Hilde Elst

Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer

This study assessed the ability of real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n=116/78%) and metastatic (stage M+, n=32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT–PCR detection method. The mean follow-up time was 786 days (+/− 487). Kaplan–Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P=0.0045 (CK-19) and P=0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P=0.551 (CK-19) and P=0.329 (MAM)). Separate analyses of the M0 and M+ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M+ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results.

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer

Background:The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardisation of technology hampers the implementation of CTC measurement in clinical routine practice.Methods:This study was designed to directly compare three techniques for detecting CTCs in blood samples taken from 76 patients with metastatic breast cancer (MBC) and from 20 healthy controls: the CellSearch CTC System, the AdnaTest Breast Cancer Select/Detect and a previously developed real-time qRT-PCR assay for the detection of CK-19 and mammaglobin transcripts.Results:As a result, 36% of patients with MBC were positive by the CellSearch System, 22% by the AdnaTest, 26% using RT–PCR for CK-19 and 54% using RT–PCR for mammaglobin. Samples were significantly more likely to be positive for at least one mRNA marker using RT–PCR than using the CellSearch System (P=0.001) or the AdnaTest (P<0.001).Conclusion:We observed a substantial variation in the detection rates of CTCs in blood from breast cancer patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and mammaglobin, which suggests that this is currently the most sensitive technique for detecting CTCs.

Nuclear Factor-κB Signature of Inflammatory Breast Cancer by cDNA Microarray Validated by Quantitative Real-time Reverse Transcription-PCR, Immunohistochemistry, and Nuclear Factor-κB DNA-Binding

Purpose: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer with high metastatic potential. In a previous study, we showed that IBC is a different form of breast cancer compared with non-IBC by cDNA microarray analysis. A list of 756 genes with significant expression differences between IBC and non-IBC was identified. In-depth functional analysis revealed the presence of a high number of nuclear factor-κB (NF-κB) target genes with elevated expression in IBC versus non-IBC. This led to the hypothesis that NF-κB contributes to the phenotype of IBC. The aim of the present study was to further investigate the role of NF-κB in IBC. Experimental Design: Immunohistochemistry and NF-κB DNA-binding experiments were done for all NF-κB subunits (RelA, RelB, cRel, NFkB1, and NFkB2) using IBC and non-IBC specimens. Transcriptionally active NF-κB dimers were identified by means of coexpression analysis. In addition, quantitative real-time reverse transcription-PCR for eight NF-κB target genes, selected upon a significant, 3-fold gene expression difference between IBC and non-IBC by cDNA microarray analysis, was done. Results: We found a significant overexpression for all of eight selected NF-κB target genes in IBC compared with non-IBC by quantitative real-time reverse transcription-PCR. In addition, we found a statistically elevated number of immunostained nuclei in IBC compared with non-IBC for RelB (P = 0.038) and NFkB1 (P < 0.001). Immunohistochemical data were further validated by NF-κB DNA-binding experiments. Significant correlations between immunohistochemical data and NF-κB DNA binding for RelA, RelB, NFkB1, and NFkB2 were found. Transcriptionally active NF-κB dimers, composed of specific combinations of NF-κB family members, were found in 19 of 44 IBC specimens compared with 2 of 45 non-IBC specimens (P < 0.001). In addition, we found evidence for an estrogen receptor (ER)–mediated inhibition of the NF-κB signaling pathway. NF-κB target genes were significantly elevated in ER− versus ER+ breast tumors. Also, the amount of immunostained nuclei for RelB (P = 0.025) and NFkB1 (P = 0.031) was higher in ER− breast tumors versus ER+ breast tumors. Conclusions: The NF-κB transcription factor pathway probably contributes to the phenotype of IBC and possibly offers new options for treatment of patients diagnosed with this aggressive form of breast cancer.

The presence of circulating total DNA and methylated genes is associated with circulating tumour cells in blood from breast cancer patients.

Circulating tumour cells (CTC) and tumour-related methylated DNA in blood have been separately assessed for their utility as a marker for subclinical metastasis in breast cancer. However, no studies have looked into the relation between the both molecular markers in this type of cancer. In this study, we investigated the correlations between total/methylated DNA and CTC in the blood from metastatic breast cancer patients. We simultaneously obtained whole blood, plasma and serum samples from 80 patients and 20 controls. The CellSearch System was used to enumerate CTC in blood samples. Plasma total DNA levels were determined by a QPCR method. Sera were analysed by methylation-specific QPCR for three markers: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A) and oestrogen receptor 1 (ESR1). Total DNA levels in patients were significantly increased when compared with controls (P<0.001) and correlated with the number of CTC (r=0.418, P<0.001). Hypermethylation of one or more genes was detected in 42 (53%) serum samples from breast cancer patients and in three (16%) serum samples from controls (P=0.003). APC was hypermethylated in 29%, RASSF1A in 35% and ESR1 in 20% of breast cancer cases. Detection of a methylated gene in serum was associated with the detection of CTC in blood (P=0.03). The detection of large amounts of circulating total/methylated DNA correlated with the presence of CTC in the blood from patients with breast cancer. This can be interpreted in two ways: (a) CTC are a potential source of circulating tumour-specific DNA; (b) high numbers of CTC and circulating methylated DNA are both a phenotypic feature of more aggressive tumour biology.

Real-time RT-PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with breast cancer

Real-time reverse transcriptase–polymerase chain reaction (RT–PCR) is a technique with the potential of improving the quantification of disseminated epithelial cells (DEC) in haematological tissues due to its exquisite sensitivity. This sensitivity may lead to false positivity. Immunocytochemistry (ICC) is regarded as the standard methodology to diagnose DEC. In this study, detection with ICC was compared with quantitative real-time RT–PCR for CK-19 and mammaglobin (hMAM) mRNA in bone marrow (BM) of patients with metastatic breast cancer (MBC). Bone marrow was aspirated from 14 control patients and from 29 patients with MBC. Mononuclear cells (MNC) were isolated. Immunostaining was carried out with the Epimet kit. Quantitative PCR was performed on the ABI Prism 7700. The CK-19 and hMAM mRNA quantities were normalised against β-Actin and calculated relative to a calibrator sample (relative gene expression). All controls were negative by ICC and for hMAM expression measured by RT–PCR, whereas the median RGE value for CK-19 was 0.57. For the MBC patients, the median RGE for hMAM was 0 and 10 out of 25 (40%) tested positive. Median RGE for CK-19 was 2.9 and 20 out of 25 (80%) tested positive. With ICC, the median value was 1 stained cell per sample, and 15 out of 24 (62%) samples were positive. A correlation was observed between CK-19 and hMAM expression (r=0.7; P=0.0003), and between hMAM expression and ICC (r=0.6; P=0.003). CK-19 expression and ICC (r=0.9; P<0.0001) showed the strongest correlation. Reverse transcriptase–polymerase chain reaction for CK-19 resulted in a higher number of positive BM samples of patients with MBC than ICC. Since an excellent correlation is observed between ICC and RT–PCR, and RT–PCR is probably more sensitive with the advantage of being less observer dependent and thus also more easy to automate, we consider our quantitative real-time RT–PCR method as validated for the detection of DEC in the bone marrow of breast cancer patients.

Relative microvessel area of the primary tumour, and not lymph node status, predicts the presence of bone marrow micrometastases detected by reverse transcriptase polymerase chain reaction in patients with clinically non-metastatic breast cancer

About 50% of patients with breast cancer have no involvement of axillary lymph nodes at diagnosis and can be considered cured after primary locoregional treatment. However, about 20–30% will experience distant relapse. The group of patients at risk is not well characterised: recurrence is probably due to the establishment of micrometastases before treatment. Given the early steps of metastasis in which tumour cells interact with endothelial cells of blood vessels, and, given the independent prognostic value in breast cancer of both the quantification of tumour vascularisation and the detection of micrometastases in the bone marrow, the aim of this study was to determine the relationship between vascularisation, measured by Chalkley morphometry, and the bone marrow content of cytokeratin-19 (CK-19) mRNA, quantified by real-time reverse transcriptase polymerase chain reaction, in a series of 68 patients with localised untreated breast cancer. The blood concentration of factors involved in angiogenesis (interleukin-6 and vascular endothelial growth factor) and of factors involved in coagulation (D-dimer, fibrinogen, platelets) was also measured. When bone marrow CK-19 relative gene expression (RGE) was categorised according to the cut-off value of 0.77 (95th centile of control patients), 53% of the patients had an elevated CK-19 RGE. Patients with bone marrow micrometastases, on the basis of an elevated CK-19 RGE, had a mean Chalkley count of 7.5 ± 1.7 (median 7, standard error [SE] 0.30) compared with a mean Chalkley count of 6.5 ± 1.7 in other patients (median 6, SE 0.3) (Mann–Whitney U-test; P = 0.04). Multiple regression analysis revealed that Chalkley count, not lymph node status, independently predicted CK-19 RGE status (P = 0.04; odds ratio 1.38; 95% confidence interval 1.009–1.882). Blood parameters reflecting angiogenesis and coagulation were positively correlated with Chalkley count and/or CK-19 RGE. Our data are in support of an association between elevated relative microvessel area of the primary tumour and the presence of bone marrow micrometastases in breast cancer patients with operable disease, and corroborate the paracrine and endocrine role of interleukin-6 and the involvement of coagulation in breast cancer growth and metastasis.

Detection of circulating tumour cells in blood by quantitative real-time RT-PCR: effect of pre-analytical time.

Abstract Background: We and others have recently explored the use of quantitative real-time RT-PCR analysis for the detection of circulating tumour cells in blood of patients with breast cancer (BC). One major problem in these experiments is the in vitro instability of the cellular RNA. The copy number of mRNA can change during storage and transport at room temperature and this may hamper accurate quantitative measurements of specific transcripts, especially when working with small numbers of target mRNAs. Methods: Peripheral blood samples were obtained from two healthy volunteers and 13 patients with BC. Blood was stored at room temperature for 0, 1, 2, 4, 6, 24, 48 and 72h. The potential alteration of gene expression for six target genes was investigated by quantitative real-time RT-PCR. Results: For β-actin, GAPDH, cytokeratin-19 (CK-19) and HER2, a significant decrease in expression level occurs after 4h (CK-19 and HER2), 6h (β-actin) or 24h (GAPDH). Mammaglobin expression was only measurable in two samples and seems to be stable for at least 6h. For vascular endothelial growth factor (VEGF), a statistically significant increase in expression level is observed in samples processed 24h after collection. Conclusions: Most transcripts were reduced in samples that were stored overnight at room temperature compared with fresh samples, but upregulation of transcripts, probably as an active response to cellular stress, also occurs when blood is removed from its in vivo environment and stored ex vivo. Optimally, blood samples and RNA should be processed or stabilised within 3h after collection to avoid interference of the in vivo gene expression signature by ex vivo stress responses. Clin Chem Lab Med 2006;44:1082–7.

Relapse-Free Survival in Breast Cancer Patients Is Associated with a Gene Expression Signature Characteristic for Inflammatory Breast Cancer

Purpose: We hypothesize that a gene expression profile characteristic for inflammatory breast cancer (IBC), an aggressive form of breast cancer associated with rapid cancer dissemination and poor survival, might be related to tumor aggressiveness in non-IBC (nIBC). Experimental Design: RNA from 17 IBC samples and 40 nIBC samples was hybridized onto Affymetrix chips. A gene signature predictive of IBC was identified and applied onto 1,157 nIBC samples with survival data of 881 nIBC samples. Samples were classified as IBC-like or nIBC-like. The IBC signature classification was compared with the classifications according to other prognostically relevant gene signatures and clinicopathologic variables. In addition, relapse-free survival (RFS) was compared by the Kaplan-Meyer method. Results: Classification according to the IBC signature is significantly (P < 0.05) associated with the cell-of-origin subtypes, the wound healing response, the invasive gene signature, the genomic grade index, the fibroblastic neoplasm signature, and the 70-gene prognostic signature. Significant associations (P < 0.01) were found between the IBC signature and tumor grade, estrogen receptor status, ErbB2 status, and patient age at diagnosis. Patients with an IBC-like phenotype show a significantly shorter RFS interval (P < 0.05). Oncomine analysis identified cell motility as an important concept linked with the IBC signature. Conclusions: We show that nIBC carcinomas having an IBC-like phenotype have a reduced RFS interval. This suggests that IBC and nIBC show comparable phenotypic traits, for example augmented cell motility, with respect to aggressive tumor cell behavior. This observation lends credit to the use of IBC to study aggressive tumor cell behavior.

Multidrug Resistance in Rat Colon Carcinoma Cell Lines CC531, CC531mdr+ and CC531rev

A rat colon carcinoma cell line, CC531, was exposed to stepwise increasing concentrations of colchicine. A cell line, CC531mdr+, which grows in the presence of 0.2 μM of colchicine was obtained. A revertant cell line, CC531rev was isolated upon colchicine withdrawal. The CC531mdr+ displayed a multidrug‐resistant phenotype. Marked resistance to the selecting agent colchicine, was found (RF= 37.5) as well as to vinblastine (RF=11.3) and actinomycin D (RF=2.6). Cross resistance to doxorubicin (RF=8) and daunorubicin (RF=13.3) was demonstrated. Verapamil was able to reverse drug resistance to colchicine and daunorubicin. The revertant cell line, CC531rev, showed increased sensitivity to colchicine (RF=0.43), vinblastine (RF = 0.13), doxorubicin (RF=0.28) and daunorubicin (RF = 0.56). Marked cross resistance to cis‐platinum (RF = 6.9) was also induced in CC531mdr+ and was maintained in CC531rev. We conclude that CC531 displays an intrinsic low‐level multidrug‐resistant phenotype, which was amplified in the CC531mdr+ variant. This correlates with a higher level of expression of P‐glycoprotein. CC531rev lacks the multidrug‐resistant phenotype and can be used as the drug‐sensitive counterpart of the latter two cell lines. Furthermore, it has been shown that in these cell lines cis‐platinum resistance is mediated through a mechanism independent of the multidrug‐resistant phenotype, since the revertant cell line CC531rev has lost the multidrug‐resistant phenotype while retaining the concomitantly induced cis‐platinum resistance of the multidrugresistant variant CC531mdr+.

Association of serum tumor-related methylated DNA with circulating tumor cells in peripheral blood of breast cancer patients.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #5017 Circulating tumor cells (CTC) and free tumor-related methylated DNA in blood have been separately associated with poorer disease outcome in breast cancer patients. However, no studies have looked into the relation between both molecular markers in breast cancer. In this study, we investigated the correlations between plasma total DNA, serum methylated DNA and CTC in blood from breast cancer patients. We simultaneously obtained matched triplets of peripheral blood, plasma and serum samples from 4 patients with localized breast cancer (group A), 59 patients with metastatic disease under treatment (group B), 16 untreated patients with metastatic disease (group C) and 20 healthy controls. CTC levels in blood were measured with the CellSearch System (Veridex). Plasma total DNA levels were determined by a qPCR method. Sera were analyzed by methylation-specific qPCR for three methylated markers: APC , RASSF1A and ESR1 . Plasma total DNA levels in breast cancer patients were significantly increased when compared to healthy controls (P<0.001). Differences between total DNA levels in different patient groups also reached statistical significance (P=0.04). The largest differences were measurable between groups A and C (median of 7 vs 29 ng/ml) (P=0.02) and groups B and C (median of 12 vs 29 ng/ml) (P=0.06). The sensitivity and the specificity of the total DNA assay for predicting malignancy was respectively 72.5% and 85%. Total DNA levels correlated with CTC ( r =0.418, P<0.001) and patient age ( r =0.298, P=0.01), but not with other clinical variables. Hypermethylation of one or more genes was detected in 42 (53%) serum samples from breast cancer patients and in 3 (16%) serum samples of healthy controls (P=0.003). APC was hypermethylated in 23 (29%), RASSF1A in 28 (35%) and ESR1 in 16 (20%) breast cancer cases. CTC were detected in 19 (70%) patients with serum methylated DNA and in 8 (30%) patients without methylated DNA (P=0.03). The presence of methylated DNA markers was only associated with ER status. On univariate analysis, the detection of CTC, high levels of circulating DNA, methylation of RASSF1A and the combinations of APC or RASSF1A methylation with ESR1 methylation were significantly associated with progressive disease. Multivariate analysis indicated that only RASSF1A methylation, high total DNA levels, HER2 and PR status were significantly associated with disease status. The number of CTC was significantly increased in patients with high levels of total DNA and in patients with at least one methylated DNA marker in serum, suggesting that CTC might be a potential source of circulating tumor-related DNA. Only RASSF1A methylation and high DNA levels were independently associated with progressive disease. The combination assay of CTC and serum methylated DNA did not enable a more informative assessment of disease status. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5017.