Violetta Filas

Expression of estrogen receptor beta in the breast carcinoma of BRCA1 mutation carriers.

BackgroundBreast cancers (BC) in women carrying mutations in BRCA1 gene are more frequently estrogen receptor negative than the nonhereditary BC. Nevertheless, tamoxifen has been found to have a protective effect in preventing contralateral tumors in BRCA1 mutation carriers. The identification of the second human estrogen receptor, ERβ, raised a question of its role in hereditary breast cancer. The aim of this study was to assess the frequency of ERα, ERβ, PgR (progesterone receptor) and HER-2 expression in breast cancer patients with mutated BRCA1 gene and in the control group.MethodsThe study group consisted of 48 women with BRCA1 gene mutations confirmed by multiplex PCR assay. The patients were tested for three most common mutations of BRCA1 affecting the Polish population (5382insC, C61G, 4153delA). Immunostaining for ERα, ERβ and PgR (progesterone receptor) was performed using monoclonal antibodies against ERα, PgR (DakoCytomation), and polyclonal antibody against ERβ (Chemicon). The EnVision detection system was applied. The study population comprised a control group of 120 BC operated successively during the years 1998–99.ResultsThe results of our investigation showed that BRCA1 mutation carriers were more likely to have ERα-negative breast cancer than those in the control group. Only 14.5% of BRCA1-related cancers were ERα-positive compared with 57.5% in the control group (P < 0.0001). On the contrary, the expression of ERβ protein was observed in 42% of BRCA1-related tumors and in 55% of the control group. An interesting finding was that most hereditary cancers (75% of the whole group) were triple-negative: ERα(-)/PgR(-)/HER-2(-) but almost half of this group (44.4%) showed the expression of ERβ.ConclusionIn the case of BRCA1-associated tumors the expression of ERβ was significantly higher than the expression of ERα. This may explain the effectiveness of tamoxifen in preventing contralateral breast cancer development in BRCA1 mutation carriers.

Study on breast carcinoma Her2/neu and hormonal receptors status assessed by automated images analysis systems: ACIS III (Dako) and ScanScope (Aperio).

UNLABELLED Her-2/neu is overexpressed in 20-30% of breast cancer patients and is associated with a more aggressive disease. Identification of Her-2/c-erbB-2-neu overexpression is based on immunohistochemical [ihc] detection of protein and/or gene amplification in fluorescence in situ hybridization test (FISH). Also Estrogen receptors [ER] and Progesterone receptors [PR] are the prognostic and predictive biomarkers, recently analysed by ihc methods. Subjective, manual scoring of the ihc Her-2/neu expression and expression of the ER/PR reported as the percentage of immunopositive cells are the most common mode of interpretation among pathologists. Automated microscopy and computerised processing have provided increased accuracy in quantification and standardisation. THE AIMS OF OUR STUDY WERE to evaluate the scoring reproducibility of Her-2 /neu ihc expression tested by two automated systems: ACIS (Dako) and ScanScope (Aperio); to estimate the ER/PR expression in ihc staining methods with different anti-ER/anti-PR antibodies (the monoclonal and the ER/PR pharmDx TM Kit) by the ACIS system. Her-2/neu ihc expression was measured in 114 primary invasive breast carcinomas by the manual and the automated scoring (ACIS and Aperio system). 106 slides stained ihc with two types of anti-ER/anti-PR antibodies entered the quantisation. The results of our investigations showed very high reproducibility of Her-2/neu scores in intra- and interobserver analysis by ACIS evaluation. The major concordance was present in strong 3+ ihc cases; very small discordance was shown by cases with low expression of Her-2/neu. The accuracy of scoring by the Aperio was little lower in comparison to ACIS but it might result from the smaller and variable series of samples analysed by Aperio. The concordance in scoring of two automated systems was 86.5% (p<0.0001; gamma=0.887); the discordance was referred only to the lower expression of Her-2/neu. The concordance in manual scoring performed by the single observer and the panel was 84.2% (p<0.0001, gamma = 0.99); the discordance comprised a few cases with strong expression (2+ vs 3+). Very high intra- and interobserver reproducibility of the ER/PR ihc measurements was present in the readers results (referred to the percentage of immunoreactive carcinomatous cell population in the breast carcinomas acc. to the ACIS algorithm). No differences were disclosed in the percentage of ER-immunoreactive and PR-immunoreactive carcinomatous cell populations when used 2 different type of antibodies, in the ACIS automated method.

Does Metformin affect ER, PR, IGF-1R, β-catenin and PAX-2 expression in women with diabetes mellitus and endometrial cancer?

ObjectiveDiabetes mellitus, as a risk factor for endometrial cancer (EC), causes an increase in insulin and IGF-1 concentrations in the blood serum. The increase in insulin and IGF-1 are considered mitogenic factors contributory to cancer development. Studies suggest that metformin has preventive activity, decreasing mortality and the risk of neoplasms. Since estrogen (ER), progesterone (PR) and IGF-1 (IGF-1R) receptor expression and β-catenin and PAX-2 mutations are significant in the development of endometrial cancer, it was decided to study these factors in patients with endometrial cancer and type 2 diabetes mellitus (DM2), and to establish the effects of metformin on their expression.MethodsThe expression of ER, PR, IGF-1R, β-catenin and PAX-2 have been immunohistochemically investigated in 86 type I endometrial cancer specimens. Patients were grouped according to the presence of DM2 and the type of hypoglycemic treatment administered.ResultsComparing EC patients with DM2 and normal glycemic status, we found increased IGF-1R expression in women with DM2. A decrease in ER expression was noted in women with EC and DM2 receiving metformin as compared to women treated with insulin (p = 0.004). There was no statistically significant difference in PR, IGF-1R, β-catenin and PAX-2 expression among women receiving metformin and other hypoglycemic treatment.ConclusionAlthough epidemiological studies suggest the beneficial role of metformin in many human cancers, there are still few studies confirming its favorable effect on endometrial cancer. Decreased ER expression in patients receiving metformin needs further research to allow evaluation of its clinical significance.

A case of primary testicular germ cell tumor with rhabdomyosarcoma metastases as an example of applying the FISH method to diagnostic pathology.

We present the interesting case of a 38‐year‐old man with a primary malignant tumor of the right testis that metachronously metastasized to the urinary bladder and the stomach. Histologically, the testicular tumor was a mixed germ cell tumor composed of teratoma and embryonal carcinoma, but it also contained a sarcoma component of somatic type malignancy. Metastases showed rhabdomyoblastic differentiation histologically identical to the sarcoma component of the testicular tumor that was diagnosed as rhabdomyosarcoma. By applying fluorescence in situ hybridization (FISH) to the cytogenetic examination of cells taken from the periventricular lymph node metastases, we demonstrated a structural chromosomal aberration characteristic of testicular neoplasms, i.e. the presence of isochromosome 12p (i(12p)). Additionally, the diagnosis was supported by immunohistochemistry.

The Determination of VEGF and MVD, among Patients with Primary Breast Cancer

The purpose of the study was to ascertain the value of assessment of vascular endothelial growth factor (VEGF) levels and microvessel density, and to search for correlations between them, in women with breast cancer. The assessment considered factors such as the stage of clinical disease advancement—according to International Union Against Cancer, the grade of histological malignancy, status of axillary lymph nodes and the size of the primary tumour. The concentration of VEGF was assessed in the plasma of 103 women with breast cancer, using an immunoenzymatic method (Quantikine test of R&D Systems). Assessment of microvessel density was performed using histopathological immunoperoxidase methods, using an anti-von Willebrand factor antibody (DAKO A/S). A statistically significant relationship was found between rising VEGF levels and microvessel density in women with breast cancer, when compared to values from a control group. A correlation was found between VEGF concentration and microvessel density (MVD) values. Statistically significant differences were found between VEGF levels of patients in stages I, II and III of clinical disease advancement. For MVD, differences were found only between stages I and III. A statistical relationship was also found between VEGF and MVD and tumour size. Similar results were found between VEGF concentrations in women with metastases to the axillary lymph nodes and cytokine levels in women with no metastases. The results of the study suggest that the degree of tumour vascularization and the concentration of VEGF may represent valuable indicators for the assessment of the angiogenic process in women with breast cancer.

Evaluation of the Sentinel Node Biopsy in Colorectal Carcinoma Including the Results of Immunohistochemical Examinations

Abstract Aim: To evaluate the method and results of sentinel node biopsy including immunohistochemical examinations in resectable colorectal cancer. Material and methods: From April 2004 to April 2005, sentinel node biopsy was carried out with the dye method in 27 patients operated on for colorectal cancer. The standard examination of sentinel nodes consisted in the evaluation of individual H&E-dyed specimens from bisection of the node. The negative sentinel nodes were examined with the use of immunohistochemistry against cytokeratins AE1/AE3. Findings: The sentinel node was identified in 25 patients (92.6%). In a routine histopathological examination it included metastases in 3 cases. The sentinel node was clean in one patient whereas other regional nodes resected “en bloc” with the tumour included metastases. The sensitivity of the method was 75%, and the number of falsely negative results was 25%. Metastases (micrometastases) in the sentinel node were found in 2 other patients (8%) in the immunohisto-chemical examination. This examination did not change the results of the analysis in the patient with positive non-sentinel nodes and with the negative sentinel node in H&E dyeing. Nevertheless, the sensitivity of the method rose to 83.3% and the number of falsely negative results dropped to 16.7%. Conclusions : 1. The sentinel node biopsy using the dye method is a safe and relatively easy technique showing a high success rate (92.6%). 2. Low sensitivity of the method, as reflected in the literature, may result from lack of extended histopathological examinations performed on the sentinel node (e.g. immunohistochemistry). 3. Further research is necessary to determine the role and importance of the sentinel node biopsy in colorectal cancer.

TRIM28 multi-domain protein regulates cancer stem cell population in breast tumor development

The expression of Tripartite motif-containing protein 28 (TRIM28)/Krüppel-associated box (KRAB)-associated protein 1 (KAP1), is elevated in at least 14 tumor types, including solid and hematopoietic tumors. High level of TRIM28 is associated with triple-negative subtype of breast cancer (TNBC), which shows higher aggressiveness and lower survival rates. Interestingly, TRIM28 is essential for maintaining the pluripotent phenotype in embryonic stem cells. Following on that finding, we evaluated the role of TRIM28 protein in the regulation of breast cancer stem cells (CSC) populations and tumorigenesis in vitro and in vivo. Downregulation of TRIM28 expression in xenografts led to deceased expression of pluripotency and mesenchymal markers, as well as inhibition of signaling pathways involved in the complex mechanism of CSC maintenance. Moreover, TRIM28 depletion reduced the ability of cancer cells to induce tumor growth when subcutaneously injected in limiting dilutions. Our data demonstrate that the downregulation of TRIM28 gene expression reduced the ability of CSCs to self-renew that resulted in significant reduction of tumor growth. Loss of function of TRIM28 leads to dysregulation of cell cycle, cellular response to stress, cancer cell metabolism, and inhibition of oxidative phosphorylation. All these mechanisms directly regulate maintenance of CSC population. Our original results revealed the role of the TRIM28 in regulating the CSC population in breast cancer. These findings may pave the way to novel and more effective therapies targeting cancer stem cells in breast tumors.

One hundred consecutive cases of sentinel lymph node mapping in colon cancer—the results of prospective, single-centre feasibility study with implementation of immunohistochemical staining

PurposeAlthough the importance of sentinel node biopsy (SNB) in colon cancer (CC) has not been clearly established, this method is proposed as potentially enabling more appropriate staging by means of immunohistochemistry (IHS). The aim of the study was to evaluate the SNB method used in CC treatment taking into consideration the results of the IHS examination.Materials and methodsIn the period from May 2005 to September 2010 in the 1st Department of Surgical Oncology and General Surgery, Wielkopolska Cancer Centre, 100 SNB in CC were performed. Sentinel nodes (SN) were identified intraoperatively with the use of Patent Blue dye. In the case of negative hematoxylin and eosin staining, the SN material was subjected to immunohistochemical examination. Finally, the histopathological findings of sentinel and non-sentinel lymph nodes were compared with the results of the immunohistochemical staining.ResultsAt least one SN was identified in 99 of 100 patients (99%). The SN was the only place of metastases in 12.1% (12/99) of the patients. The accuracy of SNB in determining the regional lymph node status was 93.9% (93/99). The sensitivity of the method was 83.3% (30/36). The false-negative rate amounted to 16.7% (6/36). Upstaging obtained by the implementation of the immunohistochemical method was 10% (7/70).ConclusionsThe application of the immunohistochemical staining enables upstaging of some patients, potentially benefiting from adjuvant chemotherapy. For full and definitive assessment of SNB in CC, further research is required especially in terms of additional factors determining a patient’s eligibility for this procedure.

2D and 3D cell cultures – a comparison of different types of cancer cell cultures

Cell culture is a widely used in vitro tool for improving our understanding of cell biology, tissue morphology, and mechanisms of diseases, drug action, protein production and the development of tissue engineering. Most research regarding cancer biology is based on experiments using two-dimensional (2D) cell cultures in vitro. However, 2D cultures have many limitations, such as the disturbance of interactions between the cellular and extracellular environments, changes in cell morphology, polarity, and method of division. These disadvantages led to the creation of models which are more closely able to mimic conditions in vivo. One such method is three-dimensional culture (3D). Optimisation of the culture conditions may allow for a better understanding of cancer biology and facilitate the study of biomarkers and targeting therapies. In this review, we compare 2D and 3D cultures in vitro as well as different versions of 3D cultures.

Oncogenic BRAF mutations and p16 expression in melanocytic nevi and melanoma in the Polish population

Introduction Twenty-five – fifty percent of skin melanomas arise from nevi. Melanocyte proliferation is activated by BRAF V600E, then is arrested, but single nevi transform to melanomas. p16 controls arrest, and p16 loss may promote transformation. Aim To analyze BRAF V600E, p16 expression and melanocyte proliferation in dermal, compound and dysplastic nevi, cells of primary and metastatic melanoma in the Polish population. Material and methods One hundred and thirty-two nevi (dermal, compound, dysplastic) and 41 melanomas (in situ, primary, metastatic) were studied. BRAF was assessed by cobas® 4800 BRAFV600 Mutation Test, High Resolution Melting Assay validated with: pyrosequencing and immunohistochemistry. p16 and Ki67 expression was analyzed by IHC. Results Eighty-two percent of nevi and 57% of melanomas display BRAF V600E expression. Most dermal and compound nevi had > 50% of p16(+) cells. BRAF V600E dysplastic nevi had a low number of p16(+) cells. Nevi without BRAF V600E (WT), had 90% of cells p16(+). In 60% of in situ and primary melanomas, there was a low number of cells of p16(+). Fifty percent of WT metastatic melanoma and 33% of BRAF V600E showed a high level of p16. The number of Ki67(+) cells in dysplastic nevi was very low. In 25% of BRAF V600E melanomas in situ and 55% of WT, > 10% cells were Ki67(+). All BRAF V600E primary melanomas and 66% of WT had > 10% Ki67(+) cells. Twenty percent of BRAF V600E and WT metastases had > 10% of Ki67(+), however, 62% of BRAF V600E and 32% of WT samples had > 50% of Ki67(+) cells. Conclusions BRAFV600E and p16 are more frequent in nevi than in melanoma in vivo. A significantly higher p16 expression was observed in mutated nevi than in WT, while in melanoma it was just the opposite. The proliferation rate of melanoma cells negatively correlated with p16 expression.