RET proto-oncogene in Sardinia: V804M is the most frequent mutation and may be associated with FMTC/MEN-2A phenotype

OBJECTIVE: Genetic screening of RET proto-oncogene is a powerful tool for the early identification of familial cases of medullary thyroid carcinoma (MTC), comprising isolated familial thyroid medullary carcinoma (FMTC) and multiple endocrine neoplasia syndromes 2A (MEN-2A) and 2B (MEN-2B). We report the results obtained by RET mutation analysis of subjects living in Sardinia, an Italian island whose inhabitants display a peculiar genetic background due to geographic isolation and low immigration rate for several centuries. DESIGN: Retrospective study reporting data on 67 patients referred during the last 5 years for RET analysis because affected by MTC or first degree relatives of MTC patients. MAIN OUTCOME: Only three mutations were identified affecting codons 620 (exon 10), 634 (exon 11), and 804 (exon 14); surprisingly, the most prevalent mutation found was V804M (overall prevalence: 59%). This finding is quite different from previous studies carried out in other Caucasian and non-Caucasian populations, in which the frequency of the V804M mutation is consistently reported less than 5%. The phenotype associated to V804M mutation was mostly FMTC (16/17 cases = 94.1%), but in one case (5.9%) primary hyperparathyroidism was found, suggesting a MEN-2A. CONCLUSIONS: These results underline the importance of the genetic background in the distribution of RET mutations and should be taken into consideration when performing genetic evaluation of MTC patients

RET/PTC rearragements in Hashimoto’s thyroiditis nodules.

An association between Hashimoto thyroiditis (HT) and thyroid carcinoma, particularly papillary thyroid carcinoma (PTC), has been postulated for decades, although definitive molecular links remain elusive. The majority of PTC is characterized by alterations or rearrangements along the MAPK signaling cascade. Activation of RET/PTC oncogene has been reported in both non-neoplastic and neoplastic HT thyroid follicular cells (Tallini et al., 2006), but the issue is still controversial. We have investigated cells from HT nodules for RET rearrangement, using interphase fluorescence in situ hybridization (I-FISH). Dual-color I-FISH experiments, using home-brew breakapart DNA probe, able to detect RET disruptions were performed on 207 fine needle aspiration biopsies on unselected nodules from 168 patients and 38 corresponding surgical samples. Normal tissue nuclei cutoff value (mean ±3DS) was ≥ 3%. In addition, RET/PTC was investigated by RT-PCR. Coexistent overt autoimmune thyroid disease (AITD) was assessed by serological (antithyroid autoantibodies, ATA), clinical and echographic data. RET breakage (FISH+) was observed in 13/207 (6.3%) samples. RET/PTC was confirmed by RT-PCR (RT-PCR+) in 2/12 available FISH+ nodules (16.6%). RT-PCR+ corresponded to FISH+ value ≥ 8%. No correlation between RET rearrangement and ATA (5/92 patients ATA +; 8/115 ATA-), or between AITD (6/78 AITD+; 7/129 AITD-) was observed. Nodules with RET disruption were observed along the whole cytological spectrum, with prevalence significantly higher in suspect or malignant cytology (13.6% in 66 TIR 3-5 vs 3.1% in 129 TIR 2 nodules: p=0.005 by X2test). 7 of 13 FISH+ nodules were surgically removed and available for I-FISH. RET breakage was confirmed in 3 samples, all of which were classified as classic PTC type. According to our results, RET rearrangement does not seem to correlate with the HT phenotype. The number of FISH+ nodules within the cytological classes, increases according to the risk of malignancy. Sensitivity of breakapart I-FISH strategy, able to detect gene disruption in single nuclei, could explain the difference between I-FISH and RT-PCR results. Since in our experimental condition, RET-PTC m-RNA transcript can be detected in ≥ 8% FISH+ samples , the oncogenic potential of nodules below this value remains to be clarified, and FISH+ nodules with benign cytology should be considered for a careful follow-up

RET/PTC rearrangement in Hashimoto’s thyroiditis nodules: contribution to an ongoing debate.

An association between Hashimoto thyroiditis (HT) and thyroid carcinoma, has been postulated for decades, although definitive molecular links remain elusive. Activation of RET/PTC oncogene (typical of papillary thyroid carcinoma) has been reported in both non-neoplastic and neoplastic HT thyroid follicular cells (Tallini et al., 2006), but the issue is still controversial. To contribute new insight to this issue, we have investigated RET rearrangement on 207 fine needle aspiration biopsies on unselected nodules from 168 patients and 38 corresponding surgical samples. I-FISH with a homebrew-breakpart DNA probe was used. Normal tissue nuclei cutoff value (mean ± 3DS) was 3%. In addition, RET/PTC was investigated by RT-PCR. Coexistent overt autoimmune thyroid disease (AITD) was assessed by serological (antithyroid autoantibodies, ATA), clinical and echographic data. RET breakage (FISH+) was observed in 6.3% samples. RET/PTC was confirmed by RT-PCR (RT-PCR+) in 23% FISH+ nodules. RT-PCR+ corresponded to FISH+ value ≥ 6,8%. No correlation between RET rearrangement and ATA (5/92 nodules ATA +; 8/115 ATA-), or between AITD (6/78 AITD+; 7/129 AITD-) was observed. Nodules with FISH+ were observed along the whole cytological spectrum, with prevalence significantly higher in suspect or malignant cytology (13.6% TIR 3-5 vs 3.1% TIR 2 nodules). 7/13 FISH+ nodules were surgically removed and available for I-FISH. FISH+ was confirmed in 3 samples, classified as PTC classic type. Our results suggest no association between RET rearrangement and HT. The percentage of FISH+ nodules increases according to the risk of malignancy. Difference between I-FISH and RT-PCR results was observed, presumibly due to the sensitivity of I-FISH strategy. Since we detected RET-PTC m-RNA transcript in ≥ 6,8% FISH+ samples, the oncogenic potential of nodules below this value remains to be clarified, and FISH+ nodules with benign cytology should be considered for a careful follow-up