Liquid Biopsy Samples Research Articles

7 Flexible probabilistic methods to unlock the clinical potential of liquid biopsy sampling

OBJECTIVES/GOALS: Decoding the origins of cell-free DNA (cfDNA) released from dying cells in a liquid biopsy sample (e.g. blood) offers the potential to provide insight into the dynamic, organism-wide changes reflective of health and disease. Thus, making cfDNA an ideal target for serial, minimally invasive monitoring of disease-related changes. METHODS/STUDY POPULATION: We develop a probabilistic method that leverages the co-regulation of neighboring CpG sites on individual methylome-wide sequencing (WGBS) reads to more flexibly model cell-specific methylation compared to prior methods that focus on the methylation rate of a single CpG site. We then extend our cross-sectional model to account for sequential sampling within the same subject. The increased sampling frequency is critical to identifying the evolutionary dynamics of disease progression influencing treatment response and resistance, and disease recurrence. We utilize Bayesian inference techniques to model patient-specific longitudinal profiles of cell-type turnover in simulated serial samples. RESULTS/ANTICIPATED RESULTS: We found our model more effective at capturing a range of methylation patterns on cfDNA fragments with lower Root Mean Square Error across simulations compared to a single CpG model. We apply our model to detect significant (p < 0.05, Friedman’s test) increases in cellular contributions from lung and cardiac tissue in breast cancer patients (n=15) undergoing radiation therapy compared to baseline. We also identify signals of radiation induced toxicity to the liver in right-sided breast cancer patients (n=8) receiving radiation treatment compared to left-sided breast cancer patients (n=7). Finally, we show our extended model results in more efficient estimates of simulated cell-type turnover profiles compared to analyzing serial samples cross-sectionally, ignoring the longitudinal nature of the data. DISCUSSION/SIGNIFICANCE: Here we address an unmet need in developing novel statistical methodologies to decode the origins of methylated cfDNA obtained from liquid biopsy samples. We demonstrate the far-ranging clinical utility of serial liquid biopsy sampling to complement and advance the standards of clinical care in oncology and other pathologies.

Abstract 2424: Detection of prostate cancer in urine by fragment-level DNA methylation analysis

Abstract Introduction: Screening for prostate cancer (PCa) with molecular markers is hindered by low tumor allele fraction (TAF) in liquid biopsy samples such as blood or urine. Others have suggested that tumor allele enrichment can be achieved by evaluating fragment-level methylation patterns generated by next generation sequencing (NGS). Here, we test this concept using target capture NGS on cfDNA from prediagnostic collected urine samples to discriminate PCa from otherwise healthy men. Methods: Buffered urines were collected from 23 patients with biopsy confirmed PCa (Gleason score 6-9) and 24 otherwise healthy age matched cancer-free controls. cfDNA was isolated and enzymatic methyl-seq libraries were prepared and enriched by hybrid capture using probes mapping to 197 differentially methylated regions (DMRs) from a previous PCa/normal tissue marker discovery study in our lab. These DMRs were subsequently vetted independently using a methylome-wide dataset from a cohort of 36 clean and disease control urines to ensure low non-cancer background signal (<1%). Indexed libraries were sequenced at 560X deduplicated coverage and fragment-level CpG methylation information was extracted from individual sample .bam files. Mapped sequencing reads exhibiting concordant methylation were used to derive a quantitative α-score. Reads had to contain at minimum 5 CpGs and have an α-value (% 5mC per read) > 60%. Positivity for the 197 markers required an α-score greater than 6 sigma (σ) above the mean of the control arm. Values were variance-adjusted to ensure equal weighting. Beta (β) analysis (mean % 5mC across all reads) of the data was also performed for comparison. Results: Of the 197 markers, 187 were positive for one or more PCa patients. Tumor-derived fragments were detected in 22 of 23 cases, which included 8 G-6, 9 G-7, 2 G-8, and 3 G-9 patients. 1 G-7 was missed, however the sequencing coverage for this sample was significantly lower than the others due to poor library enrichment. 2 of the 24 healthy controls were positive, one with extremely high fragment counts. Upon re-review of the full control cohort, this patient had been diagnosed with bladder cancer 2 years after urine collection. The 2nd patient had was diagnosed with lung cancer in a similar timeframe. The other 22 confirmed cancer-free controls had α-scores below the 6σ threshold. Using a co-captured reference gene (B3GALT6) to mimic genomic equivalents, the TAFs ranged from 0.015% to 8%. β analysis of the data detected only 13 PCa patients (>6σ) and marker positivity was lower than the fragment-level approach. Conclusion: Target capture NGS coupled with fragment-level analysis is a rational approach for sensitive and specific detection of low tumor-derived DNA in liquid biopsy samples. For PCa assay in urine, we were able to uncover positivity in cancer patients approaching 1 in 10,000 ctDNA dilution levels. These results warrant further validation studies with this and other cancers. Citation Format: Tibor Bedekovics, William R. Taylor, Zhifu Sun, Saurabh Baheti, Xiaoming Cao, Kelli N. Burger, Karen A. Doering, Patrick H. Foote, Benjamin Gochanour, Douglas, W. Mahoney, Matthew T. Gettman, John B. Kisiel. Detection of prostate cancer in urine by fragment-level DNA methylation analysis [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 2424.

Abstract 5023: Analytical validation of the Oncomine™ Dx Express Test using liquid biopsy samples as an IUO assay in a CAP/CLIA lab

Abstract Introduction: The Oncomine™ Dx Express Test (ODxET) is a next-generation sequencing (NGS) test. It can be used as an investigational use only (IUO**) test to detect somatic DNA and RNA alterations in human cell-free total nucleic acid (cfTNA) isolated from plasma samples. This study outlines the analytical validation of the ODxET for relevant single nucleotide variants (SNV) and insertion/deletions (INDEL) in liquid biopsy specimens. The Ion Torrent Genexus™ platform has a short turnaround time with minimal operator input. This study was performed in a College of American Pathologists (CAP™) accredited and Clinical Laboratory Improvement Act (CLIA) licensed laboratory. Methods: Forty-seven unique samples (39 clinical, 3 derived, 2 reference and 3 commercially available DNA controls) were analyzed. All experiments began with sample extraction, except the lower input limit of detection (LLOD). We evaluated limit of detection, variant calling accuracy, and precision. Two control NIST™ samples were sequenced with 3 technical replicates each for INDEL and SNVs analytical accuracy. The LOD was evaluated at both the target input of 2ng/µl (40ng), and the minimum input,0.33ng/µl (6.6ng) with allele frequency (AF) levels at 1%, 0.5%, and 0.25%. The 39 clinical plasma samples were used for variant calling accuracy. Inter-assay reproducibility was assessed by sequencing DNA controls across four runs by different operators over multiple days/systems. Intra-assay repeatability was assessed by sequencing 4 samples in replicate on the same run by one operator on the same day/system. Results: The analytical accuracy and NPV was 100% for SNV/INDEL variants. The 40ng input LOD sensitivity was 96% for SNVs at 0.5% AF and 100% for INDELs at 0.25% Minimum AF. The lower input LOD sensitivity was 89% for SNVs and 100% for INDEL at 1% AF. Variant calling accuracy on samples with pre-characterized data was 100% for the 13 positive samples. Specificity was >99% for all samples including both healthy donor and positive samples. Repeatability was 100% for INDELs and >96% for SNVs, reproducibility was 100% for both variant classes. Conclusion: This validation study to estimate analytical accuracy, limit of detection, variant calling accuracy and precision has demonstrated that the ODxET assay is highly robust in detecting SNV and INDEL mutations in plasma samples and is suitable for use in a CAP/CLIA laboratory as an IUO assay.**=In Vitro Diagnostic use only. Not available in all regions including North America. Citation Format: Juan C. Espinoza, Rajendra Ramsamooj, Akemi Yuki, John Williamson, Diarra Hassell, Madhu Jasti, Suzanne Salazar, David Ginzinger. Analytical validation of the Oncomine™ Dx Express Test using liquid biopsy samples as an IUO assay in a CAP/CLIA lab [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 5023.

Abstract 4626: A kit targeting detection of ESR1 mutations from circulating exosomal RNA and cell-free DNA supports longitudinal studies into endocrine therapy resistance in a broadly accessible RT-qPCR format

Abstract Introduction: The most prevalent subtype of breast cancer is HR+/HER2- (positive for hormone receptor, negative for human epidermal growth factor receptor 2). For metastatic breast cancer (mBC), a frequent occurrence is resistance to the aromatase inhibitors used in endocrine therapy (ET). Resistance is mediated through mutations in the ligand binding domain of estrogen receptor 1 (ESR1). Approval of elacestrant, a second-line agent for HR+/HER2-/ESR1 mutated mBC , underscores the need for early detection of ESR1 resistance mutations. We have developed sensitive RT-qPCR reagents for targeted longitudinal studies into the emergence of ESR1 resistance mutations in plasma. This all-inclusive kit analyzes circulating exosomal RNA (exoRNA) and cell-free DNA (cfDNA) to accurately identify low copy number mutations with a rapid turnaround time. Methods: Challenge panels were constructed using blends of synthetic nucleic acids of known mutational status in a background of partially fragmented wild-type DNA from cell lines. Additionally, plasma samples collected from subjects with stage IV mBC (HR+/HER2-) on active aromatase inhibitor therapy, +/- CDK 4/6 inhibitor for a minimum of one year, were subjected to an in-house method optimized to co-enrich exoRNA and cfDNA. Multiplex RT-qPCR-based target enrichment performed on 11 ESR1 mutations and an internal control were evaluated on widely used thermal cycler and qPCR instruments. Results: We developed technology that interrogates 11 key ESR1 mutations associated with ET resistance in HR+/HER2- mBC cases. Earlier studies performed in simplex analyzed plasmid-based ESR1 mutations with detection of 10 or fewer mutant copies, estimating analytical sensitivity of at least 0.1% (5 mutant copies in a background of 5,000 WT copies). Herein, we describe the subsequent work to complete the methodology employed in the kit, including the incorporation of an endogenous control, external batch run control materials (positive, negative), deeply optimized reagents (RT, pre-amplification, qPCR), and automated analysis. We report on the results of studies into the kit’s performance characteristics against their targets: analytical sensitivity of the equivalent of 5 copies of mutation per mL plasma, high precision across replicates at 0.1% allele fraction, analytical specificity of ≥90% negative results in mutation-negative replicates, as well as workflow attributes (reduced pipetting steps and turnaround time). Conclusion: Our solution to detect ESR1 resistance mutations facilitates the generation of nucleic acid sample to results within one day using liquid biopsy samples and a multiplexed RT-qPCR. Enhancing mutation detection sensitivity by complementing cfDNA with exoRNA will facilitate future research into breast cancer treatment options. Citation Format: Justin T. Brown, Julie R. Thibert, Liangjing Chen, Melissa Church, Holli Dale, Jamie Myers, Elliot Hallmark, Megan Yociss, J Aquiles Sanchez, Kurt Franzen, Johan Skog, Gary Latham, Brian Haynes, Sarah Statt. A kit targeting detection of ESR1 mutations from circulating exosomal RNA and cell-free DNA supports longitudinal studies into endocrine therapy resistance in a broadly accessible RT-qPCR format [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 4626.

Abstract 3663: Extending and analytical validation of a hematological cancer panel for simultaneous detection of driver mutations, copy number variations and translocations in tissue and blood samples

Abstract Background Hematological cancers represent approximately 9.4% of all new cancer cases in the US, with varying prevalence among different age groups. For instance, Acute Lymphocytic Leukemia (ALL) is more prevalent in children (less than 15 years), while Multiple Myeloma and Non-Hodgkin Lymphoma are commonly identified in older population (65 or older). Each hematological cancer subtype has distinct mutation and expression profiles, necessitating the coverage of various variant types, including point mutations, gene copy number variations (CNVs), and translocations. In this study, we extend the PredicineHEME panel to encompass long-range translocation and CNV detections. Methods The extension of the original PredicineHEME panel involves three key modifications. First, a genome-wide SNP backbone was incorporated to enhance the sensitive detection of both gene and chromosomal-level CNVs and Loss of Heterozygosity (LOH). Second, additional coverage of translocation breakpoint regions was added, considering the panel's application in liquid biopsy samples. Coverage included the most frequently found translocations in major hematological cancers. Third, the driver gene list was supplemented to include genes and mutations treated as active therapeutic targets in clinical studies. Analytical validation was conducted using contrived reference materials, cell lines with known biomarker status, and healthy and clinical samples with known mutations or translocation status. Results The expanded panel underwent balanced testing with healthy normal saliva gDNA samples to ensure even capture of targeted genomic regions. Assay testing followed CLIA/CAP guidelines on analytical validation materials. The Limit of Detection for SNV/INDEL and known translocation breakpoint reached as low as 0.3%. Our assay detected over 90% of covered and reported translocation breakpoints in the validation materials. Chromosome arm level CNVs reported in the validation materials were detected with 100% sensitivity and specificity. Conclusions The expansion of the PredicineHEME panel underwent analytical validation, meeting design expectations. With this extension, the assay can effectively detect driver mutations (SNVs/INDELs), CNVs at gene and chromosomal levels, and targeted large translocations in major hematological cancers. Citation Format: Yong Huang, Tom Zhang, Feng Xie, Jianrong Dong, Xiaohong Wang, Zhixin Zhao, Shujun Luo, Kemin Zhou, Shidong Jia, Pan Du. Extending and analytical validation of a hematological cancer panel for simultaneous detection of driver mutations, copy number variations and translocations in tissue and blood samples [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 3663.

Abstract 2426: Liquid biopsy for a predictive biomarker of resistance to immunotherapy

Abstract Immunotherapy has become a mainstay for cancer treatment. However, immune editing is a well-known resistance paradigm that limits the efficacy of immunotherapy in many patients. One type of immune editing that occurs in 1/6 of all cancer patients, with prevalence over 40% in some cancer types, is human leukocyte antigen loss of heterozygosity (HLA LOH). Liquid biopsies provide a non-invasive and inexpensive way of guiding treatment decision-making. We have developed a liquid biopsy assay for detecting HLA LOH in cancer patients, with the goal of utilizing the assay for deciding whether immunotherapy is the right course of treatment for a given patient. The assay was tested utilizing sample sets from cancer patients, where each sample set from a patient included a solid tumor biopsy, a liquid biopsy plasma sample, and PBMC sample for control. Tumor HLA LOH results were used for ground truth, and HLA LOH results from the liquid biopsy sample were compared to those from the tumor sample from the same patient. Whole exome sequencing (WES) was performed on tumor DNA, cell-free DNA (cfDNA) from plasma, and germline DNA from PBMCs, and we ran our novel liquid biopsy HLA LOH detection algorithm on the plasma WES data. Standard copy number variant (CNV) calling tools were utilized for calling HLA LOH from the tumor sequencing data. In both cases - tumor and plasma - germline PBMC DNA was used as control. We tested the liquid biopsy HLA LOH detection method on sample sets from 2 HPV-negative primary head and neck cancer patients, 14 castration-resistant prostate cancer patients, and 27 metastatic breast cancer patients. The samples from the head and neck cancer patients were obtained from patients on clinical trials at NCI, while WES data from prostate cancer patient samples and breast cancer patient samples were downloaded from NCBI Database of Genotypes and Phenotypes (dbGaP) after applying for access to dataset phs001417.v1.p1 (Adalsteinsson, et al., Nature Communications, 2017). The method for liquid biopsy HLA LOH detection utilizes a novel mathematical framework that takes into account biological and experimental variables to generate allele-specific HLA LOH information from sequencing data. In the first two cohorts tested - the head and neck cancer cohort and the prostate cancer cohort - the method was 100% consistent when comparing liquid biopsy HLA LOH results from our method to CNV calling results from matched tumors (4 true positives and 36 true negatives; 0 false negatives and 0 false positives). In the breast cancer cohort, some false negatives and false positives were observed (6 true positives and 48 true negatives; 5 false negatives and 8 false positives). Experiments are ongoing in a CLIA lab to establish sensitivity, specificity, and limit of detection (LoD) for the assay. Upon improvements and validation of the assay, the goal is to utilize the liquid biopsy HLA LOH detection method in the clinic for treatment selection in the context of immunotherapy. Citation Format: Andrew Sinkoe, James L. Gulley. Liquid biopsy for a predictive biomarker of resistance to immunotherapy [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 2426.

Abstract 2407: Detection of hotspot PIK3CA mutations in primary tumors and plasma-cfDNA of breast cancer patients using a novel highly sensitive CE-IVD kit

Abstract Introduction: Detection of PIK3CA mutations in primary tumors (FFPEs) and liquid biopsy samples is of increasing importance for treatment decisions in breast cancer. Oncolipsy PIK3CA kit (Pharmassist Ltd, Greece) is based on the combination of allele-specific priming, asymmetric PCR, and melting curve analysis. The CE-IVD kit can detect the presence of four PIK3CA hotspot mutations (E542K, E545K, E545Q, H1047R). The aim of the present study was to: a) evaluate the performance of this novel CE-IVD kit both in FFPEs and plasma-cfDNA samples from patients with metastatic breast cancer (MBC) and b) directly compare the derived results using the Oncolipsy PIK3CA kit with the cobas® PIK3CA mutation test (CE-IVD, Roche Diagnostics, Germany) in identical gDNAs isolated from FFPEs samples. Patients and methods: We analyzed 42 primary tumors (FFPEs) and 29 plasma-cfDNA samples from patients with estrogen receptor (ER) positive metastatic breast cancer. Genomic DNA was isolated from FFPEs using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), while cfDNA was extracted from 2.00 mL of plasma using the QIAamp DSP cNA Kit (Qiagen, Hilden, Germany). PIK3CA hotspot mutations were detected in the LightCycler® 2.0 (IVD instrument, Roche Diagnostics, Germany) using the highly sensitive Oncolipsy PIK3CA kit (analytical sensitivity 0.05%). A direct comparison study was performed using the cobas® PIK3CA Mutation Test on the cobas z 480 analyzer (IVD instrument, Roche Diagnostics, Germany). Results: In primary tumors the E545K mutation was detected in 22/42 (52.4%), the E542K and H1047R mutations were detected in 11/42 (26.2%) and 4/42 (9.5%) of primary tumor samples, respectively, while the E545Q mutation was not detected in any FFPE sample tested. At least one PIK3CA mutation was detected in 14/29 (48.3%) plasma-cfDNA samples. More specifically the E545K mutation was detected in 11/29 (38%), the H1047R mutation in 3/29 (10.4%) and the E542K mutation in 2/29 (6.9%) while the E545Q mutation was not detected in any plasma-cfDNA sample tested. In FFPEs, as expected, PIK3CA mutation frequency was higher compared to plasma-cfDNA. Direct comparison of the results derived using these two commercially available kits, the Oncolipsy CE-IVD kit and the cobas PIK3CA mutation test, using the same gDNA isolated from 42 FFPEs revealed a concordance of 34/42 (81%). Conclusions: The commercially available Oncolipsy PIK3CA CE-IVD kit is highly sensitive and specific for the detection of four PIK3CA hotspot mutations in primary tumors and plasma-cfDNA. Acknowledgement: This study has been financially supported by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T1RCI-02935). Citation Format: Stavroula Smilkou, Dimitra Stergiopoulou, Eleni Tzanikou, Loukas Kaklamanis, Ioanna Balgkouranidou, Helena Linardou, Flora Zagouri, Evangelia Razi, Stylianos Kakolyris, Amanda Psyrri, Christos Papadimitriou, Ioanna Koukli, Athina Markou, Evi Lianidou. Detection of hotspot PIK3CA mutations in primary tumors and plasma-cfDNA of breast cancer patients using a novel highly sensitive CE-IVD kit [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 2407.

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.

Abstract 2293: Comparisons of saliva versus plasma cfDNA and exosomal DNA as potential biomarkers for head and neck cancer

Abstract Purpose: Liquid biopsy approaches are revolutionizing cancer monitoring and have been considered potential mediators for therapeutic efficacy in multiple cancers. The investigation of different liquid biopsy biomarkers may lead to a better understanding of the cancers they are designed to detect and facilitate optimal management. This study aimed to identify and compare the epigenomic and genomic profiles of tumor biomarkers in circulation using cell-free DNA (cfDNA) and exosomal DNA (exoDNA) in plasma and saliva samples collected from patients with head and neck squamous cell carcinoma (HNSCC). Methods: In a prospective trial, we performed whole-genome bisulfite sequencing on paired cfDNA and exoDNA from saliva versus plasma of 16 HNSCC patients. cfDNA was directly extracted from plasma and saliva, while exoDNA was extracted from purified exosomes after characterization. DNAs were then converted by Zymo EZ DNA Methylation-Direct kit and libraries were constructed with xGen™ Methyl-Seq Kit and sequenced on the Illumina NovaSeq platform. Sequencing data analyses were performed using Bismark, GATK, and methylKit for methylation pattern and cfTools for Tumor Fraction Estimation (TFE). Results: Methylation data demonstrated heterogeneities across sample types. Comparisons of ctDNA as a reference for exoDNA showed 7147 hypermethylated and 9984 hypomethylated regions for plasma samples but showed 131 hypermethylated and 123 hypomethylated regions for saliva samples. Comparisons of plasma as a reference for saliva, 37 hypermethylated and 95 hypomethylated regions were noted in exoDNA, while 117 hypermethylated and 262 hypomethylated regions in ctDNA. The mean level of tumor-derived DNA was above 3%, the lower limit of sensitivity to indicate the presence of tumor cutoff as suggested by the cfTools. There were 3.24%, 3.14%, and 3.14% tumor-derived DNA in plasma cfDNA, plasma exoDNA, and saliva exoDNA, respectively. However, the tumor fraction in saliva cfDNA was below this cutoff (2.88%). A statistically significant (P < 0.001) difference in TFE was observed between combined cfDNA and exoDNA data from plasma and saliva, but the difference between combined sample types cfDNA and exoDNA did not reach statistical significance (P = 0.306). The linear mixed-effects model for TFE influence between the different groups revealed a significant interaction between sample sources (plasma and saliva) and sample types (cfDNA and exoDNA) (P = 0.005), as well as a significant effect of sample source on TFE (P < 0.001). Conclusions: This pilot investigation provides evidence that exoDNA and cfDNA have different molecular profiles in plasma and saliva samples from the same HNSCC patient, and indicates that saliva carries additional tumor markers for HNSCC. These results help to advance our understanding of the potential impact of different liquid biopsy sample types in cancer management. Citation Format: Mouadh Barbirou, Amanda Miller, David Baek, Allison Hameier, Ashley Wetzel, Alban Linnenbach, Kathryn Nunes, Pablo Llerena, Voichita Bar-Ad, Adam Luginbuhl, Ubaldo Martinez Outschoorn, Ronald Myers, Joseph Curry, Chun Wang, Hushan Yang. Comparisons of saliva versus plasma cfDNA and exosomal DNA as potential biomarkers for head and neck 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 2293.

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 2324: Leveraging a comprehensive genomic data library for detecting clonal hematopoiesis in liquid biopsy

Abstract Background: Clonal Hematopoiesis (CH) is a well established confounder in next-generation sequencing (NGS)-based liquid biopsy cancer diagnostics. Misclassification of CH variants as tumor variants can lead to false positive actionable variant detection, potentially resulting in incorrect interpretation of results and therapy selection. Moreover, CH variants may also interfere with quantitative variant monitoring leading to inaccurate assessment of treatment response. While filtering of CH variants is possible via matched sequencing of white blood cell and plasma DNA, emerging algorithmic approaches may enable a more resource-effective, time-sensitive approach with high precision. Methods: A random forest classifier was trained and validated on 1321 advanced, pan-solid tumor cancer samples (training n=660, validation n=661) sequenced using both the Tempus xF+ (liquid biopsy) and Tempus xT (solid tumor with matched buffy coat) NGS assays. Variants were labeled as CH or tumor-derived based on solid-tissue results in 39 genes that are known to be associated with CH (e.g., DNMT3A, TET2, TP53). The classifier was trained to classify SNV and indel variants detected via liquid biopsy as circulating-tumor or non-tumor (CH + germline) in origin. Features used by the classifier include the fragment size of reads overlapping each variant, prevalence in solid-tumor samples from the Tempus multimodal database, and variant allele fraction relative to estimated tumor fraction. Model classifications were validated against Tempus xT. Results: Our training set (n=660 liquid biopsy samples) included 680 pathogenic variants. 50% (n=342/680) were determined to be tumor-derived, while 50% (338/680) are likely due to CH. Our independent validation (n=661 samples) included 600 pathogenic variants. Model prediction accuracy on these validation set variants was 91.7%, with an ROC-AUC of 0.97. Sensitivity was 88.3% (n=257/291 true positives correctly labeled as non-tumor variants), specificity was 94.8% (n=293/309 true negatives correctly labeled as tumor variants), and precision was 94.1%. The model ranked “gene” as the most informative feature for variant classification, followed by measurements of fragment distribution and historical prevalence of individual variants within the Tempus Database. Conclusion: A novel classifier trained on multiple orthogonal bioinformatics features can reliably distinguish CH from tumor-derived variants using only liquid biopsy data with high accuracy, including high sensitivity and high specificity. Citation Format: Anne Sonnenschein, Maysun Hasan, Sandra Hui, Bob Tell, Halla Nimeiri, Jonathan Freaney, Kate Sasser, Wei Zhu, Christine Lo. Leveraging a comprehensive genomic data library for detecting clonal hematopoiesis in liquid biopsy [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 2324.

Abstract 6562: Comprehensive liquid biopsy: Multi-analyte evaluation of cellular and plasma components of blood in prostate cancer from a single blood collection tube

Abstract Background Liquid biopsy samples offer a non-invasive method for investigating drug targets and biomarkers in solid tumors. In this study, we describe a comprehensive liquid biopsy analysis of whole blood to identify circulating tumor cells (CTCs), large extracellular vesicles and CTC fragments, and CTC-white blood cell hybrids; quantify protein biomarker expression on CTCs; perform targeted mutation profiling on CTCs and cell-free DNA (cfDNA) from plasma; and analyze protein expression on white blood cells (WBCs) from a single 7.5 mL blood sample. This array of tests was applied to blood samples from patients with Stage IV prostate cancer to demonstrate clinical feasibility. Methods A novel dual biomarker CTC assay was developed to measure protein expression of PSMA and ARv7 on CTCs from stage IV prostate cancer patients. Nucleated cells from clinical samples were processed to microscope slides using AccuCyte®, an unbiased, density-based separation method that also allows for plasma collection for downstream analysis. Slides were fixed and stained with antibodies to cytokeratin, EpCAM, and CD45 to detect CTCs and exclude WBCs, and with PSMA and ARv7 to evaluate protein biomarker expression on identified CTCs. The slides were imaged with the CyteFinder® automated immunofluorescence scanning microscope that identifies CTCs for confirmation by trained reviewers and quantitatively measures protein mean fluorescence intensity (MFI) on any cells detected on the slides (CTCs, CTC-WBC hybrids, large extracellular vesicles and CTC fragments and WBCs). Individual CTCs and control pools of WBCs from clinical samples with at least 4 CTCs were picked using CytePicker®, a needle-based system used to isolate single cells. cfDNA was isolated from plasma removed during the AccuCyte® process using a QIAamp MinElute ccfDNA kit. Picked CTCs, WBC pools, and isolated ctDNA were sequenced using the OncoZoom Cancer HotSpot panel from Paragon Genomics. Results Nine progressing Stage IV prostate cancer patient blood samples were stained with a novel PSMA/ARv7 dual biomarker assay. CTCs were identified in all clinical samples (median 4, range 1 - 384). PSMA positivity in clinical samples ranged from 0% to 75%, and ARv7 positivity ranged from 33% to 100%, and. Additional analysis using QuPath was performed to determine PSMA/ARv7 protein expression on WBCs and CTC fragments. ctDNA analysis from 5 individual samples was compared to sequencing results from individual CTCs from the same samples. Conclusions Using a single 7.5 mL blood liquid biopsy, we can measure protein expression on enumerated CTCs and quantify these same protein biomarkers on WBCs and CTC fragments/oncosomes, determine the genetic makeup of the tumor through the sequencing of cfDNA, and monitor genetic mutations in intact CTCs in circulation. Citation Format: Jon Ladd, Erin Bayer, Brock Bartels, Arista Tischner, Rachel Ponting, Eric Kaldjian, Arturo Ramirez. Comprehensive liquid biopsy: Multi-analyte evaluation of cellular and plasma components of blood in prostate cancer from a single blood collection tube [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 6562.

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 6258: Inference of gene expression using fragmentation patterns from targeted high-depth sequencing of cell-free DNA

Abstract Cell-free DNA (cfDNA) circulating in blood plasma arises primarily from cellular chromatin fragmentation and release due to cell death. Studies have demonstrated that the assessment of fragmentomic features of cfDNA enables gene expression inference and tissue-of-origin classification with potential applications for noninvasive cancer detection. However, due to low depth of coverage of sites of interest, current whole genome sequencing (WGS) methods have not been able to infer expression of individual genes or limited gene sets. Therefore, we developed a targeted sequencing approach that enables individual gene expression inference from cfDNA fragmentation patterns based on transcriptional start-site gene activation probability (TSS-GAP). To assess the sensitivity of this method for cfDNA-inferred gene expression profiling, we applied TSS-GAP to blends of micrococcal nuclease (MNase)-digested DNA from a cancer cell line (LS180) and sorted peripheral blood mononuclear cells (PBMC), with LS180 DNA at varying concentrations (0.1-10%). This enabled the determination of the limit of detection (LoD), relative to healthy donor background levels, on the basis of the genes’ activation probabilities (TSS-GAP scores). We found 936 genes with high inferred expression in the LS180 cancer cell line and low inferred expression in non-cancer samples (i.e. CD4, CD14, and plasma cfDNA from 4 healthy donors) and used those for healthy donor background and LoD assessment. Using these gene-level background values as cutoffs, blends were assessed to determine the lowest blend level at which expression of the gene was able to be differentiated from background. Out of the 936 genes, 326 (34.8%) were detectable at a blend level of 0.1%, 59 (6.3%) at 0.3%, 31 (3.3%) at 1%, 19 (2.0%) at 3%, and 39 (4.2%) at 10%. In total, 416 genes could be detected by TSS-GAP at levels 1% of or lower above background, highlighting the potential of our method for detecting expression of certain genes even from a small fraction of cells contributing cfDNA to liquid biopsy samples. Further expansion of this work could consider the utility of such a method for circulating tumor DNA (ctDNA) detection, cfDNA tissue-of-origin classification, and deconvolution of cell types contributing to cfDNA found in blood. Citation Format: Emily Leff, Alexander Tseng, Victoria Cheung, Ehsan Tabari, Kimberly Walter, Alex Lovejoy, Cheng-Ho J. Lin. Inference of gene expression using fragmentation patterns from targeted high-depth sequencing of cell-free DNA [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 6258.

Unlocking the promise of liquid biopsies in precision oncology

Liquid biopsies have emerged as a promising and minimally invasive alternative to traditional tissue biopsies for detecting and monitoring cancer. Liquid biopsies offer a comprehensive analysis of cancer genetics and tumor burden by examining circulating cells and cell-derived analytes using a variety of assays, including conventional PCR methods and cutting-edge tools like long-read sequencing and nanotechnology. However, there are still some limitations and challenges that need to be overcome for their implementation in clinical routine, including the need for further research on their sensitivity and specificity, cost-effectiveness, standardization, and regulatory approval. Despite these challenges, liquid biopsies have the potential to become widely used tools in oncology. Here we provide an overview of the current state of liquid biopsies, highlighting recent advancements in the field and their potential benefits in clinical settings for cancer patients. The article further discusses the challenges that need to be addressed in order to facilitate their application worldwide. Prompt resolution of these challenges can be achieved by fostering international research collaborations and establishing standardized guidelines for liquid biopsy sample management and studies.

Prognostic significance of circulating tumor cells and tumor related transcripts in small cell lung cancer: A step further to clinical implementation.

Small-cell lung cancer (SCLC) is a fatal disease with limited treatment options. Circulating tumor cells (CTCs) in liquid biopsy samples may serve as predictive and prognostic biomarkers; but the analysis of CTCs is still challenging. By using microfluidic or density gradient CTC enrichment in combination with immunofluorescent (IF) staining or qPCR of CTC-related transcripts, we achieved a 60.8% to 88.0% positivity in SCLC blood samples. Epithelial and neuroendocrine transcripts including the druggable target DLL3 were associated with shorter overall survival (OS), indicating the clinical value of these markers in terms of differential diagnosis and treatment decisions. High CTC counts and the presence of CTC duplets detected by IF staining were prognostic for OS, and thus may serve as indicators of disease progression or therapy failure. In patient samples with high CTC load detected by IF staining, a concordance of the transcripts positivity in circulating free plasma RNA and CTCs was observed. Our data emphasize the role of CTCs and CTC-related transcripts and underline the clinical value of liquid biopsy analysis in SCLC.

Towards effectiveness of cell free DNA based liquid biopsy in head and neck squamous cell carcinoma

Liquid biopsy is a minimally invasive procedure, that uses body fluids sampling to detect and characterize cancer fingerprints. It is of great potential in oncology, however there are challenges associated with the proper handling of liquid biopsy samples that need to be addressed to implement such analysis in patients’ care. Therefore, in this study we performed optimization of pre-analytical conditions and detailed characterization of cfDNA fraction (concentration, length, integrity score) in surgically treated HNSCC patients (n = 152) and healthy volunteers (n = 56). We observed significantly higher cfDNA concentration in patients compared to healthy controls (p < 0.0001) and a time dependent decrease of cfDNA concentration after tumor resection. Our results also revealed a significant increase of cfDNA concentration with age in both, healthy volunteers (p = 0.04) and HNSCC patients (p = 0.000002). Moreover, considering the multitude of HNSCC locations, we showed the lack of difference in cfDNA concentration depending on the anatomical location. Furthermore, we demonstrated a trend toward higher cfDNA length (range 35–10380 and 500–10380 bp) in the group of patients with recurrence during follow-up. In conclusion, our study provide a broad characterization of cfDNA fractions in HNSCC patients and healthy controls. These findings point to several aspects necessary to consider when implementing liquid biopsy in clinical practice including: (I) time required for epithelial regeneration to avoid falsely elevated levels of cfDNA not resulting from active cancer, (II) age-related accumulation of nucleic acids accompanied by less efficient elimination of cfDNA and (III) higher cfDNA length in patients with recurrence during follow-up, reflecting predominance of tumor necrosis.

Genomic features of lung cancer patients in Indonesia’s national cancer center

IntroductionAdvances in molecular biology bring advantages to lung cancer management. Moreover, high-throughput molecular tests are currently useful for revealing genetic variations among lung cancer patients. We investigated the genomics profile of the lung cancer patients at the National Cancer Centre of Indonesia.MethodsA retrospective study enrolled 627 tissue biopsy samples using real time polymerase chain reaction (RT-PCR) and 80 circulating tumour DNA (ctDNA) liquid biopsy samples using next-generation sequencing (NGS) from lung cancer patients admitted to the Dharmais Cancer Hospital from January 2018 to December 2022. Data were obtained from medical records. Data statistically analysed with p < 0.05 is considered significant.ResultThe EGFR test results revealed by RT-PCR were wild type (51.5%), single variant (38.8%), double variant (8.3%), and triple variant (1.4%), with 18.66% L85R, 18.22% Ex19del, and 11.08% L861Q variant. Liquid biopsy ctDNA using NGS showed only 2.5% EGFR wild type, 62.5% single variant and 35% co-variant, with EGFR/TP53 and EGFR/PIK3CA as the highest.ConclusionEGFR variants are the most found in our centre. Liquid biopsy with ctDNA using NGS examination could detect broad variants and co-variants that will influence the treatment planning.

Prognostic and predictive role of liquid biopsy in lung cancer patients.

Lung cancer (LC) is a leading cause of cancer-related mortality worldwide. Approximately 80% of LC cases are of the non-small cell lung cancer (NSCLC) type, and approximately two-thirds of these cases are diagnosed in advanced stages. Only systemic treatment methods can be applied to patients in the advanced stages when there is no chance of surgical treatment. Identification of mutations that cause LC is of vital importance in determining appropriate treatment methods. New noninvasive methods are needed to repeat and monitor these molecular analyses. In this regard, liquid biopsy (LB) is the most promising method. This study aimed to determine the effectiveness of LB in detecting EGFR executive gene mutations that cause LC. One hundred forty-six patients in stages IIIB and IV diagnosed with non-squamous cell non-small cell LC were included. Liquid biopsy was performed as a routine procedure in cases where no mutation was detected in solid tissue or in cases with progression after targeted therapy. Liquid biopsy samples were also obtained for the second time from 10 patients who showed progression under the applied treatment. Mutation analyses were performed using the Cobas® EGFR Test, a real-time PCR test designed to detect mutations in exons 18, 20, and 21 and changes in exon 19 of the EGFR gene. Mutation positivity in paraffin blocks was 21.9%, whereas it was 32.2% in LB. Solids and LB were compatible in 16 patients. Additionally, while no mutation was found in solid tissue in the evaluation of 27 cases, it was detected in LB. It has been observed that new mutations can be detected not only at the time of diagnosis, but also in LB samples taken during the follow-up period, leading to the determination of targeted therapy. The results showed that "liquid biopsy" is a successful and alternative non-invasive method for detecting cancer-causing executive mutations, given the limitations of conventional biopsies.

Microfluidics-based patient-derived disease detection tool for deep learning-assisted precision medicine.

Cancer spatial and temporal heterogeneity fuels resistance to therapies. To realize the routine assessment of cancer prognosis and treatment, we demonstrate the development of an Intelligent Disease Detection Tool (IDDT), a microfluidic-based tumor model integrated with deep learning-assisted algorithmic analysis. IDDT was clinically validated with liquid blood biopsy samples (n = 71) from patients with various types of cancers (e.g., breast, gastric, and lung cancer) and healthy donors, requiring low sample volume (∼200 μl) and a high-throughput 3D tumor culturing system (∼300 tumor clusters). To support automated algorithmic analysis, intelligent decision-making, and precise segmentation, we designed and developed an integrative deep neural network, which includes Mask Region-Based Convolutional Neural Network (Mask R-CNN), vision transformer, and Segment Anything Model (SAM). Our approach significantly reduces the manual labeling time by up to 90% with a high mean Intersection Over Union (mIoU) of 0.902 and immediate results (<2 s per image) for clinical cohort classification. The IDDT can accurately stratify healthy donors (n = 12) and cancer patients (n = 55) within their respective treatment cycle and cancer stage, resulting in high precision (∼99.3%) and high sensitivity (∼98%). We envision that our patient-centric IDDT provides an intelligent, label-free, and cost-effective approach to help clinicians make precise medical decisions and tailor treatment strategies for each patient.