Regine Dahse

Protein profiling of oral brush biopsies: S100A8 and S100A9 can differentiate between normal, premalignant, and tumor cells

In oral mucosa lesions it is frequently difficult to differentiate between precursor lesions and already manifest oral squamous cell carcinoma. Therefore, multiple scalpel biopsies are necessary to detect tumor cells already in early stages and to guarantee an accurate follow‐up. We analyzed oral brush biopsies (n = 49) of normal mucosa, inflammatory and hyperproliferative lesions, and oral squamous cell carcinoma with ProteinChip Arrays (SELDI) as a non‐invasive method to characterize putative tumor cells. Three proteins were found that differentiated between these three stages. These three proteins are able to distinguish between normal cells and tumor cells with a sensitivity of 100% and specificity of 91% and can distinguish inflammatory/hyperproliferative lesions from tumor cells with a sensitivity of up to 91% and specificity of up to 90%. Two of these proteins have been identified by immunodepletion as S100A8 and S100A9 and this identification was confirmed by immunocytochemistry. For the first time, brush biopsies have been successfully used for proteomic biomarker discovery. The identified protein markers are highly specific for the distinction of the three analyzed stages and therewith reflect the progression from normal to premalignant non‐dysplastic and finally to tumor tissue. This knowledge could be used as a first diagnostic step in the monitoring of mucosal lesions.

The prognostic relevance of p16 inactivation in head and neck cancer.

The inactivation of the tumor suppressor gene p16 plays an important role in the development of malignant tumors. p16 loss can result from point mutations, loss of heterozygosity (LOH) or methylation of the promoter region. A total of 67 samples of tumor tissue from squamous cell carcinoma of the oral cavity, the pharynx and the larynx were analyzed for an inactivation of p16. The results of the molecular-biological investigations were correlated with the known clinical prognostic parameters after a follow-up period of approximately 3 years. Methylation of the promoter region and LOH were the main mechanisms of p16 inactivation. Point mutations presented as rare events. An inactivation of p16 did not have any statistical influence on tumor prognosis. Patients with a p16 gene inactivated by promoter methylation appeared to have a slightly lower tendency for local and regional recurrences. The inactivation of the tumor suppressor gene p16 plays a role in the carcinogenesis of head and neck cancer.

KRAS status and epidermal growth factor receptor expression as determinants for anti-EGFR therapies in salivary gland carcinomas

Salivary gland carcinomas (SGC) are rare cancers with poor prognosis and limited response to conventional chemotherapy. New strategies based on molecular targeted therapy are needed and the EGFR signaling cascade is considered a possible key pathway for therapeutic molecules. We have analyzed 65 SGC of the main histopathological types for the expression of EGFR and and the mutation status of its downstream effector KRAS. EGFR overexpression (+2, +3) has been identified by immunohistochemistry in 75.4%. KRAS mutation analysis was performed by direct genomic sequencing and revealed a KRAS wildtype in 98.5% except of one adenoid cystic carcinoma with a GGT-GAT transition at codon 12 (Gly12Asp). EGFR overexpression and KRAS wildtype are prerequisites for a successful anti-EGFR therapy. The results of this study plead in favor of further therapeutic trials with EGFR-targeting monoclonal antibodies in SGC.

Stromal laminin chain distribution in normal, hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation.

BACKGROUND The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains alpha2, alpha3, alpha4, alpha5 and gamma2 in the stromal compartment/vascular structures in OSCC was analysed. METHODS Frozen tissue of OSCC (9x G1, 24x G2, 8x G3) and normal (2x)/hyperplastic (11x) oral mucosa was subjected to laminin chain and alpha-smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. RESULTS Stromal laminin alpha2 chain significantly decreases and alpha3, alpha4, alpha5 and gamma2 chains and also ASMA significantly increase with rising grade. The amount of stromal alpha3, alpha4 and gamma2 chains significantly increased with rising ASMA positivity. There is a significant decrease in alpha3 chain positive vessels with neoplastic transformation. CONCLUSIONS Mediated by myofibroblasts, OSCC development is associated with a stromal up-regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning alpha3 and gamma2 chain laminins during tumour angioneogenesis is suggested.

No Incidence of BRAF Mutations in Salivary Gland Carcinomas—Implications for Anti-EGFR Therapies

BRAF is the main effector of KRAS in the RAS-RAF-MAPK axis, a signaling pathway downstream of EGFR. The activation of this cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules. Recent studies in metastatic colorectal cancer found that an oncogenic activation of BRAF by a point mutation in exon 15 (V600E) could bypass the EGFR-initiated signaling cascade with the effect that patients bearing the mutant BRAF allele are not likely to benefit from EGFR-targeted therapies. We designed an allele-specific PCR and screened 65 salivary gland carcinoma (SGC) of the main histopathological types for the BRAF V600E mutation. All 65 SGC in this cohort (100%) presented the BRAF wildtype. In a previous study, we found a KRAS wildtype in 98.5% of SGC. These findings imply that SGC rarely acquires mutations that result in a constitutive activation of the signaling cascade downstream of EGFR and this pleads in favor of further therapeutic trials with EGFR-targeting monoclonal antibodies.

Telomerase in human tumors: molecular diagnosis andclinical significance

Shortening of structures known as telomeres, which cap the ends of chromosomes, is postulated to limit the lifespan of human cells. Activation of telomerase, an enzyme that synthesizes telomeric DNA, is an essential step in cell immortalization. Telomerase is ordinarily inactive in most somatic cells, but can be detected in nearly all tumors. The activation of telomerase in malignant cancers seems to be an important step in tumorigenesis, whereby the cell gains the ability of indefinite proliferation. Due to the association between telomerase expression and malignancy, the enzyme is expected to be a useful tumor marker and a new anticancer therapeutic target. However, recent results scale down to some extent the initial enthusiastic expectations for telomerase as the ideal malignancy marker.

A semi-automated highly sensitive approach for mutation detection

▼Single-strand conformation polymorphism (SSCP) is the most common screening method for unknown mutations, but its sensitivity ranges, depending on fragment size and sequence, and mutations can therefore be missed (Ref. 1, 2). To enhance the mutation-detection efficiency, variations of SSCP have been developed. Dideoxyfingerprinting (ddF) and Bi-directional dideoxyfingerprinting (Bi-ddF) are hybrid techniques between SSCP and Sanger dideoxy sequencing. They combine a Sanger dideoxy termination reaction with only one of the dideoxy terminators with an electrophoresis under SSCP conditions through a nondenaturing gel. Mutations can be detected as a result of the gain or loss of a dideoxy termination segment and/or by an altered mobility of at least one of the termination segments containing the mutation (Ref. 3, 4). Bi-ddF is used performing the cycle-sequencing of the PCR product with opposing primers. In this way Bi-ddF has two important advantages over ddF: (i) the dideoxy terminator can detect 10 of 12 types of possible single-base changes; and (ii) the SSCP component is enhanced because mobility alterations can be detected in either the downstream or upstream direction. ddF and Bi-ddF proved to be 100% sensitive in blinded mutation analyses in different genes (Ref. 4, 5, 6). Gel matrix and gel temperature were found to be important experimental parameters that influence the sensitivity of the SSCP component in ddF (Ref. 7). Here, we present an optimized non-radioactive Bi-ddF protocol for application on an automated DNA sequencer. We tested different gel matrices and electrophoresis buffer systems. To determine the sensitivity of our technique we used 1.8 kb PCR fragments of exon 5–9 of the p53 (TP53) gene as a test sytem. Figure 1 This PCR fragment was used as a template for three Bi-ddF primer pairs to screen exon 516 (480 bp), exon 7 (330 bp) and exon 819 (410 bp). This protocol is fast and simple, with the following advantages:

3 – Microarray-Based Gene Expression Analysis of Defined Tumor Areas

One of the most important applications for DNA microarrays is the study of differential gene expression. Gene expression analysis using microarrays requires the development and successful implementation of a variety of laboratory strategies. Microarray technology is a powerful technology that will substantially increase the speed at which differential gene expression can be analyzed and gene functions are elucidated. To fulfil these expectations, improvements of the technology with respect to reproducibility, sensitivity, cost, and speed will be needed. The problems of sample collection, experimental procedure, and data analysis should not be underestimated. An independent verification method as a follow-up to the array results should also be considered, such as reverse transcription polymerase chain reaction, Northern or Western Blot analyses, or in situ hybridization. Ideally, microarray studies, limited to the messenger RNA level, should be accompanied by analyses at the protein level. Proteomics, large-scale complex analysis of the proteins that are present in a cell, is developing rapidly. Both technologies are complementary while focusing on different steps of the same process—the expression of genetic information into functional molecules.