Gene Mutations of Esophageal Squamous Cell Carcinoma Based on Next-Generation Sequencing
Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with limited therapeutic options and poor survival outcomes. It accounts for nearly 90% of esophageal cancer cases worldwide, with a pronounced incidence in Asian countries, particularly in China’s Hebei Province. Despite advancements in conventional treatments, the prognosis for ESCC remains dismal, necessitating a deeper understanding of its molecular mechanisms to identify novel therapeutic targets. This study utilized next-generation sequencing (NGS) to profile somatic mutations, copy number variations (CNVs), and mutational signatures in 29 surgically resected ESCC samples from a high-incidence region, while also exploring correlations between genomic alterations, clinicopathological features, and biomarkers relevant to immunotherapy.
Study Design and Methodology
Tumor tissues and adjacent normal tissues were collected from 29 ESCC patients who underwent surgical resection between 2015 and 2019 at the Fourth Hospital of Hebei Medical University. DNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples using QIAamp DNA FFPE Tissue Kits. A targeted NGS panel covering 520 cancer-related genes was employed to detect single nucleotide variants (SNVs), insertions/deletions (INDELs), CNVs, and microsatellite instability (MSI). Tumor mutational burden (TMB) was calculated as mutations per megabase (mut/Mb), and MSI status was determined based on the number of microsatellite site errors. Programmed death-ligand 1 (PD-L1) expression was assessed using the 22C3 pharmDx assay, with tumor proportion scores (TPS) ≥1% considered positive. Mutational signatures were inferred using non-negative matrix factorization (NMF) and compared to the COSMIC database. Statistical analyses, including Spearman rank correlation, were conducted to evaluate associations between genomic alterations, clinicopathological variables, and PD-L1 expression.
Key Genomic Alterations in ESCC
The analysis revealed 421 genomic alterations across the 29 samples, including 230 SNVs, 35 INDELs, 147 copy number amplifications, and 9 deletions. TP53 emerged as the most frequently mutated gene, altered in 96.6% (28/29) of tumors. Recurrent mutations were also observed in NOTCH1 (27.6%, 8/29), EP300 (17.2%, 5/29), and KMT2C (17.2%, 5/29). Novel mutation sites not previously documented in COSMIC were identified, including TP53 hotspot mutations (p.R273H, p.R248Q, p.R175H) and a PIK3CA hotspot mutation (p.H1047L).
Copy number amplifications were prevalent in 69.0% (20/29) of cases. The CCND1/FGF3/FGF4/FGF19 cluster on chromosome 11q13.3 was amplified in 41.4% (12/29) of tumors, making it the most frequent CNV. Other amplified genes included NKX2-1 (17.2%), NFKBIA (13.8%), and ERBB2 (10.3%). Deletions were less common, with CDKN2A/2B (10.3%) and MET (6.9%) being the most affected loci.
Mutational Signatures and Underlying Mechanisms
The study identified four distinct mutational signatures (A–D) through NMF. Signature A showed a predominance of C>T transitions, while Signature B was characterized by C>G and C>A substitutions. Signature C exhibited T>G mutations, and Signature D displayed a mix of C>T and T>G changes. Although these signatures had low similarity to COSMIC signatures (maximum cosine similarity: 0.67), further analysis quantified the contribution of known COSMIC signatures to the ESCC mutation spectrum. Signature 1 (associated with spontaneous 5-methylcytosine deamination) and APOBEC-mediated signatures (Signatures 2 and 13) were predominant, contributing to 58.6% and 51.7% of samples, respectively. Signatures 10 (POLE-related), 12 (etiology unknown), and 17 (unknown etiology) also showed notable contributions.
Two samples exhibited high similarity to COSMIC Signature 1, reflecting age-related mutagenesis. Another sample aligned closely with Signature 13, implicating APOBEC enzyme activity in generating C>G mutations. Notably, a patient with a history of alcohol consumption displayed a profile resembling Signature 16 (T>C mutations), suggesting a potential link between alcohol exposure and specific mutagenic processes in ESCC.
Dysregulated Signaling Pathways
Several oncogenic pathways were recurrently altered:
- Cell Cycle Regulation: Disrupted in 96.6% of cases, primarily via TP53 mutations and CCND1 amplifications. Additional alterations included EP300 (17.2%), CREBBP (10.3%), and CDKN2A/2B deletions (10.3%).
- Chromatin Remodeling: Mutations in ARID1A (13.8%), ATRX (10.3%), and CHD4 (13.8%) were observed, affecting the SWI/SNF and NuRD complexes.
- Notch Signaling: Alterations in NOTCH1 (27.6%), NOTCH3 (6.9%), and epigenetic regulators EP300 and CREBBP were frequent.
- JAK-STAT Pathway: Mutations in JAK1/2/3, STAT3, and PIK3CA (13.8%), along with IL7R amplifications (10.3%), were identified.
Biomarkers for Immunotherapy
PD-L1 expression (TPS ≥1%) was detected in 13.8% (4/29) of tumors. The median TMB was 5.6 mut/Mb (range: 0.8–42.9), with 17.2% (5/29) of cases classified as TMB-high (≥10 mut/Mb). All samples were microsatellite stable (MSS). TMB correlated positively with lymph node metastasis (r = 0.468, P = 0.010) but not with PD-L1 expression (r = 0.246, P = 0.198). No significant associations were found between PD-L1 expression and specific gene mutations.
Clinical and Therapeutic Implications
The study highlights CCND1/FGF3/FGF4/FGF19 amplification as a potential resistance marker to PD-1 inhibitors, consistent with prior findings that such amplifications correlate with poorer immunotherapy outcomes. CDKN2A deletions and KMT2D mutations were linked to deeper tumor infiltration (r = -0.654, P < 0.001) and lymph node metastasis (r = 0.407, P = 0.028), respectively, suggesting their roles in disease progression.
The predominance of APOBEC-related mutational signatures underscores the importance of DNA repair mechanisms in ESCC, while the heterogeneity of mutational processes emphasizes the need for personalized therapeutic strategies. Targeting chromatin remodeling complexes (e.g., SWI/SNF) or leveraging synthetic lethality in ARID1A-deficient tumors may offer novel avenues for treatment.
Limitations and Future Directions
The study’s small sample size and focus on a predefined gene panel limit the detection of novel drivers and pathways. Future whole-genome or exome sequencing studies in larger cohorts are warranted to validate these findings and explore additional biomarkers. Furthermore, functional studies are needed to elucidate the mechanistic roles of identified mutations and their interplay with immunotherapy response.
In conclusion, this work delineates the genomic landscape of ESCC in a high-incidence population, identifying recurrent mutations, CNVs, and mutational signatures with clinical relevance. These insights pave the way for targeted therapies and improved patient stratification in immunotherapy trials.
doi.org/10.1097/CM9.0000000000001411
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