Sir Peter MacCallum Department of Oncology - Research Publications

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    Modelling aggressive prostate cancers of young men in immune-competent mice, driven by isogenic Trp53 alterations and Pten loss
    Mejia-Hernandez, JO ; Keam, SP ; Saleh, R ; Muntz, F ; Fox, SB ; Byrne, D ; Kogan, A ; Pang, L ; Huynh, J ; Litchfield, C ; Caramia, F ; Lozano, G ; He, H ; You, JM ; Sandhu, S ; Williams, SG ; Haupt, Y ; Haupt, S (SPRINGERNATURE, 2022-09-08)
    Understanding prostate cancer onset and progression in order to rationally treat this disease has been critically limited by a dire lack of relevant pre-clinical animal models. We have generated a set of genetically engineered mice that mimic human prostate cancer, initiated from the gland epithelia. We chose driver gene mutations that are specifically relevant to cancers of young men, where aggressive disease poses accentuated survival risks. An outstanding advantage of our models are their intact repertoires of immune cells. These mice provide invaluable insight into the importance of immune responses in prostate cancer and offer scope for studying treatments, including immunotherapies. Our prostate cancer models strongly support the role of tumour suppressor p53 in functioning to critically restrain the emergence of cancer pathways that drive cell cycle progression; alter metabolism and vasculature to fuel tumour growth; and mediate epithelial to mesenchymal-transition, as vital to invasion. Importantly, we also discovered that the type of p53 alteration dictates the specific immune cell profiles most significantly disrupted, in a temporal manner, with ramifications for disease progression. These new orthotopic mouse models demonstrate that each of the isogenic hotspot p53 amino acid mutations studied (R172H and R245W, the mouse equivalents of human R175H and R248W respectively), drive unique cellular changes affecting pathways of proliferation and immunity. Our findings support the hypothesis that individual p53 mutations confer their own particular oncogenic gain of function in prostate cancer.
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    Targeting MDM4 as a Novel Therapeutic Approach in Prostate Cancer Independent of p53 Status
    Mejia-Hernandez, JO ; Raghu, D ; Caramia, F ; Clemons, N ; Fujihara, K ; Riseborough, T ; Teunisse, A ; Jochemsen, AG ; Abrahmsen, L ; Blandino, G ; Russo, A ; Gamell, C ; Fox, SB ; Mitchell, C ; Takano, EA ; Byrne, D ; Miranda, PJ ; Saleh, R ; Thorne, H ; Sandhu, S ; Williams, SG ; Keam, SP ; Haupt, Y ; Haupt, S (MDPI, 2022-08)
    Metastatic prostate cancer is a lethal disease in patients incapable of responding to therapeutic interventions. Invasive prostate cancer spread is caused by failure of the normal anti-cancer defense systems that are controlled by the tumour suppressor protein, p53. Upon mutation, p53 malfunctions. Therapeutic strategies to directly re-empower the growth-restrictive capacities of p53 in cancers have largely been unsuccessful, frequently because of a failure to discriminate responses in diseased and healthy tissues. Our studies sought alternative prostate cancer drivers, intending to uncover new treatment targets. We discovered the oncogenic potency of MDM4 in prostate cancer cells, both in the presence and absence of p53 and also its mutation. We uncovered that sustained depletion of MDM4 is growth inhibitory in prostate cancer cells, involving either apoptosis or senescence, depending on the cell and genetic context. We identified that the potency of MDM4 targeting could be potentiated in prostate cancers with mutant p53 through the addition of a first-in-class small molecule drug that was selected as a p53 reactivator and has the capacity to elevate oxidative stress in cancer cells to drive their death.
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    Tumor immune microenvironment of primary prostate cancer with and without germline mutations in homologous recombination repair genes
    Trigos, AS ; Pasam, A ; Banks, P ; Wallace, R ; Guo, C ; Keam, S ; Thorne, H ; Mitchell, C ; Lade, S ; Clouston, D ; Hakansson, A ; Liu, Y ; Blyth, B ; Murphy, D ; Lawrentschuk, N ; Bolton, D ; Moon, D ; Darcy, P ; Haupt, Y ; Williams, SG ; Castro, E ; Olmos, D ; Goode, D ; Neeson, P ; Sandhu, S (BMJ PUBLISHING GROUP, 2022-06)
    BACKGROUND: Aberrations in homologous recombination repair (HRR) genes are emerging as important biomarkers for personalized treatment in prostate cancer (PCa). HRR deficiency (HRD) could affect the tumor immune microenvironment (TIME), potentially contributing to differential responses to poly ADP-ribose polymerase (PARP) inhibitors and immune checkpoint inhibitors. Spatial distribution of immune cells in a range of cancers identifies novel disease subtypes and is related to prognosis. In this study we aimed to determine the differences in the TIME of PCa with and without germline (g) HRR mutations. METHODS: We performed gene expression analysis, multiplex immunohistochemistry of T and B cells and quantitative spatial analysis of PCa samples from 36 patients with gHRD and 26 patients with sporadic PCa. Samples were archival tumor tissue from radical prostatectomies with the exception of one biopsy. Results were validated in several independent cohorts. RESULTS: Although the composition of the T cell and B cells was similar in the tumor areas of gHRD-mutated and sporadic tumors, the spatial profiles differed between these cohorts. We describe two T-cell spatial profiles across primary PCa, a clustered immune spatial (CIS) profile characterized by dense clusters of CD4+ T cells closely interacting with PD-L1+ cells, and a free immune spatial (FIS) profile of CD8+ cells in close proximity to tumor cells. gHRD tumors had a more T-cell inflamed microenvironment than sporadic tumors. The CIS profile was mainly observed in sporadic tumors, whereas a FIS profile was enriched in gHRD tumors. A FIS profile was associated with lower Gleason scores, smaller tumors and longer time to biochemical recurrence and metastasis. CONCLUSIONS: gHRD-mutated tumors have a distinct immune microenvironment compared with sporadic tumors. Spatial profiling of T-cells provides additional information beyond T-cell density and is associated with time to biochemical recurrence, time to metastasis, tumor size and Gleason scores.
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    Tissue-resident memory T cells from a metastatic vaginal melanoma patient are tumor-responsive T cells and increase after anti-PD-1 treatment
    Pizzolla, A ; Keam, SP ; Vergara, IA ; Caramia, F ; Thio, N ; Wang, M ; Kocovski, N ; Tantalo, D ; Jabbari, J ; Au-Yeung, G ; Sandhu, S ; Gyorki, DE ; Weppler, A ; Perdicchio, M ; McArthur, GA ; Papenfuss, AT ; Neeson, PJ (BMJ PUBLISHING GROUP, 2022-05)
    BACKGROUND: Vaginal melanoma (VM) is a rare cancer and has a poor response to immune checkpoint blockade (ICB). CD8+Tissue Resident Memory (TRM) T cells proliferate in response to ICB and correlate with longer survival in metastatic cutaneous melanoma. However, their capacity to respond to VM and their neoantigens is not known. METHODS: Using longitudinal samples, we explored the evolution of VM mutations by whole-exome sequencing and RNAseq, we also defined the immune context using multiplex immunohistochemistry and nanostring pan cancer immune profile. Then using fresh single cell suspensions of the metastatic samples, we explored VM T cells via mass cytometry and single cell RNAseq and T cell receptor sequencing (TCRseq). Finally, we investigated TRM, pre-TRM and exhausted T cell function against melanoma neo-antigens and melanoma differentiation antigens in vitro. RESULTS: Primary VM was non-inflamed and devoid of CD8+ TRM cells. In contrast, both metastases showed proliferating CD8+ TRM were clustered at the tumor margin, with increased numbers in the second ICB-refractory metastasis. The first metastasis showed dense infiltration of CD8+ T cells, the second showed immune exclusion with loss of melanoma cell Major histocompatibility complex (MHC)-I expression associated with downregulation of antigen presentation pathway gene expression. CD8+ TRM from both metastases responded to autologous melanoma cells more robustly than all other CD8+ T cell subsets. In addition, CD8+ TRM shared TCR clones across metastases, suggesting a response to common antigens, which was supported by recognition of the same neoantigen by expanded tumor infiltrating lymphocytes. CONCLUSIONS: In this study, we identified TRM clusters in VM metastases from a patient, but not primary disease. We showed TRM location at the tumor margin, and their superior functional response to autologous tumor cells, predicted neoantigens and melanoma differentiation antigens. These CD8+ TRM exhibited the highest tumor-responsive potential and shared their TCR with tumor-infiltrating effector memory T cells. This suggests VM metastases from this patient retain strong antitumor T cell functional responses; however, this response is suppressed in vivo. The loss of VG MHC-I expression is a common immune escape mechanism which was not addressed by anti-PD-1 monotherapy; rather an additional targeted approach to upregulate MHC-I expression is required.
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    MAIT cells regulate NK cell-mediated tumor immunity
    Petley, E ; Koay, H-F ; Henderson, MA ; Sek, K ; Todd, KL ; Keam, SP ; Lai, J ; House, IG ; Li, J ; Zethoven, M ; Chen, AXY ; Oliver, AJ ; Michie, J ; Freeman, AJ ; Giuffrida, L ; Chan, JD ; Pizzolla, A ; Mak, JYW ; McCulloch, TR ; Souza-Fonseca-Guimaraes, F ; Kearney, CJ ; Millen, R ; Ramsay, RG ; Huntington, ND ; McCluskey, J ; Oliaro, J ; Fairlie, DP ; Neeson, PJ ; Godfrey, D ; Beavis, PA ; Darcy, PK (NATURE PORTFOLIO, 2021-08-06)
    The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.
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    Transcriptome sequencing and multi-plex imaging of prostate cancer microenvironment reveals a dominant role for monocytic cells in progression
    Mangiola, S ; McCoy, P ; Modrak, M ; Souza-Fonseca-Guimaraes, F ; Blashki, D ; Stuchbery, R ; Keam, SP ; Kerger, M ; Chow, K ; Nasa, C ; Le Page, M ; Lister, N ; Monard, S ; Peters, J ; Dundee, P ; Williams, SG ; Costello, AJ ; Neeson, PJ ; Pal, B ; Huntington, ND ; Corcoran, NM ; Papenfuss, AT ; Hovens, CM (BMC, 2021-07-22)
    BACKGROUND: Prostate cancer is caused by genomic aberrations in normal epithelial cells, however clinical translation of findings from analyses of cancer cells alone has been very limited. A deeper understanding of the tumour microenvironment is needed to identify the key drivers of disease progression and reveal novel therapeutic opportunities. RESULTS: In this study, the experimental enrichment of selected cell-types, the development of a Bayesian inference model for continuous differential transcript abundance, and multiplex immunohistochemistry permitted us to define the transcriptional landscape of the prostate cancer microenvironment along the disease progression axis. An important role of monocytes and macrophages in prostate cancer progression and disease recurrence was uncovered, supported by both transcriptional landscape findings and by differential tissue composition analyses. These findings were corroborated and validated by spatial analyses at the single-cell level using multiplex immunohistochemistry. CONCLUSIONS: This study advances our knowledge concerning the role of monocyte-derived recruitment in primary prostate cancer, and supports their key role in disease progression, patient survival and prostate microenvironment immune modulation.
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    Myeloma natural killer cells are exhausted and have impaired regulation of activation
    D'Souza, C ; Keam, SP ; Yeang, HXA ; Neeson, M ; Richardson, K ; Hsu, AK ; Canfield, R ; Bezman, N ; Robbins, M ; Quach, H ; Ritchie, DS ; Harrison, SJ ; Trapani, JA ; Prince, HM ; Beavis, PA ; Darcy, PK ; Neeson, PJ (FERRATA STORTI FOUNDATION, 2021-09)
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    Regulatory T Cells Shape the Differential Impact of Radiation Dose-Fractionation Schedules on Host Innate and Adaptive Antitumor Immune Defenses
    Sia, J ; Hagekyriakou, J ; Chindris, I ; Albarakati, H ; Leong, T ; Schlenker, R ; Keam, SP ; Williams, SG ; Neeson, PJ ; Johnstone, RW ; Haynes, NM (ELSEVIER SCIENCE INC, 2021-10-01)
    PURPOSE: We examined how radiation dose per fraction (DPF) and total dose, as represented by biological effective dose (BED), can independently and differentially affect the immunomodulatory capacity of radiation therapy (RT). METHODS AND MATERIALS: AT3-OVA mammary and MC38 colorectal tumors in C57BL/6 mice were irradiated with rationally selected dose-fractionation schedules, alone or with immune-modulating or -depleting agents. Tumor growth was monitored as a readout of therapeutic response. Flow cytometry and RNA sequencing of mouse tumors and analysis of transcriptomic data sets from irradiated human cancers were used to examine the immunomodulatory effects of the different radiation schedules. RESULTS: In AT3-OVA tumors, radiation DPF rather than BED determined the ability of RT to evoke local antitumor CD8+ T cell responses and synergize with anti-PD-1 therapy. Natural killer cell-mediated control of irradiated tumors was more sensitive to radiation BED. Radiation-induced regulatory T cell (Treg) responses, which were detected in both mouse and human tumors, were a major factor underlying the differential activation of adaptive immunity by radiation DPF and the activity of natural killer cells during the early phase of response to RT. Targeted inhibition of Treg responses within irradiated tumors rescued and enhanced local tumor control by RT and permitted the generation of abscopal and immunologic memory responses, irrespective of radiation schedule. MC38 tumors did not support the induction of an amplified Treg response to RT and were highly vulnerable to its immunoadjuvant effects. CONCLUSIONS: Local radiation-induced Treg responses are influenced by radiation schedule and tumor type and are a critical determinant of the immunoadjuvant potential of RT and its ability to synergize with T cell-targeted immunotherapy.
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    HIGH DOSE-RATE BRACHYTHERAPY OF LOCALIZED PROSTATE CANCER CONVERTS TUMORS FROM COLD TO HOT
    Keam, S ; Halse, H ; ThuNgoc, N ; Wang, M ; Losio, NVK ; Mitchell, C ; Caramia, F ; Byrne, D ; Haupt, S ; Ryland, G ; Darcy, P ; Sandhu, S ; Blombery, P ; Haupt, Y ; Williams, S ; Neeson, P (BMJ PUBLISHING GROUP, 2020-11)
    Background Prostate cancer is frequently cured with high dose-rate brachytherapy (HDRBT) radiation as a front-line treatment. Although considered to be an immune-excluded tissue, immune responses to radiation are implicated in driving tumour-eradication in prostate cancer.1 This has not been proven, and yet is used as the rationale for clinical trials combining radiation and immunotherapies.2 We hypothesise that there is a predictable relationship between radiation and the immune responses in prostate cancer that could be used to provide sound rationale for specific immune interventions in solid tumours that are made possible by radiation therapy. Methods We present here new results stemming from our recently published immunoprofiling study of world-unique pre- and post-radiation tissues from 24 prostate cancer patients (figure 1A), RadBank cohort).3 These samples were assessed using immune cell multiplex IHC, gene expression profiling, digital spatial profiling (DSP) and computational analysis of cell distribution. Results This study unequivocally revealed that high dose-rate radiation converts predominately ‘cold’ prostate tumour tissue to a more activated ‘hot’ state comprised of two sub-types (high and a less activated intermediate state). These changes were evident in increased tumour inflammation gene signatures and immune checkpoint expression, immune cell composition changes, and alterations in spatial interactions. However, as 20% of the patients did not respond, we also explored pre-treatment gene signatures of patient responses to radiation – identifying potential mechanisms that prime tissues to respond more favourably. Most recently, we have explored three other important facets of the immune response to HDRBT: (i) putative differential drivers of high and intermediate responses (figure 1B), (ii) TCR clonality changes (figure 1C), and (iii) the influence of clinical features (e.g. Gleason grade) and treatment (e.g. androgen deprivation) (figure 1D). Differential expression analysis has identified key molecules (e.g. CD40LG and Lck expression) which are associated with higher activation responses. TCR sequencing of pre- and post-HDRBT tissue and peripheral circulating cells is also suggestive of engagement of the adaptive immune system and the emergence of tumor-specific T cells. Finally, multivariate analysis has also revealed that higher grade tumours exhibit higher basal levels of activation and IC expression – making them less sensitive to immune activation by HDRBT. Abstract 580 Figure 1The effect of prostate brachytherapy on immune contexts(A) Study of immune response in 24 patients treated with HDRBT at Peter MacCallum Cancer Center ((DOI:10.1136/jitc2020-000792). Examples of new insights including (B) molecules associated with higher activation levels (e.g. Lck and CD40LG/CD154), (C) changes in T cell receptor dominance and diversity in tissue and peripheral circulation, and (D) effects of clinical attributes on immune modulators (e.g. TGFbeta) and TIS activation states. Conclusions We have begun to resolve clear patient and clinical classifiers based on immune responses to radiation, and identified patient groups likely to benefit from immune therapy alongside radiation. Importantly, these classifications are associated with baseline gene expression profiles that may be used for pre-clinical stratification and more sophisticated treatment paradigms. Ethics Approval All participants provided consent covering tissue research as part of a prospective tissue collection study for prostate radiobiology research, approved by the Human Research Ethics Committee at the Peter MacCallum Cancer Centre (PMCC; HREC approvals 10/68, 13/167, 18/204). Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal. References Dudzinski SO, et al., Combination immunotherapy and radiotherapy causes an abscopal treatment response in a mouse model of castration resistant prostate cancer. J Immunother Cancer 2019. 7(1): p. 218. Kwon E.D., et al., Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after docetaxel chemotherapy (CA184-043): a multicentre, randomised, double-blind, phase 3 trial. Lancet Oncol 2014;15(7): p. 700–12. Keam SP, et al., High dose-rate brachytherapy of localized prostate cancer converts tumors from cold to hot. J Immunother Cancer 2020;8(1).
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    A TOOLKIT FOR THE QUANTITATIVE ANALYSIS OF THE SPATIAL DISTRIBUTION OF CELLS OF THE TUMOR IMMUNE MICROENVIRONMENT
    Trigos, A ; Yang, T ; Feng, Y ; Ozcoban, V ; Doyle, M ; Pasam, A ; Kocovski, N ; Pizzolla, A ; Huang, Y-K ; Bass, G ; Keam, S ; Speed, T ; Neeson, P ; Sandhu, S ; Goode, D (BMJ PUBLISHING GROUP, 2020-11)