Nucleosome repositioning in chronic lymphocytic leukemia.

The location of nucleosomes in the human genome determines the primary chromatin structure and regulates access to regulatory regions. However, genome-wide information on deregulated nucleosome occupancy and its implications in primary cancer cells is scarce. Here, we conducted a genome-wide comparison of high-resolution nucleosome maps in peripheral blood B cells from patients with chronic lymphocytic leukemia (CLL) and healthy individuals at single-base-pair resolution. Our investigation uncovered significant changes of nucleosome positioning in CLL. Globally, the spacing between nucleosomes-the nucleosome repeat length (NRL)-is shortened in CLL. This effect is stronger in the more aggressive IGHV-unmutated CLL subtype than in the IGHV-mutated CLL subtype. Changes in nucleosome occupancy at specific sites are linked to active chromatin remodeling and reduced DNA methylation. Nucleosomes lost or gained in CLL marks differential binding of 3D chromatin organizers such as CTCF as well as immune response-related transcription factors and delineated mechanisms of epigenetic deregulation. The principal component analysis of nucleosome occupancy in cancer-specific regions allowed the classification of samples between cancer subtypes and normal controls. Furthermore, patients could be better assigned to CLL subtypes according to differential nucleosome occupancy than based on DNA methylation or gene expression. Thus, nucleosome positioning constitutes a novel readout to dissect molecular mechanisms of disease progression and to stratify patients. Furthermore, we anticipate that the global nucleosome repositioning detected in our study, such as changes in the NRL, can be exploited for liquid biopsy applications based on cell-free DNA to stratify patients and monitor disease progression.

The role of Bruton's tyrosine kinase in the immune system and disease.

Bruton's tyrosine kinase (BTK) is a TEC kinase with a multifaceted role in B-cell biology and function, highlighted by its position as a critical component of the B-cell receptor signalling pathway. Due to its role as a therapeutic target in several haematological malignancies including chronic lymphocytic leukaemia, BTK has been gaining tremendous momentum in recent years. Within the immune system, BTK plays a part in numerous pathways and cells beyond B cells (i.e. T cells, macrophages). Not surprisingly, BTK has been elucidated to be a driving factor not only in lymphoproliferative disorders but also in autoimmune diseases and response to infection. To extort this role, BTK inhibitors such as ibrutinib have been developed to target BTK in other diseases. However, due to rising levels of resistance, the urgency to develop new inhibitors with alternative modes of targeting BTK is high. To meet this demand, an expanding list of BTK inhibitors is currently being trialled. In this review, we synopsize recent discoveries regarding BTK and its role within different immune cells and pathways. Additionally, we discuss the broad significance and relevance of BTK for various diseases ranging from haematology and rheumatology to the COVID-19 pandemic. Overall, BTK signalling and its targetable nature have emerged as immensely important for a wide range of clinical applications. The development of novel, more specific and less toxic BTK inhibitors could be revolutionary for a significant number of diseases with yet unmet treatment needs.

An entity evolving into a community: defining the common ancestor and evolutionary trajectory of chronic lymphocytic leukemia stereotyped subset #4.

Patients with chronic lymphocytic leukemia (CLL) assigned to stereotyped subset #4 express highly homologous B-cell receptor immunoglobulin (BcR IG) sequences with intense intraclonal diversification (ID) in the context of ongoing somatic hypermutation (SHM). Their remarkable biological and clinical similarities strongly support derivation from a common ancestor. We here revisited ID in subset #4 CLL to reconstruct their evolutionary history as a community of related clones. To this end, using specialized bioinformatics tools we assessed both IGHV-IGHD-IGHJ rearrangements (n = 511) and IGKV-IGKJ rearrangements (n = 397) derived from eight subset #4 cases. Due to high sequence relatedness, a number of subclonal clusters from different cases lay very close to one another, forming a core from which clusters exhibiting greater variation stemmed. Minor subclones from individual cases were mutated to such an extent that they now resembled the sequences of another patient. Viewing the entire subset #4 data set as a single entity branching through diversification enabled inference of a common sequence representing the putative ancestral BcR IG expressed by their still elusive common progenitor. These results have implications for improved understanding of the ontogeny of CLL subset #4, as well as the design of studies concerning the antigenic specificity of the clonotypic BcR IGs.

Temporal dynamics of clonal evolution in chronic lymphocytic leukemia with stereotyped IGHV4-34/IGKV2-30 antigen receptors: longitudinal immunogenetic evidence.

Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset 4 possess distinctive patterns of intraclonal diversification (ID) within their immunoglobulin (IG) genes. Although highly indicative of an ongoing response to antigen(s), the critical question concerning the precise timing of antigen involvement is unresolved. Hence, we conducted a large-scale longitudinal study of eight subset 4 cases totaling 511 and 398 subcloned IG heavy and kappa sequences. Importantly, we could establish a hierarchical pattern of subclonal evolution, thus revealing which somatic hypermutations were negatively or positively selected. In addition, distinct clusters of subcloned sequences with cluster-specific mutational profiles were observed initially; however, at later time points, the minor cluster had often disappeared and hence not been selected. Despite the high intensity of ID, it was remarkable that certain residues remained essentially unaltered. These novel findings strongly support a role for persistent antigen stimulation in the clonal evolution of CLL subset 4.

Emerging Roles of TRIM Family Proteins in Gliomas Pathogenesis.

Gliomas encompass a vast category of CNS tumors affecting both adults and children. Treatment and diagnosis are often impeded due to intratumor heterogeneity and the aggressive nature of the more malignant forms. It is therefore essential to elucidate the molecular mechanisms and explore the intracellular signaling pathways underlying tumor pathology to provide more promising diagnostic, prognostic, and therapeutic tools for gliomas. The tripartite motif-containing (TRIM) superfamily of proteins plays a key role in many physiological cellular processes, including brain development and function. Emerging evidence supports the association of TRIMs with a wide variety of cancers, exhibiting both an oncogenic as well as a tumor suppressive role depending on cancer type. In this review, we provide evidence of the pivotal role of TRIM proteins in gliomagenesis and exploit their potential as prognostic biomarkers and therapeutic targets.

Efficient Treg depletion induces T-cell infiltration and rejection of large tumors.

There are a number of factors that hamper immunotherapy of cancer. For example, tumors exhibit an aberrant vasculature that appears to form a barrier against T-cell infiltration. Another major obstacle is created by Treg. So far, conventional depletion of Treg with anti-CD25 antibodies, which eliminate only 70% of Treg, has failed to significantly reduce the growth of established tumors. Using Foxp3.LuciDTR-4 mice, we show here that 90-95% Treg depletion resulted in complete regression of large established tumors, whereas 70% depletion was ineffective. The extensive Treg depletion induced a number of processes that are critical for tumor rejection, including activation of tumor-specific CD8(+) T cells and enhanced infiltration of these cells into the tumor. The precise mechanism of enhanced infiltration is not known, but normalization of the tumor vasculature is assumed to assist infiltration. Indeed, we observed that 90% Treg depletion caused normalization of the tumor vasculature as indicated by a reduction in leakiness and the number of dilated vessels. These results suggest that for clinical immunotherapy of cancer, it would be desirable to have reagents that allow high-level depletion of Treg, which, in conjunction with treatment modalities such as vaccination, may concomitantly increase T-cell activation and infiltration.

Extensive intraclonal diversification in a subgroup of chronic lymphocytic leukemia patients with stereotyped IGHV4-34 receptors: implications for ongoing interactions with antigen.

Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen recognition through the clonotypic B-cell receptors (BCRs). However, it is still unclear whether antigen involvement is restricted to the malignant transformation phase or whether the putative antigen(s) may continuously trigger the CLL clone and affect not only the progenitor cell but also the leukemic cells themselves. To address this issue, we conducted a large-scale subcloning study of rearranged immunoglobulin heavy variable (IGHV) genes of diverse mutational status from 71 CLL cases (total, 1496 subcloned sequences), belonging to both the common IgM/IgD variant and the rare IgG-positive variant. Although most cases showed no or low levels of intraclonal diversification (ID), we report intense ID in the IGHV genes of selected cases, especially a subgroup of 13 IgG-switched cases expressing stereotyped, mutated IGHV4-34 rearrangements (subset 4). We demonstrate that the ID evident in subset 4 cases cannot be attributed to IGHV4-34 usage, IGHV gene-mutated status, class-switch recombination, or BCR stereotypy in general; rather, it represents a unique phenomenon strongly correlated with the distinctive BCR of subset 4. In such cases, the observed ID patterns may imply a stereotyped response to an active, ongoing interaction with antigen(s).

A Sandwich-model experiment with personal response systems on epigenetics: insights into learning gain, student engagement and satisfaction.

Current trends in Higher Education Pedagogies include an ongoing discussion about active learning strategies. Technology-based interventions such as personal response systems (PRS) have gained momentum, especially since the advent of cloud-/web-based solutions. One model that supports the transition from traditional lecturing towards active learning by maintaining a balance between instruction and self-learning is the 'Sandwich Model'. In the present study, we investigated the impact of the Sandwich Model combined with PRS in student learning, engagement and satisfaction by a randomised trial in a large undergraduate biomedical/medical sciences class. A teaching session on epigenetics was delivered either as a traditional lecture (C-group) or as a PRS-including Sandwich-based session (S-group). The major finding of our experiment was the significantly enhanced performance of the S-group over the control, suggesting that the Sandwich Model improves learning gain. We also provide strong evidence that the Sandwich Model enhances student engagement and satisfaction. However, the effect of the Sandwich Model in learning gain and student attitudes was not dependent on PRS incorporation per se and students seemed to favour non-PRS activities over PRS, as evidenced by their feedback. Although further experimental research is needed in order to conclusively compare and contrast PRS and non-PRS activities regarding learning gain, we propose the usage of the Sandwich Model with a variety of in-class learning activities, both PRS and non-PRS-based. Altogether, our work shows that the Sandwich Model is a powerful pedagogical approach that exerts a positive impact on student perceptions for learning and satisfaction and that can support the teaching of challenging biomedical concepts, such as epigenetics.

Gene promoter methylation signature predicts survival of head and neck squamous cell carcinoma patients.

Infection with high-risk types of human papilloma virus (HPV) is currently the best-established prognostic marker for head and neck squamous cell carcinoma (HNSCC), one of the most common and lethal human malignancies worldwide. Clinical trials have been launched to address the concept of treatment de-escalation for HPV-positive HNSCC with the final aim to reduce treatment related toxicity and debilitating long-term impacts on the quality of life. However, HPV-related tumors are mainly restricted to oropharyngeal SCC (OPSCC) and there is an urgent need to establish reliable biomarkers for all patients at high risk for treatment failure. A patient cohort (n = 295) with mainly non-OPSCC (72.9%) and a low prevalence of HPV16-related tumors (8.8%) was analyzed by MassARRAY to determine a previously established prognostic methylation score (MS). Kaplan-Meier revealed a highly significant correlation between a high MS and a favorable survival for OPSCC (P = 0.0004) and for non-OPSCC (P<0.0001), which was confirmed for all HNSCC by multivariate Cox regression models (HR: 9.67, 95% CI [4.61-20.30], P<0.0001). Next, we established a minimal methylation signature score (MMSS), which consists of ten most informative of the originally 62 CpG units used for the MS. The prognostic value of the MMSS was confirmed by Kaplan-Meier analysis for all HNSCC (P<0.0001) and non-OPSCC (P = 0.0002), and was supported by multivariate Cox regression models for all HNSCC (HR: 2.15, 95% CI [1.36-3.41], P = 0.001). In summary, the MS and the MMSS exhibit an excellent performance as prognosticators for survival, which is not limited by the anatomical site, and both could be implemented in future clinical trials.

HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas.

High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.

New Concepts for Translational Head and Neck Oncology: Lessons from HPV-Related Oropharyngeal Squamous Cell Carcinomas.

Human papillomavirus (HPV) infection is well established as an etiological agent responsible for a number of pathologies affecting the stratified epithelia of skin and anogenital sites. More recently, the infection by (mucosal) high-risk HPV types has also been found to be causally associated with squamous cell carcinoma in the head and neck region (HNSCC), especially in the oropharynx. Intriguingly, HPV-related oropharyngeal squamous cell carcinomas (OPSCC) represent a distinct clinical entity compared to HPV-negative tumors with particular regard to treatment-response and survival outcome. The association between HPV infection and OPSCC may therefore have important implications for the prevention and/or treatment of OPSCC. The improved survival of patients with HPV-related tumors also raises the question, as to whether a better understanding of the underlying differences may help to identify new therapeutic concepts that could be used in targeted therapy for HPV-negative and improved therapy for HPV-positive cancers. This review summarizes the most recent advances in our understanding of the molecular principles of HPV-related OPSCC, mainly based on functional genomic approaches, but also emphasizes the significant role played by the tumor microenvironment, especially the immune system, for improved clinical outcome and differential sensitivity of HPV-related tumors to current treatment options.

Immunoglobulin gene repertoire in chronic lymphocytic leukemia: insight into antigen selection and microenvironmental interactions.

Immunogenetic analysis of the B cell receptors (BCRs) has been a richly rewarding field for unraveling the pathogenesis of human lymphomas, including CLL. A biased immunoglobulin gene repertoire is seen as evidence for selection of CLL progenitor cells by antigen. Additional corroborative evidence is provided by the differential prognosis of cases with distinct mutational status of the clonotypic BCRs. However, perhaps the strongest immunogenetic evidence for the importance of interactions with microenvironment in driving CLL development and evolution is the existence of subsets of patients with quasi-identical, stereotyped BCRs, collectively accounting for a remarkable one-third of the entire cohort. These observations have been instrumental in shaping the notion that CLL ontogeny is functionally driven and dynamic, rather than a simple stochastic process. From a clinical perspective, ample evidence indicates that immunogenetic information can be used for the biologically and clinically rational categorization of CLL, with important potential implications for basic, translational and clinical research.

Cytogenetic evaluation and DNA interaction studies of the food colorants amaranth, erythrosine and tartrazine.

Food coloring agents, amaranth, erythrosine and tartrazine have been tested at 0.02-8mM in human peripheral blood cells in vitro, in order to investigate their genotoxic, cytotoxic and cytostatic potential. Amaranth at the highest concentration (8mM) demonstrates high genotoxicity, cytostaticity and cytotoxicity. The frequency of SCEs/cell was increased 1.7 times over the control level. Additionally, erythrosine at 8, 4 and 2mM shows a high cytotoxicity and cytostaticity. Finally, tartrazine seems to be toxic at 8 and 4mM. No signs of genotoxicity were observed. Reversely, tartrazine showed cytotoxicity at 1 and 2mM. Furthermore, spectroscopic titration studies for the interaction of these food additives with DNA showed that these dyes bind to calf thymus DNA and distinct isosbestic points are observed clearly suggesting binding of the dyes to DNA. Additionally DNA electrophoretic mobility experiments showed that these colorants are obviously capable for strong binding to linear dsDNA causing its degradation. PCR amplification of all DNA fragments (which previously were pre-treated with three different concentrations of the colorants, extracted from agarose gel after separation and then purified), seems to be attenuated with a manner dye concentration-dependent reflecting in a delayed electrophoretic mobility due to the possible binding of some molecules of the dyes. Evaluation of the data and curves were obtained after quantitative and qualitative analysis of the lanes of the gel by an analyzer computer program. Our results indicate that these food colorants had a toxic potential to human lymphocytes in vitro and it seems that they bind directly to DNA.

Chronic lymphocytic leukaemia: an immunobiology approach.

B cell chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia that follows an extremely variable clinical course. Several important prognostic parameters defining pathogenic and clinical subgroups of CLL have been identified and validated recently. The biological significance of immunoglobulin (Ig) heavy chain variable region gene (IgHV) mutational status and associated ZAP-70 over-expression, CD38 and chromosomal aberrations have enabled to identify patients at high risk for early disease progression and inferior survival. Moreover, studies of the B cell antigen receptor (BCR) structure and receptor signalling have been most helpful in revealing some new aspects of the biology of this disease. In particular, the analysis of IG genes has revealed that the expressed IgHV/IgKV/IgLV gene repertoires of CLL cells differ from those of normal B cells. A further unique feature of the CLL IG repertoire is the existence of subsets of cases with "stereotyped" BCRs. Accumulating molecular and phenotypic data support the notion that CLL development and evolution is not a simple scholastic event and strongly indicates a role for antigen in driving the cell of origin for at least some subsets of CLL cases.