Increase In ctDNA Research Articles

Impact of circulating tumor DNA (ctDNA) detection on survival outcomes of patients (pts) treated with immune-checkpoint inhibitors (ICIs) in early clinical trials.

2542 Background: Detection of ctDNA is a promising tool for managing pts in oncology. Most methods require whole-genome sequencing of tumor samples followed by the design of personalized panels for tracking purposes. In this work, we evaluated the prognostic and predictive value of total ctDNA quantification, using shallow whole-genome sequencing (shWGS) exclusively from plasma samples, in a prospective cohort of pts treated with ICIs in early clinical trials. Methods: IchorCNA pipeline was used to quantify ctDNA of shWGS from plasma ctDNA samples of pts treated with ICIs in phase 1 trials, collected at baseline and prior to cycle 2 (prec2). We investigated the association and correlation of ctDNA levels with surrogate markers for tumor burden (LDH levels, summatory of target lesions (TL), liver metastasis) using Spearman and Kruskal-Wallis tests. Kaplan-Meier estimates of overall survival (OS) of pts with baseline detectable ctDNA levels versus undetectable ctDNA were calculated. A multivariate Cox proportional hazards model, including continuous classical prognostic factors (LDH, albumin, hemoglobin, derived Neutrophil-to-Lymphocyte ratio (dNLR), platelets, number of metastases sites, ECOG PS) and ctDNA was performed. An estimate of progression free survival (PFS) of pts with ctDNA increase levels in preC2 versus a non-increase group was evaluated. Results: Since January 2018, 113 pts with no standard-treatment options were included. Median (m) follow up was 14.8 months (mo). Baseline ctDNA levels correlated significantly with baseline TL (R = 0.4, p < 0.001) and LDH levels (R = 0.61, p < 0.001). Pts with liver metastasis had higher levels of ctDNA (11,68 ng/ml) versus pts with no liver disease (2,31 ng/ml) (p < 0,001). In the survival analysis pts with detectable baseline ctDNA (74 pts) had significantly shorter OS compared with pts with undetectable ctDNA (39 pts); median 9.6 m (8.4 – 16.4) and NA m (13.6-NA), respectively (HR = 2.25 [1.18-4.29] p < 0.01). In the multivariate analysis, only ctDNA and albumin levels maintained the impact in OS (HR = 1.03, p < 0.05 and HR = 0.22, p < 0.05, respectively). Pts with early increases in ctDNA had a shorter PFS compared with those with a stabilization or decrease, median 1.9 m (1.6 – 4.0) and 3.0 m (2.6 – 3.8), respectively (HR = 2.19 [1.31-3.67], p < 0.01). Conclusions: Quantification of baseline ctDNA using shWGS is a strong independent prognostic factor. Early dynamic changes of ctDNA could be a useful tool to predict PFS outcomes.

Circulating tumor DNA is a sensitive marker for routine monitoring of treatment response in advanced colorectal cancer

Accurate assessment of chemotherapy response provides the means to terminate ineffective treatment, trial alternative drug regimens or schedules and reduce dose to minimize toxicity. Here, we have compared circulating tumor DNA (ctDNA) with carcinoembryonic antigen (CEA) for the cycle by cycle assessment of chemotherapy response in 30 patients with metastatic colorectal cancer. CtDNA (quantified using individualized digital droplet PCR (ddPCR) assays) and CEA levels were determined immediately prior to each chemotherapy cycle over time periods ranging from 42-548 days (average of 10 time points/patient). Twenty-nine/thirty (97%) patients had detectable ctDNA compared with 83% whose tumors were CEA-positive (>5 ng/ml) during the monitoring course. Over the course of treatment, 20 disease progression events were detected by computed tomography; ctDNA predicted significantly more of these events than CEA (16 (80%) versus 6 (30%), respectively; P-value = 0.004). When progression was detected by both ctDNA and CEA, the rise in ctDNA occurred significantly earlier than CEA (P-value = 0.046). Partial responses to chemotherapy were also detected more frequently by ctDNA, although this was not significant (P-value = 0.07). In addition, another 28 colorectal cancer patients who underwent potentially curative surgery and showed no evidence of residual disease were monitored with ctDNA for up to 2 years. Clinical relapse was observed in 6/28 (21%) patients. Four out of 6 of these patients showed a significant increase in ctDNA at or prior to relapse. Overall, ctDNA analyses were able to be performed in a clinically relevant timeline and were a more sensitive and responsive measure of tumor burden than CEA.

Circulating tumor DNA (ctDNA) dynamics associate with treatment response and radiological progression-free survival (rPFS): Analyses from a randomized phase II trial in metastatic castration-resistant prostate cancer (mCRPC).

5508 Background: ctDNA can inform on prognosis, treatment response and survival. We evaluated ctDNA in serial plasma samples from patients enrolled in A.MARTIN (NCT01485861), a randomized phase II study of abiraterone with or without ipatasertib in patients with mCRPC. Methods: Blood was collected in cell-free DNA Streck tubes from 216 patients at 3 time points; baseline, C3D1 and end of treatment. Cell-free DNA (cfDNA) was extracted from plasma using a Circulating DNA Kit (Qiagen) on a QIASymphony machine (Qiagen). 25ng of extracted cfDNA was used in library preparation, constructed with a custom designed, 58 gene, QIAseq Targeted DNA panel (Qiagen) enriched for PI3K/AR pathway genes. Samples were sequenced to mean depth of 3394x on a NextSeq500 machine. Unless otherwise noted, all analyses combine patients across the 3 study arms, and reported p-values are unadjusted. Results: Baseline (BL) ctDNA positivity correlated with radiological progression-free survival (rPFS; HR: 1.8 [95% CI 1.3-2.6], p < 0.01); this association with rPFS was maintained in a multivariate cox model with > 5 baseline clinical variables (HR: 1.6 [95% CI 1.1-2.4]; p = 0.011). Patients with a C3D1 reduction in ctDNA had superior rPFS compared to patients with a C3D1 increase in ctDNA (HR: 2 [95% CI 1.3-3.2], p < 0.01). The rate of ctDNA clearance at C3D1 was higher in the Ipatasertib 400mg arm compared to placebo (56.3% versus 24.4%, p < 0.01). We find that changes in ctDNA associated with best confirmed overall response (p = 0.024); CR patients had the greatest reduction in ctDNA (mean of -23.4%), followed by PR (-16.3%), then SD (-4.1%), and lastly PD patients (-1.3%). Changes in ctDNA levels correlated with SLD changes (rs = 0.289, p = 0.05), and also PSA changes (rs = 0.33, p < 0.01). Changes in ctDNA were associated with rPFS in a multivariate cox analysis that included PSA change (p < 0.01), as well as in a separate multivariate analysis that included SLD change (p < 0.01). Lastly, we explored CNVs and observed emerging resistance mutations in progression samples, including alterations in TP53, AR, FOXA, PTEN, and PI3K/AKT pathway genes. Conclusions: ctDNA analyses may help (i) identify poorer prognosis disease at baseline, (ii) inform on treatment response (CR/PR/SD/PD) and radiological progression free survival (rPFS) in on-treatment (C3D1) samples, and (iii) can elucidate emerging resistance mechanisms at disease progression. Clinical trial information: NCT01485861 .

A randomized study evaluating tailoring of advanced/metastatic colorectal cancer (mCRC) therapy using circulating cell-free tumor DNA (ctDNA) (TACT-D).

TPS277 Background: Identifying non-responders to expensive salvage therapies with modest benefits and substantial treatment related adverse events (TRAEs) (e.g. regorafenib/TAS102 in mCRC) is necessary to maximize benefits and limit toxicities. Serial ctDNA sequencing is reliable for tracking tumor dynamics and appears to predict resistance to therapy earlier than radiographic progression. Methods: TACT-D is a randomized study (N = 100) to validate the ability of changes in ctDNA (ΔctDNA) to predict resistance early and in limiting toxicities. We hypothesize that increase in ctDNA (measured by variant allele fraction) at 2 weeks (wk) into treatment can predict resistance earlier than standard radiographic means [at 8-12 wk] and detecting resistance early can enable prompt change in therapy resulting in reduction of TRAEs. Pts with mCRC eligible for either regorafenib/TAS102 are randomized 2:1 to either standard of care (SOC) or ctDNA arm. On SOC arm, treatment is given as per current paradigm i.e. for 8 wk and then restaging. On ctDNA arm, decision to continue therapy is based on ctDNA change between baseline and 2 weeks [ΔctDNA = ctDNA (C1D15 – C1D1)]. Increase in ctDNA triggers early radiographic staging (4 wk). Treatment is continued for disease stability/regression and discontinued for progression. Study has 2 co-primary endpoints: 1) Association of Δ ctDNA and radiographic progression [62 pts on SOC arm, have 94% power (2-sided α 0.05) to detect difference of 95% vs. 58% in progressive disease between pts with increase vs decrease in ctDNA] and 2) Compare proportion of pts experiencing TRAEs within 4 months between study arms [67 in SOC arm and 33 in ctDNA arm have 82% power (2-sided α 0.05) to detect a 30% decrease in toxicity]. Key secondary endpoints include: patient-reported outcomes (MD Anderson Symptom Inventory and PRO-CTCAE), OS, clinical events of special interest (hospitalizations/ER visits/medical interventions such as blood transfusions/IV hydration), clinical trial referral and cost effectiveness. Study is now actively accruing pts (NCT03844620). Funding: MD Anderson Cancer Center, Houston, TX & Guardant Health Inc., Redwood City, CA. Clinical trial information: NCT03844620.

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.

Abstract 2293: Is a single driver gene mutation sufficient for monitoring early response in advanced colorectal cancer

Abstract Purpose: Circulating tumor DNA (ctDNA) monitoring based on an individual mutation profile during therapy is under intense investigation in modern oncology. We previously reported that the increase of ≥50% of at least one somatic mutation among multiple monitored mutations per patient is associated with a significantly worse outcome1. This study investigates whether the ctDNA monitoring of one driver gene mutation, provides enough information as compared to multiple mutations to assess response to regorafenib in advanced chemorefractory colorectal cancer (aCRC) at an early timepoint. Experimental procedures: Archival tumor tissue and plasma samples (PL) at baseline (BL) and 14 days (D14) after treatment initiation in aCRC pts (n=141) were prospectively collected in the RegARd-C multicenter clinical trial (NCT01929616). Somatic mutations were identified based on a CRC-oriented targeted gene sequencing of tumor tissue. All available (median 2 (1-4)) driver gene mutations were monitored per patient in PL at BL and D14 via droplet digital PCR (Bio-Rad QX200 ddPCR system) to assess ctDNA dynamics. Results: In 96 evaluable patients, the most frequently monitored mutated genes were APC (73%), TP53 (72%), KRAS (66%), and PI3KCA (23%). Among patients with ≥2 monitored mutations (73/96), one was selected at random and compared to previous methodology taking in account dynamics of all followed mutations. Optimal cutoff (CO) evaluation (Contal & O’Quigley method) separated patients (n=96) according to a ctDNA increase of ≥50% versus an increase of <50% or a decrease. The concordance of ctDNA dynamics based on one randomly selected mutation and multiple monitored mutations was 91%. Our data demonstrated that a ctDNA increase based on one single mutation taken at random is significantly associated with a worse clinical outcome in terms of progression-free survival (HR 2.42, 95% CI (1.56-3.74), P<0.001) and overall-survival (HR 2.17, 95% CI (1.41-3.34), P<0.001). In addition, when combining patients’ ctDNA dynamics to BL ctDNA levels (≥ or < 5 ng/mL optimal CO) or BL cell-free DNA (cfDNA) levels (≥ or < 50 ng/mL optimal CO), we could distinguish 4 patients’ subgroups with different prognosis. However, when performing a multivariate analysis including clinical parameters, BL ctDNA and BL cfDNA levels, BL ctDNA was not relevant in the presence of BL cfDNA. Conclusion: The monitoring of ctDNA dynamics based on only one randomly selected driver gene mutation versus multiple is equally informative to describe adequately aCRC patients’ outcome under regorafenib after 14 days of treatment onset. Especially, combined with pre-treatment ctDNA levels, this simplifies a personalized patient monitoring. 1 P. Kehagias, et al. Circulating tumor DNA detects early response to regorafenib in advanced colorectal cancer [abstract]. AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-221 Citation Format: Pashalina Kehagias, Caroline Vandeputte, Lieveke Ameye, Hakim El Housni, Amélie Deleporte, Karen Geboes, Thierry Delaunoit, Gauthier Demolin, Marc Peeters, Lionel D'Hondt, Jos Janssens, Javier Carrasco, Maria Gomez Galdon, Pierre Heimann, Marianne Paesmans, Patrick Flamen, Alain Hendlisz. Is a single driver gene mutation sufficient for monitoring early response in advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2293.

Detection of circulating tumor cells and circulating tumor DNA before and after mammographic breast compression in a cohort of breast cancer patients scheduled for neoadjuvant treatment

PurposeIt is not known if mammographic breast compression of a primary tumor causes shedding of tumor cells into the circulatory system. Little is known about how the detection of circulating biomarkers such as circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) is affected by breast compression intervention.MethodsCTCs and ctDNA were analyzed in blood samples collected before and after breast compression in 31 patients with primary breast cancer scheduled for neoadjuvant therapy. All patients had a central venous access to allow administration of intravenous neoadjuvant chemotherapy, which enabled blood collection from superior vena cava, draining the breasts, in addition to sampling from a peripheral vein.ResultsCTC and ctDNA positivity was seen in 26% and 65% of the patients, respectively. There was a significant increase of ctDNA after breast compression in central blood (p = 0.01), not observed in peripheral testing. No increase related with breast compression was observed for CTC. ctDNA positivity was associated with older age (p = 0.05), and ctDNA increase after breast compression was associated with high Ki67 proliferating tumors (p = 0.04). CTCs were more abundant in central compared to peripheral blood samples (p = 0.04).ConclusionsThere was no significant release of CTCs after mammographic breast compression but more CTCs were present in central compared to peripheral blood. No significant difference between central and peripheral levels of ctDNA was observed. The small average increase in ctDNA after breast compression is unlikely to be clinically relevant. The results give support for mammography as a safe procedure from the point of view of CTC and ctDNA shedding to the blood circulation. The results may have implications for the standardization of sampling procedures for circulating tumor markers.

Circulating tumor DNA fraction (ctDNA) as a surrogate predictive biomarker in metastatic castration-resistant prostate cancer (mCRPC).

5039 Background: Plasma ctDNA is a promising minimally invasive biomarker in mCRPC. Pre-treatment high levels of ctDNA reflect poor prognosis (Romanel et al, Sci Transl Med 2015; Annala et al, Cancer Discov 2018). However, the role of plasma ctDNA in prostate tumour monitoring is largely unexplored. We aimed to determine if monitoring tumour response by quantifying ctDNA levels in plasma could enable early assessment of therapy efficacy for mCRPC. Methods: Between January 2011 and June 2016, 132 sequential plasma samples from 54 mCRPC patients (pts) (30 pre- and 24 post-chemotherapy) treated with abiraterone (abi) were collected. Targeted next-generation sequencing was performed on the PGM Ion Torrent using a 316 or 318 Chip to account for 1000X expected coverage per target. We estimated the global tumour content for each sequential plasma sample from study patients by using the approach developed in (Carreira et al, Sci Trasl Med 2014; Romanel et al, Sci Transl Med 2015 ), which extends the CLONET framework (Prandi et al, Genome Biol 2014). Prostate Cancer Working Group -3 (PCWG3) criteria were used to assess clinical, biochemical (PSA) and radiographic (RAD) progression disease (PD). We considered ctDNA PD any increase of ctDNA from baseline value. Results: In our cohort of 54 pts (median age: 75 years, range 70-78), we observed 17 (31.5%) PD, 14 (25.9%) stable disease, and 23 (42.6%) partial/complete response after the first 3 months (mo) abi therapy. The odds ratio (OR) for PD having any increase in ctDNA and a PSA decline < 50% at ~3-mo therapy was 10.83, 95% CI 2.55-45.95, P = 0.001, and 3.27, 95% CI 0.89-12.3, P = 0.074, respectively. In addition, we assessed all 3 types of median PD time from starting abi treatment, suggesting the ability of ctDNA variation to predict overall PD [RAD PD = 6.8 mo, PSA PD = 4.4 mo, and ctDNA PD = 3.0 mo, P = 0.008). An increase of ctDNA levels during the first 3-mo abi treatment was significantly associated with a long-term androgen deprivation therapy (ADT) before plasma sample collection (previous ADT > 24 mo vs 12 > previous ADT ≤23 mo vs < 12 mo: P = 0.036). Conclusions: In mCRPC, an early change in ctDNA fraction may be considered as a predictive biomarker playing a key role in individualized disease monitoring. Prospective evaluation of treatment decisions based on ctDNA is now required.

Abstract P4-08-08: Genomic progression, detected by circulating tumor DNA (ctDNA) sequencing, as an early predictor of disease progression in metastatic breast cancer (MBC)

Abstract Background: The availability of multiple therapies has transformed the landscape of MBC, but also brought the challenge of selecting the right therapy for an individual patient. Furthermore, in patients with Hormone Receptor positive (HR+) breast cancer who have bone metastases only it may be difficult to assess effectiveness of therapy via imaging. Peripheral ctDNA detection and analysis by next-generation sequencing (NGS) has gained popularity in cancer diagnosis and therapeutics due to its relative noninvasiveness, ease of use, and high sensitivity. Here, we explore the utility of ctDNA change as a predictor for disease progression in MBC. We hypothesized that genomic progression is a harbinger of subsequent radiologic progression in patients with MBC. Methods: We analyzed change from pre-treatment (baseline) to on-treatment ctDNA mutant allele fraction (MAF) among patients with MBC. Patients receiving standard-of-care therapies or investigational agents on clinical trials at our institution were included. All patients were followed from the date of baseline test until death or data cutoff (6/20/2018). All peripheral blood specimens were collected and analyzed between 1/7/2016 and 3/1/2018 via NGS (Guardant360®). Peripheral blood specimens were sequenced prior to initiation of a new therapeutic regimen (baseline) and subsequently at least once while on-treatment, on average 4-12 weeks later. All patients had a follow-up CT scan of chest, abdomen and pelvis 2-4 weeks after the on-treatment NGS. A priori, we defined genomic progression as increase in ctDNA total MAF of at least 20% from baseline. We utilized Cox regression analysis to identify whether genomic progression was a predictor of radiologic progression, adjusting for common prognostic variables. Results: All patients (N= 77) were female, predominantly White (83.1%), and median age was 57 (range 32 to 77). Fifty one out of 77 patients (66.2%) were ER+, 5 HER2+, and 9 had triple negative breast cancer. The median MAF at baseline was 2.2% (range 0% - 61.7%). Common genomic alterations in ctDNA included PIK3CA, TP53, ESR1, AKT1, NF1. 27 out of 77 (35%) patients showed disease progression on the first subsequent CT scan, while 59 out of 77 (76.6%) progressed during the follow up time. We found that an increase in ctDNA MAF of at least 20% was a strong predictor of disease progression (HR =2.46, CI [1.14-5.32], p=0.02), compared to those who had a MAF increase of less than 20% or a decrease in total MAF. In multi-variable analysis, adjusting for age, number of prior therapies, type of therapy, and visceral metastases, increase in ctDNA remained a significant predictor for subsequent disease progression (HR =3.84, CI [1.63-9.07], p=0.002). Subset results in patients with bone metastases only, and relative comparison of ctDNA with standard tumor markers will be presented at the meeting. Conclusions: Genomic progression, identified by an increase in ctDNA MAF, is potentially an early predictor of subsequent disease progression in patients with MBC. Further research is needed to prospectively evaluate the clinical utility of ctDNA change as a surrogate marker in guiding treatment decision-making for patients with MBC. Citation Format: Velimirovic M, Juric D, Niemierko A, Spring LM, Vidula N, Malvarosa G, Yuen M, Moy B, Isakoff SJ, Ellisen LW, Bardia A. Genomic progression, detected by circulating tumor DNA (ctDNA) sequencing, as an early predictor of disease progression in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-08.

Abstract 3668: ctDNA and TCR dynamics predict response toimmune checkpoint blockade in non-small cell lung cancer

Abstract The dynamic nature of the cancer-immune system crosstalk has rendered single biomarker approaches insufficient to predict outcome from immune-targeted agents. These therapies also pose a challenge to radiographic response assessments that may underestimate the therapeutic benefit. There is therefore an urgent clinical need to develop molecular assays of response and resistance.In order to capture the dynamic nature of the anti-tumor immune response under checkpoint blockade, we devised an integrative non-invasive approach that captures the evolving neoantigen landscape and the associated T-cell receptor (TCR) repertoire. We employed whole exome sequencing to determine the genomic landscape of tumors and identify tumor-derived alterations in subsequent analyses of circulating cell-free tumor DNA (ctDNA), for 14 patients with metastatic NSCLC treated with immune checkpoint blockade. Liquid biopsies were obtained prior to treatment, at week 4-6 and at additional timepoints until disease progression. We used the ultrasensitive targeted error correction sequencing approach to analyze 58 cancer driver genes in the circulation of these patients and assessed the value of longitudinal ctDNA monitoring as a surrogate for response. In parallel, we evaluated dynamics changes in the TCR repertoire by TCR next generation sequencing of serially collected peripheral T cells and tumor infiltrating lymphocytes for each patient. Radiographic response assessments were performed using the RECIST 1.1 criteria. These analyses revealed that ctDNA dynamics predict outcome significantly earlier than imaging. Patients that responded to therapy had a significant drop in ctDNA early during the treatment course whereas non-responders had either limited changes or significant increase in ctDNA. For patients with acquired resistance, ctDNA kinetics revealed clearing of ctDNA at the time of response followed by a new peak at the time of progression. ctDNA detection early during the treatment course was a significant prognostic factor for progression-free and overall survival (log rank p=.004 and .002 respectively) and ctDNA elimination was superior to tumor mutation burden as a predictor of response to therapy. In parallel, we identified changes in the TCR repertoire that are predictive of outcome: peripheral T cell expansion of a subset of intra-tumoral clones was noted at the time of response whereas there was no evidence of T cell expansion in non-responders. Peripheral T cell expansion of a subset of intra-tumoral clones was noted to peak at the time of response and decrease to baseline levels at the time of resistance for patients with acquired resistance. Our findings suggest that a dynamic assay able to capture the tumor-immune system equilibrium would be superior to conventional analyses of static time points. Such an integrated approach would be highly relevant to tailored cancer immunotherapy strategies. Citation Format: Valsamo Anagnostou, Patrick Forde, Jarushka Naidoo, Kristen Marrone, Vilmos Adleff, James White, Jillian Phallen, Alessandro Leal, Carolyn Hruban, Ashok Sivakumar, Franco Verde, Rachel Karchin, Julie Brahmer, Victor Velculescu. ctDNA and TCR dynamics predict response toimmune checkpoint blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3668.

Oncological Assessment of Stent Placement for Obstructive Colorectal Cancer from Circulating Cell-Free DNA and Circulating Tumor DNA Dynamics.

The self-expanding metallic stent (SEMS) provides effective decompression for patients with malignant large bowel obstruction (MLBO); however, mechanical damage to malignant cells from insertion may negatively affect prognosis, similar to surgical manipulation, and its oncological safety is unclear. We examined mechanical damage from SEMS placement using circulating cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA). Between 1 November 2014 and 30 June 2017, 35 MLBO patients were analyzed, comprising 25 SEMS patients and 10 transanal decompression tube (TDT) patients (control). Blood samples were collected before and after decompression on days 0, 1, 3, and 7. cfDNA, ctDNA, white blood cells, C-reactive protein, and lactate dehydrogenase were analyzed. The clinical success rates of SEMS and TDT were 88 and 90%, respectively (p=1.0). The cfDNA concentration on day 7 was significantly higher in the SEMS group than in the TDT group (992 vs. 308ng/mL; p=0.005). A significant increase in ctDNA was observed in the SEMS group compared with the TDT group (83% vs. 22%; p=0.002). The cfDNA concentration showed strong positive correlations with ctDNA and lactate dehydrogenase (R 2=0.838 and 0.593, respectively), and a weak positive correlation with C-reactive protein (R 2=0.263). Despite equivalent clinical success rates, SEMS placement increased plasma levels of cfDNA and ctDNA by tumor manipulation, but TDT did not. Colonic stenting showed oncological risk in terms of molecular analysis.

Variations of circulating KRAS amount as a biomarker to monitor chemotherapy response in pancreatic cancer.

e15794 Background: Current treatment of pancreatic ductal adenocarcinoma (PDAC) is FOLFIRINOX or gemcitabine + nab-paclitaxel, and CA19-9 is the only approved biomarker to monitor tumor response. However, CA19-9 has several limitations (i.e. sensitivity and specificity), highlighting the need for new biomarkers. Being the majority of PDAC (75-95%) KRAS mutated (mutKRAS), the present study developed an analysis tool based on circulating tumor DNA (ctDNA) to monitor the variation of mutKRAS in PDAC undergoing first-line chemotherapy. Early variation of ctDNA mutKRAS after 15 days of treatment was correlated with response to therapy. Methods: Three ml of plasma were collected from patients with PDAC undergoing first-line FOLFIRINOX or gemcitabine + nab-paclitaxel. Patients underwent standard disease evaluation by imaging according to RECIST 1.1 criteria. Blood samples were drawn prior to cycle 1, after 14 days and at each radiological evaluation. ctDNA was extracted using a QIAmp Circulating nucleic acid Kit (Qiagen, Valencia, CA) and the KRAS codon 12 and p.G13D analysis was performed by digital droplet PCR (ddPCR, BioRad, Hercules, CA). Results: A total of 27 patients with locally advanced (15%) and metastatic (85%) PDAC were included in this study. mutKRAS in ctDNA was detected in 19 patients (70.3%) at baseline. There were no statistically significant differences in median PFS and OS in patients with baseline positive or negative ctDNA mutKRAS (p = 0.15). However, there was a statistically significant difference in PFS and OS between patients with increase vs. stability/reduction of ctDNA at the 14 day-evaluation (median PFS: 2.5 vs 7.5 months, p = 0.03; median OS: 9 vs 11.5 months p = 0.009). The imaging evaluation performed at 2 months after initiation of therapy demonstrated that all patients with an increase in ctDNA at 14 days had radiological progression of disease. Conclusions: The results of this pilot study support the use of ctDNA as a new marker for monitoring treatment outcome and disease progression in PDAC, suggesting that ctDNA mutKRAS is a non-invasive predictive factor of response in PDAC.

Abstract P1-02-06: Serial monitoring of circulating tumor DNA in patients with metastatic breast cancer

Abstract Background: For patients with MBC, there is currently no evidence that changing therapy on the basis of biomarker results improves outcome. Clinical benefit of treatment is defined as complete response, objective response, or stable disease as determined by RECIST criteria on radiological evaluation. Serial measurements of serum biomarkers such as CA2729 and CTCs have proven unsuccessful in predicting clinical benefit. Circulating tumor DNA(ctDNA) has emerged as a potential biomarker that may predict response to therapy or progression of disease. The present retrospective study was conducted to evaluate the relationship between change in ctDNA with clinical benefit determined by clinical and radiological evaluations of patients with MBC patients. Methods: We conducted a retrospective, single-institutional study to determine if serial monitoring of ctDNA allele frequency levels predict clinical benefit of a treatment. 55 patients with measurable MBC who had serial monitoring of ctDNA between August 2014 and May 2016 were included. The median age was 55.9 (27–94) years). Clinical outcomes were determined as per standard guidelines. The analysis was performed on all cases that had serial measurements of ctDNA with no change in therapy in between and the repeat blood draw was done within 30 days of repeat radiographic evaluation. The dataset contained 125 observations from 48 unique patients. The relationship between the change in ctDNA and clinical benefit was analyzed using a generalized linear model with a random subject effect to account for the intrapatient dependence occurring from obtaining multiple evaluations from the same patient. A logit link function was used akin to logistic regression and a compound symmetric correlation structure was assumed. Results: 68.8% of the cases were hormone receptor-positive, 18.8% HER2-positive, and 27.1% TNBC. The treatments received were 58.4% hormonal therapy, 31.2% chemotherapy, 26.4% included anti-HER2 therapy, 2 cases were on targeted therapy, and 1 case was not on any treatment. Three patients had stage 4 disease in complete remission. ctDNA analysis was repeated on average 4 days prior to radiological evaluation. The average time between repeat assessments was 108.5 days. 93% of the patients had a genomic alteration detected at some point during their course of disease. The most common mutations detected were TP53 41.7%, PIK3CA 35.4%, ESR1 18.8%, and ERBB2 amplifications 6.3%. A dichotomized change in ctDNA is a significant predictor of clinical benefit (p < 0.0001). The intrapatient correlation is estimated to be 0.273 for the transformed variable. The model yields a predicted probability of clinical benefit of 26.9% when the increase in ctDNA is greater than or equal to 0.5 and when the increase in ctDNA is less than 0.5, the a predicted probability of clinical benefit is 78.4%. The concordance of change in ctDNA and change in CA 27-29 was 76.2%. Conclusions: Serial evaluation of serum ctDNA may be useful to evaluate molecular response to treatment which may correlate with clinical benefit and potentially guide treatment decisions. Early indication that a chosen therapy is not effective may lead to avoidance of overtreatment and initiation of an alternative regimen. Further, prospective studies are needed. Citation Format: Patel A, Mukherjee A, Hwang D, Ensor J, Patel TA, Chang JC, Rodriguez AA. Serial monitoring of circulating tumor DNA in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-06.

Phase 2 study of panobinostat with or without rituximab in relapsed diffuse large B-cell lymphoma

AbstractThe majority of diffuse large B-cell lymphoma (DLBCL) tumors contain mutations in histone-modifying enzymes (HMEs), indicating a potential therapeutic benefit of histone deacetylase inhibitors (HDIs), and preclinical data suggest that HDIs augment the effect of rituximab. In this randomized phase 2 study, we evaluated the response rate and toxicity of panobinostat, a pan-HDI administered 30 mg orally 3 times weekly, with or without rituximab, in 40 patients with relapsed or refractory de novo (n = 27) or transformed (n = 13) DLBCL. Candidate genes and whole exomes were sequenced in relapse tumor biopsies to search for molecular correlates, and these data were used to quantify circulating tumor DNA (ctDNA) in serial plasma samples. Eleven of 40 patients (28%) responded to panobinostat (95% confidence interval [CI] 14.6-43.9) and rituximab did not increase responses. The median duration of response was 14.5 months (95% CI 9.4 to “not reached”). At time of data censoring, 6 of 11 patients had not progressed. Of the genes tested for mutations, only those in MEF2B were significantly associated with response. We detected ctDNA in at least 1 plasma sample from 96% of tested patients. A significant increase in ctDNA at day 15 relative to baseline was strongly associated with lack of response (sensitivity 71.4%, specificity 100%). We conclude that panobinostat induces very durable responses in some patients with relapsed DLBCL, and early responses can be predicted by mutations in MEF2B or a significant change in ctDNA level at 15 days after treatment initiation. This clinical trial was registered at www.ClinicalTrials.gov (#NCT01238692).

Abstract 5245: Identification and quantification of somatic structural variations in tumor, urine and plasma from bladder cancer patients

Abstract Bladder tumors can be divided into two clinically divergent groups, non-muscle invasive (NMIBC) and muscle invasive bladder tumors (MIBC). NMIBCs have a recurrence rate of 50-70%, while MIBCs often develop metastases. No molecular biomarkers are yet available to directly guide treatment and follow-up regimes for patients with BC. Aim of this study was to identify tumor specific, large somatic structural variations (SSV) in DNA from tumors and quantify SSVs in DNA from urine and in circulating free DNA (cfDNA) in plasma from successive visits during treatment and follow-up. Methods: Whole genome paired-end (WGS) or mate-pair (MPS) sequencing on HiSeq2000 and Nextseq sequencers. SSVs called by algorithms “CREST” or “Breakdancer”. cfDNA was extracted from 1-3 ml plasma. Quantification of SSVs was done by droplet digital PCR (ddPCR) using probes covering the breakpoint. We selected six patients with progressive/metastatic disease, followed for 4 to 19 years with consecutive sampling of tumors, urine and plasma samples (4 to 27 visits). WGS was carried out on DNA from germ-line and tumors from four patients (57x and 45x read depth). CREST identified 20-226 SSVs per tumor. PCR assays (n = 170, 21-64 per patient) showed validation rates of 60-75% (CTX), 75-90% (ITX), 88-100% (INS) and 50% (INV) among the eight tumors. MPS was carried out on DNA from two patients (22-30x physical read depth). Breakdancer identified 82 and 120 SSVs, 24 and 13 were potential somatic with a validation rate of 69% and 79%. For each patient, 3-5 fluorescent probes covering the breakpoint were used to quantify the level of tumor specific by ddPCR. SSVs were quantified in tumor DNA from several visits. Their frequency varied considerably (0.08-0.54) between the individual tumors, indicating a large inter-tumor heterogeneity. SSVs detected in DNA from urine pellets were correlated to the presence of tumor and treatment for some cases. Inter-individual cfDNA level ranged from 600-3500 genomic equivalents (GEs) per ml plasma. SSVs were identified in plasma. The level of ctDNA ranged between 5-300 GEs/ml plasma and an increase in ctDNA was already detectable up to 21 months before progression to muscle invasive disease. For metastasized disease, a 42-fold increase of the ctDNA level was observed compared to localized disease. No SSVs were detected in cfDNA from patients cured from BC (3 and 7 years post-recurrence and 5 years post-cystectomy). Conclusions: Tumor derived SSVs were identified and quantified in cfDNA from plasma of patients with progressive and metastatic BC. Detection of SSVs in cfDNA was possible at early time points before progression and metastasis of the disease were discovered by cystoscopy or image diagnostic. The tumor specific and highly sensitive detection and quantification of SSVs in cfDNA from plasma may offer a unique tool to monitor disease course, residual tumor burden, disease progression and treatment response in BC. Citation Format: Karin Birkenkamp-Demtröder, Iver Nordentoft, Emil Christensen, Søren Hoyer, Thomas Reinert, Søren Vang, Jørgen Bjerggaard Jensen, Torben F. Ørntoft, Lars Dyrskjøt. Identification and quantification of somatic structural variations in tumor, urine and plasma from bladder cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5245. doi:10.1158/1538-7445.AM2015-5245