Annals of Oncology Advance Access published online on May 21, 2007
Annals of Oncology, doi:10.1093/annonc/mdm208
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© 2007 European Society for Medical Oncology
Clinical relevance of circulating CK-19 mRNA-positive cells detected during the adjuvant tamoxifen treatment in patients with early breast cancer
1 Department of Medical Oncology,University General Hospital of Heraklion, Crete
2 Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Heraklion
3 Department of Pathology, University General Hospital of Heraklion, Crete
4 Department of Medical Oncology, University General Hospital of Alexandroupolis, Alexandroupolis, Greece
* Correspondence to: Dr V. Georgoulias, Department of Medical Oncology, University General Hospital of Heraklion, PO Box 1352, 711 10 Heraklion, Crete, Greece. Tel: +30-2810-392823; Fax: +30-2810-392802; E-mail: georgsec{at}med.uoc.gr
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Abstract |
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 Top Abstract introductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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Background: The purpose of this study was to evaluate the effect of adjuvant treatment with tamoxifen on the CK-19 mRNA+ cells in patients with early-stage breast cancer.
Patients and methods: CK-19 mRNA+ cells were prospectively and longitudinally detected using a specific real-time PCR assay for CK-19 mRNA in 119 patients with estrogen and/or progesterone receptor-positive tumors during the period of tamoxifen administration.
Results: Twenty-two (18.5%) patients had detectable CK-19 mRNA+ cells after the completion of adjuvant chemotherapy and in 15 (68.2%) of them adjuvant tamoxifen could not eliminate these cells (persistently positive). In 68 (57.1%) patients, no CK-19 mRNA+ cells could be detected throughout the follow-up period (persistently negative). Seven (46.7%) of the 15 persistently positive and six (8.8%) of the 68 persistently negative patients developed disease recurrence (P = 0.00026). Persistency of CK-19 mRNA+ cells was associated with a significantly lower median disease-free interval (P = 0.0001) and overall survival (P = 0.0005). Multivariate analysis revealed that the detection of CK-19 mRNA+ cells during the administration of tamoxifen was associated with an increased risk of relapse [hazard ratio (HR) = 22.318, P = 0.00006] and death (HR = 13.954, P < 0.00001).
Conclusions: The detection of CK-19 mRNA+ cells throughout the period of adjuvant tamoxifen treatment is an independent poor prognostic factor in patients with early breast cancer.
breast cancer, circulating tumor cells, CK-19, tamoxifen
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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In patients with early-stage breast cancer, metastasis is characterized by tumor cell migration from the original tumor to distant organs. Several clinicopathological parameters [i.e. menopausal status, tumor size, histopathological grade, number of involved axillary lymph nodes, expression of HER2 and estrogen receptors (ERs)] are established independent prognostic factors associated with a poor disease-free interval (DFI) and overall survival (OS) [1,2]. Similarly, the detection of ‘disseminated’ tumor cells (DTCs) or ‘circulating’ tumor cells (CTCs) in bone marrow aspirates and peripheral blood of otherwise metastases-free breast cancer patients was shown to be an independent prognostic factor associated with a poor clinical outcome [3–7].
Adjuvant chemotherapy followed by hormone treatment is the standard of care for the majority of patients with estrogen- and/or progesterone-positive tumors. The survival benefit associated with the adjuvant treatment should be attributed to the eradication of occult DTCs and CTCs in these patients. However, adjuvant chemotherapy could ‘eliminate’ the DTCs or the CTCs in only 50% of patients; moreover, 
30% of patients may present detectable DTCs or/and CTCs during the period of adjuvant chemotherapy [8, 9]. The ‘resistance’ of occult tumor cells to the various chemotherapy regimens has been attributed to the ‘dormant’ and ‘nonproliferating’ status of these cells [10] which may be related to their genetic [11, 12] and/or biologic [13, 14] heterogeneity.
Our group decided to conduct a prospective and longitudinal study of CK-19 mRNA+ cells since there is no information concerning the effect of tamoxifen, given in the adjuvant setting, on the peripheral blood CTCs. The data presented here indicate that in a substantial proportion of patients, tamoxifen failed to eliminate ‘chemotherapy resistant’ CK-19 mRNA+ cells; in addition, their detection during the period of adjuvant tamoxifen administration was an independent prognostic factor associated with an unfavorable clinical outcome.
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patients and methods |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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patients and treatment
One hundred and nineteen patients with ER- and/or progesterone receptor (PR)-positive early (stage I and II) breast cancer were enrolled onto the study. Eligible patients had to have ‘primary’ tumor surgically resected, adjuvant chemotherapy and radiotherapy completed with no clinical or radiological evidence of metastatic disease at the time of enrollment. All patients received adjuvant chemotherapy in the context of research protocols of the Hellenic Oncology Research Group consisting of either FEC (5-fluorouracil 700 mg/m2 on day 1 + epirubicin 75 mg/m2 on day 1 + cyclophosphamide 700 mg/m2 on day 1 every 3 weeks for six cycles) or EC-T (epirubicin 75 mg/m2 on day 1 + cyclophosphamide 700 mg/m2 on day 1 every 3 weeks for four cycles followed by docetaxel (Taxotere) 100 mg/m2 on day 1 every 3 weeks for four additional cycles) or classical CMF (cyclophosphamide 100 mg/m2 orally on days 1–14, methotrexate 40 mg/m2 on days 1 and 8, fluorouracil 600 mg/m2 on days 1 and 8, every 4 weeks for six cycles). Patients who underwent breast-conserving surgery received adjuvant radiation therapy. All patients with ER- and/or PR-positive tumors received tamoxifen 20 mg daily for 5 years; premenopausal women also received luteinizing hormone-releasing hormone analogs for 2 years. Patient follow-up consisted of clinical examination with laboratory and imaging studies every 3 months for the first 2 years, every 6 months for the next 3 years and yearly thereafter.
Peripheral blood (10 ml in EDTA) was obtained at the middle of vein puncture in order to avoid contamination of blood with skin epithelial cells, at the following time points: (i) before and after the completion of adjuvant chemotherapy, (ii) every 6 months during the 5 years of the adjuvant tamoxifen administration and (iii) every 12 months thereafter. All patients gave their informed consent to participate in the study, which had been approved by the Ethics and Scientific Committees of our Institution.
RNA extraction and real-time RT-PCR assay for CK-19 mRNA-positive cells
Peripheral blood mononuclear cells were obtained by gradient density centrifugation using Ficoll-Hypaque as previously described [7]. Total RNA isolation was carried out by using Trizol LS reagent (Gibco, Life Sciences, BRL, Grand Island, NY) according to the manufacturer's instructions. All RNA preparations and handling steps took place in a laminar flow hood under RNAse-free conditions. The isolated RNA was dissolved in diethylpyrocarbonate-treated water and stored at –80°C until used. RNA concentration was determined by absorbance reading at 260 nm with the Hitachi UV-VIS (U-2000) spectophotometer (Tokyo, Japan). RNA integrity was tested by PCR amplification of the ß-actin housekeeping gene. RNA samples prepared from the MCF-7 breast cancer and ARH-77 leukemic cell lines were used as positive and negative controls, respectively.
Reverse transcription of RNA was carried out with the Thermoscript RT-PCR system (Invitrogen, Paisley, UK). Complementary DNA (cDNA) was synthesized according to the manufacturer's instructions. The real-time RT-PCR assay for the detection of CK-19 mRNA-positive cells and its analytical details (specificity, sensitivity, cut-off for positivity) and the used primers have been already described [15]. In brief, 2 µl of cDNA were placed into a-18 µl reaction volume containing 1 µl of the sense primer CK-19- for (3 mM), 1 µl of the antisense primer CK-19-do (3 mM), 2.4 µl of the LighCycler Fast Start DNA Master Hybridization Probes reagent (10x concentration), 1 µl of the probe CK-19-FL (3 mM) and 1 µl of the probe CK-19-LC (3 mM). The details of the cycling protocol have been previously described [15] and the quality of cDNAs was evaluated by real-time PCR for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. The lower detection limit of the assay was set at >0.6 MCF-7 cell equivalents/5 µg RNA for the patients' blood sample [15].
Patients with detectable CK-19 mRNA+ cells after the completion of adjuvant chemotherapy as well as during the adjuvant tamoxifen treatment (at least two positive consecutive samples) were considered as CK-19 mRNA ‘persistently positive’, in contrast to those without detectable CK-19 mRNA+ cells during the same period who were considered as ‘persistently negative’. Patients with detectable CK-19 mRNA+ cells only after adjuvant chemotherapy but not during the follow-up period or the reverse were considered as ‘occasionally positive’. CK-19 mRNA+ cells detectable upon the completion of adjuvant chemotherapy were considered as chemotherapy resistant whereas those detectable during the administration of tamoxifen were considered as ‘tamoxifen resistant’.
statistical analysis
The main tools of analysis were logistic regression and the Cox proportional hazards model [16, 17] for outcomes related to point events and time variables, respectively. To select those factors with an independent significant influence on outcomes, both analyses were carried out in a stepwise (unconditional backward) fashion [16, 17]. Before the application of these methods, univariate analyses were carried out for a preliminary exploration of marked associations. Univariate analyses included contingency tables, independent-samples t-test or Mann–Whitney tests, log-rank tests and simple Cox regression analyses [16–18].
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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effect of adjuvant chemotherapy and tamoxifen on CK-19 mRNA+ cells
Table 1 summarizes the patient' characteristics at the time of primary diagnosis according to the detection of CK-19 mRNA+ cells after the completion of adjuvant chemotherapy. The detection of CK-19 mRNA+ cells after chemotherapy did not correlate with the different clinicopathological characteristics (Table 1). The median follow-up period for the whole group of patients was 65 months (range, 22–106 months).
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After the completion of adjuvant chemotherapy, 22 (18.5%) patients had detectable CK-19 mRNA+ cells; 17 (77.3%) of these patients had detectable CK-19 mRNA+ cells before the initiation of adjuvant chemotherapy whereas five did not (Figure 1, Table 2). The median number of postchemotherapy CK-19 mRNA+ cells was 4.4 (range, 0.6–116) MCF-7 equivalent cells/5 µg RNA. The administration of tamoxifen failed to eliminate the CK-19 mRNA+ cells in 15 (68.2%) of these 22 patients (Figure 1, Table 2), indicating that, in these particular patients, CK-19 mRNA+ cells were both chemotherapy-resistant and tamoxifen-resistant; according to the predefined criteria these patients were characterized as persistently positive. Moreover, during the administration of adjuvant tamoxifen, CK-19 mRNA+ cells could be detected in seven (36.8%) patients with prechemotherapy+/postchemotherapy–, in five (100%) with prechemotherapy–/postchemotherapy+ and in 10 (12.8%) with prechemotherapy–/postchemotherapy– CK-19 mRNA status (Figure 1, Table 2). The median time required for CK-19 mRNA-positive patients to become CK-19 mRNA negative during the administration of tamoxifen was 7.5 months (range, 3–34) and for the CK-19 mRNA-negative patients to become CK-19 mRNA-positive was 16 months (range, 2–42).
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clinical recurrences
Twenty-five (21%) clinical recurrences were observed among the 119 patients during the follow-up period (Table 2). Seven (46.6%) out of 15 patients with persistently positive and six (8.8%) out of 68 patients with persistently negative CK-19 mRNA status presented a clinical relapse (P = 0.00026; Table 2). Overall, the incidence of clinical recurrences was 37.3% (19 of 51 patients) in patients with detectable CK-19 mRNA+ cells (both persistently positive and occasionally positive) compared with 8.8% (six of 68 patients) with persistently negative CK-19 mRNA status during the administration of adjuvant tamoxifen treatment (P = 0.00016; Table 2). Eleven (64.7%) out of 17 patients who had undetectable CK-19 mRNA+ cells after the completion of adjuvant chemotherapy but had detectable CK-19 mRNA+ cells during the administration of adjuvant tamoxifen (six prechemotherapy+/postchemotherapy– and five prechemotherapy–/postchemotherapy–) presented a clinical recurrence; conversely, only seven (8.5%) recurrences were documented among the 82 patients who did not have detectable CK-19 cells during the same period (Pearson's x2 test, P < 0.0001).
disease-free interval
The patients' median DFI has not been reached; however, in patients without detectable CK-19 mRNA+ cells after the completion of adjuvant chemotherapy, the DFI was significantly higher [mean ± standard error (SE): 72.6 ± 2.7 months; range, 3–84] than that of patients with detectable CK-19 mRNA+ cells at the same time (mean ± SE: 55.5 ± 5.6 months; range, 6–75) (Log-rank test, P = 0.0196; Figure 2A). Similarly, the median DFI was significantly decreased in patients with (i) persistently positive compared to those with persistently negative CK-19 mRNA status (70 months versus not reached, P = 0.0001; Figure 3A), (ii) occasionally positive compared to those with persistently negative CK-19 mRNA status (P = 0.0018; not shown) and (iii) detectable CK-19 mRNA+ cells at any time during the administration of tamoxifen compared with those without detectable CK-19 mRNA+ cells during the same period (P < 0.00005; Figure 4A).
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overall survival
The median OS of patients has not yet been reached; the median OS was not statistically different in patients with or without detectable CK-19 mRNA+ cells after the completion of adjuvant chemotherapy (P = 0.2601; Figure 2B). Conversely, the median OS was significantly higher in patients with (i) persistently negative compared with those with persistently positive CK-19 mRNA status (P = 0.0005; Figure 3B), (ii) occasionally positive compared to those with persistently negative CK-19 mRNA status and (iii) detectable CK-19 mRNA+ cells at any time during the administration of tamoxifen compared with those with a persistently negative status at the same period (P < 0.00005; Figure 4B).
univariate and multivariate analysis
Univariate analysis (Table 3) revealed that the tumor size (P = 0.006), the premenopausal status (P = 0.017), the detection of CK-19 mRNA+ cells after the completion of adjuvant chemotherapy (P = 0.043) as well as during the period of tamoxifen administration (P < 0.00005) and the ‘persistence’ of CK-19 mRNA+ cells throughout the period of adjuvant treatment (P = 0.0001) were significantly associated with an increased risk of relapse. Similarly, the tumor size (P = 0.009), the detection of CK-19 mRNA+ cells during the period of tamoxifen administration (P = 0.00005) as well as the persistence of CK-19 mRNA+ cells throughout the period of adjuvant treatment (P = 0.0005) were significantly associated with a reduced OS (Table 3).
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Multivariate analysis demonstrated that the menopausal status (P = 0.010), the lymph node involvement (P = 0.023), the detection of CK-19 mRNA+ cells after the completion (P = 0.002) of adjuvant chemotherapy as well as during the period of tamoxifen administration (P = 0.001) and the persistence of CK-19 mRNA+ cells throughout the period of adjuvant treatment (P = 0.00006) were emerged as independent prognostic factors for early relapse. Conversely, only the tumor size (P = 0.039), the number of involved axillary lymph nodes (P = 0.005) and the detection of CK-19 mRNA+ cells during the period of tamoxifen administration (P < 0.00001) were emerged as independent prognostic factors for reduced survival (Table 4).
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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This is the first longitudinal study, which prospectively evaluated the effect of tamoxifen, given in the adjuvant setting, on the fate of peripheral blood CK-19 mRNA+ cells in patients with early-stage breast cancer. Our findings indicate that (i) most of the patients with detectable CK-19 mRNA+ cells after the completion of adjuvant chemotherapy had ‘persistent’ cells during the period of tamoxifen administration indicating that these cells may be both ‘chemotherapy and tamoxifen resistant’ and (ii) the detection of CK-19 mRNA+ cells during the period of adjuvant tamoxifen treatment represents an independent prognostic factor associated with a poor clinical outcome.
The malignant nature of cytokeratin-positive cells has been demonstrated using genomic analysis and specialized cell culture assays [13, 14, 19, 20]. In a previous study [21], we were able to cross-validate in a limited number of patients, the malignant nature of these cytokeratin-positive cells by carrying out FISH analysis for HER-2/neu gene amplification and aneusomy detection for chromosomes 1,8,11,17. Other investigators have also used FISH analysis for these markers to detect breast cancer cells in cytological specimens [22–24].
It has been previously shown that DTCs or CTCs could be detected in bone marrow aspirates or peripheral blood of patients with early-stage breast cancer during or after the administration of adjuvant chemotherapy and/or tamoxifen [8, 9, 25]. The detection of CTCs after the completion of adjuvant chemotherapy clearly indicates that these cells have an intrinsic resistance to the used chemotherapy regimen, which has been attributed to the non-proliferating and dormant status of these cells [10, 26]. Alternatively, given the identification of tumorigenic breast cancer cells [27], we could also hypothesize that the treatment-resistant phenotype of at least some of these cells could be attributed to their ‘stem-cell’ nature and the associated gene signature [28].
In previous studies, it has been demonstrated that DTCs and CTCs represent a heterogeneous cell population [10–14]; therefore, it cannot be excluded that chemotherapy may be active against some DTCs/CTCs clones while it fails to eliminate others. The observation that chemotherapy may persistently eliminate the CTCs in about half of the patients is in favor of this hypothesis. However, comparative genomic and phenotypic studies of DTCs and CTCs before and after completion of adjuvant chemotherapy are needed in order to further investigate the functional heterogeneity of different DTCs and/or CTCs clones.
The current study also demonstrated that tamoxifen failed to eliminate the CTCs which remained detectable after the completion of adjuvant chemotherapy (prechemotherapy+/postchemotherapy+ group) in a substantial proportion of patients, indicating that chemotherapy-resistant CTCs may be also tamoxifen-resistant. In addition, 17.5% of the patients without detectable CTCs upon completion of adjuvant chemotherapy had CK-19 mRNA+ cells during the period of adjuvant tamoxifen treatment; per definition, these cells are also considered as tamoxifen resistant. This observation seems to indicate that some undetectable, probably because of their low frequency, chemotherapy-resistant cells can persist after the completion of adjuvant chemotherapy; these cells could proliferate despite the administration of tamoxifen because they are tamoxifen resistant, thus, reaching a frequency which could be detected by the used real-time PCR assay. Similar findings have also been reported by other investigators [29–31]. Therefore, it seems reasonable to hypothesize that these cells are ‘resistant’ or/and ‘refractory’ to both chemotherapy and tamoxifen and their detection might be an unfavorable prognostic factor; the high incidence of clinical recurrences in this particular group of patients is in favor of the above hypothesis.
In the present study, five patients who had undetectable CK-19 mRNA+ cells before the initiation and after the completion of adjuvant chemotherapy presented detectable CK-19 mRNA+ cells during the period of tamoxifen administration; it is interesting that no clinical relapses were observed in these particular patients. It is difficult to elaborate a valuable explanation for the exceptional clinical outcome of these patients but we cannot exclude that their CK-19 mRNA+ cells may have an impaired clonogenic or metastatic potential. A more detailed biological analysis of these cells combined with a longer observation period is required in order to better understand the clinical relevance of this situation. Conversely, the detection of CK-19 mRNA+ cells at the time of diagnosis but not after the completion of adjuvant chemotherapy or the period of tamoxifen administration could be attributed to the effective ‘elimination’ of these cells by the various systemic adjuvant treatments. This hypothesis is also supported by the very low incidence of recurrences observed in this group of patients. In the present study, the detection of CK-19 mRNA+ cells either after the completion of adjuvant chemotherapy or during the administration of adjuvant tamoxifen was associated with an increased risk of clinical recurrence and disease-related death; multivariate analysis further confirmed that their detection at any time during the adjuvant treatment was an independent prognostic factor associated with an unfavorable outcome.
An important clinical implication of our findings could be the opportunity to develop tailored and individualized adjuvant therapeutic approaches in patients with detectable CK-19 mRNA+ cells after the administration of primary adjuvant treatment since these cells express c-erbB2, p53 and urokinase-type plasminogen activator receptor [32, 33]. Moreover, it has been shown that monoclonal antibodies such as edrecolomab and transtuzunab can eliminate the bone marrow CK+/EpCAM+ and peripheral blood CK+/HER2+ occult bone marrow or peripheral blood cells [21, 34], respectively. Therefore, the evaluation of the efficacy of different adjuvant treatment modalities on the occult tumor cells might open new ways of monitoring their effectiveness and evaluate novel ‘secondary’ targeted therapeutic approaches in patients with early-stage breast cancer.
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Acknowledgements |
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This work was partly supported by research grants from Astra Zeneca, the Hellenic Society of Medical Oncology and the Cretan Association for Biomedical Research (CABR). AP, GS, DP-P and LK were recipients of a CABR clinical or research fellowship.
Received for publication December 11, 2006. Revision received March 21, 2007. Accepted for publication April 25, 2007.
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