Risk Profile of the RET A883F Germline Mutation: An International Collaborative Study

years (median, 19 years). The earliest age of MTC, regional lymph node metastasis, and distant metastasis was 10, 20, and 20 years, respectively. Fifty percent penetrance of MTC and PHEO was achieved by age 19 and 34 years, respectively. Five- and 10-year survival rates (both overall and disease specific) were 88% and 88%, respectively. Biochemical cure for MTC at latest follow-up was achieved in 63% (five of eight carriers) with pertinent data. Conclusions: MTC of A883F carriers seems to have a more indolent natural course compared with that of M918T carriers. Our results support the classification of the A883F mutation in the ATA high-risk level. ( J Clin Endocrinol Metab 102: 2069 – 2074, 2017)

years (median, 19 years). The earliest age of MTC, regional lymph node metastasis, and distant metastasis was 10, 20, and 20 years, respectively. Fifty percent penetrance of MTC and PHEO was achieved by age 19 and 34 years, respectively. Five-and 10-year survival rates (both overall and disease specific) were 88% and 88%, respectively. Biochemical cure for MTC at latest follow-up was achieved in 63% (five of eight carriers) with pertinent data.
Shortly after these pivotal discoveries, strong genotypephenotype correlations were recognized (8,9). This provided the basis for individual risk profiles and establishment of the American Thyroid Association (ATA) MTC risk levels, according to the specific mutations (1,10).
In MEN2A, several risk profiles for mutations of codon 10, 11, and 15 have been well defined (11)(12)(13). In MEN2B, almost all published reports concern the M918T mutation, and little is known about the aggressiveness of the A883F mutation. Despite the lack of a well-defined risk profile, the A883F mutation has been reclassified from the highest to the high-risk level in the revised ATA guidelines for the management of MTC (1).
We conducted an international cohort study of A883F carriers for the purpose of creating a risk profile for appropriate classification in the ATA risk levels.

Patients
This international retrospective cohort study included 13 unique A883F carriers from eight unrelated families.
At the International Workshop on Multiple Endocrine Neoplasia (WorldMEN) 2014 in Vienna, Austria, the World-MEN 2016 workshop in Utrecht, the Netherlands, and during various other scientific meetings, all known MEN2 centers were asked to contribute relevant data to this study. Eleven A883F carriers were identified.
Data collection was performed using a uniform data sheet for each registrant. One carrier had been lost to follow-up several years before, and only data already published in relation to this carrier could be retrieved (15,18,19).
This study was approved by the respective institutional review boards for human subjects' protection in accordance with the ethical standards of each country and center.

Methods
The follow-up period was calculated from the date of MTC diagnosis to the date of death or latest follow-up. The date of MTC diagnosis was recorded as the date of initial thyroid surgery.
TNM (tumor-node-metastasis) staging was performed according to the seventh edition of the American Joint Committee on Cancer Staging Manual (26).
Biochemical cure was defined as basal serum calcitonin below the upper reference limit of the respective calcitonin assays at latest follow-up.

Statistical analysis
Continuous variables were calculated as the median and range. The Kaplan-Meier method was used for estimating agerelated penetrance of MTC and PHEO, overall survival, and disease-specific survival. All analyses were performed using Stata 14.1 (StataCorp, College Station, TX).

Results
A total of 13 carriers from eight unrelated families were included. Demographic, clinical, surgical, and follow-up data are shown in Table 1. De novo mutations were recorded in five (45%) of the 11 carriers with pertinent data.
Carriers and families originated from the United States (four carriers from one family), United Kingdom (four carriers from three families), France (two carriers from one family), Germany (one carrier from one family), Denmark (one carrier from one family), and Australia (one carrier from one family).

Normal/C-cell hyperplasia
Upon prophylactic thyroidectomy, carrier no. 4 was diagnosed with a normal thyroid, and carriers no. 1 to 3 were diagnosed with C-cell hyperplasia (Table 1). Median age at diagnosis was 7.5 years (range, 7 to 9 years).

MTC
Nine carriers were diagnosed with MTC. Median age at diagnosis was 19 years (range, 10 to 39 years).
Full TNM status at initial thyroid surgery was available in six carriers. MTC without metastasis (T1-4N0M0) was diagnosed in two carriers with a median age of 28.5 years (range, 25 to 32 years). MTC with regional lymph node metastasis but without distant metastasis (T1-4N1M0) was diagnosed in three carriers with a median age of 13 years (range, 10 to 39 years). Distant metastasis (T1-4N0M1 or T1-4N1M1) was diagnosed in one carrier at age 28 years. However, the earliest observed age of distant metastasis was 20 years, 10 years after initial thyroid surgery (carrier no. 6).
Median follow-up of the nine carriers with MTC was 12 years (range, 4 to 35 years). One carrier died (as a result of MTC) at 32 years of age after four years of follow-up. Eight carriers were still alive at latest followup. Five-and 10-year survival rates (both overall and disease specific) were 88% and 88%, respectively.
Basal serum calcitonin at latest follow-up was available for 12 of 13 A883F carriers. Nine of the 12 (75%) had achieved biochemical cure. Among the seven index carriers with pertinent data, four (57%) had achieved biochemical cure. Among the A883F carriers with MTC and pertinent data, biochemical cure was seen in five of eight (63%) ( Table 1).

PHEO
Five of 13 (38%) carriers were diagnosed with PHEO. The median age at diagnosis was 34 years (range, 11 to 44 years). Twenty-five percent, 50%, and 75% penetrance was achieved at 23, 34, and 39 years, respectively (Fig. 1). All PHEOs were bilateral and adrenal in location. None were malignant. All carriers had been diagnosed with MTC prior to PHEO. The median interval between the

Nonendocrine manifestations
Mucosal neuromas were shown in all but two carriers. Only the two youngest females, age 7 years, had no visible mucosal neuromas. Marfanoid habitus, thick eyelids, and narrow faces were described in six of the 13 patients. Pes cavus was diagnosed in three carriers.

Discussion
On the basis of this international retrospective cohort study of 13 A883F carriers from eight unrelated families, we created a risk profile for the A883F RET mutation. Our data show that the age-related penetrance of disease manifestations such as MTC and PHEO occur later in life than formerly thought.
Concerning age-related penetrance for MTC in our series, 50% penetrance of MTC in the A883F carriers was achieved by 19 years of age (Fig. 1). This is clearly later in life than in M918T carriers, who are grouped as having the highest risk mutation in the recently revised ATA guidelines; in this highest risk level, 50% penetrance of MTC was described at age 12 years (27). More similar to our results, the 50% penetrance for carriers of the C634R, C634W, and C634Y mutations (classified at the ATA high-risk level) has been reported at approximately 19, 30, and 33 years of age (12,28). Considerably more favorable was the age-related penetrance of MTC in exon 10 codons 609, 611, 618, and 620 (ATA moderate-risk level), ranging from age 34 to 44 years (Table 2) (11). Thus, in regard to age-related penetrance for MTC, the A883F mutation resembles the other mutations in the ATA high-risk level.
In regard to the earliest onset of MTC, a normal thyroid and C-cell hyperplasia without MTC were diagnosed in four carriers of age 7 to 9 years. The earliest age of diagnosis of MTC, regional lymph node, and distant metastasis was 10, 10, and 20 years, respectively. This is distinctively later than in the ATA highest-risk level. The earliest ages of MTC, regional, and distant metastasis in M918T carriers (ATA highest-risk level) have been reported at 0.17, 0.25, and 5 years of age, respectively (29)(30)(31). For mutation carriers of codon 634 (ATA high-risk level), the corresponding ages have been reported at 0.8, 5, and 20 years (30,32,33). The earliest ages of MTC, regional metastasis, and distant metastasis reported in carriers of mutations classified at the ATA moderate-risk level have been reported at age 1, 6, and 6 years, respectively (34,35). However, considerable variability in the age of earliest onset in regard to mutated codons and mutated amino acids has been reported in carriers belonging to the ATA moderate-risk level category (36). If one follows the rule of earliest onset, the A883F mutation appears to be most appropriately classified in the ATA high-risk or even ATA moderaterisk level.
Biochemical cure at the latest follow-up was achieved in 75% and 57% of our total cohort and our index carriers with pertinent data, respectively. In a study of M918T carriers, biochemical cure was found less frequently, in 23% (10 of 44) and 17% (seven of 41) of the total cohort and the index carriers, respectively (37). Thus, the proportion of A883F carriers achieving biochemical cure appears large compared with that of the M918T carriers. In a recent study, biochemical cure in C634R and C634Y carriers with MTC was seen in 58% (32 of 55) and 63% (22 of 35), respectively (38). Correspondingly, biochemical cure was achieved in 63% of the A883F carriers with MTC. When using biochemical cure for risk classification, the A883F cohort resembles the high-risk level.
Five-and 10-year survival rates (both overall and disease specific) were 88% and 88%, respectively. In a study of 18 patients with MEN2B and MTC, all of the 16 RET-tested patients were M918T carriers. Fiveand 10-year overall survival rates were 85% and 75%, respectively (27). Accordingly, the overall survival of A883F carriers seems favorable compared with that of M918T carriers, which also justifies classification of this mutation in the high-risk level.
PHEO was diagnosed at a median age of 34 years in the A883F carriers. This was 12 years later than seen in M918T carriers and eight years earlier than in exon 10 mutation carriers (11,27). Therefore, with respect to PHEO, stratifying the A883F mutation in the ATA highrisk level seems justified. This study shares limitations that are inherent to retrospective multinational studies of rare diseases. Small sample sizes limiting generalization are often seen when studying rare diseases, as in this study. To increase sample size, carriers of different amino acid substitutions within the same codon can be pooled (11). To the best of our knowledge, there has only been one report of a mutation in codon 883 other than the A883F mutation (39). However, inclusion of patients carrying the A883T mutation from this report was not appropriate, given that no MEN2B phenotype was described. The A883T mutation showed a very low transforming activity, as demonstrated by the absence of the MTC phenotype in heterozygous carriers. MTC was only present in two homozygous carriers (39). Retrospective studies are frequently associated with missing data. However, no data were missing with regard to key variables in this study, such as ages, procedure, and pathology at initial thyroid surgery. Data on status and biochemical cure at latest follow-up were present in 89% (eight of nine) of carriers with MTC. Full TNM status at initial thyroid surgery was available in 67% (six of nine) of carriers with MTC. MEN2B nonendocrine manifestations were present to some extent in all A883F carriers, but with this small data set, we were not able to describe if manifestations develop later or in a more discrete variant in A883F compared with M918T carriers.

Conclusion
MTC of A883F RET mutation carriers seems to have a more indolent natural course compared with that of M918T carriers. PHEO manifests later in A883F carriers than in M918T carriers. Our results support the classification of the A883F mutation in the ATA high-risk level.