CD44s and CD44v6 expression in head and neck epithelia

Abstract

Background: CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia.

Methods: Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from - to +++ (score = % x intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC.

Results: In normal epithelia CD44s and CD44v6 were expressed in 60-95% and 50-80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5+/-14.5 and 198+/-11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9+/-7.16 and 242+/-11.7; 291.8+/-5.88 and 287.3+/-6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6.

Conclusion: CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision.

Figure 1. CD44s expression pattern in head and neck epithelia.

CD44 standard form was visualised upon immunohistochemistry (reddish brown) in normal epithelium (A), oral leukoplakia (B), carcinomas in situ (C), grade 2 carcinomas (D), and grade 3 carcinomas (E). Cell nuclei are stained with hematoxylin (blue). Shown are representative sections. Arrowheads mark infiltrating lymphocytes with high-level CD44s expression.

Figure 2. CD44v6 expression pattern in head and neck epithelia.

CD44 splice variant 6 was visualised upon immunohistochemistry (reddish brown) in normal epithelium (A), oral leukoplakia (B), carcinomas in situ (C), grade 2 carcinomas (D), and grade 3 carcinomas (E). Cell nuclei are stained with hematoxylin (blue). Shown are representative sections.

Figure 3. CD44s and CD44v6 expression scores in head and neck epithelia.

The expression scores of CD44s (upper) and CD44v6 (lower) implement expression intensity and percentages of positive cells (score = staining intensity×% positive cells; max score 300). Shown are the mean scores with standard deviations. Where indicated, differences across specimen groups were significant (* p<0.05; ** p<0.005). NE: normal epithelium; LP: leukoplakia; CIS: carcinoma in situ; Ca-G2: grade 2 carcinoma; Ca-G3: grade 3 carcinoma.

Figure 4. Comparative expression of CD44s and CD44v6 in head and neck epithelia.

CD44s and CD44v6 (reddish brown) were visualised in consecutive serial sections of a carcinoma in situ (A) and one carcinoma (B). Cell nuclei are stained with hematoxylin (blue). CIS: carcinoma in situ; ve: vessel; muc: mucosa; int: interstitium; ca: carcinoma; HP: horn-pearl forming keratinised tumour cells. Shown are representative sections.

Figure 5. Comparative expression of CD44s and CD44v6 in lymphoid cells.

Cell surface expression of CD44s and CD44v6 were assessed on peripheral blood mononuclear cells (A), B blast clones (B), lymphoblastoid cell clones (C), and HNSCC FaDu cells (D) using specific antibodies and flow cytometry. Red line: control; black line: specific antibody. LCL: lymphoblastoid cell lines. Shown are representative results of two independent measurements.