The field of fluorescence-guided surgery in oncology is expanding rapidly, but standardized methods to evaluate imaging results are not widely used. Our results indicate that a standardized ex vivo fluorescence-guided imaging method for margin assessment using the pH-activated imaging agent ONM-100 shows substantial clinical potential for real-time intra-operative decision-making. We identified all tumor-positive margins by detecting fluorescent lesions at the surgical resection margin with a tumor-to-background ratio > 1.5. This easy implementable framework, which can be implemented in the surgical theatre, can be used immediately after surgical excision allowing an alteration of the surgical strategy during initial surgery. Subsequently, we showed that ONM-100 can detect remaining tumor and severe dysplasia in the surgical cavity which was otherwise missed during standard of care surgery.
We suggest the use of our standardized fluorescence framework, combined with ONM-100, for real-time surgical margin assessment in HNSCC and surgery (Fig. 4). Briefly, when a fluorescent lesion at one of the resection margins is detected (threshold TBR > 1.5), and if anatomical borders allow an additional resection in situ, an immediate re-resection can be considered . This imaging procedure can be performed in the surgical theatre to improve surgical outcome. After tumor excision, imaging of the surgical cavity is performed subsequently to fluorescence imaging of the freshly excised surgical specimen which can guide the surgeon in performing an immediate re-resection. Additionally, fluorescence imaging of the surgical cavity can detect occult tumor and/or dysplastic lesions, whether or not related to the primary tumor, as highlighted by the observations in our cohort, in which one occult additional tumor lesion and a dysplastic lesion were found using fluorescence imaging of the surgical cavity. We show that FGS using ONM-100 could guide the surgeon to perform an immediate re-resection and potentially prevent (unnecessary) additional re-resections or adjuvant treatment. This potential is underlined by the fact that after an initial tumor-positive surgical margin, two HNSCC re-resections in this series were negative for residual tumor. In the unfortunate event that it is not possible to perform an additional resection, which accounts not only for head and neck cancer patients, the surgeon receives immediately feedback on the clinical situation and is able to alter the surgical plan (i.e., no further surgery, marking of positive spot for post-operative radiotherapy).
In the current study, intra-operative ex vivo fluorescence imaging for margin assessment has been prioritized above in vivo fluorescence imaging. Ex vivo specimen imaging using a closed-field and standardized imaging device allows for standardization of measurements, which is highly relevant in fluorescence-guided imaging where multiple factors like distance to the camera, angle of illumination, and environmental light can influence imaging results severely . With the availability of a close-field imaging device at the surgical theatre, tissue can be imaged immediately after surgical excision allowing for direct surgical decision-making, as also has been described by other groups [11, 15, 16]. As the close-field imaging device used in the current study has a limited field of view, future developments in field of view, resolution, and imaging quality might further enhance intra-operative clinical decision-making.
Despite the investigated imaging agents showed promising results in previous studies, visual discrimination between tumor and non-tumor tissue remains challenging due to high, a specific background signals in non-tumor tissue [5, 7, 8, 12]. A low background signal, a consequence of the design of ONM-100, shows a clear and sharply delineated fluorescent signal in tumors in this study. This discriminative strength of ONM-100 is illustrated by the fact that the tumor-positive resection margins were clearly highlighted compared to the non-involved adjacent resection margin. Moreover, ONM-100 was administered within 24 h of surgery, providing major benefits over other fluorescence imaging agents which are administered 2–5 days prior to surgery [5, 7, 17]. Indeed, we have seen some false-positive fluorescence signal in salivary gland tissue, as is previously described as well . The exact reason for this activation needs to be further analyzed in future studies; however, as we believe that a surgeon is able to differentiate salivary gland tissue from tumor tissue, we believe this might not be a significant clinical problem.
Based on our experience as a tertiary referral hospital, performing complex surgery often in late-stage HNSCC disease (Supplemental Figure S3), we identified three possible scenarios originating from the findings in this study following the detection of a fluorescent lesion. First, fluorescent lesions can be resected with an adequate margin without compromising vital structures. Second, an identified fluorescent lesion cannot be resected due to anatomical borders or the extent of disease. Third, new unidentified lesions not directly related to the initial tumor or irresectable lesions are identified which might change or cease the surgical plan. The first and last scenario occurred in this particular study. As this study shows, the fluorescent lesions detected at the excised specimen correlated in 100 % of the cases to tumor-positive resection margin and in 60 % of the cases to close resection margins. This implicates that, in theory, in ten patients, an immediate re-resection could be considered, thereby preventing a second surgery. This is highly relevant, since not all patients were eligible for second surgery due to their intrinsic comorbidities. From the possible ten re-resections, one re-resection would result in a case of overtreatment due to a false-positive fluorescent signal according to the current guidelines on tumor involved margins. If this eventually would lead to a higher local tumor control remains to be studied. We assume that in case of a successful immediate re-resection at the primary tumor site, adjuvant therapy can at least be de-intensified from the concomitant combination of chemoradiation to adjuvant radiotherapy alone. We therefore consider these data encouraging for further exploring the use of ONM-100 for the purpose of intra-operative margin assessment in a larger phase II trial.
Recent studies in HNSCC suggest a cutoff point of 2 mm between a tumor-positive and a tumor-negative surgical margin, as > 2 mm margin shows no significant increase in disease-specific survival [3, 18]. Therefore, a method that could discriminate between tumor presence within 2 mm and > 2 mm of the surgical border would be suited for real-time surgical guidance in HNSCC patients. Interestingly, all fluorescence-negative surgical margins found in this study had a surgical margin of at least > 2 mm, again underlining the clinical applicability of this technique. It should be noted that the numbers of tumor-positive margins detected in the current study are outside the average norm . However, a tertiary referral hospital (UMCG) receives delicate and late-stage disease patients which a high a priori chance for tumor-positive surgical margins.
In conclusion, this study demonstrates the potential added value of specimen-driven fluorescence imaging of the surgical specimen in HNSCC patients after administration of the optical imaging agent ONM-100. We found a 100 % sensitivity for tumor-positive surgical margin detection using this technique and showed the potential for the intra-operative detection of occult disease. ONM-100 is easy to implement into standard clinical care due to the clear visual discrimination between tumor and non-tumor tissue, therefore having great potential in assisting in clinical decision-making during HNSCC surgery, preventing under- and overtreatment.