LETTERS AND COMMUNICATIONS Detecting Spindle Cell Squamous Cell Carcinomas With Toluidine Blue on Frozen Sections Spindle cell squamous cell carcinomas are a rare variant of squamous cell carcinomas (SCCs). They typically arise in sun-exposed or radiation-exposed areas of elderly white men. Spindle cell SCCs can be more common in patients with a history of radiation, in those with burn scars, and in organ transplant recipients.1 These tumors can be highly aggressive with rapid growth, local recurrence, and metastatic potential.2 Histologically, the tumor is composed of spindle-shaped *cells with varying degrees of classic SCC histologic features. The tumor may form nests in the dermis, with or without a connection to the overlying epidermis. At the periphery of the tumor, the cells often infiltrate singly without forming nests or cords.2 The behavior of these single cells can make it extremely difficult when reading frozen sections without the help of immunohistochemical stains (i.e., 34betaE12, p63, AE1/AE3, low-molecular weight keratin, and P KER).1 Although these immunohistochemical stains are more specific and remain the gold standard for detecting spindle cell SCCs, toluidine blue is readily available during frozen sections and can help identify tumor cells. Most Mohs surgeons generally favor hematoxylin and eosin staining over toluidine blue when reading frozen sections for SCCs.3 Here, we report the benefits of staining with toluidine blue when detecting spindle cell SCCs. Spindle cell SCCs can have single infiltrating cells that can easily go undetected with just hematoxylin and eosin stain. Toluidine blue stains the mucopolysaccharides around these single tumor cells, which enables their detection. Methods and Observations A patient with a biopsy-proven diagnosis of spindle cell SCC was treated with Mohs surgery. Frozen sections (4 mm thick) from the Stage 1 tumor positive section block were stained with both toluidine blue and © 2014 by the American Society for Dermatologic Surgery, Inc. ISSN: 1076-0512 Dermatol Surg 2014;40:1259–1272
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hematoxylin and eosin using the Shandon Automatic Stainer (ThermoFisher Scientific Inc, Waltham, MA) with slide conveyer. The frozen sections were viewed and photographs were taken at ·40 and ·100 magnifications, corresponding to Figures 1 and 2, respectively. Figure 1A highlights the stromal changes from the pink staining of the mucopolysaccharides around the tumor cells (magnification at ·40). In Figure 2A, the pink surrounding the squamous cells helps delineate them from the surrounding inflammatory cells (·100). Toluidine blue also enhances the cellular detail, allowing detection of the squamous cells with the abundant eosinophilic cytoplasm and vesicular nucleus (arrows in Figure 2A). In Figure 2B, with hematoxylin and eosin, the squamous cells blend into the background of inflammatory cells and are more difficult to isolate. To validate our findings, we conducted a short survey with 3 independent Mohs surgeons who routinely use hematoxylin and eosin but did not perform the surgery or take pictures. Each Mohs surgeon was given 4 photographs (Figure 1A,B and Figure 2A,B). They had to identify whether tumor cells were present, choose whether hematoxylin and eosin or toluidine blue was easier to detect tumor cells, and pick which stain they would prefer if they had to look at another spindle cell SCC on a frozen section. All 3 Mohs surgeons detected the spindle cells with toluidine blue, whereas only 2 of 3 could detect with just hematoxylin and eosin stain. All 3 believed that it was easier to detect with toluidine blue stain and preferred toluidine blue over hematoxylin and eosin for identifying another spindle cell SCC during frozen sections. Discussion Toluidine blue is a metachromatic stain in which specific tissue structures react with the stain to produce a color, i.e., different from the dye. The Published by Lippincott Williams & Wilkins
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Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
LETTERS AND COMMUNICATIONS
Figure 1. (A) Toluidine blue—Pink staining of the mucopolysaccharides produced by the tumor cells. (B) Hematoxylin and eosin—No obvious stromal changes (original magnification ·40).
Figure 2. (A) Toluidine blue—infiltrative squamous cells (arrows) surrounded by pink stained mucopolysaccharides. Toluidine blue enhances the cellular detail of tumor cell with the abundant eosinophilic cytoplasm and vesicular nucleus. (B) Hematoxylin and eosin—squamous cells mixed in with inflammatory cells. It is difficult to visualize the tumor cells (original magnification ·100).
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mucopolysaccharides around tumor lobules stain a distinctive pink-to-magenta color, which enables better visualization of tumor. This stromal pattern is characteristically intense surrounding basal carcinomas and tends to be fainter with SCCs. Although the stromal staining of SCCs may not appear as strong as BCCs, this faint staining aids with tumor detection. Toluidine blue is also deemed valuable when evaluating subtle tumors such as infiltrative BCCs, infiltrative SCCs, perineural tumors, and microcystic adnexal carcinomas.4
and displaying the cellular detail of the tumor cell. In conclusion, toluidine blue can be used to supplement hematoxylin and eosin when performing frozen sections for the detection of spindle cell SCCs.
Spindle cell SCCs can behave aggressively and should warrant treatment with Mohs surgery. Although Mohs surgeons typically use hematoxylin and eosin staining when reading frozen sections for SCCs, spindle cell SCCs can be more infiltrative and difficult to interpret with only using hematoxylin and eosin without immunohistochemical stains. Toluidine blue is a cheap and reliable stain, i.e., readily available during frozen sections. Toluidine blue facilitates tumor detection by means of the pink-to-magenta stromal changes
3. Silapunt S, Peterson SR, Alcalay J, Goldberg LH. Mohs tissue mapping and processing: a survey study. Dermatol Surg 2004;30:961.
References 1. Morgan M, Purohit C, Anglin T. Immunohistochemical distinction of cutaneous spindle cell carcinoma. Am J Dermatopathol 2008;30: 228–32. 2. Cassarino DS, DeRienzo DP, Barr RJ. Cutaneous squamous cell carcinoma: a comprehensive clinicopathologic classification. J Cutan Pathol 2006;33:191–206.
4. Wang SQ, Goldberg LH, Nemeth A. The merits of adding toluidine bluestained slides in Mohs surgery in the treatment of a microcystic adnexal carcinoma. J Am Acad Dermatol 2007;56:1067–9.
Diane Trieu, MD Anna Drosou, MD Leonard H. Goldberg, MD Arash Kimyai-Asadi, MD DermSurgery Associates, Houston, Texas
DERMATOLOGIC SURGERY
Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
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hematoxylin and eosin using the Shandon Automatic Stainer (ThermoFisher Scientific Inc, Waltham, MA) with slide conveyer. The frozen sections were viewed and photographs were taken at ·40 and ·100 magnifications, corresponding to Figures 1 and 2, respectively. Figure 1A highlights the stromal changes from the pink staining of the mucopolysaccharides around the tumor cells (magnification at ·40). In Figure 2A, the pink surrounding the squamous cells helps delineate them from the surrounding inflammatory cells (·100). Toluidine blue also enhances the cellular detail, allowing detection of the squamous cells with the abundant eosinophilic cytoplasm and vesicular nucleus (arrows in Figure 2A). In Figure 2B, with hematoxylin and eosin, the squamous cells blend into the background of inflammatory cells and are more difficult to isolate. To validate our findings, we conducted a short survey with 3 independent Mohs surgeons who routinely use hematoxylin and eosin but did not perform the surgery or take pictures. Each Mohs surgeon was given 4 photographs (Figure 1A,B and Figure 2A,B). They had to identify whether tumor cells were present, choose whether hematoxylin and eosin or toluidine blue was easier to detect tumor cells, and pick which stain they would prefer if they had to look at another spindle cell SCC on a frozen section. All 3 Mohs surgeons detected the spindle cells with toluidine blue, whereas only 2 of 3 could detect with just hematoxylin and eosin stain. All 3 believed that it was easier to detect with toluidine blue stain and preferred toluidine blue over hematoxylin and eosin for identifying another spindle cell SCC during frozen sections. Discussion Toluidine blue is a metachromatic stain in which specific tissue structures react with the stain to produce a color, i.e., different from the dye. The Published by Lippincott Williams & Wilkins
· 1259
Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
LETTERS AND COMMUNICATIONS
Figure 1. (A) Toluidine blue—Pink staining of the mucopolysaccharides produced by the tumor cells. (B) Hematoxylin and eosin—No obvious stromal changes (original magnification ·40).
Figure 2. (A) Toluidine blue—infiltrative squamous cells (arrows) surrounded by pink stained mucopolysaccharides. Toluidine blue enhances the cellular detail of tumor cell with the abundant eosinophilic cytoplasm and vesicular nucleus. (B) Hematoxylin and eosin—squamous cells mixed in with inflammatory cells. It is difficult to visualize the tumor cells (original magnification ·100).
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mucopolysaccharides around tumor lobules stain a distinctive pink-to-magenta color, which enables better visualization of tumor. This stromal pattern is characteristically intense surrounding basal carcinomas and tends to be fainter with SCCs. Although the stromal staining of SCCs may not appear as strong as BCCs, this faint staining aids with tumor detection. Toluidine blue is also deemed valuable when evaluating subtle tumors such as infiltrative BCCs, infiltrative SCCs, perineural tumors, and microcystic adnexal carcinomas.4
and displaying the cellular detail of the tumor cell. In conclusion, toluidine blue can be used to supplement hematoxylin and eosin when performing frozen sections for the detection of spindle cell SCCs.
Spindle cell SCCs can behave aggressively and should warrant treatment with Mohs surgery. Although Mohs surgeons typically use hematoxylin and eosin staining when reading frozen sections for SCCs, spindle cell SCCs can be more infiltrative and difficult to interpret with only using hematoxylin and eosin without immunohistochemical stains. Toluidine blue is a cheap and reliable stain, i.e., readily available during frozen sections. Toluidine blue facilitates tumor detection by means of the pink-to-magenta stromal changes
3. Silapunt S, Peterson SR, Alcalay J, Goldberg LH. Mohs tissue mapping and processing: a survey study. Dermatol Surg 2004;30:961.
References 1. Morgan M, Purohit C, Anglin T. Immunohistochemical distinction of cutaneous spindle cell carcinoma. Am J Dermatopathol 2008;30: 228–32. 2. Cassarino DS, DeRienzo DP, Barr RJ. Cutaneous squamous cell carcinoma: a comprehensive clinicopathologic classification. J Cutan Pathol 2006;33:191–206.
4. Wang SQ, Goldberg LH, Nemeth A. The merits of adding toluidine bluestained slides in Mohs surgery in the treatment of a microcystic adnexal carcinoma. J Am Acad Dermatol 2007;56:1067–9.
Diane Trieu, MD Anna Drosou, MD Leonard H. Goldberg, MD Arash Kimyai-Asadi, MD DermSurgery Associates, Houston, Texas
DERMATOLOGIC SURGERY
Copyright © American Society for Dermatologic Surgery. Unauthorized reproduction of this article is prohibited.
