Amber Anderson

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.

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

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.

Circulating Tumor Cells and Recurrence After Primary Systemic Therapy in Stage III Inflammatory Breast Cancer

Inflammatory breast cancer (IBC) is rare and aggressive, with poor survival. While circulating tumor cells (CTCs) predict outcome in non-IBC patients, little data exists regarding their prognostic significance in IBC. This prospective study analyzed blood samples for CTCs from 63 stage III IBC patients to determine if CTCs present after primary systemic chemotherapy predicted relapse. CTC identification was not associated with tumor characteristics, lymph node positivity, or complete pathologic response to systemic therapy. At mean follow-up of 38 months, multivariable analysis demonstrated that detection of one or more CTCs predicted shortened relapse-free (log-rank P = 0.005, hazard ratio [HR] = 4.22, 95% confidence interval [CI] = 1.67 to 10.67, Cox P = 0.002) but not overall survival (log-rank P = 0.54, HR = 1.53, 95% CI = 0.41 to 5.79, Cox P = 0.53). All statistical tests were two-sided. In this study, CTCs after primary chemotherapy identified IBC patients at high risk for relapse.

Detection of circulating melanoma cells in the blood of melanoma patients: a preliminary study.

Significant prognostic heterogeneity exists within the substages of melanoma; therefore, novel prognostic biomarkers are needed to provide information on the risk of recurrence. Limited available data suggest prognostic significance for circulating melanoma cells (CMCs); there is a need for a sensitive, reproducible, and standardized identification technique. Using a semiautomated technology, we sought to determine whether CMCs could be identified reliably in stage I–IV melanoma patients and whether the presence of CMC correlated with known prognostic factors. CMCs were detected in the peripheral blood (7.5 ml) of patients with stage I–IV melanoma (n=89) using the CellSearch system. CD146+ cells were immunomagnetically enriched; nucleated HMW-MAA+/CD45−/CD34− cells were considered CMCs. One or more CMCs was detected in 45% of all patients, varying with stage of disease (stages I/II, III, and IV: 35, 44, and 86%, respectively; P=0.03, for stage I/II vs. stage IV); 55% had one CMC, 32% had two CMCs, and 13% had three or more CMCs identified. The presence of CMCs in the blood was associated with histologic subtype, particularly in patients with stage I/II disease (superficial spreading 18% vs. acral lentiginous 75%). Using a semiautomated technique, CMCs can be identified in a significant number of melanoma patients. These data support further study with longer follow-up and longitudinal/serial time points to better determine the identification rates and prognostic significance of CMCs in stage I–IV melanoma patients.

Abstract 1368: Investigating the tumorigenicity of disseminated tumor cells

Background: Tumor cells that disseminate to the bone marrow (disseminated tumor cells, DTCs) have been identified in 30% of stage I - III breast cancer (BC) patients and independently predict outcome. Because DTCs are a rare, heterogeneous population of cells, little is known regarding their characteristics; such as their clinical significance with regard to disease progression, and targets that can be utilized for their eradication. DTC Identification is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), and cytokeratin (CK). However, EpCAM and CK can be down-regulated during epithelial–mesenchymal transition (EMT). To date, no published report has assessed the tumorigenicity of DTCs isolated from patient samples using an in vivo animal model. The purpose of this study was to investigate the tumorigenicity of DTCs in vivo. Methods: Thirteen clinical stage I-III BC patients provided informed consent to participate in an IRB-approved study involving collection of bone marrow at the time of primary surgery. Following Ficoll centrifugation, bone marrow aspirates were cultured using breast cancer stem cell enriching conditions for 21 days. At 21 days, viability of floating cells with high nuclear: cytoplasmic ratios were confirmed with Tryphan blue staining and cytokeratin expression was identified using pan-cytokeratin immunostaining. 200 cells were co-injected with Matrigel into the mammary fat pads (3 mice/patient) of female nude mice. Mice were monitored daily for tumor formation. Results: Viable floating cells with large nuclear: cytoplasmic ratios were observed in 10/13 cases. Both CK+ and CK- DTCs were identified in 8 out of 10 cases, and CK- DTCs only were identified in 2 out of 10 cases. One of the 10 samples was tumorigenic in vivo; all 3 mice injected with this patient9s cells developed breast tumors within ≤ 55 days. This sample was obtained from a 40-year old T2N2 patient with a grade 2 estrogen receptor positive, progesterone receptor negative, Her2/neu negative tumor. Interestingly, CK expression was not detected in this patient9s cultured cells. Conclusions: DTCs can be isolated and propagated using breast cancer stem cell enriching conditions, enabling the characterization and assessment of DTCs that are in the process of (or have undergone) EMT. In addition, the tumorigenetic potential of DTCs can be assessed on a patient-by-patient basis by employing an in vivo mouse model. Further studies are needed to develop targeted therapies for the eradication of micrometastatic disease in stage I-III BC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1368. doi:1538-7445.AM2012-1368

P4-07-11: Circulating Tumor Cells after Neoadjuvant Therapy Predict Outcome in Stage I to III Breast Cancer.

Introduction: Circulating tumor cells (CTCs) predict outcome in metastatic breast cancer, but their significance is unclear in non-metastatic patients. Furthermore, it is unclear if the presence of CTCs after completion of neoadjuvant chemotherapy (NACT) predicts worse outcome. The purpose of this study was to determine if CTCs after NACT predicts worse outcome. Methods: Clinical stage I-III breast cancer patients seen at a single tertiary cancer center provided informed consent to participate in an IRB-approved study involving collection of blood (7.5 ml x 2 tubes) at the time of surgery for their primary breast cancer. CTCs were detected using the Cell SearchTM system. A positive result was defined as the presence of one or more cells per 7.5 ml blood since the threshold for positivity has not been established in non-metastatic breast cancer. Statistical analyses used chi-square and Fischer9s exact test. Results: One hundred and twenty patients were prospectively enrolled. Median age was 50 years and median follow-up was 33 months. Eight percent of patients had T1 disease, 35% T2, 18% T3, and 39% T4. Fifty-three percent of patients (63/120) had hormone receptor positive disease. Thirty-two percent of patients (38/120) were HER-2 positive. Thirty percent (36/120) were triple negative. Seventy-eight percent (91/120) had lymph node positive disease. Two or more CTCs were present in 9% of patients (11/120). Of the 11 patients who died, 3 had 2 or more CTCs (P=0.08). Of the 20 who relapsed, 6 had 2 or more CTCs (P=0.0019). Conclusions: Presence of two or more CTCs after NACT predicted worse relapse free survival in patients with stage I-III breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-11.

P5-01-17: HER2 Amplification in Primary Tumor: A Potential Marker for Presence of Circulating Tumor Cells in Inflammatory Breast Cancer Patients?

Background: Inflammatory breast cancer (IBC) is a rare but aggressive form of invasive breast cancer accounting for 3–6% of all cases and have higher rates of distant recurrence. Circulating tumor cells (CTCs) are known to predict outcome in metastatic breast cancer (BC) patients, but little is known about their prognostic significance in non-metastatic BC. We hypothesized that CTCs can be identified in patients with IBCs and may correlate with primary tumor characteristics. Methods: All patients had blood samples collected at the time of primary surgery. CTCs (per 7.5 ml blood) were detected using the Cell Search™ system (Veridex) and were defined as nucleated cells lacking CD45 but expressing cytokeratins (CK) 8, 18, or 19. The presence of ≥ 1 epithelial cells meeting morphologic criteria for malignancy was considered a positive result. Statistical analyses employed Chi square and Fisher9s exact tests using STATA IC 11. Results: We prospectively evaluated 41 IBC patients enrolled in an IRB approved protocol undergoing surgery for stage I-III breast cancer. Median follow-up was 30 months. Mean age was 52 years. Thirty five patients (94%) had positive lymph nodes (LNs) at presentation, 30 (75%) had high-grade tumors and 20 (53%) had lymphovascular invasion. Eleven patients (28%) were ER positive, 11 (27%) were PR positive and 18 (44%) were HER2 positive. IBCs were more likely to be high grade (P Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-17.

P4-06-02: Microscopic Disease in Blood and Bone Marrow Predicts Survival in Early Stage Breast Cancer.

Background Disseminated tumor cells (DTCs) in the bone marrow have been identified in 30% of stage I - III breast cancer (BC) patients and predict survival. Circulating tumor cells (CTCs) in the blood predict outcome in metastatic BC, but their prognostic significance in primary BC is unclear. This study determined whether: 1) DTCs and CTCs could be identified in significant numbers of non-metastatic BC patients and 2) if these cells predict relapse-free (RFS) and overall survival (OS). Methods : Clinical stage I-III BC patients seen at a single tertiary cancer center provided informed consent to participate in an IRB-approved study involving collection of blood (7.5 ml x 2 tubes) and bone marrow (10 ml from bilateral iliac crests), and at the time of surgery for their primary BC. DTCs were assessed using an anti-cytokeratin antibody cocktail (MNF 116, CK 8,18, and 19, CAM 5.2, and AE1/AE3) following ficoll enrichment and cytospin. A positive result for DTCs was defined by presence of one or more CK-positive cells meeting morphologic criteria for malignancy. CTCs were detected using the Cell SearchTM system. A positive result was defined as the presence of one or more cells per 7.5 ml blood since a threshold for positivity has not been established in non-metastatic BC. Statistical analyses used chi-square and Fischer9s exact test. Results : We prospectively evaluated 313 patients. Mean age was 53 years, and median follow-up was 32 months. Forty-two percent of patients (131) had T1 tumors, 36% (112) T2, 10% (30) T3, and 13% (40) had T4 disease. Forty-five percent of patients (141/312) had positive lymph nodes. DTCs were identified in 29% (91/313) and CTCs in 25% (79/313) of all patients. Seven percent (21/313) of patients had both DTCs and CTCs. In the overall cohort, 26 (8%) patients relapsed and 15 (5%) died. Ten percent (9/91) of DTC positive patients died compared to 3% (6/222) of those who did not have DTCs (p=0.01). Similarly, 6% (7/79) of those who had CTCs died compared to 3% (8/234) of those who did not (p=0.03). Fifteen percent (12/79) of CTC positive patents relapsed compared to 6% (14/234, P=0.01) of those who were CTC negative. Simultaneous presence of DTCs and CTCs was a strong predictor of RFS (log rank p=0.030, HR= 2.8, 95% C.I. 1.20- 8.10) as well as OS (log rank p=0.026, HR= 3.66, 95% C.I. 1.03- 13.00) at 2 years. Combined presence of DTCs and CTCs was a predictor of outcome and these findings persisted after adjusting for variables including hormone receptor status, HER2 status, primary tumor size, grade, and preoperative lymph node status. There was no significant correlation between DTCs and/or CTCs with other primary tumor characteristics. Conclusions : Circulating and disseminated tumor cells can be identified in a significant number of non-metastatic breast cancer patients. Both CTCs and DTCs predicted outcome, and their combined presence was an independent predictor of survival. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-06-02.

Abstract P3-02-01: Is Ethnicity a Predictor of Micrometastatic Disease in Early Stage Breast Cancer Patients?

Background: Ethnicity plays a role in breast cancer (BC) outcome, highlighted by the fact that African-American women have a higher BC mortality rate than do Caucasian women. Microscopic disease, including disseminated tumor cells (DTCs) in bone marrow and circulating tumor cells (CTCs) in peripheral blood, has been shown to predict worse outcomes as well. We sought to determine whether ethnicity was a significant predictor for the presence of DTCs and/or CTCs in stage I-III BC patients. Methods: Patients provided informed consent to participate in an IRB-approved study involving collection of blood and bone marrow at the time of surgery for their primary BC. CTCs (per 7.5 ml blood) were detected using the Cell Search TM system (Veridex) and were defined as nucleated cells lacking CD45 but expressing cytokeratins (CK) 8, 18, or 19; for this study we considered one or more positive cells meeting these criteria a positive result. DTCs were assessed using an anti-CK antibody cocktail (AE1/AE3, CAM5.2, MNF116, CK8 and 18) following cytospin. A positive result for DTCs was defined by presence of one or more CK positive cells meeting morphologic criteria for malignancy. Information on clinicopathological factors including ethnicity was obtained from a prospective database. Statistical analyses used Chi-square test on STATA IC11. Results: We prospectively evaluated 224 patients undergoing surgery for stage I-III BC. Median follow-up was 22 months and mean age was 53 years. One hundred sixty seven patients (75%) were Caucasians, 22 (10%) were African-American (AA), 30 (14%) were Hispanic and 3 (1%) belonged to other ethnicities. CTCs were found in 25% (57/224) and DTCs in 30% (67/224) of patients. Patients of AA ethnicity were significantly more likely to have CTCs (50%, (11/22)) compared to the other ethnic groups (22%, (43/194)); {O.R. = 2.5, 95% C.I. = 1.35- 7.80, P = 0.002}, and had significantly higher numbers of CTCs (≥2 CTCs or ≥3 CTCs/7.5mL blood) than other ethnic groups (P = 0.001 and P Conclusions: Nearly one-third of primary BC patients have CTCs and/or DTCs. African-American women were much more likely to have CTCs and Hispanic patients had significantly more DTCs than did patients of other ethnicities. Ethnicity was an independent predictor of microscopic disease. These findings may shed some light on the higher BC mortality rates found in certain ethnic groups. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-01.

Abstract P3-02-02: Influence of Body Mass Index on Presence of Disseminated Tumor Cells in Clinical Stage I-III Breast Cancer Patients

Background: Disseminated tumor cells (DTCs) are found in approximately one third of clinical stage I-III breast cancer (BC) patients, and published reports show that presence of DTCs is an independent predictor of outcome. While higher body mass index (BMI) is associated with increased risk of breast cancer recurrence and lower survival rates in BC patients, women with lower BMIs may have lower bone density and higher bone turnover. We hypothesized that increases in bone turnover may result in the release of bone growth and “homing” factors that facilitate BC metastasis to bone and provide a “pre-metastatic niche” for BC cells. The purpose of this study was to determine if a correlation existed between DTCs and BMI in early stage BC patients. Methods: We obtained informed consent and collected bone marrow samples from 262 clinical stage I-III BC patients who were participants in an IRB-approved clinical study from 2/2005- 2/2010. All marrow samples were collected at the time of surgery for the primary tumor. DTCs were assessed using anti-pancytokeratin (CK) antibody cocktail (AE1/AE3, CAM5.2, MNF116, CK8 and 18) following cytospin. The presence of one or more CK positive cells meeting morphologic criteria for malignancy was considered a positive result for DTC. Patients with a BMI of (18.5 — 24.9) kg/m 2 were considered “normal weight”, those with a BMI of (25 - 29.9) kg/m 2 “overweight” and a BMI greater than 30 kg/m 2 was used to designate them as “obese”. Information on clinicopathological factors including BMI (measured on initial presentation) was obtained from a prospective database. Statistical analyses used Chi-square and non-parametric tests for trend. Results: Median follow-up was 19 months and mean age was 53 (25-80) years. Eighty-four patients (32%) were normal weight, 85 (32%) were overweight and 91 (35%) were obese. Seventy-eight (30%) patients had DTCs present at the time of assessment. Obese patients were significantly less likely to show presence of DTCs as compared to those who had a BMI 2 (20/78; 26% vs. 71/184; 39%) {O.R. = 0.55, 95% C.I. = 0.29- 0.96, P = 0.03}. DTCs were also less likely to be found in patients with BMI ≥25 kg/m 2 as compared to those with BMI 2 (40/78; 51% vs. 136/184; 74%); {O.R. = 0.42, 95% C.I. = 0.04- 1.02, P = 0.03}. No statistically significant correlation was observed between primary tumor characteristics (ER, PR, HER2, lymph node status, tumor grade) and presence of DTCs. Finally, a non-parametric analysis demonstrated a trend in occurrence of DTCs across the ordered levels of patients’ BMI values (P= 0.013). Conclusions: DTCs were much more common in patients with lower BMI. Further studies are needed to determine if patients with low BMI have unique microenvironmental factors within the bone that predisposes them to tumor cell dissemination. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-02.