Deciphering a subgroup of breast carcinomas with putative progression of grade during carcinogenesis revealed by comparative genomic hybridisation (CGH) and immunohistochemistry

Distinct parallel cytogenetic pathways in breast carcinogenesis could be identified in recent years. Nevertheless, it remained unclear as to which tumours may have progressed in grade or which patterns of cytogenetic alteration may define the switch from an in situ towards an invasive lesion. In order to gain more detailed insights into cytogenetic mechanisms of the pathogenesis of breast cancer, the chromosomal imbalances of 206 invasive breast cancer cases were characterised by means of comparative genomic hybridisation (CGH). CGH data were subjected to hierarchical cluster analysis and the results were further compared with immunohistochemical findings on tissue arrays from the same breast cancer cases. The combined analysis of immunohistochemical and cytogenetic data provided evidence that carcinomas with gains of 7p, and to a lesser extent losses of 9q and gains of 5p, are a distinct subgroup within the spectrum of ductal invasive grade 3 breast carcinomas. These aberrations were associated with a high degree of cytogenetic instability (16.6 alterations per case on average), 16q-losses in over 70% of these cases, strong oestrogen receptor expression and absence of strong expression of p53, c-erbB2 and Ck 5. These characteristics provide strong support for the hypothesis that these tumours may develop through stages of well- and perhaps intermediately differentiated breast cancers. Our results therefore underline the existence of several parallel and also stepwise progression pathways towards breast cancer

Serum HER2 Level Measured by Dot Blot: A Valid and Inexpensive Assay for Monitoring Breast Cancer Progression

Human epidermal growth factor receptor 2 (HER2) is one of the most important prognostic and predictive factors for breast cancer patients. Recently, serum HER2 ECD level of patients detected by enzyme-linked immunoabsorbent assay (ELISA) has been shown to predict tumor HER2 status and reveal its association with tumor progression, recurrence and poor prognosis. In this study, we established a new method, dot blot assay, to measure the serum HER2 level in breast cancer patients and further to evaluate the clinical value for monitoring breast cancer progression. We found that the serum HER2 level measured by dot blot assay was significantly correlated with tissue HER2 status in breast cancer patients (P = 0.001), and also significantly correlated with HER2 level measured by ELISA (P = 1.06×10−11). Compared with ELISA method, the specificity and sensitivity of dot blot assay were 95.3% and 65.0%, respectively. The serum HER2 levels of patients with grade III or ER-negative were higher than those with grade I–II (P = 0.004) and ER-positive (P = 0.033), respectively. Therefore, the novel dot blot method to detect serum HER2 level is a valid and inexpensive assay with potential application in monitoring breast cancer progression in clinical situations

Androgen receptor expression in metastatic adenocarcinoma in females favors a breast primary

BACKGROUND: The differential diagnosis of metastatic mammary adenocarcinoma and adenocarcinomas from other primary sites can be challenging, particularly in tumors that are poorly differentiated and negative for Estrogen/Progesterone receptors (ER/PR). With progression of disease, Androgen receptors (AR) are preserved with higher frequency than ER/PR in metastatic mammary carcinoma. This study was undertaken to evaluate the diagnostic significance of AR expression in adenocarcinoma of breast and other morphologically similar adenocarcinomas. DESIGN: Formalin-fixed paraffin-embedded tissue sections of 113 primary adenocarcinoma of various sites [breast (34, all females), lung (23, M- 6, F-17), colon (9, M-2, F-7), stomach (6, M-4, F-2), liver and bile duct (11, M-5, F-6), pancreas (7, M-2, F-5), ovary (10), endometrium (7), and cervix (6)] were immunostained with monoclonal antibody for AR. Except for well differentiated lobular carcinoma of breast (5) and bronchoalveolar carcinoma of lung (10), majority of the tumors were moderately to poorly differentiated. Tumors immunoreactive for ≥ 10% of nuclei were considered AR positive. However, AR immunoreactivity in the cytoplasm only was also recorded. RESULTS: 56% (19/34) mammary carcinoma and 20% (2/10) adenocarcinoma of ovary were positive for AR. Remaining 69 adenocarcinomas did not show nuclear immunoreactivity for AR in ≥ 10% nuclei; however, 52% (36/69) showed variable cytoplasmic immunoreactivity. CONCLUSION: Significant proportion of mammary carcinomas and some ovarian carcinomas express AR in the nuclei of more than 10% tumor cells. If metastatic tumor with unknown primary in a female is AR positive, breast and ovary are the most likely primary sites. Cytoplasmic immunoreactivity alone without nuclear immunoreactivity for AR was non-specific for this differential diagnosis

DAX-1 expression in human breast cancer: comparison with estrogen receptors ER-α, ER-β and androgen receptor status

BACKGROUND: So far there have been no reports on the expression pattern of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) in human breast cells and its relationship to the estrogen receptors, ER-α and ER-β, and the androgen receptor (AR). METHODS: In this study we evaluated, by immunohistochemistry and Western blot analysis, the presence and distribution of DAX-1 in benign breast disease (BBD), in situ carcinoma (CIS), and ductal and lobular breast carcinomas. RESULTS: In BBD and breast carcinomas, DAX-1 was present in both the nuclei and the cytoplasm of epithelial cells, although in infiltrative carcinomas the percentage of nuclear immunoreaction was higher than in CIS. An important relation was observed between DAX-1 and AR expression and between this orphan receptor and nodal status. CONCLUSION: DAX-1 might modify the AR and ER-β intracellular location, and because a direct positive relation between the expression of these three receptors was found it could be assumed that the presence of DAX-1 in neoplastic cells might indicate a possible failure of endocrine therapies

CAG and GGC repeat polymorphisms in the androgen receptor gene and breast cancer susceptibility in BRCA1/2 carriers and non-carriers

Variation in the penetrance estimates for BRCA1 and BRCA2 mutations carriers suggests that other genetic polymorphisms may modify the cancer risk in carriers. A previous study has suggested that BRCA1 carriers with longer lengths of the CAG repeat in the androgen receptor (AR) gene are at increased risk of breast cancer (BC). We genotyped 188 BRCA1/2 carriers (122 affected and 66 unaffected with breast cancer), 158 of them of Ashkenazi origin, 166 BC cases without BRCA1/2 mutations and 156 Ashkenazi control individuals aged over 56 for the AR CAG and GGC repeats. In carriers, risk analyses were conducted using a variant of the log-rank test, assuming two sets of risk estimates in carriers: penetrance estimates based on the Breast Cancer Linkage Consortium (BCLC) studies of multiple case families, and lower estimates as suggested by population-based studies. We found no association of the CAG and GGC repeats with BC risk in either BRCA1/2 carriers or in the general population. Assuming BRCA1/2 penetrance estimates appropriate to the Ashkenazi population, the estimated RR per repeat adjusted for ethnic group (Ashkenazi and non-Ashkenazi) was 1.05 (95%CI 0.97–1.17) for BC and 1.00 (95%CI 0.83–1.20) for ovarian cancer (OC) for CAG repeats and 0.96 (95%CI 0.80–1.15) and 0.90 (95%CI 0.60–1.22) respectively for GGC repeats. The corresponding RR estimates for the unselected case–control series were 1.00 (95%CI 0.91–1.10) for the CAG and 1.05 (95%CI 0.90–1.22) for the GGC repeats. The estimated relative risk of BC in carriers associated with ≥28 CAG repeats was 1.08 (95%CI 0.45–2.61). Furthermore, no significant association was found if attention was restricted to the Ashkenazi carriers, or only to BRCA1 or BRCA2 carriers. We conclude that, in contrast to previous observations, if there is any effect of the AR repeat length on BRCA1 penetrance, it is likely to be weak. © 2001 Cancer Research Campaign http://www.bjcancer.co

Downregulation of the anaphase-promoting complex (APC)7 in invasive ductal carcinomas of the breast and its clinicopathologic relationships

INTRODUCTION: The anaphase-promoting complex (APC) is a multiprotein complex with E3 ubiquitin ligase activity, which is required for the ubiquitination of securin and cyclin-B. Moreover, the mitotic spindle checkpoint is activated if APC activation is prevented. In addition, several APC-targeting molecules such as securin, polo-like kinase, aurora kinase, and SnoN have been reported to be oncogenes. Therefore, dysregulation of APC may be associated with tumorigenesis. However, the clinical significance and the involvement of APC in tumorigenesis have not been investigated. METHODS: The expression of APC7 was immunohistochemically investigated in 108 invasive ductal carcinomas of the breast and its relationship with clinicopathologic parameters was examined. The expression of APC7 was defined as positive when the summed scores of staining intensities (0 to 3+) and stained proportions (0 to 3+) exceeded 3+. RESULTS: Positive APC7 expression was less frequent than its negative expression when histologic (P = 0.009) or nuclear grade (P = 0.009), or mitotic number (P = 0.0016) was elevated. The frequency of APC7 negative expression was higher in high Ki-67 or aneuploid groups than in low Ki-67 or diploid groups. CONCLUSION: These data show that loss of APC7 expression is more common in breast carcinoma cases with poor prognostic parameters or malignant characteristics. They therefore suggest that dysregulation of APC activity, possibly through downregulation of APC7, may be associated with tumorigenesis in breast cancer

Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications

<p>Abstract</p> <p>Background</p> <p>A large portion of tissues stored worldwide for diagnostic purposes is formalin-fixed and paraffin-embedded (FFPE). These FFPE-archived tissues are an extremely valuable source for retrospective (genetic) studies. These include mutation screening in cancer-critical genes as well as pathogen detection. In this study we evaluated the impact of several widely used DNA extraction methods on the quality of molecular diagnostics on FFPE tissues.</p> <p>Findings</p> <p>We compared 4 DNA extraction methods from 4 identically processed FFPE mammary-, prostate-, colon- and lung tissues with regard to PCR inhibition, real time SNP detection and amplifiable fragment size. The extraction methods, with and without proteinase K pre-treatment, tested were: 1) heat-treatment, 2) QIAamp DNA-blood-mini-kit, 3) EasyMAG NucliSens and 4) Gentra Capture-Column-kit.</p> <p>Amplifiable DNA fragment size was assessed by multiplexed 200-400-600 bp PCR and appeared highly influenced by the extraction method used. Proteinase K pre-treatment was a prerequisite for proper purification of DNA from FFPE. Extractions with QIAamp, EasyMAG and heat-treatment were found suitable for amplification of fragments up to 400 bp from all tissues, 600 bp amplification was marginally successful (best was QIAamp). QIAamp and EasyMAG extracts were found suitable for downstream real time SNP detection. Gentra extraction was unsuitable. Hands-on time was lowest for heat-treatment, followed by EasyMAG.</p> <p>Conclusions</p> <p>We conclude that the extraction method plays an important role with regard to performance in downstream molecular applications.</p

Clinicopathologic study associated with long-term survival in Japanese patients with node-negative breast cancer

This study was undertaken to determine the absolute and relative value of blood vessel invasion (BVI) using both factor VIII-related antigen and elastica van Gieson staining, proliferating cell nuclear antigen (PCNA), p53, c- erb B-2, and conventional prognostic factors in predicting relapse-free survival (RFS) and overall survival (OS) rates associated with long-term survival in Japanese patients with node-negative breast cancer. Two hundred patients with histological node-negative breast cancer were studied. We investigated nine clinicopathological factors, including PCNA, p53, c- erb B-2 using permanent-section immunohistochemistry, clinical tumour size (T), histological grade (HG), mitotic index (MI), tumour necrosis (TN), lymphatic vessel invasion (LVI) and BVI, followed for a median of 10 years (range 1–20). Twenty-one patients (10.5%) had recurrence and 15 patients (7.5%) died of breast cancer. Univariate analysis showed that BVI, PCNA, T, HG, MI, p53, c- erb B-2 and LVI were significantly predictive of 20-year RFS or OS. Multivariate analysis showed that BVI (P = 0.0159, P = 0.0368), proliferating cell nuclear antigen (PCNA) (P = 0.0165, P = 0.0001), and T (P = 0.0190, P = 0.0399) were significantly independent prognostic factors for RFS or OS respectively. BVI, PCNA and T were independent prognostic indicators for RFS or OS in Japanese patients with node-negative breast cancer and are useful in selecting high-risk patients who may be eligible to receive strong adjuvant therapies. © 2000 Cancer Research Campaig

Usefulness of real time PCR for the differentiation and quantification of 652 and JP2 Actinobacillus actinomycetemcomitans genotypes in dental plaque and saliva

BACKGROUND: The aim of our study is to describe a fast molecular method, able to distinguish and quantize the two different genotypes (652 and JP2) of an important periodontal pathogen: Actinobacillus actinomycetemcomitans. The two genotypes show differences in the expression of an important pathogenic factor: the leukotoxin (ltx). In order to evidence this, we performed a real time PCR procedure on the ltx operon, able to recognize Aa clinical isolates with different leukotoxic potentials. METHODS: The specificity of the method was confirmed in subgingival plaque and saliva specimens collected from eighty-one Italian (Sardinian) subjects with a mean age of 43.9, fifty five (68 %) of whom had various clinical forms of periodontal disease. RESULTS: This procedure showed a good sensitivity and a high linear dynamic range of quantization (10(7)-10(2 )cells/ml) for all genotypes and a good correlation factor (R2 = 0.97–0.98). Compared with traditional cultural methods, this real time PCR procedure is more sensitive; in fact in two subgingival plaque and two positive saliva specimens Aa was only detected with the molecular method. CONCLUSION: A low number of Sardinian patients was found positive for Aa infections in the oral cavity, (just 10 positive periodontal cases out of 81 and two of these were also saliva positive). The highly leukotoxic JP2 strain was the most representative (60 % of the positive specimens); the samples from periodontal pockets and from saliva showed some ltx genotype for the same patient. Our experience suggests that this approach is suitable for a rapid and complete laboratory diagnosis for Aa infection