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An extensive literature on participatory design with older adults has, thus far, little to say about the support older adults need when involved in online activities. Our research suggests that to empower older adults in participatory design, scaffolding work has to be done. Scaffolding interactions - creating temporary instructional support to help the learning of participants - is a common approach in participatory design. Yet, when applied in online participatory design with older adults, the traditional understanding of the concept does not match the way older adults' learn. Hence, we argue for a new understanding of this term, which we call situated scaffolding. We illustrate our argument with a case where older adults collaborate online as part of a participatory design project. We unpack the different dimensions of situated scaffolding and discuss how this novel understanding can be used to further inform sustainable participatory design for and with older adults.

Digital literacy refers to a set of competencies related to the skilled use of computers and information technology. Low digital skills can be a barrier for older adults’ full participation in a digital society, and COVID-19 has increased this risk of social exclusion. Older adults’ digital inclusion is a complex process that consists of the interplay of structural and individual factors. The ACCESS project unwrapped the complexity of the process and developed an innovative, multilevel model that illustrates how societal, institutional, material and pedagogical aspects shape adults’ appropriation of digital literacy. A holistic model describes factors contributing to older adults’ digital literacy, acknowledging sociocultural contexts, environments, learning settings and instruction practices for learning digital literacy. Instead of seeing older adults’ reasons for learning digital skills purely as individual choice, this model recognizes the interpersonal, institutional and societal aspects that implicitly or explicitly influence older adults’ acquisition of digital literacy. The results offer a tool for stakeholders, the research community, companies, designers and other relevant stakeholders to consider digital skills and the given support. It demands diverse communication between different stakeholders about the things that should be discussed when organizing digital support in digitalized societies.

Background: HIV-positive men who have sex with men (MSM) are more likely to experience human papillomavirus (HPV) infection. The persistent HPV infection is the major factor in the development of anal and oropharyngeal neoplasms. Data on the prevalence of anal and oral HPV in MSM are almost absent from the countries of Central and Eastern Europe. We conducted a cross-sectional study focused on the prevalence of oral and anal HPV infections and the relationship between current anal and oral HPV intrapersonal infection in a Czech population of predominantly HIV-positive MSM. Methods: Oral gargle and anal swab samples from 205 predominantly HIV-positive MSM from the Czech Republic were analysed for HPV infection using PCR. Selected sociodemographic and clinical data were correlated with HPV detection using generalized linear models and multivariate analysis. Results: HPV infection was detected in 183 (96.8%) anal and 48 (23.6%) oral samples. The most common type of HR-HPV was HPV16 in both anal (25.4%) and oral (2.5%) samples. Multiple anal HPV infections and the presence of vaccine-targeted HR-HPV types were significantly correlated with abnormal anal cytology and HIV status. Conclusion: The prevalence of anal HPV infection in Czech predominantly HIV-positive MSM ranks among the highest reported, while oral HPV prevalence is consistent with MSM populations. Minimal overlap of oral and anal HPV types within a patient was observed.

B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

Categorical work in chronic care is increasingly dependent on digital technologies for remote patient care. However, remote care takes many forms and while various types of digital technologies are currently being used, we lack a nuanced understanding of how to design such technologies for specific novel usages. In this paper, we focus on digital technologies for patient-generated health data and how their use changes categorical work in chronic care. Our aim is to understand how categorical work changes, which novel forms of categorical work emerge and what the implications are for the care relation. This paper is based on an ethnographic study of healthcare professionals’ work at a pelvic cancer rehabilitation clinic and their interactions with patients. In this setting, supportive talks between patients and nurses are central. To understand the complexities of categorical work in chronic care when patient-generated health data are introduced, we contrast the traditional supportive talks with supportive talks where the nurses had access to the patients’ patient-generated health data. We identify and analyze challenges connected to novel forms of categorical work. Specifically, we focus on categorical work and how it can undergo changes. Our empirical findings show how changes occur in the way patients’ lived experience of the chronic disease aligns with the categories from chronic care, as well as in the way the nurse works with clinical categories during the talk. These insights help us further understand the implications of patient generated-data use in supportive talks. We contribute to an improved understanding of the use of patient-generated health data in clinical practice and based on this, we identify design implications for how to make categorical work more collaborative.

Developing toolkits as a support of participatory design is a common approach when designing with and for older adults. The key aspect in designing digital tools is digital literacy of the participants and how to sustain it during the project but also after its end. Yet, not enough attention has been paid to how to use such toolkits to make PD projects results sustainable. To address this issue, we are developing a mobile demo-kit, a set of didactic prototypes, which aims to foster older participants’ digital literacy and hence make findings sustainable. We illustrate it on a practice-based study, during which we conducted participatory observation, a series of interviews and organized a series of participatory workshops online with older adults. Our preliminary findings contribute to discussion on making PD with and for older adults sustainable by focusing on what older adults can learn during the PD, how to support this process but also how to communicate the findings further on.

Purpose The purpose of this study is to understand the activities in nurses’ work practices in relation to the design process of a self-monitoring application. Design/methodology/approach A design ethnographic approach was applied in this study. Findings To solve the problem of translating highly qualitative phenomena, such as pain, into the particular abstract features of a self-monitoring application, design participants had to balance these two aspects by managing complexity. In turn, the nurses’ work practices have changed because it now involves a new activity based on a different logic than the nurses’ traditional work practices. Originality/value This study describes a new activity included in nurses’ work practices when the nurses became part of a design process. This study introduces a novel way on how to gain a deeper understanding of existing professional practice through a detailed study of activities taking place in a design process. This study explores the possible implications for nurses’ professional practices when they participate in a self-monitoring application design process.

Specific cutaneous involvement in Hodgkin lymphoma is rare. In cutaneous lesions, the diagnosis is usually based on the recognition of diagnostic Reed-Sternberg cells and its variants. In nodal Hodgkin lymphoma, so-called mummified cells (cells with condensed cytoplasm and pyknotic eosinophilic or basophilic nuclei) are often seen. They are sometimes conspicuous and easy to recognize, thus serving as a clue to the diagnosis. Our objective was to study cases of cutaneous Hodgkin lymphoma to identify the occurrence of mummified cells. We studied 12 patients (4 women and 8 men; age range 23-80 years). In 6 patients, cutaneous and extracutaneous disease was identified almost simultaneously; in 4 patients, lymph node disease preceded cutaneous involvement; and in the remaining 2 patients, the skin lesions were the presenting sign, whereas lymph node involvement occurred later. Histopathological, immunohistochemical, and molecular-genetic studies, including rearrangements for TCR, IgH genes, and PCR for EBV, were performed. Cutaneous biopsy specimens revealed either a multinodular or diffuse infiltrate, included small lymphocytes, eosinophils, plasma cells, and macrophages, but in all cases, diagnostic Reed-Sternberg cells and its variants were identified. Mummified cells were detected in 9 cases, either as occasional scattered mummified cells often requiring a search (6 cases) or being conspicuous, grouped and therefore easily identified (3 cases). Immunohistochemically, in all 7 cases studied, mummified cells were positive for both CD30 and CD15. It is concluded that mummified cells are encountered in a majority of cases of cutaneous Hodgkin lymphoma.

Oct4-mediated reprogramming has recently become a novel tool for the generation of various cell types from differentiated somatic cells. Although molecular mechanisms underlying this process are unknown, it is well documented that cells over-expressing Oct4 undergo transition from differentiated state into plastic state. This transition is associated with the acquisition of stem cells properties leading to epigenetically “open” state that is permissive to cell fate switch upon external stimuli. In order to contribute to our understanding of molecular mechanisms driving this process, we characterised human fibroblasts over-expressing Oct4 and performed comprehensive small-RNAseq analysis. Our analyses revealed new interesting aspects of Oct4-mediated cell plasticity induction. Cells over-expressing Oct4 lose their cell identity demonstrated by down-regulation of fibroblast-specific genes and up-regulation of epithelial genes. Interestingly, this process is associated with microRNA expression profile that is similar to microRNA profiles typically found in pluripotent stem cells. We also provide extensive network of microRNA families and clusters allowing us to precisely determine the miRNAome associated with the acquisition of Oct4-induced transient plastic state. Our data expands current knowledge of microRNA and their implications in cell fate alterations and contributing to understanding molecular mechanisms underlying it.

Background Anal cancer (AC) screening is justified in high-risk populations, particularly HIV-positive men having sex with men (MSM). HR-HPV testing could improve the efficiency of cytologically based screening of AC, as in the screening of biologically analogical cervical cancer. The specificity of HR-HPV testing is influenced by the prevalence of HR-HPV infection in the screened population. Reported anal HR-HPV DNA prevalence in MSM is high, but HR-HPV mRNA reflects rather long-term infections and is more specific for high-grade lesions. However, no data were published about HR-HPV DNA and mRNA prevalence in the Czech AC screening population. Method Results of liquid-based anal cytology of 203 predominantly HIV-positive MSM from the Czech AC screening cohort were correlated with results of DNA and E6/E7 mRNA testing of 14 HR-HPV types, and HPV16 genotyping. Eighty-one MSM underwent a standard anoscopy. Results A total of 109 (53.7%) samples had abnormal cytology, with 12 (5.9%) ASC-H/HSIL, 67 (33.0%) samples cytologically negative, and 27 (13.3%) unsatisfactory. HR-HPV DNA was detected in 134 (66.0%) and HR-HPV RNA in 72 (35.5%) anal smears. HR-HPV mRNA and HPV16 mRNA positivity were associated with abnormal cytology (p = .0037, p = .0021). No significant association was found between HR-HPV DNA or HPV16 DNA positivity and abnormal cytology. No high-grade lesions were revealed by anoscopy. Conclusion Prevalence of anal HR-HPV DNA among Czech MSM is high, however, the prevalence of HR-HPV mRNA is half and associated with abnormal cytology. Our results indicate an increased efficiency of cytological screening when combined with HR-HPV mRNA testing.

Concrete as an important component of an engineered barrier system in deep geological repositories (DGR) for radioactive waste may come into contact with bentonite, or other clays, rich in indigenous microorganisms, with potentially harmful impacts on barrier integrity. Our study aimed to assess the effect of a concrete environment on indigenous bentonite and groundwater microbial communities as these particular conditions will select for the potentially harmful microorganisms to the concrete in the future DGR. The two-month experiment under anoxic conditions consisted of crushed, aged, low-pH concrete, Czech Ca-Mg bentonite, and anoxic groundwater, with control samples without concrete or with sterile groundwater. The microbial diversity and proliferation were estimated by qPCR and 16S rRNA gene amplicon sequencing. The presence of concrete had a strong effect on microbial diversity and reduced the increase in total microbial biomass, though low-pH concrete harbored indigenous bacteria. The growth of sulfate reducers was also limited in concrete samples. Several genera, such as Massilia, Citrifermentans, and Lacunisphaera, dominant in bentonite controls, were suppressed in concrete-containing samples. In contrast, genera such as Bacillus, Dethiobacter and Anaerosolibacter specifically proliferated in the presence of concrete. Genera such as Thermincola or Pseudomonas exhibited high versatility and proliferated well under both conditions. Because several of the detected bacterial genera are known to affect concrete integrity, further long-term studies are needed to estimate the effect of bentonite and groundwater microorganisms on concrete stability in future DGR.

Bentonite is a swelling clay, consisting mainly of montmorillonire, being planned to be used as a backfill material in the nuclear waste repository. It contains indigenous microbial populations that can negatively influence the long-term safety of the geological repository due to their metabolic activity (canister corrosion, illitization of bentonite, gas production, degradation of cementitious materials). However, reliable detection of microorganisms in clayish material is generally very difficult. Although the compactness of bentonite will undoubtedly limit the microbial activity, in the extremely long-time frame of repository lifetime this condition can fail. It is thus crucial to understand the potential of the naturally present microbial community in bentonite to compromise the safety of repository, if not limited by the compactness. Higher metabolic activity can be mainly expected at the interfaces or in the places with a lower density of bentonite.Here we present an optimized cell extraction method enabling direct estimation of bacterial density and viability in bentonite. Indigenous bacterial cells were extracted from bentonite suspensions by an improved step-wise protocol and their viability was detected using live/dead staining and epifluorescence microscopy. We used dispersant (2.5 mM natrium pyrophosphate-based solution or 1% methanol) to partially disintegrate the bentonite and detach the vital and dead microbial cells from its surface. The dispersed material was subsequently stepwise centrifuged over two high-density media (sucrose and Histodenz) to remove most of the heavy bentonite particles while keeping the light bentonite particles and cells in the final extract. We were able to detect and enumerate the cells concentrated at the surface of the light bentonite particles, which served as a sieve to retain all free cells during centrifugation. Different extraction procedures were tested and their efficiency was estimated by comparing live/dead ratios of resulting extracts and was also proved by implementing both NGS and quantitative PCR. The results show that most of the microbial genera present in the original suspension are also present in extracts but as proved by Deseq2 analysis some genera tend to settle down with heavier bentonite particles during the first centrifugation step.To conclude, we present a protocol for extraction and detection of metabolically active cells in clayish material – bentonite. The quality of the extraction procedure was estimated both by a combination of fluorescent microscopy and genetic methods. The protocol was successfully tested on different bentonite types showing general applicability of this approach for clay materials.

Our workshop will concentrate on vulnerability of specific social groups due to various reasons, including COVID-19, and the potential for technology design to result in empowerment. We want to address issues of what new forms of vulnerabilities emerge and how we can design digital environments in a way that acknowledges vulnerability but also has the potential to empower people in ways that are meaningful for them. When planning the workshop, we will also reflect on social situations that can result in vulnerabilities for participants. Therefore, we will ensure that interested participants will experience low barriers to participation include a variety of people with different backgrounds and ensure that interaction happens based on equality principles and in an atmosphere of solidarity. Participants can exchange ideas and thoughts without worrying about being exposed to biased assumptions. The workshop will allow for non-hierarchical and cooperative discussion and collaboration through interactive online exercises, resulting in a collaboratively developed zine. Finally, the social sustainability of the workshop will be ensured through a website, mailing lists, joint publications and continuous contact., NA

Research on food practices has become more common among scholars of HCI in recent years. Human-Food-Interaction (HFI) looks into the interplay of humans, food and technology. HFI, even so, has paid relatively little attention to the more collective elements of food practice, including social bonding [1]. The modest project we describe below aimed to say something about the use of digital artifacts to support community engagement for sustainable food practices. We participated, as action researchers (see [2]) in a grassroots movement that instigated a project around learning about food growing, using digital means to bring interested people together during times of physical distancing: In the project Vegetables seek a home, people from various backgrounds ‘adopted’ a chili-plant, they are invited to share what they like in a Telegram-Group, and to get learning-modules via a mailing-list. Through an analysis of the communal effort to actualize the project (video-calls, Telegram, wechange.de) and the content of the Telegram-Group for the chili-plant adopting parents and experts, we suggest some design implications for grassroots communities and sustainable food practice. In future research we intend an iterative design to support the community and its project, utilizing Holmgren's 12 principles of permaculture design.

Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2–associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the “tonic” AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

The use of the anti-CD20 antibody rituximab has improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab was shown to act via various mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct apoptosis, or sensitization to chemotherapy (reviewed in ref. [1]). However, the biological function of CD20 and the reasons for the impressive activity of rituximab and other anti-CD20 antibodies remain elusive. It has been suggested, but remains controversial, whether CD20 functions as a calcium channel, couples with CD40 and MHCII, or B-cell receptor (BCR), and whether it has a role in T cell-dependent and -independent immunity [1,2,3,4]. This is also of great clinical interest for the design of combinatorial treatment of rituximab with BCR inhibitors, DNA damaging or immunomodulatory drugs, or CAR T cells. We have previously shown that microenvironmental interactions upregulate the CD20 levels on CLL cells through the CXCR4/SDF-1 axis [5]. Here we describe that higher CD20 expression has a direct role in the BCR signaling in CLL cells, and a BCR-proficient intra-clonal CLL cell subpopulation is more efficiently eliminated by rituximab in vivo due to higher CD20 levels.

Skills, knowledge, and awareness of digital and technological tools are essential to improve the state of well-being and health of older adults and also to mitigate the condition of social isolation in the aging process. For this reason, it is necessary to implement a social learning of electronic/digital tools for health of older people to support the achievement of eHealth and digital competences. The paper reports the results of an Italian innovative eHealth training for the European project ACCESS. The training has been based on blended didactical and interactive educational techniques, aimed at collecting as many points of view as possible from older adults. A total of 58 older adults were recruited to attend a four-week training program, which included five modules. The results showed a statistical significant difference between the eHealth Literacy Scale (eHEALS) mean value before and after the course. A significant negative correlation was found between eHEALS and positive/total Survey of Technology Use (SOTU), suggesting an inverse relationship between positive/total SOTU and eHEALS. There is a strong positive and statistically significant relationship between satisfaction with the training and eHEALS. The results indicate that the intervention increased the digital competences of participants connected to health.

AIMS AND OBJECTIVES To describe and understand strategies that oncological nurses use to support self-management of radiation-induced bowel and bladder issues in pelvic-cancer rehabilitation patients. BACKGROUND Nurse-led self-management of radiation-induced bowel and bladder issues holds the potential to support cancer survivors. DESIGN An ethnographic approach was applied in this study, which adhered to Consolidated Criteria for Reporting Qualitative Research (COREQ) guidelines. METHODS Data collection was conducted in Sweden between October 2015-April 2018, involving observations of nurses' daily work, formal and informal interviews, individual and group interviews, and reviews of relevant documents used in the studied practice. Furthermore, 15 supportive nurse-patient talks were observed, and an ethnographic analysis was performed. RESULTS The analysis identified the following three categories of nursing strategies that support self-management of radiation-induced bowel and bladder issues in pelvic-cancer rehabilitation patients: encouraging self-reflection, tailoring solutions together and keeping patients motivated. Nurses and patients jointly make sense of patients' symptoms using data that patients collect about themselves. Based on their shared understanding, they can co-create solutions to meet each individual patient's needs and develop routines to keep the patient motivated in performing the devised solutions. CONCLUSIONS The results indicate that the strategies nurses use to support patients in self-management of radiation-induced bowel and bladder issues entail intertwining patients' experiences with their nurses' medical knowledge and specific clinical practice. Nurses' strategies build on their ability to connect patients' experiences and the elements of their own work practice. RELEVANCE TO CLINICAL PRACTICE A deeper understanding of nurses' strategies to support self-management of radiation-induced bowel and bladder issues in pelvic-cancer rehabilitation patients can improve other self-management programmes, inform nurses' education and aid in the design of tools for pelvic-cancer rehabilitation support.

Agents targeting B-cell receptor (BCR) signaling-associated kinases such as Bruton tyrosine kinase (BTK) or phosphatidylinositol 3-kinase can induce mobilization of neoplastic B cells from the lymphoid tissues into the blood, which makes them potentially ideal to combine with anti-CD20 monoclonal antibodies (such as rituximab, obinutuzumab, or ofatumumab) for treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL). Here we show that interactions between leukemia cells and stromal cells (HS-5) upregulate CD20 on CLL cells and that administering ibrutinib downmodulates CD20 (MS4A1) expression in vivo. We observed that CLL cells that have recently exited the lymph node microenvironment and moved into the peripheral blood (CXCR4(dim)CD5(bright) subpopulation) have higher cell surface levels of CD20 than the cells circulating in the bloodstream for a longer time (CXCR4(bright)CD5(dim) cells). We found that CD20 is directly upregulated by CXCR4 ligand stromal cell-derived factor 1 (SDF-1α, CXCL12) produced by stromal cells, and BTK-inhibitor ibrutinib and CXCR4-inhibitor plerixafor block SDF-1α-mediated CD20 upregulation. Ibrutinib also downmodulated Mcl1 levels in CLL cells in vivo and in coculture with stromal cells. Overall, our study provides a first detailed mechanistic explanation of CD20 expression regulation in the context of chemokine signaling and microenvironmental interactions, which may have important implications for microenvironment-targeting therapies.

Publisher's version (útgefin grein), Telecare has the potential to increase the quality of care while also decreasing costs. However, despite great potential, efficiency in care practices and cost reduction remain hypothetical. Within computer supported cooperative work (CSCW), one focus of telecare research has been on awareness support in distributed real-time communication in comparison to physical meetings since face-to-face consultations have been known as the "gold standard" of conducting care. Research has shown that it is hard to maintain qualities such as awareness through video-mediated meetings. In this research, the goal has not been to mimic the qualities of face-to-face consultations but rather to document the qualities of three types of patient meetings (consultations) and to understand in what kinds of situations each consultation type is a viable option. In this paper, we focus on the essential qualities of i) face-to-face consultations, ii) video-based consultations, and iii) telephone consultations and shed light on their affordances. The research contribution includes an extension of the affordance lens to incorporate socio-technical, two-sided affordances, that constitute important aspects for understanding complexity when heterogeneous actors co-existing in a practice, where affordances can differ for different "sides" in the complex practice-a view that is fruitful when dealing with heterogeneous actors and a set of analog and digital tools in a practice., "Peer Reviewed"

Recent development in self-monitoring devices indicates that using quantified data in clinical practice supporting chronic diseases management holds a big potential. However, exploration of this design space also suggests that some unattended challenges still exist, such as a low adoption rate of self-monitoring tools in existing clinical practice. In this text, we therefore focus on the ways healthcare professionals use quantified data in their practice. We draw on empirical data from an ethnographic study of a cancer rehabilitation center. Our preliminary findings suggest that the self-monitoring tool supported the nurses' work because it became a functional complement to their work by allowing them to appropriate the device to their and the patients' needs.

The variable clinical course in chronic lymphocytic leukaemia (CLL) largely depends on p53 functionality and B-cell receptor (BCR) signalling propensity; however, it is unclear if there is any crosstalk between these pathways. We show that DNA damage response (DDR) activation leads to down-modulating the transcriptional factor FOXP1, which functions as a positive BCR signalling regulator and its high levels are associated with worse CLL prognosis. We identified microRNA (miRNA) miR-34a as the most prominently upregulated miRNA during DDR in CLL cells in vitro and in vivo during FCR therapy (fludarabine, cyclophosphamide, rituximab). MiR-34a induced by DDR activation and p53 stabilization potently represses FOXP1 expression by binding in its 3'-UTR. The low FOXP1 levels limit BCR signalling partially via derepressing BCR-inhibitory molecule CD22. We also show that low miR-34a levels can be used as a biomarker for worse response or shorter progression free survival in CLL patients treated with FCR chemoimmunotherapy, and shorter overall survival, irrespective of TP53 status. Additionally, we have developed a method for the absolute quantification of miR-34a copies and defined precise prognostic/predictive cutoffs. Overall, herein, we reveal for the first time that B cells limit their BCR signalling during DDR by down-modulating FOXP1 via DDR-p53/miR-34a axis.

Im folgenden Artikel befassen wir uns mit der Sprachenpolitik sowohl auf der europäischen als auch auf der nationalen Ebene. Wir widmen Aufmerksamkeit dem Status des ö/Österreichischen Deutsch, vor allem der rechtlichen Verankerung des Sprachgebrauchs und ihren praktischen Auswirkungen. In der Linguistik wurde die Plurizentrizität der deutschen Sprache anerkannt; das österreichische Deutsch und das Schweizerdeutsch gelten aber eher als die A(nderen) Sprachvarietäten neben der D(ominanten) Varietät, nämlich des bundesdeutschen Deutsch (Clyne, 1995; Ammon, 1995). Auf der supranationalen Ebene spielte die Abgrenzung der österreichischen Varietät gegenüber den dominierenden EU-Sprachen in der Zeit des EU-Beitritts Österreichs eine starke Rolle, was durch das Protokoll Nr. 10 manifestiert ist. Auch ist die immer wachsende Position der Minderheitensprachen auf der einen Seite und die Anerkennung der Staatsprachen auf der anderen Seite ein ständiges Thema des EU-Rechtes. Auf der nationalen Ebene sind der Import der ausländischen Sprachen (Englisch und Bundesdeutsch) und die Verbreitung der inländischen Minderheitensprachen von der Bedeutung. Die Funktionen der Volksgruppensprachen sind in der Fülle Sprachgesetze nicht immer eindeutig deklariert. Neben der juristischen Lage werden einige über die Sprachpraxis aussagende Forschungsergebnisse eingebracht.

This paper is focused on measurement of some corona manifestations. In the first part is described method for measuring corona interference by antenna. Was found that corona causes interference in frequency range from 30 MHz to 1 GHz. Measurement was applied on two models wire-plate and tip-bowl model. To obtained more information about corona discharge was used partial discharge detector. Were obtained information such as amplitude of charge, inception voltage, current and number of positive and negative discharges.

We report a case of an extra nuchal-type fibroma in a 51-year-old male suspected to have attenuated familial adenomatous polyposis (Gardner's syndrome), who presented with a longstanding buttock mass excised due to enlargement and pain. Histopathologically, lobules of haphazard, hypocellular, hyalinized collagen bundles replaced the dermis and subcutis and entrapped nerve bundles, mimicking Morton neuroma. Ramifying nerve twigs found around larger nerve fascicles showed the co-existence of traumatic neuroma. Elastic tissue stain revealed elastosis characterized by large, arborizing fibers lying between and within the hyalinized collagen bundles. Modified Masson's trichrome stain showed light blue staining of collagen bundles producing the hyalinized nodules with irregular, light red staining of collagen bundles at their periphery and within tumor collagen. Compression and/or degeneration of collagen and secondary elastosis with later entrapment by tumor collagen could explain this microscopic phenotype. By immunohistochemistry, tumor spindle cells expressed nuclear β-catenin and cyclin D1, mostly within regions of fibrosis implicating activation of the adenomatous polyposis coli (APC)-Wnt pathway. Genetic analysis showed a missense mutation in APC gene (c.7504G>A, p.G2502S in exon 15) and a functional homozygous polymorphism in the MUTYH gene (c.36+325G>C, (IVS1+5G/C)). Nuchal-type fibroma has been associated with Gardner's syndrome and trauma. In this patient, genetic predisposition coupled with repetitive, localized trauma and collagen degeneration may have provided the stimulus for the development of extra nuchal-type fibroma.

Introduction: It was shown that BCR inhibitors such as ibrutinib interrupt microenvironmental interactions and mobilize malignant B cells of chronic lymphocytic leukemia (CLL) from immune niches into the blood stream. It has been suggested that this mobilization will sensitize malignant B cells to other drugs, providing a possibility to introduce synthetically lethal combinations of drugs with an acceptable toxicity profile. This might be limited to a subgroup of CLL patients with specific underlying molecular mechanisms of disease biology or reaction to BCR inhibitors. The aim of this study was to test for the effect of ibrutinib on the expression of genes and activity of pathways that could be potentially targeted by clinically available drugs to achieve highly potent combinations. Results: We performed gene expression profiling in samples obtained from CLL patients treated with ibrutinib as a single agent (pre-ibrutinib vs. day 15 and/or week 5/12/15; N=12 pairs) to identify potential therapeutic targets. We have primarily focused on analyzes of cell-surface proteins and anti-apoptotic Bcl2 family members, since therapeutic antibodies and small molecular inhibitors for these molecules are available. We have analyzed changes in >30 cell-surface molecules, 8 members of the Bcl2 family, and members of other potentially relevant pathways (chemokines, adhesion, Akt, Erk, Ras, NFkB). Notably, we have observed that the mRNA and B cell-surface levels of CD20 are strongly down-modulated with ibrutinib, which indicates that the previously suggested combination of ibrutinib and anti-CD20 antibodies (such as rituximab or ofatumumab) is likely not ideal. Indeed, we showed that CLL cells with lower levels of CD20 are more resistant to rituximab in vitro, but also in vivo (P Notably, it has been shown that addition of rituximab to ibrutinib in vivo is able to partially eliminate CLL cells despite lower levels of CD20 suggesting that there must be other mechanism of ibrutinib action that allows for the effect of rituximab. The screening of anti-apoptotic molecules revealed that Mcl1 levels were significantly down-modulated after ibrutinib treatment in vivo (P=0.002), and ibrutinib also inhibited Mcl1 induction in CLL cells co-cultured with stromal cells (HS-5). As Mcl1 was described to directly protect CLL cells from rituximab, we suggest that ibrutinib-mediated Mcl1 reduction is a mechanism that facilitates rituximab efficacy. On the other hand we have shown that several members of Bcl2 family are up-regulated in CLL samples obtained after >3 weeks on ibrutinib therapy. We have also shown that interactions of stromal cells (HS-5) with CLL cells lead to up-regulation of these molecules while ibrutinib inhibits this in vitro (P Conclusion: We have performed gene expression analysis of primary samples obtained from patients on ibrutinib, and identified gene expression changes that are associated with adaptation of B cells and/or selection of B cell resistant to BCR inhibition. We have shown that malignant B cells are dependent on integrin-signalling and the up-regulation of Bcl2 protein, which makes them synergistically lethal targets. Supported by: The results of this research have been acquired within CEITEC 2020 (LQ1601) project with financial contribution made by the Ministry of Education, Youths and Sports of the Czech Republic within special support paid from the National Programme for Sustainability II funds. Citation Format: Gabriela Pavlasova, Katerina Musilova, Vaclav Seda, Katerina Cerna, Eva Vojackova, Veronika Svobodova, Marek Mraz. The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A34.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and are frequently aberrantly expressed in cancer. We aimed to understand their role in the transformation of indolent follicular lymphoma (FL) into an aggressive diffuse large B cell lymphoma. This happens in ~3% of cases per year during the course of the disease, and is associated with median survival of only 2 years. The NGS revealed number of aberrations associated with transformed FL (tFL), including frequent high-level activity of MYC (amplifications, translocations, and mutations) or loss of DNA damage regulators (p53, CDKN2A/B). Firstly, we performed a miRNA profiling (TaqMan miRNA Arrays) in paired FL and tFL samples (N=8 pairs). This revealed a relatively small group of 5 miRNAs that are consistently differentially expressed in tFL (P1.5). Since the most frequently acquired aberration in tFL is the high-level activity of MYC we performed a correlation analysis of MYC levels and expression of these miRNAs in additional samples of FL, tFL, and CLL samples with/without MYC duplication (N=40 FL/tFL, N=39 CLL). This revealed that at least one of these miRNAs is significantly down-modulated (P Citation Format: Katerina Musilova, Gabriela Pavlasova, Vaclav Seda, Eva Vojackova, Katerina Cerna, Veronika Svobodova, Robert Pytlik, Vit Prochazka, Zuzana Prouzova, Sarka Pospisilova, Lenka Zlamalikova, Heidi Mocikova, Lenka Kruzova, Marie Jarosova, Andrew Evans, Clive Zent, Leos Kren, Marek Trneny, Jiri Mayer, Andrea Janikova, Marek Mraz. Differential expression of microRNAs in transformation of follicular lymphoma to diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1479. doi:10.1158/1538-7445.AM2017-1479

We and others have shown that deregulation of microRNAs (miRNAs) is associated with the biology of B cell malignancies, including regulation of B cell proliferation and survival (Musilova & Mraz, Leukemia, 2015). We focused on studying miRNAs that associate with the aggressiveness of FL and its transformation to diffuse large B cell lymphoma (DLBCL). First, we analyzed the expression of 380 miRNAs (TaqMan Arrays, ABI) in 8 paired primary samples of FL that subsequently transformed to DLBCL. We identified statistically significant changes (P1.8) in the expression of 5 miRNAs. The most significant change was the down-regulation of miR-150 (~5 fold, P=0.01). Similarly, we observed significantly reduced miR-150 levels in an independent cohort of non-paired samples of FL before vs. after transformation to DLBCL, and miR-150 was significantly less expressed in de novo DLBCL in comparison with FL. MicroRNA miR-150 is of particular interest as we have shown that its expression determines BCR signaling propensity in chronic lymphocytic leukemia (CLL) B cells, and low levels associated with worse survival (Mraz et al., Blood, 2014). Therefore, we analyzed miR-150 expression in a cohort of 89 FL samples. We noticed that miR-150 expression was lower in samples from patients with a FLIPI score ≥3 (P=0.03), and with high Ki67 positivity (>20%; P=0.003). Moreover, FL patients with low miR-150 levels ( To determine the potential reason for variable miR-150 levels in FL B cells, we tested the effect of microenvironmental interactions on its expression. In this experiment, a short term (48hrs) co-culture of B cell lymphoma cells with stromal cells (HS-5) led to down-regulation of miR-150 levels (P CONCLUSION: Low miR-150 levels associate with a shorter overall survival in FL. This could be used as a reasonable prognostic marker since high miRNA stability allows reliable analyses of miR-150 levels from formalin-fixed, paraffin-embedded (FFPE) samples. Interactions with stromal cells and/or soluble microenvironmental factors down-modulate miR-150 levels in B cells, which support their BCR signalling potential. We are further investigating to what extent the miR-150 down-regulation is causally connected with the aggressiveness and/or transformation of FL. This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; the research grant GACR (16-13334Y); the Ministry of Health of the Czech Republic, grant nr. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein; Masaryk university as part of the project "New approaches in research, diagnostics and therapy of hematological malignancies III", number MUNI/A/1028/2015 with the support of the Specific University Research Grant, as provided by the Ministry of Education, Youth and Sports of the Czech Republic in the year 2016; the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16); the Ministry of Education, Youth and Sports of the Czech Republic, grant nr. LD15144 (COST CZ); the research grant TACR (TEO2000058/2014-2019); and EHA Research Fellowship award granted by the European Hematology Association. G.P. is a city of Ostrava scholarship holder. Disclosures No relevant conflicts of interest to declare.

The use of anti-CD20 antibody rituximab has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab has been shown to act through several mechanisms including antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct induction of apoptosis, and sensitization to chemotherapy. However, the exact contribution of each of these mechanisms to the clinical efficacy of rituximab in vivo and the exact mechanism of its action remain unclear. Importantly, the levels of cell surface CD20 expression were shown to associate with the efficacy of rituximab. We and others have described that CLL cells that have recently exited the lymph node microenvironment to the peripheral blood express lower cell surface levels of the chemokine receptor CXCR4 and higher levels of the activation marker CD5 (Callisano et al., 2011). We observed that these CLL cells (CXCR4dimCD5bright) have also higher cell surface CD20 expression (~2-fold; P It has been previously suggested that the CD20 plays a direct role in microenvironmental interactions and especially in B cell receptor signaling (BCR; Uchida et al., 2004). Therefore, we investigated the BCR signaling propensity of CXCR4dimCD5bright subpopulation. We have observed that CXCR4dimCD5bright CLL cells have higher surface immunoglobulin (Ig) expression than CXCR4brightCD5dim cells from the same patient (~2-fold, P Altogether, this study shows that rituximab primarily and effectively eliminates, at least in the short term, the BCR signaling proficient CLL B cells. It is likely that one of the mechanisms of rituximab action is the indirect inhibition of BCR signaling by eliminating CLL cells with strongly activated BCR pathway. These observations might have important implications for combinatorial therapies with BCR signaling inhibitors such as ibrutinib or idelalisib. This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; Czech Science Foundation (project No. 16-13334Y); the Ministry of Health of the Czech Republic, grant No. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. This work was supported by the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16), MUNI/A/1028/2015, and G.P. is a city of Ostrava scholarship holder. contact: marek.mraz@email.cz Disclosures No relevant conflicts of interest to declare.

The introduction of anti-CD20 antibodies has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin lymphomas. The aim of this study was to analyze molecular pathways that influence expression of CD20 since this is largely unknown. This is of a great clinical interest, as combinatorial therapy of novel BCR-signaling inhibitors ibrutinib and idelalisib currently focuses mainly on the use with anti-CD20 antibodies. Firstly, we analyzed samples obtained from CLL patients treated with ibrutinib and showed that administration of ibrutinib in vivo leads to CD20 down-modulation (P Altogether, the CD20 levels are up-regulated within the context of microenvironmental interactions through the CXCR4/SDF-1 axis, and the impairment of microenvironmental interactions mediated by ibrutinib down-regulates CD20 expression. This study reveals a novel regulation of CD20 expression in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination. Supported by: SoMoPro II-no.4SGA8684; NGS-PTL(306242); EHA Fellowship award; Ministry of Health of CR (16-29622A); Academy of Sciences of CR (16-13334Y); Ministry of Education, Youth and Sports, grant LD15144 (COST CZ); MUNI/A/1028/2015; CZ.1.05/1.1.00/02.0068; G.P. is supported by Ostrava city. Citation Format: Gabriela Pavlasova, Marek Borsky, Vaclav Seda, Katerina Cerna, Jitka Osickova, Michael Doubek, Yvona Brychtova, Jiri Mayer, Sarka Pospisilova, Matthew S. Davids, Jennifer R. Brown, Marek Mraz. The expression of CD20 on malignant B cells is regulated by chemokine signaling through the CXCR4/SDF-1 axis: implications for targeting the microenvironmental interactions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3291.

Muir-Torre syndrome (MTS), a phenotypic variant of the more common hereditary nonpolyposis colorectal cancer syndrome, or Lynch syndrome, is an autosomal dominantly inherited condition that combines at least one cutaneous sebaceous neoplasm and at least one visceral malignancy. Most patients (~90%) with MTS carry mutations in the MSH2 gene; less than 10% of the cases are associated with a mutation MLH1 gene, and only 3 MTS patients with a pathogenic MSH6 mutation have been previously documented. We report a family affected with MTS in which 3 members (father and 2 sons) were found to harbor a missense mutation c.2633T>C (p.V878A) in exon 4 of the MSH6 gene.

The aim of this study was to investigate the role of microenvironmental interactions in the regulation of CD20 expression, since down-modulation of CD20 in the context of immune niches would be a straightforward explanation for the stroma-mediated rituximab resistance described by us and others (Mraz et al., BJH, 2011; Buchner et al., BJH, 2010; Marquez et al., BJH, 2015). Firstly, we co-cultured primary CLL cells with the bone marrow stromal cell line HS-5 for 24/48/72 hrs and analyzed CD20 surface expression by flow cytometry. Suprisingly, CD20 levels were significantly up-regulated (P=0.03) on CLL cells co-cultured with HS-5 in comparison to CLL cells cultured on plastic. The changes in CD20 expression levels are likely to play a direct role in microenvironmental interactions and especially BCR signaling in immune niches (Uchida et al., Int Immunol, 2004). To test this hypothesis we assessed the CD20 expression on CLL cell populations defined according to CXCR4 and CD5 levels. The low-level surface expression of chemokine receptor CXCR4 and high-level expression of activation molecule CD5 can characterize B cells that have recently exited the lymph nodes (Calissano et al., Mol Med, 2011). CXCR4dim CD5bright cells had ~2-fold higher surface CD20 expression when compared to CXCR4bright CD5dim CLL cells circulating in the blood of the same patient for a longer time (P Conclusion: Microenvironmental interactions up-regulate CD20 expression in CLL B cells through the CXCR4/SDF-1 axis, and the ibrutinib-mediated impairment of microenvironmental interactions down-regulates CD20 expression. This study reveals a novel regulation of CD20 levels in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination. Supported by: SoMoPro II-no.4SGA8684 (cofinanced by the European Union and the South-Moravian Region); NGS-PTL(306242); EHA Fellowship award; IGA MZ CR NT11218-6/2010; MUNI/A/1180/2014; CZ.1.05/1.1.00/02.0068; G.P. is a city of Ostrava scholarship holder. #G.P. and V.S. contributed equally to this study. contact: marek.mraz@email.cz Disclosures Mayer: Janssen: Research Funding. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy.

The biology of B cell Non-Hodgkin lymphomas (NHLs) is largely influenced by (de)regulation of B cell receptor signaling (BCR sig.) and DNA damage response pathway (DDR). We and others have shown that in NHLs, miR-34a is involved in DDR, and miR-150/miR-155 are involved in BCR sig. (Mraz et al., 2009, 2014; Cui et al., 2014). To identify miRNAs involved in DDR and BCR sig. we used NGS technology (HiSeq) in chronic lymphocytic leukemia (CLL) B cells treated with BCR inhibitor or DNA damaging drugs. To investigate the DDR-related miRNAs, primary CLL cells were treated with fludarabine in vitro and paired samples (before/after treatment, n = 20) were analyzed for miRNAs’ profile. This identified 6 differentially expressed miRNAs (FDR1.5, P Supported by: SoMoPro II-no. 4SGA8684; NGS-PTL (306242); EHA Fellowship award; IGA MZ CR NT11218-6/2010; CZ.1.07/2.4.00/17.0042; MUNI/A/0830/2013; MH CZ-DRO (FNBr, 65269705), CZ.1.05/1.1.00/02.0068, CZ.1.07/2.3.00/30.0009. Citation Format: Katerina Cerna, Jan Oppelt, Lenka Radova, Katerina Musilova, Vaclav Seda, Gabriela Pavlasova, Michal Jez, Nikola Tom, Filip Pardy, Jitka Malcikova, Karla Plevova, Boris Tichy, Yvona Brychtova, Michael Doubek, Martin Trbusek, Jiri Mayer, Jaroslav Koca, Raffaele Calogero, Sarka Pospisilova, Marek Mraz. MicroRNA involvement in DNA damage response and BCR signaling in malignant B cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2015-3084

We and others have shown that expression of miRNAs influences the biology of B cell malignancies (Calin et al., 2005; Mraz et al., 2009, 2012, 2013). The aim of this study was to identify miRNAs involved in the apoptotic response of malignant B cells. Purified primary B cells of chronic lymphocytic leukemia (CLL) patients (pts.) were treated in vitro with fludarabine (F) (LC50 dose of 3.5 μg/ml; 48 h). Five paired (n=10) samples (with and without F) were analyzed using 2 NGS platforms-SOLiD (ABI) and HiSeq (Illumina). The obtained reads were mapped to miRBase using 3 tools (CLC Genomic Workbench, SHRiMP2, miRanalyzer) and data analyzed by a pair-wise comparison with edgeR and baySeq packages (Bioconductor 2.13). The overlap of these 3+2 bioinformatic approaches identified 6 miRNAs significantly changed with DNA damage. RNA-Seq validation on 5 additional paired samples (n=10) confirmed the changed expression of all 6 previously identified miRNAs (3 down-, 3 up-regulated). The most constantly up-regulated was miR-34a, which was previously shown to be regulated by p53 (He et al., 2005; Mraz et al., 2009). To test the importance of miR-34a in vivo, we collected samples from CLL pts. (n=51) treated with F, cyclophosphamide and rituximab (FCR) regimen. miR-34a was induced in all samples after F administration (day 2, p Supported by: NGS-PTL (FP7-HEALTH-2012-INNOVATION-1, no. 306242); EHA Research Fellowship award; Grant Agency of Czech Rep.; IGA MZ CR NT11218-6/2010; CZ.1.07/2.4.00/17.0042; MUNI/A/0723/2012; CZ.1.07/2.3.00/30.0009, co-financed from EU and Czech Rep. Citation Format: Katerina Cerna, Jan Oppelt, Lenka Radova, Katerina Musilova, Nikola Tom, Filip Pardy, Jitka Malcikova, Karla Plevova, Boris Tichy, Yvona Brychtova, Michael Doubek, Martin Trbusek, Jiri Mayer, Jaroslav Koca, Raffaele Calogero, Sarka Pospisilova, Marek Mraz. Identification of microRNAs involved in DNA damage response in malignant B cells and their biological and clinical relevance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5198. doi:10.1158/1538-7445.AM2014-5198

Summary We have analyzed the miRNA sequence variations in patients with CLL and the effect of these variations on their secondary structure and expression., MicroRNA (miRNA) expression is deregulated in many tumors including chronic lymphocytic leukemia (CLL). Although the particular mechanism(s) responsible for their aberrant expression is not well characterized, the presence of mutations and single-nucleotide polymorphisms (SNPs) in miRNA genes, possibly affecting their secondary structure and expression, has been described. In CLL; however, the impact and frequency of such variations have yet to be elucidated. Using a custom resequencing microarray, we screened sequence variations in 109 cancer-related pre-miRNAs in 98 CLL patients. Additionally, the primary regions of miR-29b-2/29c and miR-16-1 were analyzed by Sanger sequencing in another cohort of 213 and 193 CLL patients, respectively. Altogether, we describe six novel miR-sequence variations and the presence of SNPs (n = 27), most of which changed the miR-secondary structure. Moreover, some of the identified SNPs have a significantly different frequency in CLL when compared with a control population. Additionally, we identified a novel variation in miR-16-1 that had not been described previously in CLL patients. We show that this variation affects the expression of mature miR-16-1. We also show that the expression of another miRNA with pathogenetic relevance for CLL, namely miR-29b-2, is influenced by the presence of a polymorphic insertion, which is more frequent in CLL than in a control population. Altogether, these data suggest that sequence variations may occur during CLL development and/or progression.

Abstract 3874 It is known that the biology of CLL and other B-cell malignancies is driven by processes dependent on immunoglobulin structure and BCR-signaling. Recently, our group and others have described the importance of miRNAs in CLL and their involvement in BCR-signaling, IgG production and V(D)J recombination. Considering the important functions of miRNAs it is remarkable that the human locus for immunoglobulin lambda light chain (IgL) contains a miR gene. The miR-650 gene is localized in exon 1 of IgL variable subgene (1st exon of V2 family members). The aim of this study was to reveal the regulation and expression of miR-650 in CLL and its relation to disease prognosis. CLL samples were separated by RosetteSep Human B Cell Enrichment Cocktail (obtained purity ≥95% of CD5+19+ cells). The expression of light chain surface immunoglobulin chain (lambda vs kappa) was determined by flow cytometry. The utilized IgL variable (V) segment was determined using BIOMED-2 protocol and sequencing. To study the relation of miR-650 expression and IgL rearrangement the surface expression of Ig light chain and the utilized V segment was determined in a CLL cohort containing 47 patients (λ n=27, κ n=19, λ+κ n=1). The gene expression was analyzed by TaqMan Assays (ABI) for mature miR-650 and protein-coding genes: CDK1, EBF3 and ING4. The western-blot for miR-650 targets was performed after transfection (48hrs/96hrs) of B-cell lines (NALM-6, MEC-1) with a short RNA mimicking miR-650 (Dharmacon). The analyses of miR-650 expression revealed that cells utilizing V2-8, V2-5, V2-14, V2-23 subgenes for IgL (n=14) had ∼10 fold higher expression of miR-650 (p We next studied the possible targets for miR-650 in B-cells. Recently, two targets were identified in solid tumors - ING4 (Inhibitor of Growth 4) and CDK1 (cyclin dependent kinase 1) (Zhang, 2010; Chien, 2010). It has been demonstrated that miR-650 is involved in the p16INK4-mediated pathway and directly regulates the CDK1. This publication suggested that up-regulation of miR-650 leads to inhibition of cell cycle progression. Moreover, the putative targets predicted by software tools (TargetScan, miRanda) include genes important for B-cell biology like EBF3 (early B-cell factor3), CLLU1, Bcl2 and cyclin D1. We therefore studied correlation between miR-650 expression and the expression of mRNAs for CDK1, ING4 and EBF3 (a predicted target with the highest score). The expression of miR-650 was not significantly associated with the expression of any of these genes on mRNA level. The lack of available material did not allow us to study the expression of CDK1, ING4, EBF3 protein levels in the original cohort. However, the transfection of B-cells with short RNA mimicking miR-650 led to down-regulation of protein levels of CDK1 and EBF3. This confirms the relevance of CDK1 as a target in B-cells and identifies a new target - EBF3, which is known to be important for B-cells development. Moreover, the expression of miR-650 was associated with overall survival (OS) and treatment free survival (TFS) in CLL (n=82). In this analysis patients were divided in two groups (based on the median of miR-650 expression). The higher expression of miR-650 was associated with statistically significant (p In conclusion, we have described a mechanism regulating miR-650 expression, identified its relevant targets in B-cells and demonstrated the association of miR-650 expression with CLL prognosis. Supported by IGA MZCR NT11218-6/2010, MSMTMSM0021622430, NS10439-3/2009, FR-TI2/254 Disclosures: Mayer: Roche: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Astellas: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Novartis: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Fresenius Medical Care: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Pfizer: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Genzyme: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; GSK: Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Amgen:.