Citations

AbstractBackgroundWe aimed to determine the feasibility of monitoring viral delivery and initial distribution to solid tumors using iodinated contrast agent and micro‐computed tomography (CT).MethodsHuman 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.ResultsA 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.ConclusionsContrast‐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. Copyright © 2012 John Wiley & Sons, Ltd.

Abstract Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5-year survival rate of 20-30%. Mitotane is the only approved drug for the treatment of patients with ACC. It often carries significant toxicities which result in the discontinuation of treatment. There are no approved second line therapies. The increased incidence of ACC in the cancer predisposition syndrome, Li-Fraumeni, suggests the involvement of the p53 pathway in ACC pathogenesis. Our analysis of the gene expression profiles of 19 ACC samples identified dysregulation of the G2/M transition and the activity of the p53 modulator, MDM2 as important in ACC pathogenesis. Polo-like kinase-1 (PLK1) is involved in the G2/M transition and acts to promote MDM2 activity through its phosphorylation. We observed that PLK1 inhibition by siRNA results in up to a 70% reduction in viability in the ACC cell lines SW-13 and H295R. Therefore, we studied PLK1 inhibition as a potential therapeutic strategy. We used the small molecule inhibitor, BI-2536, to inhibit PLK1 function in SW-13 and H295R. Drug-dose response curves demonstrated that both cell lines are sensitive to pharmacological inhibition of PLK1 (IC50 doses of 0.0094815 μM and 0.062805 μM for SW-13 and H295R, respectively.) Murine xenograft studies demonstrated that BI-2536 resulted in a statistically significant reduction in tumor growth in SW-13 but not H295R. Examination of p53 protein levels in the presence of the drug showed a dose-dependent reduction in p53 levels in SW-13, which carries a homozygous p53 mutation. The same was not true in H295R, which is p53 wild-type. To test the hypothesis that BI-2536 was decreasing mutant p53 levels by promoting its proteasomal degradation, both cell lines were treated with previously determined inhibitory concentrations of BI-2536, either alone or in combination with the proteasome inhibitor, MG132. Western blot analysis showed recovery of p53 protein when cells were concomitantly treated with MG132, supporting a role for BI-2536 as a regulator of proteasomal degradation. Both ACC cell lines are relatively insensitive to MDM2 inhibition by nutlin-3 (NCI60 GI50 values range from ∼4 uM to ∼2 uM versus ACC cell lines @13-15uM), possibly because PLK1 stimulates MDM2 and renders nutlin-3 ineffective. We therefore assessed the ability of BI-2536 to sensitize ACC cell lines to the effects of the MDM2 inhibitor. BI-2636 did show synergy with nutlin-3, shifting the IC50 in SW-13 from 19.78 μM to 6.45 μM and from 12.75 μM to 2.84 μM in H295R. These results demonstrate that inhibition of PLK1 alone or in combination with an MDM2 inhibitor warrants further investigation as a treatment for patients with ACC. Cells with mutant p53 are more sensitive to the growth inhibitory effects of BI-2536. In the context of wild-type p53, the combined inhibition of both PLK1 and MDM2 results in loss of viability. Further clinical development of PLK1 as a target should take p53 mutation status into account. 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 978. doi:1538-7445.AM2012-978

Abstract Tumor-associated hypoxia facilitates malignant progression, metastasis and poor prognosis in prostate cancer (CaP); however, effects of oxygen tension (pO2) in CaP are just beginning to be investigated. To establish mechanisms whereby hypoxia enhances malignant properties and survival of CaP and to identify pO2-regulated tumor-associated macromolecules, we propagated CaP cell lines LnCaP, VCaP and DU145 in hypoxia (pO2=2 kPa) and compared them with normoxically grown cells (pO2=20 kPa); hypoxic cells proliferated faster and secreted more vascular endothelial growth factor (p<0.05). Transcriptome studies revealed different gene expression in cells grown in hypoxia relative to those in normoxia. Interestingly, in hypoxic cells transcripts associated with cancer and urologic disease were overexpressed in comparison to normoxic cells (p<0.001) suggesting an association of low pO2 and aggressive features of CaP. Further, we analyzed the transcriptome in primary CaP cells isolated by laser-capture microdissection (LCM) and whole CaP tissue. We compared the data to those from CaP cells cultured at pO2=20 kPa or pO2=2 kPa. Notably, hypoxia increased transcript levels for pyruvate dehydrogenase kinase isozyme 1, nuclear prelamin A recognition factor, glucose phosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase in all three cell lines (p<0.05) to levels comparable to those found in primary bulk tissue and LCM isolated cells (p<0.005). This finding suggests that gene expression in hypoxically cultured cells is more akin to that in tumor cells in situ than are cells grown normoxically. By 2D-gel electrophoresis, we found that change in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Our results suggest that hypoxia affects transcriptome and proteome in CaP cells. In addition, we screened 20 patient sera and 20 healthy control sera for spontaneous antibodies cross-reactive with VCaP cells and found that the sera reacted with numerous proteins, some previously reported to elicit an immune response in CaP patients (e.g., nucleoporin 62 and transitional endoplasmic reticulum ATPase). By this method we detected numerous novel autoantigens (under validation). Currently we are clarifying the pO2 effects on the relationship of transcriptome, proteome and tumor-associated antigens in CaP cells. Support: DOD PC094680 (CRG), Minnesota Partnership for Biotechnology and Medical Genomics, Mayo Clinic Prostate SPORE 5P50CA091956 (FK); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-308. doi:10.1158/1538-7445.AM2011-LB-308

Abstract In early clinical trials, vaccination with allogeneic cancer cells enhanced the overall survival in some patients with malignancies. Because the administered vaccine cells were cultured at pO2=20 kPa, it is unclear whether they provided an adequate antigen match to tumor cells in situ where pO2 is generally much lower. Thus, we postulate that hypoxically grown vaccine cells will provide a better antigen match to tumors in situ. We are testing this hypothesis by studying the effects of hypoxia on prostate cancer (CaP) cells. LnCaP and VCaP cells were cultured at pO2=2 kPa or 20 kPa and analyzed by 2-D gel electrophoresis. The difference in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Using a threshold of fivefold change, we found that hypoxia decreased the levels of thirteen proteins and increased of four in VCaP cells. These results are in line with the reports showing that hypoxia affects expression only of a small fraction of total cellular protein and that the content of total protein is not altered significantly. To determine if the hypoxia-dependent changes affected immune reactivity of the cells, we took advantage of the spontaneous autoantibodies against tumor-associated antigens (TAAs). Hence, we compared the binding to lysates of LnCaP and VCaP cells of antibodies pooled from CaP patients (n=25), healthy controls (n=25), rheumatoid arthritis (n=17), colorectal cancer (n=10) and lung cancer (n=10). CaP patient sera plasma reacted with numerous spots, some even in control groups and thus considered nonspecific. CaP sera plasma specifically bound to seven spots; four were hypoxia-specific. All selected spots were excised from the gel, trypsin-digested, and identified by MALDI-TOF mass spectrometry as heat shock 70 kDa protein 4; protein disulfide isomerase A3; heterogeneous nuclear ribonucleoprotein L and leucine-rich repeat-containing protein 47. With the exception of the latter, the proteins have been identified or validated as TAAs before, but apparently none in CaP. CaP patient antibodies bound to lysates of LnCaP cells grown at pO2=2 kPa, but less than to such VCaP cell lysates. Our results confirmed the findings that hypoxia affects the proteome mostly quantitatively. Lysates of hypoxic CaP cells revealed novel potential TAAs suggesting the potential immunologic relevance of antigens in the hypoxic proteome. Thus, pO2 control can provide a tool for the development of more effective immunotherapy for CaP and possibly other cancers. Support: DOD PC094680 (CRG), PCF Creativity Award (CRG), Mayo Clinic Prostate SPORE 5P50CA091956 (SV-P); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C223.

Abstract Introduction: CX-4945 is a novel, orally administered small molecule designed to potently and selectively inhibit protein kinase CK2, a previously unexploited molecular target with well documented roles in many cancers. In vitro, CX-4945 selectively kills cancer cells by modulating key survival pathways, resulting in inhibition of proliferation, promotion of apoptosis and cell cycle arrest. Pre-clinically, CX-4945 has demonstrated single-agent potency in suppressing xenograft tumor growth with a wide therapeutic window. The objectives of this phase I study are to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLTs), to characterize the pharmacokinetics (PKs), and to study the pharmacodynamic effects of CX-4945. Procedures: Eligible patients with advanced solid tumors, Castleman's disease or multiple myeloma with progressive disease, or for whom there are no available standard therapies, receive CX-4945 in successive dose cohorts at: 90, 160, 300, 460, 700 and 1000 mg per dose. Oral doses are administered twice daily for twenty-one consecutive days of a four week cycle. Therapy is continued in consenting patients until signs of intolerance to CX-4945 are observed, or there is evidence of advancing disease. Response by RECIST is determined after every 2 cycles. Serial blood and plasma samples are collected on the first and final dosing days of Cycle 1 (i.e., Day 1 and Day 21) for pharmacokinetic analysis and for pharmacodynamic biomarker evaluations (specifically, total and phosphorylated forms of p21 and Akt.) Results: Thirteen patients with solid tumors (3–4 patients per cohort, from four separate dose cohorts) have received oral doses of CX-4945. These doses have been well tolerated, with no reported adverse events of grade 3 or higher. CX-4945 has demonstrated general linearity in PK parameters between the dose cohorts, with a terminal half life of approximately 25 hours at steady state. Conclusions: To date, CX-4945 has shown no drug related toxicity and has dose proportional PKs. No DLTs have yet been observed, and the MTD remains to be defined in this Phase I study. Further enrollment to the planned dose escalation cohorts is ongoing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C39.

Abstract We performed a meta-analysis of 3 genome-wide association studies to identify additional common variants influencing chronic lymphocytic leukemia (CLL) risk. The discovery phase was composed of genome-wide association study data from 1121 cases and 3745 controls. Replication analysis was performed in 861 cases and 2033 controls. We identified a novel CLL risk locus at 6p21.33 (rs210142; intronic to the BAK1 gene, BCL2 antagonist killer 1; P = 9.47 × 10−16). A strong relationship between risk genotype and reduced BAK1 expression was shown in lymphoblastoid cell lines. This finding provides additional support for polygenic inheritance to CLL and provides further insight into the biologic basis of disease development.

Summary The guideline group regarding the diagnosis and management of myelofibrosis was selected to be representative of UK ‐based medical experts, together with a contribution from a single expert from the USA . MEDLINE and EMBASE were searched systematically for publications in E nglish from 1966 until A ugust 2011 using a variety of key words. The writing group produced the draft guideline, which was subsequently revised by consensus of the members of the General Haematology and Haemato‐oncology Task Forces of the British Committee for Standards in Haematology ( BCSH ). The guideline was then reviewed by a sounding board of UK haematologists, the BCSH and the British Society for Haematology Committee and comments incorporated where appropriate. The criteria used to state levels and grades of evidence are as outlined in the Procedure for Guidelines commissioned by the BCSH ; the ‘ GRADE ‘ system was used to score strength and quality of evidence. The objective of this guideline is to provide healthcare professionals with clear guidance on the investigation and management of primary myelofibrosis, as well as post‐polycythaemic myelofibrosis (post‐ PV MF ) and post‐thrombocythemic myelofibrosis (post‐ ET MF ) in both adult and paediatric patients.

SummaryFollowing the discovery of the Janus kinase (JAK) 2 V617F mutation in 2005 the explosion of research and drug development activity has not only advanced our understanding of the pathogenesis of myeloproliferative neoplasms (MPNs) but also triggered debate about classification, allowed revised diagnostic and response criteria, provided a target for treatment and a mode of monitoring its success. These changes and the resultant clinical research are discussed in this article where we argue that discovery of the JAK2 V617F mutation has signalled the much delayed change in therapeutic paradigm for myelofibrosis and possibly other MPNs from palliation and allowing us to move closer to, but not yet attain, a cure.