Guidelines for diagnosis and pathological report of melanocytic skin lesions ― recommendations from the Brazilian Society of Pathology

Abstract

Melanoma is an aggressive form of skin cancer. Its histopathological diagnosis may be challenging and subject to significant inter- and intraobserver variability. It is also a disproportionate source of pathology malpractice lawsuits worldwide. The knowledge base on melanocytic lesions is rapidly expanding, with many recent novel classification systems, therapies and molecular targets. Hence, specimens diagnosed or suspected as melanocytic lesions should be carefully and appropriately sampled, fixed, processed and analyzed to achieve the best possible patient treatment, follow-up and counseling. Herein, we describe common important dermoscopic terms and findings, standard biopsy procedures, preanalytical procedures, microscopic criteria, useful immunohistochemical markers, predictive and diagnostic molecular findings and other ancillary tests related to melanoma diagnosis, therapy and prognostication. This article is part of a larger project from the Brazilian Society of Pathology that proposes best practice recommendations for pathologists and clinicians in different fields.

Background

Melanoma is an aggressive form of skin cancer, and its histopathological diagnosis can be challenging. The knowledge base on melanocytic lesions is rapidly expanding, with new classifications, treatments, and molecular targets emerging. This requires a careful approach in the collection, fixation, and analysis of samples to ensure the best possible treatment for the patient. This article reviews dermoscopic terms, biopsy procedures, immunohistochemical markers, and molecular tests useful for the diagnosis and prognosis of melanoma to guide pathologists while a similar paper with focus on clinicians and surgeons’ orientation will be published in Anais Brasileiros de Dermatologia. It is part of a project from the Brazilian Society of Pathology, which proposes best practices for pathologists and clinicians, aiming to improve diagnostic accuracy and clinical management of melanoma patients.

Introduction

Cutaneous melanoma (CM) is the most lethal form of skin cancer. Although it represents less than 20% of skin cancers, it causes approximately 50% of skin cancer-related deaths (Sung et al. 2021) and its burden is expected to grow in the next two decades (Arnold et al. 2022). Patients presenting with a clinically suspicious pigmented skin lesion should undergo an adequate excision to establish a definitive diagnosis (Swetter et al. 2021). The identification of risk factors and the early diagnosis should prompt immediate and appropriate referral to specialized care, which results in better outcomes for melanoma patients (Ministério da Saúde 2022). If melanoma is detected at an early stage, accurately diagnosed and appropriately treated, it is associated with an excellent prognosis (Scolyer et al. 2020). The standardization of all the steps, from sample collection, preparation, and analysis to the reporting of melanocytic tumors is essential for patient management and follow-up (Barnhill et al. 2023).

The histopathological diagnosis of melanoma is complex and subject to significant inter- and intraobserver discrepancies. This is also true for the reporting of several important histological parameters of the lesion. To overcome some of these limitations while also aiming at better pathologist-clinician communication, classification schemes have been proposed, of which MPATH-Dx, now in its v2.0, is the most widely known (Barnhill et al. 2023). Moreover, there are a disproportionate number and high costs of malpractice suits related to the diagnosis of melanoma (Troxel 2005).

The Brazilian Society of Pathology (SBP) decided to establish a working group to analyze scientific literature and recommendations from other international medical societies. The results of this effort are presented here, with the goal of assisting pathologists and other professionals involved in the diagnosis and care of melanoma patients. This paper intends to address all this information while also considering the limitations and specificities of a large low-to-mid-income developing country. We sought to describe common important dermoscopic terms and findings, standard biopsy procedures, preanalytical procedures, microscopic criteria, useful immunohistochemical markers, predictive and diagnostic molecular findings and other ancillary tests related to melanoma diagnosis, therapy and prognostication. This article is part of a larger project from the Brazilian Society of Pathology that proposes best practice recommendations for pathologists and clinicians in different fields. All authors were voluntary, with no financial or other conflicts of interest to declare. There is a bullet-point free version of this guideline available on the website of the Brazilian Society of Pathology:<https://www.sbp.org.br.>

Methods

A group of pathologists with special interest in melanocytic lesions, all members of the Brazilian Society of Pathology, independently reviewed the published literature. We searched for articles indexed in the PubMed database, international guidelines, Brazilian legislation, and textbooks published until August 2024. All the authors were responsible for reviewing the text, which was shared and discussed in online meetings during the years 2022, 2023 and 2024. Only consensual recommendations were included in this final of version of the guideline.

Results

Most common and significant dermoscopic findings

Pigmented lesions are often clinically analyzed via simple ABCDE rule (Asymmetry, Borders, Color, Diameter and Evolution) (Majem et al. 2021). Dermoscopy is a noninvasive technique that allows for the visualization of subsurface skin structures that are otherwise invisible to the naked eye. Dermoscopy greatly enhances the diagnostic accuracy of clinical examination and is widely considered a standard procedure in dermatological consultations because of its ease of application and relatively low cost (Swetter et al. 2019; Williams et al. 2021). Any lesion classified as suspicious or malignant melanocytic must be adequately sampled, analyzed and reported, according to applicable protocols (Marghoob et al. 2013; Michielin et al. 2019).

The most common dermoscopic findings in benign melanocytic lesions are as follows:

• Homogeneous pattern: preferably a monochromatic area or an area with more than one color that is symmetrically distributed. This area should be devoid of basic dermoscopic elements, or these elements should be too scarce to form a distinct pattern.

• Regular globular pattern (when predominant - includes cobblestone): commonly observed in congenital nevi, it indicates the presence of regularly distributed intradermal melanocytic nests of uniform size and shape. The nests may be larger and more compacted, resembling the mosaic fit of stones in Portuguese sidewalks (cobblestone pattern).

• Regular pigment network: a network of equidistant lines with uniform thickness and color formed by hyperpigmentation of epidermal ridges. They are often related to the presence of junctional melanocytic nests. The normopigmented areas, delimited by the lines, correspond to the suprapapillary epidermis, which is devoid of melanocytic nests. Typically, found in acquired common melanocytic nevi.

• The parallel lines in the grooves (in acral melanocytic lesions): indicate melanocytic proliferation in the deep ridges of the epidermis, which projects as pigmented columns toward the dermatoglyphic grooves (Fig. 1) (Yélamos et al. 2019).

Fig. 1

Microscopic features (H&E) of acral skin, correlating histological and dermatoscopic structures with the clinical appearance of pigmentation and melanocyte distribution. In macular benign melanocytic lesions, melanocytes are distributed along the surface furrows of acral skin (a), forming parallel lines that correspond histologically to the limitans ridges (b). In incipient acral lentiginous melanoma, melanocytic proliferation is more pronounced in the acrosyringium (c)

Dermoscopic findings suggestive of malignancy in melanocytic lesions, which should warn the pathologist of the possible need to carry out additional studies and/or deeper sections are as follows:

• Irregular pigmented network: a heterogeneous network of lines formed by epidermal ridges varying in distance, thickness and color. It is usually the result of disordered intraepidermal melanocytic proliferation.

• Globules of varying sizes and irregularly distributed: this indicates the presence of intradermal melanocytic nests with different dimensions and shapes, which are arranged in a disordered manner.

• Radiated striae: focally and asymmetrically distributed radial extensions of a pigment network, often corresponding to melanoma in the radial growth phase.

• Pseudopods: peripheral bulbous projections of a pigment network, often indicating melanoma in the radial growth phase.

• Blue-gray veil: homogeneous pattern formed by a partial area of bluish color with matte white superimposed, frequently related to areas of inflammation and/or regression. It has high specificity for melanoma, which is usually invasive.

• Parallel line on the ridges (acral melanocytic lesions): this indicates melanocytic proliferation in the suprapapillary epidermis, which projects in pigmented columns toward the dermatoglyphic ridges. As a rule, pigmented lines are devoid of white dots, corresponding to destruction of the openings of the eccrine ducts by melanoma (Fig. 2) (Ankad et al. 2021; Yélamos et al. 2019).

Fig. 2

Macroscopic and microscopic features of suspicious melanocytic lesions in the acral area. A Macroscopic appearance of a melanocytic lesion in the acral region. B Perpendicular macroscopic section through the surface lines of acral skin showing diffuse pigmentation of the epidermal surface ridges. C Histological feature (H&E) of acral skin, with a malignant melanocytic proliferation in the intermediate ridges and corresponding clinical appearance of suspected malignancy with pigmented ridges

Sampling procedure

The provision of an appropriate sample and pertinent clinical history are keys to the accurate diagnosis and prognostication of melanoma (Scolyer et al. 2020).

The biopsy techniques for a suspicious melanocytic/pigmented lesion include the following:

• Excisional biopsy with narrow margins: it is the preferred approach for most lesions.

• Incisional biopsy: it should be avoided in lesions in which melanoma is suspected. However, it may be appropriate as a first approach for larger lesions or lesions located in regions of high aesthetic and/or functional concern.

• Shave biopsies are strongly discouraged as they may impair the histological diagnosis and compromise the determination of the Breslow thickness (Grupo Brasileiro de Melanoma 2023; Nagarajan et al. 2024; Swetter et al. 2021).

The sample obtained for histopathological examination must be submerged immediately after removal in a transparent container with a wide mouth containing buffered formalin (10% buffered formaldehyde) in a volume equivalent to approximately 10 times the sample size. The ideal fixation time is between 6 and 72 h, depending on the specimen size (Lott et al. 2023). Larger specimens should be sent to the anatomic pathology laboratory and macroscopically examined as soon as possible to avoid underfixation/autolysis in the center of the specimen. The formalin solution penetrates tissues at slower rates with increasing tissue thickness. Up to 1.5 cm, it may be irrelevant but becomes very important from 2.0 cm up (Hewlett 2002).

Request for histopathological examination

Importantly, the request contains a summary description of the clinical condition, its evolution, the results of ancillary tests, and diagnostic hypotheses, as they are pivotal for a complete and contextualized report. In addition to being legally required identifiers, which must be present on the request and on the specimen container label (Anvisa RDC nº 786, 2023), the patient’s gender, age and location of the lesion are required for the appropriate pathological analysis (Garbe et al. 2020; Garbe et al. 2022; Scolyer et al. 2020; Slater and Cook, 2019, Pai 2024).

Among the main data are as follows:

• Age: newly appearing melanocytic lesions in people over 40 years of age should be considered suspicious for melanoma. On the other hand, the same microscopic findings that would lead to the diagnosis of melanoma in an elderly patient may not be sufficient evidence of malignancy in a pediatric patient.

• Location: benign melanocytic lesions located in so-called “special sites” (e.g.: scalp, anogential, breast line, ear pinna, palms, soles and flexural regions) may present alarming microscopic findings, which may lead to overdiagnosis of melanoma.

• Clinical and dermoscopic description of the lesion, as well as any other relevant information (occurrence of trauma, results of previous biopsies etc.) or complementary exams (e.g.: confocal microscopy) (Ferrara et al. 2009; Scolyer et al. 2019).

• Clinical history: personal and family history of melanoma and dysplastic nevi.

Preanalytical laboratory assessment

Promoting standardization and quality assurance of macroscopic examination in pathology is key for adequate patient care. To achieve this, it is important to follow these steps:

• Macroscopic description: a precise description of the surgical specimen allows the macroscopic characteristics to be correlated with the microscopic findings of the lesion (Fig. 3). The measurement of the surgical specimen and the macroscopic characteristics of the lesion must be described, such as color, relief, circumscription, size and distance to the margins. In the case of more than one lesion, these must be described separately and the distances between them must be measured.

• Sectioning: it is recommended to section the surgical specimen only after complete fixation in order to allow better macroscopic cuts. Preferably, sectioning should be carried out through cuts perpendicular to the epidermal plane, with uniform thicknesses, ideally 2–3 mm, along the longest specimen axis (Fig. 4). In cases of an acral melanocytic lesion, it is necessary to cut the specimen perpendicularly to the dermatoglyphic lines to allow adequate visualization of the microscopic pattern of melanocytic proliferation, which is an especially important diagnostic criterion in palmo-plantar lesions (Fig. 5).

• Sampling: all excisional samples should be preferentially submitted in their entirety. For larger specimens, at least the whole macroscopic lesion should be submitted, along with representative sections of the margins. Re-excision specimens are also preferentially submitted in their entirety. Nevertheless, larger specimens may be carefully examined, and the extent of the sampling can be judged on a case-by-case basis, with special attention given to any pigmented areas and any changes close to the scar. The number of fragments in each cassette should be limited to enable proper embedding, with all samples flat and within the same plane (Lott et al. 2023). In cases with more than one lesion, it is imperative to identify each lesion and represent the normal-appearing skin between the lesions (Fig. 6) (de Waal et al. 2014; Fives and Heffron 2018; Slater and Cook 2019).

• Embedding and microtomy: the paraffin-block embedding must allow distribution on the slide without fragment overlap and provide complete visualization of the epidermis and ink on the surgical margins. In the case of very small lesions not identified with serial cuts, reinclusion with inversion of the cut side of the paraffin block may be necessary. Routine HE sectioning should be carried out at 4–5 microns. Additional serial sections may be needed in selected cases, including atypical lesions, lesions with some diagnostic difficulty, discordance with clinical hypotheses or exams (dermoscopy and confocal microscopy) or to improve determination of the lesion’s characteristics (Stuart et al. 2014).

Fig. 3

Schematic figure correlating macroscopic appearance (A) with microscopic correspondence (B-F) of a melanocytic lesion. A Macroscopic appearance showing a flat area and a raised area. Different shades of color are observed in the flat area. B Raised area characterized by melanoma with a vertical tumorigenic growth pattern. C Whitish coloration indicates an area of regression/fibrosis in microscopy. D Brown areas at the interface between the dermis and epidermis. E Blackish coloration indicates more superficial pigmentation. F Grayish coloration indicates a deeper lesion in the dermis

Fig. 4

Excisional specimen of a melanocytic skin lesion with a representative diagram of the recommended macroscopic approach for the histological representation of melanoma. It is recommended to perform serial perpendicular sections to the surface, along the longest axis of the specimen, and to represent the lesion in its entirety for microscopic evaluation

Fig. 5

Guidance on sections to be performed in the macroscopic evaluation of acral melanocytic lesions. A It is not recommended to perform cuts parallel/longitudinal to the skin furrows. B Recommended direction for macroscopic cuts, following a perpendicular orientation to the furrows

Fig. 6

Macroscopy and histological representation of melanoma margin expansion specimens. A Macroscopic appearance of skin with expanded margins from a previous melanoma excision, showing a central scar. B-C It is recommended to perform serial perpendicular sections to the surface, along the longest axis of the specimen, and to represent the scar in its entirety for microscopic evaluation

Microscopic examination

The microscopic examination of melanoma includes diagnostic features as well as histological characteristics that may impact the prognosis and management of the patient.

The main microscopic architectural criteria applied to the diagnosis and staging/prognosis of melanocytic lesions include but are not limited to the following:

• Circumscription: benign lesions are usually well-circumscribed. However, some malignant melanocytic lesions, including nodular melanoma, epidermotropic metastatic melanoma and spitzoid lesions may be very well circumscribed. Well-circumscribed lesions usually show junctional cell nests on the lateral limits of the lesion, as opposed to a lentiginous pattern (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Symmetry: architectural similarity between the two vertical halves of the lesion and between cells located at the same horizontal level is indicative of a benign lesion (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Proliferation pattern: in most benign melanocytic lesions, the nests are distributed regularly and have similar dimensions and shapes (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Reaction pattern of the epidermis: uniform epidermal hyperplasia, which is more evident in the central part, commonly occurs in benign melanocytic lesions, whereas alternating hyperplasia and consumption of the epidermis are features that are usually related to melanoma (Harvey and Wood 2019; Massi and LeBoit 2021).

• Pagetoid dissemination: diffuse and extensive ascension of atypical cells through the epidermis and extension into the adnexal epithelium, beyond the isthmus level, are typical of melanoma. Some benign melanocytic lesions may present regular intraepidermal dissemination of monomorphic cells or nests, with lower intensity or restricted to the center of the lesion. It is especially common in the acral regions of young individuals, Spitz tumors or common acquired nevi subjected to trauma or inflammation. The predominance of single cells with or without extensive pagetoid spread is more common in melanoma (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Maturation: a decrease in the size of the nests and of the melanocytes toward the deeper part of the lesion favors a benign melanocytic lesion. This finding can also be found in some malignant lesions such as nevoid melanoma and metastatic melanoma (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Mitoses: typical, scarce, nongrouped mitotic figures restricted to the superficial part of the lesion frequently occur in benign melanocytic lesions. Increased numbers of mitotic figures present in the deeper dermis can be found in benign melanocytic lesions in children, adolescents and pregnant women, but they should raise suspicion of melanoma, especially if it is associated with atypical and grouped mitoses. There is no consensus on a specific threshold for mitotic counts in this differentiation. Although the mitotic count is not part of the current melanoma staging criteria, it is still indicative of prognosis and behavior and should be reported (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Pigmentation: alternating intensely pigmented areas with areas devoid of pigment are suspicious for melanoma. Scanty, homogeneously distributed pigment concentrated in the superficial part is typical of most benign melanocytic lesions (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Ulceration: when there is consumption of the epidermis surrounding the ulceration and there are no clear signs of trauma, the presence of ulceration is indicative of melanoma. It is part of melanoma staging, heralding a worse prognosis (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Dermal desmoplasia: its presence is more common in invasive malignant lesions (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Degree of solar elastosis: the appearance of a new melanocytic lesion in an area with significant photodamage, especially in elderly individuals, strongly suggests melanoma, even if other malignant-related findings are absent.

• Inflammatory infiltrate: melanoma more often presents with a lymphocytic infiltrate irregularly distributed throughout the lesion (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Regression: it includes the replacement of dermal tumor cells by inflammation. It is also used to indicate attenuation of the epidermis and dermal fibrosis with inflammation, pigment incontinence and telangiectasia. It is important that regression be assessed using reproducible criteria to ensure consistent identification and clinical management. How regression can influence patient prognosis remains under debate. Areas of regression on the surgical margins may prompt re-excision evaluation (Aivazian 2023; Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

• Satellites: they are defined as nodules of melanoma cells separated by normal-appearing dermis or subcutis. It is very important to exclude areas of regression within the lesion (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

The main cytological criteria applied to the diagnosis of melanocytic lesions include the following:

• Nuclear atypia: a low nuclear: cytoplasmic ratio, angulated nuclear contours, hyperchromasia, and the presence of macronucleoli are some of the worrisome features. According to the WHO classification, the degree of dysplasia in nevi is associated with nuclear size, with mild dysplasia showing melanocyte nuclei up to the size of basal keratinocyte nuclei, moderate dysplasia showing nuclei 1 to 1.5 times the size of basal keratinocyte nuclei, and severe dysplasia showing nuclei 1.5 times or more the size of basal keratinocyte nuclei (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023). Low-grade atypia connotes nevi (or other lesions) previously graded as mild and moderate (not all), and high-grade atypia nevi (or other lesions) previously graded as moderate (not all) and severe. Degree of atypia is defined by both architectural disorder and cytological atypia (Barnhill et al. 2023).

• Pleomorphism: cells in the same level of the epidermis or dermis that are very different in size and shape are not usually observed in benign lesions. Some malignant lesions may show very bland cytology with little or no pleomorphism (nevoid melanoma, for example) (Massi and LeBoit 2021; WHO Classification of Tumours Editorial Board 2023).

Importantly, none of the abovementioned findings can be used as an isolated criterion for the diagnosis of melanoma. The final diagnosis of malignancy is a result of expert training and a combination of all histological findings, which are often associated with clinical and molecular characteristics.

For a guide to structured reports, see the Manual for the Standardization of Histopathological Reports of the Brazilian Society of Pathology: https://www.sbp.org.br/manual-de-laudos-histopatologicos/pele-melanoma-cutaneo/ and other similar protocols from other institutions (CAP/ICCR/RCP/RCPA).

Immunohistochemical examination

Immunohistochemical stains may be used to confirm the melanocytic nature of the lesion, or they may contribute to the differentiation between benign and malignant lesions (Ferringer 2015). There is currently no marker that can be used in isolation for either end.

Markers used for the differential diagnosis between nevi and melanoma:

• PRAME (PReferentially expressed Antigen in MElanoma) is a recent and efficient marker for distinguishing benign nevi from melanomas (Lezcano et al., 2020). Its sensitivity varies consistently with many pitfalls (Turner et al. 2024). A smaller percentage of spindle cell and desmoplastic melanomas express PRAME (approximately 35%). Care must be taken with rare acral benign lesions and Spitzoid lesions, which may express diffuse PRAME staining. For the interpretation of PRAME as positive, more than 75% of the cells must exhibit strong nuclear staining. Weak staining, even if extensive, is not considered positive. A larger subset of benign lesions may exhibit nondiffuse PRAME expression. It is not a lineage-specific melanoma marker, as many other tumor types express the antigen.

• p16 loss is very rare in benign melanocytic lesions. It is also associated with thicker melanomas and a higher mitotic index. Some authors have suggested its use as part of a panel to help distinguish benign and malignant lesions, together with Ki-67 and Melan-A, with a sensitivity of 97.4% and a specificity of 97.3% (Uguen 2015). Since there are many clones in the market, internal validation is recommended.

• HMB-45 (Human Melanoma Black-45) benign lesions typically show stronger staining in the upper portions and weaker/negative staining in the deeper dermis. Its usefulness is restricted to thick lesions. It can also be diffusely positive in blue nevi, deep penetrating nevi and Spitz nevi. HMB45 is also useful in differentiating between nevus and subcortical metastases in sentinel lymph node evaluation.

• Double MART1/ki67 labeling is very effective in facilitating interpretation of the Ki67 index. Nevi in adult patients generally have a low rate of cell proliferation without proliferating cells in the deeper dermis.

Lineage-specific melanoma markers

Some melanocytic markers are very sensitive (e.g.: MITF, SOX10 and S100), whereas others are more specific (e.g.: MART1) (Saleem et al. 2022). These methods may also be used to detect cell density and neoplastic growth patterns when they are not possible with routine HE staining (due to the coexistence of inflammation, artifacts, etc.). However, they do not determine whether the lesion is malignant or benign: MART-1 (Melanoma Antigen Recognized by T cells-1), MiTF (Microphthalmia Transcription Factor), and SOX-10 (SRY-related HMG-bOX-10) can be negative in desmoplastic melanomas in contrast to S100 which is the most sensitive antibody in these cases. Importantly, other cell types may express those antigens, especially neural tumors. MART-1 is frequently expressed by PEComas and related lesions.

Melanin may hinder the interpretation of immunohistochemistry. Special techniques for very pigmented cases may be used, including counterstaining with magenta or Giemsa (which turns the melanin pigment black, leaving the immunohistochemical pigment tan-brown) and depigmentation with hydrogen peroxide (Petersen et al. 2018; Salvio et al. 2007).

Preoperative examination (frozen section)

Considering that intraoperative examination is not recommended, any type of frozen section analysis, including Mohs micrographic surgery, should be avoided for defining margins or diagnosing a primary melanocytic skin lesion or sentinel lymph node. The gold standard for assessing surgical margins is microscopic examination of hematoxylin and eosin-stained slides after proper fixation and histological processing (Nagarajan et al., 2024).

Nonetheless, some techniques have been reported for certain special conditions, such as pT1a melanomas on the face, ears, or acral sites, along with other surgical methods that provide comprehensive histologic assessment, such as staged excision or PDEMA (Swetter et al. 2024). In these setting, both Mohs micrographic surgery and staged excision have demonstrated high local control rates in some studies (de Vries et al. 2016; Hou et al. 2015; Walling et al. 2007). If any of these methods are performed, permanent section analysis of the central debulking specimen is strongly recommended to provide complete staging information.

These techniques are highly operator-dependent and not widely available in Brazil. The distinction between neoplastic and nonneoplastic melanocytes in frozen sections may be very difficult or impossible, especially in sun-exposed skin areas. Care should be taken, and precise communication between pathologists, surgeons and patients must be put in place before the technique can be employed. There is no validated technology clinically available for widespread use for preoperative rapid immunohistochemistry.

Sentinel lymph node

Melanoma treatment is a rapidly evolving field, and the role of sentinel nodes has been debated for certain clinical scenarios, but it remains an important tool for disease staging and prognosis. Therefore, proper handling, grossing and microscopic evaluation of the sentinel node is mandatory for guiding proper care for patients with melanoma.

The careful handling of sentinel lymph nodes is recommended to avoid damaging the lymph node capsule. The lymph node should be measured, sectioned and submitted in its entirety for histological processing and, later, microscopic evaluation by a pathologist. LN slicing is more commonly carried out perpendicularly to the largest axis (breadloafing) of the specimens, allowing better examination of the subcapsular region, although protocols using the bivalved technique have also been described and might be used. Many protocols have described how many levels should be sectioned from sentinel node paraffin blocks. Protocols include various numbers of cutting steps, including upfront sectioning of H&E and different levels of additional cuts for different immunohistochemical reactions. There is no single best protocol that should be utilized. The rationale is that the SLN should be evaluated microscopically in great detail to identify any malignant cells. As more levels are analyzed, the detection rate increases. A balance of cost, time and optimal evaluation protocol should be implemented at each institution. This group recommends that the entire lymph node must be sectioned, with tissue blocks cut into 2 mm thick levels, processed and stained with H&E, and at least three additional unstained slides included for potential immunohistochemistry staining, including markers with great sensitivity for melanoma, such as PRAME and SOX-10, be obtained. Melan-A may be used to confirm the melanocytic lineage, if in doubt (Fig. 7) (Cheng et al. 2023; Cook et al. 2019; Prieto 2017; Wong et al. 2018).

Fig. 7

Representative diagram of serial sections to be performed on the sentinel lymph node for complete microscopic evaluation. Cases negative on routine hematoxylin and eosin staining should be further evaluated by immunohistochemistry

Several parameters have been reported for microscopic evaluation of the tumor burden in the sentinel node of a melanoma patient, including the microanatomic location, diameter of the deposit, depth of the tumor, surface area and surface area ratio, volume, size/ulceration, and cell count (Cheng et al. 2023). The minimal report should include the two dimensions of the largest deposit and the presence/absence of extranodal extension.

If more than one lymph node is found, all the lymph nodes should be submitted for analysis per the same sentinel protocols. Their identification and descriptions must be kept separate.

Lymph node evaluation after adjuvant therapy

Neoadjuvant treatment with targeted or immune therapy for surgically resectable metastases has played a great role in melanoma patient care in recent years as several trials have shown its benefit. The degree of pathological response after neoadjuvant therapy is a major outcome in clinical trials and is also utilized to personalize subsequent treatment (Amaria et al. 2018, 2022; Blank et al. 2024; Patel et al. 2023; Reijers et al. 2022). The international melanoma neoadjuvant consortium has published guidelines to establish uniform practices and well-established criteria for assessing pathological tumor response in the neoadjuvant setting of melanoma (Amaria et al. 2019; Tetzlaff et al. 2018). The post-treatment lymph node should be measured, sectioned and submitted for histological processing and, subsequently, microscopic evaluation by a pathologist to assess the degree of response. If the lymph node is smaller than five centimeters (5 cm) it should be submitted in its entirety, and if it is larger than 5 cm, one slice of 2 mm thickness per centimeter should be represented for histological processing.

The pathological response is divided into 4 categories that should be stated in the final pathology report:

• Pathologic complete response (pCR): no viable tumor in the treatment area (TA).

• Major PR/near pCR: less than 10% viable tumor in the TA.

• Partial pathologic response (pPR): less than 50% viable tumor in the TA.

• Pathological nonresponse (pNR): more than 50% viable tumors in the TA.

Molecular changes

Molecular alterations in melanoma may be studied in the germline or somatic setting. They may be genetic as well as epigenetic. The most common somatic genetic alterations in melanoma are well studied and might be applied for proper classification/diagnosis of the lesions, prognosis and treatment (predictive) purposes (Fig. 8) (Hosler and Murphy 2023).

Fig. 8

Representative schema of predictive biomarkers in melanoma with summary information on frequency and recommended treatment as per current NCCN guideline

Melanomas are currently classified into three major groups and further into nine subgroups on the basis of epidemiological, clinical, pathological, and genomic findings. Each group has a specific or a combination of molecular changes that can assist in the correct classification of the lesion and potentially be applied to support therapeutic decisions. The three main categories are as folows: (1) lesions associated with low or moderate chronic sun damage (CSD), (2) high CSD and (3) lesions not caused by ultraviolet radiation (Cancer Genome Atlas Network 2015; WHO Classification of Tumours Editorial Board 2023).

Some biomarkers are currently of greater importance for the purpose of melanoma patient treatment selection. BRAF is a serine threonine kinase that, when mutated, is able to activate the mitogen-activated kinase (MAP-K) pathway. Treatment with BRAF inhibitor molecules such as dabrafenib, vemurafenib or encorafenib administered as a single agent or combined treatment might be used in different melanoma disease stages and might also include neoadjuvant or adjuvant settings. Mutations in the BRAF gene are present in approximately 50% of melanomas. The most common mutation in the BRAF gene involves codon 600, with the V600E mutation being the most frequent. Importantly, BRAF mutation does not define malignancy, as nevi also frequently present a mutation in this gene (Curtin et al. 2005; Davies et al. 2002; Flaherty et al. 2010; Long et al. 2018; Swetter et al. 2024).

BRAF mutations can be tested via many different techniques. Currently, the most commonly used techniques in routine clinical laboratories are real-time polymerase reactions (RT‒PCR) for hotspot mutation detection, next-generation sequencing (NGS) and immunohistochemistry with the VE-1 antibody. NGS is able to detect alterations in any part of the BRAF gene and might also be used to sequence a panel of different genes and to report other biomarkers such as tumor mutation burden (TMB). Therefore, NGS optimizes the usage of the tumor FFPE material available for testing (Long et al. 2013; Mosele et al. 2024).

NRAS is a GTPase that activates the MAPK signaling pathway, leading to cell growth and proliferation. Mutations in NRAS have been described in 15% of melanomas and might be used to select patients for MEK inhibitor treatments (Curtin et al. 2005; Dummer et al. 2017).

Mutations in the KIT gene are found in approximately 10–15% of melanomas, especially acral lesions, and might be used to select patients for KIT inhibitor therapy such as imatinib, sunitinib and nilotinib (Curtin et al. 2006; Guo et al. 2017; Hodi et al. 2013).

Fusion genes such as ALK, NTRK1/2/3, ROS1 and BRAF are helpful in characterizing Classical Spitz Nevus and Atypical Spitz Tumor and might be found very rarely in melanoma. Fusions can be detected by techniques such as FISH (fluorescence in situ hybridization) and NGS (next-generation sequencing). Tyrosine kinase inhibitors for ALK (crizotinib), NTRK1/2/3 (larotrectinib or entrectinib), ROS1 (crizotinib or entrectinib) or BRAF (trametinib) have a well-described clinical utility for some tumors and might be useful in the melanoma setting (Drilon et al. 2017).

Importantly, similar to any other tumor, adequate fixation of the sample with buffered formalin, strict control of preanalytical laboratory processes and sufficient representation of tumor cells are required to perform a molecular test (Assis 2020).

Discussion and conclusions

Melanoma is a very challenging disease to diagnose, classify and correctly stage due to the diversity of clinical scenarios, microscopic pictures and molecular markers available. This review carried out by members of the Brazilian Society of Pathology, gathered information on the basis of the most recent scientific evidence on melanocytic lesions to propose recommendations for pathologists and clinicians regarding topics such as dermoscopy, sample collection, grossing, histopathological diagnosis, lymph node management and molecular biomarker testing.

One of the most important challenges that clinicians face is estimating the risk of metastasis and death for any cancer and establishing a proper treatment plan for each patient. In melanoma, these include recommendations related to the definitive management of the primary tumor site such as the width of excision margins and the role of sentinel lymph node (SLN) biopsy as well as recommendations for the type, frequency and duration of treatment and clinical-radiological follow-up (Scolyer et al. 2020).

When assessing primary cutaneous melanomas, pathologists should provide a report with sufficient information to accurately stage and a reliably estimate the prognosis (Scolyer et al. 2020). Provision of an appropriate biopsy and pertinent clinical history, careful sample handling, fixing, grossing, microscopic evaluation of primary lesions, lymph nodes and metastatic lesions and correct indication and assessment of biomarkers are keys to the accurate diagnosis, prognostication and treatment of melanoma (Scolyer et al. 2020). This work is in progress as advances in melanoma diagnosis, prognosis, and therapy continue (Swetter et al. 2021).

This manuscript highlights the role of pathologists in the diagnosis of melanoma by detailing the processes of sample handling, analysis and reporting.