Presence Of ctDNA Research Articles

Circulating tumor DNA detection after neoadjuvant treatment and surgery predicts recurrence in patients with early-stage and locally advanced rectal cancer

IntroductionPatients with early-stage and locally advanced rectal cancer are often treated with neoadjuvant therapy followed by surgery or watch and wait. This study evaluated the role of circulating tumor DNA (ctDNA) to measure disease after neoadjuvant treatment and surgery to optimize treatment choices. Materials and methodsPatients with rectal cancer treated with both chemotherapy and radiotherapy were included and diagnostic biopsies were analyzed for tumor-specific mutations. Presence of ctDNA was measured in plasma by tracing the tumor-informed mutations using a next-generation sequencing panel. The association between ctDNA detection and clinicopathological characteristics and progression-free survival was measured. ResultsBefore treatment ctDNA was detected in 69% (35/51) of patients. After neoadjuvant therapy ctDNA was detected in only 15% (5/34) of patients. In none of the patients with a complete clinical response who were selected for a watch and wait strategy (0/10) or patients with ypN0 disease (0/8) ctDNA was detected, whereas it was detected in 31% (5/16) of patients with ypN + disease. After surgery ctDNA was detected in 16% (3/19) of patients, of which all (3/3) developed recurrent disease compared to only 13% (2/16) in patients with undetected ctDNA after surgery. In an exploratory survival analysis, both ctDNA detection after neoadjuvant therapy and after surgery was associated with worse progression-free survival (p = 0.01 and p = 0.007, respectively, Cox-regression). ConclusionThese data show that in patients with early-stage and locally advanced rectal cancer tumor-informed ctDNA detection in plasma using ultradeep sequencing may have clinical value to complement response prediction after neoadjuvant therapy and surgery.

Emerging trends in the prediction of pathological tumour response in rectal cancer following neoadjuvant therapy.

The treatment landscape of locally advanced rectal cancer (LARC) is moving towards total neoadjuvant therapy and potential organ preservation. Multimodality neoadjuvant therapy (NAT) and surgery remain the standard-of-care for LARC, with consideration for adjuvant chemotherapy. Neoadjuvant chemotherapy alone is another emerging strategy.1 Due to variation in multimodality treatment choice, biomarkers to predict response to NAT could personalize treatment plans for patients.2 Of particular interest are predictors of pathologic complete response (pCR) to guide selection for a watch and wait approach, or identify those who would benefit from surgery due to poor response to NAT or a higher risk of disease relapse. Tumour response to NAT is a complex interplay between variables including tumour biology, patient factors, and the sequencing and timing of the selected NAT approach. Presently available modalities (MRI and endoscopy) can aid assessment but do not predict for tumour response. There are currently no clinically actionable biomarkers to predict NAT response, however several promising contenders are emerging.2 Circulating tumour DNA (ctDNA) has been shown as a risk-stratification tool to guide adjuvant therapy choice in patients following colon cancer resection.3 The presence of ctDNA following long course chemoradiation and surgery is an adverse prognostic factor.4 Evidence for ctDNA in the neoadjuvant setting for LARC is also evolving. As a marker for minimal residual disease, studies have demonstrated that detectable ctDNA predicts for poorer response to NAT and potentially identifies patients that should proceed to surgery rather than a watch and wait strategy.5, 6 Other biochemical markers such as microRNA and tumour-infiltrating lymphocytes are also being evaluated.7, 8 The gut microbiome has a role in the carcinogenesis of colorectal cancer, and potentially predicts response to anticancer therapy through the effect of certain bacteria on pharmacokinetics, pharmacodynamics and immune response.9 However, questions remain regarding the specific microbiome biomarkers that can predict LARC response to NAT and its utility in clinical practice is currently limited.9 Immunogenomics is becoming increasingly relevant to the management of LARC, with immune checkpoint inhibitors exhibiting high response rates in mismatch repair deficient (dMMR) tumours and 100% clinical complete response in dMMR LARC.10 Initiatives such as the Cancer Genome Atlas Program are genomically sequencing colorectal cancers to identify genomic alterations beyond microsatellite instability that may predict responsiveness to immunotherapy and the likelihood of a pCR.11 Radiomics is a process whereby disease features, or ‘radiological biomarkers’ are extracted from standard patient imaging, constructing an algorithm which can then aid in predicting treatment response.12 T2 weighted MRI-based software for LARC successfully predicts pCR and nodal status post-NAT.13 Combined radiomics with both multi-parameter MRI (T1/T2/Contrast enhanced) and functional PET-CT may provide better predictive value for pCR.14 Currently, the widespread adoption of radiomics has been hindered by the technological and cost burden, limited reproducibility of data, and time-intensive manual data extraction.13 Larger prospective trials are required to validate its use in LARC management.13, 15 With the adoption of Artificial Intelligence (AI) and deep learning, data extraction will become increasingly accessible and construct reproducible algorithms,13 making clinical application feasible. Body composition measurement based on standard CT-staging is another ‘radiological biomarker’.16 Body composition is more accurate than body surface area for systemic therapy dosing, and predicting poor tolerance to chemotherapy in colorectal cancer.17 Recent evidence has suggested that skeletal muscle area pre-NAT can predict tumour response and survival outcomes for LARC, presenting a promising area for future studies.16 In summary, multiple promising biomarkers are emerging to identify the optimal individualized treatment paradigm for patients with LARC. It is hoped that further research and increasing validation of new technologies such as AI, will tailor future personalized decision making for patients with LARC. Open access publishing facilitated by The University of Melbourne, as part of the Wiley - The University of Melbourne agreement via the Council of Australian University Librarians. Matthew Y. Wei: Writing – original draft; writing – review and editing. Yasser Arafat: Validation; writing – review and editing. Margaret Lee: Validation; writing – review and editing. Suzanne Kosmider: Validation; writing – review and editing. Matthew Loft: Validation; writing – review and editing. Ian Faragher: Validation; writing – review and editing. Peter Gibbs: Conceptualization; validation; writing – review and editing. Justin Yeung: Conceptualization; supervision; writing – review and editing.

Prognostic value of postoperative ctDNA detection in patients with early non-small cell lung cancer: a systematic review and meta-analysis.

Circulating tumor DNA (ctDNA) has emerged as a potential biomarker for monitoring early non-small cell lung cancer (ENSCLC), particularly after radical surgery. However, the prognostic value of postoperative ctDNA is still being investigated due to the small sample size and heterogeneity of patients with ENSCLC in current trials. Moreover, the potential clinical utility of ctDNA assessment for administering adjuvant therapy (AT) in patients with ENSCLC is also an important area of active research. We aimed to identify the prognostic value of postoperative ctDNA detection in ENSCLC patients with stages I-III. This study type is a systematic review and meta-analysis. We conducted a search in the Cochrane Library, Embase, PubMed, and ScienceDirect for prospective or retrospective investigations involving patients with ENSCLC, gathering outcomes based on predefined end points. The literature review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, and the Newcastle-Ottawa scale was employed to carry out a quality evaluation of the included studies. The primary end point of the study was to evaluate the association of ctDNA status in two time points (within 1 month after surgery and long-term postoperative monitoring with more than 3 months) with relapse-free survival (RFS) and overall survival (OS). In addition, the study investigated the role of ctDNA in predicting the response to AT. The secondary end points of the study were to determine the impact of ctDNA on RFS and OS in different subgroups of ENSCLC patients based on pathological subtypes and TNM staging. In total, 2149 studies were screened, and 11 studies met the inclusion criteria for the analysis. The presence of ctDNA within 1 month after surgery as well as long-term postoperative ctDNA were both associated with poorer RFS [hazard ratio (HR) = 4.43; 95% CI: 3.23-6.07 and HR = 7.99; 95% CI: 3.28-19.44, respectively] and worse OS (HR = 5.07; 95% CI: 2.80-9.19 and HR = 7.49; 95% CI: 3.42-16.43, respectively). Most subgroup analyses yielded similar results. Moreover, ctDNA-positive patients could acquire survival benefits from AT (HR = 0.30; 95% CI: 0.16-0.54), while ctDNA-negative patients that received AT did not show significant improvement in RFS (HR = 1.18; 95% CI: 0.67-2.09). The postoperative ctDNA assessment is a promising approach to stratify the risk of relapse and death in ENSCLC patients. Our data suggest that patients with negative ctDNA in the postoperative setting may not benefit from AT, which warrants further investigation. This finding, if validated in prospective trials with a larger sample size, could aid in better-individualized treatment for patients and avoid potential side effects of AT. This study was designed in accordance with PRISMA and registered with PROSPERO (CRD42022311615).

Feasibility of Longitudinal ctDNA Assessment in Patients with Uterine and Extra-Uterine Leiomyosarcoma.

Background: Leiomyosarcomas (LMS) are aggressive malignancies with a propensity for early relapse. Current surveillance modalities include physical exam and imaging; however, radiological response to therapy may only manifest after 4-6 cycles of treatment. Herein, we evaluated the feasibility of longitudinal circulating tumor DNA (ctDNA) assessment in LMS patients to identify disease progression. Methods: We performed a retrospective review of patients with LMS who underwent treatment at Stanford Cancer Center between September 2019 and May 2022. ctDNA detection was performed using a personalized, tumor-informed ctDNA assay. Genomic analysis was conducted to characterize tumor mutation burden (TMB) and known driver mutations. Results: A total of 148 plasma samples were obtained from 34 patients with uterine (N = 21) and extrauterine (N = 13) LMS (median follow-up: 67.2 (19-346.3) weeks] and analyzed for ctDNA presence. Nineteen patients had metastatic disease. The most frequently mutated driver genes across sub-cohorts were TP53, RB1, and PTEN. Patients were stratified into four sub-cohorts (A-D) based on ctDNA kinetics. ctDNA levels tracked longitudinally with progression of disease and response to therapy. Conclusion: Our results indicate that while undetectable ctDNA may suggest a lower likelihood of relapse, ctDNA positivity may indicate progressive disease, enabling closer monitoring of patients for early clinical intervention.

Abstract B027: Circulating tumor DNA in colorectal carcinomatosis: Detection and prognostic value

Abstract Background: Circulating tumor DNA (ctDNA) of patients with colorectal peritoneal metastases (PM) was obtained to identify prognostic factors associated for ctDNA detection and with overall survival (OS). Prior studies have identified a correlation between ctDNA detection and OS in colorectal liver metastases (CRLM) and after resection of primary tumors. However, detection rates for PM may be lower than other metastatic sites. Methods: Peripheral blood was drawn from 152 patients after diagnosis with PM with or without other synchronous sites of disease. Patients who underwent cytoreduction were excluded. Next-generation sequencing of a panel of 70 genes commonly mutated in colorectal cancer was performed on all samples. Mutations were identified and variant allele frequencies (VAFs) calculated. Clinical data including OS from time of CRCC diagnosis, tumor grade, tumor histology, synchronous non-peritoneal metastases and patient demographics were obtained from medical records .Chi-squared tests were performed to compare differences in ctDNA detection based on clinical factors, and Cox regression analyses were performed to identify associations with ctDNA characteristics and OS. Results: The overall detection rate of any mutation in ctDNA was 74.3% (110/145 patients). The most frequently detected mutations were TP53 (55%), APC (47%), KRAS (32%), and PIK3CA (14%). Sensitivity for detecting individual mutations ranged from 50%-63%. Mucinous and signet-ring cell histology were more common in patients with PM alone (Mucinous: 37% vs 24%, p=0.0003, Signet Ring Cell: 10% vs. 2.2%, p=0.0001) and associated with lower sensitivity (mucinous: 59%, signet-ring cell: 57% and adenocarcinoma 85%, p=0.002). Synchronous CRLM were associated with increased detection compared to PM alone (88% vs. 68%, p=0.011). After adjustment for differences in histology, synchronous CRLM (OR 3.32, 95CI: 1.52-8.02, p=0.016) was associated with higher detection rates. Grade was not associated with detection (moderately differentiated: 79%, poorly differentiated: 74%, p=0.81). Detection of any ctDNA was associated with a trend towards worse OS (HR 2.18, 95% CI: 0.98-4.95, p=0.06). KRAS (HR 2.02, 95% CI: 1.14-3.60, p=0.017) and PIK3CA (HR 2.26, 95% CI: 1.07-4.78, p=0.033) ctDNA detection was associated worse OS. Detection of multiple mutations (HR: 0.85, 95% CI 0.50-1.42, p=0.52) and maximum VAF (HR 0.99, 95% CI: 0.97- 1.01, p=0.17) were not associated with OS. Conclusion: ctDNA detection is lower in patients with PM alone compared to those with PM and CRLM. Mucinous tumors had lower rates of ctDNA detection after accounting for extra-PM metastases. Presence of ctDNA is associated with a trend towards worse OS, especially for patients with detectable mutations in KRAS or PIK3CA. Quantification of ctDNA by number of mutations or VAF was not associated with OS. Interpretation of ctDNA results for PM patients should take into consideration the lower sensitivity in the absence of other metastatic sites, but may have prognostic utility. Citation Format: Abhineet Uppal, Michael G. White, George J. Chang, Scott Kopetz, John Paul Y. C. Shen. Circulating tumor DNA in colorectal carcinomatosis: Detection and prognostic value [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B027.

Detection of Early Molecular Response (EMR) and Minimal Residual Disease (MRD) in Patients with Diffuse Large B-Cell Lymphoma (DLBCL) Using a Validated Next Generation Sequencing (NGS) Assay for the Detection of Tumor Variants in Circulating Tumor (ct)DNA

DLBCL is the most common type of non-Hodgkin lymphoma in adults. Although frontline standard of care treatment is effective in 60-70% of patients, 10-15% have primary resistance and 20-30% will relapse within 18 months. There is an unmet need to predict treatment refractoriness and relapse in order to intensify treatment approaches early during induction therapy. Early molecular response (EMR), defined as the dynamics of ctDNA after one cycle of immunochemotherapy, was shown to be prognostic in a small cohort of first-line DLBCL patients (Kurtz et. al. J. Clin. Oncol 2018), and recently validated using samples from the POLARIX study (NCT03274492) in previously untreated DLBCL. To support clinical studies, the Roche Molecular CAP/CLIA Laboratory has developed and validated the AVENIO Oncology Assay Non-Hodgkin Lymphoma Test (AOA NHL Test) that uses next generation sequencing of plasma cell-free (cf)DNA and of matched genomic (g)DNA to identify tumor-specific single nucleotide variants (reporter SNVs) in a panel of > 400 genomic regions and to monitor the dynamics of the reporter SNVs on treatment using a Monte-Carlo p-value statistical algorithm (Newman et. al. Nat Biotechnol 2016). The AOA NHL Test determines ctDNA presence or absence as an indicator of MRD, and assigns high-risk or low-risk EMR status in patients with DLBCL by measuring changes in ctDNA burden on treatment and applying a previously established cutoff. The analytical validation studies for the AOA NHL Test were designed to address accuracy, sensitivity, specificity, linearity, and precision of the following 1) variant detection in plasma samples; 2) ctDNA detection (measured as mean allele fraction (AF) of reporter SNVs using a Monte-Carlo p-value statistical algorithm); and 3) classification accuracy of the log-fold change (LFC) of mean mutant molecules per mL of plasma (MMPM), using clinically relevant cutoffs. Validation samples consisted of Genome in a Bottle blended gDNAs, serial dilution of healthy donor cfDNA blends, and serial dilution of DLBCL ctDNA into healthy donor cfDNA. Key results of the analytical validation are shown in Table 1. In summary, analytical validation of the AOA NHL Test demonstrated high sensitivity for detection of individual variants and ctDNA, and the capability of accurately detecting changes in ctDNA levels as measured by LFC in MMPM. The assay sensitivity improves with the increase in cfDNA input mass as well as the number of reporter variants while specificity (i.e. ability to detect signal over noise) can be maintained from sample to sample by pre-selecting a Monte-Carlo p-value cutoff. The final recommended sample acceptance criteria as well as sequencing QC metrics will be presented. Of 858 cases of DLBCL patients examined using the initial assay prototype and the validated AOA NHL Test, including samples from the POLARIX biomarker validation study, 846 (98.6%) had identifiable reporter variants in pre-treatment plasma samples with a median number of reporter SNVs of 137 per patient (range 1-1039), demonstrating that almost 100% of patients can be monitored with this assay. Of the 12 patients with no detectable reporters, 11 had less than 10 ng cfDNA available, highlighting the importance of sufficient sample input criteria established by analytical validation. The test is currently being used in a global prospective Phase 2 clinical trial (NCT04980222) to test the safety and efficacy of glofitamab in combination with R-CHOP in patients with High Risk EMR status. Acknowledgements: Alex Lovejoy for advice on the analytical validation design, Suzanne Cheng, Jennifer Holmstrom and Jill Ray for program management, and Bowdoin Su and Yanwen Jiang for abstract preparation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Personalized Liquid Biopsy May Predict Relapse in Aggressive Endometrial Cancer

Abstract Introduction/Objective High-grade endometrial cancer (EC) recurs in 50% of patients following surgery and first-line chemotherapy. There are no established blood-based biomarkers with sufficient specificity and sensitivity to monitor EC patients for residual disease and recurrence. The ability to detect and monitor EC currently relies on serial imaging based approaches. Liquid biopsies via blood-based tests for cell-free circulating tumor DNA (ctDNA) are becoming increasingly popular, but none have been FDA approved for EC and current approaches have not been validated in patients with uncommon tumor mutations. We hypothesized that personalized testing panels based on tumor-specific chromosomal rearrangements would allow ctDNA to be monitored and that ctDNA presence correlates with other clinical indicators of recurrence. Methods/Case Report Twelve patients with FIGO Stage I-IV and biopsy confirmed high-grade EC were prospectively enrolled and underwent primary tumor resection. DNA was isolated from tumor tissue and sequenced on Illumina NGS platforms [San Diego, CA]. BIMA algorithm and SVAtools identified junctions of somatic chromosomal rearrangements and 4-5 junctions were chosen per patient, based primarily on copy number/amplification level. Blood was collected before surgery, and longitudinally between 1 and 7 timepoints. Total cell-free DNA (cfDNA) was extracted from platelet poor plasma with the Qiagen CNA kit [Germany]. Personalized ctDNA qPCR Taqman [IDT, Coralville, Iowa] probe assays were developed with pooled pre-amplification to maximize sensitivity for the multiple junctions and normalized to input. Results (if a Case Study enter NA) ctDNA was detected in 9/12 and 5/12 cases pre- and post-surgery, respectively. The detection or absence of measurable ctDNA following surgery showed high correlation with cancer recurrence as detected by CT or MRI scans. Conclusion The detection of ctDNA in high-grade EC using personalized junction panels shows promise for a blood-based assay that may help physicians better monitor and treat patients.

Detection of circulating tumour DNA after neoadjuvant chemoradiotherapy in patients with locally advanced oesophageal cancer.

Active surveillance instead of standard surgery after neoadjuvant chemoradiotherapy (nCRT) has been proposed for patients with oesophageal cancer. Circulating tumour DNA (ctDNA) may be used to facilitate selection of patients for surgery. We show that detection of ctDNA after nCRT seems highly suggestive of major residual disease. Tumour biopsies and blood samples were taken before, and 6 and 12 weeks after, nCRT. Biopsies were analysed with regular targeted next-generation sequencing (NGS). Circulating cell-free DNA (cfDNA) was analysed using targeted NGS with unique molecular identifiers and digital polymerase chain reaction. cfDNA mutations matching pre-treatment biopsy mutations confirmed the presence of ctDNA. In total, 31 patients were included, of whom 24 had a biopsy mutation that was potentially detectable in cfDNA (77%). Pre-treatment ctDNA was detected in nine of 24 patients (38%), four of whom had incurable disease progression before surgery. Pre-treatment ctDNA detection had a sensitivity of 47% (95% CI 24-71) (8/17), specificity of 85% (95% CI 42-99) (6/7), positive predictive value (PPV) of 89% (95% CI 51-99) (8/9), and negative predictive value (NPV) of 40% (95% CI 17-67) (6/15) for detecting major residual disease (>10% residue in the resection specimen or progression before surgery). After nCRT, ctDNA was detected in three patients, two of whom had disease progression. Post-nCRT ctDNA detection had a sensitivity of 21% (95% CI 6-51) (3/14), specificity of 100% (95% CI 56-100) (7/7), PPV of 100% (95% CI 31-100) (3/3), and NPV of 39% (95% CI 18-64) (7/18) for detecting major residual disease. The addition of ctDNA to the current set of diagnostics did not lead to more patients being clinically identified with residual disease. These results indicate that pre-treatment and post-nCRT ctDNA detection may be useful in identifying patients at high risk of disease progression. The addition of ctDNA analysis to the current set of diagnostic modalities may not improve detection of residual disease after nCRT. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Molecular Characterization of Circulating Tumor DNA in Pediatric Rhabdomyosarcoma: A Feasibility Study.

Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted.

Circulating tumor DNA monitoring for early recurrence detection in epithelial ovarian cancer

ObjectiveEpithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. We examined the utility of circulating tumor DNA (ctDNA) as a prognostic biomarker for EOC by assessing its relationship with patient outcome and CA-125, pre-surgically and during post-treatment surveillance. MethodsPlasma samples were collected from patients with stage I-IV EOC. Cohort A included patients with pre-surgical samples (N = 44, median follow-up: 2.7 years), cohort B and C included: patients with serially collected post-surgically (N = 12) and, during surveillance (N = 13), respectively (median follow-up: 2 years). Plasma samples were analyzed using a tumor-informed, personalized multiplex-PCR NGS assay; ctDNA status and CA-125 levels were correlated with clinical features and outcomes. ResultsGenomic profiling was performed on the entire cohort and was consistent with that seen in TCGA. In cohort A, ctDNA-positivity was observed in 73% (32/44) of presurgical samples and was higher in high nuclear grade disease. In cohort B and C, ctDNA was only detected in patients who relapsed (100% sensitivity and specificity) and preceded radiological findings by an average of 10 months. The presence of ctDNA at a single timepoint after completion of surgery +/− adjuvant chemotherapy and serially during surveillance was a strong predictor of relapse (HR:17.6, p = 0.001 and p < 0.0001, respectively), while CA-125 positivity was not (p = 0.113 and p = 0.056). ConclusionsThe presence of ctDNA post-surgically is highly prognostic of reduced recurrence-free survival. CtDNA outperformed CA-125 in identifying patients at highest risk of recurrence. These results suggest that monitoring ctDNA could be beneficial in clinical decision-making for EOC patients.

Circulating Tumor DNA-Based Disease Monitoring of Patients with Locally Advanced Esophageal Cancer.

Simple SummaryThe standard of care for patients diagnosed with locally advanced esophageal cancer is neoadjuvant chemoradiotherapy followed by surgery. There is a high clinical need to monitor response to neoadjuvant treatment and recognize patients at risk for recurrence to enable individual treatment strategies. Ultradeep sequencing-based detection of circulating tumor DNA in preoperative plasma of patients with locally advanced esophageal cancer can predict which patients have a high risk of recurrence after neoadjuvant chemoradiotherapy and surgery. Circulating tumor DNA-based prediction of the presence of distant metastasis might eventually be used to reconsider surgery and its associated morbidity in patients with detected circulating tumor DNA or stratify patients for adjuvant treatment.Patients diagnosed with locally advanced esophageal cancer are often treated with neoadjuvant chemoradiotherapy followed by surgery. This study explored whether detection of circulating tumor DNA (ctDNA) in plasma can be used to predict residual disease during treatment. Diagnostic tissue biopsies from patients with esophageal cancer receiving neoadjuvant chemoradiotherapy and surgery were analyzed for tumor-specific mutations. These tumor-informed mutations were used to measure the presence of ctDNA in serially collected plasma samples using hybrid capture-based sequencing. Plasma samples were obtained before chemoradiotherapy, and prior to surgery. The association between ctDNA detection and progression-free and overall survival was measured. Before chemoradiotherapy, ctDNA was detected in 56% (44/78) of patients and detection was associated with tumor stage and volume (p = 0.05, Fisher exact and p = 0.02, Mann-Whitney, respectively). After chemoradiotherapy, ctDNA was detected in 10% (8/78) of patients. This preoperative detection of ctDNA was independently associated with recurrent disease (hazard ratio 2.8, 95% confidence interval 1.1–6.8, p = 0.03, multivariable Cox-regression) and worse overall survival (hazard ratio 2.9, 95% confidence interval 1.2–7.1, p = 0.02, multivariable Cox-regression).Ultradeep sequencing-based detection of ctDNA in preoperative plasma of patients with locally advanced esophageal cancer may help to assess which patients have a high risk of recurrence after neoadjuvant chemoradiotherapy and surgery.

Circulating Tumor DNA After Neoadjuvant Chemotherapy in Breast Cancer Is Associated With Disease Relapse.

Detection of circulating tumor DNA (ctDNA) after neoadjuvant chemotherapy in patients with early-stage breast cancer may allow for early detection of relapse. In this study, we analyzed ctDNA using a personalized, tumor-informed multiplex polymerase chain reaction-based next-generation sequencing assay. Plasma samples (n = 157) from 44 patients were collected before neoadjuvant therapy (baseline), after neoadjuvant therapy and before surgery (presurgery), and serially postsurgery including a last follow-up sample. The primary end point was event-free survival (EFS) analyzed using Cox regression models. Thirty-eight (86%), 41 (93%), and 38 (86%) patients had baseline, presurgical, and last follow-up samples, respectively. Twenty patients had hormone receptor-positive/human epidermal growth factor receptor 2-negative, 13 had triple-negative breast cancer, and 11 had human epidermal growth factor receptor 2-positive disease. Baseline ctDNA detection was observed in 22/38 (58%) patients and was significantly associated with Ki67 > 20% (P = .036) and MYC copy-number gain (P = .0025, false discovery rate = 0.036). ctDNA detection at presurgery and at last follow-up was observed in 2/41 (5%) and 2/38 (5%) patients, respectively. Eight relapses (seven distant and one local) were noted (median follow-up 3.03 years [range, 0.39-5.85 years]). After adjusting for pathologic complete response (pCR), ctDNA detection at presurgery and at last follow-up was associated with shorter EFS (hazard ratio [HR], 53; 95% CI, 4.5 to 624; P < .01, and HR, 31; 95% CI, 2.7 to 352; P < .01, respectively). Association between baseline detection and EFS was not observed (HR, 1.4; 95% CI, 0.3 to 5.9; P = .67). The presence of ctDNA after neoadjuvant chemotherapy is associated with relapse in early-stage breast cancer, supporting interventional trials for testing the clinical utility of ctDNA monitoring in this setting.

High-Sensitivity Mutation Analysis of Cell-Free DNA for Disease Monitoring in Endometrial Cancer.

We sought to determine whether sequencing analysis of circulating cell-free DNA (cfDNA) in patients with prospectively accrued endometrial cancer captures the mutational repertoire of the primary lesion and allows for disease monitoring. Peripheral blood was prospectively collected from 44 newly diagnosed patients with endometrial cancer over a 24-month period (i.e., baseline, postsurgery, every 6 months after). DNA from the primary endometrial cancers was subjected to targeted next-generation sequencing (NGS) of 468 cancer-related genes, and cfDNA to a high-depth NGS assay of 129 genes with molecular barcoding. Sequencing data were analyzed using validated bioinformatics methods. cfDNA levels correlated with surgical stage in endometrial cancers, with higher levels of cfDNA being present in advanced-stage disease. Mutations in cfDNA at baseline were detected preoperatively in 8 of 36 (22%) patients with sequencing data, all of whom were diagnosed with advanced-stage disease, high tumor volume, and/or aggressive histologic type. Of the 38 somatic mutations identified in the primary tumors also present in the cfDNA assay, 35 (92%) and 38 (100%) were detected at baseline and follow-up, respectively. In 6 patients with recurrent disease, changes in circulating tumor DNA (ctDNA) fraction/variant allele fractions in cfDNA during follow-up closely mirrored disease progression and therapy response, with a lead time over clinically detected recurrence in two cases. The presence of ctDNA at baseline (P < 0.001) or postsurgery (P = 0.014) was significantly associated with reduced progression-free survival. cfDNA sequencing analysis in patients with endometrial cancer at diagnosis has prognostic value, and serial postsurgery cfDNA analysis enables disease and treatment response monitoring. See related commentary by Grant et al., p. 305.

Circulating Tumor DNA as a Biomarker for Monitoring Patients with Solid Cancers: Comparison with Standard Protein Biomarkers.

Protein-based biomarkers are widely used in monitoring patients with diagnosed cancer. These biomarkers however, lack specificity for cancer and have poor sensitivity in detecting early recurrences and monitoring therapy effectiveness. Emerging data suggest that the use of circulating tumor DNA (ctDNA) has several advantages over standard biomarkers. Following curative-intent surgery for cancer, the presence of ctDNA is highly predictive of early disease recurrence, while in metastatic cancer an early decline in ctDNA following the initiation of treatment is predictive of good outcome. Compared with protein biomarkers, ctDNA provides greater cancer specificity and sensitivity for detecting early recurrent/metastatic disease. Thus, in patients with surgically resected colorectal cancer, multiple studies have shown that ctDNA is superior to carcinoembryonic antigen (CEA) in detecting residual disease and early recurrence. Similarly, in breast cancer, ctDNA was shown to be more accurate than carbohydrate antigen 15-3 (CA 15-3) in detecting early recurrences. Other advantages of ctDNA over protein biomarkers in monitoring cancer patients include a shorter half-life in plasma and an ability to predict likely response to specific therapies and identify mechanisms of therapy resistance. However, in contrast to proteins, ctDNA biomarkers are more expensive to measure, less widely available, and have longer turnaround times for reporting. Furthermore, ctDNA assays are less well standardized. Because of their advantages, it is likely that ctDNA measurements will enter clinical use in the future, where they will complement existing biomarkers and imaging in managing patients with cancer. Hopefully, these combined approaches will lead to a better outcome for patients.

Probing the Molecular Assembly of a Metabolizer Drug with β-Cyclodextrin and Its Binding with CT-DNA in Augmenting Antibacterial Activity and Photostability by Physicochemical and Computational Methodologies.

The assembly of an inclusion complex in an aqueous medium using a metabolizer drug (dyphylline) as guest and β-cyclodextrin as host has been established, which is extremely appropriate for a variety of applications in modern biomedical sciences. The formation of the inclusion complex is established by 1H NMR, and surface tension and conductivity measurements demonstrate that the inclusion complex was produced with 1:1 stoichiometry. The thermodynamic parameters based on density, viscosity, and refractive index measurements were used to determine the nature of the complex. This research also forecasts how dyphylline will release in the presence of CT-DNA without any chemical modifications. The produced insertion complex (IC) has a higher photostability due to the drug dyphylline being protected by β-CD. The antibacterial activity of dyphylline greatly improved after complexation and exhibited higher toxicity against Gram-negative (highest against Escherichia coli) in comparison to Gram-positive bacteria. The encapsulation mode of the dyphylline molecule into the cavity of the β-CD was also investigated using DFT to confirm preliminary results.

Fluorescent Calixarene-Schiff as a Nanovehicle with Biomedical Purposes

Gene therapy is a technique that is currently under expansion and development. Recent advances in genetic medicine have paved the way for a broader range of therapies and laid the groundwork for next-generation technologies. A terminally substituted difluorene-diester Schiff Base calix[4]arene has been studied in this work as possible nanovector to be used in gene therapy. Changes to luminescent behavior of the calixarene macrocycle are reported in the presence of ct-DNA. The calixarene macrocycle interacts with calf thymus DNA (ct-DNA), generating changes in its conformation. Partial double-strand denaturation is induced at low concentrations of the calixarene, resulting in compaction of the ct-DNA. However, interaction between calixarene molecules themselves takes place at high calixarene concentrations, favoring the decompaction of the polynucleotide. Based on cytotoxicity studies, the calixarene macrocycle investigated has the potential to be used as a nanovehicle and improve the therapeutic efficacy of pharmacological agents against tumors.

Abstract 3405: Characterizing treatment response in head and neck cancer through viral ctDNA quantification and fragment length

Abstract Background: Head and neck cancer (HNC) is the sixth most common cancer type worldwide, and despite decreases in the rates of Human Papillomavirus (HPV)-negative HNC, an alarming increase in HPV+ HNC is occurring. Circulating tumor (ct)DNA isolated from liquid biopsy has been shown to have clinical utility as a biomarker in cancer, with ctDNA levels and fragment size used to monitor treatment response. Methods: The aim of this study was to determine the presence of ctDNA in liquid biopsy (plasma and saliva) of HPV+ HNC patients to non-invasively monitor treatment response and disease progression. Blood and saliva samples were collected from 45 HPV+ HNC patients and 14 HPV-negative controls at the McGill University Health Centre before and/or after surgical resection. Samples were analyzed using droplet digital PCR (ddPCR) with primers and probes for HPV16/HPV18, the most common subtypes of HPV in HNC, accounting for 97% of cases in North America. Analysis was performed for all patients with confirmed follow-up scans and/or pathology. ctDNA levels were correlated with disease course as determined through imaging (&amp;gt;6 months post-sampling) and pathological examination. Results: ctDNA was detectable in 21/23 (91%) patients prior to treatment and 8/36 (22%) post treatment. In the two patients who were not positive for ctDNA prior to treatment, tumor tissue was found to be negative for HPV DNA despite being p16-positive by immunohistochemistry. Patients showed shifts in ctDNA fragment length in longitudinal samples following treatment. All post-treatment patients with no detectable ctDNA had no signs of recurrence/residual disease on follow-up imaging. In contrast, all 8 patients who tested positive for ctDNA post-treatment showed signs of recurrence on follow-up imaging. In our cohort, 14 patients had matched pre- and post-treatment samples and all showed major reductions in ctDNA post treatment compared to pre-treatment, with 12/14 patients having a complete loss of detectable ctDNA. Concordance between the presence of ctDNA in saliva and plasma was found in 86% of patients. ctDNA was only detected in one analyte in 6 patients (3 in plasma only and 3 in saliva only). Conclusion: HPV ctDNA was successfully detected in saliva and blood of patients with HPV-positive HNC, with a sensitivity of 92% for ctDNA prior to treatment, and a sensitivity of 100% when adjusted for tumor HPV status as opposed to p16 staining. The inclusion of both plasma and saliva in analysis increases the sensitivity of assays, with the use of both analytes allowing for the detection of ctDNA in patients with low plasma DNA levels and for patients who did not have salivary ctDNA, potentially due to inability to produce sufficient saliva or tumor location. The presence of ctDNA correlated with treatment response, indicating the potential of HPV ctDNA for monitoring tumor progression in HPV-positive HNC patients. Citation Format: Sarah Tadhg Ferrier, Anthony Zeitouni, Nader Sadeghi, Thupten Tsering, Julia V. Burnier. Characterizing treatment response in head and neck cancer through viral ctDNA quantification and fragment length [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3405.

Abstract 3403: Multiomic, plasma-only circulating tumor DNA (ctDNA) assay identifies breast cancer patients with minimal residual disease (MRD) and predicts distant recurrence

Abstract Background: Detection of MRD after curative intent treatment may identify patients at high risk for recurrence. Most ctDNA-based MRD assays require a priori knowledge of genomic alterations from tumor tissue to achieve acceptable sensitivity and specificity. However, tissue availability may be limited in some patients with breast cancer, particularly following neoadjuvant therapy. Here we report results of a pilot study evaluating a plasma-only multiomic ctDNA MRD assay and its association with breast cancer recurrence. Methods: 47 plasma samples from 38 patients with early-stage breast cancer were collected through the BRandO BiO Registry at 12- or 36-months post-diagnosis and/or at the time of clinical recurrence. BRandO BiO is a multi-center regional registry with longitudinal biobanking from patients with newly diagnosed breast cancer at 20 affiliated network hospitals in Germany. The presence of ctDNA was evaluated using the Guardant Reveal multi-cancer assay, a next generation sequencing panel covering ~500 genes and ~4Mb of epigenomic regions that undergo differential methylation in multiple solid tumor types. The presence/absence of ctDNA is determined by a custom bioinformatics classifier that identifies tumor-derived somatic variants and methylation profiles specific to individual cancer types without the need for tumor tissue or peripheral blood mononuclear cell analysis. Samples were analyzed blinded to recurrence status.Results: 20 patients were confirmed to have clinical recurrence (7 local, 13 distant). ctDNA was detected at or prior to distant recurrence in 11/13 (85%) patients and in 1/7 (14%) patients with local recurrence. Five patients with distant recurrence had a sample available that was collected prior to distant recurrence; ctDNA was detected in 4/5 with a 3.8-18.6 month lead time. Among 15 ctDNA+ samples, 5 were positive for both methylation and somatic variant calls, 1 by somatic variants only, and 9 by methylation only. Somatic mutations were identified in AKT, RB1, KRAS, PIK3CA, ERBB2, MAP3K1, GATA3, and ESR1. The ESR1 mutation occurred in a patient with endocrine therapy resistance, confirming the ability of the assay to identify acquired treatment resistance mutations. ctDNA was not detected in samples from the 14 patients who had no confirmed clinical recurrence (100% specificity). Conclusions: This is the first study to demonstrate the feasibility of MRD detection in breast cancer using a plasma-only multiomic ctDNA-based approach. The Guardant Reveal assay demonstrated high sensitivity and specificity to detect distant breast cancer recurrence. Larger studies in this population are ongoing to further validate the clinical performance of the assay and demonstrate its applications in the management of early-stage breast cancer. Citation Format: Wolfgang Janni, Jens Huober, Tatjana Braun, Volkmar Müller, Angelina Fink, Amelie de Gregorio, Brigitte Rack, Thomas W. Friedl, Lisa Wiesmüller, Klaus Pantel, Thereasa Rich, Princy Parsana, Elena Zotenko, Shile Zhang, Sophia Huesmann. Multiomic, plasma-only circulating tumor DNA (ctDNA) assay identifies breast cancer patients with minimal residual disease (MRD) and predicts distant recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3403.

Abstract 5112: Investigating the use of circulating tumor DNA for sarcoma management

Abstract Circulating tumor DNA (ctDNA) has the potential to detect sarcoma recurrence and metastasis but requires highly sensitive methods to detect and quantify genetic variants present in very low quantities. Plasma was isolated from 20mL peripheral blood samples collected from over 400 pre-operative sarcoma patients, and matched tumor samples from surgical resection were frozen and stored. Cell-free DNA (cfDNA) extracted from plasma was quantified using qPCR, and the quality was assessed using capillary electrophoresis. A subset of these cases were selected for whole exome sequencing (WES). WES of bulk tumor and whole blood samples identified tumor-specific genetic alterations, which then serve as personalized biomarkers of tumor DNA in patient plasma. We previously showed that droplet digital PCR (ddPCR) can detect and quantify ctDNA, by targeting patient-specific variants. However, ddPCR is limited in that it can only investigate one tumor variant sequence at a time. The purpose of the present study is to investigate methods of targeting multiple tumor variants simultaneously, increasing the chances of detecting ctDNA in patient blood. To this end, four cases were selected for multiplex PCR (mPCR) followed by targeted amplicon sequencing. For each case, six to eight of the tumor variants identified by WES were selected as targets, and primers were designed to amplify these sequences concurrently by mPCR. The amplicons will then be sequenced to detect the tumor variants. Additionally, two of the four cases have plasma collected at two different time points. To assess the viability of this method as a way to monitor disease surveillance, these cfDNA samples will be compared to determine how the abundance and nature of ctDNA changes over time. To date, cfDNA has been extracted from over 100 cases, the majority of which were positive for cfDNA. For each of the cases whole exome sequenced, a variety of tumor-specific variations were identified. The variants chosen as targets were selected based on having the highest variant allele frequency (VAF), with priority being given to mutations that alter the protein coding sequence. Thus far, mPCR primers have been designed and optimized for four separate cases. Across all cases analyzed by amplicon sequencing, the variant sequences could be detected in the amplicons generated by mPCR of tumor DNA. Furthermore, amplicon sequencing was able to recapitulate the variant allele frequency observed in WES. This indicates that the mPCR successfully amplified the sequences of interest in the tumor DNA, and that the sequencing results are accurate. Furthermore, no tumor variants were detected in the amplicons generated from blood DNA, which is to be expected. The cfDNA amplicons for these cases will be sequenced in this manner to investigate the presence of ctDNA. If successful, the ability to detect ctDNA in plasma will be an important first step in developing a testing protocol for clinical use. Citation Format: Paige Darville-O'Quinn, Nalan Gokgoz, Kim M. Tsoi, Jay S. Wunder, Irene L. Andrulis. Investigating the use of circulating tumor DNA for sarcoma management [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5112.

Evaluating the utility of ctDNA in detecting residual cancer and predicting recurrence in patients with serous ovarian cancer.

e17588 Background: Ovarian cancer remains the most fatal gynecological malignancy. Patients who have no visible residual disease after surgical resection have a relatively good prognosis. Among these patients, those who later succumb to their cancer are believed to harbour non-detectable cancer cells in the peritoneal cavity after treatment which later lead to recurrence. Analyzing circulating tumour DNA (ctDNA) in the blood may offer a sensitive method to detect occult (non-visible) residual disease after surgery and to predict disease recurrence. We proposed to determine the proportion of ovarian cancer patients with and without visible residual disease documented after surgery who had detectable ctDNA from their primary tumour in their blood and to evaluate if the presence of ctDNA is associated with survival. Methods: We included biological samples and clinical information from 48 women diagnosed with high-grade serous ovarian cancer. Plasma, formalin-fixed paraffin-embedded (FFPE) tumour tissue, and white blood cells were used to extract circulating free DNA (cfDNA), tumour DNA and germline DNA, respectively. The plasma sample was collected after surgery and before initiating chemotherapy. We sequenced DNA samples for a panel of 59 breast and ovarian cancer driver genes. DNA variants in matched germline and tumour DNAs were compared to determine tumour specific variants (TSVs) and cfDNA was searched for TSVs to identify the presence of ctDNA in the plasma. The Kaplan-Meier method was used to estimate overall and recurrence-free survival according to the presence or absence of ctDNA. Results: We found TSVs in 47 patients that were used for detecting ctDNA in their post-surgery plasma. Fifteen (31.9%) of the 47 patients had visible residual disease; of these, all 15 had detectable ctDNA. Among the 31 (68.1%) pateints with no visible residual disease, 24 (77.4%) patients had detectable ctDNA. Of those with no visible residual disease, those patients with detectable ctDNA in post-surgery samples had a higher mortality risk compared to those without detectable ctDNA (HR 2.32; 95% CI: 0.67-8.05), although this difference was not statistically significant (p = 0.18). Conclusions: These findings suggest potential clinical utility in ctDNA to improve upon surgical classification of the residual disease status and a potential predictor of recurrence among women with ovarian cancer. Larger studies are necessary to validate these findings.