Andrew C. Schofield

A new histological grading system to assess response of breast cancers to primary chemotherapy: prognostic significance and survival

The clinical and complete pathological response of a primary breast cancer to chemotherapy has been shown to be an important prognostic for survival. However, the majority of patients do not experience a complete pathological response to primary chemotherapy and the significance of lesser degrees of histological response is uncertain and the prognostic significance is unknown. The purpose of this study was to evaluate a new histological grading system to assess response of breast cancers to primary chemotherapy and to determine if such a system has prognostic value.A consecutive series of 176 patients with large (> or =4cm) and locally advanced breast cancers were treated with multimodality therapy comprising primary chemotherapy, surgery, radiotherapy and tamoxifen. All underwent assessment of the primary breast tumour before and after completion of chemotherapy. Residual tumour was excised after completion of chemotherapy (mastectomy or wide local excision with axillary surgery). The removed tissue was assessed and response to chemotherapy graded using a five-point histological grading system based with the fundamental feature being a reduction in tumour cellularity; comparison being made with a pre-treatment core biopsy. All patients were followed up for 5 years or more. Pathological responses were compared to 5 year overall survival and disease-free survival using log rank tests. The overall 5-year survival for all patients was 71%, and 5 year disease free interval was 60%. There was a significant correlation between pathological response using this new grading system and both overall survival (P=0.02) and disease-free interval (P=0.04). In a multivariate analysis of known prognostic factors, the Miller/Payne grading system was an independent predictor of overall patient survival. This grading system, which assesses the histological response to primary chemotherapy, can predict overall survival and disease-free interval in patients with large and locally advanced breast cancers treated with such therapy. The relationship of degree of histological response to overall and disease-free survival has been shown in univariate and multivariate analyses and could potentially have an important role in the clinical management of patients with locally advanced breast cancer undergoing primary chemotherapy.

miRNA-34a is associated with docetaxel resistance in human breast cancer cells

Docetaxel is a chemotherapy drug to treat breast cancer, however as with many chemotherapeutic drugs resistance to docetaxel occurs in 50% of patients, and the underlying molecular mechanisms of drug resistance are not fully understood. Gene regulation through microRNAs (miRNA) has been shown to play an important role in cancer drug resistance. By directly targeting mRNA, miRNAs are able to inhibit genes that are necessary for signalling pathways or drug induced apoptosis rendering cells drug resistant. This study investigated the role of differential miRNA expression in two in vitro breast cancer cell line models (MCF-7, MDA-MB-231) of acquired docetaxel resistance. MiRNA microarray analysis identified 299 and 226 miRNAs altered in MCF-7 and MDA-MB-231 docetaxel-resistant cells, respectively. Docetaxel resistance was associated with increased expression of miR-34a and miR-141 and decreased expression of miR-7, miR-16, miR-30a, miR-125a-5p, miR-126. Computational target prediction revealed eight candidate genes targeted by these miRNAs. Quantitative PCR and western analysis confirmed decreased expression of two genes, BCL-2 and CCND1, in docetaxel-resistant cells, which are both targeted by miR-34a. Modulation of miR-34a expression was correlated with BCL-2 and cyclin D1 protein expression changes and a direct interaction of miR-34a with BCL-2 was shown by luciferase assay. Inhibition of miR-34a enhanced response to docetaxel in MCF-7 docetaxel-resistant cells, whereas overexpression of miR-34a conferred resistance in MCF-7 docetaxel-sensitive cells. This study is the first to show differences in miRNA expression, in particular, increased expression of miR-34a in an acquired model of docetaxel resistance in breast cancer. This serves as a mechanism of acquired docetaxel resistance in these cells, possibly through direct interactions with BCL-2 and CCND1, therefore presenting a potential therapeutic target for the treatment of docetaxel-resistant breast cancer.

Conjugated linoleic acids (CLAs) regulate the expression of key apoptotic genes in human breast cancer cells

Conjugated linoleic acid (CLA) reduces mammary tumorigenesis in rodent models, induces apoptosis in rodent mammary tumor cell lines, and decreases expression of antiapoptotic bcl‐2 in rat mammary tissue. This investigation focused on the cell mechanisms underlying the antitumor effects of CLA. Changes (mRNA, protein) in expression of major proapoptotic p53, p21WAF1/CIP1, bax, bcl‐Xs genes, and the antiapoptotic bcl‐2 gene were observed in malignant MCF‐7 and MDA‐MB‐231 cells and in benign MCF‐10a human mammary tumor cells in culture. CLA, but not linoleic acid (LA), inhibited proliferation in all cells; CLA mix was most effective. CLA increased DNA damage (apoptosis). CLA increased mRNA expression of p53 and p21WAF1/CIP1 (three‐ to fivefold and twofold, respectively) but either decreased bcl‐2 by 20–30% or had no effect in MCF‐7 and MCF‐10a cells, respectively; protein expression reflected mRNA values. In MDA‐MBA‐231 (mutant p53) cells, mRNA for p53 was not changed, but p21WAF1/CIP1 and bcl‐2 mRNA was increased. Protein expression largely reflected mRNA changes but, surprisingly, CLA completely suppressed mutant p53 protein in MDA‐MB‐231 cells. Apparent antiapoptotic effects of increased bcl‐2 expression in MDAMBA‐231 cells were countered by increased proapoptotic p21WAF1/CIP1, Bax, and Bcl‐Xs proteins. Findings indicate that CLA elicits mainly proapoptotic effects in human breast tumor cells through both p53‐dependent and p53‐independent pathways, according to cell type.

Alterations of β-tubulin isotypes in breast cancer cells resistant to docetaxel

Docetaxel is one of the most active drugs used to treat breast cancer. The cellular target of docetaxel is the microtubule, specifically the β‐tubulin subunit, that comprises a series of isotypes and that can modulate function. This study has examined the role of alteration in β‐tubulin isotypes in vitro and has sequenced the β‐tubulin gene to determine if there were mutations, both of which may represent important mechanisms of acquired resistance to docetaxel. Breast cancer cells, MCF‐7 (oestrogen‐receptor positive) and MDA‐MB‐231, (oestrogen‐receptor negative) were made resistant to docetaxel in vitro. Expression of β‐tubulin isotypes (class I, II, III, IVa, IVb, and VI) was determined at the RNA and protein level using RT‐PCR and western analysis, respectively. DNA sequencing evaluated the β‐tubulin gene. At the mRNA level, class I, II, III, and IVa β‐tubulin mRNA isotypes were over‐expressed in docetaxel‐resistant MCF‐7 cells when compared with the docetaxel‐sensitive parental cells. However, class VI β‐tubulin mRNA isotype expression was decreased in resistant cells. In MDA‐MB‐231 cells, there was a decrease in expression of the class I and class IVa β‐tubulin mRNA. However, there were increased expressions in class II, IVb, and VI β‐tubulin mRNA isotypes in resistant cells. Western analysis has confirmed corresponding increases in β‐tubulin protein levels in MCF‐7 cells. However, in MDA‐MB‐231 cells, there were decreased protein levels for class II and class III β‐tubulin. This study demonstrates that altered expression of mRNA β‐tubulin isotypes and modulation of β‐tubulin protein levels are associated with acquired docetaxel resistance in breast cancer cells. This allows further understanding and elucidation of mechanisms involved in resistance to docetaxel.

Reduced expression of p27 is a novel mechanism of docetaxel resistance in breast cancer cells

IntroductionDocetaxel is one of the most effective chemotherapeutic agents in the treatment of breast cancer. Breast cancers can have an inherent or acquired resistance to docetaxel but the causes of this resistance remain unclear. However, apoptosis and cell cycle regulation are key mechanisms by which most chemotherapeutic agents exert their cytotoxic effects.MethodsWe created two docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) and performed cDNA microarray analysis to identify candidate genes associated with docetaxel resistance. Gene expression changes were validated at the RNA and protein levels by reverse transcription PCR and western analysis, respectively.ResultsGene expression cDNA microarray analysis demonstrated reduced p27 expression in docetaxel-resistant breast cancer cells. Although p27 mRNA expression was found to be reduced only in MCF-7 docetaxel-resistant sublines (2.47-fold), reduced expression of p27 protein was noted in both MCF-7 and MDA-MB-231 docetaxel-resistant breast cancer cells (2.83-fold and 3.80-fold, respectively).ConclusionsThis study demonstrates that reduced expression of p27 is associated with acquired resistance to docetaxel in breast cancer cells. An understanding of the genes that are involved in resistance to chemotherapy may allow further development in modulating drug resistance, and may permit selection of those patients who are most likely to benefit from such therapies.

Can patients' likelihood of benefiting from primary chemotherapy for breast cancer be predicted before commencement of treatment?

AbstractPurpose. Primary chemotherapy is commonly used in patients with breast cancer to downstage the primary tumour prior to surgery. There is a need to establish, prior to commencement of chemotherapy, predictors of clinical and pathological response, which may then be surrogate markers for patient survival and thus allow identification of patients who are most likely to benefit from such treatment. Patients and methods. A total of 104 patients with large and locally advanced breast cancers received an anthracycline/docetaxel-based regimen prior to surgery. Immunohistochemistry was carried out on pre-treatment core biopsies of the tumour to detect hormone receptors (oestrogen-ER; progesterone-PR), a proliferation marker (MIB-1), the oncoprotein Bcl-2, an extracellular matrix degradation enzyme (cathepsin D), p53, and an oestrogen associated protein (pS2). Both clinical and pathological response were assessed following completion of chemotherapy. Results. Patients whose tumours did not express oestrogen receptor (p= 0.02) or did not express Bcl-2 (p < 0.01) had a better pathological response in a univariate analysis. However, in a multivariate model, it was only the absence of detectable Bcl-2 protein that predicted a better pathological response (p= 0.001). Conclusions. This study has identified that patients whose breast cancers are most likely to experience the greatest degree of tumour destruction by primary chemotherapy do not express either oestrogen receptors or Bcl-2. This may have important implications in the selection of patients with breast cancer for primary chemotherapy who are most likely to gain a survival benefit.

Nutrition and the surgical patient: Triumphs and challenges

The understanding of the role of nutrition in the surgical patient has lead to major developments in the nutritional support of patients undergoing surgery. Reductions in morbidity by ensuring that patients receive optimal nutritional support can be achieved. Furthermore, the use of nutrients to modify immune, inflammatory and metabolic processes also offers new possibilities for reducing morbidity following major surgery. However, we are only at an embryonic stage in our understanding of how nutrients and nutrition affect the genome and this knowledge offers exciting possibilities in the future for modulating many key intracellular processes, particularly in the patient with cancer.

Effects of decitabine on the expression of selected endogenous control genes in human breast cancer cells.

To quantify gene expression levels, appropriate controls have to be used to adjust for experimental variation. Endogenous control genes are widely used as they are stably expressed independent of cell cycle and experimental conditions, however, they can be altered upon drug treatment. DNA methylation is widely studied in chemotherapy drug resistance and the DNA methylation inhibitor decitabine showed promising results reversing drug resistance in cancer. We aimed to investigate the effect of different decitabine concentrations on the expression of selected endogenous control genes (GAPDH, 18S rRNA, PPIA, RPL13A, OAZ1) in two docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) compared to untreated cells. In MCF-7 cells, 18S rRNA remained stable, however, GAPDH, PPIA and OAZ1 gene expression was increased after treatment. RPL13A was stably expressed at 8 muM decitabine but was increased at lower drug concentrations. In MDA-MB-231 cells, GAPDH levels remained relatively stable following decitabine treatment and so was PPIA expression at low decitabine concentrations. Decitabine increased 18S rRNA, RPL13A and OAZ1 gene expression. In this study, we observed cell line specific effects of decitabine and suggest that 18S rRNA is most suitable to use in MCF-7 cells, while GAPDH is recommended to use in MDA-MB-231 cells during decitabine treatment.

Inhibition of gastric cancer cell growth by arginine: molecular mechanisms of action.

BACKGROUND & AIMS Immunonutrition, containing arginine as a key component, has been shown to enhance the immune system and significantly reduce infectious complications in patients undergoing upper gastrointestinal surgery. Arginine, however, may also influence tumour cell behaviour. The aim of this study was to investigate the effects of arginine on tumour cell growth, invasion and modulation of expression of genes involved in these aspects of cell behaviour. METHODS A human gastric cancer cell line (AGS) was grown in vitro and supplemented with arginine (2, 4, 8, 16 and 32 mM) for 24, 48 and 72 h. The effect of arginine on cell growth (MTT assay), apoptosis (DAPI staining), invasion (Matrigel assay), gene expression (cDNA microarray analysis and RT-PCR) and protein expression (western analysis) was determined. RESULTS These studies demonstrated that arginine caused a decrease in AGS cell growth via induction of apoptosis. Whilst arginine decreased cell growth, no significant effect on the invasive potential of AGS cells was noted. Subsequent gene expression analysis demonstrated that arginine increased the expression of caspase 8, which was validated at the protein level. CONCLUSIONS These results suggest that that inhibition of AGS cell growth by arginine is mediated through caspase 8 activation of apoptosis.

From peas to "chips" – the new millennium of molecular biology: a primer for the surgeon

The mechanisms underlying the basis of heredity and the beginning of understanding of the genetic basis of life began to be unravelled some 160 years ago. These fundamental concepts, which have paved the way for the current explosion in our understanding of the genetic basis of cellular function, were established from the study of pea plants by an Augustinian monk, Gregor Mendel. The next major development in genetics was 100 years later when Watson and Crick discovered the structure of DNA. Following on from their seminal work there has been an exponential growth in knowledge regarding the structure and function of DNA and its functional unit, the gene [1]. The study of DNA itself is just a broad overview of the human genome (genomics). When trying to understand more complex genetic-based traits and diseases, such as cancer, this is inadequate because it does not allow a thorough understanding of the complex inter-related processes occurring within the cell. In order to take this further, the functions of the individual genes, the messenger RNA resulting from the gene and the subsequent protein, which is produced, need to be examined. Furthermore, there are complex interactions between the cellular environment and the genes, which can affect genetic and cellular function. The measurement of gene expression can, therefore, provide information on regulatory mechanisms, biochemical pathways, cellular control mechanisms and potential targets for intervention and therapy in a variety of disease states. One technique, which allows this to be studied, is DNA microarray technology, which is now used to monitor the expression of thousands of genes simultaneously. This paper outlines briefly the applications, limitations and the possible future of microarray techniques in oncological research.