Evaluation of applying IHC4 as a prognostic model in the translational study of Intergroup Exemestane Study (IES): PathIES.

BACKGROUND: Intergroup Exemestane Study (IES) was a randomised study that showed a survival benefit of switching adjuvant endocrine therapy after 2-3 years from tamoxifen to exemestane. This PathIES aimed to assess the role of immunohistochemical (IHC)4 score in determining the relative sensitivity to either tamoxifen or sequential treatment with tamoxifen and exemestane. PATIENTS AND METHODS: Primary tumour samples were available for 1274 patients (27% of IES population). Only patients for whom the IHC4 score could be calculated (based on oestrogen receptor, progesterone receptor, HER2 and Ki67) were included in this analysis (N = 430 patients). The clinical score (C) was based on age, grade, tumour size and nodal status. The association of clinicopathological parameters, IHC4(+C) scores and treatment effect with time to distant recurrence-free survival (TTDR) was assessed in univariable and multivariable Cox regression analyses. A modified clinical score (PathIEscore) (N = 350) was also estimated. RESULTS: Our results confirm the prognostic importance of the original IHC4, alone and in conjunction with clinical scores, but no significant difference with treatment effects was observed. The combined IHC4 + Clinical PathIES score was prognostic for TTDR (P < 0.001) with a hazard ratio (HR) of 5.54 (95% CI 1.29-23.70) for a change from 1st quartile (Q1) to Q1-Q3 and HR of 15.54 (95% CI 3.70-65.24) for a change from Q1 to Q4. CONCLUSION: In the PathIES population, the IHC4 score is useful in predicting long-term relapse in patients who remain disease-free after 2-3 years. This is a first trial to suggest the extending use of IHC4+C score for prognostic indication for patients who have switched endocrine therapies at 2-3 years and who remain disease-free after 2-3 years.

Prognostic and predictive value of ERß1 and ERß2 in the Intergroup Exemestane Study (IES)-first results from PathIES†.

BACKGROUND: Intergroup Exemestane Study (IES) was a randomised study that showed a survival benefit of switching adjuvant endocrine therapy after 2-3 years from tamoxifen to exemestane. PathIES aimed to assess the potential prognostic and predictive value of ERß1 and ERß2 expression in primary tumours in order to determine benefit in the two treatment arms. PATIENTS AND METHODS: Primary tumour samples were available for 1256 patients (27% IES population). ERß1 and ERß2 expression was dichotomised at the median IHC score (high if ERß1 ≥ 191, ERß2 ≥ 164). Hazard ratios (HRs) were estimated by multivariable Cox proportional hazards models adjusting for clinicopathological factors. Treatment effects with biomarker expressions were determined by interaction tests. Analysis explored effects of markers both as a continuous variable and with dichotomised cut-offs. RESULTS: Neither ERß1 nor ERß2 were associated with disease-free survival (DFS) or overall survival (OS) in the whole cohort. In patients treated with continued tamoxifen, high ERß1 expression compared with low was associated with better DFS [HR = 0.38:95% confidence interval (CI) 0.21-0.68, P = 0.001]. DFS benefit of exemestane over tamoxifen (HR = 0.40:95% CI 0.22-0.70) was found in the low ERß1 subgroup (interaction P = 0.01). No significant difference with treatment was observed for ERß2 expression in either DFS or OS. CONCLUSION: In the PathIES population, exemestane appeared to be superior to tamoxifen among patients with low ERß1 expression but not in those with high ERß1 expression. This is the first trial of its kind to report a parameter potentially predicting benefit of an aromatase inhibitor when compared with tamoxifen and an independent validation is warranted.

The prognostic significance of tumour-stroma ratio in oestrogen receptor-positive breast cancer.

BACKGROUND: A high percentage of stroma predicts poor survival in triple-negative breast cancers but is diminished in studies of unselected cases. We determined the prognostic significance of tumour-stroma ratio (TSR) in oestrogen receptor (ER)-positive male and female breast carcinomas. METHODS: TSR was measured in haematoxylin and eosin-stained tissue sections (118 female and 62 male). Relationship of TSR (cutoff 49%) to overall survival (OS) and relapse-free survival (RFS) was analysed. RESULTS: Tumours with ≥49% stroma were associated with better survival in female (OS P=0.008, HR=0.2-0.7; RFS P=0.006, HR=0.1-0.6) and male breast cancer (OS P=0.005, HR=0.05-0.6; RFS P=0.01, HR=0.87-5.6), confirmed in multivariate analysis. CONCLUSIONS: High stromal content was related to better survival in ER-positive breast cancers across both genders, contrasting data in triple-negative breast cancer and highlighting the importance of considering ER status when interpreting the prognostic value of TSR.

Mechanisms of FGFR3 actions in endocrine resistant breast cancer.

Although endocrine therapy has dramatically improved the treatment of breast cancer therapeutic resistance and tumour recurrence occurs, even in estrogen receptor (ER) positive cases. Identifying and understanding the molecular mechanisms which underpin endocrine resistance is therefore important if future therapeutic strategies are to be developed. Members of the fibroblast growth factor (FGF) and fibroblast growth factor receptor (FGFR) families have been implicated in breast cancer development and progression. Our results demonstrate that culture of michigan cancer foundation - 1 (MCF)7 cells with FGF1 results in reduced sensitivity to tamoxifen in vitro. Furthermore, our tissue microarray expression data demonstrates that FGFR3 expression is increased in tamoxifen resistant breast tumours. To confirm that activation of FGFR3 reduced sensitivity to tamoxifen we used an inducible activation system and a constitutively active mutant of FGFR3 expressed in MCF7 cells. Activation of FGFR3 reduced sensitivity to tamoxifen and Fulvestrant but did not lead to phosphorylation of ER demonstrating that FGFR3 does not feedback to modulate ER activity. FGFR3 activation in MCF7 cells stimulated activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signalling pathways, both of which have been implicated in tamoxifen resistance in breast cancer. Furthermore, our data indicates that activation of phospholipase C gamma is a key-signalling event regulating MAPK and PI3K activation and that its activation reduces sensitivity to tamoxifen. Therefore, we hypothesise that FGFRs could play an integral part, not only in breast cancer development but also in resistance to endocrine-therapy.

Expression of COX-2, NF-kappaB-p65, NF-kappaB-p50 and IKKalpha in malignant and adjacent normal human colorectal tissue.

BACKGROUND: Cyclooxygenase-2 (COX-2) is selectively over-expressed in colorectal tumours. The mechanism of COX-2 induction in these tumours is not fully understood, although evidence suggests a possible link between nuclear factor (NF)-kappaB and COX-2. We hypothesised an association between COX-2 expression and NF-kappaB-p65, NF-kappaB-p50 and IkappaB-kinase-alpha (IKKalpha) in both epithelial and stromal cells in human colorectal cancer. METHODS: Using immunohistochemistry, we measured COX-2, NF-kappaB-p65, NF-kappaB-p65 nuclear localisation sequence (NLS), NF-kappaB-p50, NF-kappaB-p50 NLS and IKKalpha protein expression in matched colorectal biopsy samples comprising both non-tumour and adjacent tumour tissue from 32 patients with colorectal cancer. RESULTS: We have shown that stromal cells of malignant and surrounding normal colorectal tissue express COX-2. In all cell types of malignant tissue, and in vascular endothelial cells (VECs) of neighbouring normal tissue, COX-2 expression was strongly associated with NF-kappaB-p65 expression (Pearson's correlation, P=0.019 for macrophages, P=0.001 for VECs, P=0.002 for fibroblasts (malignant tissue), and P=0.011 for VECs (non-malignant tissue)) but not NF-kappaB-p50 or IKKalpha. CONCLUSIONS: These data suggest that in these cells COX-2 induction may be mediated through activation of the canonical NF-kappaB pathway. Finally, the lack of association between COX-2, NF-kappaB-p65 or IKKalpha in stromal cells with the clinical severity of colorectal cancer as determined by Duke's stage, suggests that COX-2, NF-kappaB-p65 and IKKalpha expression are possibly early post-initiation events, which could be involved in tumour progression.

Combined analysis of eIF4E and 4E-binding protein expression predicts breast cancer survival and estimates eIF4E activity.

Increased eukaryotic translation initiation factor 4E (eIF4E) expression occurs in many cancers, and makes fundamental contributions to carcinogenesis by stimulating the expression of cancer-related genes at post-transcriptional levels. This key role is highlighted by the facts that eIF4E levels can predict prognosis, and that eIF4E is an established therapeutic target. However, eIF4E activity is a complex function of expression levels and phosphorylation statuses of eIF4E and eIF4E-binding proteins (4E-BPs). Our hypothesis was that the combined analyses of these pathway components would allow insights into eIF4E activity and its influence on cancer. We have determined expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 within 424 breast tumours, and have carried out analyses to combine these and relate the product to patient survival, in order to estimate eIF4E activity. We show that this analysis gives greater prognostic insights than that of eIF4E alone. We show that eIF4E and 4E-BP expression are positively associated, and that 4E-BP2 has a stronger influence on cancer behaviour than 4E-BP1. Finally, we examine eIF4E, estimated eIF4E activity, and phosphorylated 4E-BP1 as potential predictive biomarkers for eIF4E-targeted therapies, and show that each determines selection of different patient groups. We conclude that eIF4E's influence on cancer survival is modulated substantially by 4E-BPs, and that combined pathway analyses can estimate functional eIF4E.

The potential role of ER beta isoforms in the clinical management of breast cancer.

The discovery of a second oestrogen receptor, ER beta, was a subject of much interest, as this suggested a means to improve the prognostic stratification of invasive breast cancer, better predict response to endocrine therapy, develop new chemotherapeutic/chemopreventative drugs and perhaps prevent inappropriate treatment. However, this has not proved to be straightforward with the discovery of five ER beta isoforms and numerous exon deletion variants. This review sets out to identify the present state of knowledge regarding the clinicopathological role of ER beta isoforms and discusses possible reasons for conflicting results arising from recent research findings.

Changes in expression of steroid receptors, their downstream target genes and their associated co-regulators during the sequential acquisition of tamoxifen resistance in vitro.

Tamoxifen resistance (TAMr) in breast cancer is a serious clinical dilemma, with no satisfactory explanation. We hypothesised that changes in the expression of steroid hormone receptors (ERalpha, ERbeta), their downstream target genes (PR, pS2) and their associated co-regulators (AIB-1, SRC-1, SRA, NCoR-1, SMRT and REA) could be related to the acquisition of TAMr. To test this hypothesis, we developed in vitro TAMr cell line models by continuous exposure of MCF-7 cells to 4-hydroxytamoxifen (4-HT) over 12 (MCF-7MMU1) and 21 (MCF-7MMU2) months, respectively and examined the expression of the above by Western blotting and immunohistochemistry. In addition, we further examined the changes in global gene expression in TAMr cells in comparison with TAM-sensitive cells by microarray analysis. We report here that acquisition of TAMr is associated with changes in the expression of PR, pS2 and several co-activators, but not ERs. In addition, genes associated with cell cycle, cell adhesion and extracellular matrix, were up-regulated while those associated with apoptosis or growth factors/hormones were down-regulated. Based on our results, it appears that increased co-activator expression, in concert with alterations in genes associated with controlling cell proliferation and survival contribute to TAMr in breast cancer.

Clinicians' guide to microarrays.

Cancer is a heterogeneous disease with wide-ranging subsets of patients who have different prognoses and who respond differently to treatments. Accordingly, deciding the best treatment strategy has become a priority in cancer care. The development of microarray technology over the last decade has caused great excitement, with the promise that these new tools may provide molecular signatures to help predict patient outcome and direct therapeutic approaches. Here, we describe the different types of microarrays available and discuss their pros and cons from a clinical perspective, with respect to breast cancer.

Steroid hormone receptor expression in male breast cancer.

AIMS: To investigate expression of the steroid hormone receptors estrogen receptor (ER)-alpha and -beta, progesterone receptor (PR) and androgen receptor (AR) in male breast cancer. METHODS: Specimens from 16 male breast cancers were immunostained for ERalpha, ERbeta, PR and AR. FINDINGS: Eighty-seven percent of tumours expressed ERalpha, 93% PR, 87% ERbeta and 87% AR. Staining for ERalpha and PR was confined exclusively to the nuclei of epithelial cells with some heterogeneity. Nuclear immunoreactivity was also observed with AR. Again this was restricted to epithelial cells but tended to be more uniform. ERbeta was seen in the nuclei of epithelial cells and also in stromal fibroblasts and lymphocytes. Analysis of serial sections revealed a similar pattern of staining with ERbeta and AR in epithelial cells. CONCLUSIONS: In addition to expression of the better known steroid receptors, ERalpha, PR and AR, we have demonstrated a high rate of expression of ERbeta in male breast cancer. This is in keeping with the generally high steroid receptor expression seen in males. However, the abundance of ERbeta expressed in this small series of male breast cancer is in contrast to female breast cancer where ERbeta expression is often reduced.

Properties and characteristics of the dyes injected to assist axillary sentinel node localization in breast surgery.

AIMS: A review of the safety profile of dyes injected to assist in sentinel lymph node biopsy (SLNB) in breast cancer. METHODS: A literature search was performed of the medline database 1966-2005 using the Ovid web Gateway detailing the words sentinel node, breast cancer, allergic reactions, blue dye, isosulfan blue, patent blue and methylene blue. RESULTS: There are reported side-effects from the parenteral administration of dyes, which range from minor to life threatening in severity. There are differences between the dyes as regards their effects. These aspects are discussed. CONCLUSION: Many dyes have been used for SLNB with acceptable identification rates. There are variable side-effects for each of those dyes. Further research is needed to clarify the safety profile of these dyes.

Increased expression of estrogen receptor beta mRNA in tamoxifen-resistant breast cancer patients.

Tamoxifen is currently the first-line therapy for treatment of hormone-dependent breast cancer. However, despite initial benefits, most patients eventually relapse. Two groups of patients were identified: (a) a tamoxifen-sensitive group (n = 8); and (b) a tamoxifen-resistant group (n = 9). Using reverse transcription-PCR, the relative expression of mRNA for both estrogen receptor (ER) beta and transforming growth factor beta1 was determined in each patient group and quantified against a known reference standard. ER-beta mRNA was significantly up-regulated in the tamoxifen-resistant group as compared with the tamoxifen-sensitive group (P = 0.001 by Fisher's exact test), and, consistent with previous findings, transforming growth factor beta1 was also up-regulated in the tamoxifen-resistant cohort (P = 0.02). The importance of ER-beta in tamoxifen resistance was validated using tamoxifen-sensitive and -resistant cell lines, in which it was demonstrated that ER-beta mRNA was significantly up-regulated in the resistant cells. These results lend further support to a role for ER-beta as a poor prognostic factor in breast cancer.

Coexpression of estrogen receptor alpha and beta: poor prognostic factors in human breast cancer?

The cloning of a second estrogen receptor (ER), ER beta, has prompted a reevaluation of the role of ERs in breast cancer. The aim of this study was to determine the expression of both ER isoforms in normal (n = 23) and malignant (n = 60) human breast tissue by reverse transcription-PCR and correlate this information with known prognostic factors including tumor grade and node status. In normal breast tissue, expression of ER beta predominated, with 22% of samples exclusively expressing ER beta; this was not observed in any of the breast tumor samples investigated. Most breast tumors expressed ER alpha, either alone or in combination with ER beta. Interestingly, those tumors that coexpressed ER alpha and ER beta were node positive (P = 0.02; Fisher's exact test) and tended to be of higher grade. Because antiestrogens are agonists when signaling through the AP1 element, overexpression of ER beta in tumors expressing both ER subtypes may explain the failure of antiestrogen therapy in some breast cancer patients. Thus, ER beta may be a useful prognostic factor in patients with breast cancer.

Expression of prostate specific antigen in male breast cancer.

Male breast cancer is uncommon, accounting for less than 1% of all breast cancers. Carcinoma metastatic to the male breast is also unusual, with metastatic prostatic carcinoma being among the most common primary sites from which such tumours derive. Metastatic prostatic cancer and primary breast cancer may be histologically indistinguishable without immunohistochemistry because both often infiltrate with a cribriform architecture. Distinguishing between primary and metastatic disease within the breast is important because the treatment options for each are radically different. Following a case in which metastatic prostatic disease was initially wrongly diagnosed as primary breast cancer, a small series of male breast cancers was examined for expression of prostate specific antigen (PSA) and prostatic acid phosphatase to assess the usefulness of these markers in making this distinction. Focal expression of PSA was found in one of 11 cases of male breast cancer. These results indicate that PSA should be used with caution in this context.

Loss of expression of oestrogen receptor beta in colon cancer and its association with Dukes' staging.

Gender differences in the incidence and behaviour of colon cancer suggest a hormonal influence and epidemiological data suggest a protective effect for hormone replacement therapy. Recently, it has been shown that oestrogen receptor (ER) beta is the predominant ER in colon tissue. The aim of this study was to examine the expression and distribution of ERbeta in normal and colorectal cancer samples, using immunohistochemistry and (in a subset of patients) real-time quantitative reverse transcriptase polymerase chain reaction in a well-defined patient cohort and to correlate this with clinico-pathological outcome. Immunohistochemical analyses of normal colon revealed strong specific nuclear immuno-reactivity in all epithelial cells lining the colonic crypts. In colon cancer, ERbeta expression was lost in 21% of samples irrespective of patient age or gender. Interestingly loss of ERbeta expression was higher in left colon and rectal cancers (27%) compared to right colon cancers (8%). A correlation between loss of ERbeta expression and advanced Dukes stage was observed. Loss of ERbeta with increased Dukes' stage suggests that it may be affording a protective effect against colon carcinogenesis. Its presence may be a favourable prognostic marker in this disease and could explain the protective effect of oestrogens against colon cancer development.

Angiogenesis in breast cancer: how should we measure this? (Review).

Despite the established importance of angiogenesis in the pathogenesis of solid tumours, there is still no consensus on how this is best measured or which method is the most appropriate in the determination of prognosis. Here we review the pros and cons of current methods of assessing angiogenesis, both clinical and in the laboratory, and discuss with respect to breast cancer.

A novel cell array technique for high-throughput, cell-based analysis.

Microarray technology has burgeoned over the past few years from nucleic acid-based arrays to tissue microarrays (TMAs). This study aimed to develop a technique to incorporate cell lines into an array and to demonstrate the usefulness of this technique by performing immunohistochemistry for beta-catenin. Cell suspensions were prepared from 23 tumor cell lines. These were fixed in formalin, suspended in agar, and embedded in paraffin to produce a cell block. A "tissue microarrayer" was used to remove triplicate, 0.6 mm-cores from each cell block and to transfer these into a recipient paraffin block at precise coordinates. Immunohistochemistry was used to identify cell lines positive for beta-catenin. Cultured cells were successfully incorporated into the microarray, with preservation of cell architecture and even distribution of cells within each core. A total of 18 of 69 cores (26%) were lost in processing. A total of 16 of 23 cell lines were identified as positive for membrane and cytoplasmic beta-catenin, and 6 of 23 were negative. Only one cell line was unscorable because of complete core loss. We have developed a "cell microarray" technique for analyzing antigen expression by immunohistochemistry in multiple cell lines in a single experiment. This novel application of microarrays permits high-throughput, cost-efficient analysis, with the potential to rapidly identify markers with potential diagnostic and therapeutic implications in human disease.

Identification of wild-type and exon 5 deletion variants of estrogen receptor beta in normal human mammary gland.

We have examined messenger RNA (mRNA) expression of estrogen receptor (ER) alpha, wild-type ERbeta (mRNA and protein), and ERbeta exon 5 deletion variants (ERbeta delta5) in samples of normal human mammary gland obtained from 37 premenopausal subjects undergoing reduction mammoplasty. Comparing individual expression, ERbetaP mRNA predominated, expressed in 34 of 37 samples (91%), whereas ERalpha was found in 21 of 37 cases (57%). Receptor combinations were then analyzed and compared. Most samples either coexpressed ERalpha with ERbeta (54%) or expressed just ERbeta (38%). Immunohistochemical analysis revealed that ERbeta mRNA expression mirrored that of protein. Immunoreactivity was observed in the nucleus with additional evidence of cytoplasmic staining in those epithelial cells lining the breast ducts. Sporadic immunoreactivity was also detected in stromal cells. Expression of wild type and ERbeta delta5 was analyzed, and their association with ERalpha was compared. Most samples coexpressed wild-type ERbeta and the splice variant (62%; P = 0.05), with 30% exclusively expressing wild-type ERbeta. Although samples coexpressing wild type and variant ERbeta showed no statistical association with ERalpha, those samples expressing only wild-type ERP, showed a trend toward associations with ERalpha (P = 0.07). In conclusion, our data would support a role for ERbeta in the normal human mammary gland, where we propose it may be the dominant receptor.