Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis.

OBJECTIVE: Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. DESIGN: We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. RESULTS: Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not BrafV600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. CONCLUSION: We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.

An immunohistochemical comparison of two TTF-1 monoclonal antibodies in atypical squamous lesions and sarcomatoid carcinoma of the lung, and pleural malignant mesothelioma.

UNLABELLED: Immunohistochemical detection of thyroid transcription factor-1 (TTF-1) plays an important role in the diagnosis and subclassification of non-small cell carcinomas of the lung in biopsy and some cytology samples, specifically for identification of squamous cell carcinoma (classically negative) and non-mucinous adenocarcinoma (positive in most cases) and for discrimination between lung adenocarcinoma and pleural malignant mesothelioma (classically negative). AIMS AND METHODS: We carried out a comparison of the widely used mouse monoclonal TTF-1 antibody based on the 8G7G3/1 clone versus the more recently introduced rabbit monoclonal antibody (MAb) based on the SP141 clone. RESULTS: Both antibodies labelled alveolar epithelium in normal lung parenchyma, but the SP141 antibody also labelled bronchial mucosal basal cells. All 13 cases of atypical squamous lesions (including one case of bronchial squamous dysplasia) were negative with the 8G7G3/1 antibody, but 6/13 cases of squamous carcinoma/dysplasia showed positive nuclear labelling with the SP141 antibody in the same tissue biopsy. All 35 cases of adenocarcinoma of the lung were positive with both antibodies. For 12 cases of sarcomatoid carcinoma of the lung, two cases were labelled with the 8G7G3/1 antibody, whereas positive labelling of 4/12 cases was observed with SP141. All 66 cases of epithelioid malignant mesothelioma were negative with both antibodies, but 8/19 cases of sarcomatoid mesothelioma showed positive nuclear labelling with the SP141 antibody (0/19 with 8G7G3/1). CONCLUSIONS: Our findings indicate differences in the rates of positive and negative labelling with these two antibodies, and suggest the potential for misclassification of a proportion of squamous carcinomas of the lung as adenocarcinoma, and for misdiagnosis of some sarcomatoid mesotheliomas as sarcomatoid carcinoma of the lung. If the results of SP141 are assigned overriding significance, our findings further indicate that in isolation, neither negative labelling with either 8G7G3/1 or SP141 nor positive labelling with the SP141 MAb discriminates between sarcomatoid carcinoma and sarcomatoid mesothelioma, whereas positive labelling with the 8G7G3/1 MAb favours a diagnosis of sarcomatoid carcinoma. The literature suggests that these seemingly 'aberrant' results with the SP141 antibody are not 'false' positives, but rather real detection of low levels of TTF-1 protein in a broader range of tumours than is widely recognised.

Identification of LOXL1 protein and Apolipoprotein E as components of surgically isolated pseudoexfoliation material by direct mass spectrometry.

Pseudoexfoliation (PEX) syndrome is the commonest cause of secondary glaucoma. Many extracellular matrix proteins and elastic fibre structure components are present in the pathological PEX deposits in the anterior segment of the eye including the anterior lens capsule. Common coding variants in the lysyl oxidase-like 1 (LOXL1) gene, involved in cross-linking elastin, have been reported to be strongly associated with PEX syndrome in various human populations. The mechanism by which the LOXL1 protein contributes to the formation of PEX material is unknown. A comprehensive map of the component proteins of PEX deposits can aid the understanding of disease pathogenesis. The purpose of this study was to identify additional protein constituents of pathological PEX deposits. We employed a novel proteomics approach by performing mass spectrometry on "isolated" PEX material surgically removed from the anterior lens capsule of affected eyes. This approach led to the identification of LOXL1 protein and Apolipoprotein E (ApoE) in PEX material. Previously identified protein constituents, latent-transforming growth factor beta-binding protein-2, complement 3 and clusterin were also detected. Immunohistochemical analysis of lens capsules from affected eyes confirmed the presence of both LOXL1 and ApoE in pathological PEX deposits. ApoE is a novel component of these deposits. This is the first report where a direct analytical approach has led to the identification of LOXL1 in PEX deposits and is consistent with its detection in these deposits by immunolabelling in another recent report. LOXL1 is both genetically associated with PEX syndrome and present in pathological PEX deposits. Hence it clearly has an important and direct role in pathophysiology of the disease. In conclusion, additional as yet unknown components are present in pathological PEX deposits and mass spectrometry of "isolated" PEX material is an effective strategy for their identification.