Evaluation of Fast Molecular Detection of Lymph Node Metastases in Prostate Cancer Patients Using One-Step Nucleic Acid Amplification (OSNA)

Abstract

Background: In clinical routine, only fractions of lymph nodes (LNs) are examined histopathologically, often resulting in missed (micro-)metastases and incorrect staging of prostate cancer (PCa). One-step nucleic acid amplification (OSNA) analyzes the entire LN by detecting cytokeratin 19 (CK19) mRNA as a surrogate for LN metastases requiring less effort than conventional biomolecular techniques. We aimed to evaluate performance of OSNA in detecting sentinel LN (SLN) metastases in PCa. Methods: SLNs (n = 534) of 64 intermediate- or high-risk PCa patients undergoing radical prostatectomy with extended and sentinel-guided lymphadenectomy were cut into slices and alternatingly assigned to OSNA and histopathology (hematoxylin-eosin staining, CK19, and CK AE1/AE3 immunohistochemistry). Sensitivity and specificity of OSNA and concordance and measure of agreement (Cohen's kappa (κ)) between OSNA and histopathology were assessed. Results: Histopathology revealed metastases in 76 SLNs. Sensitivity and specificity of OSNA were 84.2% and 96.1%, respectively. Discordant results were recorded for 30 of 534 SLNs, revealing high concordance (94.4%). Twenty-four discordant cases were classified as micrometastases, indicating a possible allocation bias. In 18 cases, positive results were conferred only by OSNA resulting in seven LN-positive patients who were missed by histopathology. Overall, the level of agreement was high (κ = 0.78). Conclusions: OSNA provided a diagnosis that was as least as accurate as detailed histological examination and might improve LN staging in PCa.

Keywords: OSNA; cytokeratin 19; metastases; prostate cancer; sentinel lymph node.

Figure 1

Detection of CK19 expression in one LN with macrometastasis (a,b) and in one LN with micrometastasis (c,d), each from the same PCa patient. (a,c) Amplification curves from OSNA analysis, which determines CK19 mRNA copy numbers (micrometastasis: 250–4999 c/μL, macrometastasis: ≥5000 c/μL) as a surrogate for metastasis. Vertical dotted lines plotted in the amplification curves express the rise time (time needed for precipitation of magnesium pyrophosphate to reach a turbidity of 0.1 OD at 465 nm). Rise time is shorter in macro- (06:32 min, (a)) than in micrometastasis (07:01 min, (c)). (b,d) Images show CK19 IHC stainings (total magnification for macrometastasis (b) is 400× and for micrometastasis (d) 2000×).

Figure 2

CK19 mRNA expression in SLNs (n = 534) revealed by OSNA analysis according to histopathological examination. The dotted line depicts the 250 copies/µL cut off value.