Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer.

Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer.

Monitoring of circulating tumour DNA in advanced pancreatic ductal adenocarcinoma predicts clinical outcome and reveals disease progression earlier than radiological imaging.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a need for better tools to guide treatment selection and follow-up. The aim of this prospective study was to investigate the prognostic value and treatment monitoring potential of longitudinal circulating tumour DNA (ctDNA) measurements in patients with advanced PDAC undergoing palliative chemotherapy. Using KRAS peptide nucleic acid clamp-PCR, we measured ctDNA levels in plasma samples obtained at baseline and every 4 weeks during chemotherapy from 81 patients with locally advanced and metastatic PDAC. Cox proportional hazard regression showed that ctDNA detection at baseline was an independent predictor of progression-free and overall survival. Joint modelling demonstrated that the dynamic ctDNA level was a strong predictor of time to first disease progression. Longitudinal ctDNA measurements during chemotherapy successfully revealed disease progression in 20 (67%) of 30 patients with ctDNA detected at baseline, with a median lead time of 23 days (P = 0.01) over radiological imaging. Here, we confirmed the clinical relevance of ctDNA in advanced PDAC with regard to both the prediction of clinical outcome and disease monitoring during treatment.

Comprehensive ctDNA Measurements Improve Prediction of Clinical Outcomes and Enable Dynamic Tracking of Disease Progression in Advanced Pancreatic Cancer.

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 (CNA) 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 with 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.

Prognostic value of disseminated tumor cells in unresectable pancreatic ductal adenocarcinoma: a prospective observational study.

BACKGROUND: Although pancreatic ductal adenocarcinoma (PDAC) rarely metastasizes to the skeleton, disseminated tumor cells have been detected in bone marrow samples from patients with this disease. The prognostic value of such findings is currently unclear. Thus, the current study aimed to clarify the prognostic information associated with disseminated tumor cell detection in samples from patients with PDAC. METHODS: Bone marrow aspirates were obtained from 48 patients with locally advanced (n = 11) or metastatic (n = 37) PDAC, before and after 2 months of chemotherapy. Disseminated tumor cells were detected with an mRNA panel and quantitative reverse transcription PCR. We used the highest levels measured in healthy bone marrow (n = 30) as a threshold to define the positive detection of disseminated tumor cells. Progression-free and overall survival were analyzed with Kaplan-Meier and Cox proportional hazards regression analyses. RESULTS: Disseminated tumor cells were detected in 15/48 (31%) bone marrow samples obtained before starting chemotherapy and in 8/25 (32%) samples obtained during chemotherapy. Patients with disseminated tumor cells detected before therapy had significantly shorter progression-free (p = 0.03; HR = 2.0) and overall survival (p = 0.03; HR = 2.0), compared to those without disseminated tumor cells in the bone marrow. When restricting disseminated tumor cell detection to keratins KRT7 and KRT8, the prognostic information was substantially stronger (p = 1 × 10-6; HR = 22, and p = 2 × 10-5; HR = 7.7, respectively). The multivariable Cox regression analysis demonstrated that disseminated tumor cell detection prior to treatment had independent prognostic value. In contrast, disseminated tumor cells detected during treatment did not have prognostic value. CONCLUSIONS: Disseminated tumor cells detected before commencing chemotherapy had prognostic value in patients with inoperable PDAC.

Novel hybridization- and tag-based error-corrected method for sensitive ctDNA mutation detection using ion semiconductor sequencing.

Circulating tumor DNA (ctDNA) analysis has emerged as a clinically useful tool for cancer diagnostics and treatment monitoring. However, ctDNA detection is complicated by low DNA concentrations and technical challenges. Here we describe our newly developed sensitive method for ctDNA detection on the Ion Torrent sequencing platform, which we call HYbridization- and Tag-based Error-Corrected sequencing (HYTEC-seq). This method combines hybridization-based capture with molecular tags, and the novel variant caller PlasmaMutationDetector2 to eliminate background errors. We describe the validation of HYTEC-seq using control samples with known mutations, demonstrating an analytical sensitivity down to 0.1% at > 99.99% specificity. Furthermore, to demonstrate the utility of this method in a clinical setting, we analyzed plasma samples from 44 patients with advanced pancreatic cancer, revealing mutations in 57% of the patients at allele frequencies as low as 0.23%.

Liquid biopsies and patient-reported outcome measures for integrative monitoring of patients with early-stage breast cancer: a study protocol for the longitudinal observational Prospective Breast Cancer Biobanking (PBCB) study.

INTRODUCTION: Breast cancer is still the most common malignancy among women worldwide. The Prospective Breast Cancer Biobank (PBCB) collects blood and urine from patients with breast cancer every 6 or 12 months for 11 years from 2011 to 2030 at two university hospitals in Western Norway. The project aims to identify new biomarkers that enable detection of systemic recurrences at the molecular level. As blood represents the biological interface between the primary tumour, the microenvironment and distant metastases, liquid biopsies represent the ideal medium to monitor the patient's cancer biology for identification of patients at high risk of relapse and for early detection systemic relapse.Including patient-reported outcome measures (PROMs) allows for a vast number of possibilities to compare PROM data with biological information, enabling the study of fatigue and Quality of Life in patients with breast cancer. METHODS AND ANALYSIS: A total of 1455 patients with early-stage breast cancer are enrolled in the PBCB study, which has a one-armed prospective observational design. Participants consent to contribute liquid biopsies (i.e., peripheral blood and urine samples) every 6 or 12 months for 11 years. The liquid biopsies are the basis for detection of circulating tumour cells, circulating tumour DNA (ctDNA), exosomal micro-RNA (miRNA), miRNA in Tumour Educated Platelet and metabolomic profiles. In addition, participants respond to 10 PROM questionnaires collected annually. Moreover, a control group comprising 200 women without cancer aged 25-70 years will provide the same data. ETHICS AND DISSEMINATION: The general research biobank PBCB was approved by the Ministry of Health and Care Services in 2007, by the Regional Ethics Committee (REK) in 2010 (#2010/1957). The PROM (#2011/2161) and the biomarker study PerMoBreCan (#2015/2010) were approved by REK in 2011 and 2015 respectively. Results will be published in international peer reviewed journals. Deidentified data will be accessible on request. TRIAL REGISTRATION NUMBER: NCT04488614.

Platelet Microparticles Decrease Daunorubicin-Induced DNA Damage and Modulate Intrinsic Apoptosis in THP-1 Cells.

Platelets can modulate cancer through budding of platelet microparticles (PMPs) that can transfer a plethora of bioactive molecules to cancer cells upon internalization. In acute myelogenous leukemia (AML) this can induce chemoresistance, partially through a decrease in cell activity. Here we investigated if the internalization of PMPs protected the monocytic AML cell line, THP-1, from apoptosis by decreasing the initial cellular damage inflicted by treatment with daunorubicin, or via direct modulation of the apoptotic response. We examined whether PMPs could protect against apoptosis after treatment with a selection of inducers, primarily associated with either the intrinsic or the extrinsic apoptotic pathway, and protection was restricted to the agents targeting intrinsic apoptosis. Furthermore, levels of daunorubicin-induced DNA damage, assessed by measuring gH2AX, were reduced in both 2N and 4N cells after PMP co-incubation. Measuring different BCL2-family proteins before and after treatment with daunorubicin revealed that PMPs downregulated the pro-apoptotic PUMA protein. Thus, our findings indicated that PMPs may protect AML cells against apoptosis by reducing DNA damage both dependent and independent of cell cycle phase, and via direct modulation of the intrinsic apoptotic pathway by downregulating PUMA. These findings further support the clinical relevance of platelets and PMPs in AML.

Liquid Biopsies in Solid Cancers: Implementation in a Nordic Healthcare System.

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.

Platelet Microparticles Protect Acute Myelogenous Leukemia Cells against Daunorubicin-Induced Apoptosis.

The role of platelets in cancer development and progression is increasingly evident, and several platelet-cancer interactions have been discovered, including the uptake of platelet microparticles (PMPs) by cancer cells. PMPs inherit a myriad of proteins and small RNAs from the parental platelets, which in turn can be transferred to cancer cells following internalization. However, the exact effect this may have in acute myelogenous leukemia (AML) is unknown. In this study, we sought to investigate whether PMPs could transfer their contents to the THP-1 cell line and if this could change the biological behavior of the recipient cells. Using acridine orange stained PMPs, we demonstrated that PMPs were internalized by THP-1 cells, which resulted in increased levels of miR-125a, miR-125b, and miR-199. In addition, co-incubation with PMPs protected THP-1 and primary AML cells against daunorubicin-induced cell death. We also showed that PMPs impaired cell growth, partially inhibited cell cycle progression, decreased mitochondrial membrane potential, and induced differentiation toward macrophages in THP-1 cells. Our results suggest that this altering of cell phenotype, in combination with decrease in cell activity may offer resistance to daunorubicin-induced apoptosis, as serum starvation also yielded a lower frequency of dead and apoptotic cells when treated with daunorubicin.

Detection of disseminated tumor cells in bone marrow predict late recurrences in operable breast cancer patients.

BACKGROUND: Operable breast cancer patients may experience late recurrences because of reactivation of dormant tumor cells within the bone marrow (BM). Identification of patients who would benefit from extended therapy is therefore needed. METHODS: BM samples obtained pre- and post-surgery were previously analysed for presence of disseminated tumor cells (DTC) by a multimarker mRNA quantitative reverse-transcription PCR assay. Updated survival analyses were performed on all patient data (n = 191) and in a subgroup of patients alive and recurrence-free after 5 years (n = 156). DTC data were compared to the mitotic activity index (MAI) of the primary tumors. Median follow-up time was 15.3 years. RESULTS: Among the 191 patients, 49 (25.65%) experienced systemic relapse, 24 (49%) within 5-18 years after surgery. MAI and pre- and post-operative DTC status had significant prognostic value based on Kaplan-Meier analyses and multiple Cox regression in the overall patient cohort. With exclusion of patients who relapsed or died within 5 years from surgery, only pre-operative DTC detection was an independent prognostic marker of late recurrences. High MAI (≥10) did not predict late recurrences or disease-specific mortality. CONCLUSION: Pre-operative DTC detection, but not MAI status, predicts late recurrences in operable breast cancer.

Mutation analysis by deep sequencing of pancreatic juice from patients with pancreatic ductal adenocarcinoma.

BACKGROUND: Reliable methods are needed to identify patients with early-stage cancer or high-grade precancerous lesions in the pancreas. Analysis of pancreatic juice to detect somatic mutations could represent one such approach. Here we investigated the concordance between mutations found in the primary tumor and pancreatic juice from the same patient. METHODS: Amplicon-based targeted deep sequencing was performed on samples from 21 patients with pancreatic ductal adenocarcinoma (PDAC) who had undergone Whipple's operation. Mutation profiles were determined in formalin-fixed sections of the primary tumor and in pancreatic juice sampled from the main pancreatic duct during surgery. RESULTS: Using a cut-off of 3% for variant allele frequency, KRAS mutations were detected in 20/21 primary tumors (95%) and in 15/21 (71%) juice samples. When also considering low-frequency variants, KRAS mutations were found in 20/21 juice samples. Most juice samples exhibited multiple KRAS variants not seen in the primary tumor, and only in 11 cases (52%) did the most abundant variant of the juice correspond to the KRAS mutation detected in the tumor. TP53 mutations were found in 16 tumors (76%) and six juice samples (29%). Among the positive juice samples, only one exhibited more than a single TP53 mutation. Detection of both KRAS and TP53 mutations was fully concordant in the primary tumor and juice sample in 7/21 cases (33%). CONCLUSIONS: Pancreatic juice from PDAC patients is rich in KRAS mutations often not seen in the primary tumor and possibly reflecting precancerous lesions in other regions of the pancreas. The inclusion of TP53 mutation detection and additional markers must therefore be considered for fully exploiting the clinical potential of pancreatic juice samples in early cancer detection.

Fragment size and level of cell-free DNA provide prognostic information in patients with advanced pancreatic cancer.

BACKGROUND: It 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. METHODS: Blood 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. RESULTS: Both 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). CONCLUSION: This 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.

The Prognostic Relevance of Sentinel Lymph Node Metastases Assessed by PHGR1 mRNA Quantification in Stage I to III Colon Cancer.

BACKGROUND: Regional lymph node (LN) metastasis is a strong and well-established prognostic factor in colon cancer, and recent data suggest a prognostic value of detecting micrometastases and isolated tumor cells in regional LNs. The aim of the study was to investigate the clinical relevance of detecting sentinel lymph node (SLN) metastases in colon cancer patients by measuring the novel metastasis marker PHGR1 mRNA. METHODS: Using quantitative reverse-transcription polymerase chain reaction, we measured PHGR1 mRNA levels in SLNs and primary tumors from 206 patients surgically treated for stage I to III colon cancer and 52 normal LNs from patients undergoing surgery for benign colon diseases. The prognostic impact of these findings was evaluated by Kaplan-Meier analysis and Cox proportional-hazards regression. RESULTS: Compared to normal LNs, elevated PHGR1 mRNA levels were detected in SLNs from 56 (89%) of the 63 patients with pN+ disease. Furthermore, 68 (48%) of the 143 node-negative (pN0) patients had elevated PHGR1 mRNA levels in SLNs, suggesting occult metastases. With a median follow-up of 7.2 years, a significantly shorter recurrence-free (P=.005) and disease-specific (P=.02) survival was observed in patients with elevated PHGR1 mRNA levels in SLNs. Multivariable modeling showed that the SLN PHGR1 mRNA level was an independent prognostic factor. However, when the survival analyses were restricted to pN0 patients, no significant prognostic information was found. CONCLUSION: Measuring PHGR1 mRNA in SLNs provided independent prognostic information on operable colon cancer patients but not in the pN0 subgroup.

Expression profiling and intracellular localization studies of the novel Proline-, Histidine-, and Glycine-rich protein 1 suggest an essential role in gastro-intestinal epithelium and a potential clinical application in colorectal cancer diagnostics.

BACKGROUND: The primary function of the intestines is the absorption of water and nutrients. Although our knowledge about these processes on the cellular level is extensive, a number of important intracellular elements remain unknown. Here, we characterize the novel proline-, histidine-, glycine-rich 1 (PHGR1) mRNA and protein on the molecular level and propose a functional role of the PHGR1 protein in the intestinal and gastric epithelium. METHODS: PHGR1 mRNA and protein expression in human tissues and cell lines were characterized by quantitative RT-PCR, in situ hybridization, Northern blotting, Western blotting, and immunohistochemistry. Glycosylation was assessed by a chemical deglycosylation assay, whereas intracellular localization was studied by immunofluorescent staining of cell line cells. PHGR1 mRNA levels in HT29 cells was reduced by RNA interference and the resulting global changes in gene expression assessed by microarray hybridization. RESULTS: PHGR1 mRNA and protein were found to be expressed specifically in epithelial cells of intestinal mucosa, with the highest expression in the most mature and differentiated cells. PHGR1 protein was found to be glycosylated and to localize to both the cytoplasm and nucleus. Transcript profiling and gene ontology analysis of HT29 cells subjected to PHGR1 knockdown suggested a functional relationship with transport and metabolic processes. Examination of PHGR1 mRNA and protein levels in lymph nodes with known colorectal cancer metastases indicated that they may serve as biomarkers for detection of such metastases. CONCLUSIONS: Functional analyses of the novel PHGR1 mRNA and protein suggest an essential role in gastrointestinal epithelium and a clinical application in detection of colorectal cancer lymph node metastases.

Prognostic relevance of an epigenetic biomarker panel in sentinel lymph nodes from colon cancer patients.

BACKGROUND: Patients with early colorectal cancer (stages I-II) generally have a good prognosis, but a subgroup of 15-20% experiences relapse and eventually die of disease. Occult metastases have been suggested as a marker for increased risk of recurrence in patients with node-negative disease. Using a previously identified, highly accurate epigenetic biomarker panel for early detection of colorectal tumors, we aimed at evaluating the prognostic value of occult metastases in sentinel lymph nodes of colon cancer patients. RESULTS: The biomarker panel was analyzed by quantitative methylation-specific PCR in primary tumors and 783 sentinel lymph nodes from 201 patients. The panel status in sentinel lymph nodes showed a strong association with lymph node stage (P = 8.2E-17). Compared with routine lymph node diagnostics, the biomarker panel had a sensitivity of 79% (31/39). Interestingly, among 162 patients with negative lymph nodes from routine diagnostics, 13 (8%) were positive for the biomarker panel. Colon cancer patients with high sentinel lymph node methylation had an inferior prognosis (5-year overall survival P = 3.0E-4; time to recurrence P = 3.1E-4), although not significant. The same trend was observed in multivariate analyses (P = 1.4E-1 and P = 6.7E-2, respectively). Occult sentinel lymph node metastases were not detected in early stage (I-II) colon cancer patients who experienced relapse. CONCLUSIONS: Colon cancer patients with high sentinel lymph node methylation of the analyzed epigenetic biomarker panel had an inferior prognosis, although not significant in multivariate analyses. Occult metastases in TNM stage II patients that experienced relapse were not detected.

Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

BACKGROUND: Single-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. METHODS: Single 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. RESULTS: Circulating 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. CONCLUSION: The 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.

MINDEC-An Enhanced Negative Depletion Strategy for Circulating Tumour Cell Enrichment.

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.

Assessment of clinically related outcomes and biomarker analysis for translational integration in colorectal cancer (ACROBATICC): study protocol for a population-based, consecutive cohort of surgically treated colorectal cancers and resected colorectal liver metastasis.

BACKGROUND: More accurate predictive and prognostic biomarkers for patients with colorectal cancer (CRC) primaries or colorectal liver metastasis (CLM) are needed. Outside clinical trials, the translational integration of emerging pathways and novel techniques should facilitate exploration of biomarkers for improved staging and prognosis. METHODS: An observational study exploring predictive and prognostic biomarkers in a population-based, consecutive cohort of surgically treated colorectal cancers and resected colorectal liver metastases. Long-term outcomes will be cancer-specific survival, recurrence-free survival and overall survival at 5 years from diagnosis. Beyond routine clinicopathological and anthropometric characteristics and laboratory and biochemistry results, the project allows for additional blood samples and fresh-frozen tumour and normal tissue for investigation of circulating tumour cells (CTCs) and novel biomarkers (e.g. immune cells, microRNAs etc.). Tumour specimens will be investigated by immunohistochemistry in full slides. Extracted DNA/RNA will be analysed for genomic markers using specific PCR techniques and next-generation sequencing (NGS) panels. Flow cytometry will be used to characterise biomarkers in blood. Collaboration is open and welcomed, with particular interest in mutual opportunities for validation studies. STATUS AND PERSPECTIVES: The project is ongoing and recruiting at an expected rate of 120-150 patients per year, since January 2013. A project on circulating tumour cells (CTCs) has commenced, with analysis being prepared. Investigating molecular classes beyond the TNM staging is under way, including characteristics of microsatellite instability (MSI) and elevated microsatellite alterations in selected tetranucleotides (EMAST). Hot spot panels for known mutations in CRC are being investigated using NGS. Immune-cell characteristics are being performed by IHC and flow cytometry in tumour and peripheral blood samples. The project has ethical approval (REK Helse Vest, #2012/742), is financially supported with a Ph.D.-Grant (EMAST project; Folke Hermansen Cancer Fund) and a CTC-project (Norwegian Research Council; O. Nordgård). The ACROBATICC clinical and molecular biobank repository will serve as a long-term source for novel exploratory analysis and invite collaborators for mutual validation of promising biomarker results. The project aims to generate results that can help better discern prognostic groups in stage II/III cancers; explore prognostic and predictive biomarkers, and help detail the biology of colorectal liver metastasis for better patient selection and tailored treatment. The project is registered at http://www.ClinicalTrials.gov NCT01762813.

Clinical relevance of circulating KRAS mutated DNA in plasma from patients with advanced pancreatic cancer.

We 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 Peptide-nucleic acid-clamp 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 0.6-12.9 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 ctDNA-positive before chemotherapy, nine (90%) experienced disease progression during follow-up, compared to one (25%) of four ctDNA-negative patients (P = 0.01). The pre-therapy ctDNA level was a statistically significant predictor of both progression-free and overall survival (P = 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. This pilot study supports the hypothesis that ctDNA may be used as a marker for monitoring treatment efficacy and disease progression in pancreatic cancer patients. Recruitment of more patients is ongoing to corroborate these findings.

Comparison of molecular and immunocytochemical methods for detection of disseminated tumor cells in bone marrow from early breast cancer patients.

BACKGROUND: Disseminated tumor cells (DTCs) have potential to predict the effect of adjuvant treatment. The purpose of this study was to compare two methods, reverse transcription quantitative PCR (RT-qPCR) and immunocytochemisty (ICC), for detecting breast cancer DTCs in bone marrow (BM) from early breast cancer patients. METHODS: We investigated a subset (n = 313) of BM samples obtained from 271 early breast cancer patients in the "Secondary Adjuvant Taxotere Treatment" (SATT)-trial. All patients in this study had node positive or intermediate/high-risk node negative non-metastatic disease. The DTCs were detected by ICC using AE1-AE3 anti-cytokeratin monoclonal antibodies. Patients with DTCs detected in their BM by ICC after standard adjuvant fluorouracil, cyclophosphamide, epirubicin (FEC) chemotherapy were offered docetaxel treatment. For comparison, 5 × 106 mononuclear cells from the aliquoted BM samples were also analyzed by RT-qPCR using a multimarker (MM) assay based on the tumor cell mRNA markers keratin 19 (KRT19), mammaglobin A (hMAM), and TWIST1. In the MM-assay, a sample was defined as positive for DTCs if at least one of the mRNA markers was positive. RESULTS: The MM RT-qPCR assay identified DTCs in 124 (40%) of the 313 BM samples compared with 23/313 (7%) of the samples analyzed by ICC. The concordance between the MM RT-qPCR and ICC was 61% (Kappa value = 0.04) and twelve of the BM samples were positive by both methods. By RT-qPCR, 46/313 (15%) samples were positive for KRT19, 97/313 (31%) for TWIST1, and 3/313 (1%) for hMAM mRNA. There were no statistically significant associations between the individual mRNA markers. CONCLUSION: The RT-qPCR based method demonstrated more DTC-positive samples than ICC. The relatively low concordance of positive DTC-status between the two different assessment methods suggests that they may be complementary. The clinical relevance of the methods will be evaluated based on future clinical outcome data. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00248703.