Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the ...DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation.
Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis.
On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines.
We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation.
Inflammatory breast cancer (IBC) is an aggressive disease. To date, no molecular feature reliably predicts either the response to chemotherapy (CT) or the survival. Using DNA microarrays, we searched ...for multigene predictors.
The World IBC Consortium generated whole-genome expression profiles of 137 IBC and 252 non-IBC (nIBC) samples. We searched for transcriptional profiles associated with pathological complete response (pCR) to neoadjuvant anthracycline-based CT and distant metastasis-free survival (DMFS) in respective subsets of 87 and 106 informative IBC samples. Correlations were investigated with predictive and prognostic gene expression signatures published in nIBC (nIBC-GES). Supervised analyses tested genes and activation signatures of 19 biological pathways and 234 transcription factors.
Three of five tested prognostic nIBC-GES and the two tested predictive nIBC-GES discriminated between IBC with and without pCR, as well as two interferon activation signatures. We identified a 107-gene signature enriched for immunity-related genes that distinguished between responders and nonresponders in IBC. Its robustness was demonstrated by external validation in three independent sets including two IBC sets and one nIBC set, with independent significant predictive value in IBC and nIBC validation sets in multivariate analysis. We found no robust signature associated with DMFS in patients with IBC, and neither of the tested prognostic GES, nor the molecular subtypes were informative, whereas they were in our nIBC series (220 stage I–III informative samples).
Despite the relatively small sample size, we show that response to neoadjuvant CT in IBC is, as in nIBC, associated with immunity-related processes, suggesting that similar mechanisms responsible for pCR exist. Analysis of a larger IBC series is warranted regarding the correlation of gene expression profiles and DMFS.
MicroRNAs (miRNAs) are key regulators of gene expression. In this study, we explored whether altered miRNA expression has a prominent role in defining the inflammatory breast cancer (IBC) phenotype.
...We used quantitative PCR technology to evaluate the expression of 384 miRNAs in 20 IBC and 50 non-IBC samples. To gain understanding on the biological functions deregulated by aberrant miRNA expression, we looked for direct miRNA targets by performing pair-wise correlation coefficient analysis on expression levels of 10 962 messenger RNAs (mRNAs) and by comparing these results with predicted miRNA targets from TargetScan5.1.
We identified 13 miRNAs for which expression levels were able to correctly predict the nature of the sample analysed (IBC vs non-IBC). For these miRNAs, we detected a total of 17,295 correlated miRNA-mRNA pairs, of which 7012 and 10 283 pairs showed negative and positive correlations, respectively. For four miRNAs (miR-29a, miR-30b, miR-342-3p and miR-520a-5p), correlated genes were concordant with predicted targets. A gene set enrichment analysis on these genes demonstrated significant enrichment in biological processes related to cell proliferation and signal transduction.
This study represents, to the best of our knowledge, the first integrated analysis of miRNA and mRNA expression in IBC. We identified a set of 13 miRNAs of which expression differed between IBC and non-IBC, making these miRNAs candidate markers for the IBC subtype.
To infer the prognostic value of simultaneous androgen receptor (
) and
profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of
...signaling inhibitors (ARSi).
Between March 2014 and April 2017, we recruited patients with mCRPC (
= 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight
splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of
and
was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan-Meier analysis, and independent associations were determined using multivariable Cox regression models.
Overall, no single
perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS.
inactivation harbored independent prognostic value HR 1.88; 95% confidence interval (CI), 1.18-3.00;
= 0.008, and outperformed ARV expression and detection of genomic
alterations. Using Cox coefficient analysis of clinical parameters and
status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months;
< 0.0001), which was validated in an independent mCRPC cohort (
= 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months;
< 0.0001).
In an all-comer cohort, tumor burden estimates and
outperform any
perturbation to infer prognosis.See related commentary by Rebello et al., p. 1699.
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.
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.
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.
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 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.
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.
Recently, peritumoural (lympho)vascular invasion, assessed on haematoxylin-eosin (HE)-stained slides, was added to the St Gallen criteria for adjuvant treatment of patients with operable breast ...cancer (BC). New lymphatic endothelium-specific markers, such as D2-40, make it possible to distinguish between blood (BVI) and lymph vessel invasion (LVI). The aim of this prospective study was to quantify and compare BVI and LVI in a consecutive series of patients with BC. Three consecutive sections of all formalin-fixed paraffin-embedded tissue blocks of 95 BC resection specimens were (immuno)histochemically stained in a fixed order: HE, anti-CD34 (pan-endothelium) and anti-D2-40 (lymphatic endothelium) antibodies. All vessels with vascular invasion were marked and relocated on the corresponding slides. Vascular invasion was assigned LVI (CD34 plus sign in circle or minus sign in circle/D2-40 plus sign in circle) or BVI (CD34 plus sign in circle/D2-40 minus sign in circle) and intra- (contact with tumour cells or desmoplastic stroma) or peritumoural. The number of vessels with LVI and BVI as well as the number of tumour cells per embolus were counted. Results were correlated with clinico-pathological variables. Sixty-six (69.5%) and 36 (37.9%) patients had, respectively, LVI and BVI. The presence of 'vascular' invasion was missed on HE in 20% (peritumourally) and 65% (intratumourally) of cases. Although LVI and BVI were associated intratumourally (P=0.02), only peritumoural LVI, and not BVI, was associated with the presence of lymph node (LN) metastases (p(peri)=0.002). In multivariate analysis, peritumoural LVI was the only independent determinant of LN metastases. Furthermore, the number of vessels with LVI was larger than the number of vessels with BVI (P=0.001) and lymphatic emboli were larger than blood vessel emboli (P=0.004). We demonstrate that it is possible to distinguish between BVI and LVI in BC specimens using specific lymphatic endothelium markers. This is important to study the contribution of both processes to BC metastasis. Furthermore, immunohistochemical detection of lymphovascular invasion might be of value in clinical practice.
Vascular endothelial growth factor (VEGF)-A inhibitors exhibit unseen high responses and toxicity in recurrent epithelial ovarian cancer suggesting an important role for the VEGF/VEGFR pathway. We ...studied the correlation of VEGF signalling and AKT/mTOR signalling. Using a tissue microarray of clinical samples (N=86), tumour cell immunohistochemical staining of AKT/mTOR downstream targets, pS6 and p4E-BP1, together with tumour cell staining of VEGF-A and pVEGFR2 were semi-quantified. A correlation was found between the marker for VEGFR2 activation (pVEGFR2) and a downstream target of AKT/mTOR signalling (pS6) (R=0.29; P=0.002). Additional gene expression analysis in an independent cDNA microarray dataset (N=24) showed a negative correlation (R=-0.73, P<0.0001) between the RPS6 and the VEGFR2 gene, which is consistent as the gene expression and phosphorylation of S6 is inversely regulated. An activated tumour cell VEGFR2/AKT/mTOR pathway was associated with increased incidence of ascites (chi(2), P=0.002) and reduced overall survival of cisplatin-taxane-based patients with serous histology (N=32, log-rank test, P=0.04). These data propose that VEGF-A signalling acts on tumour cells as a stimulator of the AKT/mTOR pathway. Although VEGF-A inhibitors are classified as anti-angiogenic drugs, these data suggest that the working mechanism has an important additional modality of targeting the tumour cells directly.
Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter occurs in about 40% of breast tumours and has been correlated with reduced APC protein levels. To what extent epigenetic ...alterations of the APC gene may differ according to specific breast cancer phenotypes, remains to be elucidated. Our aim was to explore the role of APC methylation in the inflammatory breast cancer (IBC) phenotype. The status of APC gene promoter hypermethylation was investigated in DNA from normal breast tissues, IBC and non-IBC by both conventional and real-time quantitative methylation-specific PCR (MSP). APC methylation levels were compared with APC mRNA and protein levels. Hypermethylation of the APC gene promoter was present in 71% of IBC samples (n=21) and 43% of non-IBC samples (n=30) by conventional MSP (P=0.047). The APC gene also showed an increased frequency of high methylation levels in IBC (in 74% of cases, n=19) vs non-IBC (in 46% of cases, n=35) using a qMSP assay (P=0.048). We observed no significant association between APC methylation levels by qMSP and APC mRNA or protein expression levels. In conclusion, for the first time, we report the association of aberrant methylation of the APC gene promoter with the IBC phenotype, which might be of biological and clinical importance.
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