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Departments of Endocrinology and Metabolism (R.E., B.C., C.R., V.B., G.R., E.M., L.A., A.V., A.P.), of Oncology (P.F., F.B.), and of Surgery (P.M., P.B.), University of Pisa 56100 Pisa, Italy; Department of Internal Medicine, Endocrinology and Metabolism, and Biochemistry (F.P.), University of Siena, 53100 Siena, Italy; and AMBISEN Center (A.P.), High Technology Center for the Study of the Environmental Damage of the Endocrine and Nervous Systems, University of Pisa, 56124 Pisa, Italy
Address all correspondence and requests for reprints to: R. Elisei, M.D., Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy. E-mail: relisei{at}endoc.med.unipi.it.
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Abstract |
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Methods: We studied 100 sporadic MTC patients with a 10.2 yr mean follow-up. RET gene exons 10–11 and 13–16 were analyzed. The correlation between the presence/absence of a somatic RET mutation, clinical/pathological features, and outcome of MTC patients was evaluated.
Results: A somatic RET mutation was found in 43 of 100 (43%) sporadic MTCs. The most frequent mutation (34 of 43, 79%) was M918T. RET mutation occurrence was more frequent in larger tumors (P = 0.03), and in MTC with node and distant metastases (P < 0.0001 and P = 0.02, respectively), thus, a significant correlation was found with a more advanced stage at diagnosis (P = 0.004). A worse outcome was also significantly correlated with the presence of a somatic RET mutation (P = 0.002). Among all prognostic factors found to be correlated with a worse outcome, at multivariate analysis only the advanced stage at diagnosis and the presence of a RET mutation showed an independent correlation (P < 0.0001 and P = 0.01, respectively). Finally, the survival curves of MTC patients showed a significantly lower percentage of surviving patients in the group with RET mutations (P = 0.006).
Conclusions: We demonstrated that the presence of a somatic RET mutation correlates with a worse outcome of MTC patients, not only for the highest probability to have persistence of the disease, but also for a lower survival rate in a long-term follow up. More interestingly, the presence of a somatic RET mutation correlates with the presence of lymph node metastases at diagnosis, which is a known bad prognostic factor for the definitive cure of MTC patients.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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A decade ago, we and others (8, 9, 10, 11) demonstrated that somatic RET gene mutations, which are present in about 40–50% of MTC, could represent a bad prognostic factor for the outcome of MTC patients. These studies were performed in small MTC series with short-term follow-ups. Furthermore, this observation has not been confirmed in several other series (12, 13, 14, 15). The aim of the present study was to reevaluate the role of somatic RET mutations as a bad prognostic factor in a large series of MTC with a median follow-up of 10 yr. In particular, we analyzed the relationship of somatic RET mutations with all the other epidemiological and pathological features of the tumors.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We studied 100 patients, 43 males and 57 females, affected by sporadic MTC with a mean age at diagnosis of 49.6 yr (range 20–83, median 49) and mean follow-up 10.2 ± 5.5 yr (range 3–32, median 10). All patients were submitted to total thyroidectomy and central neck dissection as the minimal standard procedure at the same institution. The lymphadenectomy of the lateral compartment(s) was performed during the first surgical treatment if node metastases were diagnosed before surgery.
All patients had been classified as having the sporadic form of MTC. This classification was based on the apparent absence of RET germline mutations, absence of familial history of the disease, and negative clinical and laboratory data for the presence of other endocrine neoplasia.
Clinical data were recorded in a computerized database and analyzed according to the presence or absence of a RET mutation.
Informed consent for RET genetic screening and other clinical procedures was signed by all investigated subjects.
Genomic DNA extraction and PCR amplification
After 1990, all primary MTC tissues were collected at surgery, immediately frozen in liquid nitrogen, and kept at –80 C. Paraffin-embedded primary MTC tumoral tissues were used for patients operated on before 1990. Blood from all patients was collected in EDTA. Genomic DNA was purified from peripheral blood lymphocytes using the QIAMP DNA mini kit (QIAGEN, Hilden, Germany) and from tumoral tissues using an in-house method based on overnight proteinase K digestion at 55 C, followed by phenol/chloroform extraction and ethanol precipitation. DNA was kept in Tris-EDTA at –20 C. An aliquot of DNA was also stored at –20 C with the intent of searching for new RET mutations whenever described.
The RET gene exons 10, 11, and 13–16 were analyzed in all cases using PCR and sequencing conditions, as previously reported (16). Whenever a RET point mutation altered (creating or deleting) the recognition site of a restriction enzyme, we performed the restriction analysis of the mutated exon to confirm the presence of the RET mutation.
Follow-up management
All MTC patients underwent a pentagastrin (Pg) stimulation test for Calcitonin (CT) and neck ultrasound 6 and 12 months after total thyroidectomy. Basal and stimulated CT negative patients were considered disease free with a very low risk of recurrence (17) and were submitted to another Pg stimulation test 3–5 yr later. Patients with detectable levels of basal and/or Pg-stimulated CT were evaluated with imaging techniques [computerized tomography scan, magnetic resonance imaging, and OctreoScan (Pickler, Cleveland, OH)] to reveal the localization of the metastatic disease. Patients with detectable levels of basal and/or Pg-stimulated CT and negative imaging results were defined as affected by a biochemically persistent disease (4).
Histology
MTC histological diagnosis was addressed by typical histological (i.e. tumoral cells arranged in trabecular, insular, or sheet-like growth patterns) and immunohistochemical (cells positive for calcitonin and chromogranin) findings.
Statistical analysis
The statistical analysis was performed with the 
2, t, and Mann-Whitney U tests according to the studied variables. Survival curves were analyzed using the Kaplan-Meier method, and the statistical significance was assessed by the log-rank test. The multiple logistic regression test was used to determine the independent effect of a somatic RET mutation and the other clinical and pathological features on the outcome of MTC patients. A P value less than 0.05 was considered significant.
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Results |
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Genetic analysis revealed a somatic RET mutation in 43 out of 100 (43%) sporadic MTCs (Fig. 1A
). The majority of RET mutations (34 of 43, 79%) were located at codon 918 of exon 16. The major mutation substituted a methionine (ATG) for a threonine (ACG). The other nine mutations were found in exons 10, 11, and 15. In particular, seven of 43 (16.2%) were at codon 634 of exon 11 (four C634R, two C634W, and one C634Y), one of 43 (2.3%) was a deletion of 48 bp in exon 10, and one of 43 (2.3%) was a 2-bp missense mutation at codon 883 in exon 15 that substituted an alanine (GCT) for a phenylalanine (TTT). No mutations were identified in exons 13 or 14 (Fig. 1B).
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Clinical evaluation
The mean age at diagnosis and mean follow-up of the 43 MTC patients (20 males, 23 females) carrying somatic RET mutations were 53.1 yr (median 55, range 21–83) and 9.1 yr (median 9, range 3–21), respectively. Node and distant metastases were present in 30 of 43 (70%) and 13 of 43 (30%) cases, respectively. According to the Tumor-Node-Metastasis classification (18), five patients had stage I disease (T1N0M0), eight had stage II (T2–4N0M0), 17 had stage III (T1–4N1M0), and 13 had stage IV (T1–4N1M1). At the time of the present study, seven patients were free of disease, 28 had persistent disease, and eight were dead from MTC. Among the 28 patients with persistence of the disease, 13 showed a biochemical persistence of the disease, with high levels of basal and/or Pg-stimulated CT, but no evidence of metastases, whereas 15 patients were affected by metastatic disease.
The mean age at diagnosis and mean follow-up of the 57 MTC patients (23 males and 34 females) without somatic RET mutation were 47.1 yr (median 48, range 20–71) and 10.7 yr (median 10, range 3–32), respectively. Node and distant metastases were present in 15 of 57 (26.3%) and seven of 57 (12.2%) cases, respectively. According to the Tumor-Node-Metastasis classification (18), 14 patients had stage I disease (T1N0M0), 24 had stage II (T2–4N0M0), 10 had stage III (T1–4N1M0), and nine had stage IV (T1–4N1M1). At the time of the present study, 32 patients were free of disease, 22 had persistent disease, and three were dead from MTC. Among the 22 patients with persistence of the disease, 13 showed a biochemical persistence of the disease, whereas nine patients were affected by metastatic disease.
RET mutation and MTC clinical features
We correlated the presence of somatic RET mutations with the clinical features and outcome of MTC patients. As shown in Table 1, somatic RET mutations were significantly more frequent in larger tumors (P = 0.03), in cases with node (P < 0.0001) and distant (P = 0.02) metastases. No correlation was found between the RET mutation and number of lymph node metastases (P = 0.8 and P = 0.5 by unpaired t and Mann-Whitney U tests, respectively; data not shown). Somatic RET mutations were also correlated with a more advanced stage at diagnosis (P = 0.004).
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RET mutation as a prognostic factor for the outcome of MTC patients
When comparing the presence of RET mutations and the outcome of MTC patients, we found a positive correlation between the presence of the somatic RET mutation and the persistence of the disease (disease free vs. persistence of disease or dead patients; P = 0.0002). No difference was found in the distribution of the somatic RET mutations when comparing MTC patients with only biochemical or metastatic disease (data not shown).
In addition to the presence of a RET mutation, tumor size (P = 0.0002), node metastases (P < 0.0001), distant metastases (P < 0.0001), and a more advanced stage at diagnosis (P < 0.0001) were significantly correlated with a worse outcome. At variance, no correlation of sex or age was revealed (Table 2).
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, the multivariate analysis, performed by logistic regression test, showed that only the advanced stage at diagnosis and the presence of the somatic RET mutation independently correlated with a worse outcome of MTC patients (P < 0.0001 and P = 0.01, respectively).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Among all somatic mutations, we found that 79% of RET mutations were at codon 918 of exon 16 (M918T). It is worth noting that multiple endocrine neoplasia type 2B (MEN 2B) syndrome, which is characterized by the most aggressive form of hereditary MTC, is also mainly associated with the germline M918T mutation (19, 20, 21). Among all RET mutations, M918T has had the highest penetrance and transforming activity (22, 23). On the basis of these considerations, we derive the conclusion that the worst outcome of somatic RET positive MTC patients is related to the presence of this highly transforming mutation. Unfortunately, the other somatic RET mutations are rare, and a comparison between patients with the somatic mutation at codon 918 vs. those with the other types of somatic mutations is not possible in our series. However, it is worth noting that among the other nine somatic mutations, seven were located at codon 634, which is the most frequently altered codon in MEN 2A, and one was at codon 883, which, although less frequent than M918T, also has been described as being associated with MEN 2B. On the contrary, none of the noncysteine coding codons with RET mutations, which have been demonstrated to have less transforming activity (24, 25) and to be more frequently associated with familial MTC (26), was found in this series of sporadic MTC. Another evidence supporting the hypothesis that the type of RET mutation is related to the degree of aggressiveness of the MTC is that tumors with mutation at codon 918 showed the highest prevalence of lymph node metastases (76.5%), with respect not only to negative cases (27%), but also those with RET mutation at codon 634 (43%).
The presence of metastatic nodes at the first surgical treatment is a well-known negative prognostic factor for the complete cure of the disease (6). In the present series, we demonstrated a statistically significant correlation between the mutation and lymph node metastases at diagnosis. This correlation can justify the negative role of somatic RET mutation in the outcome of these patients. In our opinion this finding also has a practical implication because the RET mutation can now be determined by analyzing DNA extracted from the tumoral thyroid nodule obtained by fine-needle aspiration biopsy (FNAB) (11). Although it is accepted that the surgical dissection of the lateral node compartment should be included whenever a presurgical diagnosis of node metastases is achieved, it is still under debate whether a modified radical neck dissection with removal of nodes in the ipsilateral or bilateral compartment should be performed on principle (27, 28). This controversy is of great significance because patients with MTC can be cured by the initial surgical treatment, thus, the most appropriate procedure should be chosen at that time. However, radical neck dissection can result in significant morbidity (29) and should be avoided if the tumor is still intrathyroid (stage I), a stage that has had a very low risk of recurrence and death (4, 5, 6, 7). The identification of a RET mutation in the DNA extracted from FNAB could allow for preoperative stratification of patients at high (RET positive) and low (RET negative) risk of developing lymph node metastases, and, as a consequence, the surgical strategy could include or omit radical neck dissection, respectively. However, because it has been reported that in some MTCs RET somatic mutations are not present in all tumoral cells (30), the possibility to obtain negative RET cells with FNAB makes less sensitive the suggested presurgical genetic screening.
In the era of the development of new targeted therapies, the demonstration that RET mutations are related to a worse outcome also has a direct therapeutic implication. RET positive cases might be treated with tyrosine kinase inhibitors and, in particular, with those having a higher affinity for RET (31). Although tumor RET gene mutation analysis could help to predict patients who will respond to RET-targeted therapies, unfortunately, the genetic test is not commercially available, and, at present, it is performed only in thyroid cancer-dedicated clinical units.
In conclusion, we demonstrated that the presence of a somatic RET mutation correlates, independent of other clinical and pathological features, with a worse outcome of MTC patients, not only in terms of the highest probability for disease persistence after surgery, but also for a lower survival rate at the long-term follow-up. This finding validates the role of somatic RET mutations as a bad prognostic factor for MTC. More interestingly, the presence of a somatic RET mutation correlates with the presence of lymph node metastases at diagnosis, thus suggesting a role of somatic RET mutation screening before surgery to have the decision of including bilateral neck node dissection in the standard surgical procedure of total thyroidectomy and central neck dissection. As far as a routine clinical application is concerned, this diagnostic approach should be cautiously suggested and eventually performed only in very specialized centers.
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Acknowledgments |
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Footnotes |
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The Department of Endocrinology and Metabolism of Pisa University is a World Health Organization Collaborating Center for the Diagnosis and Treatment of Thyroid Cancer and other Thyroid Diseases.
Disclosure Statement: The authors have nothing to disclose.
First Published Online December 11, 2007
Abbreviations: CT, Computerized tomography; FNAB, fine-needle aspiration biopsy; MEN 2B, multiple endocrine neoplasia type 2B; MTC, medullary thyroid carcinoma; Pg, prostaglandin.
Received August 1, 2007.
Accepted December 5, 2007.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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