The Experts below are selected from a list of 169290 Experts worldwide ranked by ideXlab platform

Myron S. Czuczman - One of the best experts on this subject based on the ideXlab platform.

  • CD20 Antigen Density at Time of Presentation Is An Independent Predictor of Outcome in B-Cell Lymphoproliferative Disorders
    Blood, 2008
    Co-Authors: Sapna Khubchandani, Myron S. Czuczman, Scott H. Olejniczak, Terry Mashtare, Mohammad M Chisti, Gregory E. Wilding, Francisco J. Hernandez-ilizaliturri
    Abstract:

    The integration of monoclonal antibodies (mAbs) targeting CD20 Antigen has changed the treatment paradigm of patients with B-cell non Hodgkin’s lymphomas (NHL) and chronic lymphocytic leukemia (CLL). Current scientific efforts are aimed to: identify factors that can accurately predict clinical responsiveness to rituximab, develop ways, to augment rituximab activity, and use current biotechnology to create novel and more potent anti-CD20 antibodies. Pre-clinical studies demonstrated a direct correlation between CD20 levels and rituximab-associated complement-mediated cytotoxicity. We previously demonstrated that CD20 levels varied between different subtypes of B-cell malignancies (Olejniczak SH et al. Immunol Invest. 2006; 35:93–114) raising the possibility that CD20 surface levels may correlate with the clinical behavior of B-cell lymphomas. In continuation of our work, we evaluated the correlation between quantitative CD20 expression on pathological material at diagnosis and clinical parameters/endpoints including overall survival (OS) in patients with a diagnosis of B-cell NHL and CLL. Patient’s specimens (lymph nodes, blood, or bone marrow) were identified by our tissue procurement facility and collected from 1997–2003. Surface Antigen expression of CD20 was quantitated by quantitative indirect immunofluorescence assay (QIFI). Samples were analyzed using either three or four color flow cytometry with standard antibody panels. Demographic characteristics and clinical endpoint parameters (i.e. performance status, histology, stage, LDH at diagnosis, IPI/FLIPI score, treatment history, response to therapy, time to progression, OS) were collected. CD20 expression was divided into two categories using the median level as a cut off. Kaplan Meier curves were used to predict the overall survival in patients with high and low expression of CD20. A Cox proportional hazard model was used to study the effect of CD20 in multivariate analysis adjusting for age at diagnosis, response, source of quantifying CD20 expression, stage, race (Caucasian/non Caucasian), first course of treatment (CHOP/non CHOP) and sex. A total of 324 samples were analyzed, clinical parameters were available for 135 patients (24 patients with diffuse large B-cell lymphoma, 16 patients with follicular lymphoma, 48 patients with CLL and 47 patients with small lymphocytic lymphoma). Median age of the patients was 60 years. The vast majority of the patients received rituximab immunotherapy at varying time-points during their treatment history. Overall, Kaplan-Meier analysis demonstrated a trend, not statistically significant, towards better OS among B-cell lymphoma/CLL patients with high CD20 expression. However, when adjusted for histological subtype and other known predictors of survival, higher CD20 levels were associated with better OS. Using a multivariate analysis we found that patients with high CD20 expression at presentation had a statistically significant lower hazard of death compared to patients with low CD20 expression (Hazard ratio 0.27, CI 0.11 – 0.65, p value 0.0035). In addition, and as would be expected, those who achieved complete remission, partial remission or stable disease had a lower hazard of death (HR 0.05, 0.08 and 0.12 for patients in CR, PR and SD, respectively) when compared to patients with progressive disease. In summary, our data suggests that CD20 expression by QIFI at time of presentation is an independent prognostic factor of outcome in patients with B-cell malignancies. Similarly to what has been observed with other prognostic factors, validation in a larger sample of patients treated with rituximab-based therapy is necessary and ongoing to further establish the clinical value of monitoring CD20 levels by QIFI. Table. Multivariate Analysis (OS) | | | Hazard | 95 % CI | |:--------------------------------------------------------------------------------------------------------------------------------------:| ------- | ------ | ------- | ----- | | Variable | p-value | Ratio | Lower | Upper | | Multivariate analyses adjusting for age at diagnosis, response, source, stage, race first course of treatment (CHOP/not-CHOP), and PS. | | CD20 (reference, Low) | 0.0035 | 0.27 | 0.11 | 0.65 | | Response to First Treatment (reference, PD) | | | | | | CR |

  • CD20 Antigen density at time of presentation is an independent predictor of outcome in b cell lymphoproliferative disorders
    Blood, 2008
    Co-Authors: Sapna Khubchandani, Myron S. Czuczman, Scott H. Olejniczak, Terry Mashtare, Mohammad M Chisti, Gregory E. Wilding, Francisco J Hernandezilizaliturri
    Abstract:

    The integration of monoclonal antibodies (mAbs) targeting CD20 Antigen has changed the treatment paradigm of patients with B-cell non Hodgkin’s lymphomas (NHL) and chronic lymphocytic leukemia (CLL). Current scientific efforts are aimed to: identify factors that can accurately predict clinical responsiveness to rituximab, develop ways, to augment rituximab activity, and use current biotechnology to create novel and more potent anti-CD20 antibodies. Pre-clinical studies demonstrated a direct correlation between CD20 levels and rituximab-associated complement-mediated cytotoxicity. We previously demonstrated that CD20 levels varied between different subtypes of B-cell malignancies (Olejniczak SH et al. Immunol Invest. 2006; 35:93–114) raising the possibility that CD20 surface levels may correlate with the clinical behavior of B-cell lymphomas. In continuation of our work, we evaluated the correlation between quantitative CD20 expression on pathological material at diagnosis and clinical parameters/endpoints including overall survival (OS) in patients with a diagnosis of B-cell NHL and CLL. Patient’s specimens (lymph nodes, blood, or bone marrow) were identified by our tissue procurement facility and collected from 1997–2003. Surface Antigen expression of CD20 was quantitated by quantitative indirect immunofluorescence assay (QIFI). Samples were analyzed using either three or four color flow cytometry with standard antibody panels. Demographic characteristics and clinical endpoint parameters (i.e. performance status, histology, stage, LDH at diagnosis, IPI/FLIPI score, treatment history, response to therapy, time to progression, OS) were collected. CD20 expression was divided into two categories using the median level as a cut off. Kaplan Meier curves were used to predict the overall survival in patients with high and low expression of CD20. A Cox proportional hazard model was used to study the effect of CD20 in multivariate analysis adjusting for age at diagnosis, response, source of quantifying CD20 expression, stage, race (Caucasian/non Caucasian), first course of treatment (CHOP/non CHOP) and sex. A total of 324 samples were analyzed, clinical parameters were available for 135 patients (24 patients with diffuse large B-cell lymphoma, 16 patients with follicular lymphoma, 48 patients with CLL and 47 patients with small lymphocytic lymphoma). Median age of the patients was 60 years. The vast majority of the patients received rituximab immunotherapy at varying time-points during their treatment history. Overall, Kaplan-Meier analysis demonstrated a trend, not statistically significant, towards better OS among B-cell lymphoma/CLL patients with high CD20 expression. However, when adjusted for histological subtype and other known predictors of survival, higher CD20 levels were associated with better OS. Using a multivariate analysis we found that patients with high CD20 expression at presentation had a statistically significant lower hazard of death compared to patients with low CD20 expression (Hazard ratio 0.27, CI 0.11 – 0.65, p value 0.0035). In addition, and as would be expected, those who achieved complete remission, partial remission or stable disease had a lower hazard of death (HR 0.05, 0.08 and 0.12 for patients in CR, PR and SD, respectively) when compared to patients with progressive disease. In summary, our data suggests that CD20 expression by QIFI at time of presentation is an independent prognostic factor of outcome in patients with B-cell malignancies. Similarly to what has been observed with other prognostic factors, validation in a larger sample of patients treated with rituximab-based therapy is necessary and ongoing to further establish the clinical value of monitoring CD20 levels by QIFI. Table. Multivariate Analysis (OS) | | | Hazard | 95 % CI | |:--------------------------------------------------------------------------------------------------------------------------------------:| ------- | ------ | ------- | ----- | | Variable | p-value | Ratio | Lower | Upper | | Multivariate analyses adjusting for age at diagnosis, response, source, stage, race first course of treatment (CHOP/not-CHOP), and PS. | | CD20 (reference, Low) | 0.0035 | 0.27 | 0.11 | 0.65 | | Response to First Treatment (reference, PD) | | | | | | CR | <.0001 | 0.05 | 0.02 | 0.18 | | PR | 0.0003 | 0.08 | 0.02 | 0.32 | | SD | 0.0014 | 0.12 | 0.03 | 0.44 | | Stage | 0.0199 | 0.57 | 0.35 | 0.91 | | Race (reference, non-white) | 0.0045 | 0.19 | 0.06 | 0.59 | | First Treatment (reference, non-CHOP) | 0.0378 | 3.05 | 1.07 | 8.71 | | ECOG PS | <.0001 | 7.79 | 3.41 | 17.77 | ![Figure][1] Kaplan Meier curves for survival distribution [1]: pending:yes

  • Acquirement of rituximab resistance in lymphoma cell lines is associated with both global CD20 gene and protein down-regulation regulated at the pretranscriptional and posttranscriptional levels.
    Clinical cancer research : an official journal of the American Association for Cancer Research, 2008
    Co-Authors: Myron S. Czuczman, Scott H. Olejniczak, Aruna C. Gowda, Petr Starostik, Joy Knight, Adam Kotowski, Arvinder Binder, Harman Kaur, Julie Deans, Francisco J. Hernandez-ilizaliturri
    Abstract:

    Acquirement of resistance to rituximab has been observed in lymphoma patients. To define mechanisms associated with rituximab resistance, we developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 expression/structure, lipid raft domain (LRD) reorganization, calcium mobilization, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity (CMC) between parental and RRCL. Significant changes in surface CD20 Antigen expression were shown in RRCL. Decreased calcium mobilization and redistribution of CD20 into LRD were found in RRCL. Western blotting identified a unique 35 kDa protein band in RRCL, which was not seen in parental cells and was secondary to an increase in surface and cytoplasmic expression of IgM light chains. CD20 gene expression was decreased in RRCL. In vitro exposure to PS341 increased CD20 expression in RRCL and minimally improved the sensitivity to rituximab-associated CMC. Our data strongly suggest that the acquisition of rituximab resistance is associated with global gene and protein down-regulation of the CD20 Antigen affecting LRD organization and downstream signaling. CD20 expression seems to be regulated at the pretranscriptional and posttranscriptional levels. Proteasome inhibition partially reversed rituximab resistance, suggesting the existence of additional mediators of rituximab resistance. Future research is geared to identify drugs and/or biological agents that are effective against RRCL.

  • Emergence of Rituximab-Fludarabine Resistance Is Associated with Changes in CD20 Antigen Expression and Improved Response to Almetuzumab Therapy in Patients with Chronic Lymphocytic Leukemia (CLL).
    Blood, 2007
    Co-Authors: Umeer Ashraf, Myron S. Czuczman, Anjana Elefante, Raymond Cruz, Paul K. Wallace, Francisco J. Hernandez-llizaliturri
    Abstract:

    Cancer cells, including B-cell lymphoproliferative disorders, sculpt their phenotype in an attempt to escape not only immune-surveillance but also evade the anti-tumor activity of biological agents and/or chemotherapy drugs including rituximab. In an attempt to study the mechanisms that regulate the emergence of rituximab resistance, we developed several rituximab-resistant cell lines and demonstrated that the emergence of a rituximab-resistant phenotype was associated with global down regulation of CD20 Antigen and unexpectedly an increase in CD52 Antigen. Validation of our findings in more clinically relevant settings is important to support alemtuzumab-based clinical studies in rituximab-resistant B-cell malignancies. To this end, we retrospectively studied changes in CD20 expression over time in alemtuzumab treated-patients with rituximab-fludarabine (RF) refractory CLL. Patients were identified using the institute tumor registry and pharmacy electronic database. Demographic characteristics, treatment history, outcome data were obtained for each patient. In addition, CD20 expression in CLL cells obtained from flow cytometry analysis performed on peripheral blood, bone marrow and tissue was reviewed. A total of 16 patients with RF refractory CLL treated with alemtuzumab were included in the analysis, 13 males and 3 females, the median age at the time of diagnosis was 55.5 yrs +/− 10.47stv, and most of the patients had a good PS (0) at the time of treatment with alemtuzumab (81%). The response rate to alemtuzumab was 56.3%, with 7 (43.5%) patients achieving a complete response (CR). After a median follow up period of 88.5 months, 7 patients are still alive, 3 are free of disease. Complete flow cytometry data was available only for 9 patients. A down regulation of CD20 Antigen expression was observed among 5/9 patients in blood, bone marrow and/or tissue over time when compared to baseline CD20 levels. Following alemtuzumab therapy, CR was achieved in 3/5 patients with CD20 down-regulation versus 1/4 patients with steady levels of CD20. Alemtuzumab-treated CLL patients with CD20 down-regulation had a longer overall survival (141 months) than alemtuzumab treated CLL patients with steady levels of CD20 Antigen overtime (116 months, Log Rank P = 0.026). Our data suggest, that emergence of rituximab-fludarabine resistance is associated with changes in the expression of CD20 Antigen. A response to alemtuzumab in this setting appears to be higher than historical controls (2% CR). Refractory CLL patients with decreased levels of surface CD20 appears to respond better to alemtuzumab and have a longer overall survival than patients with steady CD20 levels. Similarly to what we have observed in our pre-clinical models, an increase in CD52 expression in rituximab-fludarabine refractory patients with CD20 down regulation could explain the higher response rate observed in this small group of highly selected patients. Detailed monitoring of CD20 and CD52 levels in CLL patients before, during and after treatment with rituximab-based regimens is warranted in an attempt to identify patients that can benefit from almetuzumab-based therapies.

  • Modulation of CD20 Expression in Rituximab-Sensitive (RSCL) and Rituximab Resistant Cell Lines (RRCL) Using IL-4 and Bryostatin-1.
    Blood, 2007
    Co-Authors: Ping-chiao Tsai, Francisco J. Hernandez-ilizaliturri, Scott H. Olejniczak, Bangia Naveen, Myron S. Czuczman
    Abstract:

    While the use of rituximab in combination with chemotherapy resulted in an improved survival among various subtypes of B-cell lymphomas, a significant number of patients fail to respond or relapse as a consequence of intrinsic or acquired resistance. A change in CD20 Antigen density expression is a potential mechanism to explain rituximab resistance. Moreover, several groups of investigators are focused in understanding the mechanisms that regulate CD20 expression and to develop therapeutic strategies to up-regulate CD20 expression (i.e. IL-4, GM-CSF or Bryostatin-1). Up-regulation of CD20 in DB and Ramos cells by Bryostatin-1 was found to be PKC and Erk dependent. In an attempt to characterize the mechanisms responsible for rituximab resistance we developed several RRCL derived from rituximab-sensitive RL and Raji cells. We have demonstrated a significant down-regulation of CD20mRNA and CD20 surface Antigen in RRCL when compared to RSCL. In our present work we evaluated the mechanisms involved in the mRNA down-regulation of CD20 and the potential of IL-4 and Bryostatin-1 in modulating CD20 Antigen expression among a panel of RRCL. To this end, RSCL and RRCL were treated with either 5ng/ml of IL-4 or 3 different doses of Bryostatin-1(1, 3 or 5ng/ml). Nuclear and cytosolic extract were also obtained from RSCL RL and RRCL RL-4RH after 24, 48 and 74 hrs exposure to IL-4 (5ng/ml) or control. Differences in the expression of key regulatory transcription factors for B-cell lymphocyte development (PU.1, Oct-2, Pax5, E2A and EBF) were studied by Western Blotting. Previously, we found a significant down-regulation of CD20 Antigen in the RRCL. An up-regulation of cytosolic and surface CD20 was detected in RRCL exposed to either IL-4 or Bryostatin by western blotting and flow cytometric analysis. Besides, a higher expression of Pax5, PU.1, EBF was found in nuclear fractions of RRCL when compared to RSCL. In vitro exposure of RRCL to IL-4 decreases the expression of PU.1, Oct-2, Pax5 and EBF in nuclear extracts from RSCL or RRCL when compared with controls treated cells. Our data suggest that rituximab resistance is associated with the up-regulation of transcription factors PU.1, Oct-2, Pax5 and EBF and concomitant suppression of CD20 Antigen. Future study of how these agents induce CD20 expression in RRCL will provide a potential means to reversing resistance towards rituximab treatment.

Francisco J. Hernandez-ilizaliturri - One of the best experts on this subject based on the ideXlab platform.

  • CD20 Antigen Density at Time of Presentation Is An Independent Predictor of Outcome in B-Cell Lymphoproliferative Disorders
    Blood, 2008
    Co-Authors: Sapna Khubchandani, Myron S. Czuczman, Scott H. Olejniczak, Terry Mashtare, Mohammad M Chisti, Gregory E. Wilding, Francisco J. Hernandez-ilizaliturri
    Abstract:

    The integration of monoclonal antibodies (mAbs) targeting CD20 Antigen has changed the treatment paradigm of patients with B-cell non Hodgkin’s lymphomas (NHL) and chronic lymphocytic leukemia (CLL). Current scientific efforts are aimed to: identify factors that can accurately predict clinical responsiveness to rituximab, develop ways, to augment rituximab activity, and use current biotechnology to create novel and more potent anti-CD20 antibodies. Pre-clinical studies demonstrated a direct correlation between CD20 levels and rituximab-associated complement-mediated cytotoxicity. We previously demonstrated that CD20 levels varied between different subtypes of B-cell malignancies (Olejniczak SH et al. Immunol Invest. 2006; 35:93–114) raising the possibility that CD20 surface levels may correlate with the clinical behavior of B-cell lymphomas. In continuation of our work, we evaluated the correlation between quantitative CD20 expression on pathological material at diagnosis and clinical parameters/endpoints including overall survival (OS) in patients with a diagnosis of B-cell NHL and CLL. Patient’s specimens (lymph nodes, blood, or bone marrow) were identified by our tissue procurement facility and collected from 1997–2003. Surface Antigen expression of CD20 was quantitated by quantitative indirect immunofluorescence assay (QIFI). Samples were analyzed using either three or four color flow cytometry with standard antibody panels. Demographic characteristics and clinical endpoint parameters (i.e. performance status, histology, stage, LDH at diagnosis, IPI/FLIPI score, treatment history, response to therapy, time to progression, OS) were collected. CD20 expression was divided into two categories using the median level as a cut off. Kaplan Meier curves were used to predict the overall survival in patients with high and low expression of CD20. A Cox proportional hazard model was used to study the effect of CD20 in multivariate analysis adjusting for age at diagnosis, response, source of quantifying CD20 expression, stage, race (Caucasian/non Caucasian), first course of treatment (CHOP/non CHOP) and sex. A total of 324 samples were analyzed, clinical parameters were available for 135 patients (24 patients with diffuse large B-cell lymphoma, 16 patients with follicular lymphoma, 48 patients with CLL and 47 patients with small lymphocytic lymphoma). Median age of the patients was 60 years. The vast majority of the patients received rituximab immunotherapy at varying time-points during their treatment history. Overall, Kaplan-Meier analysis demonstrated a trend, not statistically significant, towards better OS among B-cell lymphoma/CLL patients with high CD20 expression. However, when adjusted for histological subtype and other known predictors of survival, higher CD20 levels were associated with better OS. Using a multivariate analysis we found that patients with high CD20 expression at presentation had a statistically significant lower hazard of death compared to patients with low CD20 expression (Hazard ratio 0.27, CI 0.11 – 0.65, p value 0.0035). In addition, and as would be expected, those who achieved complete remission, partial remission or stable disease had a lower hazard of death (HR 0.05, 0.08 and 0.12 for patients in CR, PR and SD, respectively) when compared to patients with progressive disease. In summary, our data suggests that CD20 expression by QIFI at time of presentation is an independent prognostic factor of outcome in patients with B-cell malignancies. Similarly to what has been observed with other prognostic factors, validation in a larger sample of patients treated with rituximab-based therapy is necessary and ongoing to further establish the clinical value of monitoring CD20 levels by QIFI. Table. Multivariate Analysis (OS) | | | Hazard | 95 % CI | |:--------------------------------------------------------------------------------------------------------------------------------------:| ------- | ------ | ------- | ----- | | Variable | p-value | Ratio | Lower | Upper | | Multivariate analyses adjusting for age at diagnosis, response, source, stage, race first course of treatment (CHOP/not-CHOP), and PS. | | CD20 (reference, Low) | 0.0035 | 0.27 | 0.11 | 0.65 | | Response to First Treatment (reference, PD) | | | | | | CR |

  • Acquirement of rituximab resistance in lymphoma cell lines is associated with both global CD20 gene and protein down-regulation regulated at the pretranscriptional and posttranscriptional levels.
    Clinical cancer research : an official journal of the American Association for Cancer Research, 2008
    Co-Authors: Myron S. Czuczman, Scott H. Olejniczak, Aruna C. Gowda, Petr Starostik, Joy Knight, Adam Kotowski, Arvinder Binder, Harman Kaur, Julie Deans, Francisco J. Hernandez-ilizaliturri
    Abstract:

    Acquirement of resistance to rituximab has been observed in lymphoma patients. To define mechanisms associated with rituximab resistance, we developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 expression/structure, lipid raft domain (LRD) reorganization, calcium mobilization, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity (CMC) between parental and RRCL. Significant changes in surface CD20 Antigen expression were shown in RRCL. Decreased calcium mobilization and redistribution of CD20 into LRD were found in RRCL. Western blotting identified a unique 35 kDa protein band in RRCL, which was not seen in parental cells and was secondary to an increase in surface and cytoplasmic expression of IgM light chains. CD20 gene expression was decreased in RRCL. In vitro exposure to PS341 increased CD20 expression in RRCL and minimally improved the sensitivity to rituximab-associated CMC. Our data strongly suggest that the acquisition of rituximab resistance is associated with global gene and protein down-regulation of the CD20 Antigen affecting LRD organization and downstream signaling. CD20 expression seems to be regulated at the pretranscriptional and posttranscriptional levels. Proteasome inhibition partially reversed rituximab resistance, suggesting the existence of additional mediators of rituximab resistance. Future research is geared to identify drugs and/or biological agents that are effective against RRCL.

  • Modulation of CD20 Expression in Rituximab-Sensitive (RSCL) and Rituximab Resistant Cell Lines (RRCL) Using IL-4 and Bryostatin-1.
    Blood, 2007
    Co-Authors: Ping-chiao Tsai, Francisco J. Hernandez-ilizaliturri, Scott H. Olejniczak, Bangia Naveen, Myron S. Czuczman
    Abstract:

    While the use of rituximab in combination with chemotherapy resulted in an improved survival among various subtypes of B-cell lymphomas, a significant number of patients fail to respond or relapse as a consequence of intrinsic or acquired resistance. A change in CD20 Antigen density expression is a potential mechanism to explain rituximab resistance. Moreover, several groups of investigators are focused in understanding the mechanisms that regulate CD20 expression and to develop therapeutic strategies to up-regulate CD20 expression (i.e. IL-4, GM-CSF or Bryostatin-1). Up-regulation of CD20 in DB and Ramos cells by Bryostatin-1 was found to be PKC and Erk dependent. In an attempt to characterize the mechanisms responsible for rituximab resistance we developed several RRCL derived from rituximab-sensitive RL and Raji cells. We have demonstrated a significant down-regulation of CD20mRNA and CD20 surface Antigen in RRCL when compared to RSCL. In our present work we evaluated the mechanisms involved in the mRNA down-regulation of CD20 and the potential of IL-4 and Bryostatin-1 in modulating CD20 Antigen expression among a panel of RRCL. To this end, RSCL and RRCL were treated with either 5ng/ml of IL-4 or 3 different doses of Bryostatin-1(1, 3 or 5ng/ml). Nuclear and cytosolic extract were also obtained from RSCL RL and RRCL RL-4RH after 24, 48 and 74 hrs exposure to IL-4 (5ng/ml) or control. Differences in the expression of key regulatory transcription factors for B-cell lymphocyte development (PU.1, Oct-2, Pax5, E2A and EBF) were studied by Western Blotting. Previously, we found a significant down-regulation of CD20 Antigen in the RRCL. An up-regulation of cytosolic and surface CD20 was detected in RRCL exposed to either IL-4 or Bryostatin by western blotting and flow cytometric analysis. Besides, a higher expression of Pax5, PU.1, EBF was found in nuclear fractions of RRCL when compared to RSCL. In vitro exposure of RRCL to IL-4 decreases the expression of PU.1, Oct-2, Pax5 and EBF in nuclear extracts from RSCL or RRCL when compared with controls treated cells. Our data suggest that rituximab resistance is associated with the up-regulation of transcription factors PU.1, Oct-2, Pax5 and EBF and concomitant suppression of CD20 Antigen. Future study of how these agents induce CD20 expression in RRCL will provide a potential means to reversing resistance towards rituximab treatment.

  • Structural Changes in the Internal Domain of CD20 Antigen Associated with the Emergence of Rituximab Resistance: Effects of Proteasome Inhibition in CD20 Structure and Rituximab Anti-Tumor Activity in Rituximab-Resistant Cell Lines (RRCL).
    Blood, 2005
    Co-Authors: Francisco J. Hernandez-ilizaliturri, Scott H. Olejniczak, Joy Knight, Myron S. Czuczman
    Abstract:

    Background: The acquirement of resistance to rituximab has been observed in lymphoma patients. To define the molecular basis for rituximab resistance we developed various RRCL and studied changes in CD20 structure, membrane reorganization and signaling events following rituximab therapy in RRCL. In addition we previously reported upregulation in gene/protein expression of members of the ubiquitin-proteasome system in RRCL. Recently, we evaluated the effects of proteasome inhibition in the structure of CD20 Antigen and rituximab sensitivity in RRCL. Methods: RRCL were generated by chronic exposure of Raji cells to escalating doses of rituximab alone (2R) or with human complement (4RH). Functional assays were performed to demonstrate a decrease in rituximab-associated activity. Changes in the structure of CD20 were determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. Following rituximab treatment, lipid raft domains were extracted from Raji and RRCL. Redistribution of CD20 Antigen following rituximab exposure was evaluated by Western blotting. RRCL were exposed to 0, 20 and 100nM of Bortezomib for 24 and 48 hrs. Changes in the internal domain of CD20 were evaluated by Western-blotting. Rituximab responses following Bortezomib exposure were determined by flow cytometric analysis in RRCL exposed to rituximab or isotype (10μg/ml) +/− human serum (dilution 1:4). Results: No significant changes were observed in the expression of the external domain of CD20 Antigen between rituximab-sensitive and RRCL as demonstrated by flow cytometric analysis and Western blotting. However, significant changes in the internal domain of CD20 were observed in RRCL. Specifically, a significant decrease in expression of the N-terminal, and to a lesser degree the C-terminal region of the internal domain of CD20 were observed in RRCL. Redistribution of CD20 into the lipid raft domains was decreased in RRCL following rituximab exposure. In vitro exposure to Bortezomib for 24 and 48 hrs resulted in an increased expression of the C-terminal region of the internal domain of CD20 in RRCL. In addition, Bortezomib-exposed RRCL regained partial sensitivity to rituximab-associated complement mediated cytotoxicity. Conclusions: Our data strongly suggests that the acquirement of rituximab resistance is associated with significant changes in the structure of the internal domain of the CD20 Antigen. The abnormal production of truncated forms of the CD20 Antigen, while not affecting rituximab binding, decreases the redistribution of CD20 Antigen into lipid raft domains and negatively impacts rituximab anti-tumor activity. The C-terminal region of CD20 expression is modulated by exposure to Bortezomib. Proteasome inhibition partially improves rituximab responsiveness suggesting the existence of mediators of rituximab-resistance.

  • Acquirement of Rituximab Resistance in Lymphoma Cell Lines Is Associated with Structural Changes in the Internal Domain of CD20 Regulated at the Post-Transcriptional Level.
    Blood, 2004
    Co-Authors: Myron S. Czuczman, Scott H. Olejniczak, Aruna C. Gowda, Petr Starostik, Francisco J. Hernandez-ilizaliturri
    Abstract:

    The acquirement of resistance to rituximab has been observed in lymphoma patients. To further define the molecular basis for rituximab-resistance we have developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 structure at the protein and gene level, membrane reorganization, and signaling events following rituximab exposure. RRCL were generated by chronic exposure of Raji cells to escalating doses of rituximab alone (2R) or concurrently with human complement (4RH). Functional assays were performed to demonstrate decrease in rituximab-associated CMC and ADCC. Changes in the structure of CD20 were determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. Sequencing of the CD20 gene from Raji parental and RRCL was performed to determine differences. Following rituximab treatment, lipid raft domains were extracted from Raji and RRCL to determine changes in polarization. Redistribution of CD20 Antigen were evaluated by Western blotting. No significant changes were observed in the expression of the external domain of CD20 Antigen between rituximab-sensitive and RRCL as demonstrated by flow cytometric analysis and Western blotting. However, significant changes in the internal domain of CD20 were observed in RRCL. Specifically, changes in the N-terminal, and to a lesser degree the C-terminal region of the internal domain of CD20 were observed in RRCL. The CD20 gene sequence was found to be identical between Raji cells and RRCL, suggesting a post-transcriptional regulatory mechanism is responsible for the changes in the structure of CD20. Redistribution of CD20 into the lipid raft domains was more evident in Raji parental cells when compared to RRCL, and a decrease in p38 activation was observed following exposure of RRCL to rituximab. In conclusion, our data strongly suggests that the acquirement of rituximab-resistance is associated with significant changes in the structure of the internal domain of the CD20 Antigen. The abnormal production of truncated forms of the CD20 Antigen, while not affecting external rituximab binding resulted in changes in the redistribution of CD20 Antigen into lipid raft domains and a decrease in signaling events. Post-transcriptional events leading to the acquirement of truncated forms of CD20 may impair signaling activation, thus contributing to rituximab-resistance seen in RRCL.

Fiorella D'auria - One of the best experts on this subject based on the ideXlab platform.

  • The clinical and biological role of CD20 membrane Antigen modulation under immunotherapy with anti-CD20 monoclonal antibody rituximab in lymphoprolipherative neoplastic disorders
    Expert Opinion on Biological Therapy, 2011
    Co-Authors: Pellegrino Musto, Fiorella D'auria
    Abstract:

    Immunotherapy using an antibody (rituximab) targeting CD20 Antigen in combination with chemotherapy has been recently associated with significantly improved response rate and survival in patients with various types of CD20-positive B-cell lymphoproliferative disorders. This treatment may induce the disappearance of CD20 surface expression on neoplastic B-cells. Several mechanisms have been proposed to explain CD20 loss after rituximab therapy, while the clinical significance (if any) of this phenomenon is still not clear. We have produced a brief overview of the biological aspects of CD20 modulation after rituximab treatment and its possible clinical implications.

  • Modulation of CD20 Antigen expression after rituximab treatment: a retrospective study in patients with chronic lymphocytic leukemia.
    Clinical therapeutics, 2010
    Co-Authors: Fiorella D'auria, Roberto Guariglia, Oreste Villani, G. Mansueto, Vitina Grieco, Antonia Zonno, Gabriella Bianchino, Luigina Di Giovannantonio, Giulia Vita, Pellegrino Musto
    Abstract:

    Abstract Background: CD20 Antigen down-modulation by anti-CD20 rituximab treatment is a well-recognized phenomenon in patients with non-Hodgkin's lymphoma. However, few data are currently available on this topic in other lymphoproliferative disorders, in particular in chronic lymphocytic leukemia (CLL). Objective: The aim of this study was to establish how many patients with CLL show a disappearance of CD20 Antigen after salvage treatment with rituximab and its possible clinical significance. Methods: We sequentially analyzed CD20 expression by flow cytometry in patients treated with rituximab in combination with other agents for relapsed/resistant disease. Results: Eleven white patients with CLL (6 females, 5 males; median age, 71.6 years [range, 60–84 years]) were included in the study. Three of the 11 patients were not positive for CD20 due to complete response at baseline. Four of the remaining 8 patients (50%) lacked CD20 Antigen on neoplastic cells after monoclonal antibody treatment. Two of them developed Richter's syndrome and died within 4 months. The phenomenon was transient in the other 2 patients, who were alive after a follow-up of 25 and 26 months, respectively, with CD20-positive recurrent disease. Conclusions: In this study, CD20 Antigen disappearance in patients with CLL treated with rituximab-containing salvage regimens occurred in 4 of 8 (50%) tested patients, half of whom developed Richter's syndrome. [Note: Since the initial writing and submission, a third patient developed Richter's syndrome.] In 2 patients (50%), CD20 returned at progression.

Pellegrino Musto - One of the best experts on this subject based on the ideXlab platform.

  • The clinical and biological role of CD20 membrane Antigen modulation under immunotherapy with anti-CD20 monoclonal antibody rituximab in lymphoprolipherative neoplastic disorders
    Expert Opinion on Biological Therapy, 2011
    Co-Authors: Pellegrino Musto, Fiorella D'auria
    Abstract:

    Immunotherapy using an antibody (rituximab) targeting CD20 Antigen in combination with chemotherapy has been recently associated with significantly improved response rate and survival in patients with various types of CD20-positive B-cell lymphoproliferative disorders. This treatment may induce the disappearance of CD20 surface expression on neoplastic B-cells. Several mechanisms have been proposed to explain CD20 loss after rituximab therapy, while the clinical significance (if any) of this phenomenon is still not clear. We have produced a brief overview of the biological aspects of CD20 modulation after rituximab treatment and its possible clinical implications.

  • Modulation of CD20 Antigen expression after rituximab treatment: a retrospective study in patients with chronic lymphocytic leukemia.
    Clinical therapeutics, 2010
    Co-Authors: Fiorella D'auria, Roberto Guariglia, Oreste Villani, G. Mansueto, Vitina Grieco, Antonia Zonno, Gabriella Bianchino, Luigina Di Giovannantonio, Giulia Vita, Pellegrino Musto
    Abstract:

    Abstract Background: CD20 Antigen down-modulation by anti-CD20 rituximab treatment is a well-recognized phenomenon in patients with non-Hodgkin's lymphoma. However, few data are currently available on this topic in other lymphoproliferative disorders, in particular in chronic lymphocytic leukemia (CLL). Objective: The aim of this study was to establish how many patients with CLL show a disappearance of CD20 Antigen after salvage treatment with rituximab and its possible clinical significance. Methods: We sequentially analyzed CD20 expression by flow cytometry in patients treated with rituximab in combination with other agents for relapsed/resistant disease. Results: Eleven white patients with CLL (6 females, 5 males; median age, 71.6 years [range, 60–84 years]) were included in the study. Three of the 11 patients were not positive for CD20 due to complete response at baseline. Four of the remaining 8 patients (50%) lacked CD20 Antigen on neoplastic cells after monoclonal antibody treatment. Two of them developed Richter's syndrome and died within 4 months. The phenomenon was transient in the other 2 patients, who were alive after a follow-up of 25 and 26 months, respectively, with CD20-positive recurrent disease. Conclusions: In this study, CD20 Antigen disappearance in patients with CLL treated with rituximab-containing salvage regimens occurred in 4 of 8 (50%) tested patients, half of whom developed Richter's syndrome. [Note: Since the initial writing and submission, a third patient developed Richter's syndrome.] In 2 patients (50%), CD20 returned at progression.

Aruna C. Gowda - One of the best experts on this subject based on the ideXlab platform.

  • Acquirement of rituximab resistance in lymphoma cell lines is associated with both global CD20 gene and protein down-regulation regulated at the pretranscriptional and posttranscriptional levels.
    Clinical cancer research : an official journal of the American Association for Cancer Research, 2008
    Co-Authors: Myron S. Czuczman, Scott H. Olejniczak, Aruna C. Gowda, Petr Starostik, Joy Knight, Adam Kotowski, Arvinder Binder, Harman Kaur, Julie Deans, Francisco J. Hernandez-ilizaliturri
    Abstract:

    Acquirement of resistance to rituximab has been observed in lymphoma patients. To define mechanisms associated with rituximab resistance, we developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 expression/structure, lipid raft domain (LRD) reorganization, calcium mobilization, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity (CMC) between parental and RRCL. Significant changes in surface CD20 Antigen expression were shown in RRCL. Decreased calcium mobilization and redistribution of CD20 into LRD were found in RRCL. Western blotting identified a unique 35 kDa protein band in RRCL, which was not seen in parental cells and was secondary to an increase in surface and cytoplasmic expression of IgM light chains. CD20 gene expression was decreased in RRCL. In vitro exposure to PS341 increased CD20 expression in RRCL and minimally improved the sensitivity to rituximab-associated CMC. Our data strongly suggest that the acquisition of rituximab resistance is associated with global gene and protein down-regulation of the CD20 Antigen affecting LRD organization and downstream signaling. CD20 expression seems to be regulated at the pretranscriptional and posttranscriptional levels. Proteasome inhibition partially reversed rituximab resistance, suggesting the existence of additional mediators of rituximab resistance. Future research is geared to identify drugs and/or biological agents that are effective against RRCL.

  • Acquirement of Rituximab Resistance in Lymphoma Cell Lines Is Associated with Structural Changes in the Internal Domain of CD20 Regulated at the Post-Transcriptional Level.
    Blood, 2004
    Co-Authors: Myron S. Czuczman, Scott H. Olejniczak, Aruna C. Gowda, Petr Starostik, Francisco J. Hernandez-ilizaliturri
    Abstract:

    The acquirement of resistance to rituximab has been observed in lymphoma patients. To further define the molecular basis for rituximab-resistance we have developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 structure at the protein and gene level, membrane reorganization, and signaling events following rituximab exposure. RRCL were generated by chronic exposure of Raji cells to escalating doses of rituximab alone (2R) or concurrently with human complement (4RH). Functional assays were performed to demonstrate decrease in rituximab-associated CMC and ADCC. Changes in the structure of CD20 were determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. Sequencing of the CD20 gene from Raji parental and RRCL was performed to determine differences. Following rituximab treatment, lipid raft domains were extracted from Raji and RRCL to determine changes in polarization. Redistribution of CD20 Antigen were evaluated by Western blotting. No significant changes were observed in the expression of the external domain of CD20 Antigen between rituximab-sensitive and RRCL as demonstrated by flow cytometric analysis and Western blotting. However, significant changes in the internal domain of CD20 were observed in RRCL. Specifically, changes in the N-terminal, and to a lesser degree the C-terminal region of the internal domain of CD20 were observed in RRCL. The CD20 gene sequence was found to be identical between Raji cells and RRCL, suggesting a post-transcriptional regulatory mechanism is responsible for the changes in the structure of CD20. Redistribution of CD20 into the lipid raft domains was more evident in Raji parental cells when compared to RRCL, and a decrease in p38 activation was observed following exposure of RRCL to rituximab. In conclusion, our data strongly suggests that the acquirement of rituximab-resistance is associated with significant changes in the structure of the internal domain of the CD20 Antigen. The abnormal production of truncated forms of the CD20 Antigen, while not affecting external rituximab binding resulted in changes in the redistribution of CD20 Antigen into lipid raft domains and a decrease in signaling events. Post-transcriptional events leading to the acquirement of truncated forms of CD20 may impair signaling activation, thus contributing to rituximab-resistance seen in RRCL.

  • Structural changes in the internal domain of the CD20 Antigen is associated with the development of rituximab resistance: Effects on signaling and redistribution of CD20 into lipid raft domains in rituximab-resistant cell lines (RRCL)
    Journal of Clinical Oncology, 2004
    Co-Authors: Aruna C. Gowda, Francisco J. Hernandez-ilizaliturri, G. Connolly, Myron S. Czuczman
    Abstract:

    2502 Backgound: Following rituximab binding, CD20 Antigen redistributes into lipid raft domains (LRD) and initiates signaling events leading to apoptosis in malignant B-cells. The development of resistance to rituximab has been observed clinically in lymphoma patients. To define the molecular basis for rituximab resistance we developed various RRCL and studied changes in CD20 structure, membrane reorganization and signaling events following rituximab therapy. Methods: RRCL were generated by chronic exposure of Raji cells to escalating doses of rituximab alone (2R) or with human complement (4RH). Changes in the structure of CD20 were determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. Following rituximab exposure, LRD were extracted from Raji and RRCL. Redistribution of CD20 Antigen and signaling events following rituximab exposure were evaluated by Western blotting. Results: No significant changes were obse...