Adrenergic Differentiation and Ret Expression in Rat Pheochromocytomas

Pheochromocytomas are catecholamine-producing tumors of the adult adrenal medulla. They are rare in humans and most other species but common in laboratory rats. However, the relevance of rat pheochromocytomas as a model for their human counterparts is uncertain. Previous studies of spontaneous and drug-induced rat pheochromocytomas and the PC12 pheochromocytoma cell line suggested a distinctive noradrenergic phenotype, possibly reflecting origin from a progenitor not present in the adult human adrenal. In this study, we studied 31 pheochromocytomas derived from test and control male and female rats in toxicologic studies for expression of the epinephrine-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) and the receptor tyrosine kinase Ret. PNMT, which defines adrenergic chromaffin cells, is frequently expressed in human pheochromocytomas, often in tumors that also overexpress RET. We also tested for the expression of the cell cycle checkpoint protein p27^Kip1, which recently was reported absent in pheochromocytomas from a strain of rats with a hereditary mixed multiple endocrine neoplasia (MEN)-like syndrome. Using immunoblots, we demonstrated PNMT expression in almost 50% of the 31 tumors, although often at lower levels than in normal rat adrenal medulla. The majority of tumors overexpressed Ret. There was no apparent correlation between PNMT and Ret. However, in this study, PNMT expression was strongly associated with tumors arising in female rats, while overexpression of Ret did not show a sex predilection. Robust expression of p27^Kip1 was seen in all tumors from the toxicologic studies and also in a small sample of pheochromocytomas from Long–Evans rats, which were reported to have a mixed MEN-like syndrome in the 1980s. The present results show that rat pheochromocytomas have greater phenotypic diversity than previously believed and greater similarity to their human counterparts with respect to these two important markers. Loss of p27^Kip1 does not appear to account for the high frequency of pheochromocytomas in commonly utilized rat strains.     

Pheochromocytomas in Nf1 knockout mice express a neural progenitor gene expression profile

Pheochromocytomas are adrenal medullary tumors that typically occur in adult patients, with increased frequency in multiple endocrine neoplasia type 2, von Hippel-Lindau disease, familial paraganglioma syndromes and neurofibromatosis type 1 (NF1). Pheochromocytomas arise in adult mice with a heterozygous knockout mutation of exon 31 of the murine Nf1 gene, providing a mouse model for pheochromocytoma development in NF1. We performed a microarray-based gene expression profiling study comparing mouse pheochromocytoma tissue to normal adult mouse adrenal medulla to develop a basis for studying the pathobiology of these tumors. The findings demonstrate that pheochromocytomas from adult neurofibromatosis knockout mice express multiple developmentally regulated genes involved in early development of both the CNS and peripheral nervous system. One of the most highly overexpressed genes is receptor tyrosine kinase Ret, which is known to be transiently expressed in the developing adrenal gland, down-regulated in adult adrenals and often overexpressed in human pheochromocytomas. Real-time polymerase chain reaction validated the microarray results and immunoblots confirmed the overexpression of Ret protein. Other highly expressed validated genes include Sox9, which is a neural crest determinant, and Hey 1, which helps to maintain the progenitor status of neural precursors. The findings are consistent with the recently proposed concept that persistent neural progenitors might give rise to pheochromocytomas in adult mouse adrenals and suggest that events predisposing to tumor development might occur before formation of the adrenal medulla or migration of cells from the neural crest. However, the competing possibility that developmentally regulated neural genes arise secondarily to neoplastic transformation cannot be ruled out. In either case, the unique profile of gene expression opens the mouse pheochromocytoma model to new applications pertinent to neural stem cells and suggests potential new targets for treatment of pheochromocytomas or eradication of their precursors.     

Differential diagnosis of pheochromocytomas and paragangliomas

Paragangliomas are of two types, sympathetic and parasympathetic, depending on the type of paraganglion in which they arise. The term pheochromocytoma is reserved for tumors arising in the adrenal medulla. These tumors are usually fairly easy to diagnose. However, several areas are the subject of debate, including the identification of malignant potential, the diagnosis of medullary hyperplasia, and the recognition of composite tumors. Some histologic features can cause problems in differential diagnosis. Paragangliomas may have spindle cell morphology or contain pigment, requiring distinction from mesenchymal tumors and melanoma, respectively. Extensive degenerative change in phenochromocytomas may mimic adrenal cortical tumors. This short review addresses the diagnosis of pheochromocytomas and paragangliomas and discusses useful approaches in the aforementioned problem areas.     

Stathmin is involved in S100A4-mediated regulation of cell cycle progression

S100A4 is a cell proliferation- and cancer metastasis-related gene. Previous studies have shown that over-expression of S100A4 drives the cells into the S-phase of the cell cycle, with concomitant enhancement of p53 detection. This has led to the postulate that S100A4 could be controlling cell cycle progression by sequestering p53 and abrogating its G₁-S checkpoint control. Cells induced by S100A4 to enter the S-phase do successfully negotiate the G₂-M checkpoint control. Here we show that S100A4 is also involved in the regulation of control at this checkpoint. Stathmin is known to be associated, together with p53 in controlling G₂-M transition. We present evidence that the expression of S100A4 and stathmin genes is up regulated in exponentially growing HeLa cells. They are down regulated in parallel when cell proliferation is inhibited by hyperthermia and 4-hydroxynonenal (4-HNE). We postulate that S100A4 might directly induce stathmin up regulation to enable cells to enter into mitosis. Since wild-type p53 is known to down regulate stathmin expression, we further postulate this might also involve S100A4-mediated sequestration of p53. The expression of heme oxygenase (HO-1), a stress-response protein, has been used to monitor effects of hyperthermia, 12-O-tetradecanoly phorbol 13-acetate (TPA) and 4-HNE. All these treatments induced HO-1 and also when cells growing in serum-deficiency were restored with full serum. HO-1 induction occurred irrespective of S100A4 expression status. HO-1 gene has responsive elements for many angiogenic agents and induces marked neovascularisation of tumours. We suggest therefore that S100A4 may not possess angiogenic properties. This revised version was published online in July 2006 with corrections to the Cover Date.     

The penetrance of paraganglioma and pheochromocytoma in SDHB germline mutation carriers

Germline mutations in succinate dehydrogenase B (SDHB) predispose to hereditary paraganglioma (PGL) syndrome type 4. The risk of developing PGL or pheochromocytoma (PHEO) in SDHB mutation carriers is subject of recent debate. In the present nationwide cohort study of SDHB mutation carriers identified by the clinical genetics centers of the Netherlands, we have calculated the penetrance of SDHB associated tumors using a novel maximum likelihood estimator. This estimator addresses ascertainment bias and missing data on pedigree size and structure. A total of 195 SDHB mutation carriers were included, carrying 27 different SDHB mutations. The 2 most prevalent SDHB mutations were Dutch founder mutations: a deletion in exon 3 (31% of mutation carriers) and the c.423+1G>A mutation (24% of mutation carriers). One hundred and twelve carriers (57%) displayed no physical, radiological or biochemical evidence of PGL or PHEO. Fifty‐four patients had a head and neck PGL (28%), 4 patients had a PHEO (2%), 26 patients an extra‐adrenal PGL (13%). The overall penetrance of SDHB mutations is estimated to be 21% at age 50 and 42% at age 70 when adequately corrected for ascertainment. These estimates are lower than previously reported penetrance estimates of SDHB‐linked cohorts. Similar disease risks are found for different SDHB germline mutations as well as for male and female SDHB mutation carriers.     

Immunohistochemical localization of endothelin-1, endothelin-3 and endothelin receptors in human pheochromocytoma and paraganglioma

Endothelln (ET) and its receptor system have been shown to exert varlous biological effects on dlfferent types of cells In addition to their well-known vasoconstrictor activity. Recently ET-1, ET-3 and the ET₃ receptor have been shown to play an Important role In the development of neural crest-derived cells and, in this context, pheochromocytomas have been reported to harbor ET-1. Endothelin-3 or ET receptor subtypes, however, have not been examined in pheochromocytoma and paraganglioma so far. In the present study the Immunohistochemical localization of ET-1/big ET-1, ET-3/big ET-3 and the ET_A and ET_B receptors were lnvestigated to clarify the biological characteristics of these two tumors using 32 pheochromocytomas and 11 extra-adrenal paragangliomas. Endothelin-lhig ET-1 was detected in 19 pheochromocytomas (59%) and eight paragangliomas (72%), while ET-3hIg ET-3 was detected in 10 pheochromocytomas (31%) and three paragangllomas (27%). The ET_A receptor was found in 21 pheochromocytomas (66%) and In eight paragangllomas (73%), whlle the ET_B receptor was found in 25 pheochromocytomas (78%) and In eight paragangllomas (73%). Normal adrenomedullary cells lacked each antigen examined. Endothelin-immunoreactive tumor cells were dlstrlbuted focally or In a manner scattered, whlle receptor-immunostained tumor cells were distributed wlth a focal pattern for the ETa receptor and wlth a focal or diffuse pattern for the ET_B receptor. Endothelln and its receptor coexlsted In the same tumor in 21 of 28 ET-posltive pheochromocytomas and in eight of 10 ET-positlve paragangliomas. In additlon, seven pheochromocytomas and two paragangllomas revealed posltivlty of the receptor(s) irrespective of the absence of ET-immunoreactlvlty. In concluslon, ET and Its receptor are frequently and concomitantly expressed in the pheochromocytoma and paraganglloma. From the highly frequent expression of this system or the receptor(s), ET-receptor-mediated slgnal transduction of these tumors concernlng growth and/or cell survival Is expected, although definite blological slgniflcance of thls llgand-receptor system in these tumors awaits further Investigation.     

RET Signaling in Endocrine Tumors: Delving Deeper into Molecular Mechanisms

The rearranged during transfection (RET) proto-oncogene encodes a receptor tyrosine kinase that is implicated in the development of endocrine tumors of the thyroid and adrenal glands. In humans, activating RET mutations are found in the inherited cancer syndrome multiple endocrine neoplasia 2 and in sporadic medullary and papillary thyroid carcinomas. The specific type and location of RET mutations are strongly correlated with the disease phenotype and have both diagnostic and prognostic value. Recent advances in the molecular characterization of the RET receptor and its mutants have begun to define the mechanisms underlying the transforming ability of the different RET mutant forms. This information has revealed key functional features of these mutant proteins that distinguish the different clinically recognized mutations and provide clues as to the functional origins of the phenotypes associated with specific RET mutations. The elucidation of molecular mechanisms involved in RET-mediated transformation is a key step in the development of much needed therapeutics that target RET’s oncogenic properties. Recent advances have begun to provide a deeper understanding of the receptor’s function, and dysfunction, in human tumors that may guide this process.     

MicroRNA Expression Profiles in Thyroid Tumors

MicroRNAs (miRNAs) constitute a recently identified class of small endogenous noncoding RNAs that act as negative regulators of the protein-coding gene expression and may impact cell differentiation, proliferation and survival, i.e., all fundamental cellular processes implicated in carcinogenesis. miRNA expression is deregulated in many types of human cancers, including thyroid cancer. The purpose of this review is to summarize the existing findings of miRNA deregulation in thyroid tumors and its potential role in thyroid cancer biology and molecular diagnostics.     

Immunoreactivity of ICAM-1 in Human Tumors, Metastases and Normal Tissues

Intercellular adhesion molecule-1 (ICAM-1) is implicated to play a role in cancer metastasis, and may serve as a diagnostic tool for tumor prognosis and progression as well as a target for therapeutic intervention. The aim of this study was to carry out a comprehensive survey of ICAM-1 immunoreactivity in normal, malignant and metastatic tissues. We assessed immunoreactivity of ICAM-1 in a total of 300 tissue cores from multiple tissue arrays of normal, malignant, and metastatic tissues by immunohistochemistry. We scored tissue samples for ICAM-1 immunoreactivity on a 0-3 scale, assessed the number of samples exhibiting infiltrating immune cells, and documented ICAM-1 immunoreactivity in some specific cell types. ICAM-1 expression in normal tissues was highest in spleen and absent in the cerebrum, peripheral nerves, pancreas, ovary, breast, uterus, cervix, prostate, lung, larynx, bone marrow, striated muscle, heart, mesothelium, esophagus, small intestine, colon and liver. In primary malignancies, lymphoid tissues received the highest average ICAM-1 score while connective tissue/skin had the lowest average ICAM-1 score. Of the metastatic tissues, those originating from the urinary tract had the highest average ICAM-1 score while those originating from glandular tissues had the lowest average ICAM-1 score. Metastases localized in lymphoid tissues had a higher average ICAM-1 score than those localized in non-lymphoid tissues. Since ICAM-1 is associated with a variety of cancer types and appears to play a role in cancer metastasis, our findings should serve as a helpful resource for investigations of ICAM-1 as a biomarker, as well as a target for therapeutic interventions.     

Stathmin overexpression cooperates with p53 mutation and osteopontin overexpression, and is associated with tumour progression, early recurrence, and poor prognosis in hepatocellular carcinoma

Stathmin, a major microtubule-depolymerizing protein, is involved in cell cycle progression and cell motility. This study aimed to elucidate its role in the progression, early tumour recurrence (ETR), and prognosis of hepatocellular carcinoma (HCC). Stathmin mRNA was overexpressed in 88/156 (56%) resected, unifocal, primary HCCs, while p53 mutation was present in 72 (46%) and osteopontin mRNA overexpression in 79 (51%). Stathmin mRNA expression exhibited high concordance (93%) with protein expression in 107 cases examined by immunohistochemistry. Stathmin overexpression correlated with high alpha-fetoprotein (>200 ng/ml, p = 0.02), larger tumour size (>5 cm, p = 0.012), high tumour grade (p < 0.0002), high tumour stage (stage IIIA-IV) with vascular invasion and various degrees of intrahepatic metastasis (p < 1 x 10(-8)), ETR (p = 0.003), and lower 5-year survival (p = 0.0007). Stathmin protein expression was often more intense in the peripheral regions of tumour trabeculae, tumour borders, and portal vein tumour thrombi. Stathmin overexpression correlated with p53 mutation (p = 0.017) and osteopontin overexpression (p = 1 x 10(-8)), both of which were associated with vascular invasion (both p < 0.0001) and poorer prognosis (p < 0.0004 and p = 0.0004, respectively). Regardless of the status of p53 mutation or osteopontin expression, stathmin overexpression was associated with higher vascular invasion (all p < 0.0001). Approximately 90% of HCCs harbouring stathmin overexpression with concomitant p53 mutation or osteopontin overexpression exhibited vascular invasion, and hence the lowest 5-year survival, p = 0.00018 and p = 0.0009, respectively. However, we did not find that stathmin overexpression exerted prognostic impact independent of tumour stage. In conclusion, stathmin expression correlates with metastatic potential, is an important prognostic factor for HCC, and may serve as a useful marker to predict ETR.     

Proliferation Markers and Their Uses in the Study of Endocrine Tumors

A growing body of literature supports the view that the proliferative activity (PA) of tumor cells is an important prognostic indicator for a variety of different tumors. We examined the role of PA in diagnosis and prediction or malignancy of endocrine tumors (ETs) of pituitary gland, pancreas, thyroid, parathyroid glands, adrenal glands, paraganglia, gastroenteric tract, and lung. The data in the literature indicate that the assessment of PA is not a diagnostic indicator of malignancy especially at the individual case level, whereas it can be useful for identifying subsets of malignant tumors with different aggressiveness potential, as well as for choosing therapeutic options in metatstatic lesions. We hope that, in the near future, multiparametric approaches including PA markers, cell growth and differentiation factors, and oncogenes will yield valuable information for diagnosis and prognosis of ETs also in individual cases.     

Tumor risks and genotype–phenotype–proteotype analysis in 358 patients with germline mutations in SDHB and SDHD

Succinate dehydrogenase B (SDHB) and D (SDHD) subunit gene mutations predispose to adrenal and extraadrenal pheochromocytomas, head and neck paragangliomas (HNPGL), and other tumor types. We report tumor risks in 358 patients with SDHB (n=295) and SDHD (n=63) mutations. Risks of HNPGL and pheochromocytoma in SDHB mutation carriers were 29% and 52%, respectively, at age 60 years and 71% and 29%, respectively, in SDHD mutation carriers. Risks of malignant pheochromocytoma and renal tumors (14% at age 70 years) were higher in SDHB mutation carriers; 55 different mutations (including a novel recurrent exon 1 deletion) were identified. No clear genotype–phenotype correlations were detected for SDHB mutations. However, SDHD mutations predicted to result in loss of expression or a truncated or unstable protein were associated with a significantly increased risk of pheochromocytoma compared to missense mutations that were not predicted to impair protein stability (most such cases had the common p.Pro81Leu mutation). Analysis of the largest cohort of SDHB/D mutation carriers has enhanced estimates of penetrance and tumor risk and supports in silicon protein structure prediction analysis for functional assessment of mutations. The differing effect of the SDHD p.Pro81Leu on HNPGL and pheochromocytoma risks suggests differing mechanisms of tumorigenesis in SDH‐associated HNPGL and pheochromocytoma. Hum Mutat 31:41–51, 2010. © 2009 Wiley‐Liss, Inc.     

自噬相关基因LC3、Beclin-1与凋亡相关基因p53、BCL-2在大肠癌中的表达及意义

目的检测自噬相关基因LC3、Beclin-1与凋亡相关基因p53、BCL-2在大肠癌、大肠腺瘤及正常大肠黏膜中的表达情况,探讨其与大肠癌发生、发展的相关性及意义。方法应用组织芯片技术和免疫组化SP法检测12例正常大肠黏膜、29例大肠腺瘤及115例大肠癌组织中LC3、Beclin-1、p53及BCL-2蛋白的表达水平,并结合临床病理因素进行分析。结果 LC3在大肠癌中的阳性率高于大肠腺瘤和正常大肠黏膜(P<0.01),且与大肠癌的组织学分化程度相关(P<0.05)。Beclin-1在大肠癌、大肠腺瘤组织中的表达高于正常大肠黏膜(P<0.01),但与大肠癌的组织学分化程度、Dukes分期及淋巴结转移无关(P均>0.05)。p53及BCL-2在正常大肠黏膜、大肠腺瘤及大肠癌中的表达逐渐增高(P<0.01),且与大肠癌的Dukes分期和淋巴结转移相关(P均<0.05)。经Spearman秩相关检验,Beclin-1蛋白与p53蛋白相关系数为0.224 3,成正相关表达(P<0.05)。结论在大肠癌中,自噬活性的上调与凋亡能力的下调并存;自噬相关基因LC3、Beclin-1与凋亡相关基因p53、BCL-2在大肠癌的发生、发展中起协调作用,对其联合检测有助于了解和判断大肠癌的进展程度及患者的预后情况。     

Simple and rapid characterization of novel large germline deletions in SDHB,SDHC and SDHD‐related paraganglioma

Germline mutations in genes encoding subunits of succinate dehydrogenase (SDH) are associated with hereditary paraganglioma and pheochromocytoma. Although most mutations in SDHB, SDHC and SDHD are intraexonic variants, large germline deletions may represent up to 10% of all variants but are rarely characterized at the DNA sequence level. Additional phenotypic effects resulting from deletions that affect neighboring genes are also not understood. We performed multiplex ligation‐dependent probe amplification, followed by a simple long‐range PCR ‘chromosome walking’ protocol to characterize breakpoints in 20 SDHx‐linked paraganglioma‐pheochromocytoma patients. Breakpoints were confirmed by conventional PCR and Sanger sequencing. Heterozygous germline deletions of up to 104 kb in size were identified in SDHB, SDHC, SDHD and flanking genes in 20 paraganglioma‐pheochromocytoma patients. The exact breakpoint could be determined in 16 paraganglioma‐pheochromocytoma patients of which 15 were novel deletions. In six patients proximal genes were also deleted, including PADI2, MFAP2, ATP13A2 (PARK9), CFAP126, TIMM8B and C11orf57. These genes were either partially or completely deleted, but did not modify the phenotype. This study increases the number of known SDHx deletions by over 50% and demonstrates that a significant proportion of large gene deletions can be resolved at the nucleotide level using a simple and rapid method.     

Prognostic Significance of p27, Ki-67, and Topoisomerase lla Expression in Clinically Nonfunctioning Pancreatic Endocrine Tumors

Nonfunctioning islet cell tumors or pancreatic endocrine tumors are the most common type of malignant islet cell tumor. Although previously detected usually at an advanced stage because of mass effect, the early detection rate of small localized disease has been increasing. To date it has been difficult to predict the clinical behavior in localized regional nonfunctioning tumors. To investigate potential markers predicting malignancy and poor prognosis in nonfunctioning pancreatic endocrine tumors, we analyzed the expression of Ki-67, topoisomerase IIα (Topollα), and p27, as well as a variety of clinicopathologic parameters in 76 cases of nonfunctioning islet cell tumors (23 benign cases and 53 malignant cases). Ki-67, Topollα, and p27 labeling indices were significantly different between benign and malignant tumors. Expression of Ki-67, Topollα, and p27 were associated with survival in patients with a malignant tumor in a univariate setting. However, only p27 and Topollα were jointly associated with survival in multivariate analysis. Immunohistochemical staining for p27, Topollα, and Ki-67 can be helpful in the diagnosis of nonfunctioning pancreatic endocrine tumor. Analysis of p27 and Topollα may also have potential utility as prognostic factors for malignant tumors.     

Sporadic type composite pheochromocytoma with neuroblastoma: Clinicomorphologic, DNA content, and ret gene analysis

Composite pheochromocytomas (CP) account for only 3% of all pheochromocytomas. We analyzed the clinical, immunohistochemical, ultrastructural, DNA content, and a 634 ret mutation feature in a 56-yr-old Mexican woman with a CP localized in the right adrenal gland and associated with a blood pressure of 140/90 mmHg. Clinical symptoms were absent after surgery. The tumor showed pheochromocytoma and neuroblastoma components. This dual phenotype was supported by light microscopy and corroborated by immunohistochemistry and ultrastructural findings. Flow cytometric analysis showed that both components were diploid. A genetic mutational analysis of the ret oncogene in exon 11 showed no 634 mutation. This case demonstrates the indolent behavior of neuroblastoma associated with a sporadic-type CP in an adult patient.