Cell-free DNA cfDNA KRAS Pancreatic cancer Liquid biopsy A B S T R A C TWe used KRAS mutations to investigate the clinical relevance of circulating tumor DNA (ctDNA) measurements in patients with advanced pancreatic cancer. Fifty-three blood samples were collected from 14 prospectively recruited patients prior to chemotherapy (gemcitabine or FOLFIRINOX) and subsequently every month during treatment. Samples were processed by density centrifugation and plasma DNA isolation. A Peptideenucleic acideclamp PCR was then used to detect KRAS mutations (present in >90% of pancreatic cancers) as a surrogate marker for ctDNA. Plasma samples from 29 healthy individuals were analyzed as a reference group. Results were compared to conventional monitoring measures and survival data. Median follow-up time was 3.7 months (range 0.6e12.9 months).Ten (71%) patients had a positive KRAS status in the plasma samples obtained prior to chemotherapy, indicating the presence of ctDNA. Among the patients who were ctDNApositive before chemotherapy, nine (90%) experienced disease progression during followup, compared to one (25%) of four ctDNA-negative patients (P ¼ 0.01). The pre-therapy ctDNA level was a statistically significant predictor of both progression-free and overall survival (P ¼ 0.014 and 0.010, respectively). Of the 14 patients, ten had !2 follow-up samples; in several of these patients, the ctDNA level changed substantially during the course of chemotherapy. Changes in ctDNA levels corresponded both with radiological follow-up data and CA19-9 levels for several patients.

BackgroundIt was recently demonstrated that the size of cell-free DNA (cfDNA) fragments that originates from tumor cells are shorter than cfDNA fragments that originates from non-malignant cells. We investigated whether cfDNA fragment size and cfDNA levels might have prognostic value in patients with advanced pancreatic cancer.MethodsBlood samples were obtained from patients with advanced pancreatic cancer, before (n = 61) initiation of chemotherapy and after the first cycle of chemotherapy (n = 39). Samples were separated with density centrifugation and plasma DNA was isolated. Mode cfDNA fragment size and cfDNA levels were then determined using a 2100 Bioanalyzer. A cohort of partially age-matched healthy volunteers (n = 28) constituted the control group.ResultsBoth a pre-treatment cfDNA fragment size of ≤ 167 bp (mode) and high pre-treatment cfDNA levels were associated with shorter progression-free survival (PFS) (p = 0.002 and p < 0.001, respectively) and overall survival (OS) (p = 0.001 and p = 0.001, respectively). Furthermore, multivariable Cox regression analyses demonstrated that pre-treatment cfDNA levels could independently predict prognosis for both PFS (HR = 3.049, p = 0.005) and OS (HR = 2.236, p = 0.028).ConclusionThis study demonstrates that cfDNA fragment size and cfDNA levels can be used to predict disease outcome in patients with advanced pancreatic cancer. The described approach, using a rapid, economic and simple test to reveal prognostic information, has potential for future treatment stratification and monitoring.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1677-2) contains supplementary material, which is available to authorized users.

Most current methods of circulating tumour cell (CTC) enrichment target the epithelial protein EpCAM, which is commonly expressed in adenocarcinoma cells. However, such methods will not recover the fraction of CTCs that have a non-epithelial phenotype due to epithelial–mesenchymal transition. For phenotype-independent CTC enrichment, we developed a new enhanced negative depletion strategy—termed MINDEC—that is based on multi-marker (CD45, CD16, CD19, CD163, and CD235a/GYPA) depletion of blood cells rather than targeted enrichment of CTCs. Here we validated the performance of MINDEC using epithelial and mesenchymal cancer cell lines, demonstrating a mean recovery of 82 ± 10%, high depletion (437 ± 350 residual white blood cells (WBCs)/mL peripheral blood), linearity between spiked and recovered cells (correlation coefficient: r = 0.995), and a low detection limit (≥1 cell recovered in all four replicates spiked with 3 cells). For clinical validation of this method, we enumerated CTCs in peripheral blood samples from patients with metastatic pancreatic cancer, detecting CTCs in 15 of 21 blood samples (71%) from 9 patients. The promising performance of the MINDEC enrichment strategy in our study encourages validation in larger clinical trials.

Background: Circulating tumor DNA (ctDNA) may originate from necrotic or apoptotic tumor cells in the primary tumor, metastatic lesions or in the circulation. There is evidence that the ctDNA level may reflect the total tumor burden in a patient. We wanted to investigate whether changes in the ctDNA level might be used to monitor disease progression in pancreatic cancer patients with locally or advanced disease. Methods: Blood samples (9 mL in EDTA tubes) were collected from 15 prospectively recruited patients with locally advanced and/or metastatic pancreatic cancer before initiation of treatment, and subsequently every month during chemotherapy. The samples were processed by LymphoprepTM (Axis Shield) density centrifugation before plasma DNA isolation using the QIAamp Circulating Nucleic Acid kit (Qiagen). A high-fidelity polymerase-based PNA clamp PCR method (Gilje et al., 2008, Oltedal et al., 2010) was then used for the detection of KRAS mutations in exon 12 and 13, as a surrogate marker for ctDNA. KRAS mutations have previously been reported to be present in &gt;90% of the pancreatic cancers. Plasma samples from 29 healthy individuals were also analysed as a reference group for the PNA clamp PCR method. The results were compared with conventional biochemical and radiological monitoring measures. Results: The majority of the patients (80%) had metastatic disease and they were treated either by gemcitabine or FOLFORINOX. Nine (60%) patients had a positive KRAS status in the plasma samples obtained before initiation of chemotherapy, indicating presence of ctDNA. Moreover, 11 of the 15 included patients had ≥2 follow-up samples, and in several of these patients the ctDNA level changed substantially during the course of chemotherapy. In total, 17/38 (44.7%) patient samples were positive for plasma DNA KRAS mutations during chemotherapy. Changes in the ctDNA level seemed to correspond both to radiological follow-up data and changes in CA19-9 for several patients. Analyses of the total plasma DNA fraction with regard to disease monitoring, gave more inconclusive results in this small pilot study. Conclusion: Our pilot study gives support to the hypothesis that ctDNA may be used as a marker for monitoring treatment efficacy and disease progression in pancreatic cancer patients. This will be further investigated. Citation Format: Kjersti Tjensvoll, Morten Lapin, Tove Buhl, Satu Oltedal, Katrine Steen-Ottosen Berry, Bjørnar Gilje, Jon Arne Søreide, Millind Javle, Oddmund Nordgård, Rune Smaaland. Clinical relevance of circulating tumor DNA in plasma from pancreatic 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 5241. doi:10.1158/1538-7445.AM2015-5241

Purpose: To investigate the prognostic value of cell-free DNA (cfDNA) fragment size and cfDNA levels in patients with advanced pancreatic cancer. Methods: Blood samples were acquired from patients with locally advanced or metastatic pancreatic cancer at baseline (n = 61) and one month (n = 48) after initiation of chemotherapy. All samples were processed by Lymphoprep™ (Axis Shield) density centrifugation followed by DNA isolation from the plasma fraction using the QIAamp Circulating Nucleic Acid kit (Qiagen). The cfDNA fragment size and cfDNA levels were then determined using the High Sensitivity DNA kit on an Agilent 2100 Bioanalyzer. A cohort of healthy age-matched volunteers (n = 28) constituted the control group. Results: Both the cfDNA fragment size (median 167 vs 176.5 bp; p &lt; 0.001) and the cfDNA levels (median 4.48 vs 0.33 ng/mL plasma; p &lt; 0.001) were significantly different between patient and healthy control samples. A pre-treatment cfDNA fragment size of ≤167 bp (median) was associated with both shorter progression-free survival (PFS) (4 vs. 7.7 months; log-rank p = 0.002) and overall survival (OS) (4.6 vs. 10.5 months; log-rank p = 0.001). Similarly, cfDNA levels above the median value were associated with both shorter PFS (3.3 vs. 7.7 months; log-rank p &lt; 0.001) and OS (5.4 vs. 8.8 months; log-rank p = 0.001) for samples obtained before initiation of chemotherapy. Survival analysis on the combination of fragment size and cfDNA levels showed a significantly longer PFS (8.1 vs 4.6 vs 2.6 months; log-rank p &lt; 0.001) and OS (10.6 vs 7.9 vs 3.8 months; log-rank p = 0.001) for patients negative for both parameters compared to patients with 1 or 2 positive parameters, respectively. Analyses of plasma samples obtained one month after initiation of chemotherapy indicated a trend towards an association with PFS (p = 0.066 and p = 0.025), but not OS (p = 0.504 and p = 0.213) for cfDNA fragment size and cfDNA levels, respectively. Univariate cox regression using both the continuous and the categorical variable for cfDNA fragment size and cfDNA levels, and the categorical variable for the combination test, confirmed the results from the Kaplan-Meier estimates. Conclusion: Determination of cfDNA fragment size and cfDNA levels is a non-invasive and simple method for predicting prognosis in patients with advanced pancreatic cancer. Further investigations in larger patient cohorts are needed to elaborate the consistency and clinical impact of this finding. Citation Format: Morten Lapin, Satu Oltedal, Kjersti Tjensvoll, Tove Buhl, Rune Smaaland, Nils Glenjen, Bjørnar Gilje, Oddmund Nordgård. The fragment size and levels of cell-free DNA provide prognostic information in patients with advanced pancreatic 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 5593.

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