OverView Circulating Nucleic Acids (CFNA) in Patients

Dave S.B. Hoon John Wayne Cancer Institute Santa Monica, CA, USA

DNA (LOH) 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, /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 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- 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