Parafibromin Expression Research Articles

Benign Parathyroid Gland Tumors and their Heterogeneity

Background: Primary hyperparathyroidism (pHPT) can be caused by different pathologic lesions – benign and malignant. The frequencies of changes vary, mainly because different criteria for diagnosis. It is especially visible in the group of benign lesions, which as it turns out, are not uniform in nature. The aim of the study was to attempt to assess the heterogeneity of benign parathyroid lesions, both adenoma and hyperplasia. Methods: Resected benign lesions from consecutive series of 125 patients treated surgically for pHPT, were studied retrospectively. We were looking for a corelation between DNA ploidy, cell proliferation activity (S phase fraction) and the expression of parafibromin and galectin-3. Results: In 74 (59.2%) of patients the diagnosis according to histopathological evaluation was benign adenoma and in 51 (40.8%) parathyroid hyperplasia. Diploid pathologies were found in 72 (57.6%) cases and aneuploid in 53(42.4%). From 74 adenomas, 39 (55%) were considered diploid and 35 (45%) aneuploid. Positive parafibromin expression was observed in 26 (49%) of aneuploid tumors and in 39 (54%) of diploid. Galectin-3 expression was found in 25 (47%) aneuploid and 30 (41%) diploid. In this changes of parathyroid glands, we did not observe any important differences between ploidy of cells and expression of parafibromin (p=0.16) and galectin (p- 0.39). Weak, but important correlation was observed between parafibromin expression and number of cells in S phase fraction in diploid pathologies (p=0.047). There is important, but weak correlation between galectin-3 expression and amount of cells in S phase fraction in aneuploid pathologies (p=0.05). In diploid pathologies, the correlation was not observed (R=0.02, p=0.83). Conclusions: 1) The nature of the cellular ploidy of benign parathyroid lesions indicates the heterogeneity. 2) A negative reactions with parafibromin and positive reactions with galectin-3 were found among adenomas as well as benign parathyroid hyperplasia. 3) In benign parathyroid lesions the expression of parafibromin and galectin-3 correlate with an increase in the number of S phase fraction cells, diploid and aneuploid, respectively.

Diagnostic and prognostic implications of parafibromin immunohistochemistry in parathyroid carcinomaT.

The aim of the present study was to elucidate the diagnostic and prognostic implications of parafibromin immunohistochemistry (IHC) in parathyroid carcinoma (PC). We performed a meta-analysis to examine the rate of loss of parafibromin expression from 18 eligible studies. In addition, a diagnostic test accuracy review was conducted to investigate the diagnostic role of parafibromin in PC. The rates of loss of parafibromin expression were 0.522 (95% CI: 0.444–0.599), 0.291 (95% CI: 0.207–0.391), 0.027 (95% CI: 0.011–0.064), and 0.032 (95% CI: 0.008–0.119) in PC, atypical parathyroid adenoma (APA), parathyroid adenoma (PA), and parathyroid hyperplasia, respectively. In the diagnostic test accuracy review for diagnosis of PC, the pooled sensitivity and specificity of parafibromin IHC was 0.53 (95% CI: 0.46–0.59) and 0.96 (95% CI: 0.95–0.97), respectively. The diagnostic odds ratio and the area under curve on summary receiver operating characteristic curve was 25.31 (95% CI: 8.91–71.87) and 0.7954, respectively. In addition, the meta-analysis demonstrated that loss of parafibromin expression was significantly correlated with worse disease-free survival (hazard ratio: 2.832; 95% CI: 1.081–7.421). Loss of parafibromin IHC expression was significantly higher in PC than in APA, PA, and parathyroid hyperplasia. Parafibromin IHC could be useful for diagnosis and prediction of prognosis of PC in daily practice.

Parafibromin-deficient (HPT-JT Type, CDC73 Mutated) Parathyroid Tumors Demonstrate Distinctive Morphologic Features.

The gene CDC73 (previously known as HRPT2) encodes the protein parafibromin. Biallelic mutation of CDC73 is strongly associated with malignancy in parathyroid tumors. Heterozygous germline mutations cause hyperparathyroidism jaw tumor syndrome,which is associated with a high life-time risk of parathyroid carcinoma. Therefore loss of parafibromin expression by immunohistochemistry may triage genetic testing for hyperparathyroidism jaw tumor syndrome and be associated with malignant behavior in atypical parathyroid tumors. We share our experience that parafibromin-negative parathyroid tumors show distinctive morphology. We searched our institutional database for parathyroid tumors demonstrating complete loss of nuclear expression of parafibromin with internal positive controls. Forty-three parafibromin-negative tumors from 40 (5.1%) of 789 patients undergoing immunohistochemistry were identified. Thirty-three (77%) were external consultation cases; the estimated incidence in unselected tumors was 0.19%. Sixteen (37.2%) fulfilled World Health Organization 2017 criteria for parathyroid carcinoma and 63% had serum calcium greater than 3mmol/L. One of 27 (3.7%) noninvasive but parafibromin-negative tumors subsequently metastasized. Parafibromin-negative patients were younger (mean, 36 vs. 63 y; P<0.001) and had larger tumors (mean, 3.04 vs. 0.62 g; P<0.001). Not all patients had full testing, but 26 patients had pathogenic CDC73 mutation/deletions confirmed in tumor (n=23) and/or germline (n=16). Parafibromin-negative tumors demonstrated distinctive morphology including extensive sheet-like rather than acinar growth, eosinophilic cytoplasm, nuclear enlargement with distinctive coarse chromatin, perinuclear cytoplasmic clearing, a prominent arborizing vasculature, and, frequently, a thick capsule. Microcystic change was found in 21 (48.8%). In conclusion, there are previously unrecognized morphologic clues to parafibromin loss/CDC73 mutation in parathyroid tumors which, given the association with malignancy and syndromic disease, are important to recognize.

Parafibromin, APC, and MIB-1 Are Useful Markers for Distinguishing Parathyroid Carcinomas From Adenomas.

Differentiation of parathyroid carcinoma (PC) from parathyroid adenoma (PA) relies solely on the pathologic determination of invasion of surrounding structures and/or distant metastasis. Parathyroid lesions with atypical histologic features with no demonstration of invasion or metastasis present a diagnostic dilemma. Different authors report a parafibromin and adenomatous polyposis coli (APC) loss or reduction in PC cases. High proliferative activity of MIB-1 and increased galectin 3 expression are reported in PC. There is no clear cutoff for the sensitivity, specificity, or predictive value for all these markers. The immunohistochemical expression of parafibromin, APC, MIB-1, and galectin 3 was studied in 73 adenomas, 21 PCs, and 3 atypical adenomas. The presence or absence of each marker was identified through the use of a comprehensive scoring system based on multiplying the percentage of tumor cells stained (0 to 100) and the staining intensity (0 to 3) on each biopsy. The highest score that any slide could reach was 300. A cutoff of >100 was used to consider the specimen positive for parafibromin, APC, or galectin 3 staining. MIB-1 proliferation indices were calculated using image cytometry; proliferation indices >5% were considered positive. We identified parafibromin loss in 7/21 (33%) carcinomas and 1/73 (1%) adenomas. Loss of APC was seen in 20/21 (95%) carcinomas and 38/73 (52%) adenomas. MIB-1 indices were elevated in 18/21 (86%) carcinomas. MIB-1 indices were <5% in all (100%) adenomas. MIB-1 indices were elevated in 2/3 (67%) atypical adenomas. Our study presents a clear cutoff to determine the practicality of using parafibromin, APC, and MIB-1 as immunohistochemical markers to differentiate between PCs and PAs. Loss of parafibromin and a high MIB-1 index are both independently sensitive and specific markers for the diagnosis of PC. Loss of APC was only specific for PC. This panel of markers provides a novel, useful approach in the diagnosis and differentiation of PCs from PAs.

The in vitro and in vivo effects of nuclear and cytosolic parafibromin expression on the aggressive phenotypes of colorectal cancer cells: a search of potential gene therapy target

Down-regulated parafibromin is positively linked to the pathogenesis of parathyroid, lung, breast, ovarian, gastric and colorectal cancers. Here, we found that wild-type (WT) parafibromin overexpression suppressed proliferation, tumor growth, induced cell cycle arrest and apoptosis in colorectal cancer cells (p<0.05), but it was the converse for mutant-type (MT, mutation in nucleus localization sequence) parafibromin (p<0.05). Both WT and MT transfectants inhibited migration and invasion, and caused better differentiation (p<0.05) of cancer cells. WT parafibromin transfectants showed the overexpression of Cyclin B1, Cyclin D1, Cyclin E, p38, p53, and AIF in HCT-15 and HCT-116 cells, while MT parafibromin only up-regulated p38 expression. There was lower mRNA expression of bcl-2 in parafibromin transfectants than the control and mock, while higher expression of c-myc, Cyclin D1, mTOR, and Raptor. According to transcriptomic analysis, WT parafibromin suppressed PI3K-Akt and FoxO signaling pathways, while MT one promoted PI3K-Akt pathway, focal adhesion, and regulation of actin cytoskeleton. Parafibromin was less expressed in colorectal cancer than paired mucosa (p<0.05), and inversely correlated with its differentiation at both mRNA and protein levels (p<0.05). These findings indicated that WT parafibromin might reverse the aggressive phenotypes of colorectal cancer cells and be employed as a target for gene therapy. Down-regulated parafibromin expression might be closely linked to colorectal carcinogenesis and cancer differentiation.

CDC73 gene mutations in sporadic ossifying fibroma of the jaws.

BackgroundThe tumor suppressor gene CDC73 was found to be associated with hyperparathyroidism-jaw tumor syndrome (HPT-JT), which is characterized by parathyroid adenoma or carcinoma, ossifying fibroma (OF) of the jaws, and renal and uterine lesions. Mutations in CDC73 have also been frequently detected in sporadic parathyroid carcinomas and renal tumors. However, the prevalence and range of CDC73 mutations in sporadic OFs have not been established.MethodsWe directly sequenced coding and flanking splice junctional regions of CDC73 in 40 cases of sporadic OF of the jaws. We also used immunohistochemistry to detect parafibromin, the protein product of CDC73, in those cases.ResultsTwo novel CDC73 mutations were identified in 2 of the 40 cases (5 %). Both were somatic mutations located in exon 1 of the coding region. Strong parafibromin expression was detected in all 40 cases, irrespective of the presence of CDC73 mutations.ConclusionsMutations inCDC73 were rare in sporadic OF of the jaws, but may affect the pathogenesis of a small subset of tumors of this type.

Significance of Parafibromin Expression in Laryngeal Squamous Cell Carcinomas.

Background:Parafibromin is a product of the tumor suppressor gene that has been studied as a potential indicator of tumor aggressiveness in the parathyroid, breast, colorectum, and stomach. However, the clinical significance and potential function of parafibromin expression in head and neck squamous cell carcinomas remain largely unknown. The aim of this study was to evaluate the expression of parafibromin in laryngeal squamous cell carcinoma (LSCC) and to verify its potential as a biomarker of tumor behavior.Methods:Parafibromin expression was evaluated in 30 cases of LSCC using immunohistochemistry. The correlations between parafibromin expression and clinicopathologic parameters were investigated.Results:Parafibromin expression was positive in 15 cases (50%) and negative in 15 cases (50%). Tumor size and T stage showed a statistically significant inverse relationship with parafibromin expression (p=.028 and p<.001, respectively). Parafibromin expression was not associated with age, sex, lymph node metastasis, tumor differentiation, or tumor location. There was no statistically significant relationship between parafibromin expression and progression-free survival in the patients (p>.05).Conclusions:Our results indicate that the downregulation or loss of parafibromin expression can be employed as a novel marker of tumor progression or aggressiveness in LSCC.

The Relation of Parafibromin Expression with Clinicopathological Factors in Invasive Breast Carcinomas.

We aimed to find the relationship between parafibromin expression and clinicopathologic variables of breast carcinoma. Ninety-seven cases of invasive breast carcinoma diagnosed at our department between the years 2010-2013 were included in the study. The parafibromin expression state was compared with the estrogen receptor, progesterone receptor, cerbB2, Ki67 results, and the clinicopathological variables. Among 97 breast carcinoma cases, 66 (68%) were invasive ductal carcinoma. The average age was 54.3 (min:25, max:100), and the average tumor size was 31.1 mm (min:7, max:120). Lymph node metastasis was detected in 58% of the cases. Eleven were diagnosed with metastasis amongst 77 cases whose distant metastasis data could be reached. Eleven cases were lost due to breast carcinoma. As the tumor grade increased, the possibility of distant metastasis and lymph node metastasis increased as well (p=0.04, p=0.05, respectively). The mean follow-up duration of the cases was 26.6 ± 9.8 (min. 6, max. 53) months, and there was no significant difference in survival between the other variables. Of the cases, 21.6% were negative, 9.3% were (+) positive, 11.3% were (++) positive and 57.7% were (+++) positive for parafibromin. It was found that there was an inverse correlation between the Ki67 proliferation index and lymph node metastasis and the parafibromin expression (p=0.018, p=0.029, respectively). We suggest that parafibromin may be a possible prognostic and predictive parameter for breast carcinomas. As the data on this matter in the literature is limited, it would be beneficial to investigate the matter and evaluate its relationship with survival in larger series.

The Role of Parafibromin, Galectin-3, HBME-1, and Ki-67 in the Differential Diagnosis of Parathyroid Tumors.

Parathyroid carcinoma (PC) is a rare parathyroid tumor compared to parathyroid adenoma (PA) and atypical parathyroid adenoma (APA). Recent studies have suggested parafibromin has a role in the differential diagnosis of parathyroid tumors. We sought to determine the role of parafibromin as well as galectin-3, Ki-67, and HBME-1 as diagnostic markers in the differential diagnosis of parathyroid tumors. A total of 92 cases diagnosed with PA, APA, or PC at Sifa University and Private Ege Pathology Laboratory between 2006-2012 were included in the study. Parafibromin (microarray), galectin-3, Ki-67, and HBME-1 were evaluated using immunohistochemistry in all parathyroid tumors. Eighty-four cases were diagnosed with PA, six with APA, and two with PC. The study group consisted of 82 females and 10 males. Their mean age was 50.9 years, and the mean tumor diameter was 1.97 cm. Parafibromin was negative in the two PC cases but positive in all APA and PA cases. Positivity was observed with galectin-3 in 17 adenoma cases, three atypical adenomas, and two carcinoma cases. Positivity with HBME-1 was found in 26 PA cases and one PC case. Parafibromin and galectin-3 expression was significant between the three tumor groups but not for HBME-1 expression. Parafibromin expression increased in PA whereas galectin-3 expression decreased. A statistical significance was found between the three tumor groups according to the Ki-67 score (p=0.010). Additionally, the Ki-67 proliferation index was under 1% in PAs. The number of PCs in our series was small so our data mostly reflects the immunohistochemical characteristics of PAs. Parafibromin expression, galectin-3 negativity, and a Ki-67 proliferation index under 1% were estimated as beneficial in the differential diagnosis of difficult parathyroid tumors.

The roles of parafibromin expression in ovarian epithelial carcinomas: a marker for differentiation and prognosis and a target for gene therapy.

Parafibromin is a protein encoded by hyperparathyroidism 2 (HRPT2) and its downregulated expression is involved in the pathogenesis of parathyroid, breast, gastric, colorectal, lung, head and neck cancers. We aimed to investigate the roles of parafibromin expression in tumorigenesis, progression, or prognostic evaluation of ovarian cancers. HRPT2-expressing plasmid was transfected into ovarian cancer cells with the phenotypes and related molecules examined. The messenger RNA (mRNA) and protein expression of parafibromin were also examined in ovarian normal tissue, benign and borderline tumors and cancers by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, or immunohistochemistry respectively. It was found that parafibromin overexpression caused a lower growth, migration and invasion, higher sensitivity to cisplatin and apoptosis than the mock and control (P < 0.05). The transfectants showed the hypoexpression of phosphoinositide 3-kinase (PI3K), Akt, p70 ribosomal protein S6 kinase (p70s6k), Wnt5a, B cell lymphoma-extra large (Bcl-xL), survivin, vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9) than the mock and control at both mRNA and protein levels (P < 0.05). According to real-time PCR, parafibromin mRNA level was lower in ovarian benign tumors and cancers than normal ovary (P < 0.05), while parafibromin was strongly expressed in metastatic cancers in omentum than primary cancers by Western blot. Immunohistochemically, parafibromin expression was stronger in primary cancers than that in ovarian normal tissue (P < 0.05) but weaker than the metastatic cancers (P < 0.05) with a positive correlation with dedifferentiation, ki-67 expression and the lower cumulative survival rate (P < 0.05). These findings indicate that parafibromin downregulation might promote the pathogenesis, dedifferentiation and metastasis of ovarian cancers possibly by suppressing aggressive phenotypes, such as proliferation, cell cycle, apoptosis, migration and invasion.

Parafibromin Staining Characteristics in Urothelial Carcinomas and Relationship with Prognostic Parameters.

Background:Parafibromin is a recently defined tumor suppressor gene. The aim of our study was to determine the relationships of parafibromin expression in urothelial carcinomas (UCs) with prognostic parameters and to evaluate the use of parafibromin as a potential marker of UC.Methods:Parafibromin expression was assessed in 49 UC specimens using immunohistochemistry. The correlations between parafibromin expression and clinical and pathologic parameters were investigated.Results:Of the patients, 42 (85.7%) were male, and the mean age was 69.6 ± 8.2 years (range, 54 to 88 years). Morphologically, the UCs were divided into two groups: papillary (n = 27) and non-papillary (n = 22). There were seven low-grade (14.3%) and 42 high-grade (85.7%) tumors. Parafibromin was negative in 13 tumors (26.5%), partially positive in 19 tumors (38.8%), and positive in 17 tumors (34.7%). Parafibromin expression was more negative in UCs from upper urinary locations (n=17) and with muscularis propria invasion (n=28), which was statistically significant (p = .009 and p = .007, respectively). There was no statistically significant relationship between parafibromin expression and gender, age, tumor grade, survival, or disease-free survival.Conclusions:We found that UC cases with parafibromin positivity had less of a tendency to show muscularis propria invasion and were more commonly located in the lower urinary system. These results need to be confirmed with studies based on larger case series.

The clinicopathological significances and biological functions of parafibromin expression in head and neck squamous cell carcinomas.

Downregulated parafibromin expression is involved in the pathogenesis and progression of parathyroid, breast, gastric, colorectal, and lung cancers. To investigate the roles of parafibromin expression in tumorigenesis, progression, and prognostic evaluation of head and neck squamous cell carcinomas (HNSCCs), we transfected parafibromin-expressing plasmid into HNSCC cell and examined the phenotypes and their relevant molecules. Parafibromin expression was detected on tissue microarray containing squamous epithelium, dysplasia, and carcinoma of head and neck by immunohistochemistry. Parafibromin overexpression was found to suppress growth, migration, and invasion, and induce apoptosis, S arrest, and mesenchymal to epithelial transition (EMT), compared with the mock and control (P < 0.05). Both overexpression of Cyclin E1, Bax, and E-cadherin and hypoexpression of c-myc, Bcl-xL, and slug were detected in B88 transfectants, in comparison to mock and control by real-time PCR. Parafibromin expression was weaker in primary cancers than those in normal squamous tissue and dysplasia (P < 0.05), but stronger than the metastatic cancers in lymph node (P < 0.05). Parafibromin expression was negatively correlated with lymph node metastasis, tumor-node-metastasis (TNM) staging, but positively with human papillomavirus (HPV) positivity (P < 0.05). The HNSCCs in tongue showed more parafibromin expression than those in larynx (P < 0.05). There was stronger parafibromin expression in moderately-than poorly-differentiated carcinomas (P < 0.05). The significantly positive correlation was observed between parafibromin expression and relapse-free survival rate by Kaplan-Meier curves (P < 0.05). Cox's proportional hazard model indicated that distant metastasis and parafibromin expression were independent prognostic factors for overall and relapse-free survival of HNSCC, respectively (P < 0.05). These findings suggest that downregulated expression of parafibromin protein plays an important role in the pathogenesis, differentiation, and metastasis of HNSCCs possibly by inducing apoptosis, suppressing proliferation, cell cycle progression, migration, invasion, and EMT. Parafibromin expression is an independent factor for relapse-free survival of HNSCCs.

Parathyroid Carcinoma: Diagnosis and Clinical Implications.

Parathyroid carcinoma is a rare type of endocrine cancer, with significant morbidity and mortality associated with parathyroid hormone (PTH)-mediated hypercalcemia. Concerning clinical features for parathyroid cancer include severe hypercalcemia (albumin-corrected calcium > 3 mmol/L), a palpable neck mass ( > 3 cm), 3rd/2nd generation PTH assay ratio ( > 1), and intraoperative suspicion of local invasion or regional metastasis. A definite diagnosis of malignancy is rendered when a parathyroid tumor presents one of the following clinicopathological features: (1) vascular invasion, (2) perineural invasion, (3) gross invasion into adjacent anatomical structures, and/or (4) metastasis. In difficult cases, the use of ancillary biomarkers is critical to establish an accurate diagnosis. Recent advances in molecular pathology have uncovered the important role of CDC73/HRPT2, a tumor suppressor gene deregulated in parathyroid carcinomas. Loss of nuclear and/or nucleolar expression of parafibromin (the gene product of CDC73/HRPT2) is now regarded as a diagnostic, prognostic and predictive biomarker for parathyroid carcinoma. Furthermore, over 15-20% of seemingly sporadic parathyroid carcinomas have underlying germline CDC73/HRPT2 mutations. As a result, many centers have integrated the use of ancillary biomarkers, notably parafibromin staining, in their routine practise. Radical surgery with en bloc resection has emerged as a primary treatment modality in parathyroid cancer, achieving cure in some patients. However, in those with inoperable disease, there remains a dire need for new therapies, as current treatments are largely ineffective. This review provides an update on the current knowledge of parathyroid carcinoma and highlights its exciting changes in endocrine practice.

Modern Experience with Aggressive Parathyroid Tumors in a High-Volume New England Referral Center

Parathyroid carcinoma (PTCA) is an exceptionally rare malignancy, often with a clinical presentation similar to that of benign atypical parathyroid adenoma. Its low incidence portends unclear guidelines for management. Accordingly, thorough examination of clinical and pathologic variables was undertaken to distinguish between PTCA and atypical adenomas. This was a retrospective analysis of a prospective database at a tertiary academic referral center. Between September 2001 and April 2014, 3,643 patients were referred for surgical treatment of PHPT. Of these, 52 harbored aggressive parathyroid tumors: parathyroid carcinomas (n=18) and atypical adenomas (n=34). We analyzed the surgical and clinicopathologic tumor characteristics, and did a statistical analysis. We measured preoperative and intraoperative variables, and postoperative and pathologic outcomes. Parathyroid carcinoma patients present with significantly increased tumor size (3.5 cm vs 2.4 cm, respectively; p=0.002), mean serum calcium (13.0 vs 11.8 mg/dL, respectively; p=0.003) and intact parathyroid hormone (iPTH) levels (489 vs 266 pg/mL, respectively; p=0.04), and a higher incidence of hypercalcemic crisis, compared with patients with atypical adenomas (50% vs 19%, respectively; p=0.072). Parathyroid carcinoma more frequently lacks a distinct capsule (47.1% vs 12.9%, respectively; p=0.03) and adheres to adjacent structures (77.8% vs 20.6%, respectively; p=0.017). Of note, there was no significant difference in loss of parafibromin expression between groups. Clinical distinction between PTCA and atypical adenomas is of critical importance in determining the appropriate extent of resection and follow-up. Loss of parafibromin has not been shown to distinguish between PTCA and atypical adenoma; clearer definition of clinicopathologic criteria for PTCA is warranted and may lead to improved postoperative management.

Familial isolated primary hyperparathyroidism/hyperparathyroidism‐jaw tumour syndrome caused by germline gross deletion or point mutations of CDC73 gene in Chinese

Hyperparathyroidism-jaw tumour syndrome (HPT-JT) and familial isolated primary hyperparathyroidism (FIHP) are two subtypes of familial primary hyperparathyroidism, which are rarely reported in Chinese population. Here, we reported three FIHP families and one HPT-JT family with long-term follow-up and genetic analysis. A total of 22 patients, from four FIHP/HPT-JT families of Chinese descent, were recruited and genomic DNA was extracted from their peripheral blood lymphocytes. Direct sequencing for MEN1, CDC73, CASR gene was conducted. Reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) were used to study the effect of splice site mutations and gross deletion mutations. Immunohistochemistry was performed to analyse parafibromin expression in parathyroid tumours. Genotype-phenotype correlations were assessed through clinical characteristics and long-term follow-up data. Genetic analysis revealed four CDC73 germline mutations that were responsible for the four kindreds, including two novel point mutation (c.157 G>T and IVS3+1 G>A), one recurrent point mutation (c.664 C>T) and one deletion mutation (c.307+?_513-?del exons 4, 5, 6). RT-PCR confirmed that IVS3+1 G>A generated an aberrant transcript with exon3 deletion. Immunohistochemical analysis demonstrated reduced nuclear parafibromin expression in tumours supporting the pathogenic effects of these mutations. This study supplies information on mutations and phenotypes of HPT-JT/FIHP syndrome in Chinese. Screening for gross deletion and point mutations of the CDC73 gene is necessary in susceptible subjects.

Understanding the Genetic Basis of Parathyroid Carcinoma

Parathyroid carcinoma has always been difficult to diagnose pathologically. In fact, most parathyroid tumors which are classified as carcinoma do not recur after excision, and most parathyroid tumors which actually metastasize or recur repeatedly in the neck are not recognized as malignant at first presentation. In 2002, germline HRPT2 (also known as CDC73) mutation was reported as the cause of hyperparathyroidism-jaw tumor (HPT-JT) syndrome, an autosomal dominant hereditary tumor syndrome associated with a lifetime risk of parathyroid carcinoma approaching 15 %. Subsequently, bi-allelic inactivation or mutation of HRPT2 has been reported in the majority of parathyroid carcinomas that actually behave in a malignant manner but very rarely in sporadic benign parathyroid disease. Furthermore, germline testing for HRPT2 mutation in patients presenting with parathyroid carcinoma often identifies occult HPT-JT syndrome even in the absence of a family history or other syndromic manifestations. HRPT2 mutation testing is not readily available, and loss of expression of parafibromin (the protein encoded by HRPT2) as determined by immunohistochemistry has been used as a surrogate marker of HRPT2 mutation. Immunohistochemistry for parafibromin can be technically difficult and has been deployed by different investigators with variable enthusiasm and success. However, proponents have found immunohistochemistry for parafibromin useful to definitively confirm a pathological diagnosis of parathyroid carcinoma, predict a worse outcome in definite parathyroid carcinomas, triage formal genetic testing for HPT-JT syndrome, and predict the outcome of histologically atypical parathyroid adenomas.

CDC73 mutational status and loss of parafibromin in the outcome of parathyroid cancer.

Inactivating mutations of the CDC73 tumor suppressor gene have been reported in parathyroid carcinomas (PC), in association with the loss of nuclear expression of the encoded protein, parafibromin. The aim of this study was to further investigate the role of the CDC73 gene in PC and evaluate whether gene carrier status and/or the loss of parafibromin staining might have an effect on the outcome of the disease. We performed genetic and immunohistochemical studies in parathyroid tumor samples from 35 patients with sporadic PC. Nonsense or frameshift CDC73 mutations were detected in 13 samples suitable for DNA sequencing. Six of these mutations were germline. Loss of parafibromin expression was found in 17 samples. The presence of the CDC73 mutation as well as the loss of parafibromin predicted a high likelihood of subsequent recurrence and/or metastasis (92.3%, P=0.049 and 94.1%, P=0.0017 respectively), but only the latter was associated with a decreased overall 5- and 10-year survival rates (59%, P=0.107, and 23%, P=0.0026 respectively). The presence of both the CDC73 mutation and loss of parafibromin staining compared with their absence predicted a lower overall survival at 10- (18 vs 84%, P=0.016) but not at 5-year follow-up. In conclusion, loss of parafibromin staining, better than CDC73 mutation, predicts the clinical outcome and mortality rate. The added value of CDC73 mutational analysis is the possibility of identifying germline mutations, which will prompt the screening of other family members.

Decreased expression of calcium-sensing receptor and parafibromin in secondary hyperparathyroidism with an abnormal whole PTH/intact PTH ratio.

A 34-year-old female hemodialysis patient with an abnormal whole parathyroid hormone (PTH)/intact PTH ratio (ratio = 1.14) underwent parathyroidectomy for advanced secondary hyperparathyroidism. The abnormal PTH ratio indicated a relative increase in the serum N-terminus PTH, a new molecular form of PTH. The abnormal ratio normalized immediately after the largest parathyroid gland (PTG) was removed, proving the largest PTG as causative. An immunohistochemical analysis revealed extremely decreased expression of the calcium-sensing receptor and parafibromin in the largest PTG in comparison with the other PTGs. This case indicated the possible involvement of decreased calcium-sensing receptor and parafibromin signaling in the development of the abnormal PTH ratio.

Effects of Parafibromin Expression on the Phenotypes and Relevant Mechanisms in the DLD-1 Colon Carcinoma Cell Line

Parafibromin is a protein encoded by the HRPT2 (hyperparathyroidism 2) oncosuppressor gene and its down-regulated expression is involved in pathogenesis of parathyroid, breast, gastric and colorectal carcinomas. This study aimed to clarify the effects of parafibromin expression on the phenotypes and relevant mechanisms of DLD-1 colon carcinoma cells. DLD-1 cells transfected with a parafibromin-expressing plasmid were subjected to examination of phenotype, including proliferation, differentiation, apoptosis, migration and invasion. Phenotype-related proteins were measured by Western blot. Parafibromin and ki-67 expression was detected by immunohistochemistry on tissue microarrays. The transfectants showed higher proliferation by CCK-8, better differentiation by electron microscopy and ALP activity and more apoptotic resistance to cisplatin by DNA fragmentation than controls. There was no difference in early apoptosis by annexin V, capase-3 activity, migration and invasion between DLD-1 cells and their transfectants. Ectopic parafibromin expression resulted in down-regulated expression of smad4, MEKK, GRP94, GRP78, GSK3β-ser9, and Caspase-9. However, no difference was detectable in caspase-12 and -8 expression. A positive relationship was noted between parafibromin and ki-67 expression in colorectal carcinoma. Parafibromin overexpression could promote cell proliferation, apoptotic resistance, and differentiation of DLD-1 cells.

Expression of Parafibromin in Clear Cell Papillary Renal Cell Carcinoma

Clear cell papillary renal cell carcinoma (CCPRCC) is a low-grade renal cell neoplasm. In this study, we investigated the expression of parafibromin, CK7, and RCC marker (RCC-Ma) in this tumor by immunohistochemistry in a group of CCPRCC. Twenty cases of CCPRCC were stained for parafibromin and showed diffuse and strong nuclear positivity for this marker. In addition the cases of CCPRCC were stained for CK7 and RCC-Ma, respectively, and the majority of tumors were positive for cytoplasmic staining of CK7 and negative for RCC-Ma. This unique staining pattern can be useful in the differential diagnosis from conventional clear cell renal cell carcinomas, which have a higher positivity rate for RCC-Ma, but largely negative for CK7 and parafibromin, and papillary renal cell carcinomas, which are usually positive for CK7 and RCC-Ma but are largely negative for parafibromin.