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Online only Supplementary Materials
Supplementary Methods
Whole exome sequencing
The diagnostic FFPE blocks were visually inspected and the ones with the most residual tumor were used for DNA isolation. A single H&E tissue section was reviewed by a consultant histopathologist (D M) and a minimum of 2 regions from the tumor were macrodissected using a 1 mm tissue microarray core needle. DNA was extracted from the FFPE tumor cores using the Gene Read kit (Qiagen) according to the manufacturer’s instructions and DNA concentration was measured as previously described.1
Illumina HiSeq was used to perform whole-exome sequencing on 200-500 ng of pooled tumor DNA from 1-3 regions cored from each FFPE primary tumor block (sequencing was performed as a fee per service by Novogene) at an average deduplicated on-target read depth of 150x for all 49 tumor DNA and 50x for 49 matched germline samples. All sequencing data will be deposited in the European Genome–Phenome Archive on publication.
Signatera Workflow
Patient specific tumor profiles
A clinical grade, strictly-controlled semi-automated lab process was performed by trained personnel with signed-off standard operating protocols and witnessing. The process and reagent/equipment information were captured electronically and uploaded to a database with built-in integrity checks. Quality control (QC) was performed at every step of the workflow (Supplementary Figure S1). Samples and amplicons that did not pass pre-specified QC were excluded from further analyses. A set of 45 SNPs were genotyped in tumor and plasma from each patient to ensure sample concordance.
Primary tissue from each patient was sequenced by whole exome sequencing (WES). WES data was used to design bespoke 16-plex assays specific to the tumor mutational signature for each patient. For each target variant identified in the plasma, a confidence score was calculated based on the depth of read for mutant and reference alleles.
Blood sample Collection, Cell free DNA extraction and quantification
Blood samples were collected in K2-EDTA tubes. Samples were processed within 2 hours of collection by double centrifugation of the blood, first for 10 minutes at 1000g, then the plasma for 10 minutes at 2000g. Plasma was stored at −80 °C. Up to 8 ml of plasma per case was available for this study (range, 1–8 ml; median 5 mL). The entire volume of plasma was used for cfDNA extraction using the QIAamp Circulating Nucleic Acid kit (Qiagen) and eluted into 50 μL DNA Suspension Buffer (Sigma). Each cfDNA sample was quantified by Quant-iT High Sensitivity dsDNA Assay Kit (Invitrogen). In 49 patients, cfDNA was isolated from a total of 215 serial plasma samples.
Cell free DNA library preparation
Up to 66 ng (20,000 genome equivalents) of cfDNA from each plasma sample was used as input into the library preparation, as described previously.25 The cfDNA was end-repaired, A-tailed, and ligated with custom adapters. The purified ligation product was amplified for 20 cycles and purified using Ampure XP beads (Agencourt/Beckman Coulter).
Plasma multiplex-PCR NGS workflow
A 16% aliquot of each library was used as input into the associated patient-specific 16-plex PCR reaction. Samples were amplified using patient-specific assay and barcoded, followed by pooling. Sequencing was performed on an Illumina HiSeq 2500 Rapid Run with 50 cycles of paired-end reads using the Illumina Paired End v2 kit with an average read depth of >100,000X per amplicon.
Bioinformatics pipeline
All paired-end reads were merged using Pear software.3 Bases that did not match in forward and reverse reads or had a low quality score were filtered out to minimize sequencing errors. Merged reads were mapped to the hg19 reference genome with Novoalign version 2.3.4 (http://www.novocraft.com/). Amplicons with < 5,000 high quality reads were considered to have failed the QC. QC was performed using an in-house program checking for a wide list of statistics per sample that included total numbers of reads, mapped reads, on-target reads, number of failed targets and average error rate (Figure S5).
Plasma variant calling
A large set of negative control samples (~1000) were pre-processed to build a background error model. For each target variant using mutant and reference alleles depth of read, a confidence score was calculated on the basis of the error model, as described in Abbosh et al. 2017.2 As previously, a plasma sample with at least 2 variants with a confidence score above a predefined algorithm threshold (0.97) was defined as ctDNA positive.
Analytical validation
The analytical validation was performed using two different sets of titration samples built from a) mononucleosomal DNA from cancer cell lines, b) a commercially available mutation mixture “SeraseqTM ctDNA Mutation Mix v2”. Mononucleosomal DNA from three cancer cell lines, including two breast cancer cell lines (HCC2218 & HCC1395) and one lung cancer cell line (NCI-H1395) were titrated into their respective matched normal B lymphoblast-derived cell lines (HCC2218-BL, HCC1395-BL, and NCI-BL1395) matched normal counterparts (ATCC). For each cell line pair, DNA was exome-sequenced, target variants were selected, and two multiplex-PCR primer pools were designed using the Signatera pipeline. Titrations of tumor into normal mononucleosomal DNA were made at average VAFs (based on DNA input) of 1%, 0.5%, 0.3%, 0.1%, 0.05%, 0.03%, 0.01%, 0.005%, 0%. Replicate numbers were from two to nine— increasing with the dilution factor. Due to heterogeneity and aneuploidy in the tumor cell lines, the VAFs of the individual targets differ from the average input tumor fraction. To accurately calculate the nominal VAF of each target in each titration step, a calibration experiment was performed with 10% VAF mixtures. The observed VAFs were then used to calculate input correction factors that were applied to their respective targets in the titration series. Additional negative samples were run using cfDNA isolated from 16 healthy human plasmas (approximately 8 mL each). For the titration series, 66 ng cfDNA (corresponding to 20,000 haploid genome equivalents) were used as input into library preparation; for the plasma cfDNA samples all isolated DNA (13-55 ng) was used as input. These libraries were then run through the plasma workflow (two different primer pools for each titration sample and five primer pools for each cfDNA sample), sequenced, and analyzed with our analysis pipeline.
Accuracy of 100% was achieved by calling all 132 positive samples as ctDNA positive with VAF down to 0.03%, and all 134 negative control samples as ctDNA negative. We calculate the sensitivity for 180 samples with VAF down 0.01% to be 93%. Figure S6A shows the estimated sensitivity for detecting individual mutations in plasma at various levels of tumor concentration by using the confidence threshold. Sensitivity of >99% at mutation frequencies down to 0.1% was achieved. However, in a total of 134 negative samples, 410 negative base changes (out of 107, 078 assessed) and 19 negative INDELs (out of 35,651 possible) were measured above the confidence threshold. Specificity was estimated to be 99.71% for individual mutation detection. Specificity for a 16-plex assay would thus be only 95.5% if a single measurement above the threshold was used to call the plasma sample ctDNA positive. In order to achieve high specificity of > 99.8% we require 2 mutations to be measured above the confidence threshold as previously described.4
The sample-level sensitivity was derived by calculating a binomial probability for detecting at least two clonal mutations at a given ctDNA level, assuming that the majority of the custom panels have between 10 to 16 clonal variants (Figure S6A). Clonality inference analyses of the 49 patients of this study estimated the median number of clonal mutations in our assays to be 13 (Figure S2). As shown in Figure S6A, ctDNA will be detected in samples with ctDNA between 0.01% and 0.02% for > 98% of samples.
Additionally, the Signatera process was also verified using a commercially available standard control, SeraseqTM ctDNA Mutation Mix v2 (SeraCare, Milford, MA). Allele fractions for all targets in the mix were verified by SeraCare by droplet digital PCR. Two 16-plex pools were designed for the SeraCare standard using the Signatera pipeline. As for the analytical validation, the SeraCare standard was titrated at 0.5%, 0.2%, 0.1%, 0.05%, 0.03%, 0.01%, and 0.005% VAF (Figure S6B). Each titration point was run in triplicate and run on both 16-plex pools. Five negative control samples run on both 16-plex pools. 66 ng cfDNA was used as input into the Signatera plasma workflow.
Accuracy of 100% was achieved by calling all 36 positive samples as ctDNA positive with VAF down to 0.01%, and all 10 negative samples as ctDNA negative. Target-level sensitivity of >99% achieved at mutations frequencies down to 0.1%, similar to targets tested in cell-line samples (Figure S6C). There was only one target above the calling threshold in control samples.
Target and sample-level performance estimated based on cell-line derived samples were corroborated using this commercially available SNV standard control (Figures S6A-C). Figure S6D shows a titration curve from this standard for a target that was present in six of the breast cancer cases in this study. This target was called down to below 0.05% in the clinical samples which is firmly within the quantitative detection range for this target (detection down to 0.005%, quantitative detection down to 0.03%).
3
Online-Only Tables
TABLE S1: Clinical characteristics of all 49 patients
Case No
Age at diagnosis
Tumour Grade
ER Status
HER2 status
TNM staging
Time from first sample to relapse (days)
Date of relapse
Site of distant metastasis
Local Recurrence
E001
48
3
-
+
T2 N1 M0
-
NA
NA
E002
56
3
+
+
T2 N2 M0
-
NA
NA
E003
65
2
+
-
T3 N2 M0
133
25/06/2014
Pleural Effusion
E004
50
2
+
-
T2 N3 M0
-
NA
NA
E005
69
2
+
-
T3 N3 M0
263
02/01/2015
Nodal disease right hilum and mediastinum
E006
48
2
+
-
T2 N2 M0
-
NA
NA
E007
67
2
+
+
T2 N3 M0
-
NA
NA
E008
47
2
+
-
T3 N3 M0
-
NA
NA
E009
44
3
+
-
T2 N1 M0
125
28/10/2014
Sternum, pelvis, vertebrae*
E010
38
2
+
-
T2 N2 M0
561
08/01/2016
NA
Sternum
E011
69
2
+
-
T2 N2 M0
-
NA
NA
E012
66
3
+
-
T2 N1 M0
-
NA
NA
E013
80
2
+
-
T3 N2 M0
-
NA
NA
E014
57
2
+
-
T2 N3 M0
-
NA
NA
E015
81
3
-
-
T2 N1 M0
-
NA
NA
E016
80
3
-
+
T2 N2 M0
-
NA
NA
E017
67
3
+
-
T3 N3 M0
1281
31/01/2018
Sternoclavicular joint, skin, lung
E018
55
2
+
-
T3 N3 M0
-
NA
NA
E019
53
3
+
-
T2 N1 M0
-
NA
NA
Case No
Age at diagnosis
Tumour Grade
ER Status
HER2 status
TNM staging
Time from first sample to relapse (days)
Date of relapse
Site of distant metastasis
Local Recurrence
E020
80
2
+
-
T2 N2 M0
-
NA
NA
E021
44
2
+
-
T2 N3 M0
-
NA
NA
E022
54
2
+
-
T2 N2 M0
-
NA
NA
New breast primary in contralateral breast
E023
54
2
+
-
T3 N2 M0
-
NA
NA
E024
78
3
-
-
T2 N1 M0
-
NA
NA
E025
46
3
-
-
T1 N2 M0
-
NA
NA
E026
73
2
+
-
T2 N3 M0
793
17/03/2017
Spine
E027
60
2
+
-
T2 N2 M0
-
NA
NA
E028
76
3
+
+
T1 N2 M0
-
NA
NA
E029
57
3
-
-
T2 N1 M0
442
30/11/2016
Lung
E030
70
3
+
-
T2 N1 M0
-
NA
NA
E031
77
2
+
-
T2 N3 M0
1087
23/05/2018
Skin on right lower back
E032
39
3
+
-
T1 N1 M0
-
NA
NA
E033
57
2
-
-
T1 N0 M0
570
30/12/2016
Intraclavicular fossa and sentinel lymph nodes
E034
66
2
+
-
T1 N3 M0
-
NA
NA
E035
59
2
+
-
T2 N3 M0
-
NA
NA
E036
46
2
+
-
T1 N3 M0
415
13/10/2016
Bone and bladder
E037
78
3
-
+
T2 N3 M0
610
07/06/2017
Lung
E038
63
1
+
-
T2 N2 M0
-
NA
NA
E039
48
3
+
-
T2 N1 M0
-
NA
NA
E040
55
3
-
+
T2 N1 M0
313
05/09/2016
Bone
Case No
Age at diagnosis
Tumour Grade
ER Status
HER2 status
TNM staging
Time from first sample to relapse (days)
Date of relapse
Site of distant metastasis
Local Recurrence
E041
67
3
+
-
T2 N1 M0
-
NA
NA
E042
49
2
+
-
T2 N1 M0
-
NA
NA
E043
41
2
+
-
T3 N1 M0
68
23/05/2016
Liver, Lung, Bone*
E044
46
3
+
-
T2 N1 M0
323
04/02/2016
Local nodes
E045
46
3
+
+
T2 N1 M0
-
NA
NA
E046
57
3
-
-
T2 N2 M0
269
08/02/2016
Bone, liver and pleura
E047
62
3
+
-
T3 N1 M0
117
18/11/2015
Local nodes and intrapulmonal nodes
E048
52
3
-
-
T2 N1 M0
202
31/12/2015
Bone and pleura
E049
45
3
-
-
T2 N1 M0
148
08/10/2016
Not known*
NA, not applicable. Patients marked with an asterisk (*) are deceased.
Table S2 (A-D) Sample visit information for 49 patients including sample visit and treatment
Table S2A: Dates of sample visits for 49 patients
Case ID
Plasma Timepoint (date)
0
1
2
3
4
5
6
7
EoS
E001
15/01/2014
08/10/2014
07/01/2015
24/06/2015
20/01/2016
13/07/2016
04/01/2017
05/07/2017
E002
10/02/2014
08/09/2014
09/02/2015
01/09/2015*
18/02/2016
25/08/2016
16/03/2017
E003
12/02/2014
E004
14/04/2014
27/10/2014
27/04/2015
19/10/2015
09/06/2016
28/11/2016
E005
14/04/2014
16/10/2014
E006
07/05/2014
09/12/2015*
23/08/2016
01/03/2017
E007
12/05/2014
11/11/2014
17/11/2015
26/05/2016
08/12/2016
16/07/2017
E008
16/05/2014
03/11/2014
20/04/2015
19/10/2015
12/12/2016
E009
25/06/2014
E010
26/06/2014
10/12/2014
20/01/2016
E011
09/07/2014
28/01/2015
19/08/2015
06/01/2016
20/07/2016
25/01/2017
06/09/2017
E012
16/07/2014
14/01/2015
08/07/2015
27/01/2016
27/07/2016
25/01/2017
26/07/2017
E013
16/07/2014
14/01/2015
15/07/2015
13/01/2016
13/07/2016
25/01/2017
09/08/2017
E014
16/07/2014
21/01/2015
03/02/2016
12/07/2016
31/01/2017
E015
21/07/2014
14/01/2015
15/07/2015
20/01/2016
20/07/2016
08/02/2017
26/07/2017
E016
23/07/2014
21/01/2015
29/10/2015
08/06/2016
11/01/2017
05/07/2017
E017
30/07/2014
14/01/2015
08/07/2015
10/02/2016
31/08/2016
22/02/2017
04/07/2017
E018
11/08/2014
16/02/2015
04/04/2016*
28/11/2016
24/05/2017
E019
27/08/2014
11/02/2015
06/08/2015
16/03/2016
18/10/2016
21/04/2017
E020
04/09/2014
20/04/2015
15/10/2015
14/01/2016
03/11/2016
03/05/2017
E021
09/09/2014
10/03/2015
15/09/2015
24/03/2016
22/09/2016
30/03/2017
E022
10/09/2014
11/03/2015
04/08/2015
E023
11/09/2014
05/02/2015
03/08/2015
08/03/2016
25/08/2016
09/03/2017
E024
24/09/2014
18/03/2015
23/09/2015
18/05/2016
30/11/2016
28/06/2017
E025
05/11/2014
13/05/2015
23/12/2015
22/06/2016
18/01/2017
06/09/2017
E026
14/01/2015
15/07/2015
24/02/2016
13/09/2016
28/02/2017
E027
03/02/2015
04/08/2015
04/02/2016
E028
11/02/2015
30/08/2016
21/02/2017
05/09/2017
E029
15/09/2015
17/03/2016
29/09/2016
12/01/2017
E030
20/05/2015
18/11/2015
18/05/2016
19/07/2017
E031
01/06/2015
29/10/2015
28/04/2016
26/01/2017
26/07/2017
Case ID
Plasma Timepoint (date)
0
1
2
3
4
5
6
7
EoS
E032
03/06/2015
14/10/2015
08/06/2016
21/12/2016
E033
09/06/2015
22/12/2015
21/07/2016
09/01/2017
E034
10/06/2015
09/12/2015*
07/06/2016
04/07/2017
E035
18/06/2015
21/01/2016
09/09/2016
30/01/2017
12/06/2017
E036
25/08/2015
24/03/2016*
22/09/2016
E037
06/10/2015
24/05/2017#
24/05/2017#
E038
07/10/2015
13/04/2016
19/10/2016*
19/04/2017
E039
20/10/2015
18/05/2016
16/11/2016
31/05/2017
E040
28/10/2015
13/04/2016
19/10/2016
E041
20/01/2016
20/07/2016
08/02/2017
04/08/2017
E042
03/02/2016
16/08/2016
24/01/2017
E043
16/03/2016
E044
18/03/2015
23/09/2015*
17/02/2016
E045
15/12/2015
15/06/2016
14/12/2016
E046
15/05/2015
06/11/2015
12/02/2016
E047
24/07/2015
16/12/2016
E048
12/06/2015
04/12/2015
08/01/2016
E049
13/05/2016
*Samples omitted due to failed QC.
#Duplicate plasma timepoint
EoS, End of Study.
Table S2B: CA15-3 levels (U/ml) at sample visit
Case ID
CA15-3 levels at plasma timepoint
0
1
2
3
4
5
6
7
EoS
E001
7
20
19
22
20
18
17
20
E002
8
9
8
10
9
9
7
E003
17
E004
18
18
18
18
19
17
E005
22
26
E006
15
11*
14*
11
13
13
E007
15
14
14
13
14
14
E008
17
17
18
17
21*
15
E009
51
E010
13
12
16
E011
9
8
7
8
9
9
ND
E012
9
8
9
8
10
9
10
E013
31
33
23
21
19
57
28
E014
18
17
18
18
20
E015
20
20
22
22
20
23
20
E016
15
13
15
17
16
15
E017
13
13
13
14
15
17
17
E018
5
6
5
7
7
E019
29
23
29
26
29
29
E020
26
51
32
28
29
36
E021
14
14
14
14
14
14
E022
10
10
12
E023
11
13
13
12
13
14
E024
23
23
28
24
25
22
E025
20
20
20
17
19
21
E026
24
31
46
58
104
E027
24
23
20
E028
35
33
29
E029
10
13
10
12
E030
13
13
10
14
E031
34
27
31
32
ND
E032
12
10
11
11
E033
18
21
20
22
E034
26
25
23
27
E035
21
19
24
22
21
E036
25
48
131
E037
20
22
21
E038
16
17
19
16
E039
14
12
11
13
E040
33
35
36
E041
26
23
26
22
E042
5
5
5
E043
65
E044
ND
ND
ND
E045
ND
ND
ND
E046
ND
ND
ND
E047
ND
ND
E048
ND
ND
ND
E049
ND
*No plasma samples.
EoS, End of Study; ND, Not Determined.
Table S2C: Details of patient’s tests and scans
Case ID
Tests/Scans showing lack of metastasis (date)
E001
CT - 12/16, Bone scan - 01/18, LFTs - 07/16, 07/17
E002
CT - 01/18, Bone scan - 03/17, LFTs - 01/16, 03/18
E003
CT - 01/14, Bone scan - 05/13, Liver US - 05/13
E004
CT - 11/15, Liver US - 05/18, LFTs - 05/16, 12/17
E005
LFTs - 04/14
E006
CT - 02/14, Liver US - 05/14, LFTs - 02/14
E007
CT - 02/14, LFTs - 02/14
E008
Bone - 12/13, Liver US - 04/14, LFTs - 05/14
E009
CT - 01/14, Bone scan - 12/13, Chest Xray - 06/14, LFTs - 06/14
E010
Bone scan - 10/13, Chest Xray - 03/14, LFTs - 06/14
E011
CT - 12/14, LFTs - 07/14
E012
Bone scan - 05/15, Chest Xray - 02/15, LFTs - 07/14
E013
Chest Xray - 03/15, Liver US - 07/15, LFTs - 03/14, 11/14, CA15-3 - normal
E014
CT - 03/14, 02/15, LFTs - 03/14, 09/14
E015
No scans but LFTs 07/14 normal
E016
CT - 12/15, Bone scan - 02/15, LFTs - 07/14
E017
CT - 07/14, LFTs - 07/14
E018
CT - 08/14, Bone scan - 08/14, LFTs - 08/14
E019
Chest Xray - 05/14, Liver US - 05/14, LFTs - 08/14
E020
Chest Xray - 06/14, Liver US - 08/15, 03/14, LFTs - 09/14
E021
CT - 07/13, Bone scan - 07/13, LFTs - 09/14
E022
CT - 07/15, Bone scan - 06/15
E023
LFTs - 05/14 (No imaging)
E024
Bone scan - 03/14, LFTs - 09/15
E025
LFTs - 05/15 (no imaging)
E026
Steadily rising CA15-3 since 02/16. LFTs - 06/14, 02/16, Mammogram - 07/15
E027
LFTs - 12/13 (no imaging)
E028
Bone scan - 02/15, LFTs - 02/15
E029
Bone scan - 03/15, 10/16, MRI Bone - 02/15, LFTs - 09/15
E030
Bone scan - 12/15, Chest Xray - 01/14, LFTs - 05/15
E031
CT - 04/17, Bone - 08/17, LFTs - 10/15
E032
Chest Xray - 11/13, Liver US - 10/16, LFTs - transient increase AT (56)
E033
LFTs - 16/15
E034
LFTs - 10/06/15, MRI - 13/11/14
E035
CT - 10/14, Bone scan - 10/14, LFTs - 06/15, MRI - 04/15
E036
CT - 09/13, Bone - 10/13, LFTs - 05/15,09/15
E037
CT - 07/15, Bone scan - 07/15, LFTs - 06/16, 7/15
E038
CT - 08/13, LFTs - 10/15
E039
CT - 03/15, Chest Xray - 07/15, LFTs - 10/15
E040
CT - 08/12, Chest Xray - 11/12, LFTs (raised but diabetic and on statins - deemed normal) - 10/15
E041
CT - 09/15, Chest Xray - 09/15, LFTs - 01/16
E042
CT - 08/14, Bone Scan - 08/14, LFTs - 02/16
E043
CT - 11/14, Bone Scan - 11/14, Chest Xray - 15/01/15, LFTs 03/16
E044
No scans but LFTs 03/15 normal
E045
Mammogram 12/15, LFT - 12/15
E046
CT scan excluded mets at baseline
E047
CT scan excluded mets at baseline
E048
CT scan excluded mets at baseline
E049
CT scan excluded mets at baseline
Table S2D: Chemotherapy status and endocrine treatment regime in 49 patients
Case ID
Previous chemotherapy
Plasma time point
0
1
2
3
4
5
6
7
EoS
E001
Yes
None
None
None
None
None
None
None
None
None
E002
Yes
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
E003
Yes
Letrozole
Letrozole
E004
Yes
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
E005
No
Anastrozole
Anastrozole
Anastrozole
E006
Yes
Letrozole
Letrozole
Letrozole
Exemestane
Letrozole
Letrozole
E007
Yes
Anastrozole
Tamoxifen
No visit
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
E008
Yes
Anastrozole/Zoladex
Anastrozole/
Zoladex
Anastrazole
Exemestane
Exemestane
Exemestane
E009
Yes
None
E010
Yes
Tamoxifen/
Zoladex
Tamoxifen/
Zoladex
E011
Yes
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
E012
Yes
Anastrozole
Anastrozole
Anastrozole
Anastrozole
Anastrozole
Anastrozole
Anastrozole
E013
No
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
E014
Yes
Letrozole
Letrozole
Missed
Letrozole
Letrozole
Letrozole
E015
No
None
None
None
None
None
None
None
E016
No
Anastrozole
Anastrozole
Anastrozole
Anastrozole
Anastrozole
Anastrozole
E017
Yes
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
E018
Yes
Exemestane
Exemestane
Missed
None
None
None
E019
Yes
None
Tamoxifen
Tamoxifen
Tamoxifen
Letrozole
Letrozole
E020
No
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
E021
Yes
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
E022
Yes
Anastrozole
Anastrozole
E023
Yes
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
E024
Yes
None
None
None
None
None
None
E025
Yes
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
Tamoxifen
E026
Yes
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
E027
Yes
Anastrozole
Anastrozole
Anastrozole
E028
Yes
Letrozole
Missed
Missed
Letrozole
Letrozole
Letrozole
E029
Yes
None
None
None
None
Case ID
Previous chemotherapy
Plasma time point
0
1
2
3
4
5
6
7
EoS
E030
Yes
Letrozole
Letrozole
Letrozole
Letrozole
E031
No
Anastrozole
Anastrozole
Anastrozole
Anastrozole
Anastrozole
E032
Yes
None
None
None
None
E033
Yes
None
None
None
E034
Yes
Letrozole
Letrozole
Letrozole
Letrozole
Letrozole
E035
Yes
Anastrozole
Anastrozole
Anastrozole
Anastrozole
Anastrozole
E036
Yes
Tamoxifen
Tamoxifen
Tamoxifen
E037
No
None
None
E038
Yes
Letrozole
Letrozole
Letrozole
Letrozole
E039
Yes
None
None
Letrozole
Letrozole
E040
Yes
Letrozole
Letrozole
E041
Yes
None
Tamoxifen
Tamoxifen
Tamoxifen
E042
Yes
Tamoxifen
Tamoxifen
Tamoxifen
E043
Yes
Tamoxifen
E044
Yes
Tamoxifen
Tamoxifen
Tamoxifen
E045
Yes
Tamoxifen
Not recorded
Not recorded
E046
Yes
None
None
None
E047
Yes
Endocrine therapy*
Endocrine therapy*
E048
Yes
None
None
None
E049
Yes
None
None
None
‘None’ indicates not on any treatment at that time. ‘Missed’ indicates that particular visit was missed by the patient. ‘No visit’ indicates no visit was made by the patient. Endocrine therapy * indicates on endocrine therapy but not specified.
Table S2E: Herceptin Treatment Regimen in HER2+ Patients
Case ID
Herceptin Treatment
Start Date
End Date
E001
02/2013
01/2014
E002
01/2013
01/2014
E007
05/2013
12/2013
E016
11/2013
N/A
E028
05/2014
05/2015
E037
E040
10/2012
10/2013
E045
12/2014
11/2015
N/A, Not Available.
Tables S3a,b and c – please see attached excel files.
Supplemental Figure Legends:
Figure S1: Quality control was performed at every step of the workflow. Of 215 plasma samples, 208 passed the sample QC process, and out of 784 unique assays designed, 767 passed the assay QC (corresponding to a total of 3237 assays passing out of 3328 across all samples). A. The quantity of cfDNA extracted per plasma sample. Median extracted cfDNA per mL was measured to be 5.5 ng. B. Quantity of input DNA per library preparation. 1 to 66ng of cfDNA (median=25.02 ng) from each plasma sample was used as input into library prep protocol. QC was performed on purified libraries before proceeding to subsequent steps. C. The depth of read per sample. Assays with sequencing coverage less than 5000x were excluded from analyses (denoted in red). Subsequently, samples with less than 8 passing assays failed sequencing coverage QC. The median depth of read for the assays passed coverage QC was 110,000x. D. Sample concordance QC. In order to track sample integrity, SNP tracers were used to measure concordance between patient’s samples. For each plasma samples, a genotyping concordance score was calculated in comparison to the corresponding matched normal genotyping data. Samples were considered to be from the same patients when at least 85% of their SNPs had identical genotypes. Six plasma samples did not pass the sample concordance QC (denoted in red) and were excluded from ctDNA analyses.
Figure S2: Signatera variant selection strategy for the 49 patient-specific panels A. The VAF distribution of custom panel in each patient’s primary tumour tissue. B. The number of inferred clonal (red) and subclonal (blue) variants in each patient’s custom pool. The median number of clonal variants in the 49 custom panels is 13 out of 16. C. The number of inferred clonal (red) and subclonal (blue) variants in each patient’s primary tumor WES data. Different colors represent different subtypes HR+/HER2- (dark blue), and HER2+ (green), and triple negative (orange). Note that clonality inference is affected by tumor purity. Very few to none clonal variants were identified from WES samples with <20% tumor cell fraction (E042, E012, E004).
Figure S3: Detection of Driver Mutations. All somatic variants identified by WES of tumor DNA were submitted to the cancer genome interpreter (https://www.cancergenomeinterpreter.org/home) to detect TIER 1 and TIER 2 driver gene variants. Of the 49 patients, only 33 (67%) had one or more driver mutations (range of 1-11) detected. For these 33 patients, a second assay was designed to track as many driver variants as possible.
Figure S4: Plasma ctDNA levels for 12 breast cancer patients. A-L. Patient-specific plots of 11 relapsed and one non-relapsed patient. Each patient specific assay was designed to target 16 somatic variants using massively parallel sequencing (median depth >100,000X per target). Mean VAFs are denoted by a dark blue circle and the solid line represent average VAF profile over time. The lead time is calculated by the difference in the date of clinical relapse (red triangle) and ctDNA determined molecular relapse (blue triangle). CA 15-3 levels are graphed over time (teal circles).
Figure S5: Distribution of VAFs and mutant counts. In total, 251 targets were detected in ctDNA positive plasma samples. A. The VAF of detected targets ranged from 0.01%–64%, with a median of 0.82%. The observed mutant VAF and total number of DNA molecules in each sample was used to calculate the number of tumor molecules present in the patient’s plasma sample. B. The number of detected mutant molecules in the 251 positive targets ranged from 1–6500 mutant molecules, with a median of 39 molecules.
Figure S6: Analytical validation and orthogonal control results. Titrations of tumor into normal mononucleosomal DNAs (cell line-derived) were made at average VAFs of 1% to 0.005%. Six primer pools were tested with replicate numbers from two to nine (for each pool)—increasing with the dilution factor. In addition, a commercially available control SNV mixture from SeraCare (SeraseqTM ctDNA Mutation Mix v2) was titrated from 0.5% to 0.005%. Starting allele fractions were confirmed by SeraCare by droplet digital PCR. Two primer pools were tested in triplicate on these mixtures. A. Sample-level performance calculated from the analytical validation. Sample-level sensitivity was calculated when at least two mutations are detected from a set of 16 target variants, assuming majority of patient-specific panels have between 10–16 clonal variants (n = the number of target level datapoints used to calculate sample-level data). Reproducibility was calculated as the %CV of the median VAF of positive targets. B. Sample-level performance calculated from orthogonal control samples from SeraCare. C. Target-level sensitivity of the 16-plex assay was >99% at mutation frequencies down to 0.1%. Circles represent the analytical validation data (n=72-562 per data point, and squares represent SeraCare data (n=90 per datapoint). D. Graph of the SeraCare titration series for target PIK3CA chr3:178952085:A>G demonstrating quantitative detection down 0.03% and single molecule detection down to 0.005%. Closed circles represent mutation detected; Open circles represent no mutation detected. This target was present and detected down to below 0.05% in plasma samples of six patients in this cohort.
Figure S1
Figure S2
Figure S3
Figure S4
Figure S5
Figure S6.
Supplemental References
1.Adamo P, Cowley CM, Neal CP, et al. Profiling tumour heterogeneity through circulating tumour DNA in patients with pancreatic cancer. Oncotarget. 2017;8(50):87221-87233.
2.McGranahan N, Favero F, de Bruin EC, Birkbak NJ, Szallasi Z, Swanton C. Clonal status of actionable driver events and the timing of mutational processes in cancer evolution. Sci Transl Med. 2015;7(283):283ra254.
3.Zhang J, Kobert K, Flouri T, Stamatakis A. PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics. 2014;30(5):614-620.
4.Abbosh C, Birkbak NJ, Wilson GA, et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature. 2017;545(7655):446-451.
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