Plasma sequencing

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The plasma sequencing (also plasma-Seq ) indicates methods for sequencing of the genome from the plasma of a blood sample.

principle

The Plasma-Seq is used to determine circulating free DNA , mostly in the context of diagnosing tumors . So-called next-generation methods of DNA sequencing are used. This is a non-invasive method for assessing the clonal evolution of the tumor genome. Plasma sequencing can reduce incorrect therapy decisions and optimize the treatment of tumors so that maximum effectiveness can be achieved with fewer side effects. Plasma sequencing is used in personalized medicine , as it enables the tumor genome to be monitored by taking regular blood samples.

Problem and application

For the treatment of tumors, such as colorectal carcinomas and prostate carcinomas , it is important to find out in advance that genome mutations of the tumor are found, as these can have a strong influence on the therapies . In the case of colorectal abnormalities with a KRAS mutation, for example, therapy with monoclonal antibodies that target the epidermal growth factor (EGFR) receptor does not work . The therapies are based on the initial diagnosis . It turns out, however, that the tumors can develop resistance after a few months of treatment for reasons that are still inexplicable . Since a tumor biopsy a very invasive procedure, there is the possibility of cell-free DNA (cfDNA, cell-free DNA) to analyze the plasma of cancer patients / inside. Cancer cells can release their tumor DNA into the bloodstream , this DNA is called ctDNA (circulating tumor DNA). It is part of the cfDNA. With the help of sequencing this DNA, genetic mutations can be identified.

Diagnosis

On the one hand, the method of plasma sequencing is used to reveal therapy-relevant mutations (this is only carried out in the plasma if no relevant tumor tissue is available), on the other hand, the ctDNA is useful to detect tumor-specific mutations in the blood as a biomarker for follow-up observations as well as tumor recurrence early detection. Plasma sequencing thus enables the identification of new mutations (e.g. KRAS , MET or ERBB2 ) in a tumor or can be of assistance in establishing a diagnosis. This enables personal and targeted therapy for the affected patients. Due to the instability of the tumor genome, the status of biomarkers that are important for stopping therapy can change. By reacting early to new mutations in the tumor, therapies can be changed and thus the progression of the disease can be prevented or delayed.

A disadvantage of plasma sequencing is that no specific statement can be made about the development of the tumor. A differentiation of the place of origin (the primary tumor tissue or the metastases ) has not yet been possible. This is due to the fact that the ctDNA can be derived from one or the other tumor.

execution

Plasma sequencing is next-generation sequencing . At the beginning, the DNA in the plasma sample is purified and isolated. The samples are then prepared for sequencing with bridge synthesis. The template DNA is fragmented and adapter sequences are attached to the DNA fragments so that they are bound to a glass slide . The sequencing takes place on this slide and, in a PCR- like step, clusters of identical molecules are formed in cycles. Exactly one fluorescence-labeled nucleotide is incorporated per cycle, complementary to the template DNA. The light signal from the fluorophore can be detected and processed and the next nucleotide is incorporated in the next cycle.

For an analysis you need a device for a sequencing, especially for a sequencing by synthesis. There is also the option of paired-end sequencing, which can increase the accuracy of the analysis. In this case the DNA fragments are sequenced from both sides.

With an instrument for whole genome sequencing with high throughput and a sequencing depth of 0.1-0.2x from a plasma sample, a genome-wide tumor profile including copy number variations can be created within two days at low cost.

Method comparison

There is also an option to check the tumor genome using circulating tumor cells (CTC). Circulating tumor cells are released into the bloodstream by primary and metastatic tumors and can then be isolated and characterized. The genotypes of the CTCs enable assessments of drug sensitivity and resistance and can be helpful in making therapy decisions. In addition, CTCs can provide information about the prognosis .

CTC analysis ctDNA analysis
Furnishing special devices for identification and of CTCs simple blood test
insulation complex isolation of CTCs no special isolation, but a standard DNA purification
Information on heterogeneity and clonality Yes, if enough cells have been analyzed No, the results reflect the average of the ctDNA from all tumor cells

literature

  • Evelyn Kidess, Stefanie S. Jeffrey: Circulating tumor cells versus tumor-derived cell-free DNA: rivals or partners in cancer care in the era of single-cell analysis? In: Genome Medicine . tape 5 , no. 8 , August 13, 2013, p. 70 , doi : 10.1186 / gm474 , PMID 23953663 .
  • Martina Auer, Ellen Heitzer, Peter Ulz, Jochen B Geigl, Michael R Speicher: Single circulating tumor cell sequencing for monitoring . In: Oncotarget . tape 4 , no. 6 , May 10, 2013, p. 812-813 , PMID 23868872 .
  • M. Zapatka, P. Lights: Modern techniques of genetic diagnosis . In: The gynecologist . tape 45 , no. 1 , January 12, 2012, p. 11-16 , doi : 10.1007 / s00129-011-2856-x .

Web links

References

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