Can existing liquid biopsy instrument replace tissue biopsy?
Conventional tissue biopsy, as the key of the diagnostic pathology, can make a definite histopathological diagnosis over the vast majority of the cancer cases [21]. However, tissue biopsy not only needs complex, time-consuming and expensive operation processes, but also is difficult to obtain pathological tissue from body organ. In addition, the conventional tissue biopsy cannot be used to monitor the treatment response. In contrast, the liquid biopsy instrument can be easily applied to interrogate cancer-related biomarkers sustainably with minimal invasive, and then achieve real-time monitor for cancers.
The major challenge of existing LB instruments is their insufficient accuracy and sensitivity caused by the heterogeneity and rarity of cancer biomarkers [22]. To address these issues, LB instruments used for CTC analysis typically contain white blood cell (WBC) depletion modules, which employ immune- or physical isolation methods depending on the morphological, biological, or physical properties of the CTC and WBCs. The immune-isolation method mainly allows the positive selection of CTCs through antigens expressed in epithelial cells, such as EpCAM. However, subpopulations of metastatic tumor cells with low EpCAM expression or undergoing epithelial-to-mesenchymal transition (EMT) cannot be captured or identified using this method [23]. Physical isolation methods typically separate CTCs based on the differences in size, shape, or deformability between CTCs and blood cells. However, CTC subpopulations with physical features similar to those of WBCs cannot be differentiated or sorted during the separation process [24]. Furthermore, the LB instrument used for cfDNA also faces similar limitations. The ctDNA originating from CTCs can be contaminated by those derived from normal blood cells of the patient and might only account for ~0.01% of the total cfDNA. Various patient-related factors, including pregnancy, smoking, exercise, and various non-malignant conditions, also affect the cfDNA levels in blood [13]. Therefore, the association between patient-related factors and the performance of specific cfDNA assays may not be entirely accurate.
The second challenge of the existing LB instruments is their inadequate automation and integration. To achieve rapid and real-time cancer diagnosis, LB instruments have been developed to reduce biochemical operations, lower the economic burden for training hospital personnel, including pathologists, nurses, and physicians, and simplify the interpretation of the test results. However, most LB instruments can only isolate biomarkers and are unequipped to conduct necessary subsequent analyses. For example, the Parsortix and ClearCell FX platforms are designed for CTC isolation [25,26], whereas the KminTrak instrument is used for cfDNA extraction [27]. The Automatic Fraction Collector can only serve as a pretreatment unit for exosome isolation [28]. Furthermore, most LB instruments require complex biochemical operations, even during the biomarker isolation process, which further impairs their automation levels. For example, CytoSorter requires labor-intensive immunoaffinity operations to separate CTCs from blood cells [29]. For LB instruments without an analytical module, the analysis of biomarkers commonly relies on complex biochemical operations, such as fluorescent in situ hybridization (FISH), next-generation sequencing (NGS), and digital PCR (dPCR) [30]. For instance, for cfDNA detection, PCR technologies are typically used to amplify the target DNA fragment and improve analytical sensitivity. These assays are usually difficult to integrate into the corresponding LB instrument [31].
The third challenge is the validity of the detection results. CellCollector and KminTrak [32,33] as new-developed LB instruments can isolate CTCs and cfDNA, separately. Although the number of CTCs in the blood and the ctDNA content in plasma have been used as a reference for early treatment response of cancers, they do not inform patients about the cancer progression stage, as no additional clinical evidence demonstrating their diagnostic efficacy is available compared to standard imaging-based tools [34]. Thus, CTC- and ctDNA-based LB technologies are not sophisticated enough to monitor treatment response in the clinical setting [35]. In addition, early-stage cancer detected via LB instrument may be indolent or will never develop into life-threatening cancers, such as in the case of prostate and breast cancers. This may result in the overdiagnosis of incidental cancers that are unlikely to affect the overall health or lifespan of the patients [36]. Notably, the overtreatment guided by an overdiagnosis can have considerable consequences [4]. For example, complications that include incontinence or erectile dysfunction can be aroused due to overtreatment in prostate cancers.
To better understand the evolutionary changes in the genetic and epigenetic landscape of tumors, multigene assays of biomarkers obtained from LB instruments are often performed. However, these additional testing require a considerable investment of time, knowledge, and resources, such as state-of-the-art NGS machines, adequate storage space for multigene sequencing data, and a wet laboratory [37]. National and international central laboratories are better equipped to perform these assays, wherein the large-scale sample processing would be more economical. Furthermore, conducting multigene assays rather than single assays may not be worthwhile for local pathology laboratories. Therefore, LB instruments combined with multigene assays for cancer detection are difficult to apply in point-of-care (POC) settings.
Although LB instruments can significantly reduce the investment in manual operation and repeated minimally invasive detection of cancers, they have been commercially developed to a much lesser extent than conventional tissue biopsy approaches. In addition, the rapid development of tissue biopsy- and imaging-based diagnosis methods make them more accessible to researchers and clinicians than LB instruments. Considering the limitations of the existing LB instruments, an obvious challenge for broad clinical applications should be addressed. Therefore, the current LB instrument is not sophisticated enough to replace tissue biopsy.