Background
The availability of more and more targeted treatments necessitates the development of techniques to screen cancer patients for the presence of the respective therapeutic targets in the primary and metastatic lesions. However, the invasiveness of sampling of biopsies from all different metastatic lesions and the associated patient morbidity demands the need for a less invasive test. EpCAM+ circulating tumor cells (CTCs) isolated from the peripheral blood of cancer patients constitute clinically relevant and easily accessible tumor material from a single tube of blood [
1,
2]. The possibility of longitudinal measurements and the screening of CTCs for therapeutic targets can provide clinicians with the mutational status of the tumor and the presence of treatment targets in real-time facilitating their decisions on treatment monitoring [
3,
4]. The reliability of such an assessment increases with the number of CTCs available. In metastatic breast cancer, ~ 50% of patients have ≥ 5 CTCs detected with the CellSearch system and the percentage of patients with ≥ 10 and ≥ 100 CTCs decreases rapidly [
1].
The development of the open-source ACCEPT software allowed the automated enumeration of all objects in the fluorescence images and the more objective assessment of treatment targets on CTCs [
5] eliminating inter- and intra-operator variations [
6].
Large tumor-derived extracellular vesicles (tdEVs) are co-isolated with CTCs and are present in an order of magnitude higher frequencies than CTCs [
7,
8]; thus, they can increase the number of “readable” patient samples for the assessment of therapeutic targets.
The original study used in the present analysis is the CLCC-IC-2006-04 (NCT00898014) with 267 stage IV breast cancer patients receiving first-line chemotherapy [
9]. The primary objective was to predict overall survival (OS) and progression-free survival (PFS) by counting all CK+ CTCs before the initiation of the second course of chemotherapy [
9]. The secondary objective was to evaluate the HER2 status on CTCs over the course of a treatment. Ligthart et al. [
3] and Zeune et al. [
5] quantified the HER2 expression on the pre-scored by the operator CTCs in an automated manner demonstrating a more objective and less biased assessment among the different operators.
In the present study, we wanted to address two important questions:
i.)
Is HER2 expressed on tdEVs?
ii.)
Is HER2 expressed on CTCs lacking CK expression? And if so, do these populations associate with the clinical outcome of the patients?
To address the aforementioned research questions, the open-source ACCEPT software was used and the enumeration of all CTCs and tdEVs falling in the different immunophenotypic classes was performed in an automated manner.
Discussion
The HER2 is found overexpressed in around 15–30% of breast cancer cases mainly because of the HER2/neu oncogene amplification [
15,
16]. Randomized clinical trials have demonstrated its predictive and prognostic value with HER2+ breast cancer patients having a remarkably improved clinical outcome when treated with anti-HER2 therapy, such as trastuzumab or lapatinib next to chemotherapy, as compared to HER2+ patients treated with solely chemotherapy [
17‐
20]. Furthermore, HER2− breast cancer patients randomized in two treatment arms with and without anti-HER2 therapy did not show any clinical benefits further supporting the predictive value of HER2 [
21]. These observations necessitate the use of an accurate test to assess the HER2 tumor status and facilitate the clinician’s treatment decision-making. The current state of the art evaluates HER2 status on solid biopsies (either tumor needle biopsy or whole tumor after resection) by immunohistochemistry or/and FISH. Several guidelines have improved the accuracy of HER2 evaluation [
22]. Nevertheless, there are cases of discorcondant immunohistochemistry and FISH results or high HER2 heterogeneity of the tumor [
23,
24] preventing an objective consensus on HER2 status among different operators.
Non-invasive liquid biopsies have emerged to be promising alternatives to solid biopsies providing clinically relevant tumor material in real-time. The increasing load of CK+ CTCs and tdEVs as identified by the CellSearch system is strongly associated with worsening progression-free and overall survival [
1,
2,
8,
10,
25]. The CellSearch system immunomagnetically enriches CTCs targeting the epithelial cell adhesion molecule (EpCAM); consequently, all EpCAM-negative or low expressing CTCs are missed by the system. Many approaches have been introduced to overcome this limitation by enrichment of CTCs based on their physical properties such as size, density, and charge [
26], but no obvious advantages have been demonstrated in clinical studies so far. Importantly, EpCAM
low, CK+ CTCs identified after size-based separation of EpCAM-depleted blood samples did not correlate with clinical outcomes in metastatic non-small cell lung, prostate, and breast cancer [
27,
28]. In the present study, we investigated whether the expression of HER2 can be assessed through tdEVs and whether CTCs and tdEVs are missed by the system due to the lack of CK expression. The whole workflow from EpCAM enrichment to CTC and tdEV scoring can be done in a fully automated manner, once a blood sample of 7.5 mL is available. Consequently, the test is not subjected to the quality of the sample, the handling, the staining methodology, and the judgment of the technician. It can be performed in a timely fashion and can facilitate the treatment monitoring of the patient.
The present analysis validates the association of CK+ tdEVs with the overall survival of metastatic breast cancer patients since it is based on an independent patient cohort than the one that we previously reported (IMMC01 study) [
8]. HER2 expression could be detected on CK+ tdEVs that were co-isolated with CTCs using the CellSearch system.
The ACCEPT software allows the visualization and automated enumeration of CK− objects, which are not presented to the operator by the FDA-cleared CellSearch image analysis algorithm (CellTracks Analyzer II). Interestingly, the inclusion of the HER2-FITC antibody allowed the detection of CD45−, CK−, and HER2+ CTCs and tdEVs in the EpCAM-enriched blood samples of metastatic breast cancer patients increasing the percentage of patient samples with detectable CTCs from 89 to 95%. CK+ tdEVs were already present in 99% of patient samples, which increased to 100% after the inclusion of anti-HER2.
Although we have not genetically proven that these CK− CTCs and tdEVs are indeed cancerous, their similar correlation with OS (Fig.
2) tends to confirm that hypothesis. The aneuploidy of these CK− cells should be addressed by FISH in a prospective clinical study, since the cartridges, whose image datasets were used in the present analysis, are no longer available. Fehm et al. [
29] first demonstrated the malignancy of EpCAM-enriched CK+, CD45−, and DAPI+ cells (CellSearch CTC definition) by FISH from the blood of patients with carcinomas. A protocol to perform FISH directly in the CellSearch cartridges was developed later by Swennenhuis et al. [
30], who evaluated the technique in blood samples of castration-resistant prostate cancer patients.
The observation of CK−HER2+ CTCs and tdEVs raises questions about other CK−HER2− CTC and tdEV populations present in the EpCAM-enriched samples. The findings of Crespo et al. that aneuploid CD45−, CK−, and AR+ CTCs in EpCAM-enriched blood samples of castration-resistant prostate cancer patients are associated with worse OS further support our hypothesis [
31]. The detection of CK− aneuploid cells in blood samples of ovarian, breast, and colorectal cancer patients has been also described by Pecot et al. [
32]. All aforementioned studies further encourage the inclusion of additional antibodies to detect nucleated events of unknown cell lineage in the EpCAM-enriched samples and decrease false-negative rates of CTCs.
Importantly, all three immunophenotypes of CTCs (HER2+CK−, HER2−CK+, and HER2+CK+) were present in the majority of EpCAM-enriched patient samples, and the presence of heterogeneous CTC and tdEV immunophenotypes was associated with worse clinical outcome of the patients (Fig.
3).
Our next question was whereas a minimum threshold of a HER2+ population could predict the HER2 status of the tissue as a real-time liquid biopsy. The %HER2+CK+ tdEVs performed the best achieving higher sensitivity and specificity than CTCs (Fig.
4), most likely because of the higher frequencies of tdEVs better reflecting the tumor heterogeneity. Based on that test, a patient can be affirmed to have a HER2+ tumor with at least 74% accuracy if ≥ 7% of total EpCAM+ tdEVs are CK+ and HER2+. However, it is important to mention that the determination of the HER2 amplification status of the primary tissue was done at the time of diagnosis with most of the patients being as stages I–III. The CTC evaluation reported in the original study (and the present analysis) occurred after the progression of the patients to stage IV (one of the inclusion criteria of the patients enrolled in the study), which can be years after the initial diagnosis and the tumor resection. It is well known that the phenotype and genotype of the primary tumor can substantially differ from the respective genotypic and phenotypic features of the metastatic lesions as the disease progresses [
33‐
36] with CTCs resembling better the mutational status of metastatic lesions than of primary tumor [
37]. Therefore, the HER2 expression on the primary tumor can differ substantially from the status of the metastatic lesions as well the CTCs and tdEVs isolated having an impact on the concordance found. Whether the HER2 assessment via a liquid biopsy (CTCs or/and tdEVs) can better predict response to anti-HER2-targeted therapies compared to the current assessment via a solid biopsy remains to be addressed.
Molecular characterization of the EpCAM+ tdEVs of patients undergoing HER2-targeted therapies at follow-up time points can contribute to better comprehend the underlying mechanism of tumor resistance to anti-HER2 treatment [
38], which is observed in more than 70% of HER+ breast cancer patients within a year from the initiation of the treatment [
39]. Ciravolo et al. have already suggested a mechanism of anti-HER2 resistance by the increased binding efficiency of HER2+ exosomes to trastuzumab in progressive HER2+ breast cancers as compared to earlier stages of breast cancer [
40]. Another mechanism has been suggested by Al-Nedawi et al. with tdEVs transferring the oncogenic form of epidermal growth factor receptor EGFRvIII to cells without that immunophenotype [
41].
Competing interests
L.W.M.M. Terstappen is listed as an inventor on US patents (No: 5,985,153; No: 5,993,665; No: 6,013,188; No: 6,136,182; No: 6,361,749; No: 6,365,362; No: 6,551,843 B1; No: 6,623,982 B1; No: 6,620,627 B1; No: 6,623,983 B1; No: 6,645,731 B2; No: 6,660,159 B1; No: 6,790,366 B2; No: 6,890,426 B2; No: 7,056,657 B2; No: 7,332,288 B2; 7,863,012 B2; No: 8,329,422 B2) related to the CellSearch system, the rights of which are assigned to Menarini Silicon Biosystems; he is the chairman of the Department of Medical Cell BioPhysics at the University of Twente, which receives research funding related to the CellSearch system from Menarini Silicon Biosystems.
F.-C. Bidard and J.-Y. Pierga receive research funding from Menarini Silicon Biosystems.
All remaining authors have declared no conflicts of interest.
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