Redirected T-cell cytotoxicity to epithelial cell adhesion molecule-overexpressing adenocarcinomas by a novel recombinant antibody, E3Bi, in vitro and in an animal model

BACKGROUND: To redirect cytotoxic T cells to target a broad range of adenocarcinomas, the authors constructed a novel, recombinant, bispecific antibody, E3Bi, directed at the tumor-associated antigen, epithelial cell adhesion molecule (EpCAM), and the CD3 receptor on T cells. METHODS: T cells were prepared from healthy blood donors. The cytotoxicity of activated T cells (ATC) redirected to tumor cells by E3Bi was measured with in vitro (51)Cr release assays. In vivo studies were performed in a severe combined immunodeficient (SCID)/Beige mouse xenograft model. Tumor-bearing mice were treated with low doses (1 mg/kg) or high doses (10 mg/kg) of E3Bi along with ATC (2 x 10(9) cells/kg), and treatment efficacy was evaluated both by ex vivo tumor cell survival assay after in vivo treatments and by in vivo tumor growth delay studies. RESULTS: In vitro, targeting the EpCAM-overexpressing human tumor cell lines with E3Bi increased specific cytotoxicity of ATC by > 70% at an effector-to-target ratio of 2.5 (P < 0.001); this cytotoxicity was abolished competitively in the presence of an anti-EpCAM monoclonal antibody. In contrast, E3Bi did not enhance ATC cytotoxicity toward the low EpCAM-expressing tumor cell line. In ex vivo tumor cytotoxicity assays, a significant reduction in tumor cell survival (40% with low-dose E3Bi; 90% with high-dose E3Bi) was observed in E3Bi/ATC-treated mice compared with control mice that were treated with ATC only. In addition, SCID/Beige mice xenografted with LS174T tumors demonstrated a significant tumor growth delay (P = 0.0139) after receiving E3Bi/ATC/interleukin 2 (IL-2) compared with mice that received ATC/IL-2 alone. CONCLUSIONS: E3Bi specifically and very efficiently redirected T cells to destroy EpCAM-overexpressing tumors both in vitro and in an animal model. These results suggest a therapeutic utility for E3Bi in the treatment of adenocarcinomas.     

Hidden chromosomal aberrations are rare in primary myelodysplastic syndromes with evolution to acute myeloid leukaemia and normal cytogenetics

In myelodysplastic syndromes (MDS) a normal karyotype by cytogenetic analysis (CA) corresponds to a low cytogenetic risk for acute myeloid leukaemia (AML) evolution, a subset of patients however develops AML. We evaluated the use of interphase fluorescence in situ hybridisation (I-FISH) in 31 patients with evolution from primary MDS to AML and a normal CA at all stages of disease. Monosomy 7 was found in 4/31 cases, one patient had a terminal deletion 5q, each after AML evolution. The low frequency and unclear prognostic value of I-FISH anomalies in MDS related AML suggests that these alterations play a minor role for AML evolution.     

Enlarged axillary nodes and position of the arms in axillary irradiation--a computed tomography and magnetic resonance imaging evaluation

The purpose of this study was to evaluate the axillary node displacement away from chest wall and their anatomical location in relation to the humeral head, according to the position of the arms, when the axilla is the site of enlarged nodes. In 13 patients with enlarged axillary nodes, the anatomical span of the nodes according to two arms positions, akimbo (A) and up over the head (U), was prospectively evaluated using computed tomography (CT) and magnetic resonance imaging (MRI). The nodes were classified into two groups, i.e. the lower and upper groups. The mean distances of the lower node group from the chest wall when the patients were in A, and U positions were 3 cm and 6.4 cm, respectively (p=0.002). The upper group nodes showed a smaller difference in the distance from the chest wall: in A position, mean 2.1 cm; in U position 2.8 cm (p=0.03). In U position, there was always a node of the lower group that was displaced in front of the humeral head. This study demonstrates the displacement of enlarged axillary nodes according to the position of the arms. In patients with axillary node involvement, CT planning should be considered when they have their arms held up over their heads.     

AIF and cyclophilin A cooperate in apoptosis-associated chromatinolysis

Cyclophilin A (CypA) was determined to interact with apoptosis-inducing factor (AIF) by mass spectroscopy, coimmunoprecipitation, pull-down assays, and molecular modeling. During the initial, caspase-independent stage of chromatin condensation that accompanies apoptosis, AIF and CypA were found to coimmunolocalize in the nucleus. Recombinant AIF and CypA proteins synergized in vitro in the degradation of plasmid DNA, as well as in the capacity to induce DNA loss in purified nuclei. The apoptogenic cooperation between AIF and CypA did not rely on the CypA peptidyl-prolyl cis-trans isomerase activity. In Cyp-expressing cells, AIF overexpression augmented apoptotic chromatinolysis. The AIF-dependent large-scale DNA fragmentation was less pronounced in CypA knockout cells as compared to controls. AIF mutants lacking the CypA-binding domain were inefficient apoptosis sensitizers in transfection experiments. Moreover, AIF failed to sensitize CypA knockout cells to apoptosis induction, and this defect in the AIF response was reversed by reintroduction of the CypA gene into CypA-deficient cells. In summary, AIF and CypA collaborate in chromatinolysis.     

Telomere instability detected in sporadic colon cancers, some showing mutations in a mismatch repair gene

Human telomeres are essential for genome stability and are composed of long simple tandem repeat arrays (STRs), comprising the consensus TTAGGG repeat interspersed, at the proximal end, with sequence-variant repeats. While the dynamics of telomere attrition through incomplete replication has been studied extensively, the effects on telomeres of error-prone DNA repair processes, known to affect other STRs, are poorly understood. We have compared the TTAGGG and sequence-variant interspersion patterns in the proximal 720 bp of telomeres in colon cancer and normal DNA samples. The frequency of telomere mutations was 5.8% per allele in a randomly collected panel of sporadic colon cancers, showing that telomere mutations occur in vivo. The mutation frequency rose to 18.6% per allele in sporadic tumours that exhibit instability at the polyA tract in the TGFbetaRII gene and to 35% per allele in tumours with somatic mutations in the hMSH2 gene. The majority of the characterized mutations resulted in the loss of one or a few repeats. If the mutation spectrum and frequency described here is reiterated in the rest of the array, there is the potential for extensive telomere destabilization especially in mismatch repair-defective cells. This may in turn lead to a greater requirement for telomere length maintenance earlier in tumourigenesis.     

Outcome after cavo-tricuspid isthmus ablation in patients with recurrent atrial fibrillation and drug-related typical atrial flutter

This study evaluates the long-term clinical outcome of 56 consecutive patients affected by atrial fibrillation and drug-related typical atrial flutter who underwent cavo-tricuspid isthmus radiofrequency ablation. Symptomatic arrhythmic events recurred after ablation in 64% of the patients during follow-up of 19 +/- 9 months. Even in those who had recurrences, there was a substantial reduction in the incidence of episodes, quality of life was improved, and hospitalizations decreased.