Abstract A29: Next-generation sequencing of circulating tumor DNA to monitor treatment response to CDK4/6 inhibitors in breast cancer
Authors: Chin Yoon Ming , Shibayama Tomoko , Otaki Masumi , Chan Hiu Ting , Ono Makiko , Ito Yoshinori , Takahashi Shunji , Ohno Shinji , Ueno Takayuki , Nakamura Yusuke , Low Siew-Kee 
Published: June 2020

Abstract
Background: CDK4/6 inhibitors are effective treatment modalities against hormone receptor-positive metastatic breast cancer (MBC). However, monitoring of clinical responses relies solely on conventional tumor markers that are insufficiently sensitive and specific as well as imaging methods that could not be performed regularly. In this study, we aimed to evaluate the potential utility of circulating tumor DNA (ctDNA) as a complementary approach to monitor clinical response and to evaluate resistance mechanisms associated with CDK4/6 treatment in MBC.
Method: The monitoring duration of this study is up to 24 months or until patient develops disease progression. In this interim report, a total of 100 samples from 21 MBC patients who received CDK4/6 inhibitors were collected before and after treatment. Cell-free DNA/RNA was extracted from plasma and subjected to ultradeep targeted next-generation sequencing (NGS) using Oncomine Pan-Cancer cell-free assay that incorporates the use of unique molecular index. This panel covers more than 1,000 hotspot mutations, 12 CNVs, and 12 fusions in cancer-related genes. Paired NGS was performed with genomic DNA from buffy coat to exclude clonal-hematopoiesis derived mutations. The changes in mutation allele frequency of ctDNA will be compared with tumor markers CA15-3 and CEA as well as CT-imaging and PET-scan results.
Results: According to RECIST criteria, one patient showed complete response 9 months after start of treatment while 11 patients showed partial response or stable disease within 3-12 months from start of treatment. A total of 9 out of 21 MBC patients developed progression of disease within 9 months of treatment. The total and molecular depth of targeted NGS are ~50,000X and ~4000X, respectively. A total of 20 nonsynonymous mutations and 5 copy number variations (CNV) were detected in 16 of the 21 patients (76%) before treatment for response monitoring. The majority of the mutations are in TP53, PIK3CA, and ESR1 genes. The ctDNA profiles were concordant with clinical responses in the majority of the patients. Mutation allele frequencies of ctDNA showed better dynamic changes compared with tumor biomarkers CEA and CA15-3. NGS of ctDNA was able to detect disease progression 3 months in advance or concurrent with imaging data in this study. Diverse ctDNA profiles were observed, indicating the usefulness of ctDNA in detecting intra- and intertumoral heterogeneity. We observed acquired mutations in AKT1 and PIK3CA in one patient, suggesting these mutations were potential CDK4/6 resistance mutations.
Conclusion: Our results suggest that ctDNA may be applied as a robust biomarker to monitor clinical response to the CDK4/6 treatment.
Citation Format: Yoon Ming Chin, Tomoko Shibayama, Masumi Otaki, Hiu Ting Chan, Makiko Ono, Yoshinori Ito, Shunji Takahashi, Shinji Ohno, Takayuki Ueno, Yusuke Nakamura, Siew-Kee Low. Next-generation sequencing of circulating tumor DNA to monitor treatment response to CDK4/6 inhibitors in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A29.