OverView Circulating Nucleic Acids (CFNA) in Cancer Patients
Dave S.B. Hoon John Wayne Cancer Institute Santa Monica, CA, USA
DNA Microsatellite (LOH) Mutation CpG site(s) promotor hypermethylation cfNA DNA Integrity Blood Assays RNA mRNA Cell-free nucleic acids as biomarkers in cancer patients. Schwarzenbach H, Hoon DS, Pantel K. miR Nat Rev Cancer. 2011 CFNA Direct Quantitative PCR Assays
Amplified DNA/RNA
Thermocycler CFNA Genomic Sequencing Melanoma & CFNA TIMELINE Breast Ca microsatellite Hepatocellular Ca LOH & Neuroblastoma Hepatocellular Ca methylation microsatellite LOH Pancreatic Ca KRAS mt & Lung Ca Prostate Ca microsatellite Breast Ca microsatellite Breast Ca LOH TP53 mt LOH DNA integrity
Ovarian & Lung Ca Nasopharyngeal Breast Ca Cervical Ca Cervical Ca Hepatocellular EGFR mt Ca DNA integrity methylation HPV DNA methylation Sequencing
1999 2000 2001 2003 2004 2005 2006 2007 2008 2011 2012 2013
Lung Ca Ovarian Ca Colorectal Ca Breast Ca Nasopharyngeal Colorectal Ca methylation DNA integrity Sequencing Sequencing Carcinoma KRAS mt EBV DNA
Hepatocellular & Lung Ca KRAS Ovarian Ca mt & Melanoma TP53 mt BRAF mt
Oral Ca Esophageal Ca microsatellite methylation LOH
Hepatocellular Ca HBV DNA
Prostate Ca & Melanoma methylation Word Analysis of Recent Papers
More than 50 patients, newer than >2008, Focused on three categories: MSI/LOH, Mutations/CNV, DNA methylation Trends in Cancer Types and Technological Approaches of Ongoing and Complete cfDNA Clinical Trials
Marzese D, Exp Rev Mol Diagn, 2013 Utility of CFNA as Biomarkers
Detection
Prognostic
Predictive 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 Potential Advantages and Applications of cfDNA Analysis
Lung
Marzese D, 2013 Sources of Circulating Tumor-Related DNA?
Tumors: Primary/Metastasis
Cancer Cells Programmed and Non Programmed Death
Cancer Cells Secreting
CTC in blood
Silencing: Hypermethylated CpG Islands of Promoter Region of Coding and Non-Coding Sequencing
Non-methylated mRNA
Protein Hypermethylated mRNA
X X Methylated CpG island Protein
Non-methylated CpG island LINE-1 Unmethylated-Index for Melanoma According to AJCC stage
1 P < 0.0001
0.8
0.6
1 U Index U 1 -
0.4 LINE 0.2
0
Normal Normal I II IIIp IIIm IV Skin Nevi (n = 13) (n = 11) (n = 20) (n = 23) (n= 33) (n = 14) (n = 12) Melanoma AJCC stage 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
1.0
p = 0.0009 0.9 0.8 0.7 0.6 0.5 AIM1 U and LINE1 M 0.4 (n=23) 0.3 0.2 AIM1 M or LINE1 U
Proportion Surviving Proportion 0.1 (n=12) 0.0 0 10 20 30 40 50 60 OS (mo) Analysis of Time to Progression and Overall Survival: CTC and Serum DNA Methylation Detection in Stage IV Biochemotherapy
Melanoma1 Patients (koyanagi1 Cancer Res 2006
.8 RASSF1A, RARbeta .8 CTC: 4 marker PCR N=50
P = 0.025
.6 .6 free - P = 0.009 CTC (-) & M (-) .4 .4
CTC (-) & M (-) surviving Proportion .2 .2 CTC or M (+)
CTC or M (+) Proportion progression Proportion CTC (+) &M (+) CTC (+) &M (+) 0 0 0 12 24 36 48 60 72 0 12 24 36 48 60 72
Months Months Monitoring Multiple Point Mutations and Structural Variants in cfDNA
Dawson, SJ. NEJM 2013 Circulating B-RAF V600E in Stage IV Melanoma Patients’ Sera: Biochemotherapy Patients Responses
Responders Non-responders
1 x 106 ● 1 x 106 ● ● ● ● ● ● 5 ● 5 ●
1 x 10 6 1 x 10 ●
● ●
6 ● ● ● 1 x 104 1 x 104 ● ● 3 3 ● ●
1 x 10 10 x Unit 1 x 10 Unit x 10 x Unit ● ● 1 x 102 1 x 102 ● 1 x 101 1 x 101 ●
0 ● 0 ● Pre Post Pre Post
Shinozaki M et al Clin Cancer Res 2007 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 Sundarbose et al, Diagnostics, 2013 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
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
1.00
0.80 - Patients’ status and -dCq AJCC Stage IV vs. Stage I-III breast cancer 0.60 N=102 patients
0.40 Sensitivity AUC = 0.833 0.20 p<0.0001
0.00 0.00 0.20 0.40 0.60 0.80 1.00 1-Specificity
Asaga S et al, Clin Chem, 2010 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 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 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.
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.
Individually both types of biomarkers tell a different story of patient’s cancer events occurring