A novel HER2 gene body enhancer contributes to HER2 expression.

The transcriptional regulation of the human epidermal growth factor receptor-2 (HER2) contributes to an enhanced HER2 expression in HER2-positive breast cancers with HER2 gene amplification and HER2-low or HER2-negative breast cancers following radiotherapy or endocrine therapy, and this drives tumorigenesis and the resistance to therapy. Epigenetic mechanisms are critical for transcription regulation, however, such mechanisms in the transcription regulation of HER2 are limited to the involvement of tri-methylated histone 3 lysine 4 (H3K4me3) and acetylated histone 3 lysine 9 (H3K9ac) at the HER2 promoter region. Here, we report the identification of a novel enhancer in the HER2 3' gene body, which we have termed HER2 gene body enhancer (HGE). The HGE starts from the 3' end of intron 19 and extends into intron 22, possesses enhancer histone modification marks in specific cells and enhances the transcriptional activity of the HER2 promoters. We also found that TFAP2C, a known regulator of HER2, binds to HGE and is required for its enhancer function and that DNA methylation in the HGE region inhibits the histone modifications characterizing enhancer and is inversely correlated with HER2 expression in breast cancer samples. The identification of this novel enhancer sheds a light on the roles of epigenetic mechanisms in HER2 transcription, in both HER2-positive breast cancer samples and individuals with HER2-low or HER2-negative breast cancers undergoing radiotherapy or endocrine therapy.

Extracellular matrix 1 (ECM1) regulates the actin cytoskeletal architecture of aggressive breast cancer cells in part via S100A4 and Rho-family GTPases.

ECM1 overexpression is an independent predictor of poor prognosis in primary breast carcinomas, however the mechanisms by which ECM1 affects tumor progression have not been completely elucidated. ECM1 was silenced in the triple-negative breast cancer cell lines Hs578T and MDAMB231 using siRNA and the cells were evaluated for changes in morphology, migration, invasion and adhesion. Actin cytoskeleton alterations were evaluated by fluorescent staining and levels of activated Rho GTPases by pull down assays. ECM1 downregulation led to significantly diminished cell migration (p = 0.0005 for Hs578T and p = 0.02 for MDAMB231) and cell adhesion (p < 0.001 for Hs578T and p = 0.01 for MDAMB231). Cell invasion (matrigel) was reduced only in the Hs578T cells (p < 0.01). Silencing decreased the expression of the prometastatic molecules S100A4 and TGFßR2 in both cell lines and CD44 in Hs578T cells. ECM1-silenced cells also exhibited alterations in cell shape and showed bundles of F-actin across the cell (stress fibers) whereas NT-siRNA treated cells showed peripheral membrane ruffling. Downregulation of ECM1 was also associated with an increased F/G actin ratio, when compared to the cells transfected with NT siRNA (p < 0.001 for Hs578T and p < 0.00035 for MDAMB231) and a concomitant decline of activated Rho A in the Hs578T cells. Re-expression of S100A4 in ECM1-silenced cells rescued the phenotype in the Hs578T cells but not the MDAMB231 cells. We conclude that ECM1 is a key player in the metastatic process and regulates the actin cytoskeletal architecture of aggressive breast cancer cells at least in part via alterations in S100A4 and Rho A.

The role of Tcfap2c in tumorigenesis and cancer growth in an activated Neu model of mammary carcinogenesis.

TFAP2C/AP-2γ influences development of the mammary gland and regulates patterns of gene expression in luminal and HER2-amplified breast cancer. The roles of TFAP2C in mammary gland tumorigenesis and in pathways critical to cancer progression remain poorly understood. To gain greater insight into oncogenic mechanisms regulated by TFAP2C, we examined mammary tumorigenesis in MMTV-Neu transgenic female mice with or without conditional knockout (KO) of Tcfap2c, the mouse homolog of TFAP2C. Loss of Tcfap2c increased the latency of tumorigenesis and tumors that formed demonstrated reduced proliferative index and increased apoptosis. In addition, tumors formed in Tcfap2c KO animals had a significant reduction in Egfr levels without a change in the expression of the Neu oncogene. The MMneu-flAP2C cell line was established from tumor tissue derived from MMTV-Neu/Tcfap2c(L/L) control animals and parallel cell lines with and without expression of Tcfap2c were created by transduction with adenovirus-empty and adenovirus-Cre, respectively. KO of Tcfap2c in vitro reduced activated phosphorylated-Erk, decreased cell viability, repressed tumor growth and was associated with attenuation of Egfr expression. Chromatin immunoprecipitation and direct sequencing and expression analysis confirmed that Egfr was a Tcfap2c target gene in murine, as well as human, mammary carcinoma cells. Furthermore, decreased viability of mammary cancer cells was directly related to Egfr functional blockade. We conclude that TFAP2C regulates tumorigenesis, cell growth and survival in HER2-amplified breast cancer through transcriptional regulation of EGFR. The findings have important implications for targeting the EGFR pathway in breast cancer.

TFAP2C governs the luminal epithelial phenotype in mammary development and carcinogenesis.

Molecular subtypes of breast cancer are characterized by distinct patterns of gene expression that are predictive of outcome and response to therapy. The luminal breast cancer subtypes are defined by the expression of estrogen receptor-alpha (ERα)-associated genes, many of which are directly responsive to the transcription factor activator protein 2C (TFAP2C). TFAP2C participates in a gene regulatory network controlling cell growth and differentiation during ectodermal development and regulating ESR1/ERα and other luminal cell-associated genes in breast cancer. TFAP2C has been established as a prognostic factor in human breast cancer, however, its role in the establishment and maintenance of the luminal cell phenotype during carcinogenesis and mammary gland development have remained elusive. Herein, we demonstrate a critical role for TFAP2C in maintaining the luminal phenotype in human breast cancer and in influencing the luminal cell phenotype during normal mammary development. Knockdown of TFAP2C in luminal breast carcinoma cells induced epithelial-mesenchymal transition with morphological and phenotypic changes characterized by a loss of luminal-associated gene expression and a concomitant gain of basal-associated gene expression. Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in mouse mammary epithelium driven by MMTV-Cre promoted aberrant growth of the mammary tree leading to a reduction in the CD24(hi)/CD49f(mid) luminal cell population and concomitant gain of the CD24(mid)/CD49f(hi) basal cell population at maturity. Our results establish TFAP2C as a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma. Furthermore, Tcfap2c influences development of the luminal cell type during mammary development. The data suggest that TFAP2C has an important role in regulated luminal-specific genes and may be a viable therapeutic target in breast cancer.

Transcriptional regulation of the GPX1 gene by TFAP2C and aberrant CpG methylation in human breast cancer.

The complexity of gene regulation has created obstacles to defining mechanisms that establish the patterns of gene expression characteristic of the different clinical phenotypes of breast cancer. TFAP2C is a transcription factor that has a critical role in the regulation of both estrogen receptor-alpha (ERα) and c-ErbB2/HER2 (Her2). Herein, we performed chromatin immunoprecipitation and direct sequencing (ChIP-seq) for TFAP2C in four breast cancer cell lines. Comparing the genomic binding sites for TFAP2C, we identified that glutathione peroxidase (GPX1) is regulated by TFAP2C through an AP-2 regulatory region in the promoter of the GPX1 gene. Knockdown of TFAP2C, but not the related factor TFAP2A, resulted in an abrogation of GPX1 expression. Selenium-dependent GPX activity correlated with endogenous GPX1 expression and overexpression of exogenous GPX1 induced GPX activity and significantly increased resistance to tert-butyl hydroperoxide. Methylation of the CpG island encompassing the AP-2 regulatory region was identified in cell lines where TFAP2C failed to bind the GPX1 promoter and GPX1 expression was unresponsive to TFAP2C. Furthermore, in cell lines where GPX1 promoter methylation was associated with gene silencing, treatment with 5'-aza-2-deoxycytidine (5'-aza-dC) (an inhibitor of DNA methylation) allowed TFAP2C to bind to the GPX1 promoter resulting in the activation of GPX1 RNA and protein expression. Methylation of the GPX1 promoter was identified in ∼20% of primary breast cancers and a highly significant correlation between the TFAP2C and GPX1 expression was confirmed when considering only those tumors with an unmethylated promoter, whereas the related factor, TFAP2A, failed to demonstrate a correlation. The results demonstrate that TFAP2C regulates the expression of GPX1, which influences the redox state and sensitivity to oxidative stress induced by peroxides. Given the established role of GPX1 in breast cancer, the results provide an important mechanism for TFAP2C to further influence oncogenesis and progression of breast carcinoma cells.

AP2alpha alters the transcriptional activity and stability of p53.

AP2alpha and p53 form nuclear complexes that establish a functional partnership, which regulates the expression of certain genes involved in cell growth and metastasis. The growth effects of AP2alpha are mediated through p21WAF1/CIP1 and the ability for AP2alpha to coactivate p21 requires p53. Herein, we have localized the AP2-binding region of p53 to amino acids 305-375. Analysis of 26 distinct p53 alleles established a correlation between AP2alpha binding and transcriptional coactivation. The L350P point mutation was the only nonbinding allele that retained normal transcriptional activity by reporter assay. Although both wild-type and L350P alleles facilitated binding of AP2alpha to the p21 promoter, the L350P allele was significantly reduced in its ability to induce the endogenous p21 gene, demonstrating a striking difference in activity comparing reporter assays with activation of endogenous p53 target genes. Interestingly, expression of AP2 in the absence of radiation repressed p53-mediated induction of p21 and this effect was explained by a reduction in p53 stability induced by AP2alpha overexpression. We conclude that AP2alpha has competing effects on p53 activity through coactivation and decreased stability. These findings may provide a mechanism to account for the discrepancies reported for the association between AP2 and p21 expression in tumor tissue.

A spike in parathyroid hormone during neck exploration may cause a false-negative intraoperative assay result.

HYPOTHESIS: We hypothesize that false-negative results using the rapid intraoperative parathyroid hormone (IOPTH) assay can be caused by spikes in the level of parathyroid hormone that occur during mobilization of the adenoma. DESIGN: Retrospective analysis of a case series. SETTING: University tertiary care center. PATIENTS: Ten consecutive patients with primary hyperparathyroidism. INTERVENTIONS: All patients underwent neck exploration with IOPTH monitoring. Using a sampling protocol described in the literature, IOPTH values were checked at the time of incision, during mobilization of the adenoma, and 10 minutes after resection of the adenoma. MAIN OUTCOME MEASURES: Patients were evaluated for adequate parathyroid tissue excision as determined by IOPTH levels and examination of ipsilateral glands. All patients had normal serum calcium values documented postoperatively. Parathyroid hormone half-life was calculated assuming first-order kinetic decay. RESULTS: Nine patients had an appropriate decline in IOPTH with a mean +/- SD parathyroid hormone half-life of 3.9 +/- 1.08 minutes. Mobilization of the adenoma resulted in a spike in the IOPTH value, with 1 patient's value increasing from a baseline of 95.5 pg/mL (10.1 pmol/L) to 751 pg/mL (79.1 pmol/L). Another patient who was confirmed to have a solitary adenoma had a false-negative postexcision value. A spike in IOPTH that occurred during neck dissection was not detected by the sampling protocol and explains the false-negative value. A literature review revealed that most protocols check baseline values early in the operation and are at risk for false-negative results due to a spike from mobilization of the adenoma. CONCLUSIONS: These data demonstrate that false-negative IOPTH assay findings can result from a spike in parathyroid hormone level during exploration, which may go unrecognized if baseline values are measured during the early stages of mobilization of the adenoma. We have altered our assay protocol and have begun measuring IOPTH at the time of neck incision, at the time the adenoma is completely removed (time zero [t(0)]), and 10 minutes after excision.

Ligand-dependent interaction of estrogen receptor-alpha with members of the forkhead transcription factor family.

Estrogen acting through the estrogen receptor (ER) is able to regulate cell growth and differentiation of a variety of normal tissues and hormone-responsive tumors. Ligand-activated ER binds DNA and transactivates the promoters of estrogen target genes. In addition, ligand-activated ER can interact with other factors to alter the physiology and growth of cells. Using a yeast two-hybrid screen, we have identified an interaction between ER alpha and the proapoptotic forkhead transcription factor FKHR. The ER alpha-FKHR interaction depends on beta-estradiol and is reduced significantly in the absence of hormone or the presence of Tamoxifen. A glutathione S-transferase pull-down assay was used to confirm the interaction and localized two interaction sites, one in the forkhead domain and a second in the carboxyl terminus. The FKHR interaction was specific to ER alpha and was not detected with other ligand-activated steroid receptors. The related family members, FKHRL1 and AFX, also bound to ER alpha in the presence of beta-estradiol. FKHR augmented ER alpha transactivation through an estrogen response element. Conversely, ER alpha repressed FKHR-mediated transactivation through an insulin response sequence, and cell cycle arrest induced by FKHRL1 in MCF7 cells was abrogated by estradiol. These results suggest a novel mechanism of estrogen action that involves regulation of the proapoptotic forkhead transcription factors.

Parathyroid crisis in a 20 year old--an unusual cause of hypercalcaemic crisis.

Since the advent of automated serum analysis, patients with primary hyperparathyroidism (PHPT) are often asymptomatic at presentation or have mild symptoms attributable to the disease. Parathyroid crisis is a rare and potentially fatal complication of PHPT in which patients develop severe hypercalcaemia with signs and symptoms of multiple organ dysfunction. A case of parathyroid crisis in a 20 year old man who presented with brown tumours and renal stones is described.

Genetic analysis of a papillary thyroid carcinoma in a patient with MEN1.

BACKGROUND: MENI is an inherited tumor syndrome characterized by the development of tumors of the parathyroid, the anterior pituitary and the pancreatic islets. Tumors of these endocrine glands in MEN1 patients demonstrate loss of heterozygosity (LOH) at the locus of the MEN1 tumor suppressor gene. Menin, the protein encoded by the MEN1 gene, is ubiquitously expressed in endocrine tissue, and less commonly these patients can present with tumors of other endocrine tissues, including thyroid and adrenal. We hypothesize that MEN1 gene mutation may be involved in the oncogenesis of other less common tumors. METHODS: We report a MEN1 patient who was found to have metastatic papillary thyroid cancer at the time of neck exploration for hyperparathyroidism. Genetic analysis of tumor tissue was performed using one intragenic (D11S4946) and two flanking (D11S4945 and D11S4940) polymorphic markers. RESULTS: Two of the markers were informative. Consistent with previous studies, there was LOH in the parathyroid adenoma identified with the intragenic marker D11S4946. However, the papillary cancer was found to be heterozygous at two informative markers. CONCLUSIONS: The lack of obvious LOH of the MEN1 locus in the papillary cancer suggests that, in contrast to parathyroid adenoma, deletion of the MEN1 tumor suppressor gene is not etiologically related to the oncogenesis of the papillary cancer in this patient.

Intraoperative ultrasonography for localization of recurrent thyroid cancer.

Advances in measurement of thyroglobulin (Tg) and in imaging techniques including high resolution ultrasound, magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET) scan have increased our ability to detect thyroid cancer recurrences at an earlier stage. (1,2) After thyroidectomy, patients are often treated with radioiodine, but the recurrent cancers may not image with radioiodine. In these instances, the only definitive treatment is surgical resection. Reoperative neck surgery can be challenging, especially when trying to find a small cancer nodule within the central neck that contains dense fibrotic scar tissue. Herein we describe the use of intraoperative ultrasonography to identify the location of recurrent thyroid cancer. This technique can aid in tumor localization and may help to avoid complications such as recurrent nerve injury.

Genomic structure of the promoters of the human estrogen receptor-alpha gene demonstrate changes in chromatin structure induced by AP2gamma.

Expression of human estrogen receptor-alpha (ERalpha) involves the activity from several promoters that give rise to alternate untranslated 5' exons. However, the genomic locations of the alternate 5' exons have not been reported previously. We have developed a contig map of the human ERalpha gene that includes all of the known alternate 5' exons. By using S1 nuclease and 5'- rapid amplification of cDNA ends, the cap sites for the alternate ERalpha transcripts E and H were identified. DNase I-hypersensitive sites specific to ERalpha-positive cells were associated with each of the cap sites. A DNase I-hypersensitive site, HS1, was localized to binding sites for AP2 in the untranslated region of exon 1 and was invariably present in the chromatin structure of ERalpha-positive cells. Overexpression of AP2gamma in human mammary epithelial cells generated the HS1-hypersensitive site. The ERalpha promoter was induced by AP2gamma in mammary epithelial cells, and trans-activation was dependent upon the region of the promoter containing the HS1 site. These results demonstrate that AP2gamma trans-activates the ERalpha gene in hormone-responsive tumors by inducing changes in the chromatin structure of the ERalpha promoter. These data are further evidence for a critical role for AP2 in the oncogenesis of hormone-responsive breast cancers.

Nonoperative management of hyperparathyroidism: present and future.

Parathyroidectomy provides effective treatment for primary and secondary hyperparathyroidism with a predictable response of symptoms related to hypercalcemia and elevated parathyroid hormone. Calcium and vitamin D supplementation has reduced the need for parathyroidectomy in dialysis patients with secondary hyperparathyroidism. However, surgery continues to be the only effective treatment of primary hyperparathyroidism. Potential nonoperative treatments for hyperparathyroidism have included the use of estrogen replacement, bisphosphonates, and a new class of drugs known as calcimimetics. Hormone replacement therapy with estrogen has been reported to improve cortical bone density in postmenopausal women with asymptomatic or mildly symptomatic primary hyperparathyroidism. Calcimimetic agents are a new class of drugs that increase the sensitivity of the calcium receptor to ionized calcium. Initial studies have shown that calcimimetics can acutely lower parathyroid hormone levels in patients with primary and secondary hyperparathyroidism. These drugs are currently being evaluated in phase II clinical trials. Ultimately, these medical modalities will need to be compared to parathyroidectomy in randomized controlled clinical trials.

Recombinant human thyrotropin in the management of thyroid cancer.

Radioiodine has been shown to reduce recurrences and improve survival in well-differentiated thyroid cancer. To maximize the effectiveness of radioiodine therapy, patients are first treated by total thyroidectomy and then allowed to become hypothyroid. The elevation of thyroid-stimulating hormone, or thyrotropin (TSH), that occurs with hypothyroidism stimulates uptake of radioiodine in normal and cancerous thyroid tissues. A recent advance has been the introduction of recombinant human TSH (rhTSH), which is administered intramuscularly prior to testing with radioiodine. Phase III trials have demonstrated that rhTSH stimulates both uptake in and production of thyroglobulin by thyroid cells and the results are comparable to those of hypothyroid protocols in the majority of patients. Patients prefer the rhTSH protocol because they continue to ingest exogenous thyroid hormone and the symptoms of hypothyroidism are avoided. The rhTSH protocol is preferable in patients with pituitary dysfunction and in those who cannot tolerate hypothyroidism. RhTSH can also allow treatment of patients who have not had an adequate thyroidectomy and who are poor candidates for reoperation.

PDZK1 and GREB1 are estrogen-regulated genes expressed in hormone-responsive breast cancer.

The function of estrogen in breast cancer proliferation and progression is likely to be due to the expression of a repertoire of genes regulated by estrogen receptor (ER). Using suppression subtractive hybridization, we have isolated a set of 14 estrogen-responsive genes that was differentially expressed in MCF7 cells stimulated by beta-estradiol as compared with unstimulated cells. Tamoxifen repressed the expression of all 14 estrogen-responsive genes. Thirteen of the genes were induced within 6 h of estrogen treatment, indicating that these were early response genes in the ER-regulated pathway. PDZK1 and a new gene, GREB1, demonstrated a significant correlation with ER phenotype in a panel of breast cancer cell lines. Treatment with cycloheximide indicated that ER directly controls GREB1 expression. Three cDNAs (GREB1a, GREB1b, and GREB1c) were isolated by screening a MCF7 cDNA library. These three cDNAs of GREB1 shared extensive sequences through the open reading frame but had divergent 5' untranslated regions, indicating the possibility of multiple promoters regulated by beta-estradiol. Studies in primary breast cancers showed that PDZK1 and GREB1 were overexpressed in ER-positive breast cancers as compared with ER-negative breast cancers by 19-fold and 3.5-fold, respectively. GREB1 was also induced by beta-estradiol in the ER-positive endometrial cell line ECC-1. The pattern of expression suggests a critical role for these two genes in the response of tissues and tumors to beta-estradiol.

Recombinant human thyrotropin (rhTSH) in the management of differentiated thyroid cancer.

Recombinant human thyrotropin (rhTSH) has been evaluated in 38 patients with differentiated thyroid cancer. The patients had all been treated previously by operation and 31 had received radioiodine 131I. The patients continued to take thyroid hormone and changed to a low iodine diet for 14 days before and throughout the week of testing. The rhTSH was injected intramuscularly on two consecutive days, 74 MBq 131I was administered on the next day and scintigraphy completed 48 h after that. TSH was measured before administration of 131I, and thyroglobulin after the scan. All patients preferred this method to withdrawal of thyroid hormone, but 45% had mild symptoms including headache and nausea. The average TSH was 127 mU x l(-1), and was inversely related to the weight of the patients. Thirty-four had negative scans with a mean uptake of 0.06%. Thyroglobulin values above 10 ng x ml(-1) were found in seven patients, of whom four had similar findings when scanned after withdrawal of thyroid hormone. Of four with positive scans, two had undetectable thyroglobulin. The rate of clearance of 131I was compared in patients studied at 72 h who were hypothyroid and at 48 h in euthyroid patients given rhTSH and was found to be longer in the latter. We conclude that rhTSH can be used to stimulate thyroid tissue to trap 131I and secrete thyroglobulin. Both scan and thyroglobulin should be obtained. The method is well tolerated.

Monoallelic amplification of estrogen receptor-alpha expression in breast cancer.

Gene amplification and loss of heterozygosity are alterations to chromosomal structure whereby tumor cells alter patterns of gene expression. We have identified a novel mechanism of gene regulation in which cancer cells predominantly express one of the two alleles of a gene. Estrogen receptor (ER)-alpha is overexpressed in hormone-responsive breast cancer compared with normal breast epithelial cells. Using a polymorphism of codon 10, we examined allele-specific expression of the four different ER promoters in MCF-7 breast cancer cells and primary tumors. Monoallelic amplification of expression (MAX) for all four ER promoters was identified, resulting in an allelic preference of > 100-fold. MAX was the result of an amplification of allele copy number and a preference to transcribe the amplified allele. The effect of MAX was most significant for the promoters clustered near the 1' exon, whereas the expression from the distant H promoter mirrored template copy number. MAX of the ER gene was not found to occur in normal endometrial or breast tissue. As a novel mechanism in cancer genetics, MAX can result in functional homozygosity at a gene locus.

AP2alpha and AP2gamma: a comparison of binding site specificity and trans-activation of the estrogen receptor promoter and single site promoter constructs.

The AP2 transcription factors exhibit a high degree of homology in the DNA binding and dimerization domains. In this study, we methodically compared the binding specificity of AP2alpha and AP2gamma using PCR-assisted binding site selection and competitive gel shift assay and determined that the consensus binding site for both factors is(G)/(C)CCNN(A/)C(/G)(G)/(A)G(G/)C(/T.)The use of single site promoter constructs with either a high or low affinity site demonstrated a direct relationship between site affinity and transcriptional activation. Overexpression of AP2alpha and AP2gamma resulted in the activation of a low affinity binding site construct to levels comparable to those seen with a high affinity site construct at lower amounts of protein expression. Both AP2alpha and AP2gamma were able to trans-activate the cloned human estrogen receptor alpha promoter in ER-negative MDA-MB-231 cells through high affinity AP2 sites in the untranslated leader sequence. This provides a functional mechanism to explain the correlation between AP2 activity and estrogen receptor expression in breast cancer. Since there is overexpression of AP2 factors in breast cancer compared to normal breast epithelium, our results suggest that increased factor expression may activate a set of target genes containing lower affinity binding sites that would normally not be expressed in normal breast epithelium.

Parathyroid localization with high-resolution ultrasound and technetium Tc 99m sestamibi.

HYPOTHESIS: High-resolution ultrasound and technetium Tc 99m sestamibi scanning can be used for preoperative localization of abnormal parathyroid glands in patients with hyperparathyroidism. DESIGN: Ultrasound and sestamibi scanning were performed in patients undergoing neck exploration for hyperparathyroidism. If the 2 scans agreed in identifying a single adenoma, and surgery confirmed the location of a single adenoma and an ipsilateral normal gland, a unilateral exploration was performed. SETTING: University tertiary care center. PATIENTS: Sixty-one consecutive patients undergoing surgery for hyperparathyroidism from September 1, 1994, through September 30, 1997. INTERVENTIONS: High-resolution ultrasound was performed in 59 patients and sestamibi scanning in 58 patients; all patients underwent neck exploration by a single surgeon. MAIN OUTCOME MEASURES: The results of preoperative ultrasound and sestamibi scanning were compared with operative and histological findings. RESULTS: All patients were cured of hypercalcemia. Specificity of ultrasound and sestamibi scanning was 98% and 99%, respectively; however, their sensitivity was only 57% and 54%, respectively. Both imaging modalities had lower sensitivities in the setting of multigland disease. If both imaging studies were considered as a single test, sensitivity for imaging in patients with primary hyperparathyroidism reached 78%. Our localization protocol allowed a unilateral approach in 43% of patients (23 of 53). CONCLUSIONS: These results confirm the value of preoperative localization in patients with hyperparathyroidism. A unilateral approach can be used with a high degree of success in cases when ultrasound and sestamibi scanning agree in the identification of a single adenoma confirmed by surgical exploration with the identification of a normal ipsilateral gland.