Cardiorespiratory Optimisation By Arteriovenous fistula Ligation after renal Transplantation (COBALT): study protocol for a multicentre randomised interventional feasibility trial.

INTRODUCTION: Cardiovascular events are a major cause of mortality following successful kidney transplantation.Arteriovenous fistulas (AVFs) are considered the best option for haemodialysis, but may contribute to this excess mortality because they promote adverse cardiac remodelling and ventricular hypertrophy. This raises the question whether recipients with a well-functioning kidney transplant should undergo elective AVF ligation. METHODS AND ANALYSIS: The COBALT feasibility study is a multicentre interventional randomised controlled trial (RCT) that will randomise renal transplant patients with stable graft function and a working AVF on a 1:1 basis to standard care (continued conservative management) or to AVF ligation. All patients will perform cardiopulmonary exercise testing (CPET) on recruitment and 6 months later. Daily functioning and quality of life will be additionally assessed by questionnaire completion and objective measure of physical activity. The primary outcome-the proportion of approached patients who complete the study (incorporating rates of consent, receipt of allocated intervention and completion of both CPETs without withdrawal)-will determine progression to a full-scale RCT. Design of the proposed RCT will be informed by an embedded qualitative assessment of participant and healthcare professional involvement. ETHICS AND DISSEMINATION: This study has been approved by the East Midlands-Derby Research Ethics Committee (22/EM/0002) and the Health Research Authority. The results of this work will be disseminated academically through presentation at national and international renal meetings and via open access, peer-reviewed outputs. Existing networks of renal patient groups will also be used to disseminate the study findings to other key stakeholders. TRIAL REGISTRATION NUMBER: ISRCTN49033491.

Estrogen regulation of myokines that enhance osteoclast differentiation and activity.

Osteoporosis and sarcopenia are maladies of aging that negatively affect more women than men. In recent years, it has become apparent that bone and muscle are coupled not only mechanically as muscle pulls on bone, but also at a higher level with myokines, biochemical and molecular signaling occurring between cells of the two tissues. However, how estrogen deficiency in females impacts the chemical crosstalk between bone and muscle cells is not understood. We hypothesize that changes in estrogen signaling alters myokine expression and intensifies bone loss in women. In our present study, we demonstrate that conditioned media from ovariectomized or skeletal muscle deficient in estrogen receptor α (ERα) expression enhances osteoclast differentiation and activity. Using a cytokine array, we identified myokines that have altered expressions in response to loss of estrogen signaling in muscle. Lastly, we demonstrate that conditional deletion of ERα in skeletal muscle results in osteopenia due to an increase in the osteoclast surface per bone surface. Our results suggest that estrogen signaling modulates expression of myokines that regulate osteoclast differentiation and activity.

Phlpp1 is induced by estrogen in osteoclasts and its loss in Ctsk-expressing cells does not protect against ovariectomy-induced bone loss.

Prior studies demonstrated that deletion of the protein phosphatase Phlpp1 in Ctsk-Cre expressing cells enhances bone mass, characterized by diminished osteoclast activity and increased coupling to bone formation. Due to non-specific expression of Ctsk-Cre, the definitive mechanism for this observation was unclear. To further define the role of bone resorbing osteoclasts, we performed ovariectomy (Ovx) and Sham surgeries on Phlpp1 cKOCtsk and WT mice. Micro-CT analyses confirmed enhanced bone mass of Phlpp1 cKOCtsk Sham females. In contrast, Ovx induced bone loss in both groups, with no difference between Phlpp1 cKOCtsk and WT mice. Histomorphometry demonstrated that Ovx mice lacked differences in osteoclasts per bone surface, suggesting that estradiol (E2) is required for Phlpp1 deficiency to have an effect. We performed high throughput unbiased transcriptional profiling of Phlpp1 cKOCtsk osteoclasts and identified 290 differentially expressed genes. By cross-referencing these differentially expressed genes with all estrogen response element (ERE) containing genes, we identified IGFBP4 as potential estrogen-dependent target of Phlpp1. E2 induced PHLPP1 expression, but reduced IGFBP4 levels. Moreover, genetic deletion or chemical inhibition of Phlpp1 was correlated with IGFBP4 levels. We then assessed IGFBP4 expression by osteoclasts in vivo within intact 12-week-old females. Modest IGFBP4 immunohistochemical staining of TRAP+ osteoclasts within WT females was observed. In contrast, TRAP+ bone lining cells within intact Phlpp1 cKOCtsk females robustly expressed IGFBP4, but levels were diminished within TRAP+ bone lining cells following Ovx. These results demonstrate that effects of Phlpp1 conditional deficiency are lost following Ovx, potentially due to estrogen-dependent regulation of IGFBP4.

Tissue selective effects of bazedoxifene on the musculoskeletal system in female mice.

The actions of selective estrogen receptor modulators are tissue dependent. The primary objective of the current study was to determine the tissue selective effects of bazedoxifene (BZA) on the musculoskeletal system of ovariectomized (OVX) female mice, focusing on the strengths of muscle-bone pairs in the lower hindlimb. Treatment with BZA after ovariectomy (OVX+BZA) did not prevent body or fat mass gains (P < 0.05). In vivo plantarflexor muscle isometric torque was not affected by treatment with BZA (P = 0.522). Soleus muscle peak isometric, concentric and eccentric tetanic force production were greater in OVX+BZA mice compared to OVX+E2 mice (P ≤ 0.048) with no effect on maximal isometric specific force (P = 0.228). Tibia from OVX+BZA mice had greater cortical cross-sectional area and moment of inertia than OVX mice treated with placebo (P < 0.001), but there was no impact of BZA treatment on cortical bone mineral density, cortical thickness, tibial bone ultimate load or stiffness (P ≥ 0.086). Overall, these results indicate that BZA may be an estrogen receptor agonist in skeletal muscle, as it has previously been shown in bone, providing minor benefits to the musculoskeletal system.

Loss of myocyte enhancer factor 2 expression in osteoclasts leads to opposing skeletal phenotypes.

Osteoclasts are multinuclear cells that resorb bone. Osteoclast differentiation is regulated by multiple transcription factors which may be acting in a single or multiple factor complex to regulate gene expression. Myocyte enhancer factor 2 (MEF2) is a family of transcription factors whose role during osteoclast differentiation has not been well characterized. Because MEF2A and MEF2D are the family members most highly expressed during osteoclast differentiation, we created conditional knockout mice models for MEF2A and/or MEF2D. In vitro cultures of A- and D-KO osteoclasts were smaller and less numerous than wild type cultures, while AD-KO osteoclasts were almost completely devoid of TRAP positive mononuclear cells. Female A-KO mice are osteopetrotic while male A- and D-KO mice of either sex had no significant in vivo skeletal phenotype, suggesting a sex-specific regulation of osteoclasts by MEF2A. Lastly, in vivo male AD-KO mice are osteopenic, indicating while MEF2 is required for M-CSF and RANKL-stimulated osteoclastogenesis in vitro, osteoclasts can form in the absence of MEF2 in vivo via a RANKL-alternative pathway.

The role of proteoglycans in the reactive stroma on tumor growth and progression.

The stroma surrounding tumors can either restrict or promote tumor growth and progression, and both the cellular and non-cellular components of the stroma play an active role. The cellular components in the surrounding stroma include tumor-associated fibroblasts, host tissue cells and immune cells. The non-cellular components, which form the extracellular matrix (ECM) scaffold, include proteoglycans, collagen, proteinases, growth factors and cytokines. For tumorigenesis to occur it is necessary for tumor cells to modify the surrounding stroma. Tumor cells have mechanisms for achieving this, such as co-opting fibroblasts and modifying the ECM they produce, degrading the surrounding ECM and/or synthesizing a favorable ECM to support invasion. Proteoglycans are an important component of the ECM and play an active role in tumor growth and progression. The expression and glycosylation patterns of proteoglycans are altered in the stroma surrounding tumors and these molecules may support or restrict tumor growth and progression depending on the type and stage of tumor. In the present review we discuss the difference between the tumor promoting and restricting stromal reactions surrounding tumors and the role proteoglycans play.

Integrated genomic analysis identifies recurrent mutations and evolution patterns driving the initiation and progression of follicular lymphoma.

Follicular lymphoma is an incurable malignancy, with transformation to an aggressive subtype representing a critical event during disease progression. Here we performed whole-genome or whole-exome sequencing on 10 follicular lymphoma-transformed follicular lymphoma pairs followed by deep sequencing of 28 genes in an extension cohort, and we report the key events and evolutionary processes governing tumor initiation and transformation. Tumor evolution occurred through either a 'rich' or 'sparse' ancestral common progenitor clone (CPC). We identified recurrent mutations in linker histone, JAK-STAT signaling, NF-κB signaling and B cell developmental genes. Longitudinal analyses identified early driver mutations in chromatin regulator genes (CREBBP, EZH2 and KMT2D (MLL2)), whereas mutations in EBF1 and regulators of NF-κB signaling (MYD88 and TNFAIP3) were gained at transformation. Collectively, this study provides new insights into the genetic basis of follicular lymphoma and the clonal dynamics of transformation and suggests that personalizing therapies to target key genetic alterations in the CPC represents an attractive therapeutic strategy.

Transformation of follicular lymphoma to diffuse large B-cell lymphoma may occur by divergent evolution from a common progenitor cell or by direct evolution from the follicular lymphoma clone.

To investigate the cell of origin linking follicular (FL) and transformed (t-FL) lymphomas, we analyzed the somatic hypermutation (SHM) pattern of the variable region of the immunoglobulin heavy gene (IgH-VH) in 18 sequential FL/t-FL samples and a father (donor) and son (recipient), who developed FL and t-FL, after transplantation. Genealogic trees showed a pattern compatible with a common progenitor cell (CPC) origin in 13 cases. The identification of the t-FL clonotype in the previous FL sample and of the putative CPC sequence in both the FL/t-FL biopsies showed that the intraclonal diversity of FL and t-FL germinal centers (GCs) is more intricate than previously described, and all 3 clonotypes (CPC, FL, t-FL) may occur simultaneously within the same lymph node. On the basis of the father/son model, this CPC must be long-lived, providing a possible explanation for the incurable nature of this disease.

Risk and clinical implications of transformation of follicular lymphoma to diffuse large B-cell lymphoma.

PURPOSE: To study the clinical significance of transformation to diffuse large B-cell lymphoma (DLBCL) in patients with follicular lymphoma (FL). PATIENTS AND METHODS: From 1972 to 1999, 325 patients were diagnosed with FL at St Bartholomew's Hospital (London, United Kingdom). With a median follow-up of 15 years, progression occurred in 186 patients and biopsy-proven transformation in 88 of the 325. The overall repeat biopsy rate was 70%. RESULTS: The risk of histologic transformation (HT) by 10 years was 28%, HT not yet having been observed after 16.2 years. The risk was higher in patients with advanced stage (P = .02), high-risk Follicular Lymphoma International Prognostic Index (FLIPI; P = .01), and International Prognostic Index (IPI; P = .04) scores at diagnosis. Expectant management (as opposed to treatment being initiated at diagnosis) also predicted for a higher risk of HT (P = .008). Older age (P = .005), low hemoglobin level (P = .03), high lactate dehydrogenase (P < .0001), and high-risk FLIPI (P = .01) or IPI (P = .003) score at the time of first recurrence were associated with the diagnosis of HT in a biopsy performed at that time. The median survival from transformation was 1.2 years. Patients with HT had a shorter overall survival (P < .0001) and a shorter survival from progression (P < .0001) than did those in whom it was not diagnosed. CONCLUSION: Advanced stage and high-risk FLIPI and IPI scores at diagnosis correlate with an increased risk of HT. This event strongly influences the outcome of patients with FL by shortening their survival. There may be a subgroup of patients in whom HT does not occur.

Immunohistochemical prognostic markers in diffuse large B-cell lymphoma: validation of tissue microarray as a prerequisite for broad clinical applications--a study from the Lunenburg Lymphoma Biomarker Consortium.

PURPOSE: The results of immunohistochemical class prediction and prognostic stratification of diffuse large B-cell lymphoma (DLBCL) have been remarkably various thus far. Apart from biologic variations, this may be caused by differences in laboratory techniques, scoring definitions, and inter- and intraobserver variations. In this study, an international collaboration of clinical lymphoma research groups from Europe, United States, and Canada concentrated on validation and standardization of immunohistochemistry of the currently potentially interesting prognostic markers in DLBCL. PATIENTS AND METHODS: Sections of a tissue microarray from 36 patients with DLBCL were stained in eight laboratories with antibodies to CD20, CD5, bcl-2, bcl-6, CD10, HLA-DR, MUM1, and MIB-1 according to local methods. The study was performed in two rounds firstly focused on the evaluation of laboratory staining variation and secondly on the scoring variation. RESULTS: Different laboratory staining techniques resulted in unexpectedly highly variable results and very poor reproducibility in scoring for almost all markers. No single laboratory stood out as uniformly poor or excellent. With elimination of variation due to staining, high agreement was found for CD20, HLA-DR, and CD10. Poor agreement was found for bcl-6 and Ki-67. Optimization of techniques and uniformly agreed on scoring criteria improved reproducibility. CONCLUSION: This study shows that semiquantitative immunohistochemistry for subclassification of DLBCL is feasible and reproducible, but exhibits varying rates of concordance for different markers. These findings may explain the wide variation of biomarker prognostic impact reported in the literature. Harmonization of techniques and centralized consensus review appears mandatory when using immunohistochemical biomarkers for treatment stratification.

Transformation of follicular lymphoma to diffuse large B-cell lymphoma proceeds by distinct oncogenic mechanisms.

This study was undertaken to further elucidate the biological mechanisms underlying the frequent event of transformation of follicular lymphoma (FL) to diffuse large B-cell lymphoma (t-FL). The gene expression profiles of 20 paired lymph node biopsies, derived from the same patient pre- and post-transformation, were analysed using the Lymphochip cDNA microarray. TP53 mutation analysis was performed and copy number alterations at the c-REL and CDNK2A examined. Immunohistochemistry was performed on an independent panel of paired transformation paraffin-embedded samples. Transformed follicular lymphoma was predominantly of the germinal centre B-like phenotype both at the mRNA and protein level. Despite this homogeneity, transformation proceeded by at least two pathways. One mechanism was characterised by high proliferation, as assessed by the co-ordinately expressed genes of the proliferation signature. This group was associated with the presence of recurrent oncogenic abnormalities. In the remaining cases, proliferation was not increased and transformation proceeded by alternative routes as yet undetermined. Genes involved in cellular proliferation prevailed amongst those that were significantly increased upon transformation and T cell and follicular dendritic-associated genes predominated amongst those that decreased. t-FL is a germinal centre B (GCB)-like malignancy that evolves by two pathways, one that is similar in proliferation rate to the antecedent FL and the other that has a higher proliferation rate and is characterised by the presence of recognised oncogenic abnormalities.

Number of CD4+ cells and location of forkhead box protein P3-positive cells in diagnostic follicular lymphoma tissue microarrays correlates with outcome.

PURPOSE: To examine the immune microenvironment in diagnostic follicular lymphoma (FL) biopsies and evaluate its prognostic significance. PATIENTS AND METHODS: Immunohistochemistry was used to study numbers and location of cells staining positive for immune cell markers CD4, CD7, CD8, CD25, CD68, forkhead box protein P3 (FOXP3), T-cell intracellular antigen-1, and Granzyme B in tissue microarrays of paraffin-embedded, diagnostic lymph node biopsies taken from 59 FL patients who lived less than 5 years (short-survival group; n = 34) and more than 15 years (long-survival group; n = 25). RESULTS: CD4 and FOXP3 expression were significantly different between the two groups. Samples from the long-survival group were more likely than those from the short-survival group to have CD4+ staining cells and to have FOXP3-positive cells in a perifollicular location. CONCLUSION: This study has identified differences in immune cell composition of the diagnostic FL lymph node immune microenvironment and these have the potential for use as prognostic biomarkers in a routine histopathology setting.

Histopathology, cell proliferation indices and clinical outcome in 304 patients with mantle cell lymphoma (MCL): a clinicopathological study from the European MCL Network.

Mantle cell lymphoma (MCL) is a distinct lymphoma subtype with a particularly poor clinical outcome. The clinical relevance of the morphological characteristics of these tumours remains uncertain. The European MCL Network reviewed 304 cases of MCL to determine the prognostic significance of histopathological characteristics. Cytomorphological subtypes, growth pattern and markers of proliferation (mitotic and Ki-67 indices) were analysed. In addition to the known cytological subtypes, classical (87.5%), small cell (3.6%), pleomorphic (5.9%) and blastic (2.6%), we identified new pleomorphic subgroups with mixtures of cells (classical + pleomorphic type; 1.6%) or transitions (classical/pleomorphic type; 1.6%), which, however, did not differ significantly in overall survival time. Exactly 80.5% of cases displayed a diffuse growth pattern, whereas 19.5% of cases had a nodular growth pattern, which was associated with a slightly more favourable prognosis. A high proliferation rate (mitotic or Ki-67 indices) was associated with shorter overall survival. Cut-off levels were defined that allowed three subgroups with different proliferation rates to be discriminated, which showed significantly different clinical outcomes (P < 0.0001). Based on this large clinicopathological study of prospective clinical trials, multivariate analysis confirmed the central prognostic role of cell proliferation and its superiority to all other histomorphological and clinical criteria.

Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells.

BACKGROUND: Patients with follicular lymphoma may survive for periods of less than 1 year to more than 20 years after diagnosis. We used gene-expression profiles of tumor-biopsy specimens obtained at diagnosis to develop a molecular predictor of the length of survival. METHODS: Gene-expression profiling was performed on 191 biopsy specimens obtained from patients with untreated follicular lymphoma. Supervised methods were used to discover expression patterns associated with the length of survival in a training set of 95 specimens. A molecular predictor of survival was constructed from these genes and validated in an independent test set of 96 specimens. RESULTS: Individual genes that predicted the length of survival were grouped into gene-expression signatures on the basis of their expression in the training set, and two such signatures were used to construct a survival predictor. The two signatures allowed patients with specimens in the test set to be divided into four quartiles with widely disparate median lengths of survival (13.6, 11.1, 10.8, and 3.9 years), independently of clinical prognostic variables. Flow cytometry showed that these signatures reflected gene expression by nonmalignant tumor-infiltrating immune cells. CONCLUSIONS: The length of survival among patients with follicular lymphoma correlates with the molecular features of nonmalignant immune cells present in the tumor at diagnosis.

Post transplant T-cell lymphoma: a case series of four patients from a single unit and review of the literature.

Post-transplant lymphoproliferative disorders (PTLDs) occur in approximately 1% of renal graft recipients. Of these, up to 15 percent are of the T-cell type. In this study, we present four cases of T-cell lymphoma from our renal transplant population, each of whom presented with non-specific symptoms, pancytopenia and/or liver dysfunction, with no obvious lymphadenopathy. They were all diagnosed with rare subsets of T-cell PTLD that included hepato-splenic T-cell lymphoma and anaplastic large cell lymphoma (ALCL). At the time of presentation, the patients were too ill for treatment to be initiated and succumbed to their illness. Increased awareness of this condition may allow for earlier diagnosis and improve its prognosis.

Reliable detection of clonal IgH/Bcl2 MBR rearrangement in follicular lymphoma: methodology and clinical significance.

The prognostic significance of IgH/Bcl2 rearrangement in follicular lymphoma (FL) remains contentious; polymerase chain reaction (PCR) methodology and tissue source variability may account for some inconsistencies. As IgH/Bcl2 major breakpoint region (MBR) sequences may be found in normal blood, an MBR+ result by conventional PCR in blood/bone marrow may not indicate FL. To establish tumour MBR status, 190 lymphoid tissue samples with histologically evident FL (and therefore >1% tumour cells) were examined by real-time quantifiable PCR; 50% (95/190) had clonal MBR IgH/Bcl2 (MBR was considered clonal when >1%). Overall survival (median = 11.5 years) of MBR+ and MBR- patients was not significantly different.

Presentation serum selenium predicts for overall survival, dose delivery, and first treatment response in aggressive non-Hodgkin's lymphoma.

PURPOSE: This study was undertaken to test the hypothesis that serum selenium concentration at presentation correlates with dose delivery, first treatment response, and overall survival in patients with aggressive B-cell non-Hodgkin's lymphoma. PATIENTS AND METHODS: The patients presented between July 1986 and March 1999 and received anthracycline-based chemotherapy, radiotherapy, or both. The total selenium content was retrospectively analyzed in 100 sera, frozen at presentation, using inductively coupled plasma mass spectrometry. RESULTS: The serum selenium concentration ranged from 0.33 to 1.51 micromol/L (mean, 0.92 micromol/L; United Kingdom adult reference range, 1.07 to 1.88 micromol/L). Serum selenium concentration correlated closely with performance status but with no other clinical variable. Multivariate analysis revealed that increased dose delivery, summarized by an area under the curve, correlated positively with younger age (P <.001), advanced stage (P =.001), and higher serum selenium concentration (P =.032). Selenium level also correlated positively with response (odds ratio, 0.62; 95% confidence interval [CI], 0.43 to 0.90; P =.011) and achievement of long-term remission after first treatment (log-rank test, 4.38; P =.036). On multivariate analysis, selenium concentration was positively predictive of overall survival (hazard ratio [HR], 0.76 for 0.2 micromol/L increase; 95% CI, 0.60 to 0.95; P =.018), whereas age indicated negative borderline significance (HR, 1.09; 95% CI, 0.99 to 1.18; P =.066). CONCLUSION: Serum selenium concentration at presentation is a prognostic factor, predicting positively for dose delivery, treatment response, and long-term survival in aggressive non-Hodgkin's lymphoma. Unlike most existing prognostic factors in aggressive non-Hodgkin's lymphoma, selenium supplementation may offer a novel therapeutic strategy in this frequently curable malignancy.

JH probe real-time quantitative polymerase chain reaction assay for Bcl-2/IgH rearrangements.

Follicular lymphoma (FL) characteristically bears the t(14;18)(q32;q21). However, only approximately 75% of the consequent Bcl-2 breakpoints lie within the major breakpoint region (MBR) or the minor cluster region (mcr). While these can be quantified by cluster region-specific real-time quantitative polymerase chain reaction (RQ-PCR), a significant proportion of cases are left requiring a customized approach. Therefore, an RQ-PCR assay for the quantification of Bcl-2/IgH breakpoints has been developed that uses germline JH TaqMan probes and germline JH primers in combination with customized forward primers. Validation of this approach by comparison with an established MBR RQ-PCR showed both techniques to be concordant across a wide range of copy numbers with a sensitivity of five copies per 10(5) cells. In addition, to generate standard curves equating to diverse Bcl-2/IgH rearrangements, a strategy for using placental DNA as a surrogate standard was devised. The performance of the assay in detecting molecular evidence of disease in sequential biopsies from five patients (three with atypical Bcl-2/IgH breakpoints identified by long-range or inverse PCR, one MBR+ and one mcr+) was tested. This alternative approach represents a sensitive and specific means of quantifying common and atypical Bcl-2/IgH rearrangements and maximizes the number of patients with FL suitable for molecular monitoring.

The relative role of peripheral blood and bone marrow for monitoring molecular evidence of disease in follicular lymphoma by quantitative real-time polymerase chain reaction.

Peripheral blood (PB) and bone marrow (BM) are used interchangeably for t(14;18) (IgH/BCL-2) molecular monitoring in follicular lymphoma (FL) and detection of rearrangement after treatment has been correlated to increased risk of relapse. To determine the relative value of each tissue, MBR t(14;18) was quantified by real-time polymerase chain reaction in 52 simultaneous paired PB and BM samples from 38 FL patients. In total, 79% of sample pairs taken in remission (n = 19) or when no morphological disease was evident in the BM (n = 29) had t(14;18) copy number within one log difference and the median difference was small. These findings suggest that, in remission, PB may be adequately monitored. In general, however, higher copy number was detected in BM than in the corresponding PB sample.