Possible role of ALDH1 and CD44 in lip carcinogenesis

Abstract

Lip squamous cell carcinoma (LSCC) accounts for 12% of all head and neck cancers. It is caused by chronic exposure to ultraviolet light solar radiation and related to previous actinic cheilitis (AC). This study aimed to investigate the immunostaining of the putative cancer stem cells (CSC) markers ALDH1 and CD44 in AC (n=30) and LSCC (n=20). ALDH1 positivity was found to be statistically higher in LSCC than in AC lesions (p=0.0045), whilst CD44 expression was statistically higher in AC than in LSCC lesions (p=0.0155). ALDH1+ cells in AC lesions were associated with specific clinical features: a younger age (<60 years old), the female gender, white skin, not smoking or consuming alcohol, and a fast evolution, and not associated with the chronic exposure to UV radiation (p<0.0001). CD44 positivity was associated with patients who were male, feoderm, smoked, consumed alcohol, underwent occupational exposure to UV-radiation, and demonstrated lesions with log-time evolution (p<0.0001). ALDH1 + cells were associated with mild dysplasia using a system from the World Health Organization (WHO), and with a low risk of malignant transformation, according to the binary system (p<0.0001). CD44+ cells were also associated with moderated dysplasia, according to the WHO system. In LSCC, ALDH1 + cells were positively associated with patients who were older (≥ 60 years old), smokers, and with those who consumed alcohol (p<0.0001). CD44 + cells in LSCC were associated with older (≥ 60 years old) patients as well, but also with female patients, white skin, non-smokers, and individuals who did not consume alcohol (p<0.0001), all of whom showed distinct patterns in pre- and malignant lesions of both markers. Additionally, in LSCC, both ALDH1 and CD44 staining were associated with smaller tumor sizes (T1/T2; p<0.0001). In summary, although both ALDH1 and CD44 were associated with the presence of dysplasia in AC lesions, the present findings suggest that ALDH1 and CD44 may be activated by different etiopathogenic pathways, predominantly in distinct steps of oral carcinogenesis. CD44 would thus be more significantly related to the potentially malignant lesion, while ALDH1 would be closely linked to malignancy.

Figure 1. A representative case demonstrating some architectural cytological features of reclassified AC dysplasia

Figure 2. Immunohistochemical Analysis of AC Lesions with Varying Dysplastic Changes. (A) Nondysplastic AC lesions classified as having a low risk of malignant transformation show no ALDH1 expression. (B) In the same low-risk AC lesions, a uniform granular CD44 staining pattern can be seen along the membranes of epithelial cells within the basal and spinous layers. (C) High view of the low-risk AC lesion confirming the absence of ALDH1 expression. (D) CD44-positive lymphocytes primarily localized in the subepithelial region of the low-risk AC lesions. (E) AC lesions demonstrating mild dysplasia exhibit focal areas with ALDH1-positive epithelial cells within the spinous and granular layers of the epithelium. (F) CD44 staining pattern in mild dysplasia AC lesions closely resembles that of nondysplastic AC lesions, observed in the membranes of epithelial cells within the basal and spinous layers. (G) High view of the mild dysplasia AC lesion showing ALDH1-positive epithelial cells in focal areas. (H) CD44 staining in mild dysplasia AC lesions continues to resemble that of nondysplastic AC lesions. (I) AC lesions categorized as having a high risk of malignancy exhibit ALDH1-positive epithelial cells concentrated in focal areas, with robust cytoplasmic staining in both normal and altered cells. (J) In high-risk AC lesions, CD44 shows a strong and uniform positivity throughout the entire population of epithelial cells, except in parakeratinized layers, where CD44 expression is notably absent. (K) High view of the high-risk AC lesion showing ALDH1-positive epithelial cells in focal areas. Perilesional macrophages exhibit a pronounced ALDH1-positive phenotype. (L) The CD44 staining in high-risk AC lesions remains strong, except in the parakeratinized layers, where it is absent

Figure 3. Immunohistochemical Analysis of LSCC Tumor Cells. (A) Within the central regions of LSCC lesions, predominantly ALDH1-positive tumor cells form niches extending from the basal layers to the superficial ones. (B) In these central regions, CD44 staining closely resembles that observed in AC dysplastic lesions. (C) High view of the central regions of LSCC lesions, highlighting the ALDH1-positive tumor cells extending from the basal layers to the superficial ones. (D) CD44 staining in the central regions continues to resemble that observed in AC dysplastic lesions. (E) In the invasive areas of LSCC, a distinctive staining pattern emerges, with mild staining for ALDH1. (F) In these invasive areas, there is strong staining for CD44, particularly within the core regions of tumor islands. (G) In the more invasive tumor regions, there is a notable shift in the staining profile, with invasive tumor cells exhibiting strong ALDH1 positivity. (H) Also in the more invasive tumor regions, there is a decrease in CD44 positivity. (I) High view of the invasive areas of LSCC, emphasizing the mild staining for ALDH1. (J) In these invasive areas, strong staining for CD44 is particularly evident within the core regions of tumor islands. (K) A close-up view of the more invasive tumor regions, showing invasive tumor cells with strong ALDH1 positivity. (L) In these more invasive tumor regions, CD44 positivity decreases as compared to less invasive areas

Figure 5. Overall survival of patients with LSCC according to ALDH1 and CD44 immunostaining. (A) Overall survival of patients with LSCC. (B) ALDH1high patients had an overall survival rate > 5 years (average of 8 years; p = 0.1736). (C) CD44low patients had an overall survival rate ≤ 5 years (p = 0.7675). However, none of these values demonstrated statistical significance

Figure 4. Comparison between the immunoexpression level of CSC markers in AC and LSCC. (A) ALDH1+ cell concentrations were statistically higher in LSCC (5.944 ± 1.867) than in AC (4,826 ± 2.862) (p= 0.0045). (B) CD44+ cell concentrations were statistically higher in AC (89.6 ± 1.197) than in LSCC (77.56 ± 6.575) (p= 0.0155). (C) CD44 and ALDH were negatively correlated in AC (r= -0.191; p=0.350), and (D) positively correlated in LSCC (r=0.438; p=0.206), although without statistically significance. A-B: Mann-Whitney Test; C-D: Person correlation