A Study of Pre-Analytical Variables and Optimization of Extraction Method for Circulating Tumor DNA Measurements by Digital Droplet PCR

Abstract

Background: Circulating free DNA (cfDNA) is an exciting novel method to diagnose, monitor, and predict resistance and response to cancer therapies, with the potential to radically alter the management of cancer patients. To fulfill its potential, greater knowledge about preanalytical variables is required to optimize and standardize the collection process, and maximize the yield and utility of the small quantities of cfDNA extracted. Methods: To this end, we have compared the cfDNA extraction efficiency of three different protocols, including a protocol developed in house (Jewish General Hospital). We evaluated the impact on cfDNA levels of preanalytical variables including speed and timing of the second centrifugation and the use of k-EDTA and CTAD blood collection tubes. Finally, we analyzed the impact on fractional abundance of targeted pre-amplification and whole genome amplification on tumor and circulating tumor DNA (ctDNA) from patients with breast cancer. Results: Making use of a novel protocol for cfDNA extraction we increased cfDNA quantities, up to double that of commercial kits. We found that a second centrifugation at 3,000 g on frozen plasma is as efficient as a high-speed (16,000 g) centrifugation on fresh plasma and does not affect cfDNA levels. Conclusions: These results allow for the implementation of protocols more suitable to the clinical setting. Finally, we found that, unlike targeted gene amplification, whole genome amplification resulted in altered fractional abundance of selected ctDNA variants. Impact: Our study of the preanalytical variables affecting cfDNA recovery and testing will significantly enhance the quality and application of ctDNA testing in clinical oncology.

Prognostic and predictive value of circulating tumor DNA during neoadjuvant chemotherapy for triple negative breast cancer

“…These targeted pre-amplifications, which involved the generation of specific PCR primers for each DNA fragment containing the selected variant, were performed for each variant in a multiplex PCR, enabling the testing of multiple variants from the same DNA sample with limited usage of the precious DNA per variant tested. As we reported, such "targeted pre-amplification" shows consistently similar MAFs compared to non-pre-amplified samples, even in samples with MAFs as low as 0.05% 15 . In order to ensure the reliability of the calling of "detection", we established stringent criteria using a pool of normal samples: a positive ctDNA value had to be 2 standard deviations above the mean of values in 3 normal pools.…”

“…To better understand the sensitivity of ctDNA in TNBCs, we used whole exome sequencing data in 26 TNBCs to generate an average of 5 personalized assays per patient, which we tested against a panel of 30 control plasma samples obtained from cancer-free patients in order to ascertain their specificity. We found a range of MAF from undetectable to 0.32%, with a median of 0.024%, in the normal plasma pools, which are likely due to the pre-amplification step used in our published protocol 15 .…”

“…We observed a broad range of MAF in normal plasma, from 0 (27% of variants) to 0.32%, with a median of 0.02% (Supplementary Table S4). The levels of MAF detected may be due to the pre-amplification step in our protocol 15 . These targeted pre-amplifications, which involved the generation of specific PCR primers for each DNA fragment containing the selected variant, were performed for each variant in a multiplex PCR, enabling the testing of multiple variants from the same DNA sample with limited usage of the precious DNA per variant tested.…”

“…cfDnA extraction. Extraction was performed from 3 ml of plasma using our previously reported protocol 15 .…”

“…We recently completed the Q-CROC-03 clinical trial, in which patients with TNBC undergoing NAC were consented for biopsies pre-chemotherapy and post-chemotherapy as well as serial bloods before, during and after NAC 14 to determine molecular factors of response or resistance to standard NAC both in tumor tissue and plasma. We also recently reported on optimized protocols for cfDNA extraction and digital droplet polymerase chain reaction (ddPCR) analysis 15 . We applied these protocols to the analysis of serial plasma from 26 patients enrolled in Q-CROC-03, by developing personalized ddPCR assays based on whole exome sequencing (WES) of each tumor.…”