In the early 20th century, research findings about the nature of microbes upended the traditional understanding of science and medicine. The acceptance of germ theory revolutionized medical care, and ultimately led to improved public health and longer life spans. A similar microbe-driven revolution may be afoot in oncology, as researchers uncover and untangle the relationships between the microbiome and cancer.

Between 2005 and 2015, the number of articles published about the microbiome and cancer increased by nearly 2000%.

 Researchers have found relationships between the oral microbiome and esophageal cancer; between the vaginal and uterine microbiomes and endometrial cancer; and between the gut microbiome and the efficacy of checkpoint inhibitors. And, although the research is young, the results so far suggest that the microbiome may, at least in part, help answer 2 of oncology’s most persistent questions: What causes cancer? Why do some people respond favorably to cancer treatment whereas others do not?

The microbiome is broadly defined as the “collective genomes of the microbes, composed of bacteria, bacteriophage, fungi, protozoa, and viruses, that live inside and on the human body.”

 Altogether, these bacteria and microorganisms are characterized as the microbiota, an “ensemble” that inhabits the epithelial barrier surfaces of the human body and influences a range of physiological functions, including metabolism and immunity.

Researchers from the University of North Carolina at Chapel Hill describe interactions between the microbiota and bodily systems within the context of the “Hallmarks of Cancer” that Douglas Hanahan, PhD, and Robert A. Weinberg, PhD, have elucidated (FIGURE).

 Fulbright and colleagues maintain that these interactions are so complex that the focus should be on the microbial metabolome, the collection of low-molecular-weight molecules in the cell, rather than on individual metabolites.

Figure. How Microbial Signals Modulate Hallmarks of Cancer

Two decades ago, it was nearly impossible to systematically study the human microbiome because most human bacteria cannot grow outside of the human body. Research initiatives such as the Human Microbiome Project, which the National Institutes of Health launched in 2007 as an extension of the Human Genome Project,

 have helped expand knowledge about the microbiota. Advances in genomic sequencing also have brought the microbiome into reach, scientifically speaking. Now, researchers can sequence the DNA of cells and identify the bacterial profile based on DNA formation. As a result, researchers have identified distinct microbiomes of different organs within the body, as well as differences between the microbiomes of individuals.

Ninety-nine percent of the microbial mass in the human body is found within the gastrointestinal (GI) tract,

 but the microbiota exerts systemic as well as local effects on physiological functions. In terms of carcinogenesis, the GI microbiota has been linked to cancers in the GI tract and in other organs (TABLE).

 Although GI activity of these microorganisms has been extensively studied, understanding is also growing about the microbiota in other epithelial barriers, such as the mouth and skin.

Scientists and physicians, for instance, have long been aware of a link between poor oral health and cancer. “We know that periodontal disease mysteriously is related to increased risk of upper gastrointestinal cancer,” said Jiyoung Ahn, PhD, an associate professor of Population Health and Environmental Medicine at NYU School of Medicine, and the associate director of Population Science at NYU Perlmutter Cancer Center in New York City. “But people didn’t have a good explanation why. Some people didn’t believe it at all.”

Research into the microbiome reveals that the same bacteria responsible for poor oral health may also be related to the development of cancer. A 2016 study published in Gut found that the presence and concentration of particular types of oral bacteria may increase or decrease the risk of developing cancers of the upper GI tract.

The oral pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are associated with an increased risk of pancreatic cancer, while a greater abundance of Fusobacteria and its genus Leptotrichia is associated with decreased risk. Study participants whose oral microbiomes included Porphyromonas gingivalis had a 59% greater risk of developing pancreatic cancer than those whose microbiomes did not contain the bacterium. Participants whose oral microbiomes contained Aggregatibacter actinomycetemcomitans were at least 50% more likely to develop the disease.

A 2017 study published in Cancer Research provides further evidence of a link between the oral microbiome and GI cancer.

 Researchers analyzed the oral microbes of more than 120,000 people and tracked their health over the next decade, noting which participants developed esophageal cancer and which did not. “We then retrospectively went through their samples to see whether the bacteria profile from when they were healthy carried bacteria related to the subsequent development of cancer,” said Ahn, who was a principal investigator.

The presence of Tannerella forsythia, a pathogen commonly linked to gum disease, was associated with a 21% increased risk of esophageal adenocarcinoma; an abundance of Porphyromonas gingivalis was linked to a higher risk of esophageal squamous cell carcinoma. Streptococcus and Neisseria were associated with a 24% decrease in risk of esophageal cancer.

Another study by the same team, published in JAMA Oncology online in January 2018, demonstrates a connection between the oral microbiome and head and neck cancers.

 It also shows that “carrying Neisseria abundance is associated with a decrease of head and neck cancer,” said Ahn. Neisseria, known to play a role in carcinogenic metabolism, was found in lower concentrations in heavy smokers, suggesting a biologic explanation for the link between smoking and the eventual development of cancer. “This is the first line of research to show how bacteria is related to the development of preventable cancers,” said Ahn.

Other studies have demonstrated a connection between the microbiome and colorectal cancer (CRC). High levels of Fusobacterium nucleatum, a microbe normally found in the mouth and associated with periodontal disease, are often present in CRC tissue,

 and is related to an increased risk of CRC.

 A 2017 study by researchers at Dana-Farber Cancer Institute suggests that the microbiome plays a role in cancer metastasis as well, because Fusobacterium was found both in CRC tumors and liver metastases.

 “Our findings suggest that the microbiome is a consistent feature of colorectal cancers, regardless of whether it is a primary cancer or a metastasis of that cancer,” said the study’s lead author, Susan Bullman, PhD, in a statement.

The microbiome makeup remained consistent even when researchers transplanted CRC tumor tissue into mouse models. In additon, when researchers treated the mice with antibiotics that target Fusobacterium, tumor cell proliferation and growth declined.

“The fact that tumor cell growth and proliferation slowed in mice treated with a targeted antibiotic may indicate that the bacteria help drive tumor cell growth, but that is very much an open question,” said Matthew L. Meyerson, MD, PhD, the study’s senior author and director of Cancer Genomics at DanaFarber, in a statement.

Several studies now suggest that the composition of patients’ gut microbiomes may explain differences in patients’ response to treatment. Irinotecan is a first-line treatment for patients with metastatic CRC, but can cause severe diarrhea in up to 40% of patients.

 A 2017 study published in npj Biofilms and Microbiomes links the gut microbiome to irinotecan toxicity; patients with “a high turnover microbiota metabotype” had an elevated risk for irinotecan-dependent adverse drug responses.

Similarly, work by Jennifer A. Wargo, MD, MMSc, an associate professor in the departments of Surgical Oncology and Genomic Medicine at The University of Texas MD Anderson Cancer Center, suggests that differences in individuals’ microbiomes may account for varying responses to immunotherapy. Wargo and her team examined oral, gut, and fecal microbiome samples of 112 patients with melanoma treated with anti—PD-1 immunotherapy through whole-genome sequencing of the microbiome, molecular profiling through whole-exome and RNA sequencing, and immune profiling through immunohistochemistry, flow cytometry, and cytokine analysis.

They then compared significant differences in the microbiomes of responders versus nonresponders.

“There is a relationship between the gut, the tumor microenvironment, and the bacteria within our intestine that can shape our immune response, and even shape how patients respond to therapy,” Wargo said in an interview with 

. “Patients who responded had a much higher diversity of bacteria within their gut and a different composition of the gut bacteria, with more Faecalibacterium, Ruminococcaceae, and Clostridiales. Patients who failed to respond to PD-1 blockade had a low diversity of the gut microbiome and a high abundance of Bacteroides,” Wargo said.

“We also found that both the diversity and the composition [of the microbiome] were associated with a prolonged progression-free survival. And that was an independent prognostic variable.” Additionally, investigators gathered fecal samples from responders to anti—PD-1 therapy and transplanted them into germ-free mice, according to findings presented at the 2017 Society for Immunotherapy of Cancer Annual Meeting by Vancheswaran Gopalakrishnan, PhD, a postdoctoral fellow in Wargo’s laboratory.

 They first evaluated the impact on the mice immune system, and then implanted a melanoma tumor.

The experiment’s results indicated that the mice that received the transplants had delayed tumor growth and a better response to therapy. Notably, the mice had a significantly higher presence of Faecalibacterium in stool samples. Additionally, they had enhanced CD8 immune infiltrates and fewer suppressive myeloid cells.

A phase II clinical trial currently in progress will further assess the link between the microbiome and response to immunotherapy. The open label, single-arm study (NCT02853318) is examining how patients with ovarian, fallopian tube, or peritoneal cancer respond to combination therapy with pembrolizumab (Keytruda) plus bevacizumab (Avastin) and cyclophosphamide.

Researchers also are analyzing blood, stool, vaginal, and skin microbial samples to see whether changes in microbiome composition may be related to response to treatment and patient outcomes. Study participants will be assessed 30 days after treatment, then every 3 months for a year, and every 12 weeks and 6 months thereafter. The hope is that such research will allow physicians to more precisely target treatment. “Should we be profiling the gut microbiome in patients going on immunotherapy? That is something to consider, but my hunch is yes,” said Wargo.

Since the microbiota plays such an extensive role in human physiology, it is not surprising that many factors would influence its composition, including an individual’s genetic profile, lifestyle, incidence of disease, and use of antibiotics.3 For example, research findings presented at the 2017 Genitourinary Cancers Symposium raised suspicions about the relationship between gut microbiota and antibiotics and their effect on responses to immune checkpoint blockade agents. Retrospective study results demonstrated that the use of antibiotics up to a month before treatment with a checkpoint inhibitor may decrease the efficacy of the immunotherapy agent.16

In patients with metastatic renal cell carcinoma, those who had received broad-spectrum antibiotics had a shorter median progression-free survival (PFS) rate of 2.3 versus 8.1 months for those who had not received antibiotics when treated with checkpoint inhibitor immunotherapy (

<.001). In a group of 62 patients who were treated with nivolumab (Opdivo) monotherapy, patients who had not taken antibiotics showed a higher PFS rate, which also achieved statistical significance (

 <.009). Overall survival also was significantly higher in patients who had not recently taken antibiotics (

“Immune-based therapies for cancer may have a complex interplay with the host’s microbiome,” said Sumanta K. Pal, MD, co-director of the Kidney Cancer Program at City of Hope in Duarte, California, commenting on the findings. “Antibiotics may influence the bacterial composition of our gut, and this could in turn impact how effective immune therapy is.”

Information about the microbiome may also eventually be used to assess an individual’s risk of certain cancers, and possibly enable healthcare providers to intervene and interrupt disease progression.

A 2016 study published in Genome Medicine examined the vaginal and uterine microbiomes of women with endometrial cancer, endometrial hyperplasia, and benign uterine conditions.

 Researchers discovered a structural microbiome shift in the cancer and hyperplasia cases; they also found differences in the bacterial composition of the vaginal microbiome in women with and without endometrial cancer. 

“These findings provide important insights into the etiology or manifestation of the disease with broad implications for biomarker development in the early detection of, and screening for, endometrial cancer,” said Marina R. WaltherAntónio, PhD, assistant professor of surgery at Mayo Clinic in Rochester, Minnesota, and lead author of the study, in a statement. “There’s a possibility that we can do a vaginal swab in a patient and have an indication as to whether or not the patient has cancer.”

Ahn’s research, which demonstrated a link between certain types of oral microbes and pancreatic cancer and head and neck cancer, could point the way toward early detection of cancers that currently claim many lives.

“Right now, there is no way we can detect pancreatic cancer early,” said Ahn, noting that a lack of early detection is one reason that the 5-year survival rate for pancreatic cancer is only 7%.

 “I’m hoping this research will help lead the way to detect this disease earlier in the future,” she said.

Ahn’s team plans to study whether the use of probiotic supplements can alter the oral microbiome and decrease the risk of upper GI cancers in humans. “Right now, we have identified specific bacteria related to cancer. The second step is designing a preventive trial for the future,” she said. “With antibiotics or probiotics, we can modify the bacteria. Then, we can do a randomized trial.”

Administration of probiotics has already been shown to slow disease in mouse models. A 2016 study by Finnish and Chinese researchers found that mice that were fed a microbial cocktail containing Lactobacillus rhamnosus GG, Escherichia coli Nissle, and heat-inactivated VSL#3, a pharmaceutical composed of multiple bacterial strains, experienced much less tumor growth than mice that were not fed the cocktail.

 Both groups of mice were injected with liver cancer; the tumors of mice that received the cocktail demonstrated a 40% reduction in tumor weight and size compared with tumors affecting the control group.

Administering antibiotics that target specific bacteria to mice with cancerous tumors has also been shown to slow disease. After researchers at Dana-Farber implanted CRC tumor tissue into mouse models, they treated the animals with metronidazole, an antibiotic that kills Fusobacterium, a microbe that is now known to be associated with colon cancer. In treated animals, tumor cell proliferation and overall tumor growth declined. These observations, investigators said, “argue for further investigation of antimicrobial interventions as a potential treatment for patients with Fusobacterium-associated colorectal cancer.”

It is also possible that research into the gut microbiome may reveal bacterial strains that could be developed into immunotherapies. Vedanta Biosciences and NYU Langone Medical Center are currently collaborating on the development of microbiome-derived immunotherapies for patients with cancer being treated with checkpoint inhibitors.

No one is yet sure what accounts for differences in individuals’ microbiomes, or even which changes are clinically significant. Findings from research conducted so far strongly suggest that the microbiome influences the immune system, and that such influence may contribute to the development of disease or the response to treatment. However, much work remains to be done.

“We are now in a discovery phase,” said Ahn. “In the next 5 or 10 years, I really want to know whether we can use this information to actually prevent some cancer.”

The microbiome and cancer: what’s all the fuss about? Cancer Research UK. http://www.cancerresearchuk.org/funding-for-researchers/research-features/2017-06-13-the-microbiome-and-cancer-whats-all-the-fuss-about. Published June 13, 2017. Accessed January 11, 2018.

Yang J. The Human Microbiome Project: extending the definition of what constitutes a human. National Human Genome Research Institute website. genome.gov/27549400/the-human-microbiome-project-extending-the-definition-of-what-constitutes-a-human/. Published July 16, 2012. Accessed January 11, 2018.

Roy S, Trinchieri G. Microbiota: a key orchestrator of cancer therapy.

2017;17(5):271-285. doi: 10.1038/nrc.2017.13.

Fulbright LE, Ellermann M, Arthur JC. The microbiome and the hallmarks of cancer. 

. 2017;13(9):e1006480. doi: 10.1371/journal.ppat.1006480.

Schwabe RF, Jobin C. The microbiome and cancer. Nat Rev Cancer. 2013;13(11):800—812. doi: 10.1038/nrc3610.

Fan X, Alekseyenko AV, Wu J, et al. Human oral microbiome and prospective risk for pancreatic cancer: a population-based nested case-control study. 

2018;67(1):120-127. doi: 10.1136/gutjnl-2016-312580.

Peters BA, Wu J, Pei Z, et al. Oral microbiome composition reflects prospective risk for esophageal cancers. 

 2017;77(23):6777-6787. doi: 10.1158/0008-5472.CAN-17-1296.

Hayes RB, Ahn J, Fan X, et al. Association of oral microbiome with risk for incident head and neck squamous cell cancer [published online January 11, 2018]. 

Castellarin M, Warren R, Freeman J, et al. Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. 

2012;22(2):299-306. doi: 10.1101/gr.126516.111.

Ahn J, Sinha R, Pei Z, et al. Human gut microbiome and risk for colorectal cancer. J Natl Cancer Inst. 2013;105(24):1907-1911. doi: 10.1093/jnci/djt300.

Bullman S, Pedamallu C, Sicinska E, et al. Analysis of fusobacterium persistence and antibiotic response in colorectal cancer.

2017;358(6369):1443-1448. doi: 10.1126/science.aal5240.

Gut microbiome may make chemo drug toxic to patients [press release]. Bronx, NY: Albert Einstein College of Medicine; November 1, 2017. http://www.einstein.yu.edu/news/releases/1277/gut-microbiome-may-make-chemo-drug-toxic-to-patients/. Accessed January 11, 2018.

Guthrie L, Gupta S, Daily J, Kelly L. Human microbiome signatures of differential colorectal cancer drug metabolism. 

 2017;3(1):27. doi: 10.1038/s41522-017-0034-1.

Gopalakrishnan V, Spencer CN, Nezi L, et al. Gut microbiome modulates response to anti—PD-1 immunotherapy in melanoma patients.

2018; 359(6371):97-103. doi: 10.1126/science.aan4236. 

Gopalakrishnan V, Spencer CN, Nezi L, et al. Gut microbiome modulates response to anti—PD-1 immunotherapy in melanoma patients. 

 2018; 359(6371):97-103. doi: 10.1126/science.aan4236. 

Derosa L, Routy B, Enot D, et al. Impact of antibiotics on outcome in patients with metastatic renal cell carcinoma treated with immune checkpoint inhibitors. Abstract presented at: 2017 Genitourinary Cancers Symposium; February 16-18, 2017; Orlando, FL. Abstract 462. https://meetinglibrary.asco.org/record/140186/abstract.

Walther-António MR, Chen J, Multinu F, et al. Potential contribution of the uterine microbiome in the development of endometrial cancer [published online November 25, 2016]. 

Prognosis. Hirshberg Foundation for Pancreatic Cancer Research website. http://pancreatic.org/pancreatic-cancer/about-the-pancreas/prognosis/. Accessed January 11, 2018.

Li J, Sung C, Lee N, et al. Probiotics modulated gut microbiota suppresses hepatocellular carcinoma growth in mice. Proc Natl Acad Sci U S A. 2016;113(9):E1306-E1315. doi: 10.1073/pnas.1518189113.

 2017;358(6369):1443-1448. doi: 10.1126/science.aal5240.

Vedanta Biosciences announces collaboration with the NYU Langone Medical Center to develop microbiome-derived immunotherapies for cancer [press release]. Cambridge, MA: Business Wire; August 11, 2016. https://www.businesswire.com/news/home/20160810006199/en/Vedanta-Biosciences-Announces-Collaboration-NYU-Langone-Medica. Accessed January 11, 2018.

Articles

Researchers have found relationships between the oral microbiome and esophageal cancer; between the vaginal and uterine microbiomes and endometrial cancer; and between the gut microbiome and the efficacy of checkpoint inhibitors.

Microbiome Emerges as a Hot Topic in Cancer Research

Checkpoint blockade immunotherapy has been hailed as a significant advance in anticancer treatment. Yet only a subset of patients experience long-term cancer remission as a result of these therapies, because a significant number of those who initially respond eventually develop resistance.

Why do some patients and not others develop resistance? How can primary and acquired resistance to immunotherapy be overcome? These questions are drawing intense interest from the research community. As investigators seek to unravel the mechanisms of acquired resistance, they also hope to shed light on the components of primary resistance.

The biological and genetic processes that may contribute to relapses were a lively area of inquiry during the 32nd Annual Meeting of the Society for Immunotherapy of Cancer (SITC), held November 8-12 in National Harbor, Maryland. Investigators reported findings about a variety of diagnostic tools to monitor immune responses throughout the treatment cycle in preclinical settings, and about the results of explorations into novel therapies and combinations.

"There’s very little so far that we understand about mechanisms of acquired resistance to immune checkpoint inhibitors,” said Katerina Politi, PhD, an associate professor in Yale University’s Department of Pathology and the Yale Cancer Center, in an interview with OncologyLive®.

Since these therapies are a relatively new modality in clinical practice, the prevalence of acquired resistance among patients treated with these agents is unclear. “I’m not sure we know how many patients are affected by acquired resistance,” Politi said. “It might be different for different cancers.”

Politi, who served as a co-chair of a session on immunotherapy resistance at the SITC conference, has used genetically engineered mouse models to study genetic changes in lung cancer that cause resistance to targeted therapies; now, she is exploring cellular changes that may cause or contribute to resistance to immunotherapy.

Politi’s team is studying tumor specimens before treatment is administered and after resistance develops to determine differences at the DNA or RNA level, and in proteins and immune cells. They are using patient-derived tumor xenografts to understand the impact of the alterations they identify. “We want to be able to validate them functionally,” she said.

Antoni Ribas, MD, PhD, a leading authority on immunotherapy and a Giants of Cancer Care® awardwinner, and colleagues have defined 3 types of resistance to immunotherapy that have been manifested in clinical scenarios: (1) primary resistance, in which the cancer fails to respond to therapy; (2) adaptive immune resistance, in which cancer cells are recognized by the immune system but develop ways to protect themselves from attack (may be primary resistance, mixed response, or acquired resistance); and (3) acquired resistance, in which a cancer responds to immunotherapy but progresses after a period of time.

The mechanisms that cause these responses are dynamic; they can evolve in response to environmental and genetic factors and to treatment interventions including surgery, chemotherapy, radiation therapy, and immunotherapy. Notably, they may exist prior to the administration of immunotherapy.

As of November 12, the FDA had approved 6 checkpoint blockade immunotherapy agents: ipilimumab (Yervoy), an anti—CTLA-4 antibody; nivolumab (Opdivo) and pembrolizumab (Keytruda), both anti–PD-1 antibodies; and atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi), all anti–PD-L1 antibodies. These agents were approved based on clinical trials that demonstrated overall or objective response rates ranging from approximately 11% to nearly 70%; in many of the studies, the median duration of response was not reached (Table 1).

able. Checkpoint Immunotherapy Response Rates and Duration in Pivotal Trials

Long-term survival rates for patients with melanoma or non—small cell lung cancer (NSCLC) who were treated with anti–PD-1 inhibitors in early clinical trials exceed what would have been expected with standard therapies in these disease settings. Forty percent of patients with advanced melanoma who started taking pembrolizumab during the clinical trial that led to its initial approval were alive after 3 years, researchers reported at the 2016 American Association of Clinical Oncology Annual Meeting.

The results from the KEYNOTE-001 study showed that 15% of the patients experienced complete remissions according to immune-related criteria (irRC) after taking the PD-1 inhibitor and that nearly 90% of these responders remain in remission. The median overall survival (OS) was 24.2 months.

At the 2017 American Association for Cancer Research Annual Meeting in April, investigators presented updated findings from CA209-003 that they described as the first report of long-term OS rates in patients with metastatic NSCLC treated with an immune checkpoint inhibitor. After Katerina Politi, PhD approximately 5 years of follow-up (minimum follow-up, 58.25 months) the OS rates for patients treated with nivolumab were 42% at 1 year, 24% at 2 years, 18% at 3 years, and 16% at 5 years.

Researchers said those rates were about 4 times higher than the survival that would have been expected with docetaxel.

Nevertheless, acquired resistance has manifested itself in a significant subset. “It is becoming clear that approximately one-fourth to one-third of patients with metastatic melanoma who have objective responses to checkpoint blockade therapy with anti—CTLA-4 or anti–PD-1 will relapse over time, even despite receiving continued therapy,” Ribas and colleagues wrote.

During a presentation at the SITC conference, Valsamo Anagnostou, MD, PhD, included an analysis of progression after response among patients with NSCLC who received checkpoint blockade immunotherapy during 4 clinical trials (Table 2).5 The rates of progression among responders ranged from 19.4% to 48%. Notably, initial response and later progression rates were markedly lower among patients who had received docetaxel, the standard comparator in each trial.

Table 2. Progression Among Responders in Selected NSCLC Trials

Strategies to improve primary response rates and the duration of responses to checkpoint blockage immunotherapies are needed, said Anagnostou, an oncology instructor and researcher at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University in Baltimore, Maryland. “Despite the clinical benefit, a fraction of patients respond to these therapies and, among the patients who do respond, the majority of them develop acquired resistance. There is therefore an unmet need for understanding mechanisms of response and resistance to these therapies,” she said.

Appropriately selected patients who do not experience tumor shrinkage or regression after initial treatment with an immunotherapy exhibit primary resistance. “In that case, it’s most likely that the cancer is not immunogenic enough to have induced an immune response, so we cannot use its checkpoints to turn it on,” said Ribas, director of the Tumor Immunology Program at the Jonsson Comprehensive Cancer Center at UCLA, in an interview. Factors intrinsic and extrinsic to the tumor cell have been identified in ongoing studies as contributing to primary and adaptive resistance mechanisms to immunotherapy.

Intrinsic factors include an absence of tumor antigens and genetic factors that prevent immune cell infiltration, while extrinsic factors may be found in the tumor microenvironment; these include myeloid-derived suppressor cells and inhibitory immune checkpoints.1 In acquired resistance, possible mechanisms that have been explored include the loss of T-cell function, lack of T-cell recognition by downregulation of tumor antigen presentation, and development of escape mutation variants in the cancer.1 Ribas and colleagues postulate that each of these mechanism may promote acquired resistance to checkpoint blockade antibodies and to adoptive cell transfer therapies, a category that includes chimeric antigen receptor (CAR) and T-cell receptor therapies.

Researchers are discovering possible overlap of mechanisms of primary and acquired resistance to immunotherapy. “The studies analyzing resistance to immunotherapy identified several genetic alterations that are shared between tumors that are primary resistant and those that acquire resistance under therapy,” said Annette Paschen, MD, an associate professor in the Department of Dermatology at the University Duisburg-Essen and German Cancer Consortium, in an email interview. She also discussed resistance mechanisms at the SITC conference.

One important mechanism of acquired resistance appears to be genomic changes in T-cell receptor clonality that prevent continued recognition of tumor antigens after an initial response, according to research Anagnostou and colleagues conducted at Johns Hopkins.

Investigators analyzed the genomic evolution of 4 patients with NSCLC who had initially responded to anti—PD-1 or anti–PD-1/CTLA-4 therapy and then developed resistance.6 To do so, they performed genome-wide sequencing of protein coding genes and T-cell receptor clonotype analysis on paired tumor specimens, followed by evaluation of eliminated neoantigens in peripheral blood T cells. They also analyzed neoantigens in 1 patient with head and neck squamous cell carcinoma who had been treated with a combination of PD-1 and KIR blockade therapy.

The investigators found that NSCLC tumors that acquired resistance shed between 7 and 18 mutation-associated neoantigens (MANAs) through the elimination of tumor subclones or through the chromosomal loss of truncal alterations.6 Some of the MANAs that were eliminated were in genes that encode peptides recognized by T cells in peripheral blood. This “therapy-induced immune editing of MANAs” appeared to allow the tumor to avoid attack, the investigators wrote, although they noted that larger studies would have to be conducted at various times of relapse and response to confirm these findings.

The focus on neoantigen changes during therapy is consistent with prior studies that have described tumor mutational load as an indicator of response to checkpoint blockade antibodies, Anagnostou noted during her SITC presentation.

“Responses are expected in tumors with a very high mutational load such as mismatch repair— deficient colon cancers as well as in tumors with known environmental exposures such as lung cancer and melanoma, whereas responses are less expected in tumors with a low mutational load such as [pediatric] neuroblastoma,” she said. The next step for the research team was to determine whether changes in the neoantigen landscape could be accurately assessed through circulating cell-free DNA (cfDNA) isolated from the plasma of patients at any stage in the treatment timeline. Investigators used a method called targeted error correction sequencing, or TEC-Seq, that was developed at Johns Hopkins to evaluate sequencing changes in cfDNA.

By comparing cfDNA and TCR dynamics during and after treatment, investigators delineated patterns of response. “For a patient with an ongoing response, there is early clearance of cfDNA [by week 4] and evidence of expansion of TCR clones in the circulation,” said Anagnostou. “For a patient with primary resistance, there is no clearance of cfDNA and no change in productive frequencies.” For patients who initially respond but develop acquired resistance, there is a clearance of cfDNA at the time of response followed by outgrowth at the time of resistance. “There is some expansion of TCR clones at time of response followed by a decrease to pretreatment levels at the time of therapeutic resistance,” said Anagnostou.

She said the findings have implications for the development of immunotherapies that target multiple clonal neoantigens to prevent immune escape. Additionally, she noted that liquid biopsies should be incorporated into the design of future clinical trials.

The evidence thus far suggests that tumors that develop resistance to checkpoint immunotherapy are highly genetically similar to tumor samples taken prior to the development of resistance. A study published in Clinical Cancer Research used genetic and immunologic analysis to study tumor evolution over 5 years in a patient with cutaneous melanoma.

The 60-year-old male patient received anti— PD-1 therapy from the time of metastasis to death; at that time, some metastases were responding to therapy, while others were progressing. Researchers conducted whole-genome sequencing on 26 responsive and nonresponsive tumor specimens (4 premortem, 22 postmortem), but they found few genetic differences.

“At the genomic level, the tumors did not display a significant number of genetic subclones with respect to the cancer cell fraction,” wrote Ascierto and collaborators. “…We found remarkable genetic similarities among regressive/progressing cutaneous metastases, indicating that all were derived from a single subclone that had not diverged significantly.” Investigators concluded that the patient’s disease was driven by a functional loss of NF1, which has been associated in earlier studies with RAS activation in melanoma cells and with MAPK pathway reactivation in resistance to BRAF inhibitors.

Notably, they did not find significant differences in progressing versus regressing metastases either in PD-L1 expression on tumor cells or infiltrating immune cells or in CD8 T-cell densities in intratumoral or peritumoral locations. However, global gene expression profiling revealed distinct gene signatures in progressing and regressing tissue samples. The most differentially expressed gene, LAMA3, a subunit of laminin-5, was also overexpressed at the protein level in progressing metastases. Laminin-5 is associated with epithelial-to-mesenchymal transition and poor prognosis in lung cancer and with the development of aggressive melanoma in culture.

Interruptions in the Interferon-Gamma Pathway

The interferon-gamma (IFNγ) signaling pathway is a critical part of the immune response, and multiple research studies now suggest that defects along the pathway play a key role in immunotherapy resistance. Notably, JAK1/2 mutations, which affect IFNγ signaling, have been found in melanoma samples exhibiting primary resistance, as well as in tumors that developed acquired resistance.

A study published in the New England Journal of Medicine analyzed paired tumor samples of 4 patients with metastatic melanoma who relapsed while receiving pembrolizumab. (The mean time to relapse was 624 days, with a range of 419-888 days). The researchers compared tissue samples from baseline and relapsing lesions through whole-exome sequencing. In the relapse samples of 2 patients, they identified acquired loss-of-function mutations in JAK1 and JAK2, resulting in a lack of response to IFNγ. A truncating mutation in B2M, which encodes the antigen-presenting protein beta-2-microglobulin, was identified in a third patient.

Paschen and colleagues analyzed exome data derived from the cell lines of patients with metastatic melanoma to demonstrate a “genetic evolution” of resistance to IFNγ. 9 By screening cultured melanoma cell lines for genes in the IFNγ signaling pathway, investigators found mutations in JAK1, JAK2, and STAT1 that undermined the immune response and resulted in T-cell—resistant human leukocyte antigen (HLA) class I–negative lesions.

“Mutations abrogating IFNγ signaling in tumor cells protect from cytokine-induced cell death and cell cycle arrest, and allow the tumor cells to maintain an HLA class I—low phenotype, which can protect against T-cell recognition. PD-L1 expression is no longer inducible by IFNγ, which makes the tumor cells resistant to anti–PD-1 therapy,” Paschen said in the interview.

Additionally, the study suggested that JAK mutations may occur early on—or even prior to&mdash;the development of resistance. Paschen and colleagues identified 10 JAK1/2 mutations in 59 tissue samples from patients undergoing anti-PD1 therapy; investigators also identified mutations in IFN signaling pathway genes in 10 of 110 pretreatment biopsies from patients who went on to receive anti—CTLA-4 therapy.

Although the authors “could not detect significant differences in responses to anti—PD1 or anti-CTLA-4 treatment between patients with and without mutations,” they noted that 2 of 3 patients with “clearly inactivating mutations” experienced cancer progression while undergoing anti–PD-1 immunotherapy. The third patient showed a partial response to treatment. Importantly, investigators determined that melanoma cells with JAK1/2 mutations “progress to a ‘higher level’ of immunotherapy resistance,” evolving from IFNγ-resistant melanoma into HLA class I–negative lesions with complete resistance to CD8-positive T-cells.

Novel research into mechanisms of resistance to immunotherapies presented at SITC included findings about the dual role of type II IFNγ signaling in tumor cells and in the tumor microenvironment. Investigators from the University of Chicago explored the role of mutations in the type II interferon pathway affecting IFNγR2 and JAK1 activity.10 They identified these mutations for study through the use of CRISPR gene-editing technology; they then generated B16.SIY cells that selectively lacked IFNγR2 or JAK1 and implanted these cells into mice.

Although these aberrations are associated with resistance to immunotherapy in vitro, the mutant tumors were controlled better when implanted into mice. The investigators repeated the experiment with multiple IFNγR2- and JAK1-deleted cell lines using different guide-RNAs and with another tumor cell line with similar results. The team also reintroduced IFNγR2 and found that tumor growth was restored and antigen-specific CD8-positive T cells increased in the tumor microenvironment.

The investigators theorize that IFNγ produced by CD8-positive T cells in the tumor microenvironment “might have a dominant negative effect through upregulation of inhibitory factors such as PD-L1 on tumor cells,” they wrote in their conference abstract. They observed that tumor cells that were positive for IFNγR2 and JAK1 in vivo reduced expression of PD-L1 and IDO, a tumor suppressor checkpoint, compared with wild-type tumors and that transduction to express PD-L1 restored the ability of IFNγR2-negative tumors to subvert the host immune response.

They concluded that whether IFNγ on tumor cells has a positive or negative impact on tumor control “may depend on whether inhibitory factors like PD-L1 are dominantly expressed and functional through tumor cells versus host immune cells.”

As researchers learn more about the underlying causes of immunotherapy resistance, they are also working to develop strategies to overcome resistance.

“The data suggest that if we were better at treating cancer up-front, there would be less acquired resistance,” Ribas said. “I think an important strategy will be to kill more cancer cells with more immunotherapy. If there are no cancer cells left, none can become resistant.”

Exactly how to do so is a question that is being studied. “That [strategy] may be different from one person to another,” Ribas said. In their paper, Ribas and colleagues cited a broad range of strategies being considered.

These include partnering a checkpoint blockade antibody with other inhibitory and stimulatory immune-targeting agents, emerging strategies such as adoptive T-cell therapy, and standard chemotherapy or radiotherapy. “Several combination therapies are now being studied in clinical trials,” Paschen said, noting that results so far indicate that “it seems to be more efficient to attack the tumor from ‘multiple sides’ to prevent or overcome resistance.”

As more is learned about genetic changes related to resistance, “it might be possible to identify the patient population that has an enhanced risk of developing JAK1/2 deficiency based on their chromosomal alterations,” Paschen said. Ultimately, “it should become routine to take biopsies from patient lesions in order to define the molecular characteristics of tumor cells and the cellular composition of the tumor microenvironment,” she noted.

“Based on this knowledge, a personalized treatment strategy could then be developed,” Paschen said. “If a tumor shows an irreversible loss of the surface expression of HLA class I antigen, then therapeutic approaches that are dependent on the activity of CD8-positive T-cells—recognizing tumor antigens are no longer applicable. But these tumor cells still could be killed by CAR-modified CD8-positive T cells, and therapeutic approaches based on CD4-positive T-cells recognizing tumor antigens in the context of HLA class II molecules could be followed.”

For now, clinicians can help by obtaining biopsy samples before, during ,and after treatment. These samples will help researchers clarify mechanisms of resistance and advance understanding of acquired resistance.

“We’re going to need a lot more data to be able to really tackle and understand the frequency of different mechanisms of resistance, and then [understand]how to treat them,” Ribas said.

Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell. 2017;168(4);707- 723. doi:10.1016/j.cell.2017.01.017.

Drugs@FDA: FDA approved drug products. FDA website. accessdata.fda.gov/scripts/cder/daf. Accessed October 29, 2017

Robert C, Ribas A, Hamid O, et al. 3-year overall survival for patients with advanced melanoma treated with pembrolizumab in KEYNOTE-001. J Clin Oncol . 2016;34(suppl; abstr 9503). meetinglibrary. asco.org/record/123428/abstract.

Brahmer JR, Horn L, Jackman D, et al. Five-year follow-up from the CA209-003 study of nivolumab in previously treated advanced non-small cell lung cancer (NSCLC): clinical characteristics of long-term survivors. Presented at: 2017 American Association for Cancer Research Annual Meeting; April 1-5, 2017; Washington, DC. Abstract CT077. cancerres.aacrjournals.org/ content/77/13_Supplement/CT077

Anagnostou V. Evolution of neoantigen landscapes. Presented at: 32nd Annual Meeting of the Society for Immunotherapy of Cancer; November 9-12, 2017; National Harbor, MD.

Anagnostou V, Smith KN, Forde PM, et al. (2016). Evolution of neoantigen landscape during immune checkpoint blockade in non—small cell lung cancer. Cancer Discov. 2016;7(3):264-276. doi:10.1158/2159-8290.cd-16-0828.

Ascierto ML, Makohon-Moore A, Lipson EJ, et al. Transcriptional mechanisms of resistance to anti—PD-1 therapy. Clin Cancer Res. 2017;23(12): 3168-3180. doi:10.1158/1078-0432.ccr-17-0270.

Zaretsky JM, Garcia-Diaz A, Shin DS, et al. Mutations associated with acquired resistance to PD-1 blockade in melanoma. N Engl J Med. 2016;375(9): 819-829. doi: 10.1056/NEJMoa1604958.

Sucker A, Zhao F, Pieper N, et al. Acquired IFNγ resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions. Nat Commun. 2017;31:15440. doi: 10.1038/ncomms15440.

. Williams J, Li S, Gajewski T, Huang H. A dual and opposing role for tumor intrinsic type-II interferon sensing in tumor establishment and acquired resistance. 32nd Annual Meeting and Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2017): Part One. J Immunother Cancer. 2017;5(suppl 2):86. Abstract O29. doi: 10.1186/s40425-017-0289-3.

Jennifer L.W. Fink

Articles

Microbiome Emerges as a Hot Topic in Cancer Research

Immunotherapy Resistance Remains a Puzzle