Recurrent mutations that accompany tumors arouse suspicion. Even if recurrent mutations have nothing against them but circumstantial evidence, they may be assumed to be drivers of cancer development, and not just passengers. And yet, sometimes, they are just passengers. If passengers are mistakenly regarded as drug targets, drug development resources could be wasted. Worse, action against cancer’s real culprits could be delayed.

We can recognize which recurrent mutations are relatively innocuous—and keep them off drug developers’ “most wanted” lists—if we exercise caution when interpreting the results of high-throughput sequencing projects. These results, argue scientists based at the Massachusetts General Hospital (MGH) Cancer Center, may need additional context.

Led by Michael Lawrence, PhD, and Lee Zou, PhD, the MGH Cancer Center team surveyed the landscape of background mutations in cancer genomes. “A typical cancer genome will have five to ten driver mutations and thousands or even millions of passenger mutations that are just along for the ride,” said Lawrence. “The thinking has been that, if the exact same mutation occurs in many different patients’ cancers, it must confer a fitness advantage to the cancer cells. While the recurrence-based approach to identifying cancer driver genes has been successful, it’s also possible that certain positions in the genome are just very easy to mutate.”

Although much is known about how patterns of gene mutation are affected by small-scale structures—such as the groups of three bases called trinucleotides—or by the large-scale “compartments” into which DNA is organized in the nucleus, relatively little is known about the effects of “mesoscale” DNA structures that may extend 30 base pairs around the site of a mutation.

Mesoscale mutational processes, the MGH Cancer Center team reasoned, merit close attention because they can target unique stretches of DNA that occur infrequently in the genome. Consequently, these processes could lead to individual base pairs becoming recurrently mutated across many patients, imitating the effects of functional selection, and masquerading as driver hotspots.

To explore this line of thought, the MGH Cancer Center team evaluated APOBEC-signature mutations. APOBEC proteins help protect against viruses that enter cells by altering the viral genome. Many types of cancer cells are known to activate APOBEC enzymes.

Previous investigation by others into breast cancer mutation hotspots associated with APOBEC enzymes identified DNA palindromes, features in which a specific sequence on one side of a mutation is repeated in reverse on the other side, suggesting the stem-loop hairpin structure. In contrast to other cancer-associated mutations that preferentially accumulate in a specific region of the genome, APOBEC-associated mutations are distributed evenly throughout the genome, frequently occurring in DNA hairpins.

The scientists’ experiments revealed that the APOBEC3A enzyme commonly mutates cytosine bases located at the end of a hairpin loop, converting them into uracils, even in genes that have little or no association with cancer. In contrast, recurrent APOBEC-associated mutations in known driver genes were at ordinary sites in the genome, not the special hairpin sites that are especially easy for APOBEC3A to mutate. This suggests that the driver mutations, while possibly being difficult to generate, do confer a survival advantage on cancer cells, and that is why they are frequently observed in patients with cancer.

Detailed findings appeared June 28 in the journal Nature, in an article entitled, “Passenger hotspot mutations in cancer driven by APOBEC3A and mesoscale genomic features.”

“We have shown that DNA stem-loops, a mesoscale genomic feature, are associated with recurrence of mutations outside of known cancer drivers,” the article’s authors wrote. “This finding not only challenges the presumption that recurrent mutations must be drivers, but also highlights the importance of incorporating mesoscale features into the analysis of cancer genomes.”

“Several APOBEC3A hairpin hotspots have already been claimed to be drivers, based solely on their frequency and with no functional evidence,” noted Zou, a senior author of the Science paper. “Our findings suggest that these are simply passenger hotspots—a term that would have been considered an oxymoron until now—and that researchers’ time would be better spent on mutations that have been proven to alter the properties of cells in ways that drive their malignant proliferation.”

Lawrence, the paper’s other senior author, added, “There are so many important questions in cancer research, and anything we can do to avoid investigators’ pursuing false leads would save time and money. But the challenge of distinguishing drivers from passengers is also central to other important questions: How many drivers does it take to make a cancer? What is the process by which a normal cell becomes cancer? Why do some cancers appear to have no driver mutations?

“Our colleague professor Gad Getz has shown that there must be considerable ‘dark matter’ in the genome to explain these driver-negative cases. Having an accurate inventory of drivers requires being able to see through passenger mutations that masquerade as drivers, and our results suggest there might be even more genomic dark matter!”

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