Role of specific transcription factors in NE tumors
The mechanisms leading to NE differentiation in tumors is still controversial. The hypothesis that NE tumors in various organs derive from the NE cell normal counterpart eventually present has been partially overcome by the evidence of peculiar epithelial gene expression profiles, at least in some NE carcinomas such as small cell lung carcinomas (Anbazhagan R et al, 1999). A positive and negative role of specific transcription factors acting in the promotion or repression of NE phenotype has been postulated, and two major classes have been identified. The first one includes proteins analogue to the achaete-scute complex, which promotes neuronal differentiation in the Drosophyla. The human homologue, hASH1, has been cloned from medullary thyroid carcinoma cDNA libraries, is highly expressed in NE tumors (Ball DW et al, 1993), and displays properties in tumor development, in a complex pathway upstream regulated by several other effectors, such as L- and N-myc (Yazawa T et al, 2002). On the other side, a negative action on neuronal gene transcription is promoted by a family of silencing factors (so-called Neuron Restrictive Silencing Factors – NRSF); splice variants of their messenger RNA have been identified in neuroendocine tumors (Coulson JM et al, 2000) and have been proposed as useful clinical markers.

Detection of mutations and/or deletions of genes involved in familial NE tumor syndromes.
Several NE tumors may arise in sporadic form or in the setting of inherited cancer syndromes. The most frequent of the latter are multiple endocrine neoplasia (MEN), types 1 and 2. Less frequently NE tumors are features of Von Hippel Lindau disease or neurofibromatosis type 1.

The cloning of the genes responsible for these inherited syndromes led to the development of sensitive tests for genetic testing of affected individuals and their relatives. These tests, especially for MEN 1 and 2 syndromes, are widely used in both clinical and pathological practice, and therefore accurate standardization of the methodologies employed in different laboratories should be achieved (Brandi ML et al, 2001). Currently, these tests are appliable even to paraffin-embedded material (Komminoth P et al, 1995), thus allowing the retrospective study of tumor series.

In addition, the analysis of molecular alterations in the genes involved in hereditary syndromes eventually occurring in sporadic NE tumors shed some light on the oncogenetic mechanisms of NE tumorigenesis (Komminoth P et al, 1996; Gortz B et al, 1999).

Analysis of clonal composition of NE tumors.
Assessment of the clonal composition of a single or multifocal tumor may be reached combining different techniques. Alternatively, detection of specific molecular alterations (namely allelic imbalances, point mutations, or others) in the tumor cell population, or analysis, in the case of female patients, the pattern of inactivation of the X chromosome (so-called Human Androgen Receptor Assay – HUMARA) (van Dijk JP et al, 2002) can be performed.

NE tumors represent one of the major exceptions to the theory of the monoclonal origin of tumors. Controversial, and sometimes surprising, data have been extensively reported in the literature. Focusing on few examples, medullary thyroid carcinoma has been demonstrated to be monoclonal when occurring sporadically (Marques AR et al, 2001), while polyclonal when associated to MEN2 syndrome, possibly as the result of the fusion of multiple independent tumor foci (Ferraris AM et al, 1997). On the other hand, parathyroid adenoma grows as a monoclonal population in MEN1 patients (Friedman E et al 1989), while is polyclonal in the sporadic form (Sanjuan X et al, 1998). Monoclonality in endocrine pancreatic tumors is associated with malignant potential, being possibly the result of a selection of a more aggressive clone in an initially polyclonal tumor cell population (Perren A. et al, 1998). On the contrary, gastric carcinoids appear to be monoclonal in origin even when small in size and clinically benign (D'Adda T. et al, 1999; Goebel SU et al, 2000).

Molecular procedures to detect NE markers in poorly differentiated NE carcinomas and in micrometastases
The detection of the NE phenotype in poorly differentiated NE tumors may be a very difficult diagnostic area, since conventional techniques such as immunohistochemistry may fail to addres their correct definition. The failure to detect NE markers in poorly differentiated NE carcinomas by the immunophenotyping approach may be due to either or both defective production or increased escretion, without appropriate storage, of the specific protein. Alternative sensitive methods, as in situ hybridization and RT-PCR, detecting the synthesizing machinery and the specific mRNA, may implement the diagnosis of NE phenotype in poorly differentiated carcinomas and are widely employed in diagnostic practice. These sensitive procedures may also carry novel interpretation problems, since the significance of the detection of focal NE phenotype in poorly differentiated, not otherwise NE, carcinomas of various sites (Grabowski P et al, 2002) remains to be clarified.

Another applycation of sensitive molecular techniques in NE tumor poathology is represented by the early recognition of micrometastases in the diagnosis and follow up of affected patients. Several pilot studies, have been published (Weber T et al, 2001; Pagani A et al, 2002; Saller B et al 2002) and have to be validated by wide clinical use. The impact of such approach might be highly valuable in the staging definition of such aggressive and highly metastatizing tumors as neuroblastomas and small cell lung carcinomas.

Detection of receptors for specific polypeptide hormones in NE tumors.
NE cells and tumors express a variety of peptidyl and non-peptidyl hormone receptors, acting in both autocrine and paracrine circuits. Among the former, major interest has been dedicated to somatostatin receptors, which are highly expressed in NE tumors of various sites. The identification of the receptor status in NE tumors has both diagnostic (Octreoscan scintigraphy) and therapeutical (treatment with somatostatin analogs) implications. Alternative and complementary methods to receptor detection include tissue binding assays (autoradiography), specific messenger RNA (in situ hibridization or RT-PCR), protein (immunohistochemistry, Western blot) identification, or in vivo (scintigraphic) detection. The molecular approach in this specific field offers the advantage of a high specificity and sensitivity, thus improving our diagnostic and interpretation prospects, especially when used in combination with other imaging procedures (Papotti M et al, 2001). Future diagnostic and clinical applications would probably include other receptors highly expressed in NE tumors and currently under investigation, such as receptors for oxytocin (Cassoni P et al, 1998), bombesin/GRP (Reubi JC et al, 2002), dopamine (Lemmer K et al, 2002) and growth hormone secretagogues (de Keizer Y et al, 1997).

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