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Simple Summary IDH1/2 mutations are a common event in acute myeloid leukemia (AML) and represent a therapeutic target. We designed the GIMEMA AML1516 observational protocol to examine the prevalence of IDH1/2 mutations and the associations between IDH mutations and clinico-biological parameters in a cohort of Italian patients affected by AML. By analyzing 284 consecutive adult AML patients, we confirmed that IDH1 and IDH2 mutations are frequently detected-14% and 18%, respectively-at diagnosis. IDH1/2 mutations were significantly associated with an inferior performance status and non-complex karyotype when compared to IDH1/2-WT. With regards to the outcome, in the subset of IDH1/2-mutated patients the rate of complete remission achievement was 60.5% and overall survival at 2 years was 44.5%: these percentages did not significantly differ from IDH1/2-WT patients. However, given the availability of IDH1/2 inhibitors, it is important to recognize IDH1/2-mutated cases up-front to offer patients the most appropriate therapeutic strategy. IDH1/2 mutations are common in acute myeloid leukemia (AML) and represent a therapeutic target. The GIMEMA AML1516 observational protocol was designed to study the prevalence of IDH1/2 mutations and associations with clinico-biological parameters in a cohort of Italian AML patients. We analyzed a cohort of 284 AML consecutive patients at diagnosis, 139 females and 145 males, of a median age of 65 years (range: 19-86). Of these, 38 (14%) harbored IDH1 and 51 (18%) IDH2 mutations. IDH1/2 mutations were significantly associated with WHO PS >2 (p < 0.001) and non-complex karyotype (p = 0.021) when compared to IDH1/2-WT. Furthermore, patients with IDH1 mutations were more frequently NPM1-mutated (p = 0.007) and had a higher platelet count (p = 0.036). At relapse, IDH1/2 mutations were detected in 6 (25%) patients. As per the outcome, 60.5% of IDH1/2-mutated patients achieved complete remission; overall survival and event-free survival at 2 years were 44.5% and 36.1%, respectively: these rates were similar to IDH1/2-WT. In IDH1/2-mutated patients, high WBC proved to be an independent prognostic factor for survival. In conclusion, the GIMEMA AML1516 confirms that IDH1/2 mutations are frequently detected at diagnosis and underlines the importance of recognizing IDH1/2-mutated cases up-front to offer the most appropriate therapeutic strategy, given the availability of IDH1/2 inhibitors.

Background. Ponatinib is a third-generation tyrosine kinase inhibitor indicated for adults with resistant or intolerant chronic phase (CP), accelerated phase (AP), or blast phase (BP) chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia, or those carrying the T315I mutation. Ponatinib has been commercially available since January 2015, yet there is a paucity of data on its use in the real-world setting. The goal of the Observational study of Iclusig ® (ponatinib) Treatment in patients with CML in Italy (OITI) was to evaluate treatment patterns and outcomes, including safety and efficacy, in patients with CML treated in Italy since the approval of ponatinib. Methods. This noninterventional study included patients aged ≥18 years with CP, AP, or BP CML who initiated ponatinib treatment in routine clinical practice across 26 Italian centers (academic and hospital settings). The study population comprised a prospective cohort, including patients who started treatment with ponatinib after site activation during the 12-month enrollment period; a retrospective cohort, including patients who started treatment with ponatinib but died or were lost to follow-up prior to site activation; and a retrospective/prospective cohort, including patients who started treatment with ponatinib prior to site activation and were still on treatment or in follow-up at the end of the study. Demographic, efficacy, and safety data were collected from patient medical charts at study entry and routine visits. The primary endpoint was the complete cytogenetic response (CCyR) rate in CP CML patients 6 months after starting ponatinib. In the absence of cytogenetic evaluation, molecular assessment was used, considering patients in MR2 or better to be in CCyR. Here, the primary analysis of all evaluable patients for the primary endpoint (median follow-up, 23.7 months [range, 1.3-70.8]) is presented. Results. A total of 119 patients (110 CP, 6 AP, and 3 BP CML) were analyzed. Fifty-nine (49.6%) received ponatinib in second-line (2L), 41 (34.4%) in 3L, and 19 (16.0%) in ≥4L. Prior cardiovascular disease/hypertension was recorded in 56 (47.1%) patients. Median age at ponatinib start was 60 years (range, 19-93). Of 68 evaluated patients, 24 (35.3%) had a confirmed ABL1 mutation, including 7 (10.3%) with the T315I mutation. Starting doses of ponatinib were 45 mg (37.0%), 30 mg (41.2%), or 15 mg (21.8%). Median treatment duration was 22.8 months (range, 1.4-73.6). At baseline, 56 patients with CP CML had less than CCyR and 53 were in CCyR. For 1 patient, assessment was not available. At 6 months, 80/107 evaluable patients with CP CML were in CCyR; 29/56 (51.8%) achieved and 50/50 (100%) maintained CCyR compared to baseline, respectively. For 1 patient, baseline data were unavailable. Additionally, 37 (34.6%) and 19 (17.8%) patients with CP CML achieved a major molecular response (MMR; MR3) and a deep molecular response (MR4-MR5), respectively (Table 1). Progression-free survival rates estimated for patients with CP CML at 12 and 24 months were 92.6% (95% CI, 87.8-97.7%) and 84.6% (95% CI, 77.2-92.6%), respectively. Corresponding overall survival rates were 95.4% (95% CI, 91.6-99.4%), and 88.4% (95% CI, 81.7-95.7%). Seventy-one (59.7%) patients had treatment-related adverse events (AEs), most commonly rash, hypertension (Grade 1-2), and thrombocytopenia (Grade 3). Treatment-related arterial occlusive events occurred in 2 (1.7%) patients. Dose modifications occurred in 77 patients: 37.1% were due to AEs, 13.5% were reductions after at least major cytogenetic response, 10.6% were increases due to lack of efficacy, 1.7% were medical decisions, and 37.1% were for other reasons. Thirty-three patients discontinued ponatinib due to AEs (33.3%), medical decisions (24.2%), and other reasons (42.4%), such as death, progression, and hematopoietic stem cell transplantation. Conclusions. This observational study demonstrates that ponatinib has a favorable efficacy and safety profile in patients with CML treated in standard clinical practice. By 6 months, 74.8% of evaluable patients were in CCyR and 52.3% achieved at least MMR. Further, the probability of survival at 2 years was >88%. No new safety signals emerged with ponatinib treatment compared to prior studies. The early use of ponatinib and careful dose selection appear key to the safety and efficacy outcomes observed in this real-world study. Figure 1 Figure 1. Disclosures Breccia: Abbvie: Honoraria; Bristol Myers Squibb/Celgene: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Abruzzese: Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Bonifacio: Pfizer: Honoraria; Amgen: Honoraria; Bristol Myers Squibb: Honoraria; Novartis: Honoraria. Castagnetti: Novartis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Galimberti: Inctye Bioscience Italy Srl: Current Employment. Iurlo: Incyte: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau.

Background BK virus (BKV)–associated nephropathy is definitely involved in allograft failure after kidney transplant. Thus, the need for an early control of viral reactivation in immunocompromised patients is well established. Determination of urinary release of decoy cells (DC) and BK viral load in plasma and urine by polymerase chain reaction (PCR) usually precedes renal biopsy. The aim of the study is to assess viral reactivation by BKV-DNA PCR and DC detection in urinary sediment using automated intelligent microscopy. Methods Seventy-eight kidney transplant patients were analyzed for the presence of plasma BKV-DNA by quantitative TaqMan real-time PCR. Additionally, automated intelligent microscopy was used for urine sediment analysis, allowing to count cells with decoy feature, confirmed by phase contrast microscopic review. Results Plasma BKV-DNA PCR was detected in 14 (17.9%) patients. DC were identified in 19 (24.3%) urine sediments by automated analyzers and confirmed by microscopic observation. Two patients were BKV-DNA–positive/DC-negative; conversely, 7 subjects were DC-positive/BKV-DNA–negative. Conclusions Plasma quantification of BK viral load is currently the best noninvasive method for the detection of viral reactivation. Nevertheless, automated methods to screen for the presence of DC in urine could facilitate early BK virus replication diagnosis and patient follow-up by quantitative and visual results.

Serum protein electrophoresis (SPE), used to identify monoclonal gammopathies, is generally performed using the “gold standard” agarose gel as migration support (AGE). However, despite being semi-automated, this electrophoresis technique remains labour-intensive, meaning analytical performance and throughput are limited. Hence fully automated capillary electrophoresis (CE), which enables rapid separation, has been adapted for use in clinical laboratories within the last decade [1–5]. Any qualitative abnormalities suggestive of monoclonal immunoglobulin detected by either AGE or CE are further analysed by immunofixation electrophoresis (IFE), to confirm the presence of monoclonal bands and to identify the type of monoclonal component (MC) [6]. In order to increase throughput, a fully automated innovative AGE technique—Agarose Gel Easy Interlab G26 electrophoresis (Interlab, Rome, Italy)—has recently been launched, to be used with purpose-designed IFE kits. The aim of this study was to compare the AGE system with the Capillarys 2® CE equipment (Sebia, Evry, France) currently in use in our laboratory, to detect serum MCs subsequently confirmed, characterized and quantified by IFE on

Group I CD1 proteins are specialized antigen-presenting molecules that present both microbial and self lipid antigens to CD1-restricted α/β T lymphocytes. The production of high levels of gamma interferon and lysis of infected macrophages by lipid-specific T lymphocytes are believed to play pivotal roles mainly in the defense against mycobacterial infections. We previously demonstrated that Mycobacterium tuberculosis and bacillus Calmette-Guérin ( Mycobacterium bovis BCG) induce human monocytes to differentiate into CD1 − dendritic cells (DC), which cannot present lipid antigens to specific T cells. Here, we show that in human monocytes mycobacteria trigger phosphorylation of p38 mitogen-activated protein kinase to inhibit CD1 expression in DC derived from infected monocytes. Pretreatment with a specific p38 inhibitor renders monocytes insensitive to mycobacterial subversion and allows them to differentiate into CD1 + DC, which are fully capable of presenting lipid antigens to specific T cells. We also report that one of the pathogen recognition receptors triggered by BCG to activate p38 is complement receptor 3 (CR3), as shown by reduced p38 phosphorylation and partial reestablishment of CD1 membrane expression obtained by CR3 blockade before infection. In conclusion, we propose that p38 signaling is a novel pathway exploited by mycobacteria to affect the expression of CD1 antigen-presenting cells and avoid immune recognition.

Summary We previously described an escape mechanism exploited by Mycobacterium tuberculosis (Mtb) to prevent the generation of fully competent dendritic cells (DC). We have now tested the effect of isolated mycobacterial components on human monocyte dif- ferentiation into DC and demonstrated that cell wall (CW)-associated alpha-glucan induces monocytes to differentiate into DC (Glu-MoDC) with the same altered phenotype and functional behaviour of DC derived from Mtb-infected monocytes (Mt-MoDC). In fact, Glu- MoDC lack CD1 molecule expression, fail to upregu- late CD80 and produce IL-10 but not IL-12. We also showed that Glu-MoDC are not able to prime effector T cells or present lipid antigens to CD1-restricted T-cell clones. Thus, we propose a mechanism of Mtb- monocyte interaction mediated by CW-associated alpha-glucan, which allows the bacterium to evade both innate and acquired immune responses.

The functional consequences of treating human monocytes with purified and chemically characterized Candida albicans β-glucan—a major microbial pathogen associated molecular pattern—on their differentiation into dendritic cells (DC) were investigated. We show here that β-glucan-treated monocytes differentiated into mature DC (Glu-MoDC) with altered phenotype and functional behavior, similarly to DC derived from C. albicans germ-tubes-infected monocytes (Gt-MoDC). They failed to express CD1a and to up-regulate CD80 and DR molecules. Moreover, they produced IL-10 but not IL-12 and primed naive T cells without inducing their functional polarization into effector cells. Since C. albicans β-glucan is a mixture of both β-(1,3) and β-(1,6) glucan, we investigated their relative contribution by the use of non-Candida β-glucan structural analogs. We found that high molecular weight (MW) glucans β−(1,6) pustulan and β-(1,3) curdlan totally mimicked the effect of C. albicans β-glucan, while the low MW β-(1,3) glucan laminarin did not have any effect. Because β-glucan is a common constituent of all fungi and is abundantly released in vivo during systemic fungal infection, this novel effect of β-glucan has potential implications for host-parasite relationship in candidiasis and other mycoses. In particular, our data suggest that β-glucan could bias noninfected, bystander monocytes, thus aggravating the general immunodeficiency, predisposing to systemic fungal infection.

Systemic bacterial infection carries a high risk of mortality in critical care patients. Improvements in diagnostic procedures are required for effective management of sepsis. Recently, the soluble CD14 subtype, or presepsin, has been suggested as a reliable marker of sepsis, and we set out to compare its diagnostic performance with that of procalcitonin (PCT). We focused on a cohort of septic patients who, during their hospitalization, relapsed after a period of clinical relief from symptoms.In total 21 adult patients were studied during their hospitalization in the Critical Care Unit of Policlinico Umberto I hospital; 74 plasma samples were collected at multiple time points, and presepsin levels were measured using a PATHFASTPresepsin and PCT were significantly lower in healthy controls than in sepsis or severe sepsis (p1000 pg/mL) remained high.This study confirms the importance of monitoring a combination of several biomarkers in order to obtain a reliable diagnosis. Maximal presepsin levels could alert clinicians not to suspend antibiotic treatments and to carefully monitor septic patients’ state of health, even after clinical symptoms have disappeared and PCT levels returned to normal.

The host immune response can limit Mycobacterium tuberculosis (Mtb) spreading in primary tuberculosis (TB) without eradicating all bacilli, which can persist causing latent TB infection and are responsible for reactivation TB. Persistent Mtb is confined to granulomas within phagocytes, but it is also found in other non-immune cells. We focused on fibroblasts since these cells participate to the granuloma formation and were shown to be infected in latent TB infections. We show that in vitro both Mtb and Bacille Calmette-Guérin actively replicate in human fibroblasts. Mycobacterial infection of fibroblasts causes a significant inhibition of interferon (IFN)-γ induced membrane expression of major histocompatibility complex class II molecules in these cells. The functional consequence of in vitro infection is a significant reduction of the fibroblast capacity to present peptides and soluble proteins to autologous specific CD4(+) T cell clones. Moreover, fibroblasts are capable of presenting antigen derived from the processing of heat-killed Mtb, but not from viable Mtb. Data indicate that IFN-γ treated fibroblasts are capable of presenting antigens derived from the processing of whole bacteria in addition to the capacity to present peptides and isolated proteins. Interestingly, Mtb infected fibroblasts lose this capacity, suggesting that Mtb may evade T helper immune surveillance by infecting fibroblasts.

BackgroundThe purpose of this study was to further characterize the immune response toMycobacterium tuberculosis(Mtb) antigens, in order to provide new insight into host-pathogen interactions in tuberculosis (TB), and to offer tools for a more accurate diagnosis of the different stages of TB.MethodsT-cell responses to Bacillus Calmette-Guérin (BCG), purified protein derivative (PPD), early secretory antigenic target-6 (ESAT-6) protein and culture filtrate protein-10 kDa (CFP-10) were measured in terms of interferon (IFN)-γ and interleukin (IL)-2 release, using a novel flow cytometric cell-secreting cytokine detection technique. The study was conducted on peripheral blood mononuclear cells (PBMC) obtained from active TB patients, latently TB infected individuals, and healthy donors. IL-10 and IL-17 were also measured to test their possible role as indicators of disease activity.ResultsWe confirmed that the enumeration of IFN-γ releasing cells upon Mtb-specific stimulation is sufficient to identify TB patients and that CD8+ T cells concur to IFN-γ secretion. IL-2 secreting cells were more frequently observed in latent TB infected individuals compared to active TB patients, suggesting that measurement of cells secreting this cytokine could be a marker of disease stage. No discriminating role was associated to IL-10 and IL-17 release in TB patients.ConclusionOur data indicate that the flow cytometric cytokine-secreting cell detection technique may be envisaged as an additional tool for TB diagnosis allowing the analysis of the immune response toM. tuberculosis-related antigens in the different stages of TB.

It is well established that human monocytes differentiate into dendritic cells (DCs) when cultured with certain cytokine cocktails, such as granulocyte-macrophage colony-stimulating factor and interleukin-4. Conversely, it is not completely established which cell population synthesizes the cytokines required for monocyte differentiation and how their secretion is regulated. We show that on specific activation T cells induce the differentiation into DCs of antigen-presenting and bystander monocytes. Monocytes exposed to cytokines released by Th1 and Th0 lymphocytes differentiate into DCs with a reduced antigen uptake and antigen presentation capacity. Moreover, these DCs show a limited capacity to induce Th1 polarization of naive T cells but are capable of priming interleukin-10-secreting T cells. Conversely, DCs derived from monocytes sensing cytokines released by Th2 lymphocytes are antigen-presenting-cell (APC) endowed with a marked Th1 polarization capacity. Monocytes are corecruited with lymphocytes in chronic inflammation sites; thus our results suggest that functionally different DCs can be generated in environments characterized by the prevalent release of Th1-, Th0-, or Th2-associated cytokines. Because the APC capacities of these DCs have opposite functional consequences, a contribution in the regulation of the ongoing immune response by monocyte-derived inflammatory DCs is envisaged.

The only available vaccine against tuberculosis is Mycobacterium bovis Bacillus Calmette Guérin (BCG), although its efficacy in preventing pulmonary tuberculosis is controversial. Early interactions between dendritic cells (DC) and BCG or Mycobacterium tuberculosis (Mtb) are thought to be critical for mounting a protective antimycobacterial immune response. Recent studies have shown that BCG and Mtb target the DC-specific C-type lectin intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) to infect DC and inhibit their immunostimulatory function. This would occur through the interaction of the mycobacterial mannosylated lipoarabinomannan to DC-SIGN, which would prevent DC maturation and induce the immunosuppressive cytokine interleukin (IL)-10 synthesis. Here, we confirm that DC-SIGN is expressed in DC derived from monocytes cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4 and show that it is not expressed in DC derived from monocytes cultured in GM-CSF and interferon-α (IFN-α). We also demonstrate that DC-SIGN– DC cultured in GM-CSF and IFN-α are able to phagocytose BCG and to undergo a maturation program as well as DC-SIGN+ DC cultured in IL-4 and GM-CSF. We also show that BCG causes the impairment of IL-12 and the induction of IL-10 secretion by DC, irrespective of DC-SIGN expression. Finally, we demonstrate that the capacity to stimulate a mixed leukocyte reaction of naïve T lymphocytes is not altered by the treatment of both DC populations with BCG. These data suggest that DC-SIGN cannot be considered as the unique DC receptor for BCG internalization, and it is more interesting that the mycobacteria-induced immunosuppression cannot be attributed to the engagement of a single receptor.