A novel multimodal diagnostic framework integrating hyperspectral imaging and deep learning for predicting RET gene mutations in medullary thyroid carcinoma.

The clinical management of patients with persistent or recurrent medullary thyroid carcinoma (MTC) is still under debate, because these patients either have a long-term survival, due to an indolent course of the disease, or develop rapidly progressing disease leading to death from distant metastases. At this moment, it cannot be predicted what will happen within most individual cases. Biomarkers, indicators which can be measured objectively, can be helpful in MTC diagnosis, molecular imaging and treatment, and/or identification of MTC progression. Several MTC biomarkers are already implemented in the daily management of MTC patients. More research is being aimed at the improvement of molecular imaging techniques and the development of molecular systemic therapies. Recent discoveries, like the prognostic value of plasma calcitonin and carcino-embryonic antigen doubling-time and the presence of somatic RET mutations in MTC tissue, may be useful tools in clinical decision making in the future. In this review, we provide an overview of different MTC biomarkers and their applications in the clinical management of MTC patients.

Identification of the germline mutation in the RET proto-oncogene is important for the diagnosis of hereditary medullary thyroid carcinoma (MTC). Hereditary forms account for approximately 25%-30% of all cases of MTC. The objective of this study was to evaluate the prevalence of the RET mutation and the genotype-phenotype relation in Korean patients with MTC. Genomic DNAs were obtained from 33 patients with MTC (M:F = 10:23, 39.8 +/- 12.0 years) who underwent total thyroidectomy between 1997 and 2003 at the Samsung Medical Center. Exons 10, 11, 13, 14, 15 and 16 of the RET proto-oncogene were amplified with specific primers using polymerase chain reaction (PCR). Sequence analysis was performed on the polymerase chain reaction (PCR) product using an automatic sequence analyzer. Nine of the 33 patients (M:F = 3:6, 33.3 +/- 10.0 years) were identified as having RET mutations. Six patients had multiple endocrine neoplasia (MEN) 2A and one had familial medullary thyroid carcinoma (FMTC). The remaining two patients were thought to have sporadic MTC. Five of the patients with MEN 2A had RET mutations in codon 634 of exon 11 (3 patients, C634Y; 2 patients, C634R) and the other patient with MEN 2A had a RET mutation in codon 618 of exon 10 (C618R). The patient with FMTC had a mutation in codon 634 (C634W). The two patients with sporadic MTC had RET mutations in codon 634 (1 patient, C634Y; 1 patient, C634S). We were not able to identify any genotype-phenotype relations because of the limited number of patients. Twenty-seven percent (9/33) of the patients with MTC in this study had RET mutations. Taking other studies into account, 77% (10/13) of Korean families with MEN 2A, including 7 other families in three reports from Korea, had RET mutations in codon 634 (5 families, C634Y; 4 families, C634R; 1 family, C634W), and 23% (3/13) had RET mutations in codon 618 (2 families, C618R; 1 family, C618S). RET proto-oncogene mutations were restricted to codon 634 and 618 in Korean families with MEN 2A.

Background. Medullary thyroid cancer (MTC) is a rare, endocrine tumour that occurs as part of hereditary cancer syndrome in approximately 25% of cases and develops as a sporadic form in 75%. The aim of the study was to evaluate the frequency and type of RET mutation occurrence in the Slovenian population between 1995 and 2015. Methods. A retrospective analysis of the National Cancer Registry of the Republic of Slovenia and the Registry of Patients with MTC of the Institute of Oncology in Ljubljana between 1995 and 2015 was made. The data of 143 patients with confirmed MTC and 43 healthy relatives referred to genetic counselling and testing was analyzed. Genomic DNA was isolated from peripheral blood leukocytes. Exons 10, 11, 13, 14, 15 and 16 of the RET proto-oncogene were amplified in the polymerase chain reaction (PCR). Point mutations of the RET gene were detected by single strand confirmation analysis (SSCA) and DNA sequencing. Detected mutations were confirmed by restriction enzymes. Results. A germline mutation in the RET proto-oncogene was identified in 51 individuals, 37 among them were patients with MTC, which accounts for 25.9% of all patients with MTC. Eight different germline RET mutations were found. Codons 634 and 618 were the most frequently altered codons, followed by codon 790, codon 804 and codon 918. The average crude incidence rate of MTC (between 1995 and 2015) is 0.34 per 100,000. Conclusions. We estimated that the crude incidence rate of MTC in Slovenia is 0.34 per 100,000. We discovered that 25.9% of Slovenian patients with MTC are RET mutation carriers. The most common mutations were found on codons 634 and 618.

Background: Tumor-associated macrophages (TAMs) are alternatively activated or M2 macrophages that infiltrate cancer tissues. It has been suggested that TAMs exert several pro-tumor functions by releasing several growth factors and proangiogenic cytokines that favor tumor progression. In fact, TAM density has been associated with poor prognosis. Although it is well known the pro-tumor roles of TAM in several tumor types, its role in medullary thyroid cancer (MTC) is still unknown. We here evaluated the M2 macrophage marker CD163 in MTC and correlated with RET mutational status and clinical and pathological features. Because it has been suggested TAMs could interact with CD44-positive tumor cells to during tumorigenesis process, we also investigated the expression of CD44 in MTC. Patients and Methods: CD163 and CD44 expression we investigated in 78 MTC by immunohistochemistry (IHC). Because hereditary form is caused by germline mutations in the RET gene, all patients were screened for RET mutations (exons 8, 10, 11, 13-16) in the DNA isolated from blood samples. Those MTC that were negative for RET germline mutations, were also screened for somatic mutations in the RET gene (exons 10, 11 e 16). Results: Germline mutation in the RET gene were identified in 29 patients (p.G533C, p.C609Y, p.C611Y, p.C620Y, p.C634R, p.C634Y/Y791F, p.S891A and p.M918V). Approximately 49 cases are sporadic cases in which somatic RET mutations were identified in about 6 cases (p.M918T, p.C634R and p.C634Y). CD163 expression was mainly found in sporadic MTC when compared to hereditary MTC (p=0.021), suggesting that TAM may play a role in the progression of sporadic MTC. On the other hand CD44 expression was mainly found in hereditary MTC than in sporadic cases (p=0.017), particularly in patients with RET germlime mutation at codon C634. Conclusion: TAM seems to be more important for sporadic MTC tumorigenesis when compared to hereditary MTC. On the other hand, CD44 expression correlated with hereditary MTC, particularly in patients with mutation at codon C634. Citation Format: MARTA MIYAZAWA, MARIA JOSE C. P. SANTOS, LARISSA V. BIM, ERIKA S. L. ORTI-RADUAN, FLÁVIA O. F. VALENTE, ADAUTO F. NUNES, ROSANA DELCELO, RUI M. B. MACIEL, JANETE M. CERUTTI. TAM and CD44 in medullary thyroid carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1072. doi:10.1158/1538-7445.AM2014-1072

6000 Background: Cabo extends progression-free survival (PFS) in patients (pts) with progressive, metastatic MTC (Schöffski, J Clin Oncol 30, 2012). Mutations in the RET oncogene are associated with most hereditary cases and ~half of sporadic cases of MTC. RAS gene mutations have recently been identified in subsets of RET wild type (wt) cases. Therefore, we investigated the association of RET (a prospectively defined endpoint) and RAS mutations (a post hoc analysis) with efficacy outcomes in the phase 3 study of cabo in MTC. Methods: Pts enrolled into the double-blind, placebo-controlled phase III trial were evaluated for the presence of somatic and germline RET mutations using Sanger and next generation methods. A subset of pts determined to be RET wt (44 pts) or RET unknown (41 pts) were then evaluated for tumor-associated mutations in KRAS, NRAS, and HRAS in codons 12, 13, and 61 by next generation sequencing. Impact of RET and RAS gene mutation status was evaluated with respect to PFS and tumor response rate (RR) according to RECIST. Results: RET status was determined in 65% of the study pts (215/330), of which 79% harbored an activating mutation, and 21% were RET wt. All RET mutational subgroups (RET mutated, RET wt, and RET unknown) showed hazard ratios indicating PFS benefit from cabo treatment, and demonstrated RRs between 22% and 32%. However pts harboring a RET mutation had longer median PFS on cabo (60 wks) than pts with wt RET (25 wks, PFS difference p=0.0001). Also, pts with the poor prognosis mutation RET M918T showed a longer median PFS on cabo treatment (61 wks) than pts with any other RET mutation (36 wks, PFS difference p=0.009). Patients with hereditary MTC had similar PFS to those with sporadic disease, and the presence of the common RET polymorphism G691S had no effect on either PFS or RR. Sixteen of 85 tested pts (5% of total study pts) with wt or unknown RET status were found to harbor a RAS gene mutation. The RAS-mutated pts showed a similar RR (31%) and PFS (47 wks) as RET mutated pts (32% and 60 wks). Conclusions: While hazard ratios indicate PFS improvement for all RET subgroups on cabo, the extent of benefit may depend in part on RET genotype. Cabo treatment benefit is also seen in pts harboring a RAS mutation. Clinical trial information: NCT00704730.

Germline mutations in the RET proto‐oncogene are the cause of multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Some cases of sporadic medullary thyroid carcinoma (MTC) have also been reported to have mutations in the RET gene. However, two previous reports have given discrepant results on the frequency of the mutations in RET in sporadic MTCs in Japan. To clarify this problem, we analyzed mutations in RET exon 16 in 72 sporadic MTCs by means of the two methods used in the previous studies, direct sequencing and polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP). Mutations in exon 16 were detected in only 2 of 72 cases of sporadic MTC. These results suggest that when a MTC has a mutation in RET exon 16, it is more likely to be a hereditary MTC than a sporadic one in Japan.

RET proto-oncogene (RET) mutations were proved to be related to the development of medullary thyroid carcinoma (MTC). We aimed to analyze the role of RET mutations in cervical lymph node metastasis in patients with MTC. Forty-nine patients with preoperatively diagnosed MTC by fine-needle aspiration cytology (FNAC) who underwent bilateral total thyroidectomy with cervical lymphadenectomy were included. Postoperative RET gene test and pathological analysis were performed with the surgical specimens; serum calcitonin (Ctn) and carcinoembryonic antigen (CEA) levels were tested pre- and postoperatively, to evaluate the association between RET mutations and cervical lymph node metastasis in MTC. In these 49 patients, the RET mutation rates of Exon 11, Exon 10, Exon 11&13, Exon 13, and Exon 16 were 20.4%, 4.1%, 38.8%, 22.4%, and 0%, respectively. The lymph node metastasis rates of patients with RET mutation in the central and lateral compartments were 71.4% and 64.3%, respectively, versus 28.6% and 14.3% of patients without RET mutation. The preoperative basal serum levels of Ctn (234.8 ± 188.4 vs. 44.4 ± 27.5, p< 0.01) and postoperative Ctn (49.8 ± 86.4 vs. 3.7 ± 2.2, p= 0.001) in MTC patients with RET mutations were significantly higher than those in MTC patients without RET mutation. In addition, the preoperative (50.2 ± 76.7 vs. 7.4 ± 6.8, p= 0.001) and postoperative serum levels of CEA (13.2 ± 19.5 vs. 1.3 ± 1.6, p< 0.01) in MTC patients with RET mutations were significantly higher than those in MTC patients without RET mutation (p< 0.05). RET mutation was related to cervical lymph node metastasis in patients with MTC, especially the mutation in Exon 11&13. Patients with RET mutation in Exon 11&13 might be regarded as the predictor for prophylactic ipsilateral total cervical lymphadenectomy even without clear evidence of lateral cervical lymph node metastasis.

RET Oncogene Mutations in Medullary Thyroid Carcinoma in Mexican Families

Thyroid cancer is the most common endocrine malignancy and accounts for nearly 1% of all of human cancer. Thyroid cancer has four main histological types: papillary, follicular, medullary, and anaplastic. Papillary, follicular, and anaplastic thyroid carcinomas are derived from follicular thyroid cells, whereas medullary thyroid carcinoma (MTC) originates from the neural crest parafollicular cells or C-cells of the thyroid gland. MTC represents a neuroendocrine tumor and differs considerably from differentiated thyroid carcinoma. MTC is one of the aggressive types of thyroid cancer, which represents 3–10% of all thyroid cancers. It occurs in hereditary (25%) and sporadic (75%) forms. The hereditary form of MTC has an autosomal dominant mode of inheritance. According to the present classification, hereditary MTC is classified as a multiple endocrine neoplasi type 2 A & B (MEN2A & MEN2B) and familial MTC (FMTC). The RET proto-oncogene is located on chromosome 10q11.21. It is composed of 21 exons and encodes a transmembrane receptor tyrosine kinase. RET regulates a complex network of signal transduction pathways during development, survival, proliferation, differentiation, and migration of the enteric nervous system progenitor cells. Gain of function mutations in RET have been well demonstrated in MTC development. Variants of MTC result from different RET mutations, and they have a good genotype–phenotype correlation. Various MTC related mutations have been reported in different exons of the RET gene. We proposed that RET genetic mutations may be different in distinct populations. Therefore, the aim of this study was to find a geographical pattern of RET mutations in different populations.

Our understanding of the genomics and epigenomics of medullary thyroid carcinoma (MTC) has advanced since the initial recognition of RET as a driver of MTC tumorigenesis in familial MTC. We now have insight into the frequency and prognostic significance of specific RET mutations in sporadic MTC. For example, the most common RET mutation in sporadic MTC is the RET Met918Thr mutation, the same mutation that underlies MEN2B and a poor prognosticator. This mutation is relatively infrequent in medullary thyroidmicrocarcinomas but is over-represented in advanced-stage disease. RAS mutations are detected in 70% of sporadic, RET wild-type MTC. Although next-generation and whole-exome sequencing studies have shown that tumors that are wild-type for RET and RAS mutations essentially lack other recurrent mutations, additional pathways and epigenetic alterations have been implicated in MTC tumorigenesis. Increased insight into the clinical course of patients with familial MTC with specific RET mutations has guided treatment recommendations for these patients. Finally, an understanding of the genomics has informed treatment for patients with advanced MTC. In this review, we will examine the genomics and epigenomics of sporadic and familial MTC, along with the prognostic significance of molecular alterations, management of patients with germline RET mutations, and treatment strategies for MTC patients.

Genetically caused medullary thyroid cancer (MTC) is associated with unfavorable survival prognosis, so it makes necessary to develop new diagnostic techniques to reveal pre-clinical stage of disease as well as to introduce into clinical practice the effective method of tumor prevention. The article represents first in Russia summary clinical experience of prophylactic thyroidectomy have been executed in the period 1998 – 2015 yeas among ten bearers of RET gene germlinemutation in families with hereditary disease including syndrome MEN2A and familial MTC. Aim : to evaluate the results of surgical treatment of asymptomatic carriers of germinal mutations in the RET gene. Materials and methods . In the period from 1998 to 2015, in two centers: N.N. Blokhin Russian Cancer Research Center, Moscow and A. Tsyb Medical Radiological Research Centre – branch of the National Medical Research Radiological Centre, Obninsk was conducted prophylactic surgical treatment in 10 patients – asymptomatic carriers of germinal mutations in RET. Age of patients – from 2 to 23 years old. 9 patients – from families with the syndrome of multiple endocrineneoplasia type 2A (MEN2A), one – with the family of MTC. According to genealogy in 9 families of patients there have been cases of death from MTC or pheochromocytoma (PC). In all cases, surgical treatment was performed in a volume of TE, two patients additionally performed lymph node dissection VI level. The observation period after surgery ranged from 6 months to 16 years. Results . DNA diagnostics in 8 patients identified a mutation in exon 11, in one case – in exon 10 and one patient had revealed two mutations in exons 13 and 14. The age of patients ranged from 2 to 23 years. Basal calcitonin level was elevated in 7 of 10 patients. Such prophylactic TE in 2 patients was supplemented by selective lymph node dissection. Histological examination of the removal of the thyroid gland (TG) revealed foci of medullary cancer in 6 of 10. At 2 patients revealed a C-cell hyperplasia and at 2 patients were found signs of the well expressed and weakly expressed sclerosis in thyroid tissue. The earliest age to identify MTC was a child 3 years old, mutations in codon 634, from a family where relatives observed for aggressive MTC. In the course of follow-recurrence was detected in one patient, a child of 15 years, a similar mutation carrier. Conclusion . In view of the risk of MTC developing identifying a mutation in RET gene and preventive TE should be carried out as soon as possible. The high risk of MTC developing in RET-gene positive subjects was confirmed in this study (6 cases of cancer from 10 patients).

Hirschsprung's disease (HD) can be associated with the development of neuroendocrine tumours such as medullary thyroid carcinoma (MTC). The RET proto-oncogene is the major gene responsible for both HD and MTC. Mutations in exon 10 (codons 609, 611, 618, 620) were found in patients with co-occurrence of HD and MTC. The aim of the study was to screen the MTC risk in patients with HD. The prospective and retrospective genetic analyses comprised 56 HD patients (41 males, 15 females, aged 0-47). The prospective subgroup of patients consisted of 34 patients (25 boys, 9 girls) operated on between June 2003 and December 2005. The retrospective subgroup comprised 22 patients (16 boys, 6 girls) of 194 patients who were operated on between December 1979 and May 2003, non-systematically chosen preferably for total colonic aganglionosis (TCA). DNAs were isolated from blood and resected segments of aganglionic bowel. The HD patients and nine available family members (2 HD) were tested for RET mutations in exons 10, 11, 13, 14, 15 and 16. Direct double-stranded fluorescent sequencing revealed typical germline heterozygous MTC risk RET mutations in 3/56 (5.4%) female HD patients: Cys609Tyr, Cys620Arg (both exon 10) and Tyr791Phe (exon 13). Two of these patients had TCA and one patient had classical type of HD. One TCA patient developed clinical stage of MTC and underwent total thyroidectomy (TTE). The other two RET positive HD patients (aged 7 and 25 years) are screened for calcitonin level and they are without TTE till now. Two family members (mothers of TCA patients) with detected RET mutation underwent prophylactic TTE with MTC finding. Results showed the benefit of systematic RET mutation screening in HD patients in order to identify the risk of MTC in preclinical stage of the disease in patients with HD and their family members. We recommend to investigate not only exon 10 but also exon 13.

Genetics of thyroid cancer and effects of thyroid hormone on tumor progress in vivo

The Oncogenic Activity of RET Point Mutants for Follicular Thyroid Cells May Account for the Occurrence of Papillary Thyroid Carcinoma in Patients Affected by Familial Medullary Thyroid Carcinoma

Medullary thyroid cancer (MTC) is a rare thyroid cancer that can occur sporadically or as part of a hereditary syndrome. To explore the genetic origin of MTC, we sequenced the protein coding exons of approximately 21,000 genes in 17 sporadic MTCs. We sequenced the exomes of 17 sporadic MTCs and validated the frequency of all recurrently mutated genes and other genes of interest in an independent cohort of 40 MTCs comprised of both sporadic and hereditary MTC. We discovered 305 high-confidence mutations in the 17 sporadic MTCs in the discovery phase, or approximately 17.9 somatic mutations per tumor. Mutations in RET, HRAS, and KRAS genes were identified as the principal driver mutations in MTC. All of the other additional somatic mutations, including mutations in spliceosome and DNA repair pathways, were not recurrent in additional tumors. Tumors without RET, HRAS, or KRAS mutations appeared to have significantly fewer mutations overall in protein coding exons. Approximately 90% of MTCs had mutually exclusive mutations in RET, HRAS, and KRAS, suggesting that RET and RAS are the predominant driver pathways in MTC. Relatively few mutations overall and no commonly recurrent driver mutations other than RET, HRAS, and KRAS were seen in the MTC exome.