BACKGROUND.In patients with cancer, one of the main mechanism of resistance to antimetabolite drugs is related to higher levels of thymidylate synthase (TS) activity.METHODS.To investigate the association between TS expression and histopathologic data, 56 resection specimens from patients with nonsmall cell lung carcinoma (NSCLC) were collected consecutively. TS messenger RNA (mRNA) was evaluated in tumor specimens by using real‐time polymerase chain reaction (PCR) analysis; protein expression was evaluated by using immunohistochemistry (IHC) in formalin‐fixed, paraffin‐embedded (FFPE) specimens; and the analysis of TS transcriptional regulation activity was performed by using real‐time PCR analysis in snap‐frozen normal and tumor specimens.RESULTS.The amplification of the TS gene from FFPE tissues was obtained from all samples, with a median level (unit‐less ratio) of 1.45 (range, 0.34–5.24); whereas positive TS status at IHC (>10% positive cells) was detected in 56% of samples. It is noteworthy that TS expression was significantly higher in squamous cell carcinoma compared with adenocarcinoma when both mRNA levels (2.17 vs. 1.16; P < .0001) and protein levels (P = .0269) were considered in FFPE specimens, and a strong association was observed between mRNA and protein expression (P = .00017). Moreover, higher TS levels were observed in high‐grade tumors (P = .0389 and P = .0068 for mRNA and protein quantification, respectively). The analysis in snap‐frozen samples revealed that the TS gene was up‐regulated strongly in tumors (P = 3.8 × 10−12), and an 8‐fold increase (as a cut‐off value) in the TS mRNA ratio between tumor and corresponding normal tissue was detected in 32 of 56 patients (57%) bearing preferentially squamous cell tumors (P = .0022) and high‐grade tumors (P < .001).CONCLUSIONS.Data from the current study consistently indicated higher TS expression levels in squamous cell and in high‐grade carcinomas. This information may be useful in selecting which patients with NSCLC should receive treatment with TS‐inhibiting agents. Cancer 2006. © 2006 American Cancer Society.

The distinction of benign from malignant follicular thyroid neoplasms remains a difficult task in diagnostic fine-needle aspiration cytology, and some discrepant results have been reported for the individual immunocytochemical markers of malignancy proposed so far. The aim of this study was to test if the combined use of a panel of markers could improve the diagnostic accuracy in the preoperative cytological evaluation of 'follicular neoplasms' in an attempt to reduce the number of thyroidectomies performed for benign lesions. The immunocytochemical expression of galectin-3, HBME-1, thyroperoxidase, cytokeratin-19 and keratan-sulfate was retrospectively analyzed in 125 consecutive fine-needle aspiration samples (cell blocks) of indeterminate diagnoses of 'follicular thyroid neoplasm', and compared with their corresponding surgical specimens, including 33 follicular carcinomas, 42 papillary carcinomas and 50 follicular adenomas. Statistical analysis on each marker confirmed that galectin-3 and HBME-1 were the most sensitive (92% and 80% respectively) and specific (94% and 96% respectively) molecules. The use of these two markers sequentially in non-oncocytic lesions (testing HBME-1 as a second marker whenever galectin-3 proved negative) increased the sensitivity and specificity up to 97% and 95% respectively. In oncocytic lesions, HBME-1 proved to be less sensitive, and the sequential combination of galectin-3 and cytokeratin-19 reached 100% of both specificity and sensitivity. Our data showed that, as compared with the use of single markers, the sequential combination of two markers represents the most accurate immunohistochemical panel in managing patients with a fine-needle aspiration biopsy diagnosis of 'follicular neoplasms', especially in otherwise controversial categories such as oncocytic tumours. The combination of three or more markers did not substantially improve the diagnostic accuracy of the test.

Although preclinical work with rapalogs suggests potential in treatment of multiple myeloma (MM), they have been less successful clinically. These drugs allostearically inhibit the mammalian target of rapamycin kinase primarily curtailing activity of the target of rapamycin complex (TORC)1. To assess if the mammalian target of rapamycin within the TORC2 complex could be a better target in MM, we tested a new agent, pp242, which prevents activation of TORC2 as well as TORC1. Although comparable to rapamycin against phosphorylation of the TORC1 substrates p70S6kinase and 4E-BP-1, pp242 could also inhibit phosphorylation of AKT on serine 473, a TORC2 substrate, while rapamycin was ineffective. pp242 was also more effective than rapamycin in achieving cytoreduction and apoptosis in MM cells. In addition, pp242 was an effective agent against primary MM cells in vitro and growth of 8226 cells in mice. Knockdown of the TORC2 complex protein, rictor, was deleterious to MM cells further supporting TORC2 as the critical target for pp242. TORC2 activation was frequently identified in primary specimens by immunostaining for AKT phosphorylation on serine 473. Potential mechanisms of up-regulated TORC2 activity in MM were stimulation with interleukin-6 or insulin-like growth factor 1, and phosphatase and tensin homolog or RAS alterations. IntroductionPreclinical data with mammalian target of rapamycin (mTOR) inhibitors such as rapamycin, temsirolimus, and everolimus suggest these drugs may have therapeutic potential in multiple myeloma (MM). [1][2][3] These mTOR inhibitors associate with the FKBP12 protein and together they bind to mTOR adjacent to its kinase domain. At this site, rapamycin allostearically inhibits the kinase, primarily that which is functional within the multiprotein complex kinase called target of rapamycin complex (TORC)1. 4 The TORC1 complex consists of mTOR associated with mLST8 and Raptor. 4 TORC1 phoshorylates the p70S6kinase (p70) and factor 4E binding protein 1 (4E-BP1) translational repressor and both these events stimulate translation of cell cycle proteins, thus promoting cell cycle transit. [5][6][7] By inactivating TORC1, these first generation mTOR inhibitors prevent cell cycle protein translation and induce G1 arrest. 8 Although some early results of phase I/II trials that use these mTOR inhibitors in combination with other anti-MM agents suggest modest efficacy, 9,10 use of tensilorimus as a single agent was relatively ineffective in MM patients. 11 Some possible reasons for these disappointing results are suggested by previous mechanistic studies. In particular, treatment of MM cells with rapamycin or temsilorimus only induces cell cycle arrest without induction of apoptosis. 1 Thus, as treated MM cells maintain viability, they may resume tumor growth during the time intervals between drug administration. One potential reason for lack of apoptosis is that there is a feedback activation of AKT when MM cells are treated with mTOR inhibitors. 12 Activated AKT could serve as an antiapoptotic pr...

Thyroid nodules are a common occurrence in the general population, but only a small number of them are eventually diagnosed as cancers. Fine-needle aspiration biopsy (FNAB) is the most accurate and cost-effective method for the presurgical management of thyroid nodules, but it misses the differential diagnosis between thyroid follicular adenomas and follicular carcinomas. Among them, minimally invasive follicular carcinoma (MIC), also defined as encapsulated tumor, only differs from follicular adenoma for the exhibition of minimal, but entire thickness, infiltration of the capsule and/or vascular invasion. This feature cannot be assessed in FNAB and can occasionally be hard to recognize in surgical specimens. As reported in several studies, galectin-3 is a reliable marker of thyroid malignancy, but no data are available on MICs. We analyzed the immunohistochemical expression of galectin-3 in 17 MICs and 52 follicular adenomas in both preoperative paraffin-embedded cytological human thyroid sediments (cell blocks) obtained by FNAB and in the corresponding surgical specimens. Among the MICs, all surgical samples showed galectin-3 immunoreactivity in the cytoplasm, whereas 16 of 17 corresponding FNAB cell blocks were positive. No evidence of cytoplasmic galectin-3 expression was observed in 48 of 52 adenomas in both cell blocks and histological tissues. These findings indicate that galectin-3 is a reliable presurgical molecular marker of MIC, improving the accuracy of conventional FNAB. It also proves to be useful in the histopathological assessment of resected tumors having suspected malignant features.

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