# Exploring the Dual Analysis of Circulating Tumour Cells and Circulating Tumour DNA **Anne-Sophie Pailhes-Jimenez**\ Senior Director, R&D\ ANGLE Europe Ltd ------------------------------------------------------------------------ ## Disclaimer I am an employee from ANGLE Europe Limited. The opinions expressed during this presentation are my own and ANGLE's and may not represent the opinions of Illumina. Any uses of ANGLE and Illumina products described in this presentation may be uses that have not been cleared or approved by the FDA or any other applicable regulatory body. Illumina is compensating me to speak at this event. ------------------------------------------------------------------------ ## The Potential of Liquid Biopsy Throughout the Patient Care Pathway \[Image placeholder: The potential of liquid biopsy throughout the patient care pathway.\] ------------------------------------------------------------------------ ## Liquid Biopsy and CTCs **Blood as liquid biopsy:** - Minimally-invasive\ - Repeatable\ - Representative of cancer heterogeneity\ - Versatile\ - Informative [Image placeholder: Klaus Pantel and Catherine Alix-Panabieres. Liquid biopsy and minimal residual disease — latest advances and implications for cure. Nat Rev Clin Oncol 16, 409–424 (2019). https://doi.org/10.1038/s41571-019-0187-3] ------------------------------------------------------------------------ ## CTCs and ctDNA Provide Complementary Information ### Hypothesis Dual analysis provides critical information not otherwise available on cancer progression and for targeted treatment selection. > "CTCs, as living cells that are active in the metastatic process, can > provide prospective insight into a patient's cancer.\ > In comparison, ctDNA derived mainly from dead and dying cells, > provides important but historical information on patient disease."\ > --- Prof. Evi Lianidou\ > Head of the Molecular Diagnostics Laboratory\ > National and Kapodistrian University of Athens ### Cancer Progression Over Time **Retrospective -- ctDNA**\ Patient immune system and/or therapies have successfully addressed these variants. Cease these therapies. **Active cancer -- ctDNA and ctc-DNA**\ Active disease being addressed by current therapies but not complete. Continue current therapies or consider new therapies. **Prospective -- ctc-DNA**\ Clonal evolution, new variants. Immune system and therapies not addressing. Requires active intervention with new therapies. ------------------------------------------------------------------------ ## Circulating Tumour Cells (CTCs) **CTC and CTC clusters:** - Reflective of metastatic tumor\ - Source of biomarkers (suitable for multiomics analysis)\ - Rare event: **1 CTC / 10⁹ blood cells** Importance of upstream workflow to generate quality sample. [Image placeholder: Biomarkers circulating in the bloodstream] ------------------------------------------------------------------------ ## Parsortix® PC1 System The first **FDA cleared / CE-IVD medical device** for the capture and harvest of circulating tumor cells from metastatic breast cancer patient blood for user-validated subsequent analysis. \*FDA Clearance for use with metastatic breast cancer patients only ### Intended Use "The Parsortix® PC1 system is an in vitro diagnostic device intended to enrich circulating tumor cells (CTCs) from peripheral blood collected in K2EDTA tubes from patients diagnosed with metastatic breast cancer. The system employs a microfluidic chamber (a Parsortix cell separation cassette) to capture cells of a certain size and deformability from the population of cells present in blood. The cells retained in the cassette are harvested by the Parsortix PC1 system for use in subsequent downstream assays. The end user is responsible for the validation of any downstream assay. The standalone device, as indicated, does not identify, enumerate or characterize CTCs and cannot be used to make any diagnostic/prognostic claims for CTCs, including monitoring indications or as an aid in any disease management and/or treatment decisions." [Image placeholder: Parsortix® PC1 system] ------------------------------------------------------------------------ ## Parsortix Systems: Capturing and Harvesting Living Cancer Cells ### Platform Technology The Parsortix system harvests cancer cells from blood based on their larger size and lack of deformability. **Other cells can be captured:** - White blood cells associated with the tumor microenvironment\ - Megakaryocytes (frequency may relate to cancer)\ - Fetal cells from maternal blood \[Image placeholder: Parsortix system plan view and cross section showing critical gap and separation step and CTCT and WBC cluster.\] ------------------------------------------------------------------------ ## Workflow \[Image placeholder: Workflow overview image.\] ------------------------------------------------------------------------ ## Workflow Allowing for CTCs and ctDNA Analysis Dual analysis of biomarkers present in plasma and cellular component. ### Proof of Concept Work No CTC loss following plasma removal, prior to Parsortix enrichment. https://angleplc.com/wp-content/uploads/2023/05/PTX-Poster-A-ISLB-2022-Bidetect-002.pdf\ https://uscnorriscancer.usc.edu/liquid-biopsy-research-core/ Can we allow for dual detection of targets/variants which may have an impact on patient care? [Image placeholder: No CTC loss following plasma removal, prior to Parsortix enrichment.] For Research Use Only. Not for use in diagnostic procedures. ------------------------------------------------------------------------ ## Molecular Analysis of CTCs and ctDNA ctDNA and Parsortix-derived CTC-DNA can be analysed from single blood sample across multiple cancer types to detect mutations using **Illumina Cell-Free DNA prep with enrichment** combined with custom panel targeting Lung Cancer. **Illumina Cell-Free DNA prep with Enrichment and custom 79 gene panel** enables flexibility in the targeted genes. ### Workflow Steps 1. Blood collection in EDTA tubes\ 2. Blood separation on Parsortix® instrument\ 3. DNA extraction from Parsortix-enriched CTCs\ 4. Plasma separation by centrifugation\ 5. DNA extraction from plasma\ 6. Library preparation using Illumina Cell-Free DNA prep with enrichment\ 7. DNA sequencing by Illumina NextSeq™ 2000 For Research Use Only. Not for use in diagnostic procedures. ------------------------------------------------------------------------ ## DNA Dual Analysis ### Assay Performance (1/2) Study design for evaluation of performance of DNA dual analysis. Contrived samples were used to assess the analytical performance of the assay: - Two cell lines with known mutations were spiked into Parsortix-enriched samples.\ - DNA samples from CTC were extracted and processed following Illumina Cell-Free DNA prep with Enrichment procedure. ### Expected Mutations to be Detected in Spiked Samples | Cell line 1 | Cell line 2 | |------------------|------------------| | pTEN p.T167A | EGFR p.T790M | | BRAF p.V600E | EGFR p.L858R | | CDK4 p.R24C | PIK3CA p.G118D | | | TP53 p.R273H | \[Image placeholder: %VAF assay performance plots for BRAF, CDK4, PIK3CA, EGFR, PTEN, TP53.\] ------------------------------------------------------------------------ ## DNA Dual Analysis of Patients with Lung Cancer ### Patient Data 1/4 Two cohorts of Lung Cancer patients: 1. 1st cohort: 8 Lung Cancer patient samples (no treatment)\ 2. 2nd cohort: 19 Lung Cancer patient samples (may be under treatment) - Good quality samples\ - Overall, **93% of samples** with at least one oncogenic gene detected ------------------------------------------------------------------------ ### Patient Data 2/4 **1st cohort -- Lung cancer panel (n=8):** - CTC (average) = **49**\ - CTC (median) = **20**\ - 100% patients showed oncogenic mutations signal for one or more targets.\ - 32 genes (74 variants) specifically detected in this cohort **Example of detected mutations in specific analyte:** | CTC | cfDNA | |----------|-------| | CHEK1* | MSH6 | | FBXW7* | POLE | | MLH1 | BRAF | | ESR1 | APC | | ROS1 | ATM | \* predominant ------------------------------------------------------------------------ ### Patient Data 3/4 **2nd cohort -- Lung cancer panel (n=19):** - CTC (average) = **4**\ - CTC (median) = **3**\ - 90% of patients with at least one oncogenic mutation in CTC\ - 38 genes (214 variants) specifically detected in this cohort **Example of detected mutations in specific analyte:** | CTC | cfDNA | |---------|--------| | EpCAM | CDKN2A | | MYCN | NTRK | | FOXL2 | PIK3CA | | HRAS | APC | | CHEK1* | RB1* | \* predominant ------------------------------------------------------------------------ ### Patient Data 4/4 **Full cohort -- Lung cancer panel (n=27):** - 43 genes (247 variants) specifically detected [Image placeholder: GENES and MUTATIONS.] Analysing both analytes gives access to **twice as many mutations**, which could be investigated and/or used as "potential" biomarkers. ------------------------------------------------------------------------ ## Validation of Data Using Another Technology Validation of patient data using another cohort of patient samples (under treatment -- n=10). Use of a **pan-CANCER NGS panel**, with blocker displacement amplification. 20 genes (27 variants) specifically detected in this cohort [Image placeholder: GENES and MUTATIONS.] **Example of detected mutations in CTC fractions:** | CTC | cfDNA | |--------|-------| | PIK3CA | MYC | | MTOR | MET | | ESR1 | EGFR* | \* predominant Confirmation of the utility of using CTC as well as cfDNA to have access to a larger number of potential biomarkers. ------------------------------------------------------------------------ ## Conclusions ### Parsortix DNA Dual Analysis Strategy - Successful detection of potential actionable targets in cfDNA and CTC, from the same sample.\ - CTC samples provide a unique detection of actionable targets in most of the patient samples.\ - This study highlights the potential for multiomic analysis.\ - More data to come... Parsortix system is allowing for CTC enrichment and DNA analysis from plasma and CTC, from the same blood sample, with high analytical sensitivity and specificity. Dual analysis provides the possibility to access (new) biomarkers, which may help guide patient care. ------------------------------------------------------------------------ ## Contact Information **ANGLE plc**\ 10 Nugent Road\ Surrey Research Park\ Guildford GU2 7AF\ United Kingdom www.angleplc.com **Anne-Sophie PAILHES-JIMENEZ**\ a.pailhes-jimenez@angleplc.com