Analysis of tumor template from multiple compartments in a blood sample provides complementary access to peripheral tumor biomarkers

Targeted cancer therapeutics are promised to have a major impact on cancer treatment and survival. Successful application of these novel treatments requires a molecular definition of a patient's disease typically achieved through the use of tissue biopsies. Alternatively, allowing longitudinal monitoring, biomarkers derived from blood, isolated either from circulating tumor cell derived DNA (ctcDNA) or circulating cell-free tumor DNA (ccfDNA) may be evaluated. In order to use blood derived templates for mutational profiling in clinical decisions, it is essential to understand the different template qualities and how they compare to biopsy derived template DNA as both blood-based templates are rare and distinct from the gold-standard. Using a next generation re-sequencing strategy, concordance of the mutational spectrum was evaluated in 32 patient-matched ctcDNA and ccfDNA templates with comparison to tissue biopsy derived DNA template. Different CTC antibody capture systems for DNA isolation from patient blood samples were also compared. Significant overlap was observed between ctcDNA, ccfDNA and tissue derived templates. Interestingly, if the results of ctcDNA and ccfDNA template sequencing were combined, productive samples showed similar detection frequency (56% vs 58%), were temporally flexible, and were complementary both to each other and the gold standard. These observations justify the use of a multiple template approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and open a path to comprehensive blood derived biomarker access.


Library preparation and ion torrent sequencing
Primary library construction was performed with either ccfDNA, ctcDNA, or wbcDNA and AmpliSeq Library kit v2.0 according to the manufacturers instructions. The reaction contained ≤1ng template. Samples were barcoded and purified on Ampure beads. The quantity of libraries was assessed using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific).
The Ion AmpliSeq™ Cancer Hotspot Panel v2 is a single pool of primers consisting of 207 amplicons spanning 50 gene. It is used to perform multiplex PCR for preparation of amplicon libraries from genomic regions that are frequently mutated in cancer. The library base is ~33 kb and surveys ~3 x 10 3 COSMIC identified mutations. Following library construction, and using manufacturers best practice, libraries were barcoded, purified, and used for ISP emulsion formation. Amplified samples were quantitated using Ion Library Quantitation Kit (Thermo Fisher Scientific). ISP pools consisted of three barcoded libraries ccfDNA/ctcDNA/wbcDNA were then sequenced. ctcDNA and wbcDNA were co-sequenced on the same 318 chip using the IonTorrent PGM (Thermo Fisher Scientific). For each matched ccfDNA sample, due to the low template quality, samples were sequenced using a single dedicated 318 chip. Similarly each FFPE sample was sequenced on a single 318 chip.

Comparison of the template for amplification
ccfDNA and ctcDNA are radically different types of template, despite being derived from whole blood. Both templates offer the possibility of utility for clinical diagnostic purposes. In order to understand the relative value of each template, it was important to understand the quality of the two templates and how it impacts what information can be gathered and how they should be evaluated. Thus one goal of this study was to compare subject-matched ctcDNA and ccfDNA templates by NGS. In order to report a fair comparison, three different qualities of the DNA templates were used. The first quality was "amplicon performance" which is defined to mean how well a particular DNA template supported PCR amplification with primers spanning amplicons from the Ion AmpliSeq Cancer Hotspot Panel v2 which contains multiplexed primers for 207 amplicons. Performance for all amplicons were evaluated for a cohort of known negative normal healthy donor samples which were processed for recovery using the same EpCAM enrichment of circulating epithelial cell derived DNA (cecDNA). Comparative analysis was performed after normalizing either cecDNA or cell free DNA (cfDNA) amplicon-performance results to subject-matched germline wbcDNA control. 29 subjects were compared ( Figure 2). Amplicons are presented arranged by size with the smallest amplicons on the left and the largest amplicons on the right. As the amplicon size increases, the efficiency of amplicon generation relative to the germline control diminishes. The variance for ctcDNA samples was fairly consistent across all amplicon sizes. However for cfDNA samples the variance for larger amplicons is very low, consistent with very low amplicon coverage in the larger size range. This is a consistent with the well known fragmented nature of the ccfDNA template [1,2]. For the purposes of head to head comparison of ccfDNA and cecDNA, these data demonstrate that approximately half the amplicons in cfDNA amplify the target sequence less efficiently than control. Therefore, for a 1% threshold sequence analysis, additional sequencing capacity has to be applied to the analysis of cfDNA.
For a DNA source to be useful in clinical testing, it must support unbiased analysis. To a first approximation, in a NGS test, PCR amplification, as judged by amplicon amplification performance is a test of uniformity. A powerful NGS test should not exhibit significant amplicon to amplicon variation or biasing. In the data presented, biasing is observed for ccfDNA. That biasing is not simply a function of amplicon size is shown clearly in Figure 2.
Here the variance between identical amplicons in different samples is substantial. ctcDNA templates show very low variance across all amplicons and sizes. The amplicon performance variance for subject matched ccfDNA samples is quite broad. The smaller amplicons show very large differences in variance while the most under-represented large amplicons show very little variance. That the variance for the larger amplicons is low is not surprising as they perform poorly. What is notable is that "well" performing small amplicons exhibit a large variance. That the smaller amplicons exhibit such template variance suggests that quantitative tests (like CNV) using amplicon performance may have significant technical challenges, especially in reproducibility of measurements due to confounding factors. The pellets were engineered with a linear range between 0.75% and 50% MCL representation. The pellets were sequenced in triplicate using GM12878 cells as a germline case control sequence. Using this approach, the sequencing was shown to be 100% sensitive to alterations present at greater than 1% representation. Furthermore the false positive rate was indexed as 0.0015% across all 20000 bp of the library. Reportable range was COSMIC identified alterations in the Ampliseq panel C.

ID
Epcam ctcDNA FFPE 1 FFPE 2 C293-032 X X C293-033 TP53; p.C176F TP53; p.C176F Alterations are indicated by the target gene and the predicted impact. Samples with no detectable alteration at the limit of detection are indicated by X. Samples that were quantity not sufficient (QNS) are indicated.