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Abstract 2280: Co-detection of circulating tumor DNA and RNA in the plasma of patients with breast cancer increases the detectable number of mutated molecules

Introduction: Circulating tumor DNA (ctDNA) has been shown in several studies to be a biomarker to predict tumor relapse and prognosis in breast cancer. However, the sensitivity of detection using ctDNA is modest in early-stage breast cancer and in patients with low-burden metastatic disease. Unlike ctDNA, which is released into circulation through necrosis or apoptosis, circulating tumor RNA (ctRNA) can be released into the circulation from living cancer cells. Therefore, detecting ctRNA along with ctDNA could be a potential solution to increase the sensitivity of detection. This pilot study is aiming to investigate the contribution of ctRNA in increasing the level of mutation detection in patients with breast cancer. Methods: Plasma samples were isolated from six patients with metastatic breast cancer for this proof of concept study. Circulating nucleic acids (DNA+RNA) were extracted from plasma for each patient and split into 2 groups: ctDNA+ctRNA OR ctDNA alone. In the ctDNA/ctRNA co-detection group, circulating nucleic acids underwent reverse transcription, followed by library preparation using the Roche Avenio ctDNA Expanded Kit, a capture-based target enrichment sequencing methodology. For the control group, the same amount of circulating cell-free DNA underwent the same library preparation (with no reverse transcription) for ctDNA detection. Sequencing was performed on an Illumina NextSeq to obtain at least 45 millions reads per sample. Results: All six patients had at least one somatic mutation detected. TP53 mutations were detected in three patients, followed by PIK3CA mutations in two patients and ERBB2 mutation in one patient. In total 14 mutations were detected across the 6 patients. The distribution of allele frequency among all mutations was from 0.41% to 11.1%. Given that the molecular barcode methodology was applied, we were able to determine the number of unique mutated molecules. Notably, the numbers of mutant molecules in the ctDNA+ctRNA co-detection group were all higher than the corresponding mutations in ctDNA-only group. We observed an average percentage increase of 67% (range=2.8%-385%) in the number of mutated molecules when mutations from ctRNA were co-detected with ctDNA. Discussion: Compared to ctDNA only, incorporation of ctRNA into the sequencing assay demonstrated increased detection of mutated molecules. This study has shown that the enhancement of mutation detection by adding ctRNA could be a potential method to increase the sensitivity of circulating tumor mutations in breast cancer. A validation study in patients with early-state triple-negative breast cancer to predict relapse using ctDNA/ctRNA co-detection is currently in progress. Citation Format: Yu-Hsiang Chen, Bradley A. Hancock, Jeffrey P. Solzak, Milan Radovich. Co-detection of circulating tumor DNA and RNA in the plasma of patients with breast cancer increases the detectable number of mutated molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2280.