Feasibility of Monitoring Response to the PARP Inhibitor Rucaparib with Targeted Deep Sequencing of Circulating Tumor DNA (ctDNA) in Women With High-­Grade Serous Carcinoma on the ARIEL2 Trial Anna M. Piskorz1, Kevin K. Lin2, James Morris1, Elaina Mann2, Amit Oza3, Robert L. Coleman4, David M. O’Malley5, Michael Friedlander6, Janiel M. Cragun7, Ling Ma8, Heidi Giordano2, Mitch Raponi2, Iain A. McNeish9, Elizabeth Swisher10, James D. Brenton1
1Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka
Shing Centre, Cambridge, United Kingdom;; 2Clovis Oncology, Inc., Boulder, CO;; 3Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada;; 4The University of Texas MD Anderson Cancer Center, Houston, TX;; 5The Ohio State University, James Cancer Center, Columbus, OH;; 6Prince of Wales Clinical School, University of New South Wales, Sydney, Australia;; 7The University of Arizona Cancer Center, Tucson, AZ;; 8Rocky Mountain Cancer Centers, Lakewood, CO;; 9Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom;; 10University of Washington, Seattle, WA RESULTS -­ TP53 ASSAY QC
FEASIBILITY STUDY
FEASIBILITY STUDY for 142 patients for 18 patients
for 14 patients
641 plasma samples
164 plasma samples
28 Plasma Samples
• TP53 mutations are present in >99% cases of high-­grade
serous ovarian carcinoma (HGSOC)
• Approximately 50% of HGSOC may have homologous
recombination deficiency (HRD), comprising germline and
somatic BRCA mutations in 10-­20%, and candidate
mutations or epigenetic silencing of other homologous
recombination (HR) genes
• In clinical trials, PARP inhibitors have demonstrated
antitumor activity and extended progression-­free survival in
patients with BRCA mutations
• Rucaparib is a potent PARP inhibitor in development for
treatment of tumors with HR pathway deficiency
• Median Seq depth was 32,199x
• Concordant TP53 mutations were detected in tumor and
ctDNA from plasma in 128 patients
• TP53 mutation was not detected in tumor and ctDNA in
13 patients
• TP53 mutation was not detected in tumor but detected in
ctDNA in 1 patient
40
n patients =142
n samples = 641
• Trend for reduction of TP53 ctDNA MAF was similar to
CA-­125 changes at high and low MAF. Values below the cut
point of 0.5% MAF also followed CA-­125 trend
Figure 4. TP53 Mutant Allele Fraction (MAF) at each visit
n patients =18
n samples = 164
Figure 2. TP53 Mutant Allele Fraction (MAF)
Median=1.3%
0
T h r e s h o ld
-­ 1 0 0
SD
PD
• Noninvasive detection of TP53 mutations by TAm-­Seq is
feasible, using plasma samples collected from women with
relapsed platinum-­sensitive HGSOC participating in an
international multicenter trial
0
0.1%
10.0%
TP53 Mutant Allele Fraction (MAF)
mutation confirmed
TAm−Seq TP53 MAF for technical replicates
100%
• There was 100% concordance for TP53 mutation status in
matched tumour−plasma samples
replicate 2 (log10)
• Changes in TP53 MAF in plasma were highly similar to the
serum marker CA-­125
1%
• Patients with >50% reduction of TP53 MAF in ctDNA after cycle 1 achieved a RECIST PR and included patients with either a germline or somatic mutation in BRCA1/BRCA2. No patients with <50% reduction at cycle 2 achieved a RECIST response r 2 = 0.96, P < 0.001
0%
1%
100%
replicate 1 (log10)
ctDNA is a promising biomarker for monitoring response to the PARP inhibitor rucaparib
Figure 3. TP53 TAm-­Seq Depth
200000
Median=32199x
n patients =142
n samples = 641
150000
References 100
1. Ahmed AA, Etemadmoghadam D, Temple J, Lynch AG, Riad M, Sharma R, Stewart C, Fereday
S, Caldas C, Defazio A, Bowtell D, Brenton JD. Driver mutations in TP53 are ubiquitous in high
grade serous carcinoma of the ovary. J Pathol. 2010 May;;221(1):49-­56.
2. Forshew T, Murtaza M, Parkinson C, Gale D, Tsui DW, Kaper F, Dawson SJ, Piskorz AM,
Jimenez-­Linan M, Bentley D, Hadfield J, May AP, Caldas C, Brenton JD, Rosenfeld N. Noninvasive
identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci
Transl Med. 2012 May 30;;4(136):136ra68.
3. Murtaza M, Dawson SJ, Tsui DW, Gale D, Forshew T, Piskorz AM, Parkinson C, Chin SF,
Kingsbury Z, Wong AS, Marass F, Humphray S, Hadfield J, Bentley D, Chin TM, Brenton JD, Caldas
C, Rosenfeld N. Non-­invasive analysis of acquired resistance to cancer therapy by sequencing of
plasma DNA. Nature. 2013 May 2;;497(7447):108-­12.
count
Sequencing Depth
100
CONCLUSIONS
10
0%
100000
50
50000
• Plasma samples (n=641) were collected from 142 patients
during screening, on day 1 of each cycle, and at the end of
rucaparib treatment
0
0
0
50000
100000
150000
200000
Sequencing Depth
mutation confirmed
• DNA extracted from plasma underwent TAm-­Seq of TP53
with 2 technical replicates
• Response was assessed by RECISTv1.1 and GCIG CA-­125
criteria
B la c k = W T
20
0.1%
Figure 1. Clinical Trial Design and Experiment Workflow
TAm−Seq depth for technical replicates
200,000
n patients =142
n samples = 641
• 14 patients had ctDNA present at cycle 1 and 2 (missing
sample n=2;; TP53 MAF <0.5% n=2)
• 7/9 patients with >50% reduction of TP53 MAF in ctDNA at
cycle 2 achieved a RECIST PR (Fig 4.);; this included 5/6
patients with either a germline or somatic mutation in BRCA1/
BRCA2. No patients with <50% reduction at cycle 2 (n=5)
achieved a RECIST response
150,000
replicate 2
• FFPE tumor specimens were profiled using the Foundation
Medicine T5 NGS-­based assay with a targeted gene panel
including TP53
B l u e = B R C A 1 /2 m u t a n t
200
PR
30
n patients =142
n samples = 641
• Our estimated cutpoint for accurate MAF assessment was
>0.5% (but lower MAF values have previously been
reported)
n patients =14
n samples = 28
R E C IS T r e s p o n s e
The aim of this study was to assess TP53 mutant allele
fraction (MAF) in ctDNA from patients in ARIEL2 Part 1
(phase 2 study of rucaparib for treatment of relapsed
HGSOC [NCT01891344])
METHODS
Figure 5. TP53 ctDNA MAF>50% fall (C1-­C2) correlates with response
-­ 2 0 0
10.0%
count
• We have developed targeted amplicon deep sequencing
(TAm-­Seq) to detect low frequency mutations throughout the
TP53 gene in ctDNA
• Median TP53 MAF at screening and cycle 1 day 1 was 5.1%
(IQR 1.1–17.5, n=16) and 3.8% (IQR 0.68–10.3, n=16)
• Median MAF was 1.3% (min 0, max 63%)
TP53 Mutant Allele Fraction (MAF)
• Detection of TP53 mutations in ctDNA extracted from plasma
has the potential to monitor disease course and treatment
response
T P 5 3 M A F C h a n g e [ % ]
BACKGROUND
100,000
r 2 = 0.96, P < 0.001
50,000
0
0
50,000
100,000
replicate 1
150,000
200,000
Acknowledgments We would like to acknowledge Dr. Nitzan Rosenfeld and Dr. Florent Mouliere for generously sharing
alternative designs for TP53 primers and for their advice on the experimental plan, ARIEL2
coordinating investigators: Dr. Elizabeth Swisher, Prof Iain A. McNeish and ARIEL2 investigators: M.
Friedlander, J. Goh, P. Harnett, G. Kichenadasse, C.L. Scott;; P. Ghatage, A. Oza, D. Provencher,
A.V. Tinker, K. Tonkin, J. Weberpals, S. Welch;; A. Floquet, A. Leary, J. Medioni, I. Ray-­Coquard, F.
Selle;; A. Oaknin, A. Poveda;; S. Banerjee, J.D. Brenton, A. Clamp, Y. Drew, R. Kristeleit, J. Sanchez;;
D. Armstrong, K. Bell-­McGuinn, C. Castro, S.K. Chambers, L.-­M. Chen, R.L. Coleman, G. Colon-­
Otero, J.M. Cragun, B. DiCarlo, R. Dichmann, P. Haluska, W.A. Harb, G.E. Konecny, P.
Konstantinopoulos, L. Ma, L. Martin, K. Moore, M. Morgan, D. Mutch, D.M. O'Malley, D. Park, N.
Teng. This work was supported by Cancer Research UK (grant numbers A15973, A15601), the
University of Cambridge;; National Institute for Health Research Cambridge Biomedical Research
Centre;; National Cancer Research Network and Hutchison Whampoa Limited.