Wiljo J. F. de Leeuw

Simultaneous loss of E-cadherin and catenins in invasive lobular breast cancer and lobular carcinoma in situ

Loss of expression of the intercellular adhesion molecule E‐cadherin frequently occurs in invasive lobular breast carcinomas as a result of mutational inactivation. Expression patterns of E‐cadherin and the molecules comprising the cytoplasmic complex of adherens junctions, α‐, β‐ and γ‐catenin, were studied in a series of 38 lobular breast carcinomas with known E‐cadherin mutation status. The effect of loss of E‐cadherin by mutational inactivation (or other mechanisms) on the expression of catenins was investigated. Complete loss of plasma membrane‐associated E‐cadherin expression was observed in 32 out of 38 invasive lobular carcinomas, for which in 21 cases a mutation was found in the extracellular domain of E‐cadherin. In total, 15 frameshift mutations of small deletions or insertions, ranging from 1 to 41 bp, three non‐sense mutations, and three splice mutations were identified. Mutations were scattered over the whole coding region and no hot spots could be detected. In all cases, simultaneous loss of E‐cadherin and α‐ and β‐catenin expression was found; in 50 per cent of these cases, additional loss of γ‐catenin was observed. In six invasive lobular carcinomas, expression of both E‐cadherin and catenins was retained. In none of these carcinomas was an E‐cadherin mutation detected. Lobular carcinoma in situ adjacent to invasive lobular carcinoma showed simultaneous loss of E‐cadherin and catenins in all the cases studied—remarkably, also, in four cases positive for E‐cadherin and catenin expression in the invasive component. These results indicate that simultaneous loss of E‐cadherin and α‐, β‐ and γ‐catenin may be an important step in the formation of lobular carcinoma in situ, as a precursor of invasive lobular breast cancer. Events additional to E‐cadherin inactivation must be involved in the transition of lobular carcinoma in situ to invasive lobular carcinoma.

Prediction of a mismatch repair gene defect by microsatellite instability and immunohistochemical analysis in endometrial tumours from HNPCC patients

Instability of microsatellite repeat sequences has been observed in colorectal carcinomas and in extracolonic malignancies, predominantly endometrial tumours, occurring in the context of hereditary non‐polyposis colorectal cancer (HNPCC). Microsatellite instability (MSI) as a feature of human DNA mismatch repair (MMR)‐driven tumourigenesis of the uterine mucosa has been studied primarily in sporadic tumours showing predominantly somatic hypermethylation of MLH1. The present study shows that all endometrial carcinomas (n=12) from carriers of MLH1 and MSH2 germline mutations demonstrate an MSI‐high phenotype involving all types of repeat markers, while in endometrial carcinomas from MSH6 mutation carriers, only 36% (4 out of 11) demonstrate an MSI‐high phenotype. Interestingly, an MSI‐high phenotype was found in endometrial hyperplasias from MSH2 mutation carriers, in contrast to hyperplasias from MLH1 mutation carriers, which exhibited an MSI‐stable phenotype. Instability of only mononucleotide repeat markers was found in both endometrial carcinomas and hyperplasias from MSH6 mutation carriers. In 29 out of 31 (94%) endometrial tumour foci, combined MSI and immunohistochemical analysis of MLH1, MSH2, and MSH6 could predict the identified germline mutation. The observation of MSI in endometrial hyperplasia and of altered protein staining for the MMR genes supports the idea that inactivation of MMR genes is an early event in endometrial tumourigenesis. A correlation was found between the variation in the extent and level of MSI and the age of onset of carcinoma, suggesting differences in the rate of tumour progression. A high frequency of MSI in hyperplasias, found only in MSH2 mutation carriers, might indicate a more rapid tumour progression, correlating with an earlier age of onset of carcinoma. The present study indicates that assessment of altered protein staining combined with MSI analysis of endometrial tumours might direct the mutational analysis of MMR genes. Copyright

Molecular genetic evidence for the conversion hypothesis of the origin of malignant mixed Müllerian tumours

The origin of malignant mixed Müllerian tumours (MMMTs) has long been debated, due to the indefinite relationship between epithelial and mesenchymal malignant cells. In order to obtain insight into the clonal relationship between the two components of these tumours, molecular genetic changes were investigated at the level of loss of heterozygosity (LOH) in both cells types. LOH was studied in a series of six cases with 74 polymorphic microsatellite markers mapping to 19 different chromosomes. The epithelial and the mesenchymal neoplastic cells were separately microdissected from formalin‐fixed, paraffin‐embedded tissue, prior to DNA isolation. LOH was observed for 35 different markers mapping to chromosomes 3, 6, 8, 11, 15, 16, 17, 18, 21, and X. The most frequently involved chromosomes were 17p, 17q, 11q, 15q, and 21q. LOH was observed in five out of six cases and identical alleles were lost in the epithelial and in the mesenchymal cells. No genetic differences were observed between the two cell types for any of the informative markers. Immunohistochemistry (IHC) and TP53 mutation analysis revealed involvement of TP53 in all cases. Mutations were identified in five MMMTs. In four tumours, of which three had a missense mutation, strong nuclear staining for p53 was observed. In the remaining two cases, the mutation resulted in a stop codon, with no nuclear staining for p53 by IHC. The results support a monoclonal origin of MMMTs, with the absence of genetic changes uniquely associated with either of the phenotypes. The latter finding is compatible with current opinion that these neoplasms should be considered as metaplastic carcinomas and supports the conversion hypothesis.

Messenger RNA levels and methlation patterns of GAPDH and β-actin genes in rat liver, spleen and brain in relation to aging

Messenger RNA levels and methylation patterns of the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and beta-actin genes were studied in spleen, liver and brain of 6-, 24- and 36-month old female inbred rats. In the spleen, the mRNA levels of both housekeeping genes significantly increased between 24 and 36 months. No age-related alterations in the expression of GAPDH or beta-actin mRNA were observed in brain or liver. A considerable intertissue and interindividual variation was observed in the mRNA levels of these genes in all age-groups as compared to the level of 28 S rRNA, which was used as an internal control. In this respect the interindividual variation in the level of GAPDH mRNA paralleled the variation observed in the beta-actin mRNA level in the three tissues studied. The methylation pattern of beta-actin was found to be tissue-specific in contrast to that of GAPDH, which was identical in all three tissues. No significant age-related alterations were observed in the GAPDH methylation pattern, whereas beta-actin appeared to become slightly demethylated with age in the spleen at the CpG site for which tissue-specificity was observed.

Bias in detection of instability of the (C)8 mononucleotide repeat of MSH6 in tumours from HNPCC patients.

Recently, we and others reported instability in the (C)8 repeat in exon 5 of MSH6 as a preferential target for somatic mutations in tumours from MSH6 germline mutation carriers. Here, we report that in 45% of tumours from MLH1, MSH2 and MSH6 germline mutation carriers no sequence change in the (C)8 repeat of MSH6 was found upon DNA sequencing analysis of PCR products with a shift in electrophoresis mobility. Using ‘standard’ PCR primers a high frequency of instability (50–86%) of the (C)8 repeat was found, but using a modified PCR reverse primer, accomplishing modulation of non-templated addition of adenine during in vitro PCR amplification by the Taq polymerase, a markedly lower frequency of instability was found in tumours from MLH1, MSH2 and MSH6 mutation carriers (6, 13 and 40%, respectively). Furthermore, a significant difference of the frequency of instability of the (C)8 repeat in tumours from MSH6 mutation carriers was found compared to MLH1, MSH2 mutation carriers. These results might have important implications for the detection of instability of other short mononucleotide repeats, e.g. TGFβRII, BAX, IGFRII, PTEN, BRCA2.

mRNA levels and methylation patterns of the tyrosine aminotransferase gene in aging inbred rats.

We have examined the mRNA levels and methylation patterns of the liver‐specific tyrosine aminotransferase (TAT) gene in inbred female rats aged 6, 24 and 36 months. Northern hybridization analysis of total RNA showed a 65% decrease in the steady state transcript level of TAT in the liver of 24‐ and 36‐month old rats as compared to 6‐month old animals. The TAT gene as studied by Southern hybridization analysis using the isoschizomers Hpa II and Msp I was found to be hypomethylated in the liver as compared to spleen and brain at six CpG sites within the gene. Methylation at these sites remained unchanged during aging.

Analysis of VNTR loci amplified by the polymerase chain reaction for investigating the origin of intimal smooth muscle cells in a coronary artery lesion developing after heart transplantation in man

Focal intimal thickening is a feature of primary atherosclerotic coronary lesions and restenotic lesions following percutaneous transluminal coronary angioplasty and other forms of vascular intervention, where it is the nonspecific response of the vessel wall to injury. The principal cellular component of the coronary plaque is the smooth muscle cell.’ Whether the smooth muscle cell in human coronary lesions is derived from cells circulating in the blood or from the vessel wall itself remains a matter for debate. On the basis of animal studies, it is generally presumed that the tunica media or subintimal space is the source of intimal smooth muscle cells.2-5 However, there is sound experimental evidence that smooth muscle cells recognized in a vascular plaque may originate in mural thrombus and not in the adjacent vessel wall. 6-8 Regardless of their source, these cells would normally be indistinguishable. Atherosclerotic lesions developing in the coronary arteries after orthotopic heart transplantation provide a unique opportunity to pursue the origin of cells within the coronary atherosclerotic plaque, as the donor and recipient invariably differ in genotype. Genetic differences may be demonstrated by the electrophoretic analysis of alleles from the highly polymorphic variable number of tandem repeats (VNTR) gene loci that occur widely in the human genome.g,10 The Dl7S5/Dl7S30 VNTR locus, the DlS80/ DlS58 VNTR locus, and the Apo B 3’ VNTR locus (the hypervariable 3’ region of the apolipoprotein B gene) represent independent, highly polymorphic deoxyribonucleic