TOP2A expression predicts responsiveness to carfilzomib in myeloma and informs novel combinatorial strategies for enhanced proteasome inhibitor cell killing.

Microarray was utilized to determine if a genetic signature associated with resistance to carfilzomib (CFZ) could be identified. Twelve human myeloma (MM) cell lines (HMCLs) were treated with CFZ and a cell-viability profile was assessed categorizing HMCLs as sensitive or resistant to CFZ. The gene expression profiles (GEP) of untreated resistant versus sensitive HMCLs revealed 29 differentially expressed genes. TOP2A, an enzyme involved in cell cycle and proliferation, was overexpressed in carfilzomib-resistant HMCLs. TOP2A protein expression levels, evaluated utilizing trephine biopsy specimens acquired prior to treatment with proteasome inhibitors, were higher in patients failing to achieve a response when compared to responding patients. Logistic-regression analysis confirmed that TOP2A protein expression was a highly significant predictor of response to PIs (AUC 0.738). Further, the combination of CFZ with TOP2A inhibitors, demonstrated synergistic cytotoxic effects in vitro, providing a rationale for combining topoisomerase inhibitors with CFZ to overcome resistance in MM.

Inhibition of mTOR complex 2 restrains tumor angiogenesis in multiple myeloma.

The mammalian Target of Rapamycin (mTOR) is an intracellular serine/threonine kinase that mediates intracellular metabolism, cell survival and actin rearrangement. mTOR is made of two independent complexes, mTORC1 and mTORC2, activated by the scaffold proteins RAPTOR and RICTOR, respectively. The activation of mTORC1 triggers protein synthesis and autophagy inhibition, while mTORC2 activation promotes progression, survival, actin reorganization, and drug resistance through AKT hyper-phosphorylation on Ser473. Due to the mTOR pivotal role in the survival of tumor cells, we evaluated its activation in endothelial cells (ECs) from 20 patients with monoclonal gammopathy of undetermined significance (MGUS) and 47 patients with multiple myeloma (MM), and its involvement in angiogenesis. MM-ECs showed a significantly higher expression of mTOR and RICTOR than MGUS-ECs. These data were supported by the higher activation of mTORC2 downstream effectors, suggesting a major role of mTORC2 in the angiogenic switch to MM. Specific inhibition of mTOR activity through siRNA targeting RICTOR and dual mTOR inhibitor PP242 reduced the MM-ECs angiogenic functions, including cell migration, chemotaxis, adhesion, invasion, in vitro angiogenesis on Matrigel®, and cytoskeleton reorganization. In addition, PP242 treatment showed anti-angiogenic effects in vivo in the Chick Chorioallantoic Membrane (CAM) and Matrigel® plug assays. PP242 exhibited a synergistic effect with lenalidomide and bortezomib, suggesting that mTOR inhibition can enhance the anti-angiogenic effect of these drugs. Data to be shown indicate that mTORC2 is involved in MM angiogenesis, and suggest that the dual mTOR inhibitor PP242 may be useful for the anti-angiogenic management of MM patients.

Myeloma bone and extra-medullary disease: Role of PET/CT and other whole-body imaging techniques.

Multiple myeloma (MM) is the second most common hematological malignancy. Although it can affect different organs, the bone compartment stands out both in terms of prevalence and clinical impact. Despite the striking advances in MM therapy, bone disease can remarkably affect the patient's quality of life. The occurrence and extension of bone marrow and extra-medullary involvement should be carefully assessed to confirm the diagnosis, to locate and whenever possible prevent dreadful complications such as pathological fractures and spinal cord compression, and to establish suitable therapeutic measures. Many imaging techniques have been proposed for the detection of MM skeletal involvement. With the development of more sophisticated imaging tools, it is time to use the right technique at the right time. Based on the review of the literature and our own experience, this article discusses advantages and disadvantages of the different imaging methods in the work-up of MM patients, with particular emphasis on the role that PET/CT can play. It is emphasized that whole body low-dose computed tomography should be the preferred imaging technique at baseline. However, bone marrow infiltration and extra-medullary manifestations are better detected by whole body magnetic resonance imaging. Positron emission tomography/computed tomography, on the other hand, combines the benefits of the two mentioned imaging procedures and is particularly useful not only for the detection of osteolytic lesions unrevealed by conventional X-ray, but also in the assessment of prognosis and therapeutic response.

KCNJ11 activating mutations in Italian patients with permanent neonatal diabetes.

Permanent neonatal diabetes mellitus (PNDM) is a rare condition characterized by severe hyperglycemia constantly requiring insulin treatment from its onset. Complete deficiency of glucokinase (GCK) can cause PNDM; however, the genetic etiology is unknown in most PNDM patients. Recently, heterozygous activating mutations of KCNJ11, encoding Kir6.2, the pore forming subunit of the ATP-dependent potassium (K(ATP)) channel of the pancreatic beta-cell, were found in patients with PNDM. Closure of the K(ATP) channel exerts a pivotal role in insulin secretion by modifying the resting membrane potential that leads to insulin exocytosis. We screened the KCNJ11 gene in 12 Italian patients with PNDM (onset within 3 months from birth) and in six patients with non-autoimmune, insulin-requiring diabetes diagnosed during the first year of life. Five different heterozygous mutations were identified: c.149G>C (p.R50P), c.175G>A (p.V59M), c.509A>G (p.K170R), c.510G>C (p.K170N), and c.601C>T (p.R201C) in eight patients with diabetes diagnosed between day 3 and 182. Mutations at Arg50 and Lys170 residues are novel. Four patients also presented with motor and/or developmental delay as previously reported. We conclude that KCNJ11 mutations are a common cause of PNDM either in isolation or associated with developmental delay. Permanent diabetes of non autoimmune origin can present up to 6 months from birth in individuals with KCNJ11 and EIF2AK3 mutations. Therefore, we suggest that the acronym PNDM be replaced with the more comprehensive permanent diabetes mellitus of infancy (PDMI), linking it to the gene product (e.g., GCK-PDMI, KCNJ11-PDMI) to avoid confusion between patients with early-onset, autoimmune type 1 diabetes.

Autoimmune uveitis: a retrospective analysis of 104 patients from a tertiary reference center.

BACKGROUND: The aim of this study was to identify the main features of a cohort of Caucasian patients with idiopathic (I) and systemic disease-associated (SDA) autoimmune uveitis (AU) who were followed up at a single tertiary reference center. The study consisted of a retrospective analysis of the demographic, clinical, and laboratory features and the response to treatment of 104 patients with AU evaluated between 2004 and 2013, with a median follow-up of 4.8 years. The primary outcome measure was the response to systemic treatment after 24 months of therapy. The data are expressed as the range, percentage, or mean ± standard error. Categorical variables were assessed by Fisher's exact test. RESULTS: The mean age at diagnosis was 40.1 ± 17.8 years for men and 44.1 ± 15.3 years for women. There was a slight female predominance. Of the 104 patients, 72.1% had I-AU and 27.9% SDA-AU. The most frequent associations were with ankylosing spondyloarthritis, autoimmune thyroiditis, inflammatory bowel diseases, and Behcet's disease. Symptoms at presentation consisted of eye redness and pain (28.8%), decreased visual acuity (25.9%), and floaters (18.3%). Complications included cataracts (24%), retinal neovascularization (16.3%), chorio-retinal scars (10.6%), cystoid macular edema (8.6%), glaucoma/ocular hypertension (7.7%), epiretinal membranes (4.8%), and retinal detachment (3.8%). The prevalence of autoantibodies, mostly antinuclear antibodies, was comparable between the I-AU and SDA-AU groups. Fisher's exact test showed a direct correlation between patients with class I HLA B27, Cw8, B5 (51, 52), B51, or Cw2 and the presence of AU, whereas among patients with class II HLA, only DQ1 was a predisposing factor for AU. The therapeutic spectrum included corticosteroids and immunosuppressive agents, given either alone or in various combinations according to the severity of AU and the extent of the clinical response. Among the immunosuppressive drugs, azathioprine was preferentially used for anterior uveitis, and cyclosporine-A for intermediate and posterior uveitis. An assessment of the patients after 24 months of therapy showed a complete remission in 43.3% and a significant clinical improvement in 26.9%. CONCLUSIONS: At our tertiary reference center, the prevalence in Caucasian patients of I-AU was approximately 2.5-fold higher than that of SDA-AU. Our findings point to the need for a patient-tailored therapeutic approach according to the anatomic site and the severity of AU. Therapy should be prolonged, over a period of months and even up to 1-2 years, in order to achieve stable control of the disease and to prevent severe complications. The outcome of SDA-AU is also influenced by treatment of the underlying systemic disease. Additional controlled trials are needed to assess the efficacy and the long-term safety of both the prescribed therapeutic agents and their combinations.

Modulation of the Fcgamma receptors induced by anti-Ro and anti-La autoantibodies: observations in salivary gland cells.

Only few reports have shown protein expression of the Fcgamma receptors (FcgammaRs) molecules on human salivary gland cells. In this study we investigate a possible upregulation of FcgammaRs following anti-Ro and anti-La autoantibodies treatment. Anti-Ro and anti-La autoantibodies were purified from IgG fractions obtained from 14 patients with primary Sjögren's syndrome (SS), using Sepharose 4B-Ro and Sepharose 4B-La affinity columns. Flow cytometry and RT-PCR were used to study the FCgammaRI, FCgammaRII and FCgammaRIII receptors expression and upregulation by anti-Ro and anti-La on a salivary gland cell line. The present data document that the anti-Ro and anti-La autoantibodies determine an increase of the FcgammaRs expression on salivary gland cells, and provide evidence that both the high affinity FcgammaRI and the low affinity FcgammaRII and FcgammaRIII are overexpressed. Treatment with IgG isolated from healthy donors had no effect on the basal FCgammaRs expression.