Donggeon Kim

Detection of large deletion mutations in the SERPINC1 gene causing hereditary antithrombin deficiency by multiplex ligation‐dependent probe amplification (MLPA)

Hereditary antithrombin (AT) deficiency (MIM 107300) is an autosomal dominant disorder characterized by decreased serum AT activity and predisposition to recurrent venous thromboembolism (VTE) [1]. The genetic background is a mutation in the gene encoding antithrombin, SERPINC1, located on the chromosome band 1q23-25.1 [2]. The mutation profile of the SERPINC1 gene is heterogeneous, being mostly comprised of point mutations and small insertion/deletion mutations, while large rearrangements including deletions account for only a small proportion (7.0% of a total of 215 mutation entries in the Human Gene Mutation Database) of the mutations reported thus far [3–5]. The technical drawback in detecting large deletion or duplication mutations has been recognized, and this might have contributed to the small proportion of such mutations in AT deficiency. Recently, a rapid comparative quantification method, multiplex ligation-dependent probe amplification (MLPA), has been developed with proven reliability and sensitivity in detecting gene dosage changes from deletions or duplications involving one or more exons to the whole gene [6,7]. For the first time in the literature, here we report on the successful detection of large deletions of the SERPINC1 gene by using MLPA in Korean patients with AT deficiency who had previously shown no mutations in direct sequencing analyses. Patient 1 was a 27-year-old Korean man who had been on warfarin medication since he was 18 years old. He first experienced painful swelling of his left leg at age 17. At 18, he developed dyspnea and visited a local tertiary hospital where he was diagnosed as having pulmonary embolism (PE). On his visit to our hospital as an outpatient, his coagulation tests without anticoagulation showed decreased AT activity at 46% (STA-StachromAT III 3, Diagnostica Stago, Asnières, France; reference range determined at our institution, 83–123%). Further tests on a separate blood sample showed type I deficiency (AT activity 53% and AT antigen 54%). Other results, including protein C activity, free protein S antigen, and lupus anticoagulant profiles, were all within normal limits. Family history revealed that his 25-year-old younger brother had a history of deep vein thrombosis (DVT) and PE at the age of 19. However, his parents or other family members had no relevant medical history. A family study was carried out and indicated that the proband and his brother (AT activity 54%) had inherited AT deficiency from his father (age 52 years; AT activity 49%). However, direct sequencing of the SERPINC1 gene in the proband revealed no mutations. Patient 2 was a 49-year-old Korean man with superior mesenteric vein thrombosis. He had a history of DVT on his left leg at the age of 41. Coagulation tests revealed decreased AT activity (42%) and antigen (50%) levels, indicating type I AT deficiency. Family history revealed that his younger brother also had DVT on his left leg, but his parents had no relevant medical history. Again, direct sequencing revealed no mutations in the SERPINC1 gene in patient 2. On suspicion of gross rearrangement mutations as the underlying genetic defect in these two families with AT deficiency, we additionally performed exon dosage analysis using MLPA (SALSA MLPA KIT P227 SerpinC1, MRC Holland, Amsterdam, the Netherlands) according to the manufacturer s instructions. Five normal control DNA samples were included in each run. Data were analyzed using the GeneScan (Applied Biosystems, Foster City, CA, USA) and GeneMarker (SoftGenetics, State College, PA, USA) software. As a result, we found that themutation running in the family of patient 1 was a heterozygous large deletion spanning exons 1–6 of the SERPINC1 gene (Fig. 1). This large deletion mutation Correspondence: Hee-Jin Kim, Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710, Korea. Tel.: +82 2 341

Inherited protein S deficiency as a reszult of a large duplication mutation of the PROS1 gene detected by multiplex ligation‐dependent probe amplification

1 Repesse Y, Slaoui M, Ferrandiz D, Gautier P, Costa C, Costa JM, Lavergne JM, Borel-Derlon A. Factor VIII gene mutations in 120 hemophilia A patients: detection of 26 novel mutations and correlation with FVIII inhibitor development. J Thromb Haemost 2007; 5: 1469–76. 2 Gitschier J, Wood WI, Goralka TM, Wion KL, Chen EY, Eaton DH, Vehar GA, Capon DJ, Lawn RM. Characterization of the human factor VIII gene. Nature 1984; 312: 326–30. 3 Higuchi M, Antonarakis SE, Kasch L, Oldenburg J, EconomouPetersen A, Olek K, Arai M, Inaba H, Kazazian HH Jr. Molecular characterization of mild-to-moderate hemophilia A: detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 1991; 88: 8307–11. 4 Scanavini D, Legnani C, Lunghi B,Mingozzi F, Palareti G, Bernardi F. The factor VIII D1241E polymorphism is associated with decreased factor VIII activity and not with activated protein C resistance levels. Thromb Haemost 2005; 93: 453–6. 5 Viel KR,MachiahDK,WarrenDM,KhachidzeM, Buil A, Fernstrom K, Souto JC, Peralta JM, Smith T, Blangero J, Porter S, Warren ST, Fontcuberta J, Soria JM, Flanders WD, Almasy L, Howard TE. A sequence variation scan of the coagulation factor VIII (FVIII) structural gene and associations with plasma FVIII activity levels. Blood 2007; 109: 3713–24. 6 Windsor S, Taylor SAM, Lillicrap D. Multiplex analysis of two intragenic microsatellite repeat polymorphisms in the genetic diagnosis of haemophilia a. Br J Haematol 1994; 86: 810–5. 7 Kogan SC, Gitschier J. Genetic prediction of hemophilia A. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: A Guide to Methods and Applications. San Diego, CA: Academic Press, 1990: 288–99. 8 AcquilaM, PasinoM, Lanza T,Molinari AC, RosanoC, BicocchiMP. Exon skipping partially restores factor VIII coagulant activity in patients with mild hemophilia A with exon 13 duplication.Haematologica 2005; 90: 997–9.

Astrocyte-derived CCL20 reinforces HIF-1-mediated hypoxic responses in glioblastoma by stimulating the CCR6-NF-κB signaling pathway

During tumor development, stromal cells are co-opted to the tumor milieu and provide favorable conditions for the tumor. Hypoxia stimulates cancer cells to acquire a more malignant phenotype via activation of hypoxia-inducible factor 1 (HIF-1). Given that cancer cells and astrocytes in glioblastomas coexist in a hypoxic microenvironment, we examined whether astrocytes affect the adaptation of glioblastoma cells to hypoxia. Immunoblotting, reporter assays, quantitative RT-PCR, and chromatin immunoprecipitation were performed to evaluate HIF-1 signaling in glioblastoma cells. Astrocyte-derived chemokine C–C motif ligand 20 (CCL20) was identified using cytokine arrays, and its role in glioblastoma development was evaluated in orthotopic xenografts. Astrocytes augmented HIF-1α expression in glioblastoma cells under hypoxia. The expression of HIF-1 downstream genes, cancer colony formation, and Matrigel invasion of glioblastoma cells were stimulated by conditioned medium from astrocytes pre-exposed to hypoxia. CCL20 was secreted in a hypoxia-dependent manner from astrocytes and busted the hypoxic induction of HIF-1α in glioblastoma cells. Mechanistically, the CCL20/CCR6 signaling pathway upregulates HIF-1α by stimulating nuclear factor kappa B-driven transactivation of the HIF1A gene. Compared with the control tumors, CCR6-deficient glioblastoma xenografts grew more slowly, with poor vascularization, and expressed lower levels of HIF-1α and its downstream proteins. Furthermore, CCR6 expression was correlated with HIF-1α expression in GEO and TCGA datasets from human glioblastoma tissues. These results suggest that glioblastoma cells adapt well to hypoxic stress by virtue of CCL20 derived from neighboring astrocytes.

Pharmacogenomic landscape of patient-derived tumor cells informs precision oncology therapy

Outcomes of anticancer therapy vary dramatically among patients due to diverse genetic and molecular backgrounds, highlighting extensive intertumoral heterogeneity. The fundamental tenet of precision oncology defines molecular characterization of tumors to guide optimal patient-tailored therapy. Towards this goal, we have established a compilation of pharmacological landscapes of 462 patient-derived tumor cells (PDCs) across 14 cancer types, together with genomic and transcriptomic profiling in 385 of these tumors. Compared with the traditional long-term cultured cancer cell line models, PDCs recapitulate the molecular properties and biology of the diseases more precisely. Here, we provide insights into dynamic pharmacogenomic associations, including molecular determinants that elicit therapeutic resistance to EGFR inhibitors, and the potential repurposing of ibrutinib (currently used in hematological malignancies) for EGFR-specific therapy in gliomas. Lastly, we present a potential implementation of PDC-derived drug sensitivities for the prediction of clinical response to targeted therapeutics using retrospective clinical studies.Analysis of genomic and transcriptomic data from 462 patient-derived tumor cell (PDC) samples across 14 cancer types, along with pharmacological responses to 60 agents, indicates that PDC-derived drug sensitivities might be predictive of clinical response to targeted therapies.

Therapeutic Drug Monitoring of Mirtazapine in a Routine Outpatient Setting in Asian Psychiatric Patients

INTRODUCTION Mirtazapine is an antidepressant that acts by enhancing serotonergic and noradrenergic neurotransmission. This study aimed to evaluate mirtazapine pharmacokinetic data from Korean psychiatric patients and to identify the potential factors affecting its steady-state concentration. METHODS A total of 337 samples of steady-state mirtazapine concentrations from 188 adult psychiatric outpatients were retrospectively evaluated. Serum mirtazapine concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry. RESULTS Median mirtazapine concentration was 43.6 μg/L (164.37 nmol) at a daily dosage range of 7.5-60 mg. At the steady state, mirtazapine dose had a positive correlation with the drug concentration. Mean concentration-to-dose (C/D) ratio was 1.48 μg/L/mg/day (5.58 nmol/mg/day), which was higher than that in a previous study in Caucasian subjects. Age and paroxetine co-medication were positively associated with C/D ratio. Initial mirtazapine concentration and C/D ratio did not show an association with responsiveness in depressive patients. DISCUSSION This study presented the therapeutic drug monitoring data for mirtazapine and pharmacokinetic variations of mirtazapine in an Asian population. A further study could be helpful for clinical decision making based on the characteristics of patients.

Prevalence of overt myeloproliferative neoplasms and JAK2 V617F mutation in Korean patients with splanchnic vein thrombosis

Introduction:  Myeloproliferative neoplasm (MPN) is known to be a major risk factor of splanchnic vein thrombosis (SVT). Recent studies revealed that a significant proportion of patients with SVT harbor a gain‐of‐function mutation in the JAK2 gene (V617F) with or without MPN. In this study, the authors investigated the prevalence of MPN and JAK2 V617F mutation in Korean patients with SVT.

Abstract 3458: Wnt activation is implicated in glioblastoma radioresistance

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Glioblastoma (GBM) patients have dismal median survival even with the most rigorous treatments currently available. Radiotherapy is the most effective non-surgical therapy for GBM patients; however, patients succumb due to tumor recurrence within a year. To develop a curative therapeutic approach, we need to better understand the underlying molecular mechanism of radiation resistance in GBM. Towards this goal, we developed an in vivo orthotopic GBM model system that mimics the radiation response of human GBM, using both established-GBM cell line and patient-derived freshly dissociated GBM specimen. In-vivo ionizing radiation (IR) treatment prolonged the survival of mice with intracranical tumor derived from U373MG, but failed to prevent tumor recurrence. U373MG and GBM578 cells isolated after in-vivo IR (U373-IR and 578-IR) were more clonogenic and enriched with stem cell-like characteristics, compared with mock-treated control tumor cells. Transcriptomic analyses and quantitative real-time reverse-transcription PCR analyses using these matched GBM cells before and after radiation treatment revealed that Wnt pathways were preferentially activated in post-IR GBM cells. U373-IR cells and 578-IR were enriched with cells positive for both active b-catenin (ABC) and Sox2 population, and this subpopulation was further increased after additional in-vitro radiation treatment, suggesting that radiation resistance of GBM is mediated due, in part, to the activation of stem cell-associated pathways including Wnt. Finally, pharmacological and siRNA inhibition of Wnt pathway significantly decreased the survival and clonogenicity of GBM cells and reduced their ABCþ/Sox2þ population. Together, these data suggest that Wnt activation is a molecular mechanism to confer GBM radioresistance and an important therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3458. doi:1538-7445.AM2012-3458

Distinct genomic profile and specific targeted drug responses in adult cerebellar glioblastoma

Background Despite extensive efforts on the genomic characterization of gliomas, very few studies have reported the genetic alterations of cerebellar glioblastoma (C-GBM), a rare and lethal disease. Here, we provide a systematic study of C-GBM to better understand its specific genomic features. Methods We collected a cohort of C-GBM patients and compared patient demographics and tumor pathologies with supratentorial glioblastoma (S-GBM). To uncover the molecular characteristics, we performed DNA and mRNA sequencing and DNA methylation arrays on 19, 6, and 4 C-GBM cases, respectively. Moreover, chemical drug screening was conducted to identify potential therapeutic options for C-GBMs. Results Despite differing anatomical origins of C-GBM and S-GBM, neither histological, cytological, nor patient demographics appeared significantly different between the 2 types. However, we observed striking differences in mutational patterns, including frequent alterations of ATRX, PDGFRA, NF1, and RAS and absence of EGFR alterations in C-GBM. These results show a distinct evolutionary path in C-GBM, suggesting specific therapeutic targeted options. Targeted-drug screening revealed that C-GBMs were more responsive to mitogen-activated protein kinase kinase (MEK) inhibitor and resistant to epidermal growth factor receptor inhibitors than S-GBMs. Also, differential expression analysis indicated that C-GBMs may have originated from oligodendrocyte progenitor cells, suggesting that different types of cells can undergo malignant transformation according to their location in brain. Master regulator analysis with differentially expressed genes between C-GBM and proneural S-GBM revealed NR4A1 as a potential therapeutic target. Conclusions Our results imply that unique gliomagenesis mechanisms occur in adult cerebellum and new treatment strategies are needed to provide greater therapeutic benefits for C-GBM patients. Key Points 1. Distinct genomic profiles of 19 adult cerebellar GBMs were characterized. 2. MEK inhibitor was highly sensitive to cerebellar GBM compared with supratentorial GBM. 3. Master regulator analysis revealed NR4A1 as a potential therapeutic target in cerebellar GBM.

Identification of genomic and molecular traits that present therapeutic vulnerability to HGF-targeted therapy in glioblastoma

BACKGROUND Cancer is a complex disease with profound genomic alterations and extensive heterogeneity. Recent studies on large-scale genomics have shed light on the impact of core oncogenic pathways, which are frequently dysregulated in a wide spectrum of cancer types. Aberrant activation of the hepatocyte growth factor (HGF) signaling axis has been associated with promoting various oncogenic programs during tumor initiation, progression, and treatment resistance. As a result, HGF-targeted therapy has emerged as an attractive therapeutic approach. However, recent clinical trials involving HGF-targeted therapies have demonstrated rather disappointing results. Thus, an alternative, in-depth assessment of new patient stratification is necessary to shift the current clinical course. METHODS To address such challenges, we have evaluated the therapeutic efficacy of YYB-101, an HGF-neutralizing antibody, in a series of primary glioblastoma stem cells (GSCs) both in vitro and in vivo. Furthermore, we performed genome and transcriptome analysis to determine genetic and molecular traits that exhibit therapeutic susceptibility to HGF-mediated therapy. RESULTS We have identified several differentially expressed genes, including MET, KDR, and SOX3, which are associated with tumor invasiveness, malignancy, and unfavorable prognosis in glioblastoma patients. We also demonstrated the HGF-MET signaling axis as a key molecular determinant in GSC invasion, and we discovered that a significant association in HGF expression existed between mesenchymal phenotype and immune cell recruitment. CONCLUSIONS Upregulation of MET and mesenchymal cellular state are essential in generating HGF-mediated therapeutic responses. Our results provide an important framework for evaluating HGF-targeted therapy in future clinical settings.

Tumor Inhibitory Effect of IRCR201, a Novel Cross-Reactive c-Met Antibody Targeting the PSI Domain

Hepatocyte growth factor receptor (HGFR, c-Met) is an essential member of the receptor tyrosine kinase (RTK) family that is often dysregulated during tumor progression, driving a malignant phenotypic state and modulating important cellular functions including tumor growth, invasion, metastasis, and angiogenesis, providing a strong rationale for targeting HGF/c-Met signaling axis in cancer therapy. Based on its protumorigenic potentials, we developed IRCR201, a potent antagonistic antibody targeting the plexin-semaphorin-integrin (PSI) domain of c-Met, using synthetic human antibody phage libraries. We characterized and evaluated the biochemical properties and tumor inhibitory effect of IRCR201 in vitro and in vivo. IRCR201 is a novel fully-human bivalent therapeutic antibody that exhibits cross-reactivity against both human and mouse c-Met proteins with high affinity and specificity. IRCR201 displayed low agonist activity and rapidly depleted total c-Met protein via the lysosomal degradation pathway, inhibiting c-Met-dependent downstream activation and attenuating cellular proliferation in various c-Met-expressing cancer cells. In vivo tumor xenograft models also demonstrated the superior tumor inhibitory responsiveness of IRCR201. Taken together, IRCR201 provides a promising therapeutic agent for c-Met-positive cancer patients through suppressing the c-Met signaling pathway and tumor growth.