Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [<1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None

Characterization of novel low passage primary and metastatic colorectal cancer cell lines

MTG2 - Moleculaire genetica van gastrointestinale tumorenMolecular tumour pathology - and tumour genetic

Characterization of novel low passage primary and metastatic colorectal cancer cell lines

10.18632/oncotarget.7391Oncotarget71214499-1450

ELOVL5 immunohistochemical staining results.

<p>ELOVL5 immunohistochemical staining results.</p

Methylation associated transcriptional repression of <i>ELOVL5</i> in novel colorectal cancer cell lines

<div><p>Genetic and epigenetic alterations mark colorectal cancer (CRC). Global hypomethylation is observed in nearly all CRC, but a distinct subset of CRC show the CpG Island Methylator Phenotype (CIMP). These tumors show DNA hypermethylation of a specific subset of CpG islands, resulting in transcriptional downregulation of nearby genes. Recently we reported the establishment of novel CRC cell lines derived from primary and metastatic CRC tissues. In this study we describe the DNA methylation profiling of these low passage CRC cell lines. We generated global DNA methylation profiles with Infinium HumanMethylation450 BeadChips and analysed them in conjunction with matching gene expression profiles. Multidimensional scaling of the DNA methylation and gene expression datasets showed that <i>BRAF</i> mutated cell lines form a distinct group. In this group we investigated the 706 loci which we have previously identified to be hypermethylated in <i>BRAF</i> mutant CRC. We validated the significant findings in the The Cancer Genome Atlas colon adenocarcinoma dataset. Our analysis identified <i>ELOVL5</i>, <i>FAM127B</i>, <i>MTERF1</i>, <i>ZNF606</i> to be subject to transcriptional downregulation through DNA hypermethylation in CRC. We further investigated <i>ELOVL5</i> with qPCR and immunohistochemical staining, validating our results, but did not find a clear relation between ELOVL5 expression and tumor stage or relapse free survival. <i>ELOVL5</i>, <i>FAM127B</i>, <i>MTERF1</i>, <i>ZNF606</i> are involved in important cellular processes such as apoptosis, lipogenesis and the downstream transcriptional effect of the MAPK-pathway. We have identified a DNA methylation profile regulating key cellular processes in CRC, resulting in a growth advantage to the tumor cells.</p></div

Clustering of gene expression and DNA methylation data.

<p>To minimize overlap between cell line names, only the numbers corresponding to the cell lines were displayed. MDS analysis of DNA methylation data shows a distinct cluster of <i>BRAF</i> mutant cell lines; cluster C. Secondary clustering is formed by <i>KRAS</i> mutant samples with low <i>ZNF304</i> expression (cluster B, Figure 2A). MDS analysis of gene expression data showed <i>BRAF</i> mutant samples cluster separately from <i>KRAS</i> mutant and WT samples (2B). The cell lines in cluster B show a 5.9-fold lower <i>ZNF304</i> expression compared to the cell lines in cluster A (2C).</p

Genes showing a negative correlation between gene expression and DNA methylation.

<p>Average β-values are plotted on the x-axis, log2 gene expression values on the y-axis. Dots represent a cell line; <i>BRAF</i> mutant cell lines are marked with an x.</p

Genes showing correlation between expression and methylation in the TCGA data.

<p>RSEM gene expression values were plotted against the average β-values of the methylation loci.</p

Regulation and targeting of CIMP profiles by MAFG and ZNF304.

<p>Mutant KRAS upregulates of deubiquitinase USP28, leading to ZNF304 upregulation. ZNF304 recruits a corepressor complex which subsequently methylates the nearby CpGI. 1B: MAFG is upregulated and phosphorylated through MAPK-signalling which when bound to it’s binding site recruits a corepressor complex resulting in DNA hypermethylation (1B). Overview of DNA methylation outcomes for genes with and without MAFG and ZNF304 binding sites in CIMP0, CIMP1 and CIMP2 tumors (1C). bs = binding site, TSS = Transcription Start Site, CpGI = CpG Island.</p

<i>CD1D</i> expression analyses in patient samples.

<p>qRT-PCR expression analysis of <i>CD1D</i> in 184 paired carcinoma and normal samples. A strong transcriptional downregulation was observed in the carcinoma compared to the normal samples. Gene expression values are relative to the median of the normal samples (4A). Comparison of <i>CD1D</i> expression in 34 polyp samples compared to the 184 normal and carcinoma samples. <i>CD1D</i> is also downregulated in the polyps, but downregulation in polyps is not as strong as in the carcinoma. Gene expression values are relative to the median of the normal samples. The box represent the 25-75-quartiles, the whiskers represent the 5–95% percentiles (4B). Comparison of the <i>CD1D</i> expression normal/carcinoma ratio in methylated versus unmethylated carcinoma. <i>CD1D</i> hypermethylation does not affect the degree of transcriptional downregulation. The bars represent the mean with 95% confidence interval (4C).</p