Notch1 mutations are drivers of oral tumorigenesis

Abstract

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 pre-malignant lesions. Importantly, almost 60% of leukoplakia patients 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 OSCC patients 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 pre-malignant 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.

Introduction

Head and neck squamous cell carcinoma (HNSCC), accounts for 650,000 new cases worldwide (1-3) with more than 12% distributed in China (4). OSCC, the most common subtype of HNSCC, is notorious for poor prognosis (5), which reflects the propensity of OSCC to present as clinically advanced disease upon diagnosis (6, 7). Unfortunately, most patients in China are already in advanced stages when diagnosed and over 76,000 die each year (4).

Nearly 20% of OSCC patients harbor multiple pre-malignant lesions, often identified as leukoplakia (8). It has been postulated that leukoplakia represents an early stage in OSCC, as some lesions evolve to malignant neoplasms (8, 9). While efforts have been made to predict malignant transformation in oral leukoplakia (10-12), no reliable markers of clinical utility have been identified. OSCC is thought to progress through a series of genetic alterations. Indeed, several signaling pathways are dysregulated in OSCC through genetic and epigenetic alterations, such as those involving TP53, NOTCH1, CDKN2A, CCND1, HRAS, PIK3CA and FAT1 (13-15). NOTCH1 is particularly noteworthy. In Caucasians, potentially inactivating mutations occurred in 11%-15% of tumors, and as such, NOTCH1 is the second most frequently mutated gene in HNSCC after TP53 (13-15). Recently Song et al. showed that in Chinese OSCC patients, the NOTCH1 mutation frequency was greater than 40% and strongly associated with poor prognosis and shorter survival (16). While these data indicate that disruption of NOTCH1 signaling is involved in oral tumorigenesis of both Asian/Chinese and Caucasian populations, this study was performed with conventional PCR to manually amplify NOTCH1 through hundreds of individual reactions and the role of NOTCH1 in malignant transformation of oral leukoplakia was not addressed.

Using new enrichment technology and NGS we assessed NOTCH1 mutation-status at different stages of OSCC progression in Chinese patients. The NOTCH1 mutation frequency was 54% for OSCC and 60% for pre-neoplastic lesions. Importantly, most leukoplakia patients with mutated NOTCH1 carried mutations that were also identified in OSCC, indicating an important role of these events in the progression of early neoplasms. Moreover, we compared all known NOTCH1 mutations in Chinese OSCC patients with those reported in Caucasians to date. Although we found obvious overlaps in critical regulatory domains alterations and identified mutations shared by both cohorts, possible gain-of-function mutations were predominantly seen in the Asian population.

Materials and Methods

Samples

144 tissue samples were collected at the Ninth People's Hospital, Shanghai, China. 49 samples were normal oral mucosa from patients undergoing oral surgery and 45 represented oral leukoplakia biopsies. Fifty OSCC samples were obtained during resection. Among them, 22 paired normal tissues were gained from the adjacent areas at least 1cm away from cancer. Samples were promptly frozen at −80°C after initial pathological examination. Frozen tissue were cut into 5μm sections, stained with H&E, and examined by light microscopy. Lesions with a low neoplastic cellularity (<70%) were additionally microdissected to remove contaminating normal cells before DNA extraction. OSCC patients had not been treated with chemotherapy or radiation before their tumor biopsy, so the spectrum of changes we observed largely reflects those of tumors in their naturally occurring state. The histopathological diagnosis was made by pathologist on duty according to World Health Organization criteria (17),(18). Clinical characteristics are shown in Supp. Table 1, 2, 3 and 4. This study was approved by the Human Research Ethics Committee of Shanghai Jiaotong University and the Administration Office of the Chinese Human Genetic Resources. Informed written consent was obtained from all patients before sampling.

DNA isolation

Genomic DNA was isolated from fresh frozen samples by QIAamp DNA kit (Qiagen) and quantified with Nanodrop system (Thermo Scientific).

Notch1 amplification

108 primers pairs were designed by Fluidigm (San Francisco, USA) to cover all 34 exons of the NOTCH1 and exon–intron boundaries (Supp. Table 5). PCR amplification was performed using a Fluidigm Access-Array microfluidic chip according to the manufacturer's instructions. Each sample was combined with primer pairs in a microfluidic chip and thermal cycling on a Fluidigm FC1 Cycler was performed. PCR products were then collected using the IFC controller and transferred to a 96-well plate. In a separate PCR, Illumina sequence-specific adaptors and barcodes were attached.

Sequencing

Pooled and indexed PCR products were sequenced on the Illumina MiSeq instrument following standard protocols with the following modifications: Illumina-specific sequencing primers were substituted with a mixture of two Fluidigm-specific primers pairs (FL1 and FL2). The average coverage of each base in the targeted regions was 668-fold, and 97% of targeted bases were represented by at least 10 reads.

Data analysis

Bioinformatic analyses were performed at PGDx (Baltimore, MD). The sequences were aligned to the human genome reference sequence (hg18) using the ELAND algorithm of CASAVA 1.7 software (Illumina). The chastity filter of the BaseCall software of Illumina was used to select sequence reads for subsequent analysis. The ELANDv2 algorithm of CASAVA 1.7 software (Illumina) was then applied to identify point mutations and small insertions and deletions. Known polymorphisms recorded in dbSNP were removed from the analysis. Potential somatic mutations were filtered and visually inspected as described previously (19).

Results

NOTCH1 mutations in OSCC patients from Chinese origin

We first examined the NOTCH1 coding-regions and intron–exon boundaries in 22 OSCC tumor-normal pairs (Supp. Table 1B). Using stringent criteria, we identified 25 nonsynonymous aberrations (Figure 1A, 1B and Supp. Figure 1A) in 11 (50%) tumors (Figure 1C). Among these alterations, 24 were missense substitutions and 1 was a frameshift deletion. Twenty three missense mutations were in the coding-region and 1 was at a splice-acceptor site (Figure 1B). The average mutation rate was 2.27 mutations per patient (Figure 1B). No nonsense mutations or copy number variations were discovered in this cohort. EGF-like repeats was the most mutated region (Figure 1D), followed by Ankyrin-repeats (ANK) and RBP-Jkappa-associated module (RAM), while fewer mutations were seen in the heterodimerization-domain (HD) and Lin12-NOTCH-repeats (LNR). We next sequenced NOTCH1 in 28 OSCC tumors without matched normal controls and 49 oral mucosa samples obtained from healthy individuals (Supp. Table 2). The sequences were aligned to the reference and all identified point mutations where checked against dbSNP database for known polymorphisms. Common polymorphisms recorded in dbSNP and all mutations identified in both OSCC and normal samples were removed from the analysis (Supp. Tables 6-8). The remaining mutations (Figure 1E and Supp. Figure 1B) are most likely either somatic alterations or rare polymorphisms. Of 22 nonsynonymous alterations, 21 were single nucleotide substitutions and 1 was a frameshift mutation. Eighteen point mutations, including 1 nonsense, were at the coding-region and 3 at splice-sites (Figure 1F). NOTCH1 mutations were found in 16 (57%) of these patients (Figure 1G), which was only slightly higher than the mutated NOTCH1 prevalence identified in 22 matched tumor-normal samples, confirming the success of our conservative filtering approach (Figure 1C). The small difference may be explained by chance alone or by the presence of a few uncommon SNPs alongside somatic alterations. Since mutation distribution in this group (Figure 1E and H) closely resembled that found in the 22 tumor-normal pairs, we combined all nonsynonymous mutations identified in both groups into one ‘OSCC-tumors’ cohort (Figure 1I). Altogether, 47 nonsynonymous alterations in 27 (54%) patients were identified in the ‘OSCC-tumors’ set (Figure 1J and K). The mutation distribution across NOTCH1 functional domains is shown in Figure 1L. Interestingly, most mutations in the frequently mutated exons (Figure 1M) were located around the HD and TAD/PEST domains, where most of the activating mutations were reported in hematologic malignancies (Figure 1I). Of 43 mutations in NOTCH1 coding-region, 11 mutations in the same amino acid were previously reported in various malignancies, including HNSCC (Figure 1N), supporting their status as bona-fide somatic mutations and suggesting a potential functional role in tumor progression.

Figure 1. NOTCH1 mutations in Chinese OSCC patients.

A. Schematic depiction of mutations in NOTCH1 identified in 22 tumor-normal pairs. Indel mutation is not included. EGF: epidermal growth factor; HD: heterodimerization domain; LNR: Lin12-Notch repeats; TM: transmembrane domain; RAM: recombination signal-binding protein - Jkappa-associated module; ANK: ankyrin repeats domain; TAD: transactivation domain; PEST: region rich in proline (P), glutamine (E), serine (S) and threonine (T) residues. B. Summary of nonsynonymous mutations found in this group. C. Shows a ratio of tumors with mutated NOTCH1. D. Distribution of nonsynonymous mutations among NOTCH1 functional domains. E. Schematic depiction of NOTCH1 mutations identified in 28 tumors without matched normal control. F. Summary of nonsynonymous mutations found in this group. G. Shows a ratio of tumors with mutated NOTCH1 identified in unmatched OSCCs. H. Distribution of NOTCH1 mutations (showed in E) across functional domains. I. Schematic depiction of all NOTCH1 nonsynonymous mutations found in 50 Chinese OCSS patients (A and E combined). Black bars show regions where most gain-of-function mutations were reported in hematologic malignancies. J. Summary of nonsynonymous mutations found in 50 OSCC tumors. K. Percentage of tumors with mutated NOTCH1 among 50 Chinese patients. L. Overall mutations distribution across NOTCH1 functional domains. M. Table of most frequently mutated exons. N. List of NOTCH1 mutations found in this study that have been previously observed in solid tumors and hematopoietic malignancies.

Please note that different technical approaches used to amplify and sequence NOTCH1 may produce some bias. We used the Fluidigm high-throughput target-enrichment system which allows more consistent amplicon generation during library preparation and even distribution of sequencing reads across all samples, including capture of repetitive or high GC rich regions that are often difficult to enrich. In the NOTCH1 sequence, 17 of 34 exons contain greater than 65% GC content. There was a 9 fold difference in the amount of identified synonymous alternations between Song et al (16) and our studies. Moreover, while Song et al (16) reported several mutation ‘hotspots’, besides mutation at codon 1531L>Q/P found in two (4%) of the 50 tumors, no mutational ‘hotspots’ were found in our study. These differences can be at least partially explained by much more stringent criteria for potential somatic mutation calls used in our work (19).

NOTCH1 mutations in patients with oral leukoplakia

To address the role of NOTCH1 in the transition from pre-malignant lesions to malignant neoplasms we sequenced NOTCH1 in 45 Chinese patients with oral leukoplakia. This population represents a unique model to study this question, since the proportion of primary tumors harboring NOTCH1 mutations is much higher than in Caucasians. Patients were subgrouped as leukoplakia with low malignant potential (mild-dysplasia) or high malignant potential (moderate or severe dysplasia) (Supp. Table 3). Since corresponding normal tissues were not available for these samples, we used the same approach that was utilized for analysis of unmatched OSCC tumors. Thirty nine nonsynonymous substitutions (Figure 2A, 2B and Supp. Figure 2) were identified in 27 (60%) of 45 patients (Figure 2C). Of 36 alterations in the coding-region, 33 were missense and 3 nonsense substitutions, with an average mutation rate of 1.4 mutations per patient (Figure 2B). Overall mutation prevalence and distribution across the gene in oral leukoplakia (Figure 2D) were consistent with those found in OSCC patients. Seven mutations affecting the same codon were previously reported in various malignancies and 32 were novel (Figure 2E). Whereas NOTCH1 mutation-status was not associated with higher tumor stage (Supp. Figure 3A), in leukoplakia patients we noticed a slight progressive pattern in NOTCH1 mutations prevalence from mild to moderate and severe dysplastic lesions (44% and 69% respectively) (Figure 2F). Although this difference was not significant (p=0.08), the high frequency of NOTCH1 mutations in patients with mild dysplasia suggests that NOTCH1 mutagenesis is an early event in OSCC tumor progression.

Figure 2. NOTCH1 mutations in Chinese patients with oral leukoplakia.

A. Schematic depiction of mutations in NOTCH1 identified in 45 leukoplakia samples. B. Summary of nonsynonymous mutations found in this cohort. C. Percentage of tumors with mutated NOTCH1. D. Summary of nonsynonymous mutations distribution across NOTCH1 functional domains. E. List of NOTCH1 mutations found in leukoplakia patients that have been previously observed in solid tumors and hematopoietic malignancies. F. Table demonstrates higher prevalence of NOTCH1 mutations in patients that were diagnosed with moderate or severe dysplasia. G. Schematic depiction of nonsynonymous mutations found in 50 Chinese OSCC tumors (top) and leukoplakia patients (bottom). Blue triangles show overlapping mutations that were found in both cohorts. Dashed bracket indicates mutations in ‘ligand binding’ domain and its boundaries. Black bars show regions where most gain-of-function mutations were reported in hematologic malignancies. H. List of mutations overlapping between OSCC and leukoplakia. I. Table shows higher prevalence of overlapping mutations in patients diagnosed with moderate or severe leukoplakia. J. Table shows mutations distribution per exon in OSCC and leukoplakia. Cells highlighted with cyan indicate most frequently mutated exons in OSCC, leukoplakia or combined.

There were several mutation ‘hotspots’, including 4 cases with mutation at codon 571D>A (8.9%), three patients with mutations at codon 1115L>P and mutations at codons 1531L>P and 517 P>L/S each in two tumors (Figure 2A). Due to a lack of germline DNA, it is possible that some of these ‘hotspots’ may be rare germline polymorphisms. However, a comparison between mutations found in leukoplakia and OSCC samples revealed that all 4 ‘hotspots’ in leukoplakia were overlapping with mutations found in OSCC (Figure 2G). Thus, it is reasonable to assume that these ‘hotspots’ are most likely somatic aberrations. Surprisingly, of 27 leukoplakia patients with mutated NOTCH1, 16 (59.3%) carried the same 9 mutations that were also seen in 10 (20%) of 50 OSCC patients (Figure 2G and 2H). This observation indicates that in some cases NOTCH1 mutagenesis may underlie a progression of oral pre-malignant lesions into the primary carcinoma. Furthermore, of 16 leukoplakia patients with overlapping mutations, 12 were diagnosed with high-grade dysplasia, whereas only 4 patients had mild dysplasia upon diagnosis (Figure 2I), suggesting an evolutionary advantage during tumor progression.

Association of NOTCH1 mutation-status with smoking and/or alcohol consumption

Smoking and excessive alcohol consumption are the major risk factors for OSCC (20). Notably, high alcohol-content liquor is traditionally consumed in China compared with predominantly wine or beer in Caucasians (16). Although exposure to tobacco and alcohol was associated with high frequency of TP53 mutations in HNSCC (20, 21), the association with NOTCH1 mutagenesis has not been reported. Since history of tobacco and alcohol consumption was not available for all patients in normal cohort, we compared the amount of patients who either smoked, consumed alcohol or both between the 50 OSCC patients and 45 patients with oral leukoplakia. While the percentage of smokers or alcohol consumers was not significantly different between the two cohorts, percentage of patients with smoking and alcohol consumption history was higher among the OSCC patients compared to leukoplakia patients (28% and 13.3% respectively, p=0.07) (Supp. Figure 3B). We next compared the amount of patients who either smoked, consumed alcohol or both between the 27 OSCC patients and 27 leukoplakia patients harboring nonsynonymous mutations in NOTCH1. No significant difference in the percentage of patients with either smoking or alcohol consumption history was observed between the two groups, whereas a 3-fold increase in patients with tobacco and alcohol history was seen in OSCC compared to leukoplakia patients with similar background (11.1% and 33% respectively, p=0.05) (Supp. Figure 3C). The most common base-pair substitutions in non-smoking OSCC and leukoplakia patients were C-T (30.8% and 30.6%) and G-A (27% and 19.4% respectively) (Supp. Figure 3D). In OSCC and leukoplakia patients with a smoking background, the A to G transition mutations were much more prominent in the pre-neoplastic lesions. Unfortunately, we were unable to determine the association between NOTCH1 mutation-status and survival as the follow-up time from tumor diagnosis until death was available for only 12 of 50 OSCC patients.

Spectrum of NOTCH1 mutations in OSCC patients of Asian and Caucasian origin

Collective analysis of all patients (leukoplakia and OSCC) revealed the most common mutated NOTCH1 exons in oral lesions (Figure 2J). Although the NOTCH1 mutation spectrum in oral lesions was fundamentally different from that seen in leukemia, 21 (27.3%) of 77 mutations found in this study were located around the HD domain (exons 26-28) and 8 (10.4%) were scattered throughout the TAD/PEST region (exon 34), where most gain-of-function mutations were reported in hematopoietic malignancies (22, 23). Consistent with previous reports (13, 15), 37 (48%) of all mutations found in oral lesions were in the EGF-like repeats. Interestingly, 15 (40.5%) of these mutations, including 2 ‘hotspots’, were within or around the ‘ligand-binding’ domain (Figure 2G), suggesting a loss-of-function potential for these mutations. These data suggest a dual biological role of the NOTCH1 as either a cancer promoting or tumor suppressor gene in OSCC.

To better understand the NOTCH1 mutation spectrum in Asian OSCC patients we combined mutations found in this study with nonsynonymous substitutions identified by Song et al. (16) (Figure 3A top and Supp. Table 9). Moreover, to evaluate the differences in NOTCH1 mutations spectrum between Chinese and Caucasian oral tumors we gathered NOTCH1 nonsynonymous variants in western population from all NGS papers known to date (13-15, 24) and the recently reported TCGA data (Figure 3A bottom and Supp. Table 10). Our combined data-set contained 101 Chinese and 595 Caucasian patients. The HPV status in 50 Chinese patients was not defined, but HPV infection does not appear to be a significant risk factor for OSCC, and the prevalence of HPV-positive oral cavity cancers is relatively low (5.9% or lower) (25, 26). Among Caucasian tumors, 93% were HPV-negative and more than 70% were OSCC (Supp. Table 11). Although the mutation prevalence was slightly higher in Chinese OSCC patients (Figure 3B), the overall NOTCH1 mutation frequency was 3-fold greater than in Caucasians, 48.5% vs. 15.8% respectively (p=0.0002) (Figure 3C). Interestingly, we found a disproportionate prevalence of nonsense (23.4% and 9.2%, p=0.009) and frameshift (18.7 and 4.6%, p=0.003) mutations in Caucasian versus Chinese patients respectively (Figure 3B).

Figure 3. Spectrum of NOTCH1 mutations in Chinese and Caucasian OSCC patients.

A. Schematic depiction of all NOTCH1 mutations identified in Chinese OSCC tumors to date (Top-triangles) and all mutations identified in Caucasian patients with HNSCC (Bottom-diamonds). B. Summary of nonsynonymous mutations within the codding region found in both ethnicities. C. Number and percentage of tumors with mutated NOTCH1 in Chinese and Caucasian patients. D. Table displays differences in mutations distribution across functional domains of NOTCH1 in Chinese and Caucasians samples. E. Mutations distribution per exon in Chinese and Caucasian patients. Cells highlighted in cyan indicate most frequently mutated exons in Chinese (lighter) and Caucasian (darker) tumors. F. Mutations clusterization in distinct functional modules of NOTCH1 in Chinese (red bars, top) and Caucasian (purple bars, bottom) tumors. G. List of mutations that were found in Caucasian tumors by two or more independent studies (left). Mutations in the same codon that were seen in Chinese and Caucasians patients (right).

While EGF-like was the most mutated domain in Chinese (49.4%) and western (77%) populations, the mutation distribution among NOTCH1 functional domains significantly varied (Figure 3D and 3E). Based on the distribution spectrum we identified three distinct functional clusters in Chinese and Caucasian patients (Figure 3F). In Chinese tumors, cluster-I partially spreads across the ‘ligand-binding’ domain (exons 8-11) and contains 9 (11%) of 83 mutations. Cluster-II overlaps with the Abruptex region (exons 19-23) and consists of 19 (23%) mutations, while cluster-III covers the negative regulatory region (NRR) and the area at the boundary between the extracellular and transmembrane domains and consists of 36 (41.4%) mutations (Figure 3F top). By comparison in Caucasian patients, 35 (40%) of 87 nonsynonymous substitutions are gathered into cluster-I (exons 6-9), (Figure 3F bottom). Cluster-II (6 mutations) overlaps with the C-terminal of the Abruptex region and 15 (17.3%) mutations (including 8 nonsense) are found in the RAM/ANK area of the NOTCH1 intracellular domain (NICD).

Among 87 single-nucleotide variants found in Caucasian patients with mutated NOTCH1, there were several recurrent mutations identified by at least two independent research groups in different OSCC cohorts (Figure 3G). Four mutations identified in Caucasian tumors, G481S/V, Q1214* and Q1080* were also seen in Chinese OSCC patients. While another set of two mutations, E424K and D142H, were found in leukoplakia patients (Figure 3G), suggesting their potential tumorigenic role in both groups.

Discussion

In this study, we examined NOTCH1 mutation-status in OSCC and oral leukoplakia from Chinese patients and observed a mutation prevalence of 54% and 60% respectively. In Asians, more than a third of mutations are located within the EGF-like repeats, particularly around the ‘ligand-binding’ and Abruptex regions. Mutations in these areas may significantly impact NOTCH1 activity. The extracellular domain of NOTCH1 contains 36 EGF-repeats. Repeats 11-13 constrain the ‘ligand-binding’ domain and are necessary for direct interactions between NOTCH1 with the Delta/Serrate/Lag-2 family (27, 28). Since NOTCH1 signaling relies on these interactions, mutations in this region may inhibit ligand–receptor interactions and subsequently signal transduction. Although little is known about the contribution of EGF-repeats to NOTCH1 function, it was shown that the integrity of the Abruptex (EGF-repeats 24-29) is required for suppression of NOTCH1 activity (29) and that mutations within this region enhance NOTCH1 signaling (30). While EGF-repeats are involved in ligand-mediated receptor activation, mutations in NRR, another frequently mutated area in Asian tumors, can lead to ligand-independent activity (31). The membrane-proximal NRR acts as a receptor activation switch and it is one of most mutated regions found in T-cell acute lymphoblastic leukemia (32). Taken together, the NOTCH1 mutation spectrum in Asian tumors suggests that significant proportion of alterations may result in a gain-of-function. A low prevalence of nonsense and frameshift mutations among Asian tumors further supports this notion. In Caucasian patients, the vast-majority of mutations clustered around the ‘ligand-binding’ domain, indicating that averting NOTCH1-ligand interaction may be the most prevalent cause for NOTCH1-related oral tumorigenesis in the Western populations. The next most mutated area was the RAM/ANK region of the NICD. Together with the DNA-binding factor CSL and co-activator proteins of MAML family, NICD is a core component of NOTCH1 nuclear complexes (33). RAM region constitutes a high-affinity binding module for CSL, and the ANK domain together with the CSL create a composite binding site required for MAML recruitment into these complexes (33, 34). Therefore, mutations in the RAM/ANK module may disrupt proper nuclear complexes assembly and subsequently prevent transcription of NOTCH1-dependent genes. Overall, almost 60% of mutations identified in Caucasians were located in areas associated with loss-of-function phenotype, concurrent with a recent report (13). Consequently, while there is an evidence of some clustering overlap between Caucasian and Asian tumors, the overall spectrum of NOTCH1 mutations is profoundly different between these cohorts.

Leukoplakia is considered to be an intermediate-step in OSCC histopathological progression (35, 36), a multi-step process which involves accumulation of alterations from normal epithelium to invasive cancer. However, since some low-grade dysplasias progress to HNSCC while some high-grade lesions regress to a normal state, dysplasia grade remains a poor predictor of malignant progression. Twenty years ago, we reported that the incidence of TP53 mutations in noninvasive lesions was 19% and increased to 43% in invasive carcinomas (37). With the advent of deep sequencing and the identification of NOTCH1 common mutations in Chinese tumors, we took the opportunity to detect the prevalence of NOTCH1 mutations in oral dysplasias. Although a higher incidence of mutated NOTCH1 was seen in lesions with high malignant potential, leukoplakia patients diagnosed with mild-dysplasia also demonstrated a substantial frequency of NOTCH1 alterations, confirming a crucial role of NOTCH1 in early pre-malignant transition. Moreover, 59.3% of leukoplakia patients with mutated NOTCH1 carried mutations overlapping with OSCC, further supporting an important role of these events in the progression of early neoplasms. While these data suggest that NOTCH1 mutagenesis may be sufficient for the malignant transformation in some Chinese patients, the risk of invasive transformation associated with NOTCH1 mutations in Asian population is yet to be determined. Similar to TP53, NOTCH1 mutations may exist in benign oral lesions for many years without progression to malignancy (38), and it is difficult to obtain longitudinal collection of leukoplakias and subsequent OSCCs that developed in the same patients. Nevertheless, larger confirmatory studies using longitudinal samples are warranted to further validate the role of NOTCH1 mutations in OSCC progression. Loss of heterozygosity (LOH) on 3p and/or 9p was reported to predict progression for oral leukoplakias, however only 20% of leukoplakia cases with LOH progressed to malignant disease (12). Consequently, concurrent evaluation of NOTCH1 mutagenesis alongside LOH status may further stratify the risk and improve diagnostic sensitivity.

Major OSCC-associated risk factors are tobacco and alcohol consumption and their effect is considered to be multiplicative rather than additive (39). Higher prevalence of individuals with smoking and alcohol consumption history among the OSCC patients parallels epidemiological evidence that alcohol acts as a co-carcinogen, enhancing the carcinogenicity of tobacco smoke (20) and confirms that the joint effect between tobacco and alcohol use increase the head and neck cancer risk (40). Increase in TP53 and RB mutations in OSCC have been associated with these carcinogens (21, 41, 42) and impact survival (43). Our notion that the percentage of OSCC patients with mutated NOTCH1 who smoked and drank was significantly higher compared to leukoplakia patients with similar background suggest that NOTCH1 mutations in OSCC tumors can be attributed in part, to DNA damage from cigarette smoke and alcohol consumption. However, NOTCH1 mutagenesis cannot be entirely attributed to tobacco and alcohol use, since a substantial proportion of OSCC and leukoplakia patients harboring mutated NOTCH1 neither smoked nor drank. Suggesting that exposure to other carcinogens and environmental factors also plays role in NOTCH1 mutagenesis. While non-smoking OSCC and leukoplakia patients display a similar pattern, the spectrum of base-pair substitutions between OSCC and leukoplakia patients with smoking history is clearly different. Consistent with complex carcinogen exposure (42), in OSCC and leukoplakia lesions with smoking background the most frequent mutations were scattered with a very interesting peak in A to G transitions, especially in the pre-neoplastic lesions, and a slightly higher G to A transition frequency in non-smokers as noted in lung cancer (44). In accordance with Song et al. (16), we also did not observe an association between tumor stage and NOTCH1 mutation-status, further supporting the role of NOTCH1 in OSCC malignant transformation as an early driver event.

Our integrated meta-analysis revealed a much higher NOTCH1 mutations prevalence among Asian patients. While the etiology remains to be determined, it may include an inherited genetic background, exposure to different carcinogens and environmental factors (45). The impact of race/ethnicity on molecular alterations in cancer is a subject of important attention, as more studies reveal that groups sharing a common biological background also acquire distinct carcinogenic molecular pathways. In lung adenocarcinoma, activating mutations in EGFR, important factors to determine small molecule inhibitor response, commonly occur in Asians, around 50%, versus only 20% in Caucasians (46-48). Moreover, a higher NOTCH1 mutation frequency was reported in esophageal adenocarcinoma from western patients when compared to Chinese (49), suggesting distinct tumorigenic mechanisms in each population. Significantly higher prevalence of nonsense and indel mutations in Caucasians compared to Asian patients supports the hypothesis that in Caucasians oral tumorigenesis more prominently relies on inactivation of NOTCH1 signaling than in the Chinese.

Interestingly, 10 recurrent mutations affecting the same codon were identified in Caucasians by at least two research groups. Although each group (13-15, 24) used a unique sample identification number, there is a chance that some samples were shared between these groups and that recurrent mutations are result of the re-sequencing of the same clinical sample. While we can't eliminate this possibility, given the low amount of overlapping variants (6.5%), the probability of parallel re-sequencing is very small. Three nonsense mutations result in premature translation termination while all missense variants are located within or around the ‘ligand-binding’ domain suggesting an inactivating role for these alterations. Since these mutations were not identified in Chinese patients or any other tumor type, it is tempting to speculate that these variants play a crucial role in OSCC tumorigenesis and may represent “core signature” alterations among Caucasian patients. Moreover, three missense and two nonsense mutations identified in Caucasians were also seen in Chinese OSCC or leukoplakia patients. Yet again, two of the three missense variants identified in both ethnic groups, G481S and E424K, fall within the ‘ligand-binding’ domain. These findings further support our observation that disruption of ‘ligand-binding’ domain architecture plays a prominent role in the oral tumorigenesis of both Chinese and Caucasian populations.

Although we used stringent analytical criteria to identify alterations present in the tumors and not intrinsic in the population, we acknowledge that some mutation candidates need to be further validated in paired normal-tumor samples. However, taken together, our findings provide a new insight into the role of NOTCH1 in OSCC evolution, showing for the first time that pre-malignant lesions display mutations that may drive malignant transformation and are likely to precede p53 inactivation in OSCC progression (37). Large sequencing studies struggle with the identification of newly mutated genes as true biologic drivers vs just passenger mutations, and our work reminds us that the study of pre-neoplastic lesions has been overlooked as another way to define key drivers of cancer progression in actual patients. Our work also emphatically emphasizes the need to extensively test tumors for genetic alterations from distinct ethnic and geographic areas.