Genetic Alterations of Esophageal Squamous Cell Carcinoma in Korean Patients

<jats:title>Abstract</jats:title>         <jats:p><jats:bold>Background</jats:bold>:<jats:bold> </jats:bold>Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers in Korea, although its incidence is lower in Korea than in China and Japan. There are no data on genetic alterations associated with ESCC in Korea. Our study is the first report of the mutational landscape of ESCC in a Korean cohort.<jats:bold>Methods</jats:bold>: We performed whole exome sequencing analysis of 43 ESCC tumor samples and germline DNA from normal esophageal mucosa or whole blood. Using electronic medical records, the patients’ clinical characteristics were reviewed carefully.<jats:bold>Results</jats:bold>: We found a mutually exclusive mutation pattern in <jats:italic>NFE2L2</jats:italic>–<jats:italic>ZNF750</jats:italic> gene pairs. Non-silent variants of <jats:italic>KMT2D</jats:italic> (<jats:italic>MLL2</jats:italic>) were associated with a poor prognosis. An analysis to identify potentially druggable genes revealed <jats:italic>NOTCH1</jats:italic> as a potential therapeutic target. We observed a high frequency of C:G → T:A transitions regardless of smoking history. In our cohort, deconstruction of the mutation signature revealed enrichment of COSMIC single base substitution (SBS) 13, SBS 39, SBS 2, SBS 40, and SBS 1.<jats:bold>Conclusion</jats:bold>: We did not find a Korean-specific signature of genetic alterations, but our data suggest that <jats:italic>KMT2D</jats:italic> is a molecular prognostic marker. Further studies need to examine the role of <jats:italic>KMT2D </jats:italic>in ESCC prognosis due to our small sample size.</jats:p>

section_content: Worldwide, esophageal cancer causes 544,076 deaths annually and is the sixth leading cause of cancer death. 1 East Asia has the highest regional incidence rates for both men and women, in part because of the large burden in China. In 2017, there were 232,255 new cases of cancer in Korea, of which 2483 (1. 1% of all cancers) were esophageal cancer. 2 The crude incidence rate of esophageal cancer was 4. 8 cases per 100,000 population in 2017. The age-standardized incidence rate decreased from 4. 0 per 100,000 population in 1999 to 2. 6 in 2017, with annual percent changes of -2. 8% in men and -1. 3% in women. 2 The incidence of esophageal cancer in Korea is relatively low compared with that in Japan and China, but the reasons for this are not clear. 3 In 2013, 90. 2% of esophageal carcinoma cases was squamous cell carcinoma (ESCC) histologically, followed by adenocarcinoma (3%). 4 The proportions of localized and regional cancer tended to increase compared with that of distant cancer, and the 5-year relative survival rate of esophageal cancer improved from 14. 0% in 1993-1995 to 38. 0% in 2013-2017. 2 ] [7] [8] [9] [10] For instance, a study of ESCC in a Chinese ESCC cohort of 139 reported known mutated genes, such as TP53, PIK3CA, and NOTCH1, and previously uncharacterized mutated genes including FAT1, FAT2, ZNF750, and KMT2D. 7 Song et al. reported mutual exclusivity between mutations in NOTCH1 and PIK3CA, and that patients with NOTCH1 mutations had shorter survival times than patients without mutations. 10 In a whole exome sequencing (WES) analysis of 144 Japanese patients with ESCC, Sawada et al. identi ed mutations in EP300 and TET2 that correlated with a shorter survival time. 8 In a WES analysis of 28 Indians with ESCC, Kiran et al. reported higher frequencies of C:G → A:T transversion and mutation signature 4 compared to smokers and non-users of tobacco. 5 However, the genomic alterations associated with ESCC in Korea are not well-characterized. In this study, we used WES to characterize the mutational landscape of ESCC in Korea.
section_name: Introduction

section_name: Results

section_content: Tumor and paired normal DNA from 43 ESCC patients was subjected to WES. The mean read depth of the target regions was 134. 4⊆, and 99. 0% of the target bases were covered by >10 independent reads after alignment. This identi ed 6,557 variants in 4,752 genes: 2,090 silent variants (synonymous and deep intronic variants) and 4,467 non-silent variants (Fig. 1 ). The non-silent variants include 3807 missense, 288 nonsense, 222 frameshift, 84 splicing, 51 in-frame, eight nonstop, and seven start codon variants. We found a median of 74 non-silent variants (range 18-307) per sample with a median tumor mutation burden of 2. 47 (range 0. 60-10. 23) non-silent variants/MB.
section_name: Results of whole exome sequencing analysis

section_content: Our ESCC cohort included the recurrently mutated genes TP53 (non-silent variants, 88% of samples), TTN (37%), NFE2L2 (30%), ZNF750 (23%), NOTCH1 (21%), FSIP2 (19%), KMT2D (16%), and LRP1B (16%) (Fig. 1, Supplementary Fig. S1 ). The top ve affected oncogenic signaling pathways were TP53 (88% of samples), NOTCH (58%), RTK-RAS (53%), Hippo (44%), and NRF2 (37%) (Supplementary Fig. S2 ). We identi ed NFE2L2 as a cancer driver gene with a single cluster, and all seven mutations were in that cluster based on the OncodriveCLUST algorithm run in maftools (p <0. 05). 13 We performed Pfam annotation using maftools, and the top ve mutated protein domains were COG5048, 7tm_1, FN3, Cadherin_repeat, and P53 (Supplementary Table S1 ). We observed a mutually exclusive mutation pattern in NFE2L2-ZNF750 gene pairs in ESCC (Fig. 2 ). In our cohort, NFE2L2 was mutated in 30% (13/43) of the samples and ZNF750 in 23% (10/43) (Supplementary Fig. S1B, C ). (19/43), con rmed pathologically. All patients were followed until either death or the last follow-up date.
section_name: Somatic mutation spectrum of esophageal squamous cell carcinoma

section_content: During the follow-up period, 7 (16. 3%) patients died and 13 (30. 2%) suffered recurrence (Table 1 ). 

In the clinical enrichment analysis, there were no genes signi cantly associated with sex, a history of alcohol or tobacco consumption, the grade of tumor differentiation, the grade of tumor invasion, the presence of nodal metastasis, lymphovascular invasion, or perineural invasion. 

In the overall survival analysis of the top 30 genes, non-silent variants of KMT2D (MLL2) were associated with a poor prognosis (hazard ratio 6. 18, p <0. 05) (Fig. 3A ). KMT2D was mutated in 16. 3% (7/43) of the samples in our cohort (Supplementary Fig. S1D ). The mortality rates in the mutated and non-mutated KMT2D groups were 42. 9% and 11. 1%, respectively (p = 0. 072). In the disease-free survival analysis of the top 30 genes, non-silent variants of KMT2D (MLL2) were associated with poor disease-free survival (hazard ratio 2. 07) (Fig. 3B ), but this was not signi cant (p = 0. 191). 

In the disease-free survival analysis of the top 30 genes, non-silent variants of LRP1B were associated with a greater likelihood of recurrence (hazard ratio 2. 6), without statistical signi cance (p = 0. 083) (Supplementary Fig. S3 ). The recurrence rates in the mutated and non-mutated LRP1B groups were 71. 4% and 22. 2%, respectively (p <0. 05). LRP1B was mutated in 16. 3% (7/43) of the samples in our cohort, and all non-silent variants of LRP1B were missense variants (Supplementary Fig. S1E ).
section_name: Clinical and prognostic relevance of each mutation in esophageal squamous cell carcinoma

section_content: To identify potential druggable targets, we screened genes bearing non-silent variants detected in at least 20% of the samples against FDA-approved antineoplastic drugs in DGIdb. 14 This revealed NOTCH1 as a potential therapeutic target. In our cohort, NOTCH1 was mutated in 21% (9/43) of the samples (Supplementary Fig. S1F ).
section_name: Identi cation of potentially druggable genes

section_content: In this study, seven samples were from non-smokers. The median mutation burden was 2. 47 and 2. 43 non-silent variants/MB in smokers and non-smokers, respectively. We observed a high frequency of C:G → T:A transitions regardless of smoking history. Further, deconstruction of the mutation signature of our cohort revealed the enrichment of COSMIC single base substitution (SBS) 13, SBS 39, SBS 2, SBS 40, and SBS 1 (Fig. 4 ). We did not observe distinct signatures associated with ESCC in patients with a history of smoking or alcohol consumption.
section_name: Mutation signatures associated with smoking history

section_content: This study investigated the mutational landscape of ESCC in a Korean cohort. We identi ed several known and novel variants in ESCC. 7] [8] [9] 15 We identi ed several frequently mutated genes, such as TP53, TTN, NFE2L2, ZNF750, NOTCH1, FSIP2, and KMT2D, which have been reported in ESCC. Mutated genes commonly found in over 10% of the ESCC samples in previous reports and this study are described in Supplementary Table S2. Except for TTN, NFE2L2, and FSIP2, most of these genes had nonsense, frameshift, or splicing variants, suggesting their tumor suppressor roles (Fig. 1 ). 

The NRF2 pathway consisting of NFE2L2, KEAP1, and CUL3 was genetically deregulated in 37. 2% of our cohort (Supplementary Fig. S1 ). NFE2L2 encodes a transcription factor that induces the production of a cytoprotective enzyme in response to oxidative stress, whereas the KEAP1/CUL-dependent proteasomal mechanism degrades the NFE2L2 protein under non-stressed circumstances. 16 All NFE2L2 variants in our cohort involved two hot spots within the DLG and ETGE motifs that bind to KEAP1, as observed in a Japanese cohort. 8 These mutations are thought to contribute to tumor development by stabilizing the NFE2L2 protein. 17 There were NFE2L2 mutations in 9. 6-16. 7% of the cases in previous reports. 6, 8, 18 ecently, in a Chinese cohort of 508 patients, NFE2L2 mutations were reported to be signi cantly associated with a worse prognosis of ESCC. 19 But there was no signi cant association between the prognosis of ESCC and NFE2L2 mutations based on our data. 

ZNF750, an epidermal differentiation regulator, is thought to be an ESCC tumor suppressor gene, 7, 8, 15 as supported by our data, which showed that most ZNF750 variants were null variants (Fig. 1 ). ZNF750 is mutated in 3. 9-16. 7% of ESCC cases. 6, 18, 20 In Japanese ESCC datasets, ZNF750 variants were positively associated with the APOBEC signature, 8 but not in our study (p = 0. 069). 

We observed a mutually exclusive mutation pattern in NFE2L2-ZNF750 gene pairs in ESCC, although this mutual exclusivity has not been implicated in any type of cancer. 21 Mutual exclusivity has been widely observed in cancer genomes and there are two major hypotheses associated with mutually exclusive mutated genes in cancers: the functional redundancy in downstream pathways and synthetic lethality hypotheses. 22 Because these two genes do not share the same pathway, the mutually exclusive mutation in NFE2L2-ZNF750 gene pairs supports the synthetic lethality hypothesis. 

NOTCH1 is frequently disrupted by loss-of-function mutations, implying that the loss of NOTCH pathway activity is critical for the growth of tumor cells with squamous differentiation characteristics. 23 NOTCH1

is mutated in about 16% of cases. 6, 8, 18 There are several reports that NOTCH1 is associated with a poor prognosis, and its mutations are mutually exclusive with PIK3CA mutations. 5, 10, 24 However, there was no signi cant association between the prognosis of ESCC and NOTCH1 mutations based on our data. Nevertheless, the analysis to identify potentially druggable genes revealed NOTCH1 as a potential therapeutic target. 

KMT2D is tumor suppressor gene that encodes histone methyltransferase and promotes the transcriptional activation of target genes by modifying histone H3 lysine 4 trimethylation (H3K4me3). LRP1B is putative tumor suppressor gene that encodes low-density lipoprotein receptor-related protein 1b 27. LRP1B is frequently deleted in various tumors and was deleted in 20. 8% of a Japanese cohort 8 and mutated in 25% of an Indian cohort. 5 A study of LRP1B gene expression in ESCC showed that 42. 9% of primary esophageal cancer cases have homozygous LRP1B deletions, and LRP1B mRNA expression was frequently silenced in cell lines without homozygous deletions (37. 8%). 28 Bisul te-PCR analysis and sequencing showed that LRP1B-nonexpressing cells without homozygous deletions were highly methylated at a LRP1B CpG island. Recently, Brown et al. reported better immune checkpoint inhibitor treatment responses in a group with likely pathogenic/pathogenic variants in LRP1B genes compared with a group carrying variant of unknown signi cance, indicating that mutations in the LRP1B gene have signi cant implications for the prognosis and treatment of multiple cancer types. 27 All LRP1B variants in our study were missense variants. Further studies of their expression and methylation are needed to clarify the roles of these LRP1B missense mutations. 

Kiran et al. showed that tobacco chewers had a higher frequency of mutation signature 4 than did smokers and non-users of tobacco in an Indian ESCC cohort. 5 There were no tobacco chewers in our study and no distinct signatures associated with ESCC in patients with a smoking history. 

Our study is the rst report of the mutational landscape of ESCC in a Korean cohort. It provides insight into molecular alterations in ESCC in Koreans and reveals potential candidates for therapeutic targeting. 

Our data suggest that KMT2D is a molecular prognostic marker. Further studies need to examine the role of KMT2D in ESCC prognosis due to the small size of our cohort. Additionally, copy number and expression studies of ESCC in Koreans are needed, as none were conducted here.
section_name: Discussion

section_content: ESCC is one of the deadliest cancers in Korea, although its incidence in Korea is lower than in China and Japan. There are no data on genomic alterations associated with ESCC in Korea. Our study is the rst report of the mutational landscape of ESCC in a Korean cohort. We found a mutually exclusive mutation pattern in NFE2L2-ZNF750 gene pairs. Non-silent variants of KMT2D (MLL2) were associated with a poor prognosis. An analysis to identify potentially druggable genes revealed NOTCH1 as a potential therapeutic target. We observed a high frequency of C:G → T:A transitions regardless of smoking history. 

Further, deconstruction of the mutation signature of our cohort revealed the enrichment of COSMIC SBS 13, SBS 39, SBS 2, SBS 40, and SBS 1. In summary, this study characterized the genomic alterations of ESCC with highly mutational heterogeneity, which provides the understanding of the molecular pathophysiology of ESCC, and it can be useful for nding potential therapeutic target in the aspect of precision medicine.
section_name: Conclusion

section_name: Materials And Methods

section_content: We performed WES analysis of 43 ESCC tumor samples and germline DNA from normal esophageal mucosal tissue or whole blood obtained during surgical resection. The patients' clinical characteristics were reviewed using electronic medical records (Table 1 ). The study protocol was approved by the Institutional Review Board of Yonsei University College of Medicine (approval no. IRB 4-2018-1210). All subjects gave informed consent when they were enrolled and all methods were performed in accordance with the relevant guidelines and regulations of Institutional Review Board of Yonsei University College of Medicine.
section_name: Patients and samples

section_content: A WES DNA library was constructed using an Agilent SureSelect V6-Post kit (Agilent Technologies, Santa Clara, CA) with genomic DNA extracted from fresh-frozen tissue using the Qiagen DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany). Genomic DNA was sheared into 150-200-bp fragments using an ultrasonic sonicator. These sheared fragments were end-repaired, phosphorylated, adenylated at the 3' ends, and ligated using paired-end adaptors. The hybridization reaction was done at 65℃ for 16 hours using a DNA library with added biotinylated RNA library bait (Agilent Technologies). Cluster ampli cation was performed according to the manufacturer's protocol (Illumina, San Diego, CA). Paired-end sequencing was performed on an Illumina NovaSeq 6000 system (Illumina) by Macrogen Inc. (Seoul, South Korea). 

Raw reads acquired in FASTQ format were trimmed using the Trimmomatic tool in paired-end mode to increase the mapping accuracy and speci city. 11 These were mapped against the reference genome hg19 (GRCh37) using the Burrows-Wheeler aligner with default parameters. Binary alignment map (bam) les were further processed using GATK (Genome Analysis Toolkit, Broad Institute, Cambridge, MA), which included the removal of duplicates using MarkDuplicates in Picard, IndelRealigner, and BaseRecalibrator. High-con dence somatic single-nucleotide variants (SNVs) were ltered using HaplotypeCaller, Mutect2, VarScan, and Pindel and annotated using AnnoVar and VEP. The variant call format (vcf) les were converted into mutation annotation format (maf) les using vcf2maf tools [Cyriac Kandoth (2020); mskcc/vcf2maf: vcf2maf v1. 6. 19, doi:10. 5281/zenodo. 593251]. 

Oncoplot, lollipop plots of somatic SNVs, the identities of genes with mutually exclusive variants and cancer driver genes, and actionable therapeutic target and mutational signatures were generated using the R package maftools. 12 To predict clinically actionable therapeutic targets, the Drug-Gene Interaction Database (DGIdb) was accessed, and the "Druggable Genome" category was used to identify potentially druggable targets in ESCC. 5
section_name: Whole exome sequencing analysis

section_content: The overall and disease-free survival distributions were described with Kaplan-Meier curves, and statistical signi cance was calculated using the log-rank test in STATA 16. Overall survival was evaluated from the time of diagnosis to death or the last follow-up. Censored cases were de ned as patients who lost contact during follow-up and who were still alive at the end of the study. Disease-free survival was evaluated from the time of diagnosis to death, the recurrence of ESCC, or the last follow-up. Censored cases were de ned as patients who were still alive and had no recurrent tumors at the end of the study. 

Clinical enrichment analysis was conducted using the R package maftools, with which pairwise and group-wise Fisher's exact tests can be performed to nd differentially enriched genes for clinical features such as sex and determine whether metastasis has occurred, the patient survives, or the cancer has recurred.
section_name: Statistical analysis

section_content: Oncoplots
section_name: Declarations Figures

section_name: Acknowledgments

section_content: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education ( 2019R1I1A1A01055513 ).
section_name: Acknowledgments

section_name: Data availability

section_content: The data that support the ndings of study are available from the corresponding author upon reasonable request.
section_name: Data availability

section_name: Author contributions

section_content: S. P curated, interpreted the data, and wrote the paper. S. Y. P and S. T. L conceptualized this research, review, editing this paper, and supervised entire project. S. Y. P acquired the funding. All authors reviewed the results and approved the nal version of the manuscript.
section_name: Author contributions

section_content: The authors declare no competing interests.
section_name: Competing interests

section_content: This is a list of supplementary les associated with this preprint. Click to download. Supplementalinfromation20211013. docx
section_name: Supplementary Files