Allelic loss of cyclophilin 40, an estrogen receptor-associated immunophilin, in breast carcinomas

Purpose: Cyclophilin 40 (CyP40) is an estrogen receptor-associated protein which appears to modify receptor function. The aim of this study was to determine the extent of allelic loss at the CyP40 locus in a panel of breast carcinomas using a newly characterized microsatellite marker located upstream of the CyP40 gene and then to correlate this with losses at chromosomal sites for cancer-associated genes. Methods: Allelic loss at CyP40 was determined from patients' matched tumor and normal breast tissue using Genescan 672 software analysis of fluorescently labeled, PAGE-separated PCR products incorporating the marker. For each patient, allelic loss at CyP40 was then assessed and compared with losses at markers for various cancer-associated genes. Results: Allelic loss was detected in 30% of breast carcinomas from patients heterozygous for the CyP40 marker. All carcinomas demonstrating allelic loss were grade II or III invasive ductal carcinomas and generally showed multiple losses at other sites near known cancer-associated genes. Conclusions: The polymorphic marker which we characterized was useful in determining allelic loss at the CyP40 locus in breast cancer patients and when applied in these studies in conjunction with various cancer-associated gene markers, suggests that deletions in the region of the CyP40 gene might be a late event in breast tumor progression.

Immunosuppressive effects of melanoma-derived heavy-chain ferritin are dependent on stimulation of IL-10 production

Cultured melanoma cells release soluble factors that influence immune responses. Screening of a cDNA library with anti-sera from a melanoma patient identified an immunoreactive plaque, which encoded heavy-chain ferritin (H-ferritin), Previous studies have drawn attention to the immunosuppressive effects of this molecule and prompted further studies on its biochemical and functional properties in human melanoma, These studies demonstrated, firstly, that H-ferritin appeared to be secreted by melanoma cells, as shown by immunoprecipitation of a 21.5 kDa band from supernatants. It was also detected in extracts of melanoma cells by Western blotting as 43 and 64 kDa dimers and trimers of the 21.5 kDa fraction. Secondly, flow-cytometric analysis of H- and light-chain ferritin (L-ferritin) expression on melanoma showed a wide variation in L-ferritin expression and consequently of the ratio of H- to L-ferritin expression. Suppression of mitogenic responses of lymphocytes to anti-CD3 showed a correlation with the ratio of H- to L-ferritin in the supernatants and was specific for H-ferritin, as shown by inhibition studies with a monoclonal antibody (MAb) against H-ferritin, Similar results were obtained with H- and L-ferritin from other sources. Suppression of mitogenic responses of lymphocytes to anti-CD3 by H-ferritin was inhibited using a MAb against IL-IO, which suggested that the immunosuppressive effect of H-ferritin was mediated by IL-IO, Assays of cytokine production from anti-CD3-stimulated lymphocytes showed that H-ferritin markedly increased production of IL-10 and IFN-gamma and had only slight effects on IL-2 and IL-4 production, Our results suggest that melanoma cells may be a major source of H-ferritin and that production of the latter may account for some of the immunosuppressive effects of melanoma, (C) 2001 Wiley-Liss. Inc.

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by Smac/DIABLO release from mitochondria

In previous studies we have shown that the sensitivity of melanoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)induced apoptosis was determined largely by the level of expression of death receptor TRAIL receptor 2 on the cells. However, approximately one-third of melanoma cell lines were resistant to TRAIL, despite expression of high levels of TRAIL receptor 2. The present studies show that these cell lines had similar levels of TRAIL-induced activated caspase-3 as the TRAIL-sensitive lines, but the activated caspase-3 did not degrade substrates downstream of caspase-3 [inhibitor of caspase-activated DNase and poly(ADP-ribose) polymerase]. This appeared to be due to inhibition of caspase-3 by X-linked inhibitor of apoptosis (XIAP) because XIAP was bound to activated caspase-3, and transfection of XIAP into TRAIL-sensitive cell lines resulted in similar inhibition of TRAIL-induced apoptosis. Conversely, reduction of XIAP levels by overexpression of Smac/ DIABLO in the TRAIL-resistant melanoma cells was associated with the appearance of catalytic activity by caspase-3 and increased TRAIL-induced apoptosis. TRAIL was shown to cause release of Smac/DIABLO from mitochondria, but this release was greater in TRAIL-sensitive cell lines than in TRAIL-resistant cell lines and was associated with downregulation of XIAP levels. Furthermore, inhibition of Smac/DIABLO release by overexpression of Bcl-2 inhibited down-regulation of XIAP levels. These results suggest that Smac/DIABLO release from mitochondria and its binding to XIAP are an alternative pathway by which TRAIL induces apoptosis of melanoma, and this pathway is dependent on the release of activated caspase-3 from inhibition by XIAP and possibly other inhibitor of apoptosis family members.

Treatment implications of a positive sentinel lymph node biopsy for patients with early-stage breast carcinoma

BACKGROUND. Sentinel lymph node (SLN) mapping and biopsy is emerging as an alternative to axillary lymph node dissection (ALND) in determining the lymph node status of patients with early-stage breast carcinoma. The hypothesis of the technique is that the SLN is the first lymph node in the regional lymphatic basin that drains the primary tumor. Non-SLN (NSLN) metastasis in the axilla is unlikely if the axillary SLN shows no tumor involvement, and, thus, further axillary interference may be avoided. However, the optimal treatment of the axilla in which an SLN metastasis is found requires ongoing evaluation. The objectives of this study were to evaluate the predictors for NSLN metastasis in the presence of a tumor-involved axillary SLN and to examine the treatment implications for patients with early-stage breast carcinoma. METHODS. Between June 1998 and May 2000, 167 patients participated in the pilot study of SLN mapping and biopsy at Westmead Hospital. SLNs were identified successfully and biopsied in 140 axillae. All study patients also underwent ALND. The incidence of NSLN metastasis in the 51 patients with a SLN metastasis was correlated with clinical and pathologic characteristics. RESULTS. Of 51 patients with a positive SLN, 24 patients (47%) had NSLN metastases. The primary tumor size was the only significant predictor for NSLN involvement. NSLN metastasis occurred in 25% of patients (95% confidence interval [95%CI], 10-47%) with a primary tumor size less than or equal to 20 mm and in 67% of patients (95%CI, 46-83%) with a primary tumor size > 20 mm (P = 0.005). The size of the SLN metastasis was not associated significantly with NSLN involvement. Three of 7 patients (43%) with an SLN micrometastasis (< 1 mm) had NSLN involvement compared with 38 of 44 patients (48%) with an SLN macrometastasis (greater than or equal to 1 mm). CONCLUSIONS. The current study did not identify a subgroup of SLN positive patients in whom the incidence of NSLN involvement was low enough to warrant no further axillary interference. At present, a full axillary dissection should be performed in patients with a positive SLN. (C) 2001 American Cancer Society.

Weight reduction improves biochemistry and histology in patients with chronic hepatitis C

Hepatitis C virus (HCV) is a major cause of chronic liver disease that may progress to cirrhosis. Antiviral treatment is successful in less than 50% of patients, is costly and causes debilitating side effects. For these reasons, additional therapies to limit the progression of liver disease are urgently required. Steatosis is found in 60% of patients with HCV and is strongly associated with more severe fibrosis. Improvements in biochemical parameters may be seen with weight reduction, however the effects on liver histology have not been investigated. We propose that in patients with chronic HCV and steatosis, obesity contributes to fat in the liver, which results in increased fibrosis and progression to cirrhosis. This study investigated the effect of weight reduction on liver biochemistry and histology in patients with HCV and the success of weight maintenance after an intensive intervention. We examined the effect of a 12 week diet and exercise program where all subjects were seen weekly by the Dietician, with the goal of achieving a 0.5 kg weight loss per week. Biochemistry was monitored monthly and a liver biopsy was performed prior to and 3-6 months after the intervention period. Patients then entered a 12 month weight maintenance program with monthly dietetic review. After 12 weeks there was a mean weight loss of 5.9 ± 3.2 kg and a mean reduction in waist circumference of 9.0 ± 5.0 cm. In 16 of the 19 patients, serum ALT levels fell progressively with weight loss. Mean fasting insulin fell from 16 to 11 mmol/L (p

Long-term survival of women with breast cancer in New South Wales

Several long-term studies of breast cancer survival have shown continued excess mortality from breast cancer up to 20-40 years following treatment. The purpose of this report was to investigate temporal trends in long-term survival from breast cancer in all New South Wales (NSW) women. Breast cancer cases incident in 1972-1996 (54,228) were derived from the NSW Central Cancer Registry a population-based registry which began in 1972. All cases of breast cancer not known to be dead were matched against death records. The expected survival for NSW women was derived from published annual life tables. Relative survival analysis compared the survival of cancer cases with the age, sex and period matched mortality of the total population. Cases were considered alive at the end of 1996, except when known to be dead. Proportional hazards regression was employed to model survival on age, period and degree of spread at diagnosis. Survival at 5, 10, 15, 20 and 25 years of follow-up was 76 per cent, 65 per cent, 60 per cent, 57 per cent and 56 per cent. The annual hazard rate for excess mortality was 4.3 per cent in year 1, maximal at 6.5 per cent in year 3, declining to 4.7 per cent in year 5, 2.7 per cent in year 10, 1.4 per cent in year 15, 1.0 per cent for years 16-20, and 0.4 per cent for years 20-25 of follow-up. Relative survival was highest in 40-49 year-olds. Cases diagnosed most recently (1992-1996) had the highest survival, compared with cases diagnosed in previous periods. Five-year survival improved over time, especially from the late 1980s for women in the screening age group (50-69 years). Survival was highest for those with localised cancer at diagnosis: 88.4 per cent, 79.1 per cent, 74.6 per cent, 72.7 per cent and 72.8 per cent at 5, 10, 15, 20 and 25 years follow-up (excluding those aged greater than or equal to 70 years). There was no significant difference between the survival of the breast cancer cases and the general population at 20-25 years follow-up. Degree of spread was less predictive of survival 5-20 years after diagnosis, compared with 0-5 years after diagnosis, and was not significant at 20-25 years of follow-up. Relative survival from breast cancer in NSW women continues to decrease to 25 years after diagnosis, but there is little excess mortality after 15 years follow-up, especially for those with localised cancer at diagnosis, and the minimal excess mortality at 20-25 years of follow-up is not statistically significant. (C) 2002 Elsevier Science Ltd. All rights reserved.

Cancer knowledge and skills of interns in Australia and New Zealand in 2001: comparison with 1990, and between course types

Objective: To compare the cancer knowledge and skills of interns in 2001 who graduated from graduate medical program (GMP) courses with those from non-GMP courses, and to compare the cancer knowledge and skills of interns in 2001 with those who completed a similar survey in 1990. Design: Questionnaire survey of recently graduated interns in a random sample of Australian and New Zealand hospitals. The questionnaire was designed to allow direct comparison with the 1990 survey, and was guided by the Australian Cancer Society's Ideal Oncology Curriculum for Medical Schools. Results: 443 interns completed the survey (response rate, 62%; 42 were excluded, leaving 401 surveys for analysis: 118 from GMP courses and 283 from non-GMP courses). Interns from GMP courses felt more competent than those from non-GMP courses at discussing death (P= 0.02), breaking bad news (P= 0.04) and advising on smoking cessation (P= 0.02), but less competent at preparing a patient for a hazardous procedure (P= 0.02). Mote GMP interns would refer a breast cancer patient to a multidisciplinary clinic (83% versus 70%; P= 0.03). Knowledge about cancer risks and prognosis was significantly less in GMP interns, but GMP interns rated their clinical skills, such as taking a Pap smear, higher than non-GMP interns. The GMP and non-GMP groups did not differ in their exposure to cancer patients, but compared with 1990 interns recent graduates had less exposure to patients with cancer. Conclusions: GMP curricula appear to have successfully introduced new course material and new methods of teaching, but have not always succeeded in producing doctors with better knowledge about cancer. Recent graduates have less exposure to cancer patients than those who trained 10 years ago.

Constitutively Active Mutant D816VKit Induces Megakayocyte and Mast Cell Differentiation of Early Haemopoietic Cells from Murine Foetal Liver

Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF. (C) 2003 Elsevier Science Ltd. All rights reserved.

Association of increased levels of heavy-chain ferritin with increased CD4(+) CD25(+) regulatory T-Cell levels in patients with melanoma

We have shown previously that melanoma cells in culture release heavy-chain ferritin (H-Ferritin) into supernatants and that this is responsible for the suppression of responses of peripheral blood lymphocytes stimulated by anti-CD3. These effects were mediated by activation of regulatory T cells to produce interleukin (IL)-10. In the present study, we examined whether a similar relation might exist between levels of H-Ferritin and activation of regulatory T cells in patients with melanoma. Ferritin levels were evaluated by ELISA and regulatory T-cell numbers were assessed by three-color flow cytometry to identify CD4(+) CD25(+) CD69(-) T cells. CD69 positive cells were excluded to avoid inclusion of normal activated CD4, CD25 expressing T cells. Measurements of H- and light-chain (L)-Ferritin by ELISA revealed that H- but not L-Ferritin was elevated in the circulation of melanoma patients. In addition, these studies revealed a marked increase in the number of CD4+ CD25+ CD69- T cells in such patients, compared with age-matched controls. The ratio of H-Ferritin:L-Ferritin correlated with the levels of regulatory T cells consistent with a causal relation between unbound H-Ferritin levels and the activation of regulatory T cells. H-Ferritin or regulatory T cells did not, however, correlate with the stage of the melanoma. These results provide evidence for the importance of H-Ferritin in the induction of regulatory T cells in patients with melanoma and provide additional insight into the suppression of immune responses in such patients.

EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human orthologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs' and encodes a HECT domain E3 ubiquitin protein-ligase. To investigate the possible involvement of EDD in human cancer, several cancers from diverse tissue sites were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus using an EDD-specific microsatellite, CEDD, and other polymorphic microsatellites mapped in the vicinity of the 8q22.3 locus. Of 143 cancers studied, 38 had AI at CEDD (42% of 90 informative cases). In 14 of these cases, discrete regions of imbalance encompassing 8q22.3 were present, while the remainder had more extensive 8q aberrations. AI of CEDD was most frequent in ovarian cancer (22/47 informative cases, 47%), particularly in the serous subtype (16/22, 73%), but was rare in benign and borderline ovarian tumours. AI was also common in breast cancer (31%), hepatocellular carcinoma (46%), squamous cell carcinoma of the tongue (50%) and metastatic melanoma (18%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers, while among breast cancer cell lines EDD overexpression and increased gene copy number were correlated. These results demonstrate that AI at the EDD locus is common in a diversity of carcinomas and that the EDD gene is frequently overexpressed in breast and ovarian cancer, implying a potential role in cancer progression.

Myb-binding Protein 1a is a Nucleocytoplasmic Shuttling Protein that Utilizes CRM1-dependent and Independent Nuclear Export Pathways

Myb-binding protein 1a (Mybbp1a) is a novel nuclear protein localized predominantly, but not exclusively, in nucleoli. Although initially isolated as a c-Myb interacting protein, Mybbp1a is expressed ubiquitously, associates with a number of different transcription factors, and may play a role in both RNA polymerase I- and II-mediated transcriptional regulation. However, its precise function remains unclear. In this study we show that Mybbp1a is a nucleocytoplasmic shuttling protein and investigate the mechanisms responsible for both nuclear import and export. The carboxyl terminus of Mybbp1a, which contains seven short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization, and this activity can be transferred to a heterologous protein. Deletion mapping demonstrated that these repeat sequences appear to act incrementally, with successive deletions resulting in a corresponding increase in the proportion of protein localized in the cytoplasm. Glutathione S-transferase pulldown experiments showed that the nuclear receptor importin-alpha/beta mediates Mybbp1a nuclear import. Interspecies heterokaryons were used to demonstrate that Mybbp1a was capable of shuttling between the nucleus and the cytoplasm. Deletion analysis and in vivo export studies using a heterologous assay system identified several nuclear export sequences which facilitate Mybbp1a nuclear export of Mybbp1a by CRM1-dependent and -independent pathways. (C) 2003 Elsevier Science (USA). All rights reserved.

Client satisfaction and empowerment through social work intervention

This Study examines the utility of satisfaction questionnaires in gauging the effectiveness of social work services in a paediatric hospital setting. Participants completed an empowerment scale before seeing a social worker. Approximately four weeks later, participants completed the empowerment scale again, at which time they also completed a satisfaction questionnaire. The difference between the pre- and post-test empowerment scores was compared with the satisfaction scores, and the influence of some demographic and intervention variables was examined. The results indicated that there was no significant relationship between participants' reported level of satisfaction with the social work service provided and the change in participants' empowerment scores before and after intervention. Most demographic and intervention. variables tested did not yield any significant associations with satisfaction or change in empowerment. However, it was found that those who received both counselling and practical assistance (rather than only one or the other) and those with a higher level of education were more likely to report an increase in their level of empowerment after receiving social work intervention. This study lends further support to the contention that satisfaction questionnaires alone may not provide reliable information with regard to the utility and effectiveness of paediatric hospital social work intervention.