Reply to the Letter to the Editor: Can a Liquid Biopsy Detect Circulating Tumor DNA With Low-passage Whole-genome Sequencing in Patients With a Sarcoma? A Pilot Evaluation

To the Editor,

We thank Fu et al. [4] for their interest in our study and thoughtful questions regarding, “Can a Liquid Biopsy Detect Circulating Tumor DNA With Low-passage Whole-genome Sequencing in Patients With a Sarcoma? A Pilot Evaluation” [1].

With regard to the sensitivity of low-passage whole-genome sequencing (LP-WGS) for minimal residual disease or early-stage sarcomas as well as a quantifiable tumor size threshold for reliable circulating tumor DNA (ctDNA) identification, our study [1] was not able to answer these questions due to the insufficient clinical information. Specifically, this was designed as a pilot study, and we were not provided clinical data about minimal residual disease for the patients whose specimens we tested. While staging and tumor size data were provided, our sample size was too small to draw any meaningful conclusions. However, we hope our upcoming prospective clinical trial evaluating the LP-WGS liquid biopsy (NCT06958107), which we are in the process of opening later this year, will put us in a position to provide answers to these types of questions in the near future.

Second, Fu et al. [4] asked how storage and transport conditions might influence results. We believe this is an important topic to discuss, especially for other centers interested in performing research with ctDNA. There are multiple factors capable of influencing the quality and quantity of ctDNA such as the choice of blood collection tube, time to processing, processing methods, plasma quality, and storage [6]. With regard to blood collection tubes, for blood samples collected in EDTA tubes, it is generally recommended for them to be processed into plasma as soon as possible, and not longer than 4 hours, to avoid contamination with germline DNA from lysis of leukocytes. In our upcoming clinical trial, we are planning to use Streck Cell-Free DNA blood collection tubes (Streck) because they allow for processing times up to 5 to 7 days and are stable at room temperature [6]. Additionally, it has been reported that cell-free DNA (cfDNA) degrades over time in storage, either as plasma or purified cfDNA [11]. We were not given information about the age of the patient-derived samples that we tested, but if some of them were stored for prolonged periods of time (many months or longer), this could have impacted the results. Furthermore, if specimens require long distance transportation, any unintended temperature variation would also likely reduce the quality and/or quantity of ctDNA.

Third, Fu et al. [4] inquired whether parallel profiling of primary tumors, metastatic sites, and plasma ctDNA should be implemented to validate discordances. Because of tumoral heterogeneity, it is known that genomic changes may be present in the primary tumor and not in a metastatic site, or in a metastatic site and not in the primary tumor or other metastatic sites. A perceived benefit of a liquid biopsy is that it can provide a snapshot in time of the entire tumor genome. While we believe it would be academically intriguing to understand whether parallel profiling could better characterize discordances, there would be limitations, including increased costs with additional specimen acquisition as well as ethical factors pertaining to additional invasive biopsies.

Lastly, Fu et al. [4] asked whether combining LP-WGS with methylation analysis or fragmentomics—which is the study of the size, distribution, and end characteristics of DNA fragments, particularly in the blood—could enhance the detection rate of translocation-driven sarcomas. We expect that both techniques would, as well as others, such as paired targeted translocation panels that have been utilized by other groups [2, 5, 9]. We are continuing to develop our own hybrid capture sequencing translocation panel for this purpose. Based on recent favorable results with methylation, we believe it could also be useful, although this may not overcome the issue of tumors that excrete low amounts of ctDNA. Fragmentomics analysis also appears to be promising, both as a standalone liquid biopsy technique and as a way to improve the sensitivity of LP-WGS by focusing on shorter fragments that are more likely tumor derived [3, 7, 8, 10]. We are planning to reanalyze data from our pilot study [1] using a fragmentomics-informed approach to see if it improves the detection ability of this assay in addition to incorporating fragmentomics into our upcoming prospective clinical trial.