Search

Udgivelsesdato: 2010 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.

Recently, we presented evidence – based on the analysis of benign hyperproliferative lesions of the breast – for the presence of cells that express the stem cell marker cytokeratin (CK) 15 in combination with CK19, a protein widely expressed by mammary epithelial cells. Here we report the finding of a subset of breast carcinomas characterized by expression of CK15. CK15 expressing tumors constituted 5% (6 out of 120; 4 of ductal type and 2 of lobular type) of the high‐risk breast carcinomas examined by gel‐based proteomics and immunohistochemistry. Five out of the six CK15+ carcinomas were CK15+/CK19−. The remaining tumor was mainly composed of cells expressing both CK15 and CK19 (CK15+/CK19+), but it also contained invasive areas with cells expressing only one of these makers (CK15+/CK19− and CK15−/CK19+ cells). To address the relationship between putative luminal progenitor/amplified CK15+ cells and malignant disease, and to determine whether cells/lesions lose expression of CK15 as a result of tumour initiation and/or progression, we searched among our sample set for carcinomas in which invasive tumor areas co‐existed with non‐malignant cells and hyperproliferative and known pre‐malignant lesions. Only one such tumour was found (T71), a CK15−/CK19+/p53+ carcinoma that contained p53 negative non‐malignant epithelial cells exhibiting a variety of, CK15/CK19 cellular phenotypes (CK15+/CK19+; CK15+/CK19−; CK15−/CK19+; CK15−/CK19−), often associated with simple columnar cells. Single layers of epithelial cells exhibiting all four CK15/CK19 phenotypes were observed contiguous to areas of atypical ductal hyperplasia that contained p53 positive cells that lost CK15 expression (CK15−/CK19+) and had a very similar phenotype to those of the neighboring ductal carcinoma in situ (DCIS) and invasive cells. The undifferentiated CK15+/CK19+ cells, which had the phenotype CK15+/CK19+/CK14+/CK8+ and −/ER−/PgR−/AR−/CD44+ (weak)/CK17−/p63−/vimentin+/Ki67−/Bcl‐2+ (weak)/GATA‐3−/p53−, most likely correspond to lineage‐restricted luminal progenitor cells able to generate the other more differentiated CK15/CK19 cellular phenotypes, thus giving rise to the daunting intratumour heterogeneity displayed by carcinoma T71. Cells with a very similar phenotype to the CK15+/CK19+ progenitor cells were observed in a juvenile fibroadenoma as well as in the large collecting ducts of the breast. The latter, however, expressed in addition CK14 and had a phenotype (CK15+/CK19+/CK14+/CK8+ (weak)/ER−/PgR−/AR−/CD44+ (weak)/CK17−/p63−/vimentin−/Ki67−/Bcl‐2+/GATA‐3−/p53−) that resembled that of the putative normal adult breast stem cells as inferred from published data. Further molecular characterization of these progenitor cells as well as unraveling of the signaling pathways that regulate their growth and differentiation may prove invaluable for developing novel therapeutic strategies that target cancer at an early stage.

We have previously reported the 2D PAGE-based proteomic profiling of a prospective cohort of 78 triple negative breast cancer (TNBC) patients, and the establishment of a cumulative TNBC protein database. Analysis of this database identified a number of proteins as being specifically overexpressed in TNBC samples. One such protein was D-3-phosphoglycerate dehydrogenase (Phgdh), a candidate oncogene. We analysed expression of Phgdh in normal and TNBC mammary tissue samples by 2D gel-based proteomics and immunohistochemistry (IHC), and show here that high-level expression of Phgdh in mammary epithelial cells is primarily associated with cell lineage, as we found that Phgdh expression was predominant in CK5-positive cells, normal as well as malignant, thus identifying an association of this protein with the basal phenotype. Quantitative IHC analysis of Phgdh expression in normal breast tissue showed high-level expression of Phgdh in normal CK5-positive mammary epithelial cells, indicating that expression of this protein was not associated with malignancy, but rather with cell lineage. However, proteomic profiling of Phgdh showed it to be expressed in two major protein forms, and that the ratio of expression between these variants was associated with malignancy. Overexpression of Phgdh in CK5-positive cell lineages, and differential protein isoform expression, was additionally found in other tissues and cancer types, suggesting that overexpression of Phgdh is generally associated with CK5 cells, and that oncogenic function may be determined by isoform expression.

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.

Invasive apocrine carcinomas (IACs), as defined by morphological features, correspond to 0.3-4% of all invasive ductal carcinomas (IDC), and despite the fact that they are histologically distinct from other breast lesions there are currently no standard molecular criteria available for their diagnosis and no unequivocal information as to their prognosis. In an effort to address these concerns we have been using protein expression profiling technologies in combination with mass spectrometry and immunohistochemistry (IHC) to discover specific biomarkers that could allow us to molecularly characterize these lesions as well as to dissect some of the steps in the processes underlying breast apocrine metaplasia and development of precancerous apocrine lesions. Establishing these apocrine-specific markers as best practice for the routine pathology evaluation of breast cancer, however, will require their validation in large cohorts of patients. Towards this goal we have composed a panel of antibodies against components of an apocrine protein signature that includes probes against the apocrine-specific markers 15-prostaglandin dehydrogenase (15-PGDH), and acyl-CoA synthetase medium-chain family member 1 (ACSM1), in addition to a set of categorizing markers that are consistently expressed (AR, CD24) or not expressed (ERα, PgR, Bcl-2, and GATA-3) by apocrine metaplasia in benign breast lesions and apocrine sweat glands. This panel was used to analyze a well-defined cohort consisting of 14 apocrine ductal carcinoma in situ (ADCIS), and 33 IACs diagnosed at the Cancer Institute Hospital, Tokyo between 1997 and 2001. Samples were originally classified on the basis of cellular morphology with all cases having more than 90% of the tumour cells exhibiting cytological features typical of apocrine cells. Using the expression of 15-PGDH and/or ACSM1 as the main criterion, but taking into account the expression of other markers, we were able to identify unambiguously 13 out of 14 ADCIS (92.9%) and 20 out of 33 (60.6%) IAC samples, respectively, as being of apocrine origin. Our results demonstrate that IACs correspond to a distinct, even if heterogeneous, molecular subgroup of breast carcinomas that can be readily identified in an unbiased way using a combination of markers that recapitulate the phenotype of apocrine sweat glands (15-PGDH(+), ACSM1(+), AR(+), CD24(+), ERα(-), PgR(-), Bcl-2(-), and GATA-3(-)). These results pave the way for addressing issues such as prognosis of IACs, patient stratification for targeted therapeutics, as well as research strategies for identifying novel therapeutic targets for developing new cancer therapies.