ctDNA Measurements Research Articles

Circulating tumor DNA analysis for assessment of recurrence risk, benefit of adjuvant therapy, and early relapse detection after treatment in colorectal cancer patients.

11 Background: Timely detection of recurrence, as well as identification of patients at high risk of recurrence after surgery and after completion of adjuvant therapy, are major challenges in the treatment of colorectal cancer (CRC). Postsurgical circulating tumor DNA (ctDNA) analysis is a promising tool for the identification of patients with minimal residual disease (MRD) and a high risk of recurrence. The objective of this prospective, multicenter study was to determine whether serial postsurgical ctDNA analysis could identify the patients at high risk of recurrence, provide an assessment of adjuvant therapy efficacy and detect relapse earlier than standard-of-care radiological imaging. Methods: The cohort comprises 265 stage I-III CRC patients, the to-date largest cohort assessed for ctDNA. All patients had the tumor resected and a subset of 62.6% (166 /265) was additionally treated with ACT. Plasma samples (n = 1503) were collected at various time points for a median follow-up of 28.4 months (range: 1.2-51.0 months). Individual tumors and matched germline DNA were whole-exome sequenced and somatic single nucleotide variants (SNVs) were identified. Personalized multiplex PCR assays were designed to track tumor-specific SNVs (Signatera, bespoke mPCR NGS assay) in each patient’s plasma sample. Results: Postoperative ctDNA status prior to ACT was assessed in 218 patients, of which 9.17% (20/218) were identified to be MRD-positive and 75% (15/20) eventually relapsed. The remaining 25% (5/20) of MRD-positive patients that did not relapse, received ACT. In contrast, only 13.6% (27/198) of MRD-negative cases relapsed (HR: 11: 95% CI: 5.7-20; p < 0.001). Longitudinal ctDNA-positive status, post-definitive therapy (n = 202) was associated with a HR of 36 (95% CI: 16-81; p < 0.001). For a subset of 155 patients postoperative CEA and ctDNA measurements were compared, wherein, ctDNA-positive status was found to be significantly associated with RFS (HR, 7.1; 95% CI, 3.4-15; P < 0.001) compared to CEA (HR, 1.2; 95% CI, 0.46-3.1; P = 0.73). Serial ctDNA analysis detected MRD up to a median of 8 months (0.56 - 21.6 months) ahead of radiologic relapse. Conclusion: Postoperative ctDNA positive status was associated with markedly reduced RFS compared to CEA. The study also shows that effective therapy can be curative in a portion of MRD-positive patients. In a longitudinal setting, ctDNA analysis predicted the risk of recurrence and is a more reliable biomarker for treatment response monitoring.

Circulating tumour DNA as a biomarker in resectable and irresectable stage IV colorectal cancer; a systematic review and meta-analysis.

For patients with metastatic colorectal cancer, stratification for treatment (surgery or chemotherapy) is often based on crude clinicopathological characteristics like tumour size and number of lesions. Circulating tumour DNA (ctDNA) acts as a potential biomarker of disease trajectory and biology, allowing better stratification. This study aims to systematically review ctDNA in stage IV colorectal cancer to assess its potential role as a prospective biomarker to guide management decisions. A literature search was performed to identify studies where the measurement of ctDNA in stage IV colorectal cancer was correlated with a clinical outcome (radiological response, secondary resection rate, PFS, DFS or OS). Twenty-eight studies were included, reporting on 2823 patients. Circulating tumour DNA was detectable in between 80% and 90% of patients prior to treatment. Meta-analysis identified a strong correlation between detectable ctDNA after treatment (surgery or chemotherapy) and overall survival (HR 2.2, 95% CI 1.79-2.69, p<0.00001), as well as progression-free survival (HR 3.15, 95% CI 2.10-4.73, p<0.00001). ctDNA consistently offered an early marker of long-term prognosis in irresectable disease, with changes after one cycle of systemic therapy demonstrating prognostic value. In resectable disease treated with curative intent, detection of ctDNA offered a lead time over radiological recurrence of 10 months. Circulating tumour DNA is detectable in the majority of resectable and irresectable patients. The presence of ctDNA is clearly associated with shorter overall survival, with changes in ctDNA an early biomarker of adverse disease behaviour. Prospective trials are essential to test its clinical efficacy.

CRISPR/Cas9 cleavage triggered ESDR for circulating tumor DNA detection based on a 3D graphene/AuPtPd nanoflower biosensor

Circulating tumor DNA (ctDNA) plays an important role in the early diagnosis and prognosis of several cancers and is a credible biomarker for predicting the response to therapy. Additionally, the fact that the strategy used to detect ctDNA is non-invasive also adds to the advantages of using ctDNA for predicting disease diagnosis and prognosis. However, low abundance in peripheral blood and the high background of wild-type DNA impair the precise and specific measurement of ctDNA. In this study, we developed a novel 3D GR/AuPtPd nanoflower sensing platform based on CRISPR/Cas9 cleavage-triggered entropy-driven strand displacement reaction (ESDR) for the effective detection of ctDNA. Low levels of ctDNA could be detected using this method as the ESDR amplification does require complicated operation procedures and stringent reaction conditions. By combining the advantages of the site-specific cleavage by “gene magic scissors,” Cas9/sgRNA, with those of the rapid amplification kinetics of entropy-driven strand displacement, our method resulted in amplification efficiency as well as high specificity for discriminating single-nucleotide mismatches. The 3D GR/AuPtPd nanoflower-based electrochemical biosensor displayed high specificity and worthy performance in assays with human serum. Therefore, this pioneered method provides a new paradigm for efficient ctDNA detection and shows great potential for use in clinical and diagnostic applications.

Cell-free DNA and circulating TERT promoter mutation for disease monitoring in newly-diagnosed glioblastoma

The clinical implications of plasmatic cell-free and tumor DNA (cfDNA and ctDNA) are challenging in glioblastoma. This prospective study included 52 consecutive newly diagnosed glioblastoma (n = 49) or gliosarcoma (n = 3) patients treated with concomitant temozolomide and radiotherapy (RT-TMZ), followed by a TMZ maintenance phase. Plasma samples were collected at baseline, before RT-TMZ (pre-RT-TMZ) and at the end of adjuvant TMZ, or at the time of progression in cases of progressive disease (PD). The cfDNA concentration was measured with a fluorometric method, and ctDNA was detected using targeted droplet digital PCR. The main objectives were to analyze the associations between cfDNA and ctDNA measurements during the course of treatment with PD and survival. There was a significant decrease in median cfDNA concentration from baseline to pre-RT-TMZ—19.4 versus 9.7 ng/mL (p < 0.0001)—in the entire cohort. In patients with PD, a significant increase in cfDNA concentration from pre-RT-TMZ to time of PD was observed, from 9.7 versus 13.1 ng/mL (p = 0.037), respectively, while no difference was observed for nonprogressive patients. Neither the cfDNA concentration at baseline nor its kinetics correlated with survival. ctDNA was detected in 2 patients (3.8%) and only in gliosarcoma subtypes.Trial registration ClinicalTrial, NCT02617745. Registered 1 December 2015, https://clinicaltrials.gov/ct2/show/NCT02617745?term=glioplak&draw=2&rank=1.

Coordination behavior of succinylsulfathiazole – Crystal structure of [Cu(SST).(Pyridine)3.H2O]n, DNA interaction and cytotoxic studies

Mononuclear copper complex of succinylsulfathiazole has been synthesized and characterized using relevant spectroscopic and X-ray diffraction techniques. EPR data and X-ray structural analysis confirmed that Cu(II) ion exhibits an intermediate geometry between trigonal and square bipyramid. Succinylsulfathiazole (SST) ligand behaves as a head-to-tail bridging moiety linking two metal (Cu) cations via carbonyl oxygen and thiazolate nitrogen atom, resulting one dimensional polymeric chain running along [010] direction. In the mononuclear copper complex formulated as [Cu(SST).(Pyridine)3.H2O]n, three nitrogen atoms of three secondary ligand pyridines coordinated to copper at the equatorial plane, the fourth coordination of distorted pentadentate copper is due to thiazolate nitrogen (N3) occupying the apical position and carbonyl oxygen (O1) of SST ligand at -x + 1/2, y-1/2, -z-1/2 remaining at fifth coordination site completes the coordination sphere of copper. The inversely related Cu-SST molecular complexes are held together by pair of NH…O dimer forming R22(14) graph set motif which are further connected by water oxygen (O6) via OH…O interactions. Quantitative contributions of intermolecular interactions are worked out using Hirshfeld surface (HS) and 2D fingerprint plots. Lipophilicity index (Log P), correlates well with the% contribution of CH…π interaction. To evaluate the binding capabilities and binding mode of molecular complex with CT-DNA, absorption study and viscosity measurement are carried out, confirming the intercalation mode of binding which is further supported by molecular docking studies. In vitro cytotoxicity screening of the complex have been performed on zoological species Artemia cyst.

Circulating tumor DNA guided adjuvant chemotherapy in stage II colon cancer (MEDOCC-CrEATE): study protocol for a trial within a cohort study

BackgroundAccurate detection of patients with minimal residual disease (MRD) after surgery for stage II colon cancer (CC) remains an urgent unmet clinical need to improve selection of patients who might benefit form adjuvant chemotherapy (ACT). Presence of circulating tumor DNA (ctDNA) is indicative for MRD and has high predictive value for recurrent disease. The MEDOCC-CrEATE trial investigates how many stage II CC patients with detectable ctDNA after surgery will accept ACT and whether ACT reduces the risk of recurrence in these patients.Methods/designMEDOCC-CrEATE follows the ‘trial within cohorts’ (TwiCs) design. Patients with colorectal cancer (CRC) are included in the Prospective Dutch ColoRectal Cancer cohort (PLCRC) and give informed consent for collection of clinical data, tissue and blood samples, and consent for future randomization. MEDOCC-CrEATE is a subcohort within PLCRC consisting of 1320 stage II CC patients without indication for ACT according to current guidelines, who are randomized 1:1 into an experimental and a control arm.In the experimental arm, post-surgery blood samples and tissue are analyzed for tissue-informed detection of plasma ctDNA, using the PGDx elio™ platform. Patients with detectable ctDNA will be offered ACT consisting of 8 cycles of capecitabine plus oxaliplatin while patients without detectable ctDNA and patients in the control group will standard follow-up according to guideline.The primary endpoint is the proportion of patients receiving ACT when ctDNA is detectable after resection. The main secondary outcome is 2-year recurrence rate (RR), but also includes 5-year RR, disease free survival, overall survival, time to recurrence, quality of life and cost-effectiveness. Data will be analyzed by intention to treat.DiscussionThe MEDOCC-CrEATE trial will provide insight into the willingness of stage II CC patients to be treated with ACT guided by ctDNA biomarker testing and whether ACT will prevent recurrences in a high-risk population. Use of the TwiCs design provides the opportunity to randomize patients before ctDNA measurement, avoiding ethical dilemmas of ctDNA status disclosure in the control group.Trial registrationNetherlands Trial Register: NL6281/NTR6455. Registered 18 May 2017, https://www.trialregister.nl/trial/6281

Abstract 5455: Identifying somatic variants with defined confidence level in targeted sequencing of tumor DNA without a matched normal sample

Abstract Sequencing tumor DNA without a matched normal sample is currently routine practice for many clinical diagnostic services that use NGS to identify somatic mutations in a panel of cancer-relevant genes, due to cost constraints. Various computational algorithms have been developed to distinguish somatic mutations from germline variations in this “single sample” setting. However, the sensitivity and specificity of these methods have not been rigorously investigated, and factors affecting accuracy of the somatic classification have not been thoroughly mapped out. We seek to address these inadequacies and identify avenues towards better somatic variant identification in “single sample” setting. We use tumor/matched normal pairs to establish true somatic/germline status of variants and analyze how adjustments in the algorithms for variant calling and somatic/germline classification affect the ability to identify true somatic variants. We show that somatic variants can be identified with varying degrees of certainty under different circumstances and demonstrate that our algorithmic methods can effectively identify a substantial subset of calls that have a very high likelihood of being truly somatic. Tunable filtering methods and confidence scoring for somatic likelihood has utility in the development of complex biomarkers such as tumor mutational burden and ctDNA measurements. Citation Format: Yanmei Huang, Amanda G. Young, Jason D. Hughes. Identifying somatic variants with defined confidence level in targeted sequencing of tumor DNA without a matched normal sample [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5455.

Abstract 1968: Early on-treatment genomic instability level in cell free DNA as a predictive and prognostic marker in metastatic breast cancer patients

Abstract Metastatic breast cancer is mostly incurable and patients are treated with serial chemotherapy regimens to prolong life and decrease symptoms. Treatment efficacy is usually monitored by imaging every 3 months and by the measurement of blood tumor biomarkers despite their poor sensitivity and specificity. Novel and more timely approaches to monitoring treatment response and estimating prognosis are needed to enable more rapid treatment decisions. The Genomic Instability Number (GIN) is a recently published measure of DNA copy number changes across the genome that can be derived from low pass whole genome sequencing of cell free DNA in plasma. As it is genome wide, this method does not require previous tumor sequencing and offers a non-invasive option for liquid biopsy testing. We measured the GIN in plasma from a cohort of metastatic breast cancer patients undergoing therapy and correlated GIN levels with clinical response and outcome data. Methods: Blood samples (n=69) were collected from 27 patients with metastatic breast cancer undergoing standard of care therapy. Blood was collected prior to the beginning of a new line of therapy (T0), and then at T1 within 10 days and T2 within 22 days post-treatment initiation. The GIN was calculated for all 69 samples analyzed and we used a pre-determined detection threshold of GIN&amp;gt;170. Treatment response was assessed at 3 and at 6 months post-treatment. Results: 20 of the 27 metastatic patients analyzed had detectable GIN levels in at least one sample. Pre-treatment baseline GIN levels ranged from 80 to 6599 with undetectable GIN in 33% of patients. Baseline plasma GIN was not significantly associated with clinical characteristics, clinical response, progression free survival (PFS) or overall survival (OS). However, the average GIN level at early on-treatment time points, T1 or T2, was associated with clinical response with lower levels in patients who presented tumor response at 3 months (p&amp;lt;0.05) but not at 6 months. We found that detectable GIN at T1 was associated with significantly lower OS (p=0.014) but not with PFS, while detectable GIN values at T2 were associated with both poorer PFS (P=0.017) and OS (p=0.0085). The dynamic changes in GIN during treatment were also associated with treatment response and outcome. In fact, the degree of fall in GIN from baseline to T1 and from baseline to T2 was associated with early clinical response (at 3 months) (p&amp;lt;0.001 and p=0034 respectively). Interestingly, the fall of detectable GIN at T1 was highest in TNBC patients (average 73%) compared to non TNBCs (average 36%) (p=0.016). Finally, the percentage drop of GIN at T1 but not atT2 was significantly associated with PFS (p=0.016). In conclusion, the results from our study highlight the value of early on-treatment ctDNA measurements as a predictor of therapeutic response in the metastatic setting, and provide an approach that does not need to rely on tumor sequencing, thus making it much more clinically feasible. Citation Format: Adriana Aguilar-Mahecha, Josiane Lafleur, Susie Brousse, Cristiano Ferrario, Kimberly A. Holden, Graham McLennan, Taylor J. Jensen, Mark Basik. Early on-treatment genomic instability level in cell free DNA as a predictive and prognostic marker in metastatic breast cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1968.

Liquid Biopsy as Effective Predictor of Stage III Melanoma Relapse

Liquid Biopsy as Effective Predictor of Stage III Melanoma Relapse

Serial circulating tumor DNA (ctDNA) measurement to predict progression in patients (pts) with advanced urothelial carcinoma (aUC).

558 Background: Next-generation sequencing (NGS) of plasma ctDNA is a promising non-invasive method to detect somatic genomic alterations (GAs). We investigated whether serial ctDNA measurements can predict disease progression and map the molecular evolution of advanced UC (aUC) through successive treatments. Methods: We analyzed cohorts from 2 academic centers, WCM and UNLV, with serial (≥2) ctDNA targeted NGS using the Guardant360 panel of 73 genes, ctDNA was collected within four weeks of restaging scans. Non-progressive disease (non-PD) was defined as stable disease or radiological response. Results: Our cohort included 176 individual ctDNA samples (median 3/patient) from 52 patients (38 men), the median age was 69 (range 38-88), 38 pts (73%) had metastatic disease, 20 pts (38.5%) had visceral metastases and 35 pts (67%) received first-line platinum-based combination chemotherapy for aUC. The median number of lines of therapy was 2. 49 pts (94%) had detectable GAs in at least one ctDNA sample. Most commonly identified GAs involved TP53 (67%), PIK3CA, EGFR, and ERBB2 (each in 19%). FGFR3 GAs were identified in 9 pts (17%, 2 FGFR3-TACC3 fusions, and 7 SNVs, including two novel mutations P250R and K650M). The mean variant allele fraction (VAF) significantly decreased in pts with non-PD (80 non-PD events) compared to pts with PD (45 PD events) with a mean VAF decrease of 2.06 % (p&lt; 0.0001). This effect was consistent independent of the number of prior lines of therapy. The mean number of GAs per patient at each time point was significantly decreased in non-PD vs. PD group (2.8 vs. 4.4, p=0.004). Pts with detectable VAF in somatic alterations at any time point during the disease course had shorter overall survival (HR 1.18, 95% CI: 1.08 – 1.28, p&lt;0.0001). Pts with sustained partial or complete responses were more likely to have VAF ≤0.2% suggesting that molecular remission was associated with clinical remission. Conclusions: Serial ctDNA testing is a promising dynamic tool to assess clonal evolution and predict progression in aUC pts.

Abstract P1-19-22: Pearl or the use of FDG positron emission tomography imaging as an early dynamic biomarker of everolimus efficacy in advanced luminal breast cancer

Abstract Background: The combination of everolimus and an endocrine agent has shown a progression-free survival (PFS) benefit but also significant toxicity in locally advanced and/or metastatic breast cancer (MBC) patients. Disappointingly, all translational research efforts conducted so far on primary/metastatic tissue or baseline ctDNA samples have failed to identify clinically useful biomarkers of benefit with the possible exception of p4EBP1 which requires fresh metastatic biopsies (Bachelot at al, ASCO 2019). Methods: Forty-seven patients were included in the PEARL trial in six Belgian centres. All patients underwent one baseline and one “early” 18FDG-PET/CT on day 14 (blinded to the treating oncologist) after treatment initiation. Eligible patients had prior progression on NSAI and at least one measurable lesion on baseline 18FDG-PET/CT (fulfilling PERCIST). Twenty seven of the 47 patients were treated in a pilot phase aiming to characterize 18FDG-PET/CT metabolic responses 14 and 28 days after treatment initiation. Day 14 18FDG-PET/CT was selected for the second phase in which 20 more patients were included to answer the primary endpoint of the trial, namely the potential of an early FDG-PET/CT to select patients who will not derive a long-term benefit from everolimus-exemestane combination. We considered as “metabolic responders” those patients in whom at least a 25% homogenous decrease in SUVmax was seen in all their target lesions; the so called “consistent” patient-based response, here correlated to PFS. Results: Seventy-four percent of the patients had visceral metastasis, 26% were pretreated with CDK 4/6 inhibitors and 58% with at least one line of chemotherapy (28% ≥ 2 lines) for the treatment of MBC. Metabolic non-response rate on day 14 was 64%. Two patients were considered non-evaluable because they stopped everolimus 3 and 8 days before early 18FDG-PET/CT respectively. The median PFS in non-responding patients was 2.3 months compared to 6 months in those who showed a consistent metabolic response (HR-1.96, 95%CI: 0.98-3.95, p=0.05). We’ll be able to assess whether day 14 ctDNA measurements add predictive value to imaging. Conclusion: Absence of 18FDG-PET/CT metabolic response after 14 days of everolimus-exemestane combination is associated with a low probability to derive benefit from this treatment. The advanced stage of the patients included in this trial (75% visceral metastasis) and their extensive prior treatments can explain the poorer outcome seen with exemestane-everolimus compared to the Bolero2 trial. Citation Format: Andrea Gombos, Martine Piccart-Gebhart, Lieveke Ameye, Peter Vuylsteke, Patrick Neven, Vincent Richard, Francois P Duhoux, Marianne Paesmans, Michail Ignatiadis, Patrick Flamen, Géraldine Gebhart. Pearl or the use of FDG positron emission tomography imaging as an early dynamic biomarker of everolimus efficacy in advanced luminal breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-22.

Analysis of pancreatobiliary adenocarcinoma (PBC) treatment response and resistance utilizing circulating tumor DNA (ctDNA).

758 Background: Accurate disease monitoring in PBC is instrumental for optimal therapeutic decision-making. CA 19-9 is the most utilized biomarker, though it has limited sensitivity/specificity and cannot be used in CA 19-9 non-secretors (n-S). ctDNA is a potentially helpful monitoring aid and surrogate for PBC n-S. Serial ctDNA could identify emerging resistant driver mutations. Our study prospectively examined ctDNA in PBC patients receiving treatment and retrospectively correlated it with clinical response. Methods: We performed genomic testing of ctDNA from metastatic PBC patients’ plasma from 11/2016 to 08/2019. This included 77 patients, of those, 18 had &gt;1 ctDNA measurement with 49 correlative data points in total. Demographics, serial CA 19-9 levels and imaging results were collected. ctDNA analysis by parallel sequencing of amplified target genes (74) using Guardant360 was obtained. We correlated imaging and CA 19-9 responses with molecular alterations in patients receiving systemic chemotherapy. Descriptive statistics and logistic regression of the data was performed. Results: Of those included, median age was 66 yo, 50% male, and 92% pancreatic ductal adenocarcinoma. Baseline ctDNA showed 103 mutations including TP53 12.6%, KRAS 9.7%, MET 6.8%, APC, ARID1A and NF1 4.8% each, and others &lt; 3%. 44% of patients were n-S with 75% having both TP53 and KRAS mutations. APC, ARID1A, and NF1 were only present in n-S. 91% vs 90% KRAS and 84% vs 78% TP53 of n-S and secretors (S), respectively, had correlation between ctDNA levels and imaging response. S TP53 and KRAS mutations correlated to CA19-9 levels and scans in 78% and 70% responses. New TP53 subclonal variant mutations were the most common resistance mutations for all progressions (75%). A logistic regression model of imaging progression on change in CA19-9 secretion and TP53 or KRAS expression was not statistically significant. Conclusions: Baseline ctDNA level changes ( TP53 and KRAS) can potentially act as a biomarker of response in PBC, specifically in n-S. TP53 subclonal mutations were the most common resistant alterations at progression and can be explored as future targets. This is being explored in larger prospective trials.

Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction.

Background Choosing the specimen type is the first step of the pre-analytical process. Previous reports suggested plasma as the optimal specimen for circulating tumor DNA (ctDNA) analysis. However, head-to-head comparisons between plasma and serum using platforms with high analytical sensitivity, such as droplet digital polymerase chain reaction (ddPCR), are limited, and several recent studies have supported the clinical utility of serum-derived ctDNA. This study aimed to compare the DNA profiles isolated from plasma and serum, characterize the effects of the differences between specimens on ctDNA measurement, and determine the major contributors to these differences. Methods We isolated cell-free DNA (cfDNA) from 119 matched plasma/serum samples from cancer patients and analyzed the cfDNA profiles by DNA fragment sizing. We then assessed KRAS mutations in ctDNA from matched plasma/serum using ddPCR. Results The amount of large DNA fragments was increased in serum, whereas that of cfDNA fragments (<800 bp) was similar in both specimens. ctDNA was less frequently detected in serum, and the KRAS-mutated fraction in serum was significantly lower than that in plasma. The differences in ctDNA fractions between the two specimen types correlated well with the amount of large DNA fragments and white blood cell and neutrophil counts. Conclusions Our results provided detailed insights into the differences between plasma and serum using DNA fragment sizing and ddPCR, potentially contributing to ctDNA analysis standardization. Our study also suggested that using plasma minimizes the dilution of tumor-derived DNA and optimizes the sensitivity of ctDNA analysis. So, plasma should be the preferred specimen type.

The Use of Circulating Tumor DNA to Monitor and Predict Response to Treatment in Colorectal Cancer

Background: Colorectal cancer is one of the most common cancers worldwide and has a high mortality rate following disease recurrence. Treatment efficacy is maximized by providing tailored cancer treatment, ideally involving surgical resection and personalized neoadjuvant and adjuvant therapies, including chemotherapy, radiotherapy and increasingly, targeted therapy. Early detection of recurrence or disease progression results in more treatable disease and is essential to improving survival outcomes. Recent advances in the understanding of tumor genetics have resulted in the discovery of circulating tumor DNA (ctDNA). A growing body of evidence supports the use of these sensitive biomarkers in detecting residual disease and diagnosing recurrence as well as enabling targeted and tumor-specific adjuvant therapies. Methods: A literature search in Pubmed was performed to identify all original articles preceding April 2019 that utilize ctDNA for the purpose of monitoring response to colorectal cancer treatment. Results: Ninety-two clinical studies were included. These studies demonstrate that ctDNA is a reliable measure of tumor burden. Studies show the utility of ctDNA in assessing the adequacy of surgical tumor clearance and changes in ctDNA levels reflect response to systemic treatments. ctDNA can be used in the selection of targeted treatments. The reappearance or increase in ctDNA, as well as the emergence of new mutations, correlates with disease recurrence, progression, and resistance to therapy, with ctDNA measurement allowing more sensitive monitoring than currently used clinical tools. Conclusions: ctDNA shows enormous promise as a sensitive biomarker for monitoring response to many treatment modalities and for targeting therapy. Thus, it is emerging as a new way for guiding treatment decisions—initiating, altering, and ceasing treatments, or prompting investigation into the potential for residual disease. However, many potentially useful ctDNA markers are available and more work is needed to determine which are best suited for specific purposes and for improving specific outcomes.

Circulating DNA for Molecular Response Prediction, Characterization of Resistance Mechanisms and Quantification of CAR T-Cells during Axicabtagene Ciloleucel Therapy

Circulating DNA for Molecular Response Prediction, Characterization of Resistance Mechanisms and Quantification of CAR T-Cells during Axicabtagene Ciloleucel Therapy

Short Call Abstracts

Short Call Abstracts

1559P - An exploratory analysis of on-treatment ctDNA measurement as a potential surrogate for overall survival for atezolizumab benefit in the OAK study

BackgroundOverall survival (OS) is the most relevant endpoint for evaluation of checkpoint inhibitors (CPI). Identification of early surrogates of OS is important to inform early clinical efficacy signals and treatment decisions. Here, we evaluated multiple metrics of circulating tumor DNA (ctDNA) for association with OS in the Ph3 OAK study, which demonstrated clinically significant OS benefit in≥2L NSCLC patients treated with atezolizumab vs docetaxel. MethodsPlasma from 94 patients taken at baseline and at subsequent cycles of therapy every three weeks (C2D1, C3D1, and C4D1) were analyzed retrospectively for ctDNA with the AVENIO ctDNA Surveillance Kit* (Jiang et al., 2018). Mutations detected in matched PBMC DNA were excluded in correlation analyses with clinical outcomes (Yaung et al., 2019). ctDNA was measured by allele frequency (AF) or mutant molecules per milliliter (MMPM), and summarized across multiple mutations within a sample by median, mean or maximum. Concordance between these per-sample metrics and PFS/OS were assessed using C index, which is equivalent to AUC under a Receiver Operating Characteristic (ROC) curve. *For Research Use Only; Not for use in diagnostic procedures. ResultsUsing absolute and relative change from baseline of these metrics showed various levels of association with OS for both treatment arms (atezolizumab arm C index average 0.63, range 0.47 -0.76 and docetaxel arm C index average 0.50, range 0.39 - 0.61). As noted, maximal MMPM at C3D1 or 6 weeks had comparable association with OS in both treatment arms (atezolizumab arm C index = 0.74 and docetaxel arm C index = 0.73). Dichotomized at a median of 8.75 maximal MMPM, KM curves showed the 1-year survival rate in the atezolizumab arm among MMPM low vs high patients was 0.81 vs 0.43; and in the docetaxel arm was 0.7 vs 0.07. Associations with PFS tended to be weaker than those for OS. ConclusionsOverall ctDNA levels were associated with OS in 2L+ NSCLC. ctDNA may be a promising non-invasive blood based biomarker in the metastatic setting of NSCLC and warrants further studies to demonstrate its utility in clinical development. Clinical trial identificationOAK (NCT02008227). Legal entity responsible for the studyRoche. FundingRoche. DisclosureD.R. Gandara: Advisory / Consultancy: AstraZeneca, Celgene, CellMax Life, FujiFilm, Roche-Genentech, Guardant Health, Inviata, IO Biotech, Lilly, Liquid Genomics, Merck, Samsung Bioepis, Pfizer; Research grant / Funding (institution), Research grants : Bristol-Myers Squib, Roche-Genentech, Novartis, Merck. W. Zou: Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. J. Jiang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. S. Yaung: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. F. Fuhlbrück: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. J. Wu: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. M. Peterson: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. J. Palma: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. M. Ballinger: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. E. Peters: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. D. Shames: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. N. Patil: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche.

Viral Genome Sequencing for Ultrasensitive Detection of Circulating Tumor DNA

Accurate detection of circulating tumor DNA (ctDNA) has the potential to guide personalized cancer therapy. DNA viruses such as Epstein-Barr virus (EBV) and human papillomavirus (HPV) are major oncologic drivers of nasopharyngeal cancer (NPC) and cervical cancer (CC), respectively, and provide a convenient etiological marker for measurement of ctDNA. Polymerase chain reaction (PCR)-based methods have been widely studied, but more accurate tests are needed. We hypothesized that a next generation sequencing (NGS) approach could outperform PCR-based methods for viral ctDNA detection. We developed a novel NGS method for viral ctDNA detection. Viral genome sequencing for EBV (EBV-seq) or HPV (HPV-seq) was achieved by hybrid capture NGS. Unique molecular identifiers were incorporated for accurate copy number quantification. We implemented a custom bioinformatics workflow to (1) quantify viral sequences, (2) report viral subtype, and (3) measure ctDNA fragment length. The lower limit of detection (LLOD) was compared with published PCR-based methods in cell line dilution series. To test performance on clinical samples, we applied EBV-seq to a cohort of 19 newly diagnosed EBV-associated NPC patients for whom the clinical EBV quantitative PCR test had provided a false negative result and to a control cohort of 20 healthy volunteers. We applied HPV-seq to 40 plasma samples from a cohort of 18 CC patients treated with chemoradiotherapy and to a control cohort of 5 healthy volunteers. The LLOD of EBV-Seq was ≤0.01 EBV genome-equivalents (GE), 100X lower than quantitative PCR. The LLOD of HPV-seq was ≤0.2 HPV GE, 10X lower than digital PCR. For EBV-seq, 19/19 NPC patients had detectable ctDNA (sensitivity=100%, range=0.08-141 EBV GE); 10/20 controls had detectable ctDNA (specificity=50%, range=0.02-0.71 EBV GE). 17/19 (89%) patients, including 6/6 with stage I-II disease, had EBV-seq levels that were >20X above the highest level seen in the control cohort (i.e., 89% sensitivity with 100% specificity). For HPV-seq, 6/6 baseline CC samples, 10/17 (59%) end-of-treatment samples, 1/14 (7%) 3-month post-treatment samples, and 3/3 samples taken upon recurrence had positive HPV-seq results (sensitivity=100%, range=0.02-559 HPV GE) that were highly correlated with digital PCR (Pearson R=0.97) but with 60X higher signal than digital PCR; 0/5 controls had detectable ctDNA (specificity=100%). EBV-seq and HPV-seq were able to report viral subtype in 49/49 positive samples. Viral ctDNA was consistently shorter than non-cancer-derived plasma DNA (median difference=15bp). For DNA virus-associated cancers, viral genome NGS provides dramatically greater sensitivity than PCR-based methods and enables viral subtyping and ctDNA fragment length analysis. Further evaluation is warranted to test the clinical utility of ultrasensitive ctDNA detection using this approach.

Intra-individual variation of circulating tumour DNA in lung cancer patients.

Circulating tumour DNA (ctDNA) has been increasingly incorporated into the treatment of cancer patients. ctDNA is generally accepted as a powerful diagnostic tool, whereas the utility of ctDNA to monitor disease activity needs to be fully validated. Central to this challenge is the question of whether changes in longitudinal ctDNA measurements reflect disease activity or merely biological variation. Thus, the aim of this study was to explore the intra‐individual biological variation of ctDNA in lung cancer patients. We identified tumour‐specific mutations using next‐generation sequencing. Day‐to‐day and hour‐to‐hour variations in plasma concentrations of the mutant allele and wild‐type cell‐free DNA (cfDNA) were determined using digital PCR. The levels of the mutant alleles varied by as much as 53% from day to day and 27% from hour to hour. cfDNA varied up to 19% from day to day and up to 56% from hour to hour, as determined using digital PCR. Variations were independent of the concentration. Both mutant allele concentrations and wild‐type cfDNA concentrations showed considerable intra‐individual variation in lung cancer patients with nonprogressive disease. This pronounced biological variation of the circulating DNA should be investigated further to determine whether ctDNA can be used for monitoring cancer activity.

Circulating Tumor DNA as a Clinical Test in Resected Pancreatic Cancer.

In research settings, circulating tumor DNA (ctDNA) shows promise as a tumor-specific biomarker for pancreatic ductal adenocarcinoma (PDAC). This study aims to perform analytical and clinical validation of a KRAS ctDNA assay in a Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathology-certified clinical laboratory. Digital-droplet PCR was used to detect the major PDAC-associated somatic KRAS mutations (G12D, G12V, G12R, and Q61H) in liquid biopsies. For clinical validation, 290 preoperative and longitudinal postoperative plasma samples were collected from 59 patients with PDAC. The utility of ctDNA status to predict PDAC recurrence during follow-up was assessed. ctDNA was detected preoperatively in 29 (49%) patients and was an independent predictor of decreased recurrence-free survival (RFS) and overall survival (OS). Patients who had neoadjuvant chemotherapy were less likely to have preoperative ctDNA than were chemo-naïve patients (21% vs. 69%; P < 0.001). ctDNA levels dropped significantly after tumor resection. Persistence of ctDNA in the immediate postoperative period was associated with a high rate of recurrence and poor median RFS (5 months). ctDNA detected during follow-up predicted clinical recurrence [sensitivity 90% (95% confidence interval (CI), 74%-98%), specificity 88% (95% CI, 62%-98%)] with a median lead time of 84 days (interquartile range, 25-146). Detection of ctDNA during postpancreatectomy follow-up was associated with a median OS of 17 months, while median OS was not yet reached at 30 months for patients without ctDNA (P = 0.011). Measurement of KRAS ctDNA in a CLIA laboratory setting can be used to predict recurrence and survival in patients with PDAC.