Randomized, Double-Blind, Placebo-Controlled Trial of MUC1 Peptide Vaccine for Prevention of Recurrent Colorectal Adenoma.

PURPOSE: To assess whether MUC1 peptide vaccine produces an immune response and prevents subsequent colon adenoma formation. PATIENTS AND METHODS: Multicenter, double-blind, placebo-controlled randomized trial in individuals age 40 to 70 with diagnosis of an advanced adenoma ≤1 year from randomization. Vaccine was administered at 0, 2, and 10 weeks with a booster injection at week 53. Adenoma recurrence was assessed ≥1 year from randomization. The primary endpoint was vaccine immunogenicity at 12 weeks defined by anti-MUC1 ratio ≥2.0. RESULTS: Fifty-three participants received the MUC1 vaccine and 50 placebo. Thirteen of 52 (25%) MUC1 vaccine recipients had a ≥2-fold increase in MUC1 IgG (range, 2.9-17.3) at week 12 versus 0/50 placebo recipients (one-sided Fisher exact P < 0.0001). Of 13 responders at week 12, 11 (84.6%) responded to a booster injection at week 52 with a ≥2-fold increase in MUC1 IgG measured at week 55. Recurrent adenoma was observed in 31 of 47 (66.0%) in the placebo group versus 27 of 48 (56.3%) in the MUC1 group [adjusted relative risk (aRR), 0.83; 95% confidence interval (CI), 0.60-1.14; P = 0.25]. Adenoma recurrence occurred in 3/11 (27.3%) immune responders at week 12 and week 55 (aRR, 0.41; 95% CI, 0.15-1.11; P = 0.08 compared with placebo). There was no difference in serious adverse events. CONCLUSIONS: An immune response was observed only in vaccine recipients. Adenoma recurrence was not different than placebo, but a 38% absolute reduction in adenoma recurrence compared with placebo was observed in participants who had an immune response at week 12 and with the booster injection.

Altered Expression of the Epithelial Mucin MUC1 Accompanies Endoscopic Recurrence of Postoperative Crohn's Disease.

BACKGROUND: MUC1-glycoprotein is expressed at low levels and in fully glycosylated form on epithelial cells. Inflammation causes MUC1 overexpression and hypoglycosylation. We hypothesized that overexpression of hypoglycosylated MUC1 would be found in postoperative Crohn's disease (CD) recurrence and could be considered an additional biomarker of recurrence severity. METHODS: We examined archived neo-terminal ileum biopsies from patients with prior ileocecal resection who had postoperative endoscopic assessment of CD recurrence and given a Rutgeerts ileal recurrence score. Consecutive tissue sections were stained using 2 different anti-MUC1 antibodies, HMPV that recognizes all forms of MUC1 and 4H5 that recognizes only inflammation-associated hypoglycosylated MUC1. RESULTS: A total of 71 postoperative CD patients were evaluated. There was significant increase in MUC1 expression of both glycosylated/normal (P<0.0001) and hypoglycosylated/abnormal (P<0.0001) forms in patients with severe endoscopic CD recurrence (i3+i4), ileal score i2, compared with patients in endoscopic remission (i0+i1). Results were similar regardless of anti-TNF-α use. Although MUC1 expression and Rutgeerts scores were in agreement when characterizing the majority of cases, there were a few exceptions where MUC1 expression was characteristic of more severe recurrence than implied by Rutgeerts score. CONCLUSIONS: MUC1 is overexpressed and hypoglycosylated in neo-terminal ileum tissue of patients with postoperative CD recurrence. Increased levels are associated with more severe endoscopic recurrence scores, and this is not influenced by anti-TNF-α use. Discrepancies found between Rutgeerts scores and MUC1 expression suggest that addition of MUC1 as a biomarker of severity of postoperative CD recurrence may improve categorization of recurrence status and consequently treatment decisions.

Myeloid derived suppressor cells in cancer, premalignancy and inflammation: A roadmap to cancer immunoprevention.

The ultimate success of any form of cancer therapy or cancer prevention depends on its ability to engage the power of the immune system to completely eliminate a growing tumor, lower the life-time tumor risk and establish long-term memory to prevent recurrence or future tumors. For that reason, all therapies but especially immunotherapies depend on the immune health (immunocompetence) of each treated individual. Cancer and chronic illnesses, combined with a usually more advanced age of cancer patients or those at risk for cancer are known to severely suppress multiple antitumor functions of the immune system. Understanding the critical mechanisms controlling and mediating immune suppression can lead to additional therapies to alleviate the effects of those mechanisms and improve the outcome of cancer therapy and prevention. We introduce and review here a highly immunosuppressive cell population found in cancer, precancer, and chronic inflammatory diseases, myeloid derived suppressor cells (MDSC). First described in the setting of advanced cancer, their presence and immunosuppressive activity has been seen more recently in early premalignant lesions and in chronic inflammatory diseases leading to cancer. We describe the detrimental effects of their presence on cancer immunotherapy, immunosurveillance and immunoprevention and review early attempts to develop drugs to eliminate them or reduce their negative impact.

Circulating Myeloid Derived Suppressor Cells (MDSC) That Accumulate in Premalignancy Share Phenotypic and Functional Characteristics With MDSC in Cancer.

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that accumulate in circulation of cancer patients and at tumor sites where they suppress anti-tumor immunity. We previously reported that in a colon cancer prevention trial of a MUC1 vaccine tested in individuals at increased risk for colon cancer, those who did not mount immune response to the vaccine had higher pre-vaccination levels of circulating MDSC compared to those who did. We also reported that individuals with pancreatic premalignancy, Intraductal Papillary Mucinous Neoplasm (IPMN), had increased circulating levels of MDSC that inversely correlated with spontaneous antibody responses against the pancreatic tumor associated antigen MUC1, abnormally expressed on IPMN. Accumulation of MDSC in cancer and their immunosuppressive role had been well established but their presence in premalignancy was unexpected. In this study we compared MDSC in premalignancy with those in cancer with the hypothesis that there might be differences in the composition of various MDSC subpopulations and their immunosuppressive functions due to different lengths of exposure to disease and/or different tissue microenvironments. In cohorts of patients with premalignant polyps, colon cancer, premalignant IPMN, and pancreatic cancer, we confirmed higher levels of MDSC in premalignancy compared to healthy controls, higher levels of MDSC in cancer compared to premalignancy, but no difference in their subpopulation composition or immunosuppressive capacity. We show that levels of MDSC in premalignancy correlate negatively in vivo with spontaneous MUC1-specific antibody responses and in vitro with polyclonal T cell proliferation and IFN-γ secretion.

Abnormally glycosylated MUC1 establishes a positive feedback circuit of inflammatory cytokines, mediated by NF-κB p65 and EzH2, in colitis-associated cancer.

The abnormal hypoglycosylated form of the epithelial mucin MUC1 is over-expressed in chronic inflammation and on human adenocarcinomas, suggesting its potential role in inflammation-driven tumorigenesis. The presence of human MUC1 aggravates colonic inflammation and increases tumor initiation and progression in an in vivo AOM/DSS mouse model of colitis-associated cancer (CAC). High expression levels of pro-inflammatory cytokines, including TNF-α and IL-6, were found in MUC1+ inflamed colon tissues. Exogenous TNF-α promoted the transcriptional activity of MUC1 as well as over-expression of its hypoglycosylated form in intestinal epithelial cells (IECs). In turn, hypoglycosylated MUC1 in IECs associated with p65 and up-regulated the expression of NF-κB-target genes encoding pro-inflammatory cytokines. Intestinal chronic inflammation also increased the expression of histone methyltransferase Enhancer of Zeste protein-2 (EzH2) and its interaction with cytokine promoters. Consequently, EzH2 was a positive regulator of MUC1 and p65-mediated IL-6 and TNF-α gene expression, and this function was not dependent on its canonical histone H3K27 methyltransferase activity. Our findings provide a mechanistic basis for already known tumorigenic role of the hypoglycosylated MUC1 in CAC, involving a transcriptional positive feedback loop of pro-inflammatory cytokines.

Immunobiology and immunosurveillance in patients with intraductal papillary mucinous neoplasms (IPMNs), premalignant precursors of pancreatic adenocarcinomas.

Premalignant lesions for many cancers have been identified, and efforts are currently directed toward identification of antigens expressed on these lesions that would provide suitable targets for vaccines for cancer prevention. Intraductal papillary mucinous neoplasms (IPMNs) are premalignant pancreatic cysts of which a subset has the potential to progress to cancer. Currently, there are no validated predictive markers for progression to malignancy. We hypothesized that the presence or absence of immune surveillance of these lesions would be one such factor. Here we show that the tumor antigen MUC1, which is abnormally expressed on pancreatic cancer and is a target for cancer immunosurveillance, is also abnormally expressed on premalignant IPMN. We show that some IPMN patients make MUC1-specific IgG. Moreover, we show evidence of CD4 and CD8 T cell infiltration into IPMN areas of high dysplasia suggesting an ongoing immune response within the lesions. We also found, however, increased levels of circulating myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in some IPMN patients as well as evidence of T cell exhaustion. Further studies correlating immunosurveillance or immunosuppression with IPMN progression to malignancy will help define the immune response as a biomarker of risk, leading potentially to a vaccine to boost spontaneous immunity and prevent progression to cancer.

Cancer immunoprevention.

Cancer immunotherapy is now a reality. The results are phenomenal but the cost is outrageous. Even if the cost eventually comes down and immunotherapy becomes more broadly available, using the knowledge derived from immunotherapy to apply to immunoprevention would be a good strategy. The most likely approach to cancer immunoprevention is cancer vaccines. To date, cancer vaccines have been tested mostly in the setting of advanced disease. Numerous immunosuppressive mechanisms have been identified in the tumor microenvironment as well as systemically that compromise the ability of cancer patients to respond to the vaccines. Multiple approaches are being tested to improve therapeutic cancer vaccine efficacy, including combinations with other immunotherapies. An alternative approach is to administer the vaccines to individuals without cancer but at high risk for cancer. Data in support of this approach and immunoprevention in general is accumulating and clinical testing has started.

Influenza virus infection elicits protective antibodies and T cells specific for host cell antigens also expressed as tumor-associated antigens: a new view of cancer immunosurveillance.

Most tumor-associated antigens (TAA) are self-molecules that are abnormally expressed in cancer cells and become targets of antitumor immune responses. Antibodies and T cells specific for some TAAs have been found in healthy individuals and are associated with lowered lifetime risk for developing cancer. Lower risk for cancer has also been associated with a history of febrile viral diseases. We hypothesized that virus infections could lead to transient expression of abnormal forms of self-molecules, some of which are TAAs; facilitated by the adjuvant effects of infection and inflammation, these molecules could elicit specific antibodies, T cells, and lasting immune memory simultaneously with immunity against viral antigens. Such infection-induced immune memory for TAA would be expected to provide life-long immune surveillance of cancer. Using influenza virus infection in mice as a model system, we tested this hypothesis and demonstrated that influenza-experienced mice control 3LL mouse lung tumor challenge better than infection-naive control mice. Using 2D-difference gel electrophoresis and mass spectrometry, we identified numerous molecules, some of which are known TAAs, on the 3LL tumor cells recognized by antibodies elicited by two successive influenza infections. We studied in detail immune responses against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H4, HSP90, malate dehydrogenase 2, and annexin A2, all of which were overexpressed in influenza-infected lungs and in tumor cells. Finally, we show that immune responses generated through vaccination against peptides derived from these antigens correlated with improved tumor control.

Preventing cancer by targeting abnormally expressed self-antigens: MUC1 vaccines for prevention of epithelial adenocarcinomas.

Prophylactic vaccines based on tumor-associated antigens (TAAs) have elicited concerns due to their potential toxicity. Because TAAs are considered self-antigens, the prediction is that such vaccines will induce autoimmunity. While this has been observed in melanoma, where an antitumor immune response leads to vitiligo, autoimmunity has almost never been seen following vaccination with numerous other TAAs. We hypothesized that antigen choice determines outcome and have been working to identify TAAs whose expression differs between normal and tumor tissue, and thus could elicit antitumor immunity without autoimmunity. Studies on the epithelial TAA MUC1 have revealed that, compared to MUC1 on normal cells, tumors, premalignant lesions, and noncancerous pathologies affecting epithelial cells express abnormal MUC1, which is not a self-antigen but rather an abnormal disease-associated antigen (DAA). This distinction, which can be made for many known TAAs, has broad implications for the design and acceptance of preventative cancer vaccines.

Prevention of colitis-associated colon cancer using a vaccine to target abnormal expression of the MUC1 tumor antigen.

Association between chronic inflammation and cancer development is exemplified by inflammatory bowel disease (IBD) where patients with chronic uncontrolled colitis have a significantly increased risk of developing colitis-associated colorectal cancer (CACC). CACC appears to progresses through the inflammation-dysplasia-carcinoma sequence. This highlights the need to identify targets and interventions that reduce inflammation and prevent development of dysplasia in the context of IBD. Using the dextran sulfate sodium (DSS) mouse model of chronic colitis and CACC, we show that an important target of intervention in human disease would be the epithelial cell molecule MUC1 that is aberrantly expressed on inflamed colonocytes and promotes inflammation and progression to CACC. We show that a MUC1 vaccine can ameliorate chronic colitis and prevent development of dysplasia in the colon and thus extend survival in human MUC1 transgenic mice. This study supports the potential of prophylactic vaccines to target antigens that become aberrantly expressed in chronic inflammation (e.g., IBD) and continue to be expressed on the associated cancers (e.g., colon cancer), to prevent and/or treat both diseases.

Importance of MUC1 and spontaneous mouse tumor models for understanding the immunobiology of human adenocarcinomas.

Many important aspects of cancer biology, such as cancer initiation, progression, and metastasis, have been studied in animal models, mostly mice. As long as cancer was considered primarily a genetic disease, the study of transplantable mouse tumors, or even human tumor xenografts in immunocompromised mice, appeared to suffice. Many important genetic events that lead to transformation and in vivo tumor growth were elucidated. However, many even more important factors that determine whether or not the genetic potential of a tumor cell will be realized, such as the host response to the tumor and the tumor microenvironment that influences this response over a long period of time of tumor development, remained untested and unappreciated. This is slowly changing with the advent of molecular techniques that have spurred efforts to engineer better mouse models of human tumors. In this review, we show results of our efforts to combine a genetic mouse model of spontaneous human adenocarcinomas based on a Kras mutation, with an important human molecule MUC1 that is abnormally expressed on human adenocarcinomas, promoting oncogenesis, proinflammatory tumor microenvironment, and immunosurveillance.

Tumor immunology: basic and clinical advances.

The third in a series of AACR conferences, entitled "Tumor Immunology: Basic and Clinical Advances," was held in Miami Beach, Florida from November 30 to December 3, 2010. The overall objective of this meeting was to discuss rapid developments in the understanding of basic principles of antitumor immunity and strategies for increasing the success rate of cancer immunotherapy. The key findings that emerged from the meeting included (i) that integrated approaches are required for the development of effective cancer immunotherapies and (ii) attention should be on multiple cellular and molecular components and their broader networks rather than on a single pathway or cell type.

Vaccine against MUC1 antigen expressed in inflammatory bowel disease and cancer lessens colonic inflammation and prevents progression to colitis-associated colon cancer.

Association of chronic inflammation with an increased risk of cancer is well established, but the contributions of innate versus adaptive immunity are not fully delineated. There has furthermore been little consideration of the role played by chronic inflammation-associated antigens, including cancer antigens, and the possibility of using them as vaccines to lower the cancer risk. We studied the human tumor antigen MUC1 which is abnormally expressed in colon cancers and also in inflammatory bowel disease (IBD) that gives rise to colitis-associated colon cancer (CACC). Using our new mouse model of MUC1(+) IBD that progresses to CACC, interleukin-10 knockout mice crossed with MUC1 transgenic mice, we show that vaccination against MUC1 delays IBD and prevents progression to CACC. One mechanism is the induction of MUC1-specific adaptive immunity (anti-MUC1 IgG and anti-MUC1 CTL), which seems to eliminate abnormal MUC1(+) cells in IBD colons. The other mechanism is the change in the local and the systemic microenvironments. Compared with IBD in vaccinated mice, IBD in control mice is dominated by larger numbers of neutrophils in the colon and myeloid-derived suppressor cells in the spleen, which can compromise adaptive immunity and facilitate tumor growth. This suggests that the tumor-promoting microenvironment of chronic inflammation can be converted to a tumor-inhibiting environment by increasing adaptive immunity against a disease-associated antigen.

Inflammation driven by overexpression of the hypoglycosylated abnormal mucin 1 (MUC1) links inflammatory bowel disease and pancreatitis.

OBJECTIVE: Pancreatitis occurs as an extraintestinal complication of inflammatory bowel disease (IBD), but the cause is poorly understood. Mucin 1 (MUC1) is overexpressed in an abnormal, hypoglycosylated form on the colonic epithelium in human IBD where it contributes to inflammation. MUC1 is also expressed on pancreatic ductal epithelia. We tested the possibility that in IBD, MUC1 expression on pancreatic ducts is also abnormal leading to inflammation and pancreatitis. METHODS: We used MUC1/interleukin-10 mice that develop IBD. We imaged abnormal MUC1 expression in these mice by adoptively transferring T cells from T cell receptor transgenic mice specific for abnormal MUC1. Cells were labeled with a novel perfluorocarbon tracer reagent and quantified and visualized in vivo using high-throughput F nuclear magnetic resonance spectroscopy and magnetic resonance imaging. RESULTS: MUC1-specific T cells migrated to the colon in mice with IBD and also to the pancreas. Immunohistochemistry confirmed increased expression on the pancreatic ducts of the abnormal MUC1 seen in the colon and the presence of cellular infiltrate. CONCLUSIONS: Migration of MUC1-specific T cells to the colon and the pancreas in diseased mice suggests that pancreatitis is an extraintestinal site of IBD, characterized by proinflammatory abnormal expression of MUC1. Therapies directed against abnormal MUC1 have the potential of targeting the disease in both sites.

Cutting edge: transgenic expression of human MUC1 in IL-10-/- mice accelerates inflammatory bowel disease and progression to colon cancer.

Epithelial cell MUC1 is aberrantly expressed on human epithelial adenocarcinomas where it functions as a regulator of immune responses and an oncogene. Normally expressed at low levels in healthy colonic epithelium, MUC1 was reported to be overexpressed in human inflammatory bowel disease (IBD) and thus may be expected to play an important role in regulating chronic inflammation and its progression to colitis-associated colon cancer. Studies in the immunobiology and pathology of IBD and colitis-associated colon cancer have been done in various mouse models but none could properly address the role of MUC1 due to low homology between the mouse and the human molecule. We report that IL-10(-/-) mice, a widely accepted mouse model of IBD, crossed to human MUC1-transgenic mice, develop MUC1(+) IBD characterized by an earlier age of onset, higher inflammation scores, and a much higher incidence and number of colon cancers compared with IL-10(-/-) mice.