Manabu Taguchi

A study of the correlation between morphological findings and biological activities in clinically nonfunctioning pituitary adenomas

OBJECTIVEThe aims of this study are to review the histology of the clinically nonfunctioning pituitary adenomas (CNFPAs) we have observed and to determine whether or not the frequency of cavernous sinus invasion is different among each type of morphology. METHODSIn addition, several proliferative markers, including Ki67, p53, E-cadherin, matrix metallo-proteinase 9 and pituitary tumor derived fibroblast growth factor receptor 4 (ptd-FGFR4), were also investigated in invasive and non-invasive tumors. RESULTSOur consequent 213 CNFPAs were diagnosed as follows: 64% were silent gonadotroph adenomas, 18% were null cell adenomas, 12% were silent corticotroph adenomas, 4% were silent Subtype 3 adenomas, and 1% were other types of adenomas. Female patients or younger patients showed a significant preponderance in silent corticotroph adenomas and in silent Subtype 3 adenomas, respectively. Cavernous sinus invasion occurs most frequently in silent corticotroph adenomas (85%) followed by Subtype 3 adenomas (67%), null cell adenomas (38%), and silent gonadotroph adenomas (11%). There were no significant differences in the Ki67, p53, E-cadherin, matrix metallo-proteinase 9, and ptd-FGFR4 expression between tumors with and without cavernous sinus invasion. CONCLUSIONFrom a clinical standpoint, it is quite important to differentiate morphological type in CNFPAs to aid the clinician in assessing the clinical behavior and prognosis of the tumor. Therefore, we suggest that all CNFPAs be examined not only by conventional light microscopy but also by immunohistochemistry, preferably by electron microscopy, to achieve a correct morphological diagnosis.

High incidence of low O6-methylguanine DNA methyltransferase expression in invasive macroadenomas of Cushing's disease

CONTEXT Crookes cell adenoma (CCA), characterized by massive Crookes hyaline change in corticotroph adenoma, causes a rare subtype of Cushings disease. In contrast to ordinary corticotroph adenomas, CCAs are generally aggressive and present as invasive macroadenomas, which are refractory to both surgery and radiotherapy and have a high-recurrence rate. Moreover, some patients with CCA present with distant or craniospinal metastases. Currently, there are no effective standard therapies for CCA. OBJECTIVE We report a patient with Crookes cell carcinoma who presented with local invasion and liver metastases, which was refractory to conventional therapeutic modalities including transsphenoidal surgery, radiosurgery, medications, and hepatic transcatheter arterial embolization. After all these treatments failed, the patient had monthly temozolomide administrations, resulting in gradual clinical improvement and biochemical data that were consistent with tumor shrinkage. In glioblastoma, low O(6)-methylguanine DNA methyltransferase (MGMT) expression is associated with epigenetic gene silencing and predicts a better response to temozolomide. METHODS We thus investigated MGMT expression, immunohistochemically, in seven CCAs (five invasive macroadenomas and two invasive microadenomas) and 17 ordinary-type adenomas (OTAs; three noninvasive macroadenomas, 12 noninvasive microadenomas, and two invasive microadenomas) from patients with Cushings disease. RESULTS In seven CCAs, all five invasive macroadenomas exhibited low MGMT expression, defined as <5% nuclear MGMT staining. In 17 OTAs, only one adenoma showed low MGMT expression. CONCLUSION In Cushings disease, invasive macroadenomas including CCA usually have low-MGMT expression. Temozolomide thus may be a new therapeutic option for invasive macroadenomas such as CCA particularly when conventional treatments are ineffective.

A patient with hypophosphatemic rickets and ossification of posterior longitudinal ligament caused by a novel homozygous mutation in ENPP1 gene.

X-linked hypophosphatemic rickets/osteomalacia (XLH), autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and autosomal recessive hypophosphatemic rickets/osteomalacia (ARHR1 or ARHR2) are hereditary fibroblast growth factor 23 (FGF23)-related hypophosphatemic rickets showing similar clinical features. We here show a patient with hypophosphatemic rickets and widespread ossification of posterior longitudinal ligament (OPLL). The proband is a 62-year-old female. Her parents are first cousins and showed no signs of rickets or osteomalacia. She showed hypophosphatemic rickets with elevated FGF23 level and had been clinically considered to be suffering from XLH. However, direct sequencing of all coding exons and exon-intron junctions of phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX), FGF23 and dentin matrix protein 1 (DMP1) genes, responsible genes for XLH, ADHR and ARHR1, respectively, showed no mutation. A novel homozygous splice donor site mutation was found at the exon-intron junction of exon 21 of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene responsible for ARHR2 (IVS21+1_3(GTA>CACC)). Subsequent analysis of mRNA revealed that this mutation caused skipping of exon 21 which created a premature stop codon in exon 22. These results indicate that genetic analysis is mandatory for the correct diagnosis of hereditary FGF23-related hypophosphatemic rickets. Because Enpp1 knockout mouse is a model of OPLL, this case also suggests that OPLL is associated with ARHR2.

Impaired glucose metabolism in Japanese patients with acromegaly is restored after successful pituitary surgery if pancreatic β-cell function is preserved

OBJECTIVE Impaired glucose metabolism is common in acromegaly, but it is not clear how glucose metabolism is impaired or what predicts its restoration after cure of the disease. To identify factors involved in the impairment of glucose metabolism in acromegaly, we evaluated clinical parameters before and immediately after surgical cure of the disease. DESIGN AND METHODS We retrospectively analyzed clinical data of 92 consecutive Japanese patients with acromegaly who underwent successful pituitary surgery. Patients who had received medical therapy for acromegaly or insulin treatment for diabetes were excluded. We evaluated insulin resistance (IR) and pancreatic β-cell function in addition to GH and IGF1 levels before and after surgery. Results In this study of Japanese patients with acromegaly, average body mass index (BMI) was 23.4, and no patient had a BMI>30. IR was involved in the impairment of glucose metabolism, which was restored upon surgical cure of acromegaly if β-cell function was preserved. Insufficient β-cell function did not improve after normalization of GH/IGF1 and was associated with impaired glucose metabolism before and after surgery. RESULTS of receiver operating characteristic analysis of preoperative clinical parameters suggest that insulinogenic index (IGI) >0.50 best predicts restoration of normal glucose metabolism upon cure of acromegaly in Japanese patients. CONCLUSIONS IR impairs glucose metabolism in acromegaly. Once β-cell function is impaired, abnormal glucose metabolism persists even after cure of acromegaly. IGI>0.50 indicates that β-cell function is preserved in non-obese Japanese patients with acromegaly.

Functional Activities of Mutant Calcium-Sensing Receptors Determine Clinical Presentations in Patients With Autosomal Dominant Hypocalcemia

OBJECTIVE Autosomal dominant hypocalcemia (ADH) is a congenital isolated hypoparathyroidism caused by activating mutations in the calcium-sensing receptor (CASR) gene. The clinical features of ADH are heterogeneous; some patients are asymptomatic, and others show severe hypocalcemia with Bartters syndrome. We therefore recruited 12 patients with ADH to clarify the determinants of their clinical presentation. DESIGN AND METHODS We studied two sporadic and 10 familial cases of ADH. Serum concentrations of calcium, intact PTH, and magnesium (Mg(2+)) were measured in each patient. Fractional excretion of Mg (FE(Mg)) was calculated in spot urine samples. A nuclear factor of activated T cells luciferase assay was used to analyze the responsiveness of each mutant CaSR to extracellular Ca(2+). RESULTS Genomic analysis revealed five known activating mutations and a novel mutation, E481K, in the CASR. Patients with the A843E, C131W, or F788C mutation showed hypomagnesemia with elevated FE(Mg). Intact PTH in these patients was consistently near the detection limit. In contrast, patients with the P221L, K47N, or E481K mutation exhibited normal Mg(2+) levels. In these patients, intact PTH increased in response to low calcium, and their maximum intact PTH exceeded the lower limit of the reference range. Functional analysis showed an association between the disease severity and the in vitro activity of the mutant CaSR. CONCLUSIONS The functional activity of mutant CaSR determines the serum Mg(2+) level, renal Mg(2+) handling, and intact PTH in patients with ADH. The presence of hypomagnesemia with elevated FE(Mg) may indicate the diagnosis of ADH among patients with PTH-deficient hypoparathyroidism.

Phosphate enhances Fgf23 expression through reactive oxygen species in UMR-106 cells.

Fibroblast growth factor 23 (FGF23) has been shown to work as a phosphotropic hormone. Although FGF23 reduces the serum phosphate level, it has not been established that phosphate directly regulates FGF23 production. In this study, we investigated whether phosphate can enhance Fgf23 expression using the rat osteoblastic cell line UMR-106, which has been shown to express Fgf23 in response to 1,25-dihydroxyvitamin D [1,25(OH)2D]. Phosphate increased Fgf23 expression in a dose- and time-dependent manner in the presence of 1,25(OH)2D. Phosphate also increased Fgf23 promoter activity, but showed no effect on the half-life of Fgf23 messenger RNA. Phosphonoformic acid and PD98059, an inhibitor of MEK, inhibited the effects of phosphate on Fgf23 expression and promoter activity. In addition, phosphate enhanced production of reactive oxygen species (ROS) in UMR-106 cells, and hydrogen peroxide enhanced FGF23 production in a dose- and time-dependent manner. Hydrogen peroxide also enhanced Elk1 reporter activity, a target of the MEK–extracellular-signal-regulated kinase (ERK) pathway. Furthermore, the effect of phosphate on ROS production and Fgf23 expression was inhibited by apocynin, an inhibitor of NADPH oxidase. These results indicate that phosphate directly enhances Fgf23 transcription without affecting the stability of Fgf23 messenger RNA by stimulating NADPH-induced ROS production and the MEK–ERK pathway in UMR-106 cells.

Functional analysis of mutant FAM20C in Raine syndrome with FGF23-related hypophosphatemia

Raine syndrome is an autosomal recessive disorder characterized by generalized osteosclerosis with periosteal bone formation and a distinctive facial phenotype. Either homozygous or compound heterozygous mutations in family with sequence similarity 20, member C (FAM20C) have been reported to cause this syndrome. Recently, it was reported that fibroblast growth factor 23 (FGF23)-related hypophosphatemia was found in patients with non-lethal Raine syndrome, and Fam20c conditional knockout mice presented Fgf23-related hypophosphatemic rickets. To clarify the mechanism of how FAM20C regulates FGF23, we performed functional analysis of mutant FAM20C proteins reported in Raine syndrome. We analyzed 6 mutant FAM20C proteins (T268M, P328S, R408W, D451N, D478A, and R549W) for their distributions, kinase activities, and effects on dentin matrix protein (DMP1) promoter activity. We also analyzed the effect of Fam20c knockdown on Dmp1 and Fgf23 mRNA levels in UMR-106 cells. As a result, all the mutant FAM20C proteins showed decreased kinase activities compared to wild-type (WT) FAM20C, and most of them also showed impaired secretion. Overexpression of WT FAM20C increased DMP1 promoter activity in Saos-2 cells while mutant FAM20C did not. Fam20c knockdown decreased Dmp1 mRNA and increased Fgf23 mRNA in UMR-106 cells. In conclusion, our results suggest that FAM20C suppresses FGF23 production by enhancing DMP1 expression, and inactivating mutations in FAM20C cause FGF23-related hypophosphatemia by decreasing transcription of DMP1.

Recurrence of primary hyperparathyroidism following spontaneous remission with intracapsular hemorrhage of a parathyroid adenoma

A few patients with primary hyperparathyroidism (PHPT) have a chance of spontaneous remission by either infarction of or hemorrhage into or around the parathyroid adenoma. In most cases, biochemical derangements associated with PHPT are permanently improved after spontaneous remission. Here we report a case with a recurrence of PHPT 4 months after spontaneous remission with acute intracapsular hemorrhage of parathyroid adenoma. In the literature, only two cases have been reported to have experienced a recurrence of clinical features of PHPT after infarction but not hemorrhage of parathyroid adenomas. Thus, the spontaneous remission of biological derangements in PHPT upon hemorrhage or infarction of parathyroid adenoma could be temporary. One should carefully observe such patients thereafter.

Patients with FGF23-related hypophosphatemic rickets/osteomalacia do not present with left ventricular hypertrophy

ABSTRACT Purpose: Fibroblast growth factor 23 (FGF23) is a hormone regulating phosphate metabolism. Excessive actions of FGF23 cause several types of FGF23-related hypophosphatemic rickets/osteomalacia. Recently, it was reported that FGF23 levels were independently correlated with left ventricular hypertrophy (LVH) in patients with chronic kidney disease (CKD). In addition, FGF23 was also shown to cause cardiac hypertrophy directly acting on cardiomyocytes. However, there is no study indicating the correlation between FGF23 and LVH in adult patients with FGF23-related hypophosphatemic rickets/osteomalacia. Therefore, we examined the existence of LVH in these patients. Materials and methods: We recruited consecutive 24 patients with FGF23-related hypophosphatemic diseases. Their serum intact FGF23 levels and the parameters associated with LVH, including left ventricular mass index (LVMI), relative wall thickness (RWT), Sokolow-Lyon voltage, and Cornell product, were measured. The correlations between FGF23 and these parameters were examined. Results: The participants did not show LVH on the whole. In addition, no significant correlation was observed by these examinations. Conclusion: It seems unlikely that FGF23 levels are the apparent determinant of the cardiac mass in patients with FGF23-related hypophosphatemic rickets/osteomalacia.

Mutational analysis of patients with FGF23-related hypophosphatemic rickets

OBJECTIVE X-linked hypophosphatemic rickets (XLHR) caused by mutations in the PHEX gene is considered to be the most frequent cause of fibroblast growth factor 23 (FGF23)-related congenital hypophosphatemic rickets. In previous studies, mutations in the PHEX gene were detected in 60-70% of patients with clinical diagnoses of XLHR. This leads to the question whether current screening methods for mutations in the PHEX gene are inadequate or whether there is a substantial number of patients with other genetic causes of hypophosphatemic rickets. We conducted a genetic analysis of patients with FGF23-related hypophosphatemic rickets to clarify their etiology and evaluate the prevalence of XLHR among this group. DESIGN AND METHODS We studied 27 patients with familial and sporadic congenital hypophosphatemic rickets in whom serum FGF23 was above 30 pg/ml using an assay for the full-length protein. Exons and exon-intron junctions of genomic DNA of causative genes for FGF23-related hypophosphatemic rickets were sequenced. PHEX mRNA from peripheral blood was analyzed in some patients. RESULTS Direct sequencing of genomic DNA identified 11 novel and four known mutations in the PHEX gene. Additionally, there was a large PHEX gene deletion in one case and abnormal PHEX mRNA splicing in another. In summary, 26 patients (96%) had XLHR and one patient had autosomal recessive hypophosphatemic rickets 2. CONCLUSIONS XLHR is by far the most prevalent cause of FGF23-related hypophosphatemic rickets. We propose that analysis of PHEX mRNA from peripheral blood would be appropriate for the first screening step in determining the etiology of FGF23-related hypophosphatemic rickets.