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Supplemental Figures and Tables legends

Supplemental Tables 6-11. Supp. Table 6. List of all mutations identified in each of the normal samples. Supp. Table 7. List of the mutations in the unmatched OSCC and leukoplakia samples that were filtered-out as they were present in the normal samples or in the dbSNP. Supp. Table 8. Summary of the number of mutations present in each of the sets of samples and the number that were filtered-out during analysis. Supp. Table 9. Combined list of all mutations identified in Chinese OSCC patients in this study and by Song et. al (16). Supp. Table 10. List of all NOTCH1 mutations identified in Caucasians HNSCC samples. Supp. Table 11. List of all HNSCC samples from Caucasian patients used in this study: including exact site and HPV status.

Supplemental Table 5. List of 108 primer pairs designed by Fluidigm Assay Design Group and used in this study for NOTCH1 amplification. Table summarizes the GC-content, length distribution and location of the Access Array amplicons.

Disruption of NOTCH1 signaling was recently discovered in head and neck cancer. This study aims to evaluate NOTCH1 alterations in the progression of oral squamous cell carcinoma (OSCC) and compare the occurrence of these mutations in Chinese and Caucasian populations. We used a high-throughput PCR-based enrichment technology and next-generation sequencing (NGS) to sequence NOTCH1 in 144 samples collected in China. Forty-nine samples were normal oral mucosa from patients undergoing oral surgery, 45 were oral leukoplakia biopsies, and 50 were chemoradiation-naïve OSCC samples with 22 paired-normal tissues from the adjacent unaffected areas. NOTCH1 mutations were found in 54% of primary OSCC and 60% of premalignant lesions. Importantly, almost 60% of patients with leukoplakia with mutated NOTCH1 carried mutations that were also identified in OSCC, indicating an important role of these clonal events in the progression of early neoplasms. We then compared all known NOTCH1 mutations identified in Chinese patients with OSCC with those reported in Caucasians to date. Although we found obvious overlaps in critical regulatory NOTCH1 domains alterations and identified specific mutations shared by both groups, possible gain-of-function mutations were predominantly seen in Chinese population. Our findings demonstrate that premalignant lesions display NOTCH1 mutations at an early stage and are thus bona fide drivers of OSCC progression. Moreover, our results reveal that NOTCH1 promotes distinct tumorigenic mechanisms in patients from different ethnical populations. Cancer Prev Res; 8(4); 277–86. ©2014 AACR.See related perspectives, p. 259 and p. 262

Supplemental Figure 1. List of all NOTCH1 mutations found in Chinese OSCC patients. A. Mutations identified in 22 tumor-normal pairs. B. Mutations found in 28 OSCC samples without matching normal control.

Supplemental Figure 3. A. Association between tumor stage of OSCC patients and NOTCH1 mutation-status. B. Table demonstrates differences in smoking and alcohol consumption rates between the OSCC and leukoplakia patients. C. Table demonstrates differences in smoking and alcohol consumption rates between the OSCC and leukoplakia lesions with mutated NOTCH1. D. The pattern of NOTCH1 base-pair mutations resulting from exposure to tobacco in OSCC and leukoplakia patients (in blue, base-pair mutations that found in both, smokers and non-smokers).

Supplemental Figure 2. List of all NOTCH1 mutations identified in Chinese patients with oral leukoplakia.

Supplemental Tables 1-4. Supp. Table 1. A. List of all OSCC samples used in this study and clinical/pathological data. B. List of 22 matched tumor-normal pairs. Supp. Table 2. List of all normal samples used in this study and clinical/pathological data. Supp. Table 3. List of all oral leukoplakia samples used in this study and clinical/pathological data. Supp. Table 4. Summary of clinical characteristics for OSCC, leukoplakia and normal samples used in this study.