EBV-Cancer Connection

Luftig’s lab at Duke has primarily focused on research into EBV-initiated B-cell lymphomas, including diffuse large B-cell lymphomas in the immune suppressed, such as transplant patients and HIV-infected individuals. 

He’s particularly excited, however, about having recently extended his EBV work, with the aid of Duke pilot-grant funding, to EBV-positive gastric cancer, which accounts for 10 percent of stomach cancer cases. Nasopharyngeal carcinoma, EBV-positive in 90 percent of cases, and gastric cancer are the only two epithelial cancers shown to be associated with EBV infection.

Microbial infection accounts for nearly 20 percent of all human cancers. The human papillomavirus (HPV) — a risk factor for cervical cancer and some cancers of the penis, anus, vagina, vulva, mouth, and throat — and the less widely-known Epstein-Barr virus are the most prevalent among these. 

Unlike HPV, there are no vaccines against EBV, though there’s recently been an effort to explore that possibility. 

There are also no treatments for EBV, which latently infects more than 90 percent of the world’s adult population and is responsible for one to two percent of all cancers worldwide.

People are typically infected with EBV between the ages of four and seven in the developed world. Primary infection is not usually associated with disease, Luftig explained, and the infection leads to the development of immunity (T cells and B cells) for life, though one can be re-infected with other strains. 

By high school age, 50 to 70 percent of people in the developed world will have been infected with EBV, he said. Those who aren’t infected until adolescence or post-adolescence, have a significant risk of developing infectious mononucleosis (mono) with their first EBV infection, which, studies have shown, may increase the lifetime risk of Hodgkin lymphoma. 

Like other closely related viruses, such as those that cause chicken pox and cold sores, an EBV infection lasts for the rest of the person’s life, although the virus generally remains in a dormant state. 

Only under certain conditions, and in rare cases, does EBV cause cancer. 

In most cases, once EBV infects the B cells, the antibody-producing cells of the immune system, the T cells will kill the majority of them. Post-infection the virus goes dormant, such that only one in a million B cells are EBV positive; i.e. the viral genomes are maintained in those EBV-positive (infected) B cells. 

The virus, over a lifetime, will reactivate sporadically and replicate in the cells of the mouth. It’s spread by saliva to uninfected or already-infected adults. 

About one to two percent of the T cells in the body are programmed to recognize EBV antigens when the virus reactivates in new B cells and will stop the virus in its tracks. If one is immune-suppressed, however, then their T cells might not recognize and kill those EBV-infected B cells. In that case, they’ll become immortalized or grow continuously.

The EBV-infected B cells express eight highly immunogenic viral proteins and many non-coding RNAs. It is these viral gene products that are believed to drive B-cell proliferation and suppress the cells’ natural apoptotic response (programmed cell death), leading to lymphoma. There are therapies, BH3 mimetics, that target the cell’s anti-apoptotic proteins that the virus regulates.

Mission Possible?

EBV’s role in blood cancers has been long studied and is well understood. The virus’ link to lymphoma — the first known linkage of any virus with human cancer — was discovered in 1964 in the biopsy of a Burkitt lymphoma tumor. 

EBV’s role in the epithelial cancers of nasopharyngeal carcinoma (first detected in 1978) and gastric cancer (first detected in 1990) has been less well studied and remains more of a mystery. 

Luftig and Patrick Tan, MD, Ph.D., Professor of Cancer and Stem Cell Biology at the Duke-National University Singapore who specializes in the genetic and genomic changes in gastric cancer, were awarded a $150,000 cancer and the environment pilot grant funded by the Duke Cancer Institute and the Nicholas School for the Environment in 2015-2016 and, more recently, a pilot grant of $200,000 in 2017 by Duke and Duke-NUS to help crack that code. 

Their mission — is to develop and characterize preclinical models for EBV-positive gastric cancer, a distinct subtype of gastric cancer that displays unique molecular, epigenetic, and clinicopathological features. The lack of such models, Luftig said, has thus far limited the development of therapies for this gastric cancer subtype. 

With a greater prevalence of EBV-positive cancers in Southeast Asia (primarily nasopharyngeal carcinoma and EBV-positive gastric cancer), Tan’s lab has had unique access to hundreds of banked gastric cancer cases. 

Together with co-Investigators David Hsu, MD, Ph.D., and Katherine Garman, MD, the research team has been using EBV-positive gastric cancer cell lines grown as xenografts in mice and is planning to develop 3D-based gastric organoid cultures. They are hoping to use new genome-wide screening technologies coupled with these unique cell cultures and animal models to understand the molecular basis of EBV-induced gastric cancer.

The Duke research team has so far found, after sequencing the DNA of gastric cancer, and following up with pathology, that the genomes of the EBV-positive gastric tumor cells are the most heavily methylated of any tumor cells. These results are consistent with recent studies by the NIH-supported Cancer Genome Atlas.

They're now studying whether the virus’ promotion of genome methylation in the host — turning off the tumor suppressors — is relevant for gastric cancer pathogenesis. 

“EBV-positive gastric cancers …harbor recurrent mutations activating PI3Kα, silencing ARID1A and amplifying PD-L1,” wrote Luftig and his lab research technician Lyla Stanland, in the October 2018 issue of Future Virology. “EBV-positive gastric cancers may be good candidates tumors for PD-1 blockade due to an inflammatory microenvironment dominated by IFN-γ signaling and upregulation of PD-L1 in tumor cells.” 

A recent prospective phase 2 clinical trial (not at Duke) came to a similar conclusion, finding that patients with EBV-positive metastatic gastric cancer were especially responsive to the PD-1 checkpoint inhibitor pembrolizumab. 

“This really got the attention of the clinical community,” said Luftig. 

Patients with metastatic gastric cancer at Duke who test positive for EBV can potentially be considered for a checkpoint inhibitor such as pembrolizumab if other treatments have failed. Given these exciting clinical findings, Hsu said, researchers are now seeking to harness the immune response against EBV as a means to increase the response to checkpoint inhibitors. 

Luftig said that long-term, the team is well-positioned to apply for follow-on funding to continue their EBV research and test unique therapeutic approaches to EBV-positive epithelial and blood cancers. 

“Ultimately, a detailed understanding of the virus/host interaction and regulation of EBV oncogenes will allow us to identify specific pathways for therapeutic intervention and better treat cancers that rely on these viral products for survival,” explained Luftig, “and possibly, more broadly, teach us about malignancies which rely on similar pathways for their proliferation, survival, or self-renewal.”