Professor Shi Qihui of Fudan University: Single-Cell Sequencing Opens New Opportunities in Liquid Biopsy

In May 2019, the 7th European Molecular Diagnostics Conference, hosted by the Cambridge Innovation Institute, was held in Lisbon, the capital of Portugal. More than 400 attendees from around the world gathered to share the latest advances and research findings in the field of molecular diagnostics. As one of the most influential conferences in molecular diagnostics globally, the event featured seven sub-sessions, two short courses, twelve roundtable discussions, and numerous poster presentations, covering hot topics such as prenatal molecular diagnostics, immunotherapy biomarkers, liquid biopsy, and point-of-care testing.

At this conference, Professor Shi Qihui from the Institute of Biomedical Sciences at Fudan University, as the sole invited speaker from mainland China, delivered a presentation titled “Metabolic Phenotyping and Single-Cell Sequencing of Circulating Tumor Cells in Non-Small Cell Lung Cancer.”

Arterial New Medicine (WeChat ID: biobeat1) contacted Professor Shi Qihui immediately upon his return to China and conducted an interview. Professor Shi shared with us his experience at the conference and his insights into the field of molecular diagnostics.

Prof. Shi Qihui

Circulating tumor cells (CTCs) have long been one of Professor Shi Qihui’s primary research focuses. The presentation delivered at this conference was based on his recent findings regarding the metabolic subtyping of CTCs. This work highlights the latest advances by Professor Shi’s team, uncovering the metabolic heterogeneity of CTCs and demonstrating that the proportion of CTCs with distinct metabolic phenotypes can predict patients’ treatment response, clinical manifestations, and survival rates.

Professor Shi Qihui believes that there is a strong correlation between circulating tumor cells (CTCs) and tumor metastasis. The primary focus of CTC applications in early cancer screening should not be on the early detection of cancer per se, but rather on the early identification of cancers that have already metastasized. “In general, early-stage tumors rarely release CTCs into the bloodstream. However, if a tumor is highly aggressive, CTCs may be detectable in the blood at a very early stage, even when the tumor itself is small. In such cases, using CTCs for early screening is meaningful,” said Professor Shi. He argues that, beyond prognosis and guidance for medication, a more significant application of CTCs lies in patient stratification or auxiliary staging. This approach helps complement current imaging techniques in distinguishing between tumors with high metastatic potential and those with low metastatic potential, thereby enabling patients and physicians to promptly identify highly metastatic tumors and formulate subsequent treatment plans.

“CTCs primarily provide information in two aspects: quantity and molecular characteristics. Molecular characteristics include genetic and protein information. The relationship between CTC count and tumor metastasis remains unclear; currently, it is mainly used for prognosis and cannot yet determine whether patients have developed hematogenous or distant metastases. However, it is certain that CTCs themselves are heterogeneous. If CTCs can be further subtyped using specific molecular targets, the distribution of CTCs across different subgroups may in the future be directly linked to metastatic status in cancer patients,” said Shi Qihui.

The clinical value of CTCs is beyond doubt. Data from ClinicalTrials.gov show that, among globally registered liquid biopsy clinical trials, those involving CTCs have consistently outnumbered those involving ctDNA. Shi Qihui pointed out that the main challenge in CTC research lies in the diverse and complex technical approaches employed. Different methodologies may capture CTCs at varying levels of purity or isolate distinct CTC subpopulations, making it difficult to directly compare CTC data obtained through different technical platforms.

The CellSearch platform, which was launched 15 years ago, is widely used in clinical trials of CTCs. Developed by Johnson & Johnson, the CellSearch platform enriches CTCs through EpCAM (epithelial cell adhesion molecule). Although this platform demonstrates good stability, it exhibits low sensitivity in certain cancers (such as non-small cell lung cancer), and its detection rate remains modest even in samples from patients with advanced-stage tumors.

Shi Qihui informed us that the low sensitivity may not be attributable to technical factors. When known quantities of tumor cell lines were spiked into healthy human blood and analyzed using this platform, the recovery rate of tumor cells was high. He speculated that the low detection rate is more likely related to biological reasons. For instance, some circulating tumor cells (CTCs) undergo epithelial-mesenchymal transition (EMT), resulting in reduced expression of epithelial markers and enhanced cellular deformability. These cells are difficult to detect using the CellSearch platform.

“In such circumstances, the use of single-cell sequencing in CTC research becomes essential. The number of CTCs in the blood is undoubtedly of research value. However, to fully exploit this value, it is crucial to verify that each captured CTC is indeed a tumor cell. This necessitates performing single-cell sequencing on every individual captured cell,” said Shi Qihui.

In Professor Shi Qihui’s view, the direct clinical application of single-cell sequencing faces significant challenges due to its high cost and technical complexity. Single-cell sequencing has been widely used in basic research on circulating tumor cells (CTCs), such as validating the accuracy of biomarkers employed for CTC isolation and confirming that the CTCs captured using these biomarkers are indeed tumor cells. Once a biomarker gains widespread acceptance through extensive long-term validation, there is no longer a need to routinely employ single-cell sequencing—a research-intensive method—to verify its performance in subsequent scientific and clinical applications.

In liquid biopsy for cancer, circulating tumor DNA (ctDNA) testing is just as important as circulating tumor cell (CTC) detection. By comparison, ctDNA analysis has advanced more rapidly and is closer to clinical application. Shi Qihui believes that the primary reasons are the clearer analytical targets of ctDNA (such as point mutations and methylation), a more straightforward technological pathway, and greater ease of implementation. Moreover, unlike CTCs, ctDNA is not affected by its own heterogeneity; results obtained using different methods are largely consistent, yielding strong comparability.

Shi Qihui stated, “Currently, there are two prominent research directions for ctDNA: one is predicting the efficacy of immunotherapy through TMB (tumor mutational burden), and the other is early cancer screening. In the application of early screening, several challenges remain to be overcome for ctDNA. For instance, the concentration of ctDNA in patients with early-stage disease is often too low, making it highly susceptible to interference from mutated cfDNA (cell-free DNA) originating from other sources. Additionally, organ localization represents another major challenge in the current application of ctDNA for early screening.”

As cell therapies and antibody drugs enter clinical practice, the prominence of immunotherapy in oncology has risen rapidly. Studies on tumor tissues have demonstrated that tumor mutational burden (TMB) and PD-L1 are independent biomarkers for predicting the efficacy of PD-1/PD-L1 immune checkpoint inhibitors, thereby enabling companion diagnostics for immunotherapy. In tissue sample testing, precision pathology can obtain rich information on tumor–immune interactions through various methods. However, due to the clinical difficulty of performing repeated tissue biopsies, liquid biopsy holds significant potential to play a major role in monitoring and guiding immunotherapy.

It is understood that clinical detection of blood tumor mutational burden (bTMB) via circulating tumor DNA (ctDNA) in blood is already feasible. Shi Qihui informed us that circulating tumor cells (CTCs) can also be used to assess PD-L1 expression in tumor cells; however, the concordance between CTC-based assessment and primary tissue has not yet been validated. “By detecting neoantigen-specific, activated CD8+ T cells in patients’ blood, it is also possible to predict the efficacy of immunotherapy,” he said. “This also falls under the broad category of liquid biopsy.”

Professor Shi Qihui also shares his views on the popular field of NIPT (Non-Invasive Prenatal Testing) in China. NIPT has already formed a market of considerable scale in China, sparking a wave of growth in the genetic testing industry. However, Professor Shi believes that there is still significant room for development in the NIPT sector. “Currently, NIPT primarily screens for chromosomal aneuploidies, such as Down syndrome, but these account for only a small fraction of birth defects caused by chromosomal abnormalities and gene mutations. There are many untapped opportunities for NIPT in birth defect screening,” he said.

Currently, NIPT primarily detects cell-free fetal DNA in the blood. Shi Qihui, however, believes that fetal cells hold significant promise for development in cell-based NIPT, provided that appropriate methods can be identified to isolate fetal cells in sufficient quantities and with high purity. He argues that since fetal cells contain a complete genome, cell-based testing has the potential to offer a universal solution for prenatal diagnosis. Current research in this area mainly focuses on fetal nucleated red blood cells and trophoblasts in maternal blood, as well as trophoblasts in cervical mucus. While fetal cells are scarce in the bloodstream, cervical mucus contains a relatively higher number of fetal cells.

“Current developments in fetal cell analysis closely mirror the situation with circulating tumor cells (CTCs). Effectively identifying, isolating, and sequencing fetal cells poses a significant challenge, with even higher technical requirements. For fetal cells, mere enumeration lacks clinical significance; sequencing is mandatory. Therefore, we must not only identify suitable biomarkers for fetal cell identification but also thoroughly validate them. During the biomarker validation phase, it is essential to perform single-cell sequencing on each target cell to confirm its fetal origin,” said Shi Qihui.

Shi Qihui informed us that another challenge in fetal cell testing is the poor quality of single-cell sequencing, which is currently insufficient to establish a reliable diagnosis. Unlike circulating tumor cells (CTCs), counting fetal cells lacks clear clinical significance; therefore, sequencing is an indispensable component of fetal cell detection. Given the scarcity of fetal cells, single-cell sequencing represents the optimal option available for their detection. However, the current workflow is lengthy, involving cell fixation, staining, isolation, and single-cell genome amplification, processes that are prone to introducing genetic bias and errors, thus requiring further optimization. Consequently, single-cell sequencing of fetal cells remains in the preclinical research stage.

With advances in non-invasive prenatal diagnosis and intrauterine gene editing technologies, we will be able to detect and treat birth defects at an earlier stage in the future.

Promoting exchanges between China’s liquid biopsy community and leading international experts, as well as facilitating access to the latest research, has long been a key focus of Professor Shi Qihui. Initiated by Professor Klaus Pantel of the University Medical Center Hamburg-Eppendorf in Germany, who first proposed the concept of “liquid biopsy,” the International Symposium on Minimal Residual Cancer (ISMRC) has been held for over two decades and is the longest-running and most influential academic conference in the global liquid biopsy field.

Professor Shi Qihui, Professor Lu Shun from Shanghai Chest Hospital, and Professor Klaus Pantel have collaborated to bring the 12th ISMRC (the 1st Global Liquid Biopsy Congress) to China for the first time, with plans to hold it in Shanghai in October 2020. At that time, nearly one hundred world-leading experts in the field of global liquid biopsy, particularly in areas such as CTCs, ctDNA, and companion diagnostics for immunotherapy, will gather in China to promote the development and translation of liquid biopsy technologies, facilitate the integration of Chinese liquid biopsy research and applications with global standards, and showcase more Chinese achievements in liquid biopsy on this world-class innovation platform.