Tracking in situ checkpoint inhibitor-bound target T cells in patients with checkpoint-induced colitis.

The success of checkpoint inhibitors (CPIs) for cancer has been tempered by immune-related adverse effects including colitis. CPI-induced colitis is hallmarked by expansion of resident mucosal IFNγ cytotoxic CD8+ T cells, but how these arise is unclear. Here, we track CPI-bound T cells in intestinal tissue using multimodal single-cell and subcellular spatial transcriptomics (ST). Target occupancy was increased in inflamed tissue, with drug-bound T cells located in distinct microdomains distinguished by specific intercellular signaling and transcriptional gradients. CPI-bound cells were largely CD4+ T cells, including enrichment in CPI-bound peripheral helper, follicular helper, and regulatory T cells. IFNγ CD8+ T cells emerged from both tissue-resident memory (TRM) and peripheral populations, displayed more restricted target occupancy profiles, and co-localized with damaged epithelial microdomains lacking effective regulatory cues. Our multimodal analysis identifies causal pathways and constitutes a resource to inform novel preventive strategies.

CD, or not CD, that is the question: a digital interobserver agreement study in coeliac disease.

OBJECTIVE: Coeliac disease (CD) diagnosis generally depends on histological examination of duodenal biopsies. We present the first study analysing the concordance in examination of duodenal biopsies using digitised whole-slide images (WSIs). We further investigate whether the inclusion of immunoglobulin A tissue transglutaminase (IgA tTG) and haemoglobin (Hb) data improves the interobserver agreement of diagnosis. DESIGN: We undertook a large study of the concordance in histological examination of duodenal biopsies using digitised WSIs in an entirely virtual reporting setting. Our study was organised in two phases: in phase 1, 13 pathologists independently classified 100 duodenal biopsies (40 normal; 40 CD; 20 indeterminate enteropathy) in the absence of any clinical or laboratory data. In phase 2, the same pathologists examined the (re-anonymised) WSIs with the inclusion of IgA tTG and Hb data. RESULTS: We found the mean probability of two observers agreeing in the absence of additional data to be 0.73 (±0.08) with a corresponding Cohen's kappa of 0.59 (±0.11). We further showed that the inclusion of additional data increased the concordance to 0.80 (±0.06) with a Cohen's kappa coefficient of 0.67 (±0.09). CONCLUSION: We showed that the addition of serological data significantly improves the quality of CD diagnosis. However, the limited interobserver agreement in CD diagnosis using digitised WSIs, even after the inclusion of IgA tTG and Hb data, indicates the importance of interpreting duodenal biopsy in the appropriate clinical context. It further highlights the unmet need for an objective means of reproducible duodenal biopsy diagnosis, such as the automated analysis of WSIs using artificial intelligence.

Impact of the transition to digital pathology in a clinical setting on histopathologists in training: experiences and perceived challenges within a UK training region.

AIMS: With increasing utility of digital pathology (DP), it is important to consider the experiences of histopathologists in training, particularly in view of the varied access to DP across a training region and the consequent need to remain competent in reporting on glass slides (GS), which is also relevant for the Fellowship of the Royal College of Pathologists part 2 examination. Understanding the impact of DP on training is limited but could aid development of guidance to support the transition. We sought to investigate the perceptions of histopathologists in training around the introduction of DP for clinical diagnosis within a training region, and the potential training benefits and challenges. METHODS: An anonymous online survey was circulated to 24 histopathologists in training within a UK training region, including a hospital which has been fully digitised since summer 2020. RESULTS: 19 of 24 histopathologists in training responded (79%). The results indicate that DP offers many benefits to training, including ease of access to cases to enhance individual learning and teaching in general. Utilisation of DP for diagnosis appears variable; almost half of the (10 of 19) respondents with DP experience using it only for ancillary purposes such as measurements, reporting varying levels of confidence in using DP clinically. For those yet to undergo the transition, there was a perceived anxiety regarding digital reporting despite experience with DP in other contexts. CONCLUSIONS: The survey evidences the need for provision of training and support for histopathologists in training during the transition to DP, and for consideration of their need to maintain competence and confidence with GS reporting.

RFID analysis of the complexity of cellular pathology workflow-An opportunity for digital pathology.

Digital pathology (DP) offers potential for time efficiency gains over an analog workflow however, to date, evidence supporting this claim is relatively lacking. Studies available concentrate on specific workflow points such as diagnostic reporting time, rather than overall efficiencies in slide logistics that might be expected. This is in part a result of the complexity and variation in analog working, and the challenge therefore in capturing this. We have utilized RFID technology to conduct a novel study capturing the movement of diagnostic cases within the analog pathway in a large teaching hospital setting, thus providing benchmark data for potential efficiency gains with DP. This technology overcomes the need to manually record data items and has facilitated the capture of both the physical journey of a case and the time associated with relevant components of the analog pathway predicted to be redundant in the digital setting. RFID tracking of 1,173 surgical pathology cases and over 30 staff in an analog cellular pathology workflow illustrates the complexity of the physical movement of slides within the department, which impacts on case traceability within the system. Detailed analysis of over 400 case journeys highlights redundant periods created by batching of slides at workflow points, including potentially 2-3 h for a case to become available for reporting after release from the lab, and variable lag-times prior to collection for reporting, and provides an illustration of patterns of lab and pathologist working within the analog setting. This study supports the challenge in evidencing efficiency gains to be anticipated with DP in the context of the variation and complexity of the analog pathway, but also evidences the efficiency gains that may be expected through a greater understanding of patterns of working and movement of cases. Such data may benefit other departments building a business case for DP.

Interferon-Gamma-Producing CD8+ Tissue Resident Memory T Cells Are a Targetable Hallmark of Immune Checkpoint Inhibitor-Colitis.

BACKGROUND & AIMS: The pathogenesis of immune checkpoint inhibitor (ICI)-colitis remains incompletely understood. We sought to identify key cellular drivers of ICI-colitis and their similarities to idiopathic ulcerative colitis, and to determine potential novel therapeutic targets. METHODS: We used a cross-sectional approach to study patients with ICI-colitis, those receiving ICI without the development of colitis, idiopathic ulcerative colitis, and healthy controls. A subset of patients with ICI-colitis were studied longitudinally. We applied a range of methods, including multiparameter and spectral flow cytometry, spectral immunofluorescence microscopy, targeted gene panels, and bulk and single-cell RNA sequencing. RESULTS: We demonstrate CD8+ tissue resident memory T (TRM) cells are the dominant activated T cell subset in ICI-colitis. The pattern of gastrointestinal immunopathology is distinct from ulcerative colitis at both the immune and epithelial-signaling levels. CD8+ TRM cell activation correlates with clinical and endoscopic ICI-colitis severity. Single-cell RNA sequencing analysis confirms activated CD8+ TRM cells express high levels of transcripts for checkpoint inhibitors and interferon-gamma in ICI-colitis. We demonstrate similar findings in both anti-CTLA-4/PD-1 combination therapy and in anti-PD-1 inhibitor-associated colitis. On the basis of our data, we successfully targeted this pathway in a patient with refractory ICI-colitis, using the JAK inhibitor tofacitinib. CONCLUSIONS: Interferon gamma-producing CD8+ TRM cells are a pathological hallmark of ICI-colitis and a novel target for therapy.

Quantitative multiparametric magnetic resonance imaging can aid non-alcoholic steatohepatitis diagnosis in a Japanese cohort.

BACKGROUND: Non-invasive assessment of non-alcoholic steatohepatitis (NASH) is increasing in desirability due to the invasive nature and costs associated with the current form of assessment; liver biopsy. Quantitative multiparametric magnetic resonance imaging (mpMRI) to measure liver fat (proton density fat fraction) and fibroinflammatory disease [iron-corrected T1 (cT1)], as well as elastography techniques [vibration-controlled transient elastography (VCTE) liver stiffness measure], magnetic resonance elastography (MRE) and 2D Shear-Wave elastography (SWE) to measure stiffness and fat (controlled attenuated parameter, CAP) are emerging alternatives which could be utilised as safe surrogates to liver biopsy. AIM: To evaluate the agreement of non-invasive imaging modalities with liver biopsy, and their subsequent diagnostic accuracy for identifying NASH patients. METHODS: From January 2019 to February 2020, Japanese patients suspected of NASH were recruited onto a prospective, observational study and were screened using non-invasive imaging techniques; mpMRI with LiverMultiScan ®, VCTE, MRE and 2D-SWE. Patients were subsequently biopsied, and samples were scored by three independent pathologists. The diagnostic performances of the non-invasive imaging modalities were assessed using area under receiver operating characteristic curve (AUC) with the median of the histology scores as the gold standard diagnoses. Concordance between all three independent pathologists was further explored using Krippendorff's alpha (a) from weighted kappa statistics. RESULTS: N = 145 patients with mean age of 60 (SD: 13 years.), 39% females, and 40% with body mass index ≥ 30 kg/m2 were included in the analysis. For identifying patients with NASH, MR liver fat and cT1 were the strongest performing individual measures (AUC: 0.80 and 0.75 respectively), and the mpMRI metrics combined (cT1 and MR liver fat) were the overall best non-invasive test (AUC: 0.83). For identifying fibrosis ≥ 1, MRE performed best (AUC: 0.97), compared to VCTE-liver stiffness measure (AUC: 0.94) and 2D-SWE (AUC: 0.94). For assessment of steatosis ≥ 1, MR liver fat was the best performing non-invasive test (AUC: 0.92), compared to controlled attenuated parameter (AUC: 0.75). Assessment of the agreement between pathologists showed that concordance was best for steatosis (a = 0.58), moderate for ballooning (a = 0.40) and fibrosis (a = 0.40), and worst for lobular inflammation (a = 0.11). CONCLUSION: Quantitative mpMRI is an effective alternative to liver biopsy for diagnosing NASH and non-alcoholic fatty liver, and thus may offer clinical utility in patient management.

Role of digital pathology in diagnostic histopathology in the response to COVID-19: results from a survey of experience in a UK tertiary referral hospital.

The COVID-19 pandemic has challenged our diagnostic services at a time when many histopathology departments already faced a diminishing workforce and increasing workload. Digital pathology (DP) has been hailed as a potential solution to at least some of the challenges faced. We present a survey of pathologists within a UK National Health Service cellular pathology department with access to DP, in which we ascertain the role of DP in clinical services during this current pandemic and explore challenges encountered. This survey indicates an increase in uptake of diagnostic DP during this period, with increased remote access. Half of respondents agreed that DP had facilitated maintenance of diagnostic practice. While challenges have been encountered, these are remediable, and none have impacted on the uptake of DP during this period. We conclude that in our institution, DP has demonstrated current and future potential to increase resilience in diagnostic practice and have highlighted some of the challenges that need to be considered.

Immune checkpoint inhibitor-related colitis assessment and prognosis: can IBD scoring point the way?

BACKGROUND: Immune checkpoint inhibitors (ICI) improve survival but cause immune-related adverse events (irAE). We sought to determine if CTCAE classification, IBD biomarkers/endoscopic/histological scores correlate with irAE colitis outcomes. METHODS: A dual-centre retrospective study was performed on patients receiving ICI for melanoma, NSCLC or urothelial cancer from 2012 to 2018. Demographics, clinical data, endoscopies (reanalysed using Mayo/Ulcerative Colitis Endoscopic Index of Severity (UCEIS) scores), histology (scored with Nancy Index) and treatment outcomes were analysed. RESULTS: In all, 1074 patients were analysed. Twelve percent (134) developed irAE colitis. Median patient age was 66, 59% were male. CTCAE diarrhoea grade does not correlate with steroid/ infliximab use. G3/4 colitis patients are more likely to need infliximab (p < 0.0001) but colitis grade does not correlate with steroid duration. CRP, albumin and haemoglobin do not correlate with severity. The UCEIS (p = 0.008) and Mayo (p = 0.016) scores correlate with severity/infliximab requirement. Patients with higher Nancy indices (3/4) are more likely to require infliximab (p = 0.03). CONCLUSIONS: CTCAE assessment does not accurately reflect colitis severity and our data do not support its use in isolation, as this may negatively impact timely management. Our data support utilising endoscopic scoring for patients with >grade 1 CTCAE disease, and demonstrate the potential prognostic utility of objective histologic scoring.

Experience from the first UK inter-regional specialist multidisciplinary meeting in the diagnosis and management of IgG4-related disease.

Immunoglobulin G4-related disease (IgG4-RD) is a complex multisystem fibro-inflammatory disorder, requiring diagnostic differentiation from malignancy and other immune-mediated conditions, and careful management to minimise glucocorticoid-induced toxicity and prevent progressive organ dysfunction. We describe the experience of the first inter-regional specialist IgG4-RD multidisciplinary team meeting (MDM) incorporating a broad range of generalists and specialists, held 6-weekly via web-link between Oxford University Hospitals NHS Foundation Trust and University College London Hospitals NHS Foundation Trust. Over 3 years, there were 206 discussions on 156 patients. Of these, 97 (62%) were considered to have definite or possible IgG4-RD; 67% had multi-organ involvement and 23% had a normal serum IgG4. The average number of specialist opinions sought prior to MDM was four per patient. Management was changed in the majority of patients (74%) with the treatment escalation recommended in 61 cases, including 19 for rituximab. Challenges arose from delays and misdiagnosis, cross-specialty presentation and the management of sub-clinical disease. Our cross-discipline IgG4-RD MDM enabled important diagnostic and management decisions in this complex multisystem disorder, and can be used as a model for other centres in the UK.

Pulmonary hyalinising granuloma: a rare and elusive cause of multiple lung nodules.

A 72-year-old woman was referred with incidentally detected multiple lung nodules, one of which was identified as 18F-fluorodeoxyglucose (FDG)-avid on positron emission tomography. Extensive workup followed, including numerous radiographs, surveillance scans and a CT-guided biopsy which demonstrated chronic inflammation only. Following a wedge-resection, a diagnosis of pulmonary hyalinising granuloma (PHG) was made. PHG is a cause of FDG-avid single or multiple pulmonary nodules and can mimic lung cancer or metastatic disease radiologically. The diagnosis is often difficult to make with minimally invasive techniques such as needle-guided biopsies which do not tend to yield the diagnosis and requires surgical resection for definitive diagnosis and exclusion of malignancy.

Early allograft pancreatectomy-Technical failure or acute pancreatic rejection?

INTRODUCTION: "Technical failure" is still perceived to be a frequent cause of graft loss after pancreas transplantation. However, some early graft losses currently attributed to technical failure could be due to unrecognized acute pancreas rejection (APR). METHODS: We investigated the apparent incidence of APR in cases of early allograft pancreatectomy (EAP) that had previously been attributed to technical failure. We performed an analysis of 198 patients who underwent pancreas transplantation between January 2009 and January 2016 and identified all those with EAP within 90 days of transplantation. Explanted grafts of EAP recipients were re-examined histologically to evaluate for evidence of APR using current Banff criteria. RESULTS: Twenty-three EAPs were identified (11.6%; 23/198). APR was identified histologically in 9 out of the 15 recipients who lost their grafts due to duodenal leaks or recurrent peripancreatic collections, but was not identified in any of the patients whose grafts were lost due to thrombosis or ischemia. INTERPRETATION: Unsuspected APR appears common in the explanted grafts of recipients who have undergone EAP for apparently "technical" reasons. We suggest that EAP should be defined as a technical failure only when APR of the pancreas (or duodenum) has been excluded by histological analysis.

Lupus causing small bowel obstruction.

A 20-year-old female patient was admitted to hospital in 2015 with 1 year history of recurrent abdominal pain, distension, borborygmi and nausea. The patient had a background of systemic lupus erythematous (SLE) diagnosed 4 years before, with skin, joint and renal involvement. The initial investigations have shown a long segment of ileal inflammation with upstream obstruction. Differential diagnoses were mainly SLE enteritis or concomitant Crohn's. Patient failed the initial conservative management and had a laparotomy with small bowel (SB) resection and ileostomy. The histology was suggestive of autoimmune enteritis. Although bowel involvement is a frequent feature of SLE, surgery for obstruction is extremely rare. Postoperatively, she had an emergency admission and was diagnosed with SB volvulus with perforation. She underwent further resection and stoma refashioning in 2016. As a consequence, she developed short gut syndrome. Eventually, the stoma was reversed and parenteral nutrition was stopped and weight became stable.

Exophytic cystic liver lesion: an unusual presentation of a hepatocellular carcinoma in a young female.

Hepatocellular carcinoma (HCC) is one of the most common malignant primary liver tumours. However, primary hepatic carcinomas are rare in young adults, accounting for approximately 1% of tumours in people below the age of 20. Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are the two most important aetiological agents of HCC. The average age at onset of HBV-related HCC (~50 years old) is 10 years younger than that of HCV-related HCC (61-64 years). Evidence for an association between the oral contraceptive pill (OCP) and development of HCC remains inconclusive. Here, we describe a case of a 28-year-old woman with normal background liver, who presented with a large palpable abdominal mass due to a bilobar, exophytic, cystic lesion arising from the right lobe of the liver, later diagnosed as HCC on histological analysis. We highlight the need for considering HCC even in the unusual setting of a cystic, exophytic lesion.

Interobserver Variability in Histologic Evaluation of Liver Fibrosis Using Categorical and Quantitative Scores.

Objectives: The aim of the study was to investigate the interobserver agreement for categorical and quantitative scores of liver fibrosis. Methods: Sixty-five consecutive biopsy specimens from patients with mixed liver disease etiologies were assessed by three pathologists using the Ishak and nonalcoholic steatohepatitis Clinical Research Network (NASH CRN) scoring systems, and the fibrosis area (collagen proportionate area [CPA]) was estimated by visual inspection (visual-CPA). A subset of 20 biopsy specimens was analyzed using digital imaging analysis (DIA) for the measurement of CPA (DIA-CPA). Results: The bivariate weighted κ between any two pathologists ranged from 0.57 to 0.67 for Ishak staging and from 0.47 to 0.57 for the NASH CRN staging. Bland-Altman analysis showed poor agreement between all possible pathologist pairings for visual-CPA but good agreement between all pathologist pairings for DIA-CPA. There was good agreement between the two pathologists who assessed biopsy specimens by visual-CPA and DIA-CPA. The intraclass correlation coefficient, which is equivalent to the κ statistic for continuous variables, was 0.78 for visual-CPA and 0.97 for DIA-CPA. Conclusions: These results suggest that DIA-CPA is the most robust method for assessing liver fibrosis followed by visual-CPA. Categorical scores perform less well than both the quantitative CPA scores assessed here.

Altered expression of epithelial-to-mesenchymal transition proteins in extraprostatic prostate cancer.

Epithelial to mesenchymal transition (EMT) of cancer cells involves loss of epithelial polarity and adhesiveness, and gain of invasive and migratory mesenchymal behaviours. EMT occurs in prostate cancer (PCa) but it is unknown whether this is in specific areas of primary tumours. We examined whether any of eleven EMT-related proteins have altered expression or subcellular localisation within the extraprostatic extension component of locally advanced PCa compared with other localisations, and whether similar changes may occur in in vitro organotypic PCa cell cultures and in vivo PCa models. Expression profiles of three proteins (E-cadherin, Snail, and α-smooth muscle actin) were significantly different in extraprostatic extension PCa compared with intra-prostatic tumour, and 18/27 cases had an expression change of at least one of these three proteins. Of the three significantly altered EMT proteins in pT3 samples, one showed similar significantly altered expression patterns in in vitro organotypic culture models, and two in in vivo Pten-/- model samples. These results suggest that changes in EMT protein expression can be observed in the extraprostatic extension component of locally invasive PCa. The biology of some of these changes in protein expression may be studied in certain in vitro and in vivo PCa models.

Bile duct basement membrane thickening in primary sclerosing cholangitis.

AIMS: Primary sclerosing cholangitis (PSC) is characterized histologically by portal inflammation, bile duct injury and regeneration and concentric periductal fibrosis. Although seen commonly in our experience, the significance of histological thickening of the bile duct basement membrane on periodic acid Schiff (PAS)-positive, diastase-resistant (DPAS) staining has never been analysed formally. In this paper we provide an evidence-based assessment of basement membrane thickening (BMT) reproducibility and diagnostic accuracy. METHODS AND RESULTS: A total of 128 archived medical liver core biopsies were retrieved and blinded for review by two independent histopathologists. BMT was assessed and designated as absent or present with a grade (G) of G1-G3. The sensitivity of any BMT for PSC was good at 77%, with moderate specificity at 61%. When only G3 BMT was considered positive, the specificity was high at 95% but the sensitivity was poor at 16%. The interobserver agreement (0.69) and consistency (0.72) were good. CONCLUSIONS: Basement membrane thickening is a reproducible predictor for PSC with good sensitivity and specificity. The presence of G2 and especially G3 BMT showed high specificity and could be regarded as highly predictive of PSC. The presence of more than G1 BMT should be reported and the possibility of PSC should be raised in the differential diagnosis.

A template for a clinico-pathological audit of medical liver biopsies.

AIMS: With changing indications for performing medical liver biopsies, we aimed to develop a tool to allow pathologists to evaluate the current usefulness, value and impact of their medical liver biopsy service. METHODS: We designed and piloted a questionnaire-based clinico-pathological audit for medical liver biopsies. RESULTS: The audit tool was simple to implement and provided useful information about our service. Hepatologists felt that 96% of reports were clinically useful. 56% of biopsies confirmed clinical diagnoses, 46% helped differentiate between diagnoses and 42% were able to exclude possible diagnoses. 74% resulted in a change of management and 27% of liver biopsies resulted in a diagnosis which was not clinically suspected. CONCLUSIONS: We demonstrate the usefulness of an audit tool in providing evidence of the value of the liver pathology service in a large UK regional centre.