Log-fold Reduction Research Articles

Clinical Utility of Circulating Tumor (ct)DNA Quantity and Kinetics in Patients Undergoing CAR T-Cell Therapy for Relapsed/Refractory Aggressive B-Cell Lymphoma

Background: CD19-targeted CAR T-cells are approved for relapsed and/or refractory (RR) aggressive B-cell lymphomas. While some patients experience durable remissions, others do not respond or relapse. Improved patient selection and early identification of patients who may need additional therapies are thus necessary. ctDNA is a promising biomarker for prognostication in DLBCL and has the potential to guide therapeutic decisions. We aimed to explore the possible clinical utility of ctDNA quantitation in patients with aggressive B-cell lymphomas undergoing CAR T-cell therapy. Methods: Twenty-eight patients undergoing CAR T-cell therapy as part of standard of care pathways for RR aggressive B-cell lymphoma were included. Pre- and post-CAR T-cell infusion ctDNA samples were retrospectively analyzed with a panel of over 400 genes relevant to B-NHL using a modified version of the AVENIO ctDNA analysis workflow (Roche; Research Use Only) based on the previously described CAPP-Seq workflow (Kurtz, J Clin Oncol 2018). Peripheral blood was used as a source of germline DNA to allow selection of tumor-specific variants in the ctDNA. ctDNA levels were expressed as mean mutant molecules per mL (MMPM) and ctDNA clearance was determined using a detection cutoff of p = 0.005. ctDNA results were correlated with clinical findings and outcomes including response rate, PFS and OS. Results: Eighteen male and 10 female patients were included in the analysis (median age 63). Sixteen (57%) patients had a diagnosis of DLBCL while the remainder had transformed FL or MZL (n=6), HGBL (n=4) and PMBL (n=2). Seventeen patients were treated with tisagenlecleucel and 11 with axicabtagene ciloleucel. Twenty-five (89%) patients had stage III or IV disease and 15 (54%) patients had ≥3 lines of prior therapy. The median number of tumor-specific variants in baseline ctDNA samples was 31 (range 1-252). ctDNA levels correlated with metabolic tumor volumes across all time points (Spearman r=0.69, p<0.0001). There was a greater reduction in ctDNA quantity observed in those who achieved CR/PR (n=21) at day 28 vs those who progressed (n=7) (median MMPM log-fold reduction 1.3 vs -0.9). Baseline ctDNA quantity was significantly higher in those with an elevated LDH pre-lymphodepletion compared to those with a normal LDH (median MMPM 228.5 vs 7.6, p = 0.0003). Survival also correlated with baseline ctDNA measurement with patients with a ctDNA quantity above the median of the cohort having a significantly shorter OS compared to those below the median (median OS 6.7 mo vs not reached, p=0.0012). Four patients had no detectable ctDNA at day 28 post CAR T-cell infusion including 1 patient with pseudoprogression on imaging (SUVMax 10) who had no evidence of lymphoma on biopsy and remained in CR. Of the 4 patients who were ctDNA negative at day 28, 3 remained in long-term remission. In summary, measurement of ctDNA quantity and kinetics shows promising clinical utility in the context of CAR T-cell treatment for aggressive B-cell lymphoma including prediction of outcome and resolving radiological pseudoprogression. Larger prospective cohorts and standardized methodological approaches are required to optimize predictive thresholds for clinical outcomes.

Risk Profiling of Relapsed/Refractory Diffuse Large B-Cell Lymphoma Patients By Measuring Circulating Tumor DNA

Risk Profiling of Relapsed/Refractory Diffuse Large B-Cell Lymphoma Patients By Measuring Circulating Tumor DNA

Temperature-sensitive recombinant subtilisin protease variants that efficiently degrade molecular biology enzymes.

We used error-prone PCR to generate mutations in a subtilisin protease-encoding gene, and screened for recombinants that expressed temperature-sensitive (TS) variants. From the dozens of mutations that we detected in the recombinant genes we found that those mutations that affected aspartate-75 had the most profound effect on temperature stability. We thus focused our analysis on two variants of subtilisin C, the more heat-sensitive variant 24 (V24), with amino acid changes D75G, L234M and Q274P; and variant 25 (V25), with a single amino acid change, D75A. For V24 a two log-fold reduction in activity occurs in under 10 min at 50°C. For V25, a two log-fold reduction occurs at 60°C, a temperature that reduces the activity of the wild type enzyme by about 30%. The V24 variant fully inactivates enzymes commonly used in molecular biology research and in molecular diagnostics, and is stabilized against autolysis with propylene glycol concentrations of 10% or greater. The subtilisin variants are produced by a strain of Bacillus subtilis that lacks expression of its native secreted proteases, and the variants can be isolated from the supernatants using nickel affinity chromatography.

Liver Function Test Abnormalities in Experimental and Clinical Plasmodium vivax Infection

.Liver transaminase elevations after treatment in malaria volunteer infection studies (VISs) have raised safety concerns. We investigated transaminase elevations from two human Plasmodium vivax VISs where subjects were treated with chloroquine (n = 24) or artefenomel (n = 8) and compared them with studies in Thailand (n = 41) and Malaysia (n = 76). In the VISs, alanine transaminase (ALT) increased to ≥ 2.5 × upper limit of normal (ULN) in 11/32 (34%) volunteers, peaking 5–8 days post-treatment. Transaminase elevations were asymptomatic, were not associated with elevated bilirubin, and resolved by day 42. The risk of an ALT ≥ 2.5 × ULN increased more than 4-fold (odds ratio [OR] 4.28; 95% CI: 1.26–14.59; P = 0.02) for every log10 increase in the parasite clearance burden (PCB), defined as the log-fold reduction in parasitemia 24 hours post-treatment. Although an elevated ALT ≥ 2.5 × ULN was more common after artefenomel than after chloroquine (5/8 [63%] versus 6/24 [25%]; OR 5.0; 95% CI: 0.91–27.47; P = 0.06), this risk disappeared when corrected for PCB. Peak ALT also correlated with peak C-reactive protein (R = 0.44; P = 0.012). Elevations in ALT (≥ 2.5 × ULN) were less common in malaria-endemic settings, occurring in 1/41 (2.5%) Thai patients treated with artefenomel, and in none of 76 Malaysians treated with chloroquine or artemisinin combination therapy. Post-treatment transaminase elevations are common in experimental P. vivax infection but do not appear to impact on participant safety. Although the mechanism of these changes remains uncertain, host inflammatory response to parasite clearance may be contributory.

Electroosmotically generated disinfectant from urine as a by-product of electricity in microbial fuel cell for the inactivation of pathogenic species

This work presents a small scale and low cost ceramic based microbial fuel cell, utilising human urine into electricity, while producing clean catholyte into an initially empty cathode chamber through the process of electro-osmostic drag. It is the first time that the catholyte obtained as a by-product of electricity generation from urine was transparent in colour and reached pH>13 with high ionic conductivity values. The catholyte was collected and used ex situ as a killing agent for the inactivation of a pathogenic species such as Salmonella typhimurium, using a luminometer assay. Results showed that the catholyte solutions were efficacious in the inactivation of the pathogen organism even when diluted up to 1:10, resulting in more than 5 log-fold reduction in 4 min. Long-term impact of the catholyte on the pathogen killing was evaluated by plating Salmonella typhimurium on agar plates and showed that the catholyte possesses a long-term killing efficacy and continued to inhibit pathogen growth for 10 days.

Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in Combination with Photochemical Internalization.

The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.

Novel benzimidazole-acrylonitrile hybrids and their derivatives: Design, synthesis and antimycobacterial activity

This paper reports the synthesis and evaluation of some benzimidazole-acrylonitrile hybrid derivatives for their in vitro antimycobacterial activities against Mycobacterium tuberculosis H37Rv. Among the derivatives studied, 3b was found to be the most active compound with MIC of 0.78 μg/mL against M. tuberculosis. This is a quite good activity compared with ethambutol (MIC = 1.56 μg/mL). Moreover, 3b showed 2.8 log fold reduction in bacterial count of dormant forms of mycobacterium which is more potent than first line drugs isoniazid, ciprofloxacin, rifampicin and moxifloxacin. Having activities against both active and dormant forms of M. tuberculosis,3b may be a useful candidate for the development of new drugs to treat tuberculosis.

Extensive Metabolic Correction of Hurler Disease By Hematopoietic Stem Cell-Based Gene Therapy: Preliminary Results from a Phase I/II Trial

Extensive Metabolic Correction of Hurler Disease By Hematopoietic Stem Cell-Based Gene Therapy: Preliminary Results from a Phase I/II Trial

Fate of three bioluminescent pathogenic bacteria fed through a cascade of urine microbial fuel cells

Microbial fuel cell (MFC) technology is currently gaining recognition as one of the most promising bioenergy technologies of the future. One aspect of this technology that has received little attention is the disinfection of effluents and the fate of pathogenic organisms that find their way into the waste stream. In this study, three independent trials were carried out to evaluate the fate of three bioluminescent pathogenic bacteria (Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus) introduced into the anodic chamber of a urine-fed cascade of 9 MFCs with matured, electroactive biofilms. These are common examples of enteric human pathogens, which could contaminate urine or waste streams. The results showed that the average power generation in the closed circuit cascade reached 754 ± 16 µW, with an average pathogen log-fold reduction of 6.24 ± 0.63 compared to 2.01 ± 0.26 for the open circuit cascade for all three pathogens. The results suggest that the bio-electrochemical reactions associated with electricity generation were the primary driving force for the inactivation of the introduced pathogens. These findings show that pathogenic organisms introduced into waste streams could be inactivated by the power-generating process within the MFC cascade system, thereby preventing propagation and thus rendering the effluent safer for possible reuse.

Local Delivery of Amikacin and Vancomycin from Chitosan Sponges Prevent Polymicrobial Implant-Associated Biofilm.

Military personnel have high risk for infection, particularly those with combat-related extremity trauma. Administration of multiple or broad-spectrum antibiotics provides clinicians with a strategy for preventing biofilm-based medical device infections. Selection of effective antibiotic combinations based on common pathogens may be used to improve chitosan wound dressing sponge-based local antibiotic delivery systems. In vitro assays in this study demonstrate that vancomycin and amikacin have a synergistic relationship against a strain of osteomyelitis-producing Gram-positive Staphylococcus aureus, although an indifferent relationship was observed against Gram-negative Pseudomonas aeruginosa. In an in vivo model of orthopedic hardware-associated polymicrobial (S. aureus and Escherichia coli) biofilm, chitosan sponges loaded with a combination of vancomycin and amikacin at 5 mg/mL each showed a greater percentage of complete clearance, 50%, than either antibiotic alone, 8.33%. Doubling the loading concentration of the combination achieved a complete clearance rate of 100%, a four log-fold reduction of S. aureus on the wire and a six log-fold reduction in bone. E. coli was detected in bone of untreated animals but did not form biofilm on wires. Results demonstrate the clinical potential of chitosan sponges to prevent infection and illustrates antibiotic selection and loading concentrations necessary for effective biofilm prevention.

Urine disinfection and in situ pathogen killing using a Microbial Fuel Cell cascade system.

Microbial Fuel Cells (MFCs) are emerging as an effective means of treating different types of waste including urine and wastewater. However, the fate of pathogens in an MFC-based system remains unknown, and in this study we investigated the effect of introducing the enteric pathogen Salmonella enterica serovar enteritidis in an MFC cascade system. The MFCs continuously fed with urine showed high disinfecting potential. As part of two independent trials, during which the bioluminescent S. enteritidis strain was introduced into the MFC cascade, the number of viable counts and the level of bioluminescence were reduced by up to 4.43±0.04 and 4.21±0.01 log-fold, respectively. The killing efficacy observed for the MFCs operating under closed-circuit conditions, were higher by 1.69 and 1.72 log-fold reduction than for the open circuit MFCs, in both independent trials. The results indicated that the bactericidal properties of a well performing anode were dependent on power performance and the oxidation-reduction potential recorded for the MFCs. This is the first time that the fate of pathogenic bacteria has been investigated in continuously operating MFC systems.

Cephalosporin-3'-Diazeniumdiolate NO Donor Prodrug PYRRO-C3D Enhances Azithromycin Susceptibility of Nontypeable Haemophilus influenzae Biofilms.

PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO) donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log fold reduction (10-fold reduction or 1-log-unit reduction) in viability (P < 0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log-unit reduction was observed (P < 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm-associated antibiotic tolerance.

Novel Antibiotic-loaded Point-of-care Implant Coating Inhibits Biofilm.

Orthopaedic biomaterials are susceptible to biofilm formation. A novel lipid-based material has been developed that may be loaded with antibiotics and applied as an implant coating at point of care. However, this material has not been evaluated for antibiotic elution, biofilm inhibition, or in vivo efficacy. (1) Do antibiotic-loaded coatings inhibit biofilm formation? (2) Is the coating effective in preventing biofilm in vivo? Purified phosphatidylcholine was mixed with 25% amikacin or vancomycin or a combination of 12.5% of both. A 7-day elution study for coated titanium and stainless steel coupons was followed by turbidity and zone of inhibition assays against Staphylococcus aureus and Pseudomonas aeruginosa. Coupons were inoculated with bacteria and incubated 24 hours (N = 4 for each test group). Microscopic images of biofilm were obtained. After washing and vortexing, attached bacteria were counted. A mouse biofilm model was modified to include coated and uncoated stainless steel wires inserted into the lumens of catheters inoculated with a mixture of S aureus or P aeruginosa. Colony-forming unit counts (N = 10) and scanning electron microscopy imaging of implants were used to determine antimicrobial activity. Active antibiotics with colony inhibition effects were eluted for up to 6 days. Antibiotic-loaded coatings inhibited biofilm formation on in vitro coupons (log-fold reductions of 4.3 ± 0.4 in S aureus and 3.1 ± 0 for P aeruginosa in phosphatidylcholine-only coatings, 5.6 ± 0 for S aureus and 3.1 ± 0 for P aeruginosa for combination-loaded coatings, 5.5 ± 0.3 for S aureus in vancomycin-loaded coatings, and 3.1 ± 0 for P aeruginosa for amikacin-loaded coatings (p < 0.001 for all comparisons of antibiotic-loaded coatings against uncoated controls for both bacterial strains, p < 0.001 for comparison of antibiotic-loaded coatings against phosphatidylcholine only for S aureus, p = 0.54 for comparison of vancomycin versus combination coating in S aureus, P = 0.99 for comparison of antibiotic- and unloaded phosphatidylcholine coatings in P aeruginosa). Similarly, antibiotic-loaded coatings reduced attachment of bacteria to wires in vivo (log-fold reduction of 2.54 ± 0; p < 0.001 for S aureus and 0.83 ± 0.3; p = 0.112 for P aeruginosa). Coatings deliver active antibiotics locally to inhibit biofilm formation and bacterial growth in vivo. Future evaluations will include orthopaedic preclinical models to confirm therapeutic efficacy. Clinical applications of local drug delivery coating could reduce the rate of implant-associated infections.

Chronic Myeloid Leukemia in Pediatrics — First Results From Study CML-PAED II.

Abstract 342▪▪This icon denotes an abstract that is clinically relevant. Background:Chronic myeloid leukemia (CML) is a rare malignancy in pediatrics. In this decade -like in adults- imatinib meyslate (IMA) has been established also as first line treatment for children with CML while allogeneic stem cell transplantation (SCT) as treatment option is postponed for those cases becoming intolerant or refractory to tyrosine kinase inhibitor (TKI) treatment. However, results from controlled trials in children are lacking so far. We here report an analysis of pediatric data from patients (pts) with newly diagnosed Philadelphia-chromosome positive (Ph+) CML on up-front treatment with IMA. Pts and Methods:According to protocol CML-PAED II pediatric pts with confirmed diagnosis of Ph+ CML were treated in CP with IMA 300 mg/sqm once daily, while in accelerated phase (AP) or in blastic phase (BC) the dose was increased to 400 mg/sqm and 500 mg/sqm (bis daily), respectively. Initial and long-term clinical and laboratory data, treatment response and side effects were reported to the study center on standardized forms by the treating physician. Specimen from peripheral blood (pB) and bone marrow (BM) were assessed by cytogenetics and by quantitative RT-PCR for BCR-ABL transcript rates in central laboratories for standardized monitoring in three months intervals. Results:From 1. Jan 2004 until 31. Mrch 2009 a total of 51 pts (21 female, 30 male; median age: 10.6 yrs [range:1-20 yrs]) were registered: 10 pts with ongoing IMA treatment were recruited and analyzed retrospectively while 41 pts were enrolled prospectively from centers in Austria (n=1), Czech Rep. (n=6), Germany (n=40), Italy (n=1), Netherlands (n=1), Slovak Rep. (n=2). Stages of disease were: CP n=47; AP n=1; BC n=3 (two myeloid). Those four pts diagnosed in AP and BC underwent early SCT. Observed side effects in the whole group included: nausea (n=9), muscle pain (n=7), edema (n=3), rhabdomyolysis (n=1, short interruption of IMA), reduced blood cell count (n=2, short interruption of IMA in one pt), biochemical alterations in bone metabolism [for details see: N Engl J Med 2006;354:2006] (n = 8), impaired longitudinal growth (n=1, [Haematologica 2009;94:1177]). Two pts experienced intolerance (muscle pain) or toxicity (hepatic), respectively, therefore stopped IMA and were put on dasatinib after 4 and 10 months, respectively. Having achieved complete cytogenetic response (CyR) and 2 log-fold reduction of BCR-ABL transcript rate, one pt opted for SCT from her HLA-identical brother after 15 mo of treatment. Response rates in advanced stages of CML were as follows: in BC (n=3) two pts became hematological responders (HR), one pt exhibited partial HR. The only one pt diagnosed in AC exhibited partial CyR but complete HR. A landmark analysis in pts entering CML-paed II in CP exhibited that 2/42 pts (5%) had no complete HR at month 3; 2/28 (7%) had no complete CyR at month 12, and 2/19 (15%) pts achieved no major molecular response (MMR, defined as >0.1% BCR-ABL [Blood 2006;108:28–37]) at month 18 after start of IMA. Each two of those four patients with incomplete response (one pt with no CyR at month 12, one pt with no MMR at month 18) underwent SCT from a sibling donor and the other two pts stopped IMA and were put on dasatinib. With a median follow-up of 19 months (range: 0-63 months) all 47 pts diagnosed in CP are alive. Of note none of the six pts (median age at diagnosis: 5 yrs; range 1–13 years) treated by imatinib meanwhile for >36 months have opted for SCT. Conclusion:Keeping in mind that the number of pediatric pts is still small, IMA treatment for children and adolescents with CML in CP is associated -like in adults- with high treatment response rates. Refractoriness to IMA is uncommon and side effects seem tolerable, as only 10% of the total cohort stopped imatinib and were put on 2nd generation TKI. However, disturbances of bone metabolism and longitudinal growth impairment may be of special concern in this not yet outgrown cohort [N Engl J Med 2006;354:2006, Blood 2008;111:2538; Haematologica 2008;93:1101; Lancet 2008;372:111; Int J Hematol; 2009;89:251; Haematologica 2009;94:1177]. Only 3/47 pts not diagnosed in advanced phases of CML so far underwent SCT thus underlining that also in pediatrics SCT has been shifted to a second-line strategy for high-risk patients and those who failed therapy with IMA. Disclosures:Suttorp:Novartis : Research Funding. Thiede:Novartis: Research Funding.

Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay.

The AML1/ETO fusion transcript can be detected by reverse transcription polymerase chain reaction (RT-PCR) in patients with t(8;21)-associated acute myeloid leukemia (AML) in long-term complete remission (CR). Quantitation of the amount of the fusion transcript during CR may therefore be more predictive of cure or relapse than a simple qualitative assessment. Real Time PCR, a fluorometric-based technique, allows simple and rapid quantitation of a target sequence during the extension phase of PCR amplification, in contrast to end-point quantitative methods. Six patients with t(8;21)(q22;q22) AML, who achieved CR were studied by Real Time RT-PCR at different time intervals following diagnosis and high-dose cytarabine and anthracycline-based induction therapy. Five patients had a diagnostic bone marrow (BM) sample available for molecular analysis. Each patient showed > or = 10(3) copies of the AML1/ETO fusion transcript at diagnosis, and each showed a 2- to 4-log decrease in copy number following successful induction chemotherapy. This is comparable to the log-fold reduction in leukemic blasts that is thought to occur in patients successfully cytoreduced into CR by induction chemotherapy. The sixth patient showed a relatively high copy number immediately following successful remission induction chemotherapy, which continued to increase during early CR and was later followed by relapse. Real Time RT-PCR appears to offer advantages over previously used quantitative RT-PCR methods by providing absolute quantitation of the target sequence, expanding the dynamic range of quantitation to over six orders of magnitude, eliminating the post-PCR processing, and reducing labor and carryover contamination. These features make this an attractive method to prospectively evaluate the prognostic value of AML1/ETO fusion transcript quantitation in a larger patient population with t(8;21)(q22;q22) AML in CR.