[Lobular neoplasms and invasive lobular breast cancer]. / Lobuläre Neoplasie und invasives lobuläres Mammakarzinom.

The term lobular neoplasia (LN) comprises both atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS) and thus a spectrum of morphologically heterogeneous but clinically and biologically related lesions. LN is regarded as a nonobligatory precursor lesion of invasive breast cancer and at the same time as an indicator lesion for ipsilateral and contralateral breast cancer risk of the patient. Rare pleomorphic or florid variants of LCIS must be differentiated from classical LCIS. The classical type of invasive lobular carcinoma (ILC) can be distinguished from the non-special type of invasive breast cancer (NST) by E-cadherin inactivation, loss of E-cadherin related cell adhesion and the subsequent discohesive growth pattern. Variant forms of ILC may show different molecular features, and solid and pleomorphic differentiation patterns in cases of high grade variants. Important parameters for the prognostic assessment of ILC are tumor grading and the recognition of morphological variants.

[TNM classification of breast cancer: changes and comments on the 7th edition]. / TNM-Klassifikation beim Mammakarzinom : Neuerungen und Anmerkungen zur 7. Auflage.

The 7th edition of the TNM classification includes only minor changes in the main TNM categories for breast cancer. Only ductal and lobular carcinoma in situ (DCIS, LCIS), and isolated Paget's disease of the nipple are classified as pTis, but not precursor lesions such as atypical ductal or lobular hyperplasia (ADH, ALH). AJCC emphasizes that microscopic measurement is the most accurate and preferred method to determine pT in small invasive cancers and stresses the importance of strict adherence to criteria for T4 cancers. For better distinction from micrometastases in regional lymph nodes, small clusters of cells not greater than 0.2 mm, or nonconfluent or nearly confluent clusters of cells not exceeding 200 cells in a single histologic lymph node cross section are classified as isolated tumour cells (pN0(i+)). The pN classification has otherwise remained unchanged. In the setting of patients having received neoadjuvant therapy, ypT1-ypT3 is based on the total extent of viable tumour cells, irrespective of tumour regression. Stage I breast tumours have been subdivided into Stage IA and Stage IB; Stage IB includes small tumours (TI) with lymph node micrometastases (N1mi). These changes and clarifications will contribute to maintaining the clinical and prognostic relevance of TNM in breast cancer.

[Concepts and problems of lobular neoplasia]. / Konzept und Problematik der lobulären Neoplasie.

The term lobular neoplasia (LN) includes lobular carcinoma in situ (LCIS) and atypical lobular neoplasia (ALH). It is generally considered to be a risk lesion and a non-obligatory precursor for the subsequent development of an invasive carcinoma in the ipsilateral or contralateral breast. LN has also been termed lobular intraepithelial neoplasia (LIN). A grading system (LIN 1-LIN 3) has been suggested as a tool for a more precise estimation of the individual risk. When LN is the most significant finding in a core biopsy, the probability of a higher grade lesion is about 17% in the follow-up surgical biopsy, justifying follow-up surgery in the majority of cases. A higher risk of progression is attributed to LIN 3 (pleomorphic LN, extensive LN, and signet ring cell LN) compared to LIN 1 or LIN 2. These special forms of LN may have an unusual presentation clinically or histologically. Using immunohistology, LN are characterized by the loss of E-cadherin, low proliferative activity and by positive hormone receptor status. The molecular characteristics of LN are similar to those of invasive lobular carcinomas, indicating the nature of LN as a precursor lesion.

c-myc amplifications in primary breast carcinomas and their local recurrences.

OBJECTIVE: To evaluate the role of c-myc oncogene amplifications in the progression of invasive breast carcinomas. METHODS: c-myc gene copy number was evaluated in a series of 49 primary breast carcinomas and the corresponding local recurrences using fluorescence in situ hybridisation. RESULTS: 11 of the primary carcinomas (22%) harboured c-myc amplifications; these tumours typically were hormone receptor negative and occurred in younger patients (43 v 53 years). At the time of relapse, six additional tumours had acquired a c-myc amplification. The mean recurrence-free survival was 24 months; c-myc amplified tumours relapsed significantly earlier than carcinomas without amplification (18 v 27 months). Univariate analysis showed a worse overall survival in these patients. CONCLUSIONS: While c-myc amplifications can be observed in early stage breast cancer, especially in younger patients, they often occur later in tumour development and appear to be associated with disease progression.

Strong expression of Bcl-2 in pigmented villonodular synovitis of the knee with aggressive clinical behaviour.

OBJECTIVE: To determine the expression of bcl-2, p53, and caspase 3, and measure the Ki-67 proliferation index as well as DNA content and DNA fragmentation in a case of diffuse pigmented villonodular synovitis (PVNS) of the knee with aggressive clinical behaviour. METHODS: Expression of p53, Bcl-2 and Ki-67 was investigated using immunohistochemistry. In addition, multiparametric flow cytometry was performed for expression of p53, bcl-2, and caspase 3, as well as analysis of DNA content and distribution of cell cycle phases. DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL). RESULTS: A strong cytoplasmic positivity for Bcl-2 protein, a key factor in regulation of apoptosis, was found in the majority of proliferating synovial cells. No apoptotic cell fraction was found by analysis of DNA content. DNA fragmentation was observed in 6.8% of cells. No elevated expression of p53 and caspase 3 was detected. CONCLUSION: Our results indicate a possible role of dysregulation of apoptosis in this case of PVNS. This aspect in the pathogenesis of PVNS should be clarified in further studies.

Rheumatoid arthritis and pigmented villonodular synovitis: comparative analysis of cell polyploidy, cell cycle phases and expression of macrophage and fibroblast markers in proliferating synovial cells.

AIMS: Rheumatoid arthritis (RA) and pigmented villonodular synovitis (PVNS) are aggressive diseases with progressive joint destruction. The present study aims to define cell cycle phases, polyploidy and the immunophenotype of proliferating synovial cells in both diseases. METHODS AND RESULTS: Synovial tissues from patients with proliferative-active RA, localized and diffuse PVNS were analysed by DNA flow cytometry, immunohistochemistry and double immunofluorescence with confocal laser scan microscopy. Expression of macrophage markers (CD68/CD163), fibroblast markers (h4Ph/CD55) and Ki67 antigen was examined. Synovial cells positive for either macrophage or fibroblast markers as well as double-labelled cells were found in both RA and PVNS. In RA, CD68/CD163+ synoviocytes were preferentially located in the vicinity of the synovial lining layer, while they were more randomly distributed in PVNS. Of cases with diffuse PVNS, 20% showed an aneuploid cell pattern. All samples of localized PVNS and RA were diploid. Proliferative activity was significantly higher in aneuploid PVNS. CONCLUSIONS: In spite of their histologically homogeneous appearance, proliferating synovial cells display a heterogeneous immunophenotype in both RA and PVNS, indicating functional properties of both macrophages and fibroblasts. Aneuploidy seems to be a special feature of diffuse PVNS.

Expression pattern of cell cycle-related gene products in synovial stroma and synovial lining in active and quiescent stages of rheumatoid arthritis.

OBJECTIVE: To investigate the expression pattern of cell cycle related gene products in active and quiescent Rheumatoid arthritis (RA). METHODS: Synovial tissue from 20 patients with active proliferative RA and 28 patients with RA in remission was immunohistochemically examined for expression of p53, p63, p21, p27, p16, cyclin D1, CDK4, RB, E2F, Ki-67 on tissue microarrays and by DNA flow cytometry for cell cycle phases. RESULTS: Elevated expression of p53 and p27 was found in synovial lining and in stromal cells in proliferative active RA. In the remission stage this finding was confined to the synovial lining. Most of the cells were in the G0-phase. Ki-67 proliferation index was maximum 10% in synovial cells. CONCLUSION: The p53 pathway is activated in synovial cells in active RA as well as in quiescent stage of disease. Differences in the spatial expression pattern of proteins involved in the p53 pathway in RA in remission compared to actively proliferating RA reflect the phasic nature of the disease and support in our opinion the concept of adaptive role of p53 pathway in RA.

Apoptosis resistance in pigmented villonodular synovitis.

OBJECTIVE: Pigmented villonodular synovitis (PVNS) is a proliferative lesion originating from synovial tissue with a locally aggressive behaviour. We analysed the pathogenetic role of apoptosis resistance for sustained cell proliferation in PVNS. METHODS: The expression of bcl-2, p53 and Ki-67 was examined in 80 cases of PVNS using immunohistochemistry. In 43 of these cases, DNA content and distribution of cell-cycle phases were investigated by flow cytometry. Additionally, 10 cases of PVNS were analysed by multi-parametric flow cytometry for expression of p53, caspase3, and bcl-2 and by TUNEL to detect DNA fragmentation. RESULTS: No apoptotic cell fractions were detected in any investigated cases. Expression of bcl-2 was found in 84% of cases (up to 6.5% of cells) and was significantly associated with DNA-fragmentation observed by TUNEL (p=0.037). Orthologous p53 expression was observed in 37% of cases. The level of p53 expression correlated with the proliferative activity and the expression of both caspase3 (p=0.017) and bcl-2 (p=0.0013). (No statistically significant correlations between expression of bcl-2, p53, caspase3, DNA fragmentation or proliferative index and age, sex of patients, disease recurrence, growth pattern or size of lesion were found). CONCLUSION: Apoptosis resistance is a critical event in the progression of PVNS and may contribute to the survival of the proliferating synovial cells in PVNS and to the permanent slow progression of these lesions.

Mutational activation of the RAS-RAF-MAPK and the Wnt pathway in small intestinal adenocarcinomas.

BACKGROUND: Adenocarcinomas of the small and the large intestine share risk factors and morphological features but both tumor types seem to follow different genetic pathways. The aim of this study on small intestinal carcinomas was to analyze alternative mechanisms of activation of pathways that are typically affected in colorectal cancer. METHODS: Twenty-one sporadic carcinomas were investigated for mutations in KRAS, BRAF, the beta-catenin gene CTNNB1, and the mutational cluster region of APC. Immunohistochemical analysis was performed with a monoclonal antibody for beta-catenin, the transcriptionally active downstream component of wnt signaling. RESULTS: Oncogene mutations were found in 13 (62%) small intestinal adenocarcinomas. Twelve tumors displayed a KRAS mutation, and a novel BRAF mutation at codon 603/604 was seen in one carcinoma without KRAS mutation. One tumor harbored a CTNNB1 mutation consisting of an insertion of 247 nucleotides deriving from chromosome 9. APC mutations were identified in 2 tumors. Immunohistochemistry demonstrated nuclear accumulation of beta-catenin in 5 carcinomas. These carcinomas included the tumor with a CTNNB1 mutation but not those with APC mutations. CONCLUSIONS: Our data show frequent activation of the RAS-RAF-MAPK pathway through mutations of either KRAS or, infrequently, BRAF. Activation of the wnt pathway through accumulation of beta-catenin may have a role in a subset of small intestinal adenocarcinomas but in contrast to colorectal carcinoma, accumulation of beta-catenin is generally not caused by inactivating APC or activating CTNNB1 mutations.

Comparative analysis of cell populations involved in the proliferative and inflammatory processes in diffuse and localised pigmented villonodular synovitis.

The aim of the present study was a comparative quantitative evaluation of cell populations involved in the proliferative and inflammatory compartment in both localised and diffuse pigmented synovitis villonodularis (PVNS). 15 cases of each localised and diffuse PVNS were examined by flow cytometry, immunohistochemistry, double immuno-fluorescence and confocal microscopy with quantitative evaluation of CD3-, CD4-, CD8-, CD20-, CD57-, CD55-, CD68-, CD163- and h4Ph positive (+) cells. The proliferative compartment of localised and diffuse PVNS was mainly composed of double-positive CD68+/h4Ph+ (CD163+/CD55+) synoviocytes. The number of double-positive synoviocytes for macrophage and fibroblast markers was significantly higher in diffuse compared to localised PVNS. The accompanying inflammatory infiltrate showed a predominance of cytotoxic cells (CD8+, CD57+), whereby the number of CD3+ and CD20+ cells was significantly higher in localised PVNS. The number of CD57+ NK cells was significantly higher in diffuse PVNS. The proliferating macrophage- like synovial cells and the cytotoxic lymphocytes could contribute to the aggressive behaviour of localised and diffuse PVNS. Moreover, with regard to the quantitative differences in cell composition between diffuse and localised PVNS and their different clinical behaviour, further studies should continue to analyse localised and diffuse PVNS separately.

Simultaneous quantification of oestrogen and progesterone receptors by a ligand binding assay in frozen sections.

We describe two modifications of a double-isotope assay for measuring the concentrations of oestrogen receptors and progesterone receptors in tumour cytosols and extracts of frozen tumour sections and endometrial sections. The concentrations of these receptors are derived from single-point/isoelectric focussing assays after incubation of cytosols or section supernatants either with ([125I]vinyl)-nortestosterone and [3H]oestradiol or [125I]oestradiol and [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor[6,7(3)H]pregn-4-ene-3,20-dione). The concentrations of the oestrogen and progesterone receptors found in cytosols (r greater than 0.93) and extracts from sections (r greater than 0.8) by dual label assays are highly correlated with those found by single label assays. The method described represents an approach to the determination of oestrogen and progesterone receptors biochemically in an amount of tissue which is comparable to that needed for immunocytochemical procedures.