Sticchi 12_9

Telomerase was found in cancers and immortalized cell lines, but only occasionally in normal tissues, thus suggesting that measurement of its hTERT subunit might help distinguishing benign from malignant tumors. Data on hTERT expression in adrenocortical tumors are scant and mostly confined to non-functioning tumors. Therefore, we investigated whether hTERT expression may predict malignancy in aldosterone producing adrenocortical tumors. We measured hTERT mRNA with a real-time one-step reverse transcription (RT) polymerase chain reaction (PCR) method, based on the use of hTERT-specific fluorescence resonance energy transfer (FRET) probes, in 19 adrenalectomized patients with aldosterone-producing adenomas (APAs), in whom longterm follow-up (median 7 years, range 5-14 years) data were available. We also studied two rare aldosterone-producing carcinomas (APCs), eight adrenocortical carcinomas (ACs), twelve normal adrenal cortexes, and two malignant cell lines (NCI-295H and SW-13). Telomerase activity and hTERT immunoreactivity were also investigated. Of interest, we detected hTERT mRNA in 58% of APAs at levels similar to those of malignant tumors, which all consistently showed hTERT expression. In hTERT expressing tumors, immunocytochemistry showed the nuclear expression of hTERT. No hTERT expression was found in the normal adrenocortical tissue. No histopathology differences were observed between hTERT-positive and -negative APAs; however, a patient originally held to have an hTERT-positive APA was retrospectively classified as APC because of metastatic spread. In conclusion, RT-PCR measurement of hTERT mRNA is a hallmark of malignant adrenocortical tumors, but identifies also a subset of hTERT-expressing APAs that might show metastatic spread at long-term follow-up. Introduction Telomeres, the repeated sequences at the chromosome ends, undergo shortening on continuous cell proliferation (1). Since they stabilize chromosomes and prevent DNA degradation, their progressive shortening with life is thought to provide a signal for cellular senescence. Telomerase (hTERT) is a ribonucleoprotein complex that catalyzes the addition of telomeric repeats to the 3'-end of chromosome DNA, thereby preventing the loss of telomeric sequences at each cell division (2). Albeit variously distributed in adult somatic cells, telomerase activity was found usually low in normal differentiated tissues (3). By contrast, hTERT activity is clearly detectable in cancer cell lines (5), and in most human cancer tissues (4), suggesting its usefulness as a marker of human cancers and index of poor prognosis (5-12). Adrenocortical tumors are highly prevalent in the general population (13,14); nonetheless, the differentiation of malignant from benign neoplasms is often difficult (15), not only on clinical but even on histopathology ground. Several criteria have been claimed to allow discrimination between these tumors (16,17), including some molecular markers as the DNA index (18,19) the expression of the proliferating cell antigen (20), the p53 protein (21), the adrenal 4 binding protein (22), the c-Myc protein (18), or the insulin-like growth factor II gene (23). Unfortunately, to date none of these markers has gained wide acceptance because of poor accuracy. The hTERT activity has been demonstrated in adrenal tumors, including few adrenocortical carcinomas (ACs) (24-27), but data are limited (27,28). Investigators used either the traditional semiquantitative TRAP (telomerase repeat amplification protocol) assay, or a quantitative determination of hTERT activity assay. These methods do not lend themselves to routine use on the tiny amount of tissue available through fine-needle biopsy, which is occasionally used to differentiate benign from malignant adrenocortical tumors. The technological development in molecular analysis techniques have allowed to investigate the expression of hTERT with unprecedented sensitivity and accuracy. In the present study we, therefore, exploited the use of a novel commercially available quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR) method based on fluorescence resonance energy transfer (FRET) technology INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 17: 469-474, 2006 469 Expression of telomerase (hTERT) in aldosterone-producing adrenocortical tumors DANIELE STICCHI1, AMBROGIO FASSINA2, CHIARA GANZAROLI1, CRISTIANA PASQUALETTO1, ACHILLE CESARE PESSINA1, GASTONE G. NUSSDORFER3 and GIAN PAOLO ROSSI1 Departments of 1Clinical and Experimental Medicine, 2Pathology, and 3Human Anatomy and Physiology, Section of Anatomy, University of Padua, I-35121 Padua, Italy Received September 12, 2005; Accepted October 24, 2005 _________________________________________ Correspondence to: Professor Gian Paolo Rossi, Department of Clinical and Experimental Medicine, Clinica Medica 4 University Hospital, via Giustiniani 2, I-35126 Padua, Italy E-mail: [email protected]