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Multi-step real time PCR coupled with HRM enables rapid mutation assessment prior to targeted re-sequencing
- 1. Multi-step real timePCR coupled with HRM enables rapid mutation assessment prior to targeted re-sequencing G. Mike Makrigiorgos, Ph.D Department of Radiation Oncology Dana Farber Cancer Institute Harvard Medical School, Boston MA
- 2. LIQUID BIOPSY INCANCER: RAPIDLY EXPANDING APPLICATIONS Exosomes miRNA; RNA; DNA OR aberrant methylation molecules Adapted from Haber and Velculescu, Cancer Discov. 2014
- 3. Girotti et al,Cancer Discovery 2016: Liquid biopsy in melanoma Circulating tumor DNA reveals patient responses to therapy recurrence Therapy (weeks) ctDNA Serum lactate dehydrogenase, LDH ctDNA as early biomarker of immuno-therapy for melanoma metastases ctDNA Therapy (weeks) Serum lactate dehydrogenase, LDH remission
- 4. INTRATUMOR HETEROGENEITY (Gerlinger etal, Swanton group, New England Journal of Medicine, 2012) Tumor-material is detectable in plasma
- 5. To increase sensitivity-specificity inccfDNA diagnostics, multiple targets need be interrogated • Murtaza et al, Nature 2013 Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA • Newman et al, Nature Med 2014 (CAPP sequencing) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage
- 6. Next Generation SequencingTechnology: revolutionizing personalized medicine and tumor biology -but good enough for detecting low-level mutations in heterogeneous tumors or plasma??
- 7. • PROBLEM 1: THE‘DEPTH’ The lower the mutant fraction the more times a sequence needs to be sampled
- 8. CURRENTLY IT ISNOT COST - EFFECTIVE TO SEQUENCE THE ENTIRE GENOME MULTIPLE TIMES e.g. HiSeq ~40 Gb/lane~4x each allele GENOME FRACTIONATION (targeted re-sequencing) IS MANDATORY FOR MUTATIONS AT LEVELS ~1% OR BELOW
- 9.
- 10. TP53 Exon 10,2% REG, 7574003 7574003, 0.025 0 0.005 0.01 0.015 0.02 0.025 0.03 7573980 7573990 7574000 7574010 7574020 7574030 7574040 nucleotide position observedvariantfrequency T C G A seq depth = 1629 2% mutation abundance TP53 Exon 10, 1% REG, 7574003 7574003, 0.008 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018 7573980 7573990 7574000 7574010 7574020 7574030 7574040 nucleotide position observedvariantfrequency T C G A seq depth = 1300 1% mutation abundance ILLUMINA HISEQ SEQUENCING from Milbury et al, Clin Chem 2012 Sequencing and sample preparation ‘noise’ NUCLEOTIDE POSITION
- 11. RECENT METHODS THATREDUCE THE SEQUENCING NOISE BY READING MANY TIMES THE SAME DNA MOLECULE • Error-suppressed deep sequencing Cancer Research 2012, Patel group, Yale University • Tagged-Amplicon Sequencing (TAm-Seq) Forshew et al, Sci Trans Med 2012 • Random barcoding of polonies, ‘Safe-Seq’ Kinde et al PNAS 2011 • Circle sequencing, Lou et al PNAS 2013 • Duplex sequencing, Schmitt et al PNAS 2012
- 12. Whichever method is chosen: •HIGH DEPTH TRANSLATES TO LOWER THROUGHPUT • VAST MAJORITY OF READS - and reagents - ARE ‘WASTED’ • OBTAINING FEW POSITIVE READS WITHIN MILLIONS OF NEGATIVE READS MAY NOT BE A RELIABLE WAY TO CLINICAL INTERVENTION
- 13. ANOTHER APPROACH: ENRICHMENT OFMUTATIONS prior to sequencing
- 14. 1. enrichment duringPCR: COLD-PCR - Li et al, Nature Med 2008 2. enrichment for large fingerprint NaME – PrO - Song et al, NAR 2016 (mutation) - Liu et al, NAR 2017 In Press (methylation)
- 15. Nuclease-assisted Mutation Enrichment usingProbe-Overlap Song et al, Nucleic Acid Res 2016 NaME-PrO Patent pending (DFCI)
- 16. • Extracted fromcrab hepatopancreas • Thermostable - optimal ~ 65oC • Digests double stranded DNA with high preference over single stranded DNA • Base mismatches inhibit enzyme • Previously used for real-time SNP detection; and gDNA ‘normalization’ DUPLEX-SPECIFIC NUCLEASE, DSN (Shagin et al, Genome Res 2002)
- 17. Double strand specific nuclease (DSN,optimal Tm ~ 65oC) TOP STRAND PROBES BOTTOM STRAND PROBES Partially overlapping probes Target region Nuclease-assisted Mutation Enrichment using Probe Overlap (NaME-PrO)
- 18. Mutant sequenceWild typesequence 4oC denature 95oC PCR NaME-PrO 65oC lower temperature, add oligos, add DSN no cutcut Song et al, NAR 2016
- 19. Single-target NaME-PrO onKRAS exon 2 0.92 0.52 4.72 83.75 0.69 0.50 0 10 20 30 40 50 60 70 80 90 Mutation Abundance (%) KRAS, p.G12V, c.35G>T NaME-PrO PCR KRAS ddPCR ddPCR mutation enrichment Wild type KRAS Mutant KRAS original mutation abundance ~0.5%
- 20. 0.0 20.0 40.0 60.0 80.0 100.0 120.0 Mutation Abundance (%) No‐DSN +DSN gDNA ctrl 9-plex-NaME50plex-NaME NaME-PrO multiplex assay (1% original mutation abundance) 0 20 40 60 80 100 120 Mutation Abundance (%) No‐ DSN +DSN
- 21. WT no mutation (numerous repeats) 0.1% C>T, 22% 0.03% C>T,22% 0.003% C>T, 21% 0.0001% C>T, 40% 0.00003% C>T, 38% Technical detection limit for NaME-PrO-Sanger: IDH1 mutation NaME-PrO followed by PCR and Sanger Sequencing 1 mutant IDH1 in 3 million WT is detectable
- 22. HM CDonor-9Donor-10 COLB-0491-2 CO LB-0587-2 CO LB-0422-2 COLB-0413-2 COLB-0419-2 CO LB-0524-2 CO LB-0548-2 COLB-0443-2 COLB-0482-2 0.0 0.1 0.2 1 2 3 4 5 6 7 MutationAbundance(%) A No-DSN Ctrl NaME-PrO Detection threshold0.05% Re-defining digital-PCR limits: Screening of cfDNA samples with or without applying NaME-PrO
- 23. HOMOGENOUS, CLOSED TUBE NaME-PrO ClinicalChemistry, July 2017
- 24.
- 25.
- 26. 0.0 0.1 1.0 10.0 100.0 1000.0 5% 1.00% 0.30%0.10% 0.03% 0.01% Mutation abundance after NaME‐PrO(%) Mutation abundance in the starting gDNA BRAF‐V600E EGFR‐T790M KRAS‐G12A MEK1‐P124L TP53‐R273H NOTCH1‐L1601P JAK2‐V617F NRAS‐Q61K FLT3‐delI836 CORRELATION TO THE ORIGINAL MUTATION ABUDANCE following NaME-PrO
- 27. Application of NaME-Prowith targeted re-sequencing Targeted PCR • Prepare library from enriched targets • Paired-end sequence analysis Deep sequencing required Targeted PCR NaME-PrO Deep sequencing NOT required without NaME-PrO
- 28. PRE-SCREENING VIA Multiplexed-PCR-HRM Clinical Chemistry,July 2017 (advanced online publication)
- 29. SAMPLE PREPARATION FORTARGETED RE-SEQUENCING 1. Multiplex‐PCR (eg ampliseq, trueseq, etc) Multiplexed HRM 4. MiSeq sequencing positive negative = STOP 3. multi‐plex anchor‐tail PCR 2. Multiplex ND‐NaME‐PrO (single‐tube homogeneous reaction) SINGLE TUBE REACTION
- 30. No NaME-PrO treatment ND-NaME-PrO WT(x4) 5% (x2) 2.5% (x2) 0.3% (x2) 1% (x2) 0.1% (x2) 20plex NaME-PrO-HRM: serial dilutions mutant into WT cfDNA – 8 mutations present Clinical Chemistry, July 2017 FLUORESCENCECHANGE HRM TEMPERATURE
- 31. MULTIPLEXED HRM -PROOF OF PRINCIPLE: one mutated target within 10 amplicons in a single tube Clinical Chemistry, July 2017
- 32. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Variant frequency No‐treatment ND‐NaME‐PrO ND‐NaME‐PrO‐ MiSeq data cfDNApt301 Patient‐301, multiplex‐HRM on 10 targets simultaneously WITHOUT NaME‐PrO ENRICHMENT WT (x4), ctDNA‐301(x2) WITH ND‐NaME‐PrO ENRICHMENT WT (x4) ctDNA‐301(x2) RAPID PRE-SCREENING FOR MUTATIONS PRIOR TO TARGETED RESEQUENCING VIA HIGH RES. MELTING MULTIPLEXED HRM MULTIPLEXED HRM Clinical Chemistry, July 2017
- 33. CONCLUSIONS • Use ofcirculating DNA as a biomarker for multiple clinical endpoints in oncology is increasing rapidly • Next generation sequencing is preferred for multiple markers • Digital PCR and qPCR are preferred for single markers • Sample preparation for NGS remains long and expensive process • Mutation enrichment can greatly simplify NGS sample preparation • NaME-PrO and COLD-PCR blend well with NGS sample preparation, or with qPCR-only detection of cfDNA biomarkers
- 34. DFCI-BWH-MGH Heather Parsons, MD KeithFlaherty, MD Ryan Sullivan, MD Harvey Mamon, MD, Ph.D Matthew Kulke, MD John Quackenbush, Ph.D Benjamin Ebert, MD, PhD Rafael Bejar, MD, PhD Contributors and Collaborators Funding: NIH / NCI IMAT Program; Bridge Foundation (DFHCC) THANK YOU FOR YOUR ATTENTION BROAD INSTITUTE Viktor Adalsteinsson Ph.D MIT Yaping Liu PhD MAKRIGIORGOS LAB Mariana Fitarelli-Kiehl, Ph.D Ioannis Ladas, Ph.D Fangyan Wu, Ph.D Ravina Ashtaputre, BA Carrie Leong, Ph.D