Comparative Assessment of Surface CD19 and CD20 Expression on B-Cell Lymphomas from Clinical Biopsies: Implications for Targeted Therapies

Background: CD19 and CD20 are B-cell lineage-specific antigens expressed on the cell surface of most B-cell lymphomas. While CD20 is acquired during late stages of B-cell lymphogenesis and is then lost upon differentiation into plasma cells, CD19 expression covers the entire spectrum of early B-cell genesis and maturation. CD20-targeting agents have been broadly integrated into the therapeutic armamentarium for B-cell lymphomas. More recently, CD19-targeting agents have emerged as promising alternatives with demonstrated therapeutic value. Given the imminent availability of both CD19 and CD20 targeted therapies, and potential for combinational approaches, we studied the surface expression of these antigens at the single-cell level on lymphoma cells and benign background lymphoid subsets from biopsy specimens. Methods: Flow cytometric analysis (seven-color) was performed on biopsy specimens from 47 patients with newly diagnosed B-cell lymphomas, including diffuse large B-cell lymphoma (n=15), follicular lymphoma (n=15), marginal zone lymphoma (n=9), mantle cell lymphoma (n=9), Burkitt lymphoma (n=2), and unclassifiable low-grade B-cell lymphoma (n=2). Small lymphocytic lymphoma was intentionally excluded, given its well-described loss of CD20 expression. Biopsies from eight additional patients with persistent/recurrent B-cell lymphomas after anti-CD20 therapy (greater than 6 months after last dose) were also evaluated. Thresholds for CD19 or CD20 antigen positivity were defined for each case, based on the 95th percentile fluorescence intensity of the respective marker on tumor-infiltrating T-cells (internal negative control). In addition, CD19 or CD20 fluorescence intensities of tumor cells were normalized to background benign B-cells (internal positive control) using the median fluorescence ratio (MFR). Results: Both CD19 and CD20 were highly expressed on CD20 treatment naïve tumor cells, with a slightly higher median percentage of positive tumor cells for CD19 (98%) compared with CD20 (93%) (p=0.003), and one case lacking CD20 expression. When compared with background benign B-cells, CD20 was frequently overexpressed on tumor cells (mean MFR=1.8), while CD19 expression was overall similar to background benign B-cells (mean MFR=0.9) (p=0.001). As the surface density of CD20 on benign B-cells is reportedly higher than CD19 (~100,000 vs ~20,000 molecules per cell, respectively), these findings are consistent with a higher density of surface CD20 than CD19 on most B-cell lymphomas. However, CD20 expression was more heterogeneous (within individual patient samples and across patients), with a higher median percentage of CD20-negative tumor events (median=0.5%, range=0-98%) compared with CD19-negative events (median=0%, range=0-28%) (p=0.003). Interestingly, expression of CD19 within the CD20-negative tumor subsets was largely preserved (mean % CD19-positive events=97.86%, min=40%). In addition, percentages of CD19/CD20 double-negative tumor events were very small (median=0%, range=0-4.9%), and only detectable in 15 cases (32%). Of eight additional cases studied post-anti-CD20 immunotherapy (6-84 months after last dose), the percentage of antigen-positive events by tumor cells was largely preserved for both CD19 (median=99.6%, range=93.5-100%) and CD20 (median=95.7%, range=55.6-100%), similar to the pre-therapy cohort. Conclusions: CD19 and CD20 are both highly and consistently expressed in B-cell lymphomas. While CD20 has a higher average density of surface molecules per tumor cell, CD19 expression is more homogenous and is preserved in small CD20-negative tumor subsets and after anti-CD20 targeted therapy. These findings support the clinical evaluation of anti-CD19 immunotherapies and combinational therapies targeting both surface antigens. Disclosures Horna: MorphoSys AG: Research Funding. Nowakowski:Selvita: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Curis: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Genentech, Inc.: Research Funding; MorphoSys: Consultancy, Research Funding; NanoString: Research Funding. Endell:MorphoSys AG: Employment, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties.

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CD20 Is an Unstable Target in Primary-Refractory High-Grade Lymphomas

Blood ◽

10.1182/blood-2019-127129 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1608-1608

Author(s):

Ryan N Rys ◽

Dominique Geoffrion ◽

Christopher Rushton ◽

Miguel Alcaide ◽

Raoul Santiago ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

High Grade ◽

Advisory Committees ◽

Increased Risk ◽

Cd20 Expression ◽

Anti Cd20

Introduction: A third of patients with diffuse large B cell lymphoma (DLBCL) are not cured with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (RCHOP). Approximately 20% of DLBCLs will lose CD20-protein expression (CD20-neg) at the time of relapse. The incidence of CD20-neg relapses in other aggressive B cell lymphomas is unknown. CD20 is not only targeted by rituximab but other antibody-based therapies that are being investigated in salvage regimens. We hypothesized that prolonged exposure to anti-CD20 containing regimens may lead a CD20-neg relapse. Our goal was to determine the clinical and genetic factors associated with loss of CD20 expression in high-grade lymphomas treated with curative intent. Method: We consented 374 patients with B cell high-grade lymphomas that were treated with RCHOP or more intensive regimens at the time of diagnosis or had a histological transformation from a prior indolent lymphoma (TLy). We recorded the baseline clinical characteristics, histological diagnosis, date of first relapse and the number of treatment regimens prior to their second biopsy. CD20 expression and cell of origin (COO) were determined by immunohistochemistry and, in the latter, using Hans criteria. We performed targeted sequencing of 63 lymphoma-related genes, including MS4A1, the gene that encodes CD20. Sequencing was performed using circulating tumor DNA in the plasma or DNA from the tumor biopsy, both obtained at the time of relapse. Results: Relapse occurred in 170/374 (45%) of patients: 102/253 DLBCL, 55/96 TLy, 6/16 primary mediastinal B cell lymphoma (PMBCL) and 7/9 high grade B cell lymphomas (HGBL) with or without translocations in MYC and BCL2 or BCL6. The median age at diagnosis was 62 years old, 54% were male and 85% had an elevated international prognostic index of ≥ 2. Of these, 104 had a biopsy taken at first (47%) or subsequent relapse (53%) to confirm the diagnosis or performed in the context of a clinical trial. CD20 could be assessed in 100 cases, of which 26 had CD20-neg lymphoma cells: 15/56 (27%) of DLBCL, 7/38 (18%) of TLy, 2/3 (66%) PMBCL and 2/3 (66%) HGBL. Relapsed PMBCL and HGBL combined, appeared to have an increased risk of CD20 negativity at relapse compared to DLBCL and TLy (p=0.043). A GCB phenotype in DLBCL was present in 35% of cases and was not associated with CD20 status. The mean number of therapies given before the second biopsy was similar in both groups (CD20+ = 2.2 and CD20-neg = 2.7, p=0.2). In fact, 11/26 (44%) of CD20-neg cases had biopsies taken after RCHOP alone. Additional chemo-immunotherapy (range 2 to 8) did not increase the risk of having a CD20-neg relapse (p=0.5), suggesting that unlike follicular lymphoma, the emergence of CD20-neg cells occurs early after rituximab exposure in high-grade lymphomas. Supporting this hypothesis, patients with a CD20-neg relapse had a shorter progressive free survival (PFS) after RCHOP (1,7 vs 3,2 years, p=0.025). Patients with primary treatment failure, defined here as a PFS of < 1 year, had a significantly increased risk of having a CD20-neg relapse (hazard ratio 7.9, p= 0.005). Sequencing was performed in 75/100 patients. MS4A1 mutations were present in 16% of CD20-neg cases, while none were present in the CD20+ cases (p=0.003). There was no significant difference in the mutation rate of TP53 (47%) or histone-modifying genes based on CD20 expression status. Conclusion: Decreased CD20 expression occurs early in high-grade lymphomas under the selective pressure of RCHOP, 16% of which are a consequence of MS4A1 mutations, suggesting that other mechanisms also modulate CD20 expression. In patients with a PFS of < 1 year, a repeat biopsy would be recommended if primary anti-CD20-targeted therapy is considered. Disclosures Assouline: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Johnson:Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; BMS: Consultancy, Honoraria; Merck: Consultancy, Honoraria; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Seattle Genetics: Honoraria; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding.

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Patient-Derived Xenograft Model in B-Cell Lymphoma: A Platform for a Personalized Clinical Trial

Blood ◽

10.1182/blood.v128.22.1839.1839 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1839-1839

Author(s):

Taylor Bell ◽

Hui Zhang ◽

Makhdum Ahmed ◽

Hui Guo ◽

Carrie J Li ◽

...

Keyword(s):

B Cell ◽

Tumor Cells ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Drug Resistant ◽

B Cell Lymphomas ◽

Advisory Committees ◽

Patient Derived Xenograft ◽

Pdx Model

Abstract Background: Imprecise correlation of treatment response in established cell line and cells obtained from patients (i.e., patient-primary) limits the preclinical investigation of novel compounds. Similarly, the "xenograft" models wherein human cancer cell lines are transplanted into immunocompromised mice do not represent the full spectrum of cancers. However, Patient-derived xenograft (PDX) mouse models have been shown to recapitulate the diversity of growth, metastasis, and histopathology of the original tumor. Based on our previously established mantle cell lymphoma (MCL) PDX model, we developed other B-cell lymphoma PDXs recapitulating tumor pathological and clinical characteristics, progression and response to therapeutic agents, this will provide an indispensable model system towards personalized treatment for B-cell lymphoma. Methods: We developed 34 PDX models with an implanted fetal bone chip for several B-cell lymphomas including marginal zone lymphoma (MZL), follicular lymphoma (FL), Burkitt's lymphoma (BL), and diffuse large B-cell lymphoma (DLBCL), and MCL. We tested the in vitro efficacy of a panel of drugs among freshly isolated tumor cells from patients and tumor cells from the PDX models. We also generated a drug-resistant MCL PDX model, compared the effect of targeted drugs on the tumor burden in the drug-resistant model and identified potential therapeutic opportunity with drug combinations. We validated combination therapy in vivoand conducted next generation sequencing (NGS) using a 1,212 gene panel (OncoPlus®) on DNA from primary patient cells. Results: We collected clinical samples from 34 patients with several types of B-cell lymphomas including MCL (n=21), DLBCL (n=3), FL (n=2), BL (n=1), and MZL (n=2). Of the 34 patients, 18 (53%) were newly diagnosed and untreated clinically, 11 (32%) patients were relapsed after treatment with 1-3 chemotherapy or targeted therapy treatments, and 5 (15%) patients were treated with one-dose therapy before sample collection. All of the tumor cells, from both the patient and PDXs, showed the same drug response pattern. Consecutive ibrutinib administration to PDX mice from PT1 during G4 induced the development of an ibrutinib-resistant tumor. The cell viability of isolated PDX tumor cells treated with ibrutinib was not significantly different between G1 and G2 nor was it different between G5 and G6 (p>0.05). In addition, histological features were consistent with patient tumor histology. Furthermore, whole exome sequencing revealed fidelity between the patient and PDX tumor cells as well as between subsequent generations of the PDX. [B1] We[Z2] engrafted a patients tumor cells into NSG-hu mice to create an MCL-bearing PDX mouse model (PT28-PDX). G2 PDX cells were isolated and treated with a panel of drugs. We found that G2 PDX cells were most sensitive to bortezomib (BTZ) (Velcade®). Growth inhibition of the G2 PDX cells was significantly higher with BTZ compared to ibrutinib (p=0.002) and cells were most sensitive to BTZ compared with other agents (p≤ 0.002). Based on this finding, the patient was treated with Velcade®, rituximab and dexamethasone and responded to treatment. However, the G3 PDX became resistant to BTZ. She was then treated with a 1 cycle of rituximab and cytarabine. Soon after, we initiated three-drug combination treatment with lenalidomide (Len), rituximab (RTX) and dexamethasone (DEX) in PDX G4. Len+RTX+DEX significantly prolonged mouse survival indicating that this could be an effective regimen for this patient after BTZ relapse. As guided by the PDX, PT28 underwent Len+RTX+DEX regimen and her peripheral lymphocytosis disappeared demonstrating response. Conclusions: The PDX model with implanted fetal bone chip is a valid experimental platform that recapitulates tumor characteristics of B-cell lymphomas. Leveraging the PDX platform to identify and select drugs that are likely to be efficacious for individual patients and subsequently administering the promising agents to those who relapse after an initial therapy is the next milestone. Disclosures Wang: BeiGene: Research Funding; Asana BioSciences: Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Deregulation of CCND1 or BCL2 in the Immature B-Cell Stage Determines the Resulting Lymphoma Histology

Blood ◽

10.1182/blood.v120.21.416.416 ◽

2012 ◽

Vol 120 (21) ◽

pp. 416-416

Author(s):

Masaharu Tashima ◽

Momoko Nishikori ◽

Wataru Kishimoto ◽

Ryo Yamamoto ◽

Tomomi Sakai ◽

...

Keyword(s):

B Cells ◽

Mouse Model ◽

B Cell ◽

Tumor Cells ◽

Cell Population ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

B Cell Lymphomas ◽

Cell Stage ◽

Genetic Event

Abstract Abstract 416 In B-cell lymphomas, several chromosomal translocations are associated with specific histological subtypes. CCND1/IGH is detected in more than 90 % of the cases with mantle cell lymphoma (MCL), and BCL2/IGH is characteristically observed in follicular lymphoma and diffuse large B-cell lymphoma (DLBCL) of germinal center (GC) B-cell origin. Although these strong correlations are clinically recognized, their biological mechanism is not clearly explained so far. According to the results of translocation breakpoint mapping, both CCND1 and BCL2 translocations are considered to be generated by an error during physiological VDJ rearrangement of the IGH gene in the precursor-B cell stage. We hypothesized that the occurrence of these translocations in the immature B-cell stage, probably as an initial genetic event, should have a special impact on the determination of resulting lymphoma histology. We generated a mouse model mimicking human lymphoma with CCND1 or BCL2 translocation by lentivirally introducing these genes into Tp53+/− B6 mouse bone marrow cells and transplanting them to lethally irradiated wild-type B6 mice. In this model, CCND1 or BCL2 is expressed from immature to mature B cell stage under the control of the CD19 promoter, and subsequently mutations accumulate in the background of Tp53 haploinsufficiency. Both mice developed B-cell lymphomas several months after the transplantation, but their tumors showed some different features. CCND1-Tp53+/− mice developed B220lowCD5+CD23− tumors that expand into the B-cell area with sparing the GC, whereas BCL2-Tp53+/− mice preferentially developed B220highCD5−CD23+ tumors that tend to localize in the GC. Additionally, somatic hypermutation (SHM) analysis of the IGH gene of the tumor cells revealed obviously higher mutation frequency in the BCL2-Tp53+/− mice than in the CCND1-Tp53+/- mice (p<0.004). These results indicate that the primary gene deregulation of CCND1 and BCL2 determines the upcoming lymphoma of MCL-type and GC B-cell lymphoma-type, respectively. MCL has been postulated to be derived from naïve pre-GC B cells, with few SHM introduced in the immunoglobulin variable region. But the MCL-like tumor cells generated in CCND1-Tp53+/−mice seem to be originated from B-1a B cells, a distinct B cell population that does not enter GC and has few SHM in nature. There has been a debate whether B-1 cell population exists in humans, but it is recently proposed that the phenotype of human B-1 cells is CD20+CD27+CD43+CD70− by testing sort-purified B cell fractions for fundamental B-1 cell functions based on mouse studies (J Exp Med 2011;208:67–80). Interestingly, flow cytometric analysis of the human MCL tumor cells has shown that they mostly express this provisional B-1 cell phenotype, supporting the idea that human MCL is also derived from B-1 cells. Our mouse model precisely reproduces the link between CCND1 and BCL2 translocations and the resulting lymphoma subtypes in humans. It is assumed that these translocations trigger the cell expansion of different B-cell subgroups, which consequently leads to the development of lymphoma of distinct histology. Our findings provide new insights into the mechanism of lymphoma subtype determination. Disclosures: No relevant conflicts of interest to declare.

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Monoclonal antibody 1F5 (anti-CD20) serotherapy of human B cell lymphomas

Blood ◽

10.1182/blood.v69.2.584.584 ◽

1987 ◽

Vol 69 (2) ◽

pp. 584-591 ◽

Cited By ~ 246

Author(s):

OW Press ◽

F Appelbaum ◽

JA Ledbetter ◽

PJ Martin ◽

J Zarling ◽

...

Keyword(s):

Monoclonal Antibody ◽

Lymph Node ◽

B Cell ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

B Cell Lymphomas ◽

Antigenic Modulation ◽

Minor Response ◽

High Doses ◽

Anti Cd20

Abstract Four patients with refractory malignant B cell lymphomas were treated with continuous intravenous (IV) infusions of murine monoclonal antibody (MoAb) 1F5 (anti-CD20) over five to ten days. Dose-dependent levels of free serum 1F5 were detected in all patients. Two patients had circulating tumor cells and in both cases 90% of malignant cells were eliminated from the blood stream within four hours of initiation of serotherapy. Antigenic modulation did not occur, and sustained reduction of circulating tumor cells was observed throughout the duration of the infusions. Serial bone marrow aspirations and lymph node biopsies were examined by immunoperoxidase and immunofluorescence techniques to ascertain MoAb penetration into extravascular sites. High doses (100 to 800 mg/m2/d and high serum 1F5 levels (13 to 190 micrograms/mL) were required to coat tumor cells in these compartments in contrast to the low doses that were adequate for depletion of circulating cells. Clinical response appeared to correlate with dose of MoAb administered with progressive disease (52 mg), stable disease (104 mg), minor response (1,032 mg), and partial response (2,380 mg) observed in consecutive patients. The patient treated with the highest 1F5 dose achieved a 90% reduction in evaluable lymph node disease, but the duration of this remission was brief (six weeks). This study demonstrates that high doses of 1F5 can be administered to patients with negligible toxicity by continuous infusion and that clinical responses can be obtained in patients given greater than 1 g of unmodified antibody over a ten-day period.

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Distinct roles for PARP-1 and PARP-2 in c-Myc-driven B-cell lymphoma in mice

Blood ◽

10.1182/blood.2021012805 ◽

2021 ◽

Author(s):

Miguel A Galindo-Campos ◽

Nura Lutfi ◽

Sarah Bonnin ◽

Carlos Martínez ◽

Talia Velasco-Hernandez ◽

...

Keyword(s):

Dna Damage ◽

B Cells ◽

B Cell ◽

Immune Escape ◽

Cell Lymphoma ◽

Tumour Progression ◽

Cancer Therapeutics ◽

B Cell Lymphoma ◽

B Cell Lymphomas ◽

Parp 1

Dysregulation of the c-Myc oncogene occurs in a wide variety of haematologic malignancies and its overexpression has been linked with aggressive tumour progression. Here, we show that Poly (ADP-ribose) polymerase (PARP)-1 and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphomas. PARP-1 and PARP-2 catalyse the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA-strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphomas, while PARP-1-deficiency accelerates lymphomagenesis in the Em-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in pre-leukemic Em-Myc B cells resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1-deficiency induces a proinflammatory response, and an increase in regulatory T cells likely contributing to immune escape of B-cell lymphomas, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centred therapeutic strategies with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumours.

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Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection

Blood ◽

10.1182/blood-2010-01-263533 ◽

2010 ◽

Vol 115 (25) ◽

pp. 5191-5201 ◽

Cited By ~ 209

Author(s):

Stephen A. Beers ◽

Ruth R. French ◽

H. T. Claude Chan ◽

Sean H. Lim ◽

Timothy C. Jarrett ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Type I ◽

Type Ii ◽

Antigenic Modulation ◽

Lymphatic Leukemia ◽

Human B Cells ◽

Anti Cd20

Abstract Rituximab, a monoclonal antibody that targets CD20 on B cells, is now central to the treatment of a variety of malignant and autoimmune disorders. Despite this success, a substantial proportion of B-cell lymphomas are unresponsive or develop resistance, hence more potent anti-CD20 monoclonal antibodies (mAbs) are continuously being sought. Here we demonstrate that type II (tositumomab-like) anti-CD20 mAbs are 5 times more potent than type I (rituximab-like) reagents in depleting human CD20 Tg B cells, despite both operating exclusively via activatory Fcγ receptor–expressing macrophages. Much of this disparity in performance is attributable to type I mAb-mediated internalization of CD20 by B cells, leading to reduced macrophage recruitment and the degradation of CD20/mAb complexes, shortening mAb half-life. Importantly, human B cells from healthy donors and most cases of chronic lymphatic leukemia and mantle cell lymphoma, showed rapid CD20 internalization that paralleled that seen in the Tg mouse B cells, whereas most follicular lymphoma and diffuse large B-cell lymphoma cells were far more resistant to CD20 loss. We postulate that differences in CD20 modulation may play a central role in determining the relative efficacy of rituximab in treating these diseases and strengthen the case for focusing on type II anti-CD20 mAb in the clinic.

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Discrepancy of CD20 Protein Expression In IHC and FCM Analyses In Primary B-Cell Lymphoma: Relationship Between FCM-Negative Phenotype and Rituximab Binding with Lymphoma Cells

Blood ◽

10.1182/blood.v116.21.5087.5087 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5087-5087 ◽

Cited By ~ 1

Author(s):

Takashi Tokunaga ◽

Akihiro Tomita ◽

Kazuyuki Shimada ◽

Junji Hiraga ◽

Takumi Sugimoto ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Primary Lymphoma ◽

Newly Diagnosed ◽

Rt Pcr ◽

Lymphoma Cells ◽

Anti Cd20

Abstract Abstract 5087 Background Rituximab is an anti-CD20 chimeric-monoclonal antibody, and its effectiveness for treatment of CD20-positive B-cell lymphomas has been proven over the past 10 years. Although rituximab is now a key molecular targeting drug for CD20-positive lymphomas, some patients with rituximab resistance have emerged. We previously reported that the CD20-protein-negative phenotypic change 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 have recognized that some newly-diagnosed B-cell lymphomas show CD20-protein-positive in immunohistochemistry (IHC) but -negative in flow cytometry (FCM) analyses. For these patients, so far, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance are clear. Thus, the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims Analyses of the molecular backgrounds of CD20 IHC(+)/FCM(−) phenotype in primary B-lymphoma cells, and confirmation of the effectiveness of rituximab therapy for the patients who show CD20 IHC(+)/FCM(−) phenotype. Results Primary B-cell lymphoma (diffuse large B-cell (DLBCL), follicular, MALT, mantle cell, and Burkitt) tissues and cells were analyzed by IHC and FCM. Four newly-diagnosed B-cell lymphoma patients showed IHC CD79(+)/CD20(+) and FCM CD19(+)/CD20(−) phenotype using anti-CD20 antibodies L26 for IHC and B1 for FCM, and all were diagnosed as DLBCL. Chromosomal analysis showed complex karyotypes in 3 out of 3 patients analyzed, and no shared abnormalities were confirmed. Primary lymphoma cells from 3 patients were available for further molecular analyses, and the genomic DNA, the total RNA, and the protein from whole cell lysate were obtained from these lymphoma cells. DNA sequencing analysis indicated no significant genetic mutations on the coding sequences (CDS) of MS4A1 (CD20) gene. Semi-quantitative and quantitative RT-PCR indicated that CD20 mRNA expression was almost normal in 2 patients and ≂~f10 times lower in 1 patient compared to the positive control B-lymphoma/leukemia cells. Almost the same expression tendency with RT-PCR was confirmed in immunoblot analysis using whole cell lysate and the two different anti-CD20 antibodies. The molecular weight of the CD20 protein in immunoblotting corresponded to the wild type in these patients. Rituximab binding assay in vitro was performed using primary lymphoma cells from a patient and the fluorescent-labeled rituximab (Alexa488-rituximab). Interestingly, rituximab binding on the surface of the CD19 positive lymphoma cells was confirmed in vitro. Rituximab containing combination chemotherapy was performed, resulting in complete response in all 4 cases after completing 4 to 8 courses. Conclusions and Discussion CD20 IHC(+)/FCM(−) phenotype was confirmed in newly-diagnosed DLBCL patients. Significant abnormalities in CD20 protein and mRNA expression in immunoblotting and RT-PCR were not confirmed, and genetic mutations on CDS of MS4A1 gene, resulting in the conformation change of CD20 protein, were not detected. The possibility of abnormal post-translational modification or aberrant localization of CD20 protein, leading to interference with antibody binding, can not be excluded. Rituximab binding with CD19-positive primary lymphoma cells was confirmed in a patient, suggesting that CD20 IHC(+)/FCM(-) phenotype does not directly indicate the ineffectiveness of rituximab for these cells. Further investigations, performing in vitro CDC and ADCC assay using primary lymphoma cells, are still warranted to show rituximab effectiveness and sensitivity to those cells. Disclosures: Kinoshita: Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

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Prenyl Transferases Are Involved in the Regulation of CD20 Levels and Influence Anti-CD20 Monoclonal Antibody-Mediated Activation of Complement-Dependent Cytotoxicity,

Blood ◽

10.1182/blood.v118.21.3722.3722 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3722-3722

Author(s):

Dominika Nowis ◽

Magdalena Winiarska ◽

Jacek Bil ◽

Angelika Muchowicz ◽

Malgorzata Wanczyk ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

Antitumor Effects ◽

Raji Cells ◽

Farnesyl Transferase ◽

Complement Dependent Cytotoxicity ◽

Cd20 Expression ◽

Geranylgeranyl Transferase ◽

Anti Cd20 ◽

Hodgkin's Lymphomas

Abstract Abstract 3722 Anti-CD20 monoclonal antibodies (mAbs) (rituximab or ofatumumab) are being successfully used in the treatment of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). They exert antitumor effects by triggering indirect effector mechanisms of the immune system, such as activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, upon crosslinking with secondary antibodies, anti-CD20 mAbs can induce cell death. It is frequently underscored that CD20 expression levels in various B-cell tumors is relatively constant. However, accumulating evidence indicates that CD20 can be modulated at transcriptional, posttranscriptional, and even posttranslational levels. Moreover, it has been clearly shown in vitro that CDC efficacy of anti-CD20 mAbs clearly depends on CD20 expression. We have previously observed that statins impair detection of CD20 in non-Hodgkin lymphoma cells and impair rituximab-mediated CDC and ADCC (Winiarska et al. PLoS Med 2008). Statins are inhibitors of cholesterol synthesis and decrease production of prenyl groups (farnesyl and geranylgeranyl PPi), which are necessary for posttranslational modification of approximately 1% of cellular proteins. In experiments aimed at elucidation of the molecular mechanisms of statin-mediated modulation of CD20 we observed that neither geranylgeranyl transferase (GGT) nor farnesyl transferase (FNT) inhibitors could mimic statins effects. On the contrary, prenyltransferase inhibitors improved rituximab-mediated CDC. Therefore, we decided to investigate in more detail the interaction of prenyltransferase inhibitors and anti-CD20 mAbs. In the initial experiments we evaluated the effects of three different farnesyl transferase inhibitors as well as three different geranylgeranyl transferase inhibitors. Among all FNT and GGT inhibitors, L-744,832 seemed to produce the most significant influence on both rituximab-mediated CDC and CD20 levels (Figure). Moreover, L-744,832 significantly increased rituximab-mediated CDC in 3 out of 5 primary tumor cell cultures isolated from patients with NHL or CLL. Therefore, L-744,832 was selected for further more systematic studies. Interestingly, in Raji cells L-744,832 did not improve rituximab-mediated ADCC and only at the highest non-toxic concentrations it sensitized to rituximab+crosslinking antibody-mediated cytotoxicity. In 10 out of 17 (58.8%) primary lymphoma/leukemia cells L-744,832 increased CD20 expression by at least 20% as measured with flow cytometry. Moreover, we observed that upon L-744,832 treatment CD20 is up-regulated in Raji cells at both mRNA as well as protein level. Experiments aimed at investigation of FTI influence on proteasome activity as well as CD20 endocytosis and shedding revealed that L-744,832 influences CD20 levels independently from its posttranslational regulation. To verify whether modulation of CD20 levels by L-744,832 results from specific inhibition of farnesyltransferase or is an off-target effect of this compound we performed FNT B subunit (FNTB) knock-down experiments that resulted in increased CD20 levels by almost 60%. Incubation of Raji cells with a transcription inhibitor cycloheximide completely prevented L-744,832-mediated increase of CD20 levels in WB. Therefore, a chromatin immunoprecipitation assay was performed to determine whether inhibition of FNT activity is associated with binding of transcription factors to the promoter of cd20 gene. These studies revealed that L-744,832 promotes binding of PU.1 and Oct2, but not TFE3 to target DNA sequences within cd20 promoter in Raji cells. To conclude, our studies indicate for the first time that CD20 expression can be modulated by prenyltransferase inhibitors. While inhibition of FNT activity significantly up-regulates expression of CD20, the influence of GGT inhibitors on this protein is more complex, and requires further studies. Furthermore, pre-incubation of NHL and CLL cells with L-744,832, a FNT inhibitor, potentiates anti-CD20 mAb-mediated activation of the complement-mediated cytotoxicity. Therefore, the combination seems to be promising and its efficacy should be determined in patients with NHL or CLL. Disclosures: Winiarska: Genmab A/S: Research Funding. Golab:Genmab A/S: Research Funding.

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Ublituximab (TGTX-1101), a Novel, Third-Generation Anti-CD20 Antibody Demonstrates Enhanced Antitumor Activity Compared to Rituximab in Primary CNS and Intraocular Lymphoma Murine Models.

Blood ◽

10.1182/blood.v120.21.2755.2755 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2755-2755

Author(s):

Sabrina Donnou ◽

Rym Ben Abdelwahed-Bagga ◽

Jérémie Cosette ◽

Hanane Ouakrim ◽

Lucile Crozet ◽

...

Keyword(s):

B Cell ◽

Tumor Cells ◽

Antitumor Effect ◽

Intraocular Lymphoma ◽

Third Generation ◽

Tumor Site ◽

Murine Models ◽

B Cell Lymphomas ◽

Cd20 Antibody ◽

Anti Cd20

Abstract Abstract 2755 Background: Primary Central Nervous lymphomas (PCNSL), that comprise primary cerebral lymphoma (PCL) and primary intraocular lymphoma (PIOL), are typically CD20+ diffuse large B-cell lymphomas that have no detectable disease outside the brain or eye. Rituximab (RTX), an anti-CD20 antibody, has demonstrated encouraging clinical benefit in systemic B-cell lymphomas as well as PCL and PIOL, however, the role of RTX in treatment of PCNSL/PIOL remains controversial, and these highly aggressive malignancies are often incurable with available therapies. Therefore, additional treatment options are needed. Ublituximab (UTX) is a novel, glycoengineered chimeric anti-CD20 monoclonal antibody (mAb) that has a high affinity for FcγRIIIa (CD16) receptors, and therefore greater ADCC activity than RTX (Le Garff-Tavernier et al., 2011). Herein, we assess the antitumor effects of UTX compared to RTX in murine models of PCL and PIOL. Methods: The murine lymphoma B-cell line A20.IIA-GFP-hCD20 (H-2d) was injected into the right cerebral striatum (PCL model) or the vitreous (PIOL model) of adult BALB/c mice (H-2d); 7 days later, single doses of UTX were injected either into the tumor site intracerebrally (PCL) or intravitreously (PIOL), or at distance of the tumor site (intrathecally, PCL). RTX was used as a reference compound. Survival was monitored for injected mice for up to 100 days, and flow cytometric analyses were performed to assess tumor growth and T-cell infiltration. Results: In PCL and PIOL models, single doses of UTX had a marked antitumor effect more pronounced than that obtained with an equivalent dose of RTX. In the PCL model, there was an overall survival (OS) advantage with intracerebral injections of UTX compared to RTX (50% vs. 10%, n=10 in each group, p=0.0028). The reduction in tumor cells was correlated with an increased proportion of CD8+ T cells. Moreover, intrathecal injections of UTX increased OS compared to buffer solution. In the PIOL model, the absolute number of tumor cells analyzed 8 days after treatment had decreased more significantly (p=0.027) with UTX compared to the RTX group. This finding again confirmed the superiority of UTX in this setting. Conclusions: These results confirm that the novel, third-generation mAb, UTX has a sustained and greater antitumor effect than RTX on primary cerebral and intraocular lymphomas when assessed in vivo. Additional experiments evaluating the combination of UTX + methotrexate are ongoing. Clinical trials to evaluate UTX as an innovative therapeutic approach to treat primary cerebral and intraocular B-cell lymphomas are currently being evaluated. Disclosures: Jacquet: LFB Biotechnologies: Employment. Fridman:LFB Biotechnologies: Membership on an entity's Board of Directors or advisory committees. Sportelli:TG Therapeutics, Inc.: Employment, Equity Ownership. Urbain:LFB Biotechnologies: Employment.

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Profiles Of De Novo CD25-Positive Mature B-Cell Lymphomas

Blood ◽

10.1182/blood.v122.21.4308.4308 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4308-4308

Author(s):

Shin-ichiro Fujiwara ◽

Raine Tatara ◽

Kiyoshi Okazuka ◽

Iekuni Oh ◽

Ken Ohmine ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

High Expression ◽

B Cell Lymphomas ◽

Cns Involvement ◽

Cd25 Expression

Abstract Background Interleukin 2 (IL-2) is an important cytokine that controls the proliferation and differentiation of not only T- but also B-lymphocytes. Recently, we reported that CD25 (IL-2 receptor alpha chain, IL-2R) is expressed in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and high expression of CD25 in the two types of lymphoma is correlated with a poor prognosis following chemotherapy regimens containing rituximab (ASH annual meeting, 2011 118:2666, 2012 120:1543). We evaluated the clinical significance of CD25 expression in a larger series of different mature B-cell lymphomas (BCL). Patients and Methods Four hundred and thirty-seven newly diagnosed patients who were admitted to our hospital between 2002 and 2013 were retrospectively evaluated. Lymph node or related tissue biopsy samples of BCL were analyzed using flow cytometry, as follows: 182 patients, DLBCL; 92, FL; 48, chronic lymphocytic leukemia (CLL); 21, mantle cell lymphoma (MCL); 23, marginal zone lymphoma (MZL); 8, Burkitt lymphoma (BL); 18, B-cell lymphoma unclassifiable with features intermediate between BL and DLBCL (BL/DLBCL); 5, lymphoplasmacytic lymphoma (LPL); and 39, reactive lymphadenopathy with sufficient B-cells. CD25-positivity was defined as >20% of clonal B-cells in a gated region. Results CD25 expression in patients with MCL, CLL, MZL, and DLBCL was significantly higher than that in patients with reactive lymphadenopathy (P<0.001,<0.001, =0.019, and <0.001, respectively). BL and FL, which were derived from germinal center B-cells, did not express CD25. These results indicate that pre- or post- germinal center-derived B-cells, activated by IL-2/IL-2R signaling, may give rise to CD25+ BCL such as CD25+ MCL, CLL, MZL, and DLBCL. The highest median CD25 expression (41.5%) was observed in MCL. CD25 expression was higher in MCL than CD5+ BCL (CLL and CD5+ DLBCL) (median, 41.5 vs. 16.9%, respectively; P<0.001). With a cut-off value of 60% CD25-positivity, patients with CD25-high (>60%) MCL (n=9) were not treated with aggressive chemotherapy regimens such as Hyper-CVAD due to their age and characteristics, compared with those with CD25-low (<60%) MCL (n=12) (11.1 vs. 72.7%, respectively, P=0.021). In patients with CLL, the range of CD25 expression was wide (0.4-90.7%), and 29 patients (60%) showed CD25-positivity (CD25+ CLL). CD25+ CLL showed higher soluble IL-2R (sIL-2R) levels and an inferior overall survival (OS) than CD25- CLL (median sIL-2R, 2,195 vs. 706 U/ml P=0.047; 5-year OS, 62.7 vs. 100%; P=0.037). There was a significant correlation between levels of CD25 and sIL-2R (r=0.53, P=0.0053). It is clinically important to distinguish between DLBCL and BCL involving MYC oncogene rearrangement (BL and BL/DLBCL, MYC+ BCL). The former showed higher CD25 expression than the latter (median, 10.2 vs. 2.1%, respectively, P=0.04). The progression-free survival rate (PFS) after rituximab containing chemotherapy was inferior in patients with CD25+ DLBCL (n=72) than those with CD25- DLBCL (n=110) and MYC+ BCL (5-year PFS, 49 vs. 70.4, 66.3%, respectively). In patients with DLBCL, central nerve system (CNS) involvement was observed in 15 patients (7 at diagnosis and 8 at relapse). CD25+ DLBCL showed a higher frequency of CNS involvement than CD25– DLBCL (13.8 vs. 4.5%, respectively, P=0.049). Regarding MZL, CD25 was highly expressed in nodal MZL, but it showed a low expression in splenic MZL. Regarding the sites of extranodal MZL, CD25 expression was lower in the thyroid than at other sites (median, 5.1 vs. 21.2%, respectively, P=0.37). There were some differences between CD25+ (n=9) and CD25- (n=14) MZL concerning the presence of B symptoms (33.3 vs. 0%, respectively) and advanced stage (66.6 vs. 35.7%, respectively). Conclusion CD25 expression using flow cytometry can potentially provide diagnostic and prognostic implications on BCL patient. The high expression of CD25 in MCL and CLL suggests the possibility of targeted anti-CD25 immunotherapy. These findings may shed light on the role of CD25 expression in B-cell lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.

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