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Detection of Circulating Tumor DNA in Early- and Late-Stage

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Dan Tran

on 14 July 2014

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Transcript of Detection of Circulating Tumor DNA in Early- and Late-Stage

Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies
Science Translational Medicine
19 February 2014
Vol. 6, Issue 224

Chetan Bettegowda, MD PhD
Mark Sausen, PhD
Rebecca J. Leary, PhD
Isaac Kinde
Authors
Director of Research and Development at Personal Genome Diagnostics
Assistant Professor of Neurological Surgery at Johns Hopkins Hospital
Lab Head in Next Generation Diagnostics at Novartis Institutes for Biomedical Research

MD PhD Candidate at Johns Hopkins School of Medicine
Biomarkers
Serum-based protein biomarkers
carcinoma antigen-125 (CA-125)
carcinoembryonic antigen (CEA)
prostate-specific antigen (PSA)
I. Authors
II. Background
Serum-based protein biomarkers
Nucleic acid biomarkers
III. Results
IV. Method
V. Discussion

Overview
CTCs and ctDNA
e.g.
CEA
Colorectal cancer
Gastric, pancreatic, lung, breast, medullary thyroid cancer
Ulcerative colitis, pancreatitis, cirrhosis, COPD, Crohn’s disease, hyptothyroidism
Smoking
CA-125
Ovarian cancer
Endometrial, fallopian tube, lung, breast, gastrointestinal cancer
Endometriosis, ovarian disease, menstruation, pregnancy
Abdominal inflammation
Cirrhosis, diabetes, mellitus

PSA
Itself
Sometimes prostate cancer
DNA?
Method
Crystallographic structure of human prostate-specific antigen (green/gold cartoon) covalently attached to an substrate acyl intermediate and an glucosamine–manose disaccharide [Wikipedia]

Tiered mutation detection
cancer specific genes
exome
genome
Limitations
lack of specificity
present in healthy individuals
often no differentiation between early and advanced cancers
detection
prognosis
monitoring
Non-invasive
47%
55%
69%
82%
*percent of patients with detectable ctDNA
DNA purified from macro-dissected tumor and corresponding plasma/serum sample
Somatic mutation in tumor identified, amplified via PCR, and sequenced
pancreatic: KRAS, codons 12, 13, 59, 60, 61
colorectal: KRAS, BRAF, TP53, SMAD4, PIK3CA, APC
all others: paired end libraries generated, 100 genes commonly mutated in cancers captured
Results
KRAS
V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog [Wikipedia]
GTPase KRas
Switch
Ras family
BRAF
proto-oncogene B-Raf
protein kinase
Raf family
V-raf murine sarcoma viral oncogene homolog B1
TP53
Tumor protein p53
Suppresses tumors
P53 complexed with DNA
[Wikipedia]
Construction of a paired end library
5' linker 5' seq
5' linker 5' seq 3' seq 3' linker
3' seq 3' linker
oncogene fragment
5' seq
3' seq
clone
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
5' linker 5' seq 3' seq 3' linker
digest
concatenate
transform
Mutation in ctDNA/CTC from plasma/serum detected
Discussion
Single base substitutions, insertions, and deletions
BEAMing (beads, emulsion, amplification, magnetics)
PCR-Ligation
Safe-SeqS
Rearrangements
PARE (Paired Analysis of Rearranged Ends)
Administer EGFR blockade
panitumumab/cetuximab
Sequencing assay:
KRAS 12, 13, 59, 60, 61
NRAS 12, 13, 59, 60, 61
BRAF 599, 600
EGFR 712-721, 738, 790, 800
PIK3CA 538-549, 1039-1050

Result: 70 somatic mutations in 24 patients
34/70 in KRAS 12
1/70 in BRAF
2/24 EGFR kinase domain
15/24 NRAS/KRAS 61
Monitoring disease in advanced cancer patients
Methodological comparisons
Early detection of localized cancers
Liquid biopsies
Tracking resistance
NRAS and KRAS codon 61 (46%)
BRAF codon 600
KRAS codon 61
KRAS codon 12, 13 in primary CRCs
KRAS codon 12 (88.2%)
CNS and mucinous neoplasias
higher survival probability before stage III
Limitations
47% stage I
Unknown mutation
ctDNA + imaging
Non-specific mutations (TP53)

low ctDNA metastatic CRC > high ctDNA
Mutations defined by tumor evaluation
Asymptomatic progression/response
ctDNA vs CTCs
more ctDNA than CTC
ctDNA half life < CTC half life
point mutations vs. translocations
point mutation clinically favorable
Full transcript