Phosphorylated CXCR4 is associated with poor survival in adults with B-acute lymphoblastic leukemia

BACKGROUND:

CXC chemokine receptor 4 (CXCR4) is activated by phosphorylation (pCXCR4) and is essential for the migration of hematopoietic precursors to bone marrow. CXCR4 overexpression predicts a poor prognosis in patients with acute myeloid leukemia. Data regarding the prognostic impact of CXCR4 in patients with B‐acute lymphoblastic leukemia (B‐ALL) are sparse and limited to the pediatric population.

METHODS:

The authors analyzed CXCR4 and pCXCR4 expression in 54 adults with newly diagnosed B‐ALL. CXCR4 was assessed by flow cytometry (FC) and immunohistochemistry (IHC) using an anti‐CXCR4 antibody. pCXCR4 expression was assessed using an anti‐pCXCR4 antibody.

RESULTS:

The study group included 30 men and 24 women with a median age of 42 years (range, 17‐84 years). Philadelphia chromosome was present in 19 patients. The median follow‐up was 16 months (range, 17‐84 months). Forty‐nine patients had a complete response, and 12 patients relapsed with a median relapse free survival >120 weeks. Fifteen patients (28%) died with a median survival >125 weeks. CXCR4 detected by FC and IHC was highly correlated (P < .001). CXCR4 was not associated with clinical or laboratory findings or survival. In contrast, pCXCR4 was associated with higher leukocyte count (P = .006) and serum bilirubin level (P = .03). In multivariate analysis, pCXCR4 expression (P = .027), high serum creatinine level (P < .01), presence of the Philadelphia chromosome (P = .017), and late clinical response (P < .001) were associated with worse overall survival.

CONCLUSIONS:

The current results indicated that detection of the activated form of CXCR4, pCXCR4, provides independent prognostic information in adult patients with B‐ALL. Cancer 2011;. © 2011 American Cancer Society.     

CXCR7 expression is associated with disease-free and disease-specific survival in cervical cancer patients

Background:

The CXC chemokine receptor (CXCR)7 is involved in tumour development and metastases formation. The aim of the present study was to determine protein expression of CXCR7, its putative co-receptors epidermal growth factor receptor (EGFR) and CXCR4, its predominant ligand CXCL12, their co-dependency and their association with survival in cervical cancer patients.

Methods:

CXC chemokine receptor 7, EGFR, CXCR4 and CXCL12 expression were determined immunohistochemically in 103 paraffin-embedded, cervical cancers. Subsequently, associations with patient characteristics were assessed and survival analyses were performed.

Results:

CXC chemokine receptor 7 was expressed by 43% of tumour specimens, in a large majority of cases together with either EGFR or CXCR4 (double positive), or both (triple positive). The CXCR7 expression was associated with tumour size (P=0.013), lymph node metastasis (P=0.001) and EGFR expression (P=0.009). CXC chemokine receptor 7 was independently associated with disease-free survival (hazard ratio (HR)=4.3, 95% confidence intervals (CI) 1.7–11.0, P=0.002), and strongly associated with disease-specific survival (HR=3.9, 95% CI 1.5–10.2, P=0.005).

Conclusion:

CXC chemokine receptor 7 expression predicts poor disease-free and disease-specific survival in cervical cancer patients, and might be a promising new therapeutic marker. In a large majority of cases, CXCR7 is co-expressed with CXCR4 and/or EGFR, supporting the hypothesis that these receptors assist in CXCR7 signal transduction.     

Correlation between CXCR4/CXCR7/CXCL12 chemokine axis expression and prognosis in lymph‐node‐positive lung cancer patients

The CXCR4/CXCR7/CXCL12 chemokine axis plays important roles in the migration of tumor cells during cancer development by modulating site‐specific distant metastasis including to regional lymph nodes. We investigated the correlation of these chemokine expressions to prognosis in lymph‐node‐positive non‐small‐cell lung cancer (NSCLC) patients. A total of 140 surgically resected specimens of primary site (PS) and metastatic lymph nodes (MLN) of NSCLC involving hilar and/or mediastinal lymph nodes (N1‐2) were collected. CXCR4, CXCR7 and CXCL12 expressions were evaluated. Cox regression analysis was performed to determine whether these chemokines were independent prognostic factors in N1‐2 NSCLC. High expression of CXCR4 in PS and CXCL12 in MLN was associated with poor overall survival (OS) (P = .025 and .033, respectively). Significant correlations between CXCR4 expression in PS and CXCL12 expression in MLN were observed (P = .040). There was significant difference in OS between 2 groups according to expressions of CXCR4 in PS and CXCL12 in MLN (P = .0033). Expression of CXCL12 in MLN was identified as an independent prognostic factor (HR 1.79, 95% CI 1.08‐3.04, P = .023). CXCL12 in MLN was mainly expressed by tumor cells compared with stromal cells (56% vs 25%, respectively, P < .0001). CXCR4/CXCL12 may play roles in tumor progression in MLN and is associated with poor prognosis of lymph‐node‐positive NSCLC patients.     

Chemokine receptor CXCR4 expression is correlated with VEGF expression and poor survival in soft‐tissue sarcoma

The expression of chemokine receptor CXCR4 has been associated with poor prognosis and VEGF expression in several kinds of human malignancy. We measured CXCR4 expression levels in soft‐tissue sarcoma and compared them with VEGF expression or microvessel density (MVD). We used real‐time quantitative PCR to examine the CXCR4 and VEGF expression levels in a total 176 tumors, including 24 intermediate tumors, 24 malignant round‐cell tumors (MRCTs) and 128 malignant non‐round‐cell tumors (MNRCTs). We also assessed their immunohistochemical expression of CXCR4 and VEGF, MVD and proliferative activities, as measured by the MIB‐1‐labeling index (LI). Furthermore, we evaluated their significance with respect to patient survival rates in MNRCTs, using the Cox regression model. Within the different types of tumor tissue, the expression levels of CXCR4 (p < 0.0001) and VEGF (p < 0.0001) in MNRCTs were significantly higher than those in intermediate tumors or MRCTs. Immunohistochemical expression levels of CXCR4 and VEGF were significantly correlated with their mRNA expression levels (p < 0.0001). Significant positive correlation was found between CXCR4 and VEGF expression in 112 primary MNRCTs (r = 0.434, p < 0.0001). Moreover, both univariate (p < 0.0001) and Cox multivariate analysis (p = 0.0001) revealed that overexpression of CXCR4 was an independent adverse prognostic factor, in addition to high stage according to the American Joint Committee on Cancer and a high MIB‐1‐LI. Determination of the CXCR4 expression level as a novel marker can provide useful prognostic information for patients and it could be a candidate for molecular targeting therapy in MNRCTs of soft‐tissue sarcomas. © 2008 Wiley‐Liss, Inc.     

Inhibition of CXCR7 extends survival following irradiation of brain tumours in mice and rats

Background:

In experimental models of glioblastoma multiforme (GBM), irradiation (IR) induces local expression of the chemokine CXCL12/SDF-1, which promotes tumour recurrence. The role of CXCR7, the high-affinity receptor for CXCL12, in the tumour''s response to IR has not been addressed.

Methods:

We tested CXCR7 inhibitors for their effects on tumour growth and/or animal survival post IR in three rodent GBM models. We used immunohistochemistry to determine where CXCR7 protein is expressed in the tumours and in human GBM samples. We used neurosphere formation assays with human GBM xenografts to determine whether CXCR7 is required for cancer stem cell (CSC) activity in vitro.

Results:

CXCR7 was detected on tumour cells and/or tumour-associated vasculature in the rodent models and in human GBM. In human GBM, CXCR7 expression increased with glioma grade and was spatially associated with CXCL12 and CXCL11/I-TAC. In the rodent GBM models, pharmacological inhibition of CXCR7 post IR caused tumour regression, blocked tumour recurrence, and/or substantially prolonged survival. CXCR7 expression levels on human GBM xenograft cells correlated with neurosphere-forming activity, and a CXCR7 inhibitor blocked sphere formation by sorted CSCs.

Conclusions:

These results indicate that CXCR7 inhibitors could block GBM tumour recurrence after IR, perhaps by interfering with CSCs.     

High CXC chemokine receptor 4 expression is an adverse prognostic factor in patients with clear-cell renal cell carcinoma

Background:

Aberrant CXC chemokine receptor 4 (CXCR4) expressions in malignant tissues have been reported; however, its role in kidney cancer prognosis remains unknown. The aim of this study was to determine the prognostic value of CXCR4 expression in patients with clear-cell renal cell carcinoma (ccRCC).

Methods:

The study included 225 patients with ccRCC. The cohort was split into a training set (n=125) and a validation set (n=100). CXC chemokine receptor 4 expression was analysed by immunohistochemical staining and its correlations with clinicopathologic features and prognosis were evaluated.

Results:

CXCR4-staining intensity increased gradually accompanied with disease progression from TNM stages I to IV in 225 patients with ccRCC. Moreover, high CXCR4 expression indicated reduced overall survival (OS) in the training (P<0.001) and validation (P<0.001) sets, especially for patients with early-stage (TNM stage I+II) diseases. Furthermore, CXCR4 expression was identified as an independent prognostic factor for OS, and combining TNM stage with CXCR4 expression showed a better prognostic value for OS in both sets.

Conclusions:

High CXCR4 expression, an independent adverse prognostic factor, could be combined with TNM stage to generate a predictive nomogram for clinical outcome in patients with ccRCC.     

Circulating endothelial progenitors and CXCR4-positive circulating endothelial cells are predictive markers for bevacizumab

BACKGROUND:

Bevacizumab plus chemotherapy is a standard option in the treatment of metastatic colorectal cancer (mCRC). The aim of this study was to investigate the potential of circulating endothelial cell progenitors (CEPs) and phenotypical circulating endothelial cells (CECs) as surrogate markers of clinical outcome in mCRC patients to identify responders to bevacizumab in combination with chemotherapy.

METHODS:

A total of 69 patients with measurable mCRC were enrolled in this prospective study. Whole blood samples were analyzed before initiation of treatment and on days 4 and 14. Phenotypical CECs and CEPs were then isolated and enumerated by using flow cytometry.

RESULTS:

CEP levels of less than 0.04% on day 4 were significantly associated with longer progression‐free survival (PFS) and overall survival (OS) (P < .001, P = .002, respectively) as compared with levels of 0.04% or more. In addition, CXCR4‐positive CEC levels of less than 20% at baseline were significantly associated with longer PFS and OS as compared other indicators investigated (P < .001, P = .002, respectively).

CONCLUSIONS:

Levels of CEPs on day 4 and proportion of CXCR4‐positive CECs at baseline were correlated with the prognosis of bevacizumab combination chemotherapy, suggesting that these surrogate markers may play a core role in the selection of candidates for bevacizumab treatment. Cancer 2011;. © 2011 American Cancer Society.     

Up‐regulation of interleukin‐17 expression by human papillomavirus type 16 E6 in nonsmall cell lung cancer

BACKGROUND:

Human papillomavirus (HPV) 16/18 infection is associated with nonsmoking lung cancer. In this study, the authors investigated a putative correlation between interleukin (IL)‐17 expression and HPV infection in clinical nonsmall cell lung cancer (NSCLC) tissues and examined the effects of HPV infection on a human NSCLC cell line.

METHODS:

IL‐17 expression was investigated in 79 NSCLC tumor tissues by immunohistochemistry. Growth rate, IL‐17 mRNA, and secreting protein levels were also examined in HPV 16/18 E6‐transfected H1299 human NSCLC cells.

RESULTS:

Immunohistochemical data showed that 48.1% of lung tumors had IL‐17 staining, which was significantly associated with patients' sex (P = .03), HPV infection (P = .002), and tumor stage (P = .03). Significant correlations of IL‐17 with IL‐6 (P < .001) and IL‐17 with Mcl‐1 (P < .001) expression were also observed. Cell growth rate was increased, and IL‐17/Mcl‐1 expression levels were elevated in HPV 16 E6‐transfected H1299 cells. The transfected E6 oncoproteins can significantly up‐regulate expression levels of IL‐17 and antiapoptotic protein Mcl‐1.

CONCLUSIONS:

The study suggests that HPV infection‐induced IL‐17 levels can stimulate Mcl‐1 expression through the PI3K pathway and promote lung tumor cell progression through a p53‐ and IL‐6‐independent pathway. Cancer 2010. © 2010 American Cancer Society.     

A prognostic model comprising pT stage,N status,and the chemokine receptors CXCR4 and CXCR7 powerfully predicts outcome in neoadjuvant resistant rectal cancer patients

Despite the optimization of the local treatment of advanced rectal cancer (LARC), combination of preoperative chemoradiotherapy (CRT) and surgery, approximately one third of patients will develop distant metastases. Since the chemokine receptor CXCR4 has been implicated in metastasis development and prognosis in colorectal cancer, the role of the entire axis CXCR4‐CXCL12‐CXCR7 was evaluated to identify high relapse risk rectal cancer patients. Tumor specimens of 68 LARC patients undergoing surgery after neoadjuvant‐CRT were evaluated for CXCR4, CXCR7, and CXCL12 expression through immunohistochemistry. Multivariable prognostic model was developed using classical prognostic factors along with chemokine receptor expression profiles. High CXCR4 correlated with a shorter relapse‐free survival (RFS) (p = 0.0006) and cancer specific survival (CSS) (p = 0.0004). Concomitant high CXCR4‐negative/low CXCR7 or high CXCR4‐negative/low CXCL12 significantly impaired RFS (p = 0.0003 and p = 0.0043) and CSS (p = 0.0485 and p = 0.0026). High CXCR4/N+ identified the worst prognostic category for RFS (p < 0.0001) and CSS (p = 0.0003). The optimal multivariable predictive model for RFS was a five‐variable model consisting of gender, pT stage, N status, CXCR4, and CXCR7 (AUC = 0.92, 95% CI = 0.77–0.98). The model is informative and supportive for adjuvant treatment and identifies CXCR4 as a new therapeutic target in rectal cancer.© 2013 UICC     

CXC‐chemokine/CXCR2 biological axis promotes angiogenesis in vitro and in vivo in pancreatic cancer

Angiogenesis is essential for tumor growth and metastasis. Although ELR⁺‐CXC‐chemokines and their corresponding receptor, CXC‐receptor 2 (CXCR2), are known mediators of angiogenesis, little is known about their role in pancreatic cancer (PaCa). The aim of our study was to determine the role of ELR⁺‐CXC‐chemokine/CXCR2 biological axis in promoting PaCa angiogenesis. We prospectively collected secretin‐stimulated exocrine pancreatic secretions (SSEPS) from normal individuals (NP) and PaCa patients. We showed that summed concentrations of ELR⁺‐CXC‐chemokines in SSEPS from PaCa patients were significantly higher than in those from NP (p = 0.002). We measured ELR⁺‐CXC‐chemokine levels in supernatants from multiple PaCa cell lines and confirmed that BxPC‐3, Colo‐357 and Panc‐28 had significantly higher expression compared with an immortalized human pancreatic ductal epithelial (HPDE) cell line. After confirming lack of autocrine effects of ELR⁺‐CXC‐chemokines on PaCa cells (due to absence of CXCR2 expression), we investigated paracrine effects of these chemokines on human umbilical vein endothelial cells (HUVEC). Both recombinant ELR⁺‐CXC‐chemokines and co‐culturing with BxPC‐3 significantly enhanced proliferation, invasion, and tube formation of HUVEC (p < 0.05). These biological effects were significantly inhibited by treatment with a neutralizing antibody against CXCR2 (anti‐CXCR2 Ab) (p < 0.05). Finally, anti‐CXCR2 Ab significantly reduced tumor volume (p < 0.05), Ki‐67 proliferation index (p = 0.043) and Factor VIII⁺ microvessel density (p = 0.004) in an orthotopic nude mouse PaCa model. Our results show that ELR⁺‐CXC‐chemokines promote PaCa tumor‐associated angiogenesis through CXCR2, suggesting that CXCR2 is an anti‐angiogenic target in PaCa. © 2009 UICC     

Fluorescent imaging of high‐grade bladder cancer using a specific antagonist for chemokine receptor CXCR4

We previously reported that the expression of CXC chemokine receptor‐4 (CXCR4) was upregulated in invasive bladder cancers and that the small peptide T140 was a highly sensitive antagonist for CXCR4. In this study, we identified that CXCR4 expression was induced in high‐grade superficial bladder tumors, including carcinoma in situ and invasive bladder tumors. To visualize the bladder cancer cells using urinary sediments from the patients and chemically induced mouse bladder cancer model, a novel fluorescent CXCR4 antagonist TY14003 was developed, that is a T140 derivative. TY14003 could label bladder cancer cell lines expressing CXCR4, whereas negative‐control fluorescent peptides did not label them. When labeling urinary sediments from patients with invasive bladder cancer, positive‐stained cells were identified in all patients with bladder cancer and positive urine cytology but not in controls. Although white blood cells in urine were also labeled with TY14003, they could be easily discriminated from urothelial cells by their shape and size. Finally, intravesical instillation of TY14003 into mouse bladder, using N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine (BBN)‐induced bladder cancer model, demonstrated that fluorescent signals were detected in the focal areas of bladder of all mice examined at 12 weeks of BBN drinking by confocal microscopy and fluorescent endoscopy. On the contrary, all the normal bladders were found to be negative for TY14003 staining. In conclusion, these results indicate that TY14003 is a promising diagnostic tool to visualize small or flat high‐grade superficial bladder cancer.     

Small interfering RNA‐mediated CXCR1 or CXCR2 knock‐down inhibits melanoma tumor growth and invasion

CXCR1 and CXCR2 are receptors for CXCL‐8 and are differentially expressed on melanoma and endothelial cells. In this study, we determined the functional role of these receptors in melanoma progression. We stably knock‐down the expression of CXCR1 and/or CXCR2 in A375‐SM (SM; high metastatic) human melanoma cells by short‐hairpin RNA transfection. Cell proliferation, migration, invasion, ERK phosphorlyation and cytoskeletal rearrangements were carried out in vitro. In vivo growth was evaluated using murine subcutaneous xenograft model. Our data demonstrate that knock‐down of CXCR1 and/or CXCR2 expression, inhibited melanoma cell proliferation, survival, migration and invasive potential in vitro. Moreover, we also observed inhibition of ERK phosphorylation and cytoskeltal rearrangement in SM‐shCXCR1, SM‐shCXCR2 and SM‐shCXCR1/2 cells. Furthermore, when SM‐shCXCR1 or SM‐shCXCR2 cells implanted in nude mice, tumor growth, proliferation and microvessel density was significantly inhibited as compared to SM‐control cells. In addition, we observed a significant increase in melanoma cell apoptosis in SM‐shCXCR1 and SM‐shCXCR2 tumors compared to SM‐control tumors. Together, these data demonstrate that CXCR1 and CXCR2 expression play a critical role in human melanoma tumor progression and, functional blockade of CXCR1 and CXCR2 could be potentially used for future therapeutic intervention in malignant melanoma.     

Enzalutamide and CXCR7 inhibitor combination treatment suppresses cell growth and angiogenic signaling in castration‐resistant prostate cancer models

Previous studies have shown that increased levels of chemokine receptor CXCR7 are associated with the increased invasiveness of prostate cancer cells. We now show that CXCR7 expression is upregulated in VCaP and C4‐2B cells after enzalutamide (ENZ) treatment. ENZ treatment induced apoptosis (sub‐G1) in VCaP and C4‐2B cells, and this effect was further increased after combination treatment with ENZ and CCX771, a specific CXCR7 inhibitor. The levels of p‐EGFR (Y1068), p‐AKT (T308) and VEGFR2 were reduced after ENZ and CCX771 combination treatment compared to single agent treatment. In addition, significantly greater reductions in migration were shown after combination treatment compared to those of single agents or vehicle controls, and importantly, similar reductions in the levels of secreted VEGF were also demonstrated. Orthotopic VCaP xenograft growth and subcutaneous MDA133‐4 patient‐derived xenograft (PDX) tumor growth was reduced by single agent treatment, but significantly greater suppression was observed in the combination treatment group. Although overall microvessel densities in the tumor tissues were not different among the different treatment groups, a significant reduction in large blood vessels (>100 μm²) was observed in tumors following combination treatment. Apoptotic indices in tumor tissues were significantly increased following combination treatment compared with vehicle control‐treated tumor tissues. Our results demonstrate that significant tumor suppression mediated by ENZ and CXCR7 combination treatment may be due, in part, to reductions in proangiogenic signaling and in the formation of large blood vessels in prostate cancer tumors.     

EphA2 overexpression is associated with lack of hormone receptor expression and poor outcome in endometrial cancer

BACKGROUND:

EphA2 is a tyrosine kinase receptor in the ephrin family that is implicated in oncogenesis and angiogenesis. The objective of the current investigation was to study the role of EphA2 in endometrial cancer and its relation to steroid hormone receptor expression.

METHODS:

EphA2, estrogen receptor (ER), progesterone receptor (PR), and Ki‐67 expression levels were evaluated using immunohistochemistry in 139 endometrioid endometrial carcinoma (EEC) samples and in 10 benign endometrial samples. Samples were scored by 2 investigators who were blinded to clinical outcome. The results were correlated with clinicopathologic characteristics using univariate and multivariate analysis. A P value <.05 was considered statistically significant.

RESULTS:

High expression of EphA2 was detected in 48% of EEC samples versus 10% of benign samples. EphA2 overexpression was associated significantly with high disease stage (P = .04), high tumor grade (P = .003), increased depth of myometrial invasion (P = .05), low ER expression (P = .01), low PR expression (P = .006), and high Ki‐67 expression (P = .04). Low ER and PR expression levels were associated with high tumor grade, positive lymph nodes, high Ki‐67 expression, and high EphA2 expression. On univariate analysis of all patients, high EphA2 expression was associated significantly with shorter disease‐specific survival (DSS) (P < .001). On multivariate analysis, age (P < .001), high disease stage (P = .002), and high EphA2 expression (P = .04) were independent predictors of poor DSS.

CONCLUSIONS:

EphA2 overexpression was associated with aggressive phenotypic features in EEC and was associated inversely with ER and PR expression. Thus, EphA2 may be an important therapeutic target, especially in patients with hormone receptor‐negative endometrial carcinoma. Cancer 2009. © 2009 American Cancer Society.     

The chemokine, CXCL12, is an independent predictor of poor survival in ovarian cancer

Background:

The chemokine CXCL12 and its cognate receptor, CXCR4, have been implicated in numerous tumour types where expression promotes tumour growth, angiogenesis, metastasis and suppresses tumour immunity.

Methods:

Using a tissue microarray of 289 primary ovarian cancers coupled to a comprehensive database of clinicopathological variables, the expression of CXCL12 and CXCR4 was assessed by immunohistochemistry and its impact in terms of survival and clinicopathological variables was determined.

Results:

Patients whose tumours expressed high levels of CXCL12 had significantly poorer survival (P=0.026) than patients whose tumours failed to produce this chemokine. Lack of CXCL12 expression within tumours was associated with a 51-month survival advantage for patients when compared with patients whose tumours expressed high levels of CXCL12. FIGO stage, adjuvant chemotherapy and the absence of macroscopic disease after surgery were all shown to predict prognosis independently of each other in this cohort of patients. CXCL12 was independently predictive of prognosis on multivariate analysis (P=0.016). There was no correlation between CXCL12 and any clinicopathological variable.

Conclusion:

The chemokine CXCL12 is an independent predictor of poor survival in ovarian cancer. High expression of CXCL12 was seen in only 20% of the tumours, suggesting a role for anti-CXCL12/CXCR4 therapy in the management of these patients.