Distinguishing between Benign and Malignant MelanocyticNevi by In Vivo Multiphoton Microscopy
Mihaela Balu1, Kristen M. Kelly2, Christopher B. Zachary2, Ronald M. Harris2, Tatiana B. Krasieva1,Karsten K€onig3,4, Anthony J. Durkin1, and Bruce J. Tromberg1
AbstractMonitoring of atypical nevi is an important step in early detection of melanoma, a clinical imperative in
preventing the disease progression. Current standard diagnosis is based on biopsy and histopathologicexamination, a method that is invasive and highly dependent upon physician experience. In this work, we useda clinical multiphoton microscope to image in vivo and noninvasively melanocytic nevi at three different stages:commonneviwithout dysplastic changes, dysplastic neviwith structural and architectural atypia, andmelanoma.We analyzed multiphoton microscopy (MPM) images corresponding to 15 lesions (five in each group) bothqualitatively and quantitatively. For the qualitative analysis, we identified themorphologic features characteristicof each group. MPM images corresponding to dysplastic nevi and melanoma were compared with standardhistopathology to determine correlations between tissue constituents and morphology and to evaluate whetherstandard histopathology criteria can be identified in the MPM images. Prominent qualitative correlationsincluded the morphology of epidermal keratinocytes, the appearance of nests of nevus cells surrounded bycollagen fibers, and the structure of the epidermal–dermal junction. For the quantitative analysis, we defined anumerical multiphoton melanoma index (MMI) based on three-dimensional in vivo image analysis that scoressignals derived from two-photon excited fluorescence, second harmonic generation, andmelanocytemorphologyfeatures on a continuous 9-point scale. Indices corresponding to common nevi (0–1), dysplastic nevi (1–4), andmelanoma (5–8) were significantly different (P < 0.05), suggesting the potential of the method to distinguishbetween melanocytic nevi in vivo. Cancer Res; 74(10); 1–10. �2014 AACR.
IntroductionOver the past 10 years of available data (1999–2008), cancer
mortality rates have declined by more than 10% in men andwomen (1), whereas the mortality rate for melanoma report-edly increased by 5.5% in men and remained stable in women(2). This is most likely due to an increase in the incidence ofmelanoma compared with other types of cancers (1) and todiagnosis at a late, incurable stage. Early detection is critical forgood prognosis and successful treatment of melanoma (3).Recently, noninvasive optical imaging technologies based on
laser scanningmicroscopy have emerged as promising tools forreal-time, in situ imaging of skin lesions with the potential toovercome current diagnostic limitations. These limitations are
related to: (i) the dermatologist's decision about the necessityof the biopsy after visual inspection based on dermoscopy andABCDE rule (Asymmetry, Border Irregularity, Color variega-tion, Diameter >6 mm, Evolving; ref. 4) and (ii) the dermato-pathologist's diagnosis decision based on a series of histolog-ical criteria. These methods are subjective and highly depen-dent upon the physician's experience, creating problems offalse-negatives, which delay diagnosis and treatment, andfalse-positives, which lead to unnecessary biopsies and treat-ments, emotional trauma, and increased medical costs. Thefalse-negative and false-positive rates for melanoma diagnosisare reported to be 10% to 50% (5, 6) and 40% to 80% (6, 7),respectively. Techniques such as reflectance confocal micro-scopy (8) andmultiphotonmicroscopy (MPM; ref. 9) have beenused in studies aiming to improve the accuracy of decisionsby dermatologists to perform a biopsy, whereas pump-probemicroscopy (10) and fluorescence lifetime microscopy (11)have been used to target limitations related to decisions bydermatopathologists.
MPM is a laser scanning microscopy technique that relieson nonlinear light–matter interactions such as two-photonexcited fluorescence (TPEF) and second harmonic generation(SHG) to achieve 3-dimensional (3D) images with submicronresolution. These contrast mechanisms produce images ofendogenous biomolecules in the tissue, without using specificfluorescent labels. In MPM, the main sources of fluorescenceare reduced nicotinamide adenine dinucleotide (NADH), flavin
Authors' Affiliations: 1Laser Microbeam and Medical Program, BeckmanLaser Institute, 2Department ofDermatology,University ofCalifornia, Irvine,California; 3JenLab GmbH, Schillerstrasse 1, Jena; and 4Department ofBiophotonics and Laser Technology, Saarland University, Saarbr€ucken,Germany
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Corresponding Author: Bruce J. Tromberg, Laser Microbeam and Med-ical Program,BeckmanLaser Institute,UC-Irvine, 1002HealthScienceRd.,E. Irvine, CA 92612. Phone: 949-824-8705; Fax: 949-824-8413; E-mail:[email protected]
adenine dinucleotide (FAD), keratin, melanin, collagen, andelastin fibers, whereas SHG is used to visualize collagen fibersin the dermis.
MPM has recently been used to establish sensitivity andspecificity criteria for melanoma diagnosis (12). These criteriawere identified on the basis of evaluation of distinguishingcharacteristics, measured using MPM on both in vivo andex vivo samples, linked to morphologic changes in melanomarelative to benign nevi and normal (control) skin. Benign nevican be common (without dysplasia) or dysplastic. Dysplasticnevi and their MPM features have not been described inprevious studies. In the pilot study presented here, we expandprevious measurements by using MPM in vivo to identifycharacteristic features of melanocytic nevi at three differentstages: common nevi without dysplastic changes, dysplasticnevi with structural and architectural atypia, and melanoma.A major focus of this work is to evaluate, for the first time, thepossibility to distinguish between melanoma and dysplasticnevi, a common clinical challenge, by establishing quantita-tive diagnostic criteria based on in vivo MPM signals.
MPM images corresponding to dysplastic nevi and mel-anoma are compared with standard histopathology toidentify correlations between tissue constituents and mor-phology and to evaluate whether standard histopathologycriteria can be identified in the MPM images. Several his-topathologic features characteristic of common nevi, dys-plastic nevi, and melanoma are expected to be identified inthe MPM images. For example, melanocytic nevi are com-posed of nevus cells, which, even though they are basicallyidentical to melanocytes, differ from melanocytes by beingarranged in clusters, or "nests" and by not showing dendriticprocesses (13). The nests are usually confined at the tips ofthe rete ridges (14). They are visualized in the MPM imagesas clusters of bright cells surrounded by collagen fibers atthe bottom of the epidermal–dermal junction (EDJ).
Dysplastic nevi are characterized by cytological atypia (var-iation in size and shape of nuclei) and architectural disordernot amounting tomelanoma in situ (13). Architectural disorderincludes lentiginous hyperplasia (proliferation of nevus cellseither singly or as nests along the basal layer of epidermis) andnests that are irregular in both shape and distribution and notconfined to the tips of the rete ridges. They are visualized in theMPM images as clusters of bright cells that are not fullysurrounded by collagen fibers and located along the EDJ.
There are several subtypes ofmelanoma. Their features havedifferences and similarities, but generally the following aresuggestive of malignancy: presence of melanocytes within theupper portion of the epidermis singly or in groups (Pagetoidspread); irregular junctional activity (atypical melanocytes,architectural disorder); and invasion of tumor cells into thedermis (13, 14).
We assessed qualitatively the presence of these features inthe in vivoMPM images corresponding to 15 lesions identifiedin 14 patients. We introduced three parameters related toTPEF, SHG, and melanocyte morphology to quantify thehistopathologic features identified in the MPM images. Theseparameters have been combined to obtain a numerical mul-tiphotonmelanoma index (MMI). TheMMI scale ranges from 0
to 9, where 0 and 9 represent the lowest and highest probabilityof melanoma, respectively. The MMI is a first attempt todevelop a quantitative index based on in vivo image parametersthat capture multiple relevant contrast elements unique tointrinsic signal nonlinear optical microscopy.
Materials and MethodsMPTflex clinical tomograph
The laser scanning–based clinical multiphoton tomograph,MPTflex (JenLab GmbH) consists of a compact, turn-keyfemtosecond laser (MaiTai Ti:Sappire oscillator, sub-100 fs,80MHz, tunable 690–1,020 nm; Spectra Physics), an articulatedarmwith near-infrared optics, and beam scanningmodule. Thesystem has 2 photomultiplier tube (PMT) detectors used forparallel acquisition of TPEF and SHG signals. A customizedmetallic ring taped on the subject's skin attaches magneticallyto the objective holder in the articulated arm, minimizingmotion artifacts. The excitation wavelength used for this studywas 790 nm. The TPEF signal was detected over the spectralrange of 410 to 650 nm, whereas the SHG signal was detectedover a narrow spectral bandwidth of 385 to 405 nm throughemission filters placed in the TPEF and SHG detection chan-nels, respectively. We used a Zeiss objective (40�, 1.3 NA, oilimmersion) for focusing into the tissue.
Study designWe imaged 15 melanocytic nevi (5 common nevi, 5 dysplas-
tic nevi, and 5 melanoma) in 14 patients. All in vivo measure-ments were conducted according to an approved institutionalprotocol with written informed consent obtained from allpatients. The 15 lesions were distributed in 6 primary locationsfor all patients, including back (4), arms (4), legs (1), chest (1),abdomen (3), and face (2). MPM measurements were per-formed on lesion sites as well as on adjacent normal skin.Optical sections of about 200 � 200 mm2 at different depthsranging from 0 to about 200 mm (5 mm steps) were obtained.The time required for each optical section was 6 seconds. Asthe optical section is limited to a small scan field, the overallinvestigation of the lesion required the acquisition of severalimage stacks of different skin sites. We acquired about threeimage stacks for each lesion. All lesions clinically diagnosed asdysplastic nevi and melanoma by board-certified dermatolo-gists (K.M. Kelly and C.B. Zachary) were biopsied and diag-nosed by a dermatopathologist (R.M. Harris), using standardhematoxylin and eosin (H&E) histopathology. For the quali-tative analysis, we compared MPM and histologic images todetermine whether H&E histopathology hallmarks could becorrelated with structures in in vivoMPM images. Quantitativemethods are described below.
Image analysisAll images were processed using ImageJ (15). For the quan-
titative analysis, we wrote macros for automatic measurementof key parameters characteristic of TPEF and SHG images.TPEF images were also used to identify and calculate thenumber of melanocytic dendrites. A composite MMI waslinearized on a 0 to 9 scale.
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For the TPEF contrast, we used the raw TPEF images tocalculate the ratio (F) between the spatial SD and the meanpixel intensity for each TPEF image in a z-stack correspondingto the EDJ.
F ¼ sFl
<I>
where sFl is the spatial SD of fluorescence signal intensity and<I> is the mean pixel intensity in the TPEF image. For eachlesion, F ratioswere calculated over 5 consecutive image planes(spanning a total of 20 mm in depth), starting with the image ofthe basal layer in each acquired stack and going down into theEDJ. We considered the EDJ starting at the location where thecollagen structure in the top of the papilla was visualized. Thebasal layer was defined as the first cell layer above the EDJ.A mean F value was calculated for each lesion. The mean
represents the average F value over the TPEF images analyzedin all stacks of the lesion.Parameter F is related to epidermal features assessed qual-
itatively by histopathology such as lentiginous hyperplasia andPagetoid spread. By its definition, it measures the degree ofpixel intensity homogeneity in the MPM image.For the SHG contrast, the SHG images were converted to 8-
bit images and subsequently to binary images by using theautomatic thresholding function in ImageJ (16). The automaticthresholding procedure was suitable for our analysis becausethe signal-to-background ratio (SBR) of the SHG images washigh. The average SBR for each of the SHG images included inthe analysis was at least 10:1 and typically averaged greaterthan 20:1. The "bright pixels" were defined as the pixels of value1 in the binary images. We defined the density of the brightpixels by the ratio between the number of bright pixels andtotal number of pixels in one image. The density of bright pixelswas calculated for each image in a z-stack of 8 consecutiveimage planes (spanning a total of 35mmin depth), startingwiththe first SHG image of the EDJ. For each stack, a parameter Swas defined as:
S ¼ sSHG
<r>
where sSHG is the SD and <r> is the mean density of brightpixels in the binary SHG images of the stack. A mean S valuewas calculated for each lesion. The mean represents theaverage S value over all the stacks of SHG images of the lesion.Parameter S, by its definition, is a measurement of the
change in collagen across the EDJ and, therefore, a measure-ment of histopathology features such as irregular nests ofnevus cells along the basal layer, erosion of the junction, andinvasion of melanocytes into the dermis. A large S reflects arapid increase in the collagen amount from the top of thedermal papillae to deeper layers in the papillary dermis. A smallS value reflects a slower increase in collagen content across thejunction, which is due to the presence of cells in the papillarydermis; typically nevus cells from the sides of the rete ridges indysplastic nevi and melanoma cells in melanoma lesions.A larger volume at the EDJ would comprise more informa-
tion for image analysis, but for dark, highly pigmented nevi,
TPEF and SHG signals diminish with depth due to highabsorption and scattering. Stacks of 20 and 35 mm totalthickness for TPEF and SHG images, respectively, proved tocontain relevant information in all analyzed lesions.
To determine melanocytic dendrite density, we used theNeuronJ plug-in (17) in ImageJ for tracing and counting ofmelanocytic dendrites in the TPEF images corresponding tospinosum and granulosum epidermal layers. We calculated adensity parameter D, which was defined as the number ofmelanocytes in a stack volume.
D ¼ NV
whereN is the number of melanocytic dendrites in the stratumspinosum and granulosum of the epidermis and V is thevolume (the image area� the thickness of the epidermis fromthe stratum corneum to the basal layer). A mean D value wascalculated for each lesion. The mean represents the averageD value obtained from melanocytic dendrites counted by 2independent observers.
Parameter D represents a measurement of the density ofmelanocytic dendrites in upper epidermal layers. A high den-sity number is a hallmark of melanoma, but a limited numberofmelanocytic dendrites is allowed in the stratum spinosum ofthe epidermis in dysplastic nevi (18). Parameter D was intro-duced to address this ambiguity.
ResultsTypical MPM images of normal pigmented skin are shown
in Fig. 1. MPM features are characterized by normal morphol-ogy and architecture of keratinocytes in the epidermal layers, aclearly delineated EDJ, and the presence of normal collagenand elastin fibers in the dermis. Pigmented keratinocytes arepresent in the basal layer. They appear as bright fluorescentcells along the EDJ due to their melanin content. Blood vesselscan be visualized in the dermis. These features can be noted inboth horizontal sections (x–y scan) and the correspondingcross-sectional (x–z scan) images.
We performed MPM imaging of pigmented lesions in threestages: (i) common melanocytic nevi without dysplasticchanges, (ii) dysplastic nevi with structural and architecturalatypia, and (iii) melanoma. MPM images were analyzed qual-itatively by identifying the morphologic features characteristicof each group of lesions. Quantitative analysis consisted ofmeasuring the parameters F, S, and D, which are related tosignals from TPEF, SHG, and melanocytic dendrites, respec-tively (see Materials and Methods).
without dysplastic changes), clinically diagnosed as junctionalor compound nevi.
The MPM features of melanocytic nevi were characterizedby normalmorphology of keratinocytes of the epidermal layersand well-defined nests of nevus cells surrounded by collagenfibers at the EDJ and in the dermis. Three melanocytic nevi
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imaged showed elongated rete ridges (Supplementary Fig. S1).Occasional melanocytic dendrites were visualized in the basallayer, but no dendrites were imaged in the upper epidermallayers of the common nevi imaged in this study. Melanocyticnevi were not biopsied, but the features identified by theMPMimaging were in good correlation with features generallyidentified by histopathology in junctional and compoundnevi (14).
The mean of F values for common nevi ranged between 0.86and 1.07. Themean of S values ranged between 0.42 and 0.6. Themean of D values was zero for all common nevi (Table 1).
Figure 2 shows representative MPM images of a compoundnevus at different depths. Nevus cells are visualized as brightdisk-like or oval cells among pigmented basal cells (Fig. 2B).Pigmented basal cells and melanocytes are also present inpigmented skin. Nevus cells can be distinguished from pig-mented basal cells and melanocytes by examining cellularmorphology and organization. Nevus cells appear to be iden-tical to melanocytes but differ because they are generally
arranged in clusters or "nests" and do not show dendriticprocesses (13). Thus, as individual cells, nevus cells and pig-mented keratinocytes are very difficult to identify in MPMimages because the source of contrast is the same: melaninfluorescence. However, they can be distinguished by theirmorphology. Nevus cells form nests at the EDJ or in the dermis,as shown in Fig. 2D–F. These images show a well-defined nestbecause the cluster of nevus cells is completely surrounded bycollagen fibers.
Corresponding F values for the images that were included inthe quantitative analysis and the S value for the full image stackare reported in the legend of Fig. 2.
Dysplastic neviWe imaged 5 dysplastic nevi with varying degrees of
atypia. At least one of the following features was presentin the MPM images: mild cellular atypia (enlarged nuclei),lentiginous hyperplasia (nevus cells with dense distributionalong the basal layer), acanthosis (thickening of the epider-mal layer), occasional melanocytes in the stratum spinosum,and nevus cells distributed in nests that were irregular inboth shape and distribution along the EDJ. The thickness ofthe epidermis was estimated by the depth at which the EDJwas visualized in the stack of images acquired from thestratum corneum to superficial dermis. Melanocytic den-drites in the spinosum layer of the epidermis could bevisualized in 2 of 5 dysplastic nevi imaged (SupplementaryFig. S2). Mild cytological atypia and mild architecturaldisorder were also identified in these lesions. Mean F andS values for dysplastic nevi ranged between 0.78–1.05 and0.37–0.53, respectively. The mean D values ranged between 0and 7,800 dendrites/mm3 (Table 1).
Table 1. The mean values of the quantitativeparameters F, S, D corresponding to the threestages of the pigmented lesions: common,dysplastic nevi, and melanoma
Figure 1. Pigmented normal skin.Left, horizontal sections of MPMimages (x–y scans) at differentdepths showing images of thestratum corneum (z ¼ 0 mm),keratinocytes normally distributedin the stratum spinosum (z ¼ 25mm), the basal cells (green)surrounding dermal papilla (blue;z ¼ 50 mm), collagen fibers (blue)and cross-sections of bloodvessels (white arrows; z ¼ 75 mm),collagen (blue) and elastin (green)in the dermis (z ¼ 120 mm). Right,cross-sectional view (x–z scan)corresponding to a vertical planethrough the horizontal sections onthe left.
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RepresentativeMPM images of a dysplastic nevus alongwithcorresponding histology are shown in Fig. 3. The histopatho-logic diagnosis was compound dysplastic nevus with mildatypia. The MPM images of the lesion showed lentiginoushyperplasia and irregular nests of nevus cells along the basallayer and in the papillary dermis. There was also more vari-ability in cell size compared with common nevi.Corresponding F values for the images that were included in
the quantitative analysis and the S value for the full image stackare reported in the legend of Fig. 3. The D value was 0 for thislesion.
MelanomaWe imaged 5 patients who were diagnosed with melanoma
corresponding to two subtypes of melanoma: superficialspreading melanoma (3) and lentigo maligna type (2).In superficial spreading melanoma lesions, we imaged
proliferation of atypical melanocytes (highly pleomorphicmelanocytes) at all levels within the epidermis and Pagetoidspread (presence of melanocytes within the upper portion ofthe epidermis singly or in groups Supplementary Fig. S3). Inmelanoma lentigo malignant type lesions, we imaged atyp-ical melanocytes in upper epidermal layers and invasion of
melanoma cells in the dermis. Mean F and S values formelanoma lesions ranged between 0.58–0.8. and 0.06–0.38,respectively. The mean D values ranged between 8,500 and78,000 dendrites/mm3 (Table 1).
MPM and histology images from one of the superficialspreading melanomas are shown in Fig. 4. MPM images ofthe lesion showed the presence of melanocytic dendrites in theupper layers of the epidermis (Fig. 4C), proliferation of atypicalmelanocytes, and architectural disorder in the basal layer (Fig.4D–F). Melanoma cells and suspected melanophages can bevisualized in the dermis (Fig. 4F). These features correlate wellwith the ones found in the corresponding histologic sections ofthe lesion (Fig. 4B). F values for the images that were includedin the quantitative analysis and the S value for the stack theimages belong to are reported in the legend of Fig. 4. ThemeanD value for this lesion was 63,878 dendrites/mm3.
The ranges of the mean F, S, and D values correspondingto the pigmented lesions in different stages are summarizedin Table 1. The distribution of the mean values of the quan-titative parameters F, S, and D are plotted in Fig. 5A–C.Significant differences between mean values for each groupwere evaluated using the Mann–Whitney U test. The nullhypothesis was that the mean F, S, and D parameters were
Figure 2. Compound nevus. A, clinical image (DermLite FOTO, Dermlite Inc.). B, MPM image of the basal layer showing nevus cells and pigmented basalcells; F ¼ 1.02. C, MPM image of the basal layer showing nevus cells, pigmented basal cells (green), and appearance of dermal papilla (blue); F ¼ 1.04. D,MPM images showing a nest of nevus cells (green) surrounded by collagen (blue) fibers; F¼ 0.95. E and F, MPM images at different depths showing a nest ofnevus cells (green) surrounded by collagen (blue) and elastin (green) fibers. The S value for this stack is 0.51. Scale bar, 40 mm.
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the same for two different groups. We rejected the null-hypo-thesis for a U value� 2 (the critical value for our experimentalconditions), corresponding to P < 0.05. Evaluating the U valuesfor each pair of groups, we found (i) the mean values of theF parameter corresponding to melanoma were significantlydifferent from themean values for dysplastic and commonnevi(P ¼ 0.016 and 0.009, respectively). Common and dysplasticnevi were not distinguished by themean F parameter (P¼ 0.5).(ii) The mean values of the S parameter for each group weresignificantly different from the values of the other two groups(P¼ 0.009–0.016). (iii) Parameter D separated melanoma fromcommon and dysplastic nevi (P¼ 0.009) but did not distinguishbetween common and dysplastic nevi (P ¼ 0.3).
The correlation between each of the parameters F, S, andD isshown in Fig. 5E–G. In this figure, the combination (color,marker) corresponds to an individual lesion. It can be noted,for example, that the dysplastic nevuswith themaximum valueof D (blue triangle, solid arrows)—closest to melanoma—alsohas theminimum value of S and a value of F close tominimum.In fact, from the dysplastic nevi we imaged, this was the onlyone diagnosed as nevus with "moderate to severe" dysplasia.One of the lesions diagnosed as "lentigo maligna" correspondsto the red circle in Fig. 5E–G (dashed arrows). In this lesion, weimaged only occasional ascending melanocytes in the upperepidermal layers (low D value) but severe proliferation of
atypical melanocytes at the EDJ and invasion in the dermis.Melanocytic proliferation resulted in a high degree of homo-geneity in the intensity of the TPEF images (low F value) due toa more uniform, increased pigmentation. The S value (ameasurement of dermal invasion) for this lesion was low butnot minimum. Figure 5E and F and the calculated Pearsoncorrelation coefficients (r) show that the S and F parametersare weakly correlated with D (r¼�0.47, P¼ 0.074 for S and D;r¼�0.5, P¼ 0.057 for F andD), whereas in Fig. 5G, we see thatS and F are well correlated with each other (r ¼ 0.71, P ¼0.0032). The relationship between S and F is not surprisinggiven the fact that they sample similar regions and comple-mentary processes around the EDJ. Among the three para-meters, S is the only one that can fully resolve all three states(i.e., benign, dysplastic, malignant). However, a combination ofall three parameters improves the performance by increasingthe separation between the dysplastic and melanoma groups,which we are mostly concerned about (the P value is reducedfrom 0.016 to 0.009). Therefore, to increase the performance ofthe metrics F, S, and D, we assigned each of these criteria ahistologic score from 0 to 3 on the basis of visualizing themeanvalues, as shown in Fig. 5A–C and Table 2. For each lesion, thescores of the three criteria were summed up to give a finalcontinuous MMI index, ranging from 0 to 9. Using thisapproach, common nevi scored between 0 and 1, dysplastic
Figure 3. Dysplastic nevus. Clinical image (DermLite FOTO, Dermlite Inc.). A, histologic section of the lesion. B, MPM images showing irregular nests of nevuscells (green) and collagen fibers (blue) along the basal layer at depths of 30 mm (C), 40 mm (D), 50 mm (E), and 60 mm (F). The corresponding F values are 0.89 (C)and 0.86 (D). The S value for this stack is 0.41. Scale bar, 40 mm.
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nevi between 1 and 4, and melanoma between 5 and 8. Thedistribution of these scores for the three groups is shown inFig. 5D. A Mann–Whitney U test shows that the MMI scorescorresponding to melanoma group are significantly differentfrom the other two groups (P ¼ 0.009). The difference inMMI scores of common and dysplastic nevi is marginally signi-ficant (P ¼ 0.03).
DiscussionMPM is capable of noninvasive in vivo imaging of human
skinwith sensitivity to the epidermis and superficial dermis (9).In this study, we performed qualitative and quantitative anal-yses of melanocytic nevi at three stages: common nevi with nodysplastic changes, dysplastic nevi, and melanoma. The qual-itative analysis involved identifying morphologic features ofthe lesions in the three groups and correlating MPM withhistologic features. The quantitative analysis was based onTPEF, SHG, and melanocyte parameters derived from 3Din vivo imaging.Common melanocytic nevi were characterized in MPM
images by normal morphology of keratinocytes of the epider-mal layers, well-defined nests of nevus cells surrounded bycollagen fibers at the EDJ and dermis, and elongated rete ridges.MPM images of dysplastic nevi showed lentiginous hyper-
plasia, mild cellular atypia, and nests of nevus cells that wereless defined and more irregular in shape and distribution than
the nests imaged in commonmelanocytic nevi. The presence ofmelanocytic dendrites in the stratum spinosum of the epider-mis was revealed in 2 of 5 dysplastic nevi. We found that thisfeature requires careful evaluation to avoid false-positive diag-nosis, as migration of melanocytes into the upper layers ofepidermis is a feature also present in melanoma.
Althoughmigration ofmelanocytes in the upper layers of theepidermis usually raises suspicion ofmelanoma, limitedmigra-tion of melanocytes in the stratum spinosum of the epidermisis acceptable in dysplastic nevi (18). In this case, the architec-tural disorder influences the overall grade. Indeed, in thelesions characterized by ascending melanocytes in the spino-sum layer of the epidermis and diagnosed as dysplastic nevi byhistopathology, MPM architectural disorder was milder incomparison with histopathologically confirmed melanoma.
In the pigmented lesions diagnosed as melanoma by histo-pathology, cytological atypia and architectural disorder werethe main features revealed by in vivo MPM imaging. Thisfinding is in good agreement with histopathology and theMPM diagnosis criteria identified in a previous study (12). Inaddition, we observed specific MPM features characteristic oftwo melanoma subtypes: (i) superficial spreading melanomawhere epidermis is mainly involved and there is proliferationof melanocytes and pleomorphic cells (Pagetoid spread)throughout all epidermal layers and (ii) lentigo maligna mel-anoma where the basal layer rather than the epidermis ismainly affected, atypical melanocytes were more confined to
Figure 4. Melanoma—superficial spreading type. A, clinical image (DermLite FOTO, Dermlite Inc.). B, histologic section of the lesion. C, MPM imagesshowing ascending melanocytes (arrows) in the granulosum layer of the epidermis. D, MPM images of the basal layer showing proliferation of atypicalmelanocytes (highly pleomorphic melanocytes); F ¼ 0.69. E and F, MPM images of the basal layer showing basal cells and atypical melanocytes (green)surrounding dermal papilla (blue); F ¼ 0.63 and 0.62, respectively. G, MPM images showing melanoma cells and probably melanophages invading thedermis (blue, collagen fibers). The S value for this stack is 0.35. Scale bar, 40 mm.
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the basal layer, and there was less Pagetoid spread in the upperepidermal layer along with epidermal atrophy.
Most of the histologic criteria for diagnosis of dysplastic neviandmelanoma such as cellular atypia, lentiginous hyperplasia,elongated dermal papilla, ascending melanocytes, and Page-toid spread were identified by MPM. Other histologic criteriacannot be easily identified in MPM images and they wouldneed to be correlated withMPM content. For instance, nests ofnevus cells localized on the sides of dermal papilla in histologicimages of dysplastic nevi are difficult to visualize in the MPM
Figure 5. Distribution and correlation of themean values and scores. The distribution of themean values of F (A),S (B), andD (C) parameters for common nevi,dysplastic nevi, andmelanoma. D, the distribution of theMMI scores for common nevi, dysplastic nevi, andmelanoma. E–G, correlation of F,S, andD values.The combination (color, marker) corresponds to an individual lesion. Black, blue, and red represent the common nevi, the dysplastic nevi, and the melanomagroups, respectively. Arrows, markers corresponding to the lesions discussed in the text.
Table 2. Scoring system based on visualizingthe mean values of the parameters F, S, Dplotted in Fig. 5
horizontal (i.e., x–y or en face) optical sections. These histologicfeatures can be seen in MPM images as nests of nevus cells,irregular in size and distribution, along the basal layer and inthe dermis. Common nevi were not biopsied, but we anticipatethat nests of nevus cells localized at the tips of the rete ridges inthe histologic images of common nevi are related to well-defined regular nests of nevus cells surrounded by collagenfibers in the MPM images.Morphologic changes such as cytological atypia and lenti-
ginous hyperplasia correlate with the TPEF signal. Likewise,morphologic changes at the EDJ such as appearance of nests ofnevus cells on the sides of the rete ridges or disruption of thejunction correlate with variations in the SHG signal. TPEF andSHGaremost sensitive to these processes whenmeasured overa volume of the EDJ, that is, several x–y image planes, in theregion of early-stage melanoma genesis. In the TPEF imagescorresponding to the three groups of lesions, common, dys-plastic, and melanoma, we identified a variation in the ratioof the spatial SD and the mean pixel intensity for the differentgroups. This ratio (the F score) measured over a stack ofimages (see Materials and Methods) is related to the degreeof pixel intensity homogeneity in the image. MPM images ofmelanoma lesions showed a higher degree of homogeneitydue to a more uniform, increased pigmentation in the EDJarea. The mean F ratios measured in melanomas were signif-icantly lower than the ratios measured in common (P¼ 0.009)and dysplastic nevi (P¼ 0.016). Themean ratios correspondingto common nevi and dysplastic nevi did not show a statisticallysignificant difference (P ¼ 0.5).In the SHG images of the three groups of lesions, we
identified a variation in the ratio of the SD and the meandensity of the bright pixels in the SHG binary images across astack of images corresponding to EDJ. This ratio (the S score) isrelated to the change in collagen amount across the EDJ. Alarge ratio reflects a rapid increase in the collagen amount fromthe top of the dermal papilla to deeper layers in the papillarydermis. Large ratios were characteristic of common nevi. Lowratios, which reflect a slower increase in collagen contentacross the junction, are due to the presence of cells in thepapillary dermis; typically nevus cells from the sides of therete ridges in dysplastic nevi and melanocytes in melanomalesions. The mean values of these ratios corresponding to eachgroup were significantly different from the values of the othertwo groups (P ¼ 0.009–0.016).A third measureable criterion for distinguishing dysplastic
nevi from melanoma, the D score, is related to the density ofmelanocytic dendrites in the upper epidermal layers. Dysplas-tic nevi were characterized by significantly lower density ofmelanocytic dendrites in the upper epidermal layers comparedwithmelanoma lesions. By combiningmelanocytemorphologywith TPEF and SHG features, we developed a quantitative 0 to 9point algorithm for evaluating in vivo images, structured in amanner similar to well-established histology scoring methodssuch as the Bloom–Richardson grading system (19). Thisintegrated MMI assigns unique values to each lesion. Byhaving a continuous 9-point scale, it is possible to separatecommon nevi (MMI ¼ 0–1), dysplastic nevi (MMI ¼ 1–4),and melanoma (MMI ¼ 5–8) with a high degree of statistical
significance (P ¼ 0.009–0.03). Given the relatively smallsample size, we feel that the three parameters F, S, and D,which sample different but complementary physiologic pro-cesses, are useful in describing a quantitative melanomaindex. However, as our patient population expands, it ispossible that sufficient predictive power can be achieved infuture studies with a subset of these parameters.
The results of this study provide an initial set of MPM fea-tures that are characteristic of common nevi, dysplastic nevi,and melanoma and correspond to descriptions from conven-tional histopathology. Using these criteria we have developed,for the first time, a quantitative algorithm derived from in vivoMPM measurements that shows potential to discriminatebetween these groups of melanocytic lesions. These results arecertainly limited by the small number of subjects. Dysplasticnevi, in particular, have very diverse features and a largerpopulation is necessary to validate diagnostic performance.Nevertheless, these findings and previously reported results(12) identify MPM signals that are consistent with melanomaand could be used to help guide further investigations. A morecomprehensive study of a larger number of patients is necessaryto validate the proposed scoring algorithm and evaluate howwell MPM technology can distinguish dysplastic nevi fromcommon nevi and melanoma. This could help dermatologistsincrease the accuracy of their diagnosis for pigmented lesionsthat fall into the borderline area, minimize the need for invasivebiopsies, and advance our knowledge of underlying biologicfactors that influence the appearance and progression of mel-anoma and related skin diseases.
Disclosure of Potential Conflicts of InterestKarsten K€onig has ownership Interest (including patents) in JenLabGmbH. A.J.
Durkin and B.J. Tromberg have ownership interest (including patents) in Modu-lated Imaging Inc. B.J. Tromberg has a commercial research grant from Unilever,Inc., and has ownership interest (including patents) in University of CaliforniaRegents. No potential conflicts of interest were disclosed by the other authors.
Authors' ContributionsConception and design: M. Balu, K.M. Kelly, C.B. Zachary, T.B. Krasieva,K. K€onig, A.J. Durkin, B.J. TrombergDevelopment of methodology:M. Balu, K.M. Kelly, C.B. Zachary, R.M. Harris,T.B. Krasieva, K. K€onig, A.J. Durkin, B.J. TrombergAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Balu, K.M. Kelly, C.B. ZacharyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Balu, K.M. Kelly, C.B. Zachary, R.M. Harris,B.J. TrombergWriting, review, and/or revision of the manuscript: M. Balu, K.M. Kelly,C.B. Zachary, R.M. Harris, T.B. Krasieva, A.J. Durkin, B.J. TrombergAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C.B. Zachary, K. K€onigStudy supervision: K.M. Kelly, B.J. Tromberg
Grant SupportSupport for this work was provided by the NIH NIBIB Laser Microbeam and
Medical Program (LAMMP, P41-EB015890), NCI-2P30CA62203 (University ofCalifornia, Irvine Cancer Center Support Grant), and NIH K25-EB007309. Beck-man Laser Institute programmatic support from the Arnold andMabel BeckmanFoundation and Air Force Research Laboratory Agreement No. FA9550-04-1-0101 are also acknowledged.
The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received September 10, 2013; revised February 20, 2014; accepted March 10,2014; published OnlineFirst March 31, 2014.
Benign and Malignant Melanocytic Nevi Imaging by In Vivo MPM
www.aacrjournals.org Cancer Res; 74(10) May 15, 2014 OF9
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