Tumour-derived extracellular vesicles in blood of metastatic cancer patients associate with overall survival

Background: Circulating tumour cells (CTCs) in blood associate with overall survival (OS) of cancer patients, but they are detected in extremely low numbers. Large tumour-derived extracellular vesicles (tdEVs) in castration-resistant prostate cancer (CRPC) patients are present at around 20 times higher frequencies than CTCs and have equivalent prognostic power. In this study, we explored the presence of tdEVs in other cancers and their association with OS. Methods: The open-source ACCEPT software was used to automatically enumerate tdEVs in digitally stored CellSearch® images obtained from previously reported CTC studies evaluating OS in 190 CRPC, 450 metastatic colorectal cancer (mCRC), 179 metastatic breast cancer (MBC) and 137 non-small cell lung cancer (NSCLC) patients before the initiation of a new treatment. Results: Presence of unfavourable CTCs and tdEVs is predictive of OS, with respective hazard ratios (HRs) of 2.4 and 2.2 in CRPC, 2.7 and 2.2 in MBC, 2.3 and 1.9 in mCRC and 2.0 and 2.4 in NSCLC, respectively. Conclusions: tdEVs have equivalent prognostic value as CTCs in the investigated metastatic cancers. CRPC, mCRC, and MBC (but not NSCLC) patients with favourable CTC counts can be further prognostically stratified using tdEVs. Our data suggest that tdEVs could be used in clinical decision-making.</p

Classification of Cells in CTC-Enriched Samples by Advanced Image Analysis

In the CellSearch® system, blood is immunomagnetically enriched for epithelial cell adhesion molecule (EpCAM) expression and cells are stained with the nucleic acid dye 4′6-diamidino-2-phenylindole (DAPI), Cytokeratin-PE (CK), and CD45-APC. Only DAPI+/CK+ objects are presented to the operator to identify circulating tumor cells (CTC) and the identity of all other cells and potential undetected CTC remains unrevealed. Here, we used the open source imaging program Automatic CTC Classification, Enumeration and PhenoTyping (ACCEPT) to analyze all DAPI+ nuclei in EpCAM-enriched blood samples obtained from 192 metastatic non-small cell lung cancer (NSCLC) patients and 162 controls. Significantly larger numbers of nuclei were detected in 300 patient samples with an average and standard deviation of 73,570 ± 74,948, as compared to 359 control samples with an average and standard deviation of 4191 ± 4463 (p < 0.001). In patients, only 18% ± 21% and in controls 23% ± 15% of the nuclei were identified as leukocytes or CTC. Adding CD16-PerCP for granulocyte staining, the use of an LED as the light source for CD45-APC excitation and plasma membrane staining obtained with wheat germ agglutinin significantly improved the classification of EpCAM-enriched cells, resulting in the identification of 94% ± 5% of the cells. However, especially in patients, the origin of the unidentified cells remains unknown. Further studies are needed to determine if undetected EpCAM+/DAPI+/CK-/CD45- CTC is present among these cells

Single tube liquid biopsy for advanced non-small cell lung cancer

The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAM(high) circulating tumor cells (CTC), EpCAM(low) CTC, tumor-derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA). EpCAM(high) CTC were detected with CellSearch, tdEV in the CellSearch images and EpCAM(low) CTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had >= 2 EpCAM(high) CTC, 15% had >= 2 EpCAM(low) CTC, 27% had >= 18 tdEV and 19% had ctDNA with >= 10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAM(high) CTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAM(low) CTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy

Leukocyte-Derived Extracellular Vesicles in Blood with and without EpCAM Enrichment

Large tumor-derived Extracellular Vesicles (tdEVs) detected in blood of metastatic prostate, breast, colorectal, and non-small cell lung cancer patients after enrichment for Epithelial Cell Adhesion Molecule (EpCAM) expression and labeling with 4′,6-diamidino-2-phenylindole (DAPI), phycoerythrin-conjugated antibodies against Cytokeratins (CK-PE), and allophycocyanin-conjugated antibody against the cluster of differentiation 45 (CD45-APC), are negatively associated with the overall survival of patients. Here, we investigated whether, similarly to tdEVs, leukocyte-derived EVs (ldEVs) could also be detected in EpCAM-enriched blood. Presence of ldEVs and leukocytes in image data sets of EpCAM-enriched samples of 25 healthy individuals and 75 metastatic cancer patients was evaluated using the ACCEPT software. Large ldEVs could indeed be detected, but in contrast to the 20-fold higher frequency of tdEVs as compared to Circulating Tumor Cells (CTCs), ldEVs were present in a 5-fold lower frequency as compared to leukocytes. To evaluate whether these ldEVs pre-exist in the blood or are formed during the CellSearch procedure, the blood of healthy individuals without EpCAM enrichment was labelled with the nuclear dye Hoechst and fluorescently tagged monoclonal antibodies recognizing the leukocyte-specific CD45, platelet-specific CD61, and red blood cell-specific CD235a. Fluorescence microscopy imaging using a similar setup as the CellSearch was performed and demonstrated the presence of a similar population of ldEVs present at a 3-fold lower frequency as compared to leukocytes

Defining the dimensions of circulating tumor cells in a large series of breast, prostate, colon, and bladder cancer patients

Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient‐derived CTCs acquired on cartridges of the FDA‐cleared CellSearch® method were retrospectively collected and automatically re‐analyzed using the accept software package. The median CTC diameter (μm) was computed per tumor type. The size differences between the different tumor types and references (tumor cell lines and leukocytes) were nonparametrically tested. A total of 1962 CellSearch® cartridges containing 71 612 CTCs were included. In BC, the median computed diameter (CD) of patient‐derived CTCs was 12.4 μm vs 18.4 μm for cultured cell line cells. For PC, CDs were 10.3 μm for CTCs vs 20.7 μm for cultured cell line cells. CDs for CTCs of CRC and BLC were 7.5 μm and 8.6 μm, respectively. Finally, leukocytes were 9.4 μm. CTC size differed statistically significantly between the four tumor types and between CTCs and the reference data. CTC size differences between tumor types are striking and CTCs are smaller than cell line tumor cells, whose size is often used as reference when developing CTC analysis methods. Based on our data, we suggest that the size of CTCs matters and should be kept in mind when designing and optimizing size‐based isolation methods

Tumor-derived extracellular vesicles in blood of metastatic breast, colorectal, prostate, and non-small cell lung cancer patients associate with worse survival

Circulating Tumor Cell (CTC) counts, as determined by the CellSearch® system, associate with poor overall survival (OS) in castration-resistant prostate cancer (CRPC), breast, colorectal and non-small cell lung (NSCLC) cancer patients. The cut-offs used to discriminate between patients with favorable and unfavorable prognoses are 5 CTCs / 7.5 mL of blood for breast and CRPC, 3 for colorectal and 1 for NSCLC. The low numbers of CTCs frequently limit the accurate discrimination of patients into favorable or unfavorable prognosis groups. We previously reported the presence of CK+, DNA-, CD45- tumor-derived extracellular vesicles (tdEVs) in ~20x higher frequencies in CRPC patients and showed their equivalence to CTC counts in predicting clinical outcome. In this study, we explored the presence of tdEVs in the blood of metastatic CRPC, breast, colorectal, and NSCLC patients, determined the association of tdEVs with OS and whether they aid in stratifying patients with favorable CTC counts. Digitally stored CellSearch® system images obtained from previously reported studies from 190 CRPC, 450 colorectal, 179 breast and 117 NSCLC patients before the initiation of a new treatment were used to enumerate tdEVs automatically using the open-source ACCEPT software. tdEV counts highly correlated with CTC counts in all cancer types (Spearman’s Rho tests p<0.001) and were present at 0-280-fold (Mean 20, SD 25) higher frequencies. A cut-off tdEV value of 23 (median +2SD of 93 healthy donors) was used to dichotomize patients into favorable and unfavorable groups. Kaplan-Meier analyses showed associations of CTCs and tdEVs with OS with hazard ratios (HRs) of 2.4 & 2.1 in CRPC, 2.7 & 2.1 in breast, 2.3 & 2.0 in colorectal and 1.6 & 2.0 in NSCLC, respectively. 52% of the 43% CRPC patients with favorable CTC counts had elevated tdEVs; 53% of the 50% breast cancer patients with favorable CTC counts had elevated tdEVs; 46% of the 72% colorectal cancer patients with favorable CTC counts had elevated tdEVs and 3% of the 75% NSCLC patients with favorable CTC counts had unfavorable tdEVs. CRPC, colorectal and breast cancer patients with favorable CTC counts could be further stratified using tdEV counts (log-rank tests p < 0.05). CTC and tdEV counts equally predict OS n the different cancer types studied. Use of tdEVs in patients with favorable CTC counts can further stratify patients and thereby contribute to clinical decision making. Funded by the NWO Applied and Engineering Sciences Cancer-ID project #14190, the EUFP7 CTCTrap project #305341 and the EU IMI CANCER-ID project # 115749-1