The Research Ethics Committee approved the Haroldo Juaçaba Hospital/Ceará Cancer Institute research under registration protocol number 2.191.839. The study complies with the norms that regulate research in human beings, as stated in resolution 466/12 of the National Health Council. Furthermore, the study was conducted using the scientific methodology known as the STROBE initiative.

The sample will consist of 30 Squamous Cell Carcinoma of the lip, 15 actinic cheilitis and 15 lips without pathological changes according to the sample calculation performed based on the study by Kakasheva-mazhenkovska et al.9 This work showed that the histological grade of Squamous Cell Carcinoma of the lip is important for tissue invasion through immunoexpression for lesions that present a greater vascular density in more intense histological grades (20.37 ± 4.36 vs. 24.90 ± 6.27). Thus, adopting a case-to-control ratio of 2:1, it is necessary to evaluate 30 Squamous Cell Carcinoma of the lip and 15 control tissues (15 actinic cheilitis and 15 without pathological alteration) in order to obtain a sample that represents 80% of power and 95% confidence the alternative hypothesis of this study (Kesley's method) (Fig. 1).

Figure 1.

Flowchart of sample selection from laboratories without histological changes, actinic cheilitis and SCC from laboratories.

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Inclusion criteria was: paraffinized blocks from incisional and excisional biopsies of patients treated at Hospital Haroldo Juaçaba, Instituto do Câncer do Ceará. After reviewing the amount of material available in the paraffinized block and patients' records, blocks without enough material and patients thar did not have clinical information were excluded (Patients whose paraffinized blocks came from review of histological slides).

For the present study, samples were collected from patients diagnosed and treated at the Hospital Haroldo Juaçaba, Instituto do Câncer do Ceará from 2015‒2020 who underwent surgery for lip vermilion carcinoma resection and did not undergo neoadjuvant treatment.

Tissues from healthy lip epithelia from head and neck squamous cell carcinoma (control group) were resected. Head and neck tumors that showed resection of lip were histologically analyzed, and paraffin blocks and histological slides with samples of lips from deep mucoceles.10 Additionally, tissues from Aquitic Cheilitis and Squamous Cell Carcinoma of the Lip cases from the Pathology Laboratory of Hospital Haroldo Juaçaba, Instituto do Câncer do Ceará were selected.

After revision of the slides, samples of the vermilion epithelium of the lip, free of tumor margins, from head and neck surgical resections were included in the control group. Patients with a revised histological diagnosis of Aquitic Cheilitis were included in the Aquitic Cheilitis group. Cases with a revised histological diagnosis of Squamous Cell Carcinoma were included in the Squamous Cell Carcinoma of the Lip group.

Clinicopathological data were collected from the patients' medical records, excluding patients with incomplete records (>30% of the sociodemographic and clinical-prognostic information). After evaluating the paraffin blocks, we excluded the blocks that did not present enough material to perform the Tissue Microarray (TMA) technique.

Histological, formalin-fixed, and paraffin-embedded specimens were selected, and the demographic and clinical data retrieved from the patients' medical records were analyzed. Histological slides of the excisional biopsies were evaluated and identified to fabricate the TMA blocks.

The classification of A Aquitic Cheilitis and Squamous Cell Carcinoma lips was performed by an experienced pathologist (>10-years of oral pathology), with an intra-examiner calibration coefficient kappa = 0.859.

The Aquitic Cheilitis had their epithelial dysplasia classified using a binary system of low/high risk of malignant transformation.11 The Squamous Cell Carcinoma lips samples were classified using Bryne's binary model, classifying Squamous Cell Carcinoma into high-and low-grade.12 The presence of perineural and vascular invasion was also assessed.13

All microscopic slides were analyzed, and sites of tumors with highly cellular Squamous Cell Carcinoma lips sections, areas of Aquitic Cheilitis showing epithelium and connective tissue with solar elastosis, and control areas showing epithelium and connective tissue were identified. We selected two representative circumferential areas, each 2 mm in diameter (3.14 mm2) from each sample, for the microarray tissue technique. Paraffin blocks were punched from each demarcated area using a tissue microarrayer (Quick-Ray UNITMA®, Seongnam-si, South Korea) and then transferred to a paraffin receptor block containing 70 circular wells of the same diameter (2 mm). The paraffin receptor blocks containing the oral ulcers were then cut into 3-µm thick sections placed on silanized slides.

For immunohistochemical processing, samples were deparaffinized, rehydrated, and subjected to antigen retrieval in Tris-EDTA buffer (Ph 9.0). Samples were incubated in Phosphate-Buffered Saline (PBS) with 3% H2O2 for 30 min to inactivate endogenous peroxidases. Then, they were washed in PBS and incubated for one h with primary antibodies against CD3 (Dako®️, A0452), CD8 (Dako®️, M710), CD20 (Dako®️, m0755), CD68 (Dako®️, M0876) and ki-67 (Dako®️, MIB-1) was also used.

Samples were washed in PBS, incubated in Envision Plus HRP Anti-IgG-rabbit/mouse for 30 min (ready-to-use; monoclonal; Dako® K4065), and washed again in PBS, after which diaminobenzidine chromogen (Dako® K3469) was applied to the samples for 5 min. Harris hematoxylin was used as the counterstain (10 s), after which the specimens were dehydrated in ethanol and xylene and covered with a permanent mounting medium (Enthelam®). Colorectal carcinoma sections were used as a positive control, and the negative control and positive control were treated in parallel with an antibody diluent instead of the primary antibody.

For evaluation of CD3, CD8, CD20, and CD68, ten fields per histological section intraepithelial (normal lip mucosa or Aquitic Cheilitis) and intratumoral (lip cancer and Squamous Cell Carcinoma) or subepithelial (normal lip mucosa or Aquitic Cheilitis) and peritumoral (lip cancer and Squamous Cell Carcinoma) were photographed at 400× magnification. The images were exported to ImageJ® software and counted to determine the number of immunostained positive cells (brown staining stromal cells). For evaluation of ki-67, ten fields per histological section were photographed at 400× magnification, and brown nuclear staining of epithelial cells (control group and AC group) and cancer cells (lip cancer and Squamous Cell Carcinoma group) was considered a positive immunoreaction.12 The images were also exported to ImageJ® software and counted to determine the percentage of immunostained positive cells.

Data were tabulated in Microsoft Excel (Microsoft Corporation®) and exported to the Statistical Package for the Social Sciences (SPSS) software, in which the analyses were performed with a 95% confidence level.

The mean and standard deviation of the percentages of immunostaining for each protein were calculated. These data were analyzed using the Kolmogorov-Smirnov normality test and did not have a Gaussian distribution. Therefore, the comparisons were performed using Mann-Whitney and Kruskal-Wallis or Dunn tests (between-group analysis).

The sample consisted of 15 lip mucosal epithelia without microscopic changes, 30 AC, and 45 lip cancer and Squamous Cell Carcinoma. Most Aquitic Cheilitis had low-risk dysplasia (n = 19), and the Lip Cancer and Squamous Cell Carcinoma was low-grade (n = 29). Only 5 lip cancer and Squamous Cell Carcinoma samples had lymphovascular invasion, and 13 had a perineural invasion.

There were CD3+, CD8+, and CD68+ cells in the lip epithelia without microscopic changes, but no CD20+ cells were observed. These cells were sparse among the epithelial cells without a specific topographic distribution. In the Aquitic Cheilitis, there was no significant increase in any of these cellular elements intraepithelial. Still, in the Lip Cancer and Squamous Cell Carcinoma, there was an increase in the amount of CD3+ (p < 0.001), CD8+ (p = 0.035), CD20+ (p < 0.001) and CD68+ (p < 0.001) cells within the tumor islands (intratumoral) (Table 1, Fig. 2).

Figure 2.

(a) Lip Lesions on HE stain; (b) Cheiliti on HE stain; (c) LSCC in HE stain; (d) Immunohistochemistry for CD8 in Lip Lesions; (e) Immunohistochemistry for CD8 in Cheiliti; (f) Immunohistochemistry for CD8 in LSCC; (g) Immunohistochemistry for CD3 in Lip Lesions; (h) Immunohistochemistry for CD3 in Cheilitis; (i) Immunohistochemistry for CD3 in LSCC; (j) Immunohistochemistry for CD20 in Lip Lesions; (k) Immunohistochemistry for CD20 in Cheiliti; (l) Immunohistochemistry for CD20 on LSCC; (m) Immunohistochemistry for CD68 on Lip Lesions; (n) Immunohistochemistry for CD68 in Cheilitis; (o) Immunohistochemistry for CD68 on LSCC.

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In the subepithelial tissue of the lip epithelia without microscopic changes most of the cells were CD3+ cells, but CD8+, CD68+ and to a lesser extent CD20+ cells could also be identified. The Aquitic Cheilitis showed a significant increase of CD8+ (p < 0.001) and CD68+ (p < 0.001) intraepithelial cells, but not CD3+ or CD20+ cells (p > 0.05). In lip cancer and Squamous Cell Carcinoma there was peritumoral increase in CD3+ (p = 0.037), CD8+ (p < 0.001), as well as CD20+ (p < 0.001) and CD68+ (p < 0.001) cells (Table 1).

Dysplasia gradation and lymphovascular invasion showed no significant association with the immunoexpression of any of the markers studied. Still, low-grade lip cancer and Squamous Cell Carcinoma (p = 0.019) and without perineural invasion (p = 0.026) showed higher intratumoral CD20+ cell immunoexpression (Table 1).

Among the 45 cases of lip cancer and Squamous Cell Carcinoma the majority were men (n = 34), aged less than 65 years (n = 24), T1/2 tumors (n = 30), N0 (n = 29), M0 (n = 47) and free of recurrence in five years (n = 47). The only clinical feature that showed an association with the immunoexpression of one of the markers was nodal metastasis. (Table 2).

In lip epithelia without microscopic changes there was only a positive correlation between subepithelial immunoexpression of CD68+ and CD3+ cells (p =  0.002, r = 0.883). In the subepithelial region of the Aquitic Cheilitis CD3+ and CD8+ cells showed significant direct correlation (p =  0.035, r = 0.547), as well as CD68+ and CD3+ cells (p =  0.008, r = 0.637) and CD8+ cells (p =  0.017, r = 0.625) (Table 3).

In peritumoral lip cancer and Squamous Cell Carcinoma, CD3+ and CD8+ cells showed a significant direct correlation (p =  0.004, r = 0.467), as well as CD68+ and CD8+ cells (p =  0.017, r = 0.390). In the intratumoral region CD8+ cells showed a significant correlation with CD20+ (p =  0.014, r = 0.396) and CD68+ (p =  0.013, r = 0.411) cells (Table 3).

There were no correlations between the immunoexpression of any intra- and subepithelial markers of the lip epithelia without microscopic changes. In the Aquitic Cheilitis, the immunoexpression of CD3 (p <  0.001, r = 0.878) and CD8 (p =  0.025, r = 0.641) showed a direct correlation between the intraepithelial and subepithelial regions. In lip cancer and Squamous Cell Carcinoma the immunoexpression of CD3+ (p =  0.002, r = 0.474), CD8+ (p =  0.001, r = 0.521) and CD68+ (p =  0.030, r = 0.363) showed direct correlation between intra and peritumoral region (Table 4).