ALBERT

Description: Mantle cell lymphoma (MCL) is a mostly incurable malignancy arising from naive B cells (NBCs) in the mantle zone of lymph nodes. We analyzed genomewide methylation in MCL patients with the HELP (HpaII tiny fragment Enrichment by Ligation–mediated PCR) assay and found significant aberrancy in promoter methylation patterns compared with normal NBCs. Using biologic and statistical criteria, we further identified 4 hypermethylated genes CDKN2B, MLF-1, PCDH8, and HOXD8 and 4 hypomethylated genes CD37, HDAC1, NOTCH1, and CDK5 when aberrant methylation was associated with inverse changes in mRNA levels. Immunohistochemical analysis of an independent cohort of MCL patient samples confirmed CD37 surface expression in 93% of patients, validating its selection as a target for MCL therapy. Treatment of MCL cell lines with a small modular immunopharmaceutical (CD37-SMIP) resulted in significant loss of viability in cell lines with intense surface CD37 expression. Treatment of MCL cell lines with the DNA methyltransferase inhibitor decitabine resulted in reversal of aberrant hypermethylation and synergized with the histone deacetylase inhibitor suberoylanilide hydroxamic acid in induction of the hypermethylated genes and anti-MCL cytotoxicity. Our data show prominent and aberrant promoter methylation in MCL and suggest that differentially methylated genes can be targeted for therapeutic benefit in MCL.

Description: Abstract 1579 Background: There is a growing awareness of the biological heterogeneity of MCL, which likely translates into differences in outcomes. Cytokines play an important role in the pathogenesis of lymphoma. Recent reports suggest that cytokine profiles can identify DLBCL (ASH 2010: Abstract 991) and HL pt (ASH 2011: Abstract 429) subsets with inferior outcomes. A distinct cytokine profile for MCL has not yet been defined. We therefore examined serum cytokines levels in MCL pts to gain insight into which may have biological relevance and their association with clinical outcomes. Methods: Serum samples from MCL pts (57% newly diagnosed, 43% relapsed) were prospectively collected and linked to relevant clinical data from our MCL outcomes database. Cytokine levels were determined quantitatively utilizing a commercially available protein array system. We first used Wilcoxan tests to identify relevant cytokines by comparing the distribution of 507 serum cytokines levels in MCL cases (n=97) to serum cytokines levels from normal controls (n=20). For cytokines that showed significant differences, we report the ratio of median values (cases vs. controls). To maintain a type I error rate of.05 we adjusted for multiple comparisons (Hochberg's method). Using Cox regression model and adjusting for multiplicity, we next examined the association between serum cytokines levels and outcome (PFS) in a MCL cohort uniformly treated with R-HyperCVAD (R-HCVAD). Results: 214 pts with MCL were identified of which 97 pts had available serum samples. Clinical characteristics were as follows: med age 62 yr, med MIPI score of 4, med Ki67 25% (range 5–95%) and median follow up of 35 months. Our analysis identified 22 cytokines with statistically significantly distinct levels in MCL pts (vs. normal serum controls) including (p

Description: Abstract 673 Mantle Cell Lymphoma (MCL) is an aggressive and incurable malignancy arising from naïve B cells (NBC) in the mantle zone of lymph node follicles. Murine models over-expressing Cyclin D1, the putative oncogene implicated in the majority of MCL, do not fully recapitulate the disease phenotype. We therefore hypothesize that there are additional mechanisms contributing to MCL pathogenesis and undertook an integrative approach by studying genome-wide DNA methylation and gene expression in primary MCL to uncover additional genes and pathways involved in MCL pathogenesis. We therefore compared and contrasted the DNA methylation levels of 14,000 gene promoters in MCL patients and normal tonsillar NBC controls using the HELP (HPA II tiny fragment Enrichment by Ligation mediated PCR) assay. All patient samples were obtained prior to any treatment from peripheral blood or apheresis specimens from newly diagnosed patients with histologically confirmed MCL. We found significant hypo-methylation of gene promoters in the MCL patients as compared to normal NBCs. Integrating genomic methylation data from HELP and gene expression data from Affymetrix U133 arrays, we determined a signature of differentially methylated genes with reciprocal changes in mRNA levels. Using pathway analysis and gene ontogeny, we selected genes for validation by choosing loci that were differentially methylated and fulfilling the following characteristics (i) demonstrating a clear methylation state change (from hypomethylated in normal B cells to methylated in MCL or vice-versa) using a threshold of 0.0 on the log ratio scale, (ii) Genes that function as tumor suppressors and were hypermethylated and suppressed in MCLs in our data (iii) Overexpressed/ hypomethylated genes with existing therapeutic options available or in clinical trial (iv) involved in pathways controlling biological processes with known relevance to MCL i.e. cell cycle control, apoptosis. Our panel included four differentially hypermethylated genes CDKN2B, MLF-1, PCDH8, HOXD8 and four differentially hypomethylated genes CD37, HDAC1, NOTCH1 and CDK5. MassArray Epityper analysis confirmed the presence of differential methylation at the promoter region of these genes, which was consistent between MCL patients and cell lines in all 8 genes studied. Remarkably, PCDH8 and CDKN2B have previously been shown to be silenced by methylation at their gene promoters and transfection of PCDH8 and CDKN2B have been shown to reduce tumor growth in breast cancer and MCL cell line models respectively. Based on these data, we hypothesized that the aberrantly hypermethylated and thereby silenced tumor suppressor genes in MCL could be pharmacologically induced by DNA hypomethylating agents and HDAC inhibitors for therapeutic benefit. We therefore next treated MCL cell lines MINO and Z138 with the DNA methyltransferase inhibitor Decitabine alone and in combination with the HDAC inhibitor SAHA. HELP analysis of MINO and Z138 cells treated with hypomethylating doses of decitabine (0.5uM × 3days) showed widespread reversal of aberrant gene promoter hypermethylation. Hypomethylation in these cell lines was accompanied with 3-7 fold increase in mRNA levels of tumor suppressor genes CDKN2B, MLF-1, PCDH8 and HOXD8. Concurrent treatment with SAHA (1 uM x 1 dose) synergized with Decitabine leading to 5-15 fold increase in mRNA of these tumor suppressor genes in Z138 cells. Importantly, treatment with Decitabine and SAHA as single agents decreased MCL cell viability by 60% and 40% respectively and the combination synergised in anti-MCL cytotoxicity with 〉 90% decrease in cell viability. In conclusion, our analysis shows prominent aberrant gene promoter methylation patterns in MCL genome and identifies novel differentially methylated and expressed genes in MCL cell lines and primary MCLs. Furthermore, we demonstrate that reversal of aberrant hypermethylated and silenced genes can be targeted for therapeutic benefit using epigenetic drugs in MCL. We are currently modeling our combination epigenetic drug therapy in primary MCL cells in culture and murine xenograft systems. We expect these results to support the development of clinical trials investigating the prospective use of combination epigenetic therapy in MCL. Disclosures: No relevant conflicts of interest to declare.

Description: Cytogenetic abnormalities at chromosome 1q21 are among the most common lesions in diffuse large-cell lymphoma and have been associated with a poor prognosis. A novel cell line, SKI-DLCL-1, was established from ascitic fluid that carries a t(1;14)(q21;q32) chromosomal translocation. Using pulsed-field gel electrophoresis, the breakpoint on the IgH locus mapped to a gamma locus between C1 and C2. A cosmid library was prepared from SKI-DLCL-1, and Cγ-positive clones spanning the breakpoint were identified by screening with fluorescence in situ hybridization. The breakpoint occurs 860 bp downstream of the 3′ UTR of the MUC1 gene. The break appears to be a staggered double-strand break consistent with an error in immunoglobulin class switching. The MUC1 gene is highly transcribed and translated, and the protein is highly glycosylated. It is postulated that MUC1 expression is brought under the control of the 3′E enhancer. MUC1 lies in a region of chromosome 1 characterized by an unusually high density of genes, with 7 known genes in a region of approximately 85 kb. To determine whether there was a pleiotropic effect of the expression of genes in the region as a consequence of the translocation, the expression of 6 additional genes was assessed. None of the other genes in this region (CLK2, propin, COTE1, GBA, metaxin, and thrombospondin 3) are overexpressed in SKI-DLCL-1. Thus, the translocation t(1;14)(q21;q32) seen in both the primary tumor and the derived cell line results in the marked overexpression of MUC1 without affecting the expression of other genes in the region.

Description: Bortezomib, the first of a new class of agents called proteasome inhibitors, has shown promising activity in several tumor types, especially in hematological malignancies. To further characterize its activity in Non Hodgkin Lymphoma (NHL), we determined the efficacy of bortezomib, in vitro in 19 cell lines representing five subtypes of lymphoma and in ex vivo culture of patients samples. Using an MTS cell cytotoxicity assay, bortezomib was found to induce apoptosis at lower doses (IC50 ranging from 13.2 to 19.4 nM) in Mantle Cell Lymphoma (MCL) and Diffuse Large B-Cell Lymphoma (DLBCL) cells than in Burkitt lymphoma, Primary Effusion Lymphoma (PEL) and Anaplastic Large Cell Lymphoma (ALCL). In ex vivo culture of patients samples treated with 5 or 10 nM of bortezomib, the percentage of cells undergoing apoptosis as determined by annexin V-FITC staining was greater in MCL (+28.2% ± 12 with 10nM; +12.5% ± 13.8 with 5 nM) than in DLCL samples (+13.4% ± 6.2 with 10nM; +4.9% ± 1.5 with 5nM) when compared to spontaneous apoptosis. Bortezomib also showed a much lower IC50 than conventional cytotoxic agents (doxorubicin, vincristine and 4-hydroperoxycyclophosphamide) in 4 MCL cell lines. The potential synergy between cytotoxic agents and bortezomib was studied using annexin FITC staining as well as PARP cleavage analysis. Co-treatment of MCL cell lines with bortezomib and any of these chemotherapy agents showed a synergistic effect. However, the synergy was greater if the cells were sequentially treated with doxorubicin or vincristine followed by bortezomib. On the opposite the synergistic effect was greatly reduced or abolished when the cells were pretreated with bortezomib before exposure to doxorubicin or vincristine. We then used gene expression profiling to characterize the molecular consequences of bortezomib treatment in both MCL and DLBCL lymphoma cell lines. Using a 2 fold cut-off and a p value of

Description: We tested the feasibility of performing gene expression profiling using amplified RNA from Fine Needle Aspiration (FNA) obtained from lymph nodes. Twenty-four samples from patients with a diagnosis of Follicular Lymphoma (FL) or Large B-Cell Lymphoma (LBCL) were obtained after informed consent. The diagnosis were performed by two pathologists and classified into two groups (10 LBCL and 14 FL) using conventional morphology and immunophenotyping. Total RNA was extracted using RNaqueous (Ambion, Austin, TX). Total RNA (100ng /per sample) was subjected to 2 cycles of standard double-stranded cDNA synthesis using Superscript Choice System (Invitrogen, Grand Island, NY) and in vitro transcription for target amplification as per GeneChip’s Eukaryotic Small Sample Target Labeling protocol (Affymetrix, Inc, Santa Clara, CA). The biotinylated cRNA from each sample was hybridized to Affymetrix HG-U133A chips. Gene expression profiling results were first analyzed by Principal Component Analysis (PCA) using a list of 146 probe sets representing 62 genes characteristic of Acivated B-Cell (ABC) or Germinal Center (GC) signature. This analysis identified 5 LBCL cases with ABC cell signature (Fig 1). Using a list of 207 probe sets representing 113 genes involved in FL transformation (K Elenitoba-Johnson, PNAS 2003), PCA analysis identified two overlapping clusters corresponding to FL and GC-DLBCL. To further improve this classification, we generated a list of 82 genes differentially expressed between FL and GC-LBCL. Using this list of genes, PCA analysis demonstrated a clear separation between FL and GC-LBCL (Fig 2). These results demonstrate that comprehensive transcription profiles can be performed in patients with NHL using RNA obtained from FNA and that one can distinguish among FL and LBCL lymphoma genetic subtypes. Figure Figure