Ensuring a Safe and Qualitative Diagnostic Biopsy for Retroperitoneal Sarcomas

Retroperitoneal sarcomas represent one third of neoplasms in the retroperitoneum and as such are an important entity when evaluating masses in this area. They are often identified incidentally as they present with non-specific symptoms and are only detectable on physical exam when they have grown to a large size. This group of tumours is a challenge for physicians as it encompasses over 50 different histological subtypes and the course of treatment greatly depends on histopathological diagnosis. Biopsy of these lesions has recently become standard of care when evaluating a suspected retroperitoneal sarcoma. However, historically, there has been specula- tion over whether this practice promotes needle tract seeding resulting in local recurrence which has resulted in limited research on the topic. As such, there is a lack of literature describing the best parameters for a safe and effective biopsy of these lesions. Our ongoing research aims to identify biopsy parameters which yield a safe and qualitative diagnostic biopsy while minimizing complications and local recurrence with the goal of consistent and quality care for all patients presenting with retroperitoneal lesions. RÉSUMÉ Les sarcomes rétropéritonéaux constituent un tiers des néoplasmes du rétropéritoine, et représentent ainsi une entité importante lors de l’évaluation de masses dans cet espace. Ils sont souvent découverts fortuitement puisqu’ils se manifestent par des symptômes non spécifiques et sont seulement décelables à l’examen physique lorsqu’ils atteignent une taille considérable. Ce groupe de tumeurs représente un défi pour les médecins, car il comprend plus de 50 différents sous-types histologiques et le traitement dépend largement du diagnostic histopathologique. La biopsie de ces lésions est récemment devenue la norme en matière de soins pour l’évaluation d’un sarcome rétropéritonéal soupçonné. Toutefois, par le passé, certains ont suggéré que cette pratique puisse possiblement disséminer le cancer et causer une récidive locale, ce qui a limité la recherche sur le sujet. Ainsi, il existe un manque de littérature décrivant les paramètres optimaux pour effectuer une biopsie sécuritaire et efficace de ces lésions. Notre recherche en cours vise à identifier les paramètres de biopsie qui produisent une biopsie diagnostique sécuritaire et qualitative, tout en minimisant les complications et les risques de récidive locale, dans le but de fournir des soins uniformes et de haute qualité à tous les patients avec des lésions rétropéritonéales.

The JNK- and AKT/GSK3β- signaling pathways converge to regulate Puma induction and neuronal apoptosis induced by trophic factor deprivation.

The AKT, GSK3 and JNK family kinases have been implicated in neuronal apoptosis associated with neuronal development and several neurodegenerative conditions. However, the mechanisms by which these kinase pathways regulate apoptosis remain unclear. In this study we have investigated the role of these kinases in neuronal cell death using an established model of trophic factor deprivation induced apoptosis in cerebellar granule neurons. BCL-2 family proteins are known to be central regulators of apoptosis and we have determined that the pro-apoptotic family member Puma is transcriptionally up-regulated in trophic factor deprived neurons and that Puma induction is required for apoptosis in vitro and in vivo. Importantly, we demonstrate that Puma induction is dependent on both JNK activation and AKT inactivation. AKT is known to regulate a number of downstream pathways, however we have determined that PI3K-AKT inactivation induces Puma expression through a GSK3β-dependent mechanism. Finally we demonstrate that the JNK and AKT/GSK3β pathways converge to regulate FoxO3a-mediated transcriptional activation of Puma. In summary we have identified a novel and critical link between the AKT, GSK3β and JNK kinases and the regulation of Puma induction and suggest that this may be pivotal to the regulation of neuronal apoptosis in neurodegenerative conditions

The JNK and AKT/GSK3β pathways signal independently during potassium withdrawal in CGNs.

<p><b>A,</b> CGNs were maintained in high potassium medium (K25) or subjected to potassium withdrawal (K5) in the presence or absence of the JNK inhibitor SP600125 (10 µM) or the GSK3β inhibitor SB415286 (30 µM). Protein extracts were collected at 2, 4 and 6 hours and Phospho-ATF2, Phospho-c-Jun and ATF3 levels were analyzed by western blot. <b>B,</b> CGNs were subjected to potassium withdrawal in the presence or absence of 200 nM IGF-1. Protein extracts were collected after 6 hours and were analyzed for Phospho-ATF2, Phospho-c-Jun and ATF3 protein levels by western blot. <b>C,</b> CGNs were subjected to potassium withdrawal in the presence or absence of 10 µM SP600125 (SP) and protein extracts were collected after 6 hours and analyzed by western blot for Phospho-AKT and Phospho-GSK3β levels.</p

Puma is essential for potassium-withdrawal induced apoptosis in CGNs.

<p>CGNS derived Puma+/+ and Puma−/− littermates were maintained in high potassium medium (K25) or switched to low potassium medium (K5). <b>A,</b> The fraction of apoptotic cells was determined at 24 h by assessing nuclear morphology following Hoechst staining of Puma+/+ vs Puma−/− CGNs (n = 7, *p<0.01). <b>B,</b> Representative images of Hoechst stained wild type and Puma-deficient neurons 24 hours following potassium withdrawal. <b>C,</b> CGNs were stained with Mitotracker Red to assess mitochondrial membrane potential. The fraction of Mitotracker Red labeled neurons was determined 20 hours after potassium withdrawal and compared between genotypes (n = 4, *p<0.05). <b>D,</b> Cell lysates were collected 20 hours after potassium withdrawal and assayed for caspase-3-like activity. Caspase activity is reported as relative fluorescence units of cleaved caspase substrate and was compared between genotypes (n = 4,* p<0.05). <b>E,</b> CGNs derived from Bim+/+ and Bim−/− littermates were maintained in high potassium medium or switched to low potassium medium and the fraction of apoptotic cells was determined at 24 h (n = 5).</p

GSK3β is required for Puma induction in potassium withdrawal induced apoptosis.

<p>CGNs were switched to low potassium medium in the presence or absence of the GSK3α/β inhibitor SB415286 (SB, 30 µM) or the GSK3β specific inhibitor AR-A01 4418 (AR, 50 µM). <b>A,</b> RNA was collected six hours after potassium withdrawal and analyzed for Puma mRNA expression using qRT-PCR (n = 4, *p<0.05). <b>B,</b> Protein extracts were collected 8 hours after potassium withdrawal and Puma protein levels were analyzed by western blot. <b>C,</b> The fraction of apoptotic neurons was quantified after 24 hours by Hoechst staining (n = 3, *p<0.05).</p

JNK is required for Puma induction and potassium withdrawal induced neuronal apoptosis.

<p>After 7 days in culture CGNs were either maintained in high potassium medium or switched to low potassium medium with or without 10 µM SP600125 (SP). <b>A,</b> Puma mRNA levels were assessed by qRT-PCR 6 hours after potassium withdrawal and are reported as fold increase over K25 controls (n = 7, *p<0.05). <b>B,</b> Protein extracts were collected 8 hours after potassium withdrawal and Phospho-ATF2, Phospho-c-Jun, ATF3 and Puma protein levels were assessed by western blot. Calnexin was included as a loading control. <b>C,</b> The fraction of apoptotic neurons was determined 24 hours after potassium withdrawal by examining nuclear morphology in Hoechst stained cells (n = 5, *p<0.05).</p

Puma expression is induced by potassium withdrawal in cerebellar granule neurons.

<p>After 7 days in culture CGNs were either maintained in media containing 25 mM potassium (K25) or switched to low potassium medium containing 5 mM potassium (K5). <b>A,</b> RNA was harvested after 4, 6 and 8 hours and Puma, Bim and Hrk expression was analyzed by qRT-PCR. Expression was normalized to ribosomal S12 levels and is reported as fold increase over untreated controls (n = 4, p<0.05). <b>B,</b> Protein extracts were collected from CGNs derived from Puma+/+ and Puma−/− littermates or Bim+/+ and Bim−/− littermates 8 hours after potassium withdrawal and Puma and Bim protein levels were analyzed by western blot. Actin was included as a loading control.</p

PI3K/AKT inhibition induces Puma expression and Puma-dependent apoptosis in CGNs.

<p><b>A,</b> CGNs maintained in high potassium medium (K25) were treated with the PI3K inhibitor LY294002 (30 µM) or vehicle and protein extracts were collected after 12 hours and Phosph-AKT, Phospho-GSK3β and Puma protein levels were assessed by western blot. <b>B,</b> CGNs were infected with recombinant adenovirus expressing either CA-AKT or GFP at 10 MOI and after 7 days neurons were treated with or without LY294002 (30 µM). RNA was collected at 8 hours and Puma mRNA levels were quantified by qRT-PCR (n = 4, *p<0.05). <b>C,</b> CGNs derived from Puma+/+ and Puma−/− littermates were treated with or without 30 µM LY294002 (LY) and the fraction of apoptotic neurons was quantified after 24 hours by Hoechst staining.</p

Puma contributes to CGN apoptosis during postnatal development <i>in vivo</i>.

<p>Sagittal sections from the cerebellum of postnatal day 7 Puma+/+ and Puma−/− mice were TUNEL stained to detect apoptotic cells. A, Representative images showing Tunel labeling (brown DAB+ cells) of cerebellar sections from Puma+/+ and Puma−/− mice. B, The mean number of TUNEL positive cells in the internal granule layer (IGL) per field was determined for each animal and the data is presented as the mean +/− SEM for Puma+/+ and Puma−/− mouse pups (n = 4 mice of each genotype, *p<0.05).</p