Abstract P5-01-11: Tracking anchor mutations in serial cell-free DNA using ultra-high sensitive mass spectrometry method provide risk of subsequent recurrence during surveillance after standard therapy
Authors: Kim Jisun , Jo Whee Kyung , Choi Soo Jeong , Park Hwan , Lee Jiyoung , Lee Sae Byul , Lee Hee Jin , Kim Hee Jeong , Chung Il Yong , Ko Beom Seok , Lee Jong Won , Son Byung Ho , Ahn Sei Hyun , Kim Sung-Bae , Jung Kyung Hae , Ahn Jin-Hee , Chun Sung-Min 
Published: March 2020

Cell-free DNA (cfDNA), as a non-invasive strategy, provides substantial benefit to overcome temporal/spatial tumor heterogeneity. Surveillance of recurrence after standard treatment in early breast cancer (BC) using cfDNA, enables to detect minimal residual disease (MRD), also to identify genomic alterations driving recurrences. We aimed to assess whether cfDNA can detect metastasis during surveillance after standard treatment by tracking mutations using mass spectrometry method from minimal plasma volume. Thirty breast cancer patients with serial plasma adequately drawn for cfDNA analysis were analyzed. Twenty cases were who subsequently developed clinical recurrence while ten were clinically disease free till last follow up. Primary tumor DNA extracted from paraffin blocks of surgical specimen and germline DNA were sent for deep targeted sequencing using in-house Oncopanel (382 genes, 184 hotspots, 8 fusions, 0.82Mb) Mean sequencing depth of tumor was x284.5 and somatic mutations with &gt;5-10% variant allelic fraction (VAF) were chosen as anchor mutations for serial analyses. Hotspot mutations eg. PIK3CA H1047 or E545K were selected for serial tracking. We applied in-house developed mass stectrometry UHS-platform (Ultra-high sensitive) -detects 0.01% frequency mutant allele in colorectal and lung cancer- from plasma processed from 2ml whole blood. Mean number of anchor mutations is 5.95 and 74.6% of mutation sites were successfully designed as multiplex-probe for UHS-platform. Anchor mutations were searched in each patients’ blood drawn every 3-6months after surgery or after time of recurrence. Among 20 patients who were clinically diagnosed to have recurrence after standard treatment, 14 displayed ctDNA positive of at least one of anchor mutations (sensitivity, 70%, false negative rate (FNR) 30%). Sensitivity was not associated with input DNA amount nor with disease free interval. It was not associated with whether patients were receiving systemic therapy at time of recurrence. Among the 6 false negative cases, 3 cases were local/or regional recurrences and the other three had distant metastasis (2 lung, 1 bone metastasis). The FNR was 0% (sensitivity 100%) however, when number of anchor mutations was more than 5. Among 10 patients with no evidence of recurrence after standard therapy, 1 patient had cfDNA positive during follow up. No patient had minimal residual disease (MRD), within 1 month after surgery (+/- prior neoadjuvant systemic therapy). One case displayed cfDNA positive during surveillance (DDR2; c.2411T&gt;C; p.F804S, TP53; c.743G&gt;A; p.R248Q), at time-point of 7mos after surgery. The patient undergone mammogram, ultrasound and systemic work-up and yet, no evidence of recurrence was found till 6months after cfDNA positive conversion. Follow-up cfDNA analyses are necessary to evaluate mutation status whether, positive/increase VAF or negative conversion. At the same time, systemic radiology work-up needs to be done to investigate the development of clinically relevant recurrence after positive cfDNA, to confirm whether cfDNA positive finding was a false positive call or a matter of longer lead time.

Anchor mutations selected from primary tumor was analyzed in serial cell-free DNA using mass spectrometry based UHS platform. With sufficient number of anchor mutations for tracking (&gt;5), 100% sensitivity was observed with 0-10% false positivity. While liquid biopsy enable non-invasive surveillance, and yet needs substantial amount of blood draw 5-10ml each time, our platform using mass spectrometry can detect anchor mutation from less than 2ml whole blood. Clinical utility of cfDNA surveillance using UHS platform needs further evidence with longer follow-up analyses in larger prospective cohort.

Citation Format: Jisun Kim, Whee Kyung Jo, Soo Jeong Choi, Hwan Park, Jiyoung Lee, Sae Byul Lee, Hee Jin Lee, Hee Jeong Kim, Il Yong Chung, Beom Seok Ko, Jong Won Lee, Byung Ho Son, Sei Hyun Ahn, Sung-Bae Kim, Kyung Hae Jung, Jin-Hee Ahn, Sung-Min Chun. Tracking anchor mutations in serial cell-free DNA using ultra-high sensitive mass spectrometry method provide risk of subsequent recurrence during surveillance after standard therapy [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 P5-01-11.