CD44 and ALDH1 immunoexpression as prognostic indicators of invasion and metastasis in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common cancer in the head and neck and results in high morbidity and mortality annually, being the worst prognosis related to the presence of metastasis in cervical lymph nodes. Metastasis has been associated with a subpopulation of tumor cells, called cancer stem cells (CSCs), which consists of a small population with stem-like cells properties, higher rate of migration and metastatic potential compared to other ordinary tumor cells from the tumor bulk. The aim of present study was to evaluate the immunoexpression of the CSC markers ALDH1 and CD44 in primary sites of OSCC and corresponding metastatic lymph nodes, by means of immunohistochemistry. The immunolabeling was further correlated with clinicopathological data. Archived Formalin-fixed, Paraffin-embedded tumor tissue specimens (n=50) and corresponding metastatic lymph nodes (n=25) were obtained from 50 patients with OSCC after surgical treatment. CD44 and ALDH1 immunostaining were semi-quantitatively scored according to the proportion and intensity of positive cells within the invasive front and metastatic cervical lymph nodes as a whole. The percentage of ALDH1 and CD44 positive tumor cells as well as immunostaining intensity was graded and a combined score, ranging from 0 to 9 (ALDH1) or 0 to 12 (CD44), was obtained by multiplying both parameters. Next, combined scores were dichotomized into a final score classified as low (ALDH1≤ 2; CD44≤ 4) or high (ALDH1> 2; CD44> 4) immunoexpression. ALDH1 and CD44 immunoexpression was detected in both primary and metastatic tumor sites, although with different immunolabeling pattern. ALDH1-positive tumor cells consisted of scattered patches and no immunoexpression was observed within keratin pearls. Conversely, CD44 immunopositivity was more homogeneous and widely distributed, with higher labeling in peripheral areas of the tumor islands within the tumor invasion front. Although not statistically significant, the means of ALDH1high (p= 0.0985) and CD44high (p= 0.1632; Mann- Whitney post-test) immunoexpression were higher in metastatic lymph nodes compared to primary tumors. ALDH1high was positively associated (p= 0.0184) with perivascular invasion, while CD44high was positively associated (p= 0.0186; Fisher's Exact Test) with metastasis (N+). Five-year survival rates tended to be lower in patients with ALDH1high immunoexpression compared to ALDH1low, although with no statistical significance (p= 0.1303). In summary, the present study revealed that CD44 is highly labeled in tumor cell from metastatic sites, being associated with lymph node metastasis, while ALDH1 high immunostaining was associated with perivascular invasion. Altogether, it suggests that immunoexpression of CD44 and ALDH1 links the cancer stem cell phenotype with OSCC invasion and metastasis.(AU) O carcinoma epidermoide de boca (CEB) e uma das neoplasias mais comuns da regiao de cabeca e pescoco e resulta em alta morbidade e mortalidade anualmente, estando o pior prognostico relacionado a presenca de metastase em linfonodos cervicais. O processo de metastase tem sido associado a uma subpopulacao de celulas tumorais, chamadas celulas-tronco de câncer (CSC, do ingles Cancer stem cells), que consistem em uma pequena populacao de celulas com propriedades de celulas-tronco, incluindo maior taxa de migracao e potencial metastatico em comparacao com outras celulas tumorais. O objetivo do presente estudo foi avaliar os marcadores candidatos de CSCs ALDH1 e CD44 em tumores primarios de CEB e metastases linfonodais correspondentes, por meio de imuno-histoquimica. A imunomarcacao foi posteriormente correlacionada com dados clinico-patologicos. Foram obtidas amostras de tecido tumoral parafinado fixado em formalina (n = 50) e os linfonodos metastaticos correspondentes (n = 25) de 50 pacientes com CEB submetidos somente ao tratamento cirurgico. Os marcadores CD44 e ALDH1 foram analisados de forma semi-quantitativa de acordo com a proporcao e intensidade de celulas positivas no fronte de invasao e em linfonodos cervicais metastaticos como um todo. A porcentagem de celulas tumorais ALDH1 e CD44 positivas, bem como a intensidade da imunomarcacao, foi classificada em um escore combinado obtido pela multiplicacao de ambos os parâmetros, variando de 0 a 9 (ALDH1) ou 0 a 12 (CD44). Em seguida, as pontuacoes combinadas foram dicotomizadas em um escore final classificado como baixo (do ingles low) (ALDH1 ≤ 2; CD44 ≤ 4) ou alto (do ingles high) (ALDH1> 2; CD44> 4). A imunoexpressao de ALDH1 e CD44 foi detectada tanto em tumores primarios quanto em linfonodos cervicais metastaticos, embora com padrao diferente de imunomarcacao. Celulas tumorais ALDH1-positivas foram identificadas como focais e dispersas ao longo do fronte de invasao, sem imunomarcacao nas perolas corneas. Em contraste, a imunopositividade para CD44 foi mais homogenea e amplamente distribuida, com maior imunomarcacao em areas perifericas das ilhotas tumorais presentes no fronte de invasao. Embora nao estatisticamente significativa, as medias da imunoexpressao ALDH1high (p = 0.0985) e CD44high (p = 0.1632, pos-teste de Mann-Whitney) foram maiores em linfonodos metastaticos em comparacao com tumores primarios. ALDH1high foi positivamente associado com invasao perivascular (p = 0.0184), enquanto CD44high foi com metastase (N+) (p = 0.0186; teste exato de Fisher). As taxas de sobrevida global em 5 anos tenderam a ser mais baixas em pacientes com imunoexpressao elevada de ALDH1 em comparacao com ALDH1low, embora sem significância estatistica (p = 0.1303). Em resumo, o presente estudo revelou que a elevada imunomarcacao de CD44 esta significativamente associada com metastases linfonodais, enquanto que a elevada imunomarcacao de ALDH1 esta associada com invasao perivascular. Em conjunto, sugerimos que a imunoexpressao de CD44 e ALDH1 esteja relacionada com o fenotipo de celulas tronco de câncer que tem capacidade de invasao e metastase em CEB.(AU)