Improving the informed consent process in clinical research is of constant concern to regulatory authorities in the field and presents a challenge for both the specialists and patients involved. Informed consent is a process that should adequately match the complexity of clinical research. In analyzing the behaviour of 68 patients during the informed consent process related to the clinical research performed at Neomed Clinical Center in Brasov, we found that many patients do not ask any questions (35.3%). From those who do, part of the questions (20,6%) referred to general aspects (addressed the form but not the gist) of the clinical trial, some (72,8%) referred to specific aspects of the clinical trial they will attend and others (6,6%) unrelated to the clinical trial. These results suggest a lack of interest, awareness, and understanding of the information presented in the informed consent form. The possible underlying causes of this attitude and its bureaucratic, ethic, and legal implications are discussed.

Background

Sickle cell anemia is an inherited disorder of hemoglobin that leads to a variety of acute and chronic complications. Abnormal cellular adhesion, mediated in part by selectins, has been implicated in the pathophysiology of the vaso-occlusion seen in sickle cell anemia, and selectin inhibition was able to restore blood flow in a mouse model of sickle cell disease.

Methods

We performed a Phase 1 study of the selectin inhibitor GMI 1070 in patients with sickle cell anemia. Fifteen patients who were clinically stable received GMI 1070 in two infusions.

Results

The drug was well tolerated without significant adverse events. There was a modest increase in total peripheral white blood cell count without clinical symptoms. Plasma concentrations were well-described by a two-compartment model with an elimination T1/2 of 7.7 hours and CLr of 19.6 mL/hour/kg. Computer-assisted intravital microscopy showed transient increases in red blood cell velocity in 3 of the 4 patients studied.

Conclusions

GMI 1070 was safe in stable patients with sickle cell anemia, and there was suggestion of increased blood flow in a subset of patients. At some time points between 4 and 48 hours after treatment with GMI 1070, there were significant decreases in biomarkers of endothelial activation (sE-selectin, sP-selectin, sICAM), leukocyte activation (MAC-1, LFA-1, PM aggregates) and the coagulation cascade (tissue factor, thrombin-antithrombin complexes). Development of GMI 1070 for the treatment of acute vaso-occlusive crisis is ongoing.

Trial registration

ClinicalTrials.gov NCT00911495.

A hallmark of acute promyelocytic leukemia (APL) is altered nuclear architecture, with disruption of promyelocytic leukemia (PML) nuclear bodies (NBs) mediated by the PML-retinoic acid receptor α (RARα) oncoprotein. To address whether this phenomenon plays a role in disease pathogenesis, we generated a knock-in mouse model with NB disruption mediated by 2 point mutations (C62A/C65A) in the Pml RING domain. Although no leukemias developed in Pml^C62A/C65A mice, these transgenic mice also expressing RARα linked to a dimerization domain (p50-RARα model) exhibited a doubling in the rate of leukemia, with a reduced latency period. Additionally, we found that response to targeted therapy with all-trans retinoic acid in vivo was dependent on NB integrity. PML-RARα is recognized to be insufficient for development of APL, requiring acquisition of cooperating mutations. We therefore investigated whether NB disruption might be mutagenic. Compared with wild-type cells, primary Pml^C62A/C65A cells exhibited increased sister-chromatid exchange and chromosome abnormalities. Moreover, functional assays showed impaired homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways, with defective localization of Brca1 and Rad51 to sites of DNA damage. These data directly demonstrate that Pml NBs are critical for DNA damage responses, and suggest that Pml NB disruption is a central contributor to APL pathogenesis.

Abstract Acute promyelocytic leukemia (APL) is characterised by the t(15;17)(q22;q21) leading to fusion of PML to the gene encoding the myeloid transcription factor Retinoic Acid Receptor α (RARα). Chromosomal translocations such as the t(15;17) are considered to be initiating events in leukemogenesis; however, sequencing of APL genomes has provided further evidence that the PML-RARα fusion is insufficient to induce leukemia, which depends upon the acquisition of cooperating mutations. The PML-RARα oncoprotein exerts a profound effect on nuclear architecture, disrupting multiprotein structures known as PML nuclear bodies (NBs). The function of these structures remains an enigma; however, their disruption in PML-RARα+ APL and acute lymphoblastic leukemia with the t(9;15)(p13;q24)/PAX5-PML fusion is associated with delocalisation of a number of component proteins including PML, which have been implicated in growth control and neoplastic transformation. It is now established that the PML moiety contributes to APL pathogenesis by conferring via the translocation a novel dimerisation capacity to RARα, but it has been unclear whether deregulation of PML and other NB components cooperates in leukemic transformation or impacts the response to differentiating agents. To address these questions, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues in the amino-terminal RING domain of Pml. Homozygous Pml RING mutant mice are viable, with no overt developmental defect; however, analysis of the bone marrow revealed significant expansion of the Lin(-)Sca-1(+)c-Kit(+) (LSK) population compared to wild type (WT) controls (p<0.01), accompanied by increased LSK cell proliferation (p<0.0001) as evaluated by in vivo labelling through incorporation of 5-ethynyl-2'-deoxyuridine (EdU). In addition, hematopoietic cells derived from homozygous Pml RING mutant mice exhibited markedly elevated levels of DNA damage compared to WT cells from age-matched controls, as evidenced by increased numbers of γH2AX foci (p=0.009). This was associated with significantly delayed DNA damage repair responses in Pml RING mutant cells following γ-irradiation (p=0.005). Accordingly, expression of PML-RARα in human hematopoietic cells, which led to disruption of NBs, also induced a significant increase in γH2AX foci (p=0.0023). While no leukemias arose in homozygous Pml RING mutant mice, they developed an excess of T- and B-cell lymphomas (p=0.03), consistent with the proposed tumour suppressor function of PML and the NBs. Since a key property conferred by the PML moiety required for leukemogenicity of the PML-RARα oncoprotein is the capacity to dimerise, we evaluated whether Pml NB disruption could cooperate with forced RARα homodimerisation (mediated artificially by linking RARα to the p50 dimerisation motif of NFκB). While Pml NB disruption or p50-RARA expressed under the control of the MRP8 promoter in murine hematopoietic stem/progenitor cells conferred limited replating capacity, in combination they exhibited marked cooperativity, with a significant increase in third round colonies (p=0.03). Moreover, NB disruption was found to cooperate with forced RARα homodimerisation in vivo with a doubling in the rate of leukemia development in p50-RARα mice with mutated Pml (p<0.0001), leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency to onset of leukemia was significantly shorter in p50-RARα mice with the Pml RING mutation, occurring from 213 days of age vs 310 days with WT Pml (p=0.008). While Pml NB disruption did not affect engraftment of p50-RARα leukemias in serial transplantation, the in vitro differentiation response of p50-RARα leukemias to All transretinoic acid (ATRA) as determined by nitroblue tetrazolium assay was significantly impaired in the context of NB disruption (p<0.05). Moreover, prolongation of survival following ATRA treatment in mice transplanted with p50-RARα leukemic blasts was dependent upon Pml NB integrity (p=0.03). Overall, these data suggest that the NB disruption mediated by the PML-RARα oncoprotein plays a key role in APL pathogenesis contributing to expansion of the LSK population and defective DNA repair predisposing to the acquisition of cooperating mutations, but also implicate NBs in the response to differentiating agents. Disclosures: No relevant conflicts of interest to declare.