The worldwide annual incidence of squamous cell carcinomas of the head and neck (HNSCCs) is about 500,000. The prognosis of these patients is based mainly on the clinicopathological tumor stage, in particular the lymph node status pN, even though it is generally accepted that outcomes can be different among tumors of the same stage. Therefore, our study focused on the identification of molecular parameters for the improvement of the daily clinical diagnosis, which contributes to further prognostic differentiation. Polo-like kinases (PLKs) have been implicated in the regulation of the eukaryotic cell cycle (Lane and Nigg, 1997). Expression of PLK mRNA, a novel marker for cellular proliferation, correlates with the prognosis of patients suffering from different types of tumor (Holtrich et al., 1994; Wolf et al., 1997; Knecht et al., 1999). We have examined the prognostic role of PLK-protein expression in human cancer. Primary tumors from 157 patients with HNSCC, collected over a period of 1 year, were evaluated for PLK immunoreactivity. Therapeutic and post-therapeutic investigations (5 years) of patients (115 male, 42 female) suffering from oropharyngeal carcinomas (stage I 14, stage II 30, stage III 34, stage IV 79) were performed as follows. Patients in tumor stages I and II underwent surgery, whereas patients in stages III and IV were subjected to surgery and post-operative radiotherapy (2 Gy fractions given as once-a-day treatment to a total dose of 70 Gy applied to the primary region and the neck). Chemotherapy was given non-concomitantly, using 3 cycles of cisplatin (20 mg/qm) and 5-fluorouracil (1,000 mg/qm) according to a standard schedule (Forastiere et al., 1992). Tumor recurrences were treated chemotherapeutically with the same regimen until no response or progressive disease was measured in 2 dimensions by computed tomography. The length of follow-up was between 5 and 132 months.

Paraffin-embedded tumor sections were immunolabeled with an affinity-purified PLK-specific, polyclonal rabbit serum. The primary antibody, diluted 1: 80, was visualized by the APAAP technique; sections were counterstained with hematoxylin. For comparative purposes, sections were labeled with antibodies for Ki67 (1: 50; Dako, Hamburg, Germany) and PCNA (1: 1,000; Dako) using the same technique. Three independent investigators calculated the number of tumor cells (stained nuclei) positive for PLK, Ki67 or PCNA, respectively, per 5,000 tumor cells. A minimum of 15 fields (400) per tumor were investigated. Prognostic evaluations were based on the mean PLK index (positive cells/5,000 cells 100). Levels of PLK protein in microscopically normal oropharyngeal mucosa (median 12.0%, minimum 0.1%, maximum 41.0%) surrounding a tumor were low. In 10% of the investigated cases, we observed in the surrounding tissue (within 3 to 5 cm of the tumor) dysplasia (grade I/II). Under