Genetic, epigenetic and environmental factors in diverticular disease: systematic review.

BACKGROUND: Diverticulosis is a normal anatomical variant of the colon present in more than 70% of the westernized population over the age of 80. Approximately 3% will develop diverticulitis in their lifetime. Many patients present emergently, suffer high morbidity rates and require substantial healthcare resources. Diverticulosis is the most common finding at colonoscopy and has the potential for causing a significant morbidity rate and burden on healthcare. There is a need to better understand the aetiology and pathogenesis of diverticular disease. Research suggests a genetic susceptibility of 40-50% in the formation of diverticular disease. The aim of this review is to present the hypothesized functional effects of the identified gene loci and environmental factors. METHODS: A systematic literature review was performed using PubMed, MEDLINE and Embase. Medical subject headings terms used were: 'diverticular disease, diverticulosis, diverticulitis, genomics, genetics and epigenetics'. A review of grey literature identified environmental factors. RESULTS: Of 995 articles identified, 59 articles met the inclusion criteria. Age, obesity and smoking are strongly associated environmental risk factors. Intrinsic factors of the colonic wall are associated with the presence of diverticula. Genetic pathways of interest and environmental risk factors were identified. The COLQ, FAM155A, PHGR1, ARHGAP15, S100A10, and TNFSF15 genes are the strongest candidates for further research. CONCLUSION: There is increasing evidence to support the role of genomics in the spectrum of diverticular disease. Genomic, epigenetic and omic research with demographic context will help improve the understanding and management of this complex disease.

Immune responses to citrullinated and homocitrullinated peptides in healthy donors are not restricted to the HLA SE shared allele and can be selected into the memory pool.

Citrullination and homocitrullination are stress induced post-translational modifications (siPTMs) which can be recognized by T cells. Peripheral blood mononuclear cells isolated from healthy donors and rheumatoid arthritis (RA) patients were stimulated with nine siPTM-peptides. CD45RA/CD45RO depletion was employed to determine if peptide-specific responses are naïve or memory. Human leucocyte antigen (HLA)-DP4 and HLA-DR4 transgenic mice were immunized with siPTM-peptides and immune responses were determined with ex vivo ELISpot assays. The majority (24 out of 25) of healthy donors showed CD4 T cell-specific proliferation to at least 1 siPTM-peptide, 19 to 2 siPTM-peptides, 14 to 3 siPTM-peptides, 9 to 4 siPTM-peptides, 6 to 5 siPTM-peptides and 4 to 6 siPTM-peptides. More donors responded to Vim28-49cit (68%) and Bip189-208cit (75%) compared with Vim415-433cit (33%). In RA patients, the presentation of citrullinated epitopes is associated with HLA-SE alleles; however, we witnessed responses in healthy donors who did not express the SE allele. The majority of responding T cells were effector memory cells with a Th1/cytotoxic phenotype. Responses to Vim28-49cit and Eno241-260cit originated in the memory pool, while the response to Vim415-433cit was naïve. In the HLA-DP4 and HLA-DR4 transgenic models, Vim28cit generated a memory response. Peptide-specific T cells were capable of Epstein-Barr virus transformed lymphoblastoid cell line recognition suggesting a link with stress due to infection. These results suggest siPTM-peptides are presented under conditions of cellular stress and inflammation and drive cytotoxic CD4 T cell responses that aid in the removal of stressed cells. The presentation of such siPTM-peptides is not restricted to HLA-SE in both humans and animal models.

Citrullinated glucose-regulated protein 78 is a candidate target for melanoma immunotherapy.

Introduction: Post translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy. Methods: A citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model. Results: We show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (p<0.0001) transgenic mouse model. Finally, we demonstrate the existence of a repertoire of responses to the citrullinated GRP78 peptide in healthy individuals (p=0.0023) with 13/17 (76%) individuals showing a response to this peptide. Conclusion: We propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach.

Vaccine Can Induce CD4-Mediated Responses to Homocitrullinated Peptides via Multiple HLA-Types and Confer Anti-Tumor Immunity.

Homocitrullination is the post translation modification (PTM) of the amino acid lysine to homocitrulline also referred to as carbamylation. This PTM has mainly been studied in relation to autoimmune diseases including rheumatoid arthritis. Homocitrullination of lysines alters their charge which can lead to generation of neoepitopes that are differentially presented by MHC-II and induce modification-specific immune responses. Homocitrullination is often considered a process which triggers autoimmune disease by bypassing self-tolerance however, we suggest that homocitrullination may also have an alternative role in immune responses including protection against cancer. Here we demonstrate that immune responses to homocitrullinated peptides from three different proteins can be induced via multiple HLA-types. Immunization of Balb/c or HLA-transgenic DR4 and DR1 mice can induce modification-specific CD4 mediated IFNγ responses. Healthy human donors show a clear repertoire for the homocitrullinated Vimentin peptide (Vim116-135Hcit), with modification-specific and oligoclonal responses. Importantly, in vivo homocitrulline specific Vim116-135Hcit,Cyk8 371-388Hcit and Aldo 140-157Hcit responses are able to confer an anti-tumor effect in the murine B16 melanoma model. The Vim116-135Hcit anti-tumor response was dependent upon tumor expression of MHC-II suggesting the direct recognition of PTMs on tumor is an important anti-tumor mechanism. Cancer patients also have a CD4 repertoire for Vim116-135Hcit. Together these results suggest that homocitrulline-specific immune responses can be generated in healthy mice and detected in human donors through a variety of HLA-restrictions. Immunization can induce responses to Vim116-135Hcit,Aldolase 140-157Hcit and Cyk8 371-388Hcit which provide anti-tumor therapy across several HLA-types. Our results advance our understanding of homocitrulline-specific immune responses, with implications for a number of fields beyond autoimmunity, including tumor immune surveillance.

PAD-2-mediated citrullination of nucleophosmin provides an effective target for tumor immunotherapy.

BACKGROUND: The enzymatic conversion of arginine to citrulline is involved in gene and protein regulation and in alerting the immune system to stressed cells, including tumor cells. Nucleophosmin (NPM) is a nuclear protein that plays key roles in cellular metabolism including ribosome biogenesis, mRNA processing and chromatin remodeling and is regulated by citrullination. In this study, we explored if the same citrullinated arginines within NPM are involved in gene regulation and immune activation. METHODS: HLA-DP4 and HLA-DR4 transgenic mice were immunized with 22 citrullinated NPM overlapping peptides and immune responses to the peptides were assessed by ex vivo ELISpot assays. Antitumor immunity of NPM targeted vaccination was assessed by challenging transgenic mice with B16F1 HHDII/iDP4, B16F1 HHDII/PAD2KOcDP4, B16F1 HHDII and Lewis lung carcinoma cells/cDP4 cells subcutaneously. Peripheral blood mononuclear cells isolated from healthy donors were stimulated with NPM266-285cit peptides with/without CD45RO+memory cell depletion to assess if the responses in human were naïve or memory. RESULTS: In contrast to NPM regulation, which is mediated by peptidylarginine deiminase (PAD4) citrullination of arginine at position 197, only citrullinated NPM266-285 peptide induced a citrulline-specific CD4 T cell response in transgenic mice models expressing human HLA-DP4 or HLA-DR4. Vaccinations with the NPM266-285cit peptide stimulated antitumor responses that resulted in dramatic tumor therapy, greatly improved survival, and protected against rechallenge without further vaccination. The antitumor response was lost if MHCII expression on the tumor cells was knocked out demonstrating direct presentation of the NPM266-285cit epitope in tumors. This antitumor response was lost in B16 tumors lacking PAD2 enzyme indicating NPM266cit is citrullinated by PAD2 in this model. Assessment of the T cell repertoire in healthy individuals and patients with lung cancer also showed CD4 T cells that respond to NPM266-285cit. The proliferative CD4 responses displayed a Th1 profile as they were accompanied with increased IFNγ and granzyme B expression. Depletion of CD45RO+ memory cells prior to stimulation suggested that responses originated from a naïve population in healthy donors. CONCLUSION: This study indicates PAD2 can citrullinate the nuclear antigen NPM at position 277 which can be targeted by CD4 T cells for antitumor therapy. This is distinct from PAD4 citrullination of arginine 197 within NPM which results in its transport from the nucleoli to the nucleoplasm.

Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor environment is a target for cancer immunotherapy.

BACKGROUND: Homocitrullination is the post-translational modification of lysine that is recognized by T cells. METHODS: This study identified homocitrullinated peptides from aldolase, enolase, cytokeratin and binding immunoglobulin protein and used human leukocyte antigen (HLA) transgenic mice to assess immunogenicity by enzyme-linked immunosorbent spot assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma expressing constitutive or interferon γ (IFNγ)-inducible major histocompatibility complex class II (MHC-II) as represented by most human tumors. To determine the mechanism behind the therapy, immune cell infiltrates were analyzed using flow cytometry and therapy studies in the presence of myeloperoxidase (MPO) inhibitor and T-cell depletion performed. We assessed the T-cell repertoire to homocitrullinated peptides in patients with cancer and healthy donors using flow cytometry. RESULTS: Homocitrulline (Hcit) peptide vaccination stimulated strong CD4 T-cell responses and induced significant antitumor therapy in an established tumor model. The antitumor response was dependent on CD4 T cells and the effect was driven mainly via direct tumor recognition, as responses were only observed if the tumors were induced to express MHC-II. In vitro proliferation assays show that healthy donors and patients with cancer have an oligoclonal CD4 T-cell repertoire recognizing homocitrullinated peptides. Inhibition of cyanate generation, which mediates homocitrullination, by MPO inhibition reduced tumor therapy by the vaccine induced T cells (p=0.0018). Analysis of the tumor microenvironment (TME) suggested that myeloid-derived suppressor cells (MDSCs) were a potential source of MPO. The selected B16 melanoma model showed MDSC infiltration and was appropriate to see if the Hcit vaccine could overcome the immunosuppression associated with MDSCs. The vaccine was very effective (90% survival) as the induced CD4 T cells directly targeted the homocitrullinated tumor and likely reversed the immunosuppressive environment. CONCLUSION: We propose that MPO, potentially produced by MDSCs, catalyzes the buildup of cyanate in the TME which diffuses into tumor cells causing homocitrullination of cytoplasmic proteins which are degraded and, in the presence of IFNγ, presented by MHC-II for direct CD4 T-cell recognition. Homocitrullinated proteins are a new target for cancer vaccines and may be particularly effective against tumors containing high levels of MPO expressing MDSCs.

Characteristics of Early-Onset vs Late-Onset Colorectal Cancer: A Review.

Importance: The incidence of early-onset colorectal cancer (younger than 50 years) is rising globally, the reasons for which are unclear. It appears to represent a unique disease process with different clinical, pathological, and molecular characteristics compared with late-onset colorectal cancer. Data on oncological outcomes are limited, and sensitivity to conventional neoadjuvant and adjuvant therapy regimens appear to be unknown. The purpose of this review is to summarize the available literature on early-onset colorectal cancer. Observations: Within the next decade, it is estimated that 1 in 10 colon cancers and 1 in 4 rectal cancers will be diagnosed in adults younger than 50 years. Potential risk factors include a Westernized diet, obesity, antibiotic usage, and alterations in the gut microbiome. Although genetic predisposition plays a role, most cases are sporadic. The full spectrum of germline and somatic sequence variations implicated remains unknown. Younger patients typically present with descending colonic or rectal cancer, advanced disease stage, and unfavorable histopathological features. Despite being more likely to receive neoadjuvant and adjuvant therapy, patients with early-onset disease demonstrate comparable oncological outcomes with their older counterparts. Conclusions and Relevance: The clinicopathological features, underlying molecular profiles, and drivers of early-onset colorectal cancer differ from those of late-onset disease. Standardized, age-specific preventive, screening, diagnostic, and therapeutic strategies are required to optimize outcomes.

Protocol for the UK cohort study to investigate the prevention of parastomal hernia (the CIPHER study).

AIM: Abdominal surgery sometimes necessitates the creation of a stoma, which can cause future complications including parastomal hernia (PSH), an incisional hernia adjacent to and related to the stoma. PSH affects approximately 40% of patients within 2 years of stoma formation. Complications of PSH reduce a patient's quality of life and can be severe (e.g. bowel obstruction). PSHs are difficult to manage and can recur after surgical repair. Therefore, it is very important to prevent a PSH. Surgeons create stomas in different ways and both patient and surgical factors are believed to influence the development of PSH. The aim of the CIPHER study is to investigate the influence of different surgical techniques on the development of PSH. METHOD: The UK cohort study to investigate the prevention of parastomal hernia (the CIPHER study) aims to recruit 4000 patients undergoing elective or expedited surgery with the intention of forming an ileostomy or colostomy, irrespective of the primary indication for the planned surgery. For each patient, surgeons will describe their methods of trephine formation, mesh reinforcement of the stoma trephine, use of the stoma as a specimen extraction site and wound closure. The primary outcome will be incident PSH during follow-up, defined as symptoms of PSH (custom-designed questionnaire) and anatomical PSH, ascertained by independent reading of usual care CT scans. Secondary outcomes will include surgical site infection, the Comprehensive Complication Index, quality of life (EQ-5D-5L and SF-12), PSH repair and use of NHS resources. RESULTS: Results of the study will be submitted for publication in peer-reviewed journals. All publications relating to the results of CIPHER will use a corporate authorship, 'The CIPHER Study Investigators' with named writing committee members. CONCLUSION: The CIPHER study will be the first to investigate detailed surgical methods of stoma formation in a large, representative cohort of patients with a range of primary indications, both cancer and noncancer.

Root-cause analyses of missed opportunities for the diagnosis of colorectal cancer in patients with inflammatory bowel disease.

BACKGROUND: Colonoscopic surveillance in patients with inflammatory bowel disease (IBD) leads to earlier detection of colorectal cancer (CRC) and reduces CRC-associated mortality. However, it is limited by poor adherence in practice. AIM: To identify missed opportunities to detect IBD-associated CRC at our hospital METHODS: We undertook root-cause analyses to identify patients with missed opportunities to diagnose IBD-associated CRC. We matched patients with IBD-associated CRC to patients with CRC in the general population to identify differences in staging at diagnosis and clinical outcomes. RESULTS: Compared with the general population, patients with IBD were at increased risk of developing CRC (odds ratio 2.7 [95% CI 1.6-3.9], P < 0.001). The mean incidence of IBD-associated CRC between 1998 and 2019 was 165.4 (IQR 130.4-199.4) per 100 000 patients and has not changed over the last 20 years. Seventy-eight patients had IBD-associated CRC. Forty-two (54%) patients were eligible for CRC surveillance: 12% (5/42) and 10% (4/42) patients were diagnosed with CRC at an appropriately timed or overdue surveillance colonoscopy, respectively. Interval cancers occurred in 14% (6/42) of patients; 64% (27/42) of patients had a missed opportunity for colonoscopic surveillance where root-cause analyses demonstrated that 10/27 (37%) patients known to secondary care had not been offered surveillance. Four (15%) patients had a delayed diagnosis of CRC due to failure to account for previous colonoscopic findings. Seventeen (63%) patients were managed by primary care including seven patients discharged from secondary care without a surveillance plan. Matched case-control analysis did not show significant differences in cancer staging or 10-year survival outcomes. CONCLUSION: The incidence of IBD-associated CRC has remained static. Two-thirds of patients eligible for colonoscopic surveillance had missed opportunities to diagnose CRC. Surveillance programmes without comprehensive and fully integrated recall systems across primary and secondary care are set to fail.

Quality of life in restorative versus non-restorative resections for rectal cancer: systematic review.

BACKGROUND: Low rectal cancers could be treated using restorative (anterior resection, AR) or non-restorative procedures with an end/permanent stoma (Hartmann's, HE; or abdominoperineal excision, APE). Although the surgical choice is determined by tumour and patient factors, quality of life (QoL) will also influence the patient's future beyond cancer. This systematic review of the literature compared postoperative QoL between the restorative and non-restorative techniques using validated measurement tools. METHODS: The review was registered on PROSPERO (CRD42020131492). Embase and MEDLINE, along with grey literature and trials websites, were searched comprehensively for papers published since 2012. Inclusion criteria were original research in an adult population with rectal cancer that reported QoL using a validated tool, including the European Organization for Research and Treatment of Cancer QLQ-CR30, QLQ-CR29, and QLQ-CR38. Studies were included if they compared AR with APE (or HE), independent of study design. Risk of bias was assessed using the Risk Of Bias In Non-Randomized Studies of Interventions (ROBINS-I) tool. Outcomes of interest were: QoL, pain, gastrointestinal (GI) symptoms (stool frequency, flatulence, diarrhoea and constipation), and body image. RESULTS: Nineteen studies met the inclusion criteria with a total of 6453 patients; all papers were observational and just four included preoperative evaluations. There was no identifiable difference in global QoL and pain between the two surgical techniques. Reported results regarding GI symptoms and body image documented similar findings. The ROBINS-I tool highlighted a significant risk of bias across the studies. CONCLUSION: Currently, it is not possible to draw a firm conclusion on postoperative QoL, pain, GI symptoms, and body image following restorative or non-restorative surgery. The included studies were generally of poor quality, lacked preoperative evaluations, and showed considerable bias in the data.

Combination vaccine based on citrullinated vimentin and enolase peptides induces potent CD4-mediated anti-tumor responses.

BACKGROUND: Stress-induced post-translational modifications occur during autophagy and can result in generation of new epitopes and immune recognition. One such modification is the conversion of arginine to citrulline by peptidylarginine deiminase enzymes. METHODS: We used Human leukocyte antigen (HLA) transgenic mouse models to assess the immunogenicity of citrullinated peptide vaccine by cytokine Enzyme linked immunosorbant spot (ELISpot) assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma and ID8 ovarian models expressing either constitutive or interferon-gamma (IFNγ) inducible Major Histocompatibility Complex (MHC) class II (MHC-II) as represented by most human tumors. To determine the importance of CD4 T cells in tumor therapy, we analyzed the immune cell infiltrate into murine tumors using flow cytometry and performed therapy studies in the presence of CD4 and CD8 T cell depletion. We assessed the T cell repertoire to citrullinated peptides in ovarian cancer patients and healthy donors using flow cytometry. RESULTS: The combination of citrullinated vimentin and enolase peptides (Modi-1) stimulated strong CD4 T cell responses in mice. Responses resulted in a potent anti-tumor therapy against established tumors and generated immunological memory which protected against tumor rechallenge. Depletion of CD4, but not CD8 T cells, abrogated the primary anti-tumor response as well as the memory response to tumor rechallenge. This was further reinforced by successful tumor regression being associated with an increase in tumor-infiltrating CD4 T cells and a reduction in tumor-associated myeloid suppressor cells. The anti-tumor response also relied on direct CD4 T cell recognition as only tumors expressing MHC-II were rejected. A comparison of different Toll-like receptor (TLR)-stimulating adjuvants showed that Modi-1 induced strong Th1 responses when combined with granulocyte-macrophage colony-stimulating factor (GMCSF), TLR9/TLR4, TLR9, TLR3, TLR1/2 and TLR7 agonists. Direct linkage of the TLR1/2 agonist to the peptides allowed the vaccine dose to be reduced by 10-fold to 100-fold without loss of anti-tumor activity. Furthermore, a CD4 Th1 response to the citrullinated peptides was seen in ovarian cancer patients. CONCLUSIONS: Modi-1 citrullinated peptide vaccine induces potent CD4-mediated anti-tumor responses in mouse models and a CD4 T cell repertoire is present in ovarian cancer patients to the citrullinated peptides suggesting that Modi-1 could be an effective vaccine for ovarian cancer patients.

Engineering the Human Fc Region Enables Direct Cell Killing by Cancer Glycan-Targeting Antibodies without the Need for Immune Effector Cells or Complement.

Murine IgG3 glycan-targeting mAb often induces direct cell killing in the absence of immune effector cells or complement via a proinflammatory mechanism resembling oncotic necrosis. This cancer cell killing is due to noncovalent association between Fc regions of neighboring antibodies, resulting in enhanced avidity. Human isotypes do not contain the residues underlying this cooperative binding mode; consequently, the direct cell killing of mouse IgG3 mAb is lost upon chimerization or humanization. Using the Lewisa/c/x -targeting 88mAb, we identified the murine IgG3 residues underlying the direct cell killing and increased avidity via a series of constant region shuffling and subdomain swapping approaches to create improved ("i") chimeric mAb with enhanced tumor killing in vitro and in vivo. Constant region shuffling identified a major CH3 and a minor CH2 contribution, which was further mapped to discontinuous regions among residues 286-306 and 339-378 that, when introduced in 88hIgG1, recapitulated the direct cell killing and avidity of 88mIgG3. Of greater interest was the creation of a sialyl-di-Lewisa-targeting i129G1 mAb via introduction of these selected residues into 129hIgG1, converting it into a direct cell killing mAb with enhanced avidity and significant in vivo tumor control. The human iG1 mAb, termed Avidimabs, retained effector functions, paving the way for the proinflammatory direct cell killing to promote antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity through relief of immunosuppression. Ultimately, Fc engineering of human glycan-targeting IgG1 mAb confers proinflammatory direct cell killing and enhanced avidity, an approach that could be used to improve the avidity of other mAb with therapeutic potential. SIGNIFICANCE: Fc engineering enhances avidity and direct cell killing of cancer-targeting anti-glycan antibodies to create superior clinical candidates for cancer immunotherapy.

Major surgery induces acute changes in measured DNA methylation associated with immune response pathways.

Surgery is an invasive procedure evoking acute inflammatory and immune responses that can influence risk for postoperative complications including cognitive dysfunction and delirium. Although the specific mechanisms driving these responses have not been well-characterized, they are hypothesized to involve the epigenetic regulation of gene expression. We quantified genome-wide levels of DNA methylation in peripheral blood mononuclear cells (PBMCs) longitudinally collected from a cohort of elderly patients undergoing major surgery, comparing samples collected at baseline to those collected immediately post-operatively and at discharge from hospital. We identified acute changes in measured DNA methylation at sites annotated to immune system genes, paralleling changes in serum-levels of markers including C-reactive protein (CRP) and Interleukin 6 (IL-6) measured in the same individuals. Many of the observed changes in measured DNA methylation were consistent across different types of major surgery, although there was notable heterogeneity between surgery types at certain loci. The acute changes in measured DNA methylation induced by surgery are relatively stable in the post-operative period, generally persisting until discharge from hospital. Our results highlight the dramatic alterations in gene regulation induced by invasive surgery, primarily reflecting upregulation of the immune system in response to trauma, wound healing and anaesthesia.

T cell repertoire to citrullinated self-peptides in healthy humans is not confined to the HLA-DR SE alleles; Targeting of citrullinated self-peptides presented by HLA-DP4 for tumour therapy.

Post-translational modifications are induced in stressed cells which cause them to be recognised by the system. One such modification is citrullination where the positive charged arginine is modified to a neutral citrulline. We demonstrate most healthy donors show an oligoclonal CD4 response in vitro to at least one citrullinated vimentin or enolase peptide. Unlike rheumatoid arthritis patients, these T cell responses were not restricted by HLA-DRB1 shared epitope (SE) alleles, suggesting they could be presented by other MHC class II alleles. As HLA-DP is less polymorphic than HLA-DR, we investigated whether the common allele, HLA-DP4 could present citrullinated epitopes. The modification of arginine to citrulline enhanced binding of the peptides to HLA-DP4 and induced high-frequency CD4 responses in HLA-DP4 transgenic mouse models. Our previous studies have shown that tumours present citrullinated peptides restricted through HLA-DR4 which are good targets for anti-tumour immunity. In this study, we show that citrullinated vimentin and enolase peptides also induced strong anti-tumour immunity (100% survival, p < 0.0001) against established B16 tumours and against the LLC/2 lung cancer model (p = 0.034) both expressing HLA-DP4. Since most tumours do not constitutively express MHC class II molecules, models were engineered that expressed MHC class II under the control of an IFNγ inducible promoter. Immunisation with citrullinated peptides resulted in 90% survival (p < 0.001) against established B16 HHD tumour expressing IFNγ inducible DP4. These studies show that citrullinated peptides can be presented by a range of MHC class II molecules, including for the first time HLA-DP4, and are strong targets for anti-tumour immunity.

Factors affecting local regrowth after watch and wait for patients with a clinical complete response following chemoradiotherapy in rectal cancer (InterCoRe consortium): an individual participant data meta-analysis.

BACKGROUND: In patients with rectal cancer who achieve clinical complete response after neoadjuvant chemoradiotherapy, watch and wait is a novel management strategy with potential to avoid major surgery. Study-level meta-analyses have reported wide variation in the proportion of patients with local regrowth. We did an individual participant data meta-analysis to investigate factors affecting occurrence of local regrowth. METHODS: We updated search results of a recent systematic review by searching MEDLINE and Embase from Jan 1, 2016, to May 5, 2017, and used expert knowledge to identify published studies reporting on local regrowth in patients with rectal cancer managed by watch and wait after clinical complete response to neoadjuvant chemoradiotherapy. We restricted studies to those that defined clinical complete response using criteria equivalent to São Paulo benchmarks (ie, absence of residual ulceration, stenosis, or mass within the rectum on clinical and endoscopic examination). The primary outcome was 2-year cumulative incidence of local regrowth, estimated with a two-stage random-effects individual participant data meta-analysis. We assessed the effects of clinical and treatment factors using Cox frailty models, expressed as hazard ratios (HRs). From these models, we derived percentage differences in mean θ as an approximation of the effect of measured covariates on between-centre heterogeneity. This study is registered with PROSPERO, number CRD42017070934. FINDINGS: We obtained individual participant data from 11 studies, including 602 patients enrolled between March 11, 1990, and Feb 13, 2017, with a median follow-up of 37·6 months (IQR 25·0-58·7). Ten of the 11 datasets were judged to be at low risk of bias. 2-year cumulative incidence of local regrowth was 21·4% (random-effects 95% CI 15·3-27·6), with high levels of between-study heterogeneity (I2=61%). We noted wide between-centre variation in patient, tumour, and treatment characteristics. We found some evidence that increasing cT stage was associated with increased risk of local regrowth (random-effects HR per cT stage 1·40, 95% CI 1·00-1·94; ptrend=0·048). In a subgroup of 459 patients managed after 2008 (when pretreatment staging by MRI became standard), 2-year cumulative incidence of local regrowth was 19% (95% CI 13-28) for stage cT1 and cT2 tumours, 31% (26-37) for cT3, and 37% (21-60) for cT4 (random-effects HR per cT stage 1·50, random-effects 95% CI 1·03-2·17; ptrend=0·0330). We estimated that measured factors contributed 4·8-45·3% of observed between-centre heterogeneity. INTERPRETATION: In patients with rectal cancer and clinical complete response after chemoradiotherapy managed by watch and wait, we found some evidence that increasing cT stage predicts for local regrowth. These data will inform clinician-patient decision making in this setting. Research is needed to determine other predictors of a sustained clinical complete response. FUNDING: None.

Personalized management of elderly patients with rectal cancer: Expert recommendations of the European Society of Surgical Oncology, European Society of Coloproctology, International Society of Geriatric Oncology, and American College of Surgeons Commission on Cancer.

With an expanding elderly population and median rectal cancer detection age of 70 years, the prevalence of rectal cancer in elderly patients is increasing. Management is based on evidence from younger patients, resulting in substandard treatments and poor outcomes. Modern management of rectal cancer in the elderly demands patient-centered treatment, assessing frailty rather than chronological age. The heterogeneity of this group, combined with the limited available data, impedes drafting evidence-based guidelines. Therefore, a multidisciplinary task force convened experts from the European Society of Surgical Oncology, European Society of Coloproctology, International Society of Geriatric Oncology and the American College Surgeons Commission on Cancer, with the goal of identifying the best practice to promote personalized rectal cancer care in older patients. A crucial element for personalized care was recognized as the routine screening for frailty and geriatrician involvement and personalized care for frail patients. Careful patient selection and improved surgical and perioperative techniques are responsible for a substantial improvement in rectal cancer outcomes. Therefore, properly selected patients should be considered for surgical resection. Local excision can be utilized when balancing oncologic outcomes, frailty and life expectancy. Watch and wait protocols, in expert hands, are valuable for selected patients and adjuncts can be added to improve complete response rates. Functional recovery and patient-reported outcomes are as important as oncologic-specific outcomes in this age group. The above recommendations and others were made based on the best-available evidence to guide the personalized treatment of elderly patients with rectal cancer.

Targeting gp100 and TRP-2 with a DNA vaccine: Incorporating T cell epitopes with a human IgG1 antibody induces potent T cell responses that are associated with favourable clinical outcome in a phase I/II trial.

A DNA vaccine, SCIB1, incorporating two CD8 and two CD4 epitopes from TRP-2/gp100 was evaluated in patients with metastatic melanoma. Each patient received SCIB1 via intramuscular injection with electroporation. The trial was designed to find the safest dose of SCIB1 which induced immune/clinical responses in patients with or without tumour. Fifteen patients with tumor received SCIB1 doses of 0.4-8 mg whilst 20 fully-resected patients received 2-8 mg doses. Twelve patients elected to continue immunization every 3 months for up to 39 months. SCIB1 induced dose-dependent T cell responses in 88% of patients with no serious adverse effects or dose limiting toxicities. The intensity of the T cell responses was significantly higher in patients receiving 4 mg doses without tumor when compared to those with tumor (p < 0.01). In contrast, patients with tumor showed a significantly higher response to the 8 mg dose than the 4 mg dose (p < 0.03) but there was no significant difference in the patients without tumor. One of 15 patients with measurable disease showed an objective tumor response and 7/15 showed stable disease. 5/20 fully-resected patients have experienced disease recurrence but all remained alive at the cut-off date with a median observation time of 37 months. A positive clinical outcome was associated with MHC-I and MHC-II expression on tumors prior to therapy (p = 0.027). We conclude that SCIB1 is well tolerated and stimulates potent T cell responses in melanoma patients. It deserves further evaluation as a single agent adjuvant therapy or in combination with checkpoint inhibitors in advanced disease.

A case report of squamous cell carcinoma in a suprapubic urinary catheter tract: surgical excision and simultaneous colostomy formation.

Squamous cell carcinoma (SCC) arising from a suprapubic cystostomy tract is a rare complication of long-term suprapubic catheterization (SPC). A 53-year-old man with paraplegia secondary to spina bifida presented with a painful granulomatous lesion around his SPC site that was being treated with silver nitrate cauterization in the community. Consequently, he developed a sacral pressure sore due to reduced mobility from the pain. He also had increasing difficulties with defaecation secondary to his spina bifida. His sacral pressure sore was secondary to a cryptoglandular fistula with coccygeal osteomylelitis. Post-operative pathology revealed infiltrative SCC involving full thickness of the specimen from skin to the bladder wall with clear surgical margins. We describe the first case requiring a simultaneous suprapubic tract SCC excision and colostomy formation. We recommend early investigation of lesions arising from a long-term suprapubic tract especially in patients with spinal cord injuries or congenital defects.