Sir Peter MacCallum Department of Oncology - Research Publications
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ItemMAIT cells regulate NK cell-mediated tumor immunityPetley, 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.
ItemTranscriptome sequencing and multi-plex imaging of prostate cancer microenvironment reveals a dominant role for monocytic cells in progressionMangiola, 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.
ItemNo Preview AvailableMyeloma natural killer cells are exhausted and have impaired regulation of activationD'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, 2021-09-01)Not available.
ItemRegulatory T Cells Shape the Differential Impact of Radiation Dose-Fractionation Schedules on Host Innate and Adaptive Antitumor Immune DefensesSia, J ; Hagekyriakou, J ; Chindris, I ; Albarakati, H ; Leong, T ; Schlenker, R ; Keam, SP ; Williams, SG ; Neeson, PJ ; Johnstone, RW ; Haynes, NM (ELSEVIER SCIENCE INC, 2021-08-30)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.
ItemHIGH DOSE-RATE BRACHYTHERAPY OF LOCALIZED PROSTATE CANCER CONVERTS TUMORS FROM COLD TO HOTKeam, 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, 2020-11-01)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).
ItemA TOOLKIT FOR THE QUANTITATIVE ANALYSIS OF THE SPATIAL DISTRIBUTION OF CELLS OF THE TUMOR IMMUNE MICROENVIRONMENTTrigos, 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, 2020-11-01)
ItemCD8+TISSUE-RESIDENT MEMORY T CELLS ARE TUMOUR REACTIVE AND INCREASE AFTER IMMUNOTHERAPY IN A CASE OF METASTATIC MUCOSAL MELANOMAPizzolla, A ; Keam, S ; Vergara, I ; Caramia, F ; Wang, M ; Kocovski, N ; ThuNgoc, N ; Macdonald, S ; Tantalo, D ; Petrone, P ; Yeang, HXA ; Gyorki, D ; Weppler, A ; Au-Yeung, G ; Sandhu, S ; Perdicchio, M ; McArthur, G ; Papenfuss, T ; Neeson, P (BMJ PUBLISHING GROUP, 2020-11-01)Background Mucosal melanoma is a rare subtype of melanoma originating from mucosal tissues (1), metastases are very aggressive and respond poorly to therapy, including immune checkpoint inhibitors (ICI) such as anti-CTLA4 and anti-PD1 antibodies (2–5). CD8+ T cells constitute the most abundant immune infiltrate in metastatic melanoma, of which the Tissue Resident Memory subset (TRM) is of particular interest (6). CD8+ TRM cells express the highest levels of immune checkpoint receptors, proliferate in response to ICI and correlate with longer disease-free and overall survival (6–8). The immune landscape in mucosal melanoma remains poorly characterized. We aimed to: 1) phenotype CD8+ T cells and TRM infiltrating metastatic mucosal melanoma, 2) characterize the clonality of TRM in relation to other CD8+ T cell subsets and 3) define the capacity of CD8+ T cells and TRM to respond to melanoma cells and to in vivo and in vitro anti-PD1 treatment. Methods We investigated the CD8+ T and TRM cells infiltrating two temporally- and spatially-distant subcutaneous metastases, these originated from a primary vaginal mucosal melanoma. One metastasis was excised prior to anti-PD1 treatment and one was anti-PD1 refractory, having progressed on treatment. We used mass cytometry and single-cell RNA and TCR sequencing to characterise the phenotype and clonality of the T cells, multiplex immunohistochemistry to define their spatial relationship with tumour cells and other T cells, and functional assays to determine TRM response to tumour cells (figure 1). Results CD8+ TRM frequency increased with time and anti-PD1 treatment, forming clusters at the tumour margin. T cells in the anti-PD1 refractory lesion were more activated than T cells in the first tumour and were bound by anti-PD1 antibody in vivo. T cells could not be stimulated by anti-PD1 directly ex vivo. Both metastatic lesions shared common T cell clusters including TRM. Furthermore, TRM in each tumour shared T cell clones, suggesting the presence of common antigens between metastatic sites. Indeed, the two metastases had a similar mutational profile. In vitro expanded tumour infiltrating lymphocytes from both lesions recognized tumour cells from both lesions and the same neoantigen generated from a single point mutation in the gene CDKN1C. Finally, tumour cells stimulated TRM cells more robustly than other T cells subsets. Abstract 548 Figure 1Graphical depiction of the methods used to characterise T cells in mucosal metastatic melanoma Conclusions In this patient with vaginal mucosal melanoma, subsequent melanoma metastases of clonal origin attracted CD8+ T cells of similar specificity, among which TRM cells responded more vigorously to tumour cells than other T cells subsets. Acknowledgements The authors would like to acknowledge imCORE La Hoffmann- Roche Ltd. for funding. Ethics Approval Patients diagnosed with stage 3 or 4 metastatic melanoma and undergoing clinically indicated surgery were enrolled in prospective studies approved by the Peter MacCallum Cancer Centre human ethics research committee (13/141). All experimental protocols have been approved and clinical data has been collected prospectively. References Carvajal RD, Hamid O, Ariyan C. Mucosal Melanoma. [cited 2020 Apr 1]; Available from: https://www.uptodate.com/contents/mucosal-melanoma Del Vecchio M, Di Guardo L, Ascierto PA, Grimaldi AM, Sileni VC, Pigozzo J, et al. Efficacy and safety of ipilimumab 3 mg/kg in patients with pretreated, metastatic, mucosal melanoma. Eur J Cancer Oxf Engl 1990; 2014 Jan;50(1):121–7. Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, Lawrence DP, et al. Ipilimumab for patients with advanced mucosal melanoma. The Oncologist 2013 Jun;18(6):726–32. D’Angelo SP, Larkin J, Sosman JA, Lebbé C, Brady B, Neyns B, et al. Efficacy and safety of nivolumab alone or in combination with ipilimumab in patients with mucosal melanoma: a pooled analysis. J Clin Oncol Off J Am Soc Clin Oncol. 2017 Jan 10;35(2):226–35. Hamid O, Robert C, Ribas A, Hodi FS, Walpole E, Daud A, et al. Antitumour activity of pembrolizumab in advanced mucosal melanoma: a post-hoc analysis of KEYNOTE-001, 002, 006. Br J Cancer 2018;119(6):670–4. Boddupalli CS, Bar N, Kadaveru K, Krauthammer M, Pornputtapong N, Mai Z, et al. Interlesional diversity of T cell receptors in melanoma with immune checkpoints enriched in tissue-resident memory T cells. JCI Insight [Internet]. 2016 Dec 22 [cited 2019 Apr 24];1(21). Available from: https://insight.jci.org/articles/view/88955 Edwards J, Wilmott JS, Madore J, Gide TN, Quek C, Tasker A, et al. CD103+ Tumor-resident CD8+ T cells are associated with improved survival in immunotherapy-naïve melanoma patients and expand significantly during anti-PD-1 treatment. Clin Cancer Res Off J Am Assoc Cancer Res 2018 Jul 1;24(13):3036–45. Savas P, Virassamy B, Ye C, Salim A, Mintoff CP, Caramia F, et al. Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis. Nat Med 2018 Jul;24(7):986–93.
ItemHigh dose-rate brachytherapy of localized prostate cancer converts tumors from cold to hotKeam, SP ; Halse, H ; Nguyen, T ; Wang, M ; Van Kooten Losio, N ; Mitchell, C ; Caramia, F ; Byrne, DJ ; Haupt, S ; Ryland, G ; Darcy, PK ; Sandhu, S ; Blombery, P ; Haupt, Y ; Williams, SG ; Neeson, PJ (BMJ PUBLISHING GROUP, 2020-01-01)BACKGROUND: Prostate cancer (PCa) has a profoundly immunosuppressive microenvironment and is commonly immune excluded with few infiltrative lymphocytes and low levels of immune activation. High-dose radiation has been demonstrated to stimulate the immune system in various human solid tumors. We hypothesized that localized radiation therapy, in the form of high dose-rate brachytherapy (HDRBT), would overcome immune suppression in PCa. METHODS: To investigate whether HDRBT altered prostate immune context, we analyzed preradiation versus postradiation human tissue from a cohort of 24 patients with localized PCa that received HDRBT as primary treatment (RadBank cohort). We performed Nanostring immune gene expression profiling, digital spatial profiling, and high-throughput immune cell multiplex immunohistochemistry analysis. We also resolved tumor and nontumor zones in spatial and bioinformatic analyses to explore the immunological response. RESULTS: Nanostring immune profiling revealed numerous immune checkpoint molecules (eg, B7-H3, CTLA4, PDL1, and PDL2) and TGFβ levels were increased in response to HDRBT. We used a published 16-gene tumor inflammation signature (TIS) to divide tumors into distinct immune activation states (high:hot, intermediate and low:cold) and showed that most localized PCa are cold tumors pre-HDRBT. Crucially, HDRBT converted 80% of these 'cold'-phenotype tumors into an 'intermediate' or 'hot' class. We used digital spatial profiling to show these HDRBT-induced changes in prostate TIS scores were derived from the nontumor regions. Furthermore, these changes in TIS were also associated with pervasive changes in immune cell density and spatial relationships-in particular, between T cell subsets and antigen presenting cells. We identified an increased density of CD4+ FOXP3+ T cells, CD68+ macrophages and CD68+ CD11c+ dendritic cells in response to HDRBT. The only subset change specific to tumor zones was PDL1- macrophages. While these immune responses were heterogeneous, HDRBT induced significant changes in immune cell associations, including a gained T cell and HMWCK+ PDL1+ interaction in tumor zones. CONCLUSION: In conclusion, we showed HDRBT converted "cold" prostate tumors into more immunologically activated "hot" tissues, with accompanying spatially organized immune infiltrates and signaling changes. Understanding and potentially harnessing these changes will have widespread implications for the future treatment of localized PCa, including rational use of combination radio-immunotherapy.
ItemTP53 Status, Patient Sex, and the Immune Response as Determinants of Lung Cancer Patient SurvivalFreudenstein, D ; Litchfield, C ; Caramia, F ; Wright, G ; Solomon, BJ ; Ball, D ; Keam, SP ; Neeson, P ; Haupt, Y ; Haupt, S (MDPI, 2020-06-01)Lung cancer poses the greatest cancer-related death risk and males have poorer outcomes than females, for unknown reasons. Patient sex is not a biological variable considered in lung cancer standard of care. Correlating patient genetics with outcomes is predicted to open avenues for improved management. Using a bioinformatics approach across non-small cell lung cancer (NSCLC) subtypes, we identified where patient sex, mutation of the major tumor suppressor gene, Tumour protein P53 (TP53), and immune signatures stratified outcomes in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), among datasets of The Cancer Genome Atlas (TCGA). We exposed sex and TP53 gene mutations as prognostic for LUAD survival. Longest survival in LUAD occurred among females with wild-type (wt) TP53 genes, high levels of immune infiltration and enrichment for pathway signatures of Interferon Gamma (INF-γ), Tumour Necrosis Factor (TNF) and macrophages-monocytes. In contrast, poor survival in men with LUAD and wt TP53 genes corresponded with enrichment of Transforming Growth Factor Beta 1 (TGFB1, hereafter TGF-β) and wound healing signatures. In LUAD with wt TP53 genes, elevated gene expression of immune checkpoint CD274 (hereafter: PD-L1) and also protein 53 (p53) negative-regulators of the Mouse Double Minute (MDM)-family predict novel avenues for combined immunotherapies. LUSC is dominated by male smokers with TP53 gene mutations, while a minor population of TCGA LC patients with wt TP53 genes unexpectedly had the poorest survival, suggestive of a separate etiology. We conclude that advanced approaches to LUAD and LUSC therapy lie in the consideration of patient sex, TP53 gene mutation status and immune signatures.