NFkB p50 (Nfkb1) Contributes to Disease in the Eu-TCL1 Mouse Model of Chronic Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL) is a disease of mature B-cells that are resistant to apoptosis and accumulate in the blood over time. Due to the slowly progressing nature of this disease, studies to determine the molecular events leading to defective apoptosis are challenging. Our group has previously reported that during disease progression in the Eμ-TCL1 CLL mouse model, genes become silenced in a progressive manner over the course of the disease. This silencing is due in part to a high degree of methylation at the promoters of key tumor suppressors. However, even though a high degree of methylation is observed in both this mouse model and in CLL patient samples, agents targeting methylation such as decitabine have proven ineffective in CLL therapy. Therefore other novel methods to reverse gene silencing in CLL are an attractive therapeutic option. In our previous study, we observed early transcriptional mechanisms of gene silencing (occurring prior to methylation) involving NF-κB p50 homodimers. NF-κB is a family of transcription factors which is known to play an important role in the progression of CLL. Advances in next generation sequencing have recently identified loss of function mutations in NFKBIE,a negative regulator of NF-κB signaling,in CLL. These mutations contribute to increased nuclear localization (and hence increased activation) of NF-κB subunits. In addition, a mutagenesis screen in the Eμ-TCL1 mouse found that several of the most frequently occurring mutations that contribute to disease progression occur in genes related to the NF-κB family, particularly in the p50 (Nfkb1) gene. In the present study, we have generated a new mouse model to further study the role of p50 in CLL pathogenesis. The Eμ-TCL1 mouse was crossed to the p50-/- mouse. The p50-/- mice are fertile with normal growth and development under sterile conditions; however they do exhibit a defective response to infection and decreased antibody production. As such, animals in this study are housed under pathogen free conditions. The initial cross resulted in mice that were all heterozygous for p50 (p50+/-), and either positive or negative for the TCL1 transgene. The p50+/-; TCL1+ animals were subsequently crossed with one another to generate three genotypes: p50+/+. p50+/- and p50-/-. Animals are monitored for disease by monthly flow cytometry analysis of CD19 and CD5 in whole blood, and leukemia is defined as >10% double positive cells. The p50-/- mice have a significantly lower incidence of leukemia compared to p50+/+ mice (Chi-square p <0.001), and p50+/- mice show an intermediate phenotype (p=0.024 compared to p50-/- mice). Despite pathogen free conditions, some mice within the p50-/- group die at an early age with no evidence of disease. Therefore, competing risks regression was performed to take into account the mice that die without leukemia via the CIF (cumulative incidence function) based on the Fine and Gray model. Differences in time to leukemia between the p50+/+ and p50-/- mice remained significant (Subdistribution Hazard Ratio; SHR = 8.45; 95% CI: 1.86 - 38.47; p=0.006). The p50+/- mice still exhibit an intermediate phenotype, with more separation between the p50+/- and p50-/- (SHR = 4.39; 95% CI: 1.00 - 19.29; p=0.050) than the p50+/- and p50+/+ groups (SHR = 0.52; 95% CI: 0.26 - 1.06; p=0.069). Finally, spleens from p50-/- mice are notably smaller than the p50+/+ littermates (average spleen score of 0-1 for p50-/- versus 2-3 for p50+/+, using a 0-4 scale), indicating that disease in the secondary lymphoid tissues is also affected by the loss of p50 in this model. In conclusion, these data genetically demonstrate the significant contribution of the p50 (Nfkb1) gene to disease progression in the Eµ-TCL1 mouse model. These studies highlight the importance of the NF-κB family in CLL, and suggest that p50 is a promising therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Role Of Lyn Kinase In The Pathogenesis Of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v122.21.668.668 ◽

2013 ◽

Vol 122 (21) ◽

pp. 668-668

Author(s):

Phuong-Hien Nguyen ◽

Nina Reinart ◽

Michael Hallek

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Myeloid Cells ◽

Lymphocytic Leukemia ◽

Negative Regulator ◽

Lymphoid Tissues ◽

Malignant Cells ◽

Deficient Mice

Abstract The Src family kinase Lyn is predominantly expressed in B cells and plays a central role in initiating B cell receptor (BCR) signaling. Lyn is associated with BCR complexes and is renowned for its role in B cell activation and proliferation. Active Lyn contributes to positive regulation of signalling through tyrosine phosphorylation of components of the BCR. Intriguingly, Lyn was also shown as a negative regulator of BCR signal transduction. Lyn plays an essential role in negative regulation of signalling through its unique ability to phosphorylate immunoreceptor tyrosine based inhibition motifs (ITIM) in inhibitory cell surface receptors. ITIM phosphorylation induces the recruitment of inhibitory phosphatases such as SHP-1/2 and SHIP-1, which attenuate BCR signalling. Lyn-deficient mice have reduced number of B cells and increased numbers of myeloid progenitors. It was reported that expression and activity of Lyn in human chronic lymphocytic leukemia (CLL) is elevated compared to healthy B cells. Besides, higher levels of Lyn are associated with a shorter treatment-free survival of CLL patients. This rises up a hypothesis about Lyn’s significant role in B cell tumorigenesis, malignant transformation of B cells, and the balance between myeloid cells and B lymphocytes. We generated Eµ-TCL1 transgenic LYN-deficient mice (TCL1+/wtLYN-/-) and monitored them in order to identify the population of malignant B cells and to characterize the development of malignant cells in these mice in comparison with Eµ-TCL1 transgenic mice (TCL1+/wtLYNwt/wt). In comparison to TCL1+/wtLYNwt/wt mice, TCL1+/wtLYN-/- mice show a significantly reduced number of malignant B cells in the peripheral blood, as well as a reduced leukocyte count. Besides, TCL1+/wtLYN-/- mice have significantly decreased infiltration of malignant B cells in lymphoid tissues such as spleen, liver, lymph node and bone marrow. This result is also resembled in a hepato-splenomegaly in the TCL1+/wtLYNwt/wt mice. These mice develop severe splenomegaly and hepatomegaly due to infiltration of malignant cells, while TCL1+/wtLYN-/- mice do not develop hepatomegaly. The non-transgenic LYN-/- control mice develop splenomegaly due to infiltration of myeloid cells. Although TCL1+/wtLYN-/- mice have hindered development of TCL1-induced CLL, preliminary data suggest it is not only due to LYN-deficiency in B cell compartment of these mice. Indeed, B cell of TCL1+/wtLYN-/- mice show enhanced proliferation and better survival ex vivo compared to TCL1+/wtLYNwt/wt mice. Notably, TCL1+/wtLYN-/- mice developed a skewed microenvironment which might contribute to CLL down regulation. LYN-/- microenvironment, particularly in aged mice, does not support engraftment of TCL1-induced leukemic B cell as well as LYNwt/wt mice in our transplantation model. These results point to a complex regulation of Lyn signalling in CLL involving not only leukemic cells but also cells of the micromillieu, that needs further investigation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Immune Thrombocytopenia Associated to Low-Dose Alemtuzumab Therapy in Chronic Lymphocytic Leukemia: A Single Retrospective Center Experience

Blood ◽

10.1182/blood.v120.21.4598.4598 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4598-4598

Author(s):

Gianluigi Reda ◽

Francesco Maura ◽

Giuseppe Gritti ◽

Daniele Vincenti ◽

Mariarita Sciumé ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Disease Progression ◽

Cumulative Dose ◽

Immune Thrombocytopenia ◽

Low Dose ◽

Conflicts Of Interest ◽

Autoimmune Disorder ◽

Intravenous Immunoglobulins ◽

Lymphocytic Leukemia ◽

Platelet Counts

Abstract Abstract 4598 Immune thrombocytopenia (ITP) is a common autoimmune complication in chronic lymphocytic leukemia (CLL), occurring in up to 5% of patients. The occurrence of alemtuzumab-associated ITP have been rarely reported in CLL and it has never been reported so far as a significant event complicating alemtuzumab treatment in clinical trials. Recently, a new distinctive form of secondary ITP occurring in 6 out of 215 patients with relapsing-remitting multiple sclerosis treated with alemtuzumab in a randomized, controlled phase II trial has been reported (Cuker et al, Blood 2011). We investigated the incidence of ITP in a cohort of 64 consecutive patients treated with low-dose alemtuzumab for relapsed/refractory CLL from 2003 to 2010. Subcutaneous alemtuzumab was administered at the dose of 10 mg three times a week, up to 18 weeks. ITP was documented in 12/64 patients: in 3 patients (4.7%) ITP developed before alemtuzumab treatment and no relapses of the autoimmune disorder were observed during the treatment; in 9 patients (14.8%, with an incidence of 5.7 events/100 patient-year) ITP developed after alemtuzumab treatment. Median time to ITP from initial alemtuzumab exposure was 12 months (range 1–42 months). Concomitant hemolytic anemia (Evans syndrome) was observed in one patient. At ITP onset, median platelet counts were 11×109/L (range 3–70) and anti-platelet antibodies (Capture P® Method, ImmucorGamma, Norcross GA, USA) were found in 7 of the 8 patients tested. No patients showed severe or life-threatening bleeding. Three of nine patients who developed ITP after alemtuzumab therapy, experienced CLL progression requiring chemo-immunotherapy after 3, 4 and 13 months from ITP onset, respectively. One patient achieved a partial remission of CLL with ITP resolution, while the other two died of disease progression. In the remaining six cases, ITP was not associated with disease progression and was treated with corticosteroids and/or intravenous immunoglobulins. Five patients achieved normal platelet counts, while one patient did not respond. Low-dose alemtuzumab (theoretical cumulative dose 540 mg, equal to half of the classic cumulative dose and one-third of the individual dose) is an effective, safe and well tolerated treatment for CLL, as reported by several recent studies (Cortelezzi et al, Leukemia 2009; Brit J Haematol 2011). In our cohort of CLL patients treated with alemtuzumab, the incidence of ITP was 14.8% that is almost three times higher than previously reported in CLL. These data may indicate a role of T-lymphocyte dysregulation induced by alemtuzumab in the pathogenesis of ITP. Our data also suggested the importance of monitoring platelet counts during follow-up in patients treated with low-dose alemtuzumab for both haematological and non-haematological diseases. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CD1d Expression Is Higher In Chronic Lymphocytic Leukemia Patients With Unfavorable Prognosis

Blood ◽

10.1182/blood.v122.21.5278.5278 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5278-5278

Author(s):

Agnieszka Bojarska-Junak ◽

Iwona Hus ◽

Anna Dmoszynska ◽

Jacek Rolinski

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Disease Progression ◽

Conflicts Of Interest ◽

Staining Intensity ◽

Lymphocytic Leukemia ◽

Disease Stage ◽

Inkt Cells ◽

Cd38 Expression

Abstract Chronic lymphocytic leukemia (CLL) is characterized by a very heterogeneous clinical course, which is slow and indolent in most of the patients, however some patient experience rapid disease progression and anticancer therapy is required shortly after the diagnosis. Many issues in CLL development and progression are still unclear. The functional consequences of CD1d expression on tumour cells are not well understood. However, increasing evidence suggests that they may affect iNKT cells.The role of CD1d expression in CLL immunopathogenesis remains undefined. In this study, we investigated the potential role of CD1d in CLL by analyzing the level of CD1d expression on leukemic B cells in peripheral blood of120 patients and assessed its correlation with prognostic markers such as ZAP-70 and CD38 expression, Rai stages and unfavourable cytogenetic changes.Measuring CD1d expression by flow cytometry and qRT-PCR, we showed lower CD1d molecule and CD1d mRNA expression in B cells of CLL patients than of healthy controls. The frequency of CD1d+/CD19+ cells, CD1d staining intensity and CD1d transcript levels increased with the disease stage. CD1d expression was positively associated with ZAP-70 and CD38 expressions as well as with unfavourable cytogenetic changes (17p deletion, 11q deletio),. We established the relationship between high CD1d expression and shorter time to treatment and overall survival. The percentage of CD1d+/CD19+cells inversely correlated with the percentage of iNKT cells. iNKT cells ζ-chain expression was downregulated in the high-CD1d group.These results suggest that high CD1d expression is associated with poor prognosis of CLL and might be involved in disease progression. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Reciprocal Densities of CXCR4 and CD5 Define Subfractions of Chronic Lymphocytic Leukemia Clones Differing in Phenotype and Response to Environmental Stimuli: Towards a Better Definition of Targetable Components of Leukemic Clones

Blood ◽

10.1182/blood.v124.21.3322.3322 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3322-3322

Author(s):

Rajendra N Damle ◽

Sonal Temburni ◽

Cristina Sison ◽

Jaison Jain ◽

Jacqueline C. Barrientos ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Negative Regulator ◽

Ki 67 ◽

Bcr Signaling ◽

In Vivo Labeling ◽

Definition Of ◽

The Impact

Abstract Although chronic lymphocytic leukemia (CLL) is an accumulative disorder of CD5+ B cells, a proliferative fraction exists and the extent of this proliferation correlates with clinical course. We previously demonstrated heterogeneous in vivo labeling kinetics of cells in 3 clonal fractions sorted based on reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and CD5 from CLL cases recruited on an in vivo labeling study. In that study, the CXCR4dimCD5br fraction contained more 2H-labeled DNA and hence recently divided cells (proliferative fraction) than the CXCR4brCD5dim(resting) fraction. Here, we have analyzed multiple CXCR4/CD5 based subclonal fractions and characterized their phenotypic differences and ability to respond to signals via the BCR and CXCR4 or a combination of the two. PBMCs from a cohort of 50 untreated IgM+ CLL cases were used for this study. Subclonal fractions marked based on differences in relative densities of CXCR4 and CD5 by CLL cells are referred to as CXCR4br, CXCR4int or CXCR4dim and CD5br, CD5int or CD5dim. Sub-populations adjacent to each other on flow cytometric plots differed at least 5 fold in the densities of both CXCR4 and CD5. When subcategorizing CLL clones based on these markers, 9 subfractions were identified. To permit comparisons between these fractions, each was defined as containing only 2-5% of the total clonal CD19+CD5+population while ensuring no overlap in CXCR4 or CD5 densities among each fraction. Interestingly, certain phenotypic and signaling features consistently clustered with distinct subfractions of the CLL clone. Subfraction analyses confirmed an enrichment of Ki-67+ cells in the CXCR4dimCD5br proliferative fraction of every CLL case. In addition, this subfraction showed the highest percentage of cells expressing smIgM and smIgD (p<0.01). Relative density (RD) of expression was deduced as a ratio of mean fluorescence intensity (MFI) of a molecule expressed by any subfraction compared to that expressed by CXCR4intCD5int cells within the same clone. When considering all cases the CXCR4dim CD5dim cells showed the highest RD of smIgM. However, among U-CLL cases the CXCR4dim CD5br cells expressed the highest RD for smIgM, and the CXCR4dim CD5dim subfraction showed the highest RD of smIgM expression in M-CLLs. Overall, both CXCR4dim CD5br and CXCR4int CD5br showed the highest RD for IgD expression, whereas among U-CLL and M-CLL cases the CXCR4int CD5br and CXCR4dim CD5int subfractions showed the highest RD of smIgD expression respectively. The CXCR4brCD5br subfraction was characterized by the highest % and RD of CD79b, the BCR signaling molecule, and CD22 and CTLA-4, negative regulators of signaling. However, Siglec-10, another negative regulator of BCR signaling, was most highly expressed both in % and RD by the CXCR4dim CD5br subfraction in all CLL cases. Finally, the highest percentages of cells expressing CD21, CD38, CD43, CD69, CD180 associated best with the 3 CD5br subfractions. Of note, CD38 was expressed at highest RD in the CXCR4dim CD5dim in M-CLL cases but in the CXCR4dim CD5brsubfraction in the U-CLL cases. When analyzing phosphorylation of Akt, Erk, p38MAPK and Syk by phosphoflow among all cases, the CXCR4brCD5br fraction exhibited the highest constitutive levels. Constitutive levels of phospho-Btk and - Syk were significantly higher in the CXCR4int CD5br subfraction in M-CLL than in U-CLL cases (p<0.05). Anti-BCR mAbs induced significant increases in phospho-p38MAPK and -Akt in the CXCR4br CD5br subfraction when comparing U-CLL to M-CLL cells. CXCL12 induced significant increases in phospho- Akt and -STAT-5 in CXCR4int CD5br subfraction in U-CLL cases (p<0.01), whereas a combination of signals via BCR and CXCR4 induced increases in phospho-Erk in the CXCR4int CD5br and CXCR4br CD5brpopulations in M-CLL cases. Together, these findings highlight the impact of the molecules expressed by the different subfractions on their ability to relay/inhibit signals elicited via the BCR or CXCR4. They also provide a general frame of reference to further understand functional post-replicative intraclonal heterogeneity of CLL clones and help define aggressive subfractions of the CLL clone as target populations for future therapies. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Divergence in CD19-Mediated Signaling Unfolds Intra-Clonal Diversity in Chronic Lymphocytic Leukemia Which Correlates with Disease Progression

Blood ◽

10.1182/blood.v120.21.4570.4570 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4570-4570

Author(s):

Yair Herishanu ◽

Sigi Kay ◽

Nili Dezorella ◽

Chava Perry ◽

Varda Deutsch ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Disease Progression ◽

Conflicts Of Interest ◽

Clonal Diversity ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Cell Fraction ◽

Akt Phosphorylation

Abstract Abstract 4570 Emerging data on intra-clonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with chronic lymphocytic leukemia (CLL), where subsets of the CLL clone responding more robustly to external stimuli may gain a growth and survival advantage. Here we report intra-clonal diversity resolved by responses to CD19 engagement in CLL cells, which can be classified into responding (CD19-R) and non-responding (CD19-N) sub-populations. Engagement of CD19 by anti-CD19 antibody rapidly induced cellular aggregation in the CD19-R CLL cells. The CD19-R CLL cells expressed higher surface levels of CD19 and c-myc mRNA and exhibited distinct morphological features. Both sub-populations reacted to IgM stimulation in a similar manner and exhibited similar levels of Akt phosphorylation, pointing to functional signaling divergence within the B-cell receptor. CD19 unresponsiveness was partially reversible, where CD19-N cells spontaneously recover their signaling capacity following incubation in vitro, pointing to possible in vivo CD19-signaling attenuating mechanisms. This concept was supported by the lower CD19-R occurrence in bone marrow-derived samples compared with cells derived from the peripheral blood of the same patients. CLL patients with more than 18.5% of CD19-R cell fraction had a shorter median time to treatment compared to patients with less than 18.5% of CD19-R cell fraction. Conclusions: Divergence in CD19-mediated signaling unfolds both inter-patient and intra-clonal diversity in CLL. This signaling diversity is associated with physiological implications including the location of the cells and disease progression. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Biological Impact of Monoallelic and Biallelic BIRC3 Loss in Del(11q) Chronic Lymphocytic Leukemia Progression

Blood ◽

10.1182/blood-2020-142658 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 4-4

Author(s):

Miguel Quijada Álamo ◽

Maria Hernandez-Sanchez ◽

Ana E. Rodriguez ◽

Claudia Pérez Carretero ◽

Marta Martín Izquierdo ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Line ◽

Cell Lines ◽

Nuclear Translocation ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Negative Regulator ◽

Depth Analysis ◽

The Impact

Chronic lymphocytic leukemia (CLL) patients harboring 11q22.3 deletion, del(11q), are characterized by a rapid disease progression. One of the suggested genes to be involved in the pathogenesis of this deletion is BIRC3, a negative regulator of NF-κB, which is monoallelically deleted in ~80% of del(11q) CLL cases. In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which accounts for marked reduced survival in CLL. Nevertheless, the biological mechanisms by which monoallelic or biallelic BIRC3 lesions could contribute to del(11q) CLL pathogenesis, progression and therapy response are partially unexplored. We used the CRISPR/Cas9 system to model monoallelic and biallelic BIRC3 loss in vitro. First, we generated an isogenic HG3 CLL cell line harboring monoallelic del(11q) - HG3-del(11q) - by the introduction of 2 guide RNAs targeting 11q22.1 and 11q23.3 (~17 Mb). Loss-of-function BIRC3 mutations (MUT) were introduced in the remaining allele, generating 3 HG3-del(11q) BIRC3MUT clones. In addition, single BIRC3MUT were introduced in HG3 and MEC1 CLL-derived cells for experimental validation (n = 3 clones/cell line). We first questioned whether monoallelic and biallelic BIRC3 loss had an impact in the DNA-binding activity of NF-κB transcription factors. Interestingly, HG3-del(11q) had higher p52 and RelB (non-canonical NF-κB signaling) activity than HG3WT cells (P = 0.005; P = 0.007), being this activity further increased in HG3-del(11q) BIRC3MUT cells (P < 0.001; P < 0.001). In depth analysis of the non-canonical signaling components by immunoblot revealed that HG3-del(11q) and, to a greater extent, HG3-del(11q) BIRC3MUT cells presented NF-κB-inducing kinase (NIK) cytoplasmic stabilization, high p-IKKα levels and p52-RelB nuclear translocation. Besides, HG3-del(11q) BIRC3MUT cells showed increased levels of the anti-apoptotic proteins BCL2 and BCL-xL. We next assessed this pathway ex vivo in stroma and CpG-stimulated primary CLL cells with or without BIRC3 deletion (n = 22; 11 each group). Remarkably, stimulated BIRC3-deleted primary cells showed higher p52 and RelB activity than BIRC3WT cases (P = 0.01; P = 0.07), and the percentage of BIRC3-deleted cells correlated with p52 activity in del(11q) cases (P = 0.04). We further performed western blot analyses in a homogenous cohort of del(11q) cases including (n = 4) or not including (n = 3) BIRC3 within the deleted region. Interestingly, del(11q)/BIRC3 deleted cases presented high levels of stabilized NIK, which correlated with higher p52 processing (P = 0.003). These patients also showed higher BCL2 levels than those del(11q)/BIRC3 undeleted, and we could further observe a correlation between p52 and BCL2 levels (P = 0.01). Given this p52-dependent BCL2 upregulation, we treated the CRISPR/Cas9 edited clones with venetoclax, demonstrating that HG3-del(11q) BIRC3MUT cells were more sensitive upon BCL2 inhibition than HG3WT clones (mean IC50 3.5 vs. 5.75 μM; P = 0.005). In vitro proliferation assays were performed to interrogate the impact of BIRC3 loss in CLL cell growth, revealing that HG3 BIRC3MUT cell lines had higher growth rates than BIRC3WT cells (P = 0.001). HG3-del(11q) BIRC3MUT cells also showed enhanced proliferation in comparison to HG3-del(11q) clones (P = 0.009). We further determined the clonal dynamics of del(11q) and/or BIRC3MUT cell lines in clonal competition experiments, showing that HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells progressively outgrew HG3WT and HG3-del(11q) cells, respectively, overtime (P = 0.02; P = 0.006). Furthermore, we injected these edited cell lines into NSG mice (n = 20) in vivo, showing that mice xenografted with HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells presented, by flow cytometry, an increase of human CD45+ cells in spleen 14 days after injection, compared to HG3WT and HG3-del(11q) cells (P = 0.02; P = 0.015). In summary, this work demonstrates that biallelic BIRC3 deletion through del(11q) and mutation triggers non-canonical NF-κB signaling, driving BCL2 overexpression and conferring clonal advantage, which could account for the negative predictive impact of BIRC3 biallelic inactivation in CLL. Taken together, our results suggest that del(11q) CLL patients harboring BIRC3 mutations should be considered as a CLL subgroup at a high risk of progression that might benefit from venetoclax-based therapies. Funding: PI18/01500 Disclosures No relevant conflicts of interest to declare.

Download Full-text

Dic(17;18)(p11.2;p11.2) Is a Recurring Abnormality in Chronic Lymphocytic Leukemia Associated with Aggressive Disease.

Blood ◽

10.1182/blood.v114.22.1233.1233 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1233-1233

Author(s):

Jennifer A Woyach ◽

John C. Byrd ◽

John Zhao ◽

Andrew McFaddin ◽

Amy S Ruppert ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Disease Progression ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Age At Diagnosis ◽

Occurrence Rate ◽

Cell Transplant ◽

Complex Karyotype ◽

Early Therapy

Abstract Abstract 1233 Poster Board I-255 Introduction Cytogenetic analysis has become a routine part of the evaluation of patients with chronic lymphocytic leukemia (CLL) because specific chromosomal aberrations have been shown to assist in predicting disease progression, treatment efficacy, and overall survival. One such abnormality is del(17p13.1), which has been found to be associated with a need for early therapy, poor response to conventional treatment, and shortened survival. Identification of recurring abnormalities in this region represents great interest to identify genes relevant to CLL progression and poor prognosis. Patients and Methods We performed an extensive review of 1213 patients undergoing metaphase cytogenetics study at our institution and identified 16 (1.3%) with a recurrent unbalanced translocation between the p arms of chromosomes 17 and 18 that results in a dicentric chromosome with loss of much of 17p and 18p. The clinical features of these patients were characterized. Results A total of 16 patients were identified to have dic(17;18)(p11.2;p11.2) representing a 1.3% occurrence rate. The median age at diagnosis of these patients was 57 years (range 37-68). The dic(17;18)(p11.2;p11.2) was associated with a complex (3 or more unrelated cytogenetic abnormalities) karyotype in 12 patients (75%) at the time that the abnormality was first identified, and eventually associated with a complex karyotype in 94% of patients. This abnormality was associated with trisomy 12 in 7 patients (44%) and with del(13q) in 5 patients (31%) with no overlap between these two abnormalities. IgVH mutational analysis was un-mutated in 88% of cases. Except for one patient who was diagnosed with CLL incidentally during a workup for metastatic tonsillar cancer, all patients identified with dic(17;18)(p11.2;p11.2) met criteria for disease treatment, with a median time from diagnosis to first treatment of 15 months. Of the 12 patients who received fludarabine-based therapy, 7 (58%) were refractory. Three patients have received stem cell transplant for recurrent/refractory disease, and 4 are currently undergoing chemotherapy. With a mean follow-up of 54 months, 4 patients have died, 21, 42, 49, and 92 months after diagnosis. Conclusions Our study combined with small series reported by others demonstrate that dic(17;18)(p11.2;p11.2) is a novel recurrent cytogenetic abnormality in CLL. Here we demonstrate the clinical significance of this recurring abnormality that is associated with young age at diagnosis, accelerated disease progression, decreased response to fludarabine, and shortened overall survival. The dic(17;18)(p11.2;p11.2) is frequently accompanied by other negative prognostic markers including a complex karyotype and unmutated IgVH status. Additional studies to further characterize the prevalence of this abnormality and genes that are disrupted by the translocation are warranted. Disclosures No relevant conflicts of interest to declare.

Download Full-text

The Immunophenotype Signature CD49d+CD38+ Identifies Chronic Lymphocytic Leukemia Cases with a Higher Potential for Migration Beneath Marrow Stromal Cells.

Blood ◽

10.1182/blood.v114.22.356.356 ◽

2009 ◽

Vol 114 (22) ◽

pp. 356-356 ◽

Cited By ~ 2

Author(s):

Antonina Kurtova ◽

Mariela Sivina ◽

Maite P. Quiroga ◽

William G. Wierda ◽

Michael J. Keating ◽

...

Keyword(s):

Drug Resistance ◽

Chronic Lymphocytic Leukemia ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Residual Disease ◽

Lymphocytic Leukemia ◽

Marrow Stromal Cells ◽

Lymphoid Tissues ◽

Mutational Status

Abstract Abstract 356 Adhesion of chronic lymphocytic leukemia (CLL) cells to stromal cells in the marrow and secondary lymphoid tissues confers drug resistance and may account for survival and maintenance of residual CLL cells after conventional treatments, paving the way to relapses. Therefore, targeting the cross talk between CLL cells and stromal cells represents an attractive and necessary approach to overcome minimal residual disease in CLL. We previously reported that fractions of CLL cells and other neoplastic B cell spontaneously migrate beneath marrow stromal cells (MSC) in a CXCR4- and CD49d-(VLA-4) dependent fashion, an in vitro phenomenon termed pseudoemperipolesis (PEP). Also, we reported that cells that migrated beneath and underneath MSC were largely protected from cytotoxic drugs, in contrast to cells that remained in the supernatant (Blood 113:4604-13, 2009). The aim of this study was to identify correlations between CLL surface markers and their ability to migrate beneath MSC. We tested samples from 116 different CLL patients in co-culture assays for their ability to migrate beneath MSC (KUSA H1 cells). After 6 hours of incubation, CLL cells were vigorously washed off the MSC layers, and PEP+ cases were identified by phase contrast microscopy as cases, in which CLL cells were visualized within the stromal layers, characterized by the dark appearance of CLL cells that had migrated into the same focal plane as the MSC. We characterized 45 PEP+ and 71 PEP- CLL cases. Next, we profiled the expression of adhesion molecules and chemokine receptors, including CD44, CD49d, CD62L, CXCR3, CXCR4 and CXCR5 by flow cytometry in the PEP+ and PEP- subgroups. Based on the mean fluorescence intensity ratios (MFIR), we found that both groups displayed comparable levels of CD44 (58.8±4.6 in PEP+ vs. 58.7±3.5 in PEP-), CD62L (7.8±1.3 vs. 9.3±1.3), CXCR3 (7.9±1.0 vs. 8.0±0.8), CXCR4 (87.6±9.3 vs. 81.4±4.6), and CXCR5 (47.4±2.9 vs. 49.3±2.7). In contrast, the levels of CD49d were significantly different between these two groups; the MFIR for CD49d was 14.0±2.4 in PEP+ cases and 4.0±0.7 in PEP- cases (p<0.0001, see Fig. 1). Interestingly, in PEP+ cases, higher levels of CD49d correlated with lower levels of CXCR4 (r=-0.43; p<0.003), which could be due to higher affinity to a CXCL12-rich microenvironment, with respective CXCR4 down regulation. Analysis of prognostic factors (CD38, ZAP-70 and mutational status) revealed that PEP+ cases displayed higher level of CD38 expression: 29.8±5.8% vs. 15.6±2.7% (MFIR, p<0.05), whereas there was no difference in ZAP-70 expression or mutational status between PEP+ or PEP- cases. When comparing cytogenetic profiles, favorable risk (del13q14.3 and normal karyotype) cases represented 43.6% in the PEP+ vs. 71.2% in PEP- groups; trisomy12 cases 23.1% vs. 4.5%, and poor risk cytogenetics (del11q, del17p, complex) represented 33.3% in the PEP+ vs. 24.3% in PEP- group. Collectively, the distinct immunophenotype of CD49d+CD38+ identifies CLL cases with a higher affinity for migration beneath MSC. Consequently, CLL cells in such cases are expected to be more difficult to eradicate by conventional treatments because of higher levels of stroma-mediated drug resistance. Drugs, such as Natalizumab or Plerixafor, that target CLL-stroma interactions, could be of particular benefit for such patients by disrupting CLL-stroma crosstalk and mobilization of CLL cells from protective marrow niches. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Preclinical Development Of ROR1 Peptide Based Vaccine With Activity Against Chronic Lymphocytic Leukemia In ROR1 Transgenic Mice

Blood ◽

10.1182/blood.v122.21.4174.4174 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4174-4174 ◽

Cited By ~ 1

Author(s):

Jian Yu ◽

Bing Cui ◽

George F. Widhopf ◽

Liguang Chen ◽

Laura Rassenti ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

High Titer ◽

Lymphocytic Leukemia ◽

Orphan Receptor ◽

Lymphoid Tissues ◽

Mouse Tissues ◽

Freund’S Adjuvant ◽

Freund's Adjuvant

Abstract Receptor tyrosine kinase-like orphan receptor (ROR1) is expressed on chronic lymphocytic leukemia (CLL) and other cancers, but not on normal post-partum tissues, except for a small subset of precursor B cells known as hematogones. Because of its restricted expression on cancer cells, ROR1 is an attractive target for developing novel anti-cancer therapies. In this study, we designed several different peptides, corresponding to distinct epitopes in the extracellular domain of ROR1. Each peptides was conjugated with keyhole-limpet hemocyanin (KLH) to generate a peptide-KLH vaccine, which we used to immunize C57BL/6 mice, first in complete Freund’s adjuvant (CFA) and then again 2 weeks later in incomplete Freund’s adjuvant (IFA). Two weeks following the second injection the sera from immunized mice were tested for binding activity for recombinant ROR1 protein via immunoblot analyses or ELISA or for cell-surface ROR1 by flow cytometry. We identified one peptide-KLH vaccine (R22-KLH) that induced high-titer anti-ROR1 antisera specific for ROR1-expressing cells that did not react with normal human or mouse tissues, which did not express this protein. The antisera generated by mice immunized with R22-KLH were capable of directing specific complement-dependent cytotoxicity (CDC) against neoplastic cells that expressed ROR1 (e.g. human CLL cells, EW36, or JeKo-1), but not against cells that did not express ROR1 (e.g. Jurkat). To study the capacity of R22-KLH to break self-tolerance, we immunized C57BL/6 mice transgenic for human ROR1 controlled by the immunoglobulin μ heavy-chain promoter/enhancer, designated as ROR1-Tg mice. Despite having B cells that express surface ROR1, such animals still were able to generate high-titer anti-ROR1 antisera comparable in binding and CDC activity and specificity as antisera generated in C57BL/6 mice. Production of such antisera was associated with reduced expression of ROR1 by the B cells of such ROR1-Tg mice. Cohorts of C57BL/6 mice or ROR1-Tg mice were immunized with R22-KLH or KLH and then challenged with intravenous injections of 3 × 105 or 2 × 104 ROR1-expressing primary CLL cells of double-transgenic ROR1xTCL1 C57BL/6 mice. Immunization with R22-KLH, but not KLH, significantly inhibited leukemia engraftment, as demonstrated by evaluation of the spleen and lymphoid tissues 4 weeks after adoptive transfer. To generate vaccines that might be suitable for clinical studies, we examined the relative immunogenicity of R22-KLH mixed with newly-developed agonists of toll-like receptor 4 (TLR-4; 1Z105) and TLR-7 (1V270). For this, we compared the immune responses of C57BL/6 mice or ROR1-Tg mice to R22-KLH administered s.c. in CFA/IFA to that of R22-KLH administered s.c. with 1Z105/1V270 in saline. Either C57BL/6 or ROR1-Tg mice generated higher titers of anti-ROR1 antibodies following immunization with R22-KLH in 1Z105/1V270 than did animals immunized with R22-KLH in CFA/IFA. Moreover, injection of R22-KLH in 1Z105/1V270 appeared better tolerated than injection of R22-KLH in CFA/IFA. These data indicate that immunization with R22-KLH can break tolerance to ROR1 and induce high-titer anti-ROR1 antisera. The immunogenicity of R22-KLH can be enhanced by injection with agonists of TLR4/7, obviating use of CFA/IFA. Pharmacology/toxicology studies are ongoing to determine the suitability of R22-KLH in 1Z105/1V270 for clinical trials involving patients with ROR1-expressing cancers. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Chronic lymphocytic leukemia disease progression is accelerated by APRIL-TACI interaction in the TCL1 transgenic mouse model

Blood ◽

10.1182/blood-2013-04-497693 ◽

2013 ◽

Vol 122 (24) ◽

pp. 3960-3963 ◽

Cited By ~ 15

Author(s):

Valeria Lascano ◽

Marco Guadagnoli ◽

Jan G. Schot ◽

Dieuwertje M. Luijks ◽

Jeroen E. J. Guikema ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Mouse Model ◽

Disease Progression ◽

Transgenic Mouse ◽

Transgenic Mouse Model ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Key Points

Key Points APRIL significantly accelerates CLL onset in TCL1-Tg mice. APRIL-mediated prosurvival effects in leukemic cells depend on TACI and not BCMA ligation.

Download Full-text