The Skin
Christopher Kinonen
Vijaya B. Reddy
Introduction
Skin cancer is the most common form of cancer in the United States, with basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) being the two most frequent types of dermatologic malignancies. Melanoma is the third most common type of skin cancer. In 2008, melanomas accounted for 59,695 new cancer diagnoses and 8,623 deaths (1). Surgical resection is the mainstay of treatment for these lesions, and many can be resected without frozen section analysis. In fact, routine frozen section analysis on all skin specimens is not clinically indicated, can be very time and resource intensive, and in many cases is not cost effective (2). Some skin lesions, however, require a primary diagnosis by frozen section with margin assessment to determine benign versus malignant or primary versus metastatic. Overall, accuracy of frozen section analysis of skin specimens has been reported to range from 71% to 99% (3,4). Literature evaluating the usage of frozen section for specific skin cancers primarily focuses on BCC, SCC, melanoma, and Mohs micrographic surgery.
For BCC, complete excision results in 99% cure rate while recurrence rates for incompletely excised tumors have been reported to be between 19% and 67% (5). Incompletely excised SCC has similar recurrence rates ranging from 30% to 60% (6); thus, accurate assessment of resection margins is critically important. In a study of 450 patients, Cataldo et al. reported complete excision, without the aid of frozen section, in 90% of patients with primary tumors having clinically distinct borders. However, in recurrent tumors, where microscopic foci can extend beyond clinically detectable margins, 24% of patients had incomplete excisions when frozen section analysis was not performed. The authors concluded that routine frozen section examination of primary lesions with clinically distinct borders was of little benefit. For recurrent lesions, when margins were clinically indistinct, or when tissue preservation was deemed important, frozen section may be of value (2). Similar conclusions regarding frozen section usage for skin specimens have been documented in other literature as well (3,4,7).
Thus, for nonmelanoma skin cancers, there are several general indications for frozen section. Appropriate lesions for frozen section include
those that are large, clinically ill defined, recurrent, or have an infiltrating growth pattern. Other indications include lesions located in areas where wide margin excision is not possible or tissue sparing is desirable, such as the eye, forehead, cheek, nose, and ears. Eyelid tumors, in particular, have shown benefit from frozen section margin assessment (2). In addition, frozen section diagnoses may be indicated if reconstructive surgery is to be performed immediately following resection or if there is a question as to whether the primary lesion is benign or malignant. Although metastatic disease to the skin is uncommon, it has been reported in 5% to 10% of internal malignancies (8) where it occurs as the initial manifestation of an undiscovered malignancy or potentially as the first indication of metastasis of a previously diagnosed and treated tumor. These lesions may also require frozen section for primary diagnosis.
those that are large, clinically ill defined, recurrent, or have an infiltrating growth pattern. Other indications include lesions located in areas where wide margin excision is not possible or tissue sparing is desirable, such as the eye, forehead, cheek, nose, and ears. Eyelid tumors, in particular, have shown benefit from frozen section margin assessment (2). In addition, frozen section diagnoses may be indicated if reconstructive surgery is to be performed immediately following resection or if there is a question as to whether the primary lesion is benign or malignant. Although metastatic disease to the skin is uncommon, it has been reported in 5% to 10% of internal malignancies (8) where it occurs as the initial manifestation of an undiscovered malignancy or potentially as the first indication of metastasis of a previously diagnosed and treated tumor. These lesions may also require frozen section for primary diagnosis.
Malignant melanoma represents a different type of skin cancer, and frozen section analysis should be discouraged for suspected melanocytic lesions. Depth of invasion, presence of ulceration, and margin assessment are critically important factors for diagnosis, prognostication, and treatment of melanoma. Freezing causes tissue distortion, which can preclude accurate measurement of depth of invasion on the permanent sections. Similarly, the ability to determine cytologic atypia is compromised with freezing. This can be especially problematic when assessing intraepidermal spread of single atypical melanocytes or on sun-damaged skin that frequently contains melanocytic hyperplasia. Determining the degree of atypia in these specimens can be difficult enough on permanent sections; freezing artifact should not be added as an additional pitfall. Rare cases when freezing may be appropriate include a lesion in a cosmetically important area, such as the face, or in a difficult-to-heal surgical site (e.g., acral sites such as the heel). In general, however, frozen section evaluation of melanocytic lesions should be discouraged and if performed, the surgeon should be made aware of the pitfalls and limitations. The use of frozen section in Mohs micrographic surgery to evaluate melanomas is discussed below.
Literature evaluating the role of frozen section in non-neoplastic lesions such as necrotizing fasciitis, toxic epidermal necrolysis/erythema multiforme (TEN/EM), calciphylaxis, vasculitis, and infections is less extensive. Nevertheless, skin biopsies to evaluate these life-threatening conditions are frequently requested in a tertiary care setting. Early, accurate diagnosis in these cases is essential to implement potentially life-saving therapies. In a study by Majeski and Majeski, 43 patients had bedside biopsies to evaluate for necrotizing fasciitis by frozen section. Twelve of the forty-three patients were diagnosed as such and each had immediate surgical debridement and supportive therapy. All patients survived (9).
Mohs Micrographic Surgery
Mohs micrographic surgery is a procedure for selected skin cancers intended to resect tumors with complete assessment of surgical margins and minimal wound size. It involves removing the tumor in stages and
microscopically examining the entire resection margin. Since tissue sparing is a primary goal of the surgery, it can be useful in anatomically and cosmetically important sites such as the eyes, ears, nose, and lips. It can also be used for recurrent tumors or lesions, which have a high likelihood of recurrence (10,11). Mohs surgeons are trained to histologically evaluate their resections, so general surgical pathologists are typically not involved with frozen section examination. This is not the case at all medical centers, however, so it is important for the surgical pathologists to be aware of this procedure as an alternative to traditional dermatologic resections. The exact procedure and method of cutting the tissue for frozen section is primarily dependent on the specific Mohs surgeon as well as the lesion itself; thus, the following information is intended to provide a general overview of the process. The procedure is typically done as an outpatient procedure using local anesthesia. The surgeon begins by removing visible cancer along with a thin rim of surrounding tissue extending peripherally and deep to the lesion. The specimen is then oriented, sectioned, and inked before standard frozen section processing. The specimen is cut such that 100% of the surgical margin is evaluated. The location of each positive margin is then carefully mapped so that the procedure can be repeated in those areas as many times as necessary to completely excise the tumor. A final margin can then be taken from around the tumor and sent for permanent sections to confirm the initial frozen section margin assessment (12).
microscopically examining the entire resection margin. Since tissue sparing is a primary goal of the surgery, it can be useful in anatomically and cosmetically important sites such as the eyes, ears, nose, and lips. It can also be used for recurrent tumors or lesions, which have a high likelihood of recurrence (10,11). Mohs surgeons are trained to histologically evaluate their resections, so general surgical pathologists are typically not involved with frozen section examination. This is not the case at all medical centers, however, so it is important for the surgical pathologists to be aware of this procedure as an alternative to traditional dermatologic resections. The exact procedure and method of cutting the tissue for frozen section is primarily dependent on the specific Mohs surgeon as well as the lesion itself; thus, the following information is intended to provide a general overview of the process. The procedure is typically done as an outpatient procedure using local anesthesia. The surgeon begins by removing visible cancer along with a thin rim of surrounding tissue extending peripherally and deep to the lesion. The specimen is then oriented, sectioned, and inked before standard frozen section processing. The specimen is cut such that 100% of the surgical margin is evaluated. The location of each positive margin is then carefully mapped so that the procedure can be repeated in those areas as many times as necessary to completely excise the tumor. A final margin can then be taken from around the tumor and sent for permanent sections to confirm the initial frozen section margin assessment (12).
Extensive literature exists pertaining to the indications and therapeutic success of Mohs surgery. It has been reported to be superior to standard resection for nonmelanoma malignancies that display features that increase the risk of recurrence (11). Success is probably largely dependent on the experience and skill of the Mohs surgeon as well as associated histotechnologists who prepare and process the tissue. As stated previously, it is commonly performed in sites where cosmesis is of concern. In addition, it can be used for recurrent tumors or tumors with increased risk of recurrence. The procedure has been utilized with multiple types of nonmelanoma malignancies including, but not limited to, BCC, SCC, dermatofibrosarcoma protuberans (DFSP), atypical fibroxanthoma, microcystic adnexal carcinoma, and leiomyosarcoma (13).
Mohs micrographic surgery is also increasingly performed on certain low-risk subtypes of melanoma, such as lentigo maligna melanoma and melanoma in situ, and in melanomas that are clinically ill defined. However, its usage with melanocytic lesions remains controversial primarily because of the questionable accuracy of detecting atypical melanocytes on the frozen section (11). This can be especially difficult on chronically sun-damaged skin, which frequently contains prominent melanocytes with pleomorphic hyperchromatic nuclei (14).
Specimen Types and Biopsy Techniques
Common skin biopsy techniques that may be submitted for frozen section analysis include punch biopsies, superficial and deep shave biopsies,
curettage, and excisions. There are specific clinical indications for each biopsy type, which can assist with the differential diagnosis. Additional factors that influence biopsy technique include anatomic site, size and shape of the lesion, cosmesis, and general health of the patient (8).
curettage, and excisions. There are specific clinical indications for each biopsy type, which can assist with the differential diagnosis. Additional factors that influence biopsy technique include anatomic site, size and shape of the lesion, cosmesis, and general health of the patient (8).
A punch biopsy is often performed to evaluate for inflammatory dermatoses, but may also be used to completely excise small lesions or sample large lesions. Punch biopsies can be 2 to 8 mm in diameter. The first step, as with any skin specimen, is to orient, measure, and describe the specimen, including skin color and presence or absence of lesions. If lesions are present, describe the size, type (e.g., papular, vesicular, or macular), color, borders (irregular vs. well circumscribed), and distance to the closest margin. Ink the margin of resection, a single color is adequate. For 3-mm punch biopsies, the entire lesion can be submitted without sectioning. Larger biopsies require bisecting or trisecting, so as to have sections no larger than 3 mm in thickness. Maintain vertical orientation when sectioning. Ideally, at least two levels should be obtained for permanent sections.
Shave biopsies can either be superficial or deep, and are used primarily for nonmalignant lesions in which the key histologic changes are expected to be present in the epidermis or superficial dermis, such as seborrheic keratoses, solar keratoses, and benign nevi. Shave biopsies can also be used to diagnose BCCs, since this technique will leave the lower portion of the dermis intact and amenable to subsequent complete resection. First, orient, measure, describe, and ink the resection margins of the specimen. If the specimen is more than 3 mm in diameter, bisect or trisect so that vertical orientation is maintained. Generally, the entire specimen should be submitted for frozen section. Order two levels on the permanent block of the frozen section.
Curettage specimens are meant to be diagnostic, usually for seborrheic keratosis, actinic keratosis, or BCC, and may consist of multiple variably sized tissue fragments. These specimens may be difficult or impossible to orient as they are usually superficial and without architectural landmarks. Note the number of fragments (or an estimate of the number), aggregate measurement, and color. Inking is not necessary since tissue fragmentation precludes margin evaluation. Order two levels on the permanent block of the frozen section.
Excisions are generally received as skin ellipses, although they may be round or irregularly shaped. These procedures are most commonly performed to remove malignant tumors such as BCC, SCC, melanoma, or atypical melanocytic nevi. They may also be used for nonmalignant lesions such as panniculitis. Excisions are usually oriented in some manner (e.g., with suture) and the purpose of frozen section in most instances is to assess margins. Less frequently, a primary diagnosis will be requested. Although excisions can be very large, most of the ones that are sent for frozen section evaluation are small enough so that the entire specimen can be submitted.
The initial steps to process an excision are to orient, measure (in three dimensions), and describe the specimen including skin color. Also note the presence of lesions, their size, type (e.g., papular, vesicular, or macular), color, and borders (irregular vs. well circumscribed). There is typically a suture, or another identifying mark, that is designated 12 o’clock. If no orientation is provided, speak with the surgeon to clarify if orientation is needed. Next, ink the resection margins so as to maintain orientation. If no orientation is provided, the entire margin can be inked one color, otherwise, ink so that margins in four quadrants can be identified microscopically. Inking is a crucial step because the location of involved margins must be correctly identified, as well as the distance of the tumor from the margin. Next, serially section the entire specimen along the short axis, in 3-mm intervals, to evaluate the relationship of the lesion(s) to the margins as well as to identify any additional deep lesions. In most cases, the entire specimen should be submitted for frozen section. If the specimen is larger and cannot be processed in four blocks, a cross section through the center and perpendicular margins of the tips can be submitted for frozen sections. The closest margin should always be evaluated with either an en face or perpendicular cut. The remainder of the specimen can be processed for permanent sections (8,15).
If any of the margins are positive for tumor, an additional strip of skin will often be re-excised and also sent for frozen section. It is important that the true resection margin be identified, either by the surgeon directly or by some other indication (e.g., suture), so that ink may be properly applied. The additional material may then be submitted en face or horizontally sectioned (7).
Generally, sections should be no larger than 0.3 cm in thickness, and are placed on a thin layer of media that is prefrozen on a chuck. Length and width are dependent on chuck size but should probably not be larger than 1 × 1 cm. If multiple pieces are submitted, they should each be placed in the same plane (7).
Cytologic preparations are not necessary for margin evaluation but may be required in cases of primary diagnoses. If performed, a touch preparation as well as a smear are recommended on separate slides. These are particularly useful in diagnosis of small blue cell tumors involving the skin, such as Merkel cell carcinoma and lymphoma.
The standard stain for frozen section processing in general surgical pathology laboratories is hematoxylin and eosin (H&E). Toluidine blue is an alternate stain used by some Mohs surgeons, particularly for BCC, because it is more rapid (7). Toluidine blue metachromatically stains the mucopolysaccharides and hyaluronic acid in the stroma present around BCC tumor cells. This yields a characteristic magenta color that contrasts with the dark blue stain of the actual tumor cells. This staining is said to help differentiate BCC from tangentially cut hair follicles and benign follicular tumors (16). It can also be helpful if there is obscuring inflammation. For cytologic preparations, a Romanowsky stain is the standard.
Basal Cell Carcinoma
BCC typically affects older individuals, most often occurring as single lesions on sun-exposed hair-bearing skin, particularly the face. BCC may appear as small well-circumscribed pearly tan-gray papules devoid of scale. Over time, the lesions enlarge and tend to ulcerate giving the characteristic “rodent ulcers” (8). Most often, these lesions have been previously biopsied, and a specimen will be sent for frozen section to assess excisional margins. Less frequently, a primary diagnosis may be requested.
The general histopathology of BCC shows prominent nests and islands of basaloid cells attached to the undersurface of the epidermis, invading to varying degrees into the dermis. These nests will show characteristic palisading of basaloid cells at the periphery. The tumor cells tend to be uniform with frequent mitoses. Other important features include the presence of clefting artifact between the epithelium and the stroma as well as the connective tissue stroma itself that proliferates around the tumor. This stroma tends to be composed of parallel bundles of young fibroblasts, occasionally in a mucinous background. There are multiple variants of BCC including superficial, nodular, sclerosing (morphea-like), keratotic, adenoid, and fibroepithelial (8). The most common subtype evaluated for frozen section at Rush University Medical Center is the nodular subtype and less commonly the superficial and sclerosing variants.
The nodular subtype of BCC shows a nodular proliferation of uniform basophilic tumor cells with characteristic peripheral palisading. The nodules are larger and typically extend deeper into the dermis than with the superficial variant (Fig. 14.1).
Superficial BCC shows buds and irregular proliferations of tumor cells, with peripherally palisading nuclei, attached to the undersurface of the epidermis with minimal penetration into the dermis (Fig. 14.2
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