Abstract Background Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. Methods We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. Results Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. Conclusions These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC. Graphical abstract

Background Non-human primates (NHP) such as rhesus macaques with naturally occurring cancers are a proposed model for translational cancer immunotherapy (CIT) research and have generated relevant proof-of-mechanism evidence for 3 different CIT agents. NHP spontaneously develop cancers with progression patterns, histology, and clinical symptoms similar to humans. Gene suppression by DNA hypermethylation in the promoter region is the major characteristics of the CpG-island-methylator-phenotype (CIMP) described in human CRC patients but information in rhesus macaques is scarce. To further validate these animals as translational models for CIT, we conducted a deep molecular characterization of NHP colorectal cancers and established novel qPCR panels to assess DNA methylation of marker genes published for humans. Methods Our cohort (n=16) consisted of Indian-origin rhesus macaques (Macaca mulatta) with naturally occurring CRC (n=16, female=11). Clinical examination, imaging (contrast-enhanced CT, FDG-PET) and biopsy to confirm cancer histology were performed. Molecular characterization was done by IHC for CRC-associated mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 and by PCR/electrophoresis for microsatellite instability. Ultimately, we designed DNA methylation- and rhesus-specific qPCR probes (Methylight) targeting corresponding regions as published in human patients, including CACNA1G, CDKN2A, CRABP1, MLH1, and NEUROG1 as parts of the CIMP panel and BMP3, NDRG4, and SEPTIN9 as probed for human CRC-screening. Results MLH1 deficiency by IHC, in conjunction with PMS2 absence, is observed in all NHP CRC cases, clearly exceeding frequencies reported in human CRCs (ranging from 2-15%). Moreover, we have documented microsatellite instable cases in some NHP CRCs, analog to human CRCs. DNA methylation of the MLH1 promoter region was significantly elevated in CRCs (100% of CRCs >2-fold, p<0.0001) compared to healthy colon. We hypothesized that this elevation would suppress MLH1 mRNA expression. This hypothesis of epigenetic suppression is corroborated by both qPCR and RNA sequencing which demonstrate significantly downregulated levels of MLH1 mRNA. DNA methylation of the other markers is less consistent but revealed CIMP positive and CIMP negative cases in our NHP CRC cohort. Conclusions Transcriptional suppression of MLH1 by promoter hypermethylation is a major and widespread driver of genetic instability and carcinogenesis in rhesus macaque colorectal cancer. Differential DNA methylation in the promoter regions as observed in NHP CRCs can provide a screening target for liquid biopsies. This work highlights the possible translatability of naturally occurring NHP cancers for human cancer immunotherapy research and can be further explored in future tumor-bearing monkey trials. Citation Format: Simon Deycmar, Brendan Johnson, Declan Ryan, Shane Sills, David Caudell, Greg Dugan, George Schaaf, Christopher Whitlow, Kiran Kumar Solingapuram Sai, Betsy Ferguson, Benjamin Bimber, Karina Ray, Cassandra Cullin, Brandy Dozier, Armando Burgos, Michael Hettich, Bruno Gomes, Jehad Charo, Maurizio Ceppi, Mark Cline. Naturally occurring colorectal cancer in nonhuman primates used to study human immunotherapeutic agents confirms a link between DNA methylation and mismatch repair deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1325.

Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and emerge at similar life stages as in human patients, but the carcinogenesis and molecular background remain elusive, limiting the value of the model for human disease and cancer therapy. In order to better understand this naturally occurring model, we established a diagnostic and staging pipeline including imaging (18F-FDG PET, plain and contrast-enhanced CT), histopathology, and clinical assessments. We observed cancer symptoms and co-morbidities in our cohort (n=16, all Indian-origin, 11 females, mean age at arrival 20.2y) such as hypoalbuminemia, fecal occult blood, and microcytic anemia, as frequently described in human CRC patients. Pathologically, all cancers were right-sided, involving the proximal colon and/or ileocecocolic junction, and most were densely fibrotic, restricting the colonic lumen. Most cancers appeared with glandular morphology and some (18.8%) had mucinous components. Immunohistochemistry revealed loss of MLH1 and PMS2 in 100% of investigated CRCs, indicating mismatch repair deficiency which furthermore resulted in microsatellite instability (PCR & fragment analysis). Whole exome sequencing revealed the close genetic relatedness to human CRCs, particularly exemplified by mutations affecting KRAS (37.5%, e.g., p.G12D), APC (31.3%), TP53 (18.8%, e.g. p.R175H), ARID1A (56.3%), and ALK (43.8%), as similarly annotated in the human COSMIC database. Nonetheless, somatic mutations do not explain the loss of MLH1 in the entire CRC cohort. Transcriptomics on the other hand revealed the transcriptional suppression of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRC in comparison to adjacent healthy colon. Moreover, comparison of differentially expressed gene sets of rhesus CRC with a human annotated database (IPA) confirmed the disease similarities observed clinically, genetically, and histopathologically. Subsequently, we investigated DNA methylation of the promoter region of MLH1 and retranslated markers for the CpG island methylator phenotype (CIMP) as described in human CRC. While only 56.3% of CRCs were considered CIMP positive (≥3/5 markers hypermethylated), 100% of investigated CRCs exhibited MLH1 promoter hypermethylation. As a result, epigenetic silencing is suggested to suppress MLH1 transcription, cause the loss of MLH1 protein, and drive mismatch repair deficiency and genomic instability in naturally occurring CRC in rhesus macaques. We therefore consider spontaneous, uninduced CRC in rhesus macaques, their treatment-naïve nature, and their unaltered immune competence an outstanding model for human disease and in particular for human cancer immunotherapy. Citation Format: Simon Deycmar, Brendan Johnson, Karina Ray, David Caudell Caudell, John Olson, Greg Dugan, W. Shane Sills, Declan Ryan, Christopher Whitlow, Kiran K. Solingapuram Sai, Betsy Ferguson, Benjamin Bimber, Cassandra Cullin, Brandy Dozier, Emily Romero, Francois Villinger, Armando Burgos, Jeff Chou, Bruno Gomes, Michael Hettich, Maurizio Ceppi, Jehad Charo, J. Mark Cline. Clinical and molecular characterization of naturally-occurring colorectal cancer in rhesus macaques reveals mismatch repair deficiency driven by epigenetic silencing of MLH1 [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A021.

Feline Infectious Peritonitis (FIP) is a deadly viral disease affecting cats that are carriers of the almost ubiquitous feline enteric coronavirus (FECV). Infection with FECV is mostly asymptomatic and transient, but it can mutate into FIP virus (FIPV) causing nonspecific, and ultimately fatal clinical signs. Diagnostic testing for FIP is often ambiguous and usually presumptive, with most available tests unable to distinguish between the two different pathotypes (FECV vs. FIPV). A definitive diagnosis is usually made by biopsy and examination of tissue post-mortem. The aim of this study is to develop and optimize a flow cytometric assay for the detection of FIPV within fluid macrophages. Felis catus whole fetus (fcwf) cells are grown in standard media. Cells are infected with FIPV, along with sham-infected controls. Then, cells are harvested, fixed, permeabilized, and stained with anti-vimentin and anti-feline coronavirus antibodies and analyzed by flow cytometry. Preliminary results show that flow cytometry detects stained FIPV within fcwf cells, and eventually, we expect this within fluid macrophages from feline patients suspected of having FIP. We anticipate that our flow cytometrybased assay for FIP will be more diagnostically reliable (higher specificity) than currently available assays. A definitive antemortem diagnosis is crucial to veterinarians and clients when it comes to making treatment or euthanasia decisions surrounding FIP. The next steps for this study include testing fluid samples from suspected FIP patients in a clinical diagnostic trial.

BackgroundNon-human primates (NHP) with naturally occurring cancers (also called tumor-bearing monkeys or TBM) are a proposed model for translational cancer immunotherapy (CIT) research.1 TBM spontaneously develop cancers with progression patterns similar to humans, potentially bridging the gap between preclinical models and cancers in patients. Interventional CIT trials recently conducted in colorectal (CRC) and breast cancer (BC)-bearing NHP, have generated relevant proof-of-mechanism evidence for three different CIT agents.1–3 To further validate these animals as translational models for CIT, we conducted a deep molecular characterization of tumors at baseline and reverse translated biomarker assays employed in human patients.MethodsOur cohort (n=19) consisted of Indian-origin rhesus macaques (Macaca mulatta) with naturally occurring CRC (n=14, female=9, male=5) and BC (n=5, female=5). Clinical examination, imaging (contrast-enhanced CT, PET) and biopsy to confirm cancer histology were performed. Molecular characterization was done by IHC for CRC-associated mismatch repair (MMR) proteins MLH1, MSH2, MSH6, and PMS2 and BC markers ER, PR, and HER2. We assessed microsatellite instability (MSI) by PCR and electrophoresis, and for selected cases somatic tumor mutations and tumor mutational burden (TMB) by whole exome sequencing.ResultsDeficiency in MMR proteins determines eligibility for PD-1 blockade therapy, is observed in approximately 15% of human CRCs, and surprisingly in 100% (14/14) of our NHP CRCs. The absence of MLH1 (14/14), MSH2 (1/14), MSH6 (0/14) and PMS2 (14/14) observed in NHP CRCs clearly exceeds the frequencies reported in human CRCs ranging from 2–15% for each individual MMR protein.4 5 Moreover, we have documented MSI cases in some NHP CRCs, as described in human CRCs. We sequenced 3 CRCs and observed mutations in KRAS (G12D & A59T), WNT7A (V238M), IDH2 (R362Q), AKT3 (R388H), and TMB of 4.27, 22.95, and 29.3 mut/Mbp. Regarding breast, we found hormone receptor positive (Luminal A), HER2 positive, and TNBC, as in human BC patients. Sequencing of 2 BCs revealed mutations in PTEN (G251V), TGFBR2 (L162P), and ERBB4 (R1250Q), and TMB of 2.32 and 17.22 mut/Mbp.ConclusionsNHP cancers can be similarly characterized as human cancers, both macroscopically and molecularly. In this study we demonstrated an overrepresentation of MMR deficiency in NHP CRCs. Receptor expression in NHP BCs revealed similar subtypes as in human BCs. Cancer-associated mutations described in humans are also evident in TBM. This work highlights the possible translatability of naturally occurring NHP cancers for human cancer immunotherapy research, and can be further explored in future TBM trials.ReferencesCeppi M, Hettich M, Teichgraeber V, Driessen W, Tuerck D, et al. Tumor-bearing non-human primates: an unrivaled model for translational cancer immunology research. Proceedings: AACR Annual Meeting 2020.Claus C, Ferrara C, Xu W, Sam J, Lang S, Uhlenbrock F, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med 2019;11(496).Waldhauer I, Gonzalez-Nicolini V, Freimoser-Grundschober A, Nayak TK, Fahrni L, et al. Simlukasfusp alfa (FAP-IL2v) immunocytokine is a versatile combination partner for cancer immunotherapy. MAbs 2021;13(1).Parc Y, Gueroult S, Mourra N, Serfaty L, Flejou J-F, Tiret E, Parc R. Prognostic significance of microsatellite instability determined by immunohistochemical staining of MSH2 and MLH1 in sporadic T3N0M0 colon cancer. Gut 2004;53.Chen L, Chen G, Zheng X, Chen Y. Expression status of four mismatch repair proteins in patients with colorectal cancer: clinical significance in 1238 cases. Int J Clin Exp Pathol 2019;12(10).Ethics ApprovalWake Forest University is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) and registered with the United States Department of Agriculture (USDA) to conduct research in laboratory animals. The protocols and any subsequent amendments are reviewed and approved by the Wake Forest Institutional Animal Care and Use Committee (IACUC) and in compliance with the U.S. Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, the Office of Laboratory Animal Welfare, and public health service regulations.