ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence.

Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22(phox) that produces H(2)O(2). Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H(2)O(2), we detected an increase in H(2)O(2) in the nucleus correlated with NOX4-p22(phox) perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1ß foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence.

Do histological, immunohistochemical, and metabolic (radioiodine and fluorodeoxyglucose uptakes) patterns of metastatic thyroid cancer correlate with patient outcome?

The aim of this study is to search for relationships between histology, radioiodine ((131)I) uptake, fluorodeoxyglucose (FDG) uptake, and disease outcome in patients with metastatic thyroid cancer. Eighty patients with metastatic thyroid cancer (34 males, 46 females, mean age at the time of the diagnosis of metastases: 55 years) were retrospectively studied. All patients were treated with radioactive iodine and evaluated by FDG-positron emission tomography (PET). Primary tumor tissue sample was available in all cases. Forty-five patients (56%) had a papillary, 12 (15%) a follicular, and 23 (29%) a poorly differentiated thyroid cancer. Cellular atypias, necrosis, mitoses, thyroid capsule infiltration, and vascular invasion were frequently detected (70, 44, 52, 60, and 71% respectively). Metastases disclosed FDG uptake in 58 patients (72%) and (131)I uptake in 37 patients (45%). FDG uptake was the only significant prognostic factor for survival (P=0.02). The maximum standardized uptake value and the number of FDG avid lesions were also related to prognosis (P=0.03 and 0.009). Age at the time of the diagnosis of metastases (P=0.001) and the presence of necrosis (P=0.002) were independent predictive factors of FDG uptake. Radioiodine uptake was prognostic for stable disease (P=0.001) and necrosis for progressive disease at 1 year (P=0.001). Histological subtype was not correlated with in vivo tumor metabolism and prognosis. In conclusion, FDG uptake in metastatic thyroid cancer is highly prognostic for survival. Histological subtype alone does not correlate with (131)I/FDG uptake pattern and patient outcome. Well-differentiated thyroid cancer presenting histological features such as necrosis and FDG uptake on PET scan should be considered aggressive differentiated cancers.

Adrenocortical carcinoma: is the surgical approach a risk factor of peritoneal carcinomatosis?

CONTEXT: Peritoneal carcinomatosis (PC) is a rare site of distant metastases in patients with adrenocortical cancer (ACC). One preliminary study suggests an increased risk of PC after laparoscopic adrenalectomy (LA) for ACC. OBJECTIVE: The objective of the study was to search for risk factors of PC including surgical approach. DESIGN: This was a retrospective cohort study conducted in an institutional practice. PATIENTS: Sixty-four consecutive patients with ACC seen at our institution between 2003 and 2009 were included. Mean tumor size was 132 mm. Patients had stage I disease in 2 cases, stage II disease in 32 cases, stage III disease in 7 cases, stage IV disease in 21 cases, and unknown stage disease in 2 cases. Surgery was open in 58 cases and laparoscopic in 6 cases. MAIN OUTCOME: The main outcome was the risk factors of PC. RESULTS: PC occurred in 18 (28%) patients. It was present at initial diagnosis in three cases and occurred during follow-up in 15 cases. The only risk factor of PC occurring during follow-up was the surgical approach with a 4-year rate of PC of 67% (95% confidence interval (CI), 30-90%) for LA and 27% (95% CI, 15-44%) for open adrenalectomy (P=0.016). Neither tumor size, stage, functional status, completeness of surgery, nor plasma level of op'DDD was associated with the occurrence of PC. CONCLUSION: We found an increased risk of PC after LA for ACC. Whether this is related to an inappropriate surgical approach or to insufficient experience in ACC surgery should be clarified by a prospective program.

Long-term outcome of 444 patients with distant metastases from papillary and follicular thyroid carcinoma: benefits and limits of radioiodine therapy.

AIM: The goal of this study was to estimate the cumulative activity of (131)I to be administered to patients with distant metastases from thyroid carcinoma. METHODS: A total of 444 patients were treated from 1953-1994 for distant metastases from papillary and follicular thyroid carcinoma: 223 had lung metastases only, 115 had bone metastases only, 82 had both lung and bone metastases, and 24 had metastases at other sites. Treatment consisted of the administration of 3.7 GBq (100 mCi) (131)I after withdrawal of thyroid hormone treatment, every 3-9 months during the first 2 yr and then once a year until the disappearance of any metastatic uptake. Thyroxine treatment was given at suppressive doses between (131)I treatment courses. RESULTS: Negative imaging studies (negative total body (131)I scans and conventional radiographs) were attained in 43% of the 295 patients with (131)I uptake; more frequently in those who were younger, had well-differentiated tumors, and had a limited extent of disease. Most negative studies (96%) were obtained after the administration of 3.7-22 GBq (100-600 mCi). Almost half of negative studies were obtained more than 5 yr after the initiation of the treatment of metastases. Among patients who achieved a negative study, only 7% experienced a subsequent tumor recurrence. Overall survival at 10 yr after initiation of (131)I treatment was 92% in patients who achieved a negative study and 19% in those who did not. CONCLUSION: (131)I treatment is highly effective in younger patients with (131)I uptake and with small metastases. They should be treated until the disappearance of any uptake or until a cumulative activity of 22 GBq has been administered. In the other patients, other treatment modalities should be used when tumor progression has been documented.

Diagnostic and prognostic value of 18-fluorodeoxyglucose positron emission tomography in adrenocortical carcinoma: a prospective comparison with computed tomography.

OBJECTIVE: Patients with adrenocortical cancer are submitted to multiple imaging procedures for diagnosis of recurrence and staging. The aim of this prospective study was to evaluate the diagnostic and prognostic values of fluorodeoxyglucose (FDG) using a combined positron emission tomography and computed tomography (PET/CT) modality, compared with thoracoabdominopelvic computed tomography (TAP-CT). METHODS: Twenty-eight consecutive patients with adrenocortical cancer referred from November 2003 to December 2004 to the Institut Gustave Roussy were included. Mean time between PET/CT and TAP-CT was 16 d. Independent readers analyzed images of each modality. The gold standard was progression on follow-up TAP-CT or pathology. RESULTS: A total of 269 lesions in 57 organs were depicted in 22 patients. The sensitivities for the detection of distinct lesions and the diagnosis of metastatic organs were 90 and 93% for PET/CT and 88 and 82% for TAP-CT, respectively. Twelve percent of the lesions were seen on PET/CT only and 10% on TAP-CT only. Eighteen percent of the metastatic organs were diagnosed with PET/CT only and 7% with TAP-CT only. Thirty-eight percent of the local relapses were seen only with PET/CT. PET/CT depicted three false-positive lesions. Treatment modalities were modified by PET/CT findings in five cases among which one was falsely positive. Tumor size and mitotic rate were significantly associated with FDG uptake. The intensity of FDG uptake (maximum standardized uptake value > 10) and the volume of FDG uptake (>150 ml) were significant prognostic factors for survival. CONCLUSIONS: We show that FDG-PET/CT is complementary to TAP-CT and of special interest in the diagnosis of local relapses.

Human galectin-3 immunoexpression in thyroid follicular adenomas with cell atypia.

Human galectin-3 (hgal-3) is a beta-galactoside binding protein involved in a number of physiological and pathological processes. Increasing hgal-3 immunoexpression has been reported in several human tumors, including thyroid carcinomas, but not in benign thyroid lesions. We analyzed the immunolocalization of hgal-3 in cell compartments of benign and malignant thyroid lesions. Hgal-3 immunoperoxidase reaction was carried out on 133 thyroid tissue samples obtained from 113 patients; 20 of these were normal (NT), 85 were benign thyroid lesions [20 colloid nodules (CN), 21 nodular hyperplasias (NH), 7 focal lymphocytic thyroiditis (FLT), 15 Hashimoto's thyroiditis (HT), 22 follicular adenomas (FA)], 25 differentiated carcinomas [15 papillary carcinomas (PC), 6 follicular carcinomas (FC) and 4 Hürthle cell carcinomas (HC)] and 3 anaplastic carcinomas (AC). Among the malignant thyroid lesions, hgal-3 was detected in 12/15 (80%) PC, 3/4 (75%) HC and in 4/6 (66.6%) FC, but in none of the 3 AC. Conversely, hgal-3 immunoexpression was absent in NT and in all benign thyroid lesions, but 1/15 HT and 10/22 (45.4%) FA. In the latter, hgal-3 was mostly expressed in microfollicular areas and in five of the six atypical FA. Hgal-3 cytoplasmic-perinuclear immunolocalization was observed in the majority of thyroid carcinomas and in more than half of the FA, theoretically suggesting an involvement of this protein in thyroid tumorigenesis throughout an antiapoptotic activity. Moreover, hgal-3 expression in FA might anticipate the likelihood of evolution of these benign lesions towards malignancy.

Differential expression of galectin 3 in solid cell nests and C cells of human thyroid.

AIMS: To provide new insights into characterising solid cell nests and gain information that might help distinguish between solid cell nests and C cells. METHODS: Thyroid tissue specimens from patients who had undergone prophylactic thyroidectomy for familial medullary thyroid cancer were immunostained for calcitonin, carcinoembryonic antigen, and galectin 3. RESULTS: Solid cell nests displayed a strong and diffuse staining for carcinoembryonic antigen and galectin 3, but not for calcitonin. C cells located at the periphery of solid cell nests and in neighbouring follicles expressed both calcitonin and carcinoembryonic antigen but not galectin 3. These three markers were positive in medullary thyroid carcinoma. CONCLUSION: Galectin 3 immunoreactivity permits a better characterisation and differentiation between solid cell nests and C cells, avoiding the misidentification of two biologically and clinically different thyroid structures.

Association of RET codon 691 polymorphism in radiation-induced human thyroid tumours with C-cell hyperplasia in peritumoural tissue.

The RET proto-oncogene encodes a protein structurally related to transmembrane receptors with an intracellular tyrosine kinase domain. In human thyroid gland, the RET proto-oncogene is normally expressed in parafollicular C-cells. Thyroid C-cell hyperplasia is associated with inherited medullary thyroid carcinomas and is considered as a pre-neoplastic stage of C-cells disease. It has also been observed in thyroid tissues adjacent to follicular and papillary carcinomas. In order to study the relationship between a misfunctioning of the RET proto-oncogene and the presence of C-cell hyperplasia, we compared a series of thyroid glands presenting sporadic or radiation-associated tumours, as well as samples of unrelated normal thyroid tissues, for alteration in exons 10 and 11 of the gene and for the presence or absence of C-cell hyperplasia. Here we report a significantly higher frequency of C-cell hyperplasia present in peritumoural thyroid tissues of radiation-induced epithelial thyroid tumours, than in peritumoural of sporadic thyroid tumours or in control normal thyroid tissues (P=0.001). A G691S RET polymorphism was present with a higher frequency in radiation-induced epithelial thyroid tumours (55%) than in sporadic tumours (20%) and in control normal thyroid tissues (15%). Interestingly, this polymorphism was associated in the majority (88%) of radiation-induced tumours with a C-cell hyperplasia in the peritumoural tissues. Several explanations for this association are discussed.

Medullary thyroid carcinoma as part of a multiple endocrine neoplasia type 2B syndrome: influence of the stage on the clinical course.

BACKGROUND: Multiple endocrine neoplasia type 2B (MEN 2B) is an exceptional syndrome, for which the optimal age of thyroidectomy is poorly established and the course of medullary thyroid carcinoma (MTC) is ill-defined. PATIENTS: All the 18 patients with a MEN 2B syndrome examined at the Institut Gustave Roussy were included in a single-center retrospective study. RESULTS: There were 9 men and 9 women with a mean age of 13 years (range, 2-27 years) at diagnosis. The diagnosis of MTC was based on the presence of a thyroid nodule or involved neck lymph nodes and on dysmorphic features of MEN 2B in 60% and 40% of the cases, respectively. The classic M918T mutation in exon 16 was found in the 16 patients in whom it was investigated. At diagnosis, 2 patients had Stage I MTC, 15 patients had Stage III, and 1 patient had Stage IV disease. T1 MTC was found in 4 patients aged 2.1-3.7 years. However, two of these patients already had N1 disease. One patient with Stage I MTC, aged 3.4 years and 2 patients with Stage III disease, aged 14 and 25 years, had undetectable basal calcitonin (CT) after initial surgery. During follow-up, basal CT became detectable in one of three patients. Among the 15 other patients with an elevated postoperative CT level, metastases were demonstrated in 5 patients after a mean follow-up of 2 years. Five patients died, three of MTC, one of the MEN 2B syndrome, and one of intercurrent disease. Five- and 10-year overall survival rates were 85% and 75%, respectively. CONCLUSIONS: This study confirms the need for early treatment of MTC in patients with the MEN 2B syndrome, preferably within the first 6 months of life. The phenotype of MTC occurring in the MEN 2B syndrome was not more aggressive than sporadic MTC or MTC occurring in other familial syndromes.

Sodium iodide symporter and pendrin expression in human thyroid tissues.

Thyroid cells synthesize thyroid hormones through a multistep process during which iodide is transported through the basolateral and the apical membranes of thyrocytes. Two genes that participate in these transports and the corresponding proteins, namely sodium iodide symporter (NIS) and pendrin, the product of the Pendred syndrome gene, have recently been characterized. We studied NIS and pendrin expression at the mRNA and protein levels by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method and by single and double immunostaining in normal and pathological human thyroid tissues. In normal tissue, NIS and pendrin were detected in about 20% and 40%-60% of thyrocytes, respectively. The number of NIS- and pendrin-positive cells was much higher in hyperfunctioning tissue from Graves' disease or toxic adenoma. In hypofunctioning adenomas and carcinomas, the number of NIS- and pendrin-positive cells was low or nonexistent. Three types of follicular cells were observed in positive tissues: NIS-negative/pendrin-negative cells, NIS-positive/pendrin-positive cells, and NIS-negative/pendrin-positive cells. The first two types of cells appear to be resting and active cells, respectively, but the functional status of NIS-negative/pendrin-positive thyrocytes remains to be determined.

Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase (ThoX, LNOX, Duox) genes and proteins in human thyroid tissues.

The large homolog of NADPH oxidase flavoprotein LNOX2, and probably LNOX1, are flavoproteins involved in the thyroid H(2)O(2) generator. Western blot analysis of membrane proteins from normal human thyroid, using antipeptide antibodies, indicated that LNOX1,2 are 164-kDa glycoproteins and that N-glycosylated motifs account for at least 10-20 kDa of their total apparent molecular mass. Northern blot analysis of 23 different human tissues demonstrated that LNOX2 messenger RNA (mRNA) is strongly expressed only in the thyroid gland, although blast analysis of expressed sequence tags databases indicated that LNOX genes are also expressed in some nonthyroid cells. We investigated LNOX1,2 gene and protein expressions in normal and pathological human thyroid tissues using real-time kinetic quantitative PCR and antipeptide antibodies, respectively. In normal tissue, LNOX1,2 are localized at the apical pole of thyrocytes. Immunostaining for LNOX1,2 was heterogeneous, inside a given follicle, with 40-60% of positive follicular cells. Among normal and pathological tissues, variations of LNOX1 and LNOX2 mRNA levels were parallel, suggesting a similar regulation of both gene expressions. Whereas LNOX mRNAs seemed slightly affected in benign disease, the expression of protein was highly variable. In multinodular goiters, 40-60% of cells were stained. In hypofunctioning adenomas, LNOX immunostaining was highly variable among follicles, whereas sodium/iodide (Na+/I-) symporter immunostaining was decreased. In hyperfunctioning thyroid tissues, only few cells (0-10%) were weakly stained, whereas sodium/iodide symporter staining was found in the majority of follicular cells. In conclusion, LNOX proteins are new apical glycoproteins with a regulation of expression that differs from other thyroid markers.

Na(+)/I(-) symporter and Pendred syndrome gene and protein expressions in human extra-thyroidal tissues.

OBJECTIVE: The expression of two recently identified iodide transporters, namely the sodium/iodide symporter (NIS) and pendrin, the product of the gene responsible for the Pendred syndrome (PDS), was studied in a series of various extra-thyroidal human tissues, and especially in those known to concentrate iodide. METHODS: To this end, we used real-time kinetic quantitative PCR to detect NIS and PDS transcripts and immunohistochemistry for the analysis of their protein products. RESULTS: NIS gene and protein expression was detected in most tissues known to concentrate iodine, and particularly in salivary glands and stomach. In contrast, PDS gene expression was restricted to a few tissues, such as kidney and Sertoli cells. Interestingly, in kidney, pendrin immunostaining was detected at the apical pole of epithelial cells of the thick ascending limb of the Henle's loop and of the distal convoluted tubule. CONCLUSION: This study provides new insights on the localization and expression of two genes involved in iodide transport and emphasizes the interest of combining real-time quantitative PCR and immunohistochemistry for the comparison of gene and protein expression in tissues.

Expression of nicotinamide adenine dinucleotide phosphate oxidase flavoprotein DUOX genes and proteins in human papillary and follicular thyroid carcinomas.

Duox2, and probably Duox1 are glycoflavoproteins involved in the thyroid H2O2 generator functionally associated to thyroperoxidase (TPO). We investigated both DUOX1 and DUOX2 gene expressions using quantitative reverse transcription-polymerase chain reaction (RT-PCR) in 47 thyroid carcinomas, including 10 paired normal/tumoral tissues. In carcinomas, variations of DUOX1 and DUOX2 mRNA levels were parallel, indicating that control mechanisms of both gene expressions operate in tumors as well as in normal thyroid tissues; DUOX1 expression was in the normal range in 20, was decreased up to 50-fold in 8, and increased up to 7-fold in 19 samples. DUOX2 expression was in the normal range in 15, was decreased up to 200-fold in 10, and increased up to 5-fold in 22 samples. In the 10 paired samples, variations of DUOX and TPO gene expressions were not correlated. We analyzed Duoxl/2 protein expression in 86 tumor samples using an antipeptide antiserum reacting with both Duox proteins. In normal tissue, Duox proteins are localized at the apical pole of thyrocytes, with 40% to 60% of thyrocytes being stained. In the 86 cancer tissues, immunostaining was absent in 19 samples, was low in 32, and normal or even slightly increased in the other 35 samples. The expression of Duox proteins was related to tumor differentiation, being more frequently found in neoplastic tissues that were able to pick up radioiodine, and in those with a detectable expression of sodium iodide symporter (NIS), pendrin and TPO.

Expression of Na+/I- symporter and Pendred syndrome genes in trophoblast cells.

Placental iodide transport is critical for the fetal thyroid function, but the molecular mechanisms of this transport are not understood. The expression of two recently identified iodide transporters, namely the sodium/iodide symporter (NIS) and pendrin, the product of the gene responsible for the Pendred syndrome (PDS), was studied using real-time kinetic quantitative PCR and immunohistochemistry 1) in placental tissues collected at different gestational ages and 2) in primary cultures of villous cytotrophoblast cells (VCT) that differentiate and fuse over 2-3 days in vitro to form villous syncytiotrophoblast (VSCT) cells. Both NIS and PDS genes are expressed in placenta, albeit at low levels compared with those in thyroid tissue. NIS gene expression in placental samples from first trimester and term pregnancies was similar. In contrast, the expression of PDS gene was higher in term than in first trimester pregnancy samples. In vitro, NIS gene was expressed at a high level in VCT obtained from first trimester pregnancy, and its expression decreased by 3- to 4-fold during the differentiation of VCT in VSCT. Expression of NIS was lower (up to 30-fold) in VCT obtained in placental samples from third trimester than from first trimester pregnancy. In contrast, the expression of PDS gene was low in VCT and increased by 5- to 10-fold during VSCT formation; this was observed in cells isolated from placental samples of both first trimester and term pregnancies. Immunohistochemical analysis showed that NIS protein was present on the entire membrane of VCT, whereas pendrin was mainly located at the brush border membrane of VSCT, facing the mother. In conclusion, 1) NIS and PDS genes are differently expressed in the placenta during gestation; and 2) whereas pendrin is expressed at the brush border membrane of syncytiotrophoblast cells, NIS protein is mainly located in the cytotrophoblast layer.

[Radiation-induced thyroid cancers]. / Cancers thyroïdiens radio-induits.

Human epithelial thyroid radiation-induced tumorigenesis is the most frequent radiation-induced tumorigenic process in man. Results of different studies, concerning the molecular mecanism(s) of epithelial thyroid radiation-associated tumorigenesis show : 1) that there is not a significant difference in the frequency of activation of ras, gsp and trk proto-oncogenes between radiation-associated and <> thyroid tumors; 2) the relevant role played by RET/PTC ret proto-oncogene activating rearrangements, in the development of radiation-associated thyroid tumors originated after therapeutic radiation (mainly PTC 1) or the atomic accident of Chernobyl (mainly PTC 3) and 3) suggest that the patients who develop thyroid tumors after a history of irradiation, show a genomic instability consisting in a DNA repair defect.

Expression of pendrin and the Pendred syndrome (PDS) gene in human thyroid tissues.

The gene recently cloned that is responsible for the Pendred syndrome (PDS), an autosomal recessive disease characterized by goiter and congenital sensorineural deafness, is mainly expressed in the thyroid gland. Its product, designated pendrin, was shown to transport chloride and iodide. To investigate whether the PDS gene is altered during thyroid tumorigenesis, PDS gene expression and pendrin expression were studied using real-time kinetic quantitative PCR and antipeptide antibodies, respectively, in normal, benign, and malignant human thyroid tissues. The results were then compared to those observed for sodium/iodide symporter (NIS) expression. In normal tissue, pendrin is localized at the apical pole of thyrocytes, and this in contrast to the basolateral location of NIS. Immunostaining for pendrin was heterogeneous both inside and among follicles. In hyperfunctioning adenomas, the PDS messenger ribonucleic acid level was in the normal range, although immunohistochemical analysis showed strong staining in the majority of follicular cells. In hypofunctioning adenomas, mean PDS gene expression was similar to that detected in normal thyroid tissues, but pendrin immunostaining was highly variable. In thyroid carcinomas, PDS gene expression was dramatically decreased, and pendrin immunostaining was low and was positive only in rare tumor cells. This expression profile was similar to that observed for the NIS gene and its protein product. In conclusion, our study demonstrates that pendrin is located at the apical membrane of thyrocytes and that PDS gene expression is decreased in thyroid carcinomas.

Search for NTRK1 proto-oncogene rearrangements in human thyroid tumours originated after therapeutic radiation.

Rearrangements of NTRK1 proto-oncogene were detected in 'spontaneous' papillary thyroid carcinomas with a frequency varying from 5 to 25% in different studies. These rearrangements result in the formation of chimaeric genes composed of the tyrosine kinase domain of NTRK1 fused to 5' sequences of different genes. To investigate if the NTRK1 gene plays a role in radiation-induced thyroid carcinogenesis, we looked for the presence of NTRK1-activating rearrangements in 32 human thyroid tumours (16 follicular adenomas, 14 papillary carcinomas and two lymph-node metastases of papillary thyroid carcinomas) from patients who had received external radiation, using the reverse transcription polymerase chain reaction, Southern blot and direct sequencing techniques. These data were compared with those obtained in a series of 28 'spontaneous' benign and malignant thyroid tumours, collected from patients without a history of radiation exposure and four in vitro culture cell lines derived from 'spontaneous' thyroid cancers. Our results concerning the radiation-associated tumours showed that only rearrangements between NTRK1 and TPM3 genes (TRK oncogene) were detected in 2/14 papillary carcinomas and in one lymph-node metastasis of one of these papillary thyroid carcinomas. All the radiation-associated adenomas were negative. In the 'spontaneous' tumours, only one of the 14 papillary carcinomas and one of the four in vitro culture cell lines, derived from a papillary carcinoma, presented a NTRK1 rearrangement also with the TPM3 gene. Twenty-five of this series of radiation-associated tumours were previously studied for the ras and RET/PTC oncogenes. In conclusion, our data: (a) show that the overall frequency of NTRK1 rearrangements is similar between radiation-associated (2/31: 6%) and 'spontaneous' epithelial thyroid tumours (2/32: 6%). The frequency, if we consider exclusively the papillary carcinomas, is in both cases 12%; (b) show that the TRK oncogene plays a role in the development of a minority of radiation-associated papillary thyroid carcinomas but not in adenomas; and (c) confirm that RET/PTC rearrangements are the major genetic alteration associated with ionizing radiation-induced thyroid tumorigenesis.

Sodium/iodide symporter: a key transport system in thyroid cancer cell metabolism.

The recent cloning of the gene encoding the sodium/iodide symporter (NIS) has enabled better characterization of the molecular mechanisms underlying iodide transport, thus opening the way to clarifying its role in thyroid diseases. Several studies, at both the mRNA and the protein expression levels, have demonstrated that TSH, the primary regulator of iodide uptake, upregulates NIS gene expression and NIS protein abundance, both in vitro and in vivo. However, other factors, including iodide, retinoic acid, transforming growth factor-beta, interleukin-1alpha and tumour necrosis factor alpha, may participate in the regulation of NIS expression. Investigation of NIS mRNA expression in different thyroid tissues has revealed increased levels of expression in Graves' disease and toxic adenomas, whereas a reduction or loss of NIS transcript was detected in differentiated thyroid carcinomas, despite the expression of other specific thyroid markers. NIS mRNA was also detected in non-thyroid tissues able to concentrate radioiodine, including salivary glands, stomach, thymus and breast. The production of specific antibodies against the NIS has facilitated study of the expression of the symporter protein. Despite of the presence of high levels of human (h)NIS mRNA, normal thyroid glands exhibit a heterogeneous expression of NIS protein, limited to the basolateral membrane of the thyrocytes. By immunohistochemistry, staining of hNIS protein was stronger in Graves' and toxic adenomas and reduced in thyroid carcinomas. Measurement of iodide uptake by thyroid cancer cells is the cornerstone of the follow-up and treatment of patients with thyroid cancer. However, radioiodide uptake is found only in about 67% of patients with persistent or recurrent disease. Several studies have demonstrated a decrease in or a loss of NIS expression in primary human thyroid carcinomas, and immunohistochemical studies have confirmed this considerably decreased expression of the NIS protein in thyroid cancer tissues, suggesting that the low expression of NIS may represent an early abnormality in the pathway of thyroid cell transformation, rather than being a consequence of cancer progression. The relationship between radioiodine uptake and NIS expression by thyroid cancer cells require further study. New strategies, based on manipulation of NIS expression, to obtain NIS gene reactivation or for use as NIS gene therapy in the treatment of radiosensitive cancer, are also being investigated.

Immunohistochemical analysis of Na+/I- symporter distribution in human extra-thyroidal tissues.

131Iodine concentration has been described in several extra-thyroidal tissues. Recent evidence has shown that iodine uptake is achieved by the recently cloned human Na(+)/I(-) symporter (hNIS) gene. However, conflicting results were observed in the expression of hNIS transcripts in extra-thyroidal tissues. In order to document further the distribution of hNIS, we investigated its expression using an immunohistochemical method, based on a polyclonal antibody raised against a synthetic peptide. Various extra-thyroidal tissues were examined, particularly from the digestive tract. Our results confirm that the salivary glands and the stomach express hNIS protein significantly. In contrast, hNIS was undetectable in the colon but the rectal mucosa, which has never been examined, exhibited positive immunohistochemical staining. Other digestive tissues, including the oesophagus, small intestine and appendix, were negative. Weak staining was observed in the mammary gland, indicating that hNIS is expressed in this tissue. The pancreas, skin, ovaries, spleen and kidney showed no positive immunostaining.