Tolerability and safety of EUS-injected adenovirus-mediated double-suicide gene therapy with chemotherapy in locally advanced pancreatic cancer: a phase 1 trial.

BACKGROUND AND AIMS: Locally advanced pancreatic cancer (LAPC) is challenging. Here, we aimed to evaluate the tolerability and safety of Ad5-yCD/mutTK(SR39)rep-ADP (Ad5-DS), a replication-competent adenovirus-mediated double-suicide gene therapy in combination with gemcitabine in patients with LAPC. METHODS: Patients with newly diagnosed LAPC were enrolled in this single-center, open-label, 3 + 3 dose-escalation phase 1 trial. Ad5-DS was injected into the pancreatic mass with EUS-guided fine needles combined with oral 5-fluorocytosine and valganciclovir, and a standard dose of intravenous gemcitabine. The doses of Ad5-DS in cohorts 1 to 3 were 1 × 1011, 3 × 1011, and 1 × 1012 viral particles (vp)/mL, respectively. Patients were observed for dose-limiting toxicity (DLT) for 8 weeks after Ad5-DS injection. Toxicity within 12 weeks, tumor response in 12 weeks, disease progression in 6.5 months, and detection of adenoviral DNA particles in 8 weeks were also assessed. RESULTS: Among the 11 enrolled patients, 9 completed the evaluation period and 2 withdrew their consent. No DLT was reported; thus, the maximum tolerated dose was not reached. No additional toxicity was reported in 9 to 12 weeks. One patient showed a partial response and 8 showed stable disease at 12 weeks. Two patients showed disease progression at 6.5 months (median progression-free survival, 11.4 months). At 8 weeks, serum adenoviral DNA particles were detected in 4 patients (median, 55 days). CONCLUSION: A combination of intratumoral Ad5-DS and gemcitabine is safe and well tolerated in patients with LAPC. This warrants further investigation in a larger clinical trial. (Clinical trial registration number: NCT02894944.).

Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease.

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).

Integrative radiogenomic analysis for genomic signatures in glioblastomas presenting leptomeningeal dissemination.

Despite therapeutic advances, the prognosis for glioblastoma (GBM) remains poor. In particular, leptomeningeal dissemination (LMD) has a dismal prognosis. The aim of this study was to identify tumor molecular phenotype, which has a great propensity to develop LMD. Between May 2004 and December 2012, a total of 145 GBM tumor samples were obtained from data registry. A total of 20 of the 145 patients with GBM were found to develop LMD. A specialized radiologist confirmed the diagnosis of LMD on magnetic resonance imaging. To clarify the genomic signatures in GBM with LMD, we performed integrative analysis of whole transcriptome sequencing and copy number alteration in the radiological features indicating LMD phenotypes in GBM. Eleven newly diagnosed patients with GBM with LMD had worse prognosis than those without LMD (median 5.55 vs. 12.94 months, P < 0.0001). Integrating analysis using gene expression based on the change of copy number revealed that SPOCK1, EHD2, SLC2A3, and ANXA11 were highly expressed with the gain of copy number, compared with the gene expression in the non-LMD group. In addition, it was demonstrated that NME2, TMEM100, and SIVA1 were downregulated with the loss of copy number. We also found that mesenchymal subtype accounted for 50% in LMD group, whereas mesenchymal subtype consisted of 29% in non-LMD group, even though there was no statistical significance (P = 0.06). Through this radiogenomic analysis, we suggested the possibility of finding candidate genes associated with LMD and highlighted the significance of integrating approach to clarify the molecular characteristics in LMD.

Integrative radiogenomic analysis for multicentric radiophenotype in glioblastoma.

We postulated that multicentric glioblastoma (GBM) represents more invasiveness form than solitary GBM and has their own genomic characteristics. From May 2004 to June 2010 we retrospectively identified 51 treatment-naïve GBM patients with available clinical information from the Samsung Medical Center data registry. Multicentricity of the tumor was defined as the presence of multiple foci on the T1 contrast enhancement of MR images or having high signal for multiple lesions without contiguity of each other on the FLAIR image. Kaplan-Meier survival analysis demonstrated that multicentric GBM had worse prognosis than solitary GBM (median, 16.03 vs. 20.57 months, p < 0.05). Copy number variation (CNV) analysis revealed there was an increase in 11 regions, and a decrease in 17 regions, in the multicentric GBM. Gene expression profiling identified 738 genes to be increased and 623 genes to be decreased in the multicentric radiophenotype (p < 0.001). Integration of the CNV and expression datasets identified twelve representative genes: CPM, LANCL2, LAMP1, GAS6, DCUN1D2, CDK4, AGAP2, TSPAN33, PDLIM1, CLDN12, and GTPBP10 having high correlation across CNV, gene expression and patient outcome. Network and enrichment analyses showed that the multicentric tumor had elevated fibrotic signaling pathways compared with a more proliferative and mitogenic signal in the solitary tumors. Noninvasive radiological imaging together with integrative radiogenomic analysis can provide an important tool in helping to advance personalized therapy for the more clinically aggressive subset of GBM.

Spatiotemporal Evolution of the Primary Glioblastoma Genome.

Tumor recurrence following treatment is the major cause of mortality for glioblastoma multiforme (GBM) patients. Thus, insights on the evolutionary process at recurrence are critical for improved patient care. Here, we describe our genomic analyses of the initial and recurrent tumor specimens from each of 38 GBM patients. A substantial divergence in the landscape of driver alterations was associated with distant appearance of a recurrent tumor from the initial tumor, suggesting that the genomic profile of the initial tumor can mislead targeted therapies for the distally recurred tumor. In addition, in contrast to IDH1-mutated gliomas, IDH1-wild-type primary GBMs rarely developed hypermutation following temozolomide (TMZ) treatment, indicating low risk for TMZ-induced hypermutation for these tumors under the standard regimen.

Translational validation of personalized treatment strategy based on genetic characteristics of glioblastoma.

Glioblastoma (GBM) heterogeneity in the genomic and phenotypic properties has potentiated personalized approach against specific therapeutic targets of each GBM patient. The Cancer Genome Atlas (TCGA) Research Network has been established the comprehensive genomic abnormalities of GBM, which sub-classified GBMs into 4 different molecular subtypes. The molecular subtypes could be utilized to develop personalized treatment strategy for each subtype. We applied a classifying method, NTP (Nearest Template Prediction) method to determine molecular subtype of each GBM patient and corresponding orthotopic xenograft animal model. The models were derived from GBM cells dissociated from patient's surgical sample. Specific drug candidates for each subtype were selected using an integrated pharmacological network database (PharmDB), which link drugs with subtype specific genes. Treatment effects of the drug candidates were determined by in vitro limiting dilution assay using patient-derived GBM cells primarily cultured from orthotopic xenograft tumors. The consistent identification of molecular subtype by the NTP method was validated using TCGA database. When subtypes were determined by the NTP method, orthotopic xenograft animal models faithfully maintained the molecular subtypes of parental tumors. Subtype specific drugs not only showed significant inhibition effects on the in vitro clonogenicity of patient-derived GBM cells but also synergistically reversed temozolomide resistance of MGMT-unmethylated patient-derived GBM cells. However, inhibitory effects on the clonogenicity were not totally subtype-specific. Personalized treatment approach based on genetic characteristics of each GBM could make better treatment outcomes of GBMs, although more sophisticated classifying techniques and subtype specific drugs need to be further elucidated.

NTRK1 fusion in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, yet with no targeted therapy with substantial survival benefit. Recent studies on solid tumors showed that fusion genes often play driver roles and are promising targets for pharmaceutical intervention. To survey potential fusion genes in GBMs, we analysed RNA-Seq data from 162 GBM patients available through The Cancer Genome Atlas (TCGA), and found that 3' exons of neurotrophic tyrosine kinase receptor type 1 (NTRK1, encoding TrkA) are fused to 5' exons of the genes that are highly expressed in neuronal tissues, neurofascin (NFASC) and brevican (BCAN). The fusions preserved both the transmembrane and kinase domains of NTRK1 in frame. NTRK1 is a mediator of the pro-survival signaling of nerve growth factor (NGF) and is a known oncogene, found commonly altered in human cancer. While GBMs largely lacked NTRK1 expression, the fusion-positive GBMs expressed fusion transcripts in high abundance, and showed elevated NTRK1-pathway activity. Lentiviral transduction of the NFASC-NTRK1 fusion gene in NIH 3T3 cells increased proliferation in vitro, colony formation in soft agar, and tumor formation in mice, suggesting the possibility that the fusion contributed to the initiation or maintenance of the fusion-positive GBMs, and therefore may be a rational drug target.

Patient-specific orthotopic glioblastoma xenograft models recapitulate the histopathology and biology of human glioblastomas in situ.

Frequent discrepancies between preclinical and clinical results of anticancer agents demand a reliable translational platform that can precisely recapitulate the biology of human cancers. Another critical unmet need is the ability to predict therapeutic responses for individual patients. Toward this goal, we have established a library of orthotopic glioblastoma (GBM) xenograft models using surgical samples of GBM patients. These patient-specific GBM xenograft tumors recapitulate histopathological properties and maintain genomic characteristics of parental GBMs in situ. Furthermore, in vivo irradiation, chemotherapy, and targeted therapy of these xenograft tumors mimic the treatment response of parental GBMs. We also found that establishment of orthotopic xenograft models portends poor prognosis of GBM patients and identified the gene signatures and pathways signatures associated with the clinical aggressiveness of GBMs. Together, the patient-specific orthotopic GBM xenograft library represent the preclinically and clinically valuable "patient tumor's phenocopy" that represents molecular and functional heterogeneity of GBMs.

MicroRNA-15a and -16-1 act via MYB to elevate fetal hemoglobin expression in human trisomy 13.

Many human aneuploidy syndromes have unique phenotypic consequences, but in most instances it is unclear whether these phenotypes are attributable to alterations in the dosage of specific genes. In human trisomy 13, there is delayed switching and persistence of fetal hemoglobin (HbF) and elevation of embryonic hemoglobin in newborns. Using partial trisomy cases, we mapped this trait to chromosomal band 13q14; by examining the genes in this region, two microRNAs, miR-15a and -16-1, appear as top candidates for the elevated HbF levels. Indeed, increased expression of these microRNAs in primary human erythroid progenitor cells results in elevated fetal and embryonic hemoglobin gene expression. Moreover, we show that a direct target of these microRNAs, MYB, plays an important role in silencing the fetal and embryonic hemoglobin genes. Thus we demonstrate how the developmental regulation of a clinically important human trait can be better understood through the genetic and functional study of aneuploidy syndromes and suggest that miR-15a, -16-1, and MYB may be important therapeutic targets to increase HbF levels in patients with sickle cell disease and ß-thalassemia.

Fabry disease with aortic regurgitation.

Aortic regurgitation is not so rare in patients with Fabry disease. Enzyme replacement therapy has become the standard medical care for Fabry disease in recent years. A 31-year-old man with Fabry disease, treated with recombinant alpha-galactosidase enzyme replacement for 19 months was admitted for evaluation of exertional dyspnea. Cardiac workup revealed left ventricular hypertrophy, increased left ventricular size, and moderate to severe aortic regurgitation. He underwent mechanical valvular replacement and heart biopsy. Histology of his aortic valve showed myxoid degeneration of valve leaflets. His heart muscle revealed extensive hypertrophy with vacuolization and the absence of lamellar bodies. We report a case of Fabry disease with aortic regurgitation in a man who underwent valvular replacement operation during enzyme replacement therapy.

The role of mesenchymal stem cells in the functional improvement of chronic renal failure.

The most commonly used therapeutic targets in nephrology are the reduction of injury, the delay of progression, or renal replacement therapy. Many animal and human studies demonstrated the role of stem cells in repair and regenerations of kidney. Mesenchymal stem cells (MSCs) have shown to improve outcome of acute renal injury models. It is controversial whether MSCs can reduce injury following a toxic/ischemic event and delay renal failure in chronic kidney disease. We evaluated the hypothesis that the treatment with MSCs could improve renal function and attenuate injury in chronic renal failure (CRF). Sprague-Dawley female rats (8 weeks old, 182.2 +/- 7.2 g) underwent modified 5/6 nephrectomy. Rats in the MSC group received an injection of MSCs (1 x 10(6) cells) via tail vein 1 day after nephrectomy. Blood and urine samples were collected after 7 days and every month thereafter. The kidneys of rats were removed for histologic evaluation after 24-h urine collection and blood sampling. The Y-chromosome stain using fluorescent in situ hybridization was performed to verify the presence of male MSCs in the kidney of female recipients. No significant differences in blood urea nitrogen and creatinine concentration were observed between the MSC group and the untreated CRF group. However, the weight gain in the MSC group was greater than those in the CRF group after 4 months. Proteinuria in the MSC group was less than that in the CRF group over time. Y chromosome was detected in the kidney of MSC group. Although no significances were observed between these two groups, the histologic analysis suggests that MSCs have positive effect against glomerulosclerosis. These results suggest that MSCs help preserve renal function and attenuate renal injury in CRF.

Characterization of the piRNA complex from rat testes.

Small noncoding RNAs regulate processes essential for cell growth and development, including mRNA degradation, translational repression, and transcriptional gene silencing (TGS). During a search for candidate mammalian factors for TGS, we purified a complex that contains small RNAs and Riwi, the rat homolog to human Piwi. The RNAs, frequently 29 to 30 nucleotides in length, are called Piwi-interacting RNAs (piRNAs), 94% of which map to 100 defined (< or = 101 kb) genomic regions. Within these regions, the piRNAs generally distribute across only one genomic strand or distribute on two strands but in a divergent, nonoverlapping manner. Preparations of piRNA complex (piRC) contain rRecQ1, which is homologous to qde-3 from Neurospora, a gene implicated in silencing pathways. Piwi has been genetically linked to TGS in flies, and slicer activity cofractionates with the purified complex. These results are consistent with a gene-silencing role for piRC in mammals.