Profiling of microRNAs in tumor interstitial fluid of breast tumors - a novel resource to identify biomarkers for prognostic classification and detection of cancer.

It has been hypothesized based on accumulated data that a class of small noncoding RNAs, termed microRNAs, are key factors in intercellular communication. Here, microRNAs present in interstitial breast tumor fluids have been analyzed to identify relevant markers for a diagnosis of breast cancer and to elucidate the cross-talk that exists among cells in a tumor microenvironment. Matched tumor interstitial fluid samples (TIF, n = 60), normal interstitial fluid samples (NIF, n = 51), corresponding tumor tissue specimens (n = 54), and serum samples (n = 27) were collected from patients with breast cancer, and detectable microRNAs were analyzed and compared. In addition, serum data from 32 patients with breast cancer and 22 healthy controls were obtained for a validation study. To identify potential serum biomarkers of breast cancer, first the microRNA profiles of TIF and NIF samples were compared. A total of 266 microRNAs were present at higher level in the TIF samples as compared to normal counterparts. Sixty-one of these microRNAs were present in > 75% of the serum samples and were subsequently tested in a validation set. Seven of the 61 microRNAs were associated with poor survival, while 23 were associated with the presence of immune cells and adipocytes. To our knowledge, these data demonstrate for the first time that profiling of microRNAs in TIF can identify novel biomarkers for the prognostic classification and detection of breast cancer. In addition, the present findings demonstrate that microRNAs may represent the cross-talk that occurs between tumor cells and their surrounding stroma.

High level PHGDH expression in breast is predominantly associated with keratin 5-positive cell lineage independently of malignancy.

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.

Safety considerations for transplanting cryopreserved ovarian tissue to restore fertility in female patients who have recovered from Ewing's sarcoma.

Ewing's sarcoma (EWS) is a highly malignant cancer in children, adolescents and young adults. The chemotherapy required to treat female EWS patients may cause primary ovarian insufficiency and infertility as a side effect. Cryopreservation of ovarian tissue before the start of chemotherapy can potentially preserve fertility. When the patient has been cured and primary ovarian insufficiency has developed, transplantation of frozen/thawed ovarian tissue can restore ovarian function. The tissue is usually collected before chemotherapy is initiated, and malignant cells may contaminate the stored ovarian tissue, potentially causing recrudescence of the original cancer after transplantation. The risk of EWS metastasizing to the ovary is probably low but has not been studied in great detail. This review describes the available evidence on the risk of malignant cell contamination in the ovaries of EWS patients and presents a new case of malignant cells in an ovarian biopsy from a girl with EWS.

Neuroendocrine carcinoma of the breast - a pilot study of a Danish population of 240 breast cancer patients.

Neuroendocrine carcinoma of the breast - a very recent diagnosis, which was not recognized by WHO until 2003 - has lately been the subject of increasing attention. It is defined as a primary breast cancer with morphologic features similar to other types of neuroendocrine tumors of the lung and gastrointestinal tract combined with positive neuroendocrine immunohistochemical markers. While much information has been gathered during the last decade, most studies suffer from poor statistics due to a low incidence, and there are still fundamental open questions regarding etiology and prognosis. Furthermore, apparent limitations of the WHO definition appear to influence diagnosis. Here, we present our own results obtained from 13 cases and furthermore review previous reports with particular reference to incidence, clinical, histological, and prognostic features.

Tissue microarray analysis as a screening tool for neuroendocrine carcinoma of the breast.

Neuroendocrine carcinoma of the breast (NCB) is a fairly recent diagnostic entity added by WHO in 2003. Since then, studies have indicated that NCB potentially displays a worse prognosis than invasive ductal carcinoma. However, due to a lack of standard use of immunohistochemical staining for neuroendocrine markers and the fact that NCB may only show slight neuroendocrine morphology that can easily be overlooked, NCB is often underdiagnosed. Consequently, there is a need for fast and reliable detection method for NCB. Here, we take a first step toward finding an easy way of identifying NCB by investigating the usefulness of tissue microarray (TMA) analysis as a screening tool. We present our findings with regard to sensitivity and specificity compared with whole-mount sections. The material consists of 240 cases of breast cancer divided into 20 TMA blocks that were all immunohistochemically stained for the neuroendocrine markers chromogranin A and synaptophysin. Cases positive in more than 50% of the tumor cells were accepted in accordance with WHO (2003) standards of NCB. Sensitivity and specificity for TMA sections vs whole-mount sections were found to be 100% and 97.8%, respectively, suggesting that TMA analysis is a reliable method for NCB detection.

Ovarian tissue cryopreserved for fertility preservation from patients with Ewing or other sarcomas appear to have no tumour cell contamination.

AIM: The chemotherapy required to treat patients with sarcoma may as a side-effect induce infertility in girls and young women. If these patients have ovarian cortical tissue cryopreserved prior to chemotherapy, they may, if necessary, have the tissue transplanted and restore their fertility. The aim of this study was to evaluate the risk of residual cancer cells in the ovarian cortex intended for transplantation. PATIENTS AND METHODS: Ovarian tissue stored for fertility preservation from 16 surviving patients diagnosed with sarcoma (nine with Ewing sarcomas, four with osteosarcomas, two with synovial sarcomas and one with chondrosarcoma) was evaluated for the presence of malignant cells by histology and by transplantation to immunodeficient mice for 20 weeks. A fraction of the tissue from patients with Ewing sarcoma was also evaluated for the presence of the molecular marker EWS-FLI1 by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The transplant itself and selected murine organs were analysed for the presence of malignant cells by histology. RESULTS: All the mice accommodated the human tissue for 20 weeks of transplantation period with none of the mice developing any sign of cancer. In no instance were any cancer cells detected by histology or RT-qPCR. CONCLUSION: Ovarian tissue from patients with sarcoma appears to be without metastatic malignant cells in numbers that allow detection. Although the actual pieces of ovarian tissue used for transplantation remain unchecked, the current data indicate that the procedure is safe at least in patients that survive the sarcoma disease.

Immunoexpression analysis and prognostic value of BLCAP in breast cancer.

Bladder Cancer Associated Protein (BLCAP, formerly Bc10), was identified by our laboratory as being down-regulated in bladder cancer with progression. BLCAP is ubiquitously expressed in different tissues, and several studies have found differential expression of BLCAP in various cancer types, such as cervical and renal cancer, as well as human tongue carcinoma and osteosarcoma. Here we report the first study of the expression patterns of BLCAP in breast tissue. We analyzed by immunohistochemistry tissue sections of normal and malignant specimens collected from 123 clinical high-risk breast cancer patients within the Danish Center for Translational Breast Cancer Research (DCTB) prospective study dataset. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. We observed weak immunoreactivity for BLCAP in mammary epithelial cells, almost exclusively localizing to the cytoplasm and found that levels of expression of BLCAP were generally higher in malignant cells as compared to normal cells. Quantitative IHC analysis of BLCAP expression in breast tissues confirmed this differential BLCAP expression in tumor cells, and we could establish, in a 62-patient sample matched cohort, that immunostaining intensity for BLCAP was increased in tumors relative to normal tissue, in more than 45% of the cases examined, indicating that BLCAP may be of value as a marker for breast cancer. We also analyzed BLCAP expression and prognostic value using a set of tissue microarrays comprising an independent cohort of 2,197 breast cancer patients for which we had follow-up clinical information.

Implementation of TMA and digitalization in routine diagnostics of breast pathology.

To ensure optimal treatment of breast cancer patients, breast tumours are classified based on clinico-pathological features. As part of this process, routine diagnostics of breast tumours includes histological typing and grading, as well as profiling by use of an immunohistochemistry panel of antibodies, probes and in situ hybridization. This will, as a minimum, include assessment of oestrogen receptor (OR) and HER2. The individual preparation and staining of many breast tumours in a large laboratory with this standard panel is thus time consuming and costly. Herein, we show that in breast cancer routine diagnostics the use of the tissue microarray technique in combination with digitalization of the stained multi-slides is not only economical, with a considerable cost reduction, but it also enhances standardization of tumour profiling. We demonstrate that 2 mm breast tumour cores correlate with the corresponding tumour on whole mount slides, regarding staining/hybridizing results with the biomarkers in our panel consisting of human epidermal growth factor receptor 2, OR and Topiomerase IIa. Furthermore, we show that simultaneous staining/hybridizing of multiple breast tumour specimens reduces variation of staining/hybridizing quality, hereby increasing reliability of interpretation. By scanning and digitalization of the stained and hybridized multi-slides, we could optimize documentation and filing of the results. Our work is an example of translational research by implementing a tool in daily diagnostics originally developed for high throughput analyses in the search for prognostic and predictive markers in targeted medicine.

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.