ALBERT

Description: The combination of fludarabine, cyclophosphamide and rituximab (FCR) is still currently regarded as the standard regimen for treatment of physically fit patients with chronic lymphocytic leukemia (CLL). This therapy can be associated with significant toxicity, and patient adherence to the protocol may often be difficult outside of clinical trials. This retrospective study aimed to evaluate the efficacy and safety of FCR therapy in the real life setting, with particular focus on the influence of dose reduction on treatment outcome. A total of 132 CLL patients (≤70 years of age) treated with FCR as frontline therapy from 10 medical centers, were reviewed. The majority of patients were males (73.5%, n=97) and younger than 60 years (78%, n=103). Eleven patients had Binet stage A (8.3%), 72 (54.5%) were stage B and 49 (37.1%) had Binet stage C. Results of FISH analysis were available for 99 patients, with high risk cytogenetics of del(11q) in 21 patients (21.2%) and del(17p) in 9 cases (9.1%). The majority (56.5%, n=74) received rituximab at a dose of 500mg/m2 and the rest 375mg/m2. Almost half of the patients (49.2%, n=65) were given a reduced dose of chemotherapy (

Description: Background: Severe thrombocytopenia is an uncommon event in lower risk MDS patients, but it may significantly influence the prognosis. In fact, when it occurs, major bleeding may be a life-threatening complication. No licensed pharmacologic approach is nowadays available yet for these patients. Eltrombopag seems to be a very interesting product, but its efficacy and safeness are still to be better demonstrated. Romiplostim could be suitable too, but, at present, its safety is uncertain in MDS patients. Also danazol, an attenuated androgen, seems to have some ability to increase the platelet count in this context. Patients and methods: We retrospectively reviewed 17 thrombocytopenic patients affected by MDS, treated with danazol and observed for at least 6 months. Three patients of these had a therapy-related MDS. At the starting time of danazol therapy, the IPSS was “low” or “intermediate-1” in 16 cases; “intermediate-2” in 1 case. The IPSS-R was “very low”, “low” or “intermediate” in 16 cases; “very high” in 1 case. In 14 patients the platelet count was lower than 25x109/L, in the other 3 lower than 40x109/L, but with spontaneous bleeding. The initial dose was 600 mg/day for all the patients. The IWG criteria of response (Cheson 2006) were adopted. The outcomes were observed after 3 and 6 months from the beginning of therapy. Only descriptive statistical analysis was used. Results: At the beginning of therapy, the average platelet count of the 17 patients was 22.6 x109/L (S.D. 8.8, range 6-38). After 3 months, the therapy with danazol was ongoing in 16 patients (in 1 case the drug was discontinued due to renal failure). Platelet improvement, according to IWG criteria, was observed in 8 cases (47%). The average platelet count was 45.3x109/L (S.D. 32.9, range 4-133). The only one “high risk” patient did not show response. After 6 months danazol was still ongoing in 11 patients (in 5 cases the drug was stopped for inefficacy). The response according to IWG criteria was evident in 9 patients (52% of the initial 17 patients). The average platelet count was 66x109/L (S.D. 63.9, range 11-218). Adverse events recorded were as follows: increase in transaminases in 3 cases (in 2 of these the dose was reduced to 400 mg/day); severe but reversible renal failure in 1 case (the drug was stopped); moderate increasing of serum creatinine in 1 case (the drug was reduced to 400 mg/day); reversible cutaneous rush (the drug was reduced to 400mg/day); amenorrhea in 1 case (the only fertile woman in the series); weight loss and loss of appetite in 1 case, weight gain in 1 case. Conclusions This series confirms the efficacy of danazol to improve platelet count in approximately half of patients with severe thrombocytopenia due to “low-risk” MDS. In all patients with increased platelet count, the response was clinically significant. The response may not be immediate. Actually, there was an improvement of platelet count even after three months of therapy. The toxicity profile of this drug is low. The mechanism of action of danazol in MDS patients remains unclear. Waiting for more information on the efficacy and safety of eltrombopag from the clinical trials in progress, danazol may be a good therapeutic option for these patients. Disclosures Off Label Use: Danazol in MDS patients with severe trhombocytopenia.

Description: Background CMV infection represents one of the main cause of morbility and mortality after stem cell transplantation. Type III interferons (IFNs), including IFNl1 (IL29), IFNl2 (IL28A) and IFNll3 (IL28B), are thought to display potent antiviral and immunemodulatory properties in vivo, which may overlap partially with those exerted by type I IFNs. Type I and Type III IFNsl both generate an antiviral state by triggering the JAK-STAT pathway, ultimately upregulating the expression of interferon-stimulated genes. Rs12979860 single nucleotide polymorphism (SNP) in IL28B gene region is well known to influence the spontaneous and treatment-induced clearance in HCV infection. Data on the relevance of such a SNP in other viral infections is still debated even, Bravo et al. recently documented a protective effect of the T allele against CMV infection in the Allogenic stem cell transplantation (Allo-SCT) (Journal of Medical Virology 2014.86:838). Aim of the study: the current study was aimed at investigating whether the IL28B polymorphism rs12979860 may effect on the incidence rate of clinically-relevant active CMV infection in the Autologous stem cell transplantation setting. Patients and methods: From October 2014 45 patients were included in the study because underwent a autologous stem cell transplantation for hematological diseases. The median age of the patients was 56 years (16-66 years). Patients were distributed according to Hematologic disease as following: 73% of the patients had Multiple Myeloma, 20% non Hodgkin Lymphoma, 5% Hodgkin Lymphoma e 2% Acute Myeloid Leukemia. The rs12979860 IL28B SNP (C/T) genotype was determined by Melthing analysis on DNA derived from peripheral blood samples. CMV DNAemia was determined by quantitative Real-Time PCR with a limit detection of 50 copies/mL (Artus, Qiagen). Patients were monitored for CMV DNAemia weekly for the three months after stem cell transplantation. RESULTS: CC genotype was detected in 51% of patients, CT genotype in 35.5% and TT genotype only in 13.5% of patients according to the lowest frequency of TT genotype harboring in general population. A clinically-active CMV infection was documented respectively in 66,6%, 17,4% and 12,5% of patients carrying TT, CT and CC genotype. A trend towards a higher incidence of clinically-active CMV infection was noted in the TT population with respect to CT and CC population (TT vs CC: P= 0.03 and TT vs CT: P=0.02). The duration and peak of CMV-DNAemia levels tended to be higher in patients carrying the TT genotype then in ones with CC or CT genotype, although statistical significance was not reached. A positive correlation was observed between day 7 post ASCT CMV-DNAemia and the monocytes and neutrophils count. By the contrast, a negative correlation was found between day 21 post ASCT CMV-DNAemia levels and the monocytes count on 35th and 45th days after ASCT. Conclusions In conclusion, our data suggest that patients with TT genotype have higher incidence of clinically-active CMV infection in Auto-SCT setting. Even though these results should be confirmed by a larger sample size, the lowest prevalence of TT genotype in general population and higher (66.6%) clinically-active CMV infection in TT genotype patients strongly support our data. Disclosures No relevant conflicts of interest to declare.

Description: A significant proportion of cord blood CD5+ B cells express the activation molecules CD23, CD25, and transferrin receptor; react with the cell-cycle-associated monoclonal antibody (MoAb) Ki67; can be induced to enter the S phase of cell cycle by interleukin-2 (IL-2), IL- 4, or low-molecular-weight B-cell growth factor (Imw-BCGF) and, exposed to IL-1 and IL-2, acquire the features (sIgD-, CD5-, CD10+, CD38+) of B blasts proliferating in the germinal centers of secondary follicles. These findings indicate that CD5+ B cells are preactivated and, in the proper microenvironment, may give rise to CD5- B cells.

Description: CD20 is a B-lineage–specific gene expressed at the pre–B-cell stage of B-cell development that disappears on differentiation to plasma cells. As such, it serves as an excellent paradigm for the study of lineage and developmental stage-specific gene expression. Using in vivo footprinting we identified two sites in the promoter at −45 and −160 that were occupied only in CD20+ B cells. The −45 site is an E box that binds basic helix-loop-helix-zipper proteins whereas the −160 site is a composite PU.1 and Pip binding site. Transfection studies with reporter constructs and various expression vectors verified the importance of these sites. The composite PU.1 and Pip site likely accounts for both lineage and stage-specific expression of CD20 whereas the CD20 E box binding proteins enhance overall promoter activity and may link the promoter to a distant enhancer.

Description: CD22 is a B-cell–specific adhesion molecule that modulates BCR-mediated signal transduction. Ligation of human CD22 with monoclonal antibodies (MoAbs) that block the ligand binding site triggers rapid tyrosine phosphorylation of CD22 and primary B-cell proliferation. Because extracellular signal-regulated kinases (ERKs) couple upstream signaling pathways to gene activation and are activated by B-cell antigen receptor (BCR) signaling, we examined whether CD22 ligation also activated ERKs and/or modified BCR-induced ERK activation. Ligation of CD22 on either primary B cells or B-cell lines failed to significantly activate the mitogen activated protein kinase (MAPK) ERK-2, but did activate the stress-activated protein kinases (SAPKs; c-jun NH2-terminal kinases or JNKs). In contrast, BCR ligation resulted in ERK-2 activation without significant SAPK activation. Concurrent ligation of CD22 and BCR enhanced BCR-mediated ERK-2 activation without appreciably modulating CD22-induced SAPK activation. Consistent with its induction of SAPK activity, there was a marked increase in nuclear extracts of activator protein-1 (AP-1) and c-jun levels within 2 hours of exposure of primary B cells to the CD22 MoAb. Despite their differences in ERK activation, both CD22 and BCR ligation triggered several Burkitt lymphoma cell lines to undergo apoptosis, and the 2 stimuli together induced greater cell death than either signal alone. The pro-apoptotic effects were CD22-blocking MoAb-specific and dose-dependent. Examination of expression levels of Bcl-2 protoncogene family members (Bcl-2, Bcl-xL, Mcl-1, and Bax) showed a downregulation of Bcl-xL and Mcl-1 after CD22 ligation. This study provides a plausible mechanism to explain how CD22 and BCR signaling can costimulate B-cell proliferation and induce apoptosis in Burkitt lymphoma cell lines.

Description: Hepatic veno-occlusive disease (VOD), a common complication of hematopoietic stem cell transplantation (HSCT), is associated with significant morbidity and mortality. The diagnosis is mainly based on clinical criteria that are neither sensitive nor specific enough to establish early diagnosis. CA-125 is a tumor marker, which may also increase in chronic liver disease, especially with ascites formation. Since VOD is known to result from liver injury and is associated with ascites, we examined whether CA-125 can be used as a serum marker for early diagnosis of VOD. Methods: 74 consecutive patients undergoing HSCT were prospectively followed for clinical signs and symptoms of VOD. Serum samples for CA-125 were taken before conditioning, on day 0 and then every 7 days until patient’s discharge from hospital. CA-125 serum levels were measured by ELISA assay. Results: 19 of 74 patients undergoing HSCT developed VOD according to Jones criteria. 89% of these patients underwent allogeneic stem cell transplantation, 47% received the regimen containing busulfan/cytoxan and 37% were treated with total body irradiation (TBI) and cytoxan. 26% of the patients had severe VOD. Diagnosis of VOD was established on average on day 7 (range 2–20). No difference in CA-125 was observed pre-transplant between the two groups (VOD versus non-VOD), but CA-125 levels increased in 68% of the patients with VOD as opposed to only 15% of those without VOD (p

Description: The introduction of imatinib mesylate has revolutionized the treatment of CML, but despite significant cytogenetic responses primary therapy failure and emergence of resistance pose a major obstacle to successful treatment. ASCT still has a pivotal role in the treatment of CML and reduction of morbidity and mortality remains an important focus of ongoing studies in CML. We conducted a prospective study with ablative conditioning and partial T cell depleted ASCT from a matched sibling donor with no post-transplant GvHD prophylaxis. This was followed by an escalated donor lymphocyte infusion (DLI) administered if there was evidence for the presence of MRD, as determined by FISH and PCR/RQ-PCR for bcr-abl. Patients and methods: 40 consecutive patients underwent ASCT for CML between 1999–2005. The transplant regimen contained busulfan (12mg/kg), cyclophosphamide (120 mg/kg), anti-thymocyte globulin (ATG-Fresenius) (25mg/kg) and fludarabine (200mg/kg) followed by an infusion of ≥5×106/kg of G-CSF-mobilized and positively selected CD34 cells and 1×105/kg T cells. No post-transplant GvHD prophylaxis was given. The presence of MRD was determined every 3 months. DLI was administered in escalated doses starting with 3×106/kg and escalating in increments up to 1×108/kg. Results: 40 patients, median age 38 (19–63), 35 transplanted in first chronic phase (CP), 1 in second CP and 4 in accelerated phase. Patients were transplanted at a median of 4 months from diagnosis (1–66). The median of positively selected CD34+ cells was 9×106 cells/kg (5–24). 39 patients engrafted neutrophils at a median of 11 days (9–25) and platelets at a median of 15 days (9–134). Primary non-engraftment occurred in one patient and late graft failure in two, all salvageable by back-up marrow in two patients or donor stem cell boost in a single patient. The median follow-up of the entire cohort is 44 months (6–84). Overall Survival 80% Transplant Related Mortality (TRM) day 100 5% day 〉 100 10% Acute GvHD Post transplant (n = 38) Post DLI (n= 24) grade I-II 21% 21% grade II-III 0 8% grade IV 0 0 Chronic GvHD Post transplant (n = 38) Post DLI (n= 24) limited 2.6% 16% extensive 0 0 Need for DLI One dose 63% Two doses 31% ≥ Three doses 16% Disease-Free Survival Major cytogenetic response 6% Major molecular response 6% Complete cytogenetic and molecular response 88% Disease Progression 10% None of the patients developed significant VOD. Post transplant acute GvHD developed in only 21% of patients and was confined to grade I–II, all responding to a short course of immunosuppression. Additional 7 out of the 24 patients receiving DLI developed acute GvHD. There was no grade IV or mortality from GvHD. At the time of this analysis all apart from 1 patient were able to stop immunosuppressive treatment for GvHD. There were no significant infectious complications in this group. All late TRM were secondary to autoimmune complications, unrelated to GvHD, and occurred in 5 patients (13% of all) at 6–14 months post transplant. These complications included: ITP, AIHA, autoimmune pneumonitis, nephritic syndrome, TTP and Kaposi sarcoma. The mortality from these complications was 80% (4 out of 5 patients). 4 patients developed blast crisis post transplant; 2 patients have died; and the others are currently in CR post salvage treatment, DLI and imatinib. Twenty five percent of patients attained complete molecular remission without ever needing DLI, while 75 % responded after DLI, mostly following a single dose. Two patients are in major cytogenetic response, one of whom received back-up marrow and for the other a DLI donor is not available. The two patients in major molecular response are awaiting DLI. Conclusions: Matched ASCT for CML can be performed with significantly reduced TRM and morbidity using partial T cell depletion, no GvHD prophylaxis and preemptive DLI. This regimen offers 〉 80% long-term curative potential. Programmed DLI is safe and associated with a low risk of GvHD. The late immunological complications pose a major risk. This appears to be related to an imbalance between B and T cell reconstitution and the precise mechanisms of this need to be elucidated. In the imatinib era ASCT using this approach may be reserved for patients not responding or resistant to the imatinib mesylate.

Description: Introduction: Multiple myeloma (MM) is a neoplasm of B lymphoid line that is characterized by clonal proliferation of malignant plasma cells in the bone marrow, producing monoclonal paraprotein (M) in blood and/or serum. Interleukin-6 (IL-6) is one of the key molecules related to growth, survival and proliferation of MM cells. Tocilizumab (TCZ) is a humanized monoclonal antibody directed against IL-6 receptor (IL-6R). When radiolabeled and used for tumor imaging, intact IgG exhibits high liver uptake. Antibody fragments (Fab´s) are quickly eliminated from blood and normal tissues (except kidneys), achieving high tumor-to-blood and tumor-to-normal tissue ratios with renal clearance. The aim of our work was to develop a 99mTc radiolabeled TCZ Fab´s fragment and to perform its chemical and biological evaluation in order to be used as a potential MM imaging agent for staging and restaging. Methods: Antibody fragmentation was carried out with papain and, once purified, Fab´s(TCZ) fragments were identified and derivatized with NHS-HYNIC-Tfa as bifunctional coupling agent. MALDITOF/TOF was used to confirm all procedures. A mixture of Tricine/SnCl2.2H2O was added to Fab´s(TCZ)-Tfa-HYNIC and radiolabeled with 99mTcO4-. Radiochemical purity and in-vitro stability in saline, serum and different concentration of L-cysteine up to 4 h were analyzed by ITLC and HPLC. In-vitro binding assays were performed using U266 and MM1S cell lines up to 120 min. Biodistribution and SPECT/CT images were evaluated on healthy Balb/c mice and MM1S tumor-bearing Balb/c nude mice at 0.5, 2 and 4 h. Results: Radiolabeling of HYNIC-Tfa-Fab´s(TCZ) was carried out in a fast, reproducible, easy, stable way showing high radiochemical purity and high specific activity. In vitro binding assays confirm that after its derivatization and radiolabeleing, Tfa-HYNIC-Fab`s(TCZ) does not interfere with the epitope recognition. In vivo biodistribution studies on healthy Balb/c mice and MM1S tumor-bearing Balb/c mice showed that 99mTc-HYNIC-Fab´s (TCZ) has significant renal uptake with neglectable uptake in other organs, indicating renal clearance. Tumor uptake was 12.84±1.80 %ID/g followed by 8.94±0.61 %ID/g and 3.05±1.49 %ID/g at 2 and 4 h, respectively. U266 tumor-to-muscle ratios were 5.79, 8.61 and 2.71 at 0.5, 2 and 4 h, respectively.Tumor uptake for MM1S tumor-bearing Balb/c nude mice was 10.05±1.32 %ID/g, 8.59±2.36 %ID/g and 3.88±0.68 %ID/g at 0.5, 2 and 4 h, respectively. MM1S tumor-to-muscle ratios were 6.32, 4.61 and 3.08 at 0.5, 2 and 4 h, respectively. Biodistribution data of 99mTc-HYNIC-Fab´s(TCZ) on U266 tumor-bearing Balb/c nude mice showed good tumor uptake and retention 0.5 h after its injection SPECT/CT images on healthy Balb/c mice and MM1S tumor-bearing Bal/c nude mice of 99mTc-HYNIC-Fab´s(TCZ) showed renal uptake and a discrete tumor uptake at 4 h p.i (Figure 1). Conclusions: Labeling Fab´s(TCZ) with 99mTc using HYNIC was performed in an easy, fast, stable and reproducible way preserving its biological activity. Biodistribution and SPECT/CT imaging assays allowed us to observe and evaluate its potential role as a diagnostic molecular imaging agent for MM. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Multiple Myeloma (MM) is a heterogeneous, incurable disease. Several prognostic scores have been developed to estimate response to treatment, progression free survival (PFS) and overall survival (OS). The International Staging System defines 3 stages with distinct prognostic significance, using serum beta 2 microglobulin (b2M) and serum albumin. However, due to a higher power of risk discrimination, current guidelines recommend to stratify patients according to cytogenetics (CG) and treat high-risk patients with Bortezomib (BZ)-based therapies. In Uruguay, BZ is financed and regulated by a National Funding Board and is approved for high-risk MM or renal impairment at diagnosis or at relapse. As a consequence, the patients included in this study are a selected high risk MM subgroup. Even though proteasome inhibitors have improved outcomes for many high-risk MM patients, some still present short response duration and poor survival. Identifying simple factors that can predict response to BZ would be useful in order to better select the most appropriate therapeutic choice. Patients and Methods. We conducted a retrospective study to evaluate MM response to BZ-based therapy according to b2M, serum creatinin (Cr) and CG. Forty-seven MM patients [median age 59 years (38-77), females 25 (53%)] from two public centers treated with BZ-based combinations were included. According to local regulatory policies, only patients with renal failure and/or high risk CG features received BZ as first line therapy (n=31). Other 16 individuals received BZ combinations for relapsed/refractory disease. Patients received a median of 5 cycles of BZ (3-6). Nineteen individuals (40%) received high dose melphalan and autologous stem cell transplantation after BZ-based induction. At diagnosis, 28 (59%) had an ISS 3, 24 (51%) patients had serum Cr higher than 2 mg/dL and 14 (30%) exhibited high risk CG. Responses to BZ therapy were evaluated according with International Myeloma Working Group criteria as complete response (CR), very good partial response (VGPR), partial response (PR), stable disease (SD) and progressive disease (PD). Responses were analyzed depending on b2M level, serum Cr and CG. Comparison between groups was made by chi-square test. PFS and OS were calculated by the Kaplan-Meier method. Survival was compared between groups by log-rank test. Results. No differences in responses to BZ-based therapies were found when comparing patients with low (lower than 5,5 mg/L) versus high b2M levels (CR/VGPR 44,4 vs 44% respectively, p=0.97), low versus high serum Cr (CR/VGPR 34 vs 55% respectively, p=0.18) and standard versus high risk CG (CR/VGPR 46,7 vs 38,5% respectively, p=0.62). Patients with renal failure (serum Cr 2 mg/dL or higher at diagnosis, n=23) had a median of 53% (0 - 88%) reduction in Cr levels after receiving at least three cycles of BZ therapy. Eleven of them (47,8%) had normal renal function after completing BZ treatment. From 9 patients requiring dyalisis at diagnosis, 4 were out of dyalisis after BZ treatment. No significant differences in PFS and OS were observed when patients with low versus high b2M levels and standard versus high risk CG were compared. Patients with low Cr levels at diagnosis show a significantly better OS compared with those with Cr higher than 2 mg/dL (median OS not reached versus 42 months respectively, p=0.004), with no differences in PFS. Conclusion. In a selected high risk MM patients group treated with BZ-based therapies, similar response rates were obtained in individuals with high or low b2M levels, Cr levels, and standard or high risk CG. Although individuals with renal failure at diagnosis exhibit similar quality of responses than standard patients, long term OS of this group is still impaired and further improvements in therapy are needed. Note: GB and ER have contributed in equal parts to this work. Disclosures No relevant conflicts of interest to declare.