J. E'"!docrinol. Invest. 21: 87-92,1998
Seventeen-year-Iong follow-up of a family affected by type 2A Multiple Endocrine Neoplasia (MEN 2A) A. Libroia*, U. Verga*, G. Vecchi**, F. Banfi***, F. Zurleni****, L. Quadro*****, C. Scurini*****, O. Fattoruso*****, and V. Colantuoni***** *Divisione di Endocrinologia, **Divisione di Radiologia, ***Servizio di Medicina Nucleare, ****Divisione di Chirurgia Pizzamiglio II, Ospedale Niguarda, Milano, and *****Dipartimento di Biochimica e Biotecnologie Mediche, University of Napoli, Italy
ABSTRACT. This paper reports the results of a 17-year-long follow-up covering 17 members of a family affected by multiple endocrine neoplasia (MEN) type 2 A, first diagnosed in 1980. This family is enrolled in our screening program. The thyroid, parathyroid and adrenal glands of the family members were investigated using the most sophisticated and sensitive techniques which have become available during this period, and their DNA was genetically tested for detecting RET mutations. Thanks to the combination of these two approaches it was possible to confirm the di-
agnosis in the members concerned from the genetic point of view, and to achieve an early diagnosis in the young members of the last generation before the clinical onset of the disease. The detection of a RET mutation also prompted a prophylactic thyroidectomy in a four year-old boy, in a pre-tumoral stage of the disease. Lastly, evidence is provided that genetic analysis of the DNA of the chorionic villi can be carried out as a prenatal test during routine amniocentesis. (J. Endocrinol. Invest. 21: 87-92, 1998) ©1998, Editrice Kurtis
INTRODUCTION
In 1995 all the members of the family were screened in order to detect mutation of the RET protooncogene. This gene codes for a tyrosine-kinase receptor and is expressed in the tissues deriving from the neural crest (2-3). RET exons 10 and 11 code for 5 Cysteine residues located in the extracellular domain of the protein. The punctiform mutation of one of these cysteines is almost always responsible for the MEN 2 and familial medullary thyroid carcinoma (FMTC) pathology (4-5). Thanks to the genetic analysis, it is possible to identify carriers of the mutation, who are therefore considered at risk, so as to plan thyroidectomy as a prophylactic measure. According to current trends, this surgery is performed at the age of about 5. The genetic mutation was found to be positive in all the members already believed to be affected and was absent in the unaffected members. We recently made a diagnosis of a genetic mutation on DNA extracted from chorionic villi during the 10th week of pregnancy.
In 1984 we described a MEN 2 A family, comprising the proband and 13 family members. Based on the medical histories of the family, we identified a sister who died at the age of 33 of bilateral pheochromocytoma diagnosed post-mortem (1). In 1980, the proband (1-4, Fig. 1) underwent bilateral adrenalectomy due to pheochromocytoma, total thyroidectomy due to medullary carcinoma of the thyroid and removal of three parathyroid glands due to hyperparathyroidism. The histological examination revealed the presence of bilateral pheochromocytoma, multifocal medullary carcinoma of the thyroid gland associated with hyperplasia of the parafollicular C-cells and hyperplasia of the chief cells of the parathyroid glands. All the members of the family were enrolled in the screening program available at the time which called for biochemical and instrumental monitoring of the thyroid, parathyroid and adrenal pathology.
METHODS
Key-words: Multiple endocrine neoplasia type 2A, MEN 2A, genetic analysis, RET proto-oncogene, chorionic villi DNA.
Over the 17 years of the follow-up, the techniques used for biochemical and instrumental investigation have evolved greatly in terms of sensitivity and
Correspondence: Uberta Verga, M.D., Via del Carmine 5, 20121 Milano, Italy. Accepted November 5,1997.
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MEN 2 A family follow-up
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1997
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Fig. 1 - Pedigree of the men 2A family.
enabling us to iconograph sites which are not yet identifiable by means of ultrasonic and CAT scans (7).
specificity for diagnosing the thyroid, parathyroid and adrenal functions.
Thyroid
Parathyroid glands
The evaluation of basal calcitonin (CT) and after stimulation with pentagastrin is still the most reliable biochemical marker for diagnosing MTC. Until 1992 we used a RIA method (RIA-mat Calcitonin II, Byk-Santec Diagnostica, normal values below 150 pg/ml). The use of an IRMA method (Medgenix DiagnosticsCT IRMA, normal values <15 pg/ml, peak lower than 80-100 pg/ml) has considerably increased the sensitivity of the method for early diagnosis (6). In the follow-up, we used a CEA assay (Ammerwell CEA assay, monoclonal Amersham, normal values <5 ng/ml) and a CT assay, which must be in the vicinity of zero and impossible to stimulate with pentagastrin in order to be sure that the disease will not set in again. In those patients in whom there was a risk of the disease recurring, we routinely used high-resolution ultrasonic scans of the neck and CAT scans. Use of scintigraphic methods with 99Tc (V)DMSA, anti CEA and anti Chromogranin A with the three-step avidin-biotin system, labelled 111-lndium was not found to be reliable (8), as already described by us. The more recent introduction of scintigraphy with 111-lndium pentetreotide has improved the possibility of locating metastases,
Intact PTH assays (Intact PTH parathyroid hormone RIA assay, Nichols Inst. Diagnostic; n.v. 10-55 pg/ml), calcaemia and phosphoraemia retain a good degree of reliability for the diagnosis of hyperparathyroidism. High-resolution ultrasonic scans and scintigrams with Sestamibi 123 1 (MIBI) make it possible to locate the pathology in a high percentage of cases (9).
Adrenal glands For the early diagnosis of pheochromocytoma, assaying vanillilmandelic acid in the urine has now been abandoned in favour of HPLC assays of the catecholamines and metanephrines in the urine. Ultrasonic scans, CAT scans and scintigrams with meta-iodo-benzyl-guanidine (MIBG 123 1) give rise to a sensitivity of over 90% in the detection of pheochromocytoma, which is often present but without symptoms in the initial phase (10).
Genetic analysis DNA extraction was performed using the standard procedures (11). The search for RET proto-oncogene mutations was carried out by amplifying exons
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A. Libroia, U. Verga, G. Vecch i, et al.
10, 11, 13, 14, and 16 by PCR, as already described (12). PCR reactions were performed on a final volume of 50-fJl containing 100 or 200 ng of genomic DNA, 50 mmol/l Tris-HCL (pH 8.3), 1.5 mmol/l MgC12,5 mmol/l NH4CI, 0.5 U Taq Polymerase (Polymed, Italy), 200 fJmol/l of each dNTP, and 2 fJmol/l of each primer. Five denaturation, annealing and extension cycles were carried out at 95 C for 1 min, 62 C for 1.5 min and 72 C for 2 min respectively, followed by 25 cycles of denaturation, annealing and extension at 95 C for 1 min, 62 C for 1.5 min and 72 C for 1.5 min respectively, using an automatic thermocycler (Perkin Elmer-Cetus Co, Italy). An aliquot of the PCR products was sequenced on both strands by the Sanger method, using the Sequenase PCR Products Sequencing Kit (U.S. Biochemical Corporation, Cleveland OH, USA) and the same oligonucleotides as sequencing-primers. In some cases, an aliquot of the PCR product of exon 11 was digested with Hhal restriction enzyme. Chorionic villi were sampled in week 10 of pregnancy and the DNA was prepared as described (13-14).
examination revealed a stage 3 MTC. This patient's CT levels remained high even after surgery (2000 pg/ml) and two years later, in 1990, the patient died at the age of 29 following MTC metastases. Patient 11-6 (aged 17), who initially refused any investigation in 1980, agreed to surgery for MTC and HPP in 1983 and, recently in 1993, unilateral adrenalectomy. His current CT values show that he is not diseasefree: basal CT=65, peak=445pg/ml. Patients 11-7 and 11-8, underwent MTC surgery at the ages of 17 and 14 respectively, on the basis of high pentagastrin-stimulated CT values only, while their basal levels were within the normal range (11-7=23 pg/ml, peak=205 pg/ml; 11-8=60 pg/ml, peak= 732 pg/ml-RIA assay). The histological examinations revealed the presence of C-cell hyperplasia and a microcarcinoma originating from the same cells. The current CT values show that these patients are not disease-free five years after surgery; 11-7: basal CT=3.3, peak=161 pg/ml; 11-8: basal CT= 3.40, peak=43.80 pg/ml. Patient 11-2, aged 17 in 1980, initially underwent surgery for a total thyroidectomy. His CT values rose progressively during the follow-up period until a second and subsequently a third operation were required (1993-1995) due to the involvement of the latera I-cervical lymph-nodes. The surgical approach was determined on the basis of a fine-needle biopsy in the area concerned, carried out during the course of an ultrasonic scan. This test followed a positive pentreteotide scintigraphy and computerised axial tomography. The histological examination confirmed that 4 out of 5 nodes were MTC metastases. The CT values remained high even after the third operation (500 pg/ml), and the current value is C~1200 pg/ml.
RESULTS Affected members in 1983 Patient 1-4 was the MEN 2A proband. Patient 1-2 died at the age of 33 of bilateral pheochromocytoma (PH), diagnosed at the time of the post-mortem examination (Fig. 1). Ofthe 13 patients screened, 11-1 was considered to be unaffected since the basal and pentagastrin-stimulated calcitonin (CT) levels were within the normal range. Three patients (1-3, 11-4, 11-5) with basal CT levels in the normal range refused stimulation tests; patient 111-1 had a normal basal CT value while the stimulation test could not be performed because she was only 1 year old. High basal CT levels were found in 6 members, four of whom were also positive for the stimulation test. These four patients (1-1, 1-5, 1-6, 11-2) underwent total thyroidectomy for medullary thyroid carcinoma (MTC) and hyperparathyroidism (HPP). The other two patients (11-3, 11-6) refused any further investigation.
Affected members in 1997 Patients 1-6 and 11-2, aged 32 and 17 respectively, who had already undergone surgery for MTC, were subsequently operated on for bilateral pheochromocytoma (PH) in 1983. Their current CT values, 17 years after surgery for MTC, may be considered good although slightly responsive to stimulation: basal CT =1.2, peak 65 pg/ml. Patient 11-3, who refused any investigation, developed an MTC, HPP and PH that were surgically removed in 1988, at the age of 27. The histological
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Fig . 2 - Detection of RET proto-oncogene mutations on germline
DNA. Germline DNA was peR amplified with RET specific oligonucleotides as primers and directly sequenced. On the left, the mutation in codon 634 of RET exon 11 in the DNA from a proband, along with that from a normal individual, is illustrated. The HhA/ restriction enzyme digestion of the same amp/icon is shown on the right. The mutation produces a new restriction site that generates two bands of lower molecular weight, in addition to the undigested band corresponding to the normal allele.
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MEN 2 A family follow-up
Cutaneous Iychen amyloidosis was observed in two out of ten patients, with the typical skin lesion on the upper back. The area had been noticeable since childhood, but increased in size and itchiness with age, even after total thyroidectomy (15) .
Patient 111-3 had pentagastrin-stimulated CT values approaching the upper limit (basal CT = 12 pg/ml, peak=95 pg/ml), with a positive genetic test. The DNA revealed a heterozigous mutation in codon 634 of exon 11 of the RET proto-oncogene replacing a TGC (Cys) with a CGC (Arg). An example of this analysis is shown in Figure 2. The base pair change generates a recognition site for the restriction enzyme Hhal. Thus, digestion of the amplified product with this enzyme confirmed the presence of the mutation. On the basis of this result, at the age of 4, the patient underwent total thyroidectomy. The histological examination of the surgical specimen showed a bilateral C-cells hyperplasia and a single nodule of about 0.5 cm in diameter, formed by a cluster of overgrown parafollicular C-cells. Four years after surgery he is in good health, with no signs of disease (CT = 0.8 pg/ml, peak= 12 pg/ml). The genetic analysis of the all members of the family, performed with their consent, confirmed the presence of the germline mutation in codon 634 only in the affected members. Detection of the RET mutations was also carried out on specimens taken from tumor tissues, when available. In particular, a hyperplastic node originating from patient 1-5 (basal CT =57, peak =1860 pg/ml); a thyroid with hyperplastic C-cells removed from patient 111-3 and a pheochromocytoma from patient 11-6 all revealed the same mutation found in the peripheral blood DNA. Lastly, for speculative purposes, we took a sample of chorionic villi in week 10 of pregnancy from the wife of patient 11-2, who had decided to undergo an abortion. The examination showed the presence of the same Cys634Arg mutation found in the father's DNA. Possible errors were excluded because mother's DNA, a likely source of cell contamination, did not bear the mutation. In those patients (7 out of 10) who underwent total thyroidectomy, all the detectable parathyroid glands were removed and the normal ones were autografted into the forearm. From a histological point of view, chief cell hyperplasia was observed mainly, in one or more glands. In two patients, replacement therapy was required after surgery due to persistent hypoparathyroidism. In two other patients recurrence of hyperparathyroidism was observed. In order to normalize PTH serum levels a second operation was required involving the new sites, detected by MIBI scintigraphy, one on the autograft and another on a residual gland. The adrenal glands were removed bilaterally in two patients at the time of the thyroidectomy, while in two other patients the operation was carried out two years later. One unilateral adrenalectomy was carried out 8 years later.
DISCUSSION In this paper we are reporting the results of a 17-year follow-up of a MEN 2A family. The significant improvement in the diagnostic techniques over this period of time has greatly refined diagnostic accuracy and sensitivity. An even greater breakthrough has resulted from the availability of a genetic analysis, aimed at identifying punctiform mutations in the RET proto-oncogene. Thanks to these means, a T to C base-pair change in codon 634 of exon 11 was identified in the DNA of the members of this family. The mutation reported here is located in the area of the gene coding for the cysteine-rich region of the protein and has been found in the majority of cases of MEN 2A and FMTC (4-5). In addition, this Cys to Arg substitution has been found in at least 60% of cases of MEN 2A associated with pheochromocytoma and parathyroid hyperplasia (5). In some members of the family, the RET mutation was also associated with cutaneous lichen amyloidosis lesions, described as a form of MEN 2A variant (15). Genetic screening confirmed the diagnosis in those subjects who had already been operated on or who had already been diagnosed as MEN 2A patients. In addition, it enabled early diagnosis in the young member of the last generation, before the clinical onset of the disease (16). Indeed, patient 111-3 underwent surgery following a positive genetic analysis. This decision was validated by the histological examination of the thyroid specimen, which revealed the presence of a widespread hyperplasia of the C-cells and a cluster of hyperplastic C-cells but no evidence of neoplastic degeneration. This is considered a preneoplastic condition usually preceding the appearance of transformed cells. The need for early surgery was also justified by the aggressive clinical progress of the disease in the previous generations. 11-3 died as a consequence of delaying surgery, while 11-2, as shown in this study, was obliged to undergo repeated surgery due to persistently high and increasing CT values. This further demonstrates that early and timely removal of the thyroid gland is an important therapeutic decision capable of affecting the subsequent behaviour of the disease and the quality of these patients' lives. This paper stresses the importance of the new sensitive techniques for a correct and early diagnosis, in conjunction with genetic screening . We also provide evidence that a genetic analysis can be
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2. Takahashi M., Buma Y., Taniguchi M. Identification of the RET protooncogene product in neuroblastoma and leukemia cells. Oncogene 6: 207, 1991.
carried out on the DNA extracted from the chorionic villi. This type of diagnosis was carried out for speculative purposes, with the informed consent of both parents, in order to prove that pre-natal diagnosis is possible. Generally speaking, gene-mutation carriers undergo total thyroidectomy at the age of 5 or older, after screening of the parathyroid and adrenal glands. There is general agreement that prophylactic removal of the thyroid gland should be offered for these patients and would reduce both mortality and morbidity (19). Carriers younger than 5 require a more conservative approach. Surgery on the four-year old child was decided on after 2 years of screening using the pentagastrin test, when the response began to give rise to suspicion. The discovery, at the same time, of the genetic mutation induced us to opt for early surgery (20). Our attitude in this case was supported also by the consideration of the aggressive nature of the pathology in this family, which appeared to be increasing in successive generations. As mentioned above, the Cys to Arg mutation is more frequently associated with pheochromocytoma and parathyroid hyperplasia (5). It is, therefore, worth noting that hyperparathyroidism has not occurred in the last 3 patients subjected to early surgery. The data in literature stress the possibility that early surgery may avoid the risk of developing hyperparathyroidism (17). The treatment of pheochromocytoma is essentially surgical; it is questionable whether it should be unilateral or biiateral.The preponderance of evidence supports unilateral adrenalectomy for MEN 2A patients, if normotensive. A second operation for a contralateral pheochromocytoma may be required many years later, but in the meantime the patient will be steroid independent. The introduction of laparoscopic surgery has made the possibility of two operations simpler, in view of the limited invasiveness (18). Non-carriers do not require further investigations and can be reassured that their risk of developing MTC is equal to that of the normal population .
3. Tsuzuki T., Takahashi M., Asai N., Iwashita T., Matsuyama M., Asai J. Spatial and temporal expression of the RET protooncogene product in embrionic infant and adult rat tissues. Oncogene 10: 191, 1995. 4. Donis-Keller H, Dou S., Chi D., Carlson K.M., Toshima K. ,Lairmore T.C., Howe J.R., Moley J.F., Goodfellow P., Wells SA Mutations in the RET protooncogene are associated with MEN 2A and FMTC. Hum. Mol. Genet. 2: 851, 1993. 5. Intemational RET Mutation Consortium Analysis. Eng C. et al. The relationship between specific RET protooncogene mutations and disease phenotype in Multiple Endocrine Neoplasia type 2. JAMA 276: 1575,1996. 6. Kaplan M.M., Stall G.M., Cummings T., Mac Aulay A, Motte P., Wolfe H.J., Reichlin S., Tashjian JR A High sensitivity serum calcitonin assay applied to screening for thyroid C-cell disease in Multiple Endocrine Neoplasia type 2A Henry Ford Hosp. Med. J. 40: 3, 1992. 7. Kwekkeboom D.J ., Reubi J.e., Lamberts S.W.J, Bruining HA, Mulder AH., Oei H.Y., Krenning E.P. In vivo somatostatin receptor imaging in medullary thyroid carcinoma. J.Clin Endocrinol. Metab. 76: 1413, 1993. 8. Verga U., Muratori F., Di Sacco G., Banfi F., Libroia A The role of radiopharmaceutical MIBG and M DMSA in the diagnosis of Medullary Thyroid Carcinoma. Henry Ford Hosp. Med. J. 37: 175, 1989. 9. Hindie F., Melliere D., Simon D., Perlemuter L., Galle P. Primary Hyperparathyroidism : Technetium 99m Sestamibi/lodine substraction scanning the best procedure to locate enlarged glands before surgery? J. Clin . Endocrinol. Metab. 80: 1, 1995. 10. Casanova S., Rosenberg-Bourgin M., Calmettes C. Pheochromocytoma in Multiple Endocrine Neoplasia type 2A Survey of 100 cases. Clin. Endocrinol. (Oxf.) 38: 531, 1993 .
ACKNOWLEDG MENTS Genetic counselling was supported by grants provided by CNR PF ACRO, SP$ Diagnostica Biotecnologica, and by Associazione Italiana per la Ricerca sui Cancro (AIRC).
11. Sambrook J., Fritsch E.F., Maniatis T. Molecular cloning : a laboratory manual, ed . 2. Cold Spring Harbor Lab Press, New York, 1989. 12. Quadro L., Panariello L., Salvatore D., Carlomagno F., Del Prete M., Nunziata V., Di Giovanni G., Brandi M.L., Mannelli M., Gheri R., Verga U., Libroia A, Berger N., Fusco A, Grieco M., Santoro M. Frequent RET Protooncogene Mutations in Multiple Endocrine Neoplasia type 2A J. Clin. Endocrinol. Metab. 79: 2, 1994.
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13. Bolino A, Schuffenecker I, Luo Y., Seri M., Silengo M., Tocco T., Chabrier G., Romeo G. RET mutation in exon 13 and 14 of FMTC patients. Oncogene 10: 2415,1995.
W.B. Saunders Company, Philadelphia, 1994, vol. 23,p.157. 17. Wells SA, Chi D.C., Toshima K., Dehner L.P., Coffin C.M., Dowton B., Ivanovich J.L., De Benedetti M.K., Dilley W.G., Moley J.F., Norton JA, Donis-Keller H. et al. Predictive DNA testing and prophylactic thyroidectomy in patients at risk for Multiple Endocrine Neoplasia type 2A Ann.Surg. 220:237, 1994. 18. Boulux P-M. G., Fakeeh M. Investigation of phaechromocytoma. Clin. Endocrinol. (Oxf) 43: 657, 1995.
14. Fattoruso 0., Quadro L., Libroia A, Verga U., Lupoli G., Cascone E., Colantuoni V. A GTG to ATG novel point mutation at codon 804 in exon 14 of the RET protooncogene in two families affected by familial medullary thyroid carcinoma. Human Mutat. (in press). 15. Robinson M.F., Furst E.J., Nunziata V., Brandi M.L., Ferrer M.J., Bugalho M., Di Giovanni G., Smith R.J.H., Donovan D.E., Alford B.R., Colantuoni V., Quadro L., Limbert E., Halperin I., Gagel R.F. Characterization of the clinical features of five families with hereditary primary cutaneous lichen amyloidosis and multiple endocrine neoplasia type 2. Henry Ford Hosp. Med. J. 40: 249, 1992.
19. De Lellis RA Multiple endocrine neoplasia syndromes rivisited. Lab. Invest., 72: 5, 494, 1995. 20. Pacini F., Romei c., Miccoli P., Elisei R., Molinaro E., Mancusi F., lacconi P., Basolo F., Martino E., Pinchera A Early treatment of hereditary medullary thyroid carcinoma after attribution of multiple endocrine neoplasia type 2 gene carrier status by screening for RET gene mutations. Surgery 118: 1031, 1995.
16. Snow K.J., Boyd AE. Management of individual tumor syndromes. Medullary thyroid carcinoma and hyperparthyroidism. In: Messersmith M., Gagel R.F. (Eds.), Endocrinology and Metabolism Clinics of North America.
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