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To evaluate the clinical response rate and cosmetic outcome after full-dose intraoperative electron radiotherapy (IOERT) in early breast cancer (BC) treated with conserving surgery. Inclusion criteria were: >60 years old, clinical tumor size ≤2 cm, luminal A carcinoma, patological negative lymph nodes, excluded lobular carcinoma histology. IOERT was delivered with a dose of 21 Gy at 90% isodose. Clinical, cosmetic and/or instrumental follow-up were performed 45 days after IOERT, 6 months after the first check, and every 12 months thereafter. Acute and late toxicities were assessed with the CTCAE v.4.03 and EORTC-RTOG scales, respectively. Cosmetic outcome was evaluated using the Harvard/NSABO/RTOG Breast Cosmesis Grading Scale. Overall, 162 consecutive patients were included in this analysis (median follow-up: 54 months, range: 1–98 months). The overall response rate was 97.5% (CI 95%: 0.93–0.99%). Locoragional relapse occurred in 2.5% of patients. No patient showed distant metastases. No patient showed radiation-related acute complications, with 3.7% showing late G2–3 toxicity. Only 3.7% of patients showed poor cosmetic results. Our data confirmed that IOERT is a feasible and valid therapeutic option in low-risk BC patients treated with lumpectomy. A low local recurrence rate combined with good cosmetic results validates the settings of our operative method in routinely clinical practice.

Breast cancer (BC) is a heterogeneous malignancy that still represents the second cause of cancer-related death among women worldwide. Due to the heterogeneity of BC, the correct identification of valuable biomarkers able to predict tumor biology and the best treatment approaches are still far from clear. Although molecular imaging with positron emission tomography/computed tomography (PET/CT) has improved the characterization of BC, these methods are not free from drawbacks. In recent years, radiomics and artificial intelligence (AI) have been playing an important role in the detection of several features normally unseen by the human eye in medical images. The present review provides a summary of the current status of radiomics and AI in different clinical settings of BC. A systematic search of PubMed, Web of Science and Scopus was conducted, including all articles published in English that explored radiomics and AI analyses of PET/CT images in BC. Several studies have demonstrated the potential role of such new features for the staging and prognosis as well as the assessment of biological characteristics. Radiomics and AI features appear to be promising in different clinical settings of BC, although larger prospective trials are needed to confirm and to standardize this evidence.

The development of chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematological diseases. However, approximately 60% of patients relapse after CAR-T cell therapy, and no clear cause for this failure has been identified. The objective of the Bio-CAR-T BS study (ClinicalTrials.gov: NCT05366569) is to improve our understanding of the lymphocyte harvest to maximize the quality of the CAR-T cell product. Of the 14 patients enrolled, 11 were diagnosed with DLBCL, 2 with PMBCL, and 1 with ALL. Five of 11 DLBCL patients met the criteria for “pre-emptive” Lymphocytes-apheresis (being at high risk of second relapse), and 6 were included in the standard-of-care Lymphocytes-apheresis group. Previous autologous stem cell transplantation (ASCT) and age were significantly different between the two groups. At the time of Lymphocyte-apheresis, patients in the “pre-emptive” group had more “fit” lymphocytes (higher CD4+/CD8+ ratio; higher naïve T cells levels) compared with standard group, probably due to the impact of ASCT. At the same time, also being older than 60 years results in a more “exhausted” lymphocyte profile. Overall, “pre-emptive” Ly-apheresis in DLBCL patients at high risk of relapse appears to be feasible and may allow the timely collection of “fit” lymphocytes for CAR-T cell manufacturing.

Purpose : Monte Carlo (MC) simulation in Nuclear Medicine is a powerful tool for modelling many physical phenomena which are difficult to track or to measure directly. MC simulation in SPECT/CT imaging is particularly suitable for optimizing the quantification of activity in a patient, and, consequently, the absorbed dose to each organ. To do so, it is mandatory validate MC results with real data acquired with gamma camera. The aim of this study was the validation of SIMIND Monte Carlo code for modelling a Siemens Symbia Intevo Excel SPECT-CT gamma camera both for 99m Tc and 177 Lu. Methods : Phantom experiments using 99m Tc and 177 Lu have been performed with the purpose to measure spatial resolution, sensitivity and evaluate the calibration factor (CF) and recovery coefficients (RC) from acquired data. The geometries used for 2D planar imaging were (1) Petri dish and (2) capillary source while for 3D volumetric imaging were (3) a uniform filled cylinder phantom and (4) a Jaszczack phantom with spheres with different volumes. The experimental results have been compared with the results obtained from Monte Carlo simulations performed in the same geometries. Results : Comparison shows good accordance between simulated and experimental data. The measured planar spatial resolution was 8.3±0.8mm for 99m Tc and 12.2±0.7mm for 177 Lu. The corresponding data obtained by SIMIND for 99mTc was 7.4±0.1 mm, while for 177Lu was 11.7±0.1 mm. The CF was 110.1±5.5 cps/MBq for Technetium and 18.3±1.0 cps/MBq for Lutetium. The corresponding CF obtained by SIMIND for 99mTc was 100.1±0.3 cps/MBq, while for 177 Lu 20.4±0.7 cps/MBq. Moreover, a complete curve RCs vs Volume (ml) both for Technetium and Lutetium was determined to correct the PVE for all volumes of clinical interest. In none of the cases a RC factor equal to 100 was found. Conclusions : The results of validation process show that SIMIND can be used for simulating both gamma camera planar and SPECT images of Siemens Symbia Intevo using 99mTc and 177Lu radionuclides for different medical purposes and treatments.

Real-time quantitative PCR (RT-qPCR) is the gold standard to quantify the BCR-ABL1 transcript for molecular response monitoring in chronic myeloid leukemia (CML) patients, and it plays a pivotal role in clinical decision-making process, even if it presents technical limits. Increasing data suggest that digital PCR (dPCR) is more accurate and reliable than RT-qPCR in CML minimal residual disease monitoring and in patients’ selection for treatment discontinuation. But what about the identification of treatment discontinuation failures? We present the case of a CML patient enrolled both in a study aiming to comparatively assess molecular response by RT-qPCR and dPCR and in the progressive arm of the OPTkIMA trial. This is a phase III trial including CML patients randomized to receive a fixed versus a progressive intermittent tyrosine kinase inhibitor regimen. At 24 months, because of two consecutive detections of MR2.0 by RT-qPCR, the patient resumed daily treatment. Conversely, dPCR revealed a stability of molecular response and even a slight decreasing of transcript over time. An additional specimen was sampled one month after the first MR2.0 detection because of clinical decision: RT-qPCR resulted MR3.0 and dPCR confirmed the transcript’s stability. Nowadays, the resumption of therapy is RT-qPCR-driven despite its limits in detection and robustness. In this case, according to dPCR, the patient could have continued intermittent treatment and the stability of response was then confirmed by RT-qPCR. So, dPCR could be able to better identify peculiar clinical response to therapy.

Exosomes are extracellular vesicles playing a pivotal role in the intercellular communication. They shuttle different cargoes, including nucleic acids from their cell of origin. For this reason, they have been studied as carriers of tumor markers in different liquid biopsy approaches, in particular for solid tumors. Few data are available concerning exosomes as markers of myeloid neoplasia. To better understand their real potential and the best approach to investigate leukemic exosomes, we present the results of a pilot feasibility study evaluating the application of next-generation sequencing analysis of dsDNA derived from exosomes isolated in 14 adult patients affected by acute myeloid leukemias. In particular, leukemia-derived exosome fractions have been analyzed. The concentration of dsDNA co-extracted with exosomes and the number and types of mutations detected were considered and compared with ones identified in the Bone Marrow (BM) and Peripheral Blood (PB) cells. Exosomal DNA concentration, both considering the cargo and the DNA surrounding the lipid membrane resulted in a linear correlation with leukemic burden. Moreover, exosomal DNA mutation status presented 86.5% of homology with BM and 75% with PB. The results of this pilot study confirmed the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA NGS analysis for AML biomarker detection. These data point to the use of liquid biopsy in myeloid neoplasia for the detection of active leukemic cells resident in the BM via a painless procedure.

Magnetic resonance imaging (MRI) is a non-invasive powerful modern clinical technique that is extensively used for the high-resolution imaging of soft tissues. To obtain high-definition pictures of tissues or of the whole organism this technique is enhanced by the use of contrast agents. Gadolinium-based contrast agents have an excellent safety profile. However, over the last two decades, some specific concerns have surfaced. Mn(II) has different favorable physicochemical characteristics and a good toxicity profile, which makes it a good alternative to the Gd(III)-based MRI contrast agents currently used in clinics. Mn(II)-disubstituted symmetrical complexes containing dithiocarbamates ligands were prepared under a nitrogen atmosphere. The magnetic measurements on Mn complexes were carried out with MRI phantom measurements at 1.5 T with a clinical magnetic resonance. Relaxivity values, contrast, and stability were evaluated by appropriate sequences. Studies conducted to evaluate the properties of paramagnetic imaging in water using a clinical magnetic resonance showed that the contrast, produced by the complex [Mn(II)(L’)2] × 2H2O (L’ = 1.4-dioxa-8-azaspiro[4.5]decane-8-carbodithioate), is comparable to that produced by gadolinium complexes currently used in medicine as a paramagnetic contrast agent.

Malnutrition is a common problem after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and could impair immune function. Immune dysfunction after allo-HSCT may be linked with infections, GVHD, and relapse and negatively affect the outcome. Aim of this review was to identify malnutrition biomarkers, potentially useful for immune-system monitoring, in the setting of allo-HSCT. After a systematic search, no satisfying biomarker was found, except for citrulline. Citrulline could be useful in monitoring gastrointestinal function after allo-HSCT and its role in the complex relationship with immune-system function ought to be better explored. A multi-omics approach, including biomarkers and PRO (patient reported outcomes) is, in our opinion, the optimal way to study the relationship between malnutrition and transplant outcomes.

Background Myelodysplastic syndromes and acute leukemias after allogeneic stem cell transplantation (allo-SCT) are mainly caused by recurrence of the primitive leukemic clones. More rarely, they originate from donor hematopoietic stem cells, developing the so-called donor cell leukemia (DCL) or myelodysplastic syndromes (DC-MDSs). DCL and DC-MDS can be considered as an in vivo model of leukemogenesis, and even if the pathogenetic mechanisms remain speculative, a genetic predisposition of donor progenitor cells, an altered host microenvironment, and the impairment of immune surveillance are considered the main causes. Case presentation We report a case of DC-MDS diagnosed 5 years after an allo-SCT from a matched related donor (patient's sister) in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph+ B-ALL). The sex-mismatch allowed us to identify the donor cell origin. At the onset, the DC-MDS was characterized by chromosome seven monosomy and NRAS, RUNX1, and BCOR mutations. Because of a familiar history of colorectal neoplasia and the variant allele frequency (VAF) of NRAS mutation at the onset, this mutation was searched on germline DNA in both the donor and the recipient, but the result was negative. Moreover, after transplant (+4 months), the patient developed severe and long-lasting chronic graft-versus-host disease (cGVHD), requiring multiple lines of treatments. Because of the severe immunosuppression, recurrent infections occurred and, lately, the patient died due to septic shock. Conclusion This case report highlights the need, whenever possible, to evaluate the donor origin of the posttransplant myelodysplasia and acute leukemias. The potential key role of the impaired immune surveillance and of long-lasting immunosuppression appears to be emerging in the development of this case of DC-MDS. Finally, this case reminds the importance to investigate the familiar genetic predisposition in donors with a familiar history of neoplasia.

Context Evaluation of elderly patients' eligibility for allogeneic stem cell transplantation (allo-SCT) is crucial. A comprehensive geriatric assessment (CGA) has demonstrated important prognostic value in hematology. However, its role in allo-SCT transplant setting has still to be defined. Objective To evaluate the feasibility and efficacy of the Fondazione Italiana Linfomi (FIL)-score, a combined comorbidity (CIRS-G) and functionality (ADL/IADL) score, on allo-SCT outcome. Patients and methods This is a retrospective study involving 228 elderly (>60y) patients consecutively submitted to allo-SCT at our centers from 2008 to 2018. Median follow-up 36 months (range, 1-118). Results The median age was 64 years (range, 60-76), and 155 (68%) were male. AML was the most common indication for transplant (53%), DRI was high/very-high in 42% of cases. A matched unrelated, sibling or alternative donor was utilized in 41%, 30%, and 29% of cases, respectively. Stem cell source was derived from peripheral blood in 82% of patients. Myeloablative conditioning regimen was reserved for only 20% of patients. Based on FIL score, available in 215 patients, 125 (58%) patients were classified as “fit” and 90 as “unfit/frail.” According to FIL score, fit patients were more frequently diagnosed with AML (p Conclusions A comprehensive geriatric assessment with FIL score seems to add significant prognostic information in elderly patients submitted to allo-SCT. The pre-transplant adoption of this easy-to-use tool could help the patients' selection and management.

Due to the discovery of their role in intra-cellular communications, exosomes, which carry information specific to the cell of origin, have garnered considerable attention in cancer research. Moreover, there is evidence to suggest the possibility of isolating different exosome sub-populations based on target antigens at the cell surface. Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the break-point cluster region-proto-oncogene 1 tyrosine-protein kinase (BCR-ABL1) fusion-gene, derived from the t (9;22) translocation. Tyrosine kinase inhibitors (TKIs) target BCR-ABL1 protein and induce major or deep molecular responses in the majority of patients. Despite the fact that several studies have demonstrated the persistence of leukemic cells in the bone marrow niche, even following treatment, TKIs prolong patient survival time and facilitate treatment-free remission. These characteristics render CML a plausible model for investigating the feasibility of tumor-derived exosome fraction enrichment. In the present study, patients in the chronic phase (CP) of CML were treated with TKIs, and the quantification of the BCR-ABL1 exosomal transcript was performed using digital PCR (dPCR). The possibility of tumor-derived exosomes enrichment was confirmed, and for the first time, to the best of our knowledge, the detection of the BCR-ABL1 transcript highlighted the presence of active leukemic cells in patients with CP-CML. According to these findings, tumor-derived exosomes may be considered a novel tool for the identification of active leukemic cells, and for the assessment of innovative monitoring focused on the biological functions of exosomes in CML.

We present a case of a patient with a three-month history of peripheral blood cytopenia without a confirmed diagnosis of myelodysplastic syndrome, who developed a favourable-risk acute myeloid leukemia (AML), according to the European Leukemia Net (ELN) criteria. The patient achieved a complete remission with incomplete platelet recovery (CRi) after induction. The patient achieved the morphological CR after the first consolidation and completed the first-line treatment with a syngeneic stem cell transplantation (SCT). A disease relapse occurred after one year of CR (blast cell count in the bone marrow 15%), and the patient was offered a haplo-SCT, which he refused due to personal reasons. In this paper, we discuss the interplay between clinical and biological risk factors in non-high-risk AML patients and speculate that some old clinical risk factors (e.g., age of the patient, achievement of CR after induction, and previous history of myelodysplastic syndrome) may still impact on the treatment decision algorithm of some of these patients.

CMV represents one of the most serious life-threatening complications of allogeneic stem cell transplantaion (allo-SCT). Pre-emptive treatment with anti-CMV drugs is highly effective, but toxicity and repetitive reactivation of CMV represent a major challenge in the clinical practice. The use of anti-CMV specific immunoglobulins (Megalotect) is controversial. We retrospectively collected data on 539 patients with hematological malignancies submitted to allo-SCT in 6 Italian Bone Marrow Transplant Units between 2016 and 2017. Two hundred and forty two of the 539 patients (45%) developed at least one CMV reactivation. In 94/242 cases (39%) Megalotect was used according to internal guidelines. This report focuses on this group of 94 patient. Megalotect was used as prophylaxis in 15/94 cases (16%) and as pre-emptive therapy together with a specific anti-CMV drug in 79/94 cases (84%). Fifty-six out of 94 cases (60%) received 50 UI/Kg weekly, 33/94 cases (35%) received 50 U/Kg every 2-3 weeks (the remaining patients received different doses and schedule according to the Center policy). Megalotect was used because all the 94 patients were considered at high risk of developing repetitive CMV reactivation. In the prophylaxis setting these risk factors were: haploidentical and cord blood transplantation in 5 cases and unfavorable CMV sireology combiation in 14 cases. In the pre-emptive setting, the risk factors were: haploidentical transplantation in 30 cases, aGVHD requiring high-dose steroids in 37 cases and unfavourable CMV serology combination (D-/R- in 44 cases, D+/R- in 1 case). In the pre-emptive setting the following anti-CMV drugs were used first-line: gancyclovir (31 cases), foscarnet (19 cases), valgancyclovir (14 cases), different combinations (15 cases). Overall, the treatment was well tolerated, with no reported infusion reactions nor other adverse events. Megalotect was administered for a median of 2 doses (range 1-9) and 3 doses (range 1-33) in patients treated prophylactically or pre-emptively. None of the 15 cases treated with Megalotect as prophylaxis developed CMV reactivation. Focusing on the 79 cases treated with Megalotect pre-emptively, 59 patients (75%) achieved at least one CMV viremia negativity during tratment. Median time to first negativity was 19 days (range 3-190). In 30/79 cases (38%) a subsequent CMV reactivation was observed during the follow up, which was treated with a second-line anti-CMV specific drug. Interestingly, only 6/94 cases (6%) developed CMV disease: gut localization in 4 cases and other site in 2 cases.Overall 24/94 patients died (25%) and no death was CMV-related Our preliminary experience with Megalotect suggests that it is safe and well tolerated both in the pre-emptive and prophylaxis setting. We have no conclusive data regarding the efficacy of this treatment in reducing the cumulative dose of anti-CMV specific drugs. Interestingly, when used as prophylaxis, no CMV reactivation was recorded. In the pre-emptive setting, the combination of Megalotect and anti-CMV specific drug was able to induce negativity of CMV viremia in a relatively short time (median 19 days) and 38% of these high-risk patients only developed further CMV reactivation during follow up. Further prospective trials are warrented to address the most appropriate use of Megalotec (preophylaxis vs pre-emptive) and to identify the best setting of patients who can benefit from Megalotect alone or in addition to anti-CMV specific drugs. Disclosures Foà: ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD; GILEAD: Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; INCYTE: Other: ADVISORY BOARD; NOVARTIS: Speakers Bureau; CELTRION: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY BOARD, Speakers Bureau.

T and B lymphocyte subsets have been not univocally associated to Graft-versus-host disease (GVHD) and relapse of hematological malignancies after stem cell transplantation (SCT). Their sequential assessment together with B and T cell neogenesis indexes has been not thoroughly analysed in relation to these changing and interrelated immunologic/clinic events yet. Lymphocyte subsets in peripheral blood (PB) and B and T cell neogenesis indexes were analysed together at different time points in a prospective study of 50 patients. Principal component analysis (PCA) was used as first step of multivariate analysis to address issues related to a high number of variables versus a relatively low number of patients. Multivariate analysis was completed by Fine-Gray proportional hazard regression model. PCA identified 3 clusters of variables (PC1-3), which correlated with acute GVHD: PC1 (pre-SCT: KRECs≥6608/ml, unswitched memory B 44%, CD8+TCM cells>4%; HR 1.9, p = 0.01), and PC3 (at aGVHD onset: CD4+TEMRA69%, switched memory CD19+ = 0 cells and KRECs

Monitoring of BCR-ABL1 levels by quantitative PCR (qPCR) is essential for the management of CML patients treated with TKIs. Because of intrinsic limits, qPCR does not appear to be an optimal assay to select the best candidates to TKIs discontinuation. Up to 40% of CML patients treated with TKIs can achieve a Deep Molecular Response (DMR), but only 50% maintain a stable Treatment Free Remission (TFR) after therapy discontinuation. Digital PCR (dPCR), giving an absolute quantification of BCR-ABL1, is expected to be more sensitive and accurate than qPCR in the assessment of molecular MRD. dPCR performed on a QuantStudio 3D Digital PCR System (Life Technologies) by using a TaqMan-MGB probes targeting the BCR-ABL1 transcript was used to comparatively analyse 102 CML patients with Major (MR3.0 = 31 cases) or Deep (MR4.0= 33 cases; MR4.5 = 24 cases and MR5.0 = 14 cases) molecular response. Preliminary results showed that: a) ≥77% of deep responders (MR4.0, MR4.5 and MR5.0) fell under the value of 0.468 BCR-ABL1 copies/▢l indicated by the ROC analysis as the value below which the patients with lower levels of MRD might be dissected (spec.=71%, sens.=77%; AUC=0,79); b) BCR-ABL1 transcript levels were detectable by dPCR also in cases resulted undetectable by qPCR. In this study, we analysed the BCR-ABL1 transcript levels by dPCR in 207 samples related to 102 CML patients. Fourteen (14%) out of 102 CML patients discontinued the TKIs therapy. Among them, 3 patients (21%) lost DMR and all of them showed dPCR values > 0.468 BCR-ABL1 copies/▢l (previously described as cut-off for a deep MRD), while 11 (79%) maintained a stable DMR and 9 of them (82%) fell under the value of 0.468 BCR-ABL1 copies/▢l. These latter patients stratified in different DMR classes by qPCR and all had undetectable level of BCR-ABL1 by qPCR. In 149 out of 207, qPCR revealed DMR. They were: MR4.0= 59 samples; MR4.5= 61 cases; MR5.0 = 29 cases. One hundred twenty-five (84%) fell under the value of 0.468 BCR-ABL1 copies/▢l. A linear regression analysis in these samples did not show any correlation between BCR-ABL1 copies/▢l detected by qPCR with the ones detected by dPCR (R2 < 0.01). On the basis of our preliminary results, TFR seems to be correlated to the maintenance of dPCR values < 0.468 BCR-ABL1 copies/▢l. The concordance between qPCR and dPCR quantification in patients with DMR and with BCR-ABL1 copies/▢l value < 0.468 was poor. Patients with dPCR values < 0.468 BCR-ABL1 copies/▢l may have 75% of probability to maintain TFR status. These results suggest that dPCR may be more sensitive to detect the MRD and could be more useful for DMR monitoring and for dissecting the best candidate to discontinuation of therapy with TKIs. Disclosures Tiribelli: Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Castagnetti:Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; ARIAD Pharmaceuticals: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Soverini:Novartis: Consultancy; Ariad: Consultancy; Bristol-Myers Squibb: Consultancy. Rosti:Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Martinelli:Roche: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Genentech: Consultancy; Amgen: Consultancy; MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Genentech: Consultancy; Amgen: Consultancy; Novartis: Speakers Bureau; MSD: Consultancy.

Background : Blood stream infections (BSIs) represent a major complication of allo-SCT and are a major cause of morbidity and mortality during and after bone marrow aplasia. Objectives: The objective of this study was to describe the incidence and outcome of BSIs in a cohort of patients submitted to allo-SCT, in order to track changes of the epidemiology and bacteria resistance. Methods : We retrospectively analyzed the microbiological data of 162 patients allotransplanted in Brescia University Hospital, over a period of 6 years. Results : Eighty patients experienced a BSIs for a total of 119 isolates. In 77 cases (65%) a Gram positive bacteria was isolated, being coagulase negative Staphilococci the most frequent species (77% of the cases). In 42 cases (35%) a Gram negative bacteria was isolated (E. coli 57% and P. aeruginosa 24%). Fluoroquinolones resistance was frequent (90% for S. epidermidis , 92% for E. coli , 90% for P. aeruginosa ). Methycillin resistance of S. epidermidis was 100%, 76% of E. coli were ESBL positive and among P. aeruginosa resistance to carbapenems was 40%. The 2 years overall survival of patients with BSIs vs patients without BSIs was 46% vs 60% (HR1,48, p=0,07). P. areuginosa and E. coli were the species with the highest mortality (50% and 33%, respectively). Conclusions : These data confirms that BSIs, mainly sustained by Gram positive bacteria, are frequent in allotransplanted patients (50% of the cases) and may influence the outcome of allotransplanted patients, being antibiotics resistance highly frequent among these bacteria.

Background Blood stream infections (BSIs) represent a major complication of allo-SCT and are a major cause of morbidity and mortality during and after bone marrow aplasia. Objectives The objective of this study was to describe the incidence and outcome of BSIs in a cohort of patients submitted to allo-SCT, in order to track changes of the epidemiology and bacteria resistance. Methods We retrospectively analyzed the microbiological data of 162 patients allotransplanted in Brescia University Hospital, over a period of 6 years. Results Eighty patients experienced a BSIs for a total of 119 isolates. In 77 cases (65%) a Gram positive bacteria was isolated, being coagulase negative Staphilococci the most frequent species (77% of the cases). In 42 cases (35%) a Gram negative bacteria was isolated (E. coli 57% and P. aeruginosa 24%). Fluoroquinolones resistance was frequent (90% for S. epidermidis, 92% for E. coli, 90% for P. aeruginosa). Methycillin resistance of S. epidermidis was 100%, 76% of E. coli were ESBL positive and among P. aeruginosa resistance to carbapenems was 40%. The 2 years overall survival of patients with BSIs vs patients without BSIs was 46% vs 60% (HR1,48, p=0,07). P. aeruginosa and E. coli were the species with the highest mortality (50% and 33%, respectively). Conclusions These data confirm that BSIs, mainly sustained by Gram positive bacteria, are frequent in allotransplanted patients (50% of the cases) and may influence the outcome of allotransplanted patients, being antibiotics resistance highly frequent among these bacteria.

Introduction In AML and MDS cases, the genetic lesions inherited or acquired by the hematopoietic stem cells are considered as starting events. Familial AML and MDS, recently recognized in the revised WHO classification (2016) provide a useful model for investigation of predisposing genetic mutations. Genetic analysis of several pure familial leukemia pedigrees led to the discovery of well defined syndromes associated with inherited de novo mutations on germline DNA. Growing clinical awareness as well as a widespread use of NGS have led to an enlarged description of familial MDS/AML cases, and the number of mutations involved, suggesting they are more frequent than those previously recognized. Despite the recent discovery of well-established causative gene mutations (RUNX1, GATA2, ETV6, TERT, TERC, SRP72, ANKRD26, DDX41, CEBPA), many cases remain unexplained (about 80%), suggesting that other inherited mutations could predispose to MDS/AML. It is expected that new sequencing approaches will help to the identification of more cases, more genes as well as novel syndromes. In 2017, we started a multicentric prospective study (Clinical trial.gov NCT03058588) aiming to look for predisposing mutations in patients and relatives affected by Familial AML and MDS syndromes (FAMS) by NGS and to screen for old and new mutations potentially associated with the disease. Methods At present, 12 AML/MDS patients have been enrolled. Leukemic (bone marrow) and germline (buccal swab) DNA were analyzed by NGS gene panel approach based on a 28 genes associated to myeloid leukemogenesis, including the 9 above mentioned genes associated to FAMS. NGS libraries were performed by a Nimblegen (Roche) custom panel based on gene capture strategy and the sequencing was performed by MiSeq (Illumina). Results Ten patients did not reveal any germline mutations and the candidates are undergoing to whole exome sequencing. One presented a germline mutation on RUNX1, and the analysis of the affected relatives is on going. One revealed a new mutation. She was a 70 years old woman affected by RARS and her pedigree was characterized by 9 relatives affected by hematologic and solid neoplasia and trombocytopenia (fig 1). The NGS analysis revealed the mutation c.*514C>T in 3'UTR of ETV6 with VAF of 50% on tumor DNA. The variant has never been described before, while ETV6 has been already associated with FAMS. Sanger sequencing confirmed the mutation on the germline DNA in heterozygosis. The screening of 2 affected relatives still alive confirmed the presence of the variant in heterozygosis. In silico analysis performed on PolymiRST Database revealed that c.*514C>T in 3'UTR of ETV6 results in a gain of miRNA binding site: hsa-miR- 4717-3p and hsa-miR- 942-3p. Discussion The variant c.*514C>T in 3'UTR of ETV6 seems to repress ETV6 due to RNA interference. The new binding miRNAs have been already described as over-expressed in solid and hematologic tumors. Moreover, the down-regulation of ETV6 is associated with alteration of cell growth and hematopoiesis. Due to these evidences, c.*514C>T in 3'UTR of ETV6 could be considered as a new mutation involved in FAMS predisposition. Disclosures No relevant conflicts of interest to declare.

Abstract 4029 Background: Azacitidine (AZA) at a dose of 75 mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates and prolongation of survival in high-risk MDS patients (pts) (Fenaux, 2009). However few data are hitherto available concerning the efficacy and safety of Aza in lower risk MDS. A lower dose regimen, AZA 5 (75 mg/mq daily, subcutaneously, for 5 consecutive days every 4 weeks) have shown to induce response rates consistent with the currently approved schedule (Lyons, 2009), however in this study pts were not classified according to IPSS risk. Aim: The use of AZA in the earlier phases of disease could be more effective and useful to control the expansion of MDS clone and disease progression. In our phase II, prospective, multicentric trial, AZA 5 regimen was administered to IPSS low-or-intermediate-1 risk pts, for a total of 8 courses, in order to evaluate its efficacy and safety. Furthermore pharmacogenomic studies (GEP, SNP) cytokine network and PI-PLC-beta1 methylation and gene expression, before and at the end of 4th and 8th course of Aza treatment, were planned to identify new biological markers to predict the response. Methods: From September 2008 to February 2010, 34 patients (24 males, 10 females), with a median age of 71 (56-84) yrs, with symptomatic transfusion-dependent anemia, previously unresponsive to erythropoietin (EPO) or not expected to respond to EPO, or with severe neutropenia or thrombocytopenia, were enrolled into the study. According to WHO classification, 15 pts had RA, 6 RARS, 7 RCMD and 6 RAEB-1. Results: At present time 30/34 pts are evaluable: 23/30 pts (77%) completed the treatment plan (8 courses), 3/30 pts (10%) are ongoing and 4/30 (13%) died during the treatment period. According to the 2006 International Working Group criteria, overall response rate (ORR) was 60,9 % (14/23 pts): 5 pts (21,7%) achieved complete remission (CR), while 9 pts (39,1%) showed an hematologic improvement (HI) (7 erythroid responses, 1 erythroid/platelet response and 1 neutrophil/platelet response). 9/23 pts (39%) maintained a stable disease (SD). Generally the drug was very well tolerated. The most commonly reported hematologic toxicities were neutropenia (55%) and thrombocytopenia (19%). 4 pts (11,7%) died during treatment (2 pts after the 1th cycle and 2 pts after the 4th course) because of septic shock, gastrointestinal hemorrage, pneumonia, and respiratory distress, respectively. The median duration of response was 3,5 months (range 1–14 months). Surprisingly, 3/14 patients (2 CR and 1 HI erythroid) showed a long duration of response (11, 13 and 14 months, respectively), still ongoing, after discontinuation of AZA. Preliminary data on the lipid signalling pathways suggested a direct correlation between the demethylating effect on PI-PLC-beta1 and responsiveness to treatment. Conclusion: Our study shows that AZA low-dose schedule may be a feasible and effective treatment for low-risk MDS pts and may induce durable responses. Despite AZA safety, extreme caution is needed in pts with age-related comorbidities and/or with severe neutropenia or thrombocytopenia, especially in low-risk MDS. Furthermore, PI-PLC–β1 demethylation and gene expression could represent a new biological marker to predict the clinical response to AZA Disclosures: Off Label Use: In Italy the use of Azicitidine for Low-Risk Myelodysplastic patients is off-label. The use of azacitidine in our study is part of a Phase II clinical trial. Finelli:Celgene: Consultancy.

To evaluate if WT1 expression may predict relapse after allo-SCT, we analyzed WT1 levels on peripheral blood (PB) and bone marrow (BM) before and after allo-SCT in 24 AML patients with WT1 overexpression at diagnosis. Five copies of WT1/ABL × 104from PB were identified as the threshold value that correlated with relapse after allo-SCT. The same correlation was not identified when WT1 expression was assessed from bone marrow (BM). Eight out of 11 (73%) patients with a pre-allo-SCT PB-WT1 ≥ 5 and 4/13 (31%) patients with a pre-allo-SCT PB-WT1 < 5 relapsed, respectively (P= 0.04). The incidence of relapse was higher in patients with PB-WT1 ≥ 5 measured after allo-SCT, at the 3rd (56% versus 38%;P= 0.43) and at the 6th month (71% versus 20%;P= 0.03). Patients with pretransplant PB-WT1 < 5 had significantly better 2-year OS and LFS than patients with a PB-WT1 ≥ 5 (81% versus 0% and 63% versus 20%) (P= 0.02). Our data suggest the usefulness of WT1 monitoring from PB to predict the relapse in allotransplanted AML patients and to modulate the intensity of conditioning and/or the posttransplant immunosuppression in an attempt to reduce the posttransplant relapse risk.

Toll-like receptors (TLRs) play a key role in the cross-talk between the innate and adaptive immune systems. Previous studies investigating associations between certain TLRs and acute graft-versus-host disease (aGVHD) have reported contrasting results, and no studies relating aGVHD to the expression and function of all human TLRs together have been published to date. We prospectively evaluated the expression of 9 TLRs on T lymphocytes and monocytes by flow cytometry in relation to aGVHD in 34 patients. Induction of TNF-α, IL-4, IFN-γ, and monocyte chemotactic protein 1 on TLR activation was assessed by ELISA on cell supernatants. Nineteen patients developed aGVHD, at a median time of 28 days (range, 20-50 days) after transplantation. A 2-step multivariate analysis was performed using principal component analysis and multifactor analysis of variance. The levels of TLR-5 expression on monocytes and T lymphocytes were positively correlated to aGVHD (P = .01), whereas levels of TLR-1 and -9 were negative predictors (P = .03 and .01, respectively). This profile of TLR-1, -5, and -9 can promote an overall immunostimulatory/proinflammatory response. If our findings are confirmed by further studies, this TLR profile could be a useful biomarker of aGVHD.

AMLs are clonal disorders characterized by high genomic heterogeneity and several chromosomal and molecular alterations affecting patients' outcome. In about 40% of AML patients who do not show any citogenetic alteration, sequencing analysis identified different gene mutations which play a pivotal role in leukemogenesis and have a negative prognostic impact: FLT3, ASXL1, TET2, IDH1, IDH2, RUNX1, CBL, CEBPα, DNMT3A and TP53. Conventional Sanger sequencing may detect clones representing more than 20% of the total tumor population, whereas Next Generation Sequencing (NGS) can identify mutations in less than 1% of leukemic cell burden. The detection of these variants is relevant because they can play an important role in driving drug resistance and disease relapse and for biologic risk assessment. To that purpose, we designed a 23-genes panel including: FLT3, DNMT3A, RUNX1, ASXL1, IDH1, IDH2, BCOR, NRAS, KRAS, TET2, TP53, U2AF1, ZRSR2, SF3B1, SRSF2, CBL, CEBPα, EZH2, NPM1, TERT, TERC, ETV6, GATA2. By using a 454 GS Junior by Roche Diagnostics with an amplicon-based sequencing approach and performing three sequencing runs per sample in order to reach a sensitivity of at least 1%, we settled an investigative strategy in order to assess the genomic profile of AMLs at diagnosis and possibly at the time of relapse or resistance. By this approach, we were able to confirm all the variants (i.e. FLT3, NPM1) previously documented with conventional tests; to reveal the presence of variants under the Sanger threshold of 20% in the genes resulted as wild type with routinely analysis; to evaluate the AML clonal heterogeneity, by assessing the coexistence of several mutated clones which have been described to be related to drug resistance, poor prognosis or to prior myelodysplastic syndrome. This gene panel NGS-based strategy may be proposed as a highly accurate and sensitive approach for genomic characterization of acute myeloid leukemias and as an useful tool for planning a target and personalized AML therapy. Disclosures No relevant conflicts of interest to declare.

Introduction The long-term efficacy of allogeneic haematopoietic stem cell transplantation (SCT) relies primarily on the Graft-versus-tumor (GVT) effect, which partially overlaps with Graft versus Host disease (GvHD), the most common cause of morbidity and mortality in SCT. Researches on GVHD-biomarkers are still ongoing and a set of validate markers are still lacking, especially for chronic GVHD. Furthermore, immune parameters that univocally associate with GVHD or GVT have not been identified yet. In this study, lymphocyte subsets together with TCR-repertoire analysis, and index of thymic and bone marrow output were evaluated at different time points, in order to identify possible predictors of GVHD and ineffective GVT. Methods Prospective evaluations of lymphocyte subsets, thymic and bone marrow output were performed in 40 patients before SCT, at 30, 90, 180 days and 1 year after SCT. CD4+/CD8+ naïve, central memory, effector memory, terminally differentiated effector memory (TEMRA) cells, subsets of regulatory T-lymphocytes, immature B cells, naïve, switched and unswitched memory B cells, memory double negative (IgD-CD27-) B cells were analysed by flow cytometry. Analysis of thymic and bone marrow output was performed by detection of T cell receptor excision circles (TRECs) and kappa-deleting recombination circles (KRECs). TRECs and KRECs were simultaneously quantified by a duplex quantitative Real-Time PCR. Heteroduplex assay was used to perform TCR-repertoire analysis. A 2-step multivariate analysis was performed using principal component analysis (PCA) and Cox regression analysis, to solve the problem of the high number of variables (immunological, patients- and transplant related) in comparison with the relatively limited and heterogeneous pool of patients. Results Twenty patients developed acute GVHD (median time: 28 days, range 19-120). Chronic GVHD was observed in 9 patients (median time: 6 months, range 4-10). In multivariate analysis, acute GVHD correlated positively with pre-transplant percentage of CD4+ central memory cells, and with values of regulatory effector memory T-cells and CD4+TEMRA cell at day +30 (p=0,0006). Pre-transplant percentage of unswitched memory B cells was also associated with acute GVHD, whereas pre-transplant levels of KRECs were inversely correlated (p=0,0005). Chronic GVHD was associated with matched unrelated donor and with (p -values of regulatory effector memory T-cells at +30, percentage of CD8+TEMRA cells at +90, values of immature B cells and levels of KRECs at +180 (positive correlation) -percentage of CD4+ central memory and CD8+ effector memory cells at +90 (negative correlation). The relapse rate (27%; median time: 5,5 months, range 3-12) was used as clinical index of ineffective GVT. The following cluster of immunological parameters at day +90 correlated positively with relapse: CD8+ effector memory cells, immature B cells, naïve, switched memory B cells, memory double negative (IgD-CD27-) B cells (p=0,006). Discussion Different clusters of immunological parameters at different time points were evidenced as predictors of GVHD and ineffective GVT, allowing a clear-cut distinction between these immunological reactions. Changes in pre- and post-transplant B-lymphopoietic microenvironment and specific imbalances in the subset of B-cells may be involved in acute and chronic GVHD development. The atypical association of regulatory T-cells with GVHD may be explained by the relative efficiency of different subsets of regulatory T-cells (naïve>effector memory), as shown in some experimental models. Increased values of CD8+ effector memory cells could be an early sign of ineffective GVL. Imbalance toward a lymphocyte B-response, and especially toward "senescent" memory (IgD-CD27-) B cells, could promote tolerance to tumor cells. The validation of these clusters of immunological parameters as specific early predictors of GVHD or GVT, even before SCT, could potentially allow the development of pre-emptive and targeted therapies. Disclosures No relevant conflicts of interest to declare.

Abstract 4676 Introduction Myeloproliferative diseases (MPDs) are a group of haematological disorders characterized by the hyper proliferation of different blood cells in peripheral blood and other hematopoietic organs. The clinical heterogenity of these neoplasms reflects the different gene pathways involved, most of which are only partially known. Given the genetic homology and the physiological similarity to mammals, zebrafish has emerged as an ideal model to study human normal and malignant haematopoiesis. In the last decade several oncogenes involved in the development of hematopoietic neoplasms have been used to model leukemia in zebrafish with the aim to discovery new molecular pathways involved in malignant transformation. Despite the first encouraging results these experimental models failed to fully recapitulate human myeloproliferative disorders. Methods We took advantage of the Gal4/UAS binary system to induce the expression of human oncogenic HRASV12G in the zebrafish hematopoietic compartment. We used a specific transgenic line that drives oncogene expression in zebrafish early hematopoietic progenitors under control of the FLI.1 (Friend Leukemia virus Integration 1) promoter. Results We observed the development of a myelo-erythroid proliferative disease in few days in zebrafish transgenic larva. The pathological phenotype is characterized by the expansion of the hematopoietic tissue, an increased expression of myelo-erythroid specific genes (PU.1, gata1, mpx, c-mpl) associated with a slight increase of staminality markers (lmo2, scl, c-myb, runx.1), and a higher number of l-plastin expressing cells. Moreover blood smear of pathological larva displayed leukemic blasts and the arrest of erythrocyte differentiation whereas kidney marrow of juvenile fish displayed abnormal myelopoiesis characterized by the increase of erythro-myeloid progenitors. We found that the pathological phenotype is associated with a down regulation of the Notch pathway as shown by the decreased gene expression of notch pathways target genes (notch1, notch3, her6). Furthermore we discovered a novel set of genes involved in neoplastic transformation induced by HRASV12 expression through RNA-Seq analysis of pathological larva. Conclusions The expansion of the zebrafish hematopoietic compartment characterized by the hyper-proliferation of the myelo-erythroid progenitors that we found in this model reproduces some of the pathological features of human myeloproliferative disorders. This study showed that forcing oncogene expression in the hemogenic endothelial cells induces the transdifferentiation of the early hemogenic pluripotent stem cells into abnormal myeloerythoid progenitors by repressing the Notch pathways. Transcriptome analysis identified a number of potential effectors of this transformation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4711 Background: Myeloproliferative diseases (MPDs) are a group of hematological malignancies characterized by the abnormal increase of different blood cells in peripheral blood and in other hematological organs. The molecular pathways involved in disease pathogenesis and progression as well as in drug sensitivity and resistance are not fully elucidated yet. Given the physiological similarity to mammals, the zebrafish (ZF) could accelerate the study of the molecular basis of these diseases. In the last decade several human leukaemia associated oncogenes have been transiently over-expressed in ZF embryos in order to perturb the myelo-erythroid compartment, with the aim to create experimental models to discover new molecular pathways that can became potential targets for new chemical compounds. Methodogy: We took advantage of the binary Gal4/UAS system to express oncogenic human HRAS in the ZF hematopoietic compartment. We used the tg(MAZe) driver line, a specific transgenic line that allows random mosaic expression of Gal4-VP16 after heat shock(hs) treatment(1). This driver line was mated with a tg(UAS:eGFP-HRASV12)(2) responder line in order to mimic somatic events leading to human oncogene expression. Results: Surprisingly a single heat shock at 30hpf, (referred here as HRASV12hs) induces human HRASV12 expression in hematopoietic progenitors as judged by the expansion of the hematopoietic tissue. Histological analysis showed an increased number of monocytes/macrophages. Blood smear of HRASV12hs larvae showed the expansion of blasts and myeloid precursors. Quantitative analysis of gene expression highlights a remarkable increase of myelo-erythroid restricted genes associated with a slight increase of staminality markers (pu1, mpx, lmo2, mpl, CD41 and c-myb). Moreover fish raised to adulthood developed hyper-plastic kidney marrow, the site of definitive haematopoiesis, the equivalent of mammalian bone marrow. Conclusions and Future Perspectives: The combination of hyper-proliferation of blood cells associated with the expansion of the hematopoietic compartment that we found in this model reproduces the pathological features of human myeloproliferative disorders. This study shows that it is possible to drive the expression of leukemia associated oncogenes to the hematopoietic compartment in developing ZF thus reproducing some of the features of human blood malignancies in an in vivo model. Our Group is now testing different driver lines in order to induce oncogene expression in earliest hematopoietic progenitors and is focusing on different responder lines expressing human oncogenes involved in hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract 4704 Background and aim of the study. Emerging trends emphasize the importance of both innate and adaptive immune system in the response against infections, in the pathogenesis of autoimmune diseases and graft-versus-host disease (GVHD). In the cross-talk between innate and adaptive immune system, pattern recognition receptors such as Toll-like receptors (TLRs) play a key role. TLRs recognize common protein, carbohydrate or DNA/RNA pattern motifs leading to signaling for cytokine production and T cell and dendritic cell maturation. These receptors may act as tuner of inflammatory and immunologic reactions. Very little is known about the expression and the function of TLRs in vivo in patients who underwent to allogeneic stem cell transplantation (SCT). The aim of this study was to evaluate the expression and the function of TLRs on lymphocytes and monocytes in relation to infection (CMV and HHV-6, especially) and the onset of GVHD. Patients and methods. The expression of TLRs on T cells and monocytes was analyzed by flow cytometry at day +30, +90, +180 after SCT and at the onset of GVHD. The expression of receptors for lipid-based pathogen-associated molecular patterns (PAMPs: TLR 1,2,4 and 6 surface receptors), receptors for nucleic acid based PAMPs (TLR 3,7,8 and 9 located in cytoplasmic compartments), TLR5 and, TLR10 (surface receptors) was evaluated as mean fluorescence intensity (MFI). Ex vivo induction of cytokines (TNFalpha, MCP1, IFNgamma, IL-10) by TLR ligands was analyzed in the cell supernatant by ELISA. Since the beginning of the study, we have analyzed data of 12 healthy donors and 14 patients. Median age was 46 years (range, 25–64) and 7 patients were male. Results. Acute GVHD developed in 7 patients. Patients without acute GVHD after SCT and healthy donors showed different MFI of TLR3 on T cells (5,8±1,4 vs 4,2±1,05 p=0,02), of TLR4 on monocytes (26,1±1,01 vs 15,8±4,9 p=0,004), and of TLR6 on T-lymphocytes (7,3±3,2 vs 4,6±1,1 p=0,02) and monocytes (27±12,1 vs 14,9±4,6 p=0,01). TLR3 expression was significantly decreased on T-lymphocytes and monocytes in patients with acute GVHD in comparison to those without GVHD (4,06±0,8 vs 5,8±1,4 p=0,02; 9,3±7,2 vs 38,02±30 p=0,04). The levels of TLR5 on T cells and monocytes were higher in patients with acute GVHD compared to healthy donors (8,4±2,1 vs 6,4±1,6 p=0,04; 54,2±20,2 vs 33,2±16,5 p=0,04). An increased induction of IFNgamma upon TLR1 ligand activation was observed in patients without GVHD in comparison to healthy donors and patients with GVHD (p=0,04). TLR6 ligand induced significantly the production of IFN gamma in patients with GVHD in comparison to controls and the other patients (p=0,03). Patients without GVHD showed a trend toward a decreased induction of MCP1 upon TLR4 ligand activation (p=0,07). The rate of infections (especially CMV reactivation), clinical and transplant characteristic were not significantly different between patients with and without GVHD. Conclusion In our study, a different expression profile of TLRs was found in healthy donors, in patients after SCT without acute GVHD and in those with GVHD. These results suggest that the innate immune response via TLRs activation could be involved in the development of GVHD. The assessment of a larger number of patients would be useful to understand the complex interplay between pathogens, self or non-self DNA and RNA and the immune system after SCT. Disclosures: Off Label Use: In Italy the use of azacitidine for Low-risk Myelodysplastic patients is off label. The use of azacitidine in our study is part of a Phase II clinical trial.