A Nationwide Study of Multiple Endocrine Neoplasia Type 2A in Norway: Predictive and Prognostic Factors for the Clinical Course of Medullary Thyroid Carcinoma

Abstract

Background:

Multiple endocrine neoplasia type 2A (MEN 2A) is an autosomal dominant syndrome caused by activating germline mutations in the RET (REarranged during Transfection) proto-oncogene. MEN 2A has a strong (>95%) and age-dependent (5–25 years) clinical penetrance of medullary thyroid carcinoma (MTC). Several major studies have analyzed the predictive and prognostic factors for MEN 2A to find indicators that predict the optimal timing of prophylactic thyroidectomy. The aims of this study were to describe all known RET positive MEN 2A patients diagnosed in Norway and to evaluate the clinical course of MTC, as well as its predictive and prognostic factors.

Methods:

This nationwide retrospective cohort study included data for 65 (14 index and 51 screening patients) out of a total of 67 MEN 2A patients with the RET gene mutation who were diagnosed in Norway since 1974. Data were collected by reviewing patient files. The variables analyzed were genotype, phenotype, preoperative basal calcitonin, age at thyroid surgery, central lymph node dissection and nodal status at primary surgery, number of surgical procedures, and biochemical cure. Of the 65 patients, 60 had undergone thyroid surgery. The median follow-up period was 9.9 years. The patients were divided into pre-RET-and RET-era, which included patients who had thyroid surgery before January 1, 1994, and after, respectively.

Results:

In index and screening patients, MTC was found, respectively, in 100% and 45% of cases, central lymph node dissection at primary surgery was done for 64% and 52% of patients, and the median total number of surgical procedures was two (range 1–6) and one (range 1–4). At primary surgery, all patients (n = 13) with lymph node metastases had preoperative basal calcitonin levels ≥68 pg/mL, and all patients (n = 17) without central lymph node dissection and preoperative basal calcitonin <40 pg/mL were biochemically cured. Multivariate analysis showed that preoperative basal calcitonin was a significant predictive factor for MTC superior to age at thyroid surgery when analyzing the entire period (p = 0.009) and the RET-era separately (p = 0.021). Prognostic factors for biochemical cure were preoperative basal calcitonin, central lymph node dissection, and nodal status at primary surgery (p = 0.037, p = 0.002, and p = 0.005) when analyzing the entire period, but only nodal status at primary surgery when the RET-era was considered separately (p = 0.006).

Conclusions:

Preoperative basal calcitonin alone can serve as an indicator for optimal timing and the extent of thyroid surgery for MEN 2A patients that could be considered safe. The results are consistent with previously reported data.

Introduction

Multiple endocrine neoplasia type 2 (MEN 2) is a relatively rare autosomal dominant syndrome that manifests as two different clinical syndromes: MEN 2A, including familiar medullary thyroid carcinoma (FMTC), and MEN 2B (1,2).

MEN 2A has a strong penetrance of medullary thyroid carcinoma (MTC) with a >95% risk, and can be accompanied by bilateral pheochromocytoma (PCC; 15–50% risk) (1 –5) and hyperparathyroidism (HPT; 5–30% risk) (1,2,4 –6). Among all MTC patients, about 25% are caused by germline mutations in the RET gene; the majority (90–95%) of them MEN 2A or FMTC (2,7 –9). Most clinical investigators maintain that FMTC should not be considered as a freestanding syndrome, but rather as a variant of the MEN 2A disease spectrum (10).

MEN 2A is caused by activating germline mutations in the RET (REarranged during Transfection) proto-oncogene. RET comprises eight exons located on chromosome 10 (10q11.2), and >100 pathogenic mutations in different codons have been identified in this gene (4,7,10 –13). The age-dependent penetrance of MTC differs between the mutations, but MTC is generally the first manifestation and becomes clinically apparent when patients are between 5 and 25 years old, or even later (2,5,14 –17). Predictors for biochemical cure and survival are well described in many large studies that considered genotype, preoperative calcitonin level, age at thyroid surgery, and tumor stage at diagnosis, including lymph node metastasis (2,5,9,10,18 –24).

Genetic mutation analyses of the RET proto-oncogene were established in 1993, and genetic testing, which can detect 92–98% of all mutation carriers, of all MTC patients soon became the standard of care (7,17,25,26). Since MTC occurs in 95% of MEN 2A patients and mutations in different codons result in varying penetrance and disease manifestations, major studies have been carried out to identify indicators that predict the optimal timing and extent of prophylactic thyroid surgery (22,27). Guidelines and treatment recommendations issued by the American Thyroid Association (ATA; 2009/2015) and European Society of Endocrine Surgeons (ESES; 2014) were based on these and other major studies (2,10,26). A summary of ATA 2009/2015 is given in Table 1.

Table 1.

Risk Classification in MEN 2A According to American Thyroid Association Guidelines 2009/2015, Including Recommended Management and Age at Thyroid Surgery in Children and Adults with Confirmed RET Germline Mutation

					Recommended age at thyroid surgery and follow-up ^a
ATA risk class 2009	Codon mutated (selection)	Recommended age at thyroid surgery and follow-up ^a	ATA risk class 2015	Codon mutated (selection)	Child	Adult
A	790 804	May delay surgery beyond age five years if stringent criteria are met^b	MOD (moderate)	790 804 611	Thyroidectomy to be performed when the serum calcitonin level becomes elevated or in childhood if the parents do not wish to embark on a lengthy period of evaluation which might last for years or decades	At normal serum calcitonin level: annual testing of calcitonin.
B	611 618 620	Consider surgery before five years of age. May delay surgery beyond five years of age if stringent criteria are met^b		618620		If calcitonin becomes elevated: thyroidectomy and dissection of lymph node compartments depending on ultrasound findings and preoperative calcitonin levels.
C	634	Before five years of age	H (high)	634	Thyroidectomy at or before five years of age based on serum calcitonin levels

American Thyroid Association Guidelines (2,12).

Screening for PCC is recommended from 11–16 years of age, independent of calcitonin level. PCC must always be excluded before surgery.

A normal annual basal ± stimulated serum calcitonin, normal neck ultrasound, less aggressive MTC family history, and family preference.

MEN 2A, multiple endocrine neoplasia type 2A; ATA, American Thyroid Association; PCC, pheochromocytoma; MTC, medullary thyroid carcinoma.

Norway has a well-established familiar genetic screening program and has offered RET mutation analysis since 1994. Høie et al. presented the Norwegian experience with familial MTC in 1994 (28), and the first results after prophylactic thyroidectomy for RET proto-oncogene mutation carriers in Norway were presented in 2000 (29). MEN 2A genetic screening and treatment are regionalized. The aim of the present study was to describe all known RET-positive MEN 2A patients diagnosed in Norway since 1974, and evaluate the clinical course of MTC. Predictive and prognostic factors for MEN 2A were evaluated, including preoperative basal calcitonin, genotype, age at thyroid surgery, extent at primary surgery, number of surgical procedures, and nodal status at primary surgery. Furthermore, it was determined whether the results in the Norwegian series coincided with those from previous major studies.

Materials and Methods

Patients and data collection

For this nationwide study, a systematic search was conducted within all four Norwegian departments of medical genetics. A total of 67 patients with confirmed MEN 2A-causing RET mutations were registered in Norway as of the March 1, 2015, censoring date. The study included data from 65 MEN 2A patients. Two patients declined to participate and thus were not part of the specific data presentation, but they were included in the epidemiological calculations of incidence and prevalence. RET mutation analysis was not performed for one patient. However, the patient was a carrier according to the clinical state and was therefore included in the study. One Dutch patient, whose relatives were living in Norway, had RET mutation analyses done in Norway. The patient was not surgically treated and was included in the study.

Since 1974, 60 patients underwent thyroid surgery before the censoring date, and five patients were not yet surgically treated. The median postoperative follow-up was 9.9 years (range 0–39.5 years).

The patients were divided into index and screening patients. In this study, an index patient was the first family member with diagnosed MTC and a subsequently confirmed RET mutation. Screening patients had MEN 2A detected by RET mutation screening of the index MTC patient's first-degree relatives.

In the pre-molecular era (preRET-era), which was prior to 1994, the screening patients had thyroid surgery based on family history and/or elevated basal and/or stimulated calcitonin levels, with RET mutation confirmed by genetic testing after 1994. In the molecular era (RET-era) beginning in 1994, the screening patients had thyroid surgery based on RET mutation analysis. In index patients, the indication for surgery was diagnosed MTC or PCC. The RET consortium, diagnostic tools, and surgical approach have changed during the time period. To account for these time-dependent disparities and minimize time bias, the patients and analyses were divided into preRET-era and RET-era groups, which included those who had thyroid surgery before January 1, 1994, and after, respectively.

Primary surgery at the thyroid and lymph nodes was done in one or more procedures. Central (uni- or bilateral) and/or lateral lymph node clearance at primary surgery or later was not done in a regular pattern throughout the time period. In the present study, primary surgery was defined as one or more surgical procedures within the first year. Lymph node clearance and number of surgical procedures were evaluated at the primary surgery and, if applicable, cumulatively after repeat surgeries. The total number of surgical procedures included revisional neck surgeries along with mediastinal surgery and surgeries for other metastatic disease.

Data collection from the referral centers in Norway was done by reviewing patient files. The following data were recorded for all identified patients: (i) genotype and phenotype; (ii) clinical, radiological, and biochemical parameters at diagnosis and follow-up; (iii) age at thyroid surgery and reason why thyroid surgery was not performed at the recommended age; (iv) extent of surgery and number of surgical procedures; (v) histological results, including tumor stage in MTC; and (f) outcome.

Postoperative biochemical cure was defined as not measurable basal calcitonin below detectable levels (<2.0 pg/mL) at latest follow-up, and biochemical disease was defined as measurable basal calcitonin with no visible tumor in imaging examinations. Residual disease (not biochemically cured) included both visible structural disease (regional and/or distant metastases) at imaging and/or elevated calcitonin alone (biochemical disease).

RET mutation analysis

Genetic testing was performed according to international standards using reference sequence NM_020975.3. In the majority of the patients, exons 10–16 of the RET proto-oncogene were routinely sequenced. Based on recent scientific knowledge, genetic analyses were repeated in three surgically treated patients during the present study, with no change in the clinical consequences. Risk stratification was based on the mutated codon and ATA risk classification (2). For the tandem mutation Q781R/S904C, the risk classification was not determined, but the phenotype was FMTC (MEN 2A). In the present study, the mutations are included in the A risk class as defined by the ATA.

Calcitonin

Basal calcitonin levels were analyzed in all patients except for eight patients preoperatively and in all patients postoperatively. Due to varied praxis, stimulated (pentagastrin or calcium infusion) calcitonin measurements were not routinely performed either pre- or postoperatively in the study period and are not presented. During the study period, calcitonin was analyzed by routine immunoassay in two different laboratories: Oslo University Hospital and Haukeland University Hospital, Bergen. The assay methods have changed over time. Calcitonin Immulite 1000 and 2000 were used from 2007 and 2003, respectively, with a limit of detection <2.0 pg/mL. All results are reported in pg/mL. The conversion factor to pmol/L is 0.296. Reference values are <7.2 and <11 pg/mL in females and males, respectively. In children, especially neonates, reference values of up to 40 pg/mL are reported in the literature (30). In this study, 11 pg/mL was used as the cutoff for normal range for both females and males. According to ATA recommendations, prophylactic lymph node dissection is recommended for patients with a preoperative calcitonin >40 pg/mL (2,10). As such, in this study, 40 pg/mL was used as the cutoff for possible lymph node metastases.

Carcinoembryonic antigen

The presence of carcinoembryonic antigen (CEA) was reported in <43% of the patients preoperatively and 65% at latest follow-up. These data are not included in this study.

MTC pathology

Each of the four University Hospitals performed separate pathological examinations of thyroid specimens. The histological diagnosis of MTC was implemented according to applicable World Health Organization/International Agency for Research on Cancer tumor classifications (31). In this study, all surgical specimens were histopathologically classified according to the 7th revision of the TNM classification (UICC 2010) (32). The pTNM at primary surgery was defined as the total pTNM after one or more surgical procedures within the first year after diagnosis. Patients with lymph node clearance at primary surgery and lymph node metastasis were classified as pN1, and pN0 when there was no lymph node metastasis. Patients without lymph node dissection at primary surgery were classified as pNx. The presence of C cell hyperplasia (CCH) was investigated in all patients with no histological evidence of MTC.

Statistics

Differences in univariate analysis were explored. For continuous variables, non-parametric tests for independent samples (Mann–Whitney U-tests/Kruskal–Wallis test) were used, and group differences between the numbers of subjects were analyzed using Pearson's chi-square two-sided test. Predictive and prognostic factors for MTC and biochemical cure were explored in univariate analysis, and in multivariate analysis using logistic regression. Statistical significance was set at <0.05. Data were analyzed using IBM SPSS Statistics for Windows v21 (IBM Corp., Armonk, NY).

Study approval

The Regional Committee for Medical and Health Research Ethics (REC) of Western Norway approved the study. All living patients or their parents gave written informed consent. An exception to include deceased patients was granted by the REC.

Results

Epidemiology

Among the Norwegian MEN 2A patients included in the study, 63 were born in Norway: 19 before 1965, 32 between 1965 and 1994, and 12 between 1994 and 2015. During the last 50 years, the mean number of live births annually in Norway was 58,465 (33). With 44 MEN 2A patients born in the last 50 years (1965–2015), the incidence of patients with RET gene mutations giving rise to MEN 2A was 1/66,438 live births per year. Sixty-two MEN 2A patients were living in Norway on the censoring date. With a population of 5,165,000 inhabitants on the censoring date (33), the prevalence of MEN 2A in the Norwegian population was 1:79,462. For preRET-era and RET-era patients, 14 and 46 had thyroid surgery, respectively. In total there were 14 index and 51 screening patients. Five MEN 2A families were discovered before 1995, six families in 1995–2004, and four families in 2005–2014. Only one family was identified within the last five years.

Risk classification, genotype, and phenotypes

The phenotype, genotype, and risk classification for MEN 2A was classified according to the 2009 ATA guidelines for the entire study period and for the preRET- and RET-eras separately (Table 2). The ATA risk class distributions shifted toward the lower-risk groups in the RET-era, but the differences were not statistically significant (p = 0.186).

Table 2.

ATA Risk Classification (2009), ^a Genotype, Sex, and Phenotype in 65 Norwegian MEN 2A Patients

ATA risk classification	ATA risk classification, 1974–2015, n (%)	ATA risk classification, preRET-era, n (%)	ATA risk classification, RET-era, n (%)	p-Value	Codon	Number of families involved	Index/screening patients	Female/male	Surgical treated	MTC, n	PCC, n	HPT, n
A	27 (41.5)	3 (21)	24 (47)	0.186	781/904^b	1	1/3	2/2	3	3	0	0
					790	1	1/10	6/5	9	4	0	1
					804	2	2/10	6/6	11	5	1	0
B	18 (27.5)	6 (43)	12 (24)		611	2^c	1/4	4/1	5	1	2	1
					618	1	1/2	3/0	3	2	2	0
					620	1	1/9	7/3	9	4	0	0
C	20 (31)	5 (36)	15 (29)		634	7	7/13	8/12	20	18	12	6
Total, n (%)	65 (100)	14 (22)	51 (78)		N/A	15 (N/A)	14/51 (22/78)	36/29 (55/45)	60 (92)	37 (57)	17 (26)	8 (12)

The risk classification in this table is according to ATA guidelines 2009 (2) due to the retrospective aspect of the study.

For the tandem mutation Q781R/S904C, the risk classification is not determined, but the phenotype was FMTC (MEN2A) and the mutations were included in ATA A risk class for all the calculations. In the first analysis, single Q781R was found. The analysis was repeated in the three surgical treated patients, all with MTC.

In one of the families, the index patient is living in Denmark. Data not collected.

HPT, hyperparathyroidism; preRET-era, time before RET mutation analysis was available, until 1994; RET-era, from 1994; ND, not determined; N/A, not applicable.

Clinical and follow-up data for index and screening patients

Clinical and follow-up data in index and screening patients for the entire period, as well as preRET-era and RET-era alone, are summarized in Table 3. More advanced disease and worse outcome were found in the index patients compared with the screening patients, and the differences were significant. With a median postoperative follow-up of 14.1 years (range 0.8–39.5 years) in the index patients, two (14.3%) were biochemically cured, eight (57%) had biochemical disease only, two (14.3%) were living with regional or distant metastases, one (7.1%) died with distant metastasis, although it is unknown whether the cause of death was MTC, and one patient (7.1%) died from MTC. The screening patients had a median follow-up of 8.2 years (range 0–26.2 years). Among these patients, 38 (82.6%) were biochemically cured, seven (15.2%) had biochemical disease only, and one (14.3%) died from MTC. One index and one screening patient, who both had biochemical disease, died from other causes after the latest follow-up for MTC but before the censoring date.

Table 3.

Clinical and Follow-Up Data in 65 MEN 2A, Index and Screening, Patients from 1974 to 2015 and Divided into preRET-era and RET-era

	1974–2015			preRET-era			RET-era
	Index	Screening	p-Value ^a	Index	Screening	p-Value ^a	Index	Screening	p-Value ^a
Patients	14	51^b	N/A			N/A		43^b	N/A
Thyroid surgery^c	14	46		6^c	8^c		8	38
No thyroid surgery		5						5
Female/male (n)	10/4	26/25	0.173	6/0	5/3	0.091	4/4	21/22	0.952
Basal calcitonin before surgery (pg/mL), median (range)	1282 (55–6999), n = 7	16 (<2, 0–1962), n = 49^d	<0.001	5090 (3180–6999), n = 2	494 (80–639), n = 8	0.044	408 (55–2662), n = 5	11.6 (<2.0–1962), n = 41^d	<0.001
Basal calcitonin >11 pg/mL before surgery, n (%)	7 (100), n = 7	34 (67), n = 50^d	0.052	2, n = 2	8, n = 8	N/A	5, n = 5	25, n = 42^d	0.058
60 patients had thyroid surgery
Age (years) at thyroid surgery, median (range)	39 (28–71)	18.5 (4–72)	0.001	35 (28–41)	19.5 (13–23)	0.001	40.5 (28–71)	19 (4–72)	0.012
Patients with MTC, n (%)	14 (100)	23 (45)	0.001	6 (100)	8 (100)	0.001	8 (100)	15 (39)	0.002
pT1–3, n (%)^e	8 (57)^f	23 (45)^g,h	0.084	1 (17)	8 (100)^g	0.035	7 (87.5)^f	15 (39)^h	0.013
pT4, n (%)	2 (14)ⁱ	0	0.002	1 (17)	0	0.035	1 (12.5)ⁱ	0 (0)	0.028
pTx	4 (29)^j	0	<0.001	4 (66)^j	0	0.006	0 (0)	0 (0)	N/A
CLX at primary surgery, n (%)	9 (64)	24 (52)	0.425	1 (16.5)	3 (37.5)	0.393	8 (100)	21 (55)	0.017
Unilaterally, n	1	1		0	1		1	0
Bilaterally, n	8	23		1	2		7	21
Not performed, n (%)	5 (36)	22 (48)		5 (83.5)	5 (62.5)		0	17 (45)
CLX (uni- or bilaterally) performed later than primary surgery, n (%)	3 (21)	0	0.003	3 (50)	0	0.040	0	0	N/A
Not available	1 (7)			1 (16.5)
LLX (overall, uni- or bilaterally) at primary surgery or later, n (%)	9 (64)	8 (17)	0.001	5 (83)	4 (50)	0.198	4 (50)	4 (11)	0.007
Number of surgical procedures, median (range)
At primary surgery	1 (1–3)	1 (1–3)	0.010	1 (1–2)	1 (1–3)	0.755	1 (1–3)	1 (1–3)	0.102
In total	2 (1–6)	1 (1–4)	<0.001	2.5 (2–6)	1.5 (1–4)	0.228	1 (1–5)	1 (1–3)	0.154
Nodal status at primary surgery,^e n (%)
pN0	3 (21)	17 (37)		0	0 (0)		3 (37.5)	17 (44.5)
pN1	6 (43)	7 (15)	0.086	1 (17)	3 (37.5)	0.393	5 (62.5)	4 (10.5)	0.002
pNx	5 (36)	22 (48)		5 (83)	5 (62.5)		0	17 (44.5)
Postoperative follow-up (years), median (range)	14.1 (0.8–39.4)	8.2 (0–26.2)	0.110	25 (20.8–39.5)	23.6 (15.1–26.2)	0.282	4.7 (0.8–15.8)	5 (0–20)	0.966
Basal calcitonin at last follow-up (pg/mL), median (range)	120 (<2.0–17,953), n = 14	<2.0 (<2.0–14,217), n = 46	<0.001	131 (11.6–3725), n = 6	28.2 (<2.0–14,217), n = 8	0.345	4.2 (<2.0–17,953), n = 8	<2.0 (<2.0–18.5), n = 38	0.003
Biochemical cure^k at last follow-up, n (%)	2 (14)	38 (82)^l	<0.001	0	4 (50)	0.04	2 (25)	34 (89)^l	<0.001

p-Value was calculated by independent samples Mann–Whitney test (continues variables) and Pearson's chi-square two-sided test (group variables).

46 patients underwent surgery (censoring date March 1, 2015.

Two index and one screening patient had thyroidectomy in two surgical procedures, all in the preRET-era.

Preoperative basal calcitonin analysis was done in 50 patients. The exact value was reported in 49 patients, and in one patient the value was reported as elevated in the patient's file.

UICC, 7th revision (2009).

MTC in one index patient with thyroid surgery because of goiter in the RET-era, clinically considered preoperatively and was defined as pT3.

MTC in one screening patient with total thyroidectomy alone, preRET-era, biochemical cured and was defined as pT1–3.

MTC in one screening patient with multifocal MTC, N0 in the RET-era, biochemical cured and was defined as pT1–3.

MTC in one index patient with advanced disease in the early RET-era, died of the disease, clinically considered preoperatively and was defined as pT4.

In four patients with thyroid surgery in 1974, 1977, 1987, and 1992, the MTC diameter was not available. Three are alive with biochemical disease only, and one patient died with metastasis to the hepatic of unknown origin.

Biochemical cure was defined as not measurable calcitonin (<2.0 pg/mL) at latest follow-up.

In one patient with codon 620 mutation, preoperative calcitonin was 11 pg/mL, thyroid surgery without lymph node dissection was done at 6 years of age, and basal calcitonin was 2.4 pg/mL after 14.8 years of follow-up. The patients was considered as biochemically cured.

CLX, central lymph node clearance; LLX, lateral lymph node clearance.

In five patients (ATA A, n = 4; ATA B, n = 1), thyroid surgery has not yet been performed. One 75-year-old patient wanted to postpone surgery. One patient died at 85 years of age without clinical evidence of MTC. Two children, five and eight years old, will have regular follow-ups with calcitonin measurements and neck ultrasounds until the optimal time for thyroid surgery. In one 36-year-old patient without sign of disease, thyroid surgery was postponed under close follow-up as per the patient's preference.

MTC patients in the preRET-era and RET-era were compared, and surgical data concerning lymph node clearance and number of surgical procedures were analyzed. Significant differences between preRET-era and RET-era patients were found for central lymph node clearance at primary surgery (29% vs. 100%; p < 0.001), median number of total surgical procedures (2 [range 1–6] vs. 1 [range 1–5; p = 0.011), and median number of patients with one surgical procedure (30% vs. 78%; p = 0.003), suggesting that the surgical approach improved over time. There was no significant difference in biochemical cure between preRET- and RET-era MTC patients (28.5% vs. 56.5%; p = 0.098), but naturally there was a different median follow-up time (24.5 years [range 15.1–39.5 years) vs. 5 years [range 0.1–20 years; p < 0.001).

Factors predicting MTC

Factors predicting MTC were analyzed in the preRET-era and RET-era as well as for the entire period (Table 4). All patients in the preRET-era had MTC. For the entire period and the RET-era alone, there was a significant difference by univariate analysis in preoperative basal calcitonin, genotype, and age at thyroid surgery (Table 4). However, by multivariate analysis, preoperative basal calcitonin was significant predictive factor for MTC superior to age at thyroid surgery for the entire period and the RET-era alone (Table 5). When adding genotype to the logistic regression, basal calcitonin was not statistical significant, but had the highest significance compared with age at thyroid surgery and genotype. There was no difference in CCH between the MTC and non-MTC groups. When the preRET-era and RET-era MTC patients were compared, RET-era patients showed significantly lower preoperative calcitonin levels and had a higher percentage of ATA group C patients (p = 0.015).

Table 4.

Factors Predicting MTC in 60 Surgically Treated Norwegian MEN 2A, Index and Screening, Patients from 1974 to 2015 and Divided into preRET-era and RET-era: Univariate Analysis

	1974–2015			preRET-era			RET-era
	No MTC	MTC	p-Value ^a	No MTC	MTC	p-Value ^a	No MTC	MTC	p-Value ^a	MTC in 14 preRET-era vs. 23 RET-era patients, p-value ^a
Number	23	37	N/A	0	14	N/A	23	23	N/A
Basal calcitonin before surgery (pg/mL), median (range)	6.5 (<2.0–95),^b n = 23	177 (3.7–6999),^c n = 29	<0.001		543 (78–6999),^c n = 10		6.5 (<2.0–94.7), n = 23	85 (3.7–2662),^c n = 19	<0.001	0.006
Codon (n)
611	4	1			0		4	4
618	1	2			6		1	1
620	5	4			4		5	5
634	2	18	0.015		5		2	13	0.002	0.011
781	0	3			0		0	3
790	5	4		N/A	0	N/A	4	4
804	6	5			3		2	2
ATA A	11	12			3		11	9
ATA B	10	7	0.005		6		10	1	<0.001	0.015
ATA C	2	18			5		2	13
Age (years) at thyroid surgery (median (range))	17 (4–64)	32 (6–72)	0.017		22.5 (13–41)		17 (4–64)	39 (6–72)	0.011	0.122
C-cell hyperplasia,^d n (%)	18 (78), n = 23	14 (64), n = 22	0.337		1 (N/A), n = 2		18 (78), n = 23	13 (65%), n = 20	0.334	0.679

p-Value was calculated by independent samples Kruskal–Wallis test (continuous variables) and Pearson's chi-square two-sided test (group variables).

The patient with a preoperative basal calcitonin of 95 pg/mL had thyroidectomy, central and lateral neck dissection bilaterally, and c-cell hyperplasia but not MTC.

The patient with a preoperative basal calcitonin of 3.7 pg/mL had thyroidectomy alone, and a 1 mm MTC was found.

C-cell hyperplasia was examined in 93% (43/46) of the patients in the RET-era, but only in 14% (2/14) of the patients in the preRET-era.

Table 5.

Predictive Factors for MTC in a Total of 60 Surgically Treated MEN 2A, Index and Screening, Patients from 1974 to 2015 and Divided into preRET-era and RET-era: Logistic Regression Analysis

	MTC in MEN 2A, 1974–2015 ^a				MTC in MEN 2A, preRET-era ^b				MTC in MEN 2A, RET-era ^c
		CI				CI				CI
	OR	Lower	Upper	p-Value	OR	Lower	Upper	p-Value	OR	Lower	Upper	p-Value
Basal calcitonin before surgery	1.174	1.041	1.325	0.009					1.161	1.023	1.317	0.021
Age at thyroid surgery	0.999	0.955	1.045	0.961					1.001	0.957	1.047	0.970
Age at thyroid surgery	1.053	1.007	1.101	0.024					1.068	1.004	1.136	0.038
Genotype^d				0.081								0.174
Basal calcitonin before surgery	1.194	0.995	1.433	0.057		N/A			1.187	0.986	1.428	0.070
Genotype^d				0.810								0.829
Basal calcitonin before surgery	1.208	0.959	1.522	0.109					1.197	0.946	1.515	0.134
Age at thyroid surgery	0.993	0.920	1.072	0.855					0.995	0.921	1.074	0.893
Genotype^d				0.824								0.838

Statistically significant values are shown in bold.

Complete data set in 23 patients without MTC and 29 patients with MTC (Table 4).

Complete data set in 10 patients with MTC. All preRET-era patients had MTC (Table 4).

Complete data set in 23 patients without MTC and 19 patients with MTC (Table 4).

CI and OR for the categorical variable genotype with subgroups are not shown.

OR, odds ratio; CI, confidence interval.

Prognostic factors associated with tumor stage and clinical outcome

Among the 46 surgically treated MEN 2A screening patients and following the 2009 ATA guidelines (2), only 10 patients had thyroid surgery at the recommended age (±1 year). For the other 36 patients, reasons for later thyroid surgery included: (i) index patient was diagnosed late (23 patients); (ii) patients were in the preRET-era (eight patients); and (iii) thyroid surgery was postponed under regular follow-up with calcitonin measurement and neck ultrasound under patient/doctor agreement (five patients).

Among the 36 screening patients treated later than the recommended age, the nodal status was pN0, pN1, and pNx in 15, 7, and 14 patients, respectively. The median preoperative calcitonin was 34.7 pg/mL (range <0.2–1962 pg/mL), with 33% of patients with calcitonin <11 pg/mL, 50% with calcitonin <40 pg/mL, and 78% achieving biochemical cure. Of the 14 screening patients without central lymph node dissection (pNx), 13 (93%) achieved biochemical cure, all with a preoperative basal calcitonin <40 pg/mL. Seven of the screening patients, two in the preRET-era and five in the RET-era, had lymph node metastasis at primary surgery (Table 3). The youngest was 12 years old and had a mutation at codon 634. All seven patients had thyroid surgery later than the age recommended in the 2009 ATA guidelines (2). At latest follow-up, one of these seven patients had achieved biochemical cure, five patients had biochemical disease only, and one patient had developed metastatic disease and died.

Among the 10 screening patients with thyroid surgery at the recommended age, all were treated in the RET-era, and had a median preoperative basal calcitonin of 12.6 pg/mL (range 4.4–51.3 pg/mL). The nodal status of these 10 patients at primary surgery was pN0, pN1, and pNx in two, zero, and eight patients, respectively, and almost all achieved biochemical cure. One patient had a preoperative basal calcitonin of 11 pg/mL, thyroid surgery without lymph node dissection at six years old, a mutation at codon 620, and a calcitonin of 2.4 pg/mL after 14.8 years of follow-up. This patient was classified clinically as biochemically cured. For one patient with a preoperative basal calcitonin >40 pg/mL, central lymph node clearance was performed with pN0 status, and the patient achieved biochemical cure.

Among the 13 index and screening patients (four preRET-era and nine RET-era) with detected lymph node metastasis at primary surgery (pN1), all patients with available preoperative calcitonin values (11/13) had preoperative basal calcitonin ≥68 pg/mL.

Among the 27 index and screening patients (10 preRET-era and 17 RET-era) for whom no central lymph node dissection was done at primary surgery (pNx), the median preoperative basal calcitonin in 23 patients with available calcitonin analysis results was 10.6 pg/mL (<2.0–3180 pg/mL), and 74% had calcitonin levels <40 pg/mL. Biochemical cure was achieved in 78% (21/27) of these patients, but all patients with preoperative basal calcitonin levels <40 pg/mL were biochemically cured. Calcitonin values were available for all patients at latest follow-up. All pNx patients treated in the RET-era were biochemically cured. The six pNx patients who did not achieve biochemical cure had thyroid surgery in the preRET-era (1974, 1974, 1977, 1987, 1988, and 1992), and preoperative basal calcitonin was available for only two patients (560 pg/mL, 1988; 3180 pg/mL, 1987).

Prognostic factors associated with biochemical cure in the surgically treated index and screening patients (group I) were analyzed for the entire period (Table 6). To reduce time-dependent disparities, the results were analyzed in more uniform patient groups as index and screening patients in the preRET-era (group II) and RET-era (group III), screening patients in the RET-era (group IV), and index and screening patients with MTC in the RET-era (group V); (Table 7). In groups I and III, age at thyroid surgery (p = 0.014), preoperative basal calcitonin (p < 0.001), number of surgical procedures at primary surgery and in total (refers to group III), and nodal state (p < 0.001) at primary surgery were shown to be prognostic factors for biochemical cure on univariate analysis. In group III, central lymph node dissection at primary surgery was also shown to be a significant prognostic factor for biochemical cure. In group I patients who had not achieved biochemical cure, there was a significantly higher percentage of patients treated in the preRET-era, and a relatively higher predominance of index patients compared with screening patients.

Table 6.

Prognostic Factors Associated with Biochemical Cure in a Total of 60 Surgically Treated MEN 2A, Index and Screening, Patients from 1974 to 2015, Group I: Univariate Analysis

	Biochemical cure, ^a n = 40 ^c	No biochemical cure, n = 20	p-Value ^b
Before thyroid surgery
Index patients, n (%)	2 (5)	12 (60)	<0.001
Screening patients, n (%)	38 (95)	8 (40)
Basal calcitonin before surgery (pg/mL), median (range)	12 (<2.0–1962), n = 38	494 (6.8–6999), n = 14	<0.001
Basal calcitonin <40 pg/mL before surgery, n (%)	27 (71)	0	<0.001
	n = 38	n = 14
Codon (n)
611	4	1
618	1	2
620	7	2
634	10	10	0.313
781	2	1
790	8	1
804	8	3
Era
PreRET-era	4 (10)	10 (50)	0.001
RET-era	36 (90)	10 (50)
Surgical treatment
Age (years) at thyroid surgery, (median (range)	18.5 (4–64)	35 (12–72)	0.014
Surgery at recommended age after risk level,^c n (%)	10 (25)	0	0.014
CLX at primary surgery, n (%)	19 (47.5)	14 (70)	0.099
Unilaterally, n	1	1
Bilaterally, n	18	13
Not performed, n (%)	21 (52.5)	6 (30)
Number of surgical procedures, median (range)
At primary surgery	1 (1–1)	1 (1–3)	<0.001
In total	1 (1–1)	2 (1–6)	<0.001
Histopathological results
MTC	17 (42.5)	20 (100)	<0.001
Nodal status at primary surgery^d
pN0, n (%)	18 (45)	2 (10)	<0.001
pN1, n (%)	1 (2.5)	12 (60)
pNx, n (%)	21 (52.5)	6 (30)
Follow-up
Postoperative follow-up (years), median (range)	6.5 (0–26.2)	15.8 (0.8–39.4)	0.004
Basal calcitonin at last follow-up (pg/nL), median (range)	<2.0 (<2.0–2.4),^e n = 40	87 (2.7–17,953), n = 20	<0.001

Biochemical cure was defined as <2.0 pg/mL.

p-Value was calculated by independent samples Mann–Whitney test (continuous variables) and Pearson's chi-square two-sided test (group variables).

According to ATA guidelines 2009.

UICC, 7th revision (2009)

In one screening patient with codon 620 mutation, RET-era, preoperative calcitonin was 11 pg/mL, thyroid surgery without lymph node dissection was done at six years of age, and basal calcitonin was 2.4 pg/mL after 14.8 years follow-up, considered as biochemically cured.

Table 7.

Prognostic Factors Associated with Biochemical Cure in a Total of 60 Surgically Treated Index and Screening MEN 2A Patients in the preRET-era and RET-era, Screening Patients Only in the RET-era, and Index and Screening Patients with MTC in the RET-era: Univariate Analysis

	Group II, preRET-era, index and screening patients, n = 14			Group III, RET-era, index and screening patients, n = 46			Group IV, RET-era, screening patients only, n = 38			Group V, RET-era with MTC, index and screening patients, n = 23
	BC, ^a n = 4	NBC, n = 10	p-Value ^b	BC, ^a n = 36^c	NBC, n = 10	p-Value ^b	BC, ^a n = 34	NBC, n = 4	p-Value ^b	BC, ^a n = 13	NBC, n = 10	p-Value ^b
Before thyroid surgery
Index patients, n (%)	0	6 (60)	0.040	2 (5.5)	6 (60)	<0.001	N/A	N/A	N/A	2 (15)	6 (60)	0.026
Screening-patients, n (%)	4 (100)	4 (40)		34 (94.5)	4 (40)					11 (85)	4 (40)
Basal calcitonin before surgery (pg/mL), median (range)	213 (80–629), n = 4	600 (461–6999), n = 6	0.067	11.3 (<2.0–962), n = 34	258 (68–2662), n = 8	<0.001	5.8 (<2.0–1962), n = 33	109 (68–178), n = 4	0.005	51 (3.8–1962), n = 11	258 (65–2662), n = 8	0.009
Basal calcitonin <40 pg/mL before surgery, n (%)	0, n = 4	0, n = 6	N/A	27 (75), n = 34	0, n = 8	<0.001	27 (79), n = 33	0	0.001	5, n = 11	0, n = 8	0.026
Codon (n)
611	0	0		4	1		4	0		0	1
618	0	2		1	0		1	0		0	0
620	2	2		5	0		5	0		0	0
634	2	3	0.359	8	7	0.116	7	4	0.089	6	7	0.394
781	0	0		2	1		2	0		2	1
790	0	0		8	1		8	0		3	1
804	0	3		8	0		7	0		2	0
Surgical treatment
Age (years) at thyroid surgery, median (range)	17.5	29	0.014	20	40.5	0.017	17.5	42	0.255	34	40.5	0.208
	(13–21)	(16–41)		(4–64)	(12–72)		(4–64)	(12–72)		(6–59)	(12–72)
Surgery at recommended age after risk level,^d n (%)	0	0	N/A	10 (28)	0	0.060	10 (29)	0	0.206	2 (15)	0	0.194
CLX at primary surgery, n (%)	0	4 (40)	0.134	19 (53)	10 (100)	0.006	17 (50)	4 (100)	0.057	12 (92)	10 (100)	0.370
Unilaterally, n	0	1		1	0		0	0		1	0
Bilaterally, n	0	3		18	10		17	4		11	10
Not performed, n (%)	4 (100)	6 (60)		17 (47)	0		0	0		1 (7.5)	0
Number of surgical procedures, median (range)
At primary surgery	1 (1–1)	1 (1–3)	0.454	1 (1–1)	1 (1–3)	0.156	1 (1–1)	1 (1–1)	1.0	1 (1–1)	1 (1–3)	0.232
In total	1 (1–1)	3 (2–6)	0.002	1 (1–1)	1.5 (1–5)	0.015	1 (1–1)	1.5 (1–3)	0.113	1 (1–1)	1.5 (1–5)	0.042
Histopathological examination
MTC	4 (100)	10 (100)	N/A	13 (36)	10 (100)	<0.001	11 (32)	4 (100)	0.009	13 (100)	10 (100)	N/A
Nodal status at primary surgery^e
pN0, n (%)	0	0		18 (50)	2 (20)		16 (47)	1 (25)		11 (85)	2 (20)
pN1, n (%)	0	4 (40)	0.134	1 (3)	8 (80)	<0.001	1 (3)	3 (75)	<0.001	1 (7.5)	8 (80)	0.002
pNx, n (%)	4 (100)	6 (60)		17 (47)	0		17 (50)	0		1 (7.5)	0
Follow-up
Postoperative follow-up (years), median (range)	24.8 (19.9–26.2)	23.6 (15.1–39.5)	0.733	5.5 (0–20)	5 (0.8–15.8)	0.454	5.4 (0–20)	8.9 (4–15.8)	0.343	5 (0.1–20)	5 (0.8–15.8)	0.552
Basal calcitonin at last follow-up (pg/nL), median (range)	<2.0 (<2.0–<2.0), n = 4	132 (11.6–14,217), n = 10	0.002	<2.0 (<2.0–2.4),^d n = 36	14.4 (2.7–17,953), n = 10	<0.001	<2.0 (<2.0–2.4), n = 34	5.3 (2.7–18.5), n = 4	<0.001	<2.0 (<2.0–<2.0), n = 13	7.5 (2.7–17,953)	<0.001

Biochemical cure was defined as <2.0 pg/mL.

p-Value was calculated by an independent samples Mann–Whitney test (Continues variables) and Pearson's chi-square two-sided test (group variables).

In one screening patient with codon 620 mutation, RET-era, preoperative calcitonin was 11 pg/mL, thyroid surgery without lymph node dissection was done at 6 year of age, and basal calcitonin was 2,4 pg/mL after 14,8 years follow up, considered as biochemical cured.

According to ATA guidelines 2009.

UICC, 7th revision (2009).

BC, biochemical cure; NBC, no biochemical cure.

In group IV, 15/38 screening patients in the RET-era had MTC, and significant prognostic factors were preoperative basal calcitonin and nodal status at primary surgery, while age at thyroid surgery did not have prognostic significance for this group.

In group V, patients without MTC were excluded from the analyses, and significant prognostic factors in this group were preoperative basal calcitonin, total number of surgical procedures, and nodal status at primary surgery.

Logistic regression analyses (not shown) of prognostic factors for biochemical cure were performed for groups I–V, and also for MTC patients (37 patients) in the entire period (group VI). In all groups, three sets of parameters were analyzed: (i) preoperative basal calcitonin, genotype, age at thyroid surgery, central lymph node clearance at primary surgery, number of surgical procedures at primary surgery, and nodal status at primary surgery; (ii) the same parameters as (i), except age at thyroid surgery and genotype; and (iii) same parameters as (i), except central lymph node clearance at primary surgery and number of surgical procedures at primary surgery. The only significant prognostic factor for biochemical cure in these 18 sets of multivariate analyses was central lymph node dissection at primary surgery and nodal status at primary surgery in Ib (p = 0.002/p = 0.005) and VIb (p = 0.030/p = 0.015) and nodal status at primary surgery alone in IIIb (p = 0.006), IVb (p = 0.014), and Vb (p = 0.021). Preoperative basal calcitonin was also significant in group Ib (p = 0.037).

Discussion

Epidemiology

Genetic counseling and screening procedures in Norway have been in place for many years (28,29). Only four regional centers are involved in genetic counseling, and all four centers took part in the data collection for this multicenter study. RET gene mutation analysis is also centralized, which minimized selection bias. Because of these testing protocols, it can be assumed that all registered MEN 2A patients constitute the majority of MEN 2A patients in Norway in the RET-era. Nonetheless, the possibility cannot be ruled out that MEN 2A patients who were diagnosed in the preRET-era may have been missed. The calculated incidence of RET gene mutations that produce MEN 2A of 1:66,438 live births in Norway over the last 50 years was slightly higher than that reported in a 2013 study by Machens et al. that involved German patients (25). This study found that the minimum overall incidence of RET gene mutation (both MEN 2A and B) over a 50-year period (1951–2000) was 1:100,000–1:200,000 live births per year.

The prevalence (1:79,462) in Norway was lower than the incidence. Usually, prevalence is well above incidence, as the observed prevalence is a function of several previous years of incidence. However, working with an inherited disease, it is generally considered more relevant to calculate incidence per live births than for all person years. With incidence defined as number of cases per live births, the incidence is the proportion of newborns that carry the given marker. In practice, this definition will lead to an approximately equal incidence and prevalence for stable populations, whereby only small changes in time trends or mortality could yield an incidence that exceeds the observed prevalence. In addition, the calculated incidence applies up to 50 years of age, whereas the prevalence applies to the entire lifetime. With an average annual birth rate of 58,465 in Norway over the last 50 years together with an incidence of 1:66,438, 0.88 RET individuals carrying MEN 2A gene mutations would be estimated to be born each year. Given the average life expectancy of 80 years in Norway and the low mortality rate for MEN 2A patients, the prevalence can be calculated as 1:73,786. Although the actual prevalence and calculated prevalence do not differ significantly, the possibility exists that a few MEN 2A patients born before 1965 may have been missed by this study.

The number of new families with MEN 2A has been declining asymptotically in the decades after 1994, assuming few undiscovered families.

Risk classification, genotype, and phenotype

Relative to international reports, Norwegian MEN 2A patients had a predominance of lower risk in the ATA A and B groups, and fewer patients in the higher risk ATA C group (1,7,14,21). When analyzed alone, the RET-era group had an even higher proportion of ATA A cases relative to when all patients were considered. For the preRET-era group, MTC in low-risk MEN 2A (FMTC) patients was more likely to be misclassified as sporadic MTC compared with the RET-era patients. For these reasons, making comparisons between results for the patients in this study with those from older international reports is difficult. In this study, PCC and HPT were reported for 26% and 12% of patients, respectively. This outcome, along with the ATA classifications, is consistent with previous findings showing that PCC and HPT penetrance in ATA risk classes A and B is lower than that for class C (2,3,6,15).

One family included in the study carried a single Q781R mutation. According to the 2015 ATA guidelines, a double RET mutation in the same allele involving RET codon 804 (V804M) and the RET mutations Y806C, S904C, E805K, or Q781R were reported as being atypical MEN 2B. Similar germline double mutations in RET have also been identified in families with an FMTC variant of MEN 2A (10). Meanwhile, a 2002 study by Maschek et al. reported a patient with a single Q781R mutation in RET (34), while Nakao et al. reported both a single (Q781R) and double mutation with Q781R/V804M in the same family (35). Based on these findings, the stored blood samples were reanalyzed for the three surgically treated patients who carried the Q781R RET mutation. Notably, these patients were members of the same family. Each of the three patients carried a double RET mutation in the same allele that involved Q781R and S904C but not V804M. To the authors' knowledge, this double mutation has not been previously reported, and thus this combination will be further evaluated. At primary surgery, all three patients had MTC with a phenotype of FMTC (MEN2A), but none had PCC, and thus this RET mutation was classified as ATA A (10,35,36).

Predictive and prognostic factors

In the preRET-era, patients were diagnosed later and cured less often than patients in the RET-era. Furthermore, lymph node dissection was less frequently performed in the preRET-era. Current calcitonin assays are much more sensitive than those used previously. By analyzing the data for predictive and prognostic factors for the preRET-era and the RET-era groups separately, these time-dependent disparities were taken into account.

When analyzing either the RET-era patients separately or the entire period, preoperative basal calcitonin was predictive factor for MTC superior to age at thyroid surgery. Bringing genotype into the analysis, preoperative basal calcitonin was not statistical significant but had the highest significance in the analysis. The loss of statistical significance when analyzing the three parameters together might be due to the small number of patients, type 2 error. Nevertheless, the results shown in Table 5 support that preoperative basal calcitonin is the main guiding factor for best timing of thyroid surgery, which is consistent with findings of a French cohort reported by Rohmer et al. in 2011 (18). In addition, the prospective study by Elisei et al. was the first study that discussed the relevance of serum calcitonin in the management of RET gene carriers (22). Among the surgically treated patients with detectable basal calcitonin, intrathyroidal tumors were found when calcitonin was <60 pg/mL, whereas either lymph node metastasis or larger tumors were observed when calcitonin was >60 pg/mL. No correlation between serum calcitonin, age, and RET mutation type was observed. This study concluded that personalized timing of thyroid surgery could be based on serum calcitonin levels (22).

In this study, all patients with lymph node metastases at surgery had preoperative basal calcitonin levels ≥68 pg/mL. In a 2010 study by Schreinemakers et al. involving Dutch patients, none of the patients with lymph node metastases at primary surgery had a basal serum calcitonin <40 pg/mL (21). In 2005, Machens et al. also found a cumulative risk of lymph node metastasis at primary surgery when the basal calcitonin was >40 pg/mL (36). In the 2015 ATA guidelines, the classifications and recommended age at thyroid surgery were adjusted, as summarized in Table 1 (10). Because of the high sensitivity of monomeric calcitonin measurements, the criteria for recommended age at thyroid surgery are based on serum basal calcitonin alone rather than a stimulated calcitonin (10). The ATA guidelines also recommend central lymph node dissection for patients with a calcitonin >40 pg/mL, since lymph node metastasis rarely occurs when calcitonin is <40 pg/mL (2,10,37).

In this study, all patients without central lymph node dissection and preoperative basal calcitonin <40 pg/mL were biochemically cured. Prognostic factors for biochemical cure were analyzed for different patient groups to minimize both time and biological biases. By multivariate analysis with a smaller number of parameters, only nodal status at primary surgery was a statistically significant independent prognostic factor for biochemical cure in all groups except the preRET-era group in the present study. In addition, central lymph node dissection at primary surgery was found to be a prognostic factor when data from all patients in the entire period and all patients with MTC were analyzed, but not when the data were adjusted for time bias. Preoperative basal calcitonin was significant prognostic factor when all the patients in the entire period were analyzed. In major studies, prognostic factors for biochemical cure in MEN 2A were genotype, preoperative calcitonin level, and age at thyroid surgery, with the most important predictor of survival being tumor stage at diagnosis, which includes the presence of lymph node metastasis (2,9,10,19 –21). However, by multivariate analysis, only age and stage of disease at the time of diagnosis were significant independent prognostic factors in studies from the United States (2000), Netherlands (2010), and France (1998) (19,21,37).

Although MEN 2 syndrome has been known for many years and was first described in a 1968 publication by Steiner et al. (4), the RET mutations associated with MEN 2 have been known only for the last 20 years (11,12). The first years of the genetic testing period revealed many family members with MEN 2A at a later age who consequently underwent thyroid surgery. It is known that MTC penetrance in low-risk mutations is low and that MTC develops at higher age in this subgroup (4,15,17). Consequently, future index patients and their same-generation relatives will also be diagnosed later than the recommended age for thyroid surgery. In the present study, 36 screening and 24 index patients had thyroid surgery later than the recommended age mainly because of late diagnosis (14 ATA A or B and 10 ATA C). Although the national genetic screening program in Norway functions well, future index patients will also have gene mutation analysis done at a later age, and relatives of the same generation will consequently not be diagnosed and treated at the recommended time.

Limitations

In addition to the previously mentioned possible misclassification of preRET-era MEN 2A patients as having sporadic MTC, this study has some other limitations. First, the data collection was retrospective and multicentric. While this approach provides heterogeneity in patient management, it may not reflect the entirety of the situation of today. Surgical procedures differed, and interpreting how these differences affect predictive and prognostic factors is difficult. Second, the relatively small number of patients (n = 67) included in this study reduces the statistical power. Two of these patients had to be excluded from the study due to lack of informed consent, which weakened the data calculation to a small degree. There were other missing data due to a lack of (i) preoperative calcitonin analysis for eight surgically treated patients; (ii) RET mutation analysis for one patient (carrier by clinical state); and (iii) exact data for one not surgically treated older patient who died.

Conclusion

Norway has a well-organized genetic screening program for MEN 2A. It can therefore be assumed that the registered MEN 2A patients constitute the majority of MEN 2A patients in Norway. Preoperative basal calcitonin was found to be the only predictive factor for MTC. Prognostic factors for biochemical cure by multivariate analysis were shown to be nodal status at thyroid surgery for the different groups, and central lymph node clearance at primary surgery when the analyses were done for all patients in the entire period, and for MTC patients alone, but not when adjusted for time bias. Lymph node metastasis was not found when the preoperative calcitonin was <68 pg/mL. Although this study involving Norwegian patients represents a small series and treatment strategies based on these data may thus have low validity, the outcomes do correspond to data reported by others and show that the preoperative basal calcitonin is a useful marker to define the age for thyroid surgery and the extent of surgery with or without central lymph node dissection. The findings also suggest that under conditions of close observation, surgery can be postponed if basal serum calcitonin levels are normal. Thus, the recommendations of the revised 2015 ATA guidelines will be followed concerning preoperative basal calcitonin level as a marker defining optimal age for thyroid surgery and indication for central lymph node dissection or not.

Footnotes

Acknowledgments

We are grateful to Wenche Sjursen, PhD, Department of Pathology and Medical Genetics, St. Olav University Hospital Trondheim, and Christoffer Jonsrud, MD, Department of Medical Genetics, University Hospital of North Norway, Tromsø, for help in completing patient data. Preliminary data were presented as abstracts of fewer than 350 words at the 7^th Biennial Congress of ESES 2014 in Cardiff, 14th International Workshop on Multiple Endocrine Neoplasia and other rare endocrine tumors in Vienna 2014, and at the Annual National Surgical Meeting in Norway 2014.

Author Disclosure Statement

This work was not funded by any organization and was not supported by any grant. There are no conflicts of interest. No competing financial interest exists for any of the authors.

References

Eng

, Clayton

, Schuffenecker

, Lenoir

, Cote

, Gagel

, van Amstel

, Lips

, Nishisho

, Takai

, Marsh

, Robinson

, Frank-Raue

, Raue

, Xue

, Noll

, Romei

, Pacini

, Fink

, Niederle

, Zedenius

, Nordenskjold

, Komminoth

, Hendy

, Mulligan

, et al. 1996. The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2. International RET mutation consortium analysis. JAMA, 276:1575–1579.

American Thyroid Association Guidelines Task Force, Kloos

, Eng

, Evans

, Francis

, Gagel

, Gharib

, Moley

, Pacini

, Ringel

, Schlumberger

, Wells

Jr . 2009. Medullary thyroid cancer: management guidelines of the American Thyroid Association. Thyroid, 19:565–612.

Machens

, Brauckhoff

, Holzhausen

, Thanh

, Lehnert

, Dralle

. 2005. Codon-specific development of pheochromocytoma in multiple endocrine neoplasia type 2. J Clin Endocrinol Metab, 90:3999–4003.

Steiner

, Goodman

, Powers

. 1968. Study of a kindred with pheochromocytoma, medullary thyroid carcinoma, hyperparathyroidism and Cushing's disease: multiple endocrine neoplasia, type 2. Medicine (Baltimore), 47:371–409.

Lips

, Landsvater

, Hoppener

, Geerdink

, Blijham

, van Veen

, van Gils

, de Wit

, Zewald

, Berends

, et al. 1994. Clinical screening as compared with DNA analysis in families with multiple endocrine neoplasia type 2A. N Engl J Med, 331:828–835.

Schuffenecker

, Virally-Monod

, Brohet

, Goldgar

, Conte-Devolx

, Leclerc

, Chabre

, Boneu

, Caron

, Houdent

, Modigliani

, Rohmer

, Schlumberger

, Eng

, Guillausseau

, Lenoir

. 1998. Risk and penetrance of primary hyperparathyroidism in multiple endocrine neoplasia type 2A families with mutations at codon 634 of the RET proto-oncogene. Groupe D'etude des Tumeurs a Calcitonine. J Clin Endocrinol Metab, 83:487–491.

Frank-Raue

, Rondot

, Schulze

, Raue

. 2007. Change in the spectrum of RET mutations diagnosed between 1994 and 2006. Clin Lab, 53:273–282.

Romei

, Cosci

, Renzini

, Bottici

, Molinaro

, Agate

, Passannanti

, Viola

, Biagini

, Basolo

, Ugolini

, Materazzi

, Pinchera

, Vitti

, Elisei

. 2011. RET genetic screening of sporadic medullary thyroid cancer (MTC) allows the preclinical diagnosis of unsuspected gene carriers and the identification of a relevant percentage of hidden familial MTC (FMTC). Clin Endocrinol (Oxf), 74:241–247.

Pelizzo

, Boschin

, Bernante

, Toniato

, Piotto

, Pagetta

, Nibale

, Rampin

, Muzzio

, Rubello

. 2007. Natural history, diagnosis, treatment and outcome of medullary thyroid cancer: 37 years experience on 157 patients. Eur J Surg Oncol, 33:493–497.

10.

Wells

Jr , Asa

, Dralle

, Elisei

, Evans

, Gagel

, Lee

, Machens

, Moley

, Pacini

, Raue

, Frank-Raue

, Robinson

, Rosenthal

, Santoro

, Schlumberger

, Shah

, Waguespack

. 2015. Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma. Thyroid, 25:567–610.

11.

Donis-Keller

, Dou

, Chi

, Carlson

, Toshima

, Lairmore

, Howe

, Moley

, Goodfellow

, Wells

Jr . 1993. Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet, 2:851–856.

12.

Mulligan

, Kwok

, Healey

, Elsdon

, Eng

, Gardner

, Love

, Mole

, Moore

, Papi

, et al. 1993. Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature, 363:458–460.

13.

Hofstra

, Landsvater

, Ceccherini

, Stulp

, Stelwagen

, Luo

, Pasini

, Hoppener

, van Amstel

, Romeo

, et al. 1994. A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature, 367:375–376.

14.

Machens

, Niccoli-Sire

, Hoegel

, Frank-Raue

, van Vroonhoven

, Roeher

, Wahl

, Lamesch

, Raue

, Conte-Devolx

, Dralle

. 2003. Early malignant progression of hereditary medullary thyroid cancer. N Engl J Med, 349:1517–1525.

15.

Frank-Raue

, Rybicki

, Erlic

, Schweizer

, Winter

, Milos

, Toledo

, Tavares

, Alevizaki

, Mian

, Siggelkow

, Hufner

, Wohllk

, Opocher

, Dvorakova

, Bendlova

, Czetwertynska

, Skasko

, Barontini

, Sanso

, Vorlander

, Maia

, Patocs

, Links

, de Groot

, Kerstens

, Valk

, Miehle

, Musholt

, Biarnes

, Damjanovic

, Muresan

, Wuster

, Fassnacht

, Peczkowska

, Fauth

, Golcher

, Walter

, Pichl

, Raue

, Eng

, Neumann

, International RETEC. 2011. Risk profiles and penetrance estimations in multiple endocrine neoplasia type 2A caused by germline RET mutations located in exon 10. Hum Mutat, 32:51–58.

16.

Machens

, Gimm

, Hinze

, Hoppner

, Boehm

, Dralle

. 2001. Genotype-phenotype correlations in hereditary medullary thyroid carcinoma: oncological features and biochemical properties. J Clin Endocrinol Metab, 86:1104–1109.

17.

Shaha

, Cohen

, Ghossein

, Tuttle

. 2006. Late-onset medullary carcinoma of the thyroid: need for genetic testing and prophylactic thyroidectomy in adult family members. Laryngoscope, 116:1704–1707.

18.

Rohmer

, Vidal-Trecan

, Bourdelot

, Niccoli

, Murat

, Wemeau

, Borson-Chazot

, Schvartz

, Tabarin

, Chabre

, Chabrier

, Caron

, Rodien

, Schlumberger

, Baudin

, Groupe Francais des Tumeurs E. 2011. Prognostic factors of disease-free survival after thyroidectomy in 170 young patients with a RET germline mutation: a multicenter study of the Groupe Francais d'Etude des Tumeurs Endocrines. J Clin Endocrinol Metab, 96:E509–518.

19.

Kebebew

, Ituarte

, Siperstein

, Duh

, Clark

. 2000. Medullary thyroid carcinoma: clinical characteristics, treatment, prognostic factors, and a comparison of staging systems. Cancer, 88:1139–1148.

20.

Raval

, Sturgeon

, Bentrem

, Elaraj

, Stewart

, Winchester

, Ko

, Reynolds

. 2010. Influence of lymph node metastases on survival in pediatric medullary thyroid cancer. J Pediatr Surg, 45:1947–1954.

21.

Schreinemakers

, Vriens

, Valk

, de Groot

, Plukker

, Bax

, Hamming

, van der Luijt

, Aronson

, Borel Rinkes

. 2010. Factors predicting outcome of total thyroidectomy in young patients with multiple endocrine neoplasia type 2: a nationwide long-term follow-up study. World J Surg, 34:852–860.

22.

Elisei

, Romei

, Renzini

, Bottici

, Cosci

, Molinaro

, Agate

, Cappagli

, Miccoli

, Berti

, Faviana

, Ugolini

, Basolo

, Vitti

, Pinchera

. 2012. The timing of total thyroidectomy in RET gene mutation carriers could be personalized and safely planned on the basis of serum calcitonin: 18 years experience at one single center. J Clin Endocrinol Metab, 97:426–435.

23.

Gagel

, Tashjian

Jr. , Cummings

, Papathanasopoulos

, Kaplan

, DeLellis

, Wolfe

, Reichlin

. 1988. The clinical outcome of prospective screening for multiple endocrine neoplasia type 2a. An 18-year experience. N Engl J Med, 318:478–484.

24.

Wells

Jr. , Chi

, Toshima

, Dehner

, Coffin

, Dowton

, Ivanovich

, DeBenedetti

, Dilley

, Moley

, et al. 1994. Predictive DNA testing and prophylactic thyroidectomy in patients at risk for multiple endocrine neoplasia type 2A. Ann Surg, 220:237–247; discussion 247–250.

25.

Machens

, Lorenz

, Sekulla

, Hoppner

, Frank-Raue

, Raue

, Dralle

. 2013. Molecular epidemiology of multiple endocrine neoplasia 2: implications for RET screening in the new millenium. Eur J Endocrinol, 168:307–314.

26.

Niederle

, Sebag

, Brauckhoff

. 2014. Timing and extent of thyroid surgery for gene carriers of hereditary C cell disease—a consensus statement of the European Society of Endocrine Surgeons (ESES). Langenbecks Arch Surg, 399:185–197.

27.

Machens

, Lorenz

, Dralle

. 2009. Individualization of lymph node dissection in RET (rearranged during transfection) carriers at risk for medullary thyroid cancer: value of pretherapeutic calcitonin levels. Ann Surg, 250:305–310.

28.

Høie

, Jørgensen

, Nesland

, Møller

, Bjøro

. 1994. [Medullary thyroid carcinoma—familial or sporadic disease?]. Tidsskr Nor Laegeforen, 114:2951–2954.

29.

Høie

, Heimdal

, Nesland

, Børmer

. 2000. [Prophylactic thyroidectomy in carriers of RET oncogene mutation carriers]. Tidsskr Nor Laegeforen, 120:3249–3252.

30.

Basuyau

, Mallet

, Leroy

, Brunelle

. 2004. Reference intervals for serum calcitonin in men, women, and children. Clin Chem, 50:1828–1830.

31.

World Health Organization. 1989, 2002 and 2013 WHO Classification of Tumours of Soft Tissue and Bone 2nd, 3rd and 4th ed. World Health Organization Press, Geneva, Switzerland.

32.

The American Joint Committee on Cancers. 2010. TNM Classification, AJCC Cancer Staging Manual, Union of International Cancer Control 7th edition. Springer-Verlag, New York.

33.

Statistic Norway. Available at: www.ssb.no (accessed November 2015 ).

34.

Maschek

, Pichler

, Rieger

, Weinhausel

, Berg

. 2002. A new identified germline mutation of the RET proto-oncogene responsible for familial medullary thyroid carcinoma in co-existence with a hyperfunctioning autonomous nodule. Clin Endocrinol (Oxf), 56:823.

35.

Nakao

, Usui

, Ikeda

, Mori

, Yamamoto

, Kawashima

, Nanba

, Yuno

, Tamanaha

, Tagami

, Naruse

, Asato

, Shimatsu

. 2013. Novel tandem germline RET proto-oncogene mutations in a patient with multiple endocrine neoplasia type 2B: report of a case and a literature review of tandem RET mutations with in silico analysis. Head Neck, 35:E363–368.

36.

Machens

, Schneyer

, Holzhausen

, Dralle

. 2005. Prospects of remission in medullary thyroid carcinoma according to basal calcitonin level. J Clin Endocrinol Metab, 90:2029–2034.

37.

Modigliani

, Cohen

, Campos

, Conte-Devolx

, Maes

, Boneu

, Schlumberger

, Bigorgne

, Dumontier

, Leclerc

, Corcuff

, Guilhem

. 1998. Prognostic factors for survival and for biochemical cure in medullary thyroid carcinoma: results in 899 patients. The GETC Study Group. Groupe d'etude des tumeurs a calcitonine. Clin Endocrinol (Oxf), 48:265–273.