الفهرس 
ACKNOWLEDGMENTOnly 14 pages are availabe for public view
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Abstract
Head and neck cancer is a major health problem throughout the world and the sixth most common malignancy in humans. It represents almost 95% of the head and neck cancer cases.
Recent studies on the pathobiology of oral squamous cell carcinoma (OSCC) have led to the discovery of a small population of cancer cells that is highly tumorigenic, capable of self-renewal and behave as tumor progenitor cells. Such behavior is consistent with the features of cancer stem cells (CSCs). CSCs play an important role in the development and maintenance of different tumors. CSCs are required and responsible not only for initiation, but also for maintenance and recurrence of disease.
The standard of care for patients with OSCC includes platinum-based chemotherapeutic drugs, surgery and radiotherapy. Surgical treatment and frequent local recurrences significantly reduce patients’ quality of life. Despite advances in therapy, mortality from this disease remains high. In OSCC, CSCs might have already metastasized at the time of surgery.
Future CSC-targeted therapies could lead to degeneration and involution of the tumor and lasting cure. CSCs show more resistance to chemo- and radiotherapy than non-stem cells. They share many features with physiological stem cells. A strategy commonly employed by investigators is the use of molecular markers for the identification of CSCs.
Among all CSC markers, CD44 is the most reported CSC marker in OSCC. CD44 is an integral cell membrane glycoprotein; it is involved in cell migration, adhesion and considered a key marker for identification of CSCs in squamous cell carcinoma (SCC). CD44 is of particular interest because it seems to be a relatively early marker of malignancy.
Considering the implication of CD44, it was decided to study the role soluble CD44 (CD44sol) in head and neck tumors by focusing on the identification of its soluble fraction in the peripheral blood in OSCC patients. This is a simple, noninvasive tool in cancer diagnostics and in monitoring the efficiency of treatment. CD44sol appeared to be a possible candidate as a screening marker for the early diagnosis of head and neck tumors, because of its stem-like properties.
The aim of the present study was to evaluate and correlate the expression of CD44 in different histopathological grades of OSCC, as well as to assess the diagnostic and prognostic value of CD44sol in peripheral blood of cancer and non cancer patients.
In the present study, a total of fifteen patients with OSCC were included. The patients’ age ranged between 52 and 71. The mean age was found to be (61.8 ± 6.3 years). Ten patients (66.7%) were males and five patients (33.3%) were females.
The most common site of occurrence was the lateral side of the tongue representing (46.7%) (n=7). The second common site was the alveolar mucosa representing (26.7%) (n=4). Followed by the buccal mucosa representing (13.3%) (n=2). The upper lip and lower lip were represented each by one case only (6.7%) (n=1).
Biopsies were histologically evaluated using haematoxylin and eosin (H&E) staining. The microscopical examination revealed that five cases (33.3%) were of the well differentiated type, eight cases (53.3%) were moderately differentiated OSCC and two cases (13.3 %) were of the poor differentiated type.
The primary tumor staging of the 15 patients included in the present study was found as follows; T2 was the predominant stage (53%) (n - 8), followed by T3 (27%) (n - 4), followed by T1 (13%) (n – 2) and the least common stage was T4a (7%) (n – 1).
The well differentiated OSCC revealed irregular islands of hyperchromatic and pleomorphic malignant epithelial cells forming large keratin pearls and epithelial pearls that deeply invaded into the connective tissue. The moderately differentiated OSCC exhibited nests of malignant cells with little or no keratin formation. The malignant epithelial cells showed hyperchromatism, pleomorphism, prominent nucleoli, increased nuclear cytoplasmic ratio and some mitotic figures. The poorly differentiated OSCC revealed highly anaplastic epithelial cells showing all the malignant criteria.
Serial sections were immunohistochemically stained by monoclonal antibody to CD44 using Labeled- Strept -Avidin Biotin (LSAB) complex method. The intensity of immunostaining of CD44 was calculated in terms of mean area percent and mean optical density by the computer image analyzer. Positive immunoreaction was classified into intense, moderate and weak reactions according to the intensity of staining.
All oral squamous cell papilloma cases (n=3) showed positive immunoreactivity for CD44 in the basal and suprabasal layers. The difference in mean CD44 area percent and optical density between the well, moderately and poorly differentiated groups revealed significantly significant difference at (p<0.001).
All grades of OSCC cases showed membranous immunosignaling of CD44. The nuclei, cytoplasm and keratin were free from any reaction in all the samples. The well differentiated OSCC cases showed weak positive membranous immunosignaling of CD44 in the peripheral epithelial cells forming the epithelial pearls and nests. The moderately differentiated OSCC cases demonstrated moderate positive membranous immunoreaction of CD44. A larger percentage of the peripheral malignant epithelial cells were stained. In the poorly differentiated type of OSCC, intense CD44 immunopositivity was detected in the malignant epithelial cells. It showed the highest number and greatest intensity of immunopositive cells.
Enzyme-linked immunosorbent assay (ELISA) method was used to determine the concentration of CD44sol in the serum of normal individuals, squamous cell papilloma patients and OSCC patients. CD44sol levels were significantly higher in OSCC patients than they were in patients with benign epithelial tumor and in healthy individuals (18.66 ± 3.52 IU/ml, 6.5 ± 1.32 IU/ml and 2.8 ± 0.53 IU/ml) respectively at (p<0.001).
Soluble CD44 serum levels were significantly higher in poorly differentiated than they were in moderately and well differentiated (26.07 ± 2.96 vs. 18.55 ± 1.33 vs. 15.88 ± 1 IU/ml) respectively. In moderately differentiated CD44sol serum levels were significantly higher than they were in well differentiated. CD44sol serum levels were significantly higher in OSCC patients exhibiting invasion into the surrounding structures compared to others lacking invasion at (p<0.001).
The present study results were in accordance with many other researchers. Though CD44 is generally accepted as a substitute marker for detection of CSCs in OSCC, several studies were in contrast to the present study results. The discrepancies between these results and the present study results may be due to differences in the site of origin, characteristics of the patients selected, different treatments, different methods used or different variants measured.
The current study concluded that CSCs can reliably be detected immunohistochemically using CD44 marker in fixed human tissues of OSCC. Different grades showed different Immunohistochemical expressions. CSCs detection in fixed human tissue and CD44sol detection in peripheral blood using ELISA seemed to be a promising method and may have a diagnostic and prognostic value in management of OSCC and non cancer individuals.
Further studies should be done to evaluate the therapeutic implications of CD44 in cancer treatment. Peripheral CD44sol level evaluation is recommended to provide presurgical information about the primary tumor grade and regional extension.