Debra Hawes

Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype.

Purpose: The presence of disseminated tumor cells (DTC) in the bone marrow of breast cancer patients is an acknowledged independent prognostic factor. The biological metastatic potential of these cells has not yet been shown. The presence of putative breast cancer stem cells is shown both in primary tumors and distant metastases. These cells with a CD44+CD24−/low phenotype represent a minor population in primary breast cancer and are associated with self-renewal and tumorigenic potential. Recognizing the potential effect of prevalence of putative stem cells among DTC, we evaluated the bone marrow DTC. Experimental Design: We employed the double/triple-staining immunohistochemistry protocol and modified the established bone marrow cytokeratin (CK) staining protocol by adding steps for additional antigens, CD44 and/or CD24. We evaluated 50 bone marrow specimens, previously categorized as CK+ from early breast cancer patients. CK+ cells were examined for CD44 and CD24 expression by light microscopy, fluorescence microscopy, and spectral imaging. Results: We detected the putative stem cell–like phenotype in all CK+ specimens. The mean prevalence of putative stem/progenitor cells was 72% and median prevalence was 65% (range, 33-100%) among the overall DTC per patient, compared with primary tumors where this phenotype is reported in <10% of cells. Conclusions: This is the first evidence of the existence of the putative stem-like phenotype within the DTC in bone marrow in early breast cancer patients. All patients had a putative stem cell phenotype among the DTC and most individual DTC showed such phenotype. Future molecular characterization of these cells is warranted.

Association of Occult Metastases in Sentinel Lymph Nodes and Bone Marrow With Survival Among Women With Early-Stage Invasive Breast Cancer

CONTEXT Immunochemical staining of sentinel lymph nodes (SLNs) and bone marrow identifies breast cancer metastases not seen with routine pathological or clinical examination. OBJECTIVE To determine the association between survival and metastases detected by immunochemical staining of SLNs and bone marrow specimens from patients with early-stage breast cancer. DESIGN, SETTING, AND PATIENTS From May 1999 to May 2003, 126 sites in the American College of Surgeons Oncology Group Z0010 trial enrolled women with clinical T1 to T2N0M0 invasive breast carcinoma in a prospective observational study. INTERVENTIONS All 5210 patients underwent breast-conserving surgery and SLN dissection. Bone marrow aspiration at the time of operation was initially optional and subsequently mandatory (March 2001). Sentinel lymph node specimens (hematoxylin-eosin negative) and bone marrow specimens were sent to a central laboratory for immunochemical staining; treating clinicians were blinded to results. MAIN OUTCOME MEASURES Overall survival (primary end point) and disease-free survival (a secondary end point). RESULTS Of 5119 SLN specimens (98.3%), 3904 (76.3%) were tumor-negative by hematoxylin-eosin staining. Of 3326 SLN specimens examined by immunohistochemistry, 349 (10.5%) were positive for tumor. Of 3413 bone marrow specimens examined by immunocytochemistry, 104 (3.0%) were positive for tumors. At a median follow-up of 6.3 years (through April 2010), 435 patients had died and 376 had disease recurrence. Immunohistochemical evidence of SLN metastases was not significantly associated with overall survival (5-year rates: 95.7%; 95% confidence interval [CI], 95.0%-96.5% for immunohistochemical negative and 95.1%; 95% CI, 92.7%-97.5% for immunohistochemical positive disease; P = .64; unadjusted hazard ratio [HR], 0.90; 95% CI, 0.59-1.39; P = .64). Bone marrow metastases were associated with decreased overall survival (unadjusted HR for mortality, 1.94; 95% CI, 1.02-3.67; P = .04), but neither immunohistochemical evidence of tumor in SLNs (adjusted HR, 0.88; 95% CI, 0.45-1.71; P = .70) nor immunocytochemical evidence of tumor in bone marrow (adjusted HR, 1.83; 95% CI, 0.79-4.26; P = .15) was statistically significant on multivariable analysis. CONCLUSION Among women receiving breast-conserving therapy and SLN dissection, immunohistochemical evidence of SLN metastasis was not associated with overall survival over a median of 6.3 years, whereas occult bone marrow metastasis, although rare, was associated with decreased survival. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00003854.

Expression of Stress Response Protein Grp78 Is Associated with the Development of Castration-Resistant Prostate Cancer

Background: Induction of molecular chaperone Grp78 (78-kDa glucose-regulated protein) occurs in stress conditions that often characterize tumor microenvironments. We investigated the role of Grp78 in prostate cancer progression and the development of castration resistance, where cancer cells continue to survive despite the stress of an androgen-starved environment. Experimental Design: Immunohistochemistry was done to examine Grp78 expression in 219 prostate cancers from patients with pathologic stage T3N0M0 disease [androgen ablation naive (untreated) and androgen ablation exposed (treated)] and castration-resistant prostate cancer. Classification of tumors was based on intensity of Grp78 cytoplasmic immunoreactivity and percentage of immunoreactive tumor cells. The associations of Grp78 expression with prostate cancer recurrence (clinical and/or serum prostate-specific antigen) and survival were examined in the untreated stage T3N0M0 group. Grp78 expression was also analyzed in the androgen-dependent LNCaP and castration-resistant C42B cell lines. Results: The percentage of tumor cells expressing Grp78 was strongly associated with castration-resistant status (P = 0.005). Increased Grp78 expression was consistently associated with greater risk of prostate cancer recurrence and worse overall survival in patients who had not undergone prior hormonal manipulation. Grp78 expression was also increased in the castration-resistant LNCaP-derived cell line C42B and in LNCaP cells grown in androgen-deprived conditions compared with LNCaP cells grown in androgen-rich media. Conclusion: Our findings show that up-regulation of Grp78 is associated with the development of castration resistance, possibly in part by augmenting cell survival as previously suggested, and may serve as an important prognostic indicator of recurrence in a subset of patients with T3N0M0 disease.

Detection of Occult Lymph Node Metastases in Locally Advanced Node-Negative Prostate Cancer

PURPOSE The purpose of this study was to determine the incidence and clinical significance of occult metastases in the lymph nodes of patients with prostate cancer originally considered node negative by routine histologic evaluation. METHODS Two hundred seventy four patients with pT3 prostate carcinoma treated by radical prostatectomy and bilateral lymph node dissection were included in this study. One hundred eighty patients were staged node negative (N0), while 94 patients were lymph node positive (N+), based on routine histologic evaluation. All lymph nodes from the 180 N0 patients were evaluated for occult metastases by immunohistochemistry using antibodies to cytokeratins and, if positive, prostate-specific antigen. Recurrence and overall survival were compared among patients with occult tumor cells (OLN+), with patients whose lymph nodes remained negative (OLN-), and with the 94 N+ patients. RESULTS A total of 3,914 lymph nodes were evaluated from 180 N0 patients (average, 21.7 lymph nodes per patient). Occult tumor cells were found in 24 of 180 patients (13.3%). The presence of OLN+ was significantly associated with increased recurrence and decreased survival compared with OLN- patients (P < .001 and P = .019, respectively; relative risk of recurrence, 2.27; relative risk of death 2.07, respectively). The presence of occult lymph node metastases was an independent predictor of recurrence and death in a multivariable analysis. The outcome for patients with OLN+ disease was similar to that for patients with N+ disease. CONCLUSION The detection of occult lymph node metastases in patients with pT3N0 prostate cancer identifies those with significantly increased risk of prostate cancer recurrence and death.

Expression and Significance of Vascular Endothelial Growth Factor Receptor 2 in Bladder Cancer

PURPOSE Vascular endothelial growth factor has a critical role in maintaining tumor microvasculature and, as such, is an attractive target for anti-angiogenic therapy. Aberrant expression of VEGF receptors, especially VEGFR2, on epithelial tumor cells allows VEGF to stimulate growth and migration of tumor cells in an autocrine and/or paracrine manner. Therefore, we studied the expression of VEGF and VEGFR2 in bladder cancer, and the relationship to disease characteristics. MATERIALS AND METHODS Expression of VEGF and VEGFR2 was studied in a cohort of 72 patients with transitional cell cancer of the bladder. Tumor tissues from all patients were analyzed by immunohistochemistry and examined by a pathologist blinded to patient outcome. Patient demographics and disease outcome were correlated with expression of these markers. Bladder cancer cell lines that express VEGFR2 were studied in vitro and in vivo to establish the significance of VEGF/VEGFR2 signaling. RESULTS Expression of VEGF and VEGFR2 was observed in 58% and 50% of urothelial tumor cells, respectively. VEGF expression failed to correlate with clinical variables. However, VEGFR2 expression correlated with disease stage (coefficient 0.23, p = 0.05). In addition, VEGFR2 expression increased with tumor invasion into the muscle (p <0.01). Experiments with VEGFR2 positive bladder cancer cell lines in vitro demonstrated increased invasion in response to VEGF. In addition, VEGF inhibition augmented the effect of docetaxel in a murine xenograft model of bladder cancer with a significant inhibition in proliferative index and microvascular density, and induction of apoptosis. CONCLUSIONS Increased VEGFR2 expression correlates with several features that predict progression of urothelial cancer, including disease stage and invasive phenotype. VEGF targeted therapy may enhance the efficacy of standard therapy for bladder cancer.

Preferential Induction of EphB4 over EphB2 and Its Implication in Colorectal Cancer Progression

The receptor tyrosine kinase EphB2 is expressed by colon progenitor cells; however, only 39% of colorectal tumors express EphB2 and expression levels decline with disease progression. Conversely, EphB4 is absent in normal colon but is expressed in all 102 colorectal cancer specimens analyzed, and its expression level correlates with higher tumor stage and grade. Both EphB4 and EphB2 are regulated by the Wnt pathway, the activation of which is critically required for the progression of colorectal cancer. Differential usage of transcriptional coactivator cyclic AMP-responsive element binding protein-binding protein (CBP) over p300 by the Wnt/beta-catenin pathway is known to suppress differentiation and increase proliferation. We show that the beta-catenin-CBP complex induces EphB4 and represses EphB2, in contrast to the beta-catenin-p300 complex. Gain of EphB4 provides survival advantage to tumor cells and resistance to innate tumor necrosis factor-related apoptosis-inducing ligand-mediated cell death. Knockdown of EphB4 inhibits tumor growth and metastases. Our work is the first to show that EphB4 is preferentially induced in colorectal cancer, in contrast to EphB2, whereby tumor cells acquire a survival advantage.

Dense breast stromal tissue shows greatly increased concentration of breast epithelium but no increase in its proliferative activity

IntroductionIncreased mammographic density is a strong risk factor for breast cancer. The reasons for this are not clear; two obvious possibilities are increased epithelial cell proliferation in mammographically dense areas and increased breast epithelium in women with mammographically dense breasts. We addressed this question by studying the number of epithelial cells in terminal duct lobular units (TDLUs) and in ducts, and their proliferation rates, as they related to local breast densities defined histologically within individual women.MethodWe studied deep breast tissue away from subcutaneous fat obtained from 12 healthy women undergoing reduction mammoplasty. A slide from each specimen was stained with the cell-proliferation marker MIB1. Each slide was divided into (sets of) areas of low, medium and high density of connective tissue (CT; highly correlated with mammographic densities). Within each of the areas, the numbers of epithelial cells in TDLUs and ducts, and the numbers MIB1 positive, were counted.ResultsThe relative concentration (RC) of epithelial cells in high compared with low CT density areas was 12.3 (95% confidence interval (CI) 10.9 to 13.8) in TDLUs and 34.1 (95% CI 26.9 to 43.2) in ducts. There was a much smaller difference between medium and low CT density areas: RC = 1.4 (95% CI 1.2 to 1.6) in TDLUs and 1.9 (95% CI 1.5 to 2.3) in ducts. The relative mitotic rate (RMR; MIB1 positive) of epithelial cells in high compared with low CT density areas was 0.59 (95% CI 0.53 to 0.66) in TDLUs and 0.65 (95% CI 0.53 to 0.79) in ducts; the figures for the comparison of medium with low CT density areas were 0.58 (95% CI 0.48 to 0.70) in TDLUs and 0.66 (95% CI 0.44 to 0.97) in ducts.ConclusionBreast epithelial cells are overwhelmingly concentrated in high CT density areas. Their proliferation rate in areas of high and medium CT density is lower than that in low CT density areas. The increased breast cancer risk associated with increased mammographic densities may simply be a reflection of increased epithelial cell numbers. Why epithelium is concentrated in high CT density areas remains to be explained.

Occult Metastases in Lymph Nodes Predict Survival in Resectable Non–Small-Cell Lung Cancer: Report of the ACOSOG Z0040 Trial

PURPOSE The survival of patients with non-small-cell lung cancer (NSCLC), even when resectable, remains poor. Several small studies suggest that occult metastases (OMs) in pleura, bone marrow (BM), or lymph nodes (LNs) are present in early-stage NSCLC and are associated with a poor outcome. We investigated the prevalence of OMs in resectable NSCLC and their relationship with survival. PATIENTS AND METHODS Eligible patients had previously untreated, potentially resectable NSCLC. Saline lavage of the pleural space, performed before and after pulmonary resection, was examined cytologically. Rib BM and all histologically negative LNs (N0) were examined for OM, diagnosed by cytokeratin immunohistochemistry (IHC). Survival probabilities were estimated using the Kaplan-Meier method. The log-rank test and Cox proportional hazards regression model were used to compare survival of groups of patients. P < .05 was considered significant. RESULTS From July 1999 to March 2004, 1,047 eligible patients (538 men and 509 women; median age, 67.2 years) were entered onto the study, of whom 50% had adenocarcinoma and 66% had stage I NSCLC. Pleural lavage was cytologically positive in only 29 patients. OMs were identified in 66 (8.0%) of 821 BM specimens and 130 (22.4%) of 580 LN specimens. In univariate and multivariable analyses OMs in LN but not BM were associated with significantly worse disease-free survival (hazard ratio [HR], 1.50; P = .031) and overall survival (HR, 1.58; P = .009). CONCLUSION In early-stage NSCLC, LN OMs detected by IHC identify patients with a worse prognosis. Future clinical trials should test the role of IHC in identifying patients for adjuvant therapy.

Detection of occult metastases in lung carcinomas: Progress and implications for lung cancer staging

The ability to detect occult regional and systemic metastases in patients with operable lung carcinoma could have a significant impact on the management of the disease. Here, we review the literature, including studies from our own laboratory, regarding the clinical significance of the presence of occult metastases in patients with lung cancer. The accumulated evidence strongly suggests that the detection of occult regional and systemic metastases is an important predictor of disease progression. The use of this method should be considered in the future design of lung cancer clinical trials, at the very least. The detection of occult metastases should have an impact on lung cancer mangement; to reflect this, we propose a change in the TNM staging system to indicate the presence or absence of occult regional (lymph node) and systemic (bone marrow) metastases. The proposed change is TNnMm, where n and m are occult nodal and bone marrow metastases status. J. Surg. Oncol. 1998;69:265–274.

Anti-Angiogenic Activity of Contortrostatin, a Disintegrin from Agkistrodon Contortrix Contortrix Snake Venom

Previous work in our laboratory has shown that contortrostatin (CN), a disintegrin from southern copperhead snake venom, possess anti-angiogenic activity. In the present study we further examined the anti-angiogenic activity of CN, focusing on the mechanisms of CN inhibition of angiogenesis. CN inhibited migration and invasion, and significantly altered Matrigel-induced tube formation of human umbilical vein endothelial cells (HUVEC), but did not affect cell viability, or MMP-2 and MMP-9 activity. Immunocytochemistry of HUVEC revealed that CN disrupted actin cytoskeleton and altered VE-cadherin distribution at cell–cell contacts. CN downregulated focal adhesion kinase (FAK) and paxillin tyrosine phosphorylation in adherent HUVEC. There was also significant inhibition of angiogenesis in vivo by CN as assessed by implanting Matrigel plugs in C57 mice and measuring ingrowth of blood vessels using either factor VIII staining or hemoglobin determination. In conclusion, the present findings confirm our earlier studies and demonstrate conclusively that CN possess strong anti-angiogenic activity in vitro and in vivo.