F. Länger

Epigenetic inactivation of microRNA gene hsa-mir-9-1 in human breast cancer†

MicroRNAs (miRNAs) represent a new class of small non‐coding RNAs regulating gene expression by inducing RNA degradation or interfering with translation. Aberrant miRNA expression has been described for several human malignancies and tumour suppressor functions have been ascribed to this new class of small regulatory RNAs. Accordingly, inactivation due to deletion or mutation has been found in human malignancies. Here, we describe the role of aberrant hypermethylation as an additional mechanism for miRNA gene inactivation in human breast cancer. Aberrant hypermethylation was shown for mir‐9‐1, mir‐124a3, mir‐148, mir‐152, and mir‐663 in 34–86% of cases in a series of 71 primary human breast cancer specimens. For comprehensive methylation analysis, combined bisulphite restriction analysis, bisulphite sequencing, and Pyrosequencing™ were employed. miRNA gene hypermethylation correlated strongly with methylation of known tumour suppressor genes (p = 0.003). After treatment of various breast cancer cell lines with the demethylating agent 5‐aza‐2′‐deoxycytidine, reduction of mir‐9‐1 gene methylation and concomitant reactivation of expression could be observed. For the mir‐9‐1 gene, which is already hypermethylated in pre‐invasive intraductal lesions, a good correlation between quantitative methylation level and reduction of expression could be demonstrated in a subset of primary human breast cancer specimen (r = 0.8). In conclusion, this study demonstrates that various microRNA genes are also affected by epigenetic inactivation due to aberrant hypermethylation and that this is an early and frequent event in breast cancer development. Copyright

Quantitative assessment of promoter hypermethylation during breast cancer development.

The aberrant methylation of cytosine residues in the promoter region of growth regulatory genes is now widely recognized as an additional mechanism for gene inactivation in cancer cells. In this study we analyzed the methylation status of four growth regulatory genes (p16, RASSF1A, cyclinD2, 14-3-3zeta) during breast cancer progression. For this purpose invasive and noninvasive tumor cell populations as well as hyperplastic cell proliferations were isolated from a series of archival breast tissue specimens (n = 57) using laser-assisted microdissection. A new real-time polymerase chain reaction-based assay was used for the sensitive and quantitative determination of the cell-specific methylation status. We found that aberrant promoter methylation was already prevalent in pure intraductal carcinoma with different frequencies and different methylation levels for the four genes analyzed. For RASSF1A and 14-3-3zeta promoter methylation was also demonstrated in epithelial hyperplasia and intraductal papillomas. By contrast, aberrant methylation of cyclinD2 and p16 was restricted to cancerous epithelium. Increased methylation of the cyclinD2 gene was significantly associated with a higher van Nuys grade. Furthermore, when intraductal and invasive tumor cells were compared, significant quantitative changes in the methylation level were detected primarily within the cyclinD2 gene. These results demonstrate that promoter methylation is an early and frequent event in breast cancer development, but displays great quantitative and gene-specific differences, and changes in a gene-specific manner during tumor progression.

Identification of differentially expressed microRNAs in human male breast cancer

BackgroundThe discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases.MethodsThe expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer.ResultsUnsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer.ConclusionsMale breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.

Epigenetic inactivation of tumour suppressor gene KLF11 in myelodysplastic syndromes.

The identification of aberrantly hypermethylated genes may lead to the development of new diagnostic markers and the identification of novel targets of epigenetic therapy in myelodysplastic syndromes (MDS). We therefore investigated the methylation status of transcription factor genes KLF5, KLF11, and MAFB, shown to be aberrantly methylated in myelogeneous leukaemia cells, in a series of 115 MDS patient as well as in 25 control subjects. Using quantitative high‐resolution pyrosequencing methodology, KLF11, MAFB, and KLF5 were shown for the first time to be hypermethylated in 17 (15%), 8 (7%), and 2 (1.7%) cases, respectively, but not in any of the patients with an isolated 5q‐deletion. Patient samples harbouring KLF11 methylation displayed reduced KLF11 mRNA expression and KLF11 hypermethylation correlated with a high International Prognostic Scoring System score (P < 0.05). In conclusion, epigenetic inactivation and subsequent transcriptional repression of the KLF11 gene is quite frequent in MDS. Patients with an isolated 5q‐deletion seem to harbour a distinct epigenetic profile.

Demonstration of light chain restricted clonal B-lymphoid infiltrates in archival bone marrow trephines by quantitative real-time polymerase chain reaction

Assessment of clonality either by demonstrating light chain restriction or showing monoclonal immunoglobulin gene rearrangement is a valuable and indispensable adjunct to diagnosis in hematopathology. The study of light chain restriction by immunohistochemistry on archival material is hampered by a very low sensitivity especially regarding low grade lymphomas of B cell origin. DNA rearrangement studies of the immunoglobulin locus do improve sensitivity markedly but for lymphomas of follicle center origin they are prone to false negative results due to hypermutations. Therefore we developed a new clonality assay based on the quantification of immunoglobulin light chain transcripts using real-time polymerase chain reaction technology, which is also suitable for the analysis of archival bone marrow trephines. We tested the reproducibility and sensitivity of this approach by comparatively analyzing a series of bone marrow trephines with multiple myeloma (n = 26), reactive lymphoid hyperplasia (n = 37), and focal infiltration by low grade B cell lymphoma (n = 29). We could raise the detection rate of clonality from an average of 17% by immunohistochemistry and 66% as assessed by polymerase chain reaction rearrangement studies to 83% by this new technique. Despite false negative results due to light chain hypermutation in some cases, the detection rate of clonality could be improved even for B cell lymphomas of follicle center origin (follicular lymphoma or marginal zone lymphoma) thus making this novel approach a valuable additional tool for the hematopathologist.

Detection of Gene Amplification in Intraductal and Infiltrating Breast Cancer by Laser-Assisted Microdissection and Quantitative Real-Time PCR

Gene amplification is one essential mechanism leading to oncogene activation which is supposed to play a major role in the pathogenesis of invasive breast cancer. However, using standard methodologies the detection of gene amplifications has been limited especially in small-sized lesions, like pre-invasive precursor lesions. The combination of two novel technologies, laser-based microdissection and quantitative real-time PCR, facilitates the detection of low-level amplifications in morphologically defined lesions. As a model system we investigated in situ breast cancer (ductal carcinoma in situ, DCIS) classified according to the morphology-based Van Nuys grading system for amplification of growth-regulatory genes. In this study 83 formalin-fixed, paraffin-embedded archival DCIS specimens were examined after laser-based microdissection by quantitative real-time PCR using the TaqMan® detection system for amplification of the c-erbB2, topoisomerase IIα, c-myc and cyclinD1 gene. In a subset of 17 DCIS with adjacent infiltrating tumour components we compared intraductal and invasive tumour components in parallel for differences in amplification status. The combination of these new techniques represents an excellent tool to gain new insights into carcinogenesis by analyzing genetic alterations in morphologically identified heterogeneous lesions in breast cancer progression within the very same specimen or even tissue slide.

Morphological response to therapy of breast carcinoma

ZusammenfassungDie primäre (neoadjuvante oder präoperative) Chemotherapie wird bei Mammakarzinomen eingesetzt, um die Tumormasse zu reduzieren und möglichst brusterhaltend operieren zu können. In bis zu 80% der Fälle ist zumindest ein klinisches Ansprechen des Tumors zu verzeichnen, die Chemosensitivität kann bei diesem Vorgehen in vivo überprüft werden. Zusätzlich ist das Ausmaß der Tumorregression nach Therapie als eigenständiger prognostischer Faktor inzwischen etabliert.Neben der Reduktion der Zellzahl des Tumors sind Fibrose, Vakuolisierung des Zytoplasmas und erhöhte Kernpolymorphie typische Folgen der Chemotherapie. Das Grading kann sich in beide Richtungen ändern, während das Typing und Immunprofil des Karzinoms nur in einem geringeren Prozentsatz der Tumoren durch die Therapie wesentlich verändert werden. Prädiktiv für die Chemosensitivität des Tumors sind ein hohes Grading, die Östrogenrezeptornegativität, die Überexpression von Her2-neu und ein negativer Lymphknotenstatus.AbstractNeoadjuvant chemotherapy has been extended to earlier stages of breast carcinoma in order to increase the rate of breast conservation by downstaging. Tumour regression can be observed in up to 80% of the cases and the chemosensitivity of the individual tumour can be studied in vivo. Moreover therapy induced regression has been established as an independent prognostic parameter.Characteristic effects of chemotherapy include reduction in cell number, fibrosis, vacuolization of cytoplasm and increased nuclear pleomorphism. Grading, typing and immunohistochemical properties of the carcinomas remain unchanged in the majority of cases. Predictive for the chemosensitivity of tumours are a high nuclear grade, overexpression of Her-2-neu, lack of estrogen receptor expression and lymph node metastases.

Diagnostics and treatment of primary bone tumors

Primary bone tumors can be either benign or malignant. Metastization is a characteristic feature of malignant bone tumors. Malignant tumors are characterized by a local aggressive and destructive behavior. The behavior of a tumor is dependent on its entity, the differentiation grade and localization and these factors are of decisive importance for the correct therapy. Even benign tumors can behave very aggressively. Different stages are defined. Patient history and conventional radiographs are the most powerful primary diagnostic tools. Many tumors show typical characteristics and if a malignant lesion is suspected a biopsy should be carried out. Several quality standards have to be respected when making the biopsy. The approach to malignant tumors is always interdisciplinary. Several biological as well as alloplastic reconstruction techniques exist. The treatment of primary malignant bone tumors requires a lot of experience and should only be done in specialized centers.ZusammenfassungPrimäre Knochentumoren lassen sich in maligne und benigne Läsionen unterscheiden. Die Metastasierung ist ein Merkmal maligner Tumoren. Lokal zeigen maligne Tumoren ein aggressives und destruierendes Wachstum. Dieselbe Tumorentität kann verschiedene Differenzierungsgrade aufweisen, die für die Therapie von entscheidender Bedeutung sind. Auch benigne Tumoren können sich abhängig von ihrem Stadium lokal aggressiv verhalten. Der Anamnese und dem konventionellen Röntgenbild kommt in der primären Diagnostik entscheidende Bedeutung bei. Viele Tumoren zeigen hier bereits charakteristische Merkmale. Bei Verdacht auf einen malignen Prozess ist in der Regel eine Biopsie erforderlich. Diese muss strengen Qualitätsansprüchen genügen. Die Therapie der malignen Tumoren ist interdisziplinär. Nach Resektion eines Tumors stehen verschiedene biologische und alloplastische Rekonstruktionsmöglichkeiten zur Verfügung. Speziell die Behandlung von malignen Knochentumoren bedarf einiger Erfahrung und sollte daher in ausgewählten Zentren erfolgen.AbstractPrimary bone tumors can be either benign or malignant. Metastization is a characteristic feature ofmalignant bone tumors. Malignant tumors are characterized by a local aggressive and destructive behavior. The behavior of a tumor is dependent on its entity, the differentiation grade and localization and these factors are of decisive importance for the correct therapy. Even benign tumors can behave very aggressively. Different stages are defined. Patient history and conventional radiographs are the most powerful primary diagnostic tools. Many tumors show typical characteristics and if a malignant lesion is suspected a biopsy should be carried out. Several quality standards have to be respected when making the biopsy. The approach to malignant tumors is always interdisciplinary. Several biological as well as alloplastic reconstruction techniques exist. The treatment of primary malignant bone tumors requires a lot of experience and should only be done in specialized centers.

Diagnostik und Therapie primärer Knochentumoren

Primary bone tumors can be either benign or malignant. Metastization is a characteristic feature of malignant bone tumors. Malignant tumors are characterized by a local aggressive and destructive behavior. The behavior of a tumor is dependent on its entity, the differentiation grade and localization and these factors are of decisive importance for the correct therapy. Even benign tumors can behave very aggressively. Different stages are defined. Patient history and conventional radiographs are the most powerful primary diagnostic tools. Many tumors show typical characteristics and if a malignant lesion is suspected a biopsy should be carried out. Several quality standards have to be respected when making the biopsy. The approach to malignant tumors is always interdisciplinary. Several biological as well as alloplastic reconstruction techniques exist. The treatment of primary malignant bone tumors requires a lot of experience and should only be done in specialized centers.ZusammenfassungPrimäre Knochentumoren lassen sich in maligne und benigne Läsionen unterscheiden. Die Metastasierung ist ein Merkmal maligner Tumoren. Lokal zeigen maligne Tumoren ein aggressives und destruierendes Wachstum. Dieselbe Tumorentität kann verschiedene Differenzierungsgrade aufweisen, die für die Therapie von entscheidender Bedeutung sind. Auch benigne Tumoren können sich abhängig von ihrem Stadium lokal aggressiv verhalten. Der Anamnese und dem konventionellen Röntgenbild kommt in der primären Diagnostik entscheidende Bedeutung bei. Viele Tumoren zeigen hier bereits charakteristische Merkmale. Bei Verdacht auf einen malignen Prozess ist in der Regel eine Biopsie erforderlich. Diese muss strengen Qualitätsansprüchen genügen. Die Therapie der malignen Tumoren ist interdisziplinär. Nach Resektion eines Tumors stehen verschiedene biologische und alloplastische Rekonstruktionsmöglichkeiten zur Verfügung. Speziell die Behandlung von malignen Knochentumoren bedarf einiger Erfahrung und sollte daher in ausgewählten Zentren erfolgen.AbstractPrimary bone tumors can be either benign or malignant. Metastization is a characteristic feature ofmalignant bone tumors. Malignant tumors are characterized by a local aggressive and destructive behavior. The behavior of a tumor is dependent on its entity, the differentiation grade and localization and these factors are of decisive importance for the correct therapy. Even benign tumors can behave very aggressively. Different stages are defined. Patient history and conventional radiographs are the most powerful primary diagnostic tools. Many tumors show typical characteristics and if a malignant lesion is suspected a biopsy should be carried out. Several quality standards have to be respected when making the biopsy. The approach to malignant tumors is always interdisciplinary. Several biological as well as alloplastic reconstruction techniques exist. The treatment of primary malignant bone tumors requires a lot of experience and should only be done in specialized centers.

Neuroendokrine Tumore des Magens Chirurgische Therapie und Prognose

Gastric carcinoid tumors are rare lesions characterized by hypergastrinemia that arise from enterochromaffin-like (ECL) cells of the stomach. A classification system distinguishing three types of gastric carcinoid tumors has been proposed: 1) tumors related to chronic atrophic gastritis, 2) tumors associated with Zollinger-Ellison syndrome, and 3) sporadic lesions. It is apparent that hypergastrinemia-associated gastric carcinoids show a rather benign biological behavior. Normogastrinemic sporadic lesions, on the other hand, require an aggressive surgical management. We report seven patients with gastric neuroendocrine tumors (“carcinoids”), who underwent surgical treatment in our department between 1988 and 2000. Surgical therapy included total gastrectomy with D2 lymphadenectomy in two cases with type I tumors and for one patient with type III tumor. One patient with a type II tumor was treated by distal subtotal gastrectomy and another by antrectomy. A local excision was performed on one patient with type I tumor. After a mean follow-up of 8 years, 5 of 7 patients are alive without recurrence.ZusammenfassungDie neuroendokrinen Tumore des Magens (sog. “Magenkarzinoide”) stellen eine seltene Tumorerkrankung dar, die meist von den enterochromaffinähnlichen Zellen des Magens (ECL-Zellen) ausgehen. Es wird zwischen dem Typ I, der mit einer Hypergastrinämie einhergeht und mit einer perniziösen Anämie vergesellschaft sein kann, dem Typ II, der ebenfalls mit einer Hypergastrinämie einhergeht und im Zusammenhang mit einem Zollinger-Ellison-Syndrom oder einer multiplen endokrinen Neoplasie Typ I (MEN-I) vorkommen kann, und dem sporadischen, normogastrinämischen Typ III unterschieden. Während die hypergastrinämischen Tumore ein relativ gutartiges biologisches Verhalten zeigen und somit mit eingeschränkten radikalen Maßnahmen zu therapieren sind, erfordert der sporadische Typ-III-Tumor aufgrund seines eher malignen Verhaltens ein radikales chirurgisches Vorgehen. Wir berichten über unsere Erfahrungen mit 7 Patienten, die im Zeitraum von Mai 1988 bis Juni 2000 in der Klinik für Viszeral- und Transplantationschirurgie der Medizinischen Hochschule Hannover aufgrund einer neuroendokrinen Tumorerkrankung des Magens operiert worden sind. Eine totale Gastrektomie mit D2-Lymphadenektomie wurde bei 4 Patienten durchgeführt. Bei 2 Patienten mit Magenkarzinoiden vom Typ II wurde einmal eine subtotale Magenresektion und einmal eine Antrektomie vorgenommen. Bei einem Patienten mit einem Typ-I-Tumor konnte dieser durch eine Manschettenresektion im Gesunden entfernt werden. Bei allen Patienten erfolgte eine R0-Resektion, eine perioperative Mortalität wurde nicht beobachtet. Nach einer durchschnittlichen Nachbeobachtungszeit von 8 Jahren leben 5 der 7 Patienten rezidivfrei.