For reasons that aren’t entirely clear, cancer immunotherapy works amazingly well in some patients, but fails the majority. The fortunate few may be distinguished by a new kind of biomarker—or, more accurately, a well-known biomarker that can be used in a new way. This biomarker isn’t a particular genetic mutation. It’s more of a genetic condition, a predisposition to genetic mutations. This condition, which is called microsatellite instability (MSI), occurs when mismatch repair proteins are unable to correct mistakes made when DNA is copied from parent cells and passed on to daughter cells.

MSI already guides diagnostic screening and chemotherapy treatment planning. In the future, report scientists from Memorial Sloan Kettering Cancer Center (MSKCC) and Johns Hopkins University, MSI may predict which patients will respond best to cancer immunotherapy. These scientists, led by MSKCC’s Timothy A. Chan, MD, PhD, found that certain MSI metrics—MSI intensity and insertion-deletion mutations—strongly affect therapeutic outcome. The scientists speculate that when MSI intensity is high, cancer cells generate more neoantigens that the immune system can recognize and destroy.

Detailed findings appeared May 3 in the journal Science, in an article titled, “Genetic diversity of tumors with mismatch repair deficiency influences anti–PD-1 immunotherapy response.” According to this article, MSI intensity could provide a genetic signature to better profile responses to cancer immunotherapy.

“This genetic signature could serve as a novel biomarker, akin to a crystal ball, to see which cancer patients may respond to immunotherapy,” said the article’s lead author, Rajarsi Mandal, MD, a researcher at Johns Hopkins University. He cautions that the new results need to be validated in a larger study.

“We’re not making any definitive claims at this time,” he continued, “but we believe this is the first evidence that it might be possible to use such sequencing data for patients with advanced mismatch repair deficient tumors.”

The scientists developed mouse models of microsatellite instability tumors to compare varying degrees of MSI, or MSI intensity, and its impact on anti-PD-1 response. The results suggest that the genome-wide intensity of MSI and resulting mutational load, particularly insertion-deletion mutations, play a critical role in both the evolution of mismatch repair deficient (MMR-d) tumors as well as their individual response to PD-1 immunotherapy.

Indel mutations involve a series of nucleotides, either inserted into the genetic code or removed during cell division. They can potentially generate neoantigens, or new proteins in cancer cells that the immune system can recognize and destroy.

In addition, using clinical data from human cancer patients with MSI-high tumors, Mandal and colleagues discovered similar relationships between MSI intensity and a patient’s response to anti-PD-1 therapy.

“The extent of response is particularly associated with the accumulation of insertion-deletion (indel) mutational load,” the authors of the current study wrote. “This study provides a rationale for the genome-wide characterization of MSI intensity and mutational load to better profile responses to anti–PD-1 immunotherapy across MMR-deficient human cancers.”

The scientists performed several experiments to tease out immunotherapy response in mismatch repair deficient tumors. First, they took mouse melanoma and mouse colorectal cancer cell lines and used gene-editing tools (CRISPR-Cas9) to create tumors with mismatch repair deficiency akin to those found in human tumor cells, and grew them over several weeks.

Those grown for four weeks were termed MSI-intermediate cell lines, whereas those grown for four months were termed MSI-high cell lines. On analysis using whole exome sequencing they found that the MSI-high cell lines had more indel mutations, and more of another type of mutation called missense (in which just one genetic letter is replaced during cell division) than the MSI-intermediate cell lines or the original parent cell lines created for the experiment.

Investigators then implanted these cell lines into the flanks of living mice and treated the mice with either immunotherapy drugs called anti-PD-1 checkpoint inhibitors or a sham treatment. Anti-PD-1 checkpoint inhibitors release the brakes on immune cells that would otherwise shut down the immune response against cancer cells.

Mice implanted with the MSI-high lines and treated with anti-PD-1 had a much more pronounced decrease in tumor volume than mice with MSI-intermediate lines and the parent lines. This suggested that the variable response the team saw was due to variations in the genetic characteristic of these tumors, said Mandal.

Then, 24 days after implantation, researchers analyzed the amount of tumor-infiltrating lymphocytes (immune system white blood cells that attack and destroy tumor cells) in the mouse tumors. They found a statistically significant and highly pronounced increase in these cells in the MSI-high tumors after anti-PD-1 therapy compared to the other samples, suggesting the response is related to the immune system.

Next, the team sequenced the DNA of the MSI-high tumors and found a reduction in missense and indel mutations in those treated with anti-PD-1. This suggested that these mutations were responsible for creating neoantigens recognized and eliminated by the immune system, Mandal indicated.

Building on these findings, the team looked at clinical data drawn from three independent cohorts of cancer patients to see what the relationship might be between MSI intensity and response to anti-PD-1 therapy in people. The researchers first examined baseline immune activity as measured by a previously reported and standardized immune activity score, known as CYT, measured across 14 human cancers, finding a general trend toward increased immune activity in MSI-high tumors compared to MSI-low tumors.

The researchers also say they observed a statistically significant difference—meaning the findings were not by chance—between the immune activity in MSI-high and MSI-low cancers frequently detected to have mismatch repair deficiency, such as uterine, endometrial, stomach, and colorectal cancers.

The team also analyzed DNA sequences from two separate groups of patients with mismatch repair deficient tumors treated with anti-PD-1 immunotherapy. Among the first group of 15 patients from Johns Hopkins, from the nine with mismatch repair deficiency they noted that patients with higher levels of genetic MSI generally were the ones who had complete or very good responses to immunotherapy compared to patients with lower levels.

In a second group of 33 patients from MSKCC, the team noted that patient MSI levels in the top 80th percentile were associated with improved survival after treatment with immune checkpoint inhibitors, mostly compared to those in the bottom 20th percentile.

“Together, our data demonstrate that a range of MSI types exist, and may help identify patients who will benefit from immunotherapy,” stated Mandal. “It may be possible to classify responders and nonresponders to anti-PD-1 therapy across mismatch repair deficient cancers by using precise, next-generation sequencing measures of MSI intensity.”

Mandal said the team’s next steps will explore the role of the innate immune system in the MSI-related immune response and confirm whether the baseline or the continuously generated mutations, particularly insertion-deletion mutations, are responsible for the therapeutic response observed.

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