Determination of factors affecting overall survival.

<p>Patients overall survival analyses were made between two groups: (A) patients with or without mutations in <i>NLRP</i> genes; (B) patients with or without mutations in <i>TP53</i> genes; (C) patients with tumors that harbored <i>TLR</i> mutations and those without <i>TLR</i> mutations; (D) patients with or without HPV infections; (E) patients with low stage (stage II) or advanced stage (stage III and beyond) tumors; (F) patients of less or more than 56 years old; (G) patients who received no adjuvant therapy, radiotherapy alone or both chemotherapy and radiotherapy.</p

Identification of <i>NLRP</i> mutations in FOM HNSCC.

<p>Mutations in four <i>NLRP</i> genes were identified in FOM HNSCC patients. Black triangles indicate novel mutations identified in this study. Gray triangles represent reported mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) database.</p

Mutation rates of FOM HNSCC.

<p>(A) Numbers of missense and nonsense mutations were compared between patients with FOM or non-FOM HNSCC. (B) Mutation rates were compared between patients with FOM or non-FOM HNSCC.</p

Demographic Profile of Study Subjects with Primary Conventional HNSCC (PC-SCC).

<p>Contingency tables were analyzed by Fisher's exact test. Survival distributions were analyzed by Log Rank test. Notes:</p>†<p>Typical number of alcohol drinks in 2 week period, §Mann-Whitney U test,</p>∥<p>Fisher's exact test,</p>¶<p>Log rank test,</p>*<p>Comparing patients with or without mutations in <i>NLRP</i> genes.</p

Identification of <i>NLRP</i> mutations in primary HNSCC.

<p>Novel mutations were identified in 10 <i>NLRP</i> genes in HNSCC. Most mutations were involving chromosomal locations 11p15.4 and 19q13.42-19q13.43.</p

Mutation rates comparisons.

<p>(A) Numbers of missense and nonsense mutations were compared between tumors with or without <i>NLRP</i> mutations. (B) Numbers of missense and nonsense mutations were compared between tumors with or without <i>TP53</i> mutations. (C) Numbers of missense and nonsense mutations were compared between tumors with or without <i>TLR</i> genes mutations. (D) Mutation rates [shown as mutations per million base (MB) pairs] were compared between tumors with or without <i>NLRP</i> mutations. (E) Mutation rates [shown as mutations per million base (MB) pairs] were compared between tumors with or without <i>TP53</i> mutations. (F) Mutation rates [shown as mutations per million base (MB) pairs] were compared between tumors with or without <i>TLR</i> genes mutations. P value less than 0.05 was considered significant.</p

HNSCC long range PCR of EGFR intron 1.

<p>^a The NCBI reference sequence for EGFR GRCh37.p10 was used with NT_033968.6 for DNA and NM_005228.3 for mRNA</p><p>^b mRNA with EGFRvIII transcript is denoted as “+”, wtEGFR only transcript is denoted as “-“</p><p>^c Tumor samples with EGFR gene amplification are denoted by “+”</p><p>^d Tumor samples with Exon 1 to 8 joining at the pre mRNA level are denoted as “+”.</p><p>HNSCC long range PCR of EGFR intron 1.</p

EGFRvIII correlation with EGFR amplification and HPV.

<p>A. Twenty five HNSCC tumors with known EGFR gene amplification status (via FISH) were tested for EGFRvIII positivity (28 tumors are shown, three tumors marked with N did not have gene amplification data). RNA was isolated and EGFRvIII and GAPDH were RT-PCR amplified as described in Materials and Methods. 4 of 12 EGFR amplified samples contained EGFRvIII, 5 of 12 of samples without EGFR amplification expressed EGFRvIII. All EGFRvIII bands were excised and sequenced to verify exon 1 to exon 8 joining (samples with confirmed EGFRvIII are denoted by “+”). B. A fisher’s exact test showed a lack of association between EGFRvIII expression and EGFR amplification (p = 0.56). C. A fisher’s exact test showed a lack of association between EGFRvIII expression and p16 expression (p = 0.27).</p

PCR amplification of EGFR for genomic alterations leading to EGFRvIII transcript.

<p>A. Schematic of sequencing primers and areas of interest. Arrows indicate the location of the primers used to detect EGFRvIII in genomic DNA and unspliced RNA. Bars with diamond caps indicate areas amplified for splice donor and acceptor mutations. The shaded area is lost in EGFRvIII. B. Representative PCR amplification of the splice donor/acceptor sites of <i>EGFR</i> exons 1, 2, 7 and 8 in an EGFRvIII positive HNSCC DNA sample. These bands were excised and sequenced for mutations. C. Representative long-range PCR amplification of <i>EGFR</i> intron 1 for a single EGFRvIII positive HNSCC DNA sample. L: base pair marker, W: water control, a-j: primer sets.</p

Presence of Exon 1 to 8 joining in DNA and total RNA.

<p>^a(+/-) indicate EGFRvIII status of tumor sample by RT-PCR</p><p>^bND: EGFRvIII not detected</p><p>^cvIII: EGFRvIII detected.</p><p>Presence of Exon 1 to 8 joining in DNA and total RNA.</p