ALBERT

Description: Multilevel genetic characterization of Waldenström macroglobulinemia (WM) is required to improve our understanding of the underlying molecular changes that lead to the initiation and progression of this disease. We performed microRNA-expression profiling of bone marrow–derived CD19+ WM cells, compared with their normal cellular counterparts and validated data by quantitative reverse-transcription–polymerase chain reaction (qRT-PCR). We identified a WM-specific microRNA signature characterized by increased expression of microRNA-363*/-206/-494/-155/-184/-542-3p, and decreased expression of microRNA-9* (ANOVA; P 〈 .01). We found that microRNA-155 regulates proliferation and growth of WM cells in vitro and in vivo, by inhibiting MAPK/ERK, PI3/AKT, and NF-κB pathways. Potential microRNA-155 target genes were identified using gene-expression profiling and included genes involved in cell-cycle progression, adhesion, and migration. Importantly, increased expression of the 6 miRNAs significantly correlated with a poorer outcome predicted by the International Prognostic Staging System for WM. We further demonstrated that therapeutic agents commonly used in WM alter the levels of the major miRNAs identified, by inducing downmodulation of 5 increased miRNAs and up-modulation of patient-downexpressed miRNA-9*. These data indicate that microRNAs play a pivotal role in the biology of WM; represent important prognostic marker; and provide the basis for the development of new microRNA-based targeted therapies in WM.

Description: Abstract 1407 Poster Board I-429 Purpose. To compare the requirements of physicians and patients for the Internet interactive service which allows patients to manage their own medical records and communicate with physician via the Internet. Background. USA federal rule defining “the Meaningful Use of Electronic Health Records” is similar to that of the National Standard of Russian Federation “The Electronic Case History (EHR)”, operating since 2008. This National Standard was developed based on experience of EHR system at the National Center for Hematology in Moscow (NCH). In 2009, we started the Personal Health Records service (PHR service) that allows patients to manage their own medical records and have internet-based communication with physicians. Simple interface for patients which blocks the full capacity of the PHR service is similar to that of EHR system of NCH. It permits integrated data presentations on a uniform axis of time and access to additional information (reported to ASH in 2001). The PHR service raises question of “meaningful use” requirements not only for EHR provider organization, but for the service users - patients and doctors. Methods. Using questionnaires and interviews, we compared expectations and acceptance of the PHR service by doctors and their patients. Results and Discussion. Preliminary results indicate that doctors are more likely to use the PHR service than the System of HER. Although the entire format of PHR service is familiar to physicians at NCH, they mostly use its information capabilities (viewing the results of the analysis, making appointments for research and planning patient's visits). The patients use PHR service with great enthusiasm (increasing with younger age and higher level of education). The complexity of integration interfaces, which we leave for the patients in the second term, gives them more inspiration than that the physicians. However, few patients take seriously the responsibilities that exist in relation to the accurate maintenance of their records. Conclusion. PHR can be widely used if integration of sources for medical information and unification format can simplify the “manual” work of PHR management. Key Words: Telemedicine, PHR, EHR Disclosures: No relevant conflicts of interest to declare.

Description: For over 30 years CHOP (cyclophosphamide-doxorubicin-vincristine-prednisolone) has been the standard therapy for Diffuse Large B-cell Lymphoma (DLBCL). Although DLBCL is one of the most chemotherapy-responsive human malignancies, less than 50% of newly diagnosed patients are cured with the conventional anthracycline-based chemotherapy. Novel strategies are sought out to improve the survival of these patients. Heat Shock protein 90 (HSP90) is a molecular chaperone critical for correct conformational folding of many cellular proteins. Many of those proteins are signal-transducing regulators of cell growth, DNA damage response, and survival. Cancer cells are particularly dependent on these regulators for maintenance of the transformed phenotype. Thus, HSP90 is an attractive target for cancer therapy. HSP90 inhibitors block the ATP-binding pocket and inhibit an essential ATPase activity, altering the function of the HSP90 complex and leading to destabilization and eventual degradation of mis-folded proteins. Inhibitors of HSP90 have shown promise alone or in combination with chemotherapy or radiation in preclinical studies, and are currently undergoing PhaseI/II clinical trials. Future clinical trials on their efficacy will likely explore if any additional benefit is apparent from their combination with conventional therapies. We recently demonstrated that schedule of administration of 17DMAG following Doxorubicin (DOX) ia critical for in vitro sensitivity of DLBCL cells. We examined whether the newly developed purine-scaffold HSP90 inhibitor, PU-H71, could similarly enhance the toxicity of cells to DOX when applied 24 hours after exposure to DOX. We found that sequential addition of PU-H71 to DOX resulted in reduction of DOX IC50, from 400 nM to 75 nM. To evaluate the role of schedule of drug exposure as a determinant of the overall response, we analyzed the interaction of DOX and PU-H71 on cell survival. We applied standard criteria for synergy: the median effect/Combination Index (CI) method using an equimolar (1:1) ratio of DOX:PU-H71. Addition of PU-H71 to cells pretreated with DOX for 24 hours was consistently synergistic over the entire range of drug doses, with CI = 0.75 at Fa = 0.5. Although addition of PU-H71 prior to DOX was synergistic with CI = 0.7 at Fa = 0.5, this was only evident for higher doses. Only additive-to-antagonistic effects were observed at lower drug concentrations (Fa 〈 0.3). These results raise the question on whether synergy between PU-H71 and DOX will be observed at the concentrations achievable clinically. Conclusions: our observations provide a rationale for further preclinical testing and stress the need to consider schedule of exposure as a critical determinant of the overall response when designing clinical trials that combine HSP90 inhibitors with DNA damaging agents.

Description: Background: According to the WHO, T/NK-cell large granular lymphocytic leukemia (T/NK-LGL) comprises 2 – 5 % of all T/NK tumors. In addition, T/NK-LGL is a very heterogeneous disease. T/NK-LGL has been divided along 2 basic types: T-cell and NKcell leukemias. We found further differences among these two basic types. Among our patients with T/NK-LGL, there were 5 immunophenotypic variants with differences in severity of their disease and other clinical parameters. Materials and Methods: Thirty five patients were diagnosed with T/NK-LGL from 01.1998 to 01.2008 (13 men and 22 women) in the age from 18 to 71 years (median age 58 years). The median survival has not been achieved. The median follow up is 72 months. Clinical common features: lymphocytosis in blood and bone marrow, cytopenia (80%), splenomegaly, rheumatoid arthritis and B-symptoms. Unusual features: lymphadenopathy, vasculitis and neuropathy. Diagnostic methods: histology, cytology and flow two-color cytometry with monoclonal antibodies (“Becton Dickinson Immunocytometry Systems” USA and “Dako” Denmark). Clonality was defined by a PCR- on T-cell receptor gene rearrangement. Results: Twenty six patients: 18 women and 8 men were accurately classified as «T/NK - cellular leukemia - LGL» according to WHO classification. The remaining 9 cases had rare immunophenotypes. All (35) patients were subdivided into 5 groups by immunophenotypic (IFT) features of leukemic lymphocytes. 1st group (4 women) had the following IFT of the leukemic lymphocytes: CD2+CD3−CD56+/−CD16+. In the 2d group, 9 patients (5 men and 4 women) had IFT of CD2+CD3+CD8+CD16+CD56+/−. There were 13 patients (3 men and 10 women) in the 3d group. Variant of IFT with leukemic T-cells was CD3+CD8+CD16−CD56−. There were 5 patients (2 men and 3 women) in the 4th group. 20–60 % peripheral blood lymphocytes simultaneous expressed CD8 and CD4 (double – positive T-cells). Others had 40–20% lymphocytes expressing only CD8+ marker. In the 5th group, there were 4 patients (2 men and 2 women). The pathologic T-cells were detected only on the bone marrow aspirate. There had heavy bone marrow aplasia or predominantly red cell aplasia. IFT of lymphocytes in the bone marrow was aberrant with presence of some markers of T-cells, including, CD8, absence of CD16 and CD56. The first three groups appeared stable and did not require chemotherapy for prolonged time (11 of 26 patients, overall 42%). A median of follow-up without treatment is 70 months. The patients of 4th and 5th group required immediate intervention and were treated with chemotherapy. Conclusions: Immunohistochemical characteristics of T-cells separate T/NK-LGL patients with stable disease from those that present with cytopenias and require immediate intervention. The molecular characteristics of these cells will be further defined.

Description: Background. MicroRNAs (miRNAs) constitute a class of small, non-coding, 18–24 nucleotide RNAs, that act as negative regulators of gene expression. By repressing several target mRNAs, mature miRNAs play a pivotal role in regulating development, cell differentiation, apoptosis, and cell proliferation. Moreover, miRNAs have been described to play roles in both solid tumors and hematologic malignancies, however the role of miRNAs in WM has not been yet elucidated. Methods. We performed miRNA expression profiling of bone marrow-derived CD19+ WM cells, compared to their normal cellular counterparts and validated data by qRT-PCR. In vitro and in vivo functional studies were performed on miRNA-155 knockdown WM cells. Effect of miRNA-155 on signaling cascades have been evaluated by western blot and immunofluorescence. NF-kB activity has been investigated using a DNA-binding enzyme-linked immunosorbent assay-based assay. Gene expression profile analysis has been performed on both miRNA-155 knockdown- and control probe-transfected WM cells in order to identify potential miRNA-155 targeted genes. Finally correlation between miRNA signature and prognosis has been evaluated. Results. We identified a WM specific miRNA signature characterized by increased expression of miRNA-363*/-206/-494/-155/-184/-542-3p; and decreased expression of miRNA-9* (ANOVA;P〈 0.01). Our data showed that miRNA-155 regulates proliferation and growth of WM cells in vitro and in vivo, by inhibiting MAPK/ERK, PI3/AKT, and NF-kB signaling pathways. Potential miRNA-155 target genes were identified using gene expression profiling and included genes involved in cell cycle progression, adhesion, and migration. Importantly, increased expression of the 6 miRNAs significantly correlated with a poorer outcome predicted by the International Prognostic Staging System for WM (P

Description: Introduction: According to the new WHO classification for Non-Hodgkin’s lymphomas developed in 2001, the diagnosis of many lymphoma patients has to be reevaluated. The existing information on therapeutic options for patients with these rare malignancies prior to development of WHO classification has been unreliable. The T-cell malignancies are associated with poor outcome, and it is important to identify patients that could benefit from novel intensive therapies. Materials and Methods: We conducted retrospective analysis of patients with T- and NK-cell malignancies treated from 2000–2006 at the Federal Research Hematology Center of Russian Academy of Medical Sciences. The purpose of this study was to determine the frequency, clincopathological features, and outcomes of patients in predominantly Slavic population (European part of Russia). Results: Because these malignancies present in a variety of nosological forms and their response to standard therapies is poor, multiple therapeutic regimens were employed as expected. Information on 151 patients with complete clinical and pathologic information that allowed appropriate diagnosis according to the WHO criteria was available for the analysis. Analysis of 188 patients required changes from the initial diagnosis after additional laboratory investigation: 25 cases (13.3%) previously considered to be T-cell lymphomas were excluded from the analysis, mostly because of an inadequate histopathological material. Similar to previously reported data, a significant number of T- and NK- cell lymphomas (12.4%) were excluded as compared to B-cell (3.1%) lymphomas, which emphasizes the difficulties in diagnosis of mature T- and NK-cell lymphomas. Twelve out of 163 patients with verified T-cell lymphomas after correction of diagnosis were treated and observed in other countries or regions, and were also excluded from the final analysis. The only difference in 151 remaining patients was an increased incidence of T-cell malignancies with mediastinal involvement, with overall survival similar to those reported elsewhere. Final analysis of 28 additional cases in 2006 for a total of 179 patients will be presented. Conclusions: Our survey of patients with T-cell malignancies represents a systematic analysis of these rare malignancies and establishes the communality of clinical features and response to therapies. Kaplan-Mayer Survival of all TCL Kaplan-Mayer Survival of all TCL

Description: Abstract 4758 Aim To determine the frequency of transformation of cutaneous T-cell tumors (CTCL) and effect of different therapies. Materials and methods Retrospective analysis of treatment results for 68 patients with two types of CTCL, MF and SS, selected from 263 patients T-cell lymphoma/leukemia treated at NCH during 9 years. All 68 patients have CD 3 +, CD 4+, CD 7-, CD 8-, CD 30- immunophenotype and T-cell clonality (rearranged genes for T-cell receptor). MF was diagnosed in 48 patients (24 men, 24 women) with median age 52 years (26 - 77), SS in 20 patients (12 men, 8 women), median age 61 years (23 -87). Half of the MF patients started surveillance at III - IV stages, 15 of 20 patients with SS - at stage IV. Treatment of CTCL in the indolent phase was conducted in accordance with their stage: monochemotherapy (leukeran or methotrexate) and biologically active agents (interferons, retinoids, monoclonal antibody) were used. Appearance of transformation to large cell lymphoma was consistent with tumor progression as determined by presence of two or more of the following: 1. rapid growth of tumors in MF or leukemization in SS; 2. presence of large anaplastic cells in blood or tumor biopsy; 3. increase in expression of Ki-67 (10-70%); 4. appearance of activation markers (CD30, CD25) in tumor cells, and 5. loss of certain linear T-cell markers. For treatment of patients in large cell transformation phase Promace-Cytabom or FNC protocols were used in 7 patients. Five patients received A-CHOP-14 (with alemtuzumab) and subsequent maintenance therapy (methotrexate 20 mg per week, 6-mercaptopurine 50-100 mg/day in combination with alpha interferon 3 million daily). Results and Discussion The overall median time of observation (before and during treatment) in 48 patients with MF was 96 months (8-264). For 13 patients who died it was 76 months (8-264), mortality rate was 27%. The overall median observation (before and during treatment) in 20 patients with SS was 60 months (6-123), for 8 who died, it was 62 months (6-94), mortality 40%. The median observation for treatment of MF was 30 months (from 3-122) for the SS, it was 18 months (1 - 85). Restraining aggressive therapy for an indolent process resulted in complete or partial remission, while maintaining a good quality of life. Large cell transformation phase was observed in 12 patients (7 of 48 patients with MF (14.5%) and 5 of 20 patients with SS (25%). Seven of the 12 patients died within 2-4 months due to septic complications during Promace-Cytabom or FNC protocols. The remaining 5 patients received A-CHOP-14 (6-7 courses) which reduced tumor formation and /or leukemization. However, the persistence of patches, plaque or erythrodermia was the reason for maintenance therapy. This combination led to further regression of skin manifestations and full or partial remission within 4-12 months. Conclusion Because of the lack of a generally accepted treatment for GM and SS in Large Cell Transformation phase, even small successes should be discussed. The effectiveness of combining of polychemotherapy with maintenance therapy may be effective due to the presence of several distinct clones of tumor cells with different properties. Keywords: treatment of cutaneous T-cell lymphoma, Mycosis Fungoidus, Sezary Syndrome, Large Cell Transformation Phase, immunohistochemical markers of T-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.