NEW YORK (GenomeWeb) - A team led by researchers at the University of Zurich has linked the level of a specific set of monocytes to patient response to anti-PD-1 immunotherapy in melanoma.
Using mass cytometry, the researchers profiled immune cell populations in the peripheral blood of stage IV melanoma patients before and after 12 weeks of anti-PD-1 immunotherapy, identifying the frequency of classical CD14+CD16−HLA-DRhi monocytes prior to commencing therapy as strongly predictive of progression-free and overall survival. They presented the results in a study published today in Nature Medicine.
The finding highlights the potential of mass cytometry to illuminate fields of research like cancer immunotherapy where, said Burkhard Becher, professor of immunology at the University of Zurich and senior author on the paper, "everybody is just in the dark."
Broadly speaking, cancer immunotherapy uses drugs to block interactions between T cells and immune-suppressing PD-1 ligands on tumor cells, the notion being that dampening the activity of PD-1 proteins on tumor cells will allow the patient's immune system to more effectively recognize cancer cells as foreign bodies and destroy them. The approach has had notable success in diseases including melanoma and lung cancer, but many patients do not respond, and identifying likely responders has proven a significant challenge.
Typically, patient tumors are tested for PD-1 levels, with tumors above a certain expression level deemed good candidates for immunotherapy. However, Becher noted, "while it makes sense that this should be [a good marker], it isn't." And so, particularly given that a number of drugs targeting similar immune suppressing ligands are on their way to the clinic, a better understanding of the processes involved and potential markers for guiding use of these therapies is needed.
Mass cytometry combines capabilities of flow cytometry and atomic mass spectrometry, allowing it to measure large numbers of proteins in single cells with high throughput. The technique detects proteins using antibodies linked to stable isotopes of elements, which can then be read with high resolution via mass spec.
The technology is offered commercially by Fluidigm which sells several mass cytometry instruments including the CyTOF instrument used in the Nature Medicine paper. According to the company, its platform can currently look at 37 markers per sample simultaneously, with a theoretical maximum of 135 markers per sample. Conventional flow cytometry, which is mass cytometry's main competitor, typically maxes out at around a dozen markers simultaneously, with most experiments looking at fewer than that.
By allowing for high-dimensional single-cell analyses, mass cytometry lets researchers better study the heterogeneity and composition of cell populations involved in processes like immune response.
"I think it's really a game changer in that regard," Becher said, adding that he expects it to be a particularly useful technology for identifying biomarkers of response to cancer immunotherapy.
"I think relying only on the expression [of markers like PD-1], either within the cancer or within the patient's blood, will probably prove not to work at all," he said. "But I think that an intelligently designed single-cell cytometry approach of large cohorts of patients will allow you to identify biomarkers. I think that this is just the beginning and we need to get much more and better detail and have larger cohorts and scan through the entire landscape of immunotherapy in cancer."
In the Nature Medicine study, Becher and his colleagues used the CyTOF to look at samples from 20 melanoma patients before and after anti-PD-1 immunotherapy along with samples from 10 healthy controls taken at the same time points. They measured 30 leukocyte markers which allowed them to identify the major immune cell populations and stage of T cell differentiation and activation.
Their analysis found that patients with higher frequency of classical CD14+CD16−CD33+HLA-DRhi monocytes had better response to treatment. They also found that the classical monocytes in good responders had higher levels of migration and activation markers, which, the authors wrote, suggests they "may sustain the development of an effective anti-tumor immune response during anti-PD-1 immunotherapy."
After conducting their initial discovery work on the CyTOF, the Zurich researchers developed a smaller panel for use with traditional flow cytometry, which Becher said is better suited to high-throughput analysis and, ultimately, clinical assays.
The CyTOF, is not robust enough to serve as a clinical instrument, he said, adding that running it consistently requires considerable expertise not available in a typical hospital.
"What every small hospital does have, however, is a small flow cytometer, where they can look at a couple of clinical samples quite quickly and efficiently," he said. "So that is where we think this is going. Whatever we find, the CyTOF will be just the discovery tool."
He suggested, in fact, that improvements in flow cytometry could ultimately make that technology competitive or superior to mass cytometry for discovery work, as well. While mass cytometry has the upper hand in multiplexing, flow cytometry offers advantages in throughput and dynamic range that make it attractive, Becher said.
He added that he saw better barcoding technology as key to moving flow cytometry forward.
"What I really love the most about CyTOF, is the fact that we can barcode all our samples and we can easily throw 30 or 40 patient samples together and then analyze them together," he said. "And the great advantage there is that you don't have this inter-experiment variation that you usually get with more traditional flow cytometry approaches."
"There are some [flow cytometry barcoding approaches] out there, but nothing that I think is better than CyTOF yet," he said. "But I believe in a couple of years that we'll have that."
In the meantime, Becher said he plans to expand upon the Nature Medicine work by looking at larger cohorts of patients including patients receiving combinations of different immunotherapies or immunotherapy and chemotherapy. He said he also hopes to look at cancers outside of melanoma.
An additional area of interest for his team is profiling neutrophils, which, he noted, are the most abundant kind of white blood cell but have received relatively little attention in immunotherapy research thus far due to the difficulty of preserving them.
"When blood cells are fixed or frozen, neutrophils are usually [destroyed], and you can't rescue them," he said. "But given that it's the largest population of leukocytes in the blood, it's kind of stupid that we've never looked at them."
"We are trying to mitigate that," Becher said, noting that he and his colleagues have developed a technique that allows them to preserve these cells for analysis.
"It is cumbersome and logistically much more demanding, but we're now able to, even with barcoding, get 30 samples together and actually really look at the neutrophils," he said. "It requires significantly more work, but we're getting there."