Heinz Höfler

Diffuse type gastric and lobular breast carcinoma in a familial gastric cancer patient with an E-cadherin germline mutation.

E-Cadherin alterations have been reported frequently in sporadic diffuse type gastric and lobular breast carcinomas. Germline mutations of this gene have been identified recently in several gastric cancer families. We analyzed seven patients with a family history of the disease who had diffuse type gastric cancer diagnosed before the age of 45 for germline mutations in CDH1, the gene encoding the E-cadherin protein. We identified a frameshift mutation in exon 3 in one patient with a strong family history of gastric cancer. The same germline mutation was found in the patients mother, who had metachronous development of lobular breast and diffuse type gastric carcinomas. Immunohistochemistry for E-cadherin protein expression revealed an abnormal staining pattern in both of these tumors, suggesting complete inactivation of the cell adhesion molecule. Thus, our finding suggests that besides diffuse type gastric cancer, lobular breast carcinomas may be associated with germline CDH1 mutations.

Risk-group discrimination in node-negative breast cancer using invasion and proliferation markers: 6-year median follow-up

SummaryFactors reflecting two major aspects of tumour biology, invasion (urokinase-type plasminogen activator (uPA), plasminogen activator inhibiter (PAI-1), cathepsin D) and proliferation (S-phase fraction (SPF), Ki-67, p53, HER-2/neu), were assessed in 125 node-negative breast cancer patients without adjuvant systemic therapy. Median follow-up time was 76 months. Antigen levels of uPA, PAI-1 and cathepsin D were immunoenzymatically determined in tumour tissue extracts. SPF and ploidy were determined flow-cytometrically, Ki‴-67, p53, and HER-2/neu immunohistochemically in adjacent paraffin sections. Their prognostic impact on disease-free (DFS) and overall survival (OS) was compared to that of traditional factors (tumour size, grading, hormone receptor status). Univariate analysis determined PAI-1 (P < 0.001), uPA (P = 0.008), cathepsin D (P = 0.004) and SPF (P = 0.023) as significant for DFS. All other factors failed to be of significant prognostic value. In a Cox model, only PAI-1 was significant for DFS (P < 0.001, relative risk (RR) 6.2). In CART analysis for DFS, the combination of PAI-1 and uPA gave the best risk group discrimination. For OS, PAI-1, cathepsin D, tumour size and ploidy were statistically significant in univariate, but PAI-1 was the only independently significant factor in Cox analysis (P < 0.001, RR 8.9). In particular, this analysis shows that PAI-1 is still a strong and independent prognostic factor in node-negative breast cancer after extended 6-year median follow-up.

Invasion marker PAI-1 remains a strong prognostic factor after long-term follow-up both for primary breast cancer and following first relapse.

In 1991, our group was the first to report the prognostic strength of plasminogen activator inhibitor type 1 (PAI‐1) in primary breast cancer. The prognostic impact of invasion markers PAI‐1 and urokinase‐type plasminogen activator (uPA) on disease‐free survival (DFS) and overall survival (OS) in breast cancer has since been independently confirmed. We now report on the prognostic impact of PAI‐1 and uPA after long‐term median follow‐up of 77 months for our cohort (n=316).Levels of uPA, PAI‐1, and cathepsin D were determined in tumor tissue extracts by immunoenzymatic methods. S‐phase fraction (SPF) was measured flowcytometrically in paraffin sections. Using log‐rank statistics, optimized cutoffs were found for PAI‐1 (14 ng/mg), uPA (3 ng/mg), cathepsin D (41 pmol/mg), and SPF (6%). In all patients, various factors (PAI‐1, uPA, nodal status, SPF, cathepsin D, grading, tumor size, hormone receptor status) showed significant univariate impact on DFS. In Cox analysis, only nodal status (p < 0.001, RR: 3.1) and PAI‐1 (p < 0.001, RR: 2.7) remained significant. In node‐negative patients (n = 147), PAI‐1, uPA, and SPF had significant univariate impact on DFS, whereas in Cox analysis, only PAI‐1 was significant. PAI‐1 was also significant for DFS within subgroups defined by established factors. In CART analysis, uPA enhanced the prognostic value of PAI‐1 and nodal status for determination of a very‐low‐risk subgroup. For OS, only lymph node status and PAI‐1 were significant in multivariate analysis. PAI‐1 levels in the primary tumor were also a significant prognostic marker for survival after first relapse in both univariate and multivariate analysis.

Analysis of E-Cadherin in Diffuse-Type Gastric Cancer Using a Mutation-Specific Monoclonal Antibody

In-frame deletions from the E-cadherin mRNA, coding for a homophilic cell adhesion molecule, are characteristic for diffuse-type gastric carcinomas. Using immunohistochemical analysis the mutant form cannot be distinguished from normal E-cadherin, making results difficult to interpret. In this study, a rat monoclonal antibody, designated E-cad delta 9-1, was generated against a peptide spanning the fusion junction region between exons 8 and 10. This new epitope is present in an E-cadherin variant that lacks exon 9 from the mRNA due to different splice-site gene mutations. Using Western blotting and immunohistochemistry of E-cadherin-transfected cells, we demonstrate that E-cad delta 9-1 specifically reacts with E-cadherin lacking exon 9 but not with the wild-type protein. No immunoreactivity was observed in 31 nontumorous and embryonal tissues analyzed. In gastric carcinoma specimens known to express mutant E-cadherin mRNA lacking exon 9, E-cad delta 9-1 targets exclusively tumor cells in routine formalin-fixed and paraffin-embedded material from biopsies, primary tumors, and lymph node metastases. In a retrospective series of 172 diffuse-type gastric carcinomas expressing E-cadherin, E-cad delta 9-1 reacted with 22 tumors (13%). This new tumor marker-monoclonal antibody system could open novel avenues for selective diagnosis and specific therapy of a subgroup of diffuse-type gastric cancer patients.

Identification of eleven novel tumor‐associated e‐cadherin mutations

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/ bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 9 families affected by severe hypophosphatasia. Fourteen distinct mutations were found, 3 of which were previously reported in the North American or Japanese populations. Seven of the 11 new mutations were missense mutations (M45L, R119H, G145V, C184Y and H154Y, D289V, E459K), the four others were 2 single nucleotide deletions (544delG and 1172delC), a mutation affecting donor splice site (862 + 5A) and a nonsense mutation (R411X).The cell adhesion molecule E‐cadherin (CDH1; MIM# 192090) has been implicated in numerous cellular functions, ranging from controlling morphogenesis to suppressing tumor invasion. We describe 11 previously unreported somatic E‐cadherin mutations in two subgroups of gastric and breast cancer showing markedly reduced homophilic cell‐to‐cell interactions. Using reverse transcription‐polymerase chain reaction (RT‐PCR) and direct sequencing of the entire coding region 5 mutations were detected in diffuse‐type gastric cancer specimens. The sequence alterations include 3 missense mutations affecting exons 3, 10, and 12. Furthermore, two in‐frame deletions were identified removing 63 and 9 base pairs from exon 4 and 5, respectively. In invasive lobular breast cancer 6 E‐cadherin mutations were detected after RT‐PCR amplification and direct sequencing or using single strand conformation polymorphism (SSCP) analysis followed by sequencing. In addition to two nonsense mutations affecting exon 2, four out‐of‐frame deletions removing 115 base pairs (entire exon 2), 224 base pairs (entire exon 3), 8 base pairs from exon 12 or 1 base pair from exon 13 were seen. Our report confirms the general principle that in diffuse‐type gastric cancer E‐cadherin mutations result in structurally altered proteins with possible reduced adhesive functions whereas in invasive lobular breast carcinomas complete loss‐of‐function mutations are characteristic.

Cadherin-11 is highly expressed in rhabdomyosarcomas and during differentiation of myoblasts in vitro.

Rhabdomyosarcomas bear a morphological and genetic resemblance to developing skeletal muscle. Apart from myogenic marker genes (bHLH factors, myosin, actin), cell adhesion molecules such as N‐cadherin and N‐CAM have been reported to be expressed both in rhabdomyosarcomas and during myogenesis. The present study demonstrates the expression of another cadherin, cadherin‐11, in rhabdomyosarcomas and during differentiation of myoblasts in vitro: cadherin‐11, a predominantly mesenchymal cell adhesion molecule, is highly expressed in embryonal rhabdomyosarcomas and alveolar rhabdomyosarcomas, which do not bear the Pax‐3–FKHR fusion previously described. Cadherin‐11 is down‐regulated in normal skeletal muscle and after myotube formation in vitro. The results of this study suggest that cadherin‐11 might be involved in myogenesis and that rhabdomyosarcomas may re‐express or fail to down‐regulate cadherin‐11. Since alveolar rhabdomyosarcomas bearing the t(2;13) translocation do not express cadherin‐11, it is postulated that Pax‐3 and cadherin‐11 might be linked and involved in the same myogenic pathway. Copyright

Identification of eleven novel tumor-associated E-cadherin mutations. Mutations in brief no. 215. Online.

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/ bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 9 families affected by severe hypophosphatasia. Fourteen distinct mutations were found, 3 of which were previously reported in the North American or Japanese populations. Seven of the 11 new mutations were missense mutations (M45L, R119H, G145V, C184Y and H154Y, D289V, E459K), the four others were 2 single nucleotide deletions (544delG and 1172delC), a mutation affecting donor splice site (862 + 5A) and a nonsense mutation (R411X).The cell adhesion molecule E‐cadherin (CDH1; MIM# 192090) has been implicated in numerous cellular functions, ranging from controlling morphogenesis to suppressing tumor invasion. We describe 11 previously unreported somatic E‐cadherin mutations in two subgroups of gastric and breast cancer showing markedly reduced homophilic cell‐to‐cell interactions. Using reverse transcription‐polymerase chain reaction (RT‐PCR) and direct sequencing of the entire coding region 5 mutations were detected in diffuse‐type gastric cancer specimens. The sequence alterations include 3 missense mutations affecting exons 3, 10, and 12. Furthermore, two in‐frame deletions were identified removing 63 and 9 base pairs from exon 4 and 5, respectively. In invasive lobular breast cancer 6 E‐cadherin mutations were detected after RT‐PCR amplification and direct sequencing or using single strand conformation polymorphism (SSCP) analysis followed by sequencing. In addition to two nonsense mutations affecting exon 2, four out‐of‐frame deletions removing 115 base pairs (entire exon 2), 224 base pairs (entire exon 3), 8 base pairs from exon 12 or 1 base pair from exon 13 were seen. Our report confirms the general principle that in diffuse‐type gastric cancer E‐cadherin mutations result in structurally altered proteins with possible reduced adhesive functions whereas in invasive lobular breast carcinomas complete loss‐of‐function mutations are characteristic.

Diagnosis and prognosis of neuroendocrine tumours of the lung by means of high resolution image analysis

Neuroendocrine tumours (NET) of the lung are divided in subtypes with different malignant potential. The first is the benign or low‐grade malignant tumours, well‐differentiated, called typical carcinoids (TC) and the second is the high‐grade malignant tumours, poorly differentiated of small (SCLC) or large cell type (LCLC). Between these tumour types lies the well‐differentiated carcinoma with a lower grade of malignancy (WDNEC). In clinical routine it is very important with regard to prognosis to distinguish patients with low malignant potential from those with higher ones. In this study 32 cases of SCLC, 13 of WDNEC and 14 of TC with a follow‐up time up to 7 years were collected. Sections 4 μm thick from paraffin embedded tissue were Feulgen stained. By means of high resolution image analysis 100 nuclei per case were randomly gathered to extract morphometric, densitometric and textural quantitative features. To investigate the ploidy status of the tumour the corrected DNA distribution was calculated. Stepwise linear discriminant analysis to differentiate the classes and Cox regression analysis for the survival time analysis were applied. Using chromatin textural and morphometric features in two two‐class discriminations, 11 of the 14 TC cases and 8 of the 13 WDNEC cases were correctly classified and 11/13 WDNEC cases and 28/32 SCLC cases, respectively. The WDNEC cases are more similar in chromatin structure to TC than to SCLC. For the survival analysis, only chromatin features were selected to differentiate patients with better and worse prognosis independent of staging and tumour type.

Molekularpathologie der Ösophagus- und Magenkarzinome

The upper gastrointestinal tract is the origin of a heterogeneous group of tumors comprising squamous cell carcinoma of the esophagus, adenocarcinoma of the esophagus (Barrett carcinoma), and gastric adenocarcinoma mainly. Among these three tumor entities there are differences in the pattern of genes affected during carcinogenesis. This review aims to describe the most important molecular genetic findings focusing on specific oncogenes (e. g. c-erbB2) or tumor suppressor genes (e. g. p53), as well as on genes involved in cell cycle regulation (e. g. cyclin D1, p16, retinoblastoma ), cell adhesion ( E-cadherin ), and proteolysis.