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.

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.

Die 7. Auflage der TNM-Klassifikation beinhaltet nur geringe Änderungen in den Hauptkategorien der TNM-Einteilung beim Mammakarzinom. Beim In-situ-Karzinom (pTis) wird neben dem duktalen und lobulären Carcinoma in situ auch der isolierte M. Paget der Mamille berücksichtigt, nicht jedoch die atypischen duktalen und lobulären Epithelhyperplasien. Beim invasiven Karzinom wird vom AJCC die Bedeutung der histologischen Größenbestimmung bei kleinen Tumoren hervorgehoben und die strikte Anwendung der Kriterien für T4 betont. Die Definition von Tumoreinzelzellen in regionären Lymphknoten – pN0(i+) – wurde zur besseren Abgrenzung gegenüber Mikrometastasen (pN1mi) präzisiert. Im Übrigen blieb die pN-Klassifikation unverändert. Bei Zustand nach neoadjuvanter Chemotherapie ist die Gesamtausdehnung des vitalen Tumors im Tumorbett maßgebend für ypT1–3, unbeschadet der eingetretenen Tumorregression. Im Stadium I wurde eine Unterteilung in die Stadien IA und IB vorgenommen, dabei beinhaltet das neue Stadium IB die pT1-Tumoren mit Lymphknotenmikrometastasen (pN1mi). Diese Änderungen und Klarstellungen werden dazu beitragen, dass die TNM-Klassifikation beim Mammakarzinom weiterhin ihre klinische und prognostische Relevanz behält.

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.

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.

OBJECTIVE:To characterize the gene expression profile and determine potential diagnostic markers and therapeutic targets in pigmented villonodular synovitis (PVNS).METHODS:Gene expression patterns in 11 patients with PVNS, 18 patients with rheumatoid arthritis (RA), and 19 patients with osteoarthritis (OA) were investigated using genome-wide complementary DNA microarrays. Validation of differentially expressed genes was performed by real-time quantitative polymerase chain reaction and immunohistochemical analysis on tissue arrays (80 patients with PVNS, 51 patients with RA, and 20 patients with OA).RESULTS:The gene expression profile in PVNS was clearly distinct from those in RA and OA. One hundred forty-one up-regulated genes and 47 down-regulated genes were found in PVNS compared with RA, and 153 up-regulated genes and 89 down-regulated genes were found in PVNS compared with OA (fold change > or = 1.5; Q < or = 0.001). Genes differentially expressed in PVNS were involved in apoptosis regulation, matrix degradation, and inflammation (ALOX5AP, ATP6V1B2, CD53, CHI3L1, CTSL, CXCR4, HSPA8, HSPCA, LAPTM5, MMP9, MOAP1, and SPP1).CONCLUSION:The gene expression signature in PVNS is similar to that of activated macrophages and is consistent with the local destructive course of the disease. The gene and protein expression patterns suggest that the ongoing proliferation in PVNS is sustained by apoptosis resistance. This result suggests the possibility of a potential novel therapeutic intervention against PVNS.

Die lobuläre Neoplasie (LN), zu welcher das lobuläre Carcinoma in situ (LCIS) und die atypische lobuläre Neoplasie (ALH) gezählt werden, gilt im Allgemeinen als Risikoläsion und als nichtobligater Vorläufer für die Entwicklung eines nachfolgenden invasiven Karzinoms in der ipsilateralen oder kontralateralen Mamma. Der Begriff der LN ist gleichbedeutend mit dem der lobulären intraepithelialen Neoplasie (LIN). Eine morphologische Graduierung (LIN 1 bis LIN 3) wurde zur besseren Abschätzung des individuellen Risikos vorgeschlagen. Bei stanzbioptisch gesicherter LN findet sich in etwa 17% der Fälle in der nachfolgenden offenen Biopsie eine gleichzeitig bestehende höhergradige Läsion, dies rechtfertigt in den meisten Fällen eine operative Abklärung. Den LIN 3 (pleomorphe LN, extensive LN, siegelringzellige LN) wird ein höheres Progressionsrisiko als den LIN 1 oder LIN 2 zugeschrieben. Diese Sonderformen können sich klinisch oder histologisch ungewöhnlich präsentieren. Immunhistologisch sind die LN charakterisiert durch einen E-Cadherin-Verlust, eine niedrige Proliferationsaktivität und einen positiven Hormonrezeptorstatus. Die molekularen Eigenschaften der LN ähneln denen des invasiven lobulären Karzinoms, dies unterstreicht für die LN den Charakter einer Vorläuferläsion.

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.

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.

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.

Ziele: Während Risikofaktoren für das Auftreten von Lokalrezidiven invasiver Mammakarzinome in zahlreichen Studien untersucht wurden, ist wenig bekannt über die biologischen Veränderungen während der Tumorprogression. Das Ziel dieser Studie war es, diese Veränderungen an einem klinisch-pathologisch gut charakterisierten Kollektiv zu untersuchen.

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.

To compare the analytical and clinical performance characteristics of 3 different primer sets targeting the human papillomavirus (HPV) L1 gene for detection and genotyping of HPV.Liquid-based cytology was obtained prospectively from 90 colposcopy clinic patients. After automated extraction, cellular DNA was subjected to SYBR Green quantitative polymerase chain reaction (qPCR) using GP5+/6+ primers and conventional PCR with MY09/11 and FAP59/64 primers. The resulting SYBR Green counts and melting temperatures (Tm) were used for quantification of HPV genomes and discrimination between presence and absence of amplicons. qPCR and PCR products were resolved by gel electrophoresis. HPV + amplicons were sequenced directly and BLAST® aligned for genotype identification.Of the 90 samples, 57 (63%) were qPCR + with a range of HPV viral load detected from 191 to 3.4 million genomes (~ 5 Log10). Only HPV-positives exhibited characteristic Tm (75–80 °C). The dynamic range of detection was similar for MY and FAP. Clinical net sensitivity was highest with simultaneous testing using all primer sets (93%) instead of individual primer pairs (GP (67%), MY (62%), FAP (49%)). Of the 27 HPV genotypes identified among 64 sequenced samples, the 3 most prevalent were HPV-16 (20%), − 53 (9%), − 31 (6%). The 4th rank included HPV-6, − 33, − 58, − 66 (4.7% each).The performance characteristics of 3 leading PCR-based HPV assays revealed qPCR to be sensitive and specific for HPV detection and quantification. Parallel PCR testing using the 3 primers and direct sequencing offered the greatest clinical sensitivity and breadth of detection for known HPV types.

Despite morphological similarities between adenocarcinomas of the small and the large intestine, recent evidence suggests that both tumor types follow different genetic pathways. In particular, inactivation of the APC tumor suppressor gene, a characteristic alteration of colorectal carcinomas, does not seem to play a significant role in sporadic small intestinal tumorigenesis. We could recently show that inactivating mutations of the SMAD4 gene frequently occur in small intestinal adenocarcinomas. To further elucidate the role of SMAD4 dysfunction for tumor development in the small intestine, we immunohistochemically analyzed 20 sporadic, non-ampullary carcinomas for the expression of the SMAD4 protein. We further determined homozygous SMAD4 gene deletions by real time PCR and compared SMAD4 immunohistochemical data with SMAD4 genetic data. Immunohistochemistry was negative for the tumor cells in two (10%) cases and strongly reduced in four (20%). Negative immunohistochemical staining corresponded with homozygous gene deletions. A regular or only slightly reduced staining pattern was noted in 14 carcinomas, including four tumors with previously identified SMAD4 missense and frame shift mutations. In conclusion, our data suggest a significant role of impaired SMAD4 function in the pathogenesis of small intestinal adenocarcinomas. Furthermore, our results show that SMAD4 immunohistochemistry may serve as a surrogate for analysis of homozygous gene deletions. However, the method fails to identify SMAD4 inactivation due to missense mutations.

Polyglutamine expansion in huntingtin (Htt) and the androgen receptor (AR) causes untreatable neurodegenerative diseases. Y-27632, a therapeutic lead, reduces Htt and AR aggregation in cultured cells, and Htt-induced neurodegeneration in Drosophila. Y-27632 inhibits both Rho-associated kinases ROCK and PRK-2, making its precise intracellular target uncertain. Over-expression of either kinase increases Htt and AR aggregation. Three ROCK inhibitors (Y-27632, HA-1077, and H-1152P), and a specific ROCK inhibitory peptide reduce polyglutamine protein aggregation, as does knockdown of ROCK or PRK-2 by RNAi. RNAi also indicates that each kinase is required for the inhibitory effects of Y-27632 to manifest fully. These two actin regulatory kinases are thus involved in polyglutamine aggregation, and their simultaneous inhibition may be an important therapeutic goal.

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 immunofluorescence 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.

Background: Adenocarcinomas of the small and the large intestine share risk factors and morphological features but both turner 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 CTNNBI, 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 CTNNBI 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 CTNNBI 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 CTNNBI mutations.

Expression patterns of cell cycle regulating gene products and Ki-67 in proliferating synovial cells of primary and recurrent pigmented villonodular synovitis (PVNS) in localized and diffuse lesions were examined by immunohistochemistry. Alterations of cell cycle-related proteins were seen in 98.7% of analyzed lesions. Both RB- and p53 pathways play a role in cell cycle dysregulation in PVNS. The RB pathway was more frequently altered in primary disease, while alterations of the p53 pathway seemed to be more important in recurrent lesions, regardless of the histomorphological type of disease. Ki-67 proliferation rate was elevated in recurrent tumors.