Summer Jackson

Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Metastatic Breast Cancer: A Validation Study of the CellSearch System

Purpose: The CellSearch system (Veridex, Warren, NJ) is designed to enrich and enumerate circulating tumor cells (CTCs) from peripheral blood. Here, we validated the analytic performance of this system for clinical use in patients with metastatic breast cancer. Experimental Design: This prospective multicenter study conducted at three independent laboratories involved samples from 92 patients with metastatic breast cancer. Intra- and inter-assay variability using controls containing defined numbers of cells (average, 50 and 1,000, respectively), cell stability based on varying storage and shipment conditions, recovery precision from samples spiked with 4 to 12 tumor cells, inter-instrument variability, and positivity of samples from metastatic breast cancer patients were tested. Results: Intra- and inter-assay precision for two sites were high: All eight positive controls analyzed in the same run and >95% of the run to run control values (n = 299) were within the specified ranges. Recovery rate of spiked samples averaged between 80% and 82%. CTCs were detected in ∼70% of metastatic breast cancer patients. CTC values of identical samples processed either immediately after blood drawing or after storage for 24, 48, or 72 h at room temperature or at 4°C did not differ significantly. Shipment of samples had no influence on CTC values. When analyzing identical samples in different centers, inter-instrument accordance was high. Conclusions: The CellSearch system enables the reliable detection of CTCs in blood and is suitable for the routine assessment of metastatic breast cancer patients in the clinical laboratory. Blood samples should be shipped at room temperature and CTC counts are stable for at least 72 h.

Circulating Tumor Cells and [18F]Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography for Outcome Prediction in Metastatic Breast Cancer

PURPOSE Circulating tumor cells (CTCs) and [(18)F]fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) are two new promising tools for therapeutic monitoring. In this study, we compared the prognostic value of CTC and FDG-PET/CT monitoring during systemic therapy for metastatic breast cancer (MBC). PATIENTS AND METHODS A retrospective analyses of 115 MBC patients who started a new line of therapy and who had CTC counts and FDG-PET/CT scans performed at baseline and at 9 to 12 weeks during therapy (midtherapy) was performed. Patients were categorized according to midtherapy CTC counts as favorable (ie, < five CTCs/7.5 mL blood) or unfavorable (> or = five CTCs/7.5 mL blood) outcomes. CTC counts and FDG-PET/CT response at midtherapy were compared, and univariate and multivariate analyses were performed to identify factors associated with survival. RESULTS In 102 evaluable patients, the median overall survival time was 14 months (range, 1 to > 41 months). Midtherapy CTC levels correlated with FDG-PET/CT response in 68 (67%) of 102 evaluable patients. In univariate analysis, midtherapy CTC counts and FDG-PET/CT response predicted overall survival (P < .001 and P = .001, respectively). FDG-PET/CT predicted overall survival (P = .0086) in 31 (91%) of 34 discordant patients who had fewer than five CTCs at midtherapy. Only midtherapy CTC levels remained significant in a multivariate analysis (P = .004). CONCLUSION Detection of five or more CTCs during therapeutic monitoring can accurately predict prognosis in MBC beyond metabolic response. FDG-PET/CT deserves a role in patients who have fewer than five CTCs at midtherapy. Prospective trials should evaluate the most sensitive and cost-effective modality for therapeutic monitoring in MBC.

Circulating tumor cells in metastatic breast cancer: From prognostic stratification to modification of the staging system?

The aim of the current study was to assess the prognostic value of baseline circulating tumor cells (CTCs) in a large cohort of patients with newly diagnosed metastatic breast cancer (MBC).

Circulating tumor cells as prognostic and predictive markers in metastatic breast cancer patients receiving first-line systemic treatment

IntroductionCirculating tumor cells (CTCs) represent an independent predictor of outcome in patients with metastatic breast cancer (MBC). We assessed the prognostic impact of CTCs according to different first-line systemic treatments, and explored their potential predictive value in MBC patients.MethodsWe retrospectively evaluated 235 newly diagnosed MBC patients, treated at the University of Texas MD Anderson Cancer Center. All patients had a baseline CTC assessment performed with CellSearch®. Progression-free survival and overall survival were compared with the log-rank test between groups, according to CTC count (< 5 vs. ≥ 5) and type of systemic therapy. We further explored the predictive value of baseline CTCs in patients receiving different treatments.ResultsAt a median follow-up of 18 months, the CTC count was confirmed to be a robust prognostic marker in the overall population (median progression-free survival 12.0 and 7.0 months for patients with CTC < 5 and ≥ 5, respectively; P < 0.001). Conversely, in patients with human epidermal growth factor receptor-2-overexpressed/amplified tumors receiving trastuzumab or lapatinib, the baseline CTC count was not prognostic (median progression-free survival 14.5 months for patients with CTC < 5 and 16.1 months for those with CTC ≥ 5; P = 0.947). Furthermore, in patients with human epidermal growth factor receptor-2 normal tumors, a baseline CTC count ≥ 5 identified subjects who derived benefit from more aggressive treatments, including combination chemotherapy and chemotherapy plus bevacizumab.ConclusionsThis analysis suggests that the prognostic information provided by CTC count may be useful in patient stratifications and therapeutic selection, particularly in the group with positive CTCs, in which various therapeutic choices may procure differential palliative benefit.

Expression of epithelial–mesenchymal transition-inducing transcription factors in primary breast cancer: The effect of neoadjuvant therapy†

Epithelial cancer cells are likely to undergo epithelial–mesenchymal transition (EMT) prior to entering the peripheral circulation. By undergoing EMT, circulating tumor cells (CTCs) lose epithelial markers and may escape detection by conventional methods. Therefore, we conducted a pilot study to investigate mRNA transcripts of EMT‐inducing transcription factors (TFs) in tumor cells from the peripheral blood (PB) of patients with primary breast cancer (PBC). PB mononuclear cells were isolated from 52 patients with stages I–III PBC and 30 healthy donors (HDs) and were sequentially depleted of EpCAM+ cells and CD45+ leukocytes, henceforth referred to as CD45−. The expression levels of EMT‐inducing TFs (TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2) in the CD45− cells were determined using quantitative real‐time polymerase chain reaction. The highest level of expression by the CD45− cell fraction of HD was used as “cutoff” to determine if samples from patients with PBC overexpressed any EMT‐inducing TFs. In total, 15.4% of patients with PBC overexpressed at least one of the EMT‐inducing TF transcripts. Overexpression of any EMT‐inducing TF transcripts was more likely to be detected in patients with PBC who received neoadjuvant therapies (NAT) than patients who received no NAT (p = 0.003). Concurrently, CTCs were detected in 7 of 38 (18.4%) patients by CellSearch® and in 15 of 42 (35.7%) patients by AdnaTest™. There was no association between the presence of CTCs measured by CellSearch® or AdnaTest™. In summary, our results demonstrate that CTCs with EMT phenotype may occur in the peripheral circulation of patients with PBC and that NAT is unable to eliminate CTCs undergoing EMT.

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

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.

Circulating tumor cells in immunohistochemical subtypes of metastatic breast cancer: lack of prediction in HER2-positive disease treated with targeted therapy

BACKGROUND Circulating tumor cells (CTCs) are associated with inferior prognosis in metastatic breast cancer (MBC). We hypothesized that the relationship between CTCs and disease subtype would provide a better understanding of the clinical and biologic behavior of MBC. PATIENTS AND METHODS We retrospectively analyzed 517 MBC patients treated at a single institution. Subtypes of primary tumors were analyzed by immunohistochemical (IHC) or fluorescent in situ hybridization analyses and CTCs were enumerated by CellSearch® at starting a new therapy. Overall survival (OS) and progression-free survival durations for each IHC subtype were determined. RESULTS At a median follow-up of 24.6 months, 276 of 517 (53%) patients had died. The median OS for patients with <5 and ≥5 CTCs were 32.4 and 18.3 months, respectively (P < 0.001). Except in HER2+ patients, the prognostic value of CTCs was independent of disease subtype and disease site. CONCLUSIONS In this large retrospective study, CTCs were strongly predictive of survival in all MBC subtypes except HER2+ patients who had been treated with targeted therapy. Our results clearly demonstrate the value of enumerating CTCs in MBC and strongly suggest an interesting biological implication in the HER2+ subset of patients that need to be further explored.BACKGROUND Circulating tumor cells (CTCs) are associated with inferior prognosis in metastatic breast cancer (MBC). We hypothesized that the relationship between CTCs and disease subtype would provide a better understanding of the clinical and biologic behavior of MBC. PATIENTS AND METHODS We retrospectively analyzed 517 MBC patients treated at a single institution. Subtypes of primary tumors were analyzed by immunohistochemical (IHC) or fluorescent in situ hybridization analyses and CTCs were enumerated by CellSearch(®) at starting a new therapy. Overall survival (OS) and progression-free survival durations for each IHC subtype were determined. RESULTS At a median follow-up of 24.6 months, 276 of 517 (53%) patients had died. The median OS for patients with <5 and ≥ 5 CTCs were 32.4 and 18.3 months, respectively (P < 0.001). Except in HER2+ patients, the prognostic value of CTCs was independent of disease subtype and disease site. CONCLUSIONS In this large retrospective study, CTCs were strongly predictive of survival in all MBC subtypes except HER2+ patients who had been treated with targeted therapy. Our results clearly demonstrate the value of enumerating CTCs in MBC and strongly suggest an interesting biological implication in the HER2+ subset of patients that need to be further explored.

Circulating tumor cells as early predictors of metastatic spread in breast cancer patients with limited metastatic dissemination

IntroductionTraditional factors currently used for prognostic stratification do not always adequately predict treatment response and disease evolution in advanced breast cancer patients. Therefore, the use of blood-based markers, such as circulating tumor cells (CTCs), represents a promising complementary strategy for disease monitoring. In this retrospective study, we explored the role of CTC counts as predictors of disease evolution in breast cancer patients with limited metastatic dissemination.MethodsA total of 492 advanced breast cancer patients who had a CTC count assessed by CellSearch prior to starting a new line of systemic therapy were eligible for this analysis. Using the threshold of 5 CTCs/7.5 ml of blood, pretreatment CTC counts were correlated in the overall population with metastatic site distribution, evaluated at baseline and at the time of treatment failure, using Fisher’s exact test. Time to visceral progression and time to the development of new metastatic lesions and sites were estimated in patients with nonvisceral metastases and with single-site metastatic disease, respectively, by the Kaplan-Meier method. Survival times were compared between groups according to pretreatment CTC count by logrank test.ResultsIn the overall population, a pretreatment level ≥5 CTCs/7.5 ml was associated with an increased baseline number of metastatic sites compared with <5 CTCs/7.5 ml (P = 0.0077). At the time of treatment failure, patients with ≥5 CTCs/7.5 ml more frequently developed new metastatic lesions and sites compared with those with <5 CTCs/7.5 ml (development of new lesions: P = 0.0002; development of new sites: P = 0.0031). Among patients with disease originally confined to nonvisceral sites, ≥5 CTCs/7.5 ml was associated with remarkably shorter time to visceral metastases (P = 0.0021) and overall survival (P = 0.0006) compared with <5 CTCs/7.5 ml. In patients with single-site metastatic disease, ≥5 CTCs/7.5 ml was associated with a significant reduction of the time to development of new metastatic sites (P = 0.0051) and new lesions (P = 0.0002) and with worse overall survival (P = 0.0101).ConclusionOur results suggest that baseline CTC counts can be used as an early predictor of metastatic potential in breast cancer patients with limited metastatic dissemination.

Circulating tumor cells and biomarkers: Implications for personalized targeted treatments for metastatic breast cancer

To the Editor:Metastatic breast cancer (MBC) is incurable andthe median survival of affected patients ranges from 2to 4 years with a few patients surviving for more than5 years (1–3). Palliation of metastatic disease can beachieved by considering pretreatment prognostic andpredictive factors, which can assist in choosing a ther-apy with the greatest likelihood of patient benefit (1).Another factor that may accurately predict prognosisand treatment efficacy in patients with MBC is thenumber of peripheral blood circulating tumor cells(CTC) (4), thereby providing a patient risk stratifica-tion tool.Recent studies confirmed the superiority of CTCover imaging assessment and other standard measures,such as hormone receptor status and tumor burden,for independently predicting survival (5). Thus, CTCmay indicate the existence of cancer cells that have aunique phenotype that distinguishes them from thepredominant population of cancer cells and raises thepossibility of resembling cancer-initiating stem cells.Hence, the characterization of CTC might provideinsights into the mechanisms of tumor developmentand metastases, and identify potential novel targetsfor therapy.In breast cancer, CD44(+)CD24()⁄low)Lineage())cancer-initiating cells are known to be highly tumori-genic in severe combined immunodeficient mice (6).Previous studies have shown that micrometastaticdisease detected in the bone marrow of patients withprimary breast cancer have CD44(+)CD24()⁄low)phenotype suggestive of putative cancer stem cells (7).The extension of these evaluations to cancer-initiatingcells is feasible and quite appealing for the possibletherapeutic implications. Gene expression profiles ofCTC would provide an opportunity for specific andindividualized treatment planning.We evaluated CTC from 20 women enrolled in aprospective clinical trial at The University of TexasM. D. Anderson Cancer Center for transcripts ofestrogen receptor (ER), progesterone receptor (PgR),and human epidermal growth factor receptor-2 (HER-2). Such biomarkers’ expression in CTC is currentlynot considered in the selection of treatment regimens,and hence we compared the expression patterns ofCTC with the expression of these proteins by the pri-mary tumor and metastatic lesions. Tumor character-istics, types of treatment, and responses for the 20women with MBC who contributed samples for thisanalysis are shown in Table 1. The median number ofCTC was 2.5 (range, 0–168). Four (20%) patients had0 CTC, eight (40%) patients had 1–4 CTC, and theother eight (40%) patients had ‡5 CTC. Of theprimary tumors, 16 (80%) were ER+ and 11 (55%)were PgR+, only three (15%) showed amplification ofHER-2⁄neu. Biopsies of metastatic lesions were avail-able in 15 of the 20 patients with recurrent disease(Table 1). Nine (60%) of the metastatic tumors wereER+, seven (47%) were PgR+, and three (20%)showed amplified HER-2⁄neu. Expression patterns ofER (kappa = 0.71; p = 0.004), PgR (kappa = 0.60;p = 0.02), and HER-2 (kappa = 0.61; p = 0.047) weresimilar in the primary and metastatic tumors.In addition to ER, PgR, and HER-2, we examinedthe expression of two other genes of interest, mamma-globin (MGB) and Notch-1 in CTC. MGB levels inprimary breast tumors inversely correlate with tumoraggressiveness and axillary node invasion (8). Notch-1can be aberrantly activated in human breast cancercells (9) and may regulate the self-renewal of tumori-genic stem cells. Therefore, Notch-1 may represent agenetic biomarker of a subset of breast cancer stem

Circulating Tumor Cells (CTCs) and Epithelial Mesenchymal Transition (EMT) in Breast Cancer: Describing the Heterogeneity of Microscopic Disease.

Background: Circulating tumor cells (CTCs) are an independent predictor of survival in metastatic breast cancer (BC) patients. CTC are readily detected by CellSearch System based on their expression of EpCAM. Epithelial-mesenchymal transition (EMT) gives rise to cells with stem cell-like properties with increased chemotherapy resistance. Human mammary epithelial cells (HMEC) transformed by the EMT transcription factor TWIST1 and spiked into normal peripheral blood (PB) are not detected by EpCAM enrichment based conventional detection methods compared to non-transformed HMECs. We hypothesize that CTCs undergoing EMT and resultant loss of epithelial markers may escape detection by conventional detection methods. The aim of this study was to detect CTCs based on expression of EMT genes in breast cancer patient9s peripheral blood.Methods: This prospective ongoing study of breast cancer patients consisted of 16 (57.1%) patients with metastatic disease, 19 (67.9%) patients with inflammatory breast cancer (IBC) and 12 (42.9%) patients with primary, non-IBC breast cancer, respectively. Isolated peripheral blood mononuclear cells (PBMC) were depleted of cells of hematopoietic origin (CD45+) using anti-CD45 coated magnetic beads. RNA extracted from CD45-depleted (CD45-) PBMC were interrogated for expression of TWIST1, SNAIL1, SLUG, ZEB1, FOXC2 and EpCAM gene transcripts by quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC from patients were compared to those of CD45- PBMC of healthy donors (HD). Expression of one or more gene transcripts was considered a positive result. Concurrently, a 7.5 mL PB sample was collected for determination of CTC by CellSearch.Results: Median age was 54 year (range: 34-72 years). Overall, the median CTC count by CellSearch was 2 (range; 0-750) per 7.5 mL of PB. TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2 were overexpressed in CD45- PBMC in 7%, 4 %, 4%, 0% and 14 % of patients, respectively. At least one of the EMT genes was overexpressed in 6 (21%) of patients. TWIST1 and SLUG were overexpressed only in IBC patients (10.5% and 5.3% of patients, respectively). Patients with triple negative breast cancer more commonly overexpressed EMT genes compared to non-triple negative patients (30.8% vs. 13.3%). There was no correlation between expression of EMT genes, EpCAM expression or CTC count measured by CellSearch, respectively.Conclusions: These data suggest that EMT genes may be involved in the dissemination of CTCs. Loss of epithelial antigen on CTC due to EMT, triggered by high expression of these genes, may be responsible for their undetection by conventional methods in a fraction of patients with early or advanced breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3011.