Mutational profiling of appendiceal tumors from diagnostic specimens and circulating tumor DNA.

Abstract

e14552 Background: Appendiceal cancer (AC) is a rare malignancy with a high tendency for peritoneal metastasis, and reliable radiographic and serologic markers remain elusive. Circulating tumor DNA (ctDNA) biomarkers are in high demand, yet commercial assays have shown limited sensitivity (55-70%). This study evaluates an in-house pipeline analyzing peripheral blood cell-free DNA (cfDNA) in AC patients. Methods: AC patients (n = 73) at Allegheny Health Network Cancer Institute provided matched tumor and blood samples for a circulating biomarker study. Blood was processed via differential centrifugation, and cfDNA was extracted using magnetic beads before next-generation sequencing with the ct-TSO500 assay. The OncoKB database was used to assess clinically actionable variants. cfDNA concentration and variant yields were analyzed in relation to clinical and pathologic factors. Results: cfDNA was purified in 72/73 patients (median, 6.4; IQR 3.7-9.5 ng/mL), the exception being a patient undergoing surveillance following complete cytoreduction for low-grade appendiceal mucinous neoplasm. cfDNA concentration was unrelated to age, sex and tumor histologic subtype, although a trend toward increasing cfDNA in higher grade tumors was observed (p = 0.06). cfDNA concentration was higher in patients with regional LN metastases, but was unassociated with lymphovascular or perineural invasion or treatment history (recent chemotherapy or prior cytoreductive surgery). cfDNA concentration was predictive of poor disease-specific (HR 3.4, p = 0.008 for tertile1 vs. tertiles 2/3) and progression-free survival (HR = 1.9, p = 0.02), effects that were stable even when adjusting for grade. In a subset of 50 cases we used a 523-gene panel to compare the mutational profiles of ctDNA with those from solid tumor samples, revealing mutations in KRAS (40%), GNAS (32%), TP53 (30%), SMAD4 (30%) and BRAF (8%). We identified an average of 1,132 ± 91 variants and 6.6 +/- 1.8 clinically actionable mutations per case, with 97.4% overall mutation concordance between ctDNA and solid tumor DNA with 88.4% concordance in identifying patients with an actionable therapeutic available. At least one oncogenic mutation shared between tumor and ctDNA was found in every patient (median, 7; range, 2-12), and the total number of such mutations per case was strongly associated with overall survival (HR 1.28, p = 0.009). Conclusions: Contrary to prior reports using commercial assays, ctDNA can be detected in every patient with active AC, regardless of tumor extent, histologic subtype or grade.The raw cfDNA concentration is associated with tumor extent and survival outcome, independent of grade, as is the number of shared oncogenic mutations between tumor and blood. These findings support ctDNA as a promising biomarker for real-time disease monitoring in AC, an advance long sought in this aggressive cancer.