Dr. Alexandra Boitor, EACR Scientific Officer, writes about her experience and scientific highlights from the EACR Conference on Liquid Biopsies, which was held in Bergamo, Italy from 24-26 May 2022

The EACR conference on Liquid Biopsies took place in late May 2022 in Bergamo, Italy. More than 140 participants from 37 different countries met in sunny Italy to discuss the latest advances from basic discoveries to clinical application of such novel minimally invasive methods for cancer detection and surveillance. Data presented at this conference spammed a broad range of cancer types including, but not limited to, breast, pancreatic, colorectal, ovarian, lung cancers and melanoma. Whilst most data presented at the conference explored the use of blood and urine samples as liquid biopsies, alternative body fluids that could be used for liquid biopsies and their potential clinical relevance have been discussed during one of the discussion forums from this meeting. Other discussions during this meeting explored the benefits that incorporating liquid biopsies in clinical practice would bring to personalised medicine.

Listening to the incredible talks from the speakers invited to the EACR Liquid Biopsies conference, felt like being a toddler in a toy shop. And as a consequence, I found it incredibly difficult to pick one favourite talk to bring to your attention. A few highlights were:

  • Dan Landau’s talk showcasing the development of a new and improved machine-learning-based classifier, MRD-EDGE (minimal residual disease Enhanced ctDNA Genomewide signal Enrichment), that promises increased signal-to-noise enrichment of whole genome sequencing ctDNA single nucleotide variant and copy number variant in patient plasma samples. The potential this platform holds for clinical practice was highlighted in a proof-of-concept study by monitoring tumour burden in the blood from patients diagnosed with early-stage NSCLC and subjected to neoadjuvant immunotherapy and de novo mutation calling in ctDNA from the blood of patients diagnosed with advanced melanoma (Widman, Shah et al. 2022).
  • Efrat Shema’s talk introducing EPINUC (Epigenetics of Plasma Isolated Nucleosomes), a new single-molecule multi-parametric cancer detection assay that uses immunofluorescence and Total Internal Reflection (TIRF) microscopy. This new technology relies on the detection of a series of anti- histone marks associated with gene silencing, active transcription and enhancers; secreted and non-secreted tumor-specific plasma proteins and levels of methylated DNA in cfDNA from less than 1ml of patient blood. Using a combination of these three types of measurements this technology can differentiate between healthy donors, early or late-disease. Further on, Dr. Efrat went on showing that performing additional single-molecule DNA sequencing could identify the tumour tissue-of-origin and potentially even the presence of metastasis (Fedyuk, Erez et al. 2022).
  • Caroline Dive’s talk discussing amongst other subjects, the potential of using enumeration and genomic profiling of circulating tumour cells (CTCs) as a marker for predicting relapse in early-stage non-small-cell lung cancer (NSCLC) (Chemi, Rothwell et al. 2019) and presenting new molecular insight on how Notch signaling modulates transition from neuroendocrine to non-neuroendocrine cell fate in small-cell lung cancer (Shue, Drainas et al. 2022).

One of my favourite talks at the EACR conference on Liquid Biopsies this year was Prof.  Nicola Aceto’s talk. His research focuses on characterising circulating tumour cell (CTC) clusters shed in the blood of breast cancer patients. The presence of CTC clusters in the bloodstream has been proven to increase the metastatic burden, especially when CTCs cluster together with immune cells, most often neutrophils, and the presence of CTC-neutrophil clusters in blood from breast cancer patients correlates with unfavourable outcomes. Single-cell RNA sequencing revealed that CTCs from CTC-neutrophil clusters overexpress genes associated with cell cycle progression and DNA replication. The mutational load of the CTC-neutrophil cluster is not significantly different from CTCs alone or CTC clusters, but C>T mutations are more frequent in CTC-neutrophile clusters and specific genes that promote neutrophil recruitment, such as MERTK and TLE1, are exclusively mutated in CTC-neutrophil clusters (Szczerba, Castro-Giner et al. 2019).

Another highlight of this study was that CTC abundance is markedly different in blood samples taken from upstream or downstream of capillary beds, raising the possibility of CTCs being shed earlier in the tumour development process and sitting trapped in the capillaries at first (Szczerba, Castro-Giner et al. 2019). Later, Prof. Nicola Aceto together with his group and their collaborators developed a device that transforms liquid biopsies into 2D tissue slides whilst conserving morphological features of CTCs. The use of this device on blood samples from stages IA, IIA, IIB or IIIA cancer patients revealed that CTC clusters are indeed shed in the blood even during the early stages of cancer progression (Krol, Schwab et al. 2021).

Prof. Aceto and collaborators also developed two microfluidic devices. One of them better replicates physiological conditions found in capillaries, veins and arteries and allows for monitoring and analysis of cell morphology and behaviour under similar stress conditions as those induced by the circulatory system (Marrella, Fedi et al. 2021). The other microfluidic device could be suitable for the processing of liquid biopsies from patient blood as it has been designed to allow for high-efficiency selection of CTCs and CTC clusters from other types of cells found in the blood and for quantification of secreted proteins with unprecedented sensitivity within the same platform by making use of magnetic beads and immunofluorescent labelling (Armbrecht, Rutschmann et al. 2020).

Nicola’s research also addresses the apparent paradox of hypoxia in the context of metastasis. By using a fluorescent hypoxia sensor consisting of an activity reporter vector for HIF1a, Prof. Aceto shows that functional blood vessels, although spares, are found in hypoxic tumoral regions providing an intravasation route for hypoxic cell clusters to the bloodstream. Comparative experiments where breast cancer was induced in tumour-free mice by injecting normoxic/ hypoxic circulating tumour cell (CTC) clusters or normoxic/ hypoxic CTCs demonstrated a significantly higher metastatic potential for the hypoxic CTC clusters. Single-cell RNA sequencing revealed a 25-gene signature of upregulated genes in cells originating from hypoxic CTC clusters, gene signature that can be found in human cells as well and could predict the clinical outcome of breast cancer patients (when tested in clinical samples derived from primary tumours, low expression levels of this gene signature correlated with 100% 10-year patient survival rate). Proteomic profiling of hypoxic cells from primary tumours or from clustered CTCs revealed enrichment of proteins involved in cell adhesion, cadherin and protein binding. Efforts to decipher the mechanism that couples hypoxia to metastasis revealed that the formation of CTC clusters and metastasis is dependent on the expression levels of the angiogenesis regulator VEGFA, but independent of HIF1a expression. In the experiments performed in the Aceto group, suppression of VEGFA expression in mouse breast tumours induced either by shRNA knockdown or treatment with FDA-approved mono-clonal anti-VGFA antibody increased the number of CTC clusters and the metastatic burden. As expected, based on this discovery, pro-angiogenic treatment with EphrinB2 Fc chimaera protein decreased CTC cluster shedding and metastasis. Moreover, treatment with EphrinB2 in combination with paclitaxel (a chemotherapeutic agent) showed improved results when treating metastatic breast cancer in mice when compared with paclitaxel treatment alone (Donato, Kunz et al. 2020).

Last, but not least, Nicola also presented results from a study investigating metastatic spread in relation to circadian rhythms. In short, after analysing blood samples taken during the active and rest phase from breast cancer patients, the Aceto group noticed that CTC, CTC cluster and CTC-neutrophil cluster shedding was significantly increased during the rest phase (during sleep). CTCs recovered from the blood samples had distinct gene expression profiles in line with prototypical gene expression timing in eukaryotic cells. If the blood sample was taken during the rest phase, pathways that support cell division were upregulated, whilst in blood samples taken during the active phase, the upregulated pathways were involved in translation. Experiments performed in mice confirmed an increase in CTC intravasation directly associated with rest and revealed that CTCs released during the rest phase have an increased metastatic potential in comparison to CTCs shed during the active phase. The circadian rhythm of CTC shedding is under hormonal influence, with melatonin inducing an increase in the production of CTCs and metastatic burden; testosterone treatment decreasing CTC shedding during the rest phase and the associated metastatic burden and insulin stimulation inverting the dynamics of CTC release (Diamantopoulou, Castro-Giner et al. 2022).

Armbrecht, L., O. Rutschmann, B. M. Szczerba, J. Nikoloff, N. Aceto and P. S. Dittrich (2020). “Quantification of Protein Secretion from Circulating Tumor Cells in Microfluidic Chambers.” Adv Sci (Weinh) 7(11): 1903237.

Chemi, F., D. G. Rothwell, N. McGranahan, S. Gulati, C. Abbosh, S. P. Pearce, C. Zhou, G. A. Wilson, M. Jamal-Hanjani, N. Birkbak, J. Pierce, C. S. Kim, S. Ferdous, D. J. Burt, D. Slane-Tan, F. Gomes, D. Moore, R. Shah, M. Al Bakir, C. Hiley, S. Veeriah, Y. Summers, P. Crosbie, S. Ward, B. Mesquita, M. Dynowski, D. Biswas, J. Tugwood, F. Blackhall, C. Miller, A. Hackshaw, G. Brady, C. Swanton and C. Dive (2019). “Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse.” Nat Med 25(10): 1534-1539.

Diamantopoulou, Z., F. Castro-Giner, F. D. Schwab, C. Foerster, M. Saini, S. Budinjas, K. Strittmatter, I. Krol, B. Seifert, V. Heinzelmann-Schwarz, C. Kurzeder, C. Rochlitz, M. Vetter, W. P. Weber and N. Aceto (2022). “The metastatic spread of breast cancer accelerates during sleep.” Nature 607(7917): 156-162.

Donato, C., L. Kunz, F. Castro-Giner, A. Paasinen-Sohns, K. Strittmatter, B. M. Szczerba, R. Scherrer, N. Di Maggio, W. Heusermann, O. Biehlmaier, C. Beisel, M. Vetter, C. Rochlitz, W. P. Weber, A. Banfi, T. Schroeder and N. Aceto (2020). “Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells.” Cell Rep 32(10): 108105.

Fedyuk, V., N. Erez, N. Furth, O. Beresh, E. Andreishcheva, A. Shinde, D. Jones, B. B. Zakai, Y. Mavor, T. Peretz, A. Hubert, J. E. Cohen, A. Salah, M. Temper, A. Grinshpun, M. Maoz, A. Zick, G. Ron and E. Shema (2022). “Multiplexed Single-Molecule Epigenetic Analysis of Plasma-Isolated Nucleosomes for Cancer Diagnostics.” Nat Biotechnol: 10.1038/s41587-022-01447-3.

Krol, I., F. D. Schwab, R. Carbone, M. Ritter, S. Picocci, M. L. De Marni, G. Stepien, G. M. Franchi, A. Zanardi, M. D. Rissoglio, A. Covelli, G. Guidi, D. Scarinci, F. Castro-Giner, L. Mazzarella, C. Doglioni, F. Borghi, P. Milani, C. Kurzeder, W. P. Weber and N. Aceto (2021). “Detection of clustered circulating tumour cells in early breast cancer.” Br J Cancer 125(1): 23-27.

Marrella, A., A. Fedi, G. Varani, I. Vaccari, M. Fato, G. Firpo, P. Guida, N. Aceto and S. Scaglione (2021). “High blood flow shear stress values are associated with circulating tumor cells cluster disaggregation in a multi-channel microfluidic device.” PLoS One 16(1): e0245536.

Shue, Y. T., A. P. Drainas, N. Y. Li, S. M. Pearsall, D. Morgan, N. Sinnott-Armstrong, S. Q. Hipkins, G. L. Coles, J. S. Lim, A. E. Oro, K. L. Simpson, C. Dive and J. Sage (2022). “A conserved YAP/Notch/REST network controls the neuroendocrine cell fate in the lungs.” Nature Communications 13(1): 2690.

Szczerba, B. M., F. Castro-Giner, M. Vetter, I. Krol, S. Gkountela, J. Landin, M. C. Scheidmann, C. Donato, R. Scherrer, J. Singer, C. Beisel, C. Kurzeder, V. Heinzelmann-Schwarz, C. Rochlitz, W. P. Weber, N. Beerenwinkel and N. Aceto (2019). “Neutrophils escort circulating tumour cells to enable cell cycle progression.” Nature 566(7745): 553-557.

Widman, A. J., M. Shah, N. Øgaard, C. C. Khamnei, A. Frydendahl, A. Deshpande, A. Arora, M. Zhang, D. Halmos, J. Bass, T. Langanay, S. Rajagopalan, Z. Steinsnyder, W. Liao, M. H. Rasmussen, S. Ø. Jensen, J. Nors, C. Therkildsen, J. Sotelo, R. Brand, R. H. Shah, A. P. Cheng, C. Maher, L. Spain, K. Krause, D. T. Frederick, M. S. Malbari, M. Marton, D. Manaa, L. Winterkorn, M. K. Callahan, G. Boland, J. D. Wolchok, A. Saxena, S. Turajlic, M. Imielinski, M. F. Berger, N. K. Altorki, M. A. Postow, N. Robine, C. L. Andersen and D. A. Landau (2022). bioRxiv: 2022.2001.2017.476508.