Opuntiol Inhibits Growth and Induces Apoptosis in Human Glioblastoma Cells by Upregulating Active Caspase 3 Expression.

BACKGROUND: Glioblastoma Multiforme (GBM) is a deadly tumor with poor prognosis. Resistance to apoptosis considered as an important factor in treatment failure. Therefore, identification of new compounds that facilitates apoptosis is crucial. Natural Anti-inflammatory compounds have emerged as potential anti-cancer agents and should be explored for their apoptotic activity against GBM. Therefore, the present study aims to evaluate growth inhibitory and apoptotic activity of a natural anti-inflammatory compound "Opuntiol" against GBM cell line U87. METHODS: MTT assay was performed to determine the effect of Temozolomide and Opuntiol on growth inhibition of U87 cell. While, TUNEL assay was used to assess their apoptotic activity. To further assess apoptosis, nuclear condensation and nuclear area factor (NAF) was evaluated through DAPI staining. Whereas, active caspase-3 protein expression determined using immunocytochemistry. RESULTS: Significant growth inhibition was observed in U87 cells treated with Temozolomide (IC50 380 µM) and Opuntiol (IC50 357 µM). Temozolomide (p<0.001) and Opuntiol (p<0.001) significantly improved rate of apoptosis when compared to control group. A significant decrease in NAF was also observed in Temozolomide (p < 0.05) and Opuntiol (p < 0.05) treated cells. There was a significant increase in active caspase-3 expression when observed in Temozolomide (p<0.001) and Opuntiol (p<0.05) treated groups as compared to control. CONCLUSION: In conclusion our findings suggests, Opuntiol repress cell viability and possess strong apoptotic activity against GBM cell line U-87. However, further mechanistic studies will be required to confirm whether it can be develop as a potential drug against GBM.

A Disintegrin and Metalloproteinase (ADAM) Family: Their Significance in Malignant Tumors of the Central Nervous System (CNS).

Despite the considerable advances in diagnostic methods in medicine, central nervous system (CNS) tumors, particularly the most common ones-gliomas-remain incurable, with similar incidence rates and mortality. A growing body of literature has revealed that degradation of the extracellular matrix by matrix metalloproteinases (MMPs) might be involved in the pathogenesis of CNS tumors. However, the subfamily of MMPs, known as disintegrin and metalloproteinase (ADAM) proteins are unique due to both adhesive and proteolytic activities. The objective of our review is to present the role of ADAMs in CNS tumors, particularly their involvement in the development of malignant gliomas. Moreover, we focus on the diagnostic and prognostic significance of selected ADAMs in patients with these neoplasms. It has been proven that ADAM12, ADAMTS4 and 5 are implicated in the proliferation and invasion of glioma cells. In addition, ADAM8 and ADAM19 are correlated with the invasive activity of glioma cells and unfavorable survival, while ADAM9, -10 and -17 are associated with tumor grade and histological type of gliomas and can be used as prognostic factors. In conclusion, several ADAMs might serve as potential diagnostic and prognostic biomarkers as well as therapeutic targets for malignant CNS tumors. However, future research on ADAMs biology should be performed to elucidate new strategies for tumor diagnosis and treatment of patients with these malignancies.

Clinical outcomes of newly diagnosed primary CNS lymphoma treated with ibrutinib-based combination therapy: A real-world experience of off-label ibrutinib use.

Ibrutinib-based combination therapy with high-dose methotrexate (HD-MTX) has recently shown clinical activity against relapse/refractory (R/R) primary central nervous system lymphoma (PCNSL). Herein, we report our real-world experience of treating 11 newly diagnosed PCNSL patients with the ibrutinib/MTX combination. HD-MTX was given at 3.5 g/m2 every 2-week for eight doses. Ibrutinib was held upon HD-MTX infusion until clearance and was administered daily post-induction until disease progression, intolerable toxicity, or death. Nine out of 11 patients completed the induction phase and received ibrutinib as maintenance therapy. An objective response rate (ORR) of 82% (9/11) was observed including complete response (64%) and partial response (18%). The median progression-free survival (PFS) was 7.4 months while the median overall survival (OS) was not reached. The ibrutinib/MTX combination was well tolerated in these treatment-naïve PCNSL patients with an acceptable safety profile. Moreover, the longitudinal analysis of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) revealed that CSF ctDNA detection was closely associated with tumor response, and sustained tumor responses correlated with the clearance of ctDNA from the CSF. In sum, our data not only demonstrated the clinical benefit of the ibrutinib and HD-MTX combination regimen in treating newly diagnosed PCNSL patients in a real-world setting, but also highlighted the significance of liquid biopsy including CSF ctDNA in tracing tumor burden and assessing treatment response.

Dual MGMT inactivation by promoter hypermethylation and loss of the long arm of chromosome 10 in glioblastoma.

BACKGROUND: Epigenetic inactivation of O6-methylguanine-methyltransferase (MGMT) gene by methylation of its promoter is predictive of Temozolomid (TMZ) response in glioblastoma (GBM). MGMT is located on chromosome 10q26 and the loss of chromosome 10q is observed in 70% of GBMs. In this study, we assessed the hypothesis that the dual inactivation of MGMT, by hypermethylation of MGMT promoter and by loss the long arm of chromosome 10 (10q), may confer greater sensitivity to TMZ. METHODS: A total of 149 tumor samples from patients diagnosed with GBM based on the WHO 2016 classification were included in this retrospective study between November 2016 and December 2018. Methylation status of MGMT promoter was evaluated by pyrosequencing and status of chromosome 10q was assessed by array comparative genomic hybridization. RESULTS: Glioblastoma patients with chromosome 10q loss associated with hypermethylation of MGMT promoter had significantly longer overall survival (OS) (P = .0024) and progression-free survival (PFS) (P = .031). Indeed, median OS of patients with dual inactivation of MGMT was 21.5 months compared to 12 months and 8.1 months for groups with single MGMT inactivation by hypermethylation and by 10q loss, respectively. The group with no MGMT inactivation had 9.5 months OS. Moreover, all long-term survivors with persistent response to TMZ treatment (OS ≥ 30 months) displayed dual inactivation of MGMT. CONCLUSIONS: Our data suggest that the molecular subgroup characterized by the dual inactivation of MGMT receives greater benefit from TMZ treatment. The results of our study may be of immediate clinical interest since chromosome 10q status and methylation of MGMT promoter are commonly determined in routine practice.

Granulocyte-macrophage colony-stimulating factor enhances effect of temozolomide on high-grade glioma cells.

In the present study, to delve into the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with temozolomide (TMZ) on high-grade glioma cells and related mechanism, six cases of high-grade glioma cells from patient's tumor tissues were cultured. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay was performed to detect cell proliferation and toxicity. Flow cytometry was performed to ascertain cell cycle and apoptosis rate. To detect the expressions of O6-methylguanine-DNA methyltransferase (MGMT) methylation status and MGMT protein, respectively, specific PCR and immunofluorescence were performed. According to the results of MTT assay, compared with the results of control group, GM-CSF group exhibited enhanced cell viability in varying degrees. In three cases of cells (MGMT gene methylation), the combination group [(67.67 ± 1.16), (68.13 ± 1.06), (68.42 ± 1.73)] had noticeably lower cell viability than the corresponding TMZ group [(90.00 ± 1.73), (82.33 ± 1.53), (82.67 ± 2.11)] (P < 0.01). Nevertheless, the two groups showed no significant difference in another three cases (MGMT gene unmethylated) (P > 0.05). In combination group, the apoptosis rate of the MGMT methylation cells was higher than that in the corresponding TMZ group (P < 0.01), which is consistent with MTT assay results. In all six cases of primary glioma cells, the fraction of cells in G1 phase of GM-CSF-treated group was noticeably down-regulated and was up-regulated in S phase (P < 0.01). GM-CSF could induce high-grade glioma cells to rapidly enter the cell cycle, thereby enhancing the lethal effect of TMZ on glioma cells with MGMT gene promoter methylation. However, this effect is not ideal on glioma cells with MGMT unmethylation.

Study on the role and mechanism of ß4GalT1 both in vivo and in vitro glioma.

OBJECTIVE: To discuss the role and mechanism of ß4GalT1 both in vivo and in vitro glioma, observe whether pathophysiological processes of glioma can be improved after ß4GalT1 is knocked down, and study whether ß4GalT1 plays a role in malignant biological processes of glioma by regulating the apoptosis and immune processes. PATIENTS AND METHODS: Firstly, the distribution difference of ß4GalT1 in tumor tissues and normal tissues was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) tumor analysis system to deduce the possible role of ß4GalT1 in glioma. Secondly, whether the malignant degree of glioma was related to the expression of ß4GalT1 and its immunity using human tumor tissues and blood lymphocyte subsets was analyzed. Thirdly, interfere lentivirus vector with ß4GalT1 and knockdown ß4GalT1 was analyzed to observe whether the malignant degree of glioma has changed. Fourthly, interfere lentivirus vector with recombinant ß4GalT protein and ß4GalT1 was analyzed to verify the effect of ß4GalT in vitro test. Fifth, interfere lentivirus vector with recombinant ß4GalT protein and ß4GalT1 was analyzed to verify effect of ß4GalT in vivo test. Finally, we discuss whether ß4GalT is involved in the biological process of glioma through inflammatory reaction. RESULTS: In the GEPIA tumor analysis system, the expression in tumor was significantly higher than that in normal tissues. The expression of ß4GalT1 in glioma tissues was higher than that in normal tissues, and the higher the malignancy of the tumor, the higher the expression of ß4GalT1 in the glioma tissues, and the lower the immune level was. The expression of IDH1, MGMT, and ki-67 was reduced, and the survival rate of the mice with glioma was improved after ß4GalT1 was knocked down. In vitro tests, the activity of tumor cells and their reproductive ability can be reduced after ß4GalT1 was knocked down, the immune level of the body can be improved, and the level of tissue apoptosis can be reduced. After recombinant ß4GalT1 was given alone, the result was opposite to that of ß4GalT1 knocked down group. In vivo tests, gross tumor volume can be reduced after ß4GalT1 was knocked down, the immune level of the body can be improved, and the level of tissue apoptosis can be reduced. After recombinant ß4GalT1 was given alone, the result was opposite to that of ß4GalT1 knocked down group. After knocking down ß4GalT1, the expression of inflammatory factors can be reduced both in vivo and in vitro, and the inflammatory microenvironment of tumors can be improved. After recombinant ß4GalT1 was given alone, the result was opposite to that of ß4GalT1 knocked down group. CONCLUSIONS: The level of ß4GalT1 expression in tumor tissues was increased. The malignant degree of glioma is related to the expression of ß4GalT1 and its immunity. The level of tumor marker can be decreased, and the survival rate of glioma model mice can be increased after ß4GalT1 is knocked down. Apoptosis and immune injury caused by tumor can be improved and gross tumor volume can be deduced after ß4GalT1 is knocked down. During the development of glioma, ß4GalT1 may play a malignant biological role through inflammatory response.

The loss of succinate dehydrogenase B expression is frequently identified in hemangioblastoma of the central nervous system.

Succinate dehydrogenase (SDH) is a mitochondrial enzyme that plays an important role in both the Krebs cycle and the electron transport chain. SDH inactivation is associated with tumorigenesis in certain types of tumor. SDH consists of subunits A, B, C and D (SDHA, SDHB, SDHC, and SDHD, respectively). Immunohistochemistry for SDHB is a reliable method for detecting the inactivation of SDH by mutations in SDHA, SDHB, SDHC, SDHD and SDH complex assembly factor 2 (SDHAF2) genes with high sensitivity and specificity. SDHB immunohistochemistry has been used to examine the inactivation of SDH in various types of tumors. However, data on central nervous system (CNS) tumors are very limited. In the present study, we investigated the loss of SDHB immunoexpression in 90 cases of CNS tumors. Among the 90 cases of CNS tumors, only three cases of hemangioblastoma showed loss of SDHB immunoexpression. We further investigated SDHB immunoexpression in 35 cases of hemangioblastoma and found that 28 (80%) showed either negative or weak-diffuse pattern of SDHB immunoexpression, which suggests the inactivation of SDH. Our results suggest that SDH inactivation may represent an alternative pathway in the tumorigenesis of hemangioblastoma.

Prognostic relevance of mutations and copy number alterations assessed with targeted next generation sequencing in IDH mutant grade II glioma.

BACKGROUND: At current prognostication of low grade glioma remains suboptimal and might be improved with additional markers. These may guide treatment decisions, in particular on early adjuvant therapy versus wait and see after surgery. METHODS: We used a targeted Next-Generation Sequencing panel to assess mutational and copy number status of selected genes and chromosomes in a consecutive series of adult grade II supratentorial glioma, and assessed the impact of molecular markers of interest on overall survival. RESULTS: 207 IDH mutated grade II glioma samples were analyzed with a median follow-up of 6.9 years. Loss of region 9p21.3 did not show a correlation with outcome in IDH mutated 1p/19q-codeleted oligodendroglioma or IDH mutated astrocytoma. We found a significant shorter overall survival with univariable analysis in IDH mutated astrocytoma patients with trisomy of chromosome 7 (Log rank P = 0.044) and in IDH mutated 1p/19q-codeleted oligodendroglioma patients with a PTEN mutation (Log rank P = 0.033). We could not validate these findings in multivariate analysis or in the TCGA dataset. CONCLUSIONS: Loss of 9p21.3 is not associated with outcome in a molecularly defined cohort of grade II glioma and therefore it remains unclear if loss of 9p21.3 can be used as additional marker of anaplasia or to guide treatment decisions. Trisomy of chromosome 7 in IDH mutated astrocytoma and PTEN mutations in IDH mutated oligodendroglioma are potential markers of poor prognosis, but require confirmation in larger series.

Screening for ALK abnormalities in central nervous system metastases of non-small-cell lung cancer.

Anaplastic lymphoma kinase (ALK) gene rearrangement was reported in 3%-7% of primary non-small-cell lung cancer (NSCLC) and its presence is commonly associated with adenocarcinoma (AD) type and non-smoking history. ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, alectinib and ceritinib showed efficiency in patients with primary NSCLC harboring ALK gene rearrangement. Moreover, response to ALK TKIs was observed in central nervous system (CNS) metastatic lesions of NSCLC. However, there are no reports concerning the frequency of ALK rearrangement in CNS metastases. We assessed the frequency of ALK abnormalities in 145 formalin fixed paraffin embedded (FFPE) tissue samples from CNS metastases of NSCLC using immunohistochemical (IHC) automated staining (BenchMark GX, Ventana, USA) and fluorescence in situ hybridization (FISH) technique (Abbot Molecular, USA). The studied group was heterogeneous in terms of histopathology and smoking status. ALK abnormalities were detected in 4.8% (7/145) of CNS metastases. ALK abnormalities were observed in six AD (7.5%; 6/80) and in single patients with adenosuqamous lung carcinoma. Analysis of clinical and demographic factors indicated that expression of abnormal ALK was significantly more frequently observed (P = 0.0002; χ2 = 16.783) in former-smokers. Comparison of IHC and FISH results showed some discrepancies, which were caused by unspecific staining of macrophages and glial/nerve cells, which constitute the background of CNS tissues. Their results indicate high frequency of ALK gene rearrangement in CNS metastatic sites of NSCLC that are in line with prior studies concerning evaluation of the presence of ALK abnormalities in such patients. However, they showed that assessment of ALK by IHC and FISH methods in CNS tissues require additional standardizations.

Identification of single nucleotide polymorphisms of the PI3K-AKT-mTOR pathway as a risk factor of central nervous system metastasis in metastatic breast cancer.

INTRODUCTION: The PI3K-AKT-mTOR pathway may be involved in the development of central nervous system (CNS) metastasis from breast cancer. Accordingly, herein we explored whether single nucleotide polymorphisms (SNPs) of this pathway are associated with altered risk of CNS metastasis formation in metastatic breast cancer patients. METHODS: The GENEOM study (NCT00959556) included blood sample collection from breast cancer patients treated in the neoadjuvant, adjuvant or metastatic setting. We identified patients with CNS metastases for comparison with patients without CNS metastasis, defined as either absence of neurological symptoms or normal brain magnetic resonance imaging (MRI) before death or during 5-year follow-up. Eighty-eight SNPs of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian (or mechanistic) target of rapamycin (mTOR) pathway genes were selected for analysis: AKT1 (17 SNPs), AKT2 (4), FGFR1 (2), mTOR (7), PDK1 (4), PI3KR1 (11), PI3KCA (20), PTEN (17), RPS6KB1 (6). RESULTS: Of 342 patients with metastases, 207 fulfilled the inclusion criteria: One-hundred-and-seven patients remained free of CNS metastases at last follow-up or date of death whereas 100 patients developed CNS metastases. Among clinical parameters, hormonal and human epidermal growth factor receptor-2 (HER2) status as well as vascular tumour emboli was associated with risk of CNS metastasis. Only PI3KR1-rs706716 was associated with CNS metastasis in univariate analysis after Bonferroni correction (p < 0.00085). Multivariate analysis showed associations between AKT1-rs3803304, AKT2-rs3730050, PDK1-rs11686903 and PI3KR1-rs706716 and CNS metastasis . CONCLUSION: PI3KR1-rs706716 may be associated with CNS metastasis in metastatic breast cancer patients and could be included in a predictive composite score to detect early CNS metastasis irrespective of breast cancer subtype.

[A case of primary central nervous system anaplastic lymphoma kinase positive anaplastic large cell lymphoma manifested as a unilateral pachymeningits].

There have been 23 reports of primary central nervous system anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma in the literature. Here we report the 24th case of a 40-year-old man who presented with occipital headache for one month. His contrast-enhanced brain MRI showed enhancement around the right temporal lobe, which suggested a diagnosis of hypertrophic pachymeningitis. He improved with steroid therapy. After discharge, however, he was readmitted with generalized convulsive seizures. Finally, he was diagnosed as primary central nervous system ALK-positive anaplastic large cell lymphoma by brain biopsy. Primary central nervous system lymphoma invading dura matter can rarely manifests as a unilateral pachymeningitis. Therefore, in case of pachymeningitis, we should pay attention to the possibility of infiltration of lymophoma with meticulous clinical follow-up.

Phosphatases and solid tumors: focus on glioblastoma initiation, progression and recurrences.

Phosphatases and cancer have been related for many years now, as these enzymes regulate key cellular functions, including cell survival, migration, differentiation and proliferation. Dysfunctions or mutations affecting these enzymes have been demonstrated to be key factors for oncogenesis. The aim of this review is to shed light on the role of four different phosphatases (PTEN, PP2A, CDC25 and DUSP1) in five different solid tumors (breast cancer, lung cancer, pancreatic cancer, prostate cancer and ovarian cancer), in order to better understand the most frequent and aggressive primary cancer of the central nervous system, glioblastoma.

Quantification of tumor-derived cell free DNA(cfDNA) by digital PCR (DigPCR) in cerebrospinal fluid of patients with BRAFV600 mutated malignancies.

Tumor-derived cell free DNA (cfDNA) can be detected in plasma. We hypothesized that mutated BRAF V600 cfDNA could be quantified in the cerebrospinal fluid (CSF) of patients with central nervous system (CNS) metastases. We collected CSF from patients with BRAF V600E or K-mutated melanoma (N=8) or BRAF V600E mutated Erdheim-Chester Disease (ECD) (N=3) with suspected central nervous system (CNS) involvement on the basis of neurological symptoms (10/11), MRI imaging (8/11), or both. Tumor-derived cfDNA was quantified by digital PCR in the CSF of 6/11 patients (range from 0.15-10.56 copies/µL). Conventional cytology was negative in all patients except in the two patients with markedly elevated levels of tumor-derived cfDNA. In 2 patients with serial measurements, CSF tumor-derived cfDNA levels reflected response to treatment or progressive disease. CSF tumor-derived cfDNA has the potential to serve as a diagnostic tool that complements MRI and may be more sensitive than conventional cytology.

Methylmercury alters glutathione homeostasis by inhibiting glutaredoxin 1 and enhancing glutathione biosynthesis in cultured human astrocytoma cells.

Methylmercury (MeHg) is a neurotoxin that binds strongly to thiol residues on protein and low molecular weight molecules like reduced glutathione (GSH). The mechanism of its effects on GSH homeostasis particularly at environmentally relevant low doses is not fully known. We hypothesized that exposure to MeHg would lead to a depletion of reduced glutathione (GSH) and an accumulation of glutathione disulfide (GSSG) leading to alterations in S-glutathionylation of proteins. Our results showed exposure to low concentrations of MeHg (1µM) did not significantly alter GSH levels but increased GSSG levels by ∼12-fold. This effect was associated with a significant increase in total cellular glutathione content and a decrease in GSH/GSSG. Immunoblot analyses revealed that proteins involved in glutathione synthesis were upregulated accounting for the increase in cellular glutathione. This was associated an increase in cellular Nrf2 protein levels which is required to induce the expression of antioxidant genes in response to cellular stress. Intriguingly, we noted that a key enzyme involved in reversing protein S-glutathionylation and maintaining glutathione homeostasis, glutaredoxin-1 (Grx1), was inhibited by ∼50%. MeHg treatment also increased the S-glutathionylation of a high molecular weight protein. This observation is consistent with the inhibition of Grx1 and elevated H2O2 production however; contrary to our original hypothesis we found few S-glutathionylated proteins in the astrocytoma cells. Collectively, MeHg affects multiple arms of glutathione homeostasis ranging from pool management to protein S-glutathionylation and Grx1 activity.

Central nervous system relapse in patients with untreated HER2-positive esophageal or gastroesophageal junction adenocarcinoma.

Although HER2-positive breast cancers demonstrate a propensity for central nervous system (CNS) metastasis, it is unknown whether other HER2-positive tumors, including adenocarcinomas of the esophagus/gastroesophageal junction (EAC), share this characteristic. Insight into this association may inform the development of HER2-targeted therapies that penetrate the blood-brain barrier. We examined HER2 overexpression and gene amplification in 708 patients with EAC who underwent curative-intent surgery during a time period (1980-1997) when no patient received HER2-targeted therapy. We identified patients whose site of first cancer recurrence was CNS and those who had a CNS relapse at any time. After a median follow-up of 61.2 months, 3.4% (24/708) of patients developed CNS relapse (all involved the brain). Patients with HER2-positive (vs -negative) primary tumors showed a higher 5-year cumulative incidence of CNS relapse as first recurrence (5.8% vs. 1.2%; p = 0.0058) and at any time (8.3% vs. 2.4%; p = 0.0062). In a multivariable model that included covariates previously associated with HER2 or with CNS relapse in breast cancer, HER2 positivity was the only variable that was statistically significantly associated with shorter time to CNS relapse as first recurrence (p = 0.0026) or at any time (hazard ratio 4.3 [95% confidence interval 1.8 to 10.3]; p = 0.001). These are the first data in a non-breast cancer to demonstrate an association between HER2 positivity and higher CNS relapse risk after surgery, and suggest that HER2-positive EACs have a predilection for CNS metastases.

[Critical roles of matrix metalloproteinases secreted by leukemic cells in the pathogenesis of central nervous system leukemia].

Objective: To observe the effects of matrix metalloproteinases (MMP)-2 and MMP-9 secreted by leukemic cells on tight junction proteins ZO-1, claudin-5 and occluding and the permeability of the blood-brain barrier (BBB) and explore the mechanisms of MMP-2 and MMP-9 in leukemic cell infiltration of the central nervous system (CNS). Methods: The mRNA expressions of MMP-2 and MMP-9 in leukemic cell lines SHI-1, HL-60 and U937 were detected by quantitative RT-PCR. The MMP inhibitor GM6001 was used to inhibit the secretion of MMP-2 and MMP-9. RNA interference (RNAi) was used to knock down the expression of MMP-2 and MMP-9. Zymography was used to analyze the secretion of MMP-2 and MMP-9 in the supernatant of different leukemia cell lines treated or untreated with drugs, as well as the RNAi-treated cells. An in vitro BBB model composed of human brain microvascular endothelial cells (BMVECs) was developed on a Matrigel-based insert. Cell invasion through a barrier of Matrigel-based human basement membrane and the BMVECs-based human BBB barrier was assayed to measure the invasive capacity and the capacity to breakdown the BBB of different leukemia cell lines treated or untreated with drugs, as well as the RNAi-treated cells. The morphologic changes of BMVECs after co-culture with different leukemia cell lines treated or untreated with drugs, as well as the RNAi-treated cells in vitro BBB models were observed by invert microscopy and tight junction proteins in these BMVECs were analyzed with a laser-scanning confocal microscope. Results: ①The mRNA expression in different leukemic cell lines shown a pronounced transcription of MMP-2 and - 9, and the transcriptional level in SHI-1 cells was the highest among all leukemic cell lines tested (P<0.01). The data of activities of MMP-2 and -9 were consistent with the results of mRNA expression and SHI-1 displayed higher capacity of invasion (P<0.01). ②After incubation 24h with different leukemic cells, the BMVECs disrupted to loss cell-cell contacts and grew in single cell. Confocal imaging showed down-regulations of ZO-1, claudin-5 and occluding accompanied by the disruption of BBB in vitro models. SHI-1 cells had stronger alterations to BMVECs, tight junction proteins and the permeability of the BBB than HL-60 and U937 cells. However, GM6001 and the knock-down of MMP-2 and MMP-9 altered the responses of BBB. They reduced the degradation of three tight junction proteins with a decreased permeability of BBB. Conclusion: MMP-2 and MMP-9 secreted by leukemic cells could disrupt the BBB by degrading the tight junction proteins ZO-1, claudin-5 and occluding, which contributed the infiltration of leukemic cell into CNS.

Extrinsic factors can mediate resistance to BRAF inhibition in central nervous system melanoma metastases.

Here, we retrospectively review imaging of 68 consecutive unselected patients with BRAF V600-mutant metastatic melanoma for organ-specific response and progression on vemurafenib. Complete or partial responses were less often seen in the central nervous system (CNS) (36%) and bone (16%) compared to lung (89%), subcutaneous (83%), spleen (71%), liver (85%) and lymph nodes/soft tissue (83%), P < 0.001. CNS was also the most common site of progression. Based on this, we tested in vitro the efficacy of the BRAF inhibitors PLX4720 and dabrafenib in the presence of cerebrospinal fluid (CSF). Exogenous CSF dramatically reduced cell death in response to both BRAF inhibitors. Effective cell killing was restored by co-administration of a PI-3 kinase inhibitor. We conclude that the efficacy of vemurafenib is variable in different organs with CNS being particularly prone to resistance. Extrinsic factors, such as ERK- and PI3K-activating factors in CSF, may mediate BRAF inhibitor resistance in the CNS.

A high-sensitive HMab-2 specifically detects IDH1-R132H, the most common IDH mutation in gliomas.

Isocitrate dehydrogenase 1 (IDH1) mutations have been detected in gliomas and other tumors. Although IDH1 catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate (α-KG) in cytosol, mutated IDH1 proteins possess the ability to change α-KG into the oncometabolite D-2-hydroxyglutarate (D-2HG). Several monoclonal antibodies (mAbs) specific for IDH1 mutations have been established, such as H09, IMab-1, and HMab-1 against IDH1-R132H, which is the most frequent IDH1 mutation in gliomas. In this study, we established a novel high-sensitive mAb HMab-2, which reacts with IDH1-R132H but not with wild type IDH1 in ELISA. HMab-2 reacted only with IDH1-R132H, not with wild type IDH1/2 and other IDH1/2 mutants in Western-blot analysis. Furthermore, HMab-2 recognized IDH1-R132H more sensitively compared with our previously established HMab-1. HMab-2 detected endogenous IDH1-R132H protein expressed in glioblastoma in immunohistochemical analysis. HMab-2 is expected to be useful for the diagnosis of IDH1-R132H-bearing tumors.

Specific immunohistochemical pattern of carbonic anhydrase IX is helpful for the diagnosis of CNS hemangioblastoma.

Hemangioblastomas are rare capillary-rich tumors predominantly found in the CNS. The histological appearance of these tumors varies across a broad spectrum. Several entities show considerable histomorphological similarities to hemangioblastomas. Therefore, morphological evaluation can be challenging. In this study, we evaluated the diagnostic utility of immunohistochemistry using antibodies against carbonic anhydrase IX and cytokeratin staining. Within our files, we identified 20 hemangioblastomas. A collection of 46 other tumors relevant to the differential diagnosis (12 pilocytic astrocytomas, 11 meningiomas, one pleomorphic xanthoastrocytoma, one angiomatous fibrous histiocytoma, 14 carcinoma metastases and seven gliomas grades II-IX) served as control. The pattern of strong, diffuse expression of carbonic anhydrase IX with membranous accentuation in combination with keratin negativity was considered diagnostic for hemangioblastomas. It was found in 18 out of 20 (90%) hemangioblastomas and in none of the control cases (P < 0.001). This resulted in a sensitivity of 90% and a specificity of 100%. The positive and negative predictive values were 100% and 96%, respectively. Carbonic anhydrase IX with cytokeratin is thus a highly sensitive and specific marker combination for hemangioblastomas. It is therefore very helpful in the diagnosis of these tumors and in their discrimination from other entities.

Expression of proliferation markers Ki67, cyclin A, geminin and aurora-kinase A in primary breast carcinomas and corresponding distant metastases.

AIMS: To assess the expression of the following cell cycle regulatory proteins in primary metastatic breast carcinomas (MBCs) and on availability in matched distant metastases (DMs): Ki67, cyclin A, geminin and aurora-kinase A (aurkA); and to compare the expression of these markers in early MBC (EMBC) and late MBC separated into groups according to median time point on metastatic event occurred (28 months). METHODS: The expression of the above mentioned markers was analysed in a total of 47 primary MBCs and 59 DMs (out of which 37 were pairs) by immunohistochemistry. Fourteen breast carcinomas with no relapse over a 10-year follow-up period were utilised as control cases (CBC). RESULTS: Among the MBCs, 22 metastasised to the bone, 4 to the lung and 21 to the central nervous system (CNS). Geminin (p<0.001) and Ki67 (p=0.001) were increased in the MBCs while aurkA and cyclin A showed no difference when compared with CBCs. There were no differences between aurkA, cyclin A and geminin expression in MBCs and DMs in general. Expression of Ki67 was, however, elevated (p=0.027) in DMs. In CNS metastases all markers showed elevated expression as compared to MBCs. In bone metastases, geminin was lower (p<0.001) compared with primary MBCs. In the metastases of the lung, the evaluated markers did not show different expression. According to the median follow-up until the metastatic event, Ki67 was found to be significantly elevated in EMBC (p=0.018). CONCLUSIONS: Ki67 index and geminin distinguish a fraction of MBC with worse prognosis, showing increased levels in the latter in comparison to CBC being tumour-free over a 10-year follow-up period. Ki67 could possibly identify a group of MBCs that develop early DMs.