Abstract 1406: Are circulating microRNAs (miRNAs) ready for inclusion as biomarkers in breast cancer clinical trials
Authors: Amankwatia Edward B. , Bennett Alexandra , Ou David , Jamieson David , Cresti Nicola , Cain Henry , McNeillis Rosie , Womack Jonathan , Howell Sacha , Harvie Michelle , May Felicity , Greystoke Alastair 
Published: September 2020

Abstract
Background: Circulating miRNAs have potential as surrogate measures of tumor burden to monitor treatment response in clinical trials due to their association with cancer and high stability in blood. Greystoke et al (PMID: 26654130) showed that 10 miRNAs (miRs-200a, 200b, 200c, 141, 429, 95, 195, 210, 335, 375) were elevated in plasma of patients with a range of solid tumors compared to in plasma of healthy volunteers (HVs). The miRNA levels decreased after treatment, especially in patients with larger clinical responses. In the present study, the same 10 miRNAs, and 3 breast cancer (BC) related miRNAs identified in a literature search (miRs-18b, 21, 148b), were analysed to determine if their measurement i) is more sensitive in plasma or serum, ii) distinguishes BC patients from HVs, and iii) changes after surgery or systemic therapy.
Methods: The 13-miRNA panel (13-plex) was analysed by qPCR in plasma and serum from 12 BC patients and 8 HVs, and in plasma from 19 BC patients pre- and 4 hours post-surgery with a multiplex of TaqManTM MiRNA Assays. Expression of the 13-plex was measured with custom TaqManTM Low Density Array (TLDA) cards after pre-amplification, in serum collected at cycle 1, day 1 (C1D1) and day 8 (C1D8) from 44 BC patients starting adjuvant chemotherapy on the B-AHEAD 2 study (ISRCTN04156504). Endogenous miR-16 and exogenous ath-miR-159a provided standards to assess miRNA extraction efficiency. Results are reported as the geometric means of the 40-Ct values for the 13 miRNAs (geomean13).
Results: Expression of the 13-plex was significantly higher in plasma compared to in matched serum. TLDA analysis of 381 miRNAs confirmed that the plasma to serum superiority was a general effect not restricted to the 13-plex. The superiority of plasma was reduced however by pre-amplification; in the 381-miRNA TLDA analysis, detection in serum improved compared to in the multiplex qPCR assay.
The 13-plex was elevated in plasma of BC patients compared to HVs (p = 0.015). There was a trend towards a reduction after surgery, but the difference did not reach statistical significance. However, miR-195, a BC specific marker, was significantly lower after surgery (p = 0.0001). In the B-AHEAD 2 study cohort, the 13-plex was decreased at C1D8 compared to C1D1 (p = 0.0002). The three patients who relapsed within 5 years of diagnosis had geomean13 values at C1D1 of 10.3, 10.6 and 11.8 compared to the median of 10.3 for all patients. Given the low event rate, either a larger patient cohort or a cohort with a higher event rate is required to assess association with clinical outcome.
Conclusions: This study shows that plasma is better than serum for miRNA evaluation but that measures in serum are useful if cDNA is pre-amplified after reverse transcription. The 13-plex is elevated in BC, has a trend towards a fall after surgery and is reduced after chemotherapy. The 13-plex has potential as a biomarker of tumor burden in BC trials.
Citation Format: Edward B. Amankwatia, Alexandra Bennett, David Ou, David Jamieson, Nicola Cresti, Henry Cain, Rosie McNeillis, Jonathan Womack, Sacha Howell, Michelle Harvie, Felicity May, Alastair Greystoke. Are circulating microRNAs (miRNAs) ready for inclusion as biomarkers in breast cancer clinical trials [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1406.