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.

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.

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.

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.

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.

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.

Role of BAX mutations in mismatch repair-deficient colorectal carcinogenesis.

BAX gene mutations occur in approximately 50% of RER+ colorectal cancers. To determine the role of these mutations in tumour progression we analysed multiple different tumour sites from RER+ colorectal cancers for BAX mutations. Sixty colorectal carcinomas were analysed for microsatellite instability at loci BAT-26, L-myc, TGF betaRII, D13S160 and D2S123. Twelve out of 60 tumours (20%) were RER+. Forty-five different tumour sites from the 12 RER+ carcinomas were analysed for BAX mutations at the [(G)8] tract in exon 3. Six out of 12 (50%) RER+ tumours showed BAX mutations, four of which showed a homogenous pattern of such mutations detected in all tumour sites. In the other two cases, BAX mutations were present in some but not all tumour sites sampled from the same patient. In contrast, TGF betaRII mutations were found in 9/12 cases (75%) and in each of these were present in all the sampled sites. Two cases showed neither BAX nor TGF betaRII mutation. These data suggest that mutations in TGF betaRII may occur at a very early stage in tumour progression, perhaps in the founder clone. BAX mutations, however, are clearly not necessary for formation of the founder clone and can occur for the first time later in tumour progression.

Mutant K-ras enhances apoptosis in embryonic stem cells in combination with DNA damage and is associated with increased levels of p19(ARF).

The roles of K-ras in neoplasia are not entirely understood, although there is evidence that K-ras affects susceptibility to apoptosis, modulating survival of DNA damaged cells which would otherwise be eliminated. In this study, we investigated the effects of mutant K-ras on apoptosis in vitro following DNA damage. To avoid complications resulting from mutations in other cancer-related genes and from the presence of endogenous K-ras, we derived K-ras null embryonic stem cells. Expression of either wild-type or mutant K-ras was reconstructed by stable plasmid transfection. The cell lines were treated with etoposide, cisplatin and UV radiation and apoptosis measured flow cytometrically. Mutant K-ras potentiated the effect of etoposide-derived DNA damage by increasing apoptosis, whereas absence of K-ras had the opposite effect. This pattern was similar but less marked with cisplatin, whereas UV yielded no difference in apoptosis with regard to K-ras status, suggesting that the effect of K-ras on apoptosis is dependent on the nature of the DNA damage. To investigate possible mechanisms, we examined the expression of p19(ARF) mRNA by RT-PCR. Cells expressing mutant K-ras produced elevated levels of p19(ARF) mRNA, which could link K-ras status with p53 expression and hence susceptibility to DNA damage-induced apoptosis.

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.

CDKN2A mutation and deletion status in thin and thick primary melanoma.

Human melanoma cell lines and tumor tissue from familial and sporadic melanomas have frequent, nonrandom chromosomal breaks and deletions on chromosome 9p21, a region that includes the tumor suppressor gene CDKN2A/p16INK4A. Germ-line mutations within this gene have been observed in some familial melanoma kindreds, but somatic mutation in sporadic primary melanoma is infrequent. Thirty-nine archival, paraffin-embedded, sporadic, primary cutaneous malignant melanomas (20 >3-mm-thick and 19 <0.75-mm-thick cases) were examined for mutations of the CDKN2A gene using single-strand conformational polymorphism analysis and direct sequencing. No mutations were detected. Loss of heterozygosity for the 9p21 microsatellite marker D9S942 was detected in 6 of 17 informative thick lesions (35%) but 0 of 18 thin lesions (P = 0.006). These results support other studies indicating that intragenic mutation is an infrequent mechanism of CDKN2A inactivation in primary melanoma. The finding of loss of heterozygosity for the 9p21 microsatellite D9S942 in thick but not thin primary melanoma suggests that deletion or inactivation of CDKN2A or other tumor suppressor gene(s) at this locus is involved in the progression rather than initiation of sporadic malignant melanoma.

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.

Novel histopathologic findings in a surviving case of hemolytic uremic syndrome after bone marrow transplantation.

We report novel renal histopathologic features in a patient with hemolytic uremic syndrome after allogeneic bone marrow transplantation who survived with plasma exchange therapy. This distribution of vascular lesions within the kidney differed from previously described cases, which were uniformly fatal, in that there were marked arterial changes in addition to arteriolar and glomerular microangiopathy. A high rate of frequency of apoptotic cells within glomeruli was found. These findings are discussed in terms of their pathogenetic and prognostic relevance.

Human papillomavirus type 18 associates with more advanced cervical neoplasia than human papillomavirus type 16.

A type-specific, sensitive, polymerase chain reaction-based assay for human papillomavirus (HPV) types 6b, 11, 16, 18, and 33 was applied to 47 cervical carcinomas, 60 cases of cervical intraepithelial neoplasia (CIN), and 24 samples of histologically normal cervix. As expected, the combined incidence of the common high-risk genital HPVs (types 16 and 18) was high in carcinomas (79%) and CIN 2/3 (60%), low in CIN 1 (25%), and nonexistent in the normal controls. Analysis of the data by viral type and pathology revealed statistically significant differences that consistently pointed to an association of HPV 18 with more advanced disease than HPV 16. This was exemplified by calculation of the relative HPV frequency in squamous cancers and CIN 2/3 lesions, which gave cancer to CIN prevalence ratios of 1.2 for HPV 16 and 2.3 for HPV 18, a twofold difference suggesting the possibility that there is a greater risk of progression or a more rapid transition to malignancy associated with HPV 18. Furthermore, HPV 16 was associated with 2.5-fold more cancers showing squamous differentiation (58%) than HPV 18 (23%), but both types showed an identical prevalence of 41% in the clinically more sinister adenocarcinomas, indicating that there may be an association between HPV type and cancer cell differentiation.

Aetiology, pathogenesis, and pathology of cervical neoplasia.

Early epidemiological studies of cervical neoplasia suggested a causal relation with sexual activity and human papillomaviruses (HPVs) have emerged as prime suspects as venerally transmitted carcinogens. HPVs fall into two broad camps: low risk types, associated with cervical condylomas and CIN 1; and high risk types (mostly 16 and 18), found in 50-80% of CIN 2 and CIN 3 lesions, and 90% of cancers. This association with cancer is very strong, with odds ratios of > 15 (often much higher) in case-control studies that are methodologically sound. An infrequently detected third group of intermediate risk type HPVs is associated with all grades of CIN and occasionally with cancers. HPVs have also been detected in a wide range of asymptomatic controls, indicating that other events are required for development of neoplasia such as viral persistence and/or altered expression of viral genes, often following integration of the viral genome. This leaves the two major viral oncogenes, E6 and E7, directly coupled to viral enhancers and promoters, allowing their continued expression after integration. High risk HPV E7 proteins bind and inactivate the Rb protein, whereas E6 proteins bind p53 and direct its rapid degradation. A range of putative cofactors has been implicated in progression: HLA type, immunosuppression, sex steroid hormones, and smoking; most of these cofactors appear to influence progression to CIN 3. The natural history includes progression to CIN 3 in 10% of CIN 1 and 20% of CIN 2 cases, whereas at least 12% of CIN 3 cases progress to invasive carcinoma. Cervical glandular intraepithelial neoplasia (CGIN) often coexists with squamous CIN, and the premalignant potential of high grade CGIN is not in doubt, but the natural history of low grade CGIN remains uncertain. A high proportion of CGIN lesions and adenocarcinomas are HPV positive, and HPV18 has been implicated more in glandular than in squamous lesions. A strong clinical case for the application of HPV typing of cells recovered from cervical scrapes can be made; however, a rigorous cost-benefit analysis of introducing HPV typing into the cervical screening programme is required. Prophylactic and therapeutic HPV vaccines are under development. This article reviews the aetiology, pathogenesis, and pathology of cervical neoplasia, emphasising the role of HPVs.

PCR analysis of the upstream regulatory region of human papillomavirus genomes in cervical intraepithelial neoplasia and cervical carcinoma.

AIMS: To test whether human papillomavirus (HPV) variants with large scale sequence alterations to the upstream regulatory region are present in cervical intraepithelial neoplasias (CIN) and cervical carcinomas. METHODS: New PCR based assays were designed specifically to detect large scale insertion/deletion alterations in the upstream regulatory region of HPV 16 and 18. The assays were applied to 24 cases of CIN and 34 cases of cervical carcinoma previously shown to contain these two high risk HPV types. RESULTS: No large scale sequence alterations were found in any of the HPV containing CIN or carcinomas. CONCLUSIONS: These negative findings suggest that major upstream regulatory region variants of HPV 16 and 18 do not contribute to most cervical neoplasms.

Dietary calcium supplementation increases apoptosis in the distal murine colonic epithelium.

BACKGROUND: Increased dietary calcium might reduce colorectal cancer risk, possibly by reduction of colonic epithelial hyperproliferation, but not all studies have demonstrated this. Little is known about the effects of calcium on colonic apoptosis. AIM: To quantify the effects of increasing calcium on apoptosis and cell proliferation in normal murine colonic crypt epithelium. METHODS: Twenty one day old male C57B1/6 mice were fed either control AIN-76 diet (0.5% calcium wt/wt; n = 10) or the same supplemented with calcium carbonate (1.0% calcium; n = 10) for 12 weeks. Apoptotic cells in proximal and distal segments were counted and expressed as an apoptotic index (AI: frequency of apoptosis/100 longitudinal crypts). The bromodeoxyuridine (BrdU) labelling index was also determined. Differences were analysed by the student's t test. RESULTS: In control animals, the AI was significantly higher in the caecum/proximal colon (mean, 28.6; SEM, 2.0) compared with the distal colon (mean, 19.9; SEM, 1.8; p = 0.004). In the calcium treated group, the AI in the caecum/proximal colon (mean, 30.6; SEM, 1.7) was similar to controls (p = 0.71) but the AI in the distal colon was significantly greater (mean, 32.6; SEM, 1.8; p = 0.001) than in control mice and was raised to values similar to those in the proximal colon. Calcium was also associated with reduced crypt cellularity and, in the proximal colon, a downward shift in the crypt position at which apoptosis occurred. There were no significant differences in the BrdU labelling index between groups or between proximal and distal colonic segments in each group. CONCLUSIONS: Increased dietary calcium is associated with the induction of apoptosis in normal mouse distal colonic epithelium without affecting cell proliferation. This might contribute to its putative chemopreventive role in colorectal carcinogenesis. Whether this effect is direct or indirect requires further study.

Sensitivity of digoxigenin and biotin labelled probes for detection of human papillomavirus by in situ hybridisation.

The sensitivity of digoxigenin and biotin labelled DNA probes for the detection of human papillomavirus (HPV) by dot blotting and in situ hybridisation was compared in tissues from cervical, laryngeal, and anogenital neoplasia. Probes were either labelled with digoxigenin by the random primer technique and detected with anti-digoxigenin antibody, or labelled with biotin by nick translation and detected with streptavidin, both methods having a common final visualisation procedure using alkaline phosphatase. Digoxigenin labelled probes proved two to 10-fold more sensitive by quantitative dot blotting and four-fold more sensitive in detecting HPV 16 DNA in a series of 31 anal carcinomas, compared with biotinylated probes. The digoxigenin method also produced less non-specific background staining of tissue sections than biotin labelled probes. It is concluded that digoxigenin DNA labelling and detection provides a simple, reliable, and efficient alternative to the use of biotin or radioactive isotopes for the detection of HPV DNA by in situ hybridisation. Digoxigenin labelled probes also offer the possibility of double labelling in situ hybridisation procedures when used with biotin labelled probes to provide simultaneous identification of different DNA sequences.