Calcitonin receptor expression in follicular cells-derived thyroid tumors and in medullary thyroid tumors: clinical implications

Background: Human calcitonin receptor (hCTR) is the only characterized receptor for calcitonin. It is a seven- transmembrane G-protein coupled receptor able to activate multiple intracellular signal transduction pathways, but mainly the c-AMP and PLC one. In humans, at least six different splice variants generated by alternative splicing of receptor gene localized on chromosome 7 have been described. Two are the most commonly and functionally expressed and they differ by an insert of 16 amino acids in the first intracellular loop: the hCTRC1b is characterized by the presence of the insert while the hCTRC1a is not. In adult tissues hCTR is expressed by neural networks, osteoclasts and osteocytes, renal distal epithelium, B and T-cells, testis, placenta, prostate and lung. Human CTR is also expressed by specific cell types in wound healing, cardiovascular disease and several types of diseased/malignant primary adult tissues such as breast, prostate and brain tumour, as well as in cell lines derived from lung, breast, brain, giant cell tumors of bone, osteoclast, prostate, lymphoid and myeloid neoplasms. The functions of hCTR in malignant tissues and cell lines are still not well known and are different according to the affected tissue. Regarding the thyroid gland, only few papers and data are available, both in normal thyroid and in tumoral one, represented only by medullary thyroid cancer (MTC). No data are available regarding follicular cells-derived thyroid cancer. Aims and methods: The aim of this research project were to: 1) identify and semi-quantify by immunohistochemistry the presence of hCTR protein in a series of follicular cells-derived tumor and of medullary thyroid tumor samples and their matched lymphnode metastases; 2) confirm and quantify by real-time PCR the hCTR mRNA expression in follicular cells-derived tumors frozen tissues samples, previously analyzed by immunohistochemistry, and in thyroid derived cell lines; 3) identify which hCTR isoform is expressed in our samples by using specific designed probe for real-time PCR; 4) correlate the hCTR expression score with clinicopathological and molecular features of samples. Results: hCTR expression was analyzed by immunohistochemistry, using a mouse monoclonal anti-human CTR antibody, in a series of 92 follicular cells-derived thyroid tumors, including 80 papillary thyroid cancers (PTC) and 12 follicular thyroid cancers (FTC), and of 138 MTCs and was positively detected in 74 cases (80.4%) of the first series and in 125 cases (90.6%) of the second one. Human CTR expression was significantly associated with less aggressive tumoral features: in PTC series with the absence of thyroid capsule invasion (p=0.007), tumors’ unifocality (p=0.03), the absence of psammoma bodies (p=0.05), the absence of lymphnode (p <0.0001) and distant metastases (p=0.001) and the cured final outcome of the disease (p=0.02); in MTC series with T1/T2 (p=0.03), N0 (p=0.002) and M0 stage (p=0.04), with a smaller tumor diameter (p=0.001), with the absence of thyroid capsular invasion (p=0.006) and of neoplastic vascular invasion (p=0.001). When we analyzed the hCTR immunoexpression in primary tumors and corresponding loco-regional lymphnode metastases in 18 PTC and 42 MTC cases, we observed 11/18 positive cases (61.1%) in the PTC group and all positive cases in MTCs. No statistically significant differences in clinical-pathological features were found between concordant and not concordant cases. Then, we analyzed the mRNA pattern of expression of all hCTR transcripts (hCTR total, hCTRC1b isoform, hCTRC1a isoform) in 46 PTC and 6 FTC cases, previously studied by immunohistochemistry: the hCTRC1b isoform was the most expressed in both papillary and follicular samples. In a subgroup of 7 PTC samples we could also study and compare the all transcripts expression levels both in primary tumors and adjacent thyroid tissue: only hCTRC1b isoform showed a almost significant higher expression levels in tumor samples compared to normal thyroid. Finally, the expression of all hCTR transcripts was also analyzed by real-time PCR in thyroid derived cell lines: cell lines 850C and TPC-1 were negative for all transcripts, while in K2 and C643 cell lines the hCTRC1b isoform seemed to be overexpressed compared to the “normal” thyroid cellular model (ORI cell line). Conclusions: the present study confirms that hCTR is expressed not only in MTCs but also in follicular cells derived thyroid tumors and that higher expression levels are associated with a more differentiated status and less aggressive clinical behavior. Moreover, for the first time we demonstrated that hCTR is expressed both in primary tumor tissues and loco-regional lymphnode metastases in PTCs and MTCs. We also demonstrated that both hCTR isoforms are expressed in normal and tumoral tissue, with higher expression levels in the malignant samples, and that only the isoform hCTRC1b is overexpressed in a tumoral context.