Melanoma is a relatively common malignant neoplasm that arises from melanocytes and is most commonly cutaneous in origin; however, it can arise in the eye, mucosae, and internal organs [1, 2]. Melanoma is one of the most common forms of cancer in young adults and thus it represents a major public health problem. Overall, melanoma is the fifth most common cancer in men and the sixth in women in the United States. The incidence of cutaneous melanoma has increased dramatically in the last years and remains to be the leading cause of death in cutaneous malignancies [3–6]. The Surveillance, Epidemiology, and End Results (SEER) Program reports an increase of more than 600 % in the diagnosis of cutaneous melanoma from 1950s to 2000s [7], with an average increase of 2.9 % each year between 1985 and 2009. Overall, the lifetime risk for developing melanoma in patients born in 2012 is 2.62 for men and 1.63 for women (SEER data) . The increased incidence varies by age, gender, ethnicity, and histologic subtype. Before the age of 40, the incidence is higher in women, with most melanomas located on the lower extremities and exhibiting the superficial spreading histology. After the age of 40, the incidence is much higher in men, with most melanomas presenting on the head and neck and back. Also, incidence increases are most pronounced in white populations, particularly elderly individuals, whereas the incidence among darkly pigmented populations has risen only slightly or remained stable. The incidence of melanoma in children is increasing [8–10], which can be associated with risk factor such as xeroderma pigmentosum, familial dysplastic nevus syndrome or familial melanoma, and possibly immunosuppression.

Interestingly, there appears to be discordance between incidence and mortality trends in melanoma. While mortality rates increased slightly in the United States and Europe during the 1970s and 1980s, a stabilization of mortality rates was observed in most countries during the 1990s and 2000s. This difference in incidence and mortality trends may be due to the fact that more patients present at an earlier stage with smaller lesions that are potentially curable although it is also possible that changes in histologic criteria may have resulted in overdiagnosis of melanoma [11–16]. While these factors likely play some role, they certainly cannot explain entirely the observed incidence rise in patients presenting with advanced tumors suggesting a true increase in melanoma incidence [17, 18].

Understanding the risk factors for the development of melanoma is important. Individual risk assessment influences clinical decision-making including the threshold for performing a biopsy, prevention counseling, and surveillance. The etiology of melanoma is multifactorial including demographic, environmental, and genetic risks. However, the majority of melanomas are likely related to ultraviolet radiation, since UV-related mutations are relatively common in melanoma [19].

Demographic Risk Factors : Age is a well-known risk factor for the development of melanoma as its incidence increases with age [20]. Although the median age of melanoma patients is about 50 years, it occurs in a wide age distribution [21]. The incidence is greatest in older patients, but it is also one of the most common cancers in young adults and adolescents. When compared to other cancers, due to the relatively young median age of melanoma patients, melanoma ranks among the worst in terms of lost productive years. The risk for melanoma is greatest in ethnic groups with lighter skin types, with the majority of melanomas in the United States diagnosed in non-Hispanic, white patients. On the other hand, in the United States, African Americans and Hispanics present with higher-stage melanomas and have a worse 5-year survival compared to white populations. The lifetime risk for melanoma is higher in men than in women, but it is also affected by sex. Before age 40, incidence rates are higher in women than in men; after age 40 years, rates are almost twice as high in men as in women.

A personal history of melanoma confers an increased risk for developing an additional melanoma. Patients with a prior melanoma have an approximately 2–5 % chance of developing a subsequent melanoma. Regarding histologic subtypes, patients whose first melanoma is lentigo maligna melanoma or nodular melanoma have a higher risk of a subsequent primary melanoma, compared with superficial spreading melanoma. A personal history of squamous cell carcinoma or basal cell carcinoma triples or doubles (respectively) the risk of developing melanoma. The risk is slightly lower in patients with actinic keratosis.

Environmental Risk Factors : It is well accepted that exposure to ultraviolet (UV) radiation is a major risk factor, especially among those with fair skin who are more susceptible to sunburns. As such, individuals who are most susceptible to the effects of excessive sunlight (e.g., blond or red haired, blue eyed, or with fair complexions) that tan poorly and tend toward sunburn have an increased risk of melanoma [22, 23]. While UVB has been regarded as the causative agent, UVA radiation is also carcinogenic and can induce melanoma [24, 25]. There is an increased incidence of melanoma in people that live close to the equator or at high altitudes and, in general, in persons who report increased exposure to UV radiation. The risk from UV radiation varies according to intensity, frequency, and age at the time of exposure. Intermittent and intense UV radiation exposure and sunburns are risk factors. Studies have shown UV radiation exposure during childhood is particularly associated with increased risk; however, sunburns occurring during any period of life also increase the risk of melanoma. The most common anatomic location of melanoma for men is the back and in women the legs , finding that has been suggested to be due to intermittent and intense UV radiation in each sex. Exposure to artificial UV radiation also increases melanoma risk, particularly tanning salons [26].

Presence of Nevi and/or Dysplastic Nevi : Large numbers of standard nevi, of large size, and presence of dysplastic nevi are risk factors for melanoma [27]. The presence of >10 clinically dysplastic nevi confers a 12-fold increased risk. Also increased numbers of conventional nevi and nevi greater than 6 mm in diameter are independent risk factors for the development of melanoma [27–30]. However, although the presence of non-dysplastic and dysplastic nevi predicts an increased risk for melanoma, only a small percentage of nevi progress to melanoma; approximately 25 % of melanomas arise in association with a melanocytic nevus [31]. Some authors have observed that younger age, superficial spreading histologic subtype, and trunk location are predictors of a melanoma being histologically associated with a melanocytic nevus [31].

Family History : A prior family history of melanoma increases the risk for acquiring melanoma. The risk of developing melanoma is 2.62 times higher when a parent has had melanoma and is 2.94 times higher if the melanoma was in a sibling. Overall, about 8 % of people newly diagnosed with melanoma have a first-degree relative with melanoma, and about 1–2 % has two or more close relatives with melanoma. Fewer than 10 % of patients present with true familial or hereditary melanomas in which there is a strong family history or a documented melanoma-related, germline mutation. These families have autosomal dominant inherited susceptibility mutations and develop single or multiple melanomas at early age [32].

Several genetic loci determine susceptibility to melanoma, with the most important of these being CDK4 and p16/CDKN2A located on chromosome 9p21, both being high-risk genes [33]. Both CDNKN2A and CDK4 are highly penetrant, susceptibility genes and result in the majority of familial melanomas. The CDKN2A gene encodes two proteins, p16 and p14 ARF, which are cell cycle inhibitors and are potent tumor suppressors. The CDKN2A mutation is present in up to 40 % of families with three or more cases of melanoma, whereas CDK4 mutations are present in far fewer families. The risk of developing melanoma in a patient who is a CDKN2A mutation carrier is between 30 % by age of 5 years and 67 % by age of 80 years. This risk varies by geographic location due to environmental factors, such as UV radiation, since the latter appears to play a significant role in susceptible families [34]. In addition, the same risk factors that influence the incidence of melanoma (such as increased number of nevi, sunburn, etc.) also increase the penetrance in CDKN2A mutation carriers. Melanoma-prone families with CDKN2A germline mutations have an increased risk of developing other neoplasms, primarily pancreatic cancer, with variable penetrance [33]. Germline CDK4 mutations in familial melanoma account for approximately 1 % of all familial melanomas [33]. While less frequent than CDKN2A mutations, CDK4 mutations carry an equally high risk for the development of melanoma and show a similar clinical phenotype [35]. In contrast to CDKN2A and CDK4, heritable mutations in the melanocortin-1 receptor (MC1R) gene have low penetrance [36]. MC1R mutation is commonly associated with a red hair phenotype, but it confers an increased risk of developing melanoma even in non-red-haired individuals. The risk of developing cutaneous melanoma lies between 1.5-fold and 3-fold for patients with MC1R variants, but when both alleles are mutated, it increases 17-fold for development of BRAF mutant melanomas. Some familial melanomas occur in the setting of dysplastic nevus syndrome. A family history of melanoma in multiple first-degree relatives and younger age at diagnosis are important features of this syndrome. Families who present with the dysplastic nevus syndrome may also have CDKN2A mutations. Other heritable disorders that predispose patients to melanoma include xeroderma pigmentosum, Li-Fraumeni syndrome, BRCA2, or familial retinoblastoma.

Accurate and thorough histopathological diagnosis of melanocytic lesions is essential for the clinical management of the patients, since the reporting of these histopathologic parameters, especially in the initial biopsies, is a critical component of both diagnosis and staging [37]. Institutions and dermatopathologists have different styles and variables in regard to the routine reports of histologic parameters in invasive melanomas. Some of these reports have only minimal information (such as Breslow thickness), while others reports are quite comprehensive. The information in those detailed reports might not be of immediate relevance, but its importance might become apparent later, when analyzed in prospective or retrospective studies. As an example of this, mitotic activity count is now included as a histopathologic element in the AJCC melanoma staging and classification system [37]. In our practices, we provide a report that includes all the histologic parameters that have been proved to be significant for staging and prognosis, as well as additional histologic features that may potentially be helpful (see Table 7.1).

Cutaneous melanomas are classified into four histologic subtypes : superficial spreading, lentigo maligna, acral lentiginous, and nodular type. While technically speaking the histologic subtype classification does not seem to affect prognosis, its proper classification is important for other reasons. One benefit of histologic classification is to provide subtyping for epidemiologic studies. Also, there are emerging genetic studies that show distinctive patterns of chromosomal aberrations in melanomas arising on chronically sun-exposed skin versus melanomas arising in areas with intermittent sun exposure or acral/mucosal areas. These heterogeneous molecular changes in melanoma are of clinical relevance because they are likely to result in different targeted therapies; acral lentiginous and mucosal melanomas can harbor KIT gene mutations and can potentially respond to targeted therapies with tyrosine kinase inhibitors [38–40]. It is also important to recognize that desmoplastic melanomas have higher propensity for neurotropism with high local recurrence and that in cases of pure desmoplastic melanoma, there is a lower incidence of lymph node metastasis and with an outcome different from conventional melanomas [41].

The initial classification of primary cutaneous melanoma was based on observations and descriptions of the clinical and histopathological features, which was first described over four decades ago [54, 83]. Lately, there have been some minor changes to the classification scheme, but the basic findings as described over 40 years form the foundation for the currently recognized subtypes of cutaneous melanoma [53, 84]. These authors started by describing the clinical appearance of the lesion, the anatomic site, the presence or absence of sun damage, and the patient’s demographics. All these features were considered alongside the histologic features of the tumors, including intraepidermal and dermal patterns of growth, cytology, epidermal changes, the presence of solar elastosis, the anatomic level of invasion into the dermis, the maximal tumor thickness, vascular invasion, mitotic activity, and the pattern and density of lymphocytic host response. Using these variables, the authors recognized four major subtypes : superficial spreading, lentigo maligna, nodular, and acral lentiginous melanoma.

Lentigo maligna goes through different phases. At its early stage, the histologic features of lentigo maligna are subtle and can be confusing due to the apparent bland nature of the intraepidermal melanocytes. Histologically, it is composed of single melanocytes located at the basal layer of epidermis, often with only minimal atypical features (such as mild nuclear enlargement and with or without hyperchromasia). These melanocytes tend to lose polarity of nuclei against the basement membrane, and in some cases, they show a clear halo with a moth-eaten appearance of the nuclei. This bland population of melanocytes is consistently accompanied by solar damage. A diagnostic clue at this early stage is the presence of asymmetric and confluent disposition of melanocytes in the basal layer and the involvement of hair follicles. One needs to keep in mind that the architectural changes may be much more pronounced than the degree of cytologic atypia at this stage. Importantly, smaller atypical melanocytes of lentigo maligna may be partially obscured by surrounding enlarged pigmented basal keratinocytes. Unfortunately, the presence of an early, pigmented actinic keratosis or of a pigmented macular seborrheic keratosis on chronically sun-damaged skin does not preclude concurrent lentigo maligna. The difficulty on making an unequivocal diagnosis at this stage is accentuated especially when the pathologist is dealing with a small tissue sample. As most lesions are located on the head and neck, small biopsies are usually taken to make the diagnosis before definitive treatment is given. Small tissue samples often pose diagnostic difficulty as the biopsy may not be representative of the entire lesion thus resulting in underdiagnosis of lentigo maligna.

As the lesion of lentigo maligna progresses, the histologic features are more pronounced and better appreciated on light microscopy. At this intermediate stage, lentigo maligna shows a confluent lentiginous and sometimes unevenly distributed, nested proliferation of enlarged melanocytes involving the basal layers of the epidermis and extending down appendageal epithelium, again associated with epidermal atrophy and solar elastosis. Melanocytes are commonly hyperchromatic and small with dense nuclear chromatin and unapparent nucleoli. Multinucleated melanoma cells (including “starburst” forms) are often present, but their presence is not specific for lentigo maligna since they can be seen also in benign lesions. There is often a variable superficial dermal inflammatory response with pigment incontinence. In our experience one of the most reproducible histological parameters that is used to confirm a diagnosis is the proliferation of atypical melanocytes at the dermal-epidermal junction in small nests or single cells, with involvement of the skin adnexa and coupled with underlying solar damage. Pagetoid spread of melanocytes is unusual in this type of melanoma and is generally seen later in the progression of the disease, often when there is dermal invasion. The distinction from actinic intraepidermal melanocytic hyperplasia (increased intraepidermal melanocytes secondary to chronic sun exposure) can be exceedingly difficult (also see differential diagnosis below). This reactive condition is also characterized by increased numbers of single basilar melanocytes occurring in the setting of an atrophic epidermis, with diminished rete ridges. The melanocytes tend to be hyperchromatic and slightly enlarged and do not significantly differ from those seen in lentigo maligna. The main distinguishing features are numbers of melanocytes (higher density in melanoma in situ), presence of pagetoid extension (when present in the later stages), and extension down cutaneous appendages. Immunohistochemistry may be helpful to highlight the numbers and size of intraepidermal melanocytes. Since anti-MART-1 is very sensitive, it may prominently label the cytoplasmic dendrites of melanocytes thus resulting in a relatively large area of the epidermis labeled with the immunodye (e.g., DAB), thus resulting in an overdiagnosis of melanoma [93]. HMB-45 is usually less sensitive than anti-MART-1 and thus the area covered by the immunodye is less extensive. At any rate, rather than evaluating the “positive area” highlighted by the dye, it is better to determine how many nuclei are surrounded by immunodye since such quantification will be closer to the actual number of melanocytes present in the epidermis [94].

When lentigo maligna becomes invasive in the dermis, melanoma cells are usually arranged in dermal nests and single cells, and there is frequent vascular ectasia in the superficial vascular. The dermal melanocytes most commonly display an epithelioid or spindle-shaped morphology. Melanocytes are hyperchromatic and atypical, but frequently lack the vesicular nuclei and prominent eosinophilic nucleoli that are seen in other subtypes of melanoma (such as superficial spreading or nodular melanoma). Also, in cases of early invasive lesions with only a few single cells or small nests within a fibrous or inflamed superficial dermis, these invasive cells may be difficult to identify. Immunohistochemistry (IHC) may be used to highlight the focal dermal invasion in cases with only individual invasive melanocytes or small nests admixed with inflammatory cells [94]. The most sensitive immunohistochemistry marker for melanocytes is probably S-100 protein; however, this marker has relatively low specificity, as dermal dendritic cells are positive for S-100, making specific identification of individual melanocytes in the dermis somewhat difficult. Although anti-MART-1 is more specific than anti-S100, it may be still positive in pigmented dermal macrophages [95]. Thus, HMB-45 or antibodies against some nuclear melanocytic markers (MITF or SOX10) may be more helpful in detecting dermal invasion.

As seen in other melanoma subtypes, dermal maturation in lentigo maligna is not apparent, and mitotic figures may be observed, but these are usually few in number. Melanocytes can be found individually throughout the dermis or seen along the skin adnexa. The spindle-shaped melanocytes have a predilection for nerves within the reticular dermis, and perineural invasion is relatively often in lentigo maligna. Some cases of lentigo maligna can be associated with desmoplastic melanoma, and the invading spindle tumor cells are very subtle, bearing a striking resemblance to a dermal scar. In such cases, anti-S100p, anti-MITF1, or anti-SOX10 is very helpful to make such distinction (since other markers such as MART-1 or HMB-45 antigen are often negative in desmoplastic melanoma).

Lentigo maligna has traditionally been difficult to diagnose, especially in its early stage or when a limited tissue sample is provided. Recent studies about the molecular pathogenesis of melanoma revealed that melanomas occurring on the sun-exposed skin show a distinct pattern of chromosomal instability sometimes related to UV DNA damage and are less frequently associated with BRAF mutations than superficial spreading melanoma (i.e., occurring on intermittently sun-exposed skin).

The main differential diagnosis of lentigo maligna is with lesions associated with sun damage such as early lesions of seborrheic keratosis, lichen planus-like keratosis, pigmented actinic keratosis/solar lentigo, and junctional melanocytic nevus. However, differentiating lentigo maligna from a background of increased melanocytes on chronically sun-damaged skin is one of the most difficult diagnostic challenges in diagnostic dermatopathology. In its earliest stages, lentigo maligna shows a single-cell, lentiginous proliferation of atypical melanocytes at the dermal-epidermal junction with only minimal cytologic atypia. It is well known that benign reactive melanocytic hyperplasia in sun-exposed areas or in the vicinity of surgical scars can exhibit histologic patterns similar to early lentigo maligna. In fact, the classic morphologic depiction of chronic photoactivation of melanocytes includes lentiginous melanocytic proliferation with the potential for nuclear hyperchromasia, binucleation and multinucleation, and low-level pagetoid ascent. Thus, a proliferation of melanocytes with slightly atypical nuclei in skin severely UV damaged is not sufficient for a diagnosis of lentigo maligna. Sun-exposed epidermis shows approximately twice as many melanocytes as non-sun-exposed, covered skin, and the density of melanocytes has been said to be proportional to cumulative sun exposure. As the lesion of lentigo maligna progresses, the histologic changes are more obvious, including coalescence of single melanocytes along the dermal-epidermal junction, extension into skin adnexa, focal pagetoid spread, and obvious junctional nest formation. Indeed, a very important and useful feature for the diagnosis of lentigo maligna is the presence of intraepidermal melanocytic nests. Furthermore, some authors have suggested that, if there are melanocytic nests in an entirely intraepithelial lesion on the skin in the head and neck area with severe solar elastosis, the diagnosis almost always is lentigo maligna. However, we consider that patients may develop junctional dysplastic nevi during all their life, and therefore some may occur on the face after sun damage has started.

The in situ component of lentigo maligna is usually wide and poorly circumscribed, with individual neoplastic melanocytes extending beyond the last melanocytic nest; thus, the absence or presence of melanocytic nests does not always help differentiate lentigo maligna from melanocytic hyperplasia in sun-damaged skin and is only rarely helpful in delineating the borders of a melanoma. The distribution of pigment is relevant in distinguishing these two conditions, as in cases of solar lentigo with melanocytic hyperplasia, there is an even distribution of melanin in basal keratinocytes, as opposed to the irregular distribution of pigment noted in lentigo maligna [96]. An important diagnostic clue is the presence of preserved elongation of rete ridges that are relatively uniform and relatively equidistant from each other in cases of solar lentigo/pigmented actinic keratosis, in contrast to lentigo maligna in which the rete ridges tend to be flattened. In cases of lentigo maligna in which there is focal preservation of rete ridges, they tend to be usually irregular and not equidistant from one another.

As mentioned above, immunohistochemistry plays an important role to separate incipient lentigo maligna from intraepidermal melanocytic hyperplasia on sun-damaged skin. Mere close inspection of H&E-stained section does not always allow an unequivocal diagnosis, because it is sometimes difficult to distinguish pigmented keratinocytes from melanocytes. Anti-MART-1 may overestimate the number of melanocytes because it labels the melanocytic dendrites and might also label pigmented keratinocytes, including structures mimicking junctional melanocytic nests in the setting of a lichenoid infiltrate [97–99]. This lack of specificity may result in false-positive result in the assessment of difficult intraepidermal melanocytic lesions. Both MITF-1 and SOX10 are nuclear makers that facilitate the diagnosis [100] since they avoid the cytoplasmic labeling seen with anti-MART-1 or HMB-45.

A similar challenge may occur when evaluating the peripheral margins of a melanoma in situ in a re-excision specimen, since epidermal melanocytes are usually increased in number as a reaction to the prior trauma (biopsy). Among the criteria that can be used for delineating the borders of a melanoma are presence of melanocytes above the junction, pleomorphism or hyperchromasia of melanocytes, and a high density of melanocytes with a confluent pattern.

The in situ component of superficial spreading is characterized by an asymmetrical proliferation of atypical melanocytes scattered singly and in clusters throughout all levels of the epithelium giving an appearance reminiscent of Paget disease (pagetoid upward migration or “buckshot” scatter). These pagetoid melanocytes are scattered throughout the epidermis giving a moth-eaten appearance and sometimes even infiltrating the stratum corneum. Melanocytes are epithelioid in appearance with abundant cytoplasm, often showing fine or dusty melanin pigmentation, and pleomorphic vesicular nuclei with prominent, eosinophilic nucleoli. There may be necrotic melanocytes and scattered mitotic figures including atypical forms. Melanocytic nests within epidermis tend to be irregularly distributed and have a confluent growth. An important diagnostic clue is that the amount of cellularity, pigment, and size of melanocytes vary from to nest. These melanocytic nests are large in general and have irregular contours; in occasions this pattern predominates over pagetoid melanocytes. Most of such cases of superficial spreading melanoma composed predominantly of large nests have only scant intraepidermal or junctional single melanocytes; thus, histologic recognition can be difficult [102, 103]. “Consumption” of the epidermis is present in approximately half of cases of superficial spreading melanoma. This epidermal consumption is defined as thinning of the epidermis with attenuation of basal and suprabasal layers and loss of rete ridges adjacent to collections of melanocytes. The epidermis may become separated from the dermis by an artefactual cleft (“zipper” sign; Christopher R. Shea, personal communication). Some authors believe that this consumption of the epidermis appears to represent a precursor to ulceration. In contrast to lentigo maligna, there is often little visible evidence of actinic damage. Also, in some cases there is lymphocytic infiltration in the papillary dermis and this may herald the presence of rare invasive cells. There may be fibroplasia of the papillary dermis; however, the organized (lamellar or concentric) stromal response observed in dysplastic nevi is not seen in such cases.

As per the original description by Dr. Clark, extension of the intraepidermal component for more than three rete ridges past the dermal component is a must to classify the lesion as superficial spreading (or acral lentiginous or lentigo maligna) melanoma. Progression of the radial growth phase starts with the development of a microinvasive component as single cells or small nests in the papillary dermis. These melanocytes have a similar cytomorphology as the intraepidermal component. When these dermal melanoma cells display either nests larger than observed in the overlying epidermis or have mitotic figures, these are the features of vertical growth phase. The invasive component in the dermis may be arranged in single melanocytes and solid nests or may have a fascicular growth pattern. Melanocytes may be epithelioid, spindle-shaped, small (nevus-like), and vacuolated (balloon cell-like). These melanocytes are pleomorphic and have irregular nuclei with irregular, hyperchromatic chromatin and thick nuclear membrane. The nuclei have an irregular outline that can be indented. The melanocytic density in melanomas is high, and the cells seem to overlap one to another. Melanocytes can form expansile nodules, which usually compress the adnexal structures. The presence of irregular distribution of pigment in the dermis is another diagnostic clue of melanoma. Detection of dermal mitotic figures in melanoma is a very important finding to confirm the diagnosis; however, absence of dermal mitotic figures should not be considered sufficient to exclude a diagnosis of melanoma. Particularly relevant is the presence of mitotic figures in the deeper portion of the lesion, since such a finding is only exceptionally seen in nevi. Recent studies have used immunodetection of phosphohistone H3 to facilitate the identification of mitotic figures in various neoplasms, including melanomas [104–106]. One study in particular provided definite support for the application of MART-1/phosphohistone H3 immunohistochemistry as an adjunctive test in the evaluation of primary melanomas [107]. This study concluded that adding this ancillary test could potentially add important value to the analysis and characterization of primary cutaneous melanoma, especially this is particularly true for the description of thin melanomas (≤1.0 mm) where mitotic figures can be difficult to identify and for which the identification of a mitotic figure would have the most profound implications for staging and patient management. In our practice, we apply dual immunohistochemical staining for MART-1 and phosphohistone H3 in cases where mitotic figures are seen in the infiltrate, but it is unclear if they represent melanocytes or other cells (e.g., macrophages, endothelial cells) or it is unclear if a cell is on mitosis or apoptosis.

Superficial spreading melanoma can mimic other melanocytic and non-melanocytic neoplasms. The main melanocytic lesion that needs to be considered in the differential diagnosis of melanoma is dysplastic nevus. In occasions, the distinction can be difficult and somewhat subjective, especially if the dysplastic nevus has severe architectural and cytologic features. Features that favor melanoma over a dysplastic nevus are the presence of significant pagetoid upward migration especially at the lateral borders of the lesion, diffuse and even cellular atypia, and the presence of deep-seated mitotic figures in the dermal component (see also the dysplastic nevus chapter). Another important clue is the presence of confluent nests in the dermal-epidermal junction, as it tends to abut the undersurface of the epidermis in between the rete ridges. In the differential diagnosis between melanoma and dysplastic nevi, it must be considered that focal areas of pagetoid upward migration in a nevus can be a sign of melanoma in situ originating in a dysplastic nevus. On the other hand, trauma to a nevus can induce focal pagetoid upward migration and this should not be interpreted as melanoma in situ. An important feature to distinguish between focal melanoma and prior trauma in a nevus is the presence of pigmented parakeratosis in the latter. Both melanoma and dysplastic nevus may show regression; however, in dysplastic nevi regression is usually focal as opposed to cases of melanoma in which the regressive changes are usually more widespread.

Recurrent/persistent nevi may simulate superficial spreading melanoma. The most important piece of information to make such distinction is to review the previous specimen. Histologic features that favor nevus include the sharp circumscription of the lesion, as recurrent nevi tend to preserve circumscription as opposed to melanomas, which are asymmetric. A banal intradermal component supports the diagnosis of recurrent nevus, as in melanomas the dermal component shows an atypical growth along with the presence of deep mitotic figures. In recurrent nevi, the intraepidermal growth is above the scar, while the epidermis beyond the scar is uninvolved. The presence of pagetoid upward migration beyond the area of scar in the dermis is almost always diagnostic of melanoma.

Non-melanocytic skin lesions can also mimic superficial spreading melanoma. Merkel cell carcinomas can be easily confused with melanoma, especially in superficial shave biopsies, since they can show an intraepidermal growth with marked pagetoid upward spread and forming small nests. If the tissue sample allows inspection of the dermis, it will become obvious the characteristic round blue cell morphology of Merkel cell carcinoma. Immunohistochemistry will allow easy recognition of both of these entities (keratin and CK20 in Merkel cell carcinoma, both unlikely to be seen in melanoma). Paget and extrammamary Paget disease can also mimic superficial spreading melanoma. Both of these conditions form nests and single cells with pagetoid growth in the epidermis and thus can look almost identical to superficial spreading melanoma. Cells in Paget disease tend to be located above the dermal-epidermal junction (so-called “eyeliner” sign ). Paget cells express keratins, including CK7, and only rarely can they express S100 (other melanocytic markers are consistently negative in Paget disease). Examples of metastatic gastrointestinal, prostate, and lung carcinoma can occasionally invade the epidermis and thus can easily simulate melanoma. In general such lesions have widespread vascular invasion or glandular arrangement. Also, immunohistochemistry will allow a more specific distinction depending on the type of lesion. Epidermotropic metastatic carcinomas can mimic superficial spreading melanoma; in such cases, clinical-pathologic correlation will be essential (i.e., history of prior melanoma of similar morphology and located nearby).

One of the major challenges in dermatopathology is to differentiate acral junctional nevus versus ALM in situ. Making a diagnosis of invasive melanoma on acral locations is not difficult; however, the diagnosis of acral lentiginous melanoma in situ can be quite difficult as it can mimic junctional nevi.

The early stage of ALM (in situ component) is composed of single melanocytes that are irregularly distributed along the dermal-epidermal junction. Intraepidermal nests are often absent at this stage and some lesions can even grow and reach a large size without forming nests. As the lesion evolves, nests can be observed, and when noted they are irregular in size and shape, are elongated, and are located parallel to the epidermis. Such nests show lateral confluence and spindle cell configuration. A characteristic feature when nests are present is that they alternate with single melanocytes forming skipping areas (in some cases one can notice areas that lack melanocytes altogether). Melanocytes tend to be elongated, plump, with darkly pigmented nucleus, and a surrounding halo. Dendrites are commonly visible, highlighting the presence of migrating melanocytes into mid- and higher levels of the epidermis. These dendrites are irregular in thickness, have different lengths, and have a tendency to form a web around the basal cells. Melanocytes with ample and dusky cytoplasm are rare but pagetoid spreading is not uncommonly observed. These pagetoid spread is not as noticeable as in other anatomic locations, mainly because of the small size of the nucleus in the melanocytes. Also, it is common to observe a cleft (“zipper” sign) at the dermal-epidermal junction in areas where basal melanocytes overwhelm the number of basal keratinocytes. Pigment is present irregularly along the stratum corneum (as opposed to being in columns characteristic of acral nevi). Early examples of ALM can be identified by the presence of individual melanocytes crowding around the crista profundae intermedia, especially if melanocytes are also detected along the junction between the cristae. This stage can be a diagnostic challenge as melanocytes display only minimal cytologic atypia. It is not unusual to identify melanocytes that extend irregularly into the eccrine ducts. This extension into the eccrine duct differs from that of nevi, as in nevi one can see melanocytes gathered in and around ducts forming well-defined nests as in ALM melanocytes are irregularly distributed and in a single-cell pattern. In some cases of ALM, this syringotropic spread of malignant melanocytes can reach the deep portion of the duct, and thus melanoma in situ may be present at the deep margin. The epidermis is usually acanthotic, hyperplastic, and hyperkeratotic. At this in situ stage, there may be a dermal lymphocytic infiltrate, a feature usually absent in acral nevi).

Experts in the topic have proposed that ALM in situ should be considered a clinicopathologic entity [112]. These authors have proposed that when a classic clinical picture of ALM is seen, e.g., irregularly shaped, darkly pigmented, flat lesion with a diameter >7 mm, pathologists should strongly suggest the diagnosis of ALM in situ if the histology shows a proliferation of single melanocytes along the dermal-epidermal junction.

Usually after years, the lesion, once limited to the epidermis, becomes infiltrative into the dermis. The overlying epidermis still shows the atypical lentiginous pattern and at this stage is more common to observe a nested component. The nests are irregular in size and shape and have a confluent pattern of growth and areas of epidermal “consumption.” The dermal component can show either spindle or epithelioid melanocytes (more commonly spindle). The atypical features become more obvious, and a clear diagnosis of melanoma can be easily made. When ALM is already invading in the dermis, the overlying epidermal component can sometimes be limited to an inconspicuous lentiginous melanocytic proliferation as seen in the very early stage of ALM in situ (macular, in situ component of the invasive neoplasm).

Acral skin has peculiar anatomic aspects that are important to keep in mind when evaluating acral melanocytic lesions. Rather than flat surface, it is composed of ridges and furrows, as seen in fingerprints. Ridges are wider than furrows and at their center there is an opening of the eccrine ducts. This particular anatomic feature of the acral skin often causes nevi in these locations to adopt a striped arrangement; thus, this is very important to evaluate when inspecting acral nevi. It has been recommended to cut the microscopic sections of any melanocytic lesion on acral skin perpendicular to the furrow pattern, to allow a better appreciation of the symmetry and circumscription of the lesion. The pattern most commonly seen in acral nevi is that of individual melanocytes concentrating at the base of the furrows and sparing the areas at the base of the ridge around the acrosyringium.

The clinical history is important when evaluating acral melanocytic lesions; acral melanomas are almost always seen in older patients, whereas acral melanocytic lesions in younger patients are almost always benign nevi. The early phase of ALM in situ can be very difficult to distinguish from acral nevus, especially in small samples. When dealing with small, pigmented lesions on acral locations that are clinically suspicious for ALM, clinicians should consider an excisional biopsy with a narrow margin. ALM in situ tends to have a lentiginous growth pattern with rare nest formation (seen in more advanced stages) with melanocytes that are angulated and with hyperchromatic nuclei. When junctional nests are seen, they are irregular in shape and oriented parallel to the epidermis. In acral nevi, melanocytic nests appear at early stage, have well-defined borders, and are uniform in size and shape. As mentioned above, in general, large acral melanocytic lesions with a wide lentiginous intraepidermal component without nest formation is a diagnostic clue of ALM in situ. On the other hand, small acral melanocytic lesions with predominance of symmetrically distributed nests are overwhelmingly nevi. Dendritic morphology is also important in the evaluation of acral lesions. In ALM in situ, the dendrites are thick and numerous as opposed to acral nevus in which they are rarely seen. Another important clue to inspect is the distribution of pigment in the stratum corneum. In acral nevus, the pigment is commonly present in the stratum corneum arranged in columns rather that randomly through the entire width of the lesion (as it is commonly seen in melanoma).

A lesion that needs to be separated from ALM is “MANIAC” nevus (melanocytic acral nevus with intraepidermal ascent of cells) . In these acral nevi, there is a tendency of melanocytes to be scattered throughout the entire thickness of the epidermis, giving the impression of pagetoid spreading, even at the periphery of the lesion. MANIAC nevi are usually seen in younger patients.

Recent studies have postulated that DNA repair mechanisms and cell growth pathways are involved in the development of ALM, particularly changes in the MAPK pathways. A recent study assessed the status of the MAP kinase pathways in the pathogenesis of ALM by analyzing the components of the RAS-RAF-MEK-ERK cascades by immunohistochemistry in tissue microarrays [113]. In that study the authors showed that more than half of cases were BRAF positive and most cases were also positive for MEK2, ERK1, and ERK2. RAS was not expressed in most cases and all cases were negative for MEK1. The combination of absence of RAS and expression of MEK2, ERK1, and ERK2 was most seen in invasive cases with high Breslow thickness.

Using comparative genomic hybridization (CGH) , Bastian et al. compared acral melanomas and superficial spreading melanoma, demonstrating that all acral melanomas had at least one gene amplification, significantly more than superficial spreading melanoma [114]. The most frequently amplified regions in acral melanomas occurred at bands 11q13 (47 %) and 22q11–22q13 (40 %). These results suggested that ALM is a distinct type of melanoma characterized by focused gene amplifications occurring early in tumorigenesis. A different study analyzed the frequencies of regional changes in the number of copies of DNA and mutation frequencies in BRAF groups of different melanoma groups (melanomas from the skin with chronic sun-induced damage, melanomas from the skin without such damage, acral melanomas, and mucosal melanomas) [38]. In this study, melanomas arising on the skin with signs of chronic sun-induced damage and skin without sun damage could be correctly classified with 84 % accuracy, and acral melanoma could be distinguished from mucosal melanoma with almost 90 % accuracy. Most melanomas on the skin without chronic sun-induced damage had mutations in BRAF or NRAS, and the majority of melanomas in the other groups had mutations in neither gene. These results suggest that the genetic alterations identified in melanomas at different sites and with different levels of sun exposure indicate that there are distinct genetic pathways in the development of melanoma and implicate CDK4 and CCND1 as independent oncogenes in melanomas without mutations in BRAF or NRAS.