Citations

Objective Eosinophil predominant inflammation characterises histological features of eosinophilic oesophagitis (EoE). Endoscopy with biopsy is currently the only method to assess oesophageal mucosal inflammation in EoE. We hypothesised that measurements of luminal eosinophil-derived proteins would correlate with oesophageal mucosal inflammation in children with EoE. Design The Enterotest diagnostic device was used to develop an oesophageal string test (EST) as a minimally invasive clinical device. EST samples and oesophageal mucosal biopsies were obtained from children undergoing upper endoscopy for clinically defined indications. Eosinophil-derived proteins including eosinophil secondary granule proteins (major basic protein-1, eosinophil-derived neurotoxin, eosinophil cationic protein, eosinophil peroxidase) and Charcot–Leyden crystal protein/galectin-10 were measured by ELISA in luminal effluents eluted from ESTs and extracts of mucosal biopsies. Results ESTs were performed in 41 children with active EoE (n=14), EoE in remission (n=8), gastro-oesophageal reflux disease (n=4) and controls with normal oesophagus (n=15). EST measurement of eosinophil-derived protein biomarkers significantly distinguished between children with active EoE, treated EoE in remission, gastro-oesophageal reflux disease and normal oesophagus. Levels of luminal eosinophil-derived proteins in EST samples significantly correlated with peak and mean oesophageal eosinophils/high power field (HPF), eosinophil peroxidase indices and levels of the same eosinophil-derived proteins in extracts of oesophageal biopsies. Conclusions The presence of eosinophil-derived proteins in luminal secretions is reflective of mucosal inflammation in children with EoE. The EST is a novel, minimally invasive device for measuring oesophageal eosinophilic inflammation in children with EoE.

Abstract Background: Therapy with proteasome inhibitors (PIs) is an effective strategy against multiple myeloma (MM), but a significant proportion of patients is primarily resistant or eventually becomes refractory. Paradoxical activation of phospho-Akt, induction of HSP27 and excessive proteasome activity in relation to polyubiquitinated protein burden are all mechanisms for bortezomib resistance in MM cells in vitro. Preclinical data showed investigational drug MLN9708, a specific, reversible, orally bioavailable, small molecule PI, to be active in MM cell lines, patient cells and mouse models and is currently under clinical investigation. We compared the signaling pathways induced by MLN2238, the biologically active form of MLN9708, and bortezomib to outline differences that could be employed to design rational combinations to overcome resistance. Results: We performed time course experiments in MM.1S cells with EC50 doses of MLN2238 and bortezomib and analyzed signaling pathways via western blotting. Compared to bortezomib, MLN2238 induced earlier cleavage of caspase 8, 9, 3 and PARP and a significant downregulation of XIAP starting at 12 hours. Early and persistent phosphorylation of Bad, known to protect from mitochondrial apoptosis, was observed in bortezomib-, but not MLN2238-treated cells, in which phospho-Bad was reduced at 24 hours. Similarly, MLN2238 decreased phosphorylated Akt and mTOR at 24 hours, while bortezomib-treated cells only showed subtle changes. Interestingly, both drugs caused downregulation of the mTOR downstream targets phospho-p70 S6 kinase and phospho-4E-BP1, albeit of different intensity. Induction of heat shock proteins was comparable with both PI, opening the possibilities for combination therapy of MLN2238 with HSP90 inhibitors. Importantly, the inhibitory effect on phosphorylation of Akt, mTOR and Bad persisted when MM.1S cells were treated with MLN2238 in the presence of bone marrow stromal cells (BMSC). Conclusion: We observed differences in several crucial signaling pathways upon treatment of MM.1S cells with bortezomib or MLN2238. In particular, treatment with the latter inhibited phosphorylation of Bad, Akt and mTOR, suggesting a broader pro-apoptotic and anti-proliferative profile, compared to bortezomib. MLN2238 retained the capability of targeting these substrates also in the presence of BMSC. Confirmatory data on patient-derived cells are under current investigation in our lab. We believe that these data help understanding the differences in PI pharmacodynamics and bortezomib escape. This will allow us to design new drug combinations in the future that overcome clinical resistance to PIs and improve MM patient survival. 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 5669. doi:1538-7445.AM2012-5669

Abstract High throughput sequencing technologies open up a new dimension in cancer genomics by enabling the characterization of cancer genomes at base-pair resolution. To date, the majority of large-scale genomics projects rely on fresh-frozen biospecimens in their studies, which are difficult to obtain and often lack long-term clinical data. Unlike fresh-frozen samples, archived formalin-fixed paraffin-embedded (FFPE) tissues are more readily accessible, and are often associated with known clinical outcomes and more complete clinical annotations. However, sequencing library preparation methods need to be further optimized with regard to applicability to FFPE samples. To test whether next-generation sequencing technologies could overcome previously reported artifacts associated with formalin fixation and report accurate sequencing results, we compared whole-genome and targeted enrichment DNA sequencing data obtained from five FFPE tumor samples for which matching frozen tissues were available. We successfully generated sequencing libraries using 1 µg of genomic DNA as input material and a modified TruSeq™ sample preparation protocol. We designed a custom enrichment pool targeting exons from 215 cancer-related genes and utilized this pool to pull-down and sequence approximately 1.3 Mb region of the genome with an average 400x coverage depth to test if deep sequencing would improve validation of tumor-specific somatic mutations. We used the Illumina sequence analysis pipeline and the Windows-based second generation DNA sequencing software NextGENe® to analyze the data and identify sequence variations that are different from the human reference genome. CNV detection was overall higher among FFPE samples compared with fresh-frozen samples, demonstrating that tissue processing impacts sequencing data quality. We obtained good concordance in variant calls between matched FFPE and fresh-frozen samples. Discordant variant calls were mainly due to low depth of coverage in the regions where variant calls were made. Improvements to DNA sequencing methods for archived samples will significantly enhance cancer research and will result in more reliable prediction of individual cancer therapies. 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 3179. doi:1538-7445.AM2012-3179

Abstract Monoclonal gammopathy of undetermined significance (MGUS) is a precursor to multiple myeloma and related diseases. The majority of MGUS cases are characterized by the expression of immunoglobulin heavy chain (IgH); however, approximately one-fifth of MGUS cases do not present with IgH expression. These individuals have light chain MGUS (LC-MGUS), a precursor to light-chain MM (LC-MM). LC-MGUS is characterized by an absence of IgH and an abnormal free light chain ratio (FLC-R), indicating excessive expression of either κ or ≤ free light chains. LC-MGUS has a prevalence of 0.8% in the general population over the age of 50, as compared with a prevalence of 3.2% for heavy-chain MGUS (HC-MGUS) (Dispenzieri, 2010). There is currently little understanding of the association between family history of HC-MGUS or multiple myeloma (MM) and LC-MGUS. We investigated whether the prevalence of LC-MGUS is elevated in first-degree relatives of patients with either HC-MGUS or MM. HC-MGUS probands expressed IgH and were recruited through a population-based study of Olmsted County residents; MM probands consisted of both HC-MM and LC-MM and were recruited through the Mayo Clinic Hematology practice. Serum samples were collected from first-degree relatives of probands older than 40 years of age and subjected to electrophoresis, immunofixation, and free light chain assay. Prevalence of LC-MGUS was calculated in all first-degree relatives combined, and in relatives from both HC-MGUS and MM families separately. We compared the prevalence in family members to that in a well-defined Olmsted County, MN cohort (Dispenzieri, 2010) of patients aged 50 and over. A total 911 relatives of 232 MM and 97 MGUS patients were studied. LC-MGUS was detected in 17/911 (1.9%) relatives, for an age- and sex-adjusted prevalence of 1.7% (95% CI, 0.9% to 2.6%). Prevalence among relatives of MM probands (age- and sex- adjusted rate 2.1%; 95% CI, 1.0% to 3.2%) was higher than among relatives of MGUS probands (age- and sex- adjusted rate 0.6%; 95% CI, 0.0%-1.6%). There was an increased risk of LC-MGUS in relatives compared to the reference population which was predominantly due to the increased risk among relatives of MM probands (risk ratio=3.4, 95% CI 2.0, 5.51) as opposed to relatives of MGUS probands (risk ratio=1.1, 95% CI, 0.3 to 4.5). Among MM probands, there was no significant difference between the prevalence increase in relatives of the 32 probands with LC-MM and that of the relatives of the 200 probands with HC-MM (3.8 vs 3.3, ratio=1.2, 95% CI: 0.3-5.1, p=0.85). We conclude that the prevalence of LC-MGUS is significantly higher among first degree family members of all MM probands compared to the general population. 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 5485. doi:1538-7445.AM2012-5485

Abstract Previous epidemiological studies have suggested that African Americans (AA) are twice as likely to be diagnosed with and to die from Multiple Myeloma (MM) as compared to European Americans (EA). In recent years, however, mortality rates have become more comparable between these groups. Although socioeconomic factors could play a major role to this potential disparity, biological factors may also be responsible for influencing these differences in either incidence and/or mortality. Here we examined a multi-ethnic cohort of MM tumors to assess potential differences in the frequency of molecular events in tumors derived from either African American (AA) or European American (EA) patients. First, we compared the frequency of 14q32 translocation breakpoints at the IgH locus among a series of data from 115 AA MM patients from three studies and EA MM from the Eastern Cooperative Oncology Group (ECOG). Furthermore, we analyzed matching genome-wide copy number and transcriptional data among 47 AA MM tumors and 196 EA MM tumors to determine differences in the frequency of molecular events in tumors from these populations. Univariate analyses were conducted using the Fisher's Exact Test and p-values were calculated to measure statistical significance. We observed statistically significant differences in the frequency of IgH translocations and somatic copy number alterations. Although we did not detect differences for specific translocation subtypes, we saw a statistically significant difference in the overall frequency of IgH translocations between the two groups, which occurred more frequently in EA patients (p = 0.032). When assessing genomic copy number events previously shown to be associated with poor outcome in MM, chromosome 13 monosomy was more frequent in hyperdiploid MM from AA patients and significant after correcting for multiple testing using the Bonferoni correction method (p = 0.001). Moreover, when using the Benjamini-Hochberg multiple testing correction method, chromosome 13 monosomy remained significantly different among hyperdiploid AA patients (p = 0.0121). Chromosome 1q amplification was also significant among hyperdiploid tumors from EA patients when using this correction method (p = 0.036). No differences in the frequency of hyperdiploid status or gene expression-based subtypes were detected when comparing tumors from AA and EA patients. Our study represents the first comprehensive assessment of the frequency and distribution of molecular alterations in MM tumors from both AA and EA patients and could help shed light on possible biological features associated with population differences in incidence and outcome in MM. 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 5064. doi:1538-7445.AM2012-5064

Abstract Glioblastoma is the most common and lethal type of primary brain tumor. Despite recent therapeutic advances in other cancers, the treatment of glioblastomas remains ineffective and essentially palliative due the highly invasive nature of the disease. To discern molecular mechanisms that drive GBM migration, we investigated the potential role of differential expression of microRNAs (miRNA) by comparing the matched large-scale, genome-wide miRNA expression profiles of migrating and migration-restricted human glioma cells. Using a radial migration assay, migrating and non-migrating populations from seven well-established glioma cell lines were isolated and utilized for miRNA expression analysis. Statistical analyses revealed 22 miRNAs common to all seven glioma cell lines were down-regulated in the migrating cell population (p < 0.0005) relative to cells in the non-migratory population. Specifically, we report here that the expression level of miR-23b was markedly down-regulated in migrating glioma cells as compared to migration-restricted cells. Over-expression of miR-23b significantly inhibited glioma cell migration and invasion. A bioinformatics search revealed a conserved target site within the 3’untranslated region (UTR) of Pyk2, a non-receptor tyrosine kinase that has been previously implicated in the regulation of glioma cell migration and invasion. Increased expression of miR-23b reduced the expression level of Pyk2 in glioma cells indicating that miR-23b targets Pyk2. Notably, increased expression of miR-23b did not alter the expression level of the related focal adhesion kinase FAK indicating that miR-23b specifically inhibits the expression Pyk2. Taken together, our study demonstrates that reduced expression of miR-23b facilitates the enhancement of glioma cell migration and invasion via modulation of Pyk2 expression. Thus, these data suggest that certain miRNAs may regulate glioma migration and invasion to influence the progression of this disease. 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 3994. doi:10.1158/1538-7445.AM2011-3994

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, NG-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall. (Circ Res. 1997;80:327-335.)

AbstractBackgroundCardiac complications following hematopoietic stem cell transplantation (HSCT) are emerging as a significant concern given the increasing utilization of HSCT for a variety of hematologic malignancies.MethodsWe utilized an existing database to determine the frequency of cardiac dysfunction (CD), namely a decrease in left ventricular ejection fraction, following conditioning with high‐dose melphalan (HDM) and autologous HSCT for multiple myeloma (MM) and systemic amyloidosis (AL). We then performed a case–control study to examine variables associated with increased risk of CD in this population.ResultsIn MM patients undergoing HSCT, the rate of CD was 1.6% (17/1050, 95% CI: [0.9, 2.6]). None of the examined pre‐HSCT variables or HDM dose were significantly associated with development of CD in this population. In patients with AL, the rate of CD was 5.6% (24/426, 95% CI: [3.6, 8.3]). On univariate analysis, decision to administer an HDM dose <200 mg/m2 [odds ratio (OR): 4.59 (1.27–16.57) P = 0.02], pretransplant left ventricular ejection fraction <60% [OR: 17.78 (2.29–138.33) P = 0.006], and documented amyloid involvement of ≥3 organs [OR: 4.0 (1.03–15.6) P = 0.046] were associated with the development of CD in the AL population. No other examined peri‐transplant factors were associated with development of CD.ConclusionTo our knowledge, this is the first series to report a significant rate of CD following HDM conditioning and autologous HSCT in patients with AL.

Abstract Carfilzomib is a selective proteasome inhibitor that binds irreversibly to its target. In phase 1 studies, carfilzomib elicited promising responses and an acceptable toxicity profile in patients with relapsed and/or refractory multiple myeloma (R/R MM). In the present phase 2, multicenter, open-label study, 129 bortezomib-naive patients with R/R MM (median of 2 prior therapies) were separated into Cohort 1, scheduled to receive intravenous carfilzomib 20 mg/m2 for all treatment cycles, and Cohort 2, scheduled to receive 20 mg/m2 for cycle 1 and then 27 mg/m2 for all subsequent cycles. The primary end point was an overall response rate (≥ partial response) of 42.4% in Cohort 1 and 52.2% in Cohort 2. The clinical benefit response (overall response rate + minimal response) was 59.3% and 64.2% in Cohorts 1 and 2, respectively. Median duration of response was 13.1 months and not reached, and median time to progression was 8.3 months and not reached, respectively. The most common treatment-emergent adverse events were fatigue (62.0%) and nausea (48.8%). Single-agent carfilzomib elicited a low incidence of peripheral neuropathy—17.1% overall (1 grade 3; no grade 4)—in these pretreated bortezomib-naive patients. The results of the present study support the use of carfilzomib in R/R MM patients. This trial is registered at www.clinicaltrials.gov as NCT00530816.