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Along with advances in single-cell sequencing and multiomic analysis, researchers are able to more precisely decode the stages of human cancers—from precursor to development of invasive cancer—to more accurately inform therapeutic approaches. With more than 2 million global cases of breast cancer diagnosed annually, can scientific innovation help enable a future where doctors are able to treat the biology to stave off the disease before it becomes invasive?
To put this into context, the American Cancer Society estimated more than 55,720 new cases of ductal carcinoma in situ (DCIS) would be reported in 2023. Earlier studies found that between 20–50% percent of cases progressed to invasive breast cancer, and virtually all patients with DCIS had surgery, with nearly one-third of patients undergoing a unilateral or bilateral mastectomy.
Identifying markers of progression could reduce the overtreatment of low-risk molecular lesions. To decipher the evolution in human cancer, with the goal of informing clinical outcomes, researchers must initiate a comprehensive interrogation that involves multiple omic layers of the central dogma of biology.
A Case Study
Fundamental to biology is the single cell: It is requisite for interrogating tumor evolution and somatic mosaicism. We need to move beyond the simple enumeration of cell types, which is at the center of most of the research conducted today, to inform meaningful multiomic insights for diagnosis, prognosis, and responses to therapy, and to advance the study of normal tissue mosaicism.
To conduct a multiomic analysis of an individual cell, two fundamental needs must be satisfied: a multiomic chemistry to expose the cellular story, and a way to provide healthy single cells as input into the chemistry. In this research, dual-viability fluorescence-activated cell sorting (FACS) provided the single cells needed as input into BioSkryb’s ResolveOME multiomic chemistry.
With primary breast cancer as an example, BioSkryb, in collaboration with Shelley Hwang, MD, Chief of Breast Surgery at Duke Medical Center, and Jeffrey R. Marks, PhD, a translational scientist, assayed nearly 1,000 single cells, which were analyzed at all three central dogma layers—genomically, transcriptomically and with a panel to assay surface proteins.
Beginning with surgically resected tissue, and in some cases with normal stromal tissue controls, the samples underwent enzymatic and mechanical dissociation to single cells. Whole genome amplification followed by whole exome sequencing offered clarity of the tumor signal where somatic mutation is in 50% of sequencing reads. This digital signal, obtained from individual cells, allows for the ability to identify rare clones that were previously masked, or obscured, in bulk sequencing.
The Power of Primary Template-Directed Amplification
In order to study clonal evolution, ResolveOME, powered by primary template–directed amplification to generate comprehensive and uniform genomic coverage, additionally provides the transcriptome to ascertain cell identity and state. The chemistry has now been expanded to enable extracellular protein expression monitoring from the same individual cell that was analyzed at the DNA/RNA levels. This protein information allowed the corroboration of RNA data in understanding cell type and state: a trimodal analysis.
BioSkryb’s ResolveOME single-cell multiomic analysis exposed heterogeneity in multiple tiers in breast cancer. By adding the phenotypic context to the genomic foundation, the single-cell genotype revealed how a genomic change was expressed and the how mutation affected that expression.
Moreover, the coupling of FACS enrichment and dispensing to the ResolveOME multiomic chemistry demonstrated that at both the genomic and transcriptomic levels, at 97%—or nearly complete coverage of the genome—can be obtained. Single-nucleotide-variant (SNV) detection sensitivity and precision was also robust, along with exceptional representation of both alleles.
The Path Forward
The comprehensive and rich reporting of unified DNA and RNA tells a cellular story that is not possible with uniomic datasets. With full-transcript level expression data, scientists can expose differential isoform utilization of the human genome that can be assayed for relevant drug targets, while armed with genomic variation information underpinning RNA differences.
We believe the way forward is single-cell multiomic analysis.
Jon Zawistowski, PhD, is the Senior Director of R&D at BioSkryb Genomics.
With its novel combination of single-cell methods, BioSkryb Genomics is unlocking a deeper understanding of tissue health. To learn more, visit www.bioskryb.com.
