Decreased CD1a + and CD83 + cells in tonsillar squamous cell carcinoma regardless of HPV status

Abstract Dendritic cells (DCs) are specialized antigen-presenting cells that play a critical role in the immune response against human papillomavirus (HPV) infection, and represent a therapeutic target in cancer. Objective: To identify and quantify DCs in tonsillar squamous cell carcinoma (TSCC) under the influence of HPV infection. Methodology: CD1a and CD83 antibodies were used to identify immature dendritic cells and mature dendritic cells by immunohistochemistry in 33 primary TSCC and 10 normal tonsils (NTs), respectively. For the TSCC samples, the number of DCs per area was evaluated in the intra- and peritumoral compartments. For the NTs, the quantification of DCs was evaluated in the intra- and peritonsillar compartments. HPV detection methods were determined according to the ASCO Clinical Practice Guidelines from the College of American Pathologists Guideline (2018). Results: There were fewer intratumoral CD1a+ DCs in the HPV-positive and HPV-negative TSCC groups than in the NT group (p<0.05). In the peritumoral compartment, there were fewer CD83+ DCs in the HPV-positive and HPV-negative TSCC groups than in the NT group (p<0.001). The quantification of DCs subtypes showed no statistical differences between HPV-positive and HPV-negative TSCC groups (p>0.137). Patients with HPV-positive TSCC had significantly better overall survival rate than those with HPV-negative TSCC (p=0.004). Conclusion: Tumor activity contributes to DC depletion regardless of intralesional HPV positivity. An improved prognosis has been reported in patients with HPV-positive TSCC.


Introduction
Dendritic cells (DCs) are antigen-presenting cells responsible for specific immune responses that capture, process, and present antigens to T lymphocytes. DCs can activate other cells, such as natural killer cells, B cells, macrophages, and eosinophils as well as generate immunological tolerance. 1 DCs are currently classified as immature (iDCs) or mature DCs (mDCs) according to their morphology and phenotype expression. In an immature state, DCs patrol tissue microenvironments and efficiently unleash various pathways when they encounter an antigen. DCs migrate to the draining lymph nodes through a maturation process that enables the presentation of pathogen-derived peptides to CD4+ and CD8+ T cells. 1,2 Epithelial DCs play a fundamental role in antiviral immunity since T cells depend on the presentation of viral antigens by DCs. The antigen presentation process conducted by DCs generates an effective immune defence against HPV. Epidemiological studies suggest that HPV takes advantage and interferes with the cell cycle to avoid being eliminated when the immune system is compromised or deficient, resulting in persistent infections and in the development of HPVpositive malignant lesions. 3,4 Smoking, alcohol consumption, and oncogenic HPV infection have been found to be etiological factors for tonsillar squamous cell carcinoma (TSCC). 5,6 This relationship of TSCC with HPV has been reported to have a better survival prognosis and less risk of recurrence than HPV-negative lesions. 7 Some investigations have shown a correlation between DC subsets with distinct clinical behaviors of squamous cell carcinoma of the oral cavity and larynx. [8][9][10][11] 6, 11, 16, 18, 31, 33, 35, 45, 51, and 52 coupled to a digital image acquisition system (A620, Canon, Lake Success, NY, USA) and a microcomputer (AOC, Miami, USA), in which the images were stored.
The number of DCs per area was evaluated in the intra-and peritumoral compartments in TSCC groups.
For NTs, the quantification of DCs was evaluated in the intra-and peritonsillar compartments. The number of CD1a+ and CD83+ DCs per mm 2 of each compartment was determined using ImageJ 1.51k software (Maryland, USA).

Statistical analysis
Values are expressed as medians with interquartile ranges. Comparison of DCs between the groups was performed using Kruskal-Wallis and Dunn's post-hoc tests. The Mann-Whitney test was used to analyze the association between HPV status and the clinicopathologic findings or the number of intra-and peritumoral DCs in TSCC cases. The clinical and microscopic findings in the different groups with HPVpositive and HPV-negative TSCC were compared using the Mann-Whitney U, Fisher's Exact, and Pearson chi-square tests. The log-rank test was performed to analyze the overall survival rate between HPV-positive and HPV-negative TSCC. Statistical significance was set at p<0.05. Statistical software SPSS version 23.0 was used for the analyses. (21.2%) were HPV-positive. There was no detection of p16 INK4a in the NT group. In the HPV-negative TSCC group, 24 patients were men (92.3%) and 2 were women (7.7%), with a mean age of 59.9 years (range, 45-76 years). The HPV-positive TSCC group included 5 men (71.4%) and 2 women (28.6%), with a mean age of 48.5 years (range, 43-54 years). The NT group had 6 men (60%) and 4 women (40%), with a mean age of 38.9 years (range, 15-84 years). Table 1 shows additional data.

Dendritic cells
CD1a+ DCs showed multiple dendritic extensions, mainly in the intratumoral compartment. In contrast, CD83+ DCs had an ovoid morphology with scarce dendritic extensions in the same compartment ( Figure   1). There was no difference in the quantification of DCs within the stages of the disease (Table 2)    (p<0.001) ( Table 3).

Survival rate of TSCC patients showed no improvement with increases or decreases in intra-
or peritumoral CD1a+ and CD83+ DCs ( Figure   2). However, patients with HPV-positive TSCC had significantly more favorable survival rate than patients with HPV-negative TSCC (p=0.004; Figure 3 and Table 4).     Laguens, et al. 15 (2002) showed that the densities of S100+ and CD1a+ DC in regional lymph *P-value derived from the log rank test for equality of survivor functions. TSCC, tonsillar squamous cell carcinoma.  HPV-positive tumors have a better prognosis than patients with HPV-negative tumours. 6,28 Fakhry,et al. 28 (2008) found a 2-year overall survival rate of 95% in HPV-positive patients with SCC of the oropharynx or larynx, in contrast to the 62% survival rate of HPV-negative cases. Our findings are consistent with these studies, since we found a better survival rate in the HPV-positive TSCC (100%) group than in the HPVnegative TSCC cases (42%) (p=0.004). Several studies have pointed out that the survival improvement is due to a greater radiotherapy sensitivity or a stronger antitumor immune response in these tumors. [28][29][30] TSCC patients with high expression of CD83+ DCs have been correlated with improved survival. 29 However,Jardim,et al. 30 (2018)

Conflict of interest
The authors declare no conflict of interest.