Liquid Biopsy-based Biomarkers Research Articles

Prospective of Mitochondrial DNA Variations in Cancer on Genomic Medicine.

Mitochondrial DNA (mtDNA) has emerged as a pivotal component in understanding the etiology and susceptibility of cancer. A recent study by Chen and colleagues delineated the germline genetic effect of mtDNA single-nucleotide polymorphisms (SNP) and haplogroups across pan-cancer risk. They identified a subset of mtSNPs and the corresponding risk score, as well as haplogroups A and M7 alongside their genetic interactions, conferring a protective effect against various cancers. These findings underscored the value of mtDNA variations as biomarkers for cancer etiology and as tools for cancer risk stratification. Future investigations are encouraged to integrate comprehensive omics data of genomics, transcriptomics, proteomics, and metabolomics, etc., from nuclear DNA with mtDNA variations, alongside single-cell and spatial technologies, to unravel the tumor mechanism and identify the drug targets. Moreover, the incorporation of polygenic risk score, that included mtDNA variations with both rare and common frequencies, and liquid biopsy-based biomarkers would enhance the predictive performance of cancer risk assessment and refine the risk stratification of population-based cancer screening. This commentary advocates for the validation across diverse populations to harness the full potential of mitochondrial genomics, and ultimately paves the prospective way for advancements in personalized cancer therapeutics and prevention strategies. See related article by Chen and colleagues, Cancer Epidemiol Biomarkers Prev 2024;33:381-8.

Liquid biopsy-based prediction of clinical benefit from immune checkpoint inhibitors in advanced biliary tract cancer.

e16179 Background: Immune checkpoint inhibitors (ICIs) show promising efficacy as first-line therapy for advanced biliary tract cancer (aBTC), but validated predictive biomarkers are lacking. Here, we identified liquid biopsy-based biomarkers and developed a prediction model. Methods: aBTC patients who received ICIs-based combination therapy as the first-line treatment were prospectively enrolled in this study (NCT06074029). Plasma samples were collected before the first and second immunotherapy infusion to perform cell-free DNA (cfDNA) sequencing of 1021 genes. Circulating tumor DNA (ctDNA) concentration was calculated by multiplying the mean variant allele frequency by the cfDNA concentration and ctDNA fold change was calculated by dividing on-treatment ctDNA concentration by the pre-treatment ctDNA concentration. Tumor mutation burden (TMB) was normalized using the cfDNA concentration. In addition, RNA was extracted from peripheral blood mononuclear cells to profile the leukocyte transcriptome using whole transcriptome sequencing, and the relative proportions of immune cells in leukocytes was deconvoluted using CIBERSORT algorithm. Log-rank and univariate Cox regression were used to evaluate the relationship between features and progression-free survival (PFS). Results: 37 patients were enrolled, with 62.2% having intrahepatic cholangiocarcinoma. The median age was 61 years and 54.1% of patients were females. The median PFS was 4.4 months. Variables related to PFS with the log-rank p value smaller than 0.05 (Table) were entered into a multivariate Cox regression using a stepwise approach for final variable selection. As a result, ctDNA fold change, KRAS mutation, and resting memory CD4 T cell level were used to construct a COX regression model to predict clinical benefit from ICIs. The model predicting 6-month PFS demonstrated an AUC of 0.909 (95% CI, 0.795-1.024). Internal validation, performed through a bootstrap analysis with 100 iterations, yielded an optimism-adjusted AUC estimate of 0.859. Conclusions: Our preliminary results support the excellent performance of this liquid biopsy-based multivariable model to early predict durable benefit from ICIs in aBTC. A large sample size and verifying this model with internal and external data are warranted. Clinical trial information: NCT06074029 . [Table: see text]

Assessment of Untargeted Metabolomics by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry to Define Breast Cancer Liquid Biopsy-Based Biomarkers in Plasma Samples.

An early diagnosis of cancer is fundamental not only in regard to reducing its mortality rate but also in terms of counteracting the progression of the tumor in the initial stages. Breast cancer (BC) is the most common tumor pathology in women and the second deathliest cancer worldwide, although its survival rate is increasing thanks to improvements in screening programs. However, the most common techniques to detect a breast tumor tend to be time-consuming, unspecific or invasive. Herein, the use of untargeted hydrophilic interaction liquid chromatography-mass spectrometry analysis appears as an analytical technique with potential use for the early detection of biomarkers in liquid biopsies from BC patients. In this research, plasma samples from 134 BC patients were compared with 136 from healthy controls (HC), and multivariate statistical analyses showed a clear separation between four BC phenotypes (LA, LB, HER2, and TN) and the HC group. As a result, we identified two candidate biomarkers that discriminated between the groups under study with a VIP > 1 and an AUC of 0.958. Thus, targeting the specific aberrant metabolic pathways in future studies may allow for better molecular stratification or early detection of the disease.

Abstract PO5-16-05: Integrating Circulating Tumor Cells (CTCs) and Cell-free DNA (cfDNA) Signatures for Monitoring Treatment Response in Stage III/IV Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer, comprising only between 1-5% of all breast cancer cases in the United States. Despite its low incidence, IBC accounts for 10% of breast cancer-related deaths and is often diagnosed with metastasis, resulting in shorter survival compared to non-IBC cases. The 5-year relative survival rate for all stages of IBC is 39%, dropping to 19% for patients with distant metastasis. Liquid biopsy-based biomarkers have emerged as potential analytes for predicting prognosis and monitoring response to therapies in IBC patients. We explored the prognostic role of liquid biopsy encompassing both CTCs and cfDNA in the blood of patients with stage III/IV IBC and their clinical outcomes. Methods: CTC and cfDNA analysis was performed on 90 blood samples from 47 patients with stage III and IV IBC. Samples were collected before (pre-treatment, n=43) initiation and after (post-treatment, n=47) completion of neoadjuvant chemotherapy. CTCs were enriched and enumerated using the CellSearch® platform (Menarini-Silicon Biosystems). CfDNA was extracted from plasma using MagMax™ Cell-Free Total Nucleic Acid Isolation Kit (Applied Biosystems™) and quantified using the Qubit™ dsDNA High Sensitivity kit (Invitrogen™). Differences in cfDNA concentrations were used to plot a receiver operating characteristic (ROC) curve which was then used to determine Area Under the Curve (AUC) and a cfDNA cut-off point. ROC cut-off was calculated based on Youden Index J. Survival analysis was performed by the Kaplan-Meier estimator and Cox regression models. P-values were measured using the Student t-test. Results: Prior to neoadjuvant treatment, CTC was detected in 51% of patient samples, and the median cfDNA concentration was 5.87 ng/mL (range 0.95-24.11 ng/mL). After completion of neoadjuvant treatment, CTC was detected in 25% of patient samples, and the median cfDNA concentration was 11.19 ng/mL (range 3.41-88.13 ng/mL). The cut-off points determined for pre-treatment and post-treatment cfDNA concentration was >4.2 ng/mL and >9.5 ng/mL respectively. At pre-treatment, CTC detection and cfDNA concentration >4.2 ng/mL were independently associated with disease relapse (P=0.007, 0.016, respectively). The combination of CTC detection and cfDNA concentration >4.2 ng/mL was significantly associated with disease relapse (P=0.0004), decreased relapse free survival (RFS, P=0.005) and overall survival (OS, P=0.007). At the post-neoadjuvant treatment time point, the combination of CTC detection and cfDNA concentration >9.5 ng/mL was significantly associated with relapse regardless of pCR status (P=0.02). Conclusion: The combined presence of CTCs and elevated cfDNA concentrations in patients with stage III/IV IBC after completion of neoadjuvant chemotherapy was associated with disease relapse regardless of pCR status. A combination of elevated cfDNA and CTC detection signatures after completion of neoadjuvant chemotherapy may be useful for risk stratification and adjuvant systemic therapy planning. Citation Format: Sridevi Addanki, Salyna Meas, Vanessa Sarli, Abdulkader Almosa, Wendy Woodward, Dhruvajyoti Roy, Anthony Lucci. Integrating Circulating Tumor Cells (CTCs) and Cell-free DNA (cfDNA) Signatures for Monitoring Treatment Response in Stage III/IV Inflammatory Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-05.

Biomarkers for Pre-Treatment Risk Stratification of Prostate Cancer Patients: A Systematic Review.

Prostate cancer (PCa) is one of the most frequently occurring malignancies. Although most cases are not life-threatening, approximately 20% endure an unfavorable outcome. PSA-based screening reduced mortality but at the cost of an increased overdiagnosis/overtreatment of low-risk (lrPCa) and favorable intermediate-risk (firPCa) PCa. PCa risk-groups are usually identified based on serum Prostate-Specific Antigen (PSA), the Gleason score, and clinical T stage, which have consistent although variable specificity or subjectivity. Thus, more effective and specific tools for risk assessment are needed, ideally making use of minimally invasive methods such as liquid biopsies. In this systematic review we assessed the clinical potential and analytical performance of liquid biopsy-based biomarkers for pre-treatment risk stratification of PCa patients. Studies that assessed PCa pre-treatment risk were retrieved from PubMed, Scopus, and MedLine. PCa risk biomarkers were analyzed, and the studies' quality was assessed using the QUADAS-2 tool. The final analysis comprised 24 full-text articles, in which case-control studies predominated, mostly reporting urine-based biomarkers (54.2%) and biomarker quantification by qPCR (41.7%). Categorization into risk groups was heterogeneous, predominantly making use of the Gleason score. This systematic review unveils the substantial clinical promise of using circulating biomarkers in assessing the risk for prostate cancer patients. However, the standardization of groups, categories, and biomarker validation are mandatory before this technique can be implemented. Circulating biomarkers might represent a viable alternative to currently available tools, obviating the need for tissue biopsies, and allowing for faster and more cost-effective testing, with superior analytical performance, specificity, and reproducibility.

Tissue- and liquid biopsy-based biomarkers for immunotherapy in breast cancer.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy and now represent the mainstay of treatment for many tumor types, including triple-negative breast cancer and two agnostic registrations. However, despite impressive durable responses suggestive of an even curative potential in some cases, most patients receiving ICIs do not derive a substantial benefit, highlighting the need for more precise patient selection and stratification. The identification of predictive biomarkers of response to ICIs may play a pivotal role in optimizing the therapeutic use of such compounds. In this Review, we describe the current landscape of tissue and blood biomarkers that could serve as predictive factors for ICI treatment in breast cancer. The integration of these biomarkers in a "holistic" perspective aimed at developing comprehensive panels of multiple predictive factors will be a major step forward towards precision immune-oncology.

Plasma Extracellular Vesicle-Associated microRNAs for Early Response Prediction in Patients with High-Grade B-Cell Lymphoma

Background: Early and accurate outcome prediction is essential in the clinical management of high-grade B-cell lymphoma (HGBL). An early switch to salvage treatments for those patients who are likely to develop refractory/relapsed disease may improve overall survival. Unfortunately, the positive predictive value of an interim PET-CT is not high enough to guide treatment decisions. Extracellular vesicle-associated microRNAs (EV-miRNAs) are considered promising liquid biopsy-based biomarkers for lymphomas. We performed small RNA sequencing of plasma samples collected during treatment and applied machine learning to build EV-miRNA signatures for early response prediction in patients with HGBL with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH). Methods: We analyzed PAXgene ccfDNA plasma samples, from 38 of the 97 patients included in the HOVON-152 trial (NCT03620578), a prospective, multicenter, non-randomized phase II trial. In this trial, patients with HGBL-DH/TH receive one cycle of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) followed by five cycles of dose adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R). Patients who achieve complete metabolic response (CMR) at the end-of-induction (EOI) receive one year of nivolumab consolidation. Response was assessed by an EOI PET-CT and classified as CMR (Deauville score 1-3) (responders) or no-CMR (non-responders). 20 responders and 18 non-responders (enrichment for non-responders), were selected for this analysis. We isolated plasma EVs with size exclusion chromatography as confirmed with transmission electron microscopy (TEM), tunable resistive pulse sensing (TRPS), and western blot (WB). Library preparation was done according to an unbiased, unique molecular identifier (UMI)-enhanced small RNA sequencing protocol (IsoSeek) (van Eijndhoven et al., 2021. bioRxiv) and sequenced on the NovaSeq 6000 (Illumina). We applied machine learning including the least absolute shrinkage and selection operator (LASSO), Elastic Net, and Ridge Regression to build models with EV-miRNAs for early EOI response prediction. We selected the most optimal model based on the lowest misclassification error. Then the model was internally validated and tested with bootstrapping (1000x) and over optimism estimate as well as the adjusted AUC was calculated. Results: TEM after one cycle of R-CHOP revealed abundant particles < 200 nm and TRPS measured significantly higher particle concentration in EV-enriched fractions. Plasma EVs were positive for CD63, CD81, flotillin-1, syntenin, HSP70, and negative for calnexin as determined by WB in accordance with the Minimal Information for Studies of EVs (MISEV) criteria 2018 for EV characterization. The most optimal model was an Elastic Net regression (a = 0.4) model consisting of 199 miRNAs with an area under the curve (AUC) of 0.95 (Confidence Interval (CI): 0.90 - 0.99) (Figure 1) [Sensitivity: 88%, Specificity: 90%; Positive Predictive Value (PPV): 90%, Negative Predictive Value (NPV): 87%]. We tested our model with bootstrapping (1,000x). After taking into account of over optimism estimate of 0.14, the adjusted AUC was 0.81 (CI: 0.64-0.96), which is higher than the performance of interim PET/CT [Sensitivity: 33-87%, Specificity: 49-94%; PPV: 20-74%, NPV: 64-95%] (Burggraaff 2019, PMID: 30141066). Conclusion: Machine learning models using plasma EV-miRNAs prepared with the IsoSeek small RNA sequencing protocol yielded a robust signature that can predict EOI response already after one cycle of R-CHOP with a NPV of 87% and a PPV of 90%. The next step is to validate this model in a large cohort of HGBL-DH/TH cases and explore its potential in all subtypes of diffuse large B-cell lymphoma. If validated in independent cohorts, this novel approach could potentially, in combination with other modalities such as cell-free DNA and interim PET-CT, guide early risk-adapted treatment strategies in aggressive high-grade B-cell lymphomas. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Plasma-Based microRNA Expression Analysis in Advanced Stage NSCLC Patients Treated with Nivolumab.

Simple SummaryNivolumab (anti-PD-1 inhibitor) is the first monoclonal antibody approved for the treatment of NSCLC, with research results showing that patients who had received previous lines of therapy had a better response to this treatment and better overall survival. Tissue-level analyses fail to capture the dynamic tumor-host relationship, in contrast to circulating biomarkers, which can reflect the systemic response of the tumor, allowing for repeated sampling and monitoring. In the context of liquid biopsy, microRNAs are studied as biomarkers of immunotherapy efficacy based on their role in regulating antitumor immunity. The present study suggests that miR-200c and miR-34a plasma expression levels have a prognostic role in patients with advanced NSCLC receiving Nivolumab. It further supports that the expression profile of circulating immunomodulatory microRNAs provides information on the survival of patients with advanced NSCLC receiving Nivolumab and could represent promising circulating biomarkers that may provide information about patients’ responses to immunotherapy.Since circulating microRNAs (miRNAs) are involved in the modulation of the immune response, they are tested as liquid biopsy-based biomarkers in patients with NSCLC treated with immunotherapy. We analyzed the expression levels and examined the clinical significance of immunoregulatory miRNAs involved in immune checkpoint regulation (miR-34a, miR-200b, miR-200c), T-cell activity (miR-155), and the function of myeloid-derived suppressive cells (MDSCs) (miR-223) or regulatory T lymphocytes (Tregs) (miR-146a), in patients with advanced NSCLC (N = 69) treated with anti-PD-1 (Nivolumab) immunotherapy as 2nd or 3rd line of treatment therapy. Plasma levels of circulating miRNAs were analyzed by RT-qPCR before the initiation of immunotherapy. Expression of miR-34a, miR-146a, mir-200c, and miR-223 was found to be associated with response to immunotherapy. High miR-200c expression emerged as an independent prognostic factor for inferior overall survival in all patients with NSCLC (OS, HR: 2.243, 95% CI: 1.208–4.163; p = 0.010) and in patients with non-Squamous (non-SqCC) subtype (N = 38) (HR: 2.809, 95% CI: 1.116–7.074; p = 0.028). Low miR-34a expression independently predicted for shorter OS (HR: 3.189, 95% CI: 1.193–8.527; p = 0.021) in the non-SqCC subgroup. Our findings suggest that alterations in circulating miR-200c and miR-34a expression levels are associated with the response and outcome in patients with advanced NSCLC treated with anti-PD1 immunotherapy.

Molecular Profiles of Serum-Derived Extracellular Vesicles in High-Grade Serous Ovarian Cancer.

Simple SummaryOvarian cancer is the deadliest gynecological malignancy worldwide, and the biggest issue faced by patients is disease relapse after primary treatment. Understanding up front how patients will respond to therapy is an important and compelling challenge. On these premises, our goal was to understand if a simple blood sample can give us this information, before the patients start chemotherapy. We determined that circulating extracellular vesicles isolated at diagnosis can distinguish between patients who will respond differently to the treatment. These results, if corroborated, will pave the way for a novel, much needed predictive marker of response.Patients with high-grade serous ovarian cancer (HGSC) who have no visible residual disease (R0) after primary surgery have the best clinical outcomes, followed by patients who undergo neoadjuvant chemotherapy (NACT) and have a response enabling interval cytoreductive surgery. Clinically useful biomarkers for predicting these outcomes are still lacking. Extracellular vesicles (EVs) have been recognized as liquid biopsy-based biomarkers for early cancer detection and disease surveillance in other disease settings. In this study, we performed extensive molecular characterization of serum-derived EVs and correlated the findings with therapeutic outcomes in patients with HGSC. Using EV-DNA whole-genome sequencing and EV-RNA sequencing, we identified distinct somatic EV-DNA alterations in cancer-hallmark genes and in ovarian cancer genes, as well as significantly altered oncogenic pathways between the R0 group and NACT groups. We also found significantly altered EV-RNA transcriptomic variations and enriched pathways between the groups. Taken together, our data suggest that the molecular characteristics of EVs could enable prediction of patients with HGSC who could undergo R0 surgery or respond to chemotherapy.

Prognostic value of integrating circulating tumour cells and cell-free DNA in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) often presents at an incurable stage, and majority of patients will be considered for palliative treatment at some point in their disease. Despite recent advances, the prognosis remains poor, with a median overall survival of 12–18 months. Liquid biopsy-based biomarkers have emerged as potential candidates for predicting prognosis and response to therapy in NSCLC patients. This pilot study evaluated whether combining circulating tumour cells and clusters (CTCs) and cell-free DNA (cfDNA) can predict progression-free survival (PFS) in NSCLC patients. MethodsCTC and cfDNA/ctDNA from advanced stage NSCLC patients were measured at study entry (T0) and 3-months post-treatment (T1). CTCs were enriched using a spiral microfluidic chip and characterised by immunofluorescence. ctDNA was assessed using an UltraSEEK® Lung Panel. Kaplan-Meier plots were generated to investigate the contribution of the presence of CTC/CTC clusters and cfDNA for PFS. Cox proportional hazards analysis compared time to progression versus CTC/CTC cluster counts and cfDNA levels. ResultsSingle CTCs were found in 14 out of 25 patients, while CTC clusters were found in 8 out of the 25 patients at T0. At T1, CTCs were found in 7 out of 18 patients, and CTC clusters in 1 out of the 18 patients. At T0, CTC presence and the combination of CTC cluster counts with cfDNA levels were associated with shorter PFS, p = 0.0261, p = 0.0022, respectively. ConclusionsCombining CTC cluster counts and cfDNA levels could improve PFS assessment in NSCLC patients. Our results encourage further investigation on the combined effect of CTC/cfDNA as a prognostic biomarker in a large cohort of advanced stage NSCLC patients.

Liquid Biopsy-Based Biomarkers of Inflammatory Nociception Identified in Male Rats.

Physicians are challenged in treating pain patients due to the lack of quantifiable, objective methods of measuring pain in the clinic; pain sensation is multifaceted and subjective to each individual. There is a critical need for point-of-care quantification of accessible biomarkers to provide objective analyses beyond the subjective pain scales currently employed in clinical care settings. In the present study, we employed an animal model to test the hypothesis that circulating regulators of the inflammatory response directly associate with an objective behavioral response to inflammatory pain. Upon induction of localized paw inflammation, we measured the systemic protein expression of cytokines, and activity levels of matrix metalloproteinases (MMPs) that are known to participate in the inflammatory response at the site of injury and investigated their relationship to the behavioral response across a 24 h period. Intraplantar injection with 1% λ-carrageenan induced a significant increase in paw thickness across this timespan with maximal effects observed at the 8 h timepoint when locomotor activity was also impaired. Expression of the chemokines C-X-C motif chemokine ligand 1 (CXCL1) and C-C motif chemokine ligand 2 (CCL2) positively correlated with paw inflammation and negatively correlated with locomotor activity at 8 h. The ratio of MMP9 to MMP2 activity negatively correlated with paw inflammation at the 8 h timepoint. We postulate that the CXCL1 and CCL2 as well as the ratio of MMP9 to MMP2 activity may serve as predictive biomarkers for the timecourse of inflammation-associated locomotor impairment. These data define opportunities for the future development of a point-of-care device to objectively quantify biomarkers for inflammatory pain states.

Abstract 5106: Distinct molecular profiles of serum-derived extracellular vesicles in high-grade serous ovarian cancer

Abstract Background: Patients with high-grade serous ovarian cancer (HGSC) who have no visible residual disease (CGR) after primary surgery have the best clinical outcomes, followed by patients who undergo neoadjuvant chemotherapy (NACT) and have response enabling interval cytoreductive surgery. Through comprehensive omics analyses, we previously characterized molecular and cellular profiles of highly clinically defined HGSC from patients who had CGR versus those triaged to NACT. However, clinically useful predictive biomarkers for such distinctions are still lacking. Extracellular vesicles (EVs) have been recognized as liquid biopsy-based biomarkers for early cancer detection and disease surveillance in other disease settings. Here, we performed extensive molecular characterization of serum-derived EVs and correlated them with therapeutic outcomes in patients with HGSC. Methods: Serum-derived medium/large size EVs from patients with HGSC treated under a systematic surgical algorithm were isolated following standard methods of the International Society for Extracellular Vesicles. Patients had either complete gross resection after primary surgery (R0, n=3); poor response to NACT (NACT-PR, n=4), or excellent response to NACT with carboplatin and paclitaxel (NACT-ER, n=3); tissue and matched germline blood samples have already been comprehensively analyzed (Lee et al, Cell Reports, 2020). The isolated EV-DNA and EV-RNA were subjected to whole-genome sequencing and RNA sequencing, respectively. Results: We identified somatic EV-DNA alterations in cancer hallmark genes, including FAT1 and KMT2D as the most frequent mutated genes. Interestingly, we found alterations in ovarian cancer-related genes, including BRCA2 and RAD51 in R0; BRCA2 in NACT-ER; TP53, CDK13, CDK12, BRAF, AKT2, and ARID1A in NACT-PR patients. We also identified 26,338 genes expressed in the EV-RNA transcriptomic profiles. The analysis of the enrichment of cancer hallmark pathways showed that the R0 group was more enriched in the interferon α/γ, PI3K/AKT/mTOR signaling, and DNA repair pathways. Interestingly, we also identified 6,757 differentially expressed genes (DEGs) in the CGR versus the NACT groups (absolute log2-fold change ≥2 and adjusted p-value &amp;lt;0.05). Among the DEGs, we identified 67 genes that were cancer hallmark genes, and 21 of them were overexpressed in the NACT group and 46 were overexpressed in the R0 group. Noticeably, 11 DEGs were directly related to ovarian cancer: TP53, PTEN, CCND1, BARD1, PMS2, and MLH3 were significantly overexpressed in the NACT group, while BRCA2, CSMD3, and GABRA6 were overexpressed in the R0 group. Conclusions: Taken together, our preliminary data suggest that molecular characteristics of EVs could provide an accurate prediction of patients with HGSC who can undergo CGR with surgery and respond to chemotherapy. Citation Format: Li Zhao, Shaolin Ma, Nicole D. Fleming, Joseph Celestino, Mark S. Kim, Richard A. Hajek, Nicholas B. Jennings, Erika J. Thompson, Hongli Tang, Shannon N. Westin, Amir A. Jazaeri, Jianhua Zhang, P Andrew Futreal, Anil K. Sood, Sanghoon Lee. Distinct molecular profiles of serum-derived extracellular vesicles in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5106.

The Utility of Repetitive Cell-Free DNA in Cancer Liquid Biopsies.

Liquid biopsy is a broad term that refers to the testing of body fluids for biomarkers that correlate with a pathological condition. While a variety of body-fluid components (e.g., circulating tumor cells, extracellular vesicles, RNA, proteins, and metabolites) are studied as potential liquid biopsy biomarkers, cell-free DNA (cfDNA) has attracted the most attention in recent years. The total cfDNA population in a typical biospecimen represents an immensely rich source of biological and pathological information and has demonstrated significant potential as a versatile biomarker in oncology, non-invasive prenatal testing, and transplant monitoring. As a significant portion of cfDNA is composed of repeat DNA sequences and some families (e.g., pericentric satellites) were recently shown to be overrepresented in cfDNA populations vs their genomic abundance, it holds great potential for developing liquid biopsy-based biomarkers for the early detection and management of patients with cancer. By outlining research that employed cell-free repeat DNA sequences, in particular the ALU and LINE-1 elements, we highlight the clinical potential of the repeat-element content of cfDNA as an underappreciated marker in the cancer liquid biopsy repertoire.

The era of biomarkers and precision medicine in colorectal cancer: an interview with Ajay Goel.

In this interview, Professor Ajay Goel speaks with Storm Johnson, Commissioning Editor for Epigenomics, on his work to date in the field of epigenetic biomarkers in colorectal cancer. Ajay Goel, PhD, is a Professor and Founding Chair of theDepartment of Molecular Diagnostics, at the Beckman Research Institute and Associate Director of Basic Science at the City of Hope comprehensive Cancer Center. Healso serves as Director of Biotech Innovations at the City of Hope, Duarte, CA, USA. DrGoel has spent more than 25 years researching cancer and has been the lead author or contributor to more than 350 scientific articles published in peer-reviewed international journals and several book chapters. He is also a primary inventor on more than 40 international patents aimed at developing various disease biomarkers or therapeutic targets for gastrointestinal cancers. He is currently using advanced genomic, epigenomic and transcriptomic approaches to develop novel circulating, liquid biopsy-based biomarkers (e.g., cell-free nucleic acids, exosomes) for the early detection, prognosis and determination of predictive responses to chemotherapy and targeted drugs in gastrointestinal (GI)cancers. In addition, his group is interested in the identification of novel therapeutic targets, particularly immune therapy, for variousGI cancers. His research also involves understanding the role of gut microbiome, health disparitiesand the prevention of GI cancers using integrative and alternative approaches. DrGoel is a member of the American Association for Cancer Research, American Society of Clinical Oncology and the American Gastroenterology Association. He is on the international editorial boards of several journals, including Gastroenterology, Clinical Cancer Research, Carcinogenesis, PLoS ONE, Molecular Carcinogenesis, Scientific Reports, Epigenomics, Future Oncology, Alternative Therapies in Heath and Medicine, Digestive Diseases and Sciences, and Molecular Therapy Oncolytics. He is also actively involved in peer-reviewing activities for more than 100 international scientific journals and various grant review panels of various national and international funding organizations. His research has been actively funded by various private and federal organizations, including the National Cancer Institute at the NIH, American Cancer Societyand state organizations. He has won more than a dozen national and international awards and honors and has been invited for visiting professorships by various national and international academic institutions and academic bodies.

Platinum-resistant ovarian cancer: From drug resistance mechanisms to liquid biopsy-based biomarkers for disease management.

Resistance to platinum-based chemotherapy is a major clinical challenge in ovarian cancer, contributing to the high mortality-to-incidence ratio. Management of the platinum-resistant disease has been difficult due to diverse underlying molecular mechanisms. Over the past several years, research has revealed several novel molecular targets that are being explored as biomarkers for treatment planning and monitoring of response. The therapeutic landscape of ovarian cancer is also rapidly evolving, and alternative therapies are becoming available for the recurrent platinum-resistant disease. This review provides a snapshot of platinum resistance mechanisms and discusses liquid-based biomarkers and their potential utility in effective management of platinum-resistant ovarian cancer.

Biomarkers and Lung Cancer Early Detection: State of the Art.

Simple SummaryLung cancer is the leading cause of cancer death worldwide. Detecting lung malignancies promptly is essential for any anticancer treatment to reduce mortality and morbidity, especially in high-risk individuals. The use of liquid biopsy to detect circulating biomarkers such as RNA, microRNA, DNA, proteins, autoantibodies in the blood, as well as circulating tumor cells (CTCs), can substantially change the way we manage lung cancer patients by improving disease stratification using intrinsic molecular characteristics, identification of therapeutic targets and monitoring molecular residual disease. Here, we made an update on recent developments in liquid biopsy-based biomarkers for lung cancer early diagnosis, and we propose guidelines for an accurate study design, execution, and data interpretation for biomarker development.Lung cancer burden is increasing, with 2 million deaths/year worldwide. Current limitations in early detection impede lung cancer diagnosis when the disease is still localized and thus more curable by surgery or multimodality treatment. Liquid biopsy is emerging as an important tool for lung cancer early detection and for monitoring therapy response. Here, we reviewed recent advances in liquid biopsy for early diagnosis of lung cancer. We summarized DNA- or RNA-based biomarkers, proteins, autoantibodies circulating in the blood, as well as circulating tumor cells (CTCs), and compared the most promising studies in terms of biomarkers prediction performance. While we observed an overall good performance for the proposed biomarkers, we noticed some critical aspects which may complicate the successful translation of these biomarkers into the clinical setting. We, therefore, proposed a roadmap for successful development of lung cancer biomarkers during the discovery, prioritization, and clinical validation phase. The integration of innovative minimally invasive biomarkers in screening programs is highly demanded to augment lung cancer early detection.

Liquid Biopsy and Potential Liquid Biopsy-Based Biomarkers in Philadelphia-Negative Classical Myeloproliferative Neoplasms: A Systematic Review.

Myeloproliferative neoplasms (MPNs) are rare, clonal disorders of the hematopoietic stem cell in which an uncontrolled proliferation of terminally differentiated myeloid cells is noted. Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are included in the category of Philadelphia-negative, so-called classical MPNs. The potential applications of liquid biopsy and liquid biopsy-based biomarkers have not been explored in MPNs until now. Thus, a systematic search was computed in PubMed/MEDLINE, Web of Science and The Cochrane Library and, in total, 198 potentially relevant papers were detected. Following the removal of duplicates (n = 85), 113 records were screened. After the exclusion of irrelevant manuscripts based on the screening of their titles and abstracts (n = 81), we examined the full texts of 33 manuscripts. Finally, after we applied the exclusion and inclusion criteria, 27 original articles were included in this review. Overall, the data analyzed in this review point out that liquid biopsy and liquid biopsy-based biomarkers (cell-free DNA, extracellular vesicles, microparticles, circulating endothelial cells) could be used in MPNs for diagnostic and prognostic purposes. Future research is needed to clarify whether this technique can be employed to differentiate between MPN subtypes and secondary causes of erythrocytosis, thrombocytosis and myelofibrosis, as well as to predict the development of thrombosis.

OMRT-2. Liquid biopsy for patient stratification and monitoring of dacomitinib clinical trial in patients with EGFR amplified recurrent glioblastoma

IntroductionLiquid biopsy for the detection and monitoring of brain tumors is of significant clinical interest. The ability to non-invasively profile tumors can avoid a risky biopsy and opens avenues for testing novel therapies by accurately stratifying patients to receive the right therapy. Here, we provide evidence of EV RNA-based diagnosis, patient stratification, and assessment of response to therapy in the setting of a clinical trial evaluating the efficacy of dacomitinib, an EGFR tyrosine kinase inhibitor in patients with recurrent, EGFR amplified GBM(NCT01112527).MethodsWe performed RNASeq on long RNA extracted from the serum samples, pre-treatment and 1-month post-treatment.ResultsFirstly, longRNASeq allowed the detection of thousands of mRNA, lincRNAs and antisense RNAs enabling the study of a wider repertoire of potential RNA based biomarkers.Secondly, we observed a differential expression profile in serum EV RNA of GBM patients and healthy controls. Combining our findings with TCGA data and literature screening, we generated a 25 gene signature representative of critical pathways in several hallmarks of cancer.Thirdly, we observed a differential expression profile in serum EV RNA of responders to dacomitinib compared to non-responders in pre-treatment serum. Specifically, the EV mRNAs ZNF35 and LAMTOR2 distinguish responders from non-responders (p-adjusted = 2.6E-8 and 2.4E-6, respectively) allowing potential patient stratification.Finally, we observed a differential expression profile in serum EV RNA of responders to dacomitinib compared to non-responders in post-treatment serum. EV mRNA DNMT3A is significantly enriched (p-adjusted = 1.8E-4) in post-treatment serum of responders compared to non-responders to dacomitinib allowing potential monitoring of response to therapy.ConclusionThis study represents the first longitudinal profiling of the EV transcriptome in a cohort of genomically selected GBM patients. These findings are a tantalizing step toward liquid biopsy-based biomarkers for the detection of GBM, as well as patient stratification and monitoring.

Abstract 596: Single circulating tumor cell enumeration and targeted sequencing in non-small cell lung cancer

Abstract Purpose: Circulating tumor cells (CTCs) may be novel biomarkers to predict cancer prognosis and monitor therapeutic efficacy. In the current study, we investigated the use of the time-dependent enumeration and targeted DNA sequensing of single CTCs as liquid biopsy based biomarkers of non-small cell lung cancer (NSCLC) patients for diagnosis, prognosis, therapeutic choice and outcome monitoring. Experimental Design: In a prospective study, we tested the AccuCyte® system (RareCyte®, Inc. Seattle, WA) for CTC enumeration in NSCLC using FDA criteria (CK/EpCAM+/CD45- with a nucleus). Whole blood (7.5 ml) from healthy donors was spiked with human lung adenocarcinoma (A549 cell line) cells at specific numbers (N=0, 100, 200, 1000). CTC detection and single-CTC picking was performed. NSCLC subjects' (N=20) and chronic smokers without cancer on screening low-dose CT subjects' (N=10) blood were collected (amount, method, biotechnology). For all blood samples, CTC detection and single CTC picking was performed. DNA was isolated from CTCs (N=3) and white blood cells (N=2) per sample. DNA libraries were prepared for targeted oncogene characterization using the CleanPlex® OncoZoom® panel. Somatic variant analysis and comparisons were performed. Results: AccuCyte enumeration of A549 cells spiked at four levels in healthy subjects' whole blood showed (Spiked #, Detected #): (0, 0); (100, 76); (200, 208); (1000, 1223). NSCLC subjects' (N=20; age: 64.85±6.64; BMI: 28.00±8.15sex ratio: 0.42) blood had a mean CTC count of 13.4 (SD=52, median=0, range [0, 237]). Ten screening controls without cancer (64.2±5.81, 1, 28.50±6.13, 0.66) had a mean CTC count of 0.20 (0.42, 0, [0, 1]). Significant association was noted regarding a higher number of CTCs found in NSCLC compared to screening subjects without cancer according to the (p= 0.020, Wilcoxon signed rank). CTC numbers increased with NSCLC AJCC stage. Somatic variant analysis on patient-matched single CTCs and WBCs is ongoing. Conclusions: The molecular investigation of patient matched single CTCs and white blood cells will fundamentally advance understanding on the NSCCL carcinogenic signature of these blood born cells and their potential value as cancer detection and therapy management liquid biopsy-based biomarkers. Citation Format: Mouadh Barbirou, Yariswamy Manjunath, Amanda Miller, Arturo Ramirez, Nolan Ericson, Kevin F. Kevin F. Staveley-O'Carroll, Wes Warren, Guangfu Li, Peter J. Tonellato, Jussuf T. Kaifi. Single circulating tumor cell enumeration and targeted sequencing in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 596.

Abstract 58: Serum Based Exosomal miRNAs as in Novel Biomarkers for the Initial Prediction of Early-Stage Lung Cancer

Abstract Purpose: Currently focuses on the identification of early-stage lung cancer (ESLC) is the core of the prevention and treatment of lung cancer, which is desperately needed throughout the world to improve lung cancer survival. Our study has primarily aimed at exosome-derived miRNAs in the detection of ESLC and discrimination between Benign lung nodules(BLN) and Healthy individuals. We aim to determine sensitive and specific liquid biopsy-based biomarkers in tackling ESLC. Methods: Discovering the non-invasive serum exosomal based miRNAs (sEXO-miRNAs) biomarkers through next-generation sequencing (NGS)in 9 participants (ESLC-3, BLN-3 and Healthy-3) and validated by 143 (ESLC-46, BLN-46 and Healthy-51) recruiters via RT-PCR in a cohort of Zhongda Hospital affiliated to the medical school of Southeast University, Nanjing, China. Results: By doing NGS, we have discovered 769 differently expressed miRNAs mutual between the three groups. Amid those miRNAs, we have selected the topmost highly up-or down-regulated significant sEXO-miRNAs as biomarkers following certain threshold and statistical criteria, which were sEXO-miR-424-5p, sEXO-miR-512-5p, and sEXo-miR-1271-5p for subsequent validation in 3 groups of a single-center cohort. The area under the receiver operating curve (AUC-ROC) of 3 individual miRNAs in between three groups ranged from 0.633 to 0.995 (95%CI: 0.515~1.000 ranged, p&amp;lt;0.0274~p&amp;lt;0.0001) in the validation cohort. Overall in the ESLC vs BLN group, the combination of the three sEXO-miRNAs'AUC was 0.824 (95%CI: 0.738~0.909, p&amp;lt;0.0001) whereas sensitivity, specificity, PPV, NPV and accuracy were 77.08%, 79.55%, 80.43%, 76.09% and 78.26% respectively by logistic regression analysis. Conclusion: Our study discovered significant three differentials expressed sEXO-miRNAs (hsa-miR-424-5p, hsa-miR-512-5p, and hsa-miR-1271-5p), and validated throughout in between ESLC, BLN and healthy individuals. These sEXO-miRNAs provided substantial diagnostic efficacy towards predicting ESLC and accurately discriminate with BLN and healthy recruiters. Therefore, our novel sEXO-miRNAs as biomarkers for prior detection ESLC is quite promising in clinical practice and can be used for precision-based diagnosing purposes. Citation Format: Nishant Patel, Haijun Zhang, Lili Ren. Serum Based Exosomal miRNAs as in Novel Biomarkers for the Initial Prediction of Early-Stage Lung Cancer [abstract]. In: Proceedings of the 9th Annual Symposium on Global Cancer Research; Global Cancer Research and Control: Looking Back and Charting a Path Forward; 2021 Mar 10-11. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2021;30(7 Suppl):Abstract nr 58.