Detection of Cancer DNA in Plasma of Patients with Early-Stage Breast Cancer

Abstract

Purpose Detecting circulating plasma tumor DNA (ptDNA) in early stage cancer patients has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection. Experimental Design In this prospective study, primary breast tumors and matched pre- and post-surgery blood samples were collected from early stage breast cancer patients (n=29). Tumors (n=30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR. Results Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Pre-surgery plasma samples (n=29) were then analyzed for PIK3CA mutations using ddPCR. Of the fifteen PIK3CA mutations detected in tumors by ddPCR, fourteen of the corresponding mutations were detected in pre-surgical ptDNA, while no mutations were found in plasma from patients with PIK3CA wild type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation positive ptDNA pre-surgery had ddPCR analysis of post-surgery plasma, with five patients having detectable ptDNA post-surgery. Conclusions This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in early stage breast cancer patients. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients.

WITHDRAWN: Prognostic value of circulating tumor DNA in breast cancers

“…Following whole-genome sequencing, ctDNA can be detected in more than 75% of patients with advanced disease and 50% of patients with early-stage disease [7]. Furthermore, the assay has a high sensitivity and specificity of 90% and 100%, respectively, showing the feasibility of detecting DNA mutations in the plasma of early-stage breast cancer patients, including patients with minimal, clinically undetectable disease [15]. Moreover, ctDNA can be used as a tool to monitor changes in tumor burden among metastatic breast cancer patients undergoing systemic therapy [16].…”

Detection of the <i>PIK3CA</i> Mutation in Circulating Tumor DNA as a Possible Predictive Indicator for Poor Prognosis of Early-Stage Breast Cancer

“…It is noteworthy that 85% of the fractional abundance of the mutated PIK3CA in ctDNA is lower than 0.1% even after pre-amplification in their study. In our study, by contrast, we directly quantified the ctDNA without pre-amplification by ddPCR and adopted the cut-off value of 0.1%, ten-fold higher than that in the study by Beaver et al [10], considering the difference in the detection rate of mutation in the platform by RainDance Technologies (Billerica, MA, USA) in their analysis and in that by Bio-Rad Laboratories (Hercules, CA, USA) in our analysis. It is also interesting that the detection rate of pre-surgery ctDNA in early breast cancer was reported to be 23% in another retrospective study using serum as a source of ctDNA [11], suggesting that serum would be less appropriate for this type of analysis.…”

“…They subsequently detected the mutant PIK3CA in 13 of 14 (93%) cases of ctDNA from pre-surgery plasma of the patients harboring the relevant mutation in primary tumors [10]. The higher detection rate of mutation in pre-surgery ctDNA in their study in comparison with that of 33% in our study would be caused by their unique procedure of digital PCR platform, including 22-cycles of pre-amplification of ctDNA and the low cut-off value of 0.01%.…”

“…(5'-cttacctgtgactccatagaa -3') and sequencing (5'-gctagagacaatgaattaaggg -3'), and PIK3CA H1047R; forward (5'-gatgacattgcatacattcg -3'), reverse (5'-gtggaagatccaatccattt -3'), and sequencing (5'-cgaaagaccctagccttag -3'), as described by Beaver, J. A. et al [10].…”

Is Circulating Tumor DNA (Ctdna) Use Ready For Prime Time? Applications and Challenges of Ctdna in the Era of Precision Oncology

“…Cell-free DNA consists of noncancerous nucleic acids and circulating tumor DNA (ctDNA). The proportion of ctDNA depends on the tumor cell of origin and stage of malignancy [2][3][4][5]. Peripheral blood biopsies can detect single nucleotide variants, indels, copy number variants, rearrangements and fusions, non-invasively, avoiding risk associated with repeat tissue biopsies.…”