Parafibromin immunoreactivity: its use as an additional diagnostic marker for parathyroid tumor classification

in Endocrine-Related Cancer
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  • 1 1Departments of Molecular Medicine and Surgery and
  • 2 2Oncology and Pathology, Karolinska Institutet, Karolinska University Hospital Solna, CMM L8:01, SE-171 76 Stockholm, Sweden
  • 3 3Department of Endocrine Surgery, 8-1 Kawada-Cho, Sinjuku-Ku, Tokyo 162-8666, Japan
  • 4 4Center for Molecular Medicine and the Division of Endocrinology and Metabolism, University of Connecticut School of Medicine, Farmington, Connecticut 06030-3101, USA

Parafibromin is a protein product derived from the hyperparathyroidism 2(HRPT2) tumor suppressor geneand its inactivation has been coupled to familial and sporadic forms of parathyroid malignancy. In this study, we have conducted immunohistochemistry on 33 parathyroid carcinomas (22 unequivocal and 11 equivocal) using four parafibromin antibodies directed to different parts of the protein. Furthermore, for a fraction of cases, the immunohistochemical results were compared with known HRPT2 mutational status. Our findings show that 68% (15 out of 22) of the unequivocal carcinomas exhibited reduced expression of parafibromin while the 25 sporadic adenomas used as controls were entirely positive for parafibromin expression. Additionally, three out of the six carcinomas with known HRPT2 mutations showed reduced expression of parafibromin. Using all four antibodies, comparable results were obtained on the cellular level in individual tumors suggesting that there exists no epitope of choice in parafibromin immunohistochemistry. The results agree with the demonstration of a ~60 kDa product preferentially in the nuclear fraction by western blot analysis. We conclude that parafibromin immunohistochemistry could be used as an additional marker for parathyroid tumor classification, where positive samples have low risk of malignancy, whereas samples with reduced expression could be either carcinomas or rare cases of adenomas likely carrying an HRPT2 mutation.

Abstract

Parafibromin is a protein product derived from the hyperparathyroidism 2(HRPT2) tumor suppressor geneand its inactivation has been coupled to familial and sporadic forms of parathyroid malignancy. In this study, we have conducted immunohistochemistry on 33 parathyroid carcinomas (22 unequivocal and 11 equivocal) using four parafibromin antibodies directed to different parts of the protein. Furthermore, for a fraction of cases, the immunohistochemical results were compared with known HRPT2 mutational status. Our findings show that 68% (15 out of 22) of the unequivocal carcinomas exhibited reduced expression of parafibromin while the 25 sporadic adenomas used as controls were entirely positive for parafibromin expression. Additionally, three out of the six carcinomas with known HRPT2 mutations showed reduced expression of parafibromin. Using all four antibodies, comparable results were obtained on the cellular level in individual tumors suggesting that there exists no epitope of choice in parafibromin immunohistochemistry. The results agree with the demonstration of a ~60 kDa product preferentially in the nuclear fraction by western blot analysis. We conclude that parafibromin immunohistochemistry could be used as an additional marker for parathyroid tumor classification, where positive samples have low risk of malignancy, whereas samples with reduced expression could be either carcinomas or rare cases of adenomas likely carrying an HRPT2 mutation.

Introduction

Parathyroid carcinoma is an uncommon but potentially life-threatening form of primary hyperparathyroidism (PHPT; Grimelius & Johansson 1997, Shane 2001, Rodgers & Perrier 2006). The patients can develop a palpable mass in the neck, concomitant kidney, and bone disease, and frequently high parathyroid hormone (PTH) levels along with persistent hypercalcemia in the case of locally invasive or metastatic disease ( Mittendorf & McHenry 2005). Early detection and en bloc resection of all tumor tissue is necessary for a favorable outcome ( Rodgers & Perrier 2006). However, parathyroid carcinoma often presents a diagnostic challenge both concerning presurgical distinction between a highly active adenoma and the malignant counterpart, as well as histopathological identification of carcinoma in the absence of invasive growth pattern ( Sandelin et al. 1992, DeLellis 2005). While unequivocal parathyroid carcinoma shows indisputable evidence of malignancy such as invasion, metastasis, or recurrence ( DeLellis et al. 2004), equivocal cancers only present characteristics associated with malignant behavior at histopathology ( Bondeson et al. 1993). The difficulties in parathyroid tumor diagnosis are further exemplified by the histological subgroup ‘atypical adenoma’ which is considered of uncertain malignant potential ( Levin et al. 1987). In the search of a marker for parathyroid malignancy, analysis of DNA content, and immunohistochemical expression of pRb, bcl-2, Ki-67, cyclin D1, p21, and p53 have been evaluated, but not found to be specific enough ( Bondeson et al. 1993, Cryns et al. 1994, Erickson et al. 1999, Farnebo et al. 1999, Vasef et al. 1999, Stojadinovic et al. 2003).

The hyperparathyroidism 2 (HRPT2) gene is the disease gene for hyperparathyroidism–jaw tumor syndrome (HPT–JT), characterized by benign and malignant PHPT in combination with tumors of the jaws, kidney, and uterus ( Carpten et al. 2002, Bradley et al. 2005, Gimm et al. 2006). Its encoded protein parafibromin is a member of the polymerase-associated factor 1 (PAF1) complex associated with RNA polymerase II which regulates transcription elongation and histone modification ( Carpten et al. 2002, Rozenblatt-Rosen et al. 2005, Yart et al. 2005). In vitro studies have revealed dominant negative tumor suppressor properties ( Zhang et al. 2006), a functional nuclear localization signal (NLS; Hahn & Marsh 2005, Woodard et al. 2005, Zhang et al. 2006, Bradley et al. 2007), and coupling to the wingless type (Wnt) signaling pathway ( Mosimann et al. 2006).

Inactivating HRPT2 mutations are frequently detected on the somatic and sometimes germ line levels in apparently sporadic parathyroid carcinoma ( Howell et al. 2003, Shattuck et al. 2003, Cetani et al. 2004), as opposed to a small minority of adenomas ( Carpten et al. 2002, Howell et al. 2003, Krebs et al. 2005, Bradley et al. 2006, Juhlin et al. 2006). In immunohistochemical studies of unequivocal and HPT–JT-related carcinomas, complete loss, or reduced nuclear immunoreactivity was found in the majority of cases. By contrast, 98–100% of unselected sporadic adenomas stained completely positive ( Tan et al. 2004, Gill et al. 2006). In a subset of cystic adenomas, loss of parafibromin has been associated with HRPT2 mutations ( Juhlin et al. 2006).

Here, we have evaluated immunohistochemical expression of parafibromin in parathyroid carcinomas using antibodies directed to different epitopes of the protein and further related the findings to western blot analyses and genetic information.

Materials and methods

Parathyroid carcinoma patients and tissue samples

This study includes 33 tumor samples collected worldwide from 32 patients (Table 1 ). Twenty-four samples are part of a previously published historical material ( Bondeson et al. 1993, Farnebo et al. 1999). Six cases carry inactivating HRPT2 mutations, five of which have been previously reported ( Shattuck et al. 2003). Three additional samples were newly collected with informed consent and ethical approval from two patients undergoing surgery at Karolinska University Hospital in Stockholm.

Histopathological classifications of tumor specimens were according to the WHO criteria. Twenty-two patients had definite (unequivocal) cancer with vascular invasion, and/or local invasion of surrounding organs, and/or distant metastasis, and/or evidence of recurrence after parathyroid surgery. They also exhibited tumor characteristics associated with parathyroid cancer such as fibrous bands, mitotic figures, focal necrosis, macronucleoli, and distinct cellular atypia (T1–T22, Table 1 ). The 11 cases classified as equivocal carcinomas exhibited at least one histopathological indication of malignancy mentioned above, but were without evidence of recurrence, invasion, or metastases (T23–T33, Table 1 ). Sample T13 was originally classified as an adenoma; however, this tumor relapsed 4 years after primary surgery thereby establishing the diagnosis as unequivocal carcinoma in this patient.

Control samples

The following positive controls were used: HeLa cells transfected with plasmid DNA containing full-length HRPT2 cDNA and two parathyroid carcinomas (T4 and T23) for western blot and immunohistochemistry ( Juhlin et al. 2006), four paraffin-embedded adenomas on the same slide for immunohistochemistry including one with a rim of normal parathyroid (control adenoma 1) and 21 sporadic adenomas (the majority with a normal rim) as normal reference for immunohistochemistry. For sub-cellular fractionation and western blot analysis, frozen samples of two cystic adenomas with or without HRPT2 gene mutation and parafibromin inactivation (corresponding to T20 and T23 in Juhlin et al. 2006), one secondary HPT gland, and a regular adenoma (control adenoma 2) were used. All samples were obtained with informed consent and local ethical approval.

Antibodies and blocking peptides

A rabbit polyclonal antibody TNYV was raised with ethical approval against aa 39–58 by the authors through AgriSera Co. (Umeå, Sweden) using published methodology ( Polak & Van Noorden 1986), affinity purified and eluated at pH 7.0. The mouse monoclonal antibody 2H1 targets aa 87–100 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). BL648 is an affinity-purified rabbit polyclonal antibody directed against a 24 aa sequence within aa 250–300 (Bethyl Laboratories, Montgomery, TX, USA). APVF, an affinity-purified rabbit polyclonal antibody raised against aa 509–531, have been previously published ( Juhlin et al. 2006).

Blocking peptides used as controls were synthesized by the authors through AgriSera Co. or commercially available (BP648, Bethyl Laboratories). Dot blot experiments with immobilized peptide antigen confirmed that all four parafibromin antibodies bind to their respective immunization peptide (data not shown).

Western blot analyses

Total proteins were extracted according to standard procedures, quantified using a dye-binding assay ( Bradford 1976), and used for western blot analyses using previously described methodology ( Juhlin et al. 2006). The sub-cellular fractionation and verification with anti Lamin A/C and anti Prohibitin was performed as previously described or has been reported before ( Forsberg et al. 2006). After electrophoresis and blotting, presence of protein was confirmed by staining with Ponceau Red solution (Sigma–Aldrich), and the membranes were incubated with the respective parafibromin antibody: 1.7 μg/ml TNYV (1:300), 4 μg/ml 2H1 (1:50), 0.2 μg/ml BL648 (1:5000), 1.7 μg/ml APVF (1:300), followed by the respective secondary HRP-conjugated antibody (goat anti-rabbit 1:12 500, goat anti-mouse 1:10 000 Bio-Rad Laboratories). In control experiments, the antibodies were preincubated with peptide antigen for 1 h at room temperature and 2 h at 4 °C.

Immunohistochemistry

Paraffin sections of parathyroid carcinomas and control samples were cut at 4 μm, deparaffinized, rehydrated, and immersed in preheated citrate buffer pH 6 (Dako, Glostrup, Denmark) at 95 °C for 20 min. In our experience, antigen retrieval by heating in citrate buffer was necessary to obtain a distinct nuclear signal without interfering background, in agreement with previous publications ( Tan et al. 2004, Gill et al. 2006). For the monoclonal antibody 2H1, the following parameters were tested: antibody dilution (1:20, 1:100, and 1:200), antigen retrieval time (10, 20, 30, and 50 min) as well as incubation time (1 h and overnight). These parameters were studied in four adenomas on the same slide and in carcinomas T4, T14, T15, and T23. In our laboratory settings, we obtained expected positive immunoreactivity in the adenomas and the western blot verified carcinomas using citrate heating for 20 min, antibody dilution of 1:20, and overnight incubation. Positive signals were not obtained using shorter antigen retrieval time, increased antibody dilution, or shorter incubation time.

The sections were incubated in 0.3% hydrogen peroxide in water for 30 min, blocked in 1% BSA with 0.01% sodium azide for 45 min, and incubated with primary antibody diluted in 1% BSA overnight using concentrations determined from dilution trials with positive controls: TNYV 2 μg/ml (1:250), 2H1 10 μg/ml (1:20), BL648 10 μg/ml (1:100), and APVF 2 μg/ml (1:250). The slides were then incubated with biotinylated secondary antibody (7.5 μg/ml (1:100) of goat anti-rabbit BA-1000 and horse anti-mouse B-200; Vector Laboratories, Burlingame, CA, USA) for 45 min, and the antigen–antibody complex was visualized using the avidin–biotin–peroxidase complex method (Vectastain Elite Kit, Vector Laboratories) for 45 min, followed by diaminobenzidine tetrahydrochloride for 6 min, and counterstaining in hematoxylin for 3 min. Sections were washed in five cycles of Tris-buffered saline (TBS, pH 7.6) between each step. Peptide neutralization tests for all four parafibromin antibodies were performed on all cases as negative controls.

Parafibromin expression was independently scored by two of the authors (C J and A H), whereby the levels of expression and the sub-cellular localization were evaluated. Each specimen was classified as having either ‘negative’ (staining of ≤ 10% tumor nuclei), ‘partial loss’ (staining of 11–89% of tumor nuclei), or ‘positive’ (staining of ≥ 90% of tumor nuclei) parafibromin expression. The partial loss group was further studied by scoring at least 1500 cells from a total offive randomly selected grid areas of the sections in high power magnification (40×). The cells were classified as having either positive or negative nuclear parafibromin expression. The negative controls were independently evaluated by three of the authors (C J, F H, and A H), including an author blinded of the primary positive findings. All slides were blinded of their diagnosis during the entire microscopic validation process.

Results

Antibody characterization and western blot analysis

The four parafibromin antibodies gave similar results with high affinity for a ~60 kDa band representing the predicted size of parafibromin (Figs 1 and 2 ). Furthermore blocking experiments with corresponding immunizing peptides confirmed the specificity of our findings. Strong expression was revealed in HRPT2-transfected HeLa cells, when compared with the weaker endogenous signal observed in untransfected HeLa cell controls (Fig. 2 ). The secondary HPT gland showed positive parafibromin expression in the total and nuclear extracts, while in the cytosolic extract parafibromin was absent or showed weak expression (Fig. 2 ). Similar results were obtained at analysis of nuclear and cytosolic extracts from control adenoma 2 (Fig. 3B ). In the cystic adenoma with wild-type HRPT2 ( Juhlin et al. 2006), all four antibodies detected a ~60 kDa product (Fig. 2 ), while the cystic adenoma with known HRPT2 mutation ( Juhlin et al. 2006) was negative for parafibromin by means of all four antibodies used (Fig. 2 ).

Immunohistochemical analysis of parafibromin expression

In control experiments, HRPT2-tranfected HeLa cells, the four regular parathyroid adenomas and normal parathyroid tissue (control adenoma 1) showed strong nuclear expression of parafibromin for each of the parafibromin antibodies, whereas the cytoplasm was only weakly positive in these samples (Fig. 3A ). In addition, 21 sporadic adenomas analyzed with 2H1 showed positive expression in all cases (data not shown).

Examples of tumors scored as having negative, partial loss, or positive parafibromin expression are shown in Fig. 4 . Fourteen of the 22 unequivocal carcinoma samples (64%) demonstrated partial loss of parafibromin expression with 25–83% positive nuclei, and one sample (4%) was completely negative for parafibromin immunoreactivity (T5, Table 2 , Fig. 4 ). The remaining seven samples (32%) were positive, exhibiting parafibromin expression in almost 100% of the tumor cell nuclei. Among the equivocal carcinomas, 5 out of 11 cases (45%) exhibited partial loss, ranging from 52 to 80% positive nuclei (Table 2 ), while six samples (55%) were completely positive for parafibromin nuclear expression. Samples T4 and T7, representing two relapses within 1-year time in the same patient, were both completely positive.

In general, positive samples demonstrated nuclear staining, while surrounding fibrous tissue was negative. Accompanying low levels of cytoplasmic staining were often observed at varying levels between tumors, especially with the BL648 antibody. In support of the specificity of the nuclear and cytoplasmic signals, the positive expression was completely eliminated in the subsequent blocking experiments.

The immunohistochemical findings were also supported by western blot analysis of two positive carcinomas (Fig. 2 ). Sample T4 represents a relapsing unequivocal carcinoma growing into the larynx, esophagus, and blood vessels, and sample T23 is an equivocal carcinoma from a 16-year-old boy. Both these tumors expressed the ~60 kDa product at western blot analyses.

Discussion

The concordant results obtained at immunohistochemistry and western blot analyses suggest that the four antibodies are adequate in detecting parafibromin regarding both sensitivity and specificity. Moreover, the predominant nuclear localization observed at immunohistochemistry and sub-cellular fractionation is in accordance with previous reports ( Hahn & Marsh 2005, Woodard et al. 2005, Bradley et al. 2007, Zhang et al. 2006). The specificity of the cytoplasmic signal was supported by peptide neutralization experiments (antibody preincubated with immunizing peptide) and detection of a ~60 kDa band in the cytosolic fraction of the secondary HPT gland at western blot analysis.

Optimal methodological conditions for parafibromin immunohistochemistry are crucial if the results ought to be used for diagnostic purposes. We found that the results were influenced by the intensity of antigen retrieval, antibody dilution, and incubation time. The combination of immunohistochemistry and western blot analysis was, as illustrated in case T4 and T23, used to adjust the immunohistochemical methodology to positive findings in these parathyroid carcinomas evidently expressing parafibromin demonstrated by western blot analysis.

Partial loss of parafibromin immunoreactivity was frequently detected, while negative staining was only observed in a single carcinoma. Similar results were obtained for the four antibodies used with comparable proportions of positive and negative cells in individual cases. The concordant findings with the different antibodies would suggest that full-length parafibromin is present in the positive nuclei found. Furthermore, the positive findings with BL648 and APVF would suggest that the functionally important β-catenin interaction domain (CID) and complex-binding domain (Cdc73) domains are physically present in the positive nuclei found.

Two tumors with double somatic HRPT2 mutations exhibited positive and partial loss of parafibromin expression respectively. This situation is not generally expected in the case of tumor suppressor gene inactivation, where loss of the normal function often is seen. However, a previous study has demonstrated in vitro expression of truncated parafibromin protein resulting from site-specific HRPT2 mutations introduced in a cell line, suggesting that HRPT2 mutated cells can still propel the production of parafibromin, albeit structurally defective ( Zhang et al. 2006). In our two cases with double mutations, this finding offers a possible explanation for the positive results obtained with the three most N-terminally located parafibromin antibodies. Furthermore, both tumors with double HRPT2 mutations carry one deleterious mutation in exon 8 (Table 1 ). The resulting frameshifts fortuitously creates new amino acid sequences prior to truncation that have partial homology with the APVF immunizing peptide; thus cross-binding with the APVF polyclonal antibody (although affinity purified) cannot be excluded. In general, the complexity of parafibromin immunostaining might be aggravated by other factors. For example, increased proliferation can result from co-expression of mutant and wild-type parafibromin ( Zhang et al. 2006). Furthermore, parathyroid carcinomas reveal frequent gain of 1q, including the HRPT2 gene locus ( Kytölä et al. 2000), in contrast to the frequent observations of copy number losses at other tumor suppressor gene loci such as the MEN1 locus in parathyroid adenomas.

The present study agrees with previous reports in which normal parathyroid tissue and parathyroid adenomas show positive parafibromin expression in all cells, giving a high specificity for the method. Since adenomas in turn are much more frequent than carcinomas, the negative predictive value (i.e. the chance that an adenoma is correctly identified by positive parafibromin staining) would end up as high, although probably not 100% since reduced expression of parafibromin has been reported in small subsets of parathyroid adenomas ( Gill et al. 2006, Juhlin et al. 2006). Hence, in parathyroid tumors with positive parafibromin expression, the risk of malignancy is very low, however, cannot be fully excluded. In the group with partial loss or negative staining, a putative positive predictive value (i.e. the chance that a carcinoma is correctly identified by negative parafibromin staining) would end up as low, indicating that a negative staining is not automatically consistent with parathyroid carcinoma. Cases with partial loss or negative staining could either be parathyroid carcinomas or adenomas likely carrying an HRPT2 mutation. In this group, mutation screening of tumor and blood is well motivated especially for detection of familial disease.

The question arises whether tumors diagnosed as parathyroid adenomas with partial loss or negative parafibromin staining could have a more aggressive course of disease. One tumor in our series exemplifies this situation; sample T13 was originally classified as an adenoma; however, the advent of recurrent disease 4 years after primary surgery ascertained this tumor as carcinoma. Careful histopathological reexamination of the primary lesion found no evidence for malignant disease, although our ensuing parafibromin staining of this specimen was consistent with partial loss. Further analysis of similar cases will establish whether parafibromin immunostaining could detect malignant potential before the occurrence of required histopathological characteristics.

The initial findings of frequent HRPT2 mutations in parathyroid carcinomas have inspired the development of parafibromin antibodies for immunohistochemical applications. The presently available data suggest that this approach has additive value in parathyroid diagnostics. However, loss of parafibromin immunoreactivity cannot be used as a diagnostic marker alone since the sensitivity is limited. Mutation screening of HRPT2 is expected to increase the sensitivity as illustrated in this study where three of the six cases with known HRPT2 mutations had positive expression (100% positive nuclei). Additional biomarkers for identification of parathyroid malignancy before the development of spread disease could be sought among markers for high proliferation and within the parafibromin pathways.

To summarize, parafibromin immunohistochemistry could be applied as an additional diagnostic marker. Positive expression would indicate benign disease, however, does not fully exclude a cancer diagnosis or the presence of an HRPT2 mutation. Negative or partial loss staining of parafibromin should motivate genetic analysis of the HRPT2 gene in blood and tumor tissue from the affected patient. However, our results suggest that parafibromin immunostaining cannot replace genetic testing or be used as a sole discriminator to separate adenoma from carcinoma. Our findings furthermore suggest that there exists no ‘epitope of choice’ for parafibromin detection related to malignant behavior of parathyroid tumors.

Table 1

Clinical details of the 33 unequivocal and equivocal parathyroid carcinoma cases in the study

HRPT2mutation status
Case no.SexAge at opTissue analyzedS-Ca (mmol/l)Initial diagnosisDead from diseaseRecurrent diseaseTime to recurrenceFinal diagnosisWeight (mg) or size (Ø in cm)HRPT2sequenceMutation typePredicted effect
M, male; F, female; Local rec, local recurrence; Lung met, lung metastasis; –, not known, not determined; aa, amino acid; elevated, above normal range, no known numerical value.
aCase T16 and T33 was originally described as ‘walnut’ and ‘hazelnut’ respectively.
Unequivocal cases
    T1M61 yearsLung metastasis3.5CarcinomaYesLung met30 mCarcinoma3400 mg226C > T ex2SomaticStop at aa 76
    T2FLung metastasisCarcinomaLung metCarcinoma664C > T ex7Germ lineStop aa 222
    T3M25 yearsNeck metastasisCarcinomaCarcinoma
    T4F61 yearsNeck metastasisCarcinomaYesCarcinoma
    T5F35 yearsLocal rec4.8CarcinomaLocal rec, lung met36 mCarcinoma
    T6M54 yearsLocal rec3.8CarcinomaYesLocal rec, lung met13 mCarcinoma2500 mg70G > T ex1, 746delT ex8Somatic × 2Stop at aa 24 and 256 respectively
    T7F60 yearsLocal recCarcinomaYesLocal rec5 mCarcinoma
    T8M52 yearsLocal rec5.5CarcinomaYesLocal rec16 mCarcinoma
    T9F78 yearsLocal rec5.0CarcinomaYesLocal recCarcinoma6600 mg
    T10Local rec4.0CarcinomaLocal rec24 mCarcinoma
    T11F64 yearsPrimary tumor3.0CarcinomaYesLocal rec72 mCarcinoma25 000 mg
    T12F58 yearsPrimary tumor3.3CarcinomaLocal rec24 mCarcinoma2 cm
    T13M57 yearsPrimary tumor3.3AdenomaLocal rec48 mCarcinoma2.7 cm
    T14F32 yearsPrimary tumor3.9CarcinomaCarcinoma3100 mg
    T15F63 yearsPrimary tumor3.2CarcinomaCarcinoma1.3 cm
    T16M74 yearsPrimary tumor3.2CarcinomaCarcinoma3 cma
    T17M78 yearsPrimary tumor3.1CarcinomaCarcinoma5 cm
    T18F85 yearsPrimary tumorElevatedCarcinomaCarcinoma3 cm
    T19M78 yearsPrimary tumor4.5CarcinomaCarcinoma3.1 cm
    T20F61 yearsPrimary tumor4.9CarcinomaYesLung met14 mCarcinoma3100 mg82del4 ex1, 732delT ex8Somatic × 2Stop at aa 35 and 256 respectively
    T21MPrimary tumorCarcinomaCarcinoma23TGCG > GTG ex1SomaticStop at aa 20
    T22M59 yearsPrimary tumor4.0CarcinomaNoCarcinoma5 cm
Equivocal cases
    T23M16 yearsPrimary tumor4.0CarcinomaCarcinoma1300 mgc127insC ex1Germ lineStop at aa 65
    T24M62 yearsPrimary tumorCarcinomaCarcinoma2.5 cm
    T25F66 yearsPrimary tumor3.6CarcinomaCarcinoma3 cm
    T26F29 yearsPrimary tumor3.7CarcinomaCarcinoma19 000 mg
    T27F58 yearsPrimary tumor3.5CarcinomaCarcinoma3 cm
    T28M46 yearsPrimary tumor3.5CarcinomaCarcinoma3100 mg
    T29M70 yearsPrimary tumorElevatedCarcinomaCarcinoma1.5 cm
    T30F51 yearsPrimary tumor3.0CarcinomaCarcinoma
    T31M53 yearsPrimary tumor3.3CarcinomaCarcinoma1050 mg
    T32M34 yearsPrimary tumor4.3CarcinomaCarcinoma10 800 mg
    T33F28 yearsPrimary tumorCarcinomaCarcinoma2 cma
Table 2

Results from the immunohistochemical staining of the 33 parathyroid carcinomas

Parafibromin immunohistochemistry (% positive cells)
SampleHRPT2mutationTNYV (aa 39–58)2H1 (aa 87–100)BL648 (aa 250–300)APVF (aa 509–531)Average %Staining pattern
–, not determined. Staining patterns: positive ≥90% positive nuclei, partial loss 11–89% positive nuclei, negative ≤10% positive nuclei.
Unequivocal cases
    T1Somatic100100100100100Positive
    T2Somatic81766474Partial loss
    T39610010099Positive
    T4100100100100100Positive
    T500000Negative
    T6Somatic × 28810576355Partial loss
    T7100100100100100Positive
    T83528602938Partial loss
    T94835902048Partial loss
    T1079697474Partial loss
    T114492807072Partial loss
    T126764456560Partial loss
    T132133222325Partial loss
    T146065597064Partial loss
    T155862705762Partial loss
    T165234642945Partial loss
    T171001009010098Positive
    T184964643954Partial loss
    T19100100100100100Positive
    T20Somatic × 2100100100100100Positive
    T21Somatic60639573Partial loss
    T227684779483Partial loss
SummaryPositive (n = 7) 32%
Partial loss (n = 14) 64%
Negative (n = 1) 4%
Equivocal cases
    T23Germ line100100100100100Positive
    T245587888980Partial loss
    T256081538470Partial loss
    T264880433652Partial loss
    T27100100100100100Positive
    T28811009010093Positive
    T295179687268Partial loss
    T30100100100100100Positive
    T316172688070Partial loss
    T32100100100100100Positive
    T33100100100100100Positive
SummaryPositive (n = 6) 55%
Partial loss (n = 5) 45%
Negative (n = 0) 0%
Figure 1
Figure 1

Schematic of the HRPT2 gene, parafibromin, and the antibodies studied. (A) Genomic structure of HRPT2, its 17 coding exons and the gene product parafibromin. ATG represent the initiation codon. Functionally important regions are marked: the nuclear localization signal (NLS) is encoded from exon 5, the β-catenin interaction domain (CID) by exons 7 and 8, and the evolutionary conserved PAF1 complex-binding domain (Cdc73 core) constitutes exons 12–17. The targets of the four antibodies are shown below the schematic of parafibromin. (B) Details of the four parafibromin antibodies.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 14, 2; 10.1677/ERC-07-0021

Figure 2
Figure 2

Western blot analyses of parafibromin expression. Parafibromin protein is demonstrated as a ~60 kDa protein in various parathyroid lesions using the antibodies TNYV, 2H1, BL648, and APVF. The expressions were visually classified as strong +, weak (+), or undetectable (−).

Citation: Endocrine-Related Cancer Endocr Relat Cancer 14, 2; 10.1677/ERC-07-0021

Figure 3
Figure 3

(A) Immunohistochemistry demonstrating mainly nuclear expression of parafibromin in control adenoma 1 as well as HRPT2-transfected HeLa cells using antibodies TNYV, 2H1, BL648, and APVF. Inserts are parathyroid cells from the normal glandular rim. The specificity is verified by peptide neutralization tests. All images are magnified 140×, while the insert are magnified 260×. (B) Western blot analysis after sub-cellular fractionation, showing parafibromin expression in the nuclear fraction of control adenoma 2. Prohibitin is a mitochondrial antigen.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 14, 2; 10.1677/ERC-07-0021

Figure 4
Figure 4

Photomicrographs showing immunohistochemical analyses of parafibromin expression in parathyroid carcinomas T5, T24, and T23 using antibodies TNYV, 2H1, BL648, and APVF. The unequivocal carcinoma T5 (negative) exhibits a total loss of parafibromin immunoreactivity with all four antibodies. In the equivocal carcinoma T24 (partial loss), the staining pattern represents ~80% positive tumor cells, while the equivocal cancer T23 (positive) demonstrates clear nuclear staining in 100% of the tumor cells. All images are magnified 160×, while the inserts are magnified 280×. To fully evaluate samples with partial loss, high power magnification is required as demonstrated in T24.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 14, 2; 10.1677/ERC-07-0021

The authors wish to acknowledge Prof. Lars Grimelius and Prof. Lennart Bondeson for their expertise in histopathological classification of the parathyroid tumors. The authors are also indebted to Dr Gerardo Guiter for contributing with two carcinoma specimens from the Department of Pathology, Rhode Island Hospital, Providence, RI, USA. The study was financially supported by Swedish Cancer Foundation, Cancer Society in Stockholm, Gustav V Jubilee Foundation, Swedish Society of Medical Research, Swedish Medical Association, Göran Gustavsson Foundation for Research in Natural Sciences and Medicine, the Murray-Heilig Fund in Molecular Medicine, Karolinska Institutet and Stockholm County Council. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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  • Cetani F, Pardi E, Borsari S, Viacava P, Dipollina G, Cianferotti L, Ambrogini E, Gazzerro E, Colussi G, Berti P et al. 2004 Genetic analyses of the HRPT2 gene in primary hyperparathyroidism: germline and somatic mutations in familial and sporadic parathyroid tumors. Journal of Clinical Endocrinology and Metabolism 89 5583–5591.

    • Search Google Scholar
    • Export Citation
  • Cryns VL, Thor A, Xu HJ, Hu SX, Wierman ME, Vickery AL Jr, Benedict WF & Arnold A 1994 Loss of the retinoblastoma tumor-suppressor gene in parathyroid carcinoma. New England Journal of Medicine 330 757–761.

    • Search Google Scholar
    • Export Citation
  • DeLellis RA 2005 Parathyroid carcinoma – an overview. Advances in Anatomic Pathology 12 53–61.

  • DeLellis RA, Lloyd RV, Heitz PU & Eng C 2004 Tumours of endocrine organs. In World Health Organization Classification of Tumour Pathology and Genetics, Eds RA DeLellis, RV Lloyd, PU Heitz & C Eng. Lyon: IARC Press.

  • Erickson LA, Jin L, Wollan P, Thompson GB, van Heerden JA & Lloyd RV 1999 Parathyroid hyperplasia, adenomas and carcinomas: differential expression of p27Kip1 protein. American Journal of Surgical Pathology 23 288–295.

    • Search Google Scholar
    • Export Citation
  • Farnebo F, Auer G, Farnebo L-O, The BT, Twigg S, Apendblad U, Thomson NW, Grimelius L, Larsson C & Sandelin K 1999 Evaluation of retinoblastoma and Ki-67 as diagnostic markers in benign and malignant parathyroid disease. World Journal of Surgery 23 68–74.

    • Search Google Scholar
    • Export Citation
  • Forsberg L, Larsson C, Sofiadis A, Lewensohn R, Höög A & Lehtio J 2006 Pre-fractionation of archival frozen tumours for proteomics applications. Journal of Biotechnology 126 582–586.

    • Search Google Scholar
    • Export Citation
  • Gill AJ, Clarkson A, Gimm O, Keil J, Dralle H, Howell VM & Marsh DJ 2006 Loss of nuclear expression of parafibromin distinguishes parathyroid carcinomas and hyperparathyroidism–jaw tumor (HPT–JT) syndrome-related adenomas from sporadic parathyroid adenomas and hyperplasias. American Journal of Surgical Pathology 30 1140–1149.

    • Search Google Scholar
    • Export Citation
  • Gimm O, Lorenz K, Nguyen Thanh P, Schneyer U, Howell VM, Marsh DJ, Teh BT, Krause U & Dralle H 2006 Prophylactic parathyroidectomy for familial parathyroid carcinoma. Der Chirurg; Zeitschrift für alle Gebiete der operativen Medizen 77 15–24 (In German).

    • Search Google Scholar
    • Export Citation
  • Grimelius L & Johansson H 1997 Pathology of parathyroid tumors. International Seminars in Surgical Oncology 13 142–154.

  • Hahn MA & Marsh DJ 2005 Identification of a functional bipartite nuclear localization in the tumor suppressor parafibromin. Oncogene 24 6241–6248.

    • Search Google Scholar
    • Export Citation
  • Howell VM, Haven CJ, Kahnoski K, Khoo SK, Petillo D, Chen J, Fleuren GJ, Robinson BG, Delbridge LW, Philips J et al. 2003 HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours. Journal of Medical Genetics 40 657–663.

    • Search Google Scholar
    • Export Citation
  • Juhlin C, Höög A, Yakoleva T, Leibiger I, Leibiger B, Weber G, Larsson C & Villablanca A 2006 Loss of parafibromin expression in a subset of sporadic parathyroid adenomas. Endocrine Related Cancer 13 509–523.

    • Search Google Scholar
    • Export Citation
  • Krebs LJ, Shattuck TM & Arnold A 2005 HRPT2 mutational analysis of typical sporadic parathyroid adenomas. Journal of Clinical Endocrinology and Metabolism 90 5015–5017.

    • Search Google Scholar
    • Export Citation
  • Kytölä S, Farnebo F, Obara T, Isola J, Grimelius L, Farnebo L-O, Sandelin K & Larsson C 2000 Patterns of chromosomal imbalances in parathyroid carcinomas. American Journal of Pathology 157 579–586.

    • Search Google Scholar
    • Export Citation
  • Levin KE, Galante M & Clark OH 1987 Parathyroid carcinoma versus parathyroid adenoma in patients with profound hypercalcemia. Surgery 101 649–660.

    • Search Google Scholar
    • Export Citation
  • Mittendorf EA & McHenry CR 2005 Parathyroid carcinoma. Journal of Surgical Oncology 89 136–142.

  • Mosimann C, Hausmann G & Basler K 2006 Parafibromin/Hyrax activates Wnt/Wg target gene transcription by direct association with β-catenin/Armadillo. Cell 125 327–341.

    • Search Google Scholar
    • Export Citation
  • Polak J & van Noorden S ( 1986) An introduction to immunocytochemistry: current techniques and problems. In Immunocytochemistry: Modern Methods and Applications, edn 2. Bristol: John Wright & Sons.

  • Rodgers SE & Perrier ND 2006 Parathyroid carcinoma. Current Opinion in Oncology 18 16–22.

  • Rozenblatt-Rosen O, Hughes CM, Nannepaga SJ, Shanmugam KS, Copeland TD, Guszczynski T, Resau JH & Meyerson M 2005 The parafibromin tumor suppressor protein is part of a human Paf1 complex. Molecular and Cellular Biology 25 612–620.

    • Search Google Scholar
    • Export Citation
  • Sandelin K, Auer G, Bondeson L, Grimelius L & Farnebo L-O 1992 Prognostic factors in parathyroid cancer: a review of 95 cases. World Journal of Surgery 16 724–731.

    • Search Google Scholar
    • Export Citation
  • Shane E 2001 Clinical review 122: parathyroid carcinoma. Journal of Clinical Endocrinology and Metabolism 86 485–493.

  • Shattuck TM, Välimäki S, Obara T, Gaz RD, Clark OH, Shoback D, Wierman ME, Tojo K, Robbins CM, Carpten JD et al. 2003 Somatic and germ-line mutations of the HRPT2 gene in sporadic parathyroid carcinoma. New England Journal of Medicine 349 1722–1729.

    • Search Google Scholar
    • Export Citation
  • Stojadinovic A, Hoos A, Nissan A, Dudas ME, Cordon-Cardo C, Shaha AR, Brennan MF, Singh B & Ghossein RA 2003 Parathyroid neoplasms: clinical, histopathological and tissue microarray-based molecular analysis. Human Pathology 34 54–64.

    • Search Google Scholar
    • Export Citation
  • Tan MH, Morrison C, Wang P, Yang X, Haven CJ, Zhang C, Zhao P, Tretiakova MS, Korpi-Hyovalti E, Burgess JR et al. 2004 Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clinical Cancer Research 10 6629–6637.

    • Search Google Scholar
    • Export Citation
  • Vasef MA, Brynes RK, Sturm M, Bromley C & Robinson RA 1999 Expression of cyclin D1 in parathyroid carcinomas, adenomas and hyperplasias: a Paraffin Immunohistochemical study. Modern Pathology 12 412–416.

    • Search Google Scholar
    • Export Citation
  • Woodard GE, Lin L, Zhang JH, Agarwal SK, Marx SJ & Simonds WF 2005 Parafibromin, product of the hyperparathyroidism-jaw tumor syndrome gene HRPT2, regulates cyclin D1/PRAD1 expression. Oncogene 24 1272–1276.

    • Search Google Scholar
    • Export Citation
  • Yart A, Gstaiger M, Wirbelauer C, Pecnik M, Anastasiou D, Hess D & Krek W 2005 The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II. Molecular and Cellular Biology 25 5052–5060.

    • Search Google Scholar
    • Export Citation
  • Zhang C, Kong D, Tan MH, Pappas DL Jr, Wang PF, Chen J, Farber J, Zhang N, Koo HM, Weinreich M et al. 2006 Parafibromin inhibits cancer cell growth and causes G1 phase arrest. Biochemical and Biophysical Research Communications 350 17–24.

    • Search Google Scholar
    • Export Citation

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  • View in gallery

    Schematic of the HRPT2 gene, parafibromin, and the antibodies studied. (A) Genomic structure of HRPT2, its 17 coding exons and the gene product parafibromin. ATG represent the initiation codon. Functionally important regions are marked: the nuclear localization signal (NLS) is encoded from exon 5, the β-catenin interaction domain (CID) by exons 7 and 8, and the evolutionary conserved PAF1 complex-binding domain (Cdc73 core) constitutes exons 12–17. The targets of the four antibodies are shown below the schematic of parafibromin. (B) Details of the four parafibromin antibodies.

  • View in gallery

    Western blot analyses of parafibromin expression. Parafibromin protein is demonstrated as a ~60 kDa protein in various parathyroid lesions using the antibodies TNYV, 2H1, BL648, and APVF. The expressions were visually classified as strong +, weak (+), or undetectable (−).

  • View in gallery

    (A) Immunohistochemistry demonstrating mainly nuclear expression of parafibromin in control adenoma 1 as well as HRPT2-transfected HeLa cells using antibodies TNYV, 2H1, BL648, and APVF. Inserts are parathyroid cells from the normal glandular rim. The specificity is verified by peptide neutralization tests. All images are magnified 140×, while the insert are magnified 260×. (B) Western blot analysis after sub-cellular fractionation, showing parafibromin expression in the nuclear fraction of control adenoma 2. Prohibitin is a mitochondrial antigen.

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    Photomicrographs showing immunohistochemical analyses of parafibromin expression in parathyroid carcinomas T5, T24, and T23 using antibodies TNYV, 2H1, BL648, and APVF. The unequivocal carcinoma T5 (negative) exhibits a total loss of parafibromin immunoreactivity with all four antibodies. In the equivocal carcinoma T24 (partial loss), the staining pattern represents ~80% positive tumor cells, while the equivocal cancer T23 (positive) demonstrates clear nuclear staining in 100% of the tumor cells. All images are magnified 160×, while the inserts are magnified 280×. To fully evaluate samples with partial loss, high power magnification is required as demonstrated in T24.

  • Bondeson L, Sandelin K & Grimelius L 1993 Histopathological variables and DNA cytometry in parathyroid carcinoma. American Journal of Surgical Pathology 17 820–829.

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    • Export Citation
  • Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 248–254.

    • Search Google Scholar
    • Export Citation
  • Bradley KJ, Hobbs MR, Buley ID, Carpten JD, Cavaco BM, Fares JE, Laidler P, Manek S, Robbins CM, Salti IS et al. 2005 Uterine tumours are a phenotypic manifestation of the hyperparathyroidism-jaw tumour syndrome. Journal of Internal Medicine 257 18–26.

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    • Export Citation
  • Bradley KJ, Cavaco BM, Bowl MR, Harding B, Cranston T, Fratter C, Besser GM, Conceicao Pereira M, Davie MW, Dudley N et al. 2006 Parafibromin mutations in hereditary hyperparathyroidism syndromes and parathyroid tumours. Journal of Clinical Endocrinology 64 299–306.

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    • Export Citation
  • Bradley KJ, Bowl MR, Williams SE, Ahmad BN, Partridge CJ, Patmanidi AL, Kennedy AM, Loh NY, Thakker RV 2007 Parafibromin is a nuclear protein with a functional monopartite nuclear localization signal. Oncogene 26 1213–1221.

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    • Export Citation
  • Carpten JD, Robbins CM, Villablanca A, Forsberg L, Presciuttini S, Bailey-Wilson J, Simonds WF, Gillanders EM, Kennedy AM, Chen JD et al. 2002 HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumour syndrome. Nature Genetics 32 676–680.

    • Search Google Scholar
    • Export Citation
  • Cetani F, Pardi E, Borsari S, Viacava P, Dipollina G, Cianferotti L, Ambrogini E, Gazzerro E, Colussi G, Berti P et al. 2004 Genetic analyses of the HRPT2 gene in primary hyperparathyroidism: germline and somatic mutations in familial and sporadic parathyroid tumors. Journal of Clinical Endocrinology and Metabolism 89 5583–5591.

    • Search Google Scholar
    • Export Citation
  • Cryns VL, Thor A, Xu HJ, Hu SX, Wierman ME, Vickery AL Jr, Benedict WF & Arnold A 1994 Loss of the retinoblastoma tumor-suppressor gene in parathyroid carcinoma. New England Journal of Medicine 330 757–761.

    • Search Google Scholar
    • Export Citation
  • DeLellis RA 2005 Parathyroid carcinoma – an overview. Advances in Anatomic Pathology 12 53–61.

  • DeLellis RA, Lloyd RV, Heitz PU & Eng C 2004 Tumours of endocrine organs. In World Health Organization Classification of Tumour Pathology and Genetics, Eds RA DeLellis, RV Lloyd, PU Heitz & C Eng. Lyon: IARC Press.

  • Erickson LA, Jin L, Wollan P, Thompson GB, van Heerden JA & Lloyd RV 1999 Parathyroid hyperplasia, adenomas and carcinomas: differential expression of p27Kip1 protein. American Journal of Surgical Pathology 23 288–295.

    • Search Google Scholar
    • Export Citation
  • Farnebo F, Auer G, Farnebo L-O, The BT, Twigg S, Apendblad U, Thomson NW, Grimelius L, Larsson C & Sandelin K 1999 Evaluation of retinoblastoma and Ki-67 as diagnostic markers in benign and malignant parathyroid disease. World Journal of Surgery 23 68–74.

    • Search Google Scholar
    • Export Citation
  • Forsberg L, Larsson C, Sofiadis A, Lewensohn R, Höög A & Lehtio J 2006 Pre-fractionation of archival frozen tumours for proteomics applications. Journal of Biotechnology 126 582–586.

    • Search Google Scholar
    • Export Citation
  • Gill AJ, Clarkson A, Gimm O, Keil J, Dralle H, Howell VM & Marsh DJ 2006 Loss of nuclear expression of parafibromin distinguishes parathyroid carcinomas and hyperparathyroidism–jaw tumor (HPT–JT) syndrome-related adenomas from sporadic parathyroid adenomas and hyperplasias. American Journal of Surgical Pathology 30 1140–1149.

    • Search Google Scholar
    • Export Citation
  • Gimm O, Lorenz K, Nguyen Thanh P, Schneyer U, Howell VM, Marsh DJ, Teh BT, Krause U & Dralle H 2006 Prophylactic parathyroidectomy for familial parathyroid carcinoma. Der Chirurg; Zeitschrift für alle Gebiete der operativen Medizen 77 15–24 (In German).

    • Search Google Scholar
    • Export Citation
  • Grimelius L & Johansson H 1997 Pathology of parathyroid tumors. International Seminars in Surgical Oncology 13 142–154.

  • Hahn MA & Marsh DJ 2005 Identification of a functional bipartite nuclear localization in the tumor suppressor parafibromin. Oncogene 24 6241–6248.

    • Search Google Scholar
    • Export Citation
  • Howell VM, Haven CJ, Kahnoski K, Khoo SK, Petillo D, Chen J, Fleuren GJ, Robinson BG, Delbridge LW, Philips J et al. 2003 HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours. Journal of Medical Genetics 40 657–663.

    • Search Google Scholar
    • Export Citation
  • Juhlin C, Höög A, Yakoleva T, Leibiger I, Leibiger B, Weber G, Larsson C & Villablanca A 2006 Loss of parafibromin expression in a subset of sporadic parathyroid adenomas. Endocrine Related Cancer 13 509–523.

    • Search Google Scholar
    • Export Citation
  • Krebs LJ, Shattuck TM & Arnold A 2005 HRPT2 mutational analysis of typical sporadic parathyroid adenomas. Journal of Clinical Endocrinology and Metabolism 90 5015–5017.

    • Search Google Scholar
    • Export Citation
  • Kytölä S, Farnebo F, Obara T, Isola J, Grimelius L, Farnebo L-O, Sandelin K & Larsson C 2000 Patterns of chromosomal imbalances in parathyroid carcinomas. American Journal of Pathology 157 579–586.

    • Search Google Scholar
    • Export Citation
  • Levin KE, Galante M & Clark OH 1987 Parathyroid carcinoma versus parathyroid adenoma in patients with profound hypercalcemia. Surgery 101 649–660.

    • Search Google Scholar
    • Export Citation
  • Mittendorf EA & McHenry CR 2005 Parathyroid carcinoma. Journal of Surgical Oncology 89 136–142.

  • Mosimann C, Hausmann G & Basler K 2006 Parafibromin/Hyrax activates Wnt/Wg target gene transcription by direct association with β-catenin/Armadillo. Cell 125 327–341.

    • Search Google Scholar
    • Export Citation
  • Polak J & van Noorden S ( 1986) An introduction to immunocytochemistry: current techniques and problems. In Immunocytochemistry: Modern Methods and Applications, edn 2. Bristol: John Wright & Sons.

  • Rodgers SE & Perrier ND 2006 Parathyroid carcinoma. Current Opinion in Oncology 18 16–22.

  • Rozenblatt-Rosen O, Hughes CM, Nannepaga SJ, Shanmugam KS, Copeland TD, Guszczynski T, Resau JH & Meyerson M 2005 The parafibromin tumor suppressor protein is part of a human Paf1 complex. Molecular and Cellular Biology 25 612–620.

    • Search Google Scholar
    • Export Citation
  • Sandelin K, Auer G, Bondeson L, Grimelius L & Farnebo L-O 1992 Prognostic factors in parathyroid cancer: a review of 95 cases. World Journal of Surgery 16 724–731.

    • Search Google Scholar
    • Export Citation
  • Shane E 2001 Clinical review 122: parathyroid carcinoma. Journal of Clinical Endocrinology and Metabolism 86 485–493.

  • Shattuck TM, Välimäki S, Obara T, Gaz RD, Clark OH, Shoback D, Wierman ME, Tojo K, Robbins CM, Carpten JD et al. 2003 Somatic and germ-line mutations of the HRPT2 gene in sporadic parathyroid carcinoma. New England Journal of Medicine 349 1722–1729.

    • Search Google Scholar
    • Export Citation
  • Stojadinovic A, Hoos A, Nissan A, Dudas ME, Cordon-Cardo C, Shaha AR, Brennan MF, Singh B & Ghossein RA 2003 Parathyroid neoplasms: clinical, histopathological and tissue microarray-based molecular analysis. Human Pathology 34 54–64.

    • Search Google Scholar
    • Export Citation
  • Tan MH, Morrison C, Wang P, Yang X, Haven CJ, Zhang C, Zhao P, Tretiakova MS, Korpi-Hyovalti E, Burgess JR et al. 2004 Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clinical Cancer Research 10 6629–6637.

    • Search Google Scholar
    • Export Citation
  • Vasef MA, Brynes RK, Sturm M, Bromley C & Robinson RA 1999 Expression of cyclin D1 in parathyroid carcinomas, adenomas and hyperplasias: a Paraffin Immunohistochemical study. Modern Pathology 12 412–416.

    • Search Google Scholar
    • Export Citation
  • Woodard GE, Lin L, Zhang JH, Agarwal SK, Marx SJ & Simonds WF 2005 Parafibromin, product of the hyperparathyroidism-jaw tumor syndrome gene HRPT2, regulates cyclin D1/PRAD1 expression. Oncogene 24 1272–1276.

    • Search Google Scholar
    • Export Citation
  • Yart A, Gstaiger M, Wirbelauer C, Pecnik M, Anastasiou D, Hess D & Krek W 2005 The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II. Molecular and Cellular Biology 25 5052–5060.

    • Search Google Scholar
    • Export Citation
  • Zhang C, Kong D, Tan MH, Pappas DL Jr, Wang PF, Chen J, Farber J, Zhang N, Koo HM, Weinreich M et al. 2006 Parafibromin inhibits cancer cell growth and causes G1 phase arrest. Biochemical and Biophysical Research Communications 350 17–24.

    • Search Google Scholar
    • Export Citation