Immunosuppressive Treg cells acquire the phenotype of effector-T cells in chronic lymphocytic leukemia patients.

BACKGROUND: In chronic lymphocytic leukemia (CLL) disease onset and progression are influenced by the behavior of specific CD4+ T cell subsets, such as T regulatory cells (Tregs). Here, we focused on the phenotypic and functional characterization of Tregs in CLL patients to improve our understanding of the putative mechanism by which these cells combine immunosuppressive and effector-like properties. METHODS: Peripheral blood mononuclear cells were isolated from newly diagnosed CLL patients (n = 25) and healthy volunteers (n = 25). The phenotypic and functional characterization of Tregs and their subsets was assessed by flow cytometry. In vitro analysis of TH1, TH2, TH17 and Tregs cytokines was evaluated by IFN-γ, IL-4, IL-17A and IL-10 secretion assays. The transcriptional profiling of 84 genes panel was evaluated by RT2 Profiler PCR Array. Statistical analysis was carried out using exact non parametric Mann-Whitney U test. RESULTS: In all CLL samples, we found a significant increase in the frequency of IL-10-secreting Tregs and Tregs subsets, a significant rise of TH2 IL-4+ and TH17 IL-17A+ cells, and a higher percentage of IFN-γ/IL-10 and IL-4/IL-10 double-releasing CD4+ T cells. In addition, we also observed the up-regulation of innate immunity genes and the down-regulation of adaptive immunity ones. CONCLUSIONS: Our data show that Tregs switch towards an effector-like phenotype in CLL patients. This multifaceted behavior is accompanied by an altered cytokine profiling and transcriptional program of immune genes, leading to a dysfunction in immune response in the peripheral blood environment of CLL patients.

Pentoxifylline-Induced Apoptosis in Chronic Lymphocytic Leukemia: New Insights into Molecular Mechanism.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is an indolent B-lineage neoplasm, characterized by clonal expansion of CD5 positive B cells with constitutive activation of survival pathways including NF-kB. Pentoxifylline, a xanthine-derivative compound indicated for the treatment of microvascular disturbancies, has been suggested to have anti-proliferative and anti-metastatic activities in various types of cancer. In the present study we extend these data showing one of the potential molecular mechanisms through which Pentoxifylline may promote apoptosis in CLL clonal lymphocytes. METHODS: Peripheral blood mononuclear cells were isolated from 15 CLL patients 0 RAI stage and 15 healthy volunteers and treated for 24 and 48 hours with Pentoxifylline. Apoptosis induction was evaluated through Annexin V and TUNEL assays. Mitochondrial membrane potential depolarization analysis, active Caspase-3 assay, reactive oxygen species generation and Western Blot were assessed to further investigate the alterations induced by Pentoxifylline. RESULTS: We observed a statistically significant occurrence of apoptosis, DNA fragmentation and active Caspase-3 in lymphocytes from CLL patients compared to healthy volunteers after 48 hours of Pentoxifylline treatment. To clarify the molecular mechanism of the drug, we also evaluated the expression levels of NF-kB/p65 and its related proteins. In treated CLL cells, NF-kB/p65 was significantly decreased in comparison to normal cells, whereas we observed a less marked reduction of Bcl-2 expression. The treatment also induced a decrease of Mcl-1 in CLL cells with a greater down-regulation of the anti-apoptotic alternatively spliced isoform. CONCLUSION: These findings showed that Pentoxifylline induced apoptosis in leukemic cells through a molecular mechanism that involves the NF-kB signaling.

IL-17/IL-10 double-producing T cells: new link between infections, immunosuppression and acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is an incurable disease with fatal infections or relapse being the main causes of death in most cases. In particular, the severe infections occurring in these patients before or during any treatment suggest an intrinsic alteration of the immune system. In this respect, IL-17-producing T helper (Th17) besides playing a key role in regulating inflammatory response, tumor growth and autoimmune diseases, have been shown to protect against bacterial and fungal pathogens. However, the role of Th17 cells in AML has not yet been clarified. METHODS: T cell frequencies were assessed by flow cytometry in the peripheral blood of 30 newly diagnosed AML patients and 30 age-matched healthy volunteers. Cytokine production was determined before and after culture of T cells with either Candida Albicans or AML blasts. Statistical analyses were carried out using the paired and unpaired two-tailed Student's t tests and confirmed with the non parametric Wilcoxon signed-rank test. RESULTS: A strong increase of Th17 cells producing immunosuppressive IL-10 was observed in AML patients compared with healthy donors. In addition, stimulation of AML-derived T cells with a Candida albicans antigen induced significantly lower IFN-γ production than that observed in healthy donors; intriguingly, depletion of patient Th17 cells restored IFN-γ production after stimulation. To address the role of AML blasts in inducing Th17 alterations, CD4+ cells from healthy donors were co-cultured with CD33+ blasts: data obtained showed that AML blasts induce in healthy donors levels of IL-10-producing Th17 cells similar to those observed in patients. CONCLUSIONS: In AML patients altered Th17 cells actively cause an immunosuppressive state that may promote infections and probably tumor escape. Th17 cells could thus represent a new target to improve AML immunotherapy.

Lenalidomide monotherapy in heavily pretreated patients with non-Hodgkin lymphoma: an Italian observational multicenter retrospective study in daily clinical practice.

Clinical trial results indicate that lenalidomide, an immunomodulatory drug, is a promising treatment in relapsed/refractory non-Hodgkin lymphoma (NHL). This retrospective multicenter study was conducted in patients with relapsed/refractory NHL treated with lenalidomide monotherapy through a Named Patient Program in Italy. Principal endpoints were overall response rate (ORR), safety and overall survival (OS). The ORR in 64 evaluable patients was 42.2% and was similar among patients receiving 10, 15 or 25 mg/day lenalidomide. Response rates in patients with mantle cell, diffuse large B-cell and follicular lymphoma were 45.5%, 42.1% and 20%, respectively. Among patients who responded to most recent prior therapy, ORR was 50.0% versus 36.8% in patients with refractory NHL. Mean duration of response in patients receiving any lenalidomide dose was 10.5 months; 1-year progression-free survival and OS were 50.3% and 82.6%, respectively. These findings suggest that lenalidomide is effective and safe for heavily pretreated patients with NHL in the clinical setting.

Lenalidomide plus R-CHOP21 in elderly patients with untreated diffuse large B-cell lymphoma: results of the REAL07 open-label, multicentre, phase 2 trial.

BACKGROUND: Up to 40% of elderly patients with untreated diffuse large B-cell lymphoma (DLBCL) given a regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone every 21 days (R-CHOP21) relapse or develop refractory disease. Lenalidomide has high activity in relapsed or refractory aggressive B-cell lymphomas. In phase 2 of the REAL07 trial, we aimed to establish the safety and efficacy of the combination of lenalidomide and R-CHOP21 in elderly patients with untreated DLBCL. METHODS: REAL07 was an open-label, multicentre trial that was done in 13 centres in Italy and one in Germany. Eligible patients were aged 60-80 years; had newly diagnosed, untreated, CD20-positive, Ann Arbor stage II-IV DLBCL or grade 3b follicular lymphoma; had an Eastern Cooperative Oncology Group performance status of 0-2; had an International Prognostic Index (IPI) risk of low-intermediate, intermediate-high, or high; and were fit according to comprehensive geriatric assessment. Participants were to receive 15 mg oral lenalidomide on days 1-14 of six 21-day cycles, and standard doses of R-CHOP21 chemotherapy (375 mg/m(2) intravenous rituximab, 750 mg/m(2) intravenous cyclophosphamide, 50 mg/m(2) intravenous doxorubicin, and 1·4 mg/m(2) intravenous vincristine on day 1, and 40 mg/m(2) oral prednisone on days 1-5). The primary endpoint was frequency of overall response (complete response [CR] and partial response [PR]), which was assessed by (18)F-fluorodeoxyglucose ((18)F-FDG) PET at the end of the treatment. Analyses were by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00907348. FINDINGS: 49 patients were included in phase 2: nine had been enrolled into phase 1 between Oct 23, 2008, and June 4, 2009, and had received the maximum tolerated dose of 15 mg lenalidomide; and 40 were enrolled into phase 2 between April 28, 2010, and June 3, 2011. 45 patients (92%, 95% CI 81-97) achieved a response (42 [86%] CR; three [6%] PR). Three patients (6%) did not respond and one (2%) died for reasons unrelated to treatment or disease. 277 (94%) of 294 planned cycles of lenalidomide and R-CHOP21 were completed. Grade 3-4 neutropenia was reported in 87 cycles (31%), grade 3-4 leukopenia in 77 (28%), and grade 3-4 thrombocytopenia in 35 (13%). No grade 4 non-haematological adverse events were reported. No patients died during the study as a result of toxic effects. INTERPRETATION: Lenalidomide with R-CHOP21 is effective and safe in elderly patients with untreated DLBCL. FUNDING: Fondazione Italiana Linfomi and Celgene.

Prolonged 18FDG-PET negative complete remission in a heavily pretreated, elderly patient with diffuse large B cell lymphoma treated with lenalidomide, low dose dexamethasone, and colony stimulating factor (Rd-G).

Diffuse large B-cell lymphoma (DLBCL) is a common lymphoid malignancy among adults in the developed world and accounts for about a third of all patients newly diagnosed with non-Hodgkin lymphoma each year. The prognosis of patients with DLBCL has improved over the past 10 years since the advent of chemoimmunotherapy regimens such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone). However, a significant number of patients still experience disease relapse or progression after first or second line therapy, and ~40% of patients will die within 5 years. In particular, elderly patients and those ineligible for high-dose chemotherapy due to comorbidities require effective salvage treatment options with favorable toxicity profile. Several novel therapeutic approaches have been proposed for these patients including monoclonal antibodies, radioimmunotherapy, proteasome inhibitors, mTOR inhibitors, and the immunomodulatory drugs such as thalidomide and lenalidomide.

Malignant pericardial mesothelioma. Report of two cases, review of the literature and differential diagnosis.

Malignant pericardial mesothelioma is an uncommon variety of a primary malignant cardio-pericardial tumor and it is a highly lethal and fortunately rare cardiac neoplasm. The presentation of pericardial mesothelioma is aspecific and pathologically mesothelioma is not the most common among primary tumors of the pericardium. It is characterized by atypical solid growth of mesothelium with formation of atypical cavities surrounded by fibrous stroma. Antemortem diagnosis is difficult and distant metastases are extremely rare. Radical surgery can be used to treat localized mesothelioma. The treatment for advanced primary pericardial mesothelioma is usually palliative because the tumor is resistant to radiotherapy and chemotherapy. The prognosis is unfavorable. The median survival from the onset of symptoms is six months. In this paper we report two cases of patients with primary mesothelioma of the pericardium without a definite history of asbestos exposure.

[Chemotherapy in extended small-cell lung cancer. Retrospective analysis of two different series of patients treated with carboplatin and etoposide or ciclophosphamide-epidoxorubicin and etoposide]. / La chemioterapia del microcitoma disseminato. Confronto tra carboplatino-etoposide e ciclofosfamide-epirubicina-etoposide.

Although cisplatin and etoposide seem to represent the treatment of choice in Small-Cell Lung Cancer, a lot of data exist in literature supporting both the use of anthracycline-containing regimens and the use of alternating regimens where platinum-containing regimens and anthracycline-containing regimens are alternatively used as first line in the same patient. In our paper we review the outcomes of two different series of patients treated with ciclophosphamide-epidoxorubicin-etoposide (CEVP16) or carboplatin-etoposide (CBE) for extended Small-Cell Lung Cancer. Sixty-three patients (53.4%) were treated with CEVP16 and 55 patients (46.6%) with CBE. Response Rate (complete plus partial responses) was greater in patients treated with CEVP16 (49.2%) when compared with the response rate in patients treated with CBE (30.9%) (p=0.04 using the Chi-Square test); no differences were observed in the median time to progression (235 vs 199 days, using the Log-Rank test). Overall survival was greater in the CEVP16 group when compared with the CBE one (281 vs 208 days and 35.6% vs 16.3% of patients alive after 2 years of follow up for CEVP16 and CBE respectively, p=0.02 using the Log-Rank test). Although our data present all the methodological limits of the "case-series", it is interesting to observe how an anthracycline-containing regimen seems to be more effective than a platinum-containing one and how it could still play a role in the treatment of extended Small-Cell Lung Cancer.

Fludarabine plus mitoxantrone with and without rituximab versus CHOP with and without rituximab as front-line treatment for patients with follicular lymphoma.

PURPOSE: Promising new therapeutic options for follicular lymphoma (FL) include fludarabine plus mitoxantrone (FM) and the mouse/human anti-CD20 antibody, rituximab. We performed a randomized comparative trial of FM with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) front-line chemotherapy with and without sequential rituximab. PATIENTS AND METHODS: All previously untreated CD20(+) FL patients presenting in 15 Italian cooperative institutions from October 1999 were randomly allocated to FM or CHOP. Following clinical or molecular restaging, patients in complete remission (CR) with bcl-2/IgH negativity (CR(-)) received no further treatment; those in CR with bcl-2/IgH positivity (CR(+)) received rituximab, as did those in partial remission (PR) with bcl-2/IgH negativity (PR(-)) or positivity (PR(+)); nonresponders (NR subgroup) were off study. RESULTS: After chemotherapy, the FM arm achieved higher rates of CR (68% [49 of 72 patients] v 42% [29 of 68 patients]; P =.003) and CR(-) (39% [28 of 72 patients] v 13 of 68 patients [19%]; P =.001). Rituximab elicited CR(-) in 55 of 95 treated patients (58%). The final CR(-) rate was higher in the FM arm (71% [51 of 72 patients] v 51% [35 of 68 patients]; P =.01). However, with a median follow-up of 19 months (range, 9 to 37 months), no statistically significant difference was found among the various study arms in terms of both progression-free (PFS) and overall survival (OS). CONCLUSION: These results indicate that FM is superior to CHOP for front-line treatment of FL and that rituximab is an effective sequential treatment option. However, they also confirm that this superiority is unlikely to translate into either better PFS or OS.

Carboplatin and gemcitabine in the palliative treatment of stage IV non-small cell lung cancer: definitive results of a phase II trial.

BACKGROUND: Cisplatin-containing regimens represent the gold standard in the treatment of advanced non-small cell lung cancer, but carboplatin is often preferred for its better toxic profile when palliation is the aim of the treatment. The synergistic effect and tolerability of carboplatin-gemcitabine combination are well known. In this phase II trial, we evaluated the activity and safety of a schedule with carboplatin and gemcitabine, defined in our previous phase I trial. METHODS: Thirty-seven patients with measurable stage IV non-small cell lung cancer were treated with carboplatin, AUC 4.5 mg/ml/min on day 1, and gemcitabine, 800 mg/m2 on days 1 and 8, every 21 days. All patients were treated until disease progression or intractable toxicity and were evaluated before each course of chemotherapy for toxicity and after every 3 courses for response. RESULTS: After a median follow-up of over 10 months, complete response, partial response, and stabilization of the disease were observed in 3 (8.1%), 9 (24.3%), and 15 patients (40.5%), respectively. Median time to progression was 7 months. At this writing, 27 patients have died, with a median survival of 10 months, and 29 (78.3%), 16 (43.2%), and 11 (29.7%) patients are alive after 6, 12, and 15 months of follow-up, respectively. Toxicity was mild, and mainly hematological, with a significant correlation with the number of courses of chemotherapy (P = 0.0003). CONCLUSIONS: Our results are comparable with those reported in the literature and confirm the good activity and tolerability of the carboplatin-gemcitabine combination. Up to 4 courses of chemotherapy with carboplatin and gemcitabine may represent an interesting option in the palliative treatment of non-small cell lung cancer.

Proangiogenic properties of human myeloma cells: production of angiopoietin-1 and its potential relationship to myeloma-induced angiogenesis.

Patients with multiple myeloma (MM) have increased bone marrow (BM) angiogenesis; however, the proangiogenic properties of myeloma cells and the mechanisms of MM-induced angiogenesis are not completely clarified. The angiopoietin system has been identified as critical in the regulation of vessel formation. In this study we have demonstrated that myeloma cells express several proangiogenic factors, and, in particular, we found that angiopoietin-1 (Ang-1), but not its antagonist Ang-2, was expressed by several human myeloma cell lines (HMCLs) at the mRNA and the protein levels. In a transwell coculture system, we observed that myeloma cells up-regulated the Ang-1 receptor Tie2 in human BM endothelial cells. Moreover, in an experimental model of angiogenesis, the conditioned medium of HMCLs significantly stimulated vessel formation compared with control or vascular endothelial growth factor (VEGF) treatment. The presence of anti-Tie2 blocking antibody completely blunted the proangiogenic effect of XG-6. Finally, our in vitro results were supported by the in vivo finding of Ang-1, but not Ang-2, mRNA and protein expression in purified MM cells obtained from approximately 47% of patients and by high BM angiogenesis in patients with MM positive for Ang-1, suggesting that the angiopoietin system could be involved, at least in part, in MM-induced angiogenesis.

Induction chemotherapy strategies for primary mediastinal large B-cell lymphoma with sclerosis: a retrospective multinational study on 426 previously untreated patients.

BACKGROUND AND OBJECTIVES: This multinational retrospective study compares the outcomes of patients with primary mediastinal large B-cell lymphoma (PMLBCL) with sclerosis after first-generation (dose-intensive regimens), third-generation (alternating regimens) and high-dose chemotherapy strategies, frequently with adjuvant radiation therapy. DESIGN AND METHODS: Between August 1981 and December 1999, a total of 426 previously untreated patients with confirmed diagnosis were enrolled in 20 institutions to receive combination chemotherapy with either first generation (CHOP or CHOP-like) regimens, third generation (MACOP-B, VACOP-B, ProMACE CytaBOM) regimens or high-dose chemotherapy (HDS/ABMT). RESULTS: With chemotherapy, complete response (CR) rates were 49% (50/105), 51% (142/277) and 53% (23/44) with first generation, third generation and high-dose chemotherapy strategies, respectively; partial response (PR) rates were 32%, 36% and 35%, respectively. All patients who achieved CR and 124/142 (84%) with PR had radiation therapy on the mediastinum. The final CR rates became 61% for CHOP/CHOP-like regimens, 79% for MACOP-B and other regimens, and 75% for HDS/ABMT. After median follow-ups from attaining CR of 48.5 months for CHOP/CHOP-like regimens, 51.7 months for MACOP-B type regimens and 32.4 months for HDS/ABMT, relapses occurred in 15/64 (23%), 27/218 (12%) and 0/33 (0%) patients, respectively. Projected 10-year progression-free survival rates were 35%, 67% and 78%, respectively (p=0.0000). Projected 10-year overall survival rates were 44%, 71% and 77%, respectively (p=0.0000), after median follow-ups from diagnosis of 52.3 months, 54.9 months and 35.8 months, respectively. INTERPRETATION AND CONCLUSIONS: In patients with PMLBCL with sclerosis, MACOP-B plus radiation therapy may be a better strategy than other treatments; these retrospective data need to be confirmed by prospective studies. The encouraging survival results after high dose chemotherapy require confirmation in selected high-risk patients.

Adjuvant chemotherapy in gastric cancer: a meta-analysis of randomized trials and a comparison with previous meta-analyses.

AIMS AND BACKGROUND: Up to now adjuvant chemotherapy after curative resection for gastric cancer (GC) has been considered an experimental approach. The results of existing phase III randomized trials comparing chemotherapy with control after surgery are controversial. Three meta-analyses have been published in recent years. It is likely that each of them presents a theoretical bias, mainly as regards the inclusion criteria of the trials. In this article we re-examine this potential bias, highlighting the differences between the present and past meta-analyses on adjuvant chemotherapy for GC. METHODS: Only randomized controlled clinical trials comparing systemic adjuvant chemotherapy with control after radical resection of GC were eligible. Total mortality was assessed as outcome measure of the treatment effect and a pooled odds ratio was calculated using the Peto-Mantel-Haenszel method. RESULTS: After the selection process 17 papers (18 comparisons) proved eligible for inclusion in the meta-analysis with a total of 3118 patients, of whom 1546 randomized to the treatment arms and 1572 to the control arms; 762 and 871 deaths occurred in the treatment and control arms, respectively. Statistical analysis suggests an absence of significant heterogeneity between the trials and a significant advantage in survival for adjuvant chemotherapy (pooled odds ratio, 0.72, 95% Cl, 0.62-0.84). CONCLUSIONS: Our meta-analysis would seem to indicate that adjuvant chemotherapy results in a significant survival advantage in patients with GC. However, this observation undoubtedly requires confirmation in large randomized controlled trials including cisplatin before adjuvant chemotherapy after curative resection for GC can be proposed for use in clinical practice.

Staging of breast cancer: new recommended standard procedure.

BACKGROUND: Staging procedures used to detect metastatic breast cancer at the time of diagnosis are bone scan (BS), chest X-ray (CXR), liver ultrasonography (LUS) and laboratory parameters (LP). These procedures are expensive and not all patients need them. We aimed to identify groups of patients with different risks for metastatic disease. METHODS: We reviewed data from 1,218 consecutive cases of breast cancer. Pathological and biological parameters and instrumental procedures performed at the time of diagnosis and during 6 months of follow-up were recorded. True positive and negative, false positive and negative cases were evaluated. All cases were grouped on the basis of tumour size, nodal involvement, biological characteristics, menopausal status and age. RESULTS: We observed 46 (3.8%) true positive cases with metastatic disease at the time of diagnosis. Documentation relating to BS, CXR and LUS was available for 1,193, 1,206 and 1,206 patients, respectively, with 37 (3.1%), 8 (0.7%) and 10 (0.8%) true positive tests. Logistic regression analysis showed significant odds ratio estimates for pT status and nodal status, thus highlighting the role of these morphological data. These findings suggest that breast cancer patients can be divided into two subgroups: first group pT1-3N0-1. with < or = 3 involved nodes, and second group pT1-3N1 with > or = 4 involved nodes, pT4 and pN2 (metastases detection rate 1.46 and 10.68%, respectively). In the former group the appropriate procedures of staging would only be laboratory parameters, whereas in the latter group BS, CXR, LUS, LP and tumour markers CEA and CA 15.3 would.be necessary. CONCLUSIONS: The standard staging procedures to detect metastatic disease at breast cancer diagnosis require modification. On the basis of the literature data and our findings, the full staging procedure is appropriate in the second group of patients.