Pathological diagnosis of melanocytic tumours: clues and pitfalls # Richard A. Scolyer 1,2,3* and Stanley W. McCarthy 1,2,3

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1 Pathological diagnosis of melanocytic tumours: clues and pitfalls # Richard A. Scolyer 1,2,3* and Stanley W. McCarthy 1,2,3 1 Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney, Australia. 2 Melanoma Institute Australia, Sydney, Australia. 3 Discipline of Pathology, Sydney Medical School, The University of Sydney, NSW, Australia. *Supported by a Cancer Institute New South Wales Clinical Research Fellowship In Australia and many other countries, melanoma is a major public health problem particularly in those individuals of Celtic ancestry. Other races are not immune, especially when acral and mucosal sites are taken into account. Accurate diagnosis requires the balancing of clinical data (including patient age and sex, family history, the anatomic site of the lesion, the history of the lesion and other factors such as a history of trauma, sunburn or pregnancy), histological features (including architecture, cytology and the host response), awareness of pitfalls and judgment. Several types of naevi are prone to be misdiagnosed as melanoma such as regenerating naevi, combined naevi, acral naevi, deep penetrating naevi and Spitz naevi. Melanomas often underdiagnosed include naevoid melanomas, desmoplastic melanomas, Spitzoid melanomas and regressed melanomas. The type of biopsy and suboptimal processing may also significantly influence the diagnosis. PATHOLOGICAL DIAGNOSIS Accurate pathological diagnosis of melanocytic tumours requires a suitable biopsy, assessment of many histological criteria, an awareness of potential pitfalls, relevant experience and, in difficult cases, judicious consultation 1-5. Correlation of a range of basic morphological and clinical features is necessary because many of the individual features are shared by both naevi and melanomas. In the future, molecular and genetic studies may be useful adjuncts for an accurate diagnosis, particularly for difficult lesion for which it is not possible to be certain from its histological features whether the lesion is benign or malignant Morphological features of importance (Table 1): Architectural: Good symmetry is often associated with benign behaviour. Poor symmetry and irregular margins are more often found in dysplastic, traumatised and malignant melanocytic tumours. Ulceration, vascular invasion, neurotropism and satellites are also more frequent in melanomas but can also occasionally occur in naevi. Minor perineural invasion may be found in all types of benign naevi so is not very helpful when diagnosing melanocytic tumours. Pagetoid epidermal invasion is very common and not usually significant in acral lesions, regenerating naevi ( pseudomelanoma ) or irritated naevi (trauma, sunburn etc.) 5 9.

2 Cytological: Cytological features may be deceptive especially in naevoid melanomas with small cells and uniform cytology, deep penetrating naevi with nuclear pleomorphism and regenerating naevi with epithelioid cells. Nucleoli when irregular or frequent (average > 2 nucleus) usually indicate dysplasia or malignancy. Mitoses when abnormal, frequent (>2/mm²) or deep in the lesion are always of concern and should be regarded as being of at least uncertain malignant potential especially when occurring in post pubertal patients. Host response: This is usually cellular and/or stromal. Cellular: Regressing tumours, benign or malignant, are usually infiltrated, at least initially, by lymphocytes. Scattered foci of lymphocytes are a common feature in desmoplastic melanoma. Benign naevi and some aggressive epithelioid melanomas lack a cellular response. Stromal: Periretal lamellar collagen is common in dysplastic naevi and may be very thick in severe forms. Fibrosis is variable in desmoplastic melanoma. Pure forms (100% desmoplastic) appear to have a better prognosis and less frequent nodal metastases 10. Table 1: Morphological Features of Melanoma Architectural Asymmetry Irregular margins Ulceration Vascular invasion Microsatellites Pagetoid epidermal invasion Cytological Nucleoli (especially if irregular and multiple) Deep or frequent mitoses Poor maturation Host response Lymphocytic infiltrate Stromal fibrosis (especially in desmoplastic melanoma) Regression Clinical features of importance: Age: Melanomas are uncommon in children just as Spitz naevi are rare in middle and older age groups. Vulval melanoma is rare below the age of 40 years. Sex: Melanomas with Spitzoid features appear to be more common in young males but the reverse is probably true after Females have a better prognosis than male melanoma patients. Family history: Melanomas are common in some families and first degree relatives.

3 Site: Sun-exposed areas are more likely to develop naevi and melanomas in Caucasian patients. Desmoplastic melanomas most commonly involve the head and neck region and are frequently associated with lentigo maligna 12. They may be subtle clinically and histologically and hence may not be diagnosed until they are at an advanced clinical stage. 9 History of lesion: A lesion of long duration which has changed in size or color, become itchy, or bled easily should always be investigated. Any trauma, previous biopsy, sunburn or topical agent may induce regenerative features that simulate malignancy. Pregnancy: May induce frequent mitoses in otherwise banal naevi. THE BIOPSY An excisional biopsy is preferred wherever possible Lesser specimens (punch, shave, curettings) may be distorted and may not permit adequate assessment of the vital parameters needed by the clinician for definitive treatment e.g. Breslow thickness, mitotic rate 13. Smaller specimens may also not be representative of the lesion and usually lack the edges and bases of the lesions needed to assist diagnostic interpretation 3 4. In the subsequent excision specimen it may be impossible to allocate atypical features cellular pleomorphism, mitoses and epidermal invasion to either malignancy or regeneration. A recent study highlighted that histopathological misdiagnosis is more common for melanomas that have been assessed with punch and shave biopsy than with excision biopsy 34. Adverse outcomes due to misdiagnosis were more commonly associated with punch biopsy than with shave and excision biopsy (odds ratio 16.6, p<0.001). The use of punch and shave biopsy also leads to increased microstaging inaccuracy. It is often difficult to separate traumatic fibrosis from regression fibrosis. The latter is more often associated with epidermal atrophy whereas post-traumatic fibrosis in compound naevi often has some overlying variable epidermal thickening. NAEVI PRONE TO MISDIAGNOSIS (Table 2) Table 2: Naevi Prone to Misdiagnosis Regenerating nevus Blue naevi Irritated naevus - Dendritic Post-excisional junctional hyperplasia - Cellular Cellular/hyperplastic nodule - Deep penetrating in congenital naevus - Epithelioid Combined naevus Acral naevi Ancient naevus Balloon and Clear cell naevi Spitz naevus Neurotized naevus Dysplastic naevus Desmoplastic naevus Naevus in pregnancy Halo naevus Genital naevus

4 Regenerating Naevi. Naevi often regenerate after incomplete removal (punch, shave, curette, incision, other trauma). Some naevi, especially compound melanocytic naevi of small congenital type and dysplastic compound naevi, display features often associated with melanoma and are sometimes termed pseudomelanoma. These features include Pagetoid spread of melanocytes, cytological atypia, dermal mitoses and HMB45 positivity. 14 Post-excisional junctional melanocytic hyperplasia at the edge of an excision scar may show some atypia and a tendency to epidermal invasion suggestive of in situ malignancy. Irritated melanocytic naevi may show epidermal invasion but the offending melanocytes tend to have small nuclei. Causes of irritation may be physical e.g. rubbing or scratching, topical agents or strong sunburn. Dysplastic naevi have variable lentiginous hyperplasia, periretal lamellar collagen, architectural disorder and cytological atypia. The cytological atypia may be mild, moderate or severe. Severe atypia or moderate atypia associated with irritative or Spitzoid changes may readily be mistaken for malignancy. The vast majority of dysplastic naevi (the commonest naevi in Caucasians) with mild to moderate cytological atypia are stable or grow very slowly and rarely progress to malignancy. Dysplastic naevi are regarded as intermediate lesions of tumour progression between naevi and melanoma. They show S-phase ploidy and intermediate levels of genetic unstability. Cellular/hyperplastic nodules in congenital naevi may have mitoses but they are not abnormal. The nodules usually have pushing margins and merge with the adjacent banal component of the naevus. Naevi in pregnancy. Dermal and compound naevi of small congenital type in pregnancy may show increased numbers of normal-looking mitoses in the otherwise banal dermal nevomelanocytes and appear to be without clinical significance. Combined naevi are often mistaken clinically and histologically for melanoma because of their biclonal (occasionally triclonal) nature and variability or change in color or shape Frequent problems are combinations of common naevus (common acquired, congenital or dysplastic) associated with pigmented blue naevus (common blue, cellular blue or deep penetrating) or Spitz naevus (conventional, regressing or desmoplastic) 16. The blue naevus or Spitz naevus component may dominate the lesion and show an occasional mitosis. So-called ancient naevi may represent a combined naevus with a regressing or degenerating Spitzoid component. Blue naevi variants cause special problems. Dendritic blue naevi and sclerosing cellular blue naevi may have an infiltrative pattern similar to desmoplastic melanomas but the latter often have some junctional change, are almost invariably non-pigmented and are negative with HMB45. 17

5 Deep penetrating naevi frequently show moderate nuclear pleomorphism, abut nerves and have an occasional dermal mitosis. Rare cases, either large or with scattered mitoses, may involve a regional or sentinel lymph node. Whether this is a benign metastasis or an indication of future malignant progression is controversial. 17 Epithelioid blue naevi (as in Carney s syndrome) and other so-called pigmented epithelioid melanocytomas are also low grade melanocytic tumours that apparently frequently involve regional lymph nodes yet paradoxically appear to have a favourable prognosis 18 and should probably be regarded as being of uncertain malignant potential especially those in children and young adults and when associated with pseudoepitheliomatous hyperplasia. Balloon and clear cell naevi may be confused with their malignant counterparts. The naevi usually have bland nuclei and lack mitoses. Neurotised and desmoplastic common naevi can be confused with desmoplastic melanoma especially of pure type. The naevi, however, lack atypical junctional changes, dermal mitoses and scattered clusters of lymphocytes. Halo or regressing naevi may present with alarming color change, with a darkening centre and pale periphery, especially in children. Most of these atypical compound naevi have a dermal component of smaller darker nevomelanocytes disrupted by lymphocytes, a feature suggestive of early regression. An occasional dermal mitosis does not indicate malignancy. Though most frequent in children, they also occur in middle and old age groups. Acral naevi and genital naevi resemble dysplastic naevi elsewhere in the skin. Focal Pagetoid epidermal invasion, especially in the acral naevi is alone not an indication of malignancy. See also Tables 3 and 4. Table 3: Naevi of Acral Skin: Histopathological Features Which Can Cause Concern Suprabasilar melanocytes ( Pagetoid spread ) (30-79%) Atypical size, shape, location of junctional nests A subset (acral lentiginous naevi) show - Predominantly lentiginous growth - Poor circumscription - Asymmetry Small biopsies Table 4: Acral Naevi v Acral Melanoma: Features favouring melanoma Severe cytological atypia Lymphocytes abutting junctional zone Marked architectural disorder Increased density of melanocytes Excessive Pagetoid spread

6 Poor maturation Expansile growth Dermal mitoses and lymphocytes Spitz naevus and Spitzoid tumours are a common problem and share many of the histological features of melanoma (Table 5) Those with atypical features cause most concern and many are of uncertain biological behavior (Table 6). 20 Table 5: Features of Spitz Naevi Diameter usually < 6mm Telangiectatic vessels Symmetrical Nested & single cell Diffuse epidermal hyperplasia Pagetoid spread Hypergranulosis Maturation and often merging Terminal nests with dermal collagen Uniformity of cells Spindle/epithelioid cells and nests across lesion Mitotic figures <=2/mm² Kamino bodies Absent deep or abnormal mitoses Subepidermal clefts Table 6: Atypical Features for Spitz Naevus Diameter >10mm Peripheral Pagetoid Asymmetry epidermal invasion Ulceration Sparse Kamino bodies (children) Lack of maturation Marked cytological atypia Deep extension Irregular or multiple nucleoli Expansile dermal nests Dermal mitoses >2/mm² Epidermal thinning Deep mitoses Long thin rete ridges Abnormal mitoses An Approach to Atypical Cases. Complete excision is probably mandatory Avoid overdiagnosis to avoid excessive treatment and possible medico-legal action. Give a preferred diagnosis acknowledging the degree of uncertainty. Seek further opinions. The safest course may be to manage the lesion as a melanoma. 21 However, wide local excision may not be appropriate if there is likely to be unacceptable cosmetic disfigurement and any co-morbidity When there is uncertainty about whether a melanocytic tumour is benign or malignant the pathologist should document in their report the thickness of the tumour (and other important features of the tumour such as its mitotic rate and ulcerative state). This information may be utilised by the clinician to manage the patient as for a melanoma of equivalent thickness (and other prognostic parameters) if this is deemed appropriate. MELANOMAS PRONE TO CAUSE DIAGNOSTIC PROBLEMS (Table 7)

7 Table 7: Melanomas Prone to Cause Diagnostic Problems Naevoid melanoma Spitzoid melanoma Desmoplastic melanoma Regressed melanoma Naevoid melanomas mimic a benign naevus. Small and intermediate cell types are sometimes called minimal deviation melanoma (or Lawyer s melanoma because they may not be diagnosed until after they have metastasized and medicolegal action may follow). 14 Large and spindle cell types are called Spitzoid or Spitz naevus-like melanoma. A probable indolent variant resembling a dermal naevus develops on the lower limb of elderly females. Recognition of naevoid melanomas of smaller cell types requires a high index of suspicion and recognition of subtle architectural and cytological features. Architectural features are basic symmetry, good circumscription in a nodular or verrucous pattern lacking any significant radial growth phase or epidermal invasion. Long thin rete ridges, expansile or sheet-like growth and pseudomaturation are common. Subtle cytological atypia is present throughout though mitoses may be sparse and only superficial. HMB45 and Ki67 staining may be helpful in some cases. Desmoplastic melanoma. Desmoplastic melanomas may be difficult to detect clinically as they are usually nonpigmented and may resemble a scar, a dermatofibroma, a basal cell carcinoma or a poorly healing pyogenic granuloma. 9 Histologically they may be confused with immature scars (which may also have S-100 positive spindle cells and foci of lymphocytes), desmoplastic naevi (usually symmetrical lacking atypia, mitoses and lymphocytic foci); desmoplastic Spitz naevi (plump cells and associated epidermal thickening); sclerosing cellular blue naevus (usually HMB45 positive and have foci of melanin pigment); dermatofibroma (tapering storiform margins, epidermal thickening, absent lymphocytes) and with some soft tissue spindle cell sarcomas. Desmoplastic melanomas also often show an associated atypical junctional component (lentigo maligna / Hutchinson s melanotic freckle) and neurotropism. Most are HMB45 and Melan A (MART-1) negative. Pure forms (spindle cells only) probably have a better prognosis and less frequently involve regional lymph nodes than other melanomas. Regressed melanoma. If the lesion is on the back or posterior shoulder a history of a previous lesion may be unavailable. Key features are: 1. Epidermal thinning with loss of rete ridges. 2. Minimal or absent atypical junctional change.

8 3. A subepidermal band of angiofibroplasia (often up to 0.8mm thick) which may variably contain a few lymphocytes, melanophages and an occasional melanocyte. Capillaries are usually slightly increased. Melanoma Pathology Report The report should include all factors that affect accurate diagnosis, management and prognosis Currently the most important pathological prognostic factors for clinically localized primary melanoma are tumour thickness (Breslow), dermal mitotic rate (which also influences the rate of sentinel lymph node positivity) and ulceration. 1, Other factors are Clark level, site (trunk worse than extremities), age and gender (males worse than females). Margins of Excision. These are important in determining further management of atypical melanocytic lesions and melanomas. The main problem is accurately assessing the margins of lentigo maligna (Hutchinson s melanotic freckle) and some other melanocytic tumours where atypical melanocytes trail off at the edges. 32 To avoid excessive excisions, a marginal excision around the clinically affected area is usually done initially for definitive diagnosis. Wider excision (as per current melanoma treatment guidelines) can then be performed once a diagnosis of melanoma has been confirmed. # Adapted from McCarthy SW, Scolyer RA. Pitfalls and Important Issues in the Pathologic Diagnosis of Melanocytic Tumors. The Ochsner Journal 2010 (in press). REFERENCES 1. Thompson JF, Scolyer RA, Kefford RF. Cutaneous melanoma.[see comment]. Lancet 2005;365(9460): Murali R, Thompson JF, Scolyer RA. Sentinel lymph node biopsy for melanoma: aspects of pathologic assessment. Future Oncology 2008;4(4): Scolyer RA, McCarthy SW, Elder DE. Frontiers in melanocytic pathology. Pathology 2004;36(5): Scolyer RA, Thompson JF, McCarthy SW, Strutton GM, Elder DE. Incomplete biopsy of melanocytic lesions can impair the accuracy of pathological diagnosis.[erratum appears in Australas J Dermatol Nov;47(4):308]. Australasian Journal of Dermatology 2006;47(1):71-3; author reply Scolyer RA, Thompson JF, Stretch JR, Sharma R, McCarthy SW. Pathology of melanocytic lesions: new, controversial, and clinically important issues. Journal of Surgical Oncology 2004;86(4): Murali R, Sharma RN, Thompson JF, Stretch JR, Lee CS, McCarthy SW, et al. Sentinel lymph node biopsy in histologically ambiguous melanocytic tumors with spitzoid features (so-called atypical spitzoid tumors). Annals of Surgical Oncology 2008;15(1): Morey AL, Murali R, McCarthy SW, Mann GJ, Scolyer RA. Diagnosis of cutaneous melanocytic tumours by fourcolour fluorescence in situ hybridisation. Pathology 2009;41(4): Huang SK, Darfler MM, Nicholl MB, You J, Bemis KG, Tegeler TJ, et al. LC/MS-based quantitative proteomic analysis of paraffin-embedded archival melanomas reveals potential proteomic biomarkers associated with metastasis. PLoS One 2009;4(2):e McCarthy SW, Scolyer RA, Palmer AA. Desmoplastic melanoma: a diagnostic trap for the unwary. Pathology 2004;36(5): Scolyer RA, Thompson JF. Desmoplastic melanoma: a heterogeneous entity in which subclassification as "pure" or "mixed" may have important prognostic significance.[comment]. Annals of Surgical Oncology 2005;12(3): Crotty KA, Scolyer RA, Li L, Palmer AA, Wang L, McCarthy SW. Spitz naevus versus Spitzoid melanoma: when and how can they be distinguished?[see comment]. Pathology 2002;34(1): Shaw HM, Quinn MJ, Scolyer RA, Thompson JF. Survival in patients with desmoplastic melanoma.[comment]. Journal of Clinical Oncology 2006;24(8):e12; author reply e Thompson JF, Scolyer RA. Cooperation between surgical oncologists and pathologists: a key element of multidisciplinary care for patients with cancer. Pathology 2004;36(5):

9 14. McCarthy SW, Scolyer RA. Melanocytic lesions of the face: diagnostic pitfalls. Annals of the Academy of Medicine, Singapore 2004;33(4 Suppl): Murali R, McCarthy SW, Thompson JF, Scolyer RA. Melanocytic nevus with focal atypical epithelioid components (clonal nevus) is a combined nevus.[comment]. Journal of the American Academy of Dermatology 2007;56(5): Scolyer RA, Zhuang L, Palmer AA, Thompson JF, McCarthy SW. Combined naevus: a benign lesion frequently misdiagnosed both clinically and pathologically as melanoma. Pathology 2004;36(5): Murali R, McCarthy SW, Scolyer RA. Blue nevi and related lesions: a review highlighting atypical and newly described variants, distinguishing features and diagnostic pitfalls. Adv Anat Pathol 2009;16(6): Mandal RV, Murali R, Lundquist KF, Ragsdale BD, Heenan P, McCarthy SW, et al. Pigmented epithelioid melanocytoma: favorable outcome after 5-year follow-up. Am J Surg Pathol 2009;33(12): Dahlstrom JE, Scolyer RA, Thompson JF, Jain S. Spitz naevus: diagnostic problems and their management implications. Pathology 2004;36(5): Busam KJ, Murali R, Pulitzer M, McCarthy SW, Thompson JF, Shaw HM, et al. Atypical spitzoid melanocytic tumors with positive sentinel lymph nodes in children and teenagers, and comparison with histologically unambiguous and lethal melanomas. Am J Surg Pathol 2009;33(9): Scolyer RA, Murali R, McCarthy SW, Thompson JF. Pathologic examination of sentinel lymph nodes from melanoma patients. Semin Diagn Pathol 2008;25(2): Chakera AH, Hesse B, Burak Z, Ballinger JR, Britten A, Caraco C, et al. EANM-EORTC general recommendations for sentinel node diagnostics in melanoma. Eur J Nucl Med Mol Imaging 2009;36(10): Karim RZ, van den Berg KS, Colman MH, McCarthy SW, Thompson JF, Scolyer RA. The advantage of using a synoptic pathology report format for cutaneous melanoma. Histopathology 2008;52(2): Azzola MF, Shaw HM, Thompson JF, Soong S-J, Scolyer RA, Watson GF, et al. Tumor mitotic rate is a more powerful prognostic indicator than ulceration in patients with primary cutaneous melanoma: an analysis of 3661 patients from a single center. Cancer 2003;97(6): Francken AB, Shaw HM, Thompson JF, Soong S-j, Accortt NA, Azzola MF, et al. The prognostic importance of tumor mitotic rate confirmed in 1317 patients with primary cutaneous melanoma and long follow-up.[see comment]. Annals of Surgical Oncology 2004;11(4): Scolyer RA, Shaw HM, Thompson JF, Li L-XL, Colman MH, Lo SK, et al. Interobserver reproducibility of histopathologic prognostic variables in primary cutaneous melanomas. American Journal of Surgical Pathology 2003;27(12): Scolyer RA, Thompson JF, Shaw HM, McCarthy SW. The importance of mitotic rate as a prognostic factor for localized primary cutaneous melanoma.[comment]. Journal of Cutaneous Pathology 2006;33(5):395-6; author reply Thompson JF, Scolyer RA, Kefford RF, Uren RF. Melanoma management in 2007.[comment]. Australian Family Physician 2007;36(7):487-8; author reply Thompson JF, Shaw HM, Hersey P, Scolyer RA. The history and future of melanoma staging. Journal of Surgical Oncology 2004;86(4): Murali R, Hughes MT, Fitzgerald P, Thompson JF, Scolyer RA. Interobserver variation in the histopathologic reporting of key prognostic parameters, particularly clark level, affects pathologic staging of primary cutaneous melanoma. Ann Surg 2009;249(4): Thompson JF, Scolyer RA, Kefford RF. Cutaneous melanoma in the era of molecular profiling. Lancet 2009;374(9687): Stretch JR, Scolyer RA. Surgical strategies and histopathologic issues in the management of lentigo maligna. Ann Surg Oncol 2009;16(6): Van Es SL, Colman M, Thompson JF, McCarthy SW, Scolyer RA. Angiotropism is an independent predictor of local recurrence and in-transit metastasis in primary cutaneous melanoma. Am J Surg Pathol 2008;32(9): Ng JC, Swain S, Dowling JP, Wolfe R, Simpson P, Kelly JW. The impact of partial biopsy on histopathologic diagnosis of cutaneous melanoma: experience of an Australian tertiary referral service. Archives of Dermatology 2010;146(3):234-9.