Paolo Miccoli

Introduction: CLM29, a pyrazolo[3,4-d]pyrimidine compound, inhibits several targets (including the RET tyrosine kinase, epidermal growth factor receptor, vascular endothelial growth factor receptor and has an anti-angiogenic effect). Recently it has been shown to inhibit proliferation and migration in primary papillary dedifferentiated thyroid cancer cells. The aim of this study is to evaluate the anti-tumor activity of CLM29 in medullary thyroid cancer (MTC). Methods/design: The CLM29 anti-proliferative and proapoptotic effects (5, 10, 30, 50 μmol/l) were tested in vitro in primary MTC (P-MTC) cells obtained at surgey, in TT cells harboring (C634W) RET mutation, and in human dermal microvascular endothelial cells (HMVEC-d). TT cells were injected in CD nu/nu mice which were treated with CLM29. Results: CLM29 (10 μmol/l, 30 μmol/l or 50 μmol/l) inhibited significantly (PPRET mutation. TT cells were injected s.c. in CD nu/nu mice and tumor masses became detectable between 20 and 30 days after xenotransplantation. CLM29 (50 mg/kg per die) inhibited significantly tumor growth and weight and the therapeutic effect was significant from the 48th day after cell implantation (18 days after the beginning of treatment). A significant reduction of Ki-67 immunostaining and of microvessel density was observed in the CLM29-treated tumors. Conclusion: The anti-tumor activity of a ‘pyrazolo[3,4-d]pyrimidine’ compound, CLM29, has been shown in MTC in vitro, and in vivo, opening the way to a future clinical evaluation.

The European Group generally agrees with the American guidelines on the issue of the indications for additional surgery in patients with recurrence of medullary thyroid cancer. The discussions have been focused mainly on the postoperative follow-up, where some European experts feel that a postoperative calcitonin-stimulating test is of some importance in assigning the patient to the "Cured" or "Non-cured" group immediately after surgery. A part of the European group feels that a negative calcitonin-stimulating test might lead to a less intensive follow-up in the late follow-up of these patients.

Measurement of thyroglobulin (Tg) protein in the washout of the needle used for fine needle aspiration biopsy cytology (FNAB-C) has been shown to increase the sensitivity of FNAB-C in identifying cervical lymph node (CLN) metastasis from well-differentiated thyroid cancer (TC). In this study, we evaluated whether routine measurement of Tg protein (FNAB-Tgp), Tg mRNA (FNAB-Tgm) and calcitonin (CT) mRNA (FNAB-CTm) in the FNAB washout of CLN increases the accuracy of FNAB-C in the diagnosis of suspicious metastatic CLN.

The applications of radioguided surgery, an approach to oncologic surgery involving a multidisciplinary team, are expanding at a rapid pace. The technique of radioguided occult lesion localization (ROLL) was originally introduced in the mid- 90s for applications in breast surgery, and later adapted also to other tumor lesions such as solitary pulmonary nodules (during either open or laparoscopic surgery) and colonic lesions. Concerning the latter, in particular, the technique called radioguided occult colonic lesion identification (ROCLI) consists of identifying, with the aid of intraoperative gammaprobe counts, small lesions that may escape colic intraoperative palpation, after prior tagging of the lesions performed endoscopically through peri- or intra-lesional injection of Technetium-99m-labeled human albumin macroaggregates (99mTc- MAA), a particulate radiopharmaceutical (25-100 μm) that does not migrate from the site of interstitial administration. Since September 2001, ROCLI has been employed in 12 patients, using a collimated gamma- probe measuring 11 mm in external diameter (Scintiprobe MR100 Pol.Hi.Tech.). All patients underwent preoperative colonoscopy in order to inject 0.2 mL of a 99mTc-MAA suspension (10-20 MBq) into the submucosa or intra-perilesionally; such tagging required only a few minutes. Eight of the 12 patients were then submitted to open laparotomy, while laparoscopic access was utilized in the remaining 4 patients. In all 12 patients, localization of the lesion with the ROCLI technique was technically feasible, safe, efficient and highly accurate, enabling quick detection of the lesion during surgery, with a 100% success rate. No complications occurred, and there was no risk of contamination by ionizing radiation.

In 1994, 21 Belarus children presenting papillary thyroid cancer (PTC) diagnosed after the Chernobyl disaster, and already submitted to subtotal surgery, underwent thyroid re-operation and post-operative radioiodine (131^I) therapy. All were re-evaluated after a 15-year follow-up, to evaluate the results of partial versus total thyroidectomy. Nineteen out of 21 children (mean age 9.2 years) had previously undergone a lobectomy. All cases underwent re-operation in 1994. Histology revealed a PTC in the residual lobe in three cases, three had lymph node metastases. After surgery, 20 patients underwent 131^I therapy. The post-131^I whole body scan was negative in seven cases, showed neck node metastases in five, lung metastases in three, multiple associated metastases in six. The follow-up was performed with rhTSH-stimulated serum thyroglobulin (Tg) evaluation and ultrasonography. Twenty patients showed Tg I therapy showed a thyroid remnant and a Tg of 32 ng/ml. Chi-square analysis showed significantly higher prevalences of residual cancer in the neck or lung, lymph node metastases, and re-operations (before completion) in patients who had undergone lobectomy than in those who had undergone completion thyroidectomy and 131^I therapy. The surgical complications after lobectomy were similar to those after completion thyroidectomy. A less-than-total thyroidectomy should not be indicated in patients with radiation-induced PTC, due to the high risk of residual cancer in the thyroid left in situ. The results of this study favor total thyroidectomy as the initial treatment for thyroid cancer in children exposed to fallout radiation.

The reported prevalence of RET somatic mutations in sporadic MTC is about 40-50% and the most frequent somatic RET mutation is Met918Thr in exon 16. MTC harboring a somatic RET mutation have been demonstrated to have both a more advanced stage at diagnosis and a worse outcome. Although RET mutations are believed to be driving events in the MTC tumorigenesis only the finding of somatic mutations in microMTC can confirm this hypothesis. Aim of the present work was to search for RET somatic mutations in sporadic microMTC (1 and < 2 cm; group C, > 2 and < 3 cm; group D, >3 cm. We found an overall prevalence of RET mutation of 18.24% (27/148) that was differently distributed in the four groups. In particular it was 4.6% (2/43) in group A, 12.5% (8/64) in group B, 40% (8/20) in group C and 42.8% (9/21) in group D, thus showing an increasing rate according to the increase of the tumor size. Furthermore, when comparing the prevalence of mutations in the four groups we found a high statistically lower prevalence in microMTC (p<0.0001). In conclusion these data indicate that: a) the prevalence of RET somatic mutations is lower than expected if particular attention is given in recruiting microMTC; b) the prevalence of RET somatic mutations is very low (4.5%) in microMTC suggesting that they are not an early event in MTC tumorigenesis. As an alternative to this hypothesis we have to suppose that microMTC could be caused by other oncogene(s) with a lower transforming activity. Nothing to Disclose: CR, BC, CU, VB, EM, LA, AT, FB, PM, AP, RE

Until now, to our knowledge, no data are present in literature about the regulation by cytokines (IFN-gamma and TNF-alpha) of chemokines (C-X-C motif) ligand 9 and 11 (CXCL9 and CXCL11) in primary cell cultures of papillary thyroid carcinoma (PTC), nor the effect of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activators on these chemokines, or the effect of these chemokines on PTC proliferation. The aims of this study were: a) to test the effect of IFN-gamma and TNF-alpha stimulation on the secretion of the CXC alpha-chemokines, CXCL9 and CXCL11, in primary cell cultures obtained from PTC, in comparison with normal thyroid tissue; b) to assess the effect of PPAR-gamma activation on CXCL9 and CXCL11 secretion and proliferation in these cell types; c) to evaluate the effect of CXCL9 and CXCL11 on proliferation and migration of PTC cells. In normal thyroid cells and PTC cells, CXCL9 and CXCL11 were absent basally; a similar dose-dependent secretion of CXCL9 and CXCL11 was induced by IFN-gamma in both cell types. TNF-alpha alone induced a significant CXCL9 and CXCL11 secretion only in PTC cells. The stimulation with IFN-gamma+TNF-alpha induced a synergistic CXCL9 and CXCL11 release in normal cells; a higher secretion was induced in PTC cells. Treatment of normal cells with the thiazolidinediones suppressed dose-dependently IFN-gamma+TNF-alpha-induced CXCL9 and CXCL11 release, while stimulated CXCL9 and CXCL11 secretion in PTC cells. A significant antiproliferative effect by thiazolidinediones was observed only in PTC cells. CXCL9 or CXCL11 (500 pg/mL) induced a significant (P<0.001) antiproliferative effect in PTC cells and inhibit migration of PTC cells, too. In conclusion, our study demonstrates that the treatment with IFN-gamma+TNF-alpha induces a marked release of CXCL9, and to a lesser extent of CXCL11, by primary cells from PTC. It has been shown a discrepancy between the stimulatory effect of thiazolidinediones on CXCL9 and CXCL11 secretion and the inhibitory role on PTCs proliferation. Furthermore, our study first shows that CXCL9 and CXCL11 are able to inhibit the proliferation and migration of primary PTC cells; the use of CXCL9 or CXCL11 as antineoplastic factors in PTC remains to be explored. Nothing to Disclose: AA, SMF, SF, MM, AC, CM, PF, PM