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1.  A novel report of cIg-FISH and cytogenetics in POEMS syndrome 
American journal of hematology  2008;83(11):840-841.
POEMS syndrome is a plasma cell proliferative disorder whose pathogenesis is poorly understood. We provide the first report of cytoplasmic immunoglobulin/FISH testing (cIg-FISH) in POEMS syndrome using established myeloma markers. We reviewed all 37 POEMS cases seen at our institution in which cIg-FISH testing had been obtained. Monosomy 13 was seen in 14 of the 37 (38%) cIg-FISH samples. One patient had trisomy 3 and 7. Three patients had IgH translocation t(11;14)(q13;q32). No abnormalities were seen at 17p13(p53). The monosomy 13 is in line with other plasma cell disorders while the low prevalence of hyperdiploidy and abnormalities at 14q32 is unique.
doi:10.1002/ajh.21285
PMCID: PMC3956589  PMID: 18839437
2.  Monitoring the initial delivery of an oncolytic measles virus encoding the human sodium iodide symporter to solid tumors using contrast-enhanced computed tomography 
The journal of gene medicine  2012;14(0):590-597.
Background
We aimed to determine the feasibility of monitoring viral delivery and initial distribution to solid tumors using iodinated contrast agent and micro-computed tomography (CT).
Methods
Human BxPC-3 pancreatic tumor xenografts were established in nude mice. An oncolytic measles virus with an additional transcriptional unit encoding the sodium iodide symporter (NIS), as a reporter for viral infection, was mixed with a 1:10 dilution of Omnipaque 300 (GE Healthcare, Milwaukee, WI, USA) contrast agent and injected directly into tumors. Mice were imaged with micro-CT immediately before and after injection to determine the location of contrast agent/virus mixture. Mice were imaged again on day 3 after injection with micro-single-photon emission CT/CT to determine the location of NIS-mediated 99mTcO4 transport.
Results
A 1:10 dilution of Omnipaque had no effect on viral infectivity or cell viability in vitro and was more than adequate for CT imaging of the intratumoral injectate distribution. The volume of tumor coverage with initial CT contrast agent and the 3-day postinfection measurement of virally infected tumor volume were significantly correlated. Additionally, regions of the tumor that did not receive contrast agent from the initial injection were largely devoid of viral infection at early time points.
Conclusions
Contrast-enhanced viral delivery enables a rapid and accurate prediction of the initial viral distribution within a solid tumor. This technique should enable real-time monitoring of viral propagation from initially infected tumor regions to adjacent tumor regions.
doi:10.1002/jgm.2670
PMCID: PMC3930067  PMID: 23015290
iodinated contrast agent; Omnipaque; oncolytic measles; pancreatic cancer; sodium-iodide symporter; SPECT/CT
3.  Measles virus for cancer therapy 
Measles virus offers an ideal platform from which to build a new generation of safe, effective oncolytic viruses. Occasional "spontaneous" tumor regressions have occurred during natural measles infections, but common tumors do not express SLAM, the wild-type MV receptor, and are therefore not susceptible to the virus. Serendipitously, attenuated vaccine strains of measles virus have adapted to use CD46, a regulator of complement activation that is expressed in higher abundance on human tumor cells than on their non transformed counterparts. For this reason, attenuated measles viruses are potent and selective oncolytic agents showing impressive antitumor activity in mouse xenograft models. The viruses can be engineered to enhance their tumor specificity, increase their antitumor potency and facilitate noninvasive in vivo monitoring of their spread. A major impediment to the successful deployment of oncolytic measles viruses as anticancer agents is the high prevalence of pre-existing anti measles immunity, which impedes bloodstream delivery and curtails intratumoral virus spread. It is hoped that these problems can be addressed by delivering the virus inside measles-infected cell carriers and/or by concomitant administration of immunosuppressive drugs. From a safety perspective, population immunity provides an excellent defense against measles spread from patient to carers and, in fifty years of human experience, reversion of attenuated measles to a wild type pathogenic phenotype has not been observed. Clinical trials testing oncolytic measles viruses as an experimental cancer therapy are currently underway.
PMCID: PMC3926122  PMID: 19203112
4.  Neuroattenuation of Vesicular Stomatitis Virus through Picornaviral Internal Ribosome Entry Sites 
Journal of Virology  2013;87(6):3217-3228.
Vesicular stomatitis virus (VSV) is potent and a highly promising agent for the treatment of cancer. However, translation of VSV oncolytic virotherapy into the clinic is being hindered by its inherent neurotoxicity. It has been demonstrated that selected picornaviral internal ribosome entry site (IRES) elements possess restricted activity in neuronal tissues. We therefore sought to determine whether the picornavirus IRES could be engineered into VSV to attenuate its neuropathogenicity. We have used IRES elements from human rhinovirus type 2 (HRV2) and foot-and-mouth disease virus (FMDV) to control the translation of the matrix gene (M), which plays a major role in VSV virulence. In vitro studies revealed slowed growth kinetics of IRES-controlled VSVs in most of the cell lines tested. However, in vivo studies explicitly demonstrated that IRES elements of HRV2 and FMDV severely attenuated the neurovirulence of VSV without perturbing its oncolytic potency.
doi:10.1128/JVI.02984-12
PMCID: PMC3592162  PMID: 23283963
5.  Lenalidomide, cyclophosphamide and dexamethasone (CRd) for newly diagnosed multiple myeloma: Results from a phase 2 trial 
American journal of hematology  2011;86(8):640-645.
The combination of lenalidomide and low-dose dexamethasone is an effective treatment for multiple myeloma (MM). Addition of alkylating agents to lenalidomide or thalidomide results in increased response rates and deeper responses. We designed this trial to study the combination of cyclophosphamide, lenalidomide, and dexamethasone (CRd) as initial therapy for MM. Fifty-three patients with previously untreated symptomatic MM was enrolled. Patients received 4-week treatment cycles consisting of lenalidomide (25 mg daily for 3 weeks), dexamethasone (40 mg weekly), and cyclophosphamide (300 mg/m2 weekly for 3 weeks). A partial response or better was seen in 85% of patients including 47% with a very good partial response or better. The toxicities were manageable with over 80% of planned doses delivered; six patients went off study for toxicity. The median progression free survival (PFS) for the entire group was 28 months (95% CI: 22.7–32.6) and the overall survival (OS) at 2 years was 87% (95% CI: 78–96). Importantly, 14 patients with high-risk MM had similar PFS and OS as the standard-risk patients (n = 39). CRd is an effective and well-tolerated regimen for upfront therapy of MM with high response rates and excellent 2-year OS, and is suitable for long-term therapy. Am. J. Hematol. 86:640–645, 2011.
doi:10.1002/ajh.22053
PMCID: PMC3901994  PMID: 21630308
6.  ONCOLYTIC VIROTHERAPY 
Nature biotechnology  2012;30(7):10.1038/nbt.2287.
Oncolytic virotherapy is an emerging treatment modality which uses replication competent viruses to destroy cancers. Advances in the past two years include preclinical proof of feasibility for a single-shot virotherapy cure, identification of drugs that accelerate intratumoral virus propagation, new strategies to maximize the immunotherapeutic potential of oncolytic virotherapy, and clinical confirmation of a critical viremic thereshold for vascular delivery and intratumoral virus replication. The primary clinical milestone was completion of accrual in a phase III trial of intratumoral herpes simplex virus therapy using talimogene laherparepvec for metastatic melanoma. Challenges for the field are to select ‘winners’ from a burgeoning number of oncolytic platforms and engineered derivatives, to transiently suppress but then unleash the power of the immune system to maximize both virus spread and anticancer immunity, to develop more meaningful preclinical virotherapy models and to manufacture viruses with orders of magnitude higher yields compared to established vaccine manufacturing processes.
doi:10.1038/nbt.2287
PMCID: PMC3888062  PMID: 22781695
7.  Engineering Oncolytic Measles Virus to Circumvent the Intracellular Innate Immune Response 
The innate antiviral responses of tumor cells are often impaired but may still be sufficient to impede the intratumoral spread of an oncolytic virus. Here, we establish that the oncolytic measles virus (MV-eGFP) induces interferon (IFN) production in human myeloma and ovarian cancer cells. In addition, MV gene expression and virus progeny production were inhibited by IFN treatment of these tumor cells. The P gene of wild-type measles virus encodes P/V/C proteins known to antagonize IFN induction and/or response. We therefore engineered MV-eGFP for IFN evasion and more efficient intratumoral spread by arming it with the P gene from wild-type IC-B strain MV, thus generating MV-eGFP-Pwt. The chimeric virus exhibited reduced IFN sensitivity and diminished capacity to induce IFN in BJAB lymphoma, ARH-77 myeloma cells, and activated peripheral blood mononuclear cells. Interestingly, unlike the wild-type MV, MV-eGFP-Pwt was unable to shut down IFN induction completely. In immunocompromised mice bearing human myeloma xenografts, intravenously administered MV-eGFP-Pwt showed significantly enhanced oncolytic potency compared to MV-eGFP. These results indicate that oncolytic viruses are subject to control by the innate immune defenses of human tumor cells and may therefore be more effective if their natural ability to combat innate immunity is maintained.
doi:10.1038/sj.mt.6300076
PMCID: PMC3833616  PMID: 17245355
8.  A new paradigm in viral resistance 
Cell Research  2012;22(11):1515-1517.
doi:10.1038/cr.2012.139
PMCID: PMC3494385  PMID: 23033124
9.  Oncolytic Measles Virus Encoding Thyroidal Sodium Iodide Symporter for Squamous Cell Cancer of the Head and Neck Radiovirotherapy 
Human Gene Therapy  2011;23(3):295-301.
Abstract
Oncolytic measles virus (MV) encoding the human thyroidal sodium iodide symporter (MV-NIS) has proved to be safe after intraperitoneal or intravenous administration in patients with ovarian cancer or multiple myeloma, respectively, but it has not yet been administered through intratumoral injection in humans. Squamous cell carcinoma (SCC) of the head and neck (SCCHN) usually is locally invasive and spreads to the cervical lymph nodes, which are suitable for the intratumoral administration of oncolytic viruses. To test whether oncolytic MV is an effective treatment for SCCHN, we used oncolytic MV-NIS to infect SCCHN in vitro and in vivo. The data show that SCCHN cells were infected and killed by MV-NIS in vitro. Permissiveness of the tumor cells to MV infection was not affected by irradiation after viral addition. Monitored noninvasively through radioiodine-based single-photon emission computed tomography/computed tomography, intratumorally virus-delivered NIS has concentrated the radioiodine in the MV-NIS–treated tumors in the FaDu mouse xenograft model of human SCCHN, and the antitumor effect could be boosted significantly (p<0.05) either with concomitant cyclophosphamide therapy or with appropriately timed administration of radioiodine 131I. MV-NIS could be a promising new anticancer agent that may substantially enhance the outcomes of standard therapy after intratumoral administration in patients with locally advanced SCCHN.
Li and colleagues investigate the use of oncolytic measles virus encoding human thyroidal sodium iodide symporter (MV-NIS) to treat squamous cell carcinoma of the head and neck (SCCHN) in vitro and in vivo. MV-NIS-treated tumors are able to concentrate administered radioiodine in a mouse xenograft model of human SCCHN, and the antitumor effect is significantly boosted by cyclophosphamide therapy.
doi:10.1089/hum.2011.128
PMCID: PMC3300082  PMID: 22235810
10.  PEGylation of Vesicular Stomatitis Virus Extends Virus Persistence in Blood Circulation of Passively Immunized Mice 
Journal of Virology  2013;87(7):3752-3759.
We are developing oncolytic vesicular stomatitis viruses (VSVs) for systemic treatment of multiple myeloma, an incurable malignancy of antibody-secreting plasma cells that are specifically localized in the bone marrow. One of the presumed advantages for using VSV as an oncolytic virus is that human infections are rare and preexisting anti-VSV immunity is typically lacking in cancer patients, which is very important for clinical success. However, our studies show that nonimmune human and mouse serum can neutralize clinical-grade VSV, reducing the titer by up to 4 log units in 60 min. In addition, we show that neutralizing anti-VSV antibodies negate the antitumor efficacy of VSV, a concern for repeat VSV administration. We have investigated the potential use of covalent modification of VSV with polyethylene glycol (PEG) or a function-spacer-lipid (FSL)–PEG construct to inhibit serum neutralization and to limit hepatosplenic sequestration of systemically delivered VSV. We report that in mice passively immunized with neutralizing anti-VSV antibodies, PEGylation of VSV improved the persistence of VSV in the blood circulation, maintaining a more than 1-log-unit increase in VSV genome copies for up to 1 h compared to the genome copy numbers for the non-PEGylated virus, which was mostly cleared within 10 min after intravenous injection. We are currently investigating if this increase in PEGylated VSV circulating half-life can translate to increased virus delivery and better efficacy in mouse models of multiple myeloma.
doi:10.1128/JVI.02832-12
PMCID: PMC3624195  PMID: 23325695
11.  Potent systemic therapy of Multiple Myeloma utilizing Oncolytic Vesicular stomatitis virus coding for Interferon-beta 
Cancer Gene Therapy  2012;19(7):443-450.
Multiple myeloma (MM) is an incurable malignancy of plasma secreting B-cells disseminated in the bone marrow. Successful utilization of oncolytic virotherapy for myeloma treatment requires a systemically administered virus that selectively destroys disseminated myeloma cells in an immune-competent host. Vesicular stomatitis virus (VSV) expressing Interferon-β (IFNβ) is a promising new oncolytic agent that exploits tumor-associated defects in innate immune signaling pathways to specifically destroy cancer cells. We demonstrate here that a single, intravenous dose of VSV-IFNβ specifically destroys subcutaneous and disseminated 5TGM1 myeloma in an immune competent myeloma model. VSV-IFN treatment significantly prolonged survival in mice bearing orthotopic myeloma. Viral murine IFNβ expression further delayed myeloma progression and significantly enhanced survival compared to VSV expressing human IFNβ. Evaluation of VSV-IFNβ oncolytic activity in human myeloma cell lines and primary patient samples confirmed myeloma specific oncolytic activity but revealed variable susceptibility to VSV-IFNβ oncolysis. The results indicate that VSV-IFNβ is a potent, safe oncolytic agent that can be systemically administered to effectively target and destroy disseminated myeloma in immune competent mice. IFNβ expression improves cancer specificity and enhances VSV therapeutic efficacy against disseminated myeloma. These data show VSV-IFNβ to be a promising vector for further development as a potential therapy for treatment of Multiple myeloma.
doi:10.1038/cgt.2012.14
PMCID: PMC3380174  PMID: 22522623
Oncolytic; virotherapy; myeloma; Vesicular stomatitis virus; systemic
12.  Mathematical Model for Radial Expansion and Conflation of Intratumoral Infectious Centers Predicts Curative Oncolytic Virotherapy Parameters 
PLoS ONE  2013;8(9):e73759.
Simple, inductive mathematical models of oncolytic virotherapy are needed to guide protocol design and improve treatment outcomes. Analysis of plasmacytomas regressing after a single intravenous dose of oncolytic vesicular stomatitis virus in myeloma animal models revealed that intratumoral virus spread was spatially constrained, occurring almost exclusively through radial expansion of randomly distributed infectious centers. From these experimental observations we developed a simple model to calculate the probability of survival for any cell within a treated tumor. The model predicted that small changes to the density of initially infected cells or to the average maximum radius of infected centers would have a major impact on treatment outcome, and this was confirmed experimentally. The new model provides a useful and flexible tool for virotherapy protocol optimization.
doi:10.1371/journal.pone.0073759
PMCID: PMC3770695  PMID: 24040057
13.  Induction of antiviral genes by the tumor microenvironment confers resistance to virotherapy 
Scientific Reports  2013;3:2375.
Oncolytic viruses obliterate tumor cells in tissue culture but not against the same tumors in vivo. We report that macrophages can induce a powerfully protective antiviral state in ovarian and breast tumors, rendering them resistant to oncolytic virotherapy. These tumors have activated JAK/STAT pathways and expression of interferon-stimulated genes (ISGs) is upregulated. Gene expression profiling (GEP) of human primary ovarian and breast tumors confirmed constitutive activation of ISGs. The tumors were heavily infiltrated with CD68+ macrophages. Exposure of OV-susceptible tumor cell lines to conditioned media from RAW264.7 or primary macrophages activated antiviral ISGs, JAK/STAT signaling and an antiviral state. Anti-IFN antibodies and shRNA knockdown studies show that this effect is mediated by an extremely low concentration of macrophage-derived IFNβ. JAK inhibitors reversed the macrophage-induced antiviral state. This study points to a new role for tumor-associated macrophages in the induction of a constitutive antiviral state that shields tumors from viral attack.
doi:10.1038/srep02375
PMCID: PMC3736178  PMID: 23921465
14.  Oncolytic measles virus retargeting by ligand display 
Despite significant advances in recent years, treatment of metastatic malignancies remains a significant challenge. There is an urgent need for development of novel therapeutic approaches. Virotherapy approaches have considerable potential and among them measles virus (MV) vaccine strains have emerged as one of the most promising oncolytic platforms. Retargeted MV strains deriving from the Edmonston vaccine lineage (MV-Edm) have shown comparable antitumor efficacy to unmodified strains against receptor expressing tumor cells with improved therapeutic index. Here we describe the construction, rescue, amplification and titration of fully retargeted MV-Edm derivatives displaying tumor specific receptor binding ligands on the viral surface in combination with CD46 and SLAM entry H ablating mutations.
doi:10.1007/978-1-61779-340-0_11
PMCID: PMC3691680  PMID: 21948475
oncolytic measles virus; measles retargeting; virotherapy; measles engineering
15.  Discordance between serum cardiac biomarker and immunoglobulin-free light-chain response in patients with immunoglobulin light-chain amyloidosis treated with immune modulatory drugs 
American journal of hematology  2010;85(10):757-759.
We evaluated the capability of soluble cardiac biomarkers to predict tolerability and outcomes of IMiD-containing treatments among 106 patients treated on clinical trials. Baseline elevations in troponin T (TnT) and N-terminal brain naturietic protein (NT-proBNP) predicted for an inability to tolerate IMiD-based regimens. The best predictors for early attrition during cycle 1 were TnT ≥ 0.07 μg/L and NT-proBNP ≥ 11,939 ng/L. NT-proBNP-response under-performed TnT-response as a predictor for overall survival (OS), but both predicted for early protocol attrition. Despite hematologic response, IMiD-treated patients were at higher risk for NT-proBNP rises and early drug discontinuation than a control population but not for early death. These observations prompt two questions: (1) does IMiD-based therapy lead to increased fluid retention and/or cardiac toxicity and (2) is an NT-proBNP-driven cardiac response system valid in IMiD-treated amyloidosis patients? Recognition of potential drug-induced cardiac toxicity is important so that increased cardiac surveillance and drug dose-adjustment or discontinuation may be implemented.
doi:10.1002/ajh.21822
PMCID: PMC3691013  PMID: 20872958
16.  Retargeting Vesicular Stomatitis Virus Using Measles Virus Envelope Glycoproteins 
Human Gene Therapy  2011;23(5):484-491.
Abstract
Oncolytic vesicular stomatitis virus (VSV) has potent antitumor activity, but infects a broad range of cell types. Here, we used the measles virus (MV) hemagglutinin (H) and fusion (F) envelope glycoproteins to redirect VSV entry and infection specifically to tumor-associated receptors. Replication-defective VSV, deleted of its glycoprotein gene (VSVΔG), was pseudotyped with MV-F and MV-H displaying single-chain antibodies (scFv) specific for epidermal growth factor receptor (EGFR), folate receptor (FR), or prostate membrane-specific antigen (PSMA). Viral titers were ∼105 PFU/ml, but could be concentrated to 107 PFU/ml. Immunoblotting confirmed incorporation of the MV-H-scFv and MV-F into functional VSV virions. Although VSV-G was able to infect all tumor cell lines tested, the retargeted VSV infected only cells that expressed the targeted receptor. In vivo specificities of the EGFR-, FR-, and PSMA-retargeted VSV were assessed by intratumoral injection into human tumor xenografts. Analysis of green fluorescent protein reporter gene expression indicated that VSV infection was restricted to receptor-positive tumors. In summary, we have demonstrated for the first time that VSV can be efficiently retargeted to different cellular receptors using the measles display technology, yielding retargeted VSV vectors that are highly specific for tumors that express the relevant receptor.
Ayala-Breton and colleagues use the measles virus (MV) hemagglutinin (H) and fusion (F) envelope glycoproteins to redirect vesicular stomatitis virus (VSV) entry and infection specifically to tumor-associated receptors such as epidermal growth factor receptor, folate receptor, and prostate membrane-specific antigen. In vivo expression of the all retargeted VSV was restricted to receptor-positive human tumor xenografts.
doi:10.1089/hum.2011.146
PMCID: PMC3360499  PMID: 22171635
17.  Oncolytic measles virus prolongs survival in a murine model of cerebral spinal fluid–disseminated medulloblastoma 
Neuro-Oncology  2012;14(4):459-470.
Medulloblastoma is the most common malignant brain tumor of childhood. Although the survival rate of afflicted children has improved considerably over the past several years, a subset of these patients will present with disseminated disease and face a much bleaker prognosis. In addition, patients may present with disseminated disease at recurrence. We previously demonstrated the efficacy of a recombinant oncolytic measles virus (MV) to treat localized medulloblastoma in a mouse xenograft model. In the present study, we sought to extend our findings to the treatment of disseminated disease. To this end, we developed and characterized a mouse xenograft model of disseminated medulloblastoma using serial bioluminescent imaging techniques in combination with histopathological examination. Mice injected with medulloblastoma cells into their right lateral ventricle showed tumor growth in their ventricles and in both intracranial and spinal subarachnoid spaces, closely recapitulating the human disease. Subsequent intraventricular administration of MV resulted in stabilization and shrinkage of the tumor, significantly prolonging the survival of the treated animals, compared with those treated with an inactivated virus. These data demonstrate that oncolytic MV may be of use in treating disseminated medulloblastoma. In addition, our protocol of intraventricular tumor cell injection, followed by bioluminescent imaging coupled with histopathological examination, provides a model for use in evaluating future recombinant oncolytic viruses and other preclinical therapeutic approaches for disseminated medulloblastoma.
doi:10.1093/neuonc/nor231
PMCID: PMC3309851  PMID: 22307474
bioluminescence; dissemination; measles virus; medulloblastoma; oncolytic virus
18.  Revised Prognostic Staging System for Light Chain Amyloidosis Incorporating Cardiac Biomarkers and Serum Free Light Chain Measurements 
Journal of Clinical Oncology  2012;30(9):989-995.
Purpose
Cardiac involvement predicts poor prognosis in light chain (AL) amyloidosis, and the current prognostic classification is based on cardiac biomarkers troponin-T (cTnT) and N-terminal pro–B-type natriuretic peptide (NT-ProBNP). However, long-term outcome is dependent on the underlying plasma cell clone, and incorporation of clonal characteristics may allow for better risk stratification.
Patients and Methods
We developed a prognostic model based on 810 patients with newly diagnosed AL amyloidosis, which was further examined in two other datasets: 303 patients undergoing stem-cell transplantation, and 103 patients enrolled onto different clinical trials.
Results
We examined the prognostic value of plasma cell–related characteristics (ie, difference between involved and uninvolved light chain [FLC-diff], marrow plasma cell percentage, circulating plasma cells, plasma cell labeling index, and β2 microglobulin). In a multivariate model that included these characteristics as well as cTnT and NT-ProBNP, only FLC-diff, cTnT, and NT-ProBNP were independently prognostic for overall survival (OS). Patients were assigned a score of 1 for each of FLC-diff ≥ 18 mg/dL, cTnT ≥ 0.025 ng/mL, and NT-ProBNP ≥ 1,800 pg/mL, creating stages I to IV with scores of 0 to 3 points, respectively. The proportions of patients with stages I, II, III and IV disease were 189 (25%), 206 (27%), 186 (25%) and 177 (23%), and their median OS from diagnosis was 94.1, 40.3, 14, and 5.8 months, respectively (P < .001). This classification system was validated in the other datasets.
Conclusion
Incorporation of serum FLC-diff into the current staging system improves risk stratification for patients with AL amyloidosis and will help develop risk-adapted therapies for AL amyloidosis.
doi:10.1200/JCO.2011.38.5724
PMCID: PMC3675680  PMID: 22331953
19.  Curative one-shot systemic virotherapy in murine myeloma 
Current therapy for multiple myeloma is complex and prolonged. Antimyeloma drugs are combined in induction, consolidation and/or maintenance protocols to destroy bulky disease, then suppress or eradicate residual disease. Oncolytic viruses have the potential to mediate both tumor debulking and residual disease elimination, but this curative paradigm remains unproven. Here we engineered an oncolytic vesicular stomatitis virus to minimize its neurotoxicity, enhance induction of antimyeloma immunity, and facilitate noninvasive monitoring of its intratumoral spread. Using high resolution imaging, autoradiography and immunohistochemistry, we demonstrate that the intravenously administered virus extravasates from tumor blood vessels in immunocompetent myeloma-bearing mice, nucleating multiple intratumoral infectious centers which expand rapidly and necrose at their centers, ultimately coalescing to cause extensive tumor destruction. This oncolytic tumor debulking phase lasts only for 72 hours after virus administration, and is completed before antiviral antibodies become detectable in the bloodstream. Anti-myeloma T cells, cross-primed as the virus-infected cells provoke an antiviral immune response, then eliminate residual uninfected myeloma cells. The study establishes a curative oncolytic paradigm for multiple myeloma where direct tumor debulking and immune eradication of minimal disease are mediated by a single intravenous dose of a single therapeutic agent. Clinical translation is underway.
doi:10.1038/leu.2012.70
PMCID: PMC3411853  PMID: 22425894
Oncolytic Virotherapy; multiple myeloma; Vesicular Stomatitis Virus; Intravenous; Immunotherapy
20.  Treatment of medulloblastoma using an oncolytic measles virus encoding the thyroidal sodium iodide symporter shows enhanced efficacy with radioiodine 
BMC Cancer  2012;12:508.
Background
Medulloblastoma is the most common malignant brain tumor of childhood. Although the clinical outcome for medulloblastoma patients has improved significantly, children afflicted with the disease frequently suffer from debilitating side effects related to the aggressive nature of currently available therapy. Alternative means for treating medulloblastoma are desperately needed. We have previously shown that oncolytic measles virus (MV) can selectively target and destroy medulloblastoma tumor cells in localized and disseminated models of the disease. MV-NIS, an oncolytic measles virus that encodes the human thyroidal sodium iodide symporter (NIS), has the potential to deliver targeted radiotherapy to the tumor site and promote a localized bystander effect above and beyond that achieved by MV alone.
Methods
We evaluated the efficacy of MV-NIS against medulloblastoma cells in vitro and examined their ability to incorporate radioiodine at various timepoints, finding peak uptake at 48 hours post infection. The effects of MV-NIS were also evaluated in mouse xenograft models of localized and disseminated medulloblastoma. Athymic nude mice were injected with D283med-Luc medulloblastoma cells in the caudate putamen (localized disease) or right lateral ventricle (disseminated disease) and subsequently treated with MV-NIS. Subsets of these mice were given a dose of 131I at 24, 48 or 72 hours later.
Results
MV-NIS treatment, both by itself and in combination with 131I, elicited tumor stabilization and regression in the treated mice and significantly extended their survival times. Mice given 131I were found to concentrate radioiodine at the site of their tumor implantations. In addition, mice with localized tumors that were given 131I either 24 or 48 hours after MV-NIS treatment exhibited a significant survival advantage over mice given MV-NIS alone.
Conclusions
These data suggest MV-NIS plus radioiodine may be a potentially useful therapy for the treatment of medulloblastoma.
doi:10.1186/1471-2407-12-508
PMCID: PMC3517484  PMID: 23134812
Medulloblastoma; Measles virus; Sodium iodide symporter; Targeted radiotherapy
21.  Concordant activity of transgene expression cassettes inserted into E1, E3 and E4 cloning sites in the adenovirus genome 
Background
Expression cassettes can be inserted at several positions into recombinant adenoviral genomes but the implications of this choice for transgene expression level have not been determined. Knowledge of the relative expression levels of transgenes inserted at different sites in the adenoviral genome is of particular significance for transgene expression monitoring approaches that rely on the concordant expression of a marker transgene inserted elsewhere in the viral genome.
Methods
Three expression cassettes, each comprising a cytomegalovirus promoter driving one of three marker peptides [serum carcinoembryonic antigen (sCEA), beta subunit of human chorionic gonadotropin (βhCG) or human sodium iodide symporter (hNIS)], were inserted into E1, E3 or E4 cloning sites in a recombinant adenoviral vector backbone. High titer stocks of bicistronic adenoviral vectors coding for combinations of marker peptides were prepared. A panel of human cells of various lineages was infected with the vectors and expression ratios of the transgene-encoded proteins were analysed. Serum levels of the soluble proteins and hepatic uptake of radioactive iodine were also compared in vivo in nude rats after intravenous vector infusion.
Results
High concordance of expression between the inserted transgenes was observed in all of the bicistronic vectors irrespective of whether the expression cassettes were placed in the E1, E3 or E4 regions. Concordance was maintained across multiple cell lineages. In vivo, in athymic rats, blood and urine levels of βhCG were highly concordant with serum levels of sCEA at all timepoints after intravenous infusion of the bicistronic vectors encoding both of these soluble markers. Hepatic radioiodine uptake was concordant with serum CEA concentration in mice infused with a bicistronic vector expressing CEA and NIS.
Conclusions
The expression level of a given transgene in an adenoviral vector genome can be accurately and quantitatively inferred from the expression of a marker protein encoded by a second transgene inserted elsewhere in the vector genome.
doi:10.1002/jgm.1289
PMCID: PMC2821815  PMID: 19140107
adenoviral vector; βhCG; CEA; gene expression concordance; sodium iodide symporter
22.  Tumor-associated macrophages infiltrate plasmacytomas and can serve as cell carriers for oncolytic measles virotherapy of disseminated myeloma 
American journal of hematology  2009;84(7):401-407.
In multiple myeloma, some of the neoplastic plasma cells are diffusely dispersed among the normal bone marrow cells (bone marrow resident), whereas others are located in discrete, well-vascularized solid tumors (plasmacytomas) that may originate in bone or soft tissue. Interactions between bone marrow-resident myeloma cells and bone marrow stromal cells (BMSCs) are important determinants of myeloma pathogenesis. However, little is known of the factors sustaining myeloma growth and cell viability at the centers of expanding plasmacytomas, where there are no BMSCs. Histologic sections of 22 plasmacytomas from myeloma patients were examined after immunostaining. Abundant CD68+, CD163+, S100-negative macrophage infiltrates were identified in all tumors, accompanied by scattered collections of CD3+ T lymphocytes. The CD68+ tumor-associated macrophages (TAM) accounted for 2– 12% of nucleated cells and were evenly distributed through the parenchyma. The TAM generally had dendritic morphology, and each dendrite was in close contact with multiple plasma cells. In some cases, the TAM were strikingly clustered around CD34+ blood vessels. To determine whether cells of the monocytic lineage might be exploitable as carriers for delivery of therapeutic agents to plasmacytomas, primary human CD14+ cells were infected with oncolytic measles virus and administered intravenously to mice bearing KAS6/1 human myeloma xenografts. The cell carriers localized to KAS6/1 tumors, where they transferred MV infection to myeloma cells and prolonged the survival of mice bearing disseminated human myeloma disease. Thus, TAM are a universal stromal component of the plasmacytomas of myeloma patients and may offer a promising new target for therapeutic exploitation.
doi:10.1002/ajh.21444
PMCID: PMC2805200  PMID: 19507209
23.  Pinhole Micro-SPECT/CT for Noninvasive Monitoring and Quantitation of Oncolytic Virus Dispersion and Percent Infection in Solid Tumors 
Gene Therapy  2011;19(3):279-287.
The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). NIS RNA level and dispersion pattern were determined in control and MV-NIS infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography, and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with 123I or 99TcO4 micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r2 = 0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from noninfected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection and can replace more time-consuming and expensive analyses (eg, autoradiography and IHC) that require animal sacrifice.
doi:10.1038/gt.2011.107
PMCID: PMC3198860  PMID: 21753796
NIS; measles virus; micro-SPECT/CT; oncolytic virus; quantitation; sodium iodide symporter
24.  Polyinosinic acid decreases sequestration and improves systemic therapy of measles virus 
Cancer Gene Therapy  2011;19(3):202-211.
Off target binding or vector sequestration can significantly limit the efficiency of systemic virotherapy. We report here that systemically administered oncolytic measles virus (MV) was rapidly sequestered by the mononuclear phagocytic system (MPS) of the liver and spleen in measles receptor CD46-positive and CD46-negative mice. Since scavenger receptors on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated here if MV uptake was mediated by scavenger receptors on Kupffer cells. Pretreatment of cells with poly(I), a scavenger receptor ligand, reduced MV expression by 99% in murine (J774A.1) macrophages and by 50% in human (THP-1) macrophages. Pre-dosing of mice with poly(I) reduced MPS sequestration of MV and increased circulating levels of MV by 4 to 15-folds at 2 minutes post virus administration. Circulating virus was still detectable 30 mins post infusion in mice predosed with poly(I) while no detectable MV was found at 5–10 min post infusion if mice did not receive poly(I). MPS blockade by poly(I) enhanced virus delivery to human ovarian SKOV3ip.1 and myeloma KAS6/1 xenografts in mice. Higher gene expression and improved control of tumor growth was noted early post therapy. Based on these results, incorporation of MPS blockade into MV treatment regimens is warranted.
doi:10.1038/cgt.2011.82
PMCID: PMC3288770  PMID: 22116376
oncolytic measles virus; mononuclear phagocytic system; sequestration; poly(I); scavenger receptor; SKOV3ip.1; KAS6/1
25.  The importance of Anatolian mountains as the cradle of global diversity in Arabis alpina, a key arctic–alpine species 
Annals of Botany  2011;108(2):241-252.
Background and Aims
Anatolia is a biologically diverse, but phylogeographically under-explored region. It is described as either a centre of origin and long-term Pleistocene refugium, or as a centre for genetic amalgamation, fed from distinct neighbouring refugia. These contrasting hypotheses are tested through a global phylogeographic analysis of the arctic–alpine herb, Arabis alpina.
Methods
Herbarium and field collections were used to sample comprehensively the entire global range, with special focus on Anatolia and Levant. Sequence variation in the chloroplast DNA trnL-trnF region was examined in 483 accessions. A haplotype genealogy was constructed and phylogeographic methods, demographic analysis and divergence time estimations were used to identify the centres of diversity and to infer colonization history.
Key Results
Fifty-seven haplotypes were recovered, belonging to three haplogroups with non-overlapping distributions in (1) North America/Europe/northern Africa, (2) the Caucuses/Iranian Plateau/Arabian Peninsula and (3) Ethiopia–eastern Africa. All haplogroups occur within Anatolia, and all intermediate haplotypes linking the three haplogroups are endemic to central Anatolia and Levant, where haplotypic and nucleotide diversities exceeded all other regions. The local pattern of haplotype distribution strongly resembles the global pattern, and the haplotypes began to diverge approx. 2·7 Mya, coinciding with the climate cooling of the early Middle Pleistocene.
Conclusions
The phylogeographic structure of Arabis alpina is consistent with Anatolia being the cradle of origin for global genetic diversification. The highly structured landscape in combination with the Pleistocene climate fluctuations has created a network of mountain refugia and the accumulation of spatially arranged genotypes. This local Pleistocene population history has subsequently left a genetic imprint at the global scale, through four range expansions from the Anatolian diversity centre into Europe, the Near East, Arabia and Africa. Hence this study also illustrates the importance of sampling and scaling effects when translating global from local diversity patterns during phylogeographic analyses.
doi:10.1093/aob/mcr134
PMCID: PMC3143044  PMID: 21712298
Anatolia; centre of origin; Pleistocene glaciations; chloroplast trnL-F; divergence times; alpine plants; Arabis alpina

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