As part of a continuing study aimed at assessing the prognostic value of the presence of CTCs in patients with MBC treated with HDCT followed by AHSCT at The University of Texas MD Anderson Cancer Center, a total of 21 patients with MBC were studied from September 2007 to March 2010. Inclusion criteria for the continuing study included informed consent, age 18 to 60 years, MBC, histological confirmation of invasive breast carcinoma, and a complete or partial response to pretransplantation standard-dose chemotherapy or hormonal therapy. Each patient underwent a complete diagnostic examination to determine the extent of disease. Patients with concurrent malignancy other than non-melanoma skin cancer in the previous 5 years were excluded. For each patient, age, tumor histology, hormone receptor status, HER2 status, and details of systemic therapy were recorded. The MD Anderson Institutional Review Board approved this study.
Hematopoietic progenitor cells were collected using chemo-mobilization plus G-CSF (10 patients) or G-CSF alone (11 patients).
The chemo-mobilization regimen consisted of cyclophosphamide 1500 mg/m2 given intravenously [IV] over 24 hours for 3 days for a total dose of 4500 mg/m2; etoposide 250 mg/m2 IV over 24 hours for 3 days for a total dose of 750 mg/m2; and cisplatin 40 mg/m2 IV daily for 3 days for a total dose of 120 mg/m2. Also administered for chemo-mobilization were mesna 500 mg/m2 IV over 30 minutes before the first dose of cyclophosphamide, mesna given as a continuous 24-hour infusion for 3 days for a total dose of 2000 mg/m2, and G-CSF (filgrastim) 5 µg/kg given subcutaneously twice daily from the fourth day of chemomobilization until aphaeresis was complete. For subjects receiving G-CSF alone, G-CSF 5 µg/kg was given subcutaneously twice daily until apheresis was complete. In both groups (chemo-mobilization and G-CSF alone), the dose of G-CSF could be increased up to 10 mcg/kg if collection of CD34-positive (CD34+) hematopoietic progenitor cells during apheresis was inadequate. The dose of G-CSF was rounded to 300 mcg or 480 mcg according to patient weight.
Apheresis was based on the standard collection procedure and was performed daily until 5 x 106 CD34+ cells/kg were collected. The minimal acceptable cell dose for transplantation was 2 x 106 CD34+ cells/kg. If fewer than 2 x 106 CD34+ cells/kg were collected by apheresis, additional apheresis was performed, and nonpurged stem cell product was collected for later infusion into the patient along with CD34+ cells. The stem cell graft was processed using an Isolex Magnetic Cell Separator 300i (Baxter, San Diego, CA) to select CD34+ cells.
Patients with at least the minimum acceptable number of hematopoietic progenitor cells collected underwent HDCT with cyclophosphamide, thiotepa, and carboplatin as follows: 1) cyclophosphamide 1.5 gm/m2/day IV on days -6, -5, -4, and -3 prior to stem cell infusion; 2) thiotepa 120 mg/m2/day IV on days -6, -5, -4, and -3 prior to stem cell infusion; 3) carboplatin to a total dose determined using the Calvert formula with a target area under the curve of 20, divided into 4 doses IV on days -6, -5, -4, and -3 prior to stem cell infusion; and 4) mesna 500 mg/m2 IV half an hour before the first dose of cyclophosphamide and mesna 2 gm/m2 given as a continuous 24-hour infusion for 4 days.
Post-AHSCT therapy could be started at the 1-month follow-up visit. Patients with HER2-amplified tumors received either 1) trastuzumab 2 mg/kg IV once a week or 2) trastuzumab loading dose of 8 mg/kg IV followed by trastuzumab 6 mg/kg IV every third week. Trastuzumab therapy was continued until progression of disease. Patients with tumors positive for estrogen receptor and/or progesterone receptor received hormonal therapy. If patients had responded to hormonal therapy prior to registration for this trial, the same hormonal therapy was continued. If patients had a prior history of resistance to hormonal therapy, they received a different class of hormonal therapy. Hormonal therapy was continued until progression of disease. Patients who had a single bone lesion and no prior radiation therapy underwent radiation therapy 5 weeks after AHSCT. Patients with bone disease could be treated with a bisphosphonate at the discretion of their primary physician. Candidates for bisphosphonates received them 5 weeks after AHSCT. If clinically indicated, patients could receive radiation therapy.
Enumeration of CTCs in peripheral blood
The CellSearch system (Veridex Corporation, Warren, NJ) was used to enumerate CTCs in 7.5 mL of PB, as previously reported 3
. Briefly, PB samples were subjected to enrichment with anti-EpCAM-coated magnetic ferrous particles, and CTCs were defined as nucleated cells (DAPI positive) lacking CD45 expression but expressing cytokeratin (CK)-8, -18, or -19 3
Detection of CTCs with EMT phenotype in apheresis products
Selection of CD45-depleted cell fraction
After depletion of CD34+ hematopoietic progenitor cells, mononuclear cells (MNCs) were obtained by density-gradient centrifugation using Ficoll-Hypaque 1.077 (Sigma) at 400xg for 30 minutes. The MNCs were collected, washed twice with sterile phosphate-buffered saline, and incubated with magnetic beads coated with anti-CD45 antibody (Miltenyi-Biotec, Auburn, CA) before being processed with an AutoMACS Pro Separator (Miltenyi-Biotec) to select for CD45-negative (CD45-) cells, as previously described 15
RNA extraction and cDNA synthesis
The CD45-depleted cell fraction was mixed with Trizol LS Reagent (Invitrogen Corporation, Carlsbad, CA) and stored at -80°C until RNA extraction. Total RNA isolation was performed using Trizol LS Reagent according to the manufacturer's instructions. The isolated RNA was dissolved in diethylpyrocarbonate-treated water, treated with DNAse (Ambion Inc., Austin, TX) to minimize contamination by genomic DNA, and subsequently stored at -80°C.
All RNA preparations and handling were conducted in a laminar flow hood under RNase-free conditions. RNA concentration was determined by absorbance readings at 260 nm. Total RNA isolated from the CD45-depleted cell fraction was reverse-transcribed using a cDNA archive kit (Applied Biosystems, Foster City, CA), as previously described15
Identification of EMT-inducing transcription factor gene transcripts in unselected and CD45-depleted MNC fractions
Synthesized cDNA was subjected to fluorescence-based quantitative real-time polymerase chain reaction (qRT-PCR) for the detection of EMT-inducing transcription factor (TWIST1, SNAIL1, and SLUG) gene transcripts (EMT-TFs). In brief, 2.5 μL of cDNA was diluted in 25 μL of reaction volume containing 12.5 μL of TaqMan Universal PCR Master Mix, No AmpErase UNG, 8.75 μL of water, and 1.25 μL of primers. The primers were purchased from Applied Biosystems (TWIST1: Hs00361186_m1, SNAIL1: Hs00195591_m1, SLUG: Hs00161904_m1). Primers were designed to span exon-exon boundaries to exclude possible amplification of genomic DNA.
Amplification was performed in an ABI Fast 7500 Real Time PCR system (Applied Biosystems) using the following cycling program: 95°C for 10 min; 40 cycles of 95°C for 15 seconds, 60°C for 60 seconds. All samples were analyzed in triplicate. Quantification of target genes and an internal reference gene was performed using qRT-PCR. Calibrator samples were run with every plate to ensure consistency of PCR reactions. PCR on the non-reverse-transcribed portion of each sample was performed to assess DNA contamination. For all fluorescence-based qRT-PCR assays, fluorescence was detected from 0 to 40 cycles for the control and marker genes in single-plex reactions, which provide the cycles at threshold (CT) value for each PCR product. The CT value is a PCR cycle at which a significant increase in fluorescence is detected because of exponential accumulation of PCR products. Expression of the genes of interest was calibrated against expression of the housekeeping gene GAPDH
and was quantified using the delta-CT method with the following formula: ½ ^Ct (target-GAPDH). For each gene, we calculated the ratio between the relative expression in CD45-depleted MNC and the relative expression in unselected MNC. We defined overexpression of EMT-TFs as at least twice the expression observed in the CD45-depleted fraction, as previously reported 15
Detection of epithelial cells and CSCs in MNC samples isolated from apheresis products
MNC samples were interrogated for aldehyde dehydrogenase (ALDH) activity using the Aldefluor assay and the manufacturer's protocol (STEMCELL Technologies, Vancouver, Canada). Briefly, 4 x 106
MNCs from patients were suspended in Aldefluor buffer, which contains a proprietary ATP-binding cassette transport inhibitor. One-third of the cells were reacted with 5 µL of the ALDH inhibitor diethylamino-benzaldehyde as a negative control. Both the test reaction and the negative control were incubated for 35 minutes at 37°C in a 5% CO2
atmosphere. Purified anti-CD44 monoclonal antibody (BD Pharmingen, San Diego, CA) was conjugated with Alexa700 using the Zenon antibody labeling kit (Invitrogen) prior to reaction with the Aldefluor-labeled cells. Additionally, preconjugated antibodies to CD24 (PE) and CD45 (PE-Cy7), both from BD Pharmingen (San Diego, CA), and CD326 (APC, Miltenyi Biotec) were used to label cells at room temperature protected from light for 30 minutes. An additional tube of Aldefluor-labeled cells was stained with the appropriate isotype-matched controls. The stained cells were spun down, and the cell pellet was suspended in 200 µL of Aldefluor buffer prior to analysis on an LSR-II flow cytometer capable of discriminating 6-color fluorescence (BD Biosciences, San Jose, CA). Cellular debris was excluded from the analysis based on low forward light scatter. For analysis, epithelial cells in CD34-depleted apheresis products were defined as cells exhibiting the phenotype CD326+CD45dim
. That same definition is used throughout the rest of this manuscript. Within the Aldefluor+ epithelial cell population, a subset of CSCs was defined as cells with a CD326+CD44+CD24lo
phenotype as previously reported 10
Median follow-up period, calculated as the median observation time, was determined for all patients and for patients alive at last follow-up. OS was defined as the time from AHSCT to the time of death due to any cause or to last follow-up. PFS was calculated from the time of AHSCT to the time of progression, death, or last follow-up. PFS and OS were estimated using the Kaplan-Meier product limit method and compared among groups using the log-rank test. Cox's proportional hazards regression analysis was used to test the statistical significance of several potential predictors of OS and PFS. This modeling was done in a univariable fashion. From this model, we estimated the hazard ratio for each potential prognostic factor with a 95% confidence interval. All potential prognostic factors with P < 0.10 from the univariable analysis were then included in a saturated model, and backward elimination was used to remove factors from the model on the basis of the likelihood ratio test in the multiple regression analysis. For the variables “Response to prior therapy”, “Response to HDCT with AHSCT”, and “HER2/neu status”, since in one of the categories no patient died, the Cox model could not provide a reliable estimation. Therefore, these particular variables were not included in the Cox univariable and multivariable modeling for OS. For comparison of two categorical variables, the chi-squared test or Fisher's exact test was used, as appropriate. The Mann-Whitney test was used to compare the differences between patient subsets in the percentages of cells with CSC phenotype. All statistical tests were two-sided, and P values < 0.05 were considered statistically significant.