In Vivo Labeling of Dividing Chronic Lymphocytic Leukemia B Cells Suggests That CXCR4 and CD5 Define the Clonal Subfraction That Recently Proliferatead and Emigrated from a Solid Tissue

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 358-358 ◽  
Author(s):  
Carlo calissano ◽  
Rajendra Damle ◽  
Gregory Hayes ◽  
Elisabeth J Murphy ◽  
Marc Hellerstein ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Cell Contact ◽  
Proliferating Cells ◽  
Solid Tissue ◽  
Group A ◽  
Group B ◽  
Time Point

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is due, in most of the cases, to the slow progressive invasion of phenotypically homogeneous monoclonal B cells. Several studies are clarifying that B-CLL cells preferentially proliferate in the context of lymphoid solid tissues where they receive accessory signals (chemokines, cytokines, cell to cell contact). Deuterium (2H) incorporation into newly synthesized DNA can be used as a direct in vivo measure of cell division. Using this technique in B-CLL, it has been found that although the fraction of cells dividing each day is small (0.1 – ~2% of the clones), the absolute number of proliferating cells is sizeable (~109 – 1012/day). The study also elucidated a relationship between proliferation and disease activity. By using this same approach, we have tried to refine our understanding of this small percentage of proliferating cells. Thirteen patients drank deuterated water (2H2O) for 6–12 weeks (“labeling period”) and were followed for 24 weeks (“washout period”). Blood was drawn during and at the end of labeling (1st, 2nd time points) and at the end of washout (3rd time point). Based upon proliferation kinetics patients could be divided into 2 groups (Group A and Group B). Four/13 patients (Group A) showed unusual kinetic curves with a strikingly delayed appearance of labeled cells in the blood as compared to Group B patients. This suggested that B-CLL cells of these patients were retained longer at the site of proliferation. Therefore, the two groups were compared for expression of a panel of chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR7, CXCR1, CXCR3, CXCR4, and CXCR5) by flow cytometry. Only CXCR4 was found at significant higher densities in Group A compared to Group B (P = 0.002). CXCR4 expression was then used to identify B-CLL sub-populations enriched in cells displaying Ki67 and MCM6, two cell cycle related markers. We observed a relationship between CXCR4 and CD5 where CXCR4dim cells tended to be CD5bright and vice-versa with the majority of the cells falling in an intermediate category (CXCR4intCD5int). The CXCR4dimCD5bright subpopulation contained significantly more Ki67+ and MCM6+ cells. Based on this observation, B-CLL cells of 9 patients were sorted into CXCR4brightCD5dim, CXCR4intCD5int, CXCR4dimCD5bright and 2H was measured for each fraction. A hierarchy of 2H enrichment in the fractions was found: CXCR4dimCD5bright > CXCR4intCD5int >CXCR4brightCD5dim. These differences were statistically significant during labelling period (1st and 2nd time point) but completely disappeared at the end of washout (3rd time point). The 2H enrichment measured in the unfractionated clones at the 1st, 2nd and 3rd time point was similar (0.45±0.15 vs. 0.53±0.16 vs. 0.45±0.07, P = ns), suggesting a redistribution of label among the fractions, presumably due to a change of phenotype over time. In vitro studies have suggested that CXCR4 has a crucial role in trafficking and survival of B-CLL cells, and it is emerging as a powerful predictor for bone marrow infiltration and clinical evolution. CXCR4 surface expression is downregulated after encountering its ligand SDF-1. Our data indicate a relationship between CXCR4 levels and B-CLL cells kinetics. Moreover, since CLL cell subpopulations defined by relative CXCR4 and CD5 levels differ in the number of proliferating cells, we suggest that these markers ca be used to define cells that have emigrated recently (CXCR4dimCD5bright) or much earlier (CXCR4brightCD5dim) from the solid tissue in which they divided. The results strongly suggest, in vivo, the important role of the stroma and SDF-1-producing cells for B-CLL cell proliferation providing indications for disease control strategies.

In Vivo Labeling of Newly Synthesized DNA Suggests That the CD38+ Fraction Is Enriched in Proliferating Cells within a Clone of Chronic Lymphocytic Leukemia B Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 27-27 ◽  
Author(s):  
Carlo Calissano ◽  
Rajendra Damle ◽  
Taraneh Banapour ◽  
Denise Cesar ◽  
Marc Hellerstein ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Gas Chromatography Mass Spectrometry ◽  
Deuterated Water ◽  
Proliferating Cells ◽  
Cd38 Expression ◽  
Accessory Cells ◽  
The Difference

Abstract B-cell chronic lymphocytic leukemia (B-CLL) has been considered for years as a slowly progressive invasion of phenotypically homogeneous, monoclonal B cells that divide rarely and accumulate because of a primary apoptotic defect. Recent studies are clarifying that the leukemic clone can be comprised of subpopulations of cells expressing different surface markers suggestive of intraclonal diversity. Indeed, it is now widely accepted that activation signals (i.e., antigenic stimulation, interaction with accessory cells, cytokines) can push B-CLL cells into the cell cycle or rescue them from apoptosis. Unanswered questions are whether all or only a subset of cells in the clone participates in these proliferative events and to what extent those cells that have divided can be identified. We have tried to answer this by labeling those cells that divide in vivo with the non-radioactive stable isotope deuterium (2H) in the form of deuterated water. Nine B-CLL patients with leukemic clones comprising 1.7 – 97.8% CD38+ cells drank daily, for 12 weeks, an aliquot of 2H2O. CD19+CD5+CD38+ and CD19+CD5+CD38− cells were isolated from PBMC drawn after 8 weeks (labeling period) and 24 weeks (washout period) and the incorporation of 2H into equal amounts of genomic DNA was measured by gas chromatography/mass spectrometry. At week 8, the mean 2H enrichment in the CD38+ population was higher than in the CD38− fraction (0.862 ± 0.24 vs. 0.445 ± 0.13; P = 0.034). Enhanced 2H enrichment in the CD38+ population was detected in every patient with CD38+/CD38− ratios ranging from 1.15 to 5.58. In 4 patients, the CD38+/CD38− ratio was above 2.0. In contrast, at week 24, 12 weeks after the cessation of 2H2O intake, 2H enrichment in DNA in the CD38+ vs. the CD38− population had changed. In 7 of 9 patients, the CD38+/CD38− enhancement ratios diminished, remaining essentially constant for the other 2 patients. The difference of CD38+/CD38− mean ratios at week 8 and week 24 was significant (2.254 ± 0.12 vs. 1.124 ± 0.13; P = 0.035). Moreover, the incorporation of 2H into CD38+ cells fell in 6 of 9 patients, with reciprocal increases in label incorporation in the CD38− cells for 8 of 9 patients. Thus, the differences in 2H incorporation into DNA at weeks 8 and 24 suggest either that the life spans of the two subpopulations differ (i.e., CD38+ cells may be shorter lived) or that expression of CD38 may be a dynamic process in vivo changing from positive to negative in individual cells over time after replication. These in vivo findings indicate that, within the same B-CLL patient, the leukemic subpopulation that expresses CD38 contains more cells that have recently undergone cell division than the CD38− subpopulation. This may relate to the clinical correlation of CD38 expression with poor clinical outcome. The 2H2O labeling technique provides a safe in vivo method to further fractionate the proliferative leukemic compartment as a means to better understand the pathophysiology of B-CLL and possibly provide an important target for therapy.

scholarly journals Chronic Lymphocytic Leukemia B Cells Can Undergo Somatic Hypermutation and Intraclonal Immunoglobulin VHDJH Gene Diversification

2002 ◽  
Vol 196 (5) ◽  
pp. 629-639 ◽  
Author(s):  
Carmela Gurrieri ◽  
Peter McGuire ◽  
Hong Zan ◽  
Xiao-Jie Yan ◽  
Andrea Cerutti ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Lymphocyte ◽  
Somatic Hypermutation ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Lymphocytic Leukemia ◽  
Gene Diversification

Chronic lymphocytic leukemia (CLL) arises from the clonal expansion of a CD5+ B lymphocyte that is thought not to undergo intraclonal diversification. Using VHDJH cDNA single strand conformation polymorphism analyses, we detected intraclonal mobility variants in 11 of 18 CLL cases. cDNA sequence analyses indicated that these variants represented unique point-mutations (1–35/patient). In nine cases, these mutations were unique to individual submembers of the CLL clone, although in two cases they occurred in a large percentage of the clonal submembers and genealogical trees could be identified. The diversification process responsible for these changes led to single nucleotide changes that favored transitions over transversions, but did not target A nucleotides and did not have the replacement/silent nucleotide change characteristics of antigen-selected B cells. Intraclonal diversification did not correlate with the original mutational load of an individual CLL case in that diversification was as frequent in CLL cells with little or no somatic mutations as in those with considerable mutations. Finally, CLL B cells that did not exhibit intraclonal diversification in vivo could be induced to mutate their VHDJH genes in vitro after stimulation. These data indicate that a somatic mutation mechanism remains functional in CLL cells and could play a role in the evolution of the clone.

CCR4:CCL17 Interaction Influences TLR-9 Mediated Cell Survival and Proliferation In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3593-3593
Author(s):  
Sonal C. Temburni ◽  
Ryon M. Andersen ◽  
Luke Janson ◽  
Xiao-Jie Yan ◽  
Barbara Sherry ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Dose Range ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
Novel Therapy

Abstract Abstract 3593 Unlike other hematologic disorders, chronic lymphocytic leukemia(CLL) exhibits remarkable heterogeneity in the rates of disease progression among cases. CLL cells survive by receiving signals from the microenvironment via various receptors: B-cell antigen receptor (BCR), Toll-like receptors (TLRs) and cytokine and chemokine receptors. We previously reported that CLL clones with somatically mutated IGHVs and high (≥30%) percentage of CD38 expressing cells have the highest percentage of CCR4-expressing cells. To further explore the functional contribution of the CCR4:CCL17 axis in CLL, we studied CCL17-induced chemotactic behavior in 16 CLL cases. In transwell cultures we observed a bimodal migratory response to CCL17 at 2 doses in a dose range of 0.78– 25ng/ml, in ~60% of cases; the remaining cases showed maximal migration at a single dose (1.56 or 3.12ng/ml). A comparison of phenotypes of the migrated and non-migrated cell populations was undertaken in 10 cases, analyzing CXCR3, CXCR4, CCR4 and CCR7 that are involved in homing of cells to sites favoring growth, and CD31, CD38 and CD69, activation related molecules. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 in the CCR4-mediated downstream pathway. CCR4 ligand, CCL17, is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes and fibroblasts, whereas CCL22 is produced by tumor cells and the tumor microenvironment. Serum levels of both these ligands in untreated patients were quantified by ELISA. CCL17 levels ranged between 45-1, 229 pg/ml in U-CLL cases (n=23) and between 43-1, 418 pg/ml in M-CLL cases (n=30). CCL22 levels ranged between 121-5, 497 pg/ml in U-CLL cases (n=23) and 409-5, 502 pg/ml in M-CLL cases (n=30). The percentages of CCR4- expressing B cells directly correlated with percentages of T cells expressing CCR4 in individual cases, whereas they inversely correlated with both, serum levels of CCL17 (p< 0.01) and CCL22 (p< 0.05). CCL17 produced by DCs in peripheral organs may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells. We therefore tested if CCL17 supported BCR- and TLR-mediated proliferative responses in a cohort of 31 (16 U-CLL and 15M-CLL) CLL cases. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, but only in 3/15 (20%) M-CLL cases. On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 (87%) M-CLL cases at a dose of 2ng/nl (approximating that detected in serum); it also augmented TLR-9 mediated B cell proliferation in 6/16 U-CLL cases but at a 5-fold or higher dose (10-25 ng/ml). In a subset of this cohort (8 cases) CCL17-induced modulation of molecules involved in the apoptotic process was studied. We found upregulation of anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulation of pro-apoptotic molecules Bim, PUMA, and Bid in 5 of these cases. The pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating TLR-9-mediated signaling and migration in CLL. Therefore, inhibition of CCR4:CCL17 interaction in vivo represents a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival. Disclosures: No relevant conflicts of interest to declare.

Targeting of Chronic Lymphocytic Leukemia B Cells with a Novel Monoclonal Antibody to ROR1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 984-984
Author(s):  
Bing CUi ◽  
George F. Widhopf ◽  
Jian Yu ◽  
Daniel Martinez ◽  
Esther Avery ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Adoptive Transfer ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Phosphorylated Akt

Abstract Abstract 984 ROR1 is an orphan receptor tyrosine kinase that is expressed on leukemia cells of patients with chronic lymphocytic leukemia (CLL), but not on most adult tissues of healthy adults, including CD5+ B cells. To generate anti-ROR1 antibodies, we immunized mice using different strategies employing vaccines comprised of recombinant ROR1 protein, polynucleotide-ROR1 vaccines and CD154 genetic adjuvants, or replication-defective adenovirus vectors encoding ROR1 and CD154. We extirpated the spleens of animals that developed high-titer serum anti-ROR1 antibodies and used these to generate monoclonal-antibody-(mAb)-producing hybridomas or antibody phage-display libraries that subsequently were screened for ROR1-binding. Over 70 unique mAbs were generated that each bound the extra-cellular domain of native ROR1. Most mAbs recognized an epitope(s) within the ROR1 Ig-like domain, which appears to represent the immune dominant epitope. Other mAb recognized epitopes within the conserved ROR1 Kringle domain. One mAb (UC D10-001) had distinctive binding to an intradomain epitope of human ROR1 (hROR1). UC D10-001 was the only mAb we found directly cytotoxic for hROR1-expressing leukemia cells cultured in media without complement for 6 hours. We found that UC D10-001 could induce significant reductions in basal levels of phosphorylated AKT in hROR1-expressing leukemia cells. Moreover, UC D10-001 significantly decreased the basal levels of phosphorylated AKT in freshly isolated human CLL cells (N=4) to levels comparable to that observed in co-cultures containing 10 mM LY294002, a broad-spectrum inhibitor of PI3K. We examined whether this mAb had cytotoxic activity for leukemia cell in vivo. For this we examined whether we could inhibit the adoptive transfer of human-ROR1-expressing leukemia cells to young, syngeneic recipient mice made transgenic for human ROR1 under control of a B-cell specific promoter. Cohorts of 5 animals per group were each given intravenous injections of antibody at a dose of at 10 mg/kg. Each cohort was treated with UC D10-001, control IgG, or 4A5, an anti-ROR1 mAb specific for a non-cross-reactive epitope located in the Ig-like domain of ROR1. Each animal received an intravenous injection of 5 × 105 ROR1-expressing leukemia cells and then was assessed weekly for circulating leukemia cells by flow cytometry. UC D10-001, but not control IgG or 4A5, significantly inhibited engraftment of the ROR1+ leukemia. Four weeks after adoptive transfer, animals treated with UC D10-001 had a 10-fold lower median number of leukemia B cells in the blood than animals treated with control IgG or 4A5. We also tested UC D10-001 for its capacity to induce clearance of human ROR1+ CLL cells engrafted into the peritoneal cavity of Rag-2−/−/γc−/− immune deficient mice. Each of these mice received intraperitoneal injections of equal numbers of human ROR1+ CLL cells prior to receiving D10-001, control IgG, or 4A5, each at 10 mg/kg. These animals were sacrificed seven days later and the human leukemia cells were harvested via peritoneal lavage. In mice treated with UC D10-001 we harvested an average of only 6 × 104 ± 3 × 104 CLL cells. This number of cells was significantly less than the average number of CLL cells harvested from control IgG or 4A5-treated mice (8 × 105 ± 4 × 105 or 7 × 105 ± 2 × 105, respectively, p <0.01). These studies indicate that the anti-ROR1 mAb UC D10-001 can be directly cytotoxic for ROR1-expressing leukemia cells in vitro and in vivo, a property that apparently is unique to this mAb among other anti-ROR1 mAbs. Because of the restricted expression of ROR1 on leukemia cells and the distinctive properties of this mAb, we propose that UC D10-001 might have potential utility in the treatment of patients with CLL. Disclosures: No relevant conflicts of interest to declare.

Selective Clearance of Chronic Lymphocytic Leukemia Cells in Vivo Following Treatment with UC99961, an Anti-ROR1 Monoclonal Antibody

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3886-3886
Author(s):  
Eva Hellqvist ◽  
Christina C.N. Wu ◽  
George F. Widhopf ◽  
Alice Shih ◽  
Rommel Tawatao ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cord Blood ◽  
Peritoneal Lavage ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Human Cord Blood

Abstract Abstract 3886 ROR1 is a receptor-tyrosine kinase like protein expressed on the surface of chronic lymphocytic leukemia (CLL) B cells, but not on normal mature B cells, suggesting that it may be a promising therapeutic target. We have generated a chimeric monoclonal antibody (mAb), UC99961, which binds to an intradomain epitope of human ROR1 (hROR1). UC99961 binds the same epitope as the murine anti-hROR1 mAb, UC D10–001, which has direct cytotoxic effects on hROR1 positive CLL cells. In this study we investigated the in-vivo anti-leukemic activity and tolerability of UC99961 on ROR1+ primary patient CLL cells and human cord-blood-derived B cells and T cells, respectively. For these studies, immunodeficient RAG2−/−γc−/− neonatal mice were reconstituted with a human immune system by intrahepatic xenotransplantation of 1×105 CD34+ human cord blood progenitor cells. Eight to ten weeks post transplantation, cord blood engraftment was verified by peripheral blood screening, at which point the mice received an intraperitoneal transplantation of 2×107 primary patient ROR1+ CLL cells. Twenty-four hours after CLL transplantation, five animals per group were each treated with a single intraperitoneal injection (10mg/kg) of UC99961, UC D10–001, or control IgG. Seven days following mAb treatment, the animals were sacrificed and marrow, spleen, thymus, and peritoneal lavage samples were collected and analyzed by flow cytometry for CLL cells, as well as normal cord-blood-derived B cells and T cells. To confirm mAb administration according to the study design, serial residual ROR1 plasma antibody levels were determined by ELISA. Results from three consecutive experiments using leukemia cells from two different patients showed that the vast majority of CLL B cells remained in the peritoneal cavity of the animals and did not migrate to other hematopoietic organs. Both anti-hROR1 mAbs UC99961 and UC D10–001 significantly reduced the average number of harvested CLL cells in the peritoneal lavage compared to control IgG (99% and 71% reduction respectively), while cord-blood-derived T cells (CD45+3+) in thymus remained unaffected by the mAb treatment. For the majority of cord-blood-derived B cells in marrow and spleen, no significant reduction could be observed after UC99961 or UC D10–001 mAb treatment. A small CD19+ROR1+CD34− cord-blood-derived B cell population was identified in marrow and spleen that was reduced after UC99961 and UC D10–001 mAb treatment. This study demonstrates that the anti-human ROR1 specific mAbs have in vivo anti-leukemic activity with minimal impact on human cord-blood-derived B cells and T cells. From these results, UC99961 appears to be an excellent candidate antibody for future clinical studies for patients with CLL. Disclosures: No relevant conflicts of interest to declare.

In Patient with Chronic Lymphocytic Leukemia the Overall SURVIVAL Is NOT Different WHEN Analysed by Cause of Death, Related or NOT Related to the Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4587-4587
Author(s):  
Luca Laurenti ◽  
Francesco Autore ◽  
Barbara Vannata ◽  
Idanna Innocenti ◽  
Francesco Santini ◽  
...  
Keyword(s):  
Overall Survival ◽  
Chronic Lymphocytic Leukemia ◽  
Survival Time ◽  
Cause Of Death ◽  
Causes Of Death ◽  
Lymphocytic Leukemia ◽  
Overall Survival Time ◽  
Group A ◽  
Group B ◽  
Age Of Death

Abstract Abstract 4587 Chronic Lymphocytic Leukemia (CLL) is the most common lymphoprolipherative disorder of the elderly population in Western countries. It shows a highly variable clinical course. In fact, some patients may die within few months from the diagnosis because of CLL itself or disease-related complications. Other patients do not require any treatment for many years and have a long-standing disease. Many of them could die because of disease different from CLL. The identification of subgroups of patients with peculiar features predictive of the clinical behaviour of the disease is important. This retrospective analysis has the purpose to study patients affected by CLL, diagnosed and followed at our single centre of Haematology, focusing our attention on their causes of death. We selected 340 patients affected by CLL from our data-base, diagnosed from January 1999 to December 2010 and followed until March 2012. We distinguished the causes of death in two groups: one related to CLL (as progression of the disease, evolution to Richter's Syndrome, infections due to chemotherapy) and the other not related to CLL (i.e. cardiovascular diseases, solid tumours, old age). Statistical analysis, conducted using SPSS version 16.0 for Windows and “GraphPad Prism” GraphPad Software Inc., compared these two groups and tried to select other subgroups. We recorded 69 deaths: 47 related to CLL (68.1%) and 22 unrelated to CLL (31.9%). The median age of death of our cohort of patients was 76 years (range 40–92); those patients with a CLL-related death (related pts) died at a median age of 76 years (range 40–89) and the patients with a CLL-unrelated death (unrelated pts) died at a median age of 76 years (range 61–92). No differences in terms of median age of death were found analysing the data by gender. Also, considering the overall survival time from diagnosis to death, it was 58 months (range 9–155) in the related group and 43 months (range 14–121) in the unrelated group (p=0.185). When divided our population by the disease behaviour, we obtained 3 subgroups of patients: patients who progressed and died for CLL related causes (group A), patients who progressed and died for CLL unrelated causes (group B) and patients who did not progress and died for CLL unrelated causes (group C) (Table 1). The only statistical significant difference was found among the median overall survival times in un-progressive patients who died for non CLL related causes (p=0.043). The median age of death was not affected by the cause of death in our CLL population. Moreover, patients with un-progressive CLL showed an overall survival time shorter than the progressive CLL subgroups. Patients with un-progressive CLL probably had a shorter survival because unrelated CLL diseases could are more difficult aggressive than CLL itself. Table 1. group A group B group C p N° patients 47 13 9 Median age at diagnosis (years) (range) 70 (39–85) 71 (56–83) 77 (67–86) n.s. Median age at death (years) (range) 76 (40–89) 75 (61–84) 79 (68–92) n.s. Overall survival time (months) (range) 58 (9–155) 48 (18–121) 22 (14–78) 0.043 Disclosures: No relevant conflicts of interest to declare.

Immunoliposomal Delivery of Mir-29b By Targeting Tumor Antigen ROR1 Induces Epigenetic Reprograming in Human-ROR1-Expressed Mouse Model of Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1743-1743
Author(s):  
Chi-Ling Chiang ◽  
Frank W Frissora ◽  
Zhiliang Xie ◽  
Xiaomeng Huang ◽  
Rajeswaran Mani ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Mouse Model ◽  
Cell Line ◽  
Tumor Antigen ◽  
Targeted Delivery ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL), characterized by accumulation of CD5+CD19+sIgM+ B lymphocytes in peripheral blood and lymphoid organs, is classified into indolent and aggressive forms. Patients with indolent CLL generally survive 5 to 10 years and do not require treatment until severe symptoms, while those with aggressive CLL show resistant to standard treatment and survive less than 24 months. While emerging B cell antigen receptor directed therapies are promising, resistance to such therapies pose problems warranting novel therapeutic approaches. MicroRNA (miR) profiling revealed lower expression of miR-29b in aggressive CLL associated with survival, drug resistance and poor prognosis via its up-regulation of anti-apoptotic proteins myeloid leukemia cell differentiation protein 1 (Mcl1) and oncogenic T-cell leukemia 1 (Tcl1). Thus, specific overexpression of miR-29b in B-CLL cells could be a potential therapy for aggressive CLL patients. Despite the promise, short circulation half-life, limited cellular uptake and off-target effects on non-desirable tissues pose a challenge for miR-based therapies. To promote efficiency and specificity of miR-29b delivery, we developed neutral immunonanoparticles with selectivity to CLL via targeting tumor antigen ROR1, which is expressed in over 95% of CLL but not normal B cells. We optimized a novel 2A2-immunoliposome (2A2-ILP) recognizing surface ROR1 on primary CLL cell to promote internalization and miR-29b uptake (n=6, p=0.042*). About 20-fold increased uptake of miR-29b was achieved with 2A2-ILP-miR-29b formulation compared to control. Further ROR1 targeted delivery of miR29b resulted in significant downregulation of DNMT1 and DNMT3a mRNA and protein (n=3, DNMT1: p= 0.0115*; DNMT3a: p=0.0231*, SP1; p=0.0031**) in primary CLL cells and a human CLL cell line OSU-CLL. Consistent with the downregulation of DNMTs, decreased global DNA methylation was observed in OSU-CLL cell line one week post- treatment with 2A2-ILP-miR-29b (n=3, p=0.0003***). To further study the in vivo ROR1-targeting efficiency of 2A2-ILP-miR-29b, we used our recently described Eµ-hROR1x Tcl1 CLL mouse model that develops CLL like disease with human ROR1 antigen in leukemic CD19+CD5+ B cells. Using hROR1+CD19+CD5+ leukemic cell engraftment model, we showed significant in-vivo efficacy of ROR1-ILP-miR-29b formulation associated with a) decreased number of circulating leukemic B220+CD5+ cells b) reduced splenomegaly (p=0.0461*, 2A2-29b: n=9; PBS: n=8) c) with extended survival (p=0.0075**, 2A2-29b: n=9; IgG-29b: n=7; 2A2-SC: n=7; PBS: n=8). In summary, 2A2-ILP effectively delivered functional miR-29b, resulting in downregulation of DNMT1 and DNMT3a, reduction of hypermethylation and anti-leukemic activity. Ongoing studies are aimed at understanding miR-29b mediated in-vivo methylome reprograming using our novel hROR1xTcl1 transgenic mouse model and ROR1-targeted miR-29b delivery formulation. Figure 1. Figure 1. Disclosures Byrd: Acerta Pharma BV: Research Funding.

scholarly journals A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Piers E. M. Patten ◽  
Gerardo Ferrer ◽  
Shih-Shih Chen ◽  
Jonathan E. Kolitz ◽  
Kanti R. Rai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Intraperitoneal Administration ◽  
Human Condition ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Quiescent State

Patient-derived xenograft models of chronic lymphocytic leukemia (CLL) can be created using highly immunodeficient animals, allowing analysis of primary tumor cells in an in vivo setting. However, unlike many other tumors, CLL B lymphocytes do not reproducibly grow in xenografts without manipulation, proliferating only when there is concomitant expansion of T cells. Here we show that in vitro pre-activation of CLL-derived T lymphocytes allows for a reliable and robust system for primary CLL cell growth within a fully autologous system that uses small numbers of cells and does not require pre-conditioning. In this system, growth of normal T and leukemic B cells follows four distinct temporal phases, each with characteristic blood and tissue findings. Phase 1 constitutes a period during which resting CLL B cells predominate, with cells aggregating at perivascular areas most often in the spleen. In Phase 2, T cells expand and provide T-cell help to promote B-cell division and expansion. Growth of CLL B and T cells persists in Phase 3, although some leukemic B cells undergo differentiation to more mature B-lineage cells (plasmablasts and plasma cells). By Phase 4, CLL B cells are for the most part lost with only T cells remaining. The required B-T cell interactions are not dependent on other human hematopoietic cells nor on murine macrophages or follicular dendritic cells, which appear to be relatively excluded from the perivascular lymphoid aggregates. Notably, the growth kinetics and degree of anatomic localization of CLL B and T cells is significantly influenced by intravenous versus intraperitoneal administration. Importantly, B cells delivered intraperitoneally either remain within the peritoneal cavity in a quiescent state, despite the presence of dividing T cells, or migrate to lymphoid tissues where they actively divide; this dichotomy mimics the human condition in that cells in primary lymphoid tissues and the blood are predominately resting, whereas those in secondary lymphoid tissues proliferate. Finally, the utility of this approach is illustrated by documenting the effects of a bispecific antibody reactive with B and T cells. Collectively, this model represents a powerful tool to evaluate CLL biology and novel therapeutics in vivo.

The effect of alemtuzumab maintenance therapy on B and T lymphocytes in chronic lymphocytic leukemia

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 7092-7092
Author(s):  
M. S. Kaufman ◽  
N. Driscoll ◽  
C. Johnson ◽  
A. Caramanica ◽  
D. Janson ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Maintenance Therapy ◽  
Cd4 T Cells ◽  
Opportunistic Infections ◽  
Lymphocytic Leukemia ◽  
Blood Levels ◽  
Time Point

7092 Background: We are conducting a pilot, exploratory study of the potential value of alemtuzumab(alem) in maintenance therapy of previously treated chronic lymphocytic leukemia (CLL) patients(pts) after they have achieved stable disease or partial remission with chemo or chemo-immunotherapy. We present the results of serially monitored CD19+ (B)lymphocytes and CD4+ (T) lymphocytes on eight evaluable patients. Methods: 30mg doses of alem were administered SC to all patients at the following schedule: wkly for 8 doses (8 wks), followed by q2 wks for 8 doses(16 wks), followed by q3 wks for 8 doses (24 wks). This schedule provides a total of 48 wks of maintenance treatment with alem. Patients received standard prophylaxis with sulfamethoxizole and acyclovir with regular CMV monitoring by quantitative PCR. Results: In the table we present data on the pattern of decrease in blood CD19+(B) cells and CD4+ (T) cells on eight evaluable pts at different time points after starting alem maintenance. Because flow cytometry was not done on all pts at each time point, the number of pts contributing to the calculation of mean counts at each given time point is variable. CD19+(B) cells were markedly reduced to 37% of baseline consistently, from 8 wks onward. CD4+(T) cells, on the other hand, were consistently higher than 50% of the baseline after 8 wks. No opportunistic infections were seen in any pt and treatment was well tolerated. Conclusion: These results from a single institution based pilot study demonstrate that alem used in maintenance schedule is effective in keeping the blood levels of CD19+(B) cells extremely low without concordant suppression of CD4+(T) lymphocytes. No significant financial relationships to disclose. [Table: see text]

Reduced Rates of B Cell Proliferation in Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2821-2821
Author(s):  
Julien Defoiche ◽  
Christophe Debacq ◽  
Becca Asquith ◽  
Yan Zhang ◽  
Arsène Burny ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Healthy Controls ◽  
Kinetic Measurements ◽  
B And T Lymphocytes

Abstract Whether chronic lymphocytic leukemia (CLL) represents latent or proliferating disease has been intensively debated. Whilst the dogma that CLL results from accumulation of dormant lymphocytes is supported by the unresponsiveness of leukemic cells to antigens and polyclonal activators, recent in vivo kinetic measurements show that B-lymphocytes do divide at significant rates in CLL. However, B cell kinetics were not compared between CLL patients and healthy controls so it was not possible to ascertain to what extent lymphocyte kinetics were aberrant in CLL. We compared proliferation rates of B- and T-lymphocytes in CLL patients and healthy controls, using a pulse-chase approach based on incorporation of deuterium from 6,6-2H2-glucose into DNA. We found dramatically reduced in vivo rates of CD3−CD19+ cell proliferation in CLL compared with controls (mean 0.47 versus 1.66 %/day respectively, P=0.001), equivalent to an extended half-life of circulating B-cells (147 days versus 42 days). Labeled (dividing) CD3−CD19+ cells had death rates similar to the healthy controls (2.29 versus 3.55 %/day, P=0.495). Despite such aberrant B-cells kinetics, T-cell proliferation was unaffected by CLL (1.77 versus 1.40 %/day, P=0.488). We conclude that, B-cell proliferation rates are reduced in leukemic patients compared to healthy subjects and that most circulating CD3−CD19+ cells are quiescent, long-lived cells.

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