Low CD20 Expression Research Articles

CD44 enhances tumor formation and lung metastasis in experimental osteosarcoma and is an additional predictor for poor patient outcome

Formation of metastases in the lungs is the major cause of death in patients suffering from osteosarcoma (OS). Metastases at presentation and poor response to preoperative chemotherapy are strong predictors for poor patient outcome. The elucidation of molecular markers that promote metastasis formation and/or chemoresistance is therefore of importance. CD44 is a plasma membrane glycoprotein that binds to the extracellular matrix component hyaluronan (HA) and has been shown to be involved in metastasis formation in a variety of other tumors. Here we investigated the role of CD44 expression on OS tumor formation and metastasis. High CD44 expression, evaluated with a tissue microarray including samples from 53 OS patients and stained with a pan-CD44 antibody (Hermes3), showed a tendency (p < 0.08) to shortened overall survival. However, nonresponders and patients with lung metastases and high CD44 expression had significantly poorer prognosis than patients with low CD44 expression. Overexpression of the standard CD44 isoform (CD44s) and its HA-binding defective mutant R41A in osteoblastic SaOS-2 cells resulted in HA-independent higher migration rates and increased chemoresistance, partially dependent on HA. In an orthotopic mouse model of OS, overexpression of CD44s in SaOS-2 cells resulted in an HA-dependent increased primary tumor formation and increased numbers of micrometastases and macrometastases in the lungs. In conclusion, although CD44 failed to be an independent predictor for patient outcome in this limited cohort of OS patients, increased CD44 expression was associated with even worse survival in patients with chemoresistance and with lung metastases. CD44-associated chemoresistance was also observed in vitro, and increased formation of lung metastases was found in vivo in SCID mice.

Rituximab Sensitivity to De Novo DLBCL Cells Showing the Specific Phenotype of CD20 Protein Immunohistochemistry-Positive / Flow Cytometory-Negative: Analyses of Its Clinical Significances and the Molecular Mechanisms.

AbstractAbstract 2682 Background:CD20 is a critical molecular target of a chimeric monoclonal antibody, rituximab, and expression of this protein can be detected in most of B-cell lymphoma cells by immunohistochemistry (IHC) and/or flow cytometry (FCM). Rituximab administration is considered when CD20 protein expression can be confirmed by IHC and/or FCM; however some patients show a CD20 IHC(−)/FCM(−) phenotype before and after rituximab treatment, and this phenomenon is thought to be strongly correlated with rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009). Recently, we encountered newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients with a CD20(+) phenotype on IHC, but a CD20(−) phenotype on FCM. Neither the molecular mechanism for this phenotype nor its clinical significance, including rituximab resistance and poor prognosis, have been clarified for these patients. Aims:To determine the frequency and the clinical features, including prognosis, of newly diagnosed DLBCL with the CD20 IHC(+)/FCM(−) phenotype, the molecular mechanisms of this phenotype in primary DLBCL cells, and the effectiveness of rituximab in vitro. Methods:Primary DLBCL patients (n = 100) were enrolled in this analysis. Anti-CD20 antibodies, L26 and B9E9, were used for the IHC and FCM analyses, respectively. Alexa488-labeled rituximab was also used for the FCM analysis. Genomic DNA, mRNA, and total protein were extracted from primary DLBCL cells showing the CD20 IHC(+)/FCM(−) phenotype, and subjected to DNA sequencing, quantitative RT-PCR, and immunoblotting. Primary DLBCL cells were used for an in vitro CDC assay with rituximab. Prognoses of the patients were statistically compared by Kaplan-Meier survival analysis. Results and Discussion:Both IHC and FCM analyses were performed for 38 of the 100 primary DLBCL patients; of these 38 cases, 8 cases (21.1%) showed CD79a(+)/L26(+) on IHC and CD19(+)/CD20(−)on FCM results. Quantitative RT-PCR indicated that MS4A1 (CD20) mRNA expression was significantly lower (p = 0.0005) in these patients cells than that in CD20 IHC(+)/FCM(+) primary DLBCL cells. Immunoblotting analysis showed that CD20 protein expression was relatively lower in these patient cells than that in the positive control cells, although no differences in length could be detected. No genetic mutations in the coding sequence of the MS4A1 gene were detected in any samples examined in the DNA sequencing analysis. FCM analysis using PE-labeled B9E9 antibody showed that the mean fluorescent intensity (MFI) of CD20 IHC(+)/FCM(−) cells was significantly lower than that of IHC(+)/FCM(+) cells (p = 0.03). When Alexa488-labeled rituximab was utilized, a similar result was obtained, but the difference in MFI between IHC(+)/FCM(+) and IHC(+)/FCM(−) cells was much smaller (p = 0.21), suggesting that the sensitivity of CD20 protein recognition by rituximab was much higher than that by B9E9. Primary DLBCL cells showing CD20 IHC(+)/FCM(+) (n = 13) and IHC(+)/FCM(−) (n = 5), and cell lines showing IHC(−)/FCM(−) after using rituximab (n = 2) were analyzed by in vitro CDC assay, partially confirming rituximab-induced cytotoxicity in IHC(+)/FCM(−) cells and showing that the effectiveness of rituximab on IHC(+)/FCM(−) cells was much lower than on IHC(+)/FCM(+) cells. No cytotoxicity of rituximab was detected in IHC(−)/FCM(−) cells. Prognoses of the IHC(+)/FCM(−) (n = 8) and IHC(+)/(+) (n = 28) patients were compared by Kaplan-Meyer analysis, and no significant differences were found (p = 0.40). These data suggest that rituximab is partially effective even in DLBCL cells with a CD20 IHC(+)/FCM(−) phenotype, and that combination chemotherapy with rituximab may overcome the lower effectiveness of rituximab on these cells in the clinical setting. Further accumulation of patient data and longer follow-up durations are required. Conclusion:Lower expression of MS4A1 mRNA may be one of the critical reasons for the CD20 IHC(+)/FCM(−) phenotype. FCM analysis using fluorescent-labeled rituximab may enable detection of cell populations with lower CD20 expression and partial sensitivity to rituximab. Utilization of rituximab may be recommended even for the DLBCL patients showing a CD20 IHC(+)/FCM(−) phenotype. Disclosures:Kinoshita:Chugai Pharmaceutical Co., LTD.: Honoraria, Research Funding; Zenyaku Kogyo: Honoraria. Naoe:Chugai Pharma.: Research Funding; Zenyaku-Kogyo.: Research Funding.

Ofatumumab (OFA) Is a Novel Anti-CD20 Monoclonal Antibody (mAb) with Improved Anti-Tumor Activity in Vitro and in Vivo in Mantle Cell Lymphoma (MCL) Pre-Clinical Models.

AbstractAbstract 2757MCL is typically characterized by an aggressive clinical course and inevitable development of refractory disease despite early intervention that often includes: immunotherapy (e.g., rituximab), multi-agent induction chemotherapy and consolidation with high dose chemotherapy and autologous stem cell transplant in first remission. Residual disease at the time of stem cell collection is an important cause for treatment failure. There is a need to evaluate more potent anti-CD20 mAbs capable to kill lymphoma cells with low CD20 surface levels. In Burkitt’s lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) pre-clinical models we previously demonstrated that OFA was more potent than rituximab (RIT) in vitro and in vivo. In order to characterize the activity of OFA against MCL, we evaluated the activity of OFA against cytarabine (Ara-C)-sensitive (eg. Mino, Jeko-1, Rec-1, HBL-2, Granta and Z-138); –resistant MCL cell lines (eg. MinoAraCR, Jeko-1AraCR, Rec-1AraCR, HBL-2AraCR and GrantaAraCR); and primary tumor cells derived from MCL patients (n=2). Antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) were measured by standard 51Cr release assays in MCL exposed to OFA, RIT or isotype control. OFA vs. RIT direct anti-proliferative effects were measured in by alamar blue reduction assay. Apoptosis following in vitro exposure to OFA or RIT was detected by caspase 3/PARP cleavage. Patient tumor cells were isolated from biopsy specimens by negative selection using magnetic beads and incubated with OFA or RIT +/− human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Surface CD20 and the complement inhibitory proteins (CIPs) (CD55 and CD59) density in MCL cell lines was determined by flow cytometry (Image stream) and compared to BL or DLBCL cell lines. For in vivo experiments 6–8 week-old SID mice were inoculated subcutaneously with 5×106 matrigel suspended Z-138 cells. Upon tumor engraftment, mice were assigned to RIT (10mg/kg), OFA (10mg/kg) or control groups. Tumor growth curves were calculated for each group. Mice were sacrificed if tumor size reached >2cm in any dimension. After 6 months, survival was analyzed by Kaplan-Meier analysis and compared by log-rank test. OFA induced significantly higher levels of CDC associated cell lysis compared to RIT in almost all MCL cell lines tested (10/11) (Mino: 53.2% vs 0.2%; MinoAraCR: 72.6% vs. 0.6%; Jeko-1: 33.4% vs. 9.8%; Jeko-1AraCR: 38.3% vs. 2.8%; REC-1: 17% vs 3%; Rec-1AraCR: 7.8% vs. 0.2%; HBL-2: 27.1% vs. 19.2%; HBL-2AraCR: 86.6% vs. 72.2%; GrantaAraCR: 17% vs 0.9%; Z-138: 56.4% vs. 0.65%; all p-values <0.05). No differences in RIT or OFA mediated ADCC or direct signaling was observed. As previously noted in BL and DLBCL models, OFA was capable of inducing a higher degree of CMC even at low CD20 levels in contrast to RIT. In vivo, OFA slowed tumor growth, and prolonged survival in Z-138 bearing SCID mice compared to RIT (median survival for RIT was 127 days vs. not reached for OFA treated animals; p<0.05). Our data suggest that, OFA is more potent than RIT against Ara-C-sensitive and –resistant MCL cells in vitro, delays tumor growth and prolongs survival compared to RIT in an in vivo MCL SCID mouse model, and retains CDC activity despite low CD20 and high CIP surface expression levels. OFA appears to be a promising mAb targeting CD20 in MCL and is undergoing clinical testing in the front-line setting (NCT01527149). Disclosures:Czuczman:Genmab: Unrestricted Research Grant Other.

SGN-CD33A: A Novel CD33-Directed Antibody-Drug Conjugate, Utilizing Pyrrolobenzodiazepine Dimers, Demonstrates Preclinical Antitumor Activity Against Multi-Drug Resistant Human AML

AbstractAbstract 3589Outcomes for patients with acute myeloid leukemia (AML) are poor, highlighting the need for novel treatment options. Most AML cells express the myeloid differentiation antigen CD33, making CD33-targeted therapy a potential treatment strategy. Gemtuzumab ozogamicin (GO), an anti-CD33 monoclonal antibody (mAb) conjugated to the cytotoxic agent calicheamicin, has recently been shown to improve survival in newly diagnosed patients with more favorable-risk AML but has insufficient activity in those with poor prognostic features as well as in relapsed disease.Here we report the preclinical testing of a novel CD33-directed antibody-drug conjugate, SGN-CD33A, consisting of a humanized anti-CD33 mAb with 2 engineered cysteine residues through which pyrrolobenzodiazepine (PBD) dimer drug moieties are conjugated via a maleimidocaproyl valine-alanine dipeptide linker. PBD dimers exert their biological activity by covalent binding and interstrand cross-linking of DNA. Fluorescence microscopy studies showed that SGN-CD33A is rapidly internalized and traffics to lysosomes within hours of binding to CD33-positive AML cell lines. Following uptake, SGN-CD33A induces DNA damage as measured by phosphorylation of histone 2AX, subsequently leading to G2-M cell cycle arrest, disruption of mitochondrial membrane integrity, increased caspase-3 activity, formation of cleaved poly ADP-ribose polymerase, DNA fragmentation and cell death.The anti-leukemic activity of SGN-CD33A was assessed in cytotoxicity assays against 12 AML cell lines and 18 primary AML patient samples of mixed cytogenetic origin (favorable, intermediate and unfavorable) and multidrug resistance (MDR) status. SGN-CD33A was highly active against all AML cell lines tested (mean IC50, 22 ng/ml), including 5 of 5 MDR-positive cell lines (mean IC50, 27 ng/mL). In contrast, GO was moderately active in 1 of 5 MDR-positive cell lines (IC50, 227 ng/mL) but inactive against the other 4 (IC50, >1000 ng/mL). SGN-CD33A was also active against 15 of 18 primary samples isolated from untreated AML patients at diagnosis (mean IC50 of responsive samples, 8 ng/mL) and was more potent than GO which was active in 10 of 18 AML samples (mean IC50 of responding samples, 27 ng/mL). The 3 AML specimens that were resistant to SGN-CD33A each had low or absent CD33 expression, as determined by flow cytometry. Cytogenetic abnormalities and MDR activity did not correlate with in vitro SGN-CD33A cytotoxicity.In vivo antitumor activity was evaluated in AML mouse xenograft models established with MDR-negative HL-60 and MDR-positive THP-1 and TF1-α cell lines. SGN-CD33A dosed once at 300 mcg/kg yielded durable complete regressions in THP-1 (Figure A) and TF1-α xenografts. In these models of drug-resistant AML, treatment with a single dose of 100 mcg/kg SGN-CD33A significantly delayed tumor growth compared to untreated and non-binding control ADC-treated mice (p<0.001), whereas GO was inactive even when dosed at 1000 mcg/kg. In the MDR-negative HL-60 model, a single dose of 30 mcg/kg of SGN-CD33A delayed tumor growth and 100 mcg/kg induced complete tumor regression (Figure B). In contrast, GO had minimal activity when dosed at 100 mcg/kg but did result in durable tumor regression when administered at a ten-fold higher dose (Figure B).Together, these data demonstrate that SGN-CD33A exhibits antitumor activity against a broad panel of primary AML samples and results in durable remissions in preclinical models of MDR-positive AML that are characteristically resistant to conventional chemotherapy and GO. CD33-directed delivery of PBD dimers may overcome multidrug resistance and may represent a new strategy for the treatment of patients with AML. Clinical trials are planned to further evaluate SGN-CD33A in AML. [Display omitted] Disclosures:Sutherland:Seattle Genetics, Inc.: Employment. Walter:Seattle Genetics, Inc.: Consultancy, Research Funding. Jeffrey:Seattle Genetics, Inc.: Employment. Burke:Seattle Genetics, Inc.: Employment. Yu:Seattle Genetics, Inc.: Employment. Stone:Seattle Genetics, Inc.: Employment. Ryan:Seattle Genetics, Inc.: Employment. Sussman:Seattle Genetics, Inc.: Employment. Zeng:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc.: Employment. Bernstein:Seattle Genetics, Inc.: Consultancy. Senter:Seattle Genetics, Inc.: Employment. Drachman:Seattle Genetics, Inc.: Employment. McEarchern:Seattle Genetics, Inc.: Employment.

T regulatory cells: regulating both term and preterm labor?

T regulatory cells: regulating both term and preterm labor?

An IgG3 switch variant of rituximab mediates enhanced complement-dependent cytotoxicity against tumor cells with low CD20 expression levels

An IgG3 switch variant of rituximab mediates enhanced complement-dependent cytotoxicity against tumor cells with low CD20 expression levels

Alteraciones en fagocitos mononucleares: un viraje al significado de la muerte de monocitos y macrófagos en la inmunopatogénesis de la tuberculosis

Mononuclear phagocytes, particularly monocytes and macrophages, have been considered the primary targets of infection by Mycobacterium tuberculosis and, at the same time, the main effector cells and reservoirs of mycobacteria. The heterogeneity of these cells and their plasticity, make diverse and complex the responses against the infection. The differences in mononuclear phagocytes responses in animals, the expression of genes associated with natural resistance to infection, and the discrepancy between circulating monocytes from patients with tuberculosis and healthy controls in the expression of CD14, CD36 and HLA-DR, and cell death, have suggested changes in the distribution of monocyte subpopulations or in their state of maturity.In humans, subpopulations of peripheral blood monocytes were defined according to the expression of surface molecules CD14 and CD16. There are two main types of monocytes, a group with high expression of CD14 that does not express CD16 (CD14+CD16-) and others with low expression of CD14, expressing CD16 (CD14+CD16+). In tuberculosis patients there are alterations in the counts of these subpopulations, which lead us to consider that these alterations could be capital phenomenon to understand the immunopathogenesis of this disease.

Aging is associated with chronic innate immune activation and dysregulation of monocyte phenotype and function

Chronic inflammation in older individuals is thought to contribute to inflammatory, age-related diseases. Human monocytes are comprised of three subsets (classical, intermediate and nonclassical subsets), and despite being critical regulators of inflammation, the effect of age on the functionality of monocyte subsets remains to be fully defined. In a cross-sectional study involving 91 healthy male (aged 20-84 years, median 52.4) and 55 female (aged 20-82 years, median 48.3) individuals, we found age was associated with an increased proportion of intermediate and nonclassical monocytes (P = 0.002 and 0.04, respectively) and altered phenotype of specific monocyte subsets (e.g. increased expression of CD11b and decreased expression of CD38, CD62L and CD115). Plasma levels of the innate immune activation markers CXCL10, neopterin (P < 0.001 for both) and sCD163 (P = 0.003) were significantly increased with age. Whilst similar age-related changes were observed in both sexes, monocytes from women were phenotypically different to men [e.g. lower proportion of nonclassical monocytes (P = 0.002) and higher CD115 and CD62L but lower CD38 expression] and women exhibited higher levels of CXCL10 (P = 0.012) and sCD163 (P < 0.001) but lower sCD14 levels (P < 0.001). Monocytes from older individuals exhibit impaired phagocytosis (P < 0.05) but contain shortened telomeres (P < 0.001) and significantly higher intracellular levels of TNF both at baseline and following TLR4 stimulation (P < 0.05 for both), suggesting a dysregulation of monocyte function in the aged. These data show that aging is associated with chronic innate immune activation and significant changes in monocyte function, which may have implications for the development of age-related diseases.

Transcript profiling of CD16‐positive monocytes reveals a unique molecular fingerprint

CD16-positive (CD14(++) CD16(+) and CD14(+) CD16(++) ) monocytes have unique features with respect to phenotype and function. We have used transcriptional profiling for comparison of CD16-positive monocytes and classical monocytes. We show herein that 187 genes are greater than fivefold differentially expressed, including 90 genes relevant to immune response and inflammation. Hierarchical clustering of data for monocyte subsets and CD1c(+) myeloid blood dendritic cells (DCs) demonstrate that CD16-positive cells are more closely related to classical monocytes than to DCs. Reverse transcriptase polymerase chain reaction for ten genes with the strongest differential expression confirmed the pattern including a lower messenger RNA level for CD14, CD163, and versican in CD16-positive monocytes. The pattern was similar for CD16-positive monocytes at rest and after exercise mobilization from the marginal pool. By contrast, alveolar macrophages, small sputum macrophages, breast milk macrophages, and synovial macrophages all showed a different pattern. When monocyte-derived macrophages (MDMs) were generated from CD16-positive monocytes by culture with macrophage colony-stimulating factor in vitro, then the MDMs maintained properties of their progeny with lower expression of CD14, CD163, and versican compared with CD14(++) CD16(-) MDMs. Furthermore, CD16-positive MDMs showed a higher phagocytosis for opsonized Escherichia coli. The data demonstrate that CD16-positive monocytes form a distinct type of cell, which gives rise to a distinct macrophage phenotype.

High expression of tumor-infiltrating macrophages correlates with poor prognosis in patients with diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is characterized by its clinical and biological heterogeneity. Although the International Prognostic Index (IPI) provides a clinical model for risk stratification of patients with DLBCL, notable variability in outcome is still observed within the same IPI category. Tumor-infiltrating macrophages (also called Tumor-associated macrophages) are the major component in the microenvironment of DLBCL. Their correlation with the prognosis of DLBCL remains controversial. Using a CD68 antibody in immunohistochemical analysis, we studied the expression of CD68 in 112 Chinese patients with DLBCL, with 65 patients (58%) categorized as low CD68 expression and 47 patients (42%) as high CD68 expression. The complete response (CR) rate of patients with low CD68 expression was higher than that with high CD68 expression (66.1% vs. 51.6%), but there was no statistical significance (P=0.060). The median survival time of patients with low CD68 expression was not achieved and that of high expression was 41 months (P=0.029). The results suggest that higher expression of CD68 tended to yield poor treatment outcome of DLBCL.

Type‐I IFN drives the differentiation of short‐lived effector CD8+ T cells in vivo

Two subsets of CD8(+) T cells are generated early during an immune response; one of these subsets forms the memory pool, known as memory precursor effector cells (MPECs), identified by high expression of CD127 and low expression of KLRG1, whereas the other subset forms short-lived effector cells (SLECs) identified by low expression of CD127 and high expression of KLRG1. Here, we studied in vivo the role of type-I IFN in this fate decision. We found that under priming conditions dominated by type-I IFN, as observed in lymphocytic choriomeningitis virus (LCMV) infection, type-I IFN signaling directly impacted the regulation of T-bet and thus the early fate decision of CD8(+) T cells. In the absence of type-I IFN signaling, CD8(+) T cells failed to form SLECs but could form MPECs that give rise to functional memory CD8(+) T cells. Together, these findings identify type-I IFN as an important factor driving SLEC differentiation and thus instructing the early division between the effector and memory precursor CD8(+) T-cell pool.

Probiotic bacterial strains differentially modulate macrophage cytokine production in a strain-dependent and cell subset-specific manner

Gut mucosal macrophages play a pivotal role in driving mucosal immune responses, resulting in either activation of inflammatory immune responses to pathogenic challenge or tolerance to beneficial luminal contents such as food and commensal bacteria. Macrophage responses elicited are dependent on tissue environment and the resulting cell subset, where homeostatic macrophages resemble the M2 macrophage subset and inflammatory macrophages resemble M1s. Probiotics can modulate macrophage function with outcome dependent on subset present. Using a THP-1 monocyte cell line-derived model of CD14high/low M1 and M2 macrophages, the aim of this study was to investigate the immunomodulatory effects of a panel of heat-killed probiotic bacteria and their secreted proteins on the subset-specific inflammatory marker profile of TNFα, IL-6 and NFκB. M1 and M2 cells were generated by differentiation of monocyte stable transfectants for high and low CD14 expression with phorbol 12-myristate 13-acetate and vitamin D3, respectively, where the resulting CD14lo M2 and CD14hi M1s mimicked homeostatic and inflammatory mucosal macrophages. Subsets were stimulated by enteropathic lipopolysaccharides in the presence or absence of heat-killed (HK) or secreted proteins (SP) from a panel of probiotic bacteria. Regulation of cytokine expression was measured by ELISA and NFκB activity by reporter assay. HK probiotics suppress CD14lo and augment CD14hi M1 and M2 production of TNFα whereas SPs augmented CD14hi M1 TNFα and were generally suppressive in the other subtypes. M2 macrophage IL-6 production was suppressed by both HK and SPs and differentially regulated in CD14lo and CD14hi M1s. NFκB activation failed to parallel the regulatory profiles for TNFα and IL-6 which is suggestive of probiotic bacteria exerting their regulatory effects on these cytokines in an NFκB-independent manner. In conclusion, HK and SP probiotics differentially regulate macrophage cytokines and NFκB activation in a subset-dependent manner and suggest a cautionary approach to probiotic treatment of mucosal inflammation.

Altered expressions of embryonic stem-related genes in pancreatic cancer stem cell

To explore the altered expressions of embryonic stem-related genes Oct4 and Nanog in pancreatic cancer stem cells (CSCs). The uni-cell suspension of human pancreatic cancer cell line PANC-1 was prepared and incubated with CD24 and CD44 antibodies. Flow cytometer was used to separate CD24(+)CD44(+) pancreatic cancer stem cells. Tumor cell spheres were observed under light microscope. Then CSCs were induced to differentiate with 10% fetal bovine serum and the expressions of CD24 and CD44 re-evaluated by flow cytometer. Finally the cells were divided into 2 groups, group 1: CD24(+)CD44(+) and group 2: non-separated group. RT-PCR (reverse transcription-polymerase chain reaction) and Q-PCR (quantitative-polymerase chain reaction) were used to examine the transcriptions of Oct4 and Nanog in CSCs. The immunofluorescence was employed to examine the expressions of Oct4 and Nanog. Chemo-sensitivity to gemcitabine was determined by CCK8 assay in each group. About 1%-3% CD24(+)CD44(+) CSCs were separated from cell line PANC-1. The sorted cells were cultured in a stem cell culture medium to observe the spheroid-forming capacity. And they showed a higher colony-forming efficiency than the unsorted cells [(122 ± 6)‰, P < 0.05]. When cultured in medium with serum, these cells gradually returned to the status of parental cells with a low expression of CD24 and CD44. Both Oct4 and Nanog were highly expressed in CD24(+)CD44(+) stem cells. And the CD24(+)CD44(+) subgroup demonstrated a higher resistance to gemcitabine. Subpopulation cells CD44(+)CD24(+) have the properties of tumor stem cells. The up-regulated levels of Oct4 and Nanog may be highly correlated with the multi-potency and a higher drug-resistance of pancreatic CSCs.

Clinical Relevance of Immunophenotypic Characteristics in Chronic Lymphocytic Leukemia

Abstract 4604 Background:Chronic lymphocytic leukemia (CLL) has the highest incidence and prevalence of any leukemia in the United States. It has a characteristic immunophenotype, but variant forms (typically termed “atypical CLL”) are commonly encountered. It is not known if this immunophenotypic heterogeneity in CLL is clinically relevant. We hypothesized that the immunophenotype of CLL cells correlates with both biologic parameters and clinical outcomes. Methods:We performed a retrospective review of clinical, biologic, and flow cytometric data of patients undergoing an evaluation of an absolute lymphocytosis at the Duke University and Durham VA Medical Centers. Patients meeting immunophenotypic criteria for CLL based on cell surface expression of CD5, CD19, CD20, CD23, and surface immunoglobulin light chain (sIg) were included in this analysis. Atypical CLL was defined if any of the following were observed: (1) expression of CD5, CD20, CD23, or sIg that differed from a typical CLL immunophenotype (e.g. bright CD20 or low CD23; hereafter non-standard immunophenotype), (2) expression of T cell markers CD7 or CD8, or (3) expression of CD123. Through electronic chart review, clinical and biologic parameters were abstracted. These included age; gender; ethnicity; WBC, hemoglobin, platelet count, and Rai stage at diagnosis; time to first treatment; overall survival; CD38, ZAP70, IGHV mutation status, and interphase cytogenetics (FISH). A chi square test or Fisher's exact test for small sample sizes was used to test association of categorical data. The central tendencies of continuous measurement were compared using the Wilcoxon rank sum test. Overall survival and time to treatment were calculated using the Kaplan-Meier product limit method. Survival curves were compared using the log-rank test. This study was approved by IRBs at Duke University and Durham VA Medical Centers. Results:We reviewed 189 patients, including 119 (63%) with typical CLL immunophenotype and 70 (37%) with atypical. Among patients with atypical CLL, 93% (65 of 70) had a non-standard immunophenotype, 33% (23 of 70) had expression of T cell markers, and 33% (14 of 43) had expression of CD123. We observed expression of T cell markers in 12% (23 of 189) and CD123 in 12% (14 of 114) of the entire cohort, respectively. At the time of diagnosis, there was no association between typical or atypical CLL immunophenotype and age, gender, race/ethnicity, WBC, hemoglobin, platelet count, or Rai stage (p>0.05 for all comparisons). With regard to biologic parameters, there was no association of CLL immunophenotype with the proportion of patients with CD38 ≥30%; however, the central tendency of CD38 was higher in the atypical group when analyzed as a continuous variable (median value 38% vs. 10%, p=0.04). There was no difference in the proportion of ZAP70 positive cases between typical and atypical groups. Analysis of interphase cytogenetics (FISH) data showed that a higher percentage of typical than atypical immunophenotypes had del 13q as a sole abnormality and a lower percentage had trisomy 12 (p=0.02). Despite the increased frequency of del 13q as a sole abnormality, lower frequency of trisomy 12, and lower expression of CD38, there was no difference between groups in time to first treatment (median 5.3 years for typical vs. 2.1 years for atypical; p= 0.11) or in overall survival (median 13.6 yrs64 years for typical vs. 18.0 years for atypical; p=0.11) (Figure 1). However, when patients with non-standard CLL immunophenotype were compared to those with a standard immunophenotype, a significantly shorter time to first treatment was observed (median 6.0 years for standard immunophenotype vs. 2.2 years for non-standard, p=0.03). [Display omitted] Conclusion:Different immunophenotypic subtypes are commonly encountered in the care of patients with CLL. These groups have different biological characteristics, including differences in expression of CD38 and cytogenetic abnormalities. Exploratory analysis also showed differences in clinical outcomes based on immunophenotypic parameters. Validation studies are currently underway. Disclosures:No relevant conflicts of interest to declare.

CD20 Protein Immunohistochemistry-Positive/Flow Cytometry-Negative Diffuse Large B-Cell Lymphoma—Analyses of the Molecular Mechanisms and Rituximab Effectiveness

Abstract 2659 Background:Rituximab is a key molecular targeting drug for CD20(+) B-cell lymphoma, and CD20 protein expression is essential in the treatment mechanism of rituximab. We previously reported that the change to the CD20(−) phenotypic after using rituximab is one of the critical mechanisms in rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009; Sugimoto T, Tomita A, et al., Biochem Biophys Res Commun, 2009). Recently, we encountered newly diagnosed patients with diffuse large B-cell lymphomas (DLBCL) which are CD20(+) on immunohistochemistry (IHC) but CD20(−) on flow cytometry (FCM). For these patients, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance have been clarified, and the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims:To confirm the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype using primary DLBCL cells, and to confirm the effectiveness of rituximab in vitro. Methods:Genomic DNA, mRNA, and total protein were extracted from primary DLBCL cells showing the CD20 IHC(+)/FCM(−) phenotype, and subjected to DNA sequencing, quantitative RT-PCR, and immunoblotting. L26 and B1 anti-CD20 antibodies were used in IHC and FCM analyses, respectively. Primary DLBCL cells were transplanted into NOD-SCID mice, and the engrafted lymphoma cells were used for in vitro CDC assay using rituximab. Results and Discussion:Ten patients with primary DLBCL had CD79a(+)/L26(+) results on IHC and CD19(+)/CD20(−) results on FCM. Quantitative RT-PCR indicated that CD20 mRNA expression that was significantly lower (almost 10 times) than that of positive control primary DLBCL cells. Immunoblotting analysis showed that CD20 protein expression was relatively lower than that of positive control cells, and no differences in length could be detected. No genetic mutations in the coding sequence of CD20 gene were detected in all samples examined in DNA sequencing analysis, suggesting that CD20 partial deletion is not the main reason for this phenotype. Interestingly, FCM analyses using fluorescent-labeled rituximab indicated that rituximab could partially recognize FCM CD20-B1(−)/CD19(+) B-cells, suggesting that the binding sensitivity of rituximab is much stronger than that of the B1 antibody. Lymphoma cells showing CD20 L26-IHC(+)/B1-FCM(−) phenotype proliferated in NOD-SCID mice and were partially killed by rituximab on in vitro CDC assay. These data suggest that rituximab is effective even for B-lymphoma cells with low CD20 expression if FCM using rituximab can be made to recognize those cell populations. Conclusion:Lower expression of CD20 mRNA may be one of the critical reasons for the CD20 L26-IHC(+)/B1-FCM(−) phenotype. FCM analysis using rituximab may be helpful to detect cell populations that are sensitive to rituximab treatment. Thus, rituximab therapy may be recommended if we can confirm the existence of cell populations recognized by rituximab on FCM. Disclosures:Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo.: Research Funding; Otsuka Pharma.: Research Funding.

A Phase II Trial of Ofatumumab in Subjects with Waldenstrom's Macroglobulinemia,

A Phase II Trial of Ofatumumab in Subjects with Waldenstrom's Macroglobulinemia,

Identification of Patient Subgroups Demonstrating Longer Progression-Free Survival (PFS) Benefit with Bortezomib-Rituximab Versus Rituximab in Patients with Relapsed or Refractory Follicular Lymphoma (FL): Biomarker Analyses of the Phase 3 LYM3001 Study

Abstract 265 Background: Treatment goals in patients with relapsed FL are to prolong PFS and improve overall survival (OS). To optimize treatment for individual patients, identification of subgroups most likely to benefit from a specific therapy is important. The international, randomized, phase 3 LYM3001 study in patients with relapsed or refractory FL demonstrated improved PFS with bortezomib-rituximab vs rituximab alone (median 12.8 vs 11.0 months, HR 0.822, p=0.039), plus increased overall response rate (ORR; 63% vs 49%, p=0.0004), complete response rate (CR/CRu; 25% vs 18%, p=0.035), and durable (≥6 months) response rate (50% vs 38%, p=0.002) in an unselected patient population. Here we present exploratory biomarker analyses aimed at identifying patient subgroups deriving a longer PFS benefit with bortezomib-rituximab and showing a trend for better OS. Methods: Patients received five 5-week cycles of bortezomib-rituximab (N=336) or rituximab (N=340). Response was assessed using modified International Working Group response criteria. Archived tumor tissue was collected at baseline from 502 (74%) patients; whole blood samples for germ-line DNA were collected on day 1 of cycle 1 from 619 (92%) patients. Protocol-specified candidate biomarkers were based on associations with bortezomib (NF-κB p65, PSMA5, p27, PSMB1/5/8/9) or rituximab (CD68, FCGR2A/3A) activity. Immunohistochemistry assays were used for protein analysis. Taqman SNP assays and PCR/LDR were used for genotyping. Statistical analyses included single-marker analyses, pair-wise combination analyses (n=1140 comparisons), and multiple comparison analyses of all evaluable patients in LYM3001. Clinical covariates included in the analysis were baseline FLIPI score, prior rituximab, time since last anti-lymphoma therapy, region, age, gender, race, Ann Arbor stage, high tumor burden, and number of prior lines of therapy. Results: Single markers and biomarker pairs (n=102) highlighted patient subsets that had significantly improved outcomes with bortezomib-rituximab vs rituximab. For 14 of the pairs, the PFS benefit was ≥6 months. Using false discovery rate (FDR) to control for multiple comparison corrections, one biomarker pair was significant. This pair (presence of the PSMB1 P11A C/G heterozygote, and low CD68 expression [0–50 CD68-positive macrophages in the follicular space]) was associated with significantly improved PFS in patients receiving bortezomib-rituximab vs rituximab (median 16.6 vs 9.1 months, HR 0.407, p 2 prior lines of therapy). There was also a trend towards an OS benefit (medians not reached, HR 0.426, p=0.0550), as well as a significantly higher ORR (73.7% vs 47.5%, p=0.0077), a higher CR rate (33.3% vs 23%, p=0.3044), and a significantly longer time to next therapy (median 33.1 vs 14.8 months, p=0.0013). In patients lacking this biomarker pair (N=238) no significant efficacy differences were seen. No other similar studies were available to confirm the reproducibility of these analyses. Therefore, we split the LYM3001 dataset into discovery and confirmation cohorts (7:3 ratio of biomarker-evaluable patients) to enable evaluation and confirmation in independent cohorts of patients The significant biomarker pair of PSMB1 P11A C/G heterozygote and low CD68 was identified in the discovery cohort (N=198) with a PFS advantage with bortezomib-rituximab vs rituximab of 5.7 months (median 14.2 vs 8.4 months, p=0.0003) and an indication of longer OS (HR 0.47, p=0.1291). This biomarker pair also showed a clear PFS advantage in the confirmation cohort (N=108, 8.7-month PFS benefit; median 18.2 vs 9.5 months, HR 0.44, p=0.0817). Other significant biomarker combinations, including combinations of molecular and clinical variables (e.g. high tumor burden) were identified and will be presented. Conclusions: Analyses of the phase 3 LYM3001 trial identified biomarker combinations present in a third of patients offering a significant PFS benefit with bortezomib-rituximab vs rituximab. Use of such biomarker assays in patients with relapsed or refractory FL may aid identification of subgroups deriving maximal benefit from the addition of bortezomib to rituximab therapy. Disclosures: Coiffier:Janssen-Cilag: Consultancy; Roche: Consultancy; Amgen: Consultancy; Sanofi: Consultancy; Pfizer: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy; MedImmune: Consultancy; CTI: Consultancy. Off Label Use: Bortezomib used in combination with rituximab in patients with relapsed/refractory follicular lymphoma. Li:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Henitz:Janssen Research & Development: Employment. Karkera:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Favis:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Gaffney:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Shapiro:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Theocharous:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Elsayed:Janssen Research & Development: Employment; Johson & Johnson: Equity Ownership. de Velde:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Rule:Johnson & Johnson: Advisory Board, Institutional grant, meeting attendance expenses, Honoraria. Walewski:Janssen-Cilag: Institutional/personal grants, advisory board; Hoffman La Roche: Honoraria, Institutional/personal grants, travel/accommodation expenses; Mundipharma: Honoraria; Celgene: Honoraria. de Vos:Millennium Pharmaceuticals, Inc: Consultancy. Crump:Janssen/Ortho-Biotech: Consultancy. Shpilberg:Janssen-Cilag: Consultancy, Honoraria. Cakana:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership. Esseltine:Millennium Pharmaceuticals, Inc: Employment; Johnson & Johnson: Equity Ownership. Mulligan:Millennium Pharmaceuticals, Inc.: Employment. Ricci:Janssen Research & Development: Employment; Johnson & Johnson: Equity Ownership.

CD20, CD22, CD23, but Not CD37 Expression on CD19+ B-Cells Is Altered by Exogenous Factors in a Sub-Population of Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia (CLL/SLL) Patients,

Abstract 3685 Introduction:Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia (CLL/SLL) is a lymphoproliferative disorder that is characterized by the slow accumulation of malignant B cells. Patients follow heterogeneous clinical courses. The effectiveness of Rituximab, an anti-CD20 monoclonal antibody (mAb), is limited because target B cells from CLL patients express low levels of CD20. We previously reported that human serum suppresses CD20 expression ex vivo. Therefore, understanding mechanisms of low expression of CD20 as well as other target surface receptors would benefit CLL patients. New therapies such as epratuzumab (anti-CD22 mAb), lumiliximab (anti-CD23 mAb), and TRU-016 (anti-CD37 SMIP protein) are being investigated as alternative means to treat CLL. We therefore investigated CD20, CD22, CD23, and CD37 expression during ex vivo culture of CLL B cells from thirteen patients with and without human serum albumin (HSA), normal donor serum (NS), autologous CLL serum (CS), fetal bovine serum (FBS), IL2, IL4, IL13, IL15, IL21, TNFα, IFNα, IFNγ, G-CSF, or GM-CSF. Methods: Peripheral blood (PB) from thirteen CLL patients and five healthy normal donors was obtained with IRB approval. Nine CLL patients were Rai 0/1, one Rai 4, and three unknown. Prognosis, as determined by cytogenetics and/or FISH, was: four good, three intermediate, one poor, and five unknown. Beta-2 Microglobulin (B2M) ranged from 1.4–7.1 mg/L. CD19+ cells, isolated by positive magnetic selection, were cultured in AIM-V serum-free media at 37°C, 5% CO2, 100% humidity for 0, 24, 48, and 96 hours. Cells were either untreated, treated with 5% serum, or 5ng/mL cytokine. Receptor expression was measured by multivariate flow cytometry and calculated as percent loss or increase in response to treatment. Data was presented as mean ± SEM and analyzed using the Mann-Whitney U test as compared to HSA treatment. Results: Prior to culture, CD19+ CLL cells had 11.7±1.7% CD20, 40.3±7.9% CD22, 60.6±6.8% CD23, and 81.6±2.7% CD37 positive expression (N=13). Analysis of untreated CLL cells cultured for 96 hours showed that the CLL patients subdivided into two groups based on changes in CD20 expression: variable (72.5±7.4% CD20+, N=9) and stable (27.2±4.2% CD20+, N=4). Within the variable group, losses in CD20 expression resulted from treatment as compared to HSA were: NS (40.2±3.6%, p<0.05), CS (43.6±8.8%, p<0.05), FBS (37.2±5.0%, p=0.064), IL2 (11.5±5.1%, p=0.860), IL4 (33.3±8.9%, p=0.185), and IL13 (24.2±9.1%, p=0.596). The percent loss in CD20 expression due to IL4 was significantly greater than IL2 (p<0.05). Percent losses in CD22 expression were: NS (62.4±9.4%, p<0.05), CS (63.2±8.1%, p<0.05), and FBS (66.6±6.8%, p<0.05). No loss in CD37 expression was significant. Percent increases in CD23 expression were: IL4 (253.7±79.4%, p=0.930) and IL13 (86.7±77.9%, p=0.377). Within the stable group after treatment, percent losses in CD22 were: IL2 (40.0±10.1%, p=1.000) and IL4 (29.1±6.5%, p=0.114). Percent increase in CD37 expression was: FBS (53.3±18.3%, p<0.05). Conclusions: Our findings show that cells from CLL patients form groups based on the ability of cells to maintain low CD20 expression after serum-free culture for 96 hours: variable and stable. Only CLL cells within the variable group have a high upregulation of CD20. These patient samples respond to serum through a reduction of CD20. In addition, suppression of CD20 expression due to IL4 or IL13 treatment was similar to that of serum. Both IL4 and IL13 share a common receptor that signals through the JAK/STAT pathway, suggesting downstream signals may be responsible for suppression of CD20 expression in CLL. Similarly, CLL patients belonging to the variable group also have reduced expression of CD22 in response to serum. Loss of CD23 expression in CLL cells cultured over time, in the absence of IL4 or IL13 treatment, may be predictive of poor clinical outcomes in patients treated with the anti-CD23 monoclonal antibody. CD20, CD22, and CD23 are susceptible to environmental cues, whereas expression of CD37 remained high throughout culture and independent of treatment. As a result, we predict CD37 to be a good target for treating CLL due to its stability. Ongoing clinical trials with TRU-016 or other agents that specifically target CD37 may therefore be of potential therapeutic benefit. Disclosures:No relevant conflicts of interest to declare.

Bovine mammary dendritic cells: A heterogeneous population, distinct from macrophages and similar in phenotype to afferent lymph veiled cells

Dendritic cells (DC) are a heterogeneous population of professional antigen presenting cells and are potent stimulators of naïve T-cells. However, there is little previous research describing DC in bovine mammary tissue, primarily because of the difficulty distinguishing these cells from macrophages, which possess a similar phenotype. Using immunohistofluorescence and a combination of markers (MHC-II, CD205, CD11c), DC were localized in the bovine mammary gland and supramammary lymph node. In mammary tissue DC were found within the alveolar epithelium and within the intralobular connective tissue. In the lymph node DC were found on the periphery of B-cell areas, in the cortex, and among T-cells in the paracortex and medulla. DC in mammary parenchyma and supramammary lymph nodes were quantified and further characterized using flow cytometry. DC were CD11c(hi), CD14(lo) cells that expressed MHC-II and CD205. DC could be distinguished from macrophages based on their low CD14 expression. This research provides a better understanding of mammary gland immunology, while potentially aiding in the targeting of antigens to mucosal DC for vaccine development.

1.28 Low CD20 Expression in Chronic Lymphocytic Leukemia is not Associated with CD20 Gene Mutation

1.28 Low CD20 Expression in Chronic Lymphocytic Leukemia is not Associated with CD20 Gene Mutation