Abstract P2-01-02: Capturing intra-tumor genetic heterogeneity in cell-free plasma DNA from patients with oligometastatic breast cancer
Authors: Ng CKY , Bidard F-C , Piscuoglio S , Lim RS , Pierga J-Y , Cottu P , Vincent-Salomon A , Viale A , Norton L , Sigal B , Weigelt B , Reis-Filho JS 
Published: May 2016

Abstract
Background: The analysis of cell-free tumor DNA (ctDNA) from plasma has been heralded as a non-invasive technique for disease monitoring and as a means to overcome the challenges posed by intra-tumor genetic heterogeneity. ctDNA levels have been shown to correlate with tumor burden in breast cancer patients. Hence, we sought to define whether massively parallel sequencing of cell-free plasma DNA would capture the entire repertoire of somatic mutations present in the primary tumors and/ or metastases from patients with oligometastatic breast cancer.
Methods: Frozen diagnostic biopsies from primary tumors and their distant metastases were obtained from five prospectively accrued treatment-naïve patients with stage IV breast cancer at presentation (1 estrogen receptor (ER)+/HER2+, 2 ER+/HER2-, 2 ER-/HER2+). A second, independent formalin-fixed paraffin-embedded (FFPE) diagnostic biopsy was obtained from the primary tumor and metastasis from 4 patients. Plasma samples were obtained from all patients. DNA samples from microdissected frozen tumors and peripheral blood, as well as plasma from one patient, were subjected to high-depth whole exome sequencing. DNA samples from all biopsies (frozen/FFPE), plasma and peripheral blood were subjected to targeted capture massively parallel sequencing, with baits for all somatic mutations detected by whole exome sequencing and all exons of the 100 genes most frequently mutated in breast cancer. Driver mutations were defined by state-of-the-art bioinformatic methods and literature search.
Results: We identified and confirmed a median of 54 (range 25-75) and 53 (range 26-85) non-synonymous mutations in the primary tumors and metastases from the 5 cases analyzed, respectively. By sequencing the plasma DNA to a median depth of 248x (range 92-431x), state-of-the-art mutation callers revealed 0-4 mutations (0%-8% of mutations) per patient, and direct interrogation of the sequencing data, based on prior knowledge of the mutations present in the lesions, resulted in the identification of 2-18 mutations (3%-38% of mutations) per patient. Of the bona fide driver mutations, 2/3 TP53 mutations, 0/1 PIK3CA hotspot mutation, 0/1 BRCA2 frameshift mutation, 0/1 GATA3 frameshift mutation and 0/1 ERBB3 activating mutation were captured in the plasma DNA. A SMAD4 pathogenic mutation and a TCF7L2 truncating mutation were found in two diagnostic biopsies of metastatic lesions but not in two biopsies of the primary tumors in one patient each. Whilst the SMAD4 mutation was detected in the plasma DNA from the respective patient, the TCF7L2 mutation was not. Of the 62 mutations restricted to the primary tumors (0-42 per patient) and 74 restricted to the metastatic tumors (1-41 per patient), 4 and 7, respectively, were captured in the plasma DNA.
Conclusions: Massively parallel sequencing assessment of plasma DNA allows for the identification of mutations found in primary tumors and/ or their metastases, however, only a subset of these could be detected at up to 431x depth. These observations suggest that current approaches for whole exome or targeted massively parallel sequencing may not be sufficient to capture the genetic heterogeneity of breast cancers in patients with oligometastatic disease.
Citation Format: Ng CKY, Bidard F-C, Piscuoglio S, Lim RS, Pierga J-Y, Cottu P, Vincent-Salomon A, Viale A, Norton L, Sigal B, Weigelt B, Reis-Filho JS. Capturing intra-tumor genetic heterogeneity in cell-free plasma DNA from patients with oligometastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-01-02.