“The microbiome is a highly complex and diverse community of microorganisms living in and on the human body,” describes Dr. Kate Markey, Assistant Professor in the Translational Science and Therapeutics Division. Dr. Markey is also a physician who cares for patients undergoing blood (hematopoietic) cell transplantation and is interested in the connection between the gut microbiome and the outcomes of immune-based cancer therapies. She emphasizes that careful mechanistic studies are needed to understand how microbes contribute to human health, but when conducting such studies, we also need to consider “the complexity of the immune networks that microbes can influence.” Dr. Markey explains, “I have been interested in the idea of the ‘blood microbiome’ for some time. I have wondered whether low-level bacteria and bacterial DNA that get into the bloodstream are a missing piece of the picture.”
Hematopoietic cell transplantation is a life-saving treatment for patients with leukemia and other blood cancers. “Large correlative studies have demonstrated clear connections between microbial communities of the gastrointestinal tract and patient outcomes, including overall survival, the development of graft-versus-host disease, infections, immune recovery, and relapse,” Dr. Markey states. She adds, “because we know that the intestinal microbiome is linked with patient outcome, and that translocation of whole bacteria is common (resulting in bloodstream infections), we wanted to study whether bacteria-derived genetic material in the bloodstream would be associated with patient outcome.” Dr. Markey and colleagues demonstrate that detecting microbial cell-free DNA from patient blood samples is possible in their recently published Blood Advances article, and that the levels of microbial cell-free DNA change during different stages of hematopoietic cell transplantation.
Cell-free DNA refers to DNA fragments that are released, typically into the bloodstream, when cells die. Recently, cell-free DNA shed by cancer cells into the blood has received a lot of attention for its potential to be used as a non-invasive diagnostic tool for early cancer detection. In contrast, microbial cell-free DNA can be used to identify the causative microbe in the setting of difficult-to-diagnose infections. The challenge with detecting cell-free DNA for either bacterial or cancer cells, is that there are only tiny bits of this DNA present in the blood, making it difficult to detect. For this research, Dr. Markey teamed up with the company Karius, who have developed a deep-sequencing technology able to detect and quantify circulating cell-free DNA. This technology allowed her to “measure microbial cell-free DNA in patient samples collected after allogeneic stem cell transplantation.”
To understand if bacterial cell-free DNA can be reliably detected in patient blood samples and be potentially used as a tool to predict transplantation outcome, the authors assembled a cohort of patients and healthy volunteers to be used as controls. This cohort included 71 patients being treated at Memorial Sloan Kettering Cancer Center— Dr. Markey’s previous institution— in addition to 99 healthy volunteers. Patients with a microbial bloodstream infection were excluded from the cohort to enable the researchers to explore only microbial cell-free DNA profiles correlated with noninfectious transplant outcomes. The group specifically analyzed microbial cell-free DNA detected in blood samples during the neutropenic period of the transplantation process. Neutropenia, or dangerously low levels of neutrophils, occurs after pre-transplant conditioning to ablate the patient’s immune system and prior to engraftment of the new donor cells. During the neutropenic period, “the patient experiences the toxicities of chemotherapy, especially to the intestinal tract, with limited capacity for tissue repair. This is the time window when gut integrity is most compromised,” Dr. Markey explains. She adds, “it makes sense that during this neutropenic period there is more bacterial DNA in the bloodstream, because there is probably more translocation from the gut, however this had never been measured before.”
Using microbial cell-free DNA sequencing to identify bacterial taxa at the genus level, the researchers compared levels of microbial cell-free DNA pre-transplantation, during the neutropenic phase, to a post-recovery time point. The authors found increases in several bacterial taxa during the neutropenic phase, with the highest increases in Parabacteroides, Bacteroides, and Prevotella, and smaller increases in other taxa, including Veillonella. While none of these taxa are considered dangerous pathogens, they have been correlated with clinical outcomes after allogeneic hematopoietic cell transplantation. For example, a high relative abundance of the Prevotella genus has been associated with low abundance of immune-regulatory bacteria, whereas Veillonella has been linked to a higher risk of graft-versus-host disease-related mortality. Furthermore, the research team asked how pre-transplant conditioning intensity correlated with increased microbial cell-free DNA during the neutropenic period. Here they found that patients receiving a more intense conditioning regimen, had significantly increased levels of microbial cell-free DNA in the neutropenic period compared to the post-recovery timepoint. In contrast, the researchers did not observe a significant increase in microbial cell-free DNA between these time points in patients who received conditioning regimens with minimal toxicity. These results demonstrate that microbial cell-free DNA is a measurable attribute that can be detected in blood plasma, and that it “undergoes dynamic change dictated by the phase of transplantation, and appreciably differs from that seen in healthy people,” the authors note in the paper.
Collectively, this work “demonstrates that this new type of measurement can be performed, and is a potentially important biomarker in transplantation,” states Dr. Markey. This measurement may also be informative for analyzing the gut microbiome composition and a provide a functional measure of gut barrier integrity. Additionally, this research provides an important new way of studying the microbiome in humans more generally, as well as helping define novel microbial-host interactions, such as new ways the gut microbiome may signal to other parts of the body. Currently, Dr. Markey has active and ongoing projects with Karius and she looks forward to being able to share those exciting results soon.
This work was supported by the National Institutes of Health, the Tri-Institutional Stem Cell Initiative Award, the Lymphoma Foundation, the Susan and Peter Solomon Divisional Genomics Program, the Parker Institute for Cancer Immunotherapy at Memorial Sloan Kettering Cancer Center, the American Australian Association, the Haematology Society of Australia and New Zealand, the Royal Australian College of Physicians and the American Society of Hematology.
Fred Hutch/UW/Seattle Children’s Cancer Consortium member Dr. Kate Markey contributed to this work.
Blair LM, Akhund-Zade J, Katsamakis ZA, Smibert OC, Wolfe AE, Giardina P, Slingerland J, Bercovici S, Perales MA, Taur Y, van den Brink MRM, Peled JU, Markey KA. Circulating microbial cell-free DNA is increased during neutropenia after hematopoietic stem cell transplantation. Blood Adv. 2023.