NGS-compatible whole genome amplification for characterizing CTCs

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Circulating tumor cells (CTCs) are the cancerous cells shed in the bloodstream by primary or secondary tumors. CTC counts have been correlated with patient survival rates in different forms of cancer such as lung, prostate and breast cancer. Circulating tumor cell cancer research is important for a number of reasons. In cases of metastatic and localized tumor diseases, CTC studies can help: 1) facilitate early diagnosis and detection, 2) noninvasively profile cancer for molecular characterization, and 3) monitor and predict patient response to treatment. Additionally, research on the genotype and phenotype of disseminating cancer cells can help us better understand the tumor’s biology and metastasis – as well as how the precursors lead to tumor development. Such information can allow for a more successful treatment and management of cancer patients (3).

Tumor profiling at a molecular level may also aid in detecting any therapy resistance that may have developed against a drug over the course of time, as in the case of ESR1 mutations in response to estrogen receptor-targeted therapy (2). Usually tumor tissue is analyzed at a molecular level during diagnosis and throughout the disease progression. This frequency of molecular tissue analysis means that the patient has to undergo multiple tissue biopsies over the course of time, which is a major hurdle to real-time monitoring of a patient’s disease. In comparison to traditional tissue biopsies, which involve a painful procedure – liquid biopsy is a noninvasive procedure, and an alternative solution, for isolating and studying mutations in circulating tumor cells.

The detection and characterization of CTCs from the bloodstream have been a major research focus in cancer. One of the first challenges for sequencing CTCs is its low number, but several techniques are available for isolating and characterizing CTCs.

The second challenge is the amount of DNA. A single cell contains only 6–7 pg of DNA, and most NGS protocols require ng or ugs of DNA.

To overcome this challenge, Whole Genome Amplification (WGA) has been successfully used to amplify limited amounts of starting genomic material for NGS. Yee et al. came up with a novel approach for the isolation and NGS of rare tumor cells by combining WGA and multiplex targeted resequencing (1). They used the REPLI-g Single Cell Kit along with multiplex targeted resequencing for real-time monitoring of tumor genetics over the course of a patient`s therapy. The authors used multiplex PCR based methods to assess the DNA and sequencing quality. This step let the researchers predict the sequencing success of REPLI-g and other WGA approaches – thus avoiding the cost of NGS for low quality samples. Mutations and copy number variations were detected successfully and later confirmed with Sanger sequencing.

The preservatives used in blood collection have been found to affect nucleic acid quality and to be incompatible with the WGA protocol. EDTA tubes used for blood collection need to be processed within 24 hours of blood collection to minimize cell degradation – a process that limits the opportunities for batching clinical patient samples. Another commonly used blood preservative – paraformaldehyde – often leads to higher sequencing errors (4).

For blood storage preservatives, Yee et al. compared the DNA BCTR tube with the commonly used EDTA tube and found the DNA quality to be the same. And the use of the DNA BCTR tube is also compatible with the REPLI-g WGA approach and the preparation of high-quality libraries.

Many scientists, such as myself, may particularly enjoy Yee’s study because the authors have presented a proof of concept for a clinically relevant NGS approach that is applicable to rare CTCs. The researchers impressively combined NGS with WGA and sample preservation to create a streamlined workflow for clinically relevant variant detection. This technique may be further applied to the rare CTCs present in the blood of cancer patients for real-time tumor monitoring using liquid biopsies – eliminating the need for painful tissue biopsies.

Find out more about the solutions from QIAGEN, including QIAGEN’s REPLI-g Single Cell Kit.

 

What approach do you take for studying mutations in small pools of cells? What is your experience with CTCs? Let us know in the comments section below!

 

Disclaimers

The REPLI-g Single Cell Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbooks or user manuals.

References:

  1. 1. Yee, S.S. et al. (2016) A novel approach for next-generation sequencing of circulating tumor cells. Mol Genet Genomic Med. (Link)
  2. 2. Robinson, D.R. et al. (2013) Activating ESR1 mutations in hormone-resistant metastatic breast cancer. Nat Genet. 45, 1446-51. (Link)
  3. 3. Pantel, K., Alix-Panabières, C. (2007) The clinical significance of circulating tumor cells. Nat Clin Pract Oncol. 4, 62-63. (Link)
  4. 4. Swennenhuis, J.F., Reumers, J., Thys, K., Aerssens, J., Terstappen, L.W. (2013) Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS. Genome Med. 5, 106. (Link)
Vishwadeepak Tripathi

Vishwadeepak Tripathi, PhD is a Global Market Manager at QIAGEN. He received his PhD in biochemistry at the Faculty of Medicine from Ruhr-University Bochum, Germany. Dr. Tripathi studied the role of chaperones and co-chaperones in protein folding and quality control and authored a number of scientific publications. He was also at RIKEN Institute in Japan where he studied the pathogenesis of Huntington's disease in cellular and mice models. He is currently interested in biomarker research, NGS and neurodegeneration.

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