Primary hyperparathyroidism (PHPT) refers to the inappropriate or unregulated overproduction of parathyroid hormone (PTH) leading to abnormality in calcium and phosphorus metabolism. High levels of PTH lead to increased renal resorption of calcium and excretion of phosphorus, indirect increased intestinal calcium absorption through increased synthesis of 1,25(OH)2D3, and increased resorption of bone.1 Consistent with the above mechanism, patients with PHPT have evidence of hypercalcemia, hypophosphatemia, hypercalciuria, and increased levels of PTH. Pathologically, in the USA and most Western European countries adenomas account for 80%-85% of all cases of PHPT, hyperplasias account for 10%-15%, and carcinomas account for less than 1%,2,3 while in China, carcinomas account for about 3%-6%, which is higher than in western countries.4 Most cases of PHPT can be cured by parathyroidectomy; however, a few patients do not improve or experience recurrence after surgery. Parathyroid carcinoma is an uncommon but potentially life-threatening form of PHPT;5 therefore, it is important to discriminate parathyroid carcinoma from other pathological subtypes to achieve proper treatment and prognosis. However, parathyroid carcinomas often present a diagnostic challenge especially between a carcinoma in the absence of an invasive growth characteristic and a highly active adenoma.3
A definitive histopathological diagnosis should be restricted to lesions showing vascular invasion, perineural space invasion, capsular penetration with growth into adjacent structures, and/or documented metastases.3 However, it is widely recognized that making the diagnosis of parathyroid carcinoma (PC) is often difficult because of the overlap of characteristics between PC and parathyroid adenoma (PA), especially at an early stage. For instance, fibrous bands are frequently present in PC; however, fibrous bands are not specific for carcinomas and may also be seen in large PAs, hyperplasias, and patients with secondary hyperparathyroidism that infarct or hemorrhage causing scar-like fibrous bands. Mitotic activity is present in approximately 80% of PCs, however, mitotic figures are also relatively common in PA and parathyroid hyperplasia (PH).6 Therefore, it is often difficult to identify malignant from benign growths before or at the first surgery. As such, the diagnosis of carcinoma is often retrospectively made after relapse when treatment options are limited.
Recent advances in identifying the molecular pathogenesis of parathyroid cancer could improve the evaluation and treatment of these patients. The development of a tumor is a process in which the balance of cell proliferation and apoptosis is altered. Some genes expressed in tumor tissues involved in the regulation of the cell cycle may be related to malignant transformation. In the search for a marker of parathyroid malignancy, analysis of DNA content and immunohistochemical expression of Rb, bcl-2, cyclin D1, p21, p27, and p53 have been evaluated, but differential expression of these proteins were not found to be specific to parathyroid cancers.7–10 Ki-67 is expressed in all phases of the cell cycle except G0, and it can be used to evaluate the biologic behavior and proliferation activity of cells. The overexpression of Ki-67 has been observed in many malignant tumors, and it has also been shown that Ki-67 overexpression is more common in PA or PH than in normal parathyroid tissues.11 Ki-67 overexpression has also been observed more often in PC than in PA and PH.12 The fragile histidine triad (FHIT) gene, a tumor suppressor gene located on 3p14.2, regulates the cell cycle and apoptosis, cell cytoclasis and reparation, and malignant transformation. Inactivation of the FHIT gene has also been confirmed in many human cancers, including pulmonary carcinoma. Recently, Thomopoulou et al13 demonstrated reduced FHIT protein expression in two PC tissues. Galectin-3 (Gal-3) is a member of the carbohydrate-binding protein family known as lectins. It plays a role in cell-cell and cell-matrix interactions, cell growth, cell-cycle regulation, apoptosis, cell damage and repair processes, neoplastic transformation, and metastasis.14,15 In recent years, several studies have documented overexpression of Gal-3 in different human tumors, including large-cell lymphoma, colorectal carcinoma, breast carcinoma, hepatocellular carcinoma, brain tumors, melanoma, and thyroid carcinoma.13 Some studies have also shown that overexpression of Gal-3 may be used as a marker for identifying malignant and benign parathyroid tumors.12,16 The HRPT2 gene is the pathogenic gene for hyperparathyroidism-jaw tumor syndrome (HPT-JT), characterized by benign and malignant PHPT in combination with tumors of the jaw, kidney, and uterus.17,18 The HRPT2 gene encodes a protein named as parafibromin, which works as a tumor suppressor.19–23 Parafibromin downregulates cyclin D1 expression during transcription24 and regulates transcriptional elongation and histone modification.17,19,20HRPT2 somatic mutations have been reported in 15%-100% of PCs in recent years, and up to 70% of apparently sporadic parathyroid carcinomas carry an HRPT2 mutation.25,26
In this study, we analyzed a series of sporadic PHPT patients in an effort to investigate the different immunohistochemical expressions of Ki-67, galectin-3, FHIT, and parafibromin in parathyroid carcinoma, adenoma, hyperplasia, and normal tissues. We also assessed these expression values for use in the differential diagnosis of parathyroid tumors.
Patients and specimens
For our analysis, we retrospectively obtained 42 surgical specimens of parathyroid lesions (15 parathyroid carcinomas, 19 parathyroid adenomas, and 8 hyperplasic parathyroid glands) from patients diagnosed and treated at Peking Union Medical College Hospital (Beijing, China) from January 1992 to 2005. The diagnosis of parathyroid carcinoma was confirmed in all cases by reviewing the original histology according to histopathologic and clinical criteria. The diagnosis of parathyroid adenoma was confirmed by the presence of a thinly encapsulated single hypercellular parathyroid gland, mainly composed of chief cells, with or without a compressed rim of normal parathyroid tissue. Parathyroid hyperplasia was defined as all parathyroid glands having primary chief-cell hyperplasia. All patients did not have family history of hyperparathyroidism.
Among the 15 patients with PC, the male-female ratio was 11/5, the mean age was (46.8±14.8) years, and the mean course of the disease was (5.1±5.0) years (1 month-14 years). Local or distant metastasis was found in five cases, and ten cases were in situ (two thyroid invasions, two striated muscle invasion, one peripheral fibro-fatty tissue invasion, and five capsular invasions). Six normal parathyroid tissues were inadvertently obtained from excision of thyroid cancers, all of them had no evidence of hyperparathyroidism or changes in pathology. Institutional approval was obtained from the local ethics committee of Peking Union Medical College Hospital before the study began.
Experiments were performed as previously described to investigate the expression of Ki-67, Gal-3, FHIT, and parafibromin in different tumors by immunohistochemistry,8,13,15,27 except that we did not use the amplyfing step. Briefly, serial sections from formalin-fixed, paraffin-embedded tissues were collected onto poly-L-lysine-coated slides, and 4-μm sections were used for immunostaining. Archival sections were deparaffinized in xylene and rehydrated in alcohol. Endogenous peroxide activity was blocked by incubating the slides in 3% hydrogen peroxide in dehydrated alcohol for 10 minutes. After a heat-induced antigen retrieval procedure in 10 mmol/L citrate buffer (pH=6.0) (Ki-67, Gal-3, FHIT for 15 minutes, parafibromin for 30 minutes), in order to unmask the antigen, slides were cooled at room temperature for 30 minutes and then blocked against nonspecific staining with 1% fetal bovine serum. Primary antibodies against parafibromin, Gal-3, Ki-67, or FHIT were incubated with the specimens for 1 hour at 37°C. Antibody characteristics, dilutions, and protein cellular localization are summarized in Table 1. The slides were then washed with phosphate buffer saline (PBS) (pH 7.2-7.4) and incubated with horseradish peroxidase-labeled secondary antibody for 1 hour at 37°C. Liquid 3,3′ diaminobenzidine (DAB)+ chromogen was added and incubated for another 6 minutes. Finally, sections were counterstained with hematoxylin, dehydrated, and mounted. The positive control for parafibromin and PHIT staining was normal parathyroid tissue and for Ki-67 and Galectin-3 it was breast carcinoma tissue overexpressing Ki-67 and Galectin-3. Omission of the primary antibody and replacement with 0.01 mmol/L PBS was included as a negative control.
Tumors were scored as positive if specific staining was detected, and staining was quantified according to the percentage of positive cells independent of the intensity of staining. We determined the degree of expression for the different proteins according to the references and receiver operating characteristic (ROC) curves of the data: (1) The cutoff value for Ki-67 to define low expression and high expression was 5%. (2) Gal-3 was scored using the following semiquantitative scale: 0%-24% of neoplastic cells positive was scored as (-), 25%-49% positive was scored as (+), 50%-74% positive was scored as (++), and 75%-100% positive was scored as (+++). (3) FHIT was scored as negative when no tumor cells showed any specific staining, and all others were scored as positive. (4) For parafibromin, no reactivity was scored as (-), percentage of positive cells <50% as (+), percentage of positive cells 50%-95% as (++), and percentage of positive cells >95% as (+++). Each section was evaluated by two pathologists without knowledge of the diagnosis or outcome. Images were acquired using an Olympus Bx51 microcope (Olympus Corp., Japan) with a mechanical stage, fitted with a Olympus u-CMAD3 videocamera (Olympus Corp); the latter was connected to a Pentium II computer located with the appropriate image analysis software. Slides were examined at high power magnification (original magnification ×200).
The statistical software package SPSS for Windows version 10.0 (SPSS, Chicago, IL, USA) was used to analyze the data. Sensitivity, specificity, predictive values, and 95% confidence intervals (CI) for proportions were calculated using standard methods for binomial distribution. Analysis of variance (ANOVA), χ2 test, Fisher's exact test, and the Mann-Whitney test were used as appropriate. Differences were considered statistically significant at P <0.05.
The characteristics of PC, PA, and PH are summarized in Table 2. Male patients were more prevalent among the PC samples than the PA/PH samples (P <0.05). Serum calcium and 24-hour urine calcium levels were significantly higher in PC than in PA and PH (P <0.05). The ratio of hypercalcemia crisis was higher in PC than in PA and PH (P <0.001), but the range of serum calcium levels overlapped among the different subtypes. Representative areas of the different parathyroid tumors and the staining of markers are shown in Figure 1. Among the patients with PC followed-up for (6.6±5.1) years (ranged from 0.25-14 years), five patients developed local or distal metastasis (two metastasis nodules in regional subcutaneous tissue, and two local recurrence and metastasis). The first recurrence of hypercalciemia was detected in (3.4±2.2) years (1.0-7.0 years) after the first surgery. Two patients died of intractable hypercalcemic crisis 10 and 14 years after the first parathyroidectomy, respectively. Patients with PA or PH had undergone follow-up for at least three years, and no evidence of recurrence was found.
Specific nuclear Ki-67 overexpression was observed mostly in PC. The Ki-67 proliferative index was high in 27% of PCs, but only in 3.7% of the benign samples (P <0.001) (Table 3). Overexpression of Ki-67 was not detected in any of the normal tissues or those with hyperplasia. Ki-67 overexpression in PC was observed in 1 of 5 metastatic tumors and 3 of 10 in situ PCs. Almost all adenomas demonstrated a low Ki-67 proliferative index (Table 4). As a marker for discriminating PC from benign tumors, Ki-67's positive predictive value (PPV) was 80%, its sensitivity was 27%, and its specificity was 96%.
The expression of Gal-3 was significantly different in PC compared with the other groups of parathyroid specimens (P <0.001; Table 4). Eleven of fifteen (73%) PC specimens stained positive (+ to +++), whereas only 6/27 (22%) benign specimens stained positive (Table 3). In most PCs, staining was strong and diffuse. Positive staining for Gal-3 was a useful marker for distinguishing PC from other parathyroid tumors as its PPV, sensitivity, and specificity were 65%, 73%, and 78%, respectively.
Nuclear parafibromin expression was observed in all specimens of normal parathyroid tissue and hyperplasia and most of the adenoma specimens (Table 4). One NP and one PA slice were excluded due to technical issues. Only one (6%) specimen of adenoma demonstrated complete loss of parafibromin expression. Nine of fifteen (60%) PC specimens demonstrated complete loss of parafibromin expression, and no carcinoma stained strongly. Complete loss of parafibromin expression was shown to be another useful marker for distinguishing PCs from other parathyroid tumors, as the PPV, sensitivity, and specificity were 90%, 60%, and 96%, respectively. Coincidentally, the only parathyroid adenoma that tested completely negative for parafibromin also showed a high Ki-67 proliferative index, which reached 18% (data not shown). As such, this patient needs to be followed closely.
FHIT expression was homogeneous among the different parathyroid tumor groups (Table 4). No differences were observed for any of the group comparisons (P=0.536)
Sensitivity, specificity, PPV, and negative predictive value for distinguishing parathyroid carcinoma from benign parathyroid tumors using both single and combined markers are shown in Table 5. The results indicated that both parafibromin and Ki-67 had relatively better specificity and PPV but a lower sensitivity for differentiating malignant parathyroid tumors from benign tumors. The specificity and sensitivity of Gal-3 were relatively higher, but the PPV of Gal-3 was only 65%. The combination of Ki-67 overexpression or loss of parafibromin improved the sensitivity compared to each marker alone, without the decrease in specificity and PPV associated with parafibromin alone. The combination of overexpression of Gal-3 or loss of parafibromin increased sensitivity to 87%. However, the specificity of simultaneous negative parafibromin and positive Gal-3 scoring could reach 100%. The combination of overexpression of Gal-3 or Ki-67 also showed improved sensitivity compared to each marker alone, with a decrease in specificity and PPV. However, the specificity of both positive Gal-3 and positive Ki-67 could reach 100%. The combination of all three markers produced similar results as the combination of Gal-3 and parafibromin.
To date, the study of differential diagnosis of PCs from benign tumors is still lacking. Here we summarized the clinical data of PCs in Peking Union Medical College Hospital and have analyzed the immunohistochemical results among different groups of parathyroid tumors in an effort to provide serviceable diagnostic clues at early stages of tumor development. In the current study, the expression of Ki-67, Gal-3, FHIT, and parafibromin was determined by immunohistochemistry, and we found that loss of parafibromin expression and overexpression of Ki-67 and Galectin-3 may distinguish PCs from other parathyroid tumors.
In our study, Ki-67 expression differed in PCs with low expression in 73.3% of the specimens. The overexpression of Ki-67 could be found almost exclusively in PCs, while only 1 of 19 benign tumors demonstrated overexpression of Ki-67. Data from previous studies showed that the sensitivity and specificity of Ki-67 overexpression was 40%-60% and 93.3%-100%, respectively.12,28,29 These results differ from our observations, and this may be due to the sample size and cutoff chosen in each study. Fernandez-Ranvier et al12 recently reported that the sensitivity of Ki-67 expression (cutoff >6%) was only 20%, which is similar to the results reported here, and they suggest that the index of Ki-67 overexpression has a high specificity (95.9%) and sensitivity (80%) for metastatic PCs. However, we did not find this relationship between Ki-67 expression and metastatic PC.
Gal-3 was overexpressed in more than 70% of PCs and about 20% of benign tumors. The sensitivity and specificity for PC diagnoses using a cutoff of 25% was 73.3% and 77.8%, respectively. While using a similar cutoff of 30%, in Bergero et al16 the sensitivity and specificity was shown to be 92.3% and 96.7%, respectively. Furthermore, Fernandez-Ranvier et al12 reported the sensitivity and specificity as 93.3% and 84%, respectively, when considering the specific stain as positive. These differences may be related to the different cutoffs used in each study, the analysis of the specific stain, and the sample size.
A higher prevalence of HRPT2 mutations has been shown in familial and sporadic PCs. In our study, complete loss of parafibromin expression was detected in 63% of PCs, and almost all of the benign tumors and normal parathyroid tissues stained positive for this protein. Compared to Ki-67, Gal-3, and FHIT, the differential expression of parafibromin demonstrated better sensitivity and specificity in identifying malignant from benign tumors. Our results were similar with that of Cetani et al27 and Fernandez-Ranvier et al,12 while the sensitivity of parafibromin measured in our study was not as high as that of Cetani et al (100%) and not as low as that of Fernandez-Ranvier et al (31.3%). These results indicated that a parathyroid tumor with complete deletion of parafibromin should be considered for possible malignancy. Interestingly, the only one PA tumor in this study showed complete loss of parafibromin, coincident with a high proliferative index of Ki-67 (18%) and almost a complete absence of Gal-3 (3%) staining. Although no clinical or pathologic evidence of malignancy was found, it is still necessary to follow this patient closely.
FHIT plays an important role in suppressing tumor growth, as its low expression has been observed in many malignant tumors. Reduced expression of FHIT had been reported in two cases of PC; however, we did not find any difference in its expression between benign and malignant parathyroid tumors in this study. Only one PC (in situ) showed complete loss of FHIT expression (Figure 1). As such, FHIT demonstrated little value for the differential diagnosis of PC in this group of patients.
Our study demonstrated that a single molecular marker for discriminating malignant tumors from benign parathyroid tumors might be inadequate (e.g., the low sensitivity of Ki-67 alone and rather low PPV of Gal-3). However, the combination of two or three markers might help to improve the sensitivity, specificity, and PPV. In this study, the combination of parafibromin (-)/Gal-3 (+) or Gal-3 (+)/Ki-67 (+) increased the sensitivity to more than 80%. Also, Gal-3 (+)/parafibromin (-) or Gal-3 (+)/Ki-67 (+) was found only in parathyroid carcinomas with a specificity and PPV of 100%. Therefore, we can select different combinations of these markers for different purpose. Since the combination of all three markers did not produce better results than any two of them, it is reasonable to select only two markers for differential diagnosis of parathyroid carcinoma in clinical practice. However, the semi-quantitative scoring of these markers used in the present study may attenuate the potential of our results. More quantitative studies with more recently discovered promising tumor markers (such as proliferating cell nuclear antigen,30 human telomerase reverse transcriptase) are needed.
In sum, it is important for the prognosis of patients with PC to identify malignant from benign tumors at an early stage. However, it is difficult to determine malignancy based on clinical manifestations, biochemical index, and/or tumor size. Our preliminary data suggested that immunohistochemistry of Ki-67, Galectin-3, and parafibromin may be useful for the differential diagnose of malignant and benign parathyroid tumors, and a combination of these markers may improve their diagnostic accuracy.
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