ALBERT

Description: Biologic factors that predict the survival of patients with a diffuse large B-cell lymphoma, such as cell of origin and stromal signatures, have been discovered by gene expression profiling. We attempted to simulate these gene expression profiling findings and create a new biologic prognostic model based on immunohistochemistry. We studied 199 patients (125 in the training set, 74 in the validation set) with de novo diffuse large B-cell lymphoma treated with rituximab and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or CHOP-like therapies, and immunohistochemical stains were performed on paraffin-embedded tissue microarrays. In the model, 1 point was awarded for each adverse prognostic factor: nongerminal center B cell–like subtype, SPARC (secreted protein, acidic, and rich in cysteine) 〈 5%, and microvascular density quartile 4. The model using these 3 biologic markers was highly predictive of overall survival and event-free survival in multivariate analysis after adjusting for the International Prognostic Index in both the training and validation sets. This new model delineates 2 groups of patients, 1 with a low biologic score (0-1) and good survival and the other with a high score (2-3) and poor survival. This new biologic prognostic model could be used with the International Prognostic Index to stratify patients for novel or risk-adapted therapies.

Description: Abstract 2005 Background: Concurrent translocations in MYC and BCL2 determined by fluorescence in-situ hybridization (FISH), have been associated with a poor outcome in diffuse large B cell lymphoma (DLBCL) patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). However, unlike immunohistochemistry (IHC), FISH is expensive and is not routinely available in all clinical laboratories. The aim of this study was to determine if MYC protein expression or expression of the proliferation marker Ki-67 by IHC could be used to identify samples that harbour MYC translocations. Methods: DLBCL samples, diagnosed by an expert panel of hematopathologists according to the WHO criteria of 2008 and derived from 167 patients treated with R-CHOP, have been subjected to gene expression profiling (GEP) and FISH using commercial break-apart probes for BCL2 and MYC. MYC mRNA expression was determined using log2 normalized expression values of probe set 202431_s_at. The presence of MYC, BCL2 and Ki-67 protein expression was determined by IHC using commercially available antibodies (clone Y69 (Epitomics), clone 124 (Dako) and clone Ki-67 (Dako) respectively). MYC protein is not normally expressed in germinal centers (tonsils, negative control), thus the % of tumour cells staining for MYC was noted in each case. Thresholds for BCL2 protein (50%) and MYC mRNA (〉9.4) were determined using the statistical software X-tile which determines the optimal threshold based on its association with clinical outcome. For MYC protein expression, no threshold was significant by X-tile thus a 40% threshold was used based on the bimodal distribution of the data, with a through occurring at 40%. Protein expression for MYC and Ki-67 was correlated to the presence of MYC translocations, MYC mRNA expression and outcome including overall survival (OS) and progression free survival (PFS). Results: Over-expression of MYC was present in 56/167 (34%) of DLBCL samples (18/167 translocations, 19/167 high mRNA and 47/167 high protein expression) and was not specific to the germinal center B cell (GCB) or activated B cell (ABC) molecular subtype. MYC protein expression (in ≥40% of cells) captured 13/18 MYC translocations and in 4/5 remaining cases, MYC staining was present in 20–39% of cells. MYC protein expression, alone, was not associated with OS but the presence of a MYC translocation was associated with an inferior OS in the ABC subtype only. BCL2 protein expression was also associated with an inferior OS in this cohort (p=0.002). Concurrent expression of MYC and BCL2 protein by IHC was associated with a markedly inferior OS compared to MYC protein- or MYC protein+/BCL2 protein- (median OS of 2 years, 7 years and 〉 7 years respectively, p90%) identified only 5/18 cases with MYC translocations and was not associated with outcome alone or in combination with BCL2. Conclusions: MYC deregulation in DLBCL is more common than previously reported (34%) and can occur in the absence of a MYC translocation. MYC and BCL2 protein expression could be easily determined by routine IHC in most clinical laboratories and should be prospectively tested as potential predictive biomarkers in DLBCL patients treated with R-CHOP. Disclosures: Grogan: Ventana Roche: Employment, Equity Ownership.

Description: T-cell prolymphocytic leukemia (T-PLL) is an aggressive lymphoproliferative disorder with postthymic T cell phenotype and prolymphocytic morphology. In the majority of patients, the leukemic process progresses rapidly and patients die shortly after diagnosis (median survival of 7 months). Bortezomib, the first proteasome inhibitor to be approved for use in haematological malignancies such as multiple myeloma, is beginning to be utilized as an effective anti-neoplastic agent in other hematopoietic and non-hematopoietic neoplastic disorders. We report here the in vitro apoptotic effects of bortezomib on leukemic cells isolated from three T-PLL patients. Interestingly, one of the patient’s leukemia developed in the setting of immunosupression due to transplant therapy (post-transplant lymphoproliferative disorder). Flow cytometric analysis of leukemic cells of the three patients showed CD8, double CD4+CD8+ and double CD4−CD8− immunophenotypic features. All cases showed monoclonal band pattern by T-cell receptor (TCR) gene rearrangement as analyzed by the PCR amplification of the TCR gamma heavy chain gene. Freshly isolated leukemic cells with the CD8 phenotype T-PLL analyzed for apoptosis after ficoll hypaque separation and cultured in the presence of various concentration of Bortezomib (0.001, 0.01, 0.1, 1, and 10 uM) for dose curve analysis. Apoptosis of the leukemic cells was determined by Annexin-V and 7-AAD staining and flow cytometric analysis after incubation at 24 and 72 hours, respectively. Samples treated for 72 hours showed higher rate of apoptosis compared to 24 hours: 10 uM (62% increase above the base line of control cells), 1 uM (58%), 0.1 uM (55%), 0.01 uM (40%) and 0.001 uM (0%) concentrations while samples treated for 24 hours with 10 uM showed (42% increase above the base line of control cells) and 1 uM (33% increase above the base line). Light microscopic analysis of leukemic cells treated with Bortezomib confirmed that the majority of cells undergo apoptosis with Bortezomib treatment as it revealed nuclear fragmentation and apoptotic bodies. Leukemic cells recovered from cryopreservation from the second and third T-PLL patient samples analyzed also showed significant increase in early and late apoptosis at 24 hours with Bortezomib treatment (10nm). These results suggest that Bortezomib may provide an alternate therapy in the treatment of T-PLL. Future collaborative efforts investigating efficacy with Bortezomib as a single agent or in combination with other therapeutic agents will be crucial to improving survival for patients with this disease.