Partially Ablative Body Radiotherapy (PABR): A novel approach for palliative radiotherapy of locally advanced bulky unresectable sarcomas.

BACKGROUND: Locally advanced, bulky, unresectable sarcomas cause significant tumour mass effects, leading to burdensome symptoms. We have developed a novel Partially Ablative Body Radiotherapy (PABR) technique that delivers a high, ablative dose to the tumour core and a low, palliative dose to its periphery aiming to increase overall tumour response without significantly increasing treatment toxicity. AIM: This study aims to report the safety and oncologic outcomes of PABR in patients with bulky, unresectable sarcomas. METHODS AND MATERIALS: A total of 18 patients with histologically proven sarcoma treated with PABR from January 2020 to October 2023 were retrospectively reviewed. The primary endpoints were symptomatic and structural response rates. Secondary endpoints were overall survival, freedom from local progression, freedom from distant progression, and acute and late toxicity rates. RESULTS: All patients had tumours ≥5 cm with a median tumour volume of 985 cc, and the most common symptom was pain. The median age is 72.5 years and 44.5 % were ECOG 2-3. The most common regimen used was 20 Gy in 5 fractions with an intratumoral boost dose of 50 Gy (83.3 %). After a median follow-up of 11 months, 88.9 % of patients exhibited a partial response with a mean absolute tumour volume reduction of 49.5 %. All symptomatic patients experienced symptom improvement. One-year OS, FFLP and FFDP were 61 %, 83.3 % and 34.8 %, respectively. There were no grade 3 or higher toxicities. CONCLUSION: PABR for bulky, unresectable sarcomas appears to be safe and may provide good symptomatic response, tumour debulking, and local control. Further study is underway.

Polyamine transport inhibition and cisplatin synergistically enhance tumor control through oxidative stress in murine head and neck cancer models.

Background: Surgery and/or platinum-based chemoradiation remain standard of care for patients with head and neck squamous cell carcinoma (HNSCC). While these therapies are effective in a subset of patients, a substantial proportion experience recurrence or treatment resistance. As cisplatin mediates cytotoxicity through oxidative stress while polyamines play a role in redox regulation, we posited that combining cisplatin with polyamine transport inhibitor, AMXT-1501, would increase oxidative stress and tumor cell death in HNSCC cells. Methods: Cell proliferation was measured in syngeneic mouse HNSCC cell lines treated with cisplatin ± AMXT-1501. Synergy was determined by administering cisplatin and AMXT-1501 at a ratio of 1:10 to cancer cells in vitro . Cancer cells were transferred onto mouse flanks to test the efficacy of treatments in vivo . Reactive oxygen species (ROS) were measured. Cellular apoptosis was measured with flow cytometry using Annexin V/PI staining. High-performance liquid chromatography (HPLC) was used to quantify polyamines in cell lines. Cell viability and ROS were measured in the presence of exogenous cationic amino acids. Results: The combination of cisplatin and AMXT-1501 synergize in vitro on HNSCC cell lines. In vivo combination treatment resulted in tumor growth inhibition greater than either treatment individually. The combination treatment increased ROS production and induced apoptotic cell death. HPLC revealed the synergistic mechanism was independent of intracellular polyamine levels. Supplementation of cationic amino acids partially rescued cancer cell viability and reduced ROS. Conclusion: AMXT-1501 enhances the cytotoxic effects of cisplatin in vitro and in vivo in aggressive HNSCC cell lines through a polyamine-independent mechanism.

Interrogation of T Cell-Enriched Tumors Reveals Prognostic and Immunotherapeutic Implications of Polyamine Metabolism.

Metabolic features of the tumor microenvironment (TME) antagonize anti-tumor immunity. We hypothesized that T cell infiltrated tumors with a known antigen should exhibit superior clinical outcomes, though some fare worse given unfavorable metabolic features leveraging T cell-infiltrated (Thi), human papillomavirus-related (HPV+) head and neck squamous cell carcinomas (HNSC) to test this hypothesis. Expression of 2,520 metabolic genes were analyzed among Thi HPV+ HNSCs stratified by high-risk molecular subtype. RNAseq data from The Cancer Genome Atlas (TCGA; 10 cancer types), single cell RNAseq data, and an immunotherapy-treated melanoma cohort were used to test the association between metabolic gene expression and clinical outcomes and contribution of tumor versus stromal cells to metabolic gene expression. Polyamine (PA) metabolism genes were overexpressed in high-risk, Thi HPV+ HNSCs. Genes involved in PA biosynthesis and transport were associated with T cell infiltration, recurrent or persistent cancer, overall survival status, primary site, molecular subtype, and MYC genomic alterations. PA biogenesis gene sets were associated with tumor intrinsic features while myeloid cells in HPV+ HNSCs were enriched in PA catabolism, regulatory, transport, putrescine, and spermidine gene set expression. PA gene set expression also correlated with IFNγ or cytotoxic T cell ssGSEA scores across TCGA tumor types. PA transport ssGSEA scores were associated with poor survival whereas putrescine ssGSEA scores portended better survival for several tumor types. Thi melanomas enriched in PA synthesis or combined gene set expression exhibited worse anti-PD-1 responses. These data address hurdles to anti-tumor immunity warranting further investigation of divergent polyamine metabolism in the TME.

Long-term Patterns of Failure and the Value of Blood Prognostic Markers in Anal Cancers Treated With Intensity-Modulated Radiation Therapy.

BACKGROUND: To analyze the long-term outcomes and prognostic value of hematological parameters in anal cancer patients receiving intensity-modulated radiation therapy (IMRT). MATERIALS: Hospital records of consecutive patients with anal squamous cell carcinoma who received curative-intent IMRT according to a standardized contouring protocol between 2010 and 2020 were reviewed. Locoregional failure-free survival (LRFS), distant metastasis-free survival (DMFS), progression-free survival (PFS), and overall survival (OS) were estimated using the Kaplan-Meier method. Coverage of locoregional recurrences by the initial IMRT volumes were assessed. The prognostic value of pretreatment blood counts for PFS and OS were determined using Cox regression analysis. RESULTS: A total of 166 patients were analyzed with a median follow-up of 3.3 years. Forty-six percent and 54% of patients had Stage I-II and IIIA-B cancers, respectively. The 5-year LRFS, DMFS, PFS and OS were 81%, 89%, 65% and 76% respectively. Grade ≥ 3 toxicity occurred in 5% of patients. Of all patients who relapsed, 70% had only locoregional recurrence as first site of failure. Ninety percent of locoregional recurrences were in-field. Hemoglobin, neutrophil and platelet counts were associated with PFS on univariable analysis, but only cancer stage and p16 status remained prognostic on multivariable analysis. Patients with more advanced cancer stages also had higher baseline neutrophil counts. Performance status and neutrophil counts were prognostic for OS on multivariable analysis. CONCLUSION: This study affirms the long-term efficacy and safety of IMRT. Treatment resistance, rather than radiation geographic miss, is a major issue underpinning locoregional recurrences. Pretreatment blood counts were not validated to be independently prognostic for disease recurrence.

Polyamines drive myeloid cell survival by buffering intracellular pH to promote immunosuppression in glioblastoma.

Glioblastoma is characterized by the robust infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs). It is not fully understood how TAMCs survive in the acidic tumor microenvironment to cause immunosuppression in glioblastoma. Metabolic and RNA-seq analysis of TAMCs revealed that the arginine-ornithine-polyamine axis is up-regulated in glioblastoma TAMCs but not in tumor-infiltrating CD8+ T cells. Active de novo synthesis of highly basic polyamines within TAMCs efficiently buffered low intracellular pH to support the survival of these immunosuppressive cells in the harsh acidic environment of solid tumors. Administration of difluoromethylornithine (DFMO), a clinically approved inhibitor of polyamine generation, enhanced animal survival in immunocompetent mice by causing a tumor-specific reduction of polyamines and decreased intracellular pH in TAMCs. DFMO combination with immunotherapy or radiotherapy further enhanced animal survival. These findings indicate that polyamines are used by glioblastoma TAMCs to maintain normal intracellular pH and cell survival and thus promote immunosuppression during tumor evolution.

Dual targeting of polyamine synthesis and uptake in diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine glioma (DIPG) is an incurable malignant childhood brain tumor, with no active systemic therapies and a 5-year survival of less than 1%. Polyamines are small organic polycations that are essential for DNA replication, translation and cell proliferation. Ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme in polyamine synthesis, is irreversibly inhibited by difluoromethylornithine (DFMO). Herein we show that polyamine synthesis is upregulated in DIPG, leading to sensitivity to DFMO. DIPG cells compensate for ODC1 inhibition by upregulation of the polyamine transporter SLC3A2. Treatment with the polyamine transporter inhibitor AMXT 1501 reduces uptake of polyamines in DIPG cells, and co-administration of AMXT 1501 and DFMO leads to potent in vitro activity, and significant extension of survival in three aggressive DIPG orthotopic animal models. Collectively, these results demonstrate the potential of dual targeting of polyamine synthesis and uptake as a therapeutic strategy for incurable DIPG.

Inhibition of polyamine synthesis and uptake reduces tumor progression and prolongs survival in mouse models of neuroblastoma.

Amplification of the MYCN oncogene is associated with an aggressive phenotype and poor outcome in childhood neuroblastoma. Polyamines are highly regulated essential cations that are frequently elevated in cancer cells, and the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase 1 (ODC1), is a direct transcriptional target of MYCN. Treatment of neuroblastoma cells with the ODC1 inhibitor difluoromethylornithine (DFMO), although a promising therapeutic strategy, is only partially effective at impeding neuroblastoma cell growth due to activation of compensatory mechanisms resulting in increased polyamine uptake from the surrounding microenvironment. In this study, we identified solute carrier family 3 member 2 (SLC3A2) as the key transporter involved in polyamine uptake in neuroblastoma. Knockdown of SLC3A2 in neuroblastoma cells reduced the uptake of the radiolabeled polyamine spermidine, and DFMO treatment increased SLC3A2 protein. In addition, MYCN directly increased polyamine synthesis and promoted neuroblastoma cell proliferation by regulating SLC3A2 and other regulatory components of the polyamine pathway. Inhibiting polyamine uptake with the small-molecule drug AMXT 1501, in combination with DFMO, prevented or delayed tumor development in neuroblastoma-prone mice and extended survival in rodent models of established tumors. Our findings suggest that combining AMXT 1501 and DFMO with standard chemotherapy might be an effective strategy for treating neuroblastoma.

Credentialing of radiotherapy centres in Australasia for TROG 09.02 (Chisel), a Phase III clinical trial on stereotactic ablative body radiotherapy of early stage lung cancer.

OBJECTIVE: A randomised clinical trial comparing stereotactic ablative body radiotherapy (SABR) with conventional radiotherapy for early stage lung cancer has been conducted in Australia and New Zealand under the auspices of the TransTasman Radiation Oncology Group (NCT01014130). We report on the technical credentialing program as prerequisite for centres joining the trial. METHODS: Participating centres were asked to develop treatment plans for two test cases to assess their ability to create plans according to protocol. Dose delivery in the presence of inhomogeneity and motion was assessed during a site visit using a phantom with moving inserts. RESULTS: Site visits for the trial were conducted in 16 Australian and 3 New Zealand radiotherapy facilities. The tests with low density inhomogeneities confirmed shortcomings of the AAA algorithm for dose calculation. Dose was assessed for a typical treatment delivery including at least one non-coplanar beam in a stationary and moving phantom. This end-to-end test confirmed that all participating centres were able to deliver stereotactic ablative body radiotherapy with the required accuracy while the planning study demonstrated that they were able to produce acceptable plans for both test cases. CONCLUSION: The credentialing process documented that participating centres were able to deliver dose as required in the trial protocol. It also gave an opportunity to provide education about the trial and discuss technical issues such as four-dimensional CT, small field dosimetry and patient immobilisation with staff in participating centres. Advances in knowledge: Credentialing is an important quality assurance tool for radiotherapy trials using advanced technology. In addition to confirming technical competence, it provides an opportunity for education and discussion about the trial.

NOX2 deficiency alters macrophage phenotype through an IL-10/STAT3 dependent mechanism: implications for traumatic brain injury.

BACKGROUND: NADPH oxidase (NOX2) is an enzyme system that generates reactive oxygen species (ROS) in microglia and macrophages. Excessive ROS production is linked with neuroinflammation and chronic neurodegeneration following traumatic brain injury (TBI). Redox signaling regulates macrophage/microglial phenotypic responses (pro-inflammatory versus anti-inflammatory), and NOX2 inhibition following moderate-to-severe TBI markedly reduces pro-inflammatory activation of macrophages/microglia resulting in concomitant increases in anti-inflammatory responses. Here, we report the signaling pathways that regulate NOX2-dependent macrophage/microglial phenotype switching in the TBI brain. METHODS: Bone marrow-derived macrophages (BMDMs) prepared from wildtype (C57Bl/6) and NOX2 deficient (NOX2-/-) mice were treated with lipopolysaccharide (LPS; 10 ng/ml), interleukin-4 (IL-4; 10 ng/ml), or combined LPS/IL-4 to investigate signal transduction pathways associated with macrophage activation using western immunoblotting and qPCR analyses. Signaling pathways and activation markers were evaluated in ipsilateral cortical tissue obtained from adult male wildtype and NOX2-/- mice that received moderate-level controlled cortical impact (CCI). A neutralizing anti-IL-10 approach was used to determine the effects of IL-10 on NOX2-dependent transitions from pro- to anti-inflammatory activation states. RESULTS: Using an LPS/IL-4-stimulated BMDM model that mimics the mixed pro- and anti-inflammatory responses observed in the injured cortex, we show that NOX2-/- significantly reduces STAT1 signaling and markers of pro-inflammatory activation. In addition, NOX2-/- BMDMs significantly increase anti-inflammatory marker expression; IL-10-mediated STAT3 signaling, but not STAT6 signaling, appears to be critical in regulating this anti-inflammatory response. Following moderate-level CCI, IL-10 is significantly increased in microglia/macrophages in the injured cortex of NOX2-/- mice. These changes are associated with increased STAT3 activation, but not STAT6 activation, and a robust anti-inflammatory response. Neutralization of IL-10 in NOX2-/- BMDMs or CCI mice blocks STAT3 activation and the anti-inflammatory response, thereby demonstrating a critical role for IL-10 in regulating NOX2-dependent transitions between pro- and anti-inflammatory activation states. CONCLUSIONS: These studies indicate that following TBI NOX2 inhibition promotes a robust anti-inflammatory response in macrophages/microglia that is mediated by the IL-10/STAT3 signaling pathway. Thus, therapeutic interventions that inhibit macrophage/microglial NOX2 activity may improve TBI outcomes by not only limiting pro-inflammatory neurotoxic responses, but also enhancing IL-10-mediated anti-inflammatory responses that are neuroprotective.

Tyrosine kinase inhibition reverses TDP-43 effects on synaptic protein expression, astrocytic function and amino acid dis-homeostasis.

The trans-activating response of DNA/RNA-binding protein (TDP)-43 pathology is associated with many neurodegenerative diseases via unknown mechanisms. Here, we use a transgenic mouse model over-expressing human wild-type neuronal TDP-43 to study the effects of TDP-43 pathology on glutamate metabolism and synaptic function. We found that neuronal TDP-43 over-expression affects synaptic protein expression, including Synapsin I, and alters surrounding astrocytic function. TDP-43 over-expression is associated with an increase in glutamate and γ-amino butyric acid and reduction of glutamine and aspartate levels, indicating impairment of presynaptic terminal. TDP-43 also decreases tricarboxylic acid cycle metabolism and induces oxidative stress via lactate accumulation. Neuronal TDP-43 does not alter microglia activity or significantly changes systemic and brain inflammatory markers compared to control. We previously demonstrated that brain-penetrant tyrosine kinase inhibitors (TKIs), nilotinib and bosutinib, reduce TDP-43-induced cell death in transgenic mice. Here, we show that TKIs reverse the effects of TDP-43 on synaptic proteins, increase astrocytic function and restore glutamate and neurotransmitter balance in TDP-43 mice. Nilotinib, but not bosutinib, reverses mitochondrial impairment and oxidative metabolism. Taken together, these data suggest that TKIs can attenuate TDP-43 toxicity and improve synaptic and astrocytic function, independent of microglial or other inflammatory effects. In conclusion, our data demonstrate novel mechanisms of the effects of neuronal TDP-43 over-expression on synaptic protein expression and alteration of astrocytic function.

Polypathology and dementia after brain trauma: Does brain injury trigger distinct neurodegenerative diseases, or should they be classified together as traumatic encephalopathy?

Neuropathological studies of human traumatic brain injury (TBI) cases have described amyloid plaques acutely after a single severe TBI, and tau pathology after repeat mild TBI (mTBI). This has helped drive the hypothesis that a single moderate to severe TBI increases the risk of developing late-onset Alzheimer's disease (AD), while repeat mTBI increases the risk of developing chronic traumatic encephalopathy (CTE). In this review we critically assess this position-examining epidemiological and case control human studies, neuropathological evidence, and preclinical data. Epidemiological studies emphasize that TBI is associated with the increased risk of developing multiple types of dementia, not just AD-type dementia, and that TBI can also trigger other neurodegenerative conditions such as Parkinson's disease. Further, human post-mortem studies on both single TBI and repeat mTBI can show combinations of amyloid, tau, TDP-43, and Lewy body pathology indicating that the neuropathology of TBI is best described as a 'polypathology'. Preclinical studies confirm that multiple proteins associated with the development of neurodegenerative disease accumulate in the brain after TBI. The chronic sequelae of both single TBI and repeat mTBI share common neuropathological features and clinical symptoms of classically defined neurodegenerative disorders. However, while the spectrum of chronic cognitive and neurobehavioral disorders that occur following repeat mTBI is viewed as the symptoms of CTE, the spectrum of chronic cognitive and neurobehavioral symptoms that occur after a single TBI is considered to represent distinct neurodegenerative diseases such as AD. These data support the suggestion that the multiple manifestations of TBI-induced neurodegenerative disorders be classified together as traumatic encephalopathy or trauma-induced neurodegeneration, regardless of the nature or frequency of the precipitating TBI.

Profile of ceritinib in the treatment of ALK+ metastatic non-small-cell lung cancer.

Lung cancer has become one of the leading causes of death in both men and women in the United States, with approximately 230,000 new cases and 160,000 deaths each year. Approximately 80% of lung cancer patients are diagnosed with non-small-cell lung cancer (NSCLC), a subset of epithelial lung cancers that are generally insensitive to chemotherapy. An estimated 3%-7% of NSCLC patients harbor tumors containing anaplastic lymphoma kinase (ALK) gene rearrangement as an oncogenic driver. Subsequent development of the first-generation tyrosine kinase inhibitor crizotinib demonstrated substantial initial ALK+-tumor regression, yet ultimately displayed resistance in treated patients. The recently approved tyrosine kinase inhibitor ceritinib has been shown to be an effective antitumor agent against crizotinib-naïve and -resistant ALK+-NSCLC patients. In this review, we will provide an overview of biology and management of ALK+-NSCLC with a special focus on clinical development of ceritinib.

Polyamine blockade promotes antitumor immunity.

The levels of polyamines are elevated in neoplastic lesions as compared with normal tissues, and cancer cells tend to manifest a robust dependence on these compounds for proliferation and survival. We have recently demonstrated that a novel approach to polyamine depletion suppresses tumor growth in a T cell-dependent manner, highlighting a poorly appreciated role of polyamines as strong modulators of antitumor immune responses.

Polyamine-blocking therapy reverses immunosuppression in the tumor microenvironment.

Correcting T-cell immunosuppression may unleash powerful antitumor responses; however, knowledge about the mechanisms and modifiers that may be targeted to improve therapy remains incomplete. Here, we report that polyamine elevation in cancer, a common metabolic aberration in aggressive lesions, contributes significantly to tumor immunosuppression and that a polyamine depletion strategy can exert antitumor effects that may also promote immunity. A polyamine-blocking therapy (PBT) that combines the well-characterized ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO) with AMXT 1501, a novel inhibitor of the polyamine transport system, blocked tumor growth in immunocompetent mice but not in athymic nude mice lacking T cells. PBT had little effect on the proliferation of epithelial tumor cells, but it increased the number of apoptotic cells. Analysis of CD45(+) tumor immune infiltrates revealed that PBT decreased levels of Gr-1(+)CD11b(+) myeloid suppressor cells and increased CD3(+) T cells. Strikingly, in a model of neoadjuvant therapy, mice administered with PBT one week before surgical resection of engrafted mammary tumors exhibited resistance to subsequent tumor rechallenge. Collectively, our results indicate that therapies targeting polyamine metabolism do not act exclusively as antiproliferative agents, but also act strongly to prevent immune escape by the tumor. PBT may offer a general approach to heighten immune responses in cancer.

AMXT-1501, a novel polyamine transport inhibitor, synergizes with DFMO in inhibiting neuroblastoma cell proliferation by targeting both ornithine decarboxylase and polyamine transport.

Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in many forms of cancer including NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested in NB. We propose that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501 will result in enhanced NB growth inhibition. Single and combination drug treatments were conducted on three NB cell lines. DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 µM in NB. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G1 cell cycle arrest. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hr suggested apoptosis. The combination treatment depleted intracellular polyamine pools and decreased intracellular ATP, further verifying growth inhibition. Given the current lack of effective therapies for patients with relapsed/refractory NB and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB.

Traumatic brain injury in aged animals increases lesion size and chronically alters microglial/macrophage classical and alternative activation states.

Traumatic brain injury (TBI) causes chronic microglial activation that contributes to subsequent neurodegeneration, with clinical outcomes declining as a function of aging. Microglia/macrophages (MG/Mɸ) have multiple phenotypes, including a classically activated, proinflammatory (M1) state that might contribute to neurotoxicity, and an alternatively activated (M2) state that might promote repair. In this study we used gene expression, immunohistochemical, and stereological analyses to show that TBI in aged versus young mice caused larger lesions associated with an M1/M2 balance switch and increased numbers of reactive (bushy and hypertrophic) MG/Mɸ in the cortex, hippocampus, and thalamus. Chitinase3-like 3 (Ym1), an M2 phenotype marker, displayed heterogeneous expression after TBI with amoeboid-like Ym1-positive MG/Mɸ at the contusion site and ramified Ym1-positive MG/Mɸ at distant sites; this distribution was age-related. Aged-injured mice also showed increased MG/Mɸ expression of major histocompatibility complex II and NADPH oxidase, and reduced antioxidant enzyme expression which was associated with lesion size and neurodegeneration. Thus, altered relative M1/M2 activation and an nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase)-mediated shift in redox state might contribute to worse outcomes observed in older TBI animals by creating a more proinflammatory M1 MG/Mɸ activation state.

Visual cues as a surrogate for tactile feedback during robotic-assisted laparoscopic prostatectomy: posterolateral margin rates in 1340 consecutive patients.

OBJECTIVE: To analyse consecutive cases of robotic-assisted laparoscopic prostatectomy (RALP), present the incidence of nerve-sparing-related positive surgical margins (SM+), include visual cues that might assist in smoothly changing to the robotic platform, and discuss the scientific rationale for 'intersensory integration' which might explain the 'reverse Braille' phenomenon, i.e. the ability to feel when vision is greatly enhanced, as the lack of tactile feedback during RALP is often cited as a disadvantage of robotic surgery, interfering with a surgeon's ability to make intraoperative oncological decisions. PATIENTS AND METHODS: Data from 1340 consecutive patients undergoing RALP from one institution were analysed and trends for positive posterolateral SM+ (PLSM+) were correlated with oncological variables before and after RALP. A sample of patient slides were reviewed by a extramural pathologist. Multivariate regression modelling was used to compare the projected rates of PLSM+ vs the actual rate, given the effect of a conscious effort to use visual cues. Finally, video recordings of the procedure were systematically reviewed and correlated with anatomical and histopathological images in an integrated session involving the surgeon and the pathology team. RESULTS: The incidence of PLSM+ was 2.1%, which gradually declined to 1.0% in the last 100 patients. The reduction in PLSM+ occurred despite an increased rate of high-risk tumours operated on during this period. Forecasting analysis showed that the actual PLSM+ rate declined by half in the most recent 1000 patients, due to an integrated effort involving the use of visual cues during surgery. The following visual cues were considered important; appreciation of periprostatic (lateral prostatic) fascial compartments; colour and texture of the tissue; periprostatic veins as a landmark for athermal dissection; signs of inflammation; and a freely separating bloodless plane showing loose shiny areolar tissue. CONCLUSION: Adapting to the robotic platform is easy and there is no compromise of the oncological safety of this procedure. Experienced surgeons can use visual cues to assist during nerve-sparing RALP and achieve low PLSM+ rates.

24S-hydroxycholesterol effects on lipid metabolism genes are modeled in traumatic brain injury.

Membrane damage during traumatic brain injury (TBI) alters the brain homeostasis of cholesterol and other lipids. Cholesterol 24S-hydroxylase (Cyp46) is a cholesterol metabolic enzyme that is increased after TBI. Here, we systematically examined the effects of the enzymatic product of Cyp46, 24S-hydroxycholesterol, on the cholesterol regulatory genes, SREBP-1 and 2, their posttranslational regulation, and their effects on gene transcription. 24S-hydroxycholesterol increased levels of SREBP-1 mRNA and full-length protein but did not change levels of cleaved SREBP-1, consistent with the role of 24-hydroxycholesterol as an LXR agonist. In contrast, 24S-hydroxycholesterol decreased levels of LXR-independent SREBP-2 mRNA, full-length protein, and SREBP-2 active cleavage product. We examined the downstream effects of changes to these lipid regulatory factors by studying cholesterol and fatty acid synthesis genes. In neuroblastoma cells, 24S-hydroxycholesterol decreased mRNA levels of the cholesterol synthesis genes HMG CoA reductase, squalene synthase, and FPP synthase but did not alter levels of the mRNA of fatty acid synthesis genes acetyl CoA carboxylase or fatty acid synthase. After TBI, as after 24S-hydroxycholesterol treatment in vitro, SREBP-1 mRNA levels were increased while SREBP-2 mRNA levels were decreased. Also similar to the in vitro results with 24S-hydroxycholesterol, HMG CoA reductase and squalene synthase mRNA levels were significantly decreased. Fatty acid synthase mRNA levels were not altered but acetyl CoA carboxylase mRNA levels were significantly decreased. Thus, changes to transcription of cholesterol synthesis genes after TBI were consistent with increases in Cyp46 activity, but changes to fatty acid synthesis genes must be regulated by other mechanisms.

The cytoplasmic adaptor protein X11alpha and extracellular matrix protein Reelin regulate ApoE receptor 2 trafficking and cell movement.

The goal of this study was to determine the effect of X11alpha on ApoE receptor 2 (ApoEr2) trafficking and the functional significance of this interaction on cell movement in MCF 10A epithelial cells. We found that X11alpha increased surface levels of ApoEr2 by 64% compared to vector control, as determined by surface protein biotinylation. To examine the functional significance of this effect, we tested whether ApoEr2 played a novel role in cell movement in a wound-healing assay. We found that overexpression of ApoEr2 in MCF 10A cells increased cell migration velocity by 87% (P<0.01, n=4) compared to GFP control. Cotransfection of X11alpha had an additive effect on average velocity compared to ApoEr2 alone (13%; P<0.05, n=4). In addition, we tested whether ApoEr2 ligands altered the effect of ApoEr2 on cell movement. We found that treatment with concentrated medium containing the extracellular matrix protein Reelin, but not control medium, further increased the velocity of ApoEr2- but not APP-transfected cells (20%; P<0.001, n=4). Similarly, Reelin treatment increased cell velocity in the presence of ApoEr2 and X11alpha (10%; P<0.05, n=4). In the present study, we are the first to demonstrate that ApoEr2 regulates cell movement, and both X11alpha and Reelin enhance this effect.