ECLIM-SEHOP, a new platform to set up and develop international academic clinical trials for childhood cancer and blood disorders in Spain.

INTRODUCTION: Cancer and blood disorders in children are rare. The progressive improvement in survival over the last decades largely relies on the development of international academic clinical trials that gather the sufficient number of patients globally to elaborate solid conclusions and drive changes in clinical practice. The participation of Spain into large international academic trials has traditionally lagged behind of other European countries, mainly due to the burden of administrative tasks to open new studies, lack of financial support and limited research infrastructure in our hospitals. METHODS: The objective of ECLIM-SEHOP platform (Ensayos Clínicos Internacionales Multicéntricos-SEHOP) is to overcome these difficulties and position Spain among the European countries leading the advances in cancer and blood disorders, facilitate the access of our patients to novel diagnostic and therapeutic approaches and, most importantly, continue to improve survival and reducing long-term sequelae. ECLIM-SEHOP provides to the Spanish clinical investigators with the necessary infrastructural support to open and implement academic clinical trials and registries. RESULTS: In less than 3 years from its inception, the platform has provided support to 20 clinical trials and 8 observational studies, including 8 trials and 4 observational studies where the platform performs all trial-related tasks (integral support: trial setup, monitoring, etc.) with more than 150 patients recruited since 2017 to these studies. In this manuscript, we provide baseline metrics for academic clinical trial performance that permit future comparisons. CONCLUSIONS: ECLIM-SEHOP facilitates Spanish children and adolescents diagnosed with cancer and blood disorders to access state-of-the-art diagnostic and therapeutic strategies.

Prognostic significance of FLT3 mutational status and expression levels in MLL-AF4+ and MLL-germline acute lymphoblastic leukemia.

There is barely any information about the prognostic significance of FLT3 expression and mutational status in cytogenetically distinct subgroups of acute lymphoblastic leukemia (ALL). We analyzed the presence of FLT3-tyrosine kinase domain (TKD) and FLT3-internal tandem duplication (ITD) mutations as well as FLT3 expression levels in 54 newly diagnosed patients with B-ALL (n=49) or T-ALL (n=5). All B/T-ALL samples tested negative for the presence of FLT3-TKD or FLT3-ITD. None of the T-ALL and E2A-PBX1+ B-ALL overexpressed FLT3. In contrast, mainly MLL-AF4+ B-ALL but also ETV6-RUNX1+, BCR-ABL+ or B-ALL displaying normal cytogenetics exhibited significantly higher FLT3 expression levels than normal bone marrow, supporting that aberrantly increased transcription of FLT3, rather than activating FLT3 mutations, contributes to the pathogenesis of these B-ALL. Using the median FLT3 expression as cut-off value we found that high-level FLT3 expression is associated with an extremely poor 1-year overall survival (OS; 0 vs 71%; P=0.002) and disease-free survival (DFS; 0 vs 43%; P=0.03) in MLL-AF4+ B-ALL but not in MLL-germline B-ALL. Cox regression analysis with OS/DFS as end points showed that age>14 years and high-level FLT3 expression were independent prognostic factors when all ALL patients were analyzed together. Importantly, when the MLL-AF4+ B-ALL subgroup was analyzed separately, high-level FLT3 expression was the only independent prognostic factor for OS and treatment outcome. These findings indicate that high FLT3 expression identifies MLL-AF4+ ALL patients at very high risk of treatment failure and poor survival, emphasizing the value of ongoing/future clinical trials for FLT3 inhibitors.

Detection of a t(8;21)(q22;q22) in a case of M5 acute monoblastic leukemia.

Although the translocation (8;21) is the single most common structural rearrangement reported in acute myeloblastic leukemia (AML), it is rarely seen in AML FAB type M5. We describe a case of a 51-year-old male with a diagnosis of acute monoblastic leukemia (AML M5b with hemophagocytic component) whose karyotype showed at (8;21)(q22;22). To our knowledge, this is the first report of this translocation in an AML M5b. The t(8;21) has been associated with a good prognosis, but our patient suffered a fast and fatal evolution.

Binding of new cirazoline derivative to imidazoline receptors from human brain.

Imidazoline compounds are known to interact with alpha 2-adrenoceptors as well as with specific non-adrenergic binding sites. Such binding sites are present in the brain and in peripheral tissues. Hypotensive effects of imidazolines were shown to be related to specific interaction with imidazoline binding sites within the brainstem. Heterogeneity of these sites based on differences in selectivities was reported. In order to facilitate the characterization of human brain imidazoline receptors, we synthetized new ligands by substitutions on the cirazoline phenyl ring. Affinities of these cirazoline derivatives were determined in two imidazoline binding site models, namely the human brain and the rabbit kidney. Interaction of these compounds with imidazoline binding sites from the human brain appeared more sensitive to structural variations of the imidazoline than those with rabbit kidney sites. Moreover, no correlation was found between affinities for imidazoline binding sites and those for alpha 2-adrenoceptors of the rat brain. Arylazide derivative of 2-(5-amino-2-methyl-phenoxymethyl)-imidazoline exhibited a higher affinity for human brain imidazoline binding sites than for human brain alpha 2-adrenoceptors. Photoincorporation of this azido-compound in human brain imidazoline binding sites was achieved and blockade of [3H]idazoxan imidazoline specific binding observed. These new tools may allow fine characterization of the different subtypes of imidazoline binding proteins.

Heterogeneity of imidazoline binding sites revealed by a cirazoline derivative.

The affinity of AMPI (2-[3-aminophenoxy]methyl imidazoline) for [3H]clonidine and [3H]idazoxan imidazoline binding sites was determined in various rabbit and human tissues. Although cirazoline showed a high affinity (nM range) in all the tested tissues, its derivative, AMPI, had a high affinity (nM range) in rabbit brain and kidney but a low affinity (microM range) in the human brain. These differences in affinities were very similar to those obtained with amiloride. The same results were obtained when considering [3H]clonidine or [3H]idazoxan specific imidazoline binding sites.

Isolation of a human cerebral imidazoline-specific binding protein.

The first isolation of a human brain specific imidazoline binding protein is described. This protein was obtained using affinity chromatography and was revealed with the aid of an anti-idiotypic antibody specific for imidazoline binding sites. The protein (43 kDa) differs from other imidazoline binding proteins previously isolated from peripheral tissues, in particular by being also sensitive to clonidine.

A circulating substance cross-reacting with antiimidazoline antibodies. Detection in serum in relation to essential hypertension.

It has been shown in various mammal species that clonidine, a well known centrally acting hypotensive agent, acts through the activation of imidazoline receptors (IRs) in the nucleus reticularis lateralis (NRL) of the brainstem. Specific binding sites sensitive to imidazolines and insensitive to catecholamines have been detected in rat and bovine, as well as human brains. An endogenous ligand, other than catecholamines, should exist for these IRs. Such a ligand could play a role in the pathophysiology of human essential hypertension. Therefore, we developed two RIAs with polyclonal and monoclonal anticlonidine antibodies. These antibodies presented specificity spectra similar to that of the IRs: they bound imidazolines and not catecholamines at all. These RIAs were used to detect imidazoline-like immunoreactivity in the human serum. Immunoreactive substance was measured in 26 normotensive subjects' sera, and specificity of interaction between antibodies and sera was verified. None of the known endogenous substances tested so far were able to interact with the two antibodies. Immunoreactivity in 32 essential hypertensive patients' sera proved higher in approximately 30% of cases. Values of immunoreactivity positively correlated with the mean arterial pressure values. This study demonstrates the existence of an "imidazoline-like" immunoreactive substance in the human serum with high levels in some hypertensive patients.

Production and characterization of an iminoimidazolidine specific monoclonal antibody using para-aminoclonidine as antigen.

Para-aminoclonidine coupled to hemocyanin was used to produce mouse monoclonal antibodies directed against clonidine. The properties of one of these, called mFE7, secreted by a clone of hybrid myeloma, are described. This antibody displayed total crossreactivity with imidazolidines and no crossreactivity at all with catecholamines or other known naturally occurring substances tested. A liquid phase radioimmunoassay permitted the detection of immunoreactivity in human brain extracts. The mFE7 antibody could be useful for immunopurifying the endogenous ligand of Imidazolines Preferring Receptors (IPR) which are catecholamines insensitive.

[From alpha 2-adrenergic to endazoline receptors]. / Des récepteurs alpha 2-adrénergiques aux récepteurs à l'endazoline.

Studies of the central hypotensive mode of action of the imidazolines of which clonidine is the leading molecule suggest the presence of non-catecholamine binding sites called imidazoline receptors. Our group showed that neither the endogenic ligand of alpha-adrenergic receptors, noradrenaline, nor any other tested catecholamine or phenylethylamine have hypotensive effects at the site of action of all imidazolines, the lateral reticular nucleus of the brainstem. In addition, a population of membrane binding sites which take up labelled clonidine and which are insensitive to noradrenaline have been demonstrated in the lateral reticular nucleus. An endogenic non-catecholamine substance whose structure is currently under identification and which is recognised by these receptors has been isolated from the brain tissues of various mammals. All this experimental evidence supports the hypothesis that the hypotensive effects of imidazoline-like substances are related to their action on brainstem receptors specific to this endogenic ligand which we propose to call endazoline. Rilmenidine, which has a chemical structure similar to that of the imidazolines, has a higher relative selectivity for the imidazoline binding sites than the reference molecule (clonidine). A central antihypertensive agent without the classical sedative effects associated with this class of drug could result. A study of the structure-activity relationship is needed to confirm this hypothesis.

New concepts on the central regulation of blood pressure. Alpha 2-adrenoceptors and "imidazoline receptors".

The most usual hypothesis to explain the central hypotensive effect of clonidine-like substances was to admit that these drugs stimulated alpha 2-adrenoceptors within the brainstem. Now it has been demonstrated that neither the endogenous ligand to the alpha-adrenoceptors, noradrenaline, nor any other catecholamine or phenylethylamine was hypotensive in the medullary nucleus reticularis lateralis, where all imidazolines proved to be such. Recently, a membrane receptor population sensitive to clonidine and insensitive to catecholamines was described within the nucleus reticularis lateralis; this subgroup of receptors represented 20 to 30 percent of the [3H]clonidine binding sites in the bovine nucleus reticularis lateralis and 100 percent within the human nucleus reticularis lateralis region. Thus, the existence of such imidazoline specific receptors was clearly established and the endogenous ligand for those receptors, which is neither a catecholamine nor likely a peptide, is under processing for purification. Therefore, it appeared that the hypotensive effect of substances with an imidazoline or imidazoline-like structure might be due to their action within medullary receptors specific for this endogenous ligand temporarily named "clonidine displacing substance." Rilmenidine, structurally close to imidazolines, also interfered with these receptors. The central component of its hypotensive effect was recently confirmed in rabbits, where its central cardiovascular effects were antagonized by "the clonidine displacing substance." Although exhibiting a lower affinity than the reference substance for these receptors, rilmenidine might have a higher selectivity, thus explaining its restricted side effects. A structure-activity study with this molecule would bring a confirmation to these first observations.

Rilmenidine selectivity for imidazoline receptors in human brain.

The selectivity of three centrally acting antihypertensive agents for the medullary imidazoline-preferring receptors (IPR) versus cortical alpha-adrenoceptors was investigated in human brain. [3H]Clonidine binding was studied in various membrane preparations. Competition experiments were performed. Cortical membrane preparations were used as they mainly contained classical alpha-adrenoceptors whereas medullary membrane preparations from the nucleus reticularis lateralis contained only IPR insensitive to catecholamines. Rilmenidine, a new antihypertensive agent, appeared 2.5 and 3.5 times more selective than clonidine and guanfacine, respectively, for medullary IPR sites than for cortical alpha-adrenoceptors, thus providing a possible explanation for the low sedative effects of this new molecule.

The imidazoline preferring receptor: binding studies in bovine, rat and human brainstem.

The binding of [3H]clonidine to brainstem membrane preparations was studied in an attempt to characterize imidazoline-sensitive, catecholamine-insensitive receptors. Human samples and samples from two animal species were used. [3H]Clonidine binding was always saturable, reversible and specific with a KD value of 6-7 nM. The Bmax values were 45.5 +/- 5.5, 145 +/- 34 and 65 +/- 33 fmol/mg protein in the whole rat medulla oblongata, the nucleus reticularis lateralis region of bovine and that of human, respectively. In the whole rat brainstem we could not demonstrate the presence of [3H]clonidine binding sites that were insensitive to catecholamines. In bovine and human nucleus reticularis lateralis (NRL) preparations, the amount of specifically bound labelled clonidine that was not displaced by an excess of (-)-norepinephrine was 25 and 100%, respectively. Substances that had a structure similar to that of clonidine were able to compete with [3H]clonidine binding within the human NRL. Cirazoline was the most potent to inhibit [3H]clonidine binding although yohimbine was also able to displace binding in the human NRL but with lower apparent affinity. Competition assays with idazoxan stereoisomers clearly showed that this binding was stereospecific. Therefore the human NRL region provides the first model of an homogenous population of imidazoline-preferring, non-alpha-adrenergic membrane receptors.

Production and characterization of anti-clonidine antibodies not cross-reacting with catecholamines.

Polyclonal antibodies against clonidine were developed, with para-aminoclonidine coupled to bovine serumalbumin or hemocyanine with glutaraldehyde used as antigens. The selected antibody (from rabbits) cross-reacted with high specificity with clonidine and its structurally closely related analogues but it recognized neither catecholamines nor various endogenous imidazole molecules such as histamine, purine, adenine, and adenosine, thus appearing to be specific for the aminoimidazoline structure. An interesting cross-reactivity was observed with the bovine clonidine displacing substance, the probable endogenous ligand for receptors involved in the hypotensive effect of clonidine-type substances. This suggested that this molecule should contain an aminoimidazoline or guanidine moiety.