Issue: May 10, 2021
Disclosures: Agarwal and Kumar report no relevant financial disclosures.
May 04, 2021
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Immediate preoperative FD-OCT used to measure ocular surface squamous neoplasia

Issue: May 10, 2021
Disclosures: Agarwal and Kumar report no relevant financial disclosures.
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Ocular surface squamous neoplasia encompasses the spectrum of pathological lesions ranging from preinvasive dysplasia to invasive squamous cell carcinoma.

Recent advances in the evaluation and management of ocular surface squamous neoplasia (OSSN) have changed the trends in treatment protocols. There have been newer modalities of management such as immunotherapy and chemotherapy, but the gold standard is still surgical removal. However, there have always been setbacks due to unexplained recurrences.

Amar Agarwal
Amar Agarwal

OCT has been used for various indications in diagnosis and management of ophthalmic diseases. Even though intraoperative OCT has revolutionized the surgical field in real-time visualization of planes, especially in corneal surgeries, it has not been widely used in tumor excision. Moreover, in developing and third-world countries, it is not possible for every center to have intraoperative visualization technology. Therefore, we have used an immediate preoperative Fourier-domain OCT (FD-OCT) to measure the extent of the lesion using a commonly available commercial OCT platform. Using the FD-OCT measurements, the surgical clearance was performed and compared between the corneal and the conjunctival clearance.

Fourier-domain OCT

The preoperative assessment included comprehensive slit lamp biomicroscopy, FD-OCT of the mass (iVue, Optovue), visual acuity (Snellen distant visual acuity in decimal equivalent), IOP (noncontact tonometer in mm Hg), digital slit lamp photography (Topcon), fundus examination and lymph node examination. The goal of minimum follow-up was 12 months, and all clinical assessments, including the FD-OCT, were done at subsequent postoperative follow-ups. The FD-OCT has 840 nm wavelengths of light with a pupil exposure of 750 µW and had acquired 26,000 A-scans per second with a frame rate of 256 to 1,024 frames per second. The depth and transverse resolutions in tissue were 5 µm and 15 µm, respectively.

The line scan in the anterior segment module was performed in both longitudinal (on the mass) and cross-sectional (vertical line scans across the mass) axes within 24 to 72 hours before surgery (Figure 1). Line scans (6 mm) were taken on the corneal margin with 0.2 mm spacing (Figure 2). Surgical clearance was executed with respect to the extent of the lesion on OCT. Epithelial thickness, junctional epithelial thickness, height of the lesion, length, subepithelial infiltration and tear meniscus height on the corneal limbus were evaluated. Tear meniscus height at the limbus was determined by measuring the fluid level (as a hyperreflective line) between the corneal epithelium and the limbal mass on OCT. All FD-OCT scans and subsequent surgery were performed by a single investigator (Kumar). Measurements were done manually by tool calipers in the OCT window.

line scan
Figure 1. The line scan in the anterior segment module was performed in both longitudinal (on the mass) (a) and cross-sectional (vertical line scan across the mass) axes for limbal nodule diagnosed as dysplasia (b).

Source: Dhivya Ashok Kumar, MD, FRCS, FICO, FAICO, and Amar Agarwal, MS, FRCS, FRCOphth
Serial line scans
Figure 2. Serial line scans were taken on the corneal side with 0.2 mm spacing for extent of epithelial involvement in intraepithelial neoplasia.

The American Joint Committee on Cancer classification was used for preoperative clinical grading. Surgical removal was defined as excision of the mass along with the surrounding normal tissue of 3 mm above the measured size of the lesion on OCT. The preoperative OCT timing was fixed as up to 72 hours instead of 24 hours, so that the time duration would be feasible for patients from far places of accessibility, operator appointment and instrument availability. Abnormal epithelium (abrupt transition of epithelium, thickened epithelium, hypertrophy) and subepithelial (hyperreflectivity) OCT changes on the corneal side were considered for edge determination.

Surgical excision and histopathological evaluation

Under peribulbar anesthesia, surgical marking with gentian violet with respect to the measurements obtained from the FD-OCT was performed. A cotton gauze soaked in 20% ethyl alcohol was placed in the rim of the exposed corneal epithelium adjacent to the limbal mass for 30 seconds, and then corneal epithelial debridement was performed. The mass with 3-mm margins was excised by a “no-touch technique.”

A thin lamellar scleral flap to confirm the depth of penetration was sent for histopathological examination (HPE). A double freeze-slow thaw cryotherapy of the conjunctival margins was completed. The raw surface was covered by preserved amniotic membrane graft or directly closed with 6-0 polyglactin sutures. Each specimen was then carefully mounted with respect to its anatomic location, oriented along the respective margins on a sterile paper strip.

Three separate specimens, the corneal margin, the conjunctival margins and the underneath lamellar scleral bed, were sent for HPE. Irrespective of the side, either nasal or temporal lesion, the corneal side was the corneal for all lesions, while the conjunctival side had three margins: the superior, the inferior and the lateral. Therefore, the margin positivity was documented as above in the histopathology specimen. All tissues were fixed with formalin and embedded in paraffin. Sections were cut in 5-mm thickness and stained with hematoxylin and eosin. Postoperatively, the patient was started on four hourly topical antibiotic steroid combination and two hourly topical lubricants for 2 weeks and tapered subsequently.

On HPE, the lesions were classified as preinvasive (carcinoma in situ and dysplasia), invasive (squamous cell, or SCC) or benign (no atypia seen). Immediate preoperative OCT morphology was compared with the surgical clearance and the HPE outcomes. The corneal and the conjunctival margin clearances were compared after the HPE. Surgical clearance was defined as tumor-free margins in all the margins, the tumor-free corneal side and the tumor-free deep sclera. Surgical clearance with one positive margin was considered as incomplete clearance. Recurrence was defined as any lesion appearing in the already surgically excised region. New tumor was defined as any lesion that appeared in a site not surgically excised before. Topical mitomycin C 0.04% three times per day and topical lubricants were given for eyes with margin positivity in SCC cases in a week on-week off cycle schedule for 3 months.

FD-OCT morphological analysis

Out of 17 eyes of 16 patients with suspected OSSN, 15 patients had unilateral disease and one patient had bilateral disease. The mean age of the patients was 56 ± 12.1 years (11 men and five women). The mean epithelial thickness and junctional epithelial thickness were 57 ± 9.6 µm (range, 40 µm to 76 µm) and 123.3 ± 57.8 µm (range, 63 µm to 276 µm), respectively. The mean horizontal and vertical dimensions as measured by OCT were 5.1 ± 4.4 mm and 5 ± 4.1 mm, respectively.

The mean height or elevation above the conjunctival surface was 572.5 ± 464.8 µm. Hyperkeratosis was visualized as a hyperreflective surface on FD-OCT in 17 eyes. The tear meniscus was absent in 70.5% eyes, while the mean height was 49.1 ± 50.1 µm in 29.4% eyes. Corneal involvement, seen as epithelial encroachment and subepithelial infiltration, was observed in 12 eyes on OCT. The subepithelial infiltration of growth was noted as a hyperreflective extension beneath the epithelium beyond the visible mass, measuring about 1.35 ± 1.5 mm. Surface pigmentation was noted in three of the 17 eyes (17.6%). Posterior back-shadowing was seen in all lesions on OCT. There was significant correlation between the height and the length of the lesion (P = .000). The longer the dimension, the greater the height of the mass. There was also a statistical correlation between the height of the lesion and the length of the subepithelial infiltration (P = .018) on OCT and also between the length of the lesion and the subepithelial infiltration (P = .000).

Histopathology correlation

Out of 17 eyes, 13 lesions (76.5%) were malignant and four lesions (23.5%) were benign. Among the 13 malignant lesions, nine were noninvasive and four were invasive. The histopathological classification of invasive type was well-differentiated SCC in two cases and moderately differentiated SCC in two cases. For the malignant noninvasive lesions, four were carcinoma in situ (CIN) and five had dysplasia. Of the five eyes with dysplasia, two were severe grade and three were moderate grade (Figure 3). In the CIN group, all four eyes were grade 2. There was no association between histopathology and age of the patient, mass size, external pigmentation and surface hyperkeratosis. There was no association between subepithelial infiltration extent and histopathology. Corneal involvement on histopathology was noted in seven eyes.

limbal mass
Figure 3. Preoperative OCT of limbal mass (a), which was diagnosed as severe dysplasia and the subsequent postoperative OCT (b). Preoperative (c) and immediate postoperative clinical picture (d) of the same eye.

Margin clearance and recurrence

The gross specimen as measured by the pathologist was 6.1 ± 5.8 mm, which was larger than the size measured on FD-OCT, as expected. Corneal clearance was noted to be 100% in all eyes, while conjunctival margin clearance was 77%. Out of 13 eyes (39 conjunctival margins) with malignant transformation, margin positivity in histopathology was noted as lateral in four cases, inferior in one case and superior in two cases. The four cases of CIN had 92% margin clearance as compared with four cases of SCC that had 75% clearance. Three out of four eyes that showed positive lateral margin were closed with direct conjunctival closure, and one eye was closed by amniotic membrane graft.

Two out of 13 eyes (15.3%) had recurrence. The mean recurrence time was 3 months postoperatively. The preoperative histopathologies of the recurrent lesions were invasive SCC and moderate dysplasia. Both patients were started on topical chemotherapy by eye drops MMC 0.04% four times daily as week on-week off therapy for 3 months. Additionally, the patient with invasive SCC required mass re-excision with partial keratectomy. Overall, four eyes were started on postoperative MMC 0.04% chemotherapy (even one margin was noted positive). Out of four eyes with positive lateral margin, two eyes showed recurrence. All patients with well-differentiated or invasive SCC were started on MMC 0.04% for 1 month in the postoperative period. Postoperative FD-OCT showed a regular limbal surface with absence of preoperative hyperreflective elevation (Figure 4). Scar tissue was seen as a hyperreflection on the surface with conjunctivalization of the limbus (Figure 5).

moderate dysplasia
Figure 4. Preoperative clinical picture (a) and the corresponding OCT (b) of patient who had moderate dysplasia on histopathology and the follow-up clinical picture (c) and OCT (d) at 6 months.
FD-OCT
Figure 5. Preoperative dysplasia (a) and postoperative (b) FD-OCT showing scar as a hyperreflective surface.

OCT in OSSN management

OCT is a noninvasive evaluation method of OSSN. Karp and colleagues have studied the use of high-resolution OCT in surgical management of OSSN and reported that the optical identification of tumor margins could potentially decrease the incidence of residual positive margins and minimize healthy tissue removal. They successfully used high-resolution OCT to detect preoperative anatomical extent of OSSN and concluded that it has the potential to predict histologic tumor margins. The algorithm used by them was different from our method.

The current study’s limitations are that FD-OCT has limited penetration through the abnormal thickened epithelium and that conjunctival lesions are difficult to assess compared with corneal lesions due to backscatter from the sclera. FD-OCT could not absolutely reflect the extent on the conjunctival side of the lesions due to the histological difference (underlying thick sclera) as compared with the cornea (underlying aqueous that acts as dark contrast). Other limitations include a varied group of patients (small to giant OSSN and both invasive and noninvasive pathology) included in the same analysis; the small sample size; and the exclusion of non-surgical management such as immunotherapy and chemotherapy. However, there are no studies using immediate preoperative (24 to 72 hours) FD-OCT data in surgical decision-making that have compared the corneal and the conjunctival clearance.

With the presented data, we cannot claim that the immediate OCT aided in complete corneal clearance, given that there is no comparison to having performed the surgery without immediate OCT. The study only highlights that immediate preoperative OCT can be an added value to measure the extent, predominantly corneal. We recommend having comparative trials on corneal clearance with and without OCT-guided methods in the future, which can add more light to the scenario.