Sir Peter MacCallum Department of Oncology - Research Publications
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ItemHIGH DOSE-RATE BRACHYTHERAPY OF LOCALIZED PROSTATE CANCER CONVERTS TUMORS FROM COLD TO HOT(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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ItemEARLY-PHENOTYPE LEWIS Y CAR-T CELLS PERSIST BETTER IN VIVO AND INDUCE SOLID TUMOR REGRESSION IN COMBINATION WITH ANTI-PD1(BMJ PUBLISHING GROUP, 2020-11)Background Chimeric antigen receptor (CAR-T) cells are a promising new therapy for patients with cancer. However, in contrast to their success in B cell malignancies, CAR-T cells targeting solid cancers have had limited success so far due to their poor proliferation and poor long-term persistence in vivo. To address this issue, we used naïve T cells to generate second-generation CAR-T cells recognizing the tumor antigen Lewis Y (LeY), termed ‘early’ CAR-T cells. Methods Purified naïve T cells were activated by CD3/CD28 soluble tetrameric antibody complex, retrovirally transduced (LeY scFv-CD3z-CD28 CAR) and expanded in IL-7/IL-15. The early LeY CAR-T cell function was tested in vitro for cytotoxicity (Cr-release and degranulation), proliferation, and cytokine secretion by CBA, either de novo or following chronic stimulation for 1 month. Finally, early CAR-T cell persistence and anti-tumor efficacy was assessed in the OVCAR3-NSG model, in the presence or absence of anti-PD-1. Results The early-CAR-T cells comprised stem cell memory-like (CD95+, CD62L+, CD45RA+) and central memory phenotype (CD95+, CD62L+, CD45RA-) T cells with increased expression of ICOS, Ki67, TCF7 and CD27 (Figure 1). The early-CAR-T cells retained potent antigen-specific cytotoxicity, and secreted significantly higher levels of cytokines (IFN-?, TNF-a and IL-2) and increased proliferation compared to conventional CAR-T cells. Importantly, early-CAR-T cells had a significantly higher proliferative capacity after long-term chronic stimulation compared to conventional CAR-T cells (figure 2), and CD4+ CAR-T cells were critical for effective early CD8+ CAR-T cell proliferation capacity in vitro (figure 3). Early CAR-T cells had significantly better in vivo tumor control compared to conventional CAR-T cells (Figure 4), this was associated with increased CAR-T cell persistence. Because chronically stimulated early-LeY-CAR-T cells expressed PD-1 (figure 2), and OVCAR-3 cells expressed PD-L1 when co-cultured with LeY-CAR-T cells (figure 5), we combined early LeY-CAR-T cells with anti-PD-1 therapy and observed complete tumour regression in these mice. Interestingly, early LeY-CAR-T cell plus anti-PD-1 treatment also enhanced the percentage of circulating stem-cell memory like CAR-T cells in vivo (figure 5). Abstract 126 Figure 1Early-CAR-T protocol, including Naïve-T cells purification and expansion in IL-7 and IL-15 promotes the maintenance of a TSCM and TCM phenotype. A) Scheme of the 7-day production protocol for Early-CAR-T cells. B) Phenotype by FACs of the conventional CAR-T cells and the Early-CAR-T cells. Pooled data in triplicate for 6 donors. C) Phenotype by Mass cytometry comparing the Conventional-CAR-T cells vs Early-CAR-T cells vs Early-CD8-CAR-T cells. Data for one donor representative of 3 different donors Abstract 126 Figure 2Early-CAR-T cells are comparable in vitro to conventional CAR-T cells in terms of killing but have a better proliferation capacity that persists after chronic stimulation. The long-term stimulated early- CAR-T cells maintain their memory phenotype and upregulated PD-1. A) Chromium release assay against the LeY+ cell line (OVCAR3), data for one donor representative of 3 other donors. B) Cytokine secretion evaluated by CBA after coculture with the LeY+ cell line (OVCAR3) or with the LeY- cell line (MDA-MB435). C) Division index of CAR-T cells quantified with CTV. D) Evaluation of the differentiation, proliferation and cytotoxicity of the CAR-T cells after chronic stimulation Abstract 126 Figure 3Early-CD4+- CAR-T cells are critical for the proliferation capacity of the Early-CD8+-CAR-T cells. A) Scheme of the CD4-depletion protocol to compare Early-CD8-CAR-T proliferation with or without CD4-T cells. B) Division index of CD4-depleted Early-CAR-T cells, CD8-T cells from bulk Early-CAR-T cells, and from CD4+ T cells from bulk Early-CAR-T-cells quantified with CTV Abstract 126 Figure 4Early-CAR-T cells show in vivo a better persistence and a better proliferation capacity associated with a better tumoral control. A) Design of the in vivo experiment (n=7 mice per group) B) T-cell persistence in peripheral blood was measured by FACS. C) Speakman correlation (Day 13) between Tumor size and% CAR-T- cells. D) Tumor kinetic and Kaplan-Meier analysis of survival of OVCAR-bearing NSG mice treated with Conventional CAR-T cells, or Early-CAR-T cells or Low-dose of Early-CAR-T cells Abstract 126 Figure 5Anti-PD1 treatment enhance the efficacy of the Early-CAR-T cells. A) Upregulation of PD-L1 on OVCAR3 when expanded in the supernatant from co-culture of OVCAR3 with LeY-CAR-T cells. B) Design of the in vivo experiment (n=7 mice per group). C) T-cell persistence, phenotype and anti-human IgG4 in peripheral blood were measured by FACS. D) Tumor kinetic of OVCAR-bearing NSG mice treated with Early-CAR-T cells or Early-CAR-T cells + Nivolumab Conclusions Our early CAR-T cells have better cytokine secretion and proliferation than conventional CAR-T cells. Early CAR-T cells also have superior anti-tumor efficacy in vivo, they have better persistence and maintain the circulating T cell memory pool. Importantly, low dose early-LeY-CAR-T cells combined with anti-PD1-treatment leads to complete clearance of LeY+ solid tumors in vivo. The early CAR-T cell production protocol is directly translatable for improving CAR-T cell efficacy in clinical trials for patients with solid tumors.
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ItemA TOOLKIT FOR THE QUANTITATIVE ANALYSIS OF THE SPATIAL DISTRIBUTION OF CELLS OF THE TUMOR IMMUNE MICROENVIRONMENT(BMJ PUBLISHING GROUP, 2020-11)
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ItemCD8+TISSUE-RESIDENT MEMORY T CELLS ARE TUMOUR REACTIVE AND INCREASE AFTER IMMUNOTHERAPY IN A CASE OF METASTATIC MUCOSAL MELANOMA(BMJ PUBLISHING GROUP, 2020-11)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.