- Email: [email protected]
Harnessing the Microbiome for Pancreatic Cancer Immunotherapy
Please cite this article in press as: Vitiello et al., Harnessing the Microbiome for Pancreatic Cancer Immunotherapy, Trends in Cancer (2019), https://doi.org/10.1016/j.trecan.2019.10.005
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Opinion
Harnessing the Microbiome for Pancreatic Cancer Immunotherapy Gerardo A. Vitiello,1 Deirdre J. Cohen,2 and George Miller1,* Late-stage pancreatic cancer harbors a fibrotic and immune-excluded tumor microenvironment that impedes immunotherapy success. A key to unlocking pancreatic cancer immunotherapy may be treating early-stage pancreatic cancer, when peripancreatic inflammation promoted by the microbiome potentiates oncogenic signaling and suppresses innate and adaptive immunity. Hence, understanding the role of microbiota in pancreatic cancer initiation, progression, and immunosuppression is crucial. We propose that not only are microbiota targets for immunomodulation in this disease, but also that microbiome profiling has a potential role in pancreatic cancer screening. Furthermore, combining microbiome profiling with liquid and tissue biopsy may validate the early pancreatic cancer treatment approach of microbiome modulation and immunotherapy.
Highlights
Too Little, Too Late
Microbe-induced inflammation affects oncogenic signaling, tumor cell metabolism, and immunosuppression in PDAC, making microbiota an attractive target for therapy.
Not only is pancreatic ductal adenocarcinoma (PDAC) primed to become the second deadliest malignancy in the USA by 2025 [1], but the 45 750 fatalities from PDAC each year nearly equal the annual incidence rate of 56 770 [2], highlighting the lethal nature of this disease. Similar trends in incidence and mortality rates can be seen across the globe [3]. Actuarial 5-year survival rates across all stages of PDAC peak at 9%, while only 3% of patients with distant or metastatic PDAC will be alive after 5 years [2]. Even early stage PDAC, generally considered to be the most treatable with surgical resection, only has a 5-year survival rate of 25% [4,5]. Lack of a sensitive diagnostic screening test for early PDAC, nonspecific clinical symptomatology leading to advanced-stage diagnosis, and poorly effective treatment options all contribute to the poor prognosis. While stroma-targeting therapy, immunotherapy, and neoantigen vaccines are emerging strategies to treat established PDAC, there has been limited success in early clinical trials thus far [6]. A novel approach to pancreatic cancer care is desperately needed.
PDAC is an unforgiving and lethal cancer, with a mortality rate approaching its incidence rate. Nonspecific clinical symptoms leading to late diagnosis, the lack of a reliable screening test, and antiquated and ineffective treatment options all contribute to the poor prognosis of PDAC.
Microbiome profiling and liquid biopsies have the potential to screen for pancreatic cancer. A novel approach to pancreatic cancer diagnosis and treatment involves early microbiome profiling and modulation with concurrent immunotherapy.
In this Opinion, we argue that the current pancreatic cancer treatment paradigm is too little and too late. There is evidence to suggest that at the time of PDAC diagnosis, disseminated pancreatic cancer cells have already spread and developed a premetastatic niche within the liver [7]. Accordingly, 75% of patients who undergo curative resection for PDAC will go on to develop metastatic disease within 2 years [8]. Recent work has shown that specific microbiota promote pancreatic tumor development by potentiating oncogenic signaling, altering tumor metabolism, and inciting chronic inflammation that suppresses the innate and adaptive immune system [9]. Consequently, we believe that microbiome profiling and modulation are novel approaches with the potential to improve early pancreatic cancer diagnosis and immunotherapy response, respectively. Here, we discuss an emerging concept of microbiome profiling and targeting the microbiome in combination with immunotherapy in developing and established PDAC (Figure 1, Key Figure).
Microbiota Promote Pancreatic Cancer and Shape the Tumor Environment The concept of the microbiome as a factor in cancer initiation and progression is gaining traction rapidly across all cancer types [10]. Helicobacter pylori colonizes gastric mucosa and produces a CagA oncoprotein, which reprograms gastric epithelial cells and contributes to the pathogenesis of gastric cancer [11]. Enterotoxigenic Bacteroides fragilis creates reactive oxygen species via catabolic metabolism and directly induces DNA damage that contributes to colon tumorigenesis [12]. Fusobacterium nucleatum has been implicated in not only primary colorectal carcinogenesis, but also distant liver metastases via its FadA adhesion virulence factor [13,14]. Even in cancers not associated with the gastrointestinal tract, such as breast cancer, intestinal dysbiosis alters the host inflammatory
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1Department of Surgery, NYU School of Medicine, 435 East 30th Street, 4th Floor, New York, NY 10016, USA 2Perlmutter Cancer Center, NYU Langone School of Medicine, 160 East 34th Street, New York, NY 10016, USA
*Correspondence: [email protected]
https://doi.org/10.1016/j.trecan.2019.10.005 ª 2019 Elsevier Inc. All rights reserved.
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Key Figure
Approach to Microbiome Therapy in Pancreatic Cancer
Figure 1. Graphic illustration of the stages of pancreatic cancer development. Current therapy for pancreatic cancer is focused on early and advanced PDAC (light blue box), which generally harbors a poor prognosis. At this stage, microbiota-specific ablation and immunomodulation has the potential to improve pancreatic cancer outcomes, but therapeutic effect may be limited due to additional oncogenic factors including KRAS activation and immunecell exclusion in the tumor microenvironment. Instead, microbiome modulation may prove more impactful at the earliest stages of pancreatic cancer development (light orange box), when microbiota directly contribute to tumor oncogenesis in the absence of an unfavorable tumor microenvironment. Microbiome profiling, screening and augmentation may also lead to earlier PDAC diagnosis and open more therapeutic opportunities. Abbreviation: PDAC, pancreatic ductal adenocarcinoma.
response, which enhances fibrosis and collagen deposition systemically, contributing to the metastatic dissemination of breast tumor cells [15]. The role of the microbiome in the development of pancreatic cancer is well supported by recent research in genetically engineered murine models. Germ-free mice that are designed to develop slowly progressive PDAC (p48Cre;LSL-KrasG12D, KC mice) display reduced fibrosis and pancreatic dysplasia when compared to KC mice with normal intestinal flora. Concordantly, microbiota ablation
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using antibiotics in KC mice protects against both the initiation and progression of pancreatic cancer [9]. Subsequent analyses revealed that pancreatic oncogenesis was associated with the expansion of F. nucleatum and Bifidobacterium pseudolongum within murine pancreata. Similarly, ablation of the microbiome reduces tumor burden by 25–50% in more aggressive mouse models of PDAC driven by Kras and p53 mutations. In KrasG12D/PTENlox/+ mice, which develop pancreatic adenocarcinoma through activated Kras and partial loss of PTEN tumor suppression [16], microbial ablation significantly decreases the rate of oncogenic progression from pancreatic intraepithelial neoplasia (PanIN) to PDAC [17]. In xenograft tumors, which do not contain an intratumoral microbe population [17], intestinal microbe ablation reduces the rate of xenograft engraftment and growth, suggesting that intestinal microbes also have the potential to enhance pancreatic cancer growth via systemic modulation. Further research has highlighted the role of microbes in both innate and adaptive immunosuppression. Immunohistochemical analysis of xenograft tumors grown in mice depleted of intestinal microbes and deficient in adaptive immunity (NOD.CB17-prkdcscid/J; Nod-SCID mice) has revealed an increased infiltration of CD45+ immune cells, implicating intestinal microbes in innate immunosuppression. Similarly, microbiome depletion significantly decreased both subcutaneous and metastatic hepatic tumor burden in a syngeneic murine pancreatic cancer model, an effect that was found to be dependent on T cells [18]. Thus, microbiota contribute to both the development and progression of murine pancreatic cancer. The gut microbiome of human PDAC patients also harbors specific bacteria when compared to those with normal pancreata, suggesting a role for microbiota in regulating the human pancreatic tumor microenvironment. Proteobacteria comprise 50% of the gut microbiome in PDAC patients, while only 8% of the bacteria in patients with normal pancreata [9]. Synergistetes and Euyarchaeota are similarly enriched in the microbiome among patients with PDAC. Moreover, it appears that microbe-induced inflammation primes a systemic immune response that supports pancreatic cancer growth. As a result, dysbiosis of the intestinal and oropharyngeal microbiome is associated with the development of human pancreatic cancer [19]. Smoking is a well-described risk factor for pancreatic cancer and results in increased Proteobacteria, Bacteroidetes, and Clostridia species within the intestine, along with a reduction in Firmicutes and Actinobacteria [20]. Exposure to the oropharyngeal bacteria Porphyromonas gingivalis is also associated with a twofold increased risk in pancreatic cancer [21]. The high level of plasma P. gingivalis antibody associated with an increased risk of pancreatic cancer was measured from the blood samples of patients obtained 10 years before pancreatic cancer diagnosis, suggesting the possibility of P. gingivalis testing in pancreatic cancer screening. Together, these results suggest that microbiota not only contribute directly to oncogenesis within the tumor microenvironment, but also induce a systemic inflammatory response that nourishes PDAC development. Mechanistically, microbiota induce pancreatic cancer via multiple pathways [22]. First, intestinal dysbiosis directly supports pancreatic oncogenic signaling. K-Ras mutant signaling alone is not sufficient to initiate invasive pancreatic cancer, but additional microbe-induced inflammation can potentiate oncogenesis [23]. P. gingivalis secretes peptidyl-arginine deaminase, an enzyme that degrades arginine and may produce point mutations in p53 and K-ras, two primary genetic drivers of pancreatic cancer [24]. Furthermore, microbiota-induced activation of pattern recognition receptors recruits MyD88 or TRIF adaptor proteins to enhance MAPK and NF-kB signaling in pancreatic cancer cells, which synergizes with K-ras signaling [25,26]. Second, microbial metabolism and metabolites can alter the biochemical tumor environment to affect gene regulation and transcription, cell proliferation, apoptosis, or cause DNA damage. Deoxycholic acid, for example, is a microbial byproduct that induces DNA damage and may be a risk factor for obesity-induced pancreatic cancer [27]. Third, microbe-induced inflammation itself is a major instigator of pancreatic cancer. Microbiota-induced activation of Toll-like receptors (TLRs) suppresses innate and adaptive immunity in early pancreatic neoplasia and established PDAC [25,28,29]. Specifically, TLR9 ligation induces pancreatic cancer stellate cells to become fibrogenic and attracts immunosuppressive T regulatory cells and myeloidderived suppressor cells (MDSCs) to the tumor environment [29]. Lipopolysaccharide and TLR4
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ligation exacerbate pancreatic inflammation and accelerate pancreatic oncogenesis via a dendriticcell-dependent Th2 immune response. Finally, microbe-mediated TLR2 and TLR5 ligation instructs macrophages to adopt an immunosuppressive phenotype that restricts T cell-mediated immunity [9]. Together, the peripancreatic inflammatory response contributes not only to genetic oncogenic events but also helps shape an immunosuppressive microenvironment mediated by TLR ligation [22]. Besides altering oncogenic signaling, tumor metabolism, and inflammation, microbiota can affect drug metabolism and alter responses to chemotherapy in established PDAC, supporting the rationale for microbiome modulation in advanced cancer. Cytidine deaminase is an enzyme expressed by many bacteria that converts active gemcitabine into an inactive metabolite. In murine and human PDAC, Gammaproteobacteria are present intratumorally and induce resistance to gemcitabine via cytidine deaminase [30]. Concordantly, ablation with antibiotics abolishes gemcitabine resistance. Thus, the rationale for microbiome-based therapy is clear not only for suppression of oncogenesis, but also in therapeutic resistance.
Considerations for Microbiome-based Immunotherapy in Established PDAC Immunotherapy has shown remarkable success in a variety of malignancies, but it has been ineffective for pancreatic cancer thus fari–xiv [31–33]. The prevailing hypothesis as to why immunotherapy has not been successful is that the PDAC tumor microenvironment is characterized by a ‘cold’, fibrotic, and immune-cell excluding stroma that limits the infiltration of cancer-killing CD8+ T cells [34]. Furthermore, the immune cells that manage to penetrate the dense stroma are often immunosuppressive MDSCs, protumoral macrophages, immunosuppressive T regulatory cells, and gd T cells; all of which restrain cytotoxic T cells and suppress antitumor immunity. As a result, current efforts are focused at transforming the cold tumor microenvironment into an immunogenic one more amenable to checkpoint blockade immunotherapy. For example, it has been shown that stromal fibroblasts secrete CXCL12, which limits the infiltration of cytotoxic CD8+ T cells [35]. Inhibition of CXCL12 results in rapid infiltration of CD8+ T cells, which then enable successful immune checkpoint blockade. Combination of a CXCL12 inhibitor with immunotherapy is now in human clinical trials (NCT03277209vii). Similarly, focal adhesion kinase (FAK) contributes to the dense, cold tumor fibrosis seen in PDAC [36]. FAK inhibition significantly reduced tumor fibrosis and the infiltration of MDSCs in mouse models of PDAC, which not only double overall survival, but also sensitize tumors to anti-PD-1 immune checkpoint blockade. While these approaches have considered the important concept of transforming a cold tumor environment into an immunogenic one, the role of microbiota in the initiation and maintenance of the immunosuppressive tumor environment has received less attention. The role of microbiota on immunotherapy response should be considered in immunotherapy for PDAC. A recent study from Riquelme and colleagues has illustrated the impact of pancreatic tumor microbiome composition on patient survival. Notably, a diverse intratumoral microbiome signature rich in Pseudoxanthomonas, Streptomyces, Sacchropolyspora, and Bacillus clausii predicted longterm survivorship in multiple patient cohorts [37]. In metastatic melanoma, diverse microbial composition with Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium predicted a positive immunotherapy response. Moreover, reconstitution of germ-free mice with a similar microbe profile enhanced the efficacy immune checkpoint blockade [10,38]. In renal and non-small cell lung cancer, microbial ablation reduces the efficacy of immune checkpoint blockade [39]. Patients receiving antibiotics for pneumonia or urinary tract infections within 30 days of starting anti-PD-L1 immunotherapy displayed an increased risk of disease progression and decreased overall survival. These results suggest that modulation of an unfavorable microbiome, rather than complete ablation of the microbiome, may be a reasonable approach for microbiome-based immunotherapy. Consequently, microbiome-based immunotherapy for established PDAC should be tailored to specific bacterial taxa and immune cells. The established pancreatic tumor is a complex environment with stromal, immune, and microbial components. While microbial ablation studies strongly demonstrate that the immunosuppressive microenvironment in PDAC is mediated by microbial TLR ligation, the exact functions and immunogenic properties of specific microbiota are still being elucidated. A pilot
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Phase I study looking at the effect of microbiome ablation in human PDAC may help answer some important questions regarding the role of specific microbiota in antitumor immunity (NCT03891979xii). Preclinical data show that pancreata of humans with pancreatic cancer contain more Gram-negative Proteobacteria, Euyarchaeota, and anaerobic Synergistetes than normal pancreata contain, and microbial ablation enhances the efficacy of immune checkpoint blockade in PDAC [9]. Therefore, patients with resectable pancreatic cancer will receive 4 weeks of Gram-negative and anaerobic bacteria-ablating antibiotics (ciprofloxacin and metronidazole) and anti-PD-1 therapy prior to undergoing definitive surgical resection. Tumor tissue will be analyzed for immune cell activation markers, providing the first evidence for microbiome modulation in immunotherapy of human pancreatic cancer. A notable strength of this study is the inclusion of only early-stage, treatment-naı¨ve pancreatic cancer. Restricting this pilot study to treatment-naı¨ve, primary tumors will maximize the ability to attribute changes in stromal and immune cell activity directly to microbiome ablation. Characterization of the microbiome changes, in conjunction with alterations in stromal and immune cell activity may shed light on the role of specific bacteria in each immune compartment of the established tumor, which can then direct future study. Further trials can be then performed to explore the efficacy of selective microbiome modulation and repopulation in established PDAC therapy, including the administration of immunostimulating probiotics to potentiate an antitumor response, or even a combination of microbiome modulation/ablation with stromal therapy, immunotherapy, or chemotherapy.
Considerations for Microbiome-based Immunotherapy in Developing PDAC In a developing tumor, it is well known that microbe-induced inflammation engenders a tolerant immune phenotype perpetuated by TLR signaling and the chronic inflammatory response. Treatment of pancreatic cancer at the earliest stages of development, when immunosuppression supports pancreatic cancer growth, may result in the most durable antitumor response. However, the ability to treat PDAC during this earliest stage hinges on the ability to accurately screen for pancreatic cancer, for which there is currently no reliable screening method. Microbiome and molecular profiling may be able to improve early pancreatic cancer detection. Comprehensive assessment and profiling of the salivary flora in pancreatic cancer patients and healthy controls revealed that a reduction in the levels of two bacteria, Neisseria elongate and Streptococcus mitis, showed promise as a screening test for pancreatic cancer (96% sensitive, 82% specific) [40]. Similarly, a significantly higher ratio of Lepotrichia to Porphyromonas was identified in the saliva of pancreatic cancer patients and its utility was assessed as a biomarker [41]. Sequencing of the tongue microbiome of pancreatic cancer patients showed that the prevalence of Haemophilus, Porphyromonas, Leptotrichia, and Fusobacterium could distinguish patients with pancreatic cancer from normal, healthy participants [42]. In all of these studies, microbiome profiling of oropharynx was utilized as a noninvasive approach to pancreatic cancer diagnosis. There are also additional opportunities to estimate tumor severity using microbiome profiling of the intestine. Analysis of the microbiome in slowly growing pancreatic cancer versus aggressively growing pancreatic cancer in genetically identical mice, for example, revealed distinct and definable bacterial communities between cancer cohorts. Furthermore, there is evidence to suggest that the microbiome of the duodenum correlates with intrapancreatic microbial profiles [43]. Simultaneous endoscopic duodenal bacterial sampling and pancreas biopsy, therefore, is an intriguing diagnostic approach for the early initiation of microbiome-based pancreatic cancer therapy. Liquid biopsies and genomic mutational profiling are also new approaches to the diagnosis of pancreatic cancer that could be used in conjunction with microbiome modulation and immunotherapy. Liquid biopsy is the concept of detecting pancreatic cancer via a blood test rather than pancreatic tissue biopsy [44]. Multiple liquid biopsy targets are emerging for the diagnosis of pancreatic cancer, including circulating tumor cells (CTCs), cell free DNA (cfDNA), and exosomes [45,46]. One CTC profile showing promise for the diagnosis of pancreatic cancer involves c-Met, h-Tert, CK20, and CEA, in which all four genes combined produced a sensitivity and specificity of 100% [47]. Similarly, pancreatic cancer is often characterized by genetic mutations in KRAS, P53, CDKN2A, APC, SMAD4, and FBXW7 [48], and cfDNA detection utilizing these pancreatic cancer proteins results in a diagnostic accuracy of 97.7% [49]. Finally, many circulating miRNAs are inserted into exosomes
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and shed into blood and pancreatic juice, including miR-21 and miR-155, which correlates well with angiogenesis, cell proliferation, metastasis, and chemoresistance in PDAC [46]. Ultimately, both microbiome profiling and liquid biopsies have the potential to identify patients with early pancreatic inflammation and pancreatic cancer, which would substantiate the approach of early microbiome modulation and immunotherapy.
Concluding Remarks Microbiota play an integral role in the PDAC environment. In the developing tumor, microbiome modulation plus immunotherapy is a novel approach to alter the grim prognosis of PDAC, but a reliable screening test for pancreatic cancer is first needed. Microbiome profiling and liquid biopsies have the potential to identify individuals at risk for pancreatic cancer, which would expedite early microbiome-based immunotherapy. In established PDAC, characterizing the exact functions of specific microbiota may open additional opportunities for pancreatic cancer immunotherapy, as the microbial–immune cell compartment interactions are likely microbe and immune-cell specific. Further prospective research is still needed prior to the widespread implementation of microbiome-based immunotherapy for developing and established PDAC (see Outstanding Questions).
Resources i
https://clinicaltrials.gov/ct2/show/NCT02558894
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https://clinicaltrials.gov/ct2/show/NCT00112580
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https://clinicaltrials.gov/ct2/show/NCT00729664
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https://clinicaltrials.gov/ct2/show/NCT00084383 https://clinicaltrials.gov/ct2/show/NCT00836407
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https://clinicaltrials.gov/ct2/show/NCT02451982
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https://clinicaltrials.gov/ct2/show/NCT03277209
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https://clinicaltrials.gov/ct2/show/NCT02588443 https://clinicaltrials.gov/ct2/show/NCT02777710
https://clinicaltrials.gov/ct2/show/NCT03153410
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https://clinicaltrials.gov/ct2/show/NCT03519308 https://clinicaltrials.gov/ct2/show/NCT03891979 https://clinicaltrials.gov/ct2/show/NCT03785210 https://clinicaltrials.gov/ct2/show/NCT03302637
Disclaimer Statement G.M. has research contract agreements with Puretech Health, Pfizer, and GlaxoSmithKline. The authors have no financial conflicts of interest to disclose. References 1. Rahib, L. et al. (2014) Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res. 74, 2913–2921 2. Siegel, R.L. et al. (2019) Cancer statistics, 2019. CA Cancer J. Clin. 69, 7–34 3. Ilic, M. and Ilic, I. (2016) Epidemiology of pancreatic cancer. World J. Gastroenterol. 22, 9694–9705 4. Geer, R.J. and Brennan, M.F. (1993) Prognostic indicators for survival after resection of pancreatic adenocarcinoma. Am. J. Surg. 165, 68–72 5. Yeo, C.J. et al. (1995) Pancreaticoduodenectomy for cancer of the head of the pancreas. 201 patients. Ann. Surg. 221, 721–731
6
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6. Balachandran, V.P. et al. (2019) Broadening the impact of immunotherapy to pancreatic cancer: challenges and opportunities. Gastroenterology 156, 2056–2072 7. Pommier, A. et al. (2018) Unresolved endoplasmic reticulum stress engenders immune-resistant, latent pancreatic cancer metastases. Science 360, eaao490 8. Groot, V.P. et al. (2018) ASO author reflections: do distinct patterns of recurrence impact the prognosis of patients with resected pancreatic ductal adenocarcinoma? Ann. Surg. Oncol. 25, 806–807 9. Pushalkar, S. et al. (2018) The pancreatic cancer microbiome promotes oncogenesis by induction of innate and adaptive immune suppression. Cancer Discov. 8, 403–416
Outstanding Questions What are the roles of specific bacterial taxa in maintaining the tumor microenvironment of established PDAC, and how can we target them? Is there a differential immune response to microbiome ablation versus microbiome modulation and repopulation with probiotics in established PDAC? What would define a positive microbiome screening result? How should we follow or treat patients with a positive microbiomebased screening test without pathologic evidence of pancreatic cancer?
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10. Gopalakrishnan, V. et al. (2018) The influence of the gut microbiome on cancer, immunity, and cancer immunotherapy. Cancer cell 33, 570–580 11. Amieva, M. and Peek, R.M., Jr. (2016) Pathobiology of Helicobacter pylori-induced gastric cancer. Gastroenterology 150, 64–78 12. Goodwin, A.C. et al. (2011) Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilisinduced colon tumorigenesis. Proc. Natl. Acad. Sci. U S A 108, 15354–15359 13. Bullman, S. et al. (2017) Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer. Science 358, 1443–1448 14. Rubinstein, M.R. et al. (2013) Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/beta-catenin signaling via its FadA adhesin. Cell Host Microbe 14, 195–206 15. Rosean, C.B. et al. (2019) Preexisting commensal dysbiosis is a host-intrinsic regulator of tissue inflammation and tumor cell dissemination in hormone receptor-positive breast cancer. Cancer Res. 79, 3662–3675 16. Hill, R. et al. (2010) PTEN loss accelerates KrasG12Dinduced pancreatic cancer development. Cancer Res. 70, 7114–7124 17. Thomas, R.M. et al. (2018) Intestinal microbiota enhances pancreatic carcinogenesis in preclinical models. Carcinogenesis 39, 1068–1078 18. Sethi, V. et al. (2018) Gut microbiota promotes tumor growth in mice by modulating immune response. Gastroenterology 155, 33–37 e36 19. Fan, X. et al. (2018) Human oral microbiome and prospective risk for pancreatic cancer: a populationbased nested case-control study. Gut 67, 120–127 20. Biedermann, L. et al. (2013) Smoking cessation induces profound changes in the composition of the intestinal microbiota in humans. PLoS One 8, e59260 21. Michaud, D.S. et al. (2013) Plasma antibodies to oral bacteria and risk of pancreatic cancer in a large European prospective cohort study. Gut 62, 1764– 1770 22. Wei, M.Y. et al. (2019) The microbiota and microbiome in pancreatic cancer: more influential than expected. Mol. Cancer 18, 97 23. Schulz, M.D. et al. (2014) High-fat-diet-mediated dysbiosis promotes intestinal carcinogenesis independently of obesity. Nature 514, 508–512 24. Ogrendik, M. (2017) Periodontal pathogens in the etiology of pancreatic cancer. Gastrointest. Tumors 3, 125–127 25. Ochi, A. et al. (2012) MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells. J. Exp. Med. 209, 1671– 1687 26. Zambirinis, C.P. et al. (2013) Induction of TRIF- or MYD88-dependent pathways perturbs cell cycle regulation in pancreatic cancer. Cell Cycle 12, 1153– 1154 27. Yoshimoto, S. et al. (2013) Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 499, 97–101 28. Seifert, L. et al. (2016) The necrosome promotes pancreatic oncogenesis via CXCL1 and Mincleinduced immune suppression. Nature 532, 245–249 29. Zambirinis, C.P. et al. (2015) TLR9 ligation in pancreatic stellate cells promotes tumorigenesis. J. Exp. Med. 212, 2077–2094
30. Geller, L.T. et al. (2017) Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine. Science 357, 1156–1160 31. Brahmer, J.R. et al. (2012) Safety and activity of antiPD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455–2465 32. O’Reilly, E.M. et al. (2019) Durvalumab with or without tremelimumab for patients with metastatic pancreatic ductal adenocarcinoma: a phase 2 randomized clinical trial. J. Clin. Oncol. 36 (4 Suppl), 217 33. Royal, R.E. et al. (2010) Phase 2 trial of single agent ipilimumab (anti-CTLA-4) for locally advanced or metastatic pancreatic adenocarcinoma. J. Immunother. 33, 828–833 34. Ritter, B. and Greten, F.R. (2019) Modulating inflammation for cancer therapy. J. Exp. Med. 216, 1234–1243 35. Feig, C. et al. (2013) Targeting CXCL12 from FAPexpressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc. Natl. Acad. Sci. U S A 110, 20212–20217 36. Jiang, H. et al. (2016) Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy. Nat. Med. 22, 851–860 37. Riquelme, E. et al. (2019) Tumor microbiome diversity and composition influence pancreatic cancer outcomes. Cell 178, 795–806 e712 38. Matson, V. et al. (2018) The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients. Science 359, 104–108 39. Derosa, L. et al. (2018) Negative association of antibiotics on clinical activity of immune checkpoint inhibitors in patients with advanced renal cell and non-small-cell lung cancer. Ann. Oncol. 29, 1437– 1444 40. Farrell, J.J. et al. (2012) Variations of oral microbiota are associated with pancreatic diseases including pancreatic cancer. Gut 61, 582–588 41. Torres, P.J. et al. (2015) Characterization of the salivary microbiome in patients with pancreatic cancer. PeerJ 3, e1373 42. Lu, H. et al. (2019) Tongue coating microbiome data distinguish patients with pancreatic head cancer from healthy controls. J. Oral Microbiol. 11, 1563409 43. Del Castillo, E. et al. (2019) The microbiomes of pancreatic and duodenum tissue overlap and are highly subject specific but differ between pancreatic cancer and noncancer subjects. Cancer Epidemiol. Biomarkers Prev. 28, 370–383 44. Crowley, E. et al. (2013) Liquid biopsy: monitoring cancer-genetics in the blood. Nat. Rev. Clin. Oncol. 10, 472–484 45. Qi, Z.H. et al. (2018) The significance of liquid biopsy in pancreatic cancer. J. Cancer 9, 3417–3426 46. Yadav, D.K. et al. (2018) Liquid biopsy in pancreatic cancer: the beginning of a new era. Oncotarget 9, 26900–26933 47. Zhou, J. et al. (2011) Marker expression in circulating cancer cells of pancreatic cancer patients. J. Surg. Res. 171, 631–636 48. Bailey, P. et al. (2016) Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531, 47–52 49. Zill, O.A. et al. (2015) Cell-free DNA next-generation sequencing in pancreatobiliary carcinomas. Cancer Discov. 5, 1040–1048
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Trends in Cancer
Opinion
Harnessing the Microbiome for Pancreatic Cancer Immunotherapy Gerardo A. Vitiello,1 Deirdre J. Cohen,2 and George Miller1,* Late-stage pancreatic cancer harbors a fibrotic and immune-excluded tumor microenvironment that impedes immunotherapy success. A key to unlocking pancreatic cancer immunotherapy may be treating early-stage pancreatic cancer, when peripancreatic inflammation promoted by the microbiome potentiates oncogenic signaling and suppresses innate and adaptive immunity. Hence, understanding the role of microbiota in pancreatic cancer initiation, progression, and immunosuppression is crucial. We propose that not only are microbiota targets for immunomodulation in this disease, but also that microbiome profiling has a potential role in pancreatic cancer screening. Furthermore, combining microbiome profiling with liquid and tissue biopsy may validate the early pancreatic cancer treatment approach of microbiome modulation and immunotherapy.
Highlights
Too Little, Too Late
Microbe-induced inflammation affects oncogenic signaling, tumor cell metabolism, and immunosuppression in PDAC, making microbiota an attractive target for therapy.
Not only is pancreatic ductal adenocarcinoma (PDAC) primed to become the second deadliest malignancy in the USA by 2025 [1], but the 45 750 fatalities from PDAC each year nearly equal the annual incidence rate of 56 770 [2], highlighting the lethal nature of this disease. Similar trends in incidence and mortality rates can be seen across the globe [3]. Actuarial 5-year survival rates across all stages of PDAC peak at 9%, while only 3% of patients with distant or metastatic PDAC will be alive after 5 years [2]. Even early stage PDAC, generally considered to be the most treatable with surgical resection, only has a 5-year survival rate of 25% [4,5]. Lack of a sensitive diagnostic screening test for early PDAC, nonspecific clinical symptomatology leading to advanced-stage diagnosis, and poorly effective treatment options all contribute to the poor prognosis. While stroma-targeting therapy, immunotherapy, and neoantigen vaccines are emerging strategies to treat established PDAC, there has been limited success in early clinical trials thus far [6]. A novel approach to pancreatic cancer care is desperately needed.
PDAC is an unforgiving and lethal cancer, with a mortality rate approaching its incidence rate. Nonspecific clinical symptoms leading to late diagnosis, the lack of a reliable screening test, and antiquated and ineffective treatment options all contribute to the poor prognosis of PDAC.
Microbiome profiling and liquid biopsies have the potential to screen for pancreatic cancer. A novel approach to pancreatic cancer diagnosis and treatment involves early microbiome profiling and modulation with concurrent immunotherapy.
In this Opinion, we argue that the current pancreatic cancer treatment paradigm is too little and too late. There is evidence to suggest that at the time of PDAC diagnosis, disseminated pancreatic cancer cells have already spread and developed a premetastatic niche within the liver [7]. Accordingly, 75% of patients who undergo curative resection for PDAC will go on to develop metastatic disease within 2 years [8]. Recent work has shown that specific microbiota promote pancreatic tumor development by potentiating oncogenic signaling, altering tumor metabolism, and inciting chronic inflammation that suppresses the innate and adaptive immune system [9]. Consequently, we believe that microbiome profiling and modulation are novel approaches with the potential to improve early pancreatic cancer diagnosis and immunotherapy response, respectively. Here, we discuss an emerging concept of microbiome profiling and targeting the microbiome in combination with immunotherapy in developing and established PDAC (Figure 1, Key Figure).
Microbiota Promote Pancreatic Cancer and Shape the Tumor Environment The concept of the microbiome as a factor in cancer initiation and progression is gaining traction rapidly across all cancer types [10]. Helicobacter pylori colonizes gastric mucosa and produces a CagA oncoprotein, which reprograms gastric epithelial cells and contributes to the pathogenesis of gastric cancer [11]. Enterotoxigenic Bacteroides fragilis creates reactive oxygen species via catabolic metabolism and directly induces DNA damage that contributes to colon tumorigenesis [12]. Fusobacterium nucleatum has been implicated in not only primary colorectal carcinogenesis, but also distant liver metastases via its FadA adhesion virulence factor [13,14]. Even in cancers not associated with the gastrointestinal tract, such as breast cancer, intestinal dysbiosis alters the host inflammatory
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1Department of Surgery, NYU School of Medicine, 435 East 30th Street, 4th Floor, New York, NY 10016, USA 2Perlmutter Cancer Center, NYU Langone School of Medicine, 160 East 34th Street, New York, NY 10016, USA
*Correspondence: [email protected]
https://doi.org/10.1016/j.trecan.2019.10.005 ª 2019 Elsevier Inc. All rights reserved.
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Key Figure
Approach to Microbiome Therapy in Pancreatic Cancer
Figure 1. Graphic illustration of the stages of pancreatic cancer development. Current therapy for pancreatic cancer is focused on early and advanced PDAC (light blue box), which generally harbors a poor prognosis. At this stage, microbiota-specific ablation and immunomodulation has the potential to improve pancreatic cancer outcomes, but therapeutic effect may be limited due to additional oncogenic factors including KRAS activation and immunecell exclusion in the tumor microenvironment. Instead, microbiome modulation may prove more impactful at the earliest stages of pancreatic cancer development (light orange box), when microbiota directly contribute to tumor oncogenesis in the absence of an unfavorable tumor microenvironment. Microbiome profiling, screening and augmentation may also lead to earlier PDAC diagnosis and open more therapeutic opportunities. Abbreviation: PDAC, pancreatic ductal adenocarcinoma.
response, which enhances fibrosis and collagen deposition systemically, contributing to the metastatic dissemination of breast tumor cells [15]. The role of the microbiome in the development of pancreatic cancer is well supported by recent research in genetically engineered murine models. Germ-free mice that are designed to develop slowly progressive PDAC (p48Cre;LSL-KrasG12D, KC mice) display reduced fibrosis and pancreatic dysplasia when compared to KC mice with normal intestinal flora. Concordantly, microbiota ablation
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using antibiotics in KC mice protects against both the initiation and progression of pancreatic cancer [9]. Subsequent analyses revealed that pancreatic oncogenesis was associated with the expansion of F. nucleatum and Bifidobacterium pseudolongum within murine pancreata. Similarly, ablation of the microbiome reduces tumor burden by 25–50% in more aggressive mouse models of PDAC driven by Kras and p53 mutations. In KrasG12D/PTENlox/+ mice, which develop pancreatic adenocarcinoma through activated Kras and partial loss of PTEN tumor suppression [16], microbial ablation significantly decreases the rate of oncogenic progression from pancreatic intraepithelial neoplasia (PanIN) to PDAC [17]. In xenograft tumors, which do not contain an intratumoral microbe population [17], intestinal microbe ablation reduces the rate of xenograft engraftment and growth, suggesting that intestinal microbes also have the potential to enhance pancreatic cancer growth via systemic modulation. Further research has highlighted the role of microbes in both innate and adaptive immunosuppression. Immunohistochemical analysis of xenograft tumors grown in mice depleted of intestinal microbes and deficient in adaptive immunity (NOD.CB17-prkdcscid/J; Nod-SCID mice) has revealed an increased infiltration of CD45+ immune cells, implicating intestinal microbes in innate immunosuppression. Similarly, microbiome depletion significantly decreased both subcutaneous and metastatic hepatic tumor burden in a syngeneic murine pancreatic cancer model, an effect that was found to be dependent on T cells [18]. Thus, microbiota contribute to both the development and progression of murine pancreatic cancer. The gut microbiome of human PDAC patients also harbors specific bacteria when compared to those with normal pancreata, suggesting a role for microbiota in regulating the human pancreatic tumor microenvironment. Proteobacteria comprise 50% of the gut microbiome in PDAC patients, while only 8% of the bacteria in patients with normal pancreata [9]. Synergistetes and Euyarchaeota are similarly enriched in the microbiome among patients with PDAC. Moreover, it appears that microbe-induced inflammation primes a systemic immune response that supports pancreatic cancer growth. As a result, dysbiosis of the intestinal and oropharyngeal microbiome is associated with the development of human pancreatic cancer [19]. Smoking is a well-described risk factor for pancreatic cancer and results in increased Proteobacteria, Bacteroidetes, and Clostridia species within the intestine, along with a reduction in Firmicutes and Actinobacteria [20]. Exposure to the oropharyngeal bacteria Porphyromonas gingivalis is also associated with a twofold increased risk in pancreatic cancer [21]. The high level of plasma P. gingivalis antibody associated with an increased risk of pancreatic cancer was measured from the blood samples of patients obtained 10 years before pancreatic cancer diagnosis, suggesting the possibility of P. gingivalis testing in pancreatic cancer screening. Together, these results suggest that microbiota not only contribute directly to oncogenesis within the tumor microenvironment, but also induce a systemic inflammatory response that nourishes PDAC development. Mechanistically, microbiota induce pancreatic cancer via multiple pathways [22]. First, intestinal dysbiosis directly supports pancreatic oncogenic signaling. K-Ras mutant signaling alone is not sufficient to initiate invasive pancreatic cancer, but additional microbe-induced inflammation can potentiate oncogenesis [23]. P. gingivalis secretes peptidyl-arginine deaminase, an enzyme that degrades arginine and may produce point mutations in p53 and K-ras, two primary genetic drivers of pancreatic cancer [24]. Furthermore, microbiota-induced activation of pattern recognition receptors recruits MyD88 or TRIF adaptor proteins to enhance MAPK and NF-kB signaling in pancreatic cancer cells, which synergizes with K-ras signaling [25,26]. Second, microbial metabolism and metabolites can alter the biochemical tumor environment to affect gene regulation and transcription, cell proliferation, apoptosis, or cause DNA damage. Deoxycholic acid, for example, is a microbial byproduct that induces DNA damage and may be a risk factor for obesity-induced pancreatic cancer [27]. Third, microbe-induced inflammation itself is a major instigator of pancreatic cancer. Microbiota-induced activation of Toll-like receptors (TLRs) suppresses innate and adaptive immunity in early pancreatic neoplasia and established PDAC [25,28,29]. Specifically, TLR9 ligation induces pancreatic cancer stellate cells to become fibrogenic and attracts immunosuppressive T regulatory cells and myeloidderived suppressor cells (MDSCs) to the tumor environment [29]. Lipopolysaccharide and TLR4
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ligation exacerbate pancreatic inflammation and accelerate pancreatic oncogenesis via a dendriticcell-dependent Th2 immune response. Finally, microbe-mediated TLR2 and TLR5 ligation instructs macrophages to adopt an immunosuppressive phenotype that restricts T cell-mediated immunity [9]. Together, the peripancreatic inflammatory response contributes not only to genetic oncogenic events but also helps shape an immunosuppressive microenvironment mediated by TLR ligation [22]. Besides altering oncogenic signaling, tumor metabolism, and inflammation, microbiota can affect drug metabolism and alter responses to chemotherapy in established PDAC, supporting the rationale for microbiome modulation in advanced cancer. Cytidine deaminase is an enzyme expressed by many bacteria that converts active gemcitabine into an inactive metabolite. In murine and human PDAC, Gammaproteobacteria are present intratumorally and induce resistance to gemcitabine via cytidine deaminase [30]. Concordantly, ablation with antibiotics abolishes gemcitabine resistance. Thus, the rationale for microbiome-based therapy is clear not only for suppression of oncogenesis, but also in therapeutic resistance.
Considerations for Microbiome-based Immunotherapy in Established PDAC Immunotherapy has shown remarkable success in a variety of malignancies, but it has been ineffective for pancreatic cancer thus fari–xiv [31–33]. The prevailing hypothesis as to why immunotherapy has not been successful is that the PDAC tumor microenvironment is characterized by a ‘cold’, fibrotic, and immune-cell excluding stroma that limits the infiltration of cancer-killing CD8+ T cells [34]. Furthermore, the immune cells that manage to penetrate the dense stroma are often immunosuppressive MDSCs, protumoral macrophages, immunosuppressive T regulatory cells, and gd T cells; all of which restrain cytotoxic T cells and suppress antitumor immunity. As a result, current efforts are focused at transforming the cold tumor microenvironment into an immunogenic one more amenable to checkpoint blockade immunotherapy. For example, it has been shown that stromal fibroblasts secrete CXCL12, which limits the infiltration of cytotoxic CD8+ T cells [35]. Inhibition of CXCL12 results in rapid infiltration of CD8+ T cells, which then enable successful immune checkpoint blockade. Combination of a CXCL12 inhibitor with immunotherapy is now in human clinical trials (NCT03277209vii). Similarly, focal adhesion kinase (FAK) contributes to the dense, cold tumor fibrosis seen in PDAC [36]. FAK inhibition significantly reduced tumor fibrosis and the infiltration of MDSCs in mouse models of PDAC, which not only double overall survival, but also sensitize tumors to anti-PD-1 immune checkpoint blockade. While these approaches have considered the important concept of transforming a cold tumor environment into an immunogenic one, the role of microbiota in the initiation and maintenance of the immunosuppressive tumor environment has received less attention. The role of microbiota on immunotherapy response should be considered in immunotherapy for PDAC. A recent study from Riquelme and colleagues has illustrated the impact of pancreatic tumor microbiome composition on patient survival. Notably, a diverse intratumoral microbiome signature rich in Pseudoxanthomonas, Streptomyces, Sacchropolyspora, and Bacillus clausii predicted longterm survivorship in multiple patient cohorts [37]. In metastatic melanoma, diverse microbial composition with Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium predicted a positive immunotherapy response. Moreover, reconstitution of germ-free mice with a similar microbe profile enhanced the efficacy immune checkpoint blockade [10,38]. In renal and non-small cell lung cancer, microbial ablation reduces the efficacy of immune checkpoint blockade [39]. Patients receiving antibiotics for pneumonia or urinary tract infections within 30 days of starting anti-PD-L1 immunotherapy displayed an increased risk of disease progression and decreased overall survival. These results suggest that modulation of an unfavorable microbiome, rather than complete ablation of the microbiome, may be a reasonable approach for microbiome-based immunotherapy. Consequently, microbiome-based immunotherapy for established PDAC should be tailored to specific bacterial taxa and immune cells. The established pancreatic tumor is a complex environment with stromal, immune, and microbial components. While microbial ablation studies strongly demonstrate that the immunosuppressive microenvironment in PDAC is mediated by microbial TLR ligation, the exact functions and immunogenic properties of specific microbiota are still being elucidated. A pilot
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Phase I study looking at the effect of microbiome ablation in human PDAC may help answer some important questions regarding the role of specific microbiota in antitumor immunity (NCT03891979xii). Preclinical data show that pancreata of humans with pancreatic cancer contain more Gram-negative Proteobacteria, Euyarchaeota, and anaerobic Synergistetes than normal pancreata contain, and microbial ablation enhances the efficacy of immune checkpoint blockade in PDAC [9]. Therefore, patients with resectable pancreatic cancer will receive 4 weeks of Gram-negative and anaerobic bacteria-ablating antibiotics (ciprofloxacin and metronidazole) and anti-PD-1 therapy prior to undergoing definitive surgical resection. Tumor tissue will be analyzed for immune cell activation markers, providing the first evidence for microbiome modulation in immunotherapy of human pancreatic cancer. A notable strength of this study is the inclusion of only early-stage, treatment-naı¨ve pancreatic cancer. Restricting this pilot study to treatment-naı¨ve, primary tumors will maximize the ability to attribute changes in stromal and immune cell activity directly to microbiome ablation. Characterization of the microbiome changes, in conjunction with alterations in stromal and immune cell activity may shed light on the role of specific bacteria in each immune compartment of the established tumor, which can then direct future study. Further trials can be then performed to explore the efficacy of selective microbiome modulation and repopulation in established PDAC therapy, including the administration of immunostimulating probiotics to potentiate an antitumor response, or even a combination of microbiome modulation/ablation with stromal therapy, immunotherapy, or chemotherapy.
Considerations for Microbiome-based Immunotherapy in Developing PDAC In a developing tumor, it is well known that microbe-induced inflammation engenders a tolerant immune phenotype perpetuated by TLR signaling and the chronic inflammatory response. Treatment of pancreatic cancer at the earliest stages of development, when immunosuppression supports pancreatic cancer growth, may result in the most durable antitumor response. However, the ability to treat PDAC during this earliest stage hinges on the ability to accurately screen for pancreatic cancer, for which there is currently no reliable screening method. Microbiome and molecular profiling may be able to improve early pancreatic cancer detection. Comprehensive assessment and profiling of the salivary flora in pancreatic cancer patients and healthy controls revealed that a reduction in the levels of two bacteria, Neisseria elongate and Streptococcus mitis, showed promise as a screening test for pancreatic cancer (96% sensitive, 82% specific) [40]. Similarly, a significantly higher ratio of Lepotrichia to Porphyromonas was identified in the saliva of pancreatic cancer patients and its utility was assessed as a biomarker [41]. Sequencing of the tongue microbiome of pancreatic cancer patients showed that the prevalence of Haemophilus, Porphyromonas, Leptotrichia, and Fusobacterium could distinguish patients with pancreatic cancer from normal, healthy participants [42]. In all of these studies, microbiome profiling of oropharynx was utilized as a noninvasive approach to pancreatic cancer diagnosis. There are also additional opportunities to estimate tumor severity using microbiome profiling of the intestine. Analysis of the microbiome in slowly growing pancreatic cancer versus aggressively growing pancreatic cancer in genetically identical mice, for example, revealed distinct and definable bacterial communities between cancer cohorts. Furthermore, there is evidence to suggest that the microbiome of the duodenum correlates with intrapancreatic microbial profiles [43]. Simultaneous endoscopic duodenal bacterial sampling and pancreas biopsy, therefore, is an intriguing diagnostic approach for the early initiation of microbiome-based pancreatic cancer therapy. Liquid biopsies and genomic mutational profiling are also new approaches to the diagnosis of pancreatic cancer that could be used in conjunction with microbiome modulation and immunotherapy. Liquid biopsy is the concept of detecting pancreatic cancer via a blood test rather than pancreatic tissue biopsy [44]. Multiple liquid biopsy targets are emerging for the diagnosis of pancreatic cancer, including circulating tumor cells (CTCs), cell free DNA (cfDNA), and exosomes [45,46]. One CTC profile showing promise for the diagnosis of pancreatic cancer involves c-Met, h-Tert, CK20, and CEA, in which all four genes combined produced a sensitivity and specificity of 100% [47]. Similarly, pancreatic cancer is often characterized by genetic mutations in KRAS, P53, CDKN2A, APC, SMAD4, and FBXW7 [48], and cfDNA detection utilizing these pancreatic cancer proteins results in a diagnostic accuracy of 97.7% [49]. Finally, many circulating miRNAs are inserted into exosomes
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and shed into blood and pancreatic juice, including miR-21 and miR-155, which correlates well with angiogenesis, cell proliferation, metastasis, and chemoresistance in PDAC [46]. Ultimately, both microbiome profiling and liquid biopsies have the potential to identify patients with early pancreatic inflammation and pancreatic cancer, which would substantiate the approach of early microbiome modulation and immunotherapy.
Concluding Remarks Microbiota play an integral role in the PDAC environment. In the developing tumor, microbiome modulation plus immunotherapy is a novel approach to alter the grim prognosis of PDAC, but a reliable screening test for pancreatic cancer is first needed. Microbiome profiling and liquid biopsies have the potential to identify individuals at risk for pancreatic cancer, which would expedite early microbiome-based immunotherapy. In established PDAC, characterizing the exact functions of specific microbiota may open additional opportunities for pancreatic cancer immunotherapy, as the microbial–immune cell compartment interactions are likely microbe and immune-cell specific. Further prospective research is still needed prior to the widespread implementation of microbiome-based immunotherapy for developing and established PDAC (see Outstanding Questions).
Resources i
https://clinicaltrials.gov/ct2/show/NCT02558894
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https://clinicaltrials.gov/ct2/show/NCT00112580
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https://clinicaltrials.gov/ct2/show/NCT00729664
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https://clinicaltrials.gov/ct2/show/NCT00084383 https://clinicaltrials.gov/ct2/show/NCT00836407
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https://clinicaltrials.gov/ct2/show/NCT02451982
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https://clinicaltrials.gov/ct2/show/NCT03277209
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https://clinicaltrials.gov/ct2/show/NCT02588443 https://clinicaltrials.gov/ct2/show/NCT02777710
https://clinicaltrials.gov/ct2/show/NCT03153410
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https://clinicaltrials.gov/ct2/show/NCT03519308 https://clinicaltrials.gov/ct2/show/NCT03891979 https://clinicaltrials.gov/ct2/show/NCT03785210 https://clinicaltrials.gov/ct2/show/NCT03302637
Disclaimer Statement G.M. has research contract agreements with Puretech Health, Pfizer, and GlaxoSmithKline. The authors have no financial conflicts of interest to disclose. References 1. Rahib, L. et al. (2014) Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res. 74, 2913–2921 2. Siegel, R.L. et al. (2019) Cancer statistics, 2019. CA Cancer J. Clin. 69, 7–34 3. Ilic, M. and Ilic, I. (2016) Epidemiology of pancreatic cancer. World J. Gastroenterol. 22, 9694–9705 4. Geer, R.J. and Brennan, M.F. (1993) Prognostic indicators for survival after resection of pancreatic adenocarcinoma. Am. J. Surg. 165, 68–72 5. Yeo, C.J. et al. (1995) Pancreaticoduodenectomy for cancer of the head of the pancreas. 201 patients. Ann. Surg. 221, 721–731
6
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6. Balachandran, V.P. et al. (2019) Broadening the impact of immunotherapy to pancreatic cancer: challenges and opportunities. Gastroenterology 156, 2056–2072 7. Pommier, A. et al. (2018) Unresolved endoplasmic reticulum stress engenders immune-resistant, latent pancreatic cancer metastases. Science 360, eaao490 8. Groot, V.P. et al. (2018) ASO author reflections: do distinct patterns of recurrence impact the prognosis of patients with resected pancreatic ductal adenocarcinoma? Ann. Surg. Oncol. 25, 806–807 9. Pushalkar, S. et al. (2018) The pancreatic cancer microbiome promotes oncogenesis by induction of innate and adaptive immune suppression. Cancer Discov. 8, 403–416
Outstanding Questions What are the roles of specific bacterial taxa in maintaining the tumor microenvironment of established PDAC, and how can we target them? Is there a differential immune response to microbiome ablation versus microbiome modulation and repopulation with probiotics in established PDAC? What would define a positive microbiome screening result? How should we follow or treat patients with a positive microbiomebased screening test without pathologic evidence of pancreatic cancer?
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10. Gopalakrishnan, V. et al. (2018) The influence of the gut microbiome on cancer, immunity, and cancer immunotherapy. Cancer cell 33, 570–580 11. Amieva, M. and Peek, R.M., Jr. (2016) Pathobiology of Helicobacter pylori-induced gastric cancer. Gastroenterology 150, 64–78 12. Goodwin, A.C. et al. (2011) Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilisinduced colon tumorigenesis. Proc. Natl. Acad. Sci. U S A 108, 15354–15359 13. Bullman, S. et al. (2017) Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer. Science 358, 1443–1448 14. Rubinstein, M.R. et al. (2013) Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/beta-catenin signaling via its FadA adhesin. Cell Host Microbe 14, 195–206 15. Rosean, C.B. et al. (2019) Preexisting commensal dysbiosis is a host-intrinsic regulator of tissue inflammation and tumor cell dissemination in hormone receptor-positive breast cancer. Cancer Res. 79, 3662–3675 16. Hill, R. et al. (2010) PTEN loss accelerates KrasG12Dinduced pancreatic cancer development. Cancer Res. 70, 7114–7124 17. Thomas, R.M. et al. (2018) Intestinal microbiota enhances pancreatic carcinogenesis in preclinical models. Carcinogenesis 39, 1068–1078 18. Sethi, V. et al. (2018) Gut microbiota promotes tumor growth in mice by modulating immune response. Gastroenterology 155, 33–37 e36 19. Fan, X. et al. (2018) Human oral microbiome and prospective risk for pancreatic cancer: a populationbased nested case-control study. Gut 67, 120–127 20. Biedermann, L. et al. (2013) Smoking cessation induces profound changes in the composition of the intestinal microbiota in humans. PLoS One 8, e59260 21. Michaud, D.S. et al. (2013) Plasma antibodies to oral bacteria and risk of pancreatic cancer in a large European prospective cohort study. Gut 62, 1764– 1770 22. Wei, M.Y. et al. (2019) The microbiota and microbiome in pancreatic cancer: more influential than expected. Mol. Cancer 18, 97 23. Schulz, M.D. et al. (2014) High-fat-diet-mediated dysbiosis promotes intestinal carcinogenesis independently of obesity. Nature 514, 508–512 24. Ogrendik, M. (2017) Periodontal pathogens in the etiology of pancreatic cancer. Gastrointest. Tumors 3, 125–127 25. Ochi, A. et al. (2012) MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells. J. Exp. Med. 209, 1671– 1687 26. Zambirinis, C.P. et al. (2013) Induction of TRIF- or MYD88-dependent pathways perturbs cell cycle regulation in pancreatic cancer. Cell Cycle 12, 1153– 1154 27. Yoshimoto, S. et al. (2013) Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 499, 97–101 28. Seifert, L. et al. (2016) The necrosome promotes pancreatic oncogenesis via CXCL1 and Mincleinduced immune suppression. Nature 532, 245–249 29. Zambirinis, C.P. et al. (2015) TLR9 ligation in pancreatic stellate cells promotes tumorigenesis. J. Exp. Med. 212, 2077–2094
30. Geller, L.T. et al. (2017) Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine. Science 357, 1156–1160 31. Brahmer, J.R. et al. (2012) Safety and activity of antiPD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455–2465 32. O’Reilly, E.M. et al. (2019) Durvalumab with or without tremelimumab for patients with metastatic pancreatic ductal adenocarcinoma: a phase 2 randomized clinical trial. J. Clin. Oncol. 36 (4 Suppl), 217 33. Royal, R.E. et al. (2010) Phase 2 trial of single agent ipilimumab (anti-CTLA-4) for locally advanced or metastatic pancreatic adenocarcinoma. J. Immunother. 33, 828–833 34. Ritter, B. and Greten, F.R. (2019) Modulating inflammation for cancer therapy. J. Exp. Med. 216, 1234–1243 35. Feig, C. et al. (2013) Targeting CXCL12 from FAPexpressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc. Natl. Acad. Sci. U S A 110, 20212–20217 36. Jiang, H. et al. (2016) Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy. Nat. Med. 22, 851–860 37. Riquelme, E. et al. (2019) Tumor microbiome diversity and composition influence pancreatic cancer outcomes. Cell 178, 795–806 e712 38. Matson, V. et al. (2018) The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients. Science 359, 104–108 39. Derosa, L. et al. (2018) Negative association of antibiotics on clinical activity of immune checkpoint inhibitors in patients with advanced renal cell and non-small-cell lung cancer. Ann. Oncol. 29, 1437– 1444 40. Farrell, J.J. et al. (2012) Variations of oral microbiota are associated with pancreatic diseases including pancreatic cancer. Gut 61, 582–588 41. Torres, P.J. et al. (2015) Characterization of the salivary microbiome in patients with pancreatic cancer. PeerJ 3, e1373 42. Lu, H. et al. (2019) Tongue coating microbiome data distinguish patients with pancreatic head cancer from healthy controls. J. Oral Microbiol. 11, 1563409 43. Del Castillo, E. et al. (2019) The microbiomes of pancreatic and duodenum tissue overlap and are highly subject specific but differ between pancreatic cancer and noncancer subjects. Cancer Epidemiol. Biomarkers Prev. 28, 370–383 44. Crowley, E. et al. (2013) Liquid biopsy: monitoring cancer-genetics in the blood. Nat. Rev. Clin. Oncol. 10, 472–484 45. Qi, Z.H. et al. (2018) The significance of liquid biopsy in pancreatic cancer. J. Cancer 9, 3417–3426 46. Yadav, D.K. et al. (2018) Liquid biopsy in pancreatic cancer: the beginning of a new era. Oncotarget 9, 26900–26933 47. Zhou, J. et al. (2011) Marker expression in circulating cancer cells of pancreatic cancer patients. J. Surg. Res. 171, 631–636 48. Bailey, P. et al. (2016) Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531, 47–52 49. Zill, O.A. et al. (2015) Cell-free DNA next-generation sequencing in pancreatobiliary carcinomas. Cancer Discov. 5, 1040–1048
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