PgR Levels Research Articles

Comparison of Methods for Detection of Fulvestrant-Induced Changes in Breast Tumor Estrogen and Progesterone Receptor Expression in a Neoadjuvant Trial (NEWEST).

Abstract Background: Fulvestrant downregulates breast tumor estrogen and progesterone receptor (ER and PgR) levels in a dose-dependent manner. Using an Automated Cellular Imaging System (ACIS), NEWEST (NCT00093002) reported that 4-wks' treatment with fulvestrant high-dose (HD, 500mg/month+500mg on Day 14 of Month 1) significantly reduced ER levels in primary breast tumors v approved-dose (AD, 250mg/month). However, no significant difference was detected between the two doses on PgR levels. To allow comparison with previous studies, a non-automated H-score assessment was performed and compared with ACIS.Methods: ER and PgR H-scores were derived by manual assessment of % tumor cells in each of 5 staining categories (negative, very weak, weak, moderate, strong) in the same sections scored by ACIS. This microscopic assessment was performed by 2 experienced observers blind to ACIS and clinical data. Mean % changes in H-scores were then calculated (table).Results: Both scoring methods showed a greater effect for fulvestrant HD v AD on ER expression at Wk 4, but H-score provided better dose discrimination. ACIS showed no difference between fulvestrant HD v AD on PgR expression at Wk 4, however, a significantly greater reduction in PgR expression was detected with fulvestrant HD using H-score.Wk 4Mean % change from baseline ACISH-score HDADHDADER-25.0%-13.5%-50.3%-13.7% n=60(n=63)(n=58)(n=60) p=0.0002 p<0.0001 PgR-22.7%-17.6%-80.5%-46.3% (n=43)(n=45)(n=31)(n=34) NS p=0.0018 Baseline mean H-score for HD and AD: 151.6 and 157.7 for ER; 103.8 and 122.9 for PgR.Conclusion: The choice of scoring method for determining ER and PgR expression in pharmacodynamic studies such as NEWEST is critical. Compared with H-scores, ACIS has a narrower dynamic range and reduced ability to discriminate fulvestrant HD v AD, particularly on PgR expression. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1087.

Early Acquired Resistance to Endocrine Therapy: Extending the Neoadjuvant Model.

Abstract Background: Neoadjuvant endocrine therapy allows biological studies of de novo response and resistance but not of acquired resistance based on clinical endpoints as surgery is usually carried out after around 4 months. Response is associated with a significant fall in Ki67 (proliferation) after 2 weeks treatment. However, at 12/16 weeks, c.15% of tumours show recovery of Ki67. We hypothesized that this recovery reflected rapidly acquired treatment resistance and have assessed its clinical relevance and association with tumour biomarkers.Methods: Ki67, ER, PgR and HER-2 (with FISH for 2+ cases) IHC measurements taken at baseline, 2 weeks and 12/16 weeks during neoadjuvant aromatase inhibitor (AI) treatment were available from three previously reported clinical trials [1-3] for a total of 177 ER+ patients. To allow pooling of data, ER and PgR levels were expressed as Allred scores. Long-term clinical follow-up data were available for 142 patients who received endocrine therapy within the IMPACT trial. Categories of response/resistance based on Ki67 change were created and patients classified accordingly: de novo resistance (<50% decrease in Ki67 from baseline to 2 weeks), acquired resistance (>50% decrease in Ki67 from baseline to 2 weeks, but overall fall at 12/16 weeks <50%) and maintained response (>50% decrease in Ki67 from baseline to 2 weeks and overall fall >50%).Results: Patients defined as showing acquired resistance had significantly worse RFS than those defined as showing maintained response (HR: 3.73, 95%CI: 1.18-11.79, p=0.025) and very similar to those with de novo resistance (HR: 3.66, 95% CI: 1.41-9.50, p=0.008). More HER-2+ cases showed Ki67 recovery than would be expected by chance (acquired resistance: 5/19 HER-2+, maintained response: 5/100 HER-2+; p=0.02). There was a weak suggestion that pre-treatment ER level may be lower for patients showing Ki67 recovery than those with persistent response (p=0.11), but no evidence was seen for an effect of pre-treatment PgR level (p=0.52).Conclusions: Tumours that exhibit an early Ki67 fall followed by Ki67 recovery at 12/16 weeks are associated with poorer RFS than those showing maintained suppression of Ki67 suggesting that Ki67 may act as a valid intermediate marker of acquired resistance. HER-2 positivity is associated with development of early acquired Ki67-based resistance, but other mechanisms for early acquired resistance exist.Supported by The Mary-Jean Mitchell Green Foundation.[1] Dowsett M et al., J Clin Oncol 2005. 23(11): 2477-92[2] Smith IE et al., J Clin Oncol 2007. 25(25): 3816-22[3] Miller WR et al., J Clin Oncol 2009. 27(9): 1382-7 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2005.

The Expression of Insulin-Like Growth Factor-1 Receptor (IGF-1R) in Early Breast Cancer.

Abstract Background: The insulin-like growth factor-1 receptor (IGF-1R) is expressed in normal breast epithelial cells and breast carcinomas. A number of studies have examined the over-expression of IGF-1R in breast cancer with the range of between 39% to 93%. However, many of these studies were carried out using ELISAs, radioimmunoassay with small patient cohorts. Phosphorylation of IGF-1R results in the activation of the MAPK and PI3K/Akt pathway. Activation of the PI3K/Akt pathway under the influence of IGF-1R plays an important role in maintaining the proliferation of breast cancer cells that are resistant to gefitnib, trastuzumab or chemoradiotherapy in vitro and in vivo. In the present study, we examined the expression of IGF-1R in early breast cancer and investigated the clinicopathological implications using immunohistochemistry and FISH.Methods: TMAs from a consecutive series (1981-98) of 1,812 women managed by wide local excision and postoperative radiotherapy (45Gy in 20-25 fractions) were collected following appropriate ethical review. Of 1686 cases stained, 198 received no adjuvant hormonal or chemotherapy, 1106 received tamoxifen only as adjuvant therapy and 144 received a combination of hormonal and chemotherapy. Median age at diagnosis was 56, 72% were post-menopausal, 23.9% node positive, median size was 1.5 cm. Samples were stained, using triplicate 0.6mm2 TMA cores for IGF-1R.Results: FISH for IGF1R gene amplification was successful in 343/408 cases (84%). Only 7 cases of IGF1R amplification were observed (2%). IGF1R expression was successfully evaluated in 1597 of 1686 (94.7%) cases. High levels of IGF-1R expression was weakly associated with tumour ER-a (correlation coefficient, 0.108; p=2.2 x 10-5) and PgR levels (correlation coefficient 0.162; p=1.86x10-10) and inversely correlated with EGFR expression (correlation coefficient -0.189; p=6.33x10-6). No association was noted between IGF1R expression and other molecular or clinical markers, such as tumour size. There was a trend for over-expression of IGF-1R to link with increased distant relapse free survival, particularly in ER positive breast cancers. This trend is time dependent, such that no difference in outcome is observed at 15 years.Conclusions: We have demonstrated in a large cohort of patients that IGF-1R gene amplification is a rare event, and over-expression is weakly associated with good prognostic features (ERa and PgR, lack of EGFR and better outcome). Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2128.

Comparison of methods for detection of fulvestrant-induced changes in breast tumor estrogen and progesterone receptor expression in a neoadjuvant trial (NEWEST)

e11602^ Background: Fulvestrant downregulates breast tumor estrogen and progesterone receptor (ER and PgR) levels in a dose-dependent manner. Using an Automated Cellular Imaging System (ACIS), NEWEST reported that 4-wks’ treatment with fulvestrant high-dose (HD, 500mg/month+500mg on Day 14 of Month 1) significantly reduced ER levels in primary breast tumors v approved-dose (AD, 250mg/month). However, no significant difference was detected between the two doses on PgR levels. To allow comparison with previous studies, a non-automated H-score assessment was performed and compared with ACIS. Methods: ER and PgR H-scores were derived by manual assessment of % tumor cells in each of 5 staining categories (negative, very weak, weak, moderate, strong) in the same sections scored by ACIS. This microscopic assessment was performed by 2 experienced observers blind to ACIS and clinical data. Mean % changes in H-scores were then calculated (table). Results: Both scoring methods showed a greater effect for fulvestrant HD v AD on ER expression at Wk 4, but H-score provided better dose discrimination. ACIS showed no difference between fulvestrant HD v AD on PgR expression at Wk 4, however, a significantly greater reduction in PgR expression was detected with fulvestrant HD using H-score. Conclusions: The choice of scoring method for determining ER and PgR expression in pharmacodynamic studies such as NEWEST is critical. Compared with H-scores, ACIS has a narrower dynamic range and reduced ability to discriminate fulvestrant HD v AD, particularly on PgR expression. [Table: see text] ASCO Conflict of Interest Policy and Exceptions In compliance with the guidelines established by the ASCO Conflict of Interest Policy (J Clin Oncol. 2006 Jan 20;24[3]:519–521) and the Accreditation Council for Continuing Medical Education (ACCME), ASCO strives to promote balance, independence, objectivity, and scientific rigor through disclosure of financial and other interests, and identification and management of potential conflicts. According to the ASCO Conflict of Interest Policy, the following financial and other relationships must be disclosed: employment or leadership position, consultant or advisory role, stock ownership, honoraria, research funding, expert testimony, and other remuneration (J Clin Oncol. 2006 Jan 20;24[3]:520). The ASCO Conflict of Interest Policy disclosure requirements apply to all authors who submit abstracts to the Annual Meeting. For clinical trials that began accrual on or after April 29, 2004, ASCO's Policy places some restrictions on the financial relationships of principal investigators (J Clin Oncol. 2006 Jan 20;24[3]:521). If a principal investigator holds any restricted relationships, his or her abstract will be ineligible for placement in the 2009 Annual Meeting unless the ASCO Ethics Committee grants an exception. Among the circumstances that might justify an exception are that the principal investigator (1) is a widely acknowledged expert in a particular therapeutic area; (2) is the inventor of a unique technology or treatment being evaluated in the clinical trial; or (3) is involved in international clinical oncology research and has acted consistently with recognized international standards of ethics in the conduct of clinical research. NIH-sponsored trials are exempt from the Policy restrictions. Abstracts for which authors requested and have been granted an exception in accordance with ASCO's Policy are designated with a caret symbol (^) in the Annual Meeting Proceedings. For more information about the ASCO Conflict of Interest Policy and the exceptions process, please visit www.asco.org/conflictofinterest .

Progesterone receptor levels are associated with time to aromatase inhibitor treatment failure in estrogen receptor-positive advanced breast cancer.

Abstract Abstract #6051 Background: Aromatase inhibitors (AIs) play a key role in the endocrine treatment of hormone receptor-positive breast cancer, yet surprisingly little information is available on the effectiveness of AIs in advanced breast cancer according to quantitative levels of ER and PgR or HER-2 status. In this work, we assess expression of these three biomarkers in primary breast tumours of women who subsequently received an AI for advanced disease. This extends an earlier study [1], improving the statistical power and excluding patients who received neo-adjuvant therapy.
 Methods: Tissue microarrays were constructed from 177 archival FFPE primary or locally recurrent breast tumours from women, diagnosed between 1963 and 2003, who received no neo-adjuvant therapy prior to surgery and were treated with 3rd generation AI treatment for advanced disease (anastrozole n = 92; letrozole n = 82; exemestane n = 3). ER, PgR and HER-2 protein levels were assessed by IHC with FISH performed if HER-2 staining was equivocal. The study endpoint of time to AI treatment failure (TTF) was defined as time from commencement of AI treatment to progression of disease or cessation of treatment due to toxicity or death. Disease-free interval (DFI) was defined as time from date of diagnosis to date of first relapse.
 Results: 146/177 patients were assessed as ER+ by IHC. When both ER+ and ER- patients were considered, higher ER and PgR levels were associated with increased TTF (HR: 0.90; 95% CI: 0.83-0.98; p = 0.011 and HR: 0.86; 95% CI: 0.80-0.93; p < 0.001 respectively) whereas HER-2 positivity was associated with decreased TTF (HR: 1.61; 95% CI: 1.01-2.59; p = 0.048). Longer DFI was also associated with increased TTF (HR: 0.79; 95% CI: 0.65-0.97; p = 0.022) with both PgR level and DFI remaining significant in multivariate analysis. When ER+ patients only were considered, higher PgR levels maintained an association with increased TTF (HR: 0.88; 95% CI: 0.81-0.97; p = 0.007) with this relationship appearing to be even stronger amongst the population considered PgR+ (HR: 0.73; 95% CI: 0.54-0.98; p = 0.037). No significant relationship was observed between ER levels and TTF in the ER+ subgroup. ER+HER-2+ patients (n = 13) showed a trend for decreased TTF compared to ER+HER-2- patients (n = 131), but this was not significant in this small subset of patients (HR: 1.34; 95% CI: 0.75-2.38; p = 0.32).
 Conclusions: Higher PgR level is significantly associated with increased TTF in ER+ patients receiving AI treatment for advanced disease, especially within the ER+PgR+ subgroup. ER+HER-2+ patients may show decreased TTF on AI treatment compared to ER+HER-2- patients. Larger prospective studies are required to confirm these data. These observations highlight the importance of PgR as a predictor of TTF even in advanced breast cancer patients.
 Supported by the Mary-Jean Mitchell Green Foundation.
 [1] Anderson H. et al., J Steroid Biochem Mol Biol 106 (2007) 49-54. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6051.

Evaluation of estrogen receptor α and β and progesterone receptor expression and correlation with clinicopathologic factors and proliferative marker Ki-67 in breast cancers

To elucidate the molecular profile of hormonal steroid receptor status, we analyzed ER-alpha, ER-beta, and PGR mRNA and protein expression in 80 breast carcinomas using reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunohistochemical analysis. Qualitative analysis revealed positive expression of ER-alpha, ER-beta, and PGR mRNA in 48%, 59%, and 48% of the breast carcinomas, respectively. ER-alpha, ER-beta, and PGR transcript overexpression was observed in 51%, 0%, and 12% of the cases, respectively, whereas moderate or strong protein expression was detected in 68%, 78%, and 49% of the cases, respectively. Tumor grade was negatively correlated with transcript and protein levels of ER-alpha (P = .0169 and P = .0006, respectively) and PGR (P = .0034 and P = .0005, respectively). Similarly, proliferative index Ki-67 was negatively associated with transcript and protein levels of ER-alpha (P = .0006 and P < .0001, respectively) and PGR (P = .0258 and P = .0005, respectively). These findings suggest that ER-alpha and PGR expression are associated with well-differentiated breast tumors and less directly related to cell proliferation. A significant statistical difference was observed between lymph node status and ER-beta protein expression (P = .0208). In ER-alpha-negative tumors, we detected a correlation between ER-beta protein expression and high levels of Ki-67. These data suggest that ER-beta could be a prognostic marker in human breast cancer.

Can Core Biopsy Be Used Instead of Surgical Biopsy in the Diagnosis and Prognostic Factor Analysis of Breast Carcinoma?

Purpose The aim of this article was to investigate the efficacy of ultrasonography-guided core needle biopsy and prognostic factor analysis of breast cancer to plan overall treatment strategy. Patients and Methods A consecutive series of nonpalpable and palpable breast cancers constituted our study group (n= 201 lesions; mean size, 20.4 mm) Mean number of core samples was 3.4. Malignant lesions diagnosed with core biopsy underwent therapeutic surgical excision. Core biopsy and surgical excisions were compared for histologic type, grade, estrogen receptors (ERs), progesterone receptors (PgRs), and c-erbB2 levels. Cutoff values for ER, PgR, and c-erbB2 affecting the management strategy were selected as 10%, 10%, and 50%, respectively. Results Eighty-five lesions (42.3%) were malignant in core biopsy (mean size, 18.4 mm). Among these, 11 were inoperable and 13 were surgically excised at other institutions. In 61 lesions, core and surgical excision specimens were evaluated in the same institution (mean tumor size, 18.6 mm; range 6–60 mm). Concordance between the 2 biopsy methods was 85.2% (52 of 61) for histologic type of tumor, 68.8% (33 of 48) for tumor grade, 90% (27 of 30) for ER, 86.7% (26 of 30) for PgR, and 79.3% (23 of 29) for c-erbB2 levels. Appropriate site selection for sampling was indicated to be of paramount importance, especially in determining reliable ER, PgR, and c-erbB2 levels. Conclusion Core needle biopsy of breast cancer is equally effective compared wth surgical biopsy and can be used in overall treatment planning. However, appropriate site selection for sampling should be guaranteed using ultrasonographic guidance.

Expression and Clinical Signification of Cytosolic Hyaluronan Levels in Invasive Breast Cancer

Hyaluronic acid (HA), a high-molecular weight glycosaminoglycan, has been considered to be involved in the growth and progression of malignant tumors in several experimental studies. The objective of this work was to evaluate the cytosolic HA content in breast cancer, its possible relationship with clinicopathological tumor parameters and steroid receptor status, as well as its potential prognostic significance. Cytosolic HA levels were examined by means of immunoradiometric techniques in 850 patients with invasive breast cancer. The mean follow-up period for these patients was 55.1 months. Cytosolic HA levels ranged widely in tumors (4-59767 ng/mg protein; median: 4960). Statistical analysis showed that HA levels were significantly higher in younger patients (p=0.0001), as well as in premenopausal than in postmenopausal patients (p=0.001). HA levels were also significantly higher in ductal or lobular histological type than in other histological types (coloid, medullar or papillar types) (p=0.0001). Likewise, HA correlated significantly and positively with tumoral levels of PgR (r sub S: 0.11; p=0.001) in the all group of patients. In the subgroup of patients with ductal invasive type, HA levels were also significantly higher in well differentiated tumors and in diploid tumors. In addition, in this latter group of patients, HA levels in tumors correlated also positively and significantly with the either estrogen-inducible proteins: PgR (r sub S: 0.11; p=0.001), pS2 (r sub S: 0.117; p=0.008) and tPA (r sub S: 0.314; p=0.0001). On the other hand, significant association between HA intratumoral levels and relapse-free survival and overall survival in the overall group of patients was not found. However, high HA intratumoral levels were significantly associated with longer relapse-free survival in the subgroup of patients with ductal histological type tumors (p=0.01), as well as in those patients without any type of systemic adjuvant treatment (p=0.01). Our results suggest that high intratumoral levels of HA may be associated with tumors of favorable evolution in certain subgroups of patients with breast cancer. Thus, HA may provide additional prognostic information to that given by other biochemical markers currently used in breast cancer.

Simultaneous Quantitative Detection of Relevant Biomarkers in Breast Cancer by Quantitative Real-Time PCR

The assessment of ERα, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERα, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERα, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Endogenous human prolactin and not exogenous human prolactin induces estrogen receptor α and prolactin receptor expression and increases estrogen responsiveness in breast cancer cells

Prolactin (PRL) and estrogen act synergistically to increase mammary gland growth, development, and differentiation. Based on their roles in the normal gland, these hormones have been studied to determine their interactions in the development and progression of breast cancer. However, most studies have evaluated only endocrine PRL and did not take into account the recent discovery that PRL is synthesized by human mammary cells, permitting autocrine/paracrine activity. To examine the effects of this endogenous PRL, we engineered MCF7 cells to inducibly overexpress human prolactin (hPRL). Using this Tet-On MCF7hPRL cell line, we studied effects on cell growth, PRLR, ERα, and PgR levels, and estrogen target genes. Induced endogenous hPRL, but not exogenous hPRL, increased ERα levels as well as estrogen responsiveness in these cells, suggesting that effects on breast cancer development and progression by estrogen may be amplified by cross-regulation of ERα levels by endogenous hPRL. The long PRLR isoform was also upregulated by endogenous, but not exogenous PRL. This model will allow investigation of endogenous hPRL in mammary epithelial cells and will enable further dissection of PRL effects on other hormone signaling pathways to determine the role of PRL in breast cancer.

Letrozole (femara) is a more effective inhibitor of estrogen activity than tamoxifen: Evidence from a randomized phase III trial of 4 months preoperative endocrine therapy for postmenopausal women with primary invasive breast cancer

Background: The agonist effects of tamoxifen (TAM) may limit efficacy against some breast cancers but the extent to which these agonist effects occur is unknown. To address this issue, changes in estrogen-regulated gene expression were monitored during preoperative endocrine therapy with either TAM or the aromatase inhibitor letrozole (LET). Methods: Postmenopausal breast cancer patients received 4 months preoperative endocrine therapy with either LET or TAM in a double-blind phase III study. Biopsies were obtained pre and post treatment and assessed by immunohistochemistry (IHC) for expression of the estrogen-regulated proteins progesterone receptor (PgR, n = 223) and trefoil factor I (PS2, n = 219). Allred IHC scores were assigned and categorized as low (O-2), medium (3-5) and high (6-8). A change with treatment was recorded if the post treatment score changed category from pre treatment to cause an increase or decrease in expression category. Results: A decrease in PgR category was more frequent with LET (65% LET vs 23% TAM). An increase in PgR level was more frequent with TAM (29% TAM vs 5% LET). Similar data were obtained with PS2 (p = 0.001. logistic regression). Study outcomes were superior for LET when compared with TAM. For a subset of patients with study biopsy confirmed ER and/or PgR positivity clinical response rates (CR + PR) were 60% for LET vs 41% TAM. p = 0.004 (Mantel-Haenszel (M-H)) and breast conservation occurred in 48% of patients treated with LET vs 36% on TAM, p = 0.036 (M-H). Conclusion: Agonist effects on PgR and PS2 occur in about one third of cases treated with TAM and rarely with LET. LET was more effective in inducing regression of ER and/or PgR positive primary breast cancer than TAM.

A case report of advanced male breast cancer with an objective response to tamoxifen treatment

A 70-year-old man presented with a firm tumor in his right breast first noticed eight years ago. The tumor had enlarged gradually and had produced an ulcer with bleeding. On physical examination, a huge tumor entirely occupied the right breast and extensively had infiltrated the chest wall. Chest X-ray and CT showed massive pleural effusion and multiple small nodular lesions in the lung. Invasive ductal carcinoma of the breast was diagnosed by incisional biopsy,confirming advanced breast cancer with lung metastases and bilateral pleural effusion(T4cN2M1, Stage IV). Because ER and PgR levels were 110 fmol/mg and 190 fmol/mg, respectively, and because his general condition was poor, we selected medical treatment with tamoxifen(TAM). Thirty-two weeks later, the tumor had showed pronounced reduction with scarring. The patient underwent local excision of the scar tissue. The quality of life of the patient was favorably improved and no severe adverse events were observed. The tumor in the chest wall recurred two months after the end of TAM treatment, possibly because the patient did not accept continuous TAM therapy. The patient died from complications of brain metastasis 32 months after the start of TAM treatment. We report a rare case of advanced male breast cancer and on the effectiveness of continuous TAM treatment.

Oestrogen and progesterone receptors in gastrointestinal cancer cell lines.

Expression of sex steroid receptors by gastrointestinal (GI) tumours may indicate a role for hormonal manipulation in their management. A panel of seven established cell lines derived from primary human GI tumours were assayed for oestrogen and progesterone receptor (ER and PgR) expression by ligand binding, immunocytochemistry and enzyme-immunoassay methods. It was possible to demonstrate very low levels of both ER and PgR in the GI cell lines using enzyme immunoassay (EIA), with levels of PgR generally higher than those for ER. Neither ER nor PgR were detected in cytosols made from the GI cell lines in a classical dextran coated charcoal ligand binding assay. Similarly, immunohistological analysis (Abbott ERICA, PgRICA) of cultured cell preparations or of frozen and paraffin sections of xenograft tumour failed to demonstrate receptors. This confirms that low PgR levels are measurable by EIA in GI tumour cell lines. Upregulation of PgR expression in tumours by exposure to oestradiol in vitro was not observed.

Estrogen and progesterone receptor status determined by the Ventana ES 320 automated immunohistochemical stainer and the CAS 200 image analyzer in 236 early-stage breast carcinomas: Prognostic significance

The quantitation of estrogen and progesterone receptors (ER and PgR) has become the standard of care in the evaluation of patients with primary breast carcinoma. It has been demonstrated that ER and PgR detected by immunohistochemical methods in formalin-fixed paraffin-embedded tissue can be quantified by computerized image analysis. In this study, ER and PgR levels were determined by using an automated immunochemistry stainer (Ventana ES 320) and an image analyzer (CAS 200) in a series of 236 patients with stage I/II carcinoma of the breast. The degree of correlation of the ER and PgR levels determined by the dextran-coated charcoal method (DCC) with image analysis quantitation was high (r=0.75). The agreement between both methods was 77% for ER and 73% for PgR. Hormone receptor levels were correlated with prognosis as determined by overall survival. An ER level of 30 fmol/mg as determined by image analysis was established to stratify the patient population most effectively into favorable and unfavorable prognostic groups (P=0.003). An ER level of 20 fmol/mg for prognostic stratification reached statistical significance (P=0.03). The DCC method was not able to stratify the patients into prognostic groups at the traditionally accepted cutpoint of 10 fmol/mg (P=0.52). We conclude that when used in combination, automated immunohistochemistry and quantitative image analysis offer a favorable alternative to the DCC method in assessment of ER and PgR status in human mammary carcinoma. In addition, quantitative immunocytochemistry techniques may prove superior to the DCC method in specimens in which there is limited tumor volume (including fine-needle aspirates), stroma-rich tumors, and early-stage lesions including intraductal carcinoma.

1267 Expression of her-2/NEU EGFR, hormone receptors, cathepsyn-D and ploidy in normal and neoplastic GI tract tissues

Six biochemical parameters, recognized as being prognostic factors in breast cancer, were evaluated on fresh samples of human GI tract tumors to better define the biology and natural history of such neoplasms. Levels of expression of HER-2/neu oncogene and epidermal growth factor receptor (EGFr) protein products, ER, PgR, cathepsin-D and ploidy were determined in 16 gastric and 39 colorectal tumors and compared with normal samples from the same subjects. In 56% of gastric, 61% of colon and 35% of rectum carcinomas HER-2/neu gene product p185 was significantly overexpressed as compared to normal tissues. On the contrary colon and rectum tumors expressed significantly lower levels of EGFr than normal in 60% of cases. Very low levels of ER and PgR were detected in all the samples (normal and malignant) tested. 75% of tumour tissues showed a significant higher cathepsin-D content compared to the respective normal sample while an aneuploid DNA profile was documented in 72% of neoplasms. Overall, change of the markers evaluated seems to be a specific phenomenon of certain GI carcinomas. Higher EGFR levels in normal than malignant tissues suggest that EGFR can be implicated in the process of growth and differentiation of the normal gastrointestinal mucosa. Further studies on a larger number of cases along with an adequate follow-up of patients are needed to define the role of these markers in the pathogenesis of GI tract neoplasms and its prognostic significance when considered together with other major risk factors.

Relationship of PS2 with response to tamoxifen therapy in patients with recurrent breast cancer.

PS2, an oestrogen-inducible protein, was measured in the cytosol of 230 primary tumours from patients who were subjected to first-line tamoxifen therapy for advanced disease without prior adjuvant therapy with tamoxifen. PS2 correlated positively with oestrogen receptor (ER, P < 0.01) and progesterone receptor content (PgR, P < 0.001), and with the length of progression-free survival (PFS, P = 0.05). Although not statistically significant, higher levels of PS2 (> or = 10 ng mg-1 protein) were also associated with increased probability of response to tamoxifen treatment and a longer total post-relapse survival (PRS). ER, PgR, menopausal status, site of disease and prior adjuvant chemotherapy were all associated with response to tamoxifen therapy and with PFS. In multivariate analysis for PFS, low levels of ER and PgR, visceral metastasis, a disease-free interval of less than 1 year and prior adjuvant chemotherapy were all significantly associated with an increased probability of a rapid disease progression after start of tamoxifen therapy. In the subset of 83 tumours with intermediate levels of ER and PgR (both > or = 10, but not both > or = 75 fmol mg-1 protein), PS2 was positively related with the length of PFS (P < 0.01) and PRS (P < 0.05). PS2 remained the strongest factor in multivariate analysis for PFS (P < 0.01) in this ER/PgR intermediate subgroup, but was not of predictive value in univariate or multivariate analysis for both PFS and PRS in tumours classified as ER/PgR low or high (> or = 75 fmol mg-1 protein). It is concluded that PS2 status may be used as a parameter, additional to ER and PgR, for better refinement of prediction of response to tamoxifen treatment in advanced breast cancer patients especially with intermediate ER/PgR levels in their primary tumour.

Cell biological factors associated with the response of breast cancer to systemic treatment

A large number of cell biological parameters are currently available to predict the prognosis of patients with breast cancer, but it is still difficult accurately to predict the response to treatment. A valuable prognostic factor can be a poor predictive factor for response, and vice versa. High tumor levels of ER, PgR, AR and pS2 predict a relatively good response to endocrine therapy, while EGF-R positivity, HER2/neu positivity, aneuploidy, high proliferation indices and possibly high uPA levels indicate a high chance of poor response to endocrine therapy in metastatic breast cancer. With respect to chemotherapy, a high proliferation rate and H ER2/neu amplification predict a good response to therapy in metastatic disease, while MDR gene expression and possibly c-myc amplification are related to a worse response. In conclusion, the newer cell biological parameters can be used to select high and low-risk patients, type of systemic treatment, and as targets for new treatment modalities.

Proportional hazards and recursive partitioning and amalgamation analyses of the southwest oncology group node-positive adjuvant CMFVP breast cancer data base: a pilot study

Several putative prognostic factors have been identified in node-positive breast cancer patients, but their importance needs to be clarified in a uniformly treated population. The objectives of this investigation were: 1) to describe the characteristics of a uniformly treated node-positive data base; 2) to use proportional hazards (Cox) and recursive partitioning and amalgamation (RPA) multivariate models to assess the importance of potential prognostic factors for disease-free and for overall survival; and 3) to define prognostic groups with different disease-free survival and survival outcomes with RPA. A data base of 768 node-positive patients enrolled on 1-year adjuvant CMFVP arms of four SWOG trials was formed. Variables were number of positive nodes, age, age at menopause, menopausal status, ER status, ER and PgR levels (for RPA only), tumor size, race, breast cancer in mother, and obesity index. Independent predictors of both disease-free and overall survival in the Cox models were: number of positive nodes (4-6 worse than 1-3, and better than greater than 6); the age/menopause category (age greater than or equal to 35/premenopausal better than age less than 35/premenopausal and better than postmenopausal); and ER status (patients on ER-negative study worse than others). The RPA for disease-free survival defined four subgroups based on nodes, menopausal status, tumor size, and age at menopause (5-year recurrence-free rates = 73%, 52%, 38%, and 15%). The RPA for survival found four prognostic groups, defined only by the number of positive nodes and ER and PgR levels (5-year survivals = 91%, 72%, 56%, and 37%). Both RPAs suggested interesting refinements of the results of the Cox models. In the RPA for disease-free survival, best node cutoffs differed by menopausal status, tumor size was important only in postmenopausal patients with few positive nodes, and age at menopause emerged as an independent predictor of recurrence potential. And, the RPA for survival showed that node cutoffs differed according to ER level. Thus, these analyses underscore the value of simple, clinically available prognostic factors and suggest the possible need to reconsider the definition of good and poor risk patient groups in future adjuvant trial design.

Influence of the antioestrogen tamoxifen on normal breast tissue

Immunohistochemical assays have been employed to study the expression of ER, PgR, EGFR and Ki67 immunostaining in normal breast tissue (n = 76). The expression of ER and PgR was highly variable in both pre and postmenopausal women and was characterised by large numbers of apparently negative cells. This was most evident for ER-ICA staining in tissues removed from premenopausal women. PgR levels were highest in the ducts of premenopausal women, while EGFR expression was elevated in both ducts and lobules. Ki67 expression was observed in less than 10% of all normal cells and was suppressed by the menopause in lobular tissue. Tamoxifen therapy (40 mg d-1) did not influence the expression of PgR, EGFR or Ki67 immunostaining in cancer associated normal tissue (n = 17). A significant increase, however, was observed in the mean percentage ER positivity in ductal tissue. No effect of duration of tamoxifen therapy was observed on the expression of the antigens studied.