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Background: Intraoperative remifentanil has been associated with postoperative hyperalgesia, higher visual analogic pain scores, and increased postoperative morphine consumption. However, this has not been investigated from patient's perspective by using a patient-reported outcomes (PROs) approach with a validated questionnaire., Methods: We joined the largest prospective observational study on postoperative pain, PAIN OUT Project (NCT02083835), and collected data for 2 years. We studied the effects of remifentanil (R+) vs nonremifentanil (R-) anesthesia on PROs regarding their pain management after elective thyroidectomy. We selected 5 primary PROs (worst pain experienced, time spent in severe pain, relief received by treatment, satisfaction about pain management, wish for more pain treatment) and five secondary PROs (drowsiness, itching, nausea, dizziness, waking up due to pain) from the validated International Pain Outcomes questionnaire., Results: The analysis included 317 patients, 208 in the R+ group (65.6%) and 109 in the R- group (34.4%), the latter receiving fentanyl as intraoperative opioid. Although the R+ group received more frequently intraoperative nonopioids (202/208, 97.1% vs 86/109, 78.9%; P < .0001) and opioids (184/208, 88.5% vs 38/109, 34.9%; P < .001), it reported higher worst pain (5.1±2.1 vs 4.3±2.1, P < .005), lower satisfaction (7.4±2.0 vs 8.1±2.1, P < .001), and worse results in 4 secondary PROs. A sensitivity analysis performed matching 67 couples of patients yielded similar results in primary PROs., Conclusions: Our study suggests that remifentanil-based anesthesia is associated with worse pain-related PROs in patients undergoing thyroidectomy despite more frequent intraoperative analgesic administration. This study adds further evidence to the growing literature about opioid- and remifentanil-induced hyperalgesia., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells in the bone marrow leading to bone destruction and bone marrow failure. Several molecular mechanisms underlie chemoresistance among which heme oxygenase-1 (HO-1) could play a major role. The aim of the present research was to evaluate the impact of HO-1 in MM following bortezomib (BTZ) treatment and how HO-1 is implicated in the mechanisms of chemoresistance. MM cells were treated for 24h with BTZ (15 nM), a boronic acid dipeptide inhibitor of the 26S proteasome used in the treatment of patients with MM as first-line therapy. We evaluated cell viability, reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress, HO-1 expression and compartmentalization and cellular genetic instability. Results showed that BTZ significantly reduced cell viability in different MM cell lines and induced ER-stress and ROS formation. Concomitantly, we observed a significant overexpression of both HO-1 gene and protein levels. This effect was abolished by concomitant treatment with 4-phenybutirric acid, a molecular chaperone, which is known to reduce ER-stress. Surprisingly, inhibition of HO activity with SnMP (10μM) failed to increase BTZ sensitivity in MM cells whereas inhibition of HO-1 nuclear translocation by E64d, a cysteine protease inhibitor, increased sensitivity to BTZ and decreased genetic instability as measured by cytokinesis-block micronucleus assay. In conclusion, our data suggest that BTZ sensitivity depends on HO-1 nuclear compartmentalization and not on its enzymatic activity and this finding may represent an important tool to overcome BTZ chemoresistance in MM patients., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article.

Lenalidomide-dexamethasone improved outcome in newly diagnosed elderly multiple myeloma patients. We randomly assigned 662 patients who were age ≥65 years or transplantation-ineligible to receive induction with melphalan-prednisone-lenalidomide (MPR) or cyclophosphamide-prednisone-lenalidomide (CPR) or lenalidomide plus low-dose dexamethasone (Rd). The primary end point was progression-free survival (PFS) in triplet (MPR and CPR) vs doublet (Rd) lenalidomide-containing regimens. After a median follow-up of 39 months, the median PFS was 22 months for the triplet combinations and 21 months for the doublet (P = .284). The median overall survival (OS) was not reached in either arms, and the 4-year OS was 67% for the triplet and 58% for the doublet arms (P = .709). By considering the 3 treatment arms separately, no difference in outcome was detected among MPR, CPR, and Rd. The most common grade ≥3 toxicity was neutropenia: 64% in MPR, 29% in CPR, and 25% in Rd patients (P < .0001). Grade ≥3 nonhematologic toxicities were similar among arms and were mainly infections (6.5% to 11%), constitutional (3.5% to 9.5%), and cardiac (4.5% to 6%), with no difference among the arms. In conclusion, in the overall population, the alkylator-containing triplets MPR and CPR were not superior to the alkylator-free doublet Rd, which was associated with lower toxicity. This study was registered at www.clinicaltrials.gov as #NCT01093196., (© 2016 by The American Society of Hematology.)

Background: High-dose melphalan plus autologous stem-cell transplantation (ASCT) is the standard approach in transplant-eligible patients with newly diagnosed myeloma. Our aims were to compare consolidation with high-dose melphalan plus ASCT versus chemotherapy (cyclophosphamide and dexamethasone) plus lenalidomide, and maintenance with lenalidomide plus prednisone versus lenalidomide alone., Methods: We did an open-label, randomised, multicentre, phase 3 study at 59 centres in Australia, Czech Republic, and Italy. We enrolled transplant-eligible patients with newly diagnosed myeloma aged 65 years or younger. Patients received a common induction with four 28-day cycles of lenalidomide (25 mg, days 1-21) and dexamethasone (40 mg, days 1, 8, 15, and 22) and subsequent chemotherapy with cyclophosphamide (3 g/m(2)) followed by granulocyte colony-stimulating factor for stem-cell mobilisation and collection. Using a 2 × 2 partial factorial design, we randomised patients to consolidation with either chemotherapy plus lenalidomide (six cycles of cyclophosphamide [300 mg/m(2), days 1, 8, and 15], dexamethasone [40 mg, days 1, 8, 15, and 22], and lenalidomide [25 mg, days 1-21]) or two courses of high-dose melphalan (200 mg/m(2)) and ASCT. We also randomised patients to maintenance with lenalidomide (10 mg, days 1-21) plus prednisone (50 mg, every other day) or lenalidomide alone. A simple randomisation sequence was used to assign patients at enrolment into one of the four groups (1:1:1:1 ratio), but the treatment allocation was disclosed only when the patient reached the end of the induction and confirmed their eligibility for consolidation. Both the patient and the treating clinician did not know the consolidation and maintenance arm until that time. The primary endpoint was progression-free survival assessed by intention-to-treat. The trial is ongoing and some patients are still receiving maintenance. This study is registered at ClinicalTrials.gov, number NCT01091831., Findings: 389 patients were enrolled between July 6, 2009, and May 6, 2011, with 256 eligible for consolidation (127 high-dose melphalan and ASCT and 129 chemotherapy plus lenalidomide) and 223 eligible for maintenance (117 lenalidomide plus prednisone and 106 lenalidomide alone). Median follow-up was 52·0 months (IQR 30·4-57·6). Progression-free survival during consolidation was significantly shorter with chemotherapy plus lenalidomide compared with high-dose melphalan and ASCT (median 28·6 months [95% CI 20·6-36·7] vs 43·3 months [33·2-52·2]; hazard ratio [HR] for the first 24 months 2·51, 95% CI 1·60-3·94; p<0·0001). Progression-free survival did not differ between maintenance treatments (median 37·5 months [95% CI 27·8-not evaluable] with lenalidomide plus prednisone vs 28·5 months [22·5-46·5] with lenalidomide alone; HR 0·84, 95% CI 0·59-1·20; p=0·34). Fewer grade 3 or 4 adverse events were recorded with chemotherapy plus lenalidomide than with high-dose melphalan and ASCT; the most frequent were haematological (34 [26%] of 129 patients vs 107 [84%] of 127 patients), gastrointestinal (six [5%] vs 25 [20%]), and infection (seven [5%] vs 24 [19%]). Haematological serious adverse events were reported in two (2%) patients assigned chemotherapy plus lenalidomide and no patients allocated high-dose melphalan and ASCT. Non-haematological serious adverse events were reported in 13 (10%) patients assigned chemotherapy plus lenalidomide and nine (7%) allocated high-dose melphalan and ASCT. During maintenance, adverse events did not differ between groups. The most frequent grade 3 or 4 adverse events were neutropenia (nine [8%] of 117 patients assigned lenalidomide plus prednisone vs 14 [13%] of 106 allocated lenalidomide alone), infection (eight [8%] vs five [5%]), and systemic toxicities (seven [6%] vs two [2%]). Non-haematological serious adverse events were reported in 13 (11%) patients assigned lenalidomide plus prednisone versus ten (9%) allocated lenalidomide alone. Four patients died because of adverse events, three from infections (two during induction and one during consolidation) and one because of cardiac toxic effects., Interpretation: Consolidation with high-dose melphalan and ASCT remains the preferred option in transplant-eligible patients with multiple myeloma, despite a better toxicity profile with chemotherapy plus lenalidomide., Funding: Celgene., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Osteolytic bone disease is a common manifestation of multiple myeloma (MM) that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. It results from increased osteolytic activity and decrease osteoblastic function. Activation of mammalian chitinases chitotriosidase (CHIT1) and YKL40 is associated with osteoclast (OCs) differentiation and bone digestion. In the current study, we investigated the effect of two Bortezomib's concentration (2.5 and 5 nM) on osteoclastogenesis by analyzing regulation of chitinase expression. OCs exposition to bortezomib (BO) was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP, and MMP9. In addition BO-treatment reduced CHIT1 enzymatic activity and both CHIT1 and YKL40 mRNA expression levels and cytoplasmatic and secreted protein. Moreover, immunofluorescence evaluation of mature OCs showed that BO was able to translocate YKL40 into the nucleus, while CHIT1 remained into the cytoplasm. Since MM cell lines such as U266, SKM-M1 and MM1 showed high levels of CHIT1 activity, we analyzed bone resorption ability of U266 using dentin disk assay resorption pits. Silencing chitinase proteins in U266 cell line with specific small interfering RNA, resulted in pits number reduction on dentine disks. In conclusion, we showed that BO decreases osteoclastogenesis and reduces bone resorption in OCs and U266 cell line by modulating the chitinases CHIT1 and YKL40. These results indicate that chitinases may be a therapeutic target for bone disease in MM patients.

Gastrointestinal lymphomas represent up to 10% of gastrointestinal malignancies and about one third of non-Hodgkin lymphomas. The most prominent histologies are mucosa-associated lymphoid tissue lymphoma and diffuse large B-cell lymphoma. However, the gastrointestinal tract can be the site of rarer lymphoma subtypes as a primary or secondary localization. Due to their rarity and the multifaceted histology, an endoscopic classification has not been validated yet. This review aims to analyze the endoscopic presentation of rare gastrointestinal lymphomas from disease diagnosis to follow-up, according to the involved site and lymphoma subtype. Existing, new and emerging endoscopic technologies have been examined. In particular, we investigated the diagnostic, prognostic and follow-up endoscopic features of T-cell and natural killer lymphomas, lymphomatous polyposis and mantle cell lymphoma, follicular lymphoma, plasma cell related disease, gastrointestinal lymphomas in immunodeficiency and Hodgkin's lymphoma of the gastrointestinal tract. Contrarily to more frequent gastrointestinal lymphomas, data about rare lymphomas are mostly extracted from case series and case reports. Due to the data paucity, a synergism between gastroenterologists and hematologists is required in order to better manage the disease. Indeed, clinical and prognostic features are different from nodal and extranodal or the bone marrow (in case of plasma cell disease) counterpart. Therefore, the approach should be based on the knowledge of the peculiar behavior and natural history of disease.

Introduction: A still not well defined proportion of patients with multiple myeloma (MM) and eligible for autologous stem cell transplantation (AuSCT) fails to mobilize CD34+ peripheral blood stem cells (PBSC) at all or to collect an adequate number for a safe procedure or sufficient for multiple transplants. These so-called "poor-mobilizers" are difficult to be predicted, due to marked difference across previous heterogeneous studies., Methods: We aimed to develop a method based on simple clinical parameters for predicting unsuccessful (<2×10(6)/kg) or sub-optimal (<5×10(6)/kg) collections of CD34+ PBSC in newly diagnosed MM patients eligible for AuSCT, treated with novel agents and receiving an homogeneous mobilizing therapy with cyclophosphamide and granulocyte-colony stimulating factor (G-CSF). To this purpose, 1,348 patients enrolled in five consecutive Italian clinical trials were retrospectively analysed. Age, baseline low peripheral blood cell counts, use of lenalidomide, and haematological toxicity developed during induction were taken into account as possible factors associated with poor mobilization., Results: Overall, 280 patients (20.8%) showed either sub-optimal (167 patients, 12.4%) or unsuccessful (113 patients, 8.4%) collections. All analysed parameters negatively influenced the procedure, but only age and haematological toxicity during induction maintained their significance at multivariate analysis. Based on ordinal logistic regression model, we constructed a risk heat-map where the four parameters were pooled and weighted according to their relevance as single or combined variables. This model was predictive for different probabilities of failure, suboptimal or optimal outcomes., Conclusions: We found that about one fifth of newly diagnosed MM fails to collect an adequate number of PBSC. Our model, based on a large group of patients treated frontline with novel agents and receiving the most popular mobilizing approach currently employed in Europe, is applicable in individual subjects and may contribute to the early identification of "poor mobilizer" phenotypes.

Many progresses have been done in the management of gastrointestinal (GI) lymphomas during last decades, especially after the discovery of Helicobacter pylori-dependent lymphoma development. The stepwise implementation of new endoscopic techniques, by means of echoendoscopy or double-balloon enteroscopy, enabled us to more precisely describe the endoscopic features of GI lymphomas with substantial contribution in patient management and in tailoring the treatment strategy with organ preserving approaches. In this review, we describe the recent progresses in GI lymphoma management from disease diagnosis to follow-up with a specific focus on the endoscopic presentation according to the involved site and the lymphoma subtype. Additionally, new or emerging endoscopic technologies that have an impact on the management of gastrointestinal lymphomas are reported. We here discuss the two most common subtypes of GI lymphomas: the mucosa-associated lymphoid tissue and the diffuse large B cell lymphoma. A general outline on the state-of-the-art of the disease and on the role of endoscopy in both diagnosis and follow-up will be performed.

Objectives: In vitro studies have shown synergistic anti-myeloma effects of bortezomib combined with alkylating agents or anthracycline. We tested safety and efficacy of the combination of bortezomib, doxorubicin cyclophosphamide, and dexamethasone (ABCD) in the treatment of relapsed/refractory myeloma., Methods: ABCD consisted of bortezomib given intravenous (IV) at dosage 1.3 mg/m(2) , dexamethasone 40 mg IV on days 1, 4, 8, and 15, pegylated liposomal doxorubicin (PLD) 20 mg IV on days 1 and 15, plus cyclophosphamide 100 mg/d per os for 15 d. Between January 2008 and February 2009, 24 patients received a median of four 28-d ABCD cycles (range 1-6). All patients had been already treated with a median of two previous lines of treatment (range 1-6): 38% were resistant to previous therapies and 62% were relapsed., Results: Clinical response was observed in 12 patients (50%), including 29% of very good partial remissions or better. Side effects included hematological toxicity (31% any grade), grades 3-4 thrombocytopenia (9%), grades 3-4 anemia (17%). Non-hematological toxicity affected 32% of administered cycles and included gastrointestinal disturbances (54%), peripheral neuropathy (8%), and infections (8%). After a median follow-up of 21.5 months (range 2-44 months), median of progression-free survival (PFS) was 8.7 months and median overall survival was 22.5 months. Achieving at least partial response within the second cycle was associated with a better PFS (19.5 months vs. 3.5 months), P = 0.03, HR 0.35 (CI 95% 0.13-0.90)., Conclusion: ABCD is safe and effective for relapsed/refractory MM subjects previously treated with novel agents., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This multicenter, open-label phase 2 trial determined the safety and efficacy of carfilzomib, a novel and irreversible proteasome inhibitor, in combination with cyclophosphamide and dexamethasone (CCyd) in patients with newly diagnosed multiple myeloma (NDMM) ≥65 years of age or who were ineligible for autologous stem cell transplantation. Patients (N = 58) received CCyd for up to 9 28-day cycles, followed by maintenance with carfilzomib until progression or intolerance. After a median of 9 CCyd induction cycles (range 1-9), 95% of patients achieved at least a partial response, 71% achieved at least a very good partial response, 49% achieved at least a near complete response, and 20% achieved stringent complete response. After a median follow-up of 18 months, the 2-year progression-free survival and overall survival rates were 76% and 87%, respectively. The most frequent grade 3 to 5 toxicities were neutropenia (20%), anemia (11%), and cardiopulmonary adverse events (7%). Peripheral neuropathy was limited to grades 1 and 2 (9%). Fourteen percent of patients discontinued treatment because of adverse events, and 21% of patients required carfilzomib dose reductions. In summary, results showed high complete response rates and a good safety profile. This trial was registered at clinicaltrials.gov as #NCT01346787., (© 2014 by The American Society of Hematology.)

Multiple Myeloma (MM) is a systemic hematologic disease due to uncontrolled proliferation of monoclonal plasma cells (PC) in bone marrow (BM). Emerging in other solid and liquid cancers, the host immune system and the microenvironment have a pivotal role for PC growth, proliferation, survival, migration, and resistance to drugs and are responsible for some clinical manifestations of MM. In MM, microenvironment is represented by the cellular component of a normal bone marrow together with extracellular matrix proteins, adhesion molecules, cytokines, and growth factors produced by both stromal cells and PC themselves. All these components are able to protect PC from cytotoxic effect of chemo- and radiotherapy. This review is focused on the role of immunome to sustain MM progression, the emerging role of myeloid derived suppressor cells, and their potential clinical implications as novel therapeutic target.

During the past decade, overall results of treatment of multiple myeloma (MM) have been improved and survival curves are now significantly better with respect to those obtained with historical treatment. These improvements are linked to a deeper knowledge of the biology of disease and to the introduction in clinical practice of drugs with different mechanism of action such as proteasome inhibitors and immunomodulatory drugs (IMiDs). However, MM remains in most cases an incurable disease. For patients who relapse after treatment with novel agents, the prognosis is dismal and new drugs and therapeutic strategies are required for continued disease control. In this review, we summarize new insights in salvage therapy for relapsed/refractory MM as emerging from recent clinical trials exploring the activity of bendamustine, new generation proteasome inhibitors, novel IMiDs, monoclonal antibodies, and drugs interfering with growth pathways.

CD200 is a relatively ubiquitously expressed molecule that plays a role in cancer immune evasion through interaction with its receptors. High expression levels of CD200 have been described in different human malignancies. For example, CD200 has been shown to be targeted after RAS/RAF/MEK/ERK activation in melanoma. Here we present the analysis of CD200 expression in human Multiple Myeloma (MM) samples. We found that CD200-positive cells express ERK and p-ERK. Moreover, UO126, a MEK inhibitor, reduces CD200 expression. Furthermore, we observe that CD200-positive cells show reduced immunogenicity compared to normal lymphocytes and that such immunogenicity increases when UO126 is used. We therefore hypothesize that CD200 expression in MM could suppress antitumor response and that anti-CD200 treatment might be therapeutically beneficial in CD200-expressing tumors., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Management of multiple myeloma (MM) has been drastically changed in the last 10 years thanks to the introduction of novel agents, which, combined with the backbone of classical chemotherapy, have led to a significant improvement in disease control. Bortezomib is the first reversible proteasome inhibitor approved for the treatment of MM, with wide synergism in vitro and in vivo with a plethora of drugs active for MM. In patients eligible for autologous stem cell transplantation (ASCT), the achievement of complete response or very good partial response before ASCT is associated with prolonged progression-free and overall survival. Thus, the goal of induction regimens should include, at least for younger patients, a continued improvement of the quality and depth of the achieved response. This article is focused on reviewing the major efforts in frontline therapy for MM, including bortezomib-containing induction regimens in patients either eligible or ineligible for ASCT.

Background: A combination of bortezomib (1.3 mg/m(2)), melphalan (5 mg/m(2)), and dexamethasone (40 mg) (BMD), with all three drugs given as a contemporary intravenous administration, was retrospectively evaluated., Patients and Methods: Fifty previously treated (median 2 previous lines) patients with myeloma (33 relapsed and 17 refractory) were assessed. The first 19 patients were treated with a twice-a-week (days 1, 4, 8, 11, 'base' schedule) administration while, in the remaining 31 patients, the three drugs were administered once a week (days 1, 8, 15, 22, 'weekly' schedule)., Results: Side-effects were predictable and manageable, with prominent haematological toxicity, and a better toxic profile in 'weekly' schedule (36% versus 66% in 'base' schedule). The overall response rate was 62%. After median follow-up of 24.5 months (range 2.7-50 months), the median progression-free survival (PFS) was 21.6 with no difference between the two schedules and the median overall survival (OS) was 33.8 months. Independently from the adopted schedule, we found that also in a cohort of relapsed/refractory patients achieving at least partial remission improved PFS (35.2 versus 9 months) and OS (unreached median versus 18 months)., Conclusion: Taken together, our observations suggest that BMD is an effective regimen in advanced myeloma patients with acceptable toxicity.

The reliability of endoscopic ultrasonography (EUS) in follow-up management of gastric lymphomas has not been clearly validated. We conducted a retrospective analysis on 23 patients, 12 affected by mucosa-associated lymphoid tissue (MALT) lymphoma, eight by diffuse large B-cell lymphoma, and three by high-grade lymphoma with low-grade component, all treated with a stomach-conservative approach. One hundred and twenty matched evaluations with both EUS and endoscopy with biopsy (E-Bx) were performed, according to validated guidelines and clinical judgment. At a median follow-up of 87 months ranged between 9.5 and 166 months, the analysis of progression-free survival and disease-free survival showed a strict relationship between the persistence of EUS abnormalities and the clinical outcome in patients with MALT lymphoma (p = 0.0079; p = 0.02) but not in patients with high-grade lymphoma. In conclusion, EUS evaluation does not seem reliable in follow-up management of high-grade lymphomas, although it could have a great clinical impact in the management of MALT lymphoma., (Copyright © 2011 John Wiley & Sons, Ltd.)

Disulfiram (DSF) is an aldehyde dehydrogenase inhibitor currently used for the treatment of alcoholism. Here, we show that multiple myeloma (MM) cell lines and primary cells from newly diagnosed and relapsed/resistant patients affected by MM, acute myeloid and lymphoblastic leukemia are significantly sensitive to DSF alone and in combination with copper. These effects are present at doses lower than those achievable in vivo after DSF standard administration. The cytotoxic effect achieved by this treatment is comparable to that obtained by conventional chemotherapy and is absent in normal hematopoietic cells. In addition, we found that DSF plus copper induces loss of mitochondrial membrane potential, triggers reactive oxygen species (ROS) production and activates executioner caspases. DSF-copper-induced apoptosis and caspases activation are strongly reversed by antioxidant N-acetylcysteine, thus indicating a critical role of ROS. These results might suggest the use of the old drug DSF, alone or in combination with copper, in the treatment of hematological malignancies., (Copyright © 2012 UICC.)

The BCR-ABL inhibitor imatinib is a standard first-line therapy for patients with chronic myeloid leukemia. However, it has been demonstrated that this long-term treatment is associated with altered bone metabolism. The mechanisms of this effect are not fully understood, but an inhibition of the platelet-derived growth factor receptor (PDGF-R) β axis has been suspected on the basis of some in vitro findings. We evaluated the osteoblastic differentiation of mesenchymal stem cells derived from bone marrow (hBM-MSCs) after in vitro treatment with dasatinib, nilotinib or bosutinib. Human bone marrow mesenchymal stem cells were induced to differentiate in osteoblastic cells by treatment with osteogenic medium with or without dasatinib, nilotinib or bosutinib. We found that the addition of dasatinib, and to a greater extend nilotinib, induced expression of osteogenic mRNA markers as compared with cultures with standard medium or osteogenic medium only. However, treatment with bosutinib did not induce an increase of osteogenic markers. In conclusion, we show that besides imatinib, other tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib, but not bosutinib, increase osteogenic markers in hBM-MSCs. Because bosutinib differs from the other TKIs because of its low affinity to other kinases such as PDGF-R, these experiments suggest that inhibition of PDGF-R may be involved in the induction of osteoblastogenesis by TKIs., (Copyright © 2011 John Wiley & Sons, Ltd.)

Hematopoietic stem cells transplantation has been successfully used in the treatment of patients with hematological malignances. A better knowledge of the mechanisms beyond their ability to completely repopulate the entire hematopoietic system would help in the treatment of hematological diseases. For this reason we focused our studies on a cell population that has been demonstrated to have some peculiar characteristics among the stem cells: CD34+KDR+ cells. These cells, an extremely rare population among the CD34 (0.1%-0.5%) cells, have been demonstrated from different groups to have the potential to give rise to the hematopoietic and endothelial lineage. By a subtraction library approach we found different sequences more expressed in CD34+KDR+ than their CD34+KDR- counterpart. In particular, we found an open reading frame correspondent to a newly characterized E3 ligase, MARCH-I. This gene is part of a recently described family involved in immune response modulation through the proteosomal mediated degradation. MARCH-I expression in stem cells could be important for their intrinsic immune properties., (Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

BCR/ABL positive cells are known to be resistant to DNA damage induced by chemotherapy while they are sensitive to imatinib (IM), a tyrosine kinase inhibitor (TKI). To evaluate whether this drug can increase the activity of cytotoxic drugs on BCR/ABL positive cells, we measured the toxicity of cytosine arabinoside (ARA-C), hydroxyurea (HU) and melphalan (MEL), after a pretreatment of 24 h with IM on K562 cell line. The highest cytotoxic effect was seen when the TKI was followed by MEL; our results indicate that inhibition of BCR/ABL activity by IM increased the cytotoxicity of MEL by favoring the DNA damage induced by this drug and by shortening the time for DNA repair at the G2/M checkpoint. A stronger activation of some genes involved in both intrinsic and extrinsic apoptotic pathways was also observed with IM/MEL combination compared to IM or MEL alone. The drugs association was further tested in a type of BaF3 cells (TonB.210) where the BCR-ABL expression is inducible by doxycycline; in this model it was confirmed that a reduction of BCR/ABL activity resulted in an increased susceptibility to the cytotoxic effect of MEL. Furthermore, we studied the effect of IM/MEL treatment on the proliferative potential of myeloid progenitors of six CML patients at diagnosis. The analysis of CFU-GM and BFU-E colonies demonstrated that the IM/MEL combination was more effective than IM alone in reducing the overall number of colonies and the number of copies of BCR/ABL. In conclusion, our work shows that inhibition of BCR/ABL activity increases the toxicity of MEL and allows an efficient killing of leukemic cells, suggesting that a clinical development of this approach could have therapeutic advantages for CML patients., (© 2011 John Wiley & Sons A/S.)

Several reports demonstrated that the activation of Nuclear Factor-kappa B NF-κB is essential for the pathogenesis of multiple myeloma (MM). We analyzed the nuclear localization of NF-κB in MM-cells derived from 60 different patients with MM at presentation and in relapse, as well as in three myeloma cell lines. Nuclear localization (the active form) of NF-κB was detected in only one MM-sample from a refractory patient and in two samples from relapsed patients, while all the other samples, including the MM-cell lines, almost exclusively express the cytoplasmic (inactive) form of NF-κB. In mesenchymal cells from MM-patients NF-κB was clearly present in the nucleus. In addition, the proteasome inhibitor Bortezomib, which is described to antagonize NF-κB activity, had a consistent antitumor activity against both chemoresistant and chemosensitive MM-cells, regardless the NF-κB localization, thus suggesting the existence of other molecular targets of proteasome inhibitors in MM., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A primary pleural marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT) is a very rare eventuality. Here, we report a rare case of MALT lymphoma arising in the pleura and update the literature on this topic. A 74-year-old female was hospitalized for persistent cough and weakness. A chest radiograph and total-body CT scan showed only large right-sided pleural effusion, and the coexistence of pleural thickening. Video-assisted thoracoscopic exploration and a talc pleurodesis were performed and microscopic and immunohistochemical findings showed that the tumor was a pleural MALT lymphoma. The patient received immunotherapy with Rituximab and obtained a good response that lasted 2 years. To the best of our knowledge, only seven cases of primary pleural MALT lymphoma have been documented until recently, mostly from Japan with a mean age for all patients of 60.5 years. The pathogenesis of MALT lymphomas remains unclear, although a possible chronic antigenic stimulation by microbial pathogens and/or autoantigens has been hypothesized. Surgical resection was performed in most cases, and some patients received postoperative chemotherapy or immunotherapy. The clinicopathologic characteristics and management of this extremely rare disease are also discussed.

Chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL) share many features and their differential diagnosis may be challenging, especially when a leukemic picture alone is present. Monoclonal antibody panels are often useful, with CD23 being the most reliable. However, MCL diagnosis should be confirmed by immunohistochemical cyclin D1 detection, sometimes with equivocal or even negative results. Other cytofluorimetric, cytogenetics or molecular techniques are reliable but not widely available. B-CLL leukemic cells express CD200, a membrane glycoprotein belonging to the immunoglobulin superfamily. We investigated its expression on fresh neoplastic cells of 93 patients with a CD5+ lymphoproliferative disease (79 selected B-CLL and 14 MCL in leukemic phase). Although these data cannot be generalized, all B-CLL samples we examined were positive, with CD200 present on the vast majority of the cells while, in MCL patients, CD200 was expressed by a small minority of CD5+ cells in three subjects and totally absent in the remaining 11. We then examined CD200 expression on paraffin-embedded lymphoid tissues and bone marrow (BM) trephine biopsies from 23 B-CLL and 44 MCL patients. Again, all B-CLL cells were CD200+ both in lymph nodes and in BM while all MCL cells were negative. Adding CD200 in routine panels could be of diagnostic utility in excluding MCL diagnosis.

Imatinib mesylate (IM), a tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML), may also affect the growth of other cellular systems besides CML cells. Because it has been reported that IM may affect bone tissue remodeling, we evaluated the effects of IM on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). After 21 days of culture, hBM-MSCs treated with IM (1 microM) alone or osteogenic medium (OM) + IM showed changes in morphology with evidence of extracellular mineralization and increased mRNA expression of osteogenic markers, such as RUNX2, osteocalcin (OCN), and bone morphogenetic protein (BMP-2). We also observed that levels of OCN and the osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL) ratio (OPG/RANKL ratio) were increased in the surnatant of the 21-day culture with IM or OM + IM compared to controls (p<0.005). In addition, we found that in 46 serum samples collected from CML patients treated with IM for 3 to 24 months, the OPG/RANKL ratio increased after 3 and 6 months (p<0.004) returning back to the basal level after 24 months of IM treatment. In these patients, OCN levels were low at diagnosis but they increased throughout the IM treatment, approaching normal levels at 24 months of IM therapy. In summary, our data show that IM increases mRNA expression of osteogenic markers in hBM-MSCs and increases the OPG/RANKL ratio and the OCN levels both in surnatant of hBM-MSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favors osteoblastogenesis.

Resistance to chemotherapy-induced cell death represents a major obstacle in the treatment of acute myeloid leukemia. APRIL (A Proliferation Inducing Ligand) is a member of the tumor necrosis factor superfamily that plays a key role in normal B-cell development, while promoting survival and proliferation of malignant B cells. We investigated APRIL expression and activity in acute myeloid leukemia. We found that APRIL mRNA and protein, including the secreted form, are expressed in leukemic cells of patients with M0, M2 and M4 acute myeloid leukemia subtypes but not in normal hematopoietic progenitors. Retrovirus-mediated APRIL expression in normal hematopoietic progenitors confers resistance to chemotherapeutic drugs-induced apoptosis. Conversely, blocking APRIL function by recombinant soluble APRIL receptors increased chemotherapeutic drugs-induced cell adeath in acute myeloid leukemia cells. These results indicate that APRIL acts in an autocrine fashion to protect acute myeloid leukemia cells from drug-induced death and foresee a therapeutic potential of APRIL antagonists in the treatment of acute myeloid leukemia.

Background: PTX is one of the most widely used drug in oncology due to its high efficacy against solid tumors and several hematological cancers. PTX is administered in a formulation containing 1:1 Cremophor EL (polyethoxylated castor oil) and ethanol, often responsible for toxic effects. Its encapsulation in colloidal delivery systems would gain an improved targeting to cancer cells, reducing the dose and frequency of administration., Methods: In this paper PTX was loaded in PLGA NS. The activity of PTX-NS was assessed in vitro against thyroid, breast and bladder cancer cell lines in cultures. Cell growth was evaluated by MTS assay, intracellular NS uptake was performed using coumarin-6 labelled NS and the amount of intracellular PTX was measured by HPLC., Results: NS loaded with 3% PTX (w/w) had a mean size < 250 nm and a polydispersity index of 0.4 after freeze-drying with 0.5% HP-Cyd as cryoprotector. PTX encapsulation efficiency was 30% and NS showed a prolonged drug release in vitro. An increase of the cytotoxic effect of PTX-NS was observed with respect to free PTX in all cell lines tested., Conclusion: These findings suggest that the greater biological effect of PTX-NS could be due to higher uptake of the drug inside the cells as shown by intracellular NS uptake and cell accumulation studies.

Cancer stem cells, previously identified in acute leukemias, have now been isolated from several human malignancies, such as breast, brain and gastrointestinal tumors. The origin of these cells is still questionable, but a growing body of evidence supports the idea that they are responsible for tumor growth and maintenance. For this reason, they have become a primary subject of investigation with a view to developing novel therapies that might lead to the eradication of neoplastic diseases. As previously demonstrated, cancer stem cells in haematological diseases have biological properties similar to those of normal adult stem cells. Therefore, better knowledge of the apoptotic machinery in stem cells might help in the development of targeted therapy for treating cancers. In this review, we discuss some concepts relating to stem cell biology, focusing on the apoptotic responses of haematopoietic and neural stem cells to conventional and novel cancer treatment strategies.

Lung carcinoma is often incurable and remains the leading cancer killer in both men and women. Recent evidence indicates that tumors contain a small population of cancer stem cells that are responsible for tumor maintenance and spreading. The identification of the tumorigenic population that sustains lung cancer may contribute significantly to the development of effective therapies. Here, we found that the tumorigenic cells in small cell and non-small cell lung cancer are a rare population of undifferentiated cells expressing CD133, an antigen present in the cell membrane of normal and cancer-primitive cells of the hematopoietic, neural, endothelial and epithelial lineages. Lung cancer CD133(+) cells were able to grow indefinitely as tumor spheres in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. The injection of 10(4) lung cancer CD133(+) cells in immunocompromised mice readily generated tumor xenografts phenotypically identical to the original tumor. Upon differentiation, lung cancer CD133(+) cells acquired the specific lineage markers, while loosing the tumorigenic potential together with CD133 expression. Thus, lung cancer contains a rare population of CD133(+) cancer stem-like cells able to self-renew and generates an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may provide valuable information to be exploited in the clinical setting.

Acute myeloid leukemia (AML) is a malignant disease characterized by abnormal proliferation of clonal precursor cells. Although different strategies have been adopted to obtain complete remission, the disease actually progresses in about 60-70% of patients. Bortezomib has been used in multiple myeloma and other lymphoid malignancies because of its antitumor activity. Here we examined the sensitivity of bone marrow cells from AML patients (34 patients: 25 newly diagnosed, 4 relapsed, 5 refractory) to bortezomib alone or in combination with TRAIL, a member of the TNF family that induces apoptosis in tumor cells while sparing normal cells. Bortezomib induced cell death in blasts from each patient sample. The cytotoxic effect was dose- and time-dependent (concentration from 0.001 to 10 microM for 24 and 48 h) and was associated with a downregulation of Bcl-xL and Mcl-1, an upregulation of TRAIL-R1, TRAIL-R2, p21, activation of executioner caspases and a loss of the mitochondrial membrane potential. Moreover, low doses of bortezomib primed TRAIL-resistant AML cells for enhanced TRAIL-mediated killing. These results suggest that a combination of proteasome inhibitors and TRAIL could be effective for treating AML patients, even patients who are refractory to conventional chemotherapy., (Copyright 2008 S. Karger AG, Basel.)

Context: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive types of cancer characterized by complete refractoriness to multimodal treatment approaches. Therapeutic strategies based on the simultaneous use of proteasome inhibitors and death receptor ligands have been shown to induce apoptosis in several tumor types but have not yet been explored in ATC., Objective and Methods: The aim of this study was to investigate the ability of the proteasome inhibitor Bortezomib to induce apoptosis in ATC cell lines. Bortezomib was used as a single agent or in combination with TNF-related apoptosis-induced ligand (TRAIL), a member of the TNF family that selectively induces tumor cell apoptosis. The molecular effects of Bortezomib were investigated by analyzing the expression of key regulators of cell cycle and apoptosis and the activation of different apoptotic pathways., Results: Bortezomib induced apoptosis in ATC cells at doses achieved in the clinical setting, differently from conventional chemotherapeutic agents. Simultaneous treatment with low doses of Bortezomib and TRAIL had a synergistic effect in inducing massive ATC cell apoptosis. Bortezomib increased the expression of cytotoxic TRAIL receptors, p21 (WAF/CIP1) and proapoptotic second mitochondria-derived activator of caspases/direct inhibitor of apoptosis binding protein with low pI, and reduced the expression of antiapoptotic mediators such as cellular Fas-associated death domain-like IL-1beta converting enzyme inhibitory protein, Bcl-2, Bcl-X(L), and inhibitor of apoptosis-1, thus resulting in cell death induction through the mitochondrial apoptotic pathway., Conclusions: The combination of proteasome inhibitors and TRAIL synergizes to induce the destruction of chemoresistant neoplastic thyrocytes and could represent a promising therapeutic strategy for the treatment of anaplastic thyroid carcinoma.

IFN-gamma inhibits the growth and differentiation of erythroid precursor cells and mediates hemopoietic suppression through mechanisms that are not completely understood. We found that treatment of human erythroid precursor cells with IFN-gamma up-regulates the expression of multiple members of the TNF family, including TRAIL and the recently characterized protein TWEAK. TWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) were expressed by purified erythroblasts at all the stages of maturation. Exposure to recombinant TWEAK or agonist anti-Fn14 Abs was able to inhibit erythroid cell growth and differentiation through caspase activation. Because other members of the TNF family such as TRAIL and CD95 ligand (CD95L) are known to interfere with erythroblast growth and differentiation, we investigated the role of different TNF/TNFR family proteins as potential effectors of IFN-gamma in the immature hemopoietic compartment. Treatment of erythroid precursor cells with agents that blocked either TRAIL, CD95L, or TWEAK activity was partially able to revert the effect of IFN-gamma on erythroid proliferation and differentiation. However, the simultaneous inhibition of TRAIL, TWEAK, and CD95L resulted in a complete abrogation of IFN-gamma inhibitory effects, indicating the requirement of different receptor-mediated signals in IFN-gamma-mediated hemopoietic suppression. These results establish a new role for TWEAK and its receptor in normal and IFN-gamma-mediated regulation of hematopoiesis and show that the effects of IFN-gamma on immature erythroid cells depend on multiple interactions between TNF family members and their receptors.

We recently proposed that Th1 and Th2 cytokines exert opposite effects on the pathogenesis and clinical outcome of organ-specific autoimmunity by altering the expression of genes involved in target cell survival. Because a Th2 response against tumors is associated with poor prognosis, we investigated the ability of IL-4 to protect tumor cells from death receptor- and chemotherapy-induced apoptosis. We found that IL-4 treatment significantly reduced CD95 (Fas/APO-1)- and chemotherapeutic drug-induced apoptosis in prostate, breast, and bladder tumor cell lines. Analysis of antiapoptotic protein expression revealed that IL-4 stimulation resulted in up-regulation of cellular (c) FLIP/FLAME-1 and Bcl-x(L). Exogenous expression of cFLIP/FLAME-1 inhibited apoptosis induced by CD95 and to a lesser extent by chemotherapy, while tumor cells transduced with Bcl-x(L) were substantially protected both from CD95 and chemotherapeutic drug stimulation. Moreover, consistent IL-4 production and high expression of both cFLIP/FLAME-1 and Bcl-x(L) were observed in primary prostate, breast, and bladder cancer in vivo. Finally, primary breast cancer cells acquired sensitivity to apoptosis in vitro only in the absence of IL-4. Thus, IL-4 protects tumor cells from CD95- and chemotherapy-induced apoptosis through the up-regulation of antiapoptotic proteins such as cFLIP/FLAME-1 and Bcl-x(L). These findings may provide useful information for the development of therapeutic strategies aimed at restoring the functionality of apoptotic pathways in tumor cells.

CD95 (Fas/Apo-1) is a broadly expressed death receptor involved in a variety of physiological and pathological apoptotic processes. Since its discovery, defects in CD95/CD95L system have been proposed as major pathogenic factors responsible for impaired immunological tolerance to self antigens and autoimmunity. Later, analysis of altered sensitivity to CD95-induced apoptosis in cells targeted by the immune response has revealed an unexpected role for CD95 and CD95L in organ-specific autoimmunity. CD95 has been shown to be expressed and functional in virtually all cell types that are target of the organ-specific autoimmune response. Here we review some of the major findings concerning the role of CD95 in autoimmunity, in dysfunctions due to increased or decreased CD95-induced apoptosis.