PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation

Phosphatidylinositol‐3‐kinase gamma (PI3Kγ) is a leukocyte‐specific lipid kinase with signaling function downstream of G protein‐coupled receptors to regulate cell trafficking, but its role in T cells remains unclear. To investigate the requirement of PI3Kγ kinase activity in T‐cell function, we studied T cells from PI3Kγ kinase‐dead knock‐in (PI3Kγ^KD/KD) mice expressing the kinase‐inactive PI3Kγ protein. We show that CD4⁺ and CD8⁺ T cells from PI3Kγ^KD/KD mice exhibit impaired TCR/CD28‐mediated activation that could not be rescued by exogenous IL‐2. The defects in proliferation and cytokine production were also evident in naïve and memory T cells. Analysis of signaling events in activated PI3Kγ^KD/KD T cells revealed a reduction in phosphorylation of protein kinase B (AKT) and ERK1/2, a decrease in lipid raft formation, and a delay in cell cycle progression. Furthermore, PI3Kγ^KD/KD CD4⁺ T cells displayed compromised differentiation toward Th1, Th2, Th17, and induced Treg cells. PI3Kγ^KD/KD mice also exhibited an impaired response to immunization and a reduced delayed‐type hypersensitivity to Ag challenge. These findings indicate that PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation, as well as for mounting an efficient T cell‐mediated immune response. The results suggest that PI3Kγ kinase inhibitors could be beneficial in reducing the undesirable immune response in autoimmune diseases.     

Impact of oviductal versus ovarian epithelial cell of origin on ovarian endometrioid carcinoma phenotype in the mouse

Endometrioid carcinoma (EC) is a relatively indolent ovarian carcinoma subtype that is nonetheless deadly if detected late. Existing genetically engineered mouse models (GEMMs) of the disease, based on transformation of the ovarian surface epithelium (OSE), take advantage of known ovarian EC driver gene lesions, but do not fully recapitulate the disease features seen in patients. An EC model in which the Apc and Pten tumour suppressor genes are conditionally deleted in murine OSE yields tumours that are biologically more aggressive and significantly less differentiated than human ECs. Importantly, OSE is not currently thought to be the tissue of origin of most ovarian cancers, including ECs, suggesting that tumour initiation in Müllerian epithelium may produce tumours that more closely resemble their human tumour counterparts. We have developed Ovgp1‐iCreER^T2 mice in which the Ovgp1 promoter controls expression of tamoxifen (TAM)‐regulated Cre recombinase in oviductal epithelium – the murine equivalent of human Fallopian tube epithelium. Ovgp1‐iCreER^T2;Apc^fl/fl;Pten^fl/fl mice treated with TAM or injected with adenovirus expressing Cre into the ovarian bursa uniformly develop oviductal or ovarian ECs, respectively. On the basis of their morphology and global gene expression profiles, the oviduct‐derived tumours more closely resemble human ovarian ECs than do OSE‐derived tumours. Furthermore, mice with oviductal tumours survive much longer than their counterparts with ovarian tumours. The slow progression and late metastasis of oviductal tumours resembles the relatively indolent behaviour characteristic of so‐called Type I ovarian carcinomas in humans, for which EC is a prototype. Our studies demonstrate the utility of Ovgp1‐iCreER^T2 mice for manipulating genes of interest specifically in the oviductal epithelium, and establish that the cell of origin is an important consideration in mouse ovarian cancer GEMMs. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

IFNγ‐responsiveness of endothelial cells leads to efficient angiostasis in tumours involving down‐regulation of Dll4

Although IFNγ is regarded as a key cytokine in angiostatic response, our poor understanding of its effective cellular target drastically limits its clinical trials against angiogenesis‐related disorders. Here, we investigated the effect of IFNγ on endothelial cells (ECs) and possible molecular mechanisms in angiostasis. By employing Tie2^IFNγR mice, in which IFNγR expression was reconstituted under the control of Tie2 promoter in IFNγR‐deficient mice, we found that the response of ECs to IFNγ was highly effective in inhibiting blood supply and retarding tumour growth. Interestingly, the expression of IFNγR on Tie2^? cells did not inhibit, but promoted tumour growth in control wild‐type mice. Mechanism studies showed that IFNγ reacting on ECs down‐regulated the delta‐like ligand 4 (Dll4)/Notch signalling pathway. Accordingly, overexpression of Dll4 in human ECs diminished the effect of IFNγ on ECs. This study demonstrates that the action of IFNγ on ECs, but not other cells, is highly effective for tumour angiostasis, which involves down‐regulating Dll4. It provides insights for EC‐targeted angiostatic therapy in treating angiogenesis‐associated disorders in the clinic. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

A tissue reconstitution model to study cancer cell‐intrinsic and ‐extrinsic factors in mammary tumourigenesis

The contribution of cancer cell‐intrinsic and ‐extrinsic factors to metastatic breast cancer is still poorly understood, hampering development of novel therapeutic strategies that decrease breast cancer mortality. Cre/loxP‐based conditional mouse models of breast cancer present unique opportunities to study sporadic tumour formation and progression in a controlled setting. Unfortunately, the generation of mouse strains carrying multiple mutant alleles needed for such studies is very time‐consuming. Moreover, conditional mouse tumour models do not permit independent manipulation of tumour cell‐intrinsic and ‐extrinsic factors. Although the latter can be achieved by cleared fat‐pad transplantation of mouse mammary epithelial cells (MMECs) from tumour suppressor gene (TSG) knockouts into wild‐type or mutant recipients, this procedure is not possible for mutations that cause embryonic lethality or preclude mammary gland development. Here we show that cleared fat‐pad transplantations with MMECs isolated from K14cre;Cdh1^F/F; Trp53^F/F mice expressing Cre recombinase under control of the cytokeratin‐14 promoter and carrying conditional null alleles for p53 and E‐cadherin (Cdh1) first resulted in the formation of phenotypically normal mammary glands, followed by the development of invasive metastatic mammary tumours. Tumour formation in the recipients mimicked tumour latency, spectrum, morphology, immunophenotype, and metastatic characteristics of the original mammary tumour model. This transplantation system, which can be expanded to other conditional TSG knockouts, permits independent genetic analysis of stromal factors and testing of additional cancer cell‐intrinsic mutations that would otherwise be embryonic lethal or require intensive breeding. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Whole‐exome DNA sequence analysis of Brca2‐ and Trp53‐deficient mouse mammary gland tumours

Germline mutations in the tumour suppressor BRCA2 predispose to breast, ovarian and a number of other human cancers. Brca2‐deficient mouse models are used for preclinical studies but the pattern of genomic alterations in these tumours has not yet been described in detail. We have performed whole‐exome DNA sequencing analysis of mouse mammary tumours from Blg–Cre Brca2^f/f Trp53^f/f animals, a model of BRCA2‐deficient human cancer. We also used the sequencing data to estimate DNA copy number alterations in these tumours and identified a recurrent copy number gain in Met, which has been found amplified in other mouse mammary cancer models. Through a comparative genomic analysis, we identified several mouse Blg–Cre Brca2^f/f Trp53^f/f mammary tumour somatic mutations in genes that are also mutated in human cancer, but few of these genes have been found frequently mutated in human breast cancer. A more detailed analysis of these somatic mutations revealed a set of genes that are mutated in human BRCA2 mutant breast and ovarian tumours and that are also mutated in mouse Brca2‐null, Trp53‐null mammary tumours. Finally, a DNA deletion surrounded by microhomology signature found in human BRCA1/2‐deficient cancers was not common in the genome of these mouse tumours. Although a useful model, there are some differences in the genomic landscape of tumours arising in Blg–Cre Brca2^f/f Trp53^f/f mice compared to human BRCA‐mutated breast cancers. Therefore, this needs to be taken into account in the use of this model. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Capn4 contributes to tumour growth and metastasis of hepatocellular carcinoma by activation of the FAK–Src signalling pathways

Calpain small subunit 1 (Capn4) has been identified as a major gene that promotes metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which Capn4 promotes progression of HCC is not understood. In this study, we found that Capn4 expression was increased in highly metastatic HCC cell lines and in tumour tissue from HCC patients compared to healthy patient tissue. Over‐expression of Capn4 in HCC cells enhanced tumour cell growth in vitro and increased invasiveness, tumourigenicity and lung metastasis in vivo. Protein microarray analyses showed that expression of multiple proteins was regulated by Capn4. Interestingly, Capn4 was found to physically associate with FAK and promoted hyperactivity of the FAK–Src signalling pathway via increased phosphorylation of specific tyrosine residues of FAK, Src and p130Cas. Knock‐down of Capn4 expression suppressed the malignant behaviour of HCC cells and inhibited the FAK–Src signalling pathway. Furthermore, Capn4‐mediated invasion and metastasis of HCC cells required up‐regulation of matrix metalloproteinase‐2 (MMP2) through activation of this signalling pathway. Our clinical data revealed that Capn4 expression correlated well with the levels of phospho‐FAK, and over‐expression of both Capn4 and phospho‐FAK correlates with the poorest survival outcomes in HCC. In conclusion, our data showed that Capn4 can contribute to HCC growth and metastasis via activation of the FAK–Src signalling pathway and MMP2. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Comprehensive behavioral study and proteomic analyses of CRMP2‐deficient mice

Collapsin response mediator protein 2 (CRMP2) plays a key role in axon guidance, dendritic morphogenesis and cell polarization. CRMP2 is implicated in various neurological and psychiatric disorders. However, in vivo functions of CRMP2 remain unknown. We generated CRMP2 gene‐deficient (crmp2^?/?) mice and examined their behavioral phenotypes. During 24‐h home cage monitoring, the activity level during the dark phase of crmp2^?/? mice was significantly higher than that of wild‐type (WT) mice. Moreover, the time during the open arm of an elevated plus maze was longer for crmp2^?/? mice than for WT mice. The duration of social interaction was shorter for crmp2^?/? mice than for WT mice. Crmp2^?/? mice also showed mild impaired contextual learning. We then examined the methamphetamine‐induced behavioral change of crmp2^?/? mice. Crmp2^?/? mice showed increased methamphetamine‐induced ambulatory activity and serotonin release. Crmp2^?/? mice also showed altered expression of proteins involved in GABAergic synapse, glutamatergic synapse and neurotrophin signaling pathways. In addition, SNAP25, RAB18, FABP5, ARF5 and LDHA, which are related genes to schizophrenia and methamphetamine sensitization, are also decreased in crmp2^?/? mice. Our study implies that dysregulation of CRMP2 may be involved in pathophysiology of neuropsychiatric disorders.     

Conditional abrogation of transforming growth factor‐β receptor 1 in PTEN‐inactivated endometrium promotes endometrial cancer progression in mice

Although a putative role for transforming growth factor‐β (TGFB) signalling in the pathogenesis of human endometrial cancer has long been proposed, the precise function of TGFB signalling in the development and progression of endometrial cancer remains elusive. Depletion of phosphatase and tensin homologue (PTEN) in the mouse uterus causes endometrial cancer. To identify the potential role of TGFB signalling in endometrial cancer, we simultaneously deleted TGFB receptor 1 (Tgfbr1) and Pten in the mouse uterus by using Cre‐recombinase driven by the progesterone receptor (termed Pten^d/d;Tgfbr1^d/d). We found that Pten^d/d;Tgfbr1^d/d mice developed severe endometrial lesions that progressed more rapidly than those resulting from conditional deletion of Pten alone, suggesting that TGFB signalling synergizes with PTEN to suppress endometrial cancer progression. Remarkably, Pten^d/d;Tgfbr1^d/d mice developed distant pulmonary metastases, leading to a significantly reduced lifespan. The development of metastasis and accelerated tumour progression in Pten^d/d;Tgfbr1^d/d mice are associated with increased production of proinflammatory chemokines, enhanced cancer cell motility, as shown by myometrial invasion and disruption, and an altered tumour microenvironment characterized by recruitment of tumour‐associated macrophages. Thus, conditional deletion of Tgfbr1 in PTEN‐inactivated endometrium leads to a disease that recapitulates invasive and lethal human endometrial cancer. This mouse model may be valuable for preclinical testing of new cancer therapies, particularly those targeting metastasis, one of the hallmarks of cancer and a major cause of death in endometrial cancer patients. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Genetic ablation of the alpha 6‐integrin subunit in Tie1Cre mice enhances tumour angiogenesis

Laminins are expressed highly in blood vessel basement membranes and have been implicated in angiogenesis. α6β1‐ and α6β4‐integrins are major receptors for laminins in endothelial cells, but the precise role of endothelial α6‐integrin in tumour angiogenesis is not clear. We show that blood vessels in human invasive ductal carcinoma of the breast have decreased expression of the α6‐integrin‐subunit when compared with normal breast tissue. These data suggest that a decrease in α6‐integrin‐subunit expression in endothelial cells is associated with tumour angiogenesis. To test whether the loss of the endothelial α6‐integrin subunit affects tumour growth and angiogenesis, we generated α6fl/fl‐Tie1Cre+ mice and showed that endothelial deletion of α6‐integrin is sufficient to enhance tumour size and tumour angiogenesis in both murine B16F0 melanoma and Lewis cell lung carcinoma. Mechanistically, endothelial α6‐integrin deficiency elevated significantly VEGF‐mediated angiogenesis both in vivo and ex vivo. In particular, α6‐integrin‐deficient endothelial cells displayed increased levels of VEGF‐receptor 2 (VEGFR2) and VEGF‐mediated downstream ERK1/2 activation. By developing the first endothelial‐specific α6‐knockout mice, we show that the expression of the α6‐integrin subunit in endothelial cells acts as a negative regulator of angiogenesis both in vivo and ex vivo. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

MicroRNA‐126 deficiency enhanced the activation and function of CD4+T cells by elevating IRS‐1 pathway

Recent evidence has shown that microRNA‐126 (miR‐126) has been involved in the development and function of immune cells, which contributed to the pathogenesis of related clinical diseases. However, the potential role of miR‐126 in the development and function of CD4⁺ T cells remains largely unknown. Here we first found that the activation and proliferation, as well as the expression of interferon (IFN)‐γ, of CD4⁺T cells from miR‐126 knock‐down (KD) mice using the miRNA‐sponge technique were enhanced significantly in vitro, compared with those in CD4⁺ T cells from wild‐type (WT) mice. To monitor further the possible effect of miR‐126 deficiency on the function of CD4⁺ T cells in vivo, we used dextran sulphate sodium (DSS)‐induced murine model of acute autoimmune colitis and found that miR‐126 deficiency could elevate the pathology of colitis. Importantly, the proportion of CD4⁺ T cells in splenocytes increased significantly in miR‐126KD mice. Moreover, the expression levels of CD69 and CD44 on CD4⁺ T cells increased significantly and the expression level of CD62L decreased significantly. Of note, adoptive cell transfer assay showed that the pathology of colitis was more serious in carboxyfluorescein succinimidyl ester (CFSE)‐labelled miR‐126KD CD4⁺ T cell‐transferred group, compared with that in the CFSE‐labelled WT CD4⁺ T cells transferred group. Consistently, the expression levels of CD69 and CD44 on CFSE⁺ cells increased significantly. Furthermore, both the proliferation and IFN‐γ secretion of CFSE⁺ cells also increased significantly in the CFSE‐labelled miR‐126KD CD4⁺ T cell‐transferred group. Mechanistic evidence showed that the expression of insulin receptor substrate 1 (IRS‐1), as a functional target of miR‐126, was elevated in CD4⁺ T cells from miR‐126KD mice, accompanied by altered transduction of the extracellular regulated kinase, protein B (AKT) and nuclear factor kappa B (NF‐κB) pathway. Our data revealed a novel role in which miR‐126 was an intrinsic regulator in the function of CD4⁺ T cells, which provided preliminary basis for exploring further the role of miR‐126 in the development, function of CD4⁺ T cells and related clinical diseases.     

The WT hemochromatosis protein HFE inhibits CD8+ T‐lymphocyte activation

MHC class I (MHC I) antigen presentation is a ubiquitous process by which cells present endogenous proteins to CD8⁺ T lymphocytes during immune surveillance and response. Hereditary hemochromatosis protein, HFE, is involved in cellular iron uptake but, while structurally homologous to MHC I, is unable to bind peptides. However, increasing evidence suggests a role for HFE in the immune system. Here, we investigated the impact of HFE on CD8⁺ T‐lymphocyte activation. Using transient HFE transfection assays in a model of APCs, we show that WT HFE (HFE_WT), but not C282Y‐mutated HFE, inhibits secretion of MIP‐1β from antigen‐specific CD8⁺ T lymphocytes. HFE_WT expression also resulted in major decreases in CD8⁺ T‐lymphocyte activation as measured by 4–1BB expression. We further demonstrate that inhibition of CD8⁺ T‐lymphocyte activation was independent of MHC I surface levels, β2‐m competition, HFE interaction with transferrin receptor, antigen origin, or epitope affinity. Finally, we identified the α1–2 domains of HFE_WT as being responsible for inhibiting CD8⁺ T‐lymphocyte activation. Our data imply a new role for HFE_WT in altering CD8⁺ T‐lymphocyte reactivity, which could modulate antigen immunogenicity.     

Topical treatment of all‐trans retinoic acid inhibits murine melanoma partly by promoting CD8+ T‐cell immunity

All‐trans retinoic acid (atRA), the main biologically active metabolite of vitamin A, has been implicated in immunoregulation and anti‐cancer. A recent finding that vitamin A could decrease the risk of melanoma in humans indicates the beneficial role of atRA in melanoma. However, it remains unknown whether topical application of atRA could inhibit melanoma growth by influencing tumour immunity. We demonstrate topical application of tretinoin ointment (atRA as the active ingredient) effectively inhibited B16F10 melanoma growth. This is accompanied by markedly enhanced CD8⁺ T‐cell responses, as evidenced by significantly increased proportions of effector CD8⁺ T cells expressing granzyme B, tumour necrosis factor‐α, or interferon‐γ, and Ki67⁺ proliferating CD8⁺ T cells in atRA‐treated tumours compared with vaseline controls. Furthermore, topical atRA treatment promoted the differentiation of effector CD8⁺ T cells in draining lymph nodes (DLN) of tumour‐bearing mice. Interestingly, atRA did not affect tumoral CD4⁺ T‐cell response, and even inhibited the differentiation of interferon‐γ‐expressing T helper type 1 cells in DLN. Importantly, we demonstrated that the tumour‐inhibitory effect of atRA was partly dependent on CD8⁺ T cells, as CD8⁺ T‐cell depletion restored tumour volumes in atRA‐treated mice, which, however, was still significantly smaller than those in vaseline‐treated mice. Finally, we demonstrated that atRA up‐regulated MHCI expression in B16F10 cells, and DLN cells from tumour‐bearing mice had a significantly higher killing rate when culturing with atRA‐treated B16F10 cells. Hence, our study demonstrates that topical atRA treatment effectively inhibits melanoma growth partly by promoting the differentiation and the cytotoxic function of effector CD8⁺ T cells.     

Kidney‐specific knockout of Sav1 in the mouse promotes hyperproliferation of renal tubular epithelium through suppression of the Hippo pathway

We have previously reported that Salvador homologue 1 (SAV1), a component of the Hippo pathway, is significantly down‐regulated in high‐grade clear cell renal cell carcinoma (ccRCC) due to 14q copy number loss, and that this down‐regulation contributes to the proliferation and survival of renal tubular epithelial cells through activation of Yes‐associated protein 1 (YAP1), a downstream target of the Hippo pathway. However, the impact of SAV1 loss on the proliferation and survival of kidney cells in vivo remained to be determined. To address this issue, we generated kidney‐specific Sav1‐knockout (Cdh16‐Cre;Sav1^fl/fl) mice. Sav1 deficiency enhanced the proliferation of renal tubular epithelial cells in Cdh16‐Cre;Sav1^fl/fl mice, accompanied by nuclear localization of Yap1, suggesting suppression of the Hippo pathway. Sav1 deficiency in renal tubules also caused structural and cellular abnormalities of the epithelial cells, including significant enlargement of their nuclei. Furthermore, Cdh16‐Cre;Sav1^fl/fl mice developed both glomerular and tubular cysts. Although lining cells of the glomerular cysts showed no atypia, those of the tubular cysts showed variations in cell size and nuclear shape, which became more severe as the mice aged. In aged Cdh16‐Cre;Sav1^fl/fl mice, we observed focal disruption of proximal tubules and perivascular lymphocytic infiltration. In conclusion, Sav1 is required for the maintenance of growth, nuclear size and structure of renal tubules under physiological conditions, and its deficiency leads to the acquisition of enhanced proliferation of renal epithelial cells through suppression of Hippo signalling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

IL‐15 is critical for the maintenance and innate functions of self‐specific CD8+ T cells

IL‐15 is a pleiotropic cytokine involved in host defense as well as autoimmunity. IL‐15‐deficient mice show a decrease of memory phenotype (MP) CD8⁺ T cells, which develop naturally in naïve mice and whose origin is unclear. It has been shown that self‐specific CD8⁺ T cells developed in male H‐Y antigen‐specific TCR transgenic mice share many similarities with naturally occurring MP CD8⁺ T cells in normal mice. In this study, we found that H‐Y antigen‐specific CD8⁺ T cells in male but not female mice decreased when they were crossed with IL‐15‐deficient mice, mainly due to impaired peripheral maintenance. The self‐specific TCR transgenic CD8⁺ T cells developed in IL‐15‐deficient mice showed altered surface phenotypes and reduced effector functions ex vivo. Bystander activation of the self‐specific CD8⁺ T cells was induced in vivo during infection with Listeria monocytogenes, in which proliferation but not IFN‐γ production was IL‐15‐dependent. These results indicated important roles for IL‐15 in the maintenance and functions of self‐specific CD8⁺ T cells, which may be included in the naturally occurring MP CD8⁺ T‐cell population in naïve normal mice and participate in innate host defense responses.     

Blinded histopathological characterisation of POLE exonuclease domain‐mutant endometrial cancers: sheep in wolf's clothing

Aims

POLE exonuclease domain mutations identify a subset of endometrial cancer (EC) patients with an excellent prognosis. The use of this biomarker has been suggested to refine adjuvant treatment decisions, but the necessary sequencing is not widely performed and is relatively expensive. Therefore, we aimed to identify histopathological and immunohistochemical characteristics to aid in the detection of POLE‐mutant ECs.

Methods and results

Fifty‐one POLE‐mutant endometrioid, 67 POLE‐wild‐type endometrioid and 15 POLE‐wild‐type serous ECs were included (total N = 133). An expert gynaecopathologist, blinded to molecular features, evaluated each case (two or more slides) for 16 morphological characteristics. Immunohistochemistry was performed for p53, p16, MLH1, MSH2, MSH6, and PMS2. POLE‐mutant ECs were characterised by a prominent immune infiltrate: 80% showed peritumoral lymphocytes and 59% showed tumour‐infiltrating lymphocytes, as compared with 43% and 28% of POLE‐wild‐type endometrioid ECs, and 27% and 13% of their serous counterparts (P < 0.01, all comparisons). Of POLE‐mutant ECs, 33% contained tumour giant cells; this proportion was significantly higher than that in POLE‐wild‐type endometrioid ECs (10%; P = 0.003), but not significantly different from that in serous ECs (53%). Serous‐like features were as often (focally) present in POLE‐mutant as in POLE‐wild‐type endometrioid ECs (6–24%, depending on the feature). The majority of POLE‐mutant ECs showed wild‐type p53 (86%), negative/focal p16 (82%) and normal mismatch repair protein expression (90%).

Conclusions

A simple combination of morphological and immunohistochemical characteristics (tumour type, grade, peritumoral lymphocytes, MLH1, and p53 expression) can assist in prescreening for POLE exonuclease domain mutations in EC, increasing the probability of a mutation being detected from 7% to 33%. This facilitates the use of this important prognostic biomarker in routine pathology.     

K‐RasV14I‐induced Noonan syndrome predisposes to tumour development in mice

The Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. A significant proportion of NS patients may also develop myeloproliferative disorders (MPDs), including juvenile myelomonocytic leukaemia (JMML). Surprisingly, scarce information is available in relation to other tumour types in these patients. We have previously developed and characterized a knock‐in mouse model that carries one of the most frequent KRAS‐NS‐related mutations, the K‐Ras^V14I substitution, which recapitulates most of the alterations described in NS patients, including MPDs. The K‐Ras^V14I mutation is a mild activating K‐Ras protein; thus, we have used this model to study tumour susceptibility in comparison with mice expressing the classical K‐Ras^G12V oncogene. Interestingly, our studies have shown that these mice display a generalized tumour predisposition and not just MPDs. In fact, we have observed that the K‐Ras^V14I mutation is capable of cooperating with the p16Ink4a/p19Arf and Trp53 tumour suppressors, as well as with other risk factors such as pancreatitis, thereby leading to a higher cancer incidence. In conclusion, our results illustrate that the K‐Ras^V14I activating protein is able to induce cancer, although at a much lower level than the classical K‐Ras^G12V oncogene, and that it can be significantly modulated by both genetic and non‐genetic events. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.