Luzie U. Wingen

Distinct methylation patterns of benign and malignant liver tumors revealed by quantitative methylation profiling.

Purpose: A comparative quantitative methylation profiling of hepatocellular carcinoma and the most frequent benign liver tumor, hepatocellular adenoma, was set up for the identification of tumor-specific methylation patterns. Experimental Design: The quantitative methylation levels of nine genes (RASSF1A, cyclinD2, p16INK4a, DAP-K, APC, RIZ-1, HIN-1, GSTπ1, SOCS-1) were analyzed in hepatocellular carcinoma and adjacent normal tissue (n = 41), hepatocellular adenoma and adjacent normal tissue (n = 26), focal nodular hyperplasia (n = 10), and unrelated normal liver tissue (n = 28). Accumulated methylation data were analyzed using various statistical algorithms, including hierarchical clustering, to detect tumor-specific methylation patterns. Results: Cluster analysis revealed that hepatocellular adenoma displays a methylation profile much more similar to that found in normal liver tissue and focal nodular hyperplasia than to that found in hepatocellular carcinoma. Many characteristic differences were not detected when using mere qualitative methylation assays. The cyclinD2 gene was identified as a new and frequent target for aberrant hypermethylation in hepatocellular carcinoma (68%). In the control group of 28 liver specimens from healthy donors, a clear correlation between age of patient and frequency and level of aberrant methylation was seen, which could not be detected in the group of hepatocellular carcinoma specimens. Conclusions: Methylation profiling can clearly contribute to the unequivocal classification of suspicious lesions, but only if done in a quantitative manner applying cell type and gene-specific thresholds. In hepatocellular carcinoma, the altered methylation patterns accompanying malignant transformation override the age-dependent increase in gene methylation.

Molecular genetic basis of pod corn (Tunicate maize)

Pod corn is a classic morphological mutant of maize in which the mature kernels of the cob are covered by glumes, in contrast to generally grown maize varieties in which kernels are naked. Pod corn, known since pre-Columbian times, is the result of a dominant gain-of-function mutation at the Tunicate (Tu) locus. Some classic articles of 20th century maize genetics reported that the mutant Tu locus is complex, but molecular details remained elusive. Here, we show that pod corn is caused by a cis-regulatory mutation and duplication of the ZMM19 MADS-box gene. Although the WT locus contains a single-copy gene that is expressed in vegetative organs only, mutation and duplication of ZMM19 in Tu lead to ectopic expression of the gene in the inflorescences, thus conferring vegetative traits to reproductive organs.

Analysis of array-CGH data using the R and Bioconductor software suite.

Background. Array-based comparative genomic hybridization (array-CGH) is an emerging high-resolution and high-throughput molecular genetic technique that allows genome-wide screening for chromosome alterations. DNA copy number alterations (CNAs) are a hallmark of somatic mutations in tumor genomes and congenital abnormalities that lead to diseases such as mental retardation. However, accurate identification of amplified or deleted regions requires a sequence of different computational analysis steps of the microarray data. Results. We have developed a user-friendly and versatile tool for the normalization, visualization, breakpoint detection, and comparative analysis of array-CGH data which allows the accurate and sensitive detection of CNAs. Conclusion. The implemented option for the determination of minimal altered regions (MARs) from a series of tumor samples is a step forward in the identification of new tumor suppressor genes or oncogenes.

Diagnosis and immunohistochemistry of medullary breast cancer

ZusammenfassungDas medulläre Mammakarzinom stellt wegen der paradoxen Assoziation von hohem Grad der Dedifferenzierung und Atypie mit einer eher günstigen Prognose eine diagnostische Herausforderung dar. Dabei sind makroskopischer und mikroskopischer Befund maßgeblich, die Haupt- und Nebenkriterien zu berücksichtigen haben. Dagegen ist die Bedeutung des Immunphänotyps noch nicht abschließend geklärt. Da die diagnostische Reproduzierbarkeit der morphologischen Kriterien eingeschränkt ist, haben wir überprüft, inwieweit die Immunhistologie zur Diagnosesicherung herangezogen werden kann. Bei 32 medullären Mammakarzinomen wurden 23 verschiedene Marker analysiert und mit wenig differenzierten duktal-invasiven Mammakarzinomen mit partiellen medullären Eigenschaften (so genannte „atypische medulläre Mammakarzinome“) verglichen. Dabei zeigte der sehr charakteristische, aber nicht spezifische Immunphänotyp eine starke Überlappung mit dem so genannten Basalzellphänotyp des Mammakarzinoms (Negativität für Steroidhormonrezptoren und Her2, Positivität für Basalzellzytokeratine). Mit keinem der angewandten Marker konnte eine spezifische Diskriminierung erreicht werden. Medulläre Mammakarzinome exprimierten signifikant häufiger EGF-R. Werden die charakteristischen morphologischen Kriterien, die nur am Resektat und nicht an der Stanze zu erheben sind, mit dem immunhistochemischen Nachweis des charakteristischen Immunphänotyps kombiniert, lässt sich die diagnostische Sicherheit deutlich erhöhen.AbstractMedullary carcinoma of the breast has a relatively favorable prognosis despite its malignant histopathological appearance, providing a challenge for the pathologically based diagnosis of breast cancer. Macroscopic and microscopic findings combined provide diagnostic criteria. The importance of the immunophenotype of medullary carcinoma is not well defined. Because the reproducibility of morphological criteria is limited, we conducted an immunohistochemical study in search of markers that could facilitate histopathological classification. We examined 32 medullary carcinomas in comparison with 30 high grade ductal invasive carcinomas with similar morphology using 23 different immunohistochemical markers. The results showed an overlap with the so called basal like subtype of invasive breast cancer (negativity for steroid hormone receptor, positivity for basal cytokeratins). None of the immunohistochemical markers enabled a specific discrimination between the two groups. Medullary carcinomas overexpress EGF-R more frequently (P<0.004). In combining the characteristic morphological criteria and the immunohistochemical detection of the basal like phenotype and EGFR, a higher diagnostic accuracy can be achieved. The immunophenotype alone does not allow a definite classification of medullary carcinoma.

Induction of aneuploidy by increasing chromosomal instability during dedifferentiation of hepatocellular carcinoma

To gain more insight into the role of chromosomal instability (CIN), the cytogenetic hallmark of most solid tumors, we performed fluorescence in situ hybridization (FISH) on interphase nuclei of cytological specimens enabling the correct detection of chromosome copies in intact tumor cells of 18 well (G1), moderately (G2), or poorly (G3) differentiated hepatocellular carcinomas (HCCs). A close correlation between the morphological dedifferentiation and increasing copy numbers and variation of FISH signals was seen for chromosomes 1 and 8, respectively (P < or = 0.0002). Four HCC G1 had constant chromosome patterns for chromosomes 1 and/or 8 with a mean of signals per nucleus < or =5.08 and < or =3 different signal combinations, indicating a low level of CIN, as confirmed by FISH using probes for centromeres of chromosomes 3, 7, and 17. In contrast to this, five HCC G2-3 revealed > or =8.46 signals per nucleus and 23-41 different signal combinations, indicating high levels of CIN. In the remaining cases, signal counts from 5.96-8.46 and 7-15 combinations were seen. Here, nuclei with constant aberration patterns and low copy numbers occurred alongside nuclei with inconstant patterns and high copy numbers. It is evident that in these cases a transition from well to moderately differentiated HCC developed in parallel to an increase in CIN, possibly induced by a major dysregulation of mitotic control mechanisms. In conclusion, CIN may induce a stepwise increase of aneuploidy in HCC that is mirrored by the morphological dedifferentiation of tumor cells.