PRAME expression in head and neck cancer correlates with markers of poor prognosis and might help in selecting candidates for retinoid chemoprevention in pre-malignant lesions

Abstract

Objectives: PRAME (Preferentially Expressed Antigen in Melanoma) is a tumor-associated antigen recognized by immunocytes, and it induces cytotoxic T cell-mediated responses in melanoma. PRAME expression in tumors interferes with retinoic acid receptor (RAR) signaling thus promoting tumor progression. Here, we study PRAME expression in head and neck squamous cell carcinoma (HNSCC) to determine its potential clinical significance.

Materials and methods: PRAME expression in HNSCC was evaluated by immunohistochemistry in tissue microarrays of primary tumors (n=53), metastatic lymph nodes (n=8) and normal oral mucosa (n=11). Biopsies of dysplastic oral lesions (n=12) were also examined. PRAME expression levels in tissues were correlated with markers of poor prognosis in HNSCC. PRAME mRNA in HNSCC cell lines and in normal immortalized human keratinocytes (HaCaT cell line) was measured by qRT-PCR, and the protein expression by flow cytometry and western blots.

Results: PRAME was expressed in HNSCC cell lines and HNSCC lesions. PRAME expression in dysplastic mucosa was variable. No or only weak expression was found in normal cells or tissues. PRAME expression levels significantly correlated with the tumor grade, size, nodal involvement and the clinical status of HNSCC patients.

Conclusions: Elevated PRAME expression associates with clinicopathologic markers of poor outcome in HNSCC and might identify potential candidates with pre-cancerous lesions for chemoprevention with retinoids.

Figure 1. PRAME expression in PCI-1, PCI-13, PCI-30 and HaCaT cell lines

(A) Expression of PRAME mRNA. The PRAME transcript levels were determined by qRT-PCR analysis of cDNA. The cells were maintained in complete RPMI-1640 medium. After trypsinization and cell collection, total RNA was isolated and treated with DNase I, quantified, and reverse-transcribed into cDNA. The levels of PRAME transcripts were standardized against PBGD cDNA level, and are expressed as relative levels per 1μg of total RNA. Each sample was measured in triplicate, and the results represent means ± SD from three experiments; (B) Expression of the PRAME protein as measured by flow cytometry using permeabilized PCI or HaCaT cells. The data are expressed as mean fluorescence intensity (MFI) (open peaks). Black peaks show control cells stained with isotype control Abs. The inserts on the upper right show results of western blots for PRAME. Representative data for one of three experiments performed with each cell line are shown.

Figure 2. PRAME expression in normal control (NC) oral mucosa obtained from normal donors (NC), HNSCC lesions or metastatic lymph nodes (LN)

(A) Isotype Ab control in a specimen of NC mucosa (× 200); (B) No or only weak expression of PRAME in a specimen of NC mucosa (× 200); (C) Negative (isotype Ab) control in a specimen of HNSCC (× 200); (D) Strong expression of PRAME in a representative specimen of HNSCC (× 200); (E) PRAME expression (% POSITIVE CELLS) in NC vs. HNSCC or LN, respectively. All HNSCC and LN were scored at >75% positivity; (F) PRAME expression (INTENSITY, measured as described in Methods) in NC vs. HNSCC or LN, respectively flow cytometry.

Figure 3. PRAME expression in HNSCC tissues

(A) PRAME staining intensity vs. tumor grade in all tumors; (B, C) representative PRAME expression in G1 tumor vs. G3 tumor (× 200); (D) PRAME staining intensity vs. tumor size for all tumors; (E, F) representative PRAME expression in T1 tumor vs. T3 tumor (× 200); (G) PRAME staining intensity vs. nodal involvement for N- or N+ specimens; (H, I) representative PRAME expression in tumor without nodal involvement (N-) vs. tumor with nodal involvement (N+) (× 200); (J) PRAME staining intensity vs. clinical stage for all tumors; (K, L) representative PRAME expression in stage I vs. stage III (× 200).

Figure 4. PRAME expression in dysplastic oral mucosa

(A) Negative/weak expression of PRAME in a specimen of the mild grade dysplasia (SIN II). (B) High PRAME expression in a specimen of the moderate grade dysplasia. (× 200). Representative data.