Dorthe Grabau

Background: Tamoxifen is used in endocrine treatment of breast cancer to inhibit estrogen signaling. A set of stratified ER‐positive and ER‐negative tumor sections was subjected to manual deposition of tamoxifen solution in order to investigate its spatial distribution upon exposure to interaction within thin tissue sections.Methods: The localization of tamoxifen in tumor sections was assessed by matrix assisted laser deposition/ionization mass spectrometry imaging. The images of extracted ion maps were analyzed for comparison of signal intensity distributions.Results: The precursor ion of tamoxifen (m/z 372.233) displayed heterogeneous signal intensity distributions in histological compartments of tumor tissue sections. The levels of tamoxifen in tumor cells compared with stroma were higher in ER‐positive tissues, whereas ER‐negative tissue sections showed lower signal intensities in tumor cells.Conclusions: The experimental model was successfully applied on frozen tumor samples allowing for differentiation between ER groups based on distribution of tamoxifen.

Breast cancer is a very heterogeneous disease and some patients are cured by the surgical removal of the primary tumour whilst other patients suffer from metastasis and spreading of the disease, despite adjuvant therapy. A number of prognostic and treatment predictive factors have been identified such as tumour size, oestrogen (ER) and progesterone (PgR) receptor status, human epidermal growth factor receptor type 2 (HER2) status, histological grade, Ki67 and age. Lymph node involvement is also assessed during surgery to determine if the tumour has spread which requires dissection of the axilla and adjuvant treatment. The prognostic and treatment predictive factors assessing the nature of the tumour are all routinely based on the status of the primary tumour. We have analysed a unique tumour set of fourteen primary breast cancer tumours with matched synchronous axillary lymph node metastases and a set of nine primary tumours with, later developed, matched distant metastases from different sites in the body. We used a pairwise tumour analysis (from the same individual) since the difference between the same tumour-type in different patients was greater. Glycopeptide capture was used in this study to selectively isolate and quantify N-linked glycopeptides from tumours mixtures and the captured glycopeptides were subjected to label-free quantitative tandem mass spectrometry analysis. Differentially expressed proteins between primary tumours and matched lymph node metastasis and distant metastasis were identified. Two of the top hits, ATPIF1 and tubulin β-chain were validated by immunohistochemistry to be differentially regulated. We show that the expression of a large number of glycosylated proteins change between primary tumours and matched lymph node metastases and distant metastases, confirming that cancer cells undergo a molecular transformation during the spread to a secondary site. The proteins are part of important pathways such as cell adhesion, migration pathways and immune response giving insight into molecular changes needed for the tumour to spread. The large difference between primary tumours and lymph node and distant metastases also suggest that treatment should be based on the phenotype of the lymph node and distant metastases.

Cholesterol lowering statins have been demonstrated to exert anti-tumoral effects on breast cancer by decreasing proliferation as measured by Ki67. The biological mechanisms behind the anti-proliferative effects remain elusive. The aim of this study was to investigate potential statin-induced effects on the central cell cycle regulators cyclin D1 and p27. This phase II window-of-opportunity trial (Trial registration: ClinicalTrials.gov NCT00816244 , NIH) included 50 patients with primary invasive breast cancer. High-dose atorvastatin (80 mg/day) was prescribed to patients for two weeks prior to surgery. Paired paraffin embedded pre- and post-statin treatment tumor samples were analyzed using immunohistochemistry for the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and the cell cycle regulators cyclin D1 and p27. Corresponding frozen tumor sample pairs were analyzed for expression of the genes coding for cyclin D1 and p27, CCND1 and CDKN1B, respectively. Forty-two patients completed all study parts, and immunohistochemical evaluation of ER and PR was achievable in 30 tumor pairs, HER2 in 29 tumor pairs, cyclin D1 in 30 tumor pairs and p27 in 33 tumor pairs. The expression of ER, PR and HER2 did not change significantly following atorvastatin treatment. Cyclin D1 expression in terms of nuclear intensity was significantly decreased (P = 0.008) after statin treatment in paired tumor samples. The protein expression of the tumor suppressor p27, evaluated either as the fraction of stained tumor cells or as cytoplasmic intensity, increased significantly (P = 0.03 and P = 0.02, respectively). At the transcriptional level, no significant differences in mRNA expression were detected for cyclin D1 (CCND1) and p27 (CDKN1B). However, CCND1 expression was lower in tumors responding to atorvastatin treatment with a decrease in proliferation although not significantly (P = 0.08). We have previously reported statin-induced anti-proliferative effects in breast cancer. This study suggests that cell cycle regulatory effects may contribute to these anti-proliferative effects via cyclin D1 and p27.

Background: Breast cancer exhibits significant molecular, pathological, and clinical heterogeneity. Current clinicopathological evaluation is imperfect for predicting outcome, which results in overtreatment for many patients, and for others, leads to death from recurrent disease. Therefore, additional criteria are needed to better personalize care and maximize treatment effectiveness and survival. Methods: To address these challenges, the Sweden Cancerome Analysis Network - Breast (SCAN-B) consortium was initiated in 2010 as a multicenter prospective study with longsighted aims to analyze breast cancers with next-generation genomic technologies for translational research in a population-based manner and integrated with healthcare; decipher fundamental tumor biology from these analyses; utilize genomic data to develop and validate new clinically-actionable biomarker assays; and establish real-time clinical implementation of molecular diagnostic, prognostic, and predictive tests. In the first phase, we focus on molecular profiling by next-generation RNA-sequencing on the Illumina platform. Results: In the first 3 years from 30 August 2010 through 31 August 2013, we have consented and enrolled 3,979 patients with primary breast cancer at the seven hospital sites in South Sweden, representing approximately 85% of eligible patients in the catchment area. Preoperative blood samples have been collected for 3,942 (99%) patients and primary tumor specimens collected for 2,929 (74%) patients. Herein we describe the study infrastructure and protocols and present initial proof of concept results from prospective RNA sequencing including tumor molecular subtyping and detection of driver gene mutations. Prospective patient enrollment is ongoing. Conclusions: We demonstrate that large-scale population-based collection and RNA-sequencing analysis of breast cancer is feasible. The SCAN-B Initiative should significantly reduce the time to discovery, validation, and clinical implementation of novel molecular diagnostic and predictive tests. We welcome the participation of additional comprehensive cancer treatment centers. Trial registration: ClinicalTrials.gov identifier NCT02306096.

Ki67 is currently the proliferation biomarker of choice, with both prognostic and predictive value in breast cancer. A lack of consensus regarding Ki67 use in pre-analytical, analytical and post-analytical practice may hinder its formal acceptance in the clinical setting.

The aim was to confirm a previously defined prognostic index, combining a proliferation marker, histological grade, and estrogen receptor (ER) in different subsets of primary N0/N1 chemo-naïve breast cancer patients. In the present study, including 1,854 patients, Ki67 was used in the index (KiGE), since it is the generally accepted proliferation marker in clinical routine. The low KiGE-group was defined as histological grade 1 patients and grade 2 patients which were ER-positive and had low Ki67 expression. All other patients made up the high KiGE-group. The KiGE-index separated patients into two groups with different prognosis. In multivariate analysis, KiGE was significantly associated with disease-free survival, when adjusted for age at diagnosis, tumor size and adjuvant endocrine treatment (hazard ratio: 3.5, 95% confidence interval: 2.6–4.7, P<0.0001). We have confirmed a prognostic index based on a proliferation marker (Ki67), histological grade, and ER for identification of a low-risk group of patients with N0/N1 primary breast cancer. For this low-risk group constituting 57% of the patients, with a five-year distant disease-free survival of 92%, adjuvant chemotherapy will have limited effect and may be avoided.

Background. Oestrogen receptor (ER) status is important for the choice of systemic treatment of breast cancer patients. However, most data from randomised trials on the effect of adjuvant endocrine therapy according to ER status are based on the cytosol methods. Comparisons with immunohistochemical methods have given similar results. The aim of the present study was to examine whether different ER antibodies and heat-induced epitope retrieval (HIER) methods influence the prevalence of ER-positivity in primary breast cancer. Material and methods. This study is based on patients included in a clinical trial designed to compare the effect of two years of adjuvant tamoxifen versus no adjuvant systemic treatment in premenopausal women. From 1986 to 1991, 564 patients from two study centres in Sweden were enrolled and randomised. Patients were randomised independently of ER status. In the present study, ER status was assessed on tissue microarrays with the three different ER antibody/HIER combinations: 1D5 in citrate pH 6 (n = 390), SP1 in Tris pH 9 (n = 390) and PharmDx in citrate pH 6 (n = 361). Results. At cut-offs of 1% and 10%, respectively, the prevalence of ER-positivity was higher with SP1 (75% and 72%) compared with 1D5 (68% and 66%) and PharmDx (66% and 62%). At these cut-offs, patients in the discordant groups (SP1-positive and 1D5-negative) seem to have a prognosis intermediate between those of the double-positive and double-negative groups. Comparison with the ER status determined by the cytosol-based methods in the discordant group also showed an intermediate pattern. The repeatability was good for all antibodies and cut-offs, with overall agreement ≥ 93%. Conclusion. The present study shows that the choice of antibody and HIER method influences the prevalence of ER-positivity. We suggest that this be taken into consideration when choosing a cut-off for clinical decision making.

Background: An increasing number of neo-adjuvant breast cancer studies are being conducted and a novel model for tumor biological studies, the "window-of-opportunity" model, has revealed several advantages. Change in tumor cell proliferation, estimated by Ki67-expression in pre-therapeutic core biopsies versus post-therapeutic surgical samples is often the primary end-point. The aim of the present study was to investigate potential differences in proliferation scores between core biopsies and surgical samples when patients have not received any intervening anti-cancer treatment. Also, a lack of consensus concerning Ki67 assessment may raise problems in the comparison of neo-adjuvant studies. Thus, the secondary aim was to present a novel model for Ki67 assessment.Methods: Fifty consecutive breast cancer cases with both a core biopsy and a surgical sample available, without intervening neo-adjuvant therapy, were collected and tumor proliferation (Ki67, MIB1 antibody) was assessed immunohistochemically. A theoretical model for the assessment of Ki67 was constructed based on sequential testing of the null hypothesis 20% Ki67-positive cells versus the two-sided alternative more or less than 20% positive cells..Results: Assessment of Ki67 in 200 tumor cells showed an absolute average proliferation difference of 3.9% between core biopsies and surgical samples (p = 0.046, paired t-test) with the core biopsies being the more proliferative sample type. A corresponding analysis on the log-scale showed the average relative decrease from the biopsy to the surgical specimen to be 19% (p = 0.063, paired t-test on the log-scale). The difference was significant when using the more robust Wilcoxon matched-pairs signed-ranks test (p = 0.029). After dichotomization at 20%, 12 of the 50 sample pairs had discrepant proliferation status, 10 showed high Ki67 in the core biopsy compared to two in the surgical specimen (p = 0.039, McNemar's test). None of the corresponding results for 1000 tumor cells were significant - average absolute difference 2.2% and geometric mean of the ratios 0.85 (p = 0.19 and p = 0.18, respectively, paired t-tests, p = 0.057, Wilcoxon's test) and an equal number of discordant cases after dichotomization. Comparing proliferation values for the initial 200 versus the final 800 cancer cells showed significant absolute differences for both core biopsies and surgical samples 5.3% and 3.2%, respectively (p < 0.0001, paired t-test).Conclusions: A significant difference between core biopsy and surgical sample proliferation values was observed despite no intervening therapy. Future neo-adjuvant breast cancer studies may have to take this into consideration.