Eccrine Porocarcinoma: A Challenging Diagnostic and Therapeutic Tumoral Entity

Eccrine porocarcinoma is a rare malignant cutaneous tumor with high rates of extracutaneous spread, and its diagnosis and management can be quite challenging. This is a case of an 82-year-old woman presenting with an asymptomatic and chronic pubic skin lesion for whom the work-up required many investigations and procedures to confirm the diagnosis of metastatic eccrine porocarcinoma. Indeed, the patient underwent a wide local excision of the skin lesion, imaging with an FDG-PET scan, a colonoscopy, and two inguinal node dissections. As illustrated in this case, surgery should always be considered to achieve disease remission. Other treatments such as chemotherapy and radiotherapy have also been reported in the literature without clear standard guidelines

Imagerie moléculaire des affections neurodégénératives

L’évaluation du fonctionnement cérébral par l’imagerie moléculaire, tant la tomographie à émission de positrons (PET) que la tomographie par émission monophotonique (SPECT),contribuent de plus en plus au diagnostic positif des maladies neurodégénératives, principalement des démences et des syndromes parkinsoniens. En l’absence actuelle de traitement modifiant l’évolution de la maladie, la plupart des patients sont souvent diagnostiqués cliniquement, sans recourir à l’imagerie moléculaire. Ceci explique que leur prescription, hormis l’étude de la perfusion cérébrale au SPECT, soit réservée aux médecins spécialistes: neurologues, neuropsychiatres ou gériatres. Les examens d’imagerie moléculaire possèdent une importante valeur prédictive,tant positive que négative, qui les aide dans les cas difficiles

Noninvasive measurement of liver regeneration with [2-11C] thymidine and positron emission tomography

Au contraire, d’autres organes de mammifères, le foie à la propriété de se régénérer aux dépens de l’ensemble des cellules résiduelles après une réduction de sa masse fonctionnelle. Une méthode de mesure non-invasive, sensible et spécifique de cette régénération conditionnerait le pronostic et le traitement de patients en insuffisance hépatique. Elle permettrait également de mieux comprendre les mécanismes régulateurs de la croissance cellulaire. La méthode de référence d’évaluation de la régénération hépatique consiste en la mesure de la radioactivité incorporée dans la fraction « ADN » après injection de thymidine marquée en 14C ou en 3H et n’est pas applicable en clinique humaine. Cette thèse rapporte une méthode originale et atraumatique de mesure de la régénération hépatique utilisant la thymidine marquée en 11C dont l’incorporation pourrait être suivie de manière externe à l’aide de la tomographie à émission de positron (TEP). Des études préliminaires in vitro utilisant de la thymidine marquée en 3H ou au 14C démontrèrent que pour distinguer les foies non encore en régénération (obtenus 1h après une hépatectomie de 70 % : NRM) des foies en régénération (obtenus 24h après l’hépatectomie : RL) il était nécessaire d’utiliser une thymidine marquée sur son deuxième carbone plutôt que sur son groupement méthyl. Une mCi de thymidine marquée en 11C sur son deuxième carbone, [2 11 C]-thymidine, obtenue selon une synthèse originale en trois étapes et 50 µCi de thymidine tritiée (utilisée comme contrôle de la prolifération hépato-cellulaire) ont été injectées à 12 rats en régénération et 13 contrôles. Déjà 10 minutes après l’injection, les images tomographique montrèrent une captation hépatique du traceur, double dans les foies en régénération par rapport au groupe contrôle. Cette différence se maintenait tout au long de l’expérience pour être en moyenne de 2.16 ± 0.3 à 120 minutes. A ce moment, les rats furent sacrifiés et leurs foies excisés pour être remplacés ex vivo sous la TEP. La radioactivité moyenne était 8 fois plus élevée dans les foies en régénération par rapport au groupe contrôle. Un contraste similaire fut retrouvé au compteur γ où la radiocativité du foie total exprimée en % de la dose par gramme était de 0.62 ± 0.07 dans les RL versus 0.10 ± 0.03 dans les NRL (p Comme des quantités importantes de [2-11C] thymidine peuvent être produite (actuellement jusqu’à 30 mCi), cette technique pourra être appliquée à des grand animaux et éventuellement à l’homme où elle pourra être d’une grand utilité après une hépatectomie partielle, une transplantation hépatique ou une insuffisance hépatique aigüe, vu son caractère atraumatique et reproductibleThe liver is the only parenchymal organ in mammalians having a substantial capacity to regenerate after reduction of its functional mass. Even though proliferative activity in the adult liver is very low with mytosis particularly difficult to enumerate, most of the mature functional hepatocytes still keep their capacity for cell division. The re-entry of normally quiescent adult hepatocytes into the cell cycle during liver regeneration is a distinct phenomenon from the continuously dividing cell populations such as in the bone marrow, in the intestinal crypts or the other population types (Cleaver, 1967). Nevertheless, some stem cells exist in the liver but they operate only as source of new hepatocytes when the DNA synthesis is inhibited or when hepatocytes have been totally destroyed (e.g. some forms of fulminant hepatitis) (for review, see Michalopoulos, 1990). Liver regeneration appears in response to partial, damaging action of toxins (e.g. CCl4), ischemia, viral infection, or administration of exogenous stimuli (e.g. α-hexachlorocyclohexane …) ‘Schulte-Hermann, 1974; Alison, 1986). Only the surgical procedure allows the suppression of a precise amount of hepatocyte, it also maintains entirely intact the survival cells, and avoids accumulation of inflammatory cells or necrotic tissues. Therefore, partial hepatectomy has been widely used as model of regeneration especially useful in the investigation of regulatory mechanisms of the regenerative process. The regenerative response involves both hyperplasia and some degree of hypertrophy of the residual hepatocytes (as will be discussed in chapter I). Unlike the cell proliferation in hepatoma, it is abruptly interrupted once the functional mass of the liver has been restored. Therefore, liver generation demonstrates the remarkable ability of adult differentiated cells to regulate their replications, and is one of the few in vivo physiological systems in which the mechanism controlling both regulated and neoplastic growth van be analyzed. From such studies, information can be obtained on the role of cellular proliferation in the development of chemically and virally induced liver tumours, and on the humoral or cellular factors regulating the growth response. Such factors may prove useful in the management of some acute and chronic liver diseases. In most clinical liver injuries, the resulting liver function depends both on the importance of the necrosis or resection, and on the importance of the liver regeneration. Therefore, a method allowing the quantification of liver regeneration will improve our understanding of homeostatic mechanisms governing cellular growth and would provide a means to test new potential mitogens for hepatocytes. They would also help in the clinical management and follow-up patients with impaired liver function, e.g. in the evaluation of the opportunity of a hepatic transplantation or retransplantation, by discriminating between necrosis and regeneration. In 1929, Fishback first reported morphological changes after partial hepatectomy, which consist in the growth of the remnant lobes to nearly the mass of the preoperative liver. On basis of the initial finding authors attempted to quantitate liver regeneration through the clinical or radiographic estimation the liver volume (Friedell et al., 1957; Walk, 1961; Lin et al, 1965). In 1931, Higgins and Anderson developed a reproducible experimental model, discussed below, describing histological and biochemical changes undergone during animal liver regeneration, and provided substantial information on the nature of hepatotrophic factors in animal liver regeneration. The estimation of mitotic activity by histological or autographic methods, the measurement of DNA, RNA, protein synthesis or enzyme activities remained however subject to recognized errors (for review, see Bucher, 1963). In addition, the practical usefulness of such methods was somewhat limited by the risk of liver biopsies, particularly in patients with hepatic failure. Therefore, the events in human hepatic regeneration are much less well defined and the use of hepatotrophic factors for treatment of human hepatic disease is generally based on the assumption that the mechanisms controlling hepatic regeneration in humans are identical to those in animals (Baker, 1985). Despite the absence of direct proof, several observations support the idea that the regenerative processes are similar in rat and man. Firstly, basic biological processes are similar among liver cells from different mammalian species although generalization from one species to another can be complicated by differences in hepatic structure and biochemical cytology. The development of new imaging methods such as ultrasound and methods using radionuclides also provided direct evidence of hepatic regrowth in patients with normal residual hepatic architecture as early as two weeks following partial hepatectomy (McDermont et al, 1963; Aronsen et al., 1968; Starzl et al., 1975; Kan and Hopkins, 1979; Lin et al., 1979; Joyeux et al., 1984; Faurous et al., 1989). Similar functional and hormonal patterns have been reported following hepatic resection in humans and experimental animals. However, the methods evaluating liver function are not sufficiently accurate and specific to quantitate liver regeneration. Direct confirmation that events and control mechanisms of liver regeneration, are similar in animals and humans awaits further studies of human hepatic regeneration, perhaps using human hepatocytes in culture or a new noninvasive method specific of liver regeneration, such as the PET imaging reported in this workThèse de doctorat en sciences biomédiacles -- UCL, 199

Monoaminergic neuronal integrity measured in vivo by ligands of the vesicular monoamine transporter

Reserpine requires intact vesicular energy charge and pH gradient for avid and essentially irreversible VMAT2 binding. The selection of benzisoquinoline derivates for in vivo imaging of VMAT2 was based n the uncomplicated, raid, high-affinity, reversible binding properties of these ligands with the VMAT2 bindings sites (Scherman and Henry, 1984). Based in the results obtained with [11C]tetrabenazine and its known metabolism, we investigated methoxytetrabenzine (MTBZ) as a potential tracer of VMAT2. the binding properties, distribution and pharmacological profile of high specific activity tritium-labeled ligand ([3H]MTBZ) were studied on rat brain sections. Saturation analysis revealed interaction with a homogeneous population of stritial sites (Hill coefficient = 1.00 ± 0.05), with an apparent dissociation constant (Kd) of 3.9 ± 0.4 nM and a maximal binding capacity (Bmax) of 1.2 ± 0.1 fmol/μg protein determined at equilibrium. Distribution of [3H]MTBZ binding sites was observed in regions richly innervated by the monoamine systems. In presence of 1μM of various neruoactive drugs, only reserpine inhibited significantly [3H]MTBZ binding. Chapter II Is devoted to the validation of the VMAT2 as an index of dopaminergic neuronal density (Vander Borght et al., 1995c). Presynaptic location of MTBZ bindings sites demonstrated following unilateral 6-OHDA lesion of the median forebrain bundle. The resulting decrease of [3H]MTBZ stratial binding assessed by quantitative autoradiography (average 60%, range 14 to 93%; n=8). Integrity of the postsynaptic site was demonstrated by the absence of significant modification of striatal [3H]raclopride binding compared to the contralateral side. The chapter III investigates the possible regulation of brain VMAT2 expression using both in vitro and in vivo experimental protocols. Based on the fact that the number of vesicles involved in synaptic transmission can be regulated by mobilisation from a voluminous pool of reserve, rather than by an increase of the number of transporters per vesicle (Kelly, 1993), we hypothesized that VMAT2 binding density may not be influences by disease progression or treatment. For in vitro experiments, rats were treated for 2 weeks with drugs known to influence dopaminergic neurotransmission, including those commonly used in the treatment of Parkinson’s disease (Vander Broght et al., 1995a). Autoradiographic assays were performed using [3H]MTBZ, [3H]raclopride, and [3H]WIM 35, 428 to measure VMAT2, dopamine D2 receptor and DAT bindings, respectively. None of the drug treatments significantly modified levels of VMAT2 binding. In contrast, both D2 receptors and DAT sites were altered by some of the treatment regimens. This data extends preliminary results that suggest that VMAT2 is not easily regulated and confirm the plasticity of D2 receptors and the DAT site. A second series of experiments realized in vivo in rodents (Kilbourn et al., 1996) demonstrate that dopaminergic treatments used in Parkinson’s disease do not influence VMAT2 measurements in the striatum. Striatal VMAT2 density may, thus, provide an objective estimate of monoaminergic innervation in neurodegenerative diseases, unaffected by the use of symptomatic therapies. In chapter IV, the development of specific in vivo VMAT2 radiotracers is discussed. The structure-pharmacological activity relationship of benzisoquinoles (Lee et al., 1996). Binding of α-TBZOH to VMAT2 was demonstrated to be stereospecific : the (+)-isomer showed high affinity in vitro (Ki = 0.97 ± 0.48 nM) for rat stritial VMAT2, whereas the (-)-isomer was inactive (Ki = 2.2 ± 0.3 nM). Each isomer was then synthesized in carbon-11 labeled form, and regional brain biodistriductions in mice determined after intravenous injection. Only (+)-α-TBZOH showed selective and specific accumulations in regions of dense monoaminergic innervation (e.g. striation, hypothalamus), which could be blocked by co-injection of unlabeled tetrabenazine. The prerequisites for successful in vivo applications of an in vitro radioligand were illustrated through the development of [11C]MTBZ. Its biodistribution, metabolism and in vivo specificity were first evaluated in rodents, and human dosimetry estimated. Regional rat brain localization of [3H]MTBZ 15 min post-injection was consistent with the known monoamine nerve terminal density, demonstrating highest activity in the striatum, lateral setum, substantia nigra pars compacta, the raphe nuclei, and the locus coerulus. In vivo [11C]MTBZ binding in the mouse brain was inhibited by c)injection of excess unlabeled TBZOH. Chromatography revealed over 82% of brain activity, but less than 47% of plasma activity, corresponded to authentic MTBZ. In contrast to rodents, MTBZ appears to be metabolized only to polar derivatives in primates. Subsequently, the human brain distribution of VMAT2 binding was determined in normal volunteers following administration of [11C]MTBZ. [11C]MTBZ had high initial brain uptake and rapid clearance from all regions, with longest retention in areas of high VMAT2 concentration. After correction for labelled blood metabolites, parametric transport and binding images were calculated using arterial blood sampling and a two-compartment tracer kinetic model. Parametric quantification of VMAT2 density revealed highest distribution volumes in the putamen and caudate nucleus with lower values in cerebral cortex and cerebellum. For in vivo applications, [11C]TBZOH rather than the [11C]MTBZ was selected due to its more reliable synthesis, lesser lipophilicity reducing the plasma protein binding and nonspecific activity, and easier metabolism that exclude all labeled degradation products from the brain. This choice is again strengthned by the discovery of a stereospecificity of [11C]TBZOH increasing significantly the signal-to-noise ratio (Kilbourn et al., 1995a). With [11C]TBZOH, a tree-compartment model appears to describe better the experimental data. Chapter V illustrates the first clinical applications of the [11C]TBZOH PET method. An aging effect on the striatal VMAT2 density has been observed in our population sample, with a 4% loss per decade. Two pathologies characterized by a decrease of dopaminergic neurons have been then investigated : Parkinson’s disease (PD) patients to correlate the striatal VMAT2 density to the clinical status (Frey et al., 1995, 1996), and patients with olivopontocerebellar atrophy (OPCA) to anticipate those that might evolve to a multisystemic degeneration (MSA; [Gilman et l., 1995, 1996]). PD patients demonstrate a decreased striatal VMAT2 density more pronounced in the posterior aspect of the putamen, richer in dopaminergic projections from the sustantia nigra pars compacta. While all MSA patients have a decrease of VMAT2 density in the striatum, only few aptients with OPCA reveal a similar diminution that renders them susceptible to develop symptomatic extrapyramidal disease. These examples illustrate clearly the interest of a non-invasive measurement of the monoaminergic neuronal density for diagnosis, prognosis and evaluation of therapeutic response. This last element becomes increasingly important to the emerging view that some treatments may not be solely symptomatic, but equally neuroprotectiveIn vivo measurement of dopaminergic neuronal density constitutes an objective of many neurologists interested in the non-invasive potential of nuclear medicine. The development of such a measurement would increase our understanding of neuropathophysiology and offer a unique tool to evaluate disease progression and possible modifications by therapy. After a short introduction reviewing the interest of the in vivo measurement of monoamine neuronal density, chapter I summarizes the different PET and SPET approaches developed for this purpose. 6[18F]Fluoro-L-dopa (FDOPA), a dopamine precursor analogue, was first used to image successively with Et presynaptic monoaminergic nerve terminals in human striatum (Garnett et al., 1983b). The tracer is converted to 6-[18F]fluorodopamine by dopa decarboxylase (DDC) and retained in the striatum. Its in vivo application is, however, complicated by the presence of labelled metabolites that contribute to the radioactivity measured by PET (Luxen et al., 1992). Despite the development of mathematical methods to correct for labelled degradative products or the synthesis of alternative dopamine precursors less prone to give rise to brain labelled metabolites, the uptake of dopamine analogues depends on the activity of DDC, an enzyme regulated via D2-dopamine receptors (Zhu et al, 1992; Hadjiconstantinou et al., 1993). Therefore, its uptake may reflect compensatory regulation mechanisms as well as real dopaminergic neuronal loss. The plasma membrane dopamine transporter (DAT) donctitutes an interesting alternative to measure the neuronal integrity since a diminution of their numbers has been demonstrated in vitro (Pimoule et al., 1983; Janowsky et al., 1987; Kaufman and Madras, 1991; Chinaglia et al., 1992) as well as in vivo (Leenders et al., 1990; Frost et al., 1993) following lesions of the nigrostriatal system. Nevertheless, as for the dopamine metabolite analoguesd, animal studies demonstrate regulation of the DAT site after pharmacological treatments (Scheffel et al., 1991; Kilbourn et al, 1992) or manipulation of endogenous dopamine concentration (Ikegami and Prasad, 1990; Sharpe et al., 1991; Wilson et al., 1994). The number of neuronal synaptic vesicular monoamine transporters (vesicular monoamine transporter type 2; VMAT2) has been proposed as an index of monoaminergic neuronal innervation. WMAT2 is responsible for movement of cytoplasmic monoamines into synaptic vesicles in exchange for two intravesicular protons (for reviews, see [Schuldiner, 1994]), and is the only isoform present in the brain. Reductions of VMAT2 binding density have been demonstrated in vitro after 6-OHDA lesions of the rat striatum (Darchen et al., 1989b) and in post-mortem tissues from Parkinson’s patients (Scherman et al., 1989; Lehericy et al., 1994; Ruberg et al., 1995). Despite a lack of transport selectivity, VMAT2 binding may be considered specific to dopaminergic neurons ion the striatum, which is relatively devoid of noradrenergic or serotonergic contents (Vander Borght et al., 1995c). Three major families of VMAT inhibitors have been identified: benzisoquinolines, reserpine and ketanserin. Among these the benzisoquinolines, particularly dihydrotetrabenazine (TBZOH), have been widely used for in vitro binding studies (Scherman and Henry, 1984; Scherman, 1986; Scherman et al., 1986 and 1988b; Darchen et al., 1989b) Despite a D2 dopamine receptor antagonist action at micromolar concentrations (Reches et al., 1983), benzisoquinolines binding has been shown to be a specific marker of monoamine synaptic vesicles. In contrast, ketanserin recognizes a population of serotonergic receptors (5-HT2; [Carchen et al., 1989b]).Thèse d'agrégation de l'enseignement supérieur (faculté de médecine) -- UCL, 199