Abstract Introduction: In patients with breast cancer receiving neoadjuvant therapy, treatment decisions are primarily guided by predefined imaging and clinical assessments. Circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) testing offers a real-time measure of response to help guide escalation and de-escalation strategies. Prior studies have evaluated binary MRD status at limited timepoints, providing low resolution and minimal quantitative insight. The MONITOR-Breast study uses an ultrasensitive whole-genome sequencing (WGS)-based assay with frequent ctDNA sampling across the neoadjuvant period. Here, we report results from this multi-center prospective study evaluating the association between post-treatment ctDNA status and pathologic complete response (pCR), providing quantitative, high-resolution data on ctDNA dynamics. Methods: Patients with stage I-III breast cancer planning to receive neoadjuvant chemotherapy were prospectively enrolled. WGS of core needle biopsy tissue was used to design personalized panels with up to 1,000 variants (Precise MRD, Myriad Genetics). Plasma was collected pre-treatment, at each cycle, after therapy, at surgery, and at post-op follow-up. ctDNA trajectories were evaluated by subtype and treatment relative to pCR or residual disease (RD). At the time of analysis, tumor and normal samples from 108 patients completed WGS and panel building; personalized panels were created for 99 (91.6%), and 91 had baseline plasma available. Results: MRD results were analyzed for 91 patients and 910 plasma samples (median=10). Subtypes were HR+/HER2- (n=24), HR+/HER2+ (n=22), HR-/HER2+ (n=9), and TNBC (n=36). Stages were I (n=24), II (n=49), and III (n=18). Treatments included chemotherapy (n=91), HER2-targeted therapy (n=31), and immunotherapy (n=38). Median follow-up was 17 months, and 84 cases had pathologic response data available. Baseline ctDNA was detected in 84/91 (92.3%) patients: 66 (78.6%) >100 parts per million (PPM), 12 (14.3%) 20-100 PPM, and 6 (7.1%) <20 PPM. ctDNA declined rapidly during therapy; by 50 days, 58% were ctDNA-, increasing to 85% by surgery. HER2+ and TNBC tumors showed faster clearance than HR+ disease. ctDNA status was significantly associated with pathological response (p < 0.0001); ctDNA was negative in all patients with pCR (n=43) (100% specificity), while among those with RD (n=41), 28 were ctDNA- and 13 were ctDNA+. PPV was 100%. Conclusions: Ultrasensitive, personalized ctDNA monitoring enables high-resolution tracking of neoadjuvant response. ctDNA clearance following neoadjuvant therapy was strongly associated with pCR, supporting the clinical validity of ultrasensitive MRD for assessing neoadjuvant treatment response and the feasibility of integrating MRD into neoadjuvant management. Citation Format: Julia Foldi, Greg Hogan, Matt LaBella, Brent Mabey, Marija Balic, Dale Muzzey, Katie Johansen Taber, Jeff S. Jasper. Early findings from MONITOR-Breast: ctDNA dynamics during neoadjuvant therapy using an ultrasensitive MRD assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT171.

187 Background: Circulating tumor DNA (ctDNA) has been established as a strong predictive biomarker for post-surgical molecular residual disease (MRD) detection. For colorectal cancer (CRC), several promising ctDNA-based MRD assays are under global development, with validation studies underway. The MONSTAR-SCREEN-3 study aims to evaluate an ultra-sensitive whole-genome sequencing (WGS)-based MRD assay across cancer types, including a dedicated CRC cohort. Methods: MONSTAR-SCREEN-3 (N=3,200) is a prospective multicenter study, including the “Definitive cohort” enrolling 1,100 patients with solid tumors undergoing definitive therapy. The study utilizes Precise MRD (Myriad Genetics) to detect ctDNA, incorporating up to 1,000 tumor-specific alterations identified through WGS of tumor tissue. Longitudinal plasma samples were collected before and after definitive therapy with a planned 2-year follow-up. For CRC, patients with cStage III–IV disease undergoing curative surgery were eligible. Results: As of the end of August, 2025, 184 CRC patients were enrolled, of whom 95 underwent WGS analysis for tissue samples, and 86 had MRD data 1-month post-surgery. Primary tumors were most frequently located in the sigmoid colon (32%) and ascending colon (17%). Pathologic stage I/II/III/IV disease accounted for 9%/42%/47%/1%, respectively; 60% received adjuvant chemotherapy. Panel design was successful in 99% (94/95) of patients with tissue WGS data available, with one case failing due to insufficient panel size. Customized panels had at least 954 variants (range: 954-1,000, 72% of patients had the maximum of 1,000). ctDNA positivity was 100% (94/94) at baseline and 20% (17/86) one month after surgery. Importantly, ultra-sensitive detection (tumor fraction <100 parts per million) was observed in 6% (6/94) at baseline and 41% (7/17) of MRD-positive patients postoperatively. Six patients developed recurrence, all of whom were MRD-positive at 1-month post-surgery, with a median lead time of 3.2 months (range: 0.0-6.0), highlighting the potential clinical impact of earlier detection. Meanwhile, no recurrence occurred among MRD-negative patients. Conclusions: A WGS-based MRD assay successfully identified future recurrence and shows potential for recurrence prediction. These findings support the potential clinical utility of this assay, though longer follow-up is needed to fully establish its role in colorectal cancer management.

3044 Background: While circulating tumor DNA (ctDNA) demonstrates promise as a molecular residual disease (MRD) biomarker, its clinical implementation has been primarily limited to tumors with favorable ctDNA shedding characteristics. The MONSTAR-SCREEN-3 evaluates a whole-genome sequencing (WGS)-based MRD assay to assess MRD positivity at ultra-sensitive level beyond conventional WES-based MRD, including traditionally low-shedding tumors. Methods: MONSTAR-SCREEN-3 is a prospective multicenter study targeting 1,100 patients with solid tumors undergoing curative-intent treatment. Personalized panels were constructed using Precise MRD (Myriad Genetics), incorporating up to 1,000 tumor-specific alterations identified through WGS of matched tumor tissue. Serial plasma samples were collected at baseline, post-neoadjuvant treatment (NAT) (when applicable), 1-month (1M) post-surgery, every 3 months in year 1, and every 6 months thereafter up to 2 years. Assay performance was evaluated across multiple cancer types for ctDNA detection and recurrence monitoring. Results: Between May 2024 and November 2025, 1,088 patients across over 20 cancer types were enrolled, including colorectal (n=250), breast (n=156), cervical (n=95), gastric (n=88), and pancreatic (n=69) cancers. Treatment strategies included upfront surgery (n=704), NAT (n=296), and definitive chemoradiotherapy (n=96). Median follow-up was 5.3 months (range, 0–18.1). Baseline ctDNA detection was achieved in 96.2% (684/711), with 16.4% at ultra-sensitive levels (tumor fraction <100 parts per million [ppm]). Post-operative MRD positivity was 26.4% (163/617) at 1M and 23.8% (120/504) at 3M, with 46.0% and 41.7% detected at ultra-sensitive levels, respectively. Among 91 patients who received NAT and underwent pathological assessment, post-NAT MRD status demonstrated a sensitivity of 74.3% (55/74) and a specificity of 100% (17/17) for predicting pathological complete response (P<0.01). Among patients with available survival data, post-1M MRD positivity was associated with significantly worse disease-free survival (DFS) compared with MRD negativity (HR, 16.9; 95% CI, 8.2–34.8; P < 0.001). Furthermore, post-1M MRD positivity at levels below 100 ppm was associated with significantly inferior DFS compared with MRD negativity (HR, 8.2; 95% CI, 3.4–19.4; P < 0.001), whereas post-1M MRD positivity at levels ≥100 ppm was associated with significantly worse DFS compared with MRD positivity below 100 ppm (HR, 3.4; 95% CI, 1.7–6.7; P < 0.001). Conclusions: The WGS-based MRD assay demonstrated high baseline detection sensitivity and robust ultra-sensitive detection across diverse cancer types. Our findings show that post-1M MRD positivity below 100 ppm at ultra-sensitive level is prognostic for recurrence risk. Updated molecular and clinical outcome data will be presented. Clinical trial information: UMIN000053975.

AIMS:Active surveillance (AS) allows selected men with localized prostate cancer to defer curative therapy and reduce treatment morbidity. Conversion from AS to treatment is commonly triggered by Gleason grade group (GGG) upgrading on confirmatory biopsy. We developed and validated a digital pathology artificial intelligence (DPAI)-derived risk score to predict GGG upgrading in AS-eligible patients. MATERIALS AND METHODS:The DPAI model was trained using histopathology image features from diagnostic biopsies of 998 patients and validated in an independent cohort of 296 patients meeting criteria for AS. Logistic regression estimated the probability of confirmatory-biopsy GGG increase, and feature selection identified the most predictive variables. RESULTS:AI-GUR (Artificial Intelligence-Gleason Upgrade Risk) predicted GGG reclassification at confirmatory biopsy (OR 1.60; p = 0.0003) and provided information beyond conventional stratification (risk group, CAPRA) and cribriform morphology (all p < 0.01). Predicted risks were similar across time from diagnosis (~10-15% to ~85% at 1, 1.5, or 2 years; p for time = 0.50), consistent with initial biopsy mischaracterization rather than time-dependent progression. CONCLUSIONS:AI-GUR provides individualized estimates of confirmatory-biopsy GGG upgrading for AS candidates. Using DPAI may improve shared decision-making by complementing standard clinicopathologic tools and molecular testing using the same biopsy specimen, while informing the likelihood of grade upgrade at confirmation.

BACKGROUND:The provision of prenatal genetic screening in 3 core clinical contexts-recessive conditions, aneuploidy, and serological incompatibility-involves multiple tests across reproductive partners. This complexity limits utilization and impairs adherence to guideline-recommended care, particularly in carrier screening, where male partners are frequently not tested when a female carrier is identified. Here, we describe the analytical validation of a fetomaternal integrated recessive, serological, and trisomy genetic screen (FIRSTGENE), a single assay that harnesses in silico fragment-length trajectory analysis to evaluate all 3 contexts simultaneously, identifying clinically relevant variants in both the mother and the fetus without requiring a paternal sample. METHODS:FIRSTGENE screens singleton pregnancies for mutations in 20 recessive genes; RhD compatibility; aneuploidies in chromosomes 13, 18, 21, X, and Y; and 22q11.2 microdeletion. Each part of the test was individually validated using a relevant subset of plasma samples from a curated collection (478 total samples from 456 patients) and 93 cell-line mixtures digested to resemble maternal and fetal cell-free DNA. RESULTS:FIRSTGENE demonstrated ≥98.2% sensitivity and ≥99.0% specificity for fetal alleles in recessive-disease genes in plasma and cell lines; 100% sensitivity and specificity for RhD compatibility in plasma; 100% sensitivity and ≥99.8% specificity for fetal chromosomal abnormalities in plasma; and ≥99.9% sensitivity and specificity for maternal alleles in recessive-disease genes in plasma. CONCLUSIONS:FIRSTGENE demonstrated high analytical sensitivity and analytical specificity for each component of the assay. Its capability to generate multiple prenatal screening results from a single blood draw may improve the efficiency and accessibility of prenatal genetic screening.

PURPOSE:Pathological complete response (pCR) after neoadjuvant therapy (NAT) strongly associates with reduced breast cancer relapse risk. Circulating tumor DNA (ctDNA) shows promise as a therapy-response biomarker, but prior studies had limited sampling and assay sensitivity. MONITOR-Breast characterized ctDNA dynamics across NAT at high temporal resolution using an ultrasensitive molecular residual disease (MRD) assay. EXPERIMENTAL DESIGN:In this prospective observational study, 154 enrolled patients with breast cancer (all subtypes, Stages I-III) were tested at baseline, throughout NAT, and post-surgery using a whole genome sequencing-based MRD assay tracking up to 1,000 variants per patient. RESULTS:Baseline ctDNA was detected in 93% of patients, with 20% detected in the ultrasensitive range (<100 parts per million). Post-NAT ctDNA positivity strongly associated with residual disease (RD) (odds ratio (OR)>20, p = 1 × 10-7) and post-operative positivity (OR =47, p = 1 × 10-6). All 56 patients with pCR and an evaluable sample had undetectable post-NAT ctDNA. Frequent testing revealed distinct patterns: 78% achieved rapid and/or sustained ctDNA clearance, while 22% exhibited persistent or intermittent positivity, and had significantly higher RD (OR =52, p = 3 × 10-9), and post-operative positivity (OR =25, p = 1 × 10-5). CONCLUSIONS:Frequent, ultrasensitive ctDNA assessment provided comprehensive characterization of treatment response, revealing opportunities for de-escalation in early responders and escalation in those with persistent ctDNA.

OBJECTIVE:To evaluate the feasibility of prenatal cell-free DNA (cfDNA) screening with fetal fraction amplification between 6 and 9 weeks of gestation. METHODS:Pregnant individuals underwent two blood draws: the first between 6 0/7 and 9 6/7 weeks of gestation and the second at 10 0/7 weeks or later. Samples were processed using a cfDNA screen with fetal fraction amplification. Before the implementation of fetal fraction amplification, 4.8% of samples drawn at 10 0/7 weeks of gestation had low fetal fraction , defined as less than 4%. Logistic regression was used to determine the earliest gestational age after the implementation of fetal fraction amplification at which the proportion of samples with low fetal fraction was 4.8% or less. Positive and negative percent agreements were calculated for samples collected before and after 10 0/7 weeks of gestation. RESULTS:Among 562 patients enrolled, 470 completed two blood draws and were included in the analysis. Median maternal age was 32 years (IQR 29-34 years), and median body mass index (BMI) was 25.7 (IQR 22.7-30.9). Median fetal fraction was 9.0% (IQR 6.5-13.6) for samples drawn at 6 0/7-9 6/7 weeks of gestation and 18.5% (IQR 14.4-23.9%) for those drawn at 10 0/7 weeks or later. Among patients with BMI 30 or higher (n=125), the median fetal fraction was 7.2% (IQR 5.5-10.7%) for samples drawn at 6 0/7-9 6/7 weeks of gestation and 14.7% (IQR 11.3-18.5) for those drawn at 10 0/7 weeks or later. The gestational age at which 4.8% of samples had low fetal fraction was 7 3/7 weeks. The screen failure rate was 1.5% (7/470) for samples drawn at 6 0/7-9 6/7 weeks of gestation and 0.3% (1/291) for samples drawn at 7 3/7-9 6/7 weeks. Because patients often present for routine prenatal care beginning in the 8th week of gestation, we also calculated performance at 8 0/7-9 6/7 weeks. During this period, the screen failure rate was 0.4% (1/241), and both positive and negative percent agreements were 100% for all screened aneuploidies. At 10 0/7 weeks of gestation or later, two samples were called positive for trisomy 21 and one for trisomy 7; their corresponding samples at 8 0/7-9 6/7 weeks were also called positive. Fetal sex call concordance was 100% for both XX (110/110) and XY (130/130). CONCLUSION:Fetal fraction amplification enables cfDNA screening beginning at 7 3/7 weeks of gestation. Performance and concordance data are limited but suggest the clinical feasibility of offering cfDNA screening with fetal fraction amplification beginning at 8 weeks of gestation.

Abstract Background: Prostate cancer (PCa) risk models based on clinicopathological features have been widely adopted by clinicians to inform treatment decisions. Prolaris provides greater prognostic power for evaluating risk of distant metastases and PCa-specific mortality than traditional risk models. Evidence is limited for how treatment decisions informed by genomic classifiers (GCs) impact treatment selection and intensification decisions, oncologic outcomes, and treatment-related adverse events (AEs) in a real-world setting. Methods: PROMPT-Bx is a multicenter, observational, pragmatic study in patients with newly diagnosed primary PCa. Registration was not required since study is non-interventional. IRB approval was obtained from Advarra (Pro00088502). Study sites routinely treat newly diagnosed localized PCa and accurately reflect real-world diagnosis and treatment of the disease. Patients who are eligible for and elect to pursue GC testing will be informed of the study and offered the chance to participate following provision of informed consent. Diagnostic biopsy samples will be used for Prolaris, with results reported back as part of standard clinical workflow. Providers will be surveyed about management decisions prior to and after receiving results. Treatment selection, oncological outcomes, and AEs will be collected from participating sites. The study is expected to enroll 3350 patients with 5 years of follow up. Primary objectives are to evaluate clinical utility of Prolaris to identify candidates for active surveillance as well as treatment intensity decisions, based on metastasis risk. Secondary objectives include evaluating non-inferiority of Prolaris recommendations compared to treatment-related AEs, impact on decision making, and prognostic utility for metastasis and biochemical recurrence. Metastasis, adverse pathology, biochemical recurrence, and treatment related AEs will be determined from the time of diagnostic biopsy and compared across NCCN risk groups using survival analyses. This study provides one of the first opportunities to better understand the utility of GC testing for patients and providers in a real-world setting. The trial is enrolling as planned, and first data analysis will occur 6 months after full enrolment. Citation Format: Brent Mabey, Lauren Lenz, Thaylon Davis, Alexander Gutin, Katherine Johansen Taber, Dale Muzzey, Jeff Jasper, Matthew Schiewer. A pragmatic study of the clinical utility of genomic classifiers in guiding prostate cancer treatment decisions: Impact of treatment selection, oncologic outcomes, and treatment-related adverse events (PROMPT-Bx) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT280.

Molecular residual disease (MRD) tests detect residual tumor in a patient by identifying the presence of circulating tumor DNA (ctDNA) in cell free DNA (cfDNA). We have developed a highly sensitive MRD test that utilizes panels of hybridization probes targeting somatic variants specific to each patient’s tumor. Our tumor-informed MRD assay utilizes whole genome sequencing (WGS) of tumor and normal tissue followed by a somatic calling pipeline to identify tumor variants and generate a panel targeting these variants. The sensitivity of bespoke MRD tests, like ours, is a function of each patient’s panel content. A major challenge of ctDNA detection at low tumor concentrations is differentiation of true tumor variants from technical errors. The variants that are most informative for detection of low tumor fraction residual disease are those that are the least likely to occur due to PCR errors or other artifacts during sample preparation. Here we assessed the technical error rates of single nucleotide variants (SNVs) and insertions and deletions (INDELs). In our experiments, many INDELs were less likely to occur due to PCR errors compared to many SNVs, though the error rates of individual variants depended on their genomic context. Several features impacted the error rate of individual INDEL variants in cfDNA including the number of bases inserted or deleted in the INDEL variant and the proximity of the INDEL variant to homopolymer regions. The lower error rate from many INDEL variants resulted in more signal to noise when detecting the presence of the tumor variant. We identified INDEL somatic variants using an ensemble of somatic callers with 99.8% confirmation rate. INDELs were typically less prevalent than SNVs but we observed that INDELs made up as much as 26.4% of a 1000 variant panel. To assess the impact of INDELs on our ability to detect residual tumor, we ran our MRD assay on patients with residual disease. Our detection pipeline was run using a 1000 probe panels that included both INDELs and SNVs or just SNVs alone. INDELs improved the sensitivity of our assay; we observed 60-90%lower error rates for INDELs with more than a single base inserted or deleted compared to SNVs in cfDNA. In conclusion, the likelihood of detecting low abundance ctDNA is higher for panels with INDELs included than for panels without INDELs. Overall, our assay achieved high sensitivity at low tumor concentrations by prioritizing the inclusion of low error rate variants including INDELs in bespoke patient panels. Ravi Patel, Elise Buser, Nafei Xu, Matthew LaBella, Kyle Trettin, Long Tian, Ashley Acevedo, Christopher Battey, Genevieve Gould, Dale Muzzey. Inclusion of INDEL somatic variants in MRD panels improves confidence in ctDNA residual disease detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6641.

3007 Background: While circulating tumor DNA (ctDNA) demonstrates promise as a molecular residual disease (MRD) biomarker, its clinical implementation has been primarily limited to tumors with favorable ctDNA shedding characteristics. We are evaluating an ultra-sensitive whole-genome sequencing (WGS)-based MRD assay in the MONSTAR-SCREEN-3 study to establish a comprehensive pan-cancer MRD platform inclusive of traditionally low-shedding tumors. Methods: MONSTAR-SCREEN-3, a prospective multicenter study targeting 1,100 patients with solid tumors undergoing curative-intent treatment in the definitive cohort, utilizes personalized panels constructed via Precise MRD (Myriad Genetics). These panels incorporate up to 1,000 tumor-specific alterations identified through WGS of matched tumor tissue, including both short variants and insertion-deletions. Serial plasma samples were collected at baseline, post-neoadjuvant chemotherapy (when applicable), 1-month post-surgery, quarterly in year 1, and biannually thereafter up to 2 years. The assay performance was evaluated across multiple cancer types for ctDNA detection and recurrence monitoring. Results: As of December 2024, 114 patients across 15 cancer types were enrolled, including colorectal (n = 33), gastric (n = 22), head and neck (n = 13), renal cell (n = 10), esophageal (n = 8), and pancreatic (n = 7) cancers. Treatment strategies included upfront surgery (n = 76) and neoadjuvant chemotherapy (n = 38). The median follow-up time was 2.4 months (range, 0.5–7.7). WGS analysis identified a median of 6,089 panel-eligible alterations per patient (range: 214-14,112), with high variants counts observed in a deficient mismatch-repair colorectal cancer, enabling comprehensive personalized panel design. Customized panel creation was successful in 69/71 patients (97.2%) across 8 cancer types, with two pancreatic cancer cases deferred to surgical specimens due to insufficient variants in FNA samples. The assay demonstrated 100% baseline sensitivity (41/41), detecting tumor fractions ranging from < 0.001% to 45.2% across all cancer types, including traditionally low-shedding tumors. Post-operative 1-month MRD assessment revealed 35.7% positivity (10/28), with tumor fractions ranging from < 0.001% to 0.27%. Two MRD-positive patients developed radiological recurrence with lead times of 2.5 and 3 months before conventional imaging detection. Conclusions: These interim results demonstrate successful pan-cancer implementation of WGS-based personalized ctDNA detection, achieving universal baseline sensitivity and ultra-sensitive MRD detection across tumor types, including those traditionally challenging to assess. Updated molecular and clinical outcome data will be presented. Clinical trial information: UMIN000053975 .

Background: Molecular residual disease (MRD) refers to the small number of tumor cells remaining in the body during or after cancer treatment. These tumor cells shed DNA into the bloodstream, resulting in circulating tumor DNA (ctDNA). ctDNA can be identified in plasma-derived cell-free DNA (cfDNA) via the presence of tumor-specific somatic variants. We have developed a second-generation tumor-informed MRD assay to detect the presence and quantity of ctDNA in the plasma of breast-cancer patients with residual disease or tumor recurrence. Our assay detects the presence of ctDNA using a hybrid-capture based sequencing panel, targeting tumor-specific somatic variants. Each panel is designed with up to 1000 carefully selected somatic variant targets identified based on tumor profiling by matched tumor and normal whole genome sequencing and is optimized to provide high sensitivity and specificity at low tumor fraction, which is particularly important because many breast tumors have low shedding of ctDNA. Targeted sequence data is fit to a statistical model that incorporates panel-specific and sample-specific parameters to provide a robust estimate of the ctDNA fraction with a rigorous assessment of confidence for each test result. Here, we evaluated the performance of our assay at low ctDNA fraction. Methods: Contrived mixtures of matched tumor and normal breast cancer cell lines and healthy donor plasma were were evaluated over a range of tumor DNA concentrations from 1 part per million (ppm) to 10,000 ppm, and were processed across multiple days, reagent lots, operators and instruments. Results: Analysis of healthy donor samples paired with non-patient matched panels demonstrated the Limit of Blank (LoB) at a 95% specificity threshold—i.e., the threshold at which 95% of negative samples return a negative result—to be 0.3 ppm. When the LoB was used as the detection threshold, we found the Limit of Detection with 95% sensitivity (LoD95) to be <5ppm. Using a more stringent detection threshold of 1.4 ppm—corresponding to a specificity of 99.615%—we found the LoD95 to be 10 ppm. Both PPV and NPV were 100% when assessed with 56 positive samples at 50 ppm and 126 negative samples. Additionally, quantification of ctDNA fraction was linear over the entire range of tumor DNA concentrations assessed. Conclusions: Overall, our assay demonstrated high sensitivity, specificity and measurement accuracy, which, together, will facilitate improved resolution in residual-disease detection and extend lead times in recurrence detection. Citation Format: Ashley Acevedo, Kyle Trettin, Nafei Xu, Matt LaBella, CJ Battey, Ravi Patel, Kiefer Haug, Elise Buser, Shalee Carlson, Thanh Tran, Britney Sadler, Abby Tucker, Genevieve Gould, Dale Muzzey. Analytical validation of a high-definition tumor-informed MRD assay demonstrates robust detection at low tumor fractions common in breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-04-23.

Circulating tumor DNA (ctDNA) is a highly sensitive and specific biomarker for molecular residual disease (MRD) and can be by identified by the presence of tumor-specific somatic variants. We have developed a second-generation hybrid-capture-based tumor-informed MRD assay to detect the presence and quantity of ctDNA in plasma-derived cell-free DNA (cfDNA). Our assay surveys the whole tumor genome to identify the superset of high-confidence somatic variant targets that can be used to distinguish ctDNA from non-tumor cfDNA. Each panel consists of up to 1000 targets that are selected based on target-specific and tumor-specific features that impact the representation of tumor variants or the effective molecular depth of a panel, such as somatic call confidence, zygosity, copy number, background error rate and expected molecular depth following capture and sequencing. cfDNA captured using a patient-matched panel undergoes targeted sequencing and that sequence data is fit to a statistical model that evaluates the presence and fraction of ctDNA in the sample. Here, we evaluated the performance of our target-ranking algorithm using contrived mixtures of matched tumor and normal cell lines, with tumor DNA concentrations ranging from 5 to 10, 000 parts per million (ppm) and samples processed across multiple days, reagent lots, operators and instruments. Identical sets of contrived samples were analyzed using subsets of panel targets, including top-ranking and bottom-ranking target sets. We found the Limit of Detection with 95% sensitivity (LoD95) for top-ranking targets was 40% of the LoD95 for low-ranking targets, demonstrating the effectiveness of our ranking algorithm in stratifying high and low sensitivity targets. In conclusion, the constituents of tumor-informed MRD panels have a measurable impact on the sensitivity of ctDNA detection and, with optimization, can enhance the resolution of residual disease detection. Ashley Acevedo, Ravi Patel, Kyle Trettin, Nafei Xu, Genevieve Gould, Dale Muzzey. Optimized selection of tumor-informed MRD panels enhances sensitivity of ctDNA detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6639.

22q11.2 deletion syndrome occurs in approximately 1 in 2,000-4,000 births. Prenatal cell-free DNA screening (pcfDNA) can detect fetuses affected by deletions as small as 2.5 Mb. Fetal fraction amplification (FFA), which has been shown to yield an average fetal fraction (FF) in excess of 20%, may further enhance pcfDNA detection of these deletions. Positive predictive values (PPV) of pcfDNA screening for 22q11.2 microdeletion have been reported between 20%-50%. Here, we sought to describe the impact of FFA on the PPV of 22q11.2 microdeletion screening using a whole-genome sequencing (WGS)-based pcfDNA platform. We retrospectively analyzed data from patients who underwent WGS-based pcfDNA screening with FFA (Prequel™, Myriad Genetics, Inc.) between 8/20-10/22. For screen-positive cases, pregnancy outcome data were requested via a routine HIPAA-compliant process. All samples with diagnostic confirmation were used to calculate PPV, defined as: true positives/(true positives + false positives). Confidence interval (CI) was estimated using the Exact Binomial Test. Complete outcomes were obtained for 54 cases that screened positive for 22q11.2 microdeletion; 21 underwent molecular diagnostic testing. All 21 were confirmed as true positives, for a PPV of 100% (21/21; 95% CI 83.9%-100%). Among the 33 cases that declined molecular confirmation, 17 had ultrasound findings that are either strongly or moderately associated with 22q11.2 deletion syndrome (e.g., cardiac defects, polyhydramnios, skeletal defects, intrauterine growth restriction). The average FF of true positive samples was 23.0%. For screen-positive patients who declined diagnostic testing, the average FF was 21.6%. The increased FF levels conferred by FFA yield a PPV for 22q11.2 microdeletion higher than any previous studies have reported, and comparable to that for common autosomal trisomies. Because respective FF levels were comparable in patients with and without diagnostic confirmation, PPV is likely to be comparable in patients with and without diagnostic confirmation.

Abstract Background: Minimal residual disease (MRD) testing can detect cancer recurrence months to years earlier than the current standard of care, enabling earlier treatment of recurrence and improved patient outcomes. Tumor-informed MRD assays typically utilize formalin-fixed paraffin-embedded (FFPE) tumor tissue, which is available in limited quantities for some patients, for example, following core needle biopsy (CNB), after neoadjuvant treatment or when patients need multiple tests from the same tumor sample. To determine the lower limit of tissue input, we evaluated our MRD assay performance across a range of extracted tumor volumes. Methods: Resected tumors and CNBs were sectioned, H&E stained, and macro-dissected. Tumor gDNA was extracted, quantified, prepared into libraries and sequenced. Sequenced libraries were aligned and evaluated for depth of coverage, variation of coverage, and duplication rate. Somatic calling was performed on matched tumor and normal samples. Up to 1000 target sites were selected for high-depth targeted sequencing of the tumor and normal gDNA for confirmation of somatic variant calls. The positive predictive value (PPV) was computed as the percent of putative somatic variant sites that were present in the tumor capture library and absent in the normal capture library. Results: Extracted tumor volumes varied by almost two orders of magnitude, from 0.06mm3 (equivalent to needle core or fine needle aspirate biopsies) to 5mm3 (achievable with resected tumor). gDNA amount varied linearly (3.6ng to 1549ng) with tumor tissue input, indicating the low tissue to paraffin ratio did not have an adverse effect on yield. Tumor gDNA inputs into library prep ranged from 2.5ng to 100ng. DNA amounts above 100ng into library prep had no discernable benefit. Below 10 ng, depth of coverage and the coefficient of variation in coverage indicated poor-quality libraries. Samples with ~10 ng of gDNA input into library prep showed depth of coverage comparable to higher inputs and saturated the achievable library complexity. Additionally, PPV of somatic calling was consistent across the range of gDNA inputs from 10-100 ng, demonstrating equivalent assay performance. Conclusion: Tumor-informed MRD assays have immense potential for increasingly sensitive treatment response and recurrence monitoring that can inform better treatment decisions. FFPE tumor tissue is a critical input into MRD assays but is a limited resource. This study supports a minimum DNA input of 10 nanograms for a single attempt at extraction, corresponding to a tissue volume of 0.2mm3 or a single 10µm slide with a 20mm2 area, representing one of the lowest tissue input requirements for an MRD assay. Low FFPE tissue requirements expand the patient population that benefit from MRD testing by utilizing samples that have low tumor content, are post-neoadjuvant therapy or do not meet the tumor volume requirements of competing MRD offerings. Citation Format: Matt LaBella, Ashley Acevedo, Ravi Patel, Kiefer Haug, Sangita Ganesh, Elise Buser, Nafei Xu, Shalee Carlson, Kyle Trettin, Sarah Ratzel, Kieko Hara, Pavlos Msaouel, Kanishka Sircar, Chad Tang, Dale Muzzey, Genevieve Gould. Design of high-performance tumor-informed minimal residual disease (MRD) panels from low FFPE tumor input [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 3675.

Purpose Clinically significant copy-number variants (CNVs) occur in 1% to 2% of pregnancies and are difficult to detect via prenatal cell-free DNA (cfDNA) screening because of the low fraction of fetal-derived cfDNA in maternal plasma. Here, we use fetal fraction amplification (FFA) and improved computational algorithms to enhance the resolution and sensitivity of CNV detection. Methods We implemented and characterized the performance of a hidden Markov model that identifies fetal CNVs. This CNV caller was analytically validated on 117 FFA samples, including 57 fetal-CNV-containing samples, and applied retrospectively to a cohort of more than 300k patient samples. Results Our assay was concordant with orthogonal testing and detected fetal CNVs ≥5 Mb with estimated aggregate sensitivity and specificity of >95.1% and >99.7%, respectively. The resolution of CNV detection was fetal fraction dependent, but 97.2% of samples reached ≥5-Mb resolution. Overall, CNVs ≥5 Mb were found in 1 in 500 pregnancies. Conclusion FFA improves the sensitivity and resolution of CNV detection in prenatal cfDNA screening, allowing accurate detection of fetal CNVs as small as 1 Mb. Using our approach, we found that clinically significant fetal CNVs were detected more frequently than the common trisomies 13 and 18 that are recommended as part of guideline-based screening.