Comparative study of droplet-digital PCR and absolute Q digital PCR for ctDNA detection in early-stage breast cancer patients.

BACKGROUND: Analysis of circulating tumor DNA (ctDNA) is increasingly used for clinical decision-making in oncology. However, ctDNA could represent ≤ 0.1 % of cell-free DNA in early-stage tumors and its detection requires high-sensitive techniques such as digital PCR (dPCR). METHODS: In 46 samples from patients with early-stage breast cancer, we compared two leading dPCR assays for ctDNA analysis: QX200 droplet digital PCR (ddPCR) system from Bio-Rad which is the gold-standard in the field, and Absolute Q plate-based digital PCR (pdPCR) system from Thermo Fisher Scientific which has not been reported before. We analyzed 5 mL of baseline plasma samples prior to any treatment. RESULTS: Both systems displayed a comparable sensitivity with no significant differences observed in mutant allele frequency. In fact, ddPCR and pdPCR possessed a concordance > 90 % in ctDNA positivity. Nevertheless, ddPCR exhibited higher variability and a longer workflow. Finally, we explored the association between ctDNA levels and clinicopathological features. Significantly higher ctDNA levels were present in patients with a Ki67 score > 20 % or with estrogen receptor-negative or triple-negative breast cancer subtypes. CONCLUSION: Both ddPCR and pdPCR may constitute sensitive and reliable tools for ctDNA analysis with an adequate agreement in early-stage breast cancer patients.

Increased blood draws for ultrasensitive ctDNA and CTCs detection in early breast cancer patients.

Early breast cancer patients often experience relapse due to residual disease after treatment. Liquid biopsy is a methodology capable of detecting tumor components in blood, but low concentrations at early stages pose challenges. To detect them, next-generation sequencing has promise but entails complex processes. Exploring larger blood volumes could overcome detection limitations. Herein, a total of 282 high-volume plasma and blood-cell samples were collected for dual ctDNA/CTCs detection using a single droplet-digital PCR assay per patient. ctDNA and/or CTCs were detected in 100% of pre-treatment samples. On the other hand, post-treatment positive samples exhibited a minimum variant allele frequency of 0.003% for ctDNA and minimum cell number of 0.069 CTCs/mL of blood, surpassing previous investigations. Accurate prediction of residual disease before surgery was achieved in patients without a complete pathological response. A model utilizing ctDNA dynamics achieved an area under the ROC curve of 0.92 for predicting response. We detected disease recurrence in blood in the three patients who experienced a relapse, anticipating clinical relapse by 34.61, 9.10, and 7.59 months. This methodology provides an easily implemented alternative for ultrasensitive residual disease detection in early breast cancer patients.