Up to 75% of patients with epithelial ovarian cancer (EOC) present with peritoneal spread, making cytoreductive surgery (CRS), with or without hyperthermic intraperitoneal chemotherapy (HIPEC), a cornerstone of treatment (1,2). The best disease control is achieved through the complete removal of all macroscopic tumor (3). Systemic chemotherapy is also frequently utilized, especially in patients unsuitable for upfront resection (4). The response to neoadjuvant chemotherapy (NACT) can be substantial with the peritoneum displaying scarring, adhesions, or even completely normal appearance, despite prior tumor presence (5,6).

Occult disease-microscopic lesions in normal-appearing peritoneum-remains a significant challenge in EOC, particularly in patients after NACT (7). It occurs near visible tumor nodules in up to 46% of cases and in entirely normal-looking regions in 34% (6,7). These lesions are clinically significant and can lead to gross disease recurrence and poor survival outcomes. Visual inspection and palpation during surgery are often insufficient for detecting occult EOC disease (8). Total parietal peritonectomy has been proposed by several centers to reduce the risk of residual occult tumor; however, this approach can result in substantial surgical trauma, associated with high complication rates and increased mortality (6,9,10).

Pafolacianine (Cytalux) is the first agent approved by the Food and Drug Administration (FDA) for intraoperative tumor-targeted visualization in ovarian and lung cancer. It works through a folic acid analog tracer conjugated with indocyanine green, which targets the folate receptor overexpressed in EOC cells, and is visualized intraoperatively using near-infrared (NIR) fluorescence imaging (11-13). Data from phase II and III trials showed promising results, leading to FDA approval in 2021; however, it has not yet been widely adopted, and no real-world data are currently available (14,15). We report, to our knowledge, one of the first national out-of-trial experiences using Cytalux, evaluating its utility in detecting occult disease following complete macroscopic resection during CRS/HIPEC in patients with primary and recurrent EOC with peritoneal metastases.

This report presents the first out-of-trial experience using Cytalux-based intraoperative imaging to detect occult disease in EOC with peritoneal spread during CRS/HIPEC. Cytalux uptake correlated with visible tumors and identified at least one additional non-visible lesion post-CRS in each of the three patients. The overall true positive rate for occult peritoneal lesions after CRS was 80%, with a false positive rate of 20%. Notably, Cytalux imaging revealed one area of post-chemotherapy fibrosis, later confirmed as malignant by pathology. Additionally, 2 Cytalux-positive peritoneal areas underwent extensive argon plasma fulguration for over 20 min until the NIR signal was lost, but both were still positive for cancer, suggesting caution in using Cytalux imaging for this purpose. These findings validate previously published data from clinical trials on Cytalux efficacy in real-world settings while also highlighting critical new challenges associated with its intraoperative use.

All patients in our study demonstrated Cytalux uptake in grossly visible tumors, confirming high folate receptor expression in all EOC cases. Previous studies have shown that EOC cells can express folate receptors at levels 10-100 times higher than normal tissues, making them an attractive target for imaging occult disease (12,13,19). In a phase III trial, 109 out of 150 patients who received infusion with Cytalux had confirmed folate-receptor-positive EOC (15). This high uptake is likely because of Cytalux targeting folate receptor alpha, present in up to 90% of EOC, rather than the less frequently expressed folate receptor beta, found in about 20% of EOC. Two patients in our study had high-grade serous carcinoma, and one had papillary serous carcinoma, indicating that even different EOC subtypes can express folate receptors and show appropriate Cytalux uptake. In the phase III trial, up to 18% of patients with folate-receptor-positive disease had less common EOC variants than high-grade serous carcinoma (15). Both our and previous data suggest that Cytalux has a high affinity for tumor cells in the majority of EOC patients, but its uptake should be confirmed at the beginning of the procedure before tumor removal.

Interestingly, the first antibody-drug conjugate targeting folate receptor alpha, mirvetuximab soravtansine, was recently approved by FDA for treatment of platinum-resistant ovarian cancer. Its interaction with Cytalux is unclear but competitive blockade of folic receptors alpha can potentially interfere with Cytalux uptake and accuracy of the imaging, warranting further investigation.

Achieving complete cytoreduction is crucial in EOC, as the presence of residual occult disease worsens patient outcomes. Therefore, total parietal peritonectomy has been proposed by several centers outside the United States to cope with occult disease yet it can significantly increase morbidity while the impact on survival is unclear (6,9,10). The concept of using Cytalux for targeted imaging is a precise identification of occult lesions that offers a more selective approach and potentially reduces the need for highly morbid procedures such as total peritonectomy. In our study, the TP rate for Cytalux-positive lesions was 80%, consistent with 83% reported in the clinical trial, where 395 of 531 lesions were TP (15). Notably, Cytalux identified additional occult lesions in all patients in our cohort. In the original trial, the rate of additionally detected lesions by Cytalux that were confirmed by pathology but not initially planned for resection was 39.7% for interval CRS and 17.1% for primary CRS (15). Our findings reinforce the clinical utility of Cytalux in guiding and achieving more thorough cytoreduction by enhancing intraoperative lesion detection beyond standard visual and tactile assessment, thereby potentially improving long-term EOC patient outcomes.

There are several limitations to the use of Cytalux. A significant FP rate, leading to unnecessary resections, has been previously reported. In our study, the FP rate was 20%, which is lower than the 32.7% reported in the phase III trial (15). The FP rate can vary significantly depending on the interpretation of imaging. For patient C, nearly the entire small bowel surface was highlighted with Cytalux at the beginning of the case, which resolved with a decrease in imaging gain. Fluorescence can occur in normal tissues, including areas of the bowel, kidneys, lymph nodes, lungs, and inflamed tissue, leading to potential interpretation errors. This underscores the importance of fluorescence standardization and surgeon training to optimize imaging interpretation and minimize unnecessary resections. Additionally, the presence of company representatives during the first several cases may be necessary for accurate imaging interpretation and navigation.

An interesting finding in our study is the failure of argon beam tumor destruction under Cytalux navigation in patient A. In this case, two substantial areas, each more than 10 cm in diameter, were Cytalux-positive but had no grossly visible lesions on close inspection. These areas underwent argon fulguration for over 20 min until the Cytalux signal was lost and eschar was formed. However, when resected, these areas were confirmed to be malignant on pathology. Energy-based tumor destruction methods, including electrocautery, argon beam coagulation, ultrasonic surgical aspirator, and carbon dioxide laser, are commonly used by cytoreductive surgeons to reduce tumor burden and increase the likelihood of complete cytoreduction, particularly for implants in difficult locations (20-22). However, the efficacy of thermal tumor destruction has not been systematically studied. Based on our experience, Cytalux should not be used for guidance during thermal tumor destruction until more data are available.

This study has several limitations related to its design. First, the small sample size, with only three patients, did not allow for a comprehensive statistical analysis. Larger, multicenter prospective studies with a greater number of participants are needed to validate our results, assess patient selection criteria, and better characterize Cytalux's sensitivity and specificity in real-world clinical settings. Second, the short follow-up period prevented us from assessing the impact of Cytalux-guided cytoreduction on survival outcomes. Future studies should incorporate longitudinal follow-up to determine whether Cytalux-guided resection of occult disease improves progression-free and overall survival. Lastly, we were unable to calculate true and false negative rates, as this would require performing a total parietal peritonectomy, a highly morbid procedure that is not done routinely. Future trials could integrate biopsy mapping strategies or other validation methods to provide a more accurate assessment of the extent of occult disease missed by standard imaging techniques. Despite these limitations, our study bridges the gap between clinical trial data and real-world application, offering early insights into the feasibility, benefits, and challenges of integrating Cytalux into routine EOC management.

In conclusion, the use of Cytalux in real-world settings demonstrates significant efficacy in identifying occult peritoneal disease in a substantial proportion of EOC patients with peritoneal metastases, with a true positive rate of up to 80%. However, a notable false positive rate emphasizes the need for cautious interpretation, particularly in the context of surgical decision-making. Cytalux signal navigation should not be used for guiding thermal tumor destruction until more evidence becomes available. Routine Cytalux-guided decision-making may require standardization of fluorescence approach, implementation of special training for surgeons with learning curve to correctly identify and interpret lesions, and development of real-time lesion validation by frozen pathology. Continued collection of real-world data is critical to fully assess Cytalux utility in out-of-trial settings, ultimately guiding its broader adoption in clinical practice.