Copyright © 2012 OA Publishing London

Hamdoon Z, Jerjes W, Upile T, McKenzie G, Jay A, Hopper C. Structural validation of facial skin using optical coherence tomography. Head Neck Oncol. 2012 Oct 15;4(3):67.

Com

petin

g in

tere

sts:

non

e de

clar

ed. C

onfli

ct o

f int

eres

ts: n

one

decl

ared

. Al

l aut

hors

con

trib

uted

to c

once

ptio

n an

d de

sign,

man

uscr

ipt p

repa

ratio

n, re

ad a

nd a

ppro

ved

the

final

man

uscr

ipt.

Al

l aut

hors

abi

de b

y th

e As

soci

atio

n fo

r Med

ical

Eth

ics (

AME)

eth

ical

rule

s of d

isclo

sure

.

Structural validation of facial skin using optical coherence tomography

Zaid Hamdoon1,2,3,4

Waseem Jerjes1,5,6 Tahwinder Upile4

Gordon McKenzie4

Amrita Jay4

Colin Hopper2,4,5 (1) Department of Oral and Maxillofacial Surgery, School of Dentistry, AL-Yarmouk University College, Baghdad, Iraq (2) Unit of Oral and Maxillofacial Surgery, UCL Eastman Dental Institute, London, UK (3) Department of Oral and Maxillofacial Surgery, University of Mosul, Mosul, Iraq (4) Head and Neck Centre, University College London Hospitals, London, UK (5) Department of Surgery, UCL Medical School, London, UK (6) Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK UCorresponding authors Zaid Hamdoon Department of Oral and Maxillofacial Surgery, University of Mosul, Mosul, Iraq ZH: zaid19772000@yahoo.com WJ: waseem_wk1@yahoo.co.uk GM: 2TUgordon.mckenzie@md-ltd.co.ukU2T TU: mrtupile@yahoo.com AJ: amritajk@msn.com CH: c.hopper@ucl.ac.uk

Abstract Background Skin cancer is the most common malignancy in the Western world. Optical coherence tomography (OCT), an optical imaging modality, offers real-time imaging with micrometer resolution and has great potential for mapping skin pathologies. Objectives In this immediate ex-vivo skin-OCT study we aimed to (1) identify the cellular structures of normal facial skin using OCT and compare them to the gold standard histopathology, (2) differentiate between normal and pathological facial skin, and (3) create an optical (OCT) diagnostic criterion for the detection of suspicious facial skin lesions. Material and Methods A total of 53 patients, with 57 suspicious facial lesions, participated in this clinical study. Each patient had the skin lesion fully excised. The tissue was then subjected to OCT in the immediate ex-vivo phase. Histopathological evaluation revealed that 26 patients had basal cell carcinoma (BCC), 12 had squamous cell carcinoma (SCC), 9 had actinic keratosis (AK), 7 had invasive malignant melanoma (MM) and 3 had lentigo maligna (LM). The normal microanatomical structures were identified and studied on optical biopsy. Then the common architectural changes on OCT and histopathology paraffin slides were correlated. Results Analysis of the OCT images facilitated the identification of normal architecture of the facial skin. In this study, the stratum corneum, epidermis and papillary dermis could be easily identified in macroscopically normal areas. Pathologically, OCT could differentiate between BCC subtypes due to different manifestations of backscattering. Morphological and light-scattering changes were visible in the stratum corneum layer in actinic keratosis. OCT features in SCC had more of a snowstorm appearance than BCC, but were still distinguishable. Malignant melanoma and lentigo maligna were potentially diagnosable using OCT. According to the pathology criteria, there was overall 87% OCT suspicious features of melanoma. Multicomponent specific OCT patterns were observed in 93% of all types of BCC and in 95% for the nodular type. With SCC, 92% OCT patterns were the most commonly observed and 73% and 95% for lentigo maligna and actinic keratosis, respectively. Conclusion This study proved the success of OCT in identifying structural changes in healthy and pathological facial skin. Further studies to prove its usefulness in vivo are required.

Introduction Skin cancer is the most frequent cancer in the western world. It encompasses basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and malignant melanoma (MM) (1). The incidence of skin cancer is still increasing globally, which introduces a huge health and financial burden on health systems (2,3). This trend can be counteracted by means of primary prevention (avoidance of risk factors) and secondary prevention (early diagnosis and intervention). Early diagnosis is usually achieved by regular visual inspection and a surgical biopsy when required. It is clearly impractical to biopsy everyone, and there are no consistent guidelines for taking a biopsy. Screening efforts using non-invasive real-time diagnostic tools are evolving. However, the lack of evidence for the efficacy of these tools remains a real obstacle to adequate skin cancer screening and may account for delayed diagnosis in some cases (4). In order to increase the sensitivity and specificity of early detection and monitoring, strong evidence-based studies are required before any tool can be introduced into the clinical setting. The development of optical diagnostics is a current necessity. The goal is to provide real-time non-invasive diagnoses of suspect tissues. This will help reduce tissue trauma, reduce the work load on pathology departments and reduce the time the anxious patient has to wait for a diagnosis (5,6). A light of a specific wavelength is fired into tissue and the reflected light is measured in such a way that a specific optical signature can be generated (i.e. cellular and subcellular changes in elastic scattering spectroscopy, molecular vibration in Raman spectroscopy, surface morphology by microendoscopy and biochemical changes from tissue fluorescence). The main aim of these optical techniques is, eventually, to act as an adjunct to histopathology to provide immediate diagnosis of lesions and aid in monitoring lesions with risk of malignant transformation in the clinical setting (7). Optical coherence tomography (OCT), first applied in 1991 by Huang et al., is a non-invasive interferometric (superimposing or interfering waves) tomographic imaging modality that allows millimetre penetration with micrometre-scale axial and lateral resolution, and provides morphological information similar to pathology. OCT is an optical technique that uses a low-coherence interferometer to develop a two dimensional image of optical scattering from internal tissue microstructures. OCT also allows non-invasive monitoring of morphologic changes in skin diseases and may have a particular role in the monitoring of medical treatment of non-melanoma skin cancer. Several technical modifications are being implemented to further improve the quality of images of this technology.

For OCT to become clinically interpretable and relevant, the structures visualised must be correlated with the corresponding tissue microstructures. To date, the interpretation of OCT images has been largely intuitive and empirical (8). We designed this immediate ex-vivo study to (1) identify cellular structures in normal facial skin using OCT and compare them to the gold standard histology, (2) differentiate between structural changes in normal and pathological facial skin, and (3) build on a diagnostic criterion in the use of OCT for the detection of suspicious facial skin lesions using a commercially approved machine.

Material and methods Fifty-seven facial lesions from 53 patients who presented with suspicious lesions to the UCLH Head & Neck Centre, London, were recruited for this study. The study protocol was approved by the local committees of ethics for human research. The protocol was devised in cooperation with the Departments of Pathology at University College London and Imperial College. Informed consent was obtained from each patient explaining the nature of the study. Inclusion criteria included a minimum age of 18 years and no prior skin cancer in the same area or any skin procedures (i.e., electrosurgical excision, PDT, cryotherapy…etc.). In this study we used a swept-source frequency-domain optical coherence tomography microscope (Michelson Diagnostics EX1301 OCT Microscope V1.0), the components of which are illustrated in Figure 1. The light source used was a Santec HSL-2000, with an imaging wavelength of 1310nm, axial optical resolution of <10µm, and lateral optical resolution of <10µm. The system provides an image resolution of 5.3µmpixels with a maximum image width of 6mm, a sub-surface imaging depth of 1.5mm, and a focal depth of 1mm. Samples can be manipulated to see full quality results on the screen instantly, with an image capture time of <100ms and refresh rate of >1Hz. The multi-beam swept source OCT EX1301 (Michelson Diagnostics Ltd., Orpington, UK) utilises a novel optical set-up involving multiple optical channels which does not suffer from loss of sensitivity or other serious drawbacks. The idea is to partition the depth of field into sub-fields, and provide a separately focused beam for each sub-field. The laser beam in the SS-OCT EX1301 is split into 5 ‘beamlets’ using an etalon-type ‘rattle plate’ prior to the interferometer beam splitter. Four of these beams are used to scan the specimens and are relayed back to an array of photodiodes where they interfere with four reference beams in the conventional manner. The fifth beam is imaged onto a 5th photodiode to generate a balance signal. Each patient underwent full excision followed by OCT of the excised lesion in the immediate ex-vivo phase. The specimens were kept in saline until being transferred for scanning. The OCT instrument captured b-mode scans of the tissue. Digital pictures and diagrams were produced to ensure that the histopathologist would be able to identify the scanned planes accurately and provide an exact histopathological image. Our co-registration method was enhanced by using dyes and sutures for better orientation. A histopathological diagnosis was then achieved after several steps, including embedding in paraffin wax, staining with haematoxylin and eosin, and examination by light microscopy. Close attention was paid to tissue shrinkage

in formalin when comparing microanatomical structures of immediate ex-vivo OCT images and paraffin wax slides. Each whole skin specimen was placed under the OCT imaging beam and imaged ex vivo as the transverse virtual line between the two landmarks (Figure 2). To provide a linear focal trough, a series of images were taken from edge to edge along the whole length of the specimen. All the pathology slides were examined and the most obvious architectural changes were recorded. Several variables were studied on the OCT images to assess micro-anatomical structures of normal facial skin and architectural changes in these areas. These included visibility of the stratum corneum, epidermis and papillary dermis and other micro-anatomical structures. Controlled OCT measurements were taken from the edges of the macroscopically normal facial skin of the surgical biopsy; this was compared to OCT images of the suspect area taken from the centre of the lesion. These variables were compared between OCT and pathology by a senior clinician and a senior pathologist who were trained to read OCT images and not blind to the diagnosis. Descriptive diagnostic criteria were then extracted from each OCT image and its corresponding pathology according to agreement between the two assessors.

Results All data were entered and stored in a computerized database designed with Microsoft Excel 2000. The statistical analysis was performed using the statistical software package SPSS 13.0 (SPSS, Chicago, Ill). Fifty–seven clinical and pathologically different skin lesions were obtained from 53 patients. The lesions were obtained from 34 women (64.1%) and 19 men (35.9%) ranging in age from 38 to 84 years (median age: 48.4 years). Twenty-nine (53%) were located on the cheek, 16 (28%) on the nose, 6 (10.5%) on the ear, 3 (5.0%) on the forehead and 3 (5.0%) on the lower lip. Histo-pathological evaluation revealed that 26 (45.5%) patients had basal cell carcinoma (BCC), 12 (21%) had squamous cell carcinoma (SCC), 9 (15.9%) had actinic keratosis (AK), 7 (12.3%) had invasive malignant melanoma (MM) and 3 (5.3%) were diagnosed with lentigo maligna (LM). The most common histological subtypes for the BCC were nodular (53.5%), major cystic (21.5%), and microcystic (17.8%) and the least common type was superficial (7.2%) BCC. The majority of SCCs were located on the ear, followed by cheek and forehead (Table1). OCT and histology correlation (normal margins) OCT was able to visualise and differentiate skin layers and structures such as stratum corneum, epidermis, and papillary dermis. Only a few large and prominent epidermal papilla has been correlated in 50% with gold standard histology. Describing features in normal facial skin was straightforward for the assessors (Figure 3). The dermal-epidermal junction, the epidermal layer and the stratum corneum could all be identified in 100% of the specimens (Table2). OCT and histopathology correlation With respect to actinic keratosis, correlation was achieved in 60% of the specimens by identifying thickening in the DEJ, in 100% by identifying destruction in the corneum layer and in 89% by identifying thickening in this layer. With regard to basal cell carcinoma, correlation was achieved in 100% of the specimens by identifying the dermal-epidermal junction, and in 100% by identifying the epidermal, keratin cell layer. With nodular BCC, areas beyond the dermal-epidermal junction exhibited a solid nest or honeycomb form in 93% of cases, while major or microcystic BCC showed small multiple empty spaces in 100% of cases. Superficial spreading BCC were mainly represented by cords or small buds protruding from the epidermis into the superficial dermis in 100% of cases. With regard to squamous cell carcinoma, correlation was achieved in 91% of the specimens by identifying the dermal-epidermal junction as damaged, 83% by identifying the epidermal layer as increased in thickness, and 100% by description of the stratum corneum layer and its changes. Other features

included small bright clusters (nonhomogenous) within the papillary dermis (80%). With regards to lentigo maligna, correlation was achieved in 30% of the specimens by identifying the rete ridges as elongated, and in 30% by identifying nests or lobules within the epidermal layer near the DEJ. With regards to malignant melanoma, correlation was achieved in 85% of the specimens by identifying abnormal signals within the papillary dermis layer, which exhibited diffuse or patchy reflectivity and loss of the typical bright horizontal linear structures, and in 57% the dermal-epidermal junction was described as intact with prominent junctional densities (Tables 3,4). Qualitative OCT analysis for different pathologies In normal skin, the majority of the top layer (stratum corneum (SC)) shows more signal reflection than the lower layer that represents the epidermis. The SC was fissured and wrinkled in areas on the face. The epidermis, the second layer below the SC layer, appears significantly darker (low signal reflection) until the transition with the dermis. The signal intensity at the lower boundary of the epidermis represents the dermo-epidermal junction. There is no boundary for the dermo-epidermal junction except the smooth or undulated demarcation between the less reflective/backscattering layer of the epidermis to the highly reflective/backscattering papillary dermal layer. Description of stratum corneum layer (SC) In AK, the SC layer demonstrates hyper-reflective features (bright) due to a water/SC refractive index difference, with mostly focal areas of damage. The damaged area appears as a punched out depression, with or without lifting of the stub from the epidermal layer (Figure 4). In nodular and infiltrating BCCs, this layer has normal-reflective features because the tumour infiltrates the deeper tissue, dissimilar to the ulcerative type. In LM and MM this layer either disappears or presents very thin hypo-reflective features. Description of epidermal layer (EL) Epidermal thickness is of considerable significance in AK, with homogeneity similar to the surrounding normal margins. The situation is similar in BCCs with dark spaces (solid or empty) of different sizes within the epidermal layer (Figures 5,6). Unlike with AK, BCCs have EP layer thicknesses that vary significantly for different histologic sub-types. In LM, the EP layer usually becomes atrophied in the active areas, with some hypoecoic areas that represent junctional activity in the rete ridges (Figure 7). With MM, elongated rete ridges are mainly seen (Figure 8). Description of dermal layer In AK, the dermal layer is completely normal with signal-poor snail-track like cavities corresponding to blood vessels, hair follicles and some sweat glands. Some BCC tumours aggregate as signal-poor round spots surrounded by hyporeflective bands.

For SCCs and MMs, these non-homogenous areas are signal-free globules, and represent concentrations of melanin or keratin pearl in spherical or elliptical arrangements below breached areas in the dermo-epidermal junction, usually extending from the EL so there is no band surrounding the spaces. Description of dermal-epidermal junction (DEJ) The dermo-epidermal junction is rather flat with an intact border between the epidermis and dermis, as in the case of AK and BCC. Breaches in cases of SCC (Figures 9, 10) may disappear in infiltrative growing melanoma or display a typical saw tooth appearance from damage to the junction which pushes the rete ridges in early stage cases. These appear as echo-poor, low scattering projections sharply demarcated from the echo-rich homogeneous dermis. Diagnostic criteria In the case of the stratum corneum (SC), OCT images were assessed with regard to reflectivity as hyper-reflective (thick), normal-reflective or hypo-reflective. Surface damage confined within the stratum corneum is one of the most important diagnostic criteria. Epidermal layer thickness, whether increased, decreased or no change according to normal margins was also assessed. The epidermal layer may show nonhomogeneous lobules with active single or multiple tumour nest collection lobules inside. DEJ may be demarcated without protrusion toward the dermis, demarcated with protrusion or non-demarcated (breach). Signs within the papillary dermis include homogenous or non-homogenous signal indicating connective tissue invasion. The presence of solid or empty spaces surrounded with hyper-reflective band within the dermal layer was found in BCCs. The main architectural feature for AC was hyper-reflectivity and/or disruption of the stratum corneum and demarcated DEJ. For squamous cell skin cancer, the DEJ lost its integrity; other features included disintegration of superficial epidermal layers (honeycombing and cobblestoning) with small bright clusters within the papillary dermis. With melanoma, non-edged papillae, cerebriform clusters infiltrating dermal papillae, diffuse or patchy reflectivity, loss of the typical bright horizontal linear structures were diagnostic. Lentigo maligna was characterised by predominantly uniformly elongated rete ridges as well as uniform nests within the epidermis (Table 5).

Discussion This study reports on the morphologic features observed in OCT imaging performed ex-vivo on fresh facial skin tissue specimens. Although the sample size is small, it represents the range of relevant morphologies needed for their future use for in-vivo diagnosis, and the results indicate that morphological changes on OCT images match histopathology. To interpret OCT images of abnormal skin, it is necessary to be familiar with normal skin architecture. In normal skin histology, the epidermis is comprised primarily of keratinocytes and a smaller population of dendritic cells, which are the melanocytes and Langerhans cells. The dermis is comprised of blood vessels, nerves, inflammatory cells and fibroblasts enmeshed with collagen fibres and elastin. The papillary dermis comprises projections into the epidermis, called dermal papillae. The meeting point between the epidermis and the dermis is called the dermal-epidermal junction. In the epidermis, the keratinocytes are differentiated to form four different layers. The stratum corneal is the most superficial layer of the epidermis, which is comprised of flat anucleated keratinocytes. Histopathologically, there are many histological variants of BCC, including nodular, superficial, morpheaform (or sclerosing), infiltrative and micronodular (9). A typical BCC lesion has a pearly or waxy appearance. The shape of the lesion is flat or slightly raised, white or light pink, flesh-coloured or brown, with visible blood vessels in the lesion or adjacent skin. It usually has a central ulceration and a well-defined border. It may also appear as a scar-like lesion without a history of injury to the skin in that area. The less common infiltrative type can be seen as poorly defined, lightly pigmented, indurated, flat skin lesions, occasionally with overlying telangiectasia. Squamous cell carcinoma is the second most common skin cancer, representing 20% of all cutaneous malignancies (10). The histologic spectrum of squamous cell carcinoma begins with actinic keratosis (AK). Actinic keratoses (AK) are the most common pre-malignant skin pathology (11). Histologically, the difference between AK, SCC in situ and frank SCC is described as such; AK involves only part of the epidermis, SCC in situ occupies the full thickness of the epidermis, and invasive SCC penetrates the basement membrane of the epidermis. In the current study, the results showed that traditional normal histological landmarks such as the stratum corneum, dermo-epidermal junction and papillary dermal layer can be clearly identified using OCT. This study indicates that the characteristic layering of normal skin is lost both in BCCs and SCCs, and this is identifiable on OCT. The depth of the OCT in this study was limited to a maximum of only 2mm. which is satisfactory to visualise the epidermis and dermis layers where most of the pathological changes occur. The axial resolution of 10µm would not allow detection of single cells. However, the investigation of single cells or subcellular structural changes is a part of ongoing research. Unlike other

imaging modalities, reflectance confocal microscopy enables identification of these layers based on architecture and cytological characteristics. This layer varies in depth depending on anatomical site and sun exposure. Under confocal microscopy, it produces a bright image. Large polygonal anucleated korneocytes (25–50µm) can be seen. Dermatoglyphs appear as dark linear valleys amidst the korneocyte groups. In comparison with OCT, conofocal microscopy has higher resolution than OCT. However, the depth of detection is less than OCT. One of the great advantages of OCT, which cannot be seen by confocal microscopy, is the possibility of analysing micro-anatomical structures up to a depth of 1500µm. This means any abnormalities extending from the papillary dermis to the reticular dermis are diagnosable. For this reason, tumours that invade the depths can be more properly assessed. This includes areas where the epidermis has a shallow thickness, and part of the reticular epidermis can also be assessed (12) Further technical development of OCT, such as polarization-sensitive OCT (PS-OCT) or speckle-reduced OCT may increase the diagnostic accuracy. Finally, the introduction of image analysis, machine learning algorithms or neural networks may provide a more precise classification of AK and BCC lesions than relying on the human eye alone. In the case of nodular BCC, a single significant feature (i.e. the formation of tumour aggregates in the upper dermis) was found to be characteristic of the investigated lesions. This study revealed that the diagnostic ability of OCT is ideally valuable in detecting sub-types and early stages of BCC; however, partial loss of normal OCT architecture may not be seen in various pre-BCC lesions. With regard to AK lesions, focal changes and thickening of the epidermis, OCT investigation of the AC showed another diagnostic feature in the form of buds and irregular proliferation of tumour tissue attached to the undersurface of the epidermis or in the form of a very distinct thickening of the dermo-epidermal layer. The dermo-epidermal junction appeared less well demarcated compared to healthy skin, which is probably due to cellular aggregates of this lesion. OCT’s ability to detect cell aggregation at the dermo-epidermal junction, at the pre-invasion stage, is a very profound finding in detecting the early invasive phase of skin cancer. Korde and colleagues (13) studied OCT images of sun damaged skin and AK and described characteristic dark elongated bands in the epidermis for accurate diagnosis of AK. These bands correspond to keratin deposits in a thickened stratum corneum. The main problem in AK diagnosis is the thickening of the stratum corneum layer, which reduces the penetration depth of OCT; this is attributed to the optical properties of hyperkeratosis in AK.

Features of SCC are distinct by OCT. However, this study did not evaluate the sensitivity and specificity of the OCT. Both BCCs and SCCs in the current study demonstrated very reliable diagnostic features, contrary to the finding of Mogensen et al (14), who concluded that the naked eye is superior to OCT for diagnosis of non-melanoma skin cancer. Other researchers have performed OCT studies on BCC. They found that the epidermis and associated basal cell carcinoma had identifiable structural features that were apparent in both the OCT images and histologic pictures. The lobules, islands, and infiltrating strands of basal cell carcinoma appeared similar in OCT images and histologic sections regardless of the type of tumour (15). Interestingly, this study demonstrated that OCT is capable of distinguishing malignant melanoma, with the ability to differentiate the early stage of lentigo maligna. Furthermore, OCT was still helpful in identifying the late stage of lentigo maligna. In malignant melanoma, OCT images show irregular structures in the lower epidermis with the dermo-epidermal junction. De Giorgi et al (16) recently performed a pilot study imaging nine dysplastic nevi and one superficial spreading melanoma by OCT in vivo. The authors reported that in selected cases OCT allows for an in vivo correlation to be established between surface dermatoscopic parameters and histo-pathological correlates, in particular the pigment network and brown globules. Another study conducted on melanoma skin lesions failed to detect any definitive features in OCT images enabling differentiation of tumour subtypes (i.e. junctional nevi, compound nevi) but demonstrated useful discriminating parameters between benign nevi and malignant melanoma (17). Our methodology of histological serial slices could play an important role in correlating the OCT scanning lane with the histological section. However, deformation by histological processing and slightly different sectioning levels are unavoidable and can make direct comparisons of histological pictures and OCT images appear difficult. Nevertheless, this happened in only four cases, in which it was still possible to demonstrate the same morphological structures with OCT imaging and by histological investigation. However, this is one of the limitations of this study. Precise control of the histological plane is difficult, and combined with processing artifacts in histology, this makes precise one-to-one registration of histology to OCT/ OCM images challenging. Therefore, the assessments of image and histological data are based on correspondences in feature information. Another limitation in the current study is the relatively small sample size, which prevents us from determining OCT’s sensitivity and specificity for assessment of skin malignancy. There are three basic differences in OCT in relation to the conventional histology routine. In OCT, images are obtained horizontally from the lesion, whereas in conventional histology the sections are made vertically. The

images are obtained in gray scale, similar to what happens in radiographs. Moreover, OCT imaging can provide instantaneous dynamic images of the skin and may in the case of in vivo OCT show events such as blood flow, in poor agreement with routine histology using formalin-fixed and paraffin-embedded cut sections. Our observations in this descriptive study indicate that despite the ex vivo nature of the specimens, the level of resolution of OCT seems adequate for clinical practice. Moreover, it suggests that in the future resolution using in vivo OCT systems will improve due to tissue perfusion, which may also further improve the diagnostic potential of the method for all skin cancers. OCT might also be a promising tool for the characterisation of melanoma skin lesions.

Conclusion This study proved the success of OCT in identifying structural changes in healthy and pathological facial skin. Further studies to prove its usefulness in vivo are required. Competing interests Mr Colin Hopper is an Advisory Board Member at Michelson Diagnostics, Kent, UK. Dr Gordon McKenzie is a Medical Applications Director at Michelson Diagnostics, Kent, UK. Authors’ contributions All authors designed and performed the study, carried out the literature research and manuscript preparation. All authors were responsible for critical revision of the scientific content and manuscript review. All authors approved the final version of the manuscript.

References 1- Diepgen TL, Mahler V. The epidemiology of skin cancer. Br J Dermatol 2002; 146 (Suppl. 61): 1–6. 2- Holme SA, Malinovszky K, Roberts DL. Changing trends in nonmelanoma skin cancer in South Wales 1988–98. Br J Dermatol 2000; 143:1224–9. 3- Levi F, La Vecchia C, Te V-C, et al. Descriptive epidemiology of skin cancer in the Swiss Canton of Vaud. Int J Cancer 1988;42:811-6. 4-Heymann WR. Screening for melanoma. J Am Acad Dermatol 2007;56:144-5. 5-Jerjes WK, Upile T, Wong BJ, Betz CS, Sterenborg HJ, Witjes MJ, Berg K, van Veen R, Biel MA, El-Naggar AK, Mosse CA, Olivo M, Richards-Kortum R, Robinson DJ, Rosen J, Yodh AG, Kendall C, Ilgner JF, Amelink A, Bagnato V, Barr H, Bolotine L, Bigio I, Chen Z, Choo-Smith LP, D'Cruz AK, Gillenwater A, Leunig A, MacRobert AJ, McKenzie G, Sandison A, Soo KC, Stepp H, Stone N, Svanberg K, Tan IB, Wilson BC, Wolfsen H, Hopper C. The future of medical diagnostics: review paper. Head Neck Oncol. 2011 Aug 23;3:38. 6-Upile T, Jerjes WK, Sterenborg HJ, Wong BJ, El-Naggar AK, Ilgner JF, Sandison A, Witjes MJ, Biel MA, van Veen R, Hamdoon Z, Gillenwater A, Mosse CA, Robinson DJ, Betz CS, Stepp H, Bolotine L, McKenzie G, Barr H, Chen Z, Berg K, D'Cruz AK, Sudhoff H, Stone N, Kendall C, Fisher S, MacRobert AJ, Leunig A, Olivo M, Richards-Kortum R, Soo KC, Bagnato V, Choo-Smith LP, Svanberg K, Tan IB, Wilson BC, Wolfsen H, Bigio I, Yodh AG, Hopper C. At the frontiers of surgery: review. Head Neck Oncol. 2011 Feb 9;3(1):7. 7-Upile T, Jerjes W, Sterenborg HJ, El-Naggar AK, Sandison A, Witjes MJ, Biel MA, Bigio I, Wong BJ, Gillenwater A, MacRobert AJ, Robinson DJ, Betz CS, Stepp H, Bolotine L, McKenzie G, Mosse CA, Barr H, Chen Z, Berg K, D'Cruz AK, Stone N, Kendall C, Fisher S, Leunig A, Olivo M, Richards-Kortum R, Soo KC, Bagnato V, Choo-Smith LP, Svanberg K, Tan IB, Wilson BC, Wolfsen H, Yodh AG, Hopper C. Head & neck optical diagnostics: vision of the future of surgery. Head Neck Oncol. 2009 Jul 13;1:25. 8-Hamdoon Z, Jerjes W, Al-Delayme R, McKenzie G, Jay A, Hopper C. Structural validation of oral mucosal tissue using optical coherence tomography. Head Neck Oncol. 2012 Jun 6;4(1):29. 9- Hutcheson AC, Fisher AH, Lang Jr PG. Basal cell carcinomas with unusual histological patterns. J Am Acad Dermatol 2005;53(5): 833-7. 10- Miller DL, Weinstock MA. Nonmelanoma skin cancer in the United States: incidence. J Am Acad Dermatol 1994;30(5 Pt 1):774-8.

11- Heaphy MR Jr, Ackerman AB: The nature of actinic keratosis: a critical review in a historical perspective. J Am Acad Dermatol 2000; 43: 138–150. 12-. Sauermann K, Clemann S, Jaspers S, Gambichler T et al (2002) Age related changes of human skin investigated with histometric measurements by confocal laser scanning microscopy in vivo. Skin Res Technol 8:52–56. 13. Korde VR, Bonnema GT, Xu W, Krishnamurthy C, Ranger-Moore J, Saboda K, Slayton LD, Salasche SJ, Warneke JA, Alberts DS, Barton JK. Using optical coherence tomography to evaluate skin sun damage and precancer. Lasers Surg Med 2007;39:687–95. 14. Mogensen M, Joergensen TM, Nürnberg BM, Morsy HA, Thomsen JB, Thrane L, Jemec GB. Assessment of optical coherence tomography imaging in the diagnosis of non-melanoma skin cancer and benign lesions versus normal skin: observer-blinded evaluation by dermatologists and pathologists. Dermatol Surg. 2009 Jun;35(6):965-72. 15. Gambichler T, Orlikov A, Vasa R, Moussa G, Hoffmann K, Stu¨ cker M, et al. In vivo optical coherence tomography of basal cell carcinoma. J Dermatol Sci 2007;45:167-73. 16. de Giorgi V, Stante M, Massi D, Mavilia L, Cappugi P, Carli P. Possible histopathologic correlates of dermoscopic features in pigmented melanocytic lesions identified by means of optical coherence tomography. Exp Dermatol 2005;14:56-9. 17. Gambichler T, Regeniter P, Bechara FG, Orlikov A, Vasa R, Moussa G, Stücker M, Altmeyer P, Hoffmann K. Characterization of benign and malignant melanocytic skin lesions using optical coherence tomography in vivo. J Am Acad Dermatol. 2007 Oct;57(4):629-37.

Tables

Table 1: Patient demographic information

No. (%) No. (%) Gender Signs and symptoms Male 19 (35,9) Itchiness 15 (28.3) Female 34 (64.1) Symptomless 13 (24.5) Scaly 10 (18.8) Anatomical location Lumpy 10 (18.8) Cheek 29 (53) Bleeding 5 (9.4) Nose 16 ( 28) Ear 6 (10.5) Clinical features Forehead 3 (5) Papule 17 (29.8) Lower lip 3 (5) Erosion 15 (26.3) Ulcer 12 (21) Lesion colour Nodule 10 (17.5) Mixed 30 (52.6) Others 3 (5.2) Red 11 (19.2) Brown 7 (12.2) Histopathological diagnosis Black 4 (7) BCC 26 (45.5) White 3 (5.2) SCC 12 (21) Non-specific 6 (10.5) AK 9 (15.9)

LM 3 (5.3) Skin type MM 7 (12.3) Type II 21 (39.6) Type III 18 (33.9) Type VI 14 (26.4)

Table 2: Correlation between OCT and normal histology

Normal resection margins OCT Histology Total Identification of the stratum corneum Yes 30 30 60 No 0 0 0 Identification of the epidermis Yes 30 30 60 No 0 0 0

Identification of epidermal papilla 15 30

Identification of (DEJ) Yes 28 30 58 No 2 0 2 Homogenous papillary dermal layer Yes 30 30 60 No 0 0 0

Table 3: Common descriptive features between OCT and pathology from skin lesions agreed by two observers (I)

SCC OCT Pathology Total Dermal–epidermal junction (DEJ) lost integrity Yes 11 12 23 No 1 0 1 Superficial epidermal layers (honeycombed) Yes 5 6 11 No 7 6 13 Small bright cluster at the papillary dermis Yes 4 5 9 No 8 7 15 Stratum corneum disruption or thickening Yes 12 12 24 No 0 0 0

BCC OCT Pathology Total

Solid nest or honeycomb beyond (DEJ) 14 15 29 Yes 12 11 23 No Small multiple empty spaces Yes 8 8 16 No 18 18 36 Cystic BCC, large one or two empty space Yes 6 6 12 No 20 20 40 Cords or small buds protruding from epidermis Yes 2 2 4 No 24 24 48

Table 4: Common descriptive features between OCT and pathology from skin lesions agreed by two observers (II)

AK OCT Pathology Total Parakeratosis/hyperkeratosis Yes 8 9 17 No 1 0 1 Stratum corneum disruption Yes 9 9 18 No 0 0 0 Dermal–epidermal junction thickening Yes 3 5 8 No 6 4 10 Total Yes 20 23 No 7 4

MM OCT Pathology Total Non-edged rete ridges with clusters infiltrating papillary dermal layer Yes 4 7 11 No 3 0 3 Loss of typical bright horizontal linear structures Yes 6 7 13 No 1 0 1 Intact dermo–epidermal junction Yes 7 7 14 No 0 0 0

LM OCT Pathology Total Elongated rete ridges Yes 1 3 4 No 2 0 2 Uniform nest within the epidermis Yes 1 3 4 No 2 0 2

Table 5: Descriptive interpretation of OCT image changes

SC h

yper

refle

ctiv

e

SC h

ypor

efle

ctiv

e

SC

norm

oref

lect

ive

dam

aged

EL ↑

EL ↓

EL ↔

nest

or l

obul

e in

EL

ho

mog

enou

s EL

DEJ

dem

arca

ted

with

out p

rotr

usio

n to

war

d D

DEJ

dem

arca

ted

with

pro

trus

ion

tow

ard

D

DEJ

not

de

mar

cate

d em

pty

spac

e in

D

solid

spa

ce in

D

non

-hom

ogen

ous

D

Pathology entity BCC (n= 26) 10 5 8 3 7 3 16 17 9 18 2 0 16 9 1 SCC (n=12) 8 0 2 2 10 0 2 0 12 0 1 11 6 0 6 AK (n=9) 8 0 0 1 3 0 6 1 8 9 0 0 0 0 0 LM (n=3) 0 0 3 0 1 1 1 3 0 1 2 0 0 0 0 MM (n=7) 2 0 5 0 2 3 2 1 6 1 4 2 4 1 2 Normal resection margins (n=30)

0 0 30 0 0 0 30 0 30 30 0 0 0 0 0

SC=stratum corneum layer, EL=epidermis layer, DEJ=dermo-epidermal junction ↑=increase, ↓=decrease, ↔=no change, D= dermis.

Figures Figure 1: Schmatic view of the lab based OCT machine Figure 2: Specimen with orientation suture under OCT scanning arm with laser guiding beam. The scanned area is between the two reference sutures Figure 3: Histology versus OCT scan for normal skin margin of the cheek showing three distinctive layers. A: representing the stratum corneum, B: epidermis, C: DEJ, D: Dermis, E: papilla. Correlation was good for the stratum corneum, epidermis while few large and prominent papilla has been correlated Figure 4: AK showing damage to stratum corneum layer (A) with thick epidermis (B) but intact dermo-epidermal junction Figure 5: Mixed cystic solid BCC Figure 6: Giant cystic BCC showing lobular hypo-ecoic OCT feature occupying large portion of dermis layer Figure 7: Lentigo maligna, characterized by a predominantly uniformly elongated rete ridges (Arrow). Uniform nests within the epidermis (circle) Figure 8: Malignant melanoma, non-edged papillae (arrow), cerebriform clusters infiltrating dermal papillae (circle), diffuse or patchy reflectivity, partial loss of the typical bright horizontal linear structures Figure 9: SCC in situ (Bowen's disease) from temple showing thick stratum corenum ,hyperkeratosis (arrow), thickening of the epidermal layer with broadening of DEJ as early sign of papillary dermal layer invasion (circle) Figure 10: SCC showing the transitional area between intact and damaged DEJ (red arrow)