# Absolute Quantification of Nucleotide Variants in Cell-Free DNA via Quantitative NGS: Clinical Application in Non-Small Cell Lung Cancer Patients

## TL;DR

A new sequencing method allows precise measurement of tumor DNA in blood, helping track cancer treatment response without needing prior genetic information.

## Contribution

A novel quantitative NGS method using UMIs and QSs enables absolute quantification of ctDNA variants without prior tumor genotype knowledge.

## Key findings

- The qNGS method showed strong linearity and high correlation with dPCR in both spiked and clinical samples.
- The method successfully quantified multiple ctDNA variants in a single plasma sample from NSCLC patients.
- Significant reductions in ctDNA levels were observed after three weeks of therapy in NSCLC patients.

## Abstract

Circulating tumor DNA (ctDNA) analysis offers a non-invasive method for tracking tumor burden and treatment response. Current quantification techniques face limitations: digital PCR (dPCR) requires prior knowledge of tumor-specific alterations, while next-generation sequencing (NGS) provides broader insights but is semi-quantitative. To address these challenges, a novel quantitative NGS (qNGS) method was developed, incorporating unique molecular identifiers (UMIs) and quantification standards (QSs). This approach enables the absolute quantification of nucleotide variants. Validated using spiked plasma samples and clinical samples from the ELUCID trial, the qNGS method demonstrated strong linearity and high correlation with dPCR. It successfully quantified multiple variants from a single plasma sample and revealed significant reductions in ctDNA levels after three weeks of therapy in non-small-cell lung cancer (NSCLC) patients. This robust method, independent of tumor genotype knowledge, enhances precision oncology by enabling simultaneous monitoring of multiple ctDNA variants.

Background/Objectives: Circulating tumor DNA (ctDNA) analysis is a powerful tool for non-invasive monitoring of tumor burden and treatment response. Reliable quantification methods are critical for the effective use of ctDNA as a tumor biomarker. Digital PCR (dPCR) offers high sensitivity and quantification, but requires the prior knowledge of tumor-specific genomic alterations. Next-generation sequencing (NGS) provides a more comprehensive approach but is semi-quantitative, relying on variant allelic fraction (VAF), which can be influenced by non-tumor cell-free DNA. Methods: We developed a novel quantitative NGS (qNGS) method for absolute quantification of nucleotide variants, utilizing unique molecular identifiers (UMIs) and of quantification standards (QSs), short synthetic DNA sequences modified to include characteristic mutations for unique identification in sequencing data. We evaluated the performance of this method using plasma samples spiked with mutated DNA and plasma pools from cancer patients. We further applied our technique to plasma samples from four non-small cell lung cancer (NSCLC) patients enrolled in the ELUCID trial. Results: Our qNGS approach demonstrated robust linearity and correlation with dPCR in both spiked and patient-derived plasma samples. Notably, the analysis of clinical samples from the ELUCID trial revealed the ability of our method to simultaneously quantify multiple variants in a single plasma sample. Significant differences in ctDNA levels were observed between baseline and post-treatment samples collected after three weeks of front-line therapy. Conclusions: We introduce a novel qNGS method that enables the absolute quantification of ctDNA, independent of non-tumor circulating DNA variations. This technique was applied for the first time to serial samples from NSCLC patients, demonstrating its ability to simultaneously monitor multiple variants, making it a robust and versatile tool for precision oncology.