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.

BackgroundSingle-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer.MethodsSingle circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns.ResultsCirculating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells.ConclusionThe analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3385-3) contains supplementary material, which is available to authorized users.

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3’ splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

Implementation of high-risk human papilloma virus (HPV) screening and the increasing proportion of HPV vaccinated women in the screening program will reduce the percentage of HPV positive women with oncogenic potential. In search of more specific markers to identify women with high risk of cancer development, we used RNA sequencing to compare the transcriptomic immune-profile of 13 lesions with cervical intraepithelial neoplasia grade 3 (CIN3) or adenocarcinoma in situ (AIS) and 14 normal biopsies from women with detected HPV infections. In CIN3/AIS lesions as compared to normal tissue, 27 differential expressed genes were identified. Transcriptomic analysis revealed significantly higher expression of a number of genes related to proliferation, (CDKN2A, MELK, CDK1, MKI67, CCNB2, BUB1, FOXM1, CDKN3), but significantly lower expression of genes related to a favorable immune response (NCAM1, ARG1, CD160, IL18, CX3CL1). Compared to the RNA sequencing results, good correlation was achieved with relative quantitative PCR analysis for NCAM1 and CDKN2A. Quantification of NCAM1 positive cells with immunohistochemistry showed epithelial reduction of NCAM1 in CIN3/AIS lesions. In conclusion, NCAM1 and CDKN2A are two promising candidates to distinguish whether women are at high risk of developing cervical cancer and in need of frequent follow-up.

Purpose: Circulating tumor DNA (ctDNA) has emerged as a promising tumor-specific biomarker in pancreatic cancer, but current evidence of the clinical potential of ctDNA is limited. In this study, we used comprehensive detection methodology to explore the utility of longitudinal ctDNA measurements in patients with advanced pancreatic cancer. Experimental Design: A targeted eight-gene next-generation sequencing panel was used to detect point mutations and copy number aberrations (CNAs) in ctDNA from 324 pre-treatment and longitudinal plasma samples obtained from 56 patients with advanced pancreatic cancer. The benefit of ctDNA measurements to predict clinical outcome and track disease progression was assessed. Results: We detected ctDNA in 35/56 (63%) patients at baseline and found that it was an independent predictor of shorter progression-free survival (PFS) and overall survival (OS). After initiation of treatment, ctDNA levels decreased significantly before significantly increasing by the time of progression. In some patients, ctDNA persistence was observed after the first chemotherapy cycles, and it was associated with rapid disease progression and shorter OS. Longitudinal monitoring of ctDNA levels in 27 patients for whom multiple samples were available detected progression in 19 (70%) patients. The median lead time of ctDNA measurements on radiologically determined progression/time of death was 19 days (p = 0.002), compared to 6 days (p = 0.007) using carbohydrate antigen 19-9. Conclusions: ctDNA is an independent prognostic marker that can be used to detect treatment failure and disease progression in patients with advanced pancreatic cancer.

Liquid biopsies have emerged as a potential new diagnostic tool, providing detailed information relevant for characterization and treatment of solid cancers. We here present an overview of current evidence supporting the clinical relevance of liquid biopsy assessments. We also discuss the implementation of liquid biopsies in clinical studies and their current and future clinical role, with a special reference to the Nordic healthcare systems. Our considerations are restricted to the most established liquid biopsy specimens: circulating tumor DNA (ctDNA) and circulating tumor cells (CTC). Both ctDNA and CTCs have been used for prognostic stratification, treatment choices, and treatment monitoring in solid cancers. Several recent publications also support the role of ctDNA in early cancer detection. ctDNA seems to provide more robust clinically relevant information in general, whereas CTCs have the potential to answer more basic questions related to cancer biology and metastasis. Epidermal growth factor receptor-directed treatment of non-small-cell lung cancer represents a clinical setting where ctDNA already has entered the clinic. The role of liquid biopsies in treatment decisions, standardization of methods, diagnostic performance and the need for further research, as well as cost and regulatory issues were identified as factors that influence further integration in the clinic. In conclusion, substantial evidence supports the clinical utility of liquid biopsies in cancer diagnostics, but further research is still required for a more general application in clinical practice.