Urinary ctDNA Platform for Diagnosis and Cancer … ctDNA Platform for Diagnosis and Cancer...
Transcript of Urinary ctDNA Platform for Diagnosis and Cancer … ctDNA Platform for Diagnosis and Cancer...
Urinary ctDNA Platform for Diagnosis and Cancer Treatment Monitoring
Mark G. Erlander, Ph.D., CSO CHI Next Generation Summit August 19,2015
Circulating Tumor DNA (ctDNA)
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Main Advantages of ctDNA
• Captures intra- and inter-tumor heterogeneity
• Systemic overview of cancer
• Frequent sampling options for monitoring applications
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Tumor cells
Blood stream
Trovagene Precision Cancer Monitoring (PCM ) Platform
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Ultra-Sensitive Detection and Quantitative Monitoring
>100-fold enrichment
~0.7µg ctDNA/ 100 mL of urine
Integration with Commercially Available Platforms
Single Molecule Detection
Proprietary error rate detection and normalization. ctDNA 4.9 ng/ul gDNA 0.04 ng/ul
ctDNA 2.0 ng/ul gDNA 1.4 ng/ul
Extraction and Isolation of ctDNA Mutant Allele Enrichment Platform Agnostic detection Detection and
Quantification
59%
92%
SM
Urine ctDNA Extraction
2.0 ng/ul
gDNA 1.4 ng/ul
Isolation without enrichment Isolation with enrichment
DNA extraction method enriches for short, fragmented DNA
Larger proportion of high MW DNA
Less high MW DNA
Higher proportion of small DNA
Smaller proportion of low MW DNA
2.9 ng/ul gDNA 0.07 ng/ul
bp bp
o Automated magnetic bead-based urinary ctDNA isolation method being implemented in CLIA o Magnetic bead-based urinary ctDNA isolation kit in development
Urine Contains Approximately 10 Times the Amount of cfDNA as Compared to Plasma
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Total ng DNA per sample* Total number of mutant KRAS copies per sample**
urine plasma urine plasma
Median 989.5 61.6 317.4 27.6
10th Percentile 177.2 25.0 18.8 1.5
90th Percentile 4411.0 752.4 27,789 197.8
Number of samples 58 43 34 23
* Urine and plasma samples from Stage IV colorectal cancer patients at any time point on treatment ** Samples with detectable KRAS
Proprietary Mutant Allele Enrichment Method
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Platform independent Works with ddPCR or sequencing
Sample type agnostic Works on tissue, blood and urine
Non-allele specific technology, specific to hotspot of interest
TROVAGENE TECHNOLOGY Mutants
Blocker
Wild Type
NGS Provides Generic Assay Design for Detection and Quantitation
o Kinetically-driven PCR followed by NGS (MiSeq).
o The enrichment PCR assay utilizes a 31-46 bp footprint and selectively amplifies mutant DNA fragments.
o Following sequencing on MiSeq, a proprietary analysis algorithm allows accurate quantitation of input level of mutant DNA. Results are standardized by reporting number of copies detected per 105 genome equivalents (GE).
o Enables unsupervised query for any mutation, eliminating the need for the creation of individual assays for specific mutations.
31bp product
CS1 - TS1
TS2 - CS2
Gene target
PE1 – CS1
CS2- BC- PE2
TS = target sequence CS = common sequence
PE = paired end BC = barcode
Round 1
Round 2
Sequencing
Examples of Standard Curves for Quantitation EGFR and KRAS Assays
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• Examples of master standard curve for EGFR Ex19 del and KRAS G12D
• 144 independent enrichments reactions/curve; spiked DNA input = 0-500 copies
• Accurate quantitation of absolute number of mutant DNA molecules in plasma or urine sample
ACCURATE DISEASE MONITORING WITH ANY TYPE OF THERAPIES
Unlike other NGS tests, PCM platforms counts absolute number of mutant DNA molecules in plasma or urine and not a ratio of mutant/wild-type DNA
EGFR Ex19 del KRAS G12D
Clinical Utility of ctDNA technology
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Detection Minimal Residual Disease (MRD) Quantitate Drug Response Monitoring
Tissue Biopsy Imaging (every 6-8 weeks) Surgery Adjuvant Therapy Therapy
• Monitoring for Progression and Emergence of Resistance
• Molecular Detection of Clinically Actionable Mutations
• Monitoring for Minimal Residual Disease
• Tumor Recurrence
• Week 1: Assess Tumor Cell Kill by Therapy
• Beyond Week 1: Monitor Tumor Mutation Burden
TRO
VAG
ENE
C
LIN
ICA
L U
TILT
Y TR
OVA
GEN
E PC
M
• Earlier Detection of Metastatic Disease
• Alternative to Tissue Biopsy • Right Patients Treated with
Right Therapy • Eliminates Patient Morbidity
due to Tissue Biopsy Procedure
• Anticipates and Enables Next Therapy to Target Tumor Resistance
• Immediate Assessment of Drug Effect on Tumor
• Predict Best Response Weeks in Advance of Imaging
• Enables Early Switch from Ineffective Therapy
Current Cancer Standard of Care
Trovagene PCM adds information that imaging does not currently provide at much earlier time points.
Prospective Blinded Study of BRAF V600E Mutation Detection in Cell-Free DNA of Patients with Systemic Histiocytic Disorders1
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Histiocytic Patients N=30
Urine ctDNA BRAF V600E
V600E Positive V600E Negative
Evaluate Tissue Biopsy BRAF V600E Concordance
Evaluate Plasma
BRAF V600E Concordance
Evaluate Tissue Biopsy
BRAF WT Concordance
Evaluate Plasma
BRAF WT Concordance
Background Objectives
Study Design
• Erdheim Chester disease (ECD) and Langerhans Cell Histiocytosis (LCH) are histiocytic diseases
• Histiocytic diseases harbor somatic mutations (i.e., BRAF V600E)
• Patients with BRAF V600E mutation respond to BRAF inhibitors
• Histiocytic tissue technically challenging for tissue biopsy
• Concordance:
− Tissue biopsy (gold standard) vs. urine BRAF ctDNA
− Urine vs. plasma BRAF cfDNA
• Longitudinal:
− Urine ctDNA BRAF status during treatment regimen
• Urine and Plasma from patients
• Blinded study: tissue biopsies (CLIA) correlated after ctDNA results
• Pre-specified cutpoint
1Hyman et al., Cancer Discov. 2015 Jan;5(1):64-71
ECD/LCH Tissue Biopsy First Line Treatment
Imaging (every 2-3 months)
Histiocytic Disorders: ECD/LCH Standard of Care
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Monitoring Therapeutic Response
BRAF V600E
Detection
BRAF V600E
TRO
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ECD/LCH ALL STAGES
• Molecular diagnosis of BRAF mutation – patients are placed on targeted therapy • Re-staging of cancer
CLI
NIC
AL Y
TILI
TY
MSKCC, MDACC, NIH (30 patients)
STU
DIE
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Urinary ctDNA Outperforms Tissue Biopsies1
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1Hyman et al., Cancer Discovery 2015 Jan;5(1):64-71
Urinary ctDNA N=30
Initial Tissue Results
Plasma ctDNA N=19
Initial Tissue Results Tr
ovag
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Ass
ay R
esul
ts
Trov
agen
e A
ssay
Res
ults
Wildtype Mutant Unknown On BRAF inhibitor
p=0.005 p=0.02
Wildtype Mutant Unknown On BRAF inhibitor
Urinary ctDNA BRAFV600E genotype
Tissue BRAFV600E genotype
Indeterminate Genotype BRAFV600E
Mutant
BRAF Wildtype
n=9
n=6 n=15
BRAFV600E Mutant
BRAF Wildtype
n=14 n=16
• BRAF mutational status obtained for 100% of patients by urine and only 70% of patients by tissue biopsy • 100% concordance between tissue, urine and plasma in treatment naive patients
Detection: Histiocytic Disorders
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Effect of Therapy on BRAF V600E Allele Burden in ctDNA of Patients with Systemic Histiocytosis1
Comparison of BRAFV600E allele burden in treatment-naïve urine samples and urinary samples acquired anytime during therapy
Effect of RAF inhibitors on ctDNA BRAFV600E mutant allele burden in 7 consecutive patients monitored weekly during treatment with RAF inhibitors
1Hyman et al., Cancer Discov. 2015 Jan;5(1):64-71
Monitoring: Histiocytic Disorders
Correlation between Longitudinal ctDNA and Radiographic Response1
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BRAFV600E burden in urine correlates w/ radiographic response.
BRAFV600E allele burden in urine changes dynamically with therapy.
1Hyman et al., Cancer Discov. 2015 Jan;5(1):64-71
Monitoring: Histiocytic Disorders
Dynamic Monitoring Captures Early Progression and Response1
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One week
Successful monitoring tumor dynamics independent of drug class used
Patient’s tumor progressed within 1 wk of Anakinra withdrawal
– Demonstration of need for higher frequency urine-based testing to monitor
Patient responded to Vemurafenib, BRAF inhibitor
– Demonstrates that continually monitoring patient enables optimal therapy over time
1Hyman et al., Cancer Discov. 2015 Jan;5(1):64-71
Monitoring: Histiocytic Disorders
Clinical Utility Enabled by Trovagene Technology
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Detection Minimal Residual Disease (MRD) Quantitate Drug Response Monitoring
Tissue Biopsy Imaging (every 6-8 weeks) Surgery Adjuvant Therapy Therapy
• Monitoring for Progression and Emergence of Resistance
• Molecular Detection of Clinically Actionable Mutations
• Monitoring for Minimal Residual Disease
• Tumor Recurrence
• Week 1: Assess Tumor Cell Kill by Therapy
• Beyond Week 1: Monitor Tumor Mutation Burden
TRO
VAG
ENE
C
LIN
ICA
L U
TILT
Y TR
OVA
GEN
E PC
M
• Earlier Detection of Metastatic Disease
• Alternative to Tissue Biopsy • Right Patients Treated with
Right Therapy • Eliminates Patient Morbidity
due to Tissue Biopsy Procedure
• Anticipates and Enables Next Therapy to Target Tumor Resistance
• Immediate Assessment of Drug Effect on Tumor
• Predict Best Response Weeks in Advance of Imaging
• Enables Early Switch from Ineffective Therapy
Current Cancer Standard of Care
Trovagene PCM adds information that imaging does not currently provide at much earlier time points.
Replacing Tissue Biopsy in Metastatic NSCLC with ctDNA Detecting EGFR and Monitoring ctDNA EGFR for Treatment Response and Early Acquisition of EGFR T790M Resistance Mutation
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Metastatic NSCLC Patients, Second Line Therapy
Metastatic NSCLC Patients at Diagnosis, EGFR Exons19 and 21 Status in Tissue
ctDNA Monitoring for Response and Early Acquisition of EGFR T790M
Urine Samples Serial Collections on Second Line Therapy
Metastatic NSCLC Patients, 1st Line anti-EGFR Therapy
Urine Samples Serial Collections on Erlotinib
Evaluate EGFR Concordance between
Urine and Tissue
Evaluate EGFR Concordance between
Plasma and Tissue
Matched Urine and Plasma Samples
EGFR T790M Concordance between Urine and Tissue
Urine vs Tissue Detection of EGFR T790M Mutation
at any time point 14/14 Patients 100%
Detection of T790M 3 months prior to detection of
progression by imaging*
3 mon *Preliminary Results
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NSCLC / Detection
Urine Identifies More Patients with EGFR T790M Resistance Mutation than Tissue and Earlier than Tissue1
1Interim results 2Urine positive for T790M at any time point on treatment
NSCLC / Detection
T790M FFPE Tumor
total Positive Negative Not yet tested
Urine Positive 14 3 8 25 Negative 0 0 10 10
Total 14 3 18 35
Urine % Sensitivity 100% (14/14) 2
1Husain et al., ASCO 2015
0
2
4
6
8
10
12
14
16
18
20
4/21
/14
6/10
/14
7/30
/14
9/18
/14
11/7
/14
12/2
7/14
2/15
/15
Patient 20 Patient 10 Patient 3
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1Husain et al., ELCC 2015
NSCLC / Monitoring
Early T790M Detection (Days to Radiographic Progressive)
Patient 1 111
Patient 3 52
Patient10 56
Patient 20 29
0
20
40
60
80
100
4/11
/14
5/21
/14
6/30
/14
8/9/
14
9/18
/14
0
10
20
30
40
50
6/10
/14
7/20
/14
8/29
/14
10/8
/14
0
100
200
300
400
500
8/29
/14
9/18
/14
10/8
/14
10/2
8/14
PD PD
PD
T790M detected
T790M detected PD
T790M detected
T790M detected
Urin
e T7
90M
Cop
ies/
100K
GE
Patient 1
Urin
e T7
90M
Cop
ies/
100K
GE
Urin
e T7
90M
Cop
ies/
100K
GE
Urin
e T7
90M
Cop
ies/
100K
GE
Urinary Detection of EGFR T790M Resistance Mutation in Advance of Radiographic Progression1
Clinical Utility Enabled by Trovagene Technology
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Detection Minimal Residual Disease (MRD) Quantitate Drug Response Monitoring
Tissue Biopsy Imaging (every 6-8 weeks) Surgery Adjuvant Therapy Therapy
• Monitoring for Progression and Emergence of Resistance
• Molecular Detection of Clinically Actionable Mutations
• Monitoring for Minimal Residual Disease
• Tumor Recurrence
• Week 1: Assess Tumor Cell Kill by Therapy
• Beyond Week 1: Monitor Tumor Mutation Burden
TRO
VAG
ENE
C
LIN
ICA
L U
TILT
Y TR
OVA
GEN
E PC
M
• Earlier Detection of Metastatic Disease
• Alternative to Tissue Biopsy • Right Patients Treated with
Right Therapy • Eliminates Patient Morbidity
due to Tissue Biopsy Procedure
• Anticipates and Enables Next Therapy to Target Tumor Resistance
• Immediate Assessment of Drug Effect on Tumor
• Predict Best Response Weeks in Advance of Imaging
• Enables Early Switch from Ineffective Therapy
Current Cancer Standard of Care
Trovagene PCM adds information that imaging does not currently provide at much earlier time points.
Monitoring Urinary ctDNA KRAS G12/13 Correlates with Treatment Response1
Monitor Urine KRAS ctDNA for Response or Treatment Failure
30 Metastatic CRC (mCRC) patients undergoing treatment with chemotherapy or
chemotherapy + surgery
Positive tissue KRAS G12/13
Interim analysis in 4 patients: Urine ctDNA KRAS dynamics correlates with treatment response
1Barzi et al, Cancer Markers and Liquid Biopsies 2015
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Colorectal / Monitoring
Urinary ctDNA KRAS Detects Response in Advance of Imaging and CEA
CEA
Urine KRAS G13D
CEA
Urine KRAS G12D Urine KRAS
G12S
2 wks on treatment
Patient 6: Best Response – Partial Reseponse
Urine KRAS G12D
CEA
2 wks on treatment
Patient 8: Best Response – Progressive Disease
Patient 3: Best Response – Complete Response Patient 1: Best Response – Partial Response
1Barzi et al, Cancer Markers and Liquid Biopsies 2015
2 wks on treatment
Urine KRAS G13D
CEA
Colorectal / Monitoring
Study Design
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o Patient demographics: 84 females and 92 males, median age 68, range 45-89 years o Locally advanced (n=50) or metastatic (n=132) pancreatic cancer o Palliative treatment with gemcitabine or FOLFIRINOX
Retrospective prospective study of archived samples from 182 patients with unresectable, locally advanced or metastatic PC undergoing
treatment with chemotherapy (Danish BIOPAC study)
ctDNA KRAS detection at baseline for association with OS
Plasma, Archived (≤ 6 years)
640 Plasma Samples from 182 Patients
o 176 of 182 patients with evaluable plasma at baseline
ctDNA KRAS monitoring for response to chemotherapy and association with OS
o 617 evaluable plasma samples
o Timepoints: baseline, before cycle 2 of chemotherapy, every 2-3 months at time of CT scans
Monitoring with Prognosis: Pancreatic Cancer
Significant Association Between Baseline KRAS Copies and Overall Survival
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p<0.0001
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o Estimated Kaplan-Meier survival plots for males, age < 65, receiving gemcitabine, with baseline KRAS counts above and below 5.5 copies/100K GE.
o Similar results obtained for female and older patients.
Low KRAS
High KRAS
Monitoring: Pancreatic Cancer
Longitudinal Dynamics of KRAS ctDNA Burden after 2 Weeks of Chemotherapy Correlates with OS Better than the Baseline KRAS
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o Plots of KRAS counts over time and hazard ratios relative to a patient with ≤ 5.5 cps/100K GE KRAS at all time points. Estimated and actual patient survival is shown.
Acknowledgements
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Hatim Husain, MD Razelle Kurzrock, MD Filip Janku, MD
Omar Abdel-Wahab, MD David Hyman, PhD MD Eli Diamond, MD Marik Minarik, PhD
Lucie Benešová PhD
Eric Collisson. MD Margaret Tempero. MD
Julia Johansen, MD Inna Chen, MD Afsaneh Barzi, MD
Heinz-Josef Lenz, MD