Ethical conduct of the study

The ANG-008 study was sponsored by ANGLE Europe Ltd (ANGLE) and involved the collection of whole blood samples from healthy women as well as from women with metastatic breast cancer. The study was considered to be exempt from the IDE (investigational device exemption) regulations (21 Code of Federal Regulations (CFR) Part 812.2.c.3) due to the fact that the only procedure required for participation in the study was the collection of blood samples, which is considered to be non-invasive, as well as the fact that none of the results of the research testing were reported back to the subjects and/or the investigators, or used in the diagnosis, treatment and/or care of the subjects.

This study was conducted in a manner consistent with:

United States (US) standards of Good Clinical Practice (GCP) as defined in US FDA CFR, particularly 21 CFR Part 812 (i.e. Sponsor & Investigator responsibilities), Part 50 (Informed Consent Requirements), Part 54 (Financial Disclosure), Part 56 (IRB Approval) and Part 11 (Electronic Records);

International GCP standards using the International Conference on Harmonization (ICH) guidelines on GCP;

Applicable FDA regulations;

Institutional Review Board(s) (IRB) requirements.

Sample size calculation

Based on the preliminary data and literature review, the hypothesis that the Parsortix® PC1 System would be able to harvest observable CTCs as identified by IF in ≥ 25% of the MBC patients and in ≤ 3% of the HV control group was used. Assuming an overall study failure rate of ~ 5% (e.g., ineligible subjects, insufficient volume of blood, processing failures, etc.), it was expected that ~ 80 HVs and ~ 80 MBC patients would need to be enrolled to ensure a minimum of 75 HV subjects and 75 MBC patients with evaluable IF results for evaluation of the objective.

With a sample size of N = 75 evaluable MBC patients, a two-sided 95% confidence interval (CI) for a single proportion, using the large sample normal approximation, will extend a maximum of ± 11.4% from the actual proportion of MBC patients found to have observable CTCs. With a sample size of N = 75 evaluable HVs, a two-sided 95% CI for a single proportion, using the large sample normal approximation, will extend a maximum of ± 3.9% from the actual proportion of HVs with observable CTCs for an expected proportion of ≤ 3%.

Additionally, with a sample size of 75 evaluable MBC patients and 75 HVs, a two-group continuity corrected chi-square test with a 0.05 two-sided significance level (α) will have ~ 95% power (1—β) to detect a difference between a proportion of > 25% for MBC patients with observable CTCs and a proportion of < 3% for HV subjects with observable CTCs.

Enrollment and sample collection

All study participants provided informed consent before being enrolled in the study. Each subject was only entered into the study once. All laboratory testing was performed by operators blinded to the clinical status of the participants. A total of 76 female HVs and 85 MBC patients were enrolled between July 2019 and November 2019 at the clinical study site (University of Rochester Medical Center, Rochester, NY).

Inclusion criteria for the MBC patients were as follows:

Female ≥ 22 years of age;

Documented evidence of metastatic breast cancer (i.e. primary tumor histopathology of breast cancer and documented evidence of distant sites of metastasis by imaging, biopsy, and/or other means);

Willing and able to provide informed consent and agree to complete all aspects of the study.

The inclusion criteria for the HV subjects are detailed below. The information obtained from the HVs was ‘self-reported’, as complete medical records were not available at the enrolling site for these control subjects.

Females ≥ 22 years of age;

No known fever or active infections at the time of the blood collection;

No known current diagnosis of acute inflammatory disease or chronic inflammation;

No known current and/or prior history of malignancy, excluding skin cancers (squamous cell or basal cell);

Willing and able to provide informed consent and agrees to complete all aspects of the study.

None (0%) of the 76 HVs were found to be ineligible. A total of 9 (10.5%) of the 85 MBC patients enrolled were found to be ineligible or not usable for the study, leaving a total of 76 eligible MBC patients (Fig. 1).

Fig. 1

CONSORT Diagram for ANG-008 Study Subject Eligibility. Diagram shows enrolled patients, reasons for ineligibility within the MBC group, and breakdown of MBC patients in newly diagnosed, stable/responding diseases and progressive/recurrent disease groups

Four tubes of blood (one 3 mL K2 Ethylenediaminetetraacetic acid (EDTA) vacutainer for CBC with differential and erythrocyte sedimentation rate testing, two 10 mL K2EDTA vacutainers for processing on the Parsortix® System, and one 7.5 mL Serum-separating tube (SST) vacutainer for serum chemistry and lipid panel testing) were collected by venipuncture (or, for MBC patients, if applicable, through a venous port) from each HV subject and from each MBC patient, a minimum of seven days after the administration of a cytotoxic therapy (intravenously administered) and immediately prior to the administration of any other type of therapy. For the objectives detailed in this report, an average of 8.6 ± 1.2 mL of blood from one of the 10 mL K2EDTA vacutainers was processed on Parsortix® PC1 Systems and the population of cells harvested were deposited onto cytology slides for cytopathological evaluation using IF and WG staining.

A breakdown of the ages, demographics, and clinical information for the eligible HV subjects and MBC patients is provided in Table 1.

Table 1 Summary of eligible participants’ clinical characteristics. Table shows demographics in N=76 HV subjects and N=76 MBC patients who were eligible for the study. The p-values for BSA, BMI and age are for the comparison of the medians for these parameters between the HV subjects and the MBC patient groups and were calculated using a non-parametric chi-squared test for equality of the medians (due to the non-normal distribution of the data), while the p-values for the comparison of the proportions of subjects in the various clinical and demographical groupings between the HV subjects and MBC patient groups were determined using a Fisher’s exact test

The demographics of the MBC patient population was consistent with the demographics of MBC patients described in the literature [29]. Approximately one-third of the MBC patients enrolled had progressive / recurrent metastatic disease (35.7%), 7.9% had newly diagnosed disease, with the largest proportion having stable/responding disease (57.9%). The race distribution is typical of most US based clinical trials, with the majority of patients having a white background. The breast cancer phenotype for most of the MBC patients was Estrogen Receptor (ER) /Progesterone Receptor (PR) positive and Human epidermal growth factor receptor-2 (HER2) negative, with approximately 89% being ER and/or PR positive and 21.1% having HER2 positive breast cancer. Bone was the most prevalent site of metastatic disease (67.1%), followed by the lymph nodes (26.3%), the liver (19.7%) and/or the lungs (18.4%), which are the most common sites of breast cancer metastasis reported in the literature [30, 31]. There was a significant difference observed between the age and menopausal status of the HV subjects compared to the MBC patients, as the majority of the HV subjects were much younger compared to the MBC patients. This also led to significantly lower proportions of HV subjects with comorbidities and those taking medications compared to the MBC patients.

Blood processing on Parsortix® PC1 instrument

Blood separation was performed at the Targeted Therapeutics Laboratory at the Wilmot Cancer Institute within eight hours from blood draw using Parsortix® PC1 Systems. The Parsortix® PC1 System is a bench top laboratory instrument consisting of inbuilt computer, pneumatic and hydraulic components, and other electronics to control the instrument hardware and behavior. The Parsortix® PC1’s proprietary application software runs a series of encrypted Protocol Files (Clean, Prime, Separate, and Harvest) to control the instrument fluidic and hydraulic components. The instrument utilizes a single use, non-sterile Parsortix® GEN3 Cell separation cassette, containing precision molded separation structures with ‘step’ configurations. Whole blood flows along a series of channels under controlled and constant pressure conditions (99 mbar) to enable separation. The channel height progressively decreases at each step toward the final ‘critical gap’. As a result, in the case of blood, cells are captured in the critical gap based on their size and resistance to compression. The looped cassette layout is designed to maximize the width of separating steps, which is a key factor affecting separation capability and capture capacity, providing fluid paths with minimal resistance to liquid flow. The cassette layout is intentionally omni-directional such that during a separation, the sample always flows across the step structures and then through the critical gap. To harvest cells captured in the cassette, this flow is intentionally reversed to release the cells from the critical gap and step structures and flush them out of the cassette into another receptacle using a small volume of buffer (~ 210 µL).

Cytology slide preparation

The Targeted Therapeutics Laboratory prepared the cytology slides for shipment to ANGLE Guildford central laboratory where the IF evaluations were performed. Following separation and enrichment, captured cells were harvested into a 1.5 mL microfuge tube containing 60 μL of fetal bovine serum (FBS). The harvested cells and FBS mixture was pipetted into a Cytospin® 4 Cytofunnel™ assembly (Thermo Fisher Scientific) containing a positively charged glass Shandon™ Single Cytoslides™ (Thermo Fisher Scientific). The slide assembly was cytocentrifuged at 800 rpm for 3 min on low acceleration, and the slide was removed from the assembly and allowed to air-dry at room temperature for 1 min. The air-dried slide was then submersed in ice-cold 100% acetone for 5 min at -20 °C and allowed to air-dry at room temperature for 30 min. The fixed slides were stored refrigerated (at + 2–8 °C) and shipped weekly to the ANGLE Guildford central laboratory for staining and evaluation.

Immunofluorescence staining and imaging

The development of the IF assay used in this study is described in Additional Files 1, 2, 3, 4, 5 and 6. The procedure is summarized below.

Slides were kept refrigerated until IF staining was performed. Before staining, each slide was re-hydrated with 1 × Phosphate Buffered Saline (PBS) for 60 min. After re-hydration, slides were blocked with 2.5% Normal Horse Serum (S-2012 Vector Labs) and stained with an antibody mixture against surface blood lineage markers (CD45-Allophycocyanin (APC), CD16-APC, CD11b-APC and CD61-APC diluted in 1 × PBS) followed by another antibody mixture against intracellular markers (Cytokeratin (CK) 8-Alexa Fluor 488 (AF488), CK18-AF488, CK19-AF488, EpCAM-Alexa Fluor 555 (AF555), and 4′,6-diamidino-2-phenylindole (DAPI) diluted in Inside Perm (Miltenyi Biotec)). Slides were mounted with 50 µL of 1 × PBS, a 25 mm × 25 mm glass coverslip and fixogum.

Slides were examined using a Leica LAS X fluorescence microscope or a BioView Allegro Plus imaging system, and the cells of interest (i.e. CTCs) were classified based on their staining patterns as follows: 1) EpCAM + , CK + , CD-, DAPI + ; 2) EpCAM + , CK-, CD-, DAPI + ; and 3) EpCAM-, CK + , CD-, DAPI + .

Wright-Giemsa staining and cytological evaluation

Upon completion of the IF evaluation, the coverslips were removed from each of the slides, and the slides were air dried and stored at room temperature until shipment to the Department of Hematopathology, Division of Pathology and Laboratory Medicine, at MD Anderson Cancer Center for WG staining and cytopathological evaluation. The slides underwent Richard-Allen Scientific WG staining on an automated stainer and examination by a qualified pathologist with expertise in blood evaluation and cytopathology (Dr. Joseph Khoury, JDK) using light microscopy. CTCs were identified and enumerated using conventional cytomorphologic criteria for malignancy, which included: size larger than peripheral WBCs, moderate to abundant cytoplasm, cytoplasmic vacuoles (micro or macro), irregular nuclear contours, nuclear hyperchromasia and prominent nucleoli. A CTC cluster was defined as 3 or more cohesive cells [32].