ExosomeDx – project supported by the German ZIM-BMWi*

(*Zentrales Innovationsprogramm Mittelstand des Bundesministeriums für Wirtschaft und Energie)

The project is conducted by Therawis Diagnostic GmbH (applicant) in collaboration with the Institute of Molecular Immunology/Experimental Oncology, MRI TU Munich, Germany.

Therawis Diagnostics has developed a novel, proprietary method for purification of exosomes.

Background:

With the introduction of the estrogen receptor antagonist tamoxifen for the treatment of breast cancer, a variety of targeted therapies have been developed and are now an important integral part of cancer treatment. In order to be able to use a specific targeted therapeutic agent, the determination of the expression of the target protein or mutation analysis are required.  Examples include the expression of the HER2 neu receptor for the treatment of breast cancer with trastuzumab or the genetic mutation status as a prerequisite for the treatment of non-small cell lung cancer with tyrosine kinase inhibitors.

In order to analyze the tumor histologically and molecular pathologically, a tissue sample is required. The tumor is characterized and the best targeted therapy is selected. However, these tissue biopsies have the disadvantage, especially in the metastatic stage of the disease, that these procedures are considerable and uncomfortable interventions for the patient and sometimes risky for example biopsies of lung and brain tumors. Furthermore, not always sufficient tumor material can be obtained by biopsy. For example, as many as 31% of patients with advanced or metastatic lung cancer (NSCLC) do not have accessible tissue (Kim et. Al., 2008, Ilié and Hofman, 2016). Even in cases where tissue is obtained by biopsy, formalin fixation can induce conversion of cytosine to thymine, which can lead to a false positive result of the genetic analysis (Quach et al. 2004, Wong et al., 2014).

Tumor cells release components such as DNA, RNA and exosomes into the blood, so that a primary tumor could in principle also be diagnosed and characterized from a blood sample in the form of a so-called “liquid biopsy”. These vesicles contain nucleic acids and proteins and serve as transport vehicles. Exosomes might reflect the phenotype of the primary tumor by analyzing the exosome content (mRNA, DNA, proteins). This is of particular interest if tumor tissue is not accessible. In addition to its non-invasiveness, the “Liquid Biopsy” method could have the considerable advantage that it can also be used for the early detection of cancer, for monitoring of therapy response and for the early diagnosis of recurrent tumors. Through “Liquid Biopsy”, the molecular characteristics of the recurrent tumor, which may have changed compared to the tumor at diagnosis, could also be analyzed. In the case of metastatic breast cancer, the regular collection of an up-to-date tumor biology is essential in order to determine further treatment regimes. There is significant evidence that the tumor characteristics as well as the mutation profile can change during the course of the disease.

The market demand not only for breast cancer would be considerable: In a recent study (Garcia-Murillas et al. 2015), a recurrence of breast cancer could be predicted on average eight months earlier by detecting tumor DNA in the blood than with conventional imaging methods.

Quantification of estrogen and progesterone receptor expression and HER2 expression/amplification is an important prerequisite for subtyping and treatment of breast cancer. However, the status of expression may change during the course of the disease from diagnosis to metastatic disease. In a meta-analysis (including 39 analyzed studies) it was shown that a conversion from positive to negative status of the estrogen receptor occurs in 22.5% of cases, progesterone receptor in 49.4% and HER2 status in 21.3% of cases; change from negative to positive status of the estrogen receptor in 21.5%, progesterone receptor in 15.9% and HER2 status in 9.5% of cases (Schrijver et al. 2018).

Analysis by Liquid Biopsy would be, therefore, of great clinical benefit. Liquid biopsy using exosomes has several advantages compared to circulating free DNA highlighted in the table.

* Semi-quantification of methylation maybe with limitation because healthy cfDNA can cause a significant background and impact the results.

References

  1. Kim ES, Hirsh V, Mok T, Socinski MA, Gervais R, Wu YL, Li LY, Watkins CL, Sellers MV, Lowe ES, Sun Y, Liao ML, Osterlind K, Reck M, Armour AA, Shepherd FA, Lippman SM, Douillard JY: Gefitinib versus docetaxel in previously treated non-small-cell lung cancer (INTEREST): a randomised phase III trial. Lancet 2008;372:1809-1818.

  2. Ilie M, Hofman P: Pros: Can tissue biopsy be replaced by liquid biopsy? Transl Lung Cancer Res 2016;5:420-423.

  3. Quach N, Goodman MF, Shibata D: In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR. BMC Clin Pathol 2004;4:1.

  4. Wong SQ, Li J, Tan AY, Vedururu R, Pang JM, Do H, Ellul J, Doig K, Bell A, MacArthur GA, Fox SB, Thomas DM, Fellowes A, Parisot JP, Dobrovic A: Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing. BMC Med Genomics 2014;7:23.

  5. Garcia-Murillas I, Schiavon G, Weigelt B, Ng C, Hrebien S, Cutts RJ, Cheang M, Osin P, Nerurkar A, Kozarewa I, Garrido JA, Dowsett M, Reis-Filho JS, Smith IE, Turner NC: Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci Transl Med 2015;7:302ra133.

  6. Schrijver W, Suijkerbuijk KPM, van Gils CH, van der Wall E, Moelans CB, van Diest PJ: Receptor Conversion in Distant Breast Cancer Metastases: A Systematic Review and Meta-analysis. J Natl Cancer Inst 2018;110:568-580.