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1.  Transient inhibition of the ERK pathway prevents cerebellar developmental defects and improves long-term motor functions in murine models of neurofibromatosis type 1 
eLife  null;3:e05151.
Individuals with neurofibromatosis type 1 (NF1) frequently exhibit cognitive and motor impairments and characteristics of autism. The cerebellum plays a critical role in motor control, cognition, and social interaction, suggesting that cerebellar defects likely contribute to NF1-associated neurodevelopmental disorders. Here we show that Nf1 inactivation during early, but not late stages of cerebellar development, disrupts neuronal lamination, which is partially caused by overproduction of glia and subsequent disruption of the Bergmann glia (BG) scaffold. Specific Nf1 inactivation in glutamatergic neuronal precursors causes premature differentiation of granule cell (GC) precursors and ectopic production of unipolar brush cells (UBCs), indirectly disrupting neuronal migration. Transient MEK inhibition during a neonatal window prevents cerebellar developmental defects and improves long-term motor performance of Nf1-deficient mice. This study reveals essential roles of Nf1 in GC/UBC migration by generating correct numbers of glia and controlling GC/UBC fate-specification/differentiation, identifying a therapeutic prevention strategy for multiple NF1-associcated developmental abnormalities.
eLife digest
Neurofibromatosis type 1 is a condition characterized by the growth of tumors along the nerves of the body. It is caused by mutations in a gene called NF1, which codes for a protein that normally works to inhibit the activity of another protein called Ras. In healthy cells, Ras is needed to stimulate the cells to grow and divide. However, if the Ras protein is not turned off at the right time or if it is activated at the wrong time, it can force cells to keep growing and dividing; this leads to the growth of tumors.
Along with being prone to developing cancer, individuals with neurofibromatosis type 1 also develop a range of neurodevelopmental disorders that alter their learning, motor skills and social interactions. Some also exhibit behaviors that are associated with autism. This led Kim, Wang et al. to investigate whether a region of the brain—called the cerebellum—that has recently been associated with autism is also affected in a mouse model of neurofibromatosis type 1.
The cerebellum is best known for its role in coordinating movement, although it also has functions in cognition, behavior and other processes. Ras is involved in the development of the cerebellum; and so Kim, Wang et al. asked whether the loss of the Nf1 gene from cells in the mouse cerebellum might cause the neurodevelopmental defects associated with neurofibromatosis type 1.
Loss of Nf1 during early (but not in late) development of the cerebellum disrupted the normal organization of the nerve cells (or neurons) into specific cell layers. These defects were caused, in part, by the over-growth of a type of supporting cell—called glia cells—at a specific developmental stage—that would normally form a scaffold to help neurons migrate to their correct position. Nf1 also controls the generation of the correct types of neurons in the right time and at right location during the early development of the cerebellum.
Next, Kim, Wang et al. treated newborn mice with a compound that inhibits Ras signaling via their mother's milk for 3 weeks. In mice with an inactive Nf1 gene, the treatment helped to prevent some defects in the cerebellum and the mice had improved motor coordination several months later. Whether this could form the basis of a preventative treatment for neurodevelopmental disorders associated with neurofibromatosis type 1 in humans remains a question for future work.
PMCID: PMC4297949  PMID: 25535838
neurofibromatosis type 1; cerebellum; bergmann glial cells; granule cells; unipolar brush cells; tumor suppressor gene; mouse
2.  Validation of a Molecular and Pathological Model for Five-Year Mortality Risk in Patients with Early Stage Lung Adenocarcinoma 
Journal of Thoracic Oncology  2014;10(1):67-73.
The aim of this study was to validate a molecular expression signature [cell cycle progression (CCP) score] that identifies patients with a higher risk of cancer-related death after surgical resection of early stage (I-II) lung adenocarcinoma in a large patient cohort and evaluate the effectiveness of combining CCP score and pathological stage for predicting lung cancer mortality.
Formalin-fixed paraffin-embedded surgical tumor samples from 650 patients diagnosed with stage I and II adenocarcinoma who underwent definitive surgical treatment without adjuvant chemotherapy were analyzed for 31 proliferation genes by quantitative real-time polymerase chain reaction. The prognostic discrimination of the expression score was assessed by Cox proportional hazards analysis using 5-year lung cancer-specific death as primary outcome.
The CCP score was a significant predictor of lung cancer-specific mortality above clinical covariates [hazard ratio (HR) = 1.46 per interquartile range (95% confidence interval = 1.12–1.90; p = 0.0050)]. The prognostic score, a combination of CCP score and pathological stage, was a more significant indicator of lung cancer mortality risk than pathological stage in the full cohort (HR = 2.01; p = 2.8 × 10−11) and in stage I patients (HR = 1.67; p = 0.00027). Using the 85th percentile of the prognostic score as a threshold, there was a significant difference in lung cancer survival between low-risk and high-risk patient groups (p = 3.8 × 10−7).
This study validates the CCP score and the prognostic score as independent predictors of lung cancer death in patients with early stage lung adenocarcinoma treated with surgery alone. Patients with resected stage I lung adenocarcinoma and a high prognostic score may be candidates for adjuvant therapy to reduce cancer-related mortality.
PMCID: PMC4272230  PMID: 25396679
Carcinoma; Nonsmall cell lung cancer; Real-time polymerase chain reaction; Risk stratification
3.  Comprehensive biomarker analysis and final efficacy results of sorafenib in the BATTLE (Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination) trial 
To report the clinical efficacy of sorafenib and to evaluate biomarkers associated with sorafenib clinical benefit in the BATTLE program.
Patients and Methods
Patients with previously treated non-small–cell lung cancer (NSCLC) received sorafenib until progression or unacceptable toxicity. Eight-week disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were assessed. Prespecified biomarkers included K-RAS, EGFR, and B-RAF mutations, and EGFR gene copy number. Gene expression profiles from NSCLC cell lines and patient tumor biopsies with wild-type EGFR were used to develop a sorafenib sensitivity signature (SSS).
105 patients were eligible and randomized to receive sorafenib. Among 98 patients evaluable for 8-week DCR, the observed DCR was 58.2%. The median PFS and OS were 2.83 (95% confidence interval [CI], 2.04-3.58) and 8.48 months (95% CI, 5.78-10.97), respectively. Eight-week DCR was higher in patients with wt-EGFR than patients with EGFR mutation (P=0.012), and in patients with EGFR gene copy number gain (FISH positive) versus patients FISH negative (P=0.048). In wt-EGFR tumors, the SSS was associated with improved PFS (median PFS 3.61 months in high SSS versus 1.84 months in low SSS, P=0.026) but not with 8-week DCR. Increased expression of fibroblast growth factor-1, NF-kB and hypoxia pathways were identified potential drivers of sorafenib resistance.
Sorafenib demonstrates clinical activity in NSCLC, especially with wt-EGFR. SSS was associated with improved PFS. These data identify subgroups that may derive clinical benefit from sorafenib and merit investigation in future trials. NCT00411671.
PMCID: PMC3905243  PMID: 24166906
multikinase inhibitor; non–small cell lung cancer; sorafenib; biomarkers; targeted treatment
4.  Hyperactive Ras/MAPK signaling is critical for tibial nonunion fracture in neurofibromin-deficient mice 
Human Molecular Genetics  2013;22(23):4818-4828.
Neurofibromatosis type 1 (NF1) is a common genetic disorder affecting 1 in 3500 individuals. Patients with NF1 are predisposed to debilitating skeletal manifestations, including osteopenia/osteoporosis and long bone pseudarthrosis (nonunion fracture). Hyperactivation of the Ras/mitogen-activated protein kinase (MAPK) pathway in NF1 is known to underlie aberrant proliferation and differentiation in cell lineages, including osteoclast progenitors and mesenchymal stem cells (MSCs) also known as osteoblast progenitors (pro-OBLs). Our current study demonstrates the hyper Ras/MAPK as a critical pathway underlying the pathogenesis of NF1-associated fracture repair deficits. Nf1-deficient pro-OBLs exhibit Ras/MAPK hyperactivation. Introduction of the NF1 GTPase activating-related domain (NF1 GAP-related domain) in vitro is sufficient to rescue hyper Ras activity and enhance osteoblast (OBL) differentiation in Nf1−/− pro-OBLs and NF1 human (h) MSCs cultured from NF1 patients with skeletal abnormalities, including pseudarthrosis or scoliosis. Pharmacologic inhibition of mitogen-activated protein kinase kinase (MEK) signaling with PD98059 partially rescues aberrant Erk activation while enhancing OBL differentiation and expression of OBL markers, osterix and osteocalcin, in Nf1-deficient murine pro-OBLs. Similarly, MEK inhibition enhances OBL differentiation of hMSCs. In addition, PD98059 rescues aberrant osteoclast maturation in Nf1 haploinsufficient bone marrow mononuclear cells (BMMNCs). Importantly, MEK inhibitor significantly improves fracture healing in an NF1 murine model, Col2.3Cre;Nf1flox/−. Collectively, these data indicate the Ras/MAPK cascade as a critical pathway in the pathogenesis of bone loss and pseudarthrosis related to NF1 mutations. These studies provide evidence for targeting the MAPK pathway to improve bone mass and treat pseudarthrosis in NF1.
PMCID: PMC3820137  PMID: 23863460
5.  Engineering Transcriptional Regulation to Control Pdu Microcompartment Formation 
PLoS ONE  2014;9(11):e113814.
Bacterial microcompartments (MCPs) show great promise for the organization of engineered metabolic pathways within the bacterial cytoplasm. This subcellular organelle is composed of a protein shell of 100–200 nm diameter that natively encapsulates multi-enzyme pathways. The high energy cost of synthesizing the thousands of protein subunits required for each MCP demands precise regulation of MCP formation for both native and engineered systems. Here, we study the regulation of the propanediol utilization (Pdu) MCP, for which growth on 1,2-propanediol induces expression of the Pdu operon for the catabolism of 1,2-propanediol. We construct a fluorescence-based transcriptional reporter to investigate the activation of the Ppdu promoter, which drives the transcription of 21 pdu genes. Guided by this reporter, we find that MCPs can be expressed in strains grown in rich media, provided that glucose is not present. We also characterize the response of the Ppdu promoter to a transcriptional activator of the pdu operon, PocR, and find PocR to be a necessary component of Pdu MCP formation. Furthermore, we find that MCPs form normally upon the heterologous expression of PocR even in the absence of the natural inducer 1,2-propanediol and in the presence of glucose, and that Pdu MCPs formed in response to heterologous PocR expression can metabolize 1,2-propanediol in vivo. We anticipate that this technique of overexpressing a key transcription factor may be used to study and engineer the formation, size, and/or number of MCPs for the Pdu and related MCP systems.
PMCID: PMC4245221  PMID: 25427074
6.  Validation of a Proliferation-based Expression Signature as Prognostic Marker in Early Stage Lung Adenocarcinoma 
New prognostic markers to guide treatment decisions in early stage non-small cell lung cancer are necessary to improve patient outcomes. In this report, we assess the utility of a pre-defined mRNA expression signature of cell cycle progression genes (CCP score) to define 5-year risk of lung cancer related death in patients with early stage lung adenocarcinoma.
A CCP score was calculated from the mRNA expression levels of 31 proliferation genes in stage I and II tumor samples from two public microarray data sets (Director’s Consortium (DC) and GSE31210). The same gene set was tested by quantitative PCR in 381 formalin-fixed paraffin-embedded (FFPE) primary tumors. Association of the CCP score with outcome was assessed by Cox proportional hazards analysis.
In univariate analysis the CCP score was a strong predictor of cancer-specific survival in both the DC cohort (p=0.00014, HR 2.08, 95%CI 1.43–3.02) and GSE31210 (p=0.0010, HR 2.25, 95%CI 1.42–3.56). In multivariate analysis the CCP score remained the dominant prognostic marker in the presence of clinical variables (p=0.0022, HR 2.02, 95%CI 1.29–3.17 in DC, p=0.0026, HR 2.16, 95%CI 1.32–3.53 in GSE31210). On a quantitative PCR platform the CCP score maintained highly significant prognostic value in FFPE derived mRNA from clinical samples in both univariate (p=0.00033, HR 2.10, 95%CI 1.39–3.17) and multivariate analyses (p=0.0071, HR 1.92, 95%CI 1.18–3.10).
The CCP score is a significant predictor of lung cancer death in early stage lung adenocarcinoma treated with surgery and may be a valuable tool in selecting patients for adjuvant treatment.
PMCID: PMC3834029  PMID: 24048333
non-small cell lung cancer; adenocarcinoma; prognosis; expression signature
7.  The BATTLE Trial: Personalizing Therapy for Lung Cancer 
Cancer discovery  2011;1(1):44-53.
Lung cancer is the leading cancer cause of mortality worldwide; large-scale trials have failed to improve clinical outcomes of patients with chemorefractory non-small-cell lung cancer (NSCLC).
Following an initial equal randomization period, BATTLE adaptively randomized patients with chemorefractory NSCLC to erlotinib, vandetanib, erlotinib plus bexarotene, or sorafenib based on molecular biomarkers of NSCLC pathogenesis in fresh core needle biopsy specimens. The primary end point was disease control rate (DCR) at 8 weeks.
Of 255 patients randomly assigned to erlotinib (59 patients), vandetanib (54), erlotinib plus bexarotene (37), and sorafenib (105), 244 were eligible for the DCR analysis. Pneumothorax after lung biopsy occurred in 11.5% and treatment-related toxicities grade 3–4 in 6.5% of patients. Overall results were a 46% 8-week DCR, 1.9-month median progression-free survival, 9-month median overall survival, and 35% 1-year survival. Individual markers predicting a significantly superior DCR for a treatment included: epidermal growth factor receptor (EGFR) mutation (P=0.04) for erlotinib; cyclin D1 positivity (P=0.01) or EGFR amplification (P=0.006) for erlotinib plus bexarotene; vascular endothelial growth factor receptor 2 positivity (P=0.05) for vandetanib; and absence of EGFR mutation (P=0.01) or of EGFR high polysomy (P=0.05) for sorafenib. A better 8-week DCR occurred with sorafenib versus all other regimens (64% versus 33%; P<0.001) among EGFR wild-type patients and versus all other regimens (61% versus 32%; P=0.11) among mutant-KRAS patients. The prespecified biomarker groups were less predictive than the individual biomarkers analyzed in this study.
The first completed biopsy-mandated study in pretreated NSCLC, BATTLE confirmed our pre-specified hypotheses regarding biomarker and targeted treatment interactions, establishing a new paradigm for personalizing therapy for patients with NSCLC. ( numbers, NCT00409968, NCT00411671, NCT00411632, NCT00410059, NCT00410189.)
PMCID: PMC4211116  PMID: 22586319
8.  Oral Epithelium as a Surrogate Tissue for Assessing Smoking-Induced Molecular Alterations in Lungs 
Both the lungs and oral cavity are exposed to tobacco carcinogens in smokers. We hypothesized that the oral epithelium undergoes molecular alterations similar to those in lungs and therefore may be used as a surrogate tissue to assess tobacco-induced molecular alterations.
Promoter methylation of p16 and FHIT genes was analyzed with methylation-specific PCR in 1,774 oral and bronchial brush specimens (baseline and 3 months after intervention) from 127 smokers enrolled in a prospective randomized placebo-controlled chemoprevention trial. The association between methylation patterns in oral tissues and bronchial methylation indices (methylated sites/total sites per subject) was analyzed blindly.
At baseline, promoter methylation was observed in 23%, 17%, and 35% of the bronchial tissues for p16, FHIT, and either of the two genes, respectively, which were comparable to the 19%, 15%, and 31% observed in the oral tissues. Among the 125 individuals with available data from both oral and bronchial tissues, strong correlations were observed between tissues from the two sites (P<0.0001 for both p16 and FHIT). Among the 39 individuals with oral tissue methylation in either of the two genes, the mean bronchial methylation index was 0.53 (± 0.29) compared with only 0.27 (± 0.26) for the 86 subjects without oral tissue methylation (P<0.0001). Similar correlations were also observed in samples obtained at 3 months after chemopreventive intervention.
The oral epithelium may be used as a surrogate tissue to assess tobacco-induced molecular damage in lungs, which has an important implication in conducting biomarker-based lung cancer prevention trials.
PMCID: PMC4183362  PMID: 19138934
9.  The Fine Temporal Structure of the Rat Licking Pattern: What Causes the Variabiliy in the Interlick Intervals and How is it Affected by the Drinking Solution? 
Chemical Senses  2013;38(8):685-704.
Licking is a repetitive behavior controlled by a central pattern generator. Even though interlick intervals (ILIs) within bursts of licks are considered fairly regular, the conditions that affect their variability are unknown. We analyzed the licking pattern in rats that licked water, 10% sucrose solution, or 10% ethanol solution, in 90-min recording sessions after 4h of water deprivation. The histograms of ILIs indicate that licking typically occurred at a preferred ILI of about 130−140ms with evidence of bimodal or multimodal distributions due to occasional licking failures. We found that the longer the pause between bursts of licks, the shorter was the first ILI of the burst. When bursts of licks were preceded by a pause >4 s, the ILI was the shortest (~110ms) at the beginning of the burst, and then it increased rapidly in the first few licks and slowly in subsequent licks. Interestingly, the first ILI of a burst of licks was not significantly different when licking any of the 3 solutions, but subsequent licks exhibited a temporal pattern characteristic of each solution. The rapid deceleration in intraburst licking rate was due to an increase from ~27ms to ~56ms in the tongue-spout contact duration while the intercontact interval was only slightly changed (80−90ms). Therefore, the contact duration seems to be the major factor that increases the variability in the ILIs and could be another means for the rat to adjust the amount of fluid ingested in each individual lick.
PMCID: PMC3777561  PMID: 23902635
autocorrelograms; bursting; licking; rhythmicity
10.  A Multi-Institutional Phase II Study of Neoadjuvant Gemcitabine and Oxaliplatin with Radiation Therapy in Patients with Pancreatic Cancer 
Cancer  2013;119(15):2692-2700.
The purpose of this study was to evaluate preoperative treatment with full-dose gemcitabine, oxaliplatin and radiation (RT) in localized pancreatic cancer.
Eligibility included confirmation of adenocarcinoma, resectable or borderline resectable disease, PS≤2, and adequate organ function. Treatment consisted of two, 28 day cycles of gemcitabine (1g/m2 over 30 minutes days 1, 8, 15) and oxaliplatin (85mg/m2 days 1, 15) with RT during cycle 1 (30Gy in 2Gy fractions). Patients were evaluated for surgery following cycle 2. Resected patients received 2 cycles of adjuvant chemotherapy.
Sixty-eight evaluable patients were treated at 4 centers. By central radiology review, 23 patients were resectable, 39 borderline resectable, and 6 unresectable. Sixty-six patients (97%) completed cycle 1/RT and 61 patients (90%) cycle 2. Adverse events ≥ grade 3 during preoperative therapy included neutropenia (32%), thrombocytopenia (25%), and biliary obstruction/cholangitis (14%). Forty-three patients were resected (63%) with R0 resection in 36 (84%). Median overall survival for all patients was 18.2 months (95%CI 13–26.9), those resected 27.1 months (95%CI 21.2–47.1) and those not resected 10.9 months (95%CI 6.1–12.6). A decrease in CA19-9 following neoadjuvant therapy was associated with R0 resection (p=0.02) which resulted in a median survival of 34.6 months (95% CI 20.3–47.1). Fourteen patients (21%) are alive and disease-free at a median follow-up time of 31.4 months (range, 24–47.6).
Preoperative therapy with full dose gemcitabine, oxaliplatin and RT was feasible and resulted in a high percentage of R0 resections. Results are particularly encouraging given a majority of patients with borderline resectable disease.
PMCID: PMC4174603  PMID: 23720019
pancreatic cancer; neoadjuvant; gemcitabine; oxaliplatin
11.  Phase II Chemoprevention Trial with High Dose Fenretinide for Oral Pre-Malignant Lesions 
In a previous phase II trial, we demonstrated that fenretinide 200 mg/day had limited activity in retinoid-refractory leukoplakia (34% response rate), possibly due to the lack of achievement of high serum levels which would be required to elicit retinoid-receptor independent apoptosis in pre-malignant cells. We therefore designed this single-arm, phase II trial to investigate whether fenretinide at a higher dose would improve the leukoplakia response rate from our previous study’s historical control.
Patients and Methods
Patients with high-risk leukoplakia were treated with 4 three-week cycles of fenretinide (900 mg/m2 orally twice daily, days 1–7). At week 12, objective clinical responses were determined and blood samples were collected for serum drug level assessment. The original sample size was 25 patients to detect a 55% response rate (90% power, one-sided 10% type I error rate). A futility analysis was planned after accrual of the first 16 patients to allow for early trial closure if ≤4 patients responded.
Fenretinide was well tolerated, with only one grade 3 toxicity (diarrhea) observed. However, only 3 of the initial 15 patients (20%) had a partial response, leading to early trial termination due to lack of efficacy. Serum levels of fenretinide rose from 0 (baseline) to 0.122 μM ± 0.093 (week 12), indicating that high serum levels of the drug were achieved during the initial days of the cycle.
Despite high serum levels, fenretinide for oral leukoplakia, at the dose and schedule studied herein, failed to improve historical response rates.
PMCID: PMC4161148  PMID: 19139014
fenretinide; oral pre-malignant lesion; leukoplakia; chemoprevention
12.  Polarized Axonal Surface Expression of Neuronal KCNQ Potassium Channels Is Regulated by Calmodulin Interaction with KCNQ2 Subunit 
PLoS ONE  2014;9(7):e103655.
KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4) membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface. These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.
PMCID: PMC4117524  PMID: 25077630
13.  Patient perspectives on switching disease-modifying therapies in the NARCOMS registry 
The evolving landscape of disease-modifying therapies (DMTs) for multiple sclerosis raises important questions about why patients change DMTs. Physicians and patients could benefit from a better understanding of the reasons for switching therapy.
To investigate the reasons patients switch DMTs and identify characteristics associated with the decision to switch.
The North American Research Committee on Multiple Sclerosis (NARCOMS) Registry conducted a supplemental survey among registry participants responding to the 2011 update survey. The supplemental survey investigated reasons for switching DMT, origin of the discussion of DMT change, and which factors influenced the decision. Chi-square tests, Fisher’s exact tests, and logistic regression were used for the analyses.
Of the 691 eligible candidates, 308 responded and met the inclusion criteria (relapsing disease course, switched DMT after September 2010). The responders were 83.4% female, on average 52 years old, with a median (interquartile range) Patient-Determined Disease Steps score of 4 (2–5). The most recent prior therapy included first-line injectables (74.5%), infusions (18.1%), an oral DMT (3.4%), and other DMTs (4.0%). The discussion to switch DMT was initiated almost equally by physicians and participants. The primary reason for choosing the new DMT was based most frequently on physician’s recommendation (24.5%) and patient perception of efficacy (13.7%).
Participants frequently initiated the discussion regarding changing DMT, although physician recommendations regarding the specific therapy were still weighed highly. Long-term follow-up of these participants will provide valuable information on their disease trajectory, satisfaction with, and effectiveness of their new medication.
PMCID: PMC4094626  PMID: 25045254
multiple sclerosis; health communication
14.  Phase II Trial of Carboplatin, Paclitaxel, Cetuximab and Bevacizumab Followed by Cetuximab and Bevacizumab in Advanced Non-Squamous Non-Small Cell Lung Cancer: SWOG S0536 
Cetuximab and bevacizumab have each been demonstrated to prolong survival when added to chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). However, the potential benefit of combining cetuximab and bevacizumab together with a platinum-based doublet had not been explored. We designed this phase II trial to evaluate the safety, tolerability and efficacy of the combination of carboplatin, paclitaxel, cetuximab and bevacizumab in chemotherapy-naïve patients with advanced, non-squamous NSCLC.
Patients received with up to six cycles of carboplatin (area under curve 6), paclitaxel (200 mg/m2), cetuximab (400 mg/m2 day 1 then 250 mg/m2 weekly) and bevacizumab (15 mg/kg) every 21 days. Patients with an objective response or stable disease received maintenance cetuximab (250 mg/m2 weekly) and bevacizumab (15 mg/kg every 21 days) until disease progression. The primary endpoint was safety as defined by the frequency and severity of hemorrhagic toxicities. Secondary endpoints included response rate (RR), progression-free survival (PFS), overall survival (OS), and toxicity. Molecular biomarkers were assessed in an exploratory manner.
The primary endpoint of grade 4 or higher hemorrhage of 2% (95% confidence interval: 0-7%) met prespecified criteria for safety. Oone hundred ten patients were enrolled. There were 4 treatment-related deaths including lung hemorrhage (2), infection (1), and unknown (1). Median progression-free survival was 7 months and median overall survival was 15 months. The RR was 56% with an overall disease control rate of 77%.
This regimen was safe, feasible and effective as frontline treatment of advanced NSCLC, providing the basis for the ongoing Phase III trial S0819.
PMCID: PMC4072123  PMID: 24189513
Non-small-cell lung cancer; Paclitaxel; Carboplatin; Cetuximab; Bevacizumab; Frontline
15.  Serum Fructosamine and Glycated Albumin and Risk of Mortality and Clinical Outcomes in Hemodialysis Patients 
Diabetes Care  2013;36(6):1522-1533.
Assays for serum total glycated proteins (fructosamine) and the more specific glycated albumin may be useful indicators of hyperglycemia in dialysis patients, either as substitutes or adjuncts to standard markers such as hemoglobin A1c, as they are not affected by erythrocyte turnover. However, their relationship with long-term outcomes in dialysis patients is not well described.
We measured fructosamine and glycated albumin in baseline samples from 503 incident hemodialysis participants of a national prospective cohort study, with enrollment from 1995–1998 and median follow-up of 3.5 years. Outcomes were all-cause and cardiovascular disease (CVD) mortality and morbidity (first CVD event and first sepsis hospitalization) analyzed using Cox regression adjusted for demographic and clinical characteristics, and comorbidities.
Mean age was 58 years, 64% were white, 54% were male, and 57% had diabetes. There were 354 deaths (159 from CVD), 302 CVD events, and 118 sepsis hospitalizations over follow-up. Both fructosamine and glycated albumin were associated with all-cause mortality; adjusted HR per doubling of the biomarker was 1.96 (95% CI 1.38–2.79) for fructosamine and 1.40 (1.09–1.80) for glycated albumin. Both markers were also associated with CVD mortality [fructosamine 2.13 (1.28–3.54); glycated albumin 1.55 (1.09–2.21)]. Higher values of both markers were associated with trends toward a higher risk of hospitalization with sepsis [fructosamine 1.75 (1.01–3.02); glycated albumin 1.39 (0.94–2.06)].
Serum fructosamine and glycated albumin are risk factors for mortality and morbidity in hemodialysis patients.
PMCID: PMC3661814  PMID: 23250799
16.  The tolerance of gastrointestinal organs to stereotactic body radiation therapy: what do we know so far? 
As stereotactic body radiation therapy (SBRT) for gastrointestinal (GI) gains popularity, there is a need to optimize doses and fractionation to minimize GI toxicity. GI organs that have classically developed radiation-induced toxicity include the liver & biliary system, small bowel, esophagus, and rectum. While the literature quantifies dose restrictions for these organs under standard fractionation, there is limited data regarding toxicity with the ablative dose schedules used in SBRT. We conducted a review of the literature to identify prospective and retrospective studies that detail GI toxicities when SBRT was employed. Based on the literature, the median SBRT dose for abdominal and thoracic tumors ranged from 24 to 60 Gy, at 5 to 25 Gy per fraction. The respective observed frequencies of grade 3 and 4 toxicities for the liver, biliary system, small bowel, and esophagus were variable among different studies. Typically, patients who suffered grade 3 and 4 toxicities were more likely to have had some form of systemic therapy as well. The effect of dose, fractionation, timing, and volume on GI toxicities has been described in the literature but more data is necessary to develop uniform treatment guidelines for SBRT.
PMCID: PMC4074956  PMID: 24982772
Stereotactic body radiation therapy (SBRT); gastrointestinal organs (GI organs); tolerance
17.  Biological Activity of Celecoxib in the Bronchial Epithelium of Current and Former Smokers 
Non-small-cell lung cancer (NSCLC) is the primary cause of cancer-related death in Western countries. One important approach taken to address this problem is the development of effective chemoprevention strategies. In this study, we examined whether the cyclooxygenase-2 (COX-2) inhibitor celecoxib, as evidenced by decreased cell proliferation, is biologically active in the bronchial epithelium of current and former smokers.
Patients and Methods
Current or former smokers with at least a 20 pack-year (pack-year = number of packs of cigarettes per day times number of years smoked) smoking history were randomized into one of four treatment arms (3-month intervals of celecoxib then placebo, celecoxib then celecoxib, placebo then celecoxib, or placebo then placebo) and underwent bronchoscopies with biopsies at baseline, 3 months, and 6 months. The 204 patients were primarily (79.4%) current smokers; 81 received either low-dose celecoxib or placebo and 123 received either high-dose celecoxib or placebo. Celecoxib was originally administered orally at 200 mg twice daily and the protocol subsequently increased the dose to 400 mg twice daily. The primary endpoint was change in Ki-67 labeling (from baseline to 3 months) in bronchial epithelium.
No cardiac toxicities were observed in the participants. Although the effect of low-dose treatment was not significant, high-dose celecoxib decreased Ki-67 labeling by 3.85% in former smokers and by 1.10% in current smokers—a significantly greater reduction (P = 0.02) than that seen with placebo after adjusting for metaplasia and smoking status.
A 3–6-month celecoxib regimen proved safe to administer. Celecoxib 400 mg bid was biologically active in the bronchial epithelium of current and former smokers; additional studies on the efficacy of celecoxib in NSCLC chemoprevention may be warranted.
PMCID: PMC4028718  PMID: 20103722
18.  Dynamic Treg interactions with intratumoral APCs promote local CTL dysfunction 
The Journal of Clinical Investigation  2014;124(6):2425-2440.
Tregs control various functions of effector T cells; however, where and how Tregs exert their immunomodulatory effects remain poorly understood. Here we developed a murine model of adoptive T cell therapy and found that Tregs induce a dysfunctional state in tumor-infiltrating CTLs that resembles T cell exhaustion and is characterized by low expression of effector cytokines, inefficient cytotoxic granule release, and coexpression of coinhibitory receptors PD-1 and TIM-3. Induction of CTL dysfunction was an active process, requiring local TCR signals in tumor tissue. Tregs infiltrated tumors only subsequent to Ag-dependent activation and expansion in tumor-draining LNs; however, Tregs also required local Ag reencounter within tumor tissue to induce CTL dysfunction and prevent tumor rejection. Multiphoton intravital microscopy revealed that in contrast to CTLs, Tregs only rarely and briefly interrupted their migration in tumor tissue in an Ag-dependent manner and formed unstable tethering-interactions with CD11c+ APCs, coinciding with a marked reduction of CD80 and CD86 on APCs. Activation of CTLs by Treg-conditioned CD80/86lo DCs promoted enhanced expression of both TIM-3 and PD-1. Based on these data, we propose that Tregs locally change the costimulatory landscape in tumor tissue through transient, Ag-dependent interactions with APCs, thus inducing CTL dysfunction by altering the balance of costimulatory and coinhibitory signals these cells receive.
PMCID: PMC4089459  PMID: 24812664
19.  Treatment selection and experience in multiple sclerosis: survey of neurologists 
Multiple sclerosis (MS) is a complex disease with many therapeutic options. Little is known about how neurologists select particular disease-modifying therapies (DMTs) for their patients.
To understand how neurologists make decisions regarding the prescription of DMTs for patients with MS, and to explore neurologists’ experiences with individual DMTs.
From December 2012 to January 2013, members of a nationwide physician market research panel were sent an online study invitation with a link to a survey website. Eligible neurologists were included if they currently practice medicine in the United States, and if they treat ≥20 patients with MS.
A total of 102 neurologists (n=63 general neurologists; n=39 MS specialists; 81.4% male) completed the survey. The mean (standard deviation) number of years in practice since completing medical training was 16.4 (8.6) years. Overall, the most commonly prescribed DMTs were subcutaneous interferon (IFN) β-1a and glatiramer acetate; approximately 5.5% of patients were untreated. The most important attributes of DMT medication selection were (in order of importance) efficacy, safety, tolerability, patient preference, and convenience. The DMT with the highest neurologist-reported percentage of patients who were “Very/Extremely Satisfied” with their therapy was fingolimod (31.0%), followed by glatiramer acetate (13.9%; P=0.017). Compared with fingolimod (94.0%), significantly fewer (P<0.05) neurologists reported that “All/Most” of their patients were adherent to treatment with glatiramer acetate (78.0%), subcutaneous IFN β-1a (84.0%), and IFN β-1b (75.0%); no significant differences were observed with intramuscular IFN β-1a (92.9%; P=0.75). Patients’ calls to neurologists’ offices were most commonly related to side effects for all self-injectable DMTs, whereas calls about fingolimod primarily involved insurance coverage issues.
Our survey results showed that very few patients with MS did not received any DMT. Among the DMTs available at the time of the survey, neurologists reported that patients were most satisfied with, and adherent to, fingolimod, but these patients also faced more problems with insurance coverage when compared with those taking self-injectable DMTs.
PMCID: PMC3979792  PMID: 24729689
multiple sclerosis; disease-modifying therapy; physician survey; treatment selection; treatment adherence; treatment satisfaction
20.  Feasibility of Image-Guided Transthoracic Core Needle Biopsy in the BATTLE Lung Trial 
As therapy for non-small cell lung cancer (NSCLC) patients becomes more personalized, additional tissue in the form of core needle biopsies (CNBs) for biomarker analysis is increasingly required for determining appropriate treatment and for enrollment into clinical trials. We report our experience with small-caliber percutaneous transthoracic (PT) CNBs for the evaluation of multiple molecular biomarkers in BATTLE (Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination), a personalized, targeted therapy NSCLC clinical trial.
The medical records of patients who underwent PTCNB for consideration of enrollment in BATTLE, were reviewed for diagnostic yield of 11 predetermined molecular markers, and procedural complications. Univariate and multivariate analyses of factors related to patient and lesion characteristics were performed to determine possible influences on diagnostic yield.
One hundred and seventy PTCNBs were performed using 20-gauge biopsy needles in 151 NSCLC patients screened for the trial. 82.9% of the biopsy specimens were found to have adequate tumor tissue for analysis of the required biomarkers. On multivariate analysis, metastatic lesions were 5.4 times more likely to yield diagnostic tissue as compared to primary tumors (p = 0.0079). Pneumothorax and chest tube insertion rates were 15.3% and 9.4%, respectively.
Image-guided 20-gauge PTCNB is safe and provides adequate tissue for analysis of multiple biomarkers in the majority of patients being considered for enrollment into a personalized, targeted therapy NSCLC clinical trial. Metastatic lesions are more likely to yield diagnostic tissue as compared to primary tumors.
PMCID: PMC3879952  PMID: 23442309
research biopsy; biomarker analysis; percutaneous transthoracic biopsy
21.  The Notch Pathway Is Important in Maintaining the Cancer Stem Cell Population in Pancreatic Cancer 
PLoS ONE  2014;9(3):e91983.
Pancreatic cancer stem cells (CSCs) represent a small subpopulation of pancreatic cancer cells that have the capacity to initiate and propagate tumor formation. However, the mechanisms by which pancreatic CSCs are maintained are not well understood or characterized.
Expression of Notch receptors, ligands, and Notch signaling target genes was quantitated in the CSC and non-CSC populations from 8 primary human pancreatic xenografts. A gamma secretase inhibitor (GSI) that inhibits the Notch pathway and a shRNA targeting the Notch target gene Hes1 were used to assess the role of the Notch pathway in CSC population maintenance and pancreatic tumor growth.
Notch pathway components were found to be upregulated in pancreatic CSCs. Inhibition of the Notch pathway using either a gamma secretase inhibitor or Hes1 shRNA in pancreatic cancer cells reduced the percentage of CSCs and tumorsphere formation. Conversely, activation of the Notch pathway with an exogenous Notch peptide ligand increased the percentage of CSCs as well as tumorsphere formation. In vivo treatment of orthotopic pancreatic tumors in NOD/SCID mice with GSI blocked tumor growth and reduced the CSC population.
The Notch signaling pathway is important in maintaining the pancreatic CSC population and is a potential therapeutic target in pancreatic cancer.
PMCID: PMC3960140  PMID: 24647545
22.  MicroRNA-484 is more highly expressed in serum of early breast cancer patients compared to healthy volunteers 
BMC Cancer  2014;14:200.
Previous studies have profiled breast cancer compared to normal breast tissue and identified differentially expressed microRNAs (miRNAs). These miRNAs are then assessed in serum of breast cancer patients compared to healthy volunteers. MiRNAs in serum however do not always reflect what is in tissue and important serum miRNAs may be missed. PCR arrays were therefore performed on serum samples from breast cancer patients compared to healthy volunteers with the aim of identifying circulating miRNAs that are more highly expressed in serum from early breast cancer patients compared to controls.
Taqman low density array (TLDA) cards were used to profile serum miRNAs in a discovery cohort of serum from 39 early breast cancer patients compared to 10 healthy volunteers. The results were confirmed in a validation cohort of serum from 98 early breast cancer patients compared to 25 healthy volunteers using customized qPCR plates.
Seventeen miRNAs were found to have significantly higher levels in breast cancer serum compared to serum of healthy volunteers in the discovery cohort. Fourteen of these miRNAs were studied in the validation cohort and serum miR-484 was found to be at a significantly higher level in breast cancer serum compared to healthy volunteers.
In this study, we found that miR-484 is significantly differentially expressed in serum of early breast cancer patients compared to healthy volunteers. We did not however find any correlation between miR-484 levels with histopathological parameters of the breast cancers. With further studies, miR-484 may prove useful as an adjunct to mammography for detection of early breast cancer.
PMCID: PMC3995145  PMID: 24641801
Breast cancer; microRNA; Serum
23.  Global assessment of genetic variation influencing response to retinoid chemoprevention in head and neck cancer patients 
Head and neck squamous cell carcinoma (HNSCC) patients are at an increased risk of developing a second primary tumor (SPT) or recurrence following curative treatment. 13-cis-retinoic acid (13-cRA) has been tested in chemoprevention clinical trials but the results have been inconclusive. We genotyped 9,465 SNPs in 450 patients from the Retinoid Head and Neck Second Primary Trial. SNPs were analyzed for associations with SPT/recurrence in patients receiving placebo to identify prognosis markers and further analyzed for effects of 13-cRA in patients with these prognostic loci. Thirteen loci identified a majority subgroup of patients at a high risk of SPT/recurrence and in whom 13-cRA was protective. Patients carrying the common genotype of rs3118570 in the retinoid X receptor (RXRA) were at a 3.33-fold increased risk (95% confidence interval [CI], 1.67–6.67) and represented over 70% of the study population. This locus also identified individuals who received benefit from chemoprevention with a 38% reduced risk (95% CI, 0.43–0.90). Analyses of cumulative effect and potential gene-gene interactions also implicated CDC25C:rs6596428 and JAK2:rs1887427 as two other genetic loci with major roles in prognosis and 13-cRA response. Patients with all three common genotypes had a 76% reduction in SPT/recurrence (95% CI, 0.093–0.64) following 13-cRA chemoprevention. Carriers of these common genotypes constituted a substantial percentage of the study population, indicating that a pharmacogenetics approach could help select patients for 13-cRA chemoprevention. The lack of any alternatives for reducing risk in these patients highlights the need for future clinical trials to prospectively validate our findings.
PMCID: PMC3955084  PMID: 21292633
HNSCC; SPT; single nucleotide polymorphisms; retinoids
24.  Fatigue Is Associated with Poor Sleep in People with Multiple Sclerosis and Cognitive Impairment 
Background. Fatigue is the most common symptom in people with multiple sclerosis (MS). Poor sleep also occurs in this population. Objective. The objective of this study was to determine the relationship between fatigue and sleep quality in people with MS and cognitive impairment. Method. This cross-sectional study assessed relationships among fatigue, assessed with the Modified Fatigue Impact Scale (MFIS) and the Fatigue Severity Scale (FSS), sleep quality assessed with the Pittsburg Sleep Quality Index (PSQI), and demographics in 121 people with MS and cognitive impairment. Results. Fatigue was significantly correlated with poor sleep quality (MFIS: F = 15.60, P < 0.01; FSS: F = 12.09, P < 0.01). FSS scores were also significantly correlated with the PSQI subscore for daytime dysfunction and MFIS scores were significantly correlated with disability, age, and the PSQI subscores for sleep quality, sleep duration, and daytime dysfunction. Conclusions. This study demonstrates a relationship between fatigue and sleep quality in individuals with MS and cognitive impairment.
PMCID: PMC3964892  PMID: 24734182
25.  Phase II Trial of Erlotinib Plus Concurrent Whole-Brain Radiation Therapy for Patients With Brain Metastases From Non–Small-Cell Lung Cancer 
Journal of Clinical Oncology  2013;31(7):895-902.
Brain metastasis (BM) is a leading cause of death from non–small-cell lung cancer (NSCLC). Reasoning that activation of the epidermal growth factor receptor (EGFR) contributes to radiation resistance, we undertook a phase II trial of the EGFR inhibitor erlotinib with whole-brain radiation therapy (WBRT) in an attempt to extend survival time for patients with BM from NSCLC. Additional end points were radiologic response and safety.
Patients and Methods
Eligible patients had BM from NSCLC, regardless of EGFR status. Erlotinib was given at 150 mg orally once per day for 1 week, then concurrently with WBRT (2.5 Gy per day 5 days per week, to 35 Gy), followed by maintenance. EGFR mutation status was tested by DNA sequencing at an accredited core facility.
Forty patients were enrolled and completed erlotinib plus WBRT (median age, 59 years; median diagnosis-specific graded prognostic assessment score, 1.5). The overall response rate was 86% (n = 36). No increase in neurotoxicity was detected, and no patient experienced grade ≥ 4 toxicity, but three patients required dose reduction for grade 3 rash. At a median follow-up of 28.5 months (for living patients), median survival time was 11.8 months (95% CI, 7.4 to 19.1 months). Of 17 patients with known EGFR status, median survival time was 9.3 months for those with wild-type EGFR and 19.1 months for those with EGFR mutations.
Erlotinib was well tolerated in combination with WBRT, with a favorable objective response rate. The higher-than-expected rate of EGFR mutations in these unselected patients raises the possibility that EGFR-mutated tumors are prone to brain dissemination.
PMCID: PMC3577951  PMID: 23341526

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