b157ec7acf5aa16949b9221b11957d1a cancers-17-02141-v2.pdf cfd506c96c84fe3726421f787da96f557bee7d05 cancers-17-02141-v2.pdf bd4b09555caba11b22a305ec52c1520b4d3503d123483034a7574aa91e22f6dc cancers-17-02141-v2.pdf Title: Performance and Prognostic Relevance of Lymph Node Assessment by One-Step Nucleic Acid Amplification Assay in Rectal Cancer: A Multicenter Study Subject: 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/L in all LNs from a surgical specimen, using a threshold of 250 copies/L for OSNA positivity. Cox proportional hazard regression was used to assess the effect of TTL 250 or 6000 copies/L 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/L was related to worse CSS and RFS. When dividing TTL into three groups: 250, 250–6000, and >6000 copies/L, only TTL 6000 copies/L 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/L could identify a subset of RC patients with worse CSS and RFS who might benefit from adjuvant treatment or intensive surveillance. Keywords: rectal carcinoma; lymph node metastases; staging; diagnosis; prognosis; OSNA Author: Qing Liu, Sandra Lopez-Prades, Karmele Saez de Gordoa, Maite Rodrigo-Calvo, Mireia Garcia, Juan Ruiz Martin, Angel Romo, Ignacio Pinilla, Jordi Tarragona, Begoña Otero Alen, Jordi Camps, Ivan Archilla and Miriam Cuatrecasas Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Thu Jun 26 11:34:28 2025 CEST ModDate: Thu Jun 26 11:38:36 2025 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2089898 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OVNEXW+EURM10 Type 1 Builtin yes yes yes 10 0 OLIGET+URWPalladioL-Roma Type 1 Custom yes yes yes 15 0 WANAIA+URWPalladioL-Bold Type 1 Custom yes yes yes 21 0 TQNYXX+URWPalladioL-Ital Type 1 Custom yes yes yes 26 0 LFLYLN+CMSY10 Type 1 Builtin yes yes yes 52 0 QLBIEB+URWPalladioL-BoldItal Type 1 Custom yes yes yes 66 0 BFLACH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 82 0 BFLACJ+PalatinoLinotype TrueType WinAnsi yes yes no 85 0 BFLACJ+PalatinoLinotype TrueType WinAnsi yes yes no 99 0 BFLACJ+PalatinoLinotype TrueType WinAnsi yes yes no 114 0 BFLACJ+PalatinoLinotype TrueType WinAnsi yes yes no 129 0 BFLACJ+PalatinoLinotype TrueType WinAnsi yes yes no 143 0 PLOLAK+VnURWPalladioL Type 1 Custom yes yes yes 156 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-09-09 03:10:48 CEST RepresentationInformation: cancers-17-02141-v2.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-09-08 10:06:48 CEST Size: 2089898 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 419 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Eligibility of Patients Destination: subsection.2.1 Item: Title: Lymph Node Processing and Examination Destination: subsection.2.2 Item: Title: One-Step Nucleic Acid Amplification Assay (OSNA) Destination: subsection.2.3 Item: Title: CK19 Immunohistochemistry Destination: subsection.2.4 Item: Title: Estimation of Sample Size Destination: subsection.2.5 Item: Title: Statistical Analysis Destination: subsection.2.6 Item: Title: Results Destination: section.3 Children: Item: Title: Patient Characteristics Destination: subsection.3.1 Item: Title: Diagnostic Performance of OSNA Versus H&E Destination: subsection.3.2 Item: Title: Association Between TTL and Clinicopathological Characteristics Destination: subsection.3.3 Item: Title: Prognostic Relevance of TTL Destination: subsection.3.4 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Performance and Prognostic Relevance of Lymph Node Assessment by One-Step Nucleic Acid Amplification Assay in Rectal Cancer: A Multicenter Study Author: Qing Liu, Sandra Lopez-Prades, Karmele Saez de Gordoa, Maite Rodrigo-Calvo, Mireia Garcia, Juan Ruiz Martin, Angel Romo, Ignacio Pinilla, Jordi Tarragona, Begoña Otero Alen, Jordi Camps, Ivan Archilla and Miriam Cuatrecasas Subject: 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/L in all LNs from a surgical specimen, using a threshold of 250 copies/L for OSNA positivity. Cox proportional hazard regression was used to assess the effect of TTL 250 or 6000 copies/L 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/L was related to worse CSS and RFS. When dividing TTL into three groups: 250, 250 6000, and >6000 copies/L, only TTL 6000 copies/L 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/L could identify a subset of RC patients with worse CSS and RFS who might benefit from adjuvant treatment or intensive surveillance. 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