Abstract:Up to one-third of women aged 30 -50 years have cysts in their breasts and are presumed to be at increased risk of developing breast cancer. Here we present an extensive proteomic and immunohistochemistry (IHC) study of breast apocrine cystic lesions aimed at generating specific biomarkers and elucidating the relationship, if existent, of apocrine cysts with cancer phenotype. To this end we compared the expression profiles of apocrine macrocysts obtained from mastectomies from high risk cancer patients with th… Show more
“…To determine if FABP7 and HMGCS2 antibodies are specific enough in terms of IHC staining to identify benign and malignant apocrine lesions [2], [16] we used a three-pronged strategy developed in our laboratory and described in detail elsewhere [23]: (i) analysis of potential cross-reactivity by 2D Western blotting (2D-WB), (ii) mass spectrometry validation of corresponding silver-stained protein spots superimposed with 2D-WB and (iii) IHC experiments with blocking of antibodies with a corresponding protein/epitope. This approach combines the intrinsic sensitivity and cellular resolution of IHC, with the specificity of 2D-WB and MS-based identification, and the overall protein analysis capability of gel-based proteomics [24].…”
Section: Resultsmentioning
confidence: 99%
“…With the aim to develop molecular criteria to reproducibly categorize IACs at the protein level we have undertaken a systematic proteomic analysis of well-defined set of apocrine carcinomas aimed at identifying biomarkers that may characterize and subtype these lesions to a greater detail, and to search for targets that may lead to the development of novel targeted therapies and chemoprevention strategies [2], [16], [17], [18], [19]. Accordingly, we have identified a number of apocrine protein markers that include 15-prostaglandin dehydrogenase (15-PGDH) and acyl-CoA synthetase medium-chain family member 1 (ACSM1) which together with a set of categorizing markers that are predominantly expressed (AR, CD24) or not expressed (bcl-2, GATA-3) by apocrine metaplastic lesions in benign breast lesions, were proven to be specific for both apocrine ductal carcinoma in situ (ADCIS) and IAC [2], [16], [17], [19], [20].…”
Section: Introductionmentioning
confidence: 99%
“…Accordingly, we have identified a number of apocrine protein markers that include 15-prostaglandin dehydrogenase (15-PGDH) and acyl-CoA synthetase medium-chain family member 1 (ACSM1) which together with a set of categorizing markers that are predominantly expressed (AR, CD24) or not expressed (bcl-2, GATA-3) by apocrine metaplastic lesions in benign breast lesions, were proven to be specific for both apocrine ductal carcinoma in situ (ADCIS) and IAC [2], [16], [17], [19], [20]. This apocrine signature has been shown to identify unambiguously 13 out of 14 ADCIS (92.9%) and 20 out of 33 (60.6%) IACs in a well characterized set of apocrine carcinomas [2] in which more than 90% of the tumor cells exhibited cytological features typical of apocrine cells [21].…”
Apocrine carcinoma of the breast is a distinctive malignancy with unique morphological and molecular features, generally characterized by being negative for estrogen and progesterone receptors, and thus not electable for endocrine therapy. Despite the fact that they are morphologically distinct from other breast lesions, no standard molecular criteria are currently available for their diagnosis. Using gel-based proteomics in combination with mass spectrometry and immunohistochemistry we have identified two novel markers, HMGCS2 and FABP7 that categorize the entire breast apocrine differentiation spectrum from benign metaplasia and cysts to invasive stages. Expression of HMGCS2 and FABP7 is strongly associated with apocrine differentiation; their expression is retained by most invasive apocrine carcinomas (IAC) showing positive immunoreactivity in 100% and 78% of apocrine carcinomas, respectively, as compared to non-apocrine tumors (16.7% and 6.8%). The nuclear localization of FABP7 in tumor cells was shown to be associated with more aggressive stages of apocrine carcinomas. In addition, when added to the panel of apocrine biomarkers previously reported by our group: 15-PGDH, HMGCR and ACSM1, together they provide a signature that may represent a golden molecular standard for defining the apocrine phenotype in the breast. Moreover, we show that combining HMGCS2 to the steroidal profile (HMGCS2+/Androgen Receptor (AR)+/Estrogen Receptor(ER)-/Progesteron Receptor (PR)- identifies IACs with a greater sensitivity (79%) as compared with the steroidal profile (AR+/ER-/PR-) alone (54%). We have also presented a detailed immunohistochemical analysis of breast apocrine lesions with a panel of antibodies against proteins which correspond to 10 genes selected from published transcriptomic signatures that currently characterize molecular apocrine subtype and shown that except for melanophilin that is overexpressed in benign apocrine lesions, these proteins were not specific for morphological apocrine differentiation in breast.
“…To determine if FABP7 and HMGCS2 antibodies are specific enough in terms of IHC staining to identify benign and malignant apocrine lesions [2], [16] we used a three-pronged strategy developed in our laboratory and described in detail elsewhere [23]: (i) analysis of potential cross-reactivity by 2D Western blotting (2D-WB), (ii) mass spectrometry validation of corresponding silver-stained protein spots superimposed with 2D-WB and (iii) IHC experiments with blocking of antibodies with a corresponding protein/epitope. This approach combines the intrinsic sensitivity and cellular resolution of IHC, with the specificity of 2D-WB and MS-based identification, and the overall protein analysis capability of gel-based proteomics [24].…”
Section: Resultsmentioning
confidence: 99%
“…With the aim to develop molecular criteria to reproducibly categorize IACs at the protein level we have undertaken a systematic proteomic analysis of well-defined set of apocrine carcinomas aimed at identifying biomarkers that may characterize and subtype these lesions to a greater detail, and to search for targets that may lead to the development of novel targeted therapies and chemoprevention strategies [2], [16], [17], [18], [19]. Accordingly, we have identified a number of apocrine protein markers that include 15-prostaglandin dehydrogenase (15-PGDH) and acyl-CoA synthetase medium-chain family member 1 (ACSM1) which together with a set of categorizing markers that are predominantly expressed (AR, CD24) or not expressed (bcl-2, GATA-3) by apocrine metaplastic lesions in benign breast lesions, were proven to be specific for both apocrine ductal carcinoma in situ (ADCIS) and IAC [2], [16], [17], [19], [20].…”
Section: Introductionmentioning
confidence: 99%
“…Accordingly, we have identified a number of apocrine protein markers that include 15-prostaglandin dehydrogenase (15-PGDH) and acyl-CoA synthetase medium-chain family member 1 (ACSM1) which together with a set of categorizing markers that are predominantly expressed (AR, CD24) or not expressed (bcl-2, GATA-3) by apocrine metaplastic lesions in benign breast lesions, were proven to be specific for both apocrine ductal carcinoma in situ (ADCIS) and IAC [2], [16], [17], [19], [20]. This apocrine signature has been shown to identify unambiguously 13 out of 14 ADCIS (92.9%) and 20 out of 33 (60.6%) IACs in a well characterized set of apocrine carcinomas [2] in which more than 90% of the tumor cells exhibited cytological features typical of apocrine cells [21].…”
Apocrine carcinoma of the breast is a distinctive malignancy with unique morphological and molecular features, generally characterized by being negative for estrogen and progesterone receptors, and thus not electable for endocrine therapy. Despite the fact that they are morphologically distinct from other breast lesions, no standard molecular criteria are currently available for their diagnosis. Using gel-based proteomics in combination with mass spectrometry and immunohistochemistry we have identified two novel markers, HMGCS2 and FABP7 that categorize the entire breast apocrine differentiation spectrum from benign metaplasia and cysts to invasive stages. Expression of HMGCS2 and FABP7 is strongly associated with apocrine differentiation; their expression is retained by most invasive apocrine carcinomas (IAC) showing positive immunoreactivity in 100% and 78% of apocrine carcinomas, respectively, as compared to non-apocrine tumors (16.7% and 6.8%). The nuclear localization of FABP7 in tumor cells was shown to be associated with more aggressive stages of apocrine carcinomas. In addition, when added to the panel of apocrine biomarkers previously reported by our group: 15-PGDH, HMGCR and ACSM1, together they provide a signature that may represent a golden molecular standard for defining the apocrine phenotype in the breast. Moreover, we show that combining HMGCS2 to the steroidal profile (HMGCS2+/Androgen Receptor (AR)+/Estrogen Receptor(ER)-/Progesteron Receptor (PR)- identifies IACs with a greater sensitivity (79%) as compared with the steroidal profile (AR+/ER-/PR-) alone (54%). We have also presented a detailed immunohistochemical analysis of breast apocrine lesions with a panel of antibodies against proteins which correspond to 10 genes selected from published transcriptomic signatures that currently characterize molecular apocrine subtype and shown that except for melanophilin that is overexpressed in benign apocrine lesions, these proteins were not specific for morphological apocrine differentiation in breast.
“…Our approach is along the line of the work of Celis et al [35,36], who analyzed the proteins found in the interstitial fluid that perfuses either the breast tumor microenvironment or the adipose tissue, and the work of Tammen et al [21], who analyzed the peptides and low molecular weight proteins extracted from tissues. Both methods identified also potentially "accessible" proteins (without chemical modifications) and were highly promising sources of biomarkers.…”
Targeting of tumoral tissues is one of the most promising approaches to improve both the efficacy and safety of anticancer treatments. The identification of valid targets, including proteins specifically and abundantly expressed in cancer lesions, is of utmost importance. Despite stateof-the-art technologies, the discovery of cancer-associated target proteins still faces the limitation, in human tissues, of antigen accessibility to suitable high-affinity ligands such as human mAb bound to bioactive molecules. Terminal perfusion of tumor-bearing mice or ex vivo perfusion of human cancer-bearing organs with a reactive biotin ester solution has successfully led to the identification of novel accessible biomarkers. This methodology is however restricted to perfusable organs, and excludes most of the tissues of interest to targeted therapies, e.g. primary breast cancer and metastases. Herein, we report on the development of a new chemical proteomic method that bypasses the perfusion step and thus offers the potential to identify accessible molecular targets in virtually all types of animal and human tissues. We have validated our new procedure by identifying biomarkers selectively expressed in human breast carcinoma. Overall, this powerful technology may lay the ground not only for custom-made therapies in cancer, but also for the development of therapies that need to be selectively delivered in a specific tissue.
“…It has been successfully used for protein analysis over more than three decades, and it is increasingly used in disease exploration [1][2][3][4]. Using 2-DE to map thousands of proteins presents the researcher with valuable information about a specific biological system.…”
2-DE is a powerful technique to discriminate post-translationally modified protein isoforms. However, all steps of 2-DE preparation and gel-staining may introduce unwanted artefacts, including inconsistent variation of background intensity over the entire 2-DE gel image. Background intensity variations limit the accuracy of gel orientation, overlay alignment and spot detection methods. We present a compact and efficient denoising algorithm that adaptively enhances the image contrast and then, through thresholding and median filtering, removes the gray-scale range covering the background. Applicability of the algorithm is demonstrated on immunoblots, isotope-labeled gels, and protein-stained gels. Validation is performed in contexts of (i) automatic gel orientation based on Hough transformation, (ii) overlay alignment based on cross correlation and (iii) spot detection. In gel stains with low background variability, e.g. Sypro Ruby, denoising will lower the spot detection sensitivity. In gel regions with high background levels denoising enhances spot detection. We propose that the denoising algorithm prepares images with high background for further automatic analysis, without requiring manual input on a gel-to-gel basis.
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