Therapeutic Interleukin-6 Trans-signaling Inhibition by Olamkicept (sgp130Fc) in Patients With Active Inflammatory Bowel Disease.

BACKGROUND & AIMS: A large unmet therapeutic need exists in inflammatory bowel disease (IBD). Inhibition of interleukin (IL)-6 appears to be effective, but the therapeutic benefit of a complete IL6/IL6 receptor (IL6R) blockade is limited by profound immunosuppression. Evidence has emerged that chronic proinflammatory activity of IL6 is mainly mediated by trans-signaling via a complex of IL6 bound to soluble IL6R engaging the gp130 co-receptor without the need for membrane-bound IL6R. We have developed a decoy protein, sgp130Fc, that exclusively blocks IL6 proinflammatory trans-signaling and has shown efficacy in preclinical models of IBD, without signs of immunosuppression. METHODS: We present a 12-week, open-label, prospective phase 2a trial (FUTURE) in 16 patients with active IBD treated with the trans-signaling inhibitor olamkicept (sgp130Fc) to assess the molecular mechanisms, safety, and effectiveness of IL6 trans-signaling blockade in vivo. We performed in-depth molecular profiling at various timepoints before and after therapy induction to identify the mechanism of action of olamkicept. RESULTS: Olamkicept was well tolerated and induced clinical response in 44% and clinical remission in 19% of patients. Clinical effectiveness coincided with target inhibition (reduction of phosphorylated STAT3) and marked transcriptional changes in the inflamed mucosa. An olamkicept-specific transcriptional signature, distinguishable from remission signatures of anti-tumor necrosis factor (infliximab) or anti-integrin (vedolizumab) therapies was identified. CONCLUSIONS: Our data suggest that blockade of IL6 trans-signaling holds great promise for the therapy of IBD and should undergo full clinical development as a new immunoregulatory therapy for IBD. (EudraCT no., Nu 2016-000205-36).

Aurora kinase A as a possible marker for endocrine resistance in early estrogen receptor positive breast cancer.

BACKGROUND: Cell culture studies have disclosed that the mitotic Aurora kinase A is causally involved in both tamoxifen and aromatase inhibitor resistant cell growth and thus may be a potential new marker for endocrine resistance in the clinical setting. MATERIAL AND METHODS: Archival tumor tissue was available from 1323 Danish patients with estrogen receptor (ER) positive primary breast cancer, who participated in the Breast International Group (BIG) 1-98 trial, comparing treatment with tamoxifen and letrozole and both in a sequence. The expression of Aurora A was determined by immunohistochemistry in 980 tumors and semi quantitively scored into three groups; negative/weak, moderate and high. The Aurora A expression levels were compared to other clinico-pathological parameters and outcome, defined as disease-free survival (DFS) and overall survival (OS). RESULTS: High expression of Aurora A was found in 26.9% of patients and moderate in 57.0%. High expression was significantly associated with high malignancy grade and HER2 amplification. High Aurora A expression was significantly more frequent in ductal compared to lobular carcinomas. We found no significant association between Aurora A expression and DFS or OS and no evidence of interaction between Aurora A expression and benefits from tamoxifen versus letrozole. CONCLUSIONS: Aurora A expression in breast tumors was associated with high malignancy grade III and with HER2 amplification. A trend as a prognostic factor for OS was found in patients with high Aurora A expression. No predictive property was observed in this study with early breast cancer.

Evaluation of interleukin-6 and its soluble receptor components sIL-6R and sgp130 as markers of inflammation in inflammatory bowel diseases.

PURPOSE: Interleukin-6 (IL-6) production and signalling are increased in the inflamed mucosa in inflammatory bowel diseases (IBD). As published serum levels of IL-6 and its soluble receptors sIL-6R and sgp130 in IBD are from small cohorts and partly contradictory, we systematically evaluated IL-6, sIL-6R and sgp130 levels as markers of disease activity in Crohn's disease (CD) and ulcerative colitis (UC). METHODS: Consecutive adult outpatients with confirmed CD or UC were included, and their disease activity and medication were monitored. Serum from 212 CD patients (815 measurements) and 166 UC patients (514 measurements) was analysed, and 100 age-matched healthy blood donors were used as controls. RESULTS: IL-6 serum levels were significantly elevated in active versus inactive CD and UC, also compared with healthy controls. However, only a fraction of IBD patients showed increased serum IL-6. IL-6 levels ranged up to 32.7 ng/mL in active CD (> 5000-fold higher than in controls), but also up to 6.9 ng/mL in inactive CD. Increases in active UC (up to 195 pg/mL) and inactive UC (up to 27 pg/mL) were less pronounced. Associations between IL-6 serum levels and C-reactive protein concentrations as well as leukocyte and thrombocyte counts were observed. Median sIL-6R and sgp130 levels were only increased by up to 15%, which was considered of no diagnostic significance. CONCLUSIONS: Only a minority of IBD patients shows elevated IL-6 serum levels. However, in these patients, IL-6 is strongly associated with disease activity. Its soluble receptors sIL-6R and sgp130 do not appear useful as biomarkers in IBD.

A high level of estrogen-stimulated proteins selects breast cancer patients treated with adjuvant endocrine therapy with good prognosis.

BACKGROUND: Adjuvant endocrine therapy has significantly improved survival of estrogen receptor α (ER)-positive breast cancer patients, but around 20% relapse within 10 years. High expression of ER-stimulated proteins like progesterone receptor (PR), Bcl-2 and insulin-like growth factor receptor I (IGF-IR) is a marker for estrogen-driven cell growth. Therefore, patients with high tumor levels of these proteins may have particularly good prognosis following adjuvant endocrine therapy. PATIENTS AND METHODS: Archival tumor tissue was available from 1323 of 1396 Danish breast cancer patients enrolled in BIG 1-98, a randomized phase-III clinical trial comparing adjuvant letrozole, tamoxifen or a sequence of the two drugs. Immunohistochemical staining for ER, HER-2, PR, Bcl-2 and IGF-IR was performed and determined by Allred scoring (ER, PR and Bcl-2) or HercepTest (HER-2 and IGF-IR). RESULTS: Data on all five markers were available from 969 patients with ER-positive, HER-2-negative tumors. These patients were classified in ER activity groups based on the level of PR, Bcl-2 and IGF-IR. High ER activity profile was found in 102 patients (10.5%) and compared with the remaining patients, univariate and multivariate analysis revealed HR (95% CI) and p values for disease-free survival (DFS) of 2.00 (1.20-3.22), 0.008 and 1.70 (1.01-2.84), 0.04 and for the overall survival (OS) of 2.33 (1.19-4.57), 0.01 and 1.90 (0.97-3.79), 0.06, respectively. The high ER activity profile did not disclose difference in DFS or OS according to treatment with tamoxifen or letrozole (p = .06 and .09, respectively). CONCLUSIONS: Stratifying endocrine-treated patients in ER activity profile groups disclosed that patient with high ER activity profile (10.5%) had significantly longer DFS and OS, and the profile was an independent marker for DFS. High ER activity is a marker for estrogen-driven tumor growth. We suggest further analyses to disclose whether the ER activity profile or other markers associated with estrogen-driven growth may be used to identify ER-positive high-risk breast cancer patients who can be spared adjuvant chemotherapy.

Aurora kinase B is important for antiestrogen resistant cell growth and a potential biomarker for tamoxifen resistant breast cancer.

BACKGROUND: Resistance to antiestrogen therapy is a major clinical challenge in the treatment of estrogen receptor α (ER)-positive breast cancer. The aim of the study was to explore the growth promoting pathways of antiestrogen resistant breast cancer cells to identify biomarkers and novel treatment targets. METHODS: Antiestrogen sensitive and resistant T47D breast cancer cell lines were used as model systems. Parental and fulvestrant resistant cell lines were subjected to a kinase inhibitor library. Kinase inhibitors preferentially targeting growth of fulvestrant resistant cells were identified and the growth inhibitory effect verified by dose-response cell growth experiments. Protein expression and phosphorylation were investigated by western blot analysis. Cell cycle phase distribution and cell death were analyzed by flow cytometry. To evaluate Aurora kinase B as a biomarker for endocrine resistance, immunohistochemistry was performed on archival primary tumor tissue from breast cancer patients who have received adjuvant endocrine treatment with tamoxifen. RESULTS: The selective Aurora kinase B inhibitor barasertib was identified to preferentially inhibit growth of fulvestrant resistant T47D breast cancer cell lines. Compared with parental cells, phosphorylation of Aurora kinase B was higher in the fulvestrant resistant T47D cells. Barasertib induced degradation of Aurora kinase B, caused mitotic errors, and induced apoptotic cell death as measured by accumulation of SubG1 cells and PARP cleavage in the fulvestrant resistant cells. Barasertib also exerted preferential growth inhibition of tamoxifen resistant T47D cell lines. Finally, high percentage of Aurora kinase B positive tumor cells was significantly associated with reduced disease-free and overall survival in 261 ER-positive breast cancer patients, who have received tamoxifen as first-line adjuvant endocrine treatment. CONCLUSIONS: Our results indicate that Aurora kinase B is a driving factor for growth of antiestrogen resistant T47D breast cancer cell lines, and a biomarker for reduced benefit of tamoxifen treatment. Thus, inhibition of Aurora kinase B, e.g. with the highly selective kinase inhibitor barasertib, could be a candidate new treatment for breast cancer patients with acquired resistance to antiestrogens.

Divergent estrogen receptor-positive and -negative breast cancer trends and etiologic heterogeneity in Denmark.

Long-term breast cancer trends in incidence in the United States (US) show rising estrogen receptor (ER)-positive rates and falling ER-negative rates. We hypothesized that these divergent trends reflect etiologic heterogeneity and that comparable trends should be observed in other countries with similar risk factor profiles. Therefore, we analyzed invasive female breast cancers in Denmark, a country with similar risk factors as the US. We summarized the overall trend in age-standardized rates with the estimated annual percentage change (EAPC) statistic (1993-2010) and used age-period-cohort models to estimate age-specific EAPCs, cohort rate ratios and projections for future time periods (2011-2018). In Denmark, the overall rate of ER-positive cancers rose between 1993 and 2010 by 3.0% per year (95% CI: 2.8-3.3% per year), whereas the overall rate of ER-negative cancers fell by 2.1% per year (95% CI: -2.5 to -1.6% per year). The ER-positive rate increased fastest among postmenopausal women and the ER-negative rate decreased fastest among premenopausal women, reflecting that cohorts born after 1944 were at relatively higher risk of ER-positive tumors and lower risk of ER-negative tumors. If current trends continue, ER-positive cancers will increase at least 13% by 2018 in Denmark, ER-negative cancers will fall 15% by 2018, and breast cancer overall will increase at least 7% by 2018. Divergent ER-specific trends are consistent with distinct etiologic pathways. If trends in known risk factors are responsible, the Danish and US experience may foreshadow a common pattern worldwide.

Association of tissue inhibitor of metalloproteinases-1 and Ki67 in estrogen receptor positive breast cancer.

BACKGROUND: The role of tissue inhibitor of metalloproteinases-1 (TIMP-1) in estrogen receptor (ER) positive breast cancer remains to be fully elucidated. We evaluated TIMP-1 as a prognostic marker in patients treated with adjuvant tamoxifen and investigated TIMP-1s association with Ki67 and ER/progesterone receptor (PR)/human epidermal growth factor receptor 2 (HER2) profiles. MATERIAL AND METHODS: TIMP-1 expression was evaluated by immunohistochemistry (IHC) on formalin fixed paraffin embedded primary tumor tissue in two independent cohorts comprised of 236 and 192 patients, respectively. RESULTS: No differences in disease free survival (HR 0.98; 95% CI 0.63-1.53; p = 0.92) and overall survival (HR 0.94; 95% CI 0.63-1.43; p = 0.79) were observed according to TIMP-1 status. A significant negative association between TIMP-1 and Ki67 was identified (p = 0.015). TIMP-1 expression did not differ significantly according to ER/PR/HER2 profiles. When analyzed as separate variables PR and HER2 status tended to have a positive but non-significant association with TIMP-1 (PR: p = 0.08; OR 2.54; 95% CI 0.91-7.10, HER2: p = 0.08; OR 0.48; 95% CI 0.21-1.08) whereas ER status was not associated with TIMP-1 expression (p = 0.48; OR 0.68; 95% CI 0.23-1.99). CONCLUSION: TIMP-1 does not appear to be prognostic in breast cancer patients receiving adjuvant tamoxifen. We identified a negative association between TIMP-1 and Ki67. We did not confirm our previous in vitro findings of a negative association between TIMP-1 and PR.

Prognostic value of Bcl-2 in two independent populations of estrogen receptor positive breast cancer patients treated with adjuvant endocrine therapy.

INTRODUCTION: Estrogen receptor (ER) status is not an optimal marker for response to adjuvant endocrine therapy since approximately 30% of patients with ER-positive tumors eventually relapse. Bcl-2 is regulated by ER and may thus be considered as an indicator of ER activity and a candidate supplementary marker to ER status. PATIENTS AND METHODS: Tumor tissue from 257 patients with ER-positive breast cancer treated with tamoxifen was used for determination of the best threshold for immunohistochemical Bcl-2 assessment as prognostic marker. Subsequently, samples from the Danish patients of the randomized clinical trial BIG 1-98 comprising 1191 ER-positive patients treated with tamoxifen, letrozole or a sequence of the two were immunohistochemically stained for Bcl-2 to further explore the prognostic value of Bcl-2. RESULTS: Two Bcl-2 levels were found to divide the population of the primary study into significantly different groups according to disease-free survival (DFS). Multivariate analysis confirmed the significance of the lowest level, and showed Bcl-2 to be an independent prognostic marker. Analysis of the Danish cohort of the BIG 1-98 confirmed that Bcl-2 was a significant predictor of DFS, independent of known prognostic markers. However, in an additional analysis of a subset of the Danish cohort of BIG 1-98 including only HER-2 normal patients, the effect of Bcl-2 was not statistically significant. DISCUSSION: Low Bcl-2 can predict poor outcome of patients with ER-positive tumors treated with adjuvant endocrine therapy, whereas the use of Bcl-2 for determination of addition of chemotherapy was not supported by this study.

Amplification of ESR1 may predict resistance to adjuvant tamoxifen in postmenopausal patients with hormone receptor positive breast cancer.

The estrogen receptor (ER) is the target of tamoxifen, but endocrine therapies do not benefit all patients with ER positive tumors. We therefore hypothesized that copy number changes in the ESR1 gene, encoding ER, confer resistance. Within a consecutive series of ER positive, postmenopausal patients allocated to 5 years tamoxifen, we identified 61 patients with recurrence less than 4 years and 48 patients without recurrence at least 7 years after initiation of adjuvant tamoxifen. Archival tissue containing primary tumor was collected from 97 patients (89%). Tumor samples were analyzed for ESR1 copy number changes using FISH with a probe covering the ESR1 gene at 6q25 and a reference probe covering the centromere of chromosome 6. The assay was validated in a material of 120 normal breast samples. FISH analysis for ESR1 was successful in 91 patients (94%). Amplification (ratio ESR1/CEN-6 ≥ 2.0) was observed in 11 of 50 (22%) patients with early recurrence, compared to two of 41 (5%) patients without recurrence. The difference is statistically significant (P = 0.033). In both groups, two patients with ESR1 deletion (ratio ESR1/CEN-6 < 0.8) were identified. ESR1 amplification was significantly associated with poor disease-free survival (P = 0.0054) and overall survival (P = 0.0004). This pilot study supports our hypothesis that ESR1 amplification is associated with a poorer outcome following adjuvant treatment with tamoxifen in ER positive early breast cancer. This study also revealed the existence of ESR1 deletions. The prognostic and predictive impact of ESR1 copy number changes needs further exploration in clinical trials.

In situ aromatase expression in primary tumor is associated with estrogen receptor expression but is not predictive of response to endocrine therapy in advanced breast cancer.

BACKGROUND: New, third-generation aromatase inhibitors (AIs) have proven comparable or superior to the anti-estrogen tamoxifen for treatment of estrogen receptor (ER) and/or progesterone receptor (PR) positive breast cancer. AIs suppress total body and intratumoral estrogen levels. It is unclear whether in situ carcinoma cell aromatization is the primary source of estrogen production for tumor growth and whether the aromatase expression is predictive of response to endocrine therapy. Due to methodological difficulties in the determination of the aromatase protein, COX-2, an enzyme involved in the synthesis of aromatase, has been suggested as a surrogate marker for aromatase expression. METHODS: Primary tumor material was retrospectively collected from 88 patients who participated in a randomized clinical trial comparing the AI letrozole to the anti-estrogen tamoxifen for first-line treatment of advanced breast cancer. Semi-quantitative immunohistochemical (IHC) analysis was performed for ER, PR, COX-2 and aromatase using Tissue Microarrays (TMAs). Aromatase was also analyzed using whole sections (WS). Kappa analysis was applied to compare association of protein expression levels. Univariate Wilcoxon analysis and the Cox-analysis were performed to evaluate time to progression (TTP) in relation to marker expression. RESULTS: Aromatase expression was associated with ER, but not with PR or COX-2 expression in carcinoma cells. Measurements of aromatase in WS were not comparable to results from TMAs. Expression of COX-2 and aromatase did not predict response to endocrine therapy. Aromatase in combination with high PR expression may select letrozole treated patients with a longer TTP. CONCLUSION: TMAs are not suitable for IHC analysis of in situ aromatase expression and we did not find COX-2 expression in carcinoma cells to be a surrogate marker for aromatase. In situ aromatase expression in tumor cells is associated with ER expression and may thus point towards good prognosis. Aromatase expression in cancer cells is not predictive of response to endocrine therapy, indicating that in situ estrogen synthesis may not be the major source of intratumoral estrogen. However, aromatase expression in combination with high PR expression may select letrozole treated patients with longer TTP. TRIAL REGISTRATION: Sub-study of trial P025 for advanced breast cancer.

An ER activity profile including ER, PR, Bcl-2 and IGF-IR may have potential as selection criterion for letrozole or tamoxifen treatment of patients with advanced breast cancer.

INTRODUCTION: The purpose of this study was to evaluate the association between response at recurrence to letrozole versus tamoxifen and the expression of estrogen regulated proteins individually and combined in an "ER activity profile" in primary tumor tissue. Our hypothesis is that letrozole may be more effective than tamoxifen for treatment of tumors with high intratumoral estrogen content, whereas tamoxifen may be more efficient for treatment of tumors with high levels of the estrogen receptor (ER) and low intratumoral estrogen content. MATERIALS AND METHODS: For this study, we produced tissue microarrays from formalin fixed paraffin embedded primary tumor material from a subgroup of patients (9.4%), who have participated in the international, randomized, phase III clinical trial PO25 comparing letrozole with tamoxifen in 907 patients with advanced breast cancer. The expression levels of ER, the progesterone receptor (PR), the anti-apoptotic protein Bcl-2 and the Insulin like Growth Factor Receptor I (IGF-IR) were determined by semi-quantitative immunohistochemistry. RESULTS: Response to letrozole and tamoxifen treatment, measured as time to progression (TTP), was independent of primary tumor expression level of ER, Bcl-2 and IGF-IR. However, high expression of PR as well as high expression of three different ER activity profiles; ER/PR/Bcl-2, ER/PR/IGF-IR and ER/PR/Bcl-2/IGF-IR identified letrozole treated patients with significantly longer TTP. The ER activity profile including ER, PR, Bcl-2 and IGF-IR showed a trend towards being a selection criterion for letrozole versus tamoxifen therapy. DISCUSSION: This small sub-study supports our hypothesis that letrozole is superior to tamoxifen primarily in patients expressing high levels of estrogen regulated proteins in the primary tumor tissue. Furthermore, it seems that the "ER activity profile" with high PR, IGF-IR and Bcl-2 is a promising selection criterion, regarding prediction of response to letrozole versus tamoxifen.

Adjuvant letrozole versus tamoxifen according to centrally-assessed ERBB2 status for postmenopausal women with endocrine-responsive early breast cancer: supplementary results from the BIG 1-98 randomised trial.

BACKGROUND: The Breast International Group (BIG) 1-98 trial (a randomised double-blind phase III trial) has shown that letrozole significantly improves disease-free survival (DFS) compared with tamoxifen in postmenopausal women with endocrine-responsive early breast cancer. Our aim was to establish whether the benefit of letrozole versus tamoxifen differs according to the ERBB2 status of tumours. METHODS: The BIG 1-98 trial consists of four treatment groups that compare 5 years of monotherapy with letrozole or tamoxifen, and sequential administration of one drug for 2 years followed by the other drug for 3 years. Our study includes data from the 4922 patients randomly assigned to the two monotherapy treatment groups (letrozole or tamoxifen for 5 years; 51 months median follow-up [range <1 to 90 months]). A central assessment of oestrogen receptor (ER), progesterone receptor (PgR) and ERBB2 status using paraffin-embedded primary tumour material was possible for 3650 (74%) patients. ER, PgR, and ERBB2 expression were measured by immunohistochemistry (IHC) and ERBB2-positivity was confirmed by fluorescence in-situ hybridisation (FISH). Positive staining in at least 1% of cells was considered to show presence of ER or PgR expression. Tumours were deemed ERBB2-positive if amplified by FISH, or, for the few tumours with unassessable or unavailable FISH results, if they were IHC 3+. Hazard ratios (HR) estimated by Cox modelling were used to compare letrozole with tamoxifen for DFS, which was the primary endpoint, and to assess treatment-by-covariate interactions. The BIG 1-98 trial is registered on the clinical trials site of the US National Cancer Institute website http://www.clinicaltrials.gov/ct/show/NCT00004205. FINDINGS: By central assessment 7% (257 of 3650) of tumours were classified as ERBB2-positive. In 3533 patients with tumours confirmed to express ER, DFS was poorer in patients with ERBB2-positive tumours (n=239) than in those with ERBB2-negative tumours (n=3294; HR 2.09 [95% CI 1.59-2.76]; p<0.0001). There was no statistical evidence of heterogeneity in the treatment effect according to ERBB2 status of the tumour (p=0.60 for interaction), thus, letrozole improves DFS compared with tamoxifen regardless of ERBB2 status. The observed HRs were 0.62 (95% CI 0.37-1.03) for ERBB2-positive tumours and 0.72 (0.59-0.87) for ERBB2-negative tumours. INTERPRETATION: A benefit of letrozole over tamoxifen was noted, irrespective of ERBB2 status of the tumour, and, therefore, ERBB2 status does not seem to be a selection criterion for treatment with letrozole versus tamoxifen in postmenopausal women with endocrine-responsive early breast cancer.

Estrogen Receptor analyses in the Danish Breast Cancer Cooperative Group. History, methods, prognosis and clinical implications.

INTRODUCTION: Estrogen receptor (ER) is a prognostic and predictive biomarker, which has been known for 40 years. The detection method has developed over the years from different biochemical assays (BCA) to immunohistochemistry (IHC) on paraffin embedded tissue. The aim of the present study is to describe the development in ER analysis in the Danish Breast Cancer cooperative Group (DBCG), in the period of 1977 to 2006, regarding quantity and method of analyses. To compare BCA with IHC, and to report the prognosis for low-risk breast cancer patients. PATIENTS AND METHODS: In the period of 1991-1993, BCA and IHC were both performed on 2 364 tumours from breast cancer patients in Denmark. Three central laboratories in Copenhagen, Aarhus and Aalborg, respectively, performed BCA, while IHC was done in each of the pathology departments participating in the study. Data on ER status, clinical variables and prognostic factors were obtained from the DBCG database. Prognosis is calculated from the DBCG protocol 89a, regarding recurrence free survival (RFS) and overall survival (OS). RESULTS: We find an increasing frequency of ER positive tumours over time, with correlation to patient age. There is a better RFS and OS for tumours positive in both ER determinations. However, BCA is more sensitive than IHC. We find a significant correlation between positive ER status and other low risk factors, except lymph node status. DISCUSSION: Immunohistochemistry has several advantages compared with BCA; it is decentralised, only requiring small amounts of tumour tissue, with direct light microscopic interpretation of invasive tumour cells. It is less expensive and more rapid than BCA. Results in this study show the same RFS in both ER determinations. We conclude that IHC in analysing ER is a rapid, reliable and easy method, and we recommend the use of external quality control programme.

Semi-quantitative scoring of potentially predictive markers for endocrine treatment of breast cancer: a comparison between whole sections and tissue microarrays.

AIM: To assess whether immunohistochemically stained tissue microarrays (TMA) of 2 mm cores from paraffin embedded tumour tissue may replace whole sections in semi-quantitative evaluation of selected potential markers for endocrine treatment. METHODS: Whole sections and 2 mm cores on TMA were used for immunohistochemical staining of potential markers for endocrine treatment. The Allred scoring system was used for the markers with nuclear localisation: the oestrogen receptor, the progesterone receptor, p27 and the oestrogen receptor co-regulator amplified in breast cancer 1 (AIB1). The Allred scoring system was also used for the non-nuclear markers Bcl-2, pS2 and cyclooxygenase 2 (COX-2); the membrane receptors HER-2, insulin-like growth factor I receptor (IGF-IR) and epidermal growth factor receptor were quantified according to the guidelines for the Herceptest. RESULTS: The data and statistical analyses showed that the semi-quantitative evaluation of oestrogen receptor, progesterone receptor, AIB1, COX-2, HER-2 and IGF-IR on TMA blocks was comparable with analysis on whole sections. CONCLUSIONS: This study shows that semi-quantitative scoring of 2 mm cores on TMA is feasible for several potential markers for endocrine therapy. Considering the small size of many breast tumours, the speed and cost-effectiveness of immunohistochemistry on TMA compared with whole sections, and the importance of the expression level of the proteins, semi-quantitative scoring on TMA has great potential in both retrospective and prospective studies aiming at improving the prediction of response to endocrine treatment.

retrospective analysis of topoisomerase IIa amplifications and deletions as predictive markers in primary breast cancer patients randomly assigned to cyclophosphamide, methotrexate, and fluorouracil or cyclophosphamide, epirubicin, and fluorouracil: Danish Breast Cancer Cooperative Group.

PURPOSE: The aim of the study was to evaluate the predictive value of HER2 and topoisomerase IIalpha gene (TOP2A) for the efficacy of epirubicin in the adjuvant setting of breast cancer patients. PATIENTS AND METHODS: In the Danish Breast Cancer Cooperative Group trial 89D, 980 pre- and postmenopausal primary patients were randomly allocated to either CMF (cyclophosphamide, methotrexate, and fluorouracil; n = 500) or CEF (cyclophosphamide, epirubicin, and fluorouracil; n = 480) times 9, between January 1990 and November 1999. Tumor tissue was retrospectively identified from 805 patients and was analyzed for HER2-positivity and for TOP2A-amplifications and deletions. RESULTS: HER2-positivity was found in 33% of the 805 investigated tumors and was not a predictive marker for epirubicin sensitivity. TOP2A changes were identified in 23% of the 773 investigated tumors: 12% had TOP2A amplifications and 11% had TOP2A deletions. We found that patients with TOP2A amplification had an increased recurrence-free (RFS; hazard ratio [HR], 0.43; 95% CI, 0.24 to 0.78) and overall survival (OS; HR, 0.57; 95% CI, 0.29 to 1.13), respectively if treated with CEF compared with CMF, and that patients with TOP2A deletions had an almost identical hazard ratio (RFS: HR, 0.63; 95% CI, 0.36 to 1.11; OS: HR, 0.56; 95% CI, 0.30 to 1.04). This is in contrast to patients with a normal TOP2A genotype for whom similar outcome was observed in both treatment arms (RFS: HR, 0.90; 95% CI, 0.70 to 1.17; OS: HR, 0.88; 95% CI, 0.66 to 1.17). CONCLUSION: TOP2A amplification-and possibly deletion-seems to be predictive markers for the effect of adjuvant epirubicin containing therapy in primary breast cancer, but a final conclusion has to await a confirmative study or a meta-analysis.

The interindividual differences in the 3-demthylation of caffeine alias CYP1A2 is determined by both genetic and environmental factors.

This study investigated the role of genetic factors (CYP1A2) in caffeine metabolism. The CYP1A2 activity was determined in 378 Danish twins following oral intake of a single dose of 200 mg caffeine and subsequent determination of the caffeine ratio (AFMU+1MU+1MX)/17DMU in a 6-h urine sample. The mean (+/- SD) caffeine ratio was 5.9 +/- 3.4. The caffeine ratio was statistically significantly higher in men compared to women, in smoking men and women compared to non-smoking persons of the same gender and in women not taking oral contraceptives compared with women on oral contraceptives. Thus, we confirmed that CYP1A2 is more active in men than in women, that it is induced by smoking and inhibited by oral contraceptives. In the subsequent analysis of heritability, we included 49 monozygotic twin pairs and 34 same gender dizygotic twin pairs concordant for non-smoking and non-use of oral contraceptives. The intraclass correlation coefficient was 0.798 (95% confidence interval, 0.696-0.900) and 0.394 (95% confidence interval, 0.109-0.680) in the monozygotic and dizygotic twins, respectively. The correlation was statistically significantly higher (P = 0.0015) in the former compared with the latter. A biometrical model for the caffeine ratio including only additive genetic factors and unique environmental factors was the overall best fitting model. Estimates based on this model gave a heritability estimate of 0.725 (95% confidence interval 0.577-0.822). Unique environmental effects seem to account for the remainder 0.275 (95% confidence interval, 0.178-0.423). Our study shows that the CYP1A2 activity is mainly governed by genetic factors.

Reliability of histological malignancy grade, ER and HER2 status on core needle biopsy vs surgical specimen in breast cancer.

Early determination of malignancy grade and biomarkers in primary breast cancer is important when planning the treatment with neoadjuvant therapy and breast-conserving surgery. The aim of this study was to assess the reliability of malignancy grade on core needle biopsies, and to compare the concordance of ER and HER2 status on core needle biopsies vs surgical specimens. Eighty-nine patients with invasive ductal or lobular carcinoma were included. Malignancy grade was upgraded by one degree in 23.0%. The mitotic count had most impact on the malignancy grade, and may be underestimated on core needle biopsies compared with surgical specimens. ER status was concordant in core needle biopsies and surgical specimens in 98.0%, but should be repeated when doubtfully negative. HER2 status was concordant in 84.0% prior to FISH-HER2. Including FISH-HER2 data, the core needle biopsies showed a final total concordance of 95.4%. In conclusion, ER and HER2 are useful markers in core needle biopsies, providing a reliable base for preoperative decisions in neoadjuvant chemotherapy, and may be omitted on surgical specimens with certain precautions and exceptions. Determination of malignancy grade should be repeated on surgical specimens, and one should be aware of underestimation on core needle biopsies.

One year of adjuvant tamoxifen compared with chemotherapy and tamoxifen in postmenopausal patients with stage II breast cancer.

PURPOSE: We report the long-term results of a randomised trial comparing tamoxifen with tamoxifen plus cyclophosphamide, methotrexate and fluorouracil (CMF) in postmenopausal high-risk breast cancer patients. In addition, we analyse the prognostic and predictive value of centrally assessed subtypes. METHODS: Postmenopausal patients with breast cancer and positive nodes, deep invasion or size exceeding 5 cm were randomly assigned to 1 year of tamoxifen, or cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2 and fluorouracil 600 mg/m2 intravenously on day 1 every 4 weeks for nine cycles plus tamoxifen (CMFT). Tissue microarrays were constructed retrospectively and oestrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), and proliferation-related Ki-67 antigen (Ki67) status were assessed. RESULTS: From October 1982 through March 1990 we randomised 1445 patients and 969 (67%) were eligible for the biomarker analysis. At 10-years 936 women had suffered a disease-free survival (DFS) event (tamoxifen, 495 events in 686 patients; CMFT, 441 events in 642 patients). The addition of CMF to tamoxifen significantly improved DFS (adjusted hazard ratio 0.82; 95% confidence interval (CI) 0.71-0.93; P = 0.003) but not overall survival (adjusted hazard ratio 0.95; 95% CI 0.85-1.08; P = 0.44). DFS was superior in Luminal A tumours (ER or PgR positive, HER2 negative and Ki67 ≤ 14%) when compared to Luminal B or non-luminal (ER and PgR negative) tumours. There was no statistical evidence of heterogeneity by subtype in the benefit from CMF (P(interaction) = 0.45). CONCLUSION: CMF added to 1 year of tamoxifen reduces the risk of a DFS event. The benefit from CMF was not significantly different in Luminal A and B subtypes.

Activated HER-receptors in predicting outcome of ER-positive breast cancer patients treated with adjuvant endocrine therapy.

The four human epidermal growth factor receptors (HER1-4) are involved in growth stimulation and may play a role in endocrine resistance. The receptors form dimers, leading to activation by mutual phosphorylation. Our purpose was to explore the role of the activated receptors (pHER1, pHER2, pHER3) in endocrine treated breast cancer in terms of co-expression and association with disease-free survival (DFS) in 1062 patients with ER-positive tumors. Furthermore, HER2 amplification was evaluated. We found positive associations between the phosphorylated receptors. pHER1 and pHER3 were co-expressed with one or two of the other activated receptors in 85% and 89% of tumors, respectively, whereas pHER2 was co-expressed with the other activated receptors in 54% of tumors. Except for HER2, which was associated with poor prognosis, none of the remaining markers were associated with DFS. However, frequent co-expression indicates a role of the other HER-family members in activation of HER2.

High concordance between two companion diagnostics tests: a concordance study between the HercepTest and the HER2 FISH pharmDx kit.

The present study was done to investigate the concordance between the HER2 status measured by immunohistochemical analysis (HercepTest, DAKO, Carpinteria, CA) and fluorescence in situ hybridization (FISH; HER2 FISH pharmDx, DAKO) in a large study cohort (n = 681) of patients with high-risk breast cancer. A high agreement between immunohistochemical and FISH results was demonstrated. For the whole study population, the agreement between the 2 assays was 93.1% with a corresponding κ coefficient of 0.85. When the equivocal immunohistochemical 2+ cases were excluded from the analysis (n = 79), the agreement increased to 95.0% with a κ coefficient of 0.90. When the cutoff value for amplified/nonamplified cases in the HER2 FISH assay was increased from 2.0 to 2.2 as recommended in the American Society of Clinical Oncology/College of American Pathologists guidelines, the concordance between the 2 assays was 94.3% with a κ coefficient of 0.87 in the whole study population. When the equivocal immunohistochemical 2+ cases were excluded from this analysis, the concordance is similar (95.7% with a κ coefficient of 0.91).