MiR-205 is progressively down-regulated in lymph node metastasis but fails as a prognostic biomarker in high-risk prostate cancer

The treatment of high-risk prostate cancer (HRPCa) is a tremendous challenge for uro-oncologists. The identification of predictive moleculobiological markers allowing risk assessment of lymph node metastasis and systemic progression is essential in establishing effective treatment. In the current study, we investigate the prognostic potential of miR-205 in HRPCa study and validation cohorts, setting defined clinical endpoints for both. We demonstrate miR-205 to be significantly down-regulated in over 70% of the HRPCa samples analysed and that reconstitution of miR-205 causes inhibition of proliferation and invasiveness in prostate cancer (PCa) cell lines. Additionally, miR-205 is increasingly down-regulated in lymph node metastases compared to the primary tumour indicating that miR-205 plays a role in migration of PCa cells from the original location into extraprostatic tissue. Nevertheless, down-regulation of miR-205 in primary PCa was not correlated to the synchronous presence of metastasis and failed to predict the outcome for HRPCa patients. Moreover, we found a tendency for miR-205 up-regulation to correlate with an adverse outcome of PCa patients suggesting a pivotal role of miR-205 in tumourigenesis. Overall, we showed that miR-205 is involved in the development and metastasis of PCa, but failed to work as a useful clinical biomarker in HRPCa. These findings might have implications for the use of miR-205 as a prognostic or therapeutic target in HRPCa.

Next-generation tissue microarray (ngTMA) increases the quality of biomarker studies: an example using CD3, CD8, and CD45RO in the tumor microenvironment of six different solid tumor types

Background Tissue microarray (TMA) technology revolutionized the investigation of potential biomarkers from paraffin-embedded tissues. However, conventional TMA construction is laborious, time-consuming and imprecise. Next-generation tissue microarrays (ngTMA) combine histological expertise with digital pathology and automated tissue microarraying. The aim of this study was to test the feasibility of ngTMA for the investigation of biomarkers within the tumor microenvironment (tumor center and invasion front) of six tumor types, using CD3, CD8 and CD45RO as an example. Methods Ten cases each of malignant melanoma, lung, breast, gastric, prostate and colorectal cancers were reviewed. The most representative H&E slide was scanned and uploaded onto a digital slide management platform. Slides were viewed and seven TMA annotations of 1 mm in diameter were placed directly onto the digital slide. Different colors were used to identify the exact regions in normal tissue (n = 1), tumor center (n = 2), tumor front (n = 2), and tumor microenvironment at invasion front (n = 2) for subsequent punching. Donor blocks were loaded into an automated tissue microarrayer. Images of the donor block were superimposed with annotated digital slides. Exact annotated regions were punched out of each donor block and transferred into a TMA block. 420 tissue cores created two ngTMA blocks. H&E staining and immunohistochemistry for CD3, CD8 and CD45RO were performed. Results All 60 slides were scanned automatically (total time < 10 hours), uploaded and viewed. Annotation time was 1 hour. The 60 donor blocks were loaded into the tissue microarrayer, simultaneously. Alignment of donor block images and digital slides was possible in less than 2 minutes/case. Automated punching of tissue cores and transfer took 12 seconds/core. Total ngTMA construction time was 1.4 hours. Stains for H&E and CD3, CD8 and CD45RO highlighted the precision with which ngTMA could capture regions of tumor-stroma interaction of each cancer and the T-lymphocytic immune reaction within the tumor microenvironment. Conclusion Based on a manual selection criteria, ngTMA is able to precisely capture histological zones or cell types of interest in a precise and accurate way, aiding the pathological study of the tumor microenvironment. This approach would be advantageous for visualizing proteins, DNA, mRNA and microRNAs in specific cell types using in situ hybridization techniques.

Serum IL-6: a candidate biomarker for intracranial pressure elevation following isolated traumatic brain injury.

BACKGROUND: Increased intracranial pressure (ICP) is a serious, life-threatening, secondary event following traumatic brain injury (TBI). In many cases, ICP rises in a delayed fashion, reaching a maximal level 48-96 hours after the initial insult. While pressure catheters can be implanted to monitor ICP, there is no clinically proven method for determining a patient's risk for developing this pathology. METHODS: In the present study, we employed antibody array and Luminex-based screening methods to interrogate the levels of inflammatory cytokines in the serum of healthy volunteers and in severe TBI patients (GCS RESULTS: Consistent with previous reports, we observed sustained increases in IL-6 levels in TBI patients irrespective of their ICP status. However, the group of patients who subsequently experienced ICP >or= 25 mm Hg had significantly higher IL-6 levels within the first 17 hours of injury as compared to the patients whose ICP remained 128 pg/ml correctly identified 85% of isolated TBI patients who subsequently developed elevated ICP, and values between these cut-off values correctly identified 75% of all patients whose ICP remained CONCLUSIONS: Our results suggest that serum IL-6 can be used for the differential diagnosis of elevated ICP in isolated TBI.

LDOC1, A NOVEL BIOMARKER OF PROGNOSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA

In chronic lymphocytic leukemia (CLL), one of the best predictors of outcome is the somatic mutation status of the immunoglobulin heavy chain variable region (IGHV) genes. Patients whose CLL cells have unmutated IGHV genes have a median survival of 8 years; those with mutated IGHV genes have a median survival of 25 years. To identify new prognostic biomarkers and molecular targets for therapy in untreated CLL patients, we reanalyzed the raw data from four published gene expression profiling microarray studies. Of 88 candidate biomarkers associated with IGHV somatic mutation status, we identified LDOC1 (Leucine Zipper, Down-regulated in Cancer 1), as one of the most significantly differentially expressed genes that distinguished mutated from unmutated CLL cases. LDOC1 is a putative transcription factor of unknown function in B-cell development and CLL pathophysiology. Using a highly sensitive quantitative RT-PCR (QRT-PCR) assay, we confirmed that LDOC1 mRNA was dramatically down-regulated in mutated compared to unmutated CLL cases. Expression of LDOC1 mRNA was also vii strongly associated with other markers of poor prognosis, including ZAP70 protein and cytogenetic abnormalities of poor prognosis (deletions of chromosomes 6q21, 11q23, and 17p13.1, and trisomy 12). CLL cases positive for LDOC1 mRNA had significantly shorter overall survival than negative cases. Moreover, in a multivariate model, LDOC1 mRNA expression predicted overall survival better than IGHV mutation status or ZAP70 protein, among the best markers of prognosis in CLL. We also discovered LDOC1S, a new LDOC1 splice variant. Using isoform-specific QRT-PCR assays that we developed, we found that both isoforms were expressed in normal B cells (naïve > memory), unmutated CLL cells, and in B-cell non-Hodgkin lymphomas with unmutated IGHV genes. To investigate pathways in which LDOC1 is involved, we knocked down LDOC1 in HeLa cells and performed global gene expression profiling. GFI1 (Growth Factor-Independent 1) emerged as a significantly up-regulated gene in both HeLa cells and CLL cells that expressed high levels of LDOC1. GFI1 oncoprotein is implicated in hematopoietic stem cell maintenance, lymphocyte development, and lymphomagenesis. Our findings indicate that LDOC1 mRNA is an excellent biomarker of overall survival in CLL, and may contribute to B-cell differentiation and malignant transformation.

TP53 as a Biomarker in Head and Neck Squamous Cell Carcinoma

Currently, there are no molecular biomarkers that guide treatment decisions for patients with head and neck squamous cell carcinoma (HNSCC). Several retrospective studies have evaluated TP53 in HNSCC, and results have suggested that specific mutations are associated with poor outcome. However, there exists heterogeneity among these studies in the site and stage of disease of the patients reviewed, the treatments rendered, and methods of evaluating TP53 mutation. Thus, it remains unclear as to which patients and in which clinical settings TP53 mutation is most useful in predicting treatment failure. In the current study, we reviewed the records of a cohort of patients with advanced, resectable HNSCC who received surgery and post-operative radiation (PORT) and had DNA isolated from fresh tumor tissue obtained at the time of surgery. TP53 mutations were identified using Sanger sequencing of exons 2-11 and the associated splice regions of the TP53 gene. We have found that the group of patients with either non-disruptive or disruptive TP53 mutations had decreased overall survival, disease-free survival, and an increased rate of distant metastasis. When examined as an independent factor, disruptive mutation was strongly associated with the development of distant metastasis. As a second aim of this project, we performed a pilot study examining the utility of the AmpliChip® p53 test as a practical method for TP53 sequencing in the clinical setting. AmpliChip® testing and Sanger sequencing was performed on a separate cohort of patients with HNSCC. Our study demonstrated the ablity of the AmpliChip® to call TP53 mutation from a single formalin-fixed paraffin-embedded slide. The results from AmpliChip® testing were identical with the Sanger method in 11 of 19 cases, with a higher rate of mutation calls using the AmpliChip® test. TP53 mutation is a potential prognostic biomarker among patients with advanced, resectable HNSCC treated with surgery and PORT. Whether this subgroup of patients could benefit from the addition of concurrent or induction chemotherapy remains to be evaluated in prospective clinical trials. Our pilot study of the p53 AmpliChip® suggests this could be a practical and reliable method of TP53 analysis in the clinical setting.

Identification of 2-Piperidone as a Biomarker of CYP2E1 Activity Through Metabolomic Phenotyping

Cytochrome P450 2E1 (CYP2E1) is a key enzyme in the metabolic activation of many low molecular weight toxicants and also an important contributor to oxidative stress. A noninvasive method to monitor CYP2E1 activity in vivo would be of great value for studying the role of CYP2E1 in chemical-induced toxicities and stress-related diseases. In this study, a mass spectrometry-based metabolomic approach was used to identify a metabolite biomarker of CYP2E1 through comparing the urine metabolomes of wild-type (WT), Cyp2e1-null, and CYP2E1-humanized mice. Metabolomic analysis with multivariate models of urine metabolites revealed a clear separation of Cyp2e1-null mice from WT and CYP2E1-humanized mice in the multivariate models of urine metabolomes. Subsequently, 2-piperidone was identified as a urinary metabolite that inversely correlated to the CYP2E1 activity in the three mouse lines. Backcrossing of WT and Cyp2e1-null mice, together with targeted analysis of 2-piperidone in mouse serum, confirmed the genotype dependency of 2-piperidone. The accumulation of 2-piperidone in the Cyp2e1-null mice was mainly caused by the changes in the biosynthesis and degradation of 2-piperidone because compared with the WT mice, the conversion of cadaverine to 2-piperidone was higher, whereas the metabolism of 2-piperidone to 6-hydroxy-2-piperidone was lower in the Cyp2e1-null mice. Overall, untargeted metabolomic analysis identified a correlation between 2-piperidone concentrations in urine and the expression and activity of CYP2E1, thus providing a noninvasive metabolite biomarker that can be potentially used in to monitor CYP2E1 activity.

A next-generation tissue microarray (ngTMA) protocol for biomarker studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a 'donor' block into a 'recipient' block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 'recipient' blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.

INTRODUCTION Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses. RESULTS For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patient's bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n=88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n=91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n=91), which reflect the total neoplastic burden, revealed four patient groups with different survival. CONCLUSION AND PERSPECTIVE Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.

Comprehensive validation of published immunohistochemical prognostic biomarkers of prostate cancer-what has gone wrong? A blueprint for the way forward in biomarker studies

BACKGROUND Treatment planning of localised prostate cancer remains challenging. Besides conventional parameters, a wealth of prognostic biomarkers has been proposed so far. None of which, however, have successfully been implemented in a routine setting so far. The aim of our study was to systematically verify a set of published prognostic markers for prostate cancer. METHODS Following an in-depth PubMed search, 28 markers were selected that have been proposed as multivariate prognostic markers for primary prostate cancer. Their prognostic validity was examined in a radical prostatectomy cohort of 238 patients with a median follow-up of 60 months and biochemical progression as endpoint of the analysis. Immunohistochemical evaluation was performed using previously published cut-off values, but allowing for optimisation if necessary. Univariate and multivariate Cox regression were used to determine the prognostic value of biomarkers included in this study. RESULTS Despite the application of various cut-offs in the analysis, only four (14%) markers were verified as independently prognostic (AKT1, stromal AR, EZH2, and PSMA) for PSA relapse following radical prostatectomy. CONCLUSIONS Apparently, many immunohistochemistry-based studies on prognostic markers seem to be over-optimistic. Codes of best practice, such as the REMARK guidelines, may facilitate the performance of conclusive and transparent future studies.

Long-term safety and feasibility of three-vessel multimodality intravascular imaging in patients with ST-elevation myocardial infarction: the IBIS-4 (integrated biomarker and imaging study) substudy.

We assessed the feasibility and the procedural and long-term safety of intracoronary (i.c) imaging for documentary purposes with optical coherence tomography (OCT) and intravascular ultrasound (IVUS) in patients with acute ST-elevation myocardial infarction (STEMI) undergoing primary PCI in the setting of IBIS-4 study. IBIS4 (NCT00962416) is a prospective cohort study conducted at five European centers including 103 STEMI patients who underwent serial three-vessel coronary imaging during primary PCI and at 13 months. The feasibility parameter was successful imaging, defined as the number of pullbacks suitable for analysis. Safety parameters included the frequency of peri-procedural complications, and major adverse cardiac events (MACE), a composite of cardiac death, myocardial infarction (MI) and any clinically-indicated revascularization at 2 years. Clinical outcomes were compared with the results from a cohort of 485 STEMI patients undergoing primary PCI without additional imaging. Imaging of the infarct-related artery at baseline (and follow-up) was successful in 92.2 % (96.6 %) of patients using OCT and in 93.2 % (95.5 %) using IVUS. Imaging of the non-infarct-related vessels was successful in 88.7 % (95.6 %) using OCT and in 90.5 % (93.3 %) using IVUS. Periprocedural complications occurred <2.0 % of OCT and none during IVUS. There were no differences throughout 2 years between the imaging and control group in terms of MACE (16.7 vs. 13.3 %, adjusted HR1.40, 95 % CI 0.77-2.52, p = 0.27). Multi-modality three-vessel i.c. imaging in STEMI patients undergoing primary PCI is consistent a high degree of success and can be performed safely without impact on cardiovascular events at long-term follow-up.

Taking personalized medicine seriously: Biomarker approaches in phase IIb/III studies in major depression and schizophrenia

The success rate in the development of psychopharmacological compounds is insufficient. Two main reasons for failure have been frequently identified: 1) treating the wrong patients and 2) using the wrong dose. This is potentially based on the known heterogeneity among patients, both on a syndromal and a biological level. A focus on personalized medicine through better characterization with biomarkers has been successful in other therapeutic areas. Nevertheless, obstacles toward this goal that exist are 1) the perception of a lack of validation, 2) the perception of an expensive and complicated enterprise, and 3) the perception of regulatory hurdles. The authors tackle these concerns and focus on the utilization of biomarkers as predictive markers for treatment outcome. The authors primarily cover examples from the areas of major depression and schizophrenia. Methodologies covered include salivary and plasma collection of neuroendocrine, metabolic, and inflammatory markers, which identified subgroups of patients in the Netherlands Study of Depression and Anxiety. A battery of vegetative markers, including sleep-electroencephalography parameters, heart rate variability, and bedside functional tests, can be utilized to characterize the activity of a functional system that is related to treatment refractoriness in depression (e.g., the renin-angiotensin-aldosterone system). Actigraphy and skin conductance can be utilized to classify patients with schizophrenia and provide objective readouts for vegetative activation as a functional marker of target engagement. Genetic markers, related to folate metabolism, or folate itself, has prognostic value for the treatment response in patients with schizophrenia. Already, several biomarkers are routinely collected in standard clinical trials (e.g., blood pressure and plasma electrolytes), and appear to be differentiating factors for treatment outcome. Given the availability of a wide variety of markers, the further development and integration of such markers into clinical research is both required and feasible in order to meet the benefit of personalized medicine. This article is based on proceedings from the "Taking Personalized Medicine Seriously-Biomarker Approaches in Phase IIb/III Studies in Major Depression and Schizophrenia" session, which was held during the 10th Annual Scientific Meeting of the International Society for Clinical Trials Meeting (ISCTM) in Washington, DC, February 18 to 20, 2014.

Circulating FABP4 Is a Prognostic Biomarker in Patients With Acute Coronary Syndrome but Not in Asymptomatic Individuals.

OBJECTIVE Blood-borne biomarkers reflecting atherosclerotic plaque burden have great potential to improve clinical management of atherosclerotic coronary artery disease and acute coronary syndrome (ACS). APPROACH AND RESULTS Using data integration from gene expression profiling of coronary thrombi versus peripheral blood mononuclear cells and proteomic analysis of atherosclerotic plaque-derived secretomes versus healthy tissue secretomes, we identified fatty acid-binding protein 4 (FABP4) as a biomarker candidate for coronary artery disease. Its diagnostic and prognostic performance was validated in 3 different clinical settings: (1) in a cross-sectional cohort of patients with stable coronary artery disease, ACS, and healthy individuals (n=820), (2) in a nested case-control cohort of patients with ACS with 30-day follow-up (n=200), and (3) in a population-based nested case-control cohort of asymptomatic individuals with 5-year follow-up (n=414). Circulating FABP4 was marginally higher in patients with ST-segment-elevation myocardial infarction (24.9 ng/mL) compared with controls (23.4 ng/mL; P=0.01). However, elevated FABP4 was associated with adverse secondary cerebrovascular or cardiovascular events during 30-day follow-up after index ACS, independent of age, sex, renal function, and body mass index (odds ratio, 1.7; 95% confidence interval, 1.1-2.5; P=0.02). Circulating FABP4 predicted adverse events with similar prognostic performance as the GRACE in-hospital risk score or N-terminal pro-brain natriuretic peptide. Finally, no significant difference between baseline FABP4 was found in asymptomatic individuals with or without coronary events during 5-year follow-up. CONCLUSIONS Circulating FABP4 may prove useful as a prognostic biomarker in risk stratification of patients with ACS.