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Critical Reviews in Clinical Laboratory Sciences, 29( 1): 1-30 (1992)
Primary Hyperparathyroidism: Pathology, Flow Cytometric DNA Analysis, and Surgical Treatment H. Jaap Bonjer, M.D. and Hajo A. Bruining, M.D. Department of Surgery, University Hospital (Dijkzigt), Rotterdam, The Netherlands
C. Bruce Bagwell, M.D., Ph.D. Maine Cytometry Research Institute, Maine Medical Center, Portland, Maine
Michael A. Jones, M.D. and Ronald H. Nishiyama, M.D. Department of Pathology, Maine Medical Center, Portland, Maine
I. PATHOLOGY The pathology of primary hyperparathyroidism remains a controversial and poorly understood subject, in spite of considerable interest on the part of pathologists, surgeons, endocrinologists, and molecular biologists. Most studies have focused on the morphologic features of the parathyroid glands involved in the disease, specifically with respect to the delineation of “adenomas” and “hyperplasias” , but recent studies employing molecular biologic techniques-have challenged traditional histopathologic concepts and in some instances have shed new light on the pathogenesis of hyperparathyroidism. In spite of these developments, however, the etiology of the disease remains obscure and continues to engender significant controversy. The purpose of this review is to evaluate the traditional morphologic concepts of primary hyperparathyroidism in light of more recent molecular studies and our own data on the subject, and discuss the potential implications they may have on the surgical treatment of primary hyperparathyroidism.
A. Morphologic Features of Normal, Hyperplastic, and Adenomatous Glands 1. Normal Gland The definition of a normal parathyroid gland remains controversial, and agreement will likely never be completely reached. The normal gland is described as 3040-8363/9Z$..SO 0 1992 by CRC Press, Inc.
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yellow-brown in color and oval or bean-shaped. While minor gross changes may be of little interest to the pathologist, the surgeon must focus on relatively subtle gross changes to identify abnormal glands. The upper limit for size has been said to vary from 0.5 to 0.9 cm.’**The upper limit for weight is generally stated to be 30 to 40 mg,3 but some have advocated normal weights as high as 75 mg. Significant inter- and intraindividual variability may occur. Histologically, the normal gland is surrounded by a thin fibrous capsule and contains a mixture of fat, fibrovascular stroma, and hormonally active parenchyma. Fat content, one of the key features used to evaluate the functional state of a gland, may vary considerably. The usual fat content of the adult is approximately 25 to 30%; however, this depends on the age and body habitus of the individual. Fat content is usually low in early life and increases with a d ~ l t h o o d . ~ In one study,5 nearly one third of the glands had less than 10% stromal fat, which invalidates the use of stromal fat as an indicator of functional state. Chief cells predominate in the normal gland with oxyphil and transitional cells forming a small minority of the cells. The latter cells may, however, increase with age. The cells are usually arranged in small nests or trabeculae with the rare presence of micronodules, acinar or follicular arrangements. Small areas of stromal fibrosis or scar with other reactive changes, such as hemorrhage and inflammation, may be seen. Staining for fat in the normal gland will show abundant intracytoplasmic fat droplets. ~ 5 3 ’
2. Adenoma (Single Gland Disease) Traditional dogma in primary hyperparathyroidism holds that the majority of cases (50 to 89%) are caused by parathyroid “adenomas” that, because of the lack of definitive criteria for its definition (vide infru), are better referred to as single gland d i s e a ~ e . ~ The - I ~ percentage of adenomas reported has varied depending upon the criteria to define them. Adenomas are reported to occur with equal frequency in all four glands and usually weigh between 300 and 1000 mg. A thin capsule surrounding the gland is usually present that allows for easy surgical dissection. Microscopically, a monomorphic proliferation of chief cells is most common, often with some oxyphil and/or transitional cells. Water clear cells are rarely found. Cytologic changes in the chief cells, such as nuclear enlargement and hyperchromasia, also occur. Occasionally, marked nuclear pleomorphism, a benign finding, may be seen. Any architectural arrangement, solid, nodular, trabecular, acinar, or follicular, is possible. The presence of small amounts of stromal fat is acceptable to many authors as long as the other criteria are met.8.9*’3.1”18 Degenerative changes, cysts, fibrosis, hemorrhage, etc., especially in larger ones, are not uncommon. The most important findings in the traditional definition of a parathyroid adenoma is a compressed rim of “normal” parathyroid tissue and, another biopsy-proven normal gland. most imp~rtantly,~~”~’~.’~-~~
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3. Hyperplasia (Multiple Gland Disease) In contrast to aden~ma,*.~.~O most series report a minority (15 to 20%) of primary hyperparathyroidism as caused by hyperplasia. Usually, all four parathyroid glands are affected, but the enlargements may be asymmetrical. Microscopically, a mixture of cells is common, and nodularity is much more common and prominent than in adenomas. Water clear cell hyperplasia is distinctive but rarely seen at the present time. Stromal fat is usually demonstrable in the gland but may not be seen in all sections. Architectural arrangements are as varied as those seen in adenomas. It is worth noting, however, that nodular patterns can be exaggerated and can result in an appearance similar to an adenoma.
B. The Distinction of Adenoma from Hyperplasia The traditional methods to distinguish an adenoma from hyperplasia rely on a variety of morphologic and/or histochemical features. H o w e ~ e r , ~no ~ single ~~’-~~ criterion has proven irrefutable in making this differentiation, and the consequence is the use of the terminology single or multiple gland disease. Many early studies on this subject have used either poorly described criteria or poorly sampled parathyroid glands. The marked variation in normal content of stromal fat and the acceptance by many authors that stromal fat can be present in adenomas has eliminated this finding as a criterion. The finding of a normal rim of compressed parathyroid as seen in 50 to 70% of adenomas, is unreliable, since this phenomenon may be reproduced by large dominant nodules in hyperplastic glands. Considerable overlap in the cell types may be seen in both adenomas and hyperplasias, as well as in their distribution, which renders this criterion useless. The most heavily relied upon criterion is the identification of another normal gland. However, problems then arise. The concept that parathyroid hyperplasia may be asymmetrical is well accepted. This may be marked at times. If one accepts this concept, then the distinction of the minimally enlarged or “microscopically hyperplastic” gland from the normal gland becomes critical. Since there is significant variability in fat content and parenchymal and total weight of normal glands, this task may be difficult even when an entire gland is available for examination. This distinction may be impossible to make when only small biopsies are submitted for frozen sections, which is a common practice. The definition of normality, in this context, may significantly alter the incidences of hyperplasia and adenoma. While some have suggested that “microscopic hyperplasia” and mild enlargement in a “normal gland” found with an adenoma correlates with higher rates of recurrence or persistence and that such cases should be classified as examples of hyperplasia,1s,23,2s-28 others have emphasized that such distinctions are probably more semantic than real because they correlate poorly with hyperfunction. For practical purposes, the distinction of normal from
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abnormal glands, at the present time, may best be done by gross evaluation of the glands at the time of parathyroidectomy. In addition to the morphologic features, both staining for fat and electron microscopy have been applied to distinguish hyperplasia from adenoma and normal from abnormal glands. Fat staining is based on the principle that inactive or functionally normal glands have abundant and easily demonstrable cytoplasmic fat droplets. The hyperfunctioning gland has little or no cytoplasmic fat.29The usefulness of this technique has been c o n t r o ~ e r s i a l . ~We ~ ~feel ~ - ~that ~ the significant overlap in fat content in both hyperactive and normal glands,29the meth-’ odologic differences between various studies, and the subjectivity of interpretation limit the usefulness of staining for cytoplasmic fat on a routine basis. Ultrastructural analysis has been applied to distinguish hyperplasia from ade n ~ m a . ~ . ~ The ” - ” ultrastructural features that correlate with functional activity include hypertrophic Golgi apparatuses, abundance of rough endoplasmic reticulum, scarcity of cytoplasmic lipid, and multiple interdigitations of the plasma membrane. While there are no ultrastructural features that separate adenoma from hyperplasia, electron microscopic classification of glandular activity has been proposed to be useful when one parathyroid gland is obviously enlarged, and a second gland is normal or minimally enlarged. In the study by Cinti et al.,34 ultrastructural study of 10 such normal glands revealed evidence of endocrine activity in all glands, which led to reclassification of the cases as examples of hyperplasias. Two of the ten cases recurred, which support their observations and conclusion. They proposed that the ultrastructural evidence of endocrine activity, in apparently normal glands taken from cases of apparent single gland disease, suggests that the incidence of adenomas should be much lower. This conclusion is supported by more recent histopathologic and molecular biologic evidence. C. The Histopathologic Redefinition of Adenoma Recently, an attempt has been made to define the distinction between adenoma and hyperplasia using stricter histologic criteria, and the results have been correlated with clinical outcome. Recognizing the shortcomings of the traditional criteria and accepting the concept of ‘‘focal hyperplasia” ,* Ghandur-Mnaymneh and Kimura2reclassified 172 patients with primary parathyroid hyperplasia using the following criteria. Based on the assumption that an adenoma is a clonal growth that would be expected to displace adjacent parathyroid tissue as it grew and not incorporate stromal fat and eliminate the normal lobularity of the gland, they proposed that any lesion that contained stromal fat, had a lobular pattern, or showed a “normal” rim with transition from normal parathyroid tissue to abnormal tissue was an example of hyperplasia. All three of these criteria had to be absent to consider the lesion an adenoma. Of the 172 cases classified by these criteria, 144 (83.8%) were classified as hyperplasias. Only 10 (5.8%) could be confidently classified as adenomas. In the remainder of the cases, the material
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submitted was either grossly normal glands or was considered insufficient for classification. Importantly, of the 144 cases considered to be hyperplastic, 129 (75.1%) showed only single gland enlargement and thus, were considered as examples of “focal hyperplasia”. By the traditional criteria for the diagnoses of adenoma and hyperplasia, these cases would have been classified as adenomas. With these stringent criteria, all adenomas were of one cell type and lacked the characteristic nodularity of hyperplasia. They were also well circumscribed and contained a lamellated fibrous capsule. Pleomorphic nuclei were seen in both adenomas and hyperplasias. One hundred patients in the series of Ghandur-Mnaymneh and Kimura were available for follow-up. Fifty-one cases showed either persistent elevation of parathyroid hormone, with or without elevation of serum calcium levels, or increasing levels of the hormone. Thirty-eight cases were examples of focal hyperplasia. One was interpreted as an adenoma, and 4, as examples of multiple gland disease. In 8 cases the surgeon was unable to find any enlarged glands. In the examples of focal hyperplasia, the most common cause of hyperparathyroidism in this series, 44% demonstrated postoperative evidence of parathyroid hyperfunction. The authors concluded that the inordinate number of recurrences in this category provide biologic support for the concept of focal hyperplasia and emphasized the clinical utility of using very strict criteria for the diagnosis of an adenoma. On the other hand, the authors emphasize that the “cure” of a patient with focal hyperplasia by the removal of only one gland does not invalidate this concept, since the, as yet unidentified, stimulus that causes hyperplasia may affect only one gland. In our labor at or^,^^ we have recently studied a series of cases of primary hyperparathyroidism and classified them by the criteria proposed by GhandurMnaymneh and Kimura. The purpose of the study was to microscopically reclassify cases of primary hyperparathyroidism and correlate them with: (1) definition of the disease process as adenoma (single gland disease) or hyperplasia (multigland disease) based on the surgical (gross) findings, (2) nuclear DNA analysis by flow cytometry, (3) clinical outcome. The patients in our study were selected from a registry of 693 patients operated on at the University Hospital Dizkzigt (Rotterdam)or University Hospital (Leiden) for primary hyperparathyroidism between the years 1952 and 1989. All the patients had been classified as having single or multiglandular disease based on the findings of the surgeon at the time of the operations. For this study, the cases of single gland disease were considered as adenomas and the cases of multigland disease as hyperplasias. One hundred and seventy-nine of the patients had hyperplasia, and of these 9 1 had adequate clinical follow-up and pathologic material to be included in the study. From the remaining 514 patients with single gland disease, a sample large enough to reflect a representative rate of recurrence was selected. Based on a published average recurrence rate of 2.5%,43 it was determined that a sample size of approximately 160 patients would be sufficient. Other than including only patients with a minimum of 4 years of follow-up, the group
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was randomly selected. After eliminating those patients with inadequate pathologic material, this group consisted of 130 patients. No patients with MEN syndromes or familial hyperparathyroidism were included. All patients had been operated on by one of two surgeons. A bilateral neck dissection, identification and biopsy of all parathyroid glands, and removal of only grossly enlarged glands was the surgical approach. An estimated weight of 40 mg or greater was used as the criterion for enlargement. Follow-up studies of the patients in both groups included clinical histories, documentation of medications, calcium or vitamin D supplementations, and the determinations of serum levels of calcium, albumin, creatinine, urea, and parathyroid hormone (intact hormone) in two blood samples. All biopsies and intact glands removed from the two groups were examined by two pathologists without knowledge of the operative findings, weights of the glands, gross findings, or follow-up information. The review was limited only to sections stained with hematoxylin and eosin. The presence or absence of the following microscopic features were recorded: lobularity, microscopic cellularity, cell types, stromal fat, a normal rim, transition from normal to abnormal tissue when a “normal rim” was present, a capsule, thick-walled blood vessels, nuclear pleomorphism, mitotic activity, and bloodllymphatic vascular space involvement. Each gland was categorized as adenoma or hyperplasia based on the criteria of Ghandur-Mnaymneh and Kimura,2 i.e., a gland is considered hyperplastic if any one of the following criteria are met: fat cells in the mass, lobular architecture or a transition of the abnormal tissue in the mass to a “normal rim”. After independent review by both pathologists, a consensus was reached in each case when there were disagreements. If no consensus could be reached, often due to inadequate samples of tissue, the case was considered unclassifiable. Of the 130 cases grossly classified as adenomas (single gland diseases), 58 (44.6%) were histologically classified as adenomas, 48 (36.9%), as hyperplasias, 18 (13.8%), as unclassifiable, and 6 (4.7%) as normal glands. The weights of the adenomas ranged from 115 mg to 16.7 gm, with a mean weight of 1500 mg. The follow-up in this group ranged from 4 to 27 years (mean, 12.5 years). Only 2 patients (1.5%) had postoperative hypercalcemia. One patient had a single enlarged gland removed that was interpreted as focal hyperplasia, and the other had one enlarged gland and one normal-sized gland removed, both of which were interpreted as examples of hyperplasia. Two hundred and six enlarged parathyroid glands were examined in the 91 patients classified by the surgeons as hyperplasias (multigland disease). Eightyfour (40.4%)were histologically classified as hyperplasias, 70 (33.9%) as normal glands, 30 (14.6%) as unclassifiable, and 22 (10.6%) as adenomas. The followup period for this group ranged from 4 to 26 years (mean 13.8 years). Six patients (6.5%) had persistent disease. In 4 of these 6 patients, the glands were classified as hyperplastic, and in 2 the glands were considered to be normal. Biopsies of glands, which were considered normal by the surgeon, taken from the cases of adenomas and hyperplasias, were combined and analyzed as a group. There were 339 glands in this group; 316 (93.2%) were classified as
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histologically normal, 2 (0.6%) as adenomas, 10 (2.9%) as hyperplasias, and 11 (3.2%) as unclassifiable. As demonstrated by these data, the microscopic classification of abnormal parathyroid glands as hyperplasias (multigland disease) or adenomas (single gland disease) correlates poorly with the gross/clinical classification of the disease. Our results are similar to those of Ghandur-Mnaymneh and Kimura2 in that many, nearly 50%, of our cases of single gland disease were classified as examples of hyperplasia, cases that by traditional criteria would have been considered adenomas. We agree with these authors that stricter definitions of adenoma and hyperplasia are appropriate, and when applied, result in many, and perhaps most, cases of adenoma being reclassified as examples of focal hyperplasia. While the results of our analysis show poor correlation between the clinical/ gross classification of abnormal glands and the histologic classification, the correlation is better when glands were grossly considered to be of normal size. The histologic diagnosis of “normal” predicted the gross/surgical identification of a normal gland with a sensitivity of 80% and a specificity of 87%. No significant difference was noted between the normal glands derived from cases of single and multiple gland disease. Our results show that the pathologist can distinguish normal from abnormal parathyroid glands with a fair degree of accuracy. We feel that this distinction should be the primary aim of microscopic examination of glands at the time of examination of frozen and permanent sections. The distinction of adenoma from hyperplasia (single vs . multigland disease) is essentially impossible on histologic grounds. If one selects very strict criteria for the diagnosis of adenomas, then they account for a much smaller percentage of cases of primary hyperparathyroidism than previously reported, and the number of cases of focal hyperplasia significantly increase. Whether the concept of focal hy perplasia has clinical relevance or not, as suggested by Ghandur-Mnaymneh and Kimura, is a more difficult issue to resolve. The surgical approach to primary hyperparathyroidism with reference to some of these concepts and our data will be discussed. Suffice it to say that in our series the recurrence/persistence rate was low, much lower than that in the Ghandur-Mnaymneh series (51%). As a result, we were unable to demonstrate that reclassification of many cases of single gland disease as focal hyperplasias predicted recurrences. While both of our patients with single gland disease who had recurrences were classified as focal hyperplasia, the vast majority of patients with this diagnosis never did.
D. Assessment of Clonality in Primary Hyperparathyroidism Recently, a number of different molecular biologic techniques have been applied to the task of defining the etiology of primary hyperparathyroidism. Fialkow et a1.44analyzed isoenzyme patterns in parathyroid “adenomas” (single gland disease) from three women heterozygous for the enzyme glucose-6-phos-
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phate dehydrogenase. Because of the phenomenon of random X-chromosome inactivation in women, heterozygous women will have an equal population of both type A and type B isoenzymes in normal tissues. A clonal growth (adenoma) would be expected to manifest only a single isoenzyme pattern. In contrast, a growth that is multicellular in origin (hyperplasia) would be expected to show both type A and type B isoenzyme patterns. In this study, no major differences were found between normal tissues and the abnormal parathyroid glands in these three patients. This finding would seem to support the concept of a multicellular (hyperplastic) origin for these cases. The fact that in two of these three cases only one gland was enlarged supports the concept of focal hyperplasia. However, as emphasized by these authors, these findings do not completely rule out the possibility of a clonal neoplasm. The latter, if mixed with a sufficient number of normal cells, may be undetected. In addition, although unlikely, activity of both X-chromosomes could result in the detection of both isoenzymes, although a hybrid molecule of both type A and type B enzymes would be expected, and none was detected. A subsequent study by Arnold et al.45appears to contradict that of Fialkow. Two techniques were used, both employing DNA probes, to study parathyroid “adenomas” from eight patients with primary hyperparathyroidism. All of the patients had single enlarged glands and biopsies taken of one or more normal or atrophic glands. All were cured by removal of the single enlarged gland. The first method used heterozygosity for a DNA polymorphism of the X-linked gene, hypoxanthine phosphoribosyltransferase (HPRT). In six of the eight specimens studied, evidence for clonality was found. In the other two, the findings were equivocal. Of five hyperplastic glands studied, defined by clinicopathologic criteria, all lacked evidence of clonality. In addition, in two of the eight cases studied by the above method, a tumor cell-specific restriction-fragment-length polymorphism (RFLP) involving the parathyroid hormone (PTH) gene was discovered. This permitted the assessment of clonality using a different method that employed a DNA probe for the PTH gene. In both samples, evidence for clonality was present, confirming the results obtained from using the first method. The abnormality in the PTH gene used to detect clonality raises the question whether abnormalities in this gene are important in the genesis of some forms of hyperparathyroidism. Similar recent studies from patients with the MEN-I syndrome have helped to shed light on the pathogenesis of at least some cases of hyperparathyroidism. Thakker et al.46used DNA probes for 18 different chromosome 11 gene sequences to demonstrate a loss of heterozygosity in the parathyroid tissue from 3 of 6 patients with the MEN-I syndrome. Their findings demonstrate that, in at least some patients with this syndrome, monoclonal tumors develop. In addition, their findings demonstrate that allelic deletions occur in these tumors. These authors also conducted linkage studies in two of the affected MEN-I families using a RFLP identified with a probe for the INT2(SS6) chromosome 11 gene. Strong linkage was found with the INT2 ~ncogene,~’ a proto-oncogene that encodes a
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protein of the fibroblast growth factor family. This finding is particularly intriguing in view of the known presence of a parathyroid mitogenic factor in the This mitogenic factor has plasma of some patients with the MEN-I been noted to have properties similar to fibroblast growth factors.49Arnold et al.’O have also demonstrated rearrangements between the PTH gene and the 1lq13 region, the region that contains INT2, in some nonfamilial parathyroid adenomas. In a study similar to that of Thakker, Friedman et al.51demonstrated monoclonality and allelic loss from chromosome 11 in 10 of 16 parathyroid tumors from patients with the MEN-I syndrome. They were also able to demonstrate similar allelic losses in 9 of 34 sporadic adenomas, and in 7 of the 9, the loss included the apparent MEN-I locus. Interestingly, in the patients with MEN, the monoclonal parathyroid tumors were substantially larger. This has led to the suggestion that parathyroid tumors in both familial and nonfamilial hyperparathyroidism may involve an initial hyperplastic phase, perhaps related to a first mutagenic event, followed by a second monoclonal phase of growth, stimulated by a second mutagenic event.46
II. FLOW CYTOMETRIC NUCLEAR DNA ANALYSIS The inability to discriminate reliably between primary parathyroid hyperplasia and parathyroid adenomas at the microscopic level, has led to a quest for objective means to make this differentiation. Flow cytometric DNA analysis has been used for this purpose. Little emphasis will be placed on methods and instrumentation in this discussion. For more detailed descriptions on methodology and instrur n e n t a t i ~ n , the ~ ~ .reader ~ ~ is referred to more detailed reviews on the subject. The format in this section will be to present data from our study of the patients described in the previous section and discuss the results in conjunction with those reported in the literature.
A. Methods and Materials The methods and materials in our study are described briefly. Our material included normal-sized parathyroid glands taken from patients with parathyroid disease and enlarged glands from patients with single and multigland disease that were embedded in paraffin. The specimens were stained with propidium iodide and analyzed on an Epics-C flow cytometer (Coulter Corporation, Hialeah, Florida). All histograms were analyzed with the VerityIModfit cell cycle analysis program (Verity Software House, Inc., Topsham, Maine). The data collected included the DNA index, %S and %G,M of the cell cycle and total events. DNA index is defined by the ratio of the position of the DNA aneuploid GoGIpopulation of cells in a specimen to the position of the G,G, cells of a normal population of cells. Position is the mean channel number of a modeled Gaussian peak. DNA
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diploid populations of cells had DNA indices between 0.9 and 1.1 and DNA tetraploid populations of cells had greater than 20% of the total number of cells at the 4C region and a DI between 1.9 and 2.1. The remainder of the histograms were in the DNA aneuploid category. Because of the arbitrary limits utilized to classify DNA tetraploid histograms and the current lack of standardization for this value, we have found it prudent to adopt %4C as another means of classifying ploidy. This appears to be a more practical method for comparisons of the different studies. We define %4C as the percentage of cells in the 4C region of the histogram. B. Compilation of Data Our review of the literature includes only those reports in which flow cytometry was used to examine specimens of parathyroid glands. The definitions of normal glands and of primary parathyroid hyperplasia, the methods of data analysis, and the data acquired in the different studies vary considerably. To facilitate analyses and comparisons, our data and that taken from the literature are tabulated in Table 1. 1. Description of Table and Definitions of Terms Each study in the table is identified by the first author. The number of patients, the total number of parathyroid glands examined, and the number and percentage of the total analyzed in each category are noted. The mean percentage of cells at the 4C region is indicated by %4C. This is to contrast it from DNA tetraploidy, which is defined by the presence of a population of cells in the same region that exceeds a previously defined upper limit. These limits varied from 10% to 20%.42*5”56 %Tet indicates the percentage of glands that showed DNA tetraploidy. The percentage of the total number of glands that contain aneuploid populations of cells is denoted by %Aneup and the average percentage of all cells in S-phase by %Total S. There are four categories in the table. “Normal” includes the normal parathyroid glands taken from patients without parathyroid disease. “Normal-sized’ ’ includes normal-sized glands derived from patients with parathyroid disease. “Hyperplasia (two or more enlarged glands)” appears to be self-explanatory; however, the definitions of hyperplasia vary. Some authors require microscopic confirmation of the diagnosis. Others define it by the number of enlarged glands that are grossly identified by the surgeon, but this number can also differ. ‘‘Adenomas (single gland enlargement)’’ is universally defined by the presence of a single enlarged gland. Carcinomas are not included in this study, but they are major parts of the studies of Harlow et al., Bowlby et al., and Shenton et a1.54-56
10
Hyperplasia (two or more enlarged glands)
Normal-sized glands from patients with hyperparathyroidism
Normal glands without parathyroid disease
Category
TABLE 1 Summary of Data Ref.
57 59 54 60 55 56 42 61 57 59 54 60 55 56 42 61 57 59 54 60 55 56 42
Study lrvin A lrvin B Harlow Rosen Bowlby Shenton Bonjer Joensuu lrvin A lrvin B Harlow Rosen Bowlby Shenton Bonjer Joensuu lrvin A lrvin B Harlow Rosen Bowlby Shenton Bonjer
54 54 41 26 12 76
54 54 41 26 12 76
54 41 26 12 76
No. patients
78 26 24 76 60 187 54 1 53 78 26 24 76 60 187 54 153 78 26 24 76 60 187
1 53
Total glands
5.06
21 13
49
5 10 92
2.00 18.00 7.00 9.96
5 14 33 13 32
8 10 59
11
8
6.90
0.38 7.48 31 38
12.00
35
21 48 30
5.06
0.18
%4Cb
54 8
11
%
14 2
17
No
0 17
2
0 3
0
0
0
3.48
21
1.89
14.6
1.2 14.6
YO Total S.
YO Aneupd
20
0
1
0
0
0
0
0
YoTets
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*
a
Ref.
61 57 59 54 60 55 56 42 61
Study Joensuu lrvin A lrvin B Harlow Rosen Bowlby Shenton Bonjer Joensuu
54
54 54 41 26 12 76
No. patients
%Tet Yo of glands with DNA tetraploidy. %Aneup, % of glands with DNA aneuploidy. %Total S, Mean Yo of total cells in S phase.
%4C, Mean Yo of cells at the 4C region of histograms.
N, Number of glands in each category.
Adenomas (single gland enlargement)
Category
TABLE 1 (continued) Summary of Data
54 153 78 26 24 76 60 187 54
Total glands
80 37 12 9 56 39 36 54
N=
52 47 46 38 74 65 19 1 00
%
30.00 10.02
7.00
3.30 18.00
%4Cb
21 50 3 9
8
0
%Tetc
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YO
5 0 22 26
0 15 25
Aneupd
%
3.64
21
1.5
Total So
C. Analysis and Comparison of Data
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Table 1 is the key to all of the analysis and comparisons of the data that follow in this section. A comment is required on the data contained in the first report of Irvin et al.57 (Irvin A) because the figures in the %4C category are geometric means and aggregate adjusted and not comparable to those taken from the other reports.58 1. Normal Parathyroid Glands from Patients Without Parathyroid Disease The definition of normality of glands differed in all of the studies listed. Irvin and B a g ~ e l l Irvin , ~ ~ et al.,59 and Rosen and MusclowM)required microscopic confirmation. Harlow et al.54define normal glands as those taken from patients without evidence of any abnormalities in calcium metabolism. Bowlby et al.55 defined normality by the weight of the glands, using age-related standards. The upper limit of normal in their study appears to be 60 mg. Shenton et al.56used an upper limit of normal for weight of 30 mg. Our upper limit of normal for weight was 40 mg.42 None of the figures for %4Cis comparable. Irvin and BagweIIs7report a low figure. The data of Rosen and MusclowWand Shenton et al.56 are estimates, being the sums of %S and %G,M. The figure for Shenton et al.56was calculated because their report only includes the mean percentages for G,G,. No tetraploidy or aneuploidy was reported in this group. The %S reported by Harlow et al.54is the only acceptable figure, because that of Rosen and MusclowM)is an estimate. 2. Normal-Sized Parathyroid Glands from Patients
with Parathyroid Disease The results of both studies of Irvin and c o - w o r k e r ~ deserve ~ ~ . ~ ~ comment, because they were able to correlate them with a relatively high rate of recurrence and persistent parathyroid hyperfunction postoperatively. The figure of 0.38% 4C from the first study (Irvin A) overlaps with those reported for normal glands, hyperplastic glands, and adenomas. Utilizing a computer-derived statistical analysis, they found that 14 glands (29%) taken from patients with adenomas had a greater than 50% chance of being abnormal. In the second study (Irvin B),59the %4C for normal glands was 6%. The figure listed is a mean of the %4C of all the normal-sized glands. Irvin et al. divided this group of glands into two parts. One group, consisting of 13 glands from 7 patients, had %4Cs that were greater than 6%. All of these patients had elevated levels of serum PTH postoperatively. The mean %4Cfor this group was 9.8%.One of these glands showed aneuploidy.
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The second group consisted of 17 glands from 10 patients without increased serum levels of PTH postoperatively. Four glands had %4Cs greater than 6%. The mean %4C was 6.3%. Problems are again encountered in comparing the results in this group. The %4C reported by Irvin et al. (Irvin B)59and Bonjer et al.42are comparable and appear to coincide. The figure for Rosen and MusclowM)is our estimate. One example of DNA tetraploidy is reported and is from our This is a biopsy of a normal-sized gland from a patient who had had two enlarged glands removed and who later developed hypercalcemia. The DNA index for this gland was 2.05. One DNA aneuploid gland was reported by Irvin et al.59and the gland was associated with elevated levels of serum PTH postoperatively. The only acceptable figure for %S phase is that reported by Bonjer et al.42 The other is an estimate.60
3. Primary Parathyroid Hyperplasia Hyperplasia is defined differently in the various studies. Harlow et al.54and Shenton et al.56 included only secondarily hyperplastic glands in their study. Rosen and Musclowmand Irvin and Bagwe1P7required microscopic confirmation. Bowlby et al.55 defined primary thyperplasia by the gross identification of 3 enlarged glands, each weighing more than 60 mg. Bonjer et al.42defined primary hyperplasia by the gross identification of two or more enlarged glands, each weighing more than 40 mg. The only acceptable figure for %4C is that of Bonjer et al.42The others are all estimates, including that of Irvin et al. (Irvin B).59They reported the average %4C for all enlarged glands and did not separate them into hyperplastic and adenomatous glands. Bowlby et al.55report a relatively high incidence of DNA tetraploidy; however, this includes only 2 of 10 glands. Bonjer et al.42record one gland with a tetraploid population of cells. The DNA index for this gland was 2.07. The gland was from a patient with three enlarged glands, one of which contained an aneuploid population of cells. The presence of DNA aneuploidy in one gland by Irvin et al.59is confirmed by the finding of DNA aneuploidy in ten hyperplastic glands by Bonjer et al.42 Irvin et al.59 were, to the best of our knowledge, the first to report DNA aneuploidy in a normal-sized gland from a patient with parathyroid disease and in one hyperplastic gland. Results such as these have limited the usefulness of DNA aneuploidy in differentiating non-neoplastic lesions of the parathyroid glands from parathyroid adenomas and carcinomas.
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4. Parathyroid Adenomas The definition of a parathyroid adenoma is universally accepted as the presence of one enlarged parathyroid gland. The only acceptable value for %4C is that of Bonjer et al.42The others are not comparable. all estimates or, in the case of Irvin et al. (Irvin A),55-57*59@' For the first time, the incidence of DNA tetraploidy is nearly completely recorded. However, there are defects. The figure recorded for Shenton et al."j is an estimate and reflects a statement in their article that over 50% of adenomas showed abnormal cell cycle patterns with high proportions (over 20%) of cells within the tetraploid region. Bowlby et al.55 also reported a high incidence of DNA tetraploidy, while Bonjer et aL4, report only one gland. The percentages of glands with DNA aneuploidy are reported in nearly all of the studies. They vary, but in 4 of 5 s t u d i e ~ , ~ the ~ , figures ~ ~ , ~are ~ , high ~ ~ but comparable. The only acceptable figures for %S are those reported by Harlow et al.54and Bonjer et al.42 D. Conclusions The core of this section of our review was to determine whether flow cytometric nuclear D N A analysis can discriminate between primary parathyroid hyperplasia and parathyroid adenomas. The analysis of our data showed the following A chi-square test was used to compare the incidences of DNA diploid with DNA aneuploid populations of cells in normal-sized parathyroid glands, hyperplastic glands (multigland disease), and adenomas (single gland disease). The differences were statistically significant when comparing normal-sized glands with hyperplastic @
Critical Reviews in Clinical Laboratory Sciences, 29( 1): 1-30 (1992)
Primary Hyperparathyroidism: Pathology, Flow Cytometric DNA Analysis, and Surgical Treatment H. Jaap Bonjer, M.D. and Hajo A. Bruining, M.D. Department of Surgery, University Hospital (Dijkzigt), Rotterdam, The Netherlands
C. Bruce Bagwell, M.D., Ph.D. Maine Cytometry Research Institute, Maine Medical Center, Portland, Maine
Michael A. Jones, M.D. and Ronald H. Nishiyama, M.D. Department of Pathology, Maine Medical Center, Portland, Maine
I. PATHOLOGY The pathology of primary hyperparathyroidism remains a controversial and poorly understood subject, in spite of considerable interest on the part of pathologists, surgeons, endocrinologists, and molecular biologists. Most studies have focused on the morphologic features of the parathyroid glands involved in the disease, specifically with respect to the delineation of “adenomas” and “hyperplasias” , but recent studies employing molecular biologic techniques-have challenged traditional histopathologic concepts and in some instances have shed new light on the pathogenesis of hyperparathyroidism. In spite of these developments, however, the etiology of the disease remains obscure and continues to engender significant controversy. The purpose of this review is to evaluate the traditional morphologic concepts of primary hyperparathyroidism in light of more recent molecular studies and our own data on the subject, and discuss the potential implications they may have on the surgical treatment of primary hyperparathyroidism.
A. Morphologic Features of Normal, Hyperplastic, and Adenomatous Glands 1. Normal Gland The definition of a normal parathyroid gland remains controversial, and agreement will likely never be completely reached. The normal gland is described as 3040-8363/9Z$..SO 0 1992 by CRC Press, Inc.
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yellow-brown in color and oval or bean-shaped. While minor gross changes may be of little interest to the pathologist, the surgeon must focus on relatively subtle gross changes to identify abnormal glands. The upper limit for size has been said to vary from 0.5 to 0.9 cm.’**The upper limit for weight is generally stated to be 30 to 40 mg,3 but some have advocated normal weights as high as 75 mg. Significant inter- and intraindividual variability may occur. Histologically, the normal gland is surrounded by a thin fibrous capsule and contains a mixture of fat, fibrovascular stroma, and hormonally active parenchyma. Fat content, one of the key features used to evaluate the functional state of a gland, may vary considerably. The usual fat content of the adult is approximately 25 to 30%; however, this depends on the age and body habitus of the individual. Fat content is usually low in early life and increases with a d ~ l t h o o d . ~ In one study,5 nearly one third of the glands had less than 10% stromal fat, which invalidates the use of stromal fat as an indicator of functional state. Chief cells predominate in the normal gland with oxyphil and transitional cells forming a small minority of the cells. The latter cells may, however, increase with age. The cells are usually arranged in small nests or trabeculae with the rare presence of micronodules, acinar or follicular arrangements. Small areas of stromal fibrosis or scar with other reactive changes, such as hemorrhage and inflammation, may be seen. Staining for fat in the normal gland will show abundant intracytoplasmic fat droplets. ~ 5 3 ’
2. Adenoma (Single Gland Disease) Traditional dogma in primary hyperparathyroidism holds that the majority of cases (50 to 89%) are caused by parathyroid “adenomas” that, because of the lack of definitive criteria for its definition (vide infru), are better referred to as single gland d i s e a ~ e . ~ The - I ~ percentage of adenomas reported has varied depending upon the criteria to define them. Adenomas are reported to occur with equal frequency in all four glands and usually weigh between 300 and 1000 mg. A thin capsule surrounding the gland is usually present that allows for easy surgical dissection. Microscopically, a monomorphic proliferation of chief cells is most common, often with some oxyphil and/or transitional cells. Water clear cells are rarely found. Cytologic changes in the chief cells, such as nuclear enlargement and hyperchromasia, also occur. Occasionally, marked nuclear pleomorphism, a benign finding, may be seen. Any architectural arrangement, solid, nodular, trabecular, acinar, or follicular, is possible. The presence of small amounts of stromal fat is acceptable to many authors as long as the other criteria are met.8.9*’3.1”18 Degenerative changes, cysts, fibrosis, hemorrhage, etc., especially in larger ones, are not uncommon. The most important findings in the traditional definition of a parathyroid adenoma is a compressed rim of “normal” parathyroid tissue and, another biopsy-proven normal gland. most imp~rtantly,~~”~’~.’~-~~
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3. Hyperplasia (Multiple Gland Disease) In contrast to aden~ma,*.~.~O most series report a minority (15 to 20%) of primary hyperparathyroidism as caused by hyperplasia. Usually, all four parathyroid glands are affected, but the enlargements may be asymmetrical. Microscopically, a mixture of cells is common, and nodularity is much more common and prominent than in adenomas. Water clear cell hyperplasia is distinctive but rarely seen at the present time. Stromal fat is usually demonstrable in the gland but may not be seen in all sections. Architectural arrangements are as varied as those seen in adenomas. It is worth noting, however, that nodular patterns can be exaggerated and can result in an appearance similar to an adenoma.
B. The Distinction of Adenoma from Hyperplasia The traditional methods to distinguish an adenoma from hyperplasia rely on a variety of morphologic and/or histochemical features. H o w e ~ e r , ~no ~ single ~~’-~~ criterion has proven irrefutable in making this differentiation, and the consequence is the use of the terminology single or multiple gland disease. Many early studies on this subject have used either poorly described criteria or poorly sampled parathyroid glands. The marked variation in normal content of stromal fat and the acceptance by many authors that stromal fat can be present in adenomas has eliminated this finding as a criterion. The finding of a normal rim of compressed parathyroid as seen in 50 to 70% of adenomas, is unreliable, since this phenomenon may be reproduced by large dominant nodules in hyperplastic glands. Considerable overlap in the cell types may be seen in both adenomas and hyperplasias, as well as in their distribution, which renders this criterion useless. The most heavily relied upon criterion is the identification of another normal gland. However, problems then arise. The concept that parathyroid hyperplasia may be asymmetrical is well accepted. This may be marked at times. If one accepts this concept, then the distinction of the minimally enlarged or “microscopically hyperplastic” gland from the normal gland becomes critical. Since there is significant variability in fat content and parenchymal and total weight of normal glands, this task may be difficult even when an entire gland is available for examination. This distinction may be impossible to make when only small biopsies are submitted for frozen sections, which is a common practice. The definition of normality, in this context, may significantly alter the incidences of hyperplasia and adenoma. While some have suggested that “microscopic hyperplasia” and mild enlargement in a “normal gland” found with an adenoma correlates with higher rates of recurrence or persistence and that such cases should be classified as examples of hyperplasia,1s,23,2s-28 others have emphasized that such distinctions are probably more semantic than real because they correlate poorly with hyperfunction. For practical purposes, the distinction of normal from
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abnormal glands, at the present time, may best be done by gross evaluation of the glands at the time of parathyroidectomy. In addition to the morphologic features, both staining for fat and electron microscopy have been applied to distinguish hyperplasia from adenoma and normal from abnormal glands. Fat staining is based on the principle that inactive or functionally normal glands have abundant and easily demonstrable cytoplasmic fat droplets. The hyperfunctioning gland has little or no cytoplasmic fat.29The usefulness of this technique has been c o n t r o ~ e r s i a l . ~We ~ ~feel ~ - ~that ~ the significant overlap in fat content in both hyperactive and normal glands,29the meth-’ odologic differences between various studies, and the subjectivity of interpretation limit the usefulness of staining for cytoplasmic fat on a routine basis. Ultrastructural analysis has been applied to distinguish hyperplasia from ade n ~ m a . ~ . ~ The ” - ” ultrastructural features that correlate with functional activity include hypertrophic Golgi apparatuses, abundance of rough endoplasmic reticulum, scarcity of cytoplasmic lipid, and multiple interdigitations of the plasma membrane. While there are no ultrastructural features that separate adenoma from hyperplasia, electron microscopic classification of glandular activity has been proposed to be useful when one parathyroid gland is obviously enlarged, and a second gland is normal or minimally enlarged. In the study by Cinti et al.,34 ultrastructural study of 10 such normal glands revealed evidence of endocrine activity in all glands, which led to reclassification of the cases as examples of hyperplasias. Two of the ten cases recurred, which support their observations and conclusion. They proposed that the ultrastructural evidence of endocrine activity, in apparently normal glands taken from cases of apparent single gland disease, suggests that the incidence of adenomas should be much lower. This conclusion is supported by more recent histopathologic and molecular biologic evidence. C. The Histopathologic Redefinition of Adenoma Recently, an attempt has been made to define the distinction between adenoma and hyperplasia using stricter histologic criteria, and the results have been correlated with clinical outcome. Recognizing the shortcomings of the traditional criteria and accepting the concept of ‘‘focal hyperplasia” ,* Ghandur-Mnaymneh and Kimura2reclassified 172 patients with primary parathyroid hyperplasia using the following criteria. Based on the assumption that an adenoma is a clonal growth that would be expected to displace adjacent parathyroid tissue as it grew and not incorporate stromal fat and eliminate the normal lobularity of the gland, they proposed that any lesion that contained stromal fat, had a lobular pattern, or showed a “normal” rim with transition from normal parathyroid tissue to abnormal tissue was an example of hyperplasia. All three of these criteria had to be absent to consider the lesion an adenoma. Of the 172 cases classified by these criteria, 144 (83.8%) were classified as hyperplasias. Only 10 (5.8%) could be confidently classified as adenomas. In the remainder of the cases, the material
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submitted was either grossly normal glands or was considered insufficient for classification. Importantly, of the 144 cases considered to be hyperplastic, 129 (75.1%) showed only single gland enlargement and thus, were considered as examples of “focal hyperplasia”. By the traditional criteria for the diagnoses of adenoma and hyperplasia, these cases would have been classified as adenomas. With these stringent criteria, all adenomas were of one cell type and lacked the characteristic nodularity of hyperplasia. They were also well circumscribed and contained a lamellated fibrous capsule. Pleomorphic nuclei were seen in both adenomas and hyperplasias. One hundred patients in the series of Ghandur-Mnaymneh and Kimura were available for follow-up. Fifty-one cases showed either persistent elevation of parathyroid hormone, with or without elevation of serum calcium levels, or increasing levels of the hormone. Thirty-eight cases were examples of focal hyperplasia. One was interpreted as an adenoma, and 4, as examples of multiple gland disease. In 8 cases the surgeon was unable to find any enlarged glands. In the examples of focal hyperplasia, the most common cause of hyperparathyroidism in this series, 44% demonstrated postoperative evidence of parathyroid hyperfunction. The authors concluded that the inordinate number of recurrences in this category provide biologic support for the concept of focal hyperplasia and emphasized the clinical utility of using very strict criteria for the diagnosis of an adenoma. On the other hand, the authors emphasize that the “cure” of a patient with focal hyperplasia by the removal of only one gland does not invalidate this concept, since the, as yet unidentified, stimulus that causes hyperplasia may affect only one gland. In our labor at or^,^^ we have recently studied a series of cases of primary hyperparathyroidism and classified them by the criteria proposed by GhandurMnaymneh and Kimura. The purpose of the study was to microscopically reclassify cases of primary hyperparathyroidism and correlate them with: (1) definition of the disease process as adenoma (single gland disease) or hyperplasia (multigland disease) based on the surgical (gross) findings, (2) nuclear DNA analysis by flow cytometry, (3) clinical outcome. The patients in our study were selected from a registry of 693 patients operated on at the University Hospital Dizkzigt (Rotterdam)or University Hospital (Leiden) for primary hyperparathyroidism between the years 1952 and 1989. All the patients had been classified as having single or multiglandular disease based on the findings of the surgeon at the time of the operations. For this study, the cases of single gland disease were considered as adenomas and the cases of multigland disease as hyperplasias. One hundred and seventy-nine of the patients had hyperplasia, and of these 9 1 had adequate clinical follow-up and pathologic material to be included in the study. From the remaining 514 patients with single gland disease, a sample large enough to reflect a representative rate of recurrence was selected. Based on a published average recurrence rate of 2.5%,43 it was determined that a sample size of approximately 160 patients would be sufficient. Other than including only patients with a minimum of 4 years of follow-up, the group
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was randomly selected. After eliminating those patients with inadequate pathologic material, this group consisted of 130 patients. No patients with MEN syndromes or familial hyperparathyroidism were included. All patients had been operated on by one of two surgeons. A bilateral neck dissection, identification and biopsy of all parathyroid glands, and removal of only grossly enlarged glands was the surgical approach. An estimated weight of 40 mg or greater was used as the criterion for enlargement. Follow-up studies of the patients in both groups included clinical histories, documentation of medications, calcium or vitamin D supplementations, and the determinations of serum levels of calcium, albumin, creatinine, urea, and parathyroid hormone (intact hormone) in two blood samples. All biopsies and intact glands removed from the two groups were examined by two pathologists without knowledge of the operative findings, weights of the glands, gross findings, or follow-up information. The review was limited only to sections stained with hematoxylin and eosin. The presence or absence of the following microscopic features were recorded: lobularity, microscopic cellularity, cell types, stromal fat, a normal rim, transition from normal to abnormal tissue when a “normal rim” was present, a capsule, thick-walled blood vessels, nuclear pleomorphism, mitotic activity, and bloodllymphatic vascular space involvement. Each gland was categorized as adenoma or hyperplasia based on the criteria of Ghandur-Mnaymneh and Kimura,2 i.e., a gland is considered hyperplastic if any one of the following criteria are met: fat cells in the mass, lobular architecture or a transition of the abnormal tissue in the mass to a “normal rim”. After independent review by both pathologists, a consensus was reached in each case when there were disagreements. If no consensus could be reached, often due to inadequate samples of tissue, the case was considered unclassifiable. Of the 130 cases grossly classified as adenomas (single gland diseases), 58 (44.6%) were histologically classified as adenomas, 48 (36.9%), as hyperplasias, 18 (13.8%), as unclassifiable, and 6 (4.7%) as normal glands. The weights of the adenomas ranged from 115 mg to 16.7 gm, with a mean weight of 1500 mg. The follow-up in this group ranged from 4 to 27 years (mean, 12.5 years). Only 2 patients (1.5%) had postoperative hypercalcemia. One patient had a single enlarged gland removed that was interpreted as focal hyperplasia, and the other had one enlarged gland and one normal-sized gland removed, both of which were interpreted as examples of hyperplasia. Two hundred and six enlarged parathyroid glands were examined in the 91 patients classified by the surgeons as hyperplasias (multigland disease). Eightyfour (40.4%)were histologically classified as hyperplasias, 70 (33.9%) as normal glands, 30 (14.6%) as unclassifiable, and 22 (10.6%) as adenomas. The followup period for this group ranged from 4 to 26 years (mean 13.8 years). Six patients (6.5%) had persistent disease. In 4 of these 6 patients, the glands were classified as hyperplastic, and in 2 the glands were considered to be normal. Biopsies of glands, which were considered normal by the surgeon, taken from the cases of adenomas and hyperplasias, were combined and analyzed as a group. There were 339 glands in this group; 316 (93.2%) were classified as
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histologically normal, 2 (0.6%) as adenomas, 10 (2.9%) as hyperplasias, and 11 (3.2%) as unclassifiable. As demonstrated by these data, the microscopic classification of abnormal parathyroid glands as hyperplasias (multigland disease) or adenomas (single gland disease) correlates poorly with the gross/clinical classification of the disease. Our results are similar to those of Ghandur-Mnaymneh and Kimura2 in that many, nearly 50%, of our cases of single gland disease were classified as examples of hyperplasia, cases that by traditional criteria would have been considered adenomas. We agree with these authors that stricter definitions of adenoma and hyperplasia are appropriate, and when applied, result in many, and perhaps most, cases of adenoma being reclassified as examples of focal hyperplasia. While the results of our analysis show poor correlation between the clinical/ gross classification of abnormal glands and the histologic classification, the correlation is better when glands were grossly considered to be of normal size. The histologic diagnosis of “normal” predicted the gross/surgical identification of a normal gland with a sensitivity of 80% and a specificity of 87%. No significant difference was noted between the normal glands derived from cases of single and multiple gland disease. Our results show that the pathologist can distinguish normal from abnormal parathyroid glands with a fair degree of accuracy. We feel that this distinction should be the primary aim of microscopic examination of glands at the time of examination of frozen and permanent sections. The distinction of adenoma from hyperplasia (single vs . multigland disease) is essentially impossible on histologic grounds. If one selects very strict criteria for the diagnosis of adenomas, then they account for a much smaller percentage of cases of primary hyperparathyroidism than previously reported, and the number of cases of focal hyperplasia significantly increase. Whether the concept of focal hy perplasia has clinical relevance or not, as suggested by Ghandur-Mnaymneh and Kimura, is a more difficult issue to resolve. The surgical approach to primary hyperparathyroidism with reference to some of these concepts and our data will be discussed. Suffice it to say that in our series the recurrence/persistence rate was low, much lower than that in the Ghandur-Mnaymneh series (51%). As a result, we were unable to demonstrate that reclassification of many cases of single gland disease as focal hyperplasias predicted recurrences. While both of our patients with single gland disease who had recurrences were classified as focal hyperplasia, the vast majority of patients with this diagnosis never did.
D. Assessment of Clonality in Primary Hyperparathyroidism Recently, a number of different molecular biologic techniques have been applied to the task of defining the etiology of primary hyperparathyroidism. Fialkow et a1.44analyzed isoenzyme patterns in parathyroid “adenomas” (single gland disease) from three women heterozygous for the enzyme glucose-6-phos-
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phate dehydrogenase. Because of the phenomenon of random X-chromosome inactivation in women, heterozygous women will have an equal population of both type A and type B isoenzymes in normal tissues. A clonal growth (adenoma) would be expected to manifest only a single isoenzyme pattern. In contrast, a growth that is multicellular in origin (hyperplasia) would be expected to show both type A and type B isoenzyme patterns. In this study, no major differences were found between normal tissues and the abnormal parathyroid glands in these three patients. This finding would seem to support the concept of a multicellular (hyperplastic) origin for these cases. The fact that in two of these three cases only one gland was enlarged supports the concept of focal hyperplasia. However, as emphasized by these authors, these findings do not completely rule out the possibility of a clonal neoplasm. The latter, if mixed with a sufficient number of normal cells, may be undetected. In addition, although unlikely, activity of both X-chromosomes could result in the detection of both isoenzymes, although a hybrid molecule of both type A and type B enzymes would be expected, and none was detected. A subsequent study by Arnold et al.45appears to contradict that of Fialkow. Two techniques were used, both employing DNA probes, to study parathyroid “adenomas” from eight patients with primary hyperparathyroidism. All of the patients had single enlarged glands and biopsies taken of one or more normal or atrophic glands. All were cured by removal of the single enlarged gland. The first method used heterozygosity for a DNA polymorphism of the X-linked gene, hypoxanthine phosphoribosyltransferase (HPRT). In six of the eight specimens studied, evidence for clonality was found. In the other two, the findings were equivocal. Of five hyperplastic glands studied, defined by clinicopathologic criteria, all lacked evidence of clonality. In addition, in two of the eight cases studied by the above method, a tumor cell-specific restriction-fragment-length polymorphism (RFLP) involving the parathyroid hormone (PTH) gene was discovered. This permitted the assessment of clonality using a different method that employed a DNA probe for the PTH gene. In both samples, evidence for clonality was present, confirming the results obtained from using the first method. The abnormality in the PTH gene used to detect clonality raises the question whether abnormalities in this gene are important in the genesis of some forms of hyperparathyroidism. Similar recent studies from patients with the MEN-I syndrome have helped to shed light on the pathogenesis of at least some cases of hyperparathyroidism. Thakker et al.46used DNA probes for 18 different chromosome 11 gene sequences to demonstrate a loss of heterozygosity in the parathyroid tissue from 3 of 6 patients with the MEN-I syndrome. Their findings demonstrate that, in at least some patients with this syndrome, monoclonal tumors develop. In addition, their findings demonstrate that allelic deletions occur in these tumors. These authors also conducted linkage studies in two of the affected MEN-I families using a RFLP identified with a probe for the INT2(SS6) chromosome 11 gene. Strong linkage was found with the INT2 ~ncogene,~’ a proto-oncogene that encodes a
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protein of the fibroblast growth factor family. This finding is particularly intriguing in view of the known presence of a parathyroid mitogenic factor in the This mitogenic factor has plasma of some patients with the MEN-I been noted to have properties similar to fibroblast growth factors.49Arnold et al.’O have also demonstrated rearrangements between the PTH gene and the 1lq13 region, the region that contains INT2, in some nonfamilial parathyroid adenomas. In a study similar to that of Thakker, Friedman et al.51demonstrated monoclonality and allelic loss from chromosome 11 in 10 of 16 parathyroid tumors from patients with the MEN-I syndrome. They were also able to demonstrate similar allelic losses in 9 of 34 sporadic adenomas, and in 7 of the 9, the loss included the apparent MEN-I locus. Interestingly, in the patients with MEN, the monoclonal parathyroid tumors were substantially larger. This has led to the suggestion that parathyroid tumors in both familial and nonfamilial hyperparathyroidism may involve an initial hyperplastic phase, perhaps related to a first mutagenic event, followed by a second monoclonal phase of growth, stimulated by a second mutagenic event.46
II. FLOW CYTOMETRIC NUCLEAR DNA ANALYSIS The inability to discriminate reliably between primary parathyroid hyperplasia and parathyroid adenomas at the microscopic level, has led to a quest for objective means to make this differentiation. Flow cytometric DNA analysis has been used for this purpose. Little emphasis will be placed on methods and instrumentation in this discussion. For more detailed descriptions on methodology and instrur n e n t a t i ~ n , the ~ ~ .reader ~ ~ is referred to more detailed reviews on the subject. The format in this section will be to present data from our study of the patients described in the previous section and discuss the results in conjunction with those reported in the literature.
A. Methods and Materials The methods and materials in our study are described briefly. Our material included normal-sized parathyroid glands taken from patients with parathyroid disease and enlarged glands from patients with single and multigland disease that were embedded in paraffin. The specimens were stained with propidium iodide and analyzed on an Epics-C flow cytometer (Coulter Corporation, Hialeah, Florida). All histograms were analyzed with the VerityIModfit cell cycle analysis program (Verity Software House, Inc., Topsham, Maine). The data collected included the DNA index, %S and %G,M of the cell cycle and total events. DNA index is defined by the ratio of the position of the DNA aneuploid GoGIpopulation of cells in a specimen to the position of the G,G, cells of a normal population of cells. Position is the mean channel number of a modeled Gaussian peak. DNA
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diploid populations of cells had DNA indices between 0.9 and 1.1 and DNA tetraploid populations of cells had greater than 20% of the total number of cells at the 4C region and a DI between 1.9 and 2.1. The remainder of the histograms were in the DNA aneuploid category. Because of the arbitrary limits utilized to classify DNA tetraploid histograms and the current lack of standardization for this value, we have found it prudent to adopt %4C as another means of classifying ploidy. This appears to be a more practical method for comparisons of the different studies. We define %4C as the percentage of cells in the 4C region of the histogram. B. Compilation of Data Our review of the literature includes only those reports in which flow cytometry was used to examine specimens of parathyroid glands. The definitions of normal glands and of primary parathyroid hyperplasia, the methods of data analysis, and the data acquired in the different studies vary considerably. To facilitate analyses and comparisons, our data and that taken from the literature are tabulated in Table 1. 1. Description of Table and Definitions of Terms Each study in the table is identified by the first author. The number of patients, the total number of parathyroid glands examined, and the number and percentage of the total analyzed in each category are noted. The mean percentage of cells at the 4C region is indicated by %4C. This is to contrast it from DNA tetraploidy, which is defined by the presence of a population of cells in the same region that exceeds a previously defined upper limit. These limits varied from 10% to 20%.42*5”56 %Tet indicates the percentage of glands that showed DNA tetraploidy. The percentage of the total number of glands that contain aneuploid populations of cells is denoted by %Aneup and the average percentage of all cells in S-phase by %Total S. There are four categories in the table. “Normal” includes the normal parathyroid glands taken from patients without parathyroid disease. “Normal-sized’ ’ includes normal-sized glands derived from patients with parathyroid disease. “Hyperplasia (two or more enlarged glands)” appears to be self-explanatory; however, the definitions of hyperplasia vary. Some authors require microscopic confirmation of the diagnosis. Others define it by the number of enlarged glands that are grossly identified by the surgeon, but this number can also differ. ‘‘Adenomas (single gland enlargement)’’ is universally defined by the presence of a single enlarged gland. Carcinomas are not included in this study, but they are major parts of the studies of Harlow et al., Bowlby et al., and Shenton et a1.54-56
10
Hyperplasia (two or more enlarged glands)
Normal-sized glands from patients with hyperparathyroidism
Normal glands without parathyroid disease
Category
TABLE 1 Summary of Data Ref.
57 59 54 60 55 56 42 61 57 59 54 60 55 56 42 61 57 59 54 60 55 56 42
Study lrvin A lrvin B Harlow Rosen Bowlby Shenton Bonjer Joensuu lrvin A lrvin B Harlow Rosen Bowlby Shenton Bonjer Joensuu lrvin A lrvin B Harlow Rosen Bowlby Shenton Bonjer
54 54 41 26 12 76
54 54 41 26 12 76
54 41 26 12 76
No. patients
78 26 24 76 60 187 54 1 53 78 26 24 76 60 187 54 153 78 26 24 76 60 187
1 53
Total glands
5.06
21 13
49
5 10 92
2.00 18.00 7.00 9.96
5 14 33 13 32
8 10 59
11
8
6.90
0.38 7.48 31 38
12.00
35
21 48 30
5.06
0.18
%4Cb
54 8
11
%
14 2
17
No
0 17
2
0 3
0
0
0
3.48
21
1.89
14.6
1.2 14.6
YO Total S.
YO Aneupd
20
0
1
0
0
0
0
0
YoTets
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*
a
Ref.
61 57 59 54 60 55 56 42 61
Study Joensuu lrvin A lrvin B Harlow Rosen Bowlby Shenton Bonjer Joensuu
54
54 54 41 26 12 76
No. patients
%Tet Yo of glands with DNA tetraploidy. %Aneup, % of glands with DNA aneuploidy. %Total S, Mean Yo of total cells in S phase.
%4C, Mean Yo of cells at the 4C region of histograms.
N, Number of glands in each category.
Adenomas (single gland enlargement)
Category
TABLE 1 (continued) Summary of Data
54 153 78 26 24 76 60 187 54
Total glands
80 37 12 9 56 39 36 54
N=
52 47 46 38 74 65 19 1 00
%
30.00 10.02
7.00
3.30 18.00
%4Cb
21 50 3 9
8
0
%Tetc
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YO
5 0 22 26
0 15 25
Aneupd
%
3.64
21
1.5
Total So
C. Analysis and Comparison of Data
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Table 1 is the key to all of the analysis and comparisons of the data that follow in this section. A comment is required on the data contained in the first report of Irvin et al.57 (Irvin A) because the figures in the %4C category are geometric means and aggregate adjusted and not comparable to those taken from the other reports.58 1. Normal Parathyroid Glands from Patients Without Parathyroid Disease The definition of normality of glands differed in all of the studies listed. Irvin and B a g ~ e l l Irvin , ~ ~ et al.,59 and Rosen and MusclowM)required microscopic confirmation. Harlow et al.54define normal glands as those taken from patients without evidence of any abnormalities in calcium metabolism. Bowlby et al.55 defined normality by the weight of the glands, using age-related standards. The upper limit of normal in their study appears to be 60 mg. Shenton et al.56used an upper limit of normal for weight of 30 mg. Our upper limit of normal for weight was 40 mg.42 None of the figures for %4Cis comparable. Irvin and BagweIIs7report a low figure. The data of Rosen and MusclowWand Shenton et al.56 are estimates, being the sums of %S and %G,M. The figure for Shenton et al.56was calculated because their report only includes the mean percentages for G,G,. No tetraploidy or aneuploidy was reported in this group. The %S reported by Harlow et al.54is the only acceptable figure, because that of Rosen and MusclowM)is an estimate. 2. Normal-Sized Parathyroid Glands from Patients
with Parathyroid Disease The results of both studies of Irvin and c o - w o r k e r ~ deserve ~ ~ . ~ ~ comment, because they were able to correlate them with a relatively high rate of recurrence and persistent parathyroid hyperfunction postoperatively. The figure of 0.38% 4C from the first study (Irvin A) overlaps with those reported for normal glands, hyperplastic glands, and adenomas. Utilizing a computer-derived statistical analysis, they found that 14 glands (29%) taken from patients with adenomas had a greater than 50% chance of being abnormal. In the second study (Irvin B),59the %4C for normal glands was 6%. The figure listed is a mean of the %4C of all the normal-sized glands. Irvin et al. divided this group of glands into two parts. One group, consisting of 13 glands from 7 patients, had %4Cs that were greater than 6%. All of these patients had elevated levels of serum PTH postoperatively. The mean %4Cfor this group was 9.8%.One of these glands showed aneuploidy.
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The second group consisted of 17 glands from 10 patients without increased serum levels of PTH postoperatively. Four glands had %4Cs greater than 6%. The mean %4C was 6.3%. Problems are again encountered in comparing the results in this group. The %4C reported by Irvin et al. (Irvin B)59and Bonjer et al.42are comparable and appear to coincide. The figure for Rosen and MusclowM)is our estimate. One example of DNA tetraploidy is reported and is from our This is a biopsy of a normal-sized gland from a patient who had had two enlarged glands removed and who later developed hypercalcemia. The DNA index for this gland was 2.05. One DNA aneuploid gland was reported by Irvin et al.59and the gland was associated with elevated levels of serum PTH postoperatively. The only acceptable figure for %S phase is that reported by Bonjer et al.42 The other is an estimate.60
3. Primary Parathyroid Hyperplasia Hyperplasia is defined differently in the various studies. Harlow et al.54and Shenton et al.56 included only secondarily hyperplastic glands in their study. Rosen and Musclowmand Irvin and Bagwe1P7required microscopic confirmation. Bowlby et al.55 defined primary thyperplasia by the gross identification of 3 enlarged glands, each weighing more than 60 mg. Bonjer et al.42defined primary hyperplasia by the gross identification of two or more enlarged glands, each weighing more than 40 mg. The only acceptable figure for %4C is that of Bonjer et al.42The others are all estimates, including that of Irvin et al. (Irvin B).59They reported the average %4C for all enlarged glands and did not separate them into hyperplastic and adenomatous glands. Bowlby et al.55report a relatively high incidence of DNA tetraploidy; however, this includes only 2 of 10 glands. Bonjer et al.42record one gland with a tetraploid population of cells. The DNA index for this gland was 2.07. The gland was from a patient with three enlarged glands, one of which contained an aneuploid population of cells. The presence of DNA aneuploidy in one gland by Irvin et al.59is confirmed by the finding of DNA aneuploidy in ten hyperplastic glands by Bonjer et al.42 Irvin et al.59 were, to the best of our knowledge, the first to report DNA aneuploidy in a normal-sized gland from a patient with parathyroid disease and in one hyperplastic gland. Results such as these have limited the usefulness of DNA aneuploidy in differentiating non-neoplastic lesions of the parathyroid glands from parathyroid adenomas and carcinomas.
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4. Parathyroid Adenomas The definition of a parathyroid adenoma is universally accepted as the presence of one enlarged parathyroid gland. The only acceptable value for %4C is that of Bonjer et al.42The others are not comparable. all estimates or, in the case of Irvin et al. (Irvin A),55-57*59@' For the first time, the incidence of DNA tetraploidy is nearly completely recorded. However, there are defects. The figure recorded for Shenton et al."j is an estimate and reflects a statement in their article that over 50% of adenomas showed abnormal cell cycle patterns with high proportions (over 20%) of cells within the tetraploid region. Bowlby et al.55 also reported a high incidence of DNA tetraploidy, while Bonjer et aL4, report only one gland. The percentages of glands with DNA aneuploidy are reported in nearly all of the studies. They vary, but in 4 of 5 s t u d i e ~ , ~ the ~ , figures ~ ~ , ~are ~ , high ~ ~ but comparable. The only acceptable figures for %S are those reported by Harlow et al.54and Bonjer et al.42 D. Conclusions The core of this section of our review was to determine whether flow cytometric nuclear D N A analysis can discriminate between primary parathyroid hyperplasia and parathyroid adenomas. The analysis of our data showed the following A chi-square test was used to compare the incidences of DNA diploid with DNA aneuploid populations of cells in normal-sized parathyroid glands, hyperplastic glands (multigland disease), and adenomas (single gland disease). The differences were statistically significant when comparing normal-sized glands with hyperplastic @
