Association of the Genomic Profile of Medullary Thyroid Carcinoma with Tumor Characteristics and Clinical Outcomes in an International Multicenter Study

Abstract

Purpose:

The prognostic importance of RET and RAS mutations and their relationship to clinicopathologic parameters and outcomes in medullary thyroid carcinoma (MTC) need to be clarified.

Experimental Design:

A multicenter retrospective cohort study was performed utilizing data from 290 patients with MTC. The molecular profile was determined and associations were examined with clinicopathologic data and outcomes.

Results:

RET germ line mutations were detected in 40 patients (16.3%). Somatic RET and RAS mutations occurred in 135 (46.9%) and 57 (19.8%) patients, respectively. RET ^M918T was the most common somatic RET mutation (n = 75). RET somatic mutations were associated with male sex, larger tumor size, advanced American Joint Committee Cancer (AJCC) stage, vascular invasion, and high International Medullary Thyroid Carcinoma Grading System (IMTCGS) grade. When compared with other RET somatic mutations, RET ^M918T was associated with younger age, AJCC (eighth edition) IV, vascular invasion, extrathyroidal extension, and positive margins. RET somatic or germ line mutations were significantly associated with reduced distant metastasis-free survival on univariate analysis, but there were no significant independent associations on multivariable analysis, after adjusting for tumor grade and stage. There were no significant differences in outcomes between RET somatic and RET germ line mutations, or between RET ^M918T and other RET mutations. Other recurrent molecular alterations included TP53 (4.2%), ARID2 (2.9%), SETD2 (2.9%), KMT2A (2.9%), and KMT2C (2.9%). Among them, TP53 mutations were associated with decreased overall survival (OS) and disease-specific survival (DSS), independently of tumor grade and AJCC stage.

Conclusions:

RET somatic mutations were associated with high-grade, aggressive primary tumor characteristics, and decreased distant metastatic-free survival but this relationship was not significant after accounting for tumor grade and disease stage. RET ^M918T was associated with aggressive primary tumors but was not independently associated with clinical outcomes. TP53 mutation may represent an adverse molecular event associated with decreased OS and DSS in MTC, but its prognostic value needs to be confirmed in future studies.

Introduction

Medullary thyroid carcinoma (MTC) accounts for ∼2% of all thyroid malignancies and 8% of thyroid cancer-related mortality.¹ It may occur sporadically or in the setting of a germ line RET mutation.^2

–5 A large proportion of sporadic (i.e., non-germ line RET mutated) MTCs harbor somatic RET (particularly RET ^M918T) or RAS mutations,^6

–10 making them potential candidates for kinase inhibitor-targeted therapies.¹¹ Some MTCs are wild type (WT) for RET or RAS somatic mutations.^6

–10

In 2022, we established an international MTC consortium to develop and validate a powerful prognostic tool for MTC, namely the International Medullary Thyroid Carcinoma Grading System (IMTCGS) based on mitotic count, Ki67 proliferation index, and/or tumor necrosis.² Although data on RET germ line mutations were collected, the underlying molecular profile and its correlation with clinicopathologic features and outcome of MTC were not analyzed.

In this study, we investigated the molecular signatures of a large multicentric cohort of 290 patients with primarily resected MTC using the polymerase chain reaction (PCR)-based methods and 6 different next-generation sequencing (NGS) platforms. The aims were twofold: first, to examine the prognostic significance of somatic RET or RAS mutations and their associations with various clinicopathologic parameters, including IMTCGS grade; and second, to identify additional molecular alterations that could be associated with disease outcomes.

Materials and Methods

Study cohort

This multicenter retrospective cohort study was approved by the Institutional Review Board (IRB) of each participating site (MSKCC 17-103). This study included 290 patients with primary MTC who underwent surgical resection between 1998 and 2021 at 6 tertiary centers (University of Bologna Medical Center [UB], Bologna, Italy: n = 64; Memorial Sloan Kettering Cancer Center [MSKCC], New York, NY: n = 54; Institut Gustave Roussy, Villejuif, France: n = 45; Brigham and Women's Hospital [BWH], Boston, MA: n = 44; Royal North Shore Hospital, Sydney, Australia: n = 42, and Emory University Hospital Midtown [EU], Atlanta, GA: n = 41). Of these, 280 (97%) patients were included in the prior IMTCGS cohort² or a subsequent validation study from EU.¹²

All cases with a successfully sequenced primary MTC were included in this study. One center (BWH) included only patients with sporadic MTC, whereas cases from other centers also included patients with germ line RET mutations.

Molecular platforms

Somatic molecular alterations, including RET and RAS mutations, were detected using either PCR-based methods targeting only RET and/or RAS genes (n = 99) (Fluidigm multiplex PCR Access-Array or Agilent SureSelect XT HS platform) or by NGS using platforms in place at each of the six centers (n = 191), either commercially available or as described previously (briefly summarized in Supplementary Table S1).^13

–18 The number of cases tested with each NGS platform was as follows: custom-designed multigene sequencing panel with Thermo Fisher Scientific Ion GeneStudio S5 Prime System sequencer (n = 104), MSK-IMPACT (n = 54), ion Ampliseq™ cancer hotspot v. 2 (CHP2, n = 17; Thermo Fisher Scientific), OncoPanel (n = 14), Paradigm Cancer Diagnostic (PCDx) platform (n = 1; Paradigm Diagnostics), and CARIS NGS platform (n = 1; CARIS Life Sciences).

Clinicopathologic characteristics, oncological outcomes, and statistical analyses

Clinicopathologic review was carried out at each participating site as described previously.^{2,12,19
–21} Oncological outcomes, including overall survival (OS), disease-specific survival (DSS), distant metastasis-free survival (DMFS), and locoregional recurrence-free survival (LRRFS), were calculated. Survival analyses were carried out using the Kaplan–Meier method and the log-rank test. Multivariable analyses examining associations between genomic profile and oncological outcomes were performed using the Cox proportional hazards models, adjusted for tumor grade and disease stage. In addition, comparisons of the clinicopathologic features among each molecular subgroup (RET germ line mutations, RET somatic mutations, RAS somatic mutations, and RET/RAS WT) were carried out using the chi-square test or Fisher's exact test for categorical variables and two-tailed Student's t-test for continuous variables. Statistical analyses were performed using the SPSS software 24.0 (IBM Corporation, Armonk, NY).

Results

Clinicopathologic characteristics of the study cohort

The median age of presentation was 57 years (range: 7–88 years, Table 1). The male-to-female ratio was 1:1.1. American Joint Committee Cancer (AJCC) eighth edition stage IV, vascular invasion, extrathyroidal extension, positive margin, and IMTCGS high grade were identified in 39.3%, 42.1%, 28.8%, 18.6%, and 24.8% of the cases, respectively. Twenty-one patients (8.2%) had distant metastasis at presentation. External beam radiation therapy (RT) and kinase inhibitors were given to 8.2% and 8.7% of patients, respectively. The tyrosine kinase inhibitors (TKIs) used included the following: selpercatinib (n = 8), vandetanib (n = 5), cabozantinib (n = 3), pralsetinib (n = 1), sorafenib (n = 1), RAD001 (n = 1), and RAD100 (n = 1).

Table 1.

Clinicopathologic Characteristics According to RET and RAS Mutation Status

	All cases (N = 290)	RET germ line mutations (n = 40) ^a	RET somatic mutations (n = 133) ^a	RAS somatic mutations (n = 56) ^a	RET/RAS WT (n = 61)	p-Values (RET somatic vs. WT)	p-Values (RET germ line vs. WT)	p-Values (RAS vs. WT)	p-Values (RET somatic vs. RET germ line)
Male:female ratio	138:152 (1:1.1)	18:22 (1:1.2)	77:56 (1:0.7)	18:38 (1:2.1)	35:36 (1:1.4)	0.031	NS	NS	NS
Age, years	57 (7–88)	42 (7–66)	59 (22–88)	57 (7–79)	58 (25–84)	NS	<0.001	NS	<0.001
Tumor size, cm	2.0 (0.1–11.0)	1.2 (0.1–6.0)	2.1 (0.4–8.0)	2.0 (0.3–11.0)	1.9 (0.3–6.0)	0.036	NS	NS	0.006
AJCC eighth edition overall stage						<0.001	NS	NS	0.006
I	92 (31.7)	13 (32.5)	29 (21.3)	24 (42.9)	26 (42.6)
II	43 (14.8)	4 (10.0)	13 (9.6)	12 (21.4)	14 (23.0)
III	41 (14.1)	11 (27.5)	17 (12.5)	7 (12.5)	6 (9.8)
IV	114 (39.3)	12 (30.0)	77 (56.6)	13 (23.2)	15 (24.6)
Vascular invasion	122 (42.1)	12 (30.0)	76 (55.9)	17 (30.4)	20 (32.8)	0.005	NS	NS	0.007
Extrathyroidal extension	83 (28.8)	9 (22.5)	47 (34.6)	14 (25.0)	15 (25.4)	NS	NS	NS	NS
Positive margin	54 (18.6)	5 (12.5)	32 (23.5)	11 (19.6)	8 (13.1)	NS	NS	NS	NS
First postoperative CEA, ng/mL	5 (0–38,335)	2 (0–600)	7 (0–26,300)	5 (1–539)	3 (0–38,335)	NS	NS	NS	NS
First postoperative calcitonin, pg/mL	13 (1–970,000)	48 (1–7979)	24 (1–970,000)	5 (1–68,791)	4 (1–16,000)	NS	NS	NS	NS
Calcitonin doubling time
Never doubled	105/163 (64.4)	23/32 (71.9)	40/75 (53.3)	19/27 (70.4)	23/31 (74.2)	NS	NS	NS	NS
Months if doubled	17 (3–139)	13 (3–139)	15 (3–84)	27 (8–35)	15 (3–99)	NS	NS	NS	NS
IMTCGS grade						0.027	NS	NS	NS
Low grade	218 (75.2)	33 (82.5)	88 (64.7)	47 (83.9)	50 (82.0)
High grade	72 (24.8)	7 (17.5)	48 (35.3)	9 (16.1)	11 (18.0)
Necrosis	43 (14.8)	1 (2.5)	30 (22.1)	4 (7.1)	8 (13.1)	NS	NS	NS	0.002
Mitotic index, /2 mm²	1 (0–29)	0 (0–7)	1 (0–18)	1 (0–9)	1 (0–7)	0.028	NS	NS	0.049
KI67, %	2 (0–58)	1 (0–15)	2 (0–30)	2 (0–58)	2 (0–30)	NS	NS	NS	0.016
DM at presentation	25 (8.7)	1 (2.5)	19 (14.4)	1 (1.8)	4 (6.7)	NS	NS	NS	0.047
Follow-up period, months	48 (0–285)	33 (0.4–285)	45 (0–228)	55 (1–268)	56 (0–226)	NS	NS	NS	NS
Radiation therapy	21 (8.2)	4 (10.3)	14 (12.2)	1 (2.1)	2 (3.7)	NS	NS	NS	NS
Kinase inhibitor therapy	25 (8.7)	2 (5.0)	20 (15.2)	1 (1.8)	2 (3.3)	0.015	NS	NS	NS

Values are expressed as n (column %) for categorical variables, or median (range) for continuous variables.

The three cases with both RET germ line mutations and somatic RET or RAS mutations are classified as RET germ line mutations in this table.

AJCC, American Joint Committee Cancer; CEA, carcinoembryonic antigen; DM, distant metastasis; IMTCGS, International Medullary Thyroid Carcinoma Grading System; NS, not significant; WT, wild type.

RET germ line mutations

After excluding the BWH subgroup, which underwent preselection so that it comprised only somatic MTCs, the frequency of RET germ line mutations was 16.3% (40/246, Table 1 and Fig. 1 and Supplementary Table S2). Among the 39 cases with family history available, 19 (49%) were index cases (proband), whereas the remaining 20 (51%) had a family history.

FIG. 1.

RET germ line and somatic mutations in MTC and their impact on clinical outcome. (A) Lollipop plot showing sites of mutations. (B) Oncoprint showing the clinicopathologic features and mutation profile of MTCs. (C–F) Kaplan–Meier curves for OS (C), (D), (E), and (F). RET mutations, somatic or germ line, are associated with decreased LRRFS and DMFS. OS and DSS do not differ according to RET or RAS mutation status. Cadherin, cadherin domain; DMFS, distant metastasis-free survival; DSS, disease-specific survival; LRRFS, locoregional recurrence-free survival; MTC, medullary thyroid carcinoma; OS, overall survival; Pkinase_Tyr, protein tyrosine kinase; TM, transmembrane.

The most frequently detected germ line mutations were C609Y (n = 14), C634R (n = 8), and G533C (n = 3). Among these familial MTCs, RET or RAS somatic mutations were detected in three cases: one case had germ line RET ^Y791F and somatic RET ^M918T, one had germ line RET ^V804Y and somatic RET ^M918T, and the third had RET ^C634R germ line mutation and HRAS ^G13R somatic mutation. None of these three patients received kinase inhibitor therapy.

RET somatic mutations and copy number alteration

RET somatic mutations were detected in 135 cases (46.9% overall, 53.6% in sporadic MTCs, Table 1 and Fig. 1 and Supplementary Table S2). Among them, RET ^M918T was the most common somatic mutation, being detected in 75 cases. Other common RET somatic mutations were located at codons 634 (n = 16), 630 (n = 11), 620 (n = 7), and 883 (n = 6).

RET copy number alteration was analyzed in 68 MTCs using MSK-IMPACT or OncoPanel platform. Two cases showed RET amplification (>2-fold gain), three had RET gain (1.3- to 1.9-fold gain), and two had deletion. All seven cases also harbored RET somatic mutations.

RAS somatic mutations

RAS somatic mutations were examined in 288 cases, and were identified in 57 MTCs (19.8%, Table 1 and Fig. 1 and Supplementary Table S2), including HRAS mutations in 37 cases (Q61R n = 21, G13R n = 10, Q61K n = 3, Q61L n = 2, and G13V n = 1), KRAS mutations in 20 cases (G12R n = 8, G12V n = 3, Q61R n = 3, Q61L n = 1, Q61K n = 1, D54N n = 1, A18D n = 1, P34L n = 1, and C186Mfs*16 n = 1), and NRAS ^Q61K mutation in 1 case. One MTC harbored two RAS somatic mutations, being HRAS ^G13R and KRAS ^C186Mfs*16. RAS and RET somatic mutations were mutually exclusive.

The allele frequency was available in 91 MTCs with RET somatic mutations and 43 cases with RAS somatic mutations, and it did not differ between the 2 groups (p = 0.750). The median allele frequency for RET and RAS somatic mutation was 35% and 36%, respectively.

In our cohort, the 61 MTCs (21.0%) without RET (somatic or germ line) or RAS mutations were grouped as RET/RAS WT.

Relationship between RET/RAS mutations and clinicopathologic parameters

The clinicopathologic characteristics listed according to RET and RAS mutations status are shown in Table 1.

Compared with RET/RAS WT MTCs, MTCs occurring in the familial setting were associated with younger age at presentation (median age: 42 years in RET germ line mutation group compared with 58 years in WT; p < 0.001). Other clinicopathologic characteristics did not differ between the two groups. Similarly, the only significant difference between the index cases (proband, first case identified to carry a germ line RET mutation) and sporadic MTCs was younger age at presentation in the index group (index cases: median age 43 years, range 9–65 years; sporadic MTC: median age: 58 years, range 22–88 years).

MTCs with RET somatic mutations were associated with aggressive tumor characteristics at presentation compared with WT MTC, including larger tumor size (median size: 2.1 cm in RET somatic mutations, 1.9 cm in WT; p = 0.036), AJCC stage IV (56.6% in RET somatic mutations, 24.6% in WT; p < 0.001), vascular invasion (55.9% in RET somatic mutations, 32.8% in WT; p = 0.005), and high IMTCGS grade (35.3% in RET somatic mutations, 18.0% in WT; p = 0.027). In addition, patients with RET somatic mutations were more commonly male (male-to-female ratio: 1:0.7 in RET somatic mutation group, 1:1.4 in WT) and more commonly treated with kinase inhibitor therapy (15.2% in RET somatic mutation group, 3.3% in WT group), which could be explained by selection criteria to receive such treatment.

Similarly, compared with MTCs with RET germ line mutations, the presence of RET somatic mutations was associated with more aggressive disease, as characterized by a larger tumor size (median 2.1 cm in RET somatic mutation group, 1.2 cm in RET germ line mutation group, p = 0.006), AJCC stage IV (56.6% in RET somatic mutations, 30.0% in RET germ line mutations, p = 0.006), vascular invasion (55.9% in RET somatic mutations, 30.0% in RET germ line mutations, p = 0.007), tumor necrosis (22.1% in RET somatic mutations, 2.5% in RET germ line mutations, p = 0.002), high mitotic count (median 0/2 mm² in RET somatic mutations, 1/2 mm² in RET germ line mutations, p = 0.049), high Ki67 proliferation rate (median 1% in RET somatic mutations, 2% in RET germ line mutations, p = 0.016), and distant metastasis at presentation (14.4% in RET somatic mutations, 2.5% in RET germ line mutations, p = 0.047).

Clinicopathologic features of MTCs with somatic RAS mutations and WT MTCs were not significantly different.

RET ^M918T somatic mutation was considered a high-risk mutation in this study. When compared with other RET somatic mutations, RET ^M918T was more commonly associated with younger age (median age: 52 years in RET ^M918T, 63 years in other RET somatic mutations), AJCC stage IV (69.3% in RET ^M918T, 40.0% in other RET somatic mutations; p < 0.001), vascular invasion (65.3% in RET ^M918T, 43.3% in other RET somatic mutations; p = 0.015), extrathyroidal extension (46.7% in RET ^M918T, 20.0% in other RET somatic mutations; p = 0.002), and positive surgical margin (30.7% in RET ^M918T, 15.0% in other RET somatic mutations, p = 0.042; Supplementary Table S3). Other clinicopathologic parameters did not differ between MTCs with RET ^M918T and those with other RET somatic mutations.

Lastly, when comparing MTCs with RET somatic mutations and those without RET somatic or germ line mutations (RET WT, which included tumors with RAS mutations), RET somatic mutation was significantly associated with male sex, AJCC stage IV, vascular invasion, tumor necrosis, IMTCGS high grade, distant metastasis at presentation, higher frequency of cases whose postoperative calcitonin did not double, postoperative RT, and TKI therapy (p < 0.05, Supplementary Table S4).

Relationship between RET and RAS mutations and oncological outcomes

RET or RAS mutations were not significantly associated with OS or DSS (results are shown in Table 2 and Fig. 1). Compared with RET/RAS WT MTCs, those with RET somatic or RET germ line mutations were associated with poorer DMFS (RET somatic mutations: p = 0.010, RET germ line mutations: p = 0.045) in univariate analysis. The 10-year DMFS in MTCs with RET somatic mutations, in MTCs with RET germ line mutations, and in WT MTCs was 49%, 47%, and 75%, respectively. Similarly, RET somatic mutations were associated with decreased DMFS and LRRFS when compared with RET WT MTC (p < 0.001 and p = 0.003, respectively, Supplementary Table S4) in univariate analyses. Compared with other RET somatic mutations, RET ^M918T somatic mutations were not significantly associated with OS, DSS, DMFS, or LRRFS (Supplementary Table S3).

Table 2.

Impact of RET and RAS Mutation on Prognosis in Medullary Thyroid Carcinoma (Log-Rank Test)

	OS	DSS	DMFS	LRRFS
RET somatic vs. RET germ line	0.387	0.556	0.745	0.630
RET somatic vs. RET/RAS WT	0.748	0.854	0.010	0.118
RET germ line vs. RET/RAS WT	0.422	0.557	0.045	0.156
RAS mutated vs. RET/RAS WT	0.406	0.407	0.081	0.193
RET ^M918T somatic vs. other RET somatic	0.202	0.328	0.375	0.531

Values in the table are p-values. Bold values: significant p-values.

DMFS; distant metastasis-free survival; DSS, disease-specific survival; LRRFS, locoregional recurrence-free survival; OS, overall survival.

There was also a nonstatistically significant trend for RAS-mutated MTCs to be associated with improved DMFS (10-year DMFS in RAS-mutated MTCs: 88%, p = 0.081).

The RET or RAS mutation profile was not significantly independently associated with DMFS in multivariable analyses adjusted for IMTCGS grade and AJCC stage (Supplementary Table S5, p > 0.05). IMTCGS grade and AJCC stage were significantly independently associated with DMFS (IMTCGS grade: hazard ratio 2.031 [confidence interval, CI 1.230–3.353], p = 0.006; AJCC stage IV: hazard ratio 1.478 [CI 1.221–1.789], p < 0.001).

The relationship of other gene mutations with oncological outcomes

Mutations other than RAS or RET were searched in 191 cases using 6 different NGS platforms. Their overlap with RET or RAS mutations is illustrated in Figure 1 and their features are detailed in Supplementary Table S6. In 30 of 38 cases (78%), mutations were secondary events in RET- or RAS-mutated tumors (RAS mutations: n = 8; RET somatic mutations n = 21; RET germ line mutation n = 1).

Recurrent somatic mutations or fusions included TP53 (8/191, 4.2%), PIK3CA (2/191, 1.0%), VHL (2/191, 1.0%), TERT promoter mutation (2/191, 1.0%), ATM (2/87, 2.3%), ARID2 (2/70, 2.9%), SETD2 (2/70, 2.9%), KMT2A (2/70, 2.9%), and KMT2C (2/70, 2.9%). TP53 mutations were associated with decreased OS (log-rank test, p = 0.027, Fig. 2), DSS (p = 0.008), and LRRFS (p = 0.018), with no statistically significant trend toward shortened DMFS (p = 0.074). On multivariable survival analyses adjusted for IMTCGS grade and AJCC stage, TP53 mutations remained independently associated with reduced OS (hazard ratio 5.150 [CI 1.130–23.465], p = 0.034) and DSS (hazard ratio 6.078 [CI 1.279–28.887], p = 0.023), but not LRRFS (hazard ratio 2.952 [CI 0.892–9.764], p = 0.076). There was no significant association between TP53 mutations and IMTCGS grade or AJCC stage (p = 1.000).

FIG. 2.

TP53 mutation is associated with decreased survival in MTC. Kaplan–Meier curves for OS, DSS, DMFS, and LRRFS. WT, wild type.

Other molecular alterations were not significantly associated with clinicopathologic characteristics or survival outcomes.

Discussion

The key findings of this largest MTC molecular study are as follows: (1) RET somatic mutations were associated with aggressive tumor characteristics, high IMTCGS grade, and decreased DMFS in univariate analysis but not in multivariable analyses adjusted for tumor grade and disease stage; (2) MTCs with RET ^M918T somatic mutations showed more aggressive features in the primary tumor (such as AJCC stage IV, vascular invasion, extrathyroidal extension, and positive margin), but were not independently associated with survival outcomes; (3) RET and RAS mutations were not significantly associated with OS or DSS; and (4) a TP53 mutation (identified in 4.2% of MTCs) was identified as a novel potential adverse molecular event associated with reduced survival [indicating which survival outcomes].

Germ line RET mutations were detected in 16.3% of the study cohort, lower than the ∼25% rate of familial cases generally reported in the literature.²² The relatively low frequency of germ line RET mutations may be explained by the selection bias of our study cohort toward adult patients: only four patients (1.4%) of our cohort were age ≤18 years. As the current American Thyroid Association (ATA) guideline recommended prophylactic thyroidectomy at a young age for patients with hereditary MTCs, such patients were likely not included in our cohort giving the selection bias toward adult patients.²²

Consistent with what has been previously reported,^5,23
–25 we found that RET germ line mutations affected codons 533, 609, 611, 618, 634, 791, 804, and 918, with codon 609 being the most prevalent. Overall, hereditary MTCs, including the index cases (proband), occurred at a younger age compared with sporadic MTCs, although it is known that the age of MTC manifestation may differ depending on the type of RET germ line mutations.²²

Recent studies had shown that germ line RET ^Y791F was not associated with increased risk of MTC.^26,27 It is therefore conceivable that it could represent a polymorphism. However, as the current ATA guideline regards RET ^Y791F as pathogenic,²² we classified the single case with germ line RET ^Y791F under the RET germ line mutation group.

RET somatic mutations have been identified in 45–70% of sporadic MTCs and have been shown to be associated with larger tumor size,²⁸ high T stage,²⁹ nodal metastasis,^28
–30 distant metastasis,^28
–30 advanced overall stage,^28
–30 and decreased survival (overall and disease free).^28
–30 Similarly, we showed here that MTCs with RET somatic mutations are associated with larger tumor size, advanced AJCC stage, and decreased DMFS in univariate analysis but not in multivariable analysis, adjusted for tumor grade and AJCC stage. Together, these data imply that RET somatic mutation may be associated with more aggressive tumor characteristics and adverse outcomes, but its role in MTC may not be independent of grade and stage.

In a study of 100 sporadic MTCs, Elisei et al. identified RET somatic mutation and advanced stage as the only two factors independently correlated with persistent disease.²⁹ There were several differences between their study and the current study. First, Elisei et al. included only sporadic MTCs, whereas our cohort contained both hereditary and sporadic cases. Second, RAS somatic mutation was not analyzed in the study by Elisei et al. Third, Elisei et al. examined associations between persistent disease and mutation/stage using a multivariable logistic regression model, and not a survival analysis. The fact that driver mutations are not independent from stage and other clinicopathologic factors such as grade is not unique to MTC. Indeed, the same scenario applies to follicular-derived thyroid carcinomas where the BRAF^V600E mutation was not independent from clinicopathologic features such as stage in predicting mortality.³¹

We show for the first time that RET somatic mutation is associated with high IMTCGS grade. Najdawi et al. previously reported that there was no significant association between RET somatic mutation and IMTGS grade in 44 sporadic MTCs.¹⁹ These 44 patients were also included in the current study. We were able to establish an association between IMTCGS high grade and RET somatic mutations in the current study for two reasons: first, we expanded the cohort size to 290 cases; and second, we excluded cases with RAS somatic mutations from the WT group.

Not all high IMTCGS grade tumors harbor RET somatic or germ line mutations. Among the 72 cases of high-grade MTCs, 55 (76.4%) had RET somatic or germ line mutations and 9 (12.5%) had RAS mutations, whereas the remaining 11 did not harbor RET or RAS mutations.

RET ^M918T somatic mutation, the most prevalent RET somatic mutation in MTC, has been implied in several studies to be an adverse molecular signature. Schilling et al.³² reported that RET ^M918T was associated with decreased OS and increased risk of distant metastasis in 34 patients with sporadic MTCs. However, the authors only examined RET ^M918T mutations in their study, and their control group theoretically included both MTCs with other RET somatic mutations and MTCs without RET mutations. Therefore, it was impossible to directly compare RET ^M918T somatic mutations with other RET somatic mutations in this study. Romei et al.³³ showed that RET ^M918T mutations were associated with larger tumor size and were relatively infrequent in MTCs <1 cm in size.

Moura et al. reported that MTCs with RET somatic mutations involving exons 15 and 16 (including 87% of M918T and 13% of A883F) had a higher prevalence of nodal metastasis, stage IV disease, and persistent disease compared with MTCs with other RET somatic mutations, in 52 sporadic MTCs.³⁴ In contrast, Najdawi et al.¹⁹ showed that RET somatic mutations affecting exons 15 and 16 did not impact DSS and progression-free survival and did not correlate with IMTCGS grade. In the current study including 75 cases with RET ^M918T somatic mutations and 60 cases with other RET somatic mutations, we find that RET ^M918T somatic mutations are associated with younger age, advanced AJCC stage, and other aggressive tumor characteristics (e.g., vascular invasion, extrathyroidal extension, and positive margin) compared with MTCs with other RET somatic mutations. However, RET ^M918T somatic mutations were not significantly associated with OS, DSS, DMFS, and LRRFS in MTC in this study.

RAS mutations are the predominant driver mutations in RET-WT sporadic MTC, being detected in 11% (range: 9–20%) of MTCs overall, 13% (range: 0–43%) of sporadic MTCs, and 61% (range: 0–81%) of RET-WT sporadic MTCs.^{7,9,10,35

–38} Similarly, we detected RAS mutations in 19.8% of all MTCs, 22.4% of sporadic MTCs, and 44.9% of RET-WT sporadic MTCs. Although we showed a nonsignificant trend of RAS-mutated MTCs with improved DMFS, overall RAS mutations were not associated with tumor characteristics or outcomes in MTC, confirming prior published research by Vuong et al.³⁰

The mutation data that we have presented suggest that additional molecular events other than RET or RAS mutations are required to explain the aggressive biological behavior seen in high-grade MTCs. In this respect, we showed that a TP53 mutation may represent a novel prognostic molecular alteration in MTCs. TP53 mutations are uncommon in MTCs, being reported in 9% (8/88 cases) in the literature³⁸ and 4.2% in our cohort. In our series, TP53 mutation nearly always coexisted with RET or RAS driver mutations.

The current study is the first to show that the TP53 mutation is associated with decreased OS and DSS in MTC, independent of IMTCGS grade and AJCC stage. Interestingly, in the majority of our cases harboring mutations other than RET or RAS, these mutations were secondary events (i.e., present in tumors already carrying RET or RAS mutations). These findings deserve further investigation to better understand the biology of MTC progression.

A limitation of the current study was the utilization of multiple molecular platforms resulting in a lack of uniformity on the molecular data. In addition, certain mutations may not be identified in the RET/RAS WT group due to the limitation of the testing platforms. This study may also be subject to selection bias as consecutive eligible cases in all institutions were not included and systematically collected data were not available to inform reporting of a participant flow diagram. Some selection bias may be reflected in the discrepancies in prevalence rates of some molecular events, relative to the published literature.

In summary, this international multicenter MTC consortium study enabled us to report detailed clinical, pathologic, and molecular characteristics of MTC. We have previously established and validated a prognostically relevant grading scheme.² We herein present evidence that RET somatic mutations and TP53 may also have some prognostic importance in MTC, although the prognostic relevance of RET somatic mutation is not independent of IMTCGS grade and AJCC stage and further studies are required to validate the prognostic values of TP53 mutations. Our data represent valuable preliminary information to develop a prognostic nomogram to accurately stratify risk in individual patients with MTCs.

Footnotes

Acknowledgments

The authors are grateful to Giuseppina Coraggio BSc (University of Bologna Medical Center) for technical help. The abstract was presented, in part, at the United States and Canadian Academy of Pathology (USCAP) 2022 annual meeting (New Orleans, LA).

Authors' Contributions

Study conception: B.X., K.V., B.U., J.A.B., A.A.G., A.J.G., R.G., G.T., and I.G.

Database management: B.X., K.V., T.L.F., B.U., J.A.B., A.A.G., A.J.G., R.G., and G.T.

Data collection: B.X., K.V., M.S.A., S.A., B.A., M.-A.B., E.B., D.B.B., M.C., N.G.C., F.C., A.C., R.C.-B., S.C., D.d.B., A.D., S.D., J.A.F., T.L.F., A.R.G., J.H., L.L., L.L., D.J.L., C.L., K.M., T.M., A.S.M., F.N., A.N., A.J.P., A.R., B.R., J.-Y.S., Q.S., S.S., E.S., M.S., R.M.T., B.U., J.A.B., A.A.G., A.J.G., R.G., G.T., and I.G.

Molecular analysis: K.V., A.D.L., S.D., C.L., A.S.M., J.A.B., A.A.G., A.J.G., R.G., G.T., and I.G.

Statistics: B.X.

Article drafting: B.X.

Article editing: K.V., M.S.A., S.A., B.A., M.-A.B., E.B., D.B.B., M.C., N.G.C., F.C., A.C., R. C.-B., S.C., D.d.B., A.D.L., S.D., J.A.F., T.L.F., A.R.G., J.H., L.L., L.L., D.J.L., C.L., K.M., T.M., A.S.M., F.N., A.N., A.J.P., A.R., B.R., J.-Yv.S., Q.S., S.S., E.S., M.S., R.M.T., B.U., J.A.B., A.A.G., A.J.G., R.G., G.T., and I.G.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

Research reported in this publication was supported, in part, by the Cancer Center Support Grant of the National Institutes of Health/National Cancer Institute under award number P30CA008748, Italian Government—Ministry of Health grant number RF-2011-02350857, the Cancer Tissue and Pathology, Emory Integrated Genomics Core (EIGC), NIH/NCI under award number P30CA138292, the National Center for Georgia Clinical & Translational Science Alliance of the National Institutes of Health under award number UL1TR002378. Emory Integrated Computational Core (EICC), which is subsidized by the Emory University School of Medicine and is one of the Emory Integrated Core Facilities, and all three are shared resources of Winship Cancer Institute of Emory University.

National Institutes of Health P30CA008748: Bin Xu, Snjezana Dogan, James A. Fagin, Abhinita S. Mohanty, R. Michael Tuttle, Brian Untch, Ronald Ghossein, and Ian Ganly.

Italian Government—Ministry of Health grant RF-2011-02350857: Giovanni Tallini.

Emory Integrated Genomics Core (EIGC), NIH/NCI under award number P30CA138292, the National Center for Georgia Clinical & Translational Science Alliance of the National Institutes of Health under award number UL1TR002378: Kartik Viswanathan, Kelly Magliocca, and Daniel J. Lubin.

No funding to declare: Mahsa S. Ahadi, Sara Ahmadi, Bayan Alzumaili, Mohamed-Amine Bani, Eric Baudin, David Blake Behrman, Marzia Capelletti, Nicole G. Chau, Federico Chiarucci, Angela Chou, Roderick Clifton-Bligh, Sara Coluccelli, Dario de Biase, Antonio De Leo, Talia L. Fuchs, Anthony Robert Glover, Julien Hadoux, Ludovic Lacroix, Livia Lamartina, Catherine Luxford, Thais Maloberti, Fedaa Najdawi, Aradhya Nigam, Alexander James Papachristos, Andrea Repaci, Bruce Robinson, Jean-Yves Scoazec, Qiuying Shi, Stan Sidhu, Erica Solaroli, Mark Sywak, Justine A. Barletta, Abir Al Ghuzlan, and Anthony J. Gill.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Table S5

Supplementary Table S6

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