Cyclooxygenase-2 expression in non-metastatic triple-negative breast cancer patients.

Triple-negative breast cancer (TNBC) is characterised by lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER)2/neu gene amplification. TNBC patients typically present at a younger age, with a larger average tumor size, higher grade and higher rates of lymph node positivity compared to patients with ER/PR-positive tumors. Cyclooxygenase (COX)-2 regulates the production of prostaglandins and is overexpressed in a variety of solid tumors. In breast cancer, the overexpression of COX-2 is associated with indicators of poor prognosis, such as lymph node metastasis, poor differentiation and large tumor size. Since both TNBC status and COX-2 overexpression are known poor prognostic markers in primary breast cancer, we hypothesized that the COX-2 protein is overexpressed in the primary tumors of TNBC patients. The purpose of this study was to determine whether there exists an association between TNBC status and COX-2 protein overexpression in primary breast cancer. We prospectively evaluated COX-2 expression levels in primary tumor samples obtained from 125 patients with stage I-III breast cancer treated between February, 2005 and October, 2007. Information on clinicopathological factors was obtained from a prospective database. Baseline tumor characteristics and patient demographics were compared between TNBC and non-TNBC patients using the Chi-square and Fisher's exact tests. In total, 60.8% of the patients were classified as having ER-positive tumors, 51.2% were PR-positive, 14.4% had HER-2/neu amplification and 28.0% were classified as TNBC. COX-2 overexpression was found in 33.0% of the patients. TNBC was associated with COX-2 overexpression (P=0.009), PR expression (P=0.048) and high tumor grade (P=0.001). After adjusting for age, menopausal status, body mass index (BMI), lymph node status and neoadjuvant chemotherapy (NACT), TNBC was an independent predictor of COX-2 overexpression (P=0.01). In conclusion, the association between TNBC and COX-2 overexpression in operable breast cancer supports further investigation into COX-2-targeted therapy for patients with TNBC.

Aldehyde dehydrogenase1 immunohistochemical staining in primary breast cancer cells independently predicted overall survival but did not correlate with the presence of circulating or disseminated tumors cells.

PURPOSE: We hypothesized that aldehyde dehydrogenase 1 (ALDH1) staining in breast cancer tumor cells might be a simple surrogate for the presence of circulating tumor cells (CTCs) or disseminated tumor cells (DTCs). EXPERIMENTAL DESIGN: Whole tissue primary tumor sections from 121 patients enrolled in a clinical trial assessing CTCs and DTCs at the time of surgery were stained for ALDH1 and scored by a dedicated breast pathologist blinded to outcome. Clinical data was extracted and staining was correlated to clinical variables and outcome by Fisher's exact test, the Log rank test and Cox proportional hazards regression analysis respectively. P < 0.05 was considered significant. RESULTS: ALDH1 staining in tumor cells was present in 12% of cases (15/121). In univariate analysis, ALDH1 tumor staining predicted worse overall survival (71% vs. 91% at 5 years P = 0.0074) and was an independent predictor on multivariable analysis of worse overall survival, (HR 4.93) after adjusting for stage, ER, grade, LVI, age and neoadjuvant chemotherapy (P = 0.04). ALDH1 was significantly associated with estrogen receptor (ER) negative (P value = 0.029) primary tumors but not the presence of CTCs or DTCs by multivariate logistic regression. Positive ALDH staining in non-tumor cells of any pattern or morphology was common but did not correlate with CTCs or DTCs, other clinical variables, or outcome. CONCLUSION: ALDH1 tumor staining was associated with ER -negative breast cancer and was an independent predictor of OS. However, it did not correlate to putative cancer stem cell surrogates CTCs and/or DTCs.

Bupropion-SR for smoking reduction and cessation in alcohol-dependent outpatients: a naturalistic, open-label study.

BACKGROUND: We sought to determine whether sustained-release bupropion (bupropion-SR) reduces smoking and promotes smoking cessation among alcohol-dependent (AD) smokers while they are in early recovery from alcohol. METHODS: We conducted an open-label, naturalistic study among AD smokers enrolled in an outpatient treatment program. The treatment group (n=58) was offered bupropion-SR and brief smoking cessation counseling. A control group (n=57) was matched to the treatment group by age, sex, ethnicity, cigarette use, and years of alcohol dependence. The controls received no smoking cessation intervention. We collected tobacco and alcohol abstinence data for 6 months after enrollment. RESULTS: Participants in the treatment group were more likely to abstain from smoking than controls, at any of the followup time points. The treatment group smoked less cigarettes per day (CPD) at baseline, 30 days and 180 days post-baseline, compared to controls. These findings persisted after adjusting for possible covariates. CONCLUSION: Bupropion-SR may be helpful to quit or reduce smoking for recently abstinent AD individuals.

Discordance in HER2 gene amplification in circulating and disseminated tumor cells in patients with operable breast cancer.

Human epidermal growth factor receptor 2 (HER2) gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might be useful for modifying Herceptin therapy in breast cancer. In the process of investigating the utility of a microfluidic platform for detecting HER2 gene amplification in these cells, we observed novel results on discordance of HER2 status. Peripheral blood (8.5 mL) and bone marrow (BM) (7.5-10 mL) were collected prospectively from patients with clinical stages I-IV breast cancer. Mononuclear cells were recovered, stained with cytokeratin (CK), CD45, and DAPI, and processed through microfluidic channels for fluorescence in situ hybridization (FISH). A ratio of HER2:CEP17 >2 in any CK+/CD45 or CK-/CD45 cell was regarded as positive for HER2 gene amplification. Peripheral blood from 95 patients and BM from 78 patients were studied. We found CK+/CD45-/DAPI+ CTCs in 27.3% of patients. We evaluated HER2 gene amplification by FISH in 88 blood and 78 BM specimens and found HER2+ CTCs in 1 of 9 (11.1%) and HER2+ DTCs (27.2%) in 3 of 11 patients with HER2+ primary tumor. Among patients with a HER2- primary tumor, 5 of 79 had HER2+ CTCs (6.3%) and 14 of 67 had HER2+ DTCs (20.8%). The overall rate of discordance in HER2 status was 15% between primary tumor and CTCs and 28.2% between primary tumor and DTCs. HER2 was amplified in CTCs and DTCs in a portion of both HER2+ and HER2- primary tumors. HER2 discordance was more frequent for DTCs. The clinical implications of evaluating HER2 status in CTCs and DTCs in breast cancer needs to be established in prospective clinical trials. The cell enrichment and extraction microfluidic technology provides a sensitive platform for evaluation of HER2 gene amplification in CTCs and DTCs.

Circulating tumour cells in non-metastatic breast cancer: a prospective study.

BACKGROUND: The identification of circulating tumour cells correlate with poor prognosis in metastatic breast cancer, but there are few data describing the importance of circulating tumour cells in patients with non-metastatic disease. Our aim was to establish if circulating tumour cells predicted worse outcome in patients with non-metastatic breast cancer. METHODS: We prospectively collected data on circulating tumour cells at the time of definitive surgery from chemonaive patients with stage 1-3 breast cancer from February, 2005, to December, 2010. We deemed eligible all patients with operable breast cancer presenting at The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Patients were ineligible if they had bilateral breast cancer or any other malignancy within 5 years of the diagnosis of the present cancer. We measured circulating tumour cells with the CellSearch System (Veridex, Raritan, NJ). We correlated findings of circulating tumour cells with standard tumour characteristics, including tumour size and grade; oestrogen and progesterone receptor and human epidural growth factor receptor 2 (HER2) status; and axillary lymph node status with χ(2) or Fisher exact tests. We assessed outcomes at a median follow-up of 35 months. Log-rank test and Cox regression analysis was applied to establish the association of circulating tumour cells with progression-free and overall survival. FINDINGS: No patients reported adverse events or complications from blood collections. We identified one or more circulating tumour cells in 73 (24%) of 302 patients. Detection of one or more circulating tumour cells predicted both decreased progression-free survival (log-rank p=0·005; hazard ratio [HR] 4·62, 95% CI 1·79-11·9) and overall survival (log-rank p=0·01; HR 4·04, 1·28-12·8). INTERPRETATION: The presence of one or more circulating tumour cells predicted early recurrence and decreased overall survival in chemonaive patients with non-metastatic breast cancer. These results suggest that assessment of circulating tumour cells might provide important prognostic information in these patients. FUNDING: Society of Surgical Oncology, Morgan Welch Inflammatory Breast Cancer Program, The University of Texas MD Anderson Cancer Center, and the State of Texas Rare and Aggressive Breast Cancer Research Program.

Invasive lobular carcinoma predicts micrometastasis in breast cancer.

BACKGROUND: Invasive lobular carcinomas (ILCs) are almost always estrogen receptor (ER) positive. Most delayed breast cancer recurrences occur in ER-positive patients. Disseminated tumor cells (DTCs) and circulating tumor cells (CTCs) have been implicated in recurrence. The purpose of this study was to determine whether DTCs and CTCs are associated with ILCs in stage I-III breast cancer. MATERIALS AND METHODS: Clinical stage I-III breast cancer patients consented to participate in an institutional review board-approved study involving collection of bone marrow and blood at surgery for primary breast cancer. We assessed DTCs by anti-CK antibody cocktail after cytospin. We detected CTCs using CellSearch, and defined them as nucleated cells lacking CD45 but expressing cytokeratin. One or more cells per 5 mL bone marrow or 7.5 mL blood was considered positive. We performed statistical analyses using chi-square and Fisher's exact tests. RESULTS: We prospectively enrolled 422 patients, 64 with ILC and 358 with invasive ductal carcinoma. Estrogen receptor positivity was higher in ILCs (92.2% versus 66.2%) {P = .001}. We identified DTCs to be 43.4% with ILC compared with 28.9% with IDC {P = 0.03}. The CTC rates were similar. Either DTCs or CTCs were identified in 75.6% with ILC, compared with 51.7% with invasive ductal carcinoma {P = .002}. We observed no correlation between the presence of DTCs and CTCs in ILC patients and tumor size, grade, hormone receptor status, stage, lymph node status, or administration of NACT. CONCLUSIONS: Invasive lobular carcinomas independently predicted micrometastatic disease. Because most late recurrences are ER positive, this raises the question of whether DTCs and CTCs are indeed responsible for late breast cancer recurrence.

Disseminated tumor cells predict survival after neoadjuvant therapy in primary breast cancer.

BACKGROUND: Tumor cells that disseminate to the bone marrow (disseminated tumor cells [DTCs]) have been identified in 30% of patients with stage I through II breast cancer (BC) and predict outcome. Neoadjuvant chemotherapy (NACT) is effective in reducing the size of primary tumors or eradicating lymph node metastases before surgery, but little is known regarding the presence or significance of DTCs after NACT. METHODS: The authors evaluated DTCs in 95 patients with clinical stage I through III BC. Bone marrow samples were collected after completion of NACT at the time they underwent surgery for primary BC. DTCs were assessed using an anticytokeratin antibody cocktail. Primary tumor markers, the extent of lymph node (LN) involvement, they type of NACT administered, and response to NACT were compared with presence of DTCs. Chi-square and Fisher exact tests were used for statistical analyses. RESULTS: The median patient age at diagnosis was 51 years, and the median follow-up was 24 months. Forty-six percent of patients had tumors classified as T1/T2, 20% had T3 tumors, 34.5% had T4 tumors, and 81% had lymph node metastasis before NACT. DTCs were identified in 26% of patients after NACT. No associations were observed between DTCs and primary tumor characteristics or LN involvement. A pathologic complete response was observed in 25 patients (26%) but was not predictive of DTCs after NACT (P = .83). DTCs after NACT predicted worse BC-specific survival (P < .02). CONCLUSIONS: The presence of DTCs was an independent predictor of outcome after NACT. The current results indicated that monitoring hematogenous micrometastatic disease after NACT may be useful in selecting patients who might benefit from additional systemic adjuvant therapies.

Mesenchymal stem cells expressing GD2 and CD271 correlate with breast cancer-initiating cells in bone marrow.

PURPOSE: The bone marrow microenvironment is considered a critical component in the dissemination and fate of cancer cells in the metastatic process. We explored the possible correlation between bone marrow mesenchymal stem cells (BM-MSC) and disseminated breast cancer-initiating cells (BCIC) in primary breast cancer patients. EXPERIMENTAL DESIGN: Bone marrow mononuclear cells (BM-MNC) were collected at the time of primary surgery in 12 breast cancer patients. BM-MNC was immunophenotyped and BCIC was defined as epithelial cells (CD326+CD45-) with a "stem-like" phenotype (CD44+CD24low/-, ALDH activity). BM-MSC was defined as CD34-CD45-cells that co-expressed GD2, CD271, and/or CD200 within CD326-depleted BM-MNC. RESULTS: The percentages of BCIC (Aldefluor+CD326+CD44+CD24-) correlated with the percentages of BM-MSC, either CD45-GD2+CD200+CD271+ (Kedall's tau = 0.684, p = 0.004) or CD45-GD2+CD271+ in the bone marrow (Kedall's tau = 0.464, p = 0.042). CONCLUSIONS: There was a positive correlation between mesenchymal stem cells expressing GD2 and CD271 and breast cancer-initiating cells in BM of patients with primary breast cancer.

Primary breast cancer patients with high risk clinicopathologic features have high percentages of bone marrow epithelial cells with ALDH activity and CD44⁺CD24lo cancer stem cell phenotype.

BACKGROUND: Cancer stem cells (CSCs) are purported to be epithelial tumour cells expressing CD44(+)CD24(lo) that exhibit aldehyde dehydrogenase activity (Aldefluor(+)). We hypothesised that if CSCs are responsible for tumour dissemination, disseminated cells in the bone marrow (BM) would be positive for putative breast CSC markers. Therefore, we assessed the presence of Aldefluor(+) epithelial (CD326(+)CD45(dim)) cells for the presence of the CD44(+)CD24(lo) phenotype in BM of patients with primary breast cancer (PBC). METHODS: BM aspirates were collected at the time of surgery from 66 patients with PBC. Thirty patients received neoadjuvant chemotherapy (NACT) prior to aspiration. BM was analysed for Aldefluor(+) epithelial cells with or without CD44(+)CD24(lo) expression by flow cytometry. BM aspirates from three healthy donors (HD) were subjected to identical processing and analyses and served as controls. RESULTS: Patients with triple-receptor-negative (TN) tumours had a significantly higher median percentage of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population than patients with other immunohistochemical subtypes (P=0.018). Patients with TN tumours or with pN2 or higher pathologic nodal status were more likely to have a proportion of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population above the highest level of HD. Furthermore, patients who received NACT were more likely to have percentages of Aldefluor(+) epithelial cells than the highest level of HD (P=0.004). CONCLUSION: The percentage of CD44(+)CD24(lo) CSC in the BM is higher in PBC patients with high risk tumour features. The selection or enrichment of Aldefluor(+) epithelial cells by NACT may represent an opportunity to target these cells with novel therapies.

Disseminated tumor cells in biologic subtypes of stage I-III breast cancer patients.

BACKGROUND: Triple receptor-negative breast cancers (TNBC) are higher grade and more likely to metastasize. Recurrences after 5 years are rare in TNBCs. Conversely, late recurrences are seen in estrogen receptor (ER)-positive (luminal) cancers. Disseminated tumor cells (DTCs) may be responsible for late recurrences. We compared rates of DTCs in basal and luminal subtypes. METHODS: We evaluated 205 stage I-III patients. DTCs were assessed from bone marrow aspirates using anti-cytokeratin (CK) antibody following cytospin, and the presence of ≥ 1 CK-positive cells was considered positive. Pathologic complete response (pCR) was defined as lack of invasive disease in primary tumor and regional lymph nodes after neoadjuvant chemotherapy (NAC). Statistical analyses used chi-square and Fischer's exact test. RESULTS: Median follow-up (f/u) was 27 months, and 40% of patients had NAC. Forty patients had TNBC, and 148 had luminal cancers. Seventeen percent of TNBC patients, and 27% of those with luminal subtype, had DTCs after NAC (P = NS). Following NAC, pCR occurred in 28% of TNBC and 23% of luminal patients. Luminal A subtypes were less likely to achieve pCR when compared with non-luminal A subtypes (16 versus 41%; P = 0.01). All TNBC patients who achieved pCR had complete eradication of DTCs, whereas 36% of luminal (A and B) subtypes had DTCs. CONCLUSIONS: DTCs were found in 29% of stage I-III patients. TNBCs were more likely to have complete eradication of DTCs after pCR. Further study is needed to determine whether DTCs are responsible for late recurrences in patients with luminal cancers.

Detection of minimal residual disease in blood and bone marrow in early stage breast cancer.

BACKGROUND: The significance of circulating tumor cells (CTCs) in blood and of disseminated tumor cells (DTCs) in bone marrow (BM) in patients with early stage breast cancer is unclear. In this study, the authors investigated the occurrence of CTCs and DTCs in women with early stage breast cancer and evaluated the correlation of their presence with other prognostic markers. METHODS: Blood and BM aspirations were collected at the time of primary breast surgery. CTCs were detected by using the CellSearch assay, and DTCs were detected by immunostaining BM aspirates for pancytokeratin. The presence of CTCs and DTCs was correlated with tumor classification (T1 vs T2), tumor histologic grade, estrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (HER2) status, and lymph node (LN) status. RESULTS: Of 92 patients who were included in the study, 49 had T1 tumors, and 43 had T2 tumors. CTCs were detected in 31% of patients, and DTCs were detected in 27% of patients. There was no correlation between the occurrence of CTCs and DTCs with the tumor classification (T1 vs T2) or histologic grade. CTCs were detected in 33% of patients with ER-positive disease versus 26% of patients with ER-negative disease, in 32% of patients with PR-positive disease versus 30% of patients with PR-negative disease, and in 25% of patients with HER2-positive disease versus 31% of patients with HER2-negative disease. DTCs were observed in 23% of patients with ER-positive disease versus 37% of patients with ER-negative disease, in 22% of patients with PR-positive disease versus 32% of patients with PR-negative disease, and in 0% of patients with HER2-positive disease versus 29% of patients with HER2-negative disease. CTCs and DTCs were nearly equally prevalent in both LN-positive women and LN-negative women. There was no significant correlation between the occurrence of CTCs or DTCs with tumor classification (T1 vs T2), tumor histologic grade, positive ER status, positive PR status, or positive HER2 status, and axillary LN status. CONCLUSIONS: CTCs and DTCs in women with early stage breast cancer did not correlate with the standard prognostic indicators that were considered. The implications of their occurrence in patients with early stage disease will require further large-scale studies.

Cyclooxygenase-2 expression in primary breast cancers predicts dissemination of cancer cells to the bone marrow.

PURPOSE: Cyclooxygenase-2 (COX2) plays a role in breast cancer progression at various stages starting from pre-malignant phenotype to clinical metastasis. Breast cancer metastasizes commonly to the bone and preclinical studies suggest an involvement of COX2 in this process. Detection of disseminated tumor cells in the bone marrow of patients at the time of surgery correlates with the subsequent development of clinical bone metastasis. Therefore, to investigate whether COX2 is important for breast cancer metastasis in humans, we analyzed COX2 protein expression by immunostaining of primary tumors from 112 operable stages I, II, or III patients and determined its correlation with bone marrow micrometastasis (BMM). METHODS: We detected COX2 protein in primary tumors by immunostaining with a monoclonal antibody, and tumor cells present in the bone marrow by immunostaining for epithelial cytokeratins and by morphological criteria. RESULTS: COX2 expression in primary breast cancer correlated with BMM in a highly statistically significant manner (P = 0.006). Our statistical analyses of correlations of the COX2 positivity in primary tumor with other clinically relevant indicators revealed that COX2 positivity correlates with high nuclear grade (P = 0.0004). Furthermore, we were able to detect COX2 protein in BMM by immunostaining. CONCLUSIONS: These studies indicate that COX2 produced in primary breast cancer cells may be vital to the initial development of BMM that may subsequently lead to osteolytic bone metastases in patients with breast cancer, and that COX2 inhibitors may be useful in halting this process.