Stockholm, Sweden, June 13–16, 2013

Granulocytes P1024 LIPEGFILGRASTIM–A LONG-ACTING, ONCE-PER-CYCLE, GLYCOPEGY - LATED RECOMBINANT HUMAN FILGRASTIM CREATED WITH SITE-SPE - P1022 CIFIC GLYCOPEGYLATION TECHNOLOGY C Scheckermann 1,* , K Schmidt 1, A Abdolzade-Bavil 1, H Allgaier 2, U Mueller 3, IMMUNOGENICITY OF LIPEGFILGRASTIM AND PEGFILGRASTIM IN W Shen 4, P Liu 5 BREAST CANCER PATIENTS RECEIVING CHEMOTHERAPY: INTEGRAT - 1BioGeneriX GmbH, 2Merckle Biotec GmbH, 3Teva GmbH, Ulm, Germany, ED ANALYSIS FROM PHASE II AND III STUDIES 4Teva Pharmaceuticals, West Chester, 5Teva Pharmaceuticals, Washington A Abdolzade-Bavil 1,* , L Zou 2, C Sadhu 3, A Buchner 4, P Liu 3 DC, United States 1BioGeneriX GmbH, Ulm, Germany, 2Teva Pharmaceuticals, Rockville, 3Teva Biopharmaceuticals, Washington DC, United States, 4Teva ratiopharm, Ulm, Background: Lipegfilgrastim is a once-per-cycle, fixed-dose glycoPEGylated Germany granulocyte-colony stimulating factor (G-CSF) under development for the preven - tion of severe neutropenia in cancer patients receiving chemotherapy (CTx). Tra - Background: Lipegfilgrastim is a novel, long-acting, fixed-dose, glycoPEGy - ditional PEGylation of biologic molecules has been used successfully for more lated recombinant granulocyte-colony stimulating factor (rG-CSF) under review than 10 years to extend the half-life in the body, requiring less frequent dosing for neutropenia prevention in patients receiving myelosuppressive chemother - and allowing for the administration of G-CSF once per CTx cycle, making treat - apy (CTx) for cancer. Like any biological product, lipegfilgrastim could poten - ment potentially less expensive and enhancing patient compliance and safety. tially elicit an anti-drug antibody (ADA) response, which could cause adverse However, the traditional PEGylation use of chemical conjugation through reac - events (AEs) or lack of efficacy. tive groups on amino acids can result in multiple PEGylated isoforms with reduced Aims: To assess the immunogenicity of lipegfilgrastim compared with pegfil - protein activity, as well as non-uniform chemical and pharmaceutical properties. grastim using data from a Phase II and a Phase III study in patients receiving Aims: To describe a highly site-specific glycoPEGylation technology for site- CTx for breast cancer, and to correlate ADA test results with clinical events. directed PEGylation and the implications of the technology with regard to Methods: In the Phase II study, breast cancer patients undergoing CTx improved outcomes, and to summarize preclinical findings for lipegfilgrastim received a subcutaneous (s.c.) injection of lipegfilgrastim3, 4.5, or 6 mg or peg - compared with pegfilgrastim (Neulasta ®). filgrastim 6 mg on Day 2 of each of four CTx cycles. In the Phase III study, Methods: Glycosylation sequon scanning was used to identify the glycoPEGy - breast cancer patients undergoing CTx received an s.c. injection of 6 mg lipeg - lation site with the least impact on protein activity. Unlike endogenous G-CSF, filgrastim or pegfilgrastim on Day 2 of each of four cycles. In both studies, recombinant G-CSF, expressed in Escherichia coli , is not O-glycosylated. E. serum samples were taken before each cycle, at the end of the study (Day 85), coli- expressed G-CSF was selectively glycosylated at the natural O-glycosyla - and on Day 180 and Day 360 during the 360-day follow-up period. Samples tion site, and a polyethylene glycol (PEG) sialic acid derivative was attached were analyzed using multi-tiered ADA assays based on Mesoscale Discovery using a sialyltransferase (glycoPEGylation technology). Ligand binding affinity technology. Screening-reactive samples were confirmed by immunocompeti - was assessed using the BIACORE 3000 system. Biologic activity of lipegfilgras - tion. All confirmed ADA positive (ADA+) study samples were measured for titer tim was assessed in an NFS-60 cell line proliferation assay versus filgrastim and and analyzed with the NFS-60 cell proliferation assay for neutralizing activity pegfilgrastim. Pharmacokinetic (PK) and pharmacodynamic properties were against lipegfilgrastim or pegfilgrastim and endogenous G-CSF. The confirmed studied in healthy and neutropenic animal models. ADA+ samples from lipegfilgrastim-administered patients were also character - Results: The use of the naturally present glycosylation site to achieve enzy - ized for ADA specificity towards XM21 (G-CSF moiety of lipegfilgrastim) and matic coupling of an activated PEG-sugar conjugate to the filgrastim polypep - PEG moiety, as well as cross-reactivity to endogenous G-CSF by competition tide produces a glycoPEGylated, long-acting G-CSF which is similar in struc - with XM21, PEG, and glycosylated G-CSF, respectively. ture to the native glycosylated protein. Lipegfilgrastim also showed improved Results: The incidence of ADAs is summarized in the Table 1. In the Phase II in vivo PK profiles in animal models. In vitro , lipegfilgrastim had binding affini - study, only two subjects had antibodies related to lipegfilgrastim treatment. ty and specific activity comparable to pegfilgrastim, while both were slightly Among them, one subject had antibodies binding to the PEG moiety, and titer lower than non-PEGylated filgrastim. Comparable increases in leukocytes, neu - for anti-lipegfilgrastim antibodies was found to be 0.6 on Day 85 only. Another trophilic granulocytes, and monocytes were seen with lipegfilgrastim and peg - subject had antibody titer of 0 and the antibodies did not bind to either the pro - filgrastim in rats and monkeys and were consistent with the effects expected tein or PEG moiety, indicating a weak or possibly false-positive response. In for a long-lasting G-CSF. No adverse effects on organ systems were reported pegfilgrastim-treated patients, only one subject was found to have antibody after a dose 100 times higher than the recommended human dose. response to the treatment. Antibody titer and binding specificity were not inves - Summary and Conclusions: The glycoPEGylation technology platform tigated. In the Phase III study, only one subject had antibodies related to lipeg - described here is used to produce lipegfilgrastim, a novel, biologically active G- filgrastim treatment, with the titer value being 1.2 and 2.1 on Days 180 and 360, CSF with greater structural homogeneity and comparable pharmacologic prop - respectively. Antibody specificity characterization showed that the antibodies erties to conventionally PEGylated G-CSFs, thus enabling a safe and effective bind to the protein moiety of lipegfilgrastim. In pegfilgrastim-treated patients, once-per-cycle fixed dosing regimen. only one subject was found to have antibody response to the treatment. Anti - body titer and binding specificity were not investigated. ADA+ samples did not exhibit neutralizing activity against lipegfilgrastim, pegfilgrastim, or endogenous P1025 G-CSF. Unexpected AEs or lack of efficacy were not observed for patients with SAFETY AND TOLERABILITY OF LIPEGFILGRASTIM IN BREAST CAN - confirmed ADA+. CER PATIENTS RECEIVING CHEMOTHERAPY: AN INTEGRATED ANALY - SIS OF PHASE II AND III STUDIES O Gladkov 1,* , R Elsaesser 2, A Buchner 2, P Bias 2, U Mueller 2 Table 1. Incidence of confirmed anti-drug antibodies. 1Chelyabinsk Regional Clinical Oncology Center, Chelyabinsk, Russian Fed - eration, 2Teva ratiopharm, Ulm, Germany

Background: Myelosuppresive chemotherapy (CTx) frequently causes neu - tropenia, leading to an increased risk of infections and delays in subsequent chemotherapy treatments. Covalent attachment of polyethylene glycol (PEG) has been shown to prolong the half-life of therapeutic proteins, allowing for less frequent dosing, improved patient compliance, and potentially lower production costs. However, traditional PEGylation methods rely on chemical conjugation through amino acid reactive groups, which may reduce protein activity and result in non-uniform chemical, pharmaceutical, and tolerability profiles. Lipegfilgrastim Summary and Conclusions: Immune response following lipegfilgrastim is a highly homogenous once-per-cycle, fixed-dose glycoPEGylated granulocyte- administration was comparable to pegfilgrastim and was not considered clini - colony stimulating factor (G-CSF) created with a highly site-specific glycoPEGy - cally significant. Serum ADA status did not appear to impact the clinical safety lation technology for site-directed PEGylation, which is under review for the pre - and efficacy of lipegfilgrastim. vention of neutropenia in cancer patients receiving CTx. Non-inferiority of lipeg - filgrastim versus pegfilgrastim (Neulasta ®) was previously demonstrated in a Phase III trial conducted in CTx-naïve breast cancer patients. 1 Aims: To report the safety and tolerability findings from an integrated analysis of two Phase II/III studies of lipegfilgrastim versus pegfilgrastim in CTx-naïve breast cancer patients. Methods: All patients who received full-dose CTx (doxorubicin 60 mg/m 2+doc - etaxel 75 mg/m 2) and either a single 6 mg subcutaneous injection of lipegfil - grastim or pegfilgrastim were included in this analysis. The incidence and sever - ity of treatment-emergent adverse events (TEAEs) and those the investigator deemed related to study drug (TEADRs) over all four cycles were compared.

haematologica | 2013; 98(s1) | 421 18 th Congress of the European Hematology Association

Results: TEAEs were experienced by 94.7% of patients in the lipegfilgrastim P1027 group (N=151) and 89% of patients in the pegfilgrastim group (N=155). The most common TEAEs were consistent with the effects of CTx and the under - A FEASIBILITY STUDY OF HOME MONITORING OF BLOOD NEUTROPHIL lying disease and were not necessarily due to G-CSF study medication. Fre - COUNTS IN PATIENTS WITH CHRONIC NEUTROPENIA D Dale 1,* , M Kelley 1, V Makaryan 1, E Rodger 1, A Bolyard 1, B Otto 1 quencies of TEAEs were comparable between treatment groups. As expect - 1 ed, there was a time-dependent trend across cycles within each treatment Medicine, UNIVERSITY OF WASHINGTON, Seattle, United States group; i.e. the frequencies of TEADRs decreased with each subsequent CTx cycle. Severe TEADRs were reported in one lipegfilgrastim patient and two peg - Background: Currently patients with chronic neutropenia and other hemato - filgrastim patients. There were no deaths as a result of study drug treatment in logical disorders must go to a doctor’s office, medical laboratory or hospital for either group. TEADRs were consistent with the underlying medical condition monitoring of their hematological status. For some chronic diseases, e.g. , cyclic and administration of CTx. Type, incidence, and severity of TEADRs are pro - and congenital neutropenia, an accurate diagnosis depends on obtaining a vided in the Table 1. series of daily or every-other-day counts over several weeks, but many patients find it difficult to comply with the number of laboratory visits that are required. It would be far more convenient if their blood cell counts could be monitored at home. Home monitoring may prove to be useful for monitoring blood counts for Table 1. Type, incidence, and severity of TEADRs. patients at risk of developing febrile neutropenia, for reducing number of cost - ly visits to medical laboratories and for reducing patients’ exposure to new pathogens outside the home environment. Aims: To determine if patients can learn to use a small home monitoring device to measure their own blood cell counts on a daily basis and to compare the accuracy of patient testing versus testing by a skilled laboratory technician. Methods: Ten patients with cyclic and idiopathic neutropenia currently enrolled in the Severe Chronic Neutropenia International Registry (SCNIR) will be enrolled. Initially the patients will make three visits to the laboratory to learn to collect blood using sterile finger puncture techniques and to use the counting device (XBC, Philips, Eindhoven, NL). At each visit the patient and the techni - cian perform counts using the XBC device and the technician makes a control count using a venous blood sample and a reference electronic counter (BC, Beckman Coulter, Miami, FL). After the third laboratory visit, the patient takes the XBC device home to monitor his/her own counts on a daily basis for 28 days. Patients return to the laboratory each week of the study period for fol - low-up in-laboratory counts by the patient and technician. Results: Initially we tested three normal donors, six tests on each of three XBCs and 4 repeat tests of each sample on BC and observed no significant Summary and Conclusions: Lipegfilgrastim has a favorable safety profile differences (two-way ANOVA). consistent with the G-CSF class of molecules and is an acceptable alternative We have enrolled four patients, three patients were receiving granulocyte to pegfilgrastim for the prevention of neutropenia in cancer patients receiving colony stimulating factor (G-CSF) and one was being treated with prednisone. chemotherapy. The patients and technicians both performed finger sticks and both have per - formed tests with XBC. The technician has performed counts on venous blood Reference using the BC device as the reference control. Teaching and learning proceed - 1. Bondarenko I et al. J Clin Oncol . 2012;30 (suppl; abstract e19587). ed smoothly and easily. Comparison of counts performed in the laboratory with the XBC device vs the BC counter are shown in Table 1. There were no sig - nificant differences in results for the two instruments (two-way ANOVA or Stu - P1026 dents t-test). Other comparisons were: patients vs. technician in lab with XBC and patients using XBC in lab or at home. There were no significant differences. CARDIAC INVOLVEMENT IN TYPE 3 EGYPTIAN GAUCHER DISEASE For the two patients who have completed the 28 day study, compliance rates PATIENTS: A REPORT OF 7 CASES AND A NEWLY REPORTED ANOM - for daily counts for were: 75% and 86%, the lower percentage for the first ALY M Abdelwahab 1,* , H Hamza 2, W Attia 2, A Beshlawy 1, K Eid 1 patient due to running out of testing cassettes. Error rates requiring repeat 1Pediatric Hematology, 2Pediatrics, Cairo University Pediatric Hospital, Cairo, counts were 26% and 17% and both patients regularly repeated the finger-stick Egypt sampling and counting on the second try. Patients 3 and 4 have quickly learned counting techniques, are maintaining their records and will soon complete the study. The discomfort of the finger sticks has not limited the study. The six addi - Background: Gaucher disease(GD) is due to deficiency of B- glucocerebrosi - dase leading to depodtion of lipid laden macrophages(Gaucher cells) typical - tional patients will be studied in the spring 2013. ly in the liver, spleen, bone marrow, lymp nodes, lungs, brain and very rarely the heart usually associated with D409H/D409H mutation.Cardiac involvement includes constrictive pericarditis, pericardial calcification, infiltration of the Table 1. Comparison of blood neutrophil count for four patients using test myocarduim leading to cardiomyopathy and calcification of the mitral, aortic (XBC) vs reference (CB) instrument. valves and aortic root. Aims: To report cardiac findings in 7 Egyptian type 3 GD Methods: Four D409H/D409H patients, a 20 year old male with typical phe - notype of D409H/D409H and 2 L444P/L444P children with cardiomeagly on ¥- ray were assessed for cardiac anomalies.The D409H/D409H patients did Mul - tislice CT angiography, CT brain and ophthalmologic assessment. Results: The 3 female D409H/D409H siblings(19.17.11 years old showed mitral regurge(MR); aortic regurge(AR), aortic root calcificationand ectatic prox - imal coronaries; thickened aortic valve, increased echogenicity of aortic root and MR respectively. They also had increased reflectivity of cornea, apraxia and hydrocephalic changes,The 15 year old female with cardiac symptoms Summary and Conclusions: Based on this initial experience, we believe that showed MR, MS, AR and calcification of the aorta extending to the coronaries the XBC counter is reliable and accurate for measurement of WBC and ANC. as well as apraxia and hydrocepahlic changes.The fifth patient had severe We find that patients can quickly learn to use this device to monitor their blood valvular affection, corneal opacities, apraxia and died postopeartively.The 2 leukocyte and differential counts at home. We believe that home monitoring is L444P/L444P children had dilated cardiomyopathy and coarctation that showed a promising strategy for diagnostic testing and evaluation of treatment respons - improvement. es in patients with neutropenia. Summary and Conclusions: This is the first report of coronary ectasia in GD.Cardiac affection in GD can invlove other mutations than D409H/D409H which should be screened routinely to pickup anomalies early. P1028 GRANULOCYTE FUNCTION IS NOT IMPAIRED BY ARGININE DEFICIENCY K Kapp 1, S Prüfer 2, A Habermeier 3, C Luckner-Minden 4, J Bomalaski 5, P Kropf 6, I Müller 7, E Closs 3, M Radsak 4, M Munder 4,* 1Institute of Immunology, University of Heidelberg, Heidelberg, 2Institute of Immunology, 3Department of Pharmacology, 4Third Department of Medicine

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(Hematology, Oncology, and Pneumology), University Medical Center of the RO+): disease progression n=2 and viral pneumonia n=1. The Kaplan Meier 5 year Johannes Gutenberg University, Mainz, Germany, 5Polaris Pharmaceuticals Overall Survival (OS) is (96.3%), the 5 y Event Free Survival (EFS) is (54.7%): Inc., San Diego, United States, 6Department of Immunology and Infection, Lon - Group I (93%), Group II (52%), Group III (47%), Group IV (83%) p0.013. The EFS don School of Hygiene and Tropical Medicine, 7Department of Medicine, Sec - is worse if age <2 years old (38%) vs (71%) (2-10 y), vs 90% (>10 y) p0.001. EFS tion of Immunology, Imperial College, London, United Kingdom is worse in patients received a 2 nd induction (48,7%) vs (80.5%) p0.009. Respon - se The 6 week evaluation from diagnosis revealed (75%) better. This percentage Background: Arginine deficiency is often encountered in cancer patients and rose to 89% at W12 with or without induction(s). Progression /Reactivation Reac - has all the characteristics of a double-edged sword: on the one hand, it is tivation occurred n=25 (38%), while progression post induction n=3 (4.5%). The induced by arginase expressing myeloid cells (neutrophil granulocytes or probability of reactivation or progression is 47%. Reactivation is less in prolonged myeloid-derived suppressor cells) in the context of cancer-related inflammation. maintenance as 37 patients non reactivated with a mean 10 m maintenance dura - The ensuing arginase-mediated arginine deficiency may profoundly suppress tion vs 25 reactivated with a 8 m treatment duration p0.043. the adaptive immune response by inhibiting T cell proliferation and cytokine syn - Summary and Conclusions: In Egyptian LCH children, response to induction thesis. On the other hand, arginine depletion might have anti-tumoral potential, is satisfactory. Although a greater incidence of reactivation, survival remains the especially against tumors that are unable to resynthesize their own arginine rule. Low age and slow responder carry a worse prognosis. With RO+ from the precursor amino acid citrulline via argininosuccinate synthase. Phar - methotrexate, 6MP reinforcement, EFS is nearly equivalent to RO-. However macological arginine depletion via pegylated arginine deiminase (ADI-PEG20) mortality are exclusively RO+. is already in clinical phase 1-3 human cancer trials. Aims: Given the profound suppressive effect of arginine deficiency on adap - tive immunity, we now studied the function of granulocytes in the context of argi - P1030 nine depletion. PEGFILGRASTIM AS PROPHYLAXIS OF FEBRILE NEUTROPENIA DUR - Methods: Human primary granulocytes were purified from healthy donors and important granulocyte effector functions were measured in cell culture ING CHEMOTHERAPY OF RELAPSED AND REFRACTORY MULTIPLE medium in the presence or in the absence of arginine. Arginine deficiency MYELOMA C Cerchione 1,* , N Pugliese 1, L Marano 1, G Cerciello 1, S Avilia 1, F Pane 1, itself was induced either by using arginine-free preformulated medium or by L Catalano 1 subjecting normal cell culture medium or whole blood to sonicates of human 1Hematology, AOU “Federico II”, Napoli, Italy granulocytes with a defined arginase activity, leading to the enzymatic deple - tion of arginine. The following granulocyte functions were measured in vitro : Background: Patients receiving cancer chemotherapy are at an increased risk viability (PI/annexin V), expression of various cell membrane marker pro - of neutropenia, leading to an increased risk of infections and delays in subse - teins, activation-induced shedding of CD62L (flow cytometry), phagocytosis ® quent chemotherapy treatments. Recombinant granulocyte colony stimulating (FITC-coupled E. coli : Phagotest ), microbicidal activity (XTT viability of C. factors (G-CSFs) have been developed to stimulate proliferation and differen - albicans ), generation of reactive oxygen species (cytochrome C reduction ® tiation of neutrophils in patients receiving chemotherapy. Pegfilgrastim is a and oxydation of dihydrorhodamine 123: Phagoburst ), chemotaxis (human pegylated long-acting recombinant form of G-CSF that extends the half-life and serum-induced migration in Boyden chamber), activation-induced synthesis allows for once-per-cycle dosing, requiring less frequent dosing than nonpegy - of IL-8 (ELISA) and various other gene products (Real Time PCR). Granulo - lated G-CSF. Multiple Myeloma (MM) in advanced phases of disease may be cyte function was also analysed in vivo in a murine model of Aspergillus fumi - managed by regimens combining agents not frequently employed in early phas - gatus pneumonia and arginine depletion in vivo was induced by injection of es of treatment ( e.g. Anthracyclines, Alkylating agents, etc), but myelotoxicity ADI-PEG20. is the main expected side effect.In this context, G-CSFs are often necessary Results: Arginine deficiency had no influence on human granulocyte viability, to counteract the risks of febrile neutropenia: their use is bound to a frequent expression of activation markers and constitutive cell membrane proteins, shed - evaluation of neutrophil counts that may not be easy for patients in home-care. ding of CD62L, phagocytosis, generation of reactive oxygen species, fungicid - Avoiding severe neutropenia by prophylactic pegfilgrastim seems particularly al activity, chemotaxis and induced synthesis of IL-8 or any other gene prod - useful in these cases. ucts on mRNA level. Also, profound pharmacological arginine depletion in vivo Aims: The objective of this study was to compare the efficacy and safety of peg - via ADI-PEG20 did not inhibit murine granulocyte functions: successful granu - filgrastim in patients affected by Multiple Myeloma in advanced phase of dis - locyte-dependent clearance of Aspergillus fumigatus and survival of mice were ease. In order to determine whether a single subcutaneous injection of pegfil - not impaired. grastim is as effective as daily injections of standard filgrastim, in terms of Summary and Conclusions: Granulocyte-based innate immune function is not haematological toxicity, febrile neutropenic episodes, antibiotic usage and inhibited by arginine deficiency. This novel finding adds to a better understand - hospedalization duration. ing of immunity during cancer inflammation-associated arginine depletion and Methods: We have enrolled in our study 18 patients (10 male and 8 female) is also important for the development of therapeutic arginine depletion as anti- with a median age of 62 years (range 54-82) affected by multiple myeloma, all metabolic tumor therapy. Our results are also consistent with the lack of co-exis - relapsed and refractory to a median of 6 lines of therapy (range 4-8), all previ - tent infections in humans treated with ADI-PEG20. ously exposed to Bortezomib, Lenalidomide, Melphalan and all relapsed after auBMT, treated with different chemotherapy regimens combining Bortezomib, Lenalidomide, Bendamustine, Melphalan, Doxorubicine. P1029 Results: Since first course, received in our out-patient department, patients per - formed blood counts twice weekly and received, from day 8 to day 19, prophylac - LANGERHANS CELL HISTEOCYTOSIS (LCH) IN EGYPTIAN CHILDREN. A tic oral chinolonic antibiotics and anti-fungal drugs. Filgrastim (5 μgr/kg/day for 3 SINGLE CENTER EXPERIENCE. days) was given if neutrophils count was <1500 ¥10^9 cells/L. Median number of M Sedky 1,* , H Sayed 1, E Moussa 1 filgrastim administrations was 4.5, r. 3-6; nadir neutropenia was registered after a 1Pediatric hematology Oncology, Children Cancer Hospital, Cairo, Egypt median of 11 days (r. 8-14); median of nadir neutrophil count was 1.27 ¥10^9 cells/L (range 0.4–1.8 ¥10^9 cells/L), with maximum duration of 10 days. After the Langerhans cell histiocytosis (LCH) is a rare immune multisys - Background: first course of chemotherapy, patients received prophylactic pegfilgrastim (6 mg), tem disease that behaves from spontaneous regression to rapid progression injected subcutaneously with a single administration on day 2 after last dose of and death or repeated reactivations especially upon end of treatment. Local - chemotherapy, independently from the neutrophil count at that time. Primary end - ized monostotic disease (skin, bone or lymph node) have a good prognosis with - point was the duration of neutropenia (neutrophil count <1.5 ¥10^9 cells/L), which out treatment. In contrast, multiple, especially high risk organs (liver, spleen, was never longer than 10 days. Median nadir neutrophil count, registered 11 days hemopoietic system) have a poor outcome and may need chemo reinforcement. after the end of the therapy (median, r. 9-15) was 1.576 (range 0.63-2.25 ¥10^9 To evaluate children cancer hospital Egypt experience in pediatric Aims: cells/L); two patients (11%) needed a second administration of pegfilgrastim one Langerhans cell Histeocytosis week after the first (day +9,+10 and +11, respectively) and also a supplement of In the period between 12/7/2007 and 9/12/2011, 80 LCH patients Methods: 3 administrations of filgrastim. During pegfilgrastim, neutropenia lasted less than were treated at Children Cancer Hospital (CCHE) according to the LCH III pro - 2/12 10 days. Pegfilgrastim was well tolerated in all patients: main side effects in our tocol. M/F: 43 (46%)/37 (54%) with a mean age of 4 years (3 months–13 patients were fever and bone pain, (3/18 patients,16%) 3/12 y). Patients were divided into 4 groups: I monostotic lesion n=14 (17.5%), Summary and Conclusions: In conclusions, in patients affected by MM II multisystem with no risk organs RO- n=29 (36%), III multisystem with risk exposed to myelosuppressive agents in advanced phases of myeloma dis - organs RO+: n=19 (24%) and IV unisystem multifocal lesions +/- special sites: ease, pegfilgrastim seems to reduce the incidence of neutropenia and may n=18 (22.5%). Group I did not receive systemic treatment. Vinblastine (VBL) increase the possibility to maintain the scheduled time of treatment. and Prednisone (PRED) were given as one +/- 2 nd induction in Group II and IV, and were associated to methotrexate(MTX)in group III. Maintenance includ - ed 6 m to 1 year VBL, PRED for group IV and II respectively. Group III had a 1 P1031 year VBL, PRED, MTX and 6 mercaptopurine (6MP). Results: Survival After a minimum 1 year and up to 5.5 year follow up, 70 patients CHRONIC BENIGN NEUTROPENIA IN ADULTS: LABORATORY AND CLIN - are better (87.5%) in No Active Disease (NAD) or Active Disease Regressive ICAL PARAMETERS OF A 6-YEAR FOLLOW-UP (ADR), 3 patients are intermediate(4%) in Active Disease Stable (ADS), 3 patients B Fattizzo 1,* , T Radice 1, F Guidotti 1, A Zaninoni 1, A Ciani 2, A Cortelezzi 2, are worse (4%)in Active Disease Progressive (ADP), 3 patients (4%) died (all in W Barcellini 1

haematologica | 2013; 98(s1) | 423 18 th Congress of the European Hematology Association

1U.O. Ematologia e Centro Trapianti di Midollo, 2U.O. Ematologia e Centro microorganisms; however, it may be also detrimental to the body due to tissue Trapianti di Midollo, Università degli Studi di Milano, Fondazione IRCCS Ca’ damage. The role of neutrophils in septic shock has not been well investigat - Granda Ospedale Maggiore Policlinico, Milano, Milano, Italy ed. Recently, changes were reported in the neutrophil surface antigens CD64 (high affinity receptor for IgG), CD10 (neutral endopeptidase), and CD16 (low Background: Chronic benign neutropenia (CBN) is a rare hematological con - affinity Fc receptor) during sepsis and DIC. dition, characterized by an absolute neutrophil counts (ANCs) lower than Aims: The aim of this study was to evaluate RBA and surface antigen expres - 1800/µl in white and 1500/µl in black people for more than 3 months. This con - sions of neutrophils in septoc patients to determine their clinical significance. dition can be congenital or acquired, idiopathic or secondary and may be char - Methods: EDTA peripheral blood was drawn from 105 healthy controls and con - acterized by the presence of anti-neutrophils antibodies. While congenital and secutive 71 patients (52 males and 19 females; median age, 67 years; range, acquired forms of infancy and childhood are largely studied, with particular 29–92 years) with severe sepsis (16 patients) and septic shock (55 patients) from attention to the infective diathesis, less is known about adult neutropenia. January 2011 to October 2012. Using flow cytometry, RBA was measured in non- Aims: To evaluate clinical and laboratory features of chronic benign neutrope - stimulated and PMA-stimulated states by the change of nonfluorescent DCF-DA nia in adult patients, focusing on ANCs variations, on the positivity for anti-neu - to green fluorescent DCF with the production of hydrogen peroxide. The surface trophil auto-antibodies and on the incidence of infectious episodes. expression of CD64, CD10, and CD16 on neutrophils were measured by the bind - Methods: Complete blood counts and physical examination were performed ing of fluorescence-conjugated anti-CD64, anti-CD10, or anti-CD16 antibodies. every 3-4 months in the first 2 years, then at least once a year. Anti-neutrophil Results: Compared with control neutrophils, patients’ neutrophils with sepsis antibodies were determined by direct and indirect granulocyte immunofluores - showed increased levels with non-stimulated RBA and CD64 expression, and cence test (GIFT method). Infectious episodes were defined according to Com - decreased values in CD10 and CD16 expression ( P< 0.001 for all biomarkers). mon Terminology Criteria for Adverse Events (Version 4.0 http://e vs. nci. nih.gov). Expressions of CD10 and CD16 were lower in patients with septic shock than Results: 47 patients (17 males and 30 females, median age 55 years, range 25- patients with severe sepsis ( P= 0.031, 0.002, respectively). When the patients 86 years) were followed up for a median time of 47 months (range 6-240 months). were subdivided into 2 groups according to the mortality within 28-days, PMA As Figure 1 shows, mean ANCs at enrolment and over time displayed a great stimulated RBA of the patient’s neutrophils was higher in survivor group variability, both inter and intra-subject (>500/µl in 74% and >1000/µl in 25% of (P= 0.002). CD64 expression was also elevated in survivor group, but there was patients). Considering the severity of neutropenia, we observed 23 patients (49%) no statistical significance ( P= 0.071). with neutrophils lower than 1000/µl in at least one observation (median number Summary and Conclusions: During sepsis, neutrophils are highly activated, of 470/µl neutrophils, range 100-969/µl). Finally, the mean ANCs observed dur - and the increased neutrophil RBA by PMA stimulation is associated with bet - ing the follow up was significantly lower in males than in females (610/µl, range ter prognosis. Neutrophil RBA could serve as a prognostic marker in patients 100-1380/µl versus 1070/µl, range 100-1750/µl, respectively; P=0,02). Anti-neu - with sepsis, and CD10 and CD16 expressions of neutrophils might be biomark - trophil antibodies were detected in 18/45 patients (40%), and mean ANCs were ers for sepsis severity. significantly lower in positive versus negative cases (P=0,021 for 6 and 12 months time). Bone marrow evaluation showed features of dismyelopoiesis in 14 cases (56%), hypocellularity in 3 (12%) and normal morphology in 8 (32%). Flow cytom - P1033 etry demonstrated increased Natural Killer cells in 13 patients (28%), Cytogenet - ic was normal 22 cases (88%), while in 3 male patients karyotype was 45, X0 (7, THE ULTRASOUND EXAMINATION OF SPLEEN CAN STRONGLY RAISE 6 and 3 metaphases respectively). Finally, monocyte counts were higher than THE SUSPICION OF THE GAUCHER DISEASE. 1,* 2 1 1 3 1 3 600/µl in 8 patients (17%), and 5 of them (62%) showed an NK marrow infiltrate P Pulluqi , K Pilika , A Ivanaj , A Perolla , T Dedej , T Caja , A Rucaj , 1 (P=0,015). Mild splenomegaly was present in 10 patients (21%) with a mean max - P Xhumari 1 2 3 imal diameter of11,4 cm by ultrasonography. These cases displayed lower ANCs Hematology, surgery, Laboratory, UHC “Mother Theresa” Tirana, Albania, compared with cases without splenomegaly (835/µl, 140-1400, versus 1380/µl, Tirana, Albania 200-3239, at 3 months; P=0,03). During the study 11 patients (23%) had an Background: The Gaucher disease (GD) is a lipid storage disease inherited as infection needing oral antibiotic or antiviral therapy (7 upper respiratory tract an autosomal recessive trait. It results from an inherited deficiency of acid β- glu - infections, 3 Herpes Zoster Virus infections and 1 urinary tract infection); no rela - cosidase leading to accumulation of glucosilceramide in tissue macrophages, tionship was found between the occurrence of infections and the patient’s mean causing hematologic, visceral organs, and skeletal abnormalities. ANCs value, the nadir of ANCs, and the presence of anti-neutrophil antibodies. Aims: To provide clinical and laboratorical data from GD patients and to try to find any special features that raise suspicions of diagnosis prior to the bone marrow examination, or enzyme estimation. Methods: 1996-2012 We analyzed the observational data from 11 GD patients diagnosed for the first time in our clinic and we tried to find any correlation betweem them and GD diagnosis before BM examination. Results: Mean age was 30.7 years (16-65). 81% (9/11) of patients were diagnosed before age 35. Only one was 65 years old. Three patients were from the same fam - ily, two brothers and one sister. Ratio M/F 54% (6/5). At diagnosis, anemia, HB<11gr/dl, was present in 72% (8/11), thrombocytopenia PL<100000/µL in 63% (7/11), and leucopenia WBC<4000/µL in 54% (6/11). Albuminuria was reported in 63% (7/11) of the patients. Bone marrow examination revealed the presence of Gaucher cells in all cases. Hemoglobin electrophoresis was normal in all cases except2, in which minor thalassemia was observed. Hepatomegaly was reported in all patients. Bone pain was reported in 45% (5/11) of patients.Splenomegaly was reported in all patients at 100% (11/11). Spleen size varied from 5-20 cm under the costal arch. In 63% of pts. the spleen was larger than 10 cm under costal arch. The most surprising thing was the strong correlation between spleen size and the view Figure 1. Mean ANCs values during follow up (mean±SD). of the ultrasound examination (USE) of the spleen. The USE of the spleens showed hypoechodens areas in 63% (7/11) of the pts. Hypoechodens areas are prescribed Summary and Conclusions: In spite of the alarm that produces in the gener - by some “brave” examiners as areas that suggest spleen lymphomas or spleen al practitioner, CBN in adults is a benign disease, often incidentally diagnosed, malignancy, but these were not associated with signs and symptoms of malignan - with an infectious rate comparable to that of general population and frequent cies such as temperature, sweats, weigh loose, etc. These Hypoechodens areas spontaneous ANCs variations. Bone marrow evaluation shows abnormal find - are observed in all cases (7/7) with splenomegaly>10 cm under costal arch. ings in a half of patients, without reaching the criteria for clonal hematological Summary and Conclusions: Splenomegaly was among the most common diseases, but suggesting that this condition deserves clinical follow up. signs of GD patients. The hypoechodens areas reported in USE of the spleen, mainly in the large spleen, which are not associated with signs and symptoms of malignancy, may strongly generate suspicions and lead to the GD diagnosis. P1032

NEUTROPHIL RESPIRATORY BURST ACTIVITY COULD SERVE AS A P1034 PROGNOSTIC MARKER IN SEPSIS PATIENTS, AND NEUTROPHIL CD10 AND CD16 EXPRESSIONS MIGHT BE BIOMARKERS FOR SEPSIS SEVER - LANGERHANS CELL HISTIOCYTOSIS: ONE CENTER EXPERIENCE ITY BETWEEN 1990-2012 M Bae 1,* , S Park 2, E Cho 1, C Park 1, S Park 1, Y Cho 1, S Jang 1, H Chi 1, T Celkan 1,* , G Tuysuz 1, N Ozdemir 1, H Apak 1 S Hong 2 1Pediatric Hematology-Oncology, Istanbul University, Cerrahpasa Medical Fac - 1Laboratory Medicine, 2Internal Medicine, Asan Medical Center, Seoul, Korea, ulty, Istanbul, Turkey Republic of Korea Background: Langerhanscell histiocytosis(LCH) isa disease seen in children Background: Respiratory burst activity (RBA) is important for the killing of especially.

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Aims: Diagnosis andfollow-upof Langerhanscell histiocytosis(LCH)patientsat childhood CHS and characterized by fatal infections and HLH. Whereas missense our center were evaluated in this study mutations of the same gene are assosiated with milder, late-onset CHS with slow - Methods: Retrospectively the files of the LCH patients who were treated at our ly progressive neurological impairment or an adolescent form with infections but center were analyzed no HLH. Also there is a correlation between the absence of cytotoxic T lympho - Results: During the 23 year of follow up, 80 patients (56 boys and 24 girls) were cyte citotoxicity and risk of developing HLH. Probably there may be a mutation of diagnosed with LCH. The complains of the patients were; local swelling (n:36, the LYST gene that is assosiated with normal phenotype and the specific findings 45%), bone pain (n: 24, 30%), skin eruption (n:16, 20%), discharge from the ear of the CHS on peripheral blood smear, bone marrow aspiration smear and light (n:6, 8%), polydipsia (n:6, 8%), fewer (n:3, 4%), cough-respiratory distress (n:3, microscopic image of the hair shaft but however causes to an early-onset HLH. In 4%) jaundice (n:2, 2%), abdominal distention (n:2, 2%), tooth loose (n:2, 2%) our case there was no specific treatment for CHS. Intraveneus antibiotherapy and and diarrhea (n:1, 1%). One of the patients were diagnosed with an X-Ray tak - in accordance with HLH 2004 treatment protocol dexamethasone and etoposide en after trauma. The patients were evaluated at 4 different groups; One sys - were used and the patient has been positively responsive to chemotherapy. Stem tem one organ involvement (n:28), one system multifocal involvement (n: 15), cell transplantation has been planned (Figure 1). multisystem low risk (n:21), multisystem high risk (n: 16). The mostly involved area was bone; (one system one organ involvement n:22, one system multifo - cal involvement n:14, multisystem low risk n:14). Mostly involved organ at mul - tisystem risk group were; liver (n:14), skin (n:17), bone (n:6), bone marrow (n: 6), spleen (n: 5), lungs (n:5), lymph node (n:3), soft tissue (n:1) and intestine (n:1). Ten of the 80 patients were transferred to other centers after diagnosis. The remaining 70 patients were treated at our center. Fourteen patients with one system one organ involvement and 4 patients with one system multifocal involvement were treated with curretage-excision of the tumour. Only one of these patients had a local relapse and treated succesfully with re-excision of the tumour. Three patients received systemic chemotherapy (prednisolone and etoposide) after curretage because lesion could not be removed completely. No relapse occured at any of these patients. Five of the patients with one bone involvement received radiotherapy to the involved side; and 3 of these patients relapsed and all had gone into remission with treatment (1 patient recieved systemic chemotherapy an 2 had excision of the lesion). Remission was achieved at patients with soft tissue involvement with local intralesionel steroid thereapy (n:4.) One of the patients had regression of the tumour spontaneous - ly. The chemothereapy protocole was LCH-I before 1996, LCH-II between 1997 and 2002 and LCH-III after 2002. Twenty seven of the patients with systemic involvement received prednisolone+vinblastine, 13 received prednisolone+vin - blastine+etoposide and 3 patients received prednisolone+vinblastine+metho - traxate. Respectively 7, 4 and 1 of these patients had reccurence. Fifty four of 70 patients achieved remission after treatment and did not have any relapse. Twelve of patients had (17%) had relapse. Six of these patients had 2, 4 of these Figure 1. patients had 3 and 2 of these patients had 4 times of relapses. One of these patients died after the second relapse. The rest 11 patients are in remission. Summary and Conclusions: Our case is suggestive of that it is not necessary Four patients with multi system involvement died during treatment due to pro - to find oculocutaneus albinism and silvery hair in all patients to diagnose CHS. gression of the disease. These patients were not in remission and died with an Peripheral blood smear and light microscopic image of the hair shaft findings avarage of two months after the diagnosis. may be sufficient to diagnose in some cases. And it is more cost effective than Summary and Conclusions: Langerhans cell histiocytosis is a disease that genetical analysis even for prenatal diagnosis. involves multiples organs. Systemic or local treatment has a good response but 20-25% of patients might have relapses. P1036

P1035 CHEMOTHERAPY-ASSOCIATED TREATMENT BURDEN IN BREAST CAN - CER PATIENTS RECEIVING LIPEGFILGRASTIM OR PEGFILGRASTIM: A NEW TYPE OF CHEDIAK-HIGASHI SYNDROME? CASE REPORT OF A SECONDARY EFFICACY DATA FROM A PHASE III STUDY BLACK HAIRED GIRL WITHOUT OCULOCUTANEUS ALBINISM PRE - O Gladkov 1,* , A Buchner 2, P Bias 2, U Mueller 2, R Elsaesser 2 SENTED WITH EARLY ONSET HEMOPHAGOCYTIC LYMPHOHISTIOCY - 1Chelyabinsk Regional Clinical Oncology Center, Chelyabinsk, Russian Fed - TOSIS eration, 2Teva ratiopharm, Ulm, Germany H Hatipoglu 1,* , M Elevli 1, M Civilibal 1, N Duru 1, S Ehl 2, O Akgun 1, T Celkan 3 1Pediatrics, Haseki Education and Research Hospital, Istanbul, Turkey, 2Cen - Background: Myelosuppressive chemotherapy (CTx) frequently causes neu - tre of Chronic Immunodeficiency, Univercity of Freiburg, Freiburg, Germany, tropenia, leading to an increased risk of infections and delays in subsequent 3Pediatric Hematology-Oncology, Cerrahpasa Medical Faculty Univercity of CTx treatments. PEGylation of the granulocyte-colony stimulating factor (G- Istanbul, Istanbul, Turkey CSF) molecule extends its half-life in the body, requiring less frequent dosing than traditional non-PEGylated G-CSFs, potentially making treatment less Background: Chediak-Higashi Syndrome(CHS) is a rare, autosomal recessive expensive while enhancing patient compliance and safety. Lipegfilgrastim is a disorder characterized by variable degrees of oculocutaneus albinism, severe long-acting, once-per-cycle glycoPEGylated G-CSF in development for the pre - immune deficiency, lymphoproliferative syndrome and intracytoplasmic giant vention of neutropenia in cancer patients receiving CTx. Noninferiority of lipeg - granules in leukocytes. Giant granules may be present in lmphocytes, mono - filgrastim versus pegfilgrastim (Neulasta ®)was demonstrated in a Phase III tri - cytes and melanocytes. al conducted in CTx-naïve breast cancer patients. 1 Aims: In this article, we report a case of CHS with neither oculocutaneus Aims: To present secondary data on the incidence of hospitalization and den - albinism nor silvery hair presented with early onset hemophagocytic lympho - sity and intensity of CTx in lipegfilgrastim- and pegfilgrastim-treated patients. histiocytosis(HLH). Methods: Patients with high-risk Stage II, III, or IV breast cancer were random - Methods: Case report ized to a single 6 mg subcutaneous injection of lipegfilgrastim (n=101) or peg - A five months old girl presented with high fever. She had splenomegaly, pan - filgrastim (n=101) on Day 2 of each CTx cycle. Full-dose CTx (doxorubicin 60 cytopenia, hipertrigliceridemia and high ferritin levels. Hemophagocytosis was mg/m 2+docetaxel 75 mg/m 2) was started on Day 1 of each cycle (day 22 of the observed on bone marrow aspiration smear. She was diagnosed with HLH. previous cycle) for up to four consecutive cycles. The primary endpoint was HLH-2004 treatment protocol including dexametasone and etoposide was duration of severe neutropenia. Secondary endpoints included days in the hos - administrated. Any known perforin gene defect was not determined. On both pital or intensive care unit (ICU) and incidence of treatment with intravenous bone marrow aspiration and peripheral blood smears intracytoplasmic giant antibiotics because of febrile neutropenia (FN) or related infections; actual ver - granules on lymphocytes were observed. She had neither oculocutaneus sus scheduled cumulative CTx dose per patient; reduced, omitted, or delayed albinism nor silvery hair but analysis of the hair shaft with ligth microscope was CTx doses; and duration of CTx delays. consistent with CHS (photograph). Thus she was diagnosed with CHS. Results: Key secondary endpoints are listed in the Table 1. In the intent-to-treat Results: CHS is usually known as a disease characterized by oculocutaneus population, two pegfilgrastim-treated patients and one lipegfilgrastim-treated albinism and silvery hair but our patient does not have oculocutaneus albinism and patient were hospitalized in Cycle 1 because of FN or associated infection. The she is black haired. Patients with CHS may have a variable clinical presentation two pegfilgrastim-treated patients were hospitalized for FN for 5 and 6 days, due to a different mutations in the lysosomal trafficking regulator gene (LYST), oth - respectively, and the lipegfilgrastim-treated patient spent 1 day in the ICU for er genetic factors and an exposure to other different pathogens. Nonsense and FN and a fungal infection. All hospitalized patients received antibiotics; the frameshift mutations of the LYST gene are asssosiated with severe early-onset lipegfilgrastim-treated patient also received antipyretics. An additional pegfil -

haematologica | 2013; 98(s1) | 425 18 th Congress of the European Hematology Association grastim-treated patient received antibiotics but was not hospitalized. The fre - quency and duration of CTx dose delays were low and comparable between groups; <40% of patients had CTx dose delays. The majority of patients received CTx as scheduled, with the mean percent of planned doxorubicin and docetaxel doses reaching over 98% in all cycles. Thirty-four patients in the lipegfilgrastim group and 41 patients in the pegfilgrastim group received delayed CTx in Cycles 2−4. There were no dose omissions or reductions in the lipegfilgrastim group and eight in the pegfilgrastim group in Cycles 2−4.

Table 1. Key secondary endpoints.

Summary and Conclusions: The burden of treatment associated with myelo - suppressive CTx was similar in breast cancer patients treated with lipegfilgras - tim or pegfilgrastim, with no clinically relevant differences in density and inten - sity of CTx.

Reference 1. Bondarenko I et al . J Clin Oncol 2012;30(Suppl): Abstract e19587.

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