A multimarker QPCR-based platform for the detection of circulating tumour cells in patients with early-stage breast cancer

Background:

The detection of circulating tumour cells (CTCs) has been linked with poor prognosis in advanced breast cancer. Relatively few studies have been undertaken to study the clinical relevance of CTCs in early-stage breast cancer.

Methods:

In a prospective study, we evaluated CTCs in the peripheral blood of 82 early-stage breast cancer patients. Control groups consisted of 16 advanced breast cancer patients and 45 healthy volunteers. The CTC detection was performed using ErbB2/EpCAM immunomagnetic tumour cell enrichment followed by multimarker quantitative PCR (QPCR). The CTC status and common clinicopathological factors were correlated to relapse-free, breast cancer-related and overall survival.

Results:

Circulating tumour cells were detected in 16 of 82 (20%) patients with early-stage breast cancer and in 13 out of 16 (81%) with advanced breast cancer. The specificity was 100%. The median follow-up time was 51 months (range: 17–60). The CTC positivity in early-stage breast cancer patients resulted in significantly poorer relapse-free survival (log rank test: P=0.003) and was an independent predictor of relapse-free survival (multivariate hazard ratio=5.13, P=0.006, 95% CI: 1.62–16.31).

Conclusion:

The detection of CTCs in peripheral blood of early-stage breast cancer patients provided prognostic information for relapse-free survival.     

Circulating tumour cells are associated with increased risk of venous thromboembolism in metastatic breast cancer patients

Background:

Cancer is a risk factor for venous thromboembolism (VTE). Circulating tumour cells (CTCs) are an independent predictor of survival in metastatic breast cancer (MBC) patients. The aim of this study was to test the hypothesis that CTCs are associated with the risk of VTE in MBC patients.

Methods:

This retrospective study included 290 MBC patients treated in the MD Anderson Cancer Center from January 2004 to December 2007. Circulating tumour cells were detected and enumerated using the CellSearch system before starting new lines of therapy.

Results:

At a median follow-up of 12.5 months, 25 patients experienced VTE and 53 patients died without experiencing thrombosis. Cumulative incidence of thrombosis at 12 months was 8.5% (95% confidence interval (CI)=5.5%, 12.4%). Patients with CTCs ⩾1 and ⩾5 had a higher incidence of VTE compared with patients with 0 and <5 CTCs (12-month estimate, 11.7 and 11.6% vs 3 and 6.6%; P=0.006 and P=0.076, respectively). In the multivariate model, patients with CTCs⩾1 had a hazard ratio of VTE of 5.29 (95% CI=1.58, 17.7, P=0.007) compared with patients with no CTCs.

Conclusion:

These results suggest that CTCs in MBC patients are associated with increased risk of VTE. These patients should be followed up more closely for the risk of VTE.     

Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer

Background:

To investigate the clinical relevance of CK-19mRNA-positive circulating tumour cells (CTCs) detected before the initiation of front-line treatment in patients with metastatic breast cancer (MBC).

Methods:

The presence of CTCs was detected in 298 patients with MBC using a real-time PCR (RT-PCR assay. In 44 patients, the detection of CTCs was evaluated by both the CellSearch and the RT-PCR assay. Interaction with known prognostic factors and association of CTCs with clinical outcome were investigated.

Results:

There was a strong correlation between the detection of CTCs by both assays. CK-19mRNA-positive CTCs were detected in 201 (67%) patients and their detection was independent of various patients'' clinico-pathological characteristics. The median progression-free survival (PFS; 9.2 vs 11.9 months (mo), P=0.003) and the overall survival (OS; 29.7 vs 38.9 mo, P=0.016) were significantly shorter in patients with detectable CK-19mRNA-positive CTCs compared with patients without detectable CTCs. Multivariate analysis demonstrated that oestrogen receptor status, performance status and detection of CTCs were emerged as independent prognostic factors associated with decreased PFS and OS.

Conclusion:

The detection of CK-19mRNA-positive CTCs in patients with MBC before front-line therapy could define a subgroup of patients with dismal clinical outcome.     

Frequency of HER2 expression of circulating tumour cells in patients with metastatic or recurrent gastrointestinal cancer

Background:

The clinical significance of circulating tumour cell (CTC) detection in gastrointestinal (GI) cancer remains controversial and the molecular biological characteristics of CTCs are poorly understood.

Methods:

A total of 87 patients with metastatic or recurrent GI cancer were prospectively enrolled. Circulating tumour cells and their HER2 status were assessed using the CellSearch System.

Results:

Among the 62 CTC-positive cases, we found 22 discordant cases (35.5%). Among the HER2-negative primary tumours, 17 of 54 developed HER2-positive CTCs. Five of eight had HER2-negative CTCs among the HER2-positive primary tumours.

Conclusion:

The findings in the current study suggest that it is critical to evaluate the HER2 status of not only the primary tumour but also the CTCs because the metastasising tumour cells are the primary target of systemic therapy.     

Detection of HER2 amplification in circulating free DNA in patients with breast cancer

Background:

Human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in 20–25% of breast cancers. This study investigated circulating free DNA (cfDNA) for detection of HER2 gene amplification in patients with breast cancer.

Methods:

Circulating free DNA was extracted from plasma of unselected patients with primary breast cancer (22 before surgery and 68 following treatment), 30 metastatic patients and 98 female controls using the QIAamp Blood DNA Mini Kit (Qiagen). The ratio of HER2 to an unamplified reference gene (contactin-associated protein 1 (CNTNAP1)) was measured in cfDNA samples by quantitative PCR (qPCR) using SK-BR-3 cell line DNA as a positive control.

Results:

We validated the qPCR assay with DNA extracted from 23 HER2 3+ and 40 HER2-negative tumour tissue samples; the results agreed for 60 of 63 (95.2%) tumours. Amplification was detected in cfDNA for 8 of 68 patients following primary breast cancer treatment and 5 of 30 metastatic patients, but was undetected in 22 patients with primary breast cancer and 98 healthy female controls. Of the patients with amplification in cfDNA, 10 had HER2 3+ tumour status by immunohistochemistry.

Conclusions:

The results demonstrate for the first time the existence of amplified HER2 in cfDNA in the follow-up of breast cancer patients who are otherwise disease free. This approach could potentially provide a marker in patients with HER2-positive breast cancer.     

A pilot study to explore circulating tumour cells in pancreatic cancer as a novel biomarker

Background:

Obtaining tissue for pancreatic carcinoma diagnosis and biomarker assessment to aid drug development is challenging. Circulating tumour cells (CTCs) may represent a potential biomarker to address these unmet needs. We compared prospectively the utility of two platforms for CTC enumeration and characterisation in pancreatic cancer patients in a pilot exploratory study.

Patients and methods:

Blood samples were obtained prospectively from 54 consenting patients and analysed by CellSearch and isolation by size of epithelial tumour cells (ISET). CellSearch exploits immunomagnetic capture of CTCs-expressing epithelial markers, whereas ISET is a marker independent, blood filtration device. Circulating tumour cell expression of epithelial and mesenchymal markers was assessed to explore any discrepancy in CTC number between the two platforms.

Results:

ISET detected CTCs in more patients than CellSearch (93% vs 40%) and in higher numbers (median CTCs/7.5 ml, 9 (range 0–240) vs 0 (range 0–144)). Heterogeneity observed for epithelial cell adhesion molecule, pan-cytokeratin (CK), E-Cadherin, Vimentin and CK 7 expression in CTCs may account for discrepancy in CTC number between platforms.

Conclusion:

ISET detects more CTCs than CellSearch and offers flexible CTC characterisation with potential to investigate CTC biology and develop biomarkers for pancreatic cancer patient management.     

Monitoring gastric cancer progression with circulating tumour DNA

Background:

Circulating tumour DNA (ctDNA) is an emerging candidate biomarker for malignancies and may be useful for monitoring the disease status of gastric cancer.

Methods:

We performed targeted deep sequencing of plasma cell-free DNA (cfDNA) by massively parallel sequencing in patients with tumours harbouring TP53 mutations. The quantitative values of TP53-ctDNA during the clinical course were compared with the tumour status.

Results:

Three out of ten patients with TP53 mutations in primary tumours showed detectable TP53 mutation levels in preoperative cfDNA. Although the cfDNA concentrations were not always reflective of the disease course, the ctDNA fraction correlated with the disease status.

Conclusions:

ctDNA may serve as a useful biomarker to monitor gastric cancer progression and residual disease.     

Circulating tumour cell-derived plastin3 is a novel marker for predicting long-term prognosis in patients with breast cancer

Background:

Identification of promising biomarkers that predict the prognosis of patients with breast cancer is needed. In this study, we hypothesised that the expression of the epithelial–mesenchymal transition-related biomarker plastin3 (PLS3) in peripheral blood could be a prognostic factor in breast cancer.

Methods:

We examined PLS3 expression in breast cancer cell lines with epithelial and mesenchymal traits and in circulating tumour cells (CTCs) obtained from the peripheral blood of breast cancer patients. We investigated PLS3 expression in the peripheral blood of 594 patients with breast cancer to evaluate the clinical significance of PLS3 expression.

Results:

Robust PLS3 expression was observed in different breast cancer cell lines (Hs578t, MCF-7, MDA-MB-468, and MDA-MB-231) as well as in a bone marrow derived cancer cell line (BC-M1). In both the training (n=298) and validation (n=296) sets, PLS3 expression was observed in CTCs of patients with breast cancer. PLS3-positive patients showed significantly poorer overall and disease-free survival than PLS3-negative patients (P=0.0001 and 0.003, respectively). Subset analysis revealed that this prognostic biomarker was relevant in patients with stage I–III cancer, particularly in patients with luminal-type and triple-negative-type tumours.

Conclusions:

These data demonstrated that PLS3 was expressed in CTCs undergoing the epithelial–mesenchymal transition in patients with breast cancer. Furthermore, PLS3 may be an excellent biomarker for identifying groups at risk of recurrence or with a poor prognosis.     

Differential effect of adjuvant taxane-based and taxane-free chemotherapy regimens on the CK-19 mRNA-positive circulating tumour cells in patients with early breast cancer

Background:

To determine the effect of adjuvant taxane-free and taxane-based chemotherapy regimens on the elimination of circulating tumour cells (CTCs) in patients with early breast cancer.

Methods:

The presence of CK-19 mRNA-positive CTCs in the peripheral blood was evaluated before and after chemotherapy, using a real-time RT–PCR assay, in a historical comparison of two cohorts of women with stage I–III breast cancer treated with adjuvant taxane-free (N=211; FE₇₅C or E₇₅C) and taxane-based (N=334; T/E₇₅C or T/E₇₅) chemotherapy.

Results:

Taxane-based chemotherapy resulted in a higher incidence of CTCs'' elimination than taxane-free regimens since 49.7% (74 of 149) and 33.0% (29 of 88) of patients with detectable CTCs before chemotherapy, respectively, turned negative post-chemotherapy (P=0.015). Patients treated with taxane-free regimens had a significantly lower disease-free survival (DFS) (P=0.035) than patients treated with taxane-based regimens; this difference was observed in patients with but not without detectable CTCs before chemotherapy (P=0.018 and P=0.481, respectively). The incidence of deaths was significantly higher in the taxane-free cohort of patients with but not without detectable CTCs before chemotherapy compared with that of the taxane-based cohort (P=0.002). Multivariate analysis revealed that the chemotherapy regimen was significantly associated with prolonged DFS (HR: 2.00; 95% CI=1.20–3.34).

Conclusion:

Elimination of CK-19 mRNA-positive CTCs during adjuvant chemotherapy seems to be an efficacy indicator of treatment and is associated with a favourable clinical outcome of patients with detectable CTCs before chemotherapy.     

Circulating tumour cells detected by a novel adenovirus-mediated system may be a potent therapeutic marker in gynaecological cancers

Background:

Recently developed detection system for circulating tumour cells (CTCs) using a telomerase-specific replicative adenovirus generated nonspecific green fluorescent protein (GFP) signals because of the co-presence of white blood cells (WBCs) nonspecifically infected by viruses. Here, we established a unique detection system for CTCs that completely excludes nonspecific signals.

Methods:

Blood obtained from the patients was subjected to haemolytic processes to eliminate red blood cells. The cell pellets were then infected with OBP-401, fixed, incubated with fluorescence-labelled anti-CD45 antibody to mark white blood WBCs, and examined on slides under a microscope.

Results:

Preparatory experiments with cancer cells artificially added to healthy donor samples confirmed that CD45 labelling could distinguish GFP-positive cancer cells from WBCs. In 53 patients with gynaecological cancers, CTCs were detected in 21 patients (39.6%) when CD45-positive cells were excluded as WBCs among GFP-positive cells. No CTCs were detected in samples from healthy volunteers. There was no significant correlation between CTC counts and known clinicopathological factors. The CTCs rapidly vanished after surgery or chemotherapy in most patients whose treatments were effective. In contrast, the persistence of CTCs even after treatments was tightly associated with poor response to the treatments (P<0.005).

Conclusion:

The presence of CTCs in our system may potentially be a novel therapeutic marker in gynaecological cancers.     

Detection and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes

Background:

The enumeration of circulating tumour cells (CTCs) with the EpCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). The aim of this study was to explore potential differences in the detection and prognostic significance of CTCs in MBC according to immunohistochemical subtypes of breast cancer.

Methods:

CellSearch CTC counts were obtained from 154 MBC patients before first-line systemic treatment between November 2007 and August 2012. Patients were categorised in five subgroups according to immunohistochemical surrogate definitions of intrinsic subtypes in breast cancer based on hormone receptor status, HER2/neu status and histological grade. Differences in progression-free (PFS) and overall survival (OS) were assessed relative to the cut-off value of ⩾5 CTCs per 7.5 ml blood.

Results:

No significant differences were observed in the absolute CTC counts (P=0.120) or in CTC positivity rates according to ⩾1 and ⩾5 CTCs per 7.5 ml blood detection thresholds (P=0.165 and P=0.651, respectively) between immunohistochemical subtypes. However, very high CTC counts, defined as ⩾80 CTCs per 7.5 ml, were observed more frequently in patients with Luminal A and triple negative (TN) breast cancer (P=0.024). In the total study population, the presence of ⩾5 CTCs was the single most significant prognostic factor for both PFS and OS in multivariate analysis (P<0.001). A more limited prognostic impact, not reaching statistical significance, was observed in patients with HER2-positive disease as opposed to patients with Luminal A, Luminal B–HER2-negative and TN disease.

Conclusion:

The detection of EpCAM+CTCs was not clearly associated with any of the immunohistochemical subtypes of breast cancer in patients with MBC before first-line treatment. Potentially clinically relevant differences were however observed at very high CTC counts. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2-positive patients with MBC.     

The correlation between cell-free DNA and tumour burden was estimated by PET/CT in patients with advanced NSCLC

Background:

Cell-free DNA (cfDNA) circulating in the blood holds a possible prognostic value in malignant diseases. Under malignant conditions, the level of cfDNA increases but the biological mechanism remains to be fully understood. We aimed to examine the correlation between cfDNA and total tumour burden defined by positron emission tomography (PET) parameters.

Methods:

Patients with advanced non-small cell lung cancer (NSCLC) were enrolled into a prospective biomarker trial. Before treatment, plasma was extracted and the level of cfDNA was determined by qPCR. An ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) PET/computed tomography (CT) scan was performed and evaluated in terms of metabolic tumour volume (MTV) and total lesion glycolysis (TLG). Tumour contours were delineated semi-automatically by a threshold standardised uptake value (SUV) of 2.5. The primary end point was correlation among cfDNA, MTV and TLG. The secondary end point was overall survival (OS) according to cfDNA, MTV and TLG.

Results:

Fifty-three patients were included. There were no correlations between cfDNA and MTV (r=0.1) or TLG (r=0.1). cfDNA >75th percentile was correlated with shorter OS (P=0.02), confirmed in a multivariate analysis. MTV>the median was associated with a significantly shorter OS (P=0.02). There was no significant difference in OS according to TLG (P=0.08).

Conclusion:

Cell-free DNA may not be a simple measure of tumour burden, but seems to reflect more complex mechanisms of tumour biology, making it attractive as an independent prognostic marker.     

Detection of circulating tumour cells with a hybrid (epithelial/mesenchymal) phenotype in patients with metastatic non-small cell lung cancer

Background:

Circulating tumour cells (CTC) have a crucial role in metastasis formation and can consistently provide information on patient prognosis. Epithelial-mesenchymal transition (EMT) is considered as an essential process in the metastatic cascade, but there is currently very few data demonstrating directly the existence of the EMT process in CTCs.

Methods:

CTCs were enriched by blood filtration using ISET (isolation by size of epithelial tumour cells), triply labelled with fluorescent anti-vimentin, anti-pan-keratin antibodies and SYTOX orange nuclear dye, and examined by confocal microscopy in six patients with metastatic non-small cell lung cancer (NSCLC). In parallel, CTCs were morphocytologically identified by an experienced cytopathologist.

Results:

Isolated or clusters of dual CTCs strongly co-expressing vimentin and keratin were evidenced in all patients (range 5–88/5 ml). CTCs expressing only vimentin were detected in three patients, but were less frequent (range 3–15/5 ml). No CTC expressing only keratin was detected.

Conclusion:

We showed for the first time the existence of hybrid CTCs with an epithelial/mesenchymal phenotype in patients with NSCLC. Their characterisation should provide further insight on the significance of EMT in CTCs and on the mechanism of metastasis in patients with NSCLC.     

Epigenetic alteration: new insights moving from tissue to plasma – the example of PCDH10 promoter methylation in colorectal cancer

Background:

Tumour-released DNA in blood represents a promising biomarker for cancer detection. Although epigenetic alterations such as aberrant promoter methylation represent an appealing perspective, the discordance existing between frequencies of alterations found in DNA extracted from tumour tissue and cell-free DNA (cfDNA) has challenged their practical clinical application. With the aim to explain this bias of agreement, we investigated whether protocadherin 10 (PCDH10) promoter methylation in tissue was associated with methylation pattern in matched cfDNA isolated from plasma of patients with colorectal cancer (CRC), and whether the strength of concordance may depend on levels of cfDNA, integrity index, as well as on different clinical–pathological features.

Methods:

A quantitative methylation-specific PCR was used to analyse a selected CpG site in the PCDH10 promoter of 67 tumour tissues, paired normal mucosae, and matched plasma samples. The cfDNA integrity index and cfDNA concentration were assessed using a real-time PCR assay.

Results:

The PCDH10 promoter methylation was detected in 63 out of 67 (94.0%) surgically resected colorectal tumours and in 42 out of 67 (62.7%) plasma samples. The median methylation rate in tumour tissues and plasma samples was 43.5% (6.3–97.8%) and 5.9% (0–80.9%), respectively. There was a significant correlation between PCDH10 methylation in cfDNA and tumour tissue in patients with early CRC (P<0.0001). The ratio between plasma and tissue methylation rate increases with increasing cfDNA integrity index in early-stage cancers (P=0.0299) and with absolute cfDNA concentration in advanced cancers (P=0.0234).

Conclusion:

Our findings provide new insight into biological aspects modulating the concordance between tissues and plasma methylation profiles.     

Clinical significance of disseminated tumour cells in non-small cell lung cancer

Background:

Early-stage non-small cell lung cancer (NSCLC) patients have a high risk of disease relapse despite curatively intended surgical resection, and the detection of tumour cells in the bone marrow could be one method of determining the presence of the disseminated disease in its early stages.

Methods:

Bone marrow aspirates were collected from 296 patients at the time of surgery, and the presence of disseminated tumour cells was determined with the help of immunomagnetic selection (IMS) using the MOC31-antibody recognising EpCAM and with the help of standard immunocytochemistry (ICC) using the anti-cytokeratin (CK) antibodies AE1/AE3.

Results:

Disseminated tumour cells were found in 152 of 252 (59%) bone marrow samples using IMS and in 25 of 234 (11%) samples using ICC. No association between the two detection methods was observed. The presence of EpCAM⁺ cells was not associated with any clinicopathological parameters, whereas a higher frequency of CK⁺ cells was found in patients with an advanced pT status. Disseminated tumour cells, as detected using IMS, had no prognostic impact. Patients with CK⁺ cells in the bone marrow had a reduced relapse-free survival, but the difference was not statistically significant.

Conclusion:

Our findings do not support the further development of DTC detection for clinical use in early-stage NSCLC. Future studies should include the molecular characterisation of DTCs, along with an attempt to identify subpopulations of cells with biological and clinical significance.