluated using breast tumor cell model systems, BT474 and MCF7, and CTCs metastatic breast cancer (MBC) patients. Microdissection and DNA extraction of archival formalin-fixed paraffin-embedded (FFPE) primary tumor were done as previously described in Waldman FM, DeVries S, Chew KL, Moore DH, 2nd, Kerlikowske K, Ljung BM. Chromosomal alterations in ductal carcinomas in situ and their in situ recurrences. J Natl Cancer Inst 2000;92(4):313-20.
Label
Cy5
Label protocol
BioPrime Array CGH Labeling System (Invitrogen) was used to enzymatically label approximately 600ng of test or reference DNA with Cyanine 3-dCTP or Cyanine 5-dCTP (Amersham), respectively.Briefly, DNA suspended in 11µL of water and 10µL of random prime mix was heated for 10mins at 100°C and snap cooled on ice. 2.5uL of 10x-dNTP mix (8µL of 100mM dATP, 8µL of 100mM dGTP, 8µL of 100mM dTTP, 4µL of 100mM dCTP, 2µL 1M Tris, 0.4µL of 0.5M EDTA, and water), 1µL of Cy3 or Cy5 labeled dCTP( 1mM) and 0.6µL Exo-Klenow (40 U/µL) were added to the denatured DNA and incubated for 12 -20 hours at 37°C. Unincorporated nucleotides were removed using a Sephadex G-50 spin-columns.
Hybridization protocol
We combined and precipitated 30 µL Cy3 labeled test DNA, 30 µL Cy5 labeled reference DNA and 100 µg human Cot-1 DNA by adding 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold ethanol. We collected the precipitate by centrifugation at 14000 rpm for 45 minutes at 4°C. We the final hybridization mixture containing 7ul dH20, 14µL 20% SDS, and 49 µL or Master Mix (5 mL ultra pure formamide from Invitrogen, 1g 500000MW Dextran Sulfate, 1mL 20xSSC, 1mL H20). We denatured the hybridization mixture at 73°C for 15 min and then incubated the samples at 37°C for 2hours to allow the Cot 1 DNA to anneal to repetitive DNA sequeces on both the sample and reference DNA. We exposed our printed arrays to 260000uJoules of UV using StrataLinker and applied a rubber cement ring around each array to form a well. After air drying the rubber cement we added our combined sample/reference hybridization solution onto the array. We placed a silicone gasket around the edge of the wells and laid a clean glass microscope slide on top of the gasket. We clamped the assembly together using binder clips to create an air tight environment. The arrays were incubated or 48-68 hours at 37°C on a slowly rocking table (1rpm). After disassembling the hybridization chamber, we rinsed off the excess hybridization fluid with PN buffer (PN: 0.1 M sodium phosphate, 0.1% nonidet P40, pH 8), then washed the arrays once in a solution containing 50% formamide in 2xSSC for 15 min. at 45°C and finally in PN buffer at room temperature for 15 min. We removed the ring of rubber cement, drained excess liquid from the arrays, mounted them in a solution containing 90% glycerol, 10% PBS and 1 mM DAPI, and sealed them with a cover slip.
Scan protocol
Arrays were imaged using a custom built CCD array imaging system and analyzed using the UCSF Spot Program to calculate Cy3/Cy5 ratios.
The aCGH data from our cohort were processed by the custom program SPROC in order to automatically filter out data points with low DAPI intensity, low correlation between Cy3 and Cy5 within each spot, and low reference/DAPI signal intensity. Clones whose ratios were derived from only one of the triplicate spots or with a triplicate log2 SD >0.2 were set as missing. The tumor data was filtered for unmapped clones and poorly performing ones identified as those missing in more than 50% of the tumor samples. A second set of triplicate spots for 38 of these clones were averaged resulting in a total of 1965 clones remaining after processing. The data were mapped to the May 2004 freeze of the human DNA sequence.
Submission date
Sep 05, 2012
Last update date
Apr 01, 2018
Contact name
Mark Magbanua
Organization name
UCSF/Helen Diller Familiy Comprehensive Cancer Center
Expanded Genomic Profiling of Circulating Tumor Cells in Metastatic Breast Cancer Patients to Assess Biomarker Status and Biology Over Time (CALGB 40502 and CALGB 40503, Alliance).
