Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Stroke subtype-dependent synapse elimination by reactive gliosis in mice.

The pathological role of reactive gliosis in CNS repair remains controversial. In this study, using murine ischemic and hemorrhagic stroke models, we demonstrated that microglia/macrophages and astrocytes are differentially involved in engulfing synapses in the reactive gliosis region. By specifically deleting MEGF10 and MERTK phagocytic receptors, we determined that inhibiting phagocytosis of microglia/macrophages or astrocytes in ischemic stroke improved neurobehavioral outcomes and attenuated brain damage. In hemorrhagic stroke, inhibiting phagocytosis of microglia/macrophages but not astrocytes improved neurobehavioral outcomes. Single-cell RNA sequencing revealed that phagocytosis related biological processes and pathways were downregulated in astrocytes of the hemorrhagic brain compared to the ischemic brain. Together, these findings suggest that reactive microgliosis and astrogliosis play individual roles in mediating synapse engulfment in pathologically distinct murine stroke models and preventing this process could rescue synapse loss.

Human glioblastoma arises from subventricular zone cells with low-level driver mutations.

Glioblastoma (GBM) is a devastating and incurable brain tumour, with a median overall survival of fifteen months1,2. Identifying the cell of origin that harbours mutations that drive GBM could provide a fundamental basis for understanding disease progression and developing new treatments. Given that the accumulation of somatic mutations has been implicated in gliomagenesis, studies have suggested that neural stem cells (NSCs), with their self-renewal and proliferative capacities, in the subventricular zone (SVZ) of the adult human brain may be the cells from which GBM originates3-5. However, there is a lack of direct genetic evidence from human patients with GBM4,6-10. Here we describe direct molecular genetic evidence from patient brain tissue and genome-edited mouse models that show astrocyte-like NSCs in the SVZ to be the cell of origin that contains the driver mutations of human GBM. First, we performed deep sequencing of triple-matched tissues, consisting of (i) normal SVZ tissue away from the tumour mass, (ii) tumour tissue, and (iii) normal cortical tissue (or blood), from 28 patients with isocitrate dehydrogenase (IDH) wild-type GBM or other types of brain tumour. We found that normal SVZ tissue away from the tumour in 56.3% of patients with wild-type IDH GBM contained low-level GBM driver mutations (down to approximately 1% of the mutational burden) that were observed at high levels in their matching tumours. Moreover, by single-cell sequencing and laser microdissection analysis of patient brain tissue and genome editing of a mouse model, we found that astrocyte-like NSCs that carry driver mutations migrate from the SVZ and lead to the development of high-grade malignant gliomas in distant brain regions. Together, our results show that NSCs in human SVZ tissue are the cells of origin that contain the driver mutations of GBM.

Stereotactic ablative body radiotherapy for spinal metastasis from hepatocellular carcinoma: its oncologic outcomes and risk of vertebral compression fracture.

Spinal metastases from hepatocellular carcinoma (HCC) require high-dose irradiation for durable pain and tumor control. Stereotactic ablative body radiotherapy (SABR) enables the delivery of high-dose radiation. However, but vertebral compression fracture (VCF) can be problematic. The aim of his study is to evaluate the outcome and risk of VCF after SABR for spinal metastasis from HCC. We retrospectively reviewed 33 lesions in 42 spinal segments from 29 patients who received SABR with 1 fraction (16-20 Gy), or 3 fractions (18-45 Gy) from September 2009 to January 2015. The 1-year local control (LC) rate was 68.3%. Radiographic grade of cord compression (RGCC) was the only independent prognostic factor associated with LC (P = 0.007). The 1-year ultimate LC rate including the outcome of salvage re-irradiation was 87.2%. The pain response rate was 73.3% according to the categories of the International Bone Metastases Consensus Group. The 1-year VCF-free rate was 71.5%. Pre-existing VCF (P < 0.001) and only-lytic change (P = 0.017) were associated with a higher post-SABR VCF rate. One-third of post-SABR VCFs required interventions. SABR for spinal metastases from HCC provided efficacious LC, especially for lesions with RGCC ≤ II, and showed effective and durable pain relief. As VCF after SABR occurred frequently for vertebral segments with pre-existing VCF and only-lytic change, early preventive vertebroplasty is considerable for those high-risk vertebral segments.

Scheduled interval trans-catheter arterial chemoembolization followed by radiation therapy in patients with unresectable hepatocellular carcinoma.

Combination treatment of trans-catheter arterial chemoembolization (TACE) and conformal radiation therapy (RT) reported promising results in patients with hepatocellular carcinoma (HCC), but, optimal interval was not determined. We hypothesized that a two-week interval between TACE and RT would be optimal. Therefore, we designed this study to evaluate the safety and efficacy of scheduled interval TACE followed by RT. HCC patients who were not eligible for standard therapies were enrolled for scheduled interval TACE followed by RT (START). Patients received TACE on the first day of treatment, and then RT was delivered after 14 days. The entire course of treatment took between four and five weeks. In 81 patients (96.4%), START was completed in the planned treatment period. RT was delayed in the remaining three patients because of decreased liver function or poor performance status after TACE. Of the 81 patients, objective response was observed in 57 patients (70.4%). One unexpected death occurred after START due to hepatic failure. Other toxicities were manageable. The median survival was 14.7 months. There was a significant difference in overall survival according to the response to START (P < 0.001). In conclusion, START is safe and feasible.

Analysis of prognostic factors and 5-year survival rate in patients with hepatocellular carcinoma: a single-center experience.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC), which is the third most common cancer in Korea, has a very poor prognosis. However, only a few studies have performed a comprehensive survival-related analysis in all patients who were consecutively diagnosed and treated over a given period of time. The aim of this study was to determine the 5-year survival rate and its prognostic factors among HCC patients. METHODS: In total, 257 patients who were consecutively diagnosed with HCC between January 2000 and December 2003 were followed until death or until December 2008. We analyzed their survival outcomes according to their clinical characteristics, tumor staging, and treatment modalities, and determined the independent prognostic factors affecting survival. RESULTS: The patients were aged 59±10 years (mean±SD). During the follow-up period, 223 patients (86.8%) died and the overall median survival was 10.8 months; the 1-, 3-, and 5-year survival rates were 44.4%, 21.0%, and 12.1%, respectively. The outcomes in patients with tumor node metastasis (TNM) stage I or II and Child-Pugh class A or B were significantly better with surgical resection than with other treatment modalities (P<0.01). Patients who underwent supplementary transcatheter arterial chemoembolization as a second-line treatment after surgical resection had better outcomes than those who underwent surgical resection alone (P=0.02). Initial symptoms, Child-Pugh class, serum alpha-fetoprotein, tumor size, portal vein thrombosis, and TNM stage were found to be independent prognostic factors for survival among HCC patients. CONCLUSIONS: This retrospective cohort study elucidated survival outcomes and prognostic factors affecting survival in HCC patients at a single center.

Prognostic index for portal vein tumor thrombosis in patients with hepatocellular carcinoma treated with radiation therapy.

We performed a retrospective review of 281 hepatocellular carcinoma (HCC) patients with portal vein tumor thrombosis (PVTT) treated with radiation therapy (RT) between 1998 and 2008 to develop a prognostic model for those patients. Of the 281 patients, PVTT and intrahepatic main masses completely disappeared in 10 patients (3.6%), and shown a partial response in 141 patients (50.2%). The median survival was 11.6 months. Patients who had more than PR have shown significantly longer survival than the others (22.0 months vs 5.0 months, P < 0.001). On the multivariate analysis, pre-treatment poor prognosticators for overall survival were ECOG performance status, Child-Pugh class, multiple tumors, main PVTT, complete portal vein occlusion, lymph node metastasis, and primary tumor size. Prognostic index of RT for PVTT of HCC (PITH) scores were defined as the number of pre-treatment poor prognostic factors. PITH scores correlated well with overall survival. In the analysis of 1 and 2 yr overall survival rate, patients who had PITH scores of 3 or greater showed a significantly lower rate of overall survival than the others (33.0%, 17.3% vs 70.1%, 40.8%, respectively, P < 0.001). The PITH scoring model, proposed in the current study in HCC patients with PVTT, reliably predict overall survival.

[A case of malignant gastrointestinal stromal tumor of ileum with liver abscess].

Gastrointestinal stromal tumor (GIST) is an uncommon mesenchymal tumor of the gastrointestinal tract and is generally located in the stomach and small intestine. They usually present with abdominal pain, gastrointestinal bleeding, and palpable mass. Some patients present with rare symptoms that are more common in malignant GIST. Malignant GIST combined with a liver abscess has not been reported yet in the literatures. We report a case of 67-year-old woman who suffered from liver abscess combined by malignant GIST with central necrosis and fistula in the ileum. She complained of fever, chills, and abdominal pain. Abdominal CT scan showed huge liver abscess and ileal mass with air pocket. Small bowel series showed contrast material filling into the ileal GIST mass. An operation was performed and the final diagnosis was malignant GIST of the ileum with invasion into the sigmoid colon and urinary bladder.