Liquid biopsy

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Liquid biopsy
DNA fragments from tumor cells enter the bloodstream
Extended clinical diagnostics using liquid biopsy compared to conventional biopsy

Under a liquid biopsy ( LB , German  liquid biopsy ) one understands the sampling and analysis of non-solid, biological tissue , mainly blood . One advantage is that it is largely non-invasive . Like the traditional biopsy , this type of examination is mainly used to diagnose and monitor diseases such as cancer . The method was made possible by advances in DNA sequencing of the human genome , which can detect genetic mutations in cancer cells.

Tumor diagnostics

After a blood sample , circulating tumor cells or tumor genetic material are detected in the blood.

Circulating tumor cells ( english Circulating tumor cells , CTCs), individual cancer cells from the tumor into the bloodstream reach. They can be distinguished from normal blood cells by certain properties . There are now several methods with which CTCs can be counted and in some cases also enriched. Once the cells have been isolated, their genome or protein composition can be examined.

In circulating tumor DNA ( English Circulating free DNA , cfDNA) is short gene sequences that as degradation remains of tumor cells in the blood were released. However, DNA that comes from healthy cells is also floating in the blood . The tumor DNA can be detected by completely removing the DNA from healthy cells from the blood sample.

The tumor marker cell free microRNA -375 (cf miR-375) is excessively released into the blood by Merkel cell tumor cells . The response to therapy can be checked with a blood test and a possible relapse of the disease can be detected early on.

With the liquid biopsy of the urine it could be shown that immune cells found in urine were more representative for the diagnosis of bladder cancer than immune cells from the blood, which suggests that the procedure using urine instead of blood can contribute to to closely examine the response to immunotherapy . T lymphocytes are not normally found in the urine in healthy individuals. It is crucial that the T cells match those in the tumor environment of the bladder carcinoma, regardless of the cancer stage and the course of treatment. Immunotherapy shows promise for cancers that are difficult to treat, but only 30-40 percent of patients respond to immunotherapy. These can thus be identified.

In malignant melanoma , immunotherapy with anti- CTLA-4 and anti- PD-1 antibodies, as well as therapies that target certain mutations, such as BRAF , NRAS and c ‐ KIT, can be carried out. In the case of melanoma patients, the results of the liquid biopsy can be helpful as novel predictive biomarkers in therapeutic decisions, especially in connection with mutation-based, targeted therapies.

In the case of adenocarcinoma of the pancreas ( English pancreatic ductal adenocarcinoma , PDAC), the examination of DNA-based biomarkers in exosomal salivary DNA (exoDNA) using liquid biopsy enables early detection, tumor stratification , therapy stratification and monitoring in patients with PDAC.

At the beginning of 2019, researchers from Heidelberg University Hospital announced that the first marketable blood tests for breast cancer had been developed. The new method using liquid biopsy recognizes cancer using biomarkers and can thus expand the diagnostic spectrum of imaging diagnostic methods such as mammography , ultrasound or magnetic resonance tomography (MRT). Up to 15 different biomarkers ( microRNA and methylation markers ) could be identified in the blood of women suffering from breast cancer , with the help of which even small tumors (<5 mm) could be detected. The biomarkers could also provide information on whether a treatment is responding or resistance to therapy occurs. In a joint statement by the German Cancer Society (DKG), German Society for Gynecology and Obstetrics (DGGG), DGS, AGO , AGO Mamma, AGO TraFo, German Society for Hematology and Medical Oncology (DGHO), research on liquid biopsy Technology welcomed, but at the same time warned against premature use of the test. A scientific publication is not yet available. The clinical consequences of the test in connection with the results of other diagnostic procedures, such as mammography and sonography , would first have to be checked in studies. This has now been confirmed by the University of Heidelberg. Although the diagnostic method could be used for test purposes in the laboratory at the end of the year, the test that can detect messenger substances from tumor cells in blood samples is not yet on the market. Especially with women over 50, the success rate of the tests would be just 60 percent. The results were better in younger patient groups, but women over 50 in particular have the greatest risk of breast cancer.

In the meantime, the Heidelberg public prosecutor has started preliminary investigations. Uniklinikum and spin-off Heiscreen described the test as a “milestone” in early breast cancer diagnosis, despite the lack of data. Financial entanglements by doctors, but also by celebrities, are said to have favored the PR campaign.

Further diagnostic options

In the myocardial infarction diagnostics , after circulating in the blood endothelial cells ( English Circulating endothelial cells CECs,) sought.

In prenatal diagnosis is from maternal blood or amniotic cell-free fetal DNA ( English Cell-free fetal DNA extracted cffDNA). Prenatal parentage reports (paternity tests) are based on this method, in which the genetic markers single nucleotide polymorphism ( English Single Nucleotide Polymorphism , SNP) of the adopted father and the embryo are compared. According to the Genetic Diagnostics Act , these may only be carried out in Germany if there is a criminal offense according to § 176 to § 178 StGB (sexual abuse of children).

Isolation of protoporphyrin IX from blood samples can be used as a diagnostic tool for atherosclerosis .

When examining the central nervous system , cerebrospinal fluid may be taken instead of blood .

Blood collection

Special tubes must be used to collect blood (around 10 ml). Most standard tubes for blood collection are made of positively charged plastics, so that the negatively charged cfDNA is adsorbed and can no longer be detected during the analysis. In addition, the blood cells start to break down after collection, releasing genomic DNA and distorting any analysis of the actual cfDNA. Both processes influence the sensitivity and the result of the analysis of the liquid biopsy . Therefore, proven blood collection tubes from prenatal diagnostics or those specially developed for tumor genetic examinations should be used. They consist of negatively charged materials and contain stabilizers that prevent blood cells from breaking down for about five days. In addition, cannulas of sufficient size (≥ 21 gauge ) should be used for blood collection so that nucleated blood cells remain intact, for example collection systems that are used for blood cultures and are designed to keep blood cells intact and vital. Due to the low concentration of <0.001% in the plasma , the cfDNA must be enriched using special amplification methods so that certain mutations can then be searched for.

analysis

The Fraunhofer Institute for Microtechnology and Microsystems (IMM) has developed a fully automated system for liquid biopsy to isolate individual tumor cells (CTCs) from patient's blood. First, the tumor cells are isolated from the sample by means of immunomagnetic separation (IMS) and transferred to a smaller sample volume. Then the extract is transferred to a microfluidic cartridge to remove the unspecific cell background, in which the cancer cells are recognized by flow cytometry . After detecting a tumor cell in the microchannel, the individual target cells are dispensed directly into the cavities of a microtiter plate by means of a pressure surge .

Clinical evaluation

The German Society for Pathology (DGP) regards the use of liquid biopsy with reservations. There are currently too great uncertainties with the procedure to be able to make reliable statements about the early detection, diagnosis, therapy, course or prognosis of cancer. Cell-free, circulating tumor DNA cannot be detected in all, but only in around 70 percent of metastatic tumor diseases. False negative results can therefore occur in individual cases. There are big differences between different tumor types and depending on the tumor stage. For brain tumors , cfDNA detection is unsuitable because of the blood-brain barrier , since only extremely few DNA fragments could be found in the blood. There is still a lack of standardization of existing cfDNA isolation and analysis technologies and quality management of sample processing.

The Liquid Biopsy can complement the diagnosis of tumors under certain conditions, for example when a primary tumor is found, the non-small cell lung cancer ( English non small cell lung cancer , NSCLC). Here the liquid biopsy takes place to detect an EGFR - T790M mutation. The mutation of the epidermal growth factor receptor ( English Epidermal Growth Factor Receptor , EGFR) replaces a threonine (T) with a methionine (M) at position 790 of exon 20. The activating mutations of the EGFR also include the mutation of exon 21 and exon 19. The activating mutations of the EGFR can permanently send out growth signals and thus contribute to the uncontrolled growth of the tumor. Osimertinib was approved by both the FDA and the European Commission for the treatment of NSCLC in 2017 .

further reading

Individual evidence

  1. ↑ Liquid Biopsy: Liquid Biopsy , Cancer Information Service , January 15, 2018. Accessed February 22, 2019.
  2. ↑ Merkel cell carcinoma: New biomarker discovered , University of Duisburg-Essen, Health Care in Europe, January 28, 2019.
  3. ↑ Alessia Finotti, Matteo Allegretti et al. a .: Liquid biopsy and PCR-free ultrasensitive detection systems in oncology (review). In: International Journal of Oncology. 2018, doi : 10.3892 / ijo.2018.4516 .
  4. ↑ Urine liquid biopsies could help monitor bladder cancer treatment , September 26, 2018. Retrieved February 23, 2019.
  5. ↑ A. Di Meo, J. Bartlett et al. a .: Liquid biopsy: a step forward towards precision medicine in urologic malignancies. In: Molecular cancer. Volume 16, number 1, 04 2017, p. 80, doi : 10.1186 / s12943-017-0644-5 , PMID 28410618 , PMC 5391592 (free full text) (review).
  6. ↑ Maria Rita Gaiser, Nikolas von Bubnoff, Christoffer Gebhardt, Jochen Sven Utikal: Liquid Biopsy for the monitoring of melanoma patients. In: JDDG: Journal of the German Dermatological Society. 16, 2018, p. 405, doi : 10.1111 / ddg.13461_g .
  7. ↑ importance exosomal DNA from saliva in ductal pancreatic cancer , German Research Foundation. Retrieved February 23, 2019.
  8. ↑ Dipesh Kumar Yadav, Xueli Bai u. a .: Liquid biopsy in pancreatic cancer: the beginning of a new era. In: Oncotarget. 9, 2018, doi : 10.18632 / oncotarget.24809 .
  9. ↑ Press release Heidelberg University Hospital , February 21, 2019.
  10. ↑ J. Cheng, K. Cuk, J. Heil, M. Golatta, S. Schott, C. Sohn, A. Schneeweiss, B. Burwinkel, H. Surowy: Cell-free circulating DNA integrity is an independent predictor of impending breast cancer recurrence. In: Oncotarget . Volume 8, number 33, August 2017, pp. 54537-54547, doi : 10.18632 / oncotarget.17384 , PMID 28903362 , PMC 5589601 (free full text).
  11. ↑ Jens-Uwe Blohmer, Annette Hasenburg, Wolfgang Janni: Joint statement on reporting on new blood test for early detection of breast cancer. Arbeitsgemeinschaft Gynäkologische Onkologie eV, February 27, 2019, accessed on March 8, 2019 .
  12. ↑ Cancer test advertised as "milestone" - Uniklinik apologizes , Spiegel online, March 22, 2019. Accessed March 24, 2019.
  13. ↑ University reports after blood test PR , Süddeutsche Zeitung, April 8, 2019. Retrieved April 14, 2019.
  14. ↑ K. Bethel, MS Luttgen u. a .: Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction. In: Physical biology. Volume 11, number 1, February 2014, p. 016002, doi : 10.1088 / 1478-3975 / 11/1/016002 , PMID 24406475 , PMC 4143170 (free full text).
  15. ↑ M. Allyse, MA Minear, E. Berson, S. Sridhar, M. Red, A. Hung, S. Chandrasekharan: Non-invasive prenatal testing: a review of international implementation and challenges. In: International journal of women's health. Volume 7, 2015, ISSN  1179-1411 , pp. 113–126, doi : 10.2147 / IJWH.S67124 , PMID 25653560 , PMC 4303457 (free full text).
  16. ↑ M. Nascimento da Silva, LB Sicchieri and a .: Liquid biopsy of atherosclerosis using protoporphyrin IX as a biomarker. In: The Analyst. Volume 139, Number 6, March 2014, pp. 1383-1388, doi : 10.1039 / c3an01945d , PMID 24432352 .
  17. ↑ OT Pyykkö, M. Lumela u. a .: Cerebrospinal fluid biomarker and brain biopsy findings in idiopathic normal pressure hydrocephalus. In: PLOS ONE . Volume 9, number 3, 2014, p. E91974, doi : 10.1371 / journal.pone.0091974 , PMID 24638077 , PMC 3956805 (free full text).
  18. ^ SE Norton, JM Lechner u. a .: A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR. In: Clinical biochemistry. Volume 46, Number 15, October 2013, pp. 1561-1565, doi : 10.1016 / j.clinbiochem.2013.06.002 , PMID 23769817 .
  19. ↑ Information on taking blood for T790M testing on liquid biopsies , Institute for Pathology Charité - Universitätsmedizin Berlin. Retrieved February 24, 2019.
  20. ↑ Andreas Jung, Thomas Kirchner: Liquid Biopsy in Tumor Genetic Diagnosis. In: Deutsches Aerzteblatt Online. 2018, doi : 10.3238 / arztebl.2018.0169 .
  21. ↑ Liquid Biopsy , Fraunhofer Institute for Microtechnology and Microsystems, 2019. Accessed March 5, 2019.
  22. ↑ E. Dahl, A. Jung, u. a .: Opportunities and risks of the blood-based molecular pathological analysis of circulating tumor cells (CTC) and cell-free DNA (cfDNA) in personalized cancer therapy. In: The Pathologist. 36, 2015, p. 92, doi : 10.1007 / s00292-014-2069-x .
  23. ↑ D. Ayeni, K. Politi, SB Goldberg: Emerging Agents and New Mutations in EGFR-Mutant Lung Cancer. In: Clinical Cancer Research . Volume 21, number 17, September 2015, pp. 3818-3820, doi : 10.1158 / 1078-0432.CCR-15-1211 , PMID 26169963 , PMC 4720502 (free full text).
  24. ↑ European Tagrisso information , European Medicines Commission, accessed on February 24, 2019.