Heat stress: A risk factor for skin carcinogenesis

BACKGROUND: The incidence of skin cancer in Australia has increased rapidly in the last few decades. Ultraviolet radiation (UV) is a major risk factor for skin carcinogenesis. UV, particularly the UVB spectrum, causes formation of cyclobutane pyrimidine dimers (CPD) in cellular DNA. Persistent and incorrectly repaired CPDs lead to DNA mutations and consequently, formation of cutaneous lesions. Interestingly, recent epidemiological studies have shown a significant increase in skin cancer incidence in geographical locations with high environmental temperatures. Thus, heat stress may potentiate the effects of UV exposure and act as an additional risk factor for skin cancer. Previous studies in mice have shown that repeated and concurrent exposure to UVB and heat stress, increases the rate and incidence of cutaneous tumour formation relative to UVB alone. However, the effects of UVB plus heat on human epidermal cells have yet to be determined. Furthermore, the exact mechanisms responsible for the observed effects of heat stress need to be characterised in skin keratinocytes to increase knowledge of its risk in skin cancer. Heat stress induces upregulation of heat shock proteins (HSPs), particularly HSP72 and HSP90 which are known to affect the activity of the p53 protein. Furthermore, heat stress has been linked with increased Sirtuin1 (SIRT1) protein activity. SIRT1 is an important histone deacetylase that helps maintain chromosomal integrity but can also induce post-translational modifications of the p53 protein. By mediating deacetylation of the p53 protein, SIRT1 can diminish the ability of p53 to bind to its downstream gene targets. The p53 protein is an integral mediator of the cellular stress response in skin cells, particularly keratinocytes. Thus, impairment of p53 transcription factor functions could compromise the ability of epidermal cells to mount an appropriate response to DNA damage. Moreover, loss of p53 function may induce survival of cells harbouring DNA lesions. We hypothesise, therefore, that exposure to UVB plus heat induces survival of DNA damaged keratinocytes and that these cells escape apoptosis surveillance as a result of heat-mediated alteration to the p53 signalling pathway. Thus, exposure to heat stress could exacerbate the carcinogenic effects of UV and increase the risk of skin tumour formation in humans AIMS: In this study, we aimed to determine whether repeated exposure to UVB followed immediately by heat stress (39°C) has a more damaging effect on human keratinocytes than UVB alone. In particular, we assessed the effects on DNA damage, apoptosis, cell cycle and DNA repair. Furthermore, we aimed to unravel the mechanism through which heat mediates the survival of UVB DNA-damaged keratinocytes, focusing on the effects on the p53 signalling pathway. MATERIALS AND METHODOLOGY: Primary adult human epidermal keratinocytes (NHEK) and ex vivo punch biopsies of normal human skin called NativeSkin® (Genoskin, France), were used as experimental models for this study. A UV cabinet fitted with a TLUVB Narrowband lamp (Philips, GERMANY), with a spectral output of 290 -315 nm, was used to administer UVB irradiation at a dose of 1 KJ/m². Heat stress involved culture in a normal CO2 incubator, with temperature maintained at 39°C for three hours. The temperature used in the experiments was based on previous measurements of skin surface temperature of open cut miners, who are prone to intense heat stress, in the Pilbara region of Western Australia. For UVB plus heat exposures, cells and skin models were sequentially exposed to 1 KJ/m2 of UVB, (at room temperature), followed immediately by 3 hours incubation at 39°C once per day, for four consecutive days. Unexposed skin models and NHEK, maintained at 37ºC, were used as experimental controls. Cell proliferation, apoptosis and whole genome expression profiles were analysed at four hours post day 4 exposure, to understand earlier events, and at 2 days post-exposure, to assess persistent outcomes of these exposures. Treated primary NHEK cells were counted in a Vi-CellTM Viability Analyser and the level of apoptosis for exposed primary cells was determined using Annexin V/Propidium Iodide apoptosis assay at 4 hours and 2 days post exposure. To determine the presence of DNA damage, total and active p53 protein, as well as total and active SIRT protein, in the skin models and primary NHEKs, immunohistochemistry and/or immunocytochemistry was performed. Skin FFPE and primary NHEKs were incubated with antibodies to thymine dimers (CPD, DNA damage) and p53 (total), acetylated p53-382 (active), SIRT1 (total) or SIRT1-p (active) antibodies. To measure apoptosis in skin, an anti-pan-cytokeratin marker was used to label keratinocytes and active-caspase-3 antibodies were used to identify apoptotic cells. To determine the expression of p53-downstream target genes at 4 hours, quantitative RT-PCR was performed using TaqMan probes for BAX, Survivin (BIRC5), ERCC1 and XPC genes, with Human 18S gene as the endogenous reference gene. Relative quantification of the expression levels of each transcript in each sample were calculated using the Delta-Delta CT method relative to untreated controls. A whole genome expression analysis was performed at 2 days postexposure using the Human HT-12 Expression v4 BeadChip (Illumina, USA). The Ingenuity Pathway Analysis (IPA) (Qiagen, USA) software was used to annotate the effects of altered gene expression on cell function and upstream signalling pathways. Two-way ANOVA was used to analyse differences across treatment groups, while parametric unpaired t-tests were used to detect differences between specific treatment groups in all experimental categories, i.e. proliferation, apoptosis and gene expression, with p-values RESULTS: Outcome 1 –Using ex vivo skin models and NHEKs, we show for the first time that UVB plus heat treated keratinocytes exhibit DNA damage, as observed after UVB treatment alone. However, apoptosis was significantly reduced, possibly as a result of inactivation of the p53-mediated stress response, in DNA damaged cells of UVB plus heat treated samples. Furthermore, whole genome expression and IPA upstream analysis showed that heat induces SIRT1 activation, which was confirmed via immunohistochemistry assays. Heat-induced SIRT1 expression was linked to a decrease in acetylated p53 and consequently, downregulation of p53-regulated pro-apoptotic and DNA damage repair genes. These results suggest that p53-mediated cell cycle arrest and apoptosis, known to be induced by UVB, are ablated with the addition of heat, leading to survival of DNA damaged cells after UVB plus heat treatment. Outcome 2 – We further confirmed that SIRT1 activation did not inhibit the transcription of the p53 protein but mediated deacetylation of p53, resulting in significant deregulation of expression of p53 downstream gene targets and decreased keratinocyte apoptosis in UVB plus heat treated samples. Importantly, chemical inhibition of SIRT1 by Ex-527, a known chemical inhibitor of SIRT1, in UVB plus heat exposed keratinocytes, resulted in reactivation of the p53 signalling pathway and increased apoptosis of DNA damaged keratinocytes. This clearly demonstrated the role of heat-mediated SIRT1 activation in the survival of DNA damaged keratinocytes after exposure to UVB plus heat. CONCLUSION: In this study, we showed that the efficiency of cellular stress response to UVB-induced DNA damage is diminished in the presence of heat and, for the first time, provide a molecular mechanism that explains these effects. With the novel use of an ex vivo human skin model, this study showed that heat stress prevents human keratinocytes, damaged by UV irradiation, from undergoing apoptosis and/or necrosis. We found UV plus heat exposure mediates SIRT1 activation which has been found to induce deacetylation of p53 and, consequently, the inactivation of the p53 signalling pathway. SIRT1 inhibition precluded the downregulation of p53 signalling by UV plus heat exposure, restoring apoptosis levels to those observed in UVB-only exposures. Thus, we demonstrated that SIRT1 activation is the main molecular mechanism driving UVB plus heat-induced survival of DNA damaged keratinocytes. Overall, the results of this study suggest that by allowing the survival of DNA damaged keratinocytes, via induction of SIRT1 activation, heat stress can exacerbate the carcinogenic effects of UVB radiation. Exposure to heat stress, in addition to UV, could therefore increase the accumulation of mutations in keratinocytes, possibly leading to the transformation of normal cells into pre-cancerous cells. Further research is warranted to determine the role of UVB plus heat in skin cancer pathogenesis. Such knowledge could be utilised in public health campaigns to decrease risk, particularly for people exposed to combinations of these environmental hazards in workplaces such as in the mining, construction and petroleum industries

Circulating tumour DNA (ctDNA) as a biomarker in metachronous melanoma and colorectal cancer- a case report

Background: Circulating tumour DNA (ctDNA) has emerged as a promising blood-based biomarker for monitoring disease status of patients with advanced cancers. The presence of ctDNA in the blood is a result of biological processes, namely tumour cell apoptosis and/or necrosis, and can be used to monitor different cancers by targeting cancer-specific mutation. Case presentation: We present the case of a 67 year old Caucasian male that was initially treated with BRAF inhibitors followed by anti-CTLA4 and then anti-PD1 immunotherapy for metastatic melanoma but later developed colorectal cancer. The kinetics of ctDNA derived from each cancer type were monitored targeting BRAF V600R (melanoma) and KRAS G13D (colon cancer), specifically reflected the status of the patient\u27s tumours. In fact, the discordant pattern of BRAF and KRAS ctDNA was significantly correlated with the clinical response of melanoma to pembrolizumab treatment and progression of colorectal cancer noted by PET and/or CT scan. Based on these results, ctDNA can be used to specifically clarify disease status of patients with metachronous cancers. Conclusions: Using cancer-specific mutational targets, we report here for the first time the efficacy of ctDNA to accurately provide a comprehensive outlook of the tumour status of two different cancers within one patient. Thus, ctDNA analysis has a potential clinical utility to delineate clinical information in patients with multiple cancer types

Liquid biopsy in ovarian cancer using circulating tumor DNA and cells: Ready for prime time?

Liquid biopsies hold the potential to inform cancer patient prognosis and to guide treatment decisions at the time when direct tumor biopsy may be impractical due to its invasive nature, inaccessibility and associated complications. Specifically, circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have shown promising results as companion diagnostic biomarkers for screening, prognostication and/or patient surveillance in many cancer types. In ovarian cancer (OC), CTC and ctDNA analysis allow comprehensive molecular profiling of the primary, metastatic and recurrent tumors. These biomarkers also correlate with overall tumor burden and thus, they provide minimally-invasive means for patient monitoring during clinical course to ascertain therapy response and timely treatment modification in the context of disease relapse. Here, we review recent reports of the potential clinical value of CTC and ctDNA in OC, expatiating on their use in diagnosis and prognosis. We critically appraise the current evidence, and discuss the issues that still need to be addressed before liquid biopsies can be implemented in routine clinical practice for OC management

Circulating tumour DNA (ctDNA) as a liquid biopsy for melanoma

Circulating tumour DNA (ctDNA) has emerged as a promising blood-based biomarker for monitoring disease status of patients with advanced cancers. In melanoma, ctDNA has been shown to have clinical value as an alternative tumour source for the detection clinically targetable mutations for the assessment of response to therapy. This review provides a critical summary of the evidence that gives credence to the utility of ctDNA as a biomarker for monitoring of disease status in advanced melanoma and the steps required for its implementation into clinical settings

SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes

Background Exposure to heat stress after UVB irradiation induces a reduction of apoptosis, resulting in survival of DNA damaged human keratinocytes. This heat-mediated evasion of apoptosis appears to be mediated by activation of SIRT1 and inactivation of p53 signalling. In this study, we assessed the role of SIRT1 in the inactivation of p53 signalling and impairment of DNA damage response in UVB plus heat exposed keratinocytes. Results Activation of SIRT1 after multiple UVB plus heat exposures resulted in increased p53 deacetylation at K382, which is known to affect its binding to specific target genes. Accordingly, we noted decreased apoptosis and down regulation of the p53 targeted pro-apoptotic gene BAX and the DNA repair genes ERCC1 and XPC after UVB plus heat treatments. In addition, UVB plus heat induced increased expression of the cell survival gene Survivin and the proliferation marker Ki67. Notably, keratinocytes exposed to UVB plus heat in the presence of the SIRT1 inhibitor, Ex-527, showed a similar phenotype to those exposed to UV alone; i.e. an increase in p53 acetylation, increased apoptosis and low levels of Survivin. Conclusion This study demonstrate that heat-induced SIRT1 activation mediates survival of DNA damaged keratinocytes through deacetylation of p53 after exposure to UVB plus heat

Detectable ctDNA at the time of treatment cessation of ipilimumab and nivolumab for toxicity predicts disease progression in advanced melanoma patients

BackgroundImmune checkpoint inhibition (ICI) has led to unprecedented outcomes for melanoma patients but is associated with toxicity. ICI resumption after high grade irAEs poses a significant challenge in the clinical management of melanoma patients and there are no biomarkers that can help identify patients that might benefit from resuming treatment. This study aims to determine if circulating tumor DNA (ctDNA) levels at the time of treatment-limiting irAE could guide treatment decisions in this clinical context.MethodsThis is a retrospective exploratory biomarker study from 34 patients treated with combination ICI for stage IV melanoma. Patients had a treatment-limiting toxicity and a baseline plasma collection prior to commencing ICI and within 6 weeks of stopping therapy. Blood samples were tested for ctDNA at baseline and cessation therapy.ResultsMedian progression free survival (PFS) and overall survival (OS) have not been reached (24-month PFS rate 54% and OS rate 72.3%). PD occurred in 47% (16/34) of patients. Median PFS with detectable ctDNA from plasma collected at the time of toxicity was 6.5 months while not reached (NR) with undetectable levels (HR: 4.0, 95% CI 0.95-17.5, p=0.0023). Median OS with detectable ctDNA at cessation for toxicity was 19.4 months and NR for undetectable ctDNA (HR: 3.9, 95%CI 20.8-18.6, p=0.024). Positive ctDNA at the time of cessation was highly specific (specificity 0.94, 95% CI 0.74-0.99, PPV 0.88, 95% CI 0.53-0.99). However, ctDNA negativity has low sensitivity as a predictor of ongoing disease control (sensitivity 0.437, 95% CI 0.23-0.67). Notably, 4/9 (44%) ctDNA negative patients who had disease progression had brain only disease progression.ConclusionsUndetectable ctDNA and CR on imaging after stopping immunotherapy for toxicity results in high rates of long-term durable control. For patients with immunotherapy related toxicity, who have persistent ctDNA at 8 – 12 weeks, the risk of disease progression is significant

Heat-mediated reduction of apoptosis in UVB-damaged keratinocytes in vitro and in human skin ex vivo

Background: UV radiation induces significant DNA damage in keratinocytes and is a known risk factor for skin carcinogenesis. However, it has been reported previously that repeated and simultaneous exposure to UV and heat stress increases the rate of cutaneous tumour formation in mice. Since constant exposure to high temperatures and UV are often experienced in the environment, the effects of exposure to UV and heat needs to be clearly addressed in human epidermal cells. Methods: In this study, we determined the effects of repeated UVB exposure 1kJ/m2 followed by heat (39°C) to human keratinocytes. Normal human ex vivo skin models and primary keratinocytes (NHEK) were exposed once a day to UVB and/or heat stress for four consecutive days. Cells were then assessed for changes in proliferation, apoptosis and gene expression at 2days post-exposure, to determine the cumulative and persistent effects of UV and/or heat in skin keratinocytes. Results: Using ex vivo skin models and primary keratinocytes in vitro, we showed that UVB plus heat treated keratinocytes exhibit persistent DNA damage, as observed with UVB alone. However, we found that apoptosis was significantly reduced in UVB plus heat treated samples. Immunohistochemical and whole genome transcription analysis showed that multiple UVB plus heat exposures induced inactivation of the p53-mediated stress response. Furthermore, we demonstrated that repeated exposure to UV plus heat induced SIRT1 expression and a decrease in acetylated p53 in keratinocytes, which is consistent with the significant downregulation of p53-regulated pro-apoptotic and DNA damage repair genes in these cells. Conclusion: Our results suggest that UVB-induced p53-mediated cell cycle arrest and apoptosis are reduced in the presence of heat stress, leading to increased survival of DNA damaged cells. Thus, exposure to UVB and heat stress may act synergistically to allow survival of damaged cells, which could have implications for initiation skin carcinogenesis. © 2016 The Author(s)

Human leucocyte antigen genotype association with the development of immune-related adverse events in patients with non-small cell lung cancer treated with single agent immunotherapy

Introduction: Biomarkers that predict the risk of immune-mediated adverse events (irAEs) among patients with non-small cell lung cancer (NSCLC) may reduce morbidity and mortality associated with these treatments. Methods: We carried out high resolution human leucocyte antigen (HLA)-I typing on 179 patients with NSCLC treated with anti-program death (PD)-1/program death ligand (PDL)-1. Toxicity data were collected and graded as per common terminology criteria for adverse event (CTCAE) v5.0. We used 14.8-week for landmark analysis to address lead-time bias to investigate the correlation between HLA-I/II zygosity, supertypes and alleles with irAE. Furthermore, we assessed the association for irAE with clinical benefit rate (CBR), progression-free survival (PFS) and overall survival (OS). Results: Homozygosity at one or more HLA-I loci, but not HLA-II, was associated with a reduced risk of irAE (relative risk (RR) = 0.61, 95% CI 0.33–0.95, P = 0.035) especially pneumonitis or any grade 3 toxicity. Patients with HLA-A03 supertype had a higher risk of developing irAE (RR = 1.42, 95% CI 1.02–2.01, P = 0.039). The occurrence of any irAE was significantly associated with improved CBR (RR = 1.48, P \u3c 0.0001), PFS (HR = 0.45, P = 0.0003) and OS (HR = 0.34, P \u3c 0.0001). Conclusions: Homozygosity at one or more HLA-I loci may serve as biomarker to predict patients who are unlikely to experience severe irAEs among patients with NSCLC and treated with anti-PD1/PDL1, but less likely to derive clinical benefit. Patients with HLA-I homozygous might benefit from additional therapy

Stopping targeted therapy for complete responders in advanced BRAF mutant melanoma

BRAF inhibitors revolutionised the management of melanoma patients and although resistance occurs, there is a subgroup of patients who maintain durable disease control. For those cases with durable complete response (CR) it is not clear whether it is safe to cease therapy. Here we identified 13 patients treated with BRAF +/− MEK inhibitors, who cease therapy after prolonged CR (median = 34 months, range 20–74). Recurrence was observed in 3/13 (23%) patients. In the remaining 10 patients with sustained CR off therapy, the median follow up after discontinuation was 19 months (range 8–36). We retrospectively measured ctDNA levels using droplet digital PCR (ddPCR) in longitudinal plasma samples. CtDNA levels were undetectable in 11/13 cases after cessation and remained undetectable in patients in CR (10/13). CtDNA eventually became detectable in 2/3 cases with disease recurrence, but remained undetectable in 1 patient with brain only progression. Our study suggests that consideration could be given to ceasing targeted therapy in the context of prolonged treatment, durable response and no evidence of residual disease as measured by ctDNA

Circulating tumour DNA in advanced melanoma patients ceasing PD1 Inhibition in the absence of disease progression

Immunotherapy is an important and established treatment option for patients with advanced melanoma. Initial anti-PD1 trials arbitrarily defined a two-year treatment duration, but a shorter treatment duration may be appropriate. In this study, we retrospectively assessed 70 patients who stopped anti-PD1 therapy in the absence of progressive disease (PD) to determine clinical outcomes. In our cohort, the median time on treatment was 11.8 months. Complete response was attained at time of anti-PD1 discontinuation in 61 (87%). After a median follow up of 34.2 months (range: 2–70.8) post discontinuation, 81% remained disease free. Using ddPCR, we determine the utility of circulating tumour DNA (ctDNA) to predict progressive disease after cessation (n = 38). There was a significant association between presence of ctDNA at cessation and disease progression (p = 0.012, Fisher’s exact test) and this conferred a negative and positive predictive value of 0.82 (95% CI: 0.645–0.930) and 0.80 (95% CI 0.284–0.995), respectively. Additionally, dichotomised treatment-free survival in patients with or without ctDNA at cessation was significantly longer in the latter group (p \u3c 0.001, HR: 0.008, 95% CI: 0.001–0.079). Overall, our study confirms that durable disease control can be achieved with cessation of therapy in the absence of disease progression and undetectable ctDNA at cessation was associated with longer treatment-free survival