Intraoperative fluorescence-guided fresh frozen sectioning for margin control in head and neck cancer: phase 2 clinical trial

Domain: biology
Published: 2026-03-10
Editorial outcome: minor_revision

Abstract
Tumour-free margins (>5 mm) in head and neck malignancies improve prognosis, possibly impacting the need for
adjuvant treatment. In this phase II prospective, non-randomised trial (NCT05499065), we investigate the use of
fluorescence imaging using the tumour targeting fluorescent tracer cetuximab-IRDye800CW combined with fresh
frozen sectioning to identify inadequate margins intraoperatively in patients with oral squamous cell carcinoma.
Primary objectives of our study are intraoperative detection of inadequate margins and additional resections, and
feasibility of implementing fluorescence-guided fresh frozen sectioning (FG-FFS) into the clinical workflow. In
this study, 10 out of 20 patients show inadequate margins after initial surgery; FG-FFS correctly classifies margin
in 19 patients. Additional resections increase tumour-free margin rates from 50% to 85%, where anatomical
boundaries limit further improvement in one patient. Intraoperative margin adjustment reduces the need for
adjuvant treatment in seven patients. These findings demonstrate that FG-FFS is a reliable and feasible method for
intraoperative margin assessment without delaying surgery.

Introduction
Surgery is the primary treatment modality for many head and neck malignancies. Resection margins are considered
adequate when at least 5 mm wide.1–3 Despite this well-established requirement, tumour-free surgical margins are
achieved in only 30-65% of patients.4,5 Inadequate resections adversely impact survival and locoregional control:
five-year disease-free survival rates of approximately 79%, 63% and 52% for tumour-free (≥ 5 mm), close (< 5 mm
& ≥ 1 mm) and tumour-positive margins (< 1 mm), respectively.1–3,6,7
Call-back surgery for inadequate margin correction is challenging, as accurately relocating inadequate margins is
difficult, particularly in patients who have undergone reconstructive surgery.8 Achieving tumour-free margins at the
initial surgery is of major clinical importance, as adjuvant treatment cannot fully compensate for the negative
prognostic impact of initially inadequate margins.9,10
Fresh frozen sectioning (FFS) of the excised tumour specimen is the standard for evaluating margins during
surgery.11–14 An FFS procedure involves preparation of a 4 µm tissue section from a tissue slice, which has been
rapidly frozen after fresh sectioning using a cryostat. This frozen section is subsequently haematoxylin & eosin
(H&E) stained to assess tumour margins. Although intraoperative FFS provides a high specificity for margin
assessment, sampling error substantially reduces the sensitivity of this technique.15–18 Various approaches for
improved intraoperative identification of inadequate margins have been investigated.19,20 Fluorescence-guided
surgery using tumour targeted tracers has proven to be beneficial in head and neck cancer.21–25 The use of cetuximab-
IRDye800CW to detect oral cancer cells is feasible, since these cells overexpress epidermal growth factor
receptor.22 However, the intensity of the fluorescent signal does not reliably correlate with the margin width using
current fluorescence imaging approaches.25 To overcome this issue, a combined approach, supplementing
fluorescence imaging with intraoperative fresh frozen sectioning (i.e. fluorescence-guided fresh frozen sectioning
(FG-FFS)), has been developed. Although the combination of fluorescence imaging and fresh frozen sectioning has
been suggested previously, this has not been systematically investigated.23–25 Krishnan et al. examined the
localisation of fresh frozen sectioning based on clinical suspicion compared to a sentinel margin approach, which
uses the areas of highest fluorescence intensities as indicators of potentially inadequate margins. They found a
strong correlation between these approaches, however they did not investigate margins not suspected to be at risk.24
In this phase two intervention study, we demonstrate that fluorescence imaging of the whole resection specimen
and fresh tissue slices containing cetuximab-IRDye800CW successfully localizes margins at risk, while FFS
provides margin distance measurements of these margins in millimetres. This combined approach enables
fluorescence imaging to guide intraoperative pathology assessment beyond clinical suspicion alone, allowing for
timely recognition and intraoperative adjustment of inadequate margins.

Results
The primary endpoints of this phase two study were the detection rates of inadequate margins by fluorescence
imaging only and FG-FFS, including the number of additional resection based on FG-FFS. Additionally, tracer
distribution (i.e. the spatial localisation of the cetuximab-800CW probe imaged with a fluorescence camera) was
evaluated and compared to pathology margins. Secondary endpoints for the study were patient characteristics and
feasibility of implementation and clinical application of FG-FFS into the current clinical workflow.

Patient and tumour characteristics
Between March 2023 and November 2024, 23 patients with histopathology proven oral squamous cell carcinoma
were included in this study. Study drugs consisting of unlabelled cetuximab and cetuximab-IRDye800CW were
administered intravenously two days prior to surgery. During study drug administration, two patients showed
infusion-related anaphylaxis to the unlabelled cetuximab, leading to termination of intravenous administration and
study exclusion. Both patients recovered after intramuscular adrenaline administration and an NaCl fluid challenge
and standard of care surgery could continue as scheduled. One other patient was excluded due to deviations from
the intraoperative imaging protocol, resulting in a total of 20 patients completing all study procedures. The
intraoperative FG-FFS margin assessment protocol is shown in Figure 1. An overview of the patient and tumour
characteristics is shown in Table 1 (secondary endpoint). The mean age of study participants was 57 (SD = 17.7),
13 (65%) patients were female. Tumours were primarily located in the tongue (n = 16) and staged pT1-3. Lymph
node metastases were present in four patients.

Surgical outcomes and clinical implication
Figure 1 gives an overview of the intraoperative specimen handling pipeline. Standard of care surgery resulted in
one tumour specimen per patient. This standard of care resection and subsequent pathology analysis, prior to any
study intervention, would have resulted in ten (50%) inadequate margins (Figure 2A). For study purposes, the whole
resection specimen was sliced into fresh tissue slices intraoperatively at the department of pathology of the
University Medical Centre Groningen (UMCG). The first primary endpoint of this study included the evaluation of
detection of inadequate margins by FG-FFS. FG-FFS based on the fresh tissue slices correctly identified margins
in 95% of patients (19 out of 20). One inadequate margin was missed where FG-FFS estimated a margin of 5.5
mm, but final pathology showed a margin of 4.5 mm. This resulted in a sensitivity, specificity and accuracy of
90.0%, 100.0% and 95.0%, respectively. Besides the identified closest margins by FG-FFS, no other locations with
inadequate margins were identified after complete pathology analysis.

Another primary endpoint was the immediate intraoperative additional resections that were performed in eight out
of nine patients with an inadequate margin identified by FG-FFS (Figure 2B). For one patient, anatomical
boundaries prevented an additional resection. The eight additional resections resulted in tumour-free margins at
final pathology assessment in seven patients (Figure 2C). Margins remained inadequate in one patient (Figure 2,
patient 9). In this case, the surgeon underestimated the additional resection width; a margin width of 1.1 mm was
converted into 3.3 mm. Overall, the final inadequate margin rate was reduced from 50% to 15%. Intraoperative
decision-making was performed within one hour (i.e. during treatment of the neck or preparations for reconstruction
of the defect) without delaying surgery and was therefore feasible within a clinical time-frame (secondary
endpoint).

Study related additional resections led to de-escalation or complete omission of margin-based adjuvant therapy in
seven of the ten patients who otherwise would have required therapy. Adjuvant therapy was entirely averted for
five patients, thereby preventing chemo-radiotherapy, radiotherapy or call-back surgery. For two patients, adjuvant
therapy was minimised to 56 Gy radiotherapy only instead of chemo-radiotherapy or 66 Gy radiotherapy (Figure
2A&C).

Correlation of FG-FFS margins with final pathology analysis
An important point to consider is whether the intraoperative margin provided by pathology based on FG-FFS is
comparable to the margin provided after final pathology analysis, when the tissue is formalin fixed and paraffin
embedded. In a retrospective analysis, these margin widths were compared. For this analysis, all patients with at
least one exact FFS margin in mm and final pathology margin width in mm of that same anatomical location, could
be included. Nineteen patients were included and one patient was excluded, due to a missing exact FFS margin
(recorded only to be greater than 5.1 mm). A significant difference (degrees of freedom (df) = 17, p < 0.001, effect
size = -1.07, 95% confidence interval (95% CI) = -1.16 – -0.43) was found between the FG-FFS margin estimate
and final pathology margin. Bland-Altman analysis revealed an average margin width overestimation of 0.8 mm
by FG-FFS, as shown in Figure 3. A post-hoc correction for this overestimation was performed to evaluate the
potential impact on this observed difference. After correcting FG-FFS margins for this overestimation, by
subtracting 0.8 mm from each determined FFS margin, no significant difference (df = 17, p = 0.591, effect size =
0.13, 95% CI = -0.25 – 0.43) remained between the FG-FFS margin and final pathology margin.

Analysis of other potential intraoperative assessment points
To further evaluate the first primary endpoint, data of other potential intraoperative assessment points other than
fresh tissue slice FG-FFS were separately analysed. These points included fluorescence imaging only of the whole
resection specimen, fluorescence imaging only of the fresh tissue slices and whole resection specimen FG-FFS.
Note that these assessment points were all performed for every resection as the steps did not interfere with one
another. As shown in Figure 1, fluorescence imaging of the whole resection specimen was performed first, followed
by identification of spots and demarcation with a needle (‘fluorescence imaging only of the whole resection
specimen’ in Table 2). Next, the specimen was cut into fresh tissue slices, followed by imaging of these tissue slices
(fluorescence imaging only of fresh tissue slices) to identify tissue slices with potential inadequate margins. For
whole resection specimen FG-FFS, an FFS of the tissue slice with the demarcation needle was acquired; for fresh
tissue slice FG-FFS, an FFS of the tissue slice with the estimated closest margin was acquired. Sensitivity,
specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) for all four strategies are
shown in Table 2. The endpoint tracer distribution and its correlation to final histopathology, was assessed using
fluorescence imaging of the whole resection specimen. Fluorescence imaging only of the whole resection specimen
analysis (Figure 1.2) accurately identified tumour-positive margins (< 1 mm), as all four detected spots (spot-to-
background ratios (SBRs) ranging between 1.7 and 2.1) were tumour-positive. However, absence of a spot was not
indicative for tumour-free margins (> 5 mm), as shown by the low sensitivity caused by six false negative cases.
Contrarily, fluorescence imaging only of the fresh tissue slices (Figure 1.4) was indicative for tumour-free margins,
but did show seven false inadequate margins. During fresh tissue slice FG-FFS, these false inadequate margins
were corrected by FFS, increasing specificity. Representative examples of inadequate and tumour-free resection
margins are shown in Figure 4.

Methods
Study design and participants
A phase two, non-randomized, non-blinded, prospective, and interventional study was performed at the
department of Oral & Maxillofacial Surgery of the UMCG, the Netherlands. The Institutional Review Board of the
UMCG approved the study (METc 2022/456) and it was conducted according to the Dutch Act on Medical Research
involving Human Subjects (WMO) and the principles of the Declaration of Helsinki (adapted version of Fortaleza,
Brazil, 2013). The study is registered at www.clinicaltrials.gov (NCT05499065).
Patients ≥18 years old with histopathology proven oral squamous cell carcinoma scheduled to undergo surgery, as
decided in the multidisciplinary head and neck tumour board meeting of the UMCG, were eligible for inclusion in
the study. All eligible patients were included regardless of sex or gender. Sex was registered for baseline
characteristics using electronical patient record data documented in the hospital electronic patient records, as self-
reported by each patient. After obtaining written informed consent, inclusion and exclusion criteria were checked.
Briefly, patients were included if they showed no evidence of QTc prolongation, were not pregnant or lactating,
and had no history of allergy or infusion reactions to monoclonal antibodies.

Fluorescent tracer administration
The study drug cetuximab-IRDye800CW was produced under Good Manufacturing Practice in the UMCG, and
released by a qualified person.39 Two days before surgery, study drugs were administered intravenously in
accordance with the previously published tracer administration protocol.22,25 First, an intravenous dose of 2 mg
clemastine was given, according to standard of care cetuximab treatment to reduce allergic reaction risk. Next,
patients received 75 mg unlabelled cetuximab as a pre-dose, followed by 15 mg fluorescently labelled cetuximab,
cetuximab-IRDye800CW, after one hour. Pre-dosing using unlabelled cetuximab improves fluorescent tracer
localization in the tumour, as shown previously.22

Intraoperative margin assessment
An overview of the intraoperative margin assessment protocol is shown in Figure 1. Surgery started with primary
tumour resection, followed by treatment of the neck. During tumour resection, paired tagging was performed for
orientation purposes. Paired tagging consists of stitches marked with clips placed in the mucosa of both the surgical
cavity and the resection specimen on at least four corresponding anatomical locations. Stiches at matching
anatomical locations were marked with the same number of clips. Paired tagging ensured accurate relocation of the
anatomical site within the surgical cavity in case of an inadequate margin (Figure 1.1).16 In this study, in-vivo
imaging was not performed, since this results in inadequate illumination of the resection margins and therefore
limits assessment. Imaging of the excised specimen is more straightforward, enabling proper and standardised
illumination of all surgical margins. Ex-vivo fluorescence imaging therefore served as red flag technique for fresh
frozen sectioning.36
Immediately after tumour excision, all sides of the resected specimen were imaged using a closed-field fluorescence
camera (i.e. PEARL-trilogy®, LI-COR BioSciences Inc., Lincoln, NE, USA) at the department of Pathology and
Medical Biology of the UMCG. The acquired images were scaled relative to the maximum intensity of the
tumour/mucosal side using ImageJ (version 1.53t, Fiji, Bethesda, MD, USA). Subsequently, two observers assessed
the images for fluorescent spots using the colormap MPL-inferno, indicating possible inadequate margins. When
a spot was identified, an SBR was calculated using formula (1):
SBR = I_spot / I_background (1)
with I being the mean intensity. The background region of interest was identified on the same image as the spot,
based on all tissue adjacent to the spot of a similar origin (e.g. muscle, fat) and identical distance to the camera (as
shown in Figure 4A). A spot with an SBR > 1.5 was marked on the whole resection specimen using a needle,
followed by a fluorescence image to confirm the marker location. The location of this needle served as the reference
for whole resection specimen FG-FFS, as shown in Figure 1.
Subsequently, the resection sides of the specimen were inked, and the specimen was freshly cut into 3 - 5 mm thick
tissue slices by dedicated pathology staff. Next, images of all fresh tissue slices were acquired using the
fluorescence camera. Delineation of tumour tissue on fluorescence images was performed by two observers, based
on maximum fluorescence intensity and a substantial drop-off of signal towards all resection sides. From these
estimated tumour outlines, resection margins were measured on the images of the tissue slices, identifying the
closest margins based on fluorescence imaging. In case of disagreement between observers, a third observer was
consulted. If, based on fluorescence imaging, all margins were assessed as being tumour-free, FFS was performed
of the fresh tissue slice containing the estimated closest margin. When one or more inadequate margin(s) were
suspected based on fluorescence imaging of the fresh tissue slices, FFS of these margins was performed. In case
FFS confirmed an inadequate margin, the surgeon was informed by the pathologist. An additional resection was
performed if deemed clinically feasible and safe. Determining the exact location for additional resection was
essential for effective treatment. To ensure this, the following measures were taken: continuous orientation tracking
by two dedicated researchers throughout tissue imaging and processing, paired tagging to preserve anatomical
orientation, documentation of cutting planes during tissue slicing to enable accurate relocation of tissue slices in
the original resection specimen, and collaborative reporting by pathologist and researchers to the surgeon of the
precise location of any inadequate margins identified in the FFS. An exemption to this workflow was made only
for cases in which a tumour-positive margin was apparent based on visual and tactile assessment by both pathologist
and surgeon and fluorescence imaging of the fresh tissue slices. Even though not proven by FFS, an immediate
additional resection was performed. Altogether, two FG-FFS methods were investigated: 1) FFS of the tissue slice
identified by a spot and marked with a needle on whole resection specimen fluorescence imaging, and 2) FFS of
the tissue slice identified as the slice containing the closest margin by fresh tissue slice fluorescence imaging.
Following the FFS procedure, the fresh tissue slices were formalin fixed for at least 24h, followed by paraffin
embedding and 4 µm sectioning for H&E staining, according to standard of care. Based on the standard of care
H&E staining assessment, margins before and after potential additional resections were assessed by a pathologist.
Based on pathology results before and after study intervention, adjuvant treatment was determined by the
multidisciplinary head and neck tumour board of the UMCG. During the trial, adverse events were reported to the
Institutional Review Board using the Common Terminology Criteria for Adverse Events version 6.0.

Endpoints
In this study, three primary and two secondary endpoints were evaluated. The first primary endpoint was the
comparison between fluorescence imaging alone and FG-FFS for detecting tumour-positive and close surgical
margins. Diagnostic accuracy was evaluated by calculating sensitivity, specificity, accuracy, PPV and NPV for both
methods. An additional primary endpoint was the number of additional resections performed based on FG-FFS data
and the corresponding postoperative pathology outcomes. Tracer distribution, evaluated by correlating SBR values
of the whole resection specimen with margin widths, and conversely, margin widths with SBR values, was the last
primary endpoint of this study. The secondary endpoints were patient characteristics, feasibility of on-site
intraoperative tissue analysis by the surgeon and pathologist and feasibility of novel 3D imaging methods of the
obtained additional biopsies. Feasibility of novel 3D imaging methods of the obtained additional biopsies is not
reported here as it did not align with the primary focus of the trial. Feasibility of the analysis was assessed based
on duration of surgical procedures and integration of FG-FFS into the operative workflow.

Statistics & Reproducibility
The sample size was calculated using a two-proportion power analysis, assuming a baseline proportion of 0.5, an
expected proportion of 0.2, a 95% confidence level, 80% power, and resulted in a required sample size of at least
19 patients. Patients were excluded from the analysis in case they did not undergo all study procedures. The patients
were not randomised.
Descriptive statistics of patient demographics were tested using the Anderson-Darling test, D’Agostino & Pearson
test, Shapiro-Wilk test and Kolmogorov-Smirnov test using GraphPad Prism (version 9.0, GraphPad Software Inc,
San Diego, California, USA). Normally distributed data were reported using mean with standard deviation (SD)
and non-gaussian data using the median with interquartile range (IQR). Next, sensitivity, specificity, accuracy, PPV
and NPV for margin assessment were calculated for both fluorescence imaging only and FG-FFS. Finally, FG-FFS
margin widths were compared with final pathology margins using a paired t-test and Bland-Altman analysis to
assess a potential bias. As this manuscript concerns a clinical intervention trial, all experiments could only be
performed once (during surgery).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary: The manuscript is a phase II clinical trial in surgical treatment of oral cancer using tumor targeted fluorescence\nguidance using cetuximab-IRDye800CW in the assessment of intra-operative margin detection and its clinical applicability.\nThe primary end points of the study were detection of inadequate margin rates, additional resections and their clinical\nimplications. Secondary endpoints assessed the feasibility of FG-FFS implementation into current SOC workflows were\ndiscussed.\nPrimary outcomes were as follows: Out of the 20 patients included in the study, 50% (10) patients had inadequate margins,\n8/9 patients among these underwent additional resections, 7/8 of these patients showed tumor free margins at final\npathological assessment. Overall, the final inadequate margin rate was reduced from 50% to 15%. The FG-FFS based on\nfresh tissue slices identified margins in 95% (19 out 20) patients.\nSecondary outcomes showed that FG-FFS was feasible within a clinical-time frame. The additional resections led to de-\nescalation or complete omission of margin-based adjuvant therapy in 7/10 patients who would otherwise have required\ntherapy. Out of these 7 patients, adjuvant therapy, entirely averted in 5 patients, and reduced in 2 patients.\nAuthors and the findings of the study make a strong case of incorporating fluorescence guidance into SOC surgical\nworkflows which can lead to better outcomes compared to the current methods. Review of the manuscript raised the\nfollowing points and comments for the authors to consider.\nSpecific comments to be addressed:\n1. Additional methodology clarity is needed for the whole tumor specimen FG-FFS. Was a shaving made from the whole\nspecimen for frozen section analysis prior to cutting the whole tumor into 3-5mm sections? Or did the final FG-FFS tissue\nsection serve as a comparator for the whole tumor specimen as well as for the fresh tissue slice identified by fluorescence?\n2. Please clarify if only one tissue per patient was used to evaluate close/positive margins via fluorescence (whole\nspecimen, fresh tissue, FG-FFS), and confirm whether pathology showed no additional close/positive margins in other tissue\nspecimens NOT used for FG-FFS. (The latter was not clear based on the results/methods.)\n3. Please clarify how correction was made for overestimation of margin width by FG-FFS relative to pathology; does this\nmean that tissues showing overestimation of margin width were excluded from the subsequent comparison? If so, how many\ntissues remained for comparison?\n4. Table 2: what is the difference between FG-FFS in whole resection specimen vs FG-FFS in fresh tissue splice? Please\nclarify in the methods.\n5. Table 2: needs further clarification on fluorescence imaging only and fluorescence-guided fresh frozen sectioning. What is\nmeant by each must be defined.\n6. Table 1: please include patient specific DOI and staging for each case, rather than a consolidated data. Deep margin\nstatus for each case, if available.\n7. How was orientation between the whole specimen and the fresh frozen samples maintained during fluorescence\nimaging? Is there a possibility of sampling error? Could the corrected overestimation of 0.8mm during fluorescence be\nrelative to the patient and tumor characteristics?\n8. Defining the terms like Fresh-frozen slice, tracer distribution, and fluorescence-imaging only, would be helpful.\n9. Please specify in abstract that the study is phase II prospective non randomized.\n10. Define parallel tagging (page 7, 11) for readers outside surgical oncology", "reviewer_2": "Comments for authors:\n1. The authors are to be congratulated on a novel approach to improving oncologically sound surgical resections.\n2. In the abstract. The statement “ necessitate additional therapy to reduce recurrence risk.” Is probably overly optimistic\nconcerning the assumption that additional therapy reduces risk following an oncologically unsound resection.\n3. The fact that up to 85% of SOC surgery leads to inadequate margins is a surprisingly high percentage. Is this an unusual\npatient population in the Netherlands or is “ inadequate margin” being defined in a different way, or is this group of surgeons’\nexperience with positive margins different from the typical rates?\n4. Under results, it would be helpful to explicitly define “ inadequate margin detection”\n5. How were the 23 patients selected for this study? In a center that no doubt sees many more than 23 OCSCC patients in\n20 months, this suggests the possibility of a selection bias.\n6. The patient population consisted of 65% female . This is a very unusual sex distribution.\n7. The statement “but final pathology showed a margin of 4.5 mm.” could use some clarification. It is understood that there\nwas FG- FFS done on fresh tissue slices at the time of the operation to determine fluorescence based margins, but was the\nfinal pathological margin status also incorporating fluorescence on the final preserved samples? That is, doing fluorescence\nevaluation on the final path specimens might change the “ gold standard” of routine H and E pathology for final margin\nadequacy .\n8. With the recent data concerning presurgical treatment of advanced OCSCC with ICI, do the authors want to comment on\nthe challenges of applying these data to this new sequential treatment paradigm?\n9. The statement that “ clinical implementation was uncomplicated” is perhaps too tunnel- visioned a focus on surgical time.\nSurely cetuximab related anaphylaxis in 2/23 patients should be considered somewhat complicated.\n10. Many of the statements throughout the manuscript seem to suggest a known benefit in terms of fluorescence – based\nmargin assessment and reduction in adjuvant treatment and therefore reduced relapses and toxicity. This is a very forward\nleaning assumption that cannot be validated by small datasets from uncontrolled trials. The notion that “increase in correct\nintraoperative margin assessment “ is an end goal in itself can only be made if an appropriately designed trial, likely with a\ncontrol arm, demonstrates that a clinically relevant outcome like OS, PFS, morbidity is both clinically and statistically\nsignificantly changed. The assumption that the omission or reduction in 70% of margin – based treatment did not\ncompromise clinically relevant endpoints is speculative, A tumor board endorsement of such reductions does not change the\nspeculative nature of this statement.", "reviewer_3": "see attached word doc. Overall an excellent paper that I would recommend accepting after considering the detailed\nsuggestions/specific edits in the word doc"}, "author_response": "Dear Editor,\nHereby, we would like to thank you and the reviewers for your time and the decision to\ninvite us to revise our work based on the reviewers’ comments. Below, our response to the\nreviewers is provided. First, we focused on the overall comments of the reviewer, and\nsubsequently responded to their detailed comments on a point-by-point basis. Besides,\nthe requested formatting instructions and editorial policies were addressed. The changes\nmade to our manuscript are highlighted in red. In the attachments, you can find two\nversions of the adapted manuscript, one with, and one without highlighted changes for\nyour convenience.\nReviewer #1\nOverall, the reviewer elegantly summarizes our study and the main findings. We are\ngrateful that the reviewer recognizes the strong case of incorporating fluorescence\nguidance into SOC surgical workflows, which can lead to better outcomes compared to\nthe current methods.\nOn a point-by-point basis the reviewer’s detailed comments have been addressed below:\n1. Additional methodology clarity is needed for the whole tumour specimen FG-FFS.\nWas a shaving made from the whole specimen for frozen section analysis prior to\ncutting the whole tumour into 3-5mm sections? Or did the final FG-FFS tissue\nsection serve as a comparator for the whole tumour specimen as well as for the fresh\ntissue slice identified by fluorescence?\nResponse: We thank the reviewer for pointing out this unclarity in the methodology.\nThe whole resection specimen was ex vivo imaged for fluorescent spots. If there were\nany present these fluorescent spots were marked with a needle. Subsequently, the\nresection specimen was cut into 3-5 mm tissue slices with this needle still in place.\nThere were no shavings taken from the margins, the whole specimen was cut in\nslices. A fresh frozen section of the tissue slice containing the needle was obtained\nand therefore served as the final FG-FFS outcome for whole resection specimen FG-\nFFS. Subsequently, fluorescence images of the tissue slices were obtained to\nestimate the closest margin based on fluorescence analysis of these slices. FFS of\nthe tissue slice with the estimated closest margin based on fluorescence imaging\nserved as the final FG-FFS outcome for fresh tissue slice FG-FFS. It should be noted\nthat the tissue slice identified by whole resection specimen fluorescence imaging\ncould therefore differ from the tissue slice identified based on the fresh tissue slice\nfluorescence imaging. In summary: The decision where to obtain tissue for an FFS\nwas determined in two different stages of the pathology analysis (see figure for visual\nrepresentation):\n1) The tissue slice for whole resection specimen FG-FFS was identified by whole\nresection specimen fluorescence imaging and marked with a needle;\n2) The tissue slice for fresh tissue slice FG-FFS was identified based on fresh tissue\nslice fluorescence imaging.\nFigure: Visual representation of the difference between whole resection specimen FG-FFS and fresh\ntissue slice FG-FFS within the same study patient. Whole resection specimen FG-FFS is based only\non whole specimen fluorescence imaging, whereas fresh tissue slice FG-FFS is based on the\nfluorescence images of fresh tissue slices.\nTo clarify the methodology, we have made the following changes to the manuscript\nand have adjusted our figure 1 to clarify the differences between the two techniques:\n• Manuscript pages 5-6, paragraph 5 (‘In-depth analysis of other potential\nintraoperative assessment points’): These points included fluorescence imaging\nonly of the whole resection specimen, fluorescence imaging only of the fresh\ntissue slices and whole resection specimen FG-FFS. Note that these assessment\npoints were all performed for every resection as the steps did not interfere with one\nanother. As shown in Figure 1, fluorescence imaging of the whole resection\nspecimen was performed first, followed by identification of spots and\ndemarcation with a needle (‘fluorescence imaging only of the whole resection\nspecimen’ in Table 2). Next, the specimen was cut into fresh tissue slices, followed\nby imaging of these tissue slices (fluorescence imaging only of fresh tissue slices)\nto identify tissue slices with potential inadequate margins. For whole resection\nspecimen FG-FFS, an FFS of the tissue slice with the demarcation needle was\nacquired; for fresh tissue slice FG-FFS, an FFS of the tissue slice with the\nestimated closest margin was acquired.\n• Manuscript page 10, paragraph 1: A spot with an SBR > 1.5 was marked on the\nwhole resection specimen using a needle, followed by a fluorescence image to\nconfirm the marker location. The location of this needle served as the reference\nfor whole resection specimen FG-FFS, as shown in Figure 1.\n• Manuscript page 10, paragraph 2: Even though not proven by FFS, an immediate\nadditional resection was performed. Altogether, two FG-FFS methods were\ninvestigated: 1) FFS of the tissue slice identified by a spot and marked with a\nneedle on whole resection specimen fluorescence imaging, and 2) FFS of the\ntissue slice identified as the slice containing the closest margin by fresh tissue\nslice fluorescence imaging.\n• Manuscript Figure 1: This figure was adapted to provide a clear overview of the\nstudy procedures, while also providing a clear distinction between whole tissue\nspecimen FG-FFS and fresh tissue slice FG-FFS approaches.\n2. Please clarify if only one tissue per patient was used to evaluate close/positive\nmargins via fluorescence (whole specimen, fresh tissue, FG-FFS), and confirm\nwhether pathology showed no additional close/positive margins in other tissue\nspecimens NOT used for FG-FFS. (The latter was not clear based on the\nresults/methods.)\nResponse: In this study, only one whole resection specimen was used per patient.\nEach specimen was evaluated using the proposed 1) whole resection specimen FG-\nFFS and 2) fresh tissue slice FG-FFS workflows, as we clarified in our response to the\nreviewer’s previous comment (reviewer 1, comment 1). In these specimens, the\nanatomical location of the closest margin was correctly identified using fresh tissue\nslice FG-FFS. In 19 of the 20 patients, the margin status (i.e. inadequate (≤ 5mm) or\nfree (> 5 mm)) always corresponded to final histopathology. In one patient, FG-FFS\ndeemed the margin to be free, but final pathology showed an inadequate margin (4.5\nmm). No other inadequate margins were found on other anatomical locations in\nthese specimens by final pathology analysis.\nTo prevent any misunderstanding, we have revised the manuscript as follows:\n• Manuscript, page 5, paragraph 1 (‘Surgical outcomes and clinical implication\nof intraoperative FG-FFS decision making’): Figure 1 gives an overview of the\nintraoperative specimen handling pipeline. Standard of care surgery resulted in\none tumour specimen per patient. This standard of care regular surgical resection\nand subsequent pathology analysis, prior to any study intervention, would have\nresulted in ten (50%) inadequate margins (Figure 2A).\n• Manuscript, page 5, paragraph 1 (‘Surgical outcomes and clinical implication\nof intraoperative FG-FFS decision making’): This resulted in a sensitivity,\nspecificity and accuracy of 90.0%, 100.0% and 95.0%, respectively. Besides the\nidentified closest margins by FG-FFS, no other locations with inadequate margins\nwere identified after complete pathology analysis.\n3. Please clarify how correction was made for overestimation of margin width by FG-\nFFS relative to pathology; does this mean that tissues showing overestimation of\nmargin width were excluded from the subsequent comparison? If so, how many\ntissues remained for comparison?\nResponse: We thank the reviewer for pointing out the ambiguity in the interpretation\nof the margin overestimation by FG-FFS. The intraoperative margins were\ndetermined on H&E stained fresh frozen sections and compared to formalin fixed\nfinal H&E histopathology and thus could only be compared after all study procedures\nwere completed. For the margin comparison itself, all patients with a known\n(absolute) margin in millimetres after both fresh frozen sectioning and formalin fixed\nfinal histopathology analysis, could be included. One patient was excluded from this\nanalysis, as no exact margin in mm was reported (a margin of >5.1 mm was reported),\nthe remaining nineteen patients were included. The Bland-Altman plot (figure 3) in\nour manuscript shows that the mean difference between the corresponding margin\ndetermined by fresh frozen sectioning and final pathology is 0.8 mm. Correction for\nthis overestimation was performed by subtracting 0.8 mm from the initial fresh frozen\nsection margin. These ‘new’ fresh frozen margins could subsequently be compared\nto final pathology margins.\nWe have added the following text to our manuscript to further specify the method for\ncorrection of the margin width overestimation:\n• Manuscript, page 5, paragraph 4 (‘Correlation of margin determination with\nfresh tissue slice FG-FFS and final pathology analysis’): An important point to\nconsider is whether the intraoperative margin provided by pathology based on FG-\nFFS is comparable to the margin provided after final pathology analysis, when the\ntissue is formalin fixed and paraffin embedded. In a retrospective analysis, these\nmargin widths obtained using FG-FFS were compared to the closest margin on\nfinal pathology analysis after formalin fixation and paraffin embedding. For this\nanalysis, all patients with at least one exact FFS margin in mm and final pathology\nmargin width in mm of that same anatomical location, could be included.\nNineteen patients were included and one patient was excluded, due to a missing\nexact FFS margin (recorded only to be greater than 5.1 mm). Therefore, this patient\nwas excluded from this comparison. For the remaining 19 patients, a A significant\ndifference (p < 0.001) was found between the FG-FFS margin estimate and final\npathology margin. Bland-Altman analysis revealed an average margin width\noverestimation of 0.8 mm by FG-FFS, as shown in Figure 3. A post-hoc correction\nfor this overestimation was performed to evaluate the potential impact on this\nobserved difference. After correcting FG-FFS margins for this overestimation, by\nsubtracting 0.8 mm from each determined FFS margin, no significant difference (p\n= 0.591) remained between the FG-FFS margin and final pathology margin.\n4. Table 2: what is the difference between FG-FFS in whole resection specimen vs FG-\nFFS in fresh tissue slice? Please clarify in the methods.\nResponse: We agree with the reviewer, in concordance with comment 1, that this\ntable might be difficult to interpret. The main difference between the two methods is\nthe fluorescence imaging moment. Whole resection specimen FG-FFS is based on\nfluorescence imaging of the whole resection specimen and thereby detection of\nspots as described in comment 1. Tissue slice FG-FFS is based on fluorescence\nimaging of the fresh tissue slices. As for the difference in fluorescence imaging\nmoment, the tissue slice selected for FFS could differ and therefore results of both\nmethods varied.\nTo clarify the differences between both methods, the manuscript has been changed\n(see below). We also kindly refer to the figure provided in this document in response\nto comment 1.\n• Manuscript page 10, paragraph 1: A spot with an SBR > 1.5 was marked on the\nwhole resection specimen using a needle, followed by a fluorescence image to\nconfirm the marker location. The location of this needle served as the reference\nfor whole resection specimen FG-FFS, as shown in Figure 1.\n• Manuscript page 10, paragraph 2: Even though not proven by FFS, an immediate\nadditional resection was performed. Altogether, two FG-FFS methods were\ninvestigated: 1) FFS of the tissue slice identified by a spot and marked with a\nneedle on whole resection specimen fluorescence imaging, and 2) FFS of the\ntissue slice identified as the slice containing the closest margin by fresh tissue\nslice fluorescence imaging.\n• Manuscript figure 1: This figure was adapted to provide a clear overview of the\nstudy procedures, while also providing a clear distinction between whole tissue\nspecimen FG-FFS and fresh tissue slice FG-FFS approaches.\n5. Table 2: needs further clarification on fluorescence imaging only and fluorescence-\nguided fresh frozen sectioning. What is meant by each must be defined.\nResponse: In line with previous comments, we agree that further clarification on\nterminology is necessary. First we would like to define the terms used in the table.\nFluorescence imaging only’ indicates margin classification as ‘tumour-free’ or\n‘inadequate’ by fluorescence imaging only (i.e. not followed by FFS). For the whole\nresection specimen, margins are deemed inadequate in case of a spot with a TBR\n>1.5. For the fresh tissue slice approach, a healthy margin, based on fluorescence\npatterns on the tissue slices, would be judged inadequate if the fluorescence margin\nis estimated to be situated <5.1 mm from the resection border.\nThe FG-FFS methods were used as described in the response to comment 1. Briefly,\nthe above-mentioned ‘fluorescence imaging only’ methods were both followed by an\nFFS of the selected areas to further increase specificity.\nBoth the margins determined by fluorescence imaging only and fluorescence\nimaging followed by FFS (FG-FFS) were subsequently compared to ‘gold\nstandard’/final histopathology to calculate the sensitivity, specificity, accuracy,\nPPV and NPV. Table 2 provided in the manuscript gives an overview for direct comparison\nbetween the fluorescence imaging only approach (currently used in literature) and\nour new FG-FFS approach.\nThe definition of the terms used in this table were added in the manuscript and to the\ndescription of Table 2:\n• Manuscript pages 5-6, paragraph 5 (‘In-depth analysis of other potential\nintraoperative assessment points’): These points included fluorescence\nimaging only of the whole resection specimen, fluorescence imaging only of the\nfresh tissue slices and whole resection specimen FG-FFS. Note that these assessment\npoints were all performed for every resection as the steps did not interfere with one\nanother. As shown in Figure 1, fluorescence imaging of the whole resection\nspecimen was performed first, followed by identification of spots and\ndemarcation with a needle (‘fluorescence imaging only of the whole resection\nspecimen’ in Table 2). Next, the specimen was cut into fresh tissue slices, followed\nby imaging of these tissue slices (fluorescence imaging only of fresh tissue slices)\nto identify tissue slices with potential inadequate margins. For whole resection\nspecimen FG-FFS, an FFS of the tissue slice with the demarcation needle was\nacquired; for fresh tissue slice FG-FFS, an FFS of the tissue slice with the\nestimated closest margin was acquired.\n• Manuscript page 6, Table 2: The possible assessments consist of fluorescence\nimaging only and fluorescence-guided fresh frozen sectioning (FG-FFS) of the\nwhole resection specimen and fresh tissue slices. Fluorescence imaging only of\nthe whole resection specimen identified spots, indicating a potential inadequate\nmargin. This location was marked with a needle. After the specimen was cut into\nfresh tissue slices, the tissue slice with the needle was used for whole resection\nspecimen FG-FFS. Additionally, fluorescence images of all fresh tissue slices were\nalso acquired (fluorescence imaging only of fresh tissue slices). Tissue slices with\nthe closest identified margin were subsequently used for fresh tissue slice FG-FFS.\n6. Table 1: please include patient specific DOI and staging for each case, rather than a\nconsolidated data. Deep margin status for each case, if available.\nResponse: We understand the request of the reviewer to add patient specific depth\nof invasion and deep margin status if available, and agree this is important\ninformation. We have added this information to Table 1 (Manuscript page 4, Table\n1).\n7. How was orientation between the whole specimen and the fresh frozen samples\nmaintained during fluorescence imaging? Is there a possibility of sampling error?\nCould the corrected overestimation of 0.8 mm during fluorescence be relative to the\npatient and tumour characteristics?\nResponse: Maintaining orientation during pathology processing is an important step\nas the reviewer clearly points out. Several precautions have been made to realize\nthis, consisting of:\n- Paired tagging to preserve anatomical orientation during fluorescence imaging and\npathology processing, conform previous reported studies [20];\n- Recording the exact cutting planes for tissue slicing at the Pathology Department\nreal-time by drawing lines of the cutting plane on a print-out of an image of the\nwhole resection specimen to provide feedback on the exact location of the tissue\nslice used for FFS;\n- Reporting the exact location of an inadequate margin in the FFS by the pathologist\nto the surgeon, based on the above described methods.\n- Continuity of orientation tracking by two dedicated researchers being present\nduring all study procedures: present from start of surgery, delivered excised tissue\nto pathology department, performed fluorescence imaging, present during fresh\ntissue slicing, selected slices for FFS, present during margin feedback to surgeon\nby pathologist, and finally checked region for re-excision (if applicable).\nTherefore, we believe the risk of sampling error has been minimized. We realize this\nprocess is time consuming and requires staff present at all processing steps of the\ntissue specimen. Therefore, we are currently investigating the use of 3D imaging\ntechniques to provide more detailed information on orientation without the need\nof staff/researchers being continuously present. Besides, 3D images will enhance\nthe discussion between surgeon and pathologist on the location of possible\ninadequate margins [43].\n[20] Aaboubout, Y. et al. Intraoperative assessment of resection margins in oral cavity cancer: This\nis the way. Journal of Visualized Experiments 2021, (2021).\n[43] Fassler, C. et al. Digital mapping of resected cancer specimens: The visual pathology report. J\nPathol Inform 15, (2024).\nRegarding the point on whether the corrected overestimation of 0.8 mm during\nfluorescence could be relative to the patient and tumour characteristics; We\nbelieve that if assessments were based solely fluorescence, such characteristics\ncould indeed influence the degree of overestimation. This is because tissue-\nspecific optical properties, affecting light scattering, can make it more difficult to\naccurately determine the true tumour boundaries. This is the mean reason why\nFFS was incorporated into the workflow. With the addition of FFS, we believe that\nthe observed overestimation is not attributable to patient or tumour\ncharacteristics, but is more likely the result of the processing steps (freezing versus\nformalin fixation and paraffin embedding). Sampling error in the comparison\nbetween frozen sectioning and final histopathology results were kept to a minimum\nas the frozen sections were first taken from the tissue slices. Then the tissue slice\nwas formalin fixed and paraffin embedded, after which sectioning continued at the\nsame section-plane. This ensures the possibility of adequate comparison. This\nwas also addressed in the response to comment 3, which we refer to for the\nadjustments made to the manuscript.\nBased on the reviewer’s comment on the orientation, the following changes have\nbeen made to the manuscript:\n• Manuscript page 10, paragraph 2: An additional resection was performed if\ndeemed clinically feasible and safe. Determining the exact location for additional\nresection was essential for effective treatment. To ensure this, the following\nmeasures were taken: continuous orientation tracking by two dedicated researchers\nthroughout tissue imaging and processing, paired tagging to preserve anatomical\norientation, documentation of cutting planes during tissue slicing to enable accurate\nrelocation of tissue slices in the original resection specimen, and collaborative\nreporting by pathologist and researchers to the surgeon of the precise location of any\ninadequate margins identified in the FFS.localised by the previously described\npaired tagging strategy.\n8. Defining the terms like Fresh-frozen slice, tracer distribution, and fluorescence-\nimaging only, would be helpful.\nResponse: We agree with the reviewer that defining these terms would be helpful for\nreaders, especially the ones not involved in surgical oncology or fluorescence\nimaging. Below, these terms are defined point-to-point:\n- Fresh-frozen section: This is a 4 µm tissue section prepared from a sample (tissue\nslice) which has been rapidly frozen after fresh slicing using a cryostat. This frozen\nsection is subsequently H&E stained to assess tumour margins.\n- Tracer distribution: The spatial localisation of the fluorescence probe imaged with\nthe PEARL Trilogy.\n- Fluorescence imaging only: This term has been described in point 5 raised by\nreviewer 1.\nBased on this comment, and the previous comments, the definition of these terms has\nbeen included in the manuscript as follows:\n• Manuscript page 3, paragraph 3: Fresh frozen sectioning (FFS) of the excised\ntumour specimen is the standard for evaluating margins during surgery.15–18 An\nFFS procedure involves preparation of a 4 µm tissue section from a tissue slice,\nwhich has been rapidly frozen after fresh sectioning using a cryostat. This\nfrozen section is subsequently haematoxylin & eosin (H&E) stained to assess\ntumour margins.\n• Manuscript page 4, paragraph 1: Additionally, tracer distribution (i.e. the\nspatial localisation of the cetuximab-800CW probe imaged with a\nfluorescence camera) was evaluated and compared to pathology margins.\n• Manuscript pages 5-6, paragraph 5 (‘In-depth analysis of other potential\nintraoperative assessment points’): These points included fluorescence\nimaging only of the whole resection specimen, fluorescence imaging only of\nthe fresh tissue slices and whole resection specimen FG-FFS. Note that these\nassessment points were all performed for every resection as the steps did not\ninterfere with one another. As shown in Figure 1, fluorescence imaging of the\nwhole resection specimen was performed first, followed by identification of\nspots and demarcation with a needle (‘fluorescence imaging only of the whole\nresection specimen’ in Table 2). Next, the specimen was cut into fresh tissue\nslices, followed by imaging of these tissue slices (fluorescence imaging only of\nfresh tissue slices) to identify tissue slices with potential inadequate margins.\nFor whole resection specimen FG-FFS, an FFS of the tissue slice with the\ndemarcation needle was acquired; for fresh tissue slice FG-FFS, an FFS of the\ntissue slice with the estimated closest margin was acquired.\n9. Please specify in abstract that the study is phase II prospective non randomized.\nResponse: We have added the requested information to our abstract. Additionally\nwe have reduced the wordcount back to 150:\n• Manuscript, page 2: In this phase II prospective, non-randomised trial, we\ninvestigated a novel approach for intraoperative approach inadequate margin\ndetection for detecting inadequate margins.\n10. Define parallel tagging (page 7, 11) for readers outside surgical oncology\nResponse: We agree with the reviewer that the term ‘parallel tagging’ should be\ndefined for readers outside surgical oncology. In the manuscript, both ‘parallel\ntagging’ and ‘paired tagging’ were mistakenly used for the same concept. For clarity,\nwe have adjusted all these terms to ‘paired tagging’. We agree with the reviewer that\nthe definition provided in our manuscript lacks clarity. To clarify: during paired\ntagging, stitches marked with clips were placed in the mucosa of both the surgical\ncavity and the resection specimen on at least four corresponding anatomical\nlocations. Stiches at matching anatomical locations were marked with the same\nnumber of clips. Paired tagging ensured accurate relocation of the anatomical site\nwithin the surgical cavity in case of an inadequate margin. This method aligns with a\nsimilar approach described in the study performed at Erasmus University Medical\nCenter in 2021 [20].\n[20] Aaboubout, Y. et al. Intraoperative assessment of resection margins in oral cavity cancer: This is\nthe way. Journal of Visualized Experiments 2021, (2021).\nTo increase clarity, we broadened the definition to:\n• Manuscript, page 9, paragraph 4 (‘Intraoperative margin assessment’):\nDuring tumour resection, paired tagging was performed for intraoperative\norientation purposes. Paired tagging consists of stitches marked with clips\nplaced in the mucosa of both the surgical cavity and the resection specimen\non at least four corresponding anatomical locations. Stiches at matching\nanatomical locations were marked with the same number of clips. Paired\ntagging ensured accurate relocation of the anatomical site within the surgical\ncavity in case of an inadequate marginAfter initial mucosal incision, before\nremoval of the tumour, matching of paired stitches were placed in the mucosa\non both sides of the incision. This resulted in matching markings on the excised\ntumour specimen and in the wound bed (Figure 1.1).20\nReviewer #2\nOn a point-by-point basis the comments of the reviewer are addressed below:\n1. The authors are to be congratulated on a novel approach to improving\noncologically sound surgical resections.\nResponse: We sincerely thank the reviewer for the kind words and for recognizing\nthe novelty of our approach to improve oncological sound surgical resections.\n2. In the abstract. The statement “necessitate additional therapy to reduce\nrecurrence risk.” Is probably overly optimistic concerning the assumption that\nadditional therapy reduces risk following an oncologically unsound resection.\nResponse: We agree with the reviewer that additional therapy will likely not\ncompletely prevent recurrence in patients with oncological unsound resections,\nespecially in case of positive margins (<1 mm). Several studies have demonstrated\na benefit of additional therapy after inadequate margins, but adequate margins\nduring primary surgery will contribute substantially more to reduction in\nrecurrence risk.\nCombined with the comments raised by reviewer 3, we have therefore rephrased\nthis statement in the abstract:\n• Manuscript page 2: Surgery is the primary treatment for oral cancer, with tumour-\nfree margins (> 5 mm) directly improving prognosis. Inadequate margin rates are\nhigh and necessitate additional therapy to reduce recurrence risk. Tumour-free\nmargins (> 5 mm) in head and neck malignancies improve prognosis, and may\nimpact the need for adjuvant treatment.\n3. The fact that up to 85% of SOC surgery leads to inadequate margins is a\nsurprisingly high percentage. Is this an unusual patient population in the\nNetherlands or is “ inadequate margin” being defined in a different way, or is this\ngroup of surgeons’ experience with positive margins different from the typical\nrates?\nResponse: We understand the concerns raised by the reviewer, as 85% seems a\nlarge number of inadequate margins. Inadequate margins in this context are\ndefined as margins < 5 mm. Generally, head and neck cancer studies report\ninadequate margin rates up to 70%. However, the 85% reported in the introduction\nrelates to a study performed retrospectively in two academic, tertial referral\ncentres in the Netherlands. In our centre, inadequate margin rates of 51% and 65%\nare reported, comparable to the number of inadequate margins reported in the\ncurrent study.[1,2]\n[1] van Oorschot HD, Hardillo JA, Baatenburg de Jong RJ; Dutch Head and Neck Audit-Oral Cavity\nCollaborator Group. Cohort profile: the Dutch oral cavity cancer cohort. BMJ Open. 2025. doi:\n10.1136/bmjopen-2024-092467\n[2] Sidorenkov G, Nagel J, Meijer C, et al. The OncoLifeS data-biobank for oncology: a\ncomprehensive repository of clinical data, biological samples, and the patient's perspective. J\nTransl Med. 2019;17(1):374. doi:10.1186/s12967-019-2122-x\nWe agree with the reviewer that the inadequate margin rate of up to 85% mentioned\nin our introduction is rather high. Therefore, in concordance with the comments\nmade by reviewer 3, we decided to delete the last sentence of the first paragraph\nof the introduction.\n• Manuscript, page 3, first paragraph: A retrospective study, performed by two\ntertiary centres in the Netherlands, reported inadequate margins (< 5 mm) of\nup to 85%.8\n4. Under results, it would be helpful to explicitly define “inadequate margin\ndetection”\nResponse: We agree with the reviewer that the reported term is ambiguous. Our\ndefinition for this term is the following: ‘the number of classified inadequate\nmargins by the various methods investigated in the trial (fluorescence imaging only\nof the whole resection specimen and fresh tissue slices and FG-FFS of both).\nTo elucidate this, we have adjusted the term ‘inadequate margin detection’ to\n‘detection of inadequate margins’.\n• Manuscript page 2, Abstract (two times): In this phase II prospective, non-\nrandomised trial, we investigated a novel approach for intraoperative approach\ninadequate margin detection for detecting inadequate margins. Fluorescence\nimaging using the tumour-targeting fluorescent tracer cetuximab-\nIRDye800CW guided fresh frozen sectioning (FG-FFS) to identify inadequate\nmargins. fluorescence guidance of fresh frozen sectioning (FG-FFS) Primary\nendpoints were intraoperative inadequate margin detection detection of\ninadequate margins, and additional resections, and their clinical implications.\n• Manuscript page 4, paragraph 1: The primary endpoints of this phase two\nstudy were the detection rates of inadequate margins by fluorescence imaging\nonly and FG-FFS, including the number of additional resection based on FG-\nFFS.\n• Manuscript page 5, paragraph 1: The first primary endpoint of this study\nincluded the evaluation of inadequate margin detection detection of\ninadequate margins by FG-FFS. FG-FFS based on the fresh tissue slices\ncorrectly identified margins in 95% of patients (19 out of 20).\n5. How were the 23 patients selected for this study? In a center that no doubt sees\nmany more than 23 OCSCC patients in 20 months, this suggests the possibility of\na selection bias.\nResponse: We realize the concern raised by the reviewer. The reviewer is correct\nthat our centre sees many more than 23 OSCC patients in 20 months. First, in this\nstudy we had the limitation, as discussed, of only including tumours without bone\ninvolvement. This already eliminated approximately half of the patients eligible for\nsurgery. Additionally, patients did not always wanted to participate and besides\nthis study, several other studies with contradicting inclusion criteria were\nconducted in the same period. Therefore, the current study could not include all\navailable patients and patients were randomly subdivided between studies. As\nshown in Table 2, based on the pTN-stage and tumour locations, this study cohort\nremains representative of patients with oral squamous cell carcinoma without\nbone involvement.\n6. The patient population consisted of 65% female. This is a very unusual sex\ndistribution.\nResponse: We agree with the reviewer that generally OSCC is more common in\nmale patients. The predominance of female patients reflects the composition of\npatients referred to our centre during the study period rather than any deliberate\nselection. Additionally, three male patients were excluded due to anaphylaxis in\ntwo patients and deviations from the intraoperative imaging protocol in the third,\ncontributing to the higher proportion of included female patients.\nWe have added this consideration to the discussion of our manuscript:\n• Manuscript page 8, paragraph 1: The addition of FFS substantially increased\nspecificity of margin assessment compared to fluorescence imaging only of\nfresh tissue slices (Table 2). Interestingly, the patient population in this study\nconsisted of 65% female patients, whereas male patients are known to have\nan increased risk for developing head and neck cancer.2 The predominance of\nfemale patients reflects the composition of patients referred to our centre\nduring the study period, rather than any deliberate selection. Additionally,\nthree male patients were excluded due to anaphylaxis and deviations from the\nintraoperative imaging protocol.\n7. The statement “but final pathology showed a margin of 4.5 mm.” could use some\nclarification. It is understood that there was FG- FFS done on fresh tissue slices at\nthe time of the operation to determine fluorescence based margins, but was the\nfinal pathological margin status also incorporating fluorescence on the final\npreserved samples? That is, doing fluorescence evaluation on the final path\nspecimens might change the “ gold standard” of routine H and E pathology for final\nmargin adequacy.\nResponse: We thank the reviewer for addressing this unclarity. In this study,\nmargins by FG-FFS were based on FFS margins of selected tissue slices using\nfluorescence imaging. Final pathology margins were based solely on H&E\npathology sections obtained after formalin fixation of the tissue specimen (i.e.\nstandard of care). So, in this case, the margin of 4.5 mm was based on H&E\nassessment by the pathologist only. For final pathology assessment, we believe\nmacroscopic fluorescence imaging cannot change this gold standard as\nmacroscopic fluorescence imaging is not capable of detecting individual cancer\ncells that can be identified with H&E or other stainings.\nThe following changes were made to the manuscript:\n• Manuscript, page 10, paragraph 3: Following the FFS procedure, the fresh\ntissue slices were formalin fixed for at least 24h, followed by paraffin\nembedding and 4 µm sectioning for H&E staining, according to standard of\ncare. Based on the standard of care H&E staining assessment, margins before\nand after potential additional resections were assessed by a pathologist.\n8. With the recent data concerning presurgical treatment of advanced OCSCC with\nICI, do the authors want to comment on the challenges of applying these data to\nthis new sequential treatment paradigm?\nResponse: We indeed are aware of the novel treatment strategies concerning neo-\nadjuvant treatment using immunotherapy or even induction radiotherapy\ncombined with immunotherapy. We believe it is important to emphasize that these\napproaches are not mutually exclusive. For example, previous studies have shown\nthat neoadjuvant therapy can induce a complete response, but in certain groups\nmay also result in ‘tumour crumbling’, due to heterogenous treatment response.\nFluorescence imaging may provide additional value by enabling the detection and\nremoval of residual tumour foci, undetectable by eye. Additionally, there will be a\ngroup of patients that initially were not eligible for surgery, but due to restaging they\nmay be curable by surgical removal. Therefore, we believe that still a very large\ngroup might benefit from the new fresh tissue slice FG-FFS approach.\nThis discussion has been added to the manuscript:\n• Manuscript page 8, paragraph 2: Orientation and intraoperative feedback from\npathologist to the surgeon could be enhanced further by addition of upcoming\ndigital 3-dimensional specimen mapping.44 Recent advances in oncology\ntreatment consist mainly of the use of immunotherapy (e.g. immune checkpoint\ninhibitors). We believe the use of the described FG-FFS technique is not mutually\nexclusive. Previous studies on neo-adjuvant treatment regimens suggest some\npatients obtain a complete response, while other patient groups only show a\npartial, heterogeneous, response. This second group still requires surgical\ntreatment, which may prove more complex due to presence of multiple tumour\nfields, difficult to distinguish by eye. Additionally, a group of patients who are\ninitially ineligible for surgery may be downstaged after immunotherapy and thereby\nbecome eligible. Therefore, we expect a large number of patients may benefit from\nthe new fresh tissue slice FG-FFS.\n9. The statement that “clinical implementation was uncomplicated” is perhaps too\ntunnel-visioned a focus on surgical time. Surely cetuximab related anaphylaxis in\n2/23 patients should be considered somewhat complicated.\nResponse: We agree with the reviewer that the anaphylaxis is an undesirable side-\neffect of cetuximab. Cetuximab, and thereby cetuximab-IRDye800CW, is well\nknown for this side effect, that occurs in >10% of patients receiving this antibody.\nTherefore, taking this high change of anaphylaxis into account, the administration\nof cetuximab and cetumixmab-IRDye800CW were performed under strict\nmonitoring of patients. This enabled fast intervention/medicine therapy in case a\npatient suffered from an allergic reaction. All patients with an allergic reaction fully\nrecovered after therapy and the planned surgical procedures could continue. It is\nimportant to note that the statement “clinical implementation was\nuncomplicated” refers to implementation of fresh tissue slice FG-FFS into the\nclinical workflow, that can be performed with any suitable tracer currently\navailable.\nTo clarify the intentions of the statement, we added the following text to our\nmanuscript:\n• Manuscript page 8, paragraph 3: . FG-FFS based additional resections\ndecreased inadequate margins from 50% to 15%, resulting in a subsequent\nomittance or reduction of margin-based adjuvant treatment in 70% of the\npatients who would have otherwise required additional treatment. Considering\nthe known side-effects of cetuximab such as anaphylaxis, clinical\nimplementation was uncomplicated, as the technique did not increase surgery\ntime and direct margin-feedback could be provided to the surgeon was\npossible.\n10. Many of the statements throughout the manuscript seem to suggest a known\nbenefit in terms of fluorescence – based margin assessment and reduction in\nadjuvant treatment and therefore reduced relapses and toxicity. This is a very\nforward leaning assumption that cannot be validated by small datasets from\nuncontrolled trials. The notion that “increase in correct intraoperative margin\nassessment” is an end goal in itself can only be made if an appropriately designed\ntrial, likely with a control arm, demonstrates that a clinically relevant outcome like\nOS, PFS, morbidity is both clinically and statistically significantly changed. The\nassumption that the omission or reduction in 70% of margin – based treatment did\nnot compromise clinically relevant endpoints is speculative, A tumour board\nendorsement of such reductions does not change the speculative nature of this\nstatement.\nResponse: We agree that based on our dataset we cannot draw any conclusions\non (disease-free) survival or morbidity and that such endpoints can only be\nvalidated in an appropriately designed randomised control trial. We are currently\ndesigning such a study, based on the found results. In the study reported in this\nmanuscript, our intention was not to suggest proven clinical benefit, but rather\nreport that fluorescence guidance increased intraoperative margin assessment\naccuracy and reduction in adjuvant treatment. The main conclusion is that we\nwere able the convert a rather large percentage of inadequate to tumour-free\nmargins. We fully acknowledge that the clinical impact remains speculative at this\nstage.\nTherefore we have rephrased the following in the manuscript:\n• Manuscript page 3, paragraph 4: With tThis combined approach we aim to\nuseenables fluorescence imaging to guide as guidance for intraoperative\npathology assessment beyond rather than relying solely on clinical suspicion\nalone, enabling allowing for timely recognition and intraoperative adjustment\nof inadequate marginsand decreasing the necessity for adjuvant treatment.\n• Manuscript page 8, paragraph 2: As decided by the multidisciplinary tumour\nboard, aAdjuvant treatment was substantially decreased (70.0%), reducing\npatient morbidity. Following the increased radical resection rate, an increased\n(disease-free) survival rate is could be expected. This will be monitored both in\nthe population of the current study and in a larger randomised control trial,\ncurrently being designed. should be examined during the coming years and in\na larger patient population.13\n• Manuscript page 8, paragraph 3: Thus, margin assessment using FG-FFS\nsubstantially increases correct intraoperative margin assessment and enables\nadditional resections in case of an inadequate margin, reducing patient\nburden.\nReviewer #3\nsee attached word doc. Overall an excellent paper that I would recommend accepting\nafter considering the detailed suggestions/specific edits in the word doc\nThe point of this paper is that imaging alone is not as good as using imaging to tailor frozen\nsection margins. Some papers have already said that and should be specifically reviewed,\nwith your stating what your paper adds. (see 1st 3 refs) This mss Introduction might be\nenhanced with a discussion of prior papers that use imaging to tailor frozen sections. I\nthink the three below perhaps should be discussed. (see also comment at end regarding\nexpanding on the Imaging (w/o frozen sections) in the Introduction as readers may not be\nfamiliar.\nResponse: We thank the reviewer for the compliments and the suggestions provided.\nBelow we have answered the questions and suggestions raised by the reviewer on a point-\nto-point basis, followed by the textual suggestions of the reviewer.\n1. The point of this paper is that imaging alone is not as good as using imaging to tailor\nfrozen section margins. Some papers have already said that and should be\nspecifically reviewed, with your stating what your paper adds. (see 1st 3 refs)\nResponse: We thank the reviewer for pointing out an extra paper discussing the\npossibility to add fresh frozen sectioning to fluorescence imaging.\nBased on this comment, we have added a discussion of the current use of fresh\nfrozen sectioning and fluorescence imaging to our introduction:\n• Manuscript page 3, paragraphs 3 and 4: Fresh frozen sectioning (FFS) of the\nexcised tumour specimen is the standard for evaluating margins during surgery.15–\nslice, which has been rapidly frozen after fresh sectioning using a cryostat. This\nfrozen section is subsequently haematoxylin & eosin (H&E) stained to assess\ntumour margins. Although intraoperative FFS provides a high specificity for margin\nassessment, sampling error substantially reduces the sensitivity of this\ntechnique.19–22 Various approaches for improved intraoperative identification of\ninadequate margins have been investigated.23,24 Fluorescence-guided surgery\nusing tumour targeted tracers has already proven to be beneficial in head and neck\ncancer certain cancer treatments, such as ovarian and brain cancer.25,26 For oral\ncancer, fluorescence-guided surgery has been identified as a promising technique\nfor intraoperative margin assessment as well.27–31 The use of cetuximab-\nIRDye800CW to detect oral cancer cells was demonstrated to be is feasible, since\nthese cells overexpress epidermal growth factor receptor.28 In the largest\nfluorescence-guided surgery trial in oral cancer patients thus far, our group has\nshown that fluorescence imaging using cetuximab-IRDye800CW can identify\ntumour-positive margins. Additionally, the study showed that fluorescence signals\nare weaker when the overlying tissue is thicker, even when a close margin is still\npresent. Therefore, fluorescence imaging is less reliable for accurately\ndetermining margin width.30,31 Thus However, the intensity of the fluorescent signal\ndoes not reliably correlate with the margin width using current fluorescence\nimaging approaches.31 To overcome this issue, a novel approach, combining\nsupplementing fluorescence imaging with intraoperative fresh frozen sectioning\n(i.e. fluorescence-guided fresh frozen sectioning (FG-FFS)), has been developed.\nAlthough the combination of fluorescence imaging and fresh frozen sectioning has\nbeen suggested previously, this has not been systematically investigated.29–31\nKrishnan et al. examined the localisation of fresh frozen sectioning based on\nclinical suspicion compared to a sentinel margin approach, which uses the areas of\nhighest fluorescence intensities as indicators of potentially inadequate\nmargins. They found a strong correlation between these approaches, however they\ndid not investigate margins not suspected to be at risk.30\nIn this phase two intervention study, we demonstrate that build on our previous\nfindings and aim to combine the strengths of fluorescence imaging of the whole\nresection specimen and fresh tissue slices containing cetuximab-IRDye800CW\ntosuccesfully localize a margins at risk, andwhile FFS to provides a margin of that\nspecific location margin distance measurements of these margins at risk in\nmillimetres. With tThis combined approach we aim to useenables fluorescence\nimaging to guide as guidance for intraoperative pathology assessment beyond\nrather than relying solely on clinical suspicion alone, enabling allowing for timely\nrecognition and intraoperative adjustment of inadequate marginsand decreasing\nthe necessity for adjuvant treatment.\n2. Figures:\n• Fig 1 delete, it is sufficient to simply in a sentence state that results are\navailable within x minutes\nResponse: We agree with the reviewer that in the current form, the figure did\nnot add to the paper. Due to the unclarities pointed out by reviewer 1, we have\nadjusted the paper in general, and Figure 1 specifcally, to more clearly show\nthe difference between whole resection specimen FG-FFS and fresh tissue\nslice FG-FFS. Therefore, we think that in the new form, the figure will be a good\naddition to the paper, and will provide more clarity on the concepts introduced.\n• Fig 2 delete: this too can be said in words\nResponse: As the reviewer suggests, this figure already has been described in\nthe corresponding paragraphs of the paper (‘Surgical outcomes and clinical\nimplication of intraoperative FG-FFS decision making’). We do believe that this\nfigure adds information and clarity to the manuscript, especially because this\nfigure summarizes the important main findings of this paper. For readability and\ntargeting a broader audience, we therefore think it is important to retain this\nfigure. However, if the editor believes this figure is redundant, we will delete this\nfigure.\n• Fig 3: ok\nResponse: We will keep this figure as the reviewer suggests.\n• Fig 4 is ok, though I am not sure it actually adds anything. The reader\nunderstands frozen sections\nResponse: We believe this figure will provide a visual explanation for readers\nnot familiar with fluorescence imaging. Indeed, most readers will probably\nunderstand frozen sectioning, but to be able to inform a larger audience on\nfluorescence imaging we think this image will be informative.\n3. Results ok, though in Table 22 round the % to nearest whole # and eliminate the\ndecimal point.\nResponse: We have adjusted the Table 2 to round to the nearest number.\n4. As you of course know, fluorescent guidance has already been shown to be useful,\nwith an emerging role in assessing surgical margins in head and neck cancer. It\nprovides intraoperative evaluation of tumor tissue, particularly at deep margins\nthat may otherwise be challenging. Multiple studies, reviewed below, demonstrate\nthat fluorescence-guided surgery using tumor-specific agents such as anti-EGFR\nantibodies conjugated to near-infrared dyes (e.g., cetuximab-800CW,\npanitumumab-IRDye800CW), can identify close margins with high sensitivity and\nspecificity, and can direct targeted frozen section analysis to areas at highest risk\nfor residual disease. In vivo imaging of the surgical bed can show residual disease\nnot apparent under white light. Hence, fluorescent guidance has already been\nshown to enhance intraoperative detection of margins in head and neck cancer\nsurgery, possibly improve oncologic outcomes by facilitating more accurate\ncomplete resections.\nResponse: We thank the reviewer for providing an elaborate response on the use\nof various tracers to guide frozen section analysis. We have added more\ninformation on the combination of fluorescence imaging and FFS in the\nintroduction, as discussed in the response to point 1.\n5. Shown below are the changes made to the manuscript based on the suggestions\nprovided by the reviewer:\n• Manuscript page 2, abstract Surgery is the primary treatment for oral cancer,\nwith tumour-free margins (> 5 mm) directly improving prognosis. Inadequate\nmargin rates are high and necessitate additional therapy to reduce recurrence\nrisk. Tumour-free margins (> 5 mm) in head and neck malignancies improve\nprognosis, and may impact the need for adjuvant treatment. In this phase II\nprospective, non-randomised trial, we investigated a novel approach for\nintraoperative approach inadequate margin detection for detecting\ninadequate margins. Fluorescence imaging using the tumour-targeting\nfluorescent tracer cetuximab-IRDye800CW guided fresh frozen sectioning (FG-\nFFS) to identify inadequate margins. fluorescence guidance of fresh frozen\nsectioning (FG-FFS) Primary endpoints were intraoperative inadequate margin\ndetection detection of inadequate margins, and additional resections, and\ntheir clinical implications. Additionally, fFeasibility of FG-FFS implementation\ninto the current clinical workflow was also assessed. Standard of careInitial\nsurgery led to inadequate margins in ten of the twenty included patients. Using\nFG-FFS, margins were correctly classified margins in nineteen patients. Study\nrelated Additional resections increased the tumour-free margin rates from 50%\nto 85%, anatomical boundaries prevented additional resection in one patient.\nIn seven patients, intraoperative adjustment of margins reduced or avoided the\nneed for adjuvant treatment. These findings demonstrate that FG-FFS is a\nreliable and feasible method for intraoperative margin assessment without\ndelaying surgery. Study procedures could all be performed without delaying\nsurgery, indicating implementing FG-FFS into clinical workflow is feasible\n• Manuscript page 3, paragraph 1: A retrospective study, performed by two\ntertiary centres in the Netherlands, reported inadequate margins (< 5 mm) of\nup to 85%.8\n• Manuscript page 3, paragraph 2: Even after adjuvant treatment, patient\nsurvival is demonstrated to be shorter for patients with an initial inadequate\nmargin compared to patients with initial tumour-free margins.13,14\nThereforeHence, acquiring tumour-free margins during at initial surgery is of\nmajor benefit will benefit outcomes.\n• Manuscript page 3, paragraph 3: Fresh frozen sectioning (FFS) of the excised\ntumour specimen is the standard for evaluating margins during surgery.15–18 An\nFFS procedure involves preparation of a 4 µm tissue section from a tissue slice,\nwhich has been rapidly frozen after fresh sectioning using a cryostat. This\nfrozen section is subsequently haematoxylin & eosin (H&E) stained to assess\ntumour margins. Although intraoperative FFS provides a high specificity for margin\nassessment, sampling error substantially reduces the sensitivity of this\ntechnique.19–22 Various approaches for improved intraoperative identification of\ninadequate margins have been investigated.23,24 Fluorescence-guided surgery\nusing tumour targeted tracers has already proven to be beneficial in head and\nneck cancer certain cancer treatments, such as ovarian and brain cancer.25,26\nFor oral cancer, fluorescence-guided surgery has been identified as a\npromising technique for intraoperative margin assessment as well.27–31 The use\nof cetuximab-IRDye800CW to detect oral cancer cells was demonstrated to be\nis feasible, since these cells overexpress epidermal growth factor receptor.28 In\nthe largest fluorescence-guided surgery trial in oral cancer patients thus far,\nour group has shown that fluorescence imaging using cetuximab-IRDye800CW\ncan identify tumour-positive margins. Additionally, the study showed that\nfluorescence signals are weaker when the overlying tissue is thicker, even\nwhen a close margin is still present. Therefore, fluorescence imaging is less\nreliable for accurately determining margin width.30,31 Thus However, the\nintensity of the fluorescent signal does not reliably correlate with the margin\nwidth using current fluorescence imaging approaches.31 To overcome this\nissue, a novel approach, combining supplementing fluorescence imaging with\nintraoperative fresh frozen sectioning (i.e. fluorescence-guided fresh frozen\nsectioning (FG-FFS)), has been developed. Although the combination of\nfluorescence imaging and fresh frozen sectioning has been suggested\npreviously, this has not been systematically investigated.29–31 Krishnan et al.\nexamined the localisation of fresh frozen sectioning based on clinical\nsuspicion compared to a sentinel margin approach, which uses the areas of\nhighest fluorescence intensities as indicators of potentially inadequate\nmargins. They found a strong correlation between these approaches, however\nthey did not investigate margins not suspected to be at risk.30\n• Manuscript page 3, paragraph 4: In this phase two intervention study, we\ndemonstrate that build on our previous findings and aim to combine the\nstrengths of fluorescence imaging of the whole resection specimen and fresh\ntissue slices containing cetuximab-IRDye800CW tosuccesfully localize a\nmargins at risk, andwhile FFS to provides a margin of that specific location\nmargin distance measurements of these margins at risk in millimetres. With\ntThis combined approach we aim to useenables fluorescence imaging to guide\nas guidance for intraoperative pathology assessment beyond rather than\nrelying solely on clinical suspicion alone, enabling allowing for timely\nrecognition and intraoperative adjustment of inadequate marginsand\ndecreasing the necessity for adjuvant treatment.\n• Manuscript page 6, paragraph 2: In this trial, fluorescence-guided surgery and\npathology fresh frozen sectioning led to correct intraoperative classification of\nthe margin in 95% of the included patients, without extending surgery time. The\ninadequate margin rate of 50% was decreased to 15%, after correct\nidentification of the margin using fresh tissue slice fluorescence-guided fresh\nfrozen sectioning. Subsequent margin-based adjuvant treatment was\ndecreased or omitted in seven out of ten patients with an initial inadequate\nmargin. Furthermore, when tumour resection precedes treatment of the neck,\nintraoperative assessment using FG-FFS proved feasible, without extending\nsurgery time.\n• Manuscript page 6, paragraph 3: The concept of targeted fluorescence\nimaging for tumour margin assessment to date has been based on whole\nresection specimen imaging in the surgery room and is assumed to yield\nmargin information with sufficient certainty.\n• Manuscript page 6, paragraph 3: These studies were all performed based on\nex-vivo whole resection specimens, stipulating the current standard in margin\nassessment using fluorescence imaging.\n• Manuscript page 7, paragraph 1: This is confirmed by the largest current trial\nin head and neck cancer using fluorescence imaging only of the whole\nresection specimen, performed in our centre.\n• Manuscript page 7, paragraph 5: Despite the considerable reduction in\ninadequate margins in the current study, some limitations should be taken into\naccount considered.\n• Manuscript page 8, paragraph 2: As decided by the multidisciplinary tumour\nboard, aAdjuvant treatment was substantially decreased (70.0%), reducing\npatient morbidity. Following the increased radical resection rate, an increased\n(disease-free) survival rate is could be expected. This will be monitored both in\nthe population of the current study and in a larger randomised control trial,\ncurrently being designed. should be examined during the coming years and in\na larger patient population.13 Here, the tracer cetuximab-IRDye800CW was\nused. This approach will also apply to but it can be expected that newly\ndeveloped tracers (e.g. fragmented antibodies and small peptides) will be\nuseful as well, as long as a sufficient tumour tracer accumulation in the tumour\nis achieved. We estimate that a tracer should be clinically usable when a\ntumour to background ratio of > 2.0 is obtained. Possibly, a shorter tumour\naccumulation time enables administration within an easier clinical applicable\ntimeframe.40,41\n• Manuscript page 8, paragraph 3: To conclude In conclusion, intraoperative\nmargin assessment using the fluorescence-guided fresh frozen sectioning\napproach correctly classified margins in 95% of the patients. FG-FFS based\nadditional resections decreased inadequate margins from 50% to 15%,\nresulting in a subsequent omittance or reduction of margin-based adjuvant\ntreatment in 70% of the patients who would have otherwise required additional\ntreatment. Considering the known side-effects of cetuximab such as\nanaphylaxis, clinical implementation was uncomplicated, as the technique\ndid not increase surgery time and direct margin-feedback could be provided to\nthe surgeon was possible. Thus, margin assessment using FG-FFS\nsubstantially increases correct intraoperative margin assessment and enables\nadditional resections in case of an inadequate margin, reducing patient\nburden.\nWe appreciate the decision to accept our manuscript for publication under an open\naccess license. Below, we provide our response Reviewer #3. First, we focused on the\noverall comments of the reviewer, and subsequently responded to the detailed\ncomments on a point-by-point basis. In addition, the editorial requests listed in the\nattached Author Checklist have been fully addressed. All revisions in the manuscript are\nhighlighted in red. In the attachments, you can find two versions of the adapted\nmanuscript, one with, and one without highlighted changes for your convenience.\nReviewer #3\nWe thank the reviewer for their kind words and final minor suggestions. Below we have\naddressed the proposed changes on a point-to-point basis. As some of these suggestions\ncontradict with previous review comments, we tried to incorporate all suggestions in the\nbest way possible.\n• Abstract [I suggest a shorter clearer less awkward abstract, as follows]\nTumour-free margins (>5 mm) in head and neck malignancies improve prognosis,\npossibly impacting the need for adjuvant treatment. We investigate a novel use of\nfluorescence imaging using the tumour targeting fluorescent tracer cetuximab-\nIRDye800CW (FG-FFS) to identify inadequate intraoperative margins. Primary\nendpoints of our study were intraoperative detection of inadequate margins and\nadditional resections. Feasibility of FG-FFS implementation into the clinical\nworkflow is also assessed. Initial surgery led to inadequate margins in 10 of the 20\npatients; FG-FFS correctly classified margins in 19. Additional resections\nincreased tumour-free margin rates to 85%; anatomical boundaries prevented\nadditional resection in one patient. Intraoperative margin adjustment reduced the\nneed for adjuvant treatment in seven patients. These findings demonstrate that\nFG-FFS is a reliable feasible method for intraoperative margin assessment without\ndelaying surgery.\nResponse: We thank the reviewer for the proposed new abstract. Below we\npropose a new, revised, abstract, based on the proposed abstract with previous\nreview comments incorporated.\n• Manuscript page 2 (Abstract): Tumour-free margins (>5 mm) in head and neck\nmalignancies improve prognosis, possibly impacting the need for adjuvant\ntreatment. In this phase II prospective, non-randomised trial, we investigate\nthe use of fluorescence imaging using the tumour targeting fluorescent tracer\ncetuximab-IRDye800CW combined with fresh frozen sectioning to identify\ninadequate margins intraoperatively. Primary endpoints of our study are\nintraoperative detection of inadequate margins and additional resections.\nFeasibility of implementing fluorescence-guided fresh-frozen sectioning (FG-\nFFS) into the clinical workflow is also assessed. Initial surgery led to\ninadequate margins in 10 of the 20 patients; FG-FFS correctly classified\nmargins in 19. Additional resections increased tumour-free margin rates to\n85%; anatomical boundaries prevented additional resection in one patient.\nIntraoperative margin adjustment reduced the need for adjuvant treatment in\nseven patients. These findings demonstrate that FG-FFS is a reliable and\nfeasible method for intraoperative margin assessment without delaying\nsurgery.\n2. I suggest a clearer and shorter next paragraph: as follows\nSurgery is the primary treatment modality for many head and neck malignancies.\nResection margins are considered adequate when at least 5 mm wide.1–3 Tumour-\nfree surgical margins are achieved however in only 30-65% of patients 6,7\nInadequate resections lead to lower survival and locoregional control: five-year\ndisease-free survival rates of ~ 79%, 63% and 52% for tumour-free (≥ 5 mm), close\n(< 5 mm & ≥ 1 mm) and tumour-positive margins (< 1 mm), respectively.1-5\nResponse: We thank the reviewer for the suggestions regarding the first paragraph\nof the introduction. The new paragraph is provided below:\n• Manuscript page 3 (Introduction, paragraph 1): Surgery is the primary\ntreatment modality for many head and neck malignancies. Resection margins\nare considered adequate when at least 5 mm wide.1–3 Despite this well-\nestablished requirement, tumour-free surgical margins are achieved in only 30-\n65% of patients.6,7 Inadequate resections adversely impact survival and\nlocoregional control: five-year disease-free survival rates of approximately\n79%, 63% and 52% for tumour-free (≥ 5 mm), close (< 5 mm & ≥ 1 mm) and\ntumour-positive margins (< 1 mm), respectively.1-5\n3. Delete as true, but irrelevant to your mss: Adjuvant radiotherapy is known to\nreduce tumour recurrence risk,but increases patient morbidity.\nResponse: We thank the reviewer for this suggestion and have removed the\nstatement from the manuscript.\n4. Change “Even after adjuvant treatment, patient survival is demonstrated to be\nshorter for patients with an initial inadequate margin.11,12 Hence, acquiring\ntumour-free margins at initial surgery is of major benefit.” to:\nTumour-free margins at initial surgery is of major benefit: adjuvant treatment does\nnot compensate for initial inadequate margins.11,12\nResponse: The sentence has been revised to more clearly reflect the evidence\nthat adjuvant therapy does not offset the negative prognostic effect of primary\ninadequate margins. We have updated the sentence below:\n• Manuscript page 3, paragraph 2: Achieving tumour-free margins at the initial\nsurgery is of major clinical importance, as adjuvant treatment cannot fully\ncompensate for the negative prognostic impact of initially inadequate\nmargins.12,13\n5. Table 1: minor changes for clarity place a space after maximum diameter (mm)\nchange header of column 1 to “Patient # (and remove word Patient from each row\n(keep only the #) delete in each row the redundant “p” column 2\nline up decimal points in column 3 and 4 (unless editor prefers lining up left edge).\nResponse: We have revised Table 1 based on these provided suggestions.\n6. Delete the added section regarding 65% women. I realize this was added in\nresponse to a reviewer comment, but I think it is sufficient to answer the reviewer\nverbally (and not include this parenthetical comment in the paper as it adds\nnothing and is irrelevant to your topic.)\n“Interestingly, the patient population in this study consisted of 65% female\npatients, whereas male patients are known to have an increased risk for\ndeveloping head and neck cancer.2 The predominance of female patients reflects\nthe composition of patients referred to our centre during the study period, rather\nthan any deliberate selection. Additionally, three male patients were excluded due\nto anaphylaxis and deviations from the intraoperative imaging protocol.”\nResponse: We agree with the reviewer that it is irrelevant to the topic addressed in\nthis manuscript. Therefore, we have deleted this phrase from the manuscript as\nthe reviewer suggests."}]

Decision letter
[Editorial Note: See attachment]