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Observation
July 2001

Detection of Clinically Amelanotic Malignant Melanoma and Assessmentof Its Margins by In Vivo Confocal Scanning Laser Microscopy

Author Affiliations

From the Department of Pathology (Drs Busam and Antonescu) and theDermatology Service, Department of Medicine (Drs Hester, Charles, Sachs, andHalpern), Memorial Sloan-Kettering Cancer Center, New York, NY; and WellmanLaboratories, Massachusettes General Hospital, and the Department of Dermatology,Harvard Medical School, Boston (Dr Gonzalez).

Arch Dermatol. 2001;137(7):923-929. doi:10-1001/pubs.Arch Dermatol.-ISSN-0003-987x-137-7-dob00091
Abstract

Background  Near-infrared confocal scanning laser microscopy (CSLM) represents anovel imaging technique for in vivo microscopic analysis of skin lesions,including pigmented lesions.

Objectives  To investigate the feasibility of detecting a clinically amelanoticmalignant cutaneous melanoma using CSLM and to explore the use of this techniquefor assessing its margins.

Patients and Methods  Two lesions from 2 patients were imaged and analyzed using CSLM. Sitessuspected to represent melanoma or benign skin on CSLM were marked as such;then, biopsy specimens were obtained for diagnosis using conventional histologicalanalysis. Both lesions were stained for melanin pigment and analyzed immunohistochemicallyfor the expression of melanosomal markers. In 1 case, a biopsy specimen wasalso examined with electron microscopy.

Results  The images obtained using CSLM allowed recognition of an abnormal intraepidermalmelanocytic proliferation that was distinctly different from normal skin.Comparison of the sites examined using CSLM and subsequently using conventionalhistological methods revealed that CSLM correctly identified intraepidermalmelanoma and benign skin. Fontana-Masson stains and immunohistochemical andultrastructural studies showed that clinically amelanotic melanoma cells containedmelanosomes and rare melanin granules.

Conclusions  We demonstrated, for the first time, the detection of clinically amelanoticmelanoma using CSLM. This technique may aid in the early detection of clinicallybarely visible or nonpigmented melanomas and may facilitate preoperative noninvasiveassessment of their margins.

MALIGNANT melanoma typically manifests clinically as an abnormal pigmentedlesion, with irregular contours and colors ranging from light brown to black.On occasion, melanomas lack sufficient pigment to be recognizable as suchand may be confused with other tumors, such as basal cell carcinomas, or inflammatoryprocesses. These so-called amelanotic melanomas account for 2% to 8% of cutaneousmelanomas.13 Theyrepresent an important diagnostic pitfall for clinicians and a significantmanagement problem for surgeons.48Since the boundaries of an amelanotic lesion often cannot be reliably determinedclinically, repeated surgical sessions may be needed to achieve a final negativemargin.4

A noninvasive imaging tool that would enable clinicians to detect amelanoticmelanoma could be of great benefit. Reflectance confocal scanning laser microscopy(CSLM) represents a novel technique that allows in vivo microscopic examinationof the epidermis and papillary dermis.9,10It is currently being explored for use in the diagnosis of a variety of cutaneouslesions,1117including pigmented skin lesions.18 However,since melanin pigment represents an endogenous "contrast" agent that facilitatesthe recognition of melanocytes by CSLM,9 ithas been unclear whether this technique can also detect amelanotic melanoma.

We report the real-time detection of clinically amelanotic malignantmelanomas by in vivo CSLM in 2 female patients. One patient had an unusuallylarge primary lesion on her leg. The other patient presented with an erythematouspatch on her cheek. Near-infrared CLSM, along with conventional histologicalanalysis, was used for diagnosis in both cases. In 1 case, CLSM was also investigatedfor its potential to map the lesion for subsequent surgical management.

PATIENTS AND METHODS
PATIENTS

Two patients were recruited from the pigmented lesion clinic at MemorialSloan-Kettering Cancer Center (MSKCC), New York, NY. They gave informed consentfor their lesions to be examined with CSLM.

CONFOCAL SCANNING LASER MICROSCOPY

Confocal imaging was performed with a commercially available, near-infrared,reflectance confocal laser scanning microscope (Vivascope 1000; Lucid Inc,Henrietta, NY). The instrument uses a diode laser at 830 nm with a power ofless than 35 mW at tissue level. A ×30 water-immersion objective lensof numerical aperture 0.9 was used with either water (refractive index, 1.33)or gel (refractive index, 1.3335) as an immersion medium. It images with aspatial resolution of 0.5 to 1.0 µm in the lateral dimension and 4 to5 µm in the axial dimension. An automated stepper was used to obtaina grid of 16 contiguous horizontal images in less than 20 seconds to constructa montage image with an effective in vivo field of view of 1.6 × 2.0mm. Use of a modified skin contact device also allows an oblique imaging mountwith an angle of 5.7°. At this angle, the depth of imaging increases approximately50 µm across the field of view. This allows a single frame to discriminatevertical information ±25 µm about the center of the image. Theresultant images have the appearance of elongate (oblique) vertical histologicalsections.

HISTOPATHOLOGIC ANALYSIS

The biopsy specimen or excised tissue was fixed in formalin and embeddedin paraffin. After routine processing, the slides were stained with hematoxylin-eosin.Immunohistochemical studies were performed using the avidin-biotin-peroxidasecomplex method. The antibodies used in this study included HMB-45 (anti-gp100;1:200, Dako Corp, Carpinteria, Calif) and A103, which recognizes Melan-A/MART-1and was described previously.19,20For the 2 antibodies, a heat-based antigen retrieval system was used. Melaninpigment was stained with silver granules using the Fontana-Masson method.21

For ultrastructural studies, tissue was fixed in 3% glutaraldehyde andprocessed according to routine procedures.22

RESULTS
CLINICAL HISTORY AND FINDINGS
Patient 1

A 69-year-old white woman presented to MSKCC for a second opinion aftera malignant melanoma was unexpectedly diagnosed and found to be unusuallylarge in size. She had a 1-year history of an erythematous rash on her leftshin that had been treated as eczema, to no effect. After an initial biopsyspecimen revealed melanoma, 17 subsequent biopsy specimens were obtained atan outside hospital prior to her visit to MSKCC. The biopsies were performedin an attempt to map the extent of the lesion. Review of the biopsy specimensat MSKCC revealed that they all showed in situ melanoma. Three specimens showedfocal invasion into the papillary dermis to a greatest measured thicknessof 0.5 mm (Clark level II).

At her visit to MSKCC, the patient appeared well, with no evidence ofextracutaneous disease. Her dermatologic history was notable for basal andsquamous cell carcinoma, multiple actinic keratoses, and a pigmented lesionon her shin that had been excised 30 years earlier. The biopsy specimen wasnot available for review. She also had a family history of melanoma: her brotherhad a cutaneous melanoma 2 years before her visit to MSKCC.

Various areas of erythema and ill-defined hypopigmentation with normal-appearingintervening skin were present on the lower part of the patient's left leg(Figure 1). Wood lamp examinationrevealed light, ill-defined areas of hypopigmentation, with no accentuationof pigment. The proximal and distal margins of the lesion could not reliablybe determined on clinical examination with the Wood lamp.

Figure 1.
Patient 1. Large amelanotic melanomaof the leg. The perimeter of the lesion as estimated by combined Wood lampexamination and analysis by near-infrared confocal scanning laser microscopy(CSLM) is marked with a blue line (short arrow). The central biopsy site (asterisk)corresponds to microinvasive melanoma. The peripheral biopsy sites (long arrow)represent in situ melanoma on CSLM and histological examination.

Patient 1. Large amelanotic melanomaof the leg. The perimeter of the lesion as estimated by combined Wood lampexamination and analysis by near-infrared confocal scanning laser microscopy(CSLM) is marked with a blue line (short arrow). The central biopsy site (asterisk)corresponds to microinvasive melanoma. The peripheral biopsy sites (long arrow)represent in situ melanoma on CSLM and histological examination.

Patient 2

A 66-year-old white woman with a history of biopsy-proven and narrowlyexcised in situ melanoma on her left cheek presented to MSKCC in the summerof 2000 for facial erythema near the scar from the prior biopsy and excision(Figure 2). There was no apparentpigmentation in or around the scar on routine visual examination. Wood lampexamination revealed an area of hypopigmentation medial and inferior to thescar.

Figure 2.
Patient 2. Erythematous patchon the cheek.

Patient 2. Erythematous patchon the cheek.

HISTOLOGICAL AND CSLM FINDINGS
Patient 1

Multiple foci of the skin of the leg of patient 1 were examined withCSLM. Most foci were studied at the periphery of the lesion to distinguishlesional from benign skin. On 5 foci, a preliminary diagnosis was made basedon the findings of CSLM: 3 foci were judged to represent in situ melanoma(1 in the center of the lesion and 2 at its periphery), and 2 peripheral fociwere judged to represent normal skin (negative margin). The preliminary diagnosesof in situ melanoma and normal skin were later confirmed histologically. Themorphological features of a focus of in situ melanoma on CSLM, as well asa focus of surrounding normal skin, are illustrated in en face (horizontal)sections in Figure 3. On CSLM, insitu melanoma was characterized by the presence of an increased number ofmelanocytes in solitary units at the dermoepidermal junction and above inthe spinous cell layer. Individual melanocytes appeared as bright round, oval,or elongated fusiform structures with or without recognizable dendritic processes.Also, small (<1 µm in diameter) bright structures of granular appearancewere found. No significantly increased number of melanocytes was seen in normalskin. The epidermis of normal skin showed a honeycomb pattern of keratinocyteswith easily recognized cell borders. This pattern was blurred in the fociof in situ melanoma. The histological features of the in situ melanoma correspondingto the focus examined with CSLM are shown in Figure 4. An atypical intraepidermal melanocytic proliferation wasidentified. Melanocytes were dispersed as solitary units and in nests in anirregular asymmetrical fashion along the dermoepidermal junction. Single melanocytespredominated over nests. Cytologically, the melanocytes were epithelioid (largeround to oval cells), with enlarged nuclei. Occasional melanocytes were presentabove the dermoepidermal junction in the midspinous cell layer. The dermisshowed marked solar elastosis.

Figure 3.
Patient 1. Near-infrared confocalscanning laser microscopy (horizontal sections). A, Normal skin (adjacentto in situ melanoma, imaged at the level of the dermoepidermal junction).A honeycomb pattern of epidermal keratinocytes with distinct cellular outlinesis present. The dark space (arrow) corresponds to papillary dermis. B, Insitu melanoma (imaged at similar depth). The honeycomb pattern is blurred.Bright irregular oval structures (arrows) or elongated dendritic processes(arrowhead) are present at the dermoepidermal junction and above.

Patient 1. Near-infrared confocalscanning laser microscopy (horizontal sections). A, Normal skin (adjacentto in situ melanoma, imaged at the level of the dermoepidermal junction).A honeycomb pattern of epidermal keratinocytes with distinct cellular outlinesis present. The dark space (arrow) corresponds to papillary dermis. B, Insitu melanoma (imaged at similar depth). The honeycomb pattern is blurred.Bright irregular oval structures (arrows) or elongated dendritic processes(arrowhead) are present at the dermoepidermal junction and above.

Figure 4.
An increased number of epithelioidmelanocytes are present at the dermoepidermal junction. Single cells predominate,and there is a confluency of cells.

An increased number of epithelioidmelanocytes are present at the dermoepidermal junction. Single cells predominate,and there is a confluency of cells.

A Fontana-Masson stain revealed focal deposits of silver granules withinmelanocytes at the dermoepidermal junction (Figure 5). Variable amounts of pigment deposition were also presentin basilar keratinocytes within the area of skin involved by in situ melanoma.No more and focally even less melanin was present in skin involved by in situmelanoma compared with adjacent uninvolved skin. Immunohistochemical studieswith antibodies to gp100 and Melan-A/MART-1 revealed positive staining ofthe melanoma cells for both antigens (more intense for Melan-A/MART-1 thanfor gp100).

Figure 5.
Foci of silver granules are seenwithin melanocytes (arrow) (Fontana-Masson stain).

Foci of silver granules are seenwithin melanocytes (arrow) (Fontana-Masson stain).

After the status of the lesion's periphery was preliminarily assessedby confocal microscopy, it was mapped in more detail by conventional lightmicroscopic examination of multiple punch biopsy specimens. Subsequently,an excision was performed with negative final margins. Histological reviewof the final excision confirmed the presence of widespread in situ melanomaspanning an area of approximately 25 × 20 cm, involving more than 80%of the excised tissue. Rare foci of microinvasion (Breslow thickness, 0.35mm; Clark level II) were found on histological examination. These foci ofinvasion had not been examined with confocal microscopy. No ulceration wasseen. Intraepidermal melanoma showed mixed patterns of nested, single-celllentiginous and pagetoid growth. In situ melanoma was focally separated bylichenoid inflammatory reactions, with loss of intraepidermal melanocytes.There was marked solar elastosis throughout the skin.

Patient 2

The erythematous patch, as well as a focus of clinically normal-appearingskin lateral from it, was examined with CSLM (Figure 6). The focus, judged on CSLM examination to represent insitu melanoma, was characterized by an irregular proliferation of melanocytesin the epidermis manifesting as bright solitary structures at and above thedermoepidermal junction and by a confluency of bright signals lacking distinctcellular outlines (Figure 6B). Theshape of individual melanocytes varied, ranging from round/oval to fusiform,with occasional short processes. As in patient 1, the keratinocytic honeycombpattern seen in the epidermis of normal skin was blurred in the focus of insitu melanoma, with loss of cell demarcation and architectural disarray. AfterCSLM examination of the lesion, a biopsy was performed, and the site previouslyimaged with CSLM (Figure 6) wasexamined by conventional histological methods (Figure 7A). An irregular proliferation of melanocytes in nests andsolitary units is present at all layers of the epidermis (Figure 7A). While a Fontana-Masson stain failed to demonstrate melaninpigment deposition in melanocytes, immunohistochemical studies confirmed thepresence of Melan-A/MART-1 (Figure 17B)and gp100 antigens.

Figure 6.
Patient 2. Near-infrared confocalscanning laser microscopy (horizontal section). A, Virtual section of normalskin within the spinous cell layer and focally at the dermoepidermal junction(darkened area). Polygonal keratinocytes form a honeycomb pattern. B, In situmelanoma. The honeycomb pattern of epidermal keratinocytes is blurred. Brightirregular fusiform (arrow 1) or round to oval structures with short dendriticprocesses (arrow 2) are present at the dermoepidermal junction and above.Confluent clusters of melanoma cells form an ill-defined bright structure(arrow 3).

Patient 2. Near-infrared confocalscanning laser microscopy (horizontal section). A, Virtual section of normalskin within the spinous cell layer and focally at the dermoepidermal junction(darkened area). Polygonal keratinocytes form a honeycomb pattern. B, In situmelanoma. The honeycomb pattern of epidermal keratinocytes is blurred. Brightirregular fusiform (arrow 1) or round to oval structures with short dendriticprocesses (arrow 2) are present at the dermoepidermal junction and above.Confluent clusters of melanoma cells form an ill-defined bright structure(arrow 3).

Figure 7.
Patient 2. In situ melanoma asobserved on conventional histological examination (vertical sections). A,Hematoxylin-eosin–stained section. B, Immunoreactivity for Melan-A (A103).

Patient 2. In situ melanoma asobserved on conventional histological examination (vertical sections). A,Hematoxylin-eosin–stained section. B, Immunoreactivity for Melan-A (A103).

A portion of a biopsy specimen of tissue was also examined with electronmicroscopy. The ultrastructural findings are illustrated in Figure 8. Intraepidermal epithelioid melanocytes are back-to-backat the dermoepidermal junction adjacent to keratinocytes. The latter are identifiedby the presence of desmosomes and tonofilaments. Within the cytoplasm of themelanocytes, several melanosomes were identified. Although most melanosomeswere stage II (premelanosomes), a few stage III and rare stage IV melanosomeswere also present.

Figure 8.
Patient 2. Ultrastructural studies.Melanosomes of various stages are present within melanoma cells at the dermoepidermaljunction.

Patient 2. Ultrastructural studies.Melanosomes of various stages are present within melanoma cells at the dermoepidermaljunction.

COMMENT

One of the most challenging problems in clinical dermatology is theearly detection of malignant melanoma. All noninvasive melanomas and the majorityof thin invasive melanomas are treated with complete excision. Delayed recognitionof melanoma needs to be avoided, as it puts the patient at risk of dying ofthe disease once the tumor has progressed to competence for metastasis. Earlydetection of melanoma relies on the critical visual analysis of a new or changingpigmented lesion and subsequent biopsy or excision for diagnosis and treatment.

Amelanotic melanomas often escape early detection.6Owing to the lack of distinct pigmentation, the clinical suspicion for melanomais low, and biopsy and/or excision is frequently delayed. Ancillary noninvasivescreening tools may be a significant advance for detection of these enigmatictumors at an early and curable stage.

Near-infrared reflectance CSLM is a novel imaging technique that allowsin vivo examination of the epidermis and superficial dermis at a resolutionthat permits visualization of microanatomical structures.9,10It sets a new paradigm of instant noninvasive quasihistological examinationof skin lesions in vivo.23 Reflectance CSLMoperates by detecting single back-scattered photons from the illuminated in-focusplane of interest.2426High-resolution optical sections are achieved by means of a pinhole placedin an optical conjugated plane in front of the detector that rejects photonscoming from out-of-focus planes. Contrast in confocal images is provided byrefraction index differences of organelles and other microstructures fromthe background. This means that highly refractile structures appear whiteon screen, while low-refractile structures appear dark gray to black.9,10,26 This technique hasbeen used to image keratinocytic and inflammatory skin lesions1117and is also being investigated for the diagnosis of pigmented skin lesions.18

Melanin pigment was found to provide a natural contrast for confocalscanning.9 Its presence results in a bright-whiteimage signal that illuminates the cytoplasm of melanin-containing cells (pigmentedkeratinocytes, melanocytes, and melanophages). Individual melanocytes arebest recognized by the presence of bright dendritic processes. However, manytimes, little or no dendritic processes are detectable, and the melanocytesare round to oval or elongated and fusiform. These melanocytes can be discriminatedfrom keratinocytes because of the bright finely granular signal intensitythroughout the cells and their spatial distribution as solitary or clusteredcells that are round/oval or fusiform in shape. Keratinocytes, on the otherhand, appear as cohesive polygonal cells in a characteristic honeycomb arrangement.When they are pigmented, bright granular signals highlight their cytoplasmon CSLM examination. A reliable distinction of individual pigmented keratinocytesfrom solitary melanocytes can, on occasion, be very difficult. If a melanocytelacks dendritic processes, one often needs to take architectural context intoconsideration (single-cell or nested growth pattern vs cohesive growth pattern)to distinguish it from a pigmented keratinocyte.

Although it was known that CSLM is capable of identifying melanocytesin clinically pigmented lesions,9,18it was unclear whether CSLM would also be able to visualize the cells of aclinically amelanotic tumor. In this study, we demonstrated for the firsttime that CSLM can indeed accomplish this. Melanocytes in a clinically amelanoticlesion can still be detected by CSLM, because residual melanin pigment ormelanosomes provide cytoplasmic contrast. It has been known from previousultrastructural studies that amelanotic melanomas still contain premelanosomes.22,2730Such stage II melanosomes are ellipsoidal structures with a striated core(lamellar arrangement of membranes) but no dense melanin granules.

In the first patient's lesion, foci with small amounts of melanin pigmentcould be demonstrated by Fontana-Masson stain, which sufficiently explainsthe presence of scattered bright signals in the cytoplasm of melanocytes onCSLM. The lesion of the second patient lacked melanin pigment on Fontana-Massonstain. However, in this case, immunohistochemical studies revealed that themelanoma cells were positive for melanosome-associated glycoproteins (gp100and Melan-A/MART-1), and ultrastructural studies confirmed the presence ofmelanosomes in the cytoplasm of melanoma cells. Although most melanosomeswere stage II (premelanosomes), stage III and rare stage IV ("mature") melanosomeswere also seen in occasional melanocytes. Thus, the fact that some melanocytesgave a bright signal on CSLM can be explained by 2 phenomena: (1) the highrefractile index of melanin (n = 1.7)9 relativeto that of epidermis (n = 1.34)31 and (2) thesize of melanosomes (0.6-1.2 µm) seen at the ultrastructural level,which is similar to the illumination wavelength.

The detection of rare mature melanosomes in the 2 lesions describedherein does not disqualify them from being designated amelanoticmelanoma. The term amelanotic melanoma isused to refer to the clinical appearance of a lesion, ie, its lack of shadesof tan, brown, or black on routine visual inspection. This is the clinicallymost relevant use of the term, because it corresponds to the diagnostic pitfallamelanotic melanomas represent in patient care. It is the melanomas, whosesum of pigment does not exceed the pigmentation of the surrounding normalskin, which are most problematic to detect and manage. If a lesion were completelydevoid of any pigment, it would likely receive more clinical attention asan area of marked hypopigmentation.

The lesions were, as illustrated, not completely amelanotic on specialstains or at the ultrastructural level. This finding is in keeping with priorobservations on amelanotic melanoma. In their study of 15 melanomas, whichwere amelanotic on routine histological examination, Gibson and Goellner30 found melanosomes in various stages of melanizationin all but 2 cases of metastatic melanoma. They concluded that "amelanoticmelanomas generally produce some pigment; thus ‘amelanotic' is usedbecause of convention, not because pigmentation is totally absent." To ourknowledge, complete lack of any mature melanosomes has not been documentedyet in a primary cutaneous melanoma and has been reported only for histologicallyamelanotic metastatic tumors.

Near-infrared, reflectance CSLM is currently an investigational toolapproved by the Food and Drug Administration for human use. Although initialresults are promising and encourage further exploration of CSLM, more experienceis needed to determine its future role in the practice of clinical dermatologyand dermatologic surgery. At its current state of technology, CSLM has 2 majorlimitations compared with conventional histological methods. First, its levelof resolution is inferior to that of conventional histology: nuclear features,such as chromatin pattern, cannot be evaluated by CSLM. Second, CSLM can assessmicroanatomical structures only to a depth of approximately 300 µm.Thus, reticular dermal process cannot be reliably analyzed by CSLM.

Nonetheless, CSLM merits evaluation for use in the screening of pigmentedlesions. As the cases reported herein suggest, CSLM may emerge not only asan ancillary tool for diagnosis, but may also be of value for assessing themargins of a biopsy-proven lesion. It has its best resolution within the epidermisand superficial dermis, which makes it suitable for assessing the marginsof in situ melanomas. If future studies confirm that there is good correlationbetween the findings of assessment of margins by CSLM and by histologicalexamination, the surgical planning of clinically poorly recognizable melanomaswould be greatly facilitated. Precise mapping of the perimeter of an in situmelanoma may be accomplished by noninvasive means in 1 clinical visit, andnumerous punch biopsies and subsequent visits may no longer be necessary.

In conclusion, we have shown that in 2 cases of clinically amelanoticmelanoma reflectance CSLM allowed the in vivo recognition of an abnormal intraepidermalmelanocytic proliferation by noninvasive means. The correlation between confocalimages and histological findings was excellent. Reflectance CSLM shows promiseas a noninvasive enhanced screening tool for the detection of melanoma, includingamelanotic melanoma, and it may be of great value in mapping the margins ofin situ melanoma. However, future studies are needed to determine the sensitivityand specificity of CSLM in detecting melanoma and in outlining its perimeter.

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Article Information

Accepted for publication February 22, 2001.

This study was supported in part by Lucid Inc. The authors thank JimZavislan, PhD, and Milind Rajadhaksha, PhD, Lucid Inc, for technical support.

We are grateful to Stefanie Ho, MD, for assistance in obtaining confocalimages, and we thank the technical staff of the Department of Pathology, MSKCC,for processing and staining the tissue sections.

Corresponding author and reprints: Klaus J. Busam, MD, Departmentof Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York,NY 10021 (e-mail: busamk@mskcc.org).

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