Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection.

Genomic characterization of circulating tumor cells (CTCs) may prove useful as a surrogate for conventional tissue biopsies. This is particularly important as studies have shown different mutational profiles between CTCs and ctDNA in some tumor subtypes. However, isolating rare CTCs from whole blood has significant hurdles. Very limited DNA quantities often can't meet NGS requirements without whole genome amplification (WGA). Moreover, white blood cells (WBC) germline contamination may confound CTC somatic mutation analyses. Thus, a good CTC enrichment platform with an efficient WGA and NGS workflow are needed. Here, Vortex label-free CTC enrichment platform was used to capture CTCs. DNA extraction was optimized, WGA evaluated and targeted NGS tested. We used metastatic colorectal cancer (CRC) as the clinical target, HCT116 as the corresponding cell line, GenomePlex® and REPLI-g as the WGA methods, GeneRead DNAseq Human CRC Panel as the 38 gene panel. The workflow was further validated on metastatic CRC patient samples, assaying both tumor and CTCs. WBCs from the same patients were included to eliminate germline contaminations. The described workflow performed well on samples with sufficient DNA, but showed bias for rare cells with limited DNA input. REPLI-g provided an unbiased amplification on fresh rare cells, enabling an accurate variant calling using the targeted NGS. Somatic variants were detected in patient CTCs and not found in age matched healthy donors. This demonstrates the feasibility of a simple workflow for clinically relevant monitoring of tumor genetics in real time and over the course of a patient's therapy using CTCs.

An RNAi screen identifies TRRAP as a regulator of brain tumor-initiating cell differentiation.

Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. In particular, knockdown of the adaptor protein TRRAP significantly increased differentiation of cultured BTICs, sensitized the cells to apoptotic stimuli, and negatively affected cell cycle progression. TRRAP knockdown also significantly suppressed tumor formation upon intracranial BTIC implantation into mice. Together, these findings support a critical role for TRRAP in maintaining a tumorigenic, stem cell-like state.

Neurological complications in eclampsia: a case series.

OBJECTIVES: Neurological abnormalities contribute significantly to maternal mortality in eclampsia. We studied the epidemiology and neurological and obstetric outcome of such patients. METHODS: A retrospective analysis was done at a referral center. 19 cases of eclampsia with recurrent convulsions (n = 8) or coma without convulsions (n = 5) or cerebrovascular accidents (n = 4) or blindness (n = 2) were studied. We excluded cases with primary neurological abnormalities. Management included initial stabilization followed by early delivery. Primary anticonvulsant was magnesium sulphate. RESULTS: The incidence of eclampsia was 0.71%. Among 61 cases, 19 (31.14%) had neurological abnormalities; 15 patients had no antenatal care. Three cases were postpartum. Comatose patients had the highest mean arterial pressure (MAP) (mean 154.66 mm Hg, p = 0.027). Fundoscopy was usually normal. Computerized tomography revealed mild cerebral edema in six cases and accurately diagnosed all cerebrovascular accidents. Phenytoin controlled convulsions in 7/8 cases with recurrent seizures. The cesarean section rate was 37.5% and admission to delivery interval was 10.38 hours. Five perinatal and two maternal deaths were recorded among 19 cases. Neurological recovery was complete in all survivors. CONCLUSIONS: Critical care back-up is essential at tertiary referral centers for a large proportion of neurological abnormalities in eclampsia. High MAP and accompanying thrombocytopenia may be key factors in cerebral pathology. CT scan is a simple and effective investigation in these cases. Phenytoin is an effective second-line anticonvulsant. No maternal death was related directly to cesarean section. Early delivery prevents worsening of systemic status.