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.

The poor prognosis of pancreatic cancer patients is associated with the frequent and early dissemination of the disease, as well as late detection due to unspecific and late symptoms from the primary tumor. Pancreatic cancers frequently spread to the liver, lung and skeletal system, suggesting that pancreatic tumor cells must be able to intravasate and travel through the circulation to distant organs. Circulating tumor cells (CTCs) are tumor cells that have acquired the ability to enter the circulatory system; this cell population is ultimately responsible for the development of metastases in distant organs. Clinical studies have revealed that the presence of CTCs in blood is correlated with disease progression for other cancers, such as breast, colorectal and prostate cancer. However, as CTCs are extremely rare, both enrichment and sensitive methods of detection are required for their enumeration. This review highlights various enrichment procedures and methods for the detection of CTCs. Furthermore, we systematically review previously reported studies of the clinical relevance of CTC detection in pancreatic cancer patients. There is evidence that the presence of CTCs also correlates with an unfavorable outcome in pancreatic cancer patients. However, technical/methodological issues may explain why some studies only show a trend toward an association between CTC detection and disease progression. Larger studies, as well as characterization of the CTC population, are required to achieve further insight into the clinical implications of CTC detection in pancreatic cancer patients. Pancreatic cancerPancreatic cancer is the fourth leading cause of cancer-related death in Western countries, with a patient survival rate that is among the worst of any solid cancer. The most prominent risk factors are a family history of pancreatic cancer and cigarette smoking.

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.

The transcription factor v-Myb is a potent inducer of myeloid leukemias, and its cellular homologue c-Myb plays a crucial role in the regulation of hematopoiesis. Recently, Bies and coworkers (Bies, J., Markus, J. & Wolff, L. (2002) J. Biol. Chem, 277, 8999-9009) presented evidence that murine c-Myb can be sumoylated under overexpression conditions in COS7 cells when cotransfected with FLAG-tagged SUMO-1. Here we provide independent evidence that human c-Myb is also subject to SUMO-1 conjugation under more physiological conditions as revealed by coimmunoprecipitation analysis of Jurkat cells and transfected CV-1 cells. Analysis in an in vitro conjugation system showed that modification of the two sites K503 and K527 is interdependent. A twohybrid screening revealed that the SUMO-1 conjugase Ubc9 is one of a few major Myb-interacting proteins. The moderate basal level of sumoylation was greatly enhanced by cotransfection of PIASy, an E3 ligase for SUMO-1. The functional consequence of abolishing sumoylation was enhanced activation both of a transiently transfected reporter gene and of a resident Myb-target gene. When single and double mutants were compared, we found a clear correlation between reduction in sumoylation and increase in transcriptional activation. Enhancing sumoylation by contransfection of PIASy had a negative effect on both Myb-induced and basal level reporter activation. Furthermore, PIASy caused a shift in nuclear distribution of c-Myb towards the insoluble matrix fraction. We propose that the negative influence on transactivation properties by the negative regulatory domain region of c-Myb depends on the sumoylation sites located here.Keywords: c-Myb; transcription; SUMO-1; Ubc9; PIASy.The c-Myb transcription factor plays a central role in the regulation of cell growth and differentiation, in particular in hematopoietic progenitor cells (reviewed in [1]). Homozygous null c-Myb/Rag1 chimerical mice are blocked in early T-cell development, while mice with a c-myb null mutation display severe hematopoietic defects leading to in utero death at E15 [2,3]. The c-Myb protein consists of an N-terminal DNA-binding domain (DBD), a central transactivation domain (TAD) and a C-terminal negative regulatory domain (NRD). The DBD of c-Myb is comprised of the three imperfect repeats: R 1 , R 2 and R 3 , each related to the helix-turn-helix motif [4][5][6][7].Oncogenic alterations, as found in AMV v-Myb, include both N-and C-terminal deletions as well as point mutations [8]. AMV v-myb is a potent and cell-type specific oncogene that transforms target cells in the macrophage lineage and induces monocytic leukemia [8,9]. Several studies have attempted to define oncogenic determinants of v-myb. N-and C-terminal deletions remove several sites of protein modification, including an N-terminal CK2 phosphorylation site (S11 and S12) [10], and a putative MAPK-site (S528) [11][12][13] as well as acetylation sites [14,15] located in the deleted portion of the C-terminal NRD. In addition, specific point mutations in v-My...

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