Detection Of Residual Disease Research Articles

What’s behind Margin Status in Oral Cancer?

AbstractIn the 2nd century AD, Galen argued that the failure to remove any single ‘root’ of a malignant tumor could result in a local relapse. After nearly 2 millennia, this problem appears to be even more challenging due to our increased understanding of the complexity of tumor formation and spread. Pathological analysis of tumor margins under a microscope remains the primary and only accepted method for confirming the complete tumor removal. However, this method is not an all-or-nothing test, and it can be compromised by various intrinsic and extrinsic limitations. Among the intrinsic limitations of pathological analysis we recall the pathologist handling, tissue shrinkage, the detection of minimal residual disease and the persistence of a precancerous field. Extrinsic limitations relate to surgical tools and their thermal damage, the different kinds of surgical resections and frozen sections collection. Surgeons, as well as oncologists and radiotherapists, should be well aware of and deeply understand these limitations to avoid misinterpretation of margin status, which can have serious consequences. Meanwhile, new technologies such as Narrow band imaging have shown promising results in assisting with the achievement of clear superficial resection margins. More recently, emerging techniques like Raman spectroscopy and near-infrared fluorescence have shown potential as real-time guides for surgical resection.The aim of this narrative review is to provide valuable insights into the complex process of margin analysis and underscore the importance of interdisciplinary collaboration between pathologists, surgeons, oncologists, and radiotherapists to optimize patient outcomes in oral cancer surgery.

HTERT Epigenetics Provides New Perspectives for Diagnosis and Evidence-Based Guidance of Chemotherapy in Cancer.

Strong epigenetic pan-cancer biomarkers are required to meet several current, urgent clinical needs and to further improve the present chemotherapeutic standard. We have concentrated on the investigation of epigenetic alteration of the hTERT gene, which is frequently epigenetically dysregulated in a number of cancers in specific developmental stages. Distinct DNA methylation profiles were identified in our data on early urothelial cancer. An efficient EpihTERT assay could be developed utilizing suitable combinations with sequence-dependent thermodynamic parameters to distinguish between differentially methylated states. We infer from this data set, the epigenetic context, and the related literature that a CpG-rich, 2800 bp region, a prominent CpG island, surrounding the transcription start of the hTERT gene is the crucial epigenetic zone for the development of a potent biomarker. In order to accurately describe this region, we have named it "Acheron" (Ἀχέρων). In Greek mythology, this is the river of woe and misery and the path to the underworld. Exploitation of the DNA methylation profiles focused on this region, e.g., idiolocal normalized Methylation Specific PCR (IDLN-MSP), opens up a wide range of new possibilities for diagnosis, determination of prognosis, follow-up, and detection of residual disease. It may also have broad implications for the choice of chemotherapy.

Tissue-free liquid biopsies combining genomic and methylation signals for minimal residual disease detection in patients with early colorectal cancer from the UK TRACC Part B study.

Absence of post-operative circulating tumour DNA (ctDNA) identifies resected colorectal cancer (CRC) patients with low recurrence risk for adjuvant chemotherapy (ACT) de-escalation. We present the largest resected CRC cohort to date with tissue-free minimal residual disease (MRD) detection. TRACC included stage I-III resectable CRC patients. Prospective longitudinal plasma collection for ctDNA occurred pre- and post-surgery, post-ACT, every 3m for year 1 and every 6m in years 2 and 3 with imaging annually. The Guardant Reveal assay evaluated genomic and methylation signals. The primary endpoint was 2yr recurrence free survival (RFS) by post-operative ctDNA detection. (NCT04050345) Results: Between December 2016 and August 2022, 1203 were patients enrolled. Plasma samples (n=997) from 214 patients were analysed. 143 patients were evaluable for the primary endpoint; 92 (64.3%) colon, 51 (35.7%) rectal; 2 (1.4%) stage I, 64 (44.8%) stage II, 77 (53.8%) stage III. Median follow-up was 30.3m (95% CI: 29.5-31.3). 2yr RFS was 91.1% in patients with ctDNA not detected post-operatively and 50.4% in those with ctDNA detected (HR 6.5 [2.96-14.5] p<0.0001). Landmark negative predictive value (NPV) was 91.2% (95% CI 83.9-95.9). Longitudinal sensitivity and specificity were 62.1% (95% CI 42.2-79.3) and 85.9% (95% CI 78.9-91.3) respectively. Median lead-time from ctDNA detection to radiological recurrence was 7.3m (IQR 3.3-12.5; n=9). Tissue-free MRD detection with longitudinal sampling predicts recurrence in stage I-III CRC without need for tissue sequencing. NPV is high supporting ACT de-escalation in patients with ctDNA not detected post-operatively, now being investigated in the UK TRACC Part C study.

Circulating tumor DNA (ctDNA) kinetics in colorectal cancer (CRC) treated with curative intent in the VICTORI study with an ultrasensitive MRD assay.

e15625 Background: Detection of molecular residual disease (MRD) with ctDNA is highly prognostic for recurrence in CRC. However, many cancers still recur without detectable ctDNA and increased lead time before clinical recurrence may create a window of opportunity to intervene. Here we apply an ultra-sensitive assay, NeXT Personal, to profile patients in the VICTORI study, a prospective cohort of patients with CRC managed with curative-intent treatment. Methods: To date, the first 33 patients with CRC have undergone panel creation. NeXT Personal was employed to construct personalized liquid biopsy panels for each patient with up to ~1,800 single nucleotide variants (SNV) identified via whole-genome sequencing. For each patient, plasma is collected before curative intervention (baseline), every 2 weeks for 2 months (MRD window), and every 3 months for up to 3 years (surveillance). VICTORI will enroll &gt; 175 patients and include a health economics analysis. Results: Of 33 patients enrolled to date, 25 (76%) are stage I-III and 8 are stage IV treated with curative interventions. 18 colon cancers and 15 rectal cancers are included. Pre-operative baseline sensitivity was 90.6% (29/32; 1 patient excluded as pCR at the time of baseline blood draw). Of the MRD- patients at baseline, 2 had prior neoadjuvant treatment and 1 was stage 1. Preliminary results (median 7.2 month follow-up; 5 recurrences) show 80% (4/5) of recurrences are MRD+ at the 4-week landmark and 50% (2/4; 1 missing sample) were MRD+ at the 2-week landmark. In 75% (3/4) stage II-III recurrences, the first MRD+ detection was in the ultra-low range of &lt; 100 PPM ( &lt; 0.01% tumor fraction). MRD detection at week 4 and 8 trended towards poorer recurrence-free survival (RFS) with similar separation of patients (log rank p = 0.062 and p = 0.011, respectively). Conclusions: Preliminary results from our prospective study demonstrate strong rates of ctDNA detection at week 4 landmark analysis, potentially due to detection of very low levels of ctDNA with an ultra-sensitive MRD assay. The majority of the first detections for MRD were in the ultra-low ctDNA range, indicating the importance of high sensitivity MRD testing. [Table: see text]

Prospective validation of ctHPVDNA for detection of minimal residual disease and prediction of recurrence in patients with HPV-associated head and neck cancer treated with surgery.

6010 Background: Prediction of minimal residual disease (MRD) following surgery, and thus the need for adjuvant therapy, is currently based on clinicopathologic risk factors which have poor individual prognostic capacity. We previously reported that MRD detection by ctHPVDNA droplet digital (dd)PCR as early as post-operative day (POD) 1 is predictive of recurrence in stage I-II HPV+HNSCC patients. Here, we applied a significantly more sensitive custom whole genome hybrid-capture-based next generation sequencing assay, termed HPV-DeepSeek, to validate the prognostic capacity of ctHPVDNA detection and explore the optimal timing of MRD testing in HPV+HNSCC patients. We tested the primary hypothesis that patients with MRD detection within 6 weeks of surgery would have inferior PFS at 2 years and the secondary hypothesis that patients with ctHPVDNA positivity detected at any point following treatment completion would have inferior PFS at 2 years. Methods: 98 patients with HPV+HNSCC were prospectively enrolled with a mean follow-up of 712 days. All patients underwent surgery as primary treatment and clinicopathologic adjusted adjuvant treatment. 10ml blood samples were collected before surgery, in the post-operative period (POD 1-42), and serially in follow-up. MRD was defined as a lack of ctHPVDNA clearance during the 6 weeks following surgery. Cell free DNA was extracted from plasma and run on HPV-DeepSeek, and on existing ddPCR assays for head to head comparisons. Results: 96/98 (98%) of patients had detectable ctHPVDNA pre-treatment. 30/98 patients (31%) were MRD positive. Patients who were MRD positive had significantly worse 2 years PFS compared to MRD negative patients (78% vs 98%, P=0.0009). Predictive performance improved by limiting time points to POD 7-42 (2 year PFS 60% vs 97%, P&lt;0.0001) as significantly fewer patients were MRD positive &gt;1 week after surgery, suggesting the use of an ultra-sensitive assay such as HPV-DeepSeek requires adjustment of MRD time points. Patients with detectable ctHPVDNA following completion of all treatment had significantly worse 2-year PFS compared to patients without detectable ctHPVDNA (0% vs 97%, P&lt;0.0001). 7/98 patients had cancer recurrence during follow-up that was detected by ctHPVDNA with a mean lead time of 207 days (35-518) to clinical diagnosis. Conclusions: ctHPVDNA detection by HPV-DeepSeek is a highly specific biomarker of MRD in HPV+HNSCC, accurately predicts disease progression and detects recurrence earlier than standard care clinical.

Impact of circulating tumor DNA (ctDNA) surveillance on clinical care for patients with stage I-III breast cancer: Findings from a multi-institutional study.

549 Background: Tumor-informed ctDNA-based detection of minimal residual disease (MRD) in the adjuvant setting for patients (pts) with early breast cancer (EBC) is strongly associated with recurrence. However, the clinical impact of early ctDNA detection and potential therapeutic intervention remains unclear. In this multi-institution retrospective analysis, we investigated whether ctDNA detection in the adjuvant setting impacted the care of pts with EBC. Methods: This multisite (4 geographically distinct regions in the US) study included ptswith stage I-III EBC who had adjuvant MRD testing between 11/2020 - 01/2024. A personalized, tumor-informed 16-plex PCR assay (Signatera) was used for plasma ctDNA detection. All tests were ordered in the real-world clinical setting. Data were obtained by retrospective review of electronic medical records. Results: As of 01/2024, 464 EBC pts had tumor-informed ctDNA testing to monitor MRD. Of these, 58 pts (13%) were ctDNA+ at ≥1 timepoint post-surgery: 7 (12%) with stage I EBC, 25 (43%) stage II, 26 (45%) with stage III. Tumor subtypes included HR+/HER2- (38; 65%), HR-/HER2- (17; 30%), and HER2+ (3; 5%). Of the ctDNA+ group, most pts (47; 81%) had testing at &gt;1 timepoint (median 3; range 1-20 timepoints). Overall, 28 (48%) had ctDNA+ at all timepoints, 11 (19%) were persistently ctDNA+ after initial ≥1 negative result (ctDNA-), and 19 (33%) cleared ctDNA after initial ≥1 ctDNA+ result. Adjuvant ctDNA+ results impacted the care plan in 53/58 (91%) pts. 45/58 (78%) had restaging imaging within 1 year of a ctDNA+ result, and 25/45 (56%) had radiographic evidence of recurrence at that time: 23 distant and 2 local recurrences. 10/25 (40%) of these recurrences were in pts with HR-/HER2- EBC. Of 20 pts that were ctDNA+ despite no radiographic evidence of recurrence (MRD+ corresponding to molecular recurrence), 17/20 (85%) had HR+/HER2- EBC. At least 5/20 (25%) had increased imaging surveillance after ctDNA+ results, and at least 14/20 (70%) had repeat ctDNA testing. In 9/20 (45%) pts with molecular recurrence, providers changed therapy based on the ctDNA+ result, including enrollment into a clinical trial. 5/9 (56%) of these pts had a subsequent ctDNA clearance, 4 (80%) of whom remained without radiologic evidence of disease (median follow-up 12.53 (range 6.08-21.6) months from time of ctDNA+). 3/9 pts had persistent ctDNA+ despite change in therapy and all 3/3 (100%) of these pts had a radiographic recurrence. Conclusions: In this multi-institutional study, ctDNA detection impacted clinical care in most pts. A subset of pts had a change in systemic therapy with subsequent ctDNA clearance which was associated with better prognosis compared to those without ctDNA clearance. Further validation of these findings in additional datasets and clinical trial(s) are needed to inform incorporation of ctDNA testing into routine care.

Longitudinal circulating tumor DNA monitoring for detection of molecular residual disease in patients with advanced germ cell tumors.

e17022 Background: Germ cell malignancies, commonly manifesting as testicular cancer, is one of the most common malignancies in men 15 to 45 years of age, with the incidence doubling over the past 40 years. When identified promptly and treated with a multidisciplinary approach, germ cell tumors have an excellent prognosis with cure rate &gt;90% and 5-year survival rate &gt;95%. Treatment involves radical orchiectomy for diagnostic and therapeutic purposes. Further management includes active surveillance, adjuvant chemotherapy, retroperitoneal lymph node dissection (RLND), and radiation therapy. While AFP, B-HCG, and LDH are good biomarkers, they are not sensitive or specific for detecting molecular residual disease (MRD). B-HCG is detected in &lt;30% of individuals with seminoma and B-HCG and AFP are detected in 50–60% of patients with non-seminomatous tumors. We describe the use of circulating tumor DNA (ctDNA) for detection of MRD in advanced germ cell malignancy, especially when baseline biomarkers are negative. Methods: This is a retrospective analysis of 20 patients with testicular cancer treated at Rush University Medical Center who monitored closely for MRD. Signatera (Natera Inc.), a personalized tumor-informed ctDNA assay, was utilized for detection and quantification of ctDNA in patients’ plasma samples, measured at MRD (1-12 weeks post-orchiectomy) and regular intervals thereafter, regardless of treatment modality. Event free survival (EFS) was determined, defined as the period from radical orchiectomy to date of radiographic progression of disease. Results: There was a total of 20 patients (35% seminoma, 65% non-seminoma) with 2 of the patients having extragonadal disease. The mean age was 32.2 years old with IQR (28-36). Post operatively, 20% underwent RLND, 10% were on surveillance, 53% received post-orchiectomy chemotherapy, 6.7% of patients underwent autologous stem cell transplant. The median follow up time is 1.14 years. Two patients had positive ctDNA despite negative baseline traditional biomarkers. There were 23.3% (7/30) of patients that had evidence of radiographic progression. Of the patients with progression, serial ctDNA monitoring had high rates of concordance (85%, 6/7) of ctDNA positivity with biomarker positivity and radiographic progression. Conclusions: Overall, ctDNA correlated with, and sometimes even preceded, traditional biomarkers and radiographic findings of disease progression. The ctDNA positivity of 2 patients in this cohort despite negative baseline AFP, B-HCG, and LDH, highlights the sensitivity and specificity of ctDNA for MRD compared to more traditional biomarkers. Serial monitoring of ctDNA offers valuable insights into germ cell tumor disease status, including presence of MRD, and there is a need for prospective studies to guide treatment escalation and de-escalation in germ cell tumors.

Detection of molecular residual disease in stage II and III melanoma utilizing circulating tumor DNA.

e21564 Background: Overall survival for patients with melanoma has significantly improved with advancements in immunotherapy, especially with immune checkpoint inhibitors and BRAF/MEK inhibitors becoming standard of care for advanced melanoma. However, the decision to proceed with adjuvant therapy should be considered against the risk of adverse side effects, particularly in early-stage melanoma patients. Accurate biomarkers are needed to risk stratify patients, determine who will most likely benefit from adjuvant therapy, and identify when the ideal time is to initiate treatment. This study evaluates the prognostic value of personalized, tumor-informed circulating tumor DNA (ctDNA) testing in patients with stage II and III melanoma. Methods: In this real-world study, plasma samples (n =429) were analyzed from 90 stage IIA-IIID cutaneous melanoma patients treated at Rush University Medical Center between 03/30/2021 and 01/30/2024. A clinically-validated, personalized, tumor-informed 16-plex PCR assay (SignateraTM, Natera, Inc.) was used for detection and quantification of ctDNA in plasma samples. Results: Personalized, tumor-informed ctDNA assays were created using tissue samples from either initial biopsy or surgical excision specimen. Assay design was successful for 100% (90/90) of patients with sufficient tissue; test requests for 4 patients could not proceed due to insufficient tissue. ctDNA was detectable in 28% (25/90) of patients at one or more time points. There was a higher ctDNA-positivity rate in stage III patients (20/50, 40%) compared to stage II patients (5/40, 13%). Of the 25 patients with identifiable ctDNA during surveillance, 13 tested positive on the initial test (2 stage II and 11 stage III). Eighteen of the 25 ctDNA-positive patients received immunotherapy; eight patients achieved sustained ctDNA clearance, 2 had transient clearance, 2 have remained positive on ctDNA testing, and 6 continue to receive adjuvant IO and follow-up ctDNA testing. Clinical or radiographic recurrence occurred in 11/90 patients, 10 of whom tested positive for ctDNA. Conclusions: Personalized, tumor-informed ctDNA offers an adjunct to conventional monitoring for melanoma patients to detect molecular residual disease and was found to be prognostic in our patient cohort. Prospective studies are needed to evaluate the role of ctDNA in clinical decision making for surveillance imaging intervals and appropriate initiation, intensification, or discontinuation of adjuvant therapy.

Ultra-sensitive ctDNA mutation tracking to identify molecular residual disease and predict relapse in patients with early breast cancer.

1010 Background: Circulating tumor DNA (ctDNA) based detection of Molecular Residual Disease (MRD) in breast cancer patients presents a strategy to identify patients at high risk of relapse, although current assays have limited sensitivity for low level ctDNA detection. In this study we profile early breast cancer patients with an ultra-sensitive, whole genome sequencing-based, tumor-informed ctDNA platform, and correlate the findings with clinical outcomes. Methods: We analysed 598 plasma samples (median 8 samples/patient, range 2-14) from 76 patients with early breast cancer (23 TNBC, 33 HER2+, 16 HR+ and 4 unknown) enrolled in the ChemoNEAR study. Samples were collected at diagnosis before therapy, at Cycle 2 of neoadjuvant chemotherapy (NAC), post-surgery after neoadjuvant therapy if administered, and every 3 months during follow-up for the first year, and subsequently every 6 months for up to five years. Plasma cfDNA was analysed using the NeXT Personal ctDNA based MRD platform, a tumour-informed approach leveraging whole-genome sequencing of tumor and normal samples to produce personalised ctDNA sequencing panels, with each bespoke panel consisting of up to ~1,800 selected variants that enable ultra-sensitive MRD detection. MRD detection was correlated with clinical outcomes and histopathological data. Results: We detected a broad range of ctDNA levels ranging from 204,900 PPM to 2.2 PPM (median 296 PPM). 40% of all ctDNA detections were in the ultra-low range of &lt;100 PPM. 97.8% (45/46) of patients had ctDNA detected at baseline prior to treatment, including 100% TNBC, 100% HER2+, and 83% of HR+ patients. At a median follow-up of 76 months (range 5-113) from study entry, detection of ctDNA associated with high risk of future relapse (HR undefined, p&lt;0.0001; log-rank test) and shortened overall survival (p&lt;0.0001; log-rank test) with median lead-time from ctDNA detection to clinical relapse of 12.5 months (range 1-60). MRD was identified in 100% (10/10) of patients who relapsed. Of the relapsed patients, the median level of ctDNA at first MRD detection was 18.5 PPM. No ctDNA undetected patients relapsed throughout follow up (58/58). Three patients had ctDNA detected in follow-up but had not relapsed at the time of data cut off. For patients that were ctDNA undetected at post-surgical landmark, 94% (31/33) patients did not relapse. Conclusions: NeXT Personal detected breast cancer relapse with a long lead-time over clinical relapse, and strongly associated with relapse free survival. The assay demonstrated high rates of ctDNA detection at diagnosis. The strong negative predictive value at landmark suggests potential use in de-escalation studies. Several prospective, interventional trials are being conducted to assess whether treatment based on MRD detection improves outcome.

Longitudinal circulating tumor DNA monitoring for detection of molecular residual disease in patients with advanced germ cell tumors.

e17022 Background: Germ cell malignancies, commonly manifesting as testicular cancer, is one of the most common malignancies in men 15 to 45 years of age, with the incidence doubling over the past 40 years. When identified promptly and treated with a multidisciplinary approach, germ cell tumors have an excellent prognosis with cure rate &gt;90% and 5-year survival rate &gt;95%. Treatment involves radical orchiectomy for diagnostic and therapeutic purposes. Further management includes active surveillance, adjuvant chemotherapy, retroperitoneal lymph node dissection (RLND), and radiation therapy. While AFP, B-HCG, and LDH are good biomarkers, they are not sensitive or specific for detecting molecular residual disease (MRD). B-HCG is detected in &lt;30% of individuals with seminoma and B-HCG and AFP are detected in 50–60% of patients with non-seminomatous tumors. We describe the use of circulating tumor DNA (ctDNA) for detection of MRD in advanced germ cell malignancy, especially when baseline biomarkers are negative. Methods: This is a retrospective analysis of 20 patients with testicular cancer treated at Rush University Medical Center who monitored closely for MRD. Signatera (Natera Inc.), a personalized tumor-informed ctDNA assay, was utilized for detection and quantification of ctDNA in patients’ plasma samples, measured at MRD (1-12 weeks post-orchiectomy) and regular intervals thereafter, regardless of treatment modality. Event free survival (EFS) was determined, defined as the period from radical orchiectomy to date of radiographic progression of disease. Results: There was a total of 20 patients (35% seminoma, 65% non-seminoma) with 2 of the patients having extragonadal disease. The mean age was 32.2 years old with IQR (28-36). Post operatively, 20% underwent RLND, 10% were on surveillance, 53% received post-orchiectomy chemotherapy, 6.7% of patients underwent autologous stem cell transplant. The median follow up time is 1.14 years. Two patients had positive ctDNA despite negative baseline traditional biomarkers. There were 23.3% (7/30) of patients that had evidence of radiographic progression. Of the patients with progression, serial ctDNA monitoring had high rates of concordance (85%, 6/7) of ctDNA positivity with biomarker positivity and radiographic progression. Conclusions: Overall, ctDNA correlated with, and sometimes even preceded, traditional biomarkers and radiographic findings of disease progression. The ctDNA positivity of 2 patients in this cohort despite negative baseline AFP, B-HCG, and LDH, highlights the sensitivity and specificity of ctDNA for MRD compared to more traditional biomarkers. Serial monitoring of ctDNA offers valuable insights into germ cell tumor disease status, including presence of MRD, and there is a need for prospective studies to guide treatment escalation and de-escalation in germ cell tumors.

Analytical and clinical validation of a personalized, tumor-informed circulating tumor DNA assay for minimal residual disease detection.

e14545 Background: The detection of minimal residual disease (MRD) through circulating tumor DNA (ctDNA) analysis provides an advanced molecular method for early identification of surgical patients at high risk of recurrence. In this study, we present the development of mClear, a personalized, tumor-informed next-generation sequencing (NGS) assay for MRD detection. Furthermore, we report the analytical and clinical validation processes conducted to evaluate the performance of the MRD assay. Methods: mClear is designed to assess MRD in patients with solid tumors. Whole exome sequencing (WES) was performed on tumor tissues and matched peripheral blood samples to detect somatic variants comprehensively. Personalized MRD assays were carefully designed by selecting up to 40 prioritized variants, including single nucleotide variants (SNVs), small insertions and deletions (InDels), and fusions while ensuring coverage of 21 additional key driver genes. For analytical validation, we utilized a pan-cancer MRD ctDNA quality control product (LDT-999, LDT Bioscience), characterized by known variant allele frequency (VAF) gradients of 0%, 0.005%, 0.05%, and 0.5%, confirmed through droplet digital PCR (ddPCR). We conducted 18 replicate tests at VAF gradients of 0.5% and 0.05%, 23 replicates at the 0.005% VAF gradient, and 16 replicates at the 0% VAF gradient. For clinical validation, preoperative and postoperative blood samples from a cohort of 264 pan-cancer patients were analyzed. ctDNA collected for MRD detection underwent ultra-deep next-generation sequencing (100,000× average depth of coverage). The library preparation process commenced with an input of 30ng DNA. Results: At VAF gradients of 0.5% and 0.05%, the assay achieved an excellent sensitivity of 100%, and at VAF gradients of 0.005% and 0%, it demonstrated a sensitivity of 95.7% and a specificity of 100%. Preoperative ctDNA was detected in 86.7% (72/83) of the pan-cancer patients with 71.4% (5/7), 90.9% (10/11), 85.5% (47/55), and 100.0% (10/10) in pathological stage (pStage)I, pStage II, pStage III, and pStage IV, respectively. In the pan-cancer cohort, the positive rate of longitudinal MRD was 35.7% (75/210) with 17.8% (21/118), 50.0% (13/26), 55.1% (27/49), and 82.4% (14/17) in pStage I, II, III, and IV, respectively. In four patients who relapsed, MRD detection during surveillance preceded radiological recurrence by a lead time of 1-8 months. Conclusions: Our personalized MRD assay demonstrated excellent analytical sensitivity and specificity with a limit of detection (LoD) of 0.005% VAF at the sample level. Furthermore, it displayed high clinical sensitivity for the detection of ctDNA in the pan-cancer cohort, highlighting its potential for monitoring of recurrence.

Clinical validation of a novel circulating tumor DNA methylation assay for predicting recurrence of colorectal cancer in patients with curative-intent surgery by monitoring minimal residual disease.

3620 Background: Circulating tumor DNA (ctDNA)-based liquid biopsy has emerged as a promising tool for early detection and monitoring of cancer. In this study, we developed COLO eDX, a digital-PCR based colorectal cancer (CRC) diagnostic assay using a single CRC-specific methylation biomarker. This study aimed to evaluate the prognostic value as the universal assay for recurrence monitoring in CRC patients with curative-intent surgery. Methods: A total of 116 CRC patients who underwent surgery at Seoul National University Boramae Medical Center were enrolled in the study from Mar 2017 to January 2020. For each patient, postoperative plasma specimens were collected at 7 days and at each regular clinical follow-up visit up to 2 years. The enrolled patients were followed up with radiological and clinical measures for maximally 5 years after surgery. The primary outcome was recurrence-free survival (RFS) according to ctDNA status. Results: The majority of samples collected at 7 days after surgery were ctDNA positive by COLO eDX (65/83, 78.3%) and the ctDNA status was not significantly associated with RFS, suggesting that cancer signatures can still be detected in most cancer patients at least 1 week after surgery. However, ctDNA positivity at 1 month or later was associated significantly worse RFS. In plasma taken between 1- and 9-months after surgery, ctDNA positive group had a significantly higher recurrence rate than ctDNA negative group (hazard ratio [HR], 11.4, 95% confidence interval [CI], 3.8-34.4; P&lt;0.0001). Incorporating serial longitudinal samples, ctDNA status was significantly associated with recurrence (HR, 13.4, 95% CI, 4.3-40.6; P&lt;0.0001). Notably, ctDNA analysis by COLO eDX was a more powerful predictor for predicting recurrence than carcinoembryonic antigen (CEA) assessment (HR, 4.0, 95% CI, 1.4-11.8; P=0.0107 and HR, 1.5, 95% CI, 0.3-8.2; P=0.5949). Conclusions: Postoperative ctDNA monitoring using a CRC-specific epigenetic biomarker-based digital PCR assay is a promising strategy for recurrence prediction and personalized clinical management of CRC through early detection of minimal residual disease (MRD).

Impact of positive tumor-informed margin and TP53 mutations on surgical outcomes in esophageal squamous cell carcinoma.

e16024 Background: The complete removal of esophageal squamous cell carcinoma (ESCC) with clear margins (R0 resection) is pivotal for patient prognosis. However, it is limited by the potential inaccuracies of conventional histopathology. Next-generation sequencing (NGS) for minimal residual disease (MRD) detection might improve the prediction of postoperative outcomes. Methods: The study cohort included a total of 48 operable ESCC patients, from whom resected esophageal tumors, resection margins, and normal tissues were collected and profiled by a 425-gene panel. Comparisons of mutational features were conducted across various tissue types, and their associations with disease-free survival (DFS) were explored. Results: Mutations were detected in 81.8% (27/33) of normal tissues, 95.8% (46/48) of tumors, and 50.0% (24/48) of resection margins. In general, genetic alterations in resection margins closely resembled those in tumor tissues, but were distinct from normal tissues. Both tumor and margin samples showed higher variant allele frequencies (VAFs) and fewer mutations per patient than normal samples ( p &lt; 0.0001). Moreover, eight patients exhibited identical mutations in paired margin and tumor samples. Notably, one patient out of eight with the same TP53 p.T175H mutation across all sample types exhibited the shortest DFS of only two months. Mutations in TP53 and NOTCH1 were the most common, displaying unique mutational patterns among different sample types. While NOTCH1 mutations were detected more frequently in normal tissues ( p = 0.001), TP53 mutations were enriched in tumors ( p = 0.04). Furthermore, normal tissues exhibited a distinct pattern of mutation sites for TP53 and NOTCH1 compared to those observed in tumor or margin tissues. We characterized tumor-informed margin (TiM) by the presence of identical mutations in both tumor and adjacent margins. The positive TiM status was associated with a shorter DFS, particularly when considering TP53 mutations alone ( p = 0.022) or excluding NOTCH1 mutations ( p = 0.031). Conclusions: A positive TiM status with TP53 mutations is a promising biomarker indicating poor surgical outcomes in ESCC. Mutations within NOTCH1 are common but showed no association with DFS. These findings provide novel insights into the feasibility of genetic profiling of resection margins to facilitate optimizing postoperative therapeutic regimens for ESCC.

Ultrasensitive circulating tumor DNA (ctDNA) minimal residual disease (MRD) detection in early stage non-small cell lung cancer (NSCLC).

8078 Background: Translation of ctDNA MRD in NSCLC has been hampered by suboptimal sensitivity of 1st-generation assays. Here, we explore how improvements in analytical sensitivity can drive improved clinical sensitivity for MRD after surgery in NSCLC. Methods: To understand MRD kinetics, we analyzed longitudinal ctDNA in early stage NSCLC using data from the TRACERx study. Patient-specific mathematical models were generated to predict MRD levels at the postsurgical landmark and estimate the impact of an assay’s 95% limit of detection (LOD95) on clinical sensitivity. To test these predictions, tumor-informed ctDNA testing was performed using an SNV-based assay (CAPP-Seq) and a phased variant-based assay (PhasED-Seq, Foresight Diagnostics) on 269 samples from 46 patients. We also assessed MRD performance for predicting 1) patient outcomes at the landmark timepoint and 2) the effect of adjuvant treatment. Results: ctDNA MRD dynamics were assessed in 23 patients from TRACERx with ≥3 consecutive samples with detectable ctDNA without intervening therapy. MRD kinetics strongly correlated with exponential growth, with a median ctDNA doubling time of 51 days. Extrapolating MRD levels to the postsurgical landmark predicted that improving MRD assay LOD95 from 100 ppm (0.01%) to 1 ppm could increase clinical sensitivity by 2.1-fold. We then evaluated 46 NSCLC cases using two assays. The median LOD95 was 1 ppm for PhasED-Seq and 84 ppm for CAPP-Seq. Twelve cases were MRD+ by PhasED-Seq, all of whom recurred (100%), with MRD levels as low as 0.19 ppm. In contrast, 6 cases were MRD+ by the SNV-based approach, of whom 5 (83%) recurred. Accordingly, PhasED-Seq had a higher clinical sensitivity than the SNV-based method (12/18 [67%] vs. 5/18 [28%], P = 0.022). Kaplan Meier analysis for freedom from recurrence revealed significantly worse outcomes for patients with detectable MRD regardless of the method used; however, outcomes were better stratified using PhasED-Seq (HR 3.1 vs. 11.4). Patients who were MRD- after surgery by both assays had similar outcomes regardless of adjuvant therapy (chemotherapy and/or radiotherapy). However, MRD+ patients by PhasED-Seq receiving adjuvant therapy had significantly better outcomes than those who did not (HR 8.2, P = 0.00035). A similar benefit for adjuvant therapy was not observed with the SNV-based assay. Using PhasED-Seq, 80% (4/5) of MRD+ patients receiving adjuvant therapy cleared their MRD, compared to 0% (0/3) without adjuvant treatment. Conclusions: Ultrasensitive ctDNA detection improved the clinical sensitivity of MRD at key landmarks in early stage NSCLC. PhasED-Seq detected MRD at levels below 1 ppm and was associated with significantly better outcomes, revealing potential benefits of adjuvant therapy in MRD+ patients. This suggests that ultrasensitive MRD detection is promising for use in risk-adapted trials in early stage NSCLC.

Advances in Liquid Biopsy for Diagnosis of Bladder Cancer.

Bladder cancer (BCa) is the most common malignancy of the urinary system. It has a high recurrence rate and requires longterm follow-up. Significant advances in BCa research have been made in recent years; however, the initial diagnosis and follow-up of BCa relies on cystoscopy, which is an invasive and expensive procedure. Over the past decade, liquid biopsies (e.g., blood and urine) have proven to be highly efficient methods for the discovery of BCa biomarkers. This noninvasive sampling method is used to analyze unique tumor components released into body fluids and enables serial sampling and longitudinal monitoring of tumor progression. Several liquid biopsy biomarkers have been studied extensively and have shown promising results in the clinical applications of BCa, including early detection, microscopic residual disease detection, recurrence prediction, and treatment response. Therefore, this review aims to provide an update on various new liquid biopsy markers and the advantages and current limitations of liquid biopsy in the diagnosis of BCa.

Liquid biopsy for molecular characterization of diffuse large B-cell lymphoma and early assessment of minimal residual disease.

Circulating tumour DNA (ctDNA) allows genotyping and minimal residual disease (MRD) detection in lymphomas. Using a next-generation sequencing (NGS) approach (EuroClonality-NDC), we evaluated the clinical and prognostic value of ctDNA in a series of R-CHOP-treated diffuse large B-cell lymphoma (DLBCL) patients at baseline (n = 68) and after two cycles (n = 59), monitored by metabolic imaging (positron emission tomography combined with computed tomography [PET/CT]). A molecular marker was identified in 61/68 (90%) ctDNA samples at diagnosis. Pretreatment high ctDNA levels significantly correlated with elevated lactate dehydrogenase, advanced stage, high-risk International Prognostic Index and a trend to shorter 2-year progression-free survival (PFS). Valuable NGS data after two cycles of treatment were obtained in 44 cases, and 38 achieved major molecular response (MMR; 2.5-log drop in ctDNA). PFS curves displayed statistically significant differences among those achieving MMR versus those not achieving MMR (2-year PFS of 76% vs. 0%, p < 0.001). Similarly, more than 66% reduction in ΔSUVmax by PET/CT identified two subgroups with different prognosis (2-year PFS of 83% vs. 38%; p < 0.001). Combining both approaches MMR and ΔSUVmax reduction, a better stratification was observed (2-year PFS of 84% vs. 17% vs. 0%, p < 0.001). EuroClonality-NDC panel allows the detection of a molecular marker in the ctDNA in 90% of DLBCL. ctDNA reduction at two cycles and its combination with interim PET results improve patient prognosis stratification.

FLT3-TKD Measurable Residual Disease Detection Using Droplet Digital PCR and Clinical Applications in Acute Myeloid Leukemia.

The tyrosine kinase domain of the FMS-Like tyrosine kinase 3 (FLT3-TKD) is recurrently mutated in acute myeloid leukemia (AML). Common molecular techniques used in its detection include PCR and capillary electrophoresis, Sanger sequencing and next-generation sequencing with recognized sensitivity limitations. This study aims to validate the use of droplet digital PCR (ddPCR) in the detection of measurable residual disease (MRD) involving the common FLT3-TKD mutations (D835Y, D835H, D835V, D835E). Twenty-two diagnostic samples, six donor controls, and a commercial D835Y positive control were tested using a commercial Bio-rad® ddPCR assay. All known variants were identified, and no false positives were detected in the wild-type control (100% specificity and sensitivity). The assays achieved a limit of detection suitable for MRD testing at 0.01% variant allelic fraction. Serial samples from seven intensively-treated patients with FLT3-TKD variants at diagnosis were tested. Five patients demonstrated clearance of FLT3-TKD clones, but two patients had FLT3-TKD persistence in the context of primary refractory disease. In conclusion, ddPCR is suitable for the detection and quantification of FLT3-TKD mutations in the MRD setting; however, the clinical significance and optimal management of MRD positivity require further exploration.

Increased blood draws for ultrasensitive ctDNA and CTCs detection in early breast cancer patients

Early breast cancer patients often experience relapse due to residual disease after treatment. Liquid biopsy is a methodology capable of detecting tumor components in blood, but low concentrations at early stages pose challenges. To detect them, next-generation sequencing has promise but entails complex processes. Exploring larger blood volumes could overcome detection limitations. Herein, a total of 282 high-volume plasma and blood-cell samples were collected for dual ctDNA/CTCs detection using a single droplet-digital PCR assay per patient. ctDNA and/or CTCs were detected in 100% of pre-treatment samples. On the other hand, post-treatment positive samples exhibited a minimum variant allele frequency of 0.003% for ctDNA and minimum cell number of 0.069 CTCs/mL of blood, surpassing previous investigations. Accurate prediction of residual disease before surgery was achieved in patients without a complete pathological response. A model utilizing ctDNA dynamics achieved an area under the ROC curve of 0.92 for predicting response. We detected disease recurrence in blood in the three patients who experienced a relapse, anticipating clinical relapse by 34.61, 9.10, and 7.59 months. This methodology provides an easily implemented alternative for ultrasensitive residual disease detection in early breast cancer patients.

Role of biomarkers in the diagnosis and management of HCC.

For many cancers, biomarkers have served as an important tool across the cancer care continuum from risk stratification and early detection to diagnosis and treatment. Alpha fetoprotein (AFP) remains one of the few validated biomarkers for patients with hepatocellular carcinoma (HCC). Although AFP has shown potential for each of these steps, its performance when used alone has often been suboptimal. There continues to be discordant recommendations about AFP's value when combined with ultrasound for surveillance as well as its role in diagnostic algorithms. Conversely, high AFP levels are associated with aggressive tumor biology and survival, so it remains a key factor for selection of liver transplant candidates. There have been immense efforts to identify and validate additional biomarkers for each of these steps in the HCC care continuum. Indeed, biomarker panels have shown promising data for HCC risk stratification and surveillance among patients with cirrhosis, as well as prognostication and detection of minimal residual disease in patients undergoing HCC treatment. Several large prospective studies to evaluate the role of these emerging biomarkers in clinical practice are currently ongoing.

Bulk lysis procedures alter target cell population counts.

To achieve high-sensitivity cell measurements (<1 in 105 cells) by flow cytometry (FCM), the minimum number of acquired cells must be considered and conventional immunophenotyping protocols fall short of these numbers. The bulk lysis (BL) assay is a standardized erythrocyte lysing approach that allows the analysis of the millions of cells required for high-sensitivity measurable residual disease (MRD) detection. However, this approach has been associated with significant cell loss, along with potential over or underestimates of rare cells when using this method. The aim of this study was to evaluate bulk lysis protocols and compare them with minimal sample perturbation (MSP) protocols, which are reported to better preserve the native cellular state and avoid significant cell loss due to washing steps. To achieve this purpose, we first generated an MRD model by spiking fresh peripheral blood with K562 cells, stably expressing EGFP, at known percentages of EGFP positive cells to leukocytes. Samples were then prepared with BL and MSP protocols and analyzed using FCM. For all percentages of K562 cells established and evaluated, a significant decrease of this population was detected in BL samples compared with MSP samples, even at low K562 cell percentages. Significant decreases for non-necrotic cells were also observed in BL samples relative to MSP samples. In conclusion, the evaluation of the potential effects of BL protocols in obtaining the final count is of great interest, especially for over- or under-estimation of target cells, as in the case of measurable residual disease. Since conventional flow cytometry or minimal sample perturbation assays fall short in obtaining the minimum numbers required to reach high sensitivity measurements, significant efforts may be needed to improve bulk lysis solution reagents.