Decoding EMT through liquid biopsy: A path to early detection and targeted therapy

Noninvasive technologies and methodologies are urgently needed for a population-based early detection and screening. Liquid biopsy continues to draw the attention of patients and physicians alike. Liquid biopsies have the potential to help clinicians screen for disease, stratify patients to the best treatment, and monitor treatment response and resistance mechanisms in the tumor. A liquid biopsy can be used for molecular characterization of the tumor, and its noninvasive nature allows repeat sampling to monitor genetic changes over time without the need for a tissue biopsy. Liquid biopsy generally refers to measuring molecular changes, e.g., genomic, proteomic, metabolomics, epigenetic, and other “omics”-derived changes, in circulating tumor cells (CTCs), cell-free DNA (cfDNA), and exosomes. However, the real success of liquid biopsy continues to evade us. There are several challenges in detecting tumor-derived mutations in plasma or in other body fluids. This is partly due to the fact that the amount of circulating DNA usually is very low. Recent technological developments and the analytics being applied to liquid biopsies are now capable of reproducibly detecting mutations at very low allelic frequencies. Advances have also been made in droplet digital PCR (ddPCR), next-generation sequencing (NGS), beads, emulsion, amplification and magnetics (BEAMing), and amplification of refractory mutation system (ARMS), to name but a few. Ultimately, the choice of platforms and required detection limit will depend on the clinical sample being analyzed, as the most sensitive methods are reported to detect allelic frequencies of as little as 0.01%; if achieved in a liquid biopsy setting, this level of detection would be sufficient to gain knowledge of a tumor genotype and rule out the need for a tissue biopsy. Despite these challenges, the advantage of liquid biopsies over other traditional tissue-based methodologies is very promising, including the ability for longitudinal monitoring, which could help clinical oncologists gain a broader molecular understanding of the disease. Additionally, liquid biopsy could also facilitate cancer progression and treatment monitoring in follow-up visits. Cancer detection in liquid biopsy achieves repetitive sampling that is limited in histologic examination, and it provides a more economical and practical noninvasive detection compared to radiation-based imaging methods such as computed tomography. Furthermore, liquid biopsy may also shed light on tumor heterogeneity and may provide a more comprehensive picture of the genetic and proteomic alternations of the tumor. The National Cancer Institute is supporting a number of programs that are addressing some of the challenges described above and pursuing careful selection of candidate markers (mutations, epigenomic changes, proteomic alterations, etc.), developing better analytical methods and sensitive molecular technologies that may contribute to early cancer detection and monitoring. The speaker will describe the objectives of these programs and provide updates. Citation Format: Sudhir Srivastava. The promise of liquid biopsy in the early detection of cancer: Hope or hype? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA09.

Simple SummaryBreast cancer screening is associated with benefits, such as mortality reduction and improved quality of life, and harms, such as false-positive results, overdiagnoses, and costs. Novel screen tests could be considered to reduce the harms and increase the benefits of screening. Liquid biopsies have been proposed as a novel method for the early detection of breast cancer. However, studies show that liquid biopsies based on cell-free DNA have a low sensitivity for early-stage breast cancer. Using the microsimulation model MISCAN-Fadia, we model the benefits, harms, and costs of the early detection of breast cancer using liquid biopsies for varying levels of liquid biopsy sensitivity and specificity. We found that liquid biopsies are unlikely to be an alternative to digital mammography, given the test performance based on a CCGA substudy. When liquid biopsies are unable to detect the precursor lesion of breast cancer—ductal carcinoma in situ (DCIS)—they need to be able to detect small, early-stage tumors, with high specificity, at low costs in order to be an alternative to digital mammography. We estimated a maximum liquid biopsy price of USD 187, which is substantially lower than currently listed prices.Breast cancer screening is associated with harms, such as false-positives and overdiagnoses, and, thus, novel screen tests can be considered. Liquid biopsies have been proposed as a novel method for the early detection of cancer, but low cell-free DNA tumor fraction might pose a problem for the use in population screening. Using breast cancer microsimulation model MISCAN-Fadia, we estimated the outcomes of using liquid biopsies in breast cancer screening in women aged 50 to 74 in the United States. For varying combinations of test sensitivity and specificity, we quantify the impact of the use of liquid biopsies on the harms and benefits of screening, and we estimate the maximum liquid biopsy price for cost-effective implementation in breast cancer screening at a cost-effectiveness threshold of USD 50,000. We investigate under what conditions liquid biopsies could be a suitable alternative to digital mammography and compare these conditions to a CCGA substudy. Outcomes were compared to digital mammography screening, and include mortality reduction, overdiagnoses, quality-adjusted life-years (QALYs), and the maximum price of a liquid biopsy for cost-effective implementation. When liquid biopsies are unable to detect DCIS, a large proportion of overdiagnosed cases is prevented but overall breast cancer mortality reduction and quality of life are lower, and costs are higher compared to digital mammography screening. Liquid biopsies prices should be restricted to USD 187 per liquid biopsy depending on test performance. Overall, liquid biopsies that are unable to detect ductal carcinoma in situ (DCIS) need to be able to detect small, early-stage tumors, with high specificity, at low costs in order to be an alternative to digital mammography. Liquid biopsies might be more suitable as an addition to digital mammography than as an alternative.

ES25.01 Liquid Biopsy: State of the Science

The Use of Liquid Biopsies in Patients With NSCLC

73 Background: Despite established screening guidelines, adherence to colonoscopy is approximately 60%. Blood-based screening tests, or Liquid Biopsies (LB), have the potential to close this screening gap. Our aim was to determine the cost-effectiveness of LB tests for CRC screening in the US. Methods: We developed a Markov model to compare four CRC screening strategies: no screening, or natural history (NH), colonoscopy (Colo) only, LB only, and colonoscopy to LB hybrid (C-LB). US SEER CRC incidence and mortality data were used to develop and validate the model. Many LB tests are in development awaiting validation; we used the preliminary performance characteristics for CRC screening of 82% sensitivity and 99.5% specificity (base case) from the Galleri multi-cancer early detection test (GRAIL). Screening ran from age 45 to 75; polyp surveillance ended at 85. We assumed 60.6% of the US population would undergo a screening colonoscopy, and 100% adherence for LB. In Colo, nonadherent patients were not offered other screening; in C-LB those who refused colonoscopy underwent LB. Primary outcomes were overall survival, total cost, incremental cost-effectiveness ratio (ICER), number of CRCs, and CRC deaths. Results: In the NH strategy, 5.2% of the population developed CRC and 1.8% died from CRC, with a cost of $7,802.15/individual. Compared to NH, in Colo, 40% of CRCs and 44% of CRC deaths were prevented, at a cost of $10,610.43/individual. In C-LB, 42% of CRCs and 50% of CRC deaths were prevented compared to NH, costing $13,762.61/individual. LB only prevented 2.0% of CRCs and 11% of CRC deaths compared to NH, and cost $34,339.85/individual. While LB showed an incremental benefit of 0.01 life years gained compared to NH, the ICER compared to NH is $2,653,770. Colo was most cost-effective with an ICER of $30,191.04. Although C-LB prevented the greatest number of CRCs, this strategy had an ICER of $457,057.64, above the accepted US willingness to pay threshold of $100,000/life year. We performed sensitivity analysis around base case estimates for LB. At both 70% and 90% test sensitivity, all outcomes remained largely constant. Conclusions: CRC screening with LB alone or with LB in conjunction with colonoscopy among nonadherent patients is not cost-effective in the US context. At this time, LB is unable to detect pre-cancerous colon polyps, which may limit its effectiveness as a CRC screening strategy. [Table: see text]

Part 2 of a 2-part series on liquid biopsy tests. Part 1 appeared in the May 1 issue. In 2013, scientists at the Sidney Kimmel Comprehensive Cancer Center of Johns Hopkins University in Baltimore, Maryland, announced the development of a new method to detect early ovarian and endometrial cancers through the Papanicolaou (Pap) test. A proof-of-principle pilot study demonstrated that the Pap test, which is widely used to detect cervical cancer, potentially could be used for both of the other cancers, which currently have no screening tests. Although initial results appeared promising, researchers noted at the time that larger scale studies needed to occur. Four years later, they released the results of their most recent, larger study, published in Science Translational Medicine.1 The test, PapSEEK, uses cervical fluid to search for mutations in 18 genes that are commonly mutated in endometrial or ovarian cancers, as well as aneuploidy, which is the presence of abnormal numbers of chromosomes in cells. Detecting these cancers at an earlier stage not only could result in the greater potential for curing them but possibly could help to preserve fertility for younger women, says corresponding author Amanda Nickles Fader, MD, director of the Johns Hopkins Medicine Kelly Gynecologic Oncology Service. In addition, she notes, current screening methods do not always distinguish benign conditions from cancer, which can lead to unnecessary procedures. “The fact that ovarian cancer has such a high relative mortality makes it a critically important area for progress. We have to be positive the test is reliable and can pick up the disease at an earlier, curable stage.” Elise Kohn, MD, head of gynecologic cancer therapeutics at the Cancer Therapy Evaluation Program of the National Cancer Institute and gynecologic oncology section editor of Cancer, notes that these assumptions regarding early detection still are premature and have yet to be proven. Endometrial cancers are on the rise, particularly in younger women, and scientists have attributed part of that increase to the obesity epidemic, although they are not certain of all the reasons. Gynecologic cancer deaths (approximately 25,000 per year) are the third leading cause of cancer-related mortality, and the majority are caused by tumors that metastasize, according to Nickolas Papadopoulos, PhD, senior author of the Science Translational Medicine article and a professor of oncology and pathology at Johns Hopkins. In their efforts to detect these cancers at earlier, more treatable stages, the researchers studied 1958 samples from 1658 women, including 656 patients with endometrial or ovarian cancer and 1002 healthy controls. Some participants provided 2 samples. The additional samples were collected with a Tao brush, which is not commonly used in the United States but is approved by the US Food and Drug Administration (FDA) for endometrial sampling. The brush extends further into the cervical canal and collects cells closer to where the cancers could originate, thereby improving test sensitivity. Tao brush samples were collected from 382 patients with endometrial cancer and 245 patients with ovarian cancer in the study. PapSEEK was nearly 99% specific for cancer, detecting 81% of endometrial cancers, 78% of which were of an early stage. It detected approximately 33% of ovarian cancers, 34% of which were of an early stage. Testing the plasma samples with Pap brush samples also increased the sensitivity of the test: PapSEEK identified endometrial cancer in those samples approximately 93% of the time and ovarian cancer approximately 63% of the time. There were no false-positive results noted. Newer technologies have enabled the team to detect extremely trace amounts of cells from the ovary and endometrium that previously were undetectable through traditional Pap testing, according to Dr. Papadopoulos. “Perhaps that is why we detect endometrial cancer cells better—because it is closer to the cervical canal than the ovaries,” he says. Investigators are optimistic about the test, but caution that more studies are necessary. “This is an important first step,” says Lucy Gilbert, MD, senior author and director of gynecologic oncology at McGill University Health Centre in Montreal, Quebec, Canada. “We have to validate this test in many more women with cancer and in older women without cancer before we can confidently offer it for widespread clinical use.” If a woman tests positive through this detection of microscopic cancers, the only way to confirm the diagnosis is by offering her major surgery; therefore, the bar for accuracy needs to be set very high, she adds. Investigators will be offering screening to women aged older than 50 years, who have a higher incidence of benign tumors and gynecologic diseases that are not life-threatening, Dr. Gilbert says. “We are fairly sure from this study that PapSEEK differentiates totally normal cells from cancer,” she notes. “We have to be sure that it clearly differentiates the benign gynecologic diseases such as fibroids, which are not life-threatening, from cancer. That is the next step.” Dr. Kohn agrees. Calling the data regarding PapSEEK “interesting and promising,” she says that such a test would need to be applied to the correct patient population, namely postmenopausal women who do not have fertility concerns. “It really needs to be evaluated prospectively and in a patient population that understands it's possible they may not have cancer but are still willing to undergo imaging and possible surgery,” she says. Dr. Kohn also cautions against the public getting too excited about certain tests before they have been fully proven. She recalls hearing about a grocery store in the 1990s offering the cancer antigen 125 blood test to its customers as a way to learn whether they may have ovarian cancer. However, the test is not accurate enough for ovarian cancer screening. “When you're doing these things in healthy people, the bar for due diligence needs to be higher,” she says. Whereas endometrial cancer usually can be diagnosed at early stages when it is most treatable, ovarian cancer tends to be diagnosed at advanced stages, and its incidence and mortality rates have remained stable. Although researchers have some leads on prevention, including knowing that oral contraceptives can be preventive at a population level and that women with the BRCA1 or BRCA2 genes should consider having their ovaries removed, Dr. Kohn says they have nothing to offer the general population. “The fact that ovarian cancer has such a high relative mortality makes it a critically important area for progress,” she notes. “We have to be positive the test is reliable and can pick up the disease at an earlier, curable stage.” The dramatic advances in genetic sequencing technology and the resulting liquid biopsy tests that have been developed have led to revolutionary changes in the medical field, says lung cancer specialist Bob Li, MD, MPH, a medical oncologist at Memorial Sloan Kettering Cancer Center in New York City. “Our researchers are now looking at a much broader panel of genetic mutations, such as ALK, BRAF, RAS1, and many others that may be treated with targeted therapy.” He cites the example of how the standard of care for pregnant women now involves prenatal tests that can determine everything from the baby's sex to fetal abnormalities. “These tests are much easier because you're looking at the DNA from 2 different people, and it's easier to tell which is from the mother and which is from the baby,” Dr. Li explains. “When it comes to cancer, it's much more difficult. But the field is evolving very rapidly—on a daily basis.” In lung cancer, for example, liquid biopsies that test for circulating tumor DNA have the potential to “detect minimal residual disease, depict genomic evolution, guide precision medicine to individual patients, and revolutionize the management of early-stage lung cancers,” Dr. Li and his colleagues write in Annals of Translational Medicine.2 He adds that recent advances have enabled the identification of tumorrelated mutations in plasma, urine, and cerebrospinal fluid. One key example is the FDA's 2016 approval of the epidermal growth factor receptor (EGFR) mutation test as a companion diagnostic for the cancer drug erlotinib (Tarceva; OSI Pharmaceuticals LLC, Northbrook, Illinois). It is the first FDA-approved blood-based genetic test that can detect EGFR gene mutations, which occur in approximately 10% to 20% of patients with non–small cell lung cancer. “Those cancers can dramatically respond to the drug, which switches off the EGFR oncogene,” Dr. Li says. “And our researchers are now looking at a much broader panel of genetic mutations, such as ALK, BRAF, RAS1, and many others that may be treated with targeted therapy.” Not only are liquid biopsies less invasive, they also enable rapid precision therapy because results can be obtained quickly, he adds. “These tests give us the ability to monitor and evaluate our treatments in real time and also help us to understand how to overcome resistance,” Dr. Li says. Even in the treatment of metastatic cancers, the tests are not completely precise, and more high-level evidence is needed to help oncologists determine how best to select and interpret liquid biopsies. Another challenge is that not every tumor sheds enough DNA into circulation, which can lead to the tests missing some cancers, according to Dr. Li. “After a test, I still go forward with a tissue biopsy because sometimes you will find something the liquid biopsy missed,” he says. Dr. Li and other scientists are investigating other cancer-related epigenetic changes that are not found in DNA and that potentially could be detected to improve the accuracy of liquid biopsy. Although he hesitates to say how soon liquid biopsies will be used regularly in clinical practice, Dr. Li is optimistic that in the future physicians will be able to tell from a blood test after a patient's surgery if the cancer is gone or if more treatment is necessary. Given the rapid rate of development in the field, he expects to see wider clinical use of liquid biopsies within the next 5 to 10 years. For a list of currently approved liquid biopsy tests and their indications, see the approved companion diagnostics section of the FDA's website at https://www.fda.gov/MedicalDevices/ ProductsandMedicalProcedures/InVitroDiagnostics/ucm 301431.htm.

Cancer-derived material circulating in the bloodstream and other bodily fluids, referred to as liquid biopsies (LBs), has become an appealing adjunct or alternative to tissue biopsies, showing vital promise in several clinical applications. A systematic literature review was conducted to (1) summarize the current health economic evidence for LB assays and (2) identify and analyze the studies addressed or reported on the challenges of health economic modeling in precision medicine. Relevant studies were identified in the EMBASE, MEDLINE, Cochrane Library, EconLit, and the University of Melbourne Full Text Journal databases from 1 January 2013 to 16 September 2022. Included papers were selected if they were economic evaluations and/or budget impact analyses. A total of 24 studies were included and analyzed, with the majority being full economic evaluations (n = 19, 79.2%). Four studies (16.7%) were health and budget impact analyses, and one study (4.1%) incorporated both an economic evaluation and a budget impact analysis. Cohort-level modeling techniques were the most common approach (n = 16; 80%). LB technologies were cost-effective in 15 studies (75%) considering different biomarkers, cancer types and stages, and economic analyses. These studies evaluated LBs for screening and early detection (66.7%), treatment selection (26.7%), and monitoring treatment response (6.6%). Budget impact analysis results were varied among included studies, with the majority of studies (n = 4; 80%) reporting either cost savings, minimal, or modest budget impact, while one study (20%) reported LBs as an efficient strategy. The reviewed studies often inadequately reported or addressed modeling challenges, such as patient-level processes, the combination of tests and treatments, preferences, and uncertainty. LBs could provide a cost-effective approach for treatment selection in lung cancer and aid in the screening and early detection of other cancers, including colorectal, gastric, breast, and brain cancers. This is in comparison with various alternatives, such as the standard of care (SOC) and no screening scenario. However, it is important to mention that in some comparisons, LBs were used in combination with SOC instead of replacing it. Importantly, few studies have pointed toward LBs' cost-effectiveness for monitoring treatment response. Most health and budget impact analyses, especially those focused on lung cancer, suggest potential cost savings or a minimal-to-moderate budget impact. Nevertheless, additional research is needed to ascertain their effectiveness across various stages of lung and colorectal cancer, as well as to address potential modeling challenges. PROSPERO CRD42022307939.

Simple SummaryGastrointestinal (GI) cancers are a common cancer, affecting both men and women, normally diagnosed through tissue biopsies in combination with imaging techniques and standardized biomarkers leading to patient selection for local or systemic therapies. Liquid biopsies (LBs)—due to their non-invasive nature as well as low risk—are the current focus of cancer research and could be a promising tool for early cancer detection and treatment surveillance, thus leading to better patient outcomes. In this review, we provide an overview of different types of LBs enabling early detection and monitoring of GI cancers and their clinical application.Worldwide, gastrointestinal (GI) cancers account for a significant amount of cancer-related mortality. Tests that allow an early diagnosis could lead to an improvement in patient survival. Liquid biopsies (LBs) due to their non-invasive nature as well as low risk are the current focus of cancer research and could be a promising tool for early cancer detection. LB involves the sampling of any biological fluid (e.g., blood, urine, saliva) to enrich and analyze the tumor’s biological material. LBs can detect tumor-associated components such as circulating tumor DNA (ctDNA), extracellular vesicles (EVs), and circulating tumor cells (CTCs). These components can reflect the status of the disease and can facilitate clinical decisions. LBs offer a unique and new way to assess cancers at all stages of treatment, from cancer screenings to prognosis to management of multidisciplinary therapies. In this review, we will provide insights into the current status of the various types of LBs enabling early detection and monitoring of GI cancers and their use in in vitro diagnostics.

BACKGROUND Hepatobiliary and pancreatic cancers are among the most lethal malignancies due to late-stage diagnosis and limited treatment options. Liquid biopsy has emerged as a minimally invasive tool for early cancer detection, prognosis, and therapeutic monitoring. AIM To concise the available data on liquid biopsy and establish its role in hepatobiliary surgeries. METHODS This systematic review was conducted following Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2009 guidelines. A comprehensive literature search was performed using PubMed, Scopus, Web of Science, and EMBASE for studies published up to March 2025. Studies assessing the role of circulating tumor DNA, circulating tumor cells, exosomes, and other liquid biopsy markers in hepatobiliary and pancreatic cancers were included. The risk of bias was evaluated using the Newcastle-Ottawa Scale for observational studies and the Cochrane Risk of Bias Tool for clinical trials. RESULTS Liquid biopsy demonstrated significant potential for early cancer detection, perioperative risk stratification, intraoperative surgical decision-making, and postoperative monitoring of minimal residual disease. However, challenges remain regarding standardization, sensitivity, and clinical validation. CONCLUSION Liquid biopsy represents a paradigm shift in hepatobiliary and pancreatic cancer management. Advancements in next-generation sequencing and artificial intelligence may enhance its clinical utility. Further large-scale studies are needed to establish standardized protocols for routine implementation.

The present article delved into the potential of liquid biopsy to revolutionize cancer diagnostics and treatment by addressing the current challenges in the field. Liquid biopsy, a non-invasive procedure, involves the detection and analysis of biomarkers in bodily fluids, offering advantages over traditional tissue biopsies. It provides a promising solution to the need for real-time monitoring, personalized treatment approaches, early detection, and minimal residual disease assessment in cancer care. By exploring various applications, such as early detection, treatment monitoring, minimal residual disease assessment, genetic profiling, personalized medicine, and prognostic evaluation, liquid biopsy emerges as a valuable tool in advancing cancer diagnostics and treatment. The present article also discussed current developments, future directions, and provided case studies and success stories showcasing the impact of liquid biopsy. Overall, liquid biopsy holds great promise in transforming cancer care by enabling early detection, personalized treatment decisions, and real-time monitoring of tumors. Keywords: Early cancer detection; Liquid biopsy; Minimal residual disease assessment; Traditional tissue biopsy

Despite recommendations for universal screening, adherence to colorectal cancer screening in the US is approximately 60%. Liquid biopsy tests are in development for cancer early detection, but it is unclear whether they are cost-effective for colorectal cancer screening. To estimate the cost-effectiveness of liquid biopsy for colorectal cancer screening in the US. In this economic evaluation, a Markov model was developed to compare no screening and 5 colorectal cancer screening strategies: colonoscopy, liquid biopsy, liquid biopsy following nonadherence to colonoscopy, stool DNA, and fecal immunochemical test. Adherence to first-line screening with colonoscopy, stool DNA, or fecal immunochemical test was assumed to be 60.6%, and adherence for liquid biopsy was assumed to be 100%. For colonoscopy, stool DNA, and fecal immunochemical test, patients who did not adhere to testing were not offered other screening. In colonoscopy-liquid biopsy hybrid, liquid biopsy was second-line screening for those who deferred colonoscopy. Scenario analyses were performed to include the possibility of polyp detection for liquid biopsy. No screening, colonoscopy, fecal immunochemical test, stool DNA, liquid biopsy, and colonoscopy-liquid biopsy hybrid screening. Model outcomes included life expectancy, total cost, and incremental cost-effectiveness ratios. A strategy was considered cost-effective if it had an incremental cost-effectiveness ratio less than the US willingness-to-pay threshold of $100 000 per life-year gained. This study used a simulated cohort of patients aged 45 years with average risk of colorectal cancer. In the base case, colonoscopy was the preferred, or cost-effective, strategy with an incremental cost-effectiveness ratio of $28 071 per life-year gained. Colonoscopy-liquid biopsy hybrid had the greatest gain in life-years gained but had an incremental cost-effectiveness ratio of $377 538. Colonoscopy-liquid biopsy hybrid had a greater gain in life-years if liquid biopsy could detect polyps but remained too costly. In this economic evaluation of liquid biopsy for colorectal cancer screening, colonoscopy was a cost-effective strategy for colorectal cancer screening in the general population, and the inclusion of liquid biopsy as a first- or second-line screening strategy was not cost-effective at its current cost and screening performance. Liquid biopsy tests for colorectal cancer screening may become cost-effective if their cost is substantially lowered.

Omega Therapeutics has developed a novel platform of programmable epigenomic mRNA medicines capable of modifying chromatin state to specifically tune gene expression at the pre-transcriptional level. Epigenomic controllers (ECs) unlock targets that have been historically considered “undruggable,” with one of the most elusive being the MYC oncogene. A direct MYC-targeting anti-cancer agent has previously remained intangible, largely due to the absence of a drug binding pocket and tight autoregulation. A clinical trial is underway (MYCHELANGELO, NCT05497453) to investigate pre-transcriptional inhibition of MYC with OTX-2002 in patients with hepatocellular carcinoma (HCC). OTX-2002, a first-in-class mRNA therapeutic delivered via lipid nanoparticles (LNP), encodes two proteins that durably modify chromatin, in part, through CpG DNA methylation at the MYC locus. Using both liquid and solid biopsy sampling from in vivo studies, we investigated whether target engagement for OTX-2002 could be assessed by MYC methylation. Detection of DNA methylation has long held promise as an oncology biomarker given its functional roles in various cancer types and the potential signal afforded by methylated CpGs. Indeed, DNA methylation has been shown to serve as a robust analyte in liquid biopsy-derived multi-cancer early detection (MCED) and minimal residual disease (MRD) tests that have recently been utilized in the clinic. Many of these platforms are founded on complex models that leverage methylation signals across >1 million CpGs that can span tens of megabases of genomic space. We faced a distinct challenge when compared to these MCED/MRD tests in developing a pharmacodynamic methylation assay for OTX-2002 as the target region consists of just a few kilobases. With a tissue specific LNP delivery system, ultra-high sensitivity was required to identify rare ctDNA events from the larger cfDNA population. To this end, we designed a minimal hybridization/capture panel that targeted ~50 kb of genomic space, effectively allowing for ultra-deep methylation sequencing of MYC. When this technique was paired with enzymatic (EM) conversion for methylation detection and supported by an analysis pipeline focused on epiallele identification, this assay was able to detect methylation down to the theoretical limit in a dilution series of control genomic DNA, 1 in 104 copies of MYC. This degree of sensitivity translated to successful preclinical detection of on-target methylation by OTX-2002 from DNA extracted from plasma samples collected from mice-bearing human HCC xenografts. Overall, we present a non-invasive and exquisitely sensitive method of assessing target engagement and site-specific pharmacodynamic activity of a novel epigenomic medicine that can be directly translated to the clinical setting. Citation Format: Justin Chen, William Senapedis, Stephen Siecinski, Elmer Figueroa, Adam Katz, Samuel Mildrum, Yaoyu E. Wang, Houda Belaghzal, Kayleigh Gallagher, Graeme Hodgson, Charles W. O'Donnell, Thomas G. McCauley. Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2417.

“Liquid biopsy” was introduced as a new diagnostic concept in 2010 (1) for the analysis of circulating tumor cells (CTCs) and has been now extended to material (in particular DNA) released by tumor cells in the peripheral blood of cancer patients (2,3). Over the past decade, various methods have been developed to detect CTCs and ctDNA in the peripheral blood of cancer patients (3,4). While reliable information can be easily obtained in patients with advanced disease, early-stage cancer patients usually present with very low concentrations of CTCs and ctDNA. At present, most CTC assays rely on epithelial markers and the majority of CTCs detected are single isolated cells. The clinical relevance of "mesenchymal" CTCs lacking any epithelial markers as well as CTC clusters is still under investigation. Although most published studies have been performed on patients with carcinomas and melanomas, CTCs have been also detected in the peripheral blood of patients with primary brain tumors (glioblastomas) despite the blood-brain barrier (5). Liquid biopsy assays are currently being validated for early detection of cancer, which is supposed to reduce cancer-related mortality. Despite remarkable progress, liquid biopsy-based detection of early stages of cancer remains a challenge, in particular in breast cancer. New blood-based biomarkers for early detection currently validated in clinical trials include miRNAs, exosomes, and tumor-educated platelets. In patients with diagnosed cancer, CTCs and ctDNA analyses can obtain independent information on prognosis in early and advanced stages of disease. In particular, CTC counts at initial diagnosis are able to refine the current risk stratification by TNM staging in early-stage breast cancer. Moreover, early detection of relapse by sequential ctDNA (or CTCs) analysis of blood samples obtained post-surgery during the follow-up is possible and may be used in future trials to stratify patients to “post-adjuvant” therapies (6). Another key application of liquid biopsy is to identify therapeutic targets or mechanisms of resistance of metastatic cells in individual patients (6). While the analysis of ctDNA focuses on mutations relevant for cancer therapy (e.g., EGFR, KRAS, or ESR1 mutations), CTCs offer a wide spectrum of analyses at the DNA, RNA, and protein levels (2,3). Metastatic cells might have unique characteristics that can differ from the bulk of cancer cells in the primary tumor currently used for stratification of patients to systemic therapy. Moreover, monitoring of CTCs and ctDNA before, during, and after systemic therapy (e.g., chemotherapy, hormonal therapy, antibody therapy) might provide unique information for the future clinical management of the individual cancer patient and might serve as surrogate marker for response to therapy. In the context of recent success in antibody-mediated blockade of immune checkpoint control molecules, expression of the PD-L1 on CTCs might be of interest as potential predictive marker. Moreover, the expression of androgen receptor variant 7 in CTCs may predict resistance to antiandrogen therapy in prostate cancer, while mutations in the estrogen receptor gene (ESR1) provides information on resistance to hormone therapy in breast cancer. Additional therapeutic targets detected on CTCs in cancer patients include the estrogen receptor and HER-2 oncogene (3). Single-cell RNAseq analysis of CTCs may provide more comprehensive information on relevant pathways. For functional analysis of CTCs, the development of in vitro and in vivo test systems has started, which might also serve as models for drug testing. In particular, the development of cell lines and xenografts derived from CTCs can provide novel insights into the biology of tumor cell dissemination and may be used to discover new pathways to target specifically metastatic cells. Besides CTCs and ctDNA, the analysis of circulating microRNAs, exosomes or tumor-educated platelets may provide complementary information as “liquid biopsy." For example, the integrin composition of exosomes seems to determine the organ site of metastatic niches and the RNA expression pattern of blood platelets reveals information on tumors in cancer patients. Sensitive methods have been also developed to capture disseminated tumor cells (DTCs) in the bone marrow in cancer patients (6), which provide new insights into the process of “cancer dormancy.” The nature of dormant breast cancer cells and the mechanisms leading to their outgrowth are poorly understood. Efforts to unravel the nature of cancer dormancy have been hampered by the lack of sensitive methods to detect dormant cells in cancer patients. The development of novel therapies designed to kill dormant residual tumor cells, or maintain them in a quiescent state, represents a highly attractive approach to prevent late recurrence. Such an approach, however, would require a far more detailed understanding of tumor dormancy and recurrence than exists today, as well as biomarkers to enable monitoring of this process and predict recurrence. Analysis of DTCs leads to the discovery of new molecules relevant to the biology of metastasis such as the putative metastasis-suppressor RAI2 (7). In conclusion, liquid biopsy analysis can be used to obtain new insights into metastasis biology, and as companion diagnostics to improve the stratification of therapies and to obtain insights into therapy-induced selection of cancer cells. Different approaches such as CTC or ctDNA analysis will provide complementary information. Technical and clinical assay validation is very important and can be achieved in international consortia such as the European IMI Cancer-ID network (www.cancer-id.eu).

Cancer is the leading cause of death in dogs, yet there are no established screening paradigms for early detection. Liquid biopsy methods that interrogate cancer-derived genomic alterations in cell-free DNA in blood are being adopted for multi-cancer early detection in human medicine and are now available for veterinary use. The CANcer Detection in Dogs (CANDiD) study is an international, multi-center clinical study designed to validate the performance of a novel multi-cancer early detection “liquid biopsy” test developed for noninvasive detection and characterization of cancer in dogs using next-generation sequencing (NGS) of blood-derived DNA; study results are reported here. In total, 1,358 cancer-diagnosed and presumably cancer-free dogs were enrolled in the study, representing the range of breeds, weights, ages, and cancer types seen in routine clinical practice; 1,100 subjects met inclusion criteria for analysis and were used in the validation of the test. Overall, the liquid biopsy test demonstrated a 54.7% (95% CI: 49.3–60.0%) sensitivity and a 98.5% (95% CI: 97.0–99.3%) specificity. For three of the most aggressive canine cancers (lymphoma, hemangiosarcoma, osteosarcoma), the detection rate was 85.4% (95% CI: 78.4–90.9%); and for eight of the most common canine cancers (lymphoma, hemangiosarcoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, mammary gland carcinoma, anal sac adenocarcinoma, malignant melanoma), the detection rate was 61.9% (95% CI: 55.3–68.1%). The test detected cancer signal in patients representing 30 distinct cancer types and provided a Cancer Signal Origin prediction for a subset of patients with hematological malignancies. Furthermore, the test accurately detected cancer signal in four presumably cancer-free subjects before the onset of clinical signs, further supporting the utility of liquid biopsy as an early detection test. Taken together, these findings demonstrate that NGS-based liquid biopsy can offer a novel option for noninvasive multi-cancer detection in dogs.

Liquid Biopsy Screening for Early Detection of Lung Cancer: Current State and Future Directions