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.
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.
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).
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.
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.
The causal relationship between persistent infection with high‐risk HPV and cervical cancer has resulted in the development of HPV DNA detection systems. The widely used MY09/11 consensus PCR targets ...a 450bp conserved sequence in the HPV L1 gene, and can therefore amplify a broad spectrum of HPV types. However, limitations of these consensus primers are evident, particularly in regard to the variability in detection sensitivity among different HPV types. This study compared MY09/11 PCR with type‐specific PCRs in the detection of oncogenic HPV types. The study population comprised 15, 774 patients. Consensus PCR failed to detect 522 (10.9%) HPV infections indicated by type‐specific PCRs. A significant correlation between failure of consensus PCR and HPV type was found. HPV types 51, 68 and 45 were missed most frequently. The clinical relevance of the HPV infections missed by MY09/11 PCR was reflected in the fraction of cases with cytological abnormalities and in follow‐up, showing 104 (25.4%) CIN2+ cases. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. Diagnostic accuracy of the PCR systems, in terms of sensitivity (MY09/11 PCR: 87.9%; type‐specific PCRs: 98.3%) and specificity (MY09/11 PCR: 38.7%; type‐specific PCRs: 76.14%), and predictive values for histologically confirmed CIN2+, suggest that type‐specific PCRs could be used in a clinical setting as a reliable screening tool.
The prognostic significance of serum interleukin (IL)-8 was evaluated in patients with metastatic breast cancer. The predictive value of serum IL-8 for the presence of occult metastatic tumor cells ...in bone marrow aspirates was evaluated in patients with operable and metastatic breast cancer.
Serum IL-8 was measured in healthy controls, patients with operable breast cancer, and patients with untreated, progressive metastatic breast cancer. In 69 patients with either operable or advanced breast cancer, occult cytokeratin-positive cells were counted in bone marrow aspirates.
Serum IL-8 levels are increased in 67% (52 of 77) of patients with advanced breast cancer. Overall, these levels are significantly higher in patients with breast cancer compared with healthy volunteers (P < 0.001). The IL-8 levels increase significantly in patients with more advanced disease. An elevated serum IL-8 is related to an accelerated clinical course, a higher tumor load, and the presence of liver or lymph node involvement. A multivariate analysis indicates that serum IL-8 is an independent significant factor for postrelapse survival. There was a significant difference between serum IL-8 levels in patients with or without occult cytokeratin-positive bone marrow cells (P < 0.04). Serum IL-8 levels also showed an association with the number of these cells (P < 0.01).
Serum IL-8 is increased in patients with breast cancer and has an independent prognostic significance for postrelapse survival. The observations on the relationship between occult cytokeratin-positive bone marrow cells corroborate the concept of IL-8 acting as a contributor to the process of tumor cell dissemination. Similarly, the relationship between serum IL-8 and nodal stage at presentation deserves further study. These results further expand the concept that inflammation and inflammatory cytokines are critical components of tumor progression.
The enumeration of circulating tumour cells (CTC) has prognostic significance in patients with metastatic breast cancer (MBC) and monitoring of CTC levels over time has considerable potential to ...guide treatment decisions. However, little is known on CTC kinetics in the human bloodstream.
In this study, we compared the number of CTC in both 7.5 ml central venous blood (CVB) and 7.5 ml peripheral venous blood (PVB) from 30 patients with MBC starting with a new line of chemotherapy.
The number of CTC was found to be significantly higher in CVB (median: 43.5; range: 0-4036) than in PVB (median: 33; range: 0-4013) (P=0.001). When analysing samples pairwise, CTC counts were found to be significantly higher in CVB than in PVB in 12 out of 26 patients with detectable CTC. In contrast, only 2 out of 26 patients had higher CTC counts in PVB as compared with CVB, whereas in 12 remaining patients no significant difference was seen. The pattern of CTC distribution was independent of the sites of metastatic involvement.
A substantial difference in the number of CTC was observed between CVB and PVB of patients with MBC. Registration of the site of blood collection is warranted in studies evaluating the role of CTC assessment in these patients.
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 beta-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.
Objective: Liquid‐based cytology (LBC) for cervical screening is becoming increasingly used. Together with SurePath® LBC, various collecting devices can be utilized, among which the Cervex‐Brush® is ...the most widely used. The new Rovers® Cervex‐Brush® Combi combines the advantages of the Cervex‐Brush® with the EndoCervex‐Brush® increasing sampling of the endocervical canal. The objective of this study was to analyse and to compare the Cervex‐Brush® Combi with the Cervex‐Brush® for the collection of squamous and endocervical cells, human papillomavirus (HPV) typing/quantification and disease detection in SurePath® LBC.
Methods: Using either the Cervex‐Brush® or the Cervex‐Brush® Combi 100 consecutive SurePath® LBC samples were collected using each brush type. All 200 slides were read by the FocalPointTM and screened by guided screening using slide wizards. The viral load of HPV type 16 E7, 18 E7, 31 E6, 33 L1, 33 E6, 35 E4, 39 E7, 45 E7, 51 E6, 52 L1, 52 E7, 53 E6, 56 E7, 58 L1, 58 E6, 59 E7, 66 E6 and 68 E7 was determined using a TaqMan‐based real‐time quantitative PCR analysis.
Results: The mean number of sampled squamous cells did not differ between the two brush types (54 963 versus 54 595 cells). The use of the Cervex‐Brush® Combi, however, resulted in a two‐ to threefold increase in the number of sampled endocervical cells (P < 0.00001). Using the Cervex‐Brush® Combi slightly more lesions were detected (three versus two low‐grade squamous intraepithelial lesions), and resulted in the detection of more atypical squamous cells of undetermined significance (six versus three). In the Cervex‐Brush® group, 60% (3/5) of abnormal smears were positive for oncogenic HPV types, whereas 66.7% (6/9) of abnormal smears in the Cervex‐Brush® Combi group tested positive. The median HPV viral load for samples taken with the Cervex‐Brush® Combi was 0.1825 copies/cell and was significantly higher than in samples taken with the Cervex‐Brush® (0.0042 copies/cell) (P = 0.02).
Conclusion: Sampling with the Cervex‐Brush® Combi resulted in the collection of the same amount of squamous cells, but in a two to threefold harvest of endocervical cells. This led to the detection of a higher viral load for oncogenic HPV and an increase in the number of detected abnormal smears.
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.
Drug screening in oncology, especially for triple-negative breast cancer (TNBC), has high demand but remains unsatisfactory. Currently available models are either nonrepresentative of the complex ...tumor microenvironment or only suitable for low throughput screening, resulting in a low-yield success for drug development. To tackle these issues, the L-TumorChip system is developed in this study. It is a three-layered microfluidic tumor-on-a-chip platform integrating tumor microvasculature and tumor-stromal microenvironment with high throughput screening capability. Its layered and modular design is readily scalable through simple integration of multiple units. Here, L-TumorChip is validated with a TNBC model. The L-TumorChip system emulates certain tumor-stroma complexities and tumor-endothelium interactions, including TNBC invasion through the leaky microvasculature and angiogenesis. Additionally, with this L-TumorChip, the influence of different stromal cells, including normal fibroblasts, mesenchymal stem cells, and cancer-associated fibroblasts (CAF), on cancer cell growth as well as the stromal effects on drug responses to doxorubicin treatment is investigated. The presence of CAF delays drug pharmacokinetics, while apoptotic responses indicated by caspase-3 activities are higher in coculture with normal fibroblasts. Collectively, the L-TumorChip system represents a translational high-throughput screening toolkit that enables drug screening with a scenario closer to the in vivo conditions. This potential use may therefore facilitate development of new cancer drugs.
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