Genetic determinants of micronucleus formation in vivo.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

Rapid artefact removal and H&E-stained tissue segmentation.

We present an innovative method for rapidly segmenting haematoxylin and eosin (H&E)-stained tissue in whole-slide images (WSIs) that eliminates a wide range of undesirable artefacts such as pen marks and scanning artefacts. Our method involves taking a single-channel representation of a low-magnification RGB overview of the WSI in which the pixel values are bimodally distributed such that H&E-stained tissue is easily distinguished from both background and a wide variety of artefacts. We demonstrate our method on 30 WSIs prepared from a wide range of institutions and WSI digital scanners, each containing substantial artefacts, and compare it to segmentations provided by Otsu thresholding and Histolab tissue segmentation and pen filtering tools. We found that our method segmented the tissue and fully removed all artefacts in 29 out of 30 WSIs, whereas Otsu thresholding failed to remove any artefacts, and the Histolab pen filtering tools only partially removed the pen marks. The beauty of our approach lies in its simplicity: manipulating RGB colour space and using Otsu thresholding allows for the segmentation of H&E-stained tissue and the rapid removal of artefacts without the need for machine learning or parameter tuning.

Multiple-instance-learning-based detection of coeliac disease in histological whole-slide images.

We present a multiple-instance-learning-based scheme for detecting coeliac disease, an autoimmune disorder affecting the intestine, in histological whole-slide images (WSIs) of duodenal biopsies. We train our model to detect 2 distinct classes, normal tissue and coeliac disease, on the patch-level, and in turn leverage slide-level classifications. Using 5-fold cross-validation in a training set of 1841 (1163 normal; 680 coeliac disease) WSIs, our model classifies slides as normal with accuracy (96.7±0.6)%, precision (98.0±1.7)%, and recall (96.8±2.5)%, and as coeliac disease with accuracy (96.7±0.5)%, precision (94.9±3.7)%, and recall (96.5±2.9)% where the error bars are the cross-validation standard deviation. We apply our model to 2 test sets: one containing 191 WSIs (126 normal; 65 coeliac) from the same sources as the training data, and another from a completely independent source, containing 34 WSIs (17 normal; 17 coeliac), obtained with a scanner model not represented in the training data. Using the same-source test data, our model classifies slides as normal with accuracy 96.5%, precision 98.4% and recall 96.1%, and positive for coeliac disease with accuracy 96.5%, precision 93.5%, and recall 97.3%. Using the different-source test data the model classifies slides as normal with accuracy 94.1% (32/34), precision 89.5%, and recall 100%, and as positive for coeliac disease with accuracy 94.1%, precision 100%, and recall 88.2%. We discuss generalising our approach to screen for a range of pathologies.

Jdp2 downregulates Trp53 transcription to promote leukaemogenesis in the context of Trp53 heterozygosity.

We performed a genetic screen in mice to identify candidate genes that are associated with leukaemogenesis in the context of Trp53 heterozygosity. To do this we generated Trp53 heterozygous mice carrying the T2/Onc transposon and SB11 transposase alleles to allow transposon-mediated insertional mutagenesis to occur. From the resulting leukaemias/lymphomas that developed in these mice, we identified nine loci that are potentially associated with tumour formation in the context of Trp53 heterozygosity, including AB041803 and the Jun dimerization protein 2 (Jdp2). We show that Jdp2 transcriptionally regulates the Trp53 promoter, via an atypical AP-1 site, and that Jdp2 expression negatively regulates Trp53 expression levels. This study is the first to identify a genetic mechanism for tumour formation in the context of Trp53 heterozygosity.

The activating mutation R201C in GNAS promotes intestinal tumourigenesis in Apc(Min/+) mice through activation of Wnt and ERK1/2 MAPK pathways.

Somatically acquired, activating mutations of GNAS, the gene encoding the stimulatory G-protein Gsalpha subunit, have been identified in kidney, thyroid, pituitary, leydig cell, adrenocortical and, more recently, in colorectal tumours, suggesting that mutations such as R201C may be oncogenic in these tissues. To study the role of GNAS in intestinal tumourigenesis, we placed GNAS R201C under the control of the A33-antigen promoter (Gpa33), which is almost exclusively expressed in the intestines. The GNAS R201C mutation has been shown to result in the constitutive activation of Gsalpha and adenylate cyclase and to lead to the autonomous synthesis of cyclic adenosine monophosphate (cAMP). Gpa33(tm1(GnasR201C)Wtsi/+) mice showed significantly elevated cAMP levels and a compensatory upregulation of cAMP-specific phosphodiesterases in the intestinal epithelium. GNAS R201C alone was not sufficient to induce tumourigenesis by 12 months, but there was a significant increase in adenoma formation when Gpa33(tm1(GnasR201C)Wtsi/+) mice were bred onto an Apc(Min/+) background. GNAS R201C expression was associated with elevated expression of Wnt and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) pathway target genes, increased phosphorylation of ERK1/2 MAPK and increased immunostaining for the proliferation marker Ki67. Furthermore, the effects of GNAS R201C on the Wnt pathway were additive to the inactivation of Apc. Our data strongly suggest that activating mutations of GNAS cooperate with inactivation of APC and are likely to contribute to colorectal tumourigenesis.

Atypical appendicitis: the impact of CT and its management.

Acute appendicitis is a diagnosis that can be made on clinical symptoms and signs but can often be extremely challenging. Difficulties arise particularly when the presentation is atypical, and this can lead to untoward sequelae. In this review, we present the range of presentations of atypical appendicitis, the variety of management options and the potential value of CT.

Loss of Rassf1a cooperates with Apc(Min) to accelerate intestinal tumourigenesis.

Promoter methylation of the RAS-association domain family 1, isoform A gene (RASSF1A) is one of the most frequent events found in human tumours. In this study we set out to test the hypothesis that loss of Rassf1a can cooperate with inactivation of the adenomatous polyposis coli (Apc) gene to accelerate intestinal tumourigenesis using the Apc-Min (Apc(Min/+)) mouse model, as mutational or deletional inactivation of APC is a frequent early event in the genesis of intestinal cancer. Further, loss of RASSF1A has also been reported to occur in premalignant adenomas of the bowel. RASSF1A has been implicated in an array of pivotal cellular processes, including regulation of the cell cycle, apoptosis, microtubule stability and most recently in the beta-catenin signalling pathway. By interbreeding isoform specific Rassf1a knockout mice with Apc(+/Min) mice, we showed that loss of Rassf1a results in a significant increase in adenomas of the small intestine and accelerated intestinal tumourigenesis leading to the earlier death of adenocarcinoma-bearing mice and decreased overall survival. Comparative genomic hybridization of adenomas from Rassf1a(-/-); Apc(+/Min) mice revealed no evidence of aneuploidy or gross chromosomal instability (no difference to adenomas from Rassf1a(+/+); Apc(+/Min) mice). Immunohistochemical analysis of adenomas revealed increased nuclear beta-catenin accumulation in adenomas from Rassf1a(-/-); Apc(+/Min) mice, compared to those from Rassf1a(+/+); Apc(+/Min) mice, but no differences in proliferation marker (Ki67) staining patterns. Collectively these data demonstrate cooperation between inactivation of Rassf1a and Apc resulting in accelerated intestinal tumourigenesis, with adenomas showing increased nuclear accumulation of beta-catenin, supporting a mechanistic link via loss of the known interaction of Rassf1 with beta-TrCP that usually mediates degradation of beta-catenin.

Magnetic resonance imaging findings of tamoxifen-associated uterine Müllerian adenosarcoma: a case report.

Müllerian adenosarcoma of the uterus is a rare biphasic tumor, which was first described in 1974. Recent studies have suggested an association with tamoxifen therapy, but there have been few reports with detailed imaging findings. We present a case with magnetic resonance imaging (MRI) findings of this rare tumor in a woman who received long-term tamoxifen therapy for breast cancer. In addition, myometrial invasion was detected more accurately with MRI compared to ultrasound in this one single case.

Conditional expression of mutated K-ras accelerates intestinal tumorigenesis in Msh2-deficient mice.

K-ras mutation occurs in 40-50% of human colorectal adenomas and carcinomas, but its contribution to intestinal tumorigenesis in vivo is unclear. We developed K-ras(V12) transgenic mice that were crossed with Ah-Cre mice to generate K-ras(V12)/Cre mice, which showed beta-naphthoflavone-induction of Cre-mediated LoxP recombination that activated intestinal expression of K-ras(V12) 4A and 4B transcripts and proteins. Only very occasional intestinal adenomas were observed in beta-naphthoflavone-treated K-ras(V12)/Cre mice aged up to 2 years, suggesting that mutated K-ras expression alone does not significantly initiate intestinal tumourigenesis. To investigate the effects of mutated K-ras on DNA mismatch repair (MMR)-deficient intestinal tumour formation, these mice were crossed with Msh2(-/-) mice to generate K-ras(V12)/Cre/Msh2(-/-) offspring. After beta-naphthoflavone treatment, K-ras(V12)/Cre/Msh2(-/-) mice showed reduced average lifespan of 17.3+/-5.0 weeks from 26.9+/-6.8 (control Msh2(-/-) mice) (P<0.01). They demonstrated increased adenomas in the small intestine from 1.41 (Msh2(-/-) controls) to 7.75 per mouse (increased fivefold, P<0.01). In the large intestine, very few adenomas were found in Msh2(-/-) mice (0.13 per mouse) whereas K-ras(V12)/Cre/Msh2(-/-) mice produced 2.70 adenomas per mouse (increased 20-fold, P<0.01). Over 80% adenomas from K-ras(V12)/Cre/Msh2(-/-) mice showed transgene recombination with expression of K-ras(V12) 4A and 4B transcripts and proteins. Sequencing of endogenous murine K-ras showed mutations in two out of 10 tumours examined from Msh2(-/-) mice, but no mutations in 17 tumours from K-ras(V12)/Cre/Msh2(-/-) mice. Expression of K-ras(V12) in tumours caused activation of the mitogen-activated protein kinase and Akt/protein kinase B signaling pathways, demonstrated by phosphorylation of p44MAPK, Akt and GSK3beta, as well as transcriptional upregulation of Pem, Tcl-1 and Trap1a genes (known targets of K-ras(V12) expression in stem cells). Thus, mutated K-ras cooperates synergistically with MMR deficiency to accelerate intestinal tumorigenesis, particularly in the large intestine.

K-ras 4A and 4B are co-expressed widely in human tissues, and their ratio is altered in sporadic colorectal cancer.

Ras activating mutations result in constitutive activation of Ras signalling pathways and occur in 30% of human malignancies. K-ras encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in lung, pancreatic and colorectal cancers. Using RT-PCR we examined their expression in normal adult human tissues and addressed whether K-ras splicing is altered in sporadic colorectal cancer by comparing normal colon with colon carcinoma cell lines, and 'matched' tumour and tumour-free colon tissues from the same patient. K-ras 4B was expressed ubiquitously and was the predominant splice variant. K-ras 4A was expressed differentially, with detection in colorectal tumours and cell lines, and normal colon, pancreas and lung--sites where tumours with K-ras activating mutations arise. Both K-ras splice variants were co-expressed by single colon carcinoma cells. The K-ras 4A/4B ratio was significantly reduced in all 6 cell lines examined, including two that lacked K-ras activating mutations, and in 4/9 primary adenocarcinomas. We conclude that K-ras activating mutations do not affect K-ras splicing per se, both isoforms may play a role in neoplastic progression, and altered splicing of either the K-ras proto-oncogene or oncogene, in favour of K-ras 4B, may modulate tumour development.

A conditional model of MLL-AF4 B-cell tumourigenesis using invertor technology.

MLL-AF4 fusion is the most common consequence of chromosomal translocations in infant leukaemia and is associated with a poor prognosis. MLL-AF4 is thought to be required in haematopoietic stem cells to elicit leukaemia and may be involved in tumour phenotype specification as it is only found in B-cell tumours in humans. We have employed the invertor conditional technology to create a model of MLL-AF4, in which a floxed AF4 cDNA was knocked into Mll in the opposite orientation for transcription. Cell-specific Cre expression was used to generate Mll-AF4 expression. The mice develop exclusively B-cell lineage neoplasias, whether the Cre gene was controlled by B- or T-cell promoters, but of a more mature phenotype than normally observed in childhood leukaemia. These findings show that the MLL-AF4 fusion protein does not have a mandatory role in multi-potent haematopoietic stem cells to cause cancer and indicates that MLL-AF4 has an instructive function in the phenotype of the tumour.

The RASSF1A isoform of RASSF1 promotes microtubule stability and suppresses tumorigenesis.

The RASSF1A isoform of RASSF1 is frequently inactivated by epigenetic alterations in human cancers, but it remains unclear if and how it acts as a tumor suppressor. RASSF1A overexpression reduces in vitro colony formation and the tumorigenicity of cancer cell lines in vivo. Conversely, RASSF1A knockdown causes multiple mitotic defects that may promote genomic instability. Here, we have used a genetic approach to address the function of RASSF1A as a tumor suppressor in vivo by targeted deletion of Rassf1A in the mouse. Rassf1A null mice were viable and fertile and displayed no pathological abnormalities. Rassf1A null embryonic fibroblasts displayed an increased sensitivity to microtubule depolymerizing agents. No overtly altered cell cycle parameters or aberrations in centrosome number were detected in Rassf1A null fibroblasts. Rassf1A null fibroblasts did not show increased sensitivity to microtubule poisons or DNA-damaging agents and showed no evidence of gross genomic instability, suggesting that cellular responses to genotoxins were unaffected. Rassf1A null mice showed an increased incidence of spontaneous tumorigenesis and decreased survival rate compared with wild-type mice. Irradiated Rassf1A null mice also showed increased tumor susceptibility, particularly to tumors associated with the gastrointestinal tract, compared with wild-type mice. Thus, our results demonstrate that Rassf1A acts as a tumor suppressor gene.

Persistent high risk HPV infection associated with development of cervical neoplasia in a prospective population study.

AIMS: To monitor the association between the course of high risk human papillomavirus (HR-HPV) infection and the development of cervical neoplasia over time, from a baseline of normal cervical cytology. METHODS: This paper presents the follow up data from a previous cross sectional analysis. Women from a screening population who had normal cytology and who were HR-HPV positive were recalled after two to three years for cytology and HPV genotyping. The development of cervical neoplasia at follow up was related to the course of HPV infection (clearance, persistence, or sequential infection) and the presence of single or multiple HPV infections at baseline. A comparator control group of women who were HPV and cytologically negative at baseline were selected from the same population. RESULTS: Twelve cases of dyskaryosis were found in women who were HPV positive at baseline; four were high grade. Only three cases of low grade dyskaryosis were found in the control group. Women with type specific persistent infections were significantly more likely to develop cervical neoplasia than women who cleared the infection (p = 0.0001) or were sequentially infected with different types (p = 0.001). Women with multiple HPV infections at baseline were no more likely to develop cervical dyskaryosis than those with a single infection. CONCLUSIONS: Type specific persistent HR-HPV infection as monitored by genotyping can identify women at increased risk of cervical neoplasia more accurately than a single or repeated presence/absence HPV test. The cost effectiveness of such an approach should be investigated by an appropriate, large scale cost-benefit analysis.

A novel immunohistochemical method for estimating cell cycle phase distribution in ovarian serous neoplasms: implications for the histopathological assessment of paraffin-embedded specimens.

We have investigated whether immunohistochemical markers can identify differences in cell cycle phase distribution in ovarian serous neoplasms, including borderline tumours of different grades. Sections of normal ovary (n=18), serous cystadenoma (n=21), borderline serous tumours (n=21) and serous cystadenocarcinoma (n=15) were analysed by immunohistochemistry using markers of cell cycle entry (Mcm-2) and cell cycle phase, including cyclin D1 (mid-to-late G1), cyclin A (S phase), cyclin B1 (G2 phase) and phosphohistone H3 (mitosis). Double-labelling confocal microscopy confirmed marker phase specificity and phase estimations were corroborated by flow cytometry. On progression from normal ovary through serous cystadenoma and borderline tumours to cystadenocarcinomas, expression of Mcm-2 (P<0.0001), cyclin D1 (P=0.002), cyclin A (P<0.0001), cyclin B1 (P<0.0001) and phosphohistone H3 (P<0.0001) increased, paralleled by an increase in the S-phase fraction (cyclin A : Mcm-2 ratio; P=0.002). Borderline tumours of increasing grade also showed increased Mcm-2 and cyclin A expression, together with an increase in the S-phase fraction. Immunohistochemistry can be used to estimate cell cycle phase distribution in ovarian serous neoplasms, giving results similar to flow cytometric analysis and enabling direct assessment of tumour heterogeneity. Immunohistochemical estimates of the S-phase fraction may identify serous borderline tumours likely to exhibit malignant progression and/or select serous cystadenocarcinomas likely to respond to adjuvant therapy.

Eosinophilic gastroenteritis presenting as obstructive jaundice.

Eosinophilic gastroenteritis is a rare condition of unknown aetiology, first described by Kaijser in 1937. It is manifest by eosinophilic infiltration of the gastrointestinal tract and peripheral eosinophilia. Patients have various clinical presentations depending on the region of the gastrointestinal tract involved and the depth and extent of the bowel wall involvement. Whereas gastrointestinal obstruction associated with this condition is not uncommon, isolated biliary obstruction has only been reported twice. We present a case of eosinophilic gastroenteritis with involvement of the biliary tract causing ulceration, fibrosis and obstruction. Although a rare condition, we believe the diagnosis of eosinophilic gastroenteritis should be entertained in patients with gastrointestinal symptoms and a peripheral eosinophilia who have no evidence of parasitic infection, vasculitis or neoplasms.

Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population.

AIMS: If human papillomavirus (HPV) testing is to be included within cervical screening programmes, the importance of multiple HPV infections in cervical neoplasia needs to be determined. This study investigated the diversity of multiple HPV types in a routine cervical screening population, and assessed associations with cervical neoplasia. METHODS: Overall HPV prevalence, type specific prevalence, and extent of multiple infection were assessed in residual material from 3444 liquid based cytology samples, using real time GP5+/GP6+ polymerase chain reaction for screening and linear array assay for genotyping. HPV status was studied in relation to age and concurrent cytological evidence of dyskaryosis. RESULTS: Twenty per cent of samples were HPV positive. HPV type diversity was broad, and multiple HPV infections occurred in half of the HPV positive samples. Younger women were significantly more likely to harbour multiple high risk HPV (HR-HPV) infections. Infections with multiple HR-HPV types were found in 3.4% of samples negative for neoplasia and in 33.3%, 41.8%, and 40.4% of samples with borderline, mild, or high grade dyskaryosis, respectively. Single HR-HPV infections were found in 4.9%, 38.6%, 45.0%, and 51.1% of negative, borderline, mild, or high grade dyskaryosis samples, respectively. CONCLUSIONS: Multiple HR-HPV infections were most prevalent in young women. Multiple HR-HPV infections were not more frequent in high grade than in low grade cervical neoplasia, reflecting common sexual transmission of multiple HR-HPV. Prospective cohort studies linking sequential loss or gain of HPV types with cytological analysis are required to assess the impact of multiple HR-HPV infections on neoplastic progression.

Unusual causes of colonic wall thickening on computed tomography.

Computed tomography (CT) appearances in "colitis" are often non-specific, and include mural thickening and mesenteric fat stranding. In the western world, the majority of cases will have, or be subsequently diagnosed with, inflammatory bowel disease, pseudomembranous colitis or ischaemic colitis. However, other rare conditions may also produce these rather non-specific signs. We present a number of cases demonstrating colonic wall thickening on CT due to rarer diagnoses, which are correlated with the histopathological features. Some of these CT appearances have not been described previously in the literature.

Mutation analysis of CBP and PCAF reveals rare inactivating mutations in cancer cell lines but not in primary tumours.

In this study we screened the histone acetyltransferases CBP and PCAF for mutations in human epithelial cancer cell lines and primary tumours. We identified two CBP truncations (both in cell lines), seven PCAF missense variants and four CBP intronic microdeletions. These data suggest that neither gene is commonly inactivated in human epithelial cancers.

Histopathological correlates of abnormal pericolic fat on CT in the assessment of colorectal carcinoma.

The aim of this study was to assess the pathological significance of abnormal pericolic fat shown by CT in the context of colorectal carcinoma. CT and histopathological findings of 63 resected colorectal carcinomas were retrospectively reviewed. CT examinations were assessed by two observers for the presence or absence of abnormal pericolic fat (typically linear or nodular opacities) at tumour sites. Specimens were reviewed histopathologically for depth of tumour invasion, extramuscular tissue reaction, and number and largest size of tumour-involved and tumour-free lymph nodes. The sensitivity, specificity, positive predictive value and negative predictive value of pericolic fat in identifying extension of tumour infiltration beyond the muscle coat were 79% (42/53), 33% (2/6), 91% (42/46) and 15% (2/13), respectively. Despite these indicators of efficacy, the association between the presence of pericolic fat abnormality on CT and extramuscular extension of tumour (infiltration and/or nodal disease) or tissue reaction alone or in combination did not reach statistical significance (p>0.3 in all cases). Abnormal ("misty" or "mucky") pericolic fat in the assessment of colorectal cancer on CT is not a precise indicator of extramuscular extension of tumour, as it cannot clearly distinguish between tumour infiltration and tissue reaction beyond the muscle coat, or pericolic nodal involvement. However, it is a very helpful CT sign that may draw attention to the presence and site of a potential colonic abnormality.

Inhibition of lipoprotein-associated phospholipase A2 diminishes the death-inducing effects of oxidised LDL on human monocyte-macrophages.

The death of macrophages contributes to atheroma formation. Oxidation renders low-density lipoprotein (LDL) cytotoxic to human monocyte-macrophages. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also termed platelet-activating factor acetylhydrolase, hydrolyses oxidised phospholipids. Inhibition of Lp-PLA2 by diisopropyl fluorophosphate or Pefabloc (broad-spectrum serine esterase/protease inhibitors), or SB222657 (a specific inhibitor of Lp-PLA2) did not prevent LDL oxidation, but diminished the ensuing toxicity and apoptosis induction when the LDL was oxidised, and inhibited the rise in lysophosphatidylcholine levels that occurred in the inhibitors' absence. Hydrolysis products of oxidised phospholipids thus account for over a third of the cytotoxic and apoptosis-inducing effects of oxidised LDL on macrophages.