Age at diagnosis predicts local recurrence in women treated with breast-conserving surgery and postoperative radiation therapy for ductal carcinoma in situ: a population-based outcomes analysis.

PURPOSE: The main goal of treating ductal carcinoma in situ (dcis) is to prevent the development of invasive breast cancer. Most women are treated with breast-conserving surgery (bcs) and radiotherapy. Age at diagnosis may be a risk factor for recurrence, leading to concerns that additional treatment may be necessary for younger women. We report a population-based study of women with dcis treated with bcs and radiotherapy and an evaluation of the effect of age on local recurrence (lr). METHODS: All women diagnosed with dcis in Ontario from 1994 to 2003 were identified. Treatments and outcomes were collected through administrative databases and validated by chart review. Women treated with bcs and radiotherapy were included. Survival analyses were performed to evaluate the effect of age on outcomes. RESULTS: We identified 5752 cases of dcis; 1607 women received bcs and radiotherapy. The median follow-up was 10.0 years. The 10-year cumulative lr rate was 27% for women younger than 45 years, 14% for women 45-50 years, and 11% for women more than 50 years of age (p < 0.0001). The 10-year cumulative invasive lr rate was 22% for women younger than 45 years, 10% for women 45-50 years, and 7% for women more than 50 years of age (p < 0.0001). On multivariate analyses, young age (<45 years) was significantly associated with lr and invasive lr [hazard ratio (hr) for lr: 2.6; 95% confidence interval (ci): 1.9 to 3.7; p < 0.0001; hr for invasive lr: 3.0; 95% ci: 2.0 to 4.4; p < 0.0001]. An age of 45-50 years was also significantly associated with invasive lr (hr: 1.6; 95% ci: 1.0 to 2.4; p = 0.04). CONCLUSIONS: Age at diagnosis is a strong predictor of lr in women with dcis after treatment with bcs and radiotherapy.

Role of Nek2 on centrosome duplication and aneuploidy in breast cancer cells.

Breast cancer is the most common solid tumor and the second most common cause of death in women. Despite a large body of literature and progress in breast cancer research, many molecular aspects of this complex disease are still poorly understood, hindering the design of specific and effective therapeutic strategies. To identify the molecules important in breast cancer progression and metastasis, we tested the in vivo effects of inhibiting the functions of various kinases and genes involved in the regulation/modulation of the cytoskeleton by downregulating them in mouse PyMT mammary tumor cells and human breast cancer cell lines. These kinases and cytoskeletal regulators were selected based on their prognostic values for breast cancer patient survival. PyMT tumor cells, in which a selected gene was stably knocked down were injected into the tail veins of mice, and the formation of tumors in the lungs was monitored. One of the several genes found to be important for tumor growth in the lungs was NIMA-related kinases 2 (Nek2), a cell cycle-related protein kinase. Furthermore, Nek2 was also important for tumor growth in the mammary fat pad. In various human breast cancer cell lines, Nek2 knockdown induced aneuploidy and cell cycle arrest that led to cell death. Significantly, the breast cancer cell line most sensitive to Nek2 depletion was of the triple negative breast cancer subtype. Our data indicate that Nek2 has a pivotal role in breast cancer growth at primary and secondary sites, and thus may be an attractive and novel therapeutic target for this disease.

PTPN12 promotes resistance to oxidative stress and supports tumorigenesis by regulating FOXO signaling.

It is well known that protein tyrosine phosphatases (PTPs) that become oxidized due to exposure to reactive oxygen species (ROS) undergo a conformational change and are inactivated. However, whether PTPs can actively regulate ROS levels in order to prevent PTP inhibition has yet to be investigated. Here, we demonstrate that PTP non-receptor type 12 (PTPN12) protects cells against aberrant ROS accumulation and death induced by oxidative stress. Murine embryonic fibroblasts (MEFs) deficient in PTPN12 underwent increased ROS-induced apoptosis under conditions of antioxidant depletion. Cells lacking PTPN12 also showed defective activation of FOXO1/3a, transcription factors required for the upregulation of several antioxidant genes. PTPN12-mediated regulation of ROS appeared to be mediated by phosphoinositide-dependent kinase-1 (PDK1), which was hyperstimulated in the absence of PTPN12. As tight regulation of ROS to sustain survival is a key feature of cancer cells, we examined PTPN12 levels in tumors from a cohort of breast cancer patients. Patients whose tumors showed high levels of PTPN12 transcripts had a significantly poorer prognosis. Analysis of tissues from patients with various breast cancer subtypes revealed that more triple-negative breast cancers, the most aggressive breast cancer subtype, showed high PTPN12 expression than any other subtype. Furthermore, both human breast cancer cells and mouse mammary epithelial tumor cells engineered to lack PTPN12 exhibited reduced tumorigenic and metastatic potential in vivo that correlated with their elevated ROS levels. The involvement of PTPN12 in the antioxidant response of breast cancer cells suggests that PTPN12 may represent a novel therapeutic target for this disease.

Porcine circovirus type 2 infection before and during an outbreak of postweaning multisystemic wasting syndrome on a pig farm in the UK.

The presence of porcine circovirus type 2 (PCV-2) and other pathogens before and during an outbreak of postweaning multisystemic wasting syndrome (PWMS) in pigs is evaluated in this study. At the time of the outbreak on a large commercial pig farm in the UK, serum samples and data were collected in two independent on-going research projects, one in weaned pigs and the other in sows. Serum samples of growing pigs and sows were PCV-2-antibody and PCR positive before and during the PMWS outbreak. Upon sequencing, PCV-2 isolates collected before the outbreak were identified as PCV-2a, and isolates collected during the outbreak were identified as PCV-2b, suggesting a shift of PCV-2 genotypes present on the farm. Pigs in the weaner study were from sows originating from different breeders and an association of sow origin and PCV-2 serostatus in offspring was found. Further, pigs had higher odds to be PCV-2 antigen positive if the sow was PCV-2 antibody positive around farrowing, the sow was of higher parity, and were less likely to test antigen positive if the sow was sourced from a particular breeder. The findings of this study highlight the potential role of the immune status of the sow on the occurrence of PMWS.

Mitochondrial D310 mutations in the early development of breast cancer.

BACKGROUND: The role of mitochondrial DNA (mtDNA) mutations in the development of breast cancer is largely unknown. In this study, we investigated the frequency and pattern of mutations in the D310 region, the most commonly mutated region in mtDNA, in a series of breast lesions. METHODS: Using capillary electrophoresis, we genotyped the D310 sequence of neoplastic epithelial cells from 23 patients with synchronous ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC), 26 patients with IDC only and 29 patients with DCIS only. RESULTS: A majority of DCIS (68.4%) and IDC (71.4%) lesions harbour different D310 sequences compared with their matched normal control. Specific D310 sequences were more frequently identified in tumour samples (77.1% of DCIS and 75.5% of IDC) compared with normal tissues (35.3% of normal; P<0.0001). No difference was identified between DCIS lesions with synchronous IDC and those from pure DCIS cases. In five cases, histologically normal tissue adjacent to tumour was found to share D310 sequences with the tumour, while normal tissue taken further away did not. CONCLUSION: Although D310 alterations do not seem to be related to DCIS progression, they were found in histologically normal cells adjacent to tumour. This suggests a field of genetically altered cells, thus D310 mutations could represent a potential marker for the clonal expansion of premalignant breast cancer cells.

Acquisition of metastatic tissue from patients with bone metastases from breast cancer.

Biopsies of metastatic tissue are increasingly being performed. Bone is the most frequent site of metastasis in breast cancer patients, but bone remains technically challenging to biopsy. Difficulties with both tissue acquisition and techniques for analysis of hormone receptor status are well described. Bone biopsies can be carried out by either by standard posterior iliac crest bone marrow trephine/aspiration or CT-guided biopsy of a radiologically evident bone metastasis. The differential yield of these techniques is unknown. Results from three prospective studies of similar methodology were pooled. Patients underwent both an outpatient posterior iliac crest bone marrow trephine/aspiration and a CT-guided biopsy of a radiologically evident bone metastasis. Samples were assessed for the presence of malignant cells and where possible also for estrogen (ER) and progesterone receptor (PgR) expression. 40 patients were enrolled. Bone marrow aspiration/trephine biopsy was completed in 39/40 (97.5%) and CT-guided biopsy was completed in 34/40 (85%) of patients. Sufficient tumor cells for hormone receptor analysis were available in 19/39 (48.8%) and 16/34 (47%) of and bone marrow aspiration/trephine and CT-guided biopsies, respectively. Significant discordance in ER and PgR between the primary and the bone metastasis was also seen. Nine patients had tissue available from both bone marrow and CT-guided bone biopsies. ER and PgR concordance between these sites was 100 and 78%, respectively. Performing studies on human bone metastases is technically challenging, with relatively low yields regardless of technique. Given resource issues and similar success rates when comparing both techniques, bone marrow examination may be utilized first and if inadequate tissue is obtained, CT-guided biopsies can then be used.

Loss of ALX4 expression in epithelial cells and adjacent stromal cells in breast cancer.

BACKGROUND: Loss of the stromally-restricted homeodomain transcription factor, Alx4, causes defective mouse mammary epithelial morphogenesis. AIMS: To begin to define the role of ALX4 in the human breast and in breast cancer, the expression pattern of ALX4 in the normal human breast and changes in expression in breast cancer were determined. METHODS: Cells expressing ALX4 in the human breast were identified by co-immunofluorescence using alpha-ALX4 antibodies and markers of specific mammary cell types. ALX4 expression in breast cancer was then determined by immunohistochemistry on tumour sections that also harboured regions of normal breast tissue. Using criteria that required ALX4 staining in both stromal and epithelial cells, changes in ALX4 expression in tumours on a tissue microarray were determined. RESULTS: ALX4 was expressed in both stromal and luminal epithelial cells in the human breast. Scoring tissue sections of duct carcinoma in situ (DCIS) or invasive ductal carcinoma (IDC) that also harboured regions of normal breast tissue, a loss of ALX4 (p<0.001) in stromal and epithelial cells in breast tumours was observed. Analysis of ALX4 expression in 123 sections on a tissue microarray confirmed a highly significant loss (p<0.001) of ALX4 in breast cancer in the tumours themselves and in adjacent stromal cells. CONCLUSIONS: These data show a distinct pattern of expression of ALX4 in the human breast relative to the murine mammary gland. Furthermore, characterisation of ALX4 in breast cancer showed that loss of ALX4 in tumours and the surrounding untransformed stroma is a basic characteristic of DCIS and IDC.

Mechanisms and pathways of bone metastasis: challenges and pitfalls of performing molecular research on patient samples.

The molecular mechanisms underlying the development of bone metastases in breast cancer remain unclear. Disseminated tumour cells (DTCs) in the bone marrow of breast cancer patients are commonly identified, even in early stage disease, but their potential to initiate metastases is not known. The mechanism whereby DTCs become overt metastatic tumour cells (MTCs) is therefore, an area of considerable interest. This study explored the analysable yield of genetic material from human biopsy samples in order to describe differences in gene expression between DTCs and bone MTCs. Thirteen breast cancer patients with bone metastases underwent a CT-guided bone metastasis biopsy and a bone marrow biopsy. Tumour cells were enriched and gene expression profiling was conducted to identify differentially expressed genes. The analysable yield of sufficient RNA for microarray analysis was 60% from bone metastasis biopsies and 80% from bone marrow biopsies. A signature of 133 candidate genes differentially expressed between DTCs and MTCs was identified. Several genes relevant to breast cancer metastasis to bone (osteopontin, CTGF, parathyroid hormone receptor, EGFR) were significantly overexpressed in MTCs as compared to DTCs. Biopsies of bone metastases and bone marrow rarely yield enough tissue for robust molecular biology studies using clinical samples. The findings obtained however are interesting and seem to overlap with the bone metastasis gene expression signature described in murine xenograft models. Larger biopsy specimens or improved RNA extraction techniques may improve analysable yield and feasibility of these techniques.

Nephrin is involved in podocyte maturation but not survival during glomerular development.

Nephrin, a major component of the glomerular slit diaphragm (SD), is both a structural protein as well as a signaling molecule influencing foot process (FP) formation and maintenance of podocyte integrity. Analyses of near-term embryonic kidneys showed normal cellular viability and no apoptosis in glomeruli from nephrin knockout mice. Moreover, expression and location of other SD or glomerular basement membrane components were similar in wild-type and mutant mice as was the location and levels of most podocyte-specific proteins. Transcriptional profiling showed that the lack of nephrin had minor impact on the expression of genes for FPs and SD proteins. Claudin 3, a tight-junction protein normally absent in glomeruli, was upregulated threefold in the knockout mice, suggesting a role of nephrin in claudin 3 gene expression within the glomeruli. Our results suggest that nephrin is expressed late in the process of podocyte differentiation and is a locus for the formation of SD and FP maintenance and physical integrity in vivo. Nephrin does not seem to have a primary role in cell survival but has a small impact on gene regulation during glomerular development.

Survey of pigs' kidneys with lesions consistent with PMWS and PDNS and ochratoxicosis. Part 2: pathological and histological findings.

One thousand condemned pigs' kidneys were collected in February 2002 from two pig abattoirs in England to assess the lesions due to postweaning multisystemic wasting syndrome (pmws) and porcine dermatitis and nephropathy syndrome (pdns) and the possible contribution of ochratoxicosis; 174 of the kidneys were pale, 295 were swollen and 81 were abnormally firm with the gross appearance of fibrosis. The main macroscopic finding was the presence of multifocal pale cortical lesions, observed in 446 of the kidneys, and there were large cysts in 266 of them. Histopathological lesions of non-suppurative tubulointerstitial nephritis, with degeneration and fibrosis of renal tubules, were identified in 213 of 250 (85.2 per cent) of the kidneys examined. These lesions were consistent with those reported in cases of pmws and pdns. The tubular degeneration and fibrosis were also consistent with ochratoxicosis. A higher mean concentration of ochratoxin A was significantly (P=0.020) associated with the presence of multifocal pale cortical lesions consistent with ochratoxicosis, but a causal relationship was not confirmed because histochemistry was not used to detect ochratoxin in the lesions directly. There was no significant correlation between the microscopic lesions and the concentration of ochratoxin. The degenerative lesions may have been caused by previous exposure to ochratoxin that had subsequently been excreted, but the microscopic lesions also included non-suppurative interstitial nephritis, which was unlikely to have been caused by ochratoxicosis.

Survey of pigs' kidneys with lesions consistent with PMWS and PDNS and ochratoxicosis. Part 1: concentrations and prevalence of ochratoxin A.

One thousand condemned pigs' kidneys were collected in February 2002 from two pig abattoirs in England to assess the possible contribution of ochratoxicosis to postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS); 250 of the kidneys with macroscopic lesions consistent with nephrosis/nephritis (pale or white cortical lesions) were selected, and the concentration of ochratoxin A was measured in samples of renal cortex by high-performance liquid chromatography (HPLC). Low concentrations were detected in 230 (92 per cent) of the kidneys tested, and in 41 (16.4 per cent) of them the concentration was below the limit of quantification of 0.2 microg/kg. In 187 (74.8 per cent) of the kidneys, the concentration was more than 0.2 microg/kg, and the highest concentration detected was 2.3 microg/kg. The mean (sd) concentration was 0.31 (0.33) microg/kg. The identification of ochratoxin A was confirmed by mass spectrometry. The concentrations of ochratoxin A did not exceed the threshold assessed by the Food Standards Agency to be safe for human food.

Limited tissue fixation times and whole genomic amplification do not impact array CGH profiles.

BACKGROUND: Array comparative genomic hybridisation (CGH) is a powerful method for the genetic analysis of lesional and normal tissues to identify genomic imbalances associated with malignancies. However, the use of this technique with DNA extracted from archival formalin fixed, paraffin embedded (FFPE) tissue specimens, the most widely available resource for retrospective studies, is subject to quantitative and qualitative limitations. In this report, the suitability and integrity of the DNA extracted from FFPE MCF7 breast cancer cells fixed for different periods of time for array CGH applications were examined. RESULTS: Using our established cDNA microarray protocol in conjunction with whole genome amplification methods, the genetic profiles of freshly harvested MCF7 cells and their matched FFPE counterparts were analysed. Congruent profiles between FFPE MCF7 cells and their fresh counterpart and between amplified and non-amplified FFPE MCF7 cells were observed. Our results demonstrate that formalin fixation of <20 hours has no significant adverse effect on the integrity of DNA for array CGH studies. CONCLUSIONS: Our findings attest to the fidelity of our array CGH methods to effectively examine material recovered from FFPE tissue specimens for microarray applications. This in turn has great potential to identify novel diagnostic and prognostic markers for human disease.

Clinical and pathological responses of weaned pigs to atmospheric ammonia and dust.

Nine hundred and sixty weaned pigs were exposed for five weeks to controlled concentrations of atmospheric ammonia and dust in a single, multifactorial experiment. The treatments were a mean dust concentration of either 1.2, 2.7, 5.1 or 9.9 mg/m3 (inhalable fraction) and a mean ammonia concentration of either 0.6, 10.0, 18.8 or 37.0 ppm, concentrations representative of commercial conditions. The experiment was carried out over two years and the pigs were used in eight batches, each consisting of five lots of 24 pigs. Each treatment combination was replicated once, and an additional control lot (nominally 0 mg/m3 dust and 0 ppm ammonia) was included in each batch. The dust concentration was the same in the other four lots in each batch in which the four concentrations of ammonia were used; thus, the split-plot design was more sensitive to the effects of ammonia than dust. The groups of pigs were kept separately in five rooms in a purpose-built facility, and the pollutants were injected continuously into the air supply. Ammonia was supplied from a pressurised cylinder, and the endogenous dust in each room was supplemented by an artificial dust manufactured from feed, barley straw and faeces, mixed by weight in the proportions 5:1:4; its ingredients were oven-dried, milled and mixed, and then resuspended in the air supply. The health of the pigs was assessed in terms of general pathology, respiratory tract pathology, and the microbiology of the nasal cavity, trachea and lung. In each batch, postmortem examinations were carried out on 40 pigs after five weeks' exposure to the pollutants and on 30 pigs two weeks later to test for carryover and recovery--a total of 560 pigs. These examinations revealed minimal gross pathology and widespread minor pathological changes of little significance. The pigs' turbinate and lung scores were low and unaffected by exposure to pollutants. All the putative bacterial pathogens, with the exception of toxigenic Pasteurella multocida type D, were isolated from the respiratory tract of the pigs of both ages, but there were no differences between the effects of the different concentrations of pollutants.

Porcine dermatitis and nephropathy syndrome. clinical and pathological features of cases in the United Kingdom (1993-1998).

This paper documents the salient clinical and pathological features of porcine dermatitis and nephropathy syndrome (PDNS) in 96 pigs submitted from 55 units in the UK from 1993 to 1998. This series of cases pre-dated the emergence of post-weaning multisystemic wasting syndrome (PMWS) in the UK. The morbidity during outbreaks was 1% or less. Affected pigs ranged from 14 to 70 kg in weight and most died after a short clinical illness. Fifty-five pigs had multifocal or coalescing erythematous skin lesions, some progressing to dermal necrosis. Biochemistry showed raised serum urea, creatinine and gamma globulin levels accompanied by proteinuria. All cases showed bilateral renal enlargement with petechiae throughout the cortices. Microscopically these renal lesions ranged in chronology from acute necrotizing glomerulitis and vasculitis with multiple hyaline casts in renal tubules to chronic glomerular sclerosis with interstitial inflammation and fibrosis. Haemorrhagic dermatitis when present was associated with necrotizing vasculitis in the dermal vessels. Vasculitis was sometimes detected in other tissues including subcutis, lymph nodes, spleen, liver, joint synovial membrane, gastric and intestinal submucosa or serosa and meninges but its frequency and distribution varied considerably in individual pigs. Immunostaining showed deposits of IgG and IgM in damaged glomeruli, renal casts and skin lesions. The aetiology and pathogenesis of the condition remain unknown but the histopathological and immunological findings suggest a systemic immune-complex disorder resulting in vasculitis with particular predilection for kidney and skin.