ALBERT

Description: Imatinib mesylate (Glivec; Novartis, Basel, Switzerland), which is a selective inhibitor of the Bcr/Abl tyrosine kinase, is now widely used for the treatment of Ph-positive leukemia based on the remarkable efficacy. Treatment with imatinib mesylate is generally well tolerated, and the risk for severe adverse effects is low. Hepatic toxicity is less common and usually resolves with interruption of imatinib therapy. On the other hand, although it is well known that HBV reactivations are observed in cancer patients with chronic HBV infection during chemotherapies, imatinib mesylate-induced HBV reactivation has not been reported yet. Here, we report the first case complicated by fatal fulminant HBV reactivation during imatinib mesylate treatment for CML. A 54-year-old man was diagnosed as CML on October 2003. At that time, HBsAg, HBeAb, and HBcAb were positive, whereas HBsAb was negative. HCV Ab and HCV RNA were also positive. However, hepatic examinations revealed normal findings, except slightly elevated levels of AST (50 IU/L). On November 2003, imatinib mesylate was started at the dose of 400mg/day p.o. and continued without any hepatic adverse effects. In contrast, the patient was suffered from neutropenia (grade 2) and lymphocytopenia (grade 2) on December 2003. Although dose of imatinib mesylate was reduced (300mg/day), lymphocytopenia continued. Since May 6, 2004, the patient complained general fatigue. On May 11, AST, ALT, and total bilirubin were 125 IU/L, 95 IU/L, and 0.7 mg/dl, respectively. At that time, bone marrow cytogenetics using a FISH of Bcr/Abl detected 2.0% fusion gene. Then, hepatic function deteriorated despite imatinib mesylate administration was stopped. On May 27, the patient had severe hepatic dysfunction with high AST, ALT, and total bilirubin (2098 IU/L, 1574 IU/L, and 5.6 mg/dl, respectively). In addition, prolonged prothrombin time (13.6%) and deterioration of consciousness were observed. HBV DNA polymerase was extremely increased (〉 20000 cpm; normal range:

Description: Abstract 1534 + equal contributions Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate and infrequent somatic mutations of TP53 and genes encoding cell cycle pathway components. Despite recent advances in the molecular understanding of DLBCL pathogenesis, clinical models such as the International Prognostic Index (IPI) are still used to identify high-risk patients. By integrating high-resolution high-density SNP array data with transcriptional profiles and performing pathway analysis in 180 primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary DLBCLs with single copy loss of 17p13.1 (TP53/RPL26/KDM6B) often had CNAs of an additional p53 modifier – 9p21.3 (CDKN2A/ARF), 19q13.42 (BCL2L12), 12q15 (MDM2) or 1q23.3 (MDM4/RFWD2). CNAs of the respective p53 modifiers, CDKN2A (ARF, 9p21.3), MDM2 (12q15) and MDM4/RFWD2 (1q23.3), occurred in largely separate groups of tumors. DLBCLs with CNAs of p53 pathway members frequently exhibited concurrent alterations of additional cell cycle components such as CCND3 (6p21.32), CDK6 (7q22.1), CDK2/CDK4 (12q15) and/or RB1 (13q14.2) or RBL2 (16q12.2). When the primary DLBCLs were clustered in the space of CNAs that perturb p53 pathway and cell cycle components, 66% of tumors had multiple alterations (“complex” pattern) whereas the remaining 34% of tumors lacked these lesions (“clean” signature). Tumors with “complex” alterations of p53 pathway and cell cycle components also had more total CNAs and more frequent TP53 mutations, highlighting the association between p53 deficiency, cell cycle deregulation and increased genomic instability in DLBCL. The patterns of “complex” vs. “clean” CNAs of p53 pathway and cell cycle components and the association between the “complex” signature and total CNAs were confirmed in an independent series of primary DLBCLs. To further characterize DLBCLs with “complex” vs. “clean” CNA patterns, we performed gene set enrichment analyses with publicly available series of p53 target genes and a RB-deficiency gene set which included multiple E2F targets. The p53 target transcripts were significantly less abundant in “complex” DLBCLs, directly linking their genetic signature of p53 deficiency with decreased p53 activity. Furthermore, the RB-deficiency gene set was significantly enriched in “complex” DLBCLs suggesting that these tumors had increased E2F-mediated cell cycle progression. Consistent with these observations, DLBCLs with “complex” CNA patterns also had significantly higher proliferation indices as determined by Ki67 immunostaining. We next assessed the prognostic significance of the “complex” CNA pattern in the subset of patients who were treated with R-CHOP (rituxan, cyclophosphamide, adriamycin, oncovin, prednisone) and had long-term follow-up. R-CHOP treated patients with “complex” CNA patterns had a 5-year overall survival of only 62%; in contrast, those with “clean” CNA signatures had an 100% overall survival (p =.001). The association between CN complexity and outcome was independent of transcriptionally defined categories and additive to the clinical IPI risk model. Taken together, these data provide a structural basis for deregulated cell cycle, increased cellular proliferation and unfavorable outcome in DLBCL and suggest targeted treatment strategies. Disclosures: No relevant conflicts of interest to declare.

Description: Posttransplant lymphoproliferative disorders (PTLDs) are potentially fatal, EBV-driven B-cell malignancies that develop in immunocompromised solid organ or hematopoietic stem cell recipients. In PTLD, the expression of EBV proteins, including latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance foster the proliferation of EBV-transformed B cells. Current PTLD treatment strategies include reduction of immunosuppression, which increases the risk of graft rejection, anti-CD20 treatment, combination chemotherapy, and administration of EBV-specific cytotoxic T cells. In the present study, we report that EBV-transformed lymphoblastoid B-cell lines (LCLs) and primary PTLDs overexpress galectin-1 (Gal1), a carbohydrate-binding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4+ Th1 and Th17 cells and cytotoxic T cells. In transcriptional reporter assays, LMP2A and LMP1 each increased Gal1-driven luciferase expression, and the combination of LMP2A and LMP1 was additive. In addition, small interfering RNA (siRNA)–mediated depletion of LMP2A decreased Gal1 protein abundance in EBV-transformed LCLs. Gal1 expression in LCLs was dependent on both activating protein 1 (AP-1) and PI3K. A newly developed neutralizing Gal1 mAb selectively inhibited Gal1-mediated apoptosis of EBV-specific CD8+ T cells. Given the tolerogenic and immunosuppressive function of Gal1, antibody-mediated Gal1 neutralization may represent a novel immunotherapeutic strategy for PTLD and other Gal1-expressing tumors.

Description: The strength and duration of B-cell–receptor (BCR) signaling depends upon the balance between protein tyrosine kinase (PTK) activation and protein tyrosine phosphatase (PTP) inhibition. BCR-dependent activation of the SYK PTK initiates downstream signaling events and amplifies the original BCR signal. Although BCR-associated SYK phosphorylation is clearly regulated by PTPs, SYK has not been identified as a direct PTP substrate. Herein, we demonstrate that SYK is a major substrate of a tissue-specific and developmentally regulated PTP, PTP receptor–type O truncated (PTPROt). PTPROt is a member of the PTPRO family (also designated GLEPP, PTP-Ø, PTP-oc, and PTPu2), a group of highly conserved receptor-type PTPs that are thought to function as tumor suppressor genes. The overexpression of PTPROt inhibited BCR-triggered SYK tyrosyl phosphorylation, activation of the associated adaptor proteins SHC and BLNK, and downstream signaling events, including calcium mobilization and mitogen-activated protein kinase/extracellular signal–regulated kinase (MAPK/ERK) activation. PTPROt overexpression also inhibited lymphoma cell proliferation and induced apoptosis in the absence of BCR cross-linking, suggesting that the phosphatase modulates tonic BCR signaling.

Description: Translocations of the mixed lineage leukemia (MLL) gene on chromosome 11q23 are found in over 70% of infant leukemias and associated with a poor prognosis. MLL translocations generate a new chimeric gene, in which N-terminal portion of MLL is fused to C-terminal sequence from multiple different partners. Given the variety of MLL fusion partners, molecular techniques such as FISH or Southern blotting are not able to detect all genetic abnormalities involving MLL. In addition, results from these time-consuming techniques are not always available at initial diagnosis. To identify molecular markers of MLL translocation that might be used in rapid diagnostic assays, we first compared the transcriptional profiles of primary precursor B cell acute lymphoblastic leukemias (ALLs) with and without MLL translocations. Galectin-1 (Gal1) transcripts were significantly more abundant in MLL-rearranged ALLs (MLL-ALL) from two extensive and independent datasets. Consistent with these findings, Gal1 protein was significantly more abundant in MLL-ALL cell lines than in non-MLL ALL lines by western blotting. These observations were of interest because Gal1 is a secreted immunoregulatory glycan-binding protein that induces the apoptosis of cytotoxic T cells and T helper1 cells and fosters an immunosuppressive tumor microenvironment. To assess the diagnostic utility of Gal1 expression in identifying the MLL-ALL subtype, we performed Gal1 immunostaining on a large series of primary ALLs with known MLL status. All 10 MLL-rearranged precursor-B ALLs had abundant Gal1 expression (10/10, 100%); in marked contrast, only 1 of 38 pre-B ALLs without a cytogenetically detectable MLL translocation expressed Gal1 (3%), p

Description: Abstract 3087 Poster Board III-24 Objective To evaluate the efficacy and safety profile of an intensive induction and post-remission Cx for untreated patients (pts) with adult ALL and LBL, focusing on the long-term follow-up results. Feasibility and efficacy of autologous or allogeneic SCT were also assessed. Patients and Methods We enrolled pts with untreated adult ALL or LBL aged 15 to 69 years. The Cx regimen consisted of induction Cx (vincristine [VCR], cyclophosphamide [CPA], prednisone [PSL], doxorubicin [DXR], L-asparaginase plus intrathecal methotrexate [IT-MTX]) followed by two consolidation Cx regimens (Consolidation A, consisting of daunorubicin, cytosine arabinoside [Ara-C], vindesine [VDS], PSL plus IT-MTX; Consolidation B, consisting of CPA, Ara-C, 6-mercaptopurine [6-MP], VCR plus IT-MTX) with the prophylactic use of granulocyte colony-stimulating factor. Thereafter, interim maintenance Cx including 6-MP and MTX concurrent with CNS prophylaxis (IT-MTX), intensification Cx (VCR, DXR, PSL, CPA, Ara-C and 6-MP) and maintenance Cx (VDS, CPM, PSL, DXR, MTX and 6-MP) were sequentially performed for two years. For pts with 50 years or younger who achieved complete remission (CR) after induction Cx, allogeneic SCT for ALL from HLA-matched related donor and autologous SCT for LBL were to be considered. Primary endpoint was 5-year progression-free survival (PFS). Secondary endpoints included CR rate (%CR), overall survival (OS) and adverse events. Results Among 115 pts who were enrolled between 1994 and 1999, 108 eligible pts (median age, 33.5 years [15-69]) including 96 ALL and 12 LBL pts who received induction Cx were assessed. Seven pts were judged ineligible, including four histologically ineligible pts revealed by institutional or central review. Other major characteristics of the 108 eligible pts were as follows: 54 males (50%); T-cell phenotype, 23 pts (21%); Ph, 24 pts (22%); t(4;11), 2 pts (2%); B-symptom+, 38 pts (35%); PS 2/3, 24 pts (22%). Eighty-seven pts achieved CR (%CR 81%; 95% CI, 72-88%), while five patients (5%) died during induction Cx mainly due to infections. The median OS and the median PFS of the 108 eligible pts were 1.8 years (95% CI, 1.5-2.6 years) and 1.2 years (95% CI, 0.8-1.6 years), respectively. Their 5-year OS and 5-year PFS were 29% and 28%, respectively, with the median follow-up of the censored cases of 9.3 years (range, 2.0-12.3 years). The 5-year OS from the date of SCT of 31 pts who underwent allogeneic (n=19) or autologous (n=12) SCT during 1st CR was 51% (95% CI, 32-67%). Major non-hematologic toxicities of grade 3 or greater included infections (n=24, 21%), pulmonary complications (n=7, 6%) and diarrhea (n=7, 6%). As compared with the investigators' previous phase II trials for ALL and LBL, JCOG9402 improved PFS and OS as compared with JCOG8702 (MST, 1.2 years; 7-year OS, 15%; 7-year PFS, 13%) (Jpn J Clin Oncol 1999;29:340), but did not show improvement as compared with JCOG9004 (MST, 2.2 years; 5-year OS, 32%; 5-year PFS, 26%) (Cancer Sci 2007;98:1350). Conclusion Although the intensified induction and post-remission Cx was feasible and 28% of pts achieved long-term PFS, JCOG9402 failed to show improvement in long-term follow-up results as compared with the investigators' latest historical control. To further improve the therapeutic outcomes of adult pts with ALL and LBL, novel strategies are warranted. Disclosures No relevant conflicts of interest to declare.

Description: Cancer cells require blocks in apoptosis to survive despite many abnormalities that should trigger apoptosis. Here we demonstrate that BH3 profiling is an effective tool for the systematic investigation of the types of blocks in apoptosis for which cancer cells select. BH3 profiling can segregate lymphoma cell lines into three different classes based on the type of apoptotic block each exhibits. In one class, cells are kept alive via expression of anti-apoptotic proteins of the BCL-2 family. When BCL-2 itself is detected by BH3 profiling as the protein keeping the cell alive, the cell is sensitive to ABT-737, a BCL-2 antagonist compound. Sensitivity to ABT-737 correlates highly with abundance of pro-apoptotic BIM complexed to BCL-2. Knockdown of BIM reduces sensitivity to ABT-737. We describe such cancer cells that maintain survival based on tonic sequestration of large amounts of pro-death molecules by anti-apoptotic proteins like BCL-2 as “primed for death”. “Primed” lymphoma cell lines are more sensitive than “unprimed” cancer cells to conventional agents killing via apoptosis like vincristine, adriamycin, and etoposide. It is important to note that, in general, non-malignant cells are unlikely to be “primed”. Hence “priming” may be an important determinant of the therapeutic window between normal and malignant cells. These results suggest that the degree to which a cell is “primed” can influence sensitivity to chemotherapy agents targeting distinct cellular targets. Therefore, BH3 profiling is a tool potentially useful not only for predicting sensitivity to agents targeting antiapoptotic BCL-2 proteins, but also to conventional cytotoxic agents that use the mitochondrial apoptotic pathway to kill.

Description: Abstract 746 Classical Hodgkin Lymphoma (cHL) Reed-Sternberg (RS) cells have crippling mutations of their rearranged Ig genes and lack B-cell receptor-mediated survival signals. As a consequence, cHLs rely on alternative survival and proliferation pathways including AP-1. Although cHL RS cells express high levels of the AP-1 components, cJun and JunB, and depend on AP-1-mediated proliferation signals, the mechanism of constitutive AP-1 activation in cHL remains undefined. In a screen for genetic abnormalities in cHL, we integrated microRNA (miR) expression profiles with high resolution copy number data and identified single copy loss of the miR-15a/16-1 locus (chromosome 13q14) and decreased miR-15a/16 expression in 75% of examined HL cell lines. We isolated primary Hodgkin RS cells using laser-capture microdissection and confirmed frequent single copy loss of the miR-15a/16-1 locus by quantitative PCR and associated decreased miR-15a/16 abundance by RT-PCR in 10/23 (43%) of primary tumors. Using retroviral vectors containing miR-15a and miR-16, we reconstituted miR-15a/16 expression in HL cell lines with single copy loss of chromosome 13q14. In these HL lines, miR-15a/16 reconstitution was associated with cell cycle arrest, significantly decreased cellular proliferation and induction of apoptosis (88% apoptotic cells at 48 hours). In addition, miR-15a/16 reconstitution resulted in significant downregulation of multiple survival pathway components and known miR-15a/16 targets including panAKT, BCL2 and cyclin D1. Using a stand-alone miRanda algorithm with direct sequence entry, we also identified a candidate miR-15a/16 binding site in the AP-1 component, cJun. In multiple HL cell lines, miR-15a/16 reconstitution decreased the expression of cJun and additional known AP-1 targets such as galectin-1. Therefore, miR-15a/16-1 deletion is a frequent genetic abnormality in HL cell lines and primary tumors, a candidate mechanism of AP-1 activation in HL and a modulator of multiple critical survival pathways in this disease. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 635 Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease with infrequent alterations of multiple apoptotic, developmental and signaling pathways. Inactivating somatic mutations of the p53 tumor suppressor are uncommon in DLBCL, prompting speculation regarding alternative mechanisms of modulating p53 activity in this disease. As part of a comprehensive analysis of genetic alterations in DLBCL, we recently integrated high-resolution copy number data and transcription profiles in a series of 81 newly diagnosed previously untreated DLBCLs. Copy number (CN) alterations were evaluated using Affymetrix SNP Array 6.0 data on over 1.8 million genomic loci (906,000 SNP probe sets and 946,000 copy number probes) and a data-processing pipeline incorporating the normalization, calibration, and segmentation of the CN profiles and their analysis by the GISTIC algorithm. The genes within the peak and region of each GISTIC-identified alteration were tested for association with the corresponding transcripts' expression and significance values were corrected for multiple hypothesis testing by the false discovery rate (FDR) procedure, with the correction accounting for both the multiple genes within each peak and the multiple peaks. The signature for a given copy number alteration was then defined as the set of within-peak and -region transcripts with FDR q-values 〈 0.25. The alteration signatures thus defined were then tested for pathway/gene set enrichment based on the hypergeometric distribution, using a compendium of gene sets from the MSigDB repository (Broad Institute). This analysis highlighted a genome-wide pattern whereby: i) individual pathways are targeted by multiple alterations at different loci (with different genes of the same pathway located within distinct CN alteration peaks or regions); and ii) members of multiple pathways are targeted by the same alteration (with genes co-located within a single CN alteration belonging to different pathways). The most highly significant enriched pathways (FDR 〈 .05) included apoptotic, p53 signaling, and ARF pathways and identified multiple modulators and effectors of normal p53 activity. These include: deletion of the MDM2-inhibitor, ARF; amplification of the p53 E3 ligases, MDM2 and COP1; deletion of p53 itself; deletion of the ribosomal protein that enhances p53 translation following DNA damage, RPL26; and deletion of p53 effectors including BNIP3L and DFFB. Of note, the deletion of 17p13.1 encompasses both p53 and its positive modulator, RPL26. In all cases, the observed alterations in p53 pathway components would decrease functionally active p53 protein and p53 apoptotic effectors in primary DLBCLs. Taken together, these data identify alternative genetic mechanisms for reducing p53 activity in DLBCL and highlight the value of an integrated comprehensive analysis of genetic alterations in this disease. Disclosures: No relevant conflicts of interest to declare.