Chemokine receptor CXCR4 level in primary tumors independently predicts outcome for patients with locally advanced breast cancer.

BACKGROUND: Chemokine receptor CXCR4 is a marker of metastatic disease. We found initially that CXCR4 level is a predictive marker for patients with locally advanced breast cancer (LABC). We now confirm our initial observations. METHODS: We evaluated 77 LABC patients who had neoadjuvant therapy. Specimens were taken at the time of definitive operation. CXCR4 levels were detected with Western blots. CXCR4 expression >6.6-fold over known concentration of HeLa cells was defined as high. Primary endpoints were cancer recurrence and death. Statistical analyses were Kaplan-Meier curves, log-rank test, and Cox proportional hazard model. RESULTS: Median follow-up time was 42 months; 55 patients (71%) had low CXCR4 level. The 5-year overall survival for the low and high CXCR4 group was 78% and 50%, respectively (P = .015). The 5-year disease-free survival (DFS) for the low and high CXCR4 group was 67% and 41%, respectively (P = .024). On multivariate analysis, CXCR4 overexpression (P = .003) and nodal status (P = .044) were independent predictors of overall survival; CXCR4 overexpression (P = .003) and nodal status (P = .026) were also independent predictors of DFS. CONCLUSION: We confirmed that high CXCR4 levels in cancer specimens after neoadjuvant therapy independently predict a poor outcome for patients with LABC.

Predictive value of eIF4E reduction after neoadjuvant therapy in breast cancer.

INTRODUCTION: Initiation factor 4E (eIF4E) overexpression has prognostic significance in breast cancer. Up-regulation of downstream gene products associated with high eIF4E overexpression has been linked to chemoresistance. We hypothesize patients whose tumors had eIF4E reduction after neoadjuvant chemotherapy will have lower cancer recurrence compared with those who did not. METHODS: Seventeen locally advanced breast cancer patients were accrued, and tumor specimens were obtained before and after neoadjuvant therapy. eIF4E was quantified by Western blots. Primary end-point was cancer recurrence. RESULTS: Low eIF4E was defined as < or =7.5-fold elevation and high eIF4E was >7.5-fold elevation. Of 17 patients, six tumors remained low after neoadjuvant therapy, six dropped from high to low eIF4E, and five remained high. With a median follow-up of 29 mo, four of five patients with tumors that remained high have recurred while only three of 12 patients in the low eIF4E post-therapy group have recurred (P=0.05, chi(2)). Survival analysis using the Kaplan-Meier method showed a higher rate of cancer recurrence in patients with post-treatment high eIF4E overexpression (P=0.026, log rank test). CONCLUSIONS: Breast cancer patients whose tumors had low eIF4E overexpression after neoadjuvant chemotherapy had lower cancer recurrence compared with those who did not.

Eukaryotic initiation factor 4E overexpression in triple-negative breast cancer predicts a worse outcome.

BACKGROUND: Triple-negative (estrogen receptor [ER], progesterone receptor [PR], and HER2/neu receptor negative) breast neoplasms are typically high grade and portend a higher risk for relapse. Not being amendable to ER, PR, or HER2-targeted therapy, adjuvant cytotoxic chemotherapy remains the only option. High eukaryotic Initiation Factor 4E (eIF4E) overexpression in tumor specimens is an independent predictor for relapse in breast cancer, perhaps secondary to tousled-like kinase 1B upregulation and subsequent doxorubicin resistance. In this prospective study, eIF4E elevation in triple-negative breast cancer (TNBC) specimens was studied to determine its effect on cancer outcome. METHODS: A prospective study of 103 TNBC patients was initiated. Tumor specimens were quantified for eIF4E expression using Western blots. Clinical outcomes data were collected after standardized adjuvant treatment and surveillance protocols. Primary end points were cancer recurrence and cancer-related death. The eIF4E levels in cancer specimens were quantified as x-fold over benign samples from noncancer patients. Statistical procedures performed include survival analysis by Kaplan-Meier method, log-rank test, Cox proportional hazards regression model, and the chi-square test. RESULTS: Levels of eIF4E were categorized into 3 tertiles. Among 103 patients, 36 were in the low group (< or =7.5-fold), 40 were in the intermediate group (7.5- to 15-fold), and 27 were in the high group (> or =15-fold). Patients with triple-negative neoplasms that were in the high eIF4E group had greater rates of cancer recurrence (P = .04) and cancer-related death (P = .02) than the low eIF4E group. Among patients with node-negative disease, high eIF4E overexpression in tumor specimens continues to portend a greater rate of cancer recurrence (P = .02), and a higher rate of cancer death (P = .03) than those in the low eIF4E group. CONCLUSION: TNBC patients with high eIF4E overexpression are more likely to recur and die from cancer recurrence. High eIF4E seems to be a significant prognostic marker, even in TNBC patients.

Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer.

BACKGROUND: Eukaryotic initiation factor 4E (eIF4E) is elevated in many cancers and is a prognostic indicator in breast cancer. Many pro-tumorigenic proteins are selectively translated via eIF4E, including c-Myc, cyclin D1, ornithine decarboxylase (ODC), vascular endothelial growth factor (VEGF) and Tousled-like kinase 1B (TLK1B). However, western blot analysis of these factors in human breast cancer has been limited by the availability of fresh frozen tissue and the labor-intensive nature of the multiple assays required. Our goal was to validate whether formalin-fixed, paraffin-embedded tissues arranged in a tissue microarray (TMA) format would be more efficient than the use of fresh-frozen tissue and western blot to test multiple downstream gene products. RESULTS: Breast tumor TMAs were stained immunohistochemically and quantitated using the ARIOL imaging system. In the TMAs, eIF4E levels correlated strongly with c-Myc, cyclin D1, TLK1B, VEGF, and ODC. Western blot comparisons of eIF4E vs. TLK1B were consistent with the immunohistochemical results. Consistent with our previous western blot results, eIF4E did not correlate with node status, ER, PR, or HER-2/neu. CONCLUSION: We conclude that the TMA technique yields similar results as the western blot technique and can be more efficient and thorough in the evaluation of several products downstream of eIF4E.

A prospective trial on initiation factor 4E (eIF4E) overexpression and cancer recurrence in node-negative breast cancer.

BACKGROUND: Eukaryotic Initiation Factor 4E (eIF4E) plays a crucial role in translation control. High eIF4E increase in tumor specimens independently predicted recurrence by multivariate analysis. This prospective trial of node-negative only breast cancer patients was initiated to test the hypothesis that high eIF4E increase predicts cancer recurrence and death, independent of nodal status. METHODS: The trial was powered to detect a 2.4-fold increase in relative risk for cancer recurrence in 240 node-negative patients on the basis of high versus low eIF4E increase in tumor specimens (type I error = .05, statistical power = .08). eIF4E level was quantified by using Western blot test. Treatment and surveillance regimens were standardized. Primary endpoints were cancer recurrence and cancer-related death. RESULTS: Of the 242 patients accrued, 112 were in the low eIF4E group (<7.5-fold), 82 were in the intermediate eIF4E group (7.5- to 15-fold), and 48 were in the high eIF4E group (>15-fold). Patients in the high eIF4E group had a statistically significant higher rate of cancer recurrence and cancer-related death (P = .0001 and P < or = .0001, log rank test). The relative risk for cancer recurrence was 2.2-fold higher in the high eIF4E group (P = .001, Cox model), and 3.7-fold higher for cancer-related death (P = .0009). CONCLUSIONS: In node-negative breast cancer, high eIF4E increase predicted a higher rate of cancer recurrence and death. High eIF4E patients had a >2-fold increase in relative risk for cancer recurrence and nearly a 4-fold increase in relative risk for death. This supports our hypothesis that high eIF4E is an independent predictor for breast cancer outcome independent of nodal status.

eIF4E-targeted suicide gene therapy in a minimal residual mouse model for metastatic soft-tissue head and neck squamous cell carcinoma improves disease-free survival.

BACKGROUND: Translation initiation factor eIF4E unwinds long 5'-untranslated regions of certain tightly regulated mRNAs and, thereby, facilitates their translation into proteins. eIF4E has been shown to be overexpressed in a majority of solid tumors, including head and neck cancers. To exploit this dysregulation, a long 5'-untranslated region was spliced upstream of a thymidine kinase (Tk) gene to enhance translation of this "suicide" gene within cells overexpressing eIF4E. We investigated the efficacy of therapy with an adenovirus incorporating this novel suicide gene (Ad-HSV-UTk) following cytoreductive tumor surgery in improving disease-free and overall survival in a mouse soft-tissue metastasis model for head and neck squamous cell carcinoma. MATERIALS AND METHODS: SCC-7 (orally-derived mouse SCCa) cells were treated with Ad-HSV-Tk, Ad-HSV-UTk, Ad-null, or saline and characterized for eIF4E and Tk levels by Western blot analysis. Cytotoxicities for cells treated with Ad-HSV-Tk, Ad-HSV-UTk, or Ad-null were quantified by MTS assay. Mice bearing SCC-7-induced tumors received cytoreduction followed by Ad-HSV-UTk + ganciclovir (GCV) or control treatment and were followed for disease-free and overall survival. RESULTS: SCC-7 cells showed uniformly high levels of eIF4E but elevated Tk for Ad-HSV-Tk- and Ad-HSV-UTk-treated cells over Ad-null-treated cells. Cytotoxicities for Ad-HSV-Tk- and Ad-HSV-UTk-treated cells were, correspondingly, observed to be 100-fold more sensitive than Ad-null-treated cells to GCV treatment. Cytoreduced mice receiving Ad-HSV-UTk + GCV treatment showed significantly longer disease-free survival (P = 0.0045) than control arm mice. CONCLUSIONS: Ad-HSV-UTk suicide gene therapy prolonged disease-free survival in a mouse minimal residual soft-tissue head and neck squamous cell carcinoma metastasis model.

Correlation of TLK1B in elevation and recurrence in doxorubicin-treated breast cancer patients with high eIF4E overexpression.

BACKGROUND: Tousled-like kinase 1B (TLK1B), a mammalian threonine kinase, facilitates the repair of DNA breaks. Eukaryotic initiation factor 4E (eIF4E) overexpression leads to the upregulation of TLK1B. Doxorubicin, commonly used in the adjuvant setting for breast cancer, causes DNA breaks. We hypothesized that the degree of TLK1B elevation is correlated with eIF4E overexpression and translates clinically to an increased risk for recurrence in breast cancer patients treated with doxorubicin-based adjuvant chemotherapy. STUDY DESIGN: We prospectively accrued 152 patients with stage I to III breast cancer treated with a doxorubicin-based chemotherapy in an adjuvant setting. Standardized treatment and surveillance protocols were used. eIF4E and TLK1B protein levels were quantified using Western blots, and patients were divided into tertiles based on previously reported stratification of eIF4E and TLK1B levels. Primary end points were cancer recurrence and death. Statistical analysis included Spearman's correlation, Kaplan-Meier survival analysis, log rank test, and the Cox proportional hazard model. RESULTS: The degree of TLK1B overexpression was highly correlated with the degree of eIF4E elevation (r=0.25, p=0.0025, Spearman rank correlation). Patients whose tumors were in the highest tertile for eIF4E overexpression had a higher risk for cancer recurrence and cancer death (p=0.015 and 0.049, respectively, log rank test). After adjusting for T-stage, nodal status, age, and estrogen receptor and progesterone receptor status, patients with tumors in the highest tertile of TLK1B overexpression treated with doxorubicin were 1.7-fold more likely to suffer recurrence than those in the low TLK1B group treated similarly (p=0.0078, CI, 1.17 to 2.75, Cox model). CONCLUSIONS: TLK1B overexpression was highly correlated with the level of eIF4E elevation. High TLK1B in cancer specimens was associated with a higher risk for cancer recurrence in patients treated with doxorubicin-based adjuvant chemotherapy.

High eIF4E, VEGF, and microvessel density in stage I to III breast cancer.

OBJECTIVE: In a prospective trial, to determine if eIF4E overexpression in breast cancer specimens is correlated with VEGF elevation, increased tumor microvessel density (MVD) counts, and a worse clinical outcome irrespective of nodal status. SUMMARY AND BACKGROUND DATA: In vitro, the overexpression of eukaryotic initiation factor 4E (eIF4E) up-regulates the translation of mRNAs with long 5'-untranslated regions (5'-UTRs). One such gene product is the vascular endothelial growth factor (VEGF). METHODS: A total of 114 stage I to III breast cancer patients were prospectively accrued and followed with a standardized clinical surveillance protocol. Cancer specimens were quantified for eIF4E, VEGF, and MVD. Outcome endpoints were cancer recurrence and cancer-related death. RESULTS: eIF4E overexpression was found in all cancer specimens (mean +/- SD, 12.5 +/- 7.6-fold). Increasing eIF4E overexpression correlated with increasing VEGF elevation (r = 0.24, P = 0.01, Spearman's coefficient), and increasing MVD counts (r = 0.35, P < 0.0002). Patients whose tumor had high eIF4E overexpression had shorter disease-free survival (P = 0.004, log-rank test) and higher cancer-related deaths (P = 0.002) than patients whose tumors had low eIF4E overexpression. Patients with high eIF4E had a hazard ratio for cancer recurrence and cancer-related death of 1.8 and 2.1 times that of patients with low eIF4E (respectively, P = 0.009 and P = 0.002, Cox proportional hazard model). CONCLUSIONS: In breast cancer patients, increasing eIF4E overexpression in the cancer specimens correlates with higher VEGF levels and MVD counts. Patients whose tumors had high eIF4E overexpression had a worse clinical outcome, independent of nodal status. Thus, eIF4E overexpression in breast cancer appears to predict increased tumor vascularity and perhaps cancer dissemination by hematogenous means.

A prospective trial on initiation factor 4E (eIF4E) overexpression and cancer recurrence in node-positive breast cancer.

OBJECTIVE: A previous study of patients with stage I to III breast cancer showed that those patients whose tumors were in the highest tertile of eIF4E overexpression experienced a higher risk for recurrence. This study was designed to determine whether high eIF4E overexpression predicts cancer recurrence independent of nodal status by specifically targeting patients with node-positive disease. METHODS: The prospective trial was designed to accrue 168 patients with node-positive breast cancer to detect a 2.5-fold increase in risk for recurrence. eIF4E level was quantified by Western blots as x-fold elevated compared with breast tissues from noncancer patients. End points measured were disease recurrence and cancer-related death. Statistical analyses performed include survival analysis by the Kaplan-Meier method, log-rank test, and Cox proportional hazard model. RESULTS: One hundred seventy-four patients with node-positive breast cancer were accrued. All patients fulfilled study inclusion and exclusion criteria, treatment protocol, and surveillance requirements, with a compliance rate >95%. The mean eIF4E elevation was 11.0 +/- 7.0-fold (range, 1.4-34.3-fold). Based on previously published data, tertile distribution was as follow: 1) lowest tertile (<7.5-fold) = 67 patients, 2) intermediate tertile (7.5-14-fold) = 54 patients, and 3) highest tertile (>14-fold) = 53 patients. At a median follow up of 32 months, patients with the highest tertile had a statistically significant higher cancer recurrence rate (log-rank test, P = 0.002) and cancer-related death rate (P = 0.036) than the lowest group. Relative risk calculations demonstrated that high eIF4E patients had a 2.4-fold increase in relative risk increase for cancer recurrence (95% confidence interval, 1.2-4.1; P = 0.01). CONCLUSIONS: In this prospective study designed to specifically address risk for recurrence in patients with node-positive breast cancer, the patients whose tumors were in the highest tertile of eIF4E overexpression had a 2.4-fold increase in relative risk for cancer recurrence. Therefore, eIF4E overexpression appears to be an independent predictor of a worse outcome in patients with breast cancer independent of nodal status.

Cystatin m: a novel candidate tumor suppressor gene for breast cancer.

The contribution of pericellular proteolysis to tumor progression is well documented. To better understand protease biology and facilitate clinical translation, specific proteolytic systems need to be better defined. In particular, the precise role of endogenous protease inhibitors still needs to be deciphered. We reported previously that cystatin M, a potent endogenous inhibitor of lysosomal cysteine proteases, significantly suppressed in vitro cell proliferation, migration, and Matrigel invasion. Here, we show that scid mice orthotopically implanted with breast cancer cells expressing cystatin M show significantly delayed primary tumor growth and lower metastatic burden in the lungs and liver when compared with mice implanted with mock controls. The incidence of metastasis, however, appeared to be unaltered between the cystatin M group and the control group. Experimental metastasis assays suggest that cystatin M suppressed tumor cell proliferation at the secondary site. By using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction, we found consistent expression of cystatin M in normal human breast epithelial cells, whereas expression was decreased by 86% in invasive ductal carcinoma (IDC) cells of stage I to IV patients. Complete loss of expression of cystatin M was observed in two of three IDCs from stage IV patients. Immunohistochemical studies confirmed that expression of cystatin M in IDCs was partially or completely lost. We propose cystatin M as a novel candidate tumor suppressor gene for breast cancer.

TLK1B is elevated with eIF4E overexpression in breast cancer.

INTRODUCTION: The overexpression of eukaryotic initiation factor 4E (eIF4E), a critical component of the "RNA helicase" necessary for the initiation of protein synthesis of mRNAs with long 5' prime untranslated regions (5'UTRs), can result in malignant transformation. In a prospective study on breast cancer outcome of women with stage I to III disease, eIF4E overexpression was an independent predictor of cancer recurrence (RR = 7.3, CI = 1.58-33.9). Dysregulation of Tousled-like kinase 1B (TLK1B), a threonine kinase with a highly conserved gene sequence, has been linked to defects in cell division and DNA replication. In cell lines, TLK1B overexpression has been recently associated with resistance to radiation. The 5'UTR of TLK1B is long (1088 nt) and the structure is complex. Our hypothesis is that TLK1B elevation is correlated with the overexpression of eIF4E in human breast carcinoma. MATERIAL AND METHODS: Eighty-seven patients with invasive breast cancer and 11 patients with benign breast disease were accrued prospectively. Clinical data collected include age, race, stage, grade of tumor, ER, and PR status. TLK1B and eIF4E levels were quantified by Western blot analysis. Statistical analysis was performed using Spearman correlation, paired and unpaired t test, and multivariate analysis. RESULTS: In the 87 cancer specimens from patients with breast carcinoma, eIF4E level was elevated by a mean of 9.5-fold (range = 1.8-48.4), and TLK1B was elevated by a mean of 9.4-fold (range = 1.0-58.0) when compared to the 11 specimens from noncancer patients. Multivariate analysis performed demonstrates the degree of eIF4E overexpression is independent of age, race, tumor grade, and ER or PR status of the tumor. Similarly, the degree of TLK1B elevation is independent of age, tumor grade, and ER or PR status of the tumor. Using the Spearman correlation, the degree of TLK1B elevation was strongly correlated with the degree of eIF4E overexpression (r = 0.39, P = 0.001). CONCLUSIONS: Both eIF4E and TLK1B are elevated in breast cancer specimens but not in benign breast specimens from noncancer patients. The degree of TLK1B elevation is correlated with the degree of IF4E overexpression. Both eIF4E and TLK1B overexpression are independent of tumor grade, tumor stage, and ER and PR status.