Blockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent ...anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1-revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1-to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.
Joint replacement is an effective intervention and prosthetic joint infection (PJI) is one of the most serious complications of such surgery. Diagnosis of PJI is often complex and requires multiple ...modalities of investigation. We describe a rare cause of PJI which highlights these challenges and the role of whole-genome sequencing to achieve a rapid microbiological diagnosis to facilitate prompt and appropriate management.
A 79-year-old man developed chronic hip pain associated with a soft-tissue mass, fluid collection and sinus adjacent to his eight-year-old hip prosthesis. His symptoms started after intravesical Bacillus Calmette-Guerin (BCG) therapy for bladder cancer. Synovasure™ and 16S polymerase chain reaction (PCR) tests were negative, but culture of the periarticular mass and genome sequencing diagnosed BCG infection. He underwent a two-stage joint revision and a prolonged duration of antibiotic therapy which was curative.
BCG PJI after therapeutic exposure can have serious consequences, and awareness of this potential complication, identified from patient history, is essential. In addition, requesting appropriate testing is required, together with recognition that traditional diagnostics may be negative in non-pyogenic PJI. Advanced molecular techniques have a role to enhance the timely management of these infections.
Pleomorphic adenoma is a benign salivary gland neoplasm with a diverse morphology. This is considered to be a function of the neoplastic myoepithelium, which shows histological and immunophenotypical ...variability. Wilms' tumor 1 gene (WT1) protein, involved in bidirectional mesenchymal-epithelial transition, has been detected by reverse transcription PCR in salivary gland tumors showing myoepithelial-epithelial differentiation. The aim of this study was to investigate the immunoreactivity of WT1 in pleomorphic adenomas and to compare the pattern of staining with p63 and calponin, two reliable markers of myoepithelial cells. A total of 31 cases of pleomorphic adenoma were selected. The myoepithelium was classified as myoepithelial-like (juxtatubular and spindled), modified myoepithelium (myxoid, chondroid and plasmacytoid) and transformed myoepithelium (solid epithelioid, squamous and basaloid cribriform). Immunohistochemistry for WT1, p63 and calponin was assessed in each myoepithelial component, as well as in nonneoplastic myoepithelial cells and inner tubular epithelial cells. There was no immunostaining of tubular epithelial cells by any of the markers. In contrast to p63 and calponin, WT1 did not react with normal myoepithelial cells. Cytoplasmic WT1 staining was present in all pleomorphic adenomas, and in 29 cases (94%), >50% of neoplastic myoepithelial cells were highlighted. p63 and calponin stained the myoepithelium in 30 tumors. In comparison, 50% of cells were positive in 21 (68%) and 9 (29%) cases of p63 and calponin, respectively. Staining with WT1 showed less variability across the spectrum of myoepithelial differentiation with the difference most marked in the transformed myoepithelium. WT1 is a sensitive marker of the neoplastic myoepithelial cell in pleomorphic adenomas. The role of this protein in influencing the mesenchymal-epithelial state of cells suggests that WT1 and the myoepithelial cell have an important role in the histogenesis of pleomorphic adenomas.
Background: Tumor levels of steroid hormone receptors, a factor used to select adjuvant treatment for early-stage breast cancer, are currently determined with immunohistochemical assays. These assays ...have a discordance of 10%–30% with previously used extraction assays. We assessed the concordance and predictive value of hormone receptor status as determined by immunohistochemical and extraction assays on specimens from International Breast Cancer Study Group Trials VIII and IX. These trials predominantly used extraction assays and compared adjuvant chemoendocrine therapy with endocrine therapy alone among pre- and postmenopausal patients with lymph node–negative breast cancer. Trial conclusions were that combination therapy provided a benefit to pre- and postmenopausal patients with estrogen receptor (ER)–negative tumors but not to ER-positive postmenopausal patients. ER-positive premenopausal patients required further study. Methods: Tumor specimens from 571 premenopausal and 976 postmenopausal patients on which extraction assays had determined ER and progesterone receptor (PgR) levels before randomization from October 1, 1988, through October 1, 1999, were re-evaluated with an immunohistochemical assay in a central pathology laboratory. The endpoint was disease-free survival. Hazard ratios of recurrence or death for treatment comparisons were estimated with Cox proportional hazards regression models, and discriminatory ability was evaluated with the c index. All statistical tests were two-sided. Results: Concordance of hormone receptor status determined by both assays ranged from 74% (κ = 0.48) for PgR among postmenopausal patients to 88% (κ = 0.66) for ER in postmenopausal patients. Hazard ratio estimates were similar for the association between disease-free survival and ER status (among all patients) or PgR status (among postmenopausal patients) as determined by the two methods. However, among premenopausal patients treated with endocrine therapy alone, the discriminatory ability of PgR status as determined by immunohistochemical assay was statistically significantly better (c index = 0.60 versus 0.51; P = .003) than that determined by extraction assay, and so immunohistochemically determined PgR status could predict disease-free survival. Conclusions: Trial conclusions in which ER status (for all patients) or PgR status (for postmenopausal patients) was determined by immunohistochemical assay supported those determined by extraction assays. However, among premenopausal patients, trial conclusions drawn from PgR status differed—immunohistochemically determined PgR status could predict response to endocrine therapy, unlike that determined by the extraction assay.
ObjectiveCoeliac 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.DesignWe 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.ResultsWe 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).ConclusionWe 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.
Summary Diabetic myonecrosis, also known as diabetic muscle infarction is a rare complication of diabetes mellitus usually associated with longstanding suboptimal glycaemic control. Although theories ...of atherosclerosis, diabetic microangiopathy, vasculitis, ischaemia-reperfusion injury and hypercoagulable state have been proposed to explain the pathophysiology, none of these have been able to individually explain the pathophysiology in entirety. Diabetic renal disease is the most common risk factor for developing DMN and its recurrence. The diagnosis is often missed due to lack of awareness and the presentation mimicking other conditions associated with DM. The routine laboratory investigations are often non-specific and do not provide much value in the diagnosis as well. Muscle biopsy can provide a definite diagnosis but is not currently recommended due to its invasiveness and association with prolonged time to symptoms resolution. Magnetic resonance imaging, in combination with classic history and risk factors can clinch the diagnosis. Treatment is generally analgesia and rest, although the former’s use may be limited in the presence of renal disease. Learning points: Diabetic myonecrosis is a rare complication of diabetes mellitus associated with longstanding suboptimal glycaemic control. Diabetic renal disease is a known risk factor, although the evidence is merely observational. Although muscle biopsy could provide a definite diagnosis, it is not recommended as it can prolong the disease process and should be reserved only for cases not responding to conventional treatment. Typical MRI findings in combination with classic symptoms and risk factors can clinch the diagnosis Current treatment recommendations include NSAIDs and/or aspirin (if not contraindicated) alongside bed rest. Physiotherapy is not recommended in the acute phase but should be started as soon as patient is discharged from hospital. Optimal glycaemic control is key to prevent recurrence.
CD248 or Endosialin is a transmembrane molecule expressed in stromal cells binding to extracellular matrix (ECM) components. It has been previously implicated in kidney fibrosis, rheumatoid arthritis ...as well as in tumour-stromal interactions. This study investigates the role of CD248 in the pathogenesis of fibrotic diseases in Idiopathic Pulmonary Fibrosis (IPF).
CD248 quantitative immunohistochemistry (IHC) was performed on lung samples from 22 IPF patients and its expression was assayed in cultured pulmonary fibroblasts and epithelial cells. Effects of CD248 silencing was evaluated on fibroblast proliferation and myofibroblast differentiation.
IHC revealed strong CD248 expression in mesenchymal cells of normal lung structures such as pleura and adventitia but not in epithelium. Fibrotic areas showed markedly stronger staining than unaffected lung tissue. The extent of CD248 staining showed a significant negative correlation to lung function parameters FEV1, FVC, TLC, and TLCO (r2 > 0 · 35, p < 0 · 01). CD248 protein levels were significantly greater in IPF-derived lung fibroblasts vs normal lung fibroblasts (p < 0 · 01) and CD248 silencing significantly reduced the proliferation of lung fibroblasts, but did not affected myofibroblast differentiation.
We conclude that CD248 overexpression is possibly involved in the pathogenesis of IPF and it has potential as a disease severity marker. Given that CD248 ligands are collagen type I, IV and fibronectin, we hypothesise that CD248 signalling represents a novel matrix-fibroblast interaction that may be a potential therapeutic target in IPF.
Background and Aims: Hepatotoxicity, especially liver fibrosis, is the major concern with long‐term, ‘low‐dose’ oral methotrexate (MTX) therapy for psoriasis. The histological features are ...non‐specific and resemble those of non‐alcoholic steatohepatitis (NASH). Moreover, most of the risk factors of MTX‐induced liver injury are also associated with NASH. In this study, we investigate whether NASH contributes to the prevalence and progression of MTX‐induced liver injury in patients receiving MTX for psoriasis.
Methods: Clinical details, including MTX dosage schedules and risk factors for liver injury, was documented for 24 patients on long‐term MTX therapy for psoriasis. Serial liver biopsies were graded according to the Roenigk classification scale and a recently proposed grading and staging system for NASH.
Results: Thirteen of the 17 patients who had a NASH‐like pattern of liver injury also had the risk factors for NASH obesity and/or diabetes, and all had progressive liver injury. The other four patients had no risk factors, but a mean cumulative dose of 6.5 g. Seven patients, who did not have a NASH‐like pattern of injury, had a mean cumulative dose of 3.8 g. There was a positive correlation between the cumulative dose, risk factors and progression when the biopsies were scored by the modified grading and staging classification for NASH, but not with the Roenigk system.
Conclusions: Non‐steatohepatitis, probably aggravated by MTX, is an important cause of liver injury in patients on long‐term, ‘low‐dose’ MTX treatment for psoriasis. In addition, MTX alone can cause a NASH‐like pattern of injury that is at least, in part, caused by a higher cumulative dose.
The detection of lymph node involvement is fundamental to the staging of rectal cancer, and aids in prognostication and identification of patients who will benefit from adjuvant therapy. The ...anatomical variation in distribution and size of mesorectal lymph nodes has received scant attention.
This study aimed to determine the size and distribution of lymph nodes in rectal cancer resection specimens.
This was a prospective, observational study of rectal cancer resection specimens analyzed by a single histopathologist.
This study was conducted from January 2007 to July 2013 at the authors' institution.
Two hundred forty-four consecutive patients underwent resection for rectal cancer.
The size and distribution of lymph nodes in the resection specimens and the anatomical position of mesorectal lymph nodes in relation to the peritoneal reflection, tumor, and anal verge were recorded.
A total of 10,473 lymph nodes were retrieved in 244 patients (75 women; median age, 68 years (interquartile range, 59-75 years)). One hundred seventy-three anterior resection and 71 abdominoperineal resection specimens were analyzed. Median lymph node yield was 41 lymph nodes (interquartile range, 31-52); 344 of 10,473 (3.2%) lymph nodes were positive. Lymph nodes were distributed in the mesorectum, sigmoid mesentery, and vascular pedicle in 40%, 32%, and 28% of the patients. Sixty-eight percent of mesorectal lymph nodes were above the peritoneal reflection. Mesorectal lymph node distribution in relation to the tumor was 53% above, 36% adjacent to, and only 11% below the tumor. Ninety-five of 334 (28%) positive nodes were ≤3 mm in diameter.
Resection specimens analyzed by other pathologists (<5%) have not been included, and fat clearance techniques were not used to retrieve lymph nodes.
To ensure accurate nodal staging of rectal cancer, both resection and subsequent pathological evaluation should focus on the mesorectum in close proximity to the tumor and along the superior rectal artery. Small lymph nodes (<3 mm in size) should not be overlooked, and lymph node metastasis to the sigmoid mesentery is rare (see Video, Supplemental Digital Content 1, http://links.lww.com/DCR/A177).
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