Stefan K. Grebe

Pheochromocytoma and Paraganglioma: An Endocrine Society Clinical Practice Guideline

OBJECTIVE The aim was to formulate clinical practice guidelines for pheochromocytoma and paraganglioma (PPGL). PARTICIPANTS The Task Force included a chair selected by the Endocrine Society Clinical Guidelines Subcommittee (CGS), seven experts in the field, and a methodologist. The authors received no corporate funding or remuneration. EVIDENCE This evidence-based guideline was developed using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system to describe both the strength of recommendations and the quality of evidence. The Task Force reviewed primary evidence and commissioned two additional systematic reviews. CONSENSUS PROCESS One group meeting, several conference calls, and e-mail communications enabled consensus. Committees and members of the Endocrine Society, European Society of Endocrinology, and Americal Association for Clinical Chemistry reviewed drafts of the guidelines. CONCLUSIONS The Task Force recommends that initial biochemical testing for PPGLs should include measurements of plasma free or urinary fractionated metanephrines. Consideration should be given to preanalytical factors leading to false-positive or false-negative results. All positive results require follow-up. Computed tomography is suggested for initial imaging, but magnetic resonance is a better option in patients with metastatic disease or when radiation exposure must be limited. (123)I-metaiodobenzylguanidine scintigraphy is a useful imaging modality for metastatic PPGLs. We recommend consideration of genetic testing in all patients, with testing by accredited laboratories. Patients with paraganglioma should be tested for SDHx mutations, and those with metastatic disease for SDHB mutations. All patients with functional PPGLs should undergo preoperative blockade to prevent perioperative complications. Preparation should include a high-sodium diet and fluid intake to prevent postoperative hypotension. We recommend minimally invasive adrenalectomy for most pheochromocytomas with open resection for most paragangliomas. Partial adrenalectomy is an option for selected patients. Lifelong follow-up is suggested to detect recurrent or metastatic disease. We suggest personalized management with evaluation and treatment by multidisciplinary teams with appropriate expertise to ensure favorable outcomes.

Analysis of Circulating MicroRNA: Preanalytical and Analytical Challenges

BACKGROUND There is great interest in circulating microRNAs (miRNAs) as disease biomarkers. Translating promising miRNAs into validated clinical tests requires the characterization of many preanalytical and analytical parameters. METHODS miRNAs were extracted from serum and plasma samples of healthy volunteers, and miRNAs known to be present in serum and plasma (miR-15b, miR-16, miR-24, and miR-122) were amplified by reverse-transcription quantitative PCR. Stability and the effects of hemolysis were determined. Assay variation and its components, including the effect of adding control miRNA, were assessed by nested ANOVA. RESULTS miRNA concentrations were higher in plasma than in serum. Processing of plasma to remove subcellular/cellular components reduced miRNA concentrations to those of serum. The miRNAs analyzed were stable refrigerated or frozen for up to 72 h and were stable at room temperature for 24 h. Hemolysis increased the apparent concentration of 3 of the miRNAs. The total variability of replicate miRNA concentrations was <2.0-fold, with most of the variability attributable to the extraction process and interassay imprecision. Normalizing results to those of spiked exogenous control miRNAs did not improve this variability. CONCLUSIONS Detailed validation of the preanalytical steps affecting miRNA detection and quantification is critical when considering the use of individual miRNAs as clinical biomarkers. Unless these causes of imprecision are considered and mitigated, only miRNAs that are extremely up- or downregulated will be suitable as clinical biomarkers.

Measurements of plasma methoxytyramine, normetanephrine, and metanephrine as discriminators of different hereditary forms of pheochromocytoma.

BACKGROUND Pheochromocytomas are rare catecholamine-producing tumors derived in more than 30% of cases from mutations in 9 tumor-susceptibility genes identified to date, including von Hippel-Lindau tumor suppressor (VHL); succinate dehydrogenase complex, subunit B, iron sulfur (Ip) (SDHB); and succinate dehydrogenase complex, subunit D, integral membrane protein (SDHD). Testing of multiple genes is often undertaken at considerable expense before a mutation is detected. This study assessed whether measurements of plasma metanephrine, normetanephrine, and methoxytyramine, the O-methylated metabolites of catecholamines, might help to distinguish different hereditary forms of the tumor. METHODS Plasma concentrations of O-methylated metabolites were measured by liquid chromatography with electrochemical detection in 173 patients with pheochromocytoma, including 38 with multiple endocrine neoplasia type 2 (MEN 2), 10 with neurofibromatosis type 1 (NF1), 66 with von Hippel-Lindau (VHL) syndrome, and 59 with mutations of SDHB or SDHD. RESULTS In contrast to patients with VHL, SDHB, and SDHD mutations, all patients with MEN 2 and NF1 presented with tumors characterized by increased plasma concentrations of metanephrine (indicating epinephrine production). VHL patients usually showed solitary increases in normetanephrine (indicating norepinephrine production), whereas additional or solitary increases in methoxytyramine (indicating dopamine production) characterized 70% of patients with SDHB and SDHD mutations. Patients with NF1 and MEN 2 could be discriminated from those with VHL, SDHB, and SDHD gene mutations in 99% of cases by the combination of normetanephrine and metanephrine. Measurements of plasma methoxytyramine discriminated patients with SDHB and SDHD mutations from those with VHL mutations in an additional 78% of cases. CONCLUSIONS The distinct patterns of plasma catecholamine O-methylated metabolites in patients with hereditary pheochromocytoma provide an easily used tool to guide cost-effective genotyping of underlying disease-causing mutations.

Genome-wide analysis reveals recurrent structural abnormalities of TP63 and other p53-related genes in peripheral T-cell lymphomas

Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.

The PAX8/PPARγ fusion oncoprotein transforms immortalized human thyrocytes through a mechanism probably involving wild-type PPARγ inhibition

Follicular thyroid carcinoma (FTC) frequently harbors the PAX8/PPARγ fusion gene (PPFP); however, its oncogenic role and mechanism(s) of action remain undefined. We investigated PPFPs effects on cell growth, apoptosis, cell–cell, and cell–matrix interactions in immortalized human thyroid cells (Nthy-ori 3-1) and NIH 3T3 cells. PPFP expression increased the growth of transient and stable Nthy-ori transfectants (∼threefold by 72 h). There was an 8.4% increase of cells in the S+G2/M phase, a 7.8% decrease in cells in the G0+G1 phase and a 66% decline in apoptosis at 72 h. Stable Nthy-ori PPFP transfectants grew in soft agar, and PPFP-transfected NIH 3T3 cells exhibited efficient focus formation, suggesting loss of anchorage-dependent growth and contact inhibition, respectively. Overexpression of PPARγ in Nthy-ori cells did not recapitulate PPFPs growth effects. Treatment of Nthy-ori cells with an irreversible PPARγ inhibitor mimicked the growth-promoting effects of PPFP and co-expression of PPFP and PPARγ blocked PPARγ transactivation activity. Our data provide functional evidence that PPFP acts as an oncoprotein, whose transforming properties depend in part on inhibition of PPARγ. Our data suggest that PPFP contributes to malignant transformation during FTC oncogenesis by acting on several cellular pathways, at least some of which are normally regulated by PPARγ.

Catecholamine metabolomic and secretory phenotypes in phaeochromocytoma

Phaeochromocytomas and paragangliomas (PPGLs) are highly heterogeneous tumours with variable catecholamine biochemical phenotypes and diverse hereditary backgrounds. This analysis of 18 catecholamine-related plasma and urinary biomarkers in 365 patients with PPGLs and 846 subjects without PPGLs examined how catecholamine metabolomic profiles are impacted by hereditary background and relate to variable hormone secretion. Catecholamine secretion was assessed in a subgroup of 156 patients from whom tumour tissue was available for measurements of catecholamine contents. Among all analytes, the free catecholamine O-methylated metabolites measured in plasma showed the largest tumour-related increases relative to the reference group. Patients with tumours due to multiple endocrine neoplasia type 2 and neurofibromatosis type 1 (NF1) showed similar catecholamine metabolite and secretory profiles to patients with adrenaline-producing tumours and no evident hereditary background. Tumours from these three patient groups contained higher contents of catecholamines, but secreted the hormones at lower rates than tumours that did not contain appreciable adrenaline, the latter including PPGLs due to von Hippel-Lindau (VHL) and succinate dehydrogenase (SDH) gene mutations. Large increases of plasma dopamine and its metabolites additionally characterised patients with PPGLs due to the latter mutations, whereas patients with NF1 were characterised by large increases in plasma dihydroxyphenylglycol and dihydroxyphenylacetic acid, the deaminated metabolites of noradrenaline and dopamine. This analysis establishes the utility of comprehensive catecholamine metabolite profiling for characterising the distinct and highly diverse catecholamine metabolomic and secretory phenotypes among different groups of patients with PPGLs. The data further suggest developmental origins of PPGLs from different populations of chromaffin cell progenitors.

Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry.

BACKGROUND Immunoassays specific for 1-84 parathyroid hormone (PTH) reportedly reflect the bioactivity of PTH; however, PTH immunoassays can be susceptible to interference by cross-reacting PTH fragments. In addition, these assays currently lack standardization. A methodology using immunocapture purification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection, along with a stable isotope-labeled internal standard, may help address these issues. METHODS We isolated 1-84 PTH from 1 mL serum by immunocapture on a 6.5-mm polystyrene bead. The immobilized PTH was digested in situ and analyzed by LC-MS/MS. For quantification, we used the selected reaction monitoring response from the N-terminal tryptic peptide 1-13 PTH ((1)SVSEIQLMHNLGK(13)). RESULTS The linear range of the assay was 39.1-4560 ng/L, and the limit of detection and limit of quantification were 14.5 ng/L and 39.1 ng/L, respectively. The intraassay CVs ranged from 6% to 11%, and the interassay CVs ranged from 7% to 17%. Interference by PTH fragments 1-44 PTH, 7-84 PTH, 43-68 PTH, 52-84 PTH, 64-84 PTH, and PTH-related protein (PTHrP) was <or=1% to <or=0.001%. Method comparison of LC-MS/MS vs the Roche Cobas immunoassay yielded Deming fit of LC-MS/MS = 1.01x immunoassay - 13.21. The mean bias by Bland-Altman plot was -9.4%. CONCLUSIONS In patients with hyperparathyroidism, the immunocapture in situ digestion LC-MS/MS method can provide accurate and precise PTH results compared with immunoassay.

The role of the PAX8/PPARγ fusion oncogene in the pathogenesis of follicular thyroid cancer

When identified at early stages, most well-differentiated thyroid cancers are readily treated and yield excellent outcomes. Follicular thyroid cancer (FTC) however, when diagnosed at a late stage, may be very resistant to treatment, and exhibits 10-year survival rates less than 40%. Despite substantial progress in recent years, we still have limited understanding of the molecular and biological interrelationships between the various subtypes of benign and malignant follicular thyroid neoplasms. In contrast to the wealth of information available regarding papillary thyroid carcinoma (PTC), the triggering mechanisms of FTC development and the major underlying genetic alterations leading to follicular thyroid carcinogenesis remain obscure. Recent studies have focused on a chromosomal translocation, t(2;3) (q13;p25), fusing PAX8, a transcription factor that is essential for normal thyroid gland development, with the peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the steroid/thyroid nuclear receptor family. This chromatin rearrangement results in the expression of a PAX8/PPARgamma fusion protein, designated PPFP, whose incidence is relatively common in FTC and may represent an initiating event in the genesis of FTC. Here we review progress on the studies of PPFP that assess its involvement in FTC tumorigenesis.

Infants of Women with Polycystic Ovary Syndrome Have Lower Cord Blood Androstenedione and Estradiol Levels

CONTEXT Prenatal androgen excess can cause a phenocopy of polycystic ovary syndrome (PCOS) in mammals. Retrospective studies have suggested that girls at risk for PCOS have low birth weight, and prospective studies have suggested an increased prevalence of small-for-gestational-age offspring in women with PCOS. OBJECTIVE The objective of the study was to determine whether infants of women with PCOS have reduced birth weight or increased intrauterine androgen levels. DESIGN This was a prospective case-control study. PARTICIPANTS Thirty-nine PCOS and 31 control women and their infants participated in the study. MAIN OUTCOME MEASURES Birth weight and mixed cord blood testosterone, androstenedione (A), dehydroepiandrosterone, 17-hydroxyprogesterone, estradiol (E2), and dihydrotestosterone levels were measured. RESULTS Mean birth weight did not differ, but there was a significant increase in the prevalence of large-for-gestational-age infants in the PCOS group. Cord blood E2 and A levels were lower (P < 0.05), but testosterone to E2 ratios did not differ in female PCOS compared with control offspring. There was no difference in E2 and A levels in the male PCOS and control offspring. There was no difference in 17-hydroxyprogesterone or other androgen levels in either male or female PCOS offspring compared with their respective control group. CONCLUSION Infants of women with PCOS were more likely to be large for gestational age. Female offspring of affected women have lower cord blood A levels; other cord blood androgen levels do not differ compared with female control offspring. Cord blood E2 levels are also significantly decreased in PCOS, without any difference in the testosterone to E2 ratio, suggesting decreased fetal or placental production of steroids.

Follicular cell-derived thyroid carcinomas

Cancers derived from the follicular epithelium (papillary, follicular, and poorly differentiated (anaplastic) cancers) comprise about 60–95% of all thyroid carcinomas. The rest are, in decreasing order of frequency, medullary carcinomas, lymphomas, sarcomas, squamous-cell carcinomas, and metastases from tumors elsewhere. Besides being extremely rare, all of the latter, with the exception of medullary thyroid carcinoma, are tumors derived from nonendocrine tissues, and therefore they are not the subject of this review. Medullary thyroid carcinoma is covered in Chapter 20.