• SL-401 was well tolerated, and a single course of treatment produced a high rate of objective responses in BPDCN patients.This is the first prospective study of treatment of patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive hematologic malignancy derived from plasmacytoid dendritic cells that typically involves the skin and rapidly progresses to a leukemia phase. Despite being initially responsive to intensive combination chemotherapy, most patients relapse and succumb to their disease. Because BPDCN blasts overexpress the interleukin-3 receptor (IL3R), the activity of SL-401, diptheria toxin (DT) 388 IL3 composed of the catalytic and translocation domains of DT fused to IL3, was evaluated in BPDCN patients in a phase 1-2 study. Eleven patients were treated with a single course of SL-401 at 12.5 mg/kg intravenously over 15 minutes daily for up to 5 doses; 3 patients who had initial responses to SL-401 received a second course in relapse. The most common adverse events including fever, chills, hypotension, edema, hypoalbuminemia, thrombocytopenia, and transaminasemia were transient. Seven of 9 evaluable (78%) BPDCN patients had major responses including 5 complete responses and 2 partial responses after a single course of SL-401. The median duration of responses was 5 months (range, 1-201 months). Further studies of SL-401 in BPDCN including those involving multiple sequential courses, alternate schedules, and combinations with other therapeutics are warranted. This trial is registered at clinicaltrials.gov as #NCT00397579. (Blood. 2014;124(3):385-392)

The bivalent anti-human anti-T cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T cell leukemia, autoimmune diseases, and tolerance induction for transplantation. The multi-domain structure of the bivalent immunotoxin hinders efficient production in Escherichia coli and most eukaryotes are sensitive to the toxin. However, Pichia pastoris has a tolerance to levels of DT (diphtheria toxin) that were previously observed to intoxicate wild type eukaryotic cells, including Saccharomyces cerevisiae. This tolerance has permitted the optimization of the secreted expression of A-dmDT390-bisFv(G(4)S) in P. pastoris under the control of AOX1 (alcohol oxidase 1) promoter. The original DNA sequence of A-dmDT390-bisFv(G(4)S) was not expressed in P. pastoris because of several AT-rich regions, which induce an early termination of transcription. After DNA rebuilding for abolishing AT-rich regions and codon optimization, the immunotoxin could be expressed up to 10mg/L in the shake flask culture. No differences in the expression levels of immunotoxin were observed by using different secretional signal sequences, Mut(s) (methanol utilization slow phenotype) or Mut(+) (methanol utilization plus phenotype) phenotypes. Buffered complex medium (pH 7.0) having 1% casamino acids provided the highest expression in shake flask culture and PMSF (phenylmethylsulfonyl fluoride) in the range of 1 to 3mM further improved the expression level presumably by inhibiting protein degradation. The immunotoxin was purified by DEAE (diethylaminoethyl) Sepharose ion exchange chromatography and Protein L affinity chromatography. The immunotoxin purified from P. pastoris culture was as fully functional as that expressed in a toxin resistant mutant CHO (Chinese hamster ovary) cell line. Our results demonstrate that P. pastoris is an ideal system for expression of toxin-based fusion proteins.

RLIP76 is a multifunctional membrane protein that transports glutathione conjugates of electrophilic compounds and other xenobiotics including chemotherapy agents out of cells. The protein is overexpressed in lung carcinomas, ovarian carcinomas, and melanomas. The protein also binds Ral and participates in mitotic spindle function, clathrin-dependent endocytosis, and triggers GTPase-activating protein activity. It is found throughout the cell, in membrane, cytosol, and the nucleus, and is known to shift between these compartments in response to stress. Loss of RLIP76 by antibody or antisense therapy is associated with increased sensitivity to radiation and chemotherapy. Conversely, liposomally delivered RLIP may treat poisoning and wounds.

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Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin. This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G(4)S)(3) spacer (sFv). Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3(+) cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone. Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G(4)S). In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1-CRM9 and the monovalent A-dmDT390-sFv. The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgepsilon 600, in which the T-cells express human CD3epsilon as well as murine CD3epsilon. T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct. The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.

Objectives. This study evaluated the benefits of performing prophylactic central neck dissection (CND) with total thyroidectomy (TT) in management of papillary thyroid carcinoma (PTC) patients who were clinically node-negative at presentation. Methods. A total of 257 patients with stage T1 or T2 PTC and without preoperative evidence of lymph node involvement (N0) were enrolled in this prospective study. The patients were randomly assigned to two groups: (1) a total thyroidectomy (TT) group (n = 104) or (2) a TT plus CND group (n = 153). The two groups were compared for their perioperative data, complication rates, disease recurrence rates, and clinical outcomes. Results. The two groups of patients were similar in age, sex ratio, follow-up duration, and tumor size (P = 0.227, 0.359, 0.214, and 0.878, resp.). The two groups showed similar rates of disease recurrence (3.9% in the TT group versus 3.3% in the TT plus CND group); however, complications occurred more frequently in the TT plus CND group; especially transient hypocalcemia (P = 0.043). Conclusions. Patients treated with TT plus CND had a higher rate of complications with similar recurrence rate. We believe that CND may not be routinely recommended when treating patients with PTC.

ObjectivesCoblation is operated in low temperature, so it is proposed that tonsillectomy with coblation involves less postoperative pain and allows accelerated healing of the tonsillar fossae compared with other methods involving heat driven processes. However, the results of the previous studies showed that the effect of coblation tonsillectomy has been equivocal in terms of postoperative pain and hemorrhage. Though, most of the previous studies which evaluated coblation tonsillectomy were performed in children. Recently, electrocautery tonsillectomy has been used most widely because of the reduced intraoperative blood loss and shorter operative time compared to other techniques. This prospective study compared intraoperative records and postoperative clinical outcomes in adolescents and adults following coblation and electrocautery tonsillectomies.MethodsEighty patients over 16 years of age with histories of recurrent tonsillitis were enrolled. The patients were randomly allocated into coblation (n=40) and electrocautery tonsillectomy groups (n=40). All operations were performed by one surgeon who was skilled in both surgical techniques. Intraoperative parameters and postoperative outcomes were checked.ResultsPostoperative pain and otalgia were not significantly different between the two groups; however, there was a tendency towards reduced pain and otalgia in the coblation group. More cotton balls for swabbing the operative field were used introoperatively in the electrocautery group (P=0.00). There was no significant difference in postoperative hemorrhage, wound healing, commencement of a regular diet, and foreign body sensation between the groups.ConclusionOnly cotton use, which represented the amount of blood loss, was less in the coblation tonsillectomy group. Coblation tonsillectomy warrants further study with respect to the decreased postoperative pain and otalgia.

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