ALBERT

Description: Imatinib mesylate (IM) is a tyrosine kinase inhibitor selective for Bcr-Abl, c-Kit, and platelet-derived growth factor receptor kinases. IM inhibits kinase activity through competition for ATP binding. IM is indicated for the treatment of newly diagnosed patients with Philadelphia chromosome positive (BCR-ABL positive) chronic myeloid leukemia (CML) in chronic phase. Recent publications demonstrate that this drug may also target other cellular components. In light of the important role of telomerase in malignant transformation, we evaluated the effect of IM on the signal transduction cascade leading to modulation of telomerase activity in the BCR-ABL positive K562 cell-line. In addition, we studied the effect of IM on telomerase in an IM resistant K562 cell-line (K562res). The K562res cell-line is resistant to the effect of IM on the BCR-ABL protein. However, the effect of IM on telomerase in these cells is unknown. Therefore, using these cells will enable us to study the connection between the signal transduction cascade of BCR-ABL and that of telomerase. IM caused an 80% inhibition of telomerase activity in both K562 and K562res cell-lines. Inhibition of telomerase activity was associated with 50% inhibition of proliferation. Inhibition of telomerase activity was not caused by changes in the transcription level of hTERT (the catalytic subunit of the enzyme). On the other hand, it seemed mainly to be caused by post-translational modifications only in K562 cells namely, a 2-fold dephosphorylation of AKT, known to phosphorylate telomerase. In addition, IM was found to upregulate the expression of PTEN (known to negatively regulate AKT) again only in K562 cells. IM did not affect the levels of phosphorylaetd AKT or PTEN in K562res cells. However, IM caused an approximate 2-fold upregulation of p53 (a transcription factor of PTEN) levels both in K562 and K562res cells. Our results demonstrate the ability of IM to inhibit telomerase activity in BCR-ABL expressing cell lines. Telomerase activity inhibition was demonstrated in K562 cells as well as in K562 cells resistant to high concentrations of IM. Therefore, the inhibitory effect of IM on telomerase activity isn’t necessarily mediated through the known tyrosine kinase targets of IM. The effect of IM on the signal transduction cascade leading to the down-regulation of telomerase activity seems to be different in K562 cells and K562res cells. While the signal transduction cascade in K562 cells appears to go through AKT and PTEN, the response to IM in K562res cells seems to be mediated by other yet unknown pathways. Future work is needed in order to clarify possible mechanism(s) by which IM down-regulates telomerase activity in these cells. This study supports previous studies demonstrating telomerase activity as an additional cellular target of IM. In addition, this study shows that cells known to be resistant to IM with regards to its effect on BCR-ABL could still be sensitive to IM treatment regarding other cellular components.

Description: Bone marrow (BM) involvement in diffuse large B cell non Hodgkin’s lymphoma (DLBCL) results in stage IV staging and has negative impact on survival. Up to 40% of patients with DLBCL have BM involvement by routine histology of marrow biopsy. The identification of lymphoid monoclonality is widely used in diagnosis of lymphoma, by flow cytometric and molecular methods. The clinical significance of monoclonality by these methods has not been systematically addressed in clinical setting. An International Workshop of response criteria in non Hodgkin’s lymphoma recommends that histologically normal marrow with small clonal population detected by flow cytometry should be considered normal until there are clinical studies that demonstrate a different outcome for this group. Recently it was reported that clonality detected by PCR amplification carries worse prognosis in patients with DLBCL. In our institution bone marrow biopsies of all patients with lymphoma are routinely evaluated by flow cytometric analysis since 1993. We evaluated the clinical course of all our patients with DLBCL diagnosed or followed up between 1993 and 2004 and compared the clinical course of patients with histologically positive marrow, histologically and cytometry negative marrow and histologically negative marrow and monoclonal lymphoid population detected by flow cytometry. Monoclonality was defined as ration of light chain expression kappa: lambda〉3:1 or lambda: kappa 〉 2:1 in at least 2% of the gated population. Selected cells were analyzed by two or three color combinations: CD5 versus CD19, CD20 versus CD10 and kappa chain versus lambda chain occasionally with the addition of CD19 or CD20. Among 110 patients, 80 had negative marrow histologically and on cytometry (BM-FC-), 16 were positive on both parameters (BM+FC+) and 14 were histologically negative and cytometry positive (BM-FC+). All three groups were similar in parameters as age, performance status, hemoglobin and LDH levels. BM-FC- and BM-FC+ were similar in IPI scores. BM-FC- patients had significantly longer survival time that BM+FC+ and BM-FC+ patients. There was no significant difference in survival of BM+FC+ and BM-FC+ patients. We conclude that the clinical outcome of patients with DLBCL with histologically normal bone marrow and monoclonality on flow cytometry is similar to those with histologically positive marrow. These results may imply that lymphoid monoclonality on flow cytometry should be regarded as bone marrow involvement and treated as such.

Description: Sarcopenia is defined as reduced muscle mass and loss of strength or function and can be part of normal aging as well as a component of cachexia. Multiple studies found correlation between reduced muscle mass and negative outcomes in chronic diseases and solid tumors. Little research exist on similar relationship in hematological malignancies and no studies investigated impact of longitudinal muscle mass changes on clinical outcomes in elderly lymphoma patients undergoing treatment. To evaluate the muscle mass during the course of chemotherapy in patients 70 years or older with DLBCL and its effect on survival and toxicity, we performed a retrospective cohort study. We included patients diagnosed between the years 2007-2014, and treated with RCHOP at the Rabin Medical Center. We collected data on age, sex, performance status (PS), number of extranodal involvement sites, Ann Arbor stage, international prognostic index (IPI), Charlson comorbidity index (CCI), hemoglobin (Hb), LDH, neutrophil, lymphocyte, monocyte, and platelet count, creatinine, albumin, CRP, treatment date, response and survival. We evaluated muscle mass by adding bilateral psoas muscle cross-sectional areas measured at the level of the third lumbar vertebra in a semi-automated manner on standard PET CT images. The ratio of the total psoas area normalized to height square (expressed in cm2/m2) was defined as muscle index. The change in muscle index after vs. before treatment was estimated. Correlation was estimated in a non-parametric test. The effect of pre-treatment muscle mass index and the above mentioned variables was estimated in a univariate Cox regression analysis. Variables potentially associated with mortality were entered into a Cox regression multivariate analysis. Results: Ninety three patients treated with RCHOP with baseline PETCT were included in the cohort. Median age was 78 years (range 70-90). Half of patients were female. Sixty percent had an IPI score of 3 or more. Mean muscle index before treatment was 4.66 cm2/m2, median 4.4 cm2/m2. End of treatment PETCT was available for 76 patients. Mean post treatment index was 4.2 cm2/m2, median 3.92 cm2/m2. A decrease in muscle index was observed in 76% of patients. The change in index ranged from -7.5 to 0.8, with a mean change of -0.58, SE0.12. No statistically significant correlation between pre treatment index and dose intensity was shown (p = 0.46). A negative correlation was shown between pre treatment index and days of hospitalization in cycles 1-2 (p=0.007, r=-0.28). Pre-treatment muscle index was not associated with overall survival (p = 0.43). In a sub-group analysis by sex a higher muscle index was associated with a longer overall survival among men (HR 0.59, 95% CI 0.44 to 78, p

Description: Abstract 1562 Background: PET-CT is of central importance in the staging of patients with Hodgkin (HL) and non-Hodgkin aggressive lymphoma and in the assessment of their response to therapy. Repeated imaging of patients is associated with cumulative exposure to substantial doses of radiation that have been linked to an increased life attributed risk of cancer. Reduction of the PET CT imaging region will decrease significantly the cumulative radiation dose. Objectives: This study aims to evaluate whether a limited PET-CT focused to the initially involved field of view (FOV) at diagnosis, above or below the diaphragm, is sufficient for effective follow up and decision making in patients with early stage curable lymphoma. Methods: We retrospectively reviewed all patients treated in our center for HD and non-Hodgkin aggressive lymphoma that were diagnosed with stage I or II disease according to the Ann-Arbor staging system during September 2006 to December 2010. Included were patients that had a PET-CT preformed as part of their initial staging and that were followed by interim PET-CT and/or a PET CT at the completion of therapy. Clinical and epidemiologic data were extracted from the patients' medical charts. All PET-CT were reviewed by a nuclear medicine specialist. We then analyzed whether limiting the PET-CT imaging to the initially involved FOV at diagnosis, above or below the diaphragm, would miss findings obtained by standard PET CT. Results: Out of 668 patients 44 were eligible for full analysis. Patient median age at diagnosis was 37.5 years (range 19–84) and 59% of patients were females. Twenty seven patients had HD and 17 had diffuse large B cell lymphoma (DLBCL). Two cases of primary mediastinal DLBCL were included in the DLBCL group. Forty two patients had stage II disease while 2 patients were diagnosed at stage I. Thirty five patients (79%) had their lymphoma located above the diaphragm while 9 patients (21%) had disease involvement below the diaphragm. One hundred twenty seven PET-CT examinations were analyzed. Of these, 44 were preformed at diagnosis. Of the 43 interim exams preformed 32 were compatible with complete response (CR) and 11 were compatible with partial response (PR). Of the 40 PET-CT preformed at the end of treatment 34 were compatible with CR, 4 were compatible with PR and 2 showed a stable or progressive disease. There was no single case in which disease progressed outside the initially involved FOV. Thus, elements of response to therapy were contained and available for analysis within the preliminary filed of involvement in all cases. Conclusion: Our findings suggest that limiting PET-CT analysis of the initially involved FOV, above or below the diaphragm, in patients with early stage curable lymphoma may be satisfactory for response assessment and the associated clinical decision making. The use of limited PET-CT will significantly reduce patient exposure to cumulative radiation. The time required to complete limited PET-CT is reduced by 50% and the cost by more than 15%. To our best of knowledge this is the first analysis demonstrating the potential utility of reduction of PET CT FOV in curable lymphoma in adults. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2820 Introduction: Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma (NHL), yet there are no standard guidelines regarding the best chemotherapeutic regimens for its management. The most prevalent regimens, especially in advanced disease, incorporate anthracyclines. There is no proof, however, that they are superior to non-anthracycline containing regimens, or even to single agent therapy. Methods: Systematic review and meta-analysis of randomized controlled trials comparing anthracycline-containing regimens (ACR) to non-anthracycline containing chemotherapy (non-ACR) for adult patients with FL. Trials assessing rituximab or other immunotherapy were included only if the anthracycline was the only difference between study arms. A comprehensive search was conducted with no restrictions, until July 2010. Two reviewers appraised the quality of trials and extracted data. The primary outcome was overall survival. Time to event data was extracted and pooled hazard ratios (HR) with 95% confidence intervals (CI) were calculated. Secondary outcomes included survival at 5 years, disease control defined as progression free survival (PFS) or response duration (RD), transformation rate to aggressive lymphoma, complete remission (CR), overall response (OR), and adverse events. Risk ratios (RR) for dichotomous outcomes were pooled using random effects model. Heterogeneity was assessed using the I2 measure of inconsistency. Results: We identified ten trials, conducted between the years 1974–2006 and randomizing 2422 adult patients. Nine trials included naïve patients; one included relapsed and refractory FL patients. Six trials compared between the same chemotherapeutic regimens, with the addition of anthracyclines in the intervention arm as the only difference. There was no statistically significant difference between ACR and non-ACR arms, regarding overall survival at the end of follow up or all cause mortality at five years (HR 0.97; 95% CI 0.76 – 1.23 and RR 0.94; 95% CI 0.76 – 1.17, respectively, I2=0% for both analyses). PFS and RD favored the ACR arm, with a HR of 0.69, 95% CI 0.51–0.91 for disease control, 3 studies, I2=11%. There was no significant difference with regard to CR and OR, but analyses were heterogeneous. In four out of the ten trials, different chemotherapeutic regimens were compared, and only one study-arm contained anthracyclines. Similar to the results obtained with the same regimens comparison, there was no benefit for ACR with regard to OS (HR 0.94; 95% CI 0.77 – 1.15) and mortality at five years (RR 0.98; 95% CI 0.84 – 1.16). Disease control was improved with ACR (HR 0.74; 95% CI 0.60–0.93). In all studies, there were no data regarding transformation rate to aggressive lymphoma. Toxicity data reported was insufficient for appropriate meta-analysis. Overall, ACR were more often associated with cytopenias, especially neutropenia, although the frequency of serious infections or death related to chemotherapy was similar to non-ACR. Also, alopecia and gastrointestinal side-effects, including nausea and vomiting, were more common with ACR. Cardiotoxicity was reported in five trials, and albeit rare, was associated with anthracycline use (RR 8.62; 95% CI 1.58 to 47.05). Conclusion: The use of anthracyclines in patients with FL has no demonstrable benefit on overall survival. However, ACR improve disease control, as measured by progression free survival and response duration with an increased risk for side effects, notably cardiotoxicity. Disclosures: Shpilberg: Roche: Consultancy, Honoraria.

Description: Background High dose chemo/radiotherapy with autologous hematopoietic cell transplantation (HCT) has been shown to be an effective therapy for a variety of malignancies. However, as more patients survive both the disease and the early post HCT period, it has become apparent that other potential long term complications, mainly secondary malignancies may hamper patients' prospects. Objectives We analyzed the incidence of different secondary malignancies occurring after consolidation with autologous HCT compared to other modalities for the treatment of various hematological and non-hematological malignancies. Methods Systematic review and meta-analysis of randomized controlled trials comparing high dose therapy with autologous HCT to other treatment modalities (observation, immunotherapy or chemotherapy). We searched the Cochrane Library, MEDLINE and conference proceedings. Outcomes assessed were rates of AML/MDS, solid tumors and overall secondary malignancies. Relative risks (RR) with 95% confidence intervals (CIs) were estimated and pooled. Heterogeneity is given when approporiate. Results Our search yielded 74 trials fulfilling inclusion criteria; among them 38 trials recruiting 10131 patients reporting secondary malignancies were included. Baseline diseases were lymphoproliferative diseases (n=17), plasma cell dyscrasias [PCD] (n=5) and solid tumors (n=16). The studies were conducted between the years 1987 and 2007. Median follow up was 55 (range 12-144) months. High dose therapy consisted of variety of regimens. Comparative arm regimens were either observation (n= 6), intensive chemotherapy [〉6 gr/m2 of cyclophosphamide or high doses of etoposide] (n= 6), or standard chemotherapy (n= 24). Overall, the RR for secondary malignancies was 1.25 (95% CI 0.99-1.57) (38 studies, 10131 pts). Among all patients, there was a higher rate of MDS/AML in patients given HCT compared to other treatments [RR=1.71 (95% CI 1.18-2.48)], (33 studies, 8778 patients), Figure. Subgroup analysis showed similar results in patients with lymphoproliferative diseases [RR=2.35 (95% CI 1.36-4.05), (16 studies, 3090 patients)]. However, the rate of AML/MDS was not increased in patients treated for PCD or solid malignancies [RR=1.41 (95% CI 0.28-7.08), 3 studies, 304 patients and RR=1.24 (95% CI 0.72-2.15), 14 studies, 5384 pts, respectively], Figure. Subgroup analysis based on the comparator's intensity- showed a higher rate of MDS/AML merely in patients given HCT compared to observation and to low intensity [RR=4.86 (95% CI 1.43-16.47), (5 studies, 732 patients) and RR=2.09 (95% CI 1.30-3.35), (19 studies, 6036 patients)]. Overall, there was no difference in the rate of secondary solid malignancies between patients given HCT and patients given other treatments [RR=0.95 (95% CI 0.67-1.32), (19 studies, 5925 patients)], with similar results in subgroup analysis according to the baseline diseases. Conclusions The rate of secondary MDS/AML is higher in patients given high dose therapy and autologous HCT compared to other treatment options, with statistically significant higher rates only in the subgroup of patients with lymphoproliferative diseases. The rate of secondary solid malignancies is similar among all patients given either high dose therapy or alternative therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4917 Differential downregulation of telomerase activity by bortezomib in multiple myeloma cells- regulatory pathways and clinical implications C. Weiss1, O Uziel2, O. Wolach2, S. Bulvick1, and M. Lahav2 1Laniado Medical Center, Netania; 2Felsenstein Medical Research Center, Rabin Medical Center, Sackler School of Medicine, Tel-Aviv University, Israel. Background The proteasome inhibitor bortezomib is an effective drug in Multiple Myeloma (MM). However, its exact mechanism of action is not yet fully understood. The importance of telomerase in the biology and prognosis MM is well established, but its response to bortezomib has not been assessed yet. In light of common signaling pathways of connecting telomerase regulation and bortezomib known mechanisms of action, we surmised that the drug may affect the activity of telomerase in MM cells. Results Bortezomib downregulated telomerase activity in all MM cell lines. However, the kinetics of this downregulation varied between the two cell lines examined. Whereas in ARP-1 cells telomerase activity decreased 24 hours after bortezomib treatment, in CAG cells the effect was observed only after 48 hours. These finding imply different regulatory mechanisms between these lines. In both lines bortezomib transcriptionally downregulated telomerase activity by inhibiting hTERT expression. ChIP (Chromatin Immune Precipitation) analysis showed that SP-1 and C-Myc transcription factors mediate this transcriptional downregulation of telomerase. Interestingly, the binding of NFkB to hTERT promoter is enhanced in response to the drug, consistent with a recent study reporting upregulation of NFkB by bortezomib. We are currently looking in depth at the pathways leading to these phenomena. However, the two lines differed in the post translational modification of AKT, which phosphorylates telomerase post-translationally. Whereas in ARP-1 cells the phosphorylated form of AKT is downregulated in response to bortezomib, the drug did not affect AKT phosphorylation in CAG cells. These findings explain the different kinetics of telomerase downregulation and are in keeping with previous data showing different AKT response between these two cell lines. Conclusions and future studies Our results shed light on the effects and mechanism of bortezomib on telomerase activity in MM. Te differential response of pAKT pathway is in keeping with recent studies suggesting differential dependency of MM cells on pAKT. Telomerase inhibition by bortezomib may be of varying efficacy in subsets of MM patients in relation to their pAKT dependency. A correlation may also be found between the effect of bortezomib on telomerase activity and resistance to bortezomib in MM. As such, the ex vivo assessment of telomerase activity response to treatment may serve as a prognostic feature and add to therapeutic armamentarium by addition of telomerase inhibitors in defined subsets of patients. Disclosures No relevant conflicts of interest to declare.