John R. Apps

PARK2 deletions occur frequently in sporadic colorectal cancer and accelerate adenoma development in Apc mutant mice

In 100 primary colorectal carcinomas, we demonstrate by array comparative genomic hybridization (aCGH) that 33% show DNA copy number (DCN) loss involving PARK2, the gene encoding PARKIN, the E3 ubiquitin ligase whose deficiency is responsible for a form of autosomal recessive juvenile parkinsonism. PARK2 is located on chromosome 6 (at 6q25–27), a chromosome with one of the lowest overall frequencies of DNA copy number alterations recorded in colorectal cancers. The PARK2 deletions are mostly focal (31% ∼0.5 Mb on average), heterozygous, and show maximum incidence in exons 3 and 4. As PARK2 lies within FRA6E, a large common fragile site, it has been argued that the observed DCN losses in PARK2 in cancer may represent merely the result of enforced replication of locally vulnerable DNA. However, we show that deficiency in expression of PARK2 is significantly associated with adenomatous polyposis coli (APC) deficiency in human colorectal cancer. Evidence of some PARK2 mutations and promoter hypermethylation is described. PARK2 overexpression inhibits cell proliferation in vitro. Moreover, interbreeding of Park2 heterozygous knockout mice with ApcMin mice resulted in a dramatic acceleration of intestinal adenoma development and increased polyp multiplicity. We conclude that PARK2 is a tumor suppressor gene whose haploinsufficiency cooperates with mutant APC in colorectal carcinogenesis.

Intra-Tumor Genetic Heterogeneity in Wilms Tumor: Clonal Evolution and Clinical Implications

The evolution of pediatric solid tumors is poorly understood. There is conflicting evidence of intra-tumor genetic homogeneity vs. heterogeneity (ITGH) in a small number of studies in pediatric solid tumors. A number of copy number aberrations (CNA) are proposed as prognostic biomarkers to stratify patients, for example 1q + in Wilms tumor (WT); current clinical trials use only one sample per tumor to profile this genetic biomarker. We multisampled 20 WT cases and assessed genome-wide allele-specific CNA and loss of heterozygosity, and inferred tumor evolution, using Illumina CytoSNP12v2.1 arrays, a custom analysis pipeline, and the MEDICC algorithm. We found remarkable diversity of ITGH and evolutionary trajectories in WT. 1q + is heterogeneous in the majority of tumors with this change, with variable evolutionary timing. We estimate that at least three samples per tumor are needed to detect > 95% of cases with 1q +. In contrast, somatic 11p15 LOH is uniformly an early event in WT development. We find evidence of two separate tumor origins in unilateral disease with divergent histology, and in bilateral WT. We also show subclonal changes related to differential response to chemotherapy. Rational trial design to include biomarkers in risk stratification requires tumor multisampling and reliable delineation of ITGH and tumor evolution.

Stem cell senescence drives age-attenuated induction of pituitary tumours in mouse models of paediatric craniopharyngioma

Senescent cells may promote tumour progression through the activation of a senescence-associated secretory phenotype (SASP), whether these cells are capable of initiating tumourigenesis in vivo is not known. Expression of oncogenic β-catenin in Sox2+ young adult pituitary stem cells leads to formation of clusters of stem cells and induction of tumours resembling human adamantinomatous craniopharyngioma (ACP), derived from Sox2− cells in a paracrine manner. Here, we uncover the mechanisms underlying this paracrine tumourigenesis. We show that expression of oncogenic β-catenin in Hesx1+ embryonic precursors also results in stem cell clusters and paracrine tumours. We reveal that human and mouse clusters are analogous and share a common signature of senescence and SASP. Finally, we show that mice with reduced senescence and SASP responses exhibit decreased tumour-inducing potential. Together, we provide evidence that senescence and a stem cell-associated SASP drive cell transformation and tumour initiation in vivo in an age-dependent fashion.Senescent cells can promote tumour progression through the activation of a senescenceassociated secretory phenotype (SASP). Here, the authors show that SASP activation is associated with non-cell autonomous cell transformation and tumour initiation in an in vivo model of adamantinomatous craniopharyngioma.

Imaging Invasion: Micro-CT imaging of adamantinomatous craniopharyngioma highlights cell type specific spatial relationships of tissue invasion

Tissue invasion and infiltration by brain tumours poses a clinical challenge, with destruction of structures leading to morbidity. We assessed whether micro-CT could be used to map tumour invasion in adamantinomatous craniopharyngioma (ACP), and whether it could delineate ACPs and their intrinsic components from surrounding tissue.Three anonymised archival frozen ACP samples were fixed, iodinated and imaged using a micro-CT scanner prior to the use of standard histological processing and immunohistochemical techniques.We demonstrate that micro-CT imaging can non-destructively give detailed 3D structural information of tumours in volumes with isotropic voxel sizes of 4–6 microns, which can be correlated with traditional histology and immunohistochemistry.Such information complements classical histology by facilitating virtual slicing of the tissue in any plane and providing unique detail of the three dimensional relationships of tissue compartments.

Stabbing and safeguarding in children and young people: a Pan-London service evaluation and audit

Objectives To characterize paediatric presentations of stabbing to emergency departments across London and to audit existing referral rates to the police and social services against the new standard set by the General Medical Council. Design Retrospective multi-centre service evaluation/audit. Setting All emergency departments within London. Participants Patients under 18 years of age presenting to emergency departments with non-accidental stabbing between 1 April 2007 and 30 April 2009. Main outcome measures Patient age, nature of assault, assailant, injuries and management. Rates of documented referral to police and social services, as mandated by GMC guidance. Results A total of 381 presentations were identified from 20 out of the 32 hospitals in London, 160 of whom were less than 16 years old. The majority were seen only by emergency department staff and only a minority (28%) were admitted. Three died in the departments. A knife was the commonest weapon and the limbs the most common site of injury. Referrals to police were documented in only 30% of patients (43% if <16 years old) and to social services in 16% (31% if <16 years old) of those discharged. In the majority, there was no documentation (police 64%, social services 79%). Conclusions A significant number of paediatric stabbings present to emergency departments across London. The majority of these are discharged directly from departments. Of those discharged, documentation regarding referral rates to Police and Social Services was poor, and documented referral rates low. This study covered a period prior to the introduction of new General Medical Council guidance and a repeat audit to assess subsequent documented referrals is required.

The immune environment of paediatric solid malignancies: evidence from an immunohistochemical study of clinical cases

Childhood malignancies are relatively poorly studied in terms of tumour/host interaction. Using tissue arrays of childhood cancers, we analysed immunohistochemical staining for CD68, CD3 and FOXP3 to evaluate infiltration of myeloid cells, lymphocytes and regulatory T cells. Staining for phosphorylated STAT3 was performed in a subset. The majority of paediatric tumours demonstrated a marked infiltration of CD68+ myeloid cells but, with the exception of neuroblastoma, most showed only sparse infiltration of CD3+/ FOXP3- cells. There was evidence for activation of STAT3 in pPNET (50%), ependymoma (45%) and undifferentiated sarcoma (38%), but it was rarely activated in other tumours.

Molecular Analyses Reveal Inflammatory Mediators in the Solid Component and Cyst Fluid of Human Adamantinomatous Craniopharyngioma.

Pediatric adamantinomatous craniopharyngioma (ACP) is a highly solid and cystic tumor, often causing substantial damage to critical neuroendocrine structures such as the hypothalamus, pituitary gland, and optic apparatus. Paracrine signaling mechanisms driving tumor behavior have been hypothesized, with IL-6R overexpression identified as a potential therapeutic target. To identify potential novel therapies, we characterized inflammatory and immunomodulatory factors in ACP cyst fluid and solid tumor components. Cytometric bead analysis revealed a highly pro-inflammatory cytokine pattern in fluid from ACP compared to fluids from another cystic pediatric brain tumor, pilocytic astrocytoma. Cytokines and chemokines with particularly elevated concentrations in ACPs were IL-6, CXCL1 (GRO), CXCL8 (IL-8) and the immunosuppressive cytokine IL-10. These data were concordant with solid tumor compartment transcriptomic data from a larger cohort of ACPs, other pediatric brain tumors and normal brain. The majority of receptors for these cytokines and chemokines were also over-expressed in ACPs. In addition to IL-10, the established immunosuppressive factor IDO-1 was overexpressed by ACPs at the mRNA and protein levels. These data indicate that ACP cyst fluids and solid tumor components are characterized by an inflammatory cytokine and chemokine expression pattern. Further study regarding selective cytokine blockade may inform novel therapeutic interventions.

MAPK pathway control of stem cell proliferation and differentiation in the embryonic pituitary provides insights into the pathogenesis of papillary craniopharyngioma

Despite the importance of the RAS-RAF-MAPK pathway in normal physiology and disease of numerous organs, its role during pituitary development and tumourigenesis remains largely unknown. Here, we show that the over-activation of the MAPK pathway, through conditional expression of the gain-of-function alleles BrafV600E and KrasG12D in the developing mouse pituitary, results in severe hyperplasia and abnormal morphogenesis of the gland by the end of gestation. Cell-lineage commitment and terminal differentiation are disrupted, leading to a significant reduction in numbers of most of the hormone-producing cells before birth, with the exception of corticotrophs. Of note, Sox2+ stem cells and clonogenic potential are drastically increased in the mutant pituitaries. Finally, we reveal that papillary craniopharyngioma (PCP), a benign human pituitary tumour harbouring BRAF p.V600E also contains Sox2+ cells with sustained proliferative capacity and disrupted pituitary differentiation. Together, our data demonstrate a crucial function of the MAPK pathway in controlling the balance between proliferation and differentiation of Sox2+ cells and suggest that persistent proliferative capacity of Sox2+ cells may underlie the pathogenesis of PCP. Highlighted Article: Constitutive activation of the MAPK/ERK pathway causes pituitary hyperplasia, abnormal morphogenesis, and abnormal endocrine cell specification due to the sustained proliferation of the Sox2+ stem cell compartment.

Nephrogenic rests in wilms tumors treated with preoperative chemotherapy: The UK SIOP wilms tumor 2001 trial experience

Nephrogenic rests (NRs) are abnormally persistent foci of embryonal cells, thought to be the precursor lesion of Wilms tumors (WTs). To date, their presence has not been systematically examined in WTs treated with preoperative chemotherapy.

Hypothalamic sonic hedgehog is required for cell specification and proliferation of LHX3/LHX4 pituitary embryonic precursors

Sonic hedgehog (SHH) is an essential morphogenetic signal that dictates cell fate decisions in several developing organs in mammals. In vitro data suggest that SHH is required to specify LHX3+/LHX4+ Rathkes pouch (RP) progenitor identity. However, in vivo studies have failed to reveal such a function, supporting instead a crucial role for SHH in promoting proliferation of these RP progenitors and for differentiation of pituitary cell types. Here, we have used a genetic approach to demonstrate that activation of the SHH pathway is necessary to induce LHX3+/LHX4+ RP identity in mouse embryos. First, we show that conditional deletion of Shh in the anterior hypothalamus results in a fully penetrant phenotype characterised by a complete arrest of RP development, with lack of Lhx3/Lhx4 expression in RP epithelium at 9.0 days post coitum (dpc) and total loss of pituitary tissue by 12.5 dpc. Conversely, overactivation of the SHH pathway by conditional deletion of Ptch1 in RP progenitors leads to severe hyperplasia and enlargement of the Sox2+ stem cell compartment by the end of gestation. Summary: Genetic approaches demonstrate that, during normal murine development, the SHH pathway is first required for normal specification of Rathkes pouch embryonic precursors and subsequently to control their proliferation.