ABSTRACTBackgroundDrawing genotype-to-phenotype maps in tumors is of paramount importance for und... more ABSTRACTBackgroundDrawing genotype-to-phenotype maps in tumors is of paramount importance for understanding tumor heterogeneity. Assignment of single cells to their tumor clones of origin can be approached by matching the genotypes of the clones to the mutations found in RNA sequencing of the cells. The confidence of the cell-to-clone mapping can be increased by accounting for additional measurements. Follicular lymphoma, a malignancy of mature B cells that continuously acquire mutations in parallel in the exome and in B-cell receptor loci, presents a unique opportunity to align exome-derived mutations with B-cell receptor clonotypes as an independent measure for clonal evolution.ResultsHere, we propose CACTUS, a probabilistic model that leverages the information from an independent genomic clustering of cells and exploits the scarce single cell RNA sequencing data to map single cells to given imperfect genotypes of tumor clones. We apply CACTUS to two follicular lymphoma patient sa...
B cell chronic lymphocytic leukemia (CLL), the most prevalent B cell malignancy in adults, is cha... more B cell chronic lymphocytic leukemia (CLL), the most prevalent B cell malignancy in adults, is characterized by expansion of monoclonal mature B lymphocytes. Despite advances in treatment, the disease remains incurable warranting further efforts to identify novel molecular targets in CLL. B cell receptor (BCR) signaling contributes to apoptosis resistance in CLL limiting the efficacy of therapeutic approaches. In this study we investigated the expression of spleen tyrosine kinase (SYK), a key component of the BCR signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared to healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCγ2, STAT3, and ERK1/2 in CLL compared to healthy B cells suggesting enhanced activation of these mediators in CLL. Separate analyses according to prognostic parameters revealed 1.8-fold higher SYK protein level in unmutated comp...
Idiotype vaccination refers to active immunization of B-cell lymphoma (B-NHL) patients with the c... more Idiotype vaccination refers to active immunization of B-cell lymphoma (B-NHL) patients with the clonal immunoglobulin (Ig) expressed by the tumor cells. After systemic cytoreductive therapy, idiotype vaccination has been shown to induce specific cellular and humoral immune responses; and humoral responses in particular are associated with prolonged remission and encouraging survival rates. Conventional idiotype vaccines are composed of the entire lymphoma-derived Ig coupled to the immunogenic carrier KLH and are administered subcutaneously with adjuvant. We have developed a idiotype production strategy based on bacterial expression of the lymphoma-derived idiotype as a recombinant Fab fragment (Bertinetti et al., EJH 2006). Intradermal administration of this antigen with lipid-based adjuvant and subcutaneous coadministration of GM-CSF had excellent immunogenicity in a phase I trial of advanced, heavily pretreated B-NHL patients (Bertinetti et al., Cancer Research 2006). In a subsequ...
The simultaneous appearance of B-CLL and MM in the same patient (pt) seems to be a rare phenomeno... more The simultaneous appearance of B-CLL and MM in the same patient (pt) seems to be a rare phenomenon. In those cases - our experience included - 1. the myeloma exhibits an aggressive course, 2. with CLL-treatment, MM seems to emerge, 3. which, if well treated (e.g. high-dose chemotherapy [CTx] with autologous peripheral blood stem cell transplantation [auto-PBSCT]), may lead to a slower myeloma course, improved clinical condition and CLL disapearance. Here we report on the simultaneous (n=2) and successive (n=2) occurrence of CLL and MM in four pts, who were diagnosed at our center during the last year. We suggest that the occurrence of CLL and MM, when carefully monitored, may be more frequent than assumed so far. Case 1. This 64-year-old male was evaluated due to weight loss, leukocytosis, anaemia, foamy urine and increased creatinine. Kappa (k) Bence-Jones (BJ) proteinuria and elevated k-serum free light chains (SFLC) were found. A bone survey was normal, but BM biopsy showed infil...
The hypervariable regions (idiotypes) of the immunoglobulins (Ig) expressed by lymphoma cells rep... more The hypervariable regions (idiotypes) of the immunoglobulins (Ig) expressed by lymphoma cells represent individual and specific tumor antigens. Purified or recombinant lymphoma-derived idiotype (Id) may be formulated into an individual vaccine and administered to lymphoma patients to elicit a therapeutic tumor-specific host immune response. We have developed an expression strategy based on anchored PCR amplification of lymphoma-derived cDNA. The Id variable (V) gene segments are inserted into a prokaryotic expression vector, and recombinant Fab fragment corresponding to the native Id protein is expressed in E.coli. This approach was applied to 106 biopsies with confirmed lymphoma involvement (47 follicular, 14 mantle cell, 6 diffuse large cell NHL, 22 myelomas, 6 CLL, and 6 other NHL types) and 8 non-lymphomatous control samples with the aim to investigate the feasibility of this production strategy in a large cohort of NHL patients, and to assess the yield and purity of the recombi...
Introduction & Objectives: Primary Cutaneous Follicle Center Lymphoma (PCFCL) is a very indolent ... more Introduction & Objectives: Primary Cutaneous Follicle Center Lymphoma (PCFCL) is a very indolent mature B-cell lymphoma that shares germinal center morphology with follicular lymphoma (FL) but lacks the characteristic t(14;18). Unlike FL, immunohistochemistry fails to detect expression of BCL2, CD10, and immunoglobulin in PCFCL. Therefore, we investigated expression of B-cell receptor (BCR) transcripts to gain insight into the immunobiology of PCFCL. Materials & Methods: Full-length heavy and light chain BCR transcripts of 13 histologically confirmed PCFCL were amplified using ARTISAN PCR and sequenced on the PacBio RSII system. BCR from 4 cases were sequenced to a depth of >2000 sequences per BCR transcript; the remaining cases to a median depth of 1663 sequences (range: 626-5301). BCR from 51 cases of FL and from peripheral B cells of 12 healthy donors were used as controls. Whole genome sequencing (WGS) and RNAseq were performed on 5 PCFCL on the Illumina HiSeq platform. Resul...
Viral infections and disease relapses are major complications between T cell depleted allogeneic ... more Viral infections and disease relapses are major complications between T cell depleted allogeneic stem cell transplantation (TCD alloSCT) and donor lymphocyte infusion (DLI). The prophylactic infusion of selected donor T cells may be an effective method to restore anti-viral and anti-tumor immunity early after TCD alloSCT. In this phase I/II study, we aimed to prevent these complications by the infusion of donor-derived T cell products containing CD8+ T cells directed against cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV) antigens (Ag), tumor associated Ag (TAA) and minor histocompatibility Ag (MiHA) 6-8 weeks after TCD alloSCT. The feasibility of multi-Ag specific T cell product generation and safety of early administration were investigated. Furthermore, clinical outcome and immune reconstitution were assessed. HLA-A2+ patients treated for a hematological malignancy with a ≥9/10 HLA-matched TCD graft from a CMV+ and/or EBV+ (un)related donor were eligible for ...
1633 Introduction: The individual collection of epitopes within the variable regions of the uniqu... more 1633 Introduction: The individual collection of epitopes within the variable regions of the unique immunoglobulin expressed by every mature B-cell lymphoma (idiotype, or Id) represents a tumor-specific antigen and lends itself as a target for therapeutic vaccination strategies. Immunization with tumor Id has the capacity to elicit polyclonal antibody responses as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II HLA molecules, respectively. Due to a perceived low immunogenicity of lymphoma-derived Id, most Id vaccines tested in clinical trials so far have been formulated as conjugates with the strongly immunogenic carrier keyhole limpet hemocyanin (KLH). In contrast, we have consistently observed high rates of humoral and cellular anti-Id immune responses in consecutive trials of active immunization with unconjugated recombinant Fab fragments of Id in indolent B-cell lymphomas (Bertinetti et al., Cancer Res. 2006; Navarrete et al., BLOOD...
3132 The idiotype (Id) of malignant lymphomas is a tumor-specific antigen and has been evaluated ... more 3132 The idiotype (Id) of malignant lymphomas is a tumor-specific antigen and has been evaluated for active immunotherapy predominantly in follicular lymphoma (FL). We have previously described “reverse immunology” evidence that HLA class I-dependent and hence presumably T-cell mediated selection pressure restricts the growth of malignant FL clones with comparatively highly immunogenic Ids (Strothmeyer et al., Blood, epub 2010). This effect was mostly attributable to the Id CDRs. We therefore hypothesized that somatic hypermutation, a constitutively active process in FL, is the mechanism that creates immunogenic Id epitopes and enables T-cell mediated immunosurveillance. To test this hypothesis, we analyzed Id of FL that utilized the same Vh gene but arose in individuals with different HLA types (IGHV3-23*01, n=9 patients: IGHV3-7*01, n=3). For every represented HLA type, an “immunogenicity landscape” mapping was performed by plotting the ten most immunogenic peptides of the germ-li...
1949 Poster Board I-972 Tumor-specific immune responses can be induced in patients with indolent ... more 1949 Poster Board I-972 Tumor-specific immune responses can be induced in patients with indolent B cell lymphomas (iNHL) by active immunization against the individual B cell receptor (BCR) expressed by the malignant B cell clone, the so-called “idiotype” (Id). In subsequent trials of intradermal vaccination with recombinant lymphoma-derived Fab fragment in iNHL, we have studied the specificity of MHC class I-restricted anti-Id T cell responses by epitope mapping experiments with synthetic Id-derived peptides predicted to be presented by the respective…
B-cell malignancy-derived immunoglobulin (idiotype) and survivin, a member of the inhibitor of ap... more B-cell malignancy-derived immunoglobulin (idiotype) and survivin, a member of the inhibitor of apoptosis gene family and a shared tumor-associated antigen, are expressed by B-CLL cells. Idiotype- and survivin-specific cytotoxic T cells (CTLs), capable of lysing primary autologous B-CLL cells, can be induced in patients with B-CLL. However, the leukemia cell microenvironment was shown to protect B-CLL cells from apoptosis. The protective effects of stromal cells can be reversed by CXCR4 antagonists in vitro and resensitize CLL cells to spontaneous and chemotherapy-induced apoptosis. The aim of the present study is to investigate whether stromal cell contact impairs CLL killing by CTLs raised against immunoglobulin- or survivin-derived peptides and whether the addition of CXCR4 inhibitors enhances T cell mediated cytotoxicity. To analyze the T cell response, we isolated CD8+ T cells and PBMCs from HLA-A2+ healthy donors. PBMCs were differentiated into dendritic cells (DCs) and CD40-ac...
2880 Background: The development and / or course of chronic lymphocytic leukemia (CLL) may be dri... more 2880 Background: The development and / or course of chronic lymphocytic leukemia (CLL) may be driven by the recognition of antigens through the B cell receptor (BCR). While it has been recognized that the diversity of epitope recognition may be astonishingly confined in CLL, knowledge on antigens recognized by CLL BCRs is still limited. Here, we identified and characterized an epitope recognized by a defined CLL BCR which may broaden our view on potential mechanisms of antigenic drive in CLL. Methods: The B- cell receptor of a random CLL-patient was cloned and expressed as Fab fragment in E.coli. Random phage display reptile litanies we skeletal on the immobilized Fab and landed peptides were tested for specific binding. Specific clones we sequenced and sequences were analyzed for homology to known proteins. Recognition of candidate proteins was verified in brooding assays or recombinant proteins. Results: Screening random phage display peptide libraries, we identified a CLL BCR epi...
182 The B-cell antigen receptor (BCR) plays a critical role in the development and progression of... more 182 The B-cell antigen receptor (BCR) plays a critical role in the development and progression of B-cell lymphomas. In chronic lymphocytic leukemia (CLL), the existence of stereotyped heavy-chain complementarity determining regions (HCDR3) suggested that binding of external antigens might play a role in CLL pathogenesis. In contrast, we recently reported that BCRs derived from both human CLL patients and from Eμ-TCL1tg mice have the unique function to induce antigen-independent signaling. This capacity is mediated by the HCDR3 through binding to a BCR internal motif in adjacent BCRs on the same cell (Dühren-von Minden et al., Nature 2012). Mature B cells as well as CLL B cells co-express IgM and IgD with the same HCDR3. In this study, we address the respective roles of these expressed BCR classes in lymphoma pathogenesis in Eμ-TCL1tg mice and in human CLL. By mating Eμ-TCL1tg-mice with IgM−/− mice, which lacks the μ constant heavy domain (μCH) and instead expresses IgD in all devel...
Introduction Trans-placental trafficking of maternal and fetal cells during pregnancy establishes... more Introduction Trans-placental trafficking of maternal and fetal cells during pregnancy establishes long-term, reciprocal micro-chimerism in both mother and child (Maloney et al., J Clin Invest, 104:41, 1999). As a consequence, the immune system of the mother may become sensitized to paternal histocompatibility antigens. In fact, antibodies directed against paternal HLA-antigens (van Rood JJ et al., Nature 181:1735, 1958) and T lymphocytes directed against paternal major and minor histocompatibility antigens (van Kampen CA et al., Hum Immunol 62:201, 2001; Verdijk RM et al., Blood 103:1961, 2004) were detected in multiparous women. More recently, it was hypothesized that mother's…
Follicular lymphoma (FL) is initiated by the t(14:18) that places the anti-apoptotic proto-oncoge... more Follicular lymphoma (FL) is initiated by the t(14:18) that places the anti-apoptotic proto-oncogene BCL2 under transcriptional control of the immunoglobulin heavy chain (IgH) locus during primary VDJ recombination. Neoplastic FL B cells are arrested at the germinal center stage with ongoing somatic hypermutation of B-cell receptor (BCR) genes by activation-induced deaminase (AID). Antigen recognition by the clonal BCR may facilitate malignant growth upon entry of t(14;18)-immortalized naïve B cells into secondary lymphoid organs. We have reported at ASH 2012 that FL may be classified according to the AID-mediated BCR evolution pattern into two distinct subgroups: A predominantly IgM-expressing subgroup with evidence for antigen-driven affinity maturation, and a subgroup selected for BCR sequence preservation that has often undergone class switch recombination. To investigate whether these BCR selection patterns were correlated to clinical outcome, we retrospectively analyzed somatic...
1929 Background: Allogeneic hematopoietic cell transplantation (HCT) is associated with inferior ... more 1929 Background: Allogeneic hematopoietic cell transplantation (HCT) is associated with inferior outcomes in patients with more advanced stage AML. In such cases, reducing the ablative intensity of the preparative regimen could potentially compromise its efficacy. We hereby compare transplant outcomes of two preparative regimens, known as FB2 and FB4, in patients (pts) with AML transplanted in second complete remission (CR2) or beyond at EBMT participating centers. Materials and methods: Between 2003 and 2010, 128 (FB2=88 (69%), FB4=40 (31%)) pts with a median age of (FB2=60 (22–70) years, FB4=42 (19–65) years, p<0.0001), with AML in ≥CR2 (FB2 (CR2=85 (97%), CR3=3 (3%)); FB4 (CR2=38 (95%), CR3=2 (5%)), p=0.67, underwent allogeneic HCT. FB2 comprised intravenous (IV) busulfan cumulative dose of 6.4 ± 10% mg/kg, while FB4 cumulative dose was 12.8 ± 10% mg/kg. Cytogenetic risks groups were similar: FB2 (good=17, int=31, poor=6) and FB4 (good=6, int=7, poor=0), p=0.35. For pts treate...
3867 Introduction: CXCR4 surface expression is strongly elevated on CLL cells and is important fo... more 3867 Introduction: CXCR4 surface expression is strongly elevated on CLL cells and is important for CLL cell survival and homing to the bone marrow. PIM kinases are overexpressed and mutated in various hematologic malignancies including B-cell lymphomas. Recent findings in AML suggest a direct link in between PIM1 kinase expression and the surface expression level of the CXCR4 receptor and Ser339 phosphorylation in CXCR4. Therefore the aim of the presented study was to investigate the role of PIM kinases in regulating the CXCR4-CXCL12 pathway in CLL. Results: By using a phospho-CXCR4 Ser339 antibody for both, western blot analysis of CLL cells from the peripheral blood (n=11) and IHC staining of bone marrow from CLL patients (n=30), we can show that in contrast to normal B-cells the majority of CLL cells have a hyperphosphorylated CXCR4 receptor. Furthermore high phosphorylation levels of the CXCR4 receptor in the bone marrow correlated with poor prognosis in CLL. Besides increased p...
2378 Poster Board II-355 B cell chronic lymphocytic leukemia (B-CLL), one of the most common leuk... more 2378 Poster Board II-355 B cell chronic lymphocytic leukemia (B-CLL), one of the most common leukemia in adults, is characterized by the accumulation of mature B cells expressing CD19 and CD5. Improved understanding of CLL has lead to new prognostic tools and therapeutic options. The protein kinase Syk as a key mediator of proximal B-cell receptor (BCR) signalling is constitutively phosphorylated in CLL B cells. For these novel therapeutics, functional in vivo models are highly valuable. We have established a cell line-based, disseminated B-CLL model in NOD/SCID-IL2-receptor-gamma-chain-/- (NSG) mice. In the current study, SYK inhibitor R788 (60mg/kg/day (d); applied daily) was evaluated in mono- and in combined therapy with Fludarabin (8mg/kg/day; applied 5 consecutive days in two cycles) as a well established compound in B-CLL treatment regimens in comparison with a control group. Equal parts of MEC1 cells were injected intravenously and into the peritoneal cavity of NSG mice and ...
ABSTRACTBackgroundDrawing genotype-to-phenotype maps in tumors is of paramount importance for und... more ABSTRACTBackgroundDrawing genotype-to-phenotype maps in tumors is of paramount importance for understanding tumor heterogeneity. Assignment of single cells to their tumor clones of origin can be approached by matching the genotypes of the clones to the mutations found in RNA sequencing of the cells. The confidence of the cell-to-clone mapping can be increased by accounting for additional measurements. Follicular lymphoma, a malignancy of mature B cells that continuously acquire mutations in parallel in the exome and in B-cell receptor loci, presents a unique opportunity to align exome-derived mutations with B-cell receptor clonotypes as an independent measure for clonal evolution.ResultsHere, we propose CACTUS, a probabilistic model that leverages the information from an independent genomic clustering of cells and exploits the scarce single cell RNA sequencing data to map single cells to given imperfect genotypes of tumor clones. We apply CACTUS to two follicular lymphoma patient sa...
B cell chronic lymphocytic leukemia (CLL), the most prevalent B cell malignancy in adults, is cha... more B cell chronic lymphocytic leukemia (CLL), the most prevalent B cell malignancy in adults, is characterized by expansion of monoclonal mature B lymphocytes. Despite advances in treatment, the disease remains incurable warranting further efforts to identify novel molecular targets in CLL. B cell receptor (BCR) signaling contributes to apoptosis resistance in CLL limiting the efficacy of therapeutic approaches. In this study we investigated the expression of spleen tyrosine kinase (SYK), a key component of the BCR signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared to healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCγ2, STAT3, and ERK1/2 in CLL compared to healthy B cells suggesting enhanced activation of these mediators in CLL. Separate analyses according to prognostic parameters revealed 1.8-fold higher SYK protein level in unmutated comp...
Idiotype vaccination refers to active immunization of B-cell lymphoma (B-NHL) patients with the c... more Idiotype vaccination refers to active immunization of B-cell lymphoma (B-NHL) patients with the clonal immunoglobulin (Ig) expressed by the tumor cells. After systemic cytoreductive therapy, idiotype vaccination has been shown to induce specific cellular and humoral immune responses; and humoral responses in particular are associated with prolonged remission and encouraging survival rates. Conventional idiotype vaccines are composed of the entire lymphoma-derived Ig coupled to the immunogenic carrier KLH and are administered subcutaneously with adjuvant. We have developed a idiotype production strategy based on bacterial expression of the lymphoma-derived idiotype as a recombinant Fab fragment (Bertinetti et al., EJH 2006). Intradermal administration of this antigen with lipid-based adjuvant and subcutaneous coadministration of GM-CSF had excellent immunogenicity in a phase I trial of advanced, heavily pretreated B-NHL patients (Bertinetti et al., Cancer Research 2006). In a subsequ...
The simultaneous appearance of B-CLL and MM in the same patient (pt) seems to be a rare phenomeno... more The simultaneous appearance of B-CLL and MM in the same patient (pt) seems to be a rare phenomenon. In those cases - our experience included - 1. the myeloma exhibits an aggressive course, 2. with CLL-treatment, MM seems to emerge, 3. which, if well treated (e.g. high-dose chemotherapy [CTx] with autologous peripheral blood stem cell transplantation [auto-PBSCT]), may lead to a slower myeloma course, improved clinical condition and CLL disapearance. Here we report on the simultaneous (n=2) and successive (n=2) occurrence of CLL and MM in four pts, who were diagnosed at our center during the last year. We suggest that the occurrence of CLL and MM, when carefully monitored, may be more frequent than assumed so far. Case 1. This 64-year-old male was evaluated due to weight loss, leukocytosis, anaemia, foamy urine and increased creatinine. Kappa (k) Bence-Jones (BJ) proteinuria and elevated k-serum free light chains (SFLC) were found. A bone survey was normal, but BM biopsy showed infil...
The hypervariable regions (idiotypes) of the immunoglobulins (Ig) expressed by lymphoma cells rep... more The hypervariable regions (idiotypes) of the immunoglobulins (Ig) expressed by lymphoma cells represent individual and specific tumor antigens. Purified or recombinant lymphoma-derived idiotype (Id) may be formulated into an individual vaccine and administered to lymphoma patients to elicit a therapeutic tumor-specific host immune response. We have developed an expression strategy based on anchored PCR amplification of lymphoma-derived cDNA. The Id variable (V) gene segments are inserted into a prokaryotic expression vector, and recombinant Fab fragment corresponding to the native Id protein is expressed in E.coli. This approach was applied to 106 biopsies with confirmed lymphoma involvement (47 follicular, 14 mantle cell, 6 diffuse large cell NHL, 22 myelomas, 6 CLL, and 6 other NHL types) and 8 non-lymphomatous control samples with the aim to investigate the feasibility of this production strategy in a large cohort of NHL patients, and to assess the yield and purity of the recombi...
Introduction & Objectives: Primary Cutaneous Follicle Center Lymphoma (PCFCL) is a very indolent ... more Introduction & Objectives: Primary Cutaneous Follicle Center Lymphoma (PCFCL) is a very indolent mature B-cell lymphoma that shares germinal center morphology with follicular lymphoma (FL) but lacks the characteristic t(14;18). Unlike FL, immunohistochemistry fails to detect expression of BCL2, CD10, and immunoglobulin in PCFCL. Therefore, we investigated expression of B-cell receptor (BCR) transcripts to gain insight into the immunobiology of PCFCL. Materials & Methods: Full-length heavy and light chain BCR transcripts of 13 histologically confirmed PCFCL were amplified using ARTISAN PCR and sequenced on the PacBio RSII system. BCR from 4 cases were sequenced to a depth of >2000 sequences per BCR transcript; the remaining cases to a median depth of 1663 sequences (range: 626-5301). BCR from 51 cases of FL and from peripheral B cells of 12 healthy donors were used as controls. Whole genome sequencing (WGS) and RNAseq were performed on 5 PCFCL on the Illumina HiSeq platform. Resul...
Viral infections and disease relapses are major complications between T cell depleted allogeneic ... more Viral infections and disease relapses are major complications between T cell depleted allogeneic stem cell transplantation (TCD alloSCT) and donor lymphocyte infusion (DLI). The prophylactic infusion of selected donor T cells may be an effective method to restore anti-viral and anti-tumor immunity early after TCD alloSCT. In this phase I/II study, we aimed to prevent these complications by the infusion of donor-derived T cell products containing CD8+ T cells directed against cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV) antigens (Ag), tumor associated Ag (TAA) and minor histocompatibility Ag (MiHA) 6-8 weeks after TCD alloSCT. The feasibility of multi-Ag specific T cell product generation and safety of early administration were investigated. Furthermore, clinical outcome and immune reconstitution were assessed. HLA-A2+ patients treated for a hematological malignancy with a ≥9/10 HLA-matched TCD graft from a CMV+ and/or EBV+ (un)related donor were eligible for ...
1633 Introduction: The individual collection of epitopes within the variable regions of the uniqu... more 1633 Introduction: The individual collection of epitopes within the variable regions of the unique immunoglobulin expressed by every mature B-cell lymphoma (idiotype, or Id) represents a tumor-specific antigen and lends itself as a target for therapeutic vaccination strategies. Immunization with tumor Id has the capacity to elicit polyclonal antibody responses as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II HLA molecules, respectively. Due to a perceived low immunogenicity of lymphoma-derived Id, most Id vaccines tested in clinical trials so far have been formulated as conjugates with the strongly immunogenic carrier keyhole limpet hemocyanin (KLH). In contrast, we have consistently observed high rates of humoral and cellular anti-Id immune responses in consecutive trials of active immunization with unconjugated recombinant Fab fragments of Id in indolent B-cell lymphomas (Bertinetti et al., Cancer Res. 2006; Navarrete et al., BLOOD...
3132 The idiotype (Id) of malignant lymphomas is a tumor-specific antigen and has been evaluated ... more 3132 The idiotype (Id) of malignant lymphomas is a tumor-specific antigen and has been evaluated for active immunotherapy predominantly in follicular lymphoma (FL). We have previously described “reverse immunology” evidence that HLA class I-dependent and hence presumably T-cell mediated selection pressure restricts the growth of malignant FL clones with comparatively highly immunogenic Ids (Strothmeyer et al., Blood, epub 2010). This effect was mostly attributable to the Id CDRs. We therefore hypothesized that somatic hypermutation, a constitutively active process in FL, is the mechanism that creates immunogenic Id epitopes and enables T-cell mediated immunosurveillance. To test this hypothesis, we analyzed Id of FL that utilized the same Vh gene but arose in individuals with different HLA types (IGHV3-23*01, n=9 patients: IGHV3-7*01, n=3). For every represented HLA type, an “immunogenicity landscape” mapping was performed by plotting the ten most immunogenic peptides of the germ-li...
1949 Poster Board I-972 Tumor-specific immune responses can be induced in patients with indolent ... more 1949 Poster Board I-972 Tumor-specific immune responses can be induced in patients with indolent B cell lymphomas (iNHL) by active immunization against the individual B cell receptor (BCR) expressed by the malignant B cell clone, the so-called “idiotype” (Id). In subsequent trials of intradermal vaccination with recombinant lymphoma-derived Fab fragment in iNHL, we have studied the specificity of MHC class I-restricted anti-Id T cell responses by epitope mapping experiments with synthetic Id-derived peptides predicted to be presented by the respective…
B-cell malignancy-derived immunoglobulin (idiotype) and survivin, a member of the inhibitor of ap... more B-cell malignancy-derived immunoglobulin (idiotype) and survivin, a member of the inhibitor of apoptosis gene family and a shared tumor-associated antigen, are expressed by B-CLL cells. Idiotype- and survivin-specific cytotoxic T cells (CTLs), capable of lysing primary autologous B-CLL cells, can be induced in patients with B-CLL. However, the leukemia cell microenvironment was shown to protect B-CLL cells from apoptosis. The protective effects of stromal cells can be reversed by CXCR4 antagonists in vitro and resensitize CLL cells to spontaneous and chemotherapy-induced apoptosis. The aim of the present study is to investigate whether stromal cell contact impairs CLL killing by CTLs raised against immunoglobulin- or survivin-derived peptides and whether the addition of CXCR4 inhibitors enhances T cell mediated cytotoxicity. To analyze the T cell response, we isolated CD8+ T cells and PBMCs from HLA-A2+ healthy donors. PBMCs were differentiated into dendritic cells (DCs) and CD40-ac...
2880 Background: The development and / or course of chronic lymphocytic leukemia (CLL) may be dri... more 2880 Background: The development and / or course of chronic lymphocytic leukemia (CLL) may be driven by the recognition of antigens through the B cell receptor (BCR). While it has been recognized that the diversity of epitope recognition may be astonishingly confined in CLL, knowledge on antigens recognized by CLL BCRs is still limited. Here, we identified and characterized an epitope recognized by a defined CLL BCR which may broaden our view on potential mechanisms of antigenic drive in CLL. Methods: The B- cell receptor of a random CLL-patient was cloned and expressed as Fab fragment in E.coli. Random phage display reptile litanies we skeletal on the immobilized Fab and landed peptides were tested for specific binding. Specific clones we sequenced and sequences were analyzed for homology to known proteins. Recognition of candidate proteins was verified in brooding assays or recombinant proteins. Results: Screening random phage display peptide libraries, we identified a CLL BCR epi...
182 The B-cell antigen receptor (BCR) plays a critical role in the development and progression of... more 182 The B-cell antigen receptor (BCR) plays a critical role in the development and progression of B-cell lymphomas. In chronic lymphocytic leukemia (CLL), the existence of stereotyped heavy-chain complementarity determining regions (HCDR3) suggested that binding of external antigens might play a role in CLL pathogenesis. In contrast, we recently reported that BCRs derived from both human CLL patients and from Eμ-TCL1tg mice have the unique function to induce antigen-independent signaling. This capacity is mediated by the HCDR3 through binding to a BCR internal motif in adjacent BCRs on the same cell (Dühren-von Minden et al., Nature 2012). Mature B cells as well as CLL B cells co-express IgM and IgD with the same HCDR3. In this study, we address the respective roles of these expressed BCR classes in lymphoma pathogenesis in Eμ-TCL1tg mice and in human CLL. By mating Eμ-TCL1tg-mice with IgM−/− mice, which lacks the μ constant heavy domain (μCH) and instead expresses IgD in all devel...
Introduction Trans-placental trafficking of maternal and fetal cells during pregnancy establishes... more Introduction Trans-placental trafficking of maternal and fetal cells during pregnancy establishes long-term, reciprocal micro-chimerism in both mother and child (Maloney et al., J Clin Invest, 104:41, 1999). As a consequence, the immune system of the mother may become sensitized to paternal histocompatibility antigens. In fact, antibodies directed against paternal HLA-antigens (van Rood JJ et al., Nature 181:1735, 1958) and T lymphocytes directed against paternal major and minor histocompatibility antigens (van Kampen CA et al., Hum Immunol 62:201, 2001; Verdijk RM et al., Blood 103:1961, 2004) were detected in multiparous women. More recently, it was hypothesized that mother's…
Follicular lymphoma (FL) is initiated by the t(14:18) that places the anti-apoptotic proto-oncoge... more Follicular lymphoma (FL) is initiated by the t(14:18) that places the anti-apoptotic proto-oncogene BCL2 under transcriptional control of the immunoglobulin heavy chain (IgH) locus during primary VDJ recombination. Neoplastic FL B cells are arrested at the germinal center stage with ongoing somatic hypermutation of B-cell receptor (BCR) genes by activation-induced deaminase (AID). Antigen recognition by the clonal BCR may facilitate malignant growth upon entry of t(14;18)-immortalized naïve B cells into secondary lymphoid organs. We have reported at ASH 2012 that FL may be classified according to the AID-mediated BCR evolution pattern into two distinct subgroups: A predominantly IgM-expressing subgroup with evidence for antigen-driven affinity maturation, and a subgroup selected for BCR sequence preservation that has often undergone class switch recombination. To investigate whether these BCR selection patterns were correlated to clinical outcome, we retrospectively analyzed somatic...
1929 Background: Allogeneic hematopoietic cell transplantation (HCT) is associated with inferior ... more 1929 Background: Allogeneic hematopoietic cell transplantation (HCT) is associated with inferior outcomes in patients with more advanced stage AML. In such cases, reducing the ablative intensity of the preparative regimen could potentially compromise its efficacy. We hereby compare transplant outcomes of two preparative regimens, known as FB2 and FB4, in patients (pts) with AML transplanted in second complete remission (CR2) or beyond at EBMT participating centers. Materials and methods: Between 2003 and 2010, 128 (FB2=88 (69%), FB4=40 (31%)) pts with a median age of (FB2=60 (22–70) years, FB4=42 (19–65) years, p<0.0001), with AML in ≥CR2 (FB2 (CR2=85 (97%), CR3=3 (3%)); FB4 (CR2=38 (95%), CR3=2 (5%)), p=0.67, underwent allogeneic HCT. FB2 comprised intravenous (IV) busulfan cumulative dose of 6.4 ± 10% mg/kg, while FB4 cumulative dose was 12.8 ± 10% mg/kg. Cytogenetic risks groups were similar: FB2 (good=17, int=31, poor=6) and FB4 (good=6, int=7, poor=0), p=0.35. For pts treate...
3867 Introduction: CXCR4 surface expression is strongly elevated on CLL cells and is important fo... more 3867 Introduction: CXCR4 surface expression is strongly elevated on CLL cells and is important for CLL cell survival and homing to the bone marrow. PIM kinases are overexpressed and mutated in various hematologic malignancies including B-cell lymphomas. Recent findings in AML suggest a direct link in between PIM1 kinase expression and the surface expression level of the CXCR4 receptor and Ser339 phosphorylation in CXCR4. Therefore the aim of the presented study was to investigate the role of PIM kinases in regulating the CXCR4-CXCL12 pathway in CLL. Results: By using a phospho-CXCR4 Ser339 antibody for both, western blot analysis of CLL cells from the peripheral blood (n=11) and IHC staining of bone marrow from CLL patients (n=30), we can show that in contrast to normal B-cells the majority of CLL cells have a hyperphosphorylated CXCR4 receptor. Furthermore high phosphorylation levels of the CXCR4 receptor in the bone marrow correlated with poor prognosis in CLL. Besides increased p...
2378 Poster Board II-355 B cell chronic lymphocytic leukemia (B-CLL), one of the most common leuk... more 2378 Poster Board II-355 B cell chronic lymphocytic leukemia (B-CLL), one of the most common leukemia in adults, is characterized by the accumulation of mature B cells expressing CD19 and CD5. Improved understanding of CLL has lead to new prognostic tools and therapeutic options. The protein kinase Syk as a key mediator of proximal B-cell receptor (BCR) signalling is constitutively phosphorylated in CLL B cells. For these novel therapeutics, functional in vivo models are highly valuable. We have established a cell line-based, disseminated B-CLL model in NOD/SCID-IL2-receptor-gamma-chain-/- (NSG) mice. In the current study, SYK inhibitor R788 (60mg/kg/day (d); applied daily) was evaluated in mono- and in combined therapy with Fludarabin (8mg/kg/day; applied 5 consecutive days in two cycles) as a well established compound in B-CLL treatment regimens in comparison with a control group. Equal parts of MEC1 cells were injected intravenously and into the peritoneal cavity of NSG mice and ...
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Papers by Hendrik Veelken