Genome-wide analysis of Chinese keloid patients identifies novel causative genes.

Background: Keloids are benign skin tumors that appears on skin lesions in humans. Keloids are characterized by invasive tumor growth and are highly prone to recurrence after treatment. The incidence of keloids is ethnically specific; however, the molecular mechanism underlying the incidence of keloids in the Chinese population remains unclear. To date, no reports appear to have been published on the molecular characteristics underlying keloids in the Chinese population from the perspective of whole-genome sequencing. Methods: In this study, we collected keloid samples from 9 keloid patients underwent surgery in the Department of Dermatology, The First Affiliated Hospital of Soochow University, paired them to normal skin tissues, and performed whole-exome sequencing. The average depth of the samples was 1,200×, and the average exome coverage was 98.90%. Results: The bioinformatics analysis identified 3,125 single nucleotide variants (SNVs) and 299 insertions/deletions (InDels). The major mutation characteristics of the SNVs were C > A and C > T. The non-synonymous SNV types included stopgain, and stoploss. The non-synonym InDels included frameshift deletion, frameshift insertion, and stopgain. We also found a total of 67,873 copy number variations (CNVs) in the samples. The genes with the highest mutation frequency included mucin 4 (MUC4) (55.6%), tubulin tyrosine ligase like 12 (TTLL12) (33.3%), calcium voltage-gated channel subunit alpha1 (CACNA1C) (33.3%), and mucin 12 (MUC12) (33.3%). The average tumor mutation burden (TMB) was 289 mutations/million base pair (MB). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the mutated genes were mainly concentrated in micro ribonucleic acids in cancer and the calcium signaling pathway. The Gene Ontology (GO) analysis showed that mutant genes were mainly concentrated in binding cells, cell parts, and cellular processes. Conclusions: Whole-exome sequencing was performed in the Chinese keloid patients and some potential candidate genes related to keloid occurrence and development were identified, which may provide new molecular targets for the clinical diagnosis and treatment of keloid patients.

Molecular profiles and circulating tumor-DNA detected in Chinese early stage breast cancer.

Background: With the development of gene-sequencing technology, genome biomarkers, including Erb-B2 receptor tyrosine kinase 2 (ERBB2), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (pIK3CA), BReast CAncer gene 1 (BRCA1), and BReast CAncer gene 2 (BRCA2), and immunomarkers, including the tumor mutational burden (TMB) and programmed death-ligand 1 (PD-L1), have become important in the selection of treatment. Methods: Twenty patients with early stage breast cancer who underwent surgery were enrolled in this study. Tissue samples and paired postoperative peripheral blood samples were collected and subjected to the targeted-capture sequencing of 1,021 cancer-associated genes. Results: The most frequently altered genes were tumor protein 53 (TP53; 70%), PIK3CA (40%), protooncogene MYC (35%), ERBB2 (30%), and cyclin-dependent kinase 12 (CDK12; 20%). Six (30%) patients presented with ERBB2 amplification of NGS and simultaneously were positive for human epidermal growth factor receptor 2 (HER2) of IHC. ERBB2 amplification and being HER2 positive were common in breast cancer patients without lymph node metastasis (5/6, 83.3%) and those in stages IA-IIA. Most of the somatic mutations clustered in the TP53 pathway, followed by the PI3K pathway. The TMB was lower than metastatic breast cancer in our cohort, and ranged from 0 to 9.6 mut/Mb (median: 1.92 mut/Mb). Interestingly, more patients had the ERBB2 mutation in the non-lymph node metastasis group than the lymph node metastasis group (55.6% vs. 9.1%; P=0.049). Similarly, more patients had the CDK12 mutation in the non-lymph node metastasis group than the lymph node metastasis group (44.4% vs. 0%; P=0.026). Circulating tumor deoxyribonucleic acid (ctDNA) was detected in 7 of the 20 patients (35%). Of these patients, 71.4% (5/7) were in stage I/II. In addition, no correlation was found between ctDNA detection and clinicopathological features or the driver gene mutations (e.g., PIK3CA and ERBB2). However, patients positive for ctDNA had a higher TMB than those negative for ctDNA when grouped according to the median TMB (1.92 mut/Mb; 85.7% vs. 38.5%; P=0.043). Conclusions: This study described that genomic characteristics of Chinese early stage breast cancer, and the results showed that TMB was related to the detection of ctDNA in postoperative blood.

The ctDNA-based postoperative molecular residual disease status in different subtypes of early-stage breast cancer.

Background: Breast cancer is a highly heterogeneous disease. Early-stage, non-metastatic breast cancer is considered curable after definitive treatment. Early detection of tumor recurrence and metastasis through sensitive biomarkers is helpful for guiding clinical decision-making and early intervention in second-line treatment, which could improve patient prognosis and survival. Methods: In this real-world study, we retrospectively analyzed 82 patients with stages I to III breast cancer who had been analyzed by molecular residual disease (MRD) assay. A total of 82 tumor tissues and 224 peripheral blood samples were collected and detected by next-generation sequencing (NGS) based on a 1,021-gene panel in this study. Results: MRD positivity was detected in 18 of 82 patients (22.0%). The hormone receptor-/human epidermal growth factor receptor 2+ (HR-/HER2+) subgroup had the highest postoperative MRD detection rate at 30.8% (4/13). The BRCA2 and SLX4 genes were significantly enriched in all patients in the MRD positive group and FGFR1 amplification was significantly enriched in the MRD negative group with HR+/HER2-. The number of single nucleotide variants (SNVs) in tissue samples of MRD-positive patients was higher than that of MRD-negative patients (11.94 vs. 8.50 SNVs/sample). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that there was a similar biological function of the tumor-mutated genes in the 2 MRD status groups. Conclusions: This real-world study confirmed that patient samples of primary tumor tissue with different MRD status and molecular subtypes had differential genetic features, which may be used to predict patients at high risk for recurrence.

Genome variation in colorectal cancer patient with liver metastasis measured by whole-exome sequencing.

BACKGROUND: Liver metastasis of colorectal cancer (CRC) is an important cause of death from CRC, but its molecular mechanism is still unclear. In recent years, whole-exome sequencing has played an increasingly important role in the study of the occurrence and development of diseases, especially malignant tumors. Its high throughput and low cost advantages enable researchers to explore the pathogenic genes of diseases, and screen potential molecular markers and therapeutic targets from the level of genomics. METHODS: This study collected the primary tumor tissues, matched paracancerous, normal tissues, and liver metastases of 4 CRC patients admitted to the Department of General Surgery of the First Affiliated Hospital of Soochow University, and performed high-depth whole-exome sequencing, with the sequencing depth of each sample reaching 123× on average, then filtered the sequencing data, compared them, and analyzed the bioinformatics data. RESULTS: we found 8,565 single nucleotide variants (SNV) and 429 insertions/deletions (InDel) in the primary and hepatic lesion tissues, and the genes with the highest mutation frequency were titin (TTN), obscurin (OBSCN), and homeodomain-interacting protein kinase 2 (HIPK2). The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the mutant genes was conducted, and it was found that the mutant genes were mainly concentrated in the cells, cell parts, and cellular process of GO. The results of KEGG pathway analysis showed that mutations were mainly distributed in circadian entrainment, insulin secretion, and glutamatergic synapse. Further, we identified 723 SNV and Indel genes with high frequency mutations including TTN, OBSCN, and hydrocephalus-inducing protein homolog (HYDIN) across all tissues of liver metastases. The GO analysis showed that the mutated genes in liver metastatic tissues were mainly concentrated in cell, cell part, and cellular process. The KEGG pathway analysis showed that high frequency mutation genes were focused on gastric acid secretion, bile secretion, and melanogenesis. CONCLUSIONS: This study found some candidate genes related to the occurrence of CRC and liver metastasis through whole-exome sequencing of relevant tissues in CRC patients with liver metastasis, which is expected to provide new markers and therapeutic targets for such patients.

FOLFIRINOX regulated tumor immune microenvironment to extend the survival of patients with resectable pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignant tumors worldwide due to its ineffective diagnosis and poor prognosis. The longest median overall survival (OS) to PDAC patients has been provided by FOLFIRINOX. It is essential to identify the mechanisms of FOLFIRINOX to gain new insights for the treatment of PDAC. METHODS: We compared gene expression levels of PDAC patients who received neoadjuvant FOLFIRINOX prior to surgery with those of patients who received no neoadjuvant chemotherapy. Bioinformatics analysis was applied to screen differentially expressed genes (DEGs). Three microarray data sets were downloaded to analyze gene expression data between PDAC and adjacent non-tumor tissues. Overlapping DEGs were subjected to Kaplan-Meier survival analysis. The genes relating to poor outcomes and would be decreased after FOLFIRINOX were input into the Oncomine, University of Alabama Cancer (UALCAN), and LinkedOmics databases to analyze the gene expression and regulation networks. RESULTS: A total of 83 differentially expressed genes (DEGs) were screened and subjected to bioinformatics analysis, which indicated FOLFIRINOX influenced the immune microenvironment of PDAC. Seventy-three genes significantly associated with the OS of PDAC patients. A Venn diagram revealed CXCL5 and PLAU were related to poor outcomes and would decrease after FOLFIRINOX chemotherapy of PDAC patients. It turned out that CXCL5 participated in the immune response-regulating signaling pathway in PDAC patients. CONCLUSIONS: FOLFIRINOX regulated tumor immunity by reducing expression of the immunosuppressive gene CXCL5, laying a foundation for further study of combination therapy of FOLFIRINOX and immunotherapy.

Experimental studies of erythropoietin protection following traumatic brain injury in rats.

This study aimed to explore the effect of erythropoietin (EPO) on brain tissue after traumatic brain injury in rats. Animals were divided into sham, control and EPO groups. The model was constructed using the improved Feeney's free falling weight traumatic brain injury model. The brain water content and the number of the apoptotic monocyte chemotactic protein-1(+) (MCP-1(+)) and CD68(+) cells were monitored at 12, 48 and 120 h post-trauma. The water content was lower in the EPO group at each time point compared to the control group. The number of apoptotic MCP-1(+) and CD68(+) cells surrounding the traumatic brain injury lesion was less in the EPO group compared to these values in the control group. In conclusion, following traumatic brain injury, EPO significantly decreased the number of apoptotic cells, the expression of MCP-1, the infiltration of CD68(+) cells as well as brain edema to protect the brain.

Epstein-Barr virus facilitates the malignant potential of immortalized epithelial cells: from latent genome to viral production and maintenance.

Epstein-Barr virus (EBV) is closely associated with several malignancies, including nasopharyngeal carcinoma. To investigate the EBV activity in tumor development, we tried to establish a malignant model of EBV-infected cells in nude mice. On the basis of the Maxi-EBV system, a human embryonic kidney epithelial cell line (293) with a low malignant potential was used for a stable EBV genome infection. The derived cell line, termed 293-EBV, exhibited obvious morphological transformation and significantly increased growth ability, with the cell cycle redistributed. The clonability and tumorigenicity were also substantially accelerated. In 293-EBV cells, the expression level of the transcription factor NF-kappaB and JNK2 were upregulated. The result suggested that latent membrane protein 1 (LMP1) was an important viral protein responsible for the enhanced malignant potential. Matured and budding virus particles were observed in tumor tissues, confirming the spontaneous reactivation of EBV from latent genome to lytic cycle at the site of tumor development. Primary culture of tumor tissues showed two patterns about the EBV maintenance or not in newly grown cells, and this was dependent on the thickness of the planted tissues. Moreover, the tumor cells lost EBV genome easily when subcultured at low density. Our findings revealed the cell-to-cell contact mechanism, which was required for the EBV maintenance in the tumor cells during the expansion of EBV-infected cells. This mechanism might give an explanation to the phenomenon that EBV genome in epithelial tumor cells becomes easily lost during subculture in vitro. Our results provided further evidence of a function for EBV in the etiology of tumor development.