Manuela Motta

Detection of RET/PTC, TRK and BRAF mutations in preoperative diagnosis of thyroid nodules with indeterminate cytological findings

Background  Fine‐needle aspiration biopsy (FNAB) is the primary means to distinguish benign from malignant nodules and select patients for surgery. However, adjunctive diagnostic tests are needed because in 20–40% of cases the FNAB result is uncertain.

Sorafenib blocks tumour growth, angiogenesis and metastatic potential in preclinical models of osteosarcoma through a mechanism potentially involving the inhibition of ERK1/2, MCL-1 and ezrin pathways

BackgroundOsteosarcoma (OS) is the most common primary bone tumour in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory.ResultsWe investigated phospho-ERK 1/2, MCL-1, and phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets in OS. Activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS whereas BRAF gene was found to be mutated in 4/30 OS samples from patients. Based on these results we tested the multi-kinase inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell line proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, and P-ERM in a dose-dependent manner. The dephosphorylation of ERM was not due to ERK inhibition. The downregulation of MCL-1 led to an increase in apoptosis in OS cell lines. In chick embryo chorioallantoic membranes, OS supernatants induced angiogenesis, which was blocked by sorafenib and it was also shown that sorafenib reduced VEGF and MMP2 production. In addition, sorafenib treatment dramatically reduced tumour volume of OS xenografts and lung metastasis in SCID mice.ConclusionIn conclusion, ERK1/2, MCL-1 and ERM pathways are shown to be active in OS. Sorafenib is able to inhibit their signal transduction, both in vitro and in vivo, displaying anti-tumoural activity, anti-angiogenic effects, and reducing metastatic colony formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients unresponsive to standard treatments.

Combined analysis of galectin-3 and BRAFV600E improves the accuracy of fine-needle aspiration biopsy with cytological findings suspicious for papillary thyroid carcinoma

Ten to fifteen percent of fine-needle aspiration biopsy (FNAB) of thyroid nodules are indeterminate. Galectin-3 (Gal-3) and the oncogene BRAFV600E are markers of malignancy useful to improve FNAB accuracy. The objective of this study was to determine whether the combined analysis of Gal-3 and BRAFV600E expression in thyroid aspirates could improve the diagnosis in FNAB with suspicious cytological findings. Two hundred and sixty-one surgical thyroid tissues and one hundred and forty-four thyroid aspirates were analyzed for the presence of the two markers. In surgical specimens, Gal-3 expression was present in 27.4% benign nodules, 91.9% papillary (PTC) and 75% follicular (FTC) thyroid carcinomas. BRAFV600E was not detected in 127 benign nodules, as well as in 32 FTCs, while was found in 42.9% PTC. No correlation was found between BRAF mutation and Gal-3 expression. Forty-seven consecutive FNAB suspicious for PTC were analyzed for the presence of the two markers. Of these nodules, 23 were benign at histology, 6 were positive for Gal-3, none displayed BRAFV600E, and 17 were negative for both the markers. Twenty suspicious nodules were diagnosed as PTC and four FTCs at histology. Of these 24 carcinomas, 9 resulted positive for BRAFV600E, 17 for Gal-3, and 22 for one or both the markers. The sensitivity, specificity, and accuracy for the presence of Gal-3 and/or BRAFV600E were significantly higher than those obtained for the two markers alone. Notably, the negative predictive value increased from 70.8 to 89.5%. In conclusion, the combined detection of Gal-3 and BRAFV600E improves the diagnosis in FNAB with cytological findings suspicious for PTC and finds clinical application in selected cases.

FINE-NEEDLE ASPIRATION BIOPSY OF THE THYROID: COMPARISON BETWEEN THYROID PALPATION AND ULTRASONOGRAPHY

OBJECTIVE To describe our experience with fine-needle aspiration biopsy (FNAB) of the thyroid and compare our results with direct palpation versus ultrasound scanning (USS) in an area of endemic goiter in Italy. METHODS We considered all patients submitted to ultrasound-guided FNAB of thyroid nodules during a 10-month period at our outpatient clinic and analyzed the following: (1) clinical data (number of nodules and identification of the nodule for FNAB); (2) USS data (number of nodules and identification of the nodule for FNAB on the basis of hypoechoic pattern + blurred perinodal halo + microcalcifications or intranodal color Doppler signal indicative of blood flow); (3) cytologic specimens, categorized as suspicious, malignant, negative, or nondiagnostic; and (4) histologic final report of the cytologically positive nodules. RESULTS The study group consisted of 348 female and 72 male patients who underwent FNAB of the thyroid at our institution. Among the 140 patients with no palpable thyroid nodules, USS showed that 106 had a single nodule and 34 had multinodular goiters. Among the 182 patients with a single palpable thyroid nodule, USS revealed that 138 had a single nodule, 42 had a multinodular goiter, and 2 had lobe enlargement without detectable nodules. All 98 patients with multinodular palpable goiter had a similar pattern on USS. Of the 420 cytologic specimens, 46(11.0%) were positive for thyroid cancer, 313 (74.5%)were negative, and 61 (14.5%) were nondiagnostic. Histologic malignant growth was confirmed in 27 cytologically positive nodules. Of these histologically malignant nodules, 12 (45%) were nonpalpable, 9 (33%) were single palpable nodules, and 6 (22%) were from a nodule with a suspicious ultrasound pattern within a multinodular goiter. CONCLUSION Manually guided FNAB is not feasible in nonpalpable nodules and not accurate in a multinodular goiter. Both situations are clinical challenges, and USS should be performed for accurate FNAB under these circumstances. Because 52% of histologically malignant nodules in our study were found only with the aid of ultrasound-guided FNAB, this procedure should be used where multinodular goiter is endemic. Our overall rate of nondiagnostic specimens was comparable to that reported in the literature.

High Diagnostic Accuracy and Interobserver Reliability of Real-Time Elastography in the Evaluation of Thyroid Nodules

Elastography is a new diagnostic tool in the evaluation of thyroid nodules. Aim of the study was to evaluate the accuracy and reliability of elastography in discriminating thyroid lesions and the interobserver variability. One hundred thirty-two nodules in 115 patients selected for thyroid surgery underwent conventional ultrasound and elastographic evaluation. Elastography score was divided in four categories (totally elastic nodule, mainly elastic, mainly rigid and totally rigid) according to signal distribution. Three independent operators conducted the study. Final histology showed 92 benign nodules and 40 malignant. On elastography, 77/92 benign nodules were classified as score 1 or 2 and 34/40 malignant nodules as score 3 or 4 (sensitivity 85%, specificity 83.7%, positive predictive value [PPV] 69.3%, negative predictive value [NPV] 92.7%). Rate of concordance between operators was good (K test: 0.64, p < 0.0001). Simple to use, with good interobserver agreement, elastography has all the requisites to become an important complement of conventional US examination in the near future.

Expression of Cox-2 in human breast cancer cells as a critical determinant of epithelial-to-mesenchymal transition and invasiveness

Introduction: Cyclooxygenase-2 (COX-2) is overexpressed in several malignancies and is implicated in breast cancer progression. Objectives: We investigated whether changes in COX-2 expression may affect epithelial-to-mesenchymal transition (EMT) and then invasive potential of human breast cancer cells, in relationship with hypoxia. COX-2-null MCF-7 human breast cancer cells, MCF-7 cells transiently expressing COX-2 and COX-2-expressing MDA-MB-231 cells were employed. Results: COX-2 overexpression resulted in downregulation of E-cadherin and β-catenin, upregulation of vimentin, N-cadherin and SNAI1, suggesting EMT occurrence. COX-2-overexpressing MCF-7 cells were also characterized by increased invasiveness and release of matrix-metalloproteinase-9. The above-mentioned characteristics, homologous to those detected in highly invasive MDA-MB-231 cells, were reverted by treatment of COX-2-overexpressing MCF-7 cells with celecoxib, a COX-2-specific inhibitor, partly through the inhibition of COX-2-related intracellular generation of reactive oxygen species. Hypoxia further exacerbated COX-2 expression, EMT changes and invasive ability in both COX-2-overexpressing MCF-7 cells and MDA-MB-231 cells. Finally, immunohistochemistry performed on samples from normal and neoplastic human breast tissues revealed that COX-2-positive malignant cells were also positive for EMT-related antigens, hypoxia-inducible factor (HIF)-2α and the oxidative stress marker heme oxygenase. Conclusions: These findings support the existence of a direct link between COX-2 overexpression, EMT and invasiveness in human breast cancer cells, emphasizing the role of hypoxic microenvironment.

High Growth Rate of Benign Thyroid Nodules Bearing RET/PTC Rearrangements

CONTEXT Benign thyroid nodules display a broad range of behaviors from a stationary size to a progressive growth. The RET/PTC oncogene has been documented in a fraction of benign thyroid nodules, besides papillary thyroid carcinomas, and it might therefore influence their growth. OBJECTIVE The aim of the present work was to evaluate whether RET/PTC in benign thyroid nodules associates with a different nodular growth rate. STUDY DESIGN In this prospective multicentric study, 125 subjects with benign nodules were included. RET rearrangements were analyzed in cytology samples; clinical and ultrasonographic nodule characteristics were assessed at the start and at the end of the study. RESULTS RET/PTC was present in 19 nodules. The difference between the mean baseline nodular volume of the RET/PTC- and RET/PTC+ nodules was not significant. After 36 months of follow-up, the RET/PTC+ group (n = 16) reached a volume higher than the RET/PTC- group (n = 90) (5.04 ± 2.67 vs. 3.04 ± 2.26 ml; P = 0.0028). We calculated the monthly change of nodule volumes as a percentage of baseline. After a mean follow-up of 36.6 months, the monthly volume increase of nodules bearing a RET rearrangement was 4.3-fold that of nodules with wild-type RET (1.83 ± 1.2 vs. 0.43 ± 1.0% of baseline volume; P < 0.0001). CONCLUSIONS Benign thyroid nodules bearing RET rearrangements grow more rapidly than those with wild-type RET. Searching for RET rearrangements in benign thyroid nodules might be useful to the clinician in choosing the more appropriate and timely therapeutic option.

Intertubular Capillary Changes in the Cortex and Medulla of Transplanted Kidneys and Their Relationship with Transplant Glomerulopathy: An Ultrastructural Study of 12 Transplantectomies

Twelve kidney allografts removed 3 to 98 months (mean, 44.8 months) after transplantation were investigated. The presence and severity of intertubular capillary changes, which were characterized by splitting and multilayering of the basement membrane, were carefully noted. These changes were graded as mild, moderate, and severe according to the number of basement membrane layers. They were found in both cortical and medullary capillaries in all but one kidney and were always associated with transplant glomerulopathy. Ultrastructural changes observed in the glomeruli and capillaries were similar, suggesting that they share the same pathogenetic mechanisms, probably connected to immune-mediated endothelial cell damage. Although glomeruli can be absent in small biopsy specimens, intertubular capillaries are easily detectable. Because capillary changes can be considered a marker of transplant glomerulopathy, which is known to have ominous prognostic significance, the identification of such changes acquires clinical relevance. Electron microscopic investigation is therefore strongly advisable whenever a renal biopsy is performed to identify transplant glomerulopathy.

COX-2 expression in human breast carcinomas: correlation with clinicopathological features and prognostic molecular markers.

Objective: COX-2 is implicated in carcinogenesis and tumour progression in many cancers, including breast cancer. Recently, it has been reported that human breast carcinomas aberrantly express COX-2, and that raised tissue levels of COX-2 may have prognostic value. Patients expressing high levels of COX-2 can develop local recurrence, and have reduced disease-free and disease-related overall survival. The aim of this study was to investigate COX-2 expression in human ductal and lobular breast cancers and its possible association with clinicopathological features and prognostic molecular markers. Research design and methods: Cytoplasmic COX-2 expression was detected by means of immunohistochemistry in a series of 91 breast carcinomas with ductal (n = 60) and lobular (n = 31) patterns. COX-2 expression was investigated by multivariate analyses and compared with clinicopathological features. Results and conclusions: COX-2 immune positivity and percentage of positive cells correlated significantly with the size, grading, extent of primary tumour and vascular invasion of carcinoma but not with biological parameters (estrogen receptor, progesterone receptor and human EGF receptor 2). The findings of the present study suggest that COX-2 overexpression in lobular and ductal breast cancers, which correlates with traditional clinico-pathological parameters, may be considered as a negative prognostic marker.

THYROID CYTOLOGY AND RISK OF THYROID CANCER: DIFFERENCES AMONG INDETERMINATE SPECIMENS

OBJECTIVE To assess the potential for stratification of indeterminate cytologic findings on fine-needle aspiration (FNA) of thyroid nodules in an effort to improve therapeutic strategies. METHODS We attempted to determine the malignant risk associated with various indeterminate FNA cytologic patterns by correlation of specimens with the final histologic diagnosis. For this analysis, we identified 294 computerized medical records of surgically treated thyroid nodules during a 5-year period at our institution with the corresponding FNA cytology reports available. RESULTS Of the 294 surgical cases, 162 with a positive or indeterminate cytologic report were selected, reviewed, and classified. Of 52 patients with positive cytologic findings on FNA, 51 (98%) had a final histologic report of a malignant thyroid nodule. Of 110 patients with indeterminate specimens, 30 (27%) had a final histologic diagnosis of thyroid carcinoma. The presence of nuclear atypia was predictive of thyroid carcinoma in 75% of patients, a Hürthle cell cytologic pattern was associated with a malignant thyroid nodule in 33%, and a hypercellular smear was suggestive of malignant involvement in 26% of cases. The lowest rate of malignant potential was associated with cytologic microfollicular and scant colloid alone subtype (6%). CONCLUSION The results of this study show that indeterminate thyroid cytologic specimens can be subdivided into groups with different malignant risks. A microfollicular cytologic pattern in the absence of a hypercellular smear or nuclear atypia does not support a recommendation of surgical treatment. A malignant cytologic diagnosis has a high positive predictive value for detection of thyroid cancer.