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Differential expression of RET isoforms in normal thyroid tissues, papillary and medullary thyroid carcinomas
No full textPOURPOSES:
We investigated the expression of RET9 and RET51 isoforms in medullary (MTC), papillary (PTC) thyroid carcinoma, normal thyroid tissues, and pheochromocytoma (PHEO) to verify if these isoforms are present also in follicular thyroid cell-derived tissues, and if there is a differential expression of RET9 and RET51 in MTC.
METHODS:
Nineteen patients with MTC, 18 patients with PTC, 18 samples of contralateral normal thyroid tissues, and 5 cases of PHEO were included in this study. RET isoform expression was studied by real-time RT-PCR.
RESULTS:
All MTCs and PHEOs were positive for RET9 and RET51. Fourteen/eighteen (77.7%) PTC cases were positive for RET9 and/or RET51, and four were positive for only one of the genes. In normal thyroid tissues, 3/18 (16.7%) cases were negative for both isoforms, 4/18 (22.2%) were positive for both, and 11/18 (61.1%) were positive for only one. RET isoforms were expressed at different levels in MTC, PHEO, PTC, and normal thyroid tissues: RET9 expression was higher in PHEO than in MTC, PTC, and normal thyroid tissues. RET9 expression was also higher in MTC than in PTC and normal thyroid tissues. No difference was observed between PTC and normal thyroid tissues. A similar pattern of expression was observed for RET51. In addition, RET51 was significantly more expressed than RET9 in MTC, while RET9 was the predominant isoform in PHEO.
CONCLUSIONS:
Our study documented the expression of the RET9 and RET51 isoforms in normal thyroid and PTC tissues. RET9 and RET51 isoforms were also present in MTC and PHEO. RET51 expression was higher than RET9 expression in MTC, while there was no difference in the expression of these two isoforms in PTC and normal thyroid tissues. RET9 was more highly expressed than RET51 in PHEOs
GENETIC SCREENING OF RET CAN IDENTIFY NEW MUTATIONS EVEN AFTER 20 YEARS
No full textObjectives: In the last 20 years we performed RET genetic screening in more than 1000 hereditary or sporadic MTC patients.
Methods: RET analysis was performed in constitutive and/or somatic DNA by direct sequencing. TA cloning was performed to characterize new mutations and deletions. Site-directed mutagenesis, focus formation and soft agar assays were performed to test in vitro the activity of new mutations. The Align GVGD program was employed for the in silico analysis.
Results: we identified 3 new RET alterations. The first was a 7bp “somatic” in frame deletion in exon 11 encompassing codon 629-631. The second showed the simultaneous presence of a “somatic” E616Q mutation in exon 10 and a “somatic” C630G mutation in exon 11 on different alleles. Moreover, in the same patient, we found an alternative splicing causing the in frame skip of exon 10 in the allele carrying the C630G mutation. The third alteration was a new “germline” mutation (E632K, exon 11) and was found in an apparently sporadic MTC. According to the in vitro and in silico tests, both E616Q and E632K RET mutations were not transforming while the C630G RET mutation showed a high transforming activity.
Conclusions: 1) RET genetic screening should be performed by sequencing analysis in all MTC patients to detect also new RET mutations that would be missed when looking only at the “hot spot” mutations; 2) all new mutations must be evaluated by in silico and/or in vitro analysis to define their transforming ability since in some cases they may be inactive mutations
Predictive factors of short and long-term vandetanib response in locally advanced or metastatic medullary thyroid cancer: a single center experience
No full textObjectives: Vandetanib (V) is an important drug in the metastatic medullary thyroid cancer (MTC) treatment. The objective of this study was to evaluate the presence of predictors of V response, in short and long period, in locally advanced or metastatic MTC patients (pts).
Methods: Seventy-nine locally advanced or metastatic MTC pts with progressive or symptomatic disease, referred to our Center between 2007 and 2018 and already treated surgically and with other systemic therapies, were treated with V. During follow-up it was performed clinical examination, biochemical and morphological evaluation. Twenty-five pts were treated with V for less than 12 months (short responders, Group 1), 54 patients were treated with V for at least 12 months (long responders, Group 2).
Results: The genetic screening showed that in the Group 1, 4/25 (16%) pts were inherited forms and 21/25 (84%) pts were sporadic cases. In the Group 2, 8/54 (14.8%) pts were inherited forms and 46/54 (85.2%) pts were sporadic cases. The evaluation of somatic mutations showed that RET mutation was present in 82.3% and in 95.3% of pts in Group 1 and in Group 2, respectively. However, the presence of RET mutations, it wasn’t a predictor of response to treatment. The metastases site wasn’t correlated with the outcome. Otherwise, we observed that in long responders group, 47/54 (87%) pts showed at least one adverse events (AE) during V treatment with a correlation between AE and V response (P=0.02). In this group we also observed a statistically significant correlation between the younger age (<45 yrs) at screening and a greater response to V (P=0.01) and between the absence of progression disease at screening and response to V (P<0.0001). In the long term outcome, considering the last CT scan performed at the data cut-off during the treatment, 29/54 (53.7%) pts showed a persistent response to V after a median follow-up of 41 months. Moreover, we observed that the pts in the Group1 had a more aggressive disease and a more advanced age at screening than pts in Group 2. The estimated median Progression Free-Survival of all patients was 47 months.
Conclusions: In our study, it was observed that the appearance of AE during V treatment, the younger age and the absence of progression disease at screening were predictive factors of long-term response to V in MTC pts. Moreover, RET somatic mutations were very frequent in the metastatic MTC patients but it wasn’t a predictor of response to V
AFTER 20 YEARS, RET GENETIC SCREENING STILL INDENTIFIES NEW GERMILINE AND SOMATIC MUTATIONS
No full textObjectives: In the last 20 years we performed RET genetic screening in more than 1000 MTC patients either hereditary or sporadic.
Methods: RET genetic screening was performed in DNA extracted from blood and/or tissue by direct sequencing. TA cloning was performed to characterize new mutations and deletions. Site-directed mutagenesis, focus formation and soft agar assays were performed to test in vitro the activity of the new mutations. The Align GVGD program was employed for the in silico analysis.
Results: in the last year we identified 3 MTC patients with new RET alterations. The first case had a 7bp “somatic” in frame deletion in exon 11 encompassing codon 629-631. The second case showed the simultaneous presence of a “somatic” E616Q mutation in exon 10 and a “somatic” C630G mutation in exon 11 on different alleles. Moreover, in the same patient, we found an alternative splicing causing the in frame skip of exon 10 in the allele carrying the C630G mutation. The third case harboured a new “germline” mutation (E632K in exon 11) although the MTC was apparently sporadic. According to the in vitro and the in silico tests, both E616Q and E632K RET mutations were not transforming while the C630G RET mutation showed a high transforming activity.
Conclusions: 1) RET genetic screening should be performed by sequencing analysis in all MTC patients to detect also new RET mutations that would be missed when looking only at the “hot spot” mutations; 2) all new mutations must be evaluated by in silico and/or in vitro analysis to define their transforming ability since in some cases they may be inactive mutations
SIMULTANEOUS MEDULLARY AND PAPILLARY THYROID CARCINOMAS SHOW INDEPENDENT GENETIC ORIGIN
No full textPapillary (PTC) and Medullary (MTC) thyroid carcinomas have distinct embryonic origins although they share the activation of common oncogenes (RET, RAS). The occurrence of these tumors in the same gland is not a rare event. Aim of this study was to investigate the hypothetical involvement of common genetic alterations in the symultaneous occurrence of the PTC and MTC.
We studied 24 patients presenting simultaneous PTC and MTC. DNA was obtained from both tumor components and healthy tissue and sequenced for mutations in codons 12, 13 and 61 of H-, K- and N-RAS, 600 of BRAF and 634 and 918 of RET.
Two/24 patients (8.3 %) were affected by MEN2A and carrying germline mutations of the RET gene (S981A, V804M). None of the patients showed mutations in the healthy tissue and 8/24 patients (33.4 %) had no mutations either in the tumor components. In the other cases, considering the MTC component: 8/24 (33.3 %) harboured the RET/M918T mutation; 3/24 (12.5 %) mutations in the H-RAS gene (G13R, Q61R, D69N) and 1/24 (4.2 %) showed simultaneously the H-RAS/Q61K and the RET/M918T mutations. In the PTC component: 1/24 (4.2 %) harboured the BRAF/V600E mutation, 1/24 (4.2 %) the H-RAS/T58A mutation and 1/24 (4.2 %) the K-RAS/M1T mutation. In one case, we found the RET (M918T) mutation in the MTC component and the BRAF/V600E in the PTC component. None of the mutations found were present in both tumors.
These data suggest independent genetic causes in the development of simultaneous PTC and MTC. Confirmation of this hypothesis is that one of the patients showed simultaneous mutation of RET in MTC and the BRAF mutation in PTC. In this study we also found novel H-RAS and K-RAS mutations suggesting that alterations outside the classic hot spots may play a role in the pathogenesis of these tumors
WHOLE EXOME SEQUENCING OF MEDULLARY THYROID CARCINOMA CASES IDENTIFIES 86 VARIATIONS IN GENES POSSIBLY INVOLVED IN TUMORAL TRANSFORMATION
No full textObjectives: About 40% of sporadic Medullary Thyroid Carcinomas (MTC) is still orphan of an oncogenic driver. Purpose of this study was to disclose novel genetic alterations leading to the pathogenesis of MTC using Whole Exome Sequencing (WES) of RET+ and RET- cases.
Methods: WES analysis was performed on 6 sporadic MTC cases (2 RET+, 4 RET-) using an Illumina platform. After processing and proper filtering, all non-synonymous Single Nucleotide Variations (SNV) shared by the RET- cases were listed. Validation on 135 MTC cases and 189 healthy controls by PCR and enzymatic restriction analysis was performed in one of the genes of interest identified through WES.
Results: WES analysis led to the identification of a panel of 86 non-synonymous SNV shared in the 4 RET- cases and possibly involved in tumoral transformation process. Among the 86 SNV identified, the A133S polymorphism of the RASSF1A oncosuppressor appeared to be of interest. We found A133S in 21/135 (15.6%) MTC cases and in 19/189 (10%) healthy controls (P=0.137). The incidence of A133S appeared to be slightly lower in RET mutated MTC [9/64 (14%)] than in not-mutated [12/71 (17%)], although not statistically significant.
Conclusions: Through WES analysis we were able to identify 86 non-synonymous SNV shared in RET- MTC cases. This panel represents the first list of variations containing hypothetically novel genetic drivers involved in MTC oncogenesis. The prevalence of the A133S SNP was found to be higher in MTC cases compared to healthy controls (15.6% vs 10%) although not statistically significant. Further validation of other candidate genes is on-going
I CARCINOMI PAPILLARI E MIDOLLARI DELLA TIROIDE PRESENTI CONTEMPORANEAMENTE HANNO ORIGINE GENETICA INDIPENDENTE
No full textIl Carcinoma Papillare (PTC) ed il Carcinoma Midollare (MTC) della tiroide si sviluppano entrambi nella tiroide ma hanno un’origine embrionale distinta: il PTC deriva dalle cellule follicolari mentre il MTC dalle cellule C-parafollicolari. In letteratura sono riportati casi in cui questi due tipi di tumore si presentano contemporaneamente ed una possibile causa genetica comune e’ stata ipotizzata anche se i dati a disposizione rimangono tuttora controversi. Scopo di questo studio è stato quello di analizzare la presenza di alterazioni genetiche caratteristiche dei due tipi di tumore (PTC e MTC) che si presentano contemporaneamente. Allo scopo abbiamo studiato 24 pazienti (6 maschi, 18 femmine) che presentavano simultaneamente un PTC ed un MTC. Il DNA genomico è stato estratto dalle componenti tumorali e dal parenchima sano ed e’ stato analizzato con sequenziamento diretto per le mutazioni nei codoni 12, 13 e 61 dei geni H-, K- ed N-RAS, 600 del gene BRAF e 634 e 918 dell’oncogene RET. Due/24 pazienti (8.3%) erano affetti da MEN2A con mutazione germinale del gene RET (S981A, V804M). Nessuno degli altri pazienti presentava mutazioni nel tessuto sano ed in 8/24 pazienti (33.4%) non presentavano mutazioni nemmeno nelle porzioni tumorali. Negli altri pazienti: 8/24 (33.3%) MTC presentavano la mutazione somatica di RET M918T; 3/24 (12.5%) mutazioni del gene H-RAS (G13R, Q61R, D69N) ed 1/24 (4.2%) mutazioni H-RAS (Q61K) e RET (M918T) contemporanea. Nella componente PTC: 1/24 (4.2%) presentava la mutazione BRAF (V600E), 1/24 (4.2%) la mutazione H-RAS (T58A) e 1/24 (4.2%) la mutazione K-RAS (M1T). In un caso, si aveva contemporaneamente la mutazione RET (M918T) nella componente MTC e BRAF (V600E) nella componente PTC. Nessuna delle mutazioni trovate era presente su entrambe le componenti tumorali. Questi dati suggeriscono cause indipendenti nell’insorgenza di PTC ed MTC che si presentano contemporaneamente in cui si osservano mutazioni tipiche per il tipo istologico (PTC e il MTC). A conferma di questa ipotesi, uno dei pazienti mostrava contemporaneamente una mutazione di RET nella componente MTC e la mutazione di BRAF nella componente PTC. Nel pannello di mutazioni trovate, le mutazioni D69N, T58A di H-RAS e M1T di K-RAS risultano nuove e mai descritte, suggerendo che anche mutazioni al di fuori degli hot spot classici dei geni RAS potrebbero avere un ruolo nella patogenesi di questi tumori
L’ANALISI GENETICA DI CARCINOMI PAPILLARI E MIDOLLARI DELLA TIROIDE PRESENTI CONTEMPORANEAMENTE RIVELA LA LORO NATURA CASUALE
No full textIl Carcinoma Papillare (PTC) ed il Carcinoma Midollare (MTC) della tiroide originano rispettivamente dalle cellule follicolari e parafollicolari della ghiandola. Abbastanza frequentemente, essi possono però presentarsi contemporaneamente. Diversi studi hanno tentato di trovare una causa patogenetica comune ma i dati a disposizione sono ancora controversi. Scopo di questo studio è quello di fornire un’analisi genetica dei tumori (PTC e MTC) che si presentano contemporaneamente per capire l’origine di questo fenomeno. Allo scopo abbiamo studiato 24 pazienti (6 maschi, 18 femmine) con presenza simultanea di PTC e MTC. DNA genomico è stato estratto dalle componenti PTC, MTC e parenchima sano per essere analizzato tramite PCR e sequenziamento diretto con primer specifici per mutazioni nei codoni 12, 13 e 61 dei geni H-, K- ed N-RAS, 600 del gene BRAF e 634 e 918 dell’oncogene RET. Due/24 pazienti (8.3%) erano affetti da MEN2A con mutazione germinale del gene RET (S981A, V804M), presente quindi in tutte e tre le componenti. Nessuno degli altri pazienti presentava mutazioni nel tessuto sano ed in 8/24 pazienti (33.4%) non e’ stata trovata alcuna mutazione nemmeno nelle porzioni PTC ed MTC. Negli altri pazienti, 8/24 (33.3%) MTC presentavano la mutazione somatica di RET M918T; 3/24 (12.5%) mutazioni del gene H-RAS (G13R, Q61R, D69N) ed 1/24 (4.2%) mutazioni H-RAS (Q61K) e RET (M918T) in contemporanea. Per quanto riguarda la componente PTC: 1/24 (4.2%) presentava la mutazione BRAF (V600E), 1/24 (4.2%) la mutazione H-RAS (T58A) e 1/24 (4.2%) la mutazione K-RAS (M1T). Uno dei pazienti mostrava contemporaneamente la mutazione RET (M918T) nella componente MTC e BRAF (V600E) nella componente PTC. Nessuna delle mutazioni trovate era presente su entrambe le. Da questi dati si evidenzia che le due componenti tumorali presentano mutazioni tipiche per il tipo istologico (PTC e il MTC) ed in nessun caso sono condivise, suggerendo cause indipendenti nell’insorgenza di questi tumori. A supporto di questa osservazione, uno dei pazienti mostrava una mutazione di RET nella componente MTC e la mutazione di BRAF nella componente PTC. Nel pannello di mutazioni trovate, le mutazioni D69N, T58A di H-RAS e M1T di K-RAS risultano nuove e mai descritte, suggerendo che anche mutazioni alternative agli hot spot classici dei geni RAS potrebbero avere un ruolo nella patogenesi di questi tumori
A novel fusion RET/PTC3 involving NCOA4 and RET genes in a pediatric case of Papillary Thyroid Carcinoma
No full textINTRODUCTION
Papillary thyroid carcinoma (PTC) originates from follicular cells of thyroid gland and it represents the most common type of thyroid cancer. About 10-20% of PTC cases harbor a RET/PTC rearrangement and the presence of these rearrangements is strongly associated to exposure to ionizing radiations and younger age.
METHODS/CASE PRESENTATION
In this study, we present the case of a 4 years old patient affected by classic variant PTC with lymph-node metastasis. After surgery, RNA and DNA were extracted from the primary tumor and 2 metastatic lymph-nodes. The cDNA was examined for the presence of RET/PTC 1 and 3 rearrangements by multiplex RT-PCR; amplicons obtained were then sequenced by Sanger method. The method of chromosome walking in the intronic regions of the genomic DNA of tumoral tissues was used to identify the breakpoint sites.
RESULTS/DISCUSSION
The results obtained on the primary tumor and metastatic tissues demonstrated the absence of a RET/PTC1 rearrangement but the presence of a RET/PTC3 rearrangement resulting in a shorter band length when compared to the positive control (300bp vs 430bp). Direct sequencing of the band obtained revealed a fusion between NCOA4 exon 6 and RET exon 12 with a skip of exon 7 of NCOA4 that is characteristic of the classic RET/PTC3 rearrangement. In order to further characterize the fusion on genomic DNA, we performed PCR with different primers located within intron 6-7 of NCOA4 and intron 11-12 of RET and the data obtained reveled a breakpoint within exon 11-12 of RET and within exon 7 of NCOA4, lost in the transcript after the splicing process. The patient’s personal and medical history did not show any link to radiation exposure or to other disruptors. Currently, a screening for this type of rearrangement on pediatric PTC patients with no history of radio exposure, is ongoing and chromosome fragility induced by other causes than radiation exposure are taken into consideration.
CONCLUSIONS
In conclusion, in this study we described an alternative and new RET/PTC3 rearrangement in a pediatric PTC patient. Apparently, the occurrence of this rearrangement is not linked to radiation exposure and other mechanisms are still under investigation
Whole Exome Sequencing of Medullary Thyroid Carcinomas did not identify oncogenic drivers alternative to RET and RAS genes
No full textAbout 60% of sporadic Medullary Thyroid Carcinomas (MTC) harbor somatic mutations in RET and RAS genes while roughly 40% of cases are still orphan of an oncogenic driver. Aim of this study was to investigate putative novel genetic alterations leading to pathogenesis of MTC from Whole Exome Sequencing (WES) data obtained in RET+ and RET- cases.
Methods:
WES analysis was performed on 6 sporadic MTC cases (2 RET+, 4 RET-) using an Illumina platform. Analyzing the list of Single Nucleotide Variations (SNV) shared by the RET- cases, we studied the gene CNOT1 presenting different SNV in the 4 RET- cases; among these SNV we chose a SNP, the R299Q, with a Minor Allele Frequency (MAF) ≤ 0.1. We first performed a validation panel of 83 sporadic MTC cases and 94 healthy controls. A larger case-control study was then performed on 874 MTC cases and 896 healthy controls. Genotyping was performed using a TaqMan SNP Assay (Life Technologies) on genomic DNA extracted from the blood of the patients. We also analyzed the presence of mutations in genes belonging to a panel of 344 oncogene panel list (NuGEN). We chose 7 SNV not present in dbSNP database as somatic mutation candidates: RB1 (R787Q), MSH2 (A189T), HIF1A (K334R), PCNA (V102I), FES (R764Q), THBS1 (G26A), NSD1 (P702S). These SNV were studied by direct sequencing on genomic DNA extracted from tumoral tissue and blood in order to evaluate their somatic or germinal nature.
Results:
On a first step of validation, we found that the frequency of the mutated allele (allele A: CGA/CAA) of the CNOT1 R299Q in our total series of 89 MTC cases was significantly higher than in the 94 healthy controls (0.045 vs 0.011, χ2=4.04, p=0.04); despite this, further validation on a larger case-control panel did not confirm the significant difference (0.0215 vs 0.0189, χ2=0.12, p=0.729). The validation obtained in the tissue and blood DNA of patient harboring the 7 oncogene SNV, revealed their germinal nature and they were not further studied.
Conclusions:
Despite the great potentiality of WES, in sporadic MTC cases it was unable to find any oncogenic driver alternative to RET and RAS. Nevertheless, the huge amount of data generated by WES will be further analyzed to complete the study
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