Mario Delia

CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice

Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34(+) cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34(+) cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34(+) leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34(+) subfraction exhibiting the phenotype (CD34(+)/CD38(-)/CD123(+)/CD33(+)/CD90(-)) of leukemic stem cells. When transplanted into immunocompromised mice, CD34(+) cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patients disease. These results indicate that the CD34(+) fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34(-) cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with IA.

Evaluation of the Practice of Antifungal Prophylaxis Use in Patients With Newly Diagnosed Acute Myeloid Leukemia: Results From the SEIFEM 2010-B Registry

BACKGROUND To analyze the efficacy of antifungal prophylaxis (AFP) with posaconazole and itraconazole in a real-life setting of patients with acute myeloid leukemia (AML) during the first induction of remission. METHODS From January 2010 to June 2011, all patients with newly diagnosed AML were consecutively registered and prospectively monitored at 30 Italian hematological centers. Our analysis focused on adult patients who received intensive chemotherapy and a mold-active AFP for at least 5 days. To determine the efficacy of prophylaxis, invasive fungal disease (IFD) incidence, IFD-attributable mortality, and overall survival were evaluated. RESULTS In total, 515 patients were included in the present analysis. Posaconazole was the most frequently prescribed drug (260 patients [50%]) followed by fluconazole (148 [29%]) and itraconazole (93 [18%]). When comparing the groups taking posaconazole and itraconazole, there were no significant differences in the baseline clinical characteristics, whereas there were significant differences in the percentage of breakthrough IFDs (18.9% with posaconazole and 38.7% with itraconazole, P< .001). The same trend was observed when only proven/probable mold infections were considered (posaconazole, 2.7% vs itraconazole, 10.7%, P= .02). There were no significant differences in the IFD-associated mortality rate, while posaconazole prophylaxis had a significant impact on overall survival at day 90 (P= .002). CONCLUSIONS During the last years, the use of posaconazole prophylaxis in high-risk patients has significantly increased. Although our study was not randomized, it demonstrates in a real-life setting that posaconazole prophylaxis confers an advantage in terms of both breakthrough IFDs and overall survival compared to itraconazole prophylaxis. CLINICAL TRIALS REGISTRATION NCT01315925.

Invasive fungal infections in patients with hematologic malignancies (aurora project): lights and shadows during 18-months surveillance.

The aim of this multicenter prospective study was to evaluate the incidence of invasive fungal infections (IFIs) in adult and pediatric patients with hematologic malignancies, involving nine nosocomial facilities in Southern Italy over a period of 18 months. Furthermore, results of an environmental microbial surveillance routinely carried out in some of the enrolled hospitals are reported. A total of 589 onco-hematological patients were enrolled and 27 IFIs were documented. The main infections were caused by yeasts, more than filamentous fungi (overall incidence of 2.7% and 1.9%, respectively). The yeasts were mainly represented by Candida spp. (87.5%), all isolated by blood cultures; C. parapsilosis was the most common species. Among mould infections, the most frequent site was the lung, with regard to aspergillosis (81.8%). In six of the 10 patients with suspected aspergillosis, the diagnosis was made by the detection of galactomannan and (1,3)-β-d-glucan antigens. The microbiological surveillance carried out on 156 air, 312 water and 312 surface samples revealed low environmental contamination: Alternaria alternata was the only fungus isolated from two surface samples. Our data, especially the low occurrence of filamentous fungi, suggest a particular local epidemiology. Further studies are needed to confirm this microbiological trend in onco-hematological patients in Southern Italy, the results of which might be helpful to improve the management of these patients.

High Ki67 Index and Bulky Disease Remain Significant Adverse Prognostic Factors in Patients with Diffuse Large B Cell Lymphoma before and after the Introduction of Rituximab

The aim of this study was to evaluate the impact of clinical variables and biologic features on response rate (RR), overall survival (OS) and progression-free survival (PFS) in 111 patients with de novo diffuse large B cell lymphoma (DLBCL). Fifty-three patients were treated with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) and 58 patients were treated with R-CHOP (rituximab + CHOP). The variables predictive of RR in the CHOP group were B symptoms, age, clinical stage, bone marrow involvement, bulky disease, International Prognostic Index (IPI) and Bcl-2; in the R-CHOP group, these variables were bulky disease, bone marrow involvement, IPI and Ki67 expression >80%. Multivariate analysis showed that in patients treated with CHOP, the independent prognostic factors associated with PFS were age, bulky disease, IPI and Bcl-2 and those associated with OS were performance status, clinical stage, IPI and bone marrow involvement. In contrast, in patients treated with R-CHOP, the variable shown by multivariate analysis to be an independent prognostic factor associated with PFS was bulky disease, whereas Ki67 expression >80% was associated with OS and PFS. Our data show that a high Ki67 expression and bulky disease could represent possible predictive factors of poor prognosis, which would help to identify a high-risk subgroup of newly diagnosed DLBCL.

Pre-chemotherapy risk factors for invasive fungal diseases: prospective analysis of 1,192 patients with newly diagnosed acute myeloid leukemia (SEIFEM 2010-a multicenter study)

Correct definition of the level of risk of invasive fungal infections is the first step in improving the targeting of preventive strategies. We investigated the potential relationship between pre-hospitalization exposure to sources of fungi and the development of invasive fungal infections in adult patients with newly diagnosed acute myeloid leukemia after their first course of chemotherapy. From January 2010 to April 2012, all consecutive acute myeloid leukemia patients in 33 Italian centers were prospectively registered. Upon first admission, information about possible pre-chemotherapy risk factors and environmental exposure was collected. We recorded data regarding comorbid conditions, employment, hygienic habits, working and living environment, personal habits, hobbies, and pets. All invasive fungal infections occurring within 30 days after the first course of chemotherapy were recorded. Of the 1,192 patients enrolled in this study, 881 received intensive chemotherapy and were included in the present analysis. Of these, 214 developed an invasive fungal infection, including 77 proven/probable cases (8.7%). Of these 77 cases, 54 were proven/probable invasive mold infections (6.1%) and 23 were proven yeast infections (2.6%). Upon univariate analysis, a significant association was found between invasive mold infections and age, performance status, diabetes, chronic obstructive pulmonary disease, smoking, cocaine use, job, hobbies, and a recent house renovation. Higher body weight resulted in a reduced risk of invasive mold infections. Multivariate analysis confirmed the role of performance status, job, body weight, chronic obstructive pulmonary disease, and house renovation. In conclusion, several hospital-independent variables could potentially influence the onset of invasive mold infections in patients with acute myeloid leukemia. Investigation of these factors upon first admission may help to define a patient’s risk category and improve targeted prophylactic strategies.

Multicentre surveillance study on feasibility, safety and efficacy of antifungal combination therapy for proven or probable invasive fungal diseases in haematological patients: the SEIFEM real-life combo study

This multicentre observational study evaluated the feasibility, efficacy and toxicity of antifungal combination therapy (combo) as treatment of proven or probable invasive fungal diseases (IFDs) in patients with haematological malignancies. Between January 2005 and January 2010, 84 cases of IFDs (39 proven and 45 probable) treated with combo were collected in 20 Hematological Italian Centres, in patients who underwent chemotherapy or allogeneic haematopoietic stem cell transplantation for haematological diseases. Median age of patients was 34 years (range 1–73) and 37% had less than 18 years. Acute leukaemia was the most common underlying haematological disease (68/84; 81%). The phase of treatment was as follows: first induction in 21/84 (25%), consolidation phase in 18/84 (21%) and reinduction/salvage in 45/84 (54%). The main site of infection was lung with or without other sites. The principal fungal pathogens were as follows: Aspergillus sp. 68 cases (81%), Candida sp. six cases (8%), Zygomycetes four cases (5%) and Fusarium sp. four cases (5%). The most used combo was caspofungin+voriconazole 35/84 (42%), caspofungin + liposomal amphotericin B (L‐AmB) 20/84 (24%) and L‐AmB+voriconazole 15/84 (18%). The median duration of combo was 19 days (range 3–180). The overall response rate (ORR) was 73% (61/84 responders) without significant differences between the combo regimens. The most important factor that significantly influenced the response was granulocyte (PMN) recovery (P 0.009). Only one patient discontinued therapy (voriconazole‐related neurotoxicity) and 22% experienced mild and reversible adverse events (hypokalaemia, ALT/AST increase and creatinine increase). The IFDs‐attributable mortality was 17%. This study indicates that combo was both well tolerated and effective in haematological patients. The most used combo regimens were caspofungin + voriconazole (ORR 80%) and caspofungin + L‐AmB (ORR 70%). The ORR was 73% and the mortality IFD related was 17%. PMN recovery during combo predicts a favourable outcome. Clinical Trials Registration: NCT00906633.

Jaw Osteonecrosis Associated with Use of Bisphosphonates and Chemotherapy : Paradoxical Complication of Treatment of Bone Lesions in Multiple Myeloma Patients

It has been demonstrated that bisphosphonate-based supportive therapy (pamidronate or zoledronate) reduces skeletal events (onset or progression of osteolytic lesions) both in patients with multiple myeloma (MM) and in cancer patients with bone metastasis. Bisphosphonates (eg, alendronate) are also indicated in the treatment of osteoporosis. Nevertheless, osteonecrosis of the jaw (ONJ) has been reported in some patients being treated with bisphosphonates. We present a series of 9 MM patients who developed ONJ after treatment with bisphosphonates and chemotherapy. All the patients in this case series had undergone tooth extraction for recurrent dental abscesses while taking bisphosphonates. We also review the diagnostic and therapeutic implications of this paradoxical complication associated with treatment of bone lesions in MM.

Mycosis fungoides/Sézary syndrome: a report of three cases treated with Campath-1H as salvage treatment.

We report the use of Alemtuzumab (Campath-1H) as salvage treatment in three patients with advanced mycosis fungoides/Sézary syndrome who had previously been treated with conventional chemotherapy. Two patients (case 1 and case 2), aged 42 and 68 yr, respectively, were heavily pretreated (more than three prior therapy regimens, including autologous transplant in case 2) and refractory to conventional chemotherapy, and the third patient (case 3), aged 80 yr, who had refused any chemotherapy, had been resistant to treatment with cyclosporine and steroids. Campath-1H was administered intravenously, after an escalating dose from 3 to 10 mg, at the dose of 30 mg, three times weekly, to a total dose of 1080, 223, and 480 mg, respectively. The patients with Sézary syndrome (case 2 and case 3) showed clearance of circulating Sézary cells and clinical improvement of the skin lesions after 2 wk of treatment. Two patients (case 1 and case 3) completed the treatment (12 and 6 wk) without significant toxicity, the former achieving a partial response and the latter a clinical complete response. The patient (case 2), who suffered from ischemic cardiopathy and diabetes, quickly achieved a clinical improvement of the Sézary syndrome, but he died because of a myocardial infarction after 3 wk of treatment.Our report shows that the treatment with Campath-1H is active even in patients with advanced refractory mycosis fungoides/Sézary syndrome. Further clinical observations on a larger cohort of patients are needed to establish if Campath-1H may have a role as first line therapy in addition to conventional therapy including chemotherapy.

Bloodstream infections caused by Klebsiella pneumoniae in onco-hematological patients: clinical impact of carbapenem resistance in a multicentre prospective survey.

The aim of this study was to identify risk factors for mortality in patients suffering from hematological malignancies (HMs) with bloodstream infections (BSIs) caused by Klebsiella pneumoniae (KP). We conducted a prospective cohort study on KP BSI in 13 Italian hematological units participating in the HEMABIS registry–SEIFEM group. The outcome measured was death within 21 days of BSI onset. Survivor and non‐survivor subgroups were compared and Cox regression analysis was conducted to identify independent predictors of mortality. A total of 278 episodes of KP BSI were included in the study between January 2010 and June 2014. We found that 161 (57.9%) KP isolates were carbapenem resistant (CRKP). The overall 21‐day mortality rate was 36.3%. It was significantly higher for patients with CRKP BSI (84/161, 52.2%) than for those with BSI caused by carbapenem susceptible KP (CSKP) (17/117, 14.5%; P < 0.001). Septic shock (HR 3.86), acute respiratory failure (HR 2.32), inadequate initial antimicrobial therapy (HR 1.87) and carbapenem resistance by KP isolates (HR 1.85) were independently associated with mortality. A subanalysis was conducted in only 149 patients with CRKP BSI who had received ≥48 hr of adequate antibiotic therapy, and combination therapy was independently associated with survival (HR 0.32). Our study shows that in recent years carbapenem resistance has dramatically increased in HM patients with KP BSI in Italy and is associated with a worse outcome. The optimal management of such infections and the definition of new empirical/targeted antimicrobial strategies in HM patients can still be considered unmet clinical needs. Am. J. Hematol. 91:1076–1081, 2016.