Genome-Wide Identification of Somatic Aberrations from Paired Normal-Tumor Samples

Genomic copy number alteration and allelic imbalance are distinct features of cancer cells, and recent advances in the genotyping technology have greatly boosted the research in the cancer genome. However, the complicated nature of tumor usually hampers the dissection of the SNP arrays. In this study, we describe a bioinformatic tool, named GIANT, for genome-wide identification of somatic aberrations from paired normal-tumor samples measured with SNP arrays. By efficiently incorporating genotype information of matched normal sample, it accurately detects different types of aberrations in cancer genome, even for aneuploid tumor samples with severe normal cell contamination. Furthermore, it allows for discovery of recurrent aberrations with critical biological properties in tumorigenesis by using statistical significance test. We demonstrate the superior performance of the proposed method on various datasets including tumor replicate pairs, simulated SNP arrays and dilution series of normal-cancer cell lines. Results show that GIANT has the potential to detect the genomic aberration even when the cancer cell proportion is as low as 5∼10%. Application on a large number of paired tumor samples delivers a genome-wide profile of the statistical significance of the various aberrations, including amplification, deletion and LOH. We believe that GIANT represents a powerful bioinformatic tool for interpreting the complex genomic aberration, and thus assisting both academic study and the clinical treatment of cancer.     

Nonparametric Comparison of Longitudinal Response Patterns from Paired Samples Using Configural Frequency Analysis

Nonparametric methods of Configural Frequency Analysis (CFA) for comparing longitudinal response patterns are discussed. Characteristics of diagonally symmetric frequency distributions are mapped with hypotheses on the locus of treatment effects. Test statistics for the analysis of local dependencies are discussed. In particular, applications to paired samples are considered. A biomedical example is given from research on the effects of spasmolytic drugs. Local CFA-testing is compared with global testing. Alternative strategies of testing and the respective underlying assumptions are discussed.     

Procalcitonin Predicts Real-Time PCR Results in Blood Samples from Patients with Suspected Sepsis

Background

Early diagnosis and rapid bacterial identification are of primary importance for outcome of septic patients. SeptiFast® (SF) real-time PCR assay is of potential utility in the etiological diagnosis of sepsis, but it cannot replace blood culture (BC) for routine use in clinical laboratory. Procalcitonin (PCT) is a marker of sepsis and can predict bacteremia in septic patients. The aim of the present study was to investigate whether PCT serum levels could predict SF results, and could help screening febrile patients in which a SF assay can improve the etiological diagnosis of sepsis.

Methods

From 1009 febrile patients with suspected sepsis, 1009 samples for BC, SF real-time PCR, and PCT determination were obtained simultaneously, and results were compared and statistically analysed. Receiver operating characteristic (ROC) curves were generated to determine the area under the curve and to identify which cut-off of PCT value produced the best sensitivity to detect SF results.

Results

Mean PCT values of sera drawn simultaneously with samples SF positive (35.42±61.03 ng/ml) or BC positive (23.14±51.56 ng/ml) for a pathogen were statistically higher than those drawn simultaneously with SF negative (0.84±1.67 ng/ml) or BC negative (2.79±16.64 ng/ml) samples (p<0.0001). For SF, ROC analysis showed an area under the curve of 0.927 (95% confidence interval: 0.899–0.955, p<0.0001). The PCT cut-off value of 0.37 ng/ml showed a negative predictive value of 99%, reducing the number of SF assays of 53.9%, still identifying the 96.4% of the pathogens.

Conclusion

PCT can be used in febrile patients with suspected sepsis to predict SF positive or negative results. A cut-off value of 0.37 ng/ml can be considered for optimal sensitivity, so that, in the routine laboratory activity, SF assay should not be used for diagnosis of sepsis in an unselected patient population with a PCT value <0.37 ng/ml.     

Global Protein Differential Expression Profiling of Cerebrospinal Fluid Samples Pooled from Chinese Sporadic CJD and non-CJD Patients

The shotgun proteomic based on the approach of tandem mass tag (TMT) labeling has received increasing attention for neuroproteomics analysis and becomes an effective tool for the identification and quantification of a large number of proteins for the purpose of revealing key proteins involved in the neuronal dysfunction and an inflammatory response associated with neurodegenerative disorders. To assess the potential expression difference of proteins in cerebrospinal fluids (CSF) between Creutzfeldt–Jakob disease (CJD) and non-CJD patients, the pooled CSF samples from 39 Chinese probable sporadic CJD (sCJD) patients and from 52 non-CJD cases were comparably analyzed with the methodology of TMT labeling and RP-RP-UPLC-MS/MS. Totally, 437 possible proteins were identified in the tested CSF specimen, among them, 49 proteins with 95 % confidence interval. Differential assays showed among those 49 CSF proteins, 12 were upregulated and 13 were downregulated significantly in the sCJD compared to non-CJD. The most affected pathway of the differential expression proteins in CSF of sCJD was complement and coagulation cascade. Western blots for six selected changed proteins in the pooled CSF samples revealed the similar altering profiles in the groups of sCJD and non-CJD as proteomics. Furthermore, CSF samples from 24 CJD patients and 24 non-CJD patients were randomly selected and subjected individually into the Western blots of an increased protein (phosphoglycerate mutase 1) and a decreased one (alpha-1-antichymotrysin), which also confirmed the altering tendency of these identified proteins. Those data indicate that proteomic assay of CSF is a powerful technique not only for selection of the potential biomarkers for the development of diagnostic tool of CJD but also for supplement of useful scientific clues for understanding the CSF homeostasis during the pathogenesis of prion diseases.     

Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

Hematogenous metastasis accounts for the majority of cancer-related deaths, yet the mechanism remains unclear. Circulating tumor cells (CTCs) in blood may employ different pathways to cross blood endothelial barrier and establish a metastatic niche. Several studies provide evidence that prostate cancer (PCa) cell tethering and rolling on microvascular endothelium via E-selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the development of metastases. However, it is unknown if CTCs from PCa patients interact with E-selectin expressed on endothelium, initiating a route for tumor metastases. Here we report that CTCs derived from PCa patients showed interactions with E-selectin and E-selectin expressing endothelial cells. To examine E-selectin-mediated interactions of PCa cell lines and CTCs derived from metastatic PCa patients, we used fluorescently-labeled anti-prostate specific membrane antigen (PSMA) monoclonal antibody J591-488 which is internalized following cell-surface binding. We employed a microscale flow device consisting of E-selectin-coated microtubes and human umbilical vein endothelial cells (HUVECs) on parallel-plate flow chamber simulating vascular endothelium. We observed that J591-488 did not significantly alter the rolling behavior in PCa cells at shear stresses below 3 dyn/cm². CTCs obtained from 31 PCa patient samples showed that CTCs tether and stably interact with E-selectin and E-selectin expressing HUVECs at physiological shear stress. Interestingly, samples collected during disease progression demonstrated significantly more CTC/E-selectin interactions than samples during times of therapeutic response (p=0.016). Analysis of the expression of sialyl Lewis X (sLe^x) in patient samples showed that a small subset comprising 1.9-18.8% of CTCs possess high sLe^x expression. Furthermore, E-selectin-mediated interactions between prostate CTCs and HUVECs were diminished in the presence of anti-E-selectin neutralizing antibody. CTC-Endothelial interactions provide a novel insight into potential adhesive mechanisms of prostate CTCs as a means to initiate metastasis.     

Mutational Analysis of Circulating Tumor Cells from Colorectal Cancer Patients and Correlation with Primary Tumor Tissue

Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3) mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC) patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.     

Evaluation of Antioxidants,Protein, and Lipid Oxidation Products in Blood from Sporadic Amiotrophic Lateral Sclerosis Patients

Several parameters indicators of oxidative stress were evaluated in blood from individuals with the sporadic form of amiotrophic lateral sclerosis (SALS) and compared to healthy controls. Plasma levels of 2-thiobarbituric-reactive substances (TBARS), products of lipid peroxidation, were significantly higher (p < 0.03) in the SALS patients compared to controls. The concentration of plasma antioxidants (-tocopherol, -carotene, ubiquinol-10 and glutathione) and the activity of red blood cell CuZn superoxide dismutase were not significantly different between the groups. The ratio TBARS/-tocopherol was 47% higher in the SALS individuals than in controls. Protein thiols and protein-associated carbonyls in red blood cell membranes and supernates were similar for both groups. A positive correlation (r² = 0.91) was found between the concentration of protein-associated carbonyls in red blood cells and the onset of clinical symptoms. These findings are in agreement with several reports showing higher levels of oxidative damage to cell components in ALS.     

Array CGH analysis of a cohort of Russian patients with intellectual disability

The use of array comparative genomic hybridization (array CGH) as a diagnostic tool in molecular genetics has facilitated the identification of many new microdeletion/microduplication syndromes (MMSs). Furthermore, this method has allowed for the identification of copy number variations (CNVs) whose pathogenic role has yet to be uncovered. Here, we report on our application of array CGH for the identification of pathogenic CNVs in 79 Russian children with intellectual disability (ID). Twenty-six pathogenic or likely pathogenic changes in copy number were detected in 22 patients (28%): 8 CNVs corresponded to known MMSs, and 17 were not associated with previously described syndromes. In this report, we describe our findings and comment on genes potentially associated with ID that are located within the CNV regions.     

Multiparameter Analysis, including EMT Markers, on Negatively Enriched Blood Samples from Patients with Squamous Cell Carcinoma of the Head and Neck

Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism by which cells change phenotype during carcinogenesis, as well as tumor metastasis. Whether EMT is involved in cancer metastasis has a specific, practical impact on the field of circulating tumor cells (CTCs). Since the generally accepted definition of a CTC includes the expression of epithelial surface markers, such as EpCAM, if a cancer cell loses its epithelial surface markers (which is suggested in EMT), it will not be separated and/or identified as a CTC. We have developed, and previously reported on the use of, a purely negative enrichment technology enriching for CTCs in the blood of squamous cell carcinoma of the head and neck (SCCHN). This methodology does not depend on the expression of surface epithelial markers. Using this technology, our initial data on SCCHN patient blood indicates that the presence of CTCs correlates with worse disease-free survival. Since our enrichment is not dependent on epithelial markers, we have initiated investigation of the presence of mesenchymal markers in these CTC cells to include analysis of: vimentin, epidermal growth factor receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have demonstrated not only presumed CTCs that express and/or contain: a nucleus, cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells that contain all of the aforementioned proteins except cytokeratins, suggesting that the cells have undergone the EMT process. We suggest that our negative depletion enrichment methodology provides a more objective approach in identifying and evaluating CTCs, as opposed to positive selection approaches, as it is not subjective to a selection bias and can be tailored to accommodate a variety of cytoplasmic and surface markers which can be evaluated to identify a multitude of phenotypic patterns within CTCs from individual patients, including so-called EMT as presented here.     

Comparison of ESR1 Mutations in Tumor Tissue and Matched Plasma Samples from Metastatic Breast Cancer Patients

BACKGROUND: ESR1 mutation in circulating cell-free DNA (cfDNA) is emerging as a noninvasive biomarker of acquired resistance to endocrine therapy, but there is a paucity of data comparing the status of ESR1 gene in cfDNA with that in its corresponding tumor tissue. The objective of this study is to validate the degree of concordance of ESR1 mutations between plasma and tumor tissue. METHODS: ESR1 ligand-binding domain mutations Y537S, Y537N, Y537C, and D538G were analyzed using droplet digital PCR in 35 patients with metastatic breast cancer (MBC) (35 tumor tissue samples and 67 plasma samples). RESULTS: Of the 35 paired samples, 26 (74.3%) were concordant: one patient had detectable ESR1 mutations both plasma (ESR1 Y537S/Y537N) and tumor tissue (ESR1 Y537S/Y537C), and 25 had WT ESR1 alleles in both. Nine (25.7%) had discordance between the plasma and tissue results: five had mutations detected only in their tumor tissue (two Y537S, one Y537C, one D538G, and one Y537S/Y537N/D538G), and four had mutations detected only in their plasma (one Y537S, one Y537N, and two Y537S/Y537N/D538G). Furthermore, longitudinal plasma samples from 19 patients were used to assess changes in the presence of ESR1 mutations during treatment. Eleven patients had cfDNA ESR1 mutations over the course of treatment. A total of eight of 11 patients with MBC with cfDNA ESR1 mutations (72.7%) had the polyclonal mutations. CONCLUSION: We have shown the independent distribution of ESR1 mutations between plasma and tumor tissue in 35 patients with MBC.     

High-Density Dielectrophoretic Microwell Array for Detection,Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%–90.0%) and excellent linear performance (R² = 0.8189–0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM− tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.     

Somatic TP53 Mutations Are Detectable in Circulating Tumor DNA from Children with Anaplastic Wilms Tumors

BACKGROUND: Diffuse anaplastic Wilms tumor (DAWT) is a rare, high-risk subtype that is often missed on diagnostic needle biopsy. Somatic mutations in TP53 are associated with the development of anaplasia and with poorer survival, particularly in advanced-stage disease. Early identification of DAWT harboring TP53 abnormalities could improve risk stratification of initial therapy and monitoring for recurrence. METHODS: Droplet digital polymerase chain reaction (ddPCR) was used to evaluate 21 samples from 4 patients with DAWT. For each patient, we assessed TP53 status in frozen tumor, matched germline DNA, and circulating tumor DNA (ctDNA) from plasma, serum, and urine collected throughout treatment. RESULTS: Mutant TP53 was detectable in ctDNA from plasma and serum in all patients. We did not detect variant TP53 in the same volume (200 μl) of urine. One patient displayed heterogeneity of TP53 in the tumor despite both histological sections displaying anaplasia. Concentration of ctDNA from plasma/serum taken prenephrectomy varied significantly between patients, ranging from 0.44 (0.05-0.90) to 125.25 (109.75-140.25) copies/μl. We observed variation in ctDNA throughout treatment, and in all but one patient, ctDNA levels fell significantly following nephrectomy. CONCLUSION: We demonstrate for the first time that ddPCR is an effective method for detection of mutant TP53 in ctDNA from children with DAWT even when there is intratumoral somatic heterogeneity. This should be further explored in a larger cohort of patients, as early detection of circulating variant TP53 may have significant clinical impact on future risk stratification and surveillance.     

Correlation of Trace Elemental Profiles in Blood Samples of Indian Patients with Leukoplakia and Oral Submucous Fibrosis

In order to ascertain possible correlation between alterations in trace elemental profile and the progression or regression of two most common potentially malignant disorders affecting oral cavity, namely oral submucous fibrosis and leukoplakia, blood from 60 patients from each group of patients as well from 30 healthy individuals was analyzed for elemental profiling employing EDXRF technique. Out of the 16 detected elements (K, Si, Ca, V, Cr, Ni, Mn, Fe, Cu, Zn, Se, Br, Rb, Sr, Co, and Pb), Mn, Fe, Zn, Br, and Co showed remarkable alteration in their profile in both leukoplakia and oral submucous fibrosis patients with respect to the normal healthy individuals. While Zn, Br, and Fe reflected similar changes--showing gross depletion in both the diseased groups, Mn and Co depicted inverse pattern of alterations in their concentrations in the two types of precancerous disorders when compared to the control subjects.     

化疗对恶性肿瘤患者的血糖影响的初步观察

目的:探讨化疗对恶性肿瘤患者血糖的影响。方法:取我院2005年3月至2006年12月收治的恶性肿瘤患者155例。回顾性分析其化疗前后的血糖变化情况及相关临床资料。结果:155例患者中,化疗后空腹血糖升高者占13．55%(21/155),其中糖耐量低减8人,占5．16%(8/155);诊断为糖尿病者7人,占4．52%(7/155);一过性血糖增高6人,占3．87%(6/155)。各患者化疗前后血糖值的变化有统计学意义(p＜0．05)。结论:恶性肿瘤患者接受化疗后可引起血糖增高,甚至发生糖耐量低减或2型糖尿病,且多发生于化疗的第3～4周期。     

肿瘤和非肿瘤患者合并肺栓塞的临床分析

目的:探讨肿瘤和非肿瘤患者合并肺栓塞的临床特点及危险因素。方法:收集2009年1月至2013年12月我院收治的影像学确诊肺栓塞患者78例,按是否合并恶性肿瘤分为肿瘤组(n=42)和非肿瘤组(n=36),分析两组的临床特点、危险因素及实验室指标。结果:肺癌是最容易发生肺栓塞的恶性肿瘤,腺癌是最常见的病理类型。肿瘤组肺栓塞50%发生在肿瘤确诊的3个月内,肿瘤组血清D-Dimer水平(3195.12±4835.28μg/L)高于非肿瘤组(1338.39±1320.16μg/L),两组间具有统计学差异(x2=2.172,P=0.03)。非肿瘤组中冠心病、慢性肺病、糖尿病及高脂血症和脑梗塞与肿瘤组有显著性差异(P0.01)。不明原因的呼吸困难50例(64.10%)是肺栓塞患者的主要症状,两组之间无明显差异;27例(34.6%)患者合并下肢静脉血栓,右下肢静脉血栓高于左下肢,影像学肺栓塞的类型以右肺动脉主干及其分支受累多见共39例(50.0%),影像学表现在两组间无显著差异。结论:肿瘤组和非肿瘤组的临床症状无明显差异,一旦确诊采取积极的抗凝溶栓治疗可以取得很好的疗效。     

MicroRNA Profiling of Pericardial Fluid Samples from Patients with Heart Failure

Aims

Multicellular organisms maintain vital functions through intercellular communication. Release of extracellular vesicles that carry signals to even distant target organs is one way of accomplishing this communication. MicroRNAs can also be secreted from the cells in exosomes and act as paracrine signalling molecules. In addition, microRNAs have been implicated in the pathogenesis of a large number of diseases, including cardiovascular diseases, and are considered as promising candidate biomarkers due to their relative stability and easy quantification from clinical samples. Pericardial fluid contains hormones secreted by the heart and is known to reflect the cardiac function. In this study, we sought to investigate whether pericardial fluid contains microRNAs and if so, whether they could be used to distinguish between different cardiovascular pathologies and disease stages.

Methods and Results

Pericardial fluid was collected from heart failure patients during open-heart surgery. MicroRNA profiles of altogether 51 patients were measured by quantitative real-time PCR (qPCR) using Exiqon human panels I and II. On the average, 256 microRNAs were detected per sample, and 70 microRNAs out of 742 profiled microRNAs were detected in every sample. The five most abundant microRNAs in pericardial fluid were miR-21-5p, miR-451a, miR-125b-5p, let-7b-5p and miR-16-5p. No specific signatures for cardiovascular pathologies or clinically assessed heart failure stages could be detected from the profiles and, overall, microRNA profiles of the samples were found to be very similar despite the heterogeneity in the study population.

Conclusion

Measured microRNA profiles did not separate the samples according to the clinical features of the patients. However, several previously identified heart failure marker microRNAs were detected. The pericardial fluid microRNA profile appeared to be a result of an active and selective secretory process indicating that microRNAs may act as paracrine signalling factors by mediating the local crosstalk between cardiac cells.     

Isolation of Rare Tumor Cells from Blood Cells with Buoyant Immuno-Microbubbles

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.     

Development of an Automated and Sensitive Microfluidic Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples

Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization.     

Quantitative Detection of Epstein-Barr Virus DNA in Cerebrospinal Fluid and Blood Samples of Patients with Relapsing-Remitting Multiple Sclerosis

The presence of Epstein-Barr Virus (EBV) DNA in cerebrospinal fluid (CSF) and peripheral blood (PB) samples collected from 55 patients with clinical and radiologically-active relapsing-remitting MS (RRMS) and 51 subjects with other neurological diseases was determined using standardized commercially available kits for viral nucleic acid extraction and quantitative EBV DNA detection. Both cell-free and cell-associated CSF and PB fractions were analyzed, to distinguish latent from lytic EBV infection. EBV DNA was detected in 5.5% and 18.2% of cell-free and cell-associated CSF fractions of patients with RRMS as compared to 7.8% and 7.8% of controls; plasma and peripheral blood mononuclear cells (PBMC) positivity rates were 7.3% and 47.3% versus 5.8% and 31.4%, respectively. No significant difference in median EBV viral loads of positive samples was found between RRMS and control patients in all tested samples. Absence of statistically significant differences in EBV positivity rates between RRMS and control patients, despite the use of highly sensitive standardized methods, points to the lack of association between EBV and MS disease activity.