Achaete-scute homolog 1 as a marker of poorly differentiated neuroendocrine carcinomas of different sites: a validation study using immunohistochemistry and quantitative real-time polymerase chain reaction on 335 cases

Summary

Neuroendocrine carcinomas show overlapping morphological and immunohistochemical features independently of their site of origin, which makes identification of the primary location problematic when they are diagnosed as metastases of unknown origin. Neuroendocrine carcinomas are easily morphologically differentiated from neuroendocrine tumors in surgical material, although this distinction can be difficult when using small biopsy specimens. The diagnostic usefulness of different transcription factors as site-specific markers or as discriminating markers between neuroendocrine carcinomas and neuroendocrine tumors has been previously studied with sometimes contradictory results. In this respect, the role of achaete-scute homolog 1 has been poorly investigated, although some recent findings demonstrate its expression in neuroendocrine carcinomas. Using immunohistochemistry and quantitative real-time polymerase chain reaction, we investigated the expression of achaete-scute homolog 1 in 335 neuroendocrine neoplasms (194 neuroendocrine carcinomas and 141 neuroendocrine tumors) of different sites, to check its possible utility as diagnostic marker. High concordance between immunohistochemical and molecular findings was found. Achaete-scute homolog 1 expression was identified in 82% of lung neuroendocrine carcinomas and 70% of extrapulmonary neuroendocrine carcinomas. Achaete-scute homolog 1 was not detected in any gastroenteropancreatic neuroendocrine tumor and was found in only a minority of lung carcinoids. The diagnostic sensitivity and specificity of achaete-scute homolog 1 expression were 82.4% and 89.7% in distinguishing neuroendocrine carcinomas from neuroendocrine tumors of the lung, 40.6% and 100% to differentiate extrapulmonary neuroendocrine carcinomas from neuroendocrine tumors, and 82.4% and 59.4% in distinguishing lung from extrapulmonary neuroendocrine carcinomas. Our data suggest that achaete-scute homolog 1 is not a site-specific marker. However, achaete-scute homolog 1 may be proposed as a diagnostic marker of poor differentiation and may help to differentiate neuroendocrine carcinomas from neuroendocrine tumors in difficult cases.

Introduction

High-grade poorly differentiated neuroendocrine carcinomas that, according to the 2010 World Health Organization (WHO) classification, are simply labeled neuroendocrine carcinomas (NECs) [1] and can arise at different sites including lung, gastrointestinal tract, urogenital system, and skin. They show typical histologic and immunohistochemical features and high proliferative activity. NECs can be composed of small to intermediate or large cells growing in poorly formed trabeculae, nests, or sheets but more commonly with diffuse patterns, with more or less extensive areas of necrosis. The small cell variant is characterized by small- to medium-sized cells with markedly hyperchromatic round to oval nuclei, inconspicuous nucleoli, a high nuclear/cytoplasmic ratio, and poorly defined cytoplasmic borders. The large cell subtype shows a proliferation of large cells with vesicular nuclei, prominent nucleoli, and abundant finely granular eosinophilic cytoplasm. In addition to these 2 main categories, neoplasms composed of cells showing intermediate features (intermediate cell type) can be observed. Tumor cells express general neuroendocrine markers, such as synaptophysin, neuron-specific enolase, CD56, and chromogranin A and, more rarely, hormone peptides [1], [2]. The above-described morphological and immunohistochemical features overlap among NECs irrespective of their site of origin, making impossible the identification of the primary site when they are first diagnosed as metastases of unknown origin. In recent years, several attempts have been made to solve this problem, but the identification of site-specific markers still remains a matter of study.

It is generally easy to distinguish between NECs and well- to moderately differentiated neuroendocrine tumors (NETs) in surgical material on morphological grounds, considering nuclear atypia, mitotic count, and Ki-67 proliferative index. Particularly, in NECs, mitotic count and Ki-67 proliferative index are by definition more than 20 per 10 high-power fields and 20%, respectively. However, when using small biopsy specimens, this distinction may be problematic due to frequent crush artifacts and the small number of neoplastic cells.

Achaete-scute homolog 1 (ASH1), termed mASH1 in rodents and hASH1 in humans, is a transcription factor belonging to the basic helix-loop-helix family. It plays a crucial role in neural/neuroendocrine determination and differentiation and is known to be involved in the development of neuroendocrine cells of the thyroid, adrenal medulla, and foregut [3], [4], [5], [6]. Expression of ASH1 has been demonstrated in NECs of the lung [7], [8], but its role as a site-specific diagnostic marker is not clear because ASH1 expression has been reported in NECs of other organs, including gastrointestinal tract and prostate [9], [10], [11]. By contrast, ASH1 expression in NETs is not fully understood, so its usefulness in the differential diagnosis between NECs and NETs needs to be verified. For these reasons, we have investigated the expression of ASH1 in a large series of neuroendocrine neoplasms of different sites, and we have reviewed the literature on this topic to provide an overview of the research on the distribution of ASH1 and its possible utility as a diagnostic marker. The 2 main aims of the present study were to evaluate the usefulness of ASH1 expression to differentiate NECs from NETs and to check its utility as a marker of lung origin.