Liquid Biopsy Assay Research Articles

Multi-dimensional cell-free DNA-based liquid biopsy and early detection of gastric cancer.

4060 Background: Gastric cancer is one of the most common cancer types. Most patients were diagnosed at advanced stages and experienced poor prognosis. A non-invasive assay for the detection of early-stage gastric cancer is highly desirable for reducing the gastric cancer-associated mortality. Methods: We prospectively collected a study cohort of 249 participants including 110 pathologically confirmed with stage I-II gastric cancer and 139 with non-cancerous conditions. A plasma sample was collected from each patient before conventional screening modalities for gastric cancer. We performed whole-genome sequencing with plasma samples and profiled four types of cell-free DNA (cfDNA) characteristics, fragment size pattern, copy number variation, nucleosome coverage pattern, and single nucleotide substitution. We used these differential profiles to develop an ensemble model to identify gastric cancer patients from non-cancer individuals. We further collected a validation cohort consisting of 73 gastric cancer patients and 94 non-cancer individuals to validate the cfDNA-based assay. Additionally, we compared the performance of our assay and conventional gastroscopy in a hypothetical 100,000 screening population by Monte Carlo simulation. Results: The liquid biopsy assay that incorporated four types of cfDNA characteristics was able to identify early-stage gastric cancer patients from non-cancer individuals at an AUROC of 0.962 in the study cohort and 0.972 in the validation cohort. At a specificity of 92.1% (128/139), it achieved a sensitivity of 88.2% (97/110) in the study cohort. With this threshold, in the validation cohort, 91.5% (86/94) of healthy individuals and 91.8% (67/73) of gastric cancer patients were correctly identified. In both study and validation cohorts, the inferred probabilities of cancer showed consistent trends to increase with pathological stages in the cancer group and disease statuses in the non-cancer group. Of note, our approach detected all gastric tumors located in the cardia and fundus (100.0%, 19/19), which could be challenging for gastroscopic examination. Additionally, through in-silico simulations, we showed that our cfDNA-based non-invasive assay may detect 96.3% more gastric cancer cases than conventional gastroscopy owing to higher sensitivity and anticipated better participant compliance in a large hypothetical screening population. Conclusions: We introduced a liquid biopsy assay using multiple dimensions of cfDNA characteristics that could accurately identify early-stage gastric cancer from non-cancerous conditions. As a cost-effective non-invasive approach, it may provide population-wide benefits for the early detection of gastric cancer.

Failure rates of mutation-based ctDNA assays in lung cancer treatment monitoring.

e18752 Background: Despite the wide adoption of noninvasive liquid biopsy approaches for cancer genotyping, little is known about the longitudinal performance of these assays for monitoring therapeutic responses in patients with advanced disease. Here, we report results of a pooled analysis that collects existing evidence regarding the failure rates of circulating tumor DNA (ctDNA) assays designed for therapeutic monitoring in patients with advanced or metastatic lung cancer. Methods: We searched PubMed for original studies published between February 2012 and January 2022, following these inclusion criteria: (1) Adults older than 18 years old with advanced or metastatic lung cancer treated with chemotherapy, targeted therapy, or immunotherapy; (2) Tumor-informed or plasma-only circulating tumor DNA assays based on droplet digital PCR (ddPCR) or targeted deep sequencing technologies for the detection of tumor-specific allele fractions; (3) Measurement of circulating tumor DNA levels at sequential timepoints. We defined failures as a lack of detectable mutant allele fraction (MAF) in patients with metastatic lung cancer in samples collected before treatment initiation. Two reviewers independently screened studies, extracted data, assessed the risk of bias, and evaluated the quality of evidence. Divergences were resolved by a third independent reviewer reaching a consensus. Study quality assessment covered: (1) representativeness of patient population, (2) risk of funding bias, (3) description of ctDNA analysis and outcomes, and (4) statistical quality and interpretation. Results: From 142 studies retrieved by an automated PubMed query, we extracted data from 42 studies which fulfilled the eligibility criteria - 11 registered trials, 31 prospective/retrospective cohorts. MAF levels in the circulation were measured using PCR-based assays (23/42, 55%); the remainder used NGS-based approaches (19/42, 45%). The median number of targeted genes was 1 (range 1-6) vs 73 (range 36-214), respectively. The median number of samples analyzed per study was 79 (range 3-334). On average, 31% of samples collected before treatment did not have a detectable MAF in any of the target genes analyzed, despite the high disease burden typically experienced in patients with metastatic lung cancer. Only 57% of PCR-based and 78% NGS-based studies succeeded in providing molecular response assessment metrics. We identified 10/42 studies with critical quality assessment concerns and 18 studies had funding provided by a company with investments in the commercial cfDNA assay. Conclusions: Available liquid biopsy assays that report tumor-specific mutations have a high rate of failure for measuring disease burden and response outcomes during therapy. Approaches that could be reliably used for monitoring patients with advanced cancers undergoing treatment are needed to improve patient care.

Retrospective clinical analysis of circulating tumor DNA (ctDNA)–based molecular response (MR) and baseline blood-based tumor mutational burden (bTMB) for monitoring response in phase 3 trial of bintrafusp alfa vs. pembrolizumab treatment of non-small cell lung cancer (NSCLC).

9098 Background: The clinical utility of ctDNA-based approaches, such as bTMB and MR, to monitor and predict response during chemo-immunotherapy was evaluated using longitudinal cohort samples from a Phase 3 study of bintrafusp alfa versus pembrolizumab as a first-line treatment in patients with PD-L1 expressing advanced NSCLC. Methods: ctDNA was collected at baseline and day 42 after treatment with bintrafusp alfa (Q2W, 1200mg) or pembrolizumab (Q3W, 200mg). The GuardantOMNI liquid biopsy (LBx) assay was used to detect somatic alterations in 497 genes and generate bTMB from baseline, and MR scores from baseline and day 42 (n = 424 samples). bTMB and somatic alterations influencing response were explored. MR scores were calculated using the validated Guardant Response algorithm. Associations between ctDNA metrics and PFS were assessed. Results: 418/424 samples passed sequencing QC; of these, somatic mutations were detected in 236/237 baseline samples and 177/181 day 42 samples. Of 212 NSCLC patients with eligible bTMB results at baseline, 104 (49.1%) patients received bintrafusp alpha and 108 (50.9%) received pembrolizumab. bTMB high (bTMB-H) is defined as >20mut/MB. In the bintrafusp alpha arm, PFS was significantly longer in patients with bTMB-H (median 8.3 months vs 2.7 months, p = 0.00086). In the pembrolizumab arm, PFS was also longer in patients with bTMB-H (median 5.65 months vs 5.5 months, p = 0.0898). Of 132 NSCLC patients with eligible MR results, 67 (50.8%) patients received bintrafusp alpha and 65 (49.2%) patients received pembrolizumab. In the bintrafusp alpha arm, molecular responders had significantly longer PFS (median 7.9 months vs 4.2 months, p = 0.00042). In the pembrolizumab arm, molecular responders also had significantly longer PFS (median 8.95 months vs 4.1 months, p = 0.00054). Conclusions: ctDNA analysis from plasma samples supported MR assessment in patients treated with ICI, indicating its utility as an adjunct to RECIST in monitoring of tumor response. Blood-based TMB-high was associated with immunotherapy treatment benefit. Analysis of b-TMB and MR allowed identification of patients with improved PFS in both treatment arms. Investigation of treatment specific TMB cut off selection and potential clinical utility of defined somatic mutations is ongoing. Clinical trial information: NCT03631706 .

Liquid-biopsy detection of FGFR2 and other actionable rearrangements in GI malignancies.

4085 Background: Actionable rearrangements (RE) represent an emerging therapeutic target for GI malignancies. However, there remains uncertainty whether liquid biopsy (LBx) assays testing circulating tumor DNA (ctDNA) can detect RE with the same fidelity as tissue biopsy. Here we report the performance of ctDNA-based comprehensive genomic profiling (CGP) for the detection of RE leveraging an FDA-approved >300-gene platform, with a focus on data from patients with GI malignancies. Methods: An institutional research database of clinical CGP results for tissue biopsy (TBx, FoundationOneCDx) and LBx (FoundationOneLiquid CDx) from patients with cancers of the GI tract was reviewed. Sensitivity for FGFR2 RE was studied in patients with cholangiocarcinoma (CCA) or carcinoma of unknown primary (CUP) and both TBx and LBx CGP available. Results: Across 7870 GI LBx, 1094 predicted pathogenic RE were detected in 826 cases (10%) including 283 oncogenic kinase RE, 686 inactivating RE, and 125 other gain-of-function RE. FGFR2 was the most frequently rearranged gene, enriched in CCA (4.4%, 37/833) and stomach (3.0%, 11/368). EGFR RE (38) occurred across 6 cancer types and included 8 fusions, 15 c-terminal truncations, 4 intragenic deletions/inversions, and 3 kinase domain duplications. BRAF RE (31) were detected among in colorectal (22) and pancreas (9) cases. LBx detected 44 exon 3 skipping (protein stabilizing) RE in CTNNB1 (beta-catenin) across all samples. Other frequent activating RE were detected in MYC (36), FGFR3, RET, ROS1 (21 each), and ALK (13). Potentially targetable inactivating RE were detected in BRCA1 (28), ARID1A (24), STK11 (22), PTEN (17), TSC2 (14), and BRCA2 (8). Focusing on CCA, FGFR2 RE were more prevalent in cases with elevated (≥1%) ctDNA tumor fraction (TF, 26/342, 7.6%), matching the prevalence in TBx (7.8%, 505/6,492) with a similar spectrum of fusion partners seen for both TBx and LBx (30% BICC1, 70% rare for both). In 13 CCA/CUP patients positive for FGFR2 RE by TBx, LBx detected 12 (92%) including 8 different fusion partners; the false negative case had TF <1%. In 11 CCA LBx samples with FGFR2 resistance mutations detected, LBx CGP successfully detected a driver FGFR2 RE in 10 and driver C382R mutation in 1. Conclusions: Rearrangements are represented across GI malignancies, including many that result in known oncogenic drivers that may be targetable with available therapies. We observe reliable detection of FGFR2 fusions in ctDNA, in contrast to some prior publications. ctDNA represents a pragmatic analyte for detection of rearrangements and other actionable alterations, offering timely result return when tissue is inadequate or unavailable. [Table: see text]

Oncomine Dx Express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation.

e21218 Background: Clinical diagnostics assays for oncology are becoming more readily available due to the advancement and democratization of Next-Generation Sequencing (NGS). Additionally, liquid biopsy can be used in NGS to detect genetic variants in circulating tumor DNA and RNA. Liquid biopsy is a less invasive option compared to traditional biopsy methods for early detection and continuous monitoring of cancer treatment outcomes. Here, we discuss screening of over 3,500 non-small cell lung cancer (NSCLC) samples, and the analytical validation of the Oncomine Dx Express Test (ODxET) and Genexus Dx Integrated Sequencer for detection of clinically significant variants in liquid biopsy samples. Methods: Our team screened over 3,500 NSCLC liquid biopsy samples in search of high priority clinical variants. Variants selected for analytical validation studies included ERBB2 exon 20 insertion, EGFR exon 20 insertion, EGFR exon 19 deletion, EGFR T790M, KRAS G12C, BRAF V600E, and RNA fusion isoforms including ALK, NTRK1/2/3, RET, and ROS1 oncogenic drivers. Over 550 screened samples were found to be positive for these variants of interest. This screening was performed on the Genexus Dx Integrated Sequencer according to the user guide. Results: The Genexus Dx Integrated Sequencer automates library preparation, sequencing, analysis, and reporting QC metrics and variant calls. Sequencing run setup is quick and straightforward, taking less than 15 minutes to start a run and just over 24 hours to go from nucleic acid to report. Limit of detection (LoD) for DNA SNVs, insertions, and deletions at 5 ng DNA input level ranged from 0.65% to 1.82% allelic frequency (AF), depending on the variant. The higher DNA input of 30 ng resulted in a lower LoD range, from 0.31% to 0.42% AF. RNA fusion and splice variant LoD at 5 ng sample input ranged from 9.9 to 19.6 molecular counts. The higher 30 ng input resulted in a lower LoD range for RNA variants as well, ranging from 6.4 to 8.0 molecular counts. In the analytical accuracy study, the false positive rate was found to be 0.2% for SNVs, 0% for insertions/deletions, and 0% for fusion targets. In the analytical reproducibility study, the average with-in run repeatability call rate (No Calls excluded) was 99.64% for DNA variants and 98.75% for RNA variants. Conclusions: Here, we demonstrated that the ODxET workflow on Genexus Dx Integrated Sequencer is a fast and efficient tool for testing clinical NSCLC liquid biopsy samples with high sensitivity and specificity.

Detection of homologous recombination deficiency (HRD) using a novel genomic and epigenomic liquid biopsy assay in patients with breast cancer.

556 Background: Homologous recombination and repair (HRR) deficiency (HRD) is characterized by genomic instability associated with dysfunction in BRCA1/2 or other HRR genes. Patients with breast cancer harboring an HRD phenotype with or without HRR mutations have derived clinical benefit from PARPi therapy. We have previously shown that GuardantINFINITY, a novel genomic and epigenomic liquid biopsy assay, can identify HRR SNVs, indels, rearrangements, copy number loss, reversions, and BRCA1 promoter methylation for patient selection and resistance monitoring, a major challenge for PARPi treatment. Here, we present a method of predicting HRD status by cfDNA using GuardantINFINITY in patients with advanced breast cancer. Methods: We developed a probabilistic genomic model to predict HRD status, inferred from genome-wide somatic SNV, indel, and CNV signatures indicative of BRCA1/2 deficiency, including large-scale state transitions (LST), whole-genome tumor loss of heterozygosity (LOH), telomeric allelic imbalance (TAI). A second probabilistic model, based on targeted measurements of genomic and epigenetic changes was learned from a subset of clinical samples to enhance the genomic model. The model was trained and tested on a cohort of over 12,000 GuardantOMNI and GuardantINFINITY clinical breast cancer samples to assess the sensitivity of accurately detecting deficiency in select HRR-genes ( BRCA1/2, PALB2, RAD51D). The aggregated predictive model was validated on an independent cohort of breast cancer samples. Results: The model based on genomic and epigenetic signals identified HRR-gene deficiency in a breast cancer cohort with a estimated 95% LoD of 22.5% tumor fraction and an AUC of 0.75 across all tumor fractions, an improvement in sensitivity over when only the genomic model is used (AUC of 0.7, 95% LoD of 28%). Specificity remained high for both models (100%, n = 83) in cancer free samples. Application of this model in a cohort of 1,101 patients with unselected breast cancer identified 390 patients with an HRD phenotype, of whom 192 patients had a known HRR-gene mutation. Of the 201 patients with a known pathogenic mutation in an HRR gene, 49 also had co-occurring LoH in the same gene, indicating biallelic loss. Conclusions: In this analysis, we demonstrate that a probabilistic model of genomic and methylation predictors can detect HRD status in patients with breast cancer from cfDNA using GuardantINFINITY. Additional analytical and clinical studies to further evaluate this model are ongoing. With HRD prediction, GuardantINFINITY provides a comprehensive minimally-invasive solution for PARPi and DNA damage treatment selection, longitudinal monitoring, and an exploratory platform for investigating epigenetic signals that may underpin resistance.

Methylated ctDNA as a biomarker for detecting previously undetectable clinically significant somatic mutations.

e15020 Background: Treatment selection liquid biopsies can be re-run upon clinical progression to identify the new clinically significant somatic mutations that might be contributing to resistance to therapy or that can help guide the selection of an alternative therapy. However, there are not currently any biomarkers or tests that suggest an increased likelihood for the detection of new, i.e. previously undetected, clinically significant somatic mutations in a subsequent treatment selection liquid biopsy assay. Methods: We explored whether a methylation-based liquid biopsy could indicate when new clinically significant alterations are more likely to be detected in a treatment selection liquid biopsy. As part of a retrospective study of metastatic lung cancer patients (N = 75 new treatment starts), both a treatment selection assay and a therapy monitoring methylation assay were performed on plasma collected pre-treatment for a subset of patients that underwent at least two separate treatment regimens. By quantifying the number of methylated ctDNA molecules at > 500 genomic locations, the methylation assay assessed the total burden of methylated ctDNA (represented as tumor methylation score), as well as the percent of methylated molecules originating from newly methylated genomic loci, representing a change in the methylation profile. Results: When the methylation assay detected at least a 2x increase in the tumor methylation score, indicating a significant increase in the fraction of ctDNA present in the patient’s blood, new clinically significant somatic mutations (including actionable ERBB2 alterations) were found 83% of the time. This appeared to be more frequent than among patients without an increase in tumor methylation score, where new clinically significant somatic mutations were found 33% of the time. Importantly, when the increase in the methylation score was coupled with a significant change in the methylation profile, new clinically significant mutations were identified in 100% of patients. Conclusions: The preliminary data suggests that an overall increase in methylated ctDNA, in combination with a significant change in methylation profile, is correlated with detecting new, previously undetected clinically significant somatic mutations in a treatment selection assay. Leveraging this methylated ctDNA assay may inform clinicians as to when to run another treatment selection assay, thereby potentially identifying the appearance of clinically significant somatic mutations which could influence clinical decision making and affect patient clinical outcomes.

Enhanced detection of molecular residual disease (MRD) by tumor-informed and tumor-agnostic variants in circulating tumor DNA (ctDNA).

e15586 Background: Liquid biopsies have been mainly used for cancer detection, non-invasive tumor genotyping and disease surveillance. Highly sensitive liquid biopsy assays have been developed that can now be applied to detect and characterize MRD. Several techniques have been developed for the detection of MRD, mainly by tracking ultra-low-frequency somatic tumor mutations in cell-free DNA. Methods: NGS using a 1123-gene-panel were performed on the tumor tissue and white blood cells which was designed for pan-cancer covering 1.8Mbp human genome. Moreover, we used a self-designed pan-cancer panel covering 208 kb and increasing 20 or less mutations individually known to be present in the surgical tissue based on the fixed panel for postoperative plasma or follow-up plasma. In Our study, plasma were collected from 137 healthy donors and UMI sequencing with 208 kb panel for built an database of cfDNA background noise. Furthermore, we constructed two different databases of CH(clonal hematopoietic) variants and negative-background noise with 2300+ cancer patients and 1000+ tumor-plasma paired data respectively. In this way, this approach not only could track selected tumor-specific mutations, but also provided with new variants using tumor-agnostic. MRD-positive status was defined as at least one variants in the plasma. Results: To test sensitivity and specificity of the minimal residual disease detection (MRD) method, we used 208kb panel data for plasma samples from 137 healthy samples and 150 samples with CRC untreatment. The result showed an area under the curve (AUC) of 0.95, with sensitivity of 92% and specificity of 98%. To test the limit of detection of 208 kb panel, we sequenced six different dilution levels (5%, 1%, 0.5%, 0.1%, 0.05% and 0.01%) with a positive control sample containing 98 known mutations. The result showed the detection limitation of 208 kb panel achieved 0.02%. We retrospectively analyzed 3000+ patients with colorectal cancer, 35 of whom met the eligibility criteria were enrolled in this study: 1) With stage II-III CRC undergoing radical surgery; 2)Blood specimens were collected on postoperative 7 days to 3 months; 3)Follow-up duration was 2 years or relapsed; 4)Enough samples for targeting sequencing. Twelve (34%) patients with MRD+,which all patients recurred while 23(66%) patients were MRD-, which 22/23 patient did not experience local recurrence or metastasis. The Kaplan-Meier estimates showed that the positivity of ctDNA was of high predictive value for relapse (HR 21, 95%CI 18.3-28.9;P < 0.001). Conclusions: In this study, combination of tumor-informed and tumor-agnostic strategies can effectively distinguish healthy and cancer patients. Meanwhile,our study highlights that postoperative ctDNA status can effectively detect MRD and identify patients with a high risk of relapse.

Longitudinal tracking of ALK-positive lung cancer from plasma using circulating-tumor RNA and circulating-tumor DNA in the BRIGHTSTAR clinical trial.

e21072 Background: While the administration of specific tyrosine-kinase inhibitors (TKIs) in ALK-fusion positive lung cancer has led to significant improvement in clinical outcomes, detection of gene fusions remains challenging, especially from liquid biopsies. In tissue biopsies, assays which incorporate RNA-based detection have demonstrated increased sensitivity for gene fusion detection. We therefore hypothesized that a liquid biopsy assay equally including assessment of gene fusions using circulating-tumor RNA (ctRNA) in addition to circulating-tumor DNA (ctDNA), will improve detection. Furthermore, we hypothesized that detection of gene fusions as well as mutations will also correlate with clinical treatment. Methods: We retrospectively analyzed 89 samples from 35 patients included in the BRIGHTSTAR clinical trial assessing local consolidative therapy (LCT) and brigatinib in patients with stage IV or recurrent Non-small Cell Lung Cancer and confirmed ALK rearrangement (NCT03707938). Samples were included at baseline (N = 31), prior to LCT after 8 weeks of brigatinib treatment (N = 29), after LCT ( < 3 weeks; N = 25) and at progression (N = 4). We used a targeted next generation sequencing (NGS) assay assessing both ctDNA as well as ctRNA (LiquidHALLMARK, Lucence Health, Palo Alto) to detect ALK fusions as well as mutations in 80 genes. Up to 5 ml of plasma was analyzed per sample. Results: At baseline, ALK fusions were detected in 15/31 patients (48%) of which 8 were detected using both ctDNA and ctRNA, while four were exclusively detected in ctDNA and three in ctRNA. Plasma ctDNA concentrations for patients with detectable ALK fusions at baseline were significantly higher than for those without detectable gene fusions (mean 26.1 ng/mL versus 16.6 ng/mL; p = 0.0044). ALK fusions were detected in two patients pre-LCT, exclusively in ctRNA, while ALK fusions cleared completely post-LCT. At progression, ALK fusions were detected in 2/4 samples (50%) in both ctDNA and ctRNA. Of the two negative samples tissue biopsies were available confirming the absence of an ALK rearrangement, including one transformation to squamous cell carcinoma. Overall, including analysis of ctRNA led to a 36% relative additional detection of ALK fusions compared to analyzing ctDNA alone. Furthermore, ctDNA mutation positivity in genes other than ALK was 17/31 (55%), 9/29 (31%), 8/25 (32%) and 3/4 (75%) for baseline, pre-LCT, post-LCT and progression, respectively. Conclusions: We highlight that ALK fusions can be reliably detected in plasma of lung cancer patients and detectability of ALK fusions correlate with exposure to treatment. Analyzing ctRNA in addition to ctDNA improves sensitivity of fusion detection and is a highly promising strategy in this setting.

Consensus molecular subtypes and orphan non-coding RNAs in colorectal cancer.

e15651 Background: Colorectal cancer (CRC) is a highly heterogeneous disease with variable response to different treatment strategies. The consensus molecular subtype (CMS) classification system has been proposed as a gene expression-based framework aimed at capturing the genetic and molecular heterogeneity in CRCs and potentially stratifying patients into clinically relevant subgroups for treatment selection. We previously demonstrated that orphan non-coding RNAs (oncRNAs), a novel class of cancer enriched small non-coding RNAs (smRNA), exhibit cancer-specific expression. We also showed that oncRNAs can be used in a liquid biopsy assay for accurate early-stage detection of CRCs. We hypothesize that oncRNAs may be informative of CRC subtype-specific transcriptional signatures as defined by the CMS groups. Methods: To assess the effectiveness of oncRNAs for categorization of CMS group, we examined smRNA-seq data from The Cancer Genome Atlas (TCGA) for colon and rectal adenocarcinoma tumors ( n= 504), consisting of 77 CMS1, 216 CMS2, 71 CMS3, and 140 CMS4 tumors. CRC-specific oncRNA expression profiles were generated for all tumors in the TCGA cohort. We then trained a machine learning model on oncRNA expression profiles to predict CMS groups using a 5-fold cross validation setup. For our validation cohort, we applied our model to an independent CRC cohort ( n= 348) consisting of 56 CMS1, 101 CMS2, 59 CMS3, and 132 CMS4 CRC samples. Final CMS predictions for the validation cohort were made by averaging across the five training models. Results: Within TCGA testing folds, the model micro-averaged ROC-AUC for CMS classifications were 0.879 (95% CI: 0.86–0.90). In the validation cohort, micro-averaged AUC remained high at 0.915 (0.90–0.93). Sensitivities and AUCs for each subtype are provided in Table 1. The highest model-predicted likelihood for each CMS subtype had an accuracy of 73.6% (68.6%–78.1%), and the two most probable predictions had an accuracy of 91.1% (87.6%–93.9%). Conclusions: This study demonstrates the utility of oncRNA expression profiling to predict CMS for colorectal cancer. The generalizability of our machine learning model suggests that oncRNAs are informative of the biological differences across distinct CMS groups and may be used as a proxy for gene expression signatures to stratify colorectal cancers. Because smRNAs are more likely to be secreted into blood, we posit that an oncRNA-based liquid biopsy may open new opportunities to monitor patients and track changes in their tumor biology in real-time throughout their course of disease and treatment. [Table: see text]

Multimodal detection in plasma of molecular residual disease (MRD) in locally advanced head and neck squamous cell carcinoma (LA-HNSCC).

6065 Background: Despite intensive therapy, 30% of patients (pts) with LA-HNSCC relapse. Published data shows that MRD detection during follow up (FU) may predict relapse, e.g. human papillomavirus (HPV) DNA in p16+ pts post radiation (RT) or chemoradiation (CRT); or circulating tumor DNA (ctDNA) post surgery. MRD detection using multiple assays after definitive RT/CRT has not been reported. Methods: We enrolled pts with high risk LA-HNSCC (stage III HPV+, III-IVB HPV-) treated with curative intent: surgery ± adjuvant therapy, RT or CRT. Plasma was collected pre-treatment at baseline (B), at 4-6 weeks (FU1) and 8-12 weeks (FU2) post-treatment. RaDaR, a personalized liquid biopsy assay that targets patient specific somatic variants identified in whole exome sequencing (WES) from matched tumor tissue, was used to detect ctDNA, reported as estimated Variant Allele Fraction (eVAF). Cancer Personalized Profiling by deep sequencing (CAPP-seq) was used as a tumor naïve assay for ctDNA, reported as mean VAF. HPV sequencing (HPV-seq) in all and digital PCR (dPCR) in p16+ pts were used to detect HPV DNA. Relapse free survival (RFS) was estimated using Kaplan Meier and associations of ctDNA or HPV DNA with RFS using log-rank test. Assays were compared with Spearman correlation. Results: A total of 89 plasma samples from 35 pts were collected prospectively; 32 pts with at least a FU sample were evaluable: 26 had both. Median age was 63 years (34-71). Most pts were stage III HPV + (N = 16, 50%) and received CRT (N = 25, 78%). No pts had clinical or radiological residual disease at FU2. Median FU was 18.3 months (5.1-25.9), there were 7 clinical relapses. RaDar was applied in 17 pts with WES in matched tissue, including 6 with relapse. ctDNA at B was detected in 15/17 (88%). eVAF at B was not associated with RFS (p = 0.98). Two pts relapsed < 1 year after RT/CRT and had eVAF > 0.001% in FU2 sample; lead time to relapse was 100 and 245 days. FU1 sample of a pt who relapsed > 1 year post-CRT was close to the threshold for ctDNA+ with eVAF 0.0001%; lead time was 494 days. Three pts who received CRT relapsed > 500 days from FU2 sample (ctDNA-). CAPP-seq was applied to 29 pts; mean VAF correlated with eVAF at B (r = 0.75) but not at FU. ctDNA+ at FU using CAPP-seq was not associated with RFS (p = 0.25). HPV-seq was applied to 32 pts; specificity and sensitivity was 100% at B. Among p16+ pts with FU sample (N = 15), HPV DNA was detected in 4/4 with relapse and not in those without, predicting shorter RFS (p < 0.01). dPCR detected HPV DNA in 2/4 pts with relapse. HPV-seq and dPCR correlation was high at B (r = 0.99), lower at FU2 (r = 0.66). Conclusions: HPV DNA and ctDNA can be detected in LA-HNSCC before and after definitive therapy. RaDaR but not CAPP-seq may detect MRD in pts who relapse within 1 year after RT/CRT with a significant lead time. HPV-seq may be more sensitive than dPCR to detect HPV DNA in MRD. Validation in an interception study is planned (NCT 04599309 ).

Detecting early stage breast cancer using low-depth cell-free DNA fragmentomics: A multi-center cohort study.

3075 Background: Breast cancer (BC) has contributed to the most cancer-related mortalities among females in 2020. Early detection using breast imaging methods, such as mammography and ultrasound, has significantly improved patients' survival. However, these early-screening methods suffer from high false-positive rates, resulting in many unnecessary biopsies which add to patients' discomfort. Cell-free DNA (cfDNA) fragmentomics assays have recently illustrated prominent abilities for detecting various cancer. In this multi-center prospective cohort study, we aim to develop a non-invasive liquid biopsy assay utilizing cfDNA fragmentomics and machine learning for detecting early-stage BC patients. Methods: We recruited 402 female patients (234 with BC and 168 with benign nodules [BN]) from the Yantai Yuhuangding Hospital (training cohort, N = 193, 91 BC and 102 BN) and the Cancer Hospital of the Chinese Academy of Medical Sciences (independent validation cohort, N = 209, 143 BC and 66 BN). Surgical or core needle biopsies were performed for all 402 patients following positive breast imaging results by mammography and ultrasound. Each patient's malignant or benign status was then pathologically confirmed using the tissue specimen. Women with negative biopsy results were excluded from breast cancer through a 6-month follow-up. The fragmentomics profiles, which were generated using plasma cfDNA WGS data (~5X), were used by our machine-learning algorithm. An automated machine learning approach was deployed to optimize base models before being ensembled into the final predictive model. Results: Our predictive model showed excellent performance in discriminating BCs from BNs, reaching an Area Under the Curve (AUC) of 0.816 and 0.811 in the training cohort (5-fold cross-validation) and the independent validation cohort, respectively. It achieved 74.1% sensitivity (95% CI: 66.1-81.1%) at 81.8% specificity (95% CI: 70.4-90.2%) in the validation cohort. In a potential clinical setting, our model could detect 53.0% (95% CI: 40.3-65.4%) false positives from the breast imaging methods at a targeted 90% sensitivity (89.5%, 95% CI:83.3–94.0%), which could theistically reduce more than half of the unnecessary biopsies. Moreover, our model illustrated great predictive power (AUC = 0.830) among small nodules (≤ 1cm), which were difficult to distinguish by the traditional imaging methods (AUC = 0.690). Finally, our model maintained its predictive power during a down-sampling process, even using 2X WGS data in the independent validation cohort (mean AUC: 0.789, 0.781-0.798). Conclusions: Our non-invasive liquid biopsy assay, which utilized low-depth cfDNA fragmentomics profiling and machine learning, can distinguish malignant nodules from benign nodules. The highlight of our model is its ability to prevent unnecessary biopsies among patients with false positive breast imaging results.

Validation of a 56 gene panel for the detection of somatic variants in metastatic breast cancer.

e13131 Background: The use of ctDNA and next generation sequencing has unprecedented potential to improve genomic profiling and predicting actionable drug treatment in patients with metastatic breast cancer (MBC). Liquid biopsy is uniquely suited for patients where tissue biopsy is not feasible. We specifically focus on actionable variants found in liquid biopsy for MBC including bTMB and MSI. Here we report on the validation of a 56 gene sub panel for the determination of somatic mutations in MBC. Methods: Commercially available, well-characterized, biological ctDNA reference samples with allele frequency ranging from 0.5 -2.5% and bTMB scores (7 – 20) were obtained to analytically evaluate sensitivity, specificity and precision of a range of clinically significant variants (SNV, indels, fusions and copy number variants) in the TSO-500 ctDNA NGS assay. Additionally, 48 Whole blood samples from well characterized metastatic breast cancer patients and 13 healthy donors were obtained from commercial sources. cfDNA was extracted from the plasma separated from whole blood and analyzed on both TS0-500 ctDNA assay and PGDx Elio Complete. TSO-500 ctDNA sequencing data was further analyzed with the DRAGEN pipeline for variant calling and all variants were annotated and curated with the Clinical Genomic Workspace from PierianDx. PGDx utilized a proprietary pipeline and reporting of all variants detected. Results: Analytical validation determined excellent sensitivity (PPA: 99.4 – 100%), specificity (NPA: 99.6 -99.8%) and reproducibility (95 – 98.6%) for SNV, indels, fusions and Copy number variants. bTMB, with a cutoff for bTMB high as >20 mut/Mb, exhibited a sensitivity of 75% and specificity of 93.3%. Limit of detection (LOD) was established at 0.5 % VAF for SNV, Indels and fusions at a minimum read depth of 1000x. LOD for Copy number amplifications was established at 1.26-fold change or 3 copies. Clinical samples also exhibited excellent concordance across both TS0-500 ctDNA and PGDx Elo Complete. SNV, indels and copy number variants exhibited 100% (PPA) sensitivity and 99-100% Specificity (NPA). bTMB exhibited 87.5 % sensitivity and 96.22% specificity. As expected, fusions and microsatellite instability were observed in the clinical population but were rare. No reportable mutations were found in the healthy donors. Actionable mutations were reported for PIK3CA, ESR1, BRCA1 and BRCA2, TP53 and ERBB2 variants. Rare mutations in ATM, CHEC2, PALB2, FGFR1 and CCND1 were also observed. Conclusions: Clinical utility of liquid biopsy assays requires excellent sensitivity, specificity and precision. The Epic 56 gene panel has established clinical validity and high sensitivity and specificity across all relevant variants including bTMB in MBC. This clinically available NGS panel for metastatic breast cancer allows for routine genomic profiling, specifically in patients where tissue biopsy cannot be obtained.

Extracellular vesicle-derived non-coding RNAs to predict outcome in patients with triple-negative breast cancer (TNBC) with residual disease (RD).

578 Background: TNBC patients with RD after neoadjuvant systemic therapy (NAST) have high risk of recurrence. Biomarkers to risk-stratify patients with RD could individualize adjuvant therapy and inform adjuvant therapy trials. We aim to investigate the impact of circulating extracellular vesicle (EV)-derived non-coding RNAs (exo-ncRNAs) on outcomes in TNBC patients with RD. Methods: The study population included 79 TNBC patients with RD post-NAST and available end-of- treatment plasma samples enrolled in an IRB-approved multisite prospective registry. EVs and their associated exo-ncRNAs were isolated by membrane affinity spin columns (Qiagen exoRNeasy). Exo-ncRNA was subjected to next-generation sequencing (Qiagen QIAseq miRNA library kit). N=47 served as a discovery cohort and N=32 served as validation cohort. With inclusion of transcripts expressed in ≥ 80% of samples, there were 1,123 normalized reads/sample. Hazard ratios and C-statistics for event-free survival (EFS) were computed for each exo-ncRNA. We report exo-ncRNAs that were significantly associated with EFS in both the discovery and validation cohorts. Results: Patient and tumor characteristics were balanced in discovery and validation cohorts. Three exo-ncRNAs were associated with increased recurrence risk in both the discovery dataset ( miR-200a-3p, HR=1.39, 95%CI 1.01-1.89, P=0.04, C-stat=0.55; miR-203a-3p, HR=1.77, 95%CI 1.15-2.73, P=0.01, C-stat=0.59; and miR-7845-5p, HR=1.53, 95%CI 1.15-2.05, P=0.004, C-stat=0.62) and the validation dataset ( miR-200a-3p, HR=1.83, 95%CI 1.24-2.72, P=0.003, C-stat=0.76; miR-203a-3p, HR=1.78, 95%CI 1.10-2.87, P=0.02, C-stat=0.67; and miR-7845-5p, HR=2.06, 95%CI 1.06-4.01, P=0.03, C-stat=0.52). Using the miRNA Target Prediction Database (miRDB), we identified 1,088, 1,352, and 387 predicted targets for miR-200a-3p, miR-203a-3p, and miR-7845-5p, respectively. Amongst 2,827 prediction events there were 2,526 unique target mRNAs. 2,235 mRNAs were targets for one candidate miRNAs, 281 mRNAs were predicted targets for two candidate miRNAs, and 10 mRNAs were predicted as targets for all three candidate miRNAs. Conclusions: The expression of miR-200a-3p, miR-203a-3p, and miR-7845-5p in plasma-derived EVs in TNBC patients with RD after NAST is associated with increased risk of recurrence. We identified ten mRNAs that are predicted targets of all three of these miRNAs. If validated in additional cohorts these exo-ncRNAs could be used in a liquid-biopsy assay to identify high-risk patients with RD who could benefit from adjuvant treatment intensification. [Table: see text]

Multimodal analysis of genome-wide methylation, copy number aberrations, and end motif signatures enhances detection of early-stage breast cancer.

Breast cancer causes the most cancer-related death in women and is the costliest cancer in the US regarding medical service and prescription drug expenses. Breast cancer screening is recommended by health authorities in the US, but current screening efforts are often compromised by high false positive rates. Liquid biopsy based on circulating tumor DNA (ctDNA) has emerged as a potential approach to screen for cancer. However, the detection of breast cancer, particularly in early stages, is challenging due to the low amount of ctDNA and heterogeneity of molecular subtypes. Here, we employed a multimodal approach, namely Screen for the Presence of Tumor by DNA Methylation and Size (SPOT-MAS), to simultaneously analyze multiple signatures of cell free DNA (cfDNA) in plasma samples of 239 nonmetastatic breast cancer patients and 278 healthy subjects. We identified distinct profiles of genome-wide methylation changes (GWM), copy number alterations (CNA), and 4-nucleotide oligomer (4-mer) end motifs (EM) in cfDNA of breast cancer patients. We further used all three signatures to construct a multi-featured machine learning model and showed that the combination model outperformed base models built from individual features, achieving an AUC of 0.91 (95% CI: 0.87-0.95), a sensitivity of 65% at 96% specificity. Our findings showed that a multimodal liquid biopsy assay based on analysis of cfDNA methylation, CNA and EM could enhance the accuracy for the detection of early- stage breast cancer.

Validation of a microRNA liquid biopsy assay for diagnosis and risk stratification of invasive cutaneous melanoma.

Noninvasive molecular biomarkers are needed to improve the early, accurate and precise diagnosis of invasive cutaneous melanoma. To independently validate a previously identified circulating microRNA signature of melanoma (MEL38), and, secondly, to develop a complementary microRNA signature, optimized for prognostication. MicroRNA expression profiling was performed on plasma samples from a multicentre observational case-control study, involving patients with primary or metastatic melanoma, melanoma in situ, nonmelanoma skin cancer, or benign naevi. MicroRNA profiles from patients with length of survival, treatment and sentinel lymph node biopsy (SLNB) data were used to develop the prognostic signature. The primary outcome of interest for MEL38 was its association with melanoma status, including area under the curve, binary diagnostic sensitivity and specificity, and incidence-adjusted positive and negative predictive values. The prognostic signature was assessed using rates of survival per risk group and relationship to conventional predictors of outcome. Circulating microRNA profiles of 372 patients with invasive melanoma and 210 control individuals were generated. The average age of all participants was 59 years; 49% were male. A MEL38 score > 5.5 indicated the presence of invasive melanoma. Overall, 551/582 (95%) of patients were correctly diagnosed, with 93% sensitivity and 98% specificity. MEL38 score ranged from 0 to 10 with an area under the curve of 0.98 (95% confidence interval 0.97-0.99, P < 0.001). A novel prognostic 12-microRNA signature (MEL12) developed from 232 patients identified low-, standard- or high-risk groups, with 94%, 78% and 58% rates of 10-year melanoma-specificsurvival, respectively (log-rank P < 0.001). MEL12 prognostic risk groups were significantly associated with clinical staging (χ2, P < 0.001) and SLNB status (P = 0.027). Patients who were classified as high risk by MEL12 were approximately three times more likely to have melanoma detected in their sentinel lymph nodes compared to low-risk patients. The circulating MEL38 signature may assist in diagnosing patients with invasive melanoma vs. other conditions associated with a lower - or negligible - risk of mortality. A complementary and prognostic MEL12 signature is predictive of SLNB status, clinical stage and probability of survival. Plasma microRNA profiling may help to optimize existing diagnostic pathways as well as enable personalized, risk-informed melanoma treatment decisions.

Simultaneous Detection of Tumor Derived Exosomal Protein-MicroRNA Pairs with an Exo-PROS Biosensor for Cancer Diagnosis.

Tumor derived exosomes (TEXs) have emerged as promising biomarkers for cancer liquid biopsy. Conventional methods (such as ELISA and qRT-PCR) and emerging biosensing technologies mainly detect a single type of exosomal biomarker due to the distinct properties of different biomolecules. Sensitive detection of two different types of TEX biomarkers, i.e., protein and microRNA combined biomarkers, may greatly improve cancer diagnostic accuracy. We developed an exosome protein microRNA one-stop (Exo-PROS) biosensor that not only selectively captured TEXs but also enabled in situ, simultaneous detection of TEX protein-microRNA pairs via a surface plasmon resonance mechanism. Exo-PROS assay is a fast, reliable, low sample consumption, and user-friendly test. With a total of 175 cancer patients and normal controls, we demonstrated that TEX protein-microRNA pairs measured by Exo-PROS assay detected lung cancer and breast cancer with 99% and 96% accuracy, respectively. Exo-PROS assay also showed superior diagnostic performance to conventional ELISA and qRT-PCR methods. Our results demonstrated that Exo-PROS assay is a potent liquid biopsy assay for cancer diagnosis.

Liquid Biopsy Assay for Accurate Detection of Pediatric Solid Tumors

Liquid Biopsy Assay for Accurate Detection of Pediatric Solid Tumors

Electrochemical Cellulase-Linked ELASA for Rapid Liquid Biopsy Testing of Serum HER-2/neu.

Non-invasive liquid biopsy assays for blood-circulating biomarkers of cancer allow both its early diagnosis and treatment monitoring. Here, we assessed serum levels of protein HER-2/neu, overexpressed in a number of aggressive cancers, by the cellulase-linked sandwich bioassay on magnetic beads. Instead of traditional antibodies we used inexpensive reporter and capture aptamer sequences, transforming the enzyme-linked immuno-sorbent assay (ELISA) into an enzyme-linked aptamer-sorbent assay (ELASA). The reporter aptamer was conjugated to cellulase, whose digestion of nitrocellulose film electrodes resulted in the electrochemical signal change. ELASA, optimized relative aptamer lengths (dimer vs monomer and trimer), and assay steps allowed 0.1 fM detection of HER-2/neu in the 10% human serum in 1.3 h. Urokinase plasminogen activator and thrombin as well as human serum albumin did not interfere, and liquid biopsy analysis of serum HER-2/neu was similarly robust but 4 times faster and 300 times cheaper than both electrochemical and optical ELISA. Simplicity and low cost of cellulase-linked ELASA makes it a perspective diagnostic tool for fast and accurate liquid biopsy detection of HER-2/neu and of other proteins for which aptamers are available.

Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer.

There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography(CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.