ctDNA-positive Patients Research Articles

Circulating Tumor DNA in Stage III Colorectal Cancer, beyond Minimal Residual Disease Detection, toward Assessment of Adjuvant Therapy Efficacy and Clinical Behavior of Recurrences.

Sensitive methods for risk stratification, monitoring therapeutic efficacy, and early relapse detection may have a major impact on treatment decisions and patient management for stage III colorectal cancer patients. Beyond assessing the predictive power of postoperative ctDNA detection, we explored the added benefits of serial analysis: assessing adjuvant chemotherapy (ACT) efficacy, early relapse detection, and ctDNA growth rates. We recruited 168 patients with stage III colorectal cancer treated with curative intent at Danish and Spanish hospitals between 2014 and 2019. To quantify ctDNA in plasma samples (n = 1,204), 16 patient-specific somatic single-nucleotide variants were profiled using multiplex-PCR, next-generation sequencing. Detection of ctDNA was a strong recurrence predictor postoperatively [HR = 7.0; 95% confidence interval (CI), 3.7-13.5; P < 0.001] and directly after ACT (HR = 50.76; 95% CI, 15.4-167; P < 0.001). The recurrence rate of postoperative ctDNA-positive patients treated with ACT was 80% (16/20). Only patients who cleared ctDNA permanently during ACT did not relapse. Serial ctDNA assessment after the end of treatment was similarly predictive of recurrence (HR = 50.80; 95% CI, 14.9-172; P < 0.001), and revealed two distinct rates of exponential ctDNA growth, slow (25% ctDNA-increase/month) and fast (143% ctDNA-increase/month; P < 0.001). The ctDNA growth rate was prognostic of survival (HR = 2.7; 95% CI, 1.1-6.7; P = 0.039). Serial ctDNA analysis every 3 months detected recurrence with a median lead-time of 9.8 months compared with standard-of-care computed tomography. Serial postoperative ctDNA analysis has a strong prognostic value and enables tumor growth rate assessment. The novel combination of ctDNA detection and growth rate assessment provides unique opportunities for guiding decision-making.See related commentary by Morris and George, p. 438.

Abstract 552: Circulating tumor DNA predicts disease recurrence in ovarian cancer patients

Abstract Introduction: Epithelial ovarian, fallopian tube, and peritoneal cancer (EOC) is the most lethal gynecologic malignancy with a 5-year survival rate of 47%. While primary treatment generally results in remission, most patients relapse within 3 years. CA-125 is a commonly used biomarker for recurrence detection, however, it lacks specificity and is not associated with improved survival. Here we examine the utility of circulating tumor DNA (ctDNA) as a biomarker for EOC by assessing its relationship to patient outcome and CA-125 when measured pre-surgically and during patient monitoring. Methods: This study included patients diagnosed with stage I-IV EOC with plasma samples collected pre-surgically (n=44) and a group of patients (n=22) with serially collected samples after surgery. Median follow-up for patients with pre-surgical samples and with prospectively collected samples was 29 months (range: 6-150) and 15 months (range: 0.6-26), respectively. Whole exome sequencing was performed on patient tumors and matched normal tissue to design patient-specific ctDNA assays (bespoke mPCR NGS assay) for variant detection in plasma samples. The relationship between ctDNA status, CA-125 levels, and recurrence-free survival (RFS) were evaluated (Fisher's exact, log-rank test). Results: Among patients with presurgical plasma samples high-grade serous was the most common histological subtype 66% (29/44). Endometrioid represented 11% (5/44) of tumors and 23% (10/44) were tumors of other epithelial subtypes. In this cohort 75% (33/44) had early-stage disease, 7% (3/44) were metastatic and 18% (8/44) had the unstaged disease. The presence of ctDNA was observed in 73% of samples at baseline with detection rates of 69% (20/29) for serous and 80% (4/5) for endometrioid histologies. Pre-surgical ctDNA detection was significantly associated with a higher grade (p=0.003). All patients with ctDNA negative status at baseline (n=12) survived until the end of follow-up (median: 25 months), while 3 deaths were observed among ctDNA positive patients (n=32; p=0.003). In the sub-cohort of patients with prospective post-surgical plasma collection, ctDNA was observed in samples of all patients who relapsed (7/7; 100% sensitivity). ctDNA detection preceded radiological findings by a median of 9 months (range: 2-36). None of the patients with ctDNA negative status within 6 months after enrollment experienced disease progression (13/13; 100% specificity). The presence of ctDNA was observed to be a strong predictor of relapse (HR: 12.75, 95%CI: 1.7-94 p&amp;lt;0.0001), while CA-125 was not significantly associated with RFS (HR: 1.3, 95%CI: 0.3-6.3; p=0.09). Conclusions: The presence of ctDNA post-surgically is highly prognostic of decreased RFS and was found to be a stronger predictor of disease progression than CA-125 monitoring. These results suggest that monitoring ctDNA could be a useful tool in clinical decision making for patients with EOC. Citation Format: Jocelyn S. Chapman, William E. Pierson, Karen Smith-McCune, Geovanni Pineda, Reena M. Vattakalam, Alexandra Ross, Meredith A. Mills, Carlos J. Suarez, Tracy Davis, Robert P. Edwards, Michelle Boisen, James M. Ford, June Y. Hou, Hsin-Ta Wu, Scott Dashner, Ekaterina Kalashnikova, Angel Rodriguez, Bernhard Zimmermann, Sarah Sawyer, Himanshu Sethi, Alexey Aleshin. Circulating tumor DNA predicts disease recurrence in ovarian cancer patients [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 552.

Circulating Tumour DNA as a Potential Cost-Effective Biomarker to Reduce Adjuvant Chemotherapy Overtreatment in Stage II Colorectal Cancer.

Substantial adjuvant chemotherapy (AC) overtreatment for stage II colorectal cancer results in a health and financial burden. Circulating tumour DNA (ctDNA) can improve patient selection for AC by detecting micro-metastatic disease. We estimated the health economic potential of ctDNA-guided AC for stage II colorectal cancer. A cost-utility analysis was performed to compare ctDNA-guided AC to standard of care, where 22.6% of standard of care patients and all ctDNA-positive patients (8.7% of tested patients) received AC and all ctDNA-negative patients (91.3%) did not. A third preference-sensitive ctDNA strategy was included where 6.8% of ctDNA-negative patients would receive AC. A state-transition model was populated using data from a prospective cohort study and clinical registries. Health and economic outcomes were discounted at 5% over a lifetime horizon from a 2019 Australian payer perspective. Extensive scenario and probabilistic analyses quantified model uncertainty. Compared to standard of care, the ctDNA and preference-sensitive ctDNA strategies increased quality-adjusted life-years by 0.20 (95% confidence interval - 0.40 to 0.81) and 0.19 (- 0.40 to 0.78), and resulted in incremental costs of AUD - 4055 (- 16,853 to 8472) and AUD - 2284 (- 14,685 to 10,116), respectively. Circulating tumour DNA remained cost effective at a willingness to pay of AUD 20,000 per quality-adjusted life-year gained throughout most scenario analyses in which the proportion of ctDNA-positive patients cured by AC and compliance to a ctDNA-negative test results were decreased. Circulating tumour-guided AC is a potentially cost-effective strategy towards reducing overtreatment in stage II colorectal cancer. Results from ongoing randomised clinical studies will be important to reduce uncertainty in the estimates.

Serial circulating tumor DNA analysis to assess recurrence risk, benefit of adjuvant therapy, growth rate and early relapse detection in stage III colorectal cancer patients.

3540 Background: Challenges in the postoperative management of stage III colorectal cancer include: 1) selection of high-risk patients for adjuvant chemotherapy (ACT), 2) lack of markers to assess ACT efficacy, 3) assessment of recurrence risk after ACT, and 4) lack of markers to guide treatment decisions for high-risk patients e.g. additional therapy or intensified surveillance. Circulating tumor DNA (ctDNA) is a promising marker with potential to mitigate the challenges. Here we used serial ctDNA measurements to assess the correlation between recurrence and ctDNA detection: postoperative, during and after ACT, and during surveillance; and to assess growth rates of metachronous metastases. Uniquely, we also used concurrent CT scans and ctDNA measurements to compare the sensitivity for detecting recurrence. Methods: Stage III CRC patients treated with curative intent at Danish and Spanish hospitals in 2014-2019 were recruited (n = 166). Blood samples (n = 1227) were collected prior to and immediately after surgery, and every third month for up to 36 months. Per patient 16 personal mutations were used to quantify plasma ctDNA (Signatera, bespoke mPCR NGS assay). Results: Detection of ctDNA was a strong recurrence predictor, both postoperatively (HR 7.2, 95% CI 3.8-13.8, P&lt; 0.001), directly after ACT (HR = 18.2, 95% CI 7.1-46, P &lt; 0.001), and when measured serially after end of treatment (HR = 41, 95% CI 16-100, P &lt; 0.001). The recurrence rate of postoperative ctDNA positive patients treated with ACT was 80% (16/20). Patients who stayed ctDNA positive during ACT all recurred. Serial post-treatment ctDNA measurements revealed exponential growth for all recurrence patients following either a SLOW (26%-increase/month) or a FAST (126%-increase/month) pattern (P &lt; 0.001). From ctDNA detection to radiologic recurrence, ctDNA levels of FAST patients increased by a median 117-fold, and up to 554-fold. The 3-year overall survival was 43% for FAST patients and 100% for SLOW and non-recurrence patients (HR = 41.3, 95% CI 7.5-228, P &lt; 0.001). Coinciding CT scans and ctDNA measurements (n = 113 patients, 235 coinciding events, median 2 per patient) showed a high agreement (92%) and ctDNA either detected residual disease before the CT scan (n = 7 patients) or at the same time (n = 14 patients). The median lead-time was 7.5 months. Conclusions: The study confirmed the prognostic power of serial postoperative ctDNA analysis. Moreover, it provided novel analyses demonstrating that ctDNA is more sensitive for recurrence detection than CT scans and can be used for tumor growth rate assessments. The difference between FAST and SLOW growing tumors suggest that growth rates could guide whom to start on systemic therapy rapidly and whom to send for diagnostic imaging. Altogether, the study highlights many potential utilities of ctDNA in guiding clinical decision-making.

Postoperative circulating tumor DNA as markers of recurrence risk in stages II to III colorectal cancer

BackgroundPrecise methods for postoperative risk stratification to guide the administration of adjuvant chemotherapy (ACT) in localized colorectal cancer (CRC) are still lacking. Here, we conducted a prospective, observational, and multicenter study to investigate the utility of circulating tumor DNA (ctDNA) in predicting the recurrence risk.MethodsFrom September 2017 to March 2020, 276 patients with stage II/III CRC were prospectively recruited in this study and 240 evaluable patients were retained for analysis, of which 1290 serial plasma samples were collected. Somatic variants in both the primary tumor and plasma were detected via a targeted sequencing panel of 425 cancer-related genes. Patients were treated and followed up per standard of care.ResultsPreoperatively, ctDNA was detectable in 154 of 240 patients (64.2%). At day 3–7 postoperation, ctDNA positivity was associated with remarkably high recurrence risk (hazard ratio [HR], 10.98; 95%CI, 5.31–22.72; P < 0.001). ctDNA clearance and recurrence-free status was achieved in 5 out of 17 ctDNA-positive patients who were subjected to ACT. Likewise, at the first sampling point after ACT, ctDNA-positive patients were 12 times more likely to experience recurrence (HR, 12.76; 95%CI, 5.39–30.19; P < 0.001). During surveillance after definitive therapy, ctDNA positivity was also associated with extremely high recurrence risk (HR, 32.02; 95%CI, 10.79–95.08; P < 0.001). In all multivariate analyses, ctDNA positivity remained the most significant and independent predictor of recurrence-free survival after adjusting for known clinicopathological risk factors. Serial ctDNA analyses identified recurrence with an overall accuracy of 92.0% and could detect disease recurrence ahead of radiological imaging with a mean lead time of 5.01 months.ConclusionsPostoperative serial ctDNA detection predicted high relapse risk and identified disease recurrence ahead of radiological imaging in patients with stage II/III CRC. ctDNA may be used to guide the decision-making in postsurgical management.

Circulating tumor DNA and magnetic resonance imaging to predict neoadjuvant chemotherapy response and recurrence risk

We investigated whether serial measurements of circulating tumor DNA (ctDNA) and functional tumor volume (FTV) by magnetic resonance imaging (MRI) can be combined to improve prediction of pathologic complete response (pCR) and estimation of recurrence risk in early breast cancer patients treated with neoadjuvant chemotherapy (NAC). We examined correlations between ctDNA and FTV, evaluated the additive value of ctDNA to FTV-based predictors of pCR using area under the curve (AUC) analysis, and analyzed the impact of FTV and ctDNA on distant recurrence-free survival (DRFS) using Cox regressions. The levels of ctDNA (mean tumor molecules/mL plasma) were significantly correlated with FTV at all time points (p < 0.05). Median FTV in ctDNA-positive patients was significantly higher compared to those who were ctDNA-negative (p < 0.05). FTV and ctDNA trajectories in individual patients showed a general decrease during NAC. Exploratory analysis showed that adding ctDNA information early during treatment to FTV-based predictors resulted in numerical but not statistically significant improvements in performance for pCR prediction (e.g., AUC 0.59 vs. 0.69, p = 0.25). In contrast, ctDNA-positivity after NAC provided significant additive value to FTV in identifying patients with increased risk of metastatic recurrence and death (p = 0.004). In this pilot study, we demonstrate that ctDNA and FTV were correlated measures of tumor burden. Our preliminary findings based on a limited cohort suggest that ctDNA at surgery improves FTV as a predictor of metastatic recurrence and death. Validation in larger studies is warranted.

Abstract PD15-06: Circulating TP53 mutations in TNBC after neoadjuvant chemotherapy is associated with rapid disease recurrence: Correlative analysis from clinical trial BRE12-158

Abstract Background: Neoadjuvant chemotherapy (NAC) is a standard approach for many patients with triple negative breast cancer (TNBC). Our group, and others, have previously reported that the detection of circulating tumor DNA (ctDNA) after NAC and surgery is a surrogate for the presence of minimal residual disease (MRD) leading to inferior survival outcomes. Further, our group has also reported that the emergence of somatic TP53 mutations in tissue samples post-NAC was associated with inferior survival outcomes. In this study, we sought to determine whether TP53 mutations detected in the plasma of patients with ctDNA-positivity after NAC was associated with inferior outcomes compared to non-TP53 mutated patients. Methods:BRE12-158 was a phase II clinical trial which randomized early-stage TNBC patients with residual disease after NAC to post-neoadjuvant genomically-directed therapy vs treatment of physician choice. 196 patients were enrolled. Patients had blood samples collected for ctDNA at the time of post-neoadjuvant treatment assignment. ctDNA was successfully sequenced using the FoundationACT or FoundationOneLiquid Assay in 142 patients. Patients who were ctDNA positive after NAC (N=90, 63%), were selected for this comparison. Median clinical follow-up was 22.9 months. A multivariate cox proportional-hazards model was used to compare DDFS, DFS, and OS of ctDNA-positive patients with and without TP53 mutations found in the ctDNA. Results: 90 patients in BRE12-158 were ctDNA-positive after NAC and surgery. 36/90 (40%) of those patients had TP53 mutations detected in their plasma. Patients with TP53-mutated ctDNA had significantly inferior outcomes and a significantly shorter time to recurrence when compared to those without TP53 mutations. Detection of TP53-mutated ctDNA was significantly associated with an inferior DDFS (median DDFS: 6.99 months vs. 48.69 months; HR=2.78, 95%CI: 1.41-5.49; p=0.0033). At 24 months, the DDFS probability was 36% in TP53-mutated patients as compared to 72% in non-mutated patients. Similarly, detection of TP53-mutated ctDNA was significantly associated with an inferior DFS (median DFS: 4.83 months vs. 48.69 months; HR=3.63, 95%CI: 1.76-7.48; p=0.00047). At 24 months, the DFS probability was 30% in TP53-mutated patients as compared to 72% in non-mutated patients. Lastly, detection of TP53-mutated ctDNA was significantly associated with an inferior OS (median OS: 17.8 months vs. Not Reached; HR=3.48, 95%CI: 1.51-8.01; p=0.0034). At 24 months, the OS probability was 42% in TP53-mutated patients as compared to 80% in non-mutated patients. Conclusion: In patients with ctDNA-positivity after NAC, the presence of TP53 mutations was associated with rapid disease relapse. These data suggest that TP53 mutation status may stratify outcomes among this high-risk group of patients with ctDNA positivity in the post-neoadjuvant setting. Citation Format: Milan Radovich, Nawal Kassem, Guanglong Jiang, Bradley A. Hancock, Bryan P. Schneider. Circulating TP53 mutations in TNBC after neoadjuvant chemotherapy is associated with rapid disease recurrence: Correlative analysis from clinical trial BRE12-158 [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-06.

Longitudinal tracking of 97 esophageal adenocarcinomas using liquid biopsy sampling

The incidence of esophageal adenocarcinoma (EAC) is rapidly rising and has a 5-year survival rate of <20%. Beyond TNM (tumor-node-metastasis) staging, no reliable risk stratification tools exist and no large-scale studies have profiled circulating tumor DNA (ctDNA) at relapse in EAC. Here we analyze the prognostic potential of ctDNA dynamics in EAC, taking into account clonal hematopoiesis with indeterminate potential (CHIP). A total of 245 samples from 97 patients treated with neoadjuvant chemotherapy and surgery were identified from the prospective national UK Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) consortium data set. A pan-cancer ctDNA panel comprising 77 genes was used. Plasma and peripheral blood cell samples were sequenced to a mean depth of 7082× (range 2196-28524) and ctDNA results correlated with survival. Characteristics of the 97 patients identified were as follows: 83/97 (86%) male, median age 68 years (SD 9.5 years), 100% cT3/T4, 75% cN+. EAC-specific drivers had higher variant allele fractions than passenger mutations. Using stringent quality criteria 16/79 (20%) were ctDNA positive following resection; recurrence was observed in 12/16 (75%) of these. As much as 78/97 (80%) had CHIP analyses that enabled filtering for CHIP variants, which were found in 18/78 (23%) of cases. When CHIP was excluded, 10/63 (16%) patients were ctDNA positive and 9/10 of these (90%) recurred. With correction for CHIP, median cancer-specific survival for ctDNA-positive patients was 10.0 months versus 29.9 months for ctDNA-negative patients (hazard ratio 5.55, 95% confidence interval 2.42-12.71; P= 0.0003). Similar outcomes were observed for disease-free survival. We demonstrate in a large, national, prospectively collected data set that ctDNA in plasma following surgery for EAC is prognostic for relapse. Inclusion of peripheral blood cell samples can reduce or eliminate false positives from CHIP. In future, post-operative ctDNA could be used to risk stratify patients into high- and low-risk groups for intensification or de-escalation of adjuvant chemotherapy.

CtDNA as a predictor of outcome in patients treated with neoadjuvant atezolizumab in muscle invasive urothelial cancer

To explore the clinical utility of circulating tumor DNA (ctDNA) with neoadjuvant atezolizumab and radical cystectomy (RC) in muscle invasive urothelial bladder cancer (MIUC). Forty patients from a phase II of neoadjuvant atezolizumab (2 cycles) and RC study in MIUC were included (NCT02662309). Whole-exome sequencing was performed on tumor and matched normal DNA to identify tumor-specific mutations and design a personalized multiplexed PCR Next Generation Sequencing (NGS) assay (bespoke, Signatera TM ) for ctDNA detection in plasma. Samples were taken at baseline, post-atezolizumab but pre-surgery, and 1-6 months post-surgery. Response (pathological complete response (pCR) and major pathological response (mPR)) and recurrence free survival (RFS) were correlated with ctDNA status (Pos/Neg) and level. Responders are pCR and mPR patients, whereas non-responders are SD and relapse patients. The pCR rate and 2 years RFS was 21.4% and 73.5%, respectively. ctDNA positivity at baseline, post-neoadjuvant pre-surgery (PreCx), and post-surgery (PostCx) were 25/40 (63%), 14/30 (47%), and 5/36 (14%), respectively. ctDNA status was highly prognostic at all timepoints, where PostCx had an HR of 78.11 (p<0.001), and zero events were observed for ctDNA negative patients at baseline and PreCx. An association between the change in ctDNA during neoadjuvant atezolizumab treatment and patient outcome was observed (mean of -90% ctDNA change in responders versus +27% in non-responders (p=0.04)). Patients who were PDL1 positive at baseline were more likely to be ctDNA positive at baseline (p=0.01), which was not the case for post-treatment time points. For patients who were ctDNA positive at baseline, PDL1-positive patients had better response rates than PDL1-negative (35% vs. 0%), as well as superior RFS (HR=0.11, p=0.0085). Pre-treatment ctDNA positivity was enriched in SCCL (Lund) and basal squamous (TCGA) patients. Tumors from ctDNA positive patients at baseline were enriched for immune signatures and the epithelial to mesenchymal transition (EMT) signature. ctDNA dynamics across timepoints correlates with clinical outcomes, including response rates and RFS in patients treated with neoadjuvant atezolizumab in MIUC. Immune biomarkers may be relevant in these responses. ctDNA may help the development of personalized therapy in this setting in the future.

Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival

Pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is strongly associated with favorable outcome. We examined the utility of serial circulating tumor DNA (ctDNA) testing for predicting pCR and risk of metastatic recurrence. Cell-free DNA (cfDNA) was isolated from 291 plasma samples of 84 high-risk early breast cancer patients treated in the neoadjuvant I-SPY 2 TRIAL with standard NAC alone or combined with MK-2206 (AKT inhibitor) treatment. Blood was collected at pretreatment (T0), 3 weeks after initiation of paclitaxel (T1), between paclitaxel and anthracycline regimens (T2), or prior to surgery (T3). A personalized ctDNA test was designed to detect up to 16 patient-specific mutations (from whole-exome sequencing of pretreatment tumor) in cfDNA by ultra-deep sequencing. The median follow-up time for survival analysis was 4.8 years. At T0, 61 of 84 (73%) patients were ctDNA positive, which decreased over time (T1: 35%; T2: 14%; and T3: 9%). Patients who remained ctDNA positive at T1 were significantly more likely to have residual disease after NAC (83% non-pCR) compared with those who cleared ctDNA (52% non-pCR; odds ratio 4.33, P= 0.012). After NAC, all patients who achieved pCR were ctDNA negative (n= 17, 100%). For those who did not achieve pCR (n= 43), ctDNA-positive patients (14%) had a significantly increased risk of metastatic recurrence [hazard ratio (HR) 10.4; 95% confidence interval (CI) 2.3-46.6]; interestingly, patients who did not achieve pCR but were ctDNA negative (86%) had excellent outcome, similar to those who achieved pCR (HR 1.4; 95% CI 0.15-13.5). Lack of ctDNA clearance was a significant predictor of poor response and metastatic recurrence, while clearance was associated with improved survival even in patients who did not achieve pCR. Personalized monitoring of ctDNA during NAC of high-risk early breast cancer may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival.

Circulating Tumor DNA as a Prognostic Biomarker in Localized Non-small Cell Lung Cancer.

BackgroundRoutine clinical surveillance involves serial radiographic imaging following radical surgery in localized non-small cell lung cancer (NSCLC). However, such surveillance can detect only macroscopic disease recurrence and is frequently inconclusive. We investigated if detection of ctDNA before and after resection of NSCLC identifies the patients with risk of relapse, and furthermore, informs about response to management.MethodsWe recruited a total of 77 NSCLC patients. A high-throughput 127 target-gene capture technology and a high-sensitivity circulating single-molecule amplification and resequencing technology (cSMART) assay were used to detect the somatic mutations in the tumor tissues as well as the plasma of NSCLC patients before and after surgery to monitor for minimal residual disease (MRD). Kaplan-Meier and Cox regression analysis were performed to evaluate the relapse-free survival (RFS) and overall survival (OS) of patients with predictor variables.ResultsPatients with a higher stage (III/IV) and preoperative ctDNA-positive status demonstrated a significant 2.8-3.4-fold risk and 3.8-4.0-fold risk for recurrence and death, respectively. Preoperative ctDNA-positive patients associated with a lower RFS (HR = 3.812, p = 0.0005) and OS (HR = 5.004, p = 0.0009). Postoperative ctDNA-positive patients also associated with a lower RFS (HR = 3.076, p = 0.0015) and OS (HR = 3.195, p = 0.0053). Disease recurrence occurred among 63.3% (19/30) of postoperative ctDNA-positive patients. Most of these patients 89.5% (17/19) had detectable ctDNA within 2 weeks after surgery and was identified in advance of radiographic findings by a median of 12.6 months.ConclusionAdvanced stage and preoperative ctDNA-positive are strong predictors of RFS and OS in localized NSCLC patients undergoing complete resection. Postoperative detection of ctDNA increases chance to detect early relapse, thus can fulfill an important role in stratifying patients for immediate further treatment with adjuvant and neoadjuvant therapy.

Abstract 3096: Liquid biopsy cell-free circulating tumor DNA as prognostic biomarker for stage III colon cancer patients

Abstract Introduction: Stage III colon cancer patients undergo surgery followed by adjuvant chemotherapy (ACT) according to clinical guidelines. However, 50% would be cured by surgery alone and are being overtreated, while 30-35% will relapse despite adjuvant treatment. This means that only 15-20% of the patients benefit from ACT. Therefore, there is a need for better prognostic biomarkers to stratify patients for ACT. Detection of cell-free circulating tumor DNA (ctDNA) in blood after surgery is indicative of minimal residual disease and highly prognostic for disease recurrence. Hence, detecting liquid biopsy ctDNA is a promising approach to guide treatment decisions in stage III colon cancer. Aim: Determine prognostic value of ctDNA in stage III colon cancer patients treated with ACT in order to reduce futile treatment. Methods: 315 stage III colon cancer patients treated with ACT will be included in the prospective observational study “PROVENC3” (PROgnostic Value of Early Notification by Ctdna in Colon Cancer stage 3). The Prospective Dutch Colorectal Cancer Cohort (PLCRC) infrastructure organizes accrual of colorectal cancer patients in more than 50 hospitals in the Netherlands, among which 25 hospitals that accrue patients for PROVENC3. If informed consent is provided, blood is collected at baseline, post-surgery, post-ACT and every six months up to 3 years, and shipped to a central location. Tumor-informed detection of mutations in ctDNA will be performed by combined analysis of targeted sequencing of a panel of &amp;gt;30 genes in cfDNA (PGDx elio plasma test) and a panel of &amp;gt;500 genes in DNA from formalin-fixed paraffin-embedded tumor tissue (PGDx elio tissue complete assay). The clinical, pathological, and molecular data will be handled according to the FAIR (findable, accessible, interoperable and reusable) principles and integrated in cBioPortal. Results and future directions: Around 125 patients and 400 blood samples included to date. Once sequencing data are obtained we will determine: 1) the proportion of ctDNA-positive and ctDNA-negative patients after surgery and the corresponding recurrence rates; 2) the prognostic value of ctDNA pre-surgery; and 3) the lead time between ctDNA detection and recurrence. Ultimately, the results of this study will be used to model and design an ethically acceptable and cost-effective ctDNA-guided interventional trial, to reduce futile ACT and its associated side-effects in stage III colon cancer patients. Citation Format: Carmen Rubio Alarcón, Dave E. van der Kruijssen, Lana Meiqari, Linda J. Bosch, John K. Simmons, Victor E. Velculescu, Daan van den Broek, Cornelis J. Punt, Veerle M. Coupé, Miriam Koopman, Gerrit A. Meijer, Geraldine R. Vink, Remond J. Fijneman. Liquid biopsy cell-free circulating tumor DNA as prognostic biomarker for stage III colon 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 3096.

Abstract PR08: The genomic landscape of metastatic urothelial carcinoma from circulating tumor DNA

Abstract The recent expansion of treatment options for patients with metastatic urothelial carcinoma (mUC) has emphasized the need for molecular biomarkers in this setting. However, knowledge of the somatic genome landscape in mUC is limited; large-scale sequencing efforts have relied on primary tumor tissue from muscle-invasive bladder cancer (MIBC), revealing a molecularly heterogeneous disease characterized in particular by high somatic mutation rates. Putative prognostic and predictive biomarkers described from primary tissue need validation in metastatic tumors. Given the challenge of obtaining metastatic tissue, we sought to utilize circulating tumor DNA (ctDNA) to characterize the somatic landscape in mUC. We collected 162 whole-blood samples from 90 mUC patients. Targeted next-generation sequencing was performed on cell-free DNA (cfDNA) and matched leukocyte DNA utilizing a custom 50-gene panel. Somatic alteration frequencies in our metastatic cohort were compared to those reported for primary MIBC by TCGA (Cell 2017, n=412), with data obtained via cBioPortal. Our cohort of 90 mUC patients included 14% with upper tract disease. The median cfDNA sequencing depth was 986x, with ctDNA detectable in at least one blood collection for 81% (73/90) of mUC patients. In 10/73 patients, the estimated tumor mutation burden was ≥30 mutations per Mb. TP53 was the most frequently mutated gene (46/73 ctDNA-positive patients). Chromatin modifiers were also frequently altered: ARID1A 27%, and 25% for KDM6A and KMT2D. Genes mutated in the PI3K pathway included PIK3CA (22%), PTEN (5.5%), and PIK3R1 (1.4%). FGFR3 mutations were identified in 8.2% of patients. ERBB2 mutations were present in 12.3% of patients, and amplification was detected in eight patients. Thus, the metastatic somatic landscape closely resembles primary disease; however, comparison to TCGA dataset revealed mutations in TP53 and FGFR1 were enriched for in the metastatic setting (TP53: 63.0% vs. 48.1%, p = 0.022; FGFR1: 5.5% vs. 1.5%, p = 0.049; two-sided Fisher's exact test). Profiling of ctDNA from a large mUC cohort suggests a relatively similar somatic landscape to primary MIBC. However, alterations in key driver genes are potentially over- or underrepresented in metastatic disease, which has prognostic implications. In mUC, ctDNA profiling is a powerful alternative to metastatic tissue biopsy. This abstract is also being presented as Poster B19. Citation Format: Gillian R. Vandekerkhove, Matti Annala, Jean-Michel Lavoie, Nora Sundahl, Simon Walz, Takeshi Sano, Andrew Murtha, Tilman Todenhöfer, Piet Ost, Kim N. Chi, Peter C. Black, Bernhard Eigl, Alexander W. Wyatt. The genomic landscape of metastatic urothelial carcinoma from circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr PR08.

Undetectable circulating tumor DNA levels correlate with low risk of recurrence/metastasis in postoperative pathologic stage I lung adenocarcinoma patients

ObjectivesThe application of circulating tumor DNA (ctDNA) monitoring after resection in pathologic(p) stage I lung adenocarcinoma (LUAD) patients remains controversial and it is of great clinical interest to decipher the difference of genetic features between ground-glass opacity (GGO) and solid nodules (non-GGO) subgroups. We aim to assess the utility of ctDNA in tracking early recurrence or metastasis following surgery and reveal the genetic differences between GGO and non-GGO. Materials and methodsTumor tissues and matched postoperative plasma samples were collected from a total of 82 (p)stage I LUAD patients. Comprehensive genomic profiling was performed using capture-based hybrid next generation sequencing by targeting 422 cancer relevant genes. ResultsEGFR and TP53 represent commonly mutated genes in this cohort of (p)stage I lung adenocarcinoma, followed by alterations in ALK, PIK3CA, STK11 and MYC. For a median follow-up period of 22.83 months after surgery, 65 out of 67 ctDNA-negative patients remained progression-free, while 3 out of 15 ctDNA-positive patients progressed [P = 0.040; positive predictive value = 0.20, 95 % confidence interval (CI), 0.04−0.48; negative predictive value = 0.97, 95 % CI, 0.9–1]. With time-dependent Cox regression analysis, we observed that ctDNA positivity significantly correlated with increased probability of early tumor recurrence or metastasis (P = 0.02, HR=8.5). Further comparison between GGO and non-GGO subgroups indicated the frequency of TP53 mutations in non-GGO was markedly higher than that in GGO (47 % vs 21 %, P < 0.05). Pathway analysis showed the epigenetic regulation pathway was more frequently affected in GGO subgroup, while impaired apoptosis/cell cycle pathway was more enriched in non-GGO LUADs. ConclusionsOur longitudinal ctDNA monitoring data showed that undetectable ctDNA may predict low risk of tumor recurrence or metastasis in postoperative (p)stage I LUAD patients, while it requires further investigation on how robust the positive ctDNA results could predict tumor relapse in these patients. Clinical registration numberNCT03172156.

Abstract A18: Plasma circulating tumor DNA is scarce and confounded by clonal hematopoiesis in metastatic renal cell carcinoma

Abstract Despite the recent implementation of novel targeted therapies, the 5-year survival rate for metastatic renal cell carcinoma (mRCC) remains dismal. Prognostic and predictive biomarkers are urgently required to avoid unnecessary patient morbidity and financial toxicity. Exploratory studies leveraging archival primary tumor tissue are considered suboptimal in this setting due to the considerable heterogeneity of metastatic lesions. Circulating tumor DNA (ctDNA) permits the noninvasive characterization of metastatic cancers using a simple blood draw. In this study, we sought to establish the utility of a ctDNA-based assay as a tool to profile the somatic genome of patients with mRCC. We collected whole blood from 55 progressing mRCC patients. All patients were systemic therapy naïve at the time of sample collection. Plasma cell-free DNA (cfDNA) and matched leukocyte DNA were subjected to targeted sequencing across 981 cancer-associated genes. Matched tumor tissue from 14 patients was also analyzed. The median cfDNA sequencing depth for this cohort was 938x. 33% of patients had evidence for RCC-derived ctDNA above 1% of total cfDNA; this was significantly lower than prostate or bladder cancer patients analyzed using the same approach. Among ctDNA-positive patients, ctDNA fraction averaged only 3.9% and showed no association with clinical variables or cfDNA yield. In these patients, the most commonly mutated genes were VHL, BAP1, and PBRM1, and matched tissue concordance was 77%. Evidence of somatic expansions unrelated to RCC, such as clonal hematopoiesis of indeterminate potential (CHIP), was detected in 43% of patients. Pathogenic germline mutations in DNA repair genes were detected in 11% of patients. Patients with ctDNA above 1% had shorter overall survival and progression-free survival on first-line therapy. Patients with evidence of CHIP but not ctDNA had an intermediate prognosis compared to ctDNA-positive and ctDNA-negative patients. CfDNA sequencing enables characterization of the somatic RCC genome in only a minority of metastatic RCC patients. Due to low ctDNA abundance and presence of non-RCC derived somatic clones in circulation, cfDNA sequencing may not be a simple pan-patient alternative to tissue biopsy in metastatic RCC. Citation Format: Jack V. W. Bacon, Matti Annala, Maryam Soleimani, Jean-Michel Lavoie, Alan I. So, Martin E. Gleave, Ladan Fazli, Kim N. Chi, Christian K. Kollmannsberger, Alexander W. Wyatt, Lucia Nappi. Plasma circulating tumor DNA is scarce and confounded by clonal hematopoiesis in metastatic renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A18.

Abstract A24: Identifying the genomic and clinical features of AKT1/PIK3CA mutant metastatic prostate cancer using circulating tumor DNA

Abstract Background: Hotspot activating mutations in AKT1 and PIK3CA represent a rare but potentially unique subset of metastatic prostate cancers (mPCa). The accompanying genomic and clinical features of these patients are currently unknown. Preclinical evidence suggests that unconstrained PI3K signaling via PTEN loss may render androgen receptor (AR)-targeted therapy less effective. Given the availability of agents that target nodes within the PI3K pathway, elucidating the genomic properties of AKT1/PIK3CA mutant patients and their response to AR-targeted therapy will be critical for therapeutic selection. Methods: We performed deep targeted sequencing on 1,381 cell-free DNA samples from 608 patients with mPCa. Analysis was restricted to patients with hotspot AKT1 or PI3KCA mutations of presumed clonal origin, requiring a variant allele frequency &amp;gt;25% of a sample’s ctDNA fraction. Activating AKT1 and PIK3CA mutations were defined as recurrent hotspot mutations with a cBioPortal annotation, as well as functionally equivalent variants within 5bp of these canonical hotspot positions. Patient records were reviewed for baseline clinical characteristics, as well as time from androgen deprivation therapy (ADT) initiation to castration resistance and overall survival (OS). Results: 5.9% (36/608) of patients harbored at least 1 clonal hotspot mutation in either AKT1 or PIK3CA, of which p.E17K and p.E545K/Q/A were most common. This population had a significantly higher ctDNA fraction compared to a control cohort of AKT1/PIK3CA wild-type mPCa patients (Mann-Whitney U test, 0.48 vs. 0.21, p &amp;lt; 0.001). There were no differences in PTEN copy number or mutation frequency compared to the control cohort. Although AR mutations and copy number amplifications were observed at similar frequencies, patients harboring AKT1 or PI3KCA mutations had fewer additional copies of AR (median 4.71 vs. 10.33, p=0.011, Mann-Whitney U Test). 30 patients with activating AKT1/PIK3CA mutations had clinical outcomes available. Interestingly, there were no significant differences in time to castration resistance or OS compared to ctDNA-positive patients without activating PI3K defects. Conclusions: AKT1/PIK3CA mutant mPCa is defined by a genomic landscape featuring high ctDNA-fraction and lower levels of AR amplification. Response to standard-of-care treatment among these patients is typical for ctDNA-positive mPCa. These findings may nominate patients who may benefit from PI3K-targeted therapeutics following resistance on AR-targeted therapy. Citation Format: Cameron M. Herberts, Andrew J. Murtha, Simon Fu, Gang Wang, Elena Schönlau, Anna Gleave, Steven Yip, Arkhjamil Angeles, Sebastien Hotte, Abdulraheem Alshangiti, Ben Tran, Scott North, Sinja Taavitsainen, Kevin Beja, Gillian Vandekerkhove1, Elie Ritch, Fred Saad, Nayyer Iqbal, Martin E. Gleave, Matti Annala, Kim N. Chi, Alexander W. Wyatt. Identifying the genomic and clinical features of AKT1/PIK3CA mutant metastatic prostate cancer using circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A24.

“Decision for adjuvant treatment in stage II colon cancer based on circulating tumor DNA:The CIRCULATE-PRODIGE 70 trial”

BackgroundAdjuvant treatment for stage II colon cancer remains debated. Finding a tool to select patients at risk for disease recurrence may help the clinical decision. Circulating tumor DNA (ctDNA) has been reported recently as a potential predictive marker for disease recurrence. We thus aim to test its ability to better select stage II colon cancer patients for adjuvant therapy. MethodsThis national, phase III trial (NCT 2019-000935-15) conducted in more than 100 centers in France, plans to screen around 2640 patients in order to randomize (2:1; minimization method) 198 ctDNA positive patients. Patients aged 18 to 75 years with ECOG performance status ≤1 with R0 surgical resection of a pT3-T4aN0 colon or high rectum adenocarcinoma will be randomized within 63 days after curative-intent surgery, to adjuvant mFOLFOX6 (oxaliplatin 85 mg/m², leucovorin 400 mg/m², and 5-FU bolus 400 mg/m2 then 5FU Continuous infusion 2.4 g/m²) every two weeks for 12 cycles or observation. Patients will be followed for maximum 7 years. A gain of 17.5% in 3-yr disease free survival (DFS) is expected (42.5% in the experimental arm vs. 25% in the control arm; HR:0.62; α, 5% [two-sided log-rank test]; 1-β, 80%). Secondary endpoints include 2-yr DFS, overall survival, and toxicity.Recruitement began End of January 2020.

Circulating tumor DNA (ctDNA) analysis predicts recurrence following surgery in patients with stage I-IIIA non-small-cell lung cancer (NSCLC): Results of GASTO1035 and GASTO1018.

9023 Background: Circulating tumor DNA can be detected in the plasma and serum of patients with solid tumors and has emerged as a noninvasive biomarker for dynamically monitoring tumor. Postsurgical ctDNA analysis of early-stage NSCLC may identify patients at high risk of recurrence and facilitate early intervention and personalized cancer therapy. Methods: These studies recruited 123 patients with newly diagnosed resectable stage I-IIIA NSCLC. Preoperative and postoperative plasma and postoperative tissue samples were subjected to next-generation sequencing (Nanjing Shihe Jiyin Biotechnology Inc.) using a 425 cancer-related genes panel. Peripheral blood samples were collected before surgery, postoperatively within 1 month, and every 3-6 months for up to 3 years. Plasma samples with at least 1 variants detected in tissue samples were defined as ctDNA positive. Results: After 4 exclusions, 119 eligible patients were enrolled from June 2016 to February 2019. Presurgical ctDNA was detectable in 31 of 117 (26.5%) patients and was associated with inferior recurrence-free survival (HR, 3.90, 95% CI, 1.44-10.58, P = 0.004). Similarly, ctDNA was detected in 13 of 116 (11.2%) of the first postsurgical samples and was associated with shorter RFS (HR, 3.54, 95% CI, 1.22-10.23, P = 0.002). During surveillance after surgery, ctDNA-positive patients (38/119, 31.9%) were more than 9 times more likely to experience disease recurrence than ctDNA-negative patients (HR, 9.17, 95% CI, 2.60-32.42, P &lt;0.001). Serial ctDNA detection preceded radiologic disease recurrence by a median lead time of 4.23 months (95% CI, 0.91-7.54 months). We also observed a positive correlation between the ctDNA detection rate and the disease stage. Conclusions: These results suggest that detection of ctDNA before and after surgery is associated with the identification of a high risk of disease recurrence of resectable NSCLC. Perioperative ctDNA analyses identify disease recurrence earlier than standard-of-care radiologic imaging, and thus could facilitate personalized cancer treatment at early time points. Clinical trial information: NCT03465241 and NCT03172156.

Abstract GS5-02: Detection of circulating tumor DNA (ctDNA) after neoadjuvant chemotherapy is significantly associated with disease recurrence in early-stage triple-negative breast cancer (TNBC): Preplanned correlative results from clinical trial BRE12-158

Abstract Background: A significant proportion of patients with early-stage TNBC are treated with neoadjuvant chemotherapy (NAC). Sequencing of ctDNA after surgery can be used to detect minimal residual disease and predict which patients may experience clinical recurrence. Methods: BRE12-158 is a recently completed Phase II clinical trial which randomized early-stage TNBC patients with residual disease after NAC to post-neoadjuvant genomically-directed therapy vs treatment of physician choice. 151 patients had a plasma sample collected at the time of treatment assignment (after surgery and radiation). ctDNA was successfully sequenced in 150 patients. 148 of the 150 sequenced patients had clinical follow-up. Sequencing was performed by Foundation Medicine using the FoundationOne Liquid assay which profiles for 70 commonly mutated oncogenes. Presence of mutated ctDNA was associated with distant disease free survival (DDFS) and overall survival (OS) in univariate analysis using the Log-Rank test, and in multi-variate analysis using Cox proportional hazards model. Results: Mutated ctDNA was detected in 94 of 148 sequenced patients (64%). TP53 was the most commonly mutated gene consistent with prior genomic studies of TNBC. At 16.7 months of median follow-up, detection of ctDNA was significantly associated with an inferior DDFS (median DDFS 32.5 months vs. Not Reached, p=0.0030). At 24 months, the DDFS probability was 53% in ctDNA-positive patients as compared to 81% in ctDNA-negative patients. In multi-variate analysis, when considering significant covariates, including: residual cancer burden (RCB); number of positive lymph nodes; tumor size; stage; grade; age; and race; detection of ctDNA remained independently associated with inferior DDFS (HR=3.1, CI: 1.4-6.8, p=0.0048). Similarly, detection of ctDNA was associated with inferior OS in univariate (p=0.021) and multi-variate analysis (HR=2.7, CI:1.1-6.2, p=0.022). Lastly, we observed a correlation between higher maximum somatic allele frequency and a shorter DDFS interval in multivariate analysis (HR=4.7, CI: 1.04-21.1, p=0.044) and shorter OS (HR=4.9, CI:1.06-22.4, p=0.041), suggesting that the quantitative degree of ctDNA burden is associated with clinical outcome. Conclusions: Detection of ctDNA in early-stage TNBC after neoadjuvant chemotherapy is an independent predictor of disease recurrence, and represents an important novel stratification factor for future post-neoadjuvant trials. Citation Format: Milan Radovich, Guanglong Jiang, Christopher Chitambar, Rita Nanda, Carla Falkson, Filipa C. Lynce, Christopher Gallagher, Claudine Isaacs, Marcelo Blaya, Elisavet Paplomata, Radhika Walling, Karen Daily, Reshma Mahtani, Michael A. Thompson, Robert Graham, Maureen E. Cooper, Dean C. Pavlick, Lee Albacker, Jeff Gregg, Casey L. Bales, Bradley A. Hancock, Erica Cantor, Fei Shen, Anna Maria V. Storniolo, Sunil Badve, Tarah Ballinger, Kathy D. Miller, Bryan P. Schneider. Detection of circulating tumor DNA (ctDNA) after neoadjuvant chemotherapy is significantly associated with disease recurrence in early-stage triple-negative breast cancer (TNBC): Preplanned correlative results from clinical trial BRE12-158 [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 GS5-02.

Plasma Circulating Tumor DNA and Clonal Hematopoiesis in Metastatic Renal Cell Carcinoma.

There is a lack of molecularly-informed biomarkers for patients with metastatic renal cell carcinoma (RCC). Plasma cell-free DNA (cfDNA) sequencing is a minimally-invasive alternative to tissue for profiling the genome in other cancers but relevance in metastatic RCC remains unclear. Whole blood was collected from 55 patients with metastatic RCC. Plasma cfDNA and leukocyte DNA were subjected to targeted sequencing across 981 cancer genes. Matched tumor tissue from 14 patients was analyzed. Thirty-three percent of patients had evidence for RCC-derived circulating tumor DNA (ctDNA), significantly lower than patients with metastatic prostate or bladder cancer analyzed using the same approach. Among ctDNA-positive patients, ctDNA fraction averaged only 3.9% and showed no strong association with clinical variables. In these patients, the most commonly mutated genes were VHL, BAP1, and PBRM1, and matched tissue concordance was 77%. Evidence of somatic expansions unrelated to RCC, such as clonal hematopoiesis of indeterminate potential, were detected in 43% of patients. Pathogenic germline mutations in DNA repair genes were detected in 11% of patients. CtDNA-positive patients had shorter overall survival and progression-free survival on first-line therapy. Patients with evidence of clonal hematopoiesis of indeterminate potential had an intermediate prognosis compared with ctDNA-positive and -negative patients. CfDNA sequencing enables straightforward characterization of the somatic RCC genome in a minority of patients with metastatic RCC. Owing to low ctDNA abundance, and the presence of non-RCC derived somatic clones in circulation, cfDNA sequencing may not be a simple pan-patient alternative to tissue biopsy in metastatic RCC.