| Japanese Journal of Clinical Oncology | Pages |
Genotype-Phenotype Correlation of Patients with Multiple Endocrine Neoplasia Type 2 in Japan
Introduction
Materials And Methods
Patients
DNA Testing
Genotype-Phenotype Correlation
Results
Genotype-Phenotype Correlation
Discussion
Clinical Implications
Acknowledgments
Affiliation of the Authors
References
Genotype-Phenotype Correlation of Patients with Multiple Endocrine Neoplasia Type 2 in Japan
Background: Multiple endocrine neoplasia type 2 (MEN 2) is a hereditary syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and hyperparathyroidism. MEN 2 is caused predominantly by germ-line mutations of the RET proto-oncogene. This study aimed to clarify the genotype-phenotype correlation in MEN 2 patients in Japan in order to modify the clinical management according to the genotype.
Methods: Constitutive DNA of 64 MEN 2 patients (48 kindreds) were searched for mutations at exons 10, 11, 13, 14 and 16 of the RET proto-oncogene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), direct sequencing and restriction enzyme digestion. The clinical characteristics of the patients were obtained from a previous nationwide questionnaire survey.
Results: Overall, 62 (96.9%) out of 64 patients had a germ-line point mutation at the hot spots. MTC and pheochromocytoma occurred equally in every genotype except C630S. Specific genotype had a correlation between tumor size and age at the operation for MTC or extent of MTC, i.e. C618S developed late onset type of MTC as compared with that of C634R, C634Y and M918T. Small MTC in C634R may be less aggressive than those in C634Y and M918T.
Conclusions: DNA testing has good clinical implications for the management of patients with MEN 2 and the timing and operative procedures of thyroidectomy can be modified according to the genotype.
INTRODUCTION
Multiple endocrine neoplasia type 2 (MEN 2) is a familial disorder inherited in an autosomal dominant trait. Patients with MEN 2A develop medullary thyroid carcinoma (MTC), pheochromocytoma and hyperplasia or adenoma of the parathyroid gland with hyperparathyroidism (1). Patients with MEN 2B develop MTC and pheochromocytoma but rarely parathyroid disease and are associated with Marfanoid habitus, intestinal ganglioneuromatosis and neurofibromatosis. Familial MTC (FMTC) syndrome is defined as patients whose families have at least four members with MTC alone, without a family history of either pheochromocytoma or parathyroid disease. Any patients with either MTC alone where screening for other organ involvement was not performed or FMTC families with fewer than four cases of MTC were classified as `other' to minimize confusion of disease phenotypes due to small family size or incomplete screening information (2). In addition to familial diseases, there are sporadic cases of MTC, pheochromocytoma and hyperparathyroidism.
Recently, germ-line point mutations affecting different domains of the RET proto-oncogene on chromosome 10q11.2 were found to be associated with MEN 2A (3,4), 2B (5,6), FMTC (4) and `other' (2). Accordingly, several investigators have shown that prophylactic total thyroidectomy at an early age is a useful intervention for management of MTC in MEN 2 gene carriers who are identified by RET mutational analysis (7,8). It is still controversial, however, when and on whom prophylactic thyroidectomy should be performed. There has been no report which compares the detailed clinical aggressiveness of MTC among genotypes. At first, the clinical characteristics, natural history and clinical outcome of patients in Japan were investigated by a nationwide questionnaire survey (9). This survey revealed that the prognosis of the disease depends predominantly upon the extent of MTC and that an early stage diagnosis and surgery are important. Since the risk of complications of thyroidectomy in young children should be counterbalanced by the gain from the prevention of MTC, it should be clarified which genotypes develop an aggressive MTC at an early age. This is the first study analyzing the correlation between genotype and clinical aggressiveness of MTC in order to adopt prophylactic operation for MEN 2 patients in Japan.
MATERIALS AND METHODS
Patients
From January 1995 to April 1997, physicians working in the fields of endocrinology, surgery and urology were asked to provide 5-10 ml of whole blood from patients with MEN 2 with informed consent about the benefits and risks of DNA testing. For each patient, the following data were obtained: family history, clinical features, biochemical data, location, size and other clinical characteristics of MTC, pheochromocytoma and parathyroid lesions, operative procedures and treatment outcomes (9). The patients with MEN 2 included in this study were subclassified into four categories by clinical features: MEN 2A, MEN 2B, FMTC and `other' as described above.
DNA Testing
Constitutive DNA was extracted from the blood of the patients using a QIamp blood kit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions. The mutational `hot spots' expanding exons 10, 11, 13, 14 and 16 were amplified with 32P-labeled primers (10,11) using an automated thermal cycler (Robocycler, Stratagene, La Jolla, CA). The polymerase chain reaction (PCR) was performed by 30 cycles of denaturation at 96°C for 1 min, annealing at 68°C for exons 10 and 11, 66°C for exon 13, 70°C for exon 14 and 56°C for exon 16 for 1.5 min and extension at 72°C for 1.5 min with an additional 5 min at 72°C. The PCR product was then subjected to single strand conformation polymorphism (SSCP) analysis (12). Electrophoresis was performed in at least two temperature conditions using an electrophoretic apparatus with a circulating water-bath (Genoquencer SSCP, ATTO, Tokyo). Gels were dried and exposed to X-ray film. PCR products showing single strand conformational variants (SSCV) were subjected to direct sequencing using a Sequenase II PCR product direct sequencing kit (United States Biochemical, Cleveland, OH) according to the manufacturer's instructions. If no mutation was detected by SSCP within these five exons, the PCR products were subjected to direct sequencing to avoid false-negative results of SSCP analysis. In these patients, SSCP analysis of the remaining 16 exons was performed using primers and conditions described previously (11). When the mutations created or destroyed restriction sites, the PCR products were digested by appropriate restriction enzymes to confirm the mutation. The digests were divided on 8% polyacrylamide gel and autoradiographed.
Genotype-Phenotype Correlation
The genotype was defined and described as the amino acid substitution created by the point mutation (13); for example, either AGC or TCC mutation at codon 618 which substitutes serine for cysteine (TGC) was described as the same genotype as C618S. The penetration of MTC, pheochromocytoma and hyperparathyroidism was defined as the fraction of the presence of the disease at the time of the questionnaire. All the statistical analyses were carried out with the Fisher (two-tailed) exact test and Student's t-test using StatView 4.02 software (Abacus Concepts, Berkeley, CA). Only patients with objective data (pathologically confirmed tumor diagnoses) were included in statistical analysis. In order to investigate the relationship between genotype and the aggressiveness of the MTC, the tumor size was plotted against the age at the operation for MTC in each genotype which had more than three informative cases. The correlation between the size and the age at operation was calculated using StatView software. The clinical aggressiveness of the MTC was evaluated as the presence of lymph node metastasis or persistent tumor after radical operation confirmed by a positive calcitonin test. The phenotype of the pheochromocytoma was represented by laterality, pathological findings and elevated serum catecholamines.
Table 1.
| Exon 10 codon 618 |
Exon 10 codon 630 |
Exon 11 codon 634 |
Exon 16 codon 918 |
No mutation | Total | |
| MEN 2A | 6 | 33 | 2 | 41 | ||
| MEN 2B | 1 | 2 | 3 | |||
| FMTC | 1 | 1 | ||||
| `Other' | 1 | 2 | 3 |
RESULTS
Table 2.
Genotype-Phenotype Correlation
In order to assess the relationship between RET mutations and disease features, the association of mutation and the presence or absence of MTC, pheochromocytoma and hyperparathyroidism were examined (Table 2). Although there was no statistical difference in the frequency of MTC, pheochromocytoma and hyperparathyroidism among the genotypes, the age at the operation for MTC was significantly different among the genotypes. Patients with M918T and C634R were operated at a mean age of 11.7 years old (y.o.) and 26.7 y.o. respectively, which is significantly younger than the age at operation for C618S, C618R, C634Y and C634F patients. The mean ages at the operation for pheochromocytoma were not statistically different among the genotypes. There was also not a genotype in which the mean age at the operation for MTC was statistically different from that for pheochromocytoma. Two females who had C634Y and C634S genotypes, respectively, developed only pheochromocytoma. One female with no RET mutation developed pheochromocytoma and hyperparathyroidism but not MTC. Hyperparathyroidism was present mainly in the patients with point mutation at codon 634, but the frequency was not statistically different owing to the small number of cases. The genotype C630S was found in one `other' kindred (two patients) who developed only MTC.
In order to distinguish the tumor aggressiveness associated with the genotype, the size and extent of the MTC were plotted against the age at operation. The correlation coefficient was calculated if the genotype had more than three informative cases. C618S genotype had a good correlation between size and age (R2 = 0.982). One C618S case had lymph node metastasis and residual tumor after radical operation, but three other cases were cured by the operation (Fig. 1A). C634R genotype is the most frequent type and the mean age at operation was 26.7 y.o. (the youngest case was 8 y.o.). In this genotype, there was no correlation (R2 = 0.02) between the size and the age at the operation (Fig. 1B), but the tumor seems to stay within the thyroid gland while the tumor is small; i.e. six out of seven cases smaller than 2 cm were cured by radical operation, while eight out of nine cases larger than 2 cm had metastatic disease (p < 0.01, Table 3). In contrast, MTCs of the patients with C634Y were progressive (Fig. 1C). The mean age at operation in C634Y cases was 39.9 y.o. (the youngest case was 19 y.o.) with no correlation with the size (R2 = 0.064). Ten out of 12 cases (83.3%) had metastatic disease and four out of five patients had metastatic disease even when the primary tumor was smaller than 2 cm in diameter (Table 3). The classical MEN 2B genotype, M918T, developed large and aggressive MTC at an early age with a strong correlation (R2 = 0.951, Fig. 1D). The mean age at operation for MTC in MEN 2B patients was 11.7 y.o. (the youngest case was 8 y.o.). An 11 y.o. girl and a 16 y.o. boy received radical operation for 2.0 and 4.0 cm MTCs, respectively, but the following provocative calcitonin test revealed that they had residual disease.
A
 C  |
B
 D  |
Figure 1. Relationship between the tumor size or aggressiveness of MTC and age at operation in each genotype. Open circle, MTC confined within the thyroid gland; closed circle, MTC with lymph node metastases at the time of operation or persistent disease after radical operation confirmed by provocative test. (A) C618S; (B) C634R; (C) C634Y; (D) M918T.
The phenotype of pheochromocytoma varied according to the genotype (Table 4). No pheochromocytoma developed in the C630S genotype. The laterality of involved adrenal gland was not different among the genotypes. Eleven out of 12 C634R and eight out of 12 C634Y genotypes developed multicentric lesions. No malignant pheochromocytoma was found. Ectopic pheochromocytoma developed only in a 53 y.o. male patient in whom no RET mutation was detected. The serum noradrenalin level was elevated only in two patients with no mutation, while adrenalin or both adrenalin and noradrenalin levels were elevated in the patients with C618 or C634 mutations. One patient with M918T genotype developed pheochromocytoma with a normal serum catecholamine level.
DISCUSSION
The detailed phenotypes of 64 patients (48 kindreds) with MEN 2 in Japan were compared for the first time according to the mutational analysis of the RET proto-oncogene. Overall, more than 95% of the patients possessed a mutation within the reported `hot spots' of the RET proto-oncogene encoding extracellular cysteine-rich domain and tyrosine kinase domain. No point mutation was detected within the entire coding region of the RET proto-oncogene in two MEN 2A patients whose clinical manifestations were compatible with the criteria of MEN 2A, i.e. one male developed both MTC and ectopic pheochromocytoma and one female developed pheochromocytoma and hyperparathyroidism with her brother developing MTC. Neither of them had ganglioneuromatosis. The International RET Mutation Consortium (2) collected 477 MEN 2 kindreds and reported four out of 203 MEN 2A kindreds without RET mutation. These data, including this study, suggest some causes of the negative results of the mutational search. One may come from the limited ability of the methods to detect mutations in the `hot spots' and the other part of the RET proto-oncogene or the other may result from the possibility that RET proto-oncogene is not the only gene responsible for MEN 2A. In fact, pheochromocytoma is present in [sim]15% of patients with von Hippel-Lindau disease (15,16) with few family members affected with MTC (17). Further linkage analysis will be required to find the genetic disorder of these patients.
Table 3.
| Genotype | MTC < 2 cm | MTC >= 2 cm | p value* | ||
| Metastatic disease | Localized disease | Metastatic disease | Localized disease | ||
| C618S | 0 | 1 | 1 | 2 | N.S. |
| C634R | 1 | 6 | 8 | 1 | <0.01 |
| C634Y | 4 | 1 | 6 | 1 | N.S. |
| M918T | 1 | 1 | 1 | 0 | N.S. |
Table 4.
| Genotype | Uni-:bilateral | Mono-:multicentric lesion | Serum catecholamine | |||
| Normal | Elevated ADR | Elevated NOR | Elevated ADR and NOR | |||
| C618S | 1:1 | N.I. | 1 | 1 | ||
| C618F | N.I. | N.I. | ||||
| C618R | 1:1 | 1:N.I. | 1 | |||
| C630S | ||||||
| C634R | 4:10 | 1:11 | 5 | 9 | ||
| C634G | 1:0 | 0:1 | ||||
| C634Y | 6:8 | 4:8 | 1 | 4 | 9 | |
| C634S | 1:1 | N.I.:1 | 1 | 1 | ||
| C634F | 1:2 | N.I.:1 | 1 | 1 | ||
| C634W | N.I. | N.I. | ||||
| M918T | 1:0 | 1:0 | 1 | |||
| No mutation | 1:1 (bilateral ectopic) | 1:1 | 2 | |||
All genotypes developed MTC and the overall penetration of MTC was more than 95%. Pheochromocytoma developed equally in any genotype but not in C630S, which was found only in the `other' subtype. The frequency of pheochromocytoma was not statistically different among various amino acid substitutions at codon 618 or 634 (data not shown). Moers et al. (13) investigated in a large family with a long-term and extensive screening protocol, finding that C618 genotype develops pheochromocytoma, but less frequently than C634 genotype. They suggested that MEN 2A families should not be subclassified into MEN 2A and FMTC, but rather according to their specific mutation in the RET proto-oncogene, like `MEN 2A RET C618S'. No malignant pheochromocytoma was found in the present study. The laterality of the pheochromocytoma was not statistically different among the genotypes. No RET point mutation was found in the two patients who developed ectopic pheochromocytoma and/or pheochromocytoma with an elevated noradrenalin but not adrenalin level, suggesting that another genetic disorder might be involved in tumorigenesis and catecholamine metabolism in these patients. Hyperparathyroidism developed in seven (12.3%) out of 49 patients with codon 634 mutations, but this frequency was not statistically different from that in patients without codon 634 mutations. Since limited screening in small families might bias the genotype-phenotype correlation and will only reveal symptomatic patients with manifest disease (preselection) and give an incomplete impression of the phenotype, the analysis for correlation between specific genotype and the penetration of hyperparathyroidism requires the collection of more specimens.
Different oncogenic transformations can be caused by different mutations in the RET proto-oncogene (18). Mulligan and co-workers (19,20) suggested that mutations of cysteine codon 618 or 620 lead to a milder course of disease compared with mutations of codon 634. Moers et al. (13) described that the clinical course of the disease in a family with C618 RET mutation (n = 70) is mild compared with that for members with C634 RET mutation (n = 115). In the present study, C618S genotype seems to develop slow-growing MTC compared with those in other genotypes with rapid growing MTC such as C634R, C634Y and M918T (Fig. 1). On the other hand, two cases with C618R genotype developed aggressive MTC (Table 2), i.e. a 39 y.o. female had lymph node metastases although the primary tumor was 1.2 cm in diameter and a 44 y.o. female showed a positive provocative calcitonin test after radical resection of MTC, 3 cm in diameter, with no lymph node metastasis. Since there are only two informative cases of C618R, the clinical aggressiveness of MTC with C618R mutation cannot be defined without more specimens of this genotype. The MTC in the patients with C634R genotype hardly disseminated until the tumor grew larger than 2 cm in diameter, while the patients with C634Y had metastatic disease in spite of small primary tumors (Fig. 1B,C and Table 3). The classical MEN 2B genotype, M918T, developed the most aggressive and rapidly growing MTC (Fig. 1D).
Clinical Implications
Since conventional biochemical screening of MTC, pheochromocytoma and hyperparathyroidism may lead to false-negative or false-positive results (21-23), the DNA testing should be performed first to identify MEN 2 gene carriers. However, it is still controversial when and on whom prophylactic thyroidectomy at an early age should be performed. Gill et al. (24) reported cases of a 5 y 11 m girl and her 3 y 8 m sister with C634R genotype who developed metastatic MTC, suggesting the need for prophylactic thyroidectomies in MEN 2A patients as young as 5 y.o. and strict yearly provocative screening beginning at 1 y.o. Skinner et al. (25) recommended prophylactic thyroidectomy with central neck lymph node dissection at 5 y.o. for patients with MEN 2A, while they offer thyroidectomy in the first year of life to MEN 2B gene carriers owing to the virulence and early onset of MTC. In contrast, Lips et al. (7) emphasized that the risk of complications of surgery (i.e. recurrent nerve paralysis and hypoparathyroidism) in young children is not counterbalanced by the gain that accrues from the prevention of MTC, suggesting that the surgery might be withheld until positive results of the stimulation test or the age of 12-13 years, provided that periodic examinations are conducted. Yamashita et al. (26) state that a good result would be expected if the treatment is carried out at an early stage, suggesting that thyroidectomy should be performed when an elevation of calcitonin has been confirmed after positive DNA testing.
The present study revealed that C618S mutation leads to slow-growing MTC compared with C634 and M918 mutation and that smaller MTC (1.2 cm or less in diameter) of C634R genotype still remains a local disease, while MTCs in C634Y can easily spread in spite of a young age and small tumor. C634W and M918T developed the most rapidly growing MTC at an early age. These data suggest that prophylactic thyroidectomy for the C634Y or M918T genotype carriers should be performed at an early age, whereas it can be withheld in the C618S or C634R carriers until the conventional biochemical or imaging examinations become positive. As for MEN 2A, we demonstrated that the group of MEN 2A patients with C634Y mutation who need early thyroidectomy can be distinguished from those with C618S or C634R with regard to aggressiveness of the disease. Radical lymph node dissection may be abbreviated for small MTC in C618 or C634R (less than 1 cm in diameter).
Concerning the penetration of pheochromocytoma, further screening should be conducted for the FMTC and `other' kindreds whose genotypes are the same as that of MEN 2A. Prophylactic adrenectomy is controversial because of its risk and poor probability of penetration, laterality and multicentricity. In order to obtain more correct genotype-phenotype correlations and better clinical management, continued clinical evaluation of patients and families in Japan is necessary.
Acknowledgments
The authors are indebted to the patients and families who participated in this study for their cooperation, to Dr Kiyomi Yamazaki of the Department of Endocrine Surgery, Tokyo Women's Medical College, for contributing the nationwide questionnaire survey, to Drs Koichi Nagasaki, Toshihiko Tsukada, Kazuki Sasaki and Kouji Maruyama of the Growth Factor Division, National Cancer Center Research Institute, for fruitful suggestions and to Ms Mon Ebinuma for her excellent technical assistance. This research was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for the Second-term Comprehensive 10-Year Strategy for Cancer Control and by Grants-in-Aid for Cancer Research (8-33 and 10-28) from the Ministry of Health and Welfare.
Affiliation of the Authors
1Growth Factor Division, National Cancer Center Research Institute, Tokyo, 2Department of Endocrine Surgery, Tokyo Women's Medical University, Tokyo, 3Yaizu Municipal Hospital, Yaizu, Shizuoka, 4Department of Internal Medicine, Gifu Red Cross Hospital, Gifu, 5Kuma Hospital, Kobe, 6Second Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Hokkaido, 7Department of Medicine III, Osaka University Medical School, Suita, Osaka, 8Third Department of Internal Medicine, Fukui Medical School, Yoshida-gun, Fukui, 9Saitama Cancer Center, Kitaadachi-gun, Saitama, 10Kitakyushu Municipal Medical Center, Kitakyushu, Fukuoka, 11Department of Urology, Oita Medical University, Oita-gun, Oita, 12Second Department of Surgery, Gunma University School of Medicine, Maebashi, 13Department of Urology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, 14First Department of Internal Medicine and 15First Department of Surgery, University of Occupational and Environmental Health, Kitakyushu, Fukuoka, 16Hyogo Prefecture Kaibara Hospital, Hikami-gun, Hyogo, 17Second Department of Surgery, School of Medicine, University of Tokushima, Tokushima, 18Department of Medicine, Shinko Hospital, Kobe, 19Niigata Prefecture Central Hospital, Joetsu, Niigata, 20Department of Surgery, Himeji Red Cross Hospital, Himeji, and 21First Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan
References
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