Genotype-Specific Progression of Hereditary. Medullary Thyroid Cancer

Transcription

1 Genotype-Specific Progression of Hereditary Medullary Thyroid Cancer Andreas Machens 1, Kerstin Lorenz 1, Frank Weber, 2 and Henning Dralle 1,2 1 Medical Faculty, Department of General, Visceral and Vascular Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, D Halle (Saale), Germany 2 Department of General, Visceral and Transplantation Surgery, Section of Endocrine Surgery, University of Duisburg-Essen, D Essen, Germany Running Title: Progression of Hereditary MTC Key Words RET Proto-Oncogene Germline Mutation Age-Related Progression Translational Medicine Medullary Thyroid Carcinoma Lymph Node Metastasis Word Count: 2796 Tables: 5 Figure: 2 Declaration of interest: The authors declare that there is no conflict of interest that could be Funding: perceived as prejudicing the impartiality of the research reported. This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: /humu

2 2 Address for correspondence: Andreas Machens, MD Department of General, Visceral and Vascular Surgery Martin Luther University Halle-Wittenberg Ernst-Grube-Straße 40, D Halle (Saale), Germany Phone #: Fax #: AndreasMachens@aol.com Abstract (Word count: 200) Although already 25 years into the genomic era, age-related progression of hereditary medullary thyroid cancer (MTC), the prevalence of which is estimated at 1 in 80,000 inhabitants, remains to be delineated for most unique RET (REarranged during Transfection) mutations. Included in this study were 567 RET carriers. The age-related progression of MTC across histopathological groups (normal thyroid/c-cell hyperplasia; node-negative MTC; node-positive MTC) was statistically significant for 13 unique RET mutations (p.cys611phe/c.1832g>t; p.cys611tyr; p.cys618ser/c.1852t>a; p.cys620arg; p.cys634arg; p.cys634phe; p.cys634ser; p.cys634tyr; p.glu768asp; p.leu790phe/c.2370g>t; p.val804met; p.ser891ala; p.met918thr), whereas two unique RET mutations (p.cys618phe; p.cys634gly) trended towards statistical significance. When grouped by mutational risk (highest; high; moderate high; low moderate; polymorphism), the age-related progression of MTC was significant for all four categories of RET mutations, which differed significantly across and within the three histopathological groups. For high, moderate high, and low moderate risk RET mutations, the age-related progression of MTC by mutated codon was broadly comparable across and within the three histopathological groups, and essentially unaffected by the amino acid substitutions examined. These data argue in favor of splitting the American Thyroid

3 3 Association s moderate-risk category into moderate high and low moderate risk categories, while emphasizing the need to contradistinguish the latter from rare non-pathogenic polymorphisms.

4 4 Introduction (Word Count: 2796) Medullary thyroid cancer (MTC) is a calcitonin-secreting neuroendocrine malignancy notorious for its propensity to spread beyond the thyroid gland. This biologic behavior can thwart efforts directed at surgical cure, in particular when 10 or more nodes are involved (Machens, Gimm, Ukkat, Hinze, Schneyer, & Dralle, 2000; Scollo et al., 2003) or calcitonin serum levels are >1000 pg/ml (Machens, & Dralle, 2010; Machens, & Dralle, 2013). Early diagnosis of MTC is facilitated by (i) calcitonin screening for sporadic MTC in nodular thyroid disease but controversial because of unresolved costeffectiveness issues (Hodak, & Burman, 2004; Cheung, Roman, Wang, Walker, & Sosa, 2008; Machens, & Dralle, 2016), and (ii) molecular proof of pathogenic gain of function RET (MIM# ) sequence variants in family members at risk of hereditary MTC (Wells, Pacini, Robinson, & Santoro, 2013). Familial MTC (MIM# ) can be associated with pheochromocytoma or pseudonodular parathyroid hyperplasia, commonly referred to as multiple endocrine neoplasia type 2A (MIM# ) or multiple endocrine neoplasia type 2B (MIM# ) depending on the constellation at hand (Machens, Lorenz, & Dralle, 2013; Mucha, Leidig-Bruckner, Frank-Raue, Bruckner, Kroiss, & Raue, 2017). Pathogenic RET sequence variants, inherited in an autosomal dominant fashion and estimated to affect 1 in 80,000 inhabitants (Machens et al., 2013), mainly include heterozygous missense nucleotide changes, causing substitution of one amino acid for another (non-synonymous exchange). Because they are less disruptive than frameshift mutations or deletions leading to protein truncation, non-synonymous missense sequence variants of medically actionable genes have an intrinsic element of uncertainty in relation to their clinical effects (Weber, & Eng, 2005). The rapid advance of gene sequencing technologies has produced an unprecedented rate of discovery of genome variation in humans, including new RET sequence variants (Toledo et al., 2015; Lebeault et al., 2017). Some of

5 5 those variants were misclassified as pathogenic owing to limited genetic analyses of highly selected patients and small numbers of controls often consisting of no more than 100 samples (Erlic et al., 2010). It emerged only recently that RET sequence variants p.ser649leu, p.tyr791phe, and p.ile852met were not pathogenic contrary to initial belief (Erlic et al., 2010; Toledo et al., 2015; Mathiesen et al., 2017). This came as a surprise because many, albeit not all, in vitro focus formation assays and computer-based in silico predictive models implicated a causative role for these gene variants in the progression of MTC (Cosci et al., 2011; Crocket et al., 2011; Lebeault et al., 2017;), raising concerns as to the generalizability of in vitro and in silico findings to man. Pooling unique RET mutations by mutated codon revealed a codon-specific, age-related progression of MTC, the pace of which differed in highest-risk, high-risk, and moderate-risk RET mutations (Machens, Holzhausen, Thanh, & Dralle, 2003; Machens, Elwerr, Lorenz, Weber, & Dralle, 2017). For high-risk mutations, this genotype-specific, age-related progression of early MTC has been validated for the unique RET mutations p.cys634arg and p.cys634tyr (Machens et al., 2003). No such data have been forthcoming for many other, less frequent RET variants. Twenty-five years into the molecular era, a wealth of genomic information has accrued that should permit more accurate characterization of the genotype-specific, age-related progression of MTC for unique RET mutations. These genetic-clinical data are important to refine the 2015 American Thyroid Association (ATA) categorization of RET mutations (Wells et al., 2015), specifically the comparability of unique RET sequence variants assigned to the same ATA risk category. Patients and Methods

6 6 RET Cohort This retrospective cohort study included 567 consecutive carriers of missense unique RET variants who underwent thyroidectomy based on contemporaneous national and international practice guidelines. Among these 567 patients, 505 carriers (89%) underwent standard MEN 2 related interventions at the authors unit under the direction the senior author of this study since 1985 (Dralle et al., 1992; Machens, Lorenz, & Dralle, 2013). Sixty-two relatives, having been captured in the process of taking the patients histories, had sufficient clinical-histopathological information to warrant inclusion in the present series. The development of hereditary pheochromocytoma and primary hyperparathyroidism has been detailed elsewhere based on 437 of the 567 patients described herein (Machens, Lorenz, & Dralle, 2013) and hence is not the subject matter of this report. For retrospective analysis of existing data sets from routine patient care, no institutional review board approval is required under German law and applicable institutional regulations.

7 7 Surgical Intervention in the Neck Care was rendered in accordance with the state-of-the-art clinical and therapeutic protocols in place at the time of diagnosis (Brandi et al., 2001; Wells et al., 2015). All 567 RET carriers underwent thyroidectomy with or without node dissection after informed consent. Patients who either did not undergo central lymph node dissection or did not have evidence of lymph node metastasis on pathological examination were assumed to be free from lymph node metastasis if they had documented normal postoperative calcitonin serum levels. Histopathological Evaluation of Surgical Specimens A diagnosis of medullary thyroid carcinoma was based on evidence of extension beyond the basement membrane, demonstration of lymphatic or vascular invasion on histopathological analysis, or both findings. C-cell hyperplasia was diagnosed by the hospital pathologist when each low-power field showed more than 50 intrafollicular calcitonin-positive cells, more than 6 C cells per thyroid follicle, or both. RET Gene Analysis Before undergoing genetic testing before or after the operation, all patients or their parents had given informed consent after genetic counseling. For identification of RET sequence variants listed in the web-based ARUP MEN 2 database (Margraf et al., 2009; genomic DNA was purified from peripheral blood leukocytes using standard techniques. Genomic DNA was amplified using PCR and oligonucleotide primers for exons 10, 11, 13, 14, 15, and 16. Single-strand conformation polymorphism analysis and direct sequencing were performed according to national laboratory and genetic regulations for RET analysis (Machens, Lorenz, & Dralle, 2009a).

8 8 For the purpose of the study, variant denotes any sequence change, whether pathogenic or not; mutation indicates a disease-causing, and polymorphism a non-pathogenic sequence change, irrespective of whether the latter is found in the population at a frequency of 1% or higher. Statistical Analysis For statistical analysis, the software package SPSS version 25 (IBM, Armonk, New York, USA) was used. Categorical and continuous data were tested on univariate analysis with the exact Mann- Whitney-Wilcoxon rank sum test and the exact Kruskal-Wallis rank sum tests, respectively. Statistical significance (all results were two-tailed) was set at P <0.05. Results Age-Related Progression of Medullary Thyroid Cancer by Unique RET Sequence Variant The age-related progression of MTC is depicted for 32 unique RET sequence variants, 29 of which are considered RET mutations, dispersed over 567 carriers (Table 1). The age-related progression of MTC was statistically significant for 13 unique RET mutations (p.cys611phe/c.1832g>t; p.cys611tyr; p.cys618ser/c.1852t>a; p.cys620arg; p.cys634arg; p.cys634phe; p.cys634ser; p.cys634tyr; p.glu768asp; p.leu790phe/c.2370g>t; p.val804met; p.ser891ala; p.met918thr), whereas 2 unique RET mutations (p.cys618phe; p.cys634gly) trended towards statistical significance by a narrow margin. The remaining 14 unique RET mutations, most of which involved fewer than 10 carriers, did not reach statistical significance.

9 9 To yield a statistically significant result, typically ten or more RET carriers, distributed across the three histopathological groups, were required. None of the 15 (52%) RET mutations that affected fewer than 10 carriers attained statistical significance. Eight unique RET mutations were associated with no (p.cys618tyr; p.cys620phe; p.cys620tyr; p.cys634ser) or one instance of node-positive MTC (p.cys620ser/c.1858t>a; p.cys620ser/c.1859g>c; p.cys630arg; p.cys634trp). Under these circumstances, only one RET variant (p.cys634ser) reached statistical significance (Table 1). No node-positive MTC was seen with two (p.ser649leu; p.ile852met) of the three 3 polymorphisms. Only one individual within each p.ser649leu, p.tyr791phe, or p.ile852met family developed MTC. Overall, polymorphisms were characterized by the absence of MTC in the patients thirties, whereas node-negative and node-positive MTC, if present, both occurred in the patients fifties, the typical age when sporadic MTC manifests clinically. Progression of MTC was rapid with highest-risk RET mutations (ATA category HST), fast with highrisk RET mutations (ATA category H), and slower with moderate-risk RET mutations (ATA category MOD). Certain differences existed between extracellular and intracellular moderate-risk RET mutations, perhaps reflecting inherent differences in the mode of action (receptor dimerization versus facilitation of downstream signaling) (Machens, Lorenz, & Dralle, 2009b). Age-Related Progression of Medullary Thyroid Cancer by RET Risk Category To clarify whether these differences warrant categorization into one or rather two risk groups, all 29 unique RET sequence variants were assigned to RET categories of highest (ATA HST), high (ATA H), moderate high (MOD H), and low moderate (L MOD) risk and contrasted with a fifth composite group of very rare (<1%) RET polymorphisms (PM): The age-related progression of MTC was significant for all four categories of RET mutations, which significantly differed across (Table 2, rows; Figure 1) and within (Table 2, columns; Figure 1) histopathological groups. With increasing age, the differences in tumor development dwindled away

10 10 between hereditary MTC in the low moderate RET risk category (L MOD) and sporadic MTC in the polymorphism category (PM): mean age 44.2 vs yrs. (P = 0.036) for node-negative MTC, and 55.2 vs yrs. (P = 0.39) for node-positive MTC (Table 2; Figure 1). Figure 1 delineates the window of opportunity in a worst-case scenario for early pre-emptive thyroidectomy (green area), with no need for central node dissection to keep operative morbidity to a minimum. This window of opportunity is marked off by the youngest age of carriers with nodepositive MTC in each mutational risk category (Figure 1, right pane). Age-Related Progression of Medullary Thyroid Cancer by Mutated Codon For moderate high (MOD H) (Table 3, upper pane) and low moderate (L MOD) risk mutations (Table 3, lower pane), the age-related progression of MTC by mutated codon was broadly comparable within each mutational category. Nevertheless, mutations in codon 611 tended to give rise to slower tumor progression than mutations in codons 618 and 620 (Table 3), perhaps owing to the greater distance of the former from the cell membrane spanning codons (Machens, Hauptmann, & Dralle, 2009). Age-Related Progression of Medullary Thyroid Cancer by Amino Acid Substitution For high (H) risk mutations (Table 4), the age-related progression of MTC by mutated codon was significant, or trended towards statistical significance, regardless of the amino acid substituting for the wildtype cysteine. High risk mutations were, by and large, comparable in terms of age-related tumor progression across the 3 histopathological groups.

11 11 Nodal disease developed significantly earlier, though, when cysteine was exchanged for arginine or tyrosine (mean age 28.9 vs yrs; P = 0.016; Table 4). For moderate (MOD) risk mutations (Table 5), the age-related progression of MTC by mutated codon within each histopathological group, in essence, was unaffected by the amino acids substituting for wildtype cysteine (p.cys611phe vs. p.cys611tyr; p.cys618phe vs. p.cys618ser) or wildtype valine (p.val804leu vs. p.val804met). Discussion This cohort study of 567 RET carriers, the most comprehensive study on the subject to date, illustrates the huge potential that lies in the combination of genomic and clinical-histopathological data to define the natural evolution of a rare genetic disease. Such natural history data are essential to lay a solid evidence-based foundation reflecting genuine mutational risk more accurately. Intriguingly, our comprehensive data argue in favor of splitting the 2015 ATA category MOD (Wells et al., 2015) into a moderate high (MOD H) and a low moderate risk category (L MOD). Marked individual variability was noted in the progression from C-cell hyperplasia to node-negative MTC and eventually node-positive MTC, framed by the width of the 95% confidence intervals, both within the same RET risk category and within groups carrying the same RET mutation. Age-Related Progression of MTC by Unique RET Mutation

12 12 The present cohort of RET carriers gave valuable insights into the age-related progression of MTC across a wide spectrum of unique RET mutations, laying a robust rational groundwork for genetic counseling of RET carriers. For 13 (45%) of 29 unique RET variants, the present study was able to validate the genotype-specific, age-related progression of MTC. Genotype-phenotype correlations form a frame (Figure 1) within which progression to early medullary thyroid cancer unfolds (Machens, Lorenz, & Dralle, 2009a; Machens, Elwerr, Lorenz, Weber, & Dralle, 2017). Genomic information does not allow one to pin this cellular transformation to a specific point in time (age). Biochemical monitoring delimits a window of opportunity that begins to shut down when calcitonin serum levels are crossing the upper reference limit of the assay (Machens, Lorenz, & Dralle, 2009a; Elisei et al., 2012). This signifies the last chance for pre-emptive thyroidectomy without central node dissection (Machens, Lorenz, & Dralle, 2009a; Machens, Elwerr, Lorenz, Weber, & Dralle, 2017).

13 13 Weak RET Mutation versus RET Polymorphisms To differentiate weaker, less penetrant mutations from benign polymorphisms, geneticists and clinicians depend on the segregation of genotype with the respective disease in two or more individuals of the same family (Margraf et al., 2009). Even when this genetic gold standard provides important clues as to the pathogenicity of gene variants, the clinical consequences regarding disease manifestation and progression are not straightforward. The rarer a RET sequence variant is in the general population, the more blurred becomes the line of distinction between mutation and benign polymorphism. Because they run in >1% of the general population, RET sequence variants p.gly691ser, p.leu769leu, p.ser836ser and p.ser904ser were assigned polymorphism status from inception (Margraf et al., 2009). In contrast, the three RET sequence variants p.ser649leu, p.tyr791phe, and p.ile852met, which affect 0.7% (Erlic et al., 2010), 0.31% to % (Erlic et al., 2010; Toledo et al., 2015), and 0.015% (Mathiesen et al., 2017) of the general population, were considered as pathogenic until recently. These examples are emblematic of the irreversible clinical consequences that may occur when fewer than 1000 samples (2000 chromosomes) are used as controls. In keeping with polymorphism status, the latter three RET sequence variants had no evidence of agerelated progression of MTC (Table 1). When combined into one polymorphism group, there was a divide in age between normal/cch and MTC pathologies (mean age 36.2 vs yrs; Table 2) without evidence of the age-related progression from node-negative to node-positive MTC typically encountered in hereditary disease. Because in vitro focus formation assays and computer-based in silico predictive models are no substitute to clinical validation, appropriate caution is warranted when clinicians are asked to trust computational outcomes for delivering patient care (Crockett et al., 2011). Limitations of the Study

14 14 When exploring rare genomic variants, inadequate statistical power always is an issue. Many genetic association studies are constrained in terms of the number of samples and have limited statistical power (Rotimi, & Jorde, 2010). In fact, 13 (45%) of the 29 unique RET mutations were observed in fewer than 10 carriers, attesting to the rarity of these mutations (Table 1). Validation of the agerelated progression of MTC was successful in 13 (87%) of the 15 unique RET mutations that involved 10 or more patients, whereas statistical significance trended towards statistical significance in the remaining two (13%) RET mutations with 10 or more patients (Table 1). In this particular situation, some researchers may consider correction for multiple testing as overly conservative. Notwithstanding the above, independent validation of the age-related progression of MTC remains important, especially for the 16 unique RET mutations that missed nominal statistical significance, presumably owing to low numbers. To overcome the methodological limitations of institutional settings for rare RET variants, improved data sharing among molecular geneticists and oncologists takes center stage. The inherent inability of in vitro focus formation studies and computer-based in silico analysis to separate late-onset (less penetrant) RET mutations from benign RET polymorphisms (Cosci et al., 2011; Crockett et al., 2011) calls for an international data collection effort for very rare gene variants. Future Perspectives Based on the present data, it may be reasonable to subdivide the 2015 ATA category MOD (Wells et al., 2015) into a moderate high (MOD H) and a low moderate risk category (L MOD) while emphasizing the need to contradistinguish the latter from rare non-pathogenic polymorphisms. The proposed split of the moderate 2015 ATA risk category (MOD), mirroring genotype-phenotype relationships better, would provide carriers of low moderate (L MOD) risk mutations with a five year longer expectant observation period ( 10 vs. 5 yrs. of age) than carriers of moderate high

15 15 (MOD H) risk mutations under the premise that calcitonin levels remain within normal limits (Figure 2). Owing to the global success of the concept of screening, surveillance and early pre-emptive thyroidectomy, a left shift in disease presentation patterns towards C-cell hyperplasia and early MTC confined to the thyroid gland has taken effect such that the opportunity of finding node-positive MTC in RET carriers is waning (Machens, & Dralle, 2015). Vanishing nodal disease is set to frustrate future endeavors to ascertain the pathogenicity of rare RET variants across the full spectrum of disease. The trend to earlier curative thyroidectomy is expected to enhance further when whole genomic sequencing in neonates for monogenic disorders will be able to uncover a genetic predisposition to MTC before the tumor develops.

16 16 Funding/Support This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Conflict of Interest Disclosures The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

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20 20 Machens, A., Lorenz, K., & Dralle, H. (2009a). Individualization of lymph node dissection in RET (rearranged during transfection) carriers at risk for medullary thyroid cancer: value of pretherapeutic calcitonin levels. Annals of Surgery, 250, Machens, A., Lorenz, K., & Dralle, H. (2009b). Constitutive RET tyrosine kinase activation in hereditary medullary thyroid cancer: clinical opportunities. Journal of Internal Medicine, 266, Machens, A., Lorenz, K., & Dralle, H. (2013). Peak incidence of pheochromocytoma and primary hyperparathyroidism in multiple endocrine neoplasia 2: need for age-adjusted biochemical screening. Journal of Clinical Endocrinology and Metabolism, 98, E336 E345. Machens, A., Lorenz, K., Sekulla, C., et al. (2013). Molecular epidemiology of multiple endocrine neoplasia 2: implications for RET screening in the new millenium. European Journal of Endocrinology, 168, Machens, A., Niccoli-Sire, P., Hoegel, J., et al.; European Multiple Endocrine Neoplasia (EUROMEN) Study Group (2003). Early malignant progression of hereditary medullary thyroid cancer. New England Journal of Medicine, 349, Margraf, R.L., Crockett, D.K., Krautscheid, P.M., et al. (2009). Multiple endocrine neoplasia type 2 RET protooncogene database: repository of MEN2-associated RET sequence variation and reference for genotype/phenotype correlations. Human Mutation, 30,

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23 Table and Figure Legends: Tab. 1: Age-Related Progression of Medullary Thyroid Cancer by Unique RET Sequence Variant Unique RET sequence variant Carrier age at thyroidectomy by histopathology, yr, mean [95% CI] RET risk category Frequency, % Protein change Genotype (cdna) No of carriers, total (normal and CCH/ MTCN0 / MTCN1) Normal and C-cell hyperplasia (CCH) MTC, nodenegative (MTCN0) MTC, node-positive (MTCN1) P Highest (ATA HST) 6.9 p.met918thr c.2753t>c 39 (1/13/25) [0.7;11.7] 16.2 [13.1;19.3] p.cys634arg c.1900t>c 84 (14/41/29) 4.9 [3.6;6.1] 20.9 [15.5;26.2] 30.2 [25.6;34.9] < This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: /humu p.cys634gly c.1900t>g 10 (3/3/4) [0;72.5]

24 24 High [1.9;9.5] [17.9;63.1] (ATA H) 3.9 p.cys634phe c.1901g>t 22 (9/9/4) 4.3 [0.7;8.0] 19.2 [8.2;30.2] 42.8 [20.1;65.4] < p.cys634ser c.1901g>c 15 (7/8/0) 5.4 [1.6;9.2] 15.6 [5.5;25.8] p.cys634trp c.1902c>g 1 (0/0/1) 46 N/A 9.2 p.cys634tyr c.1901g>a 52 (9/27/16) 4.7 [3.2;6.2] 17.1 [11.9;22.3] 26.4 [22.1;30.8] < p.cys609gly c.1825t>g 7 (2/3/2) 5.5 [0;11.9] [0;74.0] p.cys611phe c.1832g>t 17 (3/11/3) 23.6 [10.1;35.9] 41.9 [33.4;50.4] 55.3 [30.5;80.2] Moderate High ( MOD H ) 1.1 p.cys611phe c.1832_1833delinst T 3.4 p.cys611tyr c.1832g>a 19 (9/4/6) 11.2 [0.4;22.0] 6 (2/2/2) [6.7;83.1] 42.0 [26.2;57.8] 49.0 (31.3;66.7] (ATA MOD, extracellular) 1.1 p.cys618arg c.1852t>c 6 (1/2/3) 6 18 [0;56.1] 25.7 [4.5;46.8] 1.2 p.cys618gly c.1852t>g 7 (2/3/2) 8.5 [0;40.3] [0;48.0] p.cys618phe c.1853g>t 10 (2/3/5) 15.0 [0;65.8] 39.7 [0;87.9] 53.6 [40.7;66.5] 0.060

25 p.cys618ser c.1852t>a 22 (7/5/10) 12.6 [0;26.1] 33.4 [4.6;62.2] 40.6 [29.0;52.2] p.cys618ser c.1853g>c 6 (0/2/4) 8.0 [0;46.1] 40.8 [22.1;59.4] p.cys618tyr c.1853g>a 2 (2/0/0) 7.0 N/A 3.2 p.cys620arg c.1858t>c 18 (5/5/8) 4.8 [1.8;7.8] 29.0 [20.3;37.7] 38.4 [26.0;50.8] p.cys620phe c.1859g>t 5 (3/2/0) 8.7 [2.4;14.9] 1.2 p.cys620ser c.1858t>a 7 (5/1/1) 8.0 [0;17.0] p.cys620ser c.1859g>c 2 (1/0/1) 5 66 > p.cys620tyr c.1859g>a 6 (4/2/0) 12.8 [0;30.7] p.cys630arg c.1888t>c 4 (1/2/1) >0.99

26 26 Tab. 1: Age-Related Progression of Medullary Thyroid Cancer by Unique RET Sequence Variant (cont d) Unique RET sequence variant Carrier age at thyroidectomy by histopathology, yr, mean [95% CI] Risk category Frequency, % Protein change Genotype (cdna) No of carriers, total (normal and CCH/ MTCN0 / MTCN1) Normal and C- cell hyperplasia (CCH) MTC, node-negative (MTCN0) MTC, node-positive (MTCN1) P Low Moderate ( L MOD ) (ATA MOD, intracellular) 1.8 p.glu768asp c.2304g>c 10 (4/4/2) 13.5 [0;36.6] 48.0 [24.9;71.1] 71.0 [32.9;109.1] p.leu790phe c.2370g>c 9 (1/4/4) [25.7;82.8] 56.5 [54.9;58.1] p.leu790phe c.2370g>t 52 (18/16/18) 20.6 [12.7;28.5] 42.9 [33.2;52.5] 53.4 [44.3;62.6] < p.val804leu c.2410g>t 14 (4/8/2) 35.5 [5.0;66.0] 47.0 [34.3;59.7] p.val804met c.2410g>a 53 (26/14/13) 26.5 [20.4;32.7] 42.9 [35.5;50.2] 51.3 [43.4;59.2] < p.ser891ala c.2671t>g 25 (10/9/6) 14.4 [7.4;21.4] 39.8 [27.2;52.4] 62.5 [54.1;70.9] <0.001 Polymorphis m ( PM )* 0.7 p.ser649leu * c.1946c>t 4 (3/1/0) 53.0 [31.3;74.7] p.tyr791phe* c.2372a>t* 27 (22/3/2) 35.8 [25.3;46.3] 53.3 [2.6;104.1] 59.5 [40.4;78.6] p.ile852met* c.2556c>g* 6 (4/2/0) 26.0 [0;66.5] ATA, American Thyroid Association RET, REarranged during Transfection

27 27 Owing to rounding, not all percentages add up. : Exact Kruskal-Wallis rank sum test *: Recently considered polymorphism (Erlic et al., 2010; Toledo et. al., 2015; Mathiesen et al., 2017)

28 Tab. 2: Age-Related Progression of Medullary Thyroid Cancer by RET Risk Category Carrier age at thyroidectomy, yr, mean [95% CI], median (range) RET risk category Mutated codons with amino acid changes No of carriers, total (normal and CCH/ MTCN0 / MTCN1) Normal and C-cell hyperplasia (CCH) MTC, nodenegative (MTCN0) MTC, nodepositive (MTCN1) P Highest (ATA HST) p.met918thr (1 / 13 / 25) [0.7 ; 11.7] (0.5 31) [13.1 ; 19.3] (5 31) P <0.001 < High (ATA H) p.cys634x (42 / 88 / 54) [3.9 ; 5.8] (1 14) [15.8 ; 22.2] (1 69) [27.8 ; 34.4] (9 63) < P <0.001 <0.001 <0.001 Moderate High (ATA MOD, extracellular) p.cys609x, p.cys611x, p.cys618x, p.cys620x, p.cys630x 144 (49 / 47 / 48) 10.7 [7.8 ; 13.6] (0.9 47) 33.0 [28.2 ; 37.9] (1 67) 41.8 [37.3 ; 46.3] (15 75) < P <0.001 <0.001 <0.001 p.glu768asp, This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: /humu

29 29 Low Moderate (ATA MOD, intracellular) p.leu790phe, p.val804leu/met, p.ser891ala (63 / 55 / 45) [18.8 ; 26.7] (3 66) [39.9 ; 48.4] (12 75) [50.7 ; 59.7] (10 81) < P p.ser649leu, Polymorphisms ( PM ) p.tyr791phe, p.ile852met (29 / 6 / 2) [27.4 ; 45.1] (5 87) [41.9 ; 71.1] (30 68) [40.4 ; 78.6] (58 61) P <0.001 <0.001 <0.001 ATA, American Thyroid Association RET, REarranged during Transfection : Exact Kruskal-Wallis rank sum test : Exact Mann-Whitney-Wilcoxon rank sum test (for differences between adjacent RET risk categories)

30 30 Tab. 3: Age-Related Progression of Medullary Thyroid Cancer by Mutated Codon (ATA Moderate Risk Category) Carrier age at thyroidectomy, yr, RET mean [95% CI], median (range) Risk category Mutated codons with amino acid changes* No of carriers, total (normal and CCH/ MTCN0 / MTCN1) Normal and C- cell hyperplasia (CCH) MTC, nodenegative (MTCN0) MTC, nodepositive (MTCN1) P $ Moderate High (ATA MOD, extracellular) p.cys611x p.cys618x (14 / 17 / 11) 53 (14 / 15 / 24) [7.6 ; 22.7] (3 47) 11.1 [5.0 ; 17.2] (4 45) [33.5 ; 46.7] (14 63) 26.5 [16.2 ; 36.9] (5 67) [41.0 ; 59.0] (31 75) 40.5 $ [34.1 ; 46.9] (17 70) < < p.cys620x (18 / 10 / 10) [4.7 ; 11.5] (0.9 29) [25.6 ; 40.6] (18 54) [29.8 ; 52.4] (21 66) < P p.glu768asp (4 / 4 / 2) [0 ; 36.6] (3 35) [24.9 ; 71.1] (29 64) [32.9 ; 109.1] (68 74) Low Moderate (ATA MOD, intracellular) p.leu790phe 61 (19 / 20 / 22) 21.2 [13.7 ; 28.8] (5 53) 45.2 [36.6 ; 53.7] (12 75) 54.0 [46.6 ; 61.4] (10 81) < p.val804leu/met (30 / 22 / 15) [21.8 ; 33.6] (6 66) [38.4 ; 50.3] (21 [44.1 ; 59.7] (23 <0.00 1

31 31 66) 75) p.ser891ala (10 / 9 / 6) [7.4 ; 21.4] (4 34) [27.2 ; 52.4] (17 68) [54.1 ; 70.9] (55 78) < P ATA, American Thyroid Association RET, REarranged during Transfection *: Minimum of 10 RET carriers per codon per amino acid exchange : Exact Kruskal-Wallis rank sum test : Exact Mann-Whitney-Wilcoxon rank sum test: P = $: Exact Mann-Whitney-Wilcoxon rank sum test: P = Tab. 4: Age-Related Progression of Medullary Thyroid Cancer by Amino Acid Substitution (ATA High Risk Category) Carrier age at thyroidectomy, yr, RET mean [95% CI], median (range) Mutated codons with amino acid changes* No of carriers, total (normal and CCH/ MTCN0 / MTCN1) Normal and C- cell hyperplasia (CCH) MTC, nodenegative (MTCN0) MTC, node-positive (MTCN1) P p.cys634tyr (9 / 27 / 16) [3.2 ; 6.2] (1 7) [11.9 ; 22.3] (1 47) [22.1 ; 30.8] (16 41) < p.cys634arg (14 / 41 / 29) [3.6 ; 6.1] (1 9) [15.5 ; 26.2] (3 49) [25.6 ; 34.9] (9 57) < [17.9 ; 63.1]

32 32 p.cys634gly (3 / 3 / 4) [1.9 ; 9.5] (4 7) [0 ; 72.5] (6 44) (27 56) p.cys634phe (9 / 9 / 4) [0.7 ; 8.0] (1 14) [8.2 ; 30.2] (3 38) [20.1 ; 65.4] (30 63) < p.cys634ser (7 / 8 / 0) [1.6 ; 9.2] [5.5 ; 25.8] (2 13) (4 42) P ATA, American Thyroid Association RET, REarranged during Transfection *: Minimum of 10 RET carriers per codon per amino acid exchange : Exact Kruskal-Wallis rank sum test : Exact Mann-Whitney-Wilcoxon rank sum test (p.cys634arg/tyr vs. p.cys634gly/phe): 28.9 vs yrs; P = Tab. 5: Age-Related Progression of Medullary Thyroid Cancer by Amino Acid Substitution (ATA Moderate Risk Category) Carrier age at thyroidectomy, yr, RET mean [95% CI], median (range) Mutated codons with amino acid changes* No of carriers, total (normal and CCH/ MTCN0 / MTCN1) Normal and C- cell hyperplasia (CCH) MTC, nodenegative (MTCN0) MTC, node-positive (MTCN1) P [31.1 ;47.9] 51.2 [39.6 ; 62.8]

33 33 p.cys611phe 23 (5 / 13 / 5) [12.2 ; 32.2] (14 63) (42 65) (11 31) p.cys611tyr 19 (9 / 4 / 6) [0.4 ; 22.0] (3 47) [26.2; 57.8] (29 53) [31.3 ; 66.7] (31 75) P p.cys618phe 10 (2 / 3 / 5) [0 ; 65.8] [0 ; 87.9] [40.7;66.5] (4 47) (4 47) (4 47) p.cys618ser 28 (7 / 7 / 14) [0 ; 26.1] (4 45) [5.1 ; 47.2] (5 67) [32.2 ; 49.1] (25 70) P p.val804leu 14 ( 4 / 8 / 2) [5.0 ; 66.0] (15 61) [34.3 ; 59.7] (22 66) - (37 74) p.val804met 53 (26 /14 / 13) [20.4 ; 32.7] (6 66) [35.5 ; 50.2] (21 66) [43.4 ; 59.2] (23 75) < P ATA, American Thyroid Association RET, REarranged during Transfection *: Minimum of 10 RET carriers per codon per amino acid exchange : Exact Kruskal-Wallis rank sum test : Exact Mann-Whitney-Wilcoxon rank sum test

34 34 Figure 1: Window of Opportunity for Early Pre-emptive Thyroidectomy

35 35 Figure 2: RET Tyrosine Kinase Receptor Genotype and Clinical Implications

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