Nucleoside excretion in breast cancer: comparison with other biochemical tumour-index-substances

[We have measured four urinary nucleosides (dimethylguanosine, 1-methylinosine, pseudouridine and beta-aminoisobutyric acid)in patients with benign breast disease and patients with early and advanced breast cancer in order to assess their value as tumour-index-substances. We compared the results with other biochemical indices of breast cancer and sought and correlations between these indices. The results indicate that few abnormalities occurred in patients without overt metastases and these did not predict early relapse. In those with metastatic disease, dimethylguanosine excretion was most frequently elevated. Correlations were observed between some of the nucleosides and lysozyme and alpha1-antirypsin.     

MIB1 proliferation index in breast infiltrating carcinoma: comparison with other proliferative markers and association with new biological prognostic factors

AIMS: In breast invasive carcinoma our objectives were I) to compare cellular proliferation determined by MIB1 index with S-phase fraction (SPF) assessed by flow cytometry and with mitotic index, and II) to examine the association of MIB1 index with classical and with new biological prognostic factors [bcl-2, p53, c-erbB-2 and cathepsin D (CD)]. METHODS AND RESULTS: From 102 cases of breast invasive carcinoma, 5-microm thick serial sections were cut from formalin-fixed, paraffin-embedded tissue blocks, and processed for detection of CD, c-erbB-2, p53, bcl-2, Ki-67 antigen MIB-1 and estrogen receptors (ER) and progesterone receptors (PR). SPF was measured by flow cytometry in fresh-frozen tissue samples taken from the carcinoma in each patient. MIB1 index was correlated with SPF (rho=0.45, p<0.0001) and with mitotic index (rho=0.42, p<0.0001). The MIB-1 index was positively associated with the histological grade (p=0.001), tumor size (p=0.04) and the presence of metastases in axillary lymph nodes (p=0.01). MIB1 was associated directly with p53 (p=0.045) and inversely with bcl-2 (p=0.0002). The MIB-1 index was not statistically associated with c-erbB-2. There was a weak association between MIBI index and stromal cell CD. The median MIB1 index was higher in tumors with moderate to strong CD staining of stromal cell, but the difference did not reach statistical significance (p=0.09). CONCLUSIONS: MIB1 index correlates with well established methods for assessing tumor proliferation and with parameters of an aggressive phenotype of tumor. MIB1 index is an effective and readily accessible method for assessing tumor proliferation in breast carcinoma.     

IL-1 family in breast cancer: Potential interplay with leptin and other adipocytokines

Obesity is associated with an increased risk of breast cancer. interleukin-1 (IL-1), a pro-inflammatory cytokine secreted by adipose tissue, is involved in breast cancer development. There is also convincing evidence that other adipocytokines including leptin not only have a role in haematopoiesis, reproduction and immunity but are also growth factors in cancer. Therefore, IL-1 family and leptin family are adipocytokines which could represent a major link between obesity and breast cancer progression. This minireview provides insight into recent findings on the prognostic significance of IL-1 and leptin in mammary tumours, and discusses the potential interplay between IL-1 family members and adipocyte-derived hormones in breast cancer.     

LncRNA AWPPH and miRNA-21 regulates cancer cell proliferation and chemosensitivity in triple-negative breast cancer by interacting with each other

Long–non-coding RNAs (lncRNA) AWPPH promotes the progression of liver and bladder cancer, indicating its oncogenic role. The current study aimed to explore the involvement of AWPPH in triple-negative breast cancer (TNBC). In the current study, we found that plasma levels of lncRNA AWPPH and microRNA-21 (miRNA-21) were upregulated in patients with TNBC than in healthy controls, and the upregulation of plasma lncRNA AWPPH and miRNA-21 distinguished early-stage patients with TNBC from healthy controls. Plasma levels of lncRNA AWPPH and miRNA-21 were significantly and positively correlated in both patients with TNBC and healthy controls. LncRNA AWPPH and miRNA-21 overexpression led to promoted cancer cells proliferation and improved cancer cell viability under carboplatin treatment, while lncRNA AWPPH small interfering RNA (siRNA) silencing played an opposite role. In addition, miRNA-21 overexpression attenuated the effects of lncRNA AWPPH siRNA silencing on of cancer cell behaviors. LncRNA AWPPH overexpression led to upregulated miRNA-21 in TNBC cells, while miRNA-21 overexpression also led to significantly upregulated lncRNA AWPPH expression. Therefore, lncRNA AWPPH and miRNA-21 may regulate cancer cell proliferation and chemosensitivity in TNBC by interacting with each other.     

DNA grading of malignancy in breast cancer. Prognostic validity, reproducibility and comparison with other classifications

The prognostic significance of the "DNA malignancy grade" (DNA-MG) was tested in a series of 104 breast cancer patients in comparison with TNM staging, histomorphologic grading according to Bloom and Richardson, mean nuclear area (MNA) and DNA-histogram classification according to Auer. The reproducibility and representativity of the grading systems were investigated, and their results in primary tumors and lymph node metastases were compared. The scalar DNA-MG was assessed on monolayer smears prepared from paraffin-embedded tissues; the smears were automatically Feulgen stained and used for rapid interactive DNA cytometric evaluation by an automated microscope and a TV image-analysis system. TNM staging showed the highest correlation with survival, followed by histomorphologic grading and DNA-MG; MNA and the DNA-histogram classification failed to give statistically significant prognostic information. Both histomorphologic grading and DNA-MG were identified as parameters adding independent prognostic information to the TNM staging. However, only DNA-MG demonstrated an acceptable reliability, with small 95% ranges between repeated measurements within the primary tumor (+/- 0.3 DNA-MG) and a strong correlation between the results in the primary tumor and its lymph node metastases. These findings show that the DNA-MG is a valid and reliable prognostic index that adds significant prognostic information to TNM staging.     

Cell proliferation in prostatic carcinoma: comparative analysis of Ki-67, MIB-1 and PCNA

Summary Antibodies to assess the proliferative index of tumours are being increasingly employed together with established markers for prognostic evaluation. This study set out to compare three cell proliferation markers, Ki-67, MIB-1 and PCNA, utilizing a semiquantitative method of assessment, in 20 human prostatic carcinomas. The streptavidin-biotin immunostaining system was used for the monoclonal antibodies MIB-1 and PCNA and an indirect immunoperoxidase assay for the monoclonal antibody Ki-67. Significant correlations were found between the expression of Ki-67 in frozen tissues and MIB-1 in formal saline-fixed wax-embedded tissues (p = 0.0003); between Ki-67 and PCNA expression in Bouin's-fixed tissues (p </ 0.0001); and MIB-1 (formalin-saline-fixed tissues) and PCNA (Bouin's-fixed tissues) (p </ 0.0001). A more intense nuclear staining pattern with less heterogeneity was observed for MIB-1 compared with PCNA, suggesting the antibody of choice, on formal saline-fixed tissues, is MIB-1, which closely correlated with Ki-67, a marker we have previously shown to be of prognostic value in prostatic carcinoma.     

Thyroid cell proliferation in Graves' disease. Use of MIB-1 monoclonal antibody

OBJECTIVE: To measure thyroid cell proliferation in patients with Graves' disease (GD) before and during treatment with antithyroid drugs. STUDY DESIGN: Patients were assessed by fine needle aspiration biopsy before (n = 20) and after 4 (n = 19) and 12 months of treatment (n = 15) with propylthiouracil or methimazole. Cell proliferation index (CPI) was estimated by immunocytochemistry using MIB-1. CPI was studied in relation to the cytologic parameters of the smears; clinical parameters, such as Wayne's Clinical Index (WCI) and time without treatment; laboratory parameters, such as 131I uptake and dosage of serum free thyroxin and thyroid-stimulating hormone; and thyroid ultrasound. RESULTS: CPI varied from 0.00% to 25.00% before treatment, 0.00% to 23.00% at 4 months and 0.00% to 14.84% at 12 months. CPI median values were 6.50%, 4.30% and 3.30%, respectively (before and after 4 months and 12 months of treatment). CPI had a positive correlation with WCI and FT4 at 12 months of treatment. CONCLUSION: Thyroid CPI in GD varies from case to case. However, due to its decreasing pattern during follow-up and its positive correlation with thyrotoxicosis severity, CPI may indicate the functional status of the gland and contribute to a better understanding of GD.     

Proliferative activity in invasive breast carcinoma: a comprehensive comparison of MIB-1 immunocytochemical staining in aspiration biopsies to image analytic, flow cytometric and histologic parameters.

OBJECTIVE: To use MIB-1 antibody to assess proliferative activity in fine needle aspiration (FNA) samples of invasive breast carcinoma and compare these results to multiple other measures of proliferative activity. STUDY DESIGN: FNA slides from 62 patients with invasive breast carcinoma were subjected to staining with MIB-1. Quantitative MIB-1 values were compared to image analytic proliferative fractions (IPF) obtained from the same FNAs. MIB-1 values were also compared to flow cytometric S-phase fractions (SPF) and S + G2/M-phase fractions (FPF) and to histologic assessment of mitotic count (MC) in resected tumors. RESULTS: MIB-1 values, IPF, SPF, FPF and MC were suitable for evaluation in 55, 53, 50, 50 and 56 cases, respectively. MIB-1 values showed good correlation with IPF in FNAs (correlation coefficient = .57, P <.00001). MIB-1 values also showed correlation with SPF (correlation coefficient =.447, P = .003), FPF (correlation coefficient = .325, P = .023) and MC (correlation coefficient = .402, P = .01) in resected tumors. CONCLUSION: This study supports the use of MIB-1 values obtained from FNA samples for assessment of proliferative activity in invasive breast carcinoma, based on correlation of these values with multiple other parameters of proliferative activity. Assessment of these values can play a role in predicting prognosis and in selecting patients with invasive carcinoma of the breast for preoperative or adjuvant chemotherapy.     

Immunohistochemical distribution of mucinous-like carcinoma associated antigen (MCA) in breast and non-breast cancer: comparison with other biological parameters

The present study reports the immunohistochemical reactivity of the monoclonal antibody b-12 (MAb b-12) with malignant human tissues. 173 neoplastic tissues were tested: MAb b-12 stained all breast carcinomas independently of their histology, with different patterns within the various type of cancer. Some other carcinomas (stomach, bowel, ovary, lung, endometrium), were also reactive even if the fraction of positive cells was lower. A comparison between the histological localization of MCA and that of CEA was performed; anti-CEA antibodies stained the cancer tissues with different reactivity and showed different percentages of positivity. MCA expression was also compared with other biological parameters such as the presence of estrogen receptors (ER), progesterone receptors (PgR), epithelial growth factor receptors (EGF-R), and oncoprotein p-53 which is encoded by the oncogene N-myc. The proliferative activity was also evaluated by measuring the growth fraction (GF) using the antibody Ki67. Any correlation was demonstrated between MCA and these parameters except for growth fraction as revealed by Ki67 antibody.     

BRCA1 inhibits membrane estrogen and growth factor receptor signaling to cell proliferation in breast cancer

BRCA1 mutations and estrogen use are risk factors for the development of breast cancer. Recent work has identified estrogen receptors localized at the plasma membrane that signal to cell biology. We examined the impact of BRCA1 on membrane estrogen and growth factor receptor signaling to breast cancer cell proliferation. MCF-7 and ZR-75-1 cells showed a rapid and sustained activation of extracellular signal-related kinase (ERK) in response to estradiol (E2) that was substantially prevented by wild-type (wt) but not mutant BRCA1. The proliferation of MCF-7 cells induced by E2 was significantly inhibited by PD98059, a specific ERK inhibitor, or by dominant negative ERK2 expression and by expression of wt BRCA1 (but not mutant BRCA1). E2 induced the synthesis of cyclins D1 and B1, the activity of cyclin-dependent kinases Cdk4 and CDK1, and G(1)/S and G(2)/M cell cycle progression. The intact tumor suppressor inhibited all of these. wt BRCA1 also inhibited epidermal growth factor and insulin-like growth factor I-induced ERK and cell proliferation. The inhibition of ERK and cell proliferation by BRCA1 was prevented by phosphatase inhibitors and by interfering RNA knockdown of the ERK phosphatase, mitogen-activated kinase phosphatase 1. Our findings support a novel tumor suppressor function of BRCA1 that is relevant to breast cancer and identify a potential interactive risk factor for women with BRCA1 mutations.     

Skp2 is over-expressed in breast cancer and promotes breast cancer cell proliferation

The F box protein Skp2 is oncogenic. Skp2 and Skp2B, an isoform of Skp2 are overexpressed in breast cancer. However, little is known regarding the mechanism by which Skp2B promotes the occurrence and development of breast cancer. Here, we determined the expression and clinical outcomes of Skp2 in breast cancer samples and cell lines using breast cancer database, and investigated the role of Skp2 and Skp2B in breast cancer cell growth, apoptosis and cell cycle arrest. We obtained Skp2 is significantly overexpressed in breast cancer samples and cell lines, and high Skp2 expression positively correlated with poor prognosis of breast cancer. Both Skp2 and Skp2B could promote breast cancer cell proliferation, inhibit cell apoptosis, change the cell cycle distribution and induce the increased S phase cells and therefore induce cell proliferation in breast cancer cells. Moreover, the 2 isoforms could both suppress PIG3 expression via independent pathways in the breast cancer cells. Skp2 suppressed p53 and inhibited PIG3-induced apoptosis, while Skp2B attenuated the function of PIG3 by inhibiting PHB. Our results indicate that Skp2 and Skp2B induce breast cancer cell development and progression, making Skp2 and Skp2B potential molecular targets for breast cancer therapy.     

Downregulation of Wilms' tumor 1 protein inhibits breast cancer proliferation

High levels of Wilms' Tumor 1 (WT1) mRNA have been correlated with poor prognosis in breast cancer patients. However, the function of WT1 protein in breast cancer is not known. We observed that the levels of WT1 protein correlated with the proliferation of breast cancer cells. When the proliferation of breast cancer cells was stimulated by 17beta-estradiol, WT1 protein expression increased. But when the proliferation of breast cancer cells was inhibited by tamoxifen or all-trans retinoic acid (ATRA), WT1 protein expression decreased. We hypothesize that WT1 protein plays a role in regulating breast cancer cell proliferation. Using liposome-incorporated WT1 antisense oligodeoxynucleotides, we found that downregulation of WT1 protein expression led to breast cancer growth inhibition and reduced cyclin D1 protein levels. These results indicate that WT1 protein contributes to breast cancer progression by promoting breast cancer cell proliferation.     

Micrometastasis in breast cancer and other solid tumors

Hematogenous distant metastasis is the leading cause of cancer-related death in breast cancer and other solid tumors. By applying sensitive immunocytochemical and molecular assays, disseminated tumor cells (DTC) in bone marrow (BM) can be detected in 20-40% of cancer patients without any clinical or even histopathological signs of metastasis and the presence of these DTC at primary diagnosis predicts the subsequent occurrence of overt metastases in bone and other organs. cDNA-microarray analysis on primary breast carcinomas from patients with and without tumor cells in BM revealed a predominant downregulation of potential metastasis-suppressor genes in BM-positive tumors. Thus, dissemination of tumor cells appears to be an early process associated with a specific molecular signature of the primary tumor.     

Interleukin-1 directly regulates hormone-dependent human breast cancer cell proliferation in vitro

Direct in vitro effects of IL-1 on hormone-dependent (MCF-7 and ZR-75-B) and independent (HS-578-T and MDA-231) human breast cancer cell proliferation were investigated in short-term and long-term cell cultures. For short-term (48 h) studies [3H]thymidine uptake was used as an index of proliferation, while for long-term (12 day) cultures actual cell numbers were determined. Initial studies, conducted with MCF-7 cells, demonstrated that both forms of recombinant human IL-1 (alpha and beta) at 10(-11) M inhibited [3H]thymidine uptake by MCF-7 by 70%, and by day 7 of the long-term study alpha and beta IL-1 at 10(-11) M inhibited MCF-7 cell growth by 80%. IL-1, while inhibiting the growth of another hormone-dependent breast cancer cell line; ZR-75-B, had no effect on the hormone-independent cell lines MDA-231 and HS-578-T. The differing proliferative responses of the hormone-dependent and independent cells to IL-1 may, in part, be due to the expression of IL-1 receptors on these cells, in that MCF-7 cells express IL-1 receptors [dissociation constant (Kd) = 2.0 x 10(-10) M; receptor density = 2,500 sites per cell and mol wt = 80,000] while the hormone-independent MDA-231 cells do not.     

Subcategory assessment method for social life cycle assessment. Part 1: methodological framework

Purpose

The aim of this work is to propose an objective method for evaluating subcategories in social life cycle impact assessment (S-LCIA). Methods for assessing subcategories have been available since 2006, but a number of these either fail to include all the subcategories envisaged in the guidelines for S-LCA (UNEP/SETAC 2009) or are subjective in their assessment of each subcategory.

Methods

The methodology is characterized by four steps: (i) the use of the organization as unit process, in which it was decided to assess the social profile of the organization responsible for the processes involved in the product life cycle, (ii) definition of the basic requirement to assess each subcategory, (iii) definition of levels based on the environment context or organizational practice and the data availability and (iv) assignment of a quantitative value.

Results and discussion

The result of the method applied was the development of the subcategory assessment method (SAM). SAM is a characterization model that evaluates subcategories during the impact assessment phase. This method is based on the behaviour of organizations responsible for the processes along the product life cycle, thereby enabling a social performance evaluation. The method, thus, presents levels for each subcategory assessment. Level A indicates that the organization exhibits proactive behaviour by promoting basic requirement (BR) practices along the value chain. Level B means that the organization fulfils the BR. Levels C and D are assigned to organizations that do not meet the BR and are differentiated by their context. The greatest difficulty when developing SAM was the definition of the BR to be used in the evaluation of the subcategories, though many indications were present in the methodological sheets.

Conclusions

SAM makes it possible to go from inventory to subcategory assessment. The method supports evaluation across life cycle products, thereby ensuring a more objective evaluation of the social behaviour of organizations and applicable in different countries.

Recommendations

When using SAM, it is advisable to update the data for the context environment. The method might be improved by using data for the social context that would consider not only the country, but also the region, sector and product concerned. A further improvement could be a subdivision of the levels to better encompass differences between organizations. It is advisable to test SAM by applying it to a case study.     

Risk assessment issues in breast cancer

Breast cancer is the most common malignancy affecting women, and its incidence has been increasing in many countries. The aetiology of breast cancer is poorly understood, so there is concern as to which factors in our environment or lifestyle are responsible for the increase. There is a need for reliable risk assessment, which involves the steps of hazard identification, hazard evaluation, exposure evaluation and risk estimation. Short-term laboratory tests and long-term tests in animals are useful for priority-setting, but quantitative human risk assessment should preferably involve observations of humans. Epidemiological studies vary in the degree of reliance that can be placed on their results. The main types of epidemiological investigation are illustrated by recent examples from the literature on breast cancer. Careful judgement is required in assessing whether any association between a factor and a disease is likely to be causal. The injectable contraceptive, depot medroxyprogesterone acetate (DMPA, ‘Depo-Provera’), has been controversial because it caused malignant mammary tumours in beagle dogs. Two recent case-control studies found no overall association between DMPA and the risk of breast cancer in women. There was some evidence of increased risk in certain sub-groups of women, which could be interpreted with more confidence if there were a better understanding of the biology of human breast cancer. Nevertheless, the results do not support the prediction from beagle experiments that DMPA might increase the overall risk of breast cancer.     

Aromatase and other inhibitors in breast and prostatic cancer

Estrogens have an important role in the growth of breast and other hormone-sensitive cancers. We have shown that 4-hydroxyandrostenedione (4-OHA) selectively blocks estrogen synthesis by inhibiting aromatase activity in ovarian and peripheral tissues and reduces plasma estrogen levels in rat and non-human primate species. In postmenopausal men and women, estrogens are mainly of peripheral origin. When postmenopausal breast cancer patients were administered either by daily oral or parenteral weekly treatment with 4-OHA, plasma estrogen concentrations were significantly reduced. Complete or partial response to treatment occurred in 34% of 100 patients with advanced breast cancer, while the disease was stabilized in 12%. We recently studied the effects of 4-OHA and other aromatase inhibitors, 10-propargylestr-4-ene-3,17-dione (PED) and imidazo[1,5-]3,4,5,6-tetrahydropyrin-6-yl-(4-benzonitrile) (CGS 16949A) as well as 5-reductase inhibitors, N,N-diethyl-4-methyl-3-oxo-4-aza-5-androstane-17β-carboxyamide (4-MA) and 17β-hydroxy-4-aza-4-methyl-19norandrost-5-en-3-one (L651190) in prostatic tissue from 11 patients with prostatic cancer and six patients with benign prostatic hypertrophy (BPH), and from normal men at autopsy. We attempted to measure aromatase activity in tissue incubation by quantitating ³H₂O released during aromatization of androstenedione or testosterone labeled at the C-1 position. The amount of ³H₂O released from all samples was at least twice that of the heat inactivated tissue samples. The ³H₂O release was significantly inhibited by 4-OHA and 4-MA, but not by the other aromatase inhibitors. However, when HPLC and TLC were used to isolate steroid products, no estrone or estradiol was detected in the incubates. Furthermore, no aromatase mRNA was detected following amplification by PCR. The 4-OHA was found to inhibit 5-reductase in both BPH and cancer tissue, although to a lesser extent than 4-MA. The other aromatase inhibitors were without effect. Although a mechanism involving intraprostatic aromatase is not likely, inhibitors may act to reduce peripherally-formed estrogens. In postmenopausal breast cancer, the results indicate that 4-OHA is of significant benefit.     

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats

The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons; in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.