Taking on Solid Tumor MRD with cfmRNA_J Clin Biomed Res 2023 best

ISSN: 2635 - 2826 Journal of Clinical & Biomedical Research Short Communication Open Access Taking on Solid Tumor Minimal Residual Disease with Circulating Cell-Free Messenger RNA Chen Hsiung Yeh* and Shu-Ti Lin OncoDxRx, USA ABSTRACT Recent interest has grown for a measure known as minimal residual disease (MRD) status that is emerging as a key surrogate biomarker for monitoring disease recurrence. Residual cancerous cells following treatment, known as MRD, can linger in tiny numbers that evade detection by traditional tests and slowly regrow and are responsible for the relapses seen in many types of cancer. Identification of the presence or absence of residual tumor cells (i.e., MRD-positive or -negative) with blood-based testing can provide a gauge of the extent of a patient’s response to treatment as well as the risk of relapse more accurate and earlier than traditional means. Compared to DNA molecules, RNA biomarkers possess higher sensitivity and specificity, providing dynamic and deeper insights into tempo-spatial clonal evolution and tumor microenvironment. Consequently, messenger RNA (mRNA) can potentially expand the opportunities for MRD detection through the combined screening of both tumor and non-tumor analytes beyond the DNA mutations. For those cancer patients currently in remission, the threat of relapse looms like a predestined path. Too often, tumor cells will evolve during treatment, accumulating mutations that enable therapeutic resistance and survival when the rest of the malignancy has gone. Though therapy can appear successful, residual cancerous cells may persist below detection thresholds and give rise to new tumors. Early detection and warning of recurrence requires a suite of molecular diagnostics that not only can sensitively trace tumor cells, but also can obtain genetic information to guide personalized treatment. Particularly, physicians need the tools to know when cancer is present and to know what treatments are most likely to benefit each individual patient. In this aspect, medical community today is heavily relying on the non-invasive, ultra-sensitive and longitudinal liquid biopsy technology of circulating cell-free DNA (cfDNA) and mRNA (cfmRNA). These small fragments of tumor-specific genetic material are released and enriched in circulation over time due to high turnover of tumor cells, when analyzed using PCR or next-generation sequencing (NGS) technology, can provide comprehensive profile regarding the genome and origin of cancer. The application of cfDNA/cfmRNA as biomarkers in MRD testing has the potential to dramatically improve clinical outcomes. But like any tumor biomarker, they are useful only when they can be accurately and reliably detected. Here, we give a brief overview of cfmRNA, its potential as a biomarker in solid tumor monitoring and MRD testing, and the technologies that are necessary to realize this potential. *Corresponding author Chen Hsiung Yeh, OncoDxRx, LLC,150 N Santa Anita Ave., Suite 300 Arcadia, CA 91006, USA. Received: Feb 22, 2023; Accepted: March 27, 2023; Published: April 05, 2023 Keywords: Solid Tumor, Minimal Residual Disease, Cell-free MRD testing emphasizes the efforts to identify residual cancer mRNA, Liquid Biopsy cells that have survived treatment but remain below the detection threshold of conventional surveillance technologies, such as A Key Role for cfmRNA in Managing Solid Tumors radiographic imaging. In this context, residual cancer cells may The transformation, proliferation and turnover of cancerous cells be very few in number hence will require highly specialized and are associated with both active and passive biological processes sensitive assays to detect. It is hopeful that cfmRNA analyses may to release genetic materials into circulation [1]. The bloodstream be exactly that, but tapping into this biomarker is far from trivial. thus is enriched with tumor-specific driver and passenger mutations, as well as epigenetic biomarkers, and overexpressed Advantages of cfmRNA Biomarkers transcripts over time. Through plasma transcriptomic Detecting cfmRNA from solid tumors is a particularly difficult profiling, especially with cell-free mRNA (cfmRNA), challenge [3]. Unlike BCR-ABL1 fusion transcripts of chronic researchers can not only home in on the malignant tissue’s myelogenous leukemia which are readily detectable in blood, subcellular state, they can also build a temporal-spatial profile cfmRNA released from solid tumors will have to permeate tissue of tumor microenvironment. Such insights can provide us and cross vascular barriers before entering circulation. Therefore, with the tumor’s emergence, therapeutic weaknesses and cfmRNA from solid tumors is rare in liquid biopsies. forewarn of potential resistance mechanisms [2, 3]. Owing to the higher-copy-number and broader functional spectrum of Changes in cfmRNA expression in tumor cells are a highly RNA molecules over DNA mutation, cfmRNA has the dynamic and regulated process that can reflect cellular state as well potential to be a powerfully sensitive biomarker, particularly for as tumor microenvironment [4, 5]. Moreover, the study of cfmRNA MRD testing. is not merely based on the differential expression of a set of specific J Clin Biomed Res, 2023 Volume 5(2): 1-3

Citation: Chen Hsiung Yeh, Shu-Ti Lin (2023) Taking on Solid Tumor Minimal Residual Disease with Circulating Cell-Free Messenger RNA. J Clin Biomed Res 5(2): 1-3. genes, but also on multilayers of variables such as pathogenic sufficient sensitivity to detect rare MRD molecules, target alternative splicing, RNA editing or methylation, changes that enrichment approaches using gene-specific PCR amplicons or are not detectable in the genome, but only in the transcriptome. hybrid capture have been employed to significantly boost the Due to these functionalities, there has been a rising interest in the sensitivity of sequencing assays. As a result, assays using target field of cfmRNA over cfDNA in the recent years. Although enrichment have been shown to have comparable sensitivity to more technically challenging, there are major advantages digital PCR and 10,000 times more sensitive than whole genome associated with the study of cfmRNA: the copy number of sequencing [1, 2]. Currently, many MRD tests utilize off-the-shelf, cfmRNA is much higher than that of cfDNA; cfmRNA either tumor-agnostic or tumor-informed, panels to specifically biomarkers can provide dynamic and deeper insights into amplify mutation-containing cfDNA within patient samples. tempo-spatial distribution, regulatory processes and cell-to- Although these custom panels are designed to cover relevant cell communication from tumor and non-tumor tissues [6-8]. driver mutations, they’re not specific for each individual patient. The cfDNA-based tests can lead to false positive results and is When there are very few cancer cells present, such as the residual less effective against tumors harboring rare evolving subclones. cells that remain after treatment, informative cfmRNA species could represent 1-5% of the total cell-free RNA pool in a given Digital PCR (dPCR), another cutting-edge technology, improves sample [9]. In cancer patients, enriched and overexpressed cfmRNA on conventional allele-specific PCR amplification by partitioning biomarkers should be easily detected by highly sensitive qPCR- a DNA sample into a high density of tiny reactions to provide based technologies. Further, cancer type-specific gene expression an absolute quantification of mutant copy number [13]. Digital panels and patient-derived signatures could be established that PCR primers and probes can be designed to achieve very high are often linked to malignancy (Figure. 1). Circulating cfmRNA specificity, and droplet-based clonal amplification have maximized signals from both tumor and non-tumor tissues could represent the number of individual DNA molecules that can be analyzed the earliest event and lead to the earliest possible time point for from a single sample [14]. As a result, the detection limit is limited cancer early detection and MRD monitoring. Specificity could also in practice by the amount of cfDNA that can be isolated from a shift the balance toward cfmRNA side as certain driver somatic blood draw [15]. Digital PCR is very effective for tracking a small mutations in cfDNA assay could be from aging normal tissues. number of mutations identified from sequencing of tumor tissue or hotspot mutations with a high prevalence in the cancer of interest. Accordingly, dPCR is not a commonly preferred approach for solid tumor MRD detection in most contexts [16, 17]. Figure 1: Establishment of a cancer type-specific cfmRNA panel To overcome cfDNA limitations, MRD tests can instead make as a stepstone toward personalized gene expression signature for use of customized cfmRNA panels designed to specifically target MRD detection certain cancer types that are known to be uniquely present in a patient’s tumor (a tumor-informed, patient-derived gene expression signature for MRD) (Figure. 2). In this scenario, transcriptomic profiling of a tumor biopsy prior to or during treatment can provide a detailed gene expression profile of the tumor. That data can then be used to build liquid biopsy cfmRNA panels that precisely match the tumor’s functional genome and its microenvironment. The use of cfmRNA biomarkers and tumor-informed MRD panels is gaining momentum, particularly as companies continue to redefine what’s possible in liquid biopsy and precision oncology. Overcoming Challenges in Solid Tumor MRD Figure 2: Clinical utility of cfmRNA biomarkers in detecting The cfmRNA obtained from a blood sample represents a snapshot MRD. Schematic depicting scenarios and stages during a typical of the tumor transcriptome of an individual patient. Accordingly, cancer progression course the efficiency of RNA isolation becomes critical. Classical methods tend to favor the isolation of selective RNA populations and often Future Directions lead to reduced quantities of RNA [10]. The quantity and quality For solid tumor cfmRNA-based MRD assay, establishment of of input RNA has a strong impact on downstream processes. cancer type-specific and patient-derived gene expression signature Extensive studies have highlighted the importance of selecting the is critical. Coupling with a pre-validated cfmRNA panel (via best approach to isolate plasma RNA depending on the end goal transcriptomic profiling), the qPCR-based platform enables an of the study. Different strategies for RNA isolation are linked to different recovery rates and to the enrichment of selective RNA types [11, 12]. Since the field of cfmRNA is still young, there are no diagnostic tests approved for the use in the clinical practice yet. There are many challenges to be addressed to translate cfmRNA- based liquid biopsy biomarkers into the clinic, although several studies are currently undergoing clinical trials. Several sequencing methods have been developed to overcome the challenges of solid tumor MRD with a trend toward target enrichment and customized sequencing panels. To achieve J Clin Biomed Res, 2023 Volume 5(2): 2-3

Citation: Chen Hsiung Yeh, Shu-Ti Lin (2023) Taking on Solid Tumor Minimal Residual Disease with Circulating Cell-Free Messenger RNA. J Clin Biomed Res 5(2): 1-3. exceptional throughput, high-sensitivity, and quick turnaround, 9. Hulstaert E, Morlion A, Cobos FA, Verniers K, Nuytens J, et accessible and affordable workflow for MRD detection. The al. (2020) Charting extracellular transcriptomes in the human cfmRNA MRD panel can be customized based on a target tumor’s biofluid RNA atlas. Cell Rep 33: 108552. gene expression profile with multiplexing capability. The custom panel includes enough biomarkers per test, making it amenable to 10. Wright K, de Silva K, PurdieAC, Plain KM (2020) Comparison repeated testing over time (longitudinal monitoring). Detecting of methods for miRNA isolation and quantification from ovine cfmRNA transcripts in circulation is never easy, but with high- plasma. Sci Rep 10: 825. quality tools built, the MRD monitoring will get easier and faster at earlier time point than ever before. 11. Li X, Mauro M, Williams Z (2015) Comparison of plasma extracellular RNA isolation kits reveals kit-dependent biases. The field of cfmRNA liquid biopsies is promising, with a relatively BioTechniques 59: 13-17. small number of studies published, and a few candidate biomarkers undergoing clinical trials. In the recent years, there has been an 12. Schuierer S, Carbone W, Knehr J, Petitjean V, Fernandez increasing interest in liquid biopsy-based biomarkers using RNA A, et al. (2017) A comprehensive assessment of RNA-seq that the focus has started to shift from miRNA to mRNA, leading protocols for degraded and low-quantity samples. BMC to the discovery of new mRNA signatures or patterns for cancer Genomics 18: 442. diagnosis and prognosis. Although there is still much work to do to translate cfmRNA technologies into clinical practice, a 13. Zonta E, Garlan F, Pécuchet N, Perez-Toralla K, Caen number of recent promising results suggest that cfmRNA-based O, et al. (2016) Multiplex detection of rare mutations by liquid biopsies could be one of the next big revolutions in the picoliter droplet based digital PCR: sensitivity and specificity field of early detection, treatment selection, MRD, recurrence considerations. PLoS One 11: e0159094. and therapeutics. 14. Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B Due to the low abundance of cfmRNA transcripts at post- (2003) Transforming single DNA molecules into fluorescent treatment time points, optimizing cfmRNA recovery, tracking magnetic particles for detection and enumeration of genetic multiple tumor–informed cfmRNA expression, and minimizing variations. Proc Natl Acad Sci USA 100: 8817-8822. the effects of technical and biological bias are critical to achieve high sensitivity MRD for identifying patients at risk of progressive 15. Milbury CA, Zhong Q, Lin J, Williams M, Olson J, et al. disease or recurrence. Several interventional clinical trials testing (2014) Determining lower limits of detection of digital PCR the utility of cfDNA MRD for personalization of adjuvant/ assays for cancer-related gene mutations. Biomol Detect consolidation therapy are ongoing, and it is highly likely that Quantif 1: 8-22. multiple cfDNA MRD assays could soon be used in routine clinical care. Therefore, the synergy with addition of cfmRNA appears 16. Pietrasz D, Pécuchet N, Garlan F, Didelot A, Dubreuil O, et to be at the cusp of a revolution in personalized management of al. (2017) Plasma circulating tumor DNA in pancreatic cancer MRD in solid tumors. patients is a prognostic marker. Clin Cancer Res 23: 116-123. 17. Christensen E, Nordentoft I, Vang S, Birkenkamp-Demtröder K, Jensen JB, et al. (2018) Optimized targeted sequencing of cell-free plasma DNA from bladder cancer patients. Sci Rep 8: 1917. 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