Cell Lymphoma Leukemia Research Articles

Romedepsin (RM), a HDACi, Significantly Increases the Expression of NKG2D Ligands, MIC A/B, in Leukemia/Lymphoma Cells (LL), in Part through the Glycogen Synthase Kinase-3 (GSK-3) Pathway, Resulting in Enhanced NK Cytotoxicity.

Abstract 3027Poster Board II-1003 IntroductionNatural killer (NK) cells recognize malignant cells through the tumor-associated expression of NKG2D-ligands, including MIC A/B, which are known to be expressed on epithelial tumors, resulting in tumor cytotoxicity (Ayello/Cairo et al, BBMT, 2006). The expression of MIC A/B on these tumors can be induced by in vitro exposure of these cells to HDACi, specifically Romedepsin (RM) (Skov et al, Cancer Res, 2005). Glycogen synthase kinase-3 (GSK-3), a constitutively active serine-threonine kinase with numerous functions including regulation of cellular differentiation, stress and apoptosis, has also been shown to be an important regulatory enzyme in the expression of MIC A/B in response to RM (Doble et al, J cell Sci 2003; Frame et al, Biochem J 2001; Skov et al, Cancer Res, 2005). ObjectiveWe sought to determine of the expression of MIC A/B in response to RM in various leukemia and lymphoma cells (LL), its influence on NK cell mediated cytotoxicity and to investigate the role of the GSK-3 pathway in the regulation of expression of MIC A/B in response to RM. MethodsLL cells (106/ml, RS 4:11 [MLL-ALL], REH [pre-B cell ALL], Jurkat [T-cell ALL], Toledo [DLBCL], Ramos [Burkitt's Lymphoma]) were exposed to RM (10 ng/mL) for 24 hours, followed by FACS staining with PE-conjugated anti-MIC A/B antibody to determine surface expression of MIC A/B. Peripheral blood NK cells (CD3-/56+) were isolated via magnetic separation followed by IL-2 activation (3000IU/ml, 18 hrs). LL cells exposed to RM (generously supplied by Gloucester Pharmaceuticals) were subjected to NK cell mediated cytotoxicity assays (using an europium assay) at effector:target (E:T) ratio of 10:1, as we had previously described (Ayello/Cairo et al, BBMT 2006). LL cells were also pre-treated for 1 hour with 100mM lithium chloride (LiCl), a potent inhibitor of GSK-3 activity (Davies et al, Biochem J, 2000), to determine the role of this regulatory enzyme in the RM mediated expression of MIC A/B in these LL cells. Finally, blocking studies were also performed with anti-NKG2D receptor blockers to determine the specific role of NKG2D signal transduction pathway in NK cell mediated cytotoxicity. ResultsMIC A/B expression significantly increased in LL cells in response to RM ([RS4:11 0.2% vs 19.2%, p< 0.0001], [REH 0.2% vs 46%, p= 0.0003], [Jurkat 1.12% vs 44.7%, p< 0.0001], [Toledo 0.5% vs 15.8%, p=0.0001], [Ramos 0.57% vs 33.6%, p=0.0003]). In addition, the expression of MIC A/B in response to RM was inhibited when LL cells are pre-treated with LiCl (Jurkat [RM vs RM+LiCl] 85% vs 18%, p<0.0001; RS 4:11 [RM vs RM+LiCl] 82% vs 5%, p<0.0001; Ramos [RM vs RM+LiCl] 67% vs 35%, p<0.0001). Cytotoxicity assays revealed significant increases in-vitro cytotoxicty in RS 4:11, Ramos and REH cells at E:T ratio of 10:1. Mean specific release (MSR)(±SEM) was measured for four conditions in the assay: NK cells (A) vs NK cells with 10 ng/ml RM (C) vs IL-2 activated NK cells (B) IL-2 activated NK cells +10 ng/ml of RM (D). MSR for RS 4:11 10%±3.6% vs 108%±47% and 12%±4% vs 145%±45% p<0.05(A vs C, B vs D) for RS 4;11. MSR for REH was 13.2%±14.4% vs 50%±17.6% and 22%±18% vs 132%±29% p<0.01(A vs C, B vs D). MSR for Ramos was 7.6%±7.6% vs 79%±15.6% and 35%±13% vs 90%±22% p<0.05(A vs C, B vs D). NKG2D receptor-blocking experiments resulted in significant decrease in NK cell mediated cytotoxicity in REH (p<0.03) and Ramos cells (p<0.001). ConclusionOur data suggests that the surface expression of MICA/B in LL cells is significantly increased by RM leading to enhanced susceptibility for NKG2D-mediated cytotoxicity by NK cells. Furthermore, up-regulation of MICA/B in LL cells secondary to RM exposure is in part regulated by the GSK-3 signal transduction pathway. DisclosuresNo relevant conflicts of interest to declare.

Methyljasmonate, An Inhibitor of Glycolytic Energy Production, Displays Pre-Clinical Activity against Multiple Myeloma Cells.

Abstract 1741Poster Board I-767 BackgroundIn contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and ResultsWe tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of < 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of < 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades.Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. ConclusionsBased on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. DisclosuresLaubach:Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

A 56‐Year‐Old Woman with Rash, Paralytic Ileus, and Massive Gastrointestinal Bleeding

Diagnosis: Strongyloides stercoralis hyperinfection syndrome in a patient with human T lymphotropic virus type 1 (HTLV 1) infection. The duodenal sample obtained through a nasogastric tube showed multiple filariform larvae of Strongyloides stercoralis (figure 1; figure 2). Enzyme-linked immunosorbent assay (ELISA) test for toxins of Clostridium difficile had negative results, and stool cultures for bacteria showed no growth. Cultures of blood samples obtained during the abdominal catastrophe were positive for Escherichia coli. Strongyloidiasis is a parasitic disease that is caused by the soil-transmitted nematode 5. stercoralis, which is estimated to affect almost 300 million people worldwide. Once infected, humans may harbor the parasite for decades. Most of the in fections due to S. stercoralis are asymptomatic or mildly symp tomatic. However, in the presence of immunosuppressive con ditions (such as high-dose steroid use, use of cytotoxic drugs, malignancies, human immunodeficiency virus infection, mal nutrition, and more recently, HTLV-1 infection), an autoin fective cycle may ensue that amplifies the infection and leads to the hyperinfection syndrome [1], Multiple erosions in the colonic mucosa result from the migration of the filariform lar va across the intestinal wall and may induce massive bleed ing, which is associated with significant mortality [2]. The family history of T cell leukemia lymphoma in this patient raised the suspicion of HTLV-1 infection, which was confirmed by means of an ELISA and Western blot tests. The patient recalled that her sister had also been infected with HTLV-1. Further evaluation of her relatives revealed that 2 of

Rethinking the Pathogenesis of Asthma

Asthma research has focused primarily on allergic pathways on the basis that the majority of asthma is associated with atopy, the recruitment of the Th2-type T cell, the cytokines, and the chemokines that are released on exposure to allergens, and the IgE pathway. However, despite considerable investment by industry, targeting these pathways has not resulted in new treatments being developed beyond blockade of cysteinyl leukotrienes and IgE and improvements in inhaled corticosteroids and beta(2)-adrenoceptor bronchodilators. Increasingly, it is recognized that asthma is a heterogeneous disorder, and while important, allergen sensitization is only one component of the disease, with many other environmental and genetic factors playing a role. In addition, these factors act locally on a susceptible airway epithelium that is both structurally and functionally deficient. It may be worthwhile to focus on increasing the resilience of the airways to environmental insults in addition to improving strategies that modify adaptive immunity or suppress inflammation.

Lenalidomide-induced upregulation of CD80 on tumor cells correlates with T-cell activation, the rapid onset of a cytokine release syndrome and leukemic cell clearance in chronic lymphocytic leukemia

In chronic lymphocytic leukemia lenalidomide causes striking immune activation, possibly leading to clearance of tumor cells. We conducted this study to investigate the mechanism of action of lenalidomide and the basis for its unique toxicities in chronic lymphocytic leukemia. Patients with relapsed chronic lymphocytic leukemia were treated with lenalidomide 20 mg (n=10) or 10 mg (n=8) daily for 3 weeks on a 6-week cycle. Correlative studies assessed expression of co-stimulatory molecules on tumor cells, T-cell activation, cytokine levels, and changes in lymphocyte subsets. Lenalidomide upregulated the co-stimulatory molecule CD80 on chronic lymphocytic leukemia and mantle cell lymphoma cells but not on normal peripheral blood B cells in vitro. T-cell activation was apparent in chronic lymphocytic leukemia, weak in mantle cell lymphoma, but absent in normal peripheral blood mononuclear cells and correlated with the upregulation of CD80 on B cells. Strong CD80 upregulation and T-cell activation predicted more severe side effects, manifesting in 83% of patients as a cytokine release syndrome within 8-72 h after the first dose of lenalidomide. Serum levels of various cytokines, including tumor necrosis factor-alpha, increased during treatment. CD80 upregulation on tumor cells correlated with rapid clearance of leukemic cells from the peripheral blood. In contrast, neither the severity of the cytokine release syndrome nor the degree of T-cell activation in vitro correlated with clinical response. Upregulation of CD80 on tumor cells and T-cell activation correlate with unique toxicities of lenalidomide in chronic lymphocytic leukemia. However, T-cell activation appears to be dispensable for the drug's anti-tumor effects. This provides a rationale for combinations of lenalidomide with fludarabine or alemtuzumab.

Genetic fingerprinting of the development and progression of T-cell lymphoma in a murine model of atypical myeloproliferative disorder initiated by the ZNF198–fibroblast growth factor receptor-1 chimeric tyrosine kinase

AbstractA mouse model of human ZNF198–fibroblast growth factor receptor-1 (FGFR1) stem cell leukemia lymphoma has been developed to investigate mechanisms of oncogenesis and progression. Using array-based comparative genomic hybridization, we followed disease progression after serial transplantation of ZNF198-FGFR1–transformed stem cells that give rise to a distinct myeloproliferative disorder and T-lymphoblastic leukemia. A consistent, frequently homozygous, chr14:53880459-55011545 deletion, containing the T-cell receptor α and δ genes, was identified in the bone marrow, spleen, and lymph nodes in all cases. The absence of cell-surface T-cell receptor α in tumor cells precludes CD3 recruitment, resulting in loss of a functional T-cell receptor complex, supporting the idea that prevention of maturation of CD4+/CD8+ double-positive immature T cells is important in ZNF198-FGFR1 disease development. Up-regulation of the B-cell line 2, interleukin 7 receptor α and interleuking 2 receptor α prosurvival genes in these undifferentiated tumor precursor cells suggests one mechanism that allows them to escape apoptosis in the thymus. Thus, we have defined an important event in the process of ZNF198-FGFR1–induced T-cell leukemia.

Bayesian models identify specific lymphoproliferative disorders associated with hepatitis C virus infection

Bayesian models identify specific lymphoproliferative disorders associated with hepatitis C virus infection

Identification of a Homeobox-Like Protein Immune Response in Bone Marrow Transplant Patients

The identification of novel immune targets following hematopoietic cell transplantation (HCT) promises to identify clinically important graft-v-leukemia and graft-v-host disease targets. High-density protein microarrays present thousands of human proteins and facilitate broad-band multiplexed quantitative auto and alloreactive antibody screening. Our studies utilized both male and female patient plasma which were collected pre-transplant and one year post-transplant to identify new immune response targets. The tetra-peptide repeat homeobox-like protein (TPRXL) was initially identified by protein microarray as a potential biomarker in one MDS and four AML transplant patients. We developed an ELISA assay by affinity purifying TPRXL expressed in E.coli, and tested plasma samples collected from 80 HCT patients 1–2 years after HCT. A 42% sero-positive incidence was found across the group of post transplant samples (p-value <0.001). Located at 3p25.1 of the human genome, TPRXL encodes a 257 amino acid and 25 kDa protein containing a significant percentage of serine tetra-peptide repeats. Being a member of the TPRX homeobox gene family, the protein's secondary structure and function lead to direct interaction with DNA and tight regulation of gene expression. Presently, of the 18 reported TPRXL transcript variants a majority are nearly identical and are found to be highly expressed in early embryonic development, in the placenta, oral tissues and the ascites. Results indicate disease specificity with prevalence of post transplant response in patients with Mantle cell lymphoma (MCL). In addition, patients with chronic GVHD of the oral pharyngeal tissues are predominant in our analysis. In silico studies show that TPRXL is indeed differentially expressed in acute pro-myelocytic leukemia (PML) and acute lymphocytic lymphoma (ALL) cell lines as compared to controls. Since bioinformatics expression data available for MCL cell lines are scarce, perhaps due to fewer reports or studies in this area, it is clear that our novel findings will contribute to currently available information for this disease type. In sum, protein microarray technology represents a powerful tool in facilitating the identification of novel disease associated antigenic targets and that TPRXL is potentially one such prognostic marker.

Rapamycin Causes Reversible Cell Cycle Slowing, Rather Than Cell Cycle Arrest, in Myeloid and Lymphoid Cell Lines and in Acute Leukemia Cells

The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that regulates cell survival and proliferation, is constitutively activated in diverse malignancies, and constitutive mTOR activation has been implicated in both malignant transformation and resistance to chemotherapy. The mTOR inhibitor rapamycin, which inhibits mTOR signaling and has immunosuppressive effects at nanomolar concentrations, also has antineoplastic activity, but its reported effects on cell cycle and on apoptosis have been variable. Several studies evaluated lymphoid and myeloid cell lines after 24 or 48 hours of exposure to rapamycin and reported cell cycle arrest in G1, while others reported apoptosis. Rapamycin is being incorporated into treatment regimens in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and other hematologic malignancies, and knowledge of its growth inhibitory and cytotoxic effects will be useful in designing combination regimens. To this end, the concentration-dependent effects of rapamycin on survival, proliferation, cell cycle and apoptosis of myeloid and lymphoid cell lines and acute leukemia cells were studied. Assays were performed at serial time points extending to 96 hours. Cells studied included HL60 myeloid and Jurkat T-lymphoblastic leukemia, 8226 myeloma and U937 histiocytic lymphoma cells and ML-1 myeloid leukemia cells, which have intact p53, as well as multidrug resistant HL60/VCR, HL60/ADR and 8226/MR20 cells overexpressing the ATP-binding cassette (ABC) proteins ABCB1, ABCC1 and ABCG2, respectively. Pretreatment blasts from 9 patients with AML and 4 with ALL were also studied. Cell survival was measured following 96-hour (cell lines) or 48-hour (patient samples) culture with rapamycin at picomolar to micromolar concentrations using the WST-1 colorimetric assay. Cells grown in suspension culture without rapamycin and with 1 nanomolar to 100 micromolar rapamycin were counted at serial time points. Cell cycle was analyzed by propidium iodide labeling and bromodeoxyuridine incorporation, cell division by carboxyfluorescein succinimidyl ester (CFSE) staining and apoptosis by Annexin V labeling, all measured by flow cytometry. Concentration-dependent effects of rapamycin on cell lines and acute leukemia cells in the WST-1 assay were biphasic, occurring at nanomolar and again at micromolar concentrations, with a plateau from approximately 10 nanomolar to 10 micromolar. At nanomolar concentrations rapamycin prolonged cell doubling times by 20–40%, as evidenced by CFSE staining at serial time points, but it did not cause cell cycle arrest. Examined at serial time points, cells continuously exposed to rapamycin at nanomolar concentrations did not accumulate in any single phase of the cell cycle, nor did they become apoptotic. Doubling times increased, resulting in decreased cell numbers at each time point in relation to control cultures without rapamycin. When cells cultured with rapamycin were transferred to rapamycin-free medium, doubling times reverted to those in rapamycin-free cultures, demonstrating that cell cycle slowing resulted from continuous exposure to rapamycin and was fully reversible in the absence of rapamycin. In contrast to rapamycin at nanomolar concentrations, rapamycin at micromolar concentrations caused apoptosis of cell lines and acute leukemia cells. Thus rapamycin at nanomolar concentrations increases the doubling time of myeloid and lymphoid cell lines and acute leukemia cells, while rapamycin at micromolar concentrations causes apoptosis. Rapamycin doses used in immunosuppressive regimens are well tolerated and yield nanomolar plasma concentrations. Our data suggest that continuous oral administration of rapamycin or its analogs at the doses used in immunosuppressive regimens may slow growth of leukemia cells and might have utility as maintenance therapy in the minimal residual disease setting, given the known lack of effect on normal cells. Higher doses of rapamycin are being studied in phase I clinical trials. Finally, increased understanding of rapamycin's effect on leukemia cell growth will help direct its incorporation into combination therapy.

Micro-RNA Gene Deregulation by Chromosome Translocations with Fragile Genomic Regions in B-Cell Lymphoma Cells

Micro-RNA (miR) genes posttranscriptionally modulate target gene expression via imperfect 3′-UTR matching sequences and play key roles in development, homeostasis and cancer. Little is known how miR genes are themselves regulated, or deregulated in cancer. Chief paradigm for neoplastic miR deregulation concerns miR-17/92 cluster members subject to genomic amplification in B-cell lymphoma. While the repeated occurrence of oncogenic miR genes at or near chromosomal breakpoints in cancer links chromosome fragility to oncogenic miR deregulation, direct evidence of a causal connection remains tenuous. We found that t(3;7)(q27;q32) in a B-cell lymphoma cell line joins 5′-BCL6 to a noncoding region of chromosome 7 inside a common chromosomal fragile site (FRA7H). In these cells hybrid mRNA was absent, unlike canonical BCL6 translocations which involve promoter exchange yielding hybrid mRNA. Affected cells instead showed downregulation of miR-29b-1, the only gene located within FRA7H - a recurrent transcriptional feature of B-cell lymphoma subsets. In another BCL6 translocation, t(3;13)(q27;q31)t(13;12)(q31;p11), which 5′-RACE also showed to be non-fusogenic, long distance inverse (LDI)-PCR revealed junction of 5′-BCL6 to chromosome 13 sequences inside the miR-17/92 host gene MIRH1 (alias c13orf25). FISH using a sensitive tyramide amplification protocol with c13orf25 clones confirmed the presence of a cryptic BCL6-MIRH1 rearrangement. Surprisingly, reverse transcriptase quantitative (q) PCR assay revealed weak MIRH1 expression using 3′-primers. In contrast, repeating the assay using more central primers covering the miR-17/92 coding region showed massive upregulation. 3′-RACE confirmed a novel high level MIRH1 transcript truncated by 3.1 kbp. Quantitative genomic PCR and FISH excluded miR-17/92 genomic copy number alteration, while LDI-PCR analysis showed that formation of truncated MIRH1 involved multiple DNA cuts at 3q27 (x1), 12p11 (x1), and 13q31 (x5) – the last including a complex excision/inversion/insertion rearrangement. Stress induced DNA duplex destabilization (SIDD) analysis revealed that 6 of 7 breaks precisely coincided with fragility peaks. Taken together, these data suggest a novel role for BCL6 translocations in the deregulation of miR genes near sites of chromosome or DNA instability. BCL6 has been shown to suppress p53 in germinal center B-cells thus protecting B-cells from apoptosis induced by DNA damage, offering a possible explanation for chromosome rearrangements associated with genomic fragility therein. Chromosomal MIRH1 dysregulation is not limited to BCL6 expressing lymphomas, however: cytogenetic investigations performed on diverse leukemia-lymphoma cell lines, including those derived from multiple myeloma and plasma cell leukemia, showed 11/50 with cytogenetic rearrangements at or near MIRH1. In sister cell lines sequentially established at diagnosis and relapse of multiple myeloma, only the latter showed miR-17/92 chromosomal rearrangement and upregulation. Interestingly miR overexpression was limited to miR-92, while miR-17/18 were barely expressed. FISH analysis and qPCR showed that discrepant expression was associated with rearrangement upstream of MIRH1. In brief, our data show that like other cancer genes, oncogenic miRs are subject to dysregulation mediated by structural chromosome rearrangements.

PotentIn vitroandIn vivoActivity of an Fc-Engineered Anti-CD19 Monoclonal Antibody against Lymphoma and Leukemia

CD19 is a pan B-cell surface receptor expressed from pro-B-cell development until its down-regulation during terminal differentiation into plasma cells. CD19 represents an attractive immunotherapy target for cancers of lymphoid origin due to its high expression levels on the vast majority of non-Hodgkin's lymphomas and some leukemias. A humanized anti-CD19 antibody with an engineered Fc domain (XmAb5574) was generated to increase binding to Fcgamma receptors on immune cells and thus increase Fc-mediated effector functions. In vitro, XmAb5574 enhanced antibody-dependent cell-mediated cytotoxicity 100-fold to 1,000-fold relative to an anti-CD19 IgG1 analogue against a broad range of B-lymphoma and leukemia cell lines. Furthermore, XmAb5574 conferred antibody-dependent cell-mediated cytotoxicity against patient-derived acute lymphoblastic leukemia and mantle cell lymphoma cells, whereas the IgG1 analogue was inactive. XmAb5574 also increased antibody-dependent cellular phagocytosis and apoptosis. In vivo, XmAb5574 significantly inhibited lymphoma growth in prophylactic and established mouse xenograft models, and showed more potent antitumor activity than its IgG1 analogue. Comparisons with a variant incapable of Fcgamma receptor binding showed that engagement of these receptors is critical for optimal antitumor efficacy. These results suggest that XmAb5574 exhibits potent tumor cytotoxicity via direct and indirect effector functions and thus warrants clinical evaluation as an immunotherapeutic for CD19(+) hematologic malignancies.

Extended follow‐up of the Finnish cartilage‐hair hypoplasia cohort confirms high incidence of non‐Hodgkin lymphoma and basal cell carcinoma

Cartilage-hair hypoplasia (CHH) is an autosomal recessive chondrodysplasia with short stature, sparse hair and defective cell-mediated immunity. It is caused by mutations in the RMRP (ribonuclease mitochondrial RNA processing) gene, encoding the RNA component of the ribonuclease complex RNase MRP. The aim of this study was to further elucidate the risk and spectrum of cancer in CHH. A cohort of 123 Finnish patients with CHH (51 males) was followed for malignancy through the Finnish Cancer Registry. The number of identified cancers was compared with expected numbers of cancer using population-based data to obtain standardized incidence ratios (SIR). Hospital records were reviewed for clinical data related to the malignancies. During the follow-up (2,365 person-years; mean 19.2 years), 14 cases of cancer were diagnosed in the CHH cohort (expected number 2.0; SIR 7.0, CI 3.8-12). Non-Hodgkin lymphoma was the most frequent cancer type (n = 9; SIR 90.2, CI 39.0-180) followed by squamous cell carcinoma (3), leukemia (1) and Hodgkin lymphoma (1). One tumor was not histologically classified. Nine of the 14 cancers were diagnosed in patients less than 45 years of age. In addition, ten patients had basal cell carcinoma of the skin (expected number 0.3; SIR 33.2, CI 16-61). Patients with CHH have significantly increased risk for developing non-Hodgkin lymphoma or basal cell carcinoma at early age; the overall prognosis is poor. The underlying pathogenetic mechanisms remain to be elucidated in future studies. Careful follow-up, extending beyond pediatric age, is warranted for early diagnosis of malignancies.

Leukaemia cell lines are robust in vitro models

Approximately 1500 leukaemia–lymphoma (LL) cell lines have been established from haematological neoplasms, of which approximately 600 may be deemed well characterized and, therefore, ‘fit for purpose’ (Drexler, 2005). LL cell lines are widely used as disease models and, increasingly, in the wake of the Human Genome Project, as resources for discovering oncogenes and the oncological pathways they govern, to serve as potential targets for ‘intelligent’ therapies, e.g. by facilitating the development of imatinib (Gleevec) now used widely to treat chronic myeloid leukaemia (CML). Indeed, 40% of the hits scored by ‘leuk(a)emia’ as a keyword on the HighWire search engine (http://highwire.stanford.edu/) also retrieve ‘cell line’. Recently, Leupin et al (2006) reported differing transcriptional profiles in parallel series of leukaemia cell lines and primary tumours. These authors ascribed these discrepancies to the capacity of cell lines to grow indefinitely in culture – i.e. immortalization artefacts. Their conclusions (based on three cell lines only) contradicted several larger studies. These studies respectively covered the National Cancer Institute (NCI-) panel of 60 mixed tumour cell lines (Ross et al, 2000), 17 myeloid cell lines (Rucker et al, 2006), and a panel of 40 acute myeloid leukaemia (AML), mature and precursor B-cell CML in blast crisis, and T-cell acute lymphoblastic leukaemia cell lines (Andersson et al, 2005). Another study of a wide range of solid tumour and leukaemic cell lines concluded that the latter recapitulate their supposed tumours of origin best of all (Sandberg & Ernberg, 2005) – presumably because the uniquely close correspondence between gene/chromosome rearrangements and LL subtypes facilitates their characterization. Similarly, upbeat conclusions have been reached by genomic array studies comparing primary leukaemia cells versus established cell lines (Horsley et al, 2006; Feys et al, 2007; Greshock et al, 2007). Why then, might the study of Leupin et al (2006) have alone yielded discrepant conclusions? It seems that in their study, at least 2/3 leukaemic patient characteristics did not closely match those of their comparison cell lines. First, cells from an acute blastic phase CML cell line (K-562) were compared with white blood cells from patients with chronic phase CML. Second, HL-60 – a cell line known to be established from a patient with AML-M2, was compared with cells from AML-M3 patients. Though we have no reason to suspect the cell lines used by Leupin et al (2006), a major source of discrepancies between cell line and patient data is likely to involve the use of false cell lines. Unlike other key resources, such as tilepath clones, cell lines have little more than the labels on their culture flasks to identify them. Once misidentified, there is little hope of detecting false cell lines, save by cell repositories equipped for DNA profiling and with comprehensive access to the relevant data. Discrepant cell line versus primary leukaemia profiles due to the inclusion of false cell lines has been observed by the present authors. We previously showed that c. 15% of circulating LL cell lines – mainly obtained from colleagues and collaborators – are false and might well yield discrepant transcriptional profiles (Drexler et al, 2003). The molecular dissection of LL cells that is now possible by transcriptional profiling enables stratification into increasingly smaller entities. Accordingly, those adopting LL cell lines as in vitro models must exercise correspondingly finer choices to find appropriate models. To assist investigators we have compiled both a list of false LL cell lines (Drexler et al, 2003), and a catalogue raisonée of well characterized examples (Drexler, 2005).

An alternatively spliced isoform of PECAM-1 is expressed at high levels in human and murine tissues, and suggests a novel role for the C-terminus of PECAM-1 in cytoprotective signaling

The Ig-ITIM family member PECAM-1 is expressed in vascular and endothelial cells, and its functions include suppression of mitochondria-dependent apoptosis. Previous studies have identified distinct PECAM-1 cytoplasmic domain splice variants at the mRNA, but not protein, level. Several relatively abundant mRNA isoforms lack exon 15 (Delta15) and would theoretically encode a protein with a truncated cytoplasmic domain and a unique C-terminal sequence. Using a novel rabbit polyclonal antibody that specifically recognizes Delta15 PECAM-1, we found that the Delta15 PECAM-1 isoform was expressed in human tissues, including brain, testes and ovary. This isoform was also expressed on the cell surface of human platelets, human umbilical vein endothelial cells (HUVECs) and the Jurkat T-cell leukemia, human erythroleukemia (HEL) and U937 histiocytic lymphoma cell lines. Furthermore, murine platelets and lung lysates demonstrated abundant amounts of exon-15-deficient PECAM-1. Functional studies revealed that Delta15 PECAM-1 retains both its homophilic binding capacity and its ability to signal by means of its immunoreceptor tyrosine-based inhibitory motif (ITIM) domains. Delta15 PECAM-1 was unable, however, to protect against apoptosis induced by overexpression of Bax or treatment with the chemotherapy agent etoposide. These studies suggest a novel role for the PECAM-1 C-terminus in cytoprotective signaling and highlight a need for further characterization of expression of PECAM-1 isoforms in normal and malignant tissues.

Bcl‐2 Regulates Endothelial Cell Migration and Capillary Morphogenesis

Apoptosis plays a critical role during development and in the maintenance of multicellular organisms. B cell leukemia lymphoma 2 (bcl‐2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen‐induced ischemic retinopathy in bcl‐2 deficient (bcl‐2 −/−) mice. How bcl‐2 affects EC function during these processes require further investigation. To gain further insight into the functions of bcl‐2 in the endothelium we isolated retinal EC from bcl‐2 +/+ and bcl‐2 −/− mice. Retinal EC lacking bcl‐2 demonstrated reduced cell migration, reduced tenascin‐C expression, and reduced adhesion to vitronectin and fibronectin. The bcl‐2 −/− retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay we observed reduced sprouting from aortic rings grown in culture from bcl‐2 −/− mice compared to bcl‐2 +/+ mice. Furthermore, re‐expression of bcl‐2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl‐2−/− retinal EC. Thus, bcl‐2‐mediated functions play an important role in EC migration and capillary morphogenesis during retinal vascular development and angiogenesis.

Human Leukemia and Lymphoma Cell Lines as Models and Resources

Tumor cell lines are widely used as oncologic models and resources, forming, along with primary patient material and animal models, one of three major subjects for cancer investigation. With the advent of the Human Genome Project (HGP) and the ensuing provision of sequencing data and mapped clones, human cancer cell lines, notably those derived from leukemia-lymphoma (LL) have become increasingly productive tools for cancer gene ascertainment and characterization. Hence, the roles of putative novel cancer genes may be investigated using diverse panels of LL cell lines, both individually by PCR-based methods, and globally by transcriptional chip-profiling. Similar studies have also enabled the faithfulness with which cancer cell lines model their supposed in vivo counterparts to be quantified at last. Several recent transcriptional profiling studies indicate that of all tumor types well characterized human LL cell lines most accurately model the gene expression patterns of their corresponding primary tumors. Analysis using genomic arrays tells a similar story for the stability of chromosome rearrangements in LL cell lines. Well characterized LL cell lines also provide ideal tools for investigating the druggability of individual gene products, e.g. by measuring their transcript levels using q(uantitative)-PCR methods in cells subjected to treatments with small interfering (si)-RNAs. We provide a list of authentic, well characterized examples for prospective investigators, since many circulating cell lines have been cross-contaminated and describe DNA profiling methods which, together with classic and molecular cytogenetic analyses, inform authentication. We also review the problem of mycoplasma contamination and means for its eradication.

Increased microvessel density in involved organs from patients with HTLV-I associated adult T cell leukemia lymphoma

Adult T-cell leukemia-lymphoma (ATLL) is a rapidly progressive lymphoproliferative disorder secondary to infection with the human T cell lymphotropic virus type I (HTLV-I). The role of angiogenesis in the development and prognosis of many hematologic malignancies is established. We have previously shown that ATLL derived cells secrete high levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF), induce endothelial tube formation in vitro and establish functional gap junction-mediated communication with endothelial cells. We also demonstrated that plasma from ATLL and tropical spastic paraparesis/HTLV-I associated myelopathy patients exhibit very high levels of VEGF and b-FGF. Recently, we showed that treatment with the combination of zidovudine and interferon alpha reduced both HTLV-I proviral load and importantly VEGF plasma levels suggesting a potential anti-angiogenic effect of this therapy. In this report, we evaluated microvessel density (MVD) in involved organs from 20 patients with ATLL, as compared to normal organs from matched controls. We show evidence of significantly increased MVD in all tested involved organs from ATLL patients, suggesting that angiogenesis plays an important role in the development or organ invasion of ATLL, and could represent a potentially interesting target for anti-angiogenic therapy of ATLL.

Immune Targeting of the Phosphoproteome in Lymphoma and Leukemia.

CD8+ T cells have been identified as potent effectors of the adaptive anti-tumor immune response. So far, only a small number of tumor antigens have been objectively linked to the oncogenic processes. Alteration in phosphorylation status of cellular proteins is a hallmark of malignant transformation and a proven important therapeutic target. Phosphorylated antigens thus represent attractive targets for immunotherapeutic development based on the hypothesis that immune escape through mutation may be more difficult without compromising the malignant phenotype. Using a mass spectrometry approach phosphopeptide display was analyzed in CLL, AML, ALL, hairy cell leukemia and mantle cell lymphoma samples and compared against normal tissue. 109 or more primary tumor cells from HLA-A*0201+ or HLA-B*0702+ patients were lysed and peptides extracted from the surface HLA molecules and phosphopeptides isolated using in-line immobilized metal ion affinity chromatography and analyzed using tandem mass spectrometry using previously established methodology (Zarling A.L. et al PNAS 2006). 55 HLA-B*0702 phosphopeptides and 12 HLA-A*0201 phosphopeptides have been characterized. Many of these phosphopeptides are derived from phosphoproteins known to function in signaling cascades implicated in neoplastic transformation including MAP kinase pathway signaling proteins, c-Myc, NFAT and Bcl-11 with many representing novel phosphorylation sites. Peptide binding assays reveal IC50 binding affinities ranging between 4 and 300nM within the immunogenic range for the subset of phosphopeptides investigated. To investigated the mechanism of binding X-ray crystallography was performed in combination of peptide binding assays. Six HLA-phosphopeptide structures were resolved and demonstrate upward facing phosphate moiety directly interacting with HLA alpha helix residues yet with potential for direct recognition by the TCR. Peptide binding data support contributions from phosphate group in binding to the HLA in some but not all phosphopeptides tested and these data support a novel non-canonical binding mechanism for phosphopeptides complexed with HLA-A*0201. We have used mice transgenic for HLA-A*0201 and HLA-B*0702 to demonstrate in vivo immunogenicity of a subset of these peptides in a dendritic cell-based vaccination model. T cells generated differentially recognized phosphopeptides over non-phosphopeptide counterparts. Phosphopeptide-specific primary T cell lines were also generated ex-vivo from healthy lab donors which bound HLA-phosphopeptide tetramers and recognized HLA-matched primary tumor samples. In summary this work characterizes phosphopeptides that are differentially presented on primary leukemia and lymphoma samples by class I MHC molecules. These post-translationally modified peptide antigens represent distinct antigenic determinants which may overcome barriers of immune tolerance and autoimmunity inherent with other tumor antigens. These phosphopeptides therefore represent attractive novel candidates for future cancer immunotherapy.

Eltrombopag Decreases Proliferation of Human Leukemia and Lymphoma Cell Lines In Vitro.

Eltrombopag is a novel, oral platelet growth factor that interacts with the thrombopoietin receptor on bone marrow progenitors to stimulate megakaryocyte and platelet production. Clinical trials have demonstrated that eltrombopag increases platelet counts in healthy volunteers and in patients with idiopathic thrombocytopenic purpura (ITP) and hepatitis C. Because thrombopoietin (TPO) receptors are expressed on lymphoma and leukemia cell lines and recombinant human TPO (rhTPO) has been shown to stimulate their proliferation in vitro, experiments were conducted to evaluate the effects of eltrombopag on the proliferation of leukemia and lymphoma cells in vitro. Five leukemia and lymphoma cell lines (ie, CCRF-CEM, K562, MOLT-4, RPMI-8226 and SR) representing lymphoblastic T cell leukemia, chronic myelogenous leukemia, acute lymphoblastic T cell leukemia, plasmacytoma and immunoblastic large cell lymphoma, were exposed to eltrombopag (0.1–40 ug/mL), rhTPO (100 ng/mL) or both for 3 days. Proliferation was measured by 18-hour incubation with tritiated thymidine incorporation. As expected, rhTPO alone led to a small, statistically significant increase in the proliferation of CCRF-CEM and RPMI-8226 cells, while it had no effect on the other cell lines. However, the addition of eltrombopag to rhTPO negated the increased proliferation seen with rhTPO alone. Even more remarkable, treatment with eltrombopag alone inhibited the proliferation of all five leukemia and lymphoma cell lines with an IC50 range of 0.56 to 5.9 ug/mL and 100% inhibition (IC100) of thymidine incorporation at 10 ug/mL (Table). Previous studies have shown that eltrombopag induces proliferation and differentiation of bone marrow progenitor cells in vitro and increases platelet counts in vivo. While eltrombopag and rhTPO interact with the TPO receptor to stimulate the production of platelets, there are differences in the site of receptor interaction and their signaling pathways. The findings of the current study suggest that eltrombopag may inhibit the proliferation of leukemia and lymphoma cell lines unlike the effect that has been demonstrated with rhTPO. Interestingly, eltrombopag may also serve to mitigate rhTPO-mediated proliferation of malignant hematologic cell lines. These findings merit further evaluation of the effects of eltrombopag on leukemia, lymphoma, and solid tumor cell proliferation.Cell lineIC50 (ug/mL)IC100 (ug/mL)CCRF-CEM0.7410K5621.8010MOLT-40.564RPMI-82265.9010SR0.774

HTLV-1 Adult T Cell Leukemia-Lymphoma Presenting with Refractory Hypercalcemia, Cranial Neuropathy and Diagnosis by Flow Cytometry and Cytogenetic Findings.

Introduction: ATLL) is a postthymic lymphoproliferative neoplasm of T cells caused by human T-cell lymphotropic virus (HTLV-1). Most cases are found in Japan, the Caribbean basin, and West Africa.ATLL is essentially a disease of adults, characterized by generalized lymphadenopathy, hepatosplenomegaly, skin lesions, and hypercalcemia. The prognosis of most patients is quite poor, with a median survival time of only 13 months, even with combination chemotherapy. The acute form had a significantly shorter survival (2 months) than lymphoma type (13 months)Advanced performance status, high lactic dehydrogenase, age > 40 years, increased total involved lesions and hypercalcemia, are associated with shortened survival (p<0.01). Hypercalcemia is observed in >80% of ATL. CASE Report:A 43 years old carribean male presented with refractory hypercalcemia 18 g/dl wihtout hematological manifestations,lymphadenopathy or hepatosplenomegaly. He had normal skeletal survey,serum immunoglobulins,immunofixation.The patient also had normal PTH,PTHrp,Vitamin D3,angiotensin converting enzyme levels and 24 hour urinary calcium.The patient had Hemoglobin 13.5 and absolute lymphocytosis 5000–6000/cubic mm.Peripheral smear showed clover T cells Bone marrow had normal morphology but flow cytometry revealed T cells immunoreactive for CD3, CD4,CD8, CD5, and CD25 and were nonimmunoreactive for CD7 .The patient developed visual disturbances, swllowing difficulty and facial palsy during hospital stay.MRI brain was normal but CSF flow cytometry was positive for CD2, CD3, CD4, CD8,CD5, and CD25 and were negative for CD7. Cytogenetic studies showed complex karyotype with: 47, XY,add(4)(p12),+add(4)(p12),add(5)(q11.2),add(7)(q31),–12,Add(12)(q22),der(13)t(5;13)(q11.2;q22),add(14)(q32),add(17)(q23) +mar1(11)/48,idem,+mar1(5)/46,XY(4).A Hyperdiploid male karyotype with monosomy for chromosome No.12,unbalanced translocation involving the long chromosome Nos. 5 and 13, additional material on long arm of 5,7, 12, 14(14q+chromosome) and 17 Complex karyotypes with numerical and structural abnormalities with six or more breaks involving the long arms of chromosomes Nos. 1, 3,6,7,12 and 14q 32 and the short arm of chromosome Nos. 10, 17 and 21 are reported in ATLL .T cell gene rearrangement confirmed the diagnosis.The patient had history of Strongyloides stercoralis infection 8 years ago.He was HIV negativebut had HTLV-1 antibodies(gp19,gp21).He was treated with EPOCH regimen along with intrathecal treatment with normal repeat cytology and CSF flow cytometry.The patientis planned for allogeneic transplant after completion of chemotherapy.Discussion: Adult T-cell lymphoma virus type 1, is implicated diseases including HTLV 1 associated myelopathy,Strongyloides stercoralis hyperinfection, and adult T leukemia/lymphoma. The virus activates a subset of T helper Th1cells with suppression of the Th2 lymphocytes and a reduction of Th2 cytokine production of IL-4, IL-5, IL-10 and IL-13 with an inadequate immune response to invading parasitic infections such as Strongyloides stercoralis. Dual positivity for CD4 and CD8 cells thus confers better prognosis. [Display omitted]