Targeting Melanoma-Associated Fibroblasts (MAFs) with Activated γδ (Vδ2) T Cells: An In Vitro Cytotoxicity Model.

The tumor microenvironment (TME) has gained considerable scientific attention by playing a role in immunosuppression and tumorigenesis. Besides tumor cells, TME is composed of various other cell types, including cancer-associated fibroblasts (CAFs or MAFs when referring to melanoma-derived CAFs) and tumor-infiltrating lymphocytes (TILs), a subpopulation of which is labeled as γδ T cells. Since the current anti-cancer therapies using γδ T cells in various cancers have exhibited mixed treatment responses, to better understand the γδ T cell biology in melanoma, our research group aimed to investigate whether activated γδ T cells are capable of killing MAFs. To answer this question, we set up an in vitro platform using freshly isolated Vδ2-type γδ T cells and cultured MAFs that were biobanked from our melanoma patients. This study proved that the addition of zoledronic acid (1-2.5 µM) to the γδ T cells was necessary to drive MAFs into apoptosis. The MAF cytotoxicity of γδ T cells was further enhanced by using the stimulatory clone 20.1 of anti-BTN3A1 antibody but was reduced when anti-TCR γδ or anti-BTN2A1 antibodies were used. Since the administration of zoledronic acid is safe and tolerable in humans, our results provide further data for future clinical studies on the treatment of melanoma.

Landscape of BCL2 Resistance Mutations in a Real-World Cohort of Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia Treated with Venetoclax.

The oral, highly selective Bcl2 inhibitor venetoclax has substantially improved the therapeutic landscape of chronic lymphocytic leukemia (CLL). Despite the remarkable response rates in patients with relapsed/refractory (R/R) disease, acquired resistance is the leading cause of treatment failure, with somatic BCL2 mutations being the predominant genetic drivers underpinning venetoclax resistance. To assess the correlation between disease progression and the most common BCL2 mutations G101V and D103Y, sensitive (10-4) screening for the most common BCL2 mutations G101V and D103Y was performed in 67 R/R CLL patients during venetoclax single-agent or venetoclax-rituximab combination therapy. With a median follow-up time of 23 months, BCL2 G101V and D103Y were detected in 10.4% (7/67) and 11.9% (8/67) of the cases, respectively, with four patients harboring both resistance mutations. Ten out of eleven patients carrying BCL2 G101V and/or D103Y experienced relapse during the follow-up period, representing 43.5% of the cases (10/23) showing clinical signs of disease progression. All BCL2 G101V or D103Y variants were detected in patients receiving venetoclax as a continuous single-agent treatment while these mutations were not observed during or after fixed-duration venetoclax therapy. Targeted ultra-deep sequencing of BCL2 uncovered three additional variants in four patient samples obtained at relapse, suggesting convergent evolution and implying a cooperating role of BCL2 mutations in driving venetoclax resistance. This cohort is the largest R/R CLL patient population reported to date in which BCL2 resistance mutations were investigated. Our study demonstrates the feasibility and clinical value of sensitive screening for BCL2 resistance mutations in R/R CLL.

Limitations of VS38c labeling in the detection of plasma cell myeloma by flow cytometry.

Plasma cell myeloma (multiple myeloma [MM]) is a malignant neoplasm originating from the plasma cells. Besides other methods, flow cytometric analysis of the patient's bone marrow aspirate has an important role in the diagnosis and also in the response assessment. Since the cell surface markers, used for identifying abnormal plasma cells, are expressed diversely and the treatment can also alter the phenotype of the plasma cells, there is an increasing demand for new plasma cell markers. VS38c is a monoclonal antibody that recognizes the CLIMP-63 protein in the membrane of the endoplasmic reticulum. CLIMP-63 is known to be expressed at high levels in normal and pathologic plasma cells in the bone marrow, thus VS38c antibody can be used to identify them. Although VS38c staining of plasma cells is reported to be constant and strong even in myeloma, we were wondering whether sample preparation can affect the staining. We have investigated the effect of different permeabilization agents and washing of the cells on the quality of the VS38c staining and found that in many cases the staining is inadequate to identify the plasma cells. We measured the VS38c staining of the bone marrow aspirates of 196 MM patients and observed that almost all cases showed bright staining with VS38c. However, permeabilization with mild detergent resulted in the appearance of a significant VS38cdim subpopulation, which showed increased sensitivity to mechanical stress (centrifugation). Our results indicate that VS38cdim MM cells can appear due to the improper permeabilization of the endoplasmic reticulum and this finding raises the possibility of the existence of a plasma cell subpopulation with different membrane properties. The significance of this population is unclear yet, but these cells can be easily missed with VS38c staining and can be lost due to centrifugation-induced lysis during sample preparation.

Screening and monitoring of the BTKC481S mutation in a real-world cohort of patients with relapsed/refractory chronic lymphocytic leukaemia during ibrutinib therapy.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has revolutionised the therapeutic landscape of chronic lymphocytic leukaemia (CLL). Acquired mutations emerging at position C481 in the BTK tyrosine kinase domain are the predominant genetic alterations associated with secondary ibrutinib resistance. To assess the correlation between disease progression, and the emergence and temporal dynamics of the most common resistance mutation BTKC481S , sensitive (10-4 ) time-resolved screening was performed in 83 relapsed/refractory CLL patients during single-agent ibrutinib treatment. With a median follow-up time of 40 months, BTKC481S was detected in 48·2% (40/83) of the patients, with 80·0% (32/40) of them showing disease progression during the examined period. In these 32 cases, representing 72·7% (32/44) of all patients experiencing relapse, emergence of the BTKC481S mutation preceded the symptoms of clinical relapse with a median of nine months. Subsequent Bcl-2 inhibition therapy applied in 28/32 patients harbouring BTKC481S and progressing on ibrutinib conferred clinical and molecular remission across the patients. Our study demonstrates the clinical value of sensitive BTKC481S monitoring with the largest longitudinally analysed real-world patient cohort reported to date and validates the feasibility of an early prediction of relapse in the majority of ibrutinib-treated relapsed/refractory CLL patients experiencing disease progression.

Lenalidomide abrogates the survival effect of bone marrow stromal cells in chronic lymphocytic leukemia.

In the pathogenesis of chronic lymphocytic leukemia (CLL) the microenvironment plays an important role, as it produces survival signals and mediates drug resistance. Lenalidomide, which has immunomodulatory effect, can enhance the activation of T-, NK-cells and endothelial cells, however there are no data available whether it can modulate bone marrow stromal cells (BMSCs). In our study, we investigated the effects of lenalidomide on BMSCs and CLL cells. CLL cells were cultured alone or with BMSCs and were treated with lenalidomide. Apoptosis, immunophenotype, and cytokine secretion of BMSCs and CLL cells were determined by flow cytometry. Lenalidomide slightly increased the apoptosis of CLL cells and abrogated the anti-apoptotic effect of BMSCs on CLL cells. Lenalidomide treatment decreased the expression of antigens on CLL cells, which mediate the interactions with the microenvironment. Interestingly, lenalidomide enhanced the expression of IRF4 and the co-stimulatory molecule CD86. The secretion of several cytokines was not changed significantly by lenalidomide. CD49d-negative CLL cases were more sensitive to lenalidomide treatment. Our results suggest that lenalidomide has a limited effect on BMSCs, but it renders CLL cells more immunogenic and unresponsive to survival signals provided by BMSCs.

Identification of a novel resistance mechanism in venetoclax treatment and its prediction in chronic lymphocytic leukemia.

BACKGROUND: The Bcl-2 inhibitor venetoclax has been recently introduced into the treatment of chronic lymphocytic leukemia. Venetoclax is a highly effective drug, however acquired resistance may make long-term treatment challenging. In our study, we present potential novel resistance mechanisms and prognostic markers that are potentially able to predict the early appearance of the resistance. MATERIAL AND METHODS: Repeated complete blood counts, flow cytometric measurements, and physical examinations were performed during the patient follow-up. Clinical and laboratory parameters showed that the patient developed clinical resistance to venetoclax on day 450 of therapy. Resistance mutation analysis (D103Y) and apoptosis arrays from samples at the time of resistance were done. RESULTS: We were able to identify the resistance mutations just a very low variant allele frequency level from the resistant samples. Furthermore we detected increased Bcl-2 expression in peripheral blood (PB), and XIAP overexpression in bone marrow (BM) that could lead to venetoclax resistance. We examined the immunophenotype of CLL cells and recognized that while the expression of CD86 did not change until day 270 of the treatment, since then its expression steadily increased. Moreover, we compared the expression of CD86 in the resistant PB and BM samples and did not find a notable difference between the compartments. CONCLUSION: Our results imply that CLL cells may try to avoid the apoptotic effect of venetoclax through increased CD86 expression by activating antiapoptotic mechanisms. Confirmatory experiments are still required to unequivocally prove that CD86 is a prognostic marker, however, its predictive property during the venetoclax treatment is promising.

Rapamycin Plus Doxycycline Combination Affects Growth Arrest and Selective Autophagy-Dependent Cell Death in Breast Cancer Cells.

Metabolic alteration is characteristic during tumour growth and therapy; however, targeting metabolic rewiring could overcome therapy resistance. mTOR hyperactivity, autophagy and other metabolic processes, including mitochondrial functions, could be targeted in breast cancer progression. We investigated the growth inhibitory mechanism of rapamycin + doxycycline treatment in human breast cancer model systems. Cell cycle and cell viability, including apoptotic and necrotic cell death, were analysed using flow cytometry, caspase activity measurements and caspase-3 immunostainings. mTOR-, autophagy-, necroptosis-related proteins and treatment-induced morphological alterations were analysed by WesTM, Western blot, immunostainings and transmission electron microscopy. The rapamycin + doxycycline combination decreased tumour proliferation in about 2/3rd of the investigated cell lines. The continuous treatment reduced tumour growth significantly both in vivo and in vitro. The effect after short-term treatment was reversible; however, autophagic vacuoles and degrading mitochondria were detected simultaneously, and the presence of mitophagy was also observed after the long-term rapamycin + doxycycline combination treatment. The rapamycin + doxycycline combination did not cause apoptosis or necrosis/necroptosis, but the alterations in autophagy- and mitochondria-related protein levels (LC3-B-II/I, p62, MitoTracker, TOM20 and certain co-stainings) were correlated to autophagy induction and mitophagy, without mitochondria repopulation. Based on these results, we suggest considering inducing metabolic stress and targeting mTOR hyperactivity and mitochondrial functions in combined anti-cancer treatments.

Reduced GLP-1 response to a meal is associated with the CTLA4 rs3087243 G/G genotype.

Although insulitis is the characteristic main feature of type 1 diabetes mellitus (T1DM), many aspects of ß cell loss still remain elusive. Immune dysregulation and alterations in the dipeptidyl-peptidase-4-incretin system might have a role in disease development, but their connection is poorly understood. We assessed the associations of a few selected, immunologically relevant single nucleotide gene variants with the DPP-4-incretin system in individuals with T1DM and in healthy controls. Prandial plasma (total, active) GLP-1 levels, serum DPP-4 activity, CD25 and CTLA-4 expression of T cells and DPP4 rs6741949, CTLA4 rs3087243, CD25 rs61839660 and PTPN2 rs2476601 SNPs were assessed in 33 T1DM patients and 34 age-, gender-, BMI-matched non-diabetic controls without a family history of T1DM. CTLA-4 expression was lower in the Foxp3+CD25+ regulatory T cells from individuals homozygous for the CTLA4 rs3087243-G variant compared to those who carry an A allele. Prandial plasma total GLP-1 levels 45 min after a standardized meal were reduced in individuals homozygous for the CTLA4 rs3087243 G major allele compared to A allele carriers both in the entire study population (with statistical power over 90%) and within the T1DM group. Here we report for the first time a reduced total prandial GLP-1 plasma concentration in individuals with the CTLA4 rs3087243 G/G genotype. One may speculate that immune response-related L cell damage might possibly explain this novel association.

In contrast to high CD49d, low CXCR4 expression indicates the dependency of chronic lymphocytic leukemia (CLL) cells on the microenvironment.

CD49d and CXCR4 are key determinants of interactions between chronic lymphocytic leukemia (CLL) tumor cells and their microenvironment. In this study, we investigated the effect of CD49d and CXCR4 expressions on survival of CLL cells. Primary CLL cells were cultured with CD49d ligand, VCAM-1, or bone marrow stromal cells (BMSCs); then, apoptosis and immunophenotype analyses were performed. VCAM-1 treatment could not induce direct apoptosis protection or immunophenotype change on the CD49d-expressing CLL cells, but resulted in actin reorganization. The BMSC-induced apoptosis protection was independent from the presence of CD49d expression of CLL cells, but showed an inverse correlation with their CXCR4 expression level. We suppose that CD49d contributes to enhanced survival of leukemic cells by mediating migration to the protective microenvironment, not by direct prevention of apoptosis. Moreover, CLL cells with low CXCR4 expression represent a subpopulation that is more dependent on the microenvironmental stimuli for survival, and show increased "death by neglect" when separated from the supportive niche.

The effect of microenvironmental factors on the development of myeloma cells.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of monoclonal plasma cells (PCs) in the bone marrow and other tissues. Although there are several new therapies, MM remains fatal. The interaction between MM cells and the bone marrow microenvironment promotes drug resistance and cancer cells survival. In our present work, we compared the antigen expression pattern of normal and pathological PCs and investigated the possible connections between various surface receptors, adhesion molecules, and recurrent genetic aberrations. We showed that the expression of CD29, CD27, and CD81 is lower in MM cells than in normal PCs. We found correlation of chromosome 11 hyperdiploidity and the decrease of CD27 expression. We demonstrated that MM cells with CD20 positivity also have CD28 expression. Multiple myeloma patients with active CD29 showed better response to treatment. Our results suggest that these changes may result in an alteration of the interaction between stromal cell and MM cell facilitating cell survival and the development of a more aggressive and resistant phenotype.

Low CD23 expression correlates with high CD38 expression and the presence of trisomy 12 in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by a neoplastic B-cell population coexpressing CD5 and CD23; however, the expression of CD23 is variable. In human, two isotypes of CD23 have been identified and related to different functions. The aim of our study was to investigate the relative expression of the two CD23 isotypes in CLL and find possible correlation with other prognostic factors. The expression of CD23 isotypes was analyzed in 54 cases of CLL by polymerase chain reaction (PCR) and quantitative real-time PCR. The immunophenotype of CLL cells was characterized by flow cytometry. We demonstrated a higher CD23a than CD23b expression of CLL cells. Our results also revealed two subsets of CLL cases with a distinct CD23 isotype expression pattern. Thirty-two percent of the cases (group CLL1) showed both low mRNA level of CD23 isotypes and high protein levels of CD20 and CD38 in contrast to group CLL2 with high CD23 mRNA levels. By correlating these results to the presence of prognostic factors determined by fluorescence in situ hybridization, we found that the majority of the cases of group CLL1 (14/17) carried trisomy 12. In summary, our results confirm a high CD23a/CD23b ratio of the CLL cells and demonstrate that in a subset of CLL cases, low CD23 expression together with high CD20 and CD38 expressions may serve as a surrogate for trisomy 12. Copyright © 2015 John Wiley & Sons, Ltd.

[About the association between antipsychotic medication and cardiovascular morbidity: epidemiology and possible background mechanisms]. / Az antipszichotikum kezelés és a kardiovaszkuláris morbiditás közötti kapcsolatról: epidemiológiai összefüggések és azok lehetséges magyarázatai.

The history of antipsychotics began with the discovery of chlorpromazine in the 1950s. Since then this group of medications has become one of the most important element of the armamentarium of psychopharmacology. While initially these pharmacons were used in the treatment of psychotic states (including psychotic mania) in the last approximately 10-15 years new indications - such as treatment of depressive, manic and mixed states and also mood-stabilization in bipolar disorder and also the treatment of major depressive disorder - for several second-generation antipsychotic (SGA) agents have been introduced. Taking this fact into consideration it is not surprising that the market of SGAs has been broadened in several countries. At the same time, safety issues have been raised related to some SGAs, mainly because of their adverse cardiometabolic effects (e.g. weight gain; dyslipidemia; disturbances of glucose metabolism). Related to this, it is worthy of note that the lifespan of patients with serious mental illness (SMIs, such as schizophrenia; bipolar disorder; major depression) is shorter than their healthy counterparts and that somatic comorbidities (mainly cardiovascular disorders) of these patients are primarily responsible for this fact. In this paper, firstly we briefly discuss the history and features of APs then we present data on the shorter than expected lifespan of patients with SMIs and also the possible background mechanisms of it (including the supposed role of AP treatment). Then we provide a short discussion on endothelial progenitor cells (EPC), their role in cardiovascular system and related clinical relevance. Eventually, we also discuss our pilot study with the aim to reveal whether there is any effect of AP therapy on the number of CD34/KDR double-positive EPCs.

Rapamycin can restore the negative regulatory function of transforming growth factor beta 1 in high grade lymphomas.

TGF-ß1 (transforming growth factor beta 1) is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal lymphoid cells. Lymphoma cells often lose their sensitivity to proapoptotic/anti-proliferative regulators such as TGF-ß1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-ß signaling, and through these pathways it is able to enhance TGF-ß induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-ß1 combination on cell growth and on TGF-ß and mTOR signalling events in lymphoma cells. Rapamycin, an inhibitor of mTORC1 (mTOR complex 1) did not elicit apoptosis in lymphoma cells; however, the combination of rapamycin with exogenous TGF-ß1 induced apoptosis and restored TGF-ß1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-ß sensitivity in vitro. In parallel, the phosphorylation of p70 ribosomal S6 kinase (p70S6K) and ribosomal S6 protein, targets of mTORC1, was completely eliminated. Knockdown of Smad signalling by Smad4 siRNA had no influence on apoptosis induced by the rapamycin+TGF-ß1, suggesting that this effect is independent of Smad signalling. However, apoptosis induction was dependent on early protein phosphatase 2A (PP2A) activity, and in part on caspases. Rapamycin+TGF-ß1 induced apoptosis was not completely eliminated by a caspase inhibitor. These results suggest that high mTOR activity contributes to TGF-ß resistance and lowering mTORC1 kinase activity may provide a tool in high grade B-cell lymphoma therapy by restoring the sensitivity to normally available regulators such as TGF-ß1.

Cutaneous lymphocyte-associated antigen as a novel predictive marker of TNF-alpha inhibitor biological therapy in psoriasis.

A considerable number of patients with psoriasis show secondary resistance during long-term TNF-alpha inhibitor therapy, necessitating the identification of reliable predictive markers. Predictive role of cutaneous lymphocyte-associated antigen (CLA) was investigated. Thirty-eight severe patients with psoriasis were treated for a 24-week-long study period. Clinical responsiveness (PASI) and changes in flow cytometry-measured peripheral lymphocyte CLA expression (week 0-2-6) were statistically analysed. Regarding 24-week-long treatment outcome patients were divided into two groups: During the first 6 weeks, mean CLA expression showed significant (P = 0.034604) increase among responders (32/38), while after a preliminary increase, it was significantly (P = 0.012539) decreasing in the relapsing group (6/38). Pearson's correlation analysis showed significant negative correlation between PASI and CLA changes. Responders showed (not significantly) lower initial CLA expression than relapsing patients. Our observations suggest change in CLA expression during the first 6 weeks of induction period to serve as a potential predictive marker of TNF-alpha inhibitor therapy in psoriasis.

Next-Generation Sequencing-Based Genomic Profiling of Children with Acute Myeloid Leukemia.

Pediatric acute myeloid leukemia (AML) represents a major cause of childhood leukemic mortality, with only a limited number of studies investigating the molecular landscape of the disease. Here, we present an integrative analysis of cytogenetic and molecular profiles of 75 patients with pediatric AML from a multicentric, real-world patient cohort treated according to AML Berlin-Frankfurt-Münster protocols. Targeted next-generation sequencing of 54 genes revealed 17 genes that were recurrently mutated in >5% of patients. Considerable differences were observed in the mutational profiles compared with previous studies, as BCORL1, CUX1, KDM6A, PHF6, and STAG2 mutations were detected at a higher frequency than previously reported, whereas KIT, NRAS, and KRAS were less frequently mutated. Our study identified novel recurrent mutations at diagnosis in the BCORL1 gene in 9% of the patients. Tumor suppressor gene (PHF6, TP53, and WT1) mutations were found to be associated with induction failure and shorter event-free survival, suggesting important roles of these alterations in resistance to therapy and disease progression. Comparison of the mutational landscape at diagnosis and relapse revealed an enrichment of mutations in tumor suppressor genes (16.2% versus 44.4%) and transcription factors (35.1% versus 55.6%) at relapse. Our findings shed further light on the heterogeneity of pediatric AML and identify previously unappreciated alterations that may lead to improved molecular characterization and risk stratification of pediatric AML.

Heparin can liberate high molecular weight DNA from secondary necrotic cells.

The borderline between necrosis and apoptosis is indistinct, but that between types of cell death is important because necrosis may lead to local inflammation, whereas apoptosis usually does not. In certain autoimmune disorders, inhibition of cell death is crucial, since macromolecules released from the dead cells may accelerate the autoimmune processes. We have used various cell death inhibitors to block cell death induced by 4HPR [N-(4-hydroxyphenil)-retinamide] the BL41 and U937 cell lines. VD-FMK, a general caspase inhibitor, inhibited DNA fragmentation induced by 4HPR, but not PI (propidium iodide) uptake and necrosis. Interestingly heparin, a serine-protease inhibitor, lowered the PI fluorescence of the dead cell population and increased the sub-G1 population as measured by flow cytometry. Regarding these changes, we found that heparin failed to increase DNA fragmentation, but merely liberated high molecular mass DNA fragments from dead cells. The exact mechanism is unclear, but heparin during secondary necrosis might enter the cells, bind RNPs (ribonucleoproteins), and pull them out with the attached DNA, where they would be sensitive to enzymatic degradation. Thus, the results suggest that heparin treatment helps in the clearance of cell debris and decreases the immunogenity of secondary necrotic cells.

Successful thrombolytic therapy is associated with increased granulocyte CD15 expression and reduced stroke-induced immunosuppression.

OBJECTIVES: Stroke-induced immunosuppression (SIIS) increases the risk of poststroke infections. We aimed to determine whether failed versus successful thrombolytic therapy (TT) resulted in SIIS-associated changes in peripheral granulocyte markers at 1 week following the insult. METHODS: We collected peripheral blood samples from 19 patients with acute ischemic stroke undergoing TT within 6 h after the onset of their first symptoms and 7 days after the insult. Age-matched controls were sampled on one occasion. We compared the expression of CD15 and CD64 on monocytes, granulocytes, and lymphocytes using flow cytometry. RESULTS: The proportion of granulocytes and CD15+ granulocytes was comparable between controls and stroke patients at both time points. While the proportion of CD15bright granulocytes was also comparable, the mean fluorescence intensity (MFI) of CD15 on this subset was reduced in stroke patients by day 7 but was overall higher at both time points compared to controls. The MFI of CD15 on granulocytes was lower in stroke patients with failed TT than in those with successful TT 1 week after the insult. CONCLUSIONS: Our current results indicate that TT may not only acutely reduce the systemic inflammatory response following stroke but may also play a role in reversing SIIS at a later stage following the insult, as reflected by the higher expression of the CD15 marker on granulocytes following successful TT.

Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines.

Folic acid (FA) is a synthetic form of vitamin B9, generally used as a nutritional supplement and an adjunctive medication in cancer therapy. FA is involved in genetic and epigenetic regulation; therefore, it has a dual modulatory role in established neoplasms. We aimed to investigate the effect of short-term (72 h) FA supplementation on colorectal cancer; hence, HT-29 and SW480 cells were exposed to different FA concentrations (0, 100, 10,000 ng/mL). HT-29 cell proliferation and viability levels elevated after 100 ng/mL but decreased for 10,000 ng/mL FA. Additionally, a significant (p ≤ 0.05) improvement of genomic stability was detected in HT-29 cells with micronucleus scoring and comet assay. Conversely, the FA treatment did not alter these parameters in SW480 samples. RRBS results highlighted that DNA methylation changes were bidirectional in both cells, mainly affecting carcinogenesis-related pathways. Based on the microarray analysis, promoter methylation status was in accordance with FA-induced expression alterations of 27 genes. Our study demonstrates that the FA effect was highly dependent on the cell type, which can be attributed to the distinct molecular background and the different expression of proliferation- and DNA-repair-associated genes (YWHAZ, HES1, STAT3, CCL2). Moreover, new aspects of FA-regulated DNA methylation and consecutive gene expression were revealed.

Revealing a Phenotypical Appearance of Ibrutinib Resistance in Patients With Chronic Lymphocytic Leukaemia by Flow Cytometry.

Background: Ibrutinib is widely known as an effective and well-tolerated therapeutical choice of the chronic lymphocytic leukaemia (CLL). However, acquired resistance may occur during the treatment, causing relapse. Early detection of ibrutinib resistance is an important issue, therefore we aimed to find phenotypic markers on CLL cells the expression of which may correlate with the appearance of ibrutinib resistance. Methods: We examined 28 patients' peripheral blood (PB) samples (treatment naïve, ibrutinib sensitive, clinically ibrutinib resistant). The surface markers' expression (CD27, CD69, CD86, CD184, CD185) were measured by flow cytometry. Furthermore, the BTKC481S resistance mutation was assessed by digital droplet PCR. Moreover, the CLL cells' phenotype of a patient with acquired ibrutinib resistance was observed during the ibrutinib treatment. Results: The expression of CD27 (p = 0.030) and CD86 (p = 0.031) became higher in the clinically resistant cohort than in the ibrutinib sensitive cohort. Besides, we found that high CD86 and CD27 expressions were accompanied by BTKC481S mutation. Our prospective study showed that the increase of the expression of CD27, CD69 and CD86 was noticed ahead of the clinical resistance with 3 months. Conclusion: Our study suggests that the changes of the expression of these markers could indicate ibrutinib resistance and the examination of these phenotypic changes may become a part of the patients' follow-up in the future.

Mitotic lymphoma cells are characterized by high expression of phosphorylated ribosomal S6 protein.

Growth factors and mitogens influence signaling pathways and often induce the activity of p70S6 kinase (p70S6K), which in turn phosphorylates the ribosomal S6 protein (S6). Although recent data are rather conflicting, the overall view suggests that phosphorylated S6 is a regulator of global protein synthesis, cell proliferation, cell size and glucose homeostasis. In the present work, emphasis was given to cell cycle-dependent activation of S6 focusing mainly on human lymphoid and lymphoma cells. Paraffin-embedded human tissue blocks from lymph node and different tumor biopsies as well as in vitro cell lines were investigated by immunohistochemistry, immunocytochemistry, flow cytometry and Western blotting using antibodies directed against phospho-S6, phospho-mTOR, phospho-p70S6K and phospho-Histone H3. To enrich the cell number in different phases of the cell cycle, nocodazole, staurosporine or rapamycin were used in cell cultures. We observed strong phospho-S6 positivity by immunostainings in the dividing lymphoid cells of reactive lymph nodes and in lymphoma cells cultured in vitro. Phospho-S6 protein levels were shown to be elevated throughout mitosis in lymphoma cells; however, the high expression of phospho-S6 in mitotic cells was not a general hallmark of tumor cell types studied so far: phospho-S6-negative mitotic cells were detected in several carcinoma and sarcoma biopsies. These observations may have practical implications as they raise the possibility to consider p70S6K and/or S6 as a potential therapeutic target-besides mTOR-in certain lymphomas and perhaps in clinical immunosuppression.