Abstract 437: Combining circulating stromal cells with cell free DNA for increased sensitivity in profiling oncogenic mutations and indicates highly aggressive non small cell lung cancer
Authors: Adams Daniel L. , Lin Steven H. , Raghavakaimal Ashvathi , Weiss Glenn , Ford Andrew , Brown Charmaine , Yeh Chen-Hsiung 
Published: August 2019

Abstract
Background: Circulating cell free DNA (cfDNA) in the plasma of cancer patients may provide oncogenic mutation status for NSCLC. However, the low sensitivity of current cfDNA has clinical utility limited to late stage multifocal or some recurrent diseases. Recently, specific phagocytic stromal cells found in blood, i.e. cancer associated macrophage-like cells (CAMLs), were shown to contain large quantities of tumor DNA. We hypothesized that a single blood sample can provide both cfDNA and parallel CAML DNA, providing more sensitive tumor mutation screening in a broader array of patients. We screened untreated NSCLC patients with a range of stages (stage I=3, stage II=5, stage IIIa=10, stage IIIb=7, & stage IV=5), that had available primary tissue for NGS. Blood was drawn prior to induction of radiotherapy, and plasma was sequenced using a 50 gene oncopanel. Separately, CAMLs were isolated from the same blood sample, lysed and sequenced using the same panel. Our data suggests that CAMLs contain clinically relevant oncogenic variants that correspond to both the primary tumor and the matched plasma.
Methods: Whole peripheral blood was taken from 30 newly diagnosed NSCLC patients with confirmed stage I-IV disease in a preservative collection tube. Plasma was separated and frozen at -80C. CAMLs were purified from the cell pellet using a standard CellSieveTM Assay, which isolates CAMLs based on size separation. Purified CAMLs were enumerated, then removed from the filter and lysed in 150 µL of buffer. The primary tumor biopsies, plasma and lysed CAMLs were blinded and anonymized for sequencing against a standard 50 oncogene NGS panel. Total mutations in tumor/plasma/CAMLs were used to evaluate progression free survival (PFS) by censored univariate analysis.
Results: Forty three mutations were identified from primary tumor DNA (averaging 1.4 variants in 87% of patients) vs 28 mutations from cfDNA (0.9 variants in 47% of patients) with no exon variant matches, although a nonmatching variant was found on the TP53 loci. CAML lysate had 2.8X more mutations than cfDNA, with 78 variants averaging 2.6 variants in 80% of patients. Further, 2 CAML variants exactly matched the primary tumor and 3 more matched at the gene level. cfDNA and CAMLs had the best concordance, with 6 matching exon variants and 6 more matching on the same gene. Notably, patients with cfDNA or CAML mutations had lower PFS, with ≥1 variants in cfDNA (HR=4.0, 95%CI=1.4-11.3, p=0.021) and ≥4 variants in CAMLs (HR=4.3, 95%CI=1.4-12.9, p=0.020).
Conclusions: Our data suggests that a single blood sample to screen for oncogenic tumor mutations from cfDNA and CAMLs provides increased sensitivity and specificity than cfDNA alone. Further, while preliminary, we find that high mutation burden in cfDNA or CAMLs may be indicative of aggressive NSCLC that is more likely to progress within 2 years.
Citation Format: Daniel L. Adams, Steven H. Lin, Ashvathi Raghavakaimal, Glenn Weiss, Andrew Ford, Charmaine Brown, Chen-Hsiung Yeh. Combining circulating stromal cells with cell free DNA for increased sensitivity in profiling oncogenic mutations and indicates highly aggressive non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 437.