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Although the ELN MRD Working Party (Schuurhuis, 2018) recommends that Acute Myeloid Leukemia (AML) patients (pts) with mutated NPM1 (NPM1+) should have molecular assessment of measurable/minimal residual disease (MRD), the most clinically significant timepoints (TPs), the thresholds, the best source of samples, bone marrow (BM) or peripheral blood (PB) and especially the management of molecular relapse, remain controversial. We evaluated the prognostic significance of NPM1 molecular monitoring, its impact on disease recurrence and the outcome of salvage treatment. From Jul 2010 to dec 2018, 83 consecutive NPM1+ AML pts (M/F: 44/39; median age 59 y, 27-74) eligible for intensive chemotherapy (i-T) were treated, according to the NILG-AML00 protocol (ClinicalTrial.gov: NCT00400673): ICE (idarubicine-ARAC-etoposide) as induction, followed by IC consolidation and 3 further consolidation cycles of high-dose ARAC. Allogeneic stem cell transplant (HSCT) was considered at relapse. Quantitative RT-PCR was performed to detect NPM1 mutation (Gorello, 2006) on BM and PB samples at diagnosis (TP0) and at given TPs: at complete remission (CR) (TP1), post-IC (TP2), post-1st ARAC consolidation (TP3) and at the end of treatment (TP4). Serial MRD monitoring during follow-up was made on PB every 3 months (mo) for 5 years (y) or until relapse. Molecular relapse (mR) was defined as the NPM1 level increase at least 1 log in 2 consecutive samples, in absence of hematological relapse (hR). At baseline, karyotype (K) was abnormal in 8 pts (9.6%); 25 (30%) were FLT3-ITD mutated with a median Allelic Ratio (AR) of 0.35 (range: 0.2 to 2.18) (Pratcorona, 2013). During treatment, 608 samples were studied (383 BM and 225 PB), for a median of 8 per patient (range: 3 to 10). At TP1, a higher level of NPM1 unfavorably impacted on relapse (p 0.02) and a cut-off >200 *10^4/ABL was significantly associated with a higher relapse risk (RR) (68.7%) and lower RFS (p 0.006). Moreover, MRD positivity (value >0*10^4 NPM1/ABL) at TP2 on PB was associated to a higher RR (38.8%; p 0. 041) and a level >0.5*10^4/ABL allowed to predict relapse in 75% of pts, also impacting on RFS (p 0.0001) (Figure 1).Molecular NPM1 relapse/progression occurred in 10 pts early during treatment, after a median of 3.5 mo (1.4-6) from diagnosis and in 8 of them simultaneously with hR. In 16 pts mR occurred after a median CR duration of 18.5 mo (10.5-61.5) (late relapse). Hence a mR was more frequent in late than in early relapse (87.5% vs 20%; p 0.001). Median survival was 8.3 mo in early relapsing pts and was not reached in late relapsing pts (p. 0.0002). Age, NPM1 level at TP0, FLT3-ITD mutation, ITD-AR or abnormal K did not impact on type of relapse (mR or hR), but FLT3-ITD was more frequent in early than late relapse (60% vs 18.7%; p 0.04). After a median follow-up of 42 mo (4-108), 5-y relapse-free survival (RFS) and overall survival were 64.2% (+/-6.5% SE) and 71.1% (+/-6.1% SE), respectively. Overall, considering both mR and hR, 26 pts (31.3%) relapsed after a median of 13.5 mo after CR; 24/26 pts received a salvage treatment and 14 (53.8%) (median age 49y, 27-64) could proceed to HSCT after a median of 2.9 mo (5 too old, 7 early progression). Salvage treatment before HSCT was i-T in 7/14 and non-i-T in 7/14 pts (2 ATRA and 5 D-actinomycin, Falini 2015) for a median of 3 cycles (range 2-4). Disease status at HSCT was: mCR in 3 pts, CR with detectable MRD (median NPM1: 125*10^4/ABL, range 17.8-3849) in 9, refractory in 2 pts. Nine/14 HSCT pts (64.2%) are alive and in remission at a median of 29.6 mo (13-49) from HSCT, 2 pts died of sepsis in c-GVDH at 6.2 and 37.5 mo after HSCT, 1 died of VOD and 2 of disease progression. At 3 months after HSCT all MRD positive pts were NPM1 negative. mR occurred in 3 pts at 8, 12 and 15 mo after HSCT and was successfully treated with early pre-emptive donor lymphocyte infusions. With the limits of small numbers, age, type (mR or hR) or timing of relapse (early or late), disease status at HSCT, donor source and conditioning intensity didn't influence survival. Our study shows that PCR monitoring during treatment can identify pts at higher RR according to the transcript levels in BM and PB samples. In NPM1+ AML, molecular monitoring in PB during follow-up is of crucial importance in detecting late molecular relapse allowing to use less toxic molecular-oriented treatments as a bridge to HSCT. Further studies on larger cohorts hopefully will help to confirm its usefulness to guide the treatment approach. Disclosures Rossi: Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: D-actinomicin

Risk stratification in acute myeloid leukemia (AML) patients using prognostic parameters at diagnosis is effective, but may be significantly improved by the use of on treatment parameters which better define the actual sensitivity to therapy in the single patient. Minimal residual disease (MRD) monitoring has been demonstrated crucial for the identification of AML patients at high risk of relapse, but the best method and timing of MRD detection are still discussed. Thus, we retrospectively analyzed 104 newly diagnosed AML patients, consecutively treated and monitored by quantitative polymerase chain reactions (Q-PCR) on WT1 and by multiparametric flow cytometry (MFC) on leukemia-associated immunophenotypes (LAIPs) at baseline, after induction, after 1st consolidation and after 1st intensification. By multivariate analysis, the factors independently associated with adverse relapse-free survival (RFS) were: bone marrow (BM)-WT1 ≥ 121/10(4) ABL copies (P = 0.02) and LAIP ≥ 0.2% (P = 0.0001) (after 1st consolidation) (RFS at the median follow up of 12.5 months: 51% vs. 82% [P

HIST1H1T, one of the eleven members of the histone H1 gene family (H1A, H1B, H1C, H1D, H1E, H1T, H1F0, H1FNT, H1FOO, HILS1 and H1FX), is considered a germinal variant as it is specifically expresse...

In 2003 the Fondazione Italiana Linfomi (FIL) started a clinical research program for investigating initial treatment of frail elderly patients with diffuse large B-cell lymphoma (DLBCL) identified by Comprehensive Geriatric Assessment (CGA). From 2003 to 2006, 334 elderly patients underwent CGA assessment, and 99 patients were classified as frail. Frail patients had a median age of 78 years, stage III-IV disease in 62% and age-adjusted International Prognostic Index (aaIPI) of 2-3 in 53%. Treatment consisted of several different regimens according to physician discretion. After a median follow-up of 36 months, 5-year overall survival (OS) was 28%. In multivariate analysis, aaIPI 2-3 (p = 0.005) and the presence of respiratory comorbidity (p = 0.044) were the only factors that showed independent correlation with OS. Frail patients had a poorer outcome compared with fit patients also if they were treated with rituximab-containing combination chemotherapy (hazard ratio 2.37, 95% confidence interval 1.48-3.78; p < 0.001). CGA is a valid tool to prospectively identify frail subjects among elderly patients with DLBCL.

Background: As new biomarkers are validated and their significance in the natural history of specific diseases is established, these technologies can be rapidly transferred to clinical application. Since it has been shown that a single post-interferon-β (IFNβ) injection measurement of myxovirus-protein-A (MxA) mRNA correlates with IFNβ bioactivity in IFNβ treated patients with multiple sclerosis (MS), we had previously validated an assay for its quantification. Methods: We introduced a real-time PCR relative quantification assay into routine clinical practice and measured MxA mRNA expression in 564 samples from 500 unselected IFNβ treated MS patients over a 4-year period. Results: We confirmed that the assay is reproducible over time, and found that the percentage of patients lacking MxA mRNA induction is comparable to that described in studies performed worldwide after patient selection by pre-screening for the presence of anti-IFNβ antibodies. Conclusions: In view of its simplicity and reproducibility, this MxA assay is an alternative to anti-IFNβ antibody determinations for use in routine clinical practice. Clin Chem Lab Med 2010;48:1235–8.

"Real life" data are needed to complement published trials on the efficacy of lenalidomide in patients with myelodysplastic syndrome (MDS) and del(5q) and on the risk of inducing acute myeloid leukemia (AML) progression. Here, we present results of lenalidomide treatment in a consecutive, population-based series of 21 red blood cell (RBC) transfusion-dependent elderly patients with multiple comorbidities. Of 18 evaluable patients (median follow-up: 22 months), 17 achieved an erythroid hematologic response (HI-E) and 16 an RBC transfusion independence. Cytogenetic response (CyR) rate was 80%, median overall survival was 48 months (range 3-164), and 5-year leukemia-free survival was 84%. Three patients progressed to AML; one, with baseline TP53 mutation, achieved HI-E, partial CyR, and did not progress to AML. Eighteen patients experienced hematological adverse events. Overall, lenalidomide was very effective and well tolerated even in unselected elderly patients with multiple comorbidities and did not appear to increase the risk of AML.

The usefulness of posaconazole therapeutic drug monitoring (TDM) is still a matter of debate. A correlation between posaconazole serum levels and breakthrough invasive fungal infections (IFI) has not been clearly demonstrated so far. We analysed posaconazole serum levels in patients with acute myeloid leukaemia (AML) during induction therapy and correlated them with the incidence of breakthrough IFI and the need of systemic antifungal therapy. Overall, 77 AML patients receiving posaconazole were evaluated for serum levels; breakthrough IFI were observed in five with at least one posaconazole TDM (6.5%). Median serum level was 534 ng ml(-1) (IQ range: 298.5-750.5 ng ml(-1) ) and did not change significantly over time. Four of the 40 patients with median posaconazole levels

Background: The immune defects occurring in chronic lymphocytic leukemia are responsible for the frequent occurrence of infections and autoimmune phenomena, and may be involved in the initiation and maintenance of the malignant clone. Here, we evaluated the quantitative defects of newly produced B and T lymphocytes. Methods: The output of B and T lymphocytes from the production and maturation sites was analyzed in chronic lymphocytic leukemia patients and healthy controls by quantifying kappa-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs) by a Real-Time PCR assay that simultaneously detects both targets. T-lymphocyte subsets were analyzed by six-color flow cytometric analysis. Data comparison was performed by two-sided Mann-Whitney test. Results: KRECs level was reduced in untreated chronic lymphocytic leukemia patients studied at the very early stage of the disease, whereas the release of TRECs + cells was preserved. Furthermore, the observed increase of CD4 + lymphocytes could be ascribed to the accumulation of CD4 + cells with effector memory phenotype. Conclusions: The decreased number of newly produced B lymphocytes in these patients is likely related to a homeostatic mechanism by which the immune system balances the abnormal B-cell expansion. This feature may precede the profound defect of humoral immunity characterizing the later stages of the disease. Background Profound defects of both humoral and cell-mediated immunity have been described in patients with chronic lymphocytic leukemia (CLL), a disease characterized by the accumulation of mature, malignant, monoclonal B lymphocytes in blood, lymph nodes, spleen, liver, and bone marrow [1]. The disease is characterized by the presence of immune defects, responsible for the frequent occurrence of infections and autoimmune phenomena, that may be involved in the initiation and maintenance of the malignant clone. The immune abnormalities include reduced immunoglobulin (Ig) levels, as well as qualitative and quantitative defects of B, T, NK cells, neutrophils, and the monocyte/

BACKGROUND: Median age of patients (pts) with acute myeloid leukemia (AML) ranges between 69 and 72 years in population-based series (SEER, 2012; Juliusson, 2009). The prognosis of elderly AML is exceedingly poor with overall 2-year survival of 10% (Juliusson, 2011). The use of intensive chemotherapy (i-CT) in elderly AML patients as opposed to non-intensive chemotherapy (ni-CT) or best supportive care (BSC) is still debated. Age is still the major driver of treatment choice but, in recent years, the role of pts' fitness and comorbidities was underscored. In 2013, a panel of experts proposed a set of objective criteria to define pts fit or unfit to conventional i-CT, ni-CT or BSC (Ferrara et al, Leukemia, 2013). Since such criteria derived from experts opinion, they need to be validated in the clinical setting to become an useful tool for therapy decision making. AIMS: Fitness criteria were retrospectively applied to a population-based series of pts with AML (no M3), to evaluate a) their actual applicability; b) the concordance between “fitness” categorization of pts and the type of treatment they actually received; c) the outcome of pts according to “fitness”, to the prognostic stratification of European Leukemianet (ELN) and to the treatment received. PATIENTS and METHODS. We evaluated 362 pts aged > 65 y, diagnosed at five Hematology Centres of the Hematological Network of Lombardy in Northern Italy (REL). Pts were diagnosed between January 2008 and May 2014. Their median age was 73 y (range 65-94 y). Their categorization according to “fitness” criteria was carried out retrospectively by the teams of physicians who had followed pts and through medical files. Pts were classified as fit to i-CT (FIT), unfit to i-CT (UNFIT), or unfit even to ni-CT (FRAIL). According to ELN prognostic criteria, applied in 201 “de novo” AML, pts were at low-risk (36 pts: 18%), of which 7 (3,4%) with CBF leukemia, intermediate-I (75 pts: 37%), intermediate-II (30 pts: 15%), or high risk (60 pts: 30%). Criteria were not applicable in 34 pts (14%) lacking karyotype or molecular data. The group of 127 pts (35%) with AML therapy-related or secondary to myeloid neoplasms was considered at high clinical risk (cHR). According to physicians decision, 139 pts (38.4%) had received i-CT, 65 pts (18%) ni-CT, including low-dose arac, azacytidine or experimental non-myelotoxic drugs, and 158 pts (43.6%) BSC. RESULTS. Fitness criteria were not applicable in 12 pts (3%), because of insufficient data. Among 350 evaluable pts (97%), 170 (46.9%) were FIT, 140 (38.7%) were UNFIT, and 40 (11%) were FRAIL. Their median age was 69, 79 and 75 years, respectively (p CONCLUSIONS. The “fitness criteria” proposed were easily applicable even retrospectively and in a multicenter setting. Fitness was significantly related to patient's survival. In FIT and UNFIT pts outcome was similar with either i-CT or ni-CT, but these results need to be tested prospectively on larger cohorts. Integrating fitness with ELN criteria identifies a subgroup of FIT pts at low/Int-I ELN risk with a CR rate of 76.6% and a median OS of 20 months when treated with i-CT. Therefore fitness criteria seem to be a good tool to identify pts who can benefit from i-CT or ni-CT in alternative to BSC and to assist in the decision to use i-CT in elderly AML patients. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Abstract 3228 Objective: Progressive multifocal leukoencephalopathy (PML) is a rare, but often fatal demyelinating brain disease caused by the JC virus, which usually occurs in immunosuppressed patients, including those with hematological malignancies or receiving monoclonal antibodies-based immunotherapies. PML is likely the result of a complex combination of several pathogenic mechanisms, such as alterations of peripheral cell-mediated immunity and mobilization of JC virus-carrying CD34+-hematopoetic stem cells and pre-B-cells. Taking advantage of the availability of samples from a multiple sclerosis (MS) patient treated with the anti-α4β1 monoclonal antibody natalizumab who developed PML, which was monitored for 35 months since before therapy initiation, we investigated the role of B and T lymphocytes in PML onset. Methods: Real-Time PCR was used to measure the release of T and B cells from the production sites by means of T-cell receptor excision circles (TRECs) and K-deleting excision circles (KRECs) analysis and to quantify transcripts for immature hematopoietic cells such as terminal deoxynucleotidyl transferase, CD34, and pre-B lymphocyte gene 1. Naïve and mature T- and B-cell subsets were identified by flow cytometry, T-cell heterogeneity was quantified by spectratyping and IgA, IgG and IgM by turbidimetric assay. Data were compared to those of untreated and natalizumab-treated MS patients and healthy donors. Results: After 34 months of natalizumab therapy, a 42 years old female developed PML, diagnosed on the basis of magnetic resonance imaging and JC virus positivity in cerebrospinal fluid. Before therapy, her thymus and bone marrow produced a significant low number of TRECs+ and KRECs+ cells. While TRECs remained low during all therapy period, KRECs and transcripts for pre-B lymphocyte gene 1, which is selectively expressed in pre-B cells, peaked after 6 months of therapy, remained high at 12 and 15 months of treatment, and then decreased at the moment of PML onset. Flow cytometry confirmed a deficient production of CD4+CD45RA+CCR7+CD31+ recent T emigrants, counterbalanced by an increased number of CD8+CCR7–CD45RA+ TEMRA cells for all observation period, but showed a modification of peripheral CD4 and CD8 cell number only at the moment of PML. While the percentage of naïve B cells increased by about 70% after 6 months of therapy, the number of B lymphocytes within each B-cell subpopulations remained low for the entire treatment period. T-cell repertoire and immunoglobulin production were altered. Interpretation: Although performed in a single patient, data all agree in demonstrating that a deficient production of new TRECs+ T lymphocytes, together with an increase of newly produced KRECs+ B cells just few months after therapy beginning may predispose to PML. These findings encourage further researches on the utility of the TRECs/KRECs assay as a potential tool for the identification of patients at risk of developing PML after monoclonal antibodies-based therapies. Disclosures: No relevant conflicts of interest to declare.