Flujos de Eficiencia

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Analysis of the Distribution of CD8+ T-cells in Lichen Planus vs Epithelial Dysplasia

The full results of the morphological distribution of CD8+ cells are summarized in table 3. The biggest difference in the comparison of oral lichen planus with epithelial dysplasia was the pattern of infiltration of CD8+ cells.

Diagnosis Case Observations

LP 1 CD8+ cells in basal layer and below epithelium or Layer 1 LP 2 CD8+ cells below epithelium and infiltrating basal layer of epithelium

LP 3 CD8+ cells below epithelium

LP 4 CD8+ cells below epithelium and infiltrating basal layer of epithelium LP 5 CD8+ cells below epithelium and infiltrating basal layer of epithelium Dysplasia 6 Multiple CD8+ cells found in upper half of the epithelium Dysplasia 7 Atrophic epithelium, minimal amount of CD8+ cells in upper half

Dysplasia 8 CD8+ cells found in upper half

Dysplasia 9 Upper half of epithelium, some infiltrating normal appearing epithelium Dysplasia 10 CD8+ in upper half throughout later 1, and infiltrating salivary gland ducts

Table 3. Distribution of CD8+ cells in the Epithelium in Oral Lichen Planus vs Epithelial Dysplasia.

The greatest infiltration of CD8+ T-cells was observed in the OED group, where the cytotoxic cells appeared to infiltrate the dysplastic epithelium and follow the atypical features. Since all the samples selected for this group consisted of moderate to severe dysplasia, the CD8+ cells were localized to the area from the basal layer to the upper half of the epithelium or Zone 1. This because the CD8+ T-cells follow the malignant epithelial changes in the tissue.

25 DISCUSSION

This study attempted to evaluate the population of T-cells comprising the inflammatory lichenoid infiltrate underlying the epithelium in oral lichen planus and epithelial dysplasia, specifically regarding the quantity and distribution of two major subtypes of lymphocytes. Phenotype of Inflammatory Cells in Oral Lichen Planus

Established oral lichen planus lesions are typically found to contain T-cells with alpha- beta receptors, including CD4+ and CD8+, designated helper and cytotoxic respectively. Both subtypes can be found within the epithelium and lamina propria infiltrates, and are involved in a type 1 immune response, where CD4+ T-cells produce Th1 soluble factors. This response likely includes cell-mediated cytotoxicity, as CD8+ T-cells have been described to be localized to the epithelial-connective tissue interface and sometimes adjacent to apoptotic keratinocytes(6). In our study, the majority of CD4+ cells were found in the connective tissue underlying the involved epithelium, and CD8+ cytotoxic cells were mostly found infiltrating the keratinocytes of the basal layer (Figure 8). Khan et al. (2003) reported that CD4+ helper T- cells are prominent in early lesions, mainly in the superficial connective tissue with occasional cells in the epithelium (Figure 9), which is also consistent with the study of Firth et al (2015)(10) study, where regulatory T-cells expressing FoxP3 and IL-7, such as helper CD4+ lymphocytes were mostly found in the superficial connective tissue underlying the epithelium.

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Figure 8. Distribution of CD4+ and CD8+ lymphocytes detected by IF/IHC. Blue – Nuclei stained with Hoechst. Red – CD4. Green – CD8. Note the scattered CD8+ lymphocytes infiltrating the lower half of the epithelium (400x

magnification).

Figure 9. Presence of CD4+ lymphocytes mainly in the connective tissue and basal membrane some. Red – CD4+ cells (400x magnification).

Also, most of the intraepithelial lymphocytes were CD8+ cells and the proportion of these lymphocytes was higher in the superficial than the deeper lamina propria, in consonance with what has been previously established in the literature(54). CD8+ T-lymphocytes were usually found in close proximity to the epithelial basement membrane. This finding correlates with the studies of Khan et al. (2003), where the presence of CD8+ lymphocytes in oral lichen

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planus, were associated with basal epithelial cells undergoing apoptosis(13,15). It has been also described, that the presence of activated CD8+ cells expressing human leukocytic antigen surface antigen, is detected not only close to damaged epithelial cells but also to the basement membrane and epithelial areas of erosion (55). No significant differences in the ratio of CD4+/CD8+ (2:3) in the infiltrating and underlying t-lymphocytes were encountered in the present study.

The trigger for keratinocyte apoptosis in oral lichen planus is unknown. However, the localization of CD8+ cells can be explained by the previously mentioned studies and by Sugerman et al. (2000), who reported that CD8+ lesional cells recognize antigen associated MHC class I on lesional keratinocytes. Following antigen recognition and activation, CD8+ cytotoxic T-cells trigger keratinocyte apoptosis, resulting in the clinical and histological appearance of LP (16).

Phenotype of the Inflammatory Infiltrate in Oral Epithelial Dysplasia with Lichenoid Features In the oral cavity, the most common lesions clinically recognized as potentially malignant are leukoplakia and erythroplakia, but it is also apparent that as many as 50% of oral SCCs arise from apparently clinically normal mucosa (56). The prognostic significance of an individual lesion is difficult to determine. None of the currently available molecular markers have proved to be prognostically significant nor have yet been evaluated in large prospective studies. At present, therefore, the diagnosis and grading of OED is based on a combination of architectural and cytological changes, but these are subjective and there is considerable variability between pathologists in grading of the lesions. In a study by Fitzpatrick et al. (2014), 29% of 352 OED observed, have lichenoid features, which consisted of a band-like inflammatory cell infiltrate underlying and infiltrating the epithelium and basal cell degeneration(29). This finding is consistent with all the specimens encountered in our study, since the majority of these features were identified and used in case selection (Figure 10).

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Figure 10. Hematoxylin and Eosin tissue section of a sample with moderate to severe epithelial dysplasia. (Magnification 400X)

As far as the CD4+/CD8+ ratio of cells underlying moderate to severe OED was concerned, no statistically significant differences were encountered in any of the layers. There was a moderate increase in the inflammatory cell infiltrate in the OED group compared to oral LP. The infiltrates of both of the CD8+ and CD4+ cells were significantly greater in number than in LP and deeper into the tissue Zone 1 as seen in Figure 11.

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Figure 11. CD4+ and CD8+ cell infiltrate within Zone 1. Yellow line highlights the epithelium-connective tissue interphase.

CD4+/CD8+ cell ratio in Oral Lichen Planus vs Epithelial Dysplasia with Lichenoid Features Regarding the CD4+/CD8+ ratio, the CD8+ lymphocytes were found to be slightly increased in comparison to the CD4+ cell population in OED, whereas the ratio was slightly tipped in favor of CD4+ lymphocytes in LP. This suggests an important change in the behavior of the inflammatory infiltrate from a cytotoxic activity that is aimed to eliminate the lesion in OED to a more regulatory and suppressive in LP. This change can be observed in Figure 12.

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Figure 12. Graphic representation of averages of percentages of cells present in Layer 1 and 2 of oral lichen planus vs dysplasia.

An important change can be appreciated in the date for premalignant lesions where the CD4+/CD8+ cell ratio changes from the position of CD8+ lymphocytes. It is already known CD4+/CD8+ cell infiltrate is significantly higher in moderate to severe dysplasia in comparison with benign lesions and in SCC (9,40). This can be explained by the relationship between immunosurveillance and tumor progression, in which carcinogenesis results from crosstalk of cancer-cell-intrinsic factors and host immune system or “cell-intrinsic” effects(57). Hirota et al. (1990), also demonstrated an increased number of T-helper cells over T-cytotoxic cells within the T-cell subpopulation of oral SCC lesions. The above suggests an overall switching of the immune response in favor of pro-tumor immunity dominated by a CD4+ cell population that might help in tumor initiation and progression as suggested by Strauss et al. (2007). Further studies need to be conducted to identify the subpopulations of CD4+ cells that are directly responsible for such increase in the CD8+ population.

There was no statistically significant difference between the numbers of cells encountered in the different zones analyzed in our study. This indicates that the CD4+/CD8+ T-cell ratio is similar between both lesions and, therefore cannot be used to differentiate them.

Epithelium Connective Tissue Epithelium Connective Tissue

CD4+ Cells CD8+ Cells Lichen Planus 44.2 49.5 55.8 50.5 Epithelial Displasia 36.3 47.9 62.4 52.1 44.2 49.5 55.8 50.5 36.3 47.9 62.4 52.1 PE R CE N TA G E O F P O SIT IV E CE LL S

CELLS BY LAYER

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However, the localization of CD8+ cells infiltrating the epithelium can be useful in differentiating these dysplastic lesions with lichenoid features and LP.

In our study, in all the cases with moderate to severe epithelial dysplasia, a striking population of CD8+ lymphocytes were found following the dysplastic changes in the epithelial zone. Since cytotoxic CD8+ cells will try to eliminate the cells undergoing malignant transformation within the epithelial layer, it makes sense that in moderate to severe OED, these types of inflammatory cells follow the malignant changes throughout the thickness of this morphological and cytological changes within the tissue. For this reason, the localization of CD8+ cells could be used to differentiate OED and LP in equivocal cases. Of course, more proven cases are necessary, along with clinical follow-up of the patient’s progression to

confirm this theory. Also, tangential sectioning of tissue can obscure the true position of these CD8+ lymphocytes, biasing the results.

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CONCLUSIONS

The inflammatory component of the oral cavity is a complex system that, at this point in time, is still not well understood, especially with respect to potentially premalignant mucosal lesions. The diagnostic dilemma addressed in this study may be eventually solved by identifying cells with specific functions within the inflammatory population that can be used to track early malignant changes within the mucosal epithelium. However, there is no specific marker or panel of markers available yet that allow reliable prediction of malignant transformation of leukoplakia in the oral cavity. Further studies regarding specific subtypes of CD4+ and CD8+ T-cells are necessary to distinguish between OLP and OED and reliably predict the malignant transformation of OED and those equivocal cases previously mentioned.

Results of this study suggests that the distribution of CD8+ by immunohistochemistry can help in differentiating moderate/severe oral epithelial dysplasia, from LP and potentially other inflammatory conditions. Further research with more cases in comparison with diverse inflammatory models, is necessary to establish conclusive results, which will be part of the continuation of this pilot study.

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