Matrigel modulates a stem cell phenotype and promotes tumor cell formation in a mantle cell lymphoma cell line

Tumors may be maintained by subpopulations of cells possessing stem cell-like properties. We evaluated the stem cell-like and tumor-forming properties of side population (SP) and CD133+/ CD44+ cells in Granta 519, a human mantle cell lymphoma cell line. The in-vitro Cobblestone Area Forming Cell (CAFC) assay, designed to detect stem and progenitor cells, revealed that SP cells contained the greatest proportion of stem cell-like cells. The addition of Matrigel to CAFC assays of SP and non-SP cells both increased their respective stem cell frequencies in comparison to those cultures without Matrigel, and additionally resulted in observed stem cell frequencies which were the same between SP and non-SP cells. Contrary, Matrigel decreased the stem cell frequencies of CD133+/CD44+ or CD133-/CD44- cells. In-vivo assays revealed tumor formation from Matrigel-mixed SP and non-SP cells, and in one instance, occurred with as few as one Matrigel-mixed SP cell. Vehicle-mixed injections of SP and non-SP tumor cells resulted in tumor formation from SP cells only. Tumor formation did not occur from Matrigel nor hyaluronan (cellular substrate for CD44-expressing cells)-mixed populations of CD133+/CD44+ or CD133-/CD44- cells. These data demonstrate that Matrigel modulates a stem cell phenotype and promotes tumor formation from SP and non-SP cells. The tumor micro-environmental niche and tumor cell to micro-environmental interactions may be important future targets for novel chemotherapeutic agent

PRE-binding sites in the MDR of CLL: Potential Tumor Suppressor Regulation

Chronic lymphocytic leukemia [CLL] is the most common adult leukemia and is heterogeneous in clinical presentation. CLL cases present with various chromosomal aberrations, including 11q23, 14q32, 17p, and trisomy 12, with the most common abnormality being deletion of 13q14 [1]. Although monoallelic deletion of 13q14 is common, there is a subset of patients who have complete nullisomy at 13q14, a locus that has been hypothesized to contribute to CLL pa thogenesis [2] due to loss of tumor suppressors [DLEU and miR-15a/16-1].We hypothesized that deletion of both copies of 13q14 would lead to uncontrollable proliferation of CLL cells and a poor prognosis. We examined our 13q14 nullisomy for survival, treatment-free survival, lymphocyte doubling time, and the presence of lymphadenopathy. Furthermore, we compared the gene expression profiles between patients with 13q14 monosomy, nullisomy, or normal karyotype. Our results suggest that patients with 13q nullisomy have a higher incidence of bulky lymphadenopathy [16.6% compared to 10% of monosomy patients], a higher frequency of lymphocyte doubling time [27.7% compared to 7.4% of monosomy patients], and a higher rate of needing treatment [50% compared to 18.5% of monosomy patients]. We observed deletion of DLEU1 and HTR2A, consistent with a gene dosage effect, and observed PRE-binding sites on DLEU1. Patients with homozygous deletion of 13q14 had a worse prognosis compared to heterozygotes. Lastly, the DLEU1 locus is a possible “second hit” loss for CLL progression

Agricultural Pesticide Use and Risk of t(14;18)-Defined Subtypes of Non-Hodgkin Lymphoma

Pesticides have been specifically associated with the t(14;18)(q32;q21) chromosomal translocation. To investigate whether the association between pesticides and risk of non-Hodgkin lymphoma (NHL) differs for molecular subtypes of NHL defined by t(14; 18) status, we obtained 175 tumor blocks from case subjects in a population-based case-control study conducted in Nebraska between 1983 and 1986. The t(14;18) was determined by interphase fluorescence in situ hybridization in 172 of 175 tumor blocks. We compared exposures to insecticides, herbicides, fungicides, and fumigants in 65 t(14;18)-positive and 107 t(14;18)-negative case subjects with those among 1432 control subjects. Multivariate polytomous logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Compared with farmers who never used pesticides, the risk of t(14;18)-positive NHL was significantly elevated among farmers who used animal insecticides (OR = 2.6; 95%CI, 1.0-6.9), crop insecticides (OR = 3.0; 95% CI, 1.1-8.2), herbicides (OR = 2.9; 95% CI, 1.1-7.9), and fumigants (OR = 5.0; 95% CI, 1.7-14.5). None of these pesticides were associated with t(14;18)-negative NHL. The risk of t(14;18)-positive NHL associated with insecticides and herbicides increased with longer duration of use. We conclude that insecticides, herbicides, and fumigants were associated with risk of t(14;18)-positive NHL but not t(14;18)-negative NHL. These results suggest that defining subsets of NHL according to t(14;18) status is a useful approach for etiologic research. (Blood. 2006; 108:1363-1369

Long-Term Outcomes of Autologous Stem Cell Transplantation for Follicular Non-Hodgkin Lymphoma: Effect of Histological Grade and Follicular International Prognostic Index

AbstractAlthough results of autologous stem cell transplantation (SCT) for recurrent follicular non-Hodgkin lymphoma (NHL) have been previously reported, the long-term results and evaluation of prognostic factors in a large patient population receiving this therapy are difficult to find in the literature. To address these issues, we evaluated 248 patients with recurrent follicular NHL treated with high-dose chemotherapy and autologous SCT between 7/87 and 6/03. According to the World Health Organization (WHO) classification system, 64 patients (26%) had follicular NHL grade 1 (FL 1), 98 (40%) had FL 2, and 86 (35%) had FL 3. At the time of transplantation, 88 of the patients (35%) had a Follicular Lymphoma International Prognostic Index (FLIPI) score of low risk, 87 (35%) had an intermediate-risk FLIPI score, 37 (15%) had a high-risk FLIPI score, and 36 (15%) had at least 1 missing value, preventing calculation of the FLIPI score. The 5-year overall survival (OS) for all patients was 63%, and the 5-year progression-free survival (PFS) was 44%. In a multivariate analysis, a histological grade of FL 3, a high-risk FLIPI score at the time of transplantation, and having received 3 or more previous chemotherapy regimens were significant factors for predicting a worse OS. In addition, the use of a transplantation regimen including a monoclonal antibody decreased the relative risk of progressive lymphoma. These data suggest that transplantation earlier in the course of the disease for patients with follicular lymphoma with use of a monoclonal antibody–based regimen may lead to improved outcomes

Autologous hematopoietic stem cell transplantation for mantle cell lymphoma

AbstractThis study evaluated the outcomes of patients who underwent high-dose chemotherapy (HDC) and autologous hematopoietic stem cell transplantation (autoHSCT) for mantle cell non-Hodgkin's lymphoma and the effect of clinical and treatment characteristics. The clinical outcome and prognostic factors in 40 patients who underwent HDC and autoHSCT for mantle cell lymphoma between June 1991 and August 1998 were analyzed. With a median follow-up of 24 months for the surviving patients (range, 4-68 months), the 2-year overall survival was 65% and the 2-year event-free survival (EFS) was 36%. In univariate analysis, characteristics predictive of a poor EFS were blastic morphology (P = .019) and the patient having received 3 or more prior chemotherapy regimens (P = .004). In a multivariate analysis, the only factor associated with a poor EFS was the number of prior chemotherapy regimens. Those patients who received 3 or more prior therapies had a 2-year EFS of 0%, and those who received <3 therapies had a 2-year EFS of 45% (P = .004). Patients with mantle cell lymphoma can obtain prolonged EFS with HDC and autoHSCT; however, this strategy for prolonged EFS appears to work optimally in patients who are less heavily pretreated. Whether this therapy will increase the overall survival or EFS in patients receiving transplants in first complete remission will need to be tested in prospective randomized clinical trials.Biol Blood Marrow Transplant 2000;6(6):640-5

A Fast Modular Semiconductor-Based Marx Generator for Driving Dynamic Loads

The challenging demands of pulsed electron beam devices (such as the GESA device) with respect to their pulsed power supply have led to the development of a new semiconductor-based Marx generator. At a maximum output voltage of 120 kV and 600-A pulse current for a duration of up to 100 μS, stepwise arbitrary output waveforms are desired. A fast rise time of the generator is achieved by using fast switching circuitry, low inductance capacitors, and a low inductance stage arrangement. For low jitter triggering of all stages and efficient signal transmission, the generator uses an optical bus system for communication. Due to the inherent dynamic load characteristics of the GESA device, the generator features a fast overcurrent protection scheme. This paper presents selected design aspects of the generator and their validation in a small-scale assembly able of delivering up to 8 kV at 600-A load current

Chronic Lymphocytic Leukemia Cells in a Lymph Node Microenvironment Depict Molecular Signature Associated with an Aggressive Disease

Chronic lymphocytic leukemia (CLL) cells survive longer in vivo than in vitro, suggesting that the tissue microenvironment provides prosurvival signals to tumor cells. Primary and secondary lymphoid tissues are involved in the pathogenesis of CLL, and the role of these tissue microenvironments has not been explored completely. To elucidate host-tumor interactions, we performed gene expression profiling (GEP) of purified CLL cells from peripheral blood (PB; n = 20), bone marrow (BM; n = 18), and lymph node (LN; n = 15) and validated key pathway genes by real-time polymerase chain reaction, immunohistochemistry and/or TCL1 trans-genic mice. Gene signatures representing several pathways critical for survival and activation of B cells were altered in CLL cells from different tissue compartments. Molecules associated with the B-cell receptor (BCR), B cell-activating factor/a proliferation-inducing ligand (BAFF/APRIL), nuclear factor (NF)-κB pathway and immune suppression signature were enriched in LN-CLL, suggesting LNs as the primary site for tumor growth. Immune suppression genes may help LN-CLL cells to modulate antigen-presenting and T-cell behavior to suppress antitumor activity. PB CLL cells overexpressed chemokine receptors, and their cognate ligands were enriched in LN and BM, suggesting that a chemokine gradient instructs B cells to migrate toward LN or BM. Of several chemokine ligands, the expression of CCL3 was associated with poor prognostic factors. The BM gene signature was enriched with antiapoptotic, cytoskeleton and adhesion molecules. Interestingly, PB cells from lymphadenopathy patients shared GEP with LN cells. In Eμ-TCL1 transgenic mice (the mouse model of the disease), a high percentage of leukemic cells from the lymphoid compartment express key BCR and NF-κB molecules. Together, our findings demonstrate that the lymphoid microenvironment promotes survival, proliferation and progression of CLL cells via chronic activation of BCR, BAFF/APRIL and NF-κB activation while suppressing the immune response

Regulation of SOX11 expression through CCND1 and STAT3 in mantle cell lymphoma

The neural transcription factor SOX11 is usually highly expressed in typical mantle cell lymphoma (MCL), but it is absent in the more indolent form of MCL. Despite being an important diagnostic marker for this hard-to-treat malignancy, the mechanisms of aberrant SOX11 expression are largely unknown. Herein, we describe 2 modes of SOX11 regulation by the cell-cycle regulator cyclin D1 (CCND1) and the signal transducer and activator of transcription 3 (STAT3). We found that ectopic expression of CCND1 in multiple human MCL cell lines resulted in increased SOX11 transcription, which correlated with increased acetylated histones H3K9 and H3K14 (H3K9/14Ac). Increased H3K9/14Ac and SOX11 expression was also observed after histone deacetylase 1 (HDAC1) or HDAC2 was depleted by RNA interference or inhibited by the HDAC inhibitor vorinostat. Mechanistically, we showed that CCND1 interacted with and sequestered HDAC1 and HDAC2 from the SOX11 locus, leading to SOX11 upregulation. Interestingly, our data revealed a potential inverse relationship between phosphorylated Y705 STAT3 and SOX11 expression in MCL cell lines, primary tumors, and patient-derived xenografts. Functionally, inactivation of STAT3 by inhibiting the upstream Janus kinase (JAK) 1 or JAK2 or by STAT3 knockdown was found to increase SOX11 expression, whereas interleukin-21 (IL-21)–induced STAT3 activation or overexpression of the constitutively active form of STAT3 decreased SOX11 expression. In addition, targeting SOX11 directly by RNA interference or indirectly by IL-21 treatment induced toxicity in SOX11^+ MCL cells. Collectively, we demonstrate the involvement of CCND1 and STAT3 in the regulation of SOX11 expression, providing new insights and therapeutic implications in MCL

Genome-wide MicroRNA profiling of mantle cell lymphoma reveal a distinct subgroup with poor prognosis

MicroRNA (miRNA) deregulation has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Using a high-throughput quantitative real-time PCR platform, we performed miRNA profiling on cyclin D1- positive MCL (n=30) and cyclin D1-negative MCL (n=7) and compared them with small lymphocytic leukemia/lymphoma (SLL, n=12), aggressive B-cell lymphomas (n=138), normal B-cell subsets and stromal cells. We identified a 19-miRNA classifier which included six upregulated miRNAs (miR-135a, miR-708, miR-150, miR-363, miR-184, miR-342-5p) and 13 downregulated miRNAs, that was able to distinguish MCL from other aggressive lymphomas with \u3e90% probability. Some of these upregulated miRNAs are highly expressed in naïve B-cells. MicroRNA classifier showed consistent results in FFPE tissues and was able to distinguish cyclin D1-negative MCL from other lymphomas. A 26-miRNA classifier could distinguish MCL from SLL, dominated by 23 upregulated miRNAs in MCL. Unsupervised hierarchical clustering of MCL cases demonstrated a cluster characterized by high expression of miRNAs from polycistronic miR17~92 cluster and its paralogs miR-106a-363 and miR-106b-25, which was distinct from the other clusters showing enrichment of stroma associated miRNAs. The corresponding gene-expressionprofiling (GEP) data showed that the former cluster of MCL had significantly higher proliferation genesignature (PS), while the other subsets had higher expression of stroma associated genes. Clinical outcome analysis suggests that miRNAs can serve as prognosticators