Bernhard Heine

Telomerase as a prognostic marker in breast cancer: high‐throughput tissue microarray analysis of hTERT and hTR

Telomerase activity (TA) has been shown to correlate with poor clinical outcome in various tumour entities, indicating that tumours expressing this enzyme may be more aggressive and that TA may be a useful prognostic marker. For breast cancer, however, TA is a controversial prognostic marker; whereas some studies suggest an association between TA and disease outcome, others do not find this association. This study used tissue microarrays (breast carcinoma prognosis arrays) containing 611 samples (each 0.6 mm in diameter) from the tumour centre of paraffin‐embedded breast carcinomas to analyse the catalytic subunit of telomerase, human telomerase reverse‐transcriptase (hTERT), and the internal RNA component (hTR), which are the core components of the telomerase holoenzyme complex. hTERT protein expression was obtained by immunohistochemistry (human anti‐telomerase antibody Ab‐2, Calbiochem), and hTR RNA was measured by radioactive in situ hybridization. hTERT and hTR expression were determined semi‐quantitatively and graded (scores 1–4). Clinical data, such as histological subtype, pT stage, tumour diameter, pN stage, BRE grade, tumour‐specific survival (in months), patients age and others, were available for statistical analysis. A statistically significant correlation was found between tumour‐specific survival (overall survival) and hTERT expression (p < 0.0001) or hTR expression (p = 0.00110). Tumours with higher scores (scores 3, 4) for hTR and/or hTERT were associated with a worse prognosis. In multivariate analysis, hTERT expression was an independent prognostic factor. Previous studies, focusing on analysis of TA in smaller numbers of fresh‐frozen breast carcinomas by the TRAP assay, gave controversial results with respect to TA as a prognostic marker. Using tissue microarrays from 611 breast carcinomas, this study has demonstrated that increased expression levels of the telomerase core components, hTERT and hTR, are associated with lower overall survival. These findings suggest that TA should be included in future validation studies as a prognostic marker in breast cancer. Copyright

Demonstration of constant upregulation of the telomerase RNA component in human gastric carcinomas using in situ hybridization.

Upregulation of the ribonucleoprotein telomerase seems to be a prerequisite for immortality, a feature of malignant cells. Using a polymerase chain reaction (PCR)‐based assay, it is possible to demonstrate telomerase activity (TA) in specimens of most human malignancies, whereas it is absent from most normal tissues. It remains unclear, however, why between 5 and 50 per cent of various malignant tumour samples give negative results when TA is measured by the telomeric repeat amplification protocol (TRAP). The expectation that reverse transcription (RT)‐PCR for detection of the telomerase RNA component (hTR) would be able to complement or to replace the TRAP assay failed, since malignant as well as non‐malignant tissue samples gave positive results in most instances. In the present study, in situ hybridization (ISH) was developed to demonstrate the RNA component of human telomerase at the single cell level. With this method, 13 specimens of fresh frozen gastric carcinoma and four of normal, dysplastic, or inflamed gastric mucosa were investigated and the results were compared with those obtained by RT‐PCR and the TRAP assay. In addition, ISH was performed on formalin‐fixed sections of the same cases. The TRAP assay revealed positive results in 8 out of 13 gastric carcinomas and was negative in all non‐malignant tissues. RT‐PCR led to amplification of the telomerase RNA component in all specimens tested, irrespective of the presence or absence of malignant cells. By ISH, all gastric carcinomas showed strong telomerase RNA component‐specific signals over malignant cells, whereas only a few grains were detectable over some types of normal somatic cells, including activated lymphocytes. In conclusion, high expression of the telomerase RNA component was restricted to the malignant cells of all the gastric carcinomas investigated, as shown by ISH. This indicates that the absence of TA in a proportion of carcinomas is due to methodological problems of the TRAP assay and is not caused by biological factors. The detection of high levels of the telomerase RNA component by ISH is thus a useful technique for demonstrating malignant cells in frozen and formalin‐fixed pathological specimens.

Up-Regulation of the Peripheral Benzodiazepine Receptor during Human Colorectal Carcinogenesis and Tumor Spread

The peripheral benzodiazepine receptor (PBR) is overexpressed in a variety of cancers. In Unio Internationale Contra Cancrum (UICC) III colorectal cancers, a high level of PBR overexpression correlates with poor prognosis. However, little is known about the role of PBR in the development and progression of colorectal cancer. This study addresses the up-regulation of PBR during colorectal carcinogenesis and tumor spread. One hundred sixteen consecutive patients undergoing surgery for colorectal cancer with either regional (59 patients) or distant metastases (57 patients) were followed-up for 5 years or until death. Twenty-four of the 59 patients with initial UICC stage III cancers later developed distant metastases. PBR overexpression in tumor specimens was determined by immunohistochemistry. UICC stage III patients with colorectal primaries highly overexpressing PBR developed metastases significantly more often than patients with low PBR overexpression in their primary carcinoma. In 54 of the 116 patients adenomas and/or metastases and/or recurrences were available to be studied for PBR up-regulation during colorectal carcinogenesis and tumor spread. PBR was found to be overexpressed in 86% of early and late adenomas. Furthermore, 85% of primaries and of 86% of metastases displayed PBR overexpression. PBR overexpression was also detected at the mRNA level as revealed by real-time PCR. The extent of PBR protein overexpression was equivalent in colorectal adenomas and carcinomas but slightly increased in metastases. These data suggest a functional role of PBR during colorectal carcinogenesis and tumor spread. Thus, PBR qualifies as a target for innovative diagnostic and therapeutic approaches.

Telomerase expression in uveal melanoma.

BACKGROUND/AIMS Accumulating evidence indicates that telomerase activity is repressed in normal human somatic cells but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity has a role in carcinogenesis and immortalisation. To date, telomerase in uveal melanoma and, whether, it may have a role in the development or progression of these tumours has not been described. The expression patterns and the activity of telomerase were investigated in 14 uveal melanoma and these results were correlated with histological and immunohistological features of these tumours. METHODS A modified PCR based telomeric repeat amplification protocol (TRAP) assay was used to demonstrate telomerase activity in 14 uveal melanomas. In addition, in situ hybridisation was used to demonstrate the expression pattern of the telomerase RNA component (hTR) at the single cell level in eight of these globes. RESULTS The TRAP assay revealed moderate telomerase activity in all uveal melanomas examined. In situ hybridisation visualised a moderate to high upregulation of hTR in the melanoma cells but not in the admixed reactive cells. There was no correlation among tumour location, cell type, or growth fraction and the amount of telomerase activity. In addition, the cells of the germinative zone of the lens demonstrated a strong hTR expression. CONCLUSION Telomerase activity is upregulated in uveal melanomas. The expression of hTR was located to the tumour cells and not the reactive tumour infiltrating cells. Strong telomerase expression was also demonstrated in cells of the germinative zone of the lens.

Hodgkin and Reed–Sternberg cells of classical Hodgkin's disease overexpress the telomerase RNA template (hTR)

There is accumulating evidence to suggest that Hodgkin and Reed–Sternberg (HRS) cells represent the malignant cell population in Hodgkins disease (HD). A recent report that HD tissue is in most instances devoid of telomerase activity was therefore unexpected. Since telomerase activity was determined in whole tissue extracts and HRS cells comprise only a small minority of the cells in the affected tissue, the telomerase activity of the HRS cells might have escaped detection. To test this possibility and to clarify whether HRS cells contain the enzyme telomerase, 13 cases of classical HD were analysed by three different methods. The presence of telomerase was studied at the single cell level by a sensitive radioactive in situ hybridization method employing a probe specific for the telomerase RNA template (hTR). In addition, tissue extracts were studied for telomerase activity by a modified TRAP assay and for hTR by reverse transcription‐polymerase chain reaction (RT‐PCR). The extractive methods revealed telomerase activity in eight and hTR in all of the 13 HD cases studied. In situ hybridization located large amounts of hTR in the HRS cells of all 13 HD cases and low to medium amounts in some of the non‐malignant lymphoid bystander cells. These results indicate that HRS cells constitutively overexpress telomerase and thus use this enzyme for stabilizing their telomeres. This substantiates the malignant nature of HRS cells. Furthermore, the results confirm that normal lymphoid cells can express telomerase. In consequence, methods of measuring telomerase in tissue extracts are not suitable for determining the presence of this molecule in lymphoma cells, since the vast majority of lymphoid neoplasms contain significant amounts of non‐neoplastic lymphoid cells. Copyright

Prognostic value of nuclear survivin-expression in esophageal squamous cell carcinoma

Survivin, a new member of the family of apoptosis inhibitors, is expressed almost exclusively in proliferating cells, above all in cancers. Subcellular localisation and prognostic implications of the survivin protein have not yet been determined in oesophageal squamous cell carcinoma. The survival of 84 patients with oesophageal squamous cell carcinomas was correlated with the extent of immunohistochemical survivin expression in tumour cell nuclei. Tumours were scored positive when 45% cells stained positive. Patients were followed up for at least 5 years or until death. In normal oesophageal squamous cell epithelium, some cytoplasmic survivin expression was detected in the basal cells, whereas proliferating cells showed nuclear staining of survivin. Nuclear expression of survivin was also detected in 67 cancers (80%). The mean survival for patients of this group (28 months, range 20 – 36) was significantly less than that for patients without survivin expression in the tumour cell nuclei (108 months, range 62 – 154, P ¼ 0.003). Using univariate analysis, nuclear survivin expression (P ¼ 0.003), tumour depth (P ¼ 0.001), lymph node metastasis (P ¼ 0.003) and stage (Po0.001) were the best predictors of survival. In contrast, cytoplasmic survivin staining was noted in 53 (63%) tumours and had no prognostic relevance. In conclusion, the analysis of nuclear survivin expression identifies subgroups in oesophageal squamous cell cancer with favourable (survivin � ) or with poor prognosis (survivin + ). We suggest that the determination of nuclear survivin expression could be used to individualise therapeutic strategies in oesophageal squamous cell cancer in the future.

A 62‐YEAR‐OLD MAN WITH CHRONIC PYOTHORAX

June 2005. Pyothorax-associated lymphoma (PAL) is a rare B-cell non-Hodgkin lymphoma (B-NHL) which develops in the pleural cavity after a 20- to 64-year history of chronic pyothorax. We present here the case of a 62-year-old man who suffered from chronic pyothorax after pneumectomy 44 years ago, and complained of progressive ataxia. A MRI of the head revealed a solitary lesion in the vermis cerebelli, and a biopsy showed a lymphoma displaying immunoblastic features. Immunohistochemistry revealed an aberrant dual B/T phenotype and an Epstein-Barr virus (EBV) type III LMP-1+/EBNA-2+ latency profile. In-situ hybridization disclosed EBV-encoded RNAs in the tumor cells. PCR for the detection of rearranged immunoglobulin heavy chain (IgH) genes followed by GeneScan analysis demonstrated a clonal B-cell population with DNA amplificates of identical size in the brain manifestation, and a large mediastinal tumor analyzed post mortem. Among the largest series of 106 PALs collected through a nationwide survey in Japan, central nervous system (CNS) involvement was detected in 5 (14%) of 36 patients where an autopsy had been performed. To best of our knowledge, this is the first case of a pyothorax-associated lymphoma initially diagnosed on brain biopsy.