A novel, noninvasive imaging technique for intraoperative assessment of parathyroid glands: confocal reflectance microscopy.

BACKGROUND: Successful surgical management of primary hyperparathyroidism requires the ability to identify and distinguish normal from abnormal parathyroid tissue. Microscopic pathologic confirmation often helps with the diagnoses and decisions regarding the extent of parathyroid resection. Confocal reflectance microscopy (CRM) is an optical method of noninvasively imaging tissue without fixation, sectioning, and staining as in standard histopathology. The goal of this study was to determine if CRM imaging could be used to distinguish normal from diseased parathyroid tissue intraoperatively. METHODS: In this study, 44 parathyroid glands from 21 patients undergoing operations for primary hyperparathyroidism were imaged immediately after excision. CRM images were compared with conventional hematoxylin-and-eosin stained sections obtained from the same gland. The percentage area occupied by fat cells was calculated in images of both normal and diseased glands. RESULTS: Characteristic microscopic features of parathyroid glands were distinguishable by CRM and correlated well with histopathology. The stromal fat content of normal and diseased glands could easily be determined. The percentage area occupied by fat cells differed significantly (P <.00001) in normal glands (average, 23.0% +/- 10.9%) and adenomatous glands (average, 0.4% +/- 0.7%). CONCLUSIONS: CRM imaging rapidly revealed microscopic features that reliably differentiated normal and diseased parathyroid glands. The success of this preliminary ex vivo study promotes interest in further development of an in situ probe for in vivo clinical diagnostic use.

RAD51 as a candidate parathyroid tumour suppressor gene on chromosome 15q: absence of somatic mutations.

OBJECTIVE: Loss of heterozygosity (LOH) at chromosome 15q is frequent in parathyroid adenomas, but no tumour suppressor gene of importance to parathyroid tumour development has been isolated from this region. The RAD51 gene has been localized to chromosome 15q and possesses regulatory functions involving DNA stability and cell proliferation, suggesting its possible role in tumorigenesis. Additionally, mutations in the RAD51 gene cause reduced resistance to ionizing radiation, which is a major risk factor for primary hyperparathyroidism. RAD51 was therefore analysed as a candidate tumour suppressor gene in a group of parathyroid adenomas for which mutations in a 15q tumour suppressor should be most readily detectable. PATIENTS AND DESIGN: From a total of 55 parathyroid adenomas, nine were selected based on their LOH pattern showing DNA loss at chromosome 15q in the vicinity of the RAD51 gene. RAD51 mRNA expression was investigated by reverse transcription-polymerase chain reaction (RT-PCR), and sequence analysis of the entire coding region of the RAD51 cDNA was performed in all nine adenomas. RESULTS: RAD51 mRNA expression was substantiated in all parathyroid adenomas. Compared with the normal RAD51 cDNA sequence, no point mutations or microdeletions could be found in the parathyroid tumor cDNA. CONCLUSION: These observations suggest that somatic inactivating mutations of the RAD51 gene are uncommonly, if ever, associated with parathyroid tumourigenesis.

Analysis of the RAD54 gene on chromosome 1p as a potential tumor-suppressor gene in parathyroid adenomas.

Parathyroid adenomas causing primary hyperparathyroidism (pHPT) frequently exhibit allelic loss of DNA markers on the short arm of chromosome 1, indicating the presence of one or more tumor-suppressor genes on 1p. Since the development of pHPT is enhanced in individuals exposed to ionizing radiation to the neck, it could be anticipated that genes involved in DNA repair and recombination may be special targets for mutation in parathyroid tumorigenesis, whether irradiation-associated or not. RAD54 is a member of a family of genes involved in such functions, and RAD54 knockout mice show increased sensitivity to ionizing radiation. The localization of the RAD54 gene to 1p32 has therefore elevated it to a most compelling candidate parathyroid tumor-suppressor gene. Twelve parathyroid adenomas demonstrating allelic loss at chromosome 1p were selected from 55 parathyroid adenomas previously analyzed for loss of heterozygosity using polymorphic microsatellite markers. All 18 exons of the RAD54 gene were fully analyzed by automated sequencing for detection of point mutations or micro-deletions in each parathyroid adenoma. No mutational aberrations were detected in the RAD54 gene, strongly suggesting that complete somatic inactivation of RAD54 is infrequently, if ever, associated with the development of parathyroid adenomas. Whether genes controlling DNA repair and recombination are involved in parathyroid neoplasia remains to be determined.

Parathyroid tumor suppressor on 1p: analysis of the p18 cyclin-dependent kinase inhibitor gene as a candidate.

Loss of chromosome arm 1p DNA is the most common molecular defect thus far observed in human parathyroid adenomas, suggesting that 1p is the location of a putative tumor suppressor gene (or genes) whose inactivation contributes frequently to parathyroid tumorigenesis. To narrow the genomic location of this tumor suppressor gene, we analyzed 25 sporadic parathyroid adenomas for allelic loss of polymorphic DNA loci on chromosome 1 using 11 microsatellite markers not previously scored for this set of tumors. Allelic loss on chromosome arm 1p DNA was observed in 8 of 25 adenomas. Marker deletion patterns showed some complexity, with the regions most commonly deleted in these tumors being 1p36 and 1p35-p31. The 1p35-p31 region contains an excellent candidate tumor suppressor gene, p18, whose product is a cell cycle regulator that inhibits the cyclin D1-associated kinase CDK6. Given that cyclin D1 is a parathyroid oncogene, inactivation of an inhibitor of cyclin D1 function, like p18, might also cause excessive parathyroid growth. To examine the involvement of p18 in parathyroid tumorigenesis, we analyzed 25 parathyroid adenomas for mutations of the p18 coding exons by single strand conformational polymorphism analysis and sequencing. No point mutations were found in any of the 25 adenomas. These observations indicate that inactivating mutation of the p18 gene occurs uncommonly, if at all, in parathyroid adenomas. In addition, the data raise the important possibility that more than a single tumor suppressor gene on 1p could contribute to parathyroid neoplasia.

Loss of chromosome arm 9p DNA and analysis of the p16 and p15 cyclin-dependent kinase inhibitor genes in human parathyroid adenomas.

Rearrangement and overexpression of the PRAD1/cyclin D1 oncogene, a cell cycle regulator, have been implicated in the pathogenesis of a subset of parathyroid adenomas. Recently, two cell cycle regulators that inhibit the cyclin D1-associated kinases cdk4 and cdk6 have been identified: p16 and p15, the products of the INK4A (also known as CDKN2, MTS1) and INK4B (also known as MTS2) putative tumor suppressor genes located on 9p21. Because inactivation of the p16 or p15 genes might be expected to result in oncogenic consequences similar to those from cyclin D1 overexpression, we examined 25 parathyroid adenomas for 1) allelic loss of polymorphic DNA loci on chromosome arm 9p, 2) homozygous deletions of the p16 and p15 genes by Southern blot analysis, and 3) mutations of the p16 and p15 genes by single strand conformational polymorphism analysis. Heterozygous allelic loss at 9p was observed in 4 of 25 adenomas (16%); their smallest shared region of deletion was 9p21-pter, which includes both the p16 and p15 genes. However, single strand conformational polymorphism analysis of all 3 exons of the p16 gene and both exons of the p15 gene failed to demonstrate mutation in any of the 25 cases, and homozygous deletions of the p16 and p15 genes, which are present in some human cancers, were not found in any parathyroid tumors. These observations indicate that inactivating mutations or homozygous deletions of the p16 and p15 genes occur uncommonly, if ever, in parathyroid adenomas; however, loss of a different tumor suppressor gene (or genes) on 9p appears to contribute to the pathogenesis of a significant percentage of these tumors.

Frequent loss of chromosome arm 1p DNA in parathyroid adenomas.

Two molecular defects have been described in parathyroid adenomas: rearrangement and overexpression of the PRAD1/cyclin D1 oncogene and allelic loss of chromosome 11 DNA, often including the multiple endocrine neoplasia type 1 (MEN1) putative tumor suppressor gene region. In an effort to identify additional parathyroid tumor suppressor genes, we examined 25 parathyroid adenomas for tumor-specific allelic loss of polymorphic DNA loci located near known or candidate tumor suppressor genes. Control leukocyte DNA from all 25 patients was heterozygous for 1 or more of the 9 chromosome 1 markers examined. Allelic loss at 1 or more of these informative loci on chromosome 1 was observed in 10 of 25 (40%) adenomas. Although many tumors lost extensive regions on chromosome 1, all but one of these tumors had allelic loss of distal 1p (1p32-pter); four tumors also lost loci on 1q. Allelic loss at 11q13, the site of the MEN1 gene, was detected in 5 of 21 (24%) informative cases, including 3 with 1p loss. In contrast, allelic loss was rarely observed at loci on 9q and 10p and was not observed at loci on 3p, 3q, 4p, 5q, 12q, 14q, 18q, 22q, or Xp. In summary, clonal allelic loss of loci on chromosome arm 1p is a frequent feature of parathyroid adenomas, implying that inactivation of a tumor suppressor gene(s) on 1p commonly contributes to their pathogenesis.

Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia.

The pathogeneses of parathyroid disease in patients with uremia and nonfamilial primary parathyroid hyperplasia are poorly understood. Because of multigland involvement, it has been assumed that these common diseases predominantly involve polyclonal (non-neoplastic) cellular proliferations, but an overall assessment of their clonality has not been done. We examined the clonality of these hyperplastic parathyroid tumors using X-chromosome inactivation analysis with the M27 beta (DXS255) DNA polymorphism and by searching for monoclonal allelic losses at M27 beta and at loci on chromosome band 11q13. Fully 7 of 11 informative hemodialysis patients (64%) with uremic refractory hyperparathyroidism harbored at least one monoclonal parathyroid tumor (with a minimum of 12 of their 19 available glands being monoclonal). Tumor monoclonality was demonstrable in 6 of 16 informative patients (38%) with primary parathyroid hyperplasia. Histopathologic categories of nodular versus generalized hyperplasia were not useful predictors of clonal status. These observations indicate that monoclonal parathyroid neoplasms are common in patients with uremic refractory hyperparathyroidism and also develop in a substantial group of patients with sporadic primary parathyroid hyperplasia, thereby changing our concept of the pathogenesis of these diseases. Neoplastic transformation of preexisting polyclonal hyperplasia, apparently due in large part to genes not yet implicated in parathyroid tumorigenesis and possibly including a novel X-chromosome tumor suppressor gene, is likely to play a central role in these disorders.

Ras oncogene mutations in benign and malignant thyroid neoplasms.

Current models for tumorigenesis propose that a series of genetic alterations occur during the progression from the normal cell to the malignant phenotype. Mutations in each of the three ras genes (K-ras, H-ras, and N-ras) have been identified in many human neoplasms, including thyroid cancer. In this study we examined genomic DNA from benign and malignant thyroid neoplasms for mutations that are known to activate the ras oncogenes (codons 12, 13, and 61). DNA from frozen surgically excised tissue (n = 8) and from formalin-fixed paraffin-embedded tissue (n = 30) was amplified by the polymerase chain reaction and screened for mutations using oligonucleotide-specific hybridization. No mutations were identified in follicular adenomas (n = 9). In follicular carcinomas, 2 of 14 tumors contained mutations (N-ras 61, Gln to Arg), and both of these patients had bone metastases. One of 15 papillary carcinomas had a ras mutation (H-ras 12, Gly to Ser). In contrast to other studies, we found that ras mutations are relatively uncommon in both benign and malignant thyroid neoplasms. Studies of larger numbers of tumors and comparisons of different patient populations will be required to assess a possible association of mutations in N-ras 61 with clinically aggressive follicular cancer.

Lack of evidence for estrogen receptors in human and bovine parathyroid tissue.

It has been hypothesized that estrogen may have a direct effect to reduce the set-point for PTH secretion which may be implicated in its hypocalcemic action. If this is so, estrogen receptors should be demonstrable within parathyroid tissue. Seven human parathyroid adenomas and five samples of normal bovine parathyroid tissue were examined using classical hormone-binding techniques. In no case was there evidence of displaceable estrogen binding of high affinity and low capacity. To exclude the presence of receptors within a small subset of the cells, five of the human adenomas were further studied by immunohistochemistry using a monoclonal antibody to the human estrogen receptor. In no case was there evidence of estrogen receptors. We conclude that the hypocalcemic action of estrogen replacement is unlikely to be mediated via a classical estrogen receptor within the parathyroid, although normal parathyroid tissue was not studied.

Molecular cloning and chromosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma.

Parathyroid adenomas are common benign neoplasms for which no chromosomal defects have been described. We recently found two parathyroid adenomas bearing clonal restriction fragment abnormalities involving the PTH locus, and now show that in one of these tumors: (a) a DNA rearrangement occurred at the PTH locus; (b) the rearrangement separated the PTH gene's 5' flanking region from its coding exons, conceivably placing a newly adjacent gene under the influence of PTH regulatory elements; (c) the DNA that recombined with PTH normally maps to 11q13, the known chromosomal location of several oncogenes and the gene for multiple endocrine neoplasia type I; and (d) the rearrangement was a reciprocal, conservative recombination of the locus on 11q13 (Human Gene Mapping Library assignment D11S287) with PTH (on 11p15). These data provide molecular cytogenetic evidence for the clonal occurrence of a major chromosome 11 aberrancy in this benign parathyroid tumor. The D11S287 clone could prove useful in genetic linkage analyses, in determining precise 11q13 breakpoints in other neoplasms, and in identifying a gene on chromosome 11 that may participate in parathyroid tumor development.

Intraoperative measurement of parathyroid hormone in the surgical management of hyperparathyroidism.

The operative management of patients with hyperparathyroidism is controversial. Higher rates for persistent hypercalcemia and postoperative hypoparathyroidism are seen in multiple-gland hyperplasia and in bilateral neck exploration. Hyperparathyroid patients undergoing unilateral neck exploration with removal of a single parathyroid adenoma have a rapid clearance of parathyroid hormone (PTH) that declines to undetectable levels within hours after successful parathyroid surgery. We have taken advantage of a sensitive immunoradiometric assay (IRMA) for the secreted biologically active, intact PTH-(1-84) molecule and demonstrated a decline of PTH to less than 40% of baseline values 15 minutes after successful parathyroid adenomectomy in 12 patients. Intraoperative measurement of PTH by modification of this IRMA may complement surgical skill and histopathologic information and has the potential for providing guidance regarding the extent of neck exploration necessary for determining surgical care of hyperparathyroidism.

Monoclonality and abnormal parathyroid hormone genes in parathyroid adenomas.

Previous work based on the relative tissue content of glucose-6-phosphate dehydrogenase isoenzymes suggested that parathyroid adenomas, like primary hyperplasia, may be multicellular (not clonal) in origin. We have reexamined this issue by using two independent molecular genetic methods. We report tumor-cell-specific restriction-fragment-length alterations involving the parathyroid hormone gene from two human parathyroid adenomas. These abnormal restriction fragments indicate that in each case a clonal proliferation of cells was present and also suggest that DNA alterations involving the parathyroid hormone locus may be important in the tumorigenesis or clonal evolution of some parathyroid adenomas. In addition, we used a restriction-fragment-length polymorphism in an X-linked gene (hypoxanthine phosphoribosyltransferase) to examine the clonality of eight parathyroid adenomas in women. Of these eight adenomas, six had the DNA hybridization pattern of monoclonality, and two had an equivocal pattern. None of five hyperplastic parathyroid glands had a monoclonal pattern. We conclude that some (and perhaps many) single parathyroid adenomas are monoclonal neoplasms. Our observations suggest that there is a fundamental biologic difference between parathyroid adenomas and primary hyperplasia--a difference that could prove useful in distinguishing these entities clinically.

The management of 50 unusual hyperfunctioning parathyroid glands.

From 1926 to 1984, 1200 patients with hyperparathyroidism were treated surgically at Massachusetts General Hospital (MGH). This series included 50 (4%) unusual cases that involved anomalous parathyroid locations or supernumerary hyperfunctioning parathyroid glands. In 42 cases the diseased glands were found in unusual locations: In three patients, glands were high in the neck behind the angle of the jaw; nine patients' glands were entirely encapsulated within the thyroid gland; and 30 patients required sternotomy for removal of mediastinal tumors. The eight remaining patients (as well as five of the mediastinal cases) had supernumerary hyperfunctioning glands. The three undescended parapharyngeal neoplasms, five of the cervical supernumerary tumors, and the majority of the mediastinal glands were associated with the thymus gland or thymic remnants. These glands appeared to arise from undescended parapharyngeal vestiges, partially descended parathymus remnants deposited along the path of developmental migration, or hyperdescended mediastinal inferior glands from branchial pouch III. The nine intrathyroid parathyroids were totally enclosed within the thyroid parenchyma. These appeared to arise from superior parathyroid glands that were trapped during fusion of the lateral wing portion from branchial pouch IV with the developing lateral lobes of the median thyroid primordium. Of these fifty cases, 39 patients had undergone a total of 60 previous operations (57 cervical and three mediastinal explorations) at MGH (16 patients) or other institutions (23 patients). In eleven patients the unusual hyperfunctioning gland was successfully identified at the time of the initial operation. Forty-four patients (88%) were surgically cured, as evidenced by eucalcemia. There were six patients with permanent hypoparathyroidism and none with persistent or recurrent hyperparathyroidism.

Cell-specific expression of the human parathyroid hormone gene in rat pituitary cells.

The promoter region of the human parathyroid hormone gene was fused to the Escherichia coli neo gene and introduced into GH4C1 rat pituitary and human HeLa cells. Both TATA boxes of the human parathyroid hormone gene accurately directed transcription in GH4C1 cells; the parathyroid hormone promoter was inactive in HeLa cells.

Natural history of parathyroid carcinoma. Diagnosis, treatment, and results.

From 1948 to 1983, 28 patients with parathyroid carcinoma underwent treatment and analysis at the Massachusetts General Hospital. This represents about 2 percent of the 1,200 patients with hyperparathyroidism managed during the period. Patient ages ranged from 18 to 72 years (mean 45 years) with equal numbers of both sexes (14 women and 14 men). There are several hallmarks that are clues to an increased index of suspicion preoperatively. Nine of the patients (32 percent) presented with palpable neck masses. Eleven patients (39 percent) had a serum calcium level greater than 14 mg/100 ml. Significant elevations of the parathyroid hormone level were noted with values two to three times normal. The incidence and severity of metabolic complications were prominent. These complications included renal stones in 18 patients (64 percent), bone disease in 14 patients (50 percent), peptic ulcer disease in 5 patients (18 percent), parathyroid crisis in 4 patients (14 percent), and pancreatitis in 2 patients (7 percent). Eleven of the patients underwent previous surgical therapy at other institutions, and 17 patients had their initial operation at our institution. Cervical parathyroid carcinomas that ranged from 1.5 to 27 g and 1.5 to 6 cm were excised. The characteristic appearance was a gray-white, stone hard parathyroid mass with invasion of adjacent tissue. The outcome was favorable for 16 surviving patients, with 14 (50 percent) showing no evidence of recurrence 2 to 17 years postoperatively and 2 alive with persistent disease 3 years after operation. Twelve patients died. Of these, eight had unsuccessful initial operative intervention with capsular rupture and dissemination of cancer, one had advanced disease with mediastinal extension which was unresectable, and three died from unrelated causes. Recurrences became apparent within 6 months to 3 years after operation and unfortunately denoted incurable disease. The mean survival time after operation in patients with recurrent disease was 7.6 years, ranging from 1 to 22 years. Carcinoma of the parathyroid gland is a rare entity. Although it is difficult to diagnose preoperatively, there should be an increased index of suspicion in those hyperparathyroid patients with palpable neck masses, profound hypercalcemia (greater than 14 mg/100 ml), marked increase in the parathyroid hormone level to greater than twice normal, and significant metabolic complications. The initial operation must be aggressive yet meticulous with en bloc resection of the parathyroid tumor and all adjacent invaded tissues, avoiding capsular violation or tumor spillage.(ABSTRACT TRUNCATED AT 400 WORDS)

Management of asymptomatic hyperparathyroidism.

From January 1, 1971, to December 31, 1982, 242 patients with uncomplicated biochemical or asymptomatic hyperparathyroidism underwent operative therapy at the Massachusetts General Hospital. They represent 36.1 percent of the 670 total operative cases during this period. Before 1971, from 1941 to 1970, there were only 33 hyperparathyroid patients with asymptomatic hyperparathyroidism who underwent surgery. The initial surgical procedures included 88 unilateral and 134 bilateral cervical explorations. There were 19 patients who underwent reexploration, including 15 referrals and 4 patients who had their primary operation at the Massachusetts General Hospital. There were no deaths, no recurrent nerve injuries, and only one patient with protracted but temporary postoperative hypocalcemia. Four patients (1.7 percent) had persistent hypercalcemia and therefore, must be considered treatment failures. The procedure resulted in normocalcemia in 238 of the patients (98.3 percent). The mean serum calcium level decreased from a preoperative value of 11.1 to 8.9 mg/100 ml the serum phosphorus level increased from 2.8 to 3.9 mg/100 ml postoperatively. Pathologic examination revealed 201 adenomas (83.1 percent), 39 hyperplasias (16.1 percent), 2 patients with normal glands (0.8 percent), and no carcinomas. The size of the abnormal glands appeared to correlate with the degree of hypercalcemia. Patients with marked hypercalcemia generally had a large gland that was more easily identified than the gland patients who had milder disease in whom it was smaller, harder to locate, and more difficult to distinguish pathologically from a normal gland. In general, patients with milder disease (serum calcium less than 11 mg/100 ml) should be followed expectantly. In some of these patients, there is doubtless progressive exacerbation of hypercalcemia, increases in the parathyroid hormone level, osteopenia, or renal insufficiency which ultimately requires surgical intervention. In others, there is apparently severe biochemical, asymptomatic hyperparathyroidism as manifested by a serum calcium level greater than 11 mg/100 ml, an increased parathyroid hormone level, increased 24 hour urinary calcium excretion greater than 150 mg, progressive loss of bone mass, or deterioration of renal function. These latter patients should be operated on without delay. For patients who risk the long-term complications of hyperparathyroidism and menopausal patients who are potentially threatened by postmenopausal osteoporosis, surgery is likely to be beneficial. For those whose compliance with therapy or follow-up poses a significant logistic problem, surgical therapy is often the best solution.