Kan Xiong

Purpose: Circulating tumor DNA (ctDNA) could be a powerful prognostic biomarker for patients with locally advanced esophageal cancer but higher sensitivity is needed. An ultrasensitive test could enable improved treatment decision-making by accurately identifying patients with residual disease. Experimental Design: 331 plasma samples from 36 patients were collected both preoperatively and postoperatively. We applied MAESTRO-Pool (Minor Allele Enriched Sequencing Through Recognition Oligonucleotides) of the Broad Institute and the Oncodetect® V2 test of Exact Sciences, two MAESTRO-based, ultrasensitive, tumor-informed, mutation enrichment sequencing tests, to all samples. Results: With the Oncodetect V2 test, we found 9 of 14 patients (64%) with recurrence and postoperative plasma available tested ctDNA-positive at their first postoperative sample and all 14 patients tested ctDNA-positive for at least one postoperative sample prior to recurrence. ctDNA was detected below the limit of detection for first-generation assays in 59% of all samples in which we detected ctDNA. By tracking a median of 2.1-fold more mutations per patient (up to 5,000 total mutations per patient), the Oncodetect V2 test showed highly concordant results (98.9% positive percent agreement), improved ctDNA detection, and longer lead times to recurrence as compared to MAESTRO-Pool. Conclusion: ctDNA detection after curative-intent therapy for locally advanced esophageal cancer had strong positive and negative predictive values for recurrence. Given the low ctDNA levels observed, and that all patients with recurrence were eventually ctDNA-positive, ctDNA testing in esophageal cancer may benefit from ultrasensitive tests that detect part-per-million levels of ctDNA.

Despite responses of chimeric antigen receptor (CAR)-T cells in patients with relapsed/refractory large B-cell lymphoma (LBCL), over half of patients eventually relapse. Methods to detect early disease persistence are needed to identify patients at high risk of treatment failure. We recently developed MAESTRO, an ultrasensitive, tumor-informed measurable residual disease (MRD) assay, which can detect parts-per-million (ppm) levels of circulating tumor DNA (ctDNA) using minimal sequencing. We applied MAESTRO to 140 samples from 28 patients (15 durable responders at 12 months and 13 nonresponders) to identify treatment failure after axicabtagene ciloleucel (axi-cel), administered at our institution between 2018 and 2022. Responder and nonresponder patients had similar baseline tumor burden. By 1 week after infusion, responders had marked ctDNA reduction compared to nonresponders (P < .001). At weeks 2 and 4, responders had ctDNA levels approaching 0 ppm, whereas nonresponders had persistence of ctDNA (each P < .001). At day 0, up to 21% of patients had ctDNA fractions <0.01%; hence, these individuals would not have qualified for ctDNA monitoring with a less sensitive test. Our results confirm the feasibility of highly sensitive MRD detection by ctDNA for early identification of patients at high risk of disease progression from axi-cel.

Purpose:Although ctDNA is a promising biomarker for minimal residual disease (MRD) detection in head and neck squamous cell carcinoma (HNSCC), more sensitive assays are needed for accurate MRD detection at clinically relevant time points. Ultrasensitive MRD detection early after surgery could guide adjuvant therapy decisions, but early ctDNA dynamics are poorly understood.Experimental Design:We applied minor allele–enriched sequencing through recognition oligonucleotides (MAESTRO), a whole-genome, tumor-informed, mutation enrichment sequencing assay, in a pooled testing format called MAESTRO-Pool, to plasma samples from patients with HNSCC collected shortly after surgery and during surveillance. We evaluated whether early MRD detection could predict outcomes.Results:Among 24 patients with predominantly human papillomavirus–independent (95.8%) HNSCC, rapid ctDNA clearance occurred by the first postoperative sample (1–3 days postoperatively) in nine patients without an event (recurrence or death). Thirteen of fifteen patients with an event were MRD-positive (positive predictive value = 92.9%; negative predictive value = 80%) with a median tumor fraction (TFx) of 54 parts per million (ppm; range 6–1,177 ppm). In the first and last samples of the early postoperative window, 8/13 and 10/13 patients, respectively, had TFx below 100 ppm, the detection limit of leading commercial assays. Early MRD detection correlated with worse overall survival (HR, 8.3; 95% confidence interval, 1.1–66.1; P = 0.02) and event-free survival (HR, 27.4; 95% confidence interval, 3.5–214.5; P < 0.0001) independent of high-risk pathology.Conclusions:Early postoperative MRD detection by MAESTRO was associated with recurrence and worse survival. Given the ultralow TFxs observed, ultrasensitive assays will be essential for reliable MRD detection during early postoperative time points to enable personalized adjuvant therapy decision-making in HNSCC.See related article by Bryan et al., p. 3483

DNA mutations and methylation often contribute to disease development in a synergistic manner. While duplex sequencing is the most accurate method for detecting DNA mutations, it typically lacks the ability to simultaneously assess methylation or requires many reads. Here, we developed Methyl-CODEC to enable simultaneous methylation sequencing and duplex sequencing using single read pairs. To achieve this, Methyl-CODEC links an enzymatically deaminated sense strand to the reverse complement of the antisense strand, which is protected from conversion by using conversion-resistant dCTPs in the strand linking step. Methyl-CODEC shows high concordance with standard enzymatic or bisulfite-based whole genome methylation sequencing, while also uniquely preserving the original DNA sequence. We show that hydroxy-methyl-dCTP is superior in this regard relative to other conversion-resistant dCTPs. Methyl-CODEC improves genetic sequencing accuracy, enables better read alignment for next-generation sequencing, and distinguishes C > T mutations from unmethylated Cs. It also identifies rare mutations including those producing methylated Cs, which are enriched in CpG contexts. Methyl-CODEC opens new horizons for enhanced detection of biomarkers in cancer and molecular medicine.

Purpose: Although ctDNA is a promising biomarker for minimal residual disease (MRD) detection in head and neck squamous cell carcinoma (HNSCC), more sensitive assays are needed for accurate MRD detection at clinically relevant time points. Ultrasensitive MRD detection early after surgery could guide adjuvant therapy decisions, but early ctDNA dynamics are poorly understood. Experimental Design: We applied minor allele–enriched sequencing through recognition oligonucleotides (MAESTRO), a whole-genome, tumor-informed, mutation enrichment sequencing assay, in a pooled testing format called MAESTRO-Pool, to plasma samples from patients with HNSCC collected shortly after surgery and during surveillance. We evaluated whether early MRD detection could predict outcomes. Results: Among 24 patients with predominantly human papillomavirus–independent (95.8%) HNSCC, rapid ctDNA clearance occurred by the first postoperative sample (1–3 days postoperatively) in nine patients without an event (recurrence or death). Thirteen of fifteen patients with an event were MRD-positive (positive predictive value = 92.9%; negative predictive value = 80%) with a median tumor fraction (TFx) of 54 parts per million (ppm; range 6–1,177 ppm). In the first and last samples of the early postoperative window, 8/13 and 10/13 patients, respectively, had TFx below 100 ppm, the detection limit of leading commercial assays. Early MRD detection correlated with worse overall survival (HR, 8.3; 95% confidence interval, 1.1–66.1; P = 0.02) and event-free survival (HR, 27.4; 95% confidence interval, 3.5–214.5; P < 0.0001) independent of high-risk pathology. Conclusions: Early postoperative MRD detection by MAESTRO was associated with recurrence and worse survival. Given the ultralow TFxs observed, ultrasensitive assays will be essential for reliable MRD detection during early postoperative time points to enable personalized adjuvant therapy decision-making in HNSCC. See related article by Bryan et al., p. XX .

Purpose: While circulating tumor DNA (ctDNA) is a promising biomarker for minimal residual disease (MRD) detection in head and neck squamous cell carcinoma (HNSCC), more sensitive assays are needed for accurate MRD detection at clinically-relevant timepoints. Ultrasensitive MRD detection immediately after surgery could guide adjuvant therapy decisions, but early ctDNA dynamics are poorly understood. Experimental Design: We applied MAESTRO, a whole-genome, tumor-informed, mutation-enrichment sequencing assay, in a pooled testing format called MAESTRO-Pool, to plasma samples from HNSCC patients collected immediately after surgery and during surveillance. We evaluated whether early MRD detection could predict outcomes. Results: Among 24 predominantly HPV-independent (95.8%) HNSCC patients, rapid ctDNA clearance occurred by the first postoperative sample (1-3 days postoperatively) in 9 patients without an event (recurrence or death). 13/15 patients with an event were MRD+ (PPV = 92.9%; NPV = 80%) with a median tumor fraction (TFx) of 54 ppm (range 6-1,177 ppm). In the first and last sample of the immediate postoperative window, 8/13 and 10/13 patients had TFx below 100 ppm, respectively, the detection limit of leading commercial assays. Early MRD detection correlated with worse overall survival (HR = 8.3; 95% CI: 1.1-66.1; P = 0.02) and event-free survival (HR = 27.4; 95% CI: 3.5-214.5; P < 0.0001) independent of high-risk pathology. Conclusions: Immediate postoperative MRD detection by MAESTRO was predictive of recurrence and death. Given the ultralow TFxs observed, ultrasensitive assays will be essential for reliable MRD detection during early postoperative timepoints to enable personalized adjuvant therapy decision-making in HNSCC.

Background: Minimally invasive molecular profiling using cell-free DNA (cfDNA) is increasingly important to the management of cancer patients; however, low sensitivity remains a major limitation, particularly for brain tumor patients. Transiently attenuating cfDNA clearance from the body—thereby, allowing more cfDNA to be sampled—has been proposed to improve the performance of liquid biopsy diagnostics. However, there is a paucity of clinical data on the effect of higher cfDNA recovery. Here, we investigated the impact of collecting greater quantities of cfDNA on circulating tumor DNA (ctDNA) sensitivity in the “low-shedding” cancer type glioblastoma by analyzing up to approximately 15-fold more plasma than routinely obtained clinically. Methods: We tested 70 plasma samples (median 17.0 mL, range 2.5–66.5) from 8 IDH-wild-type glioblastoma patients using an optimized version of the MAESTRO-Pool ctDNA assay. Results were compared with simulated single-blood-tube equivalents of cfDNA. ctDNA results were then compared with magnetic resonance imaging (MRI) and pathology assessments of true progression vs pseudoprogression in glioblastoma patients. Results: Larger cfDNA yields exhibited a doubling in ctDNA-positivity while achieving a median specificity of 99% and more precise ctDNA quantification. In 8 glioblastoma patients, ctDNA was detected in 88%, including at multiple timepoints in 6/7. In the setting of indeterminate progression by MRI, our data suggested that MAESTRO-Pool with large plasma volumes can help distinguish true glioblastoma progression from pseudoprogression. Conclusions: Our findings provide a proof-of-principle that most glioblastomas shed ctDNA into plasma and that greater ctDNA yields could help improve liquid biopsies for “low-shedding” cancer types such as glioblastoma.

Liquid biopsies enable early detection and monitoring of diseases such as cancer, but their sensitivity remains limited by the scarcity of analytes such as cell-free DNA (cfDNA) in blood. Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. We sought to transiently augment the level of circulating tumor DNA (ctDNA) in a blood draw by attenuating its clearance in vivo. We report two intravenous priming agents given 1 to 2 hours before a blood draw to recover more ctDNA. Our priming agents consist of nanoparticles that act on the cells responsible for cfDNA clearance and DNA-binding antibodies that protect cfDNA. In tumor-bearing mice, they greatly increase the recovery of ctDNA and improve the sensitivity for detecting small tumors.

Background: Tracing patient-specific tumor mutations in cell-free DNA (cfDNA) for minimal residual disease (MRD) detection is promising but challenging. Assaying more mutations and cfDNA stands to improve MRD detection but requires highly accurate, efficient sequencing methods and proper calibration to prevent false detection with bespoke tests. Methods: MAESTRO (Minor Allele Enriched Sequencing Through Recognition Oligonucleotides) uses mutation-specific oligonucleotide probes to enrich cfDNA libraries for tumor mutations and enable their accurate detection with minimal sequencing. A new approach, MAESTRO-Pool, which entails pooling MAESTRO probes for all patients and applying these to all samples from all patients, was used to screen for 22 333 tumor mutations from 9 melanoma patients in 98 plasma samples. This enabled quantification of MRD detection in patient-matched samples and false detection in unmatched samples from other patients. To detect MRD, a new dynamic MRD caller was used that computes a probability for MRD detection based on the number of mutations and cfDNA molecules sequenced, thereby calibrating for variations in each bespoke test. Results: MAESTRO-Pool enabled sensitive detection of MRD down to 0.78 parts per million (ppm), reflecting a 10- to 100-fold improvement over existing tests. Of the 8 MRD positive samples with ultra-low tumor fractions <10 ppm, 7 were either in upward-trend preceding recurrence or downward-trend aligning with response. Of 784 patient-unmatched tests, only one was found as MRD positive (tumor fraction = 2.7 ppm), suggesting high specificity. Conclusions: MAESTRO-Pool enables massively parallel, tumor-informed MRD testing with concurrent benchmarking of bespoke MRD tests. Meanwhile, our new MRD caller enables more mutations and cfDNA molecules to be tested without compromising specificity. These improve the ability for detecting traces of MRD from blood.