Elizabeth Anderson

Estrogen Receptor-Positive Proliferating Cells in the Normal and Precancerous Breast

Recently it has been shown that epithelial cell expression of the estrogen receptor (ER) and that of the proliferation-associated marker Ki-67 are almost mutually exclusive in the normal premenopausal human breast but that coexpression frequently occurs in estrogen receptor-positive (ER+) breast cancers. This coexpression may indicate disordered expression of ER in the cell cycle or failure to suppress division of ER+ cells and could be important in neoplastic transformation. The purpose of this study was to determine whether in situ proliferations known to be associated with different levels of risk for developing breast cancer contain these coexpressing cells and, if so, the stage at which they occur. We found that ER+ proliferating cells were rare in premenopausal lobules but increased with age in the normal breast. There was no difference in nonlesional tissue between cancerous and noncancerous breasts. The percentage of dual-expressing cells was significantly increased, however, in all of the in situ proliferations and correlated positively with the level of risk of developing breast cancer. We suggest that development of at least some human breast cancers is associated with increasing failure to down-regulate ER as cells enter the cycle or to suppress division of ER+ cells. The mechanism may involve the loss of a tumor suppressor gene.

Prospective comparison of switches in biomarker status between primary and recurrent breast cancer: the Breast Recurrence In Tissues Study (BRITS)

IntroductionImmunohistochemistry of primary breast cancer is routinely used to guide changes in therapy at the time of relapse. Retrospective reviews suggest that the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor type 2 (HER2) receptor may differ between the primary and loco-regional recurrence or distant metastases. The Breast Recurrence In Tissues Study (BRITS) was a large, multicentre, prospective study to examine changes in ER, PR and HER2.MethodsMatched primary and recurrent breast cancer tissue samples were prospectively collected from 205 women attending 20 institutions. Central laboratory immunohistochemical analysis of core biopsies and tissue microarrays of ER and PR using the Allred and Quickscore methods and HER2 (confirmed by fluorescence in situ hybridisation (FISH) for HER2 2+) were performed.ResultsFrom 205 consenting women, 18 (8.8%) did not have recurrent disease on biopsy, 35 were ineligible, 13 had insufficient paired tissue and 2 were excluded for safety reasons. Paired samples from 137 women, mean age 62.6 years (range 27-87 years), 83/137 (60.6%) postmenopausal with a median 92.2 months (range 5-327 months) from primary to recurrence and 88 (64.2%) as locoregional recurrence were successfully analysed. A switch in receptor status, in either direction, by Allred score, was identified for ER in 14 patients (10.2%; P = 0.983 Wilcoxon sign rank test), PR in 34 (24.8%; P = 0.003 Wilcoxon sign rank test) and HER2 in 4 (2.9%; P = 0.074 Wilcoxon sign rank test). There was no difference between locoregional or distant recurrence in the proportion who switched. The switch in receptor status led to a change in the subsequent treatment plan for 24 patients (17.5%).ConclusionsThis prospective study confirms retrospective evidence that the management of relapsed breast cancer should include confirmatory tissue sampling and identify switches of ER, PR or HER2 which change therapeutic management for one in six patients.

Phase II, randomized trial to compare anastrozole combined with gefitinib or placebo in postmenopausal women with hormone receptor-positive metastatic breast cancer

Purpose: This phase II randomized trial evaluated the efficacy and tolerability of anastrozole combined with gefitinib or anastrozole with placebo in women with hormone receptor–positive metastatic breast cancer (MBC). Experimental Design: Postmenopausal women with hormone receptor–positive measurable or evaluable MBC who had not received prior endocrine therapy for this disease stage or who developed metastatic disease during/after adjuvant tamoxifen were eligible. The primary response variable was progression-free survival (PFS) and secondary response variables included clinical benefit rate, objective response rate, overall survival, safety and tolerability, and pharmacokinetics. Tumor biomarker evaluation was an exploratory objective. Results: Forty-three patients were randomized to anastrozole plus gefitinib and 50 patients were randomized to anastrozole plus placebo of a planned total of 174 patients (enrollment was prematurely discontinued due to slow recruitment). PFS for patients receiving the combination of anastrozole and gefitinib was longer than for patients receiving anastrozole plus placebo [hazard ratio (gefitinib/placebo), 0.55; 95% confidence interval, 0.32-0.94; median PFS, 14.7 versus 8.4 months]. The clinical benefit rate was 49% versus 34%, and the objective response rate was 2% versus 12% with anastrozole plus gefitinib and anastrozole plus placebo, respectively. No evidence of interaction between baseline biomarker levels and relative treatment effect was found. No unexpected adverse events were observed. Conclusion: This small randomized study showed that anastrozole in combination with gefitinib is associated with a marked advantage in PFS compared with anastrozole plus placebo, and that the combination was tolerated in postmenopausal women with hormone receptor–positive MBC. Further investigation of epidermal growth factor receptor inhibition in combination with endocrine therapy may be warranted. Clin Cancer Res; 16(6); 1904–14

Expression of activated gelatinase in human invasive breast carcinoma.

The expression of both 92- and 72-kDa gelatinases has been studied in 20 samples of human breast carcinoma by the technique of gelatin zymography. This technique allowed the relative amount of each gelatinase to be determined in small samples of tissue (<10 mg). More importantly, active and latent forms of the two gelatinases were resolved. Two samples (10–20 mg) were cut from each piece of tumour in order to monitor the variability of gelatinase distribution within that section of tumour. The 72-kDa latent progelatinase was present in 15 of the 20 tumours, with trace amounts in two others. The 62-kDa activated form of this gelatinase was detected in all 15 of the tumours in which the latent form was present. The 92-kDa latent progelatinase was present in 11 of the 20 tumours, with trace amounts in four others. However, the 82-kDa activated form of this gelatinase was only clearly detected in two tumours, although three others showed the presence of trace amounts. The ratio of active to latent forms of the 72-kDa gelatinase ranged from 0.9 to 3.6. There were no marked correlations between gelatinase expression and established staging and prognostic markers. Analysis of three samples of fibroadenoma revealed only very low levels of gelatinase expression. On the basis of these results, activation of the 72-kDa progelatinase appears to be a more common event in invasive breast carcinoma than activation of the 92-kDa progelatinase. However, neither proteinase showed a correlation with metastatic progression, as measured by lymph node involvement.

Regulation of human breast epithelial stem cells

Abstract.u2002 Breast epithelial stem cells are thought to be the primary targets in the aetiology of breast cancer. As breast cancers are predominantly oestrogen and progesterone receptor‐positive (ERα/PR+), we investigated the biology of ERα/PR+ cells and their relationship to stem cells in normal human breast epithelium. Several complementary approaches were used to characterize the stem‐cell population and relate it to ERα/PR+ cells, including dual label colocalization on tissue sections, isolation of a Hoechst dye‐effluxing ‘side population’ using flow cytometry, and examination of DNA label retention. The intermediate or suprabasal population suggested by others to be breast stem cells comprises ERα/PR+ cells that coexpress the putative stem‐cell markers including cytokeratin 19. Human breast epithelial cells with Hoechst dye‐effluxing ‘side population’ properties characteristic of mammary stem cells in mice were demonstrated by lack of expression of myoepithelial and luminal cell‐specific antigens such as CALLA and MUC1 to be undifferentiated cells. Using DNA radiolabelling of human tissue implanted into athymic nude mice, a population of label‐retaining putative stem cells (LRC) were shown to be enriched for cells expressing the putative stem‐cell markers p21CIP1/WAF1 and Musashi‐1, which, interestingly, were expressed in separate subpopulations of ERα/PR+ cells. Finally, expression patterns of Musashi‐1 and Notch‐1 in relation to ERα/PR+ and adjacent proliferating cells suggest that the evolutionarily conserved Delta/Notch signalling pathway regulates asymmetric division of the putative stem‐cell population. The data suggest a model in which ERα/PR+ cells scattered through the epithelium are stem cells that self‐renew through asymmetric cell division and generate patches of transit amplifying and differentiated cells. ERα/PR+ breast cancers exhibit loss of the two key regulators of asymmetric cell division, Musashi‐1 and Notch‐1 and thus may arise from symmetric division of the ERα/PR+ stem cell.

Evidence for reciprocity of bcl-2 and p53 expression in human colorectal adenomas and carcinomas

Evidence of accumulating for the failure of apoptosis as an important factor in the evolution of colorectal cancer and its poor response to adjuvant therapy. The proto-oncogene bcl-2 suppresses apoptosis. Its expression could provide an important survival advantage permitting the development of colorectal cancer. The expression of bcl-2 and p53 was determined by immunohistochemistry in 47 samples of histologically normal colonic mucosa, 19 adenomas and 53 adenocarcinomas. Expression of bcl-2 in colonic crypts > 5 cm from the tumours was confined to crypt bases but was more extensive and intense in normal crypts < 5 mm from cancers. A higher proportion of adenomas (63.2%) than carcinomas (36.5%) expressed bcl-2 (P < 0.05). A lower proportion of adenomas (31.6%) than carcinomas (62.3%) expressed p53 (P < 0.02). A total of 26.3% of adenomas and 22% of carcinomas expressed both bcl-2 and p53. To determine whether these samples contained cells which expressed both proteins, a dual staining technique for bcl-2 and p53 was used. Only 1/19 adenomas and 2/53 carcinomas contained cells immunopositive for both bcl-2 and p53. Moreover there was evidence of reciprocity of expression of bcl-2 and p53 in these three double staining neoplasms. We suggest that bcl-2 provides a survival advantage in the proliferative compartment of normal crypts and colorectal neoplasms. However, its expression is lost during the evolution from adenoma to carcinoma, whereas p53 expression is increased, an event generally coincident with the expression of stabilised p53, which we presume to represent the mutant form.

Effect of tamoxifen on Ki67 labelling index in human breast tumours and its relationship to oestrogen and progesterone receptor status.

This study aimed to investigate the effect of tamoxifen on breast tumour levels of oestrogen and progesterone receptor (ER and PR) and proliferation as defined by the Ki67 antibody. A group of primary breast cancer patients was randomised to receive either tamoxifen (n = 59) or placebo (n = 44) treatment in the interval between clinic and surgery (median 21 days). Frozen sections of breast tumour biopsies obtained before and after treatment were stained immunocytochemically to obtain the percentage of nuclei containing ER and PR, and a Ki67 labelling index (LI). Tamoxifen-treated patients had a median Ki67 LI of 5.6% in the first biopsy falling to 3.0% in the second biopsy (P < 0.001 by Wilcoxons matched pairs test), whereas placebo-treated patients had a median Ki67 LI of 5.4% in the first biopsy and 5.75% in the second (no significant difference). No significant differences were observed when the median %ER or %PR staining before and after treatment were compared. The Ki67 LI tended to increase with increasing histological grade and was greater in tumours that were ER - ve compared to those that were ER + ve (> 5% nuclei stained), median 7.8% and 4.3% respectively (P = 0.011 by Mann-Whitney U-test). However, the decline in tumour Ki67 LI following anti-oestrogen treatment failed to correlate with ER and PR status or to predict recurrence over a short follow-up period. To our knowledge, this is the first time that tamoxifen treatment has been shown to reduce the Ki67 LI in human breast tumours in vivo. These data indicate that staining with the Ki67 antibody may be useful in monitoring response to anti-oestrogen therapy.

Abnormal regulation of the oestrogen receptor in benign breast lesions

Background—In normal breast tissue the oestrogen receptor (ER) and the proliferation associated antigen Ki67 are negatively associated, indicating that ER+ cells are non-dividing, or that the receptor is downregulated as cells enter cycle. This relation is completely or partially lost in many ER+ breast cancers and in in situ proliferations associated with an increased cancer risk, where coexpression of the two markers is often found. Aims—To determine whether similar changes can be identified in other risk associated breast lesions. Patients/Methods—Paraffin wax blocks from 12 cases of lactational change, 21 apocrine metaplasias, 22 duct ectasias, 20 sclerosing adenosis, 20 fibroadenomas, 19 phyllodes tumours, 20 radial scars, 21 papillomas (15 solitary and six multiple), 15 gynaecomastias, and nine postmortem male breast tissues were retrieved. Immunohistochemistry was used to determine the expression of ER and dual labelling immunofluorescence was used to detect cells expressing both ER and Ki67. Results—Increased numbers of ER+ cells were seen in sclerosing adenosis, radial scars, papillomas, fibroadenomas, and phyllodes tumours but not in apocrine cysts (where no ER+ cells were detected) or duct ectasia (where normal numbers were found). As in the normal breast, the proportion of ER+ cells increased with age in all lesions with the exception of fibroadenomas. Coexpression of ER and Ki67 was found in an increased proportion of cells of all risk associated lesions studied. ER+ cells were less likely to be dividing than ER− cells in all cases, although this was significant only for sclerosing adenosis. The data on sclerosing adenosis, radial scars, papillomas, and fibroadenomas are comparable with those reported previously in hyperplasia of usual type, whereas those in duct ectasia are similar to those of the normal breast. The findings in all lesions, however, differed from those in ductal carcinoma in situ, where proportions of ER+ and ER+/Ki67+ cells are higher and the relation between ER+ cell numbers and age is lost. Thus, the nature and degree of dysregulation of ER in benign breast lesions is broadly in accordance with the degree of risk of developing breast cancer with which they are associated. In gynaecomastia, the proportions of ER+ and ER+/Ki67+ cells were comparable with those seen in benign female breast lesions, but changes with age were not observed. However, the changes in gynaecomastia were similar to those seen in normal male breast. Conclusion—These findings are in keeping with the contention that the dissociation of ER and Ki67 expression is a very early change in the pathway to many breast cancers. However, this change might only have preneoplastic importance in the hormonal milieu of the female breast.

Dose-dependent change in biomarkers during neoadjuvant endocrine therapy with fulvestrant: results from NEWEST, a randomized Phase II study

NEWEST (Neoadjuvant Endocrine Therapy for Women with Estrogen-Sensitive Tumors) is the first study to compare biological and clinical activity of fulvestrant 500 versus 250xa0mg in the neoadjuvant breast cancer setting. We hypothesized that fulvestrant 500xa0mg may be superior to 250xa0mg in blocking estrogen receptor (ER) signaling and growth. A multicenter, randomized, open-label, Phase II study was performed to compare fulvestrant 500xa0mg (500xa0mg/month plus 500xa0mg on day 14 of month 1) versus fulvestrant 250xa0mg/month for 16xa0weeks prior to surgery in postmenopausal women with ER+ locally advanced breast cancer. Core biopsies at baseline, week 4, and surgery were assessed for biomarker changes. Primary endpoint: change in Ki67 labeling index (LI) from baseline to week 4 determined by automated computer imaging system (ACIS). Secondary endpoints: ER protein expression and function; progesterone receptor (PgR) expression; tumor response; tolerability. ER and PgR were examined retrospectively using the H score method. A total of 211 patients were randomized (fulvestrant 500xa0mg: nxa0=xa0109; 250xa0mg: nxa0=xa0102). At week 4, fulvestrant 500xa0mg resulted in greater reduction of Ki67 LI and ER expression versus 250xa0mg (−78.8 vs. −47.4% [pxa0<xa00.0001] and −25.0 vs. −13.5% [pxa0=xa00.0002], respectively [ACIS]); PgR suppression was not significantly different (−22.7 vs. −17.6; pxa0=xa00.5677). However, H score detected even greater suppression of ER (−50.3 vs. −13.7%; pxa0<xa00.0001) and greater PgR suppression (−80.5 vs. −46.3%; pxa0=xa00.0018) for fulvestrant 500 versus 250xa0mg. At week 16, tumor response rates were 22.9 and 20.6% for fulvestrant 500 and 250xa0mg, respectively, with considerable decline in all markers by both ACIS and H score. No detrimental effects on endometrial thickness or bone markers and no new safety concerns were identified. This provides the first evidence of greater biological activity for fulvestrant 500 versus 250xa0mg in depleting ER expression, function, and growth.

Prolactin receptor antagonism reduces the clonogenic capacity of breast cancer cells and potentiates doxorubicin and paclitaxel cytotoxicity

IntroductionExogenous prolactin is mitogenic and antiapoptotic in breast cancer cells, and overexpression of autocrine prolactin cDNA in breast cancer cell lines has been shown to stimulate their growth and to protect against chemotherapy-induced apoptosis. We examined the effects of the pure prolactin receptor antagonist Δ1–9-G129R-hPrl (Δ1–9) on the breast cancer cell number and clonogenicity, alone and in combination with chemotherapy.MethodsThe effects of doxorubicin, paclitaxel and Δ1–9 on the growth of breast cancer cell lines (MCF-7, T47D, MDA-MB-453, MDA-MB-468 and SK-BR-3) in monolayer culture were assessed by the sulphorhodamine B assay. Effects on clonogenicity were assessed by soft agar assay for the cell lines and by the mammosphere assay for disaggregated primary ductal carcinoma in situ samples. Dual-fluorescence immunocytochemistry was used to identify subpopulations of cells expressing the prolactin receptor and autocrine prolactin.ResultsΔ1–9 as a single agent had no effect on the cell number in monolayer culture, but potentiated the cytotoxic effects of doxorubicin and paclitaxel. Doxorubicin accordingly induced expression of prolactin mRNA and protein in all five breast cancer cell lines tested. Δ1–9 alone inhibited the clonogenicity in soft agar of cell lines by ~90% and the mammosphere forming efficiency of six disaggregated primary ductal carcinoma in situ samples by a median of 56% (range 32% to 88%). Subpopulations of cells could be identified in the cell lines based on the prolactin receptor and prolactin expression.ConclusionAutocrine prolactin appears to act as an inducible survival factor in a clonogenic subpopulation of breast cancer cells. The rational combination of cytotoxics and Δ1–9 may therefore improve outcomes in breast cancer therapy by targeting this cell population.