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Abstract S6-5: Prognostic Relevance of Circulating Tumor Cells in the Peripheral Blood of Primary Breast Cancer Patients
Abstract
The prognostic relevance of disseminated tumor cells in bone marrow of breast cancer patients at the time of primary diagnosis and during recurrence-free follow-up has been confirmed by large pooled analyses. Furthermore, circulating tumor cells (CTC) in peripheral blood have been shown as predictor of shortened progression-free and overall survival in metastatic disease. In view of the lack of data in early breast cancer, we evaluated whether the presence of CTC before the start of systemic adjuvant treatment increases the risk of subsequent relapse and death.
Patients and Methods The SUCCESS A-Study is an open-label randomized controlled, phase III study comparing the disease-free survival after randomisation in patients treated with 3 cycles of Epirubicin (100 mg/m²)-Fluorouracil(500)- Cyclophosphamide (500, FEC)-chemotherapy, followed by 3 cycles of Docetaxel (100 mg/mg², D) versus 3 cycles of FEC, followed by 3 cycles of Gemcitabine (1,000mg/m² d1,8)-Docetaxel (75 mg/m²)(DG). In 2026 patients CTC were analyzed using the CellSearchSystem (Veridex, USA). 23 ml of peripheral blood were drawn after R0-resection of the primary tumor but before the start of adjuvant systemic treatment. After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labelled with anti-Ck8/18/19 and anti-CD45 antibodies to distinguish between epithelial cells and leukocytes. Patients were followed for a median of 35 months. Univariate and multivariable proportional hazards models were estimated to assess the prognostic significance of CTC for disease-free and overall survival. Patients with evidence of at least 1 CTC were counted as positive.
Results In 21.5% of patients (n = 435) CTC were detected before the start of systemic treatment (median 1.3, range 1 - 827). Patients with CTC before treatment were more frequently node-positive (P<0.001), but no correlation to tumor size, grading and HR-Status could be found. 114 recurrences occurred and 66 patients died of their disease. The presence of CTC before systemic treatment predicted poor disease-free (p < 0.0001), distant disease-free survival (p < 0.001) and overall survival (p = 0.0002). In multivariate analysis, detection of CTC before treatment was confirmed as independent predictor for both disease-free (HR 1.88) and overall survival (HR 1.91) next to tumor size, grading, lymph node involvement and hormone receptor status (p for all < 0.05). Outcome of patients was correlated to the number of CTC. Prognosis was worst in patients with 5 CTC or more with a four-fold increased risk for recurrence and and a three-fold increased risk for death (HR 4.04 for DFS and 3.05 for OAS; p < 0.05).
This is the first study to prospectively demonstrate the prognostic relevance of CTC in peripheral blood of early breast cancer patients before the start of systemic treatment in a large patient cohort. CTC detection could serve as clinically useful prognostic marker and treatment monitoring tool and should be tested as indicator for secondary adjuvant treatment interventions within clinical trials.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S6-5.
... Data are emerging regarding the predictive significance of CTCs in patients with non-metastatic breast cancer. Results from the French REMAGUS trial [12], the German SUCCESS-A trial [13], and a recent report from our group [14] indicate that the identification of one or more circulating tumor cell predicted progression-free and overall survival in patients with non-metastatic breast cancer. However, no data have been published about the prognostic significance of circulating tumor cell identification specifically in patients with nonmetastatic TNBC. ...
... This is one of the first reports demonstrating that the identification of two or more CTCs independently predicted both progression-free interval and overall survival in non-metastatic triple-negative breast cancer patients. Previously published studies included a smaller number of triple-negative patients [11,13] or did not assess the prognostic significance of CTCs specifically for the triplenegative subtype [13,19]. In agreement with Kim et al. [20], we found that standard prognostic indicators such as [2 cm tumor size and high tumor grade (Grade 3) did not often predict outcome in TNBC patients. ...
... This is one of the first reports demonstrating that the identification of two or more CTCs independently predicted both progression-free interval and overall survival in non-metastatic triple-negative breast cancer patients. Previously published studies included a smaller number of triple-negative patients [11,13] or did not assess the prognostic significance of CTCs specifically for the triplenegative subtype [13,19]. In agreement with Kim et al. [20], we found that standard prognostic indicators such as [2 cm tumor size and high tumor grade (Grade 3) did not often predict outcome in TNBC patients. ...
Circulating tumor cells (CTCs) can be identified in approximately 25 % of stage I-III breast cancer patients; CTCs presence is a predictor of poor outcome in metastatic breast cancer, but little is known regarding the prognostic significance of CTCs in non-metastatic triple-negative breast cancer (TNBC) patients. The aim of this study was to determine whether CTCs predict worse outcome in non-metastatic TNBC patients. We evaluated CTCs in 113 patients with stages I-III TNBC at the time of definitive surgery. CTCs were assessed using the CellSearch System(®). Progression-free and overall survival were defined as time elapsed between date of diagnosis and either date of clinical disease progression, death, or last follow-up. Log-rank test and Cox regression analysis were used to determine associations of CTCs with progression-free and overall survival. The median follow-up was 40 months. CTCs were identified in 23/113 (20 %) of patients. No primary tumor characteristic or lymph node status predicted the presence of CTCs. The identification of ≥2 CTCs predicted shorter progression-free (log rank P ≤ 0.001; hazard ratio 8.30, 95 % CI 2.61-26.37) and overall survival (log rank P = 0.0004; hazard ratio 7.19, 95 % CI 1.98-26.06) versus survival for patients with <2 CTCs. Two or more CTCs predict shorter progression-free and overall survival in TNBC patients. Larger studies are needed to determine whether CTC assessment provides beneficial information that could be used in stratifying TNBC patients at increased risk for disease progression. Finally, CTCs characterization could facilitate the development of novel treatment approaches for TNBC.
... The mean and standard deviation of the doubling time was determined from the time to DM. CTC concentration was fit to available literature values; patients with metastatic disease have 3.0 CTC/mL (5-95 percentile: 0.02-417 CTC/mL) [8][9][10][11], and patients with early stage breast cancer have CTC at a mean concentration of 0.03 CTC/mL (range of estimates 0.01-0.05 CTC/mL) [12][13][14][15]. ...
... The CTC concentration reported for primary breast cancer before surgery is 0.03 CTC/mL (range 0.01-0.05 CTC/mL [12][13][14][15]). We now find a dissemination rate for an 8 mm tumor of 280 CTC/h · g tumor (range 90-470 CTC/h · g tumor) and a metastatic efficiency of 1.7 · 10 -8 metastases formed per disseminated cell (range 1.3 · 10 -8 -4.2 · 10 -8 ), or approximately 60 million disseminated cells per formed macrometastasis. ...
... Although improvement in the risk assessment helps to identify the patients that need additional therapy after surgical removal of the primary tumor, detection of the actual presence of tumor cells beyond the primary tumor is preferred. Indeed the presence of micrometastases in bone marrow [52,53] and tumor cells in blood [12][13][14]54] of breast cancer patients have been associated with an increased risk for disease recurrence, but have not become part of clinical practice partly because the current technology lacks sufficient sensitivity and specificity. The observations that CTC have been detected in patients years after a diagnosis and treatment of breast cancer with curative intent further challenges the technology to identify those CTC characteristics that predict imminent relapse [55,56]. ...
Background
To establish a distant metastasis (DM) cells must disseminate from the primary tumor and overcome a series of obstacles, the metastatic cascade. In this study we develop a mathematical model for this cascade to estimate the tumor size and the circulating tumor cell (CTC) load before the first metastasis has formed from a primary breast cancer tumor.
Methods
The metastatic cascade is described in discrete steps: 1. local tumor growth; 2. dissemination into circulation; 3. survival in circulation; 4. extravasation into tissue; and 5. growth into a metastasis. The model was built using data and relationships described in the literature to predict the relationship between tumor size and probability of distant metastasis for 38715 patients with surgically removed TXNXM0 primary breast cancer from the Netherlands Cancer Registry. The model was calibrated using primary tumor size, probability of distant metastasis and time to distant metastasis for 1489 patients with stage T1BNXM0 (25% of total patients with T1BNXM0). Validation of the model was done with data for all patients.
Results
From the time to distant metastasis of these 38715 breast cancer patients, we determined a tumor doubling time of 1.7 ± 0.9 months. Fitting the data for 25% of T1B patients estimates a metastatic efficiency of 1 metastasis formed per 60 million disseminated tumor cells. Validation of the model to data of patients in all T-stages shows good agreement between model and epidemiological data. To reduce the 5-year risk of distant metastasis for TXNXM0 from 9.2% to 1.0%, the primary tumor needs to be detected and removed before it reaches a diameter of 2.7 ± 1.6 mm. At this size, the model predicts that there will be 9 ± 6 CTC/L blood.
Conclusions
To reduce the rate of distant metastasis in surgically treated TXNXM0 breast cancer to 1%, imaging technology will need to be able to detect lesions of 2.7 mm in diameter or smaller. Before CTC detection can be applied in the early disease setting, sensitivity will need to be improved by at least 15-fold and combined with technology that minimizes false positives.
... In patients prior to pre-neoadjuvant chemotherapy and definitive surgery the frequency is even lower and 1 or more CTC were detected in 7.5 ml of blood in 23 to 24% of these patients [29,33]. In the ongoing German SUCCESS study, 21% of patients at primary diagnosis of breast cancer, 1 or more CTC were detected in 23 ml of blood [34,35]. In this study, four 7.5 ml aliquots of blood from four blood collection tubes were investigated for the presence of CTC to increase the sensitivity of CTC detection with the FDA-cleared CellSearch CTC test. ...
... Still for the identification of patients with cancer the specificity of the CTC test at this low number needs to be improved. Some improvement can be obtained by elimination of the variation between operators in assigning the identified objects as CTC [35]. Although automation of the CTC assignment can eliminate this error, it does not imply that the correct CTC definition is used [38]. ...
... This, however, will not obliterate the specificity issue, as it is clear that the specificity of the CTC assay will need to be increased to assure that the detected objects are indeed cancer cells. The specificity of CTC detection was brought to light by the present study through the inclusion of patients with DCIS and benign breast disease in a completely blinded fashion in contrast to other CTC studies using the CellSearch system for the enumeration of CTC in the early disease setting in which no such controls were included [29,[33][34][35]. Moreover, one would like to discriminate between dormant, viable and tumor cells that have metastatic potential. ...
Introduction
The presence of circulating tumor cells (CTC) is an independent prognostic factor for progression-free survival and breast cancer-related death (BRD) for patients with metastatic breast cancer beginning a new line of systemic therapy. The current study was undertaken to explore whether the presence of CTC at the time of diagnosis was associated with recurrence-free survival (RFS) and BRD.
Methods
In a prospective single center study, CTC were enumerated with the CellSearch system in 30 ml of peripheral blood of 602 patients before undergoing surgery for breast cancer. There were 97 patients with a benign tumor, 101 did not meet the inclusion criteria of which there were 48 patients with DCIS, leaving 404 stage I to III patients. Patients were stratified into unfavorable (CTC ≥1) and favorable (CTC = 0) prognostic groups.
Results
≥1 CTC in 30 ml blood was detected in 15 (15%) benign tumors, in 9 DCIS (19%), in 28 (16%) stage I, 32 (18%) stage II and in 16 (31%) patients with stage III. In stage I to III patients 76 (19%) had ≥1 CTC of whom 16 (21.1%) developed a recurrence. In 328 patients with 0 CTC 38 (11.6%) developed a recurrence. Four-year RFS was 88.4% for favorable CTC and 78.9% for unfavorable CTC (P = 0.038). A total of 25 patients died of breast cancer-related causes and 11 (44%) had ≥1 CTC. BRD was 4.3% for favorable and 14.5% for unfavorable CTC (P = 0.001). In multivariate analysis ≥1 CTC was associated with distant disease-free survival, but not for overall recurrence-free survival. CTC, progesterone receptor and N-stage were independent predictors of BRD in multivariate analysis.
Conclusions
Presence of CTC in breast cancer patients before undergoing surgery with curative intent is associated with an increased risk for breast cancer-related death.
... In both these studies, however, neither CTC detection before or after NAC, nor changes in CTC detection during treatment , were predictive of pathological complete response [53,55]. Rack and colleagues [56] detected ≥1 CTC/22.5 ml before the start of adjuvant treatment in 21.5% of 2,026 patients with early BC [56] . In this study, pretreatment detection of CTCs was confi rmed as an indepen dent predictor for both disease-free survival and overall survival [56]. ...
... In both these studies, however, neither CTC detection before or after NAC, nor changes in CTC detection during treatment , were predictive of pathological complete response [53,55]. Rack and colleagues [56] detected ≥1 CTC/22.5 ml before the start of adjuvant treatment in 21.5% of 2,026 patients with early BC [56] . In this study, pretreatment detection of CTCs was confi rmed as an indepen dent predictor for both disease-free survival and overall survival [56]. ...
... Rack and colleagues [56] detected ≥1 CTC/22.5 ml before the start of adjuvant treatment in 21.5% of 2,026 patients with early BC [56] . In this study, pretreatment detection of CTCs was confi rmed as an indepen dent predictor for both disease-free survival and overall survival [56]. Several other investigators have detected CTCs by CellSearch® in 9% to 38% of patients with early BC without reporting survival data575859. ...
Tumor cell dissemination in bone marrow or other organs is thought to represent an important step in the metastatic process. The detection of bone marrow disseminated tumor cells is associated with worse outcome in early breast cancer. Moreover, the detection of peripheral blood circulating tumor cells is an adverse prognostic factor in metastatic breast cancer, and emerging data suggest that this is also true for early disease. Beyond enumeration, the characterization of these cells has the potential to improve risk assessment, treatment selection and monitoring, and the development of novel therapeutic agents, and to advance our understanding of the biology of metastasis.
... Also, the CTC count in peripheral blood of metastatic cancer patients during therapy directly reflects the patient's response to therapy [16, 17]. The prognostic role of CTC in primary (non-metastatic) cancer has not been widely investigated [18], but a few studies have shown that the presence of CTCs can predict poor prognosis also in patients with primary breast cancer [15, 19, 20] . Furthermore , a prospective study by Lucci et al. [21] has shown that the presence of one or more CTCs predicted both early recurrence and decreased overall survival in 302 nonmetastatic breast cancer patients independent of prognostic factors such as tumor size or grade. ...
... In this pilot study, we found no evidence of tumor cell shedding to the peripheral blood, as opposed to a substantial number of animal studies9101112, surgery shedding studies45678, and opinions expressed in letters of concern123 regarding tumor cell shedding following manipulation of the primary tumor. The presence of C1 CTC in 17 % of our subjects is close to the span of other studies (using different volumes of blood) of primary breast cancer patients (19–31 %) [15, 18, 20, 22, 31]. We could not find any relationship between the presence of CTCs and tumor characteristics (Table 1), which is consistent with other studies that have also found a lack of correlation between CTCs and histopathological factors [18, 29, 32]. ...
This pilot study aimed to investigate whether mammographic compression procedures might cause shedding of tumor cells into the circulatory system as reflected by circulating tumor cell (CTC) count in peripheral venous blood samples. From March to October 2012, 24 subjects with strong suspicion of breast malignancy were included in the study. Peripheral blood samples were acquired before and after mammography. Enumeration of CTCs in the blood samples was performed using the CellSearch(®) system. The pressure distribution over the tumor-containing breast was measured using thin pressure sensors. The median age was 66.5 years (range, 51-87 years). In 22 of the 24 subjects, breast cancer was subsequently confirmed. The difference between the average mean tumor pressure 6.8 ± 5.3 kPa (range, 1.0-22.5 kPa) and the average mean breast pressure 3.4 ± 1.6 kPa (range, 1.5-7.1 kPa) was statistically significant (p < 0.001), confirming that there was increased pressure over the tumor. The median pathological tumor size was 19 mm (range, 9-30 mm). Four subjects (17 %) were CTC positive before compression and two of these (8 %) were also CTC positive after compression. A total of seven CTCs were isolated with a mean size of 8 × 6 μm(2) (range of the longest diameter, 5-12 μm). The study supports the view that mammography is a safe procedure from the point of view of tumor cell shedding to the peripheral blood.
... The prognostic relevance of CTC detection in the peripheral blood of operable breast cancer patients, using the CellSearch ® system, has been evaluated in the SUCCESS trial. According to the most updated results, detection of at least one CTC in 23 mL of peripheral blood after surgical resection of the primary tumor and before the start of adjuvant systemic treatment was an independent predictor for worse DFS and OS in multivariate analysis [28]. The prognostic significance of CTC detection before and/or after the completion of adjuvant chemotherapy [17] , as well as the high specificity/sensitivity represent some advantages of the QPCR assay compared to the CellSearch ® . ...
... In this regard, the SWOG and the Breast Cancer Intergroup of North America have initiated a prospective trial in the metastatic setting to test whether patients with elevated CTC count (using the CellSearch ® system) after one cycle of first-line chemotherapy will benefit from a switch to a different chemotherapeutic regimen (SWOG protocol S0500). However, for patients with operable breast cancer the lower CTC detection rate post-chemotherapy makes this strategy far more challenging [28,30]. Additionally, the patients in our study received various types of adjuvant therapy based on available clinical and disease data at the time of enrolment. ...
The detection of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTC) before and/or after adjuvant chemotherapy in patients with operable breast cancer is associated with poor clinical outcome. Reliable prognostic markers for late disease relapse are not available. In this study we investigated the value of CTC detection during the first five years of follow-up in predicting late disease relapse.
Blood was analyzed from 312 women with operable breast cancer who had not experienced disease relapse during the first two years of follow-up. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) for CK-19 mRNA was used to detect CTC three months after the completion of adjuvant chemotherapy and every six months thereafter for a follow-up period of five years.
Eighty patients (25.6% of the study population) remained CTC free throughout the five-year period. A change in CTC status was observed in 133 patients (42.6%); 64 patients (20.5%) with initially CK-19 mRNA-positive CTC during the first 24 months turned CTC-negative afterwards while 69 (22.1%) who were initially CTC-negative became CTC-positive. Ninety-nine patients (31.7%) remained persistently CK-19 mRNA-positive. After a median follow-up period of 107 months (range: 38 to 161 months), the persistently CTC-positive patients with either hormonal receptor positive or negative tumors, had a higher risk of late-disease relapse compared to the persistently CTC-negative patients (36.4% versus 11.2%, P <0.001). Multivariate analysis revealed that persistently CTC-positive patients also had a shorter disease-free (P = 0.001) and overall survival (P = 0.001).
Persistent detection of CK-19 mRNA-positive CTC during the first five years of follow-up is associated with an increased risk of late relapse and death in patients with operable breast cancer and indicates the presence of chemo-and hormonotherapy-resistant residual disease. This prognostic evaluation may be useful when deciding on subsequent adjuvant systemic therapy.
... Yet, while the possibility of tumor contamination cannot be excluded for each case, it is unlikely to be the cause of the WBC BRCA1 methylation observed for several reasons. First, considering the number of circulating tumor cells, even among patients with a substantial cancer burden, such cells account for less than one in a million cells [50]. Second, as for circulating tumor DNA [51,52], the plasma volume currently required for detection of tumor-derived genomic aberrations in blood samples is far above any possible plasma remnants in our WBC assay. ...
Background
Normal cell BRCA1 epimutations have been associated with increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may have BRCA1 epimutations as their underlying cause is unknown. Neither are the time of occurrence and the potential inheritance patterns of BRCA1 epimutations established.
Methods
To address these questions, we analyzed BRCA1 methylation status in breast cancer tissue and matched white blood cells (WBC) from 408 patients with 411 primary breast cancers, including 66 TNBCs, applying a highly sensitive sequencing assay, allowing allele-resolved methylation assessment. Furthermore, to assess the time of origin and the characteristics of normal cell BRCA1 methylation, we analyzed umbilical cord blood of 1260 newborn girls and 200 newborn boys. Finally, we assessed BRCA1 methylation status among 575 mothers and 531 fathers of girls with (n = 102) and without (n = 473) BRCA1 methylation.
Results
We found concordant tumor and mosaic WBC BRCA1 epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (< 10%) tumors (combined: 14 out of 72; 19.4%; 95% CI 11.1–30.5). In contrast, we found concordant WBC and tumor methylation in only three out of 220 patients with 221 ER ≥ 10% tumors and zero out of 114 patients with 116 HER2-positive tumors. Intraindividually, BRCA1 epimutations affected the same allele in normal and tumor cells. Assessing BRCA1 methylation in umbilical WBCs from girls, we found mosaic, predominantly monoallelic BRCA1 epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals, but no correlation to BRCA1 methylation status either in mothers or fathers. A significantly lower fraction of newborn boys carried BRCA1 methylation (9/200; 4.5%) as compared to girls (p = 0.038). Similarly, WBC BRCA1 methylation was found less common among fathers (16/531; 3.0%), as compared to mothers (46/575; 8.0%; p = 0.0003).
Conclusions
Our findings suggest prenatal BRCA1 epimutations might be the underlying cause of around 20% of TNBC and low-ER expression breast cancers. Such constitutional mosaic BRCA1 methylation likely arise through gender-related mechanisms in utero, independent of Mendelian inheritance.
... Yet, tumor contamination is unlikely to be the cause of WBC BRCA1 methylation for several reasons. First; considering the number of circulating tumor cells, even among patients with a substantial cancer burden, such cells account for less than one in a million cells [46]. Second, as for circulating tumor DNA [47,48], the plasma volume currently required for detection of tumor-derived genomic aberrations in blood samples is far above any possible plasma remnants in our WBC assay. ...
Background: Normal cell BRCA1 epimutations have been associated with increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may have BRCA1 epimutations as their underlying cause is unknown.
Methods: To address this question, we analyzed BRCA1 methylation status in breast cancer tissue and matched white blood cells (WBC) from 411 patients with primary breast cancer, including 66 TNBCs, applying a highly sensitive sequencing assay, allowing allele-resolved methylation assessment. Further, to assess the time of origin and the characteristics of normal cell BRCA1 methylation, we analyzed umbilical cord blood of 1260 newborn girls.
Results: We found concordant tumor and mosaic WBC BRCA1 epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (<10%) tumors (combined: 14 out of 72; 19.4%; 95% CI 11.1–30.5). In contrast, we found concordance in only three out of 221 patients with ER≥10% tumors and zero out of 116 patients with HER2-positive tumors. Intraindividually, BRCA1 epimutations affected the same allele in normal and tumor cells. Assessing BRCA1 methylation in umbilical WBCs from girls, we found mosaic, predominantly monoallelic BRCA1 epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals.
Conclusions: Our findings reveal prenatal BRCA1 epimutations to be the underlying cause of around 20% of TNBC and low-ER expression breast cancers.
... Interestingly, after one cycle of treatment (V2), the enumeration of ≥5 CTCs in patients was also significantly associated with worse OS and PFS. Although, several studies have evaluated the prognostic value of CTCs enumeration in BC patients [8,17,18], only a number of them have explored the prognostic relevance of CTCs numbers before and after chemotherapy, supporting that the presence of persisting CTCs after treatment is associated with worse outcome, and that a decrease in the CTCs count is an early marker of individual response, during the follow-up [19,20]. ...
The study of circulating tumor cells (CTCs) has a huge clinical interest in advance and metastatic breast cancer patients. However, many approaches are biased by the use of epithelial markers, which underestimate non-epithelial CTCs phenotypes. CTCs enumeration provides valuable prognostic information; however, molecular characterization could be the best option to monitor patients throughout the disease since it may provide more relevant clinical information to the physicians. In this work, we aimed at enumerating and performing a molecular characterization of CTCs from a cohort of 20 patients with metastatic breast cancer (MBC), monitoring the disease at different time points of the therapy, and at progression when it occurred. To this end, we used a CTC negative enrichment protocol that allowed us to recover a higher variety of CTCs phenotypes. With this strategy, we were able to obtain gene expression data from CTCs from all the patients. In addition, we found that high expression levels of PALB2 and MYC were associated with a worse outcome. Interestingly, we identified that CTCs with an EpCAMhighVIMlowALDH1A1high signature showed both shorter overall survival (OS) and progression-free survival (PFS), suggesting that CTCs with epithelial-stem features had the most aggressive phenotype.
... Further, additional analyses of samples collected after chemother- apy, when many patients are considered to be diseasefree or to have a limited tumor burden, showed that methylation frequency was similar or higher than in samples collected at diagnosis. Of importance, the possibility that tumor DNA contamination (27) may have influenced our results seems negligible: The fraction of circulating tumor cells versus WBCs detected in the circulation is estimated to be less than 1 millionth (28), and the concentration of free tumor DNA in plasma is far lower than that observed in tissue and blood cells (29). Moreover, we confirmed positive methylation status in normal archival tissue from different organs in patients with positive WBC BRCA1 methylation status, adding support to the hypothesis that the epigenetic event is probably constitutive. ...
Background:
The role of normal tissue gene promoter methylation in cancer risk is poorly understood.
Objective:
To assess associations between normal tissue BRCA1 methylation and ovarian cancer risk.
Design:
2 case-control (initial and validation) studies.
Setting:
2 hospitals in Norway (patients) and a population-based study (control participants).
Participants:
934 patients and 1698 control participants in the initial study; 607 patients and 1984 control participants in the validation study.
Measurements:
All patients had their blood sampled before chemotherapy. White blood cell (WBC) BRCA1 promoter methylation was determined by using methylation-specific quantitative polymerase chain reaction, and the percentage of methylation-positive samples was compared between population control participants and patients with ovarian cancer, including the subgroup with high-grade serous ovarian cancer (HGSOC).
Results:
In the initial study, BRCA1 methylation was more frequent in patients with ovarian cancer than control participants (6.4% vs. 4.2%; age-adjusted odds ratio [OR], 1.83 [95% CI, 1.27 to 2.63]). Elevated methylation, however, was restricted to patients with HGSOC (9.6%; OR, 2.91 [CI, 1.85 to 4.56]), in contrast to 5.1% and 4.0% of patients with nonserous and low-grade serous ovarian cancer (LGSOC), respectively. These findings were replicated in the validation study (methylation-positive status in 9.1% of patients with HGSOC vs. 4.3% of control participants-OR, 2.22 [CI 1.40 to 3.52]-4.1% of patients with nonserous ovarian cancer, and 2.7% of those with LGSOC). The results were not influenced by tumor burden, storage time, or WBC subfractions. In separate analyses of young women and newborns, BRCA1 methylation was detected in 4.1% (CI, 1.8% to 6.4%) and 7.0% (CI, 5.0% to 9.1%), respectively.
Limitations:
Patients with ovarian cancer were recruited at the time of diagnosis in a hospital setting.
Conclusion:
Constitutively normal tissue BRCA1 promoter methylation is positively associated with risk for HGSOC.
Primary funding source:
Norwegian Cancer Society.
... In another study by Rack et al., 1767 patients with lymph node negative and node positive early breast cancer were evaluated for the presence of CTCs before and after treatment.[63] 10% of patients had >1 CTC before treatment, while 7% patients had >1 CTC after treatment. ...
Circulating tumor cell (CTC) measurement in peripheral blood of patients with breast cancer offers prognostic information. In this review, we will try to identify evidence that could be used for prognosis, predictive power to draw this tool to clinical utility. We reviewed 81 manuscripts, and categorized those in discovery datasets, prognostic factors in metastatic breast cancer, identification of clinical utility in early breast cancer and in novel approaches. With each patient responding differently to chemotherapy, more efficient markers would improve clinical outcome. Current CTC diagnostic techniques use epithelial markers predominantly; however, the most appropriate method is the measurement of circulating DNA. It has been hypothesized that micrometastasis occurs early in the development of tumors. That implies the presence of CTCs in nonmetastatic setting. The origin of stimulus for malignant transformation is yet unknown. The role of microenvironment as a stimulus is also being investigated. It has been shown that CTCs vary in numbers with chemotherapy. The markers, which are followed-up in the primary tumors, are also being studied on the CTCs. There is discordance of the human epidermal growth factor receptor-2 status between the primary tumor and CTCs. This review summarizes our current knowledge about the CTCs. With genetic profiling and molecular characterization of CTCs, it is possible to overcome the diagnostic difficulties. Evidence for clinical utility of CTC as prognostic and predictive marker is increasing. Appropriate patient stratification according to CTC determination among other tests, would make personalized cancer therapy more feasible.
... P = 0.010 [31]. Using CTCs (detected by CellSearch®) to predict progression in metastatic breast cancer (MBC) has been shown in a number of studies [32][33][34][35][36]. Also, several studies have shown poor early BC outcomes with the detection of CTCs before and after administration of neoadjuvant [37] or adjuvant chemotherapy [38]. However, two studies have failed to show that CTCs were predictive of pCR [39,40]. ...
The National Cancer Institute of the United States defines personalized medicine (PM) as “a form of medicine that uses information about a person’s genes, proteins and environment to prevent, diagnose and treat disease” (National Cancer Institute, Dictionary of Cancer Terms) [1]. Consequently, the ultimate dream is the generation of a “molecular fingerprint” via a simple blood test or tumor sample that allows the physician to refine an individual patient’s prognosis, select the best possible therapeutic option, and minimize the toxicity from therapies by identification of distinct genetic markers. The enormous gains to patients and ultimately health care systems are unmistakable.
... [59, 69, 70]. On en retrouve également, en pourcentage très variable en fonction de la méthode utilisée mais toujours en quantité moindre chez des patientes atteintes de maladies localisées [71, 72], de tumeurs inflammatoires ou localement avancées [31, 73]. La première méta-analyse a été publiée récemment [74] [84]. ...
Circulating tumor cells (CTCs) can be detected in the blood of patients with nearly all types of locally and metastatic adenocarcinomas. CTCs are epithelial cells whose release from a primitive tumor or a metastatic localization may be mediated by an epithelial-mesenchymal transition. Their pro-metastatic potential is still under debate because their phenotypes may be very heterogeneous, even within the same patient (expression of stem-cells markers, apoptotic status…). They often exhibit discrepancies with the primitive tumor, especially concerning the molecular basis of sensitivity/resistance to targeted therapies (expression of HER2 and hormone receptors, mutations responsible for resistance to tyrosine-kinase inhibitors). Many methods for CTCs analysis are commercially available but very few are evaluated and standardized enough for clinical applications. The CellSearch(®) device is the only one which is validated by the FDA for managing metastatic breast, prostate and colo-rectal cancer. It was used in most of the studies having demonstrated the prognostic and predictive value of CTCs in many tumoral localizations. Other studies are wanted to assess the ability of CTCs to optimize the therapeutic choices, to monitor drug efficiency in real-time as well as to become a surrogate end-point for evaluation of new therapies. Beyond CTCs enumeration, their biological features will need to be investigated.
... The clinical relevance of CTC detection in patients with carcinoma was part of extensive research and encouraging results exist for an association between CTC detection and prognosis in patients with other solid tumor entities such as metastatic breast, prostate, and colorectal cancer (16-18, 45, 46). Furthermore, the clinical relevance of CTC detection was investigated in patients with various nonmetastatic cancer diseases, for example, in the course of translational research programs within neoadjuvant and adjuvant treatment studies (47,48). For patients with OSCC there are few studies investigating the dissemination of tumor cells after surgery (20,49), but there are no studies yet investigating this issue on the impact of metastases and survival. ...
Current staging methods for squamous cell carcinomas of the oral cavity (OSCC) need to be improved to predict the risk of individual patients. Since hematogenous tumor cell dissemination is a key event in tumor progression we assessed the prognostic significance of disseminated tumor cells (DTCs) in bone marrow (BM) and circulating tumor cells (CTCs) in peripheral blood (PB) from OSCC patients.
From 110 patients with OSCC, tumors were surgically resected (R0) without neoadjuvant therapy. The CellSearch™-system was used to enumerate CTCs. BM was aspirated from the iliac crest, and mononuclear cells (MNCs) were enriched by Ficoll density gradient centrifugation. To detect DTCs, MNCs were immunostained with the pan-keratin antibody A45-B/B3. Results were correlated with clinicopathological parameters and clinical outcome such as recurrence and death during follow up time (mean 916 days).
Ten/80 patients (12.5%) harbored CTCs in PB whereas in 18/90 patients (20.0%) DTCs in BM could be detected. Surprisingly, in only two patients (1.8%) CTCs and DTCs were detected simultaneously. Significant correlations could be found regarding CTCs and tumor size (p=0.04), nodal status and DTCs (p=0.02), and distant metastasis with CTCs (p=0.004) and DTCs (p=0.005). Univariate and multivariate analyses revealed that CTCs and DTCs were significant and independent predictors of recurrence-free survival (p<0.001).
Both DTCs and CTCs are independent prognostic markers in OSCC patients, predicting relapse with higher sensitivity at various disease stages than routine staging procedures. Bone marrow might be an interesting target organ for future therapeutic interventions.
... Enumeration of CTC in breast cancer, using the FDA approved CellSearch technology, has been well established in several clinical studies, showing a correlation with decreased progression-free survival and overall survival in primary as well as in metastatic breast cancer before the initiation of treatment28293031. Monitoring studies provided valuable clinical information for the management of breast cancer patients and an ongoing prospective randomized clinical trial is now addressing the question whether to change an existing treatment protocol according to the persistence of CTC after three to five weeks of therapy instead of waiting for clinical and radiologic evidence of progression in metastatic breast cancer patients [SWOG S0500, clinicaltrials.gov, ...
The presence of circulating tumor cells (CTC) in breast cancer might be associated with stem cell-like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, to be able to disseminate and metastasize, CTC must be able to perform epithelial-mesenchymal transition (EMT). We studied the expression of three EMT markers and the stem cell marker ALDH1 in CTC from 502 primary breast cancer patients. Data were correlated with the presence of disseminated tumor cells (DTC) in the bone marrow (BM) and with clinicopathological data of the patients.
A total of 2 × 5 ml of blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1, HER2 and beta-Actin transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [TWIST1, Akt2, phosphoinositide kinase-3 (PI3Kα)] and separately for the tumor stem cell marker ALDH1. The identification of EMT markers was considered positive if at least one marker was detected in the sample. Two BM aspirates from all patients were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.
Ninety-seven percent of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts, respectively. CTC were detected in 97/502 (19%) patients. At least one of the EMT markers was expressed in 29% and ALDH1 was present in 14% of the samples, respectively. Interestingly, 5% of the ALDH1-positive and 18% of the EMT-positive patients were CTC-negative based on the cut-off level determined for CTC-positivity applying the AdnaTest BreastCancer. DTC in the BM were detected in 107/502 (21%) patients and no correlation was found between BM status and CTC positivity (P = 0.41). The presence of CTC, EMT and ALDH1 expression was not correlated to any of the prognostic clinical markers.
Our data indicate that (1) a subset of primary breast cancer patients shows EMT and stem cell characteristics and (2) the currently used detection methods for CTC are not efficient to identify a subtype of CTC which underwent EMT. (3) The clinical relevance on prognosis and therapy response has to be further evaluated in a prospective trial.
Biomarkers in Medicine is a comprehensive guide to understanding the current and future status of biomarkers. The book features 27 chapters focusing on disease biomarkers for diseases such as cancer, neurodegenerative diseases, cardiac diseases, metabolic conditions and much more.
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Silencing of tumor suppressor genes by promoter hypermethylation is a key mechanism to facilitate cancer progression in many malignancies. While promoter hypermethylation can occur at later stages of the carcinogenesis process, constitutional methylation of key tumor suppressors may be an initiating event whereby cancer is started. Constitutional BRCA1 methylation due to cis-acting germline genetic variants is associated with a high risk of breast and ovarian cancer. However, this seems to be a rare event, restricted to a very limited number of families. In contrast, mosaic constitutional BRCA1 methylation is detected in 4-7% of newborn females without germline BRCA1 mutations. While the cause of such methylation is poorly understood, mosaic normal tissue BRCA1 methylation is associated with a 2-3 fold increased risk of high-grade serous ovarian cancer (HGSOC). As such, BRCA1 methylation may be the cause of a significant number of ovarian cancers. Given the molecular similarities between HGSOC and basal-like breast cancer, the findings with respect to HGSOC suggest that constitutional BRCA1 methylation could be a risk factor for basal-like breast cancer as well. Similar to BRCA1, some specific germline variants in MLH1 and MSH2 are associated with promoter methylation and a high risk of colorectal cancers in rare hereditary cases of the disease. However, as many as 15% of all colorectal cancers are of the microsatellite instability (MSI) "high" subtype, in which commonly the tumors harbor MLH1 hypermethylation. Constitutional mosaic methylation of MLH1 in normal tissues has been detected but not formally evaluated as a potential risk factor for incidental colorectal cancers. However, the findings with respect to BRCA1 in breast and ovarian cancer raises the question whether mosaic MLH1 methylation is a risk factor for MSI positive colorectal cancer as well. As for MGMT, a promoter variant is associated with elevated methylation across a panel of solid cancers, and MGMT promoter methylation may contribute to an elevated cancer risk in several of these malignancies. We hypothesize that constitutional mosaic promoter methylation of crucial tumor suppressors may trigger certain types of cancer, similar to germline mutations inactivating the same particular genes. Such constitutional methylation events may be a spark to ignite cancer development, and if associated with a significant cancer risk, screening for such epigenetic alterations could be part of cancer prevention programs to reduce cancer mortality in the future.
PurposePrognostic effects of circulating tumor cells (CTCs) have been reported in metastatic breast cancer (MBC). However, few phase III trials have investigated the potential role of CTCs in treatment selection. We explored potential relationships between CTCs, efficacy, and differential treatment effects. Methods
Patients with HER2-negative MBC were randomized to receive either concurrent capecitabine plus docetaxel (XT) or sequential single-agent docetaxel followed by single-agent capecitabine at progression (T → X). Blood samples were collected at baseline, on day 1 of cycles 2 and 3, and at progression. CTCs were counted using the CellSearch® System. The relationship between baseline CTC count and outcomes was investigated using a pre-defined threshold of 2 CTCs/7.5 mL. ResultsAt screening, 44% of the 148 enrolled patients had positive CTC score. In multivariate analyses of pooled treatment arms, positive baseline CTC and triple-negative disease were strongly associated with worse progression-free survival (PFS) and overall survival (OS). Patients with positive CTC score at the baseline had worse OS, irrespective of change in CTC (decreased versus remaining positive) at cycle 2. The prognostic effect of baseline CTC count on OS appeared slightly less pronounced in XT-treated pts. compared with T → X. ConclusionsA baseline CTC count ≥2 CTCs/7.5 mL was associated with worse prognosis. However, some improvement in PFS and OS was shown with concurrent XT, thus baseline CTC could be a predictive marker. As the current trial was not designed to evaluate a change in chemotherapy according to on-treatment CTC changes, prospective investigation is required.
Isolierte Tumorzellen können sowohl im peripheren Blut als auch im Knochenmark (KM) von Patientinnen mit Mammakarzinom detektiert werden. Während der Nachweis von disseminierten Tumorzellen (DTZ) im Knochenmark von primären Mammakarzinompatientinnen einen unabhängigen Prognosefaktor (Level-I-Evidenz) darstellt, ist die prognostische Bedeutung von zirkulierenden Tumorzellen (ZTZ) im peripheren Blut in diesem Kollektiv noch nicht eindeutig geklärt. Im Beitrag werden die Relevanz von ZTZ beim primären wie beim metastasierten Mammakarzinom und die aktuelle Studienlage zu diesem Thema erläutert.
Abstract
Isolated tumor cells can be detected in peripheral blood and bone marrow of breast cancer patients. The prognostic significance of disseminated tumor cells (DTC) in bone marrow (BM) has been demonstrated in a large pooled analysis (level I evidence). Since BM biopsies are not well tolerated by many patients, evaluation of circulating tumor cells (CTC) in blood may represent an important alternative. Recent studies have indicated a prognostic significance of CTC in both the primary and metastatic setting. The evaluation of CTC may become one of the crucial markers for prediction of survival and therapy monitoring, and its characterization might enable specific targeting of minimal residual as well as metastatic disease. This report provides an overview of ongoing studies regarding CTC as well as the clinical relevance of CTC in primary and metastatic breast cancer.
Modern digital circuits are, with each technological evolution, increasingly affected by Single Event Upsets (SEUs). In this paper we propose a static analysis approach for the estimation of the SEU sensitivity of the system under design by identifying untestable faults. The approach relies on a formal specification language to model circuits at the gate-level and on the Linear Temporal Logic (LTL) to express untestability properties that are then evaluated using a model-checking tool. The proposed approach can be applied early during the design process, since it can be individually applied to sub-systems as soon as they are designed, before the whole system is implemented and since it does not require a specific workload to be defined. The approach has been implemented and applied to a set of circuits from the ITC99 benchmark and has been validated against fault injection experiments.
Zirkulierende Tumorzellen (CTC) lassen sich im Blut von Mammakarzinompatientinnen nachweisen. Reproduzierbare und für die Prognose aussagekräftige Ergebnisse liefert der Nachweis mit dem CellSearch®-System. Die prognostische Bedeutung von CTCs in der metastasierten Situation konnte mehrfach gezeigt werden und gilt unter Verwendung des CellSearch®-Gerätes mit einer Nachweisgrenze von ≥5CTC/7,5 ml Blut als gesichert. Auch in der adjuvanten Situation konnte die prognostische Bedeutung von CTC bereits gezeigt werden, die Datenlage ist jedoch noch gering. Sowohl die prognostische Relevanz von CTCs in der metastasierten als auch in der adjuvanten Situation konnten jedoch bereits durch eine große Metaanalyse bestätigt werden. Auf CTCs können zudem weitere prädiktive Marker (HER2, ER, PR) nachgewiesen werden. Aktuell wird untersucht, ob eine Therapieintervention aufgrund dieses Nachweises die Behandlung und damit die Prognose verbessern kann.
Circulating blood biomarkers promise to become non-invasive real-time surrogates for tumour tissue-based biomarkers. Circulating biomarkers have been investigated as tools for breast cancer diagnosis, the dissection of breast cancer biology and its genetic and clinical heterogeneity, prognostication, prediction and monitoring of therapeutic response and resistance. Circulating tumour cells and cell-free plasma DNA have been analysed in retrospective studies, and the assessment of these biomarkers is being incorporated into clinical trials. As the scope of breast cancer intratumour genetic heterogeneity unravels, the development of robust and standardized methods for the assessment of circulating biomarkers will be essential for the realization of the potentials of personalized medicine. In this Review, we discuss the current status of blood-born biomarkers as surrogates for tissue-based biomarkers, and their burgeoning impact on the management of patients with breast cancer.
Development in circulating tumor cells (CTCs) technologies represents a valuable tool for the better understanding of tumor biology. The clinical relevance of CTCs as a prognostic factor is well established both in metastatic and early-stage breast cancer patients. The eradication or decrease of CTCs following treatment is associated with improved clinical outcomes. Because of the availability of novel cancer treatments that specifically target tumor cells underlying signaling pathways, molecular characterization of CTCs has strong potential to translate into personalized treatments. A handful of studies have explored relevant markers such as the estrogen and progesterone receptor, HER2 and EGF receptor. However, there is not a single validation of a molecular marker in CTCs that provides prognostic information or predicts response to cancer therapies. This review describes the latest results on the characterization of breast cancer CTCs with a focus on CTC biology and implications in clinical practice.
The identification of patients at higher risk of recurrence after primary colorectal cancer resection is currently one of the challenges facing medical oncologists. Circulating tumor cell (CTC) may represent a surrogate marker of an early spread of disease in patients without overt metastases. Thirty-seven high-risk stages II-III colorectal cancer patients were evaluated for the presence of CTC. Enumeration of CTCs in 7.5 ml of blood was carried out with the FDA-cleared CellSearch system. CTC count was performed after primary tumor resection and before the start of adjuvant therapy. CTC was detected in 22 % of patients with a significant correlation with regional lymph nodes involvement and stage of disease. No significant correlation was found among the presence of CTC and other clinicopathological parameters. These data suggest that CTCs detection might help in the selection of high-risk stage II colorectal cancer patient candidates for adjuvant chemotherapy.
In 2004, circulating tumor cells (CTC) enumeration by the CellSearch® technique at baseline and during treatment was reported to be associated with prognosis in metastatic breast cancer patients. In 2008, the first evidence of the impact of CTC detection by this technique on survival of cM0(i+) patients were reported. These findings were confirmed by other non-interventional studies, whereas CTC were also investigated as a surrogate for tumor biology, mainly for HER2 expression/amplification. The aim of this report is to present the current prospective large interventional studies that have been specifically designed to demonstrate that CTC enumeration/characterization may improve the management of breast cancer patients: STIC CTC METABREAST (France) and Endocrine Therapy Index (USA) assess the CTC-guided hormone therapy vs chemotherapy decision in M1 patients; SWOG0500 (USA) and CirCe01 (France) assess the CTC count changes during treatment in metastatic patients; DETECT III (M1 patients, Germany) and Treat CTC (cM0(i+) patients, European Organization for Research and Treatment of Cancer/Breast International Group) assess the use of anti-HER2 treatments in HER2-negative breast cancer patients selected on the basis of CTC detection/characterization. These trials have different designs in various patient populations but are expected to be the pivotal trials for CTC implementation in the routine management of breast cancer patients.
Amastigotes of Trypanosoma cruzi were purified from overlays of infected Vero cell cultures by centrifugation over a discontinuous gradient of metrizamide. Pure amastigote preparations were usually recovered from the pellet under the layer of specific gravity 1.086. The isolated amastigotes grew in cell-free ML-15HA medium. Growth rate for the different strains of T. cruzi were in the order Y > Tulahuan > CL. The generation time of amastigotes in ML-15HA medium was 16.8, 18.0, and 26.4 h for the Y, Tulahuen, and CL strains, respectively, in the presence of 5% CO2) and 16.8, 31.2, and 36.4 h, respectively, in the absence of CO2. Intracellular amastigotes did not differ ultrastructurally from amastigotes from either the density-gradient fractionation or culture in cell-free medium.
The prognostic and predictive value of circulating tumor cells (CTCs) in primary breast cancer patients is subject of several recent publications. In the context of neoadjuvant chemotherapy CTCs were detected in 22-23% of patients before and in 10-17% after systemic treatment. These findings did not correlate with primary tumor characteristics or tumor response rates. One major trial evaluated the prognostic value of CTCs in 2.026 primary breast cancer patients after tumor resection but before adjuvant chemotherapy. The prevalence of CTCs was 22%. In multivariate analysis, the presence of CTCs before treatment was shown to be an independent predictor for both disease-free (hazard ratio; HR 1.88) and overall survival (HR 1.91). Results demonstrate that not only the mere presence but also the quantity of CTCs is associated with worse outcome. The risk for recurrence or tumor-related death increased with higher numbers of CTCs detected (≥5 CTCs: HR 4.04 for DFS and 3.05 for OAS; p < 0.05). In subsequent analyses of smaller subgroups within this trial, using a cutoff for positivity of >1 CTC, 10% of patients with the detection of CTCs before chemotherapy remained CTC-positive after completion of chemotherapy. Eight percentage of initially negative patients showed CTCs immediately after chemotherapy. Early data demonstrate that persisting CTCs after cytostatic treatment correlate with a decreased disease-free survival (p = 0.0623). Increasing evidence confirms the prognostic relevance of CTCs in primary breast cancer. CTC detection could help to identify patients with increased risk for relapse. Present trials will show whether CTCs can also be used as a valid tool for treatment monitoring or direct treatment target.
The evaluation of circulating tumor cells in peripheral blood of patients with breast cancer can be performed using different platforms. Standardization and comparison of the platforms to each other and their use in prospective clinical trials is needed for incorporation of circulating tumor cell assays into clinical practice.
The aim of this study was to develop a new method for detecting circulating tumor cells (CTCs) in breast cancer patients by using the telomerase-specific replication-selective adenovirus OBP-401. Once transfected, OBP-401 can replicate only in telomerase expressing cells and emit fluorescence as it replicates so that the transfected cells become easily recognizable. Peripheral blood samples were drawn from 50 metastatic breast cancer patients and 27 early breast cancer patients. Blood samples were subjected to both the OBP-401 and CellSearch assays for the detection of CTCs and the results were compared. The recovery rate of the OBP-401 assay was one CTC in 7.5 ml blood combined with high specificity since no CTC was observed in 80 healthy controls. In 50 metastatic patients, 21 patients (42%) were identified as positive with the OBP-401 assay and 27 patients (54%) with the CellSearch assay. The CellSearch assay showed a significantly higher positivity for hormone receptor (HR)-positive tumors (estrogen receptor and/or progesterone receptor-positive tumors) (61%, 25/41, P = 0.012) or CA15-3-positive tumors (69%, 24/35, P = 0.003) than for HR-negative tumors (13%, 1/8) or CA15-3-negative tumors (21%, 3/14), respectively. Contrary, the OBP-401 assay results were similar regardless of their HR status (positive: 44% vs. negative: 38%, P = 0.738) or CA15-3 positivity (positive: 40% vs. negative: 50%, P = 0.523). Of the 27 early stage patients, four patients (15%) were identified by the OBP-401 assay and by the CellSearch assay, respectively, but there was no overlap in the CTCs-positive patients. In conclusion, the OBP-401 assay is comparable to the CellSearch assay in the detection rate of CTCs in both metastatic and early breast cancer patients. However, there was a great discrepancy in patients with CTCs between both assays. The OBP-401 assay may isolate CTCs with other biological characteristics which CTCs detected by the CellSearch assay do not have.
The 3rd Breast-Gynecological International Cancer Conference, supported by the European Society of Medical Oncology (ESMO), was held in Cairo (Egypt) on 13-14 January 2011. The meeting was conducted under the patronage of Egypt's Ministry of Health, with the scientific committee led by Heba El Zawahry from the National Cancer Institute (Cairo, Egypt), and Yasser Abdel Kader from Cairo University (Cairo, Egypt). Several important updates and multifaceted issues in the management of various breast and gynecological cancers were addressed in this conference. Leading physicians and scientists from 12 different countries shared their expertise and views. This meeting was a good opportunity to meet fellow professionals from all parts of the world and discuss complex and interesting case studies. In this article, we highlight pertinent sessions and important topics discussed in this meeting.
More than 90% of cancer deaths result from the development of hematogenously disseminated metastasis. The presence of circulating tumor cells (CTCs) in patients with cancer was first reported in 1869. 1 More recently, methods have been developed to detect, isolate, and characterize CTCs in multiple different malignancies that arise in solid organs. 2 The most widely used method, designated CellSearch (Veridex, Raritan, NJ), relies on immunomagnetic capture of CTCs using antibodies against the epithelial cell adhesion molecule (EpCam), which is expressed on the cell surface of many epithelial malignancies, followed by additional characterization with 4,6-diamidino-2-phenylindole staining (to
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.
Blood and BM aspirations were collected at the time of primary breast surgery. CTCs were detected by using the CellSearch assay, and DTCs were detected by immunostaining BM aspirates for pancytokeratin. The presence of CTCs and DTCs was correlated with tumor classification (T1 vs T2), tumor histologic grade, estrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (HER2) status, and lymph node (LN) status.
Of 92 patients who were included in the study, 49 had T1 tumors, and 43 had T2 tumors. CTCs were detected in 31% of patients, and DTCs were detected in 27% of patients. There was no correlation between the occurrence of CTCs and DTCs with the tumor classification (T1 vs T2) or histologic grade. CTCs were detected in 33% of patients with ER-positive disease versus 26% of patients with ER-negative disease, in 32% of patients with PR-positive disease versus 30% of patients with PR-negative disease, and in 25% of patients with HER2-positive disease versus 31% of patients with HER2-negative disease. DTCs were observed in 23% of patients with ER-positive disease versus 37% of patients with ER-negative disease, in 22% of patients with PR-positive disease versus 32% of patients with PR-negative disease, and in 0% of patients with HER2-positive disease versus 29% of patients with HER2-negative disease. CTCs and DTCs were nearly equally prevalent in both LN-positive women and LN-negative women. There was no significant correlation between the occurrence of CTCs or DTCs with tumor classification (T1 vs T2), tumor histologic grade, positive ER status, positive PR status, or positive HER2 status, and axillary LN status.
CTCs and DTCs in women with early stage breast cancer did not correlate with the standard prognostic indicators that were considered. The implications of their occurrence in patients with early stage disease will require further large-scale studies.
In spite of the heterogeneity of breast cancer at the molecular level, circulating tumor cells (CTCs) may provide a novel prognostic marker. Approximately 20-40% of early breast cancer patients have detectable CTCs using reverse transcription PCR for CK19. The detection of CTCs before adjuvant chemotherapy or during tamoxifen administration has been demonstrated to be an independent adverse prognostic factor in women with early-stage breast cancer. The prognostic value of CTC detection is of great significance in subgroups of patients with estrogen receptor-negative and human EGF receptor 2-positive tumors. Prospective clinical trials are warranted in order to validate the use of CTCs as predictive and/or prognostic markers and assess their utility in individualizing therapy of patients with early breast cancer.
The search for disseminated cancer cells has become a routine procedure in many clinical centres since the pioneering work of Riethmüller and Schlimok was published in the mid 1980s. Until today, clinical studies have mostly focused on the prognostic role of disseminated cancer cells that can be detected in bone marrow samples before manifestation of metastasis. As a more recent development, the field is increasingly concentrating on the prognostic information provided by tumour cells circulating in the peripheral blood instead of analysing the nature of disseminated tumour cells that have successfully homed to a new microenvironment and may eventually grow into metastases. This review critically questions that direction and proposes exploiting the unique opportunities provided by the direct molecular analysis of metastatic precursor cells for a better understanding of metastasis, tumour dormancy, therapy target identification, and personalised medicine in an adjuvant therapy setting.
To investigate the prognostic value of the molecular detection of circulating tumor cells (CTCs) using three markers [cytokeratin 19 (CK19), mammaglobin A (MGB1), and HER2] in early breast cancer.
CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected using real-time (CK19) and nested (MGB1 and HER2) reverse transcription-PCR in the peripheral blood of 175 women with stage I to III breast cancer before the initiation of adjuvant chemotherapy. The detection of CTCs was correlated with clinical outcome. In 10 patients, immunofluorescence staining experiments were done to investigate the coexpression of cytokeratin, MGB1, and HER2 in CTCs.
CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected in 41.1%, 8%, and 28.6% of the 175 patients, respectively. Patients had one of the following molecular profiles: CK19mRNA+/MGB1mRNA+/HER2mRNA+ (n = 8), CK19mRNA+/MGB1mRNA+/HER2mRNA- (n = 1), CK19mRNA+/MGB1mRNA-/HER2mRNA+ (n = 42), CK19mRNA+/MGB1mRNA-/HER2mRNA- (n = 21), CK19mRNA-/MGB1mRNA+/HER2mRNA- (n = 5), and CK19mRNA-/MGB1mRNA-/HER2mRNA- (n = 98). Double-immunofluorescence experiments confirmed the following CTC phenotypes: CK+/MGB1+, CK+/MGB1-, CK-/MGB1+, CK+/HER2+, CK+/HER2-, MGB1+/HER2-, and MGB1+/HER2+. In univariate analysis, the detection of CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells was associated with shorter disease-free survival (DFS; P < 0.001, P = 0.001, and P < 0.001, respectively), whereas the detection of CK19mRNA+ and MGB1mRNA+ cells was associated with worse overall survival (P = 0.044 and 0.034, respectively). In multivariate analysis, estrogen receptor-negative tumors and the detection of CK19mRNA+ and MGB1mRNA+ cells were independently associated with worse DFS.
The detection of peripheral blood CK19mRNA+ and MGB1mRNA+ cells before adjuvant chemotherapy predicts poor DFS in women with early breast cancer.
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