Associations between genomic stratification of breast cancer and centrally reviewed tumour pathology in the METABRIC cohort.

The integration of genomic and transcriptomic profiles of 2000 breast tumours from the METABRIC [Molecular Taxonomy of Breast Cancer International Consortium] cohort revealed ten subtypes, termed integrative clusters (IntClust/s), characterised by distinct genomic drivers. Central histopathology (N = 1643) review was undertaken to explore the relationship between these ten molecular subtypes and traditional clinicopathological features. IntClust subtypes were significantly associated with histological type, tumour grade, receptor status, and lymphocytic infiltration (p < 0.0001). Lymph node status and Nottingham Prognostic Index [NPI] categories were also significantly associated with IntClust subtype. IntClust 3 was enriched for tubular and lobular carcinomas, the latter largely accounting for the association with CDH1 mutations in this cluster. Mucinous carcinomas were not present in IntClusts 5 or 10, but did not show an association with any of the remaining IntClusts. In contrast, medullary-like cancers were associated with IntClust 10 (15/26). Hormone receptor-positive tumours were scattered across all IntClusts. IntClust 5 was dominated by HER2 positivity (127/151), including both hormone receptor-positive (60/72) and hormone receptor-negative tumours (67/77). Triple-negative tumours comprised the majority of IntClust 10 (132/159) and around a quarter of IntClust 4 (52/217). Whilst the ten IntClust subtypes of breast cancer show characteristic patterns of association with traditional clinicopathological variables, no IntClust can be adequately identified by these variables alone. Hence, the addition of genomic stratification has the potential to enhance the biological relevance of the current clinical evaluation and facilitate genome-guided therapeutic strategies.

A distinct pre-existing inflammatory tumour microenvironment is associated with chemotherapy resistance in high-grade serous epithelial ovarian cancer.

BACKGROUND: Chemotherapy resistance is a major determinant of poor overall survival rates in high-grade serous ovarian cancer (HGSC). We have previously shown that gene expression alterations affecting the NF-κB pathway characterise chemotherapy resistance in HGSC, suggesting that the regulation of an immune response may be associated with this phenotype. METHODS: Given that intrinsic drug resistance pre-exists and is governed by both tumour and host factors, the current study was performed to examine the cross-talk between tumour inflammatory microenvironment and cancer cells, and their roles in mediating differential chemotherapy response in HGSC patients. Expression profiling of a panel of 184 inflammation-related genes was performed in 15 chemoresistant and 19 chemosensitive HGSC tumours using the NanoString nCounter platform. RESULTS: A total of 11 significantly differentially expressed genes were found to distinguish the two groups. As STAT1 was the most significantly differentially expressed gene (P=0.003), we validated the expression of STAT1 protein by immunohistochemistry using an independent cohort of 183 (52 resistant and 131 sensitive) HGSC cases on a primary tumour tissue microarray. Relative expression levels were subjected to Kaplan-Meier survival analysis and Cox proportional hazard regression models. CONCLUSIONS: This study confirms that higher STAT1 expression is significantly associated with increased progression-free survival and that this protein together with other mediators of tumour-host microenvironment can be applied as a novel response predictive biomarker in HGSC. Furthermore, an overall underactive immune microenvironment suggests that the pre-existing state of the tumour immune microenvironment could determine response to chemotherapy in HGSC.

Immunohistochemical expression of BRCA1 protein in invasive ductal carcinoma of the breast.

Immunohistochemically detected BRCA1 positivity has been reported to indicate the presence of full-length functional protein, while immunohistochemical negativity may be a result of sporadic BRCA1 mutation. We set out to study the immunohistochemical expression of BRCA1 protein in breast cancer and assess associations with lymph node status, histologic grade and expression of other biomarkers. We examined 100 patients aged from 21 to 71 years diagnosed with invasive ductal carcinoma. Immunohistochemistry was performed using anti-BRCA1, ER, PR, HER2/neu, and Ki-67 (MIB-1) antibodies. Cytoplasmic or nuclear-cytoplasmic BRCA1 expression was identified in a total of 64 breast cancer patients. None of the breast cancer tissue samples showed solely nuclear BRCA1 immunoreactivity. BRCA1 expression was associated with higher histologic grade, and majority of BRCA1-positive patients were below 50 years of age. Most BRCA1-negative patients had intermediate grade tumors. BRCA1 expression was positively associated with ER and PR positivity, and negatively associated with HER2/neu overexpression. Although immunohistochemistry can be an inexpensive and valuable preliminary method for detecting the BRCA1 status, BRCA1 protein localization is a complex issue. Well designed studies are needed to further investigate the performance of various anti-BRCA1 antibodies in formalin- fixed paraffin-embedded tissue samples, assess their association with BRCA1 gene mutations and determine clinical usefulness of BRCA1 immunohistochemistry.

BCL2 in breast cancer: a favourable prognostic marker across molecular subtypes and independent of adjuvant therapy received.

BACKGROUND: Breast cancer is heterogeneous and the existing prognostic classifiers are limited in accuracy, leading to unnecessary treatment of numerous women. B-cell lymphoma 2 (BCL2), an antiapoptotic protein, has been proposed as a prognostic marker, but this effect is considered to relate to oestrogen receptor (ER) status. This study aimed to test the clinical validity of BCL2 as an independent prognostic marker. METHODS: Five studies of 11 212 women with early-stage breast cancer were analysed. Individual patient data included tumour size, grade, lymph node status, endocrine therapy, chemotherapy and mortality. BCL2, ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) levels were determined in all tumours. A Cox model incorporating the time-dependent effects of each variable was used to explore the prognostic significance of BCL2. RESULTS: In univariate analysis, ER, PR and BCL2 positivity was associated with improved survival and HER2 positivity with inferior survival. For ER and PR this effect was time dependent, whereas for BCL2 and HER2 the effect persisted over time. In multivariate analysis, BCL2 positivity retained independent prognostic significance (hazard ratio (HR) 0.76, 95% confidence interval (CI) 0.66-0.88, P<0.001). BCL2 was a powerful prognostic marker in ER- (HR 0.63, 95% CI 0.54-0.74, P<0.001) and ER+ disease (HR 0.56, 95% CI 0.48-0.65, P<0.001), and in HER2- (HR 0.55, 95% CI 0.49-0.61, P<0.001) and HER2+ disease (HR 0.70, 95% CI 0.57-0.85, P<0.001), irrespective of the type of adjuvant therapy received. Addition of BCL2 to the Adjuvant! Online prognostic model, for a subset of cases with a 10-year follow-up, improved the survival prediction (P=0.0039). CONCLUSIONS: BCL2 is an independent indicator of favourable prognosis for all types of early-stage breast cancer. This study establishes the rationale for introduction of BCL2 immunohistochemistry to improve prognostic stratification. Further work is now needed to ascertain the exact way to apply BCL2 testing for risk stratification and to standardise BCL2 immunohistochemistry for this application.

Expression of E-cadherin and c-erbB-2/HER-2/neu oncoprotein in high-grade breast cancer.

E-cadherin (E-CD) is an epithelial-specific cell adhesion molecule, whose expression is lost in invasive lobular (ILC) but not in invasive ductal carcinoma (IDC) of the breast. This cell adhesion system can be disrupted by tyrosine kinase c-erbB-2/HER-2/neu. We examined 106 cases of high-grade invasive breast cancer, including 91 IDCs, 12 ILCs and 3 pleomorphic lobular carcinomas (PLCs). We determined Nottingham histological grade and performed immunohistochemistry for estrogen and progesterone receptors (ER/PR), Ki-67, E-CD and c-erbB-2/HER-2/neu with subsequent fluorescence in situ hybridization. Amplification of c-erbB-2/HER-2/neu gene was observed in 55/91 (60.4%) of IDCs, 3/12 (25%) of ILCs and 1/3 (33.3%) of PLCs, and associated with positive axillary lymph nodes. E-CD expression was lost in 14/91 (15.4%) of IDCs, 10/12 (83.3%) of ILCs and 2/3 (66.7%) of PLCs. The loss of E-CD immunoreactivity in IDCs appeared to be associated with c-erbB-2/HER-2/neu gene amplification, negative ER/PR status and positive lymph nodes, whereas E-CD-positive ILCs tended to be HER-2/neu-positive. The biological significance of E-CD expression seems to be different in high-grade IDC and ILC. Oncogenic pathway mediated by c-erbB-2/HER-2/neu may affect the E-CD expression in most invasive ductal breast carcinomas in vivo.

Clinical characteristics of prognostic factors in uterine endometrioid adenocarcinoma of various grade.

The aim of this study was to compare clinical characteristics of prognostic factors in uterine endometrioid adenocarcinoma of various grades. We have studied 104 postmenopausal women with a histological diagnosis of uterine endometrioid adenocarcinoma. Staging and grading of primary tumor were done according to FIGO system. The following factors were examined: family history of cancer, presence of obesity and vaginal bleeding, recurrence rate within the two years of the study (disease-free periods), vessel permeation, muscle invasion (<1/3, 2/3, >2/3), cervical involvement, lymph node metastasis, ascites cell analysis, parametrium invasion, adnexal metastasis, CA125 pre-surgery values. Histological examination has showed grade 1 endometrioid adenocarcinoma in 35 cases (33,7%, group 1), grade 2 adenocarcinoma in 44 cases (42,3%, group 2), and grade 3 adenocarcinoma in 25 cases (24%, group 3). Most of the factors we have examined seem to be associated with histological grade of uterine endometrioid carcinoma. The analysis of clinicopathological prognostic factors in G1 endometrioid adenocarcinoma cases has showed that about half of these patients are obese, vaginal bleeding is not common, no cervical involvement, parametrium invasion, adnexal metastasis and vessel permeation at the time of diagnosis, no recurrence within two years, pre-surgery value of CA125 is normal, and myometrial invasion is less than 1/3. G3 endometrioid adenocarcinoma cases have showed family history of endometrial cancer, more than half of the patients were obese, with uncommon vaginal bleeding and positive peritoneal cytology, but cervical involvement, parametrium invasion, adnexal metastasis and vessel permeation are present at the time of diagnosis, pre-surgery value of CA125 is high, and myometrial invasion is 2/3 or more than 2/3 in majority of cases, furthermore, in some cases recurrent tumors were developed within two years. G2 endometrioid adenocarcinoma can be considered as an intermediary form which should be managed according to the clinical stage. The results lead to conclude that the histological grade of uterine endometrioid adeno carcinoma seems to be an important independent prognostic indicetor as it is strongly associated with other clinical pathological prognostic factors.

The expression of cadherin e and clinical prognostic factors in uterine endometrioid adenocarcinoma.

The aim of our study was to evaluate the association between the expression of E-cadherin and clinical prognostic factors in uterine endometrioid adenocarcinoma of different histological grade. We have studied 104 postmenopausal women with diagnosis of endometrioid adenocarcinoma. We evaluated the presence of obesity and vaginal bleeding. Surgical specimens were fixed in 10% neutral buffered formalin solution and embedded in paraffin. 4 mm sections were stained by hematoxylin-eosin, von Gieson, and histological type of cancer, metastatic involvement of lymph nodes and depth of myometrial invasion were evaluated. Histological grade of cancer was assessed by FIGO grading system. All samples were analyzed by immunohistochemistry for E-cadherin (Dakocytomation). We assessed the number of E-cadherin-positive and negative tumor cells and degree of positivity (low, moderate, high). Histological study by hematoxylin-eosin has showed grade 1 endometrioid carcinoma in 35 cases (33.7%, group I), grade 2 adenocarcinoma in 44 cases (42.3%, group II), and grade 3 adenocarcinoma in 25 cases (24%, group III). Our results suggest that the loss of E-cadherin expression is associated with a higher histological grade of uterine endometrioid adenocarcinoma, depth of myometrial invasion, lymph node positivity, coexistence of obesity and vaginal bleeding. It seems that local invasion and metastatic spread of tumor should be preceded by the loss of E-cadherin expression in tumor cells, which progressively occurs in carcinogenesis. Therefore, E-cadherin negativity can be used as a poor prognostic factor and more aggressive chemotherapy regimen should be used.

Argyrophilic nucleolar organizer regions in benign and malignant mesothelial lesions.

The aim our study was to assess the usefulness of AgNOR stain in distinguishing between benign and malignant mesothelial lesions. The patients were divided into three groups: group I -- reactive mesothelium (71 cases), group II -- hyperplastic mesothelium (66 cases), group III -- epithelial type mesothelioma (52 cases). Smears were stained by Pap and AgNOR methods. After staining, all cases were randomized for blind evaluation. Each case was viewed independently by two observers. AgNORs were identified as black, usually spheric particles observed within the nucleolus. For each cell, the number of AgNOR-positive cells and the number of AgNOR-dots per nucleus were counted. Our results show that AgNOR staining is useful to differentiate epithelial type mesothelioma and benign mesothelial lesions such as reactive and hyperplastic mesothelium. This differentiation is based primarily on the mean number of AgNOR-dots per cell rather than number of AgNOR-positive cells. AgNOR is highly sensitive, specific and cost-effective technology which can be used as an ancillary diagnostic approach for distinguishing between reactive and/or hyperplastic changes of mesothelium as well as in differential diagnosis of epithelial type mesothelioma.

[The immunohistochemical characteristics of cervical glandular intraepithelial neoplasias].

The aim of our study was to identify immunohistochemical diagnostic criteria for cervical glandular intraepithelial neoplasias (CGIN). We examined 136 women with cytological diagnosis of atypical endocervical cells. These patients were divided into three groups based on a grade of the lesion: 35 patients with CGIN1 (group I), 72 patients with CGIN2 (group II), 28 patients with CGIN3 (group III). Endocervical curettages were examined by hematoxylin-eosin and immunohistochemistry using monoclonal antibodies against Ki-67, EpAg, MNF116, CEA, EMA. We used histological algorithm created by us. The comparative analysis of immunohistochemical results showed that expression of Ki-67 is seen in CGIN 2 and significantly increased in CGIN3 (p<0,05) which indicates increased proliferative activity of glandular cells in relation to increased grade of lesion. The differences in the expression of MNF116 and EMA are not statistically significant (p>0,05) which indicates that the expression of these epithelium specific markers does not change according to the grade of atypia and carcinogenesis (they can be used for determination of tumor phenotype). The expression of CEA and EpAg is strongly increased in CGIN2 and CGIN3 (p<0,05) indicating their potential role in carcinogenesis. The results suggest that evaluation of a grade of cervical glandular intraepithelial neoplasia should be based on histological and immunohistochemical studies. The morphometric algorithm should include the following criteria: type of lining epithelium (cubical, columnar), nuclear cytoplasmic index (<1, >1, =1), stratification, hyper- and hypochromasia, size and amount of nucleoli, and stromal-parenchymal ratio. The immunohistochemical study should include the expression of proliferation marker (Ki-67), carcinoembryonic antigen (CEA)and Epithelial Antigen (EpAg). We recommend the classification of CGIN into two types: low grade cervical glandular intraepithelial neoplasia (CGIN 1) and high grade cervical glandular intraepithelial neoplasia including CGIN 2 and CGIN 3.

Clinical prognostic factors and expression of cathepsin D in endometrioid adenocarcinoma.

The aim of our study was to evaluate the association between the expression of cathepsin D and clinical prognostic data in endometrioid adenocarcinoma of different histological grade. We studied 104 postmenopausal women with diagnosis of endometrioid adenocarcinoma. We evaluated the presence of obesity and vaginal bleeding. Surgical specimens were fixed in 10% neutral buffered formalin solution and embedded in paraffin. 4 mm sections were stained by hematoxylin and eosin, von Gieson, and histological type of cancer, metastatic lesion of lymph nodes and depth of myometrial invasion were evaluated. Histological grade of cancer was assessed by FIGO grading system. All samples were analysed by immunohistochemistry for cathepsin D (Dakocytomation). We assessed the number of cathepsin D-positive stromal and tumor cells and degree of positivity (low, moderate, high). Histological study by hematoxylin and eosin showed grade 1 endometrioid carcinoma in 35 cases (33,7%, group 1), grade 2 in 44 cases (42,3%, group 2), grade 3 in 25 cases (24%, grade 3). Our results suggest that the expression of cathepsin D is associated with the higher histological grade of endometrioid adenocarcinoma, depth of myometrial invasion, lymph node positivity, coexistence of obesity and vaginal bleeding. It seems that local invasion and metastatic spread of tumor should be preceeded by the expression of cathepsin D in stromal cells which can be assessed in grade 1 and 2 endometrioid adenocarcinomas. The expression of cathepsin D can be used as a prognostic factor and more aggressive chemothepery regimen should be used.

Mammary gland development and cancer.

The mammary gland is a complex organ that begins development early in gestation and constantly changes in size, shape and function from the time of puberty to menopause. The earliest stages of embryogenesis appear to be independent of steroid hormones, whereas after the 15th week breast structure is largely influenced by a variety of hormones. In most females, further breast development begins at puberty under the influence of cyclical estrogen and progesterone secretion. This process may continue into the 20s and it is enhanced by pregnancy. Growth and transcription factors contribute to the reciprocal stromal-epithelial interactions in growth, development and tumorogenesis of the mammary gland. From the embryological point of view the morphology of both mammary ductal and lobular cells results from the same developmental process. Numerous data suggest the existence of self-renewing, pluripotent mammary stem cells but their molecular characteristics and differentiation pathways are unknown. The extensive research currently being done in molecular biology and pathology, cancer genomics and proteomics will hopefully contribute to further elucidation of all the genetic and environmental factors involved in the development, differentiation, and involution of the mammary gland and this may give insight into the etiopathogenesis, early detection, treatment, and potential prevention of breast cancer.