Editors: Donna E. Hansel, MD, PhD, division head of pathology and laboratory medicine, MD Anderson Cancer Center, Houston; James Solomon, MD, PhD, assistant professor, Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York; Erica Reinig, MD, assistant professor and medical director of molecular diagnostics, University of Wisconsin-Madison; Marcela Riveros Angel, MD, molecular genetic pathology fellow, Department of Pathology, OHSU; Andrés G. Madrigal, MD, PhD, assistant professor, clinical, Ohio State University Wexner Medical Center, Columbus; Maedeh Mohebnasab, MD, assistant professor of pathology, University of Pittsburgh; and Alicia Dillard, MD, clinical pathology chief resident, New York-Presbyterian/Weill Cornell Medical Center.
Role of single-cell RNA sequencing in systemic lupus erythematosus
February 2023—Systemic lupus erythematosus is an autoimmune disease that affects multiple organ systems and is most prevalent in women of Asian, Hispanic, and African ancestries. People with the heterogeneous disease experience major organ damage, which primarily affects the kidneys, skin, heart, and joints. Transcriptomic studies of systemic lupus erythematosus (SLE) have implicated increased type 1 interferon signaling, dysregulated lymphocyte activation, and failure of apoptotic clearance as hallmarks of the disease. Many genes are near the approximately 100 loci associated with SLE. Despite the use of flow cytometry and transcriptome profiling to characterize the role of circulating immune cells in SLE, there is not a complete census of circulating immune cells in the disease, and characterizing the genetic associations has been challenging. Single-cell RNA sequencing has potential as an unbiased approach to distinguish the composition and expression of the molecular and genetic states of the circulating immune cells in lupus patients. To address the limitations of this assay, the authors previously developed a multiplexed single-cell RNA-seq (mux-seq) for obtaining cell type-specific immune traits. In this study, they used mux-seq to profile more than 1.2 million peripheral blood monocytes from a cohort of 162 SLE cases and 99 healthy people, most of whom were women of Asian or European ancestry. The composition and state of the circulating peripheral blood mononuclear cells varied among the SLE patients. The analysis of lymphocyte composition found a reduction in naïve CD4+ T cells and an increase in CD8+ T cells that express GZMHF granzyme genes. The genetic expression features of each cell type were used to predict patients’ disease status and stratify them into molecular subtypes. The analysis suggested that ancestry played an important role and determined the composition and state of the immune cells and, perhaps, the differences in symptomatology and severity seen in patients. In separate expression quantitative trait loci analyses and genomewide association studies, the authors sought to identify trait loci that could mediate SLE disease associations. Their research identified cell types specific and relevant to SLE and enabled fine-mapping and interpretation of the disease-associated variants. The authors concluded that better molecular characterization is needed to improve diagnostic modalities and treatment of SLE. They developed a molecular assay to better characterize the cellular composition of circulating cells and identify cell-specific signatures of the disease.
Perez RK, Gordon MG, Subramaniam M, et al. Single-cell RNA-seq reveals cell type-specific molecular and genetic associations to lupus. Science. 2022;376(153). doi:10.1126/science.abf1970
Correspondence: Dr. Chun Jimmie Ye at jimmie.ye@ucsf.edu
Use of liquid biopsy with a parallel mutation and methylation approach to detect liver cancer
The five-year survival rate for advanced stage hepatocellular carcinoma is 12 percent, and the most important factor in patient outcome is early disease detection. Therefore, efficient screening methods that detect hepatocellular carcinoma (HCC) in a surgically resectable stage with a high degree of accuracy are needed. One of the most promising approaches, which has been widely studied for early cancer detection, uses liquid biopsy-based cell-free DNA (cfDNA) from peripheral blood. Previous studies have identified potential biomarkers from cfDNA profiling that could be used in clinical applications. However, the ability to detect low-frequency mutations in low-yield cfDNA could be difficult because of sampling issues. The authors developed an innovative technology, called Mutation Capsule Plus (MCP), that supports parallel profiling of mutations and methylation changes in a single cfDNA sample and that can simultaneously detect genetic and epigenetic alterations. Using the technology, the authors identified a set of methylation markers that were able to distinguish 30 HCC samples from 30 non-HCC control samples. They then combined the methylation markers with mutation patterns in a training set of 60 HCC samples and 60 non-HCC controls to establish an algorithmic model that was able to outperform methylation or mutational markers alone. This resulted in an HCC detection model. The authors validated the model in an independent retrospective cohort of 58 HCC samples and 198 non-HCC samples, demonstrating a sensitivity of 90 percent and specificity of 94 percent. The model was further applied to a prospectively collected cohort of 311 people who had asymptomatic hepatitis B infection with normal liver imaging and serum AFP concentrations. The model detected four of the five HCC cases in the cohort with a sensitivity of 80 percent and specificity of 94 percent. Noninvasive cancer detection using cfDNA from liquid biopsies traditionally has focused on only one type of biomarker, often due to limited testing material. The MCP method detects the alterations present in cfDNA, including methylation changes and mutations such as base changes, insertions, deletions, and even more complex structural mutations. The authors concluded that their findings demonstrate that MCP technology has potential in discovering and validating multiomic biomarkers that can be used for earlier noninvasive cancer detection.
Wang P, Song Q, Ren J, et al. Simultaneous analysis of mutations and methylations in circulating cell-free DNA for hepatocellular carcinoma detection. Sci Transl Med. 2022;14. doi:10.1126/scitranslmed.abp8704
Correspondence: Dr. Yuchen Jiao at jiaoyuchen@163.com