**6. Histopathological examination**

Essentially all melanomas begin as a proliferation of melanocytes initially confined to the epidermis. Increasing cytologic atypia of melanocytes accompanies the aberrant architectural appearance of melanomas. After the period of intraepidermal proliferation, there is often invasion of the papillary dermis, primarily as single cells and small aggregates of cells. *Breslow* thickness (in mm) of melanoma is one of the most important factors determining prognosis and theraphy. Melanomas with prominent invasive components may display polypoid morphologies.

#### **6.1. Lentigomaligna/ Lentigomaligna Melanoma (LM/LMM)**

Lentigomaligna (known as *Hutchinson*'s melanotic freckle) which is the precursor lesion of LMM is characterized by reproduction of atypical melanocytes mainly present in the basal layer of the epidermis. Tumor cells contain polygonal-shaped, pleomorphic irregularly hyperchromatic, angulated nuclei (Figure 8). In approximately 85% of cases of LM, multi‐ nucleated melanocytes are seen in the basal layer. These cells are named as "starburst giant cell". The presence of atypical melanocytes in the hair follicles and sweat duct epithelium is a characteristic feature but sometimes it may lead to difficulties in evaluation of tumor thickness [109, 113, 114]. Also there is effacement of rete ridges [115]. Due to chronically actinic damage, epidermis is usually atrophic and solar elastosis is seen in the dermal layer [116]. The upper part of the dermis usually contains melanophages and lymphocytes to a lesser extent [114]. If the lesion progresses, pagetoid spread may be observed within the epidermis [109]. When invasion occurs into the dermis, spindled cells and tumor cell pigmentation can be seen [117].

#### **6.2. Superficial spreading melanoma**

Superficial spreading melanoma is also known as pagetoid melanoma which characterized by a proliferation of atypical melanocytes singly and in nests in all layers of the epidermis [114]. Atypical melanocytes sometimes show "buckshot scatter" within the epidermis (Figure 9) [113]. The large tumor cells contain dark, atypical nuclei and abundant, pale cytoplasm [118]. The epidermis may have normal or hyperplastic appearance [119]. There is a continuous spread of tumor growth from one rete ridge to another [114]. If the tumor progresses to vertical growth phase, microinvasive tumor which contains nested and dispersed cells is seen within the dermis [109].

**Figure 9.** Superficial spreading melanoma: atypical melanocytes are scattered throughout the epidermis. Tumor cells compose cell groups at basal layer, The melanocytes have abundant eosinophilic cytoplasm and pleomorphic vesicular

**Figure 8.** Lentigo maligna: in this in situ lesion, tumor cells are hyperchromatic and distributed in a lentiginous pattern

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Nodular melanoma has no concomitant or preexisting radial growth phase [109]. It grows vertically from the beginning and thus may invade the epidermis [113, 114]. Cellular features include a large nucleoli and frequent mitosis [118]. Epidermal melanocytic proliferation is so minimal which typically extending less than three epidermal ridges on both sides of tumor

nuclei. Nucleoli are prominent (By the courtesy of Dr. Ahmet Cemil Kaur).

**6.3. Nodular melanoma**

(By the courtesy of Dr. Ahmet Cemil Kaur).

**Figure 8.** Lentigo maligna: in this in situ lesion, tumor cells are hyperchromatic and distributed in a lentiginous pattern (By the courtesy of Dr. Ahmet Cemil Kaur).

**Figure 9.** Superficial spreading melanoma: atypical melanocytes are scattered throughout the epidermis. Tumor cells compose cell groups at basal layer, The melanocytes have abundant eosinophilic cytoplasm and pleomorphic vesicular nuclei. Nucleoli are prominent (By the courtesy of Dr. Ahmet Cemil Kaur).

#### **6.3. Nodular melanoma**

dark brown to black); gray streaks surrounding the lesion (melanoma cell infarcts); red-brown globules irregular in size and color; polymorphic angiectatic base pattern and/or aneurysms; areas of polymorphic ectatic vessels running parallel to the skin surface; peripheral erythema (red corona); microscopic ovoid blood lakes; and homogeneous pattern (brown or blue to

Essentially all melanomas begin as a proliferation of melanocytes initially confined to the epidermis. Increasing cytologic atypia of melanocytes accompanies the aberrant architectural appearance of melanomas. After the period of intraepidermal proliferation, there is often invasion of the papillary dermis, primarily as single cells and small aggregates of cells. *Breslow* thickness (in mm) of melanoma is one of the most important factors determining prognosis and theraphy. Melanomas with prominent invasive components may display polypoid

Lentigomaligna (known as *Hutchinson*'s melanotic freckle) which is the precursor lesion of LMM is characterized by reproduction of atypical melanocytes mainly present in the basal layer of the epidermis. Tumor cells contain polygonal-shaped, pleomorphic irregularly hyperchromatic, angulated nuclei (Figure 8). In approximately 85% of cases of LM, multi‐ nucleated melanocytes are seen in the basal layer. These cells are named as "starburst giant cell". The presence of atypical melanocytes in the hair follicles and sweat duct epithelium is a characteristic feature but sometimes it may lead to difficulties in evaluation of tumor thickness [109, 113, 114]. Also there is effacement of rete ridges [115]. Due to chronically actinic damage, epidermis is usually atrophic and solar elastosis is seen in the dermal layer [116]. The upper part of the dermis usually contains melanophages and lymphocytes to a lesser extent [114]. If the lesion progresses, pagetoid spread may be observed within the epidermis [109]. When invasion occurs into the dermis, spindled cells and tumor cell pigmentation can be seen [117].

Superficial spreading melanoma is also known as pagetoid melanoma which characterized by a proliferation of atypical melanocytes singly and in nests in all layers of the epidermis [114]. Atypical melanocytes sometimes show "buckshot scatter" within the epidermis (Figure 9) [113]. The large tumor cells contain dark, atypical nuclei and abundant, pale cytoplasm [118]. The epidermis may have normal or hyperplastic appearance [119]. There is a continuous spread of tumor growth from one rete ridge to another [114]. If the tumor progresses to vertical growth phase, microinvasive tumor which contains nested and dispersed cells is seen within

black) [12, 51, 87].

92 Highlights in Skin Cancer

morphologies.

**6. Histopathological examination**

**6.2. Superficial spreading melanoma**

the dermis [109].

**6.1. Lentigomaligna/ Lentigomaligna Melanoma (LM/LMM)**

Nodular melanoma has no concomitant or preexisting radial growth phase [109]. It grows vertically from the beginning and thus may invade the epidermis [113, 114]. Cellular features include a large nucleoli and frequent mitosis [118]. Epidermal melanocytic proliferation is so minimal which typically extending less than three epidermal ridges on both sides of tumor [120]. The tumor mass contains small nests and aggregates of atypical melanocytes (Figure 10, 11, 12) [117].

#### **6.4. Acral lentiginous melanoma**

Histological changes are not fully clear in the early stages which may be seen irregular epidermal hyperplasia and dispersed, localized to the basal layer, atypical melanocytes [114]. Atypical cells proliferate as diffuse along to dermoepidermal junction in the radial growth phase. These cells create a lentiginous pattern by scattered severally [121]. Atypical melano‐ cytes have marked nuclear atypia and also seen around the adnexal structures [109]. In contrast to acral nevi, pigment is seen throughout the stratum corneum [105]. Other changes in the epidermis include acanthosis and elongation of the rete ridges.Tumor infiltration of lympho‐ cytes and tumor regression are common findings in ALM. Kim et al. observed that the frequencies of these findings are 75% and 25% of ALM cases, respectively [122]. The dermal invasive component is predominantly spindle cell type, but epitheloid or nevoid cells may be seen. The presence of small nevus cells may indicate a worse prognosis. Additionally lack of elastosis in dermis is prominent [105, 113, 123].

**Figure 10.** Nodular melanoma: characteristic melanoma morphology, the tumor is composed of cell groups (By the courtesy of Dr. Ahmet Cemil Kaur).

nuerotropism ranged from 16.7% to 77.8% [76]. *Kay* et al. reported that perineural invasion was 82% [128]. There are two subtypes of desmoplastic melanoma histologically; i) pure desmoplastic melanoma that characterized by desmoplasia through out the tumor, ii) and mixed desmoplastic melanoma that characterized by desmoplasia associated with non-

**Figure 12.** Nodular melanoma: in this example there is heavy melanin pigmentation (By the courtesy of Dr. Ahmet

**Figure 11.** Nodular melanoma: close view, the tumor cells are pleomorphic with abundant cytoplasm, large vesicular

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nuclei and prominent nucleoli (By the courtesy of Dr. Ahmet Cemil Kaur).

desmoplastic invasive melanoma [129].

Cemil Kaur).

#### **6.5. Desmoplastic melanoma**

Desmoplastic melanoma is characterized by intensive atypical spindle-shape melanocytes within dense collagen bundles [124]. Tumor cells have hyperchromatic, elongated nuclei but usually no contain pigment in their cytoplasm [125, 126]. There are often nodular lymphocytic aggregates that are helpful in diagnosis [114, 127]. Perineural invasion has been reported in some studies. In a study by Lens et al., the percentage of desmoplastic melanoma with

[120]. The tumor mass contains small nests and aggregates of atypical melanocytes (Figure

Histological changes are not fully clear in the early stages which may be seen irregular epidermal hyperplasia and dispersed, localized to the basal layer, atypical melanocytes [114]. Atypical cells proliferate as diffuse along to dermoepidermal junction in the radial growth phase. These cells create a lentiginous pattern by scattered severally [121]. Atypical melano‐ cytes have marked nuclear atypia and also seen around the adnexal structures [109]. In contrast to acral nevi, pigment is seen throughout the stratum corneum [105]. Other changes in the epidermis include acanthosis and elongation of the rete ridges.Tumor infiltration of lympho‐ cytes and tumor regression are common findings in ALM. Kim et al. observed that the frequencies of these findings are 75% and 25% of ALM cases, respectively [122]. The dermal invasive component is predominantly spindle cell type, but epitheloid or nevoid cells may be seen. The presence of small nevus cells may indicate a worse prognosis. Additionally lack of

**Figure 10.** Nodular melanoma: characteristic melanoma morphology, the tumor is composed of cell groups (By the

Desmoplastic melanoma is characterized by intensive atypical spindle-shape melanocytes within dense collagen bundles [124]. Tumor cells have hyperchromatic, elongated nuclei but usually no contain pigment in their cytoplasm [125, 126]. There are often nodular lymphocytic aggregates that are helpful in diagnosis [114, 127]. Perineural invasion has been reported in some studies. In a study by Lens et al., the percentage of desmoplastic melanoma with

10, 11, 12) [117].

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**6.4. Acral lentiginous melanoma**

elastosis in dermis is prominent [105, 113, 123].

courtesy of Dr. Ahmet Cemil Kaur).

**6.5. Desmoplastic melanoma**

**Figure 11.** Nodular melanoma: close view, the tumor cells are pleomorphic with abundant cytoplasm, large vesicular nuclei and prominent nucleoli (By the courtesy of Dr. Ahmet Cemil Kaur).

**Figure 12.** Nodular melanoma: in this example there is heavy melanin pigmentation (By the courtesy of Dr. Ahmet Cemil Kaur).

nuerotropism ranged from 16.7% to 77.8% [76]. *Kay* et al. reported that perineural invasion was 82% [128]. There are two subtypes of desmoplastic melanoma histologically; i) pure desmoplastic melanoma that characterized by desmoplasia through out the tumor, ii) and mixed desmoplastic melanoma that characterized by desmoplasia associated with nondesmoplastic invasive melanoma [129].

#### **6.6. Minimal deviation melanoma**

Minimal deviation melanoma is a variant of invasive melanoma that characterized by a nodule with minimal histologic deviation compared to ordinary nevus. It may be confined into the papillary dermis or may invade into the reticular dermis or beyond. This melanoma variant is divided into 6 subtypes according to cytological features (Spitz, halo-nevus like, pigmented spindle cell, desmoplastic, small cell and dermal variant). The average thickness is 3, 40 mm. The infiltration into the subcutaneous fat tissue is not often seen. Necrosis is absent while perineural invasion, mitoses and inflammation with desmoplasia can be seen. Mitotic activity is usually quite low [78, 114, 130, 131].

*Clark*'s level which is other tumor thickness indicator is described by *Clark* as anatomic levels

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**Level I** Melanomas confined to the **Level II** Penetration by melanomas into the papillary dermis

**Level III** Tumor cells fill and expand the papillary dermis **Level IV** Spreading into the reticular dermis **Level V** Penetration into the

subcutaneous fat

Ulceration is defined as disappearance of the all layers of epidermis (including basement membrane), evidence of host response, and thinning, effacement or reactive hyperplasia of adjacent epidermis. The presence of ulceration shows that the lesion has aggressive feature. Ulceration due to trauma should be excluded. The presence of ulceration is associated with a higher risk of metastases. According to the presence or absence of ulceration, each T category is divided into two as "a" and "b" in the AJCC cutaneous melanoma classification 2009 (Table 2). This system classifies melanomas on the basis of their local, regional, and distant charac‐

**•** Stage III - Metastasis to single regional lymph node basin (with or without in-transit

The mitotic rate is important prognostic indicator that is determined by the number of mitotic

Microsatellites are defined as discrete tumor aggregates separated from the main body of the tumor mass. These deposites settled to 0,05 mm or more away from the main tumor mass are

of tumor in the most mitotically active area. The increased mitotic activity is

If Clark's level increases, the mean life span is decreased [119, 134-137].

in melanoma invasion (Table 1):

**Table 1.** *Clark* level of invasion

teristics, as follows: [39, 51, 135, 138]

**•** Stage IV – Distant metastatic disease.

associated with poor prognosis [51, 114].

**•** Stage I and II - Localized primary melanoma

*6.8.2. Ulceration*

metastases)

*6.8.3. Mitotic rate*

figures/1 mm2

*6.8.4. Satellite deposites*

#### **6.7. Special variants**

#### *6.7.1. Follicular melanoma*

This rare variant is characterized by a deep-seated follicular structure in which atypical melanocytic cells extend downward along the follicular epithelium and mainly involves follicular unit as well as adjacent dermal layer [113, 132, 133].

#### *6.7.2. Myxoid melanoma*

Myxoid variant is characterized by spindle and stellate-shaped cells embedded within myxoid stroma. There is no cytoplasmic mucin in tumor cells. The stroma stains with *Alcian* blue. HMB-45 is showed less often while tumor cells strongly express S-100 protein [109, 113].

#### *6.7.3. Balloon cell melanoma*

The tumor is composed of large cells that exhibit an abundant quantity of clear or finely vacuolated cytoplasm. The other histopathological features include nuclear pleomorphism, mitotic activity, cytological atypia and necrosis [78, 113].

#### **6.8. Histopathologic prognostic factors in melanoma**

#### *6.8.1. Tumor thickness*

Primary tumor thickness is the most powerful predictor of melanoma survival. The *Breslow* thickness that is measured from the most superficial aspect of the granular cell layer to the deepest point of invasion of the tumor is the better prognostic indicator. If the tumor is ulcerated, the measurement should be from the base of the ulcer to the deepest dermal melanoma cell. In AJCC staging system (2001), the thickness thresholds have been revised as ≤1.0 mm, >1.0-2.0 mm, 2.1-4.0 mm and >4 mm. The *Breslow* thickness increases with increasing rate of sentinel lymph node involvement: 4% in melanoma smaller than 1.00 mm, 12% in melanoma 1.01 to 2.00 mm, 28% in melanoma 2.01 to 4.00 mm, and 44% in melanoma exceeding 4.00 mm.

*Clark*'s level which is other tumor thickness indicator is described by *Clark* as anatomic levels in melanoma invasion (Table 1):


#### **Table 1.** *Clark* level of invasion

If Clark's level increases, the mean life span is decreased [119, 134-137].

#### *6.8.2. Ulceration*

**6.6. Minimal deviation melanoma**

is usually quite low [78, 114, 130, 131].

follicular unit as well as adjacent dermal layer [113, 132, 133].

mitotic activity, cytological atypia and necrosis [78, 113].

**6.8. Histopathologic prognostic factors in melanoma**

**6.7. Special variants**

96 Highlights in Skin Cancer

*6.7.1. Follicular melanoma*

*6.7.2. Myxoid melanoma*

*6.7.3. Balloon cell melanoma*

*6.8.1. Tumor thickness*

4.00 mm.

Minimal deviation melanoma is a variant of invasive melanoma that characterized by a nodule with minimal histologic deviation compared to ordinary nevus. It may be confined into the papillary dermis or may invade into the reticular dermis or beyond. This melanoma variant is divided into 6 subtypes according to cytological features (Spitz, halo-nevus like, pigmented spindle cell, desmoplastic, small cell and dermal variant). The average thickness is 3, 40 mm. The infiltration into the subcutaneous fat tissue is not often seen. Necrosis is absent while perineural invasion, mitoses and inflammation with desmoplasia can be seen. Mitotic activity

This rare variant is characterized by a deep-seated follicular structure in which atypical melanocytic cells extend downward along the follicular epithelium and mainly involves

Myxoid variant is characterized by spindle and stellate-shaped cells embedded within myxoid stroma. There is no cytoplasmic mucin in tumor cells. The stroma stains with *Alcian* blue. HMB-45 is showed less often while tumor cells strongly express S-100 protein [109, 113].

The tumor is composed of large cells that exhibit an abundant quantity of clear or finely vacuolated cytoplasm. The other histopathological features include nuclear pleomorphism,

Primary tumor thickness is the most powerful predictor of melanoma survival. The *Breslow* thickness that is measured from the most superficial aspect of the granular cell layer to the deepest point of invasion of the tumor is the better prognostic indicator. If the tumor is ulcerated, the measurement should be from the base of the ulcer to the deepest dermal melanoma cell. In AJCC staging system (2001), the thickness thresholds have been revised as ≤1.0 mm, >1.0-2.0 mm, 2.1-4.0 mm and >4 mm. The *Breslow* thickness increases with increasing rate of sentinel lymph node involvement: 4% in melanoma smaller than 1.00 mm, 12% in melanoma 1.01 to 2.00 mm, 28% in melanoma 2.01 to 4.00 mm, and 44% in melanoma exceeding Ulceration is defined as disappearance of the all layers of epidermis (including basement membrane), evidence of host response, and thinning, effacement or reactive hyperplasia of adjacent epidermis. The presence of ulceration shows that the lesion has aggressive feature. Ulceration due to trauma should be excluded. The presence of ulceration is associated with a higher risk of metastases. According to the presence or absence of ulceration, each T category is divided into two as "a" and "b" in the AJCC cutaneous melanoma classification 2009 (Table 2). This system classifies melanomas on the basis of their local, regional, and distant charac‐ teristics, as follows: [39, 51, 135, 138]


#### *6.8.3. Mitotic rate*

The mitotic rate is important prognostic indicator that is determined by the number of mitotic figures/1 mm2 of tumor in the most mitotically active area. The increased mitotic activity is associated with poor prognosis [51, 114].

#### *6.8.4. Satellite deposites*

Microsatellites are defined as discrete tumor aggregates separated from the main body of the tumor mass. These deposites settled to 0,05 mm or more away from the main tumor mass are associated with an increased risk of local recurrence, regional lymph node metastases and diminished survival [114, 134].

with a better prognosis in melanoma, there are also studies reporting that no significant

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There is no survival difference among three histological subtypes (superficial spreading, nodular and acral lentiginous) when these are corrected for thickness. But lentigo malign melanoma, particularly in woman, has been reported to have a better prognosis, independent

Regression is caused by the interaction between melanoma cells and host immune system. Tumor tissue replaced with degenerative melanoma cells, melanophages, lymphocytic proliferation, haphazard fibrosis and telangiectasias. Complete regression is characterized by total absence of malignant melanoma cells in both dermis and epidermis. The correlation

Tumor cells may invade the vessel lumen. It correlates with the development of in-transit metastases, when the presence of blood vessel and lymphatic invasion should be reported. Vascular invasion is associated with poor prognosis and decreased survival in thick cutaneous malignant melanomas [109, 119]. Angiogenesis is a distinct histological prognostic indicator that is defined as the increasing development of new blood vessels at the base of the tumor mass. Increasing angiogenesis is associated with thick tumors, surface ulceration, relapse and

Immunohistochemical staining is often used for differentiate melanomas from tumors that

S-100 is a commonly used sensitive marker for melanoma. But its positivity appears some tumors such as nerve sheath and granular cell tumors and myoepitheliomas. Although its sensitivity is 97-100%, its specificity for melanocytic lesions is limited. The specificity of S-100 is ranged from 75% to 87%. S-100 A6 is one of the subtypes of S-100 protein, expressed in both benign and malignant melanocytic lesions. S-100 A6 has been reported that it is expressed in

HMB-45 is an antibody formed against the cytoplasmic premelanosomal glycoprotein gp100 while its sensitivity is lower than S-100, its specificity is greater. HMB-45 expression is maximal (77-100%) in primary melanomas. This rate is lower (58-83%) in patients with

Melan-A, also known as melanoma antigen recognized by T cells-1 (MART-1), is an important melanocytic marker. Sensitivity and specificity of Melan-A are similar to HMB-45 that ranged

approximately 62-100% of metastatic and primary melanomas [140, 141].

correlation between lymphocytic infiltration and prognosis [119].

between regression and prognosis is still controversial [119, 134].

*6.8.6. Histological subtype*

of thickness [51].

*6.8.7. Regression*

*6.8.8. Vascular invasion*

tumor related death [114].

metastatic lesions [139].

**6.9. Immunohistochemistry of melanoma**

they mimic in conventionally stained sections [139].


T=tumor size; N=node status; M=metastasis; Ta=without ulceration; Tb=with ulceration;

#### **Table 2.** Cutaneous Melanoma Staging

#### *6.8.5. Lymphocytic infiltration*

Tumor-infiltrating lymphocytes are an important indicator of host immune response against melanoma. This response is divided into 3 categories and should be reported as brisk, nonbrisk and absent. Although the presence of host inflammatory response is generally associated with a better prognosis in melanoma, there are also studies reporting that no significant correlation between lymphocytic infiltration and prognosis [119].

#### *6.8.6. Histological subtype*

There is no survival difference among three histological subtypes (superficial spreading, nodular and acral lentiginous) when these are corrected for thickness. But lentigo malign melanoma, particularly in woman, has been reported to have a better prognosis, independent of thickness [51].

#### *6.8.7. Regression*

associated with an increased risk of local recurrence, regional lymph node metastases and

**Stage T N M Clinical-Histopathological Features** 0 Tis N0 M0 In situ melanoma (ıntraepithelial) IA T1a N0 M0 ≤1 mm without ulceration IB T1b N0 M0 ≤1 mm with ulceration

IIA T2b N0 M0 1.01-2 mm with ulceration

IIB T3b N0 M0 2.01-4 mm with ulceration

IIC T4b N0 M0 "/>4 mm with ulceration

T2a N0 M0 1.01-2 mm without ulceration

T3a N0 M0 2.01-4 mm without ulceration

T4a N0 M0 4 mm without ulceration

IIIA T1-4a N1a M0 Single regional nodal micrometastasis, without ulceration

IIIB T1-4b N1a M0 Single regional nodal micrometastasis, with ulceration

IIIC T1-4b N1b M0 Single regional nodal macrometastasis, with ulceration T1-4b N2b M0 2-3 macroscopic regional nodes, with ulceration

T1-4a N2a M0 2-3 microscopic positive regional nodes, without ulceration

T1-4b N2a M0 2-3 microscopic positive regional nodes, with ulceration T1-4a N1b M0 Single regional nodal macrometastasis, without ulceration T1-4a N2b M0 2-3 macroscopic regional nodes, without ulceration T1-4a/b N2c M0 In-transit met(s)/ satellite lesion(s) without metastatic lymph nodes

Any T N3 M0 4 or more metastatic nodes, matted nodes, or in-transit met(s)/satellite

Tumor-infiltrating lymphocytes are an important indicator of host immune response against melanoma. This response is divided into 3 categories and should be reported as brisk, nonbrisk and absent. Although the presence of host inflammatory response is generally associated

lesion(s) with metastatic nodes

M1a: Distant skin, subcutaneous, or nodal mets with normal LDH levels M1b: Lung metastases with normal LDH M1c: All other visceral metastases with normal LDH or any distant metastasis with elevated LDH

diminished survival [114, 134].

98 Highlights in Skin Cancer

IV Any T Any N Any

**Table 2.** Cutaneous Melanoma Staging

*6.8.5. Lymphocytic infiltration*

M1

T=tumor size; N=node status; M=metastasis; Ta=without ulceration; Tb=with ulceration;

Regression is caused by the interaction between melanoma cells and host immune system. Tumor tissue replaced with degenerative melanoma cells, melanophages, lymphocytic proliferation, haphazard fibrosis and telangiectasias. Complete regression is characterized by total absence of malignant melanoma cells in both dermis and epidermis. The correlation between regression and prognosis is still controversial [119, 134].

#### *6.8.8. Vascular invasion*

Tumor cells may invade the vessel lumen. It correlates with the development of in-transit metastases, when the presence of blood vessel and lymphatic invasion should be reported. Vascular invasion is associated with poor prognosis and decreased survival in thick cutaneous malignant melanomas [109, 119]. Angiogenesis is a distinct histological prognostic indicator that is defined as the increasing development of new blood vessels at the base of the tumor mass. Increasing angiogenesis is associated with thick tumors, surface ulceration, relapse and tumor related death [114].

#### **6.9. Immunohistochemistry of melanoma**

Immunohistochemical staining is often used for differentiate melanomas from tumors that they mimic in conventionally stained sections [139].

S-100 is a commonly used sensitive marker for melanoma. But its positivity appears some tumors such as nerve sheath and granular cell tumors and myoepitheliomas. Although its sensitivity is 97-100%, its specificity for melanocytic lesions is limited. The specificity of S-100 is ranged from 75% to 87%. S-100 A6 is one of the subtypes of S-100 protein, expressed in both benign and malignant melanocytic lesions. S-100 A6 has been reported that it is expressed in approximately 62-100% of metastatic and primary melanomas [140, 141].

HMB-45 is an antibody formed against the cytoplasmic premelanosomal glycoprotein gp100 while its sensitivity is lower than S-100, its specificity is greater. HMB-45 expression is maximal (77-100%) in primary melanomas. This rate is lower (58-83%) in patients with metastatic lesions [139].

Melan-A, also known as melanoma antigen recognized by T cells-1 (MART-1), is an important melanocytic marker. Sensitivity and specificity of Melan-A are similar to HMB-45 that ranged from 75-92% and 95-100%, respectively. It is less positive in lesions with metastatic melanomas than primary melanoma [139].

**7.2. Evaluation for regional metastasis**

First step should always be history taking and physical examination. A careful lymph node examination of the whole body should be made, particulary the regions close to the site of the primary lesion. Also because of the aberrant lymphatic drainage pathways to unexpected nodal basins and to interval nodes between the primary site and expected regional nodal basin, a search for clinically detectable nodal disease in unexpected locations is crucial. If there is a palpable lymph node during physical examination, first a fine needle aspiration biopsy should be performed to make a histological confirmation. If the result of the fine needle aspiration is inconclusive, an excisional biospy can be made. For the detection small nodal metastases, best noninvasive methods seem to be ultrasound imaging and positron emission tomography, with

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Sentinel lymph node biopsy (SLNB) as elective lymph node dissection (ELND) does not offer a survival advantage to melanoma patients, a new approach, SLNB has become the choice of biopsy type. *Morton* developed sentinel node biopsy as a means of ensuring that the node biopsied was the one most likely to be the first draining lymph node. This technique was widely adopted although the first randomized clinical trial to evaluate it was only published in 2006. In SLNB, lymphoscintigraphy is used to identify the lymphatic drainage pattern from the site of the primary melanoma, by injecting radiolabelled colloid and/or blue dye at the site of the primary melanoma [145]. The tracer/dye is concentrated in the sentinel node and is detected using a hand-held gamma probe (neoprobe) and examination by naked eye for the blue stained node. Sometimes there is more than one sentinel node and sometimes these sentinel nodes are in different lymph node basins. The node is removed and subjected to pathological examina‐ tion using immunohistochemistry (S100 and HMB-45) The surgical technique must be learnt and false-negative results are more common in trainees, so an experienced medical team is vital. The pathological examination of the nodes is also a skilled procedure; naevus cells may be seen, for example, in the subcapsular area of the node and must be distinguished from melanoma cells. The likelihood of a positive SLNB result is correlated with *Breslow* thickness. Use of SLNB to stage patients for trials of adjuvant therapies would appear reasonable but the patient should be aware that the risk of positivity is low. In patients with melanoma of *Breslow* thickness from 1.5 to 4.0 mm, the risk of positive SLNBs is significantly higher at 23% [146]. The value of SLNB in patients with tumours of *Breslow* thickness 4 mm or thicker is question‐ able (if the intent is therapeutic as well as being a staging tool) because the risk of haematog‐ enous spread is so high. At present SLNB is of proven staging value but of no established therapeutic value. Its use in identifying patients for adjuvant therapies means that it will

sensitivity and specificity being lower than tissue diagnosis [144].

continue to be used, but its role must be evaluated in the long term.

*7.2.1. Macroscopic metastases*

*7.2.2. Microscopic metastases*

MIB-1, also known as Ki-67, is a proliferation marker that is expressed by proliferating cells. It provides guidance about presence or absence of "maturation". MIB-1 has an important role in distinguishing between melanocytic nevi and melanoma. While less than 5% of nuclei is positive in melanocytic nevi, this ratio is greater (25% or more) in melanoma [114, 141].

Tyrosinase is an enzyme that plays a role in hydroxylation of tyrosine in the synthesis of melanin. Its sensitivity for melanoma is slightly better than HMB-45 at 84-94%. The sensitivity is reduced in advanced diseases and metastatic lesions (79-93%). The specificity is very high for melanoma (97-100%) [139].

Microphthalmic transcription factor (MITF) is expressed in most benign melanocytic nevi and melanomas. Nuclear staining occurs with MITF unlike cytoplasmic markers. MITF expression has been reported in 81-100% of melanomas [51, 113].

There are also numerous immunohistochemical markers such as epithelial markers (keratin, EMA, CEA), histiocytic markers (eg. CD68, Mac 378, alpha-1 antitrypsin), Bcl-2, Cyclin D1, p16, CD40, CD44, melanoma cell adhesion molecule (Mel-CAM) [114].
