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.

Estudo da imunoexpressão do reg-gamma e da beta catenina em queilites actínicas e carcinomas de células escamosas de lábio inferior / Study of immunoexpression of reg-gamma and beta-catenin in actinic cheilitis and lower lip squamous cell carcinoma

A queilite actínica (QA) é uma lesão potencialmente maligna que ocorre principalmente em homens leucodermas com histórico de exposição crônica ao sol. Atualmente, não é possível predizer quais os casos de QA progredirão para o Carcinoma de células escamosas (CCE), portanto alguns marcadores biomoleculares têm sido alvo de pesquisas. A ß-catenina é uma proteína multifuncional que está envolvida nos processos de adesão célula-célula. A alteração do complexo caderina-catenina tem sido demonstrada no CCE e correlacionada com a invasão tumoral, metástase e com pior prognóstico dos pacientes. O REGγ é um ativador de proteassoma que pode promover a degradação de múltiplas proteínas incluindo p53 e MDM2. Estudos mostram que o REGγ está superexpresso em numerosos tipos de câncer, sugerindo que a superexpressão do REGγ está envolvida na progressão do câncer. O objetivo deste estudo foi analisar a expressão imuno- histoquímica da ß-catenina e do REGγ em casos de QA e Carcinoma de células escamosas de lábio inferior (CCELI), comparando os achados imunohistoquímicos com os dados clínicopatológicos, afim de averiguar se há uma correlação com a progressão tumoral e se as mesmas atuam de forma sinérgica nesse processo. A imunoexpressão de ß-catenina e REGγ foi analisada semi-quantativamente em 30 casos de QA e 30 casos de CCELI de acordo com os escores: 0 (sem marcação); 1 (1-25% de células positivas); 2 (26-50% de células positivas); 3 (51-75% de células positivas); 4 (> 75% células positivas). Para a análise estatística, foram realizados os testes de Mann-Whitney e de Spearman (p < 0,05). Tantos as QAs quanto os CCELIs expressaram a proteína ß-catenina, sendo evidenciado um aumento da expressão citoplasmática e nuclear nos casos de Displasias moderadas e severas. Nos CCELIs a imunoexpressão de ß-catenina membranar foi maior nos casos de baixo grau de malignidade. Tantos as QAs quanto os CCELIs expressaram a proteína REG-γ porém não verificamos significância estatística entre a sua expressão e o grau displasia epitelial, bem como, entre a imunoexpressão do REG-γ e os parâmetros clinicopatológicos analisados nos CCELIs. Os resultados do presente estudo sugerem que a superexpressão de REG-γ e a redução na expressão membranar de ß-catenina podem ser eventos importantes na carcinogênese labial. No entanto, acreditamos que esta proteína esteja envolvida no processo da carcinogênese oral. Nesse processo, correlacionando a expressão imuno-histoquímica da ß-catenina com a expressão do REG-γ, não resultados estatisticamente significativos, sugerimos então que a expressão de ßcatenina pode não ser influenciada diretamente pelos níveis de expressão de REGγ (AU).

Actinic cheilitis (QA) is a potentially malignant lesion that occurs mainly in men with light skin and a history of chronic sun exposure. Currently, it is not possible to predict which cases of QA will progress to Squamous Cell Carcinoma (SCC), so some biomolecular markers have been researched. Β-catenin is a multifunctional protein that is involved in cell-cell adhesion processes. The alteration of the cadherin-catenin complex has been demonstrated in SCC and correlated with tumor invasion, metastasis and worse prognosis of patients. REGγ is a proteasome activator that can promote the degradation of multiple proteins including p53 and MDM2. Studies show that REGγ is overexpressed in numerous cancers, suggesting that REGγ overexpression is involved in cancer progression. The aim of this study was to analyze the immunohistochemical expression of ß-catenin and REGγ in cases of QA and lower lip squamous cell carcinoma (CCELI), comparing the immunohistochemical findings with the clinical and pathological data, in order to verify if there is any a correlation with tumor progression and whether they act synergistically in this process. Β-catenin and REGγ immunoexpression was analyzed semi-quantitatively in 30 cases of QA and 30 cases of CCELI according to the scores: 0 (no labeling); 1 (1-25% positive cells); 2 (26-50% positive cells); 3 (51-75% positive cells); 4 (> 75% positive cells). For statistical analysis, Mann-Whitney and Spearman tests were performed (p <0.05). Both QAs and CCELIs expressed the ß-catenin protein, showing an increase in cytoplasmic and nuclear expression in cases of moderate and severe dysplasias. In CCELIs, membrane ß-catenin immunoexpression was higher in cases of low grade malignancy. Both QAs and CCELIs expressed the REG-γ protein but no statistical significance between its expression and the degree of epithelial dysplasia was found, as well as between the immunoexpression of REG-γ and the clinicopathological parameters analyzed in the CCELIs. The results of the present study suggest that REG-γ overexpression and reduction in membrane expression of ß-catenin may be important events in lip carcinogenesis. However, we believe that this protein is involved in the process of oral carcinogenesis. In this process, correlating the immunohistochemical expression of ß-catenin with the expression of REG-γ, as we did not obtain statistically significant results, we suggest that ß-catenin expression may not be directly influenced by the levels of REGγ expression (AU).

Comparative analysis of three histologic grading methods for squamous cell carcinoma of the lip.

OBJECTIVES: The aim of this study was to evaluate three histologic grading methods for squamous cell carcinoma (SCC) of the lip, the conventional three-grade model proposed by the World Health Organization, tumor budding and depth of invasion (BD) model, and histologic risk assessment (HRA) model, and to correlate them with prognosis. MATERIALS AND METHODS: Fifty-three patients with lip SCC were evaluated. RESULTS: The mean age was 65 years, 69.8% of the participants were men, and 66.0% of the patients had early-stage tumors. Using the BD and conventional three-grade methods, 52.8% and 64.2% of the cases were graded as low risk, respectively. The HRA model graded 54.7% of the cases as medium risk. In the BD model, the higher histologic grade was associated with worse prognosis (P = 0.045). Overall survival at 5 years was 87.8%. Tumor size (T3 + T4) and lymph node involvement (N+) were associated with reduced overall survival and recurrence-free survival (RFS) (P = 0.002 and 0.005; 0.007 and 0.01, respectively). Surgical treatment combined with radiotherapy was associated with lower RFS (P = 0.03). CONCLUSIONS: High-grade lip SCC in advanced stages is associated with a poor prognosis. The BD model is a simple and effective tool for the prognostic evaluation of lip SCC.

CD1a+ and CD83+ Langerhans cells are reduced in lower lip squamous cell carcinoma.

BACKGROUND: Actinic cheilitis (AC) is a potentially malignant lesion diagnosed in the lip of patients chronically exposed to the sun that may give rise to a fully invasive lower lip squamous cell carcinoma (LLSCC). It is known that ultraviolet radiation causes dendritic cells (DCs) depletion in the epidermis, but the role of this cellular population in lip cancer progression remains uncertain. Therefore, this study investigated the distribution of DCs in normal, dysplastic and neoplastic tissues of the lower lip. METHODS: Thirteen cases of lower lip mucocele, 42 of ACs and 21 of LLSCC were retrieved and original diagnoses confirmed by two oral pathologists, who further classified ACs as low- and high-risk lesions. Immunoreactions against CD1a and CD83 identified immature and mature DCs, respectively. RESULTS: Immature CD1a+ Langerhans cells (LCs) were significantly decreased in LLSCC when compared to morphologically normal (P < 0.009) and dysplastic epitheliums (P < 0.003), whereas mature CD83+ LCs were significantly decreased in LLSCC when compared to normal epithelium (P = 0.038). There was no significant difference between low- and high-risk ACs regarding CD1a+ and CD83+ LCs (P > 0.05), but ACs demonstrated a lower concentration of CD1a+ LCs than normal epithelium (P < 0.009). There was no significant difference in the distribution of CD1a+ and CD83+ interstitial dendritic cells (IDCs) in the connective tissue among the studied groups (P > 0.05). CONCLUSION: These results suggest that depletion of epithelial LCs, but not IDCs in the connective tissue, would represent an important step for lip cancer development.

Lip cancer and pre-cancerous lesions harbor TP53 mutations, exhibit allelic loss at 9p, 9q, and 17p, but no BRAFV600E mutations.

Molecular mechanisms of lip squamous cell carcinoma (LSCC) and actinic cheilitis (AC) are unclear. We aimed at assessing loss of heterozygosity (LOH) and TP53 and BRAF V600E mutations in these lesions. Formalin-fixed paraffin-embedded (FFPE) samples of 17 LSCC and 16 AC were included, with additional 5 fresh LSCC genotyped for TP53 mutations. LOH was assessed by six polymorphic markers located at 9p22, 9q22, and 17p13 and correlated with cell proliferation (Ki-67) and P53 immunostaining. Direct sequencing of TP53 exons 2-11 (fresh samples), and exons 5-9 (FFPE samples) was carried out. BRAF V600E mutation was genotyped in eight LSCC. LOH occurred in at least one marker in 15/17 LSCC and in 9/16 AC. The marker exhibiting the highest frequency of allelic loss (FAL) in LSCC was D9S157 (8/12 informative cases) and D9S287 in AC (4/11 informative cases). Cell proliferation was not correlated with LOH or with the FAL and no correlation between P53 IHC and 17p LOH was observed. We found TP53 missense mutations in both lesions and nonsense in LSCC, including CC>TT transition, which is a marker of UV damage. BRAF V600E mutation was not detected. LOH and TP53 mutations detected in LSCC and AC may be associated with tumorigenesis, whereas BRAF V600E mutation does not seem to significantly contribute to LSCC pathogenesis.

E-cadherin and c-Met expression in actinic cheilits and lip squamous cell carcinoma

Objective: The aim of this study was to assess epithelial expression of E-cadherin and c-Met in normal lip, in actinic cheilitis and lip squamous cell carcinoma. Study Design: Biopsies of normal lip vermillion (NL, n=18), actinic cheilitis (AC, n=37), and lip SCC (n=22) were processed for E-cadherin and c-Met immunodetection. Epithelial and tumor cell expression was scored for each sample considering staining intensity and percentage. Results: E-cadherin expression was significantly reduced in AC and lip SCC as compared to normal lip (P<0.05), with a significant reduction in lip SCC as compared to AC (P=0.003). Expression of c-Met was significantly higher in AC and lip SCC as compared to NL (P<0.05), with a significant increase in lip SCC as compared to AC (P<0.0001). Conclusion: The results showed that epithelial E-cadherin expression is reduced and c-Met expression is increased as lip carcinogenesis progresses, suggesting that these proteins may be useful markers of malignant transformation.

Comparative analysis of the mast cell density in normal oral mucosa, actinic cheilitis and lip squamous cell carcinoma

Previous studies have shown that the number of mast cells is increased in ultraviolet (UV) irradiated skin and in neoplasias. Actinic cheilitis (AC) is a lesion caused by excessive exposure to sunlight that can transform into lip squamous cell carcinoma. The aim of this study was to compare the number of mast cells in 4 groups: NOM = normal oral mucosa (n=6); MDAC = mild dysplasia in actinic cheilitis (n=13); SDAC = severe dysplasia in actinic cheilitis (n=13); and LSCC = lip squamous cell carcinoma (n=15). The sections were stained by histochemical technique of blue toluidine and visual counting was performed with the aid of a reticulum coupled to the microscope ocular. A calibrated observer performed the count in 5 fields by case at ×400 magnification. The largest mean number of mast cells per group was observed in LSCC (40.1), followed by MDAC (30.5), SDAC (28.6) and NOM (12.2). There were significant differences between NOM and MDAC (p<0.05) and between NOM and LSCC (p<0.05). The increased density of mast cells observed in AC and in LSCC compared to NOM suggests a role for the mast cells in the development of these lesions.

Estudos prévios mostram que os mastócitos estão significantemente aumentados na pele irradiada por ultra-violeta e neoplasias. A queilite actínica (QA) é uma lesão causada por excessiva exposição solar, que pode transformar-se em carcinoma espinocelular de lábio. O objetivo deste estudo foi comparar o número de mastócitos em 4 grupos: MON = mucosa oral normal (n=6); QADL = queilite actínica com displasia leve (n=13); QADS = queilite actínica com displasia severa (n=13); e CECL = carcinoma espinocelular de lábio (n=15). Os cortes foram corados pela técnica histoquímica do azul de toluidina e a contagem visual foi realizada utilizando um retículo acoplado à ocular do microscópio. Um observador calibrado realizou a contagem em 5 campos por caso em magnificação de ×400. A média de mastócitos por grupo foi maior no CECL (40,1), seguida da QADL (30,5), QADS (28,6) e MON (12,2). Houve diferença estatisticamente significante entre MON e QADL (p<0,05) e entre MON e CECL (p<0,05). A maior densidade de mastócitos na QA e no CECL em relação à MON sugere um papel para os mastócitos no desenvolvimento dessas lesões.