Katrina M. Wood

Atypical Haemolytic Uraemic Syndrome Associated with a Hybrid Complement Gene

Background Sequence analysis of the regulators of complement activation (RCA) cluster of genes at chromosome position 1q32 shows evidence of several large genomic duplications. These duplications have resulted in a high degree of sequence identity between the gene for factor H (CFH) and the genes for the five factor H-related proteins (CFHL1–5; aliases CFHR1–5). CFH mutations have been described in association with atypical haemolytic uraemic syndrome (aHUS). The majority of the mutations are missense changes that cluster in the C-terminal region and impair the ability of factor H to regulate surface-bound C3b. Some have arisen as a result of gene conversion between CFH and CFHL1. In this study we tested the hypothesis that nonallelic homologous recombination between low-copy repeats in the RCA cluster could result in the formation of a hybrid CFH/CFHL1 gene that predisposes to the development of aHUS. Methods and Findings In a family with many cases of aHUS that segregate with the RCA cluster we used cDNA analysis, gene sequencing, and Southern blotting to show that affected individuals carry a heterozygous CFH/CFHL1 hybrid gene in which exons 1–21 are derived from CFH and exons 22/23 from CFHL1. This hybrid encodes a protein product identical to a functionally significant CFH mutant (c.3572C>T, S1191L and c.3590T>C, V1197A) that has been previously described in association with aHUS. Conclusions CFH mutation screening is recommended in all aHUS patients prior to renal transplantation because of the high risk of disease recurrence post-transplant in those known to have a CFH mutation. Because of our finding it will be necessary to implement additional screening strategies that will detect a hybrid CFH/CFHL1 gene.

Enhanced Apoptotic Cell Clearance Capacity and B Cell Survival Factor Production by IL-10-Activated Macrophages: Implications for Burkitt’s Lymphoma

Burkitt’s lymphoma (BL) is typified by frequent tumor cell apoptosis and significant macrophage infiltration. Since BL cells have an inherent tendency to undergo apoptosis at a high rate, we reasoned that macrophages in BL are functionally enhanced in at least two activities that have implications for tumor pathogenesis: 1) engulfment of apoptotic cells, an anti-inflammatory process known to suppress immune responses, and 2) production of BL cell survival factors that limit the extent of tumor cell apoptosis. In this study, we show that the microenvironment of BL is rich in the pleiotropic cytokine IL-10, which can be produced by both tumor cells and macrophages, and that IL-10-activated human macrophages have enhanced capacity to engulf apoptotic cells in vitro. This was found to be dependent on the macrophage tethering receptor of apoptotic cells, CD14. Furthermore, IL-10-activated macrophages were found to produce markedly higher levels of the B cell survival factor, B cell-activating factor of the TNF family/B lymphocyte stimulator (BAFF/BLyS) than macrophages matured in the absence of IL-10. Coculture of macrophages with BL cells further enhanced BAFF secretion. Significantly, we show that enhancement of BL cell survival by IL-10-activated macrophages is mediated by a BAFF-dependent component and that BAFF is produced at high levels by tumor-associated macrophages in situ. These results indicate that macrophages, regulated by IL-10, have the potential to promote BL pathogenesis, first, through suppression of antitumor immunity following enhanced engulfment of apoptotic tumor cells and, second, through increased production of tumor cell growth/survival factors.

Hodgkin's disease in the elderly: a population‐based study

Summary. This study evaluated the incidence and outcome of Hodgkins disease (HD) in older patients using a population‐based approach. In total, 102 patients (52 men, 50 women) aged ≥ 60 years presented in the Northern Health Region of England (population of 3·09 million) between 1 January 1991 and 31 December 1998 and were studied prospectively. The age‐specific incidence was 1·97/100 000 for those aged 60–69 years, and 2·18/100 000 for those aged 70 years or over. The median age of the cohort was 70 years (range 60–91) and the median follow up was 63 months (range 20–113). Out of 95 treated patients, 70 (74%) obtained complete or good partial (> 90% response) remissions. In the 60 to 69‐year‐old group, the disease‐specific survival at 5 years was 100% for those presenting with early stage disease and 52% for those with advanced stage disease. In patients aged >70 years the 5 year disease‐specific survival was 36% in patients with early stage and 14% for patients with advanced stage disease. The survival of patients with Epstein–Barr virus (EBV)‐positive tumours was significantly poorer than that of patients with EBV‐negative tumours (P = 0·007); median survival in the former group was 20 months versus undefined in the latter group. In total, 43 deaths were due to progressive HD and five were treatment‐related. This study defined the incidence of HD in our population and demonstrated that the prognosis of elderly patients, particularly those with advanced stage disease, has not improved concurrently with that of patients aged < 60 years old. Novel approaches to assessment and treatment are necessary.

p53 Is a Direct Transcriptional Target of MYCN in Neuroblastoma

MYCN amplification occurs in approximately 25% of neuroblastomas, where it is associated with rapid tumor progression and poor prognosis. MYCN plays a paradoxical role in driving cellular proliferation and inducing apoptosis. Based on observations of nuclear p53 accumulation in neuroblastoma, we hypothesized that MYCN may regulate p53 in this setting. Immunohistochemical analysis of 82 neuroblastoma tumors showed an association of high p53 expression with MYCN expression and amplification. In a panel of 5 MYCN-amplified and 5 nonamplified neuroblastoma cell lines, and also in the Tet21N-regulatable MYCN expression system, we further documented a correlation between the expression of MYCN and p53. In MYCN-amplified neuroblastoma cell lines, MYCN knockdown decreased p53 expression. In Tet21N MYCN+ cells, higher levels of p53 transcription, mRNA, and protein were observed relative to Tet21N MYCN- cells. In chromatin immunoprecipitation and reporter gene assays, MYCN bound directly to a Myc E-Box DNA binding motif located close to the transcriptional start site within the p53 promoter, where it could initiate transcription. E-Box mutation decreased MYCN-driven transcriptional activation. Microarray analysis of Tet21N MYCN+/- cells identified several p53-regulated genes that were upregulated in the presence of MYCN, including MDM2 and PUMA, the levels of which were reduced by MYCN knockdown. We concluded that MYCN transcriptionally upregulates p53 in neuroblastoma and uses p53 to mediate a key mechanism of apoptosis.

Evaluation of treatment outcome in 175 patients with Hodgkin lymphoma aged 60 years or over: the SHIELD study.

The SHIELD program for Hodgkin lymphoma in patients 60 years of age or older, prospectively evaluated clinical features and outcome in a large patient cohort (n = 175). The central element was a phase 2 study of VEPEMB chemotherapy (n = 103, median age 73 years) incorporating comorbidity assessment. A total of 72 other patients were treated off-study but registered prospectively and treated concurrently with: ABVD (n = 35); CLVPP (n = 19), or other (n = 18). Of VEPEMB patients, 31 had early-stage disease (stage 1A/2A) and received VEPEMB 3 times plus radiotherapy. Median follow-up was 36 months. Complete remission (CR) rate (intention-to-treat) was 74% and 3-year overall survival (OS) and progression-free survival (PFS) were 81% and 74%, respectively. A total of 72 patients had advanced-stage disease (stage 1B/2B/3 or 4) and received VEPEMB 6 times. CR rate was 61% with 3-year OS and PFS of 66% and 58%, respectively. Of patients achieving CR, 13% with early-stage and 5% with advanced-stage disease progressed. Overall treatment-related mortality was 7%. In patients treated with curative intent with VEPEMB, ABVD, and CLVPP (n = 157), CR linked to several factors in univariate analysis. In a Cox regression model only, obtaining CR remained significant for OS and CR plus comorbidity and age for PFS. RS-EBV status had no significant effect on outcome.

p53 is nuclear and functional in both undifferentiated and differentiated neuroblastoma.

Aberrant cytoplasmic sequestration has been reported as an alternative mechanism of p53 inactivation to mutation in neuroblastoma. We hypothesized that p53 localization and function in neuroblastoma is related to differentiation status. Eighty-two untreated and 24 paired pre and post-chemotherapy neuroblastomas were studied by immunocytochemistry for p53, p21WAF1, BAX, Bcl2 and Ki67. Predominantly nuclear p53 was detected in undifferentiated neuroblastoma, and both nuclear and cytoplasmic p53 in differentiating neuroblastoma. The nuclear p53 labeling index (LI) correlated with the Ki67 LI (r = 0.51, p

Micro-vessel density and the expression of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PdEGF) in classical Hodgkin lymphoma (HL)

There is little information to date regarding the role of angiogenesis in Hodgkin lymphoma (HL). The present study examines micro-vessel density and the expression of vascular endothelial growth factor (VEGF) and platelet-derived endothelial growth factor (PdEGF) in lymph node biopsies of patients with HL at presentation and relapse. Using immunohistochemistry, the degree of new blood vessel formation and the expression of VEGF and PdEGF was assessed in Hodgkin-rich tissue. The micro-vessel density (MVD) increased with disease progression in seven out of 11 cases. Expression of VEGF was observed in endothelial cells (EC) of some micro-vessels and also in follicular dendritic cells. The Hodgkin/Reed-Sternberg (H-RS) cells as well as the inflammatory lymphocytes were negative for VEGF. Cytoplasmic or cytoplasmic and nuclear expression of PdEGF by the H-RS cells was observed in five of the 11 presentation cases. The expression of PdEGF increased with disease progression in seven cases. In conclusion, Hodgkin tissue shows prominent vascularization. The increased MVD and PdEGF expression with disease progression merits further investigation.

Histological Profile of Tumours from MYCN Transgenic Mice

Background: MYCN is the most commonly amplified gene in human neuroblastomas. This proto-oncogene has been overexpressed in a mouse model of the disease in order to explore the role of MYCN in this tumour. Aims: To report the histopathological features of neuroblastomas from MYCN transgenic mice. Methods: 27 neuroblastomas from hemizygous transgenic mice and four tumours from homozygous mice were examined histologically; Ki67 and MYCN immunocytochemistry was performed in 24 tumours. Results: Tumours obtained from MYCN transgenic mice resembled human neuroblastomas, displaying many of the features associated with stroma-poor neuroblastoma, including heterogeneity of differentiation (but no overt ganglionic differentiation was seen), low levels of Schwannian stroma and a high mitosis karyorrhexis index. The tumours had a median Ki67 labelling index of 70%; all tumours expressed MYCN with a median labelling index of 68%. The most striking difference between the murine and human neuroblastomas was the presence of tingible body macrophages in the transgenic mouse tumours reflecting high levels of apoptosis. This has not previously been described in human or other murine neuroblastoma models. Conclusions: These studies highlight the histological similarities between tumours from MYCN transgenic mice and human neuroblastomas, and reaffirm their role as a valuable model to study the biology of aggressive human neuroblastoma.

Inhibition of lysosomal protease cathepsin D reduces renal fibrosis in murine chronic kidney disease

During chronic kidney disease (CKD) there is a dysregulation of extracellular matrix (ECM) homeostasis leading to renal fibrosis. Lysosomal proteases such as cathepsins (Cts) regulate this process in other organs, however, their role in CKD is still unknown. Here we describe a novel role for cathepsins in CKD. CtsD and B were located in distal and proximal tubular cells respectively in human disease. Administration of CtsD (Pepstatin A) but not B inhibitor (Ca074-Me), in two mouse CKD models, UUO and chronic ischemia reperfusion injury, led to a reduction in fibrosis. No changes in collagen transcription or myofibroblasts numbers were observed. Pepstatin A administration resulted in increased extracellular urokinase and collagen degradation. In vitro and in vivo administration of chloroquine, an endo/lysosomal inhibitor, mimicked Pepstatin A effect on renal fibrosis. Therefore, we propose a mechanism by which CtsD inhibition leads to increased collagenolytic activity due to an impairment in lysosomal recycling. This results in increased extracellular activity of enzymes such as urokinase, triggering a proteolytic cascade, which culminates in more ECM degradation. Taken together these results suggest that inhibition of lysosomal proteases, such as CtsD, could be a new therapeutic approach to reduce renal fibrosis and slow progression of CKD.

Follicular dendritic cell tumour in a 9-year-old child

Follicular dendritic cell tumour (FDCT) or sarcoma is a rare tumour first described in 1986. Some 80 cases have been reported, the youngest being in teenagers. Our patient first presented at 9 years of age with a cervical mass that was removed and revealed an apparently benign, but florid reactive process. At age 14 the lump recurred and biopsy was diagnostic of FDCT. Radical block dissection showed disease to level III and 6 weeks of radiotherapy was followed by 6 months adjuvant chemotherapy. Three years after completing his final treatment he shows no signs of recurrent disease.