Disclosure. Summary. Circulating DNA and NGS technology 3/27/2017. Disclosure of Relevant Financial Relationships. JS Reis-Filho, MD, PhD, FRCPath

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1 Circulating DNA and NGS technology JS Reis-Filho, MD, PhD, FRCPath Director of Experimental Pathology, Department of Pathology Affiliate Member, Human Oncology and Pathogenesis Program Disclosure of Relevant Financial Relationships USCAP requires that all planners (Education Committee) in a position to influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their spouse/partner have, or have had, within the past 12 months, which relates to the content of this educational activity and creates a conflict of interest. Disclosure I have no financial relationships to disclose Summary Circulating cell-free DNA (cfdna) Circulating tumour DNA (ctdna) Applications of ctdna detection Challenges Circulating biomarkers Massively parallel sequencing Circulating cell-free DNA (cfdna) DNA freely circulating in bodily fluids Plasma, cerebrospinal fluid, effusions Serum-based glycosylated tumour markers (CA 15-3, CEA) CTC enumeration (prognostic marker - level I) ctdna & CTC: molecular analysis Fragments are small ( base pairs; nucleosomes) Normal: approx 166 bp Tumour: approx 144 bp Not cancer-specific Healthy individuals: ~1 ng DNA/ml Inflammatory diseases, stroke, trauma, surgery, sepsis, PE Cancer patients: 4-fold greater levels Cancer patients: variable % of circulating DNA is tumour-derived Lee et al. 2001; Schwarzenbach et al

2 Circulating tumour DNA (ctdna) ctdna varies according to tumour burden and tumour type Sources of DNA release Types of DNA alterations The tumour burden and rate of tumour cell proliferation have a substantial impact! Schwarzenbach et al. Nat Reviews Cancer ctdna detected in: - 50% - 75% of patients with localized disease - > 85% of patients with metastatic disease Bettegowda et al. Sci Transl Med 2014 ctdna & somatic genetic alterations Circulating DNA: methods Technique Sensitivity Sanger sequencing 10% Massively parallel sequencing 1% - 2% Quantitative PCR 1% Fusion genes Structural variants Point mutations Indels Amplifications Deletions Ultra-high depth massively parallel sequencing (50,000x) with UMIs 0.1% BEAMing / Digital PCR / TAM-Seq <0.01% Leary et al. Sci Trans Med 2012 Bidard et al. Sci Transl Med 2013 ctdna analysis methods Method Principle Sensitivity PCR-based techniques Require prior knowledge of the genetic aberrations ARMS PNA/ LNA PCR PAP/ bi-pap Digital PCR and BEAMing Massively parallel sequencing Whole genome/ whole exome/ targeted capture PCR-amplification followed by NGS Tam-Seq Safe-Seq smmip Does not require prior knowledge of the genetic aberrations Prior knowledge required 1% - 0.1% 0.1% % 0.01% 0.01% % Sensitivity dependent on depth 0.1% for 50,000x 0.2% - 2% 0.001% 1% - 0.2% Applications of ctdna detection Bidard et al. Sci Transl Med

3 Potential uses of ctdna Diagnosis Can all mutations in a tumour be detected in cfdna? Diagnosis Prognostication Prediction Monitoring therapeutic resistance Identifying new targets Tackling intra-tumor heterogeneity Mainly tested in metastatic disease! Metastatic breast cancer patient ER+ / HER2- Phase I study PAM4743g GDC0068 pan-akt inhibitor DNA extracted from primary tumour (FFPE) liver metastasis (FFPE) peripheral blood leukocytes four plasma samples during AKT inhibitor-based therapy (4th line) Targeted massively parallel sequencing Response - RECIST criteria (1.1) Bidard et al. Sci Transl Med 2013 De Mattos-Arruda et al. Ann Oncol 2014 ctdna analysis captures the heterogeneity of primary tumour and metastasis ctdna: prognosis in metastatic breast cancer Primary tumour Plasma 2 Progression 52 women Tumors sequenced 25 with PIK3CA or TP53 mutations 9 with structural variants Liver metastasis Plasma 1 Plasma 3 Plasma sequenced 29 women had detectable ctdna The levels of ctdna correlate with outcome in metastatic breast cancer patients De Mattos-Arruda et al. Ann Oncol 2014 Dawson et al. NEJM Prognosis and Monitoring Early stage and locally advanced breast cancer Prognosis in early stage breast cancer Tumour - Diagnostic biopsy Surgery (for patients with residual disease) Plasma for extraction of cell free DNA (EDTA) Baseline Post-surgery Every 6 months in follow-up 55 patients from the ChemoNEAR study. 43 (78%) harbored a detectable mutation in cfdna. 3

4 ctdna detection post-surgery and on follow-up, but not at baseline, is prognostic Increase in ctdna predicts the development of metastatic disease 26 disease-free patients ctdna increases and metastasis Baseline Post-Surgery At follow-up Only one case displayed detectable ctdna on follow-up who did not develop a relapse Lead time of ctdna increases and metastatic disease detection: 7.9 months Tracking secondary ( acquired ) resistance Tracking resistance and identifying new targets Identification of treatment-associated mutational changes from exome sequencing of serial plasma ctdna. Murtaza et al. Nature. 2013; Tracking secondary ( acquired ) resistance Whole exome sequencing analysis of 216 metastatic breast cancers Genes significantly more frequently mutated in metastasis ESR1 mutations 11%-32% of ER+ patients treated with AI or estrogen deprived Very rare in primary ER+ cancers 0.6% of 321 luminal A or B cancers 3% of primary tumors from BOLERO-2 ESR1 mutations affect the LBD Lefebvre et al. PLoS Med 2016 Toy et al. Nat Genet 2013; Robinson et al. Nat Genet

5 Detection of ESR1 mutations in plasma ESR1 mutations are polyclonal and may help define the type of endocrine therapy Multiple ESR1 mutations can be identified in the plasma of AI treated ER breast cancer patients Patients with ESR1 mutations in ctdna are relatively resistant to AI therapy but not fulvestrant ESR1 mutant ESR1 WT Spoerke et al. Nat Commun 2016; Fribbens et al. JCO 2016 ctdna analysis as a means to combat intra-tumour genetic heterogeneity Intra-tumour heterogeneity Murtaza et al. Nat Commun 2016 Challenges Low tumour burden 5

6 Early vs. advanced breast cancer Fraction of patients with detectable ctdna in localized breast cancer (stages I to III) Hotspot mutation detection with ddpcr in early breast cancer 29 women 30 tumours 29 pre-op plasma samples PIK3CA mutations tested in tumour and plasma by ddpcr PIK3CA in cfdna E545K H1047R WT PIK3CA in tumour E545K H1047R WT Sensitivity: 93.3% (95% CI, 75.5% 93.3%) Specificity: 100% (95% CI, 82.2% 100.0%) Bettegowda et al. Sci Transl Med 2014 Beaver et al. Clin Cancer Res 2014 De novo mutation identification in cfdna from patients with early stage breast cancer Targeted massively parallel sequencing 275 genes most frequently mutated in breast cancer T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 Stage IIIA IIB IIA IIA IIIA IIIC IIIA IIA IIB IIIC IIB IIB Depth in 492x 248x 308x 250x 162x 250x 156x 511x 312x 232x 250x 268x ctdna Gene PIK3CA Mutation E545K Plasma Depth limited by the amount of cfdna 160x to 511x Take home messages ctdna: source of genetic material from cancer cells Methods for sequencing should be carefully chosen Potential use Prognostication Longitudinal monitoring somatic genomic alterations Secondary ( acquired ) resistance ESR1 gene mutations Combating intra-tumour genetic heterogeneity De novo mutation detection in early stage is challenging Reis-Filho et al, unpublished data Take Home Messages ctdna potential surrogate of tumour tissue ctdna analysis Diagnosis Prognostication Prediction Monitoring therapeutic resistance Tackling intra-tumour heterogeneity De novo mutation calling Challenging Dependent on disease burden More sensitive techniques required 6

7 cfdna is not a good source of tumour DNA from CNS lesions Frequency of cases with detectable plasma ctdna (%) Brain tumour cells shed tumour DNA into the CSF Adapted: Bettegowda C, et al. Sci Transl Med Structural rearrangements and fusion genes require initial sequencing of the tumour Leary et al. Science Translational Medicine 2010 ctdna: monitoring metastatic Breast Cancer ctdna: monitoring Metastatic Breast Cancer Monitoring Multiple Point Mutations and Structural Variants in ctdna. Dawson et al. NEJM

8 ctdna: monitoring Metastatic Breast Cancer Tracking secondary ( acquired ) resistance Comparison of ctdna, CTC, CA 15.3 to Monitor Tumour Dynamics Identification of treatment-associated mutational changes from exome sequencing of serial plasma ctdna. Dawson et al. NEJM Murtaza et al. Nature Tracking secondary ( acquired ) resistance Potential uses of ctdna Estimation of risk for metastatic relapse or disease progression Real-time monitoring of therapy response ER+HER2+ BC: MED1 & GAS6 Identification of therapeutic targets and resistance mechanisms Combating intra-tumour genetic heterogeneity Murtaza et al. Nature Circulating DNA Isolation and Quantification Isolation of cfdna: Commercial kits Manual vs automated Types of tubes EDTA vs STRECK Early vs. advanced breast cancer Fraction of patients with detectable ctdna in localized breast cancer (stages I to III) Quantitation of cfdna: Fluorescence-based methods PicoGreen staining Ultraviolet (UV) spectrometry Quantitative PCR (SYBR Green and TaqMan) No differentiation between tumour (ctdna) and normal DNA Van der Vaart and Pretorius, 2010 Bettegowda et al. Sci Transl Med

9 De novo mutation identification in cfdna from patients with early stage breast cancer Targeted massively parallel sequencing 275 genes most frequently mutated in breast cancer Depth 160x >500x 12 patients with stage II/ III breast cancer Longitudinal monitoring of the mutant alleles in ctdna and CA 15.3 Reis-Filho et al, unpublished data De Mattos-Arruda et al. Ann Oncol