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Recent advances in human genetics have catalyzed the attention on Pendred's syndrome and its disease-gene, PDS. Studies on the expression of the PDS gene and on the function of its encoded protein, which has been named pendrin, are currently in progress. Consistent with the Pendred's syndrome phenotype, which is characterized by thyroid dysfunction associated to deafness, PDS expression has been demonstrated in the thyroid and in the inner ear. Despite its high homology to known sulfate transporters, pendrin has been shown to transport iodide and chloride, but not sulfate. Thus, it is probably devoted to regulate, at the apical membrane where it has been immunolocalized, the flux of iodide from the thyroid cell to the colloid space. The function of pendrin in the inner ear is not well understood, but it seems to function also at this level as an anion transporter. Indeed, a pronounced PDS expression has been detected in structures of the inner ear, such as the membranous labyrinth and the endolymphatic duct and sac. At this level, the possible role of pendrin could be the maintenance of the appropriate ionic composition of the endolymph. Although many questions remain to be answered, these recent achievements concerning the putative role of pendrin aid to better understand the genetic basis of the peculiar phenotype of Pendred's syndrome, which associate the dysfunction of two so different organs such as the thyroid and the inner ear.

Pendred's syndrome is a combination of congenital sensorineural hearing loss and iodine organification defect leading to a positive perchlorate test and goiter. Although it is the commonest form of syndromic hearing loss, the variable clinical presentation contributes to the difficulty in securing a diagnosis. The identification of the disease gene (PDS) prompts the need to reevaluate the syndrome to identify possible clues for the diagnosis. To this purpose, in three Italian families presenting with the clinical features of Pendred's syndrome, the molecular analysis was accompanied by full clinical, biochemical, and radiological examination. A correlation between genotype and phenotype was found in the only patient with enlargement of vestibular aqueduct and endolymphatic duct and sac at magnetic resonance imaging. This subject was a compound heterozygote for a deletion in PDS exon 10 (1197delT, FS400) and a novel insertion in exon 19 (2182-2183insG, Y728X). The present study demonstrates for the first time the value of the combination of clinical/radiological and genetic studies in the diagnosis of Pendred's syndrome. The positivity of a perchlorate discharge test and the malformations of membranous labyrinth fit well with the recent achievements on the role of pendrin in thyroid hormonogenesis and the maintenance of endolymph homeostasis.

The RET/PTC3 oncogene is an activated form of the RET protooncogene, which is frequently rearranged in papillary thyroid carcinoma. RET/PTC3 results from a structural rearrangement between the ELE1 and the RET genes, and it has been observed in both sporadic and radiation-associated post-Chernobyl tumors. To understand the molecular basis that predisposes RET and ELE1 genes to be recurrent targets of "illegitimate" recombination, we examined the genomic regions containing the ELE1/RET breakpoints of six sporadic and three post-Chernobyl tumors in two papillary carcinomas of different origins. Our data indicated, in both genes, a clustering of the breakpoints in regions designated ELE1-bcr (1.8 kb) and RET-bcr (1.9 kb). Notably, in all sporadic tumors and in one post-Chernobyl tumor the ELE1/RET recombination corresponded with short sequences of homology (3-7 nt) between the two rearranging genes. In addition, we observed an interesting distribution of the post-Chernobyl breakpoints in ELE1-bcr located within an Alu element, or in between two close Alu elements, and always in A+T-rich regions.

A post-Chernobyl papillary thyroid cancer, displaying a novel ELE1/RET oncogenic rearrangement with an anomalous fusion transcript, was molecularly characterized. In spite of the presence of a normal breakpoint in exon 5 of the activating ELE1 gene, the sequence of the rearranged genomic DNA showed a previously unreported intra-exonic breakpoint in the RET protooncogene. As a consequence, a cDNA sequence 93 nucleotides larger than the regular one, and with the exon 5 of ELE1 joined to exon 11 instead of exon 12 of RET, is formed. To characterize the product of this new oncogenic ELE1/RET rearrangement, here designated as RET/PTC4, we performed an immunoprecipitation and Western blot analysis on cell extracts from NIH3T3 transfectants. The results showed the presence of two isoforms of the chimeric protein, displaying a constitutive tyrosine phosphorylation. As expected, the molecular weight of this protein was higher than that of RET/ PTC3 protein (p80 and p85, instead of p76 and p81). Previous reports, from our and other laboratories, showed that post-Chernobyl papillary thyroid carcinomas are characterized by a high frequency (about 60%) of RET oncogenic rearrangements (Fugazzola et al., 1995; Klugbauer et al., 1995; Ito et al., 1994). These events predominantly involve ELE1 activating sequence, thus producing RET/PTC3 oncogene (Fugazzola et al., 1995; Klugbauer et al., 1995). Hence, this elevated frequency of RET rearrangements could increase the probability of selecting unusual events as that here described. Alternatively, targeted radiation effects could be responsible for the atypical RET rearrangement producing RET/PTC4 oncogene.

Oncogenic rearrangements of RET and NTRK1 proto-oncogenes are frequently detected in papillary thyroid carcinomas. Several studies have shown an association between ionizing radiation and development of this tumor type. In addition in vitro irradiation of tumor cell lines induced rearrangements of RET similar to those observed in human papillary thyroid carcinomas. These two observations could be related to the reported increased incidence of papillary thyroid carcinomas in children living in contaminated areas around Chernobyl, given that it has been demonstrated that about 60% of them presents a RET oncogenic activation. However, this high frequency of RET positivity in radiation exposed children does not rule out the possibility that age could also play a role in the development of RET positive tumors. To assess this possibility we looked for a relationship between the presence of RET and NTRK1 oncogenic rearrangements and age at surgery in a sample of 92 consecutive patients. Our results show that, in papillary thyroid carcinoma, the frequency of RET and NTRK1 activation is significantly higher in the group of patients aged 4-30 years, thas supporting the concept that age could be contributing to this thyroid specific carcinogenic process.

Since the Chernobyl nuclear reactor accident, a striking increase of thyroid carcinoma has been reported in children exposed to radiation in Belarus. Because of its unprecedented scale and its emotional implications, this finding has raised concern and called the attention of the scientific community to this major health problem. Although epidemiologically documented, a direct correlation between thyroid cancer and radiation exposure has not been definitely proven at the molecular level. On the assumption that ionizing radiation could cause specific and common cancer-associated genetic lesions, an analysis of oncogene activation and/or tumor suppressor gene inactivation would help to define radiation-induced thyroid carcinomas. Therefore, we have analyzed by different molecular approaches, including Southern blotting, DNA transfection assay on NIH-3T3 cells, and reverse transcription-PCR analysis, six papillary carcinomas from children living in the region of Belarus at the time of the Chernobyl nuclear accident to identify tumor-specific gene rearrangements of the proto-oncogenes RET and TRK, previously found activated in a tumor type-specific manner in papillary thyroid carcinoma. Using Southern blot analysis in four cases, we could detect specific rearranged bands indicating an oncogenic activation of RET that in three cases resulted in rearranged sequences provided by the same activating gene. Moreover, the DNA of the last three cases showed a biological activity in transforming NIH-3T3 cells after the DNA-mediated transfection assay, and the respective NIH-3T3 transfectants were found to express the oncogenic fusion transcripts. These results support the possibility that RET oncogenic activation could represent a major genetic lesion associated with thyroid carcinoma in children exposed to the Chernobyl nuclear accident.

Increased concentrations of serum soluble interleukin-2 (IL-2) receptor (sIL-2R), a marker of T-lymphocyte activation, have been found in several metastatic solid tumors. To our knowledge, no information is available on serum sIL-2R in differentiated thyroid carcinoma (DTC). Aim of this study was to evaluate whether disease activity and/or thyroid status may affect circulating sIL-2R in DTC, since it is has recently been demonstrated that serum thyroid hormone concentration positively modulates circulating sIL-2R. DTC patients were divided into 3 groups: Group A: 48 patients without metastases or local recurrences; Group B: 16 patients with cervical lymph node metastases; Group C: 22 patients with distant metastases. All patients were evaluated after total thyroidectomy both off and on L-thyroxine (L-T4) therapy. Control group was composed by 20 healthy euthyroid subjects. sIL-2R was assayed by solid-phase ELISA. In the hypothyroid state, sIL-2R levels of Group A were significantly lower when compared to normal controls (256 +/- 130 vs. 461 +/- 186 U/ml, p < 0.001 by Student t test); Group B and Group C patients off L-T4 therapy had sIL-2R concentrations significantly higher (479 +/- 407 U/ml, Group B; 519 +/- 746 U/ml, Group C) when compared to hypothyroid patients of Group A (p < 0.01 and p < 0.05, respectively), but not significantly different from normal euthyroid controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Unlabelled: The aim of the present study was to assess whether 131I therapy for differentiated thyroid carcinoma (DTC) can affect endocrine testicular function., Methods: Serum follicle-stimulating hormone (FSH) and testosterone (T) concentrations were measured in 103 patients periodically submitted for radioiodine therapy for residual or metastatic disease. Mean follow-up was 93.7 +/- 54 mo (range 10-243 mo)., Results: Mean FSH values in 131I-treated patients tested after their last treatment were 15.3 +/- 9.9 mU/ml, significantly higher than those of 19 untreated patients (6.5 +/- 3.1 mU/ml). Considering the mean +3 s.d. FSH of untreated subjects as the upper limit of normal range, 36.8% of the patients had an abnormal increase in serum FSH. Longitudinal analysis performed in 21 patients showed that the behavior of FSH in response to 131I therapy was not universal. Six patients had no change or a slight increase in serum FSH after 131I administration; eleven patients had a transient increase above normal values 6-12 mo after 131I treatment, with return to normal levels in subsequent months. The administration of a second dose was followed by a similar increase in FSH levels. Finally, four patients, followed for a long period of time and treated with several 131I doses, showed a progressive increase in serum FSH, which eventually became permanent. Semen analysis, performed in a small subgroup of patients, showed a consistent reduction in the number of normokinetic sperm. No change was found in serum T levels between treated and untreated patients., Conclusions: Our results indicate that 131I therapy for thyroid carcinoma is associated with transient impairment of testicular germinal cell function. The damage may become permanent for high-radiation activities delivered year after year and might pose a significant risk of infertility.

To assess whether routine measurement of serum calcitonin (CT) could improve the preoperative diagnosis of sporadic medullary thyroid carcinoma (MTC), 1385 consecutive patients presenting for nodular thyroid disease during the year 1991 were submitted to serum CT determination and fine needle aspiration cytology (FNAC). The clinical diagnosis was nontoxic nodular goiter in 1197 (86.4%) patients, toxic multinodular goiter in 65 (4.7%), autonomously functioning thyroid nodule (AFTN) in 64 (4.6%), and autoimmune thyroid disease (Graves' disease or Hashimoto's thyroiditis) with nodule(s) in 59 (4.3%). As controls, 177 patients with nonnodular thyroid disease and 32 normal subjects were also studied. Patients with FNAC suspicious of any kind of thyroid carcinoma and patients with elevated basal and pentagastrin-stimulated serum CT, regardless of the results of FNAC, were submitted to surgery. Eight (0.57%) patients (7 with nontoxic nodular goiter and 1 with AFTN) had elevated basal serum CT levels, ranging between 55-10,000 pg/mL. The pentagastrin test was abnormal in all of them. FNAC was suggestive of MTC in 2, thyroid carcinoma in 1, benign nodule in 3, and inadequate in 2. By histology, immunohistochemistry, and Northern blot analysis of total tumor RNAs, MTC was confirmed in all patients, including the 1 with AFTN, who had the association of microfollicular adenoma and a small MTC in the same lobe. After surgery, serum CT decreased to undetectable levels in 7 patients and remained undetectable in 6 of them during a mean follow-up of 22 months, although 1 of them had a positive response to pentagastrin. Forty-four patients in the group with normal serum CT levels had FNAC suspicious for differentiated thyroid carcinoma and were treated by surgery. Differentiated thyroid carcinoma, mostly papillary, was confirmed at histology in 43 subjects (3.1% of all thyroid nodules). In conclusion, the results of our study indicate that serum CT measurement is useful for the screening of sporadic MTC in patients with thyroid nodule(s). The prevalence of MTC, diagnosed by serum CT measurement in a 12-month period, among an unselected series of 1385 patients with nodular thyroid disease was surprisingly high: 0.57% of all thyroid nodules and 15.7% of all thyroid carcinomas. Serum CT measurement was superior to FNAC in suggesting the diagnosis of MTC and was devoid of falsely positive results. Increasing the diagnostic accuracy helped the surgeon to perform more radical treatment of MTC, thus achieving frequent normalization of postoperative serum CT levels. Whether this result indicates definitive cure remains to be established on the basis of longer follow-up.(ABSTRACT TRUNCATED AT 400 WORDS)

We studied the expression of the TSH receptor (TSH-R), thyroglobulin (Tg), thyroperoxidase (TPO), and calcitonin (CT) genes in a total of 53 tissues from 30 patients with thyroid carcinoma and from 9 patients with benign thyroid diseases. By Northern blot analysis of total RNA preparations, CT mRNA was expressed in all cases (n = 6) of medullary thyroid carcinoma (MTC). Surprisingly, 3 of them expressed the TSH-R mRNA, in association with the Tg and TPO mRNAs in 1. The presence of the TSH-R transcript in the neoplastic C-cells was confirmed in 1 MTC by in situ hybridization using a mixture of 3 oligonucleotide probes derived from dog TSH-R cDNA. With various degrees of expression, all differentiated thyroid carcinomas (20 papillary and 2 follicular) expressed TSH-R, Tg, and TPO, but not CT mRNAs. On the contrary, samples from 2 patients with anaplastic carcinoma did not express TSH-R, Tg, or TPO mRNA, but 1 of them expressed CT mRNA. All of the transcripts obtained from thyroid carcinomas (both primary and metastatic) were of the same size as the transcripts from normal or benign thyroid tissues, with the exception of 2 cases of differentiated thyroid cancer, in which TSH-R mRNA of lower mol wt (approximately 4.0 kilobases) was found in the absence of alteration in cDNA size and restriction map. The main conclusions of our study are that 1) the TSH-R gene is expressed in some MTC, which supports, at molecular level, the hypothesis of the existence of mixed follicular-medullary thyroid tumors; and 2) the expression of TSH-R, Tg, and TPO in undifferentiated thyroid cancer is lost.

We studied the half-life of serum calcitonin (CT) in patients subjected to total thyroidectomy for medullary thyroid carcinoma (MTC). One patient showed a rapid serum CT component with a half-life of 3 hours and a slow component with a half-life of 30 hours; in another case only the 30-hour component was found. By chromatography of tumor extracts, we found that all the immunoreactive CT had a molecular weight of 3,600. After surgery, normalization of serum CT was achieved within 15 days in 4 patients, at 3 months and at 6 months in 2 other patients, while 1 patient never normalized. Normalization of serum CT after surgery is not an index of definitive cure in MTC, as demonstrated by one patient who relapsed 3 months after normalization of serum CT. However, as a general rule, patients who reach undetectable serum CT levels soon after surgery, are those having the best prognosis.

We studied the expression of p21, the ras encoded protein, in primary tumour of 45 patients with papillary thyroid cancer (PTC). Patients were grouped according to outcome so that one group (31 patients) had a good outcome and the other (14 patients) a fatal outcome, after a follow-up of at least 5 years. The presence of p21 ras protein was assessed by immunohistochemistry with a specific monoclonal antibody (MAb Y-13259). The results were correlated with the outcome, with the expression of proliferating cell nuclear antigen (PCNA)/cyclin (as a marker of cell proliferation) and with other well established prognostic factors for PTC (age, grading, extension and tumour size; Endocrinol Metab Clin North Am 1990, 19, 545-576). p21 staining in tumours of living patients was negative in 15, weakly positive (1+) in 10 and strongly positive (2+ or more) in 6 patients. In tumours from deceased patients, p21 staining was negative in 1, weakly positive in 2 and strongly positive in the remaining 11 patients (P < 0.001, chi 2). PCNA immunostaining was increased in 63.6% (7/11) of the tumours from deceased patients compared to 17.8% (5/28) of the tumours of living patients, but no direct correlation was found between p21 and PCNA expression. Among the other prognostic factors studied, only age > or = 40 years was a significant predictor of poor outcome. The survival curve of patients with strongly positive p21 staining was similar to that of patients aged > or = 40 years at the time of diagnosis. The combination of p21 > or = 2+ and age > or = 40 was superior to age alone (P < 0.05) as a prognostic indicator of poor outcome. In conclusion, our results indicate that the p21 product of the ras (proto)oncogene is differently expressed in PTC, in relation to the degree of aggressiveness. Regardless of the pathogenetic role of the ras oncogene in thyroid tumorigenesis, our data indicate that the expression of the p21 ras protein may be regarded as a prognostic indicator in PTC. Furthermore, overexpression of p21 ras protein is associated with patients in the older age groups, and might contribute to the poor prognosis of elderly patients.

Age is the most accepted prognostic factor in differentiated thyroid cancer. Other parameters, such as tumor size, grading, extrathyroidal extension, have also been associated with the prognosis of these tumors. Since the identification of reliable prognostic factors is essential to avoid unnecessary aggressive treatment for a disease, such as thyroid carcinoma, which only rarely is fatal, we studied two indices of cell proliferation in patients with differentiated thyroid cancer, in relation to their outcome. We studied two groups of patients with differentiated thyroid cancer, selected in a way to have one group (33 patients) with a good outcome and one (16 patients) with a fatal outcome, after a follow-up of at least 5 years. By immunohistochemistry the primary tumors of all patients were analyzed for the expression of the proliferating cell nuclear antigen (PCNA)/cyclin. In 38 (77.5%) of them also the nuclear DNA content and the percentages of S-phases were analyzed by flow cytometric analysis. At diagnosis the two groups of patients differed significantly with regard to age and extrathyroidal extension, but not for tumor size and grading. A significant difference (p=0.02) was found in the positivity of PCNA/cyclin expression between the fatal outcome group (66.6%) and the surviving patients (27%), and in the percentage of cells in the S-phase, 16.4+/-7.7% in the fatal outcome group patients and 6.0+/-2.6% in the surviving patients (p=0.0001). No difference was found in the nuclear DNA content of the two groups. A positive correlation was found between PCNA expression and S-phase (r(s)=0.55; p<0.001). A positive correlation was found between age and both the percentage of S-phase cells (r(s)=0.48; p<0.002) and PCNA expression (r(s)=0.36; p<0.009). In a multivariate analysis (Cox model) age and S-phase had independent prognostic significance (regression coefficient: 3.85 and 1.70, respectively), while PCNA was not an independent variable (0.98). Our results indicate that differentiated thyroid tumors with fatal outcome are characterized by two parameters of active cell proliferation (S-phase cell fraction and PCNA expression), which can be used as useful prognostic factors.

Multiple endocrine neoplasia (MEN) 2A associated to cutaneous lichen amyloidosis is a variant of MEN 2A recently reported in few families. We describe an additional family with this syndrome. The skin lesion is a pruritic one, located over the upper back showing, at biopsy, cutaneous amyloid. The propositus of our family was a 35-year-old woman already treated with total thyroidectomy for medullary thyroid cancer and with bilateral adrenalectomy for pheochromocytoma. The patient referred that the skin lesion was present since childhood and increased with time. Skin biopsy showed negative staining for amyloid, for calcitonin and neuron-specific enolase. The paraspinal muscles corresponding to the area of affected skin showed slight neurogenic abnormalities. Ten other members of her family were affected by MEN 2A, three of whom (all females) had the same cutaneous alteration.

We studied the expression of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in 18 patients with medullary thyroid cancer (MTC) in the neoplastic (primary or metastatic) tissue by immunohistochemistry and in the plasma by radioimmunoassay. CT immunoreactivity was found in 100% of the primary and metastatic MTC, CGRP was expressed in 66% of the primary tumors and in 73% of the metastases. Both the number of positive cells and the degree of staining were always higher for CT than for CGRP staining. While plasma CT concentrations were always increased in patients with metastases, 3 patients with metastases had undetectable plasma CGRP levels. A positive correlation was found between plasma CT and CGRP levels. These data indicate that CGRP is frequently expressed in MTC sections and that plasma CGRP measurement is an additional marker for MTC, although has no advantage with respect to CT measurements in monitoring the progression of the disease.

We studied the feasibility of employing the measurement of thyroglobulin (Tg) in the washout of the needle used to perform the fine needle aspiration cytology (FNA-Tg) for the differential diagnosis of nonthyroidal neck masses of unknown etiology. We studied 35 patients presenting for 1 or more neck lumps outside the thyroid gland. A previous history of treated differentiated thyroid cancer (DTC) was given by 23 patients and of nonthyroidal malignancy by 3 patients. FNA-Tg was measured in the Tg-free serum used to wash out the needle employed for the cytology. Finally, all patients were treated by surgery. FNA-Tg was always detectable in 14 patients with thyroid cancer metastases demonstrated by histology, with a mean (+/- SD) of 27,087 +/- 37,622 ng/FNA (P less than 0.002) compared to patients without thyroid cancer metastases (mean +/- SD, 12.1 +/- 4.8 ng/FNA in 7 cases; undetectable in 14 cases). Assuming 21.7 ng/FNA (the mean +/- 2 SD of the negative patients) as the cut-off value, all patients with metastases from DTC were detected by FNA-Tg. FNA-Tg had better negative predictive value than cytology, since this last technique gave 10 inconclusive results, comprising 2 false negative results in patients with metastases from DTC. Our results indicate that elevated concentrations of FNA-Tg in nonthyroidal neck nodes strongly suggest the diagnosis of metastases from DTC.

The authors investigated the humoral and tissue expression of six antigens associated with medullary thyroid cancer (MTC): calcitonin (CT), calcitonin gene-related peptide (CGRP), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), somatostatin (SRIF), and thyroglobulin (TG). The antigens were studied in the neoplastic C cells using immunohistochemistry with specific antisera and in the plasma using specific radioimmunoassay. Eighteen patients (8 male and 10 female patients, aged 12-72 years) were studied. Mean follow-up was 70.7 months (range, 2-179 months). Nine patients (50%) died of their disease after a mean follow-up of 47.2 months (range, 2-116 months). By immunostaining, primary tumors expressed CT and CEA in all cases and NSE was positive in 90%, CGRP in 66%, SRIF in 63%, and TG in 58%. Metastatic tissues were positive in all cases of CT staining, 92.8% of CEA, 71.4% of NSE, 73.3% of CGRP, 38.5% of SRIF, and only 13.3% of TG staining. In positive cases the percentage of positive cells and the degree of staining were variable among the different antigens. The expression of an antigen in the neoplastic cells was associated with the hypersecretion of the corresponding antigen in the circulation in the case of CT and CEA. The levels of these antigens were elevated in all patients with metastases and could accurately predict the appearance of new metastases or indicate the effective treatment of previous metastases by surgery. In the case of NSE, CGRP, and SRIF, few patients had increased plasma concentrations of the antigens and these usually occurred during very advanced phases of the disease. Detectable levels of serum TG were never observed. When the outcome of the disease was compared with the expression of CT, CEA, NSE, CGRP, and TG, no correlation could be found. On the contrary, SRIF expression in the primary tumor could differentiate two groups of patients with different survival rates. SRIF-positive patients had survival rates of 100% and 50% at five and seven years, respectively, whereas SRIF-negative patients had survival rates of 40% at five years and 25% at seven years.