Association of oestrogen receptor beta 2 (ER beta 2/ER beta cx) with outcome of adjuvant endocrine treatment for primary breast cancer--a retrospective study.

BACKGROUND: Oestrogen receptor beta (ERbeta) modulates ERalpha activity; wild type ERbeta (ERbeta1) and its splice variants may therefore impact on hormone responsiveness of breast cancer. ERbeta2/ERbetacx acts as a dominant negative inhibitor of ERalpha and expression of ERbeta2 mRNA has been proposed as a candidate marker for outcome in primary breast cancer following adjuvant endocrine therapy. We therefore now assess ERbeta2 protein by immunostaining and mRNA by quantitative RT-PCR in relation to treatment outcome. METHODS: ERbeta2-specific immunostaining was quantified in 141 primary breast cancer cases receiving adjuvant endocrine therapy, but no neoadjuvant therapy or adjuvant chemotherapy. The expression of mRNA for ERbeta2/ERbetacx was measured in 100 cases by quantitative RT-PCR. Statistical analysis of breast cancer relapse and breast cancer survival was performed using Kaplan Meier log-rank tests and Cox's univariate and multivariate survival analysis. RESULTS: High ERbeta2 immunostaining (Allred score >5) and high ERbeta2 mRNA levels were independently associated with significantly better outcome across the whole cohort, including both ERalpha positive and negative cases (Log-Rank P < 0.05). However, only ERbeta2 mRNA levels were significantly associated with better outcome in the ERalpha + subgroup (Log-Rank P = 0.01) and this was independent of grade, size, nodal status and progesterone receptor status (Cox hazard ratio 0.31 P = 0.02 for relapse; 0.17 P = 0.01 for survival). High ERbeta2 mRNA was also associated with better outcome in node negative cases (Log Rank P < 0.001).ERbeta2 protein levels were greater in ERalpha positive cases (T-test P = 0.00001), possibly explaining the association with better outcome. Levels of ERbeta2 protein did not correlate ERbeta2 mRNA levels, but 34% of cases had both high mRNA and protein and had a significantly better outcome (Log-Rank relapse P < 0.005). CONCLUSION: High ERbeta2 protein levels were associated with ERalpha expression. Although most cases with high ERbeta2 mRNA had strong ERbeta2 immunostaining, mRNA levels but not protein levels were independently predictive of outcome in tamoxifen-treated ERalpha + tumours. Post-transcriptional control needs to be considered when assessing the biological or clinical importance of ERbeta proteins.

Human homologue of cement gland protein, a novel metastasis inducer associated with breast carcinomas.

A suppression subtractive cDNA library representing mRNAs expressed at a higher level in the malignant human breast cancer cell line, MCF-7, relative to a benign breast tumor-derived cell line, Huma 123, contained a cDNA, M36, which was expressed in estrogen receptor alpha (ERalpha)-positive breast carcinoma cell lines but not in cell lines from normal/benign/ERalpha-negative malignant breast lesions. M36 cDNA had an identical coding sequence to anterior gradient 2 (AGR2), the human homologue of the cement gland-specific gene (Xenopus laevis). Screening of breast tumor specimens using reverse transcription-PCR and immunocytochemistry with affinity-purified anti-AGR2 antibodies showed that the presence of AGR2 mRNA and protein were both statistically significantly associated with ERalpha-positive carcinomas (P = 0.007, Fisher's exact test) and with malignancy (P < or = 0.025). When an expression vector for AGR2 cDNA was introduced into benign nonmetastatic rat mammary tumor cells, and three separate clones and two pools of cells were transferred to the mammary glands of syngeneic hosts, there were no consistent differences in the mean latent periods of tumor formation. However, metastases occurred in the lungs of animals receiving the AGR2 transfectants in 77% to 92% of animals with primary tumors (P = 0.0001) compared with no metastases in the control groups. The AGR2 transfectants exhibited enhanced rates of adhesion to a plastic substratum and extracellular AGR2 enhanced the rate of attachment of AGR2-negative but not AGR2-positive cells. These experiments are the first to link mechanistically the developmental gene product, AGR2, with metastasis in vivo.

Gene expression profiling in bladder cancer identifies potential therapeutic targets.

Despite advances in management, bladder cancer remains a major cause of cancer related complications. Characterisation of gene expression patterns in bladder cancer allows the identification of pathways involved in its pathogenesis, and may stimulate the development of novel therapies targeting these pathways. Between 2004 and 2005, cystoscopic bladder biopsies were obtained from 19 patients and 11 controls. These were subjected to whole transcript-based microarray analysis. Unsupervised hierarchical clustering was used to identify samples with similar expression profiles. Hypergeometric analysis was used to identify canonical pathways and curated networks having statistically significant enrichment of differentially expressed genes. Osteopontin (OPN) expression was validated by immunohistochemistry. Hierarchical clustering defined signatures, which differentiated between cancer and healthy tissue, muscle-invasive or non-muscle invasive cancer and healthy tissue, grade 1 and grade 3. Pathways associated with cell cycle and proliferation were markedly upregulated in muscle-invasive and grade 3 cancers. Genes associated with the classical complement pathway were downregulated in non-muscle invasive cancer. Osteopontin was markedly overexpressed in invasive cancer compared to healthy tissue. The present study contributes to a growing body of work on gene expression signatures in bladder cancer. The data support an important role for osteopontin in bladder cancer, and identify several pathways worthy of further investigation.

Hypersensitive K303R oestrogen receptor-alpha variant not found in invasive carcinomas.

INTRODUCTION: Genetic abnormalities or mutations in premalignant breast lesions may have a role in progression toward malignancy or influence the behaviour of subsequent disease. The A908G (Lys303-->Arg) change in the gene encoding oestrogen receptor-alpha (ER-alpha) creates a hypersensitivity to oestradiol and would have significant consequences if present in breast carcinoma, especially those treated with endocrine therapy. We have therefore examined a panel of endocrine-treated invasive carcinomas for the presence of this mutation. METHODS: Sequencing of control DNA was shown to detect mutation present in as little as 15% of the starting material. Enrichment for the mutation by using MboII restriction digestion allowed the detection of mutant present at 1% or less. We applied these techniques to genomic DNA and cDNA from 136 invasive breast carcinomas. RESULTS: No evidence of the A908G mutation was found with either technique. The incidence of this mutation in our panel of tumours is therefore significantly less than previously reported. CONCLUSION: The fact that the mutation was not found leads us to believe that this mutation is absent from most cells in invasive carcinomas and furthermore that the major expression product of the ER-alpha gene in cancers does not contain the K303R mutation. It is therefore unlikely to influence the effectiveness of endocrine treatment.

Molecular and genetic abnormalities in radial scar.

Hyperplasia of usual type (HUT) may be an early precursor of breast carcinoma and has been shown to contain molecular and genetic abnormalities previously seen in more advanced breast lesions, such as allelic imbalance (AI) and coexpression of estrogen receptor-alpha (ER) and the proliferation marker Ki67. We have examined hyperplastic and other areas from within radial scar (RS) for such abnormalities, to explore whether such regions of RS are similar at the molecular and genetic level to histologically similar lesions found independent of RS. Abnormal expression of ER and Ki67 in hyperplastic foci and other histologically distinct areas within RS was detected by dual-label immunofluorescence. Subtle differences in expression patterns were seen compared to similar lesions outside RS, with a lower overall level of ER overexpression in HUT within RS (P = 0.0012) and less evidence of the abnormal ER association with Ki67 (P = 0.004). AI of chromosome 16q and 8p was detected in RS, indicating that at least some areas of RS are clonal and neoplastic, but no clear relationship to ER dysregulation was found. Different genetic losses seen in microdissected areas of the same RS indicated clonal differences between these areas. The role of RS as a marker of malignancy and relative risk of breast cancer remains uncertain. Nonetheless, here we provide evidence that some molecular and genetic changes that occur to a greater degree in breast cancer and some premalignant breast lesions are present in a minority of RS.

Gene expression in oligodendroglial tumors.

BACKGROUND: Oligodendroglial tumors with 1p/19q loss are more likely to be chemosensitive and have longer survival than those with intact 1p/19q, but not all respond to chemotherapy, warranting investigation of the biological basis of chemosensitivity. METHODS: Gene expression profiling was performed using amplified antisense RNA from 28 oligodendroglial tumors treated with chemotherapy [26 serial stereotactic biopsy, 2 resection]. Expression of differentially expressed genes was validated by real-time PCR. RESULTS: Unsupervised hierarchical clustering showed clustering of multiple samples from the same case in 14/17 cases and identified subgroups associated with tumor grade and 1p/19q status. 176 genes were differentially expressed, 164 being associated with 1p/19q loss (86% not on 1p or 19q). 94 genes differed between responders and non-responders to chemotherapy; 12 were not associated with 1p/19q loss. Significant differential expression was confirmed in 11/13 selected genes. Novel genes associated with response to therapy included SSBP2, GFRA1, FAP and RASD1. IQGAP1, INA, TGIF1, NR2F2 and MYCBP were differentially expressed in oligodendroglial tumors with 1p/19q loss. CONCLUSION: Gene expression profiling using serial stereotactic biopsies indicated greater homogeneity within tumors than between tumors. Genes associated with 1p/19q status or response were identified warranting further elucidation of their role in oligodendroglial tumors.

Gene expression in oligodendroglial tumors.

BACKGROUND: Oligodendroglial tumors with 1p/19q loss are more likely to be chemosensitive and have longer survival than those with intact 1p/19q, but not all respond to chemotherapy, warranting investigation of the biological basis of chemosensitivity. METHODS: Gene expression profiling was performed using amplified antisense RNA from 28 oligodendroglial tumors treated with chemotherapy (26 serial stereotactic biopsy, 2 resection). Expression of differentially expressed genes was validated by real-time PCR. RESULTS: Unsupervised hierarchical clustering showed clustering of multiple samples from the same case in 14/17 cases and identified subgroups associated with tumor grade and 1p/19q status. 176 genes were differentially expressed, 164 being associated with 1p/19q loss (86% not on 1p or 19q). 94 genes differed between responders and non-responders to chemotherapy; 12 were not associated with 1p/19q loss. Significant differential expression was confirmed in 11/13 selected genes. Novel genes associated with response to therapy included SSBP2, GFRA1, FAP and RASD1. IQGAP1, INA, TGIF1, NR2F2 and MYCBP were differentially expressed in oligodendroglial tumors with 1p/19q loss. CONCLUSION: Gene expression profiling using serial stereotactic biopsies indicated greater homogeneity within tumors than between tumors. Genes associated with 1p/19q status or response were identified warranting further elucidation of their role in oligodendroglial tumors.

Microarray analysis of suppression subtracted hybridisation libraries identifies genes associated with breast cancer progression.

BACKGROUND: A major challenge of cancer research is to identify key molecules which are responsible for the development of the malignant metastatic phenotype, the major cause of cancer death. METHODS: Four subtracted cDNA libraries were constructed representing mRNAs differentially expressed between benign and malignant human breast tumour cells and between micro-dissected breast carcinoma in situ and invasive carcinoma. Hundreds of differentially expressed cDNAs from the libraries were micro-arrayed and screened with mRNAs from human breast tumor cell lines and clinical specimens. Gene products were further examined by RT-PCR and correlated with clinical data. RESULTS: The combination of subtractive hybridisation and microarray analysis has identified a panel of 15 cDNAs which shows strong correlations with estrogen receptor status, malignancy or relapse. This panel included S100P, which was associated with aneuploidy in cell lines and relapse/death in patients, and AGR2 which was associated with estrogen receptor and with patient relapse. X-box binding protein-1 is also an estrogen-dependent gene and is associated with better survival for breast cancer patients. CONCLUSIONS: The combination of subtracted cDNA libraries and microarray analysis has thus identified potential diagnostic/prognostic biomarkers and targets for cancer therapy, which have not been identified from common prognostic gene signatures.

Examination of tumour histopathology and gene expression in a neu/S100A4 transgenic model of metastatic breast cancer.

Elevated levels of the calcium-binding protein S100A4 have been causally linked to the metastatic spread of breast cancer cells in several in vitro and in vivo model systems and, more recently, correlated with patient death in a series of human breast cancer specimens. In transgenic mice expressing MMTV-neu transgenes in mammary gland, additional expression of S100A4 transgenes results in an enhanced metastatic capability. Despite this phenotypic difference arising from elevated S100A4, it is now shown that the primary breast tumours in all mice examined are histopathologically very similar and resemble those human tumours associated with elevated c-erbB-2. Using a panel of genes identified by suppression subtractive hybridization of cDNAs from individual primary tumours and a metastasis, some cDNAs were found to exhibit a differential pattern of expression associated with the expression of S100A4 protein (including osteopontin, S100A9, claudin 2 and several Expressed Sequence Tags sequences). Whilst confirming differential expression of these genes, it was demonstrated that individual primary tumours of matched transgenic status, histology and grade exhibit some degree of heterogeneity at the mRNA level by reverse Northern and Northern hybridizations. This intertumour heterogeneity of mRNA level was confirmed by cDNA array analysis and suggests that even in a transgenic model, which exhibits far less variation than the human disease, there may be multiple mechanisms of disease progression.