Introduction

Medullary thyroid carcinoma (MTC) is a rare malignant tumor of the thyroid gland arising from the parafollicular cells (C cells), often associated with elevated serum calcitonin levels. It accounts for 3.5–10 % of all thyroid cancers [3–6], and for 13.4% of all thyroid cancer-related deaths [7]. It occurs in both sporadic and hereditary forms. The former accounts for approximately 25 % of all MTC cases, which often occur as part of multiple endocrine neoplasia syndromes type 2A (MEN 2A)/familial MTC (FMTC) and MEN2B [7]; whereas, the latter is more common and constitutes 70–80 % of MTC [4, 7].

Germline activating mutations in proto-oncogene rearranged during transfection (RET) are identified as the genetic basis for MEN2A, MEN2B, and FMTC syndromes. Greater than 90 different RET mutations in exons 5, 8, 10, 11, 12 to 16, and 19 have been associated with hereditary MTC. Most of these are missense mutations, which lead to constitutive activation of the RET tyrosine kinase [8]. American Thyroid Association (ATA) has published recommendations on the timing of prophylactic thyroidectomy and the extent of surgery, which were based on genotype–phenotype correlations that stratify mutations into four risk levels [9, 10].

Up to half of the sporadic MTC harbor somatic RET mutations, located in exons 10, 11, 15, and 16 [11]. Recently RAS mutations have been reported in a subset of sporadic MTCs [1, 2]. These commonly occur in exons 2, 3, and 4 of HRAS and KRAS [12], while NRAS mutations are rare [1, 12]. The knowledge of the type of gene mutation in MTC is important to determine the treatment of the patients and the management of their family members. Furthermore, recently, the type of somatic mutation of RET in tumor tissue has proved to have a prognostic value for anti-tyrosine kinase therapy, such as Vandetanib [13].

The conventional methods for detecting RET mutation are polymerase chain reaction amplification (PCR)/Sanger sequencing-based testing, such as restriction enzyme analysis, amplification-refractory mutation system (ARMS) assay, and direct DNA sequencing following PCR [13, 14]. Recently, implementing Next-generation sequencing (NGS) allows concurrent analysis of many genes/exons in a single assay instead of one DNA fragment at a time (Sanger sequencing) [15, 16]. Many clinical laboratories are now implementing targeted NGS panels covering dozens of disease-associated genes [15, 17–19].

In this study, we are reporting our experience of detecting the molecular alterations in a series of MTC using NGS in a routine clinical setting.

Materials and Medthods

This study was approved by the University of Pennsylvania institutional Review Board. A total of 12 cases of MTC were included in this study diagnosed at the University of Pennsylvania health system (2005 to 2015). Medical record review was performed to collect clinical information. This case cohort included three patients who were referred to the Hospital of the University of Pennsylvania, and the outside surgical pathology slides were reviewed; unstained slides and/or paraffin embedded blocks were received for NGS.

The hematoxylin and eosin stained slides were reviewed to confirm at least 10 % of tumor cellularity. The tumor area was extracted for genomic DNA according to manufacturer’s instructions (Qiagen, Inc.). Targeted analysis for mutations in the regions specified in this testing panel was achieved by enrichment of those genomic loci using the Illumina Truseq Amplicon Assay (Illumina, San Diego, CA). DNA was quantified using a fluorescent-based measurement (Qubit, Life Technologies), and 20 to 250 ng of DNA was used for custom target enrichment. Sequencing of enriched libraries was performed on the Illumina MiSeq platform using multiplexed, paired end reads to an average depth of coverage greater than 1000. Analysis and interpretation utilized a customized bioinformatics process. All variants listed are with reference to the hg19 genome build. The sequencing panel consists of 47 genes (ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL).

Results

The case cohort of 12 patients comprised of 7 males and 5 females with a mean age of 50.2 years (31–71 years) (Table 1). Three patients had hereditary MTC; case 1 had a history of pheochromocytoma, and case 3 had a history of parathyroid adenoma. Lymph node metastases were present in 10 out of 12 cases (10/12, 83.3 %). Five cases had distant metastasis (41.7 %). Clinical follow-up ranged from 1 to 12 years (average 4.7 years); three patients died of disease (Table 1).

Table 1 Patient clinicopathological characteristics
Full size table

Successful 47 genes NGS was performed in all 12 cases (Table 1). Eleven out of 12 cases (91.7 %) harbored RET mutation. Only one case of sporadic MTC had HRAS mutation; all the remaining sporadic MTC had RET/M918T mutation. Two of the hereditary MTC had RET/C634R mutation; while the remaining one harbored two RET mutations (L790F and S649L).

Discussion

Multiplex next-generation sequencing (NGS) analysis is capable of detecting the full range of mutation types of multiple genes on limited and/or minute specimens [21]. The gene panel used in this study contains all the common genes associated with MTC, including RET, HRAS, NRAS, and KRAS. The low input quantity of DNA acceptable for NGS makes it applicable to various types of specimens in a clinical laboratory [22, 23]. In this study, NGS detected as low as 4.15 % allele frequency (cases 5), which only requires about 10 % tumor burden in one specimen compared with 30 to 40 % of tumor content for Sanger sequencing [24].

The RET gene is composed of 21 exons located on chromosome 10 (10q11.2) [25] and plays an important role in the development of the parathyroid, urogenital system, and neural crest—including ganglia, adrenal medulla, and thyroid C cells [26]. Different mutations in the RET gene are associated with varying phenotypes of MEN2A/FMTC and MEN2B including age of onset, aggressiveness of MTC, and with or without associated pheochromocytoma or primary hyperparathyroidism [10]. All three cases of hereditary MTCs in this study had the phenotype of MEN2A/FMTC. North American Neuroendocrine Tumor Society recently published consensus guidelines for the diagnosis and management of MTC, which were developed by classifying RET mutations into three groups based on aggressiveness of MTC or level of risk [9, 20]. Hereditary MTC with mutation of p.M918T are the most aggressive tumor (level 3), in which metastasis can develop in the first year of life [9]. Several professional groups have developed guidelines for the timing of prophylactic thyroidectomy, all of which are based on the perceived clinical behavior of the specific RET mutation [10, 20]. The three cases of hereditary MTC in this study had low risk RET mutations (level 1 and 2) (Table 1).

Multiple somatic mutations of RET can occur at a very low rate [11]. In this study, one of the hereditary MTC had two RET point mutations (p.L790F, c.2370G>T and p.S649L, c.1946C>T). RET/L790F mutation is associated with a non-aggressive form of MEN2 [9]. Bihan reported 77 French patients from 19 families with a mutation in codon 790 of the RET; only one patient had pheochromocytoma, and no patient had primary hyperparathyroidism [9]. Frank-Raue reported 47 patients with mutation in codon 790 of the RET; none of them had pheochromocytoma or primary hyperparathyroidism [27]. RET/S649L is a rare mutation, which is associated with MEN2A [28]. It is generally regarded as low risk mutation. Colombo-Benkmann documented three patients with RET/S649L had MTC, but two of them carrying both the S649L mutations and a second mutation (C634W or V804L), which are high-risk mutations [28, 29]. However, RET/S649L can be associated with primary parathyroidism [29], which explains the parathyroid adenoma in our case.

RET mutations can occur in greater than half of the cases of sporadic MTC [11]. The M918T mutation is the most common mutation in sporadic MTC [11, 30]. In this study, eight out of nine cases (88.9 %) of sporadic MTC showed M918T mutation. The remaining case had HRAS mutation. The family of human RAS genes includes the highly homologous HRAS, KRAS, and NRAS genes, which encode GTPases that function as molecular switches in regulating pathways that are responsible for diverse cellular processes. Activating mutations of RAS genes are found in about 30 % of all human cancers. In thyroid carcinoma, RAS gene point mutations, mainly in NRAS, are detected in follicular adenoma and carcinoma [31]. RAS mutations were reported in a subset of sporadic MTCs [1, 2]. Mutations commonly occur in HRAS and KRAS [12], while NRAS mutations are rare [1, 12, 31]. RAS mutations are almost always mutually exclusive with RET mutations, which present in 10 to 45 % of RET wild-type sporadic tumors [12, 30].

In this study, the patient with HRAS mutation had both medullary carcinoma and parathyroid adenoma. This patient had a very aggressive disease, and died at age of 46, 3 years after first being diagnosed with MTC. Ciampi reported that the outcome of patients with a somatic RET mutation was significantly worse than the outcome of both RAS-positive/RET-negative and RAS-negative/RET-negative cases [2]. Margarida reported that there was no statistically significant difference in clinical and pathological characteristics between RAS-positive and RAS-negative cases without somatic RET mutations [1]. However, a study by Moura showed that MTC with RAS mutations have an intermediate risk between those with RET mutations in exons 15 and 16, which are associated with the worst prognosis, and cases with other RET mutations, that follows an indolent clinical course [31].

Conclusions

NGS can provide comprehensive analysis of molecular alterations in MTC in a routine clinical setting, which facilitate determining the treatment of the patient and prophylactic thyroidectomy for the patient’s family members harboring mutations. In this study, two of the hereditary MTC had RET/C634R mutation, while the other one harbored two RET mutations (L790F and S649L). All the sporadic MTC had RET/M918T mutation except one case with HRAS mutation in this series.