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 these conditions, levels of PLK-protein expression were slightly elevated. Compared to the periphery, PLK protein was over-expressed (median = 59.4%, minimum = 22.3%, maximum = 74.8%) in oropharyngeal carcinomas (Wilcoxon's test, p = 0.016). A correlation of PLK expression with clinicopathological parameters led to further significant correlations with pathological tumor stage (p = 0.007) and pN stage (p = 0.012) but not with N stage (p = 0.09, Kruskal-Wallis test). A Kaplan-Meier analysis based on a median cut-off showed that patients exhibiting moderate PLK expression (<59.4%) had longer survival times than those exhibiting high expression (≥59.4%, log rank p = 0.013; Fig. 1). Median survival times were 52 months [95% confidence interval (CI) 36–67 months] and 26 months (95% CI 14–37 months), respectively. To test the selectivity of PLK prognostic information, we performed a Cox regression analysis. During stepwise backward selection from entered variables (age, sex, pT, pN, PLK expression, therapy), only pN (eB = 1.87, 95% CI 1.14–2.89, p = 0.007) and PLK (eB = 2.31, 95% CI 0.96–5.56, p = 0.039) were significant predictors of survival. In contrast, the proliferation markers Ki67 and PCNA, which are frequently discussed as prognostic parameters for HNSCC, were not associated with tumor stage, pN or survival in our study, which is in line with the results of Sommer and Olofsson (1997). Kaplan-Meier curve of survival of patients with HNSSC (n = 157). Adjusted survival rate is given as a percentage. PLK-expression indices are grouped according to a median of 59.4% into moderate (<59.4%, n = 78) and high (≥59.4%, n = 79). Log-rank test p = 0.013. Based on immunohistochemical methods, PLK appears to be a new prognostic marker for the daily routine diagnosis of the risk of dying for patients with oropharyngeal carcinoma in addition to the pN stage. Moreover, if the pN stage is unknown and the prognostic information relies only on clinical investigations, determination of PLK expression in tumor-derived specimens could improve the prediction of survival. Yours sincerely, Rainald Knecht, Christine Oberhauser, Klaus Strebhardt
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Aug 6, 2016
Mohamed Badawy Abdel-Naser + 5
Open Access
