Koos Boeve

104 Chapter 6 positive predictive value of 46% [13]. In HNSCC, the commonly amplified region contains 9 genes that are overexpressed when amplified including FADD, CCND1, TPCN2, PPFIA1, FLJ42258, CTTN1, FGF19, ORAOV1 and ANO1 [11]. At least 3 of these oncogenes on this region ( CCND1 , CTTN and FADD) play key roles in cellular migration of epithelial cells and are, therefore, potential biomarkers for metastases in oral cancer [9,12,14,15]. Furthermore, immunohistochemical (IHC) expression of cyclin D1, FADD and cortactin have been described as potential predictors for increased disease-related mortality, for lymph node metastasis and poor prognosis in oropharyngeal carcinomas [10-12]. Until now, only 1 study investigated CCND1 amplification and expression in early oral cavity SCC, in a relatively small cohort of 45 patients [16]. To validate the value of CCND1 as a predictive biomarker for the detection of occult nodal metastasis, we correlatedgene amplification of CCND1 andprotein overexpression of 3major oncogenes (cyclin D1, FADD and cortactin) with nodal status in a large consecutive and well-documented cohort of early oral cavity SCC. Furthermore, intra-tumor heterogeneity of protein expression of these biomarkers was analyzed to see if a biopsy could represent the whole tumor for these potential biomarkers. The correlation between expression of cyclin D1 and lymph node metastasis was subsequently validated in an independent multicenter cohort of oral cavity SCC. MATERIALS AND METHODS Cohort We enrolled a consecutive cohort of 158 patients with cT1-2 cN0 tongue and floor of mouth (FOM) cancers, primarily treated by surgery between January 2004 and December 2010 at the University Medical Center Utrecht as described earlier [13,17]. All cases were clinically lymph node negative, based on extensive imaging with both CT or MRI and ultrasound with FNAC, in case of a suspicious lymph node. Patients with a medical history of HNSCC or a synchronous primary tumor were excluded from this study. Demographic, clinical, histologic and treatment data were retrieved from electronic medical records (see Table 1). For validation, 2 independent cohorts of early tongue and FOM, SCCs primarily treated by surgery at the University Medical Center Utrecht (1996-2003, n = 73) and the University Medical Center Groningen (1997-2008, n = 82) were used [18,19]. For both validation cohorts, tissue microarrays (TMAs) were available.