Koos Boeve

114 Chapter 6 of CCND1 and overexpression of its encoded protein cyclin D1. However, only one small study was performed in early oral cavity SCC to establish its value in the detection of occult nodal metastasis [14]. In this largest study so far in early oral cavity SCC, both CCND1 copy number and nuclear cyclin D1 expression are significantly correlated with occult nodal metastasis with a NPV of respectively, 83% and 84% in clinically early oral cancer. These results are in line with a NPV of 83% found by Myo et al,[22] which is the only other study investigating the correlation between CCND1 amplification and occult nodal metastasis in early oral cancer. Protein expression of both FADD and cortactin had a slightly lower NPV compared to cyclin D1. As expected, combined expression of these 3 proteins did not improve the NPV for occult nodal metastasis significantly, because the genes encoding for these proteins are situated on the same chromosomal region (11q13.3), which is often amplified as a whole in HNSCC [9]. Subsite analysis reveals a higher NPV of CCND1 amplification and cyclin D1 expression in the FOM compared with tongue tumors, 95%, 97% and 76% respectively. As a consequence, the cyclin D1 biomarker may have a complementary role to the SNB procedure, as this procedure lacks accuracy in FOM tumors (NPV of 88% instead of 98% in other subsites) because of the close relationship between the primary tumor and first draining nodes, known as ‘shine-through’ phenomenon [23,24]. Multicenter validation in the described Utrecht and Groningen cohorts, including a total of 155 early tongue and FOM tumors, confirmed the predictive value for occult nodal metastasis of cyclin D1 expression in FOM tumors but not in tongue tumors. However, the NPV was lower than in the initial cohort (79% versus 95%). This might be explained by the composition of the validation cohorts; only patients with an elective neck dissection were included in these cohorts, which lead to selection bias. For the clinical application of a biomarker predicting occult nodal metastasis, it is pivotal that a biopsy represents thewhole tumor, i.e. expression of a biomarker is consistent in the biopsy as well as the resection specimen. Because the phenomenon of intratumor heterogeneity is common in head and neck cancer, consistency of biomarkers must be checked [25]. A well-known method to analyze intratumor heterogeneity is by establishing the ICC among multiple samples, in this study multiple cores, of the same tumor [26]. IHC expression of all 3 studied proteins (cyclin D1, FADD and cortactin) showed high concordance with an ICC between 0.89 and 0.90, which indicates almost perfect agreement between the cores [20]. Therefore, IHC expression of these proteins in a biopsy is representative for expression in the whole tumor in early oral cavity SCC. Furthermore, the high interobserver agreement of cyclin D1 expression (ICC = 0.94) showed the high reproducibility of this biomarker.