# Performance and Prognostic Relevance of Lymph Node Assessment by One-Step Nucleic Acid Amplification Assay in Rectal Cancer: A Multicenter Study

## TL;DR

The OSNA assay is more sensitive than traditional methods for detecting lymph node metastases in rectal cancer and can help identify patients with worse survival outcomes.

## Contribution

This study demonstrates the OSNA assay's high sensitivity and its prognostic value in rectal cancer patients based on total tumor load thresholds.

## Key findings

- OSNA showed 91.7% sensitivity and 84.7% specificity compared to H&E for detecting lymph node metastases.
- A total tumor load (TTL) of ≥6000 copies/μL was significantly associated with worse cancer-specific and recurrence-free survival.
- Patients with TTL ≥6000 copies/μL may benefit from adjuvant treatment or closer monitoring.

## Abstract

The presence of micrometastases in lymph nodes (LNs) has been associated with unfavorable prognosis in patients with colorectal cancer. A fraction of patients with stage III disease may be under-staged due to the limited sensitivity of conventional hematoxylin and eosin (H&E) analysis in detecting lymph node metastases (LNM). The One-Step Nucleic Acid Amplification (OSNA) assay has demonstrated superior performance in detecting LNM and holds prognostic significance. We aimed to assess the performance of the OSNA assay in detecting LNM and its prognostic value in rectal cancer (RC) patients. LNs were analyzed by both standard H&E and the OSNA assay. We concluded that the OSNA assay is highly sensitive for detecting LNM in RC and allows identification of a subset of RC patients with worse cancer-specific survival and recurrence-free survival who might benefit from adjuvant treatment or intensive surveillance.

Background/Objectives: Lymph node metastases (LNM) undetected by standard hematoxylin and eosin (H&E) have been associated with unfavorable prognosis in colorectal cancer. The One-Step Nucleic Acid Amplification (OSNA) assay has demonstrated superior sensitivity in detecting LNM compared to H&E. We aimed to assess the performance of OSNA in detecting LNM, as well as its prognostic value in rectal cancer (RC) patients. Methods: Lymph nodes (LNs) of patients from 15 centers were analyzed by both H&E and OSNA. The total tumor load (TTL) was defined as the sum of cytokeratin 19 mRNA copies/µL in all LNs from a surgical specimen, using a threshold of 250 copies/μL for OSNA positivity. Cox proportional hazard regression was used to assess the effect of TTL ≥ 250 or 6000 copies/μL on cancer-specific survival (CSS) and recurrence-free survival (RFS), with Firth’s method applied to account for low event rate. Results: A total of 97 RC patients were included. Of these, 84 patients were eligible for survival analysis. The sensitivity and specificity of OSNA, compared to H&E, were 91.7% and 84.7%, respectively. TTL ≥ 6000 versus <6000 copies/μL was related to worse CSS and RFS. When dividing TTL into three groups: ≤250, 250–6000, and >6000 copies/μL, only TTL ≥ 6000 copies/μL was significantly associated with worse CSS and RFS. Conclusions: The OSNA assay is highly sensitive for detecting LNM in RC patients. A TTL of ≥6000 copies/μL could identify a subset of RC patients with worse CSS and RFS who might benefit from adjuvant treatment or intensive surveillance.