Tumor Infiltrating Lymphocytes Target HLA-I Phosphopeptides Derived From Cancer Signaling in Colorectal Cancer.

There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients' tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients' tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies.

Clinical-grade whole-genome sequencing and 3' transcriptome analysis of colorectal cancer patients.

BACKGROUND: Clinical-grade whole-genome sequencing (cWGS) has the potential to become the standard of care within the clinic because of its breadth of coverage and lack of bias towards certain regions of the genome. Colorectal cancer presents a difficult treatment paradigm, with over 40% of patients presenting at diagnosis with metastatic disease. We hypothesised that cWGS coupled with 3' transcriptome analysis would give new insights into colorectal cancer. METHODS: Patients underwent PCR-free whole-genome sequencing and alignment and variant calling using a standardised pipeline to output SNVs, indels, SVs and CNAs. Additional insights into the mutational signatures and tumour biology were gained by the use of 3' RNA-seq. RESULTS: Fifty-four patients were studied in total. Driver analysis identified the Wnt pathway gene APC as the only consistently mutated driver in colorectal cancer. Alterations in the PI3K/mTOR pathways were seen as previously observed in CRC. Multiple private CNAs, SVs and gene fusions were unique to individual tumours. Approximately 30% of patients had a tumour mutational burden of > 10 mutations/Mb of DNA, suggesting suitability for immunotherapy. CONCLUSIONS: Clinical whole-genome sequencing offers a potential avenue for the identification of private genomic variation that may confer sensitivity to targeted agents and offer patients new options for targeted therapies.

Learning Curve Analyses for Achieving Satisfactory Procedural Completion Rates in Paediatric Oesophagogastroduodenoscopy.

BACKGROUND: The learning curve in paediatric oesophagogastroduodenoscopy (OGD) is unknown. Using ≥95% D2 (second part of the duodenum) intubation rates as a marker of technical competency, we conducted learning curve analyses to identify when trainees achieve competency in paediatric OGD. Factors associated with competency were also evaluated. METHODS: This nationwide study analysed data from paediatric OGD procedures prospectively entered into the UK endoscopy training e-portfolio between 2014 and 2018. Moving average and learning curve cumulative summation analyses were performed to determine procedural numbers required to achieve ≥95% D2 intubation rates. Factors associated with D2 intubation were assessed using a multivariable binary logistic regression approach. RESULTS: A total of 8929 procedures performed by 61 trainees were identified. These 61 trainees had recorded a mean of 124.6 procedures (range 22-571, interquartile range 165). By moving average analysis, 95% D2 intubation was achieved after 79 procedures. By learning curve cumulative summation analysis, 81.6% of trainees were competent after 100 procedures. Multivariable factors associated with unassisted procedural completion included: lifetime procedure count (P < 0.001), higher trainee seniority (P < 0.001), patient age (P = 0.002), outpatient status (P < 0.001), and attendance at a national Basic Skills OGD course (P = 0.011). CONCLUSIONS: This study demonstrates that, on average, 79 procedures in paediatric OGD are required to attain the competency outcome of ≥95% D2 intubation rates. By 100 procedures, 81.6% of our sample had achieved ≥95% D2 intubation. The minimum procedural count of 100 set by the UK and international training programmes can be used alongside existing objective assessment measures to safeguard competency within a training cohort.

Performance indicators in colonoscopy after certification for independent practice: outcomes and predictors of competence.

BACKGROUND: Robust real-world performance data of newly independent colonoscopists are lacking. In the United Kingdom, provisional colonoscopy certification (PCC) marks the transition from training to newly independent practice. We aimed to assess changes in key performance indicators (KPIs) such as cecal intubation rate (CIR) in the periods pre- and post-PCC, particularly regarding rates and predictors of trainees exhibiting a drop in performance (DIP), defined as CIR <90% in the first 50 procedures post-PCC. METHODS: A prospective United Kingdom-wide observational study of Joint Advisory Group on Gastrointestinal Endoscopy Electronic Training System (JETS) e-portfolio colonoscopy entries (257,800) from trainees awarded PCC between July 2011 and 2016 was undertaken. Moving average analyses were used to study KPI trends relative to PCC. Pre-PCC trainee, trainer, and training environment factors were compared between DIP and non-DIP cohorts to identify predictors of DIP. RESULTS: Seven hundred thirty-three trainees from 180 centers were awarded PCC after a median of 265 procedures and 3.1 years. Throughout the early post-PCC period, average CIRs surpassed the national 90% standard. Despite this, not all trainees achieved this standard post-PCC, with DIP observed in 18.4%. DIP was not influenced by trainer presence and diminished after 100 additional procedures. On multivariable analysis, pre-PCC CIRs and trainer specialty were predictive of DIP. Trainees with DIP incurred higher post-PCC rates of moderate to severe discomfort despite requiring higher analgesic dosages and were more likely to require trainer assistance in failed procedures. CONCLUSIONS: The current PCC requirements are appropriate for diagnostic colonoscopy. It is possible to identify predictors of underperformance in trainees, which may be of value to training leads and could improve the patient experience.

Risk of Recurrent Disease and Surgery Following an Admission for Acute Diverticulitis.

BACKGROUND: Diverticular disease accounts for significant morbidity and mortality and may take the form of recurrent episodes of acute diverticulitis. The role of elective surgery is not clearly defined. OBJECTIVE: This study aimed to define the rate of hospital admission for recurrent acute diverticulitis and risk factors associated with recurrence and surgery. DESIGN: This is a retrospective population-based cohort study. SETTINGS: National Health Service hospital admissions for acute diverticulitis in England between April 2006 and March 2011 were reviewed. PATIENTS: Hospital Episode Statistics data identified adult patients with the first episode of acute diverticulitis (index admission), and then identified recurrent admissions and elective or emergency surgery for acute diverticulitis during a minimum follow-up period of 4 years. Exclusion criteria included previous diagnoses of acute diverticulitis, colorectal cancer, or GI bleeding, and prior colectomy or surgery or death during the index admission. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: The primary outcomes measured were recurrent admissions for acute diverticulitis and patients requiring either elective or emergency surgery during the study period. RESULTS: Some 65,162 patients were identified with the first episode of acute diverticulitis. The rate of hospital admission for recurrent acute diverticulitis was 11.2%. A logistic regression model examined factors associated with recurrent acute diverticulitis and surgery: patient age, female sex, smoking, obesity, comorbidity score >20, dyslipidemia, and complicated acute diverticulitis increased the risk of recurrent acute diverticulitis. There was an inverse relationship between patient age and recurrence. Similar factors were associated with elective and emergency surgery. LIMITATIONS: The cases of acute diverticulitis required inpatient management and the use of Hospital Episode Statistics, relying on the accuracy of diagnostic coding. CONCLUSIONS: This is the largest study assessing the rates of hospital admission for recurrent acute diverticulitis. Knowledge of the rate and risk factors for recurrent acute diverticulitis is required to aid discussion and decision making with patients regarding the need and timing of elective surgery. Some factors associated with recurrence are modifiable; therefore, weight reduction and smoking cessation can be championed. See Video Abstract at http://links.lww.com/DCR/A449.

Impaired Transmigration of Myeloid-Derived Suppressor Cells across Human Sinusoidal Endothelium Is Associated with Decreased Expression of CD13.

Human monocytic myeloid-derived suppressor cells (MO-MDSCs) within the hepatic compartment suppress inflammation and impair immune surveillance in liver cancer. It is currently not known whether recruitment of MO-MDSCs from blood via hepatic sinusoidal endothelium (HSEC) contributes to their enrichment within the hepatic compartment. We compared the transmigratory potential of MO-MDSCs and monocytes after adhesion to hepatic endothelial monolayers in flow-based assays that mimic in vivo shear stress in the sinusoids. Despite comparable binding to HSEC monolayers, proportionally fewer MO-MDSCs underwent transendothelial migration, indicating that the final steps of extravasation, where actin polymerization plays an important role, are impaired in MO-MDSCs. In this article, we found reduced levels of CD13 on MO-MDSCs, which has recently been reported to control cell motility in monocytes, alongside reduced VLA-4 expression, an integrin predominantly involved in adherence to the apical side of the endothelium. CD13 and VLA-4 blocking and activating Abs were used in flow-based adhesion assays, live-cell imaging of motility, and actin polymerization studies to confirm a role for CD13 in impaired MO-MDSC transmigration. These findings indicate that CD13 significantly contributes to tissue infiltration by MO-MDSCs and monocytes, thereby contributing to the pathogenesis of hepatic inflammation.

Giant diverticulum- A rare complication of a common surgical condition.

A gentleman presented with abdominal distension and pain. CT confirmed a 20 cm sigmoid diverticulum. A giant diverticulum, typified by diverticula greater than 4 cm, often requires colonic resection. Fewer than 200 cases have been reported, most measuring 7-15 cm. I present a rare complication of a common surgical condition with images.

Evaluation of serum and tissue levels of VAP-1 in colorectal cancer.

BACKGROUND: The endothelial adhesion molecule, vascular adhesion protein-1 (VAP-1, AOC3) promotes lymphocyte recruitment to tumours, although the contribution that VAP-1 makes to lymphocyte recruitment in human colorectal cancer (CRC) is unknown. VAP-1 exists in circulating soluble form (sVAP-1). A previous study demonstrated elevated sVAP-1 levels in CRC patients. The aim of this study was to confirm this finding and study the differences in tissue VAP-1 expression between CRC and healthy tissues. METHODS: sVAP-1 levels were measured in the serum of 31 patients with CRC and 31 age- and sex-matched controls. Tissue VAP-1 levels were measured by immunohistochemistry, quantitative real-time PCR and Western blotting. RESULTS: The mean sVAP-1 level ± SD was significantly lower in the CRC group compared with the control group (399 ± 138 ng/ml versus 510 ± 142 ng/ml, P = 0.003). Tissue VAP-1 protein and mRNA levels were significantly lower in CRC compared with normal colon tissue. VAP-1 immunostaining was practically absent from CRC. CONCLUSIONS: VAP-1 is downregulated in human CRC and although the molecular basis of this down regulation is not yet known, we suggest it may be part of a mechanism used by the tumour to prevent the recruitment of anti-tumour immune cells. Our data contradicts the findings of others with regard sVAP-1 levels in patients with CRC. Possible reasons for this are discussed.

The proto-oncogene PBF binds p53 and is associated with prognostic features in colorectal cancer.

The PTTG1-binding factor (PBF) is a transforming gene capable of eliciting tumor formation in xenograft models. However, the precise role of PBF in tumorigenesis and its prognostic value as a cancer biomarker remain largely uncharacterised, particularly in malignancies outside the thyroid. Here, we provide the first evidence that PBF represents a promising prognostic marker in colorectal cancer. Examination of a total of 39 patients demonstrated higher PBF expression at both the mRNA (P = 0.009) and protein (P < 0.0001) level in colorectal tumors compared to matched normal tissue. Critically, PBF was most abundant in colorectal tumors associated with Extramural Vascular Invasion (EMVI), increased genetic instability (GI) and somatic TP53 mutations, all features linked with recurrence and poorer patient survival. We further demonstrate by glutathione-S-transferase (GST) pull-down and coimmunoprecipitation that PBF binds to the tumor suppressor protein p53, as well as to p53 mutants (Δ126-132, M133K, V197E, G245D, I255F and R273C) identified in the colorectal tumors. Importantly, overexpression of PBF in colorectal HCT116 cells interfered with the transcriptional activity of p53-responsive genes such as mdm2, p21 and sfn. Diminished p53 stability (> 90%; P < 0.01) was also evident with a concurrent increase in ubiquitinated p53. Human colorectal tumors with wild-type TP53 and high PBF expression also had low p53 protein levels (P < 0.05), further emphasizing a putative interaction between these genes in vivo. Overall, these results demonstrate an emerging role for PBF in colorectal tumorigenesis through regulating p53 activity, with implications for PBF as a prognostic indicator for invasive tumors.

Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry.

Phosphorylation events within cancer cells often become dysregulated, leading to oncogenic signaling and abnormal cell growth. Phosphopeptides derived from aberrantly phosphorylated proteins that are presented on tumors and not on normal tissues by human leukocyte antigen (HLA) class I molecules are promising candidates for future cancer immunotherapies, because they are tumor specific and have been shown to elicit cytotoxic T cell responses. Robust phosphopeptide enrichments that are suitable for low input amounts must be developed to characterize HLA-associated phosphopeptides from clinical samples that are limited by material availability. We present two complementary mass spectrometry-compatible, iron(III)-immobilized metal affinity chromatography (IMAC) methods that use either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) in-house-fabricated columns. We developed these protocols to enrich for subfemtomole-level phosphopeptides from cell line and human tissue samples containing picograms of starting material, which is an order of magnitude less material than what is commonly used. In addition, we added a peptide esterification step to increase phosphopeptide specificity from these low-input samples. To date, hundreds of phosphopeptides displayed on melanoma, ovarian cancer, leukemia and colorectal cancer have been identified using these highly selective phosphopeptide enrichment protocols in combination with a program called 'CAD Neutral Loss Finder' that identifies all spectra containing the characteristic neutral loss of phosphoric acid from phosphorylated serine and threonine residues. This methodology enables the identification of HLA-associated phosphopeptides presented by human tissue samples containing as little as nanograms of peptide material in 2 d.

Contact-dependent depletion of hydrogen peroxide by catalase is a novel mechanism of myeloid-derived suppressor cell induction operating in human hepatic stellate cells.

Myeloid-derived suppressor cells (MDSC) represent a unique cell population with distinct immunosuppressive properties that have been demonstrated to shape the outcome of malignant diseases. Recently, human hepatic stellate cells (HSC) have been reported to induce monocytic-MDSC from mature CD14(+) monocytes in a contact-dependent manner. We now report a novel and unexpected mechanism by which CD14(+)HLADR(low/-) suppressive cells are induced by catalase-mediated depletion of hydrogen peroxide (H2O2). Incubation of CD14(+) monocytes with catalase led to a significant induction of functional MDSC compared with media alone, and H2O2 levels inversely correlated with MDSC frequency (r = -0.6555, p < 0.05). Catalase was detected in primary HSC and a stromal cell line, and addition of the competitive catalase inhibitor hydroxylamine resulted in a dose-dependent impairment of MDSC induction and concomitant increase of H2O2 levels. The NADPH-oxidase subunit gp91 was significantly increased in catalase-induced MDSC as determined by quantitative PCR outlining the importance of oxidative burst for the induction of MDSC. These findings represent a so far unrecognized link between immunosuppression by MDSC and metabolism. Moreover, this mechanism potentially explains how stromal cells can induce a favorable immunological microenvironment in the context of tissue oxidative stress such as occurs during cancer therapy.

A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells.

The suppression assay is a commonly performed assay, measuring the ability of regulatory T cells (Treg) to suppress T cell proliferation. Most frequently, Treg are obtained from the peripheral blood or spleen. Lower yields are obtained by isolation from other tissues, rendering downstream suppression assays challenging to perform. Furthermore, the importance of suppressive subpopulations of Treg favours their isolation by fluorescent-activated cell sorting. Here we describe a method to isolate Treg from human tissues, using colorectal cancer tissue as an example. Treg suppressive capacity was further examined by expression of CCR5 to demonstrate the ability of our method to assess the suppressive capacity of regulatory T cell subsets. To optimise the standard suppression assay to achieve our research aims, the following modifications were made: Treg, isolated from tissues, were sorted directly into a well-plate.Responder T cells, which had been fluorescently-labelled prior to sorting, were added directly into the well-plate.Human Treg Suppression Inspector beads (Miltenyi Biotec Ltd, UK) provided a polyclonal stimulus for proliferation and were added to each well at a bead:lymphocyte ratio of 1:2. This method quantified the suppression of responder T cell proliferation by small numbers of strictly-defined Treg populations isolated from tissues.

A ruptured infected mesenteric cyst diagnosed on laparoscopy for suspected appendicitis.

Lower abdominal pain of acute onset in young women with a negative pregnancy test is a frequent reason for referral to the general surgical team and the differential diagnoses include acute appendicitis, complicated ovarian cysts and pelvic inflammatory disease. Intestinal and mesenteric cystic disease is a rare entity and less than half of cases present acutely. We present a case of a 25-year-old woman who underwent diagnostic laparoscopy for acute lower abdominal pain and was diagnosed with a ruptured, infected mesenteric cyst.