Fernando Rodrigues

Thyroglobulin detection in fine-needle aspirates of cervical lymph nodes: a technique for the diagnosis of metastatic differentiated thyroid cancer

BACKGROUND Fine-needle aspiration cytology is frequently used for differential diagnosis of neck masses of unknown origin. Inconclusive and even false-negative results are not uncommon. AIM To evaluate the utility of thyroglobulin (Tg) measurement in fine-needle aspirates (FNA-Tg) for detecting cervical lymph node (CLNs) metastases from differentiated thyroid carcinomas. METHODS An ultrasound-guided fine-needle aspiration was done in 67 patients with 83 suspicious enlarged CLNs to obtain material for cytology and Tg measurement in the needle washout, using an immunometric chemiluminescent assay. Measurement of anti-Tg antibodies (FNA-TgAb) was also carried out in half of all the aspirates. Subjects were divided into two groups: one of 16 patients awaiting thyroidectomy and the other of 51 patients in follow-up after surgery. RESULTS The first group of patients had positive FNA biopsy (FNAB-Tg) in 14 out of the 18 studied CLNs with a range of 3.2-43 352 ng/ml, while FNAB-cytology indicated metastasis in only 8 out of the 14 CLNs with positive histology. A total of 65 CLNs were studied in the follow-up group. Lymphadenectomy was performed in 23 patients and 28 aspirated CLNs were removed. Histology confirmed the diagnosis of metastasis suggested by FNAB-Tg in 20 CLNs and of reactive lymphadenitis in the remaining 8 CLNs. FNAB-cytology was positive in only 11 CLNs. Sensitivity of FNAB-Tg was not affected by the studied FNAB-TgAb. CONCLUSIONS The FNAB-Tg achieved a sensitivity of 100% in both groups. FNAB-Tg is an easy and inexpensive technique which proved to increase the diagnostic of cytology in the early diagnosis of papillary carcinoma recurrence to CLN even in the presence of serum TgAb.

Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells

The low-density lipoprotein receptor-related protein (LRP1B), encoding an endocytic LDL-family receptor, is among the 10 most significantly deleted genes across 3312 human cancer specimens. However, currently the apparently crucial role of this lipoprotein receptor in carcinogenesis is not clear. Here we show that LRP1B inactivation (by chromosomal, epigenetic and microRNA (miR)-mediated mechanisms) results in changes to the tumor environment that confer cancer cells an increased growth and invasive capacity. LRP1B displays frequent DNA copy number loss and CpG island methylation, resulting in mRNA underexpression. By using CpG island reporters methylated in vitro, we found that DNA methylation disrupts a functional binding site for the histone-acetyltransferase p300 located at intron 1. We identified and validated an miR targeting LRP1B (miR-548a-5p), which is overexpressed in cancer cell lines as a result of 8q22 DNA gains. Restoration of LRP1B impaired in vitro and in vivo tumor growth, inhibited cell invasion and led to a reduction of matrix metalloproteinase 2 in the extracellular medium. We emphasized the role of an endocytic receptor acting as a tumor suppressor by modulating the extracellular environment composition in a way that constrains the invasive behavior of the cancer cells.

Loss of heterozygosity at 19p13.2 and 2q21 in tumours from familial clusters of non-medullary thyroid carcinoma

Linkage studies have identified susceptibility loci for familial nonmedullary thyroid cancer (FNMTC), with and without cell oxyphilia, at chromosomal regions 19p13.2 and 2q21. There are few genetic analyses of FNMTC tumours reported at the present time and the eventual gene involved was not identified yet. The aim of this study was to assess the occurrence of loss of heterozygosity (LOH) at these loci in the tumours from familial clusters of NMTC. We have analysed LOH in 14 tumours from 9 two-case familial clusters of NMTC. Using paired blood (normal) and tumour DNA samples, we have genotyped ten microsatellite and one SNP markers throughout 19p13.2 and fourteen microsatellite markers at 2q21. Overall, eight (57%) and two (14%) out of the fourteen tumours analysed exhibited LOH at 19p13.2 and 2q21, respectively. In two families (22%), LOH for the same markers was demonstrable in the tumours of the two members of the same family. In one family (11%) LOH was demonstrable at both loci analysed. In four two-case familial clusters (44%), LOH at the 19p13.2 locus was found in only one of the tumour cases analysed. Detailed haplotype analysis showed that, in two families (22%), the pattern of LOH in tumours was consistent with selective retention of the haplotype shared by affected members. In the remaining cases, it was consistent with random allelic losses. In conclusion, we report the finding of LOH at the 19p13.2 and 2q21 loci in tumours from familial clusters of NMTC, providing evidence that inactivation of putative genes in these regions, acting as tumour-suppressors, may be involved in the development of tumours in the context of FNMTC.

Occurrence of the Cys611Tyr mutation and a novel Arg886Trp substitution in the RET proto‐oncogene in multiple endocrine neoplasia type 2 families and sporadic medullary thyroid carcinoma cases originating from the central region of Portugal

Objective  Medullary thyroid carcinoma (MTC) occurs both sporadically and in the context of autosomal dominantly inherited multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC), which are caused by activating germline mutations in the RET proto‐oncogene. The aim of this study was to characterize the RET mutational spectrum in MEN2 families and apparently sporadic MTC (AS‐MTC) cases originating from the central region of Portugal.

mTOR activation in medullary thyroid carcinoma with RAS mutation

OBJECTIVE Rearranged during transfection (RET) mutations are well-known genetic events in sporadic and familial medullary thyroid carcinoma (FMTC). The presence of RAS mutations in sporadic cases, challenging the RET paradigm in these tumors, has been recently reported. We intend to evaluate mTOR pathway activation in RET- and RAS-mutated MTC. MATERIALS AND METHODS In this study, we analysed the presence of RET, H-RAS, and K-RAS mutations in a series of 87 MTCs (82 apparently sporadic and five FMTCs; five apparently sporadic MTCs were eventually found to be familial). We also evaluated mTOR activation--using the expression of its downstream effector phospho-S6 ribosomal protein (p-S6) and the expression of the mTOR inhibitor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN)--by immunohistochemistry. RESULTS Our results revealed that RET mutations were present in 52.9% of the cases (46/87) and RAS mutations in 12.6% (11/87) of the whole series of MTCs and 14.3% of the 77 sporadic MTCs. The presence of RET and RAS mutations was mutually exclusive. RAS mutations were significantly associated with higher intensity of p-S6 expression (P=0.007), suggesting that the mTOR pathway is activated in such MTCs. We observed also an increased expression of p-S6 in invasive tumors (P=0.042) and in MTCs with lymph node metastases (P=0.046). Cytoplasmic PTEN expression was detected in 58.8% of the cases; cases WT for RAS showed a significantly lower expression of PTEN (P=0.045). CONCLUSIONS We confirmed the presence of RAS mutation in 14.3% of sporadic MTCs and report, for the first time, an association between such mutations and the activation of the mTOR pathway. The evaluation of the mTOR activation by pS6 expression may serve as an indicator of invasive MTC.

Genetic polymorphism of CYP2D6 influences susceptibility to papillary thyroid cancer

Objective  Xenobiotic‐metabolizing enzymes are widely polymorphic and confer interindividual variation in the ability to detoxify carcinogens or to activate pro‐carcinogens. A common polymorphism of cytochrome P450 2D6 (CYP2D6) results in lack of enzyme activity and has been associated with an altered susceptibility to several cancers. The aim of this study was to investigate the association between the CYP2D6 poor metaboliser genotype and the risk of papillary thyroid cancer (PTC).

Combined GSTM1 and GSTT1 null genotypes are associated with a lower risk of papillary thyroid cancer

Individual susceptibility to cancer is influenced by polymorphisms of genes encoding drug-metabolizing enzymes such as the glutathione S-transferases (GST). The null polymorphisms of the GSTM1 and GSTT1 genes have been associated to a modified risk of several cancers but studies of thyroid cancer have produced conflicting results. The aim of this study was to investigate the relationship between these polymorphisms and the risk of papillary thyroid cancer (PTC). A total of 188 patients with PTC and 247 controls were genotyped using a PCR-based assay. Odds ratios (OR) and 95% confidence intervals (CI) for each homozygous null genotype were determined. The frequency of each of the GSTM1 and GSTT1 null genotypes did not differ significantly between patients and controls (OR=0.83, 95%CI: 0.56–1.21; p=0.328; and OR=0.66, 95%CI: 0.39–1.12; p=0.123, respectively), but the frequency of individuals that had the combined GSTM1 null/GSTT1 null genotypes was significantly lower in the patient group (OR=0.50, 95%CI: 0.26–0.97; p=0.040). The GSTM1 null genotype was associated with a lower risk of advanced cancer stages (III/IV) (OR=0.50, 95%CI: 0.26–0.96; p=0.036) and the GSTT1 null genotype was associated with a lower risk of the follicular variant of PTC (OR=0.31, 95%CI: 0.10–0.97; p=0.044). These results suggest that GSTM1 and GSTT1 null genotypes are weak, yet possible, modifiers of the risk of PTC. This protective effect may be due to a role of the GSTM1 and GSTT1 encoded enzymes in the metabolic activation of putative thyroid carcinogens or in other pathways involved in thyroid carcinogenesis.

How to Treat a Signal? Current Basis for RET-Genotype-Oriented Choice of Kinase Inhibitors for the Treatment of Medullary Thyroid Cancer

The significance of RET in thyroid cancer comes from solid evidence that, when inherited, an RET activating mutation primes C-cells to transform into medullary carcinomas. Moreover, environmental exposure to radiation also induces rearranged transforming RET “isoforms” that are found in papillary thyroid cancer. The RET gene codes for a tyrosine kinase receptor that targets a diverse set of intracellular signaling pathways. The nature of RET point mutations predicts differences in the mechanisms by which the receptor becomes activated and correlates with different forms of clinical presentation, age of onset, and biological aggressiveness. A number of RET-targeting Tyrosine Kinase Inhibitors (TKIs) are currently undergoing clinical trials to evaluate their effectiveness in the treatment of thyroid cancer, and it is conceivable that the RET genotype may also influence response to these compounds. The question that now emerges is whether, in the future, the rational for treatment of refractory thyroid cancer will be based on the management of an abnormal RET signal. In this paper we address the RET-targeting TKIs and review studies about the signaling properties of distinct RET mutants as a means to predict response and design combinatorial therapies for the soon to be available TKIs.

In vitro transforming potential, intracellular signaling properties and sensitivity to a kinase inhibitor (sorafenib) of RET proto-oncogene variants Glu511Lys, Ser649Leu and Arg886Trp

Multiple endocrine neoplasia type 2 and a subset of apparently sporadic medullary thyroid carcinoma (AS-MTC) are caused by germ line activating point mutations of the rearranged during transfection (RET) proto-oncogene. RET encodes a receptor with tyrosine kinase activity that targets several intracellular signaling cascades, such as RAS-RAF-ERK1/2, PIK3-AKT, and STAT transcription factors. The objective of this study was to assess the function of three germ line RET variants Arg886Trp, Ser649Leu, and Glu511Lys of undetermined pathogenic significance, which were found in three kindreds of isolated AS-MTC. For this purpose, we employed vectors expressing each of the RET variants and measured the number of NIH3T3 transformation foci and soft agar colonies, the degree of activation of known RET intracellular signaling targets (ERK1/2, STAT1, STAT3, and TCF4), and the extent of ERK1/2 inhibition on sorafenib treatment. We found that RET variants Arg886Trp and Glu511Lys have shown increased in vitro transforming potential in a glial-derived neurotrophic factor-dependent manner. In contrast, the Ser649Leu variant did not significantly increased the number of foci and agar colonies relative to wild-type RET (RET-WT). The variants Glu511Lys and Arg886Trp showed 10- and 12.5-fold ERK1/2 activation respectively, that was significantly higher than that observed for RET-WT (fivefold). Increased levels of STAT1 and TCF4 activation were only observed for RET Arg886Trp (2.5- and 3-fold versus 1.2- and 2-fold in RET-WT respectively). The three RET variants analyzed here were sensitive to treatment with sorafenib. In conclusion, our results allow to classify previously uncharacterized RET genotypes, which may be of use to define follow-up and therapeutic regimens.

Encuesta sobre el tratamiento del carcinoma diferenciado de tiroides

INTRODUCTION In January 2005, during the annual meeting of the Portuguese Society of Endocrinology, Diabetes and Metabolism, a questionnaire on the treatment and follow-up of differentiated thyroid carcinoma (DTC) was given to attendants. The aim of this study was to present the surveys results. METHODS The questionnaire addressed the following issues: the surgical treatment of the gland and cervical lymph nodes, whole body scan and ablation with (131)I, suppression with levothyroxine, and treatment of recurrence and metastases. Fifty-four completed questionnaires were obtained (79% from clinical endocrinologists). RESULTS When DTC is diagnosed, 67% of respondents reported that total thyroidectomy is always performed. When the diagnosis is made postsurgically, completion of thyroidectomy is recommended by 70% of respondents for papillary carcinoma, by 67% for papillary microcarcinoma and by 44% for minimally invasive follicular carcinoma. Most respondents recommend lymph node dissection if the nodes are involved; 61% systematically perform whole body scan with (131)I after surgery. Twenty-eight percent routinely perform ablation of the thyroid, and 59% request adjuvant radioiodine ablation of the thyroid bed if there is (131)I uptake, if thyroglobulin is increased, or if risk factors are present. The most commonly used ablation dose is 100 mCi. Consensus on the degree of TSH suppression is lacking. Twenty-two percent of the respondents recommend surgery as the first therapeutic option in recurrence and metastases, while 57% prefer (131)I for the treatment of local recurrence. When thyroglobulin levels remain high and the results of (131)I scanning are negative, 50% choose computed tomography scan for the diagnosis of disease recurrence. CONCLUSIONS The wide variability of responses in this survey and the significant percentage (11 to 41%) of non-responders demonstrates the lack of uniformity in the treatment protocols for DCT in Portugal. According to the published guidelines and the responses to a similar survey performed in Spain, the widest differences are mainly found in lymph node dissection and the treatment of disease recurrence.Introduccion En enero de 2005, durante la reunion anual de la Sociedad Portuguesa de Endocrinologia, Diabetes y Metabolismo, se entrego a los medicos presentes una encuesta sobre el modo de actuacion en el tratamiento y el seguimiento del carcinoma diferenciado de tiroides. Es objetivo de este trabajo dar a conocer los resultados de esa encuesta. Metodos El cuestionario versaba esencialmente sobre los siguientes puntos: tratamiento quirurgico de la glandula y cadenas ganglionares, gammagrafia y ablacion con 131I, supresion con levotiroxina, tratamiento de las recidivas y metastasis. Se obtuvieron 54 respuestas (el 79% de los endocrinologos). Resultados Ante el diagnostico de carcinoma diferenciado de tiroides la tiroidectomia total es siempre preferida por el 67% de los encuestados. La totalizacion, en caso de diagnostico posquirurgico, es recomendada por el 70% en el carcinoma papilar, por el 67% en el microcarcinoma papilar y por el 44% en el folicular con invasion minima. La gran mayoria recomienda vaciamiento ganglionar si los ganglios estan afectados; el 61% realiza sistematicamente gammagrafia con 131I tras la cirugia; el 28% de los interrogados hacen ablacion de restos tumorales, ademas del 59% que la hacen si hay captacion de 131I en el lecho tiroideo, si la tiroglobulina esta aumentada o si hay factores de riesgo; 100 mCi es la dosis mas usada. No hubo unanimidad en relacion con el grado de supresion de tirotropina. En caso de recidiva y metastasis, apenas el 22% de los encuestados refiere la cirugia como primera opcion terapeutica y el 57% prefiere el 131I en el tratamiento de la recidiva local. Mas del 50% escogio la tomografia computarizada para el diagnostico de recidiva de enfermedad en los casos en que la tiroglobulina permanece elevada y la gammagrafia corporal con 131I fuese negativa. Conclusiones La gran variabilidad de respuestas en este cuestionario y un porcentaje significativo (11-41%) de no respondedores evidencian la falta de uniformidad de los protocolos de tratamiento del carcinoma de tiroides en Portugal. Segun las guias publicadas y las respuestas a un cuestionario semejante realizado en Espana, las grandes diferencias de actuacion surgen principalmente en la cuestion del vaciamiento ganglionar y en el tratamiento de la recidiva de la enfermedad.