Nigel Kirkham

Evidence of Melanoma in Wild Marine Fish Populations

The increase in reports of novel diseases in a wide range of ecosystems, both terrestrial and marine, has been linked to many factors including exposure to novel pathogens and changes in the global climate. Prevalence of skin cancer in particular has been found to be increasing in humans, but has not been reported in wild fish before. Here we report extensive melanosis and melanoma (skin cancer) in wild populations of an iconic, commercially-important marine fish, the coral trout Plectropomus leopardus. The syndrome reported here has strong similarities to previous studies associated with UV induced melanomas in the well-established laboratory fish model Xiphophorus. Relatively high prevalence rates of this syndrome (15%) were recorded at two offshore sites in the Great Barrier Reef Marine Park (GBRMP). In the absence of microbial pathogens and given the strong similarities to the UV-induced melanomas, we conclude that the likely cause was environmental exposure to UV radiation. Further studies are needed to establish the large scale distribution of the syndrome and confirm that the lesions reported here are the same as the melanoma in Xiphophorus, by assessing mutation of the EGFR gene, Xmrk. Furthermore, research on the potential links of this syndrome to increases in UV radiation from stratospheric ozone depletion needs to be completed.

Prognostic Impact of p62 Expression in Cutaneous Malignant Melanoma

Accumulating evidence suggests autophagy, the principle catabolic process for lysosomal degradation of surplus macromolecules (Roy and Debnath 2010) is fundamental to tumourigenesis. Impaired autophagy results in accumulation of cellular breakdown products, increased oxidative stress and neoplastic transformation (Mathew et al. 2009); whilst efficient autophagy facilitates metastatic tumour survival through sustained metabolic activity (Roy and Debnath 2010). Consequently, the currently accepted view is that autophagy suppresses growth early in tumour development but promotes tumour survival at later stages (Garber 2011). p62 (also known as sequestosome-1/SQSTM1) is a multidomain adaptor protein transporting ubiquitinated proteins during autophagy (Moscat and Diaz-Meco 2009); as such, p62 plays a role in selective autophagic degradation of a number of substrates (Komatsu and Ichimura 2010; Ichimura and Komatsu 2010). Normally, p62 is broken down along with its cargo within the autophagolysosome. However, impaired autophagy is accompanied by p62 accumulation, resulting in large p62/ubiquitinated protein aggregates (Komatsu and Ichimura 2010); a process thought to be a key factor in tumourigenesis (Moscat and Diaz-Meco 2009). The present biomarker discovery study aimed to define immunohistochemical p62 expression as a prognostic biomarker in a retrospective cohort comprising 29 melanocytic naevi and 121 primary cutaneous melanomas (Supplementary Methods and Table S1). In keeping with the “autophagy paradox”, we hypothesised p62 levels would be elevated in melanoma compared to naevi, with the highest levels detected in early stage disease where conditions represent a pro-tumourgenic environment. Comparison of median p62 expression levels between naevi and all melanomas revealed a significant increase in expression levels in melanoma (14.82%) compared to naevi (0.51%; Mann-Whitney U, P <0.0001) (Fig 1a), and a step-wise increase in median p62 expression levels with localised tumour development (0.51% in naevi to 44.5% in AJCC II disease, Mann-Whitney P < 0.0001) (Fig 1b). A relative decrease in median p62 expression (to 10.25%) was then observed in primary tumours from patients with metastatic stage III/IV disease (Stage II vs Stage III/IV Mann-Whitney P = 0.005) (Fig 1b). This biphasic expression in primary melanomas thus mirrors the “autophagy paradox”. Figure 1 p62 expression in melanoma is consistent with the ‘autophagy paradox’ in cancer To determine the efficacy of p62 expression levels at identifying high risk tumours at diagnosis, comparison of localised and metastatic disease (eventual AJCC stages I/II versus stages III/IV) revealed significantly lower median p62 expression in the metastatic cohort (10.25% versus 26.94% in AJCC I/II; Mann-Whitney, P = 0.016). p62 levels were visibly bimodal, with a Wilcoxon signed-rank test confirming that 20% expression was an appropriate cut-point for undertaking survival curve analysis, and as such statistical modelling was based on tumour p62 expression above (“high p62”) or below 20% (“low p62”). Univariate analysis in all tumours was used to assess disease outcome over a 7-year follow up period and revealed a modest, yet statistically significant, reduction in disease free survival (DFS) in patients with “low p62” tumours (40.9% of patients in the “low p62” group developed metastases compared to 21.8% in “high p62” tumours (Log-Rank (Mantel-Cox) Test P = 0.03, HR 1.66 (95% CI 1.03 – 2.69) (Fig 2a). Figure 2 p62 as a potential prognostic biomarker in melanoma Comparable univariate analysis of melanoma specific mortality (MSM) in the whole melanoma cohort revealed a non-significant trend towards an increased MSM in patients with “low p62” tumours (MSM 24.24% “low p62” tumours, versus 14.55% in “high p62” tumours (Log-Rank (Mantel-Cox) Test P = 0.18, HR 1.5 (95% CI 0.83 – 2.74). Univariate analysis of other pre-hypothesised risk factors for disease progression were calculated (Supplementary Methods and Table S2); as expected there was a significant increased risk of metastases with increasing Breslow depth and tumour ulceration in line with AJCC staging criteria (Balch et al. 2009). Univariate analysis of tumours pre-stratified to AJCC stage II at diagnosis revealed low p62 expression levels were associated with a trend for worse DFS; with 67.74% of patients in the “low p62” cohort developing a metastasis within 7-years compared to only 39.13% in the “high p62” group (Log-Rank (Mantel-Cox) Test) P = 0.06, HR 1.7 (95% CI 0.97 – 2.96) (Fig 2b). A similar trend was seen in AJCC stage I disease, (Log-Rank (Mantel-Cox) Test P = 0.38, HR 1.53 (95% CI 0.58 – 4.09). MSM followed an identical trend, with “low p62” AJCC stage II tumours resulting in a higher mortality rate compared to “high p62” AJCC II tumours (MSM 35.48% versus 21.74% respectively (Log-Rank (Mantel-Cox) Test P = 0.27, HR 1.51 (95% CI 0.72 – 3.2). Comparison of mean p62 expression revealed no association with Breslow depth or tumour ulceration (Figure S1) and Cox proportional hazards analysis undertaken to assess whether the predictive effects of p62 may be partially attributable to covariates (Supplementary Methods and Table S3) further supported p62 expression levels as an independent stratifying prognostic variable (p < 9E-7), representing biologically distinct processes to those included in AJCC staging alone. As a marker of autophagic activity, the p62 status of the current melanoma cohort fits with the “autophagy paradox”; with the highest levels of p62 expression found in early, localised disease (AJCC stages I and II) in keeping with pro-tumourigenic dysfunctional autophagy. However, tumour cells with increased levels of autophagic activity (either through reactivation or retention of this function following tumourigenesis) are more likely to metastasise by harnessing pro-survival autophagy. Independently of its role in autophagy, p62 expression may be regulated by other signalling mechanisms including via NF-κB activation and interaction with TRAF6 and caspase-8 (Mathew et al. 2009; Komatsu and Ichimura 2010). Therefore, the differential expression of p62 within different AJCC stages of melanoma cannot be assumed to result entirely from impaired autophagic activity. Nevertheless, results from the present study add to the growing body of evidence supporting the introduction of autophagy inhibitors to chemotherapeutic regimes in melanoma, stage I trials of which are currently underway (Komatsu et al 2007), for which p62 expression will likely provide a useful stratification criterion. Crucially however, p62 represents a potential candidate biomarker that may provide additional prognostic information to AJCC disease stage. Further validation in an independent retrospective and ongoing prospective cohort will determine the prognostic significance and effect size of p62 and its application as a biomarker for refining personalised therapies, ultimately translating into improved clinical outcome for melanoma patients.

Methotrexate Toxicity During Treatment of Chronic Plaque Psoriasis: A Case Report and Review of the Literature

Methotrexate continues to be one of the most widely used systemic immunosuppressive agents in dermatology. In addition to the important, well-characterized adverse effects such as hepatotoxicity and myelosuppression, methotrexate may induce a number of rare cutaneous adverse events including methotrexate-induced ulceration. We present a case of methotrexate-induced cutaneous ulceration in a patient with chronic plaque psoriasis occurring during long-standing methotrexate therapy. Withdrawal of the drug and appropriate skin care led to rapid healing of the ulceration and the agent was later safely reintroduced for the ongoing management of the patient’s chronic plaque psoriasis. Review of the literature demonstrates cases of this important rare adverse event, primarily occurring in patients with chronic plaque psoriasis, induced by triggers such as accidental overdose or introduction of an interacting agent. Cutaneous ulceration typically precedes other markers of toxicity. Active treatment with folinic acid (calcium leucovorin) may be required. Early recognition, prompt cessation of methotrexate, and appropriate treatment minimizes morbidity. Dermatologists need to be alert to the possibility of cutaneous adverse events associated with methotrexate therapy, aware of potential drug interactions, and confident in the management of methotrexate toxicity.

Atypical findings in three patients with Pai syndrome and literature review

Pai syndrome is a rare disorder characterized by congenital nasal or facial polyp, midline cleft lip, pericallosal lipoma, ocular anomalies, and normal neuropsychological development. Here, we report on three patients with Pai syndrome and atypical findings: temporal triangular alopecia, posterior lenticonus, bilateral palatal pits, bifid uvula, hypospadias, sacral dimple, true tracheal bronchus, and epilepsy. Thirty‐three cases of Pai syndrome have been described so far. We present a review of the previously reported cases and suggest modified diagnostic criteria for Pai syndrome.

Coma Blisters in 2 Children on Anticonvulsant Medication

Blister formation and eccrine sweat gland necrosis have been recognized to occur in states of impaired consciousness and were first reported following barbiturate intoxication. Their etiology is complex and cannot simply be explained by pressure effects. Now that barbiturates are less frequently used, clinicians are likely to be less aware of the phenomenon of coma blister formation; however, newer drugs have also been associated with the occurrence of coma blisters. We describe 2 new associations of coma blisters and anticonvulsants in children. In the first child, blisters recurred on multiple occasions along with obtundation and edema. Our aims are to alert clinicians to the occurrence of coma blisters in children sedated on anticonvulsant medications and to report the new finding of recurrent coma blisters.

WIPI-1: a novel autophagy inhibitory protein in melanoma

Bioscience, University Sydney, AUPhotoaged skin is characterised by profound remodelling of the extracellular matrix (ECM). We have previously shown that UV chromophore (Cys, His, Phe, Trp and Tyr)-rich proteins are differentially degraded by UV radiation (UVR). We tested the hypothesises that; (i) UV-mediated ECM degradation occurs via reactive oxygen species (ROS), by exposing chromophore-rich fibronectin and chromophore-poor tropoelastin to UVR in depleted-O2 and D2O environments and (ii) exogenous ROS mediates differential degradation of the same proteins. As the photodynamic production of ROS can be inhibited in depleted-O2 environments we exposed purified fibronectin to a broadband UVB (TL-12: 50 and 500mJ/cm2) in ambient and depleted-O2 conditions by bubbling with N2. Solutions were analysed by reducing SDS-PAGE. Irradiation of fibronectin in ambient-O2 (50 and 500mJ/cm2) caused dose-dependent aggregation (5 and 83 fold changes in the optical density (OD) of the aggregate at 50 and 500mJ/cm2 respectively), fibronectin aggregation was abrogated (68 fold lower OD) in the depleted-O2 compared with ambient-O2. Conversely, fibronectin aggregation was increased 4 fold in D2O compared with H2O environments. UV-mediated changes to the molecular weight (Mw) of fibronectin were recapitulated by exposure to H2O2 (40mM for 2 hrs). In contrast, UVB irradiation in both H2O and D2O had no observable effect on the Mw of tropoelastin. Therefore, ROS may play an important role in the selective degradation of long-lived UV chromophore-rich ECM in both UV-exposed and protected ageing tissues. Research supported by Alliance Boots.

p62 as a biomarker of malignant melanoma

Bioscience, University Sydney, AUPhotoaged skin is characterised by profound remodelling of the extracellular matrix (ECM). We have previously shown that UV chromophore (Cys, His, Phe, Trp and Tyr)-rich proteins are differentially degraded by UV radiation (UVR). We tested the hypothesises that; (i) UV-mediated ECM degradation occurs via reactive oxygen species (ROS), by exposing chromophore-rich fibronectin and chromophore-poor tropoelastin to UVR in depleted-O2 and D2O environments and (ii) exogenous ROS mediates differential degradation of the same proteins. As the photodynamic production of ROS can be inhibited in depleted-O2 environments we exposed purified fibronectin to a broadband UVB (TL-12: 50 and 500mJ/cm2) in ambient and depleted-O2 conditions by bubbling with N2. Solutions were analysed by reducing SDS-PAGE. Irradiation of fibronectin in ambient-O2 (50 and 500mJ/cm2) caused dose-dependent aggregation (5 and 83 fold changes in the optical density (OD) of the aggregate at 50 and 500mJ/cm2 respectively), fibronectin aggregation was abrogated (68 fold lower OD) in the depleted-O2 compared with ambient-O2. Conversely, fibronectin aggregation was increased 4 fold in D2O compared with H2O environments. UV-mediated changes to the molecular weight (Mw) of fibronectin were recapitulated by exposure to H2O2 (40mM for 2 hrs). In contrast, UVB irradiation in both H2O and D2O had no observable effect on the Mw of tropoelastin. Therefore, ROS may play an important role in the selective degradation of long-lived UV chromophore-rich ECM in both UV-exposed and protected ageing tissues. Research supported by Alliance Boots.