Detection of Circulating Tumor DNA in Patients with Pancreatic Cancer Using Digital Next-Generation Sequencing

Abstract

Circulating tumor DNA (ctDNA) measurements can be used to estimate tumor burden, but avoiding false-positive results is challenging. Herein, digital next-generation sequencing (NGS) is evaluated as a ctDNA detection method. Plasma KRAS and GNAS hotspot mutation levels were measured in 140 subjects, including 67 with pancreatic ductal adenocarcinoma and 73 healthy and disease controls. To limit chemical modifications of DNA that yield false-positive mutation calls, plasma DNA was enzymatically pretreated, after which DNA was aliquoted for digital detection of mutations (up to 384 aliquots/ sample) by PCR and NGS. A digital NGS score of two SDs above the mean in controls was considered positive. Thirty-seven percent of patients with pancreatic cancer, including 31% of patients with stages I/II disease, had positive KRAS codon 12 ctDNA scores; only one patient had a positive GNAS mutation score. Two disease control patients had positive ctDNA scores. Low-normalerange digital NGS scores at mutation hotspots were found at similar levels in healthy and disease controls, usually at sites of cytosine deamination, and were likely the result of chemical modification of plasma DNA and NGS error rather than true mutations. Digital NGS detects mutated ctDNA in patients with pancreatic cancer with similar yield to other methods. Detection of low-level, true-positive ctDNA is limited by frequent low-level detection of false-positive mutation calls in plasma DNA from controls.

The genetics of ductal adenocarcinoma of the pancreas in the year 2020: dramatic progress, but far to go

“…Given the specificity of somatic mutations for the presence of a clone of cells, the detection of circulating tumor DNA (ctDNA) provides a promising foundation for the early detection of pancreatic cancer [207]. Extremely sensitive and specific tests for ctDNA have been developed, and when applied clinically these have been very specific, but only moderately sensitive in detecting ctDNA in patients known to have a cancer [205][206][207][208]. Other approaches, including those based on detecting fragmented DNA of specific lengths and the detection of aneuploidy, have been developed that should improve the accuracy and usefulness of blood tests [205,209,210].…”

Endoscopic Ultrasound-Guided Tissue Acquisition of Pancreaticobiliary Cancer Aiming for a Comprehensive Genome Profile

“…Recently, liquid biopsy testing of blood and other body-fluid specimens has been developed as a new analytic method [ 27 , 28 , 29 ], and the F-One Liquid CDx Cancer Genome Profile [ 30 ] test for blood specimens is now covered by insurance programs in Japan. Although the liquid biopsy test is very useful for pancreaticobiliary cancers, for which the collection of samples for genetic testing is difficult, its sensitivity for detecting KRAS abnormalities is lower than that of the test using tumor tissue specimens [ 31 ], and tumor tissue specimens are superior in terms of certainty.…”

Progresses in biomarkers for cancer immunotherapy

“…Recently, B cells and tertiary lymphoid structures (TLSs) in TME were identified to promote the clinical responses to ICIs 135,136 . Patients with ICIs monotherapy showed a modest increase in circulating memory B cells, CD21lo B cells, and plasma blasts 137 . Besides, memory B‐cells in gene‐expression profiling were significantly elevated in patients receiving ICIs therapy for urothelial carcinoma (UCC) and melanoma 138 (Table 3).…”