Publisher Correction: A CD146 FACS Protocol Enriches for Luminal Keratin 14/19 Double Positive Human Breast Progenitors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of organoid cultured human breast cancer.

Organoid cultures are increasingly used to model human cancers experimentally with a view to tailoring personalized medicine and predicting drug responses. Breast cancer is no exception, but in particular, primary breast cancer poses some inherent difficulties due to the frequent presence of residual non-malignant cells in the biopsies. We originally developed an assay for the distinction between malignant and non-malignant structures in primary breast cancer organoid cultures (Petersen et al., Proc Natl Acad Sci (USA) 89(19):9064-8, 1992). Here, we apply this assay to assess the frequency of normal-like organoids in primary breast carcinoma cultures and the cellular composition as a consequence of passaging. We find that in consecutively collected samples of primary human breast cancers, residual non-malignant tissues were observed histologically in five out of ten biopsies. Based on relevant morphogenesis and correct polarization as recorded by expression in luminal epithelial cells of mucin 1 (Muc1), occludin, and keratin 19 (K19) and expression in basal cells of integrin ß4, p63, and K14, non-malignant organoids were present in all primary human breast cancer-derived cultures. Furthermore, passaging in a contemporary culture medium was in favor of the selective expansion of basal-like cells. We conclude that organoid cultures of human breast cancers are most representative of the tissue origin in primary culture.

A CD146 FACS Protocol Enriches for Luminal Keratin 14/19 Double Positive Human Breast Progenitors.

Human breast cancer is believed to arise in luminal progenitors within the normal breast. A subset of these are double positive (DP) for basal and luminal keratins and localizes to a putative stem cell zone within ducts. We here present a new protocol based on a combination of CD146 with CD117 and CD326 which provides an up to thirty fold enrichment of the DP cells. We show by expression profiling, colony formation, and morphogenesis that CD146high/CD117high/CD326high DP cells belong to a luminal progenitor compartment. While these DP cells are located quite uniformly in ducts, with age a variant type of DP (vDP) cells, which is mainly CD146-negative, accumulates in lobules. Intriguingly, in specimens with BRCA1 mutations known to predispose for cancer, higher frequencies of lobular vDP cells are observed. We propose that vDP cells are strong candidates for tracing the cellular origin of breast cancer.

Prognostic significance of nuclear expression of UMP-CMP kinase in triple negative breast cancer patients.

We have previously identified UMP-CMP kinase (CMPK1) as a prognostic marker for triple negative breast cancer (TNBC) by mass spectrometry (MS). In this study we evaluated CMPK1 association to prognosis in an independent set of samples by immunohistochemistry (IHC) and assessed biological pathways associated to its expression through gene set enrichment analysis (GSEA). A total of 461 TNBC paraffin-embedded tissues were collected from different academic hospitals in Europe, incorporated into tissue micro-arrays (TMA), and stained for CMPK1 expression. We also collected gene expression data of 60 samples, which were also present in the TMA, for GSEA correlation analysis. CMPK1 IHC staining showed both cytoplasmic and nuclear components. While cytoplasmic CMPK1 did not show any association to metastasis free survival (MFS), nuclear CMPK1 was associated to poor prognosis independently from other prognostic factors in stratified Cox regression analyses. GSEA correlation analysis of the nuclear CMPK1-stratified gene expression dataset showed a significant enrichment of extracellular matrix (ECM; positive correlation) and cell cycle (negative correlation) associated genes. We have shown here that nuclear CMPK1 is indicative of poor prognosis in TNBCs and that its expression may be related to dysregulation of ECM and cell cycle molecules.

Cytokine profiling of tumor interstitial fluid of the breast and its relationship with lymphocyte infiltration and clinicopathological characteristics.

The tumor microenvironment is composed of many immune cell subpopulations and is an important factor in the malignant progression of neoplasms, particularly breast cancer (BC). However, the cytokine networks that coordinate various regulatory events within the BC interstitium remain largely uncharacterized. Moreover, the data obtained regarding the origin of cytokine secretions, the levels of secretion associated with tumor development, and the possible clinical relevance of cytokines remain controversial. Therefore, we profiled 27 cytokines in 78 breast tumor interstitial fluid (TIF) samples, 43 normal interstitial fluid (NIF) samples, and 25 matched serum samples obtained from BC patients with Luminex xMAP multiplex technology. Eleven cytokines exhibited significantly higher levels in the TIF samples compared with the NIF samples: interleukin (IL)-7, IL-10, fibroblast growth factor-2, IL-13, interferon (IFN)γ-inducible protein (IP-10), IL-1 receptor antagonist (IL-1RA), platelet-derived growth factor (PDGF)-ß, IL-1ß, chemokine ligand 5 (RANTES), vascular endothelial growth factor, and IL-12. An immunohistochemical analysis further demonstrated that IL-1RA, IP-10, IL-10, PDGF-ß, RANTES, and VEGF are widely expressed by both cancer cells and tumor-infiltrating lymphocytes (TILs), whereas IP-10 and RANTES were preferentially abundant in triple-negative breast cancers (TNBCs) compared to Luminal A subtype cancers. The latter observation corresponds with the high level of TILs in the TNBC samples. IL-1ß, IL-7, IL-10, and PDGFß also exhibited a correlation between the TIF samples and matched sera. In a survival analysis, high levels of IL-5, a hallmark TH2 cytokine, in the TIF samples were associated with a worse prognosis. These findings have important implications for BC immunotherapy research.

High-density SNP arrays improve detection of HER2 amplification and polyploidy in breast tumors.

BACKGROUND: Human epidermal growth factor receptor-2 (HER2) overexpression and gene amplification are currently established by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. This study investigates whether high-density single nucleotide polymorphism (SNP) arrays can provide additional diagnostic power to assess HER2 gene status. METHODS: DNA from 65 breast tumor samples previously diagnosed by HER2 IHC and FISH analysis were blinded and examined for HER2 copy number variation employing SNP array analysis. RESULTS: SNP array analysis identified 24 (37%) samples with selective amplification or imbalance of the HER2 region in the q-arm of chromosome 17. In contrast, only 15 (23%) tumors were found to have HER2 amplification by IHC and FISH analysis. In total, there was a discrepancy in 19 (29%) samples between SNP array and IHC/FISH analysis. In 12 of these cases, the discrepancy towards FISH could be attributed to concomitant amplification or deletion of the centromeric region, which harbors the FISH reference probe sequence. In 3 tumors, repeated IHC/FISH analysis revealed that the original IHC/FISH analysis had failed to indicate the correct HER2 expression level. Finally, the SNP array analysis revealed that more than two thirds of the samples exhibited polyploidy that was unrecognized by conventional FISH. CONCLUSIONS: Collectively, the data show that determination of HER2 copy number variations by SNP array-based genomic segmentation analysis is an effective supplement to IHC/FISH HER2 analysis that, by providing additional diagnostic sensitivity and accuracy, may elect more women for targeted treatment with HER2 inhibitors.

FABP7 and HMGCS2 are novel protein markers for apocrine differentiation categorizing apocrine carcinoma of the breast.

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.

Ferritin heavy chain in triple negative breast cancer: a favorable prognostic marker that relates to a cluster of differentiation 8 positive (CD8+) effector T-cell response.

Ferritin heavy chain (FTH1) is a 21-kDa subunit of the ferritin complex, known for its role in iron metabolism, and which has recently been identified as a favorable prognostic protein for triple negative breast cancer (TNBC) patients. Currently, it is not well understood how FTH1 contributes to an anti-tumor response. Here, we explored whether expression and cellular compartmentalization of FTH1 correlates to an effective immune response in TNBC patients. Analysis of the tumor tissue transcriptome, complemented with in silico pathway analysis, revealed that FTH1 was an integral part of an immunomodulatory network of cytokine signaling, adaptive immunity, and cell death. These findings were confirmed using mass spectrometry (MS)-derived proteomic data, and immunohistochemical staining of tissue microarrays. We observed that FTH1 is localized in both the cytoplasm and/or nucleus of cancer cells. However, high cytoplasmic (c) FTH1 was associated with favorable prognosis (Log-rank p = 0.001), whereas nuclear (n) FTH1 staining was associated with adverse prognosis (Log-rank p = 0.019). cFTH1 staining significantly correlated with total FTH1 expression in TNBC tissue samples, as measured by MS analysis (Rs = 0.473, p = 0.0007), but nFTH1 staining did not (Rs = 0.197, p = 0.1801). Notably, IFN γ-producing CD8+ effector T cells, but not CD4+ T cells, were preferentially enriched in tumors with high expression of cFTH1 (p = 0.02). Collectively, our data provide evidence toward new immune regulatory properties of FTH1 in TNBC, which may facilitate development of novel therapeutic targets.

A marker of endocrine receptor-positive cells, CEACAM6, is shared by two major classes of breast cancer: luminal and HER2-enriched.

Elucidating the phenotypic evolution of breast cancer through distinct subtypes relies heavily on defining a lineage blueprint of the normal human breast. Here, we show that in normal breast, within the luminal epithelial lineage, a subset of cells characterized by strong staining for endocrine receptors are also characterized by expression of the surface marker CEACAM6. Topographically, this pattern of staining predominates in terminal ductal lobular units, rather than in interlobular ducts. In culture, CEACAM6-expressing cells remain essentially postmitotic under conditions in which the other cells of luminal epithelial lineage are highly proliferative. We examined the pattern of expression among three major breast cancer subtypes: luminal, HER2-enriched, and basal-like. In 104 biopsies, the luminal and HER2-enriched subtypes showed a high proportion of CEACAM6(+) tumors (78% and 83%, respectively); the basal-like subtype showed a low proportion (28%). Further accentuation of this pattern was observed in 13 established breast cancer cell lines. When differentiation was induced by all-trans retinoic acid, CEACAM6 expression strongly correlated with luminal-like differentiation. Furthermore, CEACAM6(+) cancer cells were less proliferative than CEACAM6(-) cells in tumorsphere assays and were less tumorigenic in nude mice. Based on these observations, we propose that luminal and HER2-enriched breast cancers are more closely related than previously thought and may share a common cell of origin.

Tumor interstitial fluid - a treasure trove of cancer biomarkers.

Tumor interstitial fluid (TIF) is a proximal fluid that, in addition to the set of blood soluble phase-borne proteins, holds a subset of aberrantly externalized components, mainly proteins, released by tumor cells and tumor microenvironment through various mechanisms, which include classical secretion, non-classical secretion, secretion via exosomes and membrane protein shedding. Consequently, the interstitial aqueous phase of solid tumors is a highly promising resource for the discovery of molecules associated with pathological changes in tissues. Firstly, it allows one to delve deeper into the regulatory mechanisms and functions of secretion-related processes in tumor development. Secondly, the anomalous secretion of molecules that is innate to tumors and the tumor microenvironment, being associated with cancer progression, offers a valuable source for biomarker discovery and possible targets for therapeutic intervention. Here we provide an overview of the features of tumor-associated interstitial fluids, based on recent and updated information obtained mainly from our studies of breast cancer. Data from the study of interstitial fluids recovered from several other types of cancer are also discussed. This article is a part of a Special Issue entitled: The Updated Secretome.

Proteomic profiling of triple-negative breast carcinomas in combination with a three-tier orthogonal technology approach identifies Mage-A4 as potential therapeutic target in estrogen receptor negative breast cancer.

Breast cancer is a very heterogeneous disease, encompassing several intrinsic subtypes with various morphological and molecular features, natural history and response to therapy. Currently, molecular targeted therapies are available for estrogen receptor (ER)(-) and human epidermal growth factor receptor 2 (Her2)-positive breast tumors. However, a significant proportion of primary breast cancers are negative for ER, progesterone receptor (PgR), and Her2, comprising the triple negative breast cancer (TNBC) group. Women with TNBC have a poor prognosis because of the aggressive nature of these tumors and current lack of suitable targeted therapies. As a consequence, the identification of novel relevant protein targets for this group of patients is of great importance. Using a systematic two dimensional (2D) gel-based proteomic profiling strategy, applied to the analysis of fresh TNBC tissue biopsies, in combination with a three-tier orthogonal technology (two dimensional PAGE/silver staining coupled with MS, two dimensional Western blotting, and immunohistochemistry) approach, we aimed to identify targetable protein markers that were present in a significant fraction of samples and that could define therapy-amenable sub-groups of TNBCs. We present here our results, including a large cumulative database of proteins based on the analysis of 78 TNBCs, and the identification and validation of one specific protein, Mage-A4, which was expressed in a significant fraction of TNBC and Her2-positive/ER negative lesions. The high level expression of Mage-A4 in the tumors studied allowed the detection of the protein in the tumor interstitial fluids as well as in sera. The existence of immunotherapeutics approaches specifically targeting this protein, or Mage-A protein family members, and the fact that we were able to detect its presence in serum suggest novel management options for TNBC and human epidermal growth factor receptor 2 positive/estrogen receptor negative patients bearing Mage-A4 positive tumors.

Cryopreservation of ovarian tissue for fertility preservation: no evidence of malignant cell contamination in ovarian tissue from patients with breast cancer.

Cryopreserved ovarian cortical biopsies from 51 patients with breast cancer were examined by histologic and immunohistochemical analysis and showed no sign of metastases. Autotransplantation of ovarian cortex to patients with low-stage breast cancer disease appears safe, but confirmatory studies are required, including xenotransplantation studies.

Primary cardiac tumors: a clinicopathologic evaluation of four cases.

INTRODUCTION: We report the clinical, pathological, and immunohistochemical features of four primary malignant cardiac tumors identified at the Department of Pathology, Rigshospitalet, Denmark. A panel of immunohistochemical markers for classification is proposed. METHODS: Between 2000 and 2008, four patients with malignant cardiac tumors were treated at our hospital. We retrospectively reviewed the medical records and evaluated the patient characteristics and treatment. RESULTS: Three patients presented with severe dyspnea; one patient presented with chest pain. Transthoracic echocardiography demonstrated, in all four cases, abnormal masses in the atria. The cases were, based on morphological features and immunoprofile, classified as myogenic sarcoma (two cases), undifferentiated pleomorphic sarcoma, and leiomyosarcoma. Three of the patients received orthotopic heart transplantation. One patient survived 6.5 years after the diagnosis, and two patients are still alive 2 and 3 years after being diagnosed, respectively. CONCLUSIONS: All four cases were sarcomas. A limited number of immunohistochemical markers can be used in order to define a specific line of differentiation. In this small study, three of the patients were offered orthotopic heart transplantation, and the survival times were generally longer than in most series.

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.

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.

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.

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.

A high frequent BRCA1 founder mutation identified in the Greenlandic population.

Approximately 10% of all breast and ovarian cancers are dominantly inherited and mutations are mainly found in the BRCA 1 and 2 genes. The penetrance of BRCA1 mutations is reported to be between 68 and 92% and confers a 36-92% life time risk of breast cancer. Most mutations in BRCA1 are uniquely occurring mutations, but founder mutations have been described. In this study we describe a founder mutation with wide spread presence in the Inuit population. We have screened 2,869 persons from Greenland for the presence of a BRCA1 mutation (p.Cys39Gly) only found in the Inuit population. The overall carrier frequency was 1.6% in the general population, but the frequency differs geographically from 0.6% on the West coast to 9.7% in the previously isolated population of the East coast. This is to our knowledge the highest population frequency of a BRCA1 mutation ever to be described. To determine the clinical relevance of the mutation, we have examined ten breast cancer patients and nine ovarian cancer patients from Greenland for the presence of the p.Cys39Gly mutation. We found three ovarian cancer patients (33%) and one breast cancer patient (10%) carrying the mutation. The high number of women carrying a BRCA1 mutation known to trigger the development of potentially lethal diseases leads us to recommend an offer of genetic counselling and test for the mutation to all females of Inuit origin, thereby hopefully preventing a number of breast and ovarian cancer deaths.

Molecular characterization of apocrine carcinoma of the breast: validation of an apocrine protein signature in a well-defined cohort.

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.