Abstract P5-01-17: Unbiased copy number analysis of circulating tumor DNA among HER2-positive breast cancer patients
Authors: Oikawa Masahiro , Enomoto Yasuko , Hisamatsu Kazuhumi , Hamada Tetsuo , Otsubo Ryota , Yano Hiroshi , Nagayasu Takeshi , Mishima Hiroyuki , Yoshiura Koh-ichiro , Navin Nicholas 
Published: March 2020

Abstract
Background: Circulating tumor DNA (ctDNA) is shed into the blood stream from cancer cells at primary and metastatic sites. Analysis of ctDNA from plasma reveals genomic profiles of tumors in a non-invasive way, which is promising for overcoming treatment resistance caused by tumor genetic heterogeneity. However, a large amount of DNA from normal cells exists in cell-free DNA (cfDNA), which makes detection of ctDNA difficult without information on genomic aberration at primary and metastatic sites. Recently, the Nick Navin Lab developed an unbiased method for ctDNA analysis, called PEGASUS (Plasma Exome and Genome Analysis by Size-selection and Unbiased Sequencing), that enables whole-genome sequencing of copy number aberrations and somatic mutations from ctDNA. Purpose: This study applied PEGASUS to plasma samples from HER2-positive breast cancer patients, to delineate the detection rate of ctDNA and correlation between ctDNA and clinical course, concentration of cfDNA, and therapeutic outcome. Methods: Nineteen patients with stage I to IIIC of HER2-positive breast cancer who had not received any therapy for breast cancer (Cohort A) and eight HER2-positive patients with a metastatic lesion who were undergoing therapy for breast cancer (Cohort B) were enrolled in the study with written informed consent. In cohort A, plasma samples were collected before and after the therapy. In cohort B, plasma samples were collected serially up to three times. cfDNA from 1 to 5 mL of plasma was extracted by a QIAmp cell-free DNA extraction kit (QIAGEN). Concentration and size distribution of cfDNA was determined by LabChip GX (Perkin Elmer). Following size selection by magnetic beads purification, a multiplex barcoded library was constructed by the KAPA hyper prep Illumina kit (KAPA Biosystems). For genome-wide copy number analysis, the pooled library was sparsely (0.1x) sequenced by the Illumina MiSeq platform. FASTQ files were aligned to the reference human genome (hg19) by bowtie2. The copy number profiles were generated using the median bin count with the Varbin program. Tumor fraction (TFx) was calculated by the ichorCNA program and the threshold of detecting ctDNA was set to 0.03. Results: The mean concentration of cfDNA at the enrollment was 11.1 ng/mL and 25.0 ng/mL in cohort A and cohort B, respectively. The concentration of cfDNA increased during therapy in cohort A, but it correlated to neither TFx nor therapeutic outcome. The mean TFx at the enrollment was 0.019 and 0.036 in cohort A and cohort B, respectively. ctDNA was detected in four cases (21%) and five cases (62.5%) at any timepoint in cohort A and B, respectively. Among the patients in whom ctDNA was detected, two cases showed ERBB2 amplification in both cohort A (10.5%) and cohort B (25%). In an advanced case in cohort A, TFx was 0.065 and ERBB2 amplification was detected. After neoadjuvant chemotherapy with trastuzumab and pertuzumab, pCR was obtained and the ctDNA disappeared. In the case in cohort B, ctDNA and ERBB2 amplification was diminished along with the clinical CR by the therapy. In another case in cohort B, ctDNA and ERBB2 amplification emerged with the progression of disease despite HER2 targeted therapy. Conclusions: ctDNA and ERBB2 amplification was successfully detected among patients with HER2-positive breast cancer using unbiased copy number analysis by PEGASUS.
Citation Format: Masahiro Oikawa, Yasuko Enomoto, Kazuhumi Hisamatsu, Tetsuo Hamada, Ryota Otsubo, Hiroshi Yano, Takeshi Nagayasu, Hiroyuki Mishima, Koh-ichiro Yoshiura, Nicholas Navin. Unbiased copy number analysis of circulating tumor DNA among HER2-positive breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-17.