Lymph node regression after neoadjuvant chemoradiotherapy in rectal cancer.

AIMS: Lymph node metastases (LNM) are one of the most important prognostic indicators in solid tumours and a major component of cancer staging. Neoadjuvant therapy might influence nodal status by induction of regression. Our aim is to determine the prevalence and role of regression of LNM on outcomes in patients with rectal cancer. METHODS AND RESULTS: Four independent study populations of rectal cancer patients treated with similar regimens of chemoradiotherapy were pooled together to obtain a total cohort of 469 patients. Post-treatment nodal status (ypN) and signs of tumour regression (Reg) were incorporated to form three-tiered (ypN- Reg+, ypN- Reg- and ypN+) and four-tiered (ypN- Reg+, ypN- Reg-, ypN+ Reg+ and ypN+ Reg-) classifications. In our cohort, 31% of patients presented with ypN+ rectal cancer. As expected, we found significantly worse overall survival (OS) in ypN+ patients compared to ypN- patients (P = 0.002). The percentage of ypN- patients with lymph nodes with complete regression was 20% in our cohort. While node-negative patients with and without regression had similar OS (P = 0.09), disease-free survival (DFS) was significantly better in node-negative patients with regression (P = 0.009). CONCLUSIONS: Regression in lymph nodes is frequent, and node-negative patients with evidence of lymph node regression have better DFS compared to node-negative patients without such evidence.

Reduced Serological Response to COVID-19 Booster Vaccine is Associated with Reduced B Cell Memory in Patients With Inflammatory Bowel Disease; VARIATION [VAriability in Response in IBD AgainsT SARS-COV-2 ImmunisatiON].

BACKGROUND AND AIMS: Patients with inflammatory bowel disease [IBD] have an attenuated response to initial COVID-19 vaccination. We sought to characterize the impact of IBD and its treatment on responses after the third vaccine against SARS-CoV-2. METHODS: This was a prospective multicentre observational study of patients with IBD [n = 202] and healthy controls [HC, n = 92]. Serological response to vaccination was assessed by quantification of anti-spike protein [SP] immunoglobulin [Ig]G levels [anti-SPIgG] and in vitro neutralization of binding to angiotensin-converting enzyme 2 [ACE2]. Peripheral blood B-cell phenotype populations were assessed by flow cytometry. SARS-CoV-2 antigen-specific B-cell responses were assessed in ex vivo culture. RESULTS: Median anti-SP IgG post-third vaccination in our IBD cohort was significantly lower than HCs [7862 vs 19 622 AU/mL, p < 0.001] as was ACE2 binding inhibition [p < 0.001]. IBD patients previously infected with COVID-19 [30%] had similar quantitative antibody response as HCs previously infected with COVID-19 [p = 0.12]. Lowest anti-SP IgG titres and neutralization were seen in IBD patients on anti-tumour necrosis factor [anti-TNF] agents, without prior COVID-19 infection, but all IBD patients show an attenuated vaccine response compared to HCs. Patients with IBD have reduced memory B-cell populations and attenuated B-cell responses to SARS-CoV-2 antigens if not previously infected with COVID-19 [p = 0.01]. Higher anti-TNF drug levels and zinc levels <65 ng/ml were associated with significantly lower serological responses. CONCLUSIONS: Patients with IBD have an attenuated response to three doses of SARS-CoV-2 vaccine. Physicians should consider patients with higher anti-TNF drug levels and/or zinc deficiency as potentially at higher risk of attenuated response to vaccination.

Butyrate limits human natural killer cell effector function.

The gut microbiota regulates chronic inflammation and has been implicated in the pathogenesis of a broad spectrum of disease including autoimmunity and cancer. Microbial short-chain fatty acids (SCFAs) e.g., butyrate have demonstrated immunomodulatory effects and are thought to be key mediators of the host-microbiome interaction. Here, we investigated the effect of butyrate on effector functions of blood derived human NK cells stimulated for 18 h with a combination of IL-12/IL-15, a potent mix of cytokines that drive NK cell activation. We show that butyrate has a strong anti-inflammatory effect on NK cells. NK cells cultured in the presence of butyrate expressed lower levels of activating receptors (TRAIL, NKp30, NKp44) and produced lower levels of cytokines (IFNγ, TNF-α, IL-22, granzyme B, granzyme A, perforin) in response to IL-12/IL-15. Butyrate restricted NK cell function by downregulation of mTORC1 activity, c-Myc mRNA expression and metabolism. Using a shotgun proteomic approach, we confirmed the effect of butyrate on NK cell cytokine signaling and metabolism and identified BRD2, MAT2A and EHD1 as downstream mediators of these effects. This insight into the immunomodulatory activity of butyrate on human NK cell function might help to develop new ways to limit NK cell function during chronic inflammation.

Reduced Serological Response to COVID-19 Vaccines in Patients with IBD is Further Diminished by TNF Inhibitor Therapy; Early Results of the VARIATION study [VAriability in Response in IBD Against SARS-COV-2 ImmunisatiON].

BACKGROUND AND AIMS: Evidence suggests patients with inflammatory bowel disease [IBD] receiving TNF antagonists have attenuated response to vaccination against COVID-19. We sought to determine the impact of IBD and of various medications for treatment of IBD on antibody responses to vaccination against COVID-19. METHODS: Patients with IBD [n = 270] and healthy controls [HC, n = 116] were recruited prospectively, and quantitative antibody responses were assessed following COVID-19 vaccination. The impact of IBD and of medications for treatment of IBD on vaccine response rates was investigated. RESULTS: Of HC, 100% seroconverted following complete vaccination with two vaccine doses; 2% of patients with IBD failed to seroconvert. Median anti-spike protein [SP] immunoglobulin [Ig]G levels following complete vaccination in our IBD cohort was significantly lower than among HC [2613 AU/mL versus 6871 AU/mL, p ≤0.001]. A diagnosis of IBD was independently associated with lower anti-SP IgG levels [ß coefficient -0.2, p = 0.001]. Use of mRNA vaccines was independently associated with higher anti-SP IgG levels [ß coefficient 0.25, p ≤0.001]. Patients with IBD receiving TNF inhibitors had significantly lower anti-SP IgG levels [2445 AU/mL] than IBD patients not receiving TNF inhibitors [3868 AU/mL, p ≤0.001]. Patients with IBD not receiving TNF inhibitors still showed attenuated responses compared with HC [3868 AU/mL versus 8747 AU/mL, p = 0.001]. CONCLUSIONS: Patients with IBD have attenuated serological responses to SARS-CoV-2 vaccination. Use of anti-TNF therapy negatively affects anti-SP IgG levels further. Patients who do not seroconvert following vaccination are a particularly vulnerable cohort. Impaired responses to vaccination in our study highlight the importance of booster vaccination programmes for patients with IBD.

The Trypanosoma brucei-Derived Ketoacids, Indole Pyruvate and Hydroxyphenylpyruvate, Induce HO-1 Expression and Suppress Inflammatory Responses in Human Dendritic Cells.

The extracellular parasite and causative agent of African sleeping sickness Trypanosoma brucei (T. brucei) has evolved a number of strategies to avoid immune detection in the host. One recently described mechanism involves the conversion of host-derived amino acids to aromatic ketoacids, which are detected at relatively high concentrations in the bloodstream of infected individuals. These ketoacids have been shown to directly suppress inflammatory responses in murine immune cells, as well as acting as potent inducers of the stress response enzyme, heme oxygenase 1 (HO-1), which has proven anti-inflammatory properties. The aim of this study was to investigate the immunomodulatory properties of the T. brucei-derived ketoacids in primary human immune cells and further examine their potential as a therapy for inflammatory diseases. We report that the T. brucei-derived ketoacids, indole pyruvate (IP) and hydroxyphenylpyruvate (HPP), induce HO-1 expression through Nrf2 activation in human dendritic cells (DC). They also limit DC maturation and suppress the production of pro-inflammatory cytokines, which, in turn, leads to a reduced capacity to differentiate adaptive CD4+ T cells. Furthermore, the ketoacids are capable of modulating DC cellular metabolism and suppressing the inflammatory profile of cells isolated from patients with inflammatory bowel disease. This study therefore not only provides further evidence of the immune-evasion mechanisms employed by T. brucei, but also supports further exploration of this new class of HO-1 inducers as potential therapeutics for the treatment of inflammatory conditions.

Dysregulation of Metabolic Pathways in Circulating Natural Killer Cells Isolated from Inflammatory Bowel Disease Patients.

BACKGROUND AND AIMS: Inflammatory bowel diseases [IBD], comprising Crohn's disease [CD] and ulcerative colitis [UC], are chronic conditions characterized by severe dysregulation of innate and adaptive immunity resulting in the destruction of the intestinal mucosa. Natural killer [NK] cells play a pivotal role in the dynamic interaction between the innate and adaptive immune response. There is an increasing appreciation for the key role immunometabolism plays in the regulation of NK cell function, yet little remains known about the metabolic profile, cytokine secretion, and killing capacity of human NK cells during active IBD. METHODS: Peripheral blood mononuclear cells were isolated from peripheral blood of patients with moderate to severely active IBD and healthy controls. NK cells were stained with a combination of cell surface receptors, intracellular cytokines, and proteins and analyzed by flow cytometry. For measurements of NK cell cytotoxicity, the calcein-AM release assay was performed. The metabolic profile was analyzed by an extracellular flux analyzer. RESULTS: NK cells from IBD patients produce large quantities of pro-inflammatory cytokines, IL-17A and TNF-α ex vivo, but have limited killing capability. Furthermore, patient NK cells have reduced mitochondrial mass and oxidative phosphorylation. mTORC1, an important cell and metabolic regulator, demonstrated limited activity in both freshly isolated cells and cytokine-stimulated cells. CONCLUSIONS: Our results demonstrate that circulating NK cells of IBD patients have an unbalanced metabolic profile, with faulty mitochondria and reduced capacity to kill. These aberrations in NK cell metabolism may contribute to defective killing and thus the secondary infections and increased risk of cancer observed in IBD patients.

Personalised mapping of tumour development in synchronous colorectal cancer patients.

Synchronous colorectal cancers (syCRCs) are two or more primary tumours identified simultaneously in a patient. Previous studies report high inter-tumour heterogeneity between syCRCs, suggesting independent origin and different treatment response, making their management particularly challenging, with no specific guidelines currently in place. Here, we performed in-depth bioinformatic analyses of genomic and transcriptomic data of a total of eleven syCRCs and one metachronous CRC collected from three patients. We found mixed microsatellite status between and within patients. Overlap of mutations between synchronous tumours was consistently low (<0.5%) and heterogeneity of driver events across syCRCs was high in all patients. Microbial analysis revealed the presence of Fusobacterium nucleatum species in patients with MSI tumours, while quantification of tumour immune infiltration showed varying immune responses between syCRCs. Our results suggest high heterogeneity of syCRCs within patients but find clinically actionable biomarkers that help predict responses to currently available targeted therapies. Our study highlights the importance of personalised genome and transcriptome sequencing of all synchronous lesions to aid therapy decision and improve management of syCRC patients.

Characterising the prognostic potential of HLA-DR during colorectal cancer development.

HLA-DR, an MHC class II molecule that mediates antigen presentation, is a favourable prognostic indicator in colorectal cancer (CRC). However, the dynamics and location of HLA-DR expression during CRC development are unclear. We aimed to define HLA-DR expression by immunohistochemistry in colorectal epithelium and stromal tissue at different stages of cancer development, assessing non-neoplastic colorectal adenocarcinoma-adjacent tissue, adenomas and carcinoma tissues, and to associate HLA-DR levels with clinical outcomes. Patients with higher than median HLA-DR expression survived at least twice as long as patients with lower expression. This association was significant for HLA-DR staining in the colorectal carcinoma epithelium (n = 152, p = 0.011, HR 1.9, 95% CI 1.15-3.15) and adjacent non-neoplastic epithelium (n = 152, p < 0.001, HR 2.7, 95% CI 1.59-4.66), but not stroma. In stage II cases, however, the prognostic value of HLA-DR expression was significant only in adjacent non-neoplastic tissues, for both epithelium (n = 63, p = 0.015, HR 3.6, 95% CI 1.279-10.25) and stroma (n = 63, p = 0.018, HR 5.07, 95% CI 1.32-19.49). HLA-DR was lower in carcinoma tissue compared to matched adenomas (n = 35), in epithelium (p < 0.01) and stroma (p < 0.001). HLA-DR was further reduced in late-stage carcinoma (n = 101) compared to early stage (n = 105), in epithelium (p < 0.001) and stroma (p < 0.01). HLA-DR expression was lower (p < 0.05) in the adjacent non-neoplastic epithelium of patients with cancer recurrence. We demonstrate a progressive loss of HLA-DR in epithelial and stromal tissue compartments during CRC development and show prognostic ability in carcinoma-adjacent non-neoplastic tissues, highlighting the importance of this molecule in the anti-cancer immune response. These findings may have wider implications for immunotherapeutic interventions.

KH-Type Splicing Regulatory Protein Controls Colorectal Cancer Cell Growth and Modulates the Tumor Microenvironment.

KH-type splicing regulatory protein (KHSRP) is a multifunctional nucleic acid binding protein implicated in key aspects of cancer cell biology: inflammation and cell-fate determination. However, the role KHSRP plays in colorectal cancer (CRC) tumorigenesis remains largely unknown. Using a combination of in silico analysis of large data sets, ex vivo analysis of protein expression in patients, and mechanistic studies using in vitro models of CRC, we investigated the oncogenic role of KHSRP. We demonstrated KHSRP expression in the epithelial and stromal compartments of both primary and metastatic tumors. Elevated expression was found in tumor versus matched normal tissue, and these findings were validated in larger independent cohorts in silico. KHSRP expression was a prognostic indicator of worse overall survival (hazard ratio, 3.74; 95% CI, 1.43-22.97; P = 0.0138). Mechanistic data in CRC cell line models supported a role of KHSRP in driving epithelial cell proliferation in both a primary and metastatic setting, through control of the G1/S transition. In addition, KHSRP promoted a proangiogenic extracellular environment by regulating the secretion of oncogenic proteins involved in diverse cellular processes, such as migration and response to cellular stress. Our study provides novel mechanistic insight into the tumor-promoting effects of KHSRP in CRC.

Tumour vasculature immaturity, oxidative damage and systemic inflammation stratify survival of colorectal cancer patients on bevacizumab treatment.

Despite treatment of patients with metastatic colorectal cancer (mCRC) with bevacizumab plus chemotherapy, response rates are modest and there are no biomarkers available that will predict response. The aim of this study was to assess if markers associated with three interconnected cancer-associated biological processes, specifically angiogenesis, inflammation and oxidative damage, could stratify the survival outcome of this cohort. Levels of angiogenesis, inflammation and oxidative damage markers were assessed in pre-bevacizumab resected tumour and serum samples of mCRC patients by dual immunofluorescence, immunohistochemistry and ELISA. This study identified that specific markers of angiogenesis, inflammation and oxidative damage stratify survival of patients on this anti-angiogenic treatment. Biomarkers of immature tumour vasculature (% IMM, p=0.026, n=80), high levels of oxidative damage in the tumour epithelium (intensity of 8-oxo-dG in nuclear and cytoplasmic compartments, p=0.042 and 0.038 respectively, n=75) and lower systemic pro-inflammatory cytokines (IL6 and IL8, p=0.053 and 0.049 respectively, n=61) significantly stratify with median overall survival (OS). In summary, screening for a panel of biomarkers for high levels of immature tumour vasculature, high levels of oxidative DNA damage and low levels of systemic pro-inflammatory cytokines may be beneficial in predicting enhanced survival outcome following bevacizumab treatment for mCRC.