Blue Nevi and Related Lesions

A Review Highlighting Atypical and Newly Described Variants, Distinguishing Features and Diagnostic Pitfalls

Abstract

Blue nevi and related entities are a heterogenous group of congenital and acquired melanocytic tumors that includes established entities such as dendritic (“common”) blue nevus and cellular blue nevus, and their numerous clinical and pathologic variants, such as deep penetrating nevus. They share several clinical and morphologic features including their blue tinctorial properties, the presence of a dermal proliferation of spindle, fusiform or ovoid cells, associated melanin pigment (both within the melanocytic tumor cells and also within macrophages) and stromal sclerosis and, at least focal positivity for HMB-45 (Gp100). Some variants, such as deep penetrating nevus, often show considerable variation in nuclear size and shape, and, as a consequence, are at risk of being misdiagnosed as melanoma by those unfamiliar with their characteristic morphologic features. The so-called malignant blue nevus is a controversial term denoting melanomas arising in association with or exhibiting some morphologic similarities to blue nevus. There are also lesions that are probably related to blue nevi, such as the recently described pigmented epithelioid melanocytoma and the neurocristic hamartomas, whose nature, biologic behavior, and relationship to the better established entities remains to be clearly established. This review aims to present a brief overview of these lesions, highlighting their pathologic characteristics, distinguishing features and potential diagnostic pitfalls, with particular emphasis on recently described entities, molecular findings, controversial areas, and approaches to diagnosis.

DERMAL DENDRITIC MELANOCYTIC PROLIFERATIONS

Blue nevi and related melanocytic proliferations (Table 1) are a heterogenous group of congenital and acquired melanocytic lesions, which have in common several clinical, histologic, and immunochemical features. They have been termed dermal dendritic melanocytic proliferations because they are usually composed, at least in part, of dendritic melanocytes within the dermis. Clinically, many of the lesions exhibit a blue-gray color, attributed to the deep (dermal) location of abundant melanin pigment in conjunction with the Tyndall effect. The latter is the selective absorption of longer wavelength components of light by dermal melanin pigment with reflection of shorter wavelength (blue) components from the skin.

TABLE 1: Classification of Dermal Dendritic Melanocytic Proliferations

Histogenesis

Histologically, many of the lesions contain spindled and dendritic melanocytes, which are thought to resemble immature melanocytic cells migrating from the neural crest to the skin during embryonic development. It is hypothesized that the lesions arise due to arrested migration within the dermis of these immature cells. Although it is widely believed that the cells are immature melanocytes, it has also been suggested that they could be Schwann cells exhibiting melanocytic differentiation or a common neural crest-derived cell exhibiting differentiation along both melanocytic and Schwann cell lines.¹

CONGENITAL DERMAL MELANOCYTOSES

The congenital dermal melanocytoses are hamartomatous lesions and include Mongolian spot, nevus of Ito, nevus of Ota, nevus fusocaeruleus zygomaticus, acquired dermal melanocytosis of the face and extremities, and dermal melanocyte hamartoma. They all present as blue-gray macules and are histologically very similar, being characterized by scattered dendritic melanocytes within the reticular dermis. Unlike blue nevi, there is no associated stromal sclerosis. They tend to be hypocellular lesions, which are very subtle histopathologically and the diagnosis may easily be missed, particularly if inadequate clinical information is provided to the pathologist at the time the sections are interpreted or insufficient attention is given to the clinical presentation. Distinction between the various congenital dermal melanocytoses is based on their clinical features, which have been reviewed in detail elsewhere.²

BLUE NEVUS

The term “blue nevus” was originally used by Jadassohn to describe dark blue lesions of the skin and was introduced into the literature in 1906 by Max Tièche.³ In 1949, Allen⁴ first used the term “cellular blue nevus” (CBN) for a “benign variant of the blue nevus which, because of its rich cellularity and striking abundance of melanin pigment, often is misdiagnosed as melanosarcoma.” In his textbook published in 1961, Lever⁵ referred to 2 types of histologic appearance that blue nevi may present: a common type (that described by Tièche) and a cellular type (as described by Allen). Many variants of blue nevus have since been reported, but so-called common blue nevi and CBN are the major and most common subtypes.

The so-called common blue nevus is composed predominantly of dendritic melanocytes. Although the adjective “common” is applied to these lesions, they are not necessarily the most common of the blue nevus variants, and furthermore, variable degrees of overlap exist between so-called common blue nevi and CBN. Consequently, they will be referred to as dendritic blue nevi (DBN) in this review.

In our experience, blue nevi commonly show histologic overlap between DBN and CBN. It is our practice to categorize them as one or other subtype on the basis of the predominant component (ie, ≥50% of the lesion).

Dendritic Blue Nevus (So-called Common Blue Nevus, Jadassohn-Tièche Type)

Clinical Features

DBNs are usually acquired, but may be congenital in rare instances. They may arise at any age (present at birth to 79 y), but are biopsied most often during young adulthood (mean 38.6 y).⁶ They present as well-demarcated, slightly raised papules, often <1 cm in diameter, ranging in color from blue to gray to blue-black to black.^2,7 Most occur in skin, commonly on the extremities and the face. Rare cases of DBN have also been reported in extracutaneous locations, such as the subungual region,^8,9 the orbit and conjunctiva,^10–13 oral cavity,^14–20 sinonasal mucosa,^21–23 bronchus,²⁴ esophagus,²⁵ lymph nodes,^26–28 vagina,²⁹ uterine cervix,^30–37 endometrium,³⁸ penis,³⁹ and prostate^40–43 and we have observed cases at almost all of these as well as some other sites.

Amelanotic blue nevus, also referred to as hypopigmented blue nevus,^44–46 is an uncommon variant, accounting for 2.7% of all blue nevi in 1 series.⁴⁷ It shows a similar age and sex distribution to DBN and may occur at any body site.

Rare patients present with multiple DBNs which may occur in a familial setting,⁴⁸ in the setting of LAMB syndrome⁴⁹ (Lentigenes, Atrial and Mucocutaneous myxomas and multiple Blue nevi) or NAME syndrome⁵⁰ (blue Nevi, Atrial myxomas, Myxoid neurofibromas, Ephelides), or apparently sporadically, in which case they may be diffusely distributed^51–55 or grouped in a circumscribed anatomic area (so-called agminated blue nevi).^56–58 Other clinical variants include eruptive,^54,59 linear,⁶⁰ and target⁶¹ blue nevi. The relationship of the very rarely reported LAMB syndrome and NAME syndrome to the Carney complex (which shows some overlap in its phenotypic features—epithelioid blue nevi (EBN), psammomatous melanotic schwannoma, mucocutaneous lentigenes, multiple myxomas at a variety of sites, adrenal hyperplasia, and large cell calcifying Sertoli cell tumor)^62–64 has not, to our knowledge, been established but appears likely.

Pathologic Features

DBNs are characterized by a dermal or submucosal proliferation of elongated, bipolar, spindle-shaped cells, sometimes grouped in short fascicles, often with an intervening grenz zone. The melanocytes show elongated dendritic processes and contain variable amounts of melanin pigment in the cytoplasm, including within the dendritic processes. Some dermal sclerosis is present and variable amounts of collagen are interspersed with the melanocytes. Usually there are also scattered melanophages (Fig. 1). Cellular atypia is minimal and mitotic figures are almost never seen.^2,6,7 Very rare cases of DBN associated with multiple adjacent similar satellite lesions and termed “agminated blue nevi” by some authors^56–58 (postulated to be due to perivascular spread of nevus cells), but lacking evidence of malignant behavior, have been described.^65–67

FIGURE 1.: Examples of dendritic blue nevus. A, Poorly circumscribed dermal spindle cell proliferation separated from the overlying epidermis by a grenz zone. B, Bipolar melanocytes with elongated dendritic processes, some containing cytoplasmic melanin pigment, along with scattered melanophages. C, More cellular example composed of a dermal spindle cell proliferation separated from the overlying epidermis by a grenz zone. D, Bipolar melanocytes with elongated dendritic processes, many containing cytoplasmic melanin pigment, along with scattered epithelioid melanophages.

Amelanotic blue nevus may show a variety of architectural patterns: spindle cell proliferations with entrapment of collagen identical to DBN; sclerotic fibroma-like pattern composed of a proliferation of spindle cells interspersed with thick collagen bundles (similar to sclerosing blue nevi); or a pattern resembling a scar. In contrast to DBN, they are characterized by absent or minimal melanin pigment within the spindle melanocytic cells and a paucity of melanophages (Fig. 2).^45,47 Amelanotic blue nevi may be misdiagnosed (as occurred in almost three-quarters of cases in 1 series⁴⁷) as other entities such as dermatofibroma, scar, dermal Spitz nevus, desmoplastic nevus or amelanotic melanoma.^47,68 Each of these entities is readily distinguished from blue nevi with a combination of clinical correlation, careful examination of the morphologic features, and immunohistochemical studies.

FIGURE 2.: Amelanotic/hypomelanotic/hypopigmented blue nevus. Proliferation of spindle cells lacking pigment, interspersed with thick collagen bundles.

DBN in which there is a preponderance of central fibrosis and hyaline sclerosis have been termed “sclerosing blue nevi.”²

Nodal DBN are rare and are usually located in the capsule, sometimes with extension into perinodal adipose tissue. Their distinction from metastatic melanoma may be problematic, particularly in sentinel lymph node specimens.^69–71 They are composed of a combination of elongated cells with dendritic processes and pigment-laden melanophages, and histologically resemble their cutaneous counterparts.^26–28

Differential Diagnosis

Cutaneous and extracutaneous DBN exhibiting typical morphologic features including melanin pigment are not usually difficult to diagnose. The appearances of hypopigmented DBN may raise other bland spindle cell proliferations in the differential diagnosis, such as dermatofibroma or fibroblastic scar tissue. Immunohistochemistry for melanocytic markers will clarify the diagnosis in morphologically equivocal lesions. Sclerosing DBN may resemble desmoplastic melanoma or other dermal spindle cell proliferations, but can be distinguished by its negligible cytologic atypia and the characteristic HMB-45 (and usually S-100) positive dendritic cells, which contrasts with melanoma.^2,72,73

Nodal DBNs are composed of capsular aggregates of dendritic cells with associated melanophages. The most common melanocytic lesions occurring in lymph nodes are nevus cell aggregates, which are also located in the capsule, but in contrast to DBNs, are composed of small round to oval cells with round to oval nuclei and scant cytoplasm, devoid of significant cytologic atypia and largely lacking pigment.²⁶ In contrast, melanoma cells in lymph nodes are usually located in the subcapsular sinus or parenchymal regions and show greater cell size, nuclear variability, vesicular chromatin and prominent nucleoli, and may show mitotic activity.^70,71

Cellular Blue Nevus

Clinical Features

CBN may be diagnosed at any age (range, 6-85 y), but most commonly in adults under 40 years of age. Females are more frequently affected than males (ratio 2.2:1).⁶ CBN present as pigmented nodules ranging from a few millimeters to a number of centimeters in size. The commonest sites of involvement are the scalp, lower back, and buttocks, but they may occur on the limbs and in other locations,^6,74 and have also been reported in mucosal^6,75,76 and subungual⁷⁷ locations. The duration of the lesion before diagnosis ranges from months to years, and in some cases, the lesion is present at birth.^6,74 Rare cases of congenital giant CBN occurring on the scalp have been reported.^78–80

The plaque-type variant of blue nevus (PTBN) is usually considered a variant of CBN (although it is usually characterized histologically by both DBN and CBN areas). They are present at birth or arise in early childhood, and may enlarge during puberty. The majority of such lesions affect the scalp; rare cases involve the arm or hand, or mucosal sites.⁸¹ PTBN appears as a blue-gray pigmented area measuring 1 or more centimeters in diameter and is composed of a single plaque or a confluence of several small macules and/or papules.^59,81–86

Pathologic Features

Macroscopically, CBN are well-circumscribed nodular dermal tumors, which are often heavily pigmented, with intact overlying epidermis.⁶ The majority of the pigment is in macrophages (melanophages) whereas the melanocytes are usually only lightly pigmented. CBN show considerable histologic heterogeneity. A variety of patterns have been described, but a common feature is vertically oriented, sometimes bulbous extension of the lesion into the subcutaneous adipose tissue (Fig. 3A). In the commonest (mixed biphasic) pattern, the lesion occupies the superficial and mid dermis, sometimes extending to the deep dermis and subcutis. It is composed of oval to plump spindle cells intermingled with dendritic melanocytic cells and variable numbers of melanophages (Figs. 3A, B). Sclerosis is often prominent.^6,74,87 The rare amelanotic variant of CBN (Figs. 4A–C) shows similar morphologic features to classic CBN and is composed of a cellular spindle cell proliferation with little or no cytoplasmic melanin pigment and includes few, if any, admixed melanophages.^47,88

FIGURE 3.: Cellular blue nevus. Typical appearance, comprising a wedge-shaped lesion with extension into the subcutaneous adipose tissue (inset), composed of oval, spindle and rounded cells admixed with variable numbers of melanophages (A and B).

FIGURE 4.: Example of amelanotic cellular blue nevus. A, Wedge-shaped lesion with bulbous extension into the subcutaneous adipose tissue. B, Alveolar pattern, characterized by well-defined nests of amelanotic oval to spindle cells. The alveolar pattern may merge with C, areas with a fascicular pattern in which the tumor is composed of fascicles of amelanotic spindle cells with clear cytoplasm, surrounded by fibrous tissue containing very occasional melanophages.

Well-defined nests of amelanotic cells surrounded by collagen and variable numbers of dendritic melanocytes and melanophages comprise the alveolar pattern of CBN (Fig. 4B). The merging of the alveolar pattern into nevoid cell sheets or areas of a fascicular pattern is common. Occasionally, the nests may be separated by areas of loose, edematous tissue, which may represent precursors to cystic change.⁷⁴ In the fascicular or neuronevoid pattern, the tumor is composed of fascicles of amelanotic spindle cells with clear cytoplasm, surrounded by fibrous tissue containing dendritic melanocytes and melanophages. Neuroid structures are frequently interspersed with the tumor.⁷⁴ Occasional tumors may contain balloon cells with vacuolated/foamy cytoplasm.⁸⁹ Rare cases may exhibit myxoid stroma.⁹⁰ Mitotic activity is usually absent and should be low (<1/mm²).⁷⁴ Focal areas of necrosis have been reported as being present in rare cases of otherwise benign CBN,⁷⁴ although in our view, the presence of any necrosis is an atypical finding and concerning for malignancy. If isolated mitotic activity or necrosis is present but no other atypical findings (including cell crowding, nuclear atypia, hyperchromasia, or expansile growth) are seen, we report such tumors as atypical CBN and state that it is not possible to predict the biologic behavior/malignant potential of the tumor with certainty from its morphologic features but consider the risk of aggressive behavior to be low. The cells are usually positive for S-100 and HMB-45,^91,92 although we have seen some cases which are negative for S-100 and exhibit only weak or focal positivity for HMB-45. In view of the often prominent melanin pigmentation of these lesions, the use of a red chromogen for immunohistochemistry usually allows easier interpretation than a brown chromogen. A rare angiomatoid variant of CBN containing numerous dilated blood vessels has been described.⁹³

Kazakov and Michal noted the presence of “ball in mitts” structures in CBNs, composed of a single centrally placed rounded or oval melanocyte (ball) encircled by a single dendritic cell (mitt) with an oval or spindle-shaped nucleus and slender bipolar processes containing melanin and surrounding at least a quarter of the diameter of the rounded melanocyte. They also found “microalveolar structures,” composed of 2 to 10 central rounded cells surrounded by one or more dendritic cells similar to those seen in the “ball in mitts” structures. The authors noted that similar structures may occur in combined nevi, deep penetrating nevi, acquired melanocytic nevi and in common blue nevi, and in light of this, they proposed that there may be a spectrum of evolution from common acquired nevus to combined nevus to CBN and deep penetrating nevus (DPN) through a progressive expansion and prominence of the “ball in mitts” and “microalveolar structures.”⁹⁴ However, differences in the clinicopathologic characteristics of blue nevi (and their variants) and common nevi are against this hypothesis.

Histologically, PTBN shows a combination of features seen in DBN and CBN. In addition to pigmented spindle and dendritic cells, aggregates of variably pigmented epithelioid and often clear melanocytic cells are present, as are foci extending deeply into subcutaneous tissues. The dermal melanocytes are often found in a periappendageal and perivascular location (Figs. 5A, B). No significant cytologic atypia is seen and mitotic activity is rare (Fig. 5C).^85,86,95 The lesional cells are positive for S-100, HMB-45 (Gp100), and Melan-A/Mart-1 (A103).⁸⁴ Busam et al⁸⁴ suggested that onset in childhood and the presence of heterogenous (DBN-like and CBN-like) areas are features that distinguish PTBN from CBN. However, as PTBN are composed of areas resembling CBN, histologic distinction between these entities is not possible without correlation with the clinical features. For this reason, we regard PTBN as a clinicopathologic variant of CBN characterized by large size and extension along fascial planes.

FIGURE 5.: Plaque-type blue nevus. A, Plaque-like tumor centered in the dermis with extension around adnexal structures and B, extensive involvement of the subcutis, composed of C, largely nonpigmented aggregates of oval melanocytic cells, along with scattered pigmented dendritic cells. A deep component involving subcutaneous adipose tissue and deep fascia may predominate.

Differential Diagnosis

CBNs show a range of histologic features, which may cause them to be misdiagnosed as melanoma. Rare cases of melanoma arising within preexisting CBN have also been reported.^75,96 Also, congenital giant CBNs may show intracranial involvement and such tumors are associated with a high rate of malignant transformation, often occurring before puberty.^78,79,97 Like melanoma, CBN is usually composed of large epithelioid and/or spindle cells, lacks maturation with depth, may extend deeply, may be pigmented, and may contain an occasional mitotic figure.^6,74,98 Perineural extension and intralymphatic tumor may be seen in CBNs and does not indicate malignancy. Various histologic features have been proposed as being helpful for the discrimination of CBN and CBN-like melanoma. However, these criteria are not infallible and in those cases with some atypical but not overtly malignant features, confident prediction of likely biological behavior may not always be possible on the basis of the morphologic assessment of the primary tumor. Pathologic features reported as favoring malignancy include tumoral necrosis, cytologic atypia and pleomorphism, and frequent mitoses (>2/mm²).⁶ Other findings that favor melanoma over CBN include the presence of large pleomorphic epithelioid cells, cell crowding (usually associated with increased cell nuclear to cytoplasmic ratios), an “infiltrating” growth pattern (where the margins of the tumor are irregular, as opposed to the generally rounded or “pushing” margins of CBN), expansile growth, the presence of junctional activity (especially if it is atypical or shows pagetoid epidermal invasion), or the presence of a “sarcomatoid” growth component, in which sheets of spindle cells replace small fascicular aggregates and nests of melanocytes.^6,74

A recent study documented poor interobserver reproducibility among expert pathologists in the diagnosis of atypical CBN and so-called malignant blue nevus, highlighting the lack of uniformly accepted and applied criteria for the pathologic diagnosis of these lesions.⁹⁹ Further studies preferably correlated with molecular data and long patient follow-up periods are required to determine the morphologic criteria that best predict biologic behavior in these lesions.

Lymph node involvement by CBN is rare.^6,100 Nodal involvement often takes the form of subcapsular sinus and parenchymal deposits (Figs. 6A, B), in contrast to the predominantly capsular location of nodal nevus cell aggregates and DBN.^{26,74,100,101} Such deposits of CBN pose difficulties in distinction from melanoma, and the possibility that the lesion is a metastatic CBN-like melanoma cannot always be ruled out due to the relatively short follow-up period in some reported cases.¹⁰² Comparison with the histology of the primary cutaneous tumor (which is invariably present, Fig. 6C) may help in arriving at the correct diagnosis, and prevent excessive and inappropriate treatment.⁷⁴ However, unequivocal distinction between nodal CBN and CBN-like melanoma is not always possible, and therefore in such cases, a guarded prognosis should be given.

FIGURE 6.: Nodal cellular blue nevus. A, Large subcapsular and parenchymal deposits. B, Cellular tumor composed of round to oval cells containing variable amounts of cytoplasmic pigment. The cells show some cytological atypia and occasional mitoses are present. C, The corresponding cutaneous cellular blue nevus, which shows typical architectural (inset) and cytologic features and is devoid of significant cellular atypia.

Combined Nevus

Combined melanocytic nevi are composed of more than one nevus type in a single lesion. They may occur in both cutaneous and mucosal sites,^10,74,103 and may be composed of any combination of common acquired nevi, blue nevus variants, or Spitz nevi. Blue nevi, particularly deep penetrating nevi, are common components,^104–106 but DBN^10,103,105 or CBN^74,105 may also be present (Figs. 7A, C). We consider that other histopathologically similar tumors that have been reported under a variety of other terms including clonal nevus, inverted type A nevus, nevus with focal atypical epithelioid components, and atypical dermal nodule in benign melanocytic nevus may also represent examples of combined nevi.^105,107 Barnhill and colleagues have proposed the term “nevus with phenotypic heterogeneity” for this group of lesions. Although this name is probably a more accurate reflection of the pathogenesis of these melanocytic tumors, the terminology has not been widely adopted, possibly because the term “phenotypic heterogeneity” may engender clinical concern about the nature of the lesion.

FIGURE 7.: Combined nevus. A to C, Combined nevus with banal compound nevus (C) admixed with epithelioid deep penetrating nevus (D).

“Compound Blue Nevus”

Rare cases of compound blue nevus have been reported.^108–110 The junctional component was reported to be composed of single dendritic melanocytes, a morphologic description that equally applies to normal junctional melanocytes. The dermal component comprised pigmented dendritic melanocytes arranged singly and in fascicles, similar to DBN. Melanophages were also present in the dermis. No significant cytologic atypia or mitotic activity should be observed.^108–110 We have not seen a convincing example of this entity; upon review of cases that were diagnosed by others as compound blue nevus, we classified them as DPN and combined nevus.

Clinical Behavior of Blue Nevi

In general, the blue nevi described above behave in a benign fashion. Involvement of regional lymph nodes is rare, as has been discussed above. Very rare cases of persistence or recurrence of blue nevi [DBN, CBN, and combined nevi with blue nevus component(s)] have been reported, often, but not always, after incomplete excision.^6,111,112 The recurrent lesions usually resemble the original tumor, although occasionally, they may exhibit degenerative (“ancient”) changes or atypical features (pleomorphism and mitotic activity), the latter raising concern for subtle malignancy.¹¹¹ Persistent or recurrent blue nevi seem to follow a benign course, and the atypical and degenerative morphologic changes seen in some recurrent tumors were not associated with aggressive behavior in most cases. It has been suggested that these changes are likely related to the inflammatory response to the previous surgical procedure.¹¹¹ However, as clinical recurrence of blue nevi may herald malignant transformation, careful clinical follow-up and histologic examination of the recurrent lesion is warranted.^74,113,114

BLUE NEVUS-LIKE MELANOMA (SO-CALLED MALIGNANT BLUE NEVUS)

Malignant blue nevus is a term first coined by Allen and Spitz to describe blue nevus-like lesions which had resulted in metastasis and patient death in some cases.¹¹⁵ It has since been used by several authors to denote various entities: malignant change arising in a preexisting CBN,^{74,113,116,117} melanoma arising at the site of a (previously excised) blue nevus,^{113,118–120} melanoma with cytoarchitectural features resembling CBN but apparently arising de novo,^121–123 or melanoma with an admixed residual benign CBN component.^{113,118,124–126}

So-called malignant blue nevi comprise a rare group of melanocytic tumors, which in some cases are not easy to classify precisely as benign or malignant purely on the basis of the morphologic features of the primary tumor. Mones and Ackerman¹²⁷ have published a comprehensive review of cases reported in the literature bearing these diagnoses, and are strongly critical of the use of such terms. These authors are firmly of the opinion that all such lesions should be categorized into “blue nevus,” “melanoma,” and “melanoma in association with a blue nevus.” Although their interpretation is largely based on a logical analysis of the literature, precise categorization of such lesions is not always possible, nor is it possible to reliably predict from histologic features of the primary tumor the biologic behavior of atypical melanocytic lesions showing some morphologic similarities to blue nevus or CBN. Nevertheless, we agree that the term malignant blue nevus is inadvisable, given the juxtaposition of the adjective “malignant” with the noun “nevus,” which connotes a benign lesion. It is far better, we believe, to term such lesions as “blue nevus-like melanoma (BNLM) or atypical blue nevus-like lesion of uncertain malignant potential,” depending on the degree of atypia and the degree of histologic certainty of the malignant potential of the lesion. If the latter term is used, the evidence in favor of a diagnosis of nevus and melanoma respectively should be discussed in the report and, whenever possible, a favored diagnosis proffered. Furthermore, in this situation, or whenever genuine doubt exists about the biologic potential of the lesion, it is probably appropriate to seek an opinion from one or more experienced colleagues. Rather than adding new diagnostic entities to the dermatopathologic lexicon, these terms indicate the difficulty in histologically classifying such lesions as benign or malignant, and reflect the uncertainty in prediction of their behavior. This allows communication of the level of concern about a given lesion to the clinician, facilitating the formulation of an appropriate management plan.^128,129 In this difficult area, we believe that such an approach is of more value than to attempt to precisely categorize all such lesions as either benign (nevi) or melanoma when there is a significant possibility that such categorization may be inaccurate. Further study of these cases, correlated with prolonged clinical follow-up and preferably also with molecular data, may be helpful in refining diagnostic criteria for this uncommon but difficult and problematic group of tumors.

Most of the cases of BNLM are associated with CBN (either pure CBN or CBN in combination with other nevus types). Melanoma apparently arising in association with a pure DBN is very rare.^122,130,131 Connelly and Smith¹¹³ proposed 2 criteria to enable distinction of BNLM from metastatic or nodular melanoma, namely the lack of a junctional component and the presence of a background benign blue nevus. Clearly, correlation with clinical details is also important if a diagnosis of metastatic melanoma is being considered, particularly to establish whether the patient has a known history of melanoma and whether there is evidence of metastatic disease elsewhere.

Genetic studies of small numbers of cases have suggested that aberrations commonly seen in melanomas (such as mutations in CDKN2A and alterations in chromosomes 10q, 1p, and 17) are not identified in BNLM, suggesting that the molecular pathogenesis of BNLMs is distinct from that of conventional melanoma.¹²⁵ Further genetic studies are required to confirm these findings. A recent study reported the presence of frequent somatic mutations in the heterotrimeric G protein alpha-subunit, Guanine nucleotide binding protein (G protein), q polypeptide, in blue nevi (83%) and ocular melanoma of the uvea (46%).¹³² The mutations occurred exclusively in codon 209 in the Ras-like domain and resulted in constitutive activation, turning Guanine nucleotide binding protein (G protein), q polypeptide into a dominant acting oncogene. These findings provide molecular evidence of a histogenetic link between these tumors which, as we recognized many years ago, show morphologic similarities in a subset of cases.

BNLM is rare. They often occur in older individuals (often >45 y), are usually large (3-13 cm), and may show clinical evidence of a residual CBN. Many of the reported cases exhibit local invasion or widespread metastasis.^{116–118,121,123,124,133–136} The scalp is the commonest site of involvement, followed by the face, buttocks, and chest. Rarely BNLMs in other locations, such as the vulva,¹¹⁸ have also been reported.

Cases of BNLM reported in the literature have behaved very aggressively, with a majority exhibiting metastases leading to patient death.^{113,121,123,125,131,135,137–142} There are probably a number of factors contributing to this poor outcome, including delayed diagnosis and treatment of the malignant component, an overestimate due to difficulties in separating nonmetastasizing BNLM from atypical CBN, and reporting bias. However, metastasis or death may be delayed up to 19 years after the diagnosis.^118,133 Reliable prediction of the behavior of BNLM based on histopathologic features has not always been successful, although some studies have suggested that Breslow thickness may be predictive of outcome.¹²² A recent comprehensive analysis of 23 patients with BNLM treated at the Melanoma Institute Australia (incorporating the Sydney Melanoma Unit), the largest series reported to date, found that clinical outcome was similar to that of conventional melanoma patients when matched for tumor thickness and other important prognostic factors.¹⁴³

Pathologic Features and Differential Diagnosis

BNLM can generally be distinguished from CBN by the presence of a frankly malignant component (Figs. 8A, B).¹¹⁸ Histologically, BNLM is characterized by widespread necrosis in many cases (Figs. 8C, D),^{6,116,133,134,144} and palisading of cells around the areas of necrosis may be seen.^133,134 However, necrosis is not a common finding in all series.¹²² Focal necrosis has been reported to occur rarely in otherwise typical CBN,⁷⁴ although it is our opinion that, in the absence of prior biopsy or trauma, the presence of necrosis is very concerning for malignancy. Other criteria supportive of a diagnosis of BNLM include high mitotic rate (>2/mm²), abnormal mitotic figures, vascular invasion, excessive cell crowding, expansile growth and marked cytologic pleomorphism and atypia (Figs. 8D, E). Occasional cases of BNLM may, however, be associated with a low mitotic rate.^{113,118,121,122,135,136,138–142,145–147}

FIGURE 8.: Examples of blue nevus-like melanoma (so-called malignant blue nevus). A, Cellular lesion resembling amelanotic CBN at low power. B, Prominent cytologic atypia (nuclear pleomorphism and very prominent nucleoli) and isolated mitotic figures (arrow). C, Another example showing a highly cellular and nodular tumor confined to the subcutaneous adipose tissue, and showing densely cellular nodules exhibiting areas of heavy pigmentation composed primarily of melanophages and necrosis (N). D and E, The tumor cells are oval to spindle in shape, show moderate cytologic atypia, including scattered mitotic figures (arrows), and are variably pigmented. Note the presence of admixed melanophages and necrosis (N). CBN indicates cellular blue nevi.

Cutaneous metastases from melanoma may rarely mimic BNLM, and when they lack atypical cytologic features, they may mimic blue nevus. These lesions are composed of pigmented fusiform, dendritic and epithelioid melanocytes surrounding dermal collagen bundles. Occasional atypical epithelioid melanocytes, scattered mitotic figures and an associated perivascular lymphocytic infiltrate may be present, all representing subtle clues to the correct diagnosis. Nevertheless, correlation with the clinical features is critical to definitive diagnosis.¹⁴⁸

“ATYPICAL” CELLULAR BLUE NEVUS

A rare variant of blue nevus with many morphologic features of CBN but with some additional atypical features (that in isolation would be evidence raising the possibility of melanoma) without clear cut evidence of malignancy, (ie, a lesion intermediate or indeterminate between typical CBN and melanoma) has been referred to as “atypical CBN” (ACBN),^74,98,149 “locally aggressive,”^144,150 “extensive,”^151,152 or CBN-like melanocytic tumor of uncertain malignant potential.¹⁵³ ACBNs tend to occur in the buttock or sacral region of young or middle-aged adults. All lesions of ACBN reported so far have been solitary, predominantly dermal nodules, some extending into the superficial subcutis.^74,98 Clinically, such lesions resemble CBN, but histologically, they contain worrisome features, such as asymmetry, hypercellular foci, focal cytologic atypia, and occasional mitoses (<2/mm²).^74,149,153 Atypical mitotic figures are not seen.¹¹⁸ Elder and Murphy¹⁵³ proposed diagnostic criteria for ACBN as CBN that display some, but not all, of the following features: (1) size >3 cm, (2) increased cellularity, (3) cellular pleomorphism, (4) increased mitotic activity, and (5) areas of necrosis. However, a recent study involving the assessment of a series of cellular blue melanocytic neoplasms by 14 experienced dermatopathologists showed that there is substantial disagreement among pathologists about the definitions and biologic nature of such tumors, in particular those thought to have atypical features (ACBN).⁹⁹ The results highlight the lack of clear, consistent diagnostic criteria, resulting in poor interobserver reproducibility in the diagnosis of such lesions. Clearly, further study of this group of lesions correlated with clinical follow-up data (and molecular findings) is required to clarify the clinical significance of atypical morphologic features in CBN. In our view, it is likely that ACBN does not represent a distinct entity and may be composed of a mixture of CBN with atypical histologic features and a subtle CBN-like low grade form of melanoma. The recent suggestion that the latter should be referred to as melanocytoma (as a distinct intermediate entity in contrast to the traditional dichotomous diagnostic categorization of melanocytic tumors as either nevus or melanoma) is a proposal with some merit and worthy of further consideration and study. However, to prevent it becoming a “waste basket” category liable to expansion and inappropriate (over) usage, precise diagnostic criteria would need to be defined.

Some authors found that ACBNs tend to recur, and may acquire a more aggressive phenotype if incompletely excised,¹⁵³ whereas other studies found no evidence of recurrence or metastasis in their cases.¹⁴⁹ Until further data emerges, it therefore seems an appropriate course of action to regard such lesions as being of uncertain/indeterminate malignant potential and to treat them with complete resection, followed by close clinical follow-up for local recurrence or regional/distant spread.

Differential Diagnosis

The distinction of ACBN from melanoma can be difficult. High mitotic activity (>2/mm²), atypical mitotic figures, marked cytologic atypia, cell crowding, expansile growth, and necrosis favor melanoma,^113,137 but it has been suggested that the finding of atypical mitotic figures seems to be the most helpful distinguishing feature.⁶ As described above, ACBN is distinguished from CBN by the presence of one, or occasionally more, of the following features: asymmetry, hypercellular foci, focal cytologic atypia, and occasional mitoses (<2/mm²).

Maize et al¹⁵⁴ demonstrated that ACBN showed more chromosomal aberrations than unequivocally benign CBN (which showed none), but which are less frequent and fewer in number than unequivocally malignant lesions resembling blue nevi (BNLM) using comparative genomic hybridization. The small number of cases and the limited follow-up period in this study, however, did not permit prognostic stratification of the ambiguous cases (ACBN) based on their degree of chromosomal aberration.¹⁵⁴ Nevertheless, these data may provide some molecular basis for the presence of the atypical morphologic features and, furthermore, evidence that the traditional dichotomous categorization of melanocytic tumors into benign and malignant may not always be appropriate (that is, that an intermediate/borderline category of melanocytic tumors (“melanocytomas”) or perhaps low grade forms of melanoma may exist.

Deep Penetrating Nevus, Epithelioid Blue Nevus, and Pigmented Epithelioid Melanocytoma

Deep Penetrating Nevus

The term “deep penetrating nevus” was coined by Seab et al¹⁵⁵ in 1989 to describe a lesion with worrying histologic features but benign clinical behavior, which was often misdiagnosed as melanoma. In Seab et al's series, none of the lesions recurred or metastasized after a mean follow-up of 7 years. In our experience, DPN most commonly occurs as part of a combined nevus (ie, as part of a nevus composed of more than one nevus cell type).^105,156

DPN usually presents as a solitary lesion, and rarely as multiple lesions.^155,157 Patients with DPN are usually between 10 and 30 years of age when diagnosed (although they may occur at any age), with no significant sex predilection. The most common sites of involvement are the head, neck and shoulder, and less often trunk, upper arm, hip, and thigh. Most lesions are well-demarcated, darkly pigmented papules or nodules, usually <10 mm in size.¹⁵⁵ Variable pigmentation is common, particularly in those lesions forming part of a combined nevus.

Histologically, DPNs are relatively symmetric and well-demarcated lesions extending to the deep reticular dermis and sometimes into subcutis, and often exhibit an inverted wedge-shaped silhouette (Fig. 9A). They are usually composed of nests and fascicles of predominantly ovoid epithelioid cells, which often contain cytoplasmic melanin pigment, the latter being more prominent in associated melanophages. Extension around and in association with skin adnexal structures or nerves is common (Fig. 9B). They usually exhibit some nuclear pleomorphism, but mitotic activity is low or absent. Cells with “smudged” nuclear hyperchromatism and intranuclear cytoplasmic invaginations (pseudoinclusions) are often present (Figs. 9C, D). DPNs are almost invariably positive for S-100 and HMB-45.^91,155

FIGURE 9.: Examples of deep penetrating nevus. A and B, Low power views of 2 lesions showing an inverted wedge-shaped architecture and periadnexal extension in B. C, Nests and fascicles of epithelioid and spindle melanocytic cells and admixed melanophages. D, The cells contain round to oval nuclei, which show considerable pleomorphism; some cells contain intranuclear pseudoinclusions.

Differential Diagnosis

Seab et al asserted that, although there is some overlap between the clinical and morphologic features of DPN with those of DBN, CBN, and Spitz nevus, DPN constitutes a relatively homogenous group with sufficiently distinctive architectural and cytologic features to permit distinction from these other lesions.¹⁵⁵ We concur with this point of view. In contrast to DPN, DBN lacks junctional nests, exhibits a diffuse rather than a nested dermal component, is predominantly composed of more fusiform or dendritic cells and lacks nuclear pleomorphism. CBN lacks junctional nests and is composed of prominent nests of nonpigmented or scantily pigmented uniform ovoid to spindled cells, unlike DPN. Spitz nevi generally occur in younger patients and are often clinically nonpigmented.^158,159 They show prominent junctional nests and epidermal hyperplasia, and are composed of epithelioid and spindle cells with vesicular nuclei.¹⁶⁰ Superficial mitotic figures are not uncommon. Melanomas generally lack the circumscription, symmetry and wedge-shaped architecture seen in DPN. Additional features that favor melanoma include mitoses (>2/mm²), atypical mitotic figures, diffusely atypical melanocytes with coarsely granular chromatin and fine, dusty melanin pigment, solid growth pattern, stromal reaction, and an in situ melanoma component.¹⁵⁵

Epithelioid Blue Nevus

EBN is a variant of blue nevus, first described in young patients in the setting of Carney complex (myxomas, endocrine overactivity, spotty skin pigmentation, and psammomatous melanotic schwannomas among other associations).^62–64,161 EBN have also been reported as a sporadic condition in both children and adults.¹⁶² They tend to occur most often on the trunk and extremities,^{64,161,163–165} and very rarely in genital¹⁶⁶ and oral¹⁶⁷ mucosal locations.

EBN is composed of combinations of large epithelioid melanocytes with abundant cytoplasm, variable amounts of coarsely granular cytoplasmic melanin pigment and prominent nucleoli, sometimes associated with heavily pigmented spindle cell and dendritic cell components. In some epithelioid cells, the pigment is distributed evenly throughout the cytoplasm, but in others, the pigment is concentrated at the periphery of the cell, leading to a pigment-free perinuclear zone. The cells are interspersed between dermal collagen bundles. The large and pleomorphic nuclei in the epithelioid cells may contain occasional very prominent nucleoli (Fig. 10). Maturation of the lesional cells with depth is lacking and occasional mitotic figures (typically <1/50 high power fields) may be seen. Melanophages are readily identified.^{64,161,163–165} The tumor cells stain with S-100, HMB-45, Melan-A, and MiTF, as well as CD68.^161,167 Additional (junctional or intradermal) melanocytic nevi are commonly associated with EBN, such lesions therefore representing combined melanocytic nevi.<!/HEADING><!/HEADING>^161,165

FIGURE 10.: Pigmented epithelioid melanocytoma. A and B, Heavily pigmented dermal lesion with an inverted wedge-shaped architecture exhibiting hyperplasia (A) or thinning (B) of the overlying epidermis. C, This tumor is composed of variably pigmented epithelioid and spindle cells with prominent nucleoli, interspersed with melanophages. D, This example shows features of lesions previously diagnosed as “epithelioid blue nevus.”

Differential Diagnosis

DPN may display some features seen in EBN, such as pigmentation, wedge shape, presence of epithelioid and some spindled cells and common extension to subcutaneous fat. However, DPN is more common in the head and neck region than in the trunk and extremities. Nests and fascicles of cells seen in DPN are unusual in EBN. Nuclei in DPN tend to be irregular, nucleoli are not usually prominent, the nuclear chromatin is often smudged, and intranuclear pseudoinclusions are common; in contrast, the nuclei in EBN contain prominent nucleoli and the nuclear chromatin is clear.¹⁶¹ Like EBN, Spitz nevus occurs at a young age and may exhibit a dome shape. However unlike EBN, Spitz nevi are typically sparsely pigmented, are situated in the reticular dermis, contain cells arranged in nests, show cell “maturation” with dermal depth, exhibit epidermal hyperplasia, and nucleoli are only occasionally prominent; they are frequently negative for HMB-45.^161,167

“Pigmented Epithelioid Melanocytoma”

“Pigmented epithelioid melanocytoma” (PEM) is a term coined by Zembowicz et al¹⁶⁸ in 2004 to describe heavily pigmented epithelioid and spindled melanocytic tumors exhibiting histologic features similar to those seen in EBN. Similar tumors had previously been described under the rubric “animal-type melanoma” due to their morphologic similarities with heavily pigmented tumors in horses (equine melanotic disease).^169–174 Subsequently, single cases of PEM have been reported,¹⁷⁵ and Antony et al¹⁷⁶ proposed the term “pigment-synthesizing melanoma” for similar lesions. Our experience of similar heavily pigmented melanocytic lesions confirms the overlapping morphologic features between EBN and animal-type melanoma.^177,178 However, it is our view that a subgroup of EBN remains, distinct from PEM, that shows less cell density and less pigmentation which we continue to diagnose as EBN.

PEM occurs in patients over a wide age range, with a median age of 27 years. It is most often located on the extremities. Histologically, PEM is a heavily pigmented dermal melanocytic proliferation with infiltrative margins. The tumor may abut the epidermis or be separated from it by a grenz zone, and there may be associated epidermal hyperplasia (Figs. 10A, B). A junctional component is sometimes present and PEM is associated with a nevus in a minority of cases. The tumor is composed of a mixture of epithelioid and spindled melanocytes with heavy cytoplasmic pigmentation. The epithelioid cells range in size from medium-sized to large, and contain moderate to abundant cytoplasm. Nuclear atypia and nucleolar prominence is generally more pronounced in the large cells (Fig. 10C). The spindle cells are more prevalent at the periphery of the lesion and exhibit similar nuclear features to those seen in the epithelioid cells. Their cytoplasm, which is focally dendritic, is also heavily pigmented (Fig. 10D). Mitotic activity is low (0 to 3 mitotic figures/mm²) and tumor necrosis is uncommon. Numerous melanophages are often also present and lymphocytes are seen in occasional cases, usually at the periphery of the tumor.^{168,177–180}

In Zembowicz et al's series,¹⁶⁸ regional lymph node metastases occurred in 46% of cases and liver metastasis in 1 case. Nevertheless, the clinical course was found to be favorable, although there was only a relatively short follow-up period. The authors proposed that the term PEM be used provisionally to encompass entities previously described as epithelioid blue nevus and animal-type melanoma. Interestingly, many of the cases previously reported as animal-type melanoma in the literature showed a propensity for regional and distant metastasis, but only rarely resulted in death of the patients, even in lesions of considerable thickness.^172–174 In contrast, the rate of regional lymph node metastasis in other series of PEM has been much lower.¹⁷⁹ However, unlike the series reported by Zembowicz and colleagues, the latter study included few patients in whom a sentinel lymph node biopsy was performed and most nodal metastases were detected by clinical palpation.¹⁷⁹

Some authors have questioned the need for the term “PEM” given that it subsumes other well-documented entities such as animal-type melanoma and at least some examples of EBN (as mentioned previously, we believe there remain examples of EBN that do not fulfill criteria for a diagnosis of PEM).¹⁸¹ However, the originators of the term suggest that it is justified, as animal-type melanoma and EBN cannot be distinguished histologically, nor can metastasizing and nonmetastasizing cases. Furthermore, PEM seems to exhibit distinct clinicopathologic features and clinical behavior, when contrasted with conventional melanoma. Moreover, a recent study analyzed mutations of the protein kinase A regulatory subunit type 1α (coded by the PRKAR1A gene), loss of heterozygosity at the gene locus (17q22 to 24) and immunohistochemistry for the protein product (R1alpha) in PEMs, a variety of nevi and melanomas.¹⁸² Mutations of the PRKAR1A gene are found in more than half of Carney complex patients. No mutations were found in PEMs, in nevi or in melanomas, but 17q22 to 24 loss of heterozygosity was much more common and extensive in PEMs than in nevi or melanomas. In contrast, immunohistochemical expression of R1α was absent in 82% of PEMs and in all Carney complex-associated EBNs, but was present in virtually all melanomas and melanocytic nevi. These findings provide molecular evidence in support of the proposal that PEM is a distinct melanocytic tumor. Until the nature of these tumors is resolved beyond doubt, we recommend complete local excision and careful clinical follow-up. In view of the apparently favorable prognosis of patients with PEM, the unknown clinical significance of sentinel lymph node metastases in PEM and the absence of conclusive evidence that sentinel lymph node biopsy followed by complete lymphadenectomy in those patients with metastases improves overall survival, the role of sentinel lymph node biopsy in patients with PEM is probably limited. Nevertheless, lymphoscintigraphy to identify the sites of sentinel lymph nodes (which can then be regularly carefully monitored by regular high resolution ultrasound) offers an alternative noninvasive strategy that enables growing metastases to be detected at an early stage and appropriate management to be instigated.

Is There a Relationship Between Dendritic and Cellular Blue Nevus, Epithelioid Blue Nevus, Deep Penetrating Nevus, Combined Nevus and PEM, and Spitz Nevus?

As highlighted by Groben et al, there is probably a spectrum of pigmented melanocyic tumors and occasional cases may show overlapping features and may be difficult to classify. In view of this, and the presence of occasional melanocytic nevi which show cellular heterogeneity but not clear cut evidence of 2 or more distinct nevus cell types, the proposal of Barnhill and colleagues to refer to some lesions as nevi with phenotypic heterogeneity has merit and is probably a more accurate description of the pathogenesis of the morphologic features. However, in our view, the proposal of Groben et al to classify some lesions as “epithelioid combined nevi” with various subtypes (Carney complex-like, “BLITZ”, DPN-like and mixed types) seems complex and does not reflect the documented cellular heterogeneity that occurs in the previously described subtypes of blue nevi.

In the final analysis, genetic studies of large numbers of cases may aid in refining the classification of these entities (some of which may not turn out to be entities in their own right) and relationships (if any) between them.

CUTANEOUS AND PILAR NEUROCRISTIC HAMARTOMA

Cutaneous neurocristic hamartoma (CNH) refers to very rare cutaneous tumors exhibiting divergent differentiation along nevomelanocytic, neural and pigmented dendritic cell lines. They may be congenital or acquired, and present as slow-growing multinodular plaques. Histologically, they are infiltrating multinodular tumors involving the dermis and subcutis and lacking a junctional component. The tumors comprise areas resembling DBN, CBN, EBN, neurofibroma, and schwannoma, and may infiltrate along nerves. Typically, these tumors exhibit low grade (but progressive) behavior and the clinical course is characterized by slow growth punctuated by numerous recurrences.^183–185 Rare tumors may develop expansile nodular areas composed of epithelioid cells exhibiting cytologic atypia, mitotic activity and necrosis. Such tumors have led to metastasis and death and have been termed “malignant metastasizing neurocristic tumors.”¹⁸⁶ Whether or not these represent unusual forms of melanoma arising in association with a blue nevus is open to debate.

Some heavily pigmented melanocytic lesions resembling so-called animal-type melanoma have been given the appellation “pilar neurocristic hamartoma” (PNH) due to the presence of perifollicular and perieccrine ductal arrangement of the melanocytes.^142,187,188 PNH are heavily pigmented multinodular tumors resembling blue nevi with a predilection for involvement of the perifollicular adventitia. They grow slowly over many years, with some cases metastasizing decades after excision of the primary lesion.^142,187,189

We remain doubtful that these lesions represent distinct entities and challenge the appropriateness of referring to them as hamartomas. We concur with the suggestion that both PNHs and CNHs may represent variants of blue nevus with peripheral nerve sheath differentiation,¹ and a case of a dermal melanocytic proliferation exhibiting features of both PTBN and neurocristic hamartoma has been described.¹⁹⁰ There seems to be some clinical and morphologic overlap between CNH, PNH, and PEM, although the precise relationship between these entities is unclear at present. The recently described paraganglioma-like dermal melanocytic tumor has some similar features to CBN and may represent another blue nevus variant or “melanocytoma.”^191,192

CONCLUSIONS

Blue nevi exhibit a spectrum of appearances, with variation in parameters such as cellularity, cellular composition, degree of sclerosis, and pigmentation. Although such lesions are usually classified into one of the well-defined subtypes, in some cases displaying overlapping features, subclassification may not be precise. However, awareness of the clinical and histologic spectrum of blue nevi is important for their recognition and for their distinction from melanoma, particularly in the case of large blue nevi, CBN, and blue nevus variants exhibiting unusual or atypical morphologic features. In blue nevus-like tumors with some atypical features but without clear cut evidence of malignancy, it may not be possible in all cases to definitively predict the biological behavior by morphologic assessment of the primary tumor. In such cases, we recommend documenting in the pathology report the evidence in favor of (and against) a diagnosis of nevus or melanoma and providing a favored diagnosis whenever possible. It is probably appropriate in such difficult/problematic/borderline cases to seek further opinion from one or more experienced colleagues. Complete excision is probably mandatory (if clinically appropriate), however, the role of sentinel lymph node biopsy in the management of these patients remains uncertain. In the future, it is hoped that the use of molecular testing may allow more precise classification of melanocytic tumors and more accurate prediction of their biologic potential and clinical behavior.¹⁹³

ACKNOWLEDGMENTS

The authors thank their colleagues at Royal Prince Alfred Hospital and Melanoma Institute Australia (incorporating the Sydney Melanoma Unit) for ther support and assistance. The authors also thank the Melanoma Foundation of the University of Sydney, the Cancer Institute New South Wales, and the Australian National Health and Medical Research Council for support.