Liquid Biopsy Analysis Research Articles

The Diversity of Liquid Biopsies and Their Potential in Breast Cancer Management.

Analyzing blood as a so-called liquid biopsy in breast cancer (BC) patients has the potential to adapt therapy management. Circulating tumor cells (CTCs), extracellular vesicles (EVs), cell-free DNA (cfDNA) and other blood components mirror the tumoral heterogeneity and could support a range of clinical decisions. Multi-cancer early detection tests utilizing blood are advancing but are not part of any clinical routine yet. Liquid biopsy analysis in the course of neoadjuvant therapy has potential for therapy (de)escalation.Minimal residual disease detection via serial cfDNA analysis is currently on its way. The prognostic value of blood analytes in early and metastatic BC is undisputable, but the value of these prognostic biomarkers for clinical management is controversial. An interventional trial confirmed a significant outcome benefit when therapy was changed in case of newly emerging cfDNA mutations under treatment and thus showed the clinical utility of cfDNA analysis for therapy monitoring. The analysis of PIK3CA or ESR1 variants in plasma of metastatic BC patients to prescribe targeted therapy with alpesilib or elacestrant has already arrived in clinical practice with FDA-approved tests available and is recommended by ASCO. The translation of more liquid biopsy applications into clinical practice is still pending due to a lack of knowledge of the analytes' biology, lack of standards and difficulties in proving clinical utility.

623P Advances in methylation analysis of liquid biopsy in early cancer detection of colorectal and lung cancer

623P Advances in methylation analysis of liquid biopsy in early cancer detection of colorectal and lung cancer

Liquid Biopsy Analysis Shows MAGI2 Gene as Potential Negative Prognosis Biomarker in NSCLC Patients

Liquid Biopsy Analysis Shows MAGI2 Gene as Potential Negative Prognosis Biomarker in NSCLC Patients

Clinical Feasibility of NGS Liquid Biopsy Analysis in NSCLC Patients Using Oncomine Precision Assay with Genexus™ Integrated Sequencer

Clinical Feasibility of NGS Liquid Biopsy Analysis in NSCLC Patients Using Oncomine Precision Assay with Genexus™ Integrated Sequencer

Machine-Learning-Based Single-Molecule Quantification of Circulating MicroRNA Mixtures.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression and are emerging as powerful indicators of diseases. MiRs are secreted in blood plasma and thus may report on systemic aberrations at an early stage via liquid biopsy analysis. We present a method for multiplexed single-molecule detection and quantification of a selected panel of miRs. The proposed assay does not depend on sequencing, requires less than 1 mL of blood, and provides fast results by direct analysis of native, unamplified miRs. This is enabled by a novel combination of compact spectral imaging and a machine learning-based detection scheme that allows simultaneous multiplexed classification of multiple miR targets per sample. The proposed end-to-end pipeline is extremely time efficient and cost-effective. We benchmark our method with synthetic mixtures of three target miRs, showcasing the ability to quantify and distinguish subtle ratio changes between miR targets.

Pre-radiotherapy ctDNA liquid biopsy for risk stratification of oligometastatic non-small cell lung cancer

The optimal treatment paradigm for patients with oligometastatic non-small cell lung cancer (NSCLC) remains unclear. Some patients with oligometastatic disease experience prolonged remission after locally consolidative radiation therapy (RT), while others harbor micrometastatic disease (below limits of detection by imaging) and benefit from systemic therapy. To risk-stratify and identify the patients most likely to benefit from locally consolidative RT, we performed a multi-institutional cohort study of 1487 patients with oligometastatic NSCLC undergoing liquid biopsy analysis of circulating tumor DNA (ctDNA). In total, 1880 liquid biopsies were performed and approximately 20% of patients (n = 309) had ctDNA measured prior to RT and after their diagnosis of oligometastatic disease. Patients with undetectable ctDNA (pathogenic or likely pathogenic variants in plasma using the Tempus xF assay) before RT had significantly improved progression-free survival (PFS) (P = 0.004) and overall survival (OS) (P = 0.030). ctDNA maximum variant allele frequency (VAF) pre-RT and ctDNA mutational burden pre-RT were both significantly inversely correlated with PFS (maximum VAF P = 0.008, mutational burden P = 0.003) and OS (maximum VAF P = 0.007, mutational burden P = 0.045). These findings were corroborated by multivariate Cox proportional hazards models that included eight additional clinical and genomic parameters. Overall, these data suggest that in patients with oligometastatic NSCLC, pre-RT ctDNA can potentially identify the patients most likely to benefit from locally consolidative RT and experience prolonged PFS and OS. Similarly, ctDNA may be useful to identify undiagnosed micrometastatic disease where it may be appropriate to prioritize systemic therapies.

1215P Comparative analysis of cfDNA liquid biopsy and tumor-based next-generation sequencing (NGS) approaches

1215P Comparative analysis of cfDNA liquid biopsy and tumor-based next-generation sequencing (NGS) approaches

The Health Inequality Impact of Liquid Biopsy to Inform First-Line Treatment of Advanced Non–Small Cell Lung Cancer: A Distributional Cost-Effectiveness Analysis

ObjectivesTo perform a distributional cost-effectiveness analysis of liquid biopsy (LB) followed by, if needed, tissue biopsy (TB) (LB-first strategy) relative to a TB-only strategy to inform first-line treatment of advanced non–small cell lung cancer (aNSCLC) from a US payer perspective by which we quantify the impact of LB-first on population health inequality according to race and ethnicity. MethodsWith a health economic model, quality-adjusted life-years (QALYs) and costs per patient were estimated for each subgroup. Given the lifetime risk of aNSCLC, and assuming equally distributed opportunity costs, the incremental net health benefits of LB-first were calculated, which were used to estimate general population quality-adjusted life expectancy at birth (QALE) by race and ethnicity with and without LB-first. The degree of QALYs and QALE differences with the strategies was expressed with inequality indices. Their differences were defined as the inequality impact of LB-first. ResultsLB-first resulted in an additional 0.21 (95% uncertainty interval: 0.07-0.39) QALYs among treated patients, with the greatest gain observed among Asian patients (0.31 QALYs [0.09-0.61]). LB-first resulted in an increase in relative inequality in QALYs among patients, but a minor decrease in relative inequality in QALE. ConclusionsLB-first to inform first-line aNSCLC therapy can improve health outcomes. With current diagnostic performance, the benefit is the greatest among Asian patients, thereby potentially widening racial and ethnic differences in survival among patients with aNSCLC. Assuming equally distributed opportunity costs and access, LB-first does not worsen and, in fact, may reduce inequality in general population health according to race and ethnicity.

Cell free ACE2 RNA: A potential biomarker of COVID-19 severity

Despite the downward trend of COVID-19 pandemic and increased immunity of the general population, COVID-19 is still an elusive disease with risks due to emerging variants. Fast and reliable diagnosis of COVID-19 disease would allow better therapeutic interventions for patients at risk to develop more severe outcomes. Cell-free RNAs (cfRNAs) have been proven to be an effective biomarker in cancer and infectious diseases. It has been reported that cfRNAs are amplified in the bloodstream of these patients and at earlier stages of the disease, reflecting tissue damage. Hence, we hypothesize that cfRNAs may serve as a potential indicator of COVID-19 disease severity. To our knowledge, this is the first report to display a significant link between COVID-19 severity and cfRNA of angiotensin converting enzyme-2 (ACE2), the receptor for SARS-CoV-2 virus. qRT-PCR analysis of liquid biopsies from COVID-19 patients (n = 82) displayed a significant increase in ACE2-cfRNA levels in patients with severe manifestations. This finding correlated with blood biomarkers (ANC, WBC, and Creatinine) that were also significantly increased in these patients. We previously showed that bronchial cells from obese subjects express higher ACE2 levels, hence, we further analysed the involvement of obesity as a main contributor to severe outcomes. We confirm a significant increase of ACE2-cfRNA in the plasma of obese/overweight (Ob/Ov) COVID-19 patients compared to lean subjects, with no observed significant change in blood biomarkers. These findings suggest that monitoring ACE2-cfRNAs, as a biomarker, during COVID-19 infection may allow for better disease management, specifically for severe-COVID-19 patients.

A spectroscopic liquid biopsy for the earlier detection of multiple cancer types

BackgroundA rapid, low-cost blood test that can be applied to reliably detect multiple different cancer types would be transformational.MethodsIn this large-scale discovery study (n = 2092 patients) we applied the Dxcover® Cancer Liquid Biopsy to examine eight different cancers. The test uses Fourier transform infrared (FTIR) spectroscopy and machine-learning algorithms to detect cancer.ResultsArea under the receiver operating characteristic curve (ROC) values were calculated for eight cancer types versus symptomatic non-cancer controls: brain (0.90), breast (0.76), colorectal (0.91), kidney (0.91), lung (0.91), ovarian (0.86), pancreatic (0.84) and prostate (0.86). We assessed the test performance when all eight cancer types were pooled to classify ‘any cancer’ against non-cancer patients. The cancer versus asymptomatic non-cancer classification detected 64% of Stage I cancers when specificity was 99% (overall sensitivity 57%). When tuned for higher sensitivity, this model identified 99% of Stage I cancers (with specificity 59%).ConclusionsThis spectroscopic blood test can effectively detect early-stage disease and can be fine-tuned to maximise either sensitivity or specificity depending on the requirements from different healthcare systems and cancer diagnostic pathways. This low-cost strategy could facilitate the requisite earlier diagnosis, when cancer treatment can be more effective, or less toxic.Statement of translational relevanceThe earlier diagnosis of cancer is of paramount importance to improve patient survival. Current liquid biopsies are mainly focused on single tumour-derived biomarkers, which limits test sensitivity, especially for early-stage cancers that do not shed enough genetic material. This pan-omic liquid biopsy analyses the full complement of tumour and immune-derived markers present within blood derivatives and could facilitate the earlier detection of multiple cancer types. There is a low barrier to integrating this blood test into existing diagnostic pathways since the technology is rapid, simple to use, only minute sample volumes are required, and sample preparation is minimal. In addition, the spectroscopic liquid biopsy described in this study has the potential to be combined with other orthogonal tests, such as cell-free DNA, which could provide an efficient route to diagnosis. Cancer treatment can be more effective when given earlier, and this low-cost strategy has the potential to improve patient prognosis.

Advances on Liquid Biopsy Analysis for Glioma Diagnosis.

Gliomas comprise the most frequent primary central nervous system (CNS) tumors, characterized by remarkable genetic and epigenetic heterogeneity, difficulty in monitoring, and increased relapse and mortality rates. Tissue biopsy is an established method of tumor cell collection and analysis that enables diagnosis, classification of different tumor types, and prediction of prognosis upon confirmation of tumor's location for surgical removal. However, it is an invasive and often challenging procedure that cannot be used for frequent patient screening, detection of mutations, disease monitoring, or resistance to therapy. To this end, the minimally invasive procedure of liquid biopsy has emerged, allowing effortless tumor sampling and enabling continuous monitoring. It is considered a novel preferable way to obtain faster data on potential tumor risk, personalized diagnosis, prognosis, and recurrence evaluation. The purpose of this review is to describe the advances on liquid biopsy for glioma diagnosis and management, indicating several biomarkers that can be utilized to analyze tumor characteristics, such as cell-free DNA (cfDNA), cell-free RNA (cfRNA), circulating proteins, circulating tumor cells (CTCs), and exosomes. It further addresses the benefit of combining liquid biopsy with radiogenomics to facilitate early and accurate diagnoses, enable precise prognostic assessments, and facilitate real-time disease monitoring, aiming towards more optimal treatment decisions.

Advances in methylation analysis of liquid biopsy in early cancer detection of colorectal and lung cancer

Methylation patterns in cell-free DNA (cfDNA) have emerged as a promising genomic feature for detecting the presence of cancer and determining its origin. The purpose of this study was to evaluate the diagnostic performance of methylation-sensitive restriction enzyme digestion followed by sequencing (MRE-Seq) using cfDNA, and to investigate the cancer signal origin (CSO) of the cancer using a deep neural network (DNN) analyses for liquid biopsy of colorectal and lung cancer. We developed a selective MRE-Seq method with DNN learning-based prediction model using demethylated-sequence-depth patterns from 63,266 CpG sites using SacII enzyme digestion. A total of 191 patients with stage I–IV cancers (95 lung cancers and 96 colorectal cancers) and 126 noncancer participants were enrolled in this study. Our study showed an area under the receiver operating characteristic curve (AUC) of 0.978 with a sensitivity of 78.1% for colorectal cancer, and an AUC of 0.956 with a sensitivity of 66.3% for lung cancer, both at a specificity of 99.2%. For colorectal cancer, sensitivities for stages I–IV ranged from 76.2 to 83.3% while for lung cancer, sensitivities for stages I–IV ranged from 44.4 to 78.9%, both again at a specificity of 99.2%. The CSO model's true-positive rates were 94.4% and 89.9% for colorectal and lung cancers, respectively. The MRE-Seq was found to be a useful method for detecting global hypomethylation patterns in liquid biopsy samples and accurately diagnosing colorectal and lung cancers, as well as determining CSO of the cancer using DNN analysis.Trial registration: This trial was registered at ClinicalTrials.gov (registration number: NCT 04253509) for lung cancer on 5 February 2020, https://clinicaltrials.gov/ct2/show/NCT04253509. Colorectal cancer samples were retrospectively registered at CRIS (Clinical Research Information Service, registration number: KCT0008037) on 23 December 2022, https://cris.nih.go.kr, https://who.init/ictrp. Healthy control samples were retrospectively registered.

Cell State and Cell Type: Deconvoluting Circulating Tumor Cell Populations in Liquid Biopsies by Multi-Omics.

Bi-directional crosstalk between the tumor and the tumor microenvironment (TME) has been shown to increase the rate of tumor evolution and to play a key role in neoplastic progression, therapeutic resistance, and a patient's overall survival. Here, we set out to use a comprehensive liquid-biopsy analysis to study cancer and specific TME cells in circulation and their association with disease status. Cytokeratin+, CD45- circulating rare cells (CRCs) from nine breast and four prostate cancer patients were characterized through morphometrics, single-cell copy number analysis, and targeted multiplexed proteomics to delineate cancer cell lineage from other rare cells originating in the TME. We show that we can detect epithelial circulating tumor cells (EPI.CTC), CTCs undergoing epithelial-to-mesenchymal transition (EMT.CTC) and circulating endothelial cells (CECs) using a universal rare event detection platform (HDSCA). Longitudinal analysis of an index patient finds that CTCs are present at the time of disease progression, while CECs are predominately present at the time of stable disease. In a small cohort of prostate and breast cancer patients, we find high inter-patient and temporal intra-patient variability in the expression of tissue specific markers such as ER, HER2, AR, PSA and PSMA and EpCAM. Our study stresses the importance of the multi-omic characterization of circulating rare cells in patients with breast and prostate carcinomas, specifically highlighting overlapping and cell type defining proteo-genomic characteristics of CTCs and CECs.

Current status of molecular diagnostic approaches using liquid biopsy

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers, and developing an efficient and reliable approach for its early-stage diagnosis is urgently needed. Precancerous lesions of PDAC, such as pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasms (IPMN), arise through multiple steps of driver gene alterations in KRAS, TP53, CDKN2A, SMAD4, or GNAS. Hallmark mutations play a role in tumor initiation and progression, and their detection in bodily fluids is crucial for diagnosis. Recently, liquid biopsy has gained attention as an approach to complement pathological diagnosis, and in addition to mutation signatures in cell-free DNA, cell-free RNA, and extracellular vesicles have been investigated as potential diagnostic and prognostic markers. Integrating such molecular information to revise the diagnostic criteria for pancreatic cancer can enable a better understanding of the pathogenesis underlying inter-patient heterogeneity, such as sensitivity to chemotherapy and disease outcomes. This review discusses the current diagnostic approaches and clinical applications of genetic analysis in pancreatic cancer and diagnostic attempts by liquid biopsy and molecular analyses using pancreatic juice, duodenal fluid, and blood samples. Emerging knowledge in the rapidly advancing liquid biopsy field is promising for molecular profiling and diagnosing pancreatic diseases with significant diversity.

Placental Exosomes as Biomarkers for Maternal Diseases: Current Advances in Isolation, Characterization, and Detection.

Serving as the interface between fetal and maternal circulation, the placenta plays a critical role in fetal growth and development. Placental exosomes are small membrane-bound extracellular vesicles released by the placenta during pregnancy. They contain a variety of biomolecules, including lipids, proteins, and nucleic acids, which can potentially be biomarkers of maternal diseases. An increasing number of studies have demonstrated the utility of placental exosomes for the diagnosis and monitoring of pathological conditions such as pre-eclampsia and gestational diabetes. This suggests that placental exosomes may serve as new biomarkers in liquid biopsy analysis. This review provides an overview of the current understanding of the biological function of placental exosomes and their potential as biomarkers of maternal diseases. Additionally, this review highlights current barriers and the way forward for standardization and validation of known techniques for exosome isolation, characterization, and detection. Finally, microfluidic devices for exosome research are discussed.

Epidermal Growth Factor Receptor T790M Mutation Testing in Non-Small Cell Lung Cancer: An International Collaborative Study to Assess Molecular EGFR T790M Testing in Liquid Biopsy.

The detection of the EGFR T790M (T790M) mutation in non-small cell lung cancer (NSCLC) patients who progressed under treatment with first- or second-generation EGFR-tyrosine kinase inhibitors (TKIs) is important to offer a subsequent therapy with a third-generation EGFR-TKI. Liquid biopsy is a powerful tool to determine the T790M mutation status. Several liquid biopsy platforms with varying degrees of accuracy are available to test for T790M mutations, and sensitivities may differ among these methods. As no standard exists for the testing of T790M mutation in liquid biopsy, we performed a collaborative study to describe and compare the sensitivity of different in-house liquid biopsy platforms for the detection of the T790M mutation, EGFR exon 19 deletion (del19) and EGFR L858R mutation (L858R) across multiple participating laboratories in seven Central and Eastern European countries. Of the 25 invited laboratories across Central and Eastern Europe, 21 centers participated and received 10 plasma samples spiked with cell-line DNA containing the T790M, del19, or L858R mutation in different concentrations. In-house PCR-based and NGS-based methods were used accordingly, and results were reported as in routine clinical practice. Two laboratories, which used the AmoyDx® EGFR 29 Mutations Detection Kit (AmoyDx) with Cobas® cfDNA Sample Preparation Kit and QX200 Droplet Digital PCR (ddPCR) with the QIAamp Circulating Nucleic Acid Kit identified all ten samples correctly. Cobas® EGFR Mutation Test v2 (Cobas), the NGS methods, and the IdyllaTM detection method used in this study performed within the known sensitivity range of each detection method. If a negative result was obtained from methods with lower sensitivity (e.g., Cobas), repeated liquid biopsy testing and/or tissue biopsy analysis should be performed whenever possible, to identify T790M-positive patients to allow them to receive the optimal second-line treatment with a third-generation EGFR TKI.

Polypyrrole/carbon dot nanocomposite as an electrochemical biosensor for liquid biopsy analysis of tryptophan in the human serum of normal and breast cancer women

Liquid biopsy analysis represents a suitable alternative analysis procedure in several cases where no tumor tissue is available or in poor patient conditions. Amino acids can play a crucial role in aiding cancer diagnosis. Monitoring of tryptophan (Trp) catabolism can aid in tracking cancer progression. Therefore, a novel nanocomposite was fabricated using overoxidized polypyrrole film doped with nano-carbon dots (nano-CDs) on the pencil graphite electrode (PGE) surface for sensitive evaluation of Trp in human serum. Using square wave voltammetry (SWV), the overoxidized polypyrrole/carbon dots/pencil graphite electrode (Ov-Ox PPy/CDs/PGE) achieved excellent electrochemical catalytic activity for evaluating Trp. The modified electrode, known as Ov-Ox PPy/CDs/PGE, demonstrated superior electrochemical catalytic activity compared to bare PGE, CDs/PGE, PPy/PGE, and PPy/CDs/PGE for evaluation of Trp. The method’s excellent sensitivity was confirmed by the low limits of detection (LOD = 0.003 μmol L−1) and limit of quantitation (LOQ = 0.009 μmol L−1). The biosensor that was developed can measure tryptophan (Trp) levels in the serum of both healthy individuals and female breast cancer patients with high accuracy and sensitivity. The results indicate that there is a significant difference, as shown by the F-test, between healthy individuals and those with breast cancer. This suggests that Trp amino acid could be an essential biomarker for cancer diagnosis. Consequently, liquid biopsy analysis presents a valuable opportunity for early disease detection, particularly for cancer.Graphical abstract

The Role of Liquid Biopsy in the Diagnosis and Prognosis of WHO Grade 4 Astrocytoma.

Liquid biopsy, as a non-invasive diagnostic tool, has recently gained significant attention in the field of oncology. It involves the analysis of various biomarkers present in bodily fluids, such as blood or cerebrospinal fluid, to provide information about the underlying cancer. In the case of WHO grade 4 astrocytomas, liquid biopsy has the potential to significantly impact the diagnosis and prognosis of this aggressive malignant brain tumor. By detecting specific genetic mutations, such as IDH1 or EGFR, and monitoring levels of circulating tumor DNA, liquid biopsy can aid in the early detection and monitoring of disease progression. This innovative approach is gradually being acknowledged as a less invasive and cost-effective procedure for cancer diagnosis and management to improve patient outcomes and quality of life. Various kinds of biomarkers circulating in cerebrospinal fluid (CSF), such as circulating tumor cells (CTC) and different types of nucleic acids like cell-free DNA (cfDNA), cell-free RNA (ctRNA), and microRNAs (miRNA), have been identified. These biomarkers, which require dependable detection methods, are comparatively simple to obtain and allow for repeated measurements, making them significantly superior for disease monitoring. This review aims to compare the latest liquid biopsy analysis tools for both CSF and plasma in the central nervous system.

Non-invasive methods of molecular diagnosis, clinical monitoring and approaches to the personalized therapy of diffuse midline glioma

The purpose of the study was to summarize and analyze modern data about non-invasive methods of molecular diagnosis and approaches to the personalized therapy of diffuse midline glioma (DMG). Material and Methods. The search and analysis of publications was carried out using Google Scholar, Pubmed, Elsevier, Web of Science, Elibrary systems. The review includes publications published from 2011 to 2022. Of the 102 articles found, 59 were used to write the review. Results. In this review, we discuss the spectrum of somatic driver mutations present in DMG tumor cells and their relationship with the sensitivity of tumor cells to certain types of therapy - a pharmacogenetic approach to the selection of individual treatments (targeted therapy). We provide examples of new methods of targeted therapy for DMG, which are currently at the stage of preclinical laboratory development. Also, we discuss examples of the use of 3D cell cultures for the development of targeted therapies, including the use of perfusion systems. The review describes the methods of analysis of liquid biopsy, which allow the detection of tumor-specific biomarkers in the non-invasive diagnosis of DMG, including a number of methods that have not yet been tested in the clinic. The following is a list of tumor-specific biomarkers for diagnosing, monitoring, and selecting targeted therapy for DMG. Finally, we discuss the possibility of implementing these methods in the clinic and present the results of several clinical trials. Conclusion. In oncology, new methods of molecular genetics, such as analysis of liquid biopsy, allow diagnosis and monitoring of treatment in cases where classical methods that require tissue sampling are not applicable (for example, the analysis of genetically heterogeneous tumors and tumors of surgically inaccessible localization). These tumors include DMG, a primary brain tumor most common in children. The available data confirm the relevance of the search for new specific tumor biomarkers, as well as targets for targeted therapy of the paediatric-type diffuse gliomas.

Abstract B016: Liquid biopsy biomarker analysis of a phase II trial of sacituzumab govitecan in castrate resistant metastatic prostate cancer

Abstract Background: Sacituzumab Govitecan (SG) is an antibody-drug conjugate targeting Trop-2 and has been approved for use in breast and bladder cancers. Trop-2 has been shown to be overexpressed in multiple solid tumors including prostate cancer. Here we report an interim analysis of longitudinal liquid biopsies collected in a phase II clinical trial of SG to identify biomarkers of response and resistance. Methods: We collected longitudinal blood samples from 20 patients for circulating tumor cell (CTC) and cell-free DNA (cfDNA) analysis as part of an open label Phase II trial of SG in patients with mCRPC (ClinicalTrials.gov Identifier: NCT03725761). Eligibility criteria for the trial included rising PSA and radiographic progression on abiraterone acetate or enzalutamide in the mCRPC setting. Liquid biopsies were collected at baseline, C1D8, and then every 3 cycles until disease progression. cfDNA was sequenced using a custom 822 gene targeted capture panel (IDT). CTCs were isolated with antibodies to either EpCAM or Trop-2 followed by parallel enumeration, EpCAM and Trop-2 protein phenotyping and RNA extraction for AR/NEPC marker transcriptional profiling. Results: Although no PSA50 responses were observed, the 6-month rPFS rate was 45% (64% in chemotherapy naïve patients). For this analysis, these patients will be termed responding patients. The number of Trop-2-postive CTCs at baseline was significantly higher in responding (24.3 ±13.4 per 7.5 mL blood) than non-responding (10.0±3.8) patients. During treatment the number of Trop-2 positive CTCs in responders significantly decreased by C3D1 to 10.0 ±3.8. Interestingly, while the number of Trop-2 positive CTCs continued to decline to 2.8±1.99 cells/7.5 mL, EpCAM-positive CTCs increased from 12.5±10.52 cells/7.5 mL to 23.2±19.49 at end of trial. Gene expression analysis demonstrated high expression of genes specific to adenocarcinoma including AR-splice variants, TMPRSS2, KLK2 and KLK3 in the majority of patients, with conversion to AR-variant negative CTCs identified in responders. Preliminary cfDNA analysis identified one patient with a mutation acquired in the gene encoding topoisomerase 1 (TOP1) upon progression on SG, with additional analysis ongoing. Conclusions: Patients who responded to SG had a more Trop-2 positive CTCs with adenocarcinoma characteristics with expression of AR pathway genes. Longitudinal analysis of liquid biopsy biomarkers identified multiple mechanisms of acquired SG resistance. Decline in Trop2-CTC population with parallel expansion of EpCAM-CTC population in multiple patients suggests loss of Trop2 surface protein expression as resistance mechanism, while identification of a cfDNA TOP1 mutation in one patient at time of progression is consistent with acquired resistance to the topoisomerase inhibitor payload as has been reported in metastatic triple negative breast cancer. Citation Format: Jamie M. Sperger, Amy K. Taylor, Yue Shi, Charlotte N. Stahlfeld, Kyle Helzer, Matthew Bootsma, Katherine R. Kaufmann, Marina N. Sharifi, Christos E. Kyriakopoulos, Susan Slovin, Scott Tagawa, Scott Dehm, Shuang G. Zhao, Joshua M. Lang. Liquid biopsy biomarker analysis of a phase II trial of sacituzumab govitecan in castrate resistant metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B016.