Endpoint titers of plasma antibodies at week 12 amongst vaccine responders

Supplemental Figure 3: EVL methylation levels of normal colon for patients with no history of adenoma or cancer after initial colonoscopy (left group) and for levels patients with an adenoma or colorectal cancer detected after initial (middle group). Patients with no follow-up colonoscopies (right group) were not included in the analyses for Comparison Set 2. %mEVL=percentage of DNA that is methylated at the EVL locus assayed.

Supplemental Table 6: Summary statistics for methylated EVL measurements in Comparison Set 2

Supplemental Figure 1. Flowchart of patient selection. * Subjects with hereditary conditions or inflammatory bowel disease (IBD) were excluded. A flowchart showing the selection process for the subjects included in the studies is shown. The primary exclusion criteria that affected subject selection were lack of serial colonoscopy data and lack of tissue from the normal colon

Supplemental Table 7: Summary statistics for methylated EVL measurements in Comparison Set 3

Supplemental Table 2: Summary patient characteristics for Comparison Set 1

Background Information on Advanced Adenoma

MUC1 expression and immune infiltrate in baseline and recurrent adenoma pairs

Supplemental Figure 2: Methylation levels of normal colon mucosa in patients with no history of adenoma or cancer ever (group on left) and levels for patients with a history of adenoma(s) or colorectal cancer at initial or a follow-up colonoscopy (group on right) in Comparison Set 1. %mEVL=percentage of DNA that is methylated at the EVL locus assayed.

Supplemental Table 1: Primer/probe sequences used in the duplex mEVL assay

Supplemental Table 4: Summary patient characteristics for Comparison Set 3

Supplemental Figure 4: Methylation levels of normal colon for patients with no history of adenoma or cancer after initial amongst those at least 4 years of follow-up (left group) and levels for patients with an adenoma or colorectal cancer detected after initial colonoscopy (middle group). Patients without an adenoma or colorectal cancer and less than 4 years of follow-up (right group) were not included in the analyses for Comparison Set 3. %mEVL=percentage of DNA that is methylated at the EVL locus assayed.

Supplemental Table 3: Summary patient characteristics for Comparison Set 2

MDSC Values by Vaccine Immune Response vs. Non-Response

Supplemental Table 5: Summary statistics for methylated EVL measurements in Comparison Set 1

supplementary methods: trial administration

AbstractBackground:Individuals with adenomatous colorectal polyps undergo repeated colonoscopy surveillance to identify and remove metachronous adenomas. However, many patients with adenomas do not develop recurrent adenomas. Better methods to evaluate who benefits from increased surveillance are needed. We evaluated the use of altered EVL methylation as a potential biomarker for risk of recurrent adenomas.Methods:Patients with ≥1 colonoscopy had EVL methylation (mEVL) measured with an ultra-accurate methylation-specific droplet digital PCR assay on normal colon mucosa. The association between EVL methylation levels and adenoma or colorectal cancer was evaluated using three case/control definitions in three models: unadjusted (model 1), adjusting for baseline characteristics (model 2), and an adjusted model excluding patients with colorectal cancer at baseline (model 3).Results:Between 2001 and 2020, 136 patients were included; 74 healthy patients and 62 patients with a history of colorectal cancer. Older age, never smoking, and baseline colorectal cancer were associated with higher levels of mEVL (P ≤ 0.05). Each log base 10 difference in mEVL was associated with an increased risk of adenoma(s) or cancer at/after baseline for model 1 [OR, 2.64; 95% confidence interval (CI), 1.09–6.36], and adenoma(s) or cancer after baseline for models 1 (OR, 2.01; 95% CI, 1.04–3.90) and model 2 (OR, 3.17; 95% CI, 1.30–7.72).Conclusions:Our results suggest that EVL methylation level detected in the normal colon mucosa has the potential to be a biomarker for monitoring the risk for recurrent adenomas.Impact:These findings support the potential utility of EVL methylation for improving the accuracy for assigning risk for recurrent colorectal adenomas and cancer.

AbstractPurpose: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.

Objective Vaccines against antigens expressed on adenomas could prevent new adenoma formation. We assessed whether a MUC1 peptide vaccine produces an immune response and prevents subsequent colonic adenoma formation. Design Multicenter, double blind, placebo-controlled randomized trial in individuals age 40-70 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 53 participants received the MUC1 vaccine and 50 placebo. 13/52 (25%) of MUC1 vaccine recipients had a ≥2-fold increase in MUC1 IgG (range 2.9-17.3) at week 12 vs. 0/50 placebo recipients (1-sided Fisher’s exact P<0.0001). Of the 13 responders at week 12, 11 (84.6%) had a ≥2-fold increase in MUC1 IgG with the booster and were considered immune responders. A recurrent adenoma was observed in 31 of 47 (66.0%) in the placebo group vs. 27 of 48 (56.3%) participants in the MUC1 group (adjusted relative risk (aRR) = 0.83 [95% CI, 0.60-1.14], P=0.25). Adenoma recurrence occurred in 3/11 (27.3%) immune responders, (aRR = 0.41 [95% CI, 0.15-1.11], P=0.08). Vaccine recipients had more injection site reactions than placebo recipients, but there was no difference in serious adverse events. Conclusion An immune response was observed only in vaccine recipients. Overall adenoma recurrence was not different than placebo, but a 38% absolute reduction in adenoma recurrence was observed in immune responders. [ClinicalTrials.gov][1] Identifier: [NCT02134925][2]. What is already known Antigens expressed on colonic adenomas are potential targets for immunopreventive vaccines. An effective vaccine could prevent subsequent adenoma formation. What this Study Adds In this multicenter, double blind, placebo-controlled randomized trial, MUC1 vaccine recipients developed an immune response. Overall adenoma recurrence was not different than placebo, but a 38% absolute reduction in adenoma recurrence was observed in immune responders. How this study might affect research, practice or policy Vaccine immunoprevention is a potential new frontier to colorectal cancer prevention. ### Competing Interest Statement RES: Research support from Freenome, Immunovia, and Exact Sciences LAB: None MCC: None AB: Research support from Freenome and Stella Diagnostics DK: Consultant Medtronic, Research support - Exact Sciences and Freenome CH: None LD: None SFK: None LMR: None ER: None AU: None ES: None AS: Founder, CEO and CSO of Oncovir JM: None PB: None RKP: Consultant for Alimentiv ADS: None CMJ: None RB: RB declares patents: (all provisional) PCT/US15/612657 (Cancer Immunotherapy), PCT/US18/36052 (Microbiome Biomarkers for Anti-PD-1/PD-L1 Responsiveness: Diagnostic, Prognostic and Therapeutic Uses Thereof), PCT/US63/055227 (Methods and Compositions for Treating Autoimmune and Allergic Disorders) BD: None RPM: None CS: None NRF: None DMZ: None PJL: Chief Medical Officer for Screening at Exact Sciences through a contracted services agreement with the Mayo Clinic. Dr. Limburg and Mayo Clinic have contractual rights to receive royalties through this agreement. OJF: Consultant: PDS Biotech, GeoVax, Immodulon, Ardigen, Invectys, Sparc, Isa Therapeutics ### Clinical Trial NCT02134925 ### Funding Statement This work is supported by contract HHSN261201200042I to the Mayo Clinic. Bioinformatics analysis was performed by Cancer Bioinformatics Services (CBS), supported in part by NCI through the UPMC Hillman Cancer Center CCSG award (P30CA047904). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of the following institutions: Mayo Clinic: PR13-002350-08 Dept Veterans Affairs, Kansas City, KS: AB0008 U. of Pittsburgh: CR19050206 Universidad de Puerto Rico: A2210214 Jefferson: #14F.274 Dana-Farber/Harvard Cancer Center: 14-323 gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: http://ClinicalTrials.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02134925&atom=%2Fmedrxiv%2Fearly%2F2022%2F10%2F05%2F2022.10.05.22280474.atom

Background: Immunoprevention via targeting antigens aberrantly expressed on colorectal cancers and adenomatous polyps offers the potential for a less invasive prevention strategy than endoscopic surveillance. Specificity of the immune response and long-term memory provide the potential for prolonged protection. Leveraging the infrastructure of the NCI-funded Cancer Prevention Network (CPN) consortium, we are conducting a double-blind randomized trial in individuals with a diagnosis of advanced colorectal adenomas within the previous year. We are evaluating MUC1 vaccine with the TLR-3 agonist polyICLC as an adjuvant, for its immunogenicity, ability to elicit immune memory and prevent recurrence of colorectal adenomas.Aims: We previously reported on the vaccine immunogenicity at week 12 (vaccine administered at 0, 2, and 10 weeks) compared to placebo. Here we report the ability of the vaccine to elicit long-term memory by measuring increase in anti-MUC1 antibody levels following a booster administered at one year.Methods: Subjects with endoscopically resected advanced adenoma (defined as ≥1cm, tubululovillous or villous histology, or with high grade dysplasia) were randomized. The primary endpoint was the response to the vaccine at week 12 assessed by monitoring anti-MUC1 IgG antibody titer ratio defined as t12/t0, where t0 was the titer pre-vaccination, and t12 was the titer at week 12. The key secondary endpoint was to assess anti-MUC1 IgG response at week 55 to a booster at week 52 in the vaccine arm compared to placebo.Results: 102 eligible subjects were randomized at 6 centers, 52 received MUC1 vaccine and 50 placebo. The mean age was 59.4±7.0 (range 40-70), 60.8% male, 88.2% white, and 18.6% Hispanic or Latino ethnicity. At 12 weeks the IgG ratio was ≥2.0 in 13/52 (25%) of individuals receiving vaccine (ratio range -0.5-17.3), vs. 0/50 in placebo group (2-sided p=.0001), and was ≥1.5 in 19/52 (36.5%) individuals receiving vaccine compared to 1/50 (2%) in placebo group (P<.0001). To evaluate immune memory, 1-year booster data available for 95 patients (51-MUC1, 44-placebo) were analyzed. 10/13 (76.9%) of the week 12 vs. week 0 vaccine responders showed a response at week 52 vs. baseline and 11/13 (84.6%) at week 55 vs. week 52. Using an IgG ratio of 2 (per-protocol), the response rate was 33% for MUC1 vs. 5% for placebo (1-sided p=0.0003). Using a ratio of 1.5 yielded similar results (37% MUC1 vs. 9% Placebo (1-sided p=0.0008). The MUC1 arm had significantly higher IgG at week 52 (1-sided p=0.0294), week 55 (1-sided p=0.0048), and significantly higher week 55 to week 52 IgG ratio values (1-sided p=0.0293).Conclusions: Subjects with a recent history of advanced adenoma receiving a MUC1 vaccine compared with a placebo were significantly more likely to develop an immune response at week 12, confirming the vaccine's immunogenicity, and to respond again at week 55 to a booster injection on week 52, confirming the vaccine's ability to elicit immune memory. Follow-up colonoscopy to evaluate the vaccine's potential to lower adenomatous polyp recurrence rates are in progress.Citation Format: Olivera J. Finn, Lisa Boardman, Marcia Cruz-Correa, Ajay Bansal, David Kastenberg, Chin Hurr, Sharon Kaufman, Colleen Akerley, Lynda Dzubinski, April Felt, Karrie Fursa, L.M. Rodriguez, Ellen Richmond, Asad Umar, Eva Szabo, John McKolanis, Ryan McMurray, Nathan Foster, Andres Salazar, Paul Limburg, Robert Schoen. Randomized, double-blind, placebo-controlled trial of preventative MUC1 vaccine in patients with newly diagnosed advanced adenomas: Results from one-year booster [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT236.