Converting the Hype into Reality: Realizing the Potential of Cell-Free DNA Lucy Xu, Ph.D. Associate Director Clinical Biomarker Research Eisai Inc. Woodcliff Lake, NJ Precision LBx Summit San Diego, CA January 30 - February 1, 2017
Outline: History of cfdna cfdna as for prenatal diagnostic, cancer and other diseases How to measure cfdna? Charactistic, Origin and Structure of cfdna Functions of cfdna Conclusion 3
History of cfdna CNAPS: Circulating Nucleic Acid in Plasma and Serum
First Documentation of Presence of Cell-free DNA and RNA in the Plasma 5
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1966: Circulating DNA in SLE Patients 7
CirDNA as Intercellular Messenger Non sexual transmission Eggplant inter-varietal graft Stroun M 1962 8
1977: DNA in Serum of Cancer Patients 9
1989: cfdna has tumor DNA characteristics 1994: cfdna has tumor associated oncogene mutations 1996: cfdna has tumor associated microsatellite alterations 10
Noninvasive Prenatal Testing (NIPT) 1 fetal cells per 10 5 or 10 6 maternal cells Fetal DNA in maternal plasma: 3% in early pregnancy and 6% in late pregnancy As early as 7 weeks of gestation No carryover from one pregnancy to the next Lo YMD, Lancet 1997;350:485 7. Lo YMD, et al. Am J Hum Genet 1998;62:768 75. Lo YMD, et al. Am J Hum Genet 1999;64:218 24. 11
Utility of cfdna in NIPT: Sex determination Prenatal diagnosis Paternally inherited diseases Pregnancy-associated complication Sex-linked disorders 12
Noninvasive Prenatal Diagnosis for Down Syndrome Higher concentration of cell free fetal DNA Distinguish fetal from maternal DNA by epigenetic changes in different tissue Fetal mrna expression Count DNA molecules (digital PCR) NextGen Sequencing (100% sensitivity and 98% specificity) Introduced to clinical service in 2011 By 2015, >700,000 maternal plasma and in >50 countries were tested 13
Wong and Lo 2016 Annu. Rev. Med. 67:419-32 14
Plasma DNA Chimerism: DNA from cells with detectable genetic differences can be found in the circulation of an individual. Transplant: graft rejection Infectious disease: Plasmodium from malaria Trauma: prognosis Stroke: severity of the stroke Myocardial infraction Diabetes Inflammation SLE Rheumatoid arthritis Prenatal Diagnosis: Paternally inherited diseases Pregnancy-associated complication Sex-linked disorders 15
De Vlaminck et al. PNAS 2015 16
cfdna and Trauma/Stroke/Myocardial Infarction 17
cfdna Correlation to ONC Clinical Features: Single nucleotide variant mutation LOH Translocation Amplification (copy number aberrations) Tumor related viral DNA Epigenetic changes Diagnosis Prognosis Therapy Monitoring Patient follow-up 18
Thierry AR et al. 2016 Cancer Metastasis Review 35:347-376 19
How is cfdna Detected? Natural occurring anti-dna antibody from SLE patients Southern Blot Electrophoresis Microscopy qpcr/digital PCR, methylation-specific PCR Massive Parallel Sequencing (NGS) Mouliere F et al Mol Onc 2014 20
Characteristic of cfdna DNA of different size Double stranded or single stranded Nucleoprotein complex Macromolecular complexes Internalized in vesicles Attached to cell membrane Virtosome: DNA, RNA, lipid complex 21
Cellular Origins of cfdna (By methylation pattern Analysis) Healthy individuals: lymphoid and myeloid cells Insulin dependent diabetes: pancreatic beta cells Multiple sclerosis patients: oliogdendrocytes Cerebrovascalar or heart attack: neuronal/glial origin Pancreas cancer or pancreatitis patients: exocrine pancreatic cells Lehmann-Werman R et al 2016, PNAS CirDNA is not derived from CTC: 100 and 1000 times more genome equivalents in cirdna as compared to CTC 22
Distinct Cellular Origins of cfdna in the Blood of Cancer Patients Thierry AR et al. 2016 Cancer Metastasis Review 35:347-376 23
One Possibility of cfdna (Apoptosis) Jahr, S. et al. (2001) DNA fragments in the blood plasma of cancer patients: quantitation and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 61, 1659 1665 24
Core: 146 bp Linker: 20bp-90bp 10bp: directly interacts with histone 25
Potential Vesicular Structures of Circulating DNA >2.5kbp external membrane bound 100bp to 2.5kbp interior to the vesicle Thierry AR et al. 2016 Cancer Metastasis Review 35:347-376 26
Production of cfdna Apoptosis Necrosis Active Release Phagocytosis Exocytose Swarup and Rajeswari FEBS Letter 2007 27
Functions of cfdna: Intercellular messenger Genometastasis Immunological properties 28
Thierry AR et al. 2016 Cancer Metastasis Review 35:347-376 29
Ken Flemming and Dennis Lo During Dr. Lo s admission to the Royal Society in 2011 30