What is your diagnosis? Image credits: Eva A. Hurst, MD; Washington University in St. Louis

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1 Cells to Surgery Quiz: September 2018 Eric P. Sorensen, Jessica B. Dietert, Eva A. Hurst Division of Dermatology, Washington University in St. Louis, Saint Louis, MO, USA What is your diagnosis? Image credits: Eva A. Hurst, MD; Washington University in St. Louis Questions 2 & 3 refer to the article by C. Brugière et al et al. (doi: /j.jid )

2 1. What is your diagnosis? a. Infiltrative basal cell carcinoma b. Squamous cell carcinoma with perineural invasion c. Epithelial sheath neuroma d. Desmoplastic trichoepithelioma e. Granular cell tumor ANSWER: B. Squamous cell carcinoma with perineural invasion Explanation: Cutaneous squamous cell carcinoma (SCC) is the second most common form of skin cancer after basal cell carcinoma, with an estimated lifetime risk of 9-14% for men and 4-9% for women in the United States (US) (Miller et al, 1994). Cutaneous SCC commonly presents as a tender, erythematous hyperkeratotic papule or nodule on chronically sundamaged skin, in particular the head and neck. Risk factors for the development of cutaneous SCC include age, cumulative sun exposure, and immunosuppression. While the prognosis of cutaneous SCC is mostly favorable, there are an average of 3000 disease related deaths that occur annually in the US (Karia et al, 2013). The National Comprehensive Cancer Network (NCCN), American Joint Committee on Cancer (AJCC), as well as a group out of Brigham and Women s hospital (Jambusaria-Pahlajani et al, 2013), have all proposed grading and staging criteria to characterize high risk cutaneous SCC. Perineural invasion remains an important histopathologic factor for high risk cutaneous SCC, which has been defined as tumor cells infiltrating the perineural space (between the perineurium and the nerve) (Ross et al, 2009), or for T3 tumors as tumor cells within the nerve sheath of a nerve lying deeper than the dermis or measuring 0.1 mm or larger in caliber, or presenting with clinical or radiographic involvement of named nerves without skull base invasion or transgression (NCCN guidelines ). The clinical photo shows a growing nodule on the pre-auricular area of a heart transplant patient. On histopathological analysis, a proliferation of atypical keratinocytes with mitotic figures visible is seen invading the deep dermis, and encasing a small nerve (arrow). Discussion of incorrect answers a. Basal cell carcinoma (BCC) remains the most common form of skin cancer, and form of human malignancy. Surgical treatment of BCC is curative in the vast majority of cases, however there are certain high-risk features of BCC that are associated with greater local recurrence rates. Several high risk histopathological subtypes include the infiltrative, morpheaform, sclerosing, micronodular and metatypical subtypes. The infiltrative pattern of a basal cell carcinoma is characterized by spiky islands of basaloid cells with occasional areas of

3 squamous differentiation in a fibroblast-rich stroma. Perineural extension can be noted with this subtype (Elston and Ferringer, 2009). c. Epithelial sheath neuroma is a rare entity characterized clinically by a pink papule that may mimic a basal cell or squamous cell carcinoma. Histologically, its appearance may elicit concern for a carcinoma with perineural invasion. Characteristic histologic features include multiple enlarged peripheral nerve fibers ensheathed by mature squamous epithelium, generally limited to the superficial dermis, surrounded by a loose myxoid stroma, lymphocytic infiltrate and occasionally infundibular cysts (Hirano-Ali et al, 2016). The neural elements stain positive with S100 and the epithelial components stain with cytokeratins. This entity may be differentiated from perineural invasion of a keratinocyte derived carcinoma by the absence of cellular atypia and absence of neural enlargement (Hirano-Ali et al, 2016). There is ongoing discussion as to whether the epithelial sheath neuroma represents a reactive process to irritation or a form of benign neoplasia (Requena, 2016). d. Desmoplastic trichoepitheliomas are benign hamartomatous growths that are typically found as annular, firm asymptomatic white-yellow papules on the head and neck of young women. On histology, these tumors appear as thin strands of basaloid cells in a paisley-tie pattern, with associated keratinous cysts and calcifications in a desmoplastic stroma (Mamelak et al, 2010). The major histologic differential diagnosis is a morpheaform basal cell carcinoma (Elston and Ferringer, 2009). e. Granular cell tumor is a rare neoplasm of schwannian origin that primarily presents on cutaneous and mucosal sites on the head and neck. On histopathological analysis, sheets of large polygonal cells with abundant eosinophilic granular cytoplasm are seen in the dermis. On higher power, discrete giant lysosomal granules are visualized. Overlying pseudoeptheliomatous hyperplasia may be mistaken for a squamous cell carcinoma. In some cases, tumor cells may be seen encasing nerves. The neoplasm stains positive for S-100 and CD68 (Rekhi and Jambhekar, 2010).

4 2. A diagnosis of squamous cell carcinoma (SCC) with perineural invasion is made. Which of the following statements is true regarding the staging or treatment of cutaneous SCC? a. There currently exists several high quality randomized controlled trials comparing the efficacy of various excision margins in the treatment of cutaneous SCC b. The National Comprehensive Cancer Network (NCCN) practice guidelines for cutaneous neoplasms, define high risk SCC as tumors located in area L or M (low- or medium-risk zones) 10 mm or those in area H (high-risk zone) of any size c. The NCCN practice guidelines for cutaneous SCC indicates that perineural invasion is an indication for sentinel lymph node biopsy d. Patients diagnosed with cutaneous SCC with perineural invasion should only be treated with primary radiation or systemic immunotherapy, such as with the programmed cell death 1 inhibitor, pembrolizumab e. Mohs micrographic surgery remains the most curative of all current treatment modalities for patients diagnosed with cutaneous SCC with perineural invasion ANSWER: E. Mohs micrographic surgery remains the most curative of all current treatment modalities for patients diagnosed with cutaneous SCC with perineural invasion. Explanation: There are no existing randomized controlled trials or prospective cohort studies comparing the efficacy of Mohs micrographic surgery (MMS) versus other treatment modalities for cutaneous SCC (Work Group, 2018). However, a retrospective systematic review has been published that compares treatment modalities and found that the 5 year local recurrence rate was 3.1% (N=2065) for MMS, 3.7% (N=82) for electrodessication and curettage, 8.1% (N=124) for surgical excision, and 10.0% (N=160) for radiation therapy (Rowe et al, 1992). Since then, a 10 year prospective multicenter case series of cutaneous SCC treated with Mohs in Australia (N=1263), found a similarly low 5 year local recurrence rate of 3.9%. (Leibovitch et al, 2005). The management of high-risk cutaneous SCC is still under some debate (NCCN Work Group, 2018). Authors of a large single center retrospective cohort study (N=260) concluded that Mohs surgery is effective for the treatment of high-risk SCC after they found a 2 year local recurrence rate of 1.2% (Pugliano-Mauro et al, 2010). However, other authors have acknowledged that there still remains great variability in the management of high-risk SCC. According to a survey of 118 members of the American College of Mohs Surgery (approximately 25% of its members), responders were significantly divided as to which high risk features warranted radiographic nodal staging

5 (RNS), sentinel lymph node biopsy (SLNB) or adjuvant radiation therapy (ART) (Jambusaria-Pahlajani et al, 2010). However, a significant majority agreed that perineural invasion and satellite or in-transit metastasis were criteria for RNS, SLNB or ART. Indeed, perineural invasion (PNI) has been more well recognized as a poor prognostic indicator for cutaneous SCC (Carter et al, 2013). In particular, a recent systematic review noted that clinical PNI (patients with symptoms such as pain, numbness, tingling, or paralysis) or radiologic evidence of PNI, had higher rates of 5 year local recurrence (37% vs. 17%) and disease-specific death (27% vs. 6%) when compared to patients with incidental PNI noted only during time of surgery on histologic examination (Karia et al, 2017). Discussion of incorrect answers a. There does not currently exist a randomized controlled trial comparing different excision margins for the treatment of cutaneous SCC. A retrospective systematic review by Lansbury et al (2013) examined 12 studies that analyzed local recurrence rates amongst cutaneous SCC treated with various surgical margins. In that study, the average recurrence rate was 5.4% (N=1144), with margins ranging from 2 10 mm. Of note, current NCCN guidelines recommend 4 6 mm margins for the excision of low-risk cutaneous SCC. b. The National Comprehensive Cancer Network (NCCN) clinical practice guidelines stratify cutaneous SCC into low and high risk features based on a number of clinical and histopathological features. For location and size of tumor, high risk cutaneous SCC is defined as lesions in area L (low-risk zone) 20 mm, area M (medium-risk zone) 10 mm, and area H (high-risk zone) of any size. Other features of high risk cutaneous SCC include patient history of immunosuppression, neurologic symptoms, recurrent lesions, poorly differentiated tumors, perineural or lymphovascular invasion and high risk histologic subtypes, such as acantholytic, adenosquamous, desmoplastic or carcinosarcomatous subtypes (NCCN Work Group, 2018). c. Perineural invasion is amongst the histopathologic factors defining high risk cutaneous SCC. While retrospective and prospective case series have demonstrated a potential prognostic role of sentinel lymph node biopsy (SLNB) in high risk cutaneous SCC (Ross et al, 2006), there are still no definitive guidelines on the indications for SLNB in high risk cutaneous SCC by the NCCN (Work Group, 2018). This is largely because there is a lack of randomized controlled trials on the incidence of, and the prognostic significance associated with a positive SLNB in patients with a high risk cutaneous SCC. One comprehensive systematic review found an overall positive SLNB rate of 13.9% (32 of 231 patients) of SCCs having high risk features although these factors were heterogeneous and not always well defined (Navarrete-Dechent et al, 2015). Takahashi et al (2014) provide one of the only studies evaluating long-term

6 outcomes of SLNB for high risk cutaneous SCC and found a 3-year survival of 100% for SLN-negative SCC, but only 20.8% for SLN-positive cases. d. Surgery remains the first line treatment for cutaneous SCC, including those with high risk features such as perineural invasion. Primary radiation can be used in certain special situations if surgery is not feasible, or contraindicated, or if the patient does not desire surgery. Several different radiotherapy modalities include superficial radiation therapy, isotope-based brachytherapy or external electron beam radiation (Work Group, 2018). The PD1 inhibitor pembrolizumab was recently FDA approved in the treatment of advanced non-cutaneous head and neck SCC. There is ongoing interest and research in the use of PD1 inhibitors in the case of locally advanced and metastatic cutaneous SCC, and clinical trials exist ( but surgical resection of the primary tumor is still performed in cases when possible (Work Group, 2018).

7 3. Brugiere et al presented novel findings relating to the molecular factors involved in perineural invasion (PNI) in human cutaneous squamous cell carcinoma. Which of the following is true? a. Using immunohistochemistry, perineural tumor cells demonstrated a strong expression of BDNF and TrkB b. Using digital droplet PCR, perineural tumors cells demonstrated a significant upregulation of E-cadherin and downregulation of Snail1 c. Compared with tumor cells distant from PNI areas, perineural tumor cells demonstrated marked decrease in immunostaining of NCAM1 d. In perineural invasion cells, there were significantly elevated mrna expression levels for the neurotrophic receptors TrkA and TrkC e. This study demonstrates the first tumor model showing an association between the TrkB pathway and epithelial-mesenchymal transition ANSWER: A. Using immunohistochemistry, perineural tumor cells demonstrated a strong expression of BDNF and TrkB Explanation: Brugiere et al (2018) studied human cutaneous squamous cell carcinomas and isolated tumor cells demonstrating perineural invasion (PNI), as well as tumor cells distant from PNI areas by utilizing a precise laser microdissection technique. In the study, immunohistochemical analysis showed perineural tumor cells had a significantly increased expression of BDNF, TrkB, and p75ngfr, while tumors cells distant from PNI areas did not. Tropomyosin receptor kinase B (TrkB) is a membrane bound high-affinity receptor for the so-called neurotrophins, in particular BDNF. Trk receptors help to regulate key pathways modulating cellular proliferation including the PI3/AKT and RAS/MAPK pathways (Huang and Reichardt, 2003). Specifically, TrkB has been shown to suppress anoikis, the process of programmed cell death after detachment of cells from the extracellular matrix, and induce metastasis in rat intestinal epithelial cells (Douma et al, 2004). Further, some experimental data supports the therapeutic potential of disrupting Trk signaling in other cancer types where PNI is a prognostic factor, for example prostate and pancreatic cancers (Miknyoczki et al, 2002). Discussion of incorrect answers b. Utilizing digital droplet PCR, this study demonstrated a downregulation of E- cadherin and upregulation of Snail1. These are markers of epithelialmesenchymal transition (EMT), a process originally described in embryogenesis that is now used in tumor biology. To overcome the necessary barriers to metastasis, epithelial tumors must become more flexible and migratory, like the mesenchymal cell (Smit et al, 2009). The process of EMT is characterized by the

8 downregulation of epithelial proteins, such as E-cadherin, and -catenin, and upregulation of mesenchymal proteins such as fibronectin and vimentin by transcription factors in the Twist, Snail, and ZEB protein families (Smit et al, 2009). c. Immunostaining techniques demonstrated marked increase in expression of NCAM1 in perineural tumor cells when compared with tumor cells distant from PNI sites. Similarly, by digital droplet PCR, NCAM1 mrna was only detected in perineural tumor cells. Neural cell adhesion molecule 1 (NCAM1) is a cell surface glycoprotein normally found on neurons, skeletal muscle, NK cells and T cells. NCAM1 plays a role in embryogenesis, and guides neuronal regrowth during repair after nerve injury (Martini 1994). Moreover, expression of NCAM1 by Schwann cells has been implicated in the promotion of perineural invasion of other cancers, such as pancreatic cancer. In vitro, co-culturing human pancreatic cancer cells with dorsal root ganglion extracts revealed that Schwann cells direct cancer cells to move toward nerves in a contact dependent manner mediated by NCAM1 (Deborde et al. 2016). d. There was no significant difference in the expression of TrkA and TrkC receptors in perineural tumor cells when compared with tumor cells distant from PNI areas. This is in contrast with the increased expression of TrkB receptors seen in PNI cells. TrkA and TrkC are also membrane bound tyrosine kinase receptors. These receptors have structural differences that make them responsive to a different set of neurotrophins. TrkA receptor responds to NGF, while TrkC responds to NT3 (Deinhardt et al, 2014) These various receptors likely have complex differences in how they mediate downstream signaling, which is still being investigated. e. The effects of the TrkB receptor on epidermal mesenchymal transition and promotion of perineural invasion and metastasis has previously been studied in other tumor models, including epithelial salivary adenoid cystic carcinoma (SACC). Salivary adenoid cystic carcinoma is one of the most common malignant salivary gland neoplasms, and is commonly associated with invasion and distant metastasis. In 76 human SACC specimens, tumors associated with perineural invasion and metastasis were significantly more likely to have overexpression of BDNF and TrkB and downregulation of E-cadherin by immunohistochemical analysis (Jia et al, 2015).

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