Michael Sheaff

Erythropoietin Protects the Kidney against the Injury and Dysfunction Caused by Ischemia-Reperfusion

Erythropoietin (EPO) is upregulated by hypoxia and causes proliferation and differentiation of erythroid progenitors in the bone marrow through inhibition of apoptosis. EPO receptors are expressed in many tissues, including the kidney. Here it is shown that a single systemic administration of EPO either preischemia or just before reperfusion prevents ischemia-reperfusion injury in the rat kidney. Specifically, EPO (300 U/kg) reduced glomerular dysfunction and tubular injury (biochemical and histologic assessment) and prevented caspase-3, -8, and -9 activation in vivo and reduced apoptotic cell death. In human (HK-2) proximal tubule epithelial cells, EPO attenuated cell death in response to oxidative stress and serum starvation. EPO reduced DNA fragmentation and prevented caspase-3 activation, with upregulation of Bcl-X(L) and XIAP. The antiapoptotic effects of EPO were dependent on JAK2 signaling and the phosphorylation of Akt by phosphatidylinositol 3-kinase. These findings may have major implications in the treatment of acute renal tubular damage.

Secondary neoplasms of the breast

A 90‐year archive of surgical and postmortem material was reviewed to establish the incidence, presentation, and pathology of tumors secondary to the breast.

In vivo Loss of Expression of Argininosuccinate Synthetase in Malignant Pleural Mesothelioma Is a Biomarker for Susceptibility to Arginine Depletion

Purpose: Malignant pleural mesothelioma (MPM) is an increasing health burden on many societies worldwide and, being generally resistant to conventional treatment, has a poor prognosis with a median survival of <1 year. Novel therapies based on the biology of this tumor seek to activate a proapoptotic cellular pathway. In this study, we investigated the expression and biological significance of argininosuccinate synthetase (AS), a rate-limiting enzyme in arginine production. Experimental Design: Initially, we documented down-regulation of AS mRNA in three of seven MPM cell lines and absence of AS protein in four of seven MPM cell lines. We confirmed that the 9q34 locus, the site of the AS gene, was intact using a 1-Mb comparative genomic hybridization array; however, there was aberrant promoter CpG methylation in cell lines lacking AS expression, consistent with epigenetic regulation of transcription. To investigate the use of AS negativity as a therapeutic target, arginine was removed from the culture medium of the MPM cell lines. Results: In keeping with the cell line data, 63% (52 of 82) of patients had tumors displaying reduced or absent AS protein, as assessed using a tissue microarray. Cell viability declined markedly in the AS-negative cell lines 2591 and MSTO but not in the AS-positive cell line, 28. This response was apparent by day 4 and maintained by day 9 in vitro. Arginine depletion induced BAX conformation change and mitochondrial inner membrane depolarization selectively in AS-negative MPM cells. Conclusions: In summary, we have identified AS negativity as a frequent event in MPM in vivo, leading to susceptibility to cytotoxicity following restriction of arginine. A phase II clinical trial is planned to evaluate the role of arginine depletion in patients with AS-negative MPM.

Targeting Arginine-Dependent Cancers with Arginine-Degrading Enzymes: Opportunities and Challenges

Arginine deprivation is a novel antimetabolite strategy for the treatment of arginine-dependent cancers that exploits differential expression and regulation of key urea cycle enzymes. Several studies have focused on inactivation of argininosuccinate synthetase 1 (ASS1) in a range of malignancies, including melanoma, hepatocellular carcinoma (HCC), mesothelial and urological cancers, sarcomas, and lymphomas. Epigenetic silencing has been identified as a key mechanism for loss of the tumor suppressor role of ASS1 leading to tumoral dependence on exogenous arginine. More recently, dysregulation of argininosuccinate lyase has been documented in a subset of arginine auxotrophic glioblastoma multiforme, HCC and in fumarate hydratase-mutant renal cancers. Clinical trials of several arginine depletors are ongoing, including pegylated arginine deiminase (ADI-PEG20, Polaris Group) and bioengineered forms of human arginase. ADI-PEG20 is furthest along the path of clinical development from combinatorial phase 1 to phase 3 trials and is described in more detail. The challenge will be to identify tumors sensitive to drugs such as ADI-PEG20 and integrate these agents into multimodality drug regimens using imaging and tissue/fluid-based biomarkers as predictors of response. Lastly, resistance pathways to arginine deprivation require further study to optimize arginine-targeted therapies in the oncology clinic.

Statistical Validation of the EORTC Prognostic Model for Malignant Pleural Mesothelioma Based on Three Consecutive Phase II Trials

PURPOSEnMalignant pleural mesothelioma (MPM) carries a poor prognosis due to chemoresistance. The European Organisation for Research and Treatment of Cancer (EORTC) prognostic model was reported to predict survival in MPM. Our retrospective analysis set out to test the validity of the model as a prognostic tool in patients treated in three phase II trials at St Bartholomews Hospital (London, United Kingdom) between 1999 and 2003.nnnPATIENTS AND METHODSnA total of 145 patients were treated in three phase II trials; vinorelbine (VIN; 70 patients), vinorelbine/oxaliplatin (VO; 26 patients), and irinotecan/cisplatin/mitomycin C (IPM; 49 patients). Two subgroups, high-risk and low-risk, were defined by EORTC prognostic score (EPS). EPS was determined by a five-parameter model incorporating age, sex, histology, probability of diagnosis, and leukocyte count. An EPS cutoff of less than 1.27 (low risk) or more than 1.27 (high risk) was used to stratify Kaplan-Meier survival curves. Each of the EPS variables exhibited either trends or significant stratification of overall survival (OS).nnnRESULTSnMultivariate analysis confirmed leukocyte count, Eastern Cooperative Oncology Group performance status, and sarcomatous histology as independent prognostic variables. EPS stratified OS in both individual and pooled trial datasets. No association between objective tumor response and EPS classification was identified by multinomial logistic regression. EPS stratified progression-free survival for the VO and IPM cohorts, but not for VIN.nnnCONCLUSIONnThis study validates the EPS system as a robust tool for stratifying small trials into low- and high-risk subgroups. EPS should facilitate patient selection and analysis in randomized clinical trials.

Performance of anal cytology in a clinical setting when measured against histology and high-resolution anoscopy findings.

Objectives:The success of cervical cytology in screening for cervical neoplasia has led to the concept of anal cytology screening for anal neoplasia. Our objective is to study the performance of anal cytology as a screening tool. Design:We assessed anal cytology against histology and high-resolution anoscopy in a clinical setting. Methods:Anal pap test was obtained prior to high-resolution anoscopy examinations and biopsies. The results were analysed against a number of patient variables. Results:From 395 individuals (93% men), 584 anal pap tests were obtained. HIV status was positive in 212 (54%) and negative in 156 (39%) individuals. On the basis of 288 histology results, the sensitivity of anal cytology to detect disease was 70% [95% confidence interval (CI) 64–75], whereas the specificity was 67% (95% CI 38–88). For high-grade disease (anal intraepithelial neoplasia 2/3), sensitivity of anal cytology was 81% (95% CI 70–90), and the negative predictive value was 85% (95% CI 76–92). Sensitivity was dependent on the area of disease (86% for two or more quadrants vs. 69% for one or more quadrants, P = 0.002) and HIV positivity (76% in HIV positive vs. 59% in HIV negative, P = 0.009). Amongst HIV-positive patients, the sensitivity was 90% when CD4 cell count was 400 cells/μl or less compared with 67% when CD4 cell count was above 400 cells/μl (P = 0.005). Conclusion:Anal cytology performs similar to cervical cytology in a clinical setting. Sensitivity of anal smear is dependent on the area (quadrants) of disease present. Sensitivity of anal cytology is enhanced when CD4 cell count is less than 400 cells/μl in HIV-positive men. Our results may explain the variable sensitivity reported in the literature.

The immunophenotype and activation status of the lymphocytic infiltrate in human breast cancers, the role of the major histocompatibility complex in cell-mediated immune mechanisms, and their association with prognostic indicators

BACKGROUNDnThe aims of this study were to characterize, phenotypically, the immune infiltrate in human breast cancers, to assess the activation status of tumor-infiltrating lymphocytes (TIL), and to define the association of these findings with established prognostic indicators.nnnMETHODSnImmunohistochemistry was performed on frozen sections of 60 primary breast cancers by use of monoclonal antibodies to T lymphocytes (CD3), T-helper cells (CD4), cytotoxic T-cells (CD8), natural killer cells (CD56), interleukin-2 receptors (IL-2R), and major histocompatibility (MHC) class I antigen (HLA-ABC) and MHC class II antigen (HLA-DR).nnnRESULTSnAll tumors stained positive for CD3, CD4 and CD8, but with marked variation in the intensity of the infiltrate. In tumors with a moderate infiltrate of TIL, there was a trend toward a greater representation of T-helper cells. However, as the intensity of TIL increased, there was a decline in the proportion of T-helper cells and a concomitant rise in the relative proportion of cytotoxic T cells. There was a relative paucity of natural killer cells. A significant association was found between the intensity of TIL and the number of positive nodes (P=.02) and the intensity of the infiltrate of both T-helper cells and cytotoxic T cells with ER expression (P=.03 and.05, respectively). Most tumors stained positive for IL-2R. The expression of IL-2R was associated with the intensity of TIL (P<.0001), T-helper cells (P<.002), cytotoxic T cells (P=.01) and natural killer cells (P=0.04), and also with the degree of lymph node positivity (P=.02) and histologic tumor grade (P=.05). MHC class II expression was variable, and a large proportion of the tumors showed limited expression in individual cancer cells. There was an association between the expression of HLA-DR in tumor cells and the activation status of TIL (P=.03).nnnCONCLUSIONnAn immune infiltrate is an invariable finding in breast cancers, and the intensity of the infiltrate is greater in node positive tumors. Additionally, TIL may well be activated, albeit partially, in most tumors, suggesting that cell-mediated immune mechanisms are functionally intact.

High pre-transplant soluble CD30 levels are predictive of the grade of rejection.

In renal transplantation, serum soluble CD30 (sCD30) levels in graft recipients are associated with increased rejection and graft loss. We investigated whether pre‐transplant sCD30 concentrations are predictive of the grade of rejection. Pre‐transplant sera of 51 patients with tubulointerstitial rejection (TIR), 16 patients with vascular rejection (VR) and an age‐matched control group of 41 patients with no rejection (NR) were analyzed for sCD30. The transplant biopsies were immunostained for C4d. The median sCD30 level was significantly elevated in the group with VR (248 Units (U)/mL, range: 92–802) when compared with TIR (103 U/mL, range: 36–309, p < 0.001) and NR (179 U/mL, range: 70–343, p < 0.03). Moreover, patients with TIR had significantly lower sCD30 levels compared to NR. Based on C4d staining, a TH2 driven process, the median sCD30 levels were significantly raised in C4d+ patients compared with C4d− group (177 U/mL vs. 120 U/mL, p < 0.05). sCD30 levels measured at time of transplantation correlate with the grade of rejection. High pre‐transplant levels are associated with antibody‐mediated rejection which carries a poorer prognosis. sCD30 could be another tool to assess immunological risk prior to transplantation and enable a patient centered approach to immunosuppression.

Erythropoietin attenuates acute kidney dysfunction in murine experimental sepsis by activation of the β-common receptor

The β-common receptor (βcR) plays a pivotal role in the nonhematopoietic tissue-protective effects of erythropoietin (EPO). Here we determined whether EPO reduces the acute kidney injury (AKI) caused by sepsis and whether this effect is mediated by the βcR. In young (2 months old) C57BL/6 wild-type and βcR knockout mice, lipopolysaccharide caused a significant increase in serum urea and creatinine, hence AKI. This AKI was not associated with any overt morphological alterations in the kidney and was attenuated by EPO given 1u2009h after lipopolysaccharide in wild-type but not in βcR knockout mice. In the kidneys of endotoxemic wild-type mice, EPO enhanced the phosphorylation of Akt, glycogen synthase kinase-3β, and endothelial nitric oxide synthase, and inhibited the activation of nuclear factor-κB. All these effects of EPO were lost in βcR knockout mice. Since sepsis is more severe in older animals or patients, we tested whether EPO was renoprotective in 8-month-old wild-type and βcR knockout mice that underwent cecal ligation and puncture. These older mice developed AKI at 24u2009h, which was attenuated by EPO treatment 1u2009h post cecal ligation and puncture in wild-type mice but not in βcR knockout mice. Thus, activation of the βcR by EPO is essential for the observed reduction in AKI in either endotoxemic young mice or older mice with polymicrobial sepsis, and for the activation of well-known signaling pathways by EPO.

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.