A recurring challenge for brain pathologists is to diagnose whether a brain malignancy is a primary tumor or a metastasis from some other tissue. The accurate diagnosis of brain malignancies is essential for selection of proper treatment. MicroRNAs are a class of small non-coding RNA species that regulate gene expression; many exhibit tissue-specific expression and are misregulated in cancer. Using microRNA expression profiling, we found that hsa-miR-92b and hsa-miR-9/hsa-miR-9* are over-expressed, specifically in brain primary tumors, as compared to primary tumors from other tissues and their metastases to the brain. By considering the expression of only these two microRNAs, it is possible to distinguish between primary and metastatic brain tumors with very high accuracy. These microRNAs thus represent excellent biomarkers for brain primary tumors. Previous reports have found that hsa-miR-92b and hsa-miR-9/hsa-miR-9* are expressed more strongly in developing neurons and brain than in adult brain. Thus, their specific over-expression in brain primary tumors supports a functional role for these microRNAs or a link between neuronal stem cells and brain tumorigenesis.

Skin carcinogenesis is known to be a multi-step process with several stages along its malignant evolution. We hypothesized that transformation of normal epidermis to cutaneous squamous cell carcinoma (cSCC) is causally linked to alterations in microRNAs (miRNA) expression. For this end we decided to evaluate their alterations in the pathologic states ending in cSCC. Total RNA was extracted from formalin fixed paraffin embedded biopsies of five stages along the malignant evolution of keratinocytes towards cSCC: Normal epidermis, solar elastosis, actinic keratosis KIN1-2, advanced actinic keratosis KIN3 and well-differentiated cSCC. Next-generation small RNA sequencing was performed. We found that 18 miRNAs are overexpressed and 28 miRNAs are underexpressed in cSCC compared to normal epidermis. miR-424, miR-320, miR-222 and miR-15a showed the highest fold change among the overexpressed miRNAs. And miR-100, miR-101 and miR-497 showed the highest fold change among the underexpressed miRNAs. Heat map of hierarchical clustering analysis of significantly changed miRNAs and principle component analysis disclosed that the most prominent change in miRNAs expression occurred in the switch from 'early' stages; normal epidermis, solar elastosis and early actinic keratosis to the 'late' stages of epidermal carcinogenesis; late actinic keratosis and cSCC. We found several miRNAs with 'stage specific' alterations while others display a clear 'gradual', either progressive increase or decrease in expression along the malignant evolution of keratinocytes. The observed alterations focused in miRNAs involved in the regulation of AKT/mTOR or in those involved in epithelial to mesenchymal transition. We chose to concentrate on the evaluation of the molecular role of miR-497. We found that it induces reversion of epithelial to mesenchymal transition. We proved that SERPINE-1 is its biochemical target. The present study allows us to further study the pathways that are regulated by miRNAs along the malignant evolution of keratinocytes towards cSCC.

The elderly immune system is characterized by reduced responses to infections and vaccines, and an increase in the incidence of autoimmune diseases and cancer. Age-related deficits in the immune system may be caused by peripheral homeostatic pressures that limit bone marrow B-cell production or migration to the peripheral lymphoid tissues. Studies of peripheral blood B-cell receptor spectratypes have shown that those of the elderly are characterized by reduced diversity, which is correlated with poor health status. In the present study, we performed for the first time high-throughput sequencing of immunoglobulin genes from archived biopsy samples of primary and secondary lymphoid tissues in old (74 ± 7 years old, range 61-89) versus young (24 ± 5 years old, range 18-45) individuals, analyzed repertoire diversities and compared these to results in peripheral blood. We found reduced repertoire diversity in peripheral blood and lymph node repertoires from old people, while in the old spleen samples the diversity was larger than in the young. There were no differences in somatic hypermutation characteristics between age groups. These results support the hypothesis that age-related immune frailty stems from altered B-cell homeostasis leading to narrower memory B-cell repertoires, rather than changes in somatic hypermutation mechanisms. Keywords:Aging r B cells r Bone marrow r Immunoglobulin repertoire r Secondary lymphoid tissues Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe age-related changes in the structure and function of the immune system are usually manifested as increased susceptibility Correspondence: Prof. Ramit Mehr e-mail: ramit.mehr@biu.ac.il to infections (both primary and secondary responses) and cancers, poor responsiveness to new or evolving pathogens, reduced efficacy of vaccination and increased incidence of autoimmune diseases [1][2][3][4][5]. This results in increased disease burdens and health- * These authors contributed equally to this work. * * These authors contributed equally to this work as senior authors.C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2016. 46: 480-492 Molecular immunology 481 care costs [3,6,7]. Intrinsic changes in primary and secondary lymphoid tissue function, including hematopoietic stem cells in the bone-marrow (BM), are associated with aging. Literature on immunosenescence has focused mainly on T-lineage impairments, as thymus involution is probably the most well-studied age-related immune dysfunction. In addition, there are age-related defects in the ability of CD4 + and CD8 + T cells to respond to T-cell receptor engagement, a propensity of CD4 + cells to differentiate into Th17 cells at the expense of Th1 and Th2 differentiation and an increase in regulatory T-cell numbers and function [4,8].In the B-cell lineage, changes were observed in the composition of B-cell subtypes in both BM and periphery that may result from increased B-cell longe...

Chronic gastritis is characterized by gastric mucosal inflammation due to autoimmune responses or infection, frequently with Helicobacter pylori. Gastritis with H. pylori background can cause gastric mucosa-associated lymphoid tissue lymphoma (MALT-L), which sometimes further transforms into diffuse large B-cell lymphoma (DLBCL). However, gastric DLBCL can also be initiated de novo. The mechanisms underlying transformation into DLBCL are not completely understood. We analyzed immunoglobulin repertoires and clonal trees to investigate whether and how immunoglobulin gene repertoires, clonal diversification, and selection in gastritis, gastric MALT-L, and DLBCL differ from each other and from normal responses. The two gastritis types (positive or negative for H. pylori) had similarly diverse repertoires. MALT-L dominant clones (defined as the largest clones in each sample) presented higher diversification and longer mutational histories compared with all other conditions. DLBCL dominant clones displayed lower clonal diversification, suggesting the transforming events are triggered by similar responses in different patients. These results are surprising, as we expected to find similarities between the dominant clones of gastritis and MALT-L and between those of MALT-L and DLBCL.

Background: Unlike T-cell neoplasms, B-cell lymphoproliferative disorders have a limited clinical spectrum of skin involvement. Cutaneous B-cell neoplasms mimicking rosacea or rhinophyma are rare.Observations: We described 12 patients with B-cell lymphoproliferative neoplasms presenting with a facial eruption clinically mimicking rosacea or rhinophyma. Eleven patients were women; ages ranged from 36 to 81 years. The clinical presentation included small papules on the nose and cheeks and around the eyes mimicking granulomatous rosacea; nodules on the nose, cheeks, chin, or forehead mimicking phymatous rosacea; or a combination of both. Three patients had preexisting erythematotelangiectatic rosacea and 1 had rhinophyma. Based on a clinicopathologic correlation and B-cell clonality analysis, the diagnosis was primary cutaneous follicular center B-cell lymphoma in 4 cases, primary cutaneous marginal zone lymphoma in 6, and skin involvement of chronic lymphocytic leukemia in 2. All patients had an indolent course as expected for their disease.Conclusions: Cutaneous involvement of B-cell neoplasms may mimic granulomatous rosacea or rhinophyma. This unusual clinical presentation is more common in women and appears in the setting of preexisting rosacea or as a new eruption. Proliferative B-cell disorders should be added to the differential diagnosis of symmetric papular or papulonodular eruptions of the face.

In studies of inflammatory bowel diseases (IBD), research has so far focused mainly on the role of T cells. Despite evidence suggesting that B cells and the production of autoantibodies may play a significant role in IBD pathogenesis, the role of B cells in gut inflammation has not yet been thoroughly investigated. In the present study we used the new approach of lineage tree analysis for studying immunoglobulin variable region gene diversification in B cells found in the inflamed intestinal tissue of two ulcerative colitis patients as well as B cells from mucosa-associated lymph nodes (LN) in the same patients. Healthy intestinal tissue of three patients with carcinoma of the colon was used as normal control. Lineage tree shapes revealed active immune clonal diversification processes occurring in ulcerative colitis patients, which were quantitatively similar to those in healthy controls. B cells from intestinal tissues and the associated LN are shown here to be clonally related, thus supplying the first direct evidence supporting B-cell trafficking between gut and associated LN in IBD and control tissues. IntroductionUlcerative colitis (UC), one of the idiopathic inflammatory bowel diseases (IBD), is a chronic relapsing inflammatory disorder that may lead to significant impairment of gastrointestinal structure and function. UC usually involves the large intestine and extends proximally from the rectum upwards in a continuous fashion. Histologically, inflammation is usually limited to the mucosal layer and may involve ulceration and crypt abscess formation. UC has also been linked to an increased risk of gastrointestinal malignancy [1].Despite recent progress in IBD research, in human subjects as well as a wide variety of experimental animal models, many questions concerning the immunological and genetic basis of the disease remain unanswered. The mucosa-associated lymphoid 2600tissue of the gastrointestinal tract has the important tasks of, on the one hand, recognizing harmful pathogens and antigens within the gut and mounting an appropriate immune response against them and, on the other hand, knowing when and how to dampen or suppress that response once the assault has been cleared or when commensal flora or self-antigens are encountered [2]. The pathogenesis of IBD appears to be related to a disruption of that finely tuned and regulated balance which exists within the mucosa-associated lymphoid tissue, resulting in deregulated and exaggerated local immune responses. This imbalance is most probably a result of complex genetic, environmental, and immunological susceptibility factors [3].Experimental models of intestinal inflammation indicate that mucosal inflammation is probably mediated either by excessive effector T-cell function or deficient regulatory T-cell function. Exaggerated T helper 1 cell response associated with increased secretion of IL-12, IFN-g and/or TNF results in a Crohn's Disease (CD)-like colitis in these experimental models, and exaggerated T helper 2 cell response associated with increas...

Treatment of colorectal cancer (CRC) with monoclonal antibodies against epidermal growth factor receptor requires the assessment of the mutational status of exons 2, 3, and 4 of the NRAS and KRAS oncogenes. Moreover, the mutational status of exon 15 of the BRAF oncogene is a marker of poor prognosis in CRC. The Idylla NRAS-BRAF Mutation Test is a reliable, simple (<2 minutes hands-on time), and quick (<2 hours turnaround time) sample-to-result solution, enabling the detection of clinically relevant mutations in NRAS (18 mutations) and BRAF (5 mutations). A multicenter study was conducted in 14 centers using the Idylla NRAS-BRAF Mutation Test to assess the NRAS and BRAF mutational status of 418 formalin-fixed, paraffin-embedded tissue samples from CRC patients. Results were compared with those obtained earlier by routine reference methods, including next-generation sequencing, pyrosequencing, mass spectrometry-based assays, PCR-based assays, and Sanger sequencing. In case of discordance, additional tests were performed by digital droplet PCR. Overall, after testing confirmation and excluding invalids/errors by design, concordances between the Idylla NRAS-BRAF Mutation Test and the reference test results were found in almost perfect agreement. In conclusion, the Idylla NRAS-BRAF Mutation Test enables the routine detection of all NRAS and BRAF mutations deemed clinically relevant according to the latest clinical guidelines, without necessitating molecular expertise or infrastructure.

Aging is associated with increasing prevalence and severity of infections caused by a decline in bone marrow (BM) lymphopoiesis and reduced B‐cell repertoire diversity. The current study proposes a strategy to enhance immune responsiveness in aged mice and humans, through rejuvenation of the B lineage upon B‐cell depletion. We used hCD20Tg mice to deplete peripheral B cells in old and young mice, analyzing B‐cell subsets, repertoire and cellular functions in vitro, and immune responsiveness in vivo. Additionally, elderly patients, previously treated with rituximab healthy elderly and young individuals, were vaccinated against hepatitis B (HBV) after undergoing a detailed analysis for B‐cell compartments. B‐cell depletion in old mice resulted in rejuvenated B‐cell population that was derived from de novo synthesis in the bone marrow. The rejuvenated B cells exhibited a "young"‐like repertoire and cellular responsiveness to immune stimuli in vitro. Yet, mice treated with B‐cell depletion did not mount enhanced antibody responses to immunization in vivo, nor did they survive longer than control mice in "dirty" environment. Consistent with these results, peripheral B cells from elderly depleted patients showed a "young"‐like repertoire, population dynamics, and cellular responsiveness to stimulus. Nevertheless, the response rate to HBV vaccination was similar between elderly depleted and nondepleted subjects, although antibody titers were higher in depleted patients. This study proposes a proof of principle to rejuvenate the peripheral B‐cell compartment in aging, through B‐cell depletion. Further studies are warranted in order to apply this approach for enhancing humoral immune responsiveness among the elderly population.