Using cutting-edge technology, researchers analyzed triple-negative breast cancer at the single-cell level

A recent BCRF-supported study has revealed intricate variations in genes of triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC). The research team, led by BCRF investigator Dr. Samuel Aparicio and including fellow BCRF investigator Dr. Jorge Reis-Filho, published their findings in the prestigious journal Nature.

Notably, in this study as well as in other ongoing work, Dr. Aparicio’s team used high-resolution techniques to study breast cancer biology at the single-cell and genetic levels to better understand how cancer evolves and to identify novel strategies for treating it.

“Cells are the unit of evolution,” Dr. Aparicio said. “Every time a cell divides, there’s potential for a mistake to be created in the copying of its DNA, and that is what drives evolution. We have been able to catch that in the act as it happens.”  

These findings from Drs. Aparicio, Reis-Filho, and their colleagues may have therapeutic implications and bring us another step closer to more fully understanding TNBC, a harder-to-treat and aggressive form of breast cancer.

Tumor cells, mutational signatures, and “survival of the fittest”

Our cells’ DNA contains the blueprint for how a cell functions and is made up of discreet sections called genes. Genes guide the production of proteins and can provide signals to the cell to increase or decrease their own production.

When DNA damage occurs, normal cells can repair it and keep growing. But sometimes, gene mutations occur that cause cells to lose this ability and keep growing unchecked, passing along DNA damage to future cells and causing instability in the DNA or genome. Genomic instability is a hallmark of most cancers. Mutations can also occur in specific genes (oncogenes) and allow uncontrolled growth and cancer formation.

Tumors are a collection of cells, and it is widely believed that they are the result of accumulation of many cellular mutations over time. As a tumor grows, only those cells that can withstand DNA mutations and thrive in the environment surrounding the tumor will continue to grow, a process also called evolutionary selection.

Studies have shown that there are consistent patterns of mutations (called mutational signatures) within tumor cells. Mutational signatures can represent complex changes in DNA, genes, and structure and can vary from cell to cell. These include amplifications in genes (when the number of copies of a gene are selectively increased), alterations in the number of copies of a particular gene, and variations in the length of individual genes—any of which can result in genomic instability.

Therefore, researchers hope to determine what factors contribute to “survival of the fittest” in cancer cells by identifying those mutational signatures that provide a selective advantage to specific cells, allowing them to perpetuate genomic instability and form tumors.

An innovative way to study mutational signatures

Typically, mutational signatures were found by sequencing the genes from pools of DNA derived from millions of cells (what’s known as whole genome sequencing). However, significant technological advancements have made it possible to accurately sequence the genes within individual cancer cells. As cancer evolution happens at the level of the single cell, being able to detect genomic differences on a cell-to-cell level allows researchers to understand what mutations in individual cells might allow one set of cells (clones) to survive and grow while others do not. If researchers can identify mutational signatures, they may be able to then target them as a novel strategy for treating these tumors.

Dr. Aparicio and his team performed single-cell analysis coupled with cutting-edge computational methods to examine individual cells within TNBC and HGSC tumors. Doing so, they discovered three mutational signatures that may provide clues about the genomic changes that give these tumor cells selective advantage. Importantly, these characteristics, previously hidden by standard whole genome sequencing, may be potential treatment targets.

What the researchers found

High level amplifications

Oncogenes are important drivers of tumor progression and studies have shown this is particularly true when they are overproduced by cells; this is termed high-level amplification. Dr. Aparicio and his colleagues revealed an additional layer of complexity: They found that how high the amplification/overproduction was varied substantially between individual cells and may impact genomic instability. Moreover, some of these high-level amplifications were apparent in regions containing key oncogenes, a trait that likely impacts a tumor cell’s progression. Other researchers have posited that high genomic instability may be a mechanism by which individual cancer cells develop into treatment-resistant clones. Further studies will delve into this finding and potentially identify novel strategies to target oncogenes with significant high-level amplifications.

Parallel copy number alterations

Gains and losses of DNA, or copy number alterations, are common in cancer and are most likely caused by errors in DNA processes such as replication and repair. Studies have shown that high copy number alterations can lead to genomic instability in breast and other cancers.

Dr. Aparicio’s team identified parallel copy number alterations in specific and previously undefined areas of cellular DNA (alleles). Localizing these alterations within discreet alleles demonstrates the specificity of cutting-edge techniques that allow investigators to drill down and assess individual cells whereas other previous studies have relied on bulk analysis and lacked precision.

The team’s findings indicate that parallel copy number alterations are a component of tumor cells’ mutational signatures and contribute to genomic variations that occur from cell to cell—possibly providing a selective advantage to tumor cells. The role of these allele-specific copy number alterations in breast cancer progression is the focus of ongoing studies.

Copy number segment length variations

The team found that in addition to copy number alterations, the length of certain gene segments was also variable from cell to cell (termed ‘serrate structural variations’ for their serrated appearance). Furthermore, serrate structural variations were prevalent in TNBC and HGSC. Although the underlying cause for these variations is unknown, they denote a new class of modification that may contribute to the evolution of tumors.  

These three mutational processes likely generate instability and diversity in tumor cells, which may contribute to poor prognosis. These BCRF investigators and others have demonstrated that single-cell sequencing is an important tool to reveal hidden genomic diversity and instability in tumors that have otherwise not been detected with other technologies.  

What these findings mean for patients

Defining mutational signatures has transformed our understanding of cancer genomes so doctors can make more informed therapeutic decisions and better predict prognosis. Dr. Aparicio and the team behind this study have now demonstrated that single-cell sequencing can reveal previously unknown but potentially targetable features in tumor cells, and they have illuminated mutational processes that help specific tumor cells prevail and facilitate breast cancers’ progression. Strategies to target these processes will help to improve treatment outcomes and prognosis for patients.

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