Squamous cell carcinoma: variation in dermatoscopic vascular features between well and non-well differentiated tumors

Objective: Compare the dermatoscopy vascular features of well differentiated with non-well differentiated squamous cell carcinoma (SCC). Design: A prospective study of 294 consecutive cases of histopathologically confirmed invasive SCC compared the dermatoscopic vascular features of well to combined moderate and poorly differentiated tumors. These features were recorded live directly from the patients and included: the percentage of pink in the tumor, the presence of branching, serpentine, dot, hairpin, glomerular and linear vessels, and the number of these vessel types present within each tumor. Vessel types were also reviewed by tumor depth in 1 mm increments. Setting: Two medical practices in Sydney, Australia. Patients: Eighty-six female and 208 male patients (29–95 years old). Main outcome measure: Dermatoscopic vascular feature variation between grades of tumor differentiation and tumor depth. Results: Of 294 invasive SCCs, 255 (87%) were well differentiated, 32 (11%) were moderately differentiated and 7 (2%) were poorly differentiated. The percentage of pink areas within tumors varied between differentiation grades. The combined group of moderate and poorly differentiated tumors displayed more branching (28%, P<0.001) and serpentine (62%, P<0.005) blood vessels compared to well differentiated tumors (8% and 38%, respectively). Moderate and poorly differentiated tumors displayed larger numbers of vessel types (3.3) compared to well differentiated tumors (2.6, P < 0.01). Branching and serpentine vessels both increased in incidence with increasing tumor depth (p<0.05). Conclusion: Grades of tumor differentiation in SCC display varying dermatoscopic vascular features. The incidence of branching and serpentine vessels increases with increasing tumor depth and the shift towards poor differentiation.

room. Observer 2 then immediately entered the same room to record data live directly from the same patient with the same dermatoscope.

Exclusion criteria
Exclusion criteria were applied with the intention of preventing cases entering the study with dermatoscopic features confounded by a range of external factors not due to the tumor alone. These exclusion criteria were: residual or recurrent tumors at sites of previous surgical intervention (including partial biopsies); tumors with any part involving mucosal surfaces, tattoos or juxtaposed to scars; sites of previous radiotherapy, photodynamic therapy, cryotherapy, laser ablation, or topical pharmacological therapy (either patient or clinician initiated); and collision situations between invasive SCC and any non-SCC related entity evident from clinical, dermoscopic or histopathological examination and all cases of keratoacanthoma diagnosed by histopathology. Keratoacanthomas have been observed with a higher incidence of branching vessels (25%, n = 100) compared to invasive SCC (11%, n = 410, unpublished data).

Vascular features assessed
The tumor vascular features examined included: the percentage of pink area within a tumor, recorded as either no pink, less than 50% pink, or greater than 50% pink within the tumor boundary and the presence of tumor vessels with dot, hairpin (loop), glomerular (coil), branching (arborizing), serpentine (curvilinear) and linear (no branches or curves) morphology. The number of these different vessel morphologies within the tumor boundary was also recorded.
After the application of exclusion criteria, the defined vascular features of the remaining cases were recorded with a Heine Delta 20 ® nonpolarized dermatoscope (Heine, Optotechnic GmbH, Herrsching, Germany). Recording was performed prior to anesthetic injection, excision and submission for routine histopathological examination. To avoid vessel compression and enhance image resolution, all data was recorded after transparent ultrasound gel was applied between the glass plate of the dermatoscope and the skin surface.
All excised tissue was submitted for routine histopathological confirmation using hematoxylin and eosin staining.
Histopathology reports were used to exclude all entities other than invasive SCC from the study. Tumor thickness was measured in all cases to the nearest 0.1 mm and tabulated with the grade of either well, moderate or poor differentiation. Due to the low number of cases, moderate and poorly differentiated tumors were assessed combined.

Introduction
Compared to well differentiated squamous cell carcinoma (SCC), moderate and poorly differentiated SCC behave in a more aggressive manner and portend a worse prognosis [1]. Tumor differentiation has recently been incorporated into the new American Joint Committee on Cancer Staging System for Cutaneous SCC [2]. Non-well differentiated SCCs have been reported as deeper tumors [3] with greater recurrence rates [3,4]. Poorly differentiated SCC on the ear or mucosal lip has an increased risk of metastasis [4,5]. Differentiation grade in SCC has been stated as an independent adjusted predictor for overall survival [6].
Tumor differentiation in SCC presents along a spectrum from well to moderate to poor differentiation, which is determined using histopathologic criteria originally described by Broders [7]. Broders' original grading system correlates biological behaviour with differentiation in four grades. Grade 1 is characterized by abundant keratinization, little nuclear anaplasia and less than 25% undifferentiated cells. These features extend on spectra through to Grade 4 where there is little or no keratinization, extensive nuclear anaplasia and greater than 75% undifferentiated cells. Concordance in the histopathological assessment of SCC may lack consistency, even between experienced observers [8,9].
Routine histopathology reporting, as used in this study, categorizes SCC into three grades: well, moderate and poorly  [10]. Vessels associated with invasive SCC are often surrounded by a white halo, which is characteristic of keratinocytic tumors [11]. Although pigmented blood breakdown products are frequently seen with invasive cutaneous SCC, the presence of dermatoscopically identifiable melanin pigment is rare [12,13]. We speculated that grade of differentiation in SCC may have characteristic dermatoscopic appearances determined more by vascular and keratinization features rather than pigmented structures. Vascular features were the focus of this study.

Methods
Data was collected from July 2009 to December 2010 from two medical practices in Sydney, Australia. The study was approved by the Ethics Committee from The University of Queensland, Brisbane, Australia. Throughout the tend to have a higher proportional incidence as tumor depth increases (Table 1). Maximum recorded tumor depth for each grade of differentiation was well (7.5 mm), moderate (6.0 mm) and poor differentiated (6.3 mm); the range of tumor depth and mean depth by grade of tumor differentiation are displayed in Table 2.

Pink areas
Examination of the data on the percentage of pink areas does not reveal any highly distinctive association between the grade of differentiation and the proportion of pink within the tumor (Figure 1). When reviewing the poorly differentiated tumor data alone, 5 out of these 7 cases had greater than 50% pink within the tumor boundary.

Statistical assessment
Interobserver agreement between two of the authors (JP and DS) was assessed. Kappa values from 0.41 to 0.60 indicate moderate agreement between observers, 0.61 to 0.80 are regarded as substantial agreement, and from 0.81 to 1.00 almost perfect. Confidence intervals were set at 95%.
Logistic regression was carried out to test for significance of trends. Logistic regression was used to model binary response variables (i.e., vessel morphology observed or not observed) in terms of explanatory variables (tumor depth).

Vessel types and polymorphism
Well differentiated tumors had a relatively reduced incidence of branching and serpentine vessel forms. They were also characterized by dot, glomerular

Interobserver agreement on classification by vessel morphology
Kappa values for branching vessels (0.81) and pink areas (0.80) were almost perfect. Serpentine (0.78) and hairpin (0.74) vessels were in substantial agreement (Table 3).

Discussion
Increased tumor depth was associated with a higher proportion of non-well differentiated SCC. This result is consistent with earlier work [3]. Thicker non-well differentiated tumors often appeared endophytic and usually dis-

Pink areas
The study records of the crude percent-