Background & Aims Esophageal squamous cell carcinoma (ESCC) is the predominant form of esophageal cancer in Japan. Smoking and drinking alcohol are environmental risk factors for ESCC, whereas single ...nucleotide polymorphisms in ADH1B and ALDH2 , which increase harmful intermediates produced by drinking alcohol, are genetic risk factors. We conducted a large-scale genomic analysis of ESCCs from patients in Japan to determine the mutational landscape of this cancer. Methods We performed whole-exome sequence analysis of tumor and nontumor esophageal tissues collected from 144 patients with ESCC who underwent surgery at 5 hospitals in Japan. We also performed single-nucleotide polymorphism array-based copy number profile and germline genotype analyses of polymorphisms in ADH1B and ALDH2 . Polymorphisms in CYP2A6, which increase harmful effects of smoking, were analyzed. Functions of TET2 mutants were evaluated in KYSE410 and HEK293FT cells. Results A high proportion of mutations in the 144 tumor samples were C to T substitution in CpG dinucleotides (called the CpG signature) and C to G/T substitutions with a flanking 5′ thymine (called the APOBEC signature). Based on mutational signatures, patients were assigned to 3 groups, which associated with environmental (drinking and smoking) and genetic (polymorphisms in ALDH2 and CYP2A6 ) factors. Many tumors contained mutations in genes that regulate the cell cycle ( TP53, CCND1, CDKN2A , FBXW7 ); epigenetic processes ( MLL2, EP300, CREBBP , TET2 ); and the NOTCH ( NOTCH1 , NOTCH3 ), WNT ( FAT1 , YAP1 , AJUBA ) and receptor-tyrosine kinase−phosphoinositide 3-kinase signaling pathways ( PIK3CA , EGFR , ERBB2 ). Mutations in EP300 and TET2 correlated with shorter survival times, and mutations in ZNF750 associated with an increased number of mutations of the APOBEC signature. Expression of mutant forms of TET2 did not increase cellular levels of 5-hydroxymethylcytosine in HEK293FT cells, whereas knockdown of TET2 increased the invasive activity of KYSE410 ESCC cells. Computational analyses associated the mutations in NFE2L2 we identified with transcriptional activation of its target genes. Conclusions We associated environmental and genetic factors with base substitution patterns of somatic mutations and provide a registry of genes and pathways that are disrupted in ESCCs. These findings might be used to design specific treatments for patients with esophageal squamous cancers.
Radiotherapy (RT) combined with immune checkpoint inhibitors has recently produced outstanding results and is expected to be adaptable for various cancers. However, the precise molecular mechanism by ...which immune reactions are induced by fractionated RT is still controversial. We aimed to investigate the mechanism of the immune response regarding multifractionated, long‐term radiation, which is most often combined with immunotherapy. Two human esophageal cancer cell lines, KYSE‐450 and OE‐21, were irradiated by fractionated irradiation (FIR) daily at a dose of 3 Gy in 5 d/wk for 2 weeks. Western blot analysis and RNA sequencing identified type I interferon (IFN) and the stimulator of IFN genes (STING) pathway as candidates that regulate immune response by FIR. We inhibited STING, IFNAR1, STAT1, and IFN regulatory factor 1 (IRF1) and investigated the effects on the immune response in cancer cells and the invasion of surrounding immune cells. We herein revealed type I IFN‐dependent immune reactions and the positive feedback of STING, IRF1, and phosphorylated STAT1 induced by FIR. Knocking out STING, IFNAR1, STAT1, and IRF1 resulted in a poorer immunological response than that in WT cells. The STING‐KO KYSE‐450 cell line showed significantly less invasion of PBMCs than the WT cell line under FIR. In the analysis of STING‐KO cells and migrated PBMCs, we confirmed the occurrence of STING‐dependent immune activation under FIR. In conclusion, we identified that the STING‐IFNAR1‐STAT1‐IRF1 axis regulates immune reactions in cancer cells triggered by FIR and that the STING pathway also contributes to immune cell invasion of cancer cells.
We elucidated that the fractionated irradiation‐induced immune response is regulated by the STING‐IFNAR1‐STAT1‐IRF1 pathway, which includes positive feedback, and that this mechanism acts not only on cancer cells but also on surrounding PBMCs.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Understanding intratumor heterogeneity is clinically important because it could cause therapeutic failure by fostering evolutionary adaptation. To this end, we profiled the genome and epigenome in ...multiple regions within each of nine colorectal tumors. Extensive intertumor heterogeneity is observed, from which we inferred the evolutionary history of the tumors. First, clonally shared alterations appeared, in which C>T transitions at CpG site and CpG island hypermethylation were relatively enriched. Correlation between mutation counts and patients' ages suggests that the early-acquired alterations resulted from aging. In the late phase, a parental clone was branched into numerous subclones. Known driver alterations were observed frequently in the early-acquired alterations, but rarely in the late-acquired alterations. Consistently, our computational simulation of the branching evolution suggests that extensive intratumor heterogeneity could be generated by neutral evolution. Collectively, we propose a new model of colorectal cancer evolution, which is useful for understanding and confronting this heterogeneous disease.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Esophageal squamous cell carcinoma (ESCC) often recurs after chemoradiotherapy, and the prognosis of ESCC after chemoradiotherapy has not improved over the past few decades. The mutation process in ...chemoradiotherapy-resistant clones and the functional relevance of genetic alterations remain unclear. To address these problems, we performed whole-exome sequencing of 52 tumor samples from 33 patients with ESCC who received radiotherapy combined with 5-fluorouracil/platinum. In multiregion analyses of pretreatment and locally recurrent lesions from five cases, most driver gene-altered clones remained under chemoradiotherapy selection pressure, while few driver gene alterations were acquired at recurrence. The mutation signatures of recurrent ESCC, including increased deletion frequency and platinum dose-dependent base substitution signatures, were substantially different from those of primary ESCC and reflected the iatrogenic impacts of chemoradiotherapy. Single-region analysis of 28 pretreatment tumors indicated that focal copy-number gain at the
locus was significantly associated with poor progression-free survival and overall survival after chemoradiotherapy.
gain remained throughout the chemoradiotherapy course and potentially contributes to intrinsic resistance to chemoradiotherapy. Consistent with these findings,
copy number and mRNA and protein levels in ESCC cell lines correlated positively with resistance to radiotherapy, and
knockdown improved sensitivity to radiotherapy. Overall, these data characterize the clonal evolution process induced by chemoradiotherapy and clinically relevant associations for genetic alterations in ESCC. These findings increase our understanding of therapeutic resistance and support the rationale for precision chemoradiotherapy. SIGNIFICANCE: Whole-exome sequencing reveals the genetic evolution of ESCC during chemoradiotherapy, highlighting
gain in pretreatment tumors as a potential marker of therapy resistance.
Endoscopic submucosal dissection (ESD) can be used as a less invasive treatment option for superficial esophageal cancer involving the muscularis mucosae (T1a-MM) or upper third of the submucosa ...(T1b-SM1). Additional treatment after ESD is needed to prevent lymph node metastasis. However, the efficacy of radiotherapy following ESD has not been well evaluated. Moreover, the clinical outcomes of patients with large mucosal defects of the esophagus who received radiotherapy after ESD have not been reported. This study aimed to clarify the efficacy of additional radiotherapy following ESD for esophageal squamous cell cancer involving T1a-MM or T1b-SM1.
We analyzed twenty-seven patients with pathologically confirmed T1a-MM or T1b-SM1 esophageal squamous cell cancer treated by ESD. Thirteen patients received additional radiotherapy (RT group), and the remaining patients did not (non-RT group). Locoregional control (LRC), overall survival, cause-specific survival, and adverse events including treatment-related esophageal strictures were evaluated.
The three-year LRC was significantly better for the RT than the non-RT group (100% vs. 57.8%, respectively; p = 0.022). Chemotherapy following ESD did not improve LRC. Multivariate analysis showed that radiotherapy was an independent prognostic factor for better LRC (p = 0.0022). Contrary to the results in LRC, overall and cause-specific survival were not significantly different between the RT and non-RT groups. A subgroup analysis of patients with mucosal defects involving ≥ 3/4 of the esophageal circumference after ESD showed that LRC of the RT group was better than that of the non-RT group (p = 0.049). Treatment-related esophageal strictures were observed in 2 of 6 patients in the RT group with large mucosal defects after ESD. No patients with mucosal defects involving less than 3/4 of the circumference after ESD developed treatment-related strictures.
Radiotherapy after ESD contributed to better LRC in esophageal squamous cell cancer involving pT1a-MM and pT1b-SM1. Esophageal strictures were observed in some patients with large mucosal defects after ESD. Despite leading to better LRC, radiotherapy after ESD should be undertaken after careful consideration for patients with large mucosal defects after ESD.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies owing to the high frequency of tumor recurrence. The identification of markers for early ESCC diagnosis and ...prediction of recurrence is expected to improve the long-term prognosis. Therefore, we searched for associations between tumor recurrence and cell-free DNA (cfDNA) mutations in blood plasma, which contains genetic markers for various cancer types.
Genomic DNA from tumors and cfDNA from plasma were obtained from 13 patients undergoing treatment for newly diagnosed ESCC. Next-generation sequencing of cfDNA in plasma was performed to identify mutations in 53 cancer-related genes, in which recurrent mutations were previously detected in ESCC. cfDNA mutational profiles were compared before and after tumor resection in four patients. Furthermore, somatic mutations in serial plasma samples were monitored after treatment in four patients.
We identified multiple concordant somatic mutations in cfDNA and primary tumor samples from 10 patients (83.3%) and in cfDNA and metastatic tumor samples from one patient (100%). Furthermore, the allele frequency of the concordant mutations in cfDNA changed concomitantly with tumor burden and increased approximately 6 months earlier than the detection of tumor recurrences by imaging tests in two patients. Conventional biomarkers, such as SCC and p53-Ab, did not reflect tumor recurrences.
The present multigene panel, which enabled the diagnosis of tumor recurrence with greater accuracy than did using standard tumor markers or imaging methods, is expected to greatly facilitate standard, postoperative follow-up monitoring in ESCC.
Amplification of chromosome 7p (Ch.7p) is common in colorectal cancer (CRC). The aim of this study was to identify potential driver genes on Ch.7p that are overexpressed due to DNA copy number ...amplification and determine their clinical significance in CRC.
We identified phosphoserine phosphatase (PSPH) as a potential driver gene using a CRC dataset from The Cancer Genome Atlas (TCGA) using a bioinformatics approach. The expression of PSPH in 124 primary CRCs was examined by quantitative reverse transcription polymerase chain reaction (PCR) and immunohistochemistry. The biological effect of PSPH expression was explored by Gene Set Enrichment Analysis (GSEA) using the TCGA dataset.
PSPH was overexpressed in tumor tissues and PSPH positively correlated with depth of invasion and distant metastasis. On multivariate analysis, high PSPH expression was an independent poor prognostic factor. These results were supported by GSEA.
PSPH could be a novel prognostic biomarker with malignant potential on Ch.7p in CRC.
miR-146a plays important roles in cancer as it directly targets NUMB, an inhibitor of Notch signaling. miR-146a is reportedly regulated by a G>C polymorphism (SNP; rs2910164). This polymorphism ...affects various cancers, including colorectal cancer (CRC). However, the clinical significance of miR-146a polymorphism in CRC remains unclear. A total of 59 patients with CRC were divided into 2 groups: a CC/CG genotype (n = 32) and a GG genotype (n = 27), based on the miR-146a polymorphism. cDNA microarray analysis was performed using 59 clinical samples. Significantly enriched gene sets in each genotype were extracted using GSEA. We also investigated the association between miR-146a polymorphism and miR-146a, NUMB expression or migratory response in CRC cell lines. The CC/CG genotype was associated with significantly more synchronous liver metastasis (p = 0.007). A heat map of the two genotypes showed that the expression profiles were clearly stratified. GSEA indicated that Notch signaling and JAK/STAT3 signaling were significantly associated with the CC/CG genotype (p = 0.004 and p = 0.023, respectively). CRC cell lines with the pre-miR-146a/C revealed significantly higher miR-146a expression (p = 0.034) and higher NUMB expression and chemotactic activity. In CRC, miR-146a polymorphism is involved in liver metastasis. Identification of this polymorphism could be useful to identify patients with a high risk of liver metastasis in CRC.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background
Downregulation of paired related homeobox 1 (
PRRX1
) is associated with the acquisition of cancer stem cell (CSC)-like properties and poor prognosis in cancers. The purpose of this study ...is to clarify the role of
PRRX1
expression in predicting prognosis and mediating CSC-like properties in hepatocellular carcinoma (HCC).
Methods
The association between
PRRX1
expression and overall survival (OS) of patients with HCC was analyzed in three independent datasets: 62 resected primary cases, 242 cases from GSE14520, and 162 cases from The Cancer Genome Atlas (TCGA). A cell line expressing
PRRX1
(HuH7) was established for the functional analyses. The ability to form spheres, the expression levels of the hepatic CSC surface markers (
CD13
,
CD133
, and
EpCAM
), in vitro chemosensitivity to 5-fluorouracil (FU), and radiosensitivity were evaluated.
Results
Univariate and multivariate analyses showed that the 5-year OS of the low
PRRX1
expression group was significantly poorer than that of the high
PRRX1
expression group (
P
= 0.024 and
P
= 0.045, respectively). Consistent with this, the low
PRRX1
expression group in GSE14520 and TCGA datasets showed significantly shorter OS (
P
= 0.027 and
P
= 0.010, respectively). Gene set enrichment analysis on GSE14520 and TCGA datasets indicated that downregulation of
PRRX1
was correlated with the stemness signature. The number of spheres and the expression levels of CSC markers were significantly decreased when
PRRX1
was expressed. Moreover,
PRRX1
impaired resistance to 5-FU and radiation.
Conclusions
Downregulation of
PRRX1
expression contributes to the poor prognosis of patients with HCC through acquisition of CSC-like properties.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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