One Step Nucleic Acid Amplification (OSNA) - a new method for lymph node staging in colorectal carcinomas

Roland S Croner,Vera Schellerer,Thomas Papadopulos,Werner Hohenberger,Helene Demund,Anne Schlabrakowski,Klaus E Matzel,Claus Schildberg,Michael Stürzl,Elisabeth Naschberger

doi:10.1186/1479-5876-8-83

Abstract

BackgroundAccurate histopathological evaluation of resected lymph nodes (LN) is essential for the reliable staging of colorectal carcinomas (CRC). With conventional sectioning and staining techniques usually only parts of the LN are examined which might lead to incorrect tumor staging. A molecular method called OSNA (One Step Nucleic Acid Amplification) may be suitable to determine the metastatic status of the complete LN and therefore improve staging.MethodsOSNA is based on a short homogenisation step and subsequent automated amplification of cytokeratin 19 (CK19) mRNA directly from the sample lysate, with result available in 30-40 minutes. In this study 184 frozen LN from 184 patients with CRC were investigated by both OSNA and histology (Haematoxylin & Eosin staining and CK19 immunohistochemistry), with half of the LN used for each method. Samples with discordant results were further analysed by RT-PCR for CK19 and carcinoembryonic antigen (CEA).ResultsThe concordance rate between histology and OSNA was 95.7%. Three LN were histology+/OSNA- and 5 LN histology-/OSNA+. RT-PCR supported the OSNA result in 3 discordant cases, suggesting that metastases were exclusively located in either the tissue analysed by OSNA or the tissue used for histology. If these samples were excluded the concordance was 97.2%, the sensitivity 94.9%, and the specificity 97.9%. Three patients (3%) staged as UICC I or II by routine histopathology were upstaged as LN positive by OSNA. One of these patients developed distant metastases (DMS) during follow up.ConclusionOSNA is a new and reliable method for molecular staging of lymphatic metastases in CRC and enables the examination of whole LN. It can be applied as a rapid diagnostic tool to estimate tumour involvement in LN during the staging of CRC.

Highlights

Carcinomas of the colon and rectum (CRC) are the fourth most common malignancy in the US and the second most common cancer for woman (195,400 cases) as well as the third most common cancer for men (217,400 cases) in Europe, with 207,400 associated deaths in 2006 [1,2]
One Step Nucleic Acid Amplification (OSNA) and histology 184 lymph nodes (LN) from 184 patients with colon cancer were investigated with both OSNA (CK19 mRNA as a marker) and intensive histological methods (H&E and cytokeratin 19 (CK19) IHC on 5 levels for each of 2 LN slices)
RNA quality was assured by OSNA performed for beta-actin. 139 samples gave a negative result and 37 samples gave a positive result with both methods

Summary

Introduction

Carcinomas of the colon and rectum (CRC) are the fourth most common malignancy in the US and the second most common cancer for woman (195,400 cases) as well as the third most common cancer for men (217,400 cases) in Europe, with 207,400 associated deaths in 2006 [1,2]. Pathological investigation including immunohistochemistry (IHC) and step sectioning detected tumour deposits smaller than 2 mm in 20-30% of LN in stage UICC I and II CRC patients [15,17,18]. Molecular analysis of sentinel LN in colon carcinomas has resulted in the detection of micrometastatic disease which was undetected by IHC [19]. This is due to the fact that during RT-PCR the whole LN or at least the biggest part of the LN can be analysed while during histological work-up usually only a small part of the LN is screened. A molecular method called OSNA (One Step Nucleic Acid Amplification) may be suitable to determine the metastatic status of the complete LN and improve staging

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