ALBERT

Description: Abstract 1243 Poster Board I-265 Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and is clinically very heterogeneous. Following diagnosis, some patients do not require treatment for several years whereas others have a more aggressive disease thus requiring immediate treatment. Understanding the molecular basis of clinical heterogeneity in CLL will enhance our ability to treat this presently incurable disease effectively. CLL cells in the patient body proliferate/survive for a long time resulting in their accumulation in bone marrow (BM), lymph nodes (LN), and blood (PB). However, CLL cells do not survive for a long time once they are removed from the body, suggesting that an in vivo microenvironment provides essential proliferation/survival signals to CLL cells. Therefore, to elucidate the precise role of microenvironments on the CLL cell proliferation/survival and migration, in this study, we have analyzed CLL cells from PB (n=20), BM (n=18), and LN (n=15) from patients for their gene expression profiles using microarray. Differentially expressed genes and their associated cellular pathways were identified using significant analysis of microarray (SAM) and gene set enrichment analyses (GSEA). Among the six pathways/gene expression signatures identified (BCR-, BAFF/April-, NFκB-, PI3K/Akt, cytokine-, and tolerogenic) the most significant pathways in CLL biology are the BCR-, NFκB-, PI3K/Akt pathways and tolerogenic signature associated genes with immune dysregulation particularly with CLL cells from LN. We have already reported the differential expression of CLL cell proliferation and survival related genes belonging to BCR and NFαB pathways (Mittal et al, 2008 Blood-ASH Annual Meeting Abstract 546, page 112). In this report we have focused on differentially expressed genes associated with the tolerogenic signature and PI3K/Akt pathway. Among the eighty-three differentially expressed genes in the tolerogenic signature, based on their known role in immune regulation and/or level of significant expression comparing CLL cells from PB or BM, a few selected genes were further studied to understand their possible role in clinical heterogeneity of CLL. These genes are: CAV1, CD47, CCNB2, IL2Rαa, FOXP3, ZWINT, TGFβR1, IL22, IL10Rαa, INDO, APC, and STAT1. There was a significant increase in the expression of CD47, IL-10Rαa, CAV1, APC, CCNB2, and STAT1 in the LN cells from CLL patients; whereas the expression of IL-22R was decreased in the LN cells. These genes have been shown to be associated with immunosuppression indicating a lack of immune response against CLL in the lymph node. In addition, MAPK pathway associated genes are known to increase the survival/proliferation of tumor cells, including CLL cells. Specifically, genes associated with PI3K/Akt pathway, a part of MAPK pathways are also overexpressed in CLL cells from LN that includes AKT, 4E-BP1, PSMC4 and PDK1 genes indicating the importance of PI3K/Akt pathway in proliferation/survival of CLL cells in LN microenvironment. Based on these results, we hypothesize that differentially expressed genes belonging to the tolerogenic signature in LN of CLL patients down regulate the immune response against CLL, thus leading to enhanced disease progression whereas, overexpressed proliferation/survival-related genes belong to PI3K/Akt pathway promote proliferation/survival of CLL cells. (This work was supported by the CLL Foundation, Houston, TX and National Institutes of Health, Bethesda, MD, INBRE Grant # P20 RR016469). Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1781 Chronic Lymphocytic Leukemia (CLL), the most prevalent adult B-cell malignancy in western countries, is a highly heterogeneous with a very variable clinical outcome. Emerging evidence indicates that the stromal tumor microenvironment (STME) play important roles in the pathogenesis of CLL. However, the precise mechanism and molecules of STME involved in this process remain unknown. In an attempt to explore the role of STME in this process, we examined the expression levels of stromal associated genes using gene expression profiling (GEP) of CLL cells from 53 patients’ lymph node (LN) (n=15), bone marrow (BM) (n =18), and peripheral blood (PB) (n=20). Using significant analyses of microarray (SAM), gene set enrichment analyses (GSEA), and ingenuity pathway analyses (IPA), among the major pathways associated with the differentially expressed genes, a cytoskeleton genes associated with stromal signatures are the focus of this report. Of these molecules, a significant number of molecules including: LUM, MMP9, MYLK, ITGA9, CAV1, CAV2, FBN1, PARVA, CALD1, ITGB5 and EHD2 were overexpressed and ITGB2, DLC1 and ITGA6 are under expressed in LN-CLL compared to BM-CLL and PB-CLL indicating a role of LN-mediated TME in CLL cell survival/progression. Among these genes, expression of myosin light chain kinase (MYLK), caveolin 1 (CAV1) and caveolin 2 (CAV2) correlated with clinical outcome (see adjacent Figure) as determined by time to treatment. We recently reported the role of a CAV1 in LN microenvironment-induced immune tolerance in CLL and possibility of their involvement in CLL cytoskeleton (Gilling et al, 2012). In the present study we report aberrant expression of other cytoskeleton genes such as MYLK and CAV2 are involved in the regulation of CLL cell survival in the stromal microenvironment affecting other members of the cytoskeletal signature via actin cytoskeleton signaling, integrin signaling and Pak signaling. In addition, MYLK and CAV2 are also involved in regulation of CLL proliferation. Together our studies show that members of the stromal signature particularly in the CLL cells from lymph nodes regulate the CLL cell survival and proliferation and thus leukemic progression. Figure: Association of MYLK expression with time to first treatment Figure:. Association of MYLK expression with time to first treatment Disclosures: No relevant conflicts of interest to declare.

Description: B-cell chronic lymphocytic leukemia (CLL) is a heterogenous and incurable B-cell malignancy. CLL cells migrate and accumulate in different sites including the peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) in vivo, but undergo apoptosis in vitro. Therefore, we hypothesized that CLL cells at these sites are different and receive different microenvironmental signals that regulate their proliferation/survival and migration. Most reports on the microenvironmental influence on CLL cells have used in vitro models consisting of stromal and CLL cells. However, in this study, to better understand the influence of site-specific microenvironments in vivo, gene expression patterns of CLL cells obtained from PB, BM and LN were investigated. CLL cells were isolated from patients’ PB (PB-CLL, n= 20), BM (BM-CLL, n=14) and LN (LN-CLL, n=15) and used to determine the gene expression patterns by microarray analysis. In addition, we also included PB-CLL cases from our previous study (n=40) to further validate the findings of this study. Significant Analyses of Microarray (SAM) revealed differential expression of more than 500 genes among these three sites. To understand the potential roles of these differentially-expressed genes and their association with relevant functional pathways in CLL, Gene Set Enrichment Analysis (GSEA) was performed. The validation of pathway specific genes was further confirmed by quantitative real time PCR. Among the pathways identified, the most active pathways associated with the migration and proliferation/survival of CLL cells, namely chemokine-signaling, BCR signaling, BAFF/APRIL-signaling, and NFκB-signaling pathways, were selected for further analyses. We hypothesized that chemokines and their receptors mediate the migration of CLL cells between PB and LN or BM, and that molecules of the BCR, BAFF/APRIL and NFκB pathways regulate proliferation/survival. To determine the role of chemokines and their receptors in CLL cell migration, we studied the expression of 52 chemokine/chemokine receptors and found that PB-CLL cells significantly (p

Description: Diffuse Large B Cell Lymphoma (DLBCL) is an aggressive form of Non-Hodgkin’s Lymphoma (NHL) accounting for 40% of NHL in the United States. Current treatment options are ineffective in eliminating refractory lymphoma, which is responsible for patient relapse. Therefore, a carefully developed immunotherapeutic strategy to specifically target refractory lymphoma might be useful in improving therapy. In this study, anti-lymphoma immune outcome was evaluated in a syngeneic murine model using the aggressive and highly liver metastatic RAW117-H10 lymphoma cell line that expresses the murine leukemia virus protein gp70. Anti-lymphoma immunity was generated by immunizing Balb/c mice with two peptide-based vaccines in which a T cell epitope from gp70 (SSWDFITV) was covalently attached to the N-termini of two response-selective molecular adjuvants derived from complement component C5a: YSFKPMPLaR (EP54) and YSFKDMP(MeL)aR (EP67). In both vaccines, a protease-sensitive, double-Arg linker sequence (RR) was placed between the epitope and the EP54/EP67 molecular adjuvants: SSWDFITV-RR-YSFKPMPLaR (Vaccine 2) and SSWDFITV-RR-YSFKDMP(MeL)aR (Vaccine 3). To determine the protective effect of the EP54/EP67-containing vaccines against lymphoma growth, separate groups of Balb/c mice were administered 200 μg of Vaccine 2 or Vaccine 3 dissolved in water via intraperitoneal (100 μg) and intradermal (100 μg) injections. Following 4 weekly immunizations, the mice were intravenously transplanted with 5 × 103 RAW117-H10 cells to ascertain the protective benefit of the vaccines. The results showed Vaccines 2 and 3 providing complete protective benefit, with pre-vaccinated mice surviving greater than 235 days, whereas all control, unvaccinated animals died within 18 days of tumor inoculation. In addition to survival analysis, histopathological analysis was performed using liver sections from mice upon death, or after sacrifice following 60 days of survival, to evaluate tumor burden in the mice (See Figure 1). No tumor growth was found in livers taken from mice pre-vaccinated with Vaccines 2 and 3. Furthermore, to elucidate the mechanisms of vaccine-mediated protection against lymphoma, the frequency and functions of the immune effector cells from blood and spleens were analyzed at 72 hours following the second and fourth vaccinations using flow cytometry, T and B cell-specific mitogen proliferation assays, and lymphoma-specific cytotoxicity assays in vitro. The results of these analyses showed a significant increase in the frequency (p〈 0.01) of helper and cytotoxic T cells, proliferation of both T and B cells (p〈 0.05), and significant lymphoma-specific cytotoxicity (p〈 0.001) compared to non-vaccinated mice. Therefore, these results demonstrate a pronounced protective effect of Vaccines 2 and 3 against lymphoma growth in mice. These results lay the foundation for further studies in testing the therapeutic efficacy of Vaccines 2 and 3 in mice pre-inoculated with lymphoma, as well as in initiating studies employing human lymphomas. In so doing, the principles underlying these molecular adjuvant-containing vaccines could eventually be translated from the mouse model to a clinical setting. Figure 1: Hematoxylin and Eosin staining of liver sections taken from control and vaccinated mice inoculated with RAW117-H10 lymphoma. Figure 1:. Hematoxylin and Eosin staining of liver sections taken from control and vaccinated mice inoculated with RAW117-H10 lymphoma.

Description: Abstract 3866 Chronic Lymphocytic Leukemia (CLL) is a monoclonal B-cell disorder with accumulation of leukemic cells in peripheral blood, bone marrow and lymphoid organs. It presents with a heterogeneous clinical course. Many patients survive long periods of time without any need for treatment, whereas other patients show resistance to treatment or relapse soon after administration of therapy. Although some prognostic markers such as mutational status of immunoglobulin variable heavy chain, chromosomal abnormalities, CD38 levels, or ZAP-70 expression may help predict at initial diagnosis which patients will have more aggressive disease, the exact factors that can determine chances of remission in CLL are still not clear, making treatment challenging. Furthermore, CLL remains an incurable disease, necessitating a way for controlling its progression. Identifying novel molecular signatures associated with refractory CLL disease may help devise targeted treatment strategies and thus may prolong survival times and prevent the progression of CLL in relapsed patients. Considering this, we performed gene expression profiling (GEP) on peripheral blood (PB), bone marrow (BM) and lymph node (LN) samples collected at the time of diagnosis. We divided CLL samples into 3 groups based on their response to treatment; i) Stable CLL group: asymptomatic patients requiring no treatment, ii) Treated but stable CLL group: patients required treatment but had stable disease for at least one year after the end of the treatment cycle, and iii) Relapsed CLL: patients who relapsed within a year of end of the treatment cycle. Significance analysis of microarray (SAM) revealed that the heat-shock protein (HSP) signature (HSJ2, HSP70, HSP90, HSP60, HSP10, HSP 105, HSP40, HSP27, HSPA2, HSJ1, HSF4, HSPCA), BCR signaling pathway (JUN, NFATC4, NFKBIE, PPP3CB, TRAF3, CD81, CCT4), activation markers (CD81, CD83) and MMPs (MMP3, MMP9) were overexpressed in relapsed PB-CLL (n=3) compared to stable PB-CLL (n=6) and treated but stable PB-CLL (n=10). Overexpression of heat-shock protein signature genes were further observed in additional relapsed PB-CLL (n=6) group compared to other two PB-CLL (n=22) group. Interestingly, the HSP signature was consistently overexpressed in relapsed BM-CLL (n=6) and LN-CLL (n=12) compared to stable and treated but stable BM-CLL (n=11) and LN-CLL (n=3) groups. HSPs are considered chaperones of tumorigenesis and known to enhance survival, migration, and proliferation of tumor cells which may contribute to relapse in patients. Furthermore, the HSPs genes (HSP90 and HSP70) were significantly overexpressed in LN-CLL as compared to PB-CLL which implies important role of the microenviroment in rendering CLL refractory. To investigate the link between the expression of the individual genes with the aggressiveness of the disease, Kaplan-Meier log-rank tests were performed. We found that the higher expression of HSP90A, HSP90B, HSJ, and MMP9 were significantly (p

Description: B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia with heterogenous clinical course. Prognostic markers such as CD38 expression has proven to be reliable in predicting clinical outcome where high CD38 expression correlates with poor prognosis and low CD38 expression is associated with good prognosis. Recently, our lab has shown that expression of CTLA4 in CLL cells is inversely correlated with CD38 expression using microarray profiling and real time PCR. Microarray analysis showed significant differential expression of CTLA4 between high and low CD38 subgroups (n=49) (Joshi et al. Clinical Cancer Research, 2007, in press). However, whether CTLA4 plays a direct role in CLL pathogenesis or not is unknown. Therefore to study the potential role of CTLA4 in CLL, CTLA4 expression was down regulated in primary CLL cells from six different patients using antisense oligonucleotide. The down regulation of CTLA4 expression was confirmed by RT-PCR analysis. The proliferative capability of the CTLA4-down regulated CLL cells was determined using MTT assay as well as radiolabeled thymidine uptake assay at 24, 48 and 72 hours in culture. There was a significant increase in the proliferation of CTLA4-down regulated CLL cells compared to untreated control CLL cells or CLL cells treated with an irrelevant oligonucoeotide. To further understand the CTLA4 mediated regulation of CLL cell proliferation, expression levels of downstream target molecules of CTLA4 such as NFATC2 and c-FOS was studied using RT-PCR. There was a significantly increased expression of target genes NFATC2 and c-FOS, molecules in the CTLA4-downregulated CLL cells compared to control CLL cells. In addition, further analyses of the microarray data revealed that CD38 high expressing CLL cells (n=22) has significant (p

Description: Abstract 3868 Chronic lymphocytic leukemia (CLL) is a heterogeneous B-cell malignancy which is characterized by accumulation of CLL cells in the peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). We have previously reported the gene expression profiles (GEPs) of CLL cells from these tissue sites (Blood, ASH Annual Meeting Abstracts, 2008; 112: Abstract 546) and shown that genes associated with various biologically-relevant signaling pathways (BCR, BAFF/APRIL, NFB, PI3K/AKT) were overexpressed in LN-CLL cells compared to PB-CLL and BM-CLL cells. To elucidate the association of the differentially- expressed gene signatures among PB, BM and LN-CLL with disease progression, gene expression was further studied based on clinical parameters obtained at the time of diagnosis such as chromosomal abnormalities (del 11q, del 17p and trisomy 12, and del 13q and normal karyotype as poor and good prognosis markers respectively), bulky lymphadenopathy (BLA) as a poor prognosis indicator and time to treatment 1300) of overexpressed genes. Most of these genes were associated with BCR signaling (CD79b1, CD79b2, CD72, SYK, BTK, BLNK, VAV1), the MAPK pathway (ELK4, MAP4K1, MAP3K10, MAP3K3), hedgehog signaling (GLI, SUFU, PTCH), chemokines (CCL21, CXCL13), and the NFκB pathway (TANK, TNFSF8, TNFSF7), indicating that PB-CLL cells from patients with BLA are highly activated compared to PB-CLL cells from those without BLA. Since this expression profile closely resembles that of LN-CLL, we infer that PB-CLL cells with high levels of signaling molecules had migrated out of LN in patients with BLA. We measured expression of one of the key players of BCR signaling, phosphorylated SYK (p-SYK), to validate that this pathway is active in the PB-CLL from BLA patients. Expression of p-SYK was significantly (p

Description: B-cell Chronic Lymphocytic Leukemia (B-CLL) is characterized by accumulation of clonal lymphocytes resistant to apoptosis. Therefore, identification of molecules responsible for the increased resistance to apoptosis is warranted. The identified molecules could be further targeted to develop effective therapy. Emerging evidence on the mechanism of resistant to apoptosis in several cancers suggests that GLI1 transcription factor, target of hedgehog signaling, may have a potential role in the increased resistance to apoptosis in B-CLL. However such studies have not been reported for B-CLL. Therefore, we investigated the role of sonic hedgehog (Shh)-GLI signaling in the pathogenesis B-CLL using 38 different patient samples and different parameters. The results were analyzed based on good and poor clinical outcome B-CLL subgroups identified on the basis of cytogenetic abnormality and/or CD38 expression levels. Microarray and RT-PCR analyses demonstrated active Shh-GLI signaling molecules in B-CLL cells including GLI1. Our results demonstrate that GLI1 transcripts were significantly (p = 0.003) over-expressed in B-CLL cells of poor clinical outcome patient subgroups (17pdel, 11qdel, trisomy12; N = 20) compared to good clinical outcome (13qdel, normal karyotype; N = 18) as observed in microarray analysis and confirmation by real time PCR. In addition, similar observation was also made with poor prognosis (high CD38 expression) versus good prognosis (low CD38 expression) B-CLL subgroups. Furthermore, higher expression of GLI1 was associated with significantly (p = 0.02) shorter time to treatment compared to lower GLI1 expressing B-CLL patients as determined by Kaplan-Meier survival analysis using log rank test, suggesting the increased expression of GLI1 and its association with the disease progression in poor clinical outcome B-CLL patient sub-groups. Addition of exogenous Shh on the significantly (p

Description: Abstract 3882 CLL and associated stromal cells are exposed to lower oxygen tension in lymph nodes and other lymphoid organs; the impact of this on gene expression profiles (GEPs) and cellular functions is not understood. In this study the effects of low oxygen tension (1%; “hypoxia”) on stromal and CLL cell GEPs and interactions were explored using a co-culture system employing a bone marrow derived human stromal cell line (HS-5) and CLL B cells. HS-5 was grown at 1% O2 for 4 weeks (n ≥ 3 experiments) and at weekly intervals GEPs were analyzed using whole genome expression beadchips (HumanHT-12 v 4, Illumina). Differential GEPs were determined using GenomeStudio and Partek programs, and gene sets/pathways were identified using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) with significance defined as p

Description: Mantle cell lymphoma (MCL) is among the most aggressive B cell lymphomas with median patient survival of only 3–4 years. Although high dose therapies followed by hematopoietic stem cell transplantation is an option for therapy, it is difficult to successfully treat MCL until the therapeutic strategy is developed to target and eliminate the residual/relapsing tumors after existing chemotherapies. Therefore, characterization of these residual/relapsing tumor cells after chemotherapy as a basis for developing therapeutic strategy to target residual/relapsing MCL cells is essential. Accordingly, we have isolated the residual/relapsing tumor cells by transplanting the human MCL cell line, Granta519, into NOD-SCID mice followed by CHOP+Bortezomib chemotherapy. Isolated residual/relapsing tumor cells from liver and kidney characterized morphologically using microscopy, and immunophenotypically for CD45, CD19 and CD23 expression by flow-cytometry showed no differences. However these cells had a significantly higher growth rate and increased chemo-resistant potential in vitro as determined by MTT and 3[H]-thymidine incorporation assay compared to parental Granta cells. In addition, NOD-SCID mice transplanted with cells isolated from liver had significantly shorter survival compared to parental cells indicating a higher tumorigenic potential of relapsed tumor cells pre-exposed to chemotherapy. There was a higher frequency of side population (SP) cells in relapsed tumor cells compared to parental cells as determined by flow cytometry after Hoechst staining. In addition, there was an increased expression of transcripts associated with maintenance of stem cells, including GLI transcription factors, targets of hedgehog signaling, as determined by microarray and confirmation by real time PCR, suggesting that increased stem cell-like cells in the relapsing tumors may be responsible for the chemo-resistance and increased tumorigenic potential. However, these residual/relapsing cells were susceptible to in vitro generated MCL-specific cytotoxic T lymphocytes (CTLs) against parental cells. We have demonstrated the potential of inhibition of GLI to render the MCL cells sensitive to chemotherapy (Hegde et al., Mol Cancer Ther, 7:1450–60, 2008). Therefore, we have developed a strategy of targeting hedgehog signaling mediators such as GLI transcription factors using GLI-antisense oligonucleotides or the proteosome using Bortezomib, to target residual/relapsing tumor cells after CHOP chemotherapy followed by tumor-specific adoptive T cell therapy (ATT) against human MCL using the mouse model. There was a significantly improved survival and decreased tumor burden in the mice treated with CHOP+GLI-ASO+ATT or CHOP+bortezomib+ATT compared to CHOP only treated controls suggesting the potential of targeting of residual/relapsing tumor cells using this strategy. Together, these results demonstrate the potential of this novel combined therapeutic strategy against human residual/relapsing MCL, which may have future clinical application.