Blood-based Liquid Biopsies Research Articles

Clinical Utilisation and Survival Correlation of Blood-based Liquid Biopsy in Sarcoma - A Systematic Review and Meta-analysis

Clinical Utilisation and Survival Correlation of Blood-based Liquid Biopsy in Sarcoma - A Systematic Review and Meta-analysis

Abstract A053: Liquid biopsy-based detection of triple negative breast cancer using DNA methylation biomarkers

Abstract Triple-negative breast cancer (TNBC) presents significant clinical challenges due to its aggressive phenotype, rapid progression, and limited targeted treatment options. TNBC often mimics benign lesions on ultrasound and metastatic features can be missed in routine mammography screening leading to misdiagnosis and delayed treatment. These limitations led us to investigate whether a blood-based liquid biopsy could provide a non-invasive, sensitive method for early TNBC detection, potentially overcoming the constraints of conventional imaging techniques. To discover biomarkers for TNBC, we performed targeted bisulfite sequencing on a cohort of 46 breast cancer solid tissue biopsy samples, comprising 19 TNBC and 27 non-TNBC cases. We identified 150 differentially methylated regions (mean length 703bp) separating TNBC and non-TNBC samples. Upon linking each region to its nearest gene, we observed several genes previously associated with prognosis and survival (HOXB3, PAX9, SOX9) substantiating the clinical relevance of these biomarkers. Using public TCGA data (369 breast cancer samples), we integrated expression and methylation data and observed directionally consistent changes. For example, HOXB3 showed increased methylation at its associated differential regions and decreased expression in TNBC cases. To ascertain whether these biomarkers could be useful in a liquid biopsy approach, we performed targeted bisulfite- sequencing on cell-free DNA samples derived from prospectively collected patients with breast cancer. All samples had independent tissue-based assessment of ER, PR and HER2 using immunohistochemistry or in situ hybridization. We then built a feed-forward neural network classifier with classes TNBC and non-TNBC that used transfer-learning and the identified biomarkers to learn methylation signatures in the biopsy samples and map them to cell-free DNA. To emulate clinical workflow, where subtyping follows cancer diagnosis and tissue of origin identification, we evaluated cfDNA samples correctly identified as breast cancer using two independent machine learning models trained using 2,393 prospectively collected multi- indication cancer and non-cancer samples (NCT05435066). Subsequent TNBC subtyping of breast cancer-positive cases (N=56) accurately identified 77% (10/13) of TNBC samples and 91% (39/43) of non-TNBC cases, with overall accuracy of 84%. The estimated tumor content range for correctly predicted TNBC samples was 0.5-33% with the three mis-classified samples all showing very low tumor content (0.22%, 0.28%, 0.59%). Non-TNBC cases showed a tumor content range of 0.07-36% for correct and 2.3-14% for misclassified samples. In conclusion, we identified novel methylation biomarkers that distinguish TNBC in the cell-free DNA of patients without needing invasive tissue biopsies. The high sensitivity of our assay at low tumor fractions has the potential to improve outcomes through earlier intervention and may enable earlier screening in high-risk groups, monitoring of minimal residual disease and longitudinal monitoring during treatment. Citation Format: Jocelyn Charlton, Shiva Farashahi, Kade Pettie, Elie Massaad, Josh Hubbell, Feras Hantash, Kieran I Chacko. Liquid biopsy-based detection of triple negative breast cancer using DNA methylation biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A053.

High Expression of the Tumor Suppressor Protein ITIH5 in Cholangiocarcinomas Correlates with a Favorable Prognosis.

Background: Cholangiocarcinoma (CCA) are aggressive bile duct cancers with a poor prognosis for which there are only few established prognostic biomarkers and molecular targets available. The gene ITIH5, a known class II tumor suppressor gene (C2TSG), encodes a secreted protein of the extracellular matrix mediating tumor suppressive properties. Recently, it was surprisingly found that the ITIH5 protein is specifically upregulated in CCAs and that ITIH5 detection in blood could be an excellent liquid biopsy marker for indicating the presence of a CCA tumor in a patient. We therefore investigated whether patients with CCAs with abundant versus low ITIH5 protein expression also differ in their prognosis. Methods: To clarify this question, a large CCA cohort (n = 175) was examined using immunohistochemistry on a tissue microarray (TMA). Results: Abundant ITIH5 expression in CCA was associated with favorable survival, a low UICC stage and the absence of perineural invasion (PNI). Conclusions: ITIH5 has biomarker potential not only for the early detection of CCA from blood-based liquid biopsies but also as a prognostic tissue biomarker for risk stratification. Our results suggest that the upregulation of ITIH5 is particularly abundant in intrahepatic CCAs (iCCA). The mechanisms mediating the strong initial upregulation of ITIH5 during the oncogenic transformation of bile duct cells are still unclear.

Cervical Cancer Genetic Profile through Circulating Tumor DNA: What Can We Learn from Blood?

Cervical cancer (CC) is one of the deadliest gynecological cancers worldwide. Human papillomavirus is the main etiological agent responsible for the initiation and development of most CC cases. The standard method utilized for CC screening in the global population is the cytological Pap smear test. Despite its effective validity in detecting precancerous lesions and its response to layer stages of this disease, greater screening and diagnostic reliability are needed, as well as an improvement in specificity and sensitivity. In this context, the use of liquid biopsies, like blood, for the isolation of circulating tumor DNA (ctDNA) in CC screening, diagnosis, prognosis, and surveillance could fill the gaps that still exist. In the present review, we aim to study the literature in order to collect knowledge on blood-based liquid biopsy based on descriptions of its precious molecular content and its utilization as a potential tool for CC patients' management. We will mainly focus on the important role of the novel ctDNA and the unique possibilities to additionally use HPV-ctDNA in CC at various stages of clinical application.

Current Challenges of Methylation-Based Liquid Biopsies in Cancer Diagnostics.

In current clinical practice, effective cancer testing and screening paradigms are limited to specific types of cancer, exhibiting varying efficiency, acceptance, and adherence. Cell-free DNA (cfDNA) methylation profiling holds promise in providing information about the presence of malignity regardless of its type and location while leveraging blood-based liquid biopsies as a method to obtain analytical samples. However, technical difficulties, costs and challenges resulting from biological variations, tumor heterogeneity, and exogenous factors persist. This method exploits the mechanisms behind cfDNA release but faces issues like fragmentation, low concentrations, and high background noise. This review explores cfDNA methylation's origins, means of detection, and profiling for cancer diagnostics. The critical evaluation of currently available multi-cancer early detection methods (MCEDs) as well as tests targeting single genes, emphasizing their potential and limits to refine strategies for early cancer detection, are explained. The current methodology limitations, workflows, comparisons of clinically approved liquid biopsy-based methylation tests for cancer, their utilization in companion diagnostics as well as the biological limitations of the epigenetics approach are discussed, aiming to help healthcare providers as well as researchers to orient themselves in this increasingly complex and evolving field of diagnostics.

Abstract PO5-04-05: Clinical validation of the concordance performance of ERBB2 status by single-cells genomics ERBB2 (HER2) amplification Assay in Circulating Tumor Cells (CTCs) from patients with matched HER2 status from metastatic tissue biopsies

Abstract Background Liquid biopsies are a non-invasive diagnostic approach for detecting CTCs that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional biopsy is infeasible. Blood-based liquid biopsy has the potential advantage that it allows broad sampling of metastatic tumor clones because it is capable of and likely to obtain cellular and genomic contributions from more than one metastatic site. Here we report the concordance performance of HER2 positivity by single-cell CTC genomics ERBB2 (HER2) amplification in patients with matched HER2 status from metastatic tissue biopsies. Methods A prospective Clinical Experience Program for CTC ERBB2 (HER2) Assay across 25 different United States-based cancer centers, was used to recruit 128 MBC patients with documented standard-of-care metastatic tissue biopsies performed within the last 5 years (Days from tissue biopsy to liquid biopsy, Median: 19.5; interquartile range: 40.75) CTCs from blood collection at each respective clinical site were isolated from plasma, nucleated cells were plated, & slides were bio-banked for Immunofluorescent staining & subsequent imaging in a CAP CLIA lab. CTCs were identified using Epic Sciences digital imaging & machine learning algorithms. Individual CTCs were sequenced for genomic quantification of chromosomal instability (LSTs) and ERBB2 (HER2) amplification. Results Within the clinical validation cohort of 128 MBC patients, 30% (38/128) were HER2-positive by standard pathologic criteria (IHC 3+ or IHC2+/ISH+) in metastatic tissue biopsies. Among the 64 out of 128 cases with reportable results by the CTC ERBB2 (HER2) amplification on chromosomally unstable CTCs, 31% (20/64) of patients had ERBB2 amplification. Concordance between CTC single cell genomics and available tissue results for HER2 positivity showed a sensitivity of 69% and specificity of 78%. We then addressed the effects of two known biological confounders to the use of tissue biopsy for HER2 status determination: 1) tissue biopsies performed on bone metastases, 2) tumor evolution driven by treatment pressure over time leading to potential HER2 conversion and tumor heterogeneity. Thus, we performed subgroup analysis including only validation set patients with tissue biopsies performed on non-bone tissues, which showed slightly improved concordance to the tissue comparator with a Sensitivity of 86%, and Specificity of 75%. Next, to exclude the possibility of tumor evolution we selected only patients with the HER2 status comparator performed on non-bone tissue biopsies contemporaneous (Days from tissue biopsy to liquid biopsy, Median: 14; interquartile range: 22) to the blood draw for the CTC ERBB2 (HER2) amplification assay. This showed dramatically improved concordance performance with a Sensitivity of 100%, and a Specificity of 75%. Conclusions Blood and tissue are two biologically distinct sample types where variations in HER2 and other biomarker results might be expected due to analytic, temporal and clinical differences. These data indicate that determination of HER2 status by single cell genomic ERBB2 (HER2) amplification assay can aid in the clinical assessment of ERBB2 status in patients with metastatic breast cancer (MBC) for whom tissue biopsy for HER2 status is not available, or infeasible. Additionally, these results provide evidence of the contribution of site differences (bone) of metastatic biopsies and also tumor evolution to discordance between the CTC ERBB2 (HER2) Assay and tumor biopsy. Citation Format: Giuseppe Di Caro, Ernest Lam, Megan Slade, Shuguang Huang, Rick Wenstrup, Lee Schwartzberg. Clinical validation of the concordance performance of ERBB2 status by single-cells genomics ERBB2 (HER2) amplification Assay in Circulating Tumor Cells (CTCs) from patients with matched HER2 status from metastatic tissue biopsies [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 PO5-04-05.

Clinical validation of a blood-based liquid biopsy test integrating cell-free DNA quantification and next-generation sequencing for cancer screening in dogs.

To validate the performance of a novel, integrated test for canine cancer screening that combines cell-free DNA quantification with next-generation sequencing (NGS) analysis. Retrospective data from a total of 1,947 cancer-diagnosed and presumably cancer-free dogs were used to validate test performance for the detection of 7 predefined cancer types (lymphoma, hemangiosarcoma, osteosarcoma, leukemia, histiocytic sarcoma, primary lung tumors, and urothelial carcinoma), using independent training and testing sets. Cell-free DNA quantification data from all samples were analyzed using a proprietary machine learning algorithm to determine a Cancer Probability Index (High, Moderate, or Low). High and Low Probability of Cancer were final result classifications. Moderate cases were additionally analyzed by NGS to arrive at a final classification of High Probability of Cancer (Cancer Signal Detected) or Low Probability of Cancer (Cancer Signal Not Detected). Of the 595 dogs in the testing set, 89% (n = 530) received a High or Low Probability result based on the machine learning algorithm; 11% (65) were Moderate Probability, and NGS results were used to assign a final classification. Overall, 87 of 122 dogs with the 7 predefined cancer types were classified as High Probability and 467 of 473 presumably cancer-free dogs were classified as Low Probability, corresponding to a sensitivity of 71.3% for the predefined cancer types at a specificity of 98.7%. This integrated test offers a novel option to screen for cancer types that may be difficult to detect by physical examination at a dog's wellness visit.

Evaluating the impact of delayed centrifugation on protein profiles analyzed by LC/MS in serum and plasma samples

The pre-analytical steps in blood-based liquid biopsy, involving sample collection techniques and storage conditions, play a critical role in ensuring the integrity and reliability of collected samples. These steps have a directly impact on the accuracy and reliability of test results and are therefore of utmost importance. Mass spectrometry (MS)-based proteomics is an exceptionally powerful tool in the field of liquid biopsy. It enables the comprehensive analysis of the protein content within biological specimens, providing valuable insights into the underlying mechanisms and pathogenesis of diseases. In this study, we aim to explore the variations in the protein landscape between serum and plasma specimens and evaluate the impact of delayed centrifugation on LC/MS-analyzed protein profiles. We seek to provide recommendations on optimal pre-analytical protocols for MS-based proteomics studies. This will enhance the accuracy and reliability of liquid biopsy for precision medicine, ultimately leading to improved patient outcomes.

Prostate cancer and liquid biopsies: Clinical applications and challenges.

Liquid biopsy has emerged as a valuable and minimally invasive tool for real-time detection of clinically actionable abnormalities across various cancer types. Its applicability is particularly compelling in the realm of prostate cancer, where novel therapeutic agents, including those targeting DNA repair systems, are under development. Despite these advancements, challenges persist in effectively screening for prostate cancer, enhancing risk stratification, and determining optimal approaches for treating advanced disease. Consequently, there is a pressing need for improved biomarkers to aid clinicians in decision-making within these contexts. Cell-free DNA and extracellular vesicle analysis have demonstrated promise in diagnosis, prognostication, assessment of treatment responses, and identification of emerging mechanisms of resistance. Nevertheless, obstacles must be addressed before liquid biopsies can be integrated into routine clinical practice. These challenges encompass preanalytical considerations such as sample collection and storage, methods of extracellular vesicle isolation and enrichment, and the need for enhanced interpretation of generated sequencing data. This review provides a comprehensive overview of current clinical opportunities in managing prostate cancer through blood-based liquid biopsy, highlighting the progress made, and acknowledging the challenges that remain. Additionally, we discuss the next steps required for the effective implementation of liquid biopsies in guiding personalized treatment strategies for prostate cancer.

Abstract 5165: Development and validation of an assay for the characterization of SSTR2 expression on CTCs from liquid biopsies

Abstract Background: Expression of Somatostatin Receptor subtype 2 (SSTR2) is observed in multiple cancers, including breast and colorectal cancer. Its role as a prognostic or predictive marker is cancer-type dependent. Recently, there has been renewed interest in assessing this biomarker since it plays a critical role as a target for radiotherapeutics (peptide receptor radionuclide therapy - PRRT) in patients with neuroendocrine tumors (NETs). Blood-based liquid biopsies are a non-invasive tool with proven effectiveness in characterizing cancer biomarker expression in patients. In this study, we describe the development and validation of a novel assay to analyze circulating tumor cells (CTCs) for expression of SSTR2 using the RareCyte platform. Experimental Procedures: Using AccuCyte®, an unbiased density-based method for collecting nucleated cells from whole blood, we transferred nucleated cells to slides and stained for SSTR2, CD45, and CK/EpCAM to identify CTCs (CK+/EpCAM+, CD45-), and evaluate SSTR2 expression. Slides were imaged with CyteFinder®, an automated multiparameter immunofluorescent (IF) microscopy system that applies machine learning algorithms for rare cell identification. Both clinical and spike-in samples were used for assay validation. Single-cell SSTR2 mean fluorescence intensities (MFI) were analyzed to determine protein expression levels. Colorectal cancer with neuroendocrine features and Merkel cell cancer (neuroendocrine tumor of the skin) patient samples were evaluated for CTC enumeration and SSTR2 expression. Results: The assay’s performance metrics for SSTR2 expression were 90+% accuracy, 85% sensitivity, 100% specificity with repeatability and intermediate precision CVs of 14.4% and 16.4%, respectively. A small cohort of colorectal cancer (1) and Merkel cell cancer (2) patient blood samples were tested using the assay. All 3 patients had high CTC levels (300+ per 7.5 mL). The percentage of SSTR2-positive CTCs ranged from 20-60%, reflective of tumor heterogeneity. Conclusions: SSTR2 is an important target now in the advent of theranostics - PRRT. Prognostic and therapeutic implications of SSTR2 expression is an area of active clinical research. SSTR2 targeted therapeutics like 177Lu-dotatate illustrate the possible utility of SSTR2 as a predictive biomarker of treatment response. To date, most of the work has been focused on assessing SSTR2 on tissue. Herein, we show how a CTC platform can be adapted to assess a clinically relevant cell surface protein biomarker. This is now included as an important correlative in a clinical trial for Merkel Cell Cancer patients called iPRRT that is actively accruing (NCT05583708). Citation Format: Erin G. Bayer, Rachel N. Ponting, Ciara M. Kelly, Areeb Lutfi, Maaz K. Afghan, Arturo B. Ramirez, Pashtoon M. Kasi. Development and validation of an assay for the characterization of SSTR2 expression on CTCs from liquid biopsies [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 5165.

Abstract 3671: Analytical performance of the Oncomine™ Dx Express Test, a CE-IVD NGS liquid biopsy assay for identification of clinically relevant variants

Abstract For in vitro Diagnostic use only. Not available in all regions including the United States. Introduction Minimally invasive blood-based liquid biopsy using cell-free total nucleic acid (cfTNA) and next generation sequencing (NGS) has substantially evolved in the field of clinical diagnostics for oncology, for detection of molecular therapeutic targets in non-small cell lung cancer (NSCLC). Here we describe analytical validation results for cfTNA using the CE-IVD Oncomine™ Dx Express Test (ODxET) assay, a qualitative in vitro diagnostic test that uses targeted NGS technology, the Ion Torrent Genexus™ Dx Integrated Sequencer to detect SNV, indel and copy number gain present in 42 genes and fusions in 7 genes from cfTNA isolated from NSCLC plasma samples. Methods Plasma samples from NSCLC patients were screened for variants of interest on several important gene loci. Screening involved isolation of cfTNA from K2-EDTA-derived plasma using the Genexus™ Cell-Free Total Nucleic Acid Purification kit on an automated purification system and sequencing was performed on the Genexus™ Dx integrated sequencer using the ODxET assay. Plasma samples having clinically relevant variants from the set of screened plasma samples were used to perform the validation studies. Results Analytical sensitivity and specificity of NSCLC plasma samples were characterized through determination of limit of detection (LOD) and limit of blank (LOB), respectively. The LOD level tested for DNA SNVs, and indels at 5 ng (minimum) input level ranged from 0.62% to 1.82% allelic frequency (AF), while 30 ng (maximum) input shows the LOD level ranging from 0.23% to 0.42% AF. The LOD for the tested RNA fusions ranged from 4.2 to 19.6 molecular counts for 5ng input and 4.9 to 8.0 molecular counts for 30ng input. Furthermore, LOB for the ODxET assay was determined by testing wild type (WT) cfTNA extracted from 30 healthy donor blood plasma samples, which were confirmed to be negative at all variant locations. The analytical accuracy study shows the false positive rate at 0.20% for SNVs, 0% for indel locations and fusion targets. In addition, the analytical reproducibility study shows 99.64% intra-run repeatability call rate for DNA variants and 98.75% for RNA variants. Conclusion In conclusion, we establish that the ODxET assay on Ion Torrent Genexus Dx Integrated Sequencer, is fast and efficient while demonstrating high sensitivity, specificity, and reproducibility for testing NSCLC liquid biopsy samples. Citation Format: Madhuri Jasti, Swasti Raut, Diarra Hassell, Nader Ezzedine, Daniela Garcia, Stephen Wunsch, Nick Siepert, Elliott Martinez, Emilia Ostrowska, Jeff Schagemann, Jian Gu, Thilanka Jayaweera, Luming He, Angie Cheng. Analytical performance of the Oncomine™ Dx Express Test, a CE-IVD NGS liquid biopsy assay for identification of clinically relevant variants [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 3671.

Abstract 5065: Integrating blood based liquid biopsies with TNM stage improves survival prediction model for non-small cell lung cancer

Abstract Introduction:Stage is the most important prognostic factor for survival in non-small cell lung cancer (NSCLC). The current TNM (Tumor, Node, Metastasis) staging system for NSCLC uses physical exam, histology, imaging, and surgical findings to define the extent and spread of a patient’s cancer. Despite advances in molecular testing which can impact treatment and prognosis for NSCLC patients, the current system does not incorporate molecular data into staging. We proposed a novel predictive model for survival analysis which integrates molecular analysis of liquid biopsies (B) into the TNM staging system (1). In this study, we tested this new staging system and our predictive model shows TNM + B (TNMB) provides a better predictive model for survival compared to TNM alone. Methods:176 patients were identified at Atrium Health Wake Forest Baptist with Guardant 360 liquid biopsies sent at diagnosis for NSCLC. Clinical data was obtained through retrospective chart review. Molecular analysis was performed on 81 genes across all Guardant 360 liquid biopsies. Using Cox proportional hazards model, we identified significant gene mutations or genes with significant magnitude of variant allele frequency (VAF) that improved survival prediction. We then designed a novel staging system incorporating whether a patient had a positive liquid biopsy test (B1), defined by the presence of these impact gene variables, to TNM stage. Results: Three impact genes STK11 (p=0.0253), NFE2L2 (p = 0.0025), TP53 (p=0.0129) and one gene with a magnitude of VAF, ARID1A (p=0.0082) was identified which significantly improved the predictive model for survival. Patients with negative liquid biopsy test, B0, had improved survival compared to patients with a positive liquid biopsy test, B1 (p<0.005). In our cohort, TNM staging alone did not show a significant survival difference between stage II and III (p = 0.19). Whereas TNMB staging showed a significant survival difference between stage II, III and IV. Conclusions: While TNM stage remains a major prognostic factor for NSCLC patients, it fails to incorporate molecular data which has a critical impact on management and prognosis. We designed a novel method to analyze molecular data obtained by liquid biopsy to identify significant gene mutations and gene magnitude of VAF that impacts survival prediction. In our cohort, we showed 4 impact genes which we used to create a new TNMB staging system which led to improved survival prediction compared to TNM alone. Acknowledgments: Research partly supported by NIH T32 Grant (T32CA247819) and Cancer Center Support Grant (P30CA012197) Reference: (1) Yang M, Forbes ME, Bitting RL, O'Neill SS, Chou PC, Topaloglu U, Miller LD, Hawkins GA, Grant SC, DeYoung BR, Petty WJ, Chen K, Pasche BC, Zhang W. Incorporating Blood-based Liquid Biopsy Information into Cancer Staging: Time for a TNMB System? Ann Oncol. 2018 Feb 1;29(2):311-323. Citation Format: Lauren Schmalz, Cetin Urtis, Liang Liu, Elizabeth Forbes, Fang-Chi Hsu, Nury Steuerwald, Alberto de Hoyos, Thomas Lycan, Jimmy Ruiz, William Petty, Wei Zhang. Integrating blood based liquid biopsies with TNM stage improves survival prediction model for non-small cell lung cancer [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 5065.

Abstract 3678: Beyond detection: AI-based classification of breast cancer invasiveness using cell-free orphan non-coding RNAs

Abstract Background: Approximately 1 in 8 women will be impacted by breast cancer in their lifetimes. Earlier detection of breast cancer through screening has improved survival. Liquid biopsies have the potential to complement existing screening methods by enabling earlier detection and differentiating invasive cancer (IBC) from ductal carcinoma in-situ (DCIS). We previously demonstrated high sensitivity and specificity for early detection of IBC by using a blood-based liquid biopsy platform to analyze a novel category of cancer-associated small RNAs, termed orphan RNAs (oncRNAs). Here, we developed a test that could not only detect the presence of cancer, but also classify the invasiveness of breast cancer. Methods: We utilized The Cancer Genome Atlas (TCGA) small RNA profiles to discover a library of 20,538 oncRNAs that were significantly enriched among 1,103 breast tumors compared to 349 controls from normal tissues spanning multiple tissue sites, limited to female samples. The diagnostic performance of these oncRNAs was assessed in an independent cohort of serum samples from 708 women, including 380 breast cancer patients (221 IBC and 159 DCIS; mean age: 58.0 ± 13.4 years) and 328 age-matched controls (mean age: 58.4 ± 13.7 years). We sequenced the small RNA content from 1 ml of serum from these patients at an average depth of 21.6 million 50-bp single-end reads. We detected 19,736 (96%) of the breast cancer-specific oncRNA library within at least one sample in this cohort. We then trained a multi-class generative AI model using 5-fold cross-validation to predict IBC, DCIS, and absence of breast cancer (IBC or DCIS). Results: Our oncRNA-based generative AI model achieved an overall AUC of 0.95 (95% CI: 0.94-0.97) for prediction of breast cancer versus cancer-free controls. At 90% specificity, overall model sensitivity is 90.0% (86.5%-92.8%). For DCIS and stage I IBC, the model has a sensitivity of 88.1% (82.0%-92.7%) and 90.4% (81.9%-95.8%) respectively, both at 90% specificity. In the second step, restricting to samples flagged as cancer, we observed an overall AUC of 0.9 (0.87-0.93) and sensitivity of 62.4% (55.3%-69.1%) at 90% specificity for discriminating against invasive breast cancer. Conclusions: We have demonstrated the potential utility of oncRNAs as the foundation for a liquid biopsy platform for sensitive and accurate early detection of breast cancer. Our liquid biopsy assay has the potential to complement standard of care by not only detecting breast cancer but also differentiating IBC from DCIS. Citation Format: Mehran Karimzadeh, Taylor B. Cavazos, Nae-Chyun Chen, Noura K. Tbeileh, David Siegel, Amir Momen-Roknabadi, Jennifer Yen, Jeremy Ku, Selina Chen, Diana Corti, Alice Huang, Dang Nguyen, Rose Hanna, Ti Lam, Seda Kilinc, Philip Murzynowski, Jieyang Wang, Xuan Zhao, Andy Pohl, Babak Behsaz, Helen Li, Lisa Fish, Kim H. Chau, Marra S. Francis, Laura J. Van't Veer, Laura J. Esserman, Patrick A. Arensdorf, Hani Goodarzi, Fereydoun Hormozdiari, Babak Alipanahi. Beyond detection: AI-based classification of breast cancer invasiveness using cell-free orphan non-coding RNAs [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 3678.

Abstract 5042: Urine as a non-invasive proxy for plasma in prostate cancer-related liquid biopsy applications

Abstract Introduction: The non-invasive collection of urine renders it an attractive and pragmatic substitute for plasma in liquid biopsy applications, fostering the creation of diagnostic methods that are more patient-friendly and less cumbersome for clinicians. This study investigates the feasibility of urine-derived cfDNA as a non-invasive option for acquiring vital diagnostic insights, further enhancing the liquid biopsy field. Methods: Paired male first void urine (FVU) and venous blood samples were collected from healthy donors (n=40) using Colli-Pee UAS devices (Novosanis) and BD K2EDTA Vacutainer tubes, respectively. Out of these, 20 paired urine and blood samples were spiked with 10 ng of cfDNA reference standard containing the KRAS p.G12V mutation. ~35 ml of urinary cell-free supernatant and ~3.5 ml of plasma were obtained after centrifugation of FVU and blood sample, respectively, and used as input material for cell-free nucleic acids extraction using the nRichDX Revolution Max20 cfDNA Isolation Kit. The extracted cfDNAs profile was assessed on 4200 TapeStation System, Agilent, using cfDNA ScreenTape. Extracted nucleic acids from urine samples were subjected to a qPCR assay to quantify endogenous human cfDNA content using 2X iTaq Universal SYBR Mastermix (Bio-Rad). Human cfDNA quantity was assessed using Ct values obtained from the qPCR assay. The actionable cfDNA molecules recovery was determined by qPCR mutation detection assay using TaqMan Genotyping. Results: Male FVU collected in Colli-Pee UAS device showed the presence of 100-250 bp cell-free DNA fragments similar to plasma obtained from whole blood collected in 10 ml vacutainer tubes as demonstrated by the Agilent cfDNA ScreenTape analysis. The cfDNA yield was similar when comparing one tube of blood (~3.5 mL of plasma) to one Colli-Pee of urine (~35 mL) shown by endogenous cfDNA qPCR assay. Additionally, the amplifiability of the KRAS G12V mutation was consistent in both plasma and urine samples, with comparable Ct values. Conclusion: The nRichDX Revolution Sample Prep System demonstrates the ability to extract cfDNA from both plasma and urine samples, with comparable extraction efficiency observed between matched samples from the same donor. This study provides evidence supporting the viability of first void urine samples as a non-invasive alternative for prostate cancer-related liquid biopsy applications. It indicates that cfDNA can be recovered from a sample collected with a single BCT or Colli-Pee UAS urine collection device. Future investigations will be required to assess clinical samples and biomarkers to establish a robust correlation between cfDNA levels and various cancer types and stages. This study reinforces the utility of first void urine samples as a reliable proxy for blood-based liquid biopsy applications. Citation Format: Nafiseh Jafari, Jason Saenz, Lauren Lee, Carlos Hernandez, Andrew Dunnigan, Amit Arora, Rafal Iwasiow, Mayer Saidian. Urine as a non-invasive proxy for plasma in prostate cancer-related liquid biopsy applications [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 5042.

Abstract 3789: Discovery of liquid biopsy-based novel protein biomarkers for early detection of breast cancer metastasis

Abstract Introduction and Objectives: Despite recent advances in screening and therapeutic technology, breast cancer is still the first leading cause of cancer death among females. When detected early (i.e., SEER Stage Localized), the 5-yr survival rate is 99%. The 5-yr survival rate is still 86% when cancer cells have spread beyond the breast, but this spread is limited to nearby lymph nodes (SEER Stage Regional). However, when the disease is noted to be SEER Stage Distant or metastasis, the 5-yr survival rate is significantly reduced to 31%. The evidence suggests that breast cancer is manageable if distant metastasis could be prevented. Thus, the prevailing idea is that early detection of breast cancer metastasis in surveillance will likely be the best modality to address advanced breast cancer’s dismal outcomes. Thus, liquid biopsy may be suitable for “early detection” of pre- and/or micro-metastasis because of its ease of obtaining biological material. In this pilot study, we explored the potential biomarkers for the early detection of breast cancer metastasis using publicly available datasets. Methods: We obtained 2 publicly available datasets from GEO (GSE102484 and GSE234114) containing primary tumor samples taken from subjects with and without metastatic disease. Both datasets were generated by mRNA microarray (Affymetrix Human Genome U133 Plus 2.0 Array). With these datasets, we performed the differentially expressed gene (DEG) analysis using GEO2R based on GEOquery and limma (Linear Models for Microarray Analysis) with a threshold criterion of Log 2 fold change >0 and p-value <0.05 as the cut-off point. The significantly upregulated genes were further screened using VerSeDa (Vertebrate Secretome Database) to select secreted proteins. Results: In the GSE102484 (N = 683, 582 non-metastasis and 101 metastasis), DEG identified 1798 upregulated and 1472 downregulated genes. Venn diagram analysis between the upregulated genes and VerSeDa identified 337 upregulated genes coding secreted proteins. In the GSE234114 (N = 120, 60 non-metastasis and 60 metastasis), DEG identified 2873 upregulated and 931 downregulated genes. Venn diagram analysis between the upregulated genes and VerSeDa identified 563 upregulated genes coding secreted proteins. By integrating the genes from GSE102484 and GSE234114, 82 commonly upregulated genes that code secreted proteins were identified. Conclusions: In this study, we identified 82 candidate mRNA biomarkers that are upregulated in breast tumor with metastasis. The panel of biomarkers will be validated in blood-based liquid biopsy samples in the next study. Citation Format: Cynthia Jinno, Sunao Tanaka, Takeo Fujii, Hideki Furuya. Discovery of liquid biopsy-based novel protein biomarkers for early detection of breast cancer metastasis [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 3789.

Abstract 3498: A novel combination of tissue-informed, comprehensive genomic profiling (CGP) and non-bespoke blood-based profiling for quantifying circulating tumor DNA (ctDNA)

Abstract Background: Estimating quantitative circulating tumor fraction in liquid biopsy samples is a promising area of clinical development for monitoring therapeutic molecular response and correlates with patient outcomes. Here we introduce a sensitive measure of estimating ctDNA tumor fraction (ctDNA TF) using a novel combination of tissue-informed CGP with a non-bespoke, blood-based liquid biopsy panel. Methods: Advanced pan-solid tumor samples were sequenced with both the Tempus xF/xF+ (105/523 gene, liquid biopsy) and Tempus xT (648 genes, solid-tumor with matched buffy coat) NGS assays (samples collected within 90 days). A normal distribution was fit against the somatic variants detected in solid-tissue and variants with variant allele fractions (VAF) within two standard deviations were used as selected biomarkers in the corresponding liquid biopsy to calculate a tumor-informed ctDNA TF for each sample. Results: In the initial validation set (n=79 samples also sequenced with low-pass whole genome sequencing), the linearity of tumor-informed ctDNA TF was compared to mean VAF (R2=0.26, slope=0.55), ichorCNA (R2=0.84, slope=0.84), and tumor-naive ctDNA TF (R2=0.71, slope=0.90). In a larger validation set (n=12,080), 37.5% samples had no variants detected in both xF and/or xT. Of the remaining samples, the linearity of tumor-informed ctDNA TF was compared to mean VAF (R2=0.21, slope=0.55) and tumor-naive ctDNA TF (R2=0.65, slope=0.81). In a follow-up analysis, we required 5 or more variants be detected in both xF and xT (n=1,659 samples) and compared the linearity of tumor-informed ctDNA TF to mean VAF (R2=0.37, slope=0.64) and tumor-naive ctDNA TF (R2=0.68, slope=0.98). In samples that received xF+ profiling (n=567), 24.3% had no variants detected in both xF+ and xT. The linearity of tumor-informed ctDNA TF of the remaining samples was compared to mean VAF (R2=0.01, slope=0.26) and tumor-naive ctDNA TF (R2=0.63, slope=1.02). Increasing the required number of variants to 5 or more had marginal improvement in linearity of tumor-informed ctDNA TF to mean VAF (R2=0.12, slope=0.47) and tumor-naive ctDNA TF (R2=0.67, slope=1.13).The limit-of-blank of the tumor-informed ctDNA TF approach was established to be 0% in both xF and xF+, and the limit-of-detection was ≤0.1% demonstrating that tumor informed ctDNA TF is a sensitive measure in detecting tumor fraction at low concentration levels. Conclusion: Here we introduce a novel sensitive and specific tumor-informed, non-bespoke approach for estimating ctDNA TF. Linearity improves with increased panel size and increased variant number. These results suggest that a tumor-informed ctDNA TF can be utilized to improve the sensitivity of existing methods for estimating tumor fraction to help in treatment decisions using Tempus’ tissue and liquid comprehensive NGS genomic profiling platform. Citation Format: Terri Driessen, Christine Lo, Wei Zhu, Robert Tell, Jonathan Freaney, Halla Nimeiri, Kate Sasser. A novel combination of tissue-informed, comprehensive genomic profiling (CGP) and non-bespoke blood-based profiling for quantifying circulating tumor DNA (ctDNA) [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 3498.

Assay Validation of Cell-Free DNA Shallow Whole-Genome Sequencing to Determine Tumor Fraction in Advanced Cancers

Blood-based liquid biopsy is increasingly used in clinical care of patients with cancer, and fraction of tumor-derived DNA in circulation (tumor fraction; TFx) has demonstrated clinical validity across multiple cancer types. To determine TFx, shallow whole-genome sequencing of cell-free DNA (cfDNA) can be performed from a single blood sample, using an established computational pipeline (ichorCNA), without prior knowledge of tumor mutations, in a highly cost-effective manner. We describe assay validation of this approach to facilitate broad clinical application, including evaluation of assay sensitivity, precision, repeatability, reproducibility, pre-analytic factors, and DNA quality/quantity. Sensitivity to detect TFx of 3% (lower limit of detection) was 97.2% to 100% at 1× and 0.1× mean sequencing depth, respectively. Precision was demonstrated on distinct sequencing instruments (HiSeqX and NovaSeq) with no observable differences. The assay achieved prespecified 95% agreement of TFx across replicates of the same specimen (repeatability) and duplicate samples in different batches (reproducibility). Comparison of samples collected in EDTA and Streck tubes from single venipuncture in 23 patients demonstrated that EDTA or Streck tubes were comparable if processed within 8 hours. On the basis of a range of DNA inputs (1 to 50 ng), 20 ng cfDNA is the preferred input, with 5 ng minimum acceptable. Overall, this shallow whole-genome sequencing of cfDNA and ichorCNA approach offers sensitive, precise, and reproducible quantitation of TFx, facilitating assay application in clinical cancer care.

Tumor-Educated Platelet RNA and Circulating Free RNA: Emerging Liquid Biopsy Markers for Different Tumor Types.

The incidence and mortality from malignant tumors continue to rise each year. Consequently, early diagnosis and intervention are vital for improving patient' prognosis and survival. The traditional pathological tissue biopsy is currently considered the gold standard for cancer diagnosis. However, it suffers from several limitations including invasiveness, sometimes not repeatable or unsuitable, and the inability to capture the dynamic nature of tumors in terms of space and time. Consequently, these limit the application of tissue biopsies for the diagnosis of early-stage tumors and have redirected the research focus towards liquid biopsies. Blood-based liquid biopsies have thus emerged as a promising option for non-invasive assessment of tumor-specific biomarkers. These minimally invasive, easily accessible, and reproducible tests offer several advantages, such as being mostly complication-free and efficient at monitoring tumor progression and tracing drug resistance. Liquid biopsies show great potential for cancer prediction, diagnosis, and prognostic assessment. Circulating tumor-educated platelets (TEPs) possess the unique ability to absorb nucleic acids from the bloodstream and to modify transcripts derived from megakaryocytes in response to external signals. In addition, circulating free RNA (cfRNA) constitutes a significant portion of the biomolecules present in the bloodstream. This paper aims to provide a comprehensive overview of the current research status regarding TEP RNA and cfRNA in liquid biopsies from various tumor types. Our analysis includes cancers of the lung, liver, pancreas, breast, nasopharynx, ovary and colon, as well as multiple myeloma and sarcoma. By synthesizing this information, we intend to establish a solid theoretical foundation for exploring potential applications of circulating RNA as a reliable biomarker for tumor diagnosis and monitoring.

Detection of PSMA expression on circulating tumor cells by blood-based liquid biopsy in prostate cancer.

For patients with mCRPC, PSMA-targeted radioligand treatment has significantly improved the clinical outcome. A blood-based liquid biopsy assay for recognizing PSMA protein expression on circulating tumor cells may be beneficial for better informing therapeutic decision-making and identifying the patients most likely to benefit from PSMA-targeted radioligand therapy. Using high-throughput imaging and digital AI pathology algorithms, a four-color immunofluorescence assay has been developed to find PSMA protein expression on CTCs on a glass slide. Cell line cells (LNCaP/PC3s/22Rv1) spiked into healthy donor blood were used to study the precision, specificity, sensitivity, limit of detection, and overall accuracy of the assay. Clinical validation and low-pass whole-genome sequencing were performed in PSMA-PET-positive patients with high-risk mCRPC (N = 24) utilizing 3 mL of blood. The PSMA CTC IF assay achieved analytical specificity, sensitivity, and overall accuracy above 99% with high precision. In the clinical validation, 76% (16/21) of the cases were PSMA positive with CTC heterogeneity, and 88% (21/24) of the patients contained at least one conventional CTC per milliliter of blood. Thirty-six low-pass-sequenced CTCs from 11 individuals with mCRPC frequently exhibited copy number increases in AR and MYC and losses in RB1, PTEN, TP53, and BRCA2 locus. The analytical validation utilizing Epic Sciences' liquid biopsy CTC platform demonstrated the potential to detect PSMA protein expression in CTCs from patients with mCRPC. This assay is positioned as an effective research tool to evaluate PSMA expression, heterogeneity, and therapeutic response in many ongoing clinical studies to target tumors that express PSMA.

Blood-based liquid biopsy: A promising noninvasive test in diagnosis, surveillance, and prognosis of patients with upper tract urothelial carcinoma

ObjectivesTo assess the efficacy of blood-based liquid biopsy in the diagnosis, surveillance, and prognosis of upper tract urothelial carcinoma (UTUC). Methods and MaterialsIn this prospective study, peripheral blood samples were collected from patients with primary UTUC before surgery with curative intent and follow-up visits at University of Southern California between May 2021 and September 2022. The samples were analyzed using the third-generation comprehensive high-definition single-cell assay (HDSCA3.0) to detect rare events, including circulating tumor cells (CTCs) and oncosomes, based on the immunofluorescence signals of DAPI (D), cytokeratin (CK), CD45/CD31 (CD), and vimentin (V). The findings of pre-surgery liquid biopsies were compared with those of blood samples from normal donors (NDs) and matched follow-up liquid biopsies. The association between liquid biopsy findings and clinical data, including recurrence-free survival (RFS), was also assessed. ResultsTwenty-eight patients with UTUC were included, of whom 21 had follow-up samples. Significant differences in specific rare analytes were detected in the preoperative samples compared to the NDs. In the post- vs. presurgery matched analysis, a significant decrease was detected in total-, CK-, and CK|V oncosomes, as well as in D-, D|V-, and D|V|CD cells. With a median follow-up of 11 months, 8 patients had disease recurrence. Survival analysis demonstrated that patients with >1.95 preoperative CK|V oncosomes (p = 0.020) and those with >4.18 D|CK|V cells (p = 0.050) had worse RFS compared to other patients. ConclusionsThis study demonstrated promising initial evidence for the biomarker role of CTCs and oncosomes in the diagnosis and surveillance of patients with UTUC.