Publications by authors named "Mark Shackleton"

66 Publications

Evolution of late-stage metastatic melanoma is dominated by aneuploidy and whole genome doubling.

Nat Commun 2021 03 4;12(1):1434. Epub 2021 Mar 4.

Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.
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http://dx.doi.org/10.1038/s41467-021-21576-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933255PMC
March 2021

Implementation of patient-reported outcome measures and patient-reported experience measures in melanoma clinical quality registries: a systematic review.

BMJ Open 2021 Feb 11;11(2):e040751. Epub 2021 Feb 11.

NHMRC Clinical Trials Centre, The University of Sydney, Sydney, New South Wales, Australia

Objectives: To identify patient-reported outcome measures (PROMs) and patient-reported experience measures (PREMs) in clinical quality registries, for people with cutaneous melanoma, to inform a new Australian Melanoma Clinical Outcomes Registry; and describe opportunities and challenges of routine PROM/PREM collection, especially in primary care.

Design: Systematic review.

Primary And Secondary Outcome Measures: Which PROMs and PREMs are used in clinical quality registries for people with cutaneous melanoma, how they are collected, frequency of collection, participant recruitment methods and funding models for each registry.

Results: 1134 studies were identified from MEDLINE, PreMEDLINE, Embase, PsychInfo, Cochrane Database of Abstracts of Reviews of Effects databases and TUFTS Cost-Effectiveness Analysis Registry, alongside grey literature, from database inception to 5th February 2020. Following screening, 14 studies were included, identifying four relevant registries: Dutch Melanoma Registry, Adelphi Real-World Disease-Specific Programme (Melanoma), Patient-Reported Outcomes Following Initial treatment and Long-term Evaluation of Survivorship Registry, and Cancer Experience Registry. These used seven PROMs: EuroQol-5 Dimensions, Functional Assessment of Cancer-General (FACT-G) and FACT-Melanoma (FACT-M), European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Cancer 30 (EORTC QLQ-C30), Fatigue Assessment Scale Hospital Anxiety and Depression Scale, Patient-Reported Outcome Measures Information System-29 and one PREM; EORTC QLQ-Information Module 26. PROMs/PREMs in registries were reported to improve transparency of care; facilitate clinical auditing for quality assessment; enable cost-effectiveness analyses and create large-scale research platforms. Challenges included resource burden for data entry and potential collection bias toward younger, more affluent respondents. Feedback from patients with melanoma highlighted the relevance of PROMs/PREMs in assessing patient outcomes and patient experiences.

Conclusions: Clinical registries indicate PROMs/PREMs for melanoma care can be incorporated and address important gaps, however cost and collection bias may limit generalisability.

Prospero Registration Number: CRD42018086737.
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http://dx.doi.org/10.1136/bmjopen-2020-040751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880114PMC
February 2021

Reduced melanoma referrals during COVID-19 lockdown.

Aust J Gen Pract 2021 Jan 22. Epub 2021 Jan 22.

MBBS, FACD, PhD, Director, Victorian Melanoma Service, Alfred Hospital, Vic; Adjunct Associate Professor, School of Public Health and Preventive Medicine, Monash University, Vic.

As the COVID-19 pandemic has reduced face-to-face medical consultations, reduced diagnoses of thin melanomas and delayed presentations of thick melanomas were anticipated.
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http://dx.doi.org/10.31128/AJGP-COVID-45DOI Listing
January 2021

Melanoma recurrence patterns and management after adjuvant targeted therapy: a multicentre analysis.

Br J Cancer 2021 Feb 22;124(3):574-580. Epub 2020 Oct 22.

Department of Medical Oncology, Alfred Hospital, Melbourne, VIC, Australia.

Background: Adjuvant targeted therapy (TT) improves relapse free survival in patients with resected BRAF mutant stage III melanoma. The outcomes and optimal management of patients who relapse after adjuvant TT is unknown.

Methods: Patients from twenty-one centres with recurrent melanoma after adjuvant TT were included. Disease characteristics, adjuvant therapy, recurrence, treatment at relapse and outcomes were examined.

Results: Eighty-five patients developed recurrent melanoma; nineteen (22%) during adjuvant TT. Median time to first recurrence was 18 months and median follow-up from first recurrence was 31 months. Fifty-eight (68%) patients received immunotherapy (IT) or TT as 1st line systemic therapy at either first or subsequent recurrence and had disease that was assessable for response. Response to anti-PD-1 (±trial agent), combination ipilimumab-nivolumab, TT rechallenge and ipilimumab monotherapy was 63%, 62% 25% and 10% respectively. Twenty-eight (33%) patients had died at census, all from melanoma. Two-year OS was 84% for anti-PD-1 therapy (±trial agent), 92% for combination ipilimumab and nivolumab, 49% for TT and 45% for ipilimumab monotherapy (p = 0.028).

Conclusions: Patients who relapse after adjuvant TT respond well to subsequent anti-PD-1 based therapy and have outcomes similar to those seen when first line anti-PD-1 therapy is used in stage IV melanoma.
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http://dx.doi.org/10.1038/s41416-020-01121-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851118PMC
February 2021

Feasibility and Acceptability of : A Stepped-Care Program to Manage Fear of Cancer Recurrence in People with Metastatic Melanoma.

J Clin Med 2020 Sep 14;9(9). Epub 2020 Sep 14.

Psychosocial Oncology Program, Peter MacCallum Cancer Centre, Melbourne, VIC 3000, Australia.

Immunotherapies and targeted therapies have revolutionised treatment of metastatic melanoma and improved survival rates. However, survivors treated with novel therapies are vulnerable to high levels of fear of cancer recurrence or progression (FCR). Existing FCR interventions have rarely been trialled in people with advanced cancer. The current study aimed to evaluate the acceptability and feasibility of : a stepped-care model to treat FCR in people with metastatic melanoma treated with immunotherapy or targeted therapy. Sixty-one outpatients with metastatic melanoma were screened using the Fear of Cancer Recurrence Inventory Short Form (FCRI-SF) and Fear of Progression Questionnaire Short Form (FoP-Q-SF). Survivors with subthreshold FCR were stratified to a self-management intervention while those with clinical levels of FCR were provided with an individual therapy, . Survivor experience surveys and rescreening were administered post-intervention completion. Results indicated that was an acceptable and feasible FCR intervention. Results provided preliminary support for the potential impact of in reducing FCR. is a promising first step in providing an acceptable and feasible stepped-care model to treat FCR in survivors with metastatic disease.
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http://dx.doi.org/10.3390/jcm9092969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565154PMC
September 2020

Impact of Radiotherapy on the Efficacy and Toxicity of anti-PD-1 Inhibitors in Metastatic NSCLC.

Clin Lung Cancer 2020 Jun 10. Epub 2020 Jun 10.

Department of Medical Oncology, Monash Health, Clayton, Melbourne, Australia; School of Clinical Sciences, Monash University, Melbourne, Australia. Electronic address:

Background: The impact of radiotherapy (RT) on the efficacy and toxicity of immune checkpoint inhibitors (ICIs) in patients with metastatic non-small-cell lung cancer (NSCLC) is unclear.

Materials And Methods: We identified patients with metastatic NSCLC treated with the anti-programmed death 1 antibodies nivolumab or pembrolizumab between January 2016 and May 2019 at 3 tertiary centers, who were also treated with palliative RT either during or within 3 months of starting anti-programmed death 1 treatment. Patient demographics, tumor characteristics, and treatment history were collected. Response rates, progression-free survival (PFS), and overall survival (OS) were analyzed and correlated with RT use.

Results: A total of 269 patients were identified, with a median follow-up of 19.4 months. The median age was 70 years (range, 35-90 years), and they were 63% male, 60% smokers, and 65% had adenocarcinoma histology. At the commencement of ICI treatment, the majority (86%) had ≥ 1 line of prior therapy and 34% had brain metastases. A total of 102 (38%) patients received RT within 3 months of starting ICI or subsequently during ICI treatment. Of patients that received RT, 86 (84%) received conventional hypofractionated RT, and, in the majority, 81 (79%) the intent of RT was symptom control. The use of RT did not increase grade 3/4 immune-related adverse events. The overall median PFS was 2.0 months (95% confidence interval, 1.3-2.6 months) and the median OS was 9.0 months (95% confidence interval, 6.4-9.5 months). There were no significant differences in median PFS (3.0 vs. 2.0 months; P = .515) and median OS (9.0 vs. 9.0 months; P = .917) in the patients who received RT versus those that did not.

Conclusions: In patients with metastatic NSCLC, the addition of RT to ICI was not associated with increased toxicity or improved survival.
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http://dx.doi.org/10.1016/j.cllc.2020.06.001DOI Listing
June 2020

Parity reduces mammary repopulating activity but does not affect mammary stem cells defined as CD24 + CD29/CD49fhi in mice.

Breast Cancer Res Treat 2020 Oct 21;183(3):565-575. Epub 2020 Jul 21.

Breast Cancer Risk and Prevention Laboratory, Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC, Australia.

Background: Breast cancer (BCa) mortality is decreasing with early detection and improvement in therapies. The incidence of BCa, however, continues to increase, particularly estrogen-receptor-positive (ER +) subtypes. One of the greatest modifiers of ER + BCa risk is childbearing (parity), with BCa risk halved in young multiparous mothers. Despite convincing epidemiological data, the biology that underpins this protection remains unclear. Parity-induced protection has been postulated to be due to a decrease in mammary stem cells (MaSCs); however, reports to date have provided conflicting data.

Methods: We have completed rigorous functional testing of repopulating activity in parous mice using unfractionated and MaSC (CD24CD49f)-enriched populations. We also developed a novel serial transplant method to enable us to assess self-renewal of MaSC following pregnancy. Lastly, as each pregnancy confers additional BCa protection, we subjected mice to multiple rounds of pregnancy to assess whether additional pregnancies impact MaSC activity.

Results: Here, we report that while repopulating activity in the mammary gland is reduced by parity in the unfractionated gland, it is not due to a loss in the classically defined MaSC (CD24CD49f) numbers or function. Self-renewal was unaffected by parity and additional rounds of pregnancy also did not lead to a decrease in MaSC activity.

Conclusions: Our data show instead that parity impacts on the stem-like activity of cells outside the MaSC population.
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http://dx.doi.org/10.1007/s10549-020-05804-1DOI Listing
October 2020

The Hippo pathway oncoprotein YAP promotes melanoma cell invasion and spontaneous metastasis.

Oncogene 2020 07 19;39(30):5267-5281. Epub 2020 Jun 19.

Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC, 3000, Australia.

Melanoma is a deadly form of skin cancer that accounts for a disproportionally large proportion of cancer-related deaths in younger people. Compared with most other skin cancers, a feature of melanoma is its high metastatic capacity, although the mechanisms that confer this are not well understood. The Hippo pathway is a key regulator of organ growth and cell fate that is deregulated in many cancers. To analyse the Hippo pathway in cutaneous melanoma, we generated a transcriptional signature of melanoma cells that overexpressed YAP, the key downstream Hippo pathway oncoprotein. YAP-mediated transcriptional activity varied in melanoma cell lines but did not cluster with known genetic drivers of melanomagenesis such as BRAF and NRAS mutations. Instead, it correlated strongly with published gene expression profiles linked to melanoma cell invasiveness and varied throughout the metastatic cascade in melanoma patient tumours. Consistent with this, YAP was both necessary and sufficient for melanoma cell invasion in vitro. In vivo, YAP promoted spontaneous melanoma metastasis, whilst the growth of YAP-expressing primary tumours was impeded. Finally, we identified the YAP target genes AXL, THBS1 and CYR61 as key mediators of YAP-induced melanoma cell invasion. These data suggest that YAP is a critical regulator of melanoma metastasis.
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http://dx.doi.org/10.1038/s41388-020-1362-9DOI Listing
July 2020

Stereotactic Radiation Therapy Combined With Immunotherapy Against Metastatic Melanoma: Long-Term Results of a Phase 1 Clinical Trial.

Int J Radiat Oncol Biol Phys 2020 09 22;108(1):150-156. Epub 2020 May 22.

Alfred Health Radiation Oncology, Alfred Hospital, Melbourne, Australia; Monash University, Melbourne, Australia.

Purpose: To determine the maximum tolerated dose (MTD) of stereotactic ablative radiation therapy (SABR) in combination with immunotherapy for the treatment of patients with metastatic melanoma. The study also investigates the effects of timing and dosing of SABR on clinical efficacy.

Methods: Metastatic melanoma patients with at least 2 metastases received SABR to a single metastatic site. All patients had standard dose immunotherapy with anti-PD1 or anti-CTLA4 at the discretion of their treating clinician. Following a standard 3 + 3 design, patients were escalated through 3 SABR doses (10 Gy, 15 Gy, and 20 Gy) delivered at 3 different time points (with cycle 1, 2, or 3 of immunotherapy). Dose-limiting toxicities (DLT) were defined as grade 3 or higher toxicity within 3 months of first treatment and assessed by an independent data safety monitoring committee (IDSMC). Logistic or Cox regressions were used to assess the impact of SABR dose and timing on the progression free (PFS) and overall survival (OS) of this cohort.

Results: Twenty-four patients were enrolled with a median clinical follow-up of 28 months. Four patients (16.7%) developed DLTs; 1 DLT occurred at a SABR-treated site, and all patients received 15 Gy. On this basis the IDSMC recommended stopping the trial and the MTD was defined at 10 Gy. The 2-year PFS was 21.9% (95% CI, 7.1%-41.8%) and 2-year OS was 49.6% (95% CI, 28.7%-67.6%). The median PFS for those receiving 10 Gy was numerically higher than for those receiving 15 Gy, 8.3 months versus 2.1 months (P = .38). The only treatment-related factor associated with both improved PFS (HR 0.08, P < .01) and OS (HR 0.008, P ≤ .01) was receiving SABR with cycle 3. SABR dose (PFS P = .17, OS P = .50) was not significant.

Conclusions: SABR at 10 Gy can be safely combined with immunotherapy. SABR timing appears to influence efficacy more than dose and warrants consideration in research attempting to optimize synergism.
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http://dx.doi.org/10.1016/j.ijrobp.2020.05.022DOI Listing
September 2020

Metastatic melanoma presenting as intravenous tumour thrombus.

J Med Imaging Radiat Oncol 2020 Dec 30;64(6):814-816. Epub 2020 Mar 30.

Department of Medical Oncology, Alfred Health, Melbourne, Victoria, Australia.

Tumour thrombus is a complication that occurs when a malignancy invades into the vasculature, occluding its lumen. Here, we present a rare case of melanoma tumour thrombus of the great saphenous vein of the left thigh, which was diagnosed on F-fluorodeoxyglucose positron emission tomography/computed tomography and ultrasound-guided biopsy, and responded well to immunotherapy with pembrolizumab.
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http://dx.doi.org/10.1111/1754-9485.13019DOI Listing
December 2020

An update on adjuvant systemic therapies in melanoma.

Melanoma Manag 2019 Nov 13;6(3):MMT28. Epub 2019 Nov 13.

Department of Medical Oncology, The Alfred Hospital, Melbourne 3004, Australia.

There is a global increase in the incidence of melanoma, with approximately 300,000 new cases in 2018 worldwide, according to statistics from the International Agency for Research on Cancer. With this rising incidence, it is important to optimize treatment strategies in all stages of the disease to provide better patient outcomes. The role of adjuvant therapy in patients with resected stage 3 melanoma is a rapidly evolving field. Interferon was the first agent shown to have any utility in this space, however, recent advances in both targeted therapies and immunotherapies have led to a number of practice changing adjuvant trials in resected stage 3 disease.
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http://dx.doi.org/10.2217/mmt-2019-0009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891936PMC
November 2019

Bevacizumab as a steroid-sparing agent during immunotherapy for melanoma brain metastases: A case series.

Health Sci Rep 2019 Mar 1;2(3):e115. Epub 2019 Feb 1.

Department of Cancer Medicine, Melbourne Peter MacCallum Cancer Centre Australia.

Background: Brain metastases are common in advanced melanoma and often necessitate corticosteroids such as dexamethasone to control symptoms and reduce peritumoral edema. Immunotherapy improves survival in metastatic melanoma, but concomitant treatment with corticosteroids may reduce efficacy. Here, we report the use of bevacizumab, a vascular endothelial growth factor (VEGF) inhibitor, as a steroid-sparing agent in melanoma patients with brain metastases treated with immunotherapy.

Methods: Medical records and imaging were retrospectively analyzed for melanoma patients with brain metastases who received bevacizumab at our institution between 2012 and 2017.

Results: 12 melanoma patients with brain metastases received bevacizumab (5-7.5 mg/kg Q2-3 W; median 4 cycles, range 1-9). Patients were BRAF wild-type or resistant to BRAF/MEK inhibitor therapy. All had progressive intracranial disease after prior resection, stereotactic radiosurgery and/or whole brain radiotherapy, and up to four lines of previous systemic treatment. Prior to bevacizumab, all patients had radiologically defined peritumoral edema and nine required dexamethasone for symptom control. In 10 evaluable patients, six reduced their dexamethasone dose by more than 50%, and eight displayed reduced edema 4 weeks after bevacizumab. Seven patients experienced adverse events possibly related to bevacizumab, including intracranial hemorrhage, hypertension, and gastrointestinal bleeding. Ten patients received immunotherapy after bevacizumab. Five patients survived more than 6 months, including one who remained disease-free after 4 years and without neurological deficit despite being hemiplegic from edematous brain metastases prior to bevacizumab.

Conclusion: In 12 very poor prognosis melanoma patients with brain metastases, bevacizumab was well-tolerated, associated with improved symptoms and reduced peritumoral edema despite weaning steroids, and facilitated treatment with immunotherapy that provided durable survival in a substantial proportion of cases.
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http://dx.doi.org/10.1002/hsr2.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6427059PMC
March 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

Personalised surveillance after treatment for high-risk cancer.

Oncotarget 2019 Jan 22;10(7):694-695. Epub 2019 Jan 22.

Mark Shackleton: Cancer Treatment and Development Laboratory, Central Clinical School, Monash University, Victoria, Australia; Department of Oncology, Alfred Health, Victoria, Australia.

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http://dx.doi.org/10.18632/oncotarget.26619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366832PMC
January 2019

Incidence, features and management of radionecrosis in melanoma patients treated with cerebral radiotherapy and anti-PD-1 antibodies.

Pigment Cell Melanoma Res 2019 07 3;32(4):553-563. Epub 2019 Mar 3.

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

Background: Brain radiotherapy is used in the management of melanoma brain metastases (MBM) and can result in radionecrosis. Anti-PD-1 is active in the brain and may increase the risk of radionecrosis when combined with radiotherapy. We studied the incidence, associated factors and management of radionecrosis in longer-term survivors with MBM treated with this combination.

Methods: Patients with MBM treated with radiotherapy and anti-PD-1 who survived >1 year were identified to determine radionecrosis incidence (Cohort A, n = 135). Cohort A plus additional radionecrosis cases were examined for factors associated with radionecrosis and management (Cohort B, n = 148).

Results: From Cohort A, 17% developed radionecrosis, with a cumulative incidence at 2 years of 18%. Using Cohort B, multivariable analysis confirmed an association between radionecrosis and elevated lactate dehydrogenase (p = 0.0496) and prior treatment with ipilimumab (p = 0.0319). Radionecrosis was diagnosed based on MRI (100%), symptoms (69%) and pathology (56%). Treatment included corticosteroids, bevacizumab and neurosurgery.

Conclusions: Radionecrosis is a significant toxicity in longer-term melanoma survivors with MBM treated with anti-PD-1 and radiotherapy. Identification of those at risk of radionecrosis who may avoid radiotherapy is required.
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http://dx.doi.org/10.1111/pcmr.12775DOI Listing
July 2019

Rheumatic immune-related adverse events secondary to anti-programmed death-1 antibodies and preliminary analysis on the impact of corticosteroids on anti-tumour response: A case series.

Eur J Cancer 2018 12 13;105:88-102. Epub 2018 Nov 13.

Department of Medical Oncology, Peter MacCallum Cancer Centre, Melbourne, Australia; Sir Peter MacCallum Department, University of Melbourne, Melbourne, Australia. Electronic address:

Importance: Rheumatic immune-related adverse events (irAEs) occur in approximately 10-20% of anti-programmed death 1 (anti-PD1)-treated cancer patients. There are limited data on the natural history, optimal treatment and long-term oncological outcomes of patients with rheumatic irAEs.

Objective: The objective of the study was to describe the spectrum and natural history of rheumatic irAEs and the potential impact of rheumatic irAEs and immunomodulators on anti-PD1 tumour efficacy.

Methods: Cancer patients with pre-existing rheumatic disease before anti-PD1 therapy or de novo rheumatic irAEs on anti-PD1 therapy were retrospectively reviewed across three sites. Patient demographics, treatment history, anti-PD1 irAEs, and anti-PD1 responses were evaluated. Relationships between the development or pre-existence of rheumatic irAE, use of immunomodulatory agents and outcomes were evaluated.

Results: This multicenter case series describes 36 cancer patients who had rheumatic disease before anti-PD1 therapy (n = 12) or developed de novo rheumatic irAEs (n = 24). Thirty-four of the 36 patients sustained rheumatic irAEs (median time to rheumatic irAE: 14.5 weeks), including 24 de novo (18 inflammatory arthritis, three myositis, two polymyalgia rheumatica, one fasciitis) and 10 flares in 12 patients with pre-existing rheumatic disease. Corticosteroids were used in 30 of 36 patients (median duration: 10 months), and disease-modifying antirheumatic drugs were used in 14 of 36 patients (median duration: 5.5 months). The objective response rate to anti-PD1 therapy was 69% (n = 25/36) overall and 81% (n = 21/26) in the melanoma subgroup.

Conclusions: Rheumatic irAEs are often chronic and require prolonged immunomodulatory therapy. Prospective studies are required to define optimal management of rheumatic irAEs that maintain long-term anticancer outcomes.
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http://dx.doi.org/10.1016/j.ejca.2018.09.027DOI Listing
December 2018

Whole-genome landscapes of major melanoma subtypes.

Nature 2017 05 3;545(7653):175-180. Epub 2017 May 3.

Melanoma Institute Australia, The University of Sydney, North Sydney, Sydney, New South Wales 2065, Australia.

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.
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http://dx.doi.org/10.1038/nature22071DOI Listing
May 2017

Calcium-Dependent Enhancement by Extracellular Acidity of the Cytotoxicity of Mitochondrial Inhibitors against Melanoma.

Mol Cancer Ther 2017 05 21;16(5):936-947. Epub 2017 Feb 21.

Department of Regenerative Dermatology, Graduate School of Medicine, Osaka University, Osaka, Japan.

Extracellular acidity is a hallmark of cancers and is independent of hypoxia. Because acidity potentiates malignant phenotypes, therapeutic strategies that enhance the targeting of oncogenic mechanisms in an acidic microenvironment should be effective. We report here that drugs which abrogate mitochondrial respiration show enhanced cytotoxicity against melanoma cells in a normoxic but acidic extracellular pH, independent from P53 mutations, BRAF (V600E) mutations, and/or resistance against BRAF inhibitors. Conversely, the cytotoxicity against melanoma cells of mitochondrial inhibitors is impaired by a neutral or alkaline extracellular pH, and systemic alkalinization with NaHCO enhanced subcutaneous tumor growth and lung metastasis of B16F10 cells in mice treated with the mitochondrial inhibitor phenformin. Intracellular calcium (Ca) was significantly increased in melanoma cells treated with mitochondrial inhibitors at an acidic extracellular pH and an intracellular Ca chelator, BAPTA/AM, inhibited cytoplasmic Ca as well as melanoma cell death. Surprisingly, ROS scavengers synergized with increased apoptosis in cells treated with mitochondrial inhibitors, suggesting that ROS contributes to cell survival in this context. Notably, the cytotoxic enhancement of mitochondrial inhibitors by acidity was distinct from PGC1alpha-driven mitochondrial addiction, from therapy-induced senescence, and from slow, JARID1B-high-associated cell cycling, all of which have been shown to promote vulnerability to mitochondrial inhibition. These data indicate that extracellular pH profoundly modulates the cytotoxicity of mitochondrial inhibitors against cancer cells. .
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0235DOI Listing
May 2017

Synergistic effects of ion transporter and MAP kinase pathway inhibitors in melanoma.

Nat Commun 2016 08 22;7:12336. Epub 2016 Aug 22.

Department of Pediatrics, Children's Research Institute, Dallas, Texas 75390, USA.

New therapies are required for melanoma. Here, we report that multiple cardiac glycosides, including digitoxin and digoxin, are significantly more toxic to human melanoma cells than normal human cells. This reflects on-target inhibition of the ATP1A1 Na(+)/K(+) pump, which is highly expressed by melanoma. MEK inhibitor and/or BRAF inhibitor additively or synergistically combined with digitoxin to induce cell death, inhibiting growth of patient-derived melanomas in NSG mice and synergistically extending survival. MEK inhibitor and digitoxin do not induce cell death in human melanocytes or haematopoietic cells in NSG mice. In melanoma, MEK inhibitor reduces ERK phosphorylation, while digitoxin disrupts ion gradients, altering plasma membrane and mitochondrial membrane potentials. MEK inhibitor and digitoxin together cause intracellular acidification, mitochondrial calcium dysregulation and ATP depletion in melanoma cells but not in normal cells. The disruption of ion homoeostasis in cancer cells can thus synergize with targeted agents to promote tumour regression in vivo.
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http://dx.doi.org/10.1038/ncomms12336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996948PMC
August 2016

Desmoglein 2 promotes vasculogenic mimicry in melanoma and is associated with poor clinical outcome.

Oncotarget 2016 Jul;7(29):46492-46508

Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA, Australia.

Tumors can develop a blood supply not only by promoting angiogenesis but also by forming vessel-like structures directly from tumor cells, known as vasculogenic mimicry (VM). Understanding mechanisms that regulate VM is important, as these might be exploitable to inhibit tumor progression. Here, we reveal the adhesion molecule desmoglein 2 (DSG2) as a novel mediator of VM in melanoma. Analysis of patient-derived melanoma cell lines and tumor tissues, and interrogation of The Cancer Genome Atlas (TCGA) data, revealed that DSG2 is frequently overexpressed in primary and metastatic melanomas compared to normal melanocytes. Notably, this overexpression was associated with poor clinical outcome. DSG2+ melanoma cells self-organized into tube-like structures on Matrigel, indicative of VM activity, which was inhibited by DSG2 knockdown or treatment with a DSG2-blocking peptide. Mechanistic studies revealed that DSG2 regulates adhesion and cell-cell interactions during tube formation, but does not control melanoma cell viability, proliferation or motility. Finally, analysis of patient tumors revealed a correlation between DSG2 expression, VM network density and expression of VM-associated genes. These studies identify DSG2 as a key regulator of VM activity in human melanoma and suggest this molecule might be therapeutically targeted to reduce tumor blood supply and metastatic spread.
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http://dx.doi.org/10.18632/oncotarget.10216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216812PMC
July 2016

A non-canonical role for desmoglein-2 in endothelial cells: implications for neoangiogenesis.

Angiogenesis 2016 10 23;19(4):463-86. Epub 2016 Jun 23.

Centre for Cancer Biology, University of South Australia and SA Pathology, PO Box 14, Rundle Mall, Adelaide, SA, 5000, Australia.

Desmogleins (DSG) are a family of cadherin adhesion proteins that were first identified in desmosomes and provide cardiomyocytes and epithelial cells with the junctional stability to tolerate mechanical stress. However, one member of this family, DSG2, is emerging as a protein with additional biological functions on a broader range of cells. Here we reveal that DSG2 is expressed by non-desmosome-forming human endothelial progenitor cells as well as their mature counterparts [endothelial cells (ECs)] in human tissue from healthy individuals and cancer patients. Analysis of normal blood and bone marrow showed that DSG2 is also expressed by CD34(+)CD45(dim) hematopoietic progenitor cells. An inability to detect other desmosomal components, i.e., DSG1, DSG3 and desmocollin (DSC)2/3, on these cells supports a solitary role for DSG2 outside of desmosomes. Functionally, we show that CD34(+)CD45(dim)DSG2(+) progenitor cells are multi-potent and pro-angiogenic in vitro. Using a 'knockout-first' approach, we generated a Dsg2 loss-of-function strain of mice (Dsg2 (lo/lo)) and observed that, in response to reduced levels of Dsg2: (i) CD31(+) ECs in the pancreas are hypertrophic and exhibit altered morphology, (ii) bone marrow-derived endothelial colony formation is impaired, (iii) ex vivo vascular sprouting from aortic rings is reduced, and (iv) vessel formation in vitro and in vivo is attenuated. Finally, knockdown of DSG2 in a human bone marrow EC line reveals a reduction in an in vitro angiogenesis assay as well as relocalisation of actin and VE-cadherin away from the cell junctions, reduced cell-cell adhesion and increased invasive properties by these cells. In summary, we have identified DSG2 expression in distinct progenitor cell subpopulations and show that, independent from its classical function as a component of desmosomes, this cadherin also plays a critical role in the vasculature.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026727PMC
http://dx.doi.org/10.1007/s10456-016-9520-yDOI Listing
October 2016

CD271 Expression on Patient Melanoma Cells Is Unstable and Unlinked to Tumorigenicity.

Cancer Res 2016 07 20;76(13):3965-77. Epub 2016 Jun 20.

Cancer Development and Treatment Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Australia. Sir Peter MacCallum Department of Pathology, University of Melbourne, Parkville, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Australia. Department of Cancer Medicine, Peter MacCallum Cancer Centre, East Melbourne, Australia.

The stability of markers that identify cancer cells that propagate disease is important to the outcomes of targeted therapy strategies. In human melanoma, conflicting data exist as to whether hierarchical expression of CD271/p75/NGFR (nerve growth factor receptor) marks cells with enriched tumorigenicity, which would compel their specific targeting in therapy. To test whether these discrepancies relate to differences among groups in assay approaches, we undertook side-by-side testing of published methods of patient-derived melanoma xenografting (PDX), including comparisons of tissue digestion procedures or coinjected Matrigel formulations. We found that CD271(-) and CD271(+) melanoma cells from each of seven patients were similarly tumorigenic, regardless of assay variations. Surprisingly variable CD271 expression patterns were observed in the analyses of sibling PDX tumors (n = 68) grown in the same experiments from either CD271(-) or CD271(+) cells obtained from patients. This indicates unstable intratumoral lineage relationships between CD271(-) and CD271(+) melanoma cells that are inconsistent with classical, epigenetically based theories of disease progression, such as the cancer stem cell and plasticity models. SNP genotyping of pairs of sibling PDX tumors grown from phenotypically identical CD271(-) or CD271(+) cells showed large pairwise differences in copy number (28%-48%). Differences were also apparent in the copy number profiles of CD271(-) and CD271(+) cells purified directly from each of the four melanomas (1.4%-23%). Thus, CD271 expression in patient melanomas is unstable, not consistently linked to increased tumorigenicity and associated with genetic heterogeneity, undermining its use as a marker in clinical studies. Cancer Res; 76(13); 3965-77. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-2377DOI Listing
July 2016

UV-Associated Mutations Underlie the Etiology of MCV-Negative Merkel Cell Carcinomas.

Cancer Res 2015 Dec 1;75(24):5228-34. Epub 2015 Dec 1.

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. Department of Pathology, University of Melbourne, Melbourne, Victoria, Australia.

Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viral-negative tumors have been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viral-negative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n = 13) and -negative (n = 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell-infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-1877DOI Listing
December 2015

Antisense oligonucleotide-mediated MDM4 exon 6 skipping impairs tumor growth.

J Clin Invest 2016 Jan 23;126(1):68-84. Epub 2015 Nov 23.

MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient-derived xenograft (PDX) mouse models, antisense oligonucleotide-mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target.
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http://dx.doi.org/10.1172/JCI82534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701541PMC
January 2016

Tumour procurement, DNA extraction, coverage analysis and optimisation of mutation-detection algorithms for human melanoma genomes.

Pathology 2015 Dec;47(7):683-93

1Melanoma Institute Australia, North Sydney, NSW 2Sydney Medical School, The University of Sydney, Camperdown, NSW 3Immunogenomics Laboratory, Australian National University, Canberra, ACT 4Oncogenomics Laboratory, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Qld 5Centre for Cancer Research, The University of Sydney at Westmead Millennium Institute, Westmead, NSW 6Bioplatforms Australia, Macquarie University, North Ryde, NSW 7Ludwig Institute for Cancer Research, Olivia Newton-John Cancer and Wellness Centre, Austin Health, Heidelberg, Vic 8The Cancer Development and Treatment Laboratory, Peter MacCallum Cancer Centre and Sir Peter MacCallum Department of Oncology, The University of Melbourne, Vic 9Departments of Melanoma and Surgical Oncology 10Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia; these authors contributed equally.

Whole genome sequencing (WGS) of cancer patients' tumours offers the most comprehensive method of identifying both novel and known clinically-actionable genomic targets. However, the practicalities of performing WGS on clinical samples are poorly defined.This study was designed to test sample preparation, sequencing specifications and bioinformatic algorithms for their effect on accuracy and cost-efficiency in a large WGS analysis of human melanoma samples.WGS was performed on melanoma cell lines (n = 15) and melanoma fresh frozen tumours (n = 222). The appropriate level of coverage and the optimal mutation detection algorithm for the project pipeline were determined.An incremental increase in sequencing coverage from 36X to 132X in melanoma tissue samples and 30X to 103X for cell lines only resulted in a small increase (1-2%) in the number of mutations detected, and the quality scores of the additional mutations indicated a low probability that the mutations were real. The results suggest that 60X coverage for melanoma tissue and 40X for melanoma cell lines empower the detection of 98-99% of informative single nucleotide variants (SNVs), a sensitivity level at which clinical decision making or landscape research projects can be carried out with a high degree of confidence in the results. Likewise the bioinformatic mutation analysis methodology strongly influenced the number and quality of SNVs detected. Detecting mutations in the blood genomes separate to the tumour genomes generated 41% more SNVs than if the blood and melanoma tissue genomes were analysed simultaneously. Therefore, simultaneous analysis should be employed on matched melanoma tissue and blood genomes to reduce errors in mutation detection.This study provided valuable insights into the accuracy of SNV with WGS at various coverage levels in human clinical cancer specimens. Additionally, we investigated the accuracy of the publicly available mutation detection algorithms to detect cancer specific SNVs which will aid researchers and clinicians in study design and implementation of WGS for the identification of somatic mutations in other cancers.
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http://dx.doi.org/10.1097/PAT.0000000000000324DOI Listing
December 2015

The transcription cofactor c-JUN mediates phenotype switching and BRAF inhibitor resistance in melanoma.

Sci Signal 2015 Aug 18;8(390):ra82. Epub 2015 Aug 18.

Molecular Oncology Laboratory, Peter MacCallum Cancer Centre, St. Andrew's Place, East Melbourne, Victoria 3002, Australia. Oncogenic Signalling and Growth Control Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia. Sir Peter MacCallum Department of Oncology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Victoria 3010, Australia. Cancer Therapeutics Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia. Department of Pathology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Victoria 3010, Australia.

Most patients with BRAF-mutant metastatic melanoma display remarkable but incomplete and short-lived responses to inhibitors of the BRAF kinase or the mitogen-activated protein kinase kinase (MEK), collectively BRAF/MEK inhibitors. We found that inherent resistance to these agents in BRAF(V600)-mutant melanoma cell lines was associated with high abundance of c-JUN and characteristics of a mesenchymal-like phenotype. Early drug adaptation in drug-sensitive cell lines grown in culture or as xenografts, and in patient samples during therapy, was consistently characterized by down-regulation of SPROUTY4 (a negative feedback regulator of receptor tyrosine kinases and the BRAF-MEK signaling pathway), increased expression of JUN and reduced expression of LEF1. This coincided with a switch in phenotype that resembled an epithelial-mesenchymal transition (EMT). In cultured cells, these BRAF inhibitor-induced changes were reversed upon removal of the drug. Knockdown of SPROUTY4 was sufficient to increase the abundance of c-JUN in the absence of drug treatment. Overexpressing c-JUN in drug-naïve melanoma cells induced similar EMT-like phenotypic changes to BRAF inhibitor treatment, whereas knocking down JUN abrogated the BRAF inhibitor-induced early adaptive changes associated with resistance and enhanced cell death. Combining the BRAF inhibitor with an inhibitor of c-JUN amino-terminal kinase (JNK) reduced c-JUN phosphorylation, decreased cell migration, and increased cell death in melanoma cells. Gene expression data from a panel of melanoma cell lines and a patient cohort showed that JUN expression correlated with a mesenchymal gene signature, implicating c-JUN as a key mediator of the mesenchymal-like phenotype associated with drug resistance.
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http://dx.doi.org/10.1126/scisignal.aab1111DOI Listing
August 2015