Prospective study of the co-relation of ctDNA with pathologic complete remission (pCR) and other efficacy outcomes in rectal cancer patients undergoing neoadjuvant chemotherapy and radiation.
Ruobing XueVivek R. SharmaRussell FarmerN. SheshXiaoyong WuMichael KrainockBridgette E. DrummondPerry OlshanPaul R. BillingsAlexey Aleshin
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TPS235 Background: Circulating tumor DNA (ctDNA) has emerged as a biomarker for non-invasive longitudinal monitoring of tumor progression in cancer management. Through the recent advances in next generation sequencing (NGS) technologies and personalized assays, ctDNA has been heralded as a promising tool to detect residual disease, relapse, and monitor treatment response in hematologic malignancies and solid tumors. Our study aims to determine whether treatment related ctDNA dynamics can be used as a reliable indicator to predict pathologic complete response (pCR) in patients with rectal cancer receiving neoadjuvant treatment. Methods: This is a prospective observational cohort study. The primary aim is to estimate the sensitivity and specificity of ctDNA clearance in predicting pCR in patients undergoing neoadjuvant therapy. The secondary aim is to evaluate the feasibility of using ctDNA as a surveillance method to detect progression of rectal cancer during neoadjuvant therapy and relapse in the subsequent follow up period. ctDNA levels are collected from newly diagnosed rectal cancer patients at 7 discrete time points: at diagnosis or screening, during neoadjuvant therapy, after completion of neoadjuvant therapy and 1 month, 2 months, 4 months, 6 months after surgery. This will be followed by every 3 months ctDNA testing for surveillance for up to 2 years. We expect to enroll approximately 30 patients at our institution. The subjects will be sorted into two groups: responders and non-responders based on whether they achieve pCR. ctDNA level between two groups will subsequently be compared. The use of ctDNA to predict pCR in rectal cancer patients may allow for many of these patients to safely avoid surgery if undetectable ctDNA at the end of neoadjuvant therapy strongly correlates with pCR. We are actively enrolling patients into this prospective observational study and expect to report the data in the near future. The data we obtain will be combined with those from several institutions around the US doing similar studies with the same test. Data analysis will subsequently be conducted. [Table: see text]Keywords:
Neoadjuvant Therapy
Circulating tumor DNA
Minimal Residual Disease
Clinical endpoint
Circulating tumor DNA
Liquid biopsy
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Abstract In Ph+ acute lymphoblastic leukaemia (Ph+ ALL), minimal residual disease (MRD) is the most relevant prognostic factor. Currently, its evaluation is based on quantitative real‐time polymerase chain reaction (Q‐RT‐PCR). Digital droplet PCR (ddPCR) was successfully applied to several haematological malignancies. We analyzed 98 samples from 40 Ph+ ALL cases, the majority enrolled in the GIMEMA LAL2116 trial: 10 diagnostic samples and 88 follow‐up samples, mostly focusing on positive non‐quantifiable (PNQ) or negative samples by Q‐RT‐PCR to investigate the value of ddPCR for MRD monitoring. DdPCR BCR/ABL1 assay showed good sensitivity and accuracy to detect low levels of transcripts, with a high rate of reproducibility. The analysis of PNQ or negative cases by Q‐RT‐PCR revealed that ddPCR increased the proportion of quantifiable samples ( p < 0.0001). Indeed, 29/54 PNQ samples (53.7%) proved positive and quantifiable by ddPCR, whereas 13 (24.1%) were confirmed as PNQ by ddPCR and 12 (22.2%) proved negative. Among 24 Q‐RT‐PCR‐negative samples, 13 (54.1%) were confirmed negative, four (16.7%) resulted PNQ and seven (29.2%) proved positive and quantifiable by ddPCR. Four of 5 patients, evaluated at different time points, who were negative by Q‐RT‐PCR and positive by ddPCR experienced a relapse. DdPCR appears useful for MRD monitoring in adult Ph+ ALL.
Minimal Residual Disease
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Background: In Philadelphia‐positive acute lymphoblastic leukemia (Ph+ ALL), minimal residual disease (MRD) is the strongest independent prognostic factor. Q‐RT‐PCR represents the gold‐standard approach, but it is limited by the lack of standardization across laboratories. Moreover, a fraction of patients with very low MRD levels are classified as positive not‐quantifiable (PNQ), which represent a clinical gray zone. Droplet digital PCR (ddPCR) is an alternative approach which is based on an absolute quantification without the need of a standard curve, has the potential to overcome some limitations of RQ‐PCR and has shown to be advantageous in other lymphoproliferative disorders. In Ph+ ALL, few data have been reported and there are no guidelines for ddPCR MRD analysis and interpretation. Aims: 1) To develop a ddPCR assay, by establishing the reaction parameters (i.e. cDNA input and PCR conditions) for the quantification of BCR / ABL1 p190 and p210 transcripts; 2) to assess the limit of detection (LOD), specificity and reproducibility of the assay; 3) to compare ddPCR and Q‐RT‐PCR in samples from patients enrolled in the GIMEMA LAL2116 clinical trial. Methods: To perform ddPCR experiments we followed Biorad recommendations and used the same primers and probes of Q‐RT‐PCR (BIOMED1). After PCR, the plate was loaded into the QX200 Droplets Reader and data analyzed by the QuantaSoft analysis Software v1.7.4. Only replicates with a number of droplets ≥9000 were deemed acceptable. For results interpretation, we followed the FIL MRD NETWORK recommendations. Three volumes of cDNA (i.e. 1 μL, 2.5 μL and 5 μL) were tested to define the optimal input. To evaluate the specificity of the assay, we tested 4 pools of mononuclear cells from 4 healthy donors and not template controls. To assess the reproducibility, we tested 3 diagnostic Ph+ ALL samples and their 10 –1 , 10 –2 , 10 –3 dilutions in 2 independent experiments. To define the LOD, we tested serial dilutions of the lowest point (10 copies) of a plasmid standard curve corresponding to 1x10 –4 ; 5x10 –5 ; 1x10 –5 ; 5x10 –6 ; 1x10 –6 , in a logarithmic scale. Finally, we compared Q‐RT‐PCR and ddPCR in 10 samples at diagnosis and 35 collected during MRD monitoring. Results: The set‐up experiments revealed that: 1) 1 μL of undiluted cDNA for diagnostic samples and 5 μL for follow‐up samples are required; 2) the maximum sensitivity achieved by the method is 1x10 –5 and reproducible sensitivity is 5x10 –5 . We documented a high intra‐ and inter‐run reproducibility. The comparison of Q‐RT‐PCR and ddPCR on diagnostic samples highlighted a high concordance between the two methods (Pearson correlation coefficient = 0.72). The analysis of PNQ cases by Q‐RT‐PCR revealed that 55.5% (15/27) proved quantifiable by ddPCR, 25.9% (7/27) were confirmed as PNQ and 18.5% (5/27) were negative by ddPCR. The analysis of negative cases by Q‐RT‐PCR confirmed their negativity in 75% (6/8) also by ddPCR, while 25% (2/8) were classified as PNQ. Summary/Conclusion: ddPCR is as sensitive as Q‐RT‐PCR and can provide a more accurate quantification in PNQ cases by Q‐RT‐PCR, allowing to recover their quantification in 55% of cases. At present, clinical correlates analyses cannot be performed given the short follow‐up period of patients. Finally, the application of ddPCR in clinical protocols with well‐defined international guidelines is needed to define whether it can result in a refined management of Ph+ ALL outcome.
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Abstract Background: Minimal Residual Disease (MRD) detection is vital for therapy monitoring and relapse prediction in cancers. While RNA-based MRD assays are shown to be effective at diagnostic time-points, serial response monitoring does not provide additional predictive value.DNA-based assays may be more sensitive and allow longitudinal monitoring of the disease trajectory. This study aimed to develop MRD assays based on whole genome sequencing (WGS) data for high-risk neuroblastoma (HRNB) and Ewing sarcoma (ES) patients, to establish their sensitivity in quantitative PCR (qPCR) anddroplet digital PCR (ddPCR) formats, and to directly compare against RNA-based MRD assays. Methodology: We analyzed WGS data to identify patient-specific chromosomal breakpoints in HRNB (N=6) and ES (N=6) patients, then established qPCR and ddPCR assays. Assay performance was validated using patient-derived cells spiked into bone marrow samples and in serially collected clinical samples as available. The DNA-MRD markers were compared against a panel of RNA-MRD markers for HRNB (TH/DCX/PHOX2B) and ES (EWS-FLI1/ERG/ETV1). Results: DNA-MRD markers showed high sensitivity and quantitation ranging from 10-4(0.01%) to 10-5(0.001%) in qPCR assays and ddPCR assays. In clinical samples of HRNB and ES, DNA-MRD markers were detected in diagnosis specimens and predicted disease trajectory. Among RNA-MRD markers, TH & EWS-FLI1 transcripts showed the maximum detection sensitivity (0.01%), however was less sensitive than the patient specific DNA-MRD markers. Conclusion: This study identifies DNA-MRD analysis as a reliable complimentary approach to RNA-MRD assays and highlights its enhanced sensitivity and clinical utility for monitoring treatment response and disease progression in solid pediatric tumors. Citation Format: Vinod Vijay Subhash, Alvin Kamili, Marie-Wong Erasmus, Dan Chen, Libby Huang, Vanessa Tyrell, Caroline Atkinson, Nicola Venn, Mark Cowley, Glenn Marshall, Paul Ekert, Michelle Henderson, Rosemary Sutton, Murray Norris, Michelle Haber, Jamie Fletcher, Toby Trahair. Analysis of DNA minimal residual disease markers in pediatric solid cancers using quantitative real time PCR and droplet digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 639.
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Minimal Residual Disease
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Circulating tumor DNA(ctDNA) is the DNA fragment released into blood by tumor cells.Wheather it presents or not and its plasma concentration are closely related to the prognosis of patients. The common detection methods of ctDNA include digital polymerase chain reaction,second-generation sequencing,methylation detection technology and so on. Detecting specific point mutations or methylation of ctDNA can not only assist in the diagnosis of pancreatic cancer,but also be expected to identify pancreatic cancer at an early stage. Detecting ctDNA after operation can help predicting tumor recurrence and metastasis effectively,so that patients with high recurrence and metastasis risks can be intervened in advance. Accordingly,this article intends to review detection technology of ctDNA and its clinical applications in the early diagnosis of pancreatic cancer,the prediction of tumor recurrence and metastasis after surgery,and the evaluation of patient prognosis.循环肿瘤DNA（ctDNA）是肿瘤细胞释放入血的DNA片段，其存在与否、血浆浓度的高低与患者预后息息相关。常见的检测方法有数字聚合酶链式反应技术、二代测序、甲基化检测等。针对ctDNA的特异性点突变或甲基化进行检测，不但能辅助诊断胰腺癌，还有望在早期阶段识别胰腺癌。针对术后ctDNA的检测能有效预测肿瘤复发转移，对患者进行风险分层，从而得以对高复发转移风险患者提前进行干预。据此，本文对ctDNA检测技术及ctDNA在胰腺癌早期诊断、手术后复发转移的预测和患者预后评估等方面的临床应用研究进行综述。.
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The analysis of circulating tumor DNA (ctDNA) has multiple uses in oncology. In the past few years, studies with varying designs, methods, and quality have emerged that show promise for the use of ctDNA as a tool to detect minimal residual disease (MRD) across luminal gastrointestinal malignancies. This review of the current literature looks at ctDNA in relation to detecting MRD, predicting patient prognosis, and assessing risk for recurrence.
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Minimal Residual Disease
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Real-time quantitative polymerase chain reaction (RQ-PCR) is a standardized tool for minimal residual disease (MRD) monitoring in acute lymphoblastic leukaemia (ALL). The applicability of this technology is limited by the need of a standard curve based on diagnostic DNA. The digital droplet PCR (ddPCR) technology has been recently applied to various medical fields, but its use in MRD monitoring is under investigation. In this study, we analysed 50 ALL cases by both methods in two phases: in the first, we established analytical parameters to investigate the applicability of this new technique; in the second, we analysed MRD levels in 141 follow-up (FU) samples to investigate the possible use of ddPCR for MRD monitoring in ALL patients. We documented that ddPCR has sensitivity and accuracy at least comparable to those of RQ-PCR. Overall, the two methods gave concordant results in 124 of the 141 analysed MRD samples (88%, P = 0·94). Discordant results were found in 12% borderline cases. The results obtained prove that ddPCR is a reliable method for MRD monitoring in ALL, with the advantage of quantifying without the need of the calibration curves. Its application in a cohort of patients with a longer FU will conclusively define its clinical predictive value.
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Circulating tumor DNA (ctDNA) is emerging as a novel biomarker for tumor evaluation, offering advantages such as high sensitivity and specificity, minimal invasiveness, and absence of radiation. Currently, various techniques including gene sequencing and PCR are employed for ctDNA detection. The utilization of ctDNA for monitoring minimal residual disease (MRD) enables comprehensive assessment of tumor status and early identification of tumor recurrence, achieving a remarkable detection sensitivity of 0.01%. Therefore, ctDNA holds promise as a biomarker for early diagnosis, treatment response monitoring, and prognosis prediction in solid tumors. This article reviews the commonly used methods for detecting ctDNA and their advantages in evaluating tumor MRD and guiding clinical diagnosis and treatment.循环肿瘤DNA（circulating tumor DNA, ctDNA）作为评估肿瘤的新标志物，具有高灵敏度和特异度、创伤小且无放射性等优点，目前有基因测序及聚合酶链式反应等多种检测ctDNA的方法。利用ctDNA监测微小残留病（minimal residual disease, MRD），可纵向评估肿瘤情况及早期发现肿瘤复发，其灵敏度达0.01%。因此ctDNA有望成为对实体肿瘤的早期诊断、监测治疗反应和预测预后的生物标志物。该文综述了目前检测ctDNA的常用方法及其在评估肿瘤MRD和指导临床诊疗上的优势。.
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Circulating tumor DNA
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Minimal/measurable residual disease (MRD) evaluation has resulted in a fundamental instrument to guide patient management in acute lymphoblastic leukemia (ALL). From a methodological standpoint, MRD is defined as any approach aimed at detecting and possibly quantifying residual neoplastic cells beyond the sensitivity level of cytomorphology. The molecular methods to study MRD in ALL are polymerase chain reaction (PCR) amplification-based approaches and are the most standardized techniques. However, there are some limitations, and emerging technologies, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), seem to have advantages that could improve MRD analysis in ALL patients. Furthermore, other blood components, namely cell-free DNA (cfDNA), appear promising and are also being investigated for their potential role in monitoring tumor burden and response to treatment in hematologic malignancies. Based on the review of the literature and on our own data, we hereby discuss how emerging molecular technologies are helping to refine the molecular monitoring of MRD in ALL and may help to overcome some of the limitations of standard approaches, providing a benefit for the care of patients.
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