Online-Only Supplement Material
Table of Contents eMethods
References eFigures
eFigure 1: Personalized digital PCR assays for mutation tracking of circulating tumor DNA in plasma of patients with early breast cancer.
eFigure 2: Reproducibility of digital PCR between replicates. Correlation between number of FAM positive droplets in replicate1 and replicate 2. Pearson correlation coefficient.
eFigure 3: CONSORT diagram for the study.
eFigure 4: Identified mutations by Massive Parallel Sequencing. (A) Mutations distribution and Allele Frequency on the diagnostic tumor prior to any neoadjuvant chemotherapy. (B) Mutation distribution and Allele Frequency of mutations identified in the first plasma sample DNA taken at diagnosis prior to any neoadjuvant chemotherapy. (C) Mutation distribution and Allele Frequency of mutations identified by mutation tracking on plasma DNA taken at follow-up.
eFigure 5: Personalized mutation specific digital PCR assays accurately quantify DNA. eFigure 6: Patients with CHIP identified in plasma cfDNA.
eFigure 7: Time dependent relapse free survival in patients with ctDNA detected in follow up. eFigure 8: Relapse free survival and Overall Survival of extension of proof-of-principle study. eFigure 9: CONSORT diagram for the combined analysis.
eFigure 10: Time dependent relapse free survival in patients with ctDNA detected in follow up for the combined analysis (A), and (B) relapse free survival for individual patients in the combined cohort from study entry and during follow-up. Censored patients did not have a clinical relapse at the time of the data collection.
eFigure 11: Level of ctDNA in diagnosis plasma samples, prior to any treatment, according to subtype in 122 patients that received neoadjuvant chemotherapy.
eTables
eMethods
Patient Cohort and Sample Collection
One hundred and seventy patients scheduled to receive standard treatment with either neoadjuvant chemotherapy followed by surgery (N=140) or surgery followed by adjuvant chemotherapy (N=30) were identified from two prospective ctDNA sample collection studies, the ChemoNEAR study (REC Ref No: 11/EE/0063) or the Plasma DNA study (REC Ref No: 10/H0805/50) approved by Research Ethics committees (East of England – Essex and London – Bromley, respectively). Written informed consent was obtained from all participants. Staging investigations were performed at diagnosis for all node positive and/or cT3/4 patients, usually with CT scan and bone scans, and those with distant metastatic disease were excluded from the study. Patients were treated with standard neoadjuvant chemotherapy with/without trastuzumab depending of HER2 status. After completion of surgery, with or without radiotherapy, patients were treated with adjuvant hormone therapy or trastuzumab as per standard local practice.
Core biopsies were taken at diagnosis. Plasma samples for ctDNA analysis were collected in either EDTA blood collection tubes and processed within 2 hours following venipuncture (Royal Marsden Hospital), or preservative tubes (Streck), shipped to a central laboratory, and separated within 46-72 hours post venipuncture. Serial plasma samples were taken for ctDNA after completing chemotherapy and surgery (neoadjuvant chemotherapy at 2-4 weeks after surgery, adjuvant chemotherapy 2-4 weeks after the last cycle of chemotherapy), and every three months for the first year of follow-up, and subsequently every six months until five years (eFigure 1). An additional sample was taken on patients receiving neoadjuvant chemotherapy at diagnosis prior to any treatment. (eFigure 1). Clinico-pathological characteristics of the study cohort are available in eTable 1. Oestrogen receptor (ER), progesterone receptor (PR), and HER2 status were assessed as per ASCO/CAP guidelines in the participating hospitals using standard criteria.
Proof-of-Principle Cohort Patients Characteristics
Forty three patients from the proof-of-principle study1 were included in the combined analysis. The median follow-up for this cohort of patients was updated to 31.7 months. None of the patients form this cohort was lost during follow-up and all patients were included in the combined analysis. In total 218 plasma samples were analyzed from this cohort of patients, with 30 plasma samples being new samples not previously analyzed.
Processing and DNA extraction from tumor samples
Tissue, from core biopsies or surgical resection, was formalin-fixed and embedded in paraffin (FFPE). Sections (4-8 x 4µm) were stained with Nuclear Fast Red (NFR), reviewed and marked for tumor content by a qualified pathologist and micro-dissected under a stereomicroscope to achieve >80% tumor cell content. Tumor DNA was isolated using the QIAamp DNA FFPE Tissue Kit (Qiagen) as per manufacturer’s instructions. Germline DNA was extracted from buffy coat DNA using DNeasy Blood and Tissue kit (Qiagen) as per manufacturer instructions.
Processing of blood and extraction of DNA
Blood was collected in either EDTA blood collection tubes and processed within 2 hours following venipuncture (Royal Marsden Hospital), or preservative tubes (Streck), shipped to a central
laboratory, and separated within 46-72 hours post venipuncture with a single centrifugation at 1600 rpm for 20 min, with plasma stored at -80°C until DNA extraction. Following plasma aliquoting, buffy coat was separately stored at -80°C until DNA extraction.
Plasma DNA was extracted from 4 ml of plasma using the MagMAX™ Cell-Free DNA Isolation Kit (Thermo Scientific) on a KingFisher flex (Thermo Scientific) according to manufacturer instructions. The DNA was eluted into 100 μl elution buffer, quantified and stored at -20°C.
Supermix for probes (Bio-Rad) and 1 µl of TaqMan Copy Number Reference Assay, human, RNase P (Life Technologies) on a total volume of 20 µl as previously described.1
Panel sequencing of diagnosis tumor samples
Tumor DNA was sequenced with a panel targeting 14 known driver breast cancer genes1 on an Illumina MiniSeq sequencer, Sequencing libraries were prepared with a custom Ion AmpliSeq™ Breast Cancer Panel using a modified AmpliSeq Library Preparation protocol with 5ng tumor DNA. Following initial library amplification and primer digestion, amplified libraries were end-repaired and dA tailed using the NEBNext Ultra II End Prep kit according to manufacturer instructions. NEBNext Adaptor for Illumina were added to the libraries as per manufacturer instructions and libraries enriched using NEBNext Ultra II Q5 and 6 cycles of PCR. Enriched libraries were cleaned and ran on an Illumina MiniSeq sequencer to a mean read of 846X and 77.47% on target reads.
Sequencing reads were aligned and BAM files generated with BWA. Pileup was used to estimate point mutations at each base counting reads with mapping quality > 30. Bases required a base quality > 30 with a minimum of 5 reads to be called as a variant. A combination of callers including platform specific callers such as Torrent Caller and llumina Isis and Vardict2 were used to call variants. All calls were finally manually curated as an extra quality control before being called positive.
Whole Exome Sequencing
DNA was obtained from twelve tumors and their corresponding buffy coat and sequenced at BGI (Honk Kong) on a HiSeq 4000 sequencer from Illumina. 500 ng of tumor DNA and 500 ng of matched normal DNA obtained from buffy coat were used to construct libraries. The qualified DNA samples were randomly fragmented by Covaris technology, with the size of the library fragments mainly distributed between 200bp and 300bp. Adapters were ligated to both ends of the resulting fragments. Extracted DNA was amplified by ligation-mediated PCR (LM-PCR), purified, and hybridized to the exome array for enrichment. Non-hybridized fragments were then washed out. Captured LM-PCR products were quality checked on an Agilent 2100 Bioanalyzer and quantitative PCR to estimate the magnitude of enrichment. Each qualified captured library was then loaded onto an Illumina HiSeq 4000 platform. High-throughput sequencing for each captured library was performed to produce 6734.64 Mb of sequencing, mean 98.99% on-target reads, and a median de-duplicated depth of 74.5X.
Data was aligned to the human genome GRCh37 using BWA (version 0.7.12).3 Duplicates were removed using MarkDuplicates in Picard tools (version 2.0.1)4 and sequencing metrics were obtained using BedTools.5 Using the GATK toolkit (version 3.5.0)4, the tumor and germline samples were realigned around known indel sites as outlined in the GATK best practices. Somatic
insertion/deletions were called using a combination of three mutation callers: Mutect6, Mutect2 and Vardict.2. Data from the three callers was merged using a combination of the GATK toolkit and custom Perl scripts. Variants were annotated using ANNOVAR7 and any mutations in the 1000 genomes removed. Mutations included had a minimum coverage of 40X in the tumor and 20X in the germline at each position with a minimum variant allele frequency of 5%.
Development of personalized mutation specific digital PCR assays
DNA extracted from 4 ml of plasma was analyzed in 2 replicates, with each replicate represented in two wells with 1ml plasma equivalent (eFigure 2). Digital PCR was performed on a QX-200 dPCR system (Bio-Rad) using TaqMan chemistry with primers and probes at a final concentration of 900nM primers and 250nM probes. PCR reactions were prepared with ddPCR Supermix for probes (Bio-Rad) and partitioned into a median of 50,000 droplets per sample in an Auto droplet generator (Bio-Rad) according to manufacturer’s instructions. Emulsified PCR reactions were run on 96 well plates on a G-Storm GS4 thermal cycler incubating the plates at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and specific assay extension temperature for 60 sec, followed by 10 min incubation at 98°C. The temperature ramp increment was 2.5°C/sec for all steps. Plates were read on a Bio-Rad QX-200 droplet reader using QuantaSoft v1.7.4 software from Bio-Rad to assess the number of droplets positive for mutant DNA, wild type DNA, both or neither.
For control wells, at least two negative control wells with no DNA, two wells of unmatched plasma DNA with each well containing 10 ng DNA, two wells of time-point matched buffy coat DNA with each well containing 10 ng DNA, were included in each run.
Digital PCR analysis
A mutation was only considered to be present if two or more FAM positive droplets were detected in one 2 ml replicate, validated by at least another single FAM positive in the other 2 ml replicate. A mutation was considered to be present in the time-point matched buffy coat DNA if two of more FAM positive droplets were detected across two wells. Positive mutation tracking was defined as detection of ctDNA in any of the post-surgical serial plasma samples. Detection of mutation in the buffy coat DNA was attributed as being evidence of clonal hematopoiesis of indeterminate potential (CHIP), and mutations in that matching patient’s plasma DNA were prospectively excluded from MRD assessment as pre-planned.
To assess mutation fraction, the concentration of mutant DNA (copies of mutant DNA per droplet) was estimated from the Poisson distribution. Number of mutant copies per droplet Mmu = -ln (1-(nmu/n)), where nmu = number of droplets positive for mutant-FAM probe and n = total number of droplets. The DNA concentration in the reaction was estimated as follows MDNAconc = -ln (1-(nDNAcon/n)), where nDNAconc = number of droplets positive for mutant-FAM probe and/or Wild Type-VIC probe and n = total number of droplets. The Fraction Mutation = Mmu/ MDNAconc.
To assess the number of mutant copies per ml of plasma, the number of mutant-FAM positive droplets was adjusted for the number of wells run for the sample, the total number of droplets generated, the median volume of a droplet (0.89pl), and volume equivalent of plasma run, using the following formula:
Mutant copies per ml= (Total number of droplets positive for FAM) x 20,000 x (number of wells run/volume of plasma equivalents) / (total number of droplets generated*0.89).
Statistical analysis
All plasma DNA analysis was conducted blinded to clinical and pathological information.
Kaplan-A pre-planned combined analysis was conducted of the current study with our prior proof-of-principle study1 to increase power for a number of secondary analyzes. In the combined series we analyzed the relationship between ctDNA detection and relapse free survival in breast cancer subtypes (ER+ HER2-, HER2+ and TNBC), and the lead-time in each individual subtype.
In the combined series we analyzed factors that associated with the level of ctDNA in diagnosis samples prior to neoadjuvant chemotherapy, this analyzes excluded the 17 patients who received adjuvant chemotherapy. Factors were analyzed with Mann-Whitney U test for comparison of two groups, and with Kruskal-Wallis test for comparison of three or more groups.
In the combined series we also analyzed the factors associated with a failure of detect ctDNA prior to relapse, using Fisher’s exact test for two groups, and chi-squared for more than two groups.
All P values were two sided and considered to be significant at 0.05.
Study design
The sample collection studies were designed by the last two authors, and sponsored by The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research. The first two authors and the last author wrote the first draft of the manuscript. All authors reviewed the final draft and vouch for the accuracy of the data.
References
1. Garcia-Murillas I, Schiavon G, Weigelt B, et al. Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Science Translational Medicine.
2015;7(302).
2. Lai Z, Markovets A, Ahdesmaki M, et al. VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research. Nucleic Acids Research.
2016;44(11).
3. Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. BioInformatics. 2009;25(14).
4. McKenna A, Hanna M, Banks E, et al. The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Reserach.
2010;20:1297.
5. Quinlan AR. BEDTools: the Swiss-army tool for genome feature analysis. Current Protocols Bioinformatics. 2015;47.
6. Cibulskis K, Lawrence MS, Carter SL, et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature Biotechnology.
2013;21(3):213.
eFigure 1:Personalized digital PCR assays for mutation tracking of circulating tumor DNA in plasma of patients with early breast cancer.
eFigure 2: Reproducibility of digital PCR between replicates
eFigure 3: CONSORT diagram for the Study.
eFigure 4:Identified mutation by Massive Parallel Sequencing.
(A) Mutations distribution and Allele Frequency on the diagnostic tumor prior to any neoadjuvant chemotherapy. Median allele frequency (AF) was 26% (inter-quartile range IQR: 18%-37%).
(B) Mutation distribution and Allele Frequency of mutations identified in the first plasma sample DNA taken at diagnosis prior to any neoadjuvant chemotherapy. ctDNA was detected in 51% (41/80) patients, at a median AF of 0.36% (IQR: 0.13%-1.12%), and median 3.09 copies/ml (IQR: 0.8-8.68 copies/ml).
eFigure 5: Personalized mutation specific digital PCR assays accurately quantify DNA.
(A) Correlation between detected Allele Frequency (AF) in diagnosis tumor DNA by massively parallel sequencing and by mutation-specific digital PCR (Pearson’s correlation).
eFigure 6: Patients with CHIP identified in plasma cfDNA.
eFigure 8: Relapse free survival and Overall Survival of extension of proof-of-principle study.
eFigure 9: CONSORT diagram for the combined analysis.
Clinical and pathological features
N 101
Median Age (range) 52 (31-76)
Pathology
IDC 88
ILC 10
Mixed IDC/ILC 1
Other 2
Histological Grade
1 2
2 32
3 58
Not available 9
Receptor Status
ER+ HER2- 35
ER+ Her2+ 20
ER- Her2+ 21
Triple Neg 25
Clinical tumor size (cT)
1 15
2 69
3 15
4 2
Clinical nodal status
Neg 47
Positive 51
Not available 3 Adjuvant chemotherapy 17
Chemotherapy
Hazard ratio p-value 95% CI Hazard ratio p-value 95% CI
Negative 1 - - 1 -
-Positive 25.2 6.7 - 95.6 35.7 <0.001 6.0 - 212.1
ER+ HER2- 1 - - 1 -
-HER2+ 1.9 0.467 0.3 - 10.3 6.1 0.153 0.5 - 72.8
TNBC 6.4 0.027 1.2 - 33.4 7.5 0.125 0.6 - 98.2
T1-T2 1 - - 1 -
-T3-T4 2.3 0.169 0.7 - 7.7 0.2 0.179 0.0 - 2.1
No* 1 - - 1 -
-Yes 1.5 0.52 0.5 - 4.7 4.3 0.168 0.5 - 33.9
1 - 2 1 - - 1 -
-3 1.5 0.545 0.4 - 6.0 1.9 0.577 0.2 - 19.4
Unknown 2.5 0.317 0.4 - 15.0 2.6 0.464 0.2 - 33.0
No 1 - - 1 -
-Yes 0.3 0.126 0.1 - 1.4 0.2 0.120 0.0 - 1.6
Unknown - - -
-No 1 - - 1 -
-Yes 5.6 0.029 1.2 - 26.1 2.3 0.383 0.2 - 23.2
Not available 1.1 0.928 0.1 - 12.4 3.9 0.470 0.2 - 82.0
Tumour Grade
pCR
Baseline ctDNA detection
Unadjusted univariate models Adjusted multivariable model
ctDNA detection
Subtype
Staging size
Nodal involvement
eTable 2: Covariates used in the multi-variable Cox regression analysis
Main analysis Proof of Principle Metanalysis
N 101 43 144
Median Age (range) 52 (31-76) 50 (25-67) 52 (25-76)
Pathology
IDC 88 34 122
ILC 10 2 12
Mixed IDC/ILC 1 4 5
Other 2 2 4
Unknown 1 1
Histological Grade
Grade 1 2 0 2
Grade 2 32 13 45
Grade 3 58 29 87
Not available 9 1 10
Receptor Status
ER+ HER2- 35 16 51
ER+ Her2+ 20 9 29
ER- Her2+ 21 5 26
Triple Neg 25 13 38
Clinical tumor size (cT)
1 15 5 20
2 69 24 93
3 15 5 20
4 2 8 10
Not available 1 1
Clinical nodal status
Neg 47 20 67
Positive 51 23 74
Not available 3 0 3
Chemotherapy
Neoadjuvant chemotherapy 84 43 127
Residual invasive cancer 54 34 88
Pathological complete response 29 8 37
n median ctDNA level (IQR) copies/ml P value
N 122 0.76 (0-8.0)
Pathology
IDC 104 0.76 (0-7.0) 0.13
Non-IDC 8 0 (0.-0.4)
Histological Grade
Grade 2 36 0 (0-2.6) 0.045
Grade 3 77 2.0 (0-11.4)
Subtype
ER+ HER2- 39 0 (0-4.4) 0.006*
HER2+ 48 0.81 (0-5.4)
Triple Neg 35 4.96 (0-17.0)
Clinical size at presentation (cT)
T1 12 0 (0-0) 0.012*
T2 80 0.76 (7.0)
T3/4 29 3 (0.14-15.6)
Nodal status at presenation
Positive 59 0 (0-8.0) 0.13
Neg 60 2.7 (0-8.4)
Pathological response to NAC
No pathCR 35 0.42 (0-6.6) 0.39
pathCR 85 1.2 (0-9.3)
eTable 4:Clinical and pathological factors associated with ctDNA level at diagnosis prior to treatment
