39P Invasion and Metastasis Genes Expression Profile of CTCs-Enriched Blood Samples from Breast Cancer Patients

623 Serial monitoring for presence, number & characterization of circulating cancer cells (CCC) may provide valuable information that may be relevant to prognosis and treatment outcomes of breast cancer patients (BCP). We conducted a serial blood sampling study at the University of Maryland in BCP with stage 1–4 breast carcinoma. 15–20 ml of venous blood were collected before the start of systemic therapy and periodically thereafter & processed using negative selection method with double-gradient centrifugation & magnetic cell sorting to remove WBCs. Digital images of FITC-positive epithelial cells were acquired with a fluorescence microscope & counted. CCC from 41 patients (Pts) were also stained with Trastuzu-mAb-532 to quantify the HER-2/neu cell surface receptor expression relative to a fluorescence standard. 105 Pts were accrued & 415 blood samples tested (median number of samples/pt; 4 (1–8). During the 24 mos. monitoring period CCC were detected in 57 of 105 pts (54%). The Table below shows that presence of >10 CCC/sample is associated with decreased survival and increased probability of having metastatic disease.(Exact chi-square test for presence vs. absence of metastatses in A, B, C, D groups, P < 0.0001; Fisher’s exact test to compare individual groups: for B vs C+ D, P < 0.001; B vs C, P=0.001). HER-2/neu expression was assessed in CCC of 25 pts (minimum of 4 CCC per sample) as compared with strongly HER-2/neu positive control cell line SKBR-3. 10 Pts were positive & 15 negative for HER-2/neu over-expression in CCC. CCC data & primary tumor data concurred in 6 of 7 Her-2/neu primary tumor tissue positive Pts & in 12 of 13 Her-2/neu primary tissue negative Pts. For 5 Pts tissue data was not available. Conclusions: Increasing CCC numbers/sample appear to correlate with adverse outcome of BCP. Our CCC Test may provide valuable information about prognosis of stage 1–4 BCP. HER-2/neu expression could be quantified in individual CCC & concurred with primary tumor data in 90% of Pts. Supported by NCI Grant CA081903 [Table: see text] [Table: see text]

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Molecular markers for circulating tumor cells in breast cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2011.29.27_suppl.223 ◽

2011 ◽

Vol 29 (27_suppl) ◽

pp. 223-223

Author(s):

R. Zeillinger ◽

E. Obermayr ◽

A. Fink-Retter ◽

G. Heinze ◽

A. Reinthaller ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Gene Panel ◽

Control Group ◽

Gene Marker ◽

Breast Cancer Patients ◽

Gene Markers ◽

Blood Samples

223 Background: Recently, we identified a six gene panel (CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8) for the RT-qPCR based detection of circulating tumor cells (CTC) in breast cancer patients. The aim of the present study was to evaluate the gene panel in further blood samples. Methods: Blood samples were taken from breast cancer patients with metastatic disease (MBC, N=10) or with no evidence of disease (NED, N=30). Putative CTC were enriched by Oncoquick density gradient centrifugation. Total RNA was isolated with RNeasy Micro Kit (QIAgen). Template cDNA was generated with M-MLV Reverse Transcriptase, RNase H Minus (Promega) and random nonamers as primers. RT-qPCR was performed in duplicate reactions using TaqMan Assays (Applied Biosystems) with default thermal cycling parameters. Raw data were analyzed with the AB7900 Sequence Detection Software version 2.2.2 using automatic baseline correction and manual cycle threshold setting. Gene expression was normalized to GAPDH expression. A threshold value TX for each gene X was set at two standard deviations above the mean dCtX value in the healthy control group. A patient was defined as CTC-positive, if at least one gene marker was over-expressed compared to the defined threshold. Results: The gene panel consisting of CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8 identified 4/11 MBC but only 5/27 NED patients as CTC positive (p=0.163). By adding known CTC markers (SCGB2A2, TFF1, FXYD3, AGR2, S100A18, and EPCAM) to the panel, 7/11 MBC but only 6/27 NED patients were CTC positive (p=0.018). The presence of CTC in NED patients correlated with pN staging (p=0.026). Only one out of the six CTC positive NED patients relapsed within the observation period (median 35 months, range 25-39 months from blood sampling). We observed no correlation of CTC positivity and recurrence in NED patients. Conclusions: The sensitivity of the RT-qPCR based CTC detection in breast cancer patients may be enhanced by adding known CTC markers (SCGB2A2, TFF1, FXYD3, AGR2, S100A18, and EPCAM) to the six gene panel (CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8). Longer follow-up times are needed to evaluate the predictive value of the gene markers on survival.

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Effects of G-CSF on circulating tumor cells (CTC) and CA 27.29 in breast cancer patients

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.11027 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 11027-11027

Author(s):

P. Hepp ◽

B. Rack ◽

A. Schneider ◽

M. Rezai ◽

H. Tesch ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Tumor Markers ◽

Phase Iii ◽

Breast Cancer Patients ◽

Phase Iii Trial ◽

Blood Samples ◽

Significant Difference ◽

Before And After ◽

Immunomagnetic Enrichment

11027 Background: Some recent publications indicated that the use of G-CSF could be connected to an increase in CTC as well as elevated levels of tumor markers such as CA 27.29. In the SUCCESS Trial CTC and CA27.29 are examined before and after adjuvant chemotherapy (CHT) in 3754 breast cancer patients (pts). Methods: The SUCCESS Trial is a phase III trial comparing FEC-Docetaxel vs. FEC-Doc-Gemcitabine regime and 2 vs. 5 years of treatment with zoledronate in patients with primary breast cancer (BC) (N+ or high risk). Blood samples are taken before and after CHT. CTC were assessed with the CellSearchSystem (Veridex, Warren, USA). After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labeled with anti-cytokeratin (8,18,19) and anti-CD45 antibodies to distinguish epithelial cells and leukocytes. CA27.29 has been measured with ST AIA-PACK Ca27.29 reagent using MUC-1 for AIA-600II (Tosoh Bioscience, Tessenderlo, Belgium). The cutoff for CA27.29 is 32 U/ml and >1 cell for the CTC analysis. Patients were grouped to CTC/CA27.29 raise or no raise and 1 to 6 cycles with G-CSF or no G-CSF at all. Results: Data on 1510 pts are available for CTC analysis. 745 pts (49%) received at least one course of G-CSF. 117 pts (8%) showed an increase in CTC after CHT. In this group 52 (3%) pts received G-CSF and 65 (4%) did not. 693 pts with stable or decreased CTC received G-CSF (46%) and 700 did not (46%). There was no significant difference (p=0.29). The analysis of CA27.29 is based on the data of 2556 pts. 1252 pts (49%) received at least one course of G-CSF. 338 pts (13%) exceeded the threshold for CA27.29 only after CHT. In this group 209 pts (8%) received G-CSF and 129 (5%) did not. 1043 pts with stable or decreased CA27.29 received G-CSF (41%) and 1175 did not (46%). This difference was highly significant (p<0.0001). Conclusions: No evidence can be provided for a significant correlation between an increase in the number of CTC and the application of G-CSF over CHT. Nevertheless the results on CA27.29 showed a highly significant correlation between the administration of G-CSF and elevated CA27.29 levels directly after CHT. This could be a possible explanation for the often observed increase of tumor markers after CHT. [Table: see text]

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The novel cytotoxicity of neutrophils in the peripheral blood of breast cancer patients versus women without breast cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2011.29.27_suppl.44 ◽

2011 ◽

Vol 29 (27_suppl) ◽

pp. 44-44

Author(s):

Z. Granot ◽

E. A. Comen ◽

L. Norton ◽

R. Benezra

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Peripheral Blood ◽

Primary Breast Tumor ◽

Newly Diagnosed ◽

Breast Cancer Patients ◽

Blood Samples ◽

Cell Kill ◽

A Cell

44 Background: Using murine mammary tumor models, recent research conducted by our laboratory at the Sloan-Kettering Institute indicates that select neutrophils are mobilized and entrained by a primary breast tumor and uniquely have the capacity to inhibit metastatic seeding in the lung (Granot Z et al. unpublished). We sought to determine whether entrainment of cytotoxic neutrophils also occurs in blood samples from women with newly diagnosed breast cancer as contrasted to those garnered from healthy women. Methods: Subjects were 21 newly diagnosed pre-operative breast cancer patients without evidence of metastatic disease, 9 healthy female volunteers with no history of any cancer, and 3 patients with newly diagnosed DCIS. Neutrophils were purified from the blood samples. Cytotoxicity was evaluated by incubating isolated neutrophils with luciferase labeled MDA-MB-231 cells. Luciferase activity, as a reflection of % cell kill, was measured using a Bio-Tek microplate luminescence reader. Results: Significant cytotoxicity was notably observed when MDA-MB-231 cells were co-cultured with neutrophils purified from patients with invasive tumors. Pre-operative breast cancer patients (n=21) had a cell kill range of 0-30% (mean = 12.1%), whereas healthy subjects (n=9) had a cell kill range of 0.2-8% (mean = 2.6%), p<0.004. DCIS patients (N=3) had a cell kill range of 3-4% (mean = 2.7). Conclusions: To date, this preliminary work is the first to demonstrate the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with those from women without breast cancer. Further studies are needed to evaluate the prognostic and therapeutic role of cytotoxic neutrophils.

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