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Characterization of Neuroendocrine Tumors of the Pancreas by Real-Time Quantitative Polymerase Chain Reaction. A Methodological Approach

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Abstract

The aim of this study was to assess the suitability of using real-time quantitative PCR (RT-qPCR) to characterize neuroendocrine (NE) tumors of the pancreas. For a series of tumors, we evaluated several genes of interest, and the data were matched with the “classical” immunohistochemical (IHC) features. In 21 cases, we extracted RNA from formalin-fixed paraffin-embedded (FFPE) blocks, and in nine cases, we also extracted RNA from fresh-frozen tissue. The RT-qPCR procedure was performed using two sets of customized arrays. The test using the first set, covering 96 genes of interest, was focused on assessing the feasibility of the procedure, and the results were used to select 18 genes indicative of NE differentiation, clinical behavior, and therapeutic responsiveness for use in the second set of arrays. Threshold cycle (Ct) values were used to calculate the fold-changes in gene expression using the 2-∆∆Ct method. Statistical procedures were used to analyze the results, which were matched with the IHC and follow-up data. Material from fresh-frozen samples performed better in terms of the level of amplification, but acceptable and concordant results were also obtained from FFPE samples. In addition, high concordance was observed between the mRNA and protein expression levels of somatostatin receptor type 2A (R = 0.52, p = 0.016). Genes associated with NE differentiation, as well as the gastrin-releasing peptide receptor and O-6-methylguanine-DNA methyltransferase genes, were underexpressed, whereas angiogenesis-associated markers (CDH13 and SLIT2) were overexpressed in tissues with malignant behavior. The RT-qPCR procedure is practical and feasible in economic terms for the characterization of NE tumors of the pancreas and can complement morphological and IHC-based evaluations. Thus, the results of the RT-qPCR procedure might offer an objective basis for therapeutic choices.

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Abbreviations

M:

Male

F:

Female

WHO:

World Health Organization classification

NET:

Neuroendocrine tumor

NEC:

Neuroendocrine carcinoma

G:

Grade

IHC:

Immunohistochemistry

T:

Tumor

N:

Lymph node

M:

Metastasis

SSTR2A:

Somatostatin receptor subtype 2A

p-mTOR:

Phosphorylated mammalian target of rapamycin

NED:

Not evidence of disease

AWD:

Alive with disease

DOD:

Dead of disease

*Hormone IHC:

Hormonal status detected by the immunohistochemical analysis of paraffin-embedded tissue.

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Acknowledgments

The study was supported by a grant from the Ministry of Education and Research, Project PERSOTHER—SMIS-CSNR: 549/12.024; with the support of Sectoral Operational Programme “Increase of Economic Competitiveness” Priority Axis 2: Research, Technological Development and Innovation for Competitiveness and project RENET—PN-II-PT-PCCA-2011-3.2.0623 91/20012 with the support of the Executive Unit for Financing Higher Education, Research, Development and Innovation—UEFISCDI. The critical reading of the manuscript by Professor Mauro Papotti, University of Turin, is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Medical Sciences, University of Turin, Via Santena 7, 10126, Turin, Italy

    Laura Annaratone, Sofia Asioli, Nelson Rangel & Gianni Bussolati

  2. Department of Oncology, University of Turin, Turin, Italy

    Marco Volante

  3. Institut Victor Babes, Bucharest, Romania

    Gianni Bussolati

Authors
  1. Laura Annaratone
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  2. Marco Volante
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  3. Sofia Asioli
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  4. Nelson Rangel
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  5. Gianni Bussolati
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Correspondence to Gianni Bussolati.

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Annaratone, L., Volante, M., Asioli, S. et al. Characterization of Neuroendocrine Tumors of the Pancreas by Real-Time Quantitative Polymerase Chain Reaction. A Methodological Approach. Endocr Pathol 24, 83–91 (2013). https://doi.org/10.1007/s12022-013-9246-y

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  • DOI: https://doi.org/10.1007/s12022-013-9246-y

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