Abstract PS4-03-29: N-nose as a non-invasive biomarker for predicting neoadjuvant chemotherapy response in breast cancer patients

Abstract (Background) Breast cancer (BC) remains a leading cause of cancer-related deaths despite advances in diagnosis and treatment. Accurate evaluation of response to neoadjuvant chemotherapy (NAC), especially in HER2-positive and triple-negative subtypes, is critical. In recent years, liquid biopsy techniques, which analyze tumor-derived materials in body fluids, have emerged as promising non-invasive tools for cancer detection and monitoring. Nematodes-NOSE (N-NOSE) is a novel cancer screening test that utilizes the chemotaxis index of the nematode Caenorhabditis elegans (C. elegans). The N-NOSE method is a non-invasive diagnostic approach that falls within the broader category of liquid biopsy, which analyzes tumor-derived materials in body fluids. C. elegans possesses a highly sophisticated olfactory system and has been reported to exhibit attraction toward the urine of cancer patients while avoiding that of healthy individuals. In the current study, we aimed to clarify the ability of the N-NOSE method to predict the response to NAC in BC patients. (Materials and Methods) A total of 36 BC patients scheduled to undergo NAC followed by surgery at our institution between August 2020 and May 2023 were prospectively enrolled in this study. All patients were histologically confirmed to have BC and were deemed eligible for NAC based on clinical staging and risk stratification. Urine samples were collected from each patient at three distinct time points: prior to the initiation of NAC, immediately before surgery, and four weeks after surgery. For chemotactic assays, 0.5 μL of 1 M sodium azide, an anesthetic used to minimize the effects of adaptation, was spotted onto four points on the agar plate (urine side and non-urine side), and 1 μL of urine sample diluted 100-fold with ultra-pure water was added to two points (urine side). Approximately 100 adult nematodes were placed in the center of the plate. After roaming for 30 minutes, the number of nematodes present in area A (urine side) and the number of nematodes present in area B (non-urine side) were counted. The chemotaxis index (CI) was calculated using the following equation: Index = (Number of nematodes in area A - Number of nematodes in area B)/Total number of nematodes. The change in CI (Index Reduction Score, IRS) was calculated for three intervals: before and after NAC (IRS1), before and after surgery (IRS2), and before NAC and after surgery (IRS3). The association between IRS and treatment efficacy was statistically evaluated using receiver operating characteristic (ROC) curve analysis. (Results) Among the 36 patients enrolled, the median age was 51 years (range: 35-77). Thirteen patients (36.1%) achieved pathological complete response (pCR), while sixteen patients (44.4%) achieved pathological partial response (pPR). When the treatment response included CR or PR, the area under the ROC curve (AUC) for IRS1, IRS2, and IRS3 were 0.53 (95% CI: 0.19-0.78), 0.76 (95% CI: 0.56-0.96), and 0.66 (95% CI: 0.37-0.96), respectively. Notably, when only pCR cases were analyzed, the AUCs were 0.58 (95% CI: 0.34-0.82) for IRS1, 0.64 (95% CI: 0.40-0.88) for IRS2, and 0.75 (95% CI: 0.54-0.95) for IRS3, indicating that IRS3 had the highest predictive value for pCR. In this study, some pCR cases exhibited minimal residual intraductal disease before surgery, which was subsequently removed during surgery, potentially resulting in a higher IRS3 compared to IRS1. (Conclusion) The IRS measured by the N-NOSE method may accurately reflect the efficacy of NAC in BC patients. Furthermore, N-NOSE may be able to detect minimal or subclinical residual tumors after NAC, which are usually grouped clinically but may differ in their biological characteristics. Further large-scale, multicenter prospective studies are warranted. Citation Format: A. Nakakami, Y. Tokumaru, Y. Niwa, R. Mori, M. Okawa, Y. Sato, H. Hatakeyama, T. Hirotsu, E. di Luccio, N. Matsuhashi, M. Futamura. N-nose as a non-invasive biomarker for predicting neoadjuvant chemotherapy response in breast cancer patients [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-03-29.