Unintrusive multi-cancer detection by circulating cell-free DNA methylation sequencing (THUNDER): development and independent validation studies.

BACKGROUND: Early detection of cancer offers the opportunity to identify candidates when curative treatments are achievable. The THUNDER study (THe UNintrusive Detection of EaRly-stage cancers, NCT04820868) aimed to evaluate the performance of enhanced linear-splinter amplification sequencing, a previously described cell-free DNA (cfDNA) methylation-based technology, in the early detection and localization of six types of cancers in the colorectum, esophagus, liver, lung, ovary, and pancreas. PATIENTS AND METHODS: A customized panel of 161 984 CpG sites was constructed and validated by public and in-house (cancer: n = 249; non-cancer: n = 288) methylome data, respectively. The cfDNA samples from 1693 participants (cancer: n = 735; non-cancer: n = 958) were retrospectively collected to train and validate two multi-cancer detection blood test (MCDBT-1/2) models for different clinical scenarios. The models were validated on a prospective and independent cohort of age-matched 1010 participants (cancer: n = 505; non-cancer: n = 505). Simulation using the cancer incidence in China was applied to infer stage shift and survival benefits to demonstrate the potential utility of the models in the real world. RESULTS: MCDBT-1 yielded a sensitivity of 69.1% (64.8%-73.3%), a specificity of 98.9% (97.6%-99.7%), and tissue origin accuracy of 83.2% (78.7%-87.1%) in the independent validation set. For early-stage (I-III) patients, the sensitivity of MCDBT-1 was 59.8% (54.4%-65.0%). In the real-world simulation, MCDBT-1 achieved a sensitivity of 70.6% in detecting the six cancers, thus decreasing late-stage incidence by 38.7%-46.4%, and increasing 5-year survival rate by 33.1%-40.4%, respectively. In parallel, MCDBT-2 was generated at a slightly low specificity of 95.1% (92.8%-96.9%) but a higher sensitivity of 75.1% (71.9%-79.8%) than MCDBT-1 for populations at relatively high risk of cancers, and also had ideal performance. CONCLUSION: In this large-scale clinical validation study, MCDBT-1/2 models showed high sensitivity, specificity, and accuracy of predicted origin in detecting six types of cancers.

Long non-coding RNA CCAT2 promotes the breast cancer growth and metastasis by regulating TGF-ß signaling pathway.

OBJECTIVE: Long non-coding RNA (LncRNA) CCAT2 plays an important role in tumorigenesis, tumor growth and metastasis. In this study, we reported that long noncoding RNA CCAT2 (LncRNA CCAT2) could regulate TGF-ß signaling pathway in breast cancer. PATIENTS AND METHODS: The relative mRNA expression level of CCAT2 in adjacent non-cancerous and breast cancer tissues without or with metastasis were analyzed by quantitative Real-time polymerase chain reaction (qRT-PCR). The mRNA expression levels of CCAT2 in breast cancer cell lines were analyzed by qRT-PCR. Proliferation, invasion and migration of breast cancer cells were detected after infected with si-NC or si-CCAT2. Flow cytometry analysis was used to detect the cell cycle distribution and apoptosis rate in breast cancer cells after infected with si-NC or si-CCAT2. The relative protein expression level of TGF-ß, Smad2 and α-SMA in breast cancer cells after infected with si-NC or si-CCAT2 were analyzed by Western blot. RESULTS: The relative mRNA expression level of CCAT2 in breast cancer tissues was significantly increased compared with adjacent non-cancerous tissues. The expression level of CCAT2 in breast cancer without metastasis was decreased compared with breast cancer metastasis. Meanwhile, down-regulation of CCAT2 inhibited the proliferation, invasion and migration in breast cancer cells. Furthermore, down-regulation of CCAT2 caused breast cancer cells cycle arrested in G0/G1 phase and promoted cell apoptosis. Down-regulation of CCAT2 significantly down-regulated the protein expression levels of TGF-ß, Smad2 and α-SMA in breast cancer cells. CONCLUSIONS: CCAT2 was highly expressed in breast cancer. Down-regulation of CCAT2 inhibited the proliferation, invasion and migration and promoted cell apoptosis in breast cancer cells by regulating TGF-ß signaling pathway.

Long non-coding RNA LINC00628 suppresses the growth and metastasis and promotes cell apoptosis in breast cancer.

OBJECTIVE: Breast cancer is the most common malignant tumor in women. However, the detailed mechanisms of its tumorigenesis remain largely unknown. Evidence and data have shown that abnormality in expression of Long non-coding RNA (LncRNA) is closely related to tumorigenesis. The aim of this study is to identify the detailed mechanisms of LncRNA LINC00628 in breast cancer. PATIENTS AND METHODS: The expression of LINC00628 in breast cancer tissues, adjacent non-cancerous tissues and cell lines were detected by qRT-PCR. Kaplan-Meier method and log-rank test were applied to analyze the overall survival of patients with low and high expression level of LINC00628 respectively. The LCC2 and MCF-7 cells were transfected with LINC00628 and the proliferation, invasion and migration were examined. The cell cycle distribution and cell apoptosis rate in LCC2 and MCF-7 cells after transfection with LINC00628 were explored by flow cytometry. The relative expression level of Bcl-2, Bax and Caspase-3 protein in LCC2 and MCF-7 cells after transfection with LINC00628 was detected by Western blotting. RESULTS: The relative expression level of LINC00628 in breast cancer tissues and cell lines were significantly decreased and the low expression level of LINC00628 has a poorer prognosis and lower overall survival rate. The overexpression of LINC00628 suppressed breast cancer cells proliferation, invasion and migration. Further, with the overexpression of LINC00628, cell cycle was arrested in G0/G1 phase in breast cancer cells and cell apoptosis was promoted. The relative expression of Caspase-3 and Bax protein were significantly increased and the relative expression of Bcl-2 protein was significantly decreased after transfection with LINC00628. CONCLUSIONS: The expression of LINC00628 was decreased in breast cancer. The overexpression of LINC00628 suppressed the proliferation, invasion and migration of breast cancer cells and promoted cell apoptosis associated with the regulation of Bcl-2/Bax/Caspase-3 signal pathway.