Comparison of two targeted ultra-deep sequencing technologies for analysis of plasma circulating tumour DNA in endocrine-therapy-resistant breast cancer patients

Purpose There is growing interest in the application of circulating tumour DNA (ctDNA) as a sensitive tool for monitoring tumour evolution and guiding targeted therapy in patients with cancer. However, robust comparisons of different platform technologies are still required. Here we compared the InVisionSeq™ ctDNA Assay with the Oncomine™ Breast cfDNA Assay to assess their concordance and feasibility for the detection of mutations in plasma at low (< 0.5%) variant allele fraction (VAF). Methods Ninety-six plasma samples from 50 patients with estrogen receptor (ER)-positive metastatic breast cancer (mBC) were profiled using the InVision Assay. Results were compared to the Oncomine assay in 30 samples from 26 patients, where there was sufficient material and variants were covered by both assays. Longitudinal samples were analysed for 8 patients with endocrine resistance. Results We detected alterations in 59/96 samples from 34/50 patients analysed with the InVision assay, most frequently affecting ESR1, PIK3CA and TP53. Complete or partial concordance was found in 28/30 samples analysed by both assays, and VAF values were highly correlated. Excellent concordance was found for most genes, and most discordant calls occurred at VAF < 1%. In longitudinal samples from progressing patients with endocrine resistance, we detected consistent alterations in sequential samples, most commonly in ESR1 and PIK3CA. Conclusion This study shows that both ultra-deep next-generation sequencing (NGS) technologies can detect genomic alternations even at low VAFs in plasma samples of mBC patients. The strong agreement of the technologies indicates sufficient reproducibility for clinical use as prognosic and predictive biomarker. Supplementary Information The online version contains supplementary material available at 10.1007/s10549-021-06220-9.


Supplementary Methods
Blood processing and extraction of total cfDNA 20ml blood samples were collected from 50 radiologically confirmed MBC patients attending the Breast Clinic at Charing Cross Hospital in London. Patients were sampled at different times throughout the course of their treatment, aligning with their scheduled clinical appointments. Samples were taken along with concomitant clinical examination, and biochemical measurements, including serum cancer antigen 15-3 (CA15-3) and alkaline phosphatase (ALK-PHOS).

Targeted deep sequencing
Stored plasma was shipped on dry ice to the 2 laboratories in Cambridge and Leicester for independent analysis with the Inivata and Thermofisher workflows respectively. A minimum of 20ng total cfDNA isolated from 35 patients plasma samples were sequenced in duplicate, In brief, the InVision liquid biopsy panels (v1.4/v1.5) were used for sequencing 35/36 cancerrelated genes using gene specific primers designed to hotspots and entire coding regions of interest. NGS libraries were prepared using a two-step amplification process, incorporating replicate and patient specific barcodes and Illumina sequencing adaptors. Pooled libraries were quantified using Kapa Library Quantification Kit, and 1.8pM libraries analysed on an Illumina NextSeq 500 (300 cycle PE). Sequencing files were analysed using the Inivata Somatic Mutation Analysis (ISoMA) analytical pipeline (V1.15-1.17), and sequencing reads were clipped, merged and aligned. Coding and splice-site mutations in SNVs and Indels were annotated using Variant Effect Predictor (VEP) using the canonical transcript for each gene.
For the Oncomine™ Breast cfDNA Assay, library preparation was performed according to the manufacturer's instructions. In brief, targets were amplified from cfDNA, the target amplicons were purified and amplified with barcoded primers, the barcoded libraries were purified, size selected and quantified. Templating was performed on the Ion Chef using the Ion 530 Kit-Chef and 200bp-sequencing was performed using the Ion-Torrent-S5 on Ion 530 Chip (Thermo Fisher Scientific). 12 samples were multiplexed aiming for mean depth coverage on each chip, aiming for mean molecular coverage of 2500, allowing for detecting of mutations at variant allele fraction (VAF) 0.1%. Base calling, mapping and alignment were performed using Torrent-Suite-v5 software, with overall coverage and variants were called using the coverageAnalysis v5.2 and variantCaller v.5.2 plugins, respectively, with default analysis parameters.
For a full list of all genes, type of genetic alternations and the exon where the alternation is located targeted by each approach see Supplementary Table S4.

Analysis of sequential patient samples
Further, clinical and imaging assessment data detailing response to therapy was available for all 8 patients, as well as biochemical assessment measures (liver function tests, circulating tumour cell -CTC counts and CA15-3 levels) prior to, and at the time of, blood sampling.
Detection, enrichment and enumeration of CTCs of epithelial origin were performed using the CELLSEARCH ® system and the CELLSEARCH® Epithelial Cell Kit (Menarini Silicon Biosystems). Briefly, 7.5 ml of blood were collected in CellSave tubes (Menarini Silicon Biosystems), immunomagnetic capture of CTCs based on EpCAM and cell labelling were performed on the CELLTRACKS ® AUTOPREP ® System and detection of CTCs based on morphological characteristics, positive expression of cytokeratins (8,18,19) and absence of the leukocyte marker CD45 were performed using the CELLTRACKS ANALYZER II ® System. MBC samples with ≥5 CTCs were considered positive.

High concordance between ctDNA variants and variant allele fraction detected using the InVisionSeq TM ctDNA Assay and Oncomine TM Breast cfDNA Assay
If we consider only the overlapping samples and include all the alternations called by the InVision panel a total of 59 variants were identified in 16 genes including missense (n=46), frameshift (n=3), synonymous (n=3), truncating mutations (n=3), splice variants (n=1) and amplifications (n=3). The VAFs, reflected by 56 SNVs and INDELs, detected in ctDNA ranged from 0.18 to 59.61%, median VAF: 2.54%. Of these 59 variants, 28 were not represented on the Oncomine TM panel (Supplementary Table 5). All the remaining 31 variants (100%) detected by InVisionSeq TM were called by Oncomine TM . A single variant (PIK3CA p.Q546P) was detected by Oncomine TM in 2 plasma samples (VAFs 0.09% and 0.42%), that was not represented by the by InVisionSeq TM ctDNA assay. Oncomine TM also detected an additional 16 variants in 8 samples (0.05-0.46% VAF) in 3 genes (TP53, PIK3CA and ESR1) that were represented by, but not detected, by the InVision First TM First TM assay. If we consider all the variants identified in ctDNA by Oncomine TM (n=49), the median VAF was 0.35 and the range from 0.05 to 31.05%.

Longitudinal analysis of sequential patient samples on endocrine therapy
Patient 3 (Pt.56; Figure S1A) was diagnosed with ER+/PR+/HER2-IDC in May 2006, commenced neo-adjuvant chemotherapy. The patient relapsed in February 2012 with bone, liver and lung metastases, and was responding to capecitabine therapy at the time of collection of the first research blood sample, in which no mutations were detected in the plasma. One year later, endocrine therapy with everolimus and exemestane was started, on which she remained stable for 6 months (no mutations detected at this time point but total levels of ctDNA were elevated 10fold. However, the patients started progressing with new liver metastases and five months later was sampled again and a GATA3 p.408:S/SX frameshift mutation was detected at 2.06% VAF, alongside three low-level ERBB2 missense variants; p.S310Y (0.15%) p.S310F (0.33%) and p.I767M (0.13%) and a FGFR1 amplification. The patient died 2 months after the last sample.
CTC number and CA15-3 were elevated mostly at the last time point while total cfDNA levels fell by 20%.
Patient 4 (Pt.174; Figure S1B) was diagnosed with ER+/PR+/HER2-IDC 2011 and received letrozole, which she progressed on and was switched to exemestane in 2013. She had been receiving exemestane for 9 months and fulvestrant for 3 months prior to collection of the first research blood sample. At this point (stable disease), 4 mutations were detected; a most likely germline NFE2L2 p.R43W at a VAF of 50.85%, a PIK3CA p.E542K at 2.90%, a synonymous GNAS at 0.64% and an ESR1 p.Y537N at 0.34%. The patient gradually progressed despite this treatment and developed skull metastases. After 5 months NFE2L2 remained at 53.11% VAF, PIK3CA and ESR1 increased VAF and GNAS was disappeared, although a different synonymous GNAS mutation appeared at 0.18%. CTC number and CA15-3 were elevated, in contrast to cfDNA levels and ALK-PHOS.
Patient 5 (Pt.102; Figure S1C) was initially diagnosed with ER+/PR-/HER2+ IDC in 1996. In p.E545K and a NFEL2 p.E82A at low level too. All mutations levels were reduced during whole brain radiotherapy (the low levels ones disappeared) but were increased again after the start of methotrexate, while a FGFR1 amplification appeared after sample 2. Number of CTCs was increasing before a decrease at the fifth sample and CA15-3 increased before a big drop at the fourth sample.

Relationship to other markers of disease activity
We also measured levels of other blood-based markers at the same time points as the ctDNA sequencing: in all cases, at least two measures of disease activity were worse (levels increased), with the exception of patient 182 where only one measure was worse (total cfDNA level). All except one patient, patient 43 who had no CTCs at any time point, had measurable CTCs, and in all except patient 182 these showed a rise in number (7 of 8 cases; 87.5%). Supplementary Tables S1, S2,