II.1 Laser-Scanning Confocal Microscopy

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Early detection is critical to the successful treat- ment and survival of patients with melanoma. a dermatologist’s sensitivity in detecting melano- ma by visual clinical examination is reported to be in the range of 65% to 80% [26, 42]. Dermos- copy, which provides a magnified image of a le- sion, can improve diagnostic accuracy to as high as 85% for clinicians who are highly trained in the use of dermoscopy [8]. However, sensitivity is still unacceptably low for a life threatening disease. Therefore, development of non-invasive high-resolution techniques for imaging mela- nocytic lesions in situ in the patient is highly desirable since they may increase diagnostic ac-

Chapter II.1

Laser-Scanning Confocal Microscopy

Salvador González and allan Halpern II.1

Contents

II.1.1 Fundamentals and imaging Parameters for reflectance-Mode laser Scanning Confocal Microscopy . . . .39 II.1.2 in-Vivo Confocal Microscopy of Normal

and Diseased Skin . . . .40 II.1.3 in-Vivo reflectance Confocal Microscopy

Features of Melanocytic Skin lesions . . . . .40 II.1.3.1 Melanocytic Nevi . . . .40 II.1.3.2 Cutaneous Melanoma . . . .42 II.1.4 accuracy Studies on Confocal Diagnoses

of Melanocytic Malignancy . . . .43 II.1.5 Potential Clinical applications

and Current limitations of reflectance Confocal Microscopy . . . .44 II.1.6 Conclusion . . . .44 references . . . .45

curacy. These techniques may include optical coherence tomography [40], high-frequency ul- trasound [23], magnetic resonance imaging [24], and reflectance-mode laser scanning confocal microscopy (rCM) [12, 17, 28, 36]. of these, rCM offers the highest resolution imaging com- parable to routine histology.

II.1.1 Fundamentals and Imaging Parameters for Reflectance-Mode Laser Scanning Confocal

Microscopy

Confocal microscopy, first introduced to the scientific community by Marvin Minsky in 1957, is an optical technique that produces “op- tical sections” of an object under observation [27]. over time, developments in light sources and computer technologies have enabled imag- ing human and animal skin in vivo [33, 36].

Briefly, a rCM involves the use of a light source, a condenser, objective lenses, and a detector.

The light source illuminates a small skin area

that will be imaged onto the detector passing

through a small aperture (pinhole). The pinhole

aperture is matched in size to the illuminated

spot. as a result, the detector receives light from

only a thin in-focus plane in the tissue. light

from out-of-focus planes is rejected at the pin-

hole. The point source of light, the illuminated

spot in the sample, and the pinhole aperture lie

in optically conjugate focal planes – hence the

name “confocal.” To create a two-dimensional

image, the illumination spots are raster-scanned

over the area of interest within the tissue and an

image is produced point – by-point (i.e., pixel –

by-pixel), a process that is known as “optical

sectioning.”

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0 S. González, A. Halpern

II.1

Several parameters that have been optimized for high resolution, in-vivo confocal microscopy include the use of near-infrared wavelengths, high numerical aperture water-immersion ob- jective lenses, low illumination power (about 40 mW), and detection apertures equivalent to 1–5¥ lateral resolution [34].

II.1.2 In-Vivo Confocal Microscopy of Normal and Diseased Skin real-time rCM images are acquired en-face (i.e., in the horizontal tissue plane) and resolved in gray scale with the image contrast derived from naturally occurring differences in optical index of refraction within the tissue [9, 36].

Bright regions of an rCM image generally cor- respond to regions of high refraction index in the tissue. Since water is the major constituent of living cells, the nominal refractive index of most human tissue is similar to that of water, about 1.33. rajadhyaksha and coworkers [36]

have shown that melanin (refraction index of approximately 1.7) provides strong contrast in rCM images.

in normal skin, the stratum corneum is seen to be composed of islands of corneocytes within skin folds. Hair follicles and sweat duct open- ings can be identified. Going deeper, stratum granulosum and stratum spinosum consist of polygonal cells with large, centrally placed nu- clei surrounded by a grainy cytoplasm. Cells of these strata are arranged in a characteristic pat- tern with a honeycombed appearance. Below, basal keratinocytes can be seen as bright, highly refractile, round or oval cells located on the bor- ders of the dermal papilla. The high refractivity of basal keratinocytes stems from the presence of melanin and melanosomes, with endogenous variation among skin phototypes and anatomic locations. The melanin in basal keratinocytes is typically located in a supranuclear position, of- ten referred to as “melanin caps” implying their protective function. En-face dermal papillae openings are seen as dark round areas sur- rounded by a bright crown of basal keratino- cytes and melanocytes. a reticular collagen net- work and small capillaries can be seen in the center of dermal papillae.

Non invasive, in-vivo rCM has previously been used to explore a variety of non-melano- cytic and melanocytic benign and malignant neoplasms as well as inflammatory skin condi- tions. in-vivo cytoarchitectural features of pso- riasis [18], contact dermatitis [3, 4, 16, 20, 38], bacterial [17] and fungal infections [21, 25, 29], actinic keratoses [2], basal and squamous cell carcinomas [1, 10, 19, 29, 37], as well as melano- cytic nevi and melanomas [6, 14, 22, 30–32, 39]

have been evaluated by rCM. in particular, our group and others are investing significant time and effort to develop the utility of this technique for the non-invasive evaluation of contact der- matitis, basal cell carcinoma, and melanocytic lesions. recent studies of the accuracy of rCM evaluation for predicting histopathology sug- gest a promising future for the clinical use of rCM [4, 14, 32]. in melanocytic lesions, the en- dogenous contrast provided by melanin and melanosomes facilitates recognition of the rele- vant structures involved in the differentiation of benign versus malignant tissue and, thus, make rCM imaging a promising non-invasive tool in the differential diagnosis of benign and malig- nant pigmented skin lesions.

II.1.3 In-Vivo Reflectance Confocal Microscopy Features

of Melanocytic Skin Lesions in 2001, langley and colleagues [22] reported the first descriptive study on rCM features of melanocytic lesions. These confocal features are based on the cytoarchitectural analysis of the skin, including melanocyte and keratinocyte morphology, pattern, and distribution (Ta- ble ii.1.1).

II.1.3.1 Melanocytic Nevi

Common melanocytic nevi are recognized by the distribution of melanocytes on the skin.

They present populations of small, monomor-

phous round to oval bright (refractile) cells with

frequently visible, centrally positioned, nuclei

(Fig. ii.1.1) [5, 22], and preserved keratinocyte

cell borders within the overlying epidermis. in

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Laser-Scanning Confocal Microscopy Chapter II.1 1

Table II.1.1. reflectance confocal microscopy features of melanocytic skin tumors

Melanocytic lesion Features

Melanocytic nevi Uniform population of small bright cells with central nuclei in the basal layer level Preserved honey-combed pattern in suprabasal layers

occasionally small and fine dendritic projections

regular distribution and morphology of the dermal papillae Edge papillae

Clusters of refractile cells at dermal level (dense)

Dysplastic nevi Heterogeneous population of cells in terms of morphology and brightness Focal loss of cell demarcation

Bright granular structures Fine dendrites

Cutaneous melanoma Pleomorphic bright cells within the epidermis, some of them ascending the epidermis (pagetoid infiltration)

Disarray of keratinocytes

Focal/total loss of the honeycombed pattern Coarse branching dendrites

Bright grainy structures

irregular dermal papillae distribution Non-edge papillae

Dermal cerebriform clusters and/or sparse clusters Fig. II.1.1. routine hematoxylin-and-eosin (H&E)-stain-

ed histology (left) and reflectance confocal microscopy (right) images of a benign compound nevus. Confocal image obtained at the level marked in the H&E figure

shows a rim of monomorphous refractive cells around dermal papillae (asterisk), corresponding to small mela- nocytes and melanin-rich keratinocytes in the basal layer (edged papillae). Scale bar = 100 µm

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S. González, A. Halpern

II.1

some benign melanocytic tumors, superficial epidermal layers may show the presence of small round to oval cells with bright cytoplasm and dark outlines giving rise to a cobblestone ap- pearance. Dermal papillae are uniformly dis- tributed and are seen circumscribed by a rim of monomorphous refractive cells that have been termed by Pellacani and coworkers edge papil- lae [30]. These bright cells at the dermal–epider- mal junction correspond to small melanocytes and melanin-rich keratinocytes, lacking cyto- logical atypia and heterogeneous brightness [30]. in the dermis, common nevi frequently present isolated bright cells or groups of cells seen as round to oval clusters typically distrib- uted throughout the lesion and sometimes forming central conglomerates (Fig. ii.1.1) [22, 30]. atypical nevi may be difficult to diagnose under rCM since they present intermediate characteristics when compared with nevi and melanoma [22]. They usually show a greater variability in melanocyte size and shape than banal nevi, although cells still tend to be round- ed or oval rather than dendritic. in some in- stances, medium to large cells with refractile cytoplasm and peripheral nuclei, corresponding to a mild cytological atypia, are also visualized.

Focal loss of keratinocyte demarcation within the overlying epidermis and bright granules

within the epidermis, probably representing melanin bodies, are also seen in atypical nevi.

En-face view of dermal–epidermal junction in atypical nevi often demonstrate an irregular dermal papillae distribution and the papillae of- ten lack the normal demarcating rim of refrac- tile cells (non-edge papillae; Fig. ii.1.2) [30].

II.1.3.2 Cutaneous Melanoma

The rCM imaging of melanoma commonly shows large pleomorphic, atypical bright cells at various levels within the epidermis (pagetoid spread) and, sometimes, in the dermis [22, 30].

These cells are oval, stellate, or fusiform in shape, possess coarse branching dendritic pro- cesses, and present eccentrically placed large nuclei [22, 30, 39]. The presence of these bright polymorphic cells ascending in the epidermis accompanied by loss of keratinocyte demarca- tion is highly suggestive of melanoma (Fig. ii.1.3) [30, 32]; however, some benign lesions may pres- ent dendritic cells arranged in small nests or as single cells located within the upper epidermis, raising the suspicion of a malignant process. in these lesions, the keratinocytic background usually maintains the characteristic honeycomb pattern suggestive of a benign diagnosis. addi- tionally, presence of bright grainy particles,

Fig. II.1.2. Dysplastic nevus. Confocal image (right) taken at the level marked in the routine pathology image (left) shows heterogenous brightness of the epidermis,

irregular distribution of dermal nests (asterisk), and non- edged dermal papillae (arrow). Scale bar = 100 µm.

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Laser-Scanning Confocal Microscopy Chapter II.1 3

probably melanin, also can be noticed in many melanomas [22, 39]. at the dermal–epidermal junction level, dermal papillae look smaller and more irregular in melanoma than in common nevi.

at the dermal level, melanomas may demon- strate clusters of cells with a cerebriform ap- pearance composed of aggregates of low-re- fractile polygonal or elongated cells with fine dust-like granular structures [31]. additionally, low-refractile cells seen in isolation or small groups with well-defined demarcation, called

“sparse cell clusters,” may be seen.

These criteria also apply for clinically amela- notic melanoma as shown in several previously reported cases [7, 13]. Presumably, the contrast observed in these lesions is due to either the presence of non-melanized melanosomes in the cytoplasm, an endogenous source of contrast due to their size (0.6–1.2 µm), and/or the pres- ence of some clinically inapparent melanin in pre-melanosomes [7, 35]. additionally, rCM imaging has been successfully used to map and evaluate response to topical therapies in these lesions [7, 11, 13].

a limitation for the use of rCM in melanoma diagnosis is imaging depth. lesion depth has been shown to be a very important prognostic factor for patients diagnosed with melanoma;

however, available instruments can only image

down to 200–350 µm, and the presence of re- fractive structures in the dermis, such as in- flammatory cells and collagen bundles, may decrease contrast and preclude melanocyte vi- sualization; thus, although rCM has been shown to be useful for the differential diagnosis of intraepidermal processes, limited informa- tion about dermal cells and structures can be obtained, limiting the use of this technique for deeper lesions.

II.1.4 Accuracy Studies on Confocal Diagnoses of Melanocytic Malignancy

in 2004, Gerger et al. [14] at the Medical Univer- sity of Graz published the first study of the sen- sitivity and specificity of rCM for detection of melanocytic skin tumors based on a review of previously selected confocal images. This study included 117 melanocytic skin tumors (90 be- nign nevi and 27 melanomas) under rCM using Wellman confocal criteria [22] and found sensi- tivity and specificity for melanoma of 88.15 and 97.60%, respectively. The authors concluded that three characteristics – cytomorphology, ar- chitecture, and keratinocyte cell borders – have the highest diagnostic sensitivity for melanoma using reflectance confocal microscopy. a sig-

Fig. II.1.3. Melanoma. Confocal image shows the presence of enlarged (atypical) melanocytes (arrow) around a hair follicle, within a background of keratinocyte disarray. Scale bar = 100 µm.

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II.1

nificant criticism of this study is that the analy- sis of each case was performed on confocal im- ages that appear to have been selected by an observer who was not blinded to the clinical/

histological appearance of the lesion.

another study performed by Pellacani et al.

[32] from University of Modena and reggio Emilia assessed the significance of various rCM features for melanoma identification in a series of 102 melanocytic lesions (37 melanomas, 49 acquired nevi, 16 Spitz nevi) presenting equivo- cal clinical/dermoscopic findings. among epi- dermal features, the presence of disarranged keratinocyte pattern as well as the presence, morphology, and distribution of pagetoid cells was significantly associated with melanoma. at the dermal–epidermal junction, the visualiza- tion of non-edge papillae was associated with malignancy. Finally, cerebriform clusters and isolated cells with eccentric dark nuclei within the dermal compartment were seen only in mel- anomas, whereas small to medium dense clus- ters were more typically observed in acquired nevi.

II.1.5 Potential Clinical Applications and Current Limitations of Reflec- tance Confocal Microscopy

The rCM has the potential to inform clinical care by facilitating non-invasive in-vivo diagno- sis of both benign and malignant pigmented and non-pigmented skin lesions. Unlike an in- vasive biopsy, the rCM technique permits re- peated evaluation of the cellular architecture of an area of skin to monitor the progression or resolution of lesions over time. it holds signifi-

cant potential for the direction of invasive biop- sies to more accurately pre-selected lesions and to the most histologically concerning areas within large complex lesions. Specifically, in the realm of pigmented lesions, rCM holds signifi- cant promise as:

1. a guide for performing biopsies by helping to determine which areas have features suspicious for malignancy, thus reducing sampling error [39]

2. a tool for monitoring the histological response of lesions to novel non-invasive therapy [13, 15, 41]

3. a tool for mapping out the extent of involvement prior to excision [7, 13, 41]

II.1.6 Conclusion

a major limitation of the rCM technology is the

limited imaging depth of the technique, which

prevents accurate imaging below the superficial

dermis. This limitation is greatest in lesions that

are hyperpigmented or hyperkeratotic, leading

to intensity attenuation secondary to light ab-

sorption and scattering. Efforts to improve im-

aging depth with rCM include the use of more

powerful light sources, optimal immersion me-

dia, and image deconvolution algorithms. lack

of contrast in minimally pigmented skin also

poses a challenge, which might be solved by the

future development of exogenous contrast

agents. as these technical issues are being ad-

dressed, considerable additional progress is

needed in the consistent interpretation of the

en-face images derived from the current genera-

tion of rCM instruments.

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Laser-Scanning Confocal Microscopy Chapter II.1

C

Core Messages

■ reflectance confocal scanning micros- copy has proven, in principle, that non- invasive skin imaging with single cell resolution is readily achievable.

■ Significant technical and practical barriers need to be addressed prior to the common application of this technique in clinical practice.

■ The apparent advantages of ascertain- ing quasi-histological information non- invasively dictate that this, or some similar high-resolution optical tech- nique, will find its way into clinical practice in the not-too-distant future.

■ The current generation of rCM instruments provides images of sufficient quality to justify clinical research in image interpretation and diagnostic accuracy.

■ Future iterations of the technology, used in concert with improved macro- scopic imaging techniques, will likely change the nature of dermatological practice.

■ Clinical care will benefit from bedside non-invasive “histopathology” just as the interpretation of invasive biopsies will benefit from the advent of molecu- lar diagnosis.

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