Mitochondrial DNA Is a Pro-Inflammatory Damage-Associated Molecular Pattern Released During Active IBD.

Background: Due to common evolutionary origins, mitochondrial DNA (mtDNA) shares many similarities with immunogenic bacterial DNA. MtDNA is recognized as a pro-inflammatory damage-associated molecular pattern (DAMP) with a pathogenic role in several inflammatory diseases. We hypothesised that mtDNA is released during active disease, serving as a key pro-inflammatory factor in inflammatory bowel disease (IBD). Methods: Between 2014 and 2015, we collected plasma separated within 2 hours of sampling from 97 prospectively recruited IBD patients (67 ulcerative colitis [UC] and 30 Crohn's disease [CD]) and 40 non-IBD controls. We measured circulating mtDNA using quantitative polymerase chain reaction (amplifying mitochondria COXIII/ND2 genes) and also in mouse colitis induced by dextran sulfate-sodium (DSS). We used a mass spectometry approach to detect free plasma mitochondrial formylated peptides. Furthermore, we examined for mitochondrial damage using electron microscopy (EM) and TLR9 expression, the target for mtDNA, in human intestinal IBD mucosa. Results: Plasma mtDNA levels were increased in UC and CD (both P < 0.0001) compared with non-IBD controls. These levels were significantly correlated to blood (C-reactive protein, albumin, white cell count), clinical and endoscopic markers of severity, and disease activity. In active UC, we identified 5 mitochondrial formylated peptides (the most abundant being fMMYALF with known chemoattractant function) in plasma. We observed mitochondrial damage in inflamed UC mucosa and significantly higher fecal MtDNA levels (vs non-IBD controls [P < 0.0001]), which supports gut mucosal mitochondrial DAMP release as the primary source. In parallel, plasma mtDNA levels increased during induction of acute DSS colitis and were associated with more severe colitis (P < 0.05). In active IBD, TLR9+ lamina propria inflammatory cells were significantly higher in UC and CD compared with controls (P < 0.05). Conclusions: We present the first evidence to show that mtDNA is released during active IBD. MtDNA is a potential mechanistic biomarker, and our data point to mtDNA-TLR9 as a therapeutic target in IBD. 10.1093/ibd/izy095_videoizy095.video5776747659001.

Benchmarking a large regional UK HER2 testing service against current practice guidelines.

AIMS: To critically evaluate HER2 testing data for 3500 consecutive cases over 28 months for a single laboratory and review these findings in the light of current UK reporting guidelines. METHODS: We have reviewed all data relating to the HER2 testing service including reagents and analytical machine, HER2 immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) scoring profiles. We have examined the place of double counting and rapid screening of FISH and when it is and is not safe to do so. This analysis has been facilitated greatly by the inclusion of custom scripts embedded in a spreadsheet used for recording the data. RESULTS: There were no differences in scoring profiles in relation to testing machine or reagent batch for both HER2 IHC and FISH. There was excellent concordance in scoring by the biomedical scientists and pathologists providing the service. There is a significant difference between the proximity of scores in double-scored cases with HER2 copy number >6.0 compared with those with copy number <6.00. It is safe to single score with rapid screening of HER2 FISH following a first count with a HER2/CEP17 ratio <1.5 and a HER2 copy number <3.5. CONCLUSIONS: Single counting of HER2 FISH supported by a second rapid screen is safe and practicable within strict parameters. Any assessment of proximity of scores in double-scored cases should take into account the HER2 copy number, with greater spreads experienced at higher copy numbers. Centralising HER2 testing in a regional centre allows detailed audit of the entire analytical process.

Use of a genetically engineered mouse model as a preclinical tool for HER2 breast cancer.

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies presents a major clinical problem. Although preclinical studies have identified a number of possible mechanisms, clinical validation has been difficult. This is most likely to reflect the reliance on cell-line models that do not recapitulate the complexity and heterogeneity seen in human tumours. Here, we show the utility of a genetically engineered mouse model of HER2-driven breast cancer (MMTV-NIC) to define mechanisms of resistance to the pan-HER family inhibitor AZD8931. Genetic manipulation of MMTV-NIC mice demonstrated that loss of phosphatase and tensin homologue (PTEN) conferred de novo resistance to AZD8931, and a tumour fragment transplantation model was established to assess mechanisms of acquired resistance. Using this approach, 50% of tumours developed resistance to AZD8931. Analysis of the resistant tumours showed two distinct patterns of resistance: tumours in which reduced membranous HER2 expression was associated with an epithelial-to-mesenchymal transition (EMT) and resistant tumours that retained HER2 expression and an epithelial morphology. The plasticity of the EMT phenotype was demonstrated upon re-implantation of resistant tumours that then showed a mixed epithelial and mesenchymal phenotype. Further AZD8931 treatment resulted in the generation of secondary resistant tumours that again had either undergone EMT or retained their original epithelial morphology. The data provide a strong rationale for basing therapeutic decisions on the biology of the individual resistant tumour, which can be very different from that of the primary tumour and will be specific to individual patients.

Grading of breast cancer on needle core biopsy: does a reduction in mitotic count threshold improve agreement with grade on excised specimens?

BACKGROUND: Accurate assessment of invasive breast cancer grade on needle core biopsy (NCB) is important as it guides treatment. AIM: To assess the agreement between grade of invasive cancer on NCB and excision and determine whether altering the mitotic count score threshold on NCB improves it. METHODS: Pathology slides from patients with an NCB diagnosis of invasive breast cancer who underwent subsequent breast conservation surgery in 2012 were reviewed. Mitotic counts were assessed and tumour grade agreement between NCB and excision evaluated. The mitotic count thresholds were altered and grade agreement was reassessed. RESULTS: In 283/359 (79%) cases, there was concurrence on histological grade between NCB and excision. Reduction in mitotic count thresholds did not improve either overall tumour grade agreement or agreement within any subgroup of patients. CONCLUSIONS: In our experience, the current mitotic count score thresholds are appropriate and should be maintained.

Analysis of key cell-cycle checkpoint proteins in colorectal tumours.

Aberrations in the components of cell-cycle checkpoints are a common feature of many tumours and several have been shown to have prognostic significance in colorectal cancer. In this study, seven components of cell-cycle control [cyclin D1, retinoblastoma (pRb), p21, p27, p16, p53, and proliferating cell nuclear antigen (PCNA)] were examined in a large series of well-characterized colorectal adenocarcinomas using immunohistochemistry to ascertain co-regulation and influence on survival. The majority (92%) of the tumours had abnormal staining of > or =2 cell-cycle control factors. Expression of cyclin D1 protein was correlated with both p21 (p<0.001) and p27 (p=0.033), suggesting co-regulation of these proteins in colorectal tumours. Only cyclin D1 (p=0.048) and p53 (p=0.025) were directly associated with PCNA levels, suggesting a more important role in the proliferative capacity of tumour cells. Significant associations between cell cycle-related proteins and clinicopathological data were observed: cyclin D1 and p53 proteins were correlated with patient age (p=0.042 and p<0.001, respectively) and p53 (p=0.01) and p21 (p=0.024) proteins were associated with tumour site. Expression of cyclin D1 protein was the only protein examined that was related to improved outcome in these patients (p=0.0266), but it was not an independent predictor of survival. These results suggest that loss of control of cell-cycle checkpoints is a common occurrence in colorectal tumours and may be an important therapeutic target.