Early Squamous Cell Carcinoma With Perineural Invasion: A Prospective Study Examining Anatomic Site, Tumor Surface Diameter, Invasion Depth, and Grade of Differentiation in 1,772 Consecutive Cases

Background Squamous cell carcinoma (SCC) may present with perineural invasion (PNI). Objective To investigate the characteristics of early invasive SCC with or without PNI. Methods Consecutive SCC excisions were prospectively reviewed from a single Australian community-based practice for 2016-2018. Tumor characteristics recorded were anatomic site, maximum microscopic tumor surface diameter, invasion depth, grade of differentiation, and diameter of nerves with PNI. Results In total, 1,772 cases were collected. No PNI cases were found on female patients. Seven of the total 10 PNI cases were on facial sites. Maximum average microscopic tumor surface diameters ranged from 10.1 mm (well differentiated) up to 11.0 mm (moderately differentiated). Maximum average invasion depths by differentiation ranged from 1.7 (well differentiated) up to 2.6 mm (poorly differentiated). The PNI cases were as follows: well differentiated (n = 0), moderately differentiated (n = 4), or poorly differentiated (n = 6). Minimum average histopathological margins for well, moderately, and poorly differentiated SCC, respectively, were 1.4, 1.1, and 1.3 mm. Minimum microscopic tumor surface diameters for PNI cases were 7 mm for moderately and 5 mm for poorly differentiated SCC. Minimum microscopic invasion depths for PNI cases were 2.2 mm for moderate and 0.9 mm for poor differentiation. Conclusions We found early SCC with PNI displayed nerve diameters of 0.1 mm or less and were exclusively on male patients aged 60 or older, on chronically sun-exposed sites of the head and upper midline anterior chest. Histopathological features associated with PNI were moderate and poor differentiation, tumor invasion beyond 0.9 mm, and adjacent lymphocytosis.

ethics approval (project no. 2016001221). Full excision was attempted on all cases by typically using a fusiform ellipse cut down to the subcutis. Cases with prior partial biopsies and known recurrent SCC cases as well as SCC receiving prior pharmaceutical or ablative treatments were excluded.
All cases excised during the study were marked on a body map. This map was checked prior to subsequent excisions to exclude recurrent cases. There were no exclusions based on age, anatomic site, or nerve diameters with PNI.
Prior to excision, every case was examined using a Delta 20 T Heine dermatoscope to identify the surface margin of the tumor. Excisions were marked out with an in vivo surface margin of 1 to 2 mm. Excised tissue was sectioned by transverse bread loafing at 3-mm intervals along the entire specimen. Following H&E staining, all cases underwent histopathological examination. All cases of keratoacanthoma, SCC in situ, and any diagnosis other than invasive SCC were excluded. Depth of invasion was measured from the granular layer in the epidermis down to the deepest tumor cells to the nearest 0.1 mm. This invasion depth measurement was consistently performed independent from the adjacent skin surface. Assessing the grade of differentiation was based on the original description by Broders [16]. Where the grade of differentiation varied within a tumor mass, the grade present closest to poor differentiation was recorded for that case.
We defined PNI as malignant squamous cells identified within the perineural space that must be located outside the tumor mass. Cases with malignant cells in the perineural space where the involved nerve was only found inside the tumor mass were excluded. Two board-certified dermatohistopathologists blind to each other's findings had to agree PNI was present within a case for PNI classification. Cases in which only 1 of the 2 histopathologists identified PNI were excluded from PNI allocation. Mimics of PNI were well known to the reporting histopathologists. To assess whether localized lymphocytosis is a clue to PNI, the co-location of lymphocytes with PNI or juxtaposed to the PNI was recorded when seen. The qualitative density of lymphocytes per unit area juxtaposed to the PNI was compared with the lymphocyte density in the background tissue well away from the involved nerve.

Introduction
Over recent decades the prevalence of cutaneous squamous cell carcinoma (SCC) has increased [1]. A recent review [2] found multiple studies demonstrating perineural invasion (PNI) is significantly associated with a higher risk of recurrence following surgery, metastasis, and disease-specific death. Reported presentations of SCC with PNI vary from 2.5% to 6% [3]. Most studies on PNI tend to focus on advanced disease treated in tertiary facilities. PNI has been referred to as either "incidental" when asymptomatic and involving small-diameter nerves compared with "clinical" when symptomatic and involving larger nerve diameters [4].
This small nerve diameter has been defined as equal to or less than 0.1 mm [5]. Little is known of the characteristics of early-stage, smaller-dimension SCC with early-onset PNI. Over the past 2 decades, publications [2,5] have consistently stated that an increase in nerve diameters beyond 0.1 mm is associated with a poorer prognosis including disease-specific death.
Both the clinical and dermoscopic features of SCC vary with the grade of differentiation [6]. Pain and numbness have been reported as clues to PNI [7,8]. Poorly differentiated SCC tends to have a higher prevalence of PNI [9,10] compared with well-and moderately differentiated tumors. An increase in PNI has also been recorded in SCC with a desmoplastic stroma [11]. Increasing SCC tumor diameter correlates with increased depth of PNI [12]. A minimum biopsy depth of 3 to 4 mm has been recommended to optimize not missing PNI [12]. Some SCC without PNI may display features mimicking PNI [13]. Peritumoral fibrosis is a known mimic of PNI [14].
Combining epithelial membrane antigen with cytokeratin MNF116 has been stated to facilitate diagnosing PNI when both of these immunohistochemical stains are positive [15].
As PNI develops, it is not clear whether advanced PNI correlates with an increase in affected nerve diameters alone and/or with an increase in the spatial distribution of smaller affected nerve diameters. The intention of our study was to identify clinical and histopathological characteristics of early-stage SCC associated with early-appearance PNI.
Identifying clinical characteristics of SCC associated with the threshold onset of early PNI may facilitate the bedside earlier detection of PNI, thus improving patient outcomes. Furthermore, an awareness of a higher chance for PNI being present based on the histopathological characteristics of an SCC case may also assist histopathologists in the early detection of PNI.

Materials and Methods
Consecutive invasive SCC cases were prospectively collected from routine workflow in a community-based private practice in Sydney, Australia, from 2016 to 2018. The University of Queensland (Brisbane, Australia) authorized differentiation n=65/83 (78%) ( Table 2). Table 2 illustrates the shift from well to poor differentiation was also associated with increasing rates of peripheral only, deep only, and combined peripheral and deep margin involvement.
Nine of the 10 PNI cases recorded a nerve diameter of less than 0.1 mm (  Figures 1 and 2. Although the intention was to fully excise all cases, histopathologically identified margin involvement with invasive SCC did occur in some cases. By grade of differentiation, clear histopathological margins were as follows: well n = 1,178/1,253 (94%), moderate n = 392/436 (90%), and poor

Discussion
With only 10 PNI cases in a total cohort of 1,772 cases, the overall PNI presentation was approximately 0.56%. Compared with other studies, this percentage of cases with PNI is during the study was associated with a higher prevalence of PNI ( Table 4). Most of the PNI cases (6/10) were found on patients with 4 or more SCC cases.

Limitations
Poorly differentiated cases had a higher rate of involved microscopic margins. As PNI was defined as being outside the tumor mass, some PNI may have be missed with involved tumor margins. Thus PNI may be underrepresented within the total poorly differentiated cases. The number "n" lists the number of patients with squamous cell carcinoma, not the number of SCC cases.   For moderately differentiated SCC we found the threshold for increased vigilance for PNI was an invasion depth of 2.2 mm.
The relatively uncommon prevalence of both acantholysis and PNI limits investigation of their potential association.
Lymphovascular invasion was also found in another single PNI case. This case occurred on the eyelid of a man aged 80 years with an SCC displaying infiltrative architecture, multifocal PNI, and an invasion depth of just 2.4 mm.
Whether there is an association between infiltrative architecture or desmoplasia and early multifocal PNI may be worth further study.
Metastatic behavior from a primary SCC with an invasion depth of less than 2 mm and desmoplastic histology has been reported [18]. However, overall metastatic potential in SCC requires an invasion depth to exceed 2 mm [18]. As invasion depth increases beyond 2 mm so does the increase in metastatic potential. We found deeper tumor invasion of 2.2 mm or greater was also associated with increasing prevalence of PNI. Histologically identified solar elastosis was an anecdotal observation with most PNI cases in our study. The extent to which solar elastosis is a marker for PNI risk also invites future investigation.

Conclusions
Our study found early incidental PNI on men aged 60 years or older with a high prevalence on facial sites and patients with four or more SCC cases. Tumor invasion depths progressively increased in the shift to poor differentiation, consistent with previous studies [15].
All recorded cases of PNI were either on the face (n = 7/10) or anterior upper midline chest (n = 3/10). Two of the 3 PNI cases on the chest occurred on the sun-exposed area over the manubrium. Together the anatomic sites for all 10  We found that 2 of the 10 PNI cases did not display tumor-associated lymphocytes when tumor invasion and PNI presence were both identified in the subcutis. The other 8 cases all displayed tumor-associated lymphocytes when tumor invasion and PNI did not extend into the subcutis ( Table 3).
Examination of Table 4 reveals that patients with 4 or more SCC cases were more likely to have PNI.