noma of the head and neck treated with MMS have improved or equivalent 5-year disease-speciÞc survival rates when compared to historical controls treated with

Table 18.2 Surgical margins for primary cutaneous melanoma derived from MMS based on (a) tumor depth and (b) tumor location and diameter

wide local excision [66]. In these studies, the surgical margin for the excision of primary cutaneous melanoma was found to be dependent on tumor location, diameter, and depth (Table 18.2a, b) [46, 66]. Moreover, the surgical margins derived from these studies demonstrate that 59% of primary cutaneous melanomas surgically treated with the current recommended guidelines are excised with an excess margin greater than 5 mm, including 23% that would be in excess of 1 cm. Even more disconcerting is that ~8% of primary cutaneous melanomas are inadequately excised with the current recommended guidelines [46, 66] (Table 18.2a, b). It

should be noted that these Þndings have also been conÞrmed with others [67Ð71]. Most of these inadequately excised melanomas are MIS located on the head and neck, suggesting that the consensus panel recommendation of narrow margins (5 mm) is inadequate. This prediction for inadequate excision is similar to the reported recurrence rates for melanomas on the head, neck, hands, and feet after standard surgery [47Ð51].

Summary: Application of MMS for the Treatment of Cutaneous Melanoma: IHC Stains

¥Several melanocytic markers have been applied to MMS for the treatment of melanoma, with MART-1 being the most common.

¥MART-1-speciÞc monoclonal antibodies have high sensitivity (75Ð92%) and speciÞcity (95Ð100%) for melanoma.

¥Atypical melanomas, like spindle cell and desmoplastic melanomas, often pose a diagnostic dilemma due to atypical morphology, and their IHC proÞles are different from and not as distinctive as those of other types of melanomas.

¥Other melanocytic differentiation markers are currently being investigated.

18.4Application of MMS for the Treatment of Cutaneous Melanoma

18.4.1 IHC Stains

Most neoplasms treated with MMS can be identiÞed in standard hematoxylin and eosin (H&E)-stained frozen sections with very high certainty. However, in certain tumors, especially melanoma, identifying the tumor in

Table 18.3 Antigens utilized in the diagnosis of melanoma

Derived from [75, 82Ð111, 115, 116, 120]

frozen sections can be more challenging. We, as well as others, have reported that the sensitivity and speciÞcity of H&E-stained frozen sections for the evaluation of surgical margins of MIS and primary cutaneous melanoma are 100% and 90%, respectively [66Ð70, 72Ð74]. Although frozen sections are proven to be reliable in margin assessment of primary cutaneous melanoma, they are not recommended for diagnosis or prognostic staging. Moreover, it can be difÞcult to differentiate atypical melanocytic proliferations from true melanoma utilizing light microscopy.

The application of the IHC technique (Fig. 18.1) to MMS has greatly expanded our capacity to treat a wide array of malignancies using MMS, especially melanoma. In this regard, the use of IHC staining of frozen tissue sections removed during MMS overcomes the problem of interpreting atypical melanocytes in frozen sections. Furthermore, an advantage of IHC staining of the frozen sections processed during MMS rather than formalin-Þxed, parafÞn-embedded sections is the preservation of antigenic epitopes which can often be lost in Þxation of tissues with formalin and their subsequent embedding with parafÞn [75]. To date, several melanocytic markers have been applied to MMS for the treatment of melanoma (Table 18.3). Currently, the most widely utilized marker is the melanoma differentiation antigen known as melanoma antigen recognized by T cells (MART-1) or Melan-A. MART-1 is a cytoplasmic protein of melanosomal differentiation recognized by T cells [76Ð81]. Two clones of MART-1-speciÞc monoclonal antibody (mAb) are available: M2-7C10, referred to as MART-1, and A103, referred to as Melan-A. The reactivity of these MART-1-speciÞc mAb is not restricted to melanoma since both label mesenchymal tumors consisting of perivascular epithelioid cells and some clear cell sarcomas and mAb A103 also labels adrenal cortical tumors and gonadal steroid tumors [75, 82Ð94]. These MART-1-speciÞc mAb show sensitivity (75Ð92%) and speciÞcity (95Ð100%) for melanoma that is similar to

HMB-45. There is a decrease in the percentage of stained cells in metastatic melanomas relative to primary melanomas. However, these mAb generally show more diffuse and intense staining than HMB-45 and do not show reduced staining in the dermal component of melanomas; this property makes them easier to interpret especially in metastatic melanomas [76Ð81].

Of the other melanocytic markers that have been applied to MMS for the treatment of melanoma, HMB45, a marker of the cytoplasmic premelanosomal glycoprotein gp100, was one of the Þrst melanoma-speciÞc markers discovered [86, 87, 90Ð105]. The reported sensitivity of HMB-45 for melanoma ranges from 69% to 93%, and expression is maximal in primary melanoma specimens (77Ð100%) and less in metastases (58Ð83%). Staining may be patchy, and melanoma cells are less diffusely positive than with other markers. There may be strong staining with HMB-45 in the epidermal component of primary melanomas with gradually weaker staining in the deeper vertical growth phase. HMB-45 is very speciÞc for melanomas; however its expression has been detected in angiomyolipomas, lymphangiomyomatosis, sweat gland tumors, meningeal melanocytomas, clear cell sarcoma of the tendons and aponeuroses, ovarian steroid cell tumors, breast cancers as well as renal cell carcinomas [86, 87, 90Ð105]. Decreased sensitivity of HMB-45 has also been noted in metastatic melanoma.

Other melanocytic differentiation markers are currently being investigated and are not yet in widespread clinical use including multiple myeloma oncogene-1 (MUM-1), melanocortin-1, the microphthalmia transcription factor (MITF), SM5-1, TRP-1/2, and PNL2 [75, 76, 88, 106Ð120]. However, none of these newer markers have yet shown signiÞcant advantages over the immunostains currently in clinical use.

It should be noted that spindle cell or desmoplastic melanomas often pose a diagnostic dilemma because their morphology is atypical and their IHC proÞles are different from and not as distinctive as those of other types of melanomas [82, 84Ð88, 91, 95]. All the markers that are more speciÞc for melanoma also show very poor sensitivity for spindle/desmoplastic lesions (Table 18.4). Histologically, desmoplastic melanoma is characterized as a mainly intradermal ill-deÞned lesion composed of elongated hyperchromatic spindle cells distributed singly or in bundles, fascicles, or nests, between variably increased collagen Þbers of the papillary and the reticular dermis. Both primary and metastatic desmoplastic melanomas lesions usually show immunoreactivity with S-100 protein and NKI/C3 but not with HMB-45,

Table 18.4 Sensitivity of melanoma-associated antigen in spindle cell and desmoplastic melanoma

Derived from [75, 82, 84Ð88, 90, 91, 95]

MART-1, or other antibodies (Table 18.4) [82, 84Ð88, 90, 91, 95, 120, 121]. Nevertheless, negativity does not exclude the diagnosis when the clinical picture and/or histology are characteristic. In this regard, recent evidence suggests the human chondroitin sulfate proteogly- can-4 is more sensitive than HMB-45 and MART-1 for IHC diagnosis of primary and metastatic desmoplastic melanoma lesions [122]. Whether this marker can be applied to MMS remains to be determined.

Summary: Technical Application of MMS

and Interpretation of IHC Stains

¥The margins of the remaining melanoma or biopsy site should be identiÞed prior to anesthesia using bright surgical lighting.

¥Fat must be removed from the Mohs specimen prior to staining with IHC so that extremely thin (2Ð4 mm) sections can be cut by the histotechnician without artifact or distortion.

¥Well-maintained equipment, including highgrade cryostats, liquid nitrogen to lower the temperature of fatty specimens, and very sharp cryostat blades, is essential.

¥Accurately interpreting the labeling patterns of melanocytes with MART-1-speciÞc mAb using the IHC technique requires knowledge of the melanocytic staining patterns of normal and sun-damaged skin.

18.4.2Technical Application of MMS and Interpretation of IHC Stains

Prior to MMS, patients receive detailed information regarding prognosis and alternative treatments and are taught to perform both skin and lymph node selfexams. Patients should be encouraged to ask each and

every question they may have regarding their disease. All patients are staged by complete history and physical examination including palpation of pertinent lymph node basins. Chest roentgenography and serum lactate dehydrogenase (LDH) levels may be performed in patients with melanomas greater than 1 mm thick, though the utility of these tests is questionable. The surgeon should be ready to discuss topics such as sentinel lymph node (SLN) biopsy, completion nodal dissection, and systemic therapies and be familiar with local research trials patients may qualify for. For those patients requesting SLN biopsy, it should be noted that wide local excision prior to SLN biopsy does not adversely impact the ability to identify the draining SLN [123Ð125]. Moreover, the SLN has been shown to accurately reßect the status of the regional lymph node basin in patients with melanoma previously treated with wide local excision [123Ð125].

To initiate MMS for the treatment of a melanoma, the remaining tumor or surgical scar is conÞrmed with the patient. Prior to anesthesia and using bright surgical lighting along with magniÞcation, the margin of the remaining melanoma or biopsy site scar is outlined with a surgical marking pen. For melanomas with clinically indistinct margins, a WoodÕs lamp can be used to assist in establishing the clinical margins. MMS is initiated by excising the remaining visible tumor or biopsy site scar with a 3-mm surgical margin through the dermis into the subcutaneous tissue plane deep enough to remove all adnexal structures. This ÒdebulkingÓ specimen may be submitted for permanent step sectioning using conventional histopathologic techniques to determine the maximum Breslow thickness. After debulking, a second 3-mm margin is excised to the subcutis for complete examination of the margin by means of the MMS technique. Subsequently, the epidermis and dermis are trimmed from the subcutis (Fig. 18.2) since production of consistently readable melanoma slides on frozen sections is dependent on the meticulous preparation of thin 2Ð4 mm sections without artifact or distortion. Once the epidermis and dermis have been removed from the fat, the specimen is then precisely mapped and color-coded. Very thin, 2Ð4 mm sections are then stained with both H&E [126] and immunostained with MART- 1-speciÞc mAb [79]. As noted above, MART-1-speciÞc mAb are favored for IHC staining by the authors because of its superior sensitivity and speciÞcity. Summarized in Table 18.5, the authors utilize the 1-h protocol consisting of a polymer-based detection system as previously described [79]. It is noteworthy that

Fig. 18.2 Tissue processing during the application of MMS for the treatment of melanoma. (a) The remaining tumor or biopsy scar is identiÞed, and the margin is outlined. MMS is initiated by excising the remaining visible tumor or biopsy site scar with a 3-mm surgical margin through the dermis into the subcutaneous tissue plane deep enough to remove all adnexal structures. (b) After debulking, a second 3-mm margin is excised vertically for complete examination of the margin.

(c, d) Prior to tissue staining with both H&E and MART-1- speciÞc mAb, the epidermis and dermis are trimmed from the fat

Subcutis

Table 18.5 Protocol for staining frozen sections with MART- 1-speciÞc mAb

thick sectioning, freeze artifact and folding are common pitfalls when preparing tissue for frozen section evaluation as well as IHC staining. It must be stressed that even with proper tissue preparation, consistently readable melanoma slides processed with the IHC technique on frozen sections require well-trained technicians and proper, well-maintained equipment.

Accurately interpreting the labeling of melanocytes with MART-1-speciÞc mAb using the IHC technique

requires knowledge of the melanocytic staining patterns of normal and sun-damaged skin. Criteria for identifying positive margins have been published and include: nests of three or more atypical melanocytes, upward migration of melanocytes in the epidermis (pagetoid spread), and ÒnonuniformÓ contiguous melanocytic hyperplasia along the basement membrane [127] (Fig. 18.3). Atypical melanocytes are deÞned as those that contain mitoses, pleomorphic and hyperchromatic nuclei, or pleomorphic shape. Other nondiagnostic histologic Þndings that may occasionally be observed in association with melanoma include extension of atypical, crowded melanocytes down adnexal structures, nonuniform distribution of pigment, increased number of melanophages, and/or brisk inßammatory inÞltrate. Positive margins are indicated on the map, and the corresponding tissue is then excised with additional 3-mm margins. The above steps are repeated until all margins are free of tumor, and then surgical wound management is performed. Distinguishing benign melanocytic hyperplasia from melanoma in situ can prove challenging even for the most experienced Mohs surgeon. In this regard, increased melanocytic density has been noted in up to one quarter of samples of normal sun-damaged skin with a mean number of melanocytes per high power Þeld of 20.3 [127]. In the most severe areas of melanocyte conßuence, the number of adjacent melanocytes did not exceed 9 in the study population [127].