Immunocyte density in parathyroid carcinoma is correlated with disease relapse.

PURPOSE: Parathyroid carcinoma (PC) is an endocrine malignancy with a poor prognosis. The tumour immune microenvironment is a critical factor influencing the outcomes of multiple cancer types. However, knowledge of the immune microenvironment in PC remains limited. METHODS: The intratumoural density of immunocytes and the Ki-67 index were evaluated immunohistochemically in 51 PC patient samples and were compared with clinicopathological features and parafibromin staining results. The Kaplan-Meier method and Cox proportional hazards analysis were used to estimate the effects of these variables on clinical outcomes. RESULTS: Intratumoural immunocyte density was not correlated with age, gender, urolithiasis, or palpation of a neck mass. The Ki-67 index was correlated with the intratumoural density of CD3+ cells (P = 0.022) and CD8+ cells (P = 0.021) and serum calcium levels (P = 0.022). In the intratumoural area of primary foci, Kaplan-Meier method showed that the risk factors associated with recurrence/metastasis were a low density of CD3+ (P = 0.017), CD8+ (P = 0.019) and CD45+ cells (P = 0.047), a high density of CD163+ cells (P = 0.003) and a high Ki-67 index (P = 0.004). Cox regression multivariate analysis revealed that CD163+ cell density (hazard ratio (HR) 16.19, 95% confidence interval (CI) 1.99-131.66; P = 0.009) and CD8+ cell density (HR 0.13, 95% CI 0.02-0.76, P = 0.024) were independent factors associated with PC relapse. CONCLUSION: The immune microenvironment is an important factor influencing the relapse of PC. The intratumoural density of CD3+, CD8+, CD45+, and CD163+ immunocytes was correlated with disease-free survival (DFS) in patients with PC. Immunotherapy based on T lymphocytes or tumour-associated macrophages may be a promising treatment strategy.

Characterizing parathyroid carcinomas and atypical neoplasms based on the expression of programmed death-ligand 1 expression and the presence of tumor-infiltrating lymphocytes and macrophages.

BACKGROUND: Four distinct tumor microenvironments have been proposed based on the expression of programmed death-ligand 1 and the presence of tumor-infiltrating lymphocytes: immunotype I (adaptive resistance, tumor-infiltrating lymphocytes+ and programmed death-ligand 1+); immunotype II (immunologic ignorance, tumor-infiltrating lymphocytes- and programmed death-ligand 1-); immunotype III (intrinsic induction; tumor-infiltrating lymphocytes- and programmed death-ligand 1+); and immunotype IV (tolerance, tumor-infiltrating lymphocytes+ and programmed death-ligand 1-). These subtypes may predict tumor response to immunotherapy. We hypothesized that parathyroid neoplasms may have tumor immunogenic expression that can later be used to guide treatment. METHODS: We assessed retrospectively the immunohistochemical expression of programmed death-ligand 1 and the presence of tumor-infiltrating lymphocytes (CD3+ and CD8+) and macrophages (CD68+) in parathyroid carcinomas and in atypical parathyroid neoplasms treated at the M. D. Anderson Cancer Center from 1996 to 2016. Using intratumoral digital image analysis, the programmed death-ligand 1 H score was calculated with a standardized formula for predominant staining. The tumor-infiltrating lymphocytes per square millimeter of intratumoral areas were quantified. RESULTS: Within 30 specimens (17 parathyroid carcinomas and 13 atypical parathyroid neoplasms), there was no difference in the median programmed death-ligand 1 H score between the two groups (P = .57). Four parathyroid carcinoma cases had programmed death-ligand 1 H scores ≥1 associated with CD3+ and CD8+ tumor cell density; 2 of them had distant metastases. Parathyroid carcinomas had a lesser median CD3+ density (P = .04) and a lesser median CD8+ density (P =.07) than did atypical parathyroid neoplasms. Median CD68+ density did not differ between groups (P = .22). CONCLUSION: Parathyroid carcinomas tended to have immune-ignorant and immune-tolerant microenvironments within the neoplasm (immunotypes II and IV). Of the parathyroid carcinoma microenvironments, 17 had patterns of programmed death-ligand 1 and tumor-infiltrating lymphocytes expression (immunotype I), suggesting possible benefit from immunotherapy. In addition, both parathyroid carcinomas and parathyroid neoplasms expressed CD68+. Further exploration of these potential biomarkers as a target in cancer therapies is needed.

Role of calcium-sensing receptor, Galectin-3, Cyclin D1, and Ki-67 immunohistochemistry to favor in the diagnosis of parathyroid carcinoma.

BACKGROUND: As histopathological findings of parathyroid carcinoma are not certain, the diagnosis of tumors with degenerative changes may be difficult. In these cases, immunohistochemical markers are beneficial. We aimed to research the acceptability of calcium-sensing receptor (CaSR), Galactin-3, Cyclin D1, and Ki-67 as helpful markers in parathyroid tumors in cases which are difficult to diagnose. MATERIALS AND METHODS: Those cases who had been diagnosed with atypical parathyroid adenoma and parathyroid carcinoma between 2010 and 2015 were reevaluated. Immunohistochemical markers were applied to this cases. RESULTS: About 21 cases were parathyroid adenoma, 14 were atypical adenoma, and 10 cases were parathyroid carcinoma. According to the immunohistochemical results, global loss of CaSR staining was seen in 50% (5/10) of the patients with carcinoma while there was no loss of staining in those with parathyroid adenoma (P = 0,001). Global loss of CaSR staining was found in only one out of 14 cases with atypical adenoma. The expression of Galactin-3 was found to be positive in 40% (4/10) of carcinoma cases, 71.4% (10/14) of those with atypical adenoma, and 14.3% (3/21) of those with adenoma (P = 0,002). Cyclin D1 expression was determined to be positive in 70% (7/10) of patients with carcinoma, 71.4% (10/14) of atypical adenoma cases, and 23.8% (5/21) of those with adenoma. The Ki-67 proliferation index was seen to be above 5% in 50% (5/10) of carcinoma cases and 35,7% (5/14) of those with atypical adenoma. CONCLUSION: In these studies, it has been emphasized that the global loss of CaSR staining was used as a negative marker in the diagnosis of carcinoma. In this study, we have also confirmed that the global loss of CaSR staining is a useful marker to determine potential increased malignancy.

Inflammatory infiltrates in parathyroid tumors.

CONTEXT: Inflammatory infiltrates are sometimes present in solid tumors and may be coupled to clinical behavior or etiology. Infectious viruses contribute to tumorigenesis in a significant fraction of human neoplasias. OBJECTIVE: Characterize inflammatory infiltrates and possible viral transcription in primary hyperparathyroidism. DESIGN: From the period 2007 to 2016, a total of 55 parathyroid tumors (51 adenomas and 4 hyperplasias) with prominent inflammatory infiltrates were identified from more than 2000 parathyroid tumors in the pathology archives, and investigated by immunohistochemistry for CD4, CD8, CD20 and CD45 and scored as +0, +1 or +2. Clinicopathological data were compared to 142 parathyroid adenomas without histological evidence of inflammation. Transcriptome sequencing was performed for 13 parathyroid tumors (four inflammatory, 9 non-inflammatory) to identify potential viral transcripts. RESULTS: Tumors had prominent germinal center-like nodular (+2) lymphocytic infiltrates consisting of T and B lymphocytes (31%) and/or diffuse (+1-2) infiltrates of predominantly CD8+ T lymphocytes (84%). In the majority of cases with adjacent normal parathyroid tissue, the normal rim was unaffected by the inflammatory infiltrates (96%). Presence of inflammatory infiltrates was associated with higher levels of serum-PTH (P = 0.007) and oxyphilic differentiation (P = 0.002). Co-existent autoimmune disease was observed in 27% of patients with inflammatory infiltrates, which in turn was associated with oxyphilic differentiation (P = 0.041). Additionally, prescription of anti-inflammatory drugs was associated with lower serum ionized calcium (P = 0.037). CONCLUSIONS: No evidence of virus-like sequences in the parathyroid tumors could be found by transcriptome sequencing, suggesting that other factors may contribute to attract the immune system to the parathyroid tumor tissue.

Relationship Between the Neutrophil to Lymphocyte Ratio and Parathyroid Adenoma Size in Patients With Primary Hyperparathyroidism.

The aim of our study was to evaluate the relationship between neutrophil to lymphocyte ratio (NLR) and adenoma size in parathyroidectomized patients who underwent a parathyroidectomy. The neutrophil to lymphocyte ratio has recently become popular as a biomarker for malignant diseases or for estimating tumor size preoperatively. This study aimed to estimate the relationship between adenoma size and NLR. Furthermore, we assessed whether a higher level of NLR is correlated with the presence of parathyroid carcinoma. A retrospective chart review was performed for patients with parathyroid adenoma who underwent parathyroidectomy between January 2012 and August 2014. Data related to age, sex, NLR, parathyroid hormone level (PTH), preoperative calcium, phosphorus, adenoma size, and pathology reports were collected. The neutrophil to lymphocyte ratio was significantly correlated with calcium levels, PTH levels, parathyroid adenoma size, and the presence of cancer. However, there was no correlation between NLR and age, sex, and phosphorus levels. This study is the first to document a positive correlation between NLR and parathyroid adenoma size, as well as the presence of cancer, in patients who underwent surgery as a result of primary hyperparathyroidism.

Cytologic comparison of a primary parathyroid cancer and its metastatic lesions: a case report.

We describe the fine-needle aspiration cytology features of a primary parathyroid cancer and of the local recurrent and distant metastatic lesions. The presence of prognostic factors Ki-67 and proliferating cell nuclear antigen (PCNA) was compared immunohistochemically between primary parathyroid carcinoma and related metastatic and recurrent foci. Flow cytometric DNA analysis was also performed to investigate any chromosomal abnormality of the parathyroid carcinoma. Cytologic examination of the endocrine tumor showed that it comprised a loose cohesive cluster and tumor cells with granular cytoplasm and mild nuclear atypia, but for purposes of cytodiagnosis, it is difficult to determine whether such a neoplasm is malignant on the basis of morphology alone. Immunohistochemical analysis showed that Ki-67 and PCNA labeling indices were higher in the recurrent and metastasized carcinomas than in the primary cancer, suggesting that neoplastic cells become more malignant in the recurrent and metastasized foci. To our knowledge, this is the first report describing not only cytopathologic but also immunocytologic differences between primary parathyroid cancer and the metastatic lesion.

Parafibromin, product of the hyperparathyroidism-jaw tumor syndrome gene HRPT2, regulates cyclin D1/PRAD1 expression.

Parafibromin is the 531-amino-acid protein product encoded by HRPT2, a putative tumor suppressor gene recently implicated in the autosomal dominant hyperparathyroidism-jaw tumor familial cancer syndrome, sporadic parathyroid cancer, and a minority of families with isolated hyperparathyroidism. Parafibromin contains no identified functional domains but bears sequence homology to Cdc73p, a budding yeast protein component of the RNA polymerase II-associated Paf1 complex. This study addressed the expression and functional properties of human parafibromin. A survey of human and mouse tissues analysed with polyclonal antibodies to parafibromin showed specific immunoreactivity in adrenal and parathyroid glands, kidney, heart, and skeletal muscle. Subcellular fractionation and laser confocal microscopy of normal human parathyroid gland demonstrated expression of parafibromin in both the cytoplasmic and nuclear compartments. Parafibromin was expressed in four parathyroid adenomas but was absent from two parathyroid carcinomas. Transient overexpression of wild-type parafibromin, but not its Leu64Pro missense mutant implicated in parathyroid cancer and familial isolated hyperparathyroidism, inhibited cell proliferation, and blocked expression of cyclin D1, a key cell cycle regulator previously implicated in parathyroid neoplasia. These results demonstrate that human parafibromin is a nucleocytoplasmic protein with functions consistent with its postulated role as a tumor suppressor protein.

Induction of cellular immunity in a parathyroid carcinoma treated with tumor lysate-pulsed dendritic cells.

BACKGROUND: Cytotoxic T-lymphocyte-mediated tumor immunity against major histocompatibility antigen class II-negative tumors requires help from CD4(+) T-cells. The major antigen presenting cells for CD4(+) cell activation are dendritic cells. Studies in mice and humans have demonstrated the potent capacity of these cells to induce specific antitumor immunity. OBJECTIVE: To control the growth of a metastasized parathyroid carcinoma, by immunizing a patient with tumor lysate and parathyroid hormone-pulsed dendritic cells. DESIGN AND METHODS: Mature dendritic cells were generated from peripheral blood monocytes in the presence of granulocyte/macrophage colony-stimulating factor, interleukin-4 and tumor necrosis factor alpha. Antigen-loaded dendritic cells were delivered by subcutaneous and intralymphatical injections. After five cycles, we added keyhole limpet hemocyanin (KLH) as a CD4(+) helper antigen. RESULTS: After 10 vaccinations, a specific cellular immune response to tumor lysate was observed. In vitro T-cell proliferation assays revealed a dose-dependent stimulation index of 1.8-5.7 compared with 0.9-1.1 before vaccination. In vivo immune response was demonstrated by positive delayed-type hypersensitivity toward tumor lysate. Intradermal injection of tumor lysate resulted in an erythema and induration, suggesting the efficient generation of tumor lysate-specific memory T-cells. CONCLUSIONS: These data indicate that dendritic cell vaccination can induce in vitro and in vivo responses in a highly malignant endocrine carcinoma. Regardless of the clinical outcome of our patient, this approach might be generally applicable to other advanced, radio- and chemotherapy-resistant endocrine malignancies, such as adrenal carcinomas and metastasized medullary and anaplastic thyroid carcinomas.

Control of hypercalcaemia of parathyroid carcinoma by immunisation.

BACKGROUND: Patients with parathyroid tumours can develop extreme hypercalcaemia and osteitis fibrosa cystica. Clinical features result from the action of parathyroid hormone (PTH) on bone receptors. Because this hormone is produced in microgram quantities, inhibition of its metabolic effects with potent PTH antibodies should be possible. We tested whether an immunisation with synthetic human and bovine PTH peptides could stimulate autoantibodies against PTH. METHODS: A patient with metastatic parathyroid carcinoma in the lungs and pleura developed severe bone disease and extreme hypercalcaemia that proved resistant to conventional therapy. She was immunised with 200 microg human and bovine PTH peptides and 50 microg human PTH. Booster doses were also given at 4 weeks and 11 weeks. The patient was then seen every week. FINDINGS: Antibodies against PTH were produced within 4 weeks of initial immunisation and titres increased with repeated doses of immunogens. Total serum calcium concentrations, which had ranged from 3.5 mmol/L to 4.2 mmol/L over the previous 18 months, fell to between 2.5 mmol/L and 3.0 mmol/L over 6 months of therapy. This fall was accompanied by striking clinical improvement. INTERPRETATION: We believe this is the first use of immunotherapy to control remote, non-metastatic complications of malignant disease. B-cell tolerance to human PTH was broken by immunisation with PTH peptides in adjuvant. This therapeutic approach could be used to control excess hormone production in several types of endocrine tumour and may have applications in other diseases.

Evaluation of retinoblastoma and Ki-67 immunostaining as diagnostic markers of benign and malignant parathyroid disease.

Assessment of the malignant potential of parathyroid tumors in the absence of metastases can be difficult using morphologic criteria alone. In this study we have examined a total of 58 parathyroid tumors (31 benign, 15 malignant, and 12 equivocal) from 54 patients using immunohistochemistry with monoclonal antibodies directed against the retinoblastoma (RB) protein and the cell cycle-associated antigen Ki-67 to evaluate their role as diagnostic markers. RB protein immunoreactivity was not useful for distinguishing between benign and malignant parathyroid tumors. Analysis of the proliferation marker Ki-67 showed that there was a trend toward more intense staining in the malignant cases. The Ki-67 labeling index was highest in the parathyroid cancers (median 33) and lowest in the sporadic primary adenomas (median 2). An observation that might have clinical implications is that tumors from patients with familial hyperparathyroidism linked to chromosome 1q showed a high Ki-67 index, indicating strong proliferative activity (median 25). This correlates well with the clinical observation of tumors with malignant potential in this syndrome. Because of the considerable overlap between groups of tumors, Ki-67 is not suitable for definitive differentiation between benign and malignant tumors. However, Ki-67 may give valuable information about which patients should be followed more closely.;1999>

Dendritic cell immunotherapy induces antitumour response in parathyroid carcinoma and neuroendocrine pancreas carcinoma.

Parathyroid carcinomas and neuroendocrine carcinomas of the pancreas are rare malignancies in humans. Because of their low radio- and chemosensibility, they fail to respond to conventional therapy. We therefore tested a dendritic cell immunotherapy in an attempt to control the tumour growth in two patients. Studies on mice and humans have demonstrated the potent capacity of dendritic cells to induce specific antitumour immunity. Mature dendritic cells were generated from peripheral blood monocytes in the presence of granulocyte/macrophage colony-stimulating factor, interleukin 4 and tumour necrosis factor alpha. Dendritic cells were either loaded with parathyroid hormone (PTH) or with (pancreas) tumour-derived lysate (TL), respectively, and were delivered by subcutaneous injections. All immunizations were well tolerated with no side effects, and were administered on an outpatient basis. After repeated vaccinations, specific in vivo immune response was demonstrated by positive delayed-type hypersensitivity (DTH) toward PTH or TL, demonstrating the efficient generation of antigen-specific memory T-cells. DTH reactivity was accompanied by a significant decrease of tumour markers in both patients. This approach might be generally applicable to other advanced, radio- and chemotherapy-resistant endocrine malignancies.

[Antigenicity of fresh, cryopreserved, or liquid-medium-preserved human parathyroid adenomas]. / Antigénicité des adénomes parathyroïdiens humains frais, cryopréservés et conservés en milieu liquide.

We have studied the ability of the cryopreservation and culture techniques to reduce the antigenicity of human parathyroid tissue by suppressing HLA DR bearing cells. Antigenicity was studied with an immunoperoxidase technique applied on frozen sections. Antibody against HLA DR, CD1a, CD3, CD22, CD45RA, CD68 and H et Y antigens were used. In fresh parathyroid tissue, endothelial cells, histiocytes and interstitial dendritic cells expressed HLA DR antigens. Antigenicity of cryopreserved tissue were not altered. In cultured tissue, interstitial HLA DR bearing cells have disappeared but antigenicity of endothelial cells were not modified.

Major histocompatibility complex class II expression and parathyroid autoantibodies in primary hyperparathyroidism.

BACKGROUND: Autoimmune diseases are characterized by induced parenchymal expression of major histocompatibility complex (MHC) class II antigens and circulating autoantibodies directed toward surface structures on the target cells. MHC class II expressions can be modified by viral infections of potential pathogenic importance in autoimmune reactions. Primary hyperparathyroidism exhibits incompletely clarified cause. METHODS: With cryosections, human parathyroid glands were stained with monoclonal antibodies to MHC class II antigens according to a peroxidase-antiperoxidase technique. Human parathyroid adenoma tissue transplanted to nude mice and rat parathyroid glands was tested with serum from patients with hyperparathyroidism and control subjects. RESULTS: Induced MHC class II expression was demonstrated on parathyroid parenchymal cells in 13 of 54 adenomatous and eight of 23 hyperplastic glands of patients with primary hyperparathyroidism. This reactivity was absent in 12 normal glands, nine normal-sized glands associated with the adenomas, and 17 enlarged glands of patients with hyperparathyroidism caused by uremia. Staining of parathyroid tissue was found with serum from 27 of 38 patients with primary hyperparathyroidism, whereas this reactivity was absent on rat thyroid and pancreatic tissue, as well as with control sera. CONCLUSION: The concurrent induction of MHC class II antigen expression and c circulating antiparathyroid autoantibodies in 16 or 38 patients with primary hyperparathyroidism suggests hitherto unrecognized immunologic involvement in this disease.

Detection of apoptotic cells and expression of Ki-67 antigen, Bcl-2, p53 oncoproteins in human parathyroid adenoma.

Presence of apoptotic cells and immunoreactivity to Ki-67, bcl-2 and p53 were studied in 20 cases of parathyroid adenoma. To determine apoptosis, the DNA nick end labeling method was used. 85% of the parathyroid adenomas were found to harbor apoptotic cells. All of the 20 adenomas contained Ki-67 immunoreactive cells. Proliferative activity was not more confined to nodular than to diffuse areas, but there was a highly significant difference in Ki-67 immunoreactivity between adenomatous tissue and the residual rim of normal tissue outside the adenoma. No Ki-67 immunoreactive cells were found in two normal parathyroid glands used as controls. All but one of the adenomas (95%) demonstrated immunoreactivity to bcl-2, but expression of p53 was detected in only a few adenomas (15%). There was a significant relationship between the adenoma weights and both Ki-67 and bcl-2. This study suggests that parathyroid adenomas contain cell populations with proliferative activity (clonal proliferation), but the weak immunoreactive expression of p53 combined with the relatively strong expression of bcl-2 might contribute to a slow glandular growth.

Expression and function of a CD3-like molecule on normal and abnormal human parathyroid cells.

BACKGROUND: Normal and abnormal human parathyroid tissue express the T-lymphocyte protein CD4, and parathyroid and lymphocyte cells show similarities with respect to mechanisms of calcium permeability and regulation of the cytoplasmic calcium concentration. METHODS: Anti-Leu4, a monoclonal antibody recognizing the T-lymphocyte glycoprotein complex CD3, is used to immunohistochemically stain normal and abnormal human parathyroid cells and to explore influences on the parathyroid hormone (PTH) secretion of enzymatically dispersed parathyroid cells. RESULTS: Parathyroid glands of patients with different forms of hyperparathyroidism displayed variable expression of the anti-CD3 reactive complex. The stainings correlated both positively and inversely to immunoreactivity for a previously defined calcium sensor, the decreased expression of which may constitute a molecular basis for hyperparathyroidism. Incubation of parathyroid cells with the anti-Leu4 antibody inhibited PTH secretion and reduced its sensitivity to external calcium without influence on parathyroid cytoplasmic calcium concentration. CONCLUSIONS: The results suggest that the human parathyroid cells express a CD3-like molecule with the ability to interact in PTH release.

Proliferative activity in parathyroid tumors as detected by Ki-67 immunostaining.

Clear morphological criteria for differentiating benign from malignant parathyroid tumors are not yet available and unfavorable prognosis cannot be predicted by histopathological parameters alone. A retrospective study of a series of parathyroid lesions was designed to evaluate the diagnostic role of the cell cycle-associated Ki-67 antigen detected by MIB-1 monoclonal immunocytochemistry. The mean tumor proliferative fraction (TPF), expressed as the number of Ki-67-positive nuclei per 1,000 cells, was 0.8 in normal parathyroid glands (nine specimens), 26.0 in hyperplasias (11 specimens), 32.8 in adenomas (11 specimens), and 60.5 in a group of tumors with histological features consistent with carcinoma (12 specimens). The difference between the latter two values was statistically significant (P < .05). When the five most clinically aggressive tumors were considered, the difference was even more remarkable (TPF, 78.6; P < .001). Oncocytic and pleomorphic cell components were found to proliferate with a labeling pattern similar to that of the chief cells. We conclude that proliferative activity is an additional useful parameter for evaluating parathyroid tumors diagnostically. Aggressive behavior may be expected in those tumors with a TPF greater than 6%.

Lymphocytic infiltration and destruction of parathyroid adenomas: a possible tumour-specific autoimmune reaction in two cases of primary hyperparathyroidism.

Two cases of primary hyperparathyroidism with underlying parathyroid adenomas were found to be associated with lymphocytic infiltration and destruction of the neoplastic tissue. There was no inflammatory infiltrate in the adjacent rim of the remnant of parathyroid gland or in the other tumour-free glands. The lymphoid cell population within the tumours was composed of both infiltrating T-cells and compact nodule-forming B-cells. In one of the tumours there was considerable fibrosis and atrophy of the adenomatous tissue. The histological picture was consistent with an autoimmune process directed against the adenomas, indicating that this reaction had, in part, been successful in reducing the abnormal cell population.