Learning objectives Cell-free tumor DNA for cancer monitoring Christina Lockwood, PhD, DABCC, DABMGG Department of Laboratory Medicine 1. Define circulating, cell-free tumor DNA (ctdna) 2. Understand the pre-analytic and standardization challenges associated with ctdna 3. Summarize the clinical utility of ctdna through several clinical examples Speaker has no financial disclosures Outline Defining the jargon Definitions and pre-analytic considerations for circulating tumor DNA CTC, circulating tumor cells Intact tumor cells in the bloodstream CTC cell-free or circulating DNA Clinical examples: ctdna detection, monitoring treatment response, acquired resistance cfdna, cell-free DNA DNA fragments circulating in the bloodstream ctdna, circulating tumor DNA Tumor-derived fraction of cfdna Image: Williams SCP. PNAS 2013;110:4861 Images: Nat Med 2008;14:914, Nat Biotechnol 2014;32:441 1
Timeline of discovery and recognition of the significance of cfdna in oncology Current clinical applications of cell-free DNA 1948: Mandel and Metais initial description cfdna 1977: Leon et al reported higher cfdna blood of cancer patients 1990s: multiple types of alterations in cfdna 1965: Bendich et al ctdna involved in oncogenesis 1989: Stroun et al showed mutant cfdna in plasma NIPT Somatic Images: NY Times June 11, 2015, www.choosehope.com What is a liquid biopsy? ctdna particulars CTCs cfdna ctdna cfdna concentrations vary in healthy, non-pregnant individuals 1.8 44 ng/ml Trace amounts in plasma DNA fragments released are small 180-200 bp Short plasma half life Less than 2 hours Exosomes Modified from Speicher MR and Pantel K. Nat Biotechnol 2014;32:441 2
ctdna release mechanisms from tumor cells Secretion Apoptosis Necrosis Biologic factors affect ctdna concentrations Tumor size / stage Tumor localization Tumor vascularity Tumor metastasis Mucinous features Treatment (radiation, surgery, chemotherapy) CTC lysis Modified from Diaz LA and Bardelli A. J Clin Oncol 2014;32:579 Tissue biopsy Gold standard Sample stability Archived bank +/- higher concentration of tumor cells Liquid biopsy: ctdna Less invasive Serial monitoring Preservation much less damaging Molecular proxy of overall disease Numerous pre-analytical variables can confound ctdna results Matrix Collection & processing Sample storage Sample thawing DNA isolation Storage of DNA Quantification Analysis 3
Variants come in all shapes and sizes Size (bp) Variant type Example 1 Substitution, SNV BRAF p.v600e Several types of variants are poorly (or not) detected by current methods, pipelines Size (bp) 1 Variant type Substitution, SNV <15 Small indel EGFR exon 19 deletions <15 Small indel <500 Med indel FLT3 ITD, internal tandem duplication <500 Med indel >500 Large indel Li-Fraumeni syndrome exon deletion in TP53 >500 Large indel >1000 Copy number variant MET amplification >1000 Copy number alteration Variable Structural variants eg, translocation EML4-ALK Variable Structural variants eg, translocation Different detection methods/platforms offer different advantages and limitations Outline Method Sens. Mutations Mut per assay Mut info required Real-time/digital qpcr >0.1% Known SNVs 1 Yes Targeted enriched seq >0.01% Known SNVs, indels ~100 Yes Targeted seq >0.5-1% Known SNVs, indels, structural variants, copy number variants >1000 No Definitions and pre-analytic considerations for circulating tumor DNA Clinical examples: ctdna detection, monitoring treatment response, acquired resistance Image: Williams SCP. PNAS 2013;110:4861 4
Potential clinical applications of ctdna Potential clinical applications of ctdna Detection Detection CNS tumors Resistance Heterogeneity, tumor dynamics Resistance NSCLC Heterogeneity, tumor dynamics MRD detection Tx response MRD detection Tx response CRC Frequency of cases with detectable ctdna (%) ctdna detection varies among tumor types N=163 patients Advanced disease Metastatic Primary gliomas ddpcr method Fraction of patients with detectable ctdna correlates with tumor stage Frequency of cases with detectable ctdna (%) N=223 patients Localized and metastatic disease Breast Colon Pancreas Gastroesophageal Bettegowda C, et al., Sci Transl Med 2014;226 I II III IV Tumor stage Bettegowda C, et al., Sci Transl Med 2014;226 5
Trend that ctdna concentration increases with tumor size Monitoring recurrence-free survival in mcrc: ctdna better predictor than CEA N=9 patients NSCLC stages I-IV Tumor volume measured by CT or PET-CT Deep sequencing method CAPP-Seq N=18 patients Metastatic CRC Curative surgical resection Newman AM, et al., Nat Med 2014;20:548 Diehl F, et al., Nat Med 2008;14:985 Acquired resistance to EGFR inhibitors in ctdna from mcrc patients ctdna detection in CNS tumors: cfdna concentration is higher in plasma than CSF N=24 patients All acquired resistance to EGFR blockade 96% patients Ras mutation 70 mutations present in plasma post-tki treatment None present pre-tx Bettegowda C, et al., Sci Transl Med 2014;226 Pan W, et al., Clin Chem 2015;61:514 6
ctdna detection in CNS tumors: tumor mutations more detectable in CSF CNS tumors may be more amenable to ctdna detection in CSF N=7 patients Detected ctdna in 6/7 CSF samples Detected ctdna in 3/7 (+/-1) plasma samples Both targeted NGS and ddpcr Pan W, et al., Clin Chem 2015;61:514 N=12 patients 4 GBM 8 brain mets from lung or breast primary Representative example from a patient with GBM Both targeted NGS and ddpcr De Mattos-Arruda L, et al., Nat Commun 2015;10:8839 Resistance: ctdna detection of EGFR T790M in lung cancer N=117 patients with acquired TKI resistance 117 NSCLC patients ctdna measured before, after EGFR-TKI progression (PD) Patients grouped every 2 months relative to 1 st PD Zheng D, et al., Sci Rep 2016;6:20913 7
Proof of principle: OncoPlex ctdna Tissue Comprehensive somatic sequencing assay Currently v5 contains 262 cancer-related genes Detects SNVs, indels, structural rearrangements, copy number alterations Type of mutation VAF, variant allele frequency SNV 0.29* Frameshift insertion 0.18 ctdna SNV 0.18 SNV 0.17 Pritchard CC, et al., J Mol Diagn 2014;16:56 Barriers to clinical implementation of ctdna Summary Lack of standardization regarding pre-analytic factors Specimen sampling, processing, storage ctdna generally associated with high specificity, but lower sensitivity Single target assays can not identify whether ctdna is present False negatives can have a significant clinical impact Challenging to prospectively collect plasma in clinical practice in conjunction with tumor biopsies Analytical methods and their sensitivities highly variable Proof of principle data is promising Currently lack prospective, large scale studies that establish clinical value >15 ongoing clinical trials evaluating ctdna in solid tumors with therapeutic intervention 8
ctdna: hype or hope? Acknowledgements Francis Collins at the completion of the HGP in 2006 The enthusiasm cycle for new technology John Tait Colin Pritchard Mallory Beightol Eric Konnick Steve Salipante Brian Shirts David Wu Collins F, Nature 2006;S1:9-12 9