High Levels Of CXCR4 Expression Research Articles

CXCR4 (CD184) Expression in Pediatric AML Is Associated with Bone Marrow Retention, Specific Disease Characteristics, and Worse Outcomes: A Report of 1004 Patients from the Children's Oncology Group AAML1031 Protocol

Introduction: CXCR4 (CD184) is a G protein-coupled chemokine receptor specific for CXCL12 that is associated with leukocyte chemotaxis. It is expressed on normal hematopoietic cells as well as hematopoietic and epithelial cancer cells. CXCR4 and CXCL12 have been associated with bone marrow retention of AML and ALL blasts and blocking this interaction via small molecule inhibitors has been investigated as a therapeutic target. CXCR4 has also been implicated in other aspects of tumor progression including metastasis, angiogenesis, and survival. As part of Children's Oncology Group protocol AAML1031 for de novo pediatric AML we prospectively evaluated CXCR4 expression by difference from normal (ΔN) flow cytometry and correlated expression with clinical characteristics and outcome. Given the impact of the bone marrow niche on the administration of chemotherapy we hypothesized that high levels of CXCR4 expression would be associated with unfavorable outcomes. Methods: In the AAML1031 phase 3 trial pediatric patients with de novo AML were randomized to receive standard chemotherapy (Arm A) or standard chemotherapy with bortezomib (Arm B), excepting patients with high allelic ratio FLT3-ITD who received standard chemotherapy plus sorafenib (Arm C). Patients were stratified as low-risk (LR) or high-risk (HR) based on cytogenetic and molecular abnormalities, and end of induction (EOI) minimal residual disease (MRD) by flow cytometry. LR patients received four cycles of the standard chemotherapy backbone while HR patients received three followed by best hematopoietic stem cell transplant. Patients who: (1) submitted a bone marrow aspirate for ΔN at diagnosis and (2) provided consent for correlative biology studies were included in this analysis. All diagnostic specimens were centrally and prospectively evaluated for the expression of CXCR4 by ΔN. Results: Surface CXCR4 expression was available for 1004 patients and expression on AML cells varied across the cohort (range: 54-17,793 molecules/cell). For analysis, the study population was divided into quartiles (n=257-258 patients each) based on expression levels. Quartiles showed no association with sex and were distributed as expected between treatment arms. Correlation with clinical characteristics showed that patients in the top quartile of CXCR4 expression were younger (5.9 vs. 11.4 years, P<0.001) and had higher bone marrow (BM) blast counts but lower peripheral blood (PB) blast counts (76% vs. 66%, p<0.001 and 33% vs. 43%, p=0.01, respectively). These patients were highly enriched for HR KMT2A fusions (43% of total fusion positive patients), as well as t(9;11) and NPM1 mutations; while they had a lower frequency of t(8;21), inv(16), and CEBPA mutations (p<0.002 for all). There was no association between CXCR4 expression and protocol risk group assignment or complete remission rates, but patients in the high expressing CXCR4 quartile were more commonly MRD negative at EOI (p=0.03). Flow cytometry also revealed a striking increase in RAM phenotype (p<0.001), with 76% of all RAM patients allocated to the CXCR4 high expressing quartile. Patients in the top quartile of CXCR4 expression had lower 5-year OS (58% vs. 66%, p=0.036) and EFS (38% vs. 47%, p=0.009) compared to other patients. CXCR4 was strongly associated with increased 5-year relapse risk (RR) (54% vs. 39%, p<0.001). Among patients with HR KMT2A fusions high expression of CXCR4 was associated with an increased 5-year RR (85% vs. 57%, p=0.04). Multivariable analysis for 5-year RR including MRD status and prognostic genetic factors indicates CXCR4 expression is independently associated with relapse, with a hazard ratio of 1.33 (95% CI 1.04-1.7; p=0.026). Conclusions: These data demonstrate that CXCR4 expression is associated with individual HR markers (RAM phenotype and HR KMT2A fusions), but not risk status across the entire study. Despite similar risk status, patients in the top quartile of CXCR4 expression had inferior outcomes. These patients had higher BM blast counts but lower PB blast counts, which has been observed in RAM phenotype, likely due to the impact of CXCR4 on bone marrow retention of leukemia cells. This increased retention may also play a role in treatment resistance, suggesting that the addition of small molecule inhibitors of CXCR4/CXCL12 to chemotherapy may help these high expressing patients, especially in subtypes such as HR KMT2A and RAM phenotype patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dissecting the role of the CXCL12/CXCR4 axis in acute myeloid leukaemia

Acute myeloid leukaemia (AML) is the most common adult acute leukaemia with the lowest survival rate. It is characterised by a build-up of immature myeloid cells anchored in the protective niche of the bone marrow (BM) microenvironment. The CXCL12/CXCR4 axis is central to the pathogenesis of AML as it has fundamental control over AML cell adhesion into the protective BM niche, adaptation to the hypoxic environment, cellular migration and survival. High levels of CXCR4 expression are associated with poor relapse-free and overall survival. The CXCR4 ligand, CXCL12 (SDF-1), is expressed by multiple cells types in the BM, facilitating the adhesion and survival of the malignant clone. Blocking the CXCL12/CXCR4 axis is an attractive therapeutic strategy providing a 'multi-hit' therapy that both prevents essential survival signals and releases the AML cells from the BM into the circulation. Once out of the protective niche of the BM they would be more susceptible to destruction by conventional chemotherapeutic drugs. In this review, we disentangle the diverse roles of the CXCL12/CXCR4 axis in AML. We then describe multiple CXCR4 inhibitors, including small molecules, peptides, or monoclonal antibodies, which have been developed to date and their progress in pre-clinical and clinical trials. Finally, the review leads us to the conclusion that there is a need for further investigation into the development of a 'multi-hit' therapy that targets several signalling pathways related to AML cell adhesion and maintenance in the BM.

High CXCR4 Expression Predicts a Poor Prognosis in Resected Lung Adenosquamous Carcinoma.

Background: Primary adenosquamous carcinoma (ASC) is a rare malignant tumor in the lung and its biological behavior has not yet been thoroughly described. In this study, we aimed to explore the clinical and biological role of CXCR4 in patients with resected lung ASC.Methods: We retrospectively reviewed the clinical records of patients with histologically confirmed lung ASC who underwent surgical resection with systematic lymph node dissection. Immunohistochemical staining was performed to detect the expression of CXCR4 in tumor tissues. The correlation between CXCR4 expression and clinicopathological characteristics were evaluated. The association between CXCR4 expression and patients' prognosis was analyzed by Kaplan-Meier and Cox regression. Moreover, we performed in vitro studies including CCK8, transwell and cell apoptosis to explore the potential role of CXCR4 in lung ASC.Results: A total of 78 patients with resected lung ASC were reviewed. Seventy (89.7%) patient tumors expressed CXCR4, with high level of CXCR4 expression observed in 45 (57.7%) cases. In vitro, CXCR4 conferred no difference in proliferative capacity but increased invasive potential, enhanced chemoresistance and inhibited apoptosis of lung ASC. Clinically, high CXCR4 expression was significantly associated with solid ASC, lymph node metastasis and advanced TNM stage. Patients with high CXCR4 expression and solid ASC had decreased disease-free survival and overall survival.Conclusions: CXCR4 was commonly expressed in lung ASC tumors. High CXCR4 expression might be a novel marker in predicting a poor prognosis in resected lung ASC and might serve as a potential therapeutic target.

Anti-CXCR4 Antibody Combined With Activated and Expanded Natural Killer Cells for Sarcoma Immunotherapy.

Sarcoma is one of the most severe forms of pediatric cancer and current therapies -chemotherapy and surgery- fail to eradicate the disease in half of patients. Preclinical studies combining new therapeutic approaches can be useful to design better therapies. On one hand, it is known that CXCR4 expression is implicated in rhabdomyosarcoma progression, so we analyzed relapses and chemotherapy-resistant rhabdomyosarcoma tumors from pediatric patients and found that they had particularly high levels of CXCR4 expression. Moreover, in assays in vitro, anti-CXCR4 blocking antibody (MDX1338) efficiently reduced migration and invasion of alveolar rhabdomyosarcoma RH30 cells. On the other hand, activated and expanded natural killer (NKAE) cell therapy showed high cytotoxicity against sarcoma cells in vitro and completely inhibited RH30 tumor implantation in vivo. Only the combination of MDX1338 and NKAE treatments completely suppressed metastasis in mice. In this study, we propose a novel therapeutic approach based on anti-CXCR4 blocking antibody in combination with NKAE cell therapy to prevent rhabdomyosarcoma tumor implantation and lung metastasis. These results provide the first evidence for the efficacy of this combined immunotherapy for preventing sarcoma disease dissemination.

SDF-1/CXCR4 axis enhances the immunomodulation of human endometrial regenerative cells in alleviating experimental colitis

Endometrial regenerative cells (ERCs) are a new type of mesenchymal-like stromal cells, and their therapeutic potential has been tested in a variety of disease models. SDF-1/CXCR4 axis plays a chemotaxis role in stem/stromal cell migration. The aim of the present study was to investigate the role of SDF-1/CXCR4 axis in the immunomodulation of ERCs on the experimental colitis. The immunomodulation of ERCs in the presence or absence of pretreatment of SDF-1 or AMD3100 was examined in both in vitro cell culture system and dextran sulphate sodium-induced colitis in mice. The results showed that SDF-1 increased the expression of CXCR4 on the surface of ERCs. As compared with normal ERCs, the SDF-1-treated, CXCR4 high-expressing ERCs more significantly suppressed dendritic cell population as well as stimulated both type 2 macrophages and regulatory T cells in vitro and in vivo. Meanwhile, SDF-1-pretreated ERCs increased the generation of anti-inflammatory factors (e.g., IL-4, IL-10) and decreased the pro-inflammatory factors (e.g., IL-6, TNF-α). In addition, SDF-1-pretreated CM-Dil-labeled ERCs were found to engraft to injured colon. Our results may suggest that an SDF-1-induced high level of CXCR4 expression enhances the immunomodulation of ERCs in alleviating experimental colitis in mice.

The Selective Anti Leukemic Effect of BL-8040, a Peptidic CXCR4 Antagonist, Is Mediated By Induction of Leukemic Blast Mobilization, Differentiation and Apoptosis: Results of Correlative Studies from a Ph2a Trial in Acute Myeloid Leukemia

▪Background: Persistence of minimal residual disease (MRD) after induction, consolidation and salvage therapies is an independent poor prognostic marker in AML. The bone marrow (BM) microenvironment is a protective niche that promotes chemotherapy resistance of leukemia cells. This residual subset of chemotherapy-resistant cells present during remission eventually contributes to the leukemia relapse/persistence. Adhesion of AML cells to BM stroma is dependent on the CXCR4 receptor binding to its ligand CXCL12. High levels of CXCR4 expression correlate with poor survival in AML. It is postulated that blocking the CXCL12/CXCR4 axis will dislodge AML cells from the protective microenvironment and, when combined with chemotherapy, may improve the response to therapy. BL-8040 (BKT140) is a short peptide which inhibits the binding of CXCR4 to CXCL12, resulting in mobilization of leukemic blasts from the BM to the peripheral blood (PB). BL-8040 has strong affinity for the CXCR4 receptor with long receptor occupancy and as a single agent induces differentiation and apoptosis of leukemia cells in preclinical models. A clinical trial assessing the safety and efficacy of BL-8040 in combination with cytarabine (Ara-C) for the treatment of relapsed/refractory AML patients was recently completed (NCT01838395). Here we report results from correlative studies done on samples from patients who participated in this trial.Objective: To assess BL-8040 single agent anti leukemia effects in relapsed/refractory AML.Method: Patients received a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5g/m2 for patients ≥60; 3g/m2 for patients <60) per day on days 3-7. Six dose levels of BL-8040 (0.5 - 2.0 mg/kg) were tested. PD parameters such as mobilization of leukemic blasts and induction of differentiation and apoptosis were assessed after monotherapy with BL-8040 using frequent PB sampling and BM aspirates at baseline and on day 3 prior to Ara-C administration.Results: 45 patients (including 3 receiving compassionate use BL-8040) with a median age of 61 yrs were treated. The majority of patients had poor risk disease with 24% secondary AML and 17% with prior allogeneic stem-cell transplantation. Most patients were heavily pretreated with 19% relapsing after a short first remission (CR1 ≤12 months), 17% had ≥2 relapses and 45% refractory to 1-2 inductions. The CR+CRi rate was 38% in subjects receiving BL-8040 dose ≥1.0 mg/kg (n=39). Ongoing follow-up (FU) of responding patients (expansion phase; N=19) shows median EFS of 9.3 months (range 4.3-12.8 months). At the time of this analysis 10/19 patients are alive with a FU range of 0.96 - 12.8 months. FACS analysis revealed that BL-8040 monotherapy triggered an average 4.3-fold increase of immature AML blasts (CD45+Low/CD34+/CD117+/HLA-DR+) in PB (i.e., mobilization). Response to treatment was associated with more robust mobilization of AML blasts following BL-8040 monotherapy (responders 9.5 vs non-responders 1.7 fold median). Preferential mobilization of AML blasts over normal cells (4.7 fold vs. 1.4 fold, respectively) was confirmed by FISH analysis in a subset of patients with informative cytogenetic abnormalities. Induction of granulocytic differentiation of immature leukemia progenitors (2.5 fold) in the BM following BL-8040 monotherapy was noted. Finally, induction of AML blasts apoptosis (40% increase) by BL-8040 alone was evident by FACS in day 3 (pre-araC) bone marrow biopsies.Conclusions: The data demonstrate that sustained blockade of the CXCR4-CXCL12 axis with BL-8040 is safe and well tolerated and when given in combination with Ara-C improves the response rate achieved historically with Ara-C alone. In addition, treatment with BL-8040 as a single agent rapidly and efficiently induces mobilization, differentiation and cell death of AML blasts. This selective effect on chemotherapy-resistant cells may be translated into reduction of residual disease arguing for incorporation into front-line trials and such studies are ongoing. DisclosuresForan:Millennium Pharmaceuticals, Inc.: Research Funding; medscape: Honoraria; karyopharm: Honoraria; pfizer: Honoraria; novartis: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding. DiPersio:Incyte Corporation: Research Funding. Peled:Biokine Therapeutics: Employment. Abraham:Biokine Therapeutics Ltd: Employment. Pereg:BioLineRx Ltd: Employment. Vainstein:BioLineRx Ltd.: Employment. Oberkovitz:BioLineRx Ltd.: Employment. Aharon:BioLineRx Ltd.: Employment. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

The Peptidic CXCR4 Antagonist, BL-8040, Significantly Reduces Bone Marrow Immature Leukemia Progenitors By Inducing Differentiation, Apoptosis and Mobilization: Results of the Dose Escalation Clinical Trial in Acute Myeloid Leukemia

Background: The bone marrow (BM) niche protects acute myeloid leukemia (AML) cells from chemotherapy. BM homing of AML cells is dependent on CXCR4 and its ligand CXCL12; high levels of CXCR4 expression correlate with poor survival in AML. It is postulated that blocking the CXCL12/CXCR4 axis with a potent CXCR4 antagonist will disrupt the interaction of the leukemic blasts with the BM and augment the anti-leukemic effect of chemotherapy. BL-8040 (BKT140) is a short synthetic peptide which inhibits the binding of CXCR4 to CXCL12, resulting in the mobilization of leukemic blasts from the bone marrow to the peripheral blood. BL-8040 also inhibits CXCR4/CXCL12 mediated pro-survival ERK/Akt signaling and alters balance of pro/anti-apoptotic Bcl-2 proteins. BL-8040 has strong affinity for the CXCR4 receptor with long receptor occupancy, and, in contrast to other CXCR4 inhibitors, as a single agent induces apoptosis of leukemia cells ex-vivo in clinical samples. A clinical trial assessing the safety and efficacy of BL-8040 in combination with cytarabine (Ara-C) for the treatment of adult relapsed/refractory AML patients is currently ongoing (NCT01838395).Objective: To assess the safety, efficacy and pharmacodynamics/pharmacokinetic parameters of BL-8040 in combination with Ara-C in first or second relapse and refractory AML patients.Method: The study includes a dose escalation phase (3+3 design) followed by an expansion phase. Each patient receives a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5 g/m2 for patients ≥60; 3 g/m2 for patients <60) on days 3-7. Five dose levels of BL-8040 (0.5, 0.75, 1, 1.25 and 1.5 mg/kg) have been tested. Extensive pharmacodynamic parameters such as CXCR4 expression on the leukemic cells, receptor occupancy, mobilization of leukemic cells and induction of apoptosis are being assessed after monotherapy with BL-8040 using peripheral blood sampling (PB) and BM aspirates at baseline and on day 3 prior to Ara-C administration. Clinical response to treatment is evaluated by BM biopsy on day 30 (-/+ 2).Results: Twenty two patients (median age, 61 y; range, 43-74 y) with relapsed/refractory AML were treated during the escalation phase. BL-8040 was escalated up to 1.5 mg/kg (recommended phase 2 dose) without reaching the MTD. Combination of BL-8040 with Ara-C was safe and well tolerated at all doses tested. Only one SAE (Sweet Syndrome) was reported as possibly related to study drug. The primary treatment related AEs were transient injection site and systemic reactions. The composite complete remission including complete remission (CR) and complete remission with incomplete blood count recovery (CRi) rate was 38% in subjects receiving BL-8040 doses of 1 mg/kg and higher (n=16). Two days of BL-8040 monotherapy triggered an average 40.2-fold (range 1.0-350.9) mobilization of immature AML progenitors (CD45+Low/CD34+/CD117+/HLA-DR+) from the BM to the PB. Moreover, a significant apoptotic effect on the immature leukemia progenitors in the BM following 2 days of BL-8040 monotherapy was noted. Flow cytometric evidence of apoptosis by BL-8040 was corroborated by an increase in cleaved caspase 3 positive blasts in day 3 bone marrow biopsies. In addition, BL-8040 treatment resulted in a median decrease of 57.7% (range, -10.4% to -96.2%) in the number of BM Leukemia progenitor cells (CD45+Low/CD34+/CD117+/HLA-DR+ out of total CD45+/CD34+ normal/progenitor cells) compared with the baseline biopsy. This decrease was accompanied with a 3.1 fold increase in granulocytes in the day 3 BM biopsy supporting differentiation effect. Finally, long receptor occupancy (up to 24 hours post dosing) was confirmed by FACS analysis measuring surface CXCR4 staining. Pharmacokinetics analysis confirmed dose incremental BL-8040 exposure with a short plasma half-life.Conclusions: The current data demonstrate that sustained blockade of the CXCR4-CXCL12 axis with BL-8040 has potent anti leukemic activity and in combination with Ara-C may improve the response typically achieved in this advanced AML population. Mechanistically, BL-8040 was able to induce apoptosis of leukemic progenitor cells, overcoming the stroma protection, inducing mobilization and differentiation. DisclosuresRowe:BioLineRx Ltd.: Consultancy; Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Uy:Novartis: Research Funding. Altman:Seattle Genetics: Consultancy; Novartis: Consultancy; Spectrum: Consultancy; Astellas: Consultancy; Ariad: Consultancy; BMS: Consultancy. Peled:Biokine Ltd.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BiolineRX: Consultancy, Research Funding. Pereg:BioLineRx: Employment. Vainstein:BioLineRx: Employment. Aharon:BioLineRx: Employment. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Cortes:Teva: Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; BerGenBio AS: Research Funding; Novartis: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.

The Dual E-Selectin/CXCR4 Inhibitor, GMI-1359, Enhances Efficacy of Anti-Leukemia Chemotherapy in FLT3-ITD Mutated Acute Myeloid Leukemia

Aberrant activation of the FMS-like tyrosine kinase-3 (FLT3) is driven by internal tandem duplication (ITD) mutations in the FLT3 gene, which are commonly observed in patients with acute myeloid leukemia (AML). Hence, FLT3 represents an attractive therapeutic target in AML (Weisberg et al., 2002). Indeed, several small molecule FLT3 inhibitors including sorafenib have showed encouraging efficacy in reducing leukemia blasts in the peripheral blood in FLT3 mutated AML patients. However, these agents have little effect on leukemic stem cells in the bone marrow (BM) microenvironment (Borthakur et al., 2011; Fathi and Chabner, 2011; Zhang et al., 2008).The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents (Horacek et al., 2013; Peled and Tavor, 2013). In fact, treatment with sorafenib markedly upregulated CXCR4 levels in FLT3 -mutated cells. In addition, leukemia cells can activate endothelial cells (EC) that induce adhesion of a sub-set of the leukemia cells through E-selectin. The adherent AML cells are sequestered in a nonproliferative state that further protects them from chemotherapy (Pezeshkian et al., 2013). Therefore, blocking CXCR4 and E-selectin in parallel could theoretically eliminate the protection provided by the interaction of leukemic cells with their BM microenvironment and enhance effectiveness of chemotherapy in FLT3-mutant AML patients. In the present study, we evaluated the effectiveness of a dual CXCR4 and E-selectin antagonist, GMI-1359 (GlycoMimetics, Inc., Rockville, MD), in targeting FLT3-ITD-mutant AML in vitro and in vivo. High levels of CXCR4 expression were observed in several human and murine AML cell lines, which was further increased in hypoxic (i.e., 1% oxygen) conditions that mimic the BM microenvironment. These FLT3 -ITD leukemic cell lines also expressed hypoxia-responsive, functional E-selectin ligands identified by reactivity with an antibody (HECA452) that binds the same carbohydrate epitope required for binding to E-selectin. One such E-selectin ligand CD44 increased in FLT3 -ITD cells cultured in hypoxia compared to those cultured in normoxia (i.e. 21% oxygen). In addition, hypoxia also enhanced CXCR4 expression on mesenchymal stem cells (MSC) and EC such as HUVEC. In hypoxic co-cultures of the FLT3 -ITD-mutant leukemia cells MV4-11 or MOLM14 with MSCs and ECs (i.e., HUVEC or TeloHAEC), the presence of the dual E-selectin/CXCR4 inhibitor GMI-1359 effectively reduced leukemic cell adhesion by ~ 50% to the MSC/EC feeder layer compared to the PBS-treated control (p<0.05), even in the presence of TNFa, which induces E-selectin expression in EC. However, an E-selectin specific inhibitor only reduced adhesion of MV4-11 and MOLM14 by ~ 20%. GMI-1359 markedly abrogated the protection provided by the BM microenvironment (i.e., hypoxia and/or MSC and EC) of Baf3-FLT3 -ITD leukemic cells treated with the FLT3 inhibitor sorafenib. Apoptosis was induced in 36.6%, 35.6% and 48.9% of leukemic cells cultured with sorafenib alone, sorafenib and an E-selectin inhibitor or sorafenib and GMI-1359, respectively.The significance of these in vitro findings were studied in vivo. Female SCID beige mice were injected iv with MV4-11 and followed for survival. Beginning 14 days post tumor injection, cohorts of mice (n=10/group) were treated with saline, GMI-1359 (40 mg/kg), standard chemotherapy cytarabine plus daunorubicin, or a combination of GMI-1359 and chemotherapy. Combined treatment of mice with GMI-1359 (40 mg/kg) and chemotherapy demonstrated a profound survival benefit compared to controls or chemotherapy alone at day 135 after leukemia cell injection (i.e., 67% vs. 11% or 30%, p=0.0011 and 0.0406, respectively). Single agent treatment with GMI-1359 was statistically indistinguishable from saline alone or chemotherapy alone. In a separate cohort of MV4.11-engrafted mice, the single administration of GMI-1359 increased circulating WBC and leukemic MV4-11cells, which persisted for at least 8 hrs. This effect was consistent with GMI-1359 disrupting the protective effects of the tumor microenvironment and mobilizing MV4-11 cells from the BM niche.. These findings provide the pre-clinical basis for the evaluation of GMI-1359 in patients with FLT3 -mutant AML. [Display omitted] DisclosuresZhang:Karyopharm: Research Funding. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Notch pathway promotes ovarian cancer growth and migration via CXCR4/SDF1α chemokine system

Ovarian cancer is the most deadly gynecological malignancy. Understanding the molecular pathogenesis of ovarian cancer is critical to provide new targeted therapeutic strategies. Recent evidence supports a role for Notch in ovarian cancer progression and associates its dysregulation to poor overall survival. Similarly, CXCR4/SDF1α signalling correlates with ovarian cancer progression and metastasis. Recent findings indicate that Notch promotes CXCR4/SDF1α signalling and its effect on cell growth and migration; nonetheless, up to now, the association between Notch and CXCR4/SDFα in ovarian cancer has not been reported. Thereby, the aim of this study was to investigate if Notch and CXCR4/SDF1α cooperate in determining ovarian cancer growth, survival and migration.To address this issue, Notch signalling was inhibited by using γ-secretase inhibitors, or upregulated by forcing of Notch1 expression in ovarian cancer cell lines. Our results indicated that Notch activity influenced tumour cell growth and survival and positively regulated CXCR4 and SDF1α expression. CXCR4/SDF1α signalling mediated the effect of Notch pathway on ovarian cancer cell growth and SDF1α-driven migration. Additionally, for the first time, we demonstrated that Notch signalling activation can be detected in ovarian cancer specimens by immunohistochemistry analysis of the Notch transcriptional target, HES6 and is positively correlated with high expression levels of CXCR4 and SDF1α.Our results demonstrate that Notch affects ovarian cancer cell biology through the modulation of CXCR4/SDF1α signalling and suggest that Notch inhibition may be a rationale therapeutic approach to hamper ovarian cancer progression mediated by the CXCR4/SDF1α axis.

Transfection of CXCR-4 using microbubble-mediated ultrasound irradiation and liposomes improves the migratory ability of bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) have proven useful for the treatment of various human diseases and injuries. However, their reparative capacity is limited by their poor migration and homing ability, which are primarily dependent on the SDF-1/CXCR4 axis. Most subcultured BMSCs lack CXCR4 receptor expression on the cell surface and exhibit impaired migratory capacity. To increase responsiveness to SDF-1 and promote cell migration and survival of cultured BMSCs, we used a combination of ultrasound-targeted microbubble destruction (UTMD) and liposomes to increase CXCR4 expression in vitro. We isolated and cultured rat BMSCs to their third passage and transduced them with recombinant plasmid pDsRed-CXCR4 using microbubble-mediated ultrasound irradiation and liposomes. Compared to some viral vectors, the method we employed here resulted in significantly better transfection efficiency, CXCR4 expression, and technical reproducibility. The benefits of this approach are likely due to the combination of "sonoporation" caused by shockwaves and microjet flow resulting from UTMD-generated cavitation. Following transfection, we performed a transwell migration assay and found that the migration ability of CXCR4-modified BMSCs was 9-fold higher than controls. The methods we describe here provide an effective, safe, non-viral means to achieve high levels of CXCR4 expression. This is associated with enhanced migration of subcultured BMSCs and may be useful for clinical application as well.

BL-8040, a Peptidic CXCR4 Antagonist, Induces Leukemia Cell Death and Specific Leukemia Cell Mobilization in Relapsed/Refractory Acute Myeloid Leukemia Patients in an Ongoing Phase IIa Clinical Trial

Background: The bone marrow (BM) niche protects Acute Myeloid Leukemia (AML) cells from chemotherapy. BM homing of AML cells is dependent on CXCR4 and its ligand CXCL12, and high levels of CXCR4 expression correlate with poor survival in AML patients. It is postulated that blocking the CXCL12/CXCR4 axis with a potent CXCR4 antagonist will disrupt the interaction of the malignant blasts with the BM and augment the anti-leukemic effect of chemotherapy. BL-8040 (BKT140) is a 14-residue, cyclic, synthetic peptide capped with an aromatic ring that acts as a selective inhibitor of the CXCR4 chemokine receptor. BL-8040 has strong affinity for the CXCR4 receptor and long receptor occupancy, resulting in a prolonged pharmacodynamic effect. BL-8040 has superior mobilization capacity, inverse agonism activity and direct pro-apoptotic activity against leukemia cells. Preclinical studies have shown that in addition to its activity as a mobilizer of hematopoietic cells, BL-8040 exhibits a CXCR4-dependent selective anti-tumor effect against malignant cells. A clinical trial for the treatment of adult relapsed/refractory AML patients using a combination of BL-8040 and cytarabine (Ara-C) is currently ongoing (NCT01838395).Method: The phase II clinical trial in relapsed/refractory AML patients includes a dose escalation phase (3+3 design) followed by an expansion phase using the highest safe and efficacious dose group. Each patient receives a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5g/m2 for patients ≥60; 3g/m2 for patients ≤60) on days 3-7. For each of the BL-8040 tested doses (0.5, 0.75, 1, 1.25 and 1.5 mg/kg) the safety and tolerability, pharmacokinetic profile and clinical responses are being evaluated. Additionally, CXCR4 expression on leukemia cells, receptor occupancy, timing of mobilization of leukemic blasts and stem/progenitor cells and induction of apoptosis are being followed using frequent peripheral blood sampling (PB) and BM aspirates at baseline and on day 3 prior to Ara-C administration.Results: Preliminary results from the ongoing study suggest that administration of BL-8040, in combination with Ara-C, is safe and well tolerated at all doses tested to date (0.5-1 mg/kg). There have been no DLTs or SAEs and no delays in count recovery attributable to BL-8040. The primary treatment related AE has been a transient injection site reaction. FACS analysis revealed that 2 days of BL-8040 monotherapy triggered substantial mobilization of AML blasts (CD45+Low/CD34+/CD117+/HLA-DR+) from the BM to the PB. Data from the first 9 subjects revealed a 5.14 fold (range 1.0-8.09) median increase in AML blasts in the PB. Moreover, a dramatic decrease in the amount of AML blasts in the BM following 2 days of BL-8040 monotherapy was noted. FACS data from 7 subjects, for which BM aspirate samples were evaluable for analysis, revealed a median decrease of 70.65% [range(-16.32%)-(-87.68%)] in the number of AML blasts (CD45+Low/CD34+/CD117+/HLA-DR+ out of total CD45+/CD34+ normal/progenitor cells) compared with baseline. Importantly, this effect was specific to the AML blasts as the levels of normal/progenitor cells remained stable post BL-8040 monotherapy (5/7 patients). This decrease of BM AML blasts was evident regardless of whether the patient mobilized AML blasts or not. In support of induction of apoptosis by CXCR4 inhibition, an increase in the number of blasts with cleaved caspase3 was ascertained in day 3 BM biopsies from 6/8 patients. Long receptor occupancy (up to 24 hours post dosing) was observed by FACS analysis measuring surface CXCR4 staining. Pharmacokinetics analysis confirmed increase in BL-8040 exposure over doses with a short plasma half-life.Conclusion: The current data demonstrate that BL-8040 induces mobilization of AML blasts from the BM and has sustained receptor occupancy. In addition, a direct effect on AML blast viability has been observed in samples obtained during BL-8040 monotherapy. Importantly, the data suggest a differential effect of BL-8040 monotherapy on AML blasts vs. normal progenitors. BL-8040 was found to be safe and well tolerated at all doses tested to date. The updated results of the dose escalation phase of this ongoing study will be presented. DisclosuresBorthakur:BioLineRx: Research Funding. Nagler:BioLineRx: Research Funding. Peled:BioLineRx: Research Funding. Pereg:BioLineRx: Employment. Foley-Comer:BioLineRx: Consultancy. Russovsky:BioLineRx: Employment. Aharon:BioLineRx: Employment. Cortes:BioLineRx: Research Funding. Andreeff:BioLineRx: Research Funding.

Mobilization Of Blasts and Leukemia Stem Cells by Anti-CXCR4 Antibody BMS-936564 (MDX 1338) in Patients With Relapsed/Refractory Acute Myeloid Leukemia

BackgroundBone marrow homing of AML is dependent on CXCR4, and high levels of CXCR4 expression correlate with worse survival in AML (Rombouts et al 2004, Spoo et al 2007). CXCR4 antagonists overcome environment adhesion mediated drug resistance and enhance chemotherapy induced cytotoxicity (Liesveld et al 2007, Zeng et al 2009, Nervi et al 2009, Beider et al 2010). Plerixafor, a small molecular CXCR4 inhibitor, was studied in a phase I trial of newly diagnosed AML patients (Uy et al ASH 2011), and BMS-936564, a fully-human monoclonal antibody to CXCR4, in combination with MEC (mitoxantrone, etoposide, cytarabine), is currently under study in relapsed/refractory AML. MethodThe clinical trial completed a phase I dose escalation phase in AML patients, with increasing concentrations of BMS-936564 in 4 dose cohorts (0.3,1,3, and 10 mg/kg) and is currently enrolling a cohort of first salvage AML patients at the maximum dose of 10 mg/kg. The initial cohort of patients at 0.3 mg/kg received three weekly doses of antibody on days 1, 8, 15 [monotherapy period of cycle 1 (21 days)], followed by the same dose of antibody on days 1, 8, 15 of cycle 2 plus MEC chemotherapy [days 1-5 of cycle 2, (28 day cycle)]. After enrollment of the first cohort, the protocol was amended to reduce the monotherapy period to 1 week (1 dose of BMS-936564) in cohorts 1, 3, and 10 mg/kg, followed by the same combination regimen. As a companion study to this trial with the anti-CXCR4 antibody, we are investigating CXCR4 expression, timing of mobilization of leukemic blasts and leukemia stem cells (LSCs), and induction of apoptosis. Mobililzation of LSCs will be critical to eradication of leukemia, as they might serve as a reservoir for drug resistance and future relapse. We analyzed serial blast and LSC populations from blood and bone marrow samples from patients undergoing treatment by flow cytometry for phenotype, CXCR4 and annexin V expression. The putative LSCs were defined as CD34+CD38-CD123+ or by aldehyde dehydrogenase. ResultsAn independent assessment of CXCR4 expression in 56 consecutive AML patients from our institution not related to this clinical trial revealed a mean % expression of 31%, range 1-99%, with mean fluorescence intensity (MFI) of 2092, range 319-7942. A sample of 18 patients showed a correlation in CXCR4 expression between gated blasts derived from blood and bone marrow samples from the same patient (For % expression, r2=0.85, p=5e-8; MFI r2=0.45, p=0.002). Our site has enrolled 24 AML patients thus far on the above noted trial of BMS-936564. Administration of BMS-936564 resulted in brisk mobilization of leukemic blasts in 14/24 patients that initially peaked within at 2-6 hours post start of infusion in most patients, with an average of 2.1-fold increase ± 1.8 fold (range 1.06-8.96 fold), and some blasts continued to be in circulation for days. In most cases, the samples for which mobilization was not observed either did not have circulating blasts at baseline, or were from patients who received lower doses of BMS-936564. In addition, CD34+CD38-CD123+LSCs were also mobilized post-treatment with BMS-936564, and in some cases, continued to rise over the subsequent days, during which the blast population declined. The average rise in %CXCR4 was from 29.3% pre-treatment to 69.8% peak value for blasts, and 23.0% pre-treatment to 75.6% peak value for LSCs. Although a direct correlation between CXCR4 expression by blasts and fold mobilization was not apparent, the highest fold increase in mobilization (∼9-fold) did occur in the patient with a moderately high level of CXCR4 expression, 42.5%. In this patient, there was also a sharp decline in circulating CXCR4 positive cells within 2 days and the patient achieved complete remission. BMS-936564 has demonstrated apoptosis in some preclinical models (Kuhne MR et al Clin Cancer Res v19(2): 357-66 (2013)).) For most patients, there was some increase in annexin V staining observed during the first 96 hours after antibody exposure. One patient sample with initial low baseline level of apoptosis exhibited a rise in annexin V staining (from 6% to 48%) that peaked on day 3 after administration of BMS-936564. ConclusionThese data demonstrate that BMS-936564 induces mobilization of both AML blasts and LSCs, which may enhance chemotherapy-induced cytotoxicity in relapsed/refractory AML. Disclosures:Chien:Bristol-Myers Squibb: Research Funding. Cardarelli:BMS: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Becker:Bristol-Myers Squibb: Research Funding.

High-level expression of CXCR4 in breast cancer is associated with early distant and bone metastases

Metastasis is the most life-threatening complication in all cancers. The chemokine receptor 4 (CXCR4) is expressed at high levels in many breast-cancer tumors and may modulate metastasis. We compared the time-to-metastasis and the sites of metastasis between breast-cancer tumors expressing CXCR4 at high or low levels. We enrolled 191 early breast cancer patients in our study. The expression of CXCR4 was evaluated using immunohistochemical staining, and the patients were divided into low-level (CXCR4-) and high-level (CXCR4+) CXCR4 expression groups. Associations between the patients' level of CXCR4 expression and their basic clinical characteristics, time-to-metastasis, and metastatic sites were examined using a Cox proportional-hazards regression model. A total of 107 CXCR4+ patients (56 %) were identified. No statistical differences were evident in basic characteristics between the CXCR4+ and CXCR4- groups. The CXCR4+ group had a higher incidence of distant metastasis during the first year (10.3 % versus 1.1 %, P = 0.009) and shorter event-free survival (17.43 months versus 27.5 months, P = 0.026) than those of the CXCR4- group. The CXCR4+ group also had a higher incidence of bone metastasis (P = 0.008) than the CXCR4- group. No significant difference in metastasis sites in other organs was observed between the two groups. A high level of CXCR4 expression in breast cancer is associated with early distant and bone metastases. The CXCR4+ phenotype may be a useful predictor for the prevention of early treatment failure and bone metastasis in breast cancer patients. This retrospective study shows that a high expression of CXCR4 in breast cancer is associated with earlier distant metastasis and bone metastasis in breast cancer.

Comparison of 18F-labeled CXCR4 antagonist peptides for PET imaging of CXCR4 expression

CXCR4 is overexpressed on tumor cells from many types of human cancers. A high level of CXCR4 expression often correlates with poor prognosis, chemotherapy resistance, and metastasis. The development of CXCR4-specific radiotracers for positron emission tomography (PET) imaging will allow in vivo evaluation of receptor expression level for diagnosis or therapeutic evaluation. Two new (18)F-labeled radiotracers based on an Ac-TC14012 peptide, [(18)F]FP-Ac-TC14012 and [(18)F]FB-Ac-TC14012, were synthesized and characterized. The affinities of the 2-fluoropropionate (FP)-conjugated or 4-fluorobenzoate (FB)-conjugated peptides to CXCR4-transfected Chinese hamster ovarian (CHO) cells were evaluated in a competitive binding assay with [(125)I]CXCL12 radioligand. The cell uptake and retention of [(18)F]FP-labeled and [(18)F]FB-labeled peptides were measured. The tumor targetability and pharmacokinetics of these two tracers were also evaluated by microPET imaging and biodistribution studies. The labeled peptides retained high binding affinity to CXCR4 and showed much higher uptake in CXCR4-positive CHO cells than in CXCR4-negative cells in vitro. The smaller and more hydrophilic [(18)F]FP prosthetic group resulted in higher affinity and lower nonspecific cell uptake compared to the [(18)F]FB-labeled peptide. Both radiotracers showed much higher accumulation in CXCR4-positive than CXCR4-negative tumor xenografts in mice and allowed clear visualization of CXCR4 expression by PET. Among the two, [(18)F]FP-Ac-TC14012 showed higher tumor uptake and better tumor-to-background contrast. Unlike their N-terminal 4-F-benzoate analogs, these two tracers had minimal blood retention, likely due to reduced red blood cell binding. Metabolic organs, such as the liver and kidney, also showed high uptake. When blocked with low-dose cold peptide (10μg), the tumor uptake was significantly increased, most likely due to the increased concentration in blood circulation, as evidenced by decreased liver uptake. These results demonstrate that the [(18)F]FP-labeled Ac-TC14012 peptide with high tumor uptake, low nonspecific binding, and good tumor-to-background contrast promises [(18)F]FP-Ac-TC14012 as a PET tracer for in vivo PET imaging of CXCR4 expression.

Stromal cell-derived factor-1α (SDF-1α/CXCL12)-enhanced angiogenesis of human basal cell carcinoma cells involves ERK1/2–NF-κB/interleukin-6 pathway

Stromal cell-derived factor 1alpha (SDF-1alpha) (CXCL12) has been observed to enhance tumor angiogenesis. However, the comprehensive role of SDF-1alpha (CXCL12)-CXCR4 interaction, exerted during angiogenesis, has not been well understood. We have previously demonstrated that human basal cell carcinoma (BCC) tissues and a BCC cell line (BCC-1/KMC) had significant expression of CXCR4, whose level was higher in invasive than in the non-invasive BCC types. Here, we observed that human BCC tissues with high expression levels of CXCR4 had higher vascularity. Further, among the 71 BCCs diagnosed between the years 2004-2005, BCCs with high CXCR4 expression had concomitantly higher microvessel density, as compared with those with low CXCR4 expression (P < 0.001). We found that SDF-1alpha induced angiogenic activity in human BCC cells, both in vitro and in vivo. SDF-1alpha significantly upregulated several angiogenesis-associated genes such as interferon-alpha-inducible protein 27, interleukin (IL)-6, bone morphogenetic protein (BMP)-6, SOCS2 and cyclooxygenase 2 (COX)-2 in human BCC cells. Among them, IL-6 was the earliest and highest upregulated gene whose induction was observed within 6 h of the commencement of SDF-1alpha-CXCR4 interaction. The mechanisms behind the SDF-1alpha-induced time and dose-dependent upregulation of messenger RNA expression and protein secretion of IL-6 were investigated. The transcriptional regulation of IL-6 by SDF-1alpha was mediated by phosphorylation of extracellular signal-related kinase 1/2 and activation of the nuclear factor-kappaB complex. The identification of the angiogenic profiles induced through SDF-1alpha-CXCR4 interactions in human BCC cells may contribute further insights into the mechanisms involved in the angiogenic potential of SDF-1alpha (CXCL12).

Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma: pattern of expression and correlation with clinical outcome

Nasopharyngeal carcinoma (NPC) is a tumor derived from epithelial cells and Epstein-Barr virus infection has been reported to be a cause of this disease. Chemokine receptor CXCR4 was found to be involved in HIV infection and was highly expressed in human malignant breast tumors and the ligand for CXCR4, CXCL12 (SDF-1), exhibited high expression in organs in which breast cancer metastases are often found. The metastatic pattern of NPC is quite similar to that of malignant breast tumors. In this study, we investigated the expression of CXCR4 in nasopharyngeal carcinoma (NPC) tissues by immunohistostaining. We found different staining patterns, which included localization in the nucleus, membrane, cytoplasm or a combination of them. The staining intensity was also variable among samples. The metastatic rates in patients with high compared to low or absent expression was 38.6% versus 19.8%, respectively (P = 0.004). High expression of CXCR4 was associated with poor overall survival (OS = 67.05% versus 82.08%, P = 0.0225). These results suggest that CXCR4 may be involved in the progression of NPC and that a high level of CXCR4 expression could be used as a prognostic factor.

Role of high expression levels of CXCR4 in tumor growth, vascularization, and metastasis

Hormone refractory metastatic prostate cancer remains an incurable disease. We found that high expression levels of the chemokine receptor CXCR4 correlated with the presence of metastatic disease in prostate cancer patients. Positive staining for CXCL12, the ligand for CXCR4, was mainly present in the tumor-associated blood vessels and basal cell hyperplasia. Subcutaneous xenografts of PC3 and 22Rv1 prostate tumors that overexpressed CXCR4 in NOD/SCID mice were two- to threefold larger in volume and weight vs. controls. Moreover, blood vessel density, functionality, invasiveness of tumors into the surrounding tissues, and metastasis to the lymph node and lung were significantly increased in these tumors. Neutralizing the interactions of CXCL12/CXCR4 in vivo with CXCR4 specific antibodies inhibited the CXCR4-dependent tumor growth and vascularization. In vitro, CXCL12 induced the proliferation and VEGF secretion but not migration of PC3 and 22Rv1 cells overexpressing CXCR4. Similar effects of CXCR4 overexpression on tumor growth in vivo were also noted in two breast cancer lines, suggesting that the observed effect of CXCR4 is not unique to prostate tumor cells. Thus high levels of the chemokine receptor CXCR4 induce a more aggressive phenotype in prostate cancer cells and identify CXCR4 as a potential therapeutic target in advanced cases of metastatic prostate cancer.

Engraftment of acute myeloid leukemia in NOD/SCID mice is independent of CXCR4 and predicts poor patient survival.

The aim of this study was to investigate factors influencing the engraftment potential of acute myeloid leukemia (AML) CD34+ cells in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We examined the relationship between engraftment, CXCR4 expression on CD34+ and CD34+CD38- cells, and patient (Pt) clinical/laboratory characteristics in 44 samples from 11 Pts. Engraftment, evaluated by Southern blot and CD45 flow cytometric analyses, was observed in murine bone marrow of 6 of 11 Pt samples, ranging from 0.1% to 73.9% by Southern blot and from 0.1%-36.8% by flow cytometry. Poor Pt prognosis was inversely correlated with engraftment; the median overall survival was 95.9 weeks for Pts whose cells did not engraft and 26.1 weeks for those whose cells did engraft (p = 0.012, log-rank test). No other clinical/laboratory variable predicted engraftment. No correlation between the level of CXCR4 expression on AML cells and engraftment was observed. Cells with virtually absent CXCR4 expression were able to engraft, and cells from two Pts with high expression levels of CXCR4 did not engraft. Furthermore, anti-CXCR4 antibody failed to block the engraftment of AML cells into NOD/SCID mice. In conclusion, we demonstrated that CXCR4 is not critical for the engraftment of AML CD34+ cells in NOD/SCID mice. The model may, however, reflect the clinical course of the disease.

Expression of chemokine receptors, CXCR4 and CXCR5, and chemokines, BLC and SDF-1, in the eyes of patients with primary intraocular lymphoma

Objective Chemokines have a range of biologic activities, including regulation of leukocyte trafficking, modulation of hematopoietic cell proliferation, and adhesion to extracellular matrix molecules. Specifically, B-lymphocyte chemoattractant (BLC); BCA-1; CXCL13, SCYB13) and stromal cell-derived factor-1 (SDF-1, CXCL12, SCYB12) are chemotactic for human B cells, and their ligands CXCR4 and CXCR5 are differentially expressed on B cells, including malignant B cells. We investigated the expression of these chemokine/chemokine receptors in eyes with primary intraocular B-cell lymphoma (PIOL). Design Observational case series (human tissue study). Methods Three freshly enucleated eyes with PIOL and a normal autopsied eye were frozen and sectioned. The sections were evaluated using immunohistochemistry (avidin-biotin-complex immunoperoxidase technique) for CXCR4, CXCR5, BLC, and SDF-1 to detect the expression and location. Reverse transcriptase-polymerase chain reaction was used to detect chemokine transcripts of CXCR4, CXCR5, BLC, and SDF-1 in PIOL and retinal pigment epithelium (RPE) cells after microdissection—either by laser capture (Arcturus) or by manual dissection—from frozen sections. Main outcome measures and results The three PIOL eyes showed similar pathology, with typical diffuse large B-lymphoma cells subjacent to the RPE. The eyes also demonstrated a similar chemokine profile. High expression levels of CXCR4 and CXCR5 were found limited to the lymphoma cells. In contrast, BLC protein was expressed in the RPE but not located in other ocular resident cells. SDF-1 was barely detected in a few RPE cells. CXCR4 and CXCR5 transcripts were detected abundantly in lymphoma cells, whereas BLC and SDF-1 transcripts were detected only in the RPE and not the malignant cells. No chemokine expression was detected on the RPE cells in the normal control eye. Conclusions Chemokines and chemokine receptors selective for B cells were identified in RPE and malignant B cells, respectively. BLC, and possibly SDF-1, attracts both normal and malignant B-cells while promoting migration of only small numbers of T cells and macrophages. We propose that B-cell chemokines may be involved in the pathogenesis of PIOL by selectively attracting lymphoma cells to the RPE from the choroidal circulation. Our data suggest that inhibition of B-cell chemoattractants could be a future strategy for the treatment of PIOL.

Differential expression of chemokine receptors in B cell malignancies.

Chemokines are a family of 8-10 kDa proteins with a wide range of biological activities including the regulation of leukocyte trafficking, modulation of haemopoietic cell proliferation and adhesion to extracellular matrix molecules. Using a panel of chemokine receptor-specific monoclonal antibodies (MoAb) in a multicolour flow cytometry approach we analysed the expression of the lymphocyte-associated chemokine receptors CXCR4, CXCR5, CCR5 and CCR6 in B cell acute lymphoblastic leukaemia (precursor B-ALL; six cases), B cell chronic lymphocytic leukaemia (B-CLL; 31 cases), multiple myeloma (10 cases), mantle cell lymphoma (MCL, four cases), follicular lymphoma (FL, three cases) and hairy cell leukaemia (HCL, five cases). We demonstrate that CXCR4, CXCR5 and CCR6 are differentially expressed in these B lymphoproliferative disorders depending on the maturational stage of the malignant B cell population investigated. In particular, we found that CXCR4 is strongly expressed on immature ALL blasts whereas no surface immunoreactivity for CXCR5, CCR5 and CCR6 was observed. By contrast, non-Hodgkin's lymphomas (NHLs) corresponding to more mature peripheral B cell subsets (ie B-CLL and MCL) exhibited high expression levels of CXCR4 and CXCR5. Analysis of terminally differentiated myeloma cells revealed a down-regulation of CXCR4, CXCR5 and CCR6. CCR5, which is not expressed in normal B cells, was also absent from the majority of NHLs. However, CCR5 staining was seen in three of five cases of HCL, representing the first example of cross-lineage aberrant chemokine receptor expression in malignant haemopoietic cells.