ALBERT

Description: Abstract 4574 Leukocytosis has been documented as a risk factor for thrombotic events in various clinical conditions such as ischemic heart diseases, polycythemia vera, intensive chemotherapy and stem cell transplantation (SCT). Allogeneic (allo) SCT is characterized with a unique peri-engraftment phase simultaneously harboring T cell alloreactivity, endothelial damage, leukocyte elevation and an abundance of variable secreted cytokines. Whether leukocytes are a causative or surrogate marker for the underlying process is not concluded and whether leukocytosis is linked to the severity of acute graft-versus-host disease (aGVHD) has not been previously elucidated. The study included 59 consecutive patients undergoing matched related or unrelated allo SCT for hematological malignancies at the Rambam Medical Center, Haifa, Israel in 2010. Median age 45 years (21–66), 35 males and 24 females. Conditioning protocols were either myeloablative with busulfan/cyclophosphamide or reduced intensity (busulfan or melphalan based). Patients' clinical course and blood counts were followed for 2 months and categorized into 3 groups according to their post-engraftment leukocyte counts and leukocytosis duration. The outcome is summarized in the following table:GroupLeukocyte countNo. patientsMedian days to engraftmentMedian days to leukocyte 〉10x103/μl120-day mortalityCause of death1〈 10X103/μl16+154/162 respiratory failure,1 relapse, 1 septic shock2〉 10x103/μl (〈 7days)27+13+362/271 aGVHD (GI grade 4), 1 SOS*3〉 10x103/μl (〉 7 days)16+11+2510/164 aGVHD (GI grade 4), 4 thrombotic events**, 2 idiopathic pneumonia with end organ failure and infective complications*SOS- sinusoidal obstructive syndrome**2 SOS, 2 thrombotic thrombocytopenic purpura +120 days were chosen as the cutoff to include patients dying of the mentioned complication after day +60. The results summarized in the table suggest that patients with early and protracted leukocytosis post engraftment are at increased risk for severe and fatal complications (group 3). The 16 patients in this cohort included 9 in complete remission (CR); 7 with active disease; 5 sibling and 11 matched unrelated donor (MUD) SCTs; 6 myeloablative and 10 reduced intensity (RI) conditioning. This mix was similar to that of patients in groups 1 and 2. Leukocytosis occurring at a later phase (group 2) seems to have minimal or no impact on survival during the first 120 days post transplant. All deceased patients had documented platelet engraftment, although few did receive platelet concentrates as indicated clinically. In conclusion, protracted leukocytosis post engraftment in allo SCT patients may be an independent risk factor not only for fatal thrombotic events but also for aGVHD, mostly gastrointestinal, although larger studies are needed to confirm this. The peri-engraftment syndrome may have long-term prognostic and therapeutic implications, beyond the immediate clinical management. Given that such patients are still with endothelial dysfunction and within a rich milieu of cytokines, incorporation of anti-aggregating agents (such as anti p-selectin or GPIIb/IIIa) in the treatment protocol at this stage may have a role and should be investigated. Furthermore, mechanisms leading to such leukocytosis also need to be assessed. Disclosures: No relevant conflicts of interest to declare.

Description: Mucorales-specific T cells were investigated in 28 hematologic patients during the course of their treatment. Three developed proven invasive mucormycosis (IM), 17 had infections of known origin but other than IM, and 8 never had fever during the period of observation. Mucorales-specific T cells could be detected only in patients with IM, both at diagnosis and throughout the entire course of the IM, but neither before nor for long after resolution of the infection. Such T cells predominantly produced IL-4, IFN-γ, IL-10, and to a lesser extent IL-17 and belonged to either CD4+ or CD8+ subsets. The specific T cells that produced IFN-γ were able to directly induce damage to Mucorales hyphae. None of the 25 patients without IM had Mucorales-specific T cells. Specific T cells contribute to human immune responses against fungi of the order Mucorales and could be evaluated as a surrogate diagnostic marker of IM.

Description: Abstract 3229 Introduction: Several studies have reported that different components of fungi of the genera Aspergillus spp may induce protective T-cell responses in either mouse models of invasive aspergillosis (IA) or in human healthy subjects. We evaluated the occurrence of Aspergillus-specific T-cell responses to different Aspergillus recombinant antigens in patients with proven IA, during the course of the IA, to identify the antigens most frequently targeted by protective immune responses. We characterized phenotypically and functionally such specific T cells. Finally, from peripheral blood (PB) samples of the same IA proven patients, also collected during the active infection phase, we sought to expand such Aspergillus-specific T cells. Methods: 15 patients with proven IA, according to the EORTC/MSG criteria, have been enrolled into the study. Specific immune responses producing interleukin-10 (IL-10), interferon-gamma (IFN-γ), IL-4 and IL-17A were detected and characterized by enzyme linked immunospot (ELISpot) assay and cytokine secretion assay (CSA), in all the patients, during the entire course of the IA. The recombinant antigens used were GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, and galactomannan (GM). Cytotoxicity has been investigated by means of the colorimetric assay with (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxyanilide) sodium salt plus coenzyme Q0 (XTT assay). Aspergillus-specific T cells were obtained by culturing PB mononuclear cells with a mixture containing PEP1p, GEL1p, α1–3 glucan and β1–3 glucan. The infection course were divided into 4 phases, defined from t1 to t4, each of fifteen days interval, starting from the radiological diagnosis of IA. Results: Aspergillus-specific T cells producing either IL-10 or IFN-γ were detected to all the antigens, but GM. The number of antigens targeted by IFN-γ producing specific T cells progressively increased along the course of IA, being such protective responses to 3 out of 7 antigens at t1 and to 6 out of 7 antigens at t4. At t1, IFN-γ producing specific T cells were only detected to GEL1p, α1–3 glucan and β1–3 glucan. GEL1p and α1–3 glucan resulted the antigens most constantly targeted by IFN-γ producing specific T cells, persisting the responses to these antigens in all the phases of IA. No Aspergillus-specific T cells producing IL-4 to any antigens were detected by the ELISpot assay. Aspergillus-specific T cells producing IL-17A were detected in only one out of 15 patients, and targeted CRF1p. Specific T cells to GM and specific T cells producing IL-4 to all the antigens could be shown only by CSA, suggesting that they are present only at very low frequencies during the infection. After 13-day cultures, Aspergillus-specific T cells were expanded from five out of five patients. The specific T cells tested for lytic activity included a median of 95.8% CD3+ cells, either CD4+ or CD8+ T cells, and showed a median lytic activity of 9.45% either at 3/1 or at 5/1 effector/target cells ratios. Conclusions: In patients with IA, protective immune responses may be detected since the onset and increase during the infection. At the onset of IA, specific T cells producing IFN-γ target antigens involved in the cell wall biosynthesis of Aspergillus. On the other hand, at the same phase, specific protective immune responses to CRF1p, PEP1p, and SOD1p, which are all putative virulence factors for Aspergillus, are absent. Aspergillus-specific T cells may be expanded by a mixture of antigens, even in the course of IA and are able to directly kill fungal hyphae. The above mentioned antigens and the corresponding protective T cells may be exploited for therapeutic strategies of either vaccine or autologous cytotoxic cell infusions in patients at high risk for IA. Disclosures: Luppi: MSD; GILEAD: Research Funding.

Description: Abstract 4959 Few epidemiological data about IFI in chronic lymphoid malignancies are available in literature. Our aim is to describe epidemiology, clinical manifestations and outcome of IFI in these patients (pts). We reviewed the records of pts with lymphoproliferative disorders, treated between 2004 and 2010 for probable/proven IFI, according to the revised criteria of EORTC/MSG. We registered 40 probable/proven IFI. Twenty-five pts were affected by lymphoma, 9 by CLL, 3 by Waldenström macroglobulinemia, 2 by multiple myeloma and 1 by hairy cell leukemia. The median age was 57 years (r: 17–71). Twenty-nine pts (75%) had progressive/relapsed hematological disease, and 75% were treated with multiple lines of chemotherapy. Risk factors for IFI were: neutropenia (11 pts), previous solid organ transplant (3) or allogeneic HSCT (3), treatment with high dose steroids (3), monoclonal antibodies (MABs) such as rituximab (9) and alemtuzumab (3), nucleosidic analogues (3) or multiple of these factors (8). Regarding mortality the following factors were evaluated: deep and prolonged neutropenia, multiple lines chemotherapy, MABs and purine analogues administration, diagnosis and status of the underlying disease. Incidence of IFI was 2,7%; (moulds 2%, yeasts 0,4%, mixed infections 0,3%). We recorded 7 yeast infections: 6 were documented by cultures (3 C. albicans, 2 C. glabrata and 1 Blastoschyzomices capitatus), and 1 by the microscopic observation of Candida spp in the vitreum. Thirty pts developed an invasive mould infection (IMI); 3 of them had a proven pulmonary IMI diagnosed by histology (2 Aspergillus spp, 1 Mucor). Twenty-seven pts had a probable IMI: 24 had lung involvement, 2 a sinusal localization, and 1 a pulmonary infection disseminated to brain. Mycological criteria were more often provided by the positivity of the galactomannan antigen (GM, 14 pts) in serum and/or BAL fluid. Cultures resulted positive in 12 cases (5 A. fumigatus, 2 Aspergillus spp, 2 A. flavus, 1 A. niger, 1 Fusarium and 1 for both Scedosporium and Aspergillus); in 4 of them, both GM and culture positivity from the BAL fluid were present. Finally, we observed 3 mixed infections by both moulds and yeasts: in 2 pts a proven yeast infection with isolation in the blood culture (1 C. albicans, 1 C. glabrata) was associated to a probable IMI with the positivity of CT scan and GM; in 1 pt autopsy revealed a pneumonia by Candida spp and a disseminated infection by Aspergillus spp. Eight of 40 pts died due to infection, but only in one pt IFI was the unique cause of death. IFI attributable mortality rate was 20%. At the univariate analysis, the only risk factor related to mortality was the administration of multiple lines of chemotherapies (p 0.04). Disclosures: No relevant conflicts of interest to declare.