Prognostic and Predictive Biomarkers in Non-Small Cell Lung Cancer Patients on Immunotherapy-The Role of Liquid Biopsy in Unraveling the Puzzle.

In the last decade, immunotherapy has been one of the most important advances in the non-small cell lung cancer (NSCLC) treatment landscape. Nevertheless, only a subset of NSCLC patients benefits from it. Currently, the only Food and Drug Administration (FDA) approved diagnostic test for first-line immunotherapy in metastatic NSCLC patients uses tissue biopsies to determine the programmed death ligand 1 (PD-L1) status. However, obtaining tumor tissue is not always feasible and puts the patient at risk. Liquid biopsy, which refers to the tumor-derived material present in body fluids, offers an alternative approach. This less invasive technique gives real-time information on the tumor characteristics. This review addresses different promising liquid biopsy based biomarkers in NSCLC patients that enable the selection of patients who benefit from immunotherapy and the monitoring of patients during this therapy. The challenges and the opportunities of blood-based biomarkers such as cell-free DNA (cfDNA), circulating tumor cells (CTCs), exosomes, epigenetic signatures, microRNAs (miRNAs) and the T cell repertoire will be addressed. This review also focuses on the less-studied feces-based and breath-based biomarkers.

The art of obtaining a high yield of cell-free DNA from urine.

Although liquid biopsies offer many advantages over tissue biopsies, they are not yet standard practice. An important reason for the lack of implementation is the unavailability of well standardized techniques and guidelines, especially for pre-analytical conditions which are an important factor causing the current sensitivity issues. To overcome these limitations, we investigated the effect of several pre-analytical conditions on the concentration of cell-free DNA (cfDNA) and cellular genomic DNA (gDNA) contamination. Urine samples from healthy volunteers (HVs) and cancer patients were collected and processed according to specific pre-analytical conditions. Our results show that in samples with a relatively small volume more than 50% of the cfDNA can be found in the first 50 mL of the urine sample. The total DNA concentration increased again when samples were collected more than 3.5 hours apart. Adding preservative to urine samples is recommended to obtain high concentrations of cfDNA. To remove the cellular content, high speed centrifugation protocols as 4,000g 10min or 3,000g 15min are ideal for urine collected in cfDNA Urine Preserve (Streck). Although this study was a pilot study and needs to be confirmed in a larger study population, clear trends in the effect of several pre-analytical conditions were observed.

Specialized Blood Collection Tubes for Liquid Biopsy: Improving the Pre-analytical Conditions.

INTRODUCTION: The potential of circulating cell-free DNA (cfDNA) analysis as a liquid biopsy has led to the development of several specialized measuring tools. Interest in the (pre-)analytical conditions of the liquid biopsy workflow has increased over the past few years. METHODS: In this study, we performed a systematic review of the cfDNA stabilizing efficacy in standard EDTA and specialized blood collection tubes (BCTs), namely CellSave, Norgen, PAXgene, Roche, and Streck tubes, and compared the efficacy of the latter three BCTs in a situation resembling the clinical setting. Blood samples were collected from ten KRAS-mutated metastatic cancer patients and stored for 72 h. During this time, samples were shaken and kept at either 6 °C or at room temperature for 24 h to mimic transport. RESULTS: We demonstrated that while cfDNA levels in EDTA tubes are only stable for a couple of (≤ 6) hours, they could be sustained for at least 48-72 h in all three specialized BCTs, irrespective of temperature. This timespan enables a fast turnaround time, which is one of the advantages of liquid biopsy. CONCLUSIONS: The choice between these specialized BCTs is less vital when they are processed correctly within a few days.

Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol.

The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K2EDTA (EDTA) tubes or cell-free DNA BCT® (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis.

A Multicenter Study to Assess EGFR Mutational Status in Plasma: Focus on an Optimized Workflow for Liquid Biopsy in a Clinical Setting.

A multicenter study was performed to determine an optimal workflow for liquid biopsy in a clinical setting. In total, 549 plasma samples from 234 non-small cell lung cancer (NSCLC) patients were collected. Epidermal Growth Factor Receptor (EGFR) circulating cell-free tumor DNA (ctDNA) mutational analysis was performed using digital droplet PCR (ddPCR). The influence of (pre-) analytical variables on ctDNA analysis was investigated. Sensitivity of ctDNA analysis was influenced by an interplay between increased plasma volume (p < 0.001) and short transit time (p = 0.018). Multistep, high-speed centrifugation both increased plasma generation (p < 0.001) and reduced genomic DNA (gDNA) contamination. Longer transit time increased the risk of hemolysis (p < 0.001) and low temperatures were shown to have a negative effect. Metastatic sites were found to be strongly associated with ctDNA detection (p < 0.001), as well as allele frequency (p = 0.034). Activating mutations were detected in a higher concentration and allele frequency compared to the T790M mutation (p = 0.003, and p = 0.002, respectively). Optimization of (pre-) analytical variables is key to successful ctDNA analysis. Sufficient plasma volumes without hemolysis or gDNA contamination can be achieved by using multistep, high-speed centrifugation, coupled with short transit time and temperature regulation. Metastatic site location influenced ctDNA detection. Finally, ctDNA levels might have further value in detecting resistance mechanisms.