BACKGROUND:The FDA initially approved pembrolizumab and nivolumab for doses based on patient weight, but subsequently amended approval to fixed doses. We estimated savings from novel dosing strategies based on real-world patient data from a single cancer center. METHODS:We analyzed all outpatient doses of pembrolizumab and nivolumab administered at three infusion centers affiliated with our academic hospital between July 1, 2018 and Oct 31, 2018. We estimated savings from several dosing strategies with and without vial sharing between patients. RESULTS:A total of 1029 doses of pembrolizumab or nivolumab were administered for multiple cancer types. For 77% of doses, the weight-based dose was less than the fixed dose. "Dose-minimization" (DM), defined as the lesser of weight-based and fixed dose decreased nivolumab spending by 9% without affecting pembrolizumab spending. DM plus vial sharing decreased pembrolizumab spending by 19% without affecting nivolumab. The differences in savings were due to availability of multiple vial sizes for nivolumab but not pembrolizumab. DM plus vial sharing for both drugs would have saved $1.5 million USD over the 4-month study period. CONCLUSION:New dosing strategies for pembrolizumab and nivolumab can generate large savings without anticipated decrease in efficacy. Barriers include FDA dosing labels, hospital policies against vial sharing, and inaccessibility of smaller vial sizes, which are currently available in other worldwide markets.

Elevated blood ammonia (hyperammonemia) may cause delirium, brain damage, and even death. Effective treatments exist, but preventing permanent neurological sequelae requires rapid, accurate, and serial measurements of blood ammonia. Standard methods require volumes of 1 to 3 mL, centrifugation to isolate plasma, and a turn-around time of 2 h. Collection, handling, and processing requirements mean that community clinics, particularly those in low resource settings, cannot provide reliable measurements. We describe a method to measure ammonia from small-volume whole blood samples in 2 min. The method alkalizes blood to release gas-phase ammonia for detection by a fuel cell. When an inexpensive first-generation instrument designed for 100 mu L of blood was tested on adults and children in a clinical study, the method showed a strong correlation (R-2 = 0.97) with an academic clinical laboratory for plasma ammonia concentrations up to 500 mu M (16 times higher than the upper limit of normal). A second-generation hand-held instrument designed for 10-20 mu L of blood showed a near-perfect correlation (R-2 = 0.99) with healthy donor blood samples containing known amounts of added ammonium chloride up to 1000 mu M. Our method can enable rapid and inexpensive measurement of blood ammonia, transforming diagnosis and management of hyperammonemia.

211 Background: Patient reported outcomes (PROs) allow for systematic and more continuous capture of the patient experience. PROs have been associated with improved symptom management and quality of life in patients with advanced cancer; however how to do this in a real world setting is not thoroughly described. We sought to provide a description of the implementation and feasibility of a digital PRO application in patients with advanced cancer at one academic center. Methods: This is an ongoing randomized trial, comparing digital PRO intervention vs standard of care, in advanced cancer patients from three oncology groups (thoracic, gastrointestinal, genitourinary). Prior to trial initiation, a lead in period to test workflows was conducted, as well as key stakeholder interviews to optimize engagement with the platform. Clinical team members (e.g. nurses, advanced practice providers) were critical to the workflows developed, thus operational leadership buy-in was required. We were unable to integrate our PRO platform with our digital EHR. Regular sponsor meetings addressed technical concerns and patient/provider feedback. Processes were tailored for each disease group to handle varying degrees of symptoms (mild, moderate and severe). Patient and clinician satisfaction and qualitative feedback was collected. We defined feasibility as > 70% questionnaire completion on the platform. Results: There are 64 patients on the intervention arm and 64 on the control arm. Of eligible patients, 93% (128/138) enrolled in the study. During the study period, 447 digital symptom questionnaires were sent by the platform, of which 333 were completed (74%). The majority of clinicians (82%, n = 18) and patients (85%, n = 64) felt neutral or positive when asked if the tool was easy to use. Approximately half of clinicians (45%, n = 10) and patients (46%, n = 36) would probably or definitely recommend the platform. Conclusions: Web-based PRO reporting for patients with advanced cancer is feasible. Clinicians and patients found the platform both acceptable and easy to use. Future directions include integration with our EHR, expansion across disease groups and other centers, and creation of workflows that are integrated into routine clinical practice.

Background Cancer patients receiving chemotherapy often complain of "chemobrain" or cognitive impairment, but mechanisms remain elusive. Methods A patient with gastric cancer developed delirium and hyperammonemia after chemotherapy with the 5-fluorouracil pro-drug capecitabine. Exome sequencing facilitated a search for mutations among 43 genes associated with hyperammonemia and affecting the urea cycle directly or indirectly. Results The patient's urea cycle was impaired by capecitabine-induced liver steatosis, and portosystemic shunting of gut ammonia into the systemic circulation. The patient was also heterozygous for amino acid substitution mutations previously reported to create dysfunctional proteins in 2 genes, ORNT2 (ornithine transporter-2 for the urea cycle), and ETFA (electron transport flavoprotein alpha for fatty acid oxidation). The mutations explained the patient's abnormal plasma amino acid profile and exaggerated response to allopurinol challenge. Global population variations among the 43 hyperammonemia genes were assessed for inactivating mutations, and for amino acid substitutions predicted to be deleterious by complementary algorithms, SIFT and PolyPhen-2. One or 2 deleterious mutations occur among the 43 genes in 13.9% and 1% of individuals, respectively. Conclusions Capecitabine and 5-fluorouracil inhibit pyrimidine biosynthesis, decreasing ammonia utilization. These drugs can induce hyperammonemia in susceptible individuals. The risk factors of hyperammonemia, gene mutations and liver dysfunction, are not rare. Diagnosis will trigger appropriate treatment and ameliorate brain toxicity.

In human cells, efficient global genomic re- pair of DNA damage induced by ultraviolet radiation requires the p53 tumor suppressor, but the mechanism has been unclear. The p48 gene is required for expression of an ultra- violet radiation-damaged DNA binding activity and is dis- rupted by mutations in the subset of xeroderma pigmentosum group E cells that lack this activity. Here, we show that p48 mRNA levels strongly depend on basal p53 expression and increase further after DNA damage in a p53-dependent man- ner. Furthermore, like p53-/- cells, xeroderma pigmentosum group E cells are deficient in global genomic repair. These results identify p48 as the link between p53 and the nucleotide excision repair apparatus.

Nonhomologous end joining repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. Ku, XRCC4/Ligase IV (XL) and XLF have a remarkable mismatched end (MEnd) ligase activity, particularly for ends with mismatched 3' overhangs, but the mechanism has remained obscure. Here, we showed XL required Ku to bind DNA, while XLF required both Ku and XL to bind DNA. We detected cooperative assembly of one or two Ku molecules and up to 5 molecules each of XL and XLF into a Ku:XL:XLF-DNA (MEnd ligase-DNA) complex. XLF mutations that disrupted its interactions with XRCC4 or DNA also disrupted complex assembly and end joining. Together with published co-crystal structures of truncated XRCC4 and XLF proteins, our data with full-length Ku, XL and XLF bound to DNA indicate assembly of a filament containing Ku plus alternating XL and XLF molecules. By contrast, in the absence of XLF, we detected cooperative assembly of up to 6 molecules each of Ku and XL into a Ku:XL-DNA complex, consistent with a filament containing alternating Ku and XL molecules. Despite a lower molecular weight, MEnd ligase-DNA had a lower electrophoretic mobility than Ku:XLDNA. The anomalous difference in mobility and difference in XL to Ku molar ratio suggest that MEnd ligase-DNA has a distinct structure that successfully aligns mismatched DNA ends for ligation. Nonhomologous end joining (NHEJ) repairs DNA double-strand breaks created by exogenous agents such as ionizing radiation, or by the endogenous mechanism for generating immunological diversity, V(D)J recombination (1,2). NHEJ recognizes broken ends, brings them into juxtaposition, and joins the ends while optimizing the preservation of DNA sequence. NHEJ even preserves 3’-overhanging DNA sequences created during V(D)J recombination by hairpin opening (P-nucleotide addition) or by terminal deoxynucleotidyl transferase activity (Nnucleotide addition). Thus, NHEJ has a mechanism for joining DNA ends with mismatched 3’ overhangs. Core proteins for NHEJ include Ku, DNAdependent protein kinase catalytic subunit (DNAPKcs), XRCC4/Ligase IV (XL) and XRCC4-like factor (XLF, also known as Cernunnos). Ku, which consists of two subunits, Ku70 and Ku80, binds DNA ends with high affinity (3,4) and then recruits DNA-PKcs to the ends (5). DNA-PKcs brings the ends together into a synaptic complex and then undergoes activation of its kinase domain http://www.jbc.org/cgi/doi/10.1074/jbc.M113.464115 The latest version is at JBC Papers in Press. Published on April 25, 2013 as Manuscript M113.464115

BACKGROUND Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a growth factor known to have in vitro effects on the growth and invasiveness of cancer. It has been approved by the U.S. Food and Drug Administration in limited doses for single-level anterior spinal arthrodesis, but it is commonly used off-label and at high doses. The effect of rhBMP-2 on the risk of cancer has been a concern. We sought to evaluate the risk of new cancers in patients receiving high-dose rhBMP-2. METHODS We used publicly available data from a pivotal, multicenter, randomized controlled trial of patients with degenerative lumbar spine conditions who underwent a single-level instrumented posterolateral arthrodesis with either high-dose rhBMP-2 in a compression-resistant matrix (CRM) (rhBMP-2/CRM; n = 239) or autogenous bone graft (control group; n = 224). We compared the risks of new cancers in the rhBMP-2/CRM and control groups at two and five years after surgery. RESULTS At two years, with 86% follow-up, there were fifteen new cancer events in eleven patients in the rhBMP-2/CRM group compared with two new cancer events in two patients in the control group treated with autogenous bone graft. The incidence rate of new cancer events per 100 person-years was 3.37 (95% confidence interval [CI], 1.89 to 5.56) in the rhBMP-2/CRM group at two years compared with 0.50 (95% CI, 0.06 to 1.80) in the control group. The incidence rate ratio was 6.75 (95% CI, 1.57 to 60.83; p = 0.0026) at two years. Calculated in terms of the number of patients with one or more cancer events two years after the surgery, the incidence rate per 100 person-years was 2.54 (95% CI, 1.27 to 4.54) in the rhBMP-2/CRM group compared with 0.50 (95% CI, 0.06 to 1.82) in the control group at two years; the incidence rate ratio was 5.04 (95% CI, 1.10 to 46.82; p = 0.0194). At five years, there was a 37% loss of follow-up, but a significantly greater incidence of cancer events was still observed in the rhBMP-2/CRM group. CONCLUSIONS A high dose of 40 mg of rhBMP-2/CRM in lumbar spinal arthrodesis was associated with an increased risk of new cancer.

Nonhomologous end joining repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. Ku, XRCC4/Ligase IV (XL), and XLF have a remarkable mismatched end (MEnd) ligase activity, particularly for ends with mismatched 3' overhangs, but the mechanism has remained obscure. Here, we showed XL required Ku to bind DNA, whereas XLF required both Ku and XL to bind DNA. We detected cooperative assembly of one or two Ku molecules and up to five molecules each of XL and XLF into a Ku-XL-XLF-DNA (MEnd ligase-DNA) complex. XLF mutations that disrupted its interactions with XRCC4 or DNA also disrupted complex assembly and end joining. Together with published co-crystal structures of truncated XRCC4 and XLF proteins, our data with full-length Ku, XL, and XLF bound to DNA indicate assembly of a filament containing Ku plus alternating XL and XLF molecules. By contrast, in the absence of XLF, we detected cooperative assembly of up to six molecules each of Ku and XL into a Ku-XL-DNA complex, consistent with a filament containing alternating Ku and XL molecules. Despite a lower molecular mass, MEnd ligase-DNA had a lower electrophoretic mobility than Ku-XL-DNA. The anomalous difference in mobility and difference in XL to Ku molar ratio suggests that MEnd ligase-DNA has a distinct structure that successfully aligns mismatched DNA ends for ligation.

The electrophoretic mobility shift assay (EMSA) can be used to study proteins that bind to DNA structures created by DNA-damaging agents. UV-damaged DNA-binding protein (UV-DDB), which is involved in nucleotide excision repair, binds to DNA damaged by ultraviolet radiation or the anticancer drug cisplatin. Ku, XRCC4/Ligase IV, and DNA-PKcs, which are involved in the repair of DNA double-strand breaks by nonhomologous end joining, assemble in complexes at DNA ends. This chapter will describe several EMSA protocols for detecting different DNA repair protein-DNA complexes. To obtain additional information, one can apply variations of the EMSA, which include the reverse EMSA to detect binding of (35)S-labeled protein to damaged DNA, and the antibody supershift assay to detect the presence of a specific protein in the protein-DNA complex.

An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly 'purified' cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation.

Abstract : We have developed an efficient assay capable of detecting assembly of the core ligase complex involved in nonhomologous end joining (NHEJ) of DNA double-strand breaks. The assay can also detect inhibition of complex formation upon linkage to a large protein such as estrogen receptor. We have shown that Cernunnos is an ideal target for disrupting NHEJ. Using recombinant proteins, we have found regions in Cernunnos that can be targeted for disrupting repair only in cells expressing estrogen receptor. Finally, we have performed experiments to demonstrate the feasibility of each individual step in the chemical synthesis of the adapter.

The local false discovery rate (LFDR) estimates the probability of falsely identifying specific genes with changes in expression. In computer simulations, LFDR < 10% successfully identified genes with changes in expression, while LFDR > 90% identified genes without changes. We used LFDR to compare different microarray experiments quantitatively: (i) Venn diagrams of genes with and without changes in expression, (ii) scatter plots of the genes, (iii) correlation coefficients in the scatter plots and (iv) distributions of gene function. To illustrate, we compared three methods for pre-processing microarray data. Correlations between methods were high (r = 0.84-0.92). However, responses were often different in magnitude, and sometimes discordant, even though the methods used the same raw data. LFDR complements functional assessments like gene set enrichment analysis. To illustrate, we compared responses to ultraviolet radiation (UV), ionizing radiation (IR) and tobacco smoke. Compared to unresponsive genes, genes responsive to both UV and IR were enriched for cell cycle, mitosis, and DNA repair functions. Genes responsive to UV but not IR were depleted for cell adhesion functions. Genes responsive to tobacco smoke were enriched for detoxification functions. Thus, LFDR reveals differences and similarities among experiments.

The first step in the repair of DNA damage is lesion detection. In this issue, Scrima et al. (2008) report the structure of the complex of DNA Damage-Binding Protein 1 (DDB1) and DDB2 bound to a DNA photodimer, providing critical insight into the repair of DNA damage caused by ultraviolet light.

Nonhomologous end-joining ( NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. To repair the broken ends, NHEJ processes noncompatible ends into a ligatable form but suppresses processing of compatible ends. It is not known how NHEJ controls polymerase and nuclease activities to act exclusively on noncompatible ends. Here, we analyzed processing independently of ligation by using a two-stage assay with extracts that recapitulated the properties of NHEJ in vivo. Processing of noncompatible ends required wortmannin-sensitive kinase activity. Since DNA-dependent protein kinase catalytic subunit ( DNA-PKcs) brings the ends together before undergoing activation of its kinase, this suggests that processing occurred after synapsis of the ends. Surprisingly, all polymerase and most nuclease activity required XRCC4/Ligase IV. This suggests a mechanism for how NHEJ suppresses processing to optimize the preservation of DNA sequence.