Kendall's tau correlation coefficient with the associated *p* value.

### *3.3. Analysis of CCND1, ANO1, and CTTN Gene Amplification in Relation to HPV Status in HNSCC Patients*

*CCND1*, *ANO1*, and *CTTN* gene gain and amplification was assessed by real-time PCR in 88 cases selected from the same HNSCC tissue blocks, including 26 HPV-positive cases. We found gene gains and amplifications of *CCND1* in 32 (36%) tumors, *ANO1* in 42 (48%) tumors, and *CTTN* in 26 (30%) tumors (Table 2), with relative gene copy numbers ranging from 2- to 24-fold. Co-amplifications of these three genes were also frequently observed (26 cases, 30%). We also found that tumors harboring gene amplification significantly correlated with higher expression levels of each protein (Figure 3). When analyzing the correlations with HPV infection status, we consistently found that *CCND1*, *ANO1*, and *CTTN* gene amplification inversely correlated with HPV status (Table 2). Thus, amplifications of *CTTN, CCND1*, and *ANO1* were frequent in HPV-negative tumors (ranging from 42 to 61%), while absent in HPV-positive tumors.


**Table 2.** Analysis of *CCND1, ANO1*, and *CTTN* gene gain and amplification in relation to HPV status in 88 HNSCC patients.
