Tumor initiating but differentiated luminal-like breast cancer cells are highly invasive in the absence of basal-like activity.

The majority of human breast cancers exhibit luminal epithelial differentiation. However, most aggressive behavior, including invasion and purported cancer stem cell activity, are considered characteristics of basal-like cells. We asked the following questions: Must luminal-like breast cancer cells become basal-like to initiate tumors or to invade? Could luminally differentiated cells within a basally initiated hierarchy also be tumorigenic? To answer these questions, we used rare and mutually exclusive lineage markers to isolate subsets of luminal-like and basal-like cells from human breast tumors. We enriched for populations with or without prominent basal-like traits from individual tumors or single cell cloning from cell lines and recovered cells with a luminal-like phenotype. Tumor cells with basal-like traits mimicked phenotypic and functional behavior associated with stem cells assessed by gene expression, mammosphere formation and lineage markers. Luminal-like cells without basal-like traits, surprisingly, were fully capable of initiating invasive tumors in NOD SCID gamma (NSG) mice. In fact, these phenotypically pure luminal-like cells generated larger and more invasive tumors than their basal-like counterparts. The tumorigenicity and invasive potential of the luminal-like cancer cells relied strongly on the expression of the gene GCNT1, which encodes a key glycosyltransferase controlling O-glycan branching. These findings demonstrate that basal-like cells, as defined currently, are not a requirement for breast tumor aggressiveness, and that within a single tumor there are multiple "stem-like" cells with tumorigenic potential casting some doubt on the hypothesis of hierarchical or differentiative loss of tumorigenicity.

Evidence for a stem cell hierarchy in the adult human breast.

Cellular pathways that contribute to adult human mammary gland architecture and lineages have not been previously described. In this study, we identify a candidate stem cell niche in ducts and zones containing progenitor cells in lobules. Putative stem cells residing in ducts were essentially quiescent, whereas the progenitor cells in the lobules were more likely to be actively dividing. Cells from ducts and lobules collected under the microscope were functionally characterized by colony formation on tissue culture plastic, mammosphere formation in suspension culture, and morphogenesis in laminin-rich extracellular matrix gels. Staining for the lineage markers keratins K14 and K19 further revealed multipotent cells in the stem cell zone and three lineage-restricted cell types outside this zone. Multiparameter cell sorting and functional characterization with reference to anatomical sites in situ confirmed this pattern. The proposal that the four cell types are indeed constituents of an as of yet undescribed stem cell hierarchy was assessed in long-term cultures in which senescence was bypassed. These findings identify an adult human breast ductal stem cell activity and its earliest descendants.

Epithelial-stromal interaction 1 (EPSTI1) substitutes for peritumoral fibroblasts in the tumor microenvironment.

Tumor cells can activate stroma, yet the implication of this activation in terms of reciprocal induction of gene expression in tumor cells is poorly understood. Epithelial Stromal Interaction 1 (EPSTI1) is an interferon response gene originally isolated from heterotypic recombinant cultures of human breast cancer cells and activated breast myofibroblasts. Here we describe the first immunolocalization of EPSTI1 in normal and cancerous breast tissue, and we provide evidence for a role of this molecule in the regulation of tumor cell properties and epithelial-mesenchymal transition. In general, no EPSTI1 staining was observed in normal breast epithelial cells from reduction mammoplasties (n=25). However, in carcinomas, staining was positive in 22 of 40 biopsies and inversely correlated with the level of differentiation. To address the function of EPSTI1, we expressed EPSTI1 ectopically in one cell line and silenced endogenous EPSTI1 by RNA interference in another. Irrespective of the experimental approach, EPSTI1 expression led to an increase in tumorsphere formation-a property associated with breast stem/progenitor cells. Most remarkably, we show that EPSTI1, by conveying spread of tumor cells, can replace peritumoral activated fibroblasts in a tumor environment assay. These observations implicate EPSTI1 as a hitherto unappreciated regulator of tumor cell properties.

Proteomic profiling of mammary carcinomas identifies C7orf24, a gamma-glutamyl cyclotransferase, as a potential cancer biomarker.

Breast cancer is the leading cause of cancer deaths in women today and is the most common cancer (excluding skin cancers) among women in the Western world. Although cancers detected by screening mammography are significantly smaller than nonscreening ones, noninvasive biomarkers for detection of breast cancer as early as possible are an urgent need as the risk of recurrence and subsequent death is closely related to the stage of the disease at the time of primary surgery. A set of 123 primary breast tumors and matched normal tissue was analyzed by two-dimensional (2D) gel electrophoresis, and a novel protein, C7orf24, was identified as being upregulated in cancer cells. Protein expression levels of C7orf24 were evaluated by immunohistochemical assays to qualify deregulation of this protein. Analysis of C7orf24 expression showed up-regulation in 36.4 and 23.4% of cases present in the discovery sample set (123 samples) and in an independent large TMA validation data set (2197 samples) of clinically annotated breast cancer specimens, respectively. Survival analysis showed that C7orf24 overexpression defines a subgroup of breast tumors with poor clinical outcome. Up-regulation of C7orf24 was also found in other cancer types. Four of these were investigated in greater detail, and we found that a proportion of tumors (58% in cervical, 38% in lung, 72% in colon, and 46% in breast cancer) expressed C7orf24 at levels exceeding those seen in normal samples. The observed overexpression of this protein in different types of cancer suggests deregulation of C7orf24 to be a general event in epithelial carcinogenesis, indicating that this protein may play an important role in cancer cell biology and thus constitute a novel therapeutic target. Furthermore, as C7orf24 is externalized to the tissue extracellular fluid and can be detected in serum, this protein also represents a potential serological marker.

15-prostaglandin dehydrogenase expression alone or in combination with ACSM1 defines a subgroup of the apocrine molecular subtype of breast carcinoma.

Established histopathological criteria divide invasive breast carcinomas into defined groups. Ductal of no specific type and lobular are the two major subtypes accounting for around 75 and 15% of all cases, respectively. The remaining 10% include rarer types such as tubular, cribriform, mucinous, papillary, medullary, metaplastic, and apocrine breast carcinomas. Molecular profiling technologies, on the other hand, subdivide breast tumors into five subtypes, basal-like, luminal A, luminal B, normal breast tissue-like, and ERBB2-positive, that have different prognostic characteristics. An additional subclass termed "molecular apocrine" has recently been described, but these lesions did not exhibit all the histopathological features of classical invasive apocrine carcinomas (IACs). IACs make up 0.5-3% of the invasive ductal carcinomas, and despite the fact that they are morphologically distinct from other breast lesions, there are presently no standard molecular criteria available for their diagnosis and as a result no precise information as to their prognosis. Toward this goal our laboratories have embarked in a systematic proteomics endeavor aimed at identifying biomarkers that may characterize and subtype these lesions as well as targets that may lead to the development of novel targeted therapies and chemoprevention strategies. By comparing the protein expression profiles of apocrine macrocysts and non-malignant breast epithelial tissue we have previously reported the identification of a few proteins that are specifically expressed by benign apocrine lesions as well as by the few IACs that were available to us at the time. Here we reiterate our strategy to reveal apocrine cell markers and present novel data, based on the analysis of a considerably larger number of samples, establishing that IACs correspond to a distinct molecular subtype of breast carcinomas characterized by the expression of 15-prostaglandin dehydrogenase alone or in combination with a novel form of acyl-CoA synthetase medium-chain family member 1 (ACSM1). Moreover we show that 15-prostaglandin dehydrogenase is not expressed by other breast cancer types as determined by gel-based proteomics and immunohistochemistry analysis and that antibodies against this protein can identify IACs in an unbiased manner in a large breast cancer tissue microarray making them potentially useful as a diagnostic aid.

Omics-based profiling of carcinoma of the breast and matched regional lymph node metastasis.

Axillary lymph node (ALN) status is currently used as an important clinical indicator of breast cancer prognosis. However, the molecular mechanisms underlying lymph node metastasis are poorly understood and the relationship between ALN metastasis and the primary tumor remains unclear. In an effort to reveal structural changes in the genome and related protein responses that may drive regional metastatic progression we have analyzed matched pairs of primary breast tumors and ALN metastases both at the genomic and proteomic levels using comparative genomic hybridization (CGH) array, quantitative high-resolution 2-D PAGE in combination with MS, and immunohistochemistry (IHC). Array CGH revealed a remarkable similarity in genomic aberration profiles between the matched primary tumors and the ALN metastases. Quantitative profiling of 135 known proteins also revealed striking similarities in their overall expression patterns, although we observed distinct changes in the levels of individual proteins in some sample pairs. The remarkable similarities of the overall genomic and proteomic profiles between primary tumors and matched ALN metastases are taken to suggest that, in general, key biological characteristics of the primary breast tumor are maintained in the corresponding lymph node metastases. Given that the omics-based technologies are oblivious to changes that only occur in minor cellular subsets we validated the proteomic data using IHC, which provides protein expression information with a valuable topological component. Besides confirming the omics-derived data, the IHC analysis revealed that in two cases the ALN metastases may have been derived from a distinct minor cell subpopulation present in the primary tumor rather than from the bulk of it.

Breast cancer in situ. From pre-malignant lesion of uncertain significance to well-defined non-invasive malignant lesion. The Danish Breast Cancer Cooperative Group Register 1977-2007 revisited.

In addition to nationwide standardized pathology forms for operable primary invasive breast cancer, the Danish Breast Cancer cooperative Group (DBCG) in 1982 introduced pathology forms for breast cancer in situ (CIS). The histological reporting form was used primarily for ductal cancer in situ (DCIS) treated with wide local excision. The form however, also provided information on lobular carcinoma in situ (LCIS), atypia and benign lesions. In 1989 the reporting form for DCIS was extended and now provided information on histological subtype, malignancy grade, growth pattern and both Estrogen receptor (ER) and Progesteron receptor (PR) status. Also mastectomy specimens were included. In 2004 the previous malignancy grading was replaced by the Van Nuys classification, and information on microcalcifications was introduced. The axillary status now included the sentinel node technique only. In 2006 the pleomorphic subtype of LCIS was added to histological subtypes. The present work reviews the DBCG guidelines and recommendations concerning CIS adding a brief characterization of the Danish CIS population. It also refers to the introduction of modern molecular pathology and distinction between low-risk and high-risk CIS lesions. A major point is that without the thirty years of outstanding efforts by the DBCG, future research would not be able to meet expectations.

Long-term results of breast conserving surgery vs. mastectomy for early stage invasive breast cancer: 20-year follow-up of the Danish randomized DBCG-82TM protocol.

The main objective of the present study aims at comparing the long-term efficacy of breast conserving surgery (BCS) vs. mastectomy (M) based on a randomized design. The Danish Breast Cancer Cooperative Group (DBCG) conducted the trial (DBCG-82TM) from January 1983 to March 1989 recruiting 1154 patients with invasive breast carcinoma. Follow-up time ended 1(st) May 2006 with a median follow-up time of 19.6 years (time span 17.1-23.3 years). Eligibility criteria included a one-sided, unifocal, primary operable breast carcinoma, patient age below 70 years, probability of satisfactory cosmetic outcome with BCS, and no evidence of disseminated disease. The patients accrued were grouped into three subsets: correctly randomized, suspicion of randomization error, and declining randomization. The main analyses focus on the subgroup of 793 correctly randomized patients representing 70% of the complete series. 10-year recurrence free survival (RFS) and 20-year overall survival (OS) based on intent to treat did not reveal significant differences in outcome between breast conserving surgery vs. mastectomy, p=0.95 and p=0.10, respectively. Including the complete series comprising 1133 eligible patients based on treatment in fact given similarly no significant difference between surgical options could be traced in outcome of 10-year RFS and 20-year OS, p=0.94 and p=0.24, respectively. The pattern of recurrences as a first event in breast conservation vs. mastectomy did not differ significantly irrespective of site, p=0.27. Looking into the type of local relapse, viz., new primaries vs. true recurrences, it appeared that new primaries were significantly associated to BCS, while true recurrences dominated among M treated patients (p<0.001). In conclusion, long-term data indicate that BCS in eligible patients proves as effective as mastectomy both regarding local tumour control, RFS and OS. Local failures as a first event consistent with new primaries are strongly associated with BCS, whereas true recurrence predominates after mastectomy.

A role for ADAM12 in breast tumor progression and stromal cell apoptosis.

As in developmental and regenerative processes, cell survival is of fundamental importance in cancer. Thus, a tremendous effort has been devoted to dissecting the molecular mechanisms involved in understanding the resistance of tumor cells to programmed cell death. Recently, the importance of stromal fibroblasts in tumor initiation and progression has been elucidated. Here, we show that stromal cell apoptosis occurs in human breast carcinoma but is only rarely seen in nonmalignant breast lesions. Furthermore, we show that ADAM12, a disintegrin and metalloprotease up-regulated in human breast cancer, accelerates tumor progression in a mouse breast cancer model. ADAM12 does not influence tumor cell proliferation but rather confers both decreased tumor cell apoptosis and increased stromal cell apoptosis. This dual role of ADAM12 in governing cell survival is underscored by the finding that ADAM12 increases the apoptotic sensitivity of nonneoplastic cells in vitro while rendering tumor cells more resistant to apoptosis. Together, these results show that the ability of ADAM12 to influence apoptosis may contribute to tumor progression.

Molecular pathology of breast apocrine carcinomas: a protein expression signature specific for benign apocrine metaplasia.

Breast cancer is a heterogeneous disease that encompasses a wide range of histopathological types including: invasive ductal carcinoma, lobular carcinoma, medullary carcinoma, mucinous carcinoma, tubular carcinoma, and apocrine carcinoma among others. Pure apocrine carcinomas represent about 0.5% of all invasive breast cancers according to the Danish Breast Cancer Cooperative Group Registry, and despite the fact that they are morphologically distinct from other breast lesions, there are at present no standard molecular criteria available for their diagnosis. In addition, the relationship between benign apocrine changes and breast carcinoma is unclear and has been a matter of discussion for many years. Recent proteome expression profiling studies of breast apocrine macrocysts, normal breast tissue, and breast tumours have identified specific apocrine biomarkers [15-hydroxyprostaglandin dehydrogenase (15-PGDH) and hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase)] present in early and advanced apocrine lesions. These biomarkers in combination with proteins found to be characteristically upregulated in pure apocrine carcinomas (psoriasin, S100A9, and p53) provide a protein expression signature distinctive for benign apocrine metaplasias and apocrine cystic lesions. These studies have also presented compelling evidence for a direct link, through the expression of the prostaglandin degrading enzyme 15-PGDH, between early apocrine lesions and pure apocrine carcinomas. Moreover, specific antibodies against the components of the expression signature have identified precursor lesions in the linear histological progression to apocrine carcinoma. Finally, the identification of proteins that characterize the early stages of mammary apocrine differentiation such as 15-PGDH, HMG-CoA reductase, and cyclooxygenase 2 (COX-2) has opened a window of opportunity for pharmacological intervention, not only in a therapeutic manner but also in a chemopreventive setting. Here we review published and recent results in the context of the current state of research on breast apocrine cancer.

Performance of combined clinical mammography and needle biopsy: a nationwide study from Denmark.

Clinical mammography and needle biopsy are key tools for non-operative assessment of breast lesions. We evaluated the performance of all combined tests undertaken in Denmark in 2000. Clinical mammography and needle biopsy data were collected and linked to final cancer outcome, to determine sensitivity, specificity, and predictive values of clinical mammography, needle biopsy, and combined test. In 2000, 6709 combined tests were performed in 36 mammography clinics in Denmark. The combined test was consistently more sensitive than any single test, increasing the proportion of women correctly identified with breast cancer by 9% compared with clinical mammography alone. For concordant combined tests (i.e. either both benign or both malignant), specificity and positive predictive value were 100%, sensitivity was 99.1%, and positive predictive value was 99.6%. Therefore, Danish patients with a malignant concordant combined test can proceed directly to definitive surgery without fear of a false-positive diagnosis, and Danish women with a concordant benign combined test can omit surgery without fear of a false-negative diagnosis. In discordant cases, our results showed that any of the two tests with a suspicious or malignant result indicated a high risk of cancer, and excisional diagnostic biopsy therefore still has an important role to play.

Towards discovery-driven translational research in breast cancer.

Discovery-driven translational research in breast cancer is moving steadily from the study of cell lines to the analysis of clinically relevant samples that, together with the ever increasing number of novel and powerful technologies available within genomics, proteomics and functional genomics, promise to have a major impact on the way breast cancer will be diagnosed, treated and monitored in the future. Here we present a brief report on long-term ongoing strategies at the Danish Centre for Translational Breast Cancer Research to search for markers for early detection and targets for therapeutic intervention, to identify signalling pathways affected in individual tumours, as well as to integrate multiplatform 'omic' data sets collected from tissue samples obtained from individual patients. The ultimate goal of this initiative is to coalesce knowledge-based complementary procedures into a systems biology approach to fight breast cancer.

[Mammographic screening in the county of Copenhagen. Clinical consequences of the first three screening rounds]. / Mammografiscreening i Københavns Kommune. Kliniske konsekvenser efter de første tre screeningsrunder.

INTRODUCTION: Service mammography has been offered biennially to women aged 50-69 years in the municipality of Copenhagen since 1991. The results were compared to breast cancer cases before initiation of screening. The comparison concerns prognostic factors and the treatment-related consequences. MATERIAL AND METHODS: Data from the Copenhagen service mammography screening were linked to data from the DBCG database. RESULTS: Before screening, 16% of breast cancer cases had a tumour size of 10 mm or less, this percentage increased to 41 in the screen-detected cases. Sixty per cent of breast cancer cases showed no evidence of metastatic spread to axillary lymph nodes before screening; this percentage increased to 78 per cent in the screen-detected cases. Forty per cent of ductal carcinomas showed a malignancy grade I before screening, compared to 53% in the screen-detected cases. Thirteen per cent were treated with breast conserving therapy before screening, as opposed to 48% in the screen-detected cases. Forty-one per cent needed postoperative adjuvant treatment before screening, compared with 21% in the screen-detected cases. DISCUSSION: A marked improvement was seen in the prognostic and treatment-related characteristics of the screen-detected breast cancer cases, as compared to breast cancer cases from the same area before screening was initiated.

[Mammography screening in the county of Copenhagen. Results of the first three screening rounds]. / Mammografiscreening i Københavns Kommune. Populationsresultater fra de første tre screeningsrunder.

INTRODUCTION: Biennial service mammography screening for breast cancer has been offered to women aged 50-69 years in the municipality of Copenhagen since 1991. We report the results of the first three invitation rounds. MATERIAL AND METHODS: Data were collected from the Copenhagen service mammography screening database and other Danish registers. RESULTS: The average participation rate during the first three invitation rounds was 66%. The breast cancer detection rate was 10/1,000 screened in the first invitation round and 5/1,000 in the consecutive rounds. The probability of a false positive mammography was 6% at the prevalent screen, and this was reduced to 3% at incidence screens. Fifty-two cases of interval cancer were seen after the first invitation round. The expected number was 152, which gives a proportional interval cancer rate of 0.34. The sensitivity was 86% and the specificity 94% after the first round. DISCUSSION: The detection rate of breast cancer was high, especially in the prevalence round. The trend in the incidence of breast cancer at the subsequent rounds was similar to that before screening, which indicates that mammography screening does not lead to any greater over-diagnosis. The rate of false positive mammography was high at the initial screening round, but was acceptable at subsequent rounds, and a false positive mammography does not seem to have affected participation in subsequent rounds. The Copenhagen screening programme conforms to international quality assurance guidelines for process evaluation.

[Mammographic screening in the municipality of Copenhagen 1991-1997. Evaluation of the preoperative assessment]. / Mammografiscreening i Københavns Kommune 1991-1997. Evaluering af det praeoperative udredningsforløb.

INTRODUCTION: A biennial mammography screening programme started for all women aged 50-69 in the municipality of Copenhagen, Denmark, in April 1991. The aim of the present study was to evaluate the quality of the preoperative diagnostic assessment for women recalled for further examination. Quality indicators were: validity of the fine-needle aspiration cytology, rate of malignant to benign surgery, and frequency of one-step surgery for malignant lesions. MATERIAL AND METHOD: Database registries during the first three screening rounds from April 1991 to March 1997 were studied retrospectively, for fine-needle aspiration cytology tests where surgical biopsy was also performed. RESULTS: In the period 1991-1997, 4,111 women were recalled for clinical mammography and subsequently 1,086 women underwent surgery. The use of the triple test in the preoperative assessment increased from 50% in the first screening round to 72% in the third. Throughout the period of evaluation, the number of inadequate fine-needle aspiration cytology (FNAC) was reduced from 32% to 6%. Inadequate FNAC from malignant lesions declined from 27% to 6%. The sensitivity of FNAC increased from 67% to 90% and the accuracy from 60% to 81%. The malignant/benign ratio of surgery rose from 1.4 in the first screening round to 2.8 in the third. One-step surgery as definitive treatment was performed in 67% of malignant instances. DISCUSSION: The preoperative diagnostic assessment improved during the evaluation period. The triple test was used more often over time, the validity of FNAC and the malignant/benign ratio of surgery increased.

Imaging of metastatic lymph nodes by X-ray phase-contrast micro-tomography.

Invasive cancer causes a change in density in the affected tissue, which can be visualized by x-ray phase-contrast tomography. However, the diagnostic value of this method has so far not been investigated in detail. Therefore, the purpose of this study was, in a blinded manner, to investigate whether malignancy could be revealed by non-invasive x-ray phase-contrast tomography in lymph nodes from breast cancer patients. Seventeen formalin-fixed paraffin-embedded lymph nodes from 10 female patients (age range 37-83 years) diagnosed with invasive ductal carcinomas were analyzed by X-ray phase-contrast tomography. Ten lymph nodes had metastatic deposits and 7 were benign. The phase-contrast images were analyzed according to standards for conventional CT images looking for characteristics usually only visible by pathological examinations. Histopathology was used as reference. The result of this study was that the diagnostic sensitivity of the image analysis for detecting malignancy was 100% and the specificity was 87%. The positive predictive value was 91% for detecting malignancy and the negative predictive value was 100%. We conclude that x-ray phase-contrast imaging can accurately detect density variations to obtain information regarding lymph node involvement previously inaccessible with standard absorption x-ray imaging.

Tissue proteomics of the human mammary gland: towards an abridged definition of the molecular phenotypes underlying epithelial normalcy.

Our limited understanding of the biological impact of the whole spectrum of early breast lesions together with a lack of accurate molecular-based risk criteria for the diagnosis and assignment of prognostic significance to biopsy findings presents an important problem in the clinical management of patients harboring precancerous breast lesions. As a result, there is a need to identify biomarkers that can better determine the outcome of early breast lesions by identifying subpopulations of cells in breast premalignant disease that are at high-risk of progression to invasive disease. A first step towards achieving this goal will be to define the molecular phenotypes of the various cell types and precursors - generated by the stem cell hierarchy - that are present in normal and benign conditions of the breast. To date there have been very few systematic proteomic studies aimed at characterizing the phenotypes of the different cell subpopulations present in normal human mammary tissue, partly due to the formidable heterogeneity of mammary tissue, but also due to limitations of the current proteomic technologies. Work in our laboratories has attempted to address in a systematic fashion some of these limitations and here we present our efforts to search for biomarkers using normal fresh tissue from non-neoplastic breast samples. From the data generated by the 2D gel-based proteomic profiling we were able to compile a protein database of normal human breast epithelial tissue that was used to support the biomarker discovery program. We review and present new data on the putative cell-progenitor marker cytokeratin 15 (CK15), and describe a novel marker, dihydropyriminidase-related protein 3 (DRP3) that in combination with CK15 and other well known proteins were used to define molecular phenotypes of normal human breast epithelial cells and their progenitors in resting acini, lactating alveoli, and large collecting ducts of the nipple. Preliminary results are also presented concerning DRP3 positive usual ductal hyperplasias (UDHs) and on single cell layer columnar cells (CCCs). At least two bona fide biomarkers of undifferentiated ERα/PgR negative luminal cells emerged from these studies, CK15 and c-KIT, which in combination with transformation markers may lead to the establishment of a protein signature able to identify breast precancerous at risk of progressing to invasive disease.

Up-regulated proteins in the fluid bathing the tumour cell microenvironment as potential serological markers for early detection of cancer of the breast.

Breast cancer is by far the most common diagnosed form of cancer and the leading cause of cancer death in women today. Clinically useful biomarkers for early detection of breast cancer could lead to a significant reduction in mortality. Here we describe a detailed analysis using gel-based proteomics in combination with mass spectrometry and immunohistochemistry (IHC) of the tumour interstitial fluids (TIF) and normal interstitial fluids (NIF) collected from 69 prospective breast cancer patients. The goal of this study was to identify abundant cancer up-regulated proteins that are externalised by cells in the tumour microenvironment of most if not all these lesions. To this end, we applied a phased biomarker discovery research strategy to the analysis of these samples rather than comparing all samples among each other, with inherent inter and intra-sample variability problems. To this end, we chose to use samples derived from a single tumour/benign tissue pair (patient 46, triple negative tumour), for which we had well-matched samples in terms of epithelial cell numbers, to generate the initial dataset. In this first phase we found 110 proteins that were up-regulated by a factor of 2 or more in the TIF, some of which were confirmed by IHC. In the second phase, we carried out a systematic computer assisted analysis of the 2D gels of the remaining 68 TIF samples in order to identify TIF 46 up-regulated proteins that were deregulated in 90% or more of all the available TIFs, thus representing common breast cancer markers. This second phase singled out a set of 26 breast cancer markers, most of which were also identified by a complementary analysis using LC-MS/MS. The expression of calreticulin, cellular retinoic acid-binding protein II, chloride intracellular channel protein 1, EF-1-beta, galectin 1, peroxiredoxin-2, platelet-derived endothelial cell growth factor, protein disulfide isomerase and ubiquitin carboxyl-terminal hydrolase 5 were further validated using a tissue microarray containing 70 malignant breast carcinomas of various grades of atypia. A significant number of these proteins have already been detected in the blood/plasma/secretome by others. The next steps, which include biomarker prioritization based on the hierarchal evaluation of these markers, antibody and antigen development, assay development, analytical validation, and preliminary testing in the blood of healthy and breast cancer patients, are discussed.

DNA copy number aberrations in breast cancer by array comparative genomic hybridization.

Array comparative genomic hybridization (CGH) has been popularly used for analyzing DNA copy number variations in diseases like cancer. In this study, we investigated 82 sporadic samples from 49 breast cancer patients using 1-Mb resolution bacterial artificial chromosome CGH arrays. A number of highly frequent genomic aberrations were discovered, which may act as "drivers" of tumor progression. Meanwhile, the genomic profiles of four "normal" breast tissue samples taken at least 2 cm away from the primary tumor sites were also found to have some genomic aberrations that recurred with high frequency in the primary tumors, which may have important implications for clinical therapy. Additionally, we performed class comparison and class prediction for various clinicopathological parameters, and a list of characteristic genomic aberrations associated with different clinicopathological phenotypes was compiled. Our study provides clues for further investigations of the underlying mechanisms of breast carcinogenesis.