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

**Figure 1.** Representative images of p16-positive (**A**) and negative (**C**) immunostaining in the HNSCC tissue microarrays (TMAs) and HPV-positive (**B**) and HPV-negative (**D**) cases detected by in situ hybridization. Original magnification ×10.

**Figure 2.** Representative examples of negative and strong positive staining for CCND1 (**A**,**B**), ANO1 (**C**,**D**), and CTTN (**E**,**F**). Original magnification ×20.

*3.4. Analysis of CCND1, ANO1, and CTTN mRNA Expression in Relation to HPV Status in 279 HNSCC Patients from the TGCA*

In order to confirm our results, we performed an analysis of the publicly available TCGA data from 279 HNSCC patients [19] using the platform cBioPortal for Cancer Genomics (http://cbioportal. org/) [20]. The clinicopathologic characteristics of this cohort are summarized in Supplementary Table S1 and Figure S2. A total of 36 (13%) patients were positive for HPV infection, most prevalent in the oropharynx (22 cases, 61%), and associated to lower tobacco consumption, lower mutations, improved survival, and a younger age in both men and women. The results also evidenced differential changes in mRNA expression levels of *CTTN*, *CCND1* and *ANO1* depending on HPV infection status. Thus, increased mRNA expression levels of these genes were frequently and significantly observed in HPV-negative tumors (Figure 4A–D), while very rare in HPV-positive patients (Table 3). Similarly, the analysis of copy number alterations of *CTTN, CCND1*, and *ANO1* genes also revealed that both amplifications and gains of these three genes were also highly frequent in HPV-negative tumors,

whereas almost absent in HPV-positive tumors (Figure 4A,E). Furthermore, the results consistently showed that these three genes were frequently co-amplified (28%) and overexpressed at a higher frequency (39–46%), and, more importantly, these molecular alterations (in particular CTTN and ANO1 overexpression) were associated with a worse clinical outcome in the TCGA cohort of 279 HNSCC patients and also in an extended TCGA cohort which added 251 new HNSCC patients (*n* = 530) (Figure 5).


Spearman Coefficient 0.711, *p* < 0.001.

**Figure 3.** Crosstab to evaluate the correlations between gene gains and amplification and protein staining scores for CCND1, ANO1, and CTTN.

**Figure 4.** Analysis of mRNA expression and copy number alterations of *CTTN*, *CCND1* and *ANO1* in relation to HPV status related to the available HNSCC TCGA data (*n* = 279) obtained from cBioPortal. (**A**) Schematic representation and heat map showing the percentage of cases with *CTTN, CCND1*, and *ANO1* gene amplification and mRNA expression in relation to the HPV status. (**B**) CTTN, (**C**) CCND1, and (**D**) ANO1 mRNA expression distributed according to the HPV status. mRNA expression (RNA seq V2 RSEM) values were Log2 transformed (*y*-axis). Whiskers plot (min. to max.) with median values; *p* < 0.001, two-tailed Student *t*-test. (**E**) Copy number alterations of *CTTN*, *CCND1* and *ANO1* according to the HPV status using the GISTIC method.

**Figure 5.** Analysis of *CTTN*, *CCND1* and *ANO1* gene amplification and mRNA expression in the TCGA cohort of 530 HNSCC patients using cBioPortal. Schematic representation showing the percentage of cases with amplification or mRNA upregulation of each gene. Kaplan–Meier survival curves categorized by *CTTN*, *CCND1* and *ANO1* gene amplification dichotomized as positive *versus* negative; CTTN, CCND1 and ANO1 mRNA expression (RNA seq V2 RSEM, *z*-score threshold ±2) dichotomized as normal versus upregulation (UP); *p* values estimated using the Log-rank test.

**Table 3.** Analysis of *CCND1*, *ANO1*, and *CTTN* mRNA expression in relation to HPV status in 279 HNSCC TGCA patients.

