Ed Schuuring

The involvement of the chromosome 11q13 region in human malignancies: Cyclin D1 and EMS1 are two new candidate oncogenes-a review

Amplification of oncogenes has been observed frequently in various human malignancies and might be of clinical relevance. In the last decade, the exploration of oncogene activation due to DNA amplification in cancer research has mainly focussed on three aspects: (i) the assessment of oncogene amplification as a prognostic marker for survival of cancer patients, (ii) the development of reliable methods for detection of tumors which harbor DNA amplification of oncogenes and (iii) the identification of the gene or genes responsible for the biological (prognostic) significance in tumors with DNA amplification and the characterization of these candidate proto-oncogenes that might help to elucidate their normal function and the role in tumor development. In this review, these three aspects will be highlighted with regard to DNA amplification of the chromosome 11q13 region. Chromosome 11q13 amplification has been found frequently in certain human malignancies; in cancer of the breast and of the head and neck region, amplification of this region is observed in 13 and 29% of tumors, respectively. The 11q13 amplification has been reported to be of clinical relevance in these cancers, since patients with this amplification show a poor clinical course of disease. The amplified 11q13 region is estimated to be 3-5 Mb in size and to harbor many (putative) genes. Recently, two candidate genes, CCND1 and EMS1, were identified which were both over-expressed in all carcinomas with an 11q13 amplification. Therefore, the activation of these genes might confer the selective advantage to these tumors. In addition, the characterization of these two novel genes sustained their potential role in carcinomas with 11q13 amplification.

Mantle-cell lymphoma: a population-based clinical study.

PURPOSE From a population-based non-Hodgkins lymphoma (NHL) registry, 41 patients with mantle cell lymphoma (MCL) -- a recently defined distinct B-cell NHL -- were selected and compared with patients with low- or intermediate-grade NHL from the same registry. PATIENTS AND METHODS The incidence and behavior of MCL in the area of the Comprehensive Cancer Center West (CCCW) from 1981 to 1989 were analyzed. Age, performance, tumor bulk, extranodal localization, stage, response to therapy, and survival were registered. Expression of cyclin D1 protein and Ki-67 were measured in 29 patients. RESULTS MCL made up 3.7% of NHLs. The median age was 68 years, and the male-to-female ratio was 1.6:1. Seventy-eight percent presented with stage IV, with the majority having bone marrow involvement. The complete response (CR) rate was 32% (13 of 41), with a median duration of 25 months. The median overall survival time was 31.5 months. The International Prognostic Index identified five patients with a low-risk score and a median survival time of 93+ months. In 23 of 29 patients, cyclin D1 overexpression was present, without any relation to overall or disease-free survival. In contrast, a proliferative index less than 10% was significantly related to a better overall survival time (50 v 24 months). CONCLUSION MCL is a disease of the elderly, who present with widespread disease and with a poor response to therapy. Although it harbors features of an indolent NHL, it behaves clinically as an aggressive NHL with a short overall survival time.

Detection of three common translocation breakpoints in non-Hodgkin's lymphomas by fluorescence in situ hybridization on routine paraffin-embedded tissue sections

Non‐random chromosomal translocations are specifically involved in the pathogenesis of many non‐Hodgkins lymphomas and have clinical implications as diagnostic and/or prognostic markers. Their detection is often impaired by technical problems, including the distribution of the breakpoints over large genomic areas. This study reports a fluorescence in situ hybridization (FISH) method which allows the detection of specific chromosomal breakpoints in tissue sections from routinely fixed, paraffin‐embedded samples. Hybridization was performed after demasking the DNA. Previously validated locus‐specific probes (cosmids, PACs) flanking the BCL1, BCL2 regions and similar new probes for the MYC breakpoint region were used. The cases studied were five mantle cell lymphomas (MCL) and five follicular lymphomas (FL), selected on the basis of a previously proved t(11;14) and t(14;18) and five randomly chosen Burkitts lymphomas (BL), as well as 21 negative control samples. In all samples, hybridization signals of sufficient intensity were obtained. Three different algorithms were used to score the hybridization signals in tissue sections, two of them taking into account the nuclei and their signal distribution indicative of chromosomal break, and one only considering the colocalization or segregation of the signals. In control tissues, these algorithms resulted in cut‐off levels of 9.1%, 1.3%, or 10.0%. In the 15 lymphoma samples the percentages of abnormal cells/signals ranged from 28% to 80%, 13% to 49%, and 40% to 70%, respectively. The results indicate that small locus‐specific probes can be used in FISH for regular detection of translocation breakpoints on routine paraffin tissue sections. Copyright

Role of Phosphatidylinositol 4,5-Bisphosphate in Ras/Rac-Induced Disruption of the Cortactin-Actomyosin II Complex and Malignant Transformation

ABSTRACT Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac–phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, we propose that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP2-binding molecules.

The Redistribution of Cortactin into Cell-Matrix Contact Sites in Human Carcinoma Cells with 11q13 Amplification Is Associated with Both Overexpression and Post-translational Modification

The EMS1 gene, located at the chromosome 11q13 region, is the human homologue of p80/p85 cortactin, a chicken pp60src tyrosine kinase substrate. In cells derived from breast carcinomas and squamous carcinomas of the head and neck, DNA amplification of this region results in overexpression of cortactin. Overexpression is accompanied by a partial redistribution of cortactin from the cytoplasm into cell-matrix contact sites. To investigate whether overexpression only is sufficient for this redistribution, we performed biochemical analysis of human cortactin derived from carcinoma cell lines with either normal levels (UMSCC8) or with excessive levels of cortactin due to chromosome 11q13 amplification (UMSCC2). Pulse-chase experiments performed with UMSCC2 cells revealed that p85 originated from p80 by post-translational modifications. However, the conversion of p80 into p85 was hardly observed in UMSCC8 cells, indicating a different processing of the two isoforms in cells with a normal expression level of cortactin. Western blot analysis showed that treatment of UMSCC2 cells with cycloheximide, serum, epidermal growth factor, or vanadate resulted in the disappearance of the p80 form and conversion into p85. Conversion of p80 into p85 was accompanied by a redistribution of cortactin from cytoplasm to cell-matrix contact sites. In UMSCC8 cells, these treatments had no effect on the p80/p85 ratio, and cortactin remained in the cytoplasm. Conversion into p85 therefore is correlated with a relocalization of cortactin to the cell periphery. In addition, p85 from epidermal growth factor- or vanadate-treated UMSCC2 cells showed a significant enhancement in phosphorylation compared with p85 in UMSCC8 cells. Our findings demonstrate that in carcinoma cells with 11q13 amplification not only overexpression but also post-translational modifications of cortactin coincides with the redistribution from the cytoplasm into cell-matrix contact sites.

Chromosomal markers in lymphoma diagnosis

Many Band T-cell lymphomas and leukaemias are characterized by specific genetic abnormalities. These abnormalities include chromosomal abnormalities visible by banding analysis like numerical abnormalities, gross deletions, amplifications and chromosomal translocations. In addition, an increasing number of submicroscopical abnormalities such as small interstitial deletions, amplifications and mutations are identified in various types of lymphomas by molecular methods. An overview is given in Table 1. At present a wide array of (molecular) methods is available for the detection of genetic abnormalities; these methods include the detection of DNA alterations such as chromosome banding analysis; metaphase-, interphaseand DNA fibre(fluorescent) in situ hybridization (FISH); pulsed-field gel electrophoresis; Southern blot analysis; PCR, SSCP and DNA sequencing. Methods are also available for the detection of quantitative and qualitative alterations of RNA expression such as Northern blot analysis, RT-PCR and RNA in situ hybridization, and finally, for the detection of (altered) protein expression like Western blot analysis and immunohistochemistry. More than one test can often be used to detect a particular DNA or RNA alteration. It is often difficult for the pathologist to make the best choice. Many pathologists will tend to prefer PCR because it represents a relatively straightforward, simple and rapid method. However, for many targets, other methods may be much more appropriate. New FISH techniques such as interphase FISH and DNA fibre FISH have recently been developed which are superior to the established molecular methods such as Southern blotting. Some characteristics of individual tests are listed in Table 2.