MIBI scintigraphy, indicators of cell proliferation and histology of parathyroid glands in uraemic patients

Melani R. Custódio1,4, Fábio Montenegro2, André F. P. Costa1, Luciene M. dos Reis1, Carlos A. Buchpiguel3, Sabrina G. Oliveira1, Irene L. Noronha1, Rosa M. A. Moysés1 and Vanda Jorgetti1

1 Nephrology Division, 2 Head and Neck Surgery and 3 Nuclear Medicine, University of São Paulo School of Medicine, São Paulo and 4 Nephrology Division, University of Uberlândia, Minas Gerais, Brazil

Correspondence and offprint requests to: Vanda Jorgetti, MD, PhD, Renal Physiopathology Laboratory (LIM-16), University of São Paulo School of Medicine, Av. Dr Arnaldo, 455-Sala 3342, São Paulo, SP 01246-903, Brazil. Email: vandajor{at}usp.br



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Although scintigraphy with 99mTc-sestamibi (MIBI) has been used to localize parathyroid glands prior to surgery for hyperparathyroidism, using it to evaluate parathyroid function remains controversial. The purpose of this study was to evaluate the possible association of MIBI uptake with gland weight, histological pattern and proliferative activity of parathyroid cells.

Methods. We studied 18 patients with secondary hyperparathyroidism (SHP); mean age 38±3 years, 55% female, mean time on haemodialysis 7.7±0.9 years. All patients had parathyroidectomy (PTx). The weights of the removed glands were estimated, and parathyroid hyperplasia was classified as diffuse (n = 28) or nodular (n = 29). The expression of proliferative cell nuclear antigen (PCNA) was evaluated by immunohistochemistry. Before PTx, all patients underwent MIBI evaluation and were categorized using a 0–3 uptake scoring system. Low uptake (scores of 0 and 1) was seen in 39 glands and high uptake (scores of 2 and 3) in 18.

Results. Estimated gland weights, percentage of nodular hyperplasia and PCNA expression were greater in glands with high MIBI scores than in those with low scores (P<0.01). In glands with nodular hyperplasia, PCNA expression was higher (318±66 cells/mm2) than in those with diffuse hyperplasia (104±16 cells/mm2; P<0.001).

Conclusions. High MIBI scores were associated with high estimated gland weight, degree of cell proliferation and presence of nodular hyperplasia. MIBI scintigraphy is useful in clinical practice for localizing parathyroid glands, and it could guide the management of SHP by indicating the degree of its severity.

Keywords: end-stage renal disease; hyperparathyroidism; MIBI; parathyroid glands; PCNA; 99mTc-sestamibi



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Secondary hyperparathyroidism (SHP) due to chronic renal failure is one of the serious complications in patients with end-stage renal disease (ESRD) [1]. A number of factors may contribute to SHP, such as hypocalcaemia, phosphate retention, vitamin D deficiency, and the reduction of vitamin D and calcium-sensing receptors [2]. These factors enable not yet fully understood mechanisms that lead initially to diffuse and polyclonal cell proliferation. Subsequently, in some glands, nodular growth occurs within diffuse hyperplastic tissue. These nodules present autonomous and monoclonal growth, most probably corresponding to the so-called ‘tertiary hyperparathyroidism’ [3].

Dual-phase 99mTc-sestamibi (MIBI) scintigraphy has been used in recent years to localize parathyroid glands. This method has proven particularly useful in pre-operative gland detection in patients with primary hyperparathyroidism and SPH, as well as before re-operation [4]. It was shown recently that MIBI can be used to evaluate parathyroid function, as well as to quantify patient response to treatment with calcitriol [5].

The proliferative activity of parathyroid cells has been assessed by immunostaining the proliferating cell nuclear antigen (PCNA), which is an auxiliary protein of DNA polymerase and is synthesized during the late G1 and S phases of the cell cycle [6]. The cellular expression of PCNA indicates that the cells are in the proliferative phase, therefore that the tissue is growing rapidly. In this study, we analysed the parathyroid glands of ESRD patients with refractory SHP who underwent parathyroidectomy (PTx) and compared their MIBI uptakes with glandular weight, histological findings and PCNA levels. We hypothesized that MIBI uptake might correlate with parathyroid weight, parathyroid histology or even cell proliferation.



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Study population
We studied 18 chronic haemodialysis (HD) patients (10 females and eight males). Their mean age was 38±3 years, and their mean time on dialysis was 7.7±0.9 years. All patients had refractory SHP. The decision to perform PTx was made based on one or more of the following features: bone deformity, pathological fractures, uncontrolled hyperphosphataemia, calciphylaxis and ectopic calcifications. None of the patients had been treated with vitamin D during the 6 months preceding the PTx. Each subject had total PTx, followed by a forearm autograft. The Ethics Committee of the University of São Paulo School of Medicine approved the study.

Biochemical analysis
Serum concentrations of calcium (normal range, 8.5–10.5 mg/dl) and phosphate (normal range, 2.3–4.6 mg/dl) and alkaline phosphatase activity (normal range, 60–170 UI/l) were measured using a Cobas-Integra® Auto-Analyzer (Roche Diagnostics, Basel, Switzerland). Serum intact parathyroid hormone (PTH; normal range, 8–76 pg/ml) was measured by the Immunoradiometric assay (CIS bio International, Gif-sur-Yvette, France).

99mTc-sestamibi (MIBI) parathyroid scintigraphy
Patients received 25 mCi of 99mtc-hexakis-2-methoxy-isobutylisonitrile (MIBI) intravenously, after which dual-phase radionuclide scintigraphy was performed. Images of the anterior neck were obtained using a high-resolution parallel-hole collimator, and images of the chest were obtained using a large-field-of-view collimator at 15 min and 3 h after the injection of MIBI. The initial images corresponded to the thyroid phase of the test and the later images to the parathyroid phase.

Two specialists blinded to the sources of the images interpreted the scans. A patient was classified as having abnormal parathyroid glands if one or more areas of increased MIBI uptake, characterized as a relative increase in intensity from the thyroid phase to the parathyroid phase, were observed in his or her neck or mediastinum. In addition, we performed a pilot study (unpublished observation) of the scintigraphy of 20 healthy individuals (all of whom had negative scans) and of 116 patients with renal osteodystrophy, and compared parathyroid gland uptake with that of the thyroid gland, which was used as a reference organ (data not shown). The scoring system used in the present study was based on that described by Torregrosa et al. [7]. Thus, areas of MIBI uptake by the parathyroids were scored on a 4-point scale, always comparing the degree of activity in the parathyroids with that of thyroid uptake: 0 = no uptake; 1 = faint uptake; 2 = moderate uptake; and 3 = marked uptake. Due to the small number of patients, the similarity of glands regarding their weight and MIBI's uptake intensity, represented by each score group, we decided to simplify the analysis of our MIBI scan results by grouping the glands into only two scintigraphic categories: those with low MIBI uptake (scores of 0 and 1) and those with high MIBI uptake (scores of 2 and 3).

Histological and parathyroid cell proliferation studies
Of the 72 glands removed, 57 were analysed. Each gland was measured with a sterile ruler and its dimensions recorded. The amount of tissue removed was estimated through the formula: weight (g) = 0.585 x RV + 0.134, where RV is the product of the three dimensions of the gland, expressed as cm3 [8]. The other 15 glands, probably the smallest, were not evaluated because most of the tissue was used for the autograft, and their remainders were considered insufficient for histological and histochemical analysis. All glands were fixed in a 20% formalin solution for 12–24 h, dehydrated, and embedded in paraffin. Subsequently, 4 µm thick sections were stained with haematoxylin and eosin for histopathological examination, and unstained sections were utilized for immunohistochemical studies.

The histological examinations of the glands with light microscopy were performed in a blinded fashion. Diffuse hyperplasia (DH) was defined as increased numbers of parenchymal cells with normal lobular structures, and nodular hyperplasia (NH) as at least one well circumscribed, encapsulated and virtually fat cell-free accumulation of parenchymal cells.

For immunohistochemical studies, the sections were deparaffinized and dehydrated in a graded series of ethanol, then they were treated for 30 min with methanol containing 0.3% H2O2 to block endogenous peroxidase activity. The sections were next treated for 30 min with horse serum to block non-specific background staining. This step was followed by incubation overnight at 4°C with monoclonal mouse anti-PCNA antibody (PC-10, DAKO, Copenhagen, Denmark) at a dilution of 1:100. After rinsing, the slides were incubated with biotinylated horse anti-mouse antiserum (Vector Co.) and then with an avidin–biotin complex with horseradish peroxidase (Vector Co.). Slides were developed with a freshly prepared 3-amino-9 ethylcarbazole chromogen (Sigma Chemical Co.) in the presence of 0.03% H2O2. Sections were counterstained with methyl green (Merck) and covered with Kaiser's glycerin–gelatin (Merck). Brown pigmentation in the nuclei of cells was interpreted as a positive reaction. Immunopositive cells were counted (positive cells/mm2) per 25 fields in DH and per 25 fields or less, considering only the nodular areas, in NH. Another four normal parathyroid glands, resected during thyroid surgery of patients without SHP, were used as normal controls.

Statistical analysis
All quantitative data, expressed as mean±SEM, were compared using an unpaired Student t-test. Linear correlation was evaluated by the Spearman correlation test. Fisher's exact test was applied to compare glandular weight, histological type and PCNA counts between the two MIBI uptake groups. Values of P<0.05 were considered significant.



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 Subjects and methods
 Results
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Biochemical analysis
The biochemical and clinical features of the patients are presented in Table 1.


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Table 1. Biochemical and clinical features of the patient population

 
Parathyroid scintigraphy
Pre-operatively, MIBI scanning localized parathyroid glands in 16 patients, showing a sensitivity of 88.8%. The MIBI uptake of one, two, three and four glands was observed in one, seven, five and three patients, respectively. In all, MIBI was capable of detecting 36 (50%) of the 72 glands. Low MIBI uptake was found in 68.5% of the glands, and high MIBI uptake in 31.5%.

Histological and parathyroid cell proliferation studies
Estimated parathyroid gland weights ranged from 0.15 to 16.1 g (mean, 1.71±0.32 g). Of the 57 glands evaluated, NH was identified in 29 (51%) and DH in 28 (49%). We performed a semi-quantitative analysis of the glands, according to cell types. In the glands with NH, oxyphil cells predominated significantly (15 glands with oxyphil cells, seven with chief cells and seven with both) when compared with the DH glands (15 with chief cells and 13 with both chief and oxyphil cells) (P<0.0001).

Table 2 shows glandular weights, percentages of NH and DH, PCNA expression and MIBI uptake. Glands with high MIBI uptakes had higher weights, higher prevalence of NH and greater cell proliferation, as determined by PCNA expression (P<0.01).


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Table 2. Gross and histological characteristics of parathyroid glands and PCNA expression in relation to MIBI uptake

 
There was a positive correlation between MIBI uptakes and glands weights (r = 0.38, P<0.01) when we considered all 57 glands. When we analysed glandular weight according to histological type and MIBI uptake, we observed that the NH glands with high MIBI uptakes weighed more than any of the other glands (Figure 1).



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Fig. 1. Parathyroid gland weights according to their histological patterns and MIBI uptake. NH glands with high uptake weighed more than the others glands (#P<0.01). NH = nodular hyperplasia; DH = diffuse hyperplasia.

 
PCNA expression was significantly higher in NH than in DH, as shown in Figure 2a (P<0.01). The analysis of PCNA expression according to histological type and MIBI uptake (Figure 2b) showed that PCNA expression in the low uptake glands was significantly higher in the NH glands than in the DH glands (P<0.001). We also found a tendency toward higher PCNA expression in high uptake NH glands than in high uptake DH glands (P = 0.07).



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Fig. 2. (A) PCNA expression was significantly higher in NH than DH (#P<0.01). (B) PCNA expression in relation to histological type and MIBI uptake. NH glands with low uptake presented a higher PCNA expression than the DH glands with low uptake (*P<0.001). NH glands with high uptake presented an almost significantly higher PCNA expression than the DH glands with high uptake (P = 0.07). PCNA = proliferating cell nuclear antigen; NH = nodular hyperplasia; DH = diffuse hyperplasia.

 


   Discussion
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 Subjects and methods
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A major complication of ESRD, SHP, results in significant morbidity and mortality. The hallmarks of SHP are enlargement of the parathyroid glands and excess secretion of PTH [9]. The main finding of our study was that higher MIBI scores were observed in larger, heavier NH glands with greater PCNA expression. To the best of our knowledge, this study was the first attempt to evaluate MIBI uptake, cell proliferation and histological findings in one group of patients with SHP.

Various techniques can be used to locate abnormal parathyroid glands in patients with SHP, although MIBI scintigraphy appears to be the most effective of them. When we compare MIBI and ultrasound, the latter has a relatively low cost; however, MIBI is more specific and useful in the cases of recurrent or persistent SHP [4,10]. MIBI is a lipophilic radiotracer that becomes concentrated in cells and inside the mitochondria through active transport and passive diffusion. In metabolically hyperactive cells, MIBI uptake varies, reflecting the numbers of mitochondria and the degree of cellular activity. In addition, slower MIBI washout has been observed in hyperactive parathyroid glands compared with normal thyroids and parathyroids [7]. In fact, Piga et al. [11] stated that MIBI scans do not simply reveal parathyroid enlargement but rather identify hyperfunctioning parathyroid glands in patients with SHP. Indeed, Ambrosoni et al. [12] confirmed this finding, affirming that in those patients who were to have total PTx, MIBI scans can facilitate the selection of the best parathyroid tissue to use for autografting [4]. Additional evidence that MIBI shows the functional status of parathyroids is found in the correlation between MIBI and intact PTH in SHP, recently demonstrated by Hung et al. [13]. We obtained similar results in a study of 116 patients, in whom we demonstrated a positive association between PTH levels and MIBI uptake (unpublished data). Moreover, Torregrosa et al. [10] showed a positive correlation between MIBI uptake in the parathyroid glands and the phases of the cell cycle. The same study found higher degrees of uptake to be correlated with the G2/S (active growth) phase.

In our present study, MIBI detected hyperplastic glands in 88.8% of the cases, a sensitivity similar to that found previously [10]. There are many important factors that need to be taken into account when analysing false-negative MIBI results. The expression of P-glycoprotein (Pgp) and of multi-drug resistance-associated protein has been implicated in the transport of MIBI across cell membranes, and some studies have described rapid MIBI washout in malignant cells with high levels of Pgp [14]. Therefore, in addition to gland size and activity, another possible factor that influences the localization of parathyroid glands by MIBI is significant Pgp expression in the gland [15]. The reason that some hyperplastic parathyroid glands are unable to concentrate MIBI has yet to be determined.

The different MIBI scores observed in our study may also be related to physiological variations in parathyroid function, to previous vitamin D therapy or to any of a number of other factors that can affect the rate of cellular metabolism. The reasons for such variations remain to be elucidated, but they could be related to the mechanism of parathyroid cell proliferation and the different phases of the cell cycles of glands [16]. It is well known that glands with nodular growth patterns are heavier and show higher proliferative activity than diffusely hyperplastic glands, and that both types of hyperplasia are often found in the glands of the same patient [3,17].

The association between MIBI score and the high prevalence of NH was expected, since we also found a higher prevalence of oxyphil cells in these glands. Our results are similar to those reported by Melloul et al. [18], who detected an association between the degree of MIBI uptake and the presence of oxyphil cells mostly in patients with primary hyperparathyroidism.

In the present study, we show that glandular weight correlates positively with MIBI score and NH. According to recent histological studies, 90% of glands heavier than 0.5 g present NH or a single-nodule stage, most probably due to the proliferative behaviour of this type of hyperplasia [19]. These findings can be attributed to various factors that include the decreased numbers of calcitriol receptors and calcium-sensing receptors, monoclonal proliferation and the fact that hyperparathyroid patients are generally refractory to vitamin D therapy. Most parathyroid glands we studied (70%) weighed >0.5 g; however, in contrast to the findings of the histological studies described above, only 51% of glands presented the nodular histological type. These paradoxical findings may be explained by the lack of a correlation between histological type and the macroscopically visible nodules [3]. Furthermore, the clonal characteristics of excised parathyroid tissue could also be responsible for the contradictory findings [2]. Therefore, it is probable that we classified glands composed of a single nodule, the most advanced type of NH, as glands with DH, because they appear similar [3]. Unfortunately, we were not able to conduct a clonality study. When we analysed the 29 glands (51%) classified as NH glands, we found high MIBI uptakes in 14 (48.2% of NH), whereas only four (14.2%) of the 28 DH glands presented MIBI uptake. In light of these results, we can conclude that, when high MIBI uptake is detected, the patient in question is likely to have NH since those few glands we classified as DH could actually have been uninodular hyperplastic glands.

The merits of PCNA analysis were highlighted [6] in a recent description of immunohistochemical procedures for evaluating cell proliferation. The most significant advantage of using PCNA analysis is that no complex pre-treatment is required. Therefore, proliferating cells can be easily detected, if present, in histopathological specimens preserved by standard methods. Our analysis of glandular structure indicated that cellular proliferative activity was higher in NH glands than in DH glands (Figure 2a), which is in agreement with previous reports [6]. Furthermore, PCNA expression was more pronounced in the group with higher MIBI uptake, which indicates that both MIBI and PCNA detect the increased proliferative status of parathyroid cells (Table 2). Regardless of MIBI uptake, PCNA expression was greater in NH glands than in DH glands. In glands presenting high MIBI uptake, this difference reached near significance. Again, this is probably attributable to the fact that we classified glands composed of a single nodule as DH glands. Other possible explanations include the wide dispersion of values of the glands’ weight and the small number of glands analysed.

Thus, we suggest that MIBI can be a marker of proliferative activity, based on our findings of a positive correlation with PCNA expression in the parathyroid glands of patients with SHP. Furthermore, the higher MIBI uptake and higher PCNA expression in the glands that show NH confirm the associations between MIBI, indicators of cell proliferation and histological findings in SHP. In conclusion, MIBI may be a useful tool in clinical practice for predicting the response to treatment, thus helping to avoid the prolonged exposure of patients to the harms of hyperparathyroidism. The usefulness of MIBI as a diagnostic tool for localizing ectopic parathyroid glands and for pre-operative evaluation of patients in need of re-operation is undeniable. Therefore, in recent years, some authors have used this technique to assess parathyroid function for which they have set forth various criteria, such as the presence and degree of uptake, or even the number of MIBI-positive glands. Recently, Torregrosa et al. [5] suggested that this technique may even predict responses to calcitriol treatment. In this study involving patients with severe SHP, our findings are in agreement with theirs, as we demonstrated that high MIBI uptake is correlated with all three of the factors we evaluated (glandular weight, cell proliferation and NH). The most significant finding of our study, however, is that, regardless of which of these factors is predominant, if a patient with SHP presents a higher MIBI uptake, this may characterize him as being refractory to any type of non-surgical treatment. Therefore, we propose that MIBI be considered another valuable tool in the work-up and medical treatment of SHP.



   Acknowledgments
 
The authors would like to express their gratitude to Roberto Pecoits-Filho, MD, PhD, for his valuable suggestions and contributions to this work. We also would like to acknowledge the assistance given by Jefferson D. Boyles in the translation and editing of the text, and by Wagner V. Dominguez and Fabiana G. Graciolli in technical support.

Conflict of interest statement. None declared.



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 23. 1.04
Accepted in revised form: 8.12.04