Abstract P5-02-46: Targeted capture sequencing allows sensitive genomic profiling of circulating tumor DNA in advanced HR+/HER- breast cancer patients
Authors: Dandachi Nadia , Graf Ricarda , Potocnik Nina , Pancheri Lara , Klocker Eva , Suppan Christoph , Jost Philipp , Gerritsmann Hanno , Heitzer Ellen , Balic Marija 
Published: March 2023

Abstract
Introduction: CDK4/6 inhibitors provide substantial benefits as 1st or 2nd line treatments and are now the standard of care for patients with advanced HR-positive, HER2-negative breast cancer. Recently, we demonstrated that a high-resolution SiMSen-seq assay (SSS) provides a sensitive and robust method for detecting 11 PIK3CA hotspot mutations in cell-free circulating DNA, allowing the identification of patients eligible for alpelisib treatment. Unfortunately, all patients progress at some time point due to intrinsic or acquired resistance. Therefore, detecting additional genomic biomarkers for treatment resistance beyond PIK3CA mutations is crucial. Targeted panel sequencing offers a promising strategy to profile circulating tumor DNA (ctDNA) for genetic alterations in multiple genes associated with treatment response and disease progression. This ongoing study aims to show that a commercial NGS assay (AVENIO ctDNA Expanded Kit, Roche Diagnostics) can (1) detect PIK3CA mutations with similar sensitivity as our high-resolution SSS assay and can (2) simultaneously identify additional genetic alterations in multiple genes, possibly associated with treatment resistance or disease progression. Material and Methods: To this end, we collected plasma samples from 46 metastatic HR+/HER2- breast cancer patients before starting 1st (32 patients) or 2nd (14 patients) line treatment. Samples were analyzed using SSS and the AVENIO ctDNA Expanded Kit, enriching for 77 clinically relevant cancer genes. PIK3CA mutation detection and variant allele frequencies (VAF) were compared between the two methods. Additionally, mFAST-SeqS was used to estimate the tumor fractions in plasma samples. Results: The median z-score from mFAST-SeqS analyses was 2.38 [25–75th percentile: 1.23–4.5], and 17/46 (37%) patients had z-scores 3, indicating elevated tumor fractions (&gt;5%). Patients starting 2nd line treatment had significantly higher z-scores than those starting 1st line treatment (median 2.2 vs. 3.8, rank-sum p-value 0.042). One sample repeatedly failed with the SSS assay, leaving 45 samples for a head-to-head comparison. Considering only PIK3CA hotspot mutations covered by both assays, 16 alterations were detected in 14 patients (31%) by the SSS assay and 19 alterations in 17 patients (38%) by the AVENIO ctDNA Expanded Kit. Both assays detected the identical co-occurrence of two PIK3CA mutations in two samples. Two of three mutations only detected with the AVENIO ctDNA Expanded Kit were also observed with the SSS assay but below the pre-defined detection limit. One mutation was only detected by the AVENIO ctDNA Expanded Kit. Overall, we found an excellent concordance rate of 94% between the two assays, confirming the high sensitivity of the panel sequencing assay. Moreover, the VAF of SSS and AVENIO kit were highly correlated (Spearman’s rho = 0.97, p &lt; 0.001). Using the AVENIO kit, a large number of additional mutations in 40 genes could be identified in 42/46 (91%) patients with a median of 2.0 variants (range 1-13) per sample. The most frequently mutated genes included PIK3CA (43%), followed by ESR1 (20%), TP53 (20%), MET (17%), SMAD4 (17%), ERBB2 (11%), and BRCA2 (11%). The median VAF was 0.64% (range 0.1-29.8). Further analyses are still ongoing. Conclusion: The AVENIO ctDNA Expanded Kit revealed a high sensitivity and concordance rate for detecting PIK3CA hotspot mutations in plasma samples compared with the high-resolution SSS assay. A major advantage of panel sequencing over a single gene approach is that the interrogation of multiple genes can indicate a true negative PIK3CA result if other variants are present with a high VAF. Moreover, other actionable targets or mechanisms of resistance can be captured simultaneously, thus improving the effective precision treatment of metastatic breast cancer patients.
Citation Format: Nadia Dandachi, Ricarda Graf, Nina Potocnik, Lara Pancheri, Eva Klocker, Christoph Suppan, Philipp Jost, Hanno Gerritsmann, Ellen Heitzer, Marija Balic. Targeted capture sequencing allows sensitive genomic profiling of circulating tumor DNA in advanced HR+/HER- breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-46.