Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection

T Ehlert,I Gockel,W Kneist,C Werner,A Brahmer,V Neef,P Simon,S Tug,B Jansen-Winkeln,F Heid,H Lang

doi:10.1038/s41598-017-09137-w

Abstract

The analysis of blood plasma or serum as a non-invasive alternative to tissue biopsies is a much-pursued goal in cancer research. Various methods and approaches have been presented to determine a patient’s tumour status, chances of survival, and response to therapy from serum or plasma samples. We established PNB-qPCR (Pooled, Nested, WT-Blocking qPCR), a highly specific nested qPCR with various modifications to detect and quantify minute amounts of circulating tumour DNA (ctDNA) from very limited blood plasma samples. PNB-qPCR is a nested qPCR technique combining ARMS primers, blocking primers, LNA probes, and pooling of multiple first round products for sensitive quantification of the seven most frequent point mutations in KRAS exon 2. Using this approach, we were able to characterize ctDNA and total cell-free DNA (cfDNA) kinetics by selective amplification of KRAS mutated DNA fragments in the blood plasma over the course of tumour resection and the surrounding days. Whereas total cfDNA concentrations increased over the surgical and regenerative process, ctDNA levels showed a different scheme, rising only directly after tumour resection and about three days after the surgery. For the first time, we present insights into the impact of surgery on the release of ctDNA and total cfDNA.

Highlights

KRAS mutation status is crucial for treatment of colorectal cancer patients, as anti-EGFR therapy is ineffective in the presence of KRAS mutations[1]
In the quantitative real-time PCR (qPCR), 30,000 WT copies alone generated a false positive signal at a cycle of quantification (Cq) lower than the calculated Cq for one mutated copy. These false positive signals ranged from 0.14 to 24 calculated copies in the qPCR. These background signals were effectively reduced to a median of 0.04 calculated copies (0.03–0.45 calculated copies) for 30,000 WT copies using the nested qPCR with WT blocker
The establishment of plasma DNA analysis as a liquid biopsy is of major interest in cancer research[18]

Summary

Introduction

KRAS mutation status is crucial for treatment of colorectal cancer patients, as anti-EGFR therapy is ineffective in the presence of KRAS mutations[1]. The use of plasma samples as liquid biopsies based on cell-free DNA (cfDNA) circulating in the blood of tumour patients and potentially harbouring tumour derived DNA (ctDNA) has been proposed to replace conventional classification tests on tumour tissue[2]. Genome wide or hot spot sequencing approaches covering huge numbers of possible cancer related mutations have been developed[13]. Plasma samples at hand might contain no mutated sequences or only in very low copy numbers, even in the presence of a tumour bearing the sought-after mutation[14, 15]. Tumour-derived cell-free DNA fragments in the blood plasma tend to be short and are difficult to detect with conventional methods[17]. Required amount of plasma for quantification* Required amount of plasma for detection* Nr. of possible test reruns** LOQ (copies/quantification)

Methods

Results

Conclusion