Conjunctival Primary Acquired Melanosis: Is It Time for a New Terminology?

PERSPECTIVE Conjunctival Primary Acquired Melanosis: Is It Time for a New Terminology?

FREDERICK A. JAKOBIEC

PURPOSE: To review the diagnostic categories of a CONCLUSION: All pre- and postoperative biopsies of group of conditions referred to as ‘‘primary acquired flat conjunctival melanocytic disorders should be evalu- melanosis.’’ ated immunohistochemically if there is any question DESIGN: Literature review on the subject and proposal regarding atypicality. This should lead to a clearer micro- of an alternative diagnostic schema with histopathologic scopic descriptive diagnosis that is predicated on an and immunohistochemical illustrations. analysis of the participating cell types and their architec- METHODS: Standard hematoxylin-eosin–stained sec- tural patterns. This approach is conducive to a better tions and immunohistochemical stains for MART-1, appreciation of features indicating when to intervene HMB-45, microphthalmia-associated transcription factor therapeutically. An accurate early diagnosis should fore- (MiTF), and Ki-67 for calculating the proliferation index stall unnecessary later surgery. (Am J Ophthalmol are illustrated. 2016;162:3–19. Ó 2016 by Elsevier Inc. All rights RESULTS: ‘‘Melanosis’’ is an inadequate and misleading reserved.) term because it does not distinguish between conjunctival intraepithelial melanin overproduction (‘‘hyperpigmenta- ONJUNCTIVAL MELANOMAS ARE SEEN IN 2–8 INDI- tion’’) and intraepithelial melanocytic proliferation. It viduals per million in predominantly white popula- is recommended that ‘‘intraepithelial melanocytic prolif- tions and constitute only 2% of ocular eration’’ be adopted for histopathologic diagnosis. Atyp- C 1 melanomas, the remainder developing in the uveal tract. ical proliferations are characterized either by bloated There is some evidence that ultraviolet radiation plays a dendritic melanocytes with enlarged cell components role in causing many conjunctival melanocytic lesions, (dendrites, cell bodies, and nuclei) or by epithelioid mela- which develop most often in the interpalpebral zone.2 nocytes without dendrites. Atypical polygonal or epithe- Around 25% of conjunctival melanomas arise in pre- lioid pagetoid cells may reach higher levels of the existing nevi (which appears not to influence prognosis) epithelium beyond the basal layer. Immunohistochem- and 70%–75% in pre-existing ‘‘primary acquired melano- istry defines the degree of melanocytic proliferation or sis,’’ which is somewhat more common than melanomas. the cellular shape (dendritic or nondendritic) (MART-1, Primary acquired melanosis (PAM) is one of the few dis- HMB-45) or identifies the melanocytic nuclei (MiTF). eases in ophthalmology that is on a trajectory capable of Intraepithelial melanocytic proliferation without atypia culminating in fatality (13%–30% lethality for mela- represents increased numbers of normal-appearing den- nomas at 10 years). An important exercise in ophthalmic dritic melanocytes (hyperplasia or early neoplasia) that pathology, therefore, is the prediction of which patients generally remain confined to the basal/basement mem- with PAM are at high risk to develop melanoma, a pro- brane region. Intraepithelial nonproliferative melano- gressively metastasizing lesion once the invasive nodule cytic pigmentation signifies the usually small number of exceeds a thickness of 0.8 mm.3 conjunctival basal dendritic melanocytes that synthesize Despite ongoing investigations, including many clini- increased amounts of melanin that is transferred to sur- cally valuable and biologically insightful studies and re- rounding keratinocytes. views,3–12 the subject of flat, acquired pigmented lesions of the conjunctiva continues to be challenging and to See Accompanying Editorial on page 1. retain a certain degree of mystery. This is particularly Accepted for publication Nov 3, 2015. true of the group of conditions collectively referred to From the David G. Cogan Laboratory of Ophthalmic Pathology, as primary acquired melanosis, a designation with Massachusetts Eye and Ear Infirmary and the Department of Ophthalmology, Massachusetts Eye and Ear Infirmary and Harvard limitations that has been in circulation for over half a 11,12 Medical School, Boston, Massachusetts. century and that is the main focus of this article. Inquiries to Frederick A. Jakobiec, Director, David G. Cogan Zimmerman (1920–2013)12 has given a reminiscence of a Laboratory of Ophthalmic Pathology, Massachusetts Eye and Ear Infirmary, 243 Charles St – Room 328, Boston, MA 02114; e-mail: gentleman’s difference of opinion regarding the fate of [email protected] PAM between him and Algernon Reese (1900–1980),

0002-9394/$36.00 Ó 2016 BY ELSEVIER INC.ALL RIGHTS RESERVED. 3 http://dx.doi.org/10.1016/j.ajo.2015.11.003 who took a special interest in the subject as the father of MELANOCYTIC ORIGINS AND American ophthalmic oncology.13,14 It is proposed herein that the term PAM, which departs from descriptions of BIOLOGY analogous skin conditions in dermatopathology, be AS THE MIDLINE EMBRYONIC ECTODERM INVAGINATES TO abandoned because it is insufficiently specific and form the neural tube, cells also delaminate to create aggregates nondescriptive of the underlying cellular events. Over on the dorsolateral aspects of the tube called the neural crest the years, attempts have been made to grade PAM with (Figure 1, Top panel, left side).16 Among its many roles, the respect to mild, moderate, or severe degrees of atypia, but neural crest contributes Schwann cells that wrap around the intraobserver and interobserver variability has often axons of peripheral nerves; it also persists postembryonically confounded reproducible accuracy. I have therefore come as the paraspinal ganglia; and most germane to the current dis- to the conclusion that the term primary acquired cussion, it spawns melanocytes, which undergo far-flung mi- melanosis creates an unnecessary impediment for clearly grations to ultimately arrive in the skin and mucosae communicating to ophthalmologists, dermatopathologists, (including the conjunctiva) (Figure 1, Top panel),16,17 but and general pathologists the true biologic nature of the also in other sites like the uvea of the eye (the retinal family of conditions it is meant to encompass. pigment epithelium derives not from the neural crest, but The objectives of this review are 3-fold: (1) to describe instead from the outer neuroectodermal layer of the optic cup). in uncomplicated terms the origin and biology of melano- In the conjunctiva, most migrating melanocytes reach cytes; (2) to describe and illustrate the clinical, histopath- the epithelium to lodge among the basal germinal keratino- ologic, and immunohistochemical features of conjunctival cytes (squamous or epidermoid cells) at the level of the PAM; and (3) to offer an alternative pathologic diag- basement membrane. In more darkly complexioned indi- nostic terminology and schema based on current concepts viduals, some melanocytes may get ‘‘hung up’’ or arrested and language in dermatopathology. The prognosis for in their migration without ever settling in an epithelium cutaneous and conjunctival melanomas has improved (Figure 1, Top panel). Such melanocytic collections in over the years, less as a consequence of advances in ther- the episclera or conjunctival substantia propria appear slate apy, but more as a result of the treatment of lesions at gray to blue, are stationary through life, and are postmitotic the earliest stages before melanomatous evolution has and therefore not responsible for generating conjunctival occurred. For the purposes of this presentation, the term melanomas; they may, however, rarely create diffuse loose primary acquired melanosis has been episodically used, aggregates of cells resulting in the nevus of Ota or focal col- even though an argument is simultaneously made for lections such as the blue nevus or cellular blue nevus. the adoption of what is regarded as a more appropriate The density of the basal dendritic melanocytes in the diagnostic nosology based on the vocabulary that is skin (intraepidermal) differs little among the various frequently employed today for cutaneous precursor le- races, but it may vary to some degree among the races sions, namely ‘‘intraepithelial melanocytic proliferations in mucosal epithelia. Melanocytes are normally found 15 (IMP).’’ The expedient of a dual description of the singly and widely spaced along the basement membrane conjunctival disease (ie, IMP/PAM) in this exposition is rather than in nests (Figure 1, Top panel and Bottom intended to ease the intellectual transition to the new panel #1). Non-nesting hyperplasias of normal-appearing proposed diagnostic framework. dendritic melanocytes of whatever causation are referred Any effort to revise an entrenched terminology will to as manifesting a lentiginous growth pattern when inevitably be considered as quixotic or even provocative confined to the basal layer of the epithelium. Their den- by some experts and consequently encounter variable resis- drites serve to distribute melanin granules by functioning tance. This writer believes, based on the rationale provided as physiologic syringes that inject the granules into the below, that this undertaking is nonetheless worthwhile. surrounding keratinocytes (melanin–keratinocytic unit) The current conjunctival terminology is isolated and paro- (Figure 1, Top panel, inset on right side).17,18 chial, failing to reflect that used in dermatopathology, nor In more darkly complexioned individuals, bilateral, asym- does it convey a sufficiently precise impression of the un- metrical small golden or chocolate brown macules in the derlying histopathologic features. Any reaction to the pro- sun-exposed, interpalpebral epibulbar conjunctiva may posed new nosology described herein is considered a frequently be seen. Such a lesion in the skin is designated positive development, especially if it stimulates better as an ephelis or freckle. These totally benign lesions in the communication and improved diagnostic accuracy. Since conjunctiva have been referred to as epithelial melanoses it is generally conceded that there are no clinical features or hypermelanoses; because of the argument advanced below that can distinguish premalignant from totally benign flat regarding confusion engendered by the term melanosis, I pigmented conjunctival lesions (although progressive clin- would prefer to call them nonproliferative melanocytic ical spread must be regarded with high suspicion), this pigmentary spots, macules, or patches. These spots are article is focused mostly on the histopathologic aspects of movable upon the sclera and are formed as a result of the diagnosis. increased manufacture and transfer of melanin granules to

4 AMERICAN JOURNAL OF OPHTHALMOLOGY FEBRUARY 2016 FIGURE 1. Schematic summary of the origin and anatomic-physiologic features of melanocytes (Top) and their various pigmentary and proliferative conditions (Bottom). Because intraepithelial melanocytic proliferation is a more biologically descriptive and diag- nostically accurate term, it has been substituted throughout for primary acquired melanosis. (Top panel) After invaginating from the midline embryonic ectoderm, neural crest cellular aggregates form on the dorsolateral aspects of the neural tube. The neural crest is the source of melanocytes (among other cells) that undergo widespread migrations to eventually lodge among the basal germinal keratinocytes of the epithelium or epidermis, where they extend long processes or dendrites among a group of surrounding keratinocytes (squamous cells), the melanin-epithelial unit (shown in the upper right inset with the melanocytic cell body resting on the basement membrane, BM). Some dendritic cells round up and create intraepithelial nevocytic junctional nests that evolve into conventional nevi; upon dropping off into the connective tissue, the lesion becomes a compound nevus. A population of melanocytes can get ‘‘hung up’’ in the dermis or episclera to produce ocular ‘‘melanosis’’, focal blue nevi, or the more diffuse nevus of Ota. (Bottom panel) (1) Normal population of widely scattered basal dendritic melanocytes in the conjunctival epithelium. (2) Intraepithelial nonproliferative melanocytic pigmentation is due to an increased synthesis of melanin granules without an apparent increase in the usual number of melanocytes that transfer the granules to surrounding keratinocytes (called ephelis or freckle in the skin). (3) Intraepithelial melanocytic proliferation without atypia (hyperplasia) is represented by increased numbers of normal-sized dendritic melanocytes that transfer melanin and remain in their normal position along the basement membrane, but are separated by fewer basal keratinocytes (termed a lentigo in the skin). (4) Intraepithelial melanocytic proliferation with atypia is represented toward the left by enlarged bloated dendritic melanocytes with large hyperchromatic nuclei, large cell bodies, and swollen dendrites that occupy all levels of the epithelium and continue to manufacture melanin. An alternative morphologic form of atypia is depicted toward the right in the form of polygonal epithelioid cells that are crowded along the basement membrane region and additionally ascend singly to higher levels of the epithelium (pagetoid growth pattern). Owing to the absence of dendrites on the epithelioid cells, little pigment is transferred to the keratinocytes. If virtually all of the epithelium is replaced by atypical proliferating melanocytes, morphologically a melanoma in situ stage has been reached. For the more common morphologic stages of proliferation featuring better preservation of the keratinocytes, melanocytic proliferation with atypia is a recommended term.

keratinocytes rather than from melanocytic proliferation to higher-level keratinocytes and goblet cells. Unlike hyper- (Figure 1, Bottom panel #2). The pigment is most prominent plasia (Figure 1, Bottom panel #3), melanocytic neoplasia in the cytoplasm of the basal germinal keratinocytes and be- (Figure 1, Bottom panel #4) denotes an irreversible genetic comes diluted in the suprabasal layers of the epithelium, alteration that allows cells to escape the usually operative where it is aggregated as supranuclear caps to protect the negative feedbacks designed to arrest proliferation. Except keratinocytic nuclear DNA from incident ultraviolet radia- when there is frank atypia, morphology alone is probably tion.4,9,19 As long as there is preservation of functioning not adequate for separating these 2 processes when neoplasia sinuous dendrites, melanin can additionally be transferred is in its earliest phase.

VOL. 162 NEW TERMINOLOGY FOR PRIMARY ACQUIRED MELANOSIS 5 INTRAEPITHELIAL MELANOCYTIC for conjunctival melanoma, although conceptually this is a long-shot possibility. Unfortunately, ‘‘melanosis’’ is too PROLIFERATION/PRIMARY ACQUIRED noncommittal a term for a proliferative condition of the me- MELANOSIS: DEFINITIONS AND lanocytes. One can reserve the use of melanosis for the PRECEDING STUDIES strictly nonproliferative synthesis of melanin granules, for which, however, the descriptive term of ‘‘intraepithelial IT CANNOT BE OVEREMPHASIZED THAT THE LESIONS SUB- nonproliferative melanocytic pigmentation’’ would seem to sumed under the rubric of IMP/PAM share one overarching be more appropriate and accurately descriptive. feature: melanocytic proliferation (Figure 1, Bottom panel The precursor lesions to melanoma in the skin often sur- #3 and Bottom panel #4). Most melanomas (75%) arise vive as an intraepithelial radial growth phase at the edges of from the dispersed dendritic melanocytes of IMP/PAM; a the invasive nodule. As mentioned, the precise histologic minority arise from persistent intraepithelial junctional features of these radial extensions are not identically repro- mitotic activity of more rounded nevocytes that have lost duced in the conjunctival epithelium, which is thinner than their dendrites9 and are responsible for conventional nevo- the epidermis and lacks the rete pegs and adnexal structures melanocytic nevi (Figure 1, Top panel, right side). displayed in the skin. Moreover, the definition of the radial Extremely rare melanomas can develop from a small num- growth phase in the skin includes extension of atypical cells ber of melanocytes with an evanescent intraepithelial from the epithelium into the papillary dermis,15 which is phase and are called nodular. The subepithelial nevocytes not a microanatomic modification of the conjunctival are postmitotic in contrast to the junctional nevocytes, substantia propria.8,22 Any degree of involvement of the and therefore do not play a significant role in generating superficial substantia propria is thus considered to be melanomas. The stage of IMP/PAM corresponds to what synonymous with microinvasion. The nomenclature for has been characterized in dermatopathology as a variably melanocytic intraepidermal proliferations that has been prolonged radial growth phase within the epidermis, which employed in dermatopathology15 includes the following: represent in the skin precursors for lesions such as lentigo atypical melanocytic hyperplasia, pagetoid melanocytic maligna, acral lentiginous, and superficial spreading sub- proliferation, atypical lentiginous hyperplasia, pagetoid types of melanoma.15 Because of differences in anatomy, melanocytosis, precancerous melanosis, severe melanocytic they are precluded from being reliably identified in the con- dysplasia, dysplastic atypical nevus, and melanoma in situ. junctiva.8,20 A vertical growth phase develops when a Pagetoid spread, according to dermatopathologic usage, in- nodule forms in the subepithelial connective tissues (the dicates the percolation of individual atypical melanocytes dermis of skin or the substantia propria of the throughout the multilaminar epidermis (buckshot appear- conjunctiva), at which stage metastases may develop ance), typically reaching higher levels beyond the basal from increasing access to lymphatic channels as the cell/basement membrane zone. This feature is more difficult nodule thickens. As the vertical thickness of the nodule to appreciate in the conjunctival epithelium, which can be progresses beyond 0.8 mm, metastases become more as thin as 2–3 cell layers in some topographic zones such as common.5,21–23 the fornices. Two terms that are sufficiently encompassing The most pressing issue is to determine if a given lesion of and more clearly descriptive and that consequently seem IMP/PAM is likely to progress to invasive melanoma.6,10,24 preferable are ‘‘melanocytic intraepidermal neoplasia’’ and In this connection the term ‘‘primary acquired melanosis’’ ‘‘intraepithelial melanocytic proliferation,’’ with or without poses several difficulties. For example, today ‘‘primary’’ in atypia. oncology refers to the original site of a tumor as The latter term is preferred by some but not all derma- distinguished from its metastases (secondary lesions). In topathologists; it has been recommended herein for diag- conjunctival PAM, on the other hand, ‘‘primary’’ is nosing conjunctival and periocular cutaneous eyelid employed to rule out certain local extrinsic stimuli such as intraepithelial lesions manifesting increased numbers of inflammation, climatic factors, or ultraviolet radiation that melanocytes. ‘‘Proliferation’’ is less ominous in terms of provoke melanin granule overproduction or deposition in insurability issues than ‘‘neoplasia.’’ Both ‘‘proliferation’’ melanophages but generally do not stimulate a conspicuous and ‘‘neoplasia’’ confined to the epithelium signify a melanocytic proliferation. ‘‘Acquired’’ means that the nonmetastasizing condition because of a lack of access lesion is not congenital, as in congenital ocular melanosis to lymphatic channels. Other efforts to bring conjunctival and oculodermal melanosis (nevus of Ota), conditions due lesions into conformity with the criteria of dermatopa- to embryologically incompletely migrated melanocytes thology have been discussed and alternative classifications resident within the episcleral tissues (melanosis oculi) or have been offered.24–26 The closest new classification conjunctival substantia propria. Via the Tyndall effect scheme to the one outlined in this article has been (light scattering of higher-energy blue wavelengths from described by Damato and Coupland26 and restated by deep cells) these locations confer a bluish or slate-gray discol- Kenawy and associates.27 They conducted a critical re- oration that is immovable (Figure 1, Top panel). In practice, view of the term primary acquired melanosis and pointed melanocytes in these locations do not serve as precursors out its failure to convey the biologic substrate behind the

6 AMERICAN JOURNAL OF OPHTHALMOLOGY FEBRUARY 2016 histopathologic diagnosis, similar to the critique already individual cells or small cell clusters into higher levels of provided. They curiously do retain, however, the terms the epithelium, are their criteria for establishing risk for primary hypermelanosis for complexion-associated the eventual emergence of melanoma. In a defense of the pigmentation and secondary melanosis for Addison dis- term primary acquired melanosis Folberg has written: ‘‘to ease and other externally provoked melanin granule pro- change nomenclature in the pathology report interrupts duction. Even if one makes an argument for salvaging the chain of clinical logic.pathologists should strive melanosis as a histopathologic term, it is not obvious to assist surgeons in the management of their cases, what diagnostic benefit accrues from the use of the inten- not by imposing on them a classification that may be sified term hypermelanosis (hyperpigmentation), since the biologically accurate but clinically confusing.’’22 conjunctiva is normally nonpigmented, so that melanosis There is no reason, however, why clinical and pathologic (pigmentation) should suffice. diagnoses should rely on the same vocabulary. Clinicians in Damato and Coupland26 recommend adoption of the all specialties are accustomed in their practices of medicine term conjunctival melanocyte intraepithelial neoplasia to making a descriptive and vaguely interpretive diagnosis us- (C-MIN)28 when there are increased numbers of melano- ing one vocabulary, and receiving post-biopsy a more specific, cytes. The problem with invoking neoplasia rather than biologically grounded histopathologic diagnosis employing proliferation is that not all flat pigmented lesions are neces- another more technical vocabulary. In place of ‘‘primary ac- sarily autonomously neoplastic or clonal—an indetermi- quired melanosis’’ for a clinical diagnosis, one could describe nate number may in fact be preneoplastic idiopathic the lesion simply as a pigmented macule, a flat pigmented hyperplasias or reactive hyperplasias due to an external patch, or another kindred term with accompanying measure- stimulus (eg, ultraviolet radiation, postinflammatory) that ments of the greatest horizontal and vertical dimensions. A occur within a normal range of physiologic responses and clinical photograph is invaluable for documentation, espe- can be modulated by negative and suppressive feedback. cially if the lesion reflects from the epibulbar conjunctiva They therefore are nonclonal and conceivably reversible through the fornix onto the palpebral conjunctiva when or, at the most, stationary. The sharp definition of the straightforward planar measurements are more difficult to interface between hyperplasias and neoplasias awaits obtain. Furthermore, one microscopic aspect that has not further genetic studies. The presence of cytologic atypi- been heavily stressed in previous work is an objective charac- cality, however, can be employed as an indirect morpho- terization with adequate nonimpressionistic specificity of the logic sign for inferring clonality. A significant virtue of elements of cytologic atypia—eg, nuclear size, the presence of Damato and Coupland’s approach is that they have devised nucleoli, the prevalence of mitoses, etc. Until recently I a 12-category grading system of potentially unfavorable his- employed the AFIP diagnostic scheme, which I still believe topathologic features, which reflects an attempt to objec- to be fundamentally sound, but I have rethought the termi- tify microscopic risk factors, as further alluded to below. nology employed, which I believe can be redesigned to better Historically, the cornerstone of contemporary histopath- communicate what is actually going on biologically. ologic interpretation of conjunctival acquired flat pigmen- Sugiura and associates28 have followed up on the work of tations was first developed by Zimmerman in 1966 and Folberg and associates. While retaining the term primary 1978,11,12 then elaborated upon by Folberg and associates acquired melanosis with atypia, they attempted to analyze in an outstanding series of articles from the Armed in 29 cases the type of atypicality manifested by the lesions Forces Institute of Pathology (AFIP) in the 1980s4–6,21 and came up with 2 forms of cytologic atypia along with 2 and recently briefly summarized in a major pathologic categories of architectural atypia concerning growth oncologic textbook.22 Zimmerman’s first set of histopatho- pattern. They did not employ the term melanoma logic diagnostic recommendations divided acquired mela- in situ.24 Melanocytic hyperplasia along the basement nosis into benign melanosis (subgroup A, represented membrane (lentiginous or basilar hyperplasia) was detected either by pigmentation or by a few clusters of basal ‘‘nevus’’ in 13 cases considered to be low risk for developing cells) or marked ‘‘junctional’’ activity (subgroup B, exhibit- melanoma (although recurrences were often seen). It ing many nests of ‘‘disturbing’’ nevus cells). Cancerous was composed of small to medium-sized polyhedral melanosis was employed for invasive lesions.11 Subse- melanocytes with inconspicuous cytoplasm, a high quently he adopted the term benign acquired melanosis nuclear-to-cytoplasmic ratio, and small to medium-sized either with minimal or no intraepithelial melanocytic ac- hyperchromatic nuclei bereft of nucleoli. High-risk lesions tivity or moderate to marked activity with atypia, the latter (16 cases) exhibited larger epithelioid cells with pagetoid typically associated with chronic inflammation. Malignant features, patently visible eosinophilic cytoplasm, medium acquired melanosis was used for invasive lesions. The work to low nuclear-to-cytoplasm ratios, large clearly observable of Folberg and associates5,6,21,22 is based on the nucleoli, and often nuclei with a vesicular character. These determination of the presence of melanocytic atypia or lesions had a 63% rate of local recurrence and a metastatic its absence in biopsy specimens. The identification of rate of 25%. Microinvasion or frank invasion of the epithelioid cells and their creation of discohesive and substantia propria were apt to be associated with intraepi- often confluent nests, and of pagetoid spread of thelial epithelioid cells. Four cases were cytologically and

VOL. 162 NEW TERMINOLOGY FOR PRIMARY ACQUIRED MELANOSIS 7 architecturally mixed, in that disparate biopsies from sepa- CLINICAL FEATURES rate regions of the same lesion could display either low-risk or high-risk characteristics. An overall ominous behavior IMP/PAM INITIALLY APPEARS AS FLAT PIGMENTED PATCHES (metastases) was aligned with the more worrisome cytopa- (Figure 2, Top left and Top right); they can also be multi- thologic and architectural features. focal, waxing and waning sectors (presumably immune- Most authors in this field (Damato and Coupland,26 mediated, with partial regression and renewed spread Folberg,22 and Sugiura and associates28) have recommen- occurring in different directions), potentially involving ded treatment for all preinvasion lesions of primary ac- wide areas during a prolonged phase measured in years quired melanosis with atypia. Sugiura’s observations (Figure 2, Middle left to Bottom right). The disease can unaccountably were not carried forward by one of his spread up the ducts of the lacrimal gland and through co-authors and are not included in that author’s revised the puncta and canaliculi to extend into the lacrimal textbook completely dedicated to cutaneous and mucosal sac, where a melanomatous nodule may subsequently melanocytic proliferations.25 This textbook offers a few develop.29,30 This forerunner lesion displays a distinctive terminological changes. Flat pigmentations are divided golden or chocolate-brown coloration that can eventuate into 2 main groups, conjunctival hypermelanosis (either in 1 or more nodules of invasive conjunctival melanoma. without perceptible melanocytic hyperplasia or with cyto- A rare variant is IMP/PAM sine pigmento (amelan- logically ‘‘banal’’ hyperplasia) and conjunctival hypermela- otic signaling poor synthesis of melanin),31,32 which nosis with atypical melanocytic hyperplasia (either can be encountered before or after therapy and lentiginous [basal] or pagetoid). In essence, the term pri- requires a biopsy for its detection. While it is classically mary acquired melanosis was replaced by hypermelanosis, completely flat and movable upon the surface of the which continues to obscure the precise underlying patho- globe, some surface irregularities in IMP/PAM may be logic and biologic substrate as discussed above. evinced by collections of subepithelial melanophages Melanoma in situ is a morphologic entity denoting virtu- (from pigmentary incontinence) and/or exuberant ally total replacement of the squamous epithelium with infiltrates of mononuclear inflammatory cells in the sparing of a monolayer of flattened surface keratinocytes. substantia propria. Invasive disease, however, may apparently arise more The disease makes its appearance usually after age 40 commonly from less extreme forms of intraepithelial mela- (average age at presentation for IMP/PAM is 56 years33 nocytic proliferation and has also been considered to be and for melanoma arising in IMP/PAM is 61 years,34 potentially equivalent to melanoma in situ.24 This situa- lending credence to the proposition that it takes many tion parallels that of squamous lesions—namely, do squa- years for atypical IMP/PAM to culminate in melanoma). mous carcinomas obligatorily arise from full-thickness IMP/PAM with and without melanoma has occasionally carcinomas in situ, or can squamous dysplasias of moderate been observed in teenagers. White patients are most severity also serve as precursor lesions? Damato and Coup- frequently affected, although black patients can develop land26 have circumvented subjective grading of intraepi- the disease. (Curiously, melanomas of the skin develop thelial conjunctival melanocytic disease by offering a rarely in black patients because of the protective properties cogent stratification scheme in the form of a grading list of melanin, whereas they do appear in these patients in the that attempts to evaluate 12 parameters regarding atypia. relatively nonpigmented soles of the feet and mucosal Individual consideration is given to intraepithelial melano- membranes.) The proliferative disease is always unilateral cytic patterns of growth, various degrees of vertical exten- and thereby can generally be distinguished from nonproli- sion upwards within the epithelium, the characterization of ferative melanocytic pigmentation (due to overproduction nuclear size, ampleness of cytoplasm, and the presence of of melanin granules), which is virtually always bilateral nucleoli and mitoses, among other features. Since it re- even if asymmetrically so, especially in darkly complex- quires more concentrated attention for analyzing cases, ioned individuals. time will tell whether ophthalmic pathologists will expend IMP/PAM can spread to any region of the conjunctival the intellectual energy entailed in its use, which will deter- sac and may become most accentuated in the inferior mine whether it will be widely adopted. Anyone with a epibulbar forniceal and tarsal areas, while simple melanin deep interest in the subject under discussion should read pigmentation tends to fade toward the fornices, being this article.26 I came across it in preparing the bibliography most pronounced in the exposed interpalpebral zone. for this paper after I had formulated and finalized my own Pigmentation of the tarsal conjunctiva is always suspi- thoughts, which have substantial terminological overlap cious.35 The eyelids should therefore be everted for with the contribution of Damato and Coupland. My minor clinical inspection of concealed surfaces. Pigmentary quibbles should not detract from the historical precedence involvement of the peripheral or central corneal epithe- and credit that must be accorded to their work, which has lium usually indicates a proliferative melanocytic process received less attention in the United States than it ought to (Figure 2, Middle left, Middle right, Bottom right). A have, owing to its publication in the Australian ophthalmic crucial issue is how large a given lesion must be for a bi- literature. opsy to be deemed necessary. If biopsies are performed

8 AMERICAN JOURNAL OF OPHTHALMOLOGY FEBRUARY 2016 FIGURE 2. Clinical appearances of conjunctival intraepithelial melanocytic pigmentary conditions. The degree of proliferation and atypia can only be determined with accuracy by biopsy. (Top left) A small, flat, movable, light brown epibulbar pigmentary patch. (Top right) An intermediate-sized juxtalimbal pigmented area that does not extend into the peripheral corneal epithelium. (Middle left) A more extensive conjunctival/limbal golden brown lesion that extends onto the peripheral cornea. This lesion is suspicious with regard to an underlying melanocytic proliferation and should be biopsied. (Middle right) Peripheral corneal involvement is readily apparent and the conjunctival pigmentary portion spreads toward the lateral canthus. The lesion almost certainly is a proliferative melanocytic condition. (Bottom left) Lateral half of epibulbar surface is involved with flat pigmentation. (Bottom right) Clin- ical close-up of lesion on bottom left. Note the fine granularity of the epibulbar zone and extension onto the peripheral cornea. Such a large lesion should be biopsied in several places to fully sample the greatest degree of melanocytic proliferation and atypicality.

on small lesions (Figure 2, Top left and Top right), a biopsy. Lesions that occupy a quadrant or greater of the higher yield of nonproliferative, totally benign pigmenta- epibulbar surface or an analogous area in the forniceal/ tions will be obtained. In 1 series of 50 patients with palpebral conjunctiva when ﬁrst brought to attention nonbiopsied ﬂat conjunctival pigmentations, the median raise concern and should also be biopsied. For lesions in surface area was 1.0 mm2, comparatively small lesions between these size parameters, clinical judgment and that would be expected to be nonproliferative and nona- experience must be relied upon, but if there is a residual typical.36 The criteria for biopsy, however, are not widely doubt about the seriousness of the blemish, a biopsy agreed upon, but for sure any enlarging lesion deserves a would be judicious.

VOL. 162 NEW TERMINOLOGY FOR PRIMARY ACQUIRED MELANOSIS 9 RECOMMENDED HISTOPATHOLOGIC adnexal glands, and the appearance of mixed patterns in multiple biopsies of the same lesions. AND IMMUNOHISTOCHEMICAL Atypia is a nondescript term that is bandied about too APPROACH FOR ANALYSIS often without a clear definition of its various putative meanings. It can be applied to overall architectural or cyto- THE ACCURATE INTERPRETATION OF BIOPSIES OBTAINED logic features, either of which may depart from what is ex- from IMP/PAM5,6,9,10,21 is central to its management but pected (eg, a balloon cell nevus is in some sense an atypical can be frequently vexing for pathologists with limited but benign lesion)37; most often it means lesions that exposure to conjunctival pathology. The normal, small display disquieting cytologic features of the participating numbers of intraepithelial dendritic melanocytes (Figure 1, cells. Atypia has been evaluated ultramicroscopically but Bottom panel #1 and Figure 3, Top left, Top right, Middle this is labor -intensive and requires longer intervals to left) synthesizing increased numbers of melanin granules obtain results.9 One class of melanocytic lesions that has has been called an ephelis (freckle) in cutaneous pathology been intensively investigated in dermatopathology is Spitz or primary and secondary ‘‘melanoses’’ by ophthalmic pathol- nevi and related conditions.38–41 A comparison of several ogists. There are no perceptible macroscopic differences be- large series of Spitz nevi evaluated for atypicality by tween these 2 conjunctival types of melanoses (except by highly experienced experts has revealed that there are clinical history). They can, however, be differentiated from surprisingly dramatic subjective differences in cytologic mild basal proliferations of melanocytes (hyperplasias) with interpretation.39 This has led to a 5-part stratification abiospy(Figure 1, Bottom panel #3 and Figure 3,Middle scheme in an attempt to create reproducibility of the ana- right). The term lentigo is reserved for non-neoplastic (hy- lyses by the scoring of salient histopathologic parameters.41 perplasias) or early neoplastic proliferations of normal- If a conjunctival lesion displays increased numbers of den- appearing intraepithelial basal dendritic melanocytes dritic melanocytes confined to the basement membrane re- (Figure 1, Bottom panel #3 and Figure 3, Bottom left and Bot- gion of the epithelium with regular-sized nuclei, then it is tom right). It is recommended that the term ‘‘nonproliferative judged to be nonatypical (Figure 3, Bottom left and Bottom melanocytic pigmentation’’ be substituted for ephelis or right). If any of the following findings is present (Figure 1, melanosis in the conjunctiva, which can sometimes include Bottom panel #4), the lesion is considered to be atypical: an inconspicuous increase in the number of basilar dendritic bloated or enlarged dendritic melanocytes with thick den- melanocytes (minimal hyperplasia). A red chromogen stain dritic processes and large nuclei and cell bodies that percolate for the detection of melanocytic biomarkers or electron mi- throughout all levels of the epithelium (Figure 4, Top right, croscopy9 may be required to demonstrate single dendritic top and bottom panels); groups of small basal epithelioid me- melanocytes at the level of the pigmented basal keratinocytes lanocytes (Figure 4, Middle left, top and bottom panels); (Figure 3, Bottom left and right), since both the melanocytes confluent nests of larger epithelioid cells sometimes forming and keratinocytes are intensely pigmented.5,9,21 a continuous band along and above the basement membrane Delicate, inconspicuous dendritic melanocytes may not (Figure 4, Top right); single or small clusters of melanocytes be confined only to the basal region, but may be randomly at higher levels of the epithelium than the basal region (page- found in suprabasal layers and still be considered innocuous toid intraepithelial spread) (Figure 1, Bottom panel #4 and (Figure 4, Top left), particularly in darkly complexioned Figure 4, Middle right, top and bottom panels); and mitoti- patients. A mild increase in the number of basal melano- cally active cells with small, hyperchromatic nuclei, vesicular cytes in melanocytic hyperplasias as well as early neoplastic nuclei, or pleomorphic nuclei sporting prominent nucleoli. proliferations of melanocytes are probably both responsible The degree of individual cell atypia is often graded by in an indeterminate ratio constituting the category of IMP/ dermatopathologists, usually in connection with cutaneous PAM without atypia. For IMP/PAM, a fundamentally pro- dysplastic nevi (extremely rarely seen in the conjunctiva), liferative process, analysis of the biopsy must be focused on into categories of mild, moderate, and severe. In the the intensity of the proliferation, the types of cells epidermis, evaluation of the melanocytic nuclei is conduct- comprising the lesion, the position of the cells within the ed in comparison with the size of the neighboring keratino- epithelium, and the assessment of the atypia of the partici- cytic (squamous) nuclei. If the melanocytic nuclei pating melanocytes. What commences as a lentiginous (normally smaller than those of keratinocytes) are twice basal intraepithelial dendritic melanocytic proliferation as large as the keratinocytic ones, then the degree of atypia will more often than not culminate in the emergence of is judged to be severe; if the same size as keratinocytic small polygonal cells or large epithelioid cells bereft of nuclei, then moderately severe; and if increased in number visible dendrites (Figure 1, Bottom panel #4). Attempts but somewhat smaller than keratinocytic nuclei, then mild to subdivide IMP in the conjunctiva into the major types atypia. Transposing this impressionistic and subjective of radial growth phase displayed by cutaneous melanomas grading system to the conjunctival epithelium confronts (acral lentiginous, lentigo maligna, and pagetoid) have the additional problem of the smaller size of the normal been thwarted8,20,21,23 owing to the thinness of the conjunctival keratinocytic and melanocytic cell bodies conjunctival epithelium, the absence of rete pegs and and nuclei in comparison with those of the epidermis. To

10 AMERICAN JOURNAL OF OPHTHALMOLOGY FEBRUARY 2016 FIGURE 3. Spectrum of biopsied benign conjunctival melanocytic lesions. (Top left) Normal, multilaminar, nonkeratinizing conjunctival epithelium without any detectable basal melanocytes. (Top right) Top panel: Immunohistochemical staining with a brown chromogen reveals microphthalmia-associated transcription factor (MiTF)-positive nuclei of basal melanocytes somewhat erratically, but generally widely, spaced along the basement membrane region. Bottom panel: A red chromogen for MART-1 discloses the dendritic shape of the small melanocytes. In darkly complexioned individuals a slightly increased number of melanocytes may drift to suprabasal layers of the epithelium. (Middle left) Top panel: Clear melanocytes are visible along the basement membrane region (arrowheads). Bottom panel: Immunostaining for the presence of MART-1, which demonstrates narrow and blunt dendrites and the small cell bodies of the basal melanocytes. (Middle right) Top panel: The melanocytes responsible for intraepithelial nonproliferative pigmentation are often impossible to discern. Bottom panel: In a bleached section of a goblet cell–rich plical pigmentary patch there is a mild increase in basal melanocytes that are positively immunostained for the melanocytic nuclear biomarker MiTF. (Bottom left) Intraepithelial melanocytic proliferation without atypia. Prominent pigmentation is seen along the basement membrane region with the suggestion of halo formation around an increased number of basal melanocytic nuclei (lentiginous hyperplasia). Note the small amounts of pigment that have been transferred to the suprabasilar keratinocytes. (Bottom right) Red chromogen for MART-1 shows increased numbers of small dendritic melanocytes strictly limited to the basement membrane region. Top left, Middle left (top panel), Middle right (top panel), Bottom left: hematoxylin-eosin, all 3400; Top right (top panel), Middle left (bottom panel), Middle right (bottom panel): immunoperoxidase reaction, diaminobenzidine chromogen, hematoxylin counterstain, 3200, 3400, 3200; Top right (bottom panel) and Bottom right: immunoperoxidase reaction, 3-amino-9-ethylcarbazole chromogen, hematoxylin counterstain, both 3400.

VOL. 162 NEW TERMINOLOGY FOR PRIMARY ACQUIRED MELANOSIS 11 FIGURE 4. Varying grades of mostly atypical intraepithelial melanocytic proliferation. (Top left) Top panel: Barely perceptible delicate dendritic processes are distributed throughout the epithelium. Bottom panel: HMB-45 immunostaining highlights the extremely slender dendritic processes and the small cell bodies. Even though there are increased numbers of melanocytes occupying suprabasal layers of the epithelium, the lesion was diagnosed as nonatypical because of the benign characteristics of the individual melanocytes. (Top right) Top panel: Intraepithelial dendritic melanocytic proliferation with atypia. The melanocytes immunostained for HMB-45 are large (compare with Top left, bottom panel), crowded, and distributed throughout all layers of the epithelium. Bottom panel: Higher power of HMB-45-positive bloated atypical dendritic melanocytes extending to the surface. The nuclei are negative staining and enveloped by cytoplasmic melanin. (Middle left) Top panel: Ill-defined small cellular clusters of medium-sized epithelioid cells are present along the basement membrane region constituting an atypical intraepithelial proliferation. The arrow points to a discohesive cell nest. Bottom panel: microphthalmia-associated transcription factor (MiTF) nuclear immunostaining reveals the full extent of the intraepithelial melanocytes. (Middle right) Top panel: Frankly atypical intraepithelial melanocytic proliferation composed of epithelioid cells that are confluent in the basal zone and have formed a distinct but loose nest (arrow). Bottom panel: MiTF immunostaining demonstrates the mass of basal melanocytes and a single cell dispersion of pagetoid melanocytes at higher levels of the epithelium (arrows). (Bottom left) Extensive proliferation of atypical, somewhat spindled intraepithelial melanocytes that have replaced almost all of the keratinocytes except for a surface layer (arrows). This pattern approaches a melanoma in situ. (Bottom right) HMB-45 immunostaining discloses the disorganized florid intraepithelial melanocytic proliferation of large tumor cells with round, nonstaining nuclei. Top left (top panel), Middle left (top panel), Middle right (top panel), Bottom left: hematoxylin-eosin, all 3400; Top left (bottom panel), Top right (both panels), Middle left (bottom panel), Middle right (bottom panel), Bottom right: immunoperoxidase reaction, diaminobenzidine chromogen, hematoxylin counterstain, 3400, 3200, 3400, 3400, 3200, 3400.

12 AMERICAN JOURNAL OF OPHTHALMOLOGY FEBRUARY 2016 deal with such considerations, the more objective grading map so that the findings can be topographically evaluated. system based on the analysis of specific cellular and growth Decisions regarding therapy should depend on the most pattern features as outlined by Damato and Coupland26 worrisome diagnostic cytologic results obtained from multi- should be promoted. Many dermatopathologists use check ple biopsies. If the evaluation of melanocytic atypia is inde- lists for evaluating melanomas but not generally for prema- terminate (see Table), either additional biopsies should be lignant intraepithelial lesions. The eventual application of taken or a re-biopsy should be performed after some time digitized morphometry with accurate nuclear and nucleolar has elapsed. Rather than overinterpreting an inconclusive measurements and their codification with standard devia- finding or basing therapy on ‘‘gut instinct,’’ diagnosis should tions for diagnostic purposes may also help to alleviate invoke objective and describable criteria after studying the grading problems. At the moment, however, even semi- best tissue specimen available. In some cases a fresh exami- quantitative cytologic assessments for conjunctival IMP/ nation of the slide after a day’s delay may be beneficial. PAM have not been uniformly attempted. Another It could be argued that some of the foregoing features are approach in estimating atypia is to determine the prolifer- suggestive of a junctional nevus (nests of benign cells along ation index (PI) of a given lesion42; conjunctival IMP/ the basement membrane in the absence of subepithelial PAM with atypia has a 3 times higher MIB-1 positivity nevus cells in the substantia propria), a not uncommon than IMP/PAM without atypia.43 Care in such calculations dilemma in differential diagnosis. It should be remembered, must be taken to separate Ki-67 positivity derived from however, that junctional nevi are a temporary stage and basal keratinocytic activity from that of melanocytes and almost always restricted to the first 2 decades of life (although can be accomplished with double staining. very rare lesions in young adults can be observed in the third The extent of intraepithelial atypical melanocytic prolif- and fourth decades). On the other hand, IMP/PAM with eration may be so severe that virtually all vestiges of the pre- or without atypia is a disease of middle-aged or older existent squamous epithelium are replaced by neoplastic me- patients. Microscopically, the intraepithelial nests (theques) lanocytes. This appearance has been called melanoma comprising nevi are clearly demarcated in their intraepider- in situ.4,24,25 The cells responsible for this phenomenon mal cavities and the cells are usually tightly bound together, may be vaguely spindled (Figure 4, Bottom left and right), in contrast to the more discohesive cells with hyperchro- but more typically are epithelioid in character (Figure 5, matic or large vesicular nuclei in the intraepithelial cellular Top left and right). This is an exceptional morphologic collections of atypical IMP/PAM. Conjunctival intraepithe- expression of an atypical preinvasive lesion that contrasts lial spread of an eyelid sebaceous carcinoma can mimic an with the vast majority of IMP lesions that display only amelanotic IMP/PAM, except that it will stain positively incomplete degrees of keratinocytic replacement. These for nuclear androgen receptors, cytoplasmic adipophilin, latter lesions are capable of evolving into melanomas once and epithelial membrane antigen44,45 but negatively for the stage of atypia is manifested, and this has led to their S100 and melanocytic lineage biomarkers. designation by some as a form of melanoma in situ as well. The intraepithelial junctional nests in evolving nevi Their continuing mutations with emergent new clones usually coexist except at the earliest stage with a subepi- displaying more aggressive properties complete their incre- thelial component however inconspicuous (conjunctival mental evolution to invasive melanoma. The cells of atyp- compound nevi), the latter displaying maturation as the ical IMP can be uniformly nonpigmented and require a nevocytes descend deeper into the substantia propria melanocytic biomarker (microphthalmia-associated tran- (ie, the cells become smaller and invested by delicate scription factor [MiTF], HMB-45, MART-1) for their iden- collagen).16 If the intraepithelial melanocytic prolifera- tification (Figure 5, Top right, top panel). Biomarkers are tion spreads for a considerable distance beyond the especially valuable in analyzing tarsal IMP/PAM owing to substantia propria component, however, suspicion ought the thinness of the substantia propria and the prevalence to be aroused regarding the likelihood that the junctional of pseudoglands of Henle (Figure 5, Middle left and right). nests have progressed to a genuine radial IMP/PAM stage Another situation where biomarkers can be highly revela- (Figure 5, Bottom right, top panel). Individual cells in tory is in assessing biopsies after cryotherapy or topical conjunctival nevi are hardly ever found scattershot at chemotherapy, when the keratinocytes may be distorted higher levels of the epithelium (pagetoid cells). In well- andatypical(inducedbychemotherapyoranabnormal developed nevi any persistent junctional component scarred stroma) and camouflage the persistence of atypical tends to terminate close to the lateral border of the sube- melanocytes (Figure 5, Bottom left, top and bottom panels). pithelial component. Finally, truly junctional nevi about One should be mindful that, in cases of conjunctival IMP/ to become compound often cause nubbins of epithelium PAM involving large geographic zones (ie, multiple epibul- to project downward into the substantia propria bar quadrants), extensive biopsies ought to be taken of (Figure 5, Bottom right, bottom panel). These epithelial several different areas, because there can be a wide spectrum units may have nevus cell nests within them, and over of morphologic expressions as successive clones of cells time they will acquire lumens and eventually create, in emerge throughout of the disease course. Each biopsy should late-evolving compound nevi, typical epithelial cysts in be placed in a separate bottle of fixative accompanied by a the substantia propria.

VOL. 162 NEW TERMINOLOGY FOR PRIMARY ACQUIRED MELANOSIS 13 FIGURE 5. Additional patterns of severely atypical intraepithelial melanocytic proliferations. (Top left) Massive collections of intraepithelial epithelioid melanocytes. (Top right) Top panel: The discohesive epithelioid cells immunostain for HMB-45. Bottom panel: The Ki-67 proliferation index is 18%, more than 3 times the level of the junctional component of conventional nevomelanocytic nevi. (Middle left) Interpretation of the tarsal component of proliferating melanocytes can be difficult owing to the presence of epithelial pseudoglands of Henle with goblet cells (arrows). (Middle right) HMB-45 demonstrates the extent of the atypical melanocytic proliferation in the walls of the pseudoglands (arrows). (Bottom left) Top panel: Biopsy after surgical excision coupled with cryotherapy for atypical intraepithelial melanocytic proliferation. The substantia propria is scarred and the epithelium appears to be distorted without clearly identifiable melanocytes. Bottom panel: MART-1 immunostaining discloses innumerable intraepithelial atypical melanocytes. (Bottom right) Top panel: Confluent melanocytes occupy the lower half of the epithelium in a 30-year-old patient as revealed with MART-1. This appearance alone suggests atypical melanocytic proliferation. Bottom panel: A focus in the lesion portrayed in the top panel establishes early compound nevus formation with superficial subepithelial nevocytic nests (crossed arrows). Nubbins of epithelium with inconspicuous lumens possess nevocytic nests (uncrossed arrows), a feature absent in intraepithelial melanocytic proliferations/primary acquired melanosis. These 2 findings interpreted together are most consistent with an exceptional junctional nevomelanocytic nevus in an adult undergoing early transformation into a compound nevus. Top left, Middle left, Bottom left (top panel), Bottom right (bottom panel): hematoxylin-eosin, 3400, 3400, 3200, 3200; Top right (both panels), Middle right, Bottom left (bottom panel): immunoperoxidase reaction, diaminobenzidine chromogen, hematoxylin counterstain, 3600, 3600, 3200, 3200; Bottom right (top panel): immunoperoxidase reaction, 3-amino-9-ethylcarbazole chromogen, hematoxylin counterstain, 3200.

14 AMERICAN JOURNAL OF OPHTHALMOLOGY FEBRUARY 2016 TABLE. Recommended Histopathologic Classiﬁcation of Flat Conjunctival Melanocytic Disorders Contrasted With Earlier Termsa

Proposed New Terminology Alternatively Employed Terminology

1) Intraepithelial nonproliferative melanocytic pigmentationb 1) Primary acquired melanosis without atypia Primary and secondary hypermelanosis Benign acquired melanosis Ephelis or freckle Epithelial melanosis Racial or complexion-associated melanosis Metabolic melanosis (eg, Addison disease) 2) Intraepithelial melanocytic proliferation without atypiac 2) Primary acquired melanosis without atypia Benign acquired melanosis Melanocytic intraepithelial neoplasia without atypia Reese’s precancerous melanosis Melanocytic hyperplasia Hypermelanosis with banal melanocytic hyperplasia Lentinginous hyperplasia 3) Intraepithelial melanocytic proliferation with atypiad 3) Primary acquired melanosis with atypia Melanocytic intraepithelial neoplasia with atypia Benign acquired melanosis with atypia Precancerous melanosis Premalignant melanosis Hypermelanosis with atypia Hutchinson’s melanotic freckle Dubreuilh’s precancerous melanosis Reese’s precancerous melanosis Senile freckle Melanocytic dysplasia Atypical melanocytic hyperplasia Dysplastic nevus Lentigo maligna Atypical lentiginous hyperplasia Pagetoid melanosis Pagetoid melanocytosis Melanoma in situ 4) Intraepithelial melanocytic proliferation of indeterminate atypia 4) Deﬁnition: cytologic features that are indeterminate or fall between categories; microscopic interpretation hampered by small size of specimen, poor sectioning and staining, or crush artifact; rebiopsy is recommended

aExcludes congenital ocular and oculodermal melanosis and junctional nevomelanocytic nevi. bDenotes exuberant melanocytic synthesis of melanin granules that are transferred to basal and suprabasal keratinocytes (eg, complexion- associated, idiopathic). cIncludes mild melanocytic hyperplasia (eg, idiopathic, ultraviolet-induced, postinﬂammatory) and early melanocytic neoplasia with well- preserved and functioning basal dendritic melanocytes. dAtypia denotes lesions that are autonomous and clonal proliferations composed of cells with aberrant nuclear characteristics; the word ‘‘proliferation’’ could be replaced with ‘‘neoplasia’’ for these lesions with.

COMPARATIVE VALUE OF nevocytic intraepithelial nests are sharply demarcated from nearby squamous cells, sometimes by a peripheral IMMUNOHISTOCHEMICAL AND cleft. There are cases, however, in which the melanocytes GENETIC TESTS cannot be identified or separated from chemotherapeuti- cally altered keratinocytes with certainty, as mentioned IN ROUTINE HEMATOXYLIN-EOSIN–STAINED SECTIONS, ONE above in connection with post-therapy biopsies. can often appreciate the presence of basal dendritic Consequently, for selected pre- and post-therapy bi- melanocytes because they retract from the surrounding opsies suspected of harboring an atypical intraepithelial keratinocytes, creating haloes owing to the former’s lack melanocytic proliferation, immunohistochemical staining of intercellular desmosomes and tonofilaments that leads for the lineage biomarkers MART-1 (trans-membrane), to melanocytic shrinkage during tissue fixation. Likewise,

VOL. 162 NEW TERMINOLOGY FOR PRIMARY ACQUIRED MELANOSIS 15 HMB-45 (melanosomal), or microphthalmia-associated atypia (PAM with atypia) showed no differences from the (nuclear) transcription factor (MiTF) can be resorted to junctional component of compound nevi (there were only for better estimating the extent of an abnormal melano- 2 cases, however, of IMP/PAM with atypia in this series). cytic cell proliferation. HMB-45 and MART-1 will disclose This suggests preliminarily that there are qualitative and the outlines of the cells (particularly the presence or biologic differences between IMP of the conjunctiva and absence of dendrites), their sizes, and their disposition skin; in the latter site intraepidermal atypical melanocytic throughout the epithelium, whereas MiTF highlights mela- proliferations parallel the p16 staining properties of invasive nocytic nuclei and can reveal differences in nuclear sizes melanoma in the dermis. It is possible, therefore, that p16 in and shapes, thereby giving a more precise impression of the conjunctival lesions may not be of any value in distin- the numbers of melanocytes vs keratinocytes. MiTF is guishing IMP/PAM from purely junctional nevi. particularly helpful in facilitating an appreciation of single-cell, high-level pagetoid spread within the epithelium. Both brown (diaminobenzidine or DAB) and red (3-amino-9-ethylcarbazole or AEC) chromogens used in SUMMARY AND CONCLUSIONS immunohistochemistry for highlighting positive results are available, the latter being notably worthwhile when THE QUOTIDIAN LABORS OF A PRACTICING EYE PATHOLO- dealing with highly pigmented lesions. Not uncommonly, gist should be animated by his or her quest for diagnostic a greater presence of atypical melanocytes is revealed precision and by a desire to continuously develop a lively with immunohistochemistry than initially suspected from dialogue with clinicians so as to achieve the highest hematoxylin-eosin–stained sections. In brief, a more reli- quality of patient care. No matter how crystalline the diag- able detection of melanocytes and their disposition nostic terminology may be, this professional communica- throughout the epithelium is customarily provided by tion should not be foreclosed but should be cultivated to immunohistochemistry. be genuinely collegial and mutually educational. The pro- The addition of immunohistochemistry can greatly facil- fusion of terms revolving around IMP/PAM (Table) betrays itate diagnostic accuracy when estimating which IMP/PAM the extent of the conceptual ambiguity that, over time, has lesions are apt to evolve into melanomas (namely, those enshrouded the various stages of the disease. For example, with atypia). Furthermore, immunohistochemistry can be early on Reese bundled together conjunctival melanocytic an indirect aid in the distinction between IMP/PAM and hyperpigmentation and melanocytic proliferations with persistent junctional activity in a conjunctival nevus. In and without atypia as a single entity.12 This indiscriminate PAM there tends to be more intense cytoplasmic staining rubric, in retrospect, frequently resulted in unnecessarily with HMB-45, and the Ki-67 proliferation index is also radical treatment (exenteration) for lesions at the more higher than in the junctional component of conjunctival benign end of the disease spectrum. In cutaneous pathology nevi.46 Ki-67 (MIB-1) should be employed more often acceptance has more recently congealed around the for assessing the degree of atypia of IMP/PAM.42,43 construct that atypical intraepidermal melanocytic prolif- Nonmelanocytic biomarkers for elucidating neoplasia erations that partially replace the keratinocytes are, for employed in combination,46–49 plus the introduction of all intents and purposes, closely related to, or on the road fluorescence in situ hybridization technology for genetic to, melanoma in situ, a morphologic term employed analysis (particularly translocations),50,51 will undoubtedly when the epithelium has been almost completely replaced progress to further enhance diagnostic accuracy and by atypical melanocytes, which is rarely observed. facilitate therapeutic success. To date, genetic studies have The diagnostic schema described in this article rests on concentrated on distinguishing nevi from melanomas the work of many experts and departs from earlier terms rather than on grading the severity of IMP/PAM. More especially in regard to the abolition of ‘‘melanosis’’ and recently the immunohistochemical identification of p16 ‘‘hypermelanosis.’’ The trigger point for therapy is reached protein, a tumor suppressor gene product expressed in the histopathologically when conjunctival IMP/PAM with nucleus and cytoplasm of many non-neoplastic cells, has atypia exhibits 1 of 3 basic patterns: (1) involvement of shown some promise for separating cutaneous and the epithelium by swollen and crowded dendritic melano- conjunctival nevi from melanomas.38,52 An article on cytes (large dendrites, cell bodies, and nuclei); (2) conjunctival lesions52 provides an excellent tabular sum- confluent nests or a band of basal epithelioid melanocytes mary of previous immunohistochemical studies using bio- located above the basement membrane; or (3) atypical me- markers in conjunctival melanomas. Studies with these lanocytes occupying suprabasal keratinocytic levels (page- diagnostic aids have so far not been productively used to toid spread). A melanoma in situ picture is not necessary grade escalating stages of IMP/PAM. In this particular article and, in fact, is a most uncommon melanoma precursor in devoted to p16 in conjunctival melanocytic lesions, there comparison with less severe intraepithelial atypical prolif- was a higher degree of preserved p16 expression in nevi in erations. comparison with its attenuation or loss in melanomas. Un- The foregoing patterns may or may not coexist. By the fortunately, intraepithelial melanocytic proliferation with time of clinical detection and biopsy, intraepithelial atypical

16 AMERICAN JOURNAL OF OPHTHALMOLOGY FEBRUARY 2016 melanocytic proliferation composed of myriad, large den- to avoid the complications and morbidities that can dritic cells is far less common that that of polygonal or attend various therapies. epithelioid cells (Figure 1, Bottom panel #4). IMP/PAM is The management53–58 of IMP/PAM has been improved currently not graded by most ophthalmic pathologists for by the introduction of topical interferon or mitomycin C the severity of the cytologic aberrations, since once the stage chemotherapeutic drops. With interferon, there may be of atypia has been reached, as deduced from architectural de- deleterious effects on stem cells, which apparently can be tails and a superficial survey of cellular composition, it is reduced by incorporating retinoic acid into the commonly recommended that such lesions ought to be preparation.54,55 Brachytherapy with plaques (Sr-90 or completely extirpated to prevent the development of mela- more often I-125) has been employed; complete resolution nomatous nodules21,22,26,28,53—in the spirit that the earlier a without recurrences or metastases was recently found to be threatening lesion is uncovered and treated, the better the 82% at 10 years.53 Cryotherapy59,60 still remains a standby prognosis will be, since the development of a modality with its own limitations (eg, corneal scarring and melanomatous nodule will likely be averted. Pathologists symblepharon formation). Regarding melanomas,61,62 confronting a conjunctival lesion posing a diagnostic excisional vs incisional biopsy seems to reduce metastases. dilemma should feel no reluctance to seek out advice from BRAF mutations portend metastases and are detected in more experienced colleagues. younger individuals.62 Melanomas of the plica, caruncle, While it is not the subject of this perspective, a few re- fornix, and palpebral conjunctiva (all extrabulbar sites) marks should be made about therapy.53 Selecting the have a worse prognosis,3,23,59,62 probably because they are optimal therapy for each patient depends upon exercising thick at the time of discovery. Sentinel node biopsy and clinical judgment informed by an awareness of the pa- cervical lymph node dissection, unless there is a palpable tient’s overall medical and ocular conditions, which on enlargement, remain unsettled issues without widespread occasion may call for a departure from general guidelines. consensus in cases of conjunctival melanoma. Following a patient without treatment with IMP/PAM When all is said and done, careful histomorphologic with atypia because there is no associated nodule of mel- analysis remains essential for the diagnosis of conjunctival anoma poses many risks. These include failure of the pa- IMP/PAM. If the data generated by immunohistochem- tient to reappear for follow-up examination, during which ical and genetic studies are to be clinically meaningful interval the disease could progress to involve larger areas and reliable, relatively pure diagnostic groups must first of the conjunctiva with eventual nodule development be created as the indispensable points of departure for (nodular thickness heavily influences metastatic poten- additional studies. This will lead to the superior charac- tial),3,23 thereby creating a more daunting therapeutic terization of disease mechanisms and tumor behavior. challenge. There are rare exceptions, however, for The present overview has been intended, at a minimum, withholding treatment of IMP/PAM with atypia. For to stimulate further critical thinking in clarifying the example, an elderly and sickly patient with this disorder diagnosis of, and devising the most appropriate terminol- may prefer to receive no treatment of the non-nodular ogy for, conjunctival melanocytic proliferations with the disease affecting the only remaining seeing eye in order goal of improving therapy.

FUNDING/SUPPORT: NO FUNDING OR GRANT SUPPORT. FINANCIAL DISCLOSURES: FREDERICK A. JAKOBIEC HAS NO FINANCIAL disclosures. The author attests that he meets the current ICMJE criteria for authorship.

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VOL. 162 NEW TERMINOLOGY FOR PRIMARY ACQUIRED MELANOSIS 19 Biosketch

Frederick A. Jakobiec, MD, DSc, is the Director of the David G. Cogan Laboratory of Ophthalmic Pathology at the Massachusetts Eye and Ear Infirmary in Boston, Massachusetts. Dr Jakobiec is the Henry Willard Williams Professor Emeritus of Ophthalmology and Pathology, the past Chairman of Ophthalmology at the Harvard Medical School, and the past Chief of Ophthalmology at the Infirmary. His main clinical and pathologic interests are in ocular and adnexal tumors and idiopathic inflammations.

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