# Extemporaneous histological analysis according to slow-Mohs combined with Full-Field Optical Coherence Tomography evaluation (FFOCT) in cutaneous tumor pathology: Toward a digital extemporaneous analysis?

Sarah Hendriks, Eugénie Dalimier, Bernard Gasser, Olivier Camuzard, Thérèse Adawa, Elise Lupon

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Sarah Hendriks, Eugénie Dalimier, Bernard Gasser, Olivier Camuzard, Thérèse Adawa, et al. Extemporaneous histological analysis according to slow-Mohs combined with Full-Field Optical Coherence Tomography evaluation (FFOCT) in cutaneous tumor pathology: Toward a digital extemporaneous analysis?. Journal of Plastic, Reconstructive and Aesthetic Surgery, 2025, 104, pp.450-460. 10.1016/j.bjps.2025.03.025 . hal-05110208

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# Extemporaneous histological analysis according to slow-Mohs combined with Full-Field Optical Coherence Tomography evaluation (FFOCT) in cutaneous tumor pathology: Toward a digital extemporaneous analysis?

<figure><img src="image_3.png" /></figure>

Sarah Hendriks<sup>a</sup>, Eugénie Dalimier<sup>b</sup>, Bernard Gasser<sup>c</sup>, Olivier Camuzard<sup>d</sup>, Thérèse Adawa<sup>e</sup>, Elise Lupon<sup>d,f,*</sup>

Received 31 August 2024; Accepted 8 March 2025

## KEYWORDS

Mohs;
Slow Mohs;
Full-Field Optical
Coherence
Tomography;
Histology;
Cutaneous tumor

**Summary** *Background:* Slow-Mohs micrographic surgery use is limited by the logistic constraints it imposes. Full-field optical coherence tomography (FFOCT) is an emerging non-invasive imaging technique that provides skin tissue imaging at the cellular level without tissue preparation.

**Objective:** Evaluating the FFOCT technology's diagnostic possibilities in examining surgical sections in micrographic surgery for basal cell carcinomas compared to slow-Mohs.

**Materials and methods:** Two plastic surgeons provided 24 Mohs sections from 20 patients with basal cell carcinomas from a single-center. Each section was scanned using FFOCT, and a diagnosis-blinded pathologist reviewed the digital images for malignancy. The FFOCT images were compared with the standard histologic analysis of the sample sections.

<footnote><sup>a</sup> Department of Plastic, Reconstructive and Aesthetic Surgery, University Hospital of Strasbourg, France</footnote>

<footnote><sup>b</sup> LLTech SAS, Paris, France</footnote>

<footnote><sup>c</sup> Laboratoire de Pathologie, Centre Hospitalier Emile Muller, Mulhouse, France</footnote>

<footnote><sup>d</sup> Department of Plastic and Reconstructive Surgery, Institut Universitaire Locomoteur et du Sport, Pasteur 2 Hospital, University Côte d'Azur, Nice, France</footnote>

<footnote><sup>e</sup> Department of Plastic Surgery, Hospital Cochin, AP-HP, Paris, France</footnote>

<footnote><sup>f</sup> Laboratory of Molecular PhysioMedicine (LP2M), UMR 7370, CNRS, University Côte d'Azur, Nice, France</footnote>

<footnote>* Correspondence to: Plastic and Reconstructive Surgery Unit, Pasteur 2, 30, Voie Romaine, 06001 Nice, France. E-mail address: elupon@mgh.harvard.edu (E. Lupon).</footnote>
**Results:** The agreement between FFOCT imaging results and slide histology included 17 true positives (VP) and 4 true negatives (TN) for debulking and 19 TN and 2 VP for Mohs peripheral cuts. The positive predictive value (PPV) was 85% for debulking, and the negative predictive value (NPV) was 100%. For recuts, the PPV was 50% and NPV was 95%.

**Conclusion:** We developed a protocol for analyzing skin tumors ex vivo using FFOCT, providing digital images that can be transmitted remotely. This study serves as a proof of concept. The visualization of peripheral margins in a single image and the predictive values need to be improved before clinical use can be considered.

© 2025 The Author(s). Published by Elsevier Ltd on behalf of British Association of Plastic, Reconstructive and Aesthetic Surgeons. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

There is no international consensus among pathologists on the macroscopic sampling method. However, this step is essential to guide the microscopic observations.¹⁻⁶ The major limitation of the current technique of histopathological analysis is its inability to confirm the complete removal of the initial lesion, which necessitates the careful removal of healthy tissue surrounding the tumor.⁶⁻¹¹ Mohs micrographic surgery has been developed to overcome this issue in our department in 2 steps: a debulking and cup-shaped peripheral recut, removing the skin defect's bottom (deep margins) and the edges (lateral margins). The peripheral section is laid flat by eversion, oriented, marked with ink for mapping, cryogenically frozen, and sent to the pathologist for extemporaneous examination.¹²,¹³ Thus, the deep hypodermic border of the recut passes on the same plane as its lateral border, dermis, and epidermis. This last step of recutting is repeated until a malignancy-free cutting plane is obtained.⁶,¹⁴ Notably, some Mohs surgeons excise the full specimen and process it with the margins frozen flat. Using a formalin-fixed tissue technique, the slow-Mohs technique requires less technician time than a frozen section service and reduces the need for hospitalization.¹⁵,¹⁶ In this variant, surgical excision remains unchanged. However, unlike the original technique, histological analysis of this section is deferred, as it is embedded in kerosene, enabling micrographic surgery to be implemented in centers that do not have the necessary structure and equipment nearby.¹⁶ Histological analysis most often occurs around 24 to 48 h after lesion removal via direct reading of the slides by the anatomo-pathologist.¹⁵

Full-field optical coherence tomography (FFOCT) is an emerging non-invasive imaging technique for rapid histological analysis capable of visualizing details at the cellular level without tissue preparations such as freezing or using contrast agents. The entire imaged field is illuminated with low temporal and spatial coherence light produced by an initial halogen lamp. The tissue is then moved vertically to image a depth field. Image analysis and pathologic correlation on various excised tissues¹⁷⁻²⁴ have confirmed the clinical relevance of full-field OCT, including the skin.¹⁷ In the context of a tangential and, therefore, parallel analysis of the surface as practiced during micrographic surgery, FFOCT was an attractive tool to aid the implementation of slow-Mohs micrographic surgery. Indeed, digitizing images makes it possible to read sections without the anatomo-pathologist having to possess the slides physically.

This study aimed to evaluate the diagnostic possibilities and limitations of FFOCT technology in examining surgical

sections in micrographic surgery compared with slow-Mohs for basal cell carcinoma (BCC).

## Material and method

This is a prospective, single-center, comparative, blinded study. All patients were operated on in the plastic surgery department of a French Hospital between March and April 2017 after prior informed written consent was obtained. The head of the pathology department at this hospital interpreted the excisional parts. The local Institutional Review Board approved the study.

## Surgical removal using micrographic surgery

All patients were operated under local anesthesia with a 1% adrenalized xylocaine mixture by 2 board-certified plastic surgeons. A first tracing was performed at 1 mm and a second at 2 mm from the lesion borders (Figure 1A). All subsequent steps were performed under binocular loupes. The first debulking step was performed with 1 mm margins from the previously identified lesion edges, followed by intradermal tangential excision (Figure 1B). A non-absorbable thread then guided the debulking positioned at noon on the piece. According to Mohs, the second step of peripheral recutting was performed with 1 mm of margin from the debulking limits, thus 2 mm from the initial lesion borders. The peripheral recut was then oriented with a non-absorbable suture positioned at noon (Figure 1C).

To allow tangential analysis, it is necessary to obtain a complete spreading of the peripheral cut so that the lateral margins, epidermis and dermis, deep margin, and hypodermis are on the same cutting plane: the flattening was obtained because of an eversion of the lateral edges favored by radial incisions at the 4 cardinal points, at 12 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock (Figure 2A) and a counter-incision on the inner side of the lateral edges (Figure 2B). The specimen was placed on the glass slide dedicated to the Light-CT Scanner sample holder (LightCT, LLTech, Paris, France) (Figure 2C).

## Acquisition of FFOCT images from surgical specimens

For the acquisition of images to be studied using FFOCT, the debulking part was manipulated first. The deep surface
<figure><img src="image_4.png" /><figcaption>**Figure 1** Scheme of our surgical excision following the rules of Mohs Micrographic surgery (MMC). Delineation of the lesion (CBC) at 1 mm (blue line) and 2 mm from the lesion (red) (A). Debulking, i.e., tangential and intradermal removal of the lesion with orientation by a thread at 12 o'clock (B). Peripheral recutting according to Mohs, i.e., complementary peripheral recutting with orientation by a non-absorbable thread at 12 o'clock (C).</figcaption></figure>

furthest from the lesion was referred to as the "surgical section surface to be analyzed." This was positioned against a glass slide (Figure 3A) and then placed in the dedicated specimen holder (Figure 3B & 3C). The peripheral cut, according to Mohs, was handled second. The surgical section surface to be analyzed was positioned against the glass slide (Figure 4A) and placed in the dedicated specimen holder (Figure 4B & 4C). A drop of optical oil was placed on the glass slide to ensure contact between the objective and glass slide.

We acquired an image plane at a depth of 15 µm below the surface of the specimen, i.e., at -15 µm from the surgical section surface for all debulking and peripheral recuts. In addition, to assess the visibility of the lateral edges (epidermis and dermis) in 15 of the 24 lesions, we also acquired 3 additional planes at -45, -75, and -105 µm from the surgical section surface (Figure 5).

## Sample preparation technique for anatomopathological analysis

After FFOCT image acquisition using fresh tissue, the surgical specimens were oriented and marked at the cardinal points using different inks (Supplemental Figure 1), placed in cassettes, fixed in a 10% formalin solution, and sent to the pathology laboratory for paraffin embedding. The laboratory specimen technique included 5 steps: dehydration with an alcohol solution, paraffin embedding of the tissue in a purified paraffin mixture, microtome cutting of the paraffin block (containing the tissue) into 4-µm-thick ribbons, spreading on slides, and standard hematoxylin-eosin staining. For debulking specimens, the paraffin blocks were cut with a microtome perpendicular to the axis of the specimen, allowing a reading of the lesion, including the

<figure><img src="image_5.png" /><figcaption>**Figure 2** Diagram of the flattening of the lateral edges, epidermis, and dermis of the peripheral cut according to Mohs. Radial incisions at the four cardinal points, at 12 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock with the scalpel (A). Eversion of the lateral edges for flattening (B). Flattened specimen with the surgical section to be analyzed against the glass slide of the specimen holder (C).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 3** Scheme showing the placement of the debulking patch in the specimen holder. The "surgical section surface to be analyzed" is the deep surface of the piece and is positioned against a glass slide (A). It is turned upside down (B) and placed in the dedicated specimen holder so that it faces up (C) toward the Light-CT Scanner head.</figcaption></figure>

whole thickness of the specimen, epidermis, and dermis, and sometimes a part of the hypodermis (Figure 6).

For peripheral recut specimens, the paraffin blocks are cut using a microtome tangentially to the specimen's axis from the specimen's depth (surgical section surface to be

analyzed) to the surface (Figure 7). The anatomopathology department follows a well-defined protocol consisting of an initial "roughing" of the surgical section with the microtome (16 to 20 µm thickness). This level is designated R0 and corresponds to the first slide read and analyzed (Figure 7A).

<figure><img src="image_7.png" /><figcaption>**Figure 4** Scheme of the placement of the peripheral cut in the specimen holder. The "surgical section surface to be analyzed" is the deep surface of the specimen and is positioned against a glass slide (A). It is turned over (B) and placed in the dedicated specimen holder so that it faces upward (C) toward the Light-CT Scanner head.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 5** Scheme of the acquisition of the different image planes of a peripheral cut by FFOCT; Side view. Schematic of the Light-CT Scanner after placement of a fresh peripheral recut in the specimen holder; the deep surgical section of the specimen oriented toward the Light-CT Scanner (A) Enlarged schematic centered on the surgical section and the different image planes at –15 µm, –45 µm, –75 µm, and –105 µm (B). FFOCT, full-field optical coherence tomography.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 6** Histological microsections on slides after microtome cutting of a debulking specimen. For debulking specimen, the paraffin blocks are cut with a microtome perpendicular to the axis of the specimen, allowing a reading of the lesion including the entire thickness of the specimen, epidermis, and dermis (a) and sometimes part of the hypodermis (b).</figcaption></figure>

The second retained slide level, R1, is at 40 µm. The third level, R2, is at 60 µm. The fourth retained slide level, R3, is at 80 µm. In the fifth retained slide level, R4, unlike the other levels, technicians "go up in the piece" until they arrive at a cutting plane that includes all the deep (hypodermis) and lateral (epidermis and dermis) margins on the same plane, (principle of micrographic surgery). By experience, this level is between 150 and 200 µm (Figure 7B).

The specimens were always accompanied by an anatomo-pathological analysis request slip specifying the suspected type of lesion (BCC diagnosed either by biopsy or clinically by a dermatologist), its location, the orientation of the specimen by a thread at noon for debulking and by a detailed diagram with 4 different colors corresponding to the 4 cardinal points of the previously inked specimen for peripheral recutting.
<figure><img src="image_10.png" /><figcaption>**Figure 7** Histological sections on slides after microtome cutting of a peripheral cut after paraffin embedding. (A) R0: First read slide after "roughing" the surgical section to 16 to 20 µm. (B) R4: not standardized, often between 150 and 200 µm, showing on the same plane (a) epidermis and dermis laterally and (b) the deep hypodermis centrally.</figcaption></figure>

## Histological analysis and FFOCT by the pathologist

The pathologist first analyzed the histologic slides of the specimens, indicating the need for debulking and measuring the distance between the tumor and lateral (LM) and deep (DM) surgical margins of the specimen and for peripheral recuts, the presence or absence of malignancy on the surgical section (tangential). If malignancy was present, the pathologist specified the time for lateral margins and depth over the first 200 µm (after reading from R0 to R4) for deep sections. Reading of FFOCT-acquired images was performed blinded at 3 and 4 months after histological analysis by the same pathologist (for practical issues).

For the debulking specimen and peripheral recut piece, an image at 15 µm from the surgical section surface, the pathologist was asked whether he found cancerous areas and on what criteria (trabecular, nodular, or scleroderma form).

For 15 out of the 24 lesions, for which we acquired several planes depth from the periphery of the specimen toward the lesion, namely -15 µm, -45 µm, -75 µm, and -105 µm from the surgical section surface. We also determined the percentage of lateral margins (epidermis and dermis) visualized simultaneously on a single image, depending on the plane depth scored between A = 0-25%, B = 25-50%, C = 50-75%, or D = 75-100%.

After reading the FFOCT images, we proofread the standard histology slides to verify that the data found by reading the FFOCT images corresponded to what was visualized on the pathological slides. The reading was timed for each case, including the FFOCT image and histological slide reading, to establish a "learning curve."

## Results

Between March and April 2017, we included 24 lesions for 20 patients (9 women and 11 men), with a mean age of 75.8 years with extremes of 58 to 88 years, with BCC, confirmed

either via biopsy (n = 16/24; 66.6%) or clinically by a dermatologist (n = 8/24; 33.3%). The mean diameter of the lesions was 11.58 mm.

## Histological examination (Table 1)

For debulking, out of the 24 lesions, 22 were BCCs; one was a squamous cell carcinoma (lesion N°8, Table 1), and one was an actinic keratosis (lesion N°13, Table 1). Among the 8 lesions that were clinically diagnosed by a dermatologist, 1 case was actinic keratosis (lesion N°13), and 1 was a squamous cell carcinoma (lesion N°8). For 16 lesions out of the 24 lesions, we found a contact with the deep surgical section and, in 1 case (lesion N°23), a limit at 0.01 mm (10 µm).

For peripheral recutting, in 21 lesions out of the 24 lesions, the deep and lateral margins were free of malignancy. In 3 lesions out of the 24 lesions, there was a neoplastic extension at the surgical section: 2 times at the deep margin (hypodermis) (lesion N°3 and lesion N°8, Table 1) and once at the lateral margin (epidermis and dermis) 5-9 h (lesion N°11, Table 1).

## Comparison of FFOCT and histological analysis

For debulking, in the 16 lesions out of the 24, FFOCT images were interpreted as positive, and the lesion was found to be in contact with the deep surgical section. For lesion N°23, histological analysis revealed that it was at a depth of 10 µm from the deep surgical section; hence, its visualization in FFOCT, knowing that the 1st visualized depth is at 15 µm from the surgical section. We obtained 17 true positives (TP). In 3 lesions out of the 24 lesions (lesions N°13, N°14, and N°21), the FFOCT image was interpreted as positive, whereas the lesion was not in contact with the deep surgical section in anatomopathology. We obtained 3 false positives (FP). The comparison of FFOCT and histological analysis is described in Table 2.
<table><caption>**Table 1** Demographic data of the sample.</caption><thead><tr><th>Patients</th><th>Gender (M/F)</th><th>Age (years)</th><th>Lesion N° after randomization</th><th>Preoperative biopsy</th><th>Max. diameter (mm)</th><th>Localization</th><th>Post-operative histological diagnosis</th></tr></thead><tbody><tr><td rowspan="2">1</td><td rowspan="2">M</td><td rowspan="2">62</td><td>19</td><td>+</td><td>12</td><td>Forehead</td><td>Nodular BCC</td></tr><tr><td>16</td><td>+</td><td>7</td><td>Nose</td><td>Nodular BCC</td></tr><tr><td>2</td><td>M</td><td>75</td><td>21</td><td>+</td><td>15</td><td>Temple</td><td>Nodular BCC</td></tr><tr><td>3</td><td>F</td><td>77</td><td>14</td><td>-</td><td>16</td><td>Temple</td><td>Infiltrative BCC</td></tr><tr><td>4</td><td>M</td><td>73</td><td>7</td><td>+</td><td>9</td><td>Nose</td><td>Nodular BCC</td></tr><tr><td>5</td><td>M</td><td>58</td><td>22</td><td>+</td><td>7</td><td>Nose</td><td>Infiltrative BCC (dermis)</td></tr><tr><td rowspan="2">6</td><td rowspan="2">F</td><td rowspan="2">82</td><td>20</td><td>-</td><td>6</td><td>Temple</td><td>Nodular BCC</td></tr><tr><td>1</td><td>-</td><td>8</td><td>Cheek</td><td>Nodular and Infiltrative BCC</td></tr><tr><td rowspan="3">7</td><td rowspan="3">M</td><td rowspan="3">78</td><td>3</td><td>+</td><td>11</td><td>Preauricular</td><td>Nodular BCC</td></tr><tr><td>6</td><td>+</td><td>16</td><td>Cheek</td><td>Superficial and Nodular</td></tr><tr><td>5</td><td>+</td><td>15</td><td>Cheek</td><td>BCC</td></tr><tr><td>8</td><td>M</td><td>85</td><td>9</td><td>+</td><td>5</td><td>Nose</td><td>Infiltrative BCC</td></tr><tr><td>9</td><td>M</td><td>74</td><td>23</td><td>+</td><td>25</td><td>Neck</td><td>Infiltrative BCC</td></tr><tr><td>10</td><td>F</td><td>75</td><td>13</td><td>-</td><td>5</td><td>Nose</td><td>Actinic keratosis</td></tr><tr><td>11</td><td>F</td><td>72</td><td>4</td><td>+</td><td>4</td><td>Forehead</td><td>Infiltrative BCC</td></tr><tr><td>12</td><td>M</td><td>78</td><td>2</td><td>+</td><td>20</td><td>Upper eyebrow</td><td>Superficial and Infiltrative BCC</td></tr><tr><td>13</td><td>F</td><td>88</td><td>8</td><td>-</td><td>11</td><td>Nose</td><td>SCC</td></tr><tr><td>14</td><td>M</td><td>82</td><td>15</td><td>+</td><td>12</td><td>Lower eyelid</td><td>Nodular and Infiltrative BCC</td></tr><tr><td>15</td><td>F</td><td>58</td><td>10</td><td>+</td><td>6</td><td>Lower eyelid</td><td>Infiltrative BCC</td></tr><tr><td>16</td><td>M</td><td>85</td><td>11</td><td>+</td><td>25</td><td>Nose</td><td>Infiltrative BCC</td></tr><tr><td>17</td><td>F</td><td>82</td><td>17</td><td>-</td><td>16</td><td>Temple</td><td>Infiltrative BCC</td></tr><tr><td>18</td><td>F</td><td>78</td><td>12</td><td>+</td><td>7</td><td>Lower eyelid</td><td>BCC</td></tr><tr><td>19</td><td>M</td><td>76</td><td>18</td><td>-</td><td>10</td><td>Temple</td><td>Infiltrative BCC (dermis)</td></tr><tr><td>20</td><td>F</td><td>79</td><td>24</td><td>-</td><td>10</td><td>Scalp</td><td>Superficial BCC</td></tr></tbody></table>

M = Male; F = Female; BCC = Basal Cell Carcinoma; SCC = Squamous Cell Carcinoma; Max. = Maximal; mm = millimeter.

In 4 lesions out of the 24 lesions, the FFOCT images were interpreted as negative, and the lesion was not in contact with the deep surgical section. Therefore, we obtained 4 true negatives (TN).

For peripheral recutting, in 19 lesions out of the 24 lesions, the FFOCT images were interpreted as negative, and the surgical sections were free of malignancy in anatomopathology. We obtained 19 TN. In 2 lesions out of the 24 lesions (lesion N°15 and lesion N°21), the FFOCT image was interpreted as positive, whereas the lesion was not in contact with the deep surgical section. We obtained 2 FP.

In 1 case out of the 24 (lesion N°11), the FFOCT image was interpreted as negative while the lesion was in contact with the lateral surgical section between 5-9 h. We obtained a false negative (FN). In 2 lesions out of the 24, interpreted as positive in FFOCT, the surgical sections were positive in anatomopathology. We obtained 2 TP.

The sensitivity (Se), specificity (Spe), positive predictive value (PPV), and negative predictive (PNV) values calculated are summarized in Supplemental Figure 2. Figure 8 illustrates the parallel between a histological slide and an FFOCT image at 15 µm. Light-CT Scanner images of a de-bulking at 15 µm from the surgical section, centered on a nodular-shaped BCC, are available in Supplemental Figure 3.

Visibility on a single image of the lateral margins (epidermis and dermis) on the peripheral FFOCT cuttings

We analyzed the epidermis and dermis for a total of 69 FFOCT images. The totality of the lateral margins (epidermis and dermis), even at a depth of -105 µm from the surgical section surface, was never visualized in a single image in all the images examined.

At -15 µm from the surgical section surface, 0 to 25% of the lateral margins were visualized in 63% of the cases (15 of the 24 cases analyzed) and 25 to 50% of the lateral margins in 21% of the cases (5 of the 24 cases analyzed).

At -45 µm from the surgical section surface, 0 to 25% of the lateral margins were visualized in 46% of the cases (7 of the 15 cases analyzed) and 25 to 50% of the lateral margins in 47% (7 of the 15 cases analyzed).

At -75 µm from the deep surgical section surface, 0 to 25% of the lateral margins were visualized in 33% of the cases (5 of the 15 cases analyzed) and 25 to 50% of the lateral margins in 40% (6 of the 15 cases analyzed).

At -105 µm from the deep surgical section surface, 0 to 25% of the lateral margins were visualized in 33% of the
<table><caption>**Table 2** Comparison of FFOCT and histological analysis. Lateral surgical margins (LM), deep surgical margins (DM), true positives (TP), true negatives (TN), and basal cell carcinoma (BCC).</caption><thead><tr><th rowspan="2">Lesion</th><th rowspan="2">Localization</th><th colspan="3">DEBULKING</th><th colspan="3">PERIPHERAL SECTION ACCORDING TO MOHS</th><th rowspan="2">Total Reading time (min)</th></tr><tr><th>Standard histology LM (µm)</th><th>FFOCT (-15 µm)</th><th>Match</th><th>Standard histology (µm)</th><th>FFOCT (-15 µm)</th><th>Match</th></tr></thead><tbody><tr><td>1</td><td>Cheek</td><td>Nodular &amp; Infiltrative BCC DM in contact</td><td>Basaloid cell lobules, peripheral palisading, fibrotic stroma</td><td>YES TP</td><td>- DM 200</td><td>-</td><td>YES TN</td><td>55</td></tr><tr><td>2</td><td>Upper eyebrow</td><td>Superficial and Infiltrative BCC DM 500</td><td>-</td><td>YES TN</td><td>-</td><td>-</td><td>YES TN</td><td>35</td></tr><tr><td>3</td><td>Preauricular</td><td>Nodular BCC DM in contact</td><td>Lobules, no peripheral palisading nor retractions</td><td>YES TP</td><td>DM in contact</td><td>+</td><td>YES TP</td><td>45</td></tr><tr><td>4</td><td>Forehead</td><td>Infiltrative BCC DM uncertain thus in contact</td><td>Lobules</td><td>YES TP</td><td>-</td><td>-</td><td>YES TN</td><td>42</td></tr><tr><td>5</td><td>Cheek</td><td>Infiltrative BCC DM in contact</td><td>Lobules</td><td>YES TP</td><td>-</td><td>-</td><td>YES TN</td><td>20</td></tr><tr><td>6</td><td>Cheek</td><td>Superficial and Nodular BCC DM 500</td><td>-</td><td>YES TN</td><td>-</td><td>-</td><td>YES TN</td><td>25</td></tr><tr><td>7</td><td>Nose</td><td>Nodular BCC DM in contact</td><td>Lobules and dense central region</td><td>YES TP</td><td>-</td><td>-</td><td>YES TN</td><td>20</td></tr><tr><td>8</td><td>Nose</td><td>SCC DM in contact</td><td>Dense central region, no clear lobules, smaller and more infiltrating</td><td>YES TP</td><td>DM in contact Clark V</td><td>+</td><td>YES TP</td><td>12</td></tr><tr><td>9</td><td>Nose</td><td>Superficial BCC DM 200</td><td>-</td><td>YES TN</td><td>-</td><td>-</td><td>YES TN</td><td>35</td></tr><tr><td>10</td><td>Lower eyelid</td><td>Infiltrative BCC DM in contact</td><td>Unclear lobules, smaller and less distinct</td><td>YES TP</td><td>-</td><td>-</td><td>YES TN</td><td>16</td></tr><tr><td>11</td><td>Nose</td><td>Infiltrative BCC DM in contact</td><td>No clear lobules, smaller and more infiltrating, certain it is not nodular</td><td>YES TP</td><td>LM in contact At 5-9 h</td><td>-</td><td>NO FN</td><td>21</td></tr><tr><td>12</td><td>Lower eyelid</td><td>Nodular BCC DM in contact</td><td>Typical lobules with cystic appearance</td><td>YES TP</td><td>- DM 200</td><td>-</td><td>YES TN</td><td>22</td></tr><tr><td>13</td><td>Nose</td><td>Actinic keratosis DM untouched</td><td>Because of doubtful central area, but no lobules visible</td><td>NO FP</td><td>-</td><td>-</td><td>YES TN</td><td>8</td></tr><tr><td>14</td><td>Temple</td><td>Infiltrative BCC DM 250</td><td>Poorly defined lobules</td><td>NO FP</td><td>-</td><td>-</td><td>YES TN</td><td>19</td></tr><tr><td>15</td><td>Lower eyelid</td><td>Nodular &amp; Infiltrative BCC DM in contact</td><td>Unclear lobules, smaller and more infiltrating++</td><td>YES TP</td><td>- DM 150</td><td>+</td><td>NO FP</td><td>18</td></tr><tr><td>16</td><td>Nose</td><td>Nodular BCC DM in contact</td><td>Clear central lobules Nodular BCC++</td><td>YES TP</td><td>-</td><td>-</td><td>YES TN</td><td>10</td></tr><tr><td>17</td><td>Temple</td><td>Infiltrative BCC DM in contact</td><td>Unclear lobules because of fibrosis++ Infiltrative BCC++</td><td>YES TP</td><td>-</td><td>-</td><td>YES TN</td><td>10</td></tr><tr><td>18</td><td>Temple</td><td>Infiltrative BCC DM in contact</td><td>Clear lobules</td><td>YES TP</td><td>-</td><td>-</td><td>YES TN</td><td>13</td></tr><tr><td>19</td><td>Forehead</td><td>Nodular BCC DM in contact</td><td>Certain about BCC Doubt if infiltrative</td><td>YES TP</td><td>-</td><td>-</td><td>YES TN</td><td>12</td></tr><tr><td>20</td><td>Temple</td><td>Nodular BCC DM in contact</td><td>Clear lobules Nodular BCC</td><td>YES TP</td><td>-</td><td>-</td><td>YES TN</td><td>10</td></tr><tr><td>21</td><td>Temple</td><td>Nodular BCC DM 400</td><td>Uncertain thus considered positive</td><td>NO FP</td><td>-</td><td>+</td><td>NO FP</td><td>22</td></tr><tr><td>22</td><td>Nose</td><td>Infiltrative BCC DM in contact</td><td>Clear central lobules Nodular BCC</td><td>YES TP</td><td>- DM 150</td><td>-</td><td>YES TN</td><td>18</td></tr><tr><td>23</td><td>Neck</td><td>Infiltrative BCC DM 10</td><td>Certain about BCC Doubt if nodular</td><td>YES TP</td><td>-</td><td>-</td><td>YES TN</td><td>11</td></tr><tr><td>24</td><td>Scalp</td><td>Superficial BCC DM 1000</td><td>-</td><td>YES TN</td><td>-</td><td>-</td><td>YES TN</td><td>9</td></tr></tbody></table>

BCC = Basal Cell Carcinoma; SCC = Squamous Cell Carcinoma; "+" = visualized malignancy criteria; "-" = absence of visualized malignancy criteria; DM = deep margin (distance between the tumor and the deep surgical section surface); LM = lateral margin (distance between the tumor and the lateral surgical section surface); TP = true positive; TN = true negative; FP = false positive; FN = false negative.
<figure><img src="image_11.png" /><figcaption>**Figure 8** HE x 20 histological slides (A) and FFOCT image at 15 µm (B) CBC lobule (a); retraction (b); hair follicle (c).</figcaption></figure>

cases (5 of the 15 cases analyzed) and 25 to 50% of the lateral margins in 33% (5 of the 15 cases analyzed).

Therefore, multiple different FFOCT images from the same lesion were required to visualize all the lateral margins.

## Combined reading time

The mean time was 21.1 min per lesion, with a mean standard deviation of 9.3 min. We also observed an apparent decrease in time from the 10th lesion reading, with a mean of 14.6 min and a mean standard deviation of 4.5 min

## Discussion

The results of this study regarding FFOCT use to assist in implementing slow-Mohs micrographic surgery suggested its feasibility. This study serves as a proof of concept. However, the visualization of the peripheral margins in a single image and estimation of the predictive values need to be improved before clinical use can be considered. The examination did not alter tissues analyzed using FFOCT, and its subsequent histological examination was unremarkable compared with a conventional exam. The average acquisition time for 1 cm² was 1 min 30 s, i.e., 5 min for the entire surface.

With the acquisition of digital images and a medical imaging platform offering a resolution identical to that of the Light-CT Scanner, our results could be interpreted by an anatomopathologist in another city. This modality could circumvent some of the obstacles currently encountered in implementing slow-Mohs surgery.²⁵

The FFOCT image was approached as a grayscale recognition of the general tissue architecture at low magnification, followed by a more detailed analysis down to the cellular level by "hovering" over the image and zooming in on-demand with applications from the Keen Eye medical imaging platform. When comparing the FFOCT analysis and Mohs peripheral cut analysis, we observed a PPV of 0.5. However, the NPV is much better, at 0.95. These results are

comparable to those found in the only other study by Durkin et al. in 2014 that investigated the diagnostic contribution of FFOCT in Mohs peripheral cut analysis.¹⁸ In this study, 2 pathologists compared 18 peripheral FFOCT-fixed tissue cuts to extemporaneous histological sections in a double-blind fashion. The results were a PPV of 66% and an NPV of 100%.¹⁸

For debulking, we had 3 FP (lesions N°13, 14, and 21). For lesion N°13, we answered positively because we visualized architectural abnormalities without visualizing clear characteristic structures of BCC. The histology concluded with an actinic keratosis. Notably, the preoperative BCC was diagnosed based on the clinic and not on a gold standard biopsy for this lesion.²⁶ For lesion N°21, histology concluded that it was a nodular BCC with a lesion-free deep surgical section (0.4 mm distant from the surgical section). The FFOCT image was labeled as positive because we visualized patches that were considered suspicious but did not visualize the characteristic lobules suggestive of BCC. For lesion N°14, standard histology concluded with an infiltrating BCC with a DM at 250 µm. Even though it theoretically could not have been visualized using the FFOCT image, as it passed 15 µm from the surgical section, we had the impression of seeing lobules labeled "ill-defined." We had 3 discordant lesions out of the 24 lesions in the peripheral cuts, 2 FP (lesions N°15 and N°21), and 1 FN (N°11). For lesion N°15, as for reading the FFOCT images of the debulking, we visualized doubtful areas without visualizing architectural structures suggestive of BCC. For lesion N°21, the FFOCT images did not show abnormalities suggestive of BCC but rather significant areas of disorganized fibrosis, an indirect argument for the presence of BCC; hence, the positive label. For lesion N°11, the FFOCT image was interpreted as negative while the lesion was in contact with the lateral surgical section between 5-9 h. An explanation for this discrepancy is that the FFOCT image, image plane 15 µm from the surgical section, showed only 25-50% of the lateral margins, and this was irregular and incomplete between 5-9 h. For these debulked specimen, perpendicular sectioning was used for histological analysis. However, horizontal or tangential orientation is used for the FFOCT
images. This would potentially have an impact on margin assessment, i.e., some of the 4 FP obtained in the FFOCT column may have, in fact, been TP if the full surgical margin had been assessed histologically. This point could potentially increase the PPV for the debulking results.

The technique for flattening the peripheral cut should be further improved. As mentioned above, the FFOCT image is only correctly interpretable to a depth of 60 to 80 µm. However, even with our method of flattening the part, we could not visualize the entire lateral edges at once in a single image. This problem was previously discussed by Durkin et al. in 2014 (19). They did not describe the specifics of their surgical cut. However, they suggested adapting the specimen holder and giving it a concave shape to best fit the peripheral cut's cupuliform shape.¹⁸ In our study, we attempted to remedy this problem by making counter-incisions along with the entire height of the epidermis at the cardinal points and a partial counter-incision on the inner side of the epidermis (Figure 2A & 2B). However, at the micron scale, this technical trick is not sufficient. The Tübingen or “cake” method, according to V. Chaussade²⁷ may be the solution until the developments in FFOCT system provide better resolution at deeper planes (up to 200 µm).

Finally, since this study was done, advancements have been made in FFOCT technology.²⁸ Higher resolution and sensitivity improvements have allowed better cellular-level imaging in ophthalmic imaging.²⁸ Advances in the quantum efficiency of cameras have also improved image contrast and depth penetration.²⁸ An increased field of view has been developed with newer systems using stitching algorithms to expand the imaging area, making it more suitable for Mohs surgery and large tissue evaluation.²⁹ Artificial intelligence could also be integrated to help distinguish between healthy and pathological tissues with greater accuracy.

## Conclusion

With further optimization, analyzing skin tumors ex vivo using FFOCT will allow tangential histological analysis of fresh tissue, with no additional preparation for providing digital images that can be transmitted remotely.

## Ethical approval

Not required.

## Funding

No funding was received for this article.

## Financial disclosure statement

The authors have nothing to disclose.

## Conflicts of interest

None declared.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bjps.2025.03.025.

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