Simon Wilkins

Targeting the Progression of Parkinson’s Disease

By the time a patient first presents with symptoms of Parkinson’s disease at the clinic, a significant proportion (50-70%) of the cells in the substantia nigra (SN) has already been destroyed. This degeneration progresses until, within a few years, most of the cells have died. Except for rare cases of familial PD, the initial trigger for cell loss is unknown. However, we do have some clues as to why the damage, once initiated, progresses unabated. It would represent a major advance in therapy to arrest cell loss at the stage when the patient first presents at the clinic. Current therapies for Parkinson’s disease focus on relieving the motor symptoms of the disease, these unfortunately lose their effectiveness as the neurodegeneration and symptoms progress. Many experimental approaches are currently being investigated attempting to alter the progression of the disease. These range from replacement of the lost neurons to neuroprotective therapies; each of these will be briefly discussed in this review. The main thrust of this review is to explore the interactions between dopamine, alpha synuclein and redox-active metals. There is abundant evidence suggesting that destruction of SN cells occurs as a result of a self-propagating series of reactions involving dopamine, alpha synuclein and redox-active metals. A potent reducing agent, the neurotransmitter dopamine has a central role in this scheme, acting through redox metallo-chemistry to catalyze the formation of toxic oligomers of alpha-synuclein and neurotoxic metabolites including 6-hydroxydopamine. It has been hypothesized that these feed the cycle of neurodegeneration by generating further oxidative stress. The goal of dissecting and understanding the observed pathological changes is to identify therapeutic targets to mitigate the progression of this debilitating disease.

Insertion/deletion polymorphisms in the ΔNp63 promoter are a risk factor for bladder exstrophy epispadias complex.

Bladder exstrophy epispadias complex (BEEC) is a severe congenital anomaly; however, the genetic and molecular mechanisms underlying the formation of BEEC remain unclear. TP63, a member of TP53 tumor suppressor gene family, is expressed in bladder urothelium and skin over the external genitalia during mammalian development. It plays a role in bladder development. We have previously shown that p63−/− mouse embryos developed a bladder exstrophy phenotype identical to human BEEC. We hypothesised that TP63 is involved in human BEEC pathogenesis. RNA was extracted from BEEC foreskin specimens and, as in mice, ΔNp63 was the predominant p63 isoform. ΔNp63 expression in the foreskin and bladder epithelium of BEEC patients was reduced. DNA was sequenced from 163 BEEC patients and 285 ethnicity-matched controls. No exon mutations were detected. Sequencing of the ΔNp63 promoter showed 7 single nucleotide polymorphisms and 4 insertion/deletion (indel) polymorphisms. Indel polymorphisms were associated with an increased risk of BEEC. Significantly the sites of indel polymorphisms differed between Caucasian and non-Caucasian populations. A 12-base-pair deletion was associated with an increased risk with only Caucasian patients (p = 0.0052 Odds Ratio (OR) = 18.33), whereas a 4-base-pair insertion was only associated with non-Caucasian patients (p = 0.0259 OR = 4.583). We found a consistent and statistically significant reduction in transcriptional efficiencies of the promoter sequences containing indel polymorphisms in luciferase assays. These findings suggest that indel polymorphisms of the ΔNp63 promoter lead to a reduction in p63 expression, which could lead to BEEC.

Elemental bio-imaging using laser ablation-triple quadrupole-ICP-MS

Elemental bio-imaging (EBI) of trace metal distributions in tissue sections is typically performed by laser ablation-single quadrupole-inductively coupled plasma-mass spectrometry (LA-ICP-SQ-MS) in which the sensitivity of biologically relevant elements such as Fe may be inhibited by polyatomic interferences. The triple quadrupole ICP-MS (ICP-QQQ-MS) is designed to reduce interferences and was examined for its potential application to EBI. Three tune modes; a no-gas, an H 2 , and an O 2 /H 2 tune were developed and investigated for their suitability. All three performed well with high signal to noise ratios and low limits of detection. The O 2 /H 2 tune was applied to a prostate cancer biopsy sample, with high levels of Zn found in the area correlating with the cancer. The oxygen mass shift was applied to image P and Se in a mouse brain. The unique focussing properties of the ICP-QQQ-MS and the ability to use mixed cell gases provided sensitive analyses of all measured isotopes with the O 2 /H 2 tune, including those unaffected by mass shift reactions.

The novel compound PBT434 prevents iron mediated neurodegeneration and alpha-synuclein toxicity in multiple models of Parkinson's disease

Elevated iron in the SNpc may play a key role in Parkinson’s disease (PD) neurodegeneration since drug candidates with high iron affinity rescue PD animal models, and one candidate, deferirpone, has shown efficacy recently in a phase two clinical trial. However, strong iron chelators may perturb essential iron metabolism, and it is not yet known whether the damage associated with iron is mediated by a tightly bound (eg ferritin) or lower-affinity, labile, iron pool. Here we report the preclinical characterization of PBT434, a novel quinazolinone compound bearing a moderate affinity metal-binding motif, which is in development for Parkinsonian conditions. In vitro, PBT434 was far less potent than deferiprone or deferoxamine at lowering cellular iron levels, yet was found to inhibit iron-mediated redox activity and iron-mediated aggregation of α-synuclein, a protein that aggregates in the neuropathology. In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. We conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.

Colorectal Cancer Surgery in the Very Elderly: Nonagenarians.

BACKGROUND: Surgery in the very elderly is a topic that has not been well studied, despite the steady rise in this population. With the rise in this population, there is now discussion on the safety of surgery in this cohort for colorectal cancer. OBJECTIVE: The purpose of this study was to investigate elective and nonelective colorectal cancer surgery outcomes in patients aged ≥90 years at both private and public hospitals in Melbourne, Victoria, Australia. DESIGN: This was a retrospective analysis of patients aged ≥90 years who were included in the prospectively maintained Cabrini Monash University Department of Surgery colorectal neoplasia database for patients entered between January 2010 and February 2015. Comorbidity, ASA score, acuity of surgery, treatment, mortality, morbidity, and survival were analyzed. SETTINGS: This study was conducted in a tertiary referral hospital. PATIENTS: A total of 48 patients were identified from the database. The majority of these patients were women (58.0%), ASA score III to IV (91.7%), and treated in an elective setting (79.2%). The median age was 91.8 years. MAIN OUTCOME MEASURES: We measured 30-day mortality, 180-day mortality, and perioperative morbidity. RESULTS: Thirty-day mortality rate was 2.1%. The 180-day mortality rate was 10.4%. A total of 29.2% of patients had a perioperative complication. Median follow-up was 21 months (range, 13–54 months). In 180-day mortality, minimally invasive surgery was associated with a lower mortality rate vs open surgery (p = 0.043). Perioperative complications were associated with nonelective surgery (p = 0.045), open surgery procedures (p = 0.014), and higher stages of disease (p = 0.014). A total of 81.3% of patients were able to return home after surgery. LIMITATIONS: This was a retrospective study with the usual limitations; however, these have been minimized with the use of a high-quality, prospective data collection database. The median follow-up was 21 months. CONCLUSIONS: Colorectal surgery was generally safe for nonagenarians in this study. This study demonstrates that excellent outcomes can be achieved in a selected group. Additional prospective studies with larger numbers and 5-year follow-up are recommended.

ISL1 is a major susceptibility gene for classic bladder exstrophy and a regulator of urinary tract development

Previously genome-wide association methods in patients with classic bladder exstrophy (CBE) found association with ISL1, a master control gene expressed in pericloacal mesenchyme. This study sought to further explore the genetics in a larger set of patients following-up on the most promising genomic regions previously reported. Genotypes of 12 markers obtained from 268 CBE patients of Australian, British, German Italian, Spanish and Swedish origin and 1,354 ethnically matched controls and from 92 CBE case-parent trios from North America were analysed. Only marker rs6874700 at the ISL1 locus showed association (p = 2.22 × 10−08). A meta-analysis of rs6874700 of our previous and present study showed a p value of 9.2 × 10−19. Developmental biology models were used to clarify the location of ISL1 activity in the forming urinary tract. Genetic lineage analysis of Isl1-expressing cells by the lineage tracer mouse model showed Isl1-expressing cells in the urinary tract of mouse embryos at E10.5 and distributed in the bladder at E15.5. Expression of isl1 in zebrafish larvae staged 48 hpf was detected in a small region of the developing pronephros. Our study supports ISL1 as a major susceptibility gene for CBE and as a regulator of urinary tract development.

Complete Pathological Response After Neoadjuvant Long-course Chemoradiotherapy for Rectal Cancer and Its Relationship to the Degree of T3 Mesorectal Invasion

BACKGROUND: Many studies have shown significantly improved outcomes (reduced local recurrence and improved overall survival) for patients achieving a complete pathological response from neoadjuvant chemoradiotherapy. OBJECTIVE: This study aimed to document the complete pathological response rate and outcomes in patients receiving preoperative long-course chemoradiotherapy stratified for the extent of T3 mesorectal invasion measured on preoperative imaging. DESIGN: This is a retrospective study of prospectively collected data, of patients with rectal cancer in the Cabrini Monash University Department of Surgery colorectal neoplasia database, incorporating data from Cabrini Hospital and The Alfred Hospital, identifying patients entered between January 2010 and June 2014. PATIENTS AND SETTINGS: One hundred eighteen patients with T3 rectal cancer met the selection criteria for the study; 26 achieved complete pathological response (22%). MAIN OUTCOME MEASURES: Outcomes in terms of complete pathological response and oncological outcomes such as disease-free and overall survival were analyzed. RESULTS: Patients with complete pathological response had significantly less preoperative invasion than those with no complete pathological response (p < 0.001). Depth of invasion was the only variable associated with complete pathological response (p < 0.002), and the likelihood of complete pathological response decreased by 35% for every millimeter of invasion. Complete pathological response was associated with increased disease-free survival (p = 0.018) and a lower risk of cancer progression (p = 0.046). Depth of invasion was associated with an increased risk of death after surgery; HR increased by 1.07 (95% CI, 1.00–1.15) for each 1-mm increase in invasion. LIMITATIONS: This was a retrospective study with the usual limitations, although these were minimized through the use of a clinician-driven prospective database. CONCLUSIONS: The smaller the degree of T3 invasion, the higher the chance of achieving complete pathological response (up to 35%), which is associated with improved disease-free and overall survival. A higher complete pathological response rate is observed in early T3 disease in comparison with more extensive T3 invasion.

The Effect of Diabetes on the Perioperative Outcomes of Colorectal Cancer Surgery Patients

There are approximately 1.3 million patients in Australia with diabetes. Conflicting reports exist in the literature as to the effect of diabetes on the outcomes of colorectal cancer patients. We hypothesized that patients with diabetes would have poorer perioperative outcomes, and that diabetes was an independent risk factor for both 30-day mortality and perioperative morbidity. The aim of this study was to assess the impact of diabetes on perioperative colorectal cancer surgery outcomes, as compared to a diabetes-free reference population, and to examine factors affecting perioperative risk. We conducted an analysis of a prospectively collected, clinician-led colorectal cancer database of patients from 2010–2015. Patients with diabetes were compared to patients without diabetes on a range of perioperative outcomes. Pearson χ-squared tests, Wilcoxon rank sum tests and t-tests were employed for univariate analyses. Confounding factors were controlled for by separate logistic and linear regression analyses. The Huber-White Sandwich Estimator was used to calculate robust standard errors. A total of 1725 patients were analysed over 1745 treatment episodes in the study period with 267 patients (268 episodes) with diabetes studied. Diabetes contributed to medical, surgical complications, and increased length of inpatient stay in univariate analyses. Multivariable analysis adjusted for variables independently associated with each outcome revealed that diabetes was an independent contributor to an increased risk of surgical complications, with no significant effect on medical complications, return to the operating room, 30-day mortality, or readmission within 30 days. In this study, where overall baseline morbidity and mortality levels are low, the effect of diabetes alone on perioperative surgical outcomes appears to be overstated with control of associated perioperative risk factors such as cardiac, renal and respiratory factors being more important.

Immunohistochemistry testing for mismatch repair deficiency in Stage 2 colon cancer: A cohort study of two cancer centres

BACKGROUND/OBJECTIVES Adjuvant chemotherapy for Stage II colon cancer offers a small (2-3%) overall survival benefit and is not universally recommended. Mismatch repair deficiency (dMMR) confers an improved prognosis identifying patients unlikely to benefit from adjuvant chemotherapy. The aim of this study was to investigate the use of dMMR immunohistochemistry in two major cancer treatment centres. METHODS Prospective data were collected on all patients with resected Stage II colon cancer between 2010 and 2015 across two large Australian hospitals. Data collected included patient demographics, tumour histology, dMMR immunohistochemistry, chemotherapy use, and outcomes. RESULTS All 355 patients (56.1% female, median age 81) with resected Stage 2 Colon cancer entered on to the surgical database were included in this analysis. MMR testing was performed on 167 patient samples (47%), most occurred post-2013 (73.1% vs. 26.9% patients). dMMR rates were 34.1%. 25 (7.3%) received adjuvant chemotherapy, with no patient >80 years receiving treatment. Presence of ≥2 high-risk feature increased the likelihood of adjuvant chemotherapy. Only 3.6% dMMR patients received chemotherapy; both were young with high-risk features. 27/288 (7.6%) patients (with follow up) relapsed, with 7 disease-free post-resection of metastatic disease, 9 are alive with metastatic disease, and 11 deceased. CONCLUSIONS Unlike clinical trial populations, Stage 2 colon cancer patients are often elderly, have high rates of dMMR tumours, are rarely offered chemotherapy, yet still have excellent outcomes. dMMR immunohistochemistry is being increasingly used to identify Stage 2 patients who do not require chemotherapy.

Role of lymph node yield and lymph node ratio in predicting outcomes in non-metastatic colorectal cancer: Lymph node yield and ratio for outcome prediction in colorectal cancer

Lymph node yield (LNY) of 12 or more in resection of colorectal cancer is recommended in current international guidelines. Although a low LNY (less than 12) is associated with poorer outcome in some studies, its prognostic value is unclear in patients with early‐stage colorectal or rectal cancer with a complete pathological response following neoadjuvant therapy. Lymph node ratio (LNR), which reflects the proportion of positive to total nodes obtained, may be more accurate in predicting outcome in stage III colorectal cancer. This study aimed to identify factors correlating with LNY and evaluate the prognostic role of LNY and LNR in colorectal cancer.