PET/MRI for staging patients with Hodgkin lymphoma: equivalent results with PET/CT in a prospective trial.

To compare FDG-PET/unenhanced MRI and FDG-PET/diagnostic CT in detecting infiltration in patients with newly diagnosed Hodgkin lymphoma (HL). The endpoint was equivalence between PET/MRI and PET/CT in correctly defining the revised Ann Arbor staging system. Seventy consecutive patients with classical-HL were prospectively investigated for nodal and extra-nodal involvement during pretreatment staging with same-day PET/CT and PET/MRI. Findings indicative of malignancy with the imaging procedures were regarded as lymphoma infiltration; in case of discrepancy, positive-biopsy and/or response to treatment were evidenced as lymphoma. Sixty of the 70 (86%) patients were evaluable having completed the staging program. Disease staging based on either PET/MRI or PET/CT was correct for 54 of the 60 patients (90% vs. 90%), with difference between proportions of 0.0 (95% CI, -9 to 9%; P=0.034 for the equivalence test). As compared with reference standard, invasion of lymph nodes was identified with PET/MRI in 100% and with PET/CT in 100%, of the spleen with PET/MRI in 66% and PET/CT in 55%, of the lung with PET/MRI in 60% and PET/CT in 100%, of the liver with PET/MRI in 67% and PET/CT in 100%, and of the bone with PET/MRI in 100% and PET/CT in 50%. The only statistically significant difference between PET/MRI and PET/CT was observed in bony infiltration detection rates. For PET/CT, iodinate contrast medium infusions' average was 86 mL, and exposure to ionizing radiation was estimated to be 4-fold higher than PET/MRI. PET/MRI is a promising safe new alternative in the care of patients with HL.

2-deoxy-2[F-18] fluoro-D-glucose positron emission tomography Deauville scale and core-needle biopsy to determine successful management after six doxorubicin, bleomycin, vinblastine and dacarbazine cycles in advanced-stage Hodgkin lymphoma.

BACKGROUND: The clinical impact of the positivity of the Deauville scale (DS) of positron emission tomography (PET) performed at the end of doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) in patients with advanced Hodgkin lymphoma (HL), in terms of providing rationale to shift poor responders onto a more intensive regimen, remain to be validated by histopathology. PATIENTS AND METHODS: This prospective trial involved patients with stage IIB/IV HL who after six ABVD cycles underwent PET (PET6) and core-needle cutting biopsy (CNCB) of 2-deoxy-2[F-18] fluoro-d-glucose (FDG)-avid lymph nodes. Patients received high-dose chemotherapy/autologous haematopoietic stem cell rescue (HDCT/AHSCR) if CNCB was positive for HL, alternatively, if CNCB or PET was negative, received observation or consolidation radiotherapy (cRT) on residual nodal masses, as initially planned. The end-point was 5-year progression-free survival (PFS). RESULTS: In all, 43 of the 169 (25%) evaluable patients were PET6 positive (DS 4, 32; DS 5, 11). Among them, histology showed malignancy (HL) in 100% of DS 5 scores and in 12.5% of DS 4 scores. Fifteen patients with positive biopsy received HDCT/AHSCR, whereas 28 patients with negative biopsy, as well as 126 patients with negative PET6, continued the original plan (cRT, 78 patients; observation, 76 patients). The 5-year PFS in the negative PET6 group, negative biopsy group and positive biopsy group was 95.4%, 100% and 52.5%, respectively. CONCLUSION: DS positivity of end-of-ABVD PET in advanced HL carried a certain number of CNCB-proven non-malignant FDG-uptakes. The DS 4 scores which were found to have negative histology appeared to benefit from continuing the original non-intensive therapeutic plane as indicated by the successful outcome in more than 95% of them by obtaining similar 5-year PFS to the PET6-negative group. By contrast, the DS 5 score had consistently positive histology and was associated with unsuccessful conventional therapy, promptly requiring treatment intensification or innovative therapeutic approaches.

Brentuximab vedotin followed by bendamustine supercharge for refractory or relapsed Hodgkin lymphoma.

We evaluated the impact on progression-free survival (PFS) of achieving a deep metabolic response at 2-deoxy-2[18F] fluoro-d-glucose positron emission tomography (FDG-PET) in patients with refractory or relapsed (R/R) classic Hodgkin lymphoma (cHL) following a new salvage regimen named Bv+Bs (brentuximab vedotin + bendamustine supercharge), from 2013 to 2017. In this real-life study, 20 consecutive patients (aged <60 years) with R/R cHL after failure of ≥1 salvage treatments received Bv+Bs regimen consisting of 3-days outpatient IV infusions of 1.8 mg/kg of Bv on day 1 of each 3-week cycle combined in sequence to bendamustine on days 2 and 3 of the treatment cycle at a fixed dose of 120 mg/m2 per day, for a total of 4 courses. A robust primary prophylaxis approach, including premedication, antimicrobials, stimulating factors, and cytomegalovirus monitoring, was systematically performed. The 20 patients (all evaluable) underwent 4 courses of Bv+Bs with a median dose intensity of 100% for both Bv and Bs. Ten patients (50%) experienced grade ≥3 treatment-related adverse events, without requiring hospitalization. At post-Bv+Bs reevaluation, 80% of patients had deep metabolic responses with Deauville 5-point scale scores ≤2. Thereafter, 14 patients (70%) received autologous hematopoietic stem cell transplantation (HSCT; peripheral blood stem cells previously harvested in 12 cases), and 4 patients (10%) received allogeneic HSCT. At a median follow-up of 27 months from Bv+Bs regimen initiation, the 2-year PFS of the entire population was 93.7% (95% confidence interval, 62.7% to 99.6%). Our data suggest that Bv+Bs regimen-driven strategy may be a promising salvage option to improve long-term control of high-risk Hodgkin lymphoma.

The origin recognition complex subunit, ORC3, is developmentally regulated and supports the expression of biochemical markers of neuronal maturation in cultured cerebellar granule cells.

The origin recognition complex (ORC) regulates DNA replication. However, some members of the ORC core, such as ORC3 and ORC5, have been implicated in neuronal maturation. In cultured cerebellar granule cells (CGCs), ORC3 mRNA and protein levels increased from 6 to 8days in vitro, a time that coincided with the maximal development of the dendritic arbor. In contrast, expression of ORC5 remained low throughout CGC maturation. Activation of type-4 metabotropic glutamate receptors with the selective enhancer, PHCCC, during a critical time-window (from 4 to 6days in vitro) anticipated the developmental peak of ORC3, increased the expression of two proteins associated with neuronal maturation, i.e. the mitogen-associated protein-2 (MAP-2) and postsynaptic density-95 (PSD-95), as well as dendritic length. siRNA-induced ORC3 knockdown reduced MAP-2 and PSD-95 expression on its own and abrogated the action of PHCCC. We examined whether the maturational effects of ORC3 were mediated by changes in the activity of the monomeric GTP-binding protein, Rho, which is known to regulate granule cell morphology. ORC3 knockdown increased the levels of the GTP-bound active form of Rho, whereas exposure to PHCCC reduced Rho activation. The action of PHCCC was largely attenuated in cultures deprived of ORC3. Finally, granule cell exposure to the Rho-associated kinase inhibitor, Y-27632, abolished the lowering effect of ORC3 knockdown on MAP-2 expression, and increased dendritic length. These data suggest that ORC3 supports neuronal maturation by inhibiting the Rho signaling pathway, and mediates the differentiating activity of mGlu4 receptors in cultured cerebellar granule cells.

Metabotropic glutamate receptors regulate differentiation of embryonic stem cells into GABAergic neurons.

Mouse embryonic stem (ES) cells were stimulated to differentiate either as adherent monolayer cultures in DMEM/F12 supplemented with N2/B27, or as floating embryoid bodies (EBs) exposed to 1 microM retinoic acid (RA) for 4 days, starting from 4 DIV, and subsequently re-plated in DMEM/F12 medium. Adherent monolayer cultures of ES cells expressed mGlu5 receptors throughout the entire differentiation period. Selective pharmacological blockade of mGlu5 receptors with methyl-6-(phenylethynyl)-pyridine (MPEP) (1 microM, added once a day) accelerated the appearance of the neuronal marker, beta-tubulin. In addition, treatment with MPEP increased the number of cells expressing glutamate decarboxylase-65/67 (GAD(65/67)), a marker of GABAergic neurons. In floating EBs, mGlu5 receptors are progressively replaced by mGlu4 receptors. The orthosteric mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), or the selective mGlu4 receptor enhancer, PHCCC,--both combined with RA at concentrations of 30 microM--increased the expression of both beta-tubulin and GAD(65/67), inducing the appearance of fully differentiated neurons that released GABA in response to membrane depolarization. We conclude that mGlu receptor subtypes regulate neuronal differentiation of ES cells in a context-dependent manner, and that subtype-selective ligands of these receptors might be used for the optimization of in vitro protocols aimed at producing GABAergic neurons from ES cells.

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid.

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co-receptor low-density lipoprotein receptor-related protein (LRP) type 5. A 4-day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk-1, and induced the synthesis of the Wnt/Dkk-1 co-receptor LRP6. Recombinant Dkk-1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal-less homeobox gene (Dlx-2). Recombinant Dkk-1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear beta-catenin levels. Remarkably, the antisense- or small interfering RNA-induced knockdown of Dkk-1 largely reduced the expression of Dlx-2, and the neuronal marker beta-III tubulin in EBs exposed to RA. These data suggest that induction of Dkk-1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.

Characterization of single nucleotide polymorphisms in sheep and their variation as evidence of selection.

The discovery of SNPs was performed using animals from eight European sheep breeds. Eleven SNPs were further characterized using about 1,700 sheep belonging to 57 breeds. A method for the identification of loci that were likely subject to selection was applied; three of the 11 SNPs lying outside the 95% confidence region of the conditional joint distribution of F(ST) and mean heterozygosity were identified as outliers.

Assessment of population structure by single nucleotide polymorphisms (SNPs) in goat breeds.

Single nucleotide polymorphisms (SNPs) may be used in biodiversity studies and commercial tasks like traceability, paternity testing and selection for suitable genotypes. Twenty-seven SNPs were characterized and genotyped on 250 individuals belonging to eight Italian goat breeds. Multilocus genotype data were used to infer population structure and assign individuals to populations. To estimate the number of groups (K) to test in population structure analysis we used likelihood values and variance of the bootstrap samples, deriving optimal K from a drop in the likelihood and a rise in the variance plots against K.

The mGlu5 metabotropic glutamate receptor is expressed in zones of active neurogenesis of the embryonic and postnatal brain.

Metabotropic glutamate (mGlu) receptors have been implicated in the regulation of developmental plasticity. Here, we examined the expression of mGlu1a-b, -2, -3, -4a-b, and -5a receptor subtypes from embryonic day 12 (E12) to the early and late postnatal life. While all transcripts (with the exception of mGlu4 mRNA) were detected prenatally, only the mGlu5 receptor protein was found in detectable amounts in the embryonic brain. Immunohistochemical analysis showed that the mGlu5 receptor was mainly expressed by cells surrounding the ventricles at E15, whereas it was more diffusely expressed at E18. In the postnatal life, besides its classical expression sites, the mGlu5 receptor was found in zones of active neurogenesis such as the external granular layer (EGL) of the cerebellar cortex and the subventricular zone. In these regions, the presence of actively proliferating progenitor cells was detected by BrdU staining. No other subtype (among those we have examined) was found to be expressed in regions enriched of BrdU(+) cells. These data suggest a role for mGlu5 receptors in the early brain development and in basic cellular processes such as proliferation and/or differentiation.

Endogenous activation of group-II metabotropic glutamate receptors inhibits the hypothalamic-pituitary-adrenocortical axis.

Systemic injection of the mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p.), increased plasma corticosterone in mice to an extent similar to that induced by the despair test. Treatment with the mGlu2/3 receptor agonist, LY379268 (1 mg/kg, i.p.), or the non-competitive mGlu5 receptor antagonist, MPEP (5 mg/kg, i.p.), failed to induce significant changes in corticosterone levels. Searching for a site of action of LY341495, we examined the expression of mGlu receptor subtypes in the various anatomical regions of the mouse hypothalamic-pituitary-adrenal (HPA) axis. Only mGlu5 and -7 receptor mRNAs were detected in the adrenal gland by RT-PCR, whereas mGlu -1, -3, -4, -5, -7 and -8 receptor mRNAs were detected in the anterior pituitary. All transcripts (with the exception of mGlu5 and mGlu6 receptor mRNAs) were detected in the hypothalamus. However, Western blot analysis showed the presence of mGlu2/3 receptor proteins only in the hypothalamus and not in the anterior pituitary. This was consistent with functional data showing that LY341495 (0.1 and 1 microM) failed to affect ACTH secretion from isolated mouse anterior pituitaries. Moving from these observations, we examined whether LY341495 could activate the HPA axis by inhibiting mGlu2/3 receptors at hypothalamic level. We measured the release of corticotropin releasing hormone (CRH) in isolated mouse hypothalami incubated in the presence of subtype-selective mGlu receptor agonists or antagonists. Among all the drugs we have tested, only LY341495 was able to increase CRH secretion. With high concentrations of LY341495 (1 microM) this increase was similar to that induced by 50 mM K(+). The action of LY341495 was prevented by the combined application of the mGlu2/3 receptor agonist, LY379268. We conclude that group-II mGlu receptors tonically regulate the HPA axis by controlling CRH secretion at hypothalamic level.

Imipramine treatment up-regulates the expression and function of mGlu2/3 metabotropic glutamate receptors in the rat hippocampus.

We examined the effect of a chronic imipramine treatment (10 mg/kg, i.p., once daily for 21 days) on the expression and function of metabotropic glutamate (mGlu) receptors in discrete regions of the rat brain. Chronic imipiramine treatment up-regulated the expression of mGlu2/3 receptor proteins in the hippocampus, nucleus accumbens, cerebral cortex and corpus striatum. Expression of mGlu1a receptor protein was increased exclusively in the hippocampus, whereas no changes in the expression of mGlu4 and mGlu5 receptors or Homer-1a protein were detected. Using hippocampal slices, we examined the stimulation of polyphosphoinositide (PI) hydrolysis induced by mGlu receptor agonists in control and imipramine-treated rats. Imipramine treatment amplified the PI response to the non subtype-selective mGlu receptor agonist, 1S,3R-aminocyclopentane-1,3-dicarboxylated (1S,3R-ACPD) in both hippocampal and cortical slices, but failed to affect the response to the selective mGlu1/5 receptor agonist, S-3,5-dihydroxyphenylglycine (DHPG). Amplification was restored when DHPG was combined with the selective mGlu2/3 receptor agonist, LY379268. In addition, 1S,3R-ACPD-stimulated PI hydrolysis was no longer enhanced in imipramine-treated rats when the mGlu2/3 component of the PI response was abrogated by the antagonist, LY341495. In contrast, the ability of LY379268 to inhibit forskolin-stimulated cAMP formation was reduced in hippocampal slices of rats chronically treated with imipramine. Taken together, these results suggest that neuroadaptive changes in the expression and function of mGlu2/3 receptors occur in response to chronic antidepressants.

Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA.

The random amplified polymorphic DNA technique (RAPD) has found wide use in molecular genetics because of its speed and ease of use. For various reasons, with this method the amplified DNA fragments are produced at different concentrations between genotypes and even between polymerase chain reaction (PCR) runs. Since the detection of the multiple amplified fragments is performed routinely by agarose gel, and seldom by acrylamide gel electrophoresis, we have found that by capillary zone electrophoresis (CZE), which is more sensitive and accurate than gel electrophoresis, it is possible to unequivocally detect amplified fragments even at low concentration, avoiding polymorphism misinterpretation. CZE is also useful to make more potentially polymorphic fragments evident per random primer used, with obvious economical benefits.