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Monday, October 26, 2020

The Role of Fecal Microbial Transplants in Pancreatic Cancer

By Warren Froelich

In the first clinical trial of its kind, a small group of patients with pancreatic ductal adenoma carcinoma (PDAC) will receive fecal microbial transplants (FMT) to test whether this novel approach might alter the disease's unique tumor microbiome, leaving recalcitrant tumors open to immunotherapy.

As described during the September 29-30 virtual conference on pancreatic cancer held by the American Association for Cancer Research (AACR), tumor tissue from 10 PDAC patients scheduled for surgery will be examined prior to transplant, and then re-examined 1 year after surgery to see if the bacterial makeup of the patients' tumor microenvironment has been changed through the transplants.

"Within a year (after FMT), if there is a proto-tumorigenic microenvironment associated with PDAC, by reversing that we might be able to reverse immune suppression," said Florencia McAllister, MD, Professor in the Department of Clinical Cancer Prevention at MD Anderson Cancer Center, who outlined the trial, along with its pre-clinical underpinnings, during a "Rising Star" keynote address at the meeting.

Research Overview

In brief, the foundation for the trial rests on several years of pre-clinical study, including recent tests showing that FMT from healthy patients or PDAC survivors resulted in decreased immunosuppression and decreased tumor growth in mice implanted with human pancreatic tumors. This suggested that PDAC patients have gut bacteria which may be inducing and sustaining PDAC immunosuppression.

"There is crosstalk between the gut microbiome and pancreatic tumor microbiome, which provides the basis for FMT studies," McAllister said in an interview.

The clinical trial will include adult patients with resectable PDAC, using FMT from healthy donors delivered through both colonoscopy and oral pills. Similar trials are ongoing for other cancers, but this is the first for pancreatic cancer.

"By normalizing the gut microbiome, we aim to affect tumor microbiome and systemic as well as potentially local immune responses against pancreatic cancer," McAllister noted. "Initially, we want to prove that this is doable and that changes are sustainable, but perhaps we can already see some beneficial signals which will enlighten a second study that will center on efficacy."

Pancreatic cancer is one of the most aggressive malignancies with a 5-year survival of about 9 percent, with most available therapies, including immunotherapy, showing limited efficacy. However, a minor subset of patients survives beyond 5 years post-surgery, leading to the unanswered question of what's different about these patients.

After ruling out potential genetic differences, some researchers turned to an examination of microbial host factors as a possible answer to this enigma. For several years, studies have shown the presence of bacteria in pancreatic tumors, but no one knew what role, if any, these microbes played in tumor progression, prognosis or survival.

Study Details

To find out, McAllister and colleagues initially compared bacterial DNA found in long-term survivors with short-term survivors, using two independent cohorts at MD Anderson and Johns Hopkins Hospital. Long-term survival in the MD Anderson cohort of 22 patients was 10 years, and 1.6 years for 21 short-term survivors; long-term survivors in the Johns Hopkins validation cohort of 15 patients was 10 years, while 10 others survived less than 5 years.

Using 16S rRNA sequencing, the team discovered that tumors in long-term survivors had a greater microbial diversity across both cohorts. Researchers also found marked differences in the types of bacteria, with long-term survivors in both cohorts showing a relative abundance of Pseudoxanthomonas, Saccharopolyspora, and Streptomyces. The presence of these three bacterial types, as well as the species Bacillus clausii, predicted better outcomes for patients in both cohorts, suggesting they might provide a "microbial signature" to predict long-term versus short-term survival.

Immunofluorescence staining also found greater densities of CD8+T cells in the tumors of long-term survivors compared to short-term survivors, which meshed well with the finding of higher concentrations of the three bacterial species found in long-term survivors.

"These findings suggest that the tumor microbiome diversity and the presence of these three genus in the tumor may contribute to the anti-tumor immune response by favoring recruitment and activation of CD8+ T cells," the researchers wrote in the August 8, 2019 issue of the journal Cell (DOI:https://doi.org/10.1016/j.cell.2019.07.008)

"However, we have not yet proven that those bacteria indeed had a functional importance in patient prognosis," McAllister cautioned. "Further studies are needed to point out to specific bacteria species from a functional perspective."

Next, the MD Anderson team wanted to find out if the tumor microenvironment could be altered. Again, using 16S rRNA sequencing, they analyzed the stool, the tumor, and normal adjacent tissue of three patients undergoing Whipple surgery. About 20-25 percent of the tumor bacteria was similar to the bacterial species found in their stool, but absent in adjacent tissue, suggesting bacteria in the gut can colonize pancreatic tumors.

"This was interesting because the existence of crosstalk between the gut and tumor suggests that if you make changes within the gut, that may actually reflect within the tumor," McAllister said.

Following this step, McAllister and colleagues performed stool transplants into mice from patients who had advanced pancreatic cancer, patients who had survived more than 5 years and had no evidence of disease, and healthy controls.

Five weeks after tumor implementation, mice that received FMT from patients with advanced disease had much larger tumors than those that received FMT from long-term survivors (70% smaller average size) or healthy controls (50% smaller average size). The findings suggested that gut and tumor bacteria from long-term survivors of PDAC may have a protective effect against tumors.

Mice transplanted with stools from long-term survivors with no evidence of disease (LTS-NED) were given antibiotics post-FMT and were compared to mice who didn't receive any antibiotics. Those given antibiotics had larger tumors than untreated mice, demonstrating that reduction in the bacterial tumor environment can weaken the impact of FMT from the LTS-NED patients, further validating the central role of bacteria in the disease.

Flow cytometry analysis demonstrated that tumors from mice receiving FMT from LTS-NED had significantly higher numbers of CD8+ T cells, as well as activated T cells versus those with stools transferred from short-term or healthy control donors. When antibodies were given to mice transplanted with LTS-NED stools, the researchers found that CD8+T cells were depleted, effectively blocking the anti-tumoral effect induced by the transplant.

"Together, these data strongly suggest that the gut microbiome can colonize pancreatic tumors, modify its overall tumoral bacterial composition, and modulate immune function to ultimately affect the natural history and survival," the researchers wrote.

Updated Findings

During the AACR meeting, the MD Anderson team presented new findings showing that interleukin-17 (IL-17), a potent proinflammatory cytokine, plays a significant role in triggering and sustaining immunosuppression in patients with pancreatic cancer.

Using immunofluorescence staining in mice implanted with pancreatic tumor cells, the team found that IL-17 signaling triggered neutrophils into the tumor microenvironment, while excluding CD8+T cells. Further, the neutrophils now enmeshed in the tumor environment formed extracellular traps (NETs), which contributes to PDAC immunosuppression. Neutrophils, essential components of the immune system found in the blood, are one of the first responders of inflammatory cells to migrate to the site of inflammation.

When IL-17 was blocked by PD-1 antibodies, IL-17 signaling and subsequent tumor growth was reduced.

To evaluate its clinical relevance, the team analyzed sera from healthy controls and PDAC patients and measured their effect on NET formation. PDAC sera resulted in higher NET formation as well as lower NET degradation.

"Overall, what we see is that IL-17 signaling in pancreatic tumor cancer cells releases factors that recruit neutrophils and these neutrophils go on to form NETs which excluded CD8+T cells," said Vidhi Chandra, a PhD candidate in Dr. McAllister's lab who presented these results during a poster session at the AACR meeting.

"When you block IL-17 signaling, the signaling goes down and CD8+ T cells can move closer to the tumors and when you further combine this with PD-1 antibodies, you get reduction in tumor growth."

Added McAllister: "If PDAC-associated microbiome increases IL-17, then either reversing the process with either healthy FMT, antibiotics or anti-IL-17 may have similar efficacy."

Warren Froelich is a contributing writer.