Effects of miRNA-15 and miRNA-16 expression replacement in chronic lymphocytic leukemia: implication for therapy.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

lncRNA profiling in early-stage chronic lymphocytic leukemia identifies transcriptional fingerprints with relevance in clinical outcome.

Long non-coding RNAs (lncRNAs) represent a novel class of functional RNA molecules with an important emerging role in cancer. To elucidate their potential pathogenetic role in chronic lymphocytic leukemia (CLL), a biologically and clinically heterogeneous neoplasia, we investigated lncRNAs expression in a prospective series of 217 early-stage Binet A CLL patients and 26 different subpopulations of normal B-cells, through a custom annotation pipeline of microarray data. Our study identified a 24-lncRNA-signature specifically deregulated in CLL compared with the normal B-cell counterpart. Importantly, this classifier was validated on an independent data set of CLL samples. Belonging to the lncRNA signature characterizing distinct molecular CLL subgroups, we identified lncRNAs recurrently associated with adverse prognostic markers, such as unmutated IGHV status, CD38 expression, 11q and 17p deletions, and NOTCH1 mutations. In addition, correlation analyses predicted a putative lncRNAs interplay with genes and miRNAs expression. Finally, we generated a 2-lncRNA independent risk model, based on lnc-IRF2-3 and lnc-KIAA1755-4 expression, able to distinguish three different prognostic groups in our series of early-stage patients. Overall, our study provides an important resource for future studies on the functions of lncRNAs in CLL, and contributes to the discovery of novel molecular markers with clinical relevance associated with the disease.

A phase II, single-arm, prospective study of bendamustine plus melphalan conditioning for second autologous stem cell transplantation in de novo multiple myeloma patients through a tandem transplant strategy.

This phase II trial evaluates, for the first time, the safety and efficacy of bendamustine plus high-dose melphalan (HDM) as a conditioning regimen before the second autologous stem cell transplantation (ASCT) in previously untreated multiple myeloma (MM) patients. In total, 32 ASCT patients received HDM (200 mg/m(2)) as conditioning for the first ASCT. After 3-6 months from the first ASCT, responding patients underwent a second ASCT following bendamustine (200 mg/m(2)) and HDM (140 mg/m(2)). High-dose chemotherapy and ASCT were performed with complete neutrophil and platelet recovery in all patients. The median number of days to neutrophil and platelet engraftment was 11 (range 9-15) and 12 (range 10-19), respectively. Only one subject experienced grade 3 diarrhea; the rate of mucositis and vomiting was significantly lower with the bendamustine plus HDM regimen compared with the HDM-only regimen (81.2 vs 96.9%, P=0.025 and 78.1 vs 100%, P=0.008). Overall response rate (ORR) was 81.2% after the first transplant, and 90.6% after the second, while complete response rates were 46.8 and 62.5%, respectively (P=0.016). Actuarial 2-year PFS and OS were 79% (95% confidence interval (CI), 60-98) and 97% (95% CI, 91-100), respectively. Bendamustine+HDM is feasible as the conditioning regimen for second ASCT in MM patients. The present study may pave the way for phase III studies specifically aimed at further investigating this combination strategy. The role of this combination in MM for conditioning regimen in a first or single ASCT setting should be also investigated.

A phase 2 study of three low-dose intensity subcutaneous bortezomib regimens in elderly frail patients with untreated multiple myeloma.

This phase 2 trial evaluated three low-dose intensity subcutaneous bortezomib-based treatments in patients ⩾75 years with newly diagnosed multiple myeloma (MM). Patients received subcutaneous bortezomib plus oral prednisone (VP, N=51) or VP plus cyclophosphamide (VCP, N=51) or VP plus melphalan (VMP, N=50), followed by bortezomib maintenance, and half of the patients were frail. Response rate was 64% with VP, 67% with VCP and 86% with VMP, and very good partial response rate or better was 26%, 28.5% and 49%, respectively. Median progression-free survival was 14.0, 15.2 and 17.1 months, and 2-year OS was 60%, 70% and 76% in VP, VCP, VMP, respectively. At least one drug-related grade ⩾3 non-hematologic adverse event (AE) occurred in 22% of VP, 37% of VCP and 33% of VMP patients; the discontinuation rate for AEs was 12%, 14% and 20%, and the 6-month rate of toxicity-related deaths was 4%, 4% and 8%, respectively. The most common grade ⩾3 AEs included infections (8-20%), and constitutional (10-14%) and cardiovascular events (4-12%); peripheral neuropathy was limited (4-6%). Bortezomib maintenance was effective and feasible. VP, VCP and VMP regimens demonstrated no substantial difference. Yet, toxicity was higher with VMP, suggesting that a two-drug combination followed by maintenance should be preferred in frail patients.

Diagnostic and prognostic role of PET/CT in patients with chronic lymphocytic leukemia and progressive disease.

In order to evaluate the predictive value of positron emission tomography-computed tomography (PET/CT) in discriminating the presence of a Richter's syndrome (RS) or a second malignancy (SM), as well as to evaluate its prognostic value in patients with chronic lymphocytic leukemia (CLL), we retrospectively analyzed the data of 90 patients who, in the suspicion of a RS or a SM, underwent PET/CT followed by the biopsy of the involved tissue. The median maximum Standardized Uptake Value (SUV max) in the presence of a CLL/small lymphocytic lymphoma, a diffuse large B-cell lymphoma (DLBCL), a Hodgkin lymphoma (HL), a SM were 3.5, 14.6, 7.0 and 6.3, respectively (P ⩽ 0.0001). A SUV max cutoff value ⩾ 5 showed a sensitivity, specificity, positive and negative predictive values of 88.2, 71.2, 51.3 and 94%, respectively, for the presence of a more aggressive disease (DLBCL, HL and SM). A SUV max ⩾ 5 identified also a subset of treatment naive patients with an inferior progression-free survival (P = 0.011) and overall survival (P = 0.067). These findings suggest that PET/CT may helpfully integrate the biologically-based prognostic stratification of CLL. Prospective clinical trials including larger cohorts of patients are needed to conclusively define the role and prognostic impact of PET/CT in the routine management of CLL patients.

Bendamustine in combination with ofatumumab in relapsed or refractory chronic lymphocytic leukemia: a GIMEMA Multicenter Phase II Trial.

We conducted a phase II, noncomparative, open-label, multicenter GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) study (CLL0809) to assess the efficacy and safety of bendamustine in combination with ofatumumab (BendOfa) in relapsed/refractory chronic lymphocytic leukemia (CLL). Forty-seven patients from 14 centers were evaluated. Therapy consisted of bendamustine (70 mg/m(2)) for 2 consecutive days every 28 days, and ofatumumab 300 mg on day 1 and 1000 mg on day 8 during the first cycle, and 1000 mg on day 1 subsequently. Treatment was administered up to six cycles. The overall response rate (ORR), as per intention-to-treat analysis, was 72.3% (95% confidence of interval (CI), 57-84%), with 17% complete responses. After a median follow-up of 24.2 months, the overall survival was 83.6% (95% CI, 73.0-95.7%) and the progression-free survival (PFS) was 49.6% (95% CI, 35.9-68.6%). The median PFS was 23.6 months. Univariate and multivariate analyses were used to identify clinical and biological characteristics associated with ORR and PFS. Myelosuppression was the most common toxicity; grade ≥3 neutropenia was observed in 61.7% of patients; however, grade ≥3 infections occurred in 6% of patients. BendOfa is feasible and effective in relapsed/refractory CLL patients, including patients with high-risk clinical and biological features.

A prognostic model based on combining estrogen receptor expression and Ki-67 value after neoadjuvant chemotherapy predicts clinical outcome in locally advanced breast cancer: extension and analysis of a previously reported cohort of patients.

BACKGROUND: Ki-67 expression has gained attention as a breast cancer prognostic factor, however its significance in the remaining malignant cells after neoadjuvant chemotherapy (NAC) has been rarely examined. This investigation, extension and analysis of a previously reported cohort of patients, evaluates the significance of Ki-67 and estrogen receptor (ER) expression after NAC in LABC (locally advanced breast cancer). PATIENTS AND METHODS: clinical stage, tumor size, clinical and pathological lymph node involvement, Ki-67, ER, progesterone receptor (PgR), HER2 expression, grading and clinical response were evaluated before and after NAC in 110 patients with LABC. Ki-67 expression was assessed both in pre and post-therapy histological samples, using >15% positive cells as cut-off value to distinguish high from low Ki-67 expressing tumors. RESULTS: six patients (5.45%) attained pCR after NAC. A significant relationship between elevated post-CT Ki-67 and ER expression was showed at Cox multivariate analysis of disease free survival (DFS). On univariate analysis high post-chemotherapy Ki-67 and ER status were associated with worse survival; at multivariate model included these results were confirmed. Based on these two parameters, a prognostic model identified two different groups: low risk (low postchemotherapy Ki-67 and ER positive, or either high post-chemotherapy Ki-67 or ER negative), and high risk (high post-chemotherapy Ki-67 and ER negative). The low risk group showed a good prognosis (median OS still not reached), while the high risk group had a worse OS (median 41 months). CONCLUSIONS: Ki-67 value after NAC and ER status could predict a worse prognosis among LABC patients treated with NAC.

Experimental infrared measurements for hydrocarbon pollutant determination in subterranean waters.

Subterranean waters are often polluted by industrial and anthropic effluents that are drained in subsoil. To prevent and control pollution, legislations of different developed countries require an online monitoring measurement, especially for detecting organic solvents (chlorinated and unchlorinated ones). Online measurements include both real-time and no real-time measurements. In general, it is difficult to implement real-time measurements in stricto sensu for online acquisitions on aqueous effluents since they need to be processed by a modeling. This research presents an experimental measurement system based on infrared (IR) spectroscopy for aqueous effluents containing hydrocarbons and capable of displaying excellent values of pollutant concentrations even in instable conditions; the system is able to detect pollutants either in laminar or turbulent flow. The results show the possibility of avoiding the use of "Pitot tube" that is employed to create a stagnation point in order to convert kinetic energy into potential one. This conversion allows the transformation of a turbulent flow in a laminar flow making easy measurement of pollutants included in an aqueous effluent. Obviously, "Pitot tube" is also used for other fluid effluents. The obtained results have been compared with those produced by means of sophisticated IR instrumentation for laboratory applications.

miR-29b sensitizes multiple myeloma cells to bortezomib-induced apoptosis through the activation of a feedback loop with the transcription factor Sp1.

MicroRNAs (miRNAs) with tumor-suppressor potential might have therapeutic applications in multiple myeloma (MM) through the modulation of still undiscovered molecular pathways. Here, we investigated the effects of enforced expression of miR-29b on the apoptotic occurrence in MM and highlighted its role in the context of a new transcriptional loop that is finely tuned by the proteasome inhibitor bortezomib. In details, in vitro growth inhibition and apoptosis of MM cells was induced by either transient expression of synthetic miR-29b or its stable lentivirus-enforced expression. We identified Sp1, a transcription factor endowed with oncogenic activity, as a negative regulator of miR-29b expression in MM cells. Since Sp1 expression and functions are regulated via the 26S proteasome, we investigated the effects of bortezomib on miR-29b-Sp1 loop, showing that miR-29b levels were indeed upregulated by the drug. At the same time, the bortezomib/miR-29b combination produced significant pro-apoptotic effects. We also demonstrated that the PI3K/AKT pathway plays a major role in the regulation of miR-29b-Sp1 loop and induction of apoptosis in MM cells. Finally, MM xenografts constitutively expressing miR-29b showed significant reduction of their tumorigenic potential. Our findings indicate that miR-29b is involved in a regulatory loop amenable of pharmacologic intervention and modulates the anti-MM activity of bortezomib in MM cells.

N-(4-hydroxyphenyl)retinamide promotes apoptosis of resting and proliferating B-cell chronic lymphocytic leukemia cells and potentiates fludarabine and ABT-737 cytotoxicity.

The in vitro effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR, fenretinide) on primary B-cell chronic lymphocytic leukemia (CLL) cells from previously untreated CLL patients were investigated. 4HPR promoted the intrinsic apoptotic pathway by reactive oxygen species (ROS) generation and was accompanied by drop of Mcl-1 protein expression. The latter was not attributable to transcriptional downregulation but to protein degradation mediated by jun N-terminal kinase activation, and likely by NF-kB downregulation and Noxa upregulation. CLL cells stimulated in vitro with CD40L did not increase 4HPR chemoresistance if activation was accompanied by proliferation. Intra-patient analysis confirmed that the proliferating pool of CLL cells was more sensitive to the cytotoxic action of 4HPR than the activated but resting CLL subpopulation. The different 4HPR susceptibility of the two subpopulations was associated with higher Noxa expression in proliferating CLLs. Combination experiments revealed that 4HPR strongly potentiated ABT-737 cytotoxicity, especially in proliferating CLL cells that displayed amplified chemoresistance to ABT-737 alone. Synergic cytotoxicity was also demonstrated in combination with fludarabine, in both resting and stimulated CLL samples. This study entitles 4HPR to be assayed as a chemotherapeutic adjuvant for the treatment of CLL.

Prognostic factors in CLL.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, as some patients progress rapidly toward the more advanced studies, whereas others survive for a long period without the need for treatment. This heterogeneity of clinical course was somehow unexplained until studies on the CLL cell features disclosed that the CLL clones were heterogeneous and were characterized by different phenotypic and genotypic features in the different patients. On the basis of these observations, it was determined in retrospective studies that clones characterized by unmutated IGHV genes, and/or CD38 and/or ZAP-70 expression conferred a more severe prognosis to the CLL patients. Here, we present data on prospective studies carried out on Binet A-stage patients, in whom the markers were determined at diagnosis and their predictive value was assessed in comparison with chromosomal abnormalities and gene expression or micro RNA profiles. In addition, hypothesis on the potential pathogenetic role of these markers will be presented.

Primary synovial sarcoma of the kidney. A case report with pathologic appraisal investigation and literature review.

Synovial sarcoma (SS) is a soft tissue neoplasm with clearly defined histologic, immunohistochemical and molecular features that usually arises in the extremities of young adults. The occurrence of these tumors in the kidney is extremely rare and have been prevalently described in case reports. The objectives of this work were to evaluate the frequency of primary renal synovial sarcomas and the pathologic progression in recognition of this possibly under-diagnosed entity. A comprehensive review of the literature has also been performed with a focus on survival. We report the clinico-pathological features of an intrarenal SS occurring in a 67-year-old man. The tumour, measuring 4 cm in its greatest diameter, completely replaced the cortex and the medulla of the inferior region of the left kidney compressing the iliopsoas muscle. Radiological imaging was consistent with a renal cell carcinoma. Histologically, the tumour was composed of atypical monotonous vimentin+, CD99+, bcl-2+ spindle cells exhibiting a haphazard fascicular growth pattern and a high mitotic rate (3 to 5 mitoses per HPF). The diagnosis was supported by reverse transcription-polymerase chain reaction which demonstrated SYT-SSX2 gene fusion. The patient was alive with local recurrence of disease 24 months after surgery. Synovial sarcomas occurring in the kidney, in analogy to other sites, tend to have an aggressive biologic behaviour. Despite being extremely uncommon, with only 44 cases reported to date, they should be included in the differential diagnosis of benign and malignant spindle cell tumours of the kidney. This study also emphasizes the importance of a correct pathologic diagnosis for prognostic and therapeutic implications.

Rituximab for the treatment of patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that originates from antigen-experienced B lymphocytes that do not die and hence accumulate due to external survival signals or undergo apoptosis and are replenished by proliferating precursors. These neoplastic lymphocytes exhibit a characteristic immunophenotype of CD5(+)/CD19(+)/CD20(+)/HLA-DR+/CD23(+)/sIgdim. Thus, the CD20 antigen has been an appealing target for therapy. The introduction of the monoclonal antibody rituximab (anti-CD20) enabled an outstanding advance in CLL treatment. The introduction of this monoclonal antibody into chemotherapy regimens has dramatically improved complete response rates and progression-free survival in patients with both untreated and relapsed CLL. Although only preliminary data from phase III confirmatory trials have been reported, the FCR regimen, which combines fludarabine and cyclophosphamide with rituximab, is currently the most effective treatment regimen for CLL patients, and has also been demonstrated to significantly improve overall survival. The success of rituximab and the identification of other CLL lymphocyte surface antigens have spurred the development of a multitude of monoclonal antibodies targeting distinct proteins and epitopes in an attempt to target CLL cells more effectively.

Liposomal amphotericin B in the treatment of visceral leishmaniasis in immunocompromised patients.

Leishmaniasis is a zoonosis caused by a protozoan of the Leishmania genus. First-line treatment for all forms is currently represented by the use of antimony derivatives, although toxic effects and the number of resistant strains in both immunocompromised and immunocompetent patients is increasing. Liposomal amphotericin B (L-AMB) is less toxic, more effective, and better tolerated, especially in human immunodeficiency virus-negative immunocompromised patients. We present 2 cases of transplanted patients affected by visceral leishmaniasis treated successfully with L-AMB.

Prognostic value of estrogen receptor and Ki-67 index after neoadjuvant chemotherapy in locally advanced breast cancer expressing high levels of proliferation at diagnosis.

PURPOSE: Breast cancers expressing high levels of Ki-67, a nuclear marker of cell proliferation, are associated with worse outcome. Recent data from neoadjuvant studies indicate that a single measurement of the nuclear proliferation marker Ki-67 in breast carcinoma during neoadjuvant therapy is strongly predictive of long-term outcome. Secondly, recent literature data indicate that prognostic evaluation with Ki-67 may be better after pre-surgical therapy. A retrospective study from a prospectively maintained clinical database to compare the predictive and prognostic significance of biological markers, assessed before and after neoadjuvant chemotherapy, in locally advanced breast cancer, was performed. PATIENTS AND METHODS: The following parameters were considered before and after chemotherapy for their relationship with treatment response and disease-free survival in 64 patients with locally advanced breast cancer: clinical stage, clinical and pathological lymph node involvement, Ki-67, estrogen receptor (ER), progesterone receptor (Pgr), Her2, tumor grade, clinical response, type of surgery performed, and number of chemotherapy cycles administered. The expression of Ki-67 was assessed using immunohistochemistry in pre-therapy tru-cut and post-therapy surgical excision specimens after neoadjuvant chemotherapy; only patients with breast tumors expressing high baseline Ki-67 (≥ 15%) were included in the analysis. In addition, the correlation between pre-chemotherapy biological markers and clinical and pathological response was reported. RESULTS: Post-chemotherapy Ki-67 proliferation index decrease, pre-chemotherapy ER expression and post-chemotherapy ER expression were the only significant prognostic factors adversely influencing disease-free survival in univariate analysis. Her2 overexpression was the only factor to impact on the clinical response. CONCLUSIONS: Post-treatment Ki-67 and ER status were predictors of outcome for patients with locally advanced breast cancer and a high pre-chemotherapy proliferation index.