Project description:IntroductionLeptomeningeal metastasis (LM) commonly occurs in non-small cell lung cancer (NSCLC) patients and has a poor prognosis. Due to limited access to leptomeningeal lesions, the genetic characteristics of LM have not been explored to date. Cerebrospinal fluid (CSF) may be the most representative liquid biopsy medium to obtain genomic information from LM in NSCLC.MethodsCSF biopsies and matched peripheral blood biopsies were collected from 33 NSCLC patients with LM. We profiled genetic alterations from LM by comparing CSF cell-free DNA (cfDNA) with plasma cfDNA. Somatic mutations were examined using targeted sequencing. Genomic instability was analyzed by low-coverage whole-genome sequencing (WGS).ResultsDriver mutations were detected in 100% of CSF cfDNA with much higher variant allele frequency than that in matched plasma cfDNA (57.5%). Furthermore, we found that the proportions of CSF cfDNA fragments below 150 bp were significantly higher than those in plasma cfDNA. These findings indicate enrichment of circulating tumor DNA (ctDNA) in CSF and explain the high sensitivity of mutation detection in the CSF. The absence of some mutations in CSF cfDNA-especially the first-/second-generation mutation T790M, which confers resistance to epidermal growth factor receptor (EGFR)-Tyrosine kinase inhibitors (TKIs)-that were present in plasma cfDNA samples indicates different mechanisms of cancer evolution between LM and extracranial lesions. In addition, 86.6% of CSF ctDNA samples revealed high levels of genomic instability compared with 2.5% in plasma cfDNA samples. A higher number of large-scale state transitions (LSTs) in CSF cfDNA were associated with a shorter overall survival (OS).ConclusionOur results suggest that LM and extracranial lesions develop independently. Both CSF cfDNA genetic profiling and plasma cfDNA genetic profiling are necessary for clinical decision-making for NSCLC patients with LM. Through CSF-based low-coverage WGS, a high level of LSTs was identified as a potential biomarker of poor prognosis.

Project description:Pancreatic cancer is one of the most aggressive cancers worldwide with a high mortality rate. Prognosis remains poor even in this era of advanced medicine mainly due to early metastasis and invasion. The present study aimed to explore and validate predictors of distant metastasis and prognosis in pancreatic cancer. In our preliminary experiment, we established a novel metastatic pancreatic cancer cell line BxPC‑M8 from parent BxPC‑3 cells. Via whole genome sequencing, RT‑qPCR, western blotting, migration and invasion assays, we initially found that BxPC‑M8 shared similar biological characteristics to BxPC‑3, but only differed in enhanced metastatic and invasive capabilities with a significant increase in collagen type VI α1 chain (COL6A1) expression. Knockdown of COL6A1 via small interfering RNA led to a significant decrease in migration and invasion of BxPC‑M8 cells, suggesting suppressed epithelial‑mesenchymal transition. Furthermore, a significant increase in COL6A1 expression was observed in cancerous tissue compared with paracancerous tissue (40.7 vs 3.7, P=0.001). Additionally, its expression was observed to be significantly associated with distant metastasis and vascular invasion at the time of surgery. Multivariate analysis revealed that COL6A1 expression (hazard ratio 1.90, 95% confidence interval 1.04‑3.47, P=0.037) is an independent predictor of overall survival (OS). The median OS observed for COL6A1+ and COL6A1‑ patients was found to be 8±4 and 14±7 months (P=0.021), respectively. Of note, we identified that COL6A1 expression in tissue samples was associated with significantly reduced OS (P=0.001), demonstrating that COL6A1 may serve an important role in the metastatic process and could be considered as a predictor of poor outcomes in patients with pancreatic cancer. In addition, our findings suggest that COL6A1 could be an indicator of distant metastasis and a valid prognostic predictor in such patients; however, further investigation is required.

Project description:Background - Hepatocellular carcinomas (HCCs) are heterogeneous tumors with respect to etiology, cell of origin and biology. The course of the disease is unpredictable and is in part dependent on the tumor microenvironment. One of the microenvironmental factors is hypoxia, which is known to promote aggressiveness in other malignant tumors. We hypothesized that certain regions in HCC exist with chronic hypoxia and a characteristic gene expression pattern. Moreover, during the development of HCC there is an important contribution of this chronic hypoxia on prognosis via this gene expression. Until now, most research has been performed in acute hypoxic models (< 24 hours). Methods – Human hepatoblastoma cells HepG2 were cultured in either normoxic (20% O2) or hypoxic (2% O2) conditions for 72 hrs, the time it takes to adapt to chronic hypoxia. After 3 days the cells were harvested and analyzed by microarray technology. The highly significant differentially expressed genes were selected and used to assess the clinical value of our in vitro chronic hypoxia gene signature in four published patient studies. Three of these independent microarray studies on HCC patients were used as training sets to determine a minimal prognostic gene set and one study was used for validation. Gene expression analysis and correlation with clinical outcome was assessed with the bioinfomatic method of Goeman et al (). Results – In the HepG2 cells, 3592 genes were differentially expressed in cells cultured at 2% oxygen for 72 hrs. Out of these, 265 showed a high significant change (2-fold change and p=0.0001). The level of gene expression after 72 hrs was different from the acute hypoxic response (during the first 24 hours) and represented chronicity. Using computational methods we identified 7 out of the 265 highly significant genes that showed correlation with prognosis in all three different training sets and this was independently validated in a 4th dataset. With our approach we could include the largest number of HCC patients in one single study. Conclusion – We identified a 7-gene signature, which is associated with chronic hypoxia and predicts prognosis in patients with HCC. In the future this signature could be used as a diagnostic tool. In addition, chronic hypoxia gene expression information can be used in the search for new therapeutic targets. Two conditions were compared and each sample has a biological replicate. Samples are hybridized in dye-swap, resulting in 4 hybridizations.

Project description:Background - Hepatocellular carcinomas (HCCs) are heterogeneous tumors with respect to etiology, cell of origin and biology. The course of the disease is unpredictable and is in part dependent on the tumor microenvironment. One of the microenvironmental factors is hypoxia, which is known to promote aggressiveness in other malignant tumors. We hypothesized that certain regions in HCC exist with chronic hypoxia and a characteristic gene expression pattern. Moreover, during the development of HCC there is an important contribution of this chronic hypoxia on prognosis via this gene expression. Until now, most research has been performed in acute hypoxic models (< 24 hours). Methods – Human hepatoblastoma cells HepG2 were cultured in either normoxic (20% O2) or hypoxic (2% O2) conditions for 72 hrs, the time it takes to adapt to chronic hypoxia. After 3 days the cells were harvested and analyzed by microarray technology. The highly significant differentially expressed genes were selected and used to assess the clinical value of our in vitro chronic hypoxia gene signature in four published patient studies. Three of these independent microarray studies on HCC patients were used as training sets to determine a minimal prognostic gene set and one study was used for validation. Gene expression analysis and correlation with clinical outcome was assessed with the bioinfomatic method of Goeman et al (). Results – In the HepG2 cells, 3592 genes were differentially expressed in cells cultured at 2% oxygen for 72 hrs. Out of these, 265 showed a high significant change (2-fold change and p=0.0001). The level of gene expression after 72 hrs was different from the acute hypoxic response (during the first 24 hours) and represented chronicity. Using computational methods we identified 7 out of the 265 highly significant genes that showed correlation with prognosis in all three different training sets and this was independently validated in a 4th dataset. With our approach we could include the largest number of HCC patients in one single study. Conclusion – We identified a 7-gene signature, which is associated with chronic hypoxia and predicts prognosis in patients with HCC. In the future this signature could be used as a diagnostic tool. In addition, chronic hypoxia gene expression information can be used in the search for new therapeutic targets.

Project description:ObjectiveThe objective of this research is to investigate the clinical application value of cerebrospinal fluid (CSF) cytology and circulating tumor DNA (ctDNA) in lung adenocarcinoma (LUAD) meningeal metastasis-meningeal carcinomatosis (MC), and to further explore the possible molecular mechanisms and drug treatment targets of LUAD meningeal metastasis by next-generation sequencing (NGS).MethodsWe retrospectively analyzed LUAD with MC in 52 patients. CSF cytology was carried out using the slide centrifugation precipitation method and May-Grüwald-Giemsa (MGG) staining. Tumor tissue, plasma and CSF ctDNA of some MC patients were detected by NGS.ResultsOf the 52 MC patients, 46 (88.46%) were positive for CSF cytology and 34 (65.38%) were positive for imaging, with statistically significant differences in diagnostic positivity (P < 0.05). In 32 of these patients, CSF cytology, cerebrospinal fluid ctDNA, plasma ctDNA and MRI examination were performed simultaneously, and the positive rates were 84.38, 100, 56.25, and 62.50% respectively, the difference was statistically significant (P < 0.001). Analysis of the NGS profiles of tumor tissues, plasma and CSF of 12 MC patients: the mutated gene with the highest detection rate was epidermal growth factor receptor (EGFR) and the detection rate were 100, 58.33, and 100% respectively in tumor tissues, plasma and CSF, and there were 6 cases of concordance between plasma and tissue EGFR mutation sites, with a concordance rate of 50.00%, and 12 cases of concordance between CSF and tissue EGFR mutation sites, with a concordance rate of 100%. In addition, mutations not found in tissue or plasma were detected in CSF: FH mutation, SETD2 mutation, WT1 mutation, CDKN2A mutation, CDKN2B mutation, and multiple copy number variants (CNV), with the most detected being CDKN2A mutation and MET amplification.ConclusionCSF cytology is more sensitive than traditional imaging in the diagnosis of meningeal carcinomatosis and has significant advantages in the early screening and diagnosis of MC patients. CSF ctDNA can be used as a complementary diagnostic method to negative results of CSF cytology and MRI, and CSF ctDNA can be used as an important method for liquid biopsy of patients with MC, which has important clinical significance in revealing the possible molecular mechanisms and drug treatment targets of meningeal metastasis of LUAD.

Dataset Information

Genomic Instability in Cerebrospinal Fluid Cell-Free DNA Predicts Poor Prognosis in Solid Tumor Patients with Meningeal Metastasis.

Publications

Genomic Instability in Cerebrospinal Fluid Cell-Free DNA Predicts Poor Prognosis in Solid Tumor Patients with Meningeal Metastasis.

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