Introduction

Whether using immunomodulators (IMM) or biologics after a first cancer in Inflammatory Bowel Disease patients (pts) may increase the risk of new or recurrent cancer is undefined.

Aims & Methods

The primary aim was to assess the frequency of new or recurrent cancer in IBD pts treated with IMM or biologics after a previous diagnosis of cancer. The secondary aim was to evaluate the cancer-related mortality in IBD pts treated with IMM and biologics after the first cancer. In a retrospective, multicentre study, clinical records of all IBD pts treated with IMM or biologics after a previous diagnosis of cancer were reviewed. Inclusion criteria:1) Diagnosis of Crohn’s Disease (CD) or Ulcerative Colitis (UC); 2)Cancer history;3)IMM or biologics used after a first cancer;4)IBD and cancer data recorded; 5)Follow-up after cancer ≥4 months (mos). Exclusion criteria:1) IMM used for ≤3 mos. Data considered: IBD features,cancer type,IBD duration, age at diagnosis of cancer, follow-up after cancer,cancer-related death.Data expressed as median [range].

Results

75 IBD pts (57 CD,18 UC) treated with IMM or biologics after a first diagnosis of cancer were enrolled (n [%]: F 36 [48%];age 61 [26-89] yrs).CD features included (n=):L1 (37);L2 (2);L3 (16);L4 (8);B1 (17);B2 (30);B3 (10).UC extent included: E1 (2),E2 (8), E3 (8). Cancer in IBD pts treated with IMM/biologics included (n [%]):basal cell carcinoma (BCC) 14 (18.4%), breast 9 (12%),colorectal 9 (12%),melanoma 8 (10.7%),prostatic or testicular 7 (9.3%), urinary tract 7 (9.3%),thyroid 6 (8%),squamous cell carcinoma (SCC) 4 (5.3%),endometrial 2 (2.7%),hematopoietic 4 (5.3%),others 7 (9.3%).In 2 (2.7%) pts, ≥2 cancers occurred. The median time interval between index cancer and ISS/biologics use was 38 mos [0-428]. IMM after cancer were used for a median of 32.5 [4-131] mos, including: methotrexate (MTX) in 13 (17.3%),thiopurines in 8 (10.7%),mycophenolate mofetil,tacrolimus or everolimus in 3 (4%) pts.Biologics were used after cancer for a median of 36 [3-194] mos:Ustekinumab (UST) in 24 (32%),Vedolizumab (VDZ) in 31 (41.3%),Adalimumab (ADA) in 17 (22.7%), Infliximab (IFX) in 12 (16%),Tofacitinib (Tofa) in 2 (2.7%),Golimumab in 1 (1.3%) pt. Both IMM and biologics were used after a first cancer in 15 (20%),while ≥2 biologics in 16 (%) pts.In a median follow up of 95 [4-509] mos after the first cancer, 8 (10.7%) IBD pts developed a new and/or recurrent cancer after IMM/biologics use.In particular, cancer recurred in 3 (4%) pts (1 melanoma,1 prostatic,1 colonic adenocarcinoma), while new cases of cancer occurred in 6 (8%) pts,all 6 represented by non-melanoma skin cancer (NMSC) (both recurrent and new cancer in 1 pt).A not significant trend for a higher frequency of new vs recurrent cancer (p=0.47) was observed.No cancer-related death were observed.

New and recurrent cancers after IMM/biologics

Conclusion

In a retrospective multicentre study, a low frequency of recurrent cancer was observed in IBD pts using IMM or biologics after the first cancer. A not significant trend for a higher frequency of new vs recurrent cancer (p=0.47) was observed, being almost all new cancers represented by NMSC.

Introduction

Decreased vagus nerve activity and increased inflammatory immune cells are observed in ulcerative colitis (UC) (1, 2). Due to its anti-inflammatory and protective properties, vagus nerve stimulation (VNS) has garnered attention for UC therapy (3). Non-invasive transcutaneous auricular vagus nerve stimulation (NitaVNS), devoid of the side effects associated with invasive techniques, demonstrates protective anti-inflammatory effects in our mouse UC-like model of colitis (4), although the specific cellular mechanisms remain unclear.

Aims & Methods

To evaluate the effects of NitaVNS on the distribution of granulocytes, macrophages, natural killer (NK) cells, and CD4⁺ and CD8⁺ T cells in the colon tissue, spleen, and mesenteric lymph nodes of a UC-like colitis mouse model, NitaVNS was administered to C57BL/6 male mice daily for 10 min, beginning one day before colitis induction with 5% dextran sulfate sodium for 5 days in water (the control group received regular water). This continued until the end of the study, with no stimulation in sham mice. The daily disease activity index (DAI) was evaluated along with distal macroscopic scores at the time of sacrifice. The frequencies of granulocytes (Ly6G), macrophages (F4/80), NK cells (NK1.1), CD4⁺ T cells (CD3, CD4), and CD8⁺ T cells (CD3, CD8) were analyzed in the cecum, proximal, mid and distal colon, spleen, and mesenteric lymph nodes (MLNs) using flow cytometry.

Results

NitaVNS in colitic mice significantly improved the DAI and macroscopic scores related to rectal bleeding, colon bleeding, fecal consistency loss, and shortened colon length compared to sham-stimulated colitic mice. In contrast, no changes were observed in the non-colitic mice. While NitaVNS did not alter the frequency of Ly6G⁺ cells in the cecum, mid/distal colon, and MLNs, it significantly decreased the positivity of these cells in the proximal colon and spleen of mice with colitis without affecting non-colitic mice. NitaVNS significantly decreased the frequency of F4/80⁺ cells in the mid-colon but did not influence these cells in the cecum, proximal and distal colon, spleen, or MLNs. In colitic conditions, NitaVNS significantly increased NK cell frequency in the spleen without affecting non-colitic mice. Moreover, NitaVNS did not alter the frequency of NK cells in the colon or MLNs in colitic or non-colitic mice. Additionally, NitaVNS did not induce changes in CD4⁺ and CD8⁺ T cell distribution in the colon tissue, spleen, or mesenteric lymph nodes of colitic and non-colitic mice.

Conclusion

These results suggest that NitaVNS could potentially reduce inflammation in colitic mice, locally and systemically, by decreasing the frequency of granulocytes in the colon and spleen and colonic macrophages. Additionally, NitaVNS increased the number of splenic NK cells, potentially contributing to its systemic effects. These findings indicate promising therapeutic potential for NitaVNS.

References

1. Ghia JE, Blennerhassett P, Kumar-Ondiveeran H, Verdu EF, Collins SM. The vagus nerve: a tonic inhibitory influence associated with inflammatory bowel disease in a murine model. Gastroenterology. 2006;131(4):1122-30.
2. Wiese JJ, Manna S, Kühl AA, Fascì A, Elezkurtaj S, Sonnenberg E, Bubeck M, Atreya R, Becker C, Weixler B, Siegmund B, Patankar JV, Prüß MS, Schumann M. Myenteric Plexus Immune Cell Infiltrations and Neurotransmitter Expression in Crohn's Disease and Ulcerative Colitis. J Crohns Colitis. 2024 Jan 27;18(1):121-133.
3. Verma N, Mudge JD, Kasole M, Chen RC, Blanz SL, Trevathan JK, Lovett EG, Williams JC, Ludwig KA. Auricular Vagus Neuromodulation-A Systematic Review on Quality of Evidence and Clinical Effects. Front Neurosci. 2021 Apr 30;15:664740.
4. Hesampour F, Tshikudi D, Veysel Özden A, Bernstein CN, Ghia JE. A54 The impact of non-invasive transcutaneous auricular nerve stimulation (NitaVNS) on the development of experimental colitis in mice. J Can Assoc Gastroenterol. 2023 Mar 7;6(Suppl 1):30.

Disclosure

F. Hesampour: None Declared, D. Tshikudi: None Declared, A. Veysel Özden: The employee of the company providing the stimulator, C. N. Bernstein: None Declared, J-E. Ghia: None Declared.

Introduction

Fibrosis in Crohn´s disease (CD) is a complication of chronic inflammatory processes that may result in impaired gastrointestinal function and bowel strictures. However, exact pathomechanisms leading to intestinal fibrosis in CD are not fully understood. Since the de novo DNA methyltransferase DNMT3A is involved in cellular differentiation processes and genetic variants have been associated with increased risk for CD, we sought to characterize how DNMT3A loss might affect fibroblasts, the key mesenchymal cells involved in fibrosis.

Aims & Methods

We stably deleted Dnmt3a in 3T3 fibroblast cell line using CRISPR-Cas9 technology and subsequently selected GPF-positive clones using FACS sorting. Cell cultures were performed in cell culture plates with or without 0.2% gelatin coat. Reverse transcription quantitative PCR, western blot, and immunofluorescence were used to confirm Dnmt3a gene deletion and characterize wild-type (WT) and knockout (KO) cells. Cell adhesion was evaluated by fluorescence levels of calcein acetoxymethyl ester (CAM)-labelled cells that attached to cell culture plates after 1h of culture and subsequent wash. Collagen lattice assays in presence of proinflammatory and profibrotic stimuli served to assess fibroblast contractibility.

Results

3T3 fibroblasts lacking Dnmt3a acquire an activated myofibroblast phenotype (increased alpha-SMA expression) that also overexpress tissue remodeling matrix metalloproteinase Mmp3, and the inflammation-induced necroptosis marker RIPK3 when compared to their WT counterparts. In addition, Dnmt3a-depleted 3T3 cells adhere more strongly to cell culture plates. In presence of transforming growth factor (TGF)-beta, cells embedded in a 3D collagen matrix and lacking Dnmt3a build an interconnected cellular network and contract up to a 77.78% (compared to interconnected cells that reach up to 13.73% contraction by WT cells). This contraction was independent of the presence of interleukin (IL)-11. Profibrotic factors such as bleomycin prevent intercellular network formation in both WT and KO cells; hence, incapacitating cells to contract the collagen matrix.

Conclusion

We conclude that Dnmt3a is crucial for fibroblast homeostasis, including cell activation and functionality. Furthermore, TGF-beta induces higher fibroblast contractibility when Dnmt3a is lacking. Thus, additional characterization of Dnmt3a depletion in fibroblasts will deepen the understanding of fibrosis and may point towards new therapeutic approaches for this untreatable complication.

References

1. Hampe J, Franke A, Rosenstiel P, et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet. 2007;39(2):207-211. doi:10.1038/ng1954
2. Fazio A, Bordoni D, Kuiper JWP, et al. DNA methyltransferase 3A controls intestinal epithelial barrier function and regeneration in the colon. Nat Commun. 2022;13(1):6266. doi:10.1038/s41467-022-33844-2

Disclosure

The authors have no disclosures.

Introduction

Checkpoint agonism represents a promising class of therapies for the treatment of autoimmune and inflammatory diseases, including ulcerative colitis (UC), where unmet needs persist. Engagement of checkpoint receptor PD-1 by its natural ligand PD-L1 is critical for regulation of T cells. Rosnilimab is an investigational PD-1 agonist IgG1 antibody designed to optimize inhibitory signaling through PD-1, does not block PD-1 binding to PD-L1, and depletes PD-1^high pathogenic T cells while agonizing remaining PD-1+ T cells. The percentages of PD-1+ T cells in the peripheral blood of moderate and severe UC patients are higher than in mild UC patients (1-4). Reduction of elevated PD-1^high Tph cells in both UC colon and peripheral blood correlates with remission (2). Therapeutic reduction of PD-1^high Tph cells and inflammatory cytokines from PD-1+ T cells, by depletion and agonism signaling together, has the potential to reduce overactive immune responses and induce remission.

Aims & Methods

The potency of the agonism and depletion mechanisms of rosnilimab was evaluated in vitro using peripheral blood mononuclear cells (PBMCs) from UC patients. Rosnilimab is a human IgG1 antibody with Fc effector function. A version of rosnilimab that lacks Fc effector function was generated by mutating the IgG1 Fc domain at L234A and L235A (LALA). PBMCs from UC patients were activated with anti-CD3 and anti-CD28, cultured for several days with rosnilimab IgG1, rosnilimab IgG1-LALA, or isotype control and analyzed for the number of PD-1+ T cells and levels of inflammatory cytokines. In addition, the therapeutic potential of depletion and agonism of pathogenic PD-1+ T cells was evaluated in a mouse model of UC using a surrogate of rosnilimab. The surrogate consists of the fab domains of rosnilimab formatted with a mouse IgG2a Fc domain which enables immune cell depletion in mice. On Day 0, hPD1 CD4+CD45Rb+ T cells donor cells were adoptively transferred into Rag2-/- recipients to elicit UC-like disease. Starting on Day 21, 10 mpk of isotype control mIgG2a, rosnilimab-mIgG2a, or control anti-mIL-12 p40 were dosed intraperitoneally twice weekly for 4 weeks. This model system allowed assessment of the therapeutic potential of depletion and agonism mechanisms of rosnilimab on hPD1+ T cells, with a depletion-enabled mouse IgG2a isotype, in the context of T cell-driven gut inflammation and pathology.

Results

In PBMCs from UC patients, at therapeutically relevant concentrations, rosnilimab IgG1 reduced PD-1^high T cells by 90%, while rosnilimab IgG1-LALA reduced PD-1^high T cells by 14%. Isotype control did not reduce T cells. Furthermore, rosnilimab IgG1 reduced secretion of inflammatory cytokines interferon gamma (IFN-γ) and CXCL13 compared to rosnilimab IgG1-LALA or isotype control. In the hPD-1 mouse model of UC, rosnilimab mIgG2a reduced body weight loss, colon pathology, IFN-γ and CXCL13 as assessed by spatial transcriptomics of the colon, and infiltration of hPD-1 CD4+ T cells into the colon as assessed by immunofluorescence, compared to isotype control. In addition, rosnilimab mIgG2a demonstrated efficacy comparable to anti-IL-12 p40 antibody treatment.

Conclusion

Rosnilimab, a PD-1 checkpoint agonist antibody, functions to reduce pathogenic PD-1+ T cells by both depletion and agonism. These in vivo and in vitro results are consistent with the mechanisms of depletion and agonism and support the strong rationale for evaluating the therapeutic potential of rosnilimab in an ongoing phase 2 study in UC (NCT06127043).

References

1. Shi W, Zhang Y, Hao C, Guo X, Yang Q, Du J, Hou Y, Cao G, Li J, Wang H, Fang W. PeerJ. 2023; May 31;11:e15481.
2. Long Y, Xia C, Sun Y, Ma Y, Xu L, Song Y, Liu C. Immunol Lett. 2021; 233:2-10.
3. Roosenboom B, Horjus Talabur Horje CS, Smids C, Leeuwis JW, van Koolwijk E, Groenen MJM, Wahab PJ, van Lochem EG. Scand J Gastroenterol. 2021; 56(6):671-679.
4. Nguyen J, Finkelman BS, Escobar D, Xue Y, Wolniak K, Pezhouh M. Hum Pathol. 2022; 126:19-27.

Disclosure

All authors are employees and shareholders of AnaptysBio, Inc.

Introduction

Data on using Intestinal Ultrasonography (IUS) for monitoring colonic lesions and treatment response in ulcerative colitis (UC) are still limited and most results are from single-center units usually performed in centers with a special expertise in IUS.

Aims & Methods

Our multicenter study aimed to identify predictive factors and monitor early response after different therapies in UC.
Methods: From July 2022 to Oct. 2023, we conducted a multicenter study at 5 IBD centers. UC pts starting biological therapies or small molecules were included, and underwent colonoscopy (CS), IUS, clinical assessments at baseline and at 12 weeks. All pts received standard of care therapy according to ECCO guidelines. Exclusion criteria were disease limited to the rectum, active infections, and inability or contraindication to perform CS and/or IUS within the time intervals established by the study. Colonic wall thickening (CWT), vascularization (CWF), Milan Ultrasound Criteria (MUC), Mayo endoscopic score (MES), total/partial Mayo score were evaluated.

Results

Eighty-four UC patients were enrolled and eligible for starting biological therapy or small molecules. Forty-nine out of 84 pts (58.4%) were male, 40 pts (47.6%) had extensive colitis and 44 pts (52.4%) had left colitis, with a disease duration of 20 months (1-336); 53 pts (63%) were steroid-dependent/refractory. Forty-one pts (48.9%) started anti-TNFs, 14 pts (16.6%) anti-IL12/23 or anti IL 23, 16 pts (19%) vedolizumab, and 13 pts (15.5%) JAKi. After 12 weeks, 39 out of 84 patients (46.4%) had partial Mayo score<2, 31 patients (36.9%) 2-4, 10 patients (11.9%) 5-7, and 4 patients (4.76%) >7. Forty-three out of 84 patients (51.2%) had MES=0-1, while 41 out of 84 patients (48.8%) had MES=2-3. Fifteen out of 84 patients (17.9%) had ultrasonographic response (defined as CWT<3mm) and 26 out of 84 patients (30.1%) had MUC<6.2 (1). Logistic regression was performed to evaluate the relationship between clinical/endoscopic and ultrasonographic characteristics and early IUS response at 3 months. Univariable analysis showed that active smoking ([RR], 1.9; 95% CI, 1.2–3.1, p<0.005), appendectomy ([RR], 2.5; 95% CI, 1.9–3.2, p<0.0001), partial Mayo score <2 ([RR], 2.18; 95% CI, 1.5–3, p<0.001) were associated with higher risk of response at 3 months. Inversely, severe endoscopic lesions (MES 3) (RR, 0.3; 95% CI, 0.2–0.6; p<0.0001), presence of lymph-nodes, mesenteric hypertrophy and vascularization (p<0.001, RR 0.36, 0.28, 0.45 respectively) were associated with a lower risk of response at 3 months. CWT was associated with a lower risk of response at 3 months (RR 0.66, 95% CI, 0.53-0.81, p<0.0001) and every increased mm was associated with a 34% reduction of IUS response. MUC was associated with a lower risk of response at 3 months (RR 0.78, 95% CI, 0.7-0.87) and every increased of one point was associated with a 22% reduction of IUS response. In the multivariable analysis, CWT and MUC were the only predictive factors associated with IUS response at 3 months (RR 0.70, 0.78 respectively, p<0.04). A CWT<5.5 mm showed a 2.5 risk of treatment response at 3 months.

Conclusion

Our multicenter study demonstrated that IUS allows close monitoring of treatment in UC. MUC, and in particular CWT, are valuable parameters to predict early response in UC.

References

1)Allocca M, et al. Predictive value of Milan ultrasound criteria in ulcerative colitis: a prospective observational cohort study. United European Gastroenterol J. 2022; 10:190-7.