OverView Circulating Nucleic Acids (CFNA) in Cancer Patients. Dave S.B. Hoon John Wayne Cancer Institute Santa Monica, CA, USA
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1 OverView Circulating Nucleic Acids (CFNA) in Cancer Patients Dave S.B. Hoon John Wayne Cancer Institute Santa Monica, CA, USA
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3 cfna Blood Assays Cell-free nucleic acids as biomarkers in cancer patients. Schwarzenbach H, Hoon DS, Pantel K. Nat Rev Cancer DNA Microsatellite (LOH) Mutation CpG site(s) promotor hypermethylation DNA Integrity mrna mir RNA
4 CFNA Direct Quantitative PCR Assays Amplified DNA/RNA Thermocycler
5 CFNA Genomic Sequencing
6 Melanoma & Breast Ca microsatellite LOH Pancreatic Ca KRAS mt & Breast Ca TP53 mt Lung Ca microsatellite LOH CFNA TIMELINE Hepatocellular Ca microsatellite LOH Breast Ca DNA integrity Hepatocellular Ca & Neuroblastoma methylation Prostate Ca microsatellite LOH Breast Ca methylation Cervical Ca HPV DNA Ovarian & Cervical Ca methylation Lung Ca EGFR mt Nasopharyngeal Ca DNA integrity Hepatocellular Sequencing Nasopharyngeal Carcinoma EBV DNA Colorectal Ca KRAS mt Lung Ca methylation Ovarian Ca DNA integrity Colorectal Ca Sequencing Breast Ca Sequencing Hepatocellular & Ovarian Ca TP53 mt Lung Ca KRAS mt & Melanoma BRAF mt Oral Ca microsatellite LOH Esophageal Ca methylation Hepatocellular Ca HBV DNA Prostate Ca & Melanoma methylation
7 Word Analysis of Recent Papers More than 50 patients, newer than >2008, Focused on three categories: MSI/LOH, Mutations/CNV, DNA methylation
8 Trends in Cancer Types and Technological Approaches of Ongoing and Complete cfdna Clinical Trials Marzese D, Exp Rev Mol Diagn, 2013
9 Utility of CFNA as Biomarkers Detection Prognostic Predictive
10 CFNA Utility Different forms of genetic/epigenetic biomarkers can be detected in serum/plasma Have clinic utility as single or multiple biomarkers involving different forms Identify early disease recurrence Used to monitor cancer patients during treatment
11 Potential Advantages and Applications of cfdna Analysis Lung Marzese D, 2013
12 Sources of Circulating Tumor-Related DNA? Tumors: Primary/Metastasis Cancer Cells Programmed and Non Programmed Death Cancer Cells Secreting CTC in blood
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14 Silencing: Hypermethylated CpG Islands of Promoter Region of Coding and Non-Coding Sequencing Non-methylated mrna Hypermethylated Protein mrna X Methylated CpG island X Protein Non-methylated CpG island
15 LINE-1 U Index LINE-1 Unmethylated-Index for Melanoma According to AJCC stage 1 P < Normal Skin (n = 14) Normal Nevi (n = 12) I (n = 13) II (n = 11) IIIp (n = 20) Melanoma AJCC stage IIIm (n = 23) IV (n= 33)
16 CFNA Analysis of Overall Survival of Stage IV Melanoma Patients: LINE1 Unmethylated and/or AIM1 Methylated CFDNA vs. Methylated LINE-1 and Unmethylated AIM1 CFDNA in Serum Proportion Surviving p = AIM1 U and LINE1 M (n=23) AIM1 M or LINE1 U (n=12) OS (mo)
17 Proportion progression-free Proportion surviving Analysis of Time to Progression and Overall Survival: CTC and Serum DNA Methylation Detection in Stage IV Biochemotherapy Melanoma Patients (koyanagi Cancer Res RASSF1A, RARbeta CTC: 4 marker PCR N= P = P = CTC (-) & M (-).2 CTC (-) & M (-) CTC or M (+).2 CTC or M (+) 0 CTC (+) &M (+) 0 CTC (+) &M (+) Months Months
18 Monitoring Multiple Point Mutations and Structural Variants in cfdna Dawson, SJ. NEJM 2013
19 Circulating B-RAF V600E in Stage IV Melanoma Patients Sera: Biochemotherapy Patients Responses Responders Non-responders Unit x x x x x x x 10 1 Unit x x x x x x x Pre Post 0 Pre Post Shinozaki M et al Clin Cancer
20 Analysis of Acquired Resistance to Cancer Therapy by cfdna Sequencing Mutations showing evidence of genomic tumor evolution Initial allele fractions (Anchor mutations) used for initial cfdna screening decreassing and tumor burden increasing during therapy. Murtaza, M. Nature 2013
21 Sundarbose et al, Diagnostics, 2013
22 Advantages of mirs for Blood Assays Low degradation rate (mrna degrades rapidly) Stable at room temperature Does not require special blood handling logistics: limited volume Functional targets of tumor-related genes Disadvantages of mirs for Blood Assays Specificity and robustness of assays Normal healthy donors or other disease effects Cut-off quantification values; standardization Isolation/detection processes; robustness
23 Sensitivity Identification of Circulating mir-21 in Breast Cancer by RT-qPCR-DS(Direct Serum Assay) of Circulating mir-21 AJCC Stage IV vs. Stage I, II, or III Breast Cancer Patients Patients status and -dc q AJCC Stage IV vs. Stage I-III breast cancer N=102 patients AUC = p< Specificity Asaga S et al, Clin Chem, 2010
24 Detection of Chromosomal Alterations in cfdna of Cancer Patients by Whole-Genome Sequencing Detection of Chromosomal aberrations in all the cancer patients Leary, R. Sci Transl Med 2012
25 cfdna Clearance After HepatoCellular Carcinoma Surgery Tumor Tissue Pre Surgery cfdna Post Surgery cfdna Copy number aberrations detected in the tumor tissue sample (inner ring), presurgery plasma sample (middle ring), and postsurgery plasma sample (outer ring) for a HCC case Chan, A. Clin Chem 2013
26 Issues of CFNA That Need to be Addressed Degradation and Half-life of CFNA in blood Isolation of CFNA: tedious process and losses Quantification of CFNA after extraction; how much is put into each assay, robustness, reproducibility, standardization Sensitivity and specificity of assays: certain CFNA types better than others Regardless of how interesting CFNA are they must follow standard cancer biomarker validation regulatory requirements for clinical approval. Competition with other biomarkers; analytes, proteins, etc.
27 CFNA vs CTC Utility CTC represents a detection of a realtime metastasis event occurring. CFNA does not CFNA detection occurs at any stage; CTC very limited in earlier tumor stages. Tumor volume often relates to CFNA levels whereas CTC does not. Both differ in utility relative to cancer type and natural disease history CFNA analysis requires far less amount of blood and less logistic problems in multicenter trials.
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