Germline Mutations in 32 Cancer Susceptibility Genes by Next-Generation Sequencing among Breast Cancer Patients.

INTRODUCTION: BRCA1/2 germline mutations are the most well-known genetic determinants for breast cancer. However, the distribution of germline mutations in non-BRCA1/2 cancer susceptibility genes in Chinese breast cancer patients is unclear. The association between clinical characteristics and germline mutations remains to be explored. METHODS: Consecutive breast cancer patients from Peking University People's Hospital were enrolled. Clinical characteristics were collected, and next-generation sequencing was performed using blood samples of participants to identify pathogenic/likely pathogenic (P/LP) germline mutations in 32 cancer susceptibility genes including homologous recombination repair (HRR) genes. RESULTS: A total of 885 breast cancer patients underwent the detection of germline mutations. 107 P/LP germline mutations of 17 genes were identified in 116 breast cancer patients including 79 (8.9%) in BRCA1/2 and 40 (4.5%) in 15 non-BRCA1/2 genes. PALB2 was the most frequently mutated gene other than BRCA1/2 but still relatively rare (1.1%). There were 38 novel P/LP germline variants detected. P/LP germline mutations in BRCA1/2 were significantly associated with onset age (p < 0.001), the family history of breast/ovarian cancer (p = 0.010), and molecular subtype (p < 0.001), while being correlated with onset age (p < 0.001), site of breast tumor (p = 0.028), and molecular subtype (p < 0.001) in HRR genes. CONCLUSIONS: The multiple-gene panel prominently increased the detection rate of P/LP germline mutations in 32 cancer susceptibility genes compared to BRCA1/2 alone. Onset younger than or equal to 45 years of age, bilateral and triple-negative breast cancer patients may be more likely to be recommended for detecting P/LP germline mutations in HRR genes.

Novel biomarkers identified in triple-negative breast cancer through RNA-sequencing.

BACKGROUND AND AIMS: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis due to its aggressive biological behavior and lack of therapeutic targets. Here, we aimed to identify specific biomarkers for TNBC by using RNA-sequencing and bioinformatics analysis. MATERIALS AND METHODS: Fresh breast tumor tissues were obtained from 34 patients who were admitted to the Breast Center, Peking University People's Hospital, from June 2020 to December 2020; the patients were pathologically diagnosed with primary breast cancer and underwent surgery for the resection of tumor tissues. Tumor-tissue RNA was extracted and the generated cDNA libraries were sequenced using the NextSeq platform, after which the differentially expressed genes (DEGs) between TNBC and other subtypes of breast cancer were identified and DEG functional-enrichment analysis was performed. Next, weighted gene co-expression network analysis (WGCNA) was used to identify the most significant module and hub genes in TNBC, and then the correlations between the hub genes and the prognosis of TNBC patients were analyzed through survival analysis. Lastly, qRT-PCR analysis was used to validate the expression levels of hub genes in tumor tissues from TNBC and other subtypes of breast cancer. RESULTS: Comparison of TNBC tissues and tissues from other subtypes of breast cancer led to the identification of 273 DEGs in TNBC: 172 upregulated and 101 downregulated genes. In Gene Ontology analysis of the DEGs, five terms were significantly enriched, "developmental process," "anatomical structure development," "tissue development," "cell cycle," and "epithelium development," and in Kyoto Encyclopedia of Genes and Genomes pathway analysis, the most significantly enriched pathways for all DEGs were "cell cycle," "mitophagy-animal," and "autophagy-animal." Furthermore, we identified the core module related to TNBC and screened for hub genes by using WGCNA, and after verifying the top 100 genes based on survival analysis, we selected four genes as the hub genes: SERPINB4, SMR3A, FERMT1, and STARD4; elevated expression of these genes was associated with poor overall survival (OS) of TNBC patients. Notably, qRT-PCR results indicated that FERMT1 mRNA expression was significantly upregulated in TNBC samples. CONCLUSION: The DEG profiles between tissues from TNBC and other subtypes of breast cancer were identified using RNA-sequencing and bioinformatics analysis. FERMT1 was significantly upregulated in TNBC tumor tissues, and increased expression of FERMT1 was associated with poor OS of TNBC patients. FERMT1 could serve as a specific biomarker of and therapeutic target in TNBC.

Long noncoding RNA ZNFX1-AS1 promotes the invasion and proliferation of gastric cancer cells by regulating LIN28 and CAPR1N1.

BACKGROUND: Long noncoding RNA (lncRNA) ZNFX1-AS1 (ZFAS1) is a newly discovered lncRNA, but its diagnostic value in gastric cancer is unclear. AIM: To investigate the potential role of ZFAS1 in gastric cancer and to evaluate the clinical significance of ZFAS1 as a biomarker for gastric cancer screening. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to screen for gastric cancer-associated lncRNAs in gastric cancer patients, gastric stromal tumor patients, gastritis or gastric ulcer patients, and healthy controls. Correlations between ZFAS1 expression and clinicopathological features were analyzed. The biological effects of ZFAS1 on the proliferation, migration, and invasion of gastric cancer cells were studied by MTT, colony formation, and transwell mi-gration assays. The potential mechanism of ZFAS1 was demonstrated using enzyme-linked immunosorbent assay and qRT-PCR. The relationship between ZFAS1 and tumorigenesis was demonstrated using in vivo tumor formation assays. RESULTS: The plasma level of lncRNA ZFAS1 was significantly higher in preoperative patients with gastric cancer than in individuals in the other 4 groups. Increased expression of ZFAS1 was significantly associated with lymph node metastasis, advanced TNM stage, and poor prognosis. ZFAS1 regulated the proliferation, migration, and invasion of gastric cancer cells and regulated the growth of gastric cancer cells in vivo. LIN28 and CAPRIN1 were identified as key downstream mediators of ZFAS1 in gastric cancer cells. CONCLUSION: LncRNA ZFAS1 promoted the invasion and proliferation of gastric cancer cells by modulating LIN28 and CAPRIN1 expression, suggesting that ZFAS1 can be used as a potential diagnostic and prognostic biomarker in gastric cancer.

Four novel BRCA variants found in Chinese hereditary breast cancer patients by next-generation sequencing.

Breast cancer is the most frequent cancer among women worldwide. Patients carrying mutations in breast cancer susceptibility genes like BRCA1 and BRCA2 (BRCA1/2) account for 5-10% of all breast cancer patients. Therefore, screening for susceptibility genes may reduce the incidence of breast cancer and improve prognosis. To provide evidence for mutation interpretation and targeted drug use in breast cancer patients, gene mutations were screened in 78 women diagnosed with sporadic breast cancer using a next-generation sequencing panel, confirmed by Sanger sequencing. Then the pathogenicity of the identified novel variants was explored using in vitro experiments including western blotting, co-immunoprecipitation and cell-migration assays. Four novel variants (BRCA2 L1390W, BRCA2 Glu432fs, BRCA1 P706L, and BRCA1 Cys882fs) were identified. BRCA2 Glu432fs decreased the expression of BRCA2 protein, enhanced cell migration and invasion ability, and prevented the protein from interacting with RAD51, resulting in a defect in the homologous recombination pathway. The identification of these novel BRCA variants and the confirmation of their pathogenicity have enriched the genetic database of breast cancer, especially in the Chinese population. Moreover, the variants are the genetic risk factors for hereditary breast cancer. Therefore, BRCA variant detection and genetic counseling for breast cancer patients are meaningful and important.

Multiple Mutation Detection for Risk Assessment in Patients with Breast Cancer by Using Next-Generation Sequencing.

OBJECTIVE: Breast cancer is recognized as the most common cause of malignancy and cancer death worldwide; however, mutations in the cancer-related BRCA genes are detected in only 2-3% of patients with breast cancer. Because next-generation sequencing technology allows concurrent sequencing of numerous target genes, diverse cancer-susceptibility genes are now being evaluated, although their significance in clinical practice remains unclear. METHODS: In this study, we developed a sequencing panel containing the genes BRCA1, BRCA2, TP53, PIK3CA, ERBB2 (Her2), and PTEN, which are all associated with cancer risk in patients, and we enrolled 60 patients with breast cancer. RESULTS: Germline mutations were found to be carried by nine patients (15%): 3 in BRCA1, 5 in BRCA2, and 1 in TP53. The patients harboring these mutations are considered to face a high risk of developing malignant tumors, and cancer screening is thus recommended for the patients. CONCLUSION: This study demonstrates the feasibility of using Ion Torrent sequencing technology for reliably detecting gene mutations in clinical practice for guiding individualized drug therapy or combination therapies for breast cancer.

A novel heterozygous HTRA1 mutation is associated with autosomal dominant hereditary cerebral small vessel disease.

BACKGROUND: We investigated whether a heterozygous mutation that we newly identified in HTRA1 (high-temperature requirement serine protease A1 gene) in a pedigree with autosomal dominant hereditary cerebral small vessel disease (SVD) reduces the function of HTRA1 and affects the transforming growth factor-ß1 (TGF-ß1)/Smad signaling. METHODS: Whole-exome sequence from the proband and her two sisters was examined using whole-exome enrichment and sequencing. Expression of HTRA1 and TGF-ß1/Smad and HTRA1 activity were assayed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting analyses after transfecting wild-type and mutant HTRA1 genes into HEK293 cells. RESULTS: A new heterozygous mutation (c.614C>G:p.Ser205Cys) in HTRA1 was identified in the sequence encoding the trypsin-like serine protease domain. The mutation was predicted to be deleterious by in silico tools. Moreover, in vitro activity and protein analyses revealed a loss-of-function effect of the mutation: the proteolytic activity of mutant HTRA1 was decreased, and, notably, this was accompanied by an increase in TGF-ß1/Smad protein levels. CONCLUSIONS: The heterozygous mutation HTRA1 S205C causing diminished protease activity is associated with-and could represent a cause of-autosomal dominant hereditary cerebral SVD. Our results also indicate a relationship between HTRA1 and TGF-ß1/Smad signaling.

Circulating long non-coding RNAs HULC and ZNFX1-AS1 are potential biomarkers in patients with gastric cancer.

Long non-coding RNAs (lncRNAs) have been demonstrated to be involved in different types of cancer, including gastric cancer. Although altered lncRNAs profiles have been observed in or around gastric cancer tissues, the diagnostic value of circulating lncRNAs in gastric cancer remains unclear. In the present study, a number of highly expressed lncRNAs, including uc001lsz, GACAT2, ABHD11-AS1, GACAT3, SUMP1P3, CHET1, TUG1, SNHG12, GAS5, PVT1, LINC00152, HOTAIR, CCAT1, H19, HULC and ZNFX1-AS1, were investigated as potential minimally invasive biomarkers for this tumor. Preliminary screening experiments revealed that ZNFX1-AS1 and HULC were differentially expressed in the plasma of gastric cancer patients and healthy control subjects. The study further examined the relative expression of ZNFX1-AS1 and HULC in the plasma of 50 matching preoperative and postoperative patients, 50 gastrointestinal stromal tumor (GIST) patients, 50 gastritis/peptic ulcer patients and 50 healthy control subjects through reverse transcription-quantitative polymerase chain reaction. The correlation of lncRNA relative expression with the general characteristics and clinicopathological factors was analyzed. It was observed that the levels of ZNFX1-AS1 and HULC in the plasma of preoperative patients were markedly higher compared with those in the plasma of GIST patients, gastritis/peptic ulcer patients and healthy control subjects, while no significant difference was detected among these three groups. Receiver operating characteristic curve analysis was also conducted to distinguish gastric cancer patients from healthy control subjects. The area under the curve was 0.85 and 0.65 for ZNFX1-AS1 and HULC, respectively. In conclusion, the results indicated that the lncRNAs ZNFX1-AS1 and HULC are promising in the clinical diagnosis of gastric cancer.

Association between CYP2C19 and ABCB1 polymorphisms and clopidogrel resistance in clopidogrel-treated Chinese patients.

OBJECTIVE: To investigate the association between CYP2C19 and ABCB1 polymorphisms and clopidogrel resistance (CR) in patients with cardiovascular disease in Beijing district. METHODS: In total, 325 patients were enrolled in the study, including 101 experimental group patients and 224 control group patients. The experimental group was divided into CR group (n=30) and non-CR group (n=71) according to the adenosine diphosphate (ADP)-induced platelet inhibition rate in thromboelastography (TEG) (ADP-induced platelet inhibition rate of <30% was defined as CR and rate of 30%-100% was defined as non-CR). Genotypes, including CYP2C19*2, CYP2C19*3, CYP2C19*4, CYP2C19*5, CYP2C19*17, and ABCB1, were determined using time-of-flight mass spectrometry (Clin-TOF) and Sanger sequencing in all patients. RESULTS: In the experimental group, carriers of CYP2C19 heterozygous (*1/*2, n=46; *1/*3, n=7), and mutation homozygous (*2/*2, n=7; *2/*3, n=3; *3/*3, n=0) genotypes showed significantly lower ADP-induced platelet inhibition rates than noncarriers (*1/*1, n=38; p=0.035 and 0.001, respectively); the carriage of mutant CYP2C19*2 or *3 allele was significantly associated with an increased risk of CR. In contrast, carriers of ABCB1 heterozygous (TC, n=50) showed significantly lower ADP-induced platelet inhibition rates than noncarriers (CC, n=39, p=0.097), and there was no significant correlation between ABCB1 genotypes and higher CR risk. CONCLUSION: The carriage of CYP2C19*2 or *3 mutant allele was significantly associated with attenuated platelet response to clopidogrel and increased CR risk. The carriage of ABCB1 mutant allele was not significantly associated with CR risk.

Shp2 regulates migratory behavior and response to EGFR-TKIs through ERK1/2 pathway activation in non-small cell lung cancer cells.

In the clinical treatment of lung cancer, therapy failure is mainly caused by cancer metastasis and drug resistance. Here, we investigated whether the tyrosine phosphatase Shp2 is involved in the development of metastasis and drug resistance in non-small cell lung cancer (NSCLC). Shp2 was overexpressed in a subset of lung cancer tissues, and Shp2 knockdown in lung cancer cells inhibited cell proliferation and migration, downregulated c-Myc and fibronectin expression, and upregulated E-cadherin expression. In H1975 cells, which carry double mutations (L858R + T790M) in epidermal growth factor receptor (EGFR) that confers resistance toward the tyrosine kinase inhibitor gefitinib, Shp2 knockdown increased cellular sensitivity to gefitinib; conversely, in H292 cells, which express wild-type EGFR and are sensitive to gefitinib, Shp2 overexpression increased cellular resistance to gefitinib. Moreover, by overexpressing Shp2 or using U0126, a small-molecule inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2), we demonstrated that Shp2 inhibited E-cadherin expression and enhanced the expression of fibronectin and c-Myc through activation of the ERK1/2 pathway. Our findings reveal that Shp2 is overexpressed in clinical samples of NSCLC and that Shp2 knockdown reduces the proliferation and migration of lung cancer cells, and further suggest that co-inhibition of EGFR and Shp2 is an effective approach for overcoming EGFR T790M mutation acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Thus, we propose that Shp2 could serve as a new biomarker in the treatment of NSCLC.