Session Title: Cell-free DNA, Circulating Tumor Cells, Exosomes, and Bladder Cancer
(UroToday.com) Day 2 of the 8th Annual Leo & Anne Albert Institute for Bladder Cancer Care and Research (AIBCCR) Symposium was chaired by Dr Bishoy Faltas (Weill Cornell Medicine) who started the session with his talk on serial cell tumor DNA (ctDNA) analysis for evolution-directed therapy in patients with urothelial carcinoma. ctDNA represents a subset of cell-free DNA (sfDNA), where tumor DNA from cancer cells are shed in the blood stream. Given that urothelial cancer has a high tumor mutational burden (TMB), this allows for identification of ctDNA in the blood which provides a ‘window’ to mutations present in the primary tumor. The advantage of ctDNA allows for serial liquid biopsies to map evolutionally trajectories in patients with advanced bladder cancer.
Dr Faltas presented published data from his group based on 183 serial ctNDA samples from 53 patients with advanced urothelial carcinoma analyzed with the GUARDANT360 targeted sequencing panel. 1 He reported that p53 and PIK3CA mutations were identified in 50% and 19% of patients respectively with FGFR3 mutations found in 10 patients. ctDNA improved clinical prognostic models (c-statistic from 0.65 to 0.84) with serial sampling further improving these models compared to baseline ctDNA alone. The key benefit of cfDNA is the ability to dynamically track clonal cell evolution. Analysis of this patient cohort suggested that cfDNA has prognostic value and can predict oncological outcomes. Patients who were ctDNA positive prior to treatment and subsequently became ctDNA negative post treatment had a significantly lower risk of death independent to confounding factors suggesting that molecular remission predicts durable response to treatment. ctDNA was also showed to be highly correlated with radiological progression of disease. He concluded this talk with his vision of evolution-directed therapy, where tumor biopsy and liquid biopsies will undergo molecular profiling to allow for treatment selection and serial analysis of liquid biopsies would allow the prediction of tumor genomic evolution with the opportunity to intervene with therapeutics. 2
Vision of evolution-directed therapy 2
The second talk was delivered by Dr Petros Grivas (Fred Hutchinson Cancer Center) which was on the potential opportunities for screening, surveillance, monitoring and therapeutic guidance utilizing ctDNA in urothelial carcinoma. He started his talk by highlighting the relationship between cancer proliferation burden and ctDNA which can eb highly variable. Highly sensitive assays are crucial for the detection of somatic alterations in cfDNA because ctDNA only represents 0.1% of sfDNA which can limit of detection ability of assays. He highlighted important considerations in the application of ctNDA: 1) the different roles of liquid biopsies such as cancer detection (diagnostic, screening) versus genomic characterization (prognostic, predictive, therapeutic response, resistance monitoring), 2) assay characteristics (depth and breadth of sequencing, bioinformatics, large versus small panel assay and tissue specific versus agnostic assays) and 3) the different endpoints used in studies.
Dr Grivas highlighted a report which used a patient specific tumor 16-plex PCR amplicon test (Singnatera) to interrogate longitudinal ctDNA samples in patients with advanced bladder cancer. 3 Patients who were ctDNA positive prior to neoadjuvant chemotherapy and radical cystectomy as well as following radical cystectomy had a much higher risk of disease recurrence compared with patients with ctDNA negative. Similar findings were reported in patients treated with neoadjuvant immunotherapy in the single arm ABACUS trial. 4 ctDNA also had an average positive lead time of 3 months ahead of radiological identification of clinical recurrence. 3 However, it remains uncertain if earlier identification of disease recurrence would change cancer specific survival.
Translational endpoints from clinical trials may provide important data. In the INVIGOR 010, a randomized phase III trial of adjuvant atezolizumab versus observation in operable urothelial cancer, patients who were ctDNA positive had worse survival outcome (HR: 6.3, 95% CI: 4.45-8.92), but this same patient cohort had an improved disease-free survival (HR: 0.58, 95% CI: 0.43-0.79) and overall survival (HR: 0.59, 95% CI: 0.41-0.86) in the atezolizumab versus observational arm. 5 Dr Grivas concluded that ctDNA requires further validation work and well design trails with proven clinical utility is crucial before clinical adoption.
Negative ctDNA patients have better DFS and OS. In patients with positive ctDNA, treatment with atezolizumab resulted in better DFS and OS compared to observation 5
The next talk was on the utilization of extracellular vesicles (EV) as a diagnostic and therapeutic nanotool in bladder cancer delivered by Dr Yi-Fin Lee (University of Rochester Medical Center). She introduced the role of EV as an important regulator of intracellular communication. These lipid bilayer nanoparticles (30-250nm) are naturally released from all animal cells and transport protein, nucleic acid, lipids and metabolites. Cancer cells release more EV which is implicated in all cancer stages. EV may function in immune response modulation, biomarkers and vehicle for drug delivery. In bladder cancer, Dr Lee’s group has shown that EV can drive tumorigenesis by inducing unfolding of protein response in the endoplastic reticulum of non-cancer cells. 6 . Utilizing protein disulfide isomerase (PDI) as an example, she described how unfolded protein accumulates within the cancer cell resulting in endoplastic reticulum stress and unfolding of protein response. This results in the oxidized PDI which is packed into EV and released from the cancer cell. These EV are then endocytosed in normal cells and hydrogen peroxide is then released leading to DNA damage and malignant transformation. As a biomarker, her group has also reported that EV can modulate response to BCG therapy in bladder cancer, allowing for early identification of metastatic disease and disease recurrence. 7 She concludes that EV is an area of active research due to its physiological and pathological role in cancer and prognostic biomarkers to therapy although challenges exist in purification and characterization of EV as well as further validation studies.The final talk was delivered by Dr Steve Soper (University of Kansas) on the opportunities and challenges on liquid biopsies for cancer. He started his talk by highlighting difference in substrate concentration in circulating tumor cells (CTC) (6-600 pg of gDNA/ml), cfDNA (2.8 molecules/ ml) and exosomes (30 molecules/ml). CTC are dynamic due to upregulation and downregulation in EMT genes. Key limitations are the ability to detect the ‘needle in the haystack’. His team developed the BioFluidica, LiquidScan, a fully automated platform with allows interrogation of CTC, cfDNA and EVs which represents an growing area of research. 8
Presented by: Dr Bishoy Faltas, MD, Director, Bladder Cancer Research, Englander Institute of Precision Medicine, Weill Cornell Medicine; Dr Petros Grivas, MD, PhD, Professor of Medical Oncology, Fred Hutchinson Cancer Center; Dr Yi-Fin Lee, PhD, Professor of Urology, Pathology and Laboratory Medicine, University of Rochester Medical Center; Dr Steve Soper, PhD, Professor Chemistry and Mechanical Engineering, University of Kansas.
Written by: Wei Shen Tan MBBCh, PhD, FRCS (Urol), Urologic Oncology Fellow, Department of Urology, MD Anderson Cancer Center, Twitter: @drtanws during the 2022 8th Annual Leo & Anne Albert Institute for Bladder Cancer Care and Research (AIBCCR) Friday Sept 16 – Saturday Sept 17, 2022
- Shohdy KS, Villamar DM, Cao Y, et al. Serial ctDNA analysis predicts clinical progression in patients with advanced urothelial carcinoma. Br J Cancer 2022;126(3):430-39.
- Vlachostergios PJ, Faltas BM. Treatment resistance in urothelial carcinoma: an evolutionary perspective. Nature Reviews Clinical Oncology 2018;15(8):495-509.
- Christensen E, Birkenkamp-Demtröder K, Sethi H, et al. Early Detection of Metastatic Relapse and Monitoring of Therapeutic Efficacy by Ultra-Deep Sequencing of Plasma Cell-Free DNA in Patients With Urothelial Bladder Carcinoma. J Clin Oncol 2019;37(18):1547-57.
- Szabados B, Kockx M, Assaf ZJ, et al. Final Results of Neoadjuvant Atezolizumab in Cisplatin-ineligible Patients with Muscle-invasive Urothelial Cancer of the Bladder. Eur Urol 2022;82(2):212-22.
- Powles T, Assaf ZJ, Davarpanah N, et al. ctDNA guiding adjuvant immunotherapy in urothelial carcinoma. Nature 2021;595(7867):432-37.
- Wu CH, Silvers CR, Messing EM, Lee YF. Bladder cancer extracellular vesicles drive tumorigenesis by inducing the unfolded protein response in endoplasmic reticulum of nonmalignant cells. J Biol Chem 2019;294(9):3207-18.
- Liu YR, Ortiz-Bonilla CJ, Lee YF. Extracellular Vesicles in Bladder Cancer: Biomarkers and Beyond. Int J Mol Sci 2018;19(9).
- Muller R, Purushotham S, Diaz P, et al. Detection of CTCs from whole blood of lung cancer patients using the automated Liquid Scan. Cancer Research 2019;79(13_Supplement):3984-84.