Annals of Surgical Oncology 8:342-346 (2001)
© 2001 Society of Surgical Oncology
Genetic Analysis of a Papillary Thyroid Carcinoma in a Patient with MEN1
Dev Desai, MD, PhD,
Lisa A. McPherson, PhD,
John P. T. Higgins, MD and
Ronald J. Weigel, MD, PhD
From The Department of Surgery (DD, LAMc, RJW) and Pathology (JPTH), Stanford University School of Medicine, Stanford, California.
Correspondence: Address correspondence to: Ronald J. Weigel, MD, PhD, Room P214, MSLS, Stanford University School of Medicine, Stanford, CA 94305-5494; Fax: 650-724-3229; E-mail: ronald.weigel{at}stanford.edu
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ABSTRACT
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Background: MEN1 is an inherited tumor syndrome characterized by the development of tumors of the parathyroid, the anterior pituitary and the pancreatic islets. Tumors of these endocrine glands in MEN1 patients demonstrate loss of heterozygosity (LOH) at the locus of the MEN1 tumor suppressor gene. Menin, the protein encoded by the MEN1 gene, is ubiquitously expressed in endocrine tissue, and less commonly these patients can present with tumors of other endocrine tissues, including thyroid and adrenal. We hypothesize that MEN1 gene mutation may be involved in the oncogenesis of other less common tumors.
Methods: We report a MEN1 patient who was found to have metastatic papillary thyroid cancer at the time of neck exploration for hyperparathyroidism. Genetic analysis of tumor tissue was performed using one intragenic (D11S4946) and two flanking (D11S4945 and D11S4940) polymorphic markers.
Results: Two of the markers were informative. Consistent with previous studies, there was LOH in the parathyroid adenoma identified with the intragenic marker D11S4946. However, the papillary cancer was found to be heterozygous at two informative markers.
Conclusions: The lack of obvious LOH of the MEN1 locus in the papillary cancer suggests that, in contrast to parathyroid adenoma, deletion of the MEN1 tumor suppressor gene is not etiologically related to the oncogenesis of the papillary cancer in this patient.
Key Words: MEN1 • Parathyroid • Papillary thyroid cancer • Gene • LOH
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INTRODUCTION
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MEN1 is a dominantly inherited familial cancer syndrome in which affected individuals develop parathyroid adenomas, pancreatic islet cell tumors, and adenomas of the anterior pituitary with varying penetrance.1 The MEN1 gene was identified by positional cloning and was found to encode a 610 amino acid protein called menin.2 The MEN1 gene is likely to function as a tumor suppressor gene, and one mechanism of action is repression of JunD transcriptional activation.3 The most convincing evidence that MEN1 is a tumor suppressor gene is the finding that tumors occurring in MEN1 patients have loss of heterozygosity (LOH) of the MEN1 locus. In the classic paradigm of a tumor suppressor gene, elimination of both copies of the gene involves inheritance of one mutated MEN1 gene with somatic loss of the wild-type allele in the tumor cells. These genetic changes during oncogenesis result in LOH at the MEN1 gene locus. LOH has been reported in parathyroid adenomas,4,5 pituitary tumors,6 and islet cell tumors7 in MEN1 patients. Genetic studies have also indicated a role for MEN1 gene mutations in the etiology of sporadic parathyroid adenomas.5,8,9 Some studies have also indicated a role for MEN1 gene mutation in sporadic islet cell tumors.10–12 However, at least one report failed to find MEN1 gene mutations in sporadic islet cell tumors,13 and sporadic pituitary tumors have rarely demonstrated evidence for MEN1 involvement.9,14
In addition to the classic triad of endocrine tumors, MEN1 patients can less commonly develop foregut carcinoids, lipomas, esophageal leiomyomas, angiofibromas, thyroid adenomas, adrenocortical adenomas, angiomyolipomas, and spinal cord ependymomas.2,6,15 Genetic analysis of these less common tumors has reported LOH in carcinoids, lipomas, and esophageal leiomyomas, indicating a role for MEN1 mutation in the etiology of these tumors. Interestingly, tumors arising in MEN1 patients tend to lack metastatic potential with the notable exception of gastrinomas and occasionally insulinomas. Examining LOH in other tumors occurring in MEN1 patients can add to the growing body of evidence for the MEN1 gene as a tumor suppressor gene and may also help to define the tissue specificity and other important physiologic criteria involved in tumor oncogenesis.
Because menin is expressed in most endocrine tissue,16 it is reasonable to hypothesize a role for the MEN1 gene in the oncogenesis of other endocrine tumors. Specifically, normal thyroid has been shown to express menin,16 and it is possible that the occurrence of thyroid tumors in MEN1 patients might involve MEN1 gene mutations. In addition, a recent report indicated a potential tumor suppressor gene at 11q13 distinct from the MEN1 gene.17 We now report the occurrence of a metastatic papillary cancer identified in a MEN1 patient undergoing neck exploration for hyperparathyroidism. A genetic analysis of tumor tissue from this patient was performed utilizing polymorphic markers closely linked to the MEN1 gene. Although LOH was confirmed to occur in the parathyroid adenoma, no evidence for LOH was found in the papillary thyroid cancer. This finding suggests that deletion of the MEN1 gene was not etiologically related to the papillary cancer.
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PATIENTS AND METHODS
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Case Report
Patient DD was a 45-year-old man with MEN1 who had been diagnosed with a prolactin secreting pituitary adenoma 10 years earlier that had been managed medically with pergolide and bromocriptine. The patient subsequently developed nephrolithiasis and a work-up for hyperparathyroidism revealed a serum calcium of 10.5 mg/dl and an intact PTH of 160 pg/ml. Serum gastrin evaluation had been normal. The patient relates no family history of MEN 1, thyroid cancer, or hypercalcemia.
The patient underwent a standard neck exploration for hyperparathyroidism. The findings at surgery are diagrammed in Fig. 1. At the time of surgery, the patient was found to have an enlarged right upper parathyroid gland weighing 4.134 g and a slightly enlarged right lower gland weighing 179 mg. Both glands were resected and were hypercellular on frozen section examination. During the right neck dissection, a firm nodule of approximately 1 cm in size adjacent to the trachea was removed and on evaluation by frozen section revealed metastatic papillary thyroid cancer. The left superior and inferior parathyroid glands were identified and were slightly enlarged. These glands were generously biopsied and confirmed on frozen section to be normal parathyroid tissue. Multiple firm extra-thyroid nodules were noted in the left neck These were resected and on frozen section evaluation were confirmed to be metastatic papillary thyroid cancer. A total thyroidectomy and central neck dissection was performed.
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FIG. 1. Diagram of operative findings: At the time of neck exploration, an enlarged right upper parathyroid gland (parathyroid glands diagrammed in solid black) was encountered and was resected. The right lower gland was slightly enlarged and was also removed. The two left parathyroid glands were slightly enlarged and both were generously biopsied. Also noted at the time of neck exploration were multiple fibrotic nodules that were determined to be metastatic papillary cancer (cancer nodules diagrammed in hatched).
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Intraoperative PTH monitoring was performed to guide the extent of parathyroid resection. The baseline PTH before the neck incision was 73 pg/ml. Immediately after resection of the two enlarged right glands the intact PTH was 10 pg/ml, and 10 minutes after parathyroid resection the intact PTH was 4 pg/ml.
The patient had an uneventful recovery. One week postoperatively the patient’s calcium was 8.1 mg/dl with an intact PTH of 7 pg/ml, and 1 month postoperatively his calcium was 9.4 mg/dl. Pathologic evaluation revealed a 1-cm focus of papillary thyroid carcinoma with metastases to multiple lymph nodes. Final pathology confirmed the finding of parathyroid hyperplasia.
DNA Analysis
DNA was isolated from the patient’s blood using the DNeasy Tissue Kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer. Archival specimens were microdissected and DNA was extracted with the same kit using the protocol for paraffin-embedded tissue.
The polymorphic markers D11S4945, D11S4946, and D11S4940 were used in the genetic analysis. Primers used for PCR amplification were previously described for these markers.18 One of each primer pair was labeled using 
-32P-ATP and polynucleotide kinase. For D11S4946 and D11S4940, the primer chosen for labeling has been described18 and for D11S4945 the primer 5'-GCATCGATGGAGGCGCACAGCAC-3' was labeled. PCR reactions were carried out in a 50-µl reaction volume containing 10 pmoles of each primer, 0.25 pmoles of one labeled primer, 200 µM dNTPs, 1 x PCR buffer, and 1µl Advantage cDNA polymerase mix (Clontech, Palo Alto, CA). Reactions were denatured at 95°C for 4 minute, then cycled at 95°C for 1 minute, 60°C for 30 sec, 72°C for 1 minute for 30 cycles, then 72°C for 10 minute, then held at 4°C. A 2-µl reaction aliquot was analyzed on 6% denaturing polyacrylamide gel, dried, and exposed to x-ray film.
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RESULTS
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As seen in Fig. 1, patient DD was found to have metastatic papillary cancer during neck exploration for hyperparathyroidism. Histologic examination confirmed the finding of a small focus of papillary cancer in the thyroid (see Fig. 2). The cancer was metastatic with tumor nodules noted in the neck. As seen in Fig. 2, the papillary cancer completely replaced what is presumed to have been a lymph node. Also seen in Fig. 2 is the histology from the parathyroid adenoma resected from the right upper gland position. As is the case for the papillary cancer nodule, the majority of cells in the parathyroid specimen (>90%) are tumor cells.
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FIG. 2. Histology of parathyroid glands and papillary thyroid cancer: (A) Parathyroid adenoma showing typical histology with sheets of cells with small nuclei and clear cytoplasm arranged in clusters (H&E; original magnification x40). (A inset) Parathyroid adenoma (H&E; original magnification x200). (B) Papillary thyroid carcinoma in the thyroid. Non-neoplastic thyroid in the left-hand side of the panel is infiltrated by papillary carcinoma on the right (H&E; original magnification x40). (B inset) High power detail showing enlarged nuclei with grooves and intranuclear inclusions (H&E; original magnification x200). (C) Parathyroid adenoma: The box indicates the portion of the tissue removed for molecular analysis. This tissue consists almost exclusively of tumor. (H&E; original magnification x40). (D) Papillary carcinoma metastasis to the left neck: The box indicates the portion of the tissue removed for molecular analysis. This tissue consists almost exclusively of tumor. (H&E; original magnification x40).
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The parathyroid adenoma and nodule of metastatic papillary cancer were microdissected in the areas shown in Fig. 2. DNA was isolated from these archival specimens and was analyzed in parallel with DNA obtained from the patient’s blood. Three polymorphic markers were used that are closely linked to the MEN1 gene. D11S4945 and D11S4940 flank the MEN1 gene, and D11S4946 is intragenic. Figure 3 shows results of the genetic analysis with the three markers. D11S4940 was not informative because the patient was homozygous at this locus. The patient was heterozygous for marker D11S4945, but no LOH was found in either the parathyroid adenoma or papillary cancer. Analysis with the intragenic marker D11S4946 was informative and demonstrated LOH in the parathyroid adenoma. However, no LOH was identified in the papillary cancer. Failure to identify LOH in the papillary thyroid cancer with two informative makers is an indication that deletion of the MEN1 gene is not involved in the oncogenesis of this tumor. The results confirm the occurrence of LOH in parathyroid adenomas from MEN1 patients.
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FIG. 3. Genetic analysis of MEN1 Locus. Two polymorphic markers that flank the MEN1 gene, D11S4945 and D11S4940, and one intragenic marker, D11S4946, were used to examine DNA for LOH of the MEN1 locus. DNA templates analyzed were isolated from blood (B), parathyroid adenoma (PA), and papillary cancer (PC). Arrow indicates LOH noted in parathyroid adenoma with intragenic marker D11S4946.
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DISCUSSION
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Most of the familial cancer syndromes involve inheritance of a mutation in a tumor suppressor gene. In the classic Knudson’s two-hit oncogenesis model involving a tumor suppressor gene, one mutated allele of the gene is inherited and the second copy (wild-type) is deleted in the somatic target cells through a process that may also delete large regions of the locus or the entire chromosome. This molecular alteration can be identified as a loss of heterozygosity (LOH) involving the gene locus. The determination of LOH in this study used identical criteria as in similar studies, which required an examination with at least two informative markers closely linked to the MEN1 gene.6 As expected in the analysis of a parathyroid adenoma in an MEN1 patient, we confirmed the occurrence of LOH in the parathyroid adenoma with the intragenic marker D11S4946. The flanking marker D11S4945 remained heterozygous, indicating that one boundary of the deletion in the parathyroid tumor occurred between this flanking marker and the MEN1 gene. However, examination with two informative markers, including an intragenic marker, failed to identify LOH in the papillary cancer from this patient. This finding suggests that elimination of the MEN1 tumor suppressor gene is not necessary for the oncogenesis of the papillary cancer in this patient.
There is one other published study examining LOH in a papillary cancer occurring in an MEN1 patient.15 Similar to our results, this study also demonstrated retained heterozygosity at the MEN1 locus in the papillary cancer. The importance of verifying these results in additional cases is exemplified by studies of esophageal leiomyomas occurring in MEN1 patients. In the study by Dong et al.,6 the authors failed to identify LOH in tissue from an esophageal leiomyoma, whereas Vortmeyer et al.15 found evidence of LOH at the locus in these tumors. Our results strengthen the evidence that loss of the tumor suppressor function of the MEN1 gene is not required for oncogenesis in papillary thyroid cancer.
The mutations described for MEN1 are scattered throughout the gene and include intron mutations that affect splicing.19 Missense mutations have been reported throughout the entire coding region, suggesting that menin may contain multiple functional domains. It is possible that the second allele in the papillary cancer could have been inactivated by a point mutation or other alteration too subtle to detect in this analysis. It is conceivable that a more extensive analysis of the retained MEN1 allele from the papillary cancer by SSCP analysis or complete sequencing might identify an inactivating mutation. Mutations within introns or transcriptional regulatory regions may also silence expression of this retained allele and these alterations may not even be identified by complete sequence analysis of the cDNA from this allele. It is a further possibility that MEN1 patients that develop papillary thyroid cancer may have specific MEN1 mutations of the affected allele that act like dominant oncogenes with regard to thyroid cancer oncogenesis. If any of these scenarios were the case, the MEN1 gene could play a role in the development of the papillary cancer without obvious LOH of the gene locus. Therefore, the conclusions derived from this study need to be considered within the limitations of the assay used in the analysis.
One interesting observation concerning tumors in MEN1 patients that have been shown to involve LOH at the MEN1 locus is the low degree of metastatic potential. The parathyroid adenomas and tumors of the anterior pituitary are benign. In addition, most pancreatic islet cell tumors with the exception of gastrinomas are also benign and even metastatic gastrinomas have a relatively indolent course compared with other malignancies of the pancreas. This implies that the loss of the MEN1 tumor suppressor gene induces a hyperplastic response in endocrine tissues rather than a malignant transformation. The inability to find LOH of the MEN1 locus in metastatic papillary carcinoma agrees with this hypothesis. Therefore, defining the genetic alterations in endocrine tumors occurring in MEN1 patients will help to explain the mechanism of action and tissue specificity of the MEN1 gene.
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Acknowledgments
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RJW is supported in part by a George H. A. Clowes Research Career Development Award through the American College of Surgeons.
Received for publication July 3, 2000.
Accepted for publication December 18, 2000.
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ABSTRACT
INTRODUCTION
