The role of aberrant tumor suppressor gene methylation in the aggressiveness of papillary thyroid cancer (PTC) has not been documented. By showing promoter methylation-induced gene silencing in PTC-derived cell lines, we first demonstrated the functional consequence of methylation of several recently identified tumor suppressor genes, including those for tissue inhibitor of metalloproteinase-3 (TIMP3), SLC5A8, death-associated protein kinase (DAPK) and retinoic acid receptor b2 (RARb2). We then investigated the role of methylation of these genes in the aggressiveness of PTC by examining the relationship of their aberrant methylation to clinicopathological characteristics and BRAF mutation in 231 primary PTC tumors. Methylation of TIMP3, SLC5A8 and DAPK was significantly associated with several aggressive features of PTC, including extrathyroidal invasion, lymph node metastasis, multifocality and advanced tumor stages. Methylation of these genes was also significantly associated with BRAF mutation in PTC, either individually or collectively in various combinations. Methylation of these genes, either individually or collectively, occurred more frequently in more aggressive classical and tall-cell PTC subtypes than in less aggressive follicularvariant PTC, with the latter known to infrequently harbor BRAF mutation. Several other tumor suppressor genes investigated were not methylated. These results suggest that aberrant methylation and hence silencing of TIMP3, SLC5A8, DAPK and RARb2, in association with BRAF mutation, may be an important step in PTC tumorigenesis and progression.

GnRH neurons follow a carefully orchestrated journey from their birth in the olfactory placode area. Initially, they migrate along with the vomeronasal nerve into the brain at the cribriform plate, then progress caudally to sites within the hypothalamus where they halt and send projections to the median eminence to activate pituitary gonadotropes. Many factors controlling this precise journey have been elucidated by the silencing or over expression of candidate genes in mouse models. Importantly, a number of these factors may not only play a role in normal physiology of the hypothalamic-pituitary-gonadal axis but also be mis-expressed to cause human disorders of GnRH deficiency, presenting as a failure to undergo normal pubertal development. This review outlines the current cadre of candidates thought to modulate GnRH neuronal migration. The further elucidation and characterization of these factors that impact GnRH neuron development may shed new light on human reproductive disorders and provide potential targets to develop new profertility or contraceptive agents. IntroductionGonadotropin-releasing hormone (GnRH) is a hypothalamic releasing hormone that is synthesized in a small heterogenous neuronal population and secreted in an episodic fashion to control pituitary gonadotropin production and normal reproductive function(1-5). The GnRH neurons are unique among hypothalamic releasing factor neurons in that they originate in the olfactory placode/vomeronasal organ and migrate along vomeronasal nerves to the cribriform plate, the boundary between the peripheral olfactory system and the forebrain(1-5). Migrating GnRH neurons follow a branch of the vomeronasal nerve caudally into the hypothalamus(4). They then extend processes to the median eminence to release GnRH into the capillaries of the median eminence to modulate pituitary gonadotropin (luteinizing hormone, LH and follicle stimulating hormone, FSH) production and secretion. Loss of these neurons or misdirecting along the route results in failure of sexual maturation in mice and man. The underlying mechanisms that regulate the migration of GnRH neurons are incompletely understood. Recent studies have identified multiple factors extrinsic and Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptFront Neuroendocrinol. Author manuscript; available in PMC 2012 January 1. intrinsic to GnRH neurons that control specific steps along the migratory route (discussed in (6-9)). This review updates the list of candidate proteins and pathways involved in GnRH neuronal migration and highlights...

Gonadotrope and null cell pituitary tumors cause significant morbidity, often presenting with signs of hypogonadism together with visual disturbances due to mass effects. Surgery and radiation are the only therapeutic options to date. To identify dysregulated genes and pathways that may play a role in tumorigenesis and/or progression, molecular profiling was performed on 14 gonadotrope tumors, with nine normal human pituitaries obtained at autopsy serving as controls. Bioinformatic analysis identified putative downstream effectors of tumor protein 53 (p53) that were consistently repressed in gonadotrope pituitary tumors, including RPRM, P21, and PMAIP1, with concomitant inhibition of the upstream p53 regulator, PLAGL1(Zac1). Further analysis of the growth arrest and DNA damage-inducible (GADD45) family revealed no change in the p53 target, GADD45α, but identified repression of GADD45β in pituitary tumors in addition to the previously reported inhibition of GADD45γ. Overexpression of GADD45β in LβT2 mouse gonadotrope cells blocked tumor cell proliferation and increased rates of apoptosis in response to growth factor withdrawal. Stable gonadotrope cell transfectants expressing increased GADD45β showed decreased colony formation in soft agar, confirming its normal role as a tumor suppressor. Unlike previous studies of GADD45γ in pituitary tumors and α and β in other tumors, bisulfite sequencing showed no evidence of hypermethylation of the GADD45β promoter in human pituitary tumor samples to explain the repression of its expression. Thus, GADD45β is a novel pituitary tumor suppressor whose reexpression blocks proliferation, survival, and tumorigenesis. Together these studies identify new targets and mechanisms to explore in pituitary tumor initiation and progression.

Purpose Growth hormone (GH) pituitary tumors are associated with significant morbidity and mortality. Current treatments, including surgery and medical therapy with somatostatin analogues (SSA), dopamine agonists and/or a GH receptor antagonist, result in disease remission in approximately half of patients. Predictors of GH tumor response to different therapies have been incompletely defined based on histologic subtype, particularly densely (DG) versus sparsely (SG) granulated adenomas. The aim of this study was to examine our own institutional experience with GH adenomas and correlate how subtype related to clinical parameters as well as response to surgery and medical therapies. Methods A retrospective chart review of 101 acromegalic patients operated by a single neurosurgeon was performed. Clinical data were correlated with histologic subtype and disease control, as defined by IGF-1 levels, and random growth hormone levels in response to surgery and/or medical therapies. Results SG tumors, compared to DG, occurred in younger patients (p=0.0010), were 3-fold larger (p=0.0030), but showed no differences in tumor-invasion characteristics (p=0.12). DG tumors had a higher rate of remission in response to surgery compared to SG, 65.7% vs. 14.3% (p<0.0001), as well as to medical therapy with SSAs (68.8% for DG vs. 28.6% for SG tumors; p=0.028). SG tumors not controlled with SSAs consistently responded to a switch to, or addition of, a GH receptor antagonist. Conclusions Histological GH tumor subtyping implicates a different clinical phenotype and biologic behavior, and provides prognostic significance for surgical success and response to medical therapies.

Adrenocortical cancer (ACC) is an orphan malignancy that results in heterogeneous clinical phenotypes and molecular genotypes. There are no curative treatments for this deadly cancer with 35% survival at five years. Our understanding of the underlying pathobiology and our ability to test novel therapeutic targets has been limited due to the lack of preclinical models. Here, we report the establishment of two new ACC cell lines and corresponding patient-derived xenograft (PDX) models. CU-ACC1 cell line and PDX were derived from a perinephric metastasis in a patient whose primary tumor secreted aldosterone. CU-ACC2 cell line and PDX were derived from a liver metastasis in a patient with Lynch syndrome. Short tandem repeat profiling confirmed consistent matches between human samples and models. Both exomic and RNA sequencing profiling were performed on the patient samples and the models, and hormonal secretion was evaluated in the new cell lines. RNA sequencing and immunohistochemistry confirmed the expression of adrenal cortex markers in the PDXs and human tumors. The new cell lines replicate two of the known genetic models of ACC. CU-ACC1 cells had a mutation in and secreted cortisol but not aldosterone. CU-ACC2 cells had a mutation and loss of consistent with the patient's known germline mutation causing Lynch syndrome. Both cell lines can be transfected and transduced with similar growth rates. These new preclinical models of ACC significantly advance the field by allowing investigation of underlying molecular mechanisms of ACC and the ability to test patient-specific therapeutic targets.

BACKGROUND:The objective of this study was to document the differences in testosterone (T) levels between crizotinib-treated and noncrizotinib-treated patients with metastatic nonsmall cell lung cancer (NSCLC). METHODS: Testosterone levels were measured in 19 men with metastatic NSCLC who received crizotinib and in 19 men with metastatic NSCLC who did not receive crizotinib. Clinical characteristics of the patients were compared, and additional hormone assays were performed as appropriate. Two patients who began crizotinib and 4 patients who had dose interruptions or who stopped crizotinib therapy had serial hormone measurements, permitting the documentation of dynamic hormone changes on and off crizotinib treatment. RESULTS: Total T levels were low (<241 ng/dL) in 19 of 19 (100%) crizotinib-treated men and in 6 of 19 men (32%) with metastatic NSCLC who did not receive crizotinib (mean T levels, 131 ng/dL and 311 ng/dL, respectively; P ¼ .0002). Only 1 in 5 patients who had anaplastic lymphoma kinase (ALK) gene rearrangements and had not yet received crizotinib had low T. The initiation of crizotinib in 2 patients who had previously normal T levels was associated with a rapid decreases in T and in luteinizing hormone and follicle stimulating hormone levels within 14 to 21 days. Discontinuation of crizotinib led to increases back to normal T levels. CONCLUSIONS: Crizotinib therapy caused rapid suppression of T levels in men. The current results indicated that the site of action must include a central (hypothalamic or pituitary) effect, but additional direct testicular effects could not be excluded. Further work is required to assess the correlation between low T levels and crizotinib side effects as well as the exact molecular mechanism and site of drug toxicity. Cancer 2012;118:5302-9. V C 2012American Cancer Society.KEYWORDS: crizotinib, nonsmall cell lung cancer, testosterone, anaplastic lymphoma kinase gene rearrangements, hypogonadism. INTRODUCTIONApproximately 4% of patients with nonsmall cell lung cancer (NSCLC) have rearrangements in the anaplastic lymphoma kinase (ALK) gene, leading to an oncogene-addicted state from aberrant ALK activation. 1-9 Crizotinib, an oral ALK and MET-directed tyrosine kinase inhibitor, has produced a high response rate and prolonged median progression-free survival in patients with ALK-positive NSCLC. 10 Side effects of crizotinib include grade 1/2 nausea (54%), diarrhea (48%), visual disturbance (41%), constipation (24%), dizziness (15%), and fatigue (10%). 10 Crizotinib (Xalkori; Pfizer, New York, NY) recently received US Food and Drug Administration approval for the treatment of patients with advanced, ALK-rearranged NSCLC, a group that potentially represents >10,000 patients per year in the United States alone. 11 The potential for crizotinib-related hypogonadism came to our attention through an index case. A man aged 35 years with metastatic NSCLC and an ALK rearrangement who received crizotinib (250 mg twice daily in 28-day cycles) for 14 cycles reported worsening fatigue...

Context Although the development of immune checkpoint inhibitors has transformed treatment strategies of several human malignancies, research models to study immunotherapy in adrenocortical carcinoma (ACC) are lacking. Objective To explore the effect of anti-PD1 immunotherapy on the alteration of the immune milieu in ACC in a newly generated preclinical model and correlate with the response of the matched patient. Design, Setting, and Intervention To characterize the CU-ACC2-M2B patient-derived xenograft in a humanized mouse model, evaluate the effect of a PD-1 inhibitor therapy, and compare it with the CU-ACC2 patient with metastatic disease. Results Characterization of the CU-ACC2-humanized cord blood-BALB/c-Rag2nullIl2rγnullSirpaNOD model confirmed ACC origin and match with the original human tumor. Treatment of the mice with pembrolizumab demonstrated significant tumor growth inhibition (60%) compared with controls, which correlated with increased tumor infiltrating lymphocyte activity, with an increase of human CD8+ T cells (P &lt; 0.05), HLA-DR+ T cells (P &lt; 0.05) as well as Granzyme B+ CD8+ T cells (&lt;0.001). In parallel, treatment of the CU-ACC2 patient, who had progressive disease, demonstrated a partial response with 79% to 100% reduction in the size of target lesions, and no new sites of metastasis. Pretreatment analysis of the patient's metastatic liver lesion demonstrated abundant intratumoral CD8+ T cells by immunohistochemistry. Conclusions Our study reports the first humanized ACC patient-derived xenograft mouse model, which may be useful to define mechanisms and biomarkers of response and resistance to immune-based therapies, to ultimately provide more personalized care for patients with ACC.

Several recent reports indicate that the balance of skeletal muscle phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is a key determinant of muscle contractile function and metabolism. The purpose of this study was to determine relationships between skeletal muscle PC, PE and insulin sensitivity, and whether PC and PE are dynamically regulated in response to acute exercise in humans. Insulin sensitivity was measured via intravenous glucose tolerance in sedentary obese adults (OB; n = 14), individuals with type 2 diabetes (T2D; n = 15), and endurance-trained athletes (ATH; n = 15). Vastus lateralis muscle biopsies were obtained at rest, immediately after 90 min of cycle ergometry at 50% maximal oxygen consumption (V̇o2 max), and 2-h postexercise (recovery). Skeletal muscle PC and PE were measured via infusion-based mass spectrometry/mass spectrometry analysis. ATH had greater levels of muscle PC and PE compared with OB and T2D (P < 0.05), with total PC and PE positively relating to insulin sensitivity (both P < 0.05). Skeletal muscle PC:PE ratio was elevated in T2D compared with OB and ATH (P < 0.05), tended to be elevated in OB vs. ATH (P = 0.07), and was inversely related to insulin sensitivity among the entire cohort (r = -0.43, P = 0.01). Muscle PC and PE were altered by exercise, particularly after 2 h of recovery, in a highly group-specific manner. However, muscle PC:PE ratio remained unchanged in all groups. In summary, total muscle PC and PE are positively related to insulin sensitivity while PC:PE ratio is inversely related to insulin sensitivity in humans. A single session of exercise significantly alters skeletal muscle PC and PE levels, but not PC:PE ratio.