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Contains fulltext : 139287.pdf (Publisher’s version ) (Open Access) BACKGROUND: Several studies have shown that the immunophenotype of distant breast cancer metastases may differ significantly from that of the primary tumor, especially with regard to differences in the level of hormone receptor protein expression, a process known as receptor conversion. This study aimed to compare expression levels of several membrane proteins between primary breast tumors and their corresponding distant metastases in view of their potential applicability for molecular imaging and drug targeting. METHODS: Expression of Claudin-4, EGFR, CAIX, GLUT-1 and IGF1R was assessed by immunohistochemistry on tissue microarrays composed of 97 paired primary breast tumors and their distant (non-bone) metastases. RESULTS: In both the primary cancers and the metastases, Claudin-4 was most frequently expressed, followed by GLUT-1, CAIX and EGFR.From primary breast cancers to their distant metastases there was positive to negative conversion, e.g. protein expression in the primary tumor with no expression in its paired metastasis, in 6%, 19%, 12%, 38%, and 0% for Claudin-4 (n.s), GLUT-1 (n.s), CAIX (n.s), EGFR (n.s) and IGF1R (n.s) respectively. Negative to positive conversion was seen in 65%, 47%, 43%, 9% and 0% of cases for Claudin-4 (p = 0.049), GLUT-1 (p = 0.024), CAIX (p = 0.002), EGFR (n.s.) and IGF1R (n.s.) respectively. Negative to positive conversion of Claudin-4 in the metastasis was significantly associated with tumor size (p = 0.015), negative to positive conversion of EGFR with negative PR status (p = 0.046) and high MAI (p = 0.047) and GLUT-1 negative to positive conversion with (neo)adjuvant chemotherapy (p = 0.039) and time to metastasis formation (p = 0.034). CAIX and GLUT-1 expression in the primary tumor were significantly associated with high MAI (p = 0.008 and p = 0.038 respectively). CONCLUSION: Claudin-4 is frequently expressed in primary breast cancers but especially in their metastases and is thereby an attractive membrane bound molecular imaging and drug target. Conversion in expression of the studied proteins from the primary tumor to metastases was fairly frequent, except for IGF1R, implying that the expression status of metastases cannot always be reliably predicted from the primary tumor, thereby necessitating biopsy for reliable assessment.

Contains fulltext : 109187pub.pdf (Publisher’s version ) (Closed access) BACKGROUND: Changes in the receptor profile of primary breast cancers to their metastases (receptor conversion) have been described for the estrogen receptor alpha (ERalpha) and progesterone receptor (PR). The purpose of this study was to evaluate the impact of receptor conversion for ERalpha and PR on survival in a large group of distant non-bone breast cancer metastases. METHODS: Receptor conversion was studied by immunohistochemistry in a group of 233 metastatic breast cancer patients. Kaplan-Meier overall survival curves were plotted, and differences between the curves were analyzed by log-rank analysis. The additional prognostic value of conversion to established prognosticators was studied by Cox regression. RESULTS: Overall survival of patients showing conversion from positive to negative ERalpha or PR, or from negative to positive ERalpha or PR, or remaining receptor negative was comparable, and significantly worse than patients remaining receptor positive. ERalpha or PR receptor conversion from positive in the primary breast tumor to negative in distant metastases has independent negative prognostic value. CONCLUSIONS: ERalpha or PR receptor conversion from positive in the primary breast cancer to negative in distant metastases has negative prognostic value. Cancer 2012. (c) 2012 American Cancer Society.

One of the most frequent genetic changes in sporadic breast cancer is amplification of the HER2 gene, usually resulting in protein overexpression on the cell membrane and growth activation of the cells. Breast cancer patients that have this HER2 amplification have a worse prognosis but can be treated with an antibody (trastuzumab) directed against the HER2 receptor which results in a survival benefit. However, this treatment is expensive and is associated with potentially severe side effects such as cardiotoxicity and should thus only be administrated to patients that are likely to show a response. It is therefore very important that a reliable diagnostic test exists that selects patients for this targeted treatment. Although many efforts have been made to standardize current diagnostic tests, there is still a lot of variability in procedure and interpretation. In this thesis we evaluated the suitability of a new fully-automatic HER2 protein staining system for use as an aid in a more stable determination of eligibility for trastuzumab therapy, by comparing it with other already established methods, and we explored a new PCR-based technique called MLPA (multiplex ligation-dependent probe amplification) as a low cost, technically uncomplicated and quantitative method to detect amplifications of HER2 in breast cancer in comparison with other techniques used for HER2 amplification or overexpression detection (IHC, CISH). Concordance between MLPA and CISH was 94% and between MLPA and IHC 90%. It is however not very likely that analysis of a single marker like HER2 will be sufficient for proper therapeutic choice-making and perhaps multiplex assays (being able to analyze several prognostic and predictive genes simultaneously) may facilitate oncologists in clinical decision making allowing a more personalized treatment. Furthermore, several processes such as polysomy 17 and genetic variation in breast cancer are still not yet fully understood. Therefore, in the second part of this thesis, we explored MLPA as a multiplex technique: first, we analyzed a large set of patients for both HER2 and TOP2A copy number by MLPA and CISH and found a concordance of 91% for TOP2A and 96% for HER2. MLPA is thus able to simultaneously detect breast cancer HER2 and TOP2A copy number in small quantities of DNA extracted from paraffin blocks, and thereby a good alternative or supplementary technique to other gene amplification detection methods like CISH. Secondly, we investigated the presence of polysomy 17 in breast cancer by simultaneously analyzing 17 chromosome 17 genes in 111 patients using MLPA. None of these patients showed a true polysomy 17; chromosome 17 usually showed a complex pattern of gains and losses, rather unrelated to the copy number status of the centromere (CEP17). And finally, we investigated the frequencies of amplifications, co-amplifications and losses of multiple important or potential breast cancer (onco)genes (MYC, CCND1, ESR1,…) and studied their association with each other and with clinicopathological parameters such as tumor size, age etc. We found a significant correlation between the number of amplified genes per tumor and the histological grade and mitotic index of the tumor.

Columnar cell lesions of the breast are proposed as the earliest recognizable precursor lesions of low grade breast cancer. The lesions are characterized by cystically enlarged terminal duct lobular units with monotonous (monoclonal) columnar type luminal cells. Variants with and without atypia are recognized. In her thesis, Mirthe de Boer discusses the morphological, immunohistochemical and molecular features of columnar cells lesions and other benign and premalignant look-alikes. Morphologically, a new phenomenon of pagetoid “pale cells” is described in columnar cell lesions. These pale cells could potentially serve as a feature of atypia. The role of chromosome 16q losses in breast carcinogenesis is reviewed and the frequency of chromosome 16q losses is studied in columnar cell lesions, columnar cell look-alikes, other members of the low nuclear grade breast neoplasia family and blunt duct adenosis. Based on the lack of chromosome 16q losses, blunt duct adenosis is proposed as a separate entity, falling in the spectrum of the benign lesions. Morphological criteria to discriminate between blunt duct adenosis and columnar cell lesions are presented. Finally, also the overlapping characteristics of columnar cell lesions and intraductal papillomas are discussed and the first papillary atypical columnar cell lesion variants are described. The results of this thesis underline the precursor role of columnar cell lesions and have the potential to improve accuracy and reproducibility of the diagnosis of early precursor breast lesions, which is the basis for researching optimal patient care.

Breast cancer is the most common type of cancer in women, representing approximately one-fourth of all cancers diagnosed in women, both worldwide and in the Netherlands. The most recent numbers from the Netherlands show that in 2019, a total of 17,114 women were diagnosed with primary ductal carcinoma in situ (DCIS) or invasive breast cancer. This high incidence of breast cancer led to the implementation of screening programs to detect breast tumors at an early stage. A disadvantage of current programs that rely on imaging is the fact that imaging only detects breast cancer when a tumor can be visualized. In view of the surge of liquid biopsy research (i.e., research focused on biofluids as sources of tumor-derived material shed by (pre)cancerous cells), it is not unlikely that a combination of imaging and measurement of biomarkers could be an approach used in the near future. Liquid biopsies can be collected minimally invasively or non-invasively and allow an easy and regular (even serial; e.g., every half year) measurement of cancer biomarkers. A liquid biopsy of special interest for early detection of breast cancer is nipple aspirate fluid, on account of its breast-specific source for biomarkers. Nipple aspirate fluid (NAF) refers to a fluid that is present in the ductal tree of the breasts and that can be obtained non-invasively using adapted breast pumps. Given its ductal (and possibly lobular) origin, which is where breast tumors develop, the acquisition of NAF and evaluation of its biomarker content are hypothesized to provide relevant information about early changes inside the breast. Another advantageous characteristic of NAF is the ability to synchronously acquire matched pairs of bilateral NAF samples, which provides intra-patient control samples for unilateral disease. NAF samples are a feasible source for the evaluation of biomarkers. The focus of investigation in this thesis was the biomarker class of microRNAs (miRNAs). MiRNAs are short, non-coding RNAs which regulate gene expression at the post-transcriptional level by binding to messenger RNA (mRNA). Clinically, miRNAs are biomarkers of interest because, in combination, they can form a signature or fingerprint for oncological diseases. Furthermore, miRNAs can be of value as biomarkers to understand the biology of disease or disease-related risk factors, such as high breast density for breast cancer. To investigate the potential of miRNAs in NAF and blood as biomarkers for early breast cancer detection, NAF, serum, and plasma samples were collected from three cohorts of women: a longitudinal high-risk cohort with repeated sample collection, a healthy cohort, and a cohort of women with established invasive breast cancer. Blood was also collected to compare the diagnostic value of biomarkers found in each fluid. All samples were immediately biobanked after collection. The first part of this thesis (Chapters 2-4) provides background information about NAF research, summarizes the practical aspects of creating the biobank, and describes the NAF collection process from the perspective of participants. The second part (Chapters 5-6) reports the first results of miRNAs analyses in NAF. In Chapter 7, we summarize the data obtained.

Male breast cancer (BC) is a rare disease that accounts for approximately 1% of all breast cancers. Approximately 5% of these cases are diagnosed as ductal carcinoma in situ (DCIS) and 95% as invasive BC, making DCIS of the male breast very uncommon. Due to its low prevalence, dedicated male BC research is challenging, and only few studies have been able to include a large cohort. Studies on male invasive BC that have been performed showed similarities with female breast cancer but also differences on several levels. But, as a consequence of the limited number of studies, male BC management is largely extrapolated from female BC studies. Therefore, there is an urgent need for more research in this field in order to improve risk stratification and optimize patient management. For this thesis we included patients from the EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program, a large male BC cohort. Paraffin embedded male BC tissue was retrospectively collected by the Dutch Breast Cancer Research Group (BOOG). We aimed to further characterize male BC by describing and evaluating histologic features including histological subtype, grade, mitotic activity index (MAI), presence of a fibrotic focus, density of tumor-infiltrating lymphocytes (TILs) and elastosis. We found that histological grade was not significantly associated with overall survival (OS) but MAI, presence of a fibrotic focus and a low TILs density were correlated to an unfavorable OS. BC subtype was correlated with TILs density. In addition, despite high ERα expression, male BC showed significantly less elastosis than female BC. In addition we aimed at enhancing our understanding of male breast carcinogenesis by comparing male DCIS to male invasive carcinoma. For the latter we studied the activated hypoxia response by conducting immunohistochemical stainings for the hypoxia related proteins HIF-1α, CAIX and Glut-1. This revealed that the hypoxia response is already at its maximum in the pre-invasive DCIS stage. We also studied oncogene amplification using multiplex ligation-dependent probe amplification (MLPA) and promotor hypermethylation using methylation specific MLPA (MS-MLPA). These studies showed that male DCIS and invasive cancer have a similar copy number profile and methylation of the studied genes is more likely to occur early in the male breast carcinogenesis.

Background: Androgen receptor (AR) has been described to play a prominent role in male breast cancer (MBC). It maps on chromosome X, and recent reports indicate that X-chromosome polysomy is frequent in MBC. Since the response to anti-androgen therapy may depend on AR polysomy and on its overexpression similarly to prostate cancer, the aim of the present study was to investigate the DNA methylation level of AR and its coregulators, especially those mapped on the X-chromosome, that may influence the activity of AR in MBC. Methods: The DNA methylation level of AR, MAGEA2, MAGEA11, MAGEC1, MAGEC2, FLNA, HDAC6, and UXT, mapped on the X-chromosome, was evaluated by quantitative bisulfite-NGS. Bioinformatic analysis was performed in a Galaxy Project environment using BWA-METH, MethylDackel, and Methylation Plotter tools. The study population consisted of MBC (41 cases) compared with gynecomastia (17 cases). Results: MAGEA family members, especially MAGEA2, MAGEA11, MAGEC, and UXT and HDAC6 showed hypomethylation of several CpGs, reaching statistical significance by the Kruskal–Wallis test (p < 0.01) in MBC when compared to gynecomastia. AR showed almost no methylation at all. Conclusions: Our study demonstrated for the first time that MAGEA family members mapped on the X-chromosome and coregulators of AR are hypomethylated in MBC. This may lead to their overexpression, enhancing AR activity.

Breast cancer in males is a rare disease. Due to its rarity, little research has been conducted on male breast cancer (MBC), especially when compared to its female counterpart. Knowledge on MBC is therefore based on small single institutional studies, as large series are lacking. The optimal clinical management of MBC may not be known and most treatment algorithms are yet derived from studies on its female counterpart. In our institution we collaborated with different institutes to collect a large group of MBC cases. Our aim was to better identify differences between male and female breast cancer (FBC), find better prognosticators, novel targets for therapy and thereby a better understanding of the MBC carcinogenesis. Based on our findings we conclude that although MBC and FBC are similar in many ways, behind these similarities clear differences are hidden. We therefore support the notion that MBC should be considered separately when in search for novel prognosticators and targets for therapy. Our findings on protein level Other than in FBC, Bcl2 expression has no significant associations with clinicopathologic features in MBC. In addition, the Bcl2/mitotic index combination did not predict survival in MBC, as opposed to FBC. In view of the important role of the microenvironment in cancer progression, stromal CTGF is an interesting target for novel therapies and molecular imaging in MBC. In the light of our findings it would be interesting to further explore and unravel the interactions of CTGF with other members of the CCN family and with other pathway effectors. The potential role of CTGF as a therapeutic target for triple negative FBC deserves to be further studied. Prognostic models, originally developed and validated for FBC, also perform quite well for MBC. These models may therefore help in MBC prognostication and decisions on adjuvant systemic therapy. Our findings on the molecular level Copy number loss on the long arm of chromosome 16 in combined with gain of the short arm identified a group of MBC with low propensity to develop lymph node metastasis, and copy number increase of NEUROD2 emerged as an independent prognostic factor. It would be interesting to verify these findings on a larger group of MBC and using more extensive techniques to shed a light on the possible mechanisms behind these observations. Having said that, work continues on the massive parallel sequencing of our MBC cases using a next generation sequencing platform. This will enable us to thoroughly study many known and interesting oncogenes moving forward in unraveling the carcinogenesis in MBC and finding novel possible targets for therapy. We propose a role for chromosome X in male carcinogenesis. Further studies are needed to clarify this role and its possible clinical value in terms of therapy. The predictive and prognostic value of BRCA2-like profile in MBC also deserves further study in light of uncovering better regimens to treat men with breast cancer. This urges a prospective clinical trial stratifying MBC patients for PARP inhibitors/platinum-based or conventional chemotherapy according to aCGH profile.