Daniela Domínguez Damiani

Abstract The best treatment of type II mixed cryoglobulinemia (MC) has still to be defined. Antiviral treatment for the frequent underlying infectious trigger hepatitis C virus (HCV) may be ineffective, contraindicated, or not tolerated in a fraction of cases, whereas current immunosuppressive treatments may lead to relevant complications. Selective B-cell blockade with rituximab was used in this study, based on favorable results in preliminary experience. Fifteen consecutive patients with type II MC (HCV-related in 12 of 15) were treated with rituximab, 375 mg/m2 intravenously weekly for 4 weeks. Only medium- to low-dose steroids were allowed, if already administered at the time of recruitment. All patients had active disease, poorly controlled or difficult to manage with previous treatments, including corticosteroids in all. Efficacy and safety of rituximab therapy were evaluated in the following 6 months. The overall follow-up after rituximab treatment ranged from 9 to 31 months. Rituximab proved effective on skin vasculitis manifestations (ulcers, purpura, or urticaria), subjective symptoms of peripheral neuropathy, low-grade B-cell lymphoma, arthralgias, and fever. Nephritis of recent onset went into remission in one case. Laboratory features, that is, significantly decreased serum rheumatoid factor and cryoglobulins and increased C4, were consistent with the clinical efficacy. Treatment was well tolerated, with no infectious complications. Thrombosis of retinal artery or self-limiting panniculitis occurred in one patient each. Rituximab may represent a safe and effective alternative to standard immunosuppression in type II MC. Controlled studies are needed to better define drug indications and the cost-efficacy profile in the different systemic manifestations.

Abstract The aims of our study were to verify whether it was possible to generate in vitro, from different adult human tissues, a population of cells that behaved, in culture, as multipotent stem cells and if these latter shared common properties. To this purpose, we grew and cloned finite cell lines obtained from adult human liver, heart, and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs, obtained from the 3 different tissues, expressed the pluripotent state–specific transcription factors Oct-4, NANOG, and REX1, displayed telomerase activity, and exhibited a wide range of differentiation potential, as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content, and shared a common gene expression signature, compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular, the pathways regulating stem cell self-renewal/maintenance, such as Wnt, Hedgehog, and Notch, were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and functional features of mature cells even embryologically not related to the tissue of origin.

Ruxolitinib is a potent Janus kinase (JAK) 1/JAK2 inhibitor approved for the treatment of myelofibrosis (MF). Ruxolitinib was assessed in JUMP, a large (N = 2233), phase 3b, expanded-access study in MF in countries without access to ruxolitinib outside a clinical trial, which included patients with low platelet counts (<100 × 109 /l) and patients without splenomegaly - populations that have not been extensively studied. The most common adverse events (AEs) were anaemia and thrombocytopenia, but they rarely led to discontinuation (overall, 5·4%; low-platelet cohort, 12·3%). As expected, rates of worsening thrombocytopenia were higher in the low-platelet cohort (all grades, 73·2% vs. 53·5% overall); rates of anaemia were similar (all grades, 52·9% vs. 59·5%). Non-haematologic AEs, including infections, were mainly grade 1/2. Overall, ruxolitinib led to meaningful reductions in spleen length and symptoms, including in patients with low platelet counts, and symptom improvements in patients without splenomegaly. In this trial, the largest study of ruxolitinib in patients with MF to date, the safety profile was consistent with previous reports, with no new safety concerns identified. This study confirms findings from the COMFORT studies and supports the use of ruxolitinib in patients with platelet counts of 50-100 × 109 /l. (ClinicalTrials.gov identifier NCT01493414).

Transverse Momentum Dependent (TMD) parton distributions obtained from the Parton Branching (PB) method are combined with next-to-leading-order (NLO) calculations of Drell-Yan (DY) production. We apply the MCatNLO method for the hard process calculation and matching with the PB TMDs. We compute predictions for the transverse momentum, rapidity and $\phi^*$ spectra of Z-bosons. We find that the theoretical uncertainties of the predictions are dominated by the renormalization and factorization scale dependence, while the impact of TMD uncertainties is moderate. The theoretical predictions agree well, within uncertainties, with measurements at the Large Hadron Collider (LHC). In particular, we study the region of lowest transverse momenta at the LHC, and comment on its sensitivity to nonperturbative TMD contributions.

P‐glycoprotein (PGP), lung resistance‐related protein (LRP) and multidrug resistance associated protein (MRP) expression and the blast cells' intracellular daunorubicin accumulation (IDA) were evaluated in 96 previously untreated cases of de novo acute non‐lymphocytic leukaemia (ANLL). 47/96 patients (49%) were classified as PGP + , 44/96 (46%) as LRP + , and 8/96 (8%) as MRP + . The more frequent MDR clusters were PGP − /LRP − /MRP − (32/96 cases, 33%) and the PGP + /LRP + /MRP − (27/96 cases, 28%) followed by PGP + /LRP − /MRP − (15/96 cases, 16%) and PGP − /LRP + /MRP − (14/96 cases, 14%). A favourable karyotype was observed more frequently in PGP − and LRP − cases. A highly significant correlation was found between either PGP or LRP overexpression and leukaemic blast cell IDA. All the patients received standard induction and consolidation treatments containing MDR‐related (idarubicin, mitoxantrone, etoposide) and other (arabinosyl cytosine) drugs. Multivariate analysis showed that PGP overexpression was significantly associated with a poor response to treatment, both in terms of primary resistance or shorter survival. Other independent prognostic factors were age and cytogenetics. LRP overexpression did not reach statistical significance, although for LRP + cases the trend was unfavourable. Due to small numbers, no conclusion could be made regarding MRP overexpression, but 5/8 cases showed unfavourable karyotypic abnormalities, 8/8 had a defective IDA and 6/8 failed to achieve remission. This study showed that both PGP and LRP overexpression are common features in de novo ANLL at onset whereas MRP overexpression is more rare. It suggested that overexpression of one of the MDR related proteins was associated with a defective IDA, and confirmed that, in addition to age and cytogenetics, PGP retains an independent prognostic value. It also suggested that LRP did not affect clinical outcome when patients were treated with idarubicin or mitoxantrone and arabinosyl cytosine.

We evaluated the incidence, the risk factors, and the outcome of late-onset noninfectious pulmonary complications (LONIPCs) among 50 patients who underwent allogeneic stem cell transplantation from unrelated donors. Of the 39 patients surviving at least 3 months, 10 (26%) fulfilled the diagnostic criteria of LONIPCs and were further subclassified as having bronchiolitis obliterans (four patients), bronchiolitis obliterans with organizing pneumonia (four patients), and interstitial pneumonia (two patients). Two patients had a durable partial remission after treatment with prednisone and cyclosporine; the remaining eight patients did not respond to treatment and five of them died of respiratory failure. Advanced stage of disease at transplant and chronic extensive graft-versus-host disease (GVHD) were significantly associated with the development of LONIPCs. Pulmonary function test (PFT) results before transplantation were similar in all patients, but patients with LONIPCs had a significant decrease in PFT indexes at the third month after BMT compared with controls. Moreover, the rate of cyclosporine taper during the fourth and fifth months after BMT was significantly more rapid in patients with LONIPCs than in controls, suggesting that the risk of LONIPCs may be influenced by a faster reduction of GVHD prophylaxis.

Two monoclonal antibodies, MRK16 and C219, both directed at the 170 kDa P‐glycoprotein multidrug resistance agent, were applied to frozen sections or cytospin preparations from normal human tissues and 60 non‐Hodgkin's malignant lymphomas. Adrenal gland, kidney, liver and pancreas were always stained by the reagents, albeit with slightly different patterns. Brain capillaries as well as macrophages and some elements of the bone marrow, peripheral blood, ovarian stroma and colonic, gastric and jejunal mucosa were positive in all examined preparations. There were differences in the staining patterns with the two antibodies. Among the 60 non‐Hodgkin's lymphomas, 25 contained a number of positive cells, which ranged from 2% to 100%. No correlation was seen between the expression of P1 70 and histological type, stage, clinical symptoms or growth fraction. A close relationship was shown between the presence of P1 70 positive elements and the clinical course of the disease ( P &lt;0.001).

Abstract It has been observed in the literature that measurements of low-mass Drell–Yan (DY) transverse momentum spectra at low center-of-mass energies $$\sqrt{s}$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msqrt><mml:mi>s</mml:mi></mml:msqrt></mml:math> are not well described by perturbative QCD calculations in collinear factorization in the region where transverse momenta are comparable with the DY mass. We examine this issue from the standpoint of the Parton Branching (PB) method, combining next-to-leading-order (NLO) calculations of the hard process with the evolution of transverse momentum dependent (TMD) parton distributions. We compare our predictions with experimental measurements at low DY mass, and find very good agreement. In addition we use the low mass DY measurements at low $$\sqrt{s}$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msqrt><mml:mi>s</mml:mi></mml:msqrt></mml:math> to determine the width $$q_s$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msub><mml:mi>q</mml:mi><mml:mi>s</mml:mi></mml:msub></mml:math> of the intrinsic Gauss distribution of the PB-TMDs at low evolution scales. We find values close to what has earlier been used in applications of PB-TMDs to high-energy processes at the Large Hadron Collider (LHC) and HERA. We find that at low DY mass and low $$\sqrt{s}$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msqrt><mml:mi>s</mml:mi></mml:msqrt></mml:math> even in the region of $$p_\mathrm{T}/m_\mathrm{DY}\sim 1$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>T</mml:mi></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mi>m</mml:mi><mml:mi>DY</mml:mi></mml:msub><mml:mo>∼</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:math> the contribution of multiple soft gluon emissions (included in the PB-TMDs) is essential to describe the measurements, while at larger masses ( $$m_\mathrm{DY}\sim m_{{\mathrm{Z}}}$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:msub><mml:mi>m</mml:mi><mml:mi>DY</mml:mi></mml:msub><mml:mo>∼</mml:mo><mml:msub><mml:mi>m</mml:mi><mml:mi>Z</mml:mi></mml:msub></mml:mrow></mml:math> ) and LHC energies the contribution from soft gluons in the region of $$p_\mathrm{T}/m_\mathrm{DY}\sim 1$$ <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>T</mml:mi></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mi>m</mml:mi><mml:mi>DY</mml:mi></mml:msub><mml:mo>∼</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:math> is small.

Multidrug resistance is a major cause of treatment failure in acute myeloid leukemia (AML). P-glycoprotein (PGP) over-expression has an unfavorable prognostic significance, while the role of breast cancer resistance protein (BCRP) is less clear, especially in AML patients with a normal karyotype. We studied 73 consecutive AML patients with a normal karyotype. BCRP was over-expressed in 24 patients (33%) and was significantly co-expressed with PGP (13/24 vs 11/49, p=0.006) and with CD56. Only PGP, along with age and CD34, affected the achievement of complete remission (p=0.02), while BCRP-positive cases showed an increased risk of relapse (p=0.005) and a shorter disease-free survival (p=0.027). BCRP over-expression did not influence the achievement of remission, but significantly affected the duration of complete remissions. BCRP may, therefore, be regarded as a prognostic factor in patients with normal karyotype AML, for the design of risk-adapted post-remission therapy.

Abstract: Resistance to several cytotoxic agents, including anthracyclines, vinca alkaloids and epipodophylline derivatives (multidrug resistance, or MDR) can develop in tumor cells by overexpression of a 170‐kd glycoprotein (p170) which is an essential component of a membrane transport system leading to increased drug efflux and decreased intracellular drug concentration. By means of a p170‐directed monoclonal antibody (MRK‐16) and immunocytochemistry (alkaline phosphatase anti‐alkaline phosphatase technique), we investigated the expression of p170 in marrow blast cells of 59 cases (38 at diagnosis and 21 in relapse) of acute‐non‐lymphocytic leukemia (ANLL). The proportion of strongly MDR‐positive cells was higher in relapse that at diagnosis (median 15.5% vs 1.5%). Out of 31 patients who were evaluable for the results of first remission induction, failure of first‐line treatment (including Daunorubicin, standard‐dose and high‐dose Arabinosyl Cytosine, and sometimes also Mitoxantrone) occurred in 8/22 MDR‐positive cases and in 1/9 MDR‐negative ones (p = 0.21). Failure of first‐line treatment was always associated with a progressive increase of p170 expression. Total failures (no remission plus early relapse) were more frequent (p = 0.001) among MDR‐positive cases (16/22) than among the others (2/9). These data show that MDR is very frequent in ANLL also at diagnosis and suggest that MDR can contribute to early failure of standard treatment.

Tumor necrosis factor a is one of the principal cytokines involved in the pathogenesis of acute graft-versus-host-disease (GVHD). Infliximab is an antibody to this cytokine.We performed a retrospective analysis to evaluate the activity of infliximab in 32 patients with severe steroid-refractory acute GVHD. The patients received a median of 3 weekly courses of infliximab. The main organs involved in the patients were skin (n=2) liver (n=1), bowel (n=19), liver and bowel at the same stage (n=10).Nineteen out 32 patients (59%) responded to infliximab with 6 (19%) complete and 13 (40%) partial responses. Age younger than 35 years, intestinal involvement and a longer time between hematopoietic stem cell transplantation and infliximab administration were factors predicting a favorable response. Infective episodes developed in 23/32 (72%) patients. All the 13 unresponsive patients died of GVHD shortly after infliximab. Thirteen of 19 responsive patients were alive at a median follow-up of 449 days (range 155-842) after infliximab, with no signs of chronic GVHD (n=5), limited (n=5) or extensive involvement (n=3). Six patients who responded subsequently died, one of chronic lung GVHD, the others of vascular complications or infections (2 fungal diseases).We conclude that infliximab is active in the treatment of severe steroid-refractory acute GVHD, particularly when the intestine is involved. Infections commonly followed its administration. The clinical activity of infliximab and the possibility that it increases the risk of infections are worth investigating in prospective trials.

Despite recent advances, prognosis of acute myeloid leukemia (AML) remains unsatisfactory due to poor response to therapy or relapse. Among causes of resistance, over-expression of multidrug resistance (MDR) proteins represents a pivotal mechanism. ABCG2 is an efflux transporter responsible for inducing MDR in leukemic cells; through its ability to extrude many antineoplastic drugs, it leads to AML resistance and/or relapse, even if conflicting data have been reported to date. Moreover, ABCG2 may be co-expressed with other MDR-related proteins and is finely regulated by epigenetic mechanisms. Here, we review the main issues regarding ABCG2 activity and regulation in the AML clinical scenario, focusing on its expression and the role of polymorphisms, as well as on the potential ways to inhibit its function to counteract drug resistance to, eventually, improve outcomes in AML patients.

P‐glycoprotein (PGP) lung resistance protein (LRP) and multidrug resistance associated protein (MRP) expressions and function were evaluated by flow cytometry in 65 leukaemic patients (38 acute non‐lymphocytic leukaemias, eight acute lymphocytic leukaemias, 19 Ph‐positive chronic myeloid leukaemias in blastic phase). By using the MRK‐16, the LRP‐56 and the MRPm6 MoAbs, 34% of the cases did not over‐express any proteins (−); 24.5% over‐expressed (+) only PGP, 11% only LRP, 1.5% only MRP, 24.5% both PGP and LRP, and 4.5% both PGP and MRP. The mean intracellular daunorubicin accumulation (IDA) and rhodamine 123 (Rh123) retention in the presence or absence of the reversal agent SDZ PSC 833 (PSC) of the PGP − /LRP − /MRP − cases were comparable to the ones observed in normal leucocytes. With respect to the non‐over‐expressing cases, the PGP − /LRP + /MRP − cases showed only an impaired IDA (mean 204 ± 29; P &lt; 0.001). The PGP + /LRP + /MRP − cases had a defect both in IDA (mean 166 ± 47, P &lt; 0.001) and Rh123 retention (mean 0.42 ±0.14; P &lt; 0.001), which were both corrected by PSC. All the PGP + /LRP + /MRP − cases had a defect in IDA (mean daunorubicin (DNR) accumulation 192 ± 44; P &lt; 0.001. However, only in 8/16 of them an evident defect in Rh123 retention was found. In conclusion, both PGP and LRP over‐expression were common in leukaemia. An impaired IDA was found in all cases over‐expressing PGP, LRP or both. The study of Rh123 retention could give incorrect information about the blast cells’ ability to accumulate cytotoxic drugs in patients over‐expressing both PGP and LRP.

Abstract Although a large amount of data is available on the effects of filgrastim (granulocyte colony-stimulating factor [G-CSF]) on the mobilization of stem cells in the circulation, data concerning its effects on bone marrow (BM) harvesting is scarce and controversial. We have designed a randomized trial comparing filgrastim-mobilized peripheral blood stem cell (PBSC) transplantation with filgrastim-primed autologous bone marrow transplantation (ABMT). Fifty-five patients affected by non-Hodgkin's (n = 38) or Hodgkin's (n = 17) lymphoma, selected for autologous transplantation over a 12-month period in a single institution, were randomized 2:1 to undergo BM or PB harvest/collection after priming for 3 days with filgrastim, 16 μg/kg body weight daily subcutaneously. BM priming with G-CSF allowed the harvest of a significantly higher number of mononuclear cells (MNC) (0.53 × 108/kg, range, 0.32 to 1.40), as compared with a historical control of unprimed BM harvests (0.43 × 108 MNC/kg, range, 0.15 to 0.72, P = .001). After high-dose ablative therapy, median time to neutrophil recovery above 0.5 × 109/L was 12 days for BM and 11 days for PB (P = .219); median time to platelet recovery above 20 × 109/L was 13 days for BM and 11 days for PB (P = .242). The same number of red blood cells, platelet transfusions, and posttransplant G-CSF doses were required in the two groups of patients. Less patients (50% v 70%) became febrile in the group transplanted with mobilized PB, but days of fever/patient and days on antibiotics were overlapping. The median time spent in the hospital after reinfusion was 16.5 and 15.5 days after primed BM and primed PB, respectively (P = .134). These data suggest that in patients with lymphoma submitted to autologous transplantation, the reinfusion of filgrastim-primed BM or filgrastim-mobilized PB leads to similar results, with an advantage of only 1 day in the neutrophil recovery and 1 day on the time spent in the hospital in favor of primed PB. Either option can be chosen on the basis of the availability of a surgery room or cell separator facilities and considering the patients' characteristics and wishes.

Cases of B-cell chronic lymphocytic leukaemia (B-CLL) with mutated (M) IgV(H) genes have a better prognosis than unmutated (UM) cases. We analysed the IgV(H) mutational status of B-CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B-CLLs, 124 cases were examined for IgV(H) gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity-determining regions as evidence of antigen-driven selection; this identified three B-CLL subsets: significantly mutated (sM), with evidence of antigen-driven selection, not significantly mutated (nsM) and UM, without such evidence and IgV(H) gene per cent mutations above or below the 2% cut-off. sM B-CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgV(H) genes. sM, nsM and UM B-CLL were also characterized for the biased usage of IgV(H) families, intraclonal IgV(H) gene diversification, preference of mutations to target-specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase zeta and eta and activation-induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B-CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B-CLL.

Invasive Fungal Infections (IFI) remain a severe and major complication among patients with hematologic diseases, but the recent availability of new antifungal agents (echinocandins and new azoles) have improved the chance of cure. Caspofungin (Cancidas-Merck) is a large lipopeptide molecule able to inhibit the enzyme complex 1,3-d-glucan synthetase; this action specifically damages the fungal cell wall. Caspofungin (CAS) is active, in vitro and in vivo, against most Candida species and Aspergillus species. We report on our experience with this drug as first-line therapy for proven or probable pulmonary IFI in immunocompromised patients with hematologic malignancies. Thirty-two consecutive patients (20 males and 12 females, with a median age of 52 yr) have been treated with CAS (27 acute leukemias, 1 chronic leukemia, 3 lymphomas and 1 multiple myeloma). Sixteen patients (50%) had a relapsed or resistant hematologic disease, while 12 patients were in complete remission and 4 were at onset of disease; 8/32 (25%) developed IFI after a hematopoietic stem cell transplant (HSCT) procedure. Seven out of 32 patients (22%) had a proven pulmonary IFI (7/7 Aspergillosis) and 25 (78%) had a probable IFI with pulmonary localization as defined according to international consensus. Thirty-one patients (97%) had less than 1000 granulocytes/mL at onset of infection and at the start of CAS therapy. The CAS was given at the dose of 70 mg on day 1, followed by 50 mg/day. Median duration of CAS therapy was 20 d (range 8-64); all the 31 neutropenic patients received concomitant granulocyte colony-stimulating factor (G-CSF). The overall response rate was 56% (18/32) with 12/18 complete responses and 6/18 partial responses; two patients (6%) had a stable disease. Twelve out of 32 (38%) did not respond and seven died of mycotic infection. Univariate analysis showed that granulocytes recovery (>500/mL vs. <500/mL) and status of hematologic disease (remission/onset vs. refractory/relapsed) were significantly associated to favourable outcome. No clinical adverse events (AE) were reported and only a grades I and II transient increase of serum alkaline phosphatase and/or transaminases occurred in 4/32 (12%) patients. After CAS therapy six non-responders and six cases with a partial or stable response were rescued with voriconazole. Two out of six patients (33%) in the former group and 6/6 (100%) in the latter obtained a complete resolution of IFI. Our experience suggests an efficacy of CAS, in combination with G-CSF, as first-line treatment of proven or probable IFI with pulmonary localization. The drug was well tolerated and there were no significant hepatic AE even in patients receiving CAS with cyclosporine after a HSCT. A significant proportion of non-responders or partial responders to CAS can be rescued with a subsequent voriconazole-based therapy.

Scleroderma may be one of the most severe forms of chronic graft-versus-host disease (GVHD). We retrospectively evaluated its incidence, predictor variables and outcome in 133 patients who survived at least 4 months after allogeneic hematopoietic stem cell transplantation. The 5-year cumulative incidence was 15.5% in patients with chronic GVHD. The generalized form had a progressive course despite immunosuppressive therapy. Eosinophilia, autoimmune markers, and previous skin involvement by chronic GVHD with disorders of pigmentation were significantly associated with an increased probability of developing scleroderma.

CD200, a protein belonging to the immunoglobulin superfamily, has been associated with a poor prognosis in lymphoproliferative disorders and in acute leukemia. We studied the expression of CD200 in a series of 244 patients with diagnosis of acute myeloid leukemia (AML), to evaluate its impact on outcome and its possible association with other known prognostic factors. CD200 was found in 136/244 (56%) patients, in 41 of whom (30%) with high intensity of expression (MFI ≥ 11). CD200 was more frequent in secondary compared to de novo leukemia (p = 0.0006), in CD34 positive cases (p = 0.00001), in Bcl2 overexpressing cases (p = 0.01), in those wild-type Flt3 (p = 0.004) and with favorable or unfavorable compared to intermediate karyotype (p = 0.0003). CD200+ patients have a two-fold lower probability to attain complete remission, both in univariate (p = 0.006) and multivariate (p = 0.04) analysis. The negative impact of CD200 was found also in overall survival (p = 0.02) and was correlated with the intensity of expression of the molecule (p = 0.024). CD200 has an additive negative impact on survival in patients with unfavorable cytogenetic (p = 0.046) and in secondary leukemia (p = 0.05), and is associate with a worsening of outcome in patients with favorable biological markers, such as mutated NPM (p = 0.02), wild-type Flt3 (p = 0.034), negativity of CD34 (p = 0.03) and of CD56 (p = 0.03). In conclusion, CD200 is emerging as both a prognostic factor and a potential target of novel therapeutic approaches for AML, aiming to reverse the "do not eat me" signal of CD200 or to manipulate the suppressive immune microenvironment induced by CD200 binding to its receptor.

Homoharringtonine (HHT) is a cephalotaxine alkaloid that showed clinical efficacy in the chronic phase of chronic myeloid leukemia (Ph1+CML). As a single agent, it resulted in effectively controlling leukocytosis and in producing sporadic karyotypic conversions; its clinical use in combination with interferon (IFN-α) for the treatment of CML could thus be considered. In this study we evaluated the growth inhibition and the induction of apoptosis determined by HHT alone and in combination with IFN-α and cytosine arabinoside (Ara-C) on normal and CML (both in chronic, CML-CP and in blastic phase; CML-BP) hematopoietic progenitors. HHT is able to determine a dose-dependent cell growth inhibition; evaluation of cytotoxic activity on semisolid cultures showed an activity significantly higher on CML-CP than on normal cells (P = 0.02 for HHT 50 ng/ml and P = 0.01 for HHT 200 ng/ml). HHT exerted a synergistic effect with IFN-α, Ara-C and IFN-α + Ara-C in inhibiting CML-CP colony growth; the same activity was demonstrated by the combination of HHT with Ara-C and by the triple combination, but not by HHT + IFN-α, on normal myeloid progenitors. The triple combination only was able to exert a synergistic effect in CML-BP. The induction of apoptosis resulted HHT dose-dependent in CML-CP and normals; at higher drug concentrations (100–200–1000 ng/ml), HHT induced a significant increase of apoptotic cells (for normals: P = 0.04, P = 0.02 and P = 0.04; for CML-CP: P = 0.01, P = 0.01 and P = 0.04, respectively); no significant changes were observed in CML-BP. In conclusion, the differences in cytotoxic effect and apoptosis induction observed, depending on the various phases of CML, add experimental evidence to the different clinical results between the chronic phase, where the clone is responsive to HHT, and the acute phase, where the drug is ineffective. The in vitro synergism of HHT with Ara-C and IFN-α in CML-CP suggests further evaluation in the clinical setting.

The possibility that Daunoxome (DNX), a combination of daunorubicin (DNR) with a liposomal targeting system, escapes PGP was tested. Two pairs of leukaemic cell lines, each consisting of the parental non‐multidrug resistance (MDR) line and of a MDR variant, were studied for cytotoxicity (MTT test) and for cellular DNR kinetic and accumulation (flow cytometry). DNX and free DNR were equally toxic against non‐MDR cells, whereas the liposomal anthracycline was more toxic than the free drug against the MDR variant. Non‐MDR cells accumulated DNR more rapidly when they were exposed to free DNR than to DNX, but MDR cells accumulated more DNR when they were exposed to DNX. The kinetics of DNX and free DNR were also studied in the blast cells of 41 cases of acute leukaemia and they were found to be related to blast cell PGP expression. In 15 cases with a low PGP expression intracellular DNR accumulation was faster and higher with free DNR than with DNX. In 26 cases with a high PGP expression the area under the curve was similar with DNX and free DNR, but the kinetics of intracellular DNR accumulation showed an early low plateau with free DNR and a slow and continuous increase with DNX. In MDR cell lines the ratio was more favourable to DNX than to free DNR. We conclude that liposome encapsulated DNR is partially protected from PGP and that it is worth testing for the treatment of PGP‐positive acute leukaemia.

Because of the fact that tumor cell sensitivity to cytotoxic agents may play a major role in cancer treatment, and several anthracyclines are widely used for first-line treatment of leukemia, lymphoma and other tumors, and since the overexpression of the mdr-1 gene-coded 170 Kd glycoprotein (P170) decreases cell sensitivity to anthracyclines, we investigated the relationship between P170 overexpression and the cytotoxicity of two classic anthracyclines (Daunorubicin or DNR and Doxorubicin or DX) and two lipophilic anthracycline derivatives (Idarubicin or IDA and Iododoxorubicin or IDX). For these purposes, we used multidrug resistant (MDR) and non-MDR tumor and leukemia cell lines and the MTT-microcultured tetrazolium colorimetric assay. We showed that mdr-1 gene overexpression was strongly associated with the development of a high level of resistance to DNR and DX, but not to the derivatives IDA and IDX. These data suggest that more lipophilic anthracycline derivatives may also be active in MDR cell systems.

The pharmacokinetic-pharmacodynamic profile of a fixed 6-g daily continuous intravenous infusion of ceftazidime was assessed in 20 febrile neutropenic patients with acute myeloid leukemia. Mean steady-state ceftazidime concentrations averaging 40 mg/liter from day 2 on ensured maximized pharmacodynamic exposure (values close to four to five times the MIC breakpoint against Pseudomonas aeruginosa). However, large intra- and interindividual pharmacokinetic variability was documented throughout the study period.

British Journal of HaematologyVolume 77, Issue 4 p. 460-465 The expression of the multidrug resistance-associated glycoprotein in B-cell chronic lymphocytic leukaemia Mariacrazia Michieli, Corresponding Author Mariacrazia Michieli Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalyDr M. Michieli, Cattedra di Ematologia, Policlinico dell' Università di Udine, P. le S. Maria della Misericordia, 33100 Udine, Italy.Search for more papers by this authorDonatella Raspadori, Donatella Raspadori Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorDaniela Damiani, Daniela Damiani Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorAntonella Geromin, Antonella Geromin Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorCristiana Gallizia, Cristiana Gallizia Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorAngela Michelutti, Angela Michelutti Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorRenato Fanin, Renato Fanin Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorGianpiero Fasola, Gianpiero Fasola Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorDomenico Russo, Domenico Russo Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorPierluici Tazzari, Pierluici Tazzari Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorStefan0 Pileri, Stefan0 Pileri Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorFranco Mallardi, Franco Mallardi Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorMichele Baccarani, Michele Baccarani Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this author Mariacrazia Michieli, Corresponding Author Mariacrazia Michieli Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalyDr M. Michieli, Cattedra di Ematologia, Policlinico dell' Università di Udine, P. le S. Maria della Misericordia, 33100 Udine, Italy.Search for more papers by this authorDonatella Raspadori, Donatella Raspadori Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorDaniela Damiani, Daniela Damiani Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorAntonella Geromin, Antonella Geromin Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorCristiana Gallizia, Cristiana Gallizia Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorAngela Michelutti, Angela Michelutti Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorRenato Fanin, Renato Fanin Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorGianpiero Fasola, Gianpiero Fasola Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorDomenico Russo, Domenico Russo Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorPierluici Tazzari, Pierluici Tazzari Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorStefan0 Pileri, Stefan0 Pileri Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorFranco Mallardi, Franco Mallardi Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this authorMichele Baccarani, Michele Baccarani Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and Institute of Haematology ‘L. and A. Seràgnoli’, University of Bologna School of Medicine, Bologna, ItalySearch for more papers by this author First published: April 1991 https://doi.org/10.1111/j.1365-2141.1991.tb08610.xCitations: 34AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Abstract Summary. The expression of the multidrug resistance (MDR)-associated 170 kDa glycoprotein (p170) was investigated in 63 cases of B-cell chronic lymphocytic leukaemia (CLL), with two monoclonal antibodies (MRK-16 and C-219). By immunocytochemistry with MRK-16 (63 cases), the great majority of the cells was positive, with a weak reaction in 61% of cases and a strong reaction in 39% of cases. By flow cytometry, the proportion of positive cells was 39±25% with MRK–16 (63 cases), and 23±22% with C–219 (36 cases). The expression of pl70 in leukaemic B-lymphocytes suggests that also in B-CLL the development of MDR can have some therapeutic relevance. 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Over-expression of multidrug resistance (MDR) proteins PGP and BCRP has a negative prognostic impact in acute myeloid leukemia (AML) patients. Inclusion of fludarabine in induction chemotherapy increases remission rate in PGP over-expressing cases. We investigated the role of BCRP in 138 adult AML patients receiving induction therapy with fludarabine. None of the MDR-related proteins influenced complete remission attainment. Conversely, high levels of BCRP significantly affected disease-free survival, as higher relapse rates (48.5% vs 28.5%) and earlier relapse occurred in BCRP+ patients. Also overall survival was affected by BCRP positivity, and survival significantly worsened in case of concomitant PGP and BCRP over-expression.

Overexpression of brain and acute leukemia cytoplasmic (BAALC) gene confers poor prognosis in cytogenetically normal acute myeloid leukemia (AML) patients, while less defined is its role in AML with abnormal karyotype. We evaluated the effect of BAALC overexpression on outcome of 175 adult AML patients with different cytogenetic risks. Karyotype was favorable in 13, intermediate in 117 and unfavorable in 45 patients, respectively. Quantitative BAALC expression was determined by real‐time PCR, with cut off value set at 50th percentile. BAALC was overexpressed in 87/175 (50%) patients, without association with cytogenetic status. High BAALC was associated with unmutated NPM ( P = 0.006) and CD34 positivity ( P &lt; 0.0001). Complete remission (CR) was attained in 111 patients (63%), and was maintained at 5 years in 52 ± 7%. BAALC overexpression had a negative impact on CR achievement ( P = 0.04), while did not influence relapse probability. Median survival was 22 months with a 5‐years overall survival (OS) of 35%. Factors with a negative impact on OS were older age ( P = 0.0001), unfavorable cytogenetic ( P = 0.005), ABCG2 overexpression ( P = 0.03) and high BAALC levels ( P = 0.01). We observed a worse outcome in patients with high BAALC expression through all cytogenetic risk categories: 5‐years OS was 100% vs. 71% in patients with favorable cytogenetics ( P = 0.05), 55% vs. 40% in cases with intermediate karyotype ( P = 0.04) and 34% vs. 23% in unfavorable cytogenetic subgroup ( P = 0.02). BAALC overexpression identified AML patients with poor prognosis in all cytogenetic groups. Though relatively rare, BAALC positivity in patients with favorable or unfavorable karyotype significantly worsened survival. Am. J. Hematol. 88:848–852, 2013. © 2013 Wiley Periodicals, Inc.

Abstract Background Patients with hematological malignancies are at an increased risk of SARS‐CoV‐2 disease (COVID‐19) and adverse outcome. However, a low mortality rate has been reported in patients with chronic myeloid leukemia (CML). Preclinical evidence suggests that tyrosine kinase inhibitors (TKIs) may have a protective role against severe COVID‐19. Methods We conducted a cross‐sectional study of 564 consecutive patients with CML who were tested for anti‐SARS‐CoV‐2 IgG/IgM antibodies at their first outpatient visit between May and early November 2020 in five hematologic centers representative of three Italian regions. Results The estimated serological prevalence of SARS‐CoV‐2 infection in patients with CML after the first pandemic wave was similar to that in the general population (about 2%), both at national and regional levels. CML patients with positive anti‐SARS‐CoV‐2 serology were more frequently male ( p = 0.027) and active workers ( p = 0.012), while there was no significant association with TKI treatment type. Only 3 out of 11 IgG‐positive patients had previously received a molecular diagnosis of COVID‐19, while the remainders were asymptomatic or with mild symptoms. Conclusions Our data confirm that the course of SARS‐CoV‐2 infection in patients with CML is generally mild and reassure about the safety of continuing TKIs during the COVID‐19 pandemic. Furthermore, we suggest that patients with CML succeed to mount an antibody response after exposure to SARS‐CoV‐2, similar to the general population.

The prognosis of acute myeloid leukemia (AML) remains unsatisfactory. Among the reasons for the poor response to therapy and high incidence of relapse, there is tumor cell immune escape, as AML blasts can negatively influence various components of the immune system, mostly weakening T-cells. Since leukemic cells can dysregulate immune checkpoints (ICs), receptor-based signal transductors that lead to the negative regulation of T-cells and, eventually, to immune surveillance escape, the inhibition of ICs is a promising therapeutic strategy and has led to the development of so-called immune checkpoint inhibitors (ICIs). ICIs, in combination with conventional chemotherapy, hypomethylating agents or targeted therapies, are being increasingly tested in cases of AML, but the results reported are often conflicting. Here, we review the main issues concerning the immune system in AML, the main pathways leading to immune escape and the results obtained from clinical trials of ICIs, alone or in combination, in newly diagnosed or relapsed/refractory AML.

Few series of adult patients with primary systemic CD30 (Ki-1)-positive anaplastic large cell lymphoma (ALCL) are reported in the literature; most of them have been treated with combination chemotherapy (CHT), with only an occasional patient being autotransplanted, mainly after relapsing. The remission rate ranges from 60% to 90%, but relapses are frequent (up to 60%) and precocious (mainly in the first 24 months). The aim of our study was to analyze the outcome of a series of adult patients affected by primary systemic ALCL that were treated at our institution with a sequential intensive therapeutic program including CHT, radiotherapy (RT), and autologous bone marrow transplantation (ABMT). Sixteen consecutive, unselected patients with ALCL were identified. All of them were treated with the 5-fluorouracil, methotrexate, cytosine arabinoside, cyclophosphamide, doxorubicin, vincristine, and prednisone (F-MACHOP) regimen; 9 of 16 (56.2%) reached a complete remission (CR). In six cases with residual mediastinal disease, involved-field RT was performed, allowing three additional patients to become free of disease. All 16 were then autotransplanted with bone marrow stem cells after conditioning with the cytosine arabinoside, etoposide, cyclophasphamide, and carmustine (BAVC) regimen. A present, 16 of 16 patients are alive and in CR. The actuarial overall survival is 100% at a median of 45.5 months, and the actuarial disease-free survival is 100% at a median of 33.5 months. These data suggest that ALCL can be successfully managed with a sequential intensive treatment (CHT +/- RT + ABMT) that prevents early relapses and projects these patients as long-term survivors.

We analysed the expression of three drug transporter proteins [p-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP1)] involved in anthracycline resistance that are frequently overexpressed in poor-risk adult acute non-lymphocytic leukaemia (ANLL), in 23 acute promyelocytic leukaemia (APL) patients at onset managed at a single institution. Cellular daunorubicin accumulation was also evaluated. At onset, no case had PGP or MRP1 expression that exceeded that of non-multidrug-resistant (MDR) cell lines. Only one case showed LRP overexpression. No peculiar MDR features distinguished the seven patients who relapsed from those who maintained complete remission. In the onset vs. first relapse, only one patient showed an increased (threefold) PGP expression at relapse. At second relapse, three out of four patients showed a PGP expression two- to threefold higher than baseline values. These results are consistent with the view that low PGP, LRP and MRP1 expression and the absence of defects in intracellular drug accumulation may account for the peculiarly high sensitivity of APLs to anthracycline. It does not support the screening of MDR markers in APL patients at onset as predicting factors of early relapse. The results suggest that no significant changes in PGP, LRP or MRP1 expression are likely to occur at first relapse. In contrast, PGP expression is likely to increase later in the patient history as a result of additional chemotherapy courses.

Fludarabine plus cytarabine (Ara-C) and idarubicin (FLAI) is an effective and well-tolerated induction regimen for the treatment of acute myeloid leukaemia (AML). This phase III trial compared the efficacy and toxicity of FLAI versus idarubicin plus Ara-C and etoposide (ICE) in 112 newly diagnosed AML patients <60 years. Fifty-seven patients received FLAI, as the first induction-remission course, and 55 patients received ICE. Post-induction treatment consisted of high-dose Ara-C (HDAC). After HDAC, patients in complete remission (CR) received a second consolidation course (mitoxantrone, etoposide, Ara-C) and autologous stem cell transplantation (auto-SCT) or allogeneic (allo)-SCT, according to the age, disease risk and donor availability. After a single induction course, CR rate was 74% in the FLAI arm and 51% in the ICE arm (P = 0.01), while death during induction was 2% and 9% respectively. Both haematological (P = 0.002) and non-haematological (P = 0.0001) toxicities, especially gastrointestinal (i.e. nausea, vomiting, mucositis and diarrhoea), were significantly lower in FLAI arm. In both arms, relapses were more frequent in patients who were not submitted to allo-SCT. After a median follow-up of 17 months, 30% and 38% of the patients are in continuous CR in FLAI and ICE arm respectively. Our prospective randomised study confirmed the anti-leukaemic effect and the low toxic profile of FLAI as induction treatment for newly diagnosed AML patients.

P-glycoprotein (P-gp), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP) expression, and blast cell intracellular daunorubicin accumulation (IDA) were evaluated in 95 previously untreated cases of adult acute lymphoblastic leukaemia (ALL) using flow cytometry. Forty-five out of 95 (47%) patients were P-gp positive (+), 12/66 (18%) were LRP+ and 11/66 (17%) were MRP+. Eighteen out of 66 (28%) patients showed a simultaneous multidrug resistance (MDR)-related protein expression higher than controls for more than one protein, while 24/66 (36%) cases did not overexpress any protein. Twenty-one out of 24 (87%) cases overexpressing at least one MDR-related protein had a defect in accumulating daunorubicin into their blast cells, while only 4/24 (16%) cases who did not overexpress any protein had similar features. The complete remission rates were similar in MDR-positive and -negative (-) patients but relapses within 6 months were more frequent in P-gp+ cases, and therefore the disease-free survival duration was shorter in P-gp+ than in P-gp- patients (P = 0.01). The number of MRP+ and/or LRP+ cases was too small to be able to draw any conclusion on their role in affecting or predicting therapy outcome. In conclusion, P-gp overexpression associated with a defect in daunorubicin accumulation is a frequent feature in adult ALL at onset and seems to be related to poorer therapy outcome and, consequently, a shorter disease-free survival. LRP and MRP overexpression seems to be a rare event and no conclusion can be drawn on its prognostic role.

Abstract Cytogenetic abnormalities are among the most important factors affecting the outcome of patients with acute myeloid leukaemia (AML), but approximately 40–50% of AML cases display a normal karyotype at diagnosis. Multidrug resistance (MDR) proteins overexpression is associated with worse prognosis in acute leukaemias, but its role in normal karyotype AML is less defined. We analysed the expression of P‐glycoprotein (PGP), MDR‐related protein (MRP) and lung resistance protein (LRP) in 135 adult patients with normal karyotype AML and its correlation with other biological features of the disease, to evaluate the impact of MDR proteins on response to therapy and on survival. Increased PGP expression was associated with lower rate of complete remission (CR; p = 0.006), similarly to advanced age. Cases overexpressing PGP displayed also a shorter event‐free survival (EFS; 4 vs. 10 months, p = 0.035) and the increased expression of at least one MDR protein was associated with a reduced overall survival (OS; p = 0.038). Also age was predictive of worse prognosis. Our data confirm the prognostic role of MDR proteins, in particular of PGP, also in AML patients with normal karyotype at diagnosis. This finding could be used to stratify patients with different prognosis and to design risk‐adapted therapeutic strategies. Copyright © 2007 John Wiley &amp; Sons, Ltd.

The addition of gemtuzumab-ozogamicin (GO) to an induction regimen including synergistic drugs, such as intermediate dose of cytarabine (Ara-C), idarubicin and fludarabine (FLAI), could reduce treatment failure in acute myeloid leukemia (AML) patients. Nevertheless, the role and safety of this antibody target-therapy in first-line chemotherapy in patients younger than 65 years has not yet been defined. The primary goal of this prospective phase II pilot study was to evaluate the efficacy and the safety profile of FLAI plus GO as induction regimen. Thirty consecutive AML patients were included. All patients were younger than 65 with a median age of 53 years and CD33 expression exceeded 20% in all cases. The M/F ratio was 16/14 and 21/30 (70%) of patients were poor-risk at diagnosis. The induction regimen (FLAI-GO) included fludarabine (30 mg/m2) and Ara-C (2 g/m2) on days 1–5, idarubicin (10 mg/m2) on days 1, 3, and 5 and GO (3 mg/m2) on day 6. Hematopoietic stem cell transplant (HSCT) was planned for all high risk AML patients in first complete remission (CR) after consolidation with intermediate doses of Ara-C and idarubicin (IDAC-IDA). Cytogenetic, multidrug-resistance phenotype, FLT3 mutation status, and WT1 quantitative expression analyses were performed at diagnosis in all patients. WT1 expression and cytogenetic (in positive cases) analyses were performed after induction to detect and follow minimal residual disease. Patients were evaluated for response rate, treatment-related adverse events, overall survival and relapse free survival. After induction with FLAI-GO, CR rate was 90% (26 of 29 evaluable pts); one patient achieved partial remission and two were resistant. There was only one case of death during induction (DDI). After FLAI-GO, the mean value of WT1 dropped from 4200 ± 2777 copies/104ABL to 192 ± 399 copies/104ABL. The toxicity of FLAI-GO was acceptable; 57% of patients experienced transient and reversible GO infusion-related adverse events (especially fever and chills), but no cases of veno-occlusive disease occurred during CHT or after HSCT. After a median follow-up of 16 months (range 2–25), 24/30 (80%) patients are alive (24/24 in CR). The probability of 1-year OS and RFS was 90 and 85%, respectively. Allogeneic and autologus HSCT was performed in 19 (63%) and 4 (13%) patients, respectively. These preliminary results suggest that FLAI-GO is an effective and well tolerated induction regimen for CD33 positive AML patients younger than 65 years, with a high complete response rate, favourable safety profile, low DDI. These results encourage the testing of this regimen in a multicenter prospective trial.

Genetic heterogeneity in drug-metabolizing enzyme/transporter (DMET) genes affects specific drug-related cancer phenotypes. To investigate the relationships between genetic variation and response to treatment in acute myeloid leukemia (AML), we genotyped 1931 variants on DMET genes in 94 CD33-positive AML patients enrolled in a phase III multicenter clinical trial combining Gemtuzumab-Ozogamicin (GO) with Fludarabine-Cytarabine-Idarubicin (FLAI) regimen, with the DMET Plus platform. Two ADH1A variants showed statistically significant differences (odds ratio (OR)=5.68, P=0.0006; OR=5.35, P=0.0009) in allele frequencies between patients in complete/partial remission and patients without response, two substitutions on CYP2E1 (OR=0.13, P=0.001; OR=0.09, P=0.003) and one on SLCO1B1 (OR=4.68, P=0.002) were found to differently influence liver toxicity, and two nucleotide changes on SULTB1 and SLC22A12 genes correlated with response to GO (OR=0.24, P=0.0009; OR=2.75, P=0.0029). Genetic variants were thus found for the first time to be potentially associated with differential response and toxicity in AML patients treated with a combination of GO-FLAI regimen.

The successful operation of the Large Hadron Collider (LHC) and the excellent performance of the ATLAS, CMS, LHCb and ALICE detectors in Run-1 and Run-2 with $pp$ collisions at center-of-mass energies of 7, 8 and 13 TeV as well as the giant leap in precision calculations and modeling of fundamental interactions at hadron colliders have allowed an extraordinary breadth of physics studies including precision measurements of a variety physics processes. The LHC results have so far confirmed the validity of the Standard Model of particle physics up to unprecedented energy scales and with great precision in the sectors of strong and electroweak interactions as well as flavour physics, for instance in top quark physics. The upgrade of the LHC to a High Luminosity phase (HL-LHC) at 14 TeV center-of-mass energy with 3 ab$^{-1}$ of integrated luminosity will probe the Standard Model with even greater precision and will extend the sensitivity to possible anomalies in the Standard Model, thanks to a ten-fold larger data set, upgraded detectors and expected improvements in the theoretical understanding. This document summarises the physics reach of the HL-LHC in the realm of strong and electroweak interactions and top quark physics, and provides a glimpse of the potential of a possible further upgrade of the LHC to a 27 TeV $pp$ collider, the High-Energy LHC (HE-LHC), assumed to accumulate an integrated luminosity of 15 ab$^{-1}$.

Bcl-2 overexpression has been shown to be associated with several malignancies, including B-cell chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphomas (NHL), mainly low-grade and follicular in type. It has as yet not been described in hairy cell leukemia (HCL). In 30 patients with CLL and 14 with HCL who were consecutively selected for treatment with purine analogues (Fludarabine in CLL and 2-chloro-deoxy-adenosine in HCL), we evaluated bcl-2 oncoprotein expression in leukemic cells on marrow sections that were taken before treatment and stained immunohistochemically with a monoclonal antibody (Dakopatts 124 clone), by the avidin-biotin-peroxidase method. All samples were found to be bcl-2 positive, with a staining intensity that was moderate to strong in CLL and weak to moderate in HCL. 83% of CLL and 100% of HCL patients were responsive to purine analogues. These findings show that bcl-2 is overexpressed in almost all cases CLL and HCL and that bcl-2 overexpression does not predict a poor response to purine analogues, which are believed to induce apoptosis.

Abstract We assessed the hematopoietic recovery and transplantation-related mortality (TRM) of patients who had failed peripheral blood stem cell mobilization and subsequently received high-dose chemotherapy supported by granulocyte colony-stimulating factor (G-CSF)–primed bone marrow (BM). Studied were 86 heavily pretreated consecutive patients with acute leukemia (n = 21), refractory/relapsed non-Hodgkin lymphoma (n = 41) and Hodgkin disease (n = 17), and multiple myeloma (n = 7). There were 78 patients who showed insufficient mobilization of CD34+ cells (&amp;lt; 10 cells/μL), whereas 8 patients collected less than 1 × 106 CD34+ cells/kg. BM was primed in vivo for 3 days with 15 to 16 μg/kg of subcutaneous G-CSF. Median numbers of nucleated cells, colony-forming unit cells (CFU-Cs), and CD34+ cells per kilogram harvested were 3.5 × 108, 3.72 × 104, and 0.82 × 106, respectively. Following myeloablative chemotherapy, median times to achieve a granulocyte count higher than 0.5 × 109/L and an unsupported platelet count higher than 20 and 50 × 109/L were 13 (range, 8-24), 15 (range, 12-75), and 22 (range, 12-180) days, respectively, for lymphoma/myeloma patients and 23 (range, 13-53), 52 (range, 40-120), and 90 (range, 46-207) days, respectively, for leukemia patients. Median times to hospital discharge after transplantation were 17 (range, 12-40) and 27 (range, 14-39) days for lymphoma/myeloma and acute leukemia patients, respectively. TRM was 4.6%, whereas 15 patients died of disease. G-CSF–primed BM induces effective multilineage hematopoietic recovery after high-dose chemotherapy and can be safely used in patients with poor stem cell mobilization.

ABCG2 protein overexpression and FLT3 internal tandem duplication (ITD) correlate with higher relapse rate and shorter disease-free survival (DFS) in acute myeloid leukemia (AML), but no data are available on the possible effect of concomitant presence of these 2 factors.The authors analyzed the outcome of 166 cases of adult AML patients who were homogeneously treated with a fludarabine-based induction therapy.ABCG2 overexpression and FLT3-ITD were detected in 83 (50%) and 47 (28%) patients, respectively. A significant correlation was found between ABCG2 positivity and FLT3 mutation, with 33 (40%) ITD in 83 ABCG2-positive patients compared with 14 (17%) ITD in 83 ABCG2-negative patients (P = .002). Complete remission (CR) after induction therapy was achieved in 95 (57%) patients. Neither ABCG2 overexpression nor FLT3-ITD had any impact on achievement of CR. Relapse occurred in 42 of 95 (44%) patients at a median time of 28 months. Time to relapse was shortened in patients overexpressing ABCG2 (P = .0004). DFS was not affected by FLT3-ITD alone, but FLT3 mutation significantly worsened long-term outcome of ABCG2-positive patients. DFS at 1 and 3 years in patients with overexpression of both ABCG2 and FLT3-ITD was only 36% and 28%, respectively; in ABCG2-positive/FLT3-negative patients, DFS at 1 and 3 years was 65% and 48%, respectively; and in ABCG2-negative cases (regardless of FLT3 status), DFS at 1 and 3 years was greater than 85% and 75%.Concomitant overexpression of ABCG2 and FLT3-ITD is relatively frequent and identifies a subgroup of AML patients with a significantly worse prognosis. The possible interactions between these 2 prognostic factors need to be defined.

Over-expression of multidrug resistance protein ABCG2 has been associated to chemotherapy failure in solid and hematologic tumors.[1][1]–[3][2] More than 40 ‘synonymous’ and ‘non-synonymous’ single nucleotide polymorphisms (SNPs) of ABCG2 have been identified.[4][3] Among them, the 421C>A

Overexpression of CD200, a trans-membrane protein belonging to the immunoglobulin superfamily, has been associated with poor prognosis in patients with acute myeloid leukemia (AML). As few data are available in the subset of cytogenetically-normal (CN) AML, we retrospectively evaluated the correlations between CD200 expression and response to therapy in a series of 139 adults with CN-AML. CD200 was expressed in 67/139 (48%) cases; 18 of them (28%) expressed CD200 at high intensity. No differences in CD200 expression rate were observed according to age, WBC count, type of leukemia, FLT3 or NMP1 mutation, and CD56 expression. A higher incidence of CD200 expression was observed in CD34+ cases (P<0.0001) and in BCL2+ patients (P=0.04). Complete remission (CR) was evaluable achieved in 98 patients (70%): 56/71 (79%) in CD200- and 47/67 (63%) in CD200+ patients (P=0.03), with a lower CR rate in patients with high CD200 intensity (9/18, 50%). CD200 expression had a negative impact on long-term outcome. CD200 expression, per se, did not impact on disease-free survival (DFS), but cases with high CD200 expression had a lower 3-year DFS compared to CD200-negative and low-expressing ones (0% vs 65% vs 68%, P=0.019). Three-year overall survival (OS) was 51% in CD200- and 27% in CD200+ patients (P=0.01), with a significant difference among cases with low or high CD200 expression (35% vs 0%, P=0.001). CD200 high expression defined a group with very poor DFS and OS also among the 37 FLT3-/NPM1+: 3-year DFS and OS were 88% and 60% in CD200-, 50% and 32% in CD200 low and 0% and 0% in CD200 high patients, respectively (P=0.01 for DFS and P=0.05 for OS). Our data suggest a negative impact of CD200 expression in CN-AML, with a further worsening in high-expressing cases, also in the subset of FLT3-/NPM1+ patients.

ABGG2 protein overexpression in acute myeloid leukemia (AML) has been associated with poor response to conventional chemotherapy and increased relapse risk. No data are available on the role of allogeneic stem cell transplantation (SCT) in reversing its negative prognostic role. We have reviewed the outcome of 142 patients with high risk AML who underwent allogeneic SCT in complete remission (n = 94) or with active disease (n = 48). Patients with ABCG2 overexpression at AML diagnosis have lower leukemia free survival (LFS) and increased cumulative incidence of relapse (CIR) compared with ABCG2- patients (5-year LFS 50% vs. 65%, P = 0.01; 5-year CIR 46% vs. 27%, P = 0.003). Five-year overall survival was not significantly different between ABCG2+ and ABCG2- patients (39% vs. 51%, P = 0.1). However, if we consider only disease-related deaths, ABCG2 maintains its negative role (64% vs. 78%, P = 0.018). The negative impact of ABCG2 overexpression was higher in patients undergoing SCT in CR compared with patients receiving transplant with active disease. Conditioning regimen did not abrogate the effect of ABCG2 overexpression, as CIR was higher in ABCG2+ patients receiving both myeloablative (44% vs. 22%, P = 0.018) or reduced intensity conditioning (50% vs. 32%, P = 0.03). In conclusion, ABCG2 overexpression at AML diagnosis identifies a subset of patients with poor outcome also after allogeneic SCT, mainly in terms of higher relapse rates. Prospective studies employing conditioning drugs or post-transplant strategies able to target ABCG2 are needed to maximize the curative potential of stem cell transplantation.

B-cell prolymphocytic leukemia (B-PLL) is a rare mature B-cell tumor with an aggressive clinical course and poor prognosis. It is characterized by prominent splenomegaly and prolymphocytes exceeding 55% of the lymphoid cells in the blood. Purine analog-based chemo-immunotherapy is the first-line therapy for B-PLL. Owing to its rarity, there are few reports on the efficacy of bendamustine and rituximab (BR) regimen. Our study presents three cases of BR being effective in the treatment of B-PLL and provides experience for clinical treatment.This report describes the cases of three male patients (median age: 66 years old) who initially presented with abdominal discomfort. Physical examinations and imaging revealed splenomegaly, while a peripheral blood (PB) smear revealed a prolymphocyte count exceeding 70% of the lymphoid cells. Therefore, the three patients were diagnosed with B-PLL. Further molecular detection showed that they harbored P53 abnormalities (17p deletion/TP53 mutation) associated with resistance to conventional chemotherapies. In addition, one of the patients had a highly complex karyotype and multiple gene mutations. All patients underwent four cycles of BR, and two of them received two further cycles of rituximab monotherapy. Ultimately, the patients achieved a complete response (CR) that lasted for 25, 33, and 34 months, respectively, with a median follow-up time of 34 months. The adverse events of the BR mainly included a grade 3 haematological toxicities. Also, the treatment was well-tolerated.This case series suggests that BR regimen is promising for bringing deep remission to patients with B-PLL. Prospective trials are still required for further elucidation.

<h3>Background</h3> The potential risks associated with the use of levosimendan in the pediatric population has not been systematically evaluated. This study aimed to review the available evidence regarding the safety of this treatment. <h3>Methods</h3> Bio Med Central, PubMed, Embase, and the Cochrane Central Register of clinical trials were searched for studies describing levosimendan administration in the pediatric population in any setting. Relevant studies were independently screened, selected, and their data extracted by two investigators. The authors excluded: reviews, meta-analyses, as well as basic research and trials involving patients >18 years old. The primary outcome was the number and the type of adverse side effects reported during levosimendan administration. <h3>Results</h3> The updated systematic review included 48 studies, enrolling a total of 1,271 pediatric patients who received levosimendan as treatment (790 patients in the 11 studies that reported side effects). The primary adverse effects of levosimendan administration were hypotension and cardiac arrhythmias, particularly tachycardia. Hypotension occurred in approximately 28.9% of patients, while arrhythmia occurred in about 12.3% of patients. Meta analysis of RCTs revealed a rate of all-cause mortality of 2.0% (8 out of 385) in the levosimendan group compared to 3.9% (15 out of 378) in the control group (dobutamine, milrinone or placebo) (risk ratio [RR] = 0.55; 95% confidence interval [CI] = 0.25-1.21; P=0.14; I² = 0%) <h3>Conclusions</h3> Hypotension and cardiac arrhythmia are the most reported side effects of levosimendan in pediatric patients. However, adverse events remain underreported, especially in randomized trials.

Despite the progress in the knowledge of disease pathogenesis and the identification of many molecular markers as potential targets of new therapies, the cure of acute myeloid leukemia remains challenging. Disease recurrence after an initial response and the development of resistance to old and new therapies account for the poor survival rate and still make allogeneic stem cell transplantation the only curative option. Multidrug resistance (MDR) is a multifactorial phenomenon resulting from host-related characteristics and leukemia factors. Among these, the overexpression of membrane drug transporter proteins belonging to the ABC (ATP-Binding Cassette)-protein superfamily, which diverts drugs from their cellular targets, plays an important role. Moreover, a better understanding of leukemia biology has highlighted that, at least in cancer, ABC protein's role goes beyond simple drug transport and affects many other cell functions. In this paper, we summarized the current knowledge of ABCG2 (formerly Breast Cancer Resistance Protein, BCRP) in acute myeloid leukemia and discuss the potential ways to overcome its efflux function and to revert its ability to confer stemness to leukemia cells, favoring the persistence of leukemia progenitors in the bone marrow niche and justifying relapse also after therapy intensification with allogeneic stem cell transplantation.

Abstract Objectives: Infections are the major cause of morbidity and mortality in patients with acute myeloid leukaemia (AML). They primarily occur during the first course of induction chemotherapy and may increase the risk of leukaemia relapse, due to a significant delay in consolidation therapy. The intensification of induction chemotherapy and the use of non‐conventional drugs such as fludarabine are considered responsible for the increased risk of infections. Methods: In this study, we retrospectively analysed the infections occurred in 224 newly diagnosed AML patients ≤65 yr, consecutively treated between 1997 and 2002 with an induction regimen including fludarabine, arabinosyl cytosine and idarubicin, with or without etoposide (FLAI/FLAIE), in the context of three multicentric prospective trials (AML97, AML99, AML02). Results: During the induction phase, 146 (65%) patients experienced fever of undetermined origin (FUO), 30 (13%) and 47 (21%) patients had Gram‐negative and positive bacteremias, respectively, and 10 (4%) patients developed a probable/proven invasive fungal infection (IFI). The fatality rate for Gram‐negative, Gram‐positive bacteremias and probable/proven IFI was 10%, 8% and 60% respectively. During consolidation, 75 (35%) patients had FUO, 43 (20%) and 40 (19%) patients had Gram‐negative and positive bacteremias, respectively, and 5 (2%) patients developed a probable/proven IFI. The fatality rate for Gram‐negative, Gram‐positive bacteremias and probable/proven IFI was 14%, 5% and 80% respectively. Interestingly, the overall incidence of microbiologically documented infections during induction was 38% and the incidence of probable/proven IFIs during the induction/consolidation programme was 7%. No infections caused by viruses or opportunistic pathogens were observed neither during induction, nor during consolidation. Conclusions: These data, although retrospectively collected, suggest that fludarabine‐based chemotherapy is not associated with an increased incidence of infections, in particular IFIs, compared to conventional regimens commonly used for AML induction.

The immunophenotype of 110 adult patients with diagnosis of acute myeloblastic leukemia (AML) was analyzed using a wide panel of monoclonal antibodies (mAbs). Leukemic blasts were tested by applying direct immunofluorescence analysis and dual-fluorescence staining, and two groups of patients were identified: 56/110 (51%) expressing only myeloid antigens (My/AML) and 54/110 (49%) expressing both myeloid and lymphoid antigens (Ly/AML). CD13 and CD33 were expressed in almost all FAB subtypes, whereas CD14, frequently expressed in M4 and M5 subtypes (70%), was rarely expressed in M0 + M1 cases (9%). On the contrary, CD34, expressed in 77% of M0 + M1 cases, was practically absent in M3 and M5 subtypes (6% and 7%, respectively). CD2 and CD7 antigens were found in 34% and 42% of patients respectively, whereas B cell-associated antigens, such as CD10 and CD19, were found in 31% and 18% of patients. Cytogenetic abnormalities characteristically present in AML patients were also analyzed and, except for t(8;21) which was found in both groups of patients, the other abnormalities were frequently found in cases coexpressing lymphoid-associated antigens. Finally, the complete remission (CR) rate, survival and event-free survival were analyzed according to the presence of lymphoid markers and also of some specific antigens such as CD7 and CD34. The only prognostic difference was represented by CD34+ patients who showed a reduction in the CR rate compared with CD34- patients (65% versus 82%) (p = 0.05) which became more evident when the mean intensity of fluorescence was considered. In conclusion, mAbs conjugated with fluorochromes and analyzed by more sophisticated cytometers allow the identification of a higher number of AML cases bearing lymphoid-associated antigens, but this phenotypic coexpression is not associated with biologically distinct forms of leukemia.

We report on a single-center experience about the characteristics and outcome of 36 acute promyelocytic leukemia (APL) patients observed at our Department of Hematology between 1990 and 2002. The expression, of multidrug-resistance (MDR) associated proteins (PGP, LRP, MRP1) was also analyzed. There were 12 males and 24 females (median age 37 yr), 89% (32 of 36) with classic morphology, and 11% (four of 36) with a microgranular variant. Risk class (according to GIMEMA/PETHEMA): 25% (nine of 36) high risk (HR), 53% (nineteen of 36) intermediate risk (IR), 22% (eight of 36) low risk (LR). PGP, LRP, and MRP1 expression at onset and at first relapse was low. CD33 antigen expression was high in all cases. The patients were treated according to GIMEMA protocols (LAP0389 and AIDA) including ATRA in induction in 75% (27 of 36) of cases and 94% (34 of 36) achieved a complete remission (CR) after induction therapy while 6% (two of 36) died early (DDI) of hemorrhage.71% (24 of 34) of evaluable patients remain in CR at a median follow-up of 57 months (range 4-158 months) while 29% (10 of 34) relapsed at a median time of 12 months (range 8-43 months) and, of them, eight of 10 died early. The majority of patients that relapsed were in high-risk group. The overall survival (OS) of the whole population at 32 months was 66% and the DFS at 42 months was 62%. A statistically significant difference in terms of DFS was observed between HR and IR/LR patients (P = 0.04 by log-rank). DFS was not affected by age, sex, Hb levels, karyotype, and BCR isoform. At conclusion, our data confirm that despite the high rate of success with ATRA plus chemotherapy as induction (more than 90% of CR), about 30% of APL patients have a relapse (without a long-lasting second remission) and underline the importance of patient stratification in distinct risk groups at diagnosis in order to better adapt the type and intensity of treatment (risk-adapted therapy). Taking into account the high expression of CD33 and the low expression of MDR proteins in APL, new and investigational approaches like gemtuzumab-ozogamicin, with or without ATRA and other new drugs, should be strongly considered expecially in HR APL.

Abstract: The treatment of relapsed adult acute lymphoblastic leukaemia (ALL) is frequently unsuccessful with current chemotherapy regimens, and often there is an overexpression of multidrug resistance (MDR)‐related proteins. Liposomal encapsulation makes daunorubicin (DNR) less sensitive to the efflux effect of P‐glycoprotein (PGP), and in vitro data indicate that liposomal‐encapsulated DNR (Daunoxome‐DNX) is more toxic than DNR against ALL cell lines. In this study, we assessed the in vivo and in vitro efficacy and toxicity of DNX plus cytarabine (Ara‐C) as reinduction chemotherapy in 25 relapsed ALL patients (pts). The expression of MDR‐related proteins (PGP, MRP1 and LRP) was also analysed. Of the 25 pts, 12 were males and 13 females; median age was 32 yr (range 18–58). Six cases were ALL T and 19 ALL B; eight pts were Ph+ (32%), and nine Bcr‐Abl+ (36%). The expression of MDR‐related proteins, and DNR and DNX retention and induction of apoptosis in leukaemic cells were evaluated in all cases. Seventeen of 25 (68%) pts were at first relapse and eight (32%) at second or subsequent relapse. The DNX was given in a dose of 80 mg/m 2 /d (days 1–3) in 11/25 pts (44%) and in a dose of 100 mg/m 2 /d (days 1–3) in 14/25 pts (66%). In all pts, Ara‐C was administered in a dose of 2 g/m 2 (days 1–5). Twenty pts (80%) achieved a complete remission (CR) and two (8%) entered a partial remission (PR) for an overall response (OR) rate of 88% (22/25), with a tolerable toxicity and without significant cardiotoxicity. Before the start of DNX therapy, 18/25 (72%) cases overexpressed at least one MDR‐related protein compared with 9/25 (36%) cases with MDR overexpression at diagnosis ( P = 0.01). Taking into account the small number of cases, the response rate was not affected by MDR expression and the in vitro results also showed a higher uptake and apoptotic cell death by DNX compared with DNR. Twelve pts subsequently underwent allogeneic bone marrow transplantation (11 unrelated donor BMT, and one sibling BMT). The overall survival was 39% after 12 months. These data show the efficacy (OR rate 88% and CR rate 80%) of DNX plus Ara‐C as reinduction therapy in very poor‐risk ALL pts and the response rate seems not to be affected by MDR overexpression. Moreover, the high rate of remissions and the good clinical tolerance in heavily pretreated pts suggest a promising role of DNX in ALL chemotherapy regimens.

We analyzed BCR::ABL1 expression at stop and in the first month after discontinuation in 168 chronic myeloid leukemia patients who stopped imatinib or 2nd generation tyrosine kinase inhibitors (2G-TKIs) while in sustained deep molecular response. Patients were divided among those who maintained response (group 1, n = 123) and those who lost major molecular response (group 2, n = 45). Mean BCR::ABL1 RNA levels 1 month after discontinuation were higher in group 2 than in group 1 (p = 0.0005) and the difference was more evident 2 months after stop (p < 0.0001). The same trend was found both for imatinib and 2G-TKIs. A receiver operating characteristic (ROC) analysis to determine a threshold value of BCR::ABL1 at 1 month after discontinuation identified a cut-off value of 0.0051%, with 92.2% specificity, 31.7% sensitivity and a likelihood ratio of 4.087.

Large-scale CD34+ enrichment has been demonstrated a safe method in autologous transplantation for multiple myeloma. However, the high CD34+ enrichment and the consequent plasma cell purging result in concomitant T-cell and dendritic-cell (DC) depletion, theoretically increasing the risk of life-threatening infections.We evaluated immunological and dendritic reconstitution in 72 myeloma patients who had undergone CD34+-selected (n = 45) and unmanipulated (n = 27) stem cell transplant, and its correlation with infections.Haematological recovery occurred promptly in all patients. Only a slight delay in platelet recovery to >50 x 10(9)/l was observed in patients receiving CD34+-enriched graft. Natural killer (NK) cell count recovered in all patients within 2 months and B-cell count had recovered by 6 months post-transplant in both groups. CD3 cells remained lower than normal in both groups. CD8 cells increased above the normal level, reaching a peak at day 90, and lowered to normal level within 1 year post-transplant. CD4 lymphocytes remained <50% of normal, especially in selected patients. In both groups, both DC1 and DC2 counts were already significantly lower than in normal individuals before conditioning therapy. Pre-conditioning levels of DC1 were reached in unmanipulated patients at day 30 and became normal at 6 months. In selected patients, DC1 pre-transplant level was observed at day 60 and was maintained thereafter. DC2 recovery showed a similar trend. In unselected patients, DC2 count increased to pre-conditioning level at haematological recovery and was normal after 1 year. In selected transplants, DC2 increased more slowly than DC1 in the same patients: pre-transplant level was detected at day 90 but was still significantly lower than normal 1 year after transplant. The incidence of infection was similar in both groups. Sepsis had Gram+ aetiology in the majority of cases. After engraftment only viral infections were recorded, mostly due to herpes reactivation, with no difference between groups.In spite of a delay in immune recovery, CD34 enrichment is not associated with a significant increase of complications due to infection. Relatively fast NK cell recovery to pre-transplant levels and the presence of functionally efficient DCs can justify the low incidence of infections.

British Journal of HaematologyVolume 78, Issue 2 p. 288-289 mdr-1 GENE AMPLIFICATION IN ACUTE LYMPHOBLASTIC LEUKAEMIA PRIOR TO ANTILEUKAEMIC TREATMENT Mariagrazia Michieli, Mariagrazia Michieli Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this authorMauro Giacca, Mauro Giacca Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this authorRenato Fanin, Renato Fanin Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this authorDaniela Damiani, Daniela Damiani Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this authorAntonella Geromin, Antonella Geromin Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this authorMichele Baccarani, Michele Baccarani Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this author Mariagrazia Michieli, Mariagrazia Michieli Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this authorMauro Giacca, Mauro Giacca Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this authorRenato Fanin, Renato Fanin Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this authorDaniela Damiani, Daniela Damiani Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this authorAntonella Geromin, Antonella Geromin Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this authorMichele Baccarani, Michele Baccarani Chair of Haematology and Chair of Anatomy, Institute of Experimental and Clinical Morphology, University of Udine School of Medicine, Udine, and International Centre for Genetic Engineering and Biotechnology, Trieste, ItalySearch for more papers by this author First published: June 1991 https://doi.org/10.1111/j.1365-2141.1991.tb04435.xCitations: 17AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume78, Issue2June 1991Pages 288-289 RelatedInformation

Front line treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) and chemotherapy (CHT) results in molecular remission in approximately 95% of patients tested after consolidation. The small fraction of patients with persistence of molecular disease (i.e. those in whom polymerase chain reaction (PCR) is positive for PML/RARalpha) after such therapy are thought to have a dismal prognosis but has not yet been investigated in detail.We analyzed the clinico-biological features at presentation of APL patients who showed PCR-detectable residual disease and compared them to those of patients achieving molecular remission after AIDA induction and consolidation. Furthermore, we report the outcome of patients with molecularly persistent disease treated with salvage therapy.Patients attaining molecular remission (n=650) and patients who tested PCR+ve at the end of consolidation (n=23) were not statistically significantly different as regards median age, white cell and platelet counts, morphologic subtype (M3 or M3v), fibrinogen levels or PML/RARalpha transcript type. As to treatment outcome after salvage therapy, 7 patients were treated before morphologic relapse [3 with chemotherapy and autologous stem cell transplantation (SCT) and 4 with allogeneic SCT], and are alive after 64-118 months. Of 16 patients treated at the time of morphologic relapse, only 2 patients are alive, both of whom received an allogeneic SCT.Our findings indicate that APL patients who are molecularly resistant to the AIDA protocol have no distinguishing features at presentation. Their outcome suggests the need for early therapeutic intervention with aggressive treatment prior to the occurrence of hematologic relapse.

American Journal of HematologyVolume 83, Issue 6 p. 517-518 LetterFree Access Brain natriuretic peptide level as marker of cardiac function in imatinib—Treated chronic myeloid leukemia patients: No evidence of cardiotoxicity of imatinib therapy Mario Tiribelli, Mario Tiribelli Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorAntonio Colatutto, Antonio Colatutto Department of Laboratory Diagnostics, “Azienda Ospedaliero-Universitaria”, Udine, ItalySearch for more papers by this authorLuciana Marin, Luciana Marin Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorGiuseppe Barbina, Giuseppe Barbina Department of Laboratory Diagnostics, “Azienda Ospedaliero-Universitaria”, Udine, ItalySearch for more papers by this authorUgo Qualizza, Ugo Qualizza Department of Laboratory Diagnostics, “Azienda Ospedaliero-Universitaria”, Udine, ItalySearch for more papers by this authorDaniela Damiani, Daniela Damiani Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorEleonora Toffoletti, Eleonora Toffoletti Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorMarta Medeot, Marta Medeot Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorAnna Candoni, Anna Candoni Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorElio Tonutti, Elio Tonutti Department of Laboratory Diagnostics, “Azienda Ospedaliero-Universitaria”, Udine, ItalySearch for more papers by this authorPierguido Sala, Pierguido Sala Department of Laboratory Diagnostics, “Azienda Ospedaliero-Universitaria”, Udine, ItalySearch for more papers by this authorRenato Fanin, Renato Fanin Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this author Mario Tiribelli, Mario Tiribelli Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorAntonio Colatutto, Antonio Colatutto Department of Laboratory Diagnostics, “Azienda Ospedaliero-Universitaria”, Udine, ItalySearch for more papers by this authorLuciana Marin, Luciana Marin Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorGiuseppe Barbina, Giuseppe Barbina Department of Laboratory Diagnostics, “Azienda Ospedaliero-Universitaria”, Udine, ItalySearch for more papers by this authorUgo Qualizza, Ugo Qualizza Department of Laboratory Diagnostics, “Azienda Ospedaliero-Universitaria”, Udine, ItalySearch for more papers by this authorDaniela Damiani, Daniela Damiani Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorEleonora Toffoletti, Eleonora Toffoletti Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorMarta Medeot, Marta Medeot Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorAnna Candoni, Anna Candoni Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this authorElio Tonutti, Elio Tonutti Department of Laboratory Diagnostics, “Azienda Ospedaliero-Universitaria”, Udine, ItalySearch for more papers by this authorPierguido Sala, Pierguido Sala Department of Laboratory Diagnostics, “Azienda Ospedaliero-Universitaria”, Udine, ItalySearch for more papers by this authorRenato Fanin, Renato Fanin Division of Hematology and Bone Marrow Transplantation, Department of Medical and Morphological Researches, Udine, ItalySearch for more papers by this author First published: 29 January 2008 https://doi.org/10.1002/ajh.21157Citations: 12AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume83, Issue6June 2008Pages 517-518 ReferencesRelatedInformation

Overexpression of P-glycoprotein (PGP), a multidrug-related (MDR) protein, is one of the most important factors responsible for reduced drug sensitivity in acute myeloid leukemia (AML). Recently, we demonstrated that the presence of CD56 antigen, an isoform of the neural adhesion molecule, in AML cells is a negative independent prognostic factor for the achievement of complete remission (CR) and correlates with shorter survival. Since in our previous report we observed a more frequent PGP expression in CD56+ patients, we hypothesized that the reduced response to chemotherapy in this group of patients was due to increased PGP-mediated drug efflux. To confirm this hypothesis in this study PGP and CD56 expression on AML cells was correlated with other clinical and biological features and treatment response.Immunophenotypic analysis, including evaluation of CD56 and PGP expression, was performed using multiparameter flow cytometry on fresh and/or cryopreserved blast cells, obtained after informed consent, from bone marrow and/or peripheral blood of 143 consecutive newly diagnosed AML cases at the time of diagnosis. Samples expressing CD56 in at least 15% or more cells were considered as positive (CD56+). PGP expression was expressed as a mean fluorescence index (MFI) i.e. as the ratio of sample mean fluorescence channel and the isotypic control mean fluorescence channel.Overall results showed that 67/143 cases were PGP-/CD56-, 23/143 were PGP+ /CD56+, 40/143 were PGP+/CD56- and the remaining 13/143 were PGP-/CD56+. CD56+ and PGP+ on AML cells significantly reduced the CR rate (83% in the PGP-/CD56- group vs 60% in the PGP-/CD56+ group, 46% in the PGP+/CD56- group and 58% in the PGP+/CD56+ group, p = 0.002). In addition we observed a significantly higher proportion of total failures in patients expressing PGP or CD56 compared to in the group not expressing either (73% vs 27%, respectively; p = 0.0001). CD56 and PGP overexpression influenced the overall survival: in fact, the median survival of CD56+ and PGP+ patients ranged from 10 to 23 months, while the actuarial survival of CD56-/PGP- patients at 5 years is 52% (p = 0.023).Our data underline the independent negative prognostic role of PGP and CD56 expression in acute myeloid leukemia. Since the mechanism by which CD56 reduces drug sensitivity is still unknown, further investigations are required.

Epstein-Barr virus (EBV) infection and reactivation is an increasing complication in immune deficient patients, particularly after allogeneic hematopoietic stem cell transplantation (HSCT). Therapy with anti-thymocyte globulin (ATG) is associated with higher incidence of EBV-related disease in HSCT patients, but this risk is not documented in patients receiving ATG for severe aplastic anemia (SAA). We describe the case of a patient who developed an EBV infection, with the clinical features of an infectious mononucleosis, after immune suppression with cyclosporine and two courses of ATG for SAA.

Summary One hundred and six patients aged ≤60 years with newly diagnosed acute myeloid leukaemia (AML) treated with fludarabine‐based regimens (cases) were matched with 106 AML patients treated with conventional non‐fludarabine‐based regimens (controls). The cases and controls were matched by expression of the multidrug resistance P‐glycoprotein (MDR‐Pgp), measured by flow cytometry as mean fluorescence index (MFI), cytogenetics, and age. The complete remission (CR) rate of the cases was 61% among the MDR‐Pgp‐positive (pos ve ) patients (MFI ≥ 6) vs. 75% among the MDR‐Pgp‐negative (neg ve ) ones (MFI &lt; 6) ( P = 0·16). Conversely, in the controls, the CR rate was 44% among the MDR‐Pgp‐pos ve patients vs. 67% among the MDR‐Pgp‐neg ve ones ( P = 0·02). The 4‐year disease‐free survival (DFS) and overall survival (OS) of MDR‐Pgp‐pos ve cases were significantly longer than those of MDR‐Pgp‐pos ve controls (DFS, 28·1% vs. 6·5%, P = 0·004; OS, 33·5% vs. 9·6%, P = 0·01). This difference was not found among the MDR‐Pgp‐neg ve patients. By univariate ( P = 0·007) and multivariate ( P = 0·007) analysis, the MDR‐Pgp‐pos ve phenotype was negatively correlated with CR and it emerged as the most important independent negative prognostic factor, after cytogenetics. Our study confirms the prognostic impact of the MDR phenotype in AML and strongly suggests fludarabine‐based induction treatments as a promising strategy for MDR‐Pgp‐pos ve AML patients. In this setting of patients, large prospective randomised studies should be planned.

Topic: 16. Myeloproliferative neoplasms - Clinical Background: Essential thrombocythemia (ET) course is characterized by increased risk of thrombotic and hemorrhagic events, that represent their leading causes of mortality and morbidity. Treatment cornerstone are low dose aspirin and cytoreductive therapy, whose uses are proportionate to the risk of thrombosis assessed through IPSET score, the main thrombotic risk stratification tool among ET patients. Differently, there is no specific prognostic tool used to predict hemorrhagic risk in ET. Aims: The primary objective of this study was to analyze incidence and main risk factors connected to thrombotic and hemorrhagic events onset in ET patients, to provide correct prognostic stratification and optimize therapeutic strategies. Methods: We retrospectively analyzed distribution of clinical, biochemical, and molecular features among 308 patients diagnosed with ET between 1984 and 2002 and referred to the Hematology Department of Udine. Patients have been treated according to the current international guidelines. T-test and chi-squared test were performed to assess results. Results: Median age at ET diagnosis was 57.2 years (range: 17.7-87.6), with a slight prevalence of females (53.6%). According to mutational status, 201 patients (65.3%) were positive for V617F JAK2, 66 (21.4%) had a CALR mutation (type 1 in 40/66, 60.6%), and 14 (4.5%) had a MPL mutation (W515L in 12/14, 85.7%) while 21 patients (6.8%) were “triple negative”), while in 6 patients (2%) molecular analysis was not available. According to IPSET-thrombosis score, 49.7% patients were at high, 24.7% at intermediate and 25.6% at low risk, respectively. Nineteen patients (6.2%) presented an arterial or venous thrombotic event at ET diagnosis; 32 patients (10.4%) had a thrombotic event after diagnosis, after a median time of 92 months (range: 2-292): 25/32 were arterial events (10 pertaining the CNS, 8 the heart and 7 peripheral) and 7/32 were venous events (DVT). At thrombotic event, 26 patients were under cytoreductive therapy (HU in 21), 22 were in antiplatelet therapy (ASA in 20). Thrombosis-free survival (TFS) was significantly lower in high IPSET patients compared to intermediate or low risk, with a relative risk (RR) 4.1 times higher in high-risk patients compared to intermediate- or low-risk patients; estimated TFS at 10 and 20 years was 80.7% and 63.0% for high-risk, compared to 96.4% and 96.4% for intermediate-risk and 96.9% and 90.2% for low-risk, respectively (p <0.001. While we found no correlation between TFS and mutational status (p=0.18). Twenty-four patients (7.8%) experienced a hemorrhagic event after ET diagnosis, at a median time of 117 months (range: 1-316). At hemorrhage, 18 patients received cytoreduction (HU in 15), 19 antiplatelet therapy (ASA in 14) and 4 were under anticoagulants. As for thrombosis, there was a significant correlation between hemorrhagic risk and IPSET score: estimated HFS at 10 and 20 years was 90.2% and 75.0% for high-risk, compared to 97.1% and 97.1% for intermediate-risk and 96.8% and 96.8% for low-risk, respectively (p=0.002, Fig.1); RR for hemorrhage in high-risk patients was 3.3. Again, no correlation was found between hemorrhages and mutational status. Summary/Conclusion: Results of our analysis confirm the validity of IPSET score in predicting individual thrombotic risk among ET patients. More, a statistically significant correlation between the same score and hemorrhagic events incidence has been found, suggesting a possible role of IPSET scoring system as a global evaluator for vascular events.Keywords: Essential Thrombocytemia, Hemorrhage, Thromboembolic events

Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of post-thymic malignancies relatively uncommon in the Western world and their prognosis and therapeutic approach are still not well defined. The aim of this study was to retrospectively analyze the clinical, hematological and histological features at diagnosis, the relevance of the International Prognostic Index and the outcome of a group of 23 patients affected by peripheral T-cell lymphoma, unspecified (PTCL-U), according to the Revised European-American Classification of Lymphoid Neoplasms (REAL), observed between September 1985 and April 1995 at our Institution.Patients were separated into different prognostic groups according to Ann Arbor stage, cell size and International Prognostic Index. All patients had been treated with multiagent combination chemotherapy, mainly CHOP (9 cases) and F-MACHOP (9 cases), and were evaluable for response. The treatment was intensified with allogeneic bone marrow transplantation (BMT) in 1 patient and with autologous BMT in 4 patients.Median age was 55 (range 18-77) years and 70% of the patients were males. Four patients were in stage II (17%), 5 in stage III (22%) and 14 in stage IV (61%). Patient risk was classified according to the International Prognostic Index as follows: 8 cases (35%) low risk, 2 cases (9%) low-intermediate, 8 cases (35%) high-intermediate, 5 cases (21%) high. Median follow-up time was 20 months (range 2-132). Median progression-free survival (PFS) and overall survival (OS) for all the patients studied were 10 and 34 months, respectively. Stage IV was associated with a poorer response rate and a shorter PFS (median 6 months) and OS (median 32 months). No statistical correlation was found between cell size and overall response (complete + partial remission), PFS (p = 0.38) or OS (p = 0.59), although a better trend was observed for the large cell group. A less favorable outcome was observed in patients in the high-intermediate + high risk groups, where median PFS and OS were 7 and 24 months, respectively, than in patients in the low + low-intermediate risk groups. No difference in response or outcome was detected between patients treated with the CHOP and the F-MACHOP regimens, while all 5 patients given high-dose chemotherapy and BMT are alive and in CR.Our experience shows that PTCL-U are rare lymphomas frequently having an aggressive presentation. The response to conventional polychemotherapeutic regimens like CHOP or F-MACHOP is generally poor, especially in those cases with advanced stage and a high-intermediate or high International Prognostic Index. The observation that all five patients who were treated with bone marrow transplantation are alive and in complete remission suggests using this strategy, particularly in young patients with a poor International Prognostic Index.

Abstract Objective Overexpression of ABCG 2 and CD 200 has been independently associated with poor outcome in acute myeloid leukemia ( AML ). However, no data are available on the role of these two factors in patients with core‐binding factor ( CBF )‐positive or FLT 3‐negative/ NPM 1‐mutated cytogenetically normal ( CN ) AML . Methods We analyzed 65 adult AML patients with CBF + (n=16) or FLT 3−/ NPM 1+ CN (n=49), evaluating clinical and biological factors associated with complete remission attainment, leukemia‐free survival ( LFS ) and overall survival ( OS ). Results ABCG 2 was expressed in 36 (55%) cases, and CD 200 was positive in 33 (51%) cases, six at high levels. Both ABCG 2 and CD 200 positivity have a negative impact on relapse risk: 3‐year LFS was 51% vs 82% in ABCG 2+ cases ( RR 3.3), 49% vs 82% in CD 200+ patients ( RR =4.4), and 25% in CD 200− high cases ( RR =17.1). ABCG 2 and CD 200 affected also OS with 3‐year OS of 39% in ABCG 2+ (compared to 71% in ABCG 2−; RR =2.6) and CD 200+ (compared to 68% in CD 200−; RR =2.5) patients. Conclusions Our data confirm a negative impact of ABCG 2 and CD 200 overexpression also in AML patients considered at favorable risk according to ELN cytogenetic/molecular classification.

The outcome of a series of adult patients, affected by primary systemic CD30-positive anaplastic large cell lymphoma (ALCL), treated with a sequential intensive therapeutic program, has been analyzed and all data available in the literature have been reviewed. Forty consecutive, unselected patients with ALCL were treated with the F-MACHOP regimen, followed by radiotherapy (RT) for residual mediastinal disease (15 cases) and by autologous stem cell transplantation (ASCT) conditioned with BAVC (29 cases). Eighty-nine percent (32/36) of the patients younger than 60 years were eligible for completing the sequential treatment. Since then, 3 patients in CR refused ASCT, 1 was excluded for cardiac toxicity and 3 progressed and died of disease. Thus, 29 have been so far submitted to the transplant procedure. CR and PR rates were 40% and 45% respectively after CHT; 52.5% and 35% after RT; 80% and 5% after ASCT, with 78% of patients transplanted in PR convertin to a CR. Actuarial overall survival is 85% at 48.5 months (93% at 66 months for the 29 transplanted patients) and disease free survival is 100% at 54 and 64 months respectively, with no relapses observed among patients who reached a CR. Considering our data and those of the literature, it can be concluded that although the role of ASCT in the therapy of ALCL must not be considered as definitive, its efficacy in converting PR into CR and in preventing relapses, suggests that a randomized trial comparing CHT alone vs CHT+ASCT should be undertaken.

The mdr-1 gene, which codes for a 170-kd transmembrane glycoprotein (P170), is frequently overexpressed in multidrug resistant (MDR) tumor cell lines and in spontaneous tumors, including leukemia and lymphoma. However, it is also constitutively expressed as a normal gene in normal tissues.We used human mdr-1 cDNA and three anti-P170 monoclonal antibodies (MoAbs: MRK-16, C-219 and JSB-1) to investigate the normal peripheral blood leukocyte content of mdr-1 specific mRNA and of P170 through immunocytochemistry and flow cytometry.We did not find any increase in mdr-1-specific mRNA, while small amounts of P170 were easily detectable in about two thirds of the lymphocytes and monocytes and in about one third of the granulocytes. The level of P170 expression in leukocytes was similar to that found in non-MDR tumor cell lines.mdr-1 is constitutively expressed in human normal leukocytes at levels that cannot significantly affect drug resistance. Accordingly, low-level mdr-1 expression in leukemic cells should not be considered a label of pleiotropic drug resistance.

The actual prevalence and the clinical relevance of gene mutations of HFE (which are linked to hemochromatosis) have not yet been established in patients on chronic dialysis. On the basis of theoretical premises, it could be hypothesized that these genetic determinants might influence the response to iron intake and the susceptibility for iron overload in patients in parenteral iron therapy. Furthermore, carriers for these mutations might be prone to develop sporadic porphyria cutanea tarda and cardiovascular events.C282Y/H63D mutations of HFE gene were evaluated in 132 patients (34 in peritoneal dialysis, 98 in HD) and correlated with biochemical parameters of iron status (ferritin (FER) concentration and transferrin saturation (TSAT)), red cell parameters (red cell size and hemoglobin content), erythropoietin (EPO) dosage, major cardiovascular events and C-reactive protein as marker of chronic inflammation, in patients without iron therapy and after i.v. iron supplementation (< or = 60 mg/week) and with the presence of biopsy-proven porphyria.C282Y heterozygous mutation was found in 8/132 (6.6%); H63D homozygous and heterozygous mutations were found in 3/132 (2.3%) and 22/132 (16%) patients, respectively. Two patients (1.5%) showed double heterozygosis. No differences in baseline serum FER and TSAT and the other biochemical and clinical parameters were found in patients bearing mutations alleles nor after continuous iron therapy at low dosages. However, the prevalence of patients capable of maintaining normal hemoglobin (Hb) level without EPO therapy is increased in the C282Y-mutated patients. Only 1 patient out of the 4 with biopsy-proven porphyria cutanea tarda was bearing gene mutations (H63D heterozygosis).C282Y/H63D HFE gene mutations do not seem to be related to major abnormalities in biochemical parameters of iron status in dialysis patients without iron therapy or after i.v. iron supplementation, granted that low dosages are employed. Obviously, as our patients were exposed to a relatively uniform iron regimen in our clinical center (< or = 60 mg/week), it is unclear if other dosing regimens will unmask clinically significant differences between the heterozygotes and normals. The fact that the C282Y-mutated patients more frequently maintain high Hb values without EPO is interesting as could suggest a better use of available iron for erythopoiesis, but needs to be confirmed in larger samples. No clear association is demonstrated with porphyria cutanea tarda and major cardiovascular events.

We read with interest the work from Rubio et al (2003) on adult T-biphenotypic leukaemia, reporting the clinical and biological characteristics of this rare clinical entity. We have reviewed our file of acute leukaemias, both lymphoblastic and myeloid, to identify the biphenotypic acute leukaemias (BAL) and, among them, the cases co-expressing T-lymphoid and myeloid markers. In the last 10 years (1993–2003), we have diagnosed 280 acute leukaemias, 202 myeloid (AML) and 78 lymphoblastic (ALL). Using the diagnostic criteria recently proposed (Bene et al, 1995), 26 (9%) patients had a BAL. In accordance with other reports (Legrand et al, 1998; Killick et al, 1999), the most common BAL phenotype consisted of the co-expression of myeloid and B-lymphoid markers, but three of these patients (11·5%) had a T-BAL. Clinical and biological characteristics of the patients are listed in Table I. Two of the patients were female and one was male. The median age at diagnosis was 58 years (range 26–60 years). According to the French–American–British (FAB) classification, all the patients had a type 2 ALL, with co-expression of myeloid markers, with a score of at least two for both T-lymphoid and myeloid markers. Cytogenetic analysis was available for two patients. Both showed a deletion of chromosomes 7 and 12, plus other abnormalities; no Philadelphia chromosome (Ph+) metaphases were documented. When karyotype was missing, BCR-ABL transcripts were not detected by molecular biology. We analysed the multidrug-resistance (MDR) proteins and found an overexpression of P-glycoprotein with a mean of 7·2 (range 6·6–8·1), while lung resistance-related protein and multidrug resistance-related protein 1 were normally expressed. Two patients (patients 2 and 3) received an ALL-designed induction therapy (vincristine, idarubicin, prednisone and asparaginase). Patient 2 died during induction due to a haemolytic-uraemic syndrome and pneumonia. Patient 3 achieved a complete remission (CR) that was consolidated with two courses of therapy (high-dose cytarabine, then vincristine, methotrexate, cyclophosphamide and adriamicin). She relapsed after 5 months but attained a second CR with salvage therapy (liposomal daunorubicin and high-dose cytarabine). The remission lasted for another 4 months, then the patient relapsed again and died of cerebral haemorrhage. Patient 1 displayed a more undifferentiated morphology, with two blast populations. He initially received an AML-like course with idarubicin and low-dose cytosine arabinoside. Day +14 bone marrow was still completely blastic, but with more distinctive lymphoid features. He therefore was switched to our ALL protocol, attaining a CR and subsequently underwent an autologous bone marrow transplant. He is alive in CR, 9 years after transplant. Our small experience partially confirms the findings of Rubio et al (2003). In addition, our patients were morphologically classified as L2 and presented superficial adenopathies at diagnosis, but none of them had mediastinal involvement. Leucocytosis and peripheral blast count were generally low or moderate. None of our patients had the Philadelphia chromosome or BCR-ABL rearrangement, but two cases displayed unfavourable cytogenetics, with deletions of chromosome 7. In a previous report, four of six patients with T-BAL had a complex karyotype and none was Ph+ (Carbonell et al, 1996). Treatment of BAL remains controversial and these patients have generally a bad prognosis. Except in patient 2, who died during induction, we observed a good response when a mixed or ‘sequential’ AML/ALL induction therapy was used, possibly followed by intensification with transplantation. When an ALL-type course was used alone (patient 3), a CR was obtained but it was short-lived, and also a salvage therapy with high-dose cytarabine and liposomal daunorubicin was only transiently effective. The aggressive clinical course of T-BAL may be associated with adverse cytogenetic abnormalities and MDR overexpression, but these speculations require confirmation in larger studies.

BAALC expression is an indicator of aggressiveness in acute myelogenous leukemia (AML). Overexpression of this gene is associated to poor of clinical outcome. It is known that post-translational histone modifications control gene transcription. Thus, here we have investigated BAALC expression and post-translational histone modifications in leukemia cell lines. We show that Kasumi-6 and Kyo cells have high and low BAALC mRNA levels, respectively. Moreover, we demonstrate that these cell lines present distinct profiles in terms of histone post-translational modifications (H3K9K14 acetylation, H3K4 trimethylation and H3K23 trimethylation) at the level of BAALC promoter. These findings, in light of recent data on how histone post-translational modifications control gene expression, indicate that BAALC gene is “paused” and that in leukemia cells its transcription can be activated or repressed by mechanisms acting on epigenetic marks.

Verapamil (VRP) is an effective modulator of P170-associated multidrug resistance (MDR), but its clinical application is limited by cardiovascular side-effects. The D-isomer of VRP (D-VRP) is 10 times less active than the racemic mixture on the cardiovascular system, but retains a MDR modulating activity. Daunorubicin (DNR) and doxorubicin (DX) are two anthracyclines whose cytotoxicity is strongly related with the expression of P170, while their respective lipophylic derivatives idarubicin (IDA) and iododoxorubicin (IDX) are less P170-dependent. We studied the effect of D-VRP on intracellular retention and on the cytotoxicity of these four anthracyclines in two MDR cell systems (LOVO and CEM) by flow cytometry and by a microcultured tetrazolium colorimetric assay (MTT). We found that in MDR cells D-VRP increased intracellular anthracycline concentration and increased the cytotoxicity of DNR, IDA and DX but not of IDX. The effect of D-VRP was dose-related, but it was already consistent at D-VRP concentrations that can be readily maintained in vivo (2-3 microM). These data suggest that at a clinically tolerable concentration D-VRP can downmodulate the resistance to DNR and DX and can restore full sensitivity to IDA.

By using RNA slot-blot technique, the frequency and the degree of GST pi and mdr-1 gene coexpression were investigated in 23 AML patients, 9 ALL, 9 CLL and 11 cases of NHL in an attempt to study their clinical and prognostic relevance. GST pi and mdr-1 levels were expressed as arbitrary units (U) with respect to the negative controls (U = 0), MCF7 and HL60 sensitive cell lines, and the positive controls (U = 10), MCF7/DOXO and HL60/DNR resistant cell lines. The concomitant GST pi/mdr-1 gene overexpression showed a negative prognostic value in the set of newly diagnosed AML pts (10 cases), furthermore higher GST pi and mdr-1 mRNA levels were averagely detected in the relapsed/resistant ALL pts (4 cases), and in CLL (7 cases) and NHL (8 cases) heavily pretreated patients who were unresponsive to chemotherapy and with a disease progression. These preliminary data show that two different mechanisms of drug resistance can be coexpressed at the same time in those leukemias and lymphomas with a clinically unfavourable course.

Selective elimination of multidrug resistance-positive cells (LoVo/Dx) was obtained by using the monoclonal antibody MRK 16, which recognizes a surface epitope of the Mr 170,000 glycoprotein, and a sheep anti-mouse immunoglobulin antibody, conjugated to the ribosome-inactivating protein saporin 6. The killing was greatly decreased or even abolished by adding the monoclonal antibody at a 100-fold concentration. Both the MRK 16 and anti-mouse saporin 6 conjugate did not show any killing activity when they were used separately. In cell suspensions composed of 90% normal bone marrow cells and 10% multidrug resistance-positive cells, the monoclonal antibody MRK 16 followed by the anti-mouse immunotoxin caused the elimination of 99% multidrug resistance-positive cells, as revealed by immunofluorescence and immunocytochemistry as well as by a clonal assay. Human normal hematopoietic precursors (granulomonocytic colony-forming units, erythroid burst-forming units, and multipotent granulomonocytic, erythroid, and megakaryocytic-forming units) were not affected by the MRK 16 plus immunotoxin treatment. This technique might be suitable for ex vivo bone purging in an appropriate clinical setting, such as autologous bone marrow transplantation.

BCL-2 overexpression has been associated with resistance to chemotherapy and reduced survival in acute myeloid leukemia (AML), but few data are available in elderly patients, a subset accounting for majority of AML cases and with dismal prognosis. We retrospectively analyzed 113 AML patients aged ≥65 years treated with 3 + 7 chemotherapy (n = 51) or hypomethylating agents (HMAs) (n = 62), evaluating the role of BCL-2 expression on complete remission (CR) and overall survival (OS). BCL-2 was expressed in 81 patients (72%), more frequently in those with unfavorable cytogenetic-molecular risk. CR was achieved in 34.5% cases, without differences according to BCL-2 expression or induction therapy. In the whole population 1-year OS was 39%, similar in BCL-2+ and BCL-2- cases. In BCL-2 positive patients OS was superior with HMAs (56% vs. 25% with 3 + 7; p = 0.02), while no advantage for HMA was found in BCL-2 negative cases (36% vs. 27% for 3 + 7). Therapy with HMAs was the only factor associated with longer OS in BCL-2+ AML by multivariable analysis. Use of HMAs, possibly in combination with BCL-2 inhibitors, appears to be particularly appealing in BCL2+ AML, where it is associated with superior survival.

Background: Most previous studies have focused on the intrinsic carcinogenic pathways of tumors; however, little is known about the potential role of N6-methyladenosine (m6A) methylation in the tumor immune microenvironment (TIME). To better diagnose and treat acute myeloid leukemia (AML), we sought to examine the correlation between m6A regulatory factors and immune infiltration in cases of AML. At the same time, a prognostic model was constructed to predict the survival of AML. Methods: We extracted data from The Cancer Genome Atlas (TCGA) database, including ribonucleic acid sequencing (RNA-seq) transcriptome data and data on the corresponding clinical characteristics of AML patients. We identified two m6A modification patterns with distinct clinical outcomes and found a significant relationship between them. Simultaneous discovery of distinct m6A clusters associated with the tumor immune microenvironment [immune cell types and Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm] are closely related. Next, we implemented Lasso (Least Absolute Shrinkage and Selection Operator) Cox regression to build a predictive model in the 2-m6A regulator TCGA dataset to further explore m6A prognostic features in AML, and perform correlation validation. Results: We identified 2 molecular subtypes (Clusters 1 and 2) by the consistent clustering of significant m6A regulators in AML. Cluster 2 was associated with a higher immune score and obvious immune cell infiltration, and thus patients in Cluster 2 had a poorer prognosis than those in Cluster 1 (P<0.05). Additionally, the 2 m6A-related signatures representing the independent prognostic factors in AML were screened to construct a prognostic risk-score model. We found that patients with low-risk scores had higher immune scores than those with high-risk scores (P<0.05). Conclusions: Our research confirmed that m6A methylation plays an important role in AML. Further provide new directions for the prognosis and treatment of AML.

It is now well known that the bone marrow (BM) cell niche contributes to leukemogenesis, but emerging data support the role of the complex crosstalk between AML cells and the BM microenvironment to induce a permissive immune setting that protects leukemic stem cells (LSCs) from therapy-induced death, thus favoring disease persistence and eventual relapse. The identification of potential immune targets on AML cells and the modulation of the BM environment could lead to enhanced anti-leukemic effects of drugs, immune system reactivation, and the restoration of AML surveillance. Potential targets and effectors of this immune-based therapy could be monoclonal antibodies directed against LSC antigens such as CD33, CD123, and CLL-1 (either as direct targets or via several bispecific T-cell engagers), immune checkpoint inhibitors acting on different co-inhibitory axes (alone or in combination with conventional AML drugs), and novel cellular therapies such as chimeric antigen receptor (CAR) T-cells designed against AML-specific antigens. Though dozens of clinical trials, mostly in phases I and II, are ongoing worldwide, results have still been negatively affected by difficulties in the identification of the optimal targets on LSCs.

Overexpression of the mdr-1 gene that codes for a 170 Kd transmembrane glycoprotein (P170) is a factor responsible for decreased cell sensitivity to anthracyclines and other drugs, and is related to treatment failure in acute leukemia and other tumors. Several agents, including verapamil and cyclosporine derivatives, can modify P170-related resistance in vitro and can be proposed as adjuvant treatment for leukemia and cancer.To investigate the optimal conditions for adjuvant treatment, D-verapamil (D-VRP), cyclosporin-A (CyA) and the cyclosporine derivative SDZ PSC 833 (PSC) were used alone and in combination at drug concentrations that can be achieved and maintained in vivo. The drugs were tested for their capacity to restore intracellular daunorubicin (DNR) and idarubicin (IDA) accumulation in high-resistance (CEM VLB 300) and intermediate-resistance (CEM VLB 100) cells to levels found in the non-resistant parental cell line (CCRF CEM).In intermediate-resistance cells, IDA alone was more easily restored than DNR plus modifiers, and intracellular IDA concentration was returned to the level of non-resistant cells with low-dose D-VRP (1 microM), CyA (0.8 microM) and PSC (0.4 microM). In high-resistance cells no modifier or modifier combination was able to restore intracellular DNR concentration to the value of non-resistant cells. Intracellular IDA concentration was almost completely restored only with D-VRP (2-3 microM) and CyA (0.8-1.6 microM) in combination or with PSC alone (0.4 microM).These data suggest that as far as P170-related resistance is concerned, IDA alone is as efficient as or even more efficient than DNR plus modifiers, and that residual resistance to IDA can be overcome with a combination of D-VRP and Cy-A at a clinically achievable concentration, or with a more powerful modifier like SDZ PSC 833.

European senior programme (ESP) is well known for acceptable outcomes using expanded criteria donor (ECD) kidneys from donors older than 65 years for recipients older than 65 years. The incidence of end-stage renal disease (ESRD) is 229/million in India with a mean age of 45 years. We performed a retrospective analysis of transplantation of ECD versus standard criteria donor (SCD) kidneys into younger recipients.Forty-three ECD transplantations among 158 deceased donor organ transplantation (DDOT) were performed between January 2006 and December 2009. Among 43 transplantation from 30 donors, 14 were dual kidney transplantations (DKT) performed based upon biopsy evaluation. All recipients received thymoglobulin (rATG) induction followed by immunosuppression with a steroid, mycophenolate mofetil (MMF), and a calcineurin inhibitor. Statistical analysis used chi-square test and unpaired Student t test. Kaplan-Meier curves were used for survival analysis.For ECD the mean donor age was 64 ± 11 years. Cerebrovascular accidents (CVA) were the cause of death among 60% of donors, 73.13% of whom were hypertensive and 23.13% diabetic.Mean DKT donor age was 75 ± 9.17 years versus 60 ± 8.0 years for single kidney transplantation (SKT). Mean recipient age of DKT versus SKT was 44 ± 12.4 years versus 43 ± 14 years. Mean serum creatinine (SCr; mg/dL) of SKT patients was 1.64 ± 0.75 versus 1.68 ± 0.46 in DKT. Mean follow-up was 455 ± 352 days. Mean SCr of 43 ECD recipients of mean age, 43.4 ± 14.2 years was 1.61 ± 0.61 mg/dL. Among 43 recipients, 23.25% were diabetic, 41.86% displayed delayed graft function (DGF), and 23.25% experienced biopsy-proven acute rejection (BPAR). Patient survival rate was 72.09% and graft survival rate was 67.44%. For SCD transplantations (n = 115), the mean donor age was 36 ± 14 years and recipient mean age was 32.8 ± 14.07 years. Mean SCr was 1.32 ± 0.46 mg/dL with 26.95% recipients displaying DGF, whereas 20.86% had BPAR. In the SCD group the patient survival rate was 79.13% and the graft survival rate was 72.17%. Thus, although the ECD group showed poor graft function (P = .042), they had acceptable patient and graft survivals (P = .34 and P = .56, respectively).Because of the organ shortage, DDOT using ECD transplants for younger recipients is a feasible option with acceptable outcomes.

We retrospectively analyzed the data of 337 patients with cytogenetically normal (CN) acute myeloid leukemia (AML), aged ≤ 65 years (training set). A prognostic index score (PIS) was calculated by totaling the score derived from the regression coefficients of each clinical variable, significantly associated with prognosis by multivariate analysis. The variables that were independent prognostic factors for event-free survival (EFS) and overall survival (OS) in the training set were: age ≥ 50 years, secondary AML and white blood cell count (WBC) ≥ 20 × 109/L. The patients of the training set were stratified into three groups: low-, intermediate- and high-risk. The median EFS was 25, 12 and 7 months in the low-, intermediate- and high-risk groups (p < 0.0001), respectively. The median OS was not reached in the low-risk group and was 19 and 10 months in the intermediate- and high-risk groups (p < 0.0001). This PIS was validated in a series of 193 patients with CN-AML. The median EFS was 66, 16, and 3 months (p < 0.0001) and the median OS was 66, 16, and 5 months in the three risk groups, respectively (p < 0.0001). This PIS may be useful for clinical decision-making in CN-AML and may be prospectively integrated with the newest biological markers which at present are not routinely assessed and need prognostic validation.

Cell sensitivity to anthracyclines and other drugs depends on several factors, including overexpression of a 170Kd transmembrane glycoprotein (P170) that enhances drug efflux from the cells. Since the result of treatment is negatively related to the expression of P170 in leukemia, malignant lymphoma and other tumors, it is important to investigate drugs and methods that can modify multidrug resistance (MDR).Using an MTT-microcultured tetrazolium colorimetric method, we assayed sensitivity to daunorubicin (DNR) and to its 4-demethoxy derivative idarubicin (IDA) in two MDR cell lines (CEM VLB and LOVO DX) and in their respective non-MDR parental lines (CEM and LOVO 109), with and without three MDR modifiers, namely the D-isomer of verapamil (DVRP), cyclosporin A (CyA) and the new CyA derivative SDZ PSC 833.We showed that down-modulation of resistance with MDR modifiers was greater for DNR than for IDA in MDR cells. However, we also demonstrated that restoration of full sensitivity could only be achieved for IDA, not for DNR. DVRP and CyA in combination were more effective than either compound alone and could abolish P170-related resistance to IDA at concentrations of 1-2 microM and 1.6 microM, respectively. SDZ PSC 833 alone was even more effective and set MDR to zero at a concentration ranging between 0.8 and 1.6 microM.These data suggest that combinations of IDA and MDR modifiers may improve the results of cancer and leukemia treatment and that they are worth investigating in vivo, with attention to possible effects on drug pharmacokinetics and on normal tissue damage.

In cell lines, there is an ongoing debate about the role of the lung resistance-related protein (LRP) whereas the role played by P-glycoprotein (PGP) in determining a multidrug resistance is well known. The aim of this study was to evaluate the frequency and the role of a PGP and an LRP overexpression in affecting the intracellular daunorubicin accumulation (IDA) and in predicting the therapy outcome on a subset of overt secondary acute non lymphocytic leukemias (ANLL). An adjunctive point was to evaluate the efficacy of the reversal agent SDZ PSC 833 (PSC) in counteracting impaired IDA.By flow cytometry, PGP and LRP expression and the IDA were evaluated on 54 overt secondary ANLL PGP and LRP overexpressions were respectively defined by an MRK-16 mean fluorescence index (MFI) > or = 6 (PGP+) and by an LRP-56 MFI > or = 5 i.e. by MRK-16 and LRP-56 MFIs higher than the one observed in normal leukocytes. The blasts' IDA was studied after a two-hour incubation in 1000 ng/mL daunorubicin in the presence or in the absence of the MDR reversal agent SDZ PSC 833 (PSC) 1.6 mumol.A PGP overexpression was detected in 40/54 (74%) cases while an LRP overexpression was observed on 33/54 (61%) cases. No differences were found in terms of PGP and LRP expressions between ANLL developing after chemo/radiotherapy (therapy-related ANLL) or evolving from a myelodysplastic syndrome (MDS-related ANLL). Compared to the PGP-, the PGP+ cases showed a significantly lower mean IDA (DNR NMFI 196 +/- 46 vs. 267 +/- 53, p < 0.001). The co-incubation of DNR with the PSC significantly increased only the mean IDA of the PGP+ cases, that grew from a DNR NMFI of 196 +/- 46 to a DNR NMFI of 284 +/- 67 (p < 0.0001). With respect to normal leukocytes, even the PGP- cases had an impaired IDA suggesting that other mechanisms, including an LRP overexpression, could affect the IDA. A strongly negative correlation was observed between PGP overexpression and therapy outcome, in fact, 8/10 (80%) PGP- but only 2/27 (7%) PGP+ patients obtained complete remission (p = 0.0002). Moreover, 7/33 (21%) cases showing an impaired IDA (NMFI < 280) but 4/4 (100%) with NMFI > 280 had complete remission (p = 0.006). No correlation was found between therapy response and LRP or CD34 expression.This data suggests that an important role in determining therapy outcome is played by PGP in secondary leukemias. Even if the LRP is frequently overexpressed in secondary leukemias and is likely to contribute to the reduction of the intracellular drug accumulation, the role played by LRP in determining the therapy-outcome has still to be cleared.

More than 50% of adults with acute lymphoblastic leukemia (ALL) actually die with resistant leukemia. The multidrug resistance (MDR) related P170 glycoprotein may be involved in treatment failure.P170 content of leukemic cells was assayed by immunocytochemistry with the MRK-16 monoclonal antibody in 41 consecutive cases of adult ALL (27 at onset and 14 at relapse).Positive cells were found in 11/14 cases at relapse and in 18/27 cases at onset. All cases with a high WBC, FAB L3, B-mature phenotype, and t(9; 22) were positive. In the 27 patients who were studied at onset and were treated with the same protocol, the overall failure rate was 44% in negative cases and 83% in positive cases.P170 overexpression may contribute to drug resistance and to chemotherapy failure in adult ALL.

Mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes, along with CD38 expression, is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL). Configuration of IgVH genes displaying > 2% mismatch has been shown to correlate with longer survivals. In a series of 64 B-CLLs, we failed to confirm the prognostic value of the IgVH gene mutational status by using the suggested cutoff. However, the IgVH mutational status maintained its prognostic value only when evidence of antigen-driven selection could be documented. This was accomplished by applying statistical methods aimed at evaluating a significant skewing of replacement mutations from framework to complementary determining regions, as it occurs during germinal center differentiation of B cells. These data caution against wide application of the 2% somatic mutation cutoff as a prognostic determinant without demonstration of antigen-driven selection.

Abstract Background Multidrug resistance (MDR) remains a major obstacle for successful treatment in cancer, in particular in acute leukemia. In acute promyelocytic leukemia (APL), the high sensitivity to anthracyclines appears to be attributable to the low frequency of MDR proteins overexpression at onset even if 30% of patients still relapse and become resistant to therapy. In attempt to explain different blast cell sensitivity, we studied the expression of PGP, MRP1, MRP2, and LRP in 45 cases of APL, comparing onset of disease with relapse. Methods PGP, LRP, and MRP on bone marrow or peripheral blood blast cells were evaluated by flow cytometry using the MRK‐16, LRP‐56, MRP‐m6, and MRP2 antibodies and results expressed by the mean fluorescence index (MFI). The antibody binding capacity (ABC) for each MDR protein was also calculated. Results At diagnosis, only 2 of 45 patients overexpressed PGP and 1 overexpressed LRP. PGP and LRP overexpressing cases significantly grew up during disease progression and at second relapse mean PGP MFI and mean LRP MFI were significantly higher than at onset ( P = 0.001 and P = 0.008, respectively). By analyzing ABC, the same trend was more evident because a significant increment of PGP and LRP was observed at second ( P = 0.002 and P = 0.002, respectively), but even at first relapse ( P = 0.018 and P = 0.002, respectively). No changes were demonstrated in MRP1 and MRP2 expression in any phase of disease considered. Conclusions Our data confirm the low expression at diagnosis of proteins related to development of drug resistance in APL. The evidence of a relative easy induction of PGP and LRP, but not of MRP, can be useful in choosing drugs to employ for consolidation or rescue therapy. © 2004 Wiley‐Liss, Inc.

Abstract Nucleophosmin 1 (NPM1) is an abundant phosphoprotein mainly located in the nucleolus but also shuttling between the nucleus and cytoplasm. NPM1 has been proposed to be involved in synthesis and processing of ribosomal RNA, regulation of chromatin structure and transport of rRNA and ribosomal proteins. NPM1 gene is considered to be implicated in human cancer as it is a frequent target of genetic alterations, primarily in haematopoietic neoplasms. We describe a case of a therapy‐responder acute myeloid leukaemia (AML) patient bearing two novel NPM1 mutations. Cells' transfection studies indicate that the presence of one of these mutations is associated to an abnormal nucleolar structure, suggesting that NPM1 may contribute to the control of nucleolar integrity. Copyright © 2009 John Wiley &amp; Sons, Ltd.

Recent studies have shown that ABGG2 protein overexpression in acute myeloid leukemia (AML) may be associated with poor response to therapy and increased relapse risk. Few data are available in patients with AML undergoing allogeneic stem cell transplantation (SCT), particularly when in complete remission (CR). We analyzed 105 patients with AML who underwent allogeneic SCT in CR evaluating the role of ABCG2 and other pretransplantation features on subsequent transplantation outcomes. Factors negatively associated with leukemia-free survival (LFS) were unfavorable cytogenetics (3-year LFS 48% versus 80%, P = .0035) and ABCG2 positivity (65% versus 80%, P = .045). Three-year cumulative incidence of relapse (CIR) in the whole population was 20%; a higher incidence of relapse was associated with adverse cytogenetics (41% versus 16%, P = .018), ABCG2 overexpression (29% versus 15%, P = .04), and, marginally, age > 50 years (30% versus 14%, P = .06). We grouped patients according to the combination of these 3 risk factors: no patient relapsed within 3 years from SCT in the group without risk factors, whereas the 3-year CIR was 12% (95% confidence interval [CI], 2% to 25%) in the group with 1 risk factor and 47% (95% CI, 31% to 70%) in patients with 2 or 3 risk factors (P = .00005). In conclusion, allogeneic SCT does not seem to abrogate the negative prognosis associated with ABCG2 overexpression at diagnosis, specifically in terms of a higher relapse risk. ABCG2, age, and cytogenetics can predict AML relapse after SCT in patients who undergo transplantation while in CR.

Correction to: Oncogene (2014) 33, 2876–2887; doi: 10.1038/onc.2013.251; published online 8 July 2013 Since the publication of the above article, it has been noted that the affiliation for E Colombo was incorrect. The correct affiliation is given above. The publisher apologises for any inconveniencecaused by this error.

The biologic and clinical importance of the multidrug resistance (MDR) that is related with the over-expression of the PI70 glycoprotein (Pgp) is widely recognized. However, a major issue that has not yet been solved is the definition of the degree of Pgp expression which is associated with a significant decrease of the sensitivity of the cells to chemotherapy. For this reason we studied the leukemic cells from 83 cases of acute leukemia. Leukemic cells were fixed in PLP and treated with saponine. Pgp expression was assayed by flow cytometry, using the anti Pgp monoclonal antibody MRK-16. Results were expressed both as the number of positive cells and by the intensity of the reaction as defined by the mean fluorescence index (MFI), i.e. the ratio between the mean fluorescence intensity of the MRK-16 incubated cells and of the IgG2a incubated cells. Thus, Pgp expression was compared with the results of two in vitro tests of cell sensitivity to anthracyclines, daunorubicin (DNR) cell retention and DNR cytotoxicity. We found that it was not the number of MRK-16 positive cells, but the degree of the reaction with MRK-16 (MFI) that significantly related to the anthracycline toxicity tests. Therefore, we propose that for clinical purposes a quick and cheap determination of Pgp-related MDR in leukemic cells may be obtained by measuring the MFI with MRK-16 in a standard flow cytometry assay and that the assay may indeed be sufficient to estimate Pgp expression as well as the influence of Pgp on cell sensitivity to anthracyclines.