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1.  Investigating the Influence of Flip Angle and k-Space Sampling on Dynamic Contrast-Enhanced MRI Breast Examinations 
Academic Radiology  2014;21(11):1394-1401.
Rationale and Objectives
To retrospectively investigate the effect of flip angle (FA) and k-space sampling on the performance of dynamic contrast-enhanced (DCE-) magnetic resonance imaging (MRI) breast sequences.
Materials and Methods
Five DCE-MRI breast sequences were evaluated (10°, 14°, and 18° FAs; radial or linear k-space sampling), with 7–10 patients in each group (n = 45). All sequences were compliant with current technical breast screening guidelines. Contrast agent (CA) uptake curves were constructed from the right mammary artery for each examination. Maximum relative enhancement, Emax, and time-to-peak enhancement, Tmax, were measured and compared between protocols (analysis of variance and Mann–Whitney). For each sequence, calculated values of maximum relative enhancement, Ecalc, were derived from the Bloch equations and compared to Emax. Fat suppression performance (residual bright fat and chemical shift artifact) was rated for each examination and compared between sequences (Fisher exact tests).
Results
Significant differences were identified between DCE-MRI sequences. Emax increased significantly at higher FAs and with linear k-space sampling (P < .0001; P = .001). Radial protocols exhibited greater Tmax than linear protocols at FAs of both 14° (P = .025) and 18° (P < .0001), suggesting artificially flattened uptake curves. Good correlation was observed between Ecalc and Emax (r = 0.86). Fat suppression failure was more pronounced at an FA of 18° (P = .008).
Conclusions
This retrospective approach is validated as a tool to compare and optimize breast DCE-MRI sequences. Alterations in FA and k-space sampling result in significant differences in CA uptake curve shape which could potentially affect diagnostic interpretation. These results emphasize the need for careful parameter selection and greater standardization of breast DCE-MRI sequences.
doi:10.1016/j.acra.2014.06.014
PMCID: PMC4234081  PMID: 25179563
Breast imaging; MRI; contrast agents; enhancement; quality assurance
2.  Recombinant Interleukin-2 in Patients Aged Younger Than 60 Years With Acute Myeloid Leukemia in First Complete Remission 
Cancer  2013;120(7):1010-1017.
BACKGROUND
Recombinant interleukin-2 (rIL-2) induces cellular cytotoxicity against leukemia blasts. Patients with acute myeloid leukemia (AML) in first complete remission (CR) may harbor minimal residual disease that is susceptible to rIL-2–activated effector cells.
METHODS
In the Cancer and Leukemia Group B (CALGB) 19808 study, patients with AML in first CR were randomly assigned after all planned chemotherapy to receive a 90-day course of subcutaneously administered rIL-2 or no further therapy. The primary objective was to compare disease-free survival (DFS) between the 2 treatment arms. A total of 534 patients achieved a CR, 214 of whom were randomized. Six courses of low-dose daily rIL-2 were given for the expansion of cytotoxic effector cells, each followed by 3-day high-dose boluses given to trigger cytotoxicity against minimal residual disease.
RESULTS
On the protocol-specified intention-to-treat analysis, the hazards ratio for DFS was 0.75 (95% confidence interval, 0.52–1.09; P =.13); the 5-year DFS rate was 42% in the observation arm and 53% in the rIL-2 treatment arm. The hazards ratio for overall survival (OS) was 0.88 (95% confidence interval, 0.54–1.23; P =.34); the 5-year OS rate was 58% for the observation arm and 63% for the rIL-2 treatment arm. Twenty-five of the 107 patients randomized to treatment with rIL-2 either refused or were unable to initiate therapy and 30 patients did not complete their assigned therapy. However, significant toxicities were not commonly observed. The trial design did not anticipate the difficulties patients would encounter with protocol compliance.
CONCLUSIONS
The efficacy of immunotherapy with rIL-2 administered after intensive postremission treatment was not assessed as planned because of unexpected refusals by patients and/or their physicians to comply with protocol-directed therapy. Neither DFS nor OS was found to be significantly improved.
doi:10.1002/cncr.28516
PMCID: PMC4139069  PMID: 24382782
acute myeloid leukemia; recombinant interleukin-2; phase 3; immunotherapy; adjuvant therapy
3.  Feasibility Study in an in vivo Porcine Model 
Ultrasound in medicine & biology  2013;39(8):1398-1409.
Hepatocellular carcinoma (HCC) or liver cancer is one of the fastest growing cancers in the United States. Current liver ablation methods are thermal-based and share limitations due to the heat sink effect from the blood flow through the highly vascular liver. In this study, we demonstrate the feasibility of using histotripsy for non-invasive liver ablation in the treatment of liver cancer. Histotripsy is a non-thermal ablation method that fractionates soft tissue through the control of acoustic cavitation. Twelve histotripsy lesions ~1cm3 were created in the livers of six pigs through an intact abdomen and chest in vivo. Histotripsy pulses of 10 cycles, 500 Hz pulse repetition frequency (PRF), and 14-17 MPa estimated in situ peak negative pressure were applied to the liver using a 1 MHz therapy transducer. Treatments were performed through 4-6 cm of overlying tissue with 30-50% of the ultrasound pathway covered by the ribcage. Complete fractionation of liver parenchyma was observed with sharp boundaries after 16.7 minute treatments. In addition, two larger volumes of 18 cm3 and 60 cm3 were generated within 60 minutes in two additional pigs. As major vessels and gallbladder have higher mechanical strength and are more resistant to histotripsy, the major hepatic vessels and gallbladder remained intact while the liver surrounding these structures was completely fractionated. This work demonstrates that histotripsy is capable of non-invasively fractionating liver tissue while preserving critical anatomical structures within the liver. Results suggest histotripsy has potential for the non-invasive ablation of liver tumors.
doi:10.1016/j.ultrasmedbio.2013.02.005
PMCID: PMC3709011  PMID: 23683406
Liver cancer; therapeutic ultrasound; cavitation; non-invasive tissue ablation; histotripsy
4.  Nanoroughened Surfaces for Efficient Capture of Circulating Tumor Cells without Using Capture Antibodies 
ACS nano  2012;7(1):566-575.
Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization and monitoring of cancers. Here we report a simple yet effective strategy for capturing CTCs without using capture antibodies. Our method uniquely utilized the differential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cells and thus did not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques.
doi:10.1021/nn304719q
PMCID: PMC3962680  PMID: 23194329
Cancer; circulating tumor cell; nanotopography; biomaterials; microfabrication
5.  IGHV1-69 B Cell Chronic Lymphocytic Leukemia Antibodies Cross-React with HIV-1 and Hepatitis C Virus Antigens as Well as Intestinal Commensal Bacteria 
PLoS ONE  2014;9(3):e90725.
B-cell chronic lymphocytic leukemia (B-CLL) patients expressing unmutated immunoglobulin heavy variable regions (IGHVs) use the IGHV1-69 B cell receptor (BCR) in 25% of cases. Since HIV-1 envelope gp41 antibodies also frequently use IGHV1-69 gene segments, we hypothesized that IGHV1-69 B-CLL precursors may contribute to the gp41 B cell response during HIV-1 infection. To test this hypothesis, we rescued 5 IGHV1-69 unmutated antibodies as heterohybridoma IgM paraproteins and as recombinant IgG1 antibodies from B-CLL patients, determined their antigenic specificities and analyzed BCR sequences. IGHV1-69 B-CLL antibodies were enriched for reactivity with HIV-1 envelope gp41, influenza, hepatitis C virus E2 protein and intestinal commensal bacteria. These IGHV1-69 B-CLL antibodies preferentially used IGHD3 and IGHJ6 gene segments and had long heavy chain complementary determining region 3s (HCDR3s) (≥21 aa). IGHV1-69 B-CLL BCRs exhibited a phenylalanine at position 54 (F54) of the HCDR2 as do rare HIV-1 gp41 and influenza hemagglutinin stem neutralizing antibodies, while IGHV1-69 gp41 antibodies induced by HIV-1 infection predominantly used leucine (L54) allelic variants. These results demonstrate that the B-CLL cell population is an expansion of members of the innate polyreactive B cell repertoire with reactivity to a number of infectious agent antigens including intestinal commensal bacteria. The B-CLL IGHV1-69 B cell usage of F54 allelic variants strongly suggests that IGHV1-69 B-CLL gp41 antibodies derive from a restricted B cell pool that also produces rare HIV-1 gp41 and influenza hemagglutinin stem antibodies.
doi:10.1371/journal.pone.0090725
PMCID: PMC3948690  PMID: 24614505
6.  A Review of Emergency Cardiopulmonary Bypass for Severe Poisoning by Cardiotoxic Drugs 
Journal of Medical Toxicology  2012;9(1):54-60.
Cardiovascular collapse remains a leading cause of death in severe acute drug intoxication. Commonly prescribed medications such as antidysrhythmics, calcium channel antagonists, and beta adrenergic receptor antagonists can cause refractory cardiovascular collapse in massive overdose. Emergency cardiopulmonary bypass (ECPB), a modality originating in cardiac surgery, is a rescue technique that has been successfully implemented in the treatment of refractory cardiogenic shock and cardiac arrest unresponsive to traditional medical interventions. More recently a growing number of animal studies, case reports, and case series have documented its use in refractory hemodynamic collapse in poisoned patients. This article will review current ECPB techniques and explore its growing role in the treatment of severely hemodynamically compromised poisoned patients.
doi:10.1007/s13181-012-0281-8
PMCID: PMC3576497  PMID: 23238774
Emergency cardiopulmonary bypass; Extracorporeal membrane oxygenation; Extracorporeal life support; Cardiogenic shock; Cardiotoxic drugs
7.  Volunteer Bias in Recruitment, Retention, and Blood Sample Donation in a Randomised Controlled Trial Involving Mothers and Their Children at Six Months and Two Years: A Longitudinal Analysis 
PLoS ONE  2013;8(7):e67912.
Background
The vulnerability of clinical trials to volunteer bias is under-reported. Volunteer bias is systematic error due to differences between those who choose to participate in studies and those who do not.
Methods and Results
This paper extends the applications of the concept of volunteer bias by using data from a trial of probiotic supplementation for childhood atopy in healthy dyads to explore 1) differences between a) trial participants and aggregated data from publicly available databases b) participants and non-participants as the trial progressed 2) impact on trial findings of weighting data according to deprivation (Townsend) fifths in the sample and target populations. 1) a) Recruits (n = 454) were less deprived than the target population, matched for area of residence and delivery dates (n = 6,893) (mean [SD] deprivation scores 0.09[4.21] and 0.79[4.08], t = 3.44, df = 511, p<0.001). b) i)As the trial progressed, representation of the most deprived decreased. These participants and smokers were less likely to be retained at 6 months (n = 430[95%]) (OR 0.29,0.13–0.67 and 0.20,0.09–0.46), and 2 years (n = 380[84%]) (aOR 0.68,0.50–0.93 and 0.55,0.28–1.09), and consent to infant blood sample donation (n = 220[48%]) (aOR 0.72,0.57–0.92 and 0.43,0.22–0.83). ii)Mothers interested in probiotics or research or reporting infants’ adverse events or rashes were more likely to attend research clinics and consent to skin-prick testing. Mothers participating to help children were more likely to consent to infant blood sample donation. 2) In one trial outcome, atopic eczema, the intervention had a positive effect only in the over-represented, least deprived group. Here, data weighting attenuated risk reduction from 6.9%(0.9–13.1%) to 4.6%(−1.4–+10.5%), and OR from 0.40(0.18–0.91) to 0.56(0.26–1.21). Other findings were unchanged.
Conclusions
Potential for volunteer bias intensified during the trial, due to non-participation of the most deprived and smokers. However, these were not the only predictors of non-participation. Data weighting quantified volunteer bias and modified one important trial outcome.
Trial Registration
This randomised, double blind, parallel group, placebo controlled trial is registered with the International Standard Randomised Controlled Trials Register, Number (ISRCTN) 26287422. Registered title: Probiotics in the prevention of atopy in infants and children.
doi:10.1371/journal.pone.0067912
PMCID: PMC3706448  PMID: 23874465
8.  Assessment of Risk Evaluation and Mitigation Strategies in Oncology: Summary of the Oncology Risk Evaluation and Mitigation Strategies Workshop 
Journal of Oncology Practice  2012;9(2):e24-e39.
Increased provider and professional society participation should form the basis of ongoing and future REMS standardization discussions with the FDA to work toward overall improvement of risk communication.
To address oncology community stakeholder concerns regarding implementation of the Risk Evaluation and Mitigation Strategies (REMS) program, ASCO sponsored a workshop to gather REMS experiences from representatives of professional societies, patient organizations, pharmaceutical companies, and the US Food and Drug Administration (FDA). Stakeholder presentations and topical panel discussions addressed REMS program development, implementation processes, and practice experiences, as well as oncology drug safety processes. A draft REMS decision tool prepared by the ASCO REMS Steering Committee was presented for group discussion with facilitated, goal-oriented feedback.
The workshop identified several unintended consequences resulting from current oncology REMS: (1) the release of personal health information to drug sponsors as a condition for gaining access to a needed drug; (2) risk information that is not tailored—and therefore not accessible—to all literacy levels; (3) exclusive focus on drug risk, thereby affecting patient-provider treatment discussion; (4) REMS elements that do not consider existing, widely practiced oncology safety standards, professional training, and experience; and (5) administrative burdens that divert the health care team from direct patient care activities and, in some cases, could limit patient access to important therapies.
Increased provider and professional society participation should form the basis of ongoing and future REMS standardization discussions with the FDA to work toward overall improvement of risk communication.
doi:10.1200/JOP.2012.000620
PMCID: PMC3595448  PMID: 23814522
9.  Harnessing Single Cell Sorting to Identify Cell Division Genes and Regulators in Bacteria 
PLoS ONE  2013;8(4):e60964.
Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development.
doi:10.1371/journal.pone.0060964
PMCID: PMC3614548  PMID: 23565292
10.  COPY NUMBER GAINS IN EGFR AND COPY NUMBER LOSSES IN PTEN ARE COMMON EVENTS IN OSTEOSARCOMA TUMORS 
Cancer  2008;113(6):1453-1461.
Background
Osteosarcoma cell lines and tumors have been shown to express EGFR and harbor amplifications at the EGFR locus. In this study, we further analyze the genomic features of EGFR in osteosarcoma tumors and investigate whether they correlate with PTEN expression and copy number status.
Methods
EGFR and PTEN expression were assessed by immunohistochemistry (n=28), and copy number alterations at the EGFR and PTEN loci were surveyed using Affymetrix® 50K single nucleotide polymorphism (SNP) arrays (n=31) and fluorescence in situ hybridization (FISH) (n=27). The EGFR tyrosine kinase domain was sequenced to survey for activating mutations (n=34). In addition, we assessed EGFRvIII expression using RT-PCR (n=24). Results were correlated with available clinical information on 59 patients, with a median age of 14.1 years (range, 5–23 years) and median follow up of 4.4 years.
Results
EGFR expression was detected in the majority of osteosarcoma tumors surveyed (23/28; 82%). SNP arrays revealed evidence of frequent copy number gains at 7p11.2 and losses at 10q23.21. A sizeable subset (16/27 cases; 59%) showed gains at the EGFR locus using FISH – amplification in 4/27 (15%) and balanced chromosome 7 polysomy in 12/27 (44%), and 12 cases showed deletions at the PTEN locus – biallelic deletions in 4/27 (15%) and monoallelic deletion in 9/27 (33%). No activating mutations in the EGFR tyrosine kinase domain, EGFRvIII expression, or association with clinical findings were detected.
Conclusion
EGFR expression and genomic gains at the EGFR locus are prevalent in osteosarcoma tumors, which also commonly harbor deletions at the PTEN locus.
doi:10.1002/cncr.23782
PMCID: PMC3529469  PMID: 18704985
childhood cancer; osteosarcoma; epidermal growth factor receptor; tyrosine kinase; EGFRvIII; PTEN; single nucleotide polymorphism array
11.  A randomized trial to evaluate a launderable bed protection system for hospital beds 
Background
Hospital beds are potential reservoirs of bacteria in hospitals. Preventing contamination of the bed and providing a cleaner surface should help prevent hospital-acquired infections (HAIs). Most hospital beds are cleaned between patients (terminal cleaning) using quaternary ammonia compounds (quats).
Objective
The study had two objectives: identify levels of bacterial contamination on beds (including the mattress and bed deck) and evaluate a new launderable cover.
Methods
Hospital beds on a bariatric surgery ward were randomized to either receive or not receive a launderable cover (Trinity Guardion, Batesville, IN). Bacterial counts on the surface of the mattress, the bed deck, and the launderable cover were then collected using Petrifilm™ Aerobic Count Plates (Petrifilm™, 3M™, St. Paul, MN, USA) (Petrifilm™) at three time periods (before patient use, after discharge, and after terminal cleaning). Standard hospital linen was used in all rooms.
Results
The launderable cover (n = 28) was significantly cleaner prior to patient use than were the cleaned mattresses (n = 38) (1.1 CFU/30 cm2 vs. 7.7 CFU/30 cm2; p = 0.0189). The mattresses without launderable covers became significantly contaminated during use (7.7 CFU/30 cm2 on admission vs. 79.1 CFU/30 cm2 after discharge; p < 0.001). The mattresses with launderable covers did not become contaminated (3.0 CFU/30 cm2 on admission vs. 2.5 CFU/30 cm2 at discharge; p = 0.703). After terminal cleaning, the mattress surface contamination decreased to 12.8 CFU/30 cm2 (median 3 CFU/30 cm2; SD 7.8), but the bed deck was more contaminated (6.7 CFU/30 cm2 after discharge compared to 30.9 CFU/30 cm2 after terminal cleaning; p = 0.031).
Conclusions
Terminal cleaning fails to eliminate bacteria from the surface of the hospital mattress. The launderable cover provides a cleaner surface than does terminal cleaning with quats, and the cover protects the bed from contamination during use.
doi:10.1186/2047-2994-1-27
PMCID: PMC3441859  PMID: 22958605
12.  Phase-Sensitive Sodium B1 Mapping 
Quantitative sodium MRI requires accurate knowledge of factors affecting the sodium signal. One important determinant of sodium signal level is the transmit B1 field strength. However, the low signal-to-noise ratio typical of sodium MRI makes accurate B1 mapping in reasonable scan times challenging. A new phase-sensitive B1 mapping technique has recently been shown to work better than the widely used dual-angle method in low-signal-to-noise ratio situations and over a broader range of flip angles. In this work, the phase-sensitive B1 mapping technique is applied to sodium, and its performance compared to the dual-angle method through both simulation and phantom studies. The phase-sensitive method is shown to yield higher quality B1 maps at low signal-to-noise ratio and greater consistency of measurement than the dual-angle method. An in vivo sodium B1 map of the human breast is also shown, demonstrating the phase-sensitive method’s feasibility for human studies.
doi:10.1002/mrm.22700
PMCID: PMC3073006  PMID: 21413078
B1 mapping; phase-sensitive B1 mapping; sodium MRI
13.  TRANSGLUTAMINASE 2 REGULATES EARLY CHONDROGENESIS AND GLYCOSAMINOGLYCAN SYNTHESIS 
Mechanisms of development  2010;128(3-4):234-245.
The expression pattern for tissue transglutaminase (TG2) suggests that it regulates cartilage formation. We analyzed the role of TG2 in early stages of chondrogenesis using differentiating high-density cultures of mesenchymal cells from chicken limb bud as a model. We demonstrate that TG2 promotes cell differentiation towards a pre-hypertrophic stage without inducing precocious hypertrophic maturation. This finding, combined with distinctive up-regulation of extracellular TG2 in the pre-hypertrophic cartilage of the growth plate, indicates that TG2 is an autocrine regulator of chondrocyte differentiation. We also show that TG2 regulates synthesis of the cartilaginous glycosaminoglycan (GAG)-rich extracellular matrix. Elevated levels of TG2 down-regulate xylosyltransferase activity which mediates the key steps in chondroitin sulfate synthesis. On the contrary, inhibition of endogenous transglutaminase activity in differentiating chondrogenic micromasses results in increased GAG deposition and enhancement of early chondrogenic markers. Regulation of GAG synthesis by TG2 appears independent of TGF-β activity, which is a downstream mediator of the TG2 functions in some biological systems. Instead, our data suggest a major role for cAMP/PKA signaling in transmitting TG2 signals in early chondrogenic differentiation. In summary, we demonstrate that matrix synthesis and early stages of chondrogenic differentiation are regulated through a novel mechanism involving TG2-dependent inhibition of PKA. These findings further advance understanding of cartilage formation and disease, and contribute to cartilage bioengineering.
doi:10.1016/j.mod.2010.11.007
PMCID: PMC3140913  PMID: 21129482
chondrocyte; mesenchymal cells; transglutaminase 2; glycosaminoglycan; PKA; cell differentiation
14.  High Dose Cytarabine plus Gemtuzumab Ozogamicin for Patients with Relapsed or Refractory Acute Myeloid Leukemia: Cancer and Leukemia Group B Study 19902 
Leukemia research  2010;35(3):329-333.
Gemtuzumab ozogamicin (GO), an anti-CD33 immunoconjugate, was combined with high dose cytarabine (HiDAC; cytarabine 3 g/m2 over 3 hours daily for 5 days) for adults with relapsed or refractory AML. HiDAC plus GO 9 mg/m2 on day 7 and 4.5 mg/m2 on day 14 was not tolerated, but HiDAC followed by GO 9 mg/m2 on day 7 was safe: 12/37 (32%) patients with relapsed AML achieved complete remission. Median overall survival was 8.9 months. No grade 4 hepatic veno-occlusive disease was observed. This regimen merits further study, both in this setting and as a remission consolidation therapy.
doi:10.1016/j.leukres.2010.07.017
PMCID: PMC3023007  PMID: 20688393
gemtuzumab ozogamicin; acute myeloid leukemia; cytarabine; relapse
15.  Intraclonal Complexity in Chronic Lymphocytic Leukemia: Fractions Enriched in Recently Born/Divided and Older/Quiescent Cells 
Molecular Medicine  2011;17(11-12):1374-1382.
The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with more dangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lymphocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium (2H)-labeled cells as an indicator of a cell that had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and cluster designation 5 (CD5) revealed that the CXCR4dimCD5bright (proliferative) fraction contained more 2H-labeled DNA and hence divided cells than the CXCR4brightCD5dim (resting) fraction. This enrichment was confirmed by the relative expression of two cell cycle–associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisons of global gene expression between the CXCR4dimCD5bright and CXCR4brightCD5dim fractions indicated higher levels of pro-proliferation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunophenotype was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analyses suggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, enriched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cells that need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two populations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more robust understanding of the development and clonal evolution of this currently incurable disease.
doi:10.2119/molmed.2011.00360
PMCID: PMC3321822  PMID: 21968788
16.  Torque Teno Virus 10 Isolated by Genome Amplification Techniques from a Patient with Concomitant Chronic Lymphocytic Leukemia and Polycythemia Vera 
Molecular Medicine  2011;17(11-12):1338-1348.
An infectious etiology has been proposed for many human cancers, but rarely have specific agents been identified. One difficulty has been the need to propagate cancer cells in vitro to produce the infectious agent in detectable quantity. We hypothesized that genome amplification from small numbers of cells could be adapted to circumvent this difficulty. A patient with concomitant chronic lymphocytic leukemia (CLL) and polycythemia vera (PV) requiring therapeutic phlebotomy donated a large amount of phlebotomized blood to test this possibility. Using genome amplification methods, we identified a new isolate (BIS8-17) of torque teno virus (TTV) 10. The presence of blood isolate sequence 8-17 (BIS8-17) in the original plasma was confirmed by polymerase chain reaction (PCR), validating the approach, since TTV is a known plasma virus. Subsequent PCR testing of plasmas from additional patients showed that BIS8-17 had a similar incidence (~20%) in CLL (n = 48) or PV (n = 10) compared with healthy controls (n = 52). CLL cells do not harbor BIS8-17; PCR did not detect it in CLL peripheral blood genomic deoxyribonucleic acid (DNA) (n = 20). CLL patient clinical outcome or prognostic markers (immunoglobulin heavy chain variable region [IGHV ] mutation, CD38 or zeta-chain associated protein kinase 70kDa [ZAP-70]) did not correlate with BIS8-17 infection. Although not causative to our knowledge, this is the first reported isolation and detection of TTV in either CLL or PV. TTV could serve as a covirus with another infectious agent or TTV variant with rearranged genetic components that contribute to disease pathogenesis. These results prove that this method identifies infectious agents and provides an experimental methodology to test correlation with disease.
doi:10.2119/molmed.2010.00110
PMCID: PMC3321826  PMID: 21953418
17.  Comparison of Two Methods for Carboplatin Dosing in Children with Retinoblastoma 
Pediatric blood & cancer  2010;55(1):47-54.
Background
Carboplatin is the most effective drug in retinoblastoma but systemic clearance is variable in young patients. While most regimens use a flat dose, individualized targeting may provide a more adjusted systemic exposure.
Patients and Methods
We compared carboplatin doses between two groups of children with retinoblastoma that were treated using a flat dose of 560 mg/m2 or a targeted AUC of 6.5 using a modified Calvert formula.
Results
Ninety-eight patients with retinoblastoma received a total of 576 cycles of carboplatin (median 8 cycles). Fifty patients (51%) received a fixed dose per m2, 32 (33%) received a dose based on AUC, one patient received fixed dose per kilogram, and in 15 patients a combination AUC and fixed doses was used. The median cumulative carboplatin dose (mg/m2) for patients who received 8 cycles using fixed per m2 dosing was 2151.8 (range, 1414.2 – 2852.0), compared to 1104.1 for 9 patients who received 8 cycles using Calvert dosing (range, 779.0 – 1992.7)(p<0.001). For cycles given using AUC, the median percentage of the hypothetical fixed per m2 dose was 70% (range, 48%–134%). Younger patients had larger differences. Patients receiving carboplatin based on fixed per m2 dosing were 3.0 times more likely to have a platelet transfusion (95% confidence interval, 1.3–7.3).
Conclusions
Carboplatin administration needs to consider the changes in renal function occurring during the first months of life. The use of a targeted AUC provides the most accurate method; however, mg per kg of body weight dosing is a very reliable alternative method.
doi:10.1002/pbc.22467
PMCID: PMC2921445  PMID: 20486170
Carboplatin; Retinoblastoma; Glomerular filtration rate
18.  Paroxysmal Nocturnal Hemoglobinuria Following Alemtuzumab Immunosuppressive Therapy for Myelodysplastic Syndrome and Complicated by Recurrent Life-Threatening Thrombosis Despite Anticoagulation: Successful Intervention with Eculizumab and Fondaparinux 
Leukemia research  2010;34(4):e85-e87.
The pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH) is not fully understood. We report a patient with myelodysplastic syndrome who developed symptomatic PNH following treatment with alemtuzumab. A small PNH clone, identified prior to alemtuzumab, expanded resulting in hemolytic anemia and recurrent CNS thromboses despite anticoagulation. Remission was achieved with eculizumab and fondaparinux therapy. Alemtuzumab has been associated with the development of glycosylphosphotidylinositol negative cells, but its clinical significance has been unclear. Our case emphasizes its potential clinical importance. Future studies are necessary to expand our understanding of this rare disease entity and improve its management.
doi:10.1016/j.leukres.2009.09.024
PMCID: PMC2826546  PMID: 19836075
19.  Unanswered questions in the use of blood component therapy in trauma 
Recent advances in our approach to blood component therapy in traumatic hemorrhage have resulted in a reassessment of many of the tenants of management which were considered standards of therapy for many years. Indeed, despite the use of damage control techniques, the mortality from trauma induced coagulopathy has not changed significantly over the past 30 years. More specifically, a resurgence of interest in postinjury hemostasis has generated controversies in three primary areas: 1) The pathogenesis of trauma induced coagulopathy 2) The optimal ratio of blood components administered via a pre-emptive schedule for patients at risk for this condition, ("damage control resuscitation"), and 3) The appropriate use of monitoring mechanisms of coagulation function during the phase of active management of trauma induced coaguopathy, which we have previously termed "goal directed therapy". Accordingly, recent experience from both military and civilian centers have begun to address these controversies, with certain management trends emerging which appear to significantly impact the way we approach these patients.
doi:10.1186/1757-7241-19-5
PMCID: PMC3027129  PMID: 21241509
20.  Phase II Multi-Institutional Trial of the Histone Deacetylase Inhibitor Romidepsin As Monotherapy for Patients With Cutaneous T-Cell Lymphoma 
Journal of Clinical Oncology  2009;27(32):5410-5417.
Purpose
Romidepsin (depsipeptide or FK228) is a member of a new class of antineoplastic agents active in T-cell lymphoma, the histone deacetylase inhibitors. On the basis of observed responses in a phase I trial, a phase II trial of romidepsin in patients with T-cell lymphoma was initiated.
Patients and Methods
The initial cohort was limited to patients with cutaneous T-cell lymphoma (CTCL), or subtypes mycosis fungoides or Sézary syndrome, who had received no more than two prior cytotoxic regimens. There were no limits on other types of therapy. Subsequently, the protocol was expanded to enroll patients who had received more than two prior cytotoxic regimens.
Results
Twenty-seven patients were enrolled onto the first cohort, and a total of 71 patients are included in this analysis. These patients had undergone a median of four prior treatments, and 62 patients (87%) had advanced-stage disease (stage IIB, n = 15; stage III, n= 6; or stage IV, n = 41). Toxicities included nausea, vomiting, fatigue, and transient thrombocytopenia and granulocytopenia. Pharmacokinetics were evaluated with the first administration of romidepsin. Complete responses were observed in four patients, and partial responses were observed in 20 patients for an overall response rate of 34% (95% CI, 23% to 46%). The median duration of response was 13.7 months.
Conclusion
The histone deacetylase inhibitor romidepsin has single-agent clinical activity with significant and durable responses in patients with CTCL.
doi:10.1200/JCO.2008.21.6150
PMCID: PMC2773225  PMID: 19826128
21.  Cholangiocyte secretion of chemokines in experimental biliary atresia 
Journal of pediatric surgery  2009;44(3):500-507.
Biliary atresia (BA) is a disease of the newborn which results in obstruction of the biliary tree. The cause of BA remains unknown; however, recent studies using the murine model of biliary atresia have found that rotavirus infection of the biliary epithelial cell (cholangiocyte) triggers an inflammatory response. We hypothesized that rotavirus infection of cholangiocytes results in the release of chemokines, important mediators of the host immune response.
Methods
In vivo, Balb/c pups were injected with rhesus rotavirus (RRV) or saline, and, their extrahepatic bile ducts were microdissected 2,5, 7, and 14 days after injection. Next, an immortalized cholangiocyte cell line (mCl) was incubated with RRV or serum free media. Qualitative and quantitative chemokine assement was performed using ELISA, PCR, and immunohistchemistry.
Results
In vivo, increased levels of the chemokines MIP-2, MCP-1, KC and RANTES were found in RRV-infected murine bile ducts. In vitro, infected mCl cells produced increasing amounts of these same chemokines in relation to dose and time.
Conclusion
These novel results suggest that chemokine expression by RRV-infected cholangiocytes may trigger a host inflammatory process that causes bile duct obstruction. Understanding how viral infection initiates this response may shed light on the pathogenesis of biliary atresia.
doi:10.1016/j.jpedsurg.2008.07.007
PMCID: PMC2730110  PMID: 19302848
22.  Chronic Lymphocytic Leukemia Cells Recognize Conserved Epitopes Associated with Apoptosis and Oxidation 
Molecular Medicine  2008;14(11-12):665-674.
Chronic lymphocytic leukemia (CLL) represents the outgrowth of a CD5+ B cell. Its etiology is unknown. The structure of membrane Ig on CLL cells of unrelated patients can be remarkably similar. Therefore, antigen binding and stimulation could contribute to clonal selection and expansion as well as disease promotion. Initial studies suggest that CLL mAbs bind autoantigens. Since apoptosis can make autoantigens accessible for recognition by antibodies, and also create neo-epitopes by chemical modifications occurring naturally during this process, we sought to determine if CLL mAbs recognize autoantigens associated with apoptosis. In general, ~60% of CLL mAbs bound the surfaces of apoptotic cells, were polyreactive, and expressed unmutated IGHV. mAbs recognized two types of antigens: native molecules located within healthy cells, which relocated to the external cell surface during apoptosis; and/or neoantigens, generated by oxidation during the apoptotic process. Some of the latter epitopes are similar to those on bacteria and other microbes. Although most of the reactive mAbs were not mutated, the use of unmutated IGHV did not bestow autoreactivity automatically, since several such mAbs were not reactive. Particular IGHV and IGHV/D/J rearrangements contributed to autoantigen binding, although the presence and degree of reactivity varied based on specific structural elements. Thus, clonal expansion in CLL may be stimulated by autoantigens occurring naturally during apoptosis. These data suggest that CLL may derive from normal B cells whose function is to remove cellular debris, and also to provide a first line of defense against pathogens.
doi:10.2119/2008-00102.Catera
PMCID: PMC2582860  PMID: 19009014
23.  Surface Expression of Bcl-2 in Chronic Lymphocytic Leukemia and Other B-Cell Leukemias and Lymphomas Without a Breakpoint t(14;18) 
Molecular Medicine  2008;14(9-10):618-627.
Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings. Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity. Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t(14;18).Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis. Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells. It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.
doi:10.2119/2008.00061.McCarthy
PMCID: PMC2464573  PMID: 18633450
24.  Effect of Rotavirus Strain on the Murine Model of Biliary Atresia▿  
Journal of Virology  2006;81(4):1671-1679.
Biliary atresia is a devastating disorder of the newborn in which afflicted infants develop inflammation and fibrosis of the extrahepatic biliary tract, resulting in cirrhosis and end-stage liver disease. Infection with a virus is thought to be a contributing factor in the etiology of biliary atresia. In the murine model of biliary atresia, perinatal exposure to rhesus rotavirus (RRV) results in biliary epithelial cell infection causing bile duct obstruction. The purpose of this study was to determine if tropism for the biliary epithelial cell was unique to RRV. Newborn mice underwent intraperitoneal injection with five strains of rotavirus: RRV (simian), SA11-FM (simian/bovine), SA11-SM (simian), EDIM (murine), and Wa (human). RRV and SA11-FM caused clinical manifestations of bile duct obstruction and high mortality. SA11-SM caused clinical signs of hepatobiliary injury but the mortality was markedly reduced. EDIM and Wa caused no sign of hepatobiliary disease. The systemic and temporal distribution of viral protein and live virus varied according to the injected strain. Immunohistochemistry revealed that RRV and SA11-FM targeted the biliary epithelial cells. In contrast, SA11-SM was found in the liver but in not in the biliary epithelium. These results indicate that strain-specific characteristics dictate tropism for cells of hepatobiliary origin which in turn impact the ability to induce the murine model of biliary atresia.
doi:10.1128/JVI.02094-06
PMCID: PMC1797562  PMID: 17121809
25.  Similarities and Differences Between the Light and Heavy Chain Ig Variable Region Gene Repertoires in Chronic Lymphocytic Leukemia 
Molecular Medicine  2006;12(11-12):300-308.
Analyses of Ig VHDJH rearrangements expressed by B-CLL cells have provided insights into the antigen receptor repertoire of B-CLL cells and the maturation stages of B-lymphocytes that give rise to this disease. However, less information is available about the L chain V gene segments utilized by B-CLL cells and to what extent their characteristics resemble those of the H chain. We analyzed the VL and JL gene segments of 206 B-CLL patients, paying particular attention to frequency of use and association, mutation status, and LCDR3 characteristics. Approximately 40% of B-CLL cases express VL genes that differ significantly from their germline counterparts. Certain genes were virtually always mutated and others virtually never. In addition, preferential pairing of specific VL and JL segments was found. These findings are reminiscent of the expressed VH repertoire in B-CLL. However unlike the VH repertoire, VL gene use was not significantly different than that of normal B-lymphocytes. In addition, Vκ genes that lie more upstream on the germline locus were less frequently mutated than those at the 3′ end of the locus; this was not the case for Vλ genes and is not for VH genes. These similarities and differences between the IgH and IgL V gene repertoires expressed in B-CLL suggest some novel features while also reinforcing concepts derived from studies of the IgH repertoire.
doi:10.2119/2006-00080.Ghiotto
PMCID: PMC1829199  PMID: 17380195

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