Abstract Introduction: Loss of function of the PTEN tumour suppressor, which leads to elevated PI3K/Akt pathway activation, is one of the common events in breast cancer. Although the mechanisms of PTEN loss of function are diverse, the most frequently observed events are PTEN gene deletion or mutation, resulting in little or no PTEN protein expression measured by tissue-based immunohistochemical staining. Tissue-based immunostaining or genetic analysis could be challenging due to its invasive nature, whereas a robust assay for blood-based detection of copy number loss has been lacking. The aim of this pilot study was to explore the pattern of genetic alterations of PTEN and its downstream PI3K-AKT pathways in breast cancer using a minimally-invasive, blood-based next generation sequencing (NGS) technology. Methods: Plasma samples were prepared from a cohort of 244 breast cancer (BC) samples of all stages. Cell-free DNA (cfDNA) was extracted from plasma sample and further analyzed using the PredicinePLUS™ NGS 180-gene panel. Specifically, cfDNA was enriched for targeted regions using a hybridization-based protocol, and sequenced using Illumina sequencer. Mutational profiling was analyzed using Predicine’s proprietary DeepSea bioinformatics platform with focus on coding exonic regions of genes of interests in PTEN, PI3K and AKT pathway. Results: cfDNA-based PTEN copy number loss was detected in 7/244 BC samples, whereas PTEN mutations were found in 19 out of 244 BC samples leading to loss of PTEN. Specifically, there are frameshift variants (p.Ile8MetfsTer16 and p.Arg130GlnfsTer4), stop gain variant (p.Arg233Ter, p.Glu242Ter), start lost variant (p.Met1?), inframe deletion variants (p.Ile101del), frameshift deletion variant (p.Ile168SerfsTer11), missense variants (p.Cys136Arg, p.Ile122Asn, p.Phe206Leu, p.Pro30Leu, p.Leu318Phe). Along the PI3K-AKT pathway, we also observed activating mutations in AKT1 (E17K only) in 12/244 samples, PIK3CA (p.His1047Arg, p.Glu545Lys, p.Glu542Lys, etc) in 99/244 samples, PIK3CB variants (p.Gly1031Trp, p.Arg153Cys, p.Tyr888Ter, p.Ser890Cys) in 4/244 samples. Interestingly, PTEN loss and PI3K/AKT pathway alterations were not mutually exclusive events in breast cancer. Conclusions: This study demonstrated the capacity of blood-based detection of gene deletion and genomic variants associated with loss of PTEN function in breast cancer. The findings further revealed the mutational spectra of PTEN-PI3K-AKT pathway alterations that could lead to better understanding of the biological mechanisms of PTEN loss in breast cancer. This blood-based liquid biopsy assay may ultimately result in improvement of precision diagnostics for biomarker-driven treatment options and patient prognosis in breast cancer. Citation Format: Pan Du, Eric Jia, Zhixin Zhao. Blood-based detection of PTEN loss and pathway alterations for personalized treatment of breast cancer [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 P5-01-20.