Gudrun Demel

Expression of vascular endothelial growth factor in lymphomas and castleman's disease

Vascular endothelial growth factor (VEGF) is one of the main angiogenic cytokines in human solid tumours and inhibition of VEGF‐induced angiogenesis suppresses tumour growth. Some groups of malignant lymphoma, including peripheral T‐cell lymphomas and Hodgkins disease, are characterized by a conspicuous proliferation of small vessels. To test the hypothesis that VEGF may also be involved in the angiogenesis in lymphomas and other lesions of the lymphoid system, VEGF expression was analysed in tissues, employing in situ hybridization with a 35S‐labelled RNA probe specific for this cytokine. Significant expression of VEGF transcripts was observed in Hodgkins disease and peripheral T‐cell lymphomas, particularly of the angioimmunoblastic type. In contrast, expression of this cytokine was minimal or absent in follicle centre lymphoma and chronic lymphocytic leukemia of B‐cell type. VEGF was mainly observed in reactive non‐lymphoid CD68‐negative cells, which probably represent fibroblasts or myofibroblasts. In normal and ulcerated tonsils, VEGF was expressed in the squamous epithelium but only rarely found in the lymphoid tissue. Although infectious mononucleosis tonsils contained high numbers of VEGF‐positive cells in the interfollicular zone, expression of this cytokine was not found in Epstein–Barr virus (EBV)‐infected cells, as determined by simultaneous in situ hybridization for VEGF and EBV‐encoded small nuclear RNAs (EBER). In 5/8 cases of Castlemans disease, germinal centres containing small vessels also showed expression of VEGF, in contrast to normal tonsillar germinal centres which are devoid of both vessels and VEGF transcripts. It is concluded that VEGF may be involved in the induction of the angiogenesis of both peripheral T‐cell lymphomas and Hodgkins disease, but not in low‐grade B‐cell lymphomas. In contradistinction to solid tumours, in which this cytokine is commonly secreted by the tumour cells themselves, in malignant lymphoma VEGF is not a product of neoplastic cells. Vascularization of germinal centres in Castlemans disease may also be a consequence of abnormal local expression of VEGF.

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.

Expression of cytotoxic molecules in intestinal T-cell lymphomas

Intestinal T‐cell lymphoma (ITCL) represents a subgroup of peripheral T‐cell lymphomas which is thought to arise from αβ intraepithelial T‐lymphocytes. Since these lymphocytes may contain cytotoxic molecules, the question of whether this also holds true for ITCL arises. Twenty ITCL cases were examined for the presence of granzyme B, perforin, and T‐cell‐restricted intracellular antigen (TIA‐1)/granule membrane protein of 17 kD (GMP‐17). Two molecules with restricted expression in cytotoxic cells, granzyme B and perforin, were detected by immunocytochemistry and by in situ hybridization with an isotopically labelled RNA probe, respectively. Immunocytochemistry was also performed with the antibody 2G9, which recognizes two molecules, one expressed by cytotoxic cells (TIA‐1) and the other found in granulocytes and cytotoxic cells (GMP‐17). Granzyme B, TIA‐1/GMP‐17, and perforin were found in the neoplastic cells of 16/19 cases, 19/20 cases, and 16/17 cases, respectively, of ITCL, but not in the tumour cells of the control group, which consisted of intestinal B‐cell lymphomas (five cases) and CD8‐negative peripheral nodal T‐cell lymphomas (six cases). At least one of these molecules was expressed in the tumour cells of all ITCL cases. 2G9 proved to be the most sensitive immunohistological marker, since reactivity with this antibody was not only observed in the highest number of cases, but also found in high numbers of neoplastic cells in positive cases. In conclusion, ITCL appears to show cytotoxic differentiation in all cases. In conjunction with immunophenotypic and genotypic data, our results support a uniform derivation of this tumour from intraepithelial αβ cytotoxic T‐lymphocytes.

Uniform expression of cytotoxic molecules in anaplastic large cell lymphoma of null/T cell phenotype and in cell lines derived from anaplastic large cell lymphoma.

We recently provided ample evidence that anaplastic large cell lymphomas of T/null phenotype (T-/null-ALCL) genotypically represent peripheral T cell lymphomas which in up to 90% have a phenotype of cytotoxic cells with expression of granzyme B protein and perforin transcripts. However, the issue of granzyme B expression in T-/null-ALCL is still controversial due to differing results from another laboratory. To verify our earlier immunohistochemical stainings for granzyme B, we looked for granzyme B transcripts by in situ hybridization (ISH). In addition, we investigated our previously analyzed cases by immunohistology (IH) with another antibody (2G9), which reacts with two molecules known to be expressed in cytotoxic cells: T-cell-restricted intracellular antigen (TIA)-1 and granule membrane protein-17 (GMP-17). We also extended our studies to homogenous tumor cell populations provided by ALCL-derived cell lines. As evidenced by ISH, transcripts for perforin, TIA-1 and granzyme B were found in all ALCL-derived cell lines. Similarly, proteins of TIA-1/GMP-17, granzyme B and perforin were expressed in all of these lines as shown by IH. In biopsy specimens, TIA-1/GMP-17 were detected by IH in 14/16 cases of T-/null-ALCL, and granzyme B transcripts were found in 13/13 T-/null-ALCL cases, but not in 6 B-ALCL cases. The detection of granzyme B transcripts yielded results largely identical to those of IH for granzyme B protein, thus confirming our earlier data and suggesting that the regulation of the expression of this molecule largely occurs at the transcriptional level. Our data further confirm the almost uniform expression of cytotoxic molecules in both primary ALCL cases and ALCL-derived cell lines and therefore suggest that the derivation from cytotoxic T cells may be the unifying characteristic for T-/null-ALCL.

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