Immunostains

Summary: Introduction

•Mohs micrographic surgery (MMS) is a surgical technique utilized for skin cancers that allows for 100% visualization of the lateral and deep margins of the excision specimen.

•During excision of squamous cell or basal cell carcinoma with Mohs, frozen section slides may occasionally be difficult to interpret with H & E staining if there is fibrosis, inflammation, or perineural and muscle invasion.

•Permanent sections stained with H & E allow for better visualization of nuclear detail and cell morphology of melanocytes compared to frozen sections.

•Immunostaining is an indispensible adjunctive technique during MMS to visualize certain tumors in frozen section slides that are difficult to see on H & E.

15.1Introduction

Mohs micrographic surgery (MMS) has proven to be an effective technique for the excision of various skin cancers as it allows visualization of the complete peripheral and deep margins of surgery [1]. It results in excellent cure rates with minimal recurrences; for example, only 1% for primary basal cell carcinoma (BCC) and 3–8% for recurrent basal BCC. In contrast, standard excisional surgery permits examination of only 1–2% of the surgical margin and is associated with higher recurrence rates of up to 10% for primary lesions and 5–40% for recurrent tumors [2, 3].

For most cases of BCC and squamous cell carcinoma (SCC) submitted for MMS, delineating margins is relatively straightforward in frozen sections (FS). However, there are occasional cases with extensive inflammation or where there is dense fibrosis masking the tumor relative to its surrounding. Occasionally, the tumor may infiltrate skeletal muscle or nerve bundles.

The problem with Mohs for melanoma is that it may be difficult to differentiate melanocytes from atypical keratinocytes in the background of sun-dam- aged skin, and there is risk of freeze artifact. The use of formalin-fixed paraffin-embedded sections (FFPES, or “permanent sections”) permits better visualization of melanocytes than in FS and provides more detail of nuclear and cellular morphology, such as, for example,

Table 15.1 Various immunostains used during Mohs micrographic surgery

the perinuclear “halo” that pathologist rely upon to recognize melanocytes in permanent sections.

To improve the accuracy and quality of frozen sections for both melanoma and nonmelanoma skin cancers, it has become common to use immunostaining or immunohistochemistry as an adjunct to routine H & E staining for the more difficult or recurrent and highrisk tumors.

Its use for cutaneous tumors was first reported in 1984 by Drs. Robinson and Gottschalk for the staining of antibodies to fibrous keratin in deeply invasive basal and squamous cell carcinomas [4]. Now, immunohistochemistry is used mostly for patients with melanoma, basal cell carcinoma, squamous cell carcinoma, extramammary Paget’s disease, dermatofibrosarcoma protuberans, granular cell tumor, and trichilemmal carcinoma (Table 15.1).

Summary: Review of Immunofluorescence

and Immunoperoxidase Techniques

•Immunoperoxidase and immunofluorescence staining techniques are both utilized to identify specific antigens in tissue.

•Immunofluorescence is conducted on frozen section material, but is not applicable to MMS because it requires special UV microscopy and its staining is not durable.

•Immunohistochemistry using a DAB chromophore permanently stains tissue sections and can be viewed with a light microscope; ideal of MMS.

•Methods such as heating tissue sections and the use of polymers during immunohistochemistry reduce processing time, which may range from 1 h to less than 20 min per stage.

15.2Review of Immunofluorescence and Immunoperoxidase Techniques

Initially, immunohistochemistry for frozen sections was prohibitively time consuming; some cases taking over an hour for results. Now, newer techniques employ rapid and even ultrarapid protocols and can produce reliable results is as little as 19 min [2]. It is still, however, paramount that immunohistochemistry-trained laboratory personnel perform these protocols due to the complex and precise nature of these stains.

As a review, immunostaining techniques are generally divided into immunofluorescence (IF) and immunoperoxidase (IMP). Immunofluorescence includes both direct and indirect techniques. Direct IF utilizes frozen sections that are incubated at room temperature with fluorescein isothiocyanate (FITC) antisera. The resulting fluorescent green deposits are visualized by the use of special ultraviolet microscopy. Paraffin-embedded sections are generally not recommended, but perhaps could be attempted after pretreatment with proteolytic enzymes and 0.1% aluminum hydroxide. Indirect immunofluorescence detects serum polyclonal or monoclonal antibodies by incubating the serum with normal skin substrates obtained from volunteers. If the antibody is present and becomes attached to the substrate, then it is detected via incubation with FITC-labeled antisera [5].

Immunoperoxidase staining is reliable in both FS and FFPES. Techniques that have been developed include the peroxidase–antiperoxidase method (PAP), the avi- din–biotin–peroxidase complex procedure (ABC), and the alkaline phosphatase–antialkaline phosphatase procedure (APAAP). The initial studies used a direct method that involved a single antibody conjugated with an enzyme (i.e., peroxidase), which then bound to antigen. After the substrate was introduced, the peroxidase enzyme would oxidize the chromagen and produce an insoluble colorized product (Fig. 15.1). The limitation of this method is its low sensitivity and the need for higher concentrations of the antibody to produce results [6]. Indirect methods were developed to shorten the procedure, enhance its sensitivity, and decrease the quantity of antibody required. There may be increased background staining using indirect staining and a diminishment in the specificity of this method. A negative control is recommended for comparison [6].

In the PAP method, after the tissue is exposed to the primary antibody, it is then washed and incubated with a secondary antibody labeled with horseradish

Direct conjugate method

Antigen

Chromagen

Insoluble product

Melanocyte

Fig. 15.1 The peroxidase is conjugated to the primary antibody

Indirect conjugate method

Melanocyte

Fig. 15.2 The peroxidase is conjugated to the secondary antibody

peroxidase. The peroxidase oxidizes the chromagen detection system, forming an insoluble precipitate, corresponding to the localization of the entire complex (Fig. 15.2). The most commonly used chromagens include 3,3¢-diaminobenzidine hydrochloride (DAB), which forms a brown product, or 3-amino-9-ethylcar- bazole (AEC), which forms a red product.

The ABC procedure is an alternative technique that utilizes the natural attraction between biotin bound to the primary antibody, and avidin, which is conjugated to the secondary antibody, whereby creating tertiary complexes of avidin, biotin, and horseradish peroxidase. This produces a higher degree of sensitivity compared to PAP. Finally, the APAAP method utilizes intestinal-type alkaline phosphatase instead of horseradish peroxidase and uses fast red instead of DAB as the chromagen. The advantage of this technique is that endogenous tissue peroxidase does not need to be quenched unlike in the PAP and ABC methods because the alkaline phosphatase enzyme has activity specific to intestinal mucosa. A protein blocking step can also

be included to eliminate nonspecific IgG antibodies in the vicinity of Ig receptors in the cutaneous tissue, allowing for the primary antibody to more specifically bind to the antigen [7].

In order to speed up the processing time, polymerbased techniques have been developed, where the secondary antibody is bound to a spherical polymer containing horseradish peroxidase (Fig. 15.3) [7]. The increased amount of peroxidase on the polymer is correlated with the increased chromagen activation [7].

Some newer methods of immunohistochemistry use enhanced techniques of antigen retrieval to improve upon the sensitivity of the stain because antigens are often obscured by the fixation process. One such example involves using microwave heating combined with citrate buffers or heavy metal–containing solutions leading to enhanced immunoperoxidase staining of antigen. Again, there may be increased background staining and a resultant decrease in specificity [5, 8].

A major advantage of immunohistochemistry, compared to immunofluorescence, is that the resultant staining is durable if not permanent within FS and FFPES sections, rather than fading with time. The staining is clearly visible with light microscopy, rather than special ultraviolet microscopy, and the sensitivity is substantially greater than immunofluorescence [4, 8].

Summary: Melanoma

•Frozen section H & E stained slides may not always permit clear visualization of melanoma cells because they are usually recognized by an artifact seen in formalin-fixed paraffin-embedded processing.

•With specific immunostains, melanocytes are readily identified in frozen sections, which facilitate determination of surgical margins for melanoma with MMS.

•MART-1 or Melan-A is the most commonly used immunostain for melanoma; however, HMB-45, Mel-5, S100, and MITF may also be useful in some cases.

15.3Melanoma

The standard treatment of melanoma (MM) is wide local excision, and size of the margins varies with tumor depth. Application of frozen section techniques like MMS for resection of melanoma is still considered controversial, but gradually becoming mainstream for the more ill-defined and difficult to remove melanomas like lentigo maligna (LM). The most significant limitation of this technique is that melanocytes can be difficult to find and distinguish from atypical keratinocytes along the dermal epidermal junction in chronically sundamaged skin. The microscopic feature of “retraction artifact” seen in permanent sections stained with H & E, which pathologists depend on to recognize melanocytes, is not consistently present on FS, and without precise identification of melanocytes, MMS cannot be considered a viable approach to interpreting surgical margins for MIS, that is, without the aid of immunostains (Fig. 15.4). On the contrary, investigators have reported that some architectural features in FS can be used to signal the presence of MIS. The basal layer may be disordered, and nesting of cells may be present.

Fig. 15.4 Biopsy of melanoma in situ in a permanent section stained with H & E at (a) 10×, and (b) 20× magnification, compared to a frozen section obtained during Mohs surgery at (c) 20× and (d) 40× magnification. The features of melanoma in

situ, including confluence and nesting, are far more apparent in the permanent section compared to the frozen section. MART-1 highlights (e) confluence of melanocytes along the interface and nesting within a follicular unit at 20× and (f) 40×

Fig. 15.5 Negative margins during Mohs surgery on frozen section stained with (a) H & E and (b) MART-1 (20×). The distribution of melanocytes within the epidermis is difficult to discern by H & E staining, but they label with MART-1. There is a

“false positive” pattern of confluence by (c) MART-1, but a more clear depiction of the density and diameter of melanocytes by (d) MITF at 40× magnification, indicating a “negative” margin for melanoma

Also, a dense dermal infiltrate immediately beneath the interface may signal the presence of melanoma [9].

Regardless of the theoretical limitations of MMS for melanoma, the recurrence rate after the procedure for primary lesions is only 1%, and for recurrent melanoma, about 10% [10]. In a 5-year study, MMS for melanoma with FS analysis was found to be equivalent or better than a historic control of wide local excisions for melanoma, with a recurrence rate of 0.5%. The average margin for MMS was 6 mm which cleared 83% of the patients [9], and the sensitivity and specificity values for the identification of melanoma ranges from 73% to 100% and 68% to 90%, respectively [11, 12].

With the advent of immunostaining, enhanced visualization of melanocytes has facilitated the use of MMS for melanoma. The immunostain most commonly used currently for melanoma is Melan-A or MART-1 (Figs. 15.4 and 15.5). Using immunohistochemistry, the requirements for reliable MMS results have been described as follows: the tumor cells must be visually identifiable in the section, the tumor must be contiguous to avoid false negatives, the mapping and staining component must be technically feasible, and the total tissue processing time should be short enough to allow for a staged excision and repair on the same day [13].

Melan-A or MART-1 is a 22 kDa cytoplasmic mel- anosome-associated glycoprotein recognized by mouse monoclonal antibodies A-103 and M2–7 C10 [6, 14]. It is a melanocyte differentiation antigen similar to gp100, gp75, and tyrosinase [6, 14]. It is present in 80–100% of melanomas and is also found in benign melanocytes within nevi and normal skin [15].

The sensitivity of this marker for melanocytes has been reported to be 75–92%, while the specificity is 97–100% [16]. Several studies have found this marker to be more sensitive than HMB-45, or more specific compared to S100 [15, 17, 18]. However, there are reports that MART-1 may not be very helpful in regard to differentiating pigmented actinic keratoses from MIS in heavily sun-damaged skin. In one study, MART- 1, HMB-45, S100, and tyrosinase were used to stain unequivocal pigmented actinic keratosis to determine the usefulness of these markers in sun-damaged skin [18]. MART-1 was found to stain numerous melanocytes in the areas of the actinic keratoses as well as adjacent sun-damaged skin, exceeding staining observed with the other markers [18]. In addition, four of ten cases stained with MART-1 revealed focal clusters suggestive of melanocytic nests [18]. This increased staining could be explained by an increased number of melanocytes, making this stain nonspecific in patients with sun-damaged skin or possibly due to the expression of MART-1 in keratinocytes or other nonmelanocytic cells damaged by inflammation [18–20]. As it applies to MMS, this can potentially lead the surgeon to excise greater margins in areas that appear to be melanoma but are in fact a false positive [21].

In one study, investigators used MART-1 immunostaining of FS material in hopes of differentiating melanoma from melanocytic hyperplasia in individuals with severely chronically sun-damaged skin. The study found that in normal sun-exposed skin, the melanocyte density is 15–20 per high-power field, confluence of up to nine melanocytes, and melanocytes were noted to extend along hair follicles [19]. They also noted the lack of pagetoid spread and nesting, but as previously mentioned, the MART-1 staining at times may stain clusters of cytoplasmic material that can simulate nests [18, 19]. The use of negative and positive controls can aid in interpreting the specimens. Some studies have used a biopsy from a similarly exposed body site, sometimes the contralateral side of the face, while others have utilized dog ears removed during reconstruction to serve as negative controls [7, 14–16, 18, 22]. The initial debulking specimen taken from the center

of the tumor, and often processed for a permanent section to assess Breslow depth, can also serve as a positive control. In addition, some surgeons may use two or more immunostains if margins still appear equivocal after the primary immunostain; however, this is not always feasible or cost-effective [17].

The results with MART-1-stained FS were found to correlate 100% with FFPES, in a study of patient with LM [15]. The MART-1 stain can take up to 2–3.5 h; however, various investigators have been able to shorten this time considerably. In one study of 40 patients with melanoma (24 MIS, 16 MM), the authors utilized a polymer-based immunoperoxidase stain which considerably shortened the blocking step of the procedure and eliminated the linking step where the chromagen would be linked to the secondary antibody (Fig. 15.3). This shortened the protocol to just 1 h, while still maintaining quality MART-1-stained sections in frozen material [7]. There have been other reports of processing time shortened to 19 min without significant differences in histologic features, including the number of keratinocytes, nuclear diameter of keratinocytes or melanocytes, number of melanocytes, the presence of confluence, pagetoid spread, and melanocytic nesting or atypical melanocytes when compared to H & E [22].

Another marker is HMB-45, human melanoma black-45, which is one of the first melanoma-specific markers, and a mouse monoclonal antibody that recognizes the 30–35 kDa cytoplasmic premelanosomal glycoprotein gp100 [6, 16]. This marker is present in stages I and II of melanosomes in neoplastic melanocytes and stages II and III of melanosomes in fetuses and infants [23]. There is a complete absence of keratinocyte staining with HMB-45, and it does not stain normal adult benign melanocytes [14, 24].

The sensitivity for HMB-45 ranges from 69% to 93%, with a higher range for primary melanoma (77– 100%) as compared to metastatic melanoma (56–83%) [16]. This marker has a greater specificity than S100, but a decreased sensitivity. Of particular note is that desmoplastic, spindle cell, and amelanotic melanomas may be negative for HMB-45, though with newer antigen retrieval techniques, the sensitivity increases to 75% for spindle cells melanomas [14, 25]. Due to the decreased sensitivity for MM and the weaker staining of the deeper component of the tumor, some authors recommend this stain be performed in conjunction with other stains [16]. This staining technique takes 90 min [24].

In one study of 20 patients (18 MIS, 2 MM) with melanoma, HMB-45 staining was found to be positive in 11 patients, which is equivalent to results with permanent sections. The sensitivity was 100%, and the specificity was 95% as there was one false positive [24]. In a case of a patient with recurrent acral melanoma, H & E–stained FS, FFPESs, and HMB-45-stained FSs were utilized. The authors found that the HMB-45 stain was comparable to the H & E–stained FS and FFPES, but was also strongly positive in areas where the FFPESs were equivocal or negative [10]. While it appears to be useful in some cases, HMB-45 is generally not commonly used for immunostaining during MMS.

S100 is a 21 kDa acidic calcium-binding protein, given its name because of solubility in 100% saturated ammonium sulfate solution [6, 14, 16]. S100 is present in the nucleus and cytoplasm of melanocytes. It stains benign melanocytic lesions, melanomas, glial cells, Schwann cells, skeletal and cardiac muscle, histiocytes, chondrocytes, and salivary and sweat glands. The sensitivity and specificity of this marker is 92–100% and 75–87%, respectively; consequently, it is more sensitive than HMB-45 and MART-1, but less specific than MART-1 [16]. This nonspecificity leads to increased background staining making the interpretation of the slide more difficult. It is superior to HMB-45, MART-1, and Mel-5 for the deeper component of tumors, but does not stain the epidermal component as reliably. Furthermore, it is the preferred stain for desmoplastic and spindle cell melanoma [17].

Mel-5, a mouse monoclonal antibody, recognizes the most ample glycoprotein in melanocytes, gp75. It is in the tyrosinase-related family of proteins and is found in melanosomes, particularly in stages III and IV. It stains normal fetal and adult melanocytes in the epidermis, benign nevi and melanoma, as well as areas of melanosome transfer such as the basal layer of the epidermis [14]. Therefore, this stain would appear useful for in situ melanomas. The specificity was found to be better than S100 but less than HMB-45 [14]. An increase in specificity generally correlates with a decrease in background staining. In a large study of 200 patients (158 LM, 42 LM melanoma), with both primary and recurrent tumors, Mel-5 immunostaining was utilized resulting in a 99.5% cure rate over a 38.4 follow-up period. This method added 40 additional minutes per stage, with half the time required if an autostainer was utilized [26].

In a study comparing MART-1, HMB-45, Mel-5, and S-100, 68 patients with melanoma (46 MIS, 22 MM) underwent MMS. MART-1 was found to be the most crisp with regular staining of basilar epithelial cells and easily interpretable stain with 96% of tumors staining positively. While S100 stained 100% of tumors positive, it had increased background staining making it difficult to interpret. Interestingly, in this study, only 50% of patients with MIS cleared with margins of </= 6 mm, while 96% cleared with 15 mm margins [14]. This is triple that of the wide local excision margins recommended currently for MIS, highlighting the benefits of MMS and immunostaining. Another study concluded with similar results in regard to the superiority of MART-1, with HMB-45 and S100 failing to recognize melanocytic proliferations in certain cases [27].

MITF, microphthalmia transcription factor, is a phosphorylation target in the Steel/c-kit signaling pathway. Stimulation of c-Kit results in MAP kinase– mediated phosphorylation of MITF producing transcriptional superactivation [28]. It is a transcription factor of the tyrosinase gene and is involved in development, survival, and differentiation of melanocytes [29]. MITF demonstrates nuclear staining, compared to stains such as HMB-45 or S-100 which are more cytoplasmic or diffuse. In one study of 76 patients (19 MIS, 50 MM, 7 metastatic melanoma), MITF stained 100% of melanomas on FFPES, including the amelanotic variant, while HMB-45 staining and S100 were 91% and 93% positive, respectively. The authors summarize that the nuclear staining pattern is useful as it helps reduce the background staining as well as diminish architectural changes (Fig. 15.5) [28].

Recently, a 35-min protocol for MITF on FS was made possible with the elimination of the surfactant in the buffer and use of diaminobenzidine hydrochloride and Giemsa counterstaining. The group compared FFPES to FS of sun-damaged and MIS specimens stained with MITF and MART-1. They found no difference in nuclear diameter or density between frozen and permanent sections for MITF, but did find a decrease in melanocytic density on FS compared to MART-1 from previously published data by the same group using FS material. They concluded that while it was a useful stain due to the nuclear staining pattern, allowing for easier identification of the melanocytes, they prefer to use the MITF stain concomitantly with the MART-1 stain [30].