Immunohistochemical Expression of CaSR, VDR, and AMA in Parathyroid Tumors Linked to Hypercalcemia Severity in Primary Hyperparathyroidism

Immunohistochemical Expression of CaSR, VDR, and AMA in Parathyroid Tumors Linked to Hypercalcemia Severity in Primary Hyperparathyroidism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Immunohistochemical Expression of CaSR, VDR, and AMA in Parathyroid Tumors Linked to Hypercalcemia Severity in Primary Hyperparathyroidism Anna K. Eremkina, Dariya A. Pastuhova, Anastasiia P. Pershina-Miliutina, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6811379/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: Primary hyperparathyroidism (PHPT) is usually caused by parathyroid tumors, resulting in hypercalcemia due to excessive PTH secretion. While disease severity correlates with calcium blood levels, the molecular mechanisms driving clinical variability remain unclear. The role of the calcium-sensing receptor (CaSR), the vitamin D receptor (VDR) and parathyroid cells mitochondrial activity in the pathogenesis of hyperparathyroidism is of particular interest. Methods: This retrospective study included 96 patients with PHPT who underwent parathyroidectomy. Patients were stratified by albumin-corrected calcium blood levels (≤ 2.8; 2.8<Ca corr .≤ 3.0; 3.0 ≤ Ca corr. < 3.5 Ca corr. ≥ 3.5 mmol/L). Immunohistochemical expression (IHC) was estimated with CaSR, VDR, and antimitochondrial antibodies (AMA).Statistical analysis was performed using the Statistica v. 13.3 software package (TIBCO Software Inc., USA). Results: Strong CaSR expression was observed in 95.8% of tumors, whereas VDR expression was reduced in 46.9%. AMA staining was heterogeneous, with high reactivity in oxyphilic cells. No significant differences in CaSR, VDR, or AMA expression were found across groups with different hypercalcemia severity. No significant correlations were identified between IHC markers expression and parameters of calcium-phosphorus metabolism, as well as cinacalcet treatment response. Conclusions: Despite their established roles in parathyroid regulation, CaSR and VDR expression did not correlate with hypercalcemia severity in PHPT. These findings suggest that alternative molecular mechanisms, rather than receptor expression levels, contribute to clinical heterogeneity. Further research is required. primary hyperparathyroidism hypercalcemia immunohistochemistry parathyroid tumors CaSR VDR cinacalcet Figures Figure 1 Background Solitary parathyroid adenomas cause approximately 85–90% of the cases of primary hyperparathyroidism (PHPT) and present well-differentiated, benign, clonal tumors, which cause hypercalcemia through excessive secretion of parathyroid hormone (PTH). About 15–20% cases of PHPT are caused by multiple adenomas and even less (1%) by parathyroid carcinoma. [ 1 ] The PHPT severity directly depends on the degree of hypercalcemia, which is the one of the main biochemical markers of the disease. Hypercalcemia due to PHPT can be classified into mild (2.55–2.99 mmol/l), moderate (3–3.49 mmol/l), and severe with high risks of hypercalcemic crisis (more than 3.49 mmol/l). Clinical presentation of increased blood calcium ranges from asymptomatic to renal, skeletal, gastrointestinal and neuromuscular manifestations.[ 2 ] The molecular mechanisms explaining the severe course of parathyroid adenomas remains unknown. Most data on larger parathyroid tumors (those with large tumor volume) were associated with severe hypercalcemia and hypercalcemic crisis symptoms, and tumor mass effect. The contribution of distinct parathyroid cell types in tumorigenesis has rarely been considered. A recent study found, that oxyphilic cell adenomas were associated with higher preoperative serum calcium and PTH levels, as well asa higher incidenceof symptomatic disease.[ 3 ] The abnormal Ca + + sensing in hyperparathyroidism has been generally attributed to altered CaSR expression. Recent studies employing various methodologies have reported varying levels of both CaSR mRNA and protein expression in parathyroid tumors reduced СaSR expression likely contributes to impaired PTH release inhibition, but it has been suggested that low levels of G proteins from the Gq subfamily may serve as an additional contributing factor.[ 4 ] Parathyroid cells also express VDR, which additionally affects the expression of the CaSR in parathyroid cells. Expression of VDR mRNA is significantly reduced in the parathyroid adenomas due to PHPT. The decreased VDR expression can inversely correlate with parathyroid tumor weight, CaSR downregulation and increased secretory set-point.[ 5 ] These observations raise the possibility that VDR may be related to the clinically aggressive course of parathyroid adenomas. Increased secretory activity of parathyroid cells may also be linked to mitochondrial abundance which plays a crucial role in cellular energy metabolism. Mitochondrial dysfunction is frequently associated with various pathological conditions, including tumors. Evidence suggests that the oxyphil parathyroid adenomas characterized by an elevated mitochondrial count, often accompanied by enlargement and abnormal morphology. These alterations may reflect an metabolic shift, potentially contributing to both tumorigenesis and enhanced secretory activity. However, the relationship between the mitochondrial abundance, proliferative activity, and hormonal hypersecretion remains to be clarified.[ 6 ] The aim of this study wasto evaluate the immunohistochemical expression of CaSR, VDR, and AMA in parathyroid tumors and assess its correlation with the severity of hypercalcemia in patients with primary hyperparathyroidism. Materials and methods A single-center retrospective study was conducted at the Endocrinology Research Centre (ERC), Moscow from January 2018 to August 2023. The study included 96 patients with PHPT who underwent parathyroidectomy (PTE) at the Centre. Based on the severity of hypercalcemia, the patients were divided into four groups: Group 1 - Ca corr. ≤ 2.8 mmol/L (n=28); Group 2 - 2.8 < Ca corr. ≤ 3.0 mmol/L (n=25); Group 3 - 3.0 ≤ Ca corr. < 3.5 mmol/L (n=29); Group 4 - Ca corr. ≥ 3.5 mmol/L (n=14). The use of cut-off points for hypercalcemia were defined according to the Russian PHPT guidelines Furthermore, the degree of hypercalcaemia determined the use of cinacalcet either as monotherapyor in combination with other hypocalcaemic agents. Patients underwent several laboratory tests. The ARCHITECT c8000 chemistry analyzer (Abbott, Abbott Park, IL, USA) was used to determine the concentrations of serum calcium (reference interval (RI) 2.15–2.55 mmol/L); albumin (RI 34–48 g/L female, RI 35–50 g/L male); phosphorus—(RI 0.74–1.52 mmol/L); creatinine (RI 50–98 µmol/L female, 63–110 µmol/L male); and 24-h urine calcium (RI 2.5–8.0 mmol/L). PTH (range 15–65 pg/mL), osteocalcin (range 11–43 ng/mL female, 14–42 ng/mL male) and b-CrossLaps (range 0.3–1.1 ng/mL female, range 0.1–0.85 ng/mL male) were measured on the COBAS 6000 (Roche, Rotkreuz, Switzerland). Albumin-corrected calcium was calculated using the formula: albumin-corrected calcium (mmol/L) = measured plasma calcium level (mmol/L) + 0.02 × (40 − albumin level (g/L)). eGFR was calculated using the CKD-EPI 2009 formula. The examination for PHPT complications included dual-energy X-ray absorptiometry (DXA) of the lumbar spine (LS), femoral neck (FN), total femur (TH), total radius (RT), and distal third of radius (R33%) (Lunar iDXA enCORE GE Healthcare version 17; USA); X-ray of thoracic and LS (X-ray diagnostic system Optima RF420, GE Healthcare, Chicago, IL, USA); renal ultrasound (Aplio 500, Toshiba, Tokyo, Japan) and/or renal CT scan (Optima CT660, GE, Chicago, IL, USA). We additionally assessed cinacalcet dosage, tolerability, and its efficacy in reducing hypercalcemia relative to baseline values Morphological examination Tumor tissue samples were fixed in 10% buffered formalin, processed in the histological wiring system of Leica ASP6025 and poured into paraffin. Further, paraffin sections with a thickness of 3 µm were made from the paraffin-embedded tumor tissue samples on the microtome, applied to slides treated with poly (l-lysine). Then the slides were stained with hematoxylin and eosin according to the standard procedure. All tumor tissue samples were verified in accordance with the 2022 WHO classification of parathyroid tumors. Immunohistochemistry imaging Immunohistochemical analysis of tumor tissue sections was carried out according to the standard technique with a peroxidase detection system with DAB on an automatic Leica BOND III IHC staining system using Leica reagents. Antibodies to Vitamin D Receptor (VDR, GeneTex), Calcium Sensing Receptor (CaSR, 5C10, GeneTex), Anti-Mitochondrial Antigen (AMA, 113-1, BioGenex) were used. All histological slides were scanned using a Leica Aperio AT2 system at 20x magnification for further analysis. The most representative fields of view were selected. (tab.1) The expression of AMA and VDR was determined in the cytoplasm of tumor cells and evaluated as strong (1), moderate (2), weak or absent (3). CaSR expression was detected in the membrane of tumour cells and evaluated similarly (tab. 3). Statistical analysis Statistical analysis was performed using the Statistica v. 13.3 software package (TIBCO Software Inc., USA). For the description of quantitative data, medians and interquartile ranges were used, presented as Me [Q1; Q3], while absolute and relative frequencies were used for qualitative data. The comparison of four dependent groups for quantitative indicators was carried out using the Friedman test, followed by post-hoc analysis. Intergroup differences in categorical variables were assessed using the Freeman-Halton test. The significance level (p) for testing statistical hypotheses was set at 0.05. To correct the critical significance level for multiple comparisons, the Bonferroni correction (p0) was applied, after which p-values in the range between the calculated p0 and 0.05 were interpreted as a statistical trend. Results The study included 86 women and 10 men, with a median age of 57 years [47.5; 67] at the time of enrollment. The majority of patients presented with symptomatic PHPT with either bone or renal complications. A detailed description is provided in Table 2. Cinacalcet was administered in a median dose of 30 mg/day [30; 60] prior to surgery in 73.9% of cases. Exceptions included 22/28 patients in the mild hypercalcemia subgroup and 2/14 patients in the life-threatening hypercalcemia group. Treatment tolerance was satisfactory in 91.7% of cases. A significant response to cinacalcet therapy at average doses of 30-60 mg per day was noted in 35.7% of patients (defined as a reduction in calcium levels of more than 0.25 mmol/L from baseline), while no effect was observed in 42.9%. The histological examination revealed predominated adenomas, while atypical tumors and carcinoma of the parathyroid gland were found in 10.4% and 2.1% of cases, respectively. The diagnosis of hyperplasia was established only in one patient (tab.3). In our study, the vast majority of adenomas exhibited a mixed cellular composition. Chief cell-only adenomas were observed in 18.75% (18/96) of cases, while clear cell-only adenomas were rare, accounting for only 1% (1/96). No adenomas composed exclusively of oncocytic cells were identified. Among Group 1, chief cells predominated in 53.6% (15/28) of adenomas, while clear cells were the dominant type in 25% (7/28). In Group 2, chief cell adenomas were identified in 24% (6/25) of tumors, clear cell adenomas in 24% (6/25) and oncocytic cell adenomas in 8% (2/25). In Group 3, chief cells predominated in 48.3% (14/29) of adenomas, clear cells in 10.3% (3/29), and oncocytic adenomas accounted for 3.4% (1/29). Finally, in Group 4, chief cell adenomas accounted for 28.6% (4/14) of cases, pure cell adenomas predominated in 14.3% (2/14), and predominantly oncocytic adenomas were identified in 7.1% (1/14) of samples. We did not find any corellations between serum calcium levels and predominant adenoma histological type (all p>0.05, p chief cells = 0.137, p water-clear cells = 0.238, p oncocytic cells =0.276). (Figure 1) Almost all parathyroid lesions demonstrated pronounced strong expression of CaSR, comparable to that of healthy parathyroid gland tissue. No cases of absent IHC reactions were identified. Membrane-cytoplasmic staining was detected in 86.5% (83/96) of parathyroid tumour samples, whereas pure cytoplasmic staining was observed only in 11.5% (11/96). Decreased VDR expression was observed in 46.9% of cases, where tumor cells were less intensely stained compared to the unchanged parathyroid gland. Anti-mitochondrial antibodies showed heterogeneous "mosaic" expression, with alternating cells exhibiting strong and weak cytoplasmic staining throughout the examined material. When compared with histological data, it was found that the cytoplasm of oxyphilic cells was stained more intensely than that of chief and clear cells. In a separate subgroup of atypical tumors, 4 out of 8 samples showed focal or weak expression of VDR, and the expression of AMA was also decreased in 5 out of 8 samples. The study group included only 2 carcinomas, in both cases, the expression of VDR was focal, which distinguished it from healthy tissue. Table 2. Clinical features of the studied group. This table summarizes the clinical characteristics of the study group, including demographics (sex, age), biochemical markers (PTH, phosphorus, calciuria, eGFR), bone mineral density (BMD) scores, and the prevalence of fractures and nephrocalcinosis/nephrolithiasis. Sign N Me [Q 1 ; Q 3 ] / n (%) Sex Female 96 86 (89,6%) Male 10 (10,4%) Age at diagnosis, years 96 57 [47,5; 67] PTH, pg/mL 96 248,55 [138,15; 558,50] Phosphorus , mmol/L 92 0,78 [0,665; 0,93] Daily calciuria, mmol/D 88 8,70 [6,095; 10,965] eGFR (CKD-EPI), mL/min/1.73 m2 96 81,5 [64,0; 97,5] DXA scans (Z-scores), SD 28 -1,45 [-2,35; -0,75] Reduction in BMD SD (T-scores.) ≥ -1,0 / SD (Z-scores) ≥ -2,0 86 24 (27,9%) -1,0 > SD (T-scores) > -2,5 18 (20,9%) SD (T-scores) ≤-2,5 / SD (Z-scores) < -2,0 29 (33,7%) SD (T-scores) ≤-2,5 + low-energy fracture / SD (Z-scores) < -2,0 + low-energy fracture 15 (17,4%) Vertebral fractures 96 19 (19,8%) Non-vertebral low-energy fractures 96 14 (14,6%) Nephrocalcinosis/nephrolithiasis 96 68 (70,8%) Table 3. Histological features of parathyroid tumours. This table provides an overview of the histological classification of parathyroid tumors (adenoma, atypical tumor, hyperplasia, carcinoma) and the expression levels of key markers (CaSR, VDR, AMA), categorized as strong, moderate, or weak/absent expression. Sign N Me [Q 1 ; Q 3 ] / n (%) Histology Adenoma 96 83 (86,5%) Atypical tumor 10 (10,4%) Hyperplasia 1 (1%) Carcinoma 2 (2,1%) CaSR expression 1* 96 92 (95,8%) 2 4 (4,2%) 3 0 (0%) VDR expression 1 96 51 (53,1%) 2 35 (36,5%) 3 10 (10,4%) AMA expression 1 96 46 (47,9%) 2 47 (49%) 3 3 (3,1%) * 1 - strong expression, 2 - moderate expression, 3 - weak or absence expression. Further we conducted a comparative analysis of groups 1-4. As expected, the groups differed in levels of iPTH and bone remodeling markers. Among the clinical features statistically significant differences were found only for the frequency of fibrocystic osteitis which was higher in patients with Ca corr more than 3,5 mmol/l. A significant decrease in BMD and impaired renal filtration function showed a statistical trend that was more pronounced in patients with hypercalcemia greater than 3 mmol/L. We performed a correlation analysis to assess the relationship between the degree of IHC expression and key indicators of calcium-phosphorus metabolism; however, no significant correlations were identified. Additionally, no correlation was found between the expression of CaSR and VDR and the effectiveness of the cinacalcet therapy. Discussion Parathyroid cells in vivo and in vitro respond to increasing concentrations of extracellular ionized calcium by reducing PTH secretion. This effect is mediated by a direct interaction of calcium with a G protein-coupled CaSR. In patients with PHPT high concentrations of ionized calcium do not suppress autonomous PTH secretion, but the molecular mechanism for this abnormality is not clearly understood.[ 7 ] CaSR down-regulation is generally believed to be the principal reason for the abnormal calcium-PTH interactions in parathyroid tumors, i.e. calcium responsiveness should be directly dependent upon CaSR expression on cells. Corbetta et al. reported diminished CaSR expression both at mRNA (by RT-PCR) and protein level (by Western blot) in 27 parathyroid adenoma and 4 hyperplasia cases. The authors also proposed that the disrupted response of parathyroid cells to extracellular calcium concentrations in adenomas might be associated with a 2-fold reduction in the expression of the membrane G-protein.[ 4 ] Singh et al. using real-time qPCR revealed reduced CaSR mRNA expression with a fold reduction of 0.12 (P < 0.0001) in parathyroid adenomas. The obtained data were correlated with the results of IHC results showing reduced expression of CaSR in 90% of adenomas. Unlike the findings of Singh et al., we did not observe a significant reduction of CaSR expression in different tumors compared to healthy tissue. The reaction with CaSR antibodies predominantly exhibited diffuse and pronounced membrane-cytoplasmic expression, which was almost similar to intact parathyroid tissue. Moreover, strong positive IHC reactions persisted even in cases of extremely "aggressive" PHPT (including carcinomas and atypical tumors) and hypercalcemia exceeding 3.5 mmol/L. At the same time, Haven et al. did not find a significant decrease of CaSR expression in parathyroid hyperplasia or adenomas. While only 1 of 104 benign tumors (1%) exhibited irregular or absent CaSR staining, carcinomas showed loss of CaSR expression in 31% of cases (9/29).[ 8 ] In Agarwal et al.'s study[ 9 ], normal autopsy-derived parathyroid tissues showed strong but predominantly incomplete membranous CaSR expression (in 85% of cases). The cytoplasm of the normal parathyroid cells also showed strong expression of CaSR. In the PHPT group, membranous staining for CaSR was usually of moderate intensity. Besides, none of the parathyroid hyperplasia cases showed moderate cytoplasmic staining as the predominant pattern, in contrast to 16% of adenomas and 43% of the carcinomas. The authors suggested that not only diminished CaSR synthesis, but additional defects in the tracking of the receptor from the cytoplasm to the membrane may play a role in the PHPT pathogenesis. In our study, pure cytoplasmic staining was observed only in 11.5%. Interesting data was obtained by Shi Y. et al. Using flow cytometric analysis of resected adenomatous parathyroid glands, they have isolated and characterized chief and oxyphil cells, as well as tumor-infiltrating lymphocytes. While chief and oxyphil cells exhibited comparable CaSR expression at both protein and mRNA levels, oxyphil cells demonstrated greater calcium sensitivity despite showing higher basal PTH secretion. This suggests that CaSR expression alone does not determine PTH production and calcium responsiveness in the parathyroid gland. The increased PTH levels in PHPT may reflect not only a higher number of tumor cells but also the greater PTH secretion profile of oxyphil compared to chief cells. The authors further proposed that tumor-infiltrating lymphocytes might target antibodies against CaSR, PTH, or other parathyroid antigens. Such antibody-mediated mechanisms - through either CaSR inhibition or immune clearance of CaSR-expressing/PTH-producing cells - could initially trigger compensatory hyperplasia, potentially progressing to adenoma formation.[ 10 ] In our work, the majority of adenomas exhibited a mixed cellular composition. Furthermore, there were no corellations between serum calcium levels and adenoma histological type. With regard to VDR, we obtained more expected results. The VDR expression was reduced or absent in approximately 50% of cases, which is generally consistent with previously published research. Carling et al. demonstrated similar VDR mRNA levels in the adenomas and hyperplasias, which were reduced (42 +/- 2.8% and 44 +/- 4.0%) compared to the normal glands (P < 0.0001).[ 11 ] Rao et al. evaluated receptor expression by IHC in 24 parathyroid adenomas and in 11 normal glands.There was reduction of VDR expression in almost all cells of parathyroid adenomas. The decrease in CaSR expression was not so pronounced as VDR which generally does not contradict our data. The authors proposed that reduced CaSR expression may frequently occur secondary to VDR loss. While this downregulation may be moderate, it appears sufficient to elevate the PTH secretory set-point and subsequently promote adenoma development.[ 12 ] This hypothesis is supported by the presence of vitamin D response elements (VDREs) in the 5'-flanking region of the CASR gene, enabling its transcriptional regulation through 1,25-dihydroxyvitamin D3-VDR complex binding to these regulatory sequences.[ 13 ] Data on the AMA expression in the parathyroid tumors are limited. We observed heterogeneous expression of the AMA throughout the studied samples. It was noted that the cytoplasm of oxyphil cells exhibited more intense staining compared to chief and clear cells. The most likely reason for this reaction is the presence of a high number of mitochondria in the cytoplasm of oncocytes. In contrast, cells with clear cytoplasm displayed the weakest staining, with some cases showing no staining at all. The origin of these clear cells remains insufficiently studied. There is a hypothesis that clear cytoplasm cells represent the final stage of chief cell hyperplasia. Ultrastructural analysis of this cell type has shown that vacuoles may either be dilated structures of the Golgi complex or components of the endoplasmic reticulum, which could explain the observed IHC reactions with AMA. For a more accurate assessment, further analysis on a larger sample size is necessary. [ 14 ] In the study by Varshney S. et al., the expression of CaSR and VDR in parathyroid adenomas was lower than in normal tissue ( according to IHC analysis and mRNA expression). No correlations were identified between СaSR and VDR expression and preoperative levels of calcium, iPTH, and 25(OH)D. Other markers including PTH and CD1 also did not correlate with serum calcium and iPTH. Similar results were obtained in our study. We found no differences in the expression of CaSR, VDR, and AMA among groups with varying severity of hypercalcemia, as well as no correlations between the IHC phenotype and the response to calcimimetic therapy.The assessment of the immunohistochemical profile of parathyroid tumors in relation to the severity of hypercalcemia has been conducted for the first time.[ 15 ] Conclusion According to obtained results CaSR expression remained strong in the majority of tumors, including aggressive cases, suggesting that reduced CaSR expression is not the primary driver of hypercalcemia severity. In contrast, VDR expression was diminished in approximately half of the cases, aligning with previous research implicating VDR downregulation in PHPT pathogenesis. AMA expression exhibited heterogeneity, with oxyphil cells showing more intense staining, likely due to their mitochondrial-rich cytoplasm. No significant correlations were found between the IHC profiles of CaSR, VDR, or AMA and the degree of hypercalcemia, bone remodeling markers, or response to cinacalcet therapy. These results suggest that other molecular mechanisms, beyond the examined markers, may contribute to the clinical variability of PHPT. Further research is warranted to explore additional pathways and cellular interactions underlying the disease's heterogeneity. Declarations Ethical Approval: Generated Statement: The studies involving humans were approved by the Ethics Committee of the Endocrinology Research Centre (Protocol No. 1 dated January 17, 2018) (Moscow, Russia). The studies were conducted in accordance with the local legislation and institutional requirements, as well as with the Helsinki Declaration. The participants provided their written informed consent to participate in this study. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests. Authors’ contributions All authors contributed equally to the manuscript and read and approved the final version of the manuscript. Funding: This article was supported by the Russian Science Foundation, agreement №24-15-00269. Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. References Mizamtsidi M, Nastos C, Mastorakos G, Dina R, Vassiliou I, Gazouli M, et al. Diagnosis, management, histology and genetics of sporadic primary hyperparathyroidism: old knowledge with new tricks. Endocr Connect. 2018;7:R56–68. Al Hassan MS, El Ansari W, Issa N, Darweesh A, Abdelaal A. Severe hypercalcemia secondary to parathyroid adenoma: Series of four consecutive cases at a tertiary care hospital in Qatar. Int J Surg Case Rep. 2023;109:108560. Howson P, Kruijff S, Aniss A, Pennington T, Gill AJ, Dodds T, et al. Oxyphil Cell Parathyroid Adenomas Causing Primary Hyperparathyroidism: a Clinico-Pathological Correlation. Endocr Pathol. 2015;26:250–4. Corbetta S, Mantovani G, Lania A, Borgato S, Vicentini L, Beretta, et al. Calcium‐sensing receptor expression and signalling in human parathyroid adenomas and primary hyperplasia. Clin Endocrinol (Oxf). 2000;52:339–48. Sudhaker Rao, Han, Phillips ER, Palnitkar S, Parfitt. Reduced vitamin D receptor expression in parathyroid adenomas: implications for pathogenesis. Clin Endocrinol (Oxf). 2000;53:373–81. Costa-Guda J, Tokura T, Roth SI, Arnold A. Mitochondrial DNA mutations in oxyphilic and chief cell parathyroid adenomas. BMC Endocr Disord. 2007;7:8. Cetani F, Picone A, Cerrai P, Vignali E, Borsari S, Pardi E, et al. Parathyroid Expression of Calcium-Sensing Receptor Protein and in Vivo Parathyroid Hormone-Ca 2+ Set-Point in Patients with Primary Hyperparathyroidism 1 . J Clin Endocrinol Metab. 2000;85:4789–94. Haven CJ, van Puijenbroek M, Karperien M, Fleuren G, Morreau H. Differential expression of the calcium sensing receptor and combined loss of chromosomes 1q and 11q in parathyroid carcinoma. J Pathol. 2004;202:86–94. Agarwal S, Kardam S, Chatterjee P, Kumar C, Boruah M, Sharma MC, et al. CaSR expression in normal parathyroid and PHPT: new insights into pathogenesis from an autopsy-based study. J Endocrinol Invest. 2022;45:337–46. Shi Y, Hogue J, Dixit D, Koh J, Olson JA. Functional and genetic studies of isolated cells from parathyroid tumors reveal the complex pathogenesis of parathyroid neoplasia. Proceedings of the National Academy of Sciences. 2014;111:3092–7. Carling T, Rastad J, Szabó E, Westin G, Åkerström G. Reduced Parathyroid Vitamin D Receptor Messenger Ribonucleic Acid Levels in Primary and Secondary Hyperparathyroidism*. J Clin Endocrinol Metab. 2000;85:2000–3. Sudhaker Rao, Han, Phillips ER, Palnitkar S, Parfitt. Reduced vitamin D receptor expression in parathyroid adenomas: implications for pathogenesis. Clin Endocrinol (Oxf). 2000;53:373–81. Canaff L, Hendy GN. Human Calcium-sensing Receptor Gene. Journal of Biological Chemistry. 2002;277:30337–50. Duan K, Gomez Hernandez K, Mete O. Clinicopathological correlates of hyperparathyroidism. J Clin Pathol. 2015;68:771–87. Varshney S, Bhadada SK, Saikia UN, Sachdeva N, Behera A, Arya AK, et al. Simultaneous expression analysis of vitamin D receptor, calcium-sensing receptor, cyclin D1, and PTH in symptomatic primary hyperparathyroidism in Asian Indians. Eur J Endocrinol. 2013;169:109–16. Table Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. 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Gorbacheva","email":"","orcid":"","institution":"Endocrinology Research Center","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"M.","lastName":"Gorbacheva","suffix":""},{"id":489347182,"identity":"3ddcd93a-d1af-4794-9200-4264385c4059","order_by":4,"name":"Hanum V. Bagirova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYBACAwnGxgcfKmwYGJiZG4jVwtxsOONMGlALI9Fa2NukedsOA5nEajGXbmyT5jlzPpq/nbGBubKNCC2Wcw42W86puJ074zBjA+NZYrQY3EhsvPHmzO3cBpCWRiK1NEjwtp3LnU+KliZJ3rYDuRuI13LnICiQk3M3ArUcbDhHjJbb7Q+BUWmXO+/84YMPG8qI0IICDpCqYRSMglEwCkYBDgAAC1xBBSn0/3IAAAAASUVORK5CYII=","orcid":"","institution":"Endocrinology Research Center","correspondingAuthor":true,"prefix":"","firstName":"Hanum","middleName":"V.","lastName":"Bagirova","suffix":""},{"id":489347185,"identity":"846bee0e-1d0b-4d47-9cfa-ba45404ffe58","order_by":5,"name":"Liliya S. Urusova","email":"","orcid":"","institution":"Endocrinology Research Center","correspondingAuthor":false,"prefix":"","firstName":"Liliya","middleName":"S.","lastName":"Urusova","suffix":""},{"id":489347187,"identity":"fed50094-95b6-4d17-91cb-15ad28169578","order_by":6,"name":"Natalia G. Mokrysheva","email":"","orcid":"","institution":"Endocrinology Research Center","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"G.","lastName":"Mokrysheva","suffix":""}],"badges":[],"createdAt":"2025-06-03 12:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6811379/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6811379/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87550971,"identity":"3690ff27-3324-4671-88f4-fffef4ada1c5","added_by":"auto","created_at":"2025-07-25 06:19:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGroups depending on the predominant cell type.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6811379/v1/2d2dd3067758225171aca502.png"},{"id":87554494,"identity":"dea779ce-0623-4029-abaa-8b7ddef3906d","added_by":"auto","created_at":"2025-07-25 06:43:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":753470,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6811379/v1/63fb3629-d30e-46ee-90b7-7656032c0b38.pdf"},{"id":87550978,"identity":"7213286c-2866-4d31-88da-c982ae0b995e","added_by":"auto","created_at":"2025-07-25 06:19:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1363948,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6811379/v1/225ab34a65e33de20c0538d6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eImmunohistochemical Expression of CaSR, VDR, and AMA in Parathyroid Tumors Linked to Hypercalcemia Severity in Primary Hyperparathyroidism\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eSolitary parathyroid adenomas cause approximately 85\u0026ndash;90% of the cases of primary hyperparathyroidism (PHPT) and present well-differentiated, benign, clonal tumors, which cause hypercalcemia through excessive secretion of parathyroid hormone (PTH). About 15\u0026ndash;20% cases of PHPT are caused by multiple adenomas and even less (1%) by parathyroid carcinoma. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThe PHPT severity directly depends on the degree of hypercalcemia, which is the one of the main biochemical markers of the disease. Hypercalcemia due to PHPT can be classified into mild (2.55\u0026ndash;2.99 mmol/l), moderate (3\u0026ndash;3.49 mmol/l), and severe with high risks of hypercalcemic crisis (more than 3.49 mmol/l). Clinical presentation of increased blood calcium ranges from asymptomatic to renal, skeletal, gastrointestinal and neuromuscular manifestations.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThe molecular mechanisms explaining the severe course of parathyroid adenomas remains unknown. Most data on larger parathyroid tumors (those with large tumor volume) were associated with severe hypercalcemia and hypercalcemic crisis symptoms, and tumor mass effect. The contribution of distinct parathyroid cell types in tumorigenesis has rarely been considered. A recent study found, that oxyphilic cell adenomas were associated with higher preoperative serum calcium and PTH levels, as well asa higher incidenceof symptomatic disease.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThe abnormal Ca\u0026thinsp;+\u0026thinsp;+\u0026thinsp;sensing in hyperparathyroidism has been generally attributed to altered CaSR expression. Recent studies employing various methodologies have reported varying levels of both CaSR mRNA and protein expression in parathyroid tumors reduced СaSR expression likely contributes to impaired PTH release inhibition, but it has been suggested that low levels of G proteins from the Gq subfamily may serve as an additional contributing factor.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eParathyroid cells also express VDR, which additionally affects the expression of the CaSR in parathyroid cells. Expression of VDR mRNA is significantly reduced in the parathyroid adenomas due to PHPT. The decreased VDR expression can inversely correlate with parathyroid tumor weight, CaSR downregulation and increased secretory set-point.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] These observations raise the possibility that VDR may be related to the clinically aggressive course of parathyroid adenomas.\u003c/p\u003e\u003cp\u003eIncreased secretory activity of parathyroid cells may also be linked to mitochondrial abundance which plays a crucial role in cellular energy metabolism. Mitochondrial dysfunction is frequently associated with various pathological conditions, including tumors. Evidence suggests that the oxyphil parathyroid adenomas characterized by an elevated mitochondrial count, often accompanied by enlargement and abnormal morphology. These alterations may reflect an metabolic shift, potentially contributing to both tumorigenesis and enhanced secretory activity. However, the relationship between the mitochondrial abundance, proliferative activity, and hormonal hypersecretion remains to be clarified.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe aim\u003c/b\u003e of this study wasto evaluate the immunohistochemical expression of CaSR, VDR, and AMA in parathyroid tumors and assess its correlation with the severity of hypercalcemia in patients with primary hyperparathyroidism.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eA single-center retrospective study was conducted at the Endocrinology Research Centre (ERC), Moscow from January 2018 to August 2023.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study included 96 patients with PHPT who underwent parathyroidectomy (PTE) at the Centre. Based on the severity of hypercalcemia, the patients were divided into four groups: Group 1 - Ca corr. \u0026le; 2.8 mmol/L (n=28); Group 2 - 2.8 \u0026lt; Ca corr. \u0026le; 3.0 mmol/L (n=25); Group 3 - 3.0 \u0026le; Ca corr. \u0026lt; 3.5 mmol/L (n=29); Group 4 - Ca corr. \u0026ge; 3.5 mmol/L (n=14). The use of cut-off points for hypercalcemia were defined according to the Russian PHPT guidelines Furthermore, the degree of hypercalcaemia determined the use of cinacalcet either\u0026nbsp;as monotherapyor in combination with other hypocalcaemic agents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePatients underwent several laboratory tests. The ARCHITECT c8000 chemistry analyzer (Abbott, Abbott Park, IL, USA) was used to determine the concentrations of serum calcium (reference interval (RI) 2.15\u0026ndash;2.55 mmol/L); albumin (RI 34\u0026ndash;48 g/L female, RI 35\u0026ndash;50 g/L male); phosphorus\u0026mdash;(RI 0.74\u0026ndash;1.52 mmol/L); creatinine (RI 50\u0026ndash;98 \u0026micro;mol/L female, 63\u0026ndash;110 \u0026micro;mol/L male); and 24-h urine calcium (RI 2.5\u0026ndash;8.0 mmol/L). PTH (range 15\u0026ndash;65 pg/mL), osteocalcin (range 11\u0026ndash;43 ng/mL female, 14\u0026ndash;42 ng/mL male) and b-CrossLaps (range 0.3\u0026ndash;1.1 ng/mL female, range 0.1\u0026ndash;0.85 ng/mL male) were measured on the COBAS 6000 (Roche, Rotkreuz, Switzerland). Albumin-corrected calcium was calculated using the formula: albumin-corrected calcium (mmol/L) = measured plasma calcium level (mmol/L) + 0.02 \u0026times; (40 \u0026minus; albumin level (g/L)). eGFR was calculated using the CKD-EPI 2009 formula.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe examination for PHPT complications included dual-energy X-ray absorptiometry (DXA) of the lumbar spine (LS), femoral neck (FN), total femur (TH), total radius (RT), and distal third of radius (R33%) (Lunar iDXA enCORE GE Healthcare version 17; USA); X-ray of thoracic and LS (X-ray diagnostic system Optima RF420, GE Healthcare, Chicago, IL, USA); renal ultrasound (Aplio 500, Toshiba, Tokyo, Japan) and/or renal CT scan (Optima CT660, GE, Chicago, IL, USA).\u003c/p\u003e\n\u003cp\u003eWe additionally assessed cinacalcet dosage, tolerability, and its efficacy in reducing hypercalcemia relative to baseline values\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor tissue samples were fixed in 10% buffered formalin, processed in the histological wiring system of Leica ASP6025 and poured into paraffin. Further, paraffin sections with a thickness of 3 \u0026micro;m were made from the paraffin-embedded tumor tissue samples on the microtome, applied to slides treated with poly (l-lysine). Then the slides were stained with hematoxylin and eosin according to the standard procedure.\u003cbr\u003e\u0026nbsp;All tumor tissue samples were verified in accordance with the 2022 WHO classification of parathyroid tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analysis of tumor tissue sections was carried out according to the standard technique with a peroxidase detection system with DAB on an automatic Leica BOND III IHC staining system using Leica reagents. Antibodies to Vitamin D Receptor (VDR, GeneTex), Calcium Sensing Receptor (CaSR, 5C10, GeneTex), Anti-Mitochondrial Antigen (AMA, 113-1, BioGenex) were used. All histological slides were scanned using a Leica Aperio AT2 system at 20x magnification for further analysis. The most representative fields of view were selected. (tab.1)\u003c/p\u003e\n\u003cp\u003eThe expression of AMA and VDR was determined in the cytoplasm of tumor cells and evaluated as strong (1), moderate (2), weak or absent (3). CaSR expression was detected in the membrane of tumour cells and evaluated similarly (tab. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using the Statistica v. 13.3 software package (TIBCO Software Inc., USA). For the description of quantitative data, medians and interquartile ranges were used, presented as Me [Q1; Q3], while absolute and relative frequencies were used for qualitative data. The comparison of four dependent groups for quantitative indicators was carried out using the Friedman test, followed by post-hoc analysis. Intergroup differences in categorical variables were assessed using the Freeman-Halton test. The significance level (p) for testing statistical hypotheses was set at 0.05. To correct the critical significance level for multiple comparisons, the Bonferroni correction (p0) was applied, after which p-values in the range between the calculated p0 and 0.05 were interpreted as a statistical trend.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe study included 86 women and 10 men, with a median age of 57 years [47.5; 67] at the time of enrollment. The majority of patients presented with symptomatic PHPT with either bone or renal complications. A detailed description is provided in Table 2. Cinacalcet was administered in a median dose of 30 mg/day [30; 60] prior to surgery in 73.9% of cases. Exceptions included 22/28 patients in the mild hypercalcemia subgroup and 2/14 patients in the life-threatening hypercalcemia group. Treatment tolerance was satisfactory in 91.7% of cases. A significant response to cinacalcet therapy at average doses of 30-60 mg per day was noted in 35.7% of patients (defined as a reduction in calcium levels of more than 0.25 mmol/L from baseline), while no effect was observed in 42.9%.\u003c/p\u003e\n\u003cp\u003eThe histological examination revealed predominated adenomas, while atypical tumors and carcinoma\u0026nbsp;of the parathyroid gland were found in 10.4% and 2.1% of cases, respectively. The diagnosis of hyperplasia was established only in one patient (tab.3). In our study, the vast majority of adenomas exhibited a mixed cellular composition. Chief cell-only adenomas were observed in 18.75% (18/96) of cases, while clear cell-only adenomas were rare, accounting for only 1% (1/96). No adenomas composed exclusively of oncocytic cells were identified. Among Group 1, chief cells predominated in 53.6% (15/28) of adenomas, while clear cells were the dominant type in 25% (7/28). In Group 2, chief cell adenomas were identified in 24% (6/25) of tumors, clear cell adenomas in 24% (6/25) and oncocytic cell adenomas in 8% (2/25). In Group 3, chief cells predominated in 48.3% (14/29) of adenomas, clear cells in 10.3% (3/29), and oncocytic adenomas accounted for 3.4% (1/29). Finally, in Group 4, chief cell adenomas accounted for 28.6% (4/14) of cases, pure cell adenomas predominated in 14.3% (2/14), and predominantly oncocytic adenomas were identified in 7.1% (1/14) of samples. We did not find any corellations between serum calcium levels and predominant adenoma histological type (all p\u0026gt;0.05, p\u003csub\u003echief cells\u003c/sub\u003e = 0.137, p\u003csub\u003ewater-clear cells\u003c/sub\u003e = 0.238, p\u003csub\u003eoncocytic cells\u003c/sub\u003e=0.276). (Figure 1)\u003c/p\u003e\n\u003cp\u003eAlmost all parathyroid lesions demonstrated pronounced strong expression of CaSR, comparable to that of healthy parathyroid gland tissue. No cases of absent IHC reactions were identified. Membrane-cytoplasmic staining was detected in 86.5% (83/96) of parathyroid tumour samples, whereas pure cytoplasmic staining was observed only in 11.5% (11/96). \u0026nbsp; Decreased VDR expression was observed in 46.9% of cases, where tumor cells were less intensely stained compared to the unchanged parathyroid gland. Anti-mitochondrial antibodies showed heterogeneous \u0026quot;mosaic\u0026quot; expression, with alternating cells exhibiting strong and weak cytoplasmic staining throughout the examined material. When compared with histological data, it was found that the cytoplasm of oxyphilic cells was stained more intensely than that of chief and clear cells. In a separate subgroup of atypical tumors, 4 out of 8 samples showed focal or weak expression of VDR, and the expression of AMA was also decreased in 5 out of 8 samples. The study group included only 2 carcinomas, in both cases, the expression of VDR was focal, which distinguished it from healthy tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e \u003cstrong\u003eClinical features of the studied group.\u0026nbsp;\u003c/strong\u003eThis table summarizes the clinical characteristics of the study group, including demographics (sex, age), biochemical markers (PTH, phosphorus, calciuria, eGFR), bone mineral density (BMD) scores, and the prevalence of fractures and nephrocalcinosis/nephrolithiasis.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"602\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSign\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMe [Q\u003csub\u003e1\u003c/sub\u003e; Q\u003csub\u003e3\u003c/sub\u003e] / n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 220px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e86 (89,6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e10 (10,4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge at diagnosis, years\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e57 [47,5; 67]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePTH, pg/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e248,55 [138,15; 558,50]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhosphorus , mmol/L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e0,78 [0,665; 0,93]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDaily calciuria, mmol/D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e8,70 [6,095; 10,965]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eeGFR (CKD-EPI), mL/min/1.73 m2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e81,5 [64,0; 97,5]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDXA scans (Z-scores), SD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e-1,45 [-2,35; -0,75]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 220px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReduction in BMD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003eSD (T-scores.) \u0026ge; -1,0 / SD (Z-scores) \u0026ge; -2,0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e24 (27,9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003e-1,0 \u0026gt; SD (T-scores) \u0026gt; -2,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e18 (20,9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003eSD (T-scores) \u0026nbsp;\u0026le;-2,5 / SD (Z-scores) \u0026lt; -2,0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e29 (33,7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003eSD (T-scores) \u0026nbsp;\u0026le;-2,5 + low-energy fracture /\u003c/p\u003e\n \u003cp\u003eSD (Z-scores) \u0026lt; -2,0 + low-energy fracture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e15 (17,4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVertebral fractures\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e19 (19,8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNon-vertebral low-energy fractures\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e14 (14,6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNephrocalcinosis/nephrolithiasis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e68 (70,8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Histological features of parathyroid tumours.\u0026nbsp;\u003c/strong\u003eThis table provides an overview of the histological classification of parathyroid tumors (adenoma, atypical tumor, hyperplasia, carcinoma) and the expression levels of key markers (CaSR, VDR, AMA), categorized as strong, moderate, or weak/absent expression.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"602\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 430px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSign\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMe [Q\u003csub\u003e1\u003c/sub\u003e; Q\u003csub\u003e3\u003c/sub\u003e] / n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 220px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003eAdenoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e83 (86,5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003eAtypical tumor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e10 (10,4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003eHyperplasia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e1 (1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003eCarcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e2 (2,1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 220px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCaSR expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003e1*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e92 (95,8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e4 (4,2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 220px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVDR expression\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e51 (53,1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e35 (36,5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e10 (10,4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 220px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAMA expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e46 (47,9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e47 (49%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 209px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e3 (3,1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e* 1 - strong expression, 2 - moderate expression, 3 - weak or absence expression.\u003c/p\u003e\n\u003cp\u003eFurther we conducted a comparative analysis of groups 1-4. As expected, the groups differed in levels of iPTH and bone remodeling markers. Among the clinical features statistically significant differences were found only for the frequency of fibrocystic osteitis which was higher in patients with Ca\u003csub\u003ecorr\u003c/sub\u003e more than 3,5 mmol/l. A significant decrease in BMD and impaired renal filtration function showed a statistical trend that was more pronounced in patients with hypercalcemia greater than 3 mmol/L.\u003c/p\u003e\n\u003cp\u003eWe performed a correlation analysis to assess the relationship between the degree of IHC expression and key indicators of calcium-phosphorus metabolism; however, no significant correlations were identified. Additionally, no correlation was found between the expression of CaSR and VDR and the effectiveness of the cinacalcet therapy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eParathyroid cells \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e respond to increasing concentrations of extracellular ionized calcium by reducing PTH secretion. This effect is mediated by a direct interaction of calcium with a G protein-coupled CaSR. In patients with PHPT high concentrations of ionized calcium do not suppress autonomous PTH secretion, but the molecular mechanism for this abnormality is not clearly understood.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] CaSR down-regulation is generally believed to be the principal reason for the abnormal calcium-PTH interactions in parathyroid tumors, i.e. calcium responsiveness should be directly dependent upon CaSR expression on cells. Corbetta et al. reported diminished CaSR expression both at mRNA (by RT-PCR) and protein level (by Western blot) in 27 parathyroid adenoma and 4 hyperplasia cases. The authors also proposed that the disrupted response of parathyroid cells to extracellular calcium concentrations in adenomas might be associated with a 2-fold reduction in the expression of the membrane G-protein.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Singh et al. using real-time qPCR revealed reduced CaSR mRNA expression with a fold reduction of 0.12 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in parathyroid adenomas. The obtained data were correlated with the results of IHC results showing reduced expression of CaSR in 90% of adenomas. Unlike the findings of Singh et al., we did not observe a significant reduction of CaSR expression in different tumors compared to healthy tissue. The reaction with CaSR antibodies predominantly exhibited diffuse and pronounced membrane-cytoplasmic expression, which was almost similar to intact parathyroid tissue. Moreover, strong positive IHC reactions persisted even in cases of extremely \"aggressive\" PHPT (including carcinomas and atypical tumors) and hypercalcemia exceeding 3.5 mmol/L. At the same time, Haven et al. did not find a significant decrease of CaSR expression in parathyroid hyperplasia or adenomas. While only 1 of 104 benign tumors (1%) exhibited irregular or absent CaSR staining, carcinomas showed loss of CaSR expression in 31% of cases (9/29).[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eIn Agarwal et al.'s study[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], normal autopsy-derived parathyroid tissues showed strong but predominantly incomplete membranous CaSR expression (in 85% of cases). The cytoplasm of the normal parathyroid cells also showed strong expression of CaSR. In the PHPT group, membranous staining for CaSR was usually of moderate intensity. Besides, none of the parathyroid hyperplasia cases showed moderate cytoplasmic staining as the predominant pattern, in contrast to 16% of adenomas and 43% of the carcinomas. The authors suggested that not only diminished CaSR synthesis, but additional defects in the tracking of the receptor from the cytoplasm to the membrane may play a role in the PHPT pathogenesis. In our study, pure cytoplasmic staining was observed only in 11.5%.\u003c/p\u003e\u003cp\u003eInteresting data was obtained by Shi Y. et al. Using flow cytometric analysis of resected adenomatous parathyroid glands, they have isolated and characterized chief and oxyphil cells, as well as tumor-infiltrating lymphocytes. While chief and oxyphil cells exhibited comparable CaSR expression at both protein and mRNA levels, oxyphil cells demonstrated greater calcium sensitivity despite showing higher basal PTH secretion. This suggests that CaSR expression alone does not determine PTH production and calcium responsiveness in the parathyroid gland. The increased PTH levels in PHPT may reflect not only a higher number of tumor cells but also the greater PTH secretion profile of oxyphil compared to chief cells. The authors further proposed that tumor-infiltrating lymphocytes might target antibodies against CaSR, PTH, or other parathyroid antigens. Such antibody-mediated mechanisms - through either CaSR inhibition or immune clearance of CaSR-expressing/PTH-producing cells - could initially trigger compensatory hyperplasia, potentially progressing to adenoma formation.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] In our work, the majority of adenomas exhibited a mixed cellular composition. Furthermore, there were no corellations between serum calcium levels and adenoma histological type.\u003c/p\u003e\u003cp\u003eWith regard to VDR, we obtained more expected results. The VDR expression was reduced or absent in approximately 50% of cases, which is generally consistent with previously published research. Carling et al. demonstrated similar VDR mRNA levels in the adenomas and hyperplasias, which were reduced (42 +/- 2.8% and 44 +/- 4.0%) compared to the normal glands (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] Rao et al. evaluated receptor expression by IHC in 24 parathyroid adenomas and in 11 normal glands.There was reduction of VDR expression in almost all cells of parathyroid adenomas. The decrease in CaSR expression was not so pronounced as VDR which generally does not contradict our data. The authors proposed that reduced CaSR expression may frequently occur secondary to VDR loss. While this downregulation may be moderate, it appears sufficient to elevate the PTH secretory set-point and subsequently promote adenoma development.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] This hypothesis is supported by the presence of vitamin D response elements (VDREs) in the 5'-flanking region of the \u003cem\u003eCASR\u003c/em\u003e gene, enabling its transcriptional regulation through 1,25-dihydroxyvitamin D3-VDR complex binding to these regulatory sequences.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eData on the AMA expression in the parathyroid tumors are limited. We observed heterogeneous expression of the AMA throughout the studied samples. It was noted that the cytoplasm of oxyphil cells exhibited more intense staining compared to chief and clear cells. The most likely reason for this reaction is the presence of a high number of mitochondria in the cytoplasm of oncocytes. In contrast, cells with clear cytoplasm displayed the weakest staining, with some cases showing no staining at all. The origin of these clear cells remains insufficiently studied. There is a hypothesis that clear cytoplasm cells represent the final stage of chief cell hyperplasia. Ultrastructural analysis of this cell type has shown that vacuoles may either be dilated structures of the Golgi complex or components of the endoplasmic reticulum, which could explain the observed IHC reactions with AMA. For a more accurate assessment, further analysis on a larger sample size is necessary. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eIn the study by Varshney S. et al., the expression of CaSR and VDR in parathyroid adenomas was lower than in normal tissue ( according to IHC analysis and mRNA expression). No correlations were identified between СaSR and VDR expression and preoperative levels of calcium, iPTH, and 25(OH)D. Other markers including PTH and CD1 also did not correlate with serum calcium and iPTH. Similar results were obtained in our study. We found no differences in the expression of CaSR, VDR, and AMA among groups with varying severity of hypercalcemia, as well as no correlations between the IHC phenotype and the response to calcimimetic therapy.The assessment of the immunohistochemical profile of parathyroid tumors in relation to the severity of hypercalcemia has been conducted for the first time.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAccording to obtained results CaSR expression remained strong in the majority of tumors, including aggressive cases, suggesting that reduced CaSR expression is not the primary driver of hypercalcemia severity. In contrast, VDR expression was diminished in approximately half of the cases, aligning with previous research implicating VDR downregulation in PHPT pathogenesis. AMA expression exhibited heterogeneity, with oxyphil cells showing more intense staining, likely due to their mitochondrial-rich cytoplasm. No significant correlations were found between the IHC profiles of CaSR, VDR, or AMA and the degree of hypercalcemia, bone remodeling markers, or response to cinacalcet therapy. These results suggest that other molecular mechanisms, beyond the examined markers, may contribute to the clinical variability of PHPT. Further research is warranted to explore additional pathways and cellular interactions underlying the disease's heterogeneity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthical Approval:\u003c/h2\u003e\n\u003cp\u003eGenerated Statement: The studies involving humans were approved by the Ethics Committee of the Endocrinology Research Centre (Protocol No. 1 dated January 17, 2018) (Moscow, Russia). The studies were conducted in accordance with the local legislation and institutional requirements, as well as with the Helsinki Declaration. The participants provided their written informed consent to participate in this study.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCompeting interests:\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eAll authors contributed equally to the manuscript and read and approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThis article was supported by the Russian Science Foundation, agreement №24-15-00269.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials:\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMizamtsidi M, Nastos C, Mastorakos G, Dina R, Vassiliou I, Gazouli M, et al. Diagnosis, management, histology and genetics of sporadic primary hyperparathyroidism: old knowledge with new tricks. Endocr Connect. 2018;7:R56\u0026ndash;68.\u003c/li\u003e\n\u003cli\u003eAl Hassan MS, El Ansari W, Issa N, Darweesh A, Abdelaal A. Severe hypercalcemia secondary to parathyroid adenoma: Series of four consecutive cases at a tertiary care hospital in Qatar. Int J Surg Case Rep. 2023;109:108560.\u003c/li\u003e\n\u003cli\u003eHowson P, Kruijff S, Aniss A, Pennington T, Gill AJ, Dodds T, et al. Oxyphil Cell Parathyroid Adenomas Causing Primary Hyperparathyroidism: a Clinico-Pathological Correlation. Endocr Pathol. 2015;26:250\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eCorbetta S, Mantovani G, Lania A, Borgato S, Vicentini L, Beretta, et al. Calcium‐sensing receptor expression and signalling in human parathyroid adenomas and primary hyperplasia. Clin Endocrinol (Oxf). 2000;52:339\u0026ndash;48.\u003c/li\u003e\n\u003cli\u003eSudhaker Rao, Han, Phillips ER, Palnitkar S, Parfitt. Reduced vitamin D receptor expression in parathyroid adenomas: implications for pathogenesis. Clin Endocrinol (Oxf). 2000;53:373\u0026ndash;81.\u003c/li\u003e\n\u003cli\u003eCosta-Guda J, Tokura T, Roth SI, Arnold A. Mitochondrial DNA mutations in oxyphilic and chief cell parathyroid adenomas. BMC Endocr Disord. 2007;7:8.\u003c/li\u003e\n\u003cli\u003eCetani F, Picone A, Cerrai P, Vignali E, Borsari S, Pardi E, et al. Parathyroid Expression of Calcium-Sensing Receptor Protein and \u003cem\u003ein Vivo\u003c/em\u003e Parathyroid Hormone-Ca \u003csup\u003e2+\u003c/sup\u003e Set-Point in Patients with Primary Hyperparathyroidism \u003csup\u003e1\u003c/sup\u003e. J Clin Endocrinol Metab. 2000;85:4789\u0026ndash;94.\u003c/li\u003e\n\u003cli\u003eHaven CJ, van Puijenbroek M, Karperien M, Fleuren G, Morreau H. Differential expression of the calcium sensing receptor and combined loss of chromosomes 1q and 11q in parathyroid carcinoma. J Pathol. 2004;202:86\u0026ndash;94.\u003c/li\u003e\n\u003cli\u003eAgarwal S, Kardam S, Chatterjee P, Kumar C, Boruah M, Sharma MC, et al. CaSR expression in normal parathyroid and PHPT: new insights into pathogenesis from an autopsy-based study. J Endocrinol Invest. 2022;45:337\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003eShi Y, Hogue J, Dixit D, Koh J, Olson JA. Functional and genetic studies of isolated cells from parathyroid tumors reveal the complex pathogenesis of parathyroid neoplasia. Proceedings of the National Academy of Sciences. 2014;111:3092\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eCarling T, Rastad J, Szabó E, Westin G, Åkerström G. Reduced Parathyroid Vitamin D Receptor Messenger Ribonucleic Acid Levels in Primary and Secondary Hyperparathyroidism*. J Clin Endocrinol Metab. 2000;85:2000\u0026ndash;3.\u003c/li\u003e\n\u003cli\u003eSudhaker Rao, Han, Phillips ER, Palnitkar S, Parfitt. Reduced vitamin D receptor expression in parathyroid adenomas: implications for pathogenesis. Clin Endocrinol (Oxf). 2000;53:373\u0026ndash;81.\u003c/li\u003e\n\u003cli\u003eCanaff L, Hendy GN. Human Calcium-sensing Receptor Gene. Journal of Biological Chemistry. 2002;277:30337\u0026ndash;50.\u003c/li\u003e\n\u003cli\u003eDuan K, Gomez Hernandez K, Mete O. Clinicopathological correlates of hyperparathyroidism. J Clin Pathol. 2015;68:771\u0026ndash;87.\u003c/li\u003e\n\u003cli\u003eVarshney S, Bhadada SK, Saikia UN, Sachdeva N, Behera A, Arya AK, et al. Simultaneous expression analysis of vitamin D receptor, calcium-sensing receptor, cyclin D1, and PTH in symptomatic primary hyperparathyroidism in Asian Indians. Eur J Endocrinol. 2013;169:109\u0026ndash;16.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-endocrine-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bend","sideBox":"Learn more about [BMC Endocrine Disorders](http://bmcendocrdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bend/default.aspx","title":"BMC Endocrine Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"primary hyperparathyroidism, hypercalcemia, immunohistochemistry, parathyroid tumors, CaSR, VDR, cinacalcet","lastPublishedDoi":"10.21203/rs.3.rs-6811379/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6811379/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Primary hyperparathyroidism (PHPT) is usually caused by parathyroid tumors, resulting in hypercalcemia due to excessive PTH secretion. While disease severity correlates with calcium blood levels, the molecular mechanisms driving clinical variability remain unclear. The role of the calcium-sensing receptor (CaSR), the vitamin D receptor (VDR) and parathyroid cells mitochondrial activity in the pathogenesis of hyperparathyroidism is of particular interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThis retrospective study included 96 patients with PHPT who underwent parathyroidectomy. Patients were stratified by albumin-corrected calcium blood levels (≤ 2.8; 2.8\u0026lt;Ca\u003csub\u003ecorr\u003c/sub\u003e.≤ 3.0; 3.0 ≤ Ca\u003csub\u003ecorr.\u003c/sub\u003e\u0026lt; 3.5 Ca\u003csub\u003ecorr.\u003c/sub\u003e≥ 3.5 mmol/L). Immunohistochemical expression (IHC) was estimated with CaSR, VDR, and antimitochondrial antibodies (AMA).Statistical analysis was performed using the Statistica v. 13.3 software package (TIBCO Software Inc., USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Strong CaSR expression was observed in 95.8% of tumors, whereas VDR expression was reduced in 46.9%. AMA staining was heterogeneous, with high reactivity in oxyphilic cells. No significant differences in CaSR, VDR, or AMA expression were found across groups with different hypercalcemia severity. No significant correlations were identified between IHC markers expression and parameters of calcium-phosphorus metabolism, as well as cinacalcet treatment response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Despite their established roles in parathyroid regulation, CaSR and VDR expression did not correlate with hypercalcemia severity in PHPT. These findings suggest that alternative molecular mechanisms, rather than receptor expression levels, contribute to clinical heterogeneity. Further research is required.\u003c/p\u003e","manuscriptTitle":"Immunohistochemical Expression of CaSR, VDR, and AMA in Parathyroid Tumors Linked to Hypercalcemia Severity in Primary Hyperparathyroidism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-25 06:19:04","doi":"10.21203/rs.3.rs-6811379/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-25T22:55:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-03T17:57:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-01T14:09:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51054048732138961555557989717996727249","date":"2025-07-30T20:06:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156071239704462134693721631057062059697","date":"2025-07-21T11:58:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-20T18:22:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-16T06:59:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-24T06:18:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-20T13:10:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Endocrine Disorders","date":"2025-06-20T13:07:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-endocrine-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bend","sideBox":"Learn more about [BMC Endocrine Disorders](http://bmcendocrdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bend/default.aspx","title":"BMC Endocrine Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c6a9e213-e06a-462d-b6fd-4552504080ee","owner":[],"postedDate":"July 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-26T22:08:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-25 06:19:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6811379","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6811379","identity":"rs-6811379","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}