Evaluation of Nerves in Mohs Micrographic Surgery: Histologic Mimickers of Perineural Invasion and Nervous Tissue on Frozen Section IKUE SHIMIZU, MD,*

AND

VALENCIA D. THOMAS, MD†

BACKGROUND Perineural invasion (PNI) is an important histologic finding and may be a negative prognostic factor for squamous cell carcinoma (SCC). It may be associated with more-aggressive tumor behavior. Mohs surgeons encounter microscopic PNI regularly and must be able to diagnose it accurately to guide care decisions. OBJECTIVE To describe benign histologic mimickers of PNI and neural structures in SCC commonly encountered on frozen, hematoxylin and eosin–stained sections and to review how to differentiate them from PNI. METHODS AND MATERIALS Review of the literature regarding histologic mimickers of PNI and additional contributions to frozen section PNI and nerve tissue mimickers. RESULTS We describe benign findings, including arrector pili muscles, eccrine muscles, vessels, granulomatous inflammation, and eddies of SCC, that may each be mistaken for nerves or PNI. We discuss the ways in which they may be distinguished on frozen sections and review other commonly encountered entities that resemble PNI. CONCLUSION Perineural inflammation and peritumoral fibrosis are common mimickers of PNI on frozen section, although other mimickers exist on permanent sections. Normal structures may appear “neural” by way of frozen tissue orientation, processing, or inflammation and thus must be differentiated from nerve tissue and PNI during Mohs surgery. The authors have indicated no significant interest with commercial supporters.

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irst described in the 19th century by European scientists, perineural invasion (PNI) is a route of metastatic spread that plays a role in several varieties of cancer.1 It has been established as an important prognostic factor in squamous cell carcinoma (SCC) that can profoundly affect outcome.2 Disease-specific death risk of 16% has been reported for cutaneous SCC with PNI, compared with 4% for other high-risk SCC.3

In general, PNI can be broadly categorized into clinical PNI, in which neurologic symptoms or radiologic involvement are appreciated, and microscopic PNI, in which PNI is noted only on

histologic examination. Clinical PNI has a worse prognosis, but even microscopic PNI may have adverse effects.4 PNI of nerves smaller than 1 mm was shown to increase the risk of local recurrence and cervical metastasis for upper aerodigestive tract SCC.5 Miller and colleagues6 measured the extent of microscopic PNI for head and neck SCCs and observed that the maximum extent of PNI was significantly correlated with disease-free survival. There was a striking difference in recurrence, nodal metastases, distal metastases, and disease-specific death between cutaneous SCC with small-caliber (smaller than the median diameter of 0.09 mm) and large-caliber (larger than the median) nerve

*Mohs Unit, Department of Dermatology, Health Sciences Center, Texas Tech University, Lubbock, Texas; †Mohs Unit, Department of Dermatology, University of Texas MD Anderson Cancer Center, Houston, Texas © 2014 by the American Society for Dermatologic Surgery, Inc.  Published by Wiley Periodicals, Inc.  ISSN: 1076-0512  Dermatol Surg 2014;40:497–504  DOI: 10.1111/dsu.12473 497

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involvement.7 More recently, Lin and colleagues noted that nerve size based on a cut-off of 0.1 mm did not seem to affect survival for cutaneous SCC, whereas extent of nerve involvement did.8 With that in mind, dermatologic surgeons need to accurately recognize and evaluate PNI. For Mohs surgeons in particular, identifying PNI on frozen sections is fundamental to guiding decisions regarding the characterization of tumor aggressiveness and treatment. Green and colleagues recently noted that, despite concern over greater tissue distortion and artifact than with paraffin sections, frozen sections allow accurate evaluation of PNI.9 A confounding factor is that past studies have varied in their definition of PNI. Batsakis and colleagues defined it broadly as tumor cells in, around, and through nerves.10 Other definitions include epineurial, perineurial, or endoneurial tumor cells; tumor in close proximity to and involving at least 33% of nerve circumference1; tumor within the nerve sheath7; and tumor cells wrapping tightly around the perineurium, infiltrating the perineurium or nerve, or in close enough proximity to a nerve away from the main mass of tumor as to suggest neurotrophic spread.9 One review defined it simply as tumor cells in the perineural space, between the perineurium and nerve fascicle, or in equivocal cases, tumor encompassing nearly the nerve’s entire circumference at least.11 Such lack of standardization may explain variations in outcome and incidence, but whatever the definition, familiarity with the appearance of nerves, PNI, and potential confounders is critical. Here, we examine histologic mimickers of PNI and nerve tissue commonly seen on Mohs frozen sections.

PNI on Mohs Surgery Frozen Sections Peripheral Nerve Anatomy Peripheral myelinated and unmyelinated nerves are enclosed in a loose connective tissue layer termed the endoneurium (Figure 1). A second connective tissue

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Figure 1. Nerve fiber, cross-section, hematoxylin and eosin, original magnification 9400. Nerve fibers are enveloped by endoneurium (star) and grouped into fascicles surrounded by perineurium (triangle). Epineurium (circle) envelops individual fascicles (epifascicular) and the entire nerve fiber (epineurial).

layer, the perineurium, groups collections of these nerve fibers with their corresponding endoneurium into fascicles. Finally, the epineurium, divided into the epifascicular and epineurial parts, envelops individual fascicles and the entire nerve fiber, respectively. The perineural space surrounds the entire nerve.1,12 Nerves sectioned tangentially appear to have a wavy appearance, commonly with eosinophilic, granular-appearing cytoplasm. (Figure 2). Estimated measurements of nerve diameter can be based on the size of adjacent eccrine coils (50–80 lm). Perineural Invasion PNI, for the purposes of this article, is defined as tumor cells within any of the three layers of the nerve sheath or tumor foci outside of the nerve with involvement of at least 33% of the nerve’s circumference, as described by Leibig and colleagues1 (Figures 3 and 4). Perineural Inflammation and Peritumoral Fibrosis Although perineural inflammation may obscure PNI, it also may be an indication that there is nearby PNI,

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Figure 2. Nerve fiber, tangential section, hematoxylin and eosin, original magnification 9200. Note the granular appearance of the cytoplasm. From central to peripheral: endoneurium (star), perineurium (triangle), epineurium (circle).

Figure 4. Perineural squamous cell carcinoma (SCC), hematoxylin and eosin, original magnification 9200: nerve surrounded by large aggregates of SCC.

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Figure 3. Perineural squamous cell carcinoma (SCC), hematoxylin and eosin, original magnification 9200: SCC (arrowhead) noted between epineural (circle) and epifascicular (square) layers of epineurium surrounding nerve (star).

and such a case may warrant further frozen or paraffin sectioning8 (Figure 5). Perineural inflammation in the absence of hematologic malignancy commonly consists predominantly of normal-appearing lymphocytes with regular cell size, basophilic nucleus, and scant cytoplasm without cytologic atypia. Inflammation that includes neural and non-neural structures may be a clue to nonspecific inflammation rather than an indicator of possible tumor involvement, although isolated perineural inflammation may indicate nerve

Figure 5. Perineural inflammation, (A) hematoxylin and eosin (H&E), original magnification 9100; (B) H&E original magnification 9200. Note the normal epineurium without cytologic atypia.

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Figure 6. Perineural fibrosis, hematoxylin and eosin, original magnification 9200. Note the spindled fibroblasts and collagen.

involvement with tumor. Deeper sections may be required to further investigate the possibility of PNI. Peritumoral fibrosis is another entity that closely resembles PNI or can obscure it and yet be a marker for PNI (Figure 6). Hassanein and colleagues13 examined basal cell carcinomas (BCCs) and SCCs for peritumoral fibrosis, defined as concentric rings of peritumoral fibrosis within or around nests of tumor cells, and reported incidences of 5.8% in BCC and 4.5% in SCC and association with PNI. Fibrosis will show spindled fibroblasts and collagen rather than the wavy nuclei characteristic of nerve fibers. Being relatively common, peritumoral fibrosis is another finding that, much like PNI, warrants further examination of the tissue.14 Intuitively, the presence of such fibrosis or inflammation would seem to be the result of some reaction to tumor close by.

Mimickers on frozen sections during Mohs surgery Squamous Cell Carcinoma Squamous cell carcinoma is histologically characterized by aggregates of malignant, squamous

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Figure 7. Squamous cell carcinoma (SCC) mimicking perineural invasion, hematoxylin and eosin, original magnification 9400: Keratin (K); Aggregate of SCC. Note the consistently larger nuclear size of the cells at the periphery and within the SCC aggregate; nerves tend to have a smaller nuclear size.

epithelial cells with large, atypical nuclei; abundant eosinophilic cytoplasm; and intercellular bridges (in tumors without acantholysis).15 These tumors commonly form keratin pearls and squamous eddies that may be histologically similar to PNI. Examining the central aspect of the squamous pearl or eddy may provide valuable information regarding the structure. The presence of lamellar keratin, intercellular bridges, or characteristic vesicular nuclei would favor the histology of SCC. Identifying the structures of the nerve sheath, granules within the nerve fiber cytoplasm, or the smaller and more-uniform nuclear size of the cells at the center of the structure would favor the histology of a nerve (Figure 7). Pilosebaceous Units: Arrector Pili Muscles and Papillary Mesenchymal Bodies Arrector pili muscles are smooth muscle aggregates that originate in the dermis and insert into the pilosebaceous unit near the level of the sebaceous gland. These muscles consist of cells with

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eosinophilic cytoplasm and cigar-shaped nuclei (Figure 8A, B) The fascicular nature of these cells makes them a common mimicker of nerve tissue. When surrounded by inflammation, arrector pili muscles can be mistaken for PNI (Figure 9). Examination of ambiguous arrector pili muscles in serial sections should reveal adjacent hair follicles, similar structures at the same depth within the dermis, and an absence of the normal nerve sheath structures (epineurium, perineurium, endoneurium, and nerve fascicles). The papillary mesenchymal body is the cup-shaped invagination at the base of the hair follicle where the

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Figure 9. Lymphocytic inflammation surrounding an arrector pili muscle mimicking perineural invasion, hematoxylin and eosin, original magnification 9200: Muscle. Note the absence of nerve sheath structures and note an adjacent, uninvolved muscle.

elongated, follicular dermal papilla meets the vascular mesenchyme, which provides nutrients. Crosssection of this area of the follicle may appear as central epithelioid, pale cells ringed with squamous epithelium, similar to a nerve encased in carcinoma. The location high in the reticular dermis; the similarity between the outer, basophilic cells with those of adjacent hair follicles; and the absence of neural cytology favor this being a follicular structure (Figure 10). Vessels Vascular smooth muscle poses histologic challenges similar to those of the smooth muscle found in arrector pili muscles. Vascular structures are lined structures with muscle that can appear to have nerve-like cells. Examination of these structures for a lumen, red blood cells, or vascular smooth muscle with characteristic longitudinal and radial orientation of the muscle may be helpful in distinguishing these structures from nerves (Figure 11).

Figure 8. Cytology of an arrector pili muscle and a nerve fiber. (A) Cytology of an arrector pili muscle, hematoxylin and eosin (H&E), original magnification 9200, inset 9400; (B) Cytology of a nerve fiber, H&E, original magnification 9400.

Eccrine Glands Eccrine glands are composed of the coiled glandular unit located in the superficial fat, with coiled and

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(A)

Figure 10. Cross section of papillary mesenchymal body mimicking perineural invasion, hematoxylin and eosin, original magnification 9400. Note the central epithelioid, pale cells ringed with squamous epithelium reminiscent of a nerve fiber (arrows). The location high in the reticular dermis; the similarity between the outer, basophilic cells and adjacent hair follicles; and the absence of neural cytology favor this being a follicular structure.

straight ductal sections in the dermis and epidermis. These structures consist of two cell layers and are surrounded by a thick basement membrane. Examination of the ducts for lumens or a thickened basement membrane may be helpful in distinguishing these structures from neural structures. Eccrine squamous syringometaplasia, the inflammation or trauma-associated distortion of eccrine ducts, can mimic SCC.16 When it involves adjacent nerves, it can earn the name “re-excision PNI” and can mimic PNI (see discussion of re-excision PNI below)17 (Figure 12). Examination of adjacent eccrine acrosyringia will reveal similar dystrophic eccrine glands, which can help differentiate this benign metaplasia from SCC.

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Figure 11. Vessels mimicking nerve, hematoxylin and eosin, original magnification 9200: (A) Note the presence of a lumen and the alternating horizontal and vertical orientation of the vascular smooth muscle. (B) Original magnification 9400. Note the absence of the nerve fiber cytologic features.

Granulomatous Inflammation Foreign-body giant cells, with their peripheral nuclei and central pink cytoplasm, in a background of histiocytic inflammation as seen in a granuloma may resemble atypical squamous cells surrounding nerve structures. Examination of multinucleated cells for divisions between nuclei (cell membranes) will help distinguish these from discrete atypical cells surrounding pink nerve structures (Figure 13).

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Other Mimickers A variety of entities are more commonly encountered on paraffin sections that resemble PNI. Epithelial sheath neuroma, a rare benign tumor consisting of nerve bundles in the dermis surrounded by a benign squamous proliferation, can be easily mistaken for PNI.14,18 Re-excision PNI may also be seen on frozen section histology. This phenomenon

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Figure 13. Granulomatous inflammation with foreign body giant cells, hematoxylin and eosin, original magnification 9200: multinucleated cells with central pink cytoplasm may resemble atypical squamous cells surrounding a nerve structure.

Figure 12. Eccrine squamous syringometaplasia mimicking perineural invasion (re-excision perineural invasion). (A) Hematoxylin and eosin (H&E), original magnification 9200; (B) H&E original magnification 9100: Examination of adjacent eccrine acrosyringia reveals similar dystrophic eccrine glands (left arrow) to eccrine squamous syringometaplasia (right arrow).

arises when the benign squamous epithelium of adnexal structures becomes metaplastic or dystrophic in the setting of trauma or inflammation and begins to mimic carcinoma. If this squamous epithelium occurs in the vicinity of or within the perineural space through traumatic implantation, it earns the name, “re-excision PNI.”14,19 Also seen in noncutaneous malignancies, re-excision PNI has mostly been reported after an excision, although cases arising in association with prior biopsy or in the absence of preceding surgery have been

described.20 Eccrine squamous syringometaplasia involving the perineural space is an example of re-excision PNI (see discussion of eccrine squamous syringometaplasia above) (Figure 12). Reparative perineural proliferation is a proliferation of spindled cells of the perineurium secondary to prior trauma and may resemble aggregates of carcinoma in the perineural space. Morphologically, comparison of the perineural proliferation with the carcinoma undergoing excision should reveal less cellular pleomorphism and atypia than the malignant proliferation.14 Immunohistochemical staining with S100, cytokeratins, and epithelial membrane antigen aid in distinguishing these lesions.

Conclusion The ability to distinguish PNI from common mimickers based on cell morphology is critical in Mohs surgery. Histologic mimickers of PNI on hematoxylin and eosin–stained frozen sections commonly include normal skin structures (arrector pili muscles, eccrine coils, vessels), peritumoral fibrosis, perineural inflammation, and SCC. If a definitive conclusion

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cannot be reached based on morphologic findings or additional frozen-section levels, sending the Mohs tissue block for permanent sections or immunohistochemical staining with S100, cytokeratins, and epithelial membrane antigen can be of assistance.21 Although further studies are needed to better correlate nerve diameter with clinical outcome, the presence or absence of PNI is critically important regarding overall tumor behavior. When Mohs surgeons encounter microscopic PNI during surgery, patients should be appropriately staged according to the current AJCC criteria and counseled regarding their tumor prognosis. The AJCC 7th edition staging manual for cutaneous SCC includes PNI as a high-risk feature. When combined with another high-risk feature such as histologic grade or location on the ear or nonglabrous lip, PNI means upstaging from T1 to T2 and from Stage I to II.2 Given that Mohs surgeons often encounter aggressive SCCs or SCCS on the ear or nonglabrous lip, recognizing PNI and distinguishing it from benign mimickers on frozen Mohs sections is an essential component of management.

References 1. Liebig C, Ayala G, Wilks JA, Berger DH, et al. Perineural invasion in cancer: a review of the literature. Cancer 2009;115:3379–91. 2. Farasat S, Yu SS, Neel VA, Nehal KS, et al. A new American Joint Committee on Cancer staging system for cutaneous squamous cell carcinoma: creation and rationale for inclusion of tumor (T) characteristics. J Am Acad Dermatol 2011;64:1051–9. 3. Jambusaria-Pahlajani A, Miller CJ, Quon H, Smith N, et al. Surgical monotherapy versus surgery plus adjuvant radiotherapy in high-risk cutaneous squamous cell carcinoma: a systematic review of outcomes. Dermatol Surg 2009;35:574–85. 4. Mendenhall WM, Ferlito A, Takes RP, Bradford CR, et al. Cutaneous head and neck basal and squamous cell carcinomas with perineural invasion. Oral Oncol 2012;48:918–22. 5. Fagan JJ, Collins B, Barnes L, D’Amico F, et al. Perineural invasion in squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg 1998;124:637–40. 6. Miller ME, Palla B, Chen Q, Elashoff DA, et al. A novel classification system for perineural invasion in noncutaneous head

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and neck squamous cell carcinoma: histologic subcategories and patient outcomes. Am J Otolaryngol 2012;33:212–5. 7. Ross AS, Whalen FM, Elenitsas R, Xu X, et al. Diameter of involved nerves predicts outcomes in cutaneous squamous cell carcinoma with perineural invasion: an investigator-blinded retrospective cohort study. Dermatol Surg 2009;35:1859–66. 8. Lin C, Tripcony L, Keller J, Poulsen M, et al. Perineural infiltration of cutaneous squamous cell carcinoma and basal cell carcinoma without clinical features. Int J Radiat Oncol Biol Phys 2012;82:334–40. 9. Green JS, Tournas JA, Allen EJ, Youker SR, et al. Mohs frozen tissue sections in comparison to similar paraffin-embedded tissue sections in identifying perineural tumor invasion in cutaneous squamous cell carcinoma. J Am Acad Dermatol 2012;67:113–21. 10. Batsakis JG. Nerves and neurotropic carcinomas. Ann Otol Rhinol Laryngol 1985;94:426–7. 11. Dunn M, Morgan MB, Beer TW. Perineural invasion: identification, significance, and a standardized definition. Dermatol Surg 2009;35:214–21. 12. Stewart JD. Peripheral nerve fascicles: anatomy and clinical relevance. Muscle Nerve 2003;28:525–41. 13. Hassanein AM, Proper SA, Depcik-Smith ND, Flowers FP. Peritumoral fibrosis in basal cell and squamous cell carcinoma mimicking perineural invasion: potential pitfall in Mohs micrographic surgery. Dermatol Surg 2005;31:1101–6. 14. Dunn M, Morgan MB, Beer TW, Chen KT, et al. Histologic mimics of perineural invasion. J Cutan Pathol 2009;36: 937–42. 15. Pathology and Genetics of Tumours of the Skin [Internet]. Lyon: International Agency for Research on Cancer (France) 2012. Available from: http://www.iarc.fr/en/publications/pdfs-online/ pat-gen/bb6/index.php/ Accessed May 12, 2012. 16. Tan KB, Tan SH, Aw DC, Jaffar H, et al. Simulators of squamous cell carcinoma of the skin: diagnostic challenges on small biopsies and clinicopathological correlation. J Skin Cancer 2013;2013:752864. [Internet]. Available from: http://www.ncbi. nlm.nih.gov/pmc/articles/PMC3708441// Accessed August 3, 2013. 17. Serrano T, Saez A, Moreno A. Eccrine squamous syringometaplasia: a prospective clinicopathologic study. J Cutan Pathol 1993;20:61–5. 18. Husain EA, Al-Daraji WI. Epithelial sheath neuroma: be aware of benign perineural invasion!. J Cutan Pathol 2009;36:570–2. 19. Beer TW. Re-excision perineural invasion in the skin. J Clin Pathol 2011;64:936. 20. Chen KT. Reactive neuroepithelial aggregates of the skin. Int J Surg Pathol 2003;11:205–10. 21. Miller CJ, Sobanko JF, Zhu X, Nunnciato T, et al. Special stains in Mohs surgery. Dermatol Clin 2011;19:273–86.

Address correspondence and reprint requests to: Ikue Shimizu, MD, 3601 4th Street, MS 9400, Lubbock, Texas 79401, or e-mail: [email protected]

Evaluation of nerves in Mohs micrographic surgery: histologic mimickers of perineural invasion and nervous tissue on frozen section.

Perineural invasion (PNI) is an important histologic finding and may be a negative prognostic factor for squamous cell carcinoma (SCC). It may be asso...
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