Predicting local tumour progression after ablation for colorectal liver metastases: CT-based radiomics of the ablation zone.

PURPOSE: To assess whether CT-based radiomics of the ablation zone (AZ) can predict local tumour progression (LTP) after thermal ablation for colorectal liver metastases (CRLM). MATERIALS AND METHODS: Eighty-two patients with 127 CRLM were included. Radiomics features (with different filters) were extracted from the AZ and a 10 mm periablational rim (PAR)on portal-venous-phase CT up to 8 weeks after ablation. Multivariable stepwise Cox regression analyses were used to predict LTP based on clinical and radiomics features. Performance (concordance [c]-statistics) of the different models was compared and performance in an 'independent' dataset was approximated with bootstrapped leave-one-out-cross-validation (LOOCV). RESULTS: Thirty-three lesions (26 %) developed LTP. Median follow-up was 21 months (range 6-115). The combined model, a combination of clinical and radiomics features, included chemotherapy (HR 0.50, p = 0.024), cT-stage (HR 10.13, p = 0.016), lesion size (HR 1.11, p = <0.001), AZ_Skewness (HR 1.58, p = 0.016), AZ_Uniformity (HR 0.45, p = 0.002), PAR_Mean (HR 0.52, p = 0.008), PAR_Skewness (HR 1.67, p = 0.019) and PAR_Uniformity (HR 3.35, p < 0.001) as relevant predictors for LTP. The predictive performance of the combined model (after LOOCV) yielded a c-statistic of 0.78 (95 %CI 0.65-0.87), compared to the clinical or radiomics models only (c-statistic 0.74 (95 %CI 0.58-0.84) and 0.65 (95 %CI 0.52-0.83), respectively). CONCLUSION: Combining radiomics features with clinical features yielded a better performing prediction of LTP than radiomics only. CT-based radiomics of the AZ and PAR may have potential to aid in the prediction of LTP during follow-up in patients with CRLM.

Assessment and Optimisation of Lung Cancer Patients for Treatment with Curative Intent.

Over the past decade the field of lung cancer management has seen many developments. Coupled with an ageing population and increasing rates of comorbid illness, the work-up for treatments with curative intent has become more complex and detailed. As well as improvements in imaging and staging techniques, developments in both surgery and radiotherapy may now allow patients who would previously have been considered unfit or not appropriate for treatment with curative intent to undergo radical therapies. This overview will highlight published studies relating to investigation and staging techniques, together with assessments of fitness, with the aim of helping clinicians to determine the most appropriate treatments for each patient. We also highlight areas where further research may be required.

The RNA helicase p68 (DDX5) is selectively required for the induction of p53-dependent p21 expression and cell-cycle arrest after DNA damage.

The RNA helicase p68 (DDX5) is an established co-activator of the p53 tumour suppressor that itself has a pivotal role in orchestrating the cellular response to DNA damage. Although several factors influence the biological outcome of p53 activation, the mechanisms governing the choice between cell-cycle arrest and apoptosis remain to be elucidated. In the present study, we show that, while p68 is critical for p53-mediated transactivation of the cell-cycle arrest gene p21(WAF1/CIP1), it is dispensable for induction of several pro-apoptotic genes in response to DNA damage. Moreover, p68 depletion results in a striking inhibition of recruitment of p53 and RNA Pol II to the p21 promoter but not to the Bax or PUMA promoters, providing an explanation for the selective effect on p21 induction. Importantly, these findings are mirrored in a novel inducible p68 knockout mouse model in which p68 depletion results in a selective inhibition of p21 induction in several tissues. Moreover, in the bone marrow, p68 depletion results in an increased sensitivity to γ-irradiation, consistent with an increased level of apoptosis. These data highlight a novel function of p68 as a modulator of the decision between p53-mediated growth arrest and apoptosis in vitro and in vivo.

Bone grafting, orthopaedic biomaterials, and the clinical need for bone engineering.

As the population ages, the number of operations performed on bone is expected to increase. Diseases such as arthritis, tumours, and trauma can lead to defects in the skeleton requiring an operation to replace or restore the lost bone. Surgeons can use autografts, allografts, and/or bone graft substitutes to restore areas of bone loss. Surgical implants are also used in addition or in isolation to replace the diseased bone. This review considers the application of available bone grafts in different clinical settings. It also discusses recently introduced bioactive biomaterials and highlights the clinical difficulties and technological deficiencies that exist in our current surgical practice.

Outcome of palliative esophageal stenting for malignant dysphagia: a retrospective analysis.

Greater than 50% of patients with esophageal carcinoma are found to be incurable at the time of diagnosis, leaving only palliative options. Self-expanding metal stents (SEMs) are effective for relieving symptoms and complications associated with esophageal carcinoma and improving quality of life. We undertook a retrospective analysis to evaluate the experience of palliative esophageal stenting for symptomatic malignant dysphagia in our institution over a period of 7 years. Between January 1999 and January 2006, 126 patients who received SEMs for malignant dysphagia were identified using an upper gastrointestinal specialist nurse clinician database. Data were obtained from patient case notes, endoscopy, histopathology, radiology, and external agency databases. Of the 126 identified, 36 patients were excluded from the analysis. A number of variables including age, sex, presenting complaints, type of stent, indications of stenting, success or failure of stent insertion, survival rate, and complication rate were analyzed. Of the 90 patients, 55 (61%) were male and 35 (39%) were female. The mean age of patients was 70.79 (range 40-97) years. The predominant presenting complaints were dysphagia (n = 81) and weight loss (n = 48). The indication for stenting was worsening dysphagia in all patients. Tumors were confined to the distal esophagus and esophagogastric junction in 73 patients (81%), and the mid-esophagus in 17 (19%). Adenocarcinoma was identified in 61 patients (67.8%) and squamous cell carcinoma in 29 (32.2%). Stenting numbers were comparable in endoscopic and radiologic groups (47 vs. 43), with successful stent deployment in 89 patients. The 7- and 30-day mortality was 9% (n = 8) and 28% (n = 25), respectively. Comparable numbers of early deaths were seen in both radiologic (n = 13) and endoscopic (n = 12) groups. Causes of early inpatient death included hemorrhage (n = 5), pneumonia (n = 7), exhaustion (n = 2), cardiac causes (n = 3), perforation (n = 1), and sepsis (n = 1). The number of patients with complications was 41 (45.6%), 25 in the surgical group and 15 in the radiologic group; the difference was not significant (P = 0.13). The mean survival time was 92.5 (0-638) days and median survival time was 61 days. A subgroup of patients with complete dysphagia (score 4) gained a mean survival of 59 days. Those patients receiving adjuvant chemotherapy or radiotherapy survived significantly longer than those receiving stenting alone (152.8 days vs. 71.8 days). There is no significant difference in complications or survival when using endoscopic or radiologic methods to deploy SEMs in patients with inoperable esophageal cancer. Mortality is low; however, the morbidity rate is significant. Patients receiving adjuvant chemotherapy or radiotherapy, in addition to stenting, survived significantly longer than those with a stent only.

Incidence of acquired demyelination of the CNS in Canadian children.

BACKGROUND: The incidence of acquired demyelination of the CNS (acquired demyelinating syndromes [ADS]) in children is unknown. It is important that physicians recognize the features of ADS to facilitate care and to appreciate the future risk of multiple sclerosis (MS). OBJECTIVE: To determine the incidence, clinical features, familial autoimmune history, and acute management of Canadian children with ADS. METHODS: Incidence and case-specific data were obtained through the Canadian Pediatric Surveillance Program from April 1, 2004, to March 31, 2007. Before study initiation, a survey was sent to all pediatric health care providers to determine awareness of MS as a potential outcome of ADS in children. RESULTS: Two hundred nineteen children with ADS (mean age 10.5 years, range 0.66-18.0 years; female to male ratio 1.09:1) were reported. The most common presentations were optic neuritis (ON; n = 51, 23%), acute disseminated encephalomyelitis (ADEM; n = 49, 22%), and transverse myelitis (TM; n = 48, 22%). Children with ADEM were more likely to be younger than 10 years, whereas children with monolesional ADS (ON, TM, other) were more likely to be older than 10 years (p < 0.001). There were 73 incident cases per year, leading to an annual incidence of 0.9 per 100,000 Canadian children. A family history of MS was reported in 8%. Before study initiation, 65% of physicians indicated that they considered MS as a possible outcome of ADS in children. This increased to 74% in year 1, 81% in year 2, and 87% in year 3. CONCLUSION: The incidence of pediatric acquired demyelinating syndromes (ADS) is 0.9 per 100,000 Canadian children. ADS presentations are influenced by age.

The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis.

Cell-cycle transition from the G(2) phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA interference results in delayed cell-cycle progression at the onset of mitosis. Knockdown of CK2beta causes stabilization of Wee1 and increased phosphorylation of CDK1 at the inhibitory Tyr15. PLK1-Wee1 association is an essential event in the degradation of Wee1 in unperturbed cell cycle. We have found that CK2beta participates in PLK1-Wee1 complex formation whereas its cellular depletion leads to disruption of PLK1-Wee1 interaction and reduced Wee1 phosphorylation at Ser53 and 121. The data reported here reinforce the notion that CK2beta has functions that are independent of its role as the CK2 regulatory subunit, identifying it as a new component of signaling pathways that regulate cell-cycle progression at the entry of mitosis.

Transcription factor TAFII250 promotes Mdm2-dependent turnover of p53.

The p53 tumour suppressor is regulated mainly by Mdm2, an E3 ubiquitin ligase that promotes the ubiquitylation and proteasome-mediated degradation of p53. Many agents that induce p53 are inhibitors of transcription, suggesting that the p53 pathway can detect a signal(s) arising from transcriptional malfunction. Mdm2 associates with TAFII250, a component of the general transcription factor TFIID. Inactivation of TAFII250 in ts13 cells, which express a temperature-sensitive mutant of TAFII250, leads to the induction of p53 and cell cycle arrest. In the present study, we show that TAFII250 stimulates the ubiquitylation and degradation of p53 in a manner that is dependent upon Mdm2 and requires its acidic domain. Mechanistically, TAFII250 downregulates Mdm2 auto-ubiquitylation, leading to Mdm2 stabilization, and promotes p53-Mdm2 association through a recently defined second binding site in the acidic domain of Mdm2. These data provide a novel route through which TAFII250 can directly influence p53 levels and are consistent with the idea that the maintenance of p53 turnover is coupled to the integrity of RNA polymerase II transcription.

c-Abl phosphorylates Hdm2 at tyrosine 276 in response to DNA damage and regulates interaction with ARF.

The p53 tumour-suppressor protein is tightly regulated through its association with the Hdm2 E3 ligase. Activation of p53 by DNA strand breaks is orchestrated by the ataxia-telangiectasia mutated (ATM) protein kinase and involves interruption of Hdm2-mediated p53 degradation. As part of this mechanism ATM itself, and the ATM-activated protein tyrosine kinase, c-Abl, inhibit Hdm2 function through phosphorylation of serine 395 and tyrosine 394 (Y394), respectively. In the present study, we have identified a novel target of c-Abl in the Hdm2 protein, tyrosine 276 (Y276). We show that c-Abl phosphorylates this residue in vitro and confirm that Y394 is a target of c-Abl. We also show that Y276 is phosphorylated in a c-Abl-dependent manner in cultured cells and provide evidence that Y276 is phosphorylated in response to DNA damage coincident with the activation of c-Abl. Finally, we show that Y276 phosphorylation stimulates interaction with ARF, leading to increased levels of nucleolar Hdm2 and decreased turnover of p53. These data establish Y276 as a physiological target of c-Abl that contributes functionally to the induction of p53.

Critical roles for the serine 20, but not the serine 15, phosphorylation site and for the polyproline domain in regulating p53 turnover.

The p53 tumour suppressor protein is a short-lived transcription factor that becomes stabilized in response to a wide range of cellular stresses. Ubiquitination and the targeting of p53 for degradation by the proteasome are mediated by Mdm2 (mouse double minute clone 2), a negative regulatory partner of p53. Previous studies have suggested that DNA-damage-induced phosphorylation of p53 at key N-terminal sites has a pivotal role in regulating the interaction with Mdm2 but the precise role of phosphorylation of serines 15 and 20 is still unclear. Here we show that replacement of serine 15 and a range of other key N-terminal phosphorylation sites with alanine, which cannot be phosphorylated, has little effect on the ubiquitination and degradation of full-length human p53. In contrast, replacement of serine 20 makes p53 highly sensitive to Mdm2-mediated turnover. These results define distinct roles for serines 15 and 20, two sites previously demonstrated to be dependent on phosphorylation through mechanisms mediated by DNA damage and ATM (ataxia telangiectasia mutated). We also show that the polyproline region of p53, a domain that has a key role in p53-induced apoptosis, exerts a critical influence over the Mdm2-mediated turnover of p53.

Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells.

Murine double minute clone 2 oncoprotein (MDM2) is a key component in the regulation of the tumour suppressor p53. MDM2 mediates the ubiqutination of p53 in the capacity of an E3 ligase and targets p53 for rapid degradation by the proteasome. Stress signals which impinge on p53, leading to its activation, promote disruption of the p53-MDM2 complex, as in the case of ionizing radiation, or block MDM2 synthesis and thereby reduce cellular MDM2 levels, as in the case of UV radiation. It is therefore likely that MDM2, which is known to be modified by ubiquitination, SUMOylation and multi-site phosphorylation, may itself be a target for stress signalling (SUMO is small ubiquitin-related modifier-1). In the present study we show that, like p53, the MDM2 protein is a substrate for phosphorylation by the protein kinase CK2 (CK2) in vitro. CK2 phosphorylates a single major site, Ser(267), which lies within the central acidic domain of MDM2. Fractionation of cellular extracts revealed the presence of a single Ser(267) protein kinase which co-purified with CK2 on ion-exchange chromatography and, like CK2, was subject to inhibition by micromolar concentrations of the CK2-specific inhibitor 5,6-dichlororibofuranosylbenzimidazole. Radiolabelling of cells expressing tagged recombinant wild-type MDM2 or a S267A (Ser(267)-->Ala) mutant, followed by phosphopeptide analysis, confirmed that Ser(267) is a cellular target for phosphorylation. Ser(267) mutants are still able to direct the degradation of p53, but in a slightly reduced capacity. These data highlight a potential route by which one of several physiological modifications occurring within the central acidic domain of the MDM2 protein can occur.

IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects.

The p53-targeted kinases casein kinase 1delta (CK1delta) and casein kinase 1epsilon (CK1epsilon) have been proposed to be involved in regulating DNA repair and chromosomal segregation. Recently, we showed that CK1delta localizes to the spindle apparatus and the centrosomes in cells with mitotic failure caused by DNA-damage prior to mitotic entry. We provide here evidence that 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261), a novel inhibitor of CK1delta and CK1epsilon, triggers the mitotic checkpoint control. At low micromolar concentrations IC261 inhibits cytokinesis causing a transient mitotic arrest. Cells containing active p53 arrest in the postmitotic G1 phase by blockage of entry into the S phase. Cells with non-functional p53 undergo postmitotic replication developing an 8N DNA content. The increase of DNA content is accompanied by a high amount of micronucleated and apoptotic cells. Immunfluorescence images show that at low concentrations IC261 leads to centrosome amplification causing multipolar mitosis. Our data are consistent with a role for CK1delta and CK1epsilon isoforms in regulating key aspects of cell division, possibly through the regulation of centrosome or spindle function during mitosis.

Multiple sites of in vivo phosphorylation in the MDM2 oncoprotein cluster within two important functional domains.

The MDM2 oncoprotein is a negative regulatory partner of the p53 tumour suppressor. MDM2 mediates ubiquitination of p53 and targets the protein to the cytoplasm for 26S proteosome-dependent degradation. In this paper, we show that MDM2 is modified in cultured cells by multisite phosphorylation. Deletion analysis of MDM2 indicated that the sites of modification fall into two clusters which map respectively within the N-terminal region encompassing the p53 binding domain and nuclear export sequence, and the central acidic domain that mediates p14(ARF) binding, p53 ubiquitination and cytoplasmic shuttling. The data are consistent with potential regulation of MDM2 function by multisite phosphorylation.

The role of p53 in the response to mitotic spindle damage.

The p53 tumour suppressor protein has defined roles in G1/S and G2/M cell cycle checkpoints in response to a range of cellular stresses including DNA damage, dominant oncogene expression, hypoxia, metabolic changes and viral infection. In addition to these responses, p53 can also be activated when damage occurs to the mitotic spindle. Initially, spindle damage activates a p53-independent checkpoint which functions at the metaphase-anaphase transition and prevents cells from progressing through mitosis until the completion of spindle formation. Cells eventually escape from this block (a process termed 'mitotic slippage'), and an aberrant mitosis ensues in which sister chromatids fail to segregate properly. After a delay period, p53 responds to this mitotic failure by instituting a G1-like growth arrest, with an intact nucleus containing 4N DNA, but without the cells undergoing division. Cells lacking wild-type p53 are still able to arrest transiently at mitosis, and also fail to undergo division, underscoring that the delay in mitosis is p53-independent. However, these cells are not prevented from re-entering the cell cycle and can reduplicate their DNA unchecked, leading to polyploidy. Additionally, p53-null cells which experience spindle failure often show the appearance of micronuclei arising from poorly segregated chromosomes which have decondensed and been enclosed in a nuclear envelope. The ability of p53 to prevent their formation suggests an additional G2 involvement which prevents nuclear breakdown prior to mitosis. The molecular mechanism by which p53 is able to sense mitotic failure is still unknown, but may be linked to the ability of p53 to regulate duplication of the centrosome, the organelle which nucleates spindle formation.

Protein kinase CK1 is a p53-threonine 18 kinase which requires prior phosphorylation of serine 15.

p53 is a potent transcription factor which is regulated by sequential multisite phosphorylation and acetylation. In this paper, we identify threonine 18 of p53, a key site in regulating the interaction between p53 and its regulatory partner MDM2, as a novel site phosphorylated in vitro by purified recombinant casein kinase 1 (CK1) delta. Strikingly, phosphorylation of threonine 18 is dependent upon prior phosphorylation of serine 15. These data highlight an additional and physiologically important target residue for CK1 in p53 and suggest a potential mechanism by which sequential modification of a pivotal N-terminal residue in p53 may occur following stress-activated modification of serine 15.

Serine15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2.

The p53 tumour suppressor protein is a labile transcription factor that is activated and stabilized in response to a wide range of cellular stresses, through a mechanism involving disruption of its interaction with MDM2, a negative regulatory partner. Induction of p53 by DNA damage additionally involves a series of phosphorylation and acetylation modifications, some of which are thought to regulate MDM2 binding. Here we report the effects of introducing mutations at several known or putative N-terminal phosphorylation sites on the transactivation function of p53. These studies highlight phosphorylation of Ser15, a key phosphorylation target during the p53 activation process, as being critical for p53-dependent transactivation. Biochemical data indicate that the mechanism by which phosphorylation of Ser15 stimulates p53-dependent transactivation occurs through increased binding to the p300 coactivator protein. The data also indicate that Ser15-dependent regulation of transactivation is independent of any involvement in modulating MDM2 binding, and that Ser15 phosphorylation alone is not sufficient to block the p53-MDM2 interaction.

Phosphorylation of murine p53, but not human p53, by MAP kinase in vitro and in cultured cells highlights species-dependent variation in post-translational modification.

The p53 tumour suppressor protein is tightly regulated by protein-protein association, protein turnover and a variety of post-translational modifications. Multisite phosphorylation plays a major role in activating and in finely tuning p53 function. The proline rich domain of murine p53 is a substrate for phosphorylation, in vitro and in cultured cells, by the p42ERK2 and p44ERK1 mitogen-activated protein (MAP) kinases. However, to date there have been no reports of attempts to determine whether p53 from any other species is a substrate for MAP kinase. In this paper we confirm that murine p53 is targeted by recombinant MAP kinase and by MAP kinases in extracts of both murine and human cells. In contrast, human p53 is not a substrate for recombinant MAP kinase nor are there any detectable levels of protein kinase activity in stimulated human cell extracts which phosphorylate the proline rich domain of human p53 in vitro. Finally, although stimulation of murine fibroblasts with o-tetradecanolylphorbol 13-acetate (TPA), an indirect activator of the MAP kinase pathway, leads to site-specific phosphorylation of murine p53, similar treatment of human fibroblasts and epithelial cells showed no significant changes in the phosphorylation pattern. These data are consistent with accumulating evidence that significant species-dependent differences exist in the post-translational modification of p53.

Protein kinase CK2-dependent regulation of p53 function: evidence that the phosphorylation status of the serine 386 (CK2) site of p53 is constitutive and stable.

The p53 tumour suppressor protein is regulated by several mechanisms including multisite phosphorylation. One of the protein kinases which has an established role in regulating p53 function is the protein kinase CK2. The regulation by CK2 occurs both through interaction of p53 with CK2 itself (the regulatory beta subunit) and phosphorylation at the penultimate residue of p53, serine 386 (murine p53). Strikingly, this phosphorylation event controls several independent functions of p53 including site-specific DNA binding, strand renaturation, transcriptional repression and the anti-proliferative function of p53. However, CK2 is a constitutively-active enzyme and therefore the mechanism by which the phosphorylation of p53 at serine 386 is itself regulated, or indeed the question as to whether phosphorylation of this site is regulated at all, remains unresolved. In this paper we provide evidence that serine 386 is highly resistant to dephosphorylation in cultured cells, even though this site can be dephosphorylated in vitro by recombinant protein phosphatase 1. These data suggest that, once phosphorylated at the CK2 site, a p53 molecule remains in this modified form throughout its lifespan. To address the issue of whether the level of serine 386 phosphorylation may be regulated through controlling the subcellular compartmentalisation of p53 and CK2, we examined the subcellular localisation of p53 and CK2alpha in C57MG cells and Rat-1 fibroblasts by immunofluorescence staining. Both proteins were present in the cytoplasm and enriched in the nucleus, with minor variations in the intensity of subcellular location over the course of the cell cycle. Similarly, activation of p53 by UV irradiation or DNA damage-inducing drugs had no effect on either the localisation or levels of CK2alpha, even although significant nuclear p53 accumulation was observed. A striking observation arising from these studies was the intense staining of CK2alpha with the centrosomes, suggesting a potentially important role for this kinase in microtubule formation and/or chromosomal segregation.

Mechanisms of switching on p53: a role for covalent modification?

The p53 protein plays a pivotal role in activating and integrating adaptive cellular responses to a wide range of environmental stresses. Activation of p53 can occur by different molecular routes, depending on the nature of the activating signal. Central to the activation process, by whichever route, is the destabilization of the p53-MDM2 interaction. The molecular mechanisms which activate p53 involve elements of post-translational modification, protein stabilization and protein-protein interaction. Two central themes are emerging from recent work in this area. The first is that there are common events in the p53 activation process among different activating pathways. The second is that activation involves not just a single molecular event such as disruption of the p53-MDM2 interaction, but a series of sequential events the nature of which is governed by the type of activating stimulus. This review summarizes our current knowledge of the p53 activation process in response to two stimuli, DNA damage and activated oncogenes, and considers the contribution made by multisite phosphorylation in determining the nature of the p53 response.