Publications by authors named "Helen Rizos"

102 Publications

Circulating Tumor DNA Reflects Uveal Melanoma Responses to Protein Kinase C Inhibition.

Cancers (Basel) 2021 Apr 6;13(7). Epub 2021 Apr 6.

Department of Biomedical Sciences, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW 2109, Australia.

The prognosis for patients with UM is poor, and recent clinical trials have failed to prolong overall survival (OS) of these patients. Over 95% of UM harbor activating driver mutations, and this allows for the investigation of ctDNA. In this study, we investigated the value of ctDNA for adaptive clinical trial design in metastatic UM. Longitudinal plasma samples were analyzed for ctDNA in 17 metastatic UM patients treated with PKCi-based therapy in a phase 1 clinical trial setting. Plasma ctDNA was assessed using digital droplet PCR (ddPCR) and a custom melanoma gene panel for targeted next generation sequencing (NGS). Baseline ctDNA strongly correlated with baseline lactate dehydrogenase (LDH) ( < 0.001) and baseline disease burden ( = 0.002). Early during treatment (EDT) ctDNA accurately predicted patients with clinical benefit to PKCi using receiver operator characteristic (ROC) curves (AUC 0.84, [95% confidence interval 0.65-1.0, = 0.026]). Longitudinal ctDNA assessment was informative for establishing clinical benefit and detecting disease progression with 7/8 (88%) of patients showing a rise in ctDNA and targeted NGS of ctDNA revealed putative resistance mechanisms prior to radiological progression. The inclusion of longitudinal ctDNA monitoring in metastatic UM can advance adaptive clinical trial design.
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http://dx.doi.org/10.3390/cancers13071740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038771PMC
April 2021

Dosing of BRAK and MEK Inhibitors in Melanoma: No Point in Taking a Break.

Cancer Cell 2020 Dec;38(6):779-781

Faculty of Medicine, Health and Human Sciences, Macquarie University, NSW, Australia; Melanoma Institute Australia, NSW, Australia. Electronic address:

Resistance to BRAF/MEK inhibitor treatment occurs in most patients with advanced BRAF-mutant melanoma. Intermittent drug dosing had been proposed as a strategy to circumvent resistance. In a clinical trial published in Nature Medicine, continuous dosing of BRAF/MEK inhibitors produced superior progression-free survival compared to intermittent dosing in BRAF-mutant melanoma patients.
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http://dx.doi.org/10.1016/j.ccell.2020.11.010DOI Listing
December 2020

Detection of splicing variants in plasma-derived cell-free nucleic acids and extracellular vesicles of melanoma patients failing targeted therapy therapies.

Oncotarget 2020 Nov 3;11(44):4016-4027. Epub 2020 Nov 3.

School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia, Australia.

The analysis of plasma circulating tumour nucleic acids provides a non-invasive approach to assess disease burden and the genetic evolution of tumours in response to therapy. splicing variants are known to confer melanoma resistance to BRAF inhibitors. We developed a test to screen cell-free RNA (cfRNA) for the presence of splicing variants. Custom droplet digital PCR assays were designed for the detection of splicing variants p61, p55, p48 and p41 and then validated using RNA from cell lines carrying these variants. Evaluation of plasma from patients with reported objective response to BRAF/MEK inhibition followed by disease progression was revealed by increased circulating tumour DNA (ctDNA) in 24 of 38 cases at the time of relapse. Circulating splicing variants were detected in cfRNA from 3 of these 38 patients; two patients carried the BRAF p61 variant and one the p55 variant. In all three cases the presence of the splicing variant was apparent only at the time of progressive disease. p61 was also detectable in plasma of one of four patients with confirmed splicing variants in their progressing tumours. Isolation and analysis of RNA from extracellular vesicles (EV) from resistant cell lines and patient plasma demonstrated that splicing variants are associated with EVs. These findings indicate that in addition to plasma ctDNA, RNA carried by EVs can provide important tumour specific information.
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http://dx.doi.org/10.18632/oncotarget.27790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646833PMC
November 2020

Tumor MHC Expression Guides First-Line Immunotherapy Selection in Melanoma.

Cancers (Basel) 2020 Nov 14;12(11). Epub 2020 Nov 14.

Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW 2109, Australia.

Immunotherapy targeting T-cell inhibitory receptors, namely programmed cell death-1 (PD-1) and/or cytotoxic T-lymphocyte associated protein-4 (CTLA-4), leads to durable responses in a proportion of patients with advanced metastatic melanoma. Combination immunotherapy results in higher rates of response compared to anti-PD-1 monotherapy, at the expense of higher toxicity. Currently, there are no robust molecular biomarkers for the selection of first-line immunotherapy. We used flow cytometry to profile pretreatment tumor biopsies from 36 melanoma patients treated with anti-PD-1 or combination (anti-PD-1 plus anti-CTLA-4) immunotherapy. A novel quantitative score was developed to determine the tumor cell expression of antigen-presenting MHC class I (MHC-I) molecules, and to correlate expression data with treatment response. Melanoma MHC-I expression was intact in all tumors derived from patients who demonstrated durable response to anti-PD-1 monotherapy. In contrast, melanoma MHC-I expression was low in 67% of tumors derived from patients with durable response to combination immunotherapy. Compared to MHC-I high tumors, MHC-I low tumors displayed reduced T-cell infiltration and a myeloid cell-enriched microenvironment. Our data emphasize the importance of robust MHC-I expression for anti-PD-1 monotherapy response and provide a rationale for the selection of combination immunotherapy as the first-line treatment in MHC-I low melanoma.
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http://dx.doi.org/10.3390/cancers12113374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696726PMC
November 2020

Genetic Alterations in the INK4a/ARF Locus: Effects on Melanoma Development and Progression.

Biomolecules 2020 10 15;10(10). Epub 2020 Oct 15.

Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW 2109, Australia.

Genetic alterations in the (or ) locus have been reported in many cancer types, including melanoma; head and neck squamous cell carcinomas; lung, breast, and pancreatic cancers. In melanoma, loss of function CDKN2A alterations have been identified in approximately 50% of primary melanomas, in over 75% of metastatic melanomas, and in the germline of 40% of families with a predisposition to cutaneous melanoma. The CDKN2A locus encodes two critical tumor suppressor proteins, the cyclin-dependent kinase inhibitor p16 and the p53 regulator p14. The majority of CDKN2A alterations in melanoma selectively target p16 or affect the coding sequence of both p16 and p14. There is also a subset of less common somatic and germline INK4a/ARF alterations that affect p14, while not altering the syntenic p16 coding regions. In this review, we describe the frequency and types of somatic alterations affecting the CDKN2A locus in melanoma and germline CDKN2A alterations in familial melanoma, and their functional consequences in melanoma development. We discuss the clinical implications of CDKN2A inactivating alterations and their influence on treatment response and resistance.
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http://dx.doi.org/10.3390/biom10101447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602651PMC
October 2020

Circulating Tumor DNA Predicts Outcome from First-, but not Second-line Treatment and Identifies Melanoma Patients Who May Benefit from Combination Immunotherapy.

Clin Cancer Res 2020 Nov 16;26(22):5926-5933. Epub 2020 Oct 16.

School of Medical and Health Sciences, Edith Cowan University, Perth, Australia.

Purpose: We evaluated the predictive value of pretreatment ctDNA to inform therapeutic outcomes in patients with metastatic melanoma relative to type and line of treatment.

Experimental Design: Plasma circulating tumor DNA (ctDNA) was quantified in 125 samples collected from 110 patients prior to commencing treatment with immune checkpoint inhibitors (ICIs), as first- ( = 32) or second-line ( = 27) regimens, or prior to commencing first-line BRAF/MEK inhibitor therapy ( = 66). An external validation cohort included 128 patients commencing ICI therapies in the first- ( = 77) or second-line ( = 51) settings.

Results: In the discovery cohort, low ctDNA (≤20 copies/mL) prior to commencing therapy predicted longer progression-free survival (PFS) in patients treated with first-line ICIs [HR, 0.20; 95% confidence interval (CI) 0.07-0.53; < 0.0001], but not in the second-line setting. An independent cohort validated that ctDNA is predictive of PFS in the first-line setting (HR, 0.42; 95% CI, 0.22-0.83; = 0.006), but not in the second-line ICI setting. Moreover, ctDNA prior to commencing ICI treatment was not predictive of PFS for patients pretreated with BRAF/MEK inhibitors in either the discovery or validation cohorts. Reduced PFS and overall survival were observed in patients with high ctDNA receiving anti-PD-1 monotherapy, relative to those treated with combination anti-CTLA-4/anti-PD-1 inhibitors.

Conclusions: Pretreatment ctDNA is a reliable indicator of patient outcome in the first-line ICI treatment setting, but not in the second-line ICI setting, especially in patients pretreated with BRAF/MEK inhibitors. Preliminary evidence indicated that treatment-naïve patients with high ctDNA may preferentially benefit from combined ICIs.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2251DOI Listing
November 2020

Spatial and Temporal Changes in PD-L1 Expression in Cancer: The Role of Genetic Drivers, Tumor Microenvironment and Resistance to Therapy.

Int J Mol Sci 2020 Sep 27;21(19). Epub 2020 Sep 27.

Department of Biomedical Sciences, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney 2109, Australia.

Immunotherapies blocking immune inhibitory receptors programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) on T-cells have dramatically improved patient outcomes in a range of advanced cancers. However, the lack of response, and the development of resistance remain major obstacles to long-term improvements in patient outcomes. There is significant interest in the clinical use of biomarkers to improve patient selection, and the expression of PD-1 ligand 1 (PD-L1) is often reported as a potential biomarker of response. However, accumulating evidence suggests that the predictive value of PD-L1 expression in tumor biopsies is relatively low due, in part, to its complex biology. In this review, we discuss the biological consequences of PD-L1 expression by various cell types within the tumor microenvironment, and the complex mechanisms that regulate PD-L1 expression at the genomic, transcriptomic and proteomic levels.
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http://dx.doi.org/10.3390/ijms21197139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583014PMC
September 2020

Multiplex detection of ctDNA mutations in plasma of colorectal cancer patients by PCR/SERS assay.

Nanotheranostics 2020 25;4(4):224-232. Epub 2020 Aug 25.

Department of Molecular Sciences, Macquarie University, Sydney, Australia.

Molecular diagnostic testing of KRAS and BRAF mutations has become critical in the management of colorectal cancer (CRC) patients. Some progress has been made in liquid biopsy detection of mutations in circulating tumor DNA (ctDNA), which is a fraction of circulating cell-free DNA (cfDNA), but slow analysis for DNA sequencing methods has limited rapid diagnostics. Other methods such as quantitative PCR and more recently, droplet digital PCR (ddPCR), have limitations in multiplexed capacity and the need for expensive specialized equipment. Hence, a robust, rapid and facile strategy is needed for detecting multiple ctDNA mutations to improve the management of CRC patients. To address this significant problem, herein, we propose a new application of multiplex PCR/SERS (surface-enhanced Raman scattering) assay for the detection of ctDNA in CRC, in a fast and non-invasive manner to diagnose and stratify patients for effective treatment. To discriminate ctDNA mutations from wild-type cfDNA, allele-specific primers were designed for the amplification of three clinically important DNA point mutations in CRC including KRAS G12V, KRAS G13D and BRAF V600E. Surface-enhanced Raman scattering (SERS) nanotags were labelled with a short and specific sequence of oligonucleotide, which can hybridize with the corresponding PCR amplicons. The PCR/SERS assay was implemented by firstly amplifying the multiple mutations, followed by binding with multicolor SERS nanotags specific to each mutation, and subsequent enrichment with magnetic beads. The mutation status was evaluated using a portable Raman spectrometer where the fingerprint spectral peaks of the corresponding SERS nanotags indicate the presence of the mutant targets. The method was then applied to detect ctDNA from CRC patients under a blinded test, the results were further validated by ddPCR. The PCR/SERS strategy showed high specificity and sensitivity for genotyping CRC cell lines and plasma ctDNA, where as few as 0.1% mutant alleles could be detected from a background of abundant wild-type cfDNA. The blinded test using 9 samples from advanced CRC patients by PCR/SERS assay was validated with ddPCR and showed good consistency with pathology testing results. With ddPCR-like sensitivity yet at the convenience of standard PCR, the proposed assay shows great potential in sensitive detection of multiple ctDNA mutations for clinical decision-making.
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http://dx.doi.org/10.7150/ntno.48905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484630PMC
April 2021

Methylated circulating tumor DNA as a biomarker in cutaneous melanoma.

Melanoma Manag 2020 Jul 30;7(3):MMT46. Epub 2020 Jul 30.

Department of Biomedical Sciences, Faculty of Medicine, Health & Human Sciences, Macquarie University, Sydney, NSW 2109, Australia.

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http://dx.doi.org/10.2217/mmt-2020-0010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7475794PMC
July 2020

Design and Testing of a Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

Cancers (Basel) 2020 Aug 10;12(8). Epub 2020 Aug 10.

Department of Biomedical Sciences, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW 2109, Australia.

Detection of melanoma-associated mutations using circulating tumor DNA (ctDNA) from plasma is a potential alternative to using genomic DNA from invasive tissue biopsies. In this study, we developed a custom melanoma next-generation sequencing (NGS) panel which includes 123 amplicons in 30 genes covering driver and targetable mutations and alterations associated with treatment resistance. Analysis of a cohort of 74 stage III and IV treatment-naïve melanoma patients revealed that sensitivity of ctDNA detection was influenced by the amount of circulating-free DNA (cfDNA) input and stage of melanoma. At the recommended cfDNA input quantity of 20 ng (available in 28/74 patients), at least one cancer-associated mutation was detected in the ctDNA of 84% of stage IV patients and 47% of stage III patients with a limit of detection for mutant allele frequency (MAF) of 0.2%. This custom melanoma panel showed significant correlation with droplet digital PCR (ddPCR) and provided a more comprehensive melanoma mutation profile. Our custom panel could be further optimized by replacing amplicons spanning the promoter, which did not perform well due to the high GC content. To increase the detection rate to 90% of stage IV melanoma and decrease the sensitivity to 0.1% MAF, we recommend increasing the volume of plasma to 8 mL to achieve minimal recommended cfDNA input and the refinement of poorly performing amplicons. Our panel can also be expanded to include new targetable and treatment resistance mutations to improve the tracking of treatment response and resistance in melanoma patients treated with systemic drug therapies.
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http://dx.doi.org/10.3390/cancers12082228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465941PMC
August 2020

Whole genome landscapes of uveal melanoma show an ultraviolet radiation signature in iris tumours.

Nat Commun 2020 05 15;11(1):2408. Epub 2020 May 15.

QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia.

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX). We identify three other significantly mutated genes (TP53, RPL5 and CENPE).
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http://dx.doi.org/10.1038/s41467-020-16276-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229209PMC
May 2020

Longitudinal Monitoring of ctDNA in Patients with Melanoma and Brain Metastases Treated with Immune Checkpoint Inhibitors.

Clin Cancer Res 2020 08 22;26(15):4064-4071. Epub 2020 Apr 22.

Department of Biomedical Science, Macquarie University, Sydney, New South Wales, Australia.

Purpose: Brain involvement occurs in the majority of patients with metastatic melanoma. The potential of circulating tumor DNA (ctDNA) for surveillance and monitoring systemic therapy response in patients with melanoma brain metastases merits investigation.

Experimental Design: This study examined circulating , and mutations in patients with melanoma with active brain metastases receiving PD-1 inhibitor-based therapy. Intracranial and extracranial disease volumes were measured using the sum of product of diameters, and response assessment performed using RECIST. Longitudinal plasma samples were analyzed for ctDNA over the first 12 weeks of treatment (threshold 2.5 copies/mL plasma).

Results: Of a total of 72 patients, 13 patients had intracranial metastases only and 59 patients had concurrent intracranial and extracranial metastases. ctDNA detectability was 0% and 64%, respectively, and detectability was associated with extracranial disease volume ( < 0.01). Undetectable ctDNA on-therapy was associated with extracranial response ( < 0.01) but not intracranial response. The median overall survival in patients with undetectable ( = 34) versus detectable ( = 38) ctDNA at baseline was 39.2 versus 10.6 months [HR, 0.51; 95% confidence interval (CI), 0.28-0.94; = 0.03] and on-therapy was 39.2 versus 9.2 months (HR, 0.32; 95% CI, 0.16-0.63; < 0.01).

Conclusions: ctDNA remains a strong prognostic biomarker in patients with melanoma with brain metastases, especially in patients with concurrent extracranial disease. However, ctDNA was not able to detect or monitor intracranial disease activity, and we recommend against using ctDNA as a sole test during surveillance and therapeutic monitoring in patients with melanoma.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3926DOI Listing
August 2020

Transcriptional downregulation of MHC class I and melanoma de- differentiation in resistance to PD-1 inhibition.

Nat Commun 2020 04 20;11(1):1897. Epub 2020 Apr 20.

Macquarie University, Sydney, NSW, Australia.

Transcriptomic signatures designed to predict melanoma patient responses to PD-1 blockade have been reported but rarely validated. We now show that intra-patient heterogeneity of tumor responses to PD-1 inhibition limit the predictive performance of these signatures. We reasoned that resistance mechanisms will reflect the tumor microenvironment, and thus we examined PD-1 inhibitor resistance relative to T-cell activity in 94 melanoma tumors collected at baseline and at time of PD-1 inhibitor progression. Tumors were analyzed using RNA sequencing and flow cytometry, and validated functionally. These analyses confirm that major histocompatibility complex (MHC) class I downregulation is a hallmark of resistance to PD-1 inhibitors and is associated with the MITF/AXL de-differentiated phenotype and cancer-associated fibroblast signatures. We demonstrate that TGFß drives the treatment resistant phenotype (MITF/AXL) and contributes to MHC class I downregulation in melanoma. Combinations of anti-PD-1 with drugs that target the TGFß signaling pathway and/or which reverse melanoma de-differentiation may be effective future therapeutic strategies.
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http://dx.doi.org/10.1038/s41467-020-15726-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171183PMC
April 2020

Co-targeting bromodomain and extra-terminal proteins and MCL1 induces synergistic cell death in melanoma.

Int J Cancer 2020 10 24;147(8):2176-2189. Epub 2020 Apr 24.

Melanoma Immunology and Oncology, The Centenary Institute, Camperdown, New South Wales, Australia.

The treatment of melanoma has been markedly improved by the introduction of targeted therapies and checkpoint blockade immunotherapy. Unfortunately, resistance to these therapies remains a limitation. Novel anticancer therapeutics targeting the MCL1 anti-apoptotic protein have shown impressive responses in haematological cancers but are yet to be evaluated in melanoma. To assess the sensitivity of melanoma to new MCL1 inhibitors, we measured the response of 51 melanoma cell lines to the novel MCL1 inhibitor, S63845. Additionally, we assessed combination of this drug with inhibitors of the bromodomain and extra-terminal (BET) protein family of epigenetic readers, which we postulated would assist MCL1 inhibition by downregulating anti-apoptotic targets regulated by NF-kB such as BCLXL, BCL2A1 and XIAP, and by upregulating pro-apoptotic proteins including BIM and NOXA. Only 14% of melanoma cell lines showed sensitivity to S63845, however, combination of S63845 and I-BET151 induced highly synergistic apoptotic cell death in all melanoma lines tested and in an in vivo xenograft model. Cell death was dependent on caspases and BAX/BAK. Although the combination of drugs increased the BH3-only protein, BIM, and downregulated anti-apoptotic proteins such as BCL2A1, the importance of these proteins in inducing cell death varied between cell lines. ABT-199 or ABT-263 inhibitors against BCL2 or BCL2 and BCLXL, respectively, induced further cell death when combined with S63845 and I-BET151. The combination of MCL1 and BET inhibition appears to be a promising therapeutic approach for metastatic melanoma, and presents opportunities to add further BCL2 family inhibitors to overcome treatment resistance.
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http://dx.doi.org/10.1002/ijc.33000DOI Listing
October 2020

Charge Conversional Biomimetic Nanocomplexes as a Multifunctional Platform for Boosting Orthotopic Glioblastoma RNAi Therapy.

Nano Lett 2020 03 7;20(3):1637-1646. Epub 2020 Feb 7.

Henan-Macquarie Uni Joint Centre for Biomedical Innovation, School of Life Sciences, Henan University, Kaifeng, Henan 475004, China.

Nanotechnology-based RNA interference (RNAi) has shown great promise in overcoming the limitations of traditional clinical treatments for glioblastoma (GBM). However, because of the complexity of brain physiology, simple blood-brain barrier (BBB) penetration or tumor-targeting strategies cannot entirely meet the demanding requirements of different therapeutic delivery stages. Herein, we developed a charge conversional biomimetic nanoplatform with a three-layer core-shell structure to programmatically overcome persistent obstacles in siRNA delivery to GBM. The resulting nanocomplex presents good biocompatibility, prolonged blood circulation, high BBB transcytosis, effective tumor accumulation, and specific uptake by tumor cells in the brain. Moreover, red blood cell membrane (RBCm) disruption and effective siRNA release can be further triggered elegantly by charge conversion from negative to positive in the endo/lysosome (pH 5.0-6.5) of tumor cells, leading to highly potent target-gene silencing with a strong anti-GBM effect. Our study provides an intelligent biomimetic nanoplatform tailored for systemically siRNA delivery to GBM, leveraging Angiopep-2 peptide-modified, immune-free RBCm and charge conversional components. Improved therapeutic efficacy, higher survival rates, and minimized systemic side effects were achieved in orthotopic U87MG-luc human glioblastoma tumor-bearing nude mice.
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http://dx.doi.org/10.1021/acs.nanolett.9b04683DOI Listing
March 2020

Analysis of the Whole-Exome Sequencing of Tumor and Circulating Tumor DNA in Metastatic Melanoma.

Cancers (Basel) 2019 Nov 29;11(12). Epub 2019 Nov 29.

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia.

The use of circulating tumor DNA (ctDNA) to monitor cancer progression and response to therapy has significant potential but there is only limited data on whether this technique can detect the presence of low frequency subclones that may ultimately confer therapy resistance. In this study, we sought to evaluate whether whole-exome sequencing (WES) of ctDNA could accurately profile the mutation landscape of metastatic melanoma. We used WES to identify variants in matched, tumor-derived genomic DNA (gDNA) and plasma-derived ctDNA isolated from a cohort of 10 metastatic cutaneous melanoma patients. WES parameters such as sequencing coverage and total sequencing reads were comparable between gDNA and ctDNA. The mutant allele frequency of common single nucleotide variants was lower in ctDNA, reflecting the lower read depth and minor fraction of ctDNA within the total circulating free DNA pool. There was also variable concordance between gDNA and ctDNA based on the total number and identity of detected variants and this was independent of the tumor biopsy site. Nevertheless, established melanoma driver mutations and several other melanoma-associated mutations were concordant between matched gDNA and ctDNA. This study highlights that WES of ctDNA could capture clinically relevant mutations present in melanoma metastases and that enhanced sequencing sensitivity will be required to identify low frequency mutations.
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http://dx.doi.org/10.3390/cancers11121905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966626PMC
November 2019

Mitogen-activated protein kinase dependency in BRAF/RAS wild-type melanoma: A rationale for combination inhibitors.

Pigment Cell Melanoma Res 2020 03 25;33(2):345-357. Epub 2019 Sep 25.

Department of Biomedical Science, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia.

Inhibitors targeting the mitogen-activated protein kinase (MAPK) pathway and immune checkpoint molecules have dramatically improved the survival of patients with BRAF -mutant melanoma. For BRAF/RAS wild-type (WT) melanoma patients, however, immune checkpoint inhibitors remain the only effective therapeutic option with 40% of patients responding to PD-1 inhibition. In the present study, a large panel of 10 BRAF -mutant and 13 BRAF/RAS WT melanoma cell lines was analyzed to examine MAPK dependency and explore the potential utility of MAPK inhibitors in this melanoma subtype. We now show that the majority of BRAF/RAS WT melanoma cell lines (8/13) display some degree of sensitivity to trametinib treatment and resistance to trametinib in this melanoma subtype is associated with, but not mediated by NF1 suppression. Although knockdown of NF1 stimulates RAS and CRAF activity, the activation of CRAF by NF1 knockdown is limited by ERK-dependent feedback in BRAF-mutant cells, but not in BRAF/RAS WT melanoma cells. Thus, NF1 is not a dominant regulator of MAPK signaling in BRAF/RAS WT melanoma, and co-targeting multiple MAP kinase nodes provides a therapeutic opportunity for this melanoma subtype.
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http://dx.doi.org/10.1111/pcmr.12824DOI Listing
March 2020

Neoadjuvant systemic therapy in melanoma: recommendations of the International Neoadjuvant Melanoma Consortium.

Lancet Oncol 2019 07;20(7):e378-e389

Department of Pathology, Netherlands Cancer Institute-Antoni van Leeuwenhoek, Amsterdam, Netherlands.

Advances in the treatment of metastatic melanoma have improved responses and survival. However, many patients continue to experience resistance or toxicity to treatment, highlighting a crucial need to identify biomarkers and understand mechanisms of response and toxicity. Neoadjuvant therapy for regional metastases might improve operability and clinical outcomes over upfront surgery and adjuvant therapy, and has become an established role for drug development and biomarker discovery in other cancers (including locally advanced breast cancer, head and neck squamous cell carcinomas, gastroesophageal cancer, and anal cancer). Patients with clinically detectable stage III melanoma are ideal candidates for neoadjuvant therapy, because they represent a high-risk patient population with poor outcomes when treated with upfront surgery alone. Neoadjuvant therapy is now an active area of research for melanoma with numerous completed and ongoing trials (since 2014) with disparate designs, endpoints, and analyses under investigation. We have, therefore, established the International Neoadjuvant Melanoma Consortium with experts in medical oncology, surgical oncology, pathology, radiation oncology, radiology, and translational research to develop recommendations for investigating neoadjuvant therapy in melanoma to align future trial designs and correlative analyses. Alignment and consistency of neoadjuvant trials will facilitate optimal data organisation for future regulatory review and strengthen translational research across the melanoma disease continuum.
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http://dx.doi.org/10.1016/S1470-2045(19)30332-8DOI Listing
July 2019

Neoadjuvant dabrafenib combined with trametinib for resectable, stage IIIB-C, BRAF mutation-positive melanoma (NeoCombi): a single-arm, open-label, single-centre, phase 2 trial.

Lancet Oncol 2019 07 3;20(7):961-971. Epub 2019 Jun 3.

Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia; Royal North Shore Hospital, St Leonards, Sydney, NSW, Australia.

Background: Adjuvant dabrafenib plus trametinib therapy improves relapse-free survival in patients with resected stage III melanoma. We aimed to ascertain the proportion of patients who would have a pathological response and a response according to Response Evaluation Criteria in Solid Tumors (RECIST) after neoadjuvant dabrafenib plus trametinib therapy for resectable clinical stage III melanoma.

Methods: NeoCombi was a single-arm, open-label, single-centre, phase 2 study done at Melanoma Institute Australia (Sydney, NSW, Australia). Eligible patients were adults (aged ≥18 years) with histologically confirmed, resectable, RECIST-measurable, clinical stage IIIB-C (American Joint Committee on Cancer [AJCC] 7th edition), BRAF-mutant melanoma, and had an Eastern Cooperative Oncology Group performance status of 1 or lower. Patients received 150 mg dabrafenib orally, twice daily, plus 2 mg trametinib orally, once daily, for 52 weeks (12 weeks of neoadjuvant therapy before complete resection of the pre-therapy tumour bed, and 40 weeks of adjuvant therapy thereafter). CT and PET scans were done at baseline and before resection. The primary outcomes were the proportion of patients achieving a complete pathological response and the proportion of patients achieving a response according to RECIST at week 12, analysed as per protocol. This trial is registered with ClinicalTrials.gov, NCT01972347, and follow-up of patients is ongoing.

Findings: Between Aug 20, 2014, and April 19, 2017, 40 patients were screened, of whom 35 eligible patients were enrolled, received neoadjuvant dabrafenib plus trametinib, and underwent resection. At the data cutoff (Sept 24, 2018), median follow-up was 27 months (IQR 21-36). At resection, 30 (86%) patients achieved a RECIST response; 16 (46%; 95% CI 29-63) had a complete response and 14 (40%; 24-58) had a partial response. Five patients (14%; 95% CI 5-30) had stable disease, and no patients progressed. After resection and pathological evaluation, all 35 patients achieved a pathological response, of whom 17 (49%; 95% CI 31-66) patients had a complete pathological response and 18 (51%; 95% CI 34-69) had a non-complete pathological response. Treatment-related serious adverse events occurred in six (17%) of 35 patients and grade 3-4 adverse events occurred in ten (29%) patients. No treatment-related deaths were reported.

Interpretation: Neoadjuvant dabrafenib plus trametinib therapy could be considered in the management of RECIST-measurable resectable stage III melanoma as it led to a high proportion of patients achieving a complete response according to RECIST and a high proportion of patients achieving a complete pathological response, with no progression during neoadjuvant therapy.

Funding: GlaxoSmithKline; Novartis; National Health and Medical Research Council, Australia; and Melanoma Institute Australia.
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http://dx.doi.org/10.1016/S1470-2045(19)30331-6DOI Listing
July 2019

Label-Free Fluorescent Poly(amidoamine) Dendrimer for Traceable and Controlled Drug Delivery.

Biomacromolecules 2019 05 24;20(5):2148-2158. Epub 2019 Apr 24.

Department of Biomedical Sciences, Faculty of Medicine & Health Sciences , Macquarie University , Sydney , New South Wales 2109 , Australia.

Poly(amidoamine) dendrimer (PAMAM) is well-known for its high efficiency as a drug delivery vehicle. However, the intrinsic cytotoxicity and lack of a detectable signal to facilitate tracking have impeded its practical applications. Herein, we have developed a novel label-free fluorescent and biocompatible PAMAM derivative by simple surface modification of PAMAM using acetaldehyde. The modified PAMAM possessed a strong green fluorescence, which was generated by the C=N bonds of the resulting Schiff Bases via n-π* transition, while the intrinsic cytotoxicity of PAMAM was simultaneously ameliorated. Through further PEGylation, the fluorescent PAMAM demonstrated excellent intracellular tracking in human melanoma SKMEL28 cells. In addition, our PEGylated fluorescent PAMAM derivative achieved enhanced loading and delivery efficiency of the anticancer drug doxorubicin (DOX) compared to the original PAMAM. Importantly, the accelerated kinetics of DOX-encapsulated fluorescent PAMAM nanocomposites in an acidic environment facilitated intracellular drug release, which demonstrated comparable cytotoxicity to that of the free-form doxorubicin hydrochloride (DOX·HCl) against melanoma cells. Overall, our label free fluorescent PAMAM derivative offers a new opportunity of traceable and controlled delivery for DOX and other drugs of potential clinical importance.
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http://dx.doi.org/10.1021/acs.biomac.9b00494DOI Listing
May 2019

Somatic Hypermutation of the Oncogene in a Human Cutaneous Melanoma.

Mol Cancer Res 2019 07 4;17(7):1435-1449. Epub 2019 Mar 4.

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Melanoma is usually driven by mutations in BRAF or NRAS, which trigger hyperactivation of MAPK signaling. However, MAPK-targeted therapies are not sustainably effective in most patients. Accordingly, characterizing mechanisms that co-operatively drive melanoma progression is key to improving patient outcomes. One possible mechanism is the Hippo signaling pathway, which regulates cancer progression via its central oncoproteins YAP and TAZ, although is thought to be only rarely affected by direct mutation. As YAP hyperactivation occurs in uveal melanoma, we investigated this oncogene in cutaneous melanoma. YAP protein expression was elevated in most benign nevi and primary cutaneous melanomas but present at only very low levels in normal melanocytes. In patient-derived xenografts and melanoma cell lines, we observed variable reliance of cell viability on Hippo pathway signaling that was independent of TAZ activity and also of classical melanoma driver mutations such as BRAF and NRAS. Finally, in genotyping studies of melanoma, we observed the first ever hyperactivating mutations in a human cancer, manifest as seven distinct missense point mutations that caused serine to alanine transpositions. Strikingly, these mutate four serine residues known to be targeted by the Hippo pathway and we show that they lead to hyperactivation of YAP. IMPLICATIONS: Our studies highlight the YAP oncoprotein as a potential therapeutic target in select subgroups of melanoma patients, although successful treatment with anti-YAP therapies will depend on identification of biomarkers additional to YAP protein expression.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0407DOI Listing
July 2019

Distinct Immune Cell Populations Define Response to Anti-PD-1 Monotherapy and Anti-PD-1/Anti-CTLA-4 Combined Therapy.

Cancer Cell 2019 02;35(2):238-255.e6

Melanoma Institute Australia, The University of Sydney, Sydney, NSW 2065, Australia; Sydney Medical School, The University of Sydney, Sydney, NSW 2006, Australia. Electronic address:

Cancer immunotherapies provide survival benefits in responding patients, but many patients fail to respond. Identifying the biology of treatment response and resistance are a priority to optimize drug selection and improve patient outcomes. We performed transcriptomic and immune profiling on 158 tumor biopsies from melanoma patients treated with anti-PD-1 monotherapy (n = 63) or combined anti-PD-1 and anti-CTLA-4 (n = 57). These data identified activated T cell signatures and T cell populations in responders to both treatments. Further mass cytometry analysis identified an EOMESCD69CD45RO effector memory T cell phenotype that was significantly more abundant in responders to combined immunotherapy compared with non-responders (n = 18). The gene expression profile of this population was associated with longer progression-free survival in patients treated with single agent and greater tumor shrinkage in both treatments.
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http://dx.doi.org/10.1016/j.ccell.2019.01.003DOI Listing
February 2019

Integrated molecular and immunophenotypic analysis of NK cells in anti-PD-1 treated metastatic melanoma patients.

Oncoimmunology 2019;8(2):e1537581. Epub 2018 Oct 31.

Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia.

: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma. : Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line). : Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (< 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells' cytotoxicity. : A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.
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http://dx.doi.org/10.1080/2162402X.2018.1537581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343795PMC
October 2018

Pharmacokinetic and cytokine profiles of melanoma patients with dabrafenib and trametinib-induced pyrexia.

Cancer Chemother Pharmacol 2019 04 19;83(4):693-704. Epub 2019 Jan 19.

Faculty of Medicine and Health, School of Pharmacy, The University of Sydney, Sydney, NSW, Australia.

Purpose: The combination of a BRAF inhibitor dabrafenib and a MEK inhibitor trametinib (CombiDT) has improved outcomes compared with chemotherapy or BRAF inhibitor monotherapy in advanced BRAF V600E/K melanoma. However, CombiDT causes a high incidence of pyrexia and treatment interruptions. Pharmacokinetic analysis may provide an explanation for the pyrexia.

Methods: 34 patients with Stage 3 BRAF V600 melanoma were treated with CombiDT on a clinical trial between August 2014 and June 2017. Plasma concentrations of drugs and metabolites were determined using validated LC-MS assays, in addition to analysis of a panel of cytokines.

Results: Pyrexia was experienced by 71% of the patients, with an additional 17% requiring dose interruption related to a pyrexia-like prodrome. Dabrafenib concentrations ranged from 4.0 to 4628 ng/ml and trametinib from 1.0 to 45 ng/ml in 34 patients. N-desmethyl-dabrafenib was the most prevalent metabolite, followed by carboxy- and hydroxy-dabrafenib. No definitive association between pyrexia and AUC or C of the drugs, or metabolites could be observed. The level of IL-1B at the early during treatment (EDT) (as a % of pre-treatment) was higher in the pyrexia group (median 109% (range 32-681%) than in the no-incidence group [56% (26-79%)] (p = 0.029). Similarly, the level of IL-6 at EDT was higher in the pyrexia group [181% (34-3156%) vs 73% (57-101%)] (p = 0.028).

Conclusions: No apparent associations between pyrexia and exposure to the drugs or metabolites could be observed. Greater elevations in IL-1B and IL-6 were observed in patients with pyrexia during the first week of treatment compared to those without pyrexia.
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http://dx.doi.org/10.1007/s00280-019-03780-yDOI Listing
April 2019

Distinct Molecular Profiles and Immunotherapy Treatment Outcomes of V600E and V600K -Mutant Melanoma.

Clin Cancer Res 2019 02 10;25(4):1272-1279. Epub 2019 Jan 10.

Melanoma Institute Australia and The University of Sydney, Sydney, New South Wales, Australia.

Purpose: V600E and V600K melanomas have distinct clinicopathologic features, and V600K appear to be less responsive to ±. We investigated mechanisms for this and explored whether genotype affects response to immunotherapy.

Experimental Design: Pretreatment formalin-fixed paraffin-embedded tumors from patients treated with ± underwent gene expression profiling and DNA sequencing. Molecular results were validated using The Cancer Genome Atlas (TCGA) data. An independent cohort of V600E/K patients treated with anti-PD-1 immunotherapy was examined.

Results: Baseline tissue and clinical outcome with ± were studied in 93 patients (78 V600E, 15 V600K). V600K patients had numerically less tumor regression (median, -31% vs. -52%, = 0.154) and shorter progression-free survival (PFS; median, 5.7 vs. 7.1 months, = 0.15) compared with V600E. V600K melanomas had lower expression of the ERK pathway feedback regulator dual-specificity phosphatase 6, confirmed with TCGA data (116 V600E, 17 V600K). Pathway analysis showed V600K had lower expression of ERK and higher expression of PI3K-AKT genes than V600E. Higher mutational load was observed in V600K, with a higher proportion of mutations in and tumor-suppressor genes. In patients treated with anti-PD-1, V600K ( = 19) had superior outcomes than V600E ( = 84), including response rate (53% vs. 29%, = 0.059), PFS (median, 19 vs. 2.7 months, = 0.049), and overall survival (20.4 vs. 11.7 months, = 0.081).

Conclusions: V600K melanomas appear to benefit less from ± than V600E, potentially due to less reliance on ERK pathway activation and greater use of alternative pathways. In contrast, these melanomas have higher mutational load and respond better to immunotherapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015248PMC
February 2019

Evaluation of commercial kits for purification of circulating free DNA.

Cancer Genet 2018 12 29;228-229:21-27. Epub 2018 Aug 29.

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; The Poche Centre, Melanoma Institute Australia, NSW 2065, Australia. Electronic address:

Analysis of liquid biopsies and the identification of non-invasive biomarkers for the diagnosis and prognosis of solid tumors has grown exponentially over the last few years. This has led to an increasing number of commercial kits optimised for the purification of circulating free (cf) DNA and RNA/miRNA from biofluids such as plasma, serum and urine. To optimise and standardise current practices we sought to evaluate the performance of spin column-based and magnetic bead-based commercial kits. The following commercial cfDNA purification kits were analysed in this study: QIAamp circulating nucleic acid kit (Qiagen, Germany); Plasma/serum cell-free circulating DNA Purification midi kit (Norgen Biotek, Canada); QIAamp minElute ccfDNA mini kit (Qiagen); Maxwell RSC ccfDNA plasma kit (Promega, USA); MagMAX cell-free DNA isolation kit (Applied Biosystems, USA); and NextPrep-Mag cfDNA isolation kit (Bioo Scientific, USA). Extracted DNA from the plasma of healthy individuals, either nonspiked or spiked with DNA fragments or cfDNA, was evaluated for recovery using either a BioRad Experion or ddPCR analysis. This study represents the first to use a comprehensive size distribution of spiked-in DNA fragments to evaluate commercial cfDNA kits. The commonly used spin column-based Qiagen QIAamp circulating nucleic acid kit was found to be the most consistent performing kit across the two evaluation assays employed. The Qiagen QIAamp minElute ccfDNA mini kit represented the best performing magnetic bead-based kit and provides an alternative based on lower cost/sample with a simpler workflow than spin column-based kits.
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http://dx.doi.org/10.1016/j.cancergen.2018.08.005DOI Listing
December 2018

Circulating Cytokines Predict Immune-Related Toxicity in Melanoma Patients Receiving Anti-PD-1-Based Immunotherapy.

Clin Cancer Res 2019 03 8;25(5):1557-1563. Epub 2018 Nov 8.

Faculty of Medicine and Health Sciences, Macquarie University, Sydney, Australia.

Purpose: Combination PD-1 and CTLA-4 inhibitor therapy has dramatically improved the survival of patients with advanced melanoma but is also associated with significant immune-related toxicities. This study sought to identify circulating cytokine biomarkers of treatment response and immune-related toxicity.

Experimental Design: The expression of 65 cytokines was profiled longitudinally in 98 patients with melanoma treated with PD-1 inhibitors, alone or in combination with anti-CTLA-4, and in an independent validation cohort of 49 patients treated with combination anti-PD-1 and anti-CTLA-4. Cytokine expression was correlated with RECIST response and immune-related toxicity, defined as toxicity that warranted permanent discontinuation of treatment and administration of high-dose steroids.

Results: Eleven cytokines were significantly upregulated in patients with severe immune-related toxicities at baseline (PRE) and early during treatment (EDT). The expression of these 11 cytokines was integrated into a single toxicity score, the CYTOX (cytokine toxicity) score, and the predictive utility of this score was confirmed in the discovery and validation cohorts. The AUC for the CYTOX score in the validation cohort was 0.68 at PRE [95% confidence interval (CI), 0.51-0.84; = 0.037] and 0.70 at EDT (95% CI, 0.55-0.85; = 0.017) using ROC analysis.

Conclusions: The CYTOX score is predictive of severe immune-related toxicity in patients with melanoma treated with combination anti-CTLA-4 and anti-PD-1 immunotherapy. This score, which includes proinflammatory cytokines such as IL1a, IL2, and IFNα2, may help in the early management of severe, potentially life-threatening immune-related toxicity..
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http://dx.doi.org/10.1158/1078-0432.CCR-18-2795DOI Listing
March 2019

Monitoring Melanoma Using Circulating Free DNA.

Am J Clin Dermatol 2019 Feb;20(1):1-12

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, 2109, Australia.

Genetic material derived from tumours is constantly shed into the circulation of cancer patients both in the form of circulating free nucleic acids and within circulating cells or extracellular vesicles. Monitoring cancer-specific genomic alterations, particularly mutant allele frequencies, in circulating nucleic acids allows for a non-invasive liquid biopsy for detecting residual disease and response to therapy. The advent of molecular targeted treatments and immunotherapies with increasing effectiveness requires corresponding effective molecular biology methods for the detection of biomarkers such as circulating nucleic acid to monitor and ultimately personalise therapy. The use of polymerase chain reaction (PCR)-based methods, such as droplet digital PCR, allows for a very sensitive analysis of circulating tumour DNA, but typically only a limited number of gene mutations can be detected in parallel. In contrast, next-generation sequencing allows for parallel analysis of multiple mutations in many genes. The development of targeted next-generation sequencing cancer gene panels optimised for the detection of circulating free DNA now provides both the flexibility of multiple mutation analysis coupled with a sensitivity that approaches or even matches droplet digital PCR. In this review, we discuss the advantages and disadvantages of these current molecular technologies in conjunction with how this field is evolving in the context of melanoma diagnosis, prognosis, and monitoring of response to therapy.
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http://dx.doi.org/10.1007/s40257-018-0398-xDOI Listing
February 2019

Oncogenic PI3K/AKT promotes the step-wise evolution of combination BRAF/MEK inhibitor resistance in melanoma.

Oncogenesis 2018 Sep 20;7(9):72. Epub 2018 Sep 20.

Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia.

Nearly all patients with BRAF-mutant melanoma will progress on BRAF inhibitor monotherapy and combination BRAF/MEK inhibitor therapy within the first year of therapy. In the vast majority of progressing melanomas, resistance occurs via the re-activation of MAPK signalling, commonly via alterations in BRAF, NRAS and MEK1/2. A small proportion of resistant melanomas rely on the activation of the compensatory PI3K/AKT signalling cascade, although activation of this pathway does not preclude patient responses to BRAF/MEK inhibition. We now show, that PI3K/AKT signalling via potent oncogenic PIK3CA and AKT3 mutants, is not sufficient to overcome proliferative arrest induced by BRAF/MEK inhibition, but rather enables the survival of a dormant population of MAPK-inhibited melanoma cells. The evolution of resistance in these surviving tumour cells was associated with MAPK re-activation and no longer depended on the initial PI3K/AKT-activating oncogene. This dynamic form of resistance alters signalling dependence and may lead to the evolution of tumour subclones highly resistant to multiple targeted therapies.
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http://dx.doi.org/10.1038/s41389-018-0081-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148266PMC
September 2018

Interferon Signaling Is Frequently Downregulated in Melanoma.

Front Immunol 2018 21;9:1414. Epub 2018 Jun 21.

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia.

Immune checkpoint inhibitors that block the programmed cell death protein 1/PD-L1 pathway have significantly improved the survival of patients with advanced melanoma. Immunotherapies are only effective in 15-40% of melanoma patients and resistance is associated with defects in antigen presentation and interferon signaling pathways. In this study, we examined interferon-γ (IFNγ) responses in a large panel of immune checkpoint inhibitor-naïve melanoma cells with defined genetic drivers; -mutant ( = 11), -mutant ( = 10), wild type ( = 10), and -mutant uveal melanomas (UVMs) ( = 8). Cell surface expression of established IFNγ downstream targets PD-L1, PD-L2, HLA-A, -B, and -C, HLA-DR, and nerve growth factor receptor (NGFR) were analyzed by flow cytometry. Basal cellular expression levels of HLA-A, -B, -C, HLA-DR, NGFR, and PD-L2 predicted the levels of IFNγ-stimulation, whereas PD-L1 induction was independent of basal expression levels. Only 13/39 (33%) of the melanoma cell lines tested responded to IFNγ with potent induction of all targets, indicating that downregulation of IFNγ signaling is common in melanoma. In addition, we identified two well-recognized mechanisms of immunotherapy resistance, the loss of β-2-microglobulin and interferon gamma receptor 1 expression. We also examined the influence of melanoma driver oncogenes on IFNγ signaling and our data suggest that UVM have diminished capacity to respond to IFNγ, with lower induced expression of several targets, consistent with the disappointing response of UVM to immunotherapies. Our results demonstrate that melanoma responses to IFNγ are heterogeneous, frequently downregulated in immune checkpoint inhibitor-naïve melanoma and potentially predictive of response to immunotherapy.
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http://dx.doi.org/10.3389/fimmu.2018.01414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021492PMC
June 2018