Keywords

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FormalPara Key Points

  • Reflectance confocal microscopy (RCM) is a noninvasive in vivo imaging technique that relays on the natural variation of refractive indices in tissue structures at different depths of the skin to reconstruct the images.

  • Melanin has the highest refractive index and appears the brightest on RCM images. Other sources of contrast include keratin, cellular organelles, chromatin, and collagen.

  • A clinical breakthrough in RCM has been the capability to bring to the bedside an almost equivalent tool to histological evaluation without the need for invasive diagnostic procedures.

  • The RCM technology can be implemented as a diagnostic, surgical margin assessment, and monitoring of therapeutic interventions, such as cryosurgery.

  • The aid of RCM in the setting of pigmented lesions include guiding the clinicians to the areas of greater atypia to perform skin biopsies, identification of benign appearing melanocytic lesions, and assessment of lentigo maligna suspected lesions.

  • RCM is a highly sensitive and specific imaging modality for the identification and assessment of BCC cases.

1 Introduction

Reflectance confocal microscopy (RCM) is a noninvasive in vivo imaging technique intended to help the clinician diagnostically view skin lesions in real time while avoiding trauma or scar formation from surgical biopsies. First described in 1955 by Marvin Minsky [1], it was not until the late 1980s and early 1990s when light and computer technology was advanced enough to image tissue in vivo. In 1995, Rajadhyaksha and colleagues [2] were the first to describe RCM imaging of human skin in vivo, utilizing a laser light source to provide enough illumination and near-infrared wavelengths strong enough to view deeper levels of the skin in high resolution at a cellular level.

RCM’s principles rely on the natural variation of refractive indices in tissue structures at different depths of the skin [3]. Of all the molecular structures in the skin, melanin has the highest refractive index and appears the brightest on RCM images [2]. Other sources of contrast include keratin, cellular organelles, chromatin, and collagen (Fig. 14.1).

Fig. 14.1
figure 1

Refractive index. Since the human body contains over 60 % water, this eliminates the need for contrast agent. Therefore, water serves as the standard. The refractive index of water is 1.31. The closer the materials refractive index ratio is to 1.31, the darker the material looks. The further the deviation from this value (+or −), the brighter that material will appear (Reproduced and modified with permission of Caliber I.D. Inc.)

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With the use of a near-infrared diode laser, light projected from the confocal microscope focuses on a specific point in the skin. Reflected light from this point is captured through a pinhole-sized opening by a detector as shown in Fig. 14.2. The use of such a small aperture segregates reflected light from the focal section of interest from other areas of the tissue. The depth of light penetration is dependent on wavelength, laser power, reflectivity of the superficial layers of skin, and scattering properties of the dermis. Of all these factors, only laser power can be changed. At longer wavelengths, deeper penetration is gained at the expense of resolution; therefore, present single-laser confocal microscopes are fixed at 830 nm [2].

Fig. 14.2
figure 2

The basis of the technology: refraction. Confocal scanning laser microscopy technology: after emission, light passes through a beam splitter, a scanning and focusing optical lens, and a skin contact device. Cells and their morphology and cytology are clearly resolved. Diagnostic images are captured at relevant depths in the skin (Reproduced and modified with permission of Caliber I.D. Inc.)

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Each section projects as a two-dimensional image (Fig. 14.2) with dimensions of 0.5 mm laterally and 4 mm axially [3] that are oriented parallel (en face) to the skin surface. The resolution of each image is comparable to those of histological sections (Fig. 14.3).

Fig. 14.3
figure 3

Representative diagram of the skin layers and corresponding RCM images. SC Stratum corneum, SG Stratum granulosum, SS Stratum spinosum, SB Stratum basale, PD Papillary dermis, RD reticular dermis (Reproduced and modified with permission of Caliber I.D. Inc.)

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The commercially available near-infrared reflectance microscope (VivaScope 1500, Caliber I.D. Henrietta, NY) is equipped with a diode laser with peak emission at 830 nm and has a maximum power of 35 mW. With this system, each image has an effective 500 × 500 μm field of view and the imaging depth in normal skin is 200–300 μm, i.e., the level of papillary dermis and upper reticular dermis and the spatial resolution in the lateral dimension are 0.5–1.0 μm [4–6]. The new generation of this device (VivaScope 1500, Multilaser) combines reflectance and fluorescence imaging, with available wavelengths of 785 nm (near infrared), 658 nm (red), and 488 nm (blue), which are integrated into a single device amplifying the imaging potentials. These laser wavelengths are applied to the skin, one wavelength at a time, to eliminate the possibility of bleed-through from other fluorophores. The use of several fluorescent dyes has been described including the administration of Indocyanine green (ICG) and fluorescein human skin in vivo [7]. The well-defined intercellular staining pattern of ICG in the skin, with a stain duration of almost 48 hours, has allowed the development of automated cell-recognition algorithms currently under investigation. The expanded capabilities of the multilaser microscope allow the implementation of the device not only in studies seeking the assessment of morphological features in the skin (e.g., atypical honeycomb pattern, atypical keratinocytic and melanocytic nest formation, cell pleomorphism, etc.) but also in research studies aiming to develop targeted fluorescence markers in the field of diagnostic and therapeutic biomarkers, as well as drug delivery studies.

2 Clinical Application of RCM as a Diagnostic Tool

A clinical breakthrough in RCM has been the capability to bring to the bedside an almost equivalent tool to histological evaluation without the need for invasive diagnostic procedures. In the setting of cryosurgery, RCM has the capability to aid in the diagnostic decision of a lesion prior to undergoing therapy, which has the advantage of avoiding postponing the treatment until the diagnosis is histologically confirmed. The sensitivity and specificity of RCM varies significantly across tumor types, level of training of the operator, and expertise of the individual interpreting the confocal images [8]. The following skin lesions represent clinical examples in which RCM has been evaluated to date with the potential to offer a diagnostic aid in cases considered for cryosurgery intervention.

2.1 Pigmented Lesions

Because melanin and melanocytes are the easiest structures to recognize in RCM, much of the research in the field has been focused on the evaluation of pigmented lesions. RCM has been identified as a useful tool in distinguishing melanomas from benign pigmented lesions (Fig. 14.4a–d). Malignant melanoma in situ (MMIS), lentigo maligna (LM) type, shares similar RCM features such as non-edged papillae, round and large pagetoid cells, and nucleated cells in the dermal papillae with other subtypes of malignant melanoma; however, follicular localization of atypical cells has been proposed as a unique finding in LM. A 2013 systematic review examined five cohort studies on RCM diagnostic accuracy for melanoma, LM, and MMIS in clinically equivocal pigmented lesions using various RCM scoring systems and algorithms [9]. The meta-analysis for these RCM studies demonstrated a per-lesion sensitivity of 93 % and specificity of 76 % for melanoma diagnosis. Thus, RCM features of melanoma may be helpful in noninvasively differentiating equivocal pigmented lesions and delineating margins within lesions such as LM.

Fig. 14.4
figure 4

(a–d) RCM features in benign and atypical melanocytic lesions. (a) RCM mosaic (4 × 4 mm) acquired at the suprabasal/epidermal-junctional level, showing a ringed and meshwork pattern in a slightly asymmetrical overall architecture. Also milia-like cysts and comedo-like openings are visible throughout the lesion, whereas atypical cells are lacking. The ringed and meshwork pattern fade off at the border of the lesion. (b) Basic image (0.5 × 0.5 mm) showing mostly edged papillae . Arrow is showing the milia-like cyst aspect (→). (c) RCM mosaic (4 × 4 mm) of a very asymmetrical lesion, with a mixed pattern (meshwork, ringed, and clod). In the center of the lesion, a nonhomogeneous meshwork pattern is present, with junctional nests, irregular in size and shape. At the periphery, there are junctional and dermal nests (clods). Dense and sparse and a ringed structure shading off at the periphery. (d) Basic image (0.5 × 0.5 mm) of a central area of the atypical nevus showing dishomogeneous and irregular junctional thickenings and nests surrounding non-edged papillae (red → and *). Within dermal papillae dense and dense and sparse nests are seen (red *), mixed with inflammatory infiltrate, including plump bright cells (yellow *) and small bright spots. Numerous atypical cells are detectable at the DEJ, in single cells or aggregated in nests

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Overall, the aid of RCM in the setting of pigmented lesions will be in guiding the clinicians to the areas of greater atypia to perform skin biopsies for histological confirmation and exclusion of invasion [10, 11]. In some cases, invasiveness of melanoma can be readily visualized by RCM, which will aid the clinician when making a decision on whether to perform a cryosurgery intervention or not in an atypical pigmented skin lesion. On the other hand, using RCM as a screening modality for borderline pigmented skin lesions of the head and neck can be of value prior to cryosurgery since, not infrequently, lesions with a lower degree of clinical suspicion can demonstrate increasingly concerning features by dermoscopy and RCM evaluation [12]. This scenario is particularly relevant when evaluating pigmented lesions in chronically photoexposed areas such as the head and neck, as well as distal upper extremity areas [13].

2.2 Basal Cell Carcinoma

RCM features of basal cell carcinoma (BCC) have been described at two powers of microscopy: mosaic and individual image levels [14, 15]. At the lower magnification of the mosaic grid, tightly packed tumor cells with high refractility (brightness) can been seen oriented parallel to each other (palisading) at the papillary dermis, forming cord-like structures surrounded by hyporefractile darkness (clefting) that corresponds histologically to the mucinous surrounding stroma. At higher magnification of individual images [14], within the individual tumor cells, elongated hyporefractile nuclei are present, producing a polarizing nuclear appearance [14]. Adjacent to tumor cells, dilated and increased vasculature is often seen with leukocytes on the endothelial lining (Fig. 14.5a–h). Small hyperrefractile dendritic cells within and surrounding the islands of tumor cells have also been identified in pigmented BCCs [15, 16]. A 2004 retrospective study showed that the presence of elongated nuclei and polarized nuclei on RCM was 91.6 % sensitive and 97 % specific for BCC [17].

Fig. 14.5
figure 5

RCM features in benign and atypical keratinocytic neoplasms. (a) Cobblestone pattern (red arrow) and keratin-filled invaginations . (b) Bright interpapillary spaces (red arrow), melanophages within dermal papillae . (c) Round to triangular, nonnucleated bright cells within dermal papillae (red arrow). Atypical (red arrow) and typical (yellow arrow) honeycomb pattern (HCP). (d) Bright reflectance of the papillary dermis (red arrow). (e) Irregular size of nuclei and thickness of cellular outlines (red arrow); regular HCP (yellow arrow). (f) “Streaming”of spinous keratinocytes (red arrow), regular HCP (yellow arrow) pattern. (g) Tumor islands at the DEJ (red arrow), fibrotic tumor stroma (yellow arrow). (h) Round, oval and trabecular, reflective tumor islands (red arrow); aggregates of melanophages (yellow arrow). All skin lesions shown as macroscopic and corresponding dermoscopy images (10×). RCM mosaic (4 × 4 mm) was obtained for all lesions (Images provided by Curiel and Hofman)

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In differentiating between BCC and malignant melanoma (MM), the basaloid cords seen in the papillary dermis are highly specific for BCC [18]. The absence of epidermal honeycomb disarray, papillae (papillae disruption), and cerebriform nests is also useful for diagnosing BCC. Yet, to date, there have been no studies directly comparing pigmented BCCs and MM, and further investigations would be helpful in delineating the RCM features that are unique to each entity. Overall, diagnosis of BCC is one area where RCM holds a high sensitivity and specificity facilitating an RCM diagnostic tool with the potential to spare the need for skin biopsies when interpreted by trained readers.

2.3 Actinic Keratosis and Squamous Cell Carcinoma

Actinic keratoses (AK) are one of the most common forms of cutaneous malignancies. While there is debate over whether AK represents a precancerous lesion, studies have shown that without treatment, 0.2–20 % of AKs [19–21] progress to squamous cell carcinomas (SCC). Currently, the diagnostic gold standard for AKs is histopathological examination. While this method represents the most accurate form of identification, it is often time consuming and impractical in daily clinical practice. Over the past six years, a few studies have examined RCM characteristics of clinically diagnosed AK and SCC and their diagnostic value.

In some of these studies, solar elastosis, polygonal, nucleated keratinocytes in the stratum corneum (parakeratosis), scaling, and stratum corneum disruption were seen in a majority of AKs [22, 23]. In particular, certain RCM features seem to be the most reliable in diagnosing AKs. One study noted irregular architecture in the stratum spinosum and stratum granulosum (atypical honeycombing) with pleomorphic cellular features at that level being the most specific and sensitive signs of AK [22]. Another group presented similar findings but also described irregular keratinocyte borders as the main finding seen in images of high power (individual images). In this study, RCM was 93.34 % sensitive and up to 90 % specific for AK when accounting for all of these features [23]. Other features seen in AKs include blood vessel dilation in dermal papillae [24] (Fig. 14.5a–h).

To date, a single study has attempted to compare RCM features of AK to SCC [24]. Although atypical honeycombing was seen in both AKs and SCCs, SCCs showed more extensive atypia in the spinous and granular layers. Similar features in AKs were more focal or displayed milder patterns. Round, bright, nucleated cells seen in the spinous and granular layers corresponding to atypical, dyskeratotic keratinocytes on histopathology were found more frequently in SCCs (65 %) than AKs (14 %). Dilated blood vessels in the dermal papillae were also more numerous in SCC vs. AK. The findings in this study support the theory that AK, SCCIS, and invasive SCC are not separate entities, but more a continuum in the keratinocytic carcinogenesis process.

2.4 Other Applications of RCM in Cryosurgery

2.4.1 Noninvasive Determination of Lesion Margins

One of the challenges of cryosurgery and subsequent variability in reported response rates is likely related to the challenge in clinically determining the precise extension of the lesion and, therefore, treatment margins. Such limitation could result in a decreased rate of complete response to therapy.

The current standard for margin assessment is through permanent histological margin evaluation in surgical excision and by staged excisions with frozen histopathological examination used during Mohs microsurgery. RCM has been shown to have good correlation with histopathology in vivo and ex vivo and has demonstrated magnification that is equivocal to the microscopic power used during frozen histopathology examination on Mohs excisions [25]. Fluorescence RCM has also demonstrated high clinical accuracy when studied in Mohs excisions. Karen JK et al. evaluated the accuracy of fluorescence RCM in detecting residual BCC features following surgical excisions. The sensitivity of the instrument was 96.6 % with a specificity of 89.2 %, supporting the potential role of RCM in evaluating surgical margins [26].

In addition to the implementation of RCM in lesions undergoing surgical intervention, the use of RCM prior to cryosurgical application holds the potential to increase the response rate. This approach will be particularly relevant for cutaneous malignancies with ill-defined borders such as AK, superficial BCC, and LM. Future studies assessing the implementation of RCM for margin determination in the cryosurgery setting are needed.

2.4.2 Noninvasive Therapy Monitoring

Along with margin identification, response to treatment is essential to cryosurgery. As an operator-dependent treatment, response to therapy can vary according to the technique and potency of liquid nitrogen application. Although a goal temperature is desirable, cryosurgical systems that accurately assess lesion and skin temperature during each freezing and thawing cycle have not been widely implemented into practice. In addition, the system is currently limited to a device by Brymill.

RCM has demonstrated accurate assessment of residual tumor in multiple types of cutaneous malignancies undergoing surgical and nonsurgical treatments. The study by Torres et al. examined the efficacy of imiquimod 5 % as a pre-micrographic surgery treatment for BCC and demonstrated a high concordance between RCM and histological findings [27]. The study by Marra DE et al. reported three patients who had undergone shave biopsies, one of which had also undergone imiquimod treatment. In all three cases, features of residual BCC were identified by RCM in well-healed appearing scars and subsequently confirmed by histological sections during Mohs micrograph surgery [28]. A recent study using RCM to examine superficial BCC (sBCC) response to cryosurgery showed necrotic cells at basal layer after 5 h from treatment application [29]. This was indicative of the higher sensitivity of tumor cells to low temperatures. In a study evaluating the morphological changes of subclinical AKs, RCM identified subclinical AKs at baseline, features of an inflammatory response to imiquimod at 2 weeks and residual atypia in 2 patients after end of treatment without any clinical aspects of AK [30] (Fig. 14.6a–l).

Fig. 14.6
figure 6

RCM features in actinic keratosis treated with cryosurgery. (a) Disrupted epidermis (red arrow), evidence of an atypical honeycomb pattern . (b) Increased epidermal disruption (red arrows) and necrosis . (c) Epidermis with minimal disruption (red arrows) and normal honeycomb pattern . (d) Minimal disruption (red arrows). Normal honeycomb pattern . (e) Epidermal disruption present (red arrows), spongiosis, inflammation (yellow arrows). (f) Disrupted epidermis (red arrows) atypical honeycomb pattern . (g) Normalized DEJ (red arrows). No evidence of inflammation. (h) Normalized DEJ (red arrows). No evidence of inflammation. (i) Increased inflammation (yellow arrows). Polymorphous and round vessels (red arrows). Solar elastosis . (j) Increased number of polymorphous and round vessels (yellow arrows). Solar elastosis . (k) Normal density of round vessels (red arrows). Solar elastosis . (l) Normal density of round vessels (red arrows). Solar elastosis

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Given the ability of RCM to accurately identify features indicative of different cutaneous malignancies, at a resolution of histological equivalence, the implementation of this imaging tool to monitor NMSC and AK responses is a rational proposition. This will ensure complete tumor clearance in situations where cryosurgery is standard treatment or when it is an appropriate alternative to surgical modalities.

Essential Tidbits

  • RCM will give an almost equivalent information on the tumor’s nature without the need for biopsy.

  • Confirming a diagnosis of BCC by RCM will help make more accurate treatment decisions.

  • In actinic keratosis (AK), confirming diagnoses becomes particularly useful and important due to the relevance of excluding squamous cell carcinoma.

  • Regarding cryosurgery, RCM can be used to define lesion margins and measure response to treatment.

3 Conclusions

As the imaging field continues to evolve at galloping steps, noninvasive imaging tools like RCM are revolutionizing the way we practice. The capacity to visualize and instantaneously document skin findings, at a histological resolution, has the capability to increase the accuracy of our clinical diagnosis and therefore our confidence at the time of cryosurgery interventions. In addition, the capability to noninvasively delineate lesion margins and the opportunity to follow lesions over time at this level of resolutions make RCM an ideal complementary imaging tool for cryosurgery interventions.

AK:

Actinic keratosis

BCC:

Basal cell carcinoma

ICG:

Indocyanine green

LM:

Lentigo maligna

MM:

Malignant melanoma

MMIS:

Malignant melanoma in situ

RCM:

Reflectance confocal microscopy

SCC:

Squamous cell carcinoma