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Liquid Biopsy Technique Detects Circulating Tumor Cells in Patients with Non-Small Cell Lung Cancer

By LabMedica International staff writers
Posted on 16 Mar 2021
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Image: Cartoon representation of the molecular structure of the hexokinase-2 (HK2) protein (Photo courtesy of Wikimedia Commons)
Image: Cartoon representation of the molecular structure of the hexokinase-2 (HK2) protein (Photo courtesy of Wikimedia Commons)
A liquid biopsy technique based on hexokinase-2, a key enzyme in glucose metabolism, is able to detect circulating tumor cells (CTCs) in patients with non-small cell lung cancer (NSCLC).

Unlike other epithelial cancer types, CTCs are less frequently detected in the peripheral blood of NSCLC patients using epithelial marker–based detection approaches despite the aggressive nature of NSCLC, which accounts for more than 80% of all lung cancer cases.

To better detect CTCs in NSCLC patients, investigators at the Institute for Systems Biology (Seattle, WA, USA) demonstrated the value of using hexokinase-2 (HK2) as a metabolic function–associated marker for the detection of CTCs.

For this study, the investigators used the Menarini Silicon Biosystems Inc. (Huntington Valley, PA, USA) CellSearch method. CellSearch is the only [U.S.]Food and Drug Administration-approved platform for CTC isolation. This method is based on the use of iron nanoparticles coated with a polymer layer carrying biotin analogues and conjugated with antibodies against EpCAM for the capture of CTCs. Isolation is coupled to an analyzer to take images of isolated cells upon their staining with specific fluorescent antibody conjugates. Blood is sampled in an EDTA tube with an added preservative.

Upon arrival in the laboratory, 7.5 milliliters of blood is centrifuged and placed in a preparation system. This system first immunomagnetically enriches the tumor cells by means of ferrofluid nanoparticles and a magnet. Subsequently, recovered cells are permeabilized and stained with a nuclear stain, a fluorescent antibody conjugate against CD45 (leukocyte marker) and cytokeratins 8, 18, and 19 (epithelial markers). The sample is then scanned on an analyzer which takes images of the nuclear, cytokeratin, and CD45 stains.

To be considered a CTC a cell must contain a nucleus, be positive for cytoplasmic expression of cytokeratin as well as negative for the expression of CD45 marker, and have a diameter larger than five microns. If the total number of tumor cells found to meet the criteria cited above is five or more, a blood sample is positive. In studies done on prostate, breast, and colon cancer patients, median survival of metastatic patients with positive samples is about half the median survival of metastatic patients with negative samples. This system is characterized by a recovery capacity of 93% and a detection limit of one CTC per 7.5 milliliters of whole blood.

In the current study, the use of HK2 as a biomarker enabled development of a metabolic activity-based method for the identification of a novel CTC population without CK expression that was normally overlooked by conventional methods. Thus, the investigators showed that in 59 NSCLC patients bearing cytokeratin-positive (CKpos) primary tumors, HK2 enabled resolving cytokeratin-negative (HK2high/CKneg) CTCs as a prevalent population in about half of the peripheral blood samples with positive CTC counts.

"The use of HK2 as a tumor cell marker permits us to reveal a novel circulating tumor cell population which is not accessible with and is normally overlooked by current epithelial marker-based CTC detection methods," said senior author Dr. Wei Wei, an assistant professor at the Institute for Systems Biology. "Moreover, this approach can be exploited to anticipate NSCLC patient therapy response before they undergo cancer therapy, and more generally, it will be useful in identifying CTCs from patients with a wide variety of cancers, independent of epithelial traits."

The hexokinase-2 liquid biopsy study was published in the March 16, 2021, online edition of the journal Proceedings of the National Academy of Sciences of the United States of America.

Related Links:
Institute for Systems Biology
Menarini Silicon Biosystems Inc.

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